Structured Review

Affibody anti egfr affibody probe
Neck lymph node in normal mouse ( a ). The arrows indicate neck lymph nodes in dissection. H E staining showed lymph nodes in the excised cervical tissue ( a ). Two-phase combined imaging of SLNs in normal mice ( b ). 99m Tc-Phytate and <t>anti-EGFR</t> <t>affibody</t> probe were injected into the tongue of mice. Two excised neck specimens with lymph nodes from two mice are shown ( b , left). Autoradiography showed that 99m Tc radioactivity accumulated in the lymph nodes of each neck specimen and one solitary lymph node in the corresponding tissues of the left panel ( b , middle). NIR fluorescence was localized in the lymph nodes after anti-EGFR affibody probe injection into the tongue ( b , right).
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1) Product Images from "Imaging of Metastatic Cancer Cells in Sentinel Lymph Nodes using Affibody Probes and Possibility of a Theranostic Approach"

Article Title: Imaging of Metastatic Cancer Cells in Sentinel Lymph Nodes using Affibody Probes and Possibility of a Theranostic Approach

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms20020427

Neck lymph node in normal mouse ( a ). The arrows indicate neck lymph nodes in dissection. H E staining showed lymph nodes in the excised cervical tissue ( a ). Two-phase combined imaging of SLNs in normal mice ( b ). 99m Tc-Phytate and anti-EGFR affibody probe were injected into the tongue of mice. Two excised neck specimens with lymph nodes from two mice are shown ( b , left). Autoradiography showed that 99m Tc radioactivity accumulated in the lymph nodes of each neck specimen and one solitary lymph node in the corresponding tissues of the left panel ( b , middle). NIR fluorescence was localized in the lymph nodes after anti-EGFR affibody probe injection into the tongue ( b , right).
Figure Legend Snippet: Neck lymph node in normal mouse ( a ). The arrows indicate neck lymph nodes in dissection. H E staining showed lymph nodes in the excised cervical tissue ( a ). Two-phase combined imaging of SLNs in normal mice ( b ). 99m Tc-Phytate and anti-EGFR affibody probe were injected into the tongue of mice. Two excised neck specimens with lymph nodes from two mice are shown ( b , left). Autoradiography showed that 99m Tc radioactivity accumulated in the lymph nodes of each neck specimen and one solitary lymph node in the corresponding tissues of the left panel ( b , middle). NIR fluorescence was localized in the lymph nodes after anti-EGFR affibody probe injection into the tongue ( b , right).

Techniques Used: Dissection, Staining, Imaging, Mouse Assay, Injection, Autoradiography, Radioactivity, Fluorescence

Affibody molecules are shown with randomized positions in the binding site, which are indicated in blue. The molecule was labeled with ICG maleimide or IRDye 700. The figure was adopted from Wallberg H, et al. [ 46 ] ( a ). Thin-layer chromatography (TLC) showed that the fluorescence droplet spots of ICG-conjugated anti-EGFR, anti-HER2 affibody, and IR700-conjugated anti-EGFR affibody (2 µL) stayed at the starting point; in contrast, free ICG and free IR700 ran on the slides ( b ).
Figure Legend Snippet: Affibody molecules are shown with randomized positions in the binding site, which are indicated in blue. The molecule was labeled with ICG maleimide or IRDye 700. The figure was adopted from Wallberg H, et al. [ 46 ] ( a ). Thin-layer chromatography (TLC) showed that the fluorescence droplet spots of ICG-conjugated anti-EGFR, anti-HER2 affibody, and IR700-conjugated anti-EGFR affibody (2 µL) stayed at the starting point; in contrast, free ICG and free IR700 ran on the slides ( b ).

Techniques Used: Binding Assay, Labeling, Thin Layer Chromatography, Fluorescence

Near-infrared (NIR) imaging of cell lines by the addition of affibody probes to conditioned medium ( a , b ). SAS cells showed strong fluorescence signals of anti-EGFR affibody imaging probes ( a , left). SAS cells ( a , middle) expressed higher anti-epidermal growth factor receptor (EGFR) levels than MCF-7 cells ( a , right). SK-BR3 cells showed stronger fluorescence signals in anti-HER2 affibody imaging probes than MDA-MB231 cells ( b ). Histological section study ( c ). Anti-HER2 affibody probe was administered to histological sections of lymph nodes from breast cancer patients. Metastatic cancer cells are shown after hematoxylin and eosin (H E) staining ( c , upper row, left). Human epidermal growth factor receptor 2 (HER2) expression was positive in metastatic cancer cells by immunohistochemical staining ( c , upper row, middle). High-intensity NIR signals from the probe identically corresponded with the area of increased HER2 expression ( c , upper row, right). In the HER2-negative metastatic lymph node section ( c , lower row), immunohistochemical staining for HER2 and NIR signals were not visible in metastatic cancer cells ( c , lower row, middle, right). Scale bar in ( a ): 20 μm. Scale bar in ( c ): 200 μm.
Figure Legend Snippet: Near-infrared (NIR) imaging of cell lines by the addition of affibody probes to conditioned medium ( a , b ). SAS cells showed strong fluorescence signals of anti-EGFR affibody imaging probes ( a , left). SAS cells ( a , middle) expressed higher anti-epidermal growth factor receptor (EGFR) levels than MCF-7 cells ( a , right). SK-BR3 cells showed stronger fluorescence signals in anti-HER2 affibody imaging probes than MDA-MB231 cells ( b ). Histological section study ( c ). Anti-HER2 affibody probe was administered to histological sections of lymph nodes from breast cancer patients. Metastatic cancer cells are shown after hematoxylin and eosin (H E) staining ( c , upper row, left). Human epidermal growth factor receptor 2 (HER2) expression was positive in metastatic cancer cells by immunohistochemical staining ( c , upper row, middle). High-intensity NIR signals from the probe identically corresponded with the area of increased HER2 expression ( c , upper row, right). In the HER2-negative metastatic lymph node section ( c , lower row), immunohistochemical staining for HER2 and NIR signals were not visible in metastatic cancer cells ( c , lower row, middle, right). Scale bar in ( a ): 20 μm. Scale bar in ( c ): 200 μm.

Techniques Used: Imaging, Fluorescence, Multiple Displacement Amplification, Staining, Expressing, Immunohistochemistry

Imaging of metastatic cancer cells in lymph nodes. ( a ) In a mouse lymph node metastasis model, an anti-EGFR affibody probe was injected into the mouse tongue 24 h prior to sacrifice and lymph node dissection. Six lymph nodes were excised; three lymph nodes were highly fluorescent, and the remaining three lymph nodes were not fluorescent. Immunohistochemical staining for EGFR was found in fluorescence-positive lymph nodes (lymph node 2 (R), lymph node 3 (R)). EGFR expression was not visible in the nonfluorescent lymph node (lymph node 2 (L)). ( b ) Two lymph nodes, one from each side of the mouse, were dissected 24 h after anti-EGFR affibody probe injection into the tongue. The indocyanine green (ICG) fluorescence signal was obvious and corresponded to the immunohistochemically stained EGFR expression in the lymph nodes (red circles and arrows). The panel on the right is a magnified view of the two lymph nodes in the left panel of the NIR images.
Figure Legend Snippet: Imaging of metastatic cancer cells in lymph nodes. ( a ) In a mouse lymph node metastasis model, an anti-EGFR affibody probe was injected into the mouse tongue 24 h prior to sacrifice and lymph node dissection. Six lymph nodes were excised; three lymph nodes were highly fluorescent, and the remaining three lymph nodes were not fluorescent. Immunohistochemical staining for EGFR was found in fluorescence-positive lymph nodes (lymph node 2 (R), lymph node 3 (R)). EGFR expression was not visible in the nonfluorescent lymph node (lymph node 2 (L)). ( b ) Two lymph nodes, one from each side of the mouse, were dissected 24 h after anti-EGFR affibody probe injection into the tongue. The indocyanine green (ICG) fluorescence signal was obvious and corresponded to the immunohistochemically stained EGFR expression in the lymph nodes (red circles and arrows). The panel on the right is a magnified view of the two lymph nodes in the left panel of the NIR images.

Techniques Used: Imaging, Injection, Dissection, Immunohistochemistry, Staining, Fluorescence, Expressing

Dynamic imaging study. ( a ) NIR images showed changes in the signal from the anti-EGFR affibody probe in the lymph nodes of a normal control mouse (left). The fluorescence signal intensity was examined at 0.5, 1, 2, 3, 4, 6, and 24 h after the tongue injection in two mice (total four lymph nodes). The peak intensity was observed at one and two hours after the injection (right). The error bar shows the standard deviation. ( b ) The left panels are images of metastatic and nonmetastatic lymph nodes. Weak, almost equal NIR signal intensity was found in two nonmetastatic lymph nodes at 0.5 and 1 h post-injection. The signal intensity almost disappeared at 24 h after the injection (top, left). A high signal intensity remained in the metastatic lymph node (arrow) at 24 h after injection (bottom, left). The time-dependent NIR signal intensity of the anti-EGFR affibody probe is represented as a percentage of the initial signal intensity (30 min). The signal intensity ratio was greater for metastatic lymph nodes than nonmetastatic lymph nodes at 1, 2, 3, and 6 h after the injection (right panel). * p
Figure Legend Snippet: Dynamic imaging study. ( a ) NIR images showed changes in the signal from the anti-EGFR affibody probe in the lymph nodes of a normal control mouse (left). The fluorescence signal intensity was examined at 0.5, 1, 2, 3, 4, 6, and 24 h after the tongue injection in two mice (total four lymph nodes). The peak intensity was observed at one and two hours after the injection (right). The error bar shows the standard deviation. ( b ) The left panels are images of metastatic and nonmetastatic lymph nodes. Weak, almost equal NIR signal intensity was found in two nonmetastatic lymph nodes at 0.5 and 1 h post-injection. The signal intensity almost disappeared at 24 h after the injection (top, left). A high signal intensity remained in the metastatic lymph node (arrow) at 24 h after injection (bottom, left). The time-dependent NIR signal intensity of the anti-EGFR affibody probe is represented as a percentage of the initial signal intensity (30 min). The signal intensity ratio was greater for metastatic lymph nodes than nonmetastatic lymph nodes at 1, 2, 3, and 6 h after the injection (right panel). * p

Techniques Used: Imaging, Fluorescence, Injection, Mouse Assay, Standard Deviation

( a ) Image of a custom-built illuminator (light emitting diode (LED) emission: peak wavelength, 690 nm). Image of SAS xenograft tumor in the back of a mouse after anti-EGFR affibody photoimmunotherapy (PIT). The image was captured one hour after the probe was injected into the right tumor. The contralateral xenograft tumor served as a control (right). ( b ) The CCK-8 assay showed that the combination of the EGFR affibody IR700 probe and NIR irradiation decreased the survival rate of SAS cells rather than exposure to NIR or the EGFR affibody IR700 probe alone ( p
Figure Legend Snippet: ( a ) Image of a custom-built illuminator (light emitting diode (LED) emission: peak wavelength, 690 nm). Image of SAS xenograft tumor in the back of a mouse after anti-EGFR affibody photoimmunotherapy (PIT). The image was captured one hour after the probe was injected into the right tumor. The contralateral xenograft tumor served as a control (right). ( b ) The CCK-8 assay showed that the combination of the EGFR affibody IR700 probe and NIR irradiation decreased the survival rate of SAS cells rather than exposure to NIR or the EGFR affibody IR700 probe alone ( p

Techniques Used: Injection, CCK-8 Assay, Irradiation

2) Product Images from "Near‐infrared photoimmunotherapy targeting EGFR—Shedding new light on glioblastoma treatment"

Article Title: Near‐infrared photoimmunotherapy targeting EGFR—Shedding new light on glioblastoma treatment

Journal: International Journal of Cancer

doi: 10.1002/ijc.31246

Testing Z EGFR:03115 –IR700DX specificity in vivo and studying the effect of functional groups on dye pharmacokinetics. ( a ) The U87‐MGvIII tumor could easily be differentiated as early as 1 h post Z EGFR:03115 –IR700DX (6 µg/mouse) being intravenously injected, whereas minimal tumor uptake was observed when administering the same amount of the non‐specific Z Taq ‐IR700DX. ( b ) Fluorescence imaging of Z EGFR:03115 –IR700DX uptake in excised tissues (1 h post‐injection) and respective tumor‐to‐organ ratios. ( c ) Mean radiant efficiency in U87‐MGvIII tumors 1 h after administering either 6 µg Z EGFR:03115 –IR700DX, 18 µg Z EGFR:03115 –IR700DX or 6 µg of the non‐specific Z Taq ‐IR700DX. ( d ) Tumor‐to‐background ratio comparison when altering the injected dose of Z EGFR:03115 –IR700DX. ( e , f ) Fluorescence intensity and tumor‐to‐background ratio in the U87‐MGvIII tumors over time after 18 µg Z EGFR:03115 –IR700DX. ( g , h ) Fluorescence imaging of mice bearing subcutaneous U87‐MGvIII tumors. Images were acquired 30 min, 1 h or 3 h post IR700DX–maleimide, IR800CW–maleimide, IR700DX–NHS ester and IR700DX–carboxylate injection and the mean radiant efficiency was determined for each of the dyes. ( i ) An SDS‐PAGE gel of mouse blood serum imaged using the IVIS/Spectrum imaging system to visualize the fluorescent dyes’ association with blood proteins. All data are presented as mean ± SD ( n ≥ 3).
Figure Legend Snippet: Testing Z EGFR:03115 –IR700DX specificity in vivo and studying the effect of functional groups on dye pharmacokinetics. ( a ) The U87‐MGvIII tumor could easily be differentiated as early as 1 h post Z EGFR:03115 –IR700DX (6 µg/mouse) being intravenously injected, whereas minimal tumor uptake was observed when administering the same amount of the non‐specific Z Taq ‐IR700DX. ( b ) Fluorescence imaging of Z EGFR:03115 –IR700DX uptake in excised tissues (1 h post‐injection) and respective tumor‐to‐organ ratios. ( c ) Mean radiant efficiency in U87‐MGvIII tumors 1 h after administering either 6 µg Z EGFR:03115 –IR700DX, 18 µg Z EGFR:03115 –IR700DX or 6 µg of the non‐specific Z Taq ‐IR700DX. ( d ) Tumor‐to‐background ratio comparison when altering the injected dose of Z EGFR:03115 –IR700DX. ( e , f ) Fluorescence intensity and tumor‐to‐background ratio in the U87‐MGvIII tumors over time after 18 µg Z EGFR:03115 –IR700DX. ( g , h ) Fluorescence imaging of mice bearing subcutaneous U87‐MGvIII tumors. Images were acquired 30 min, 1 h or 3 h post IR700DX–maleimide, IR800CW–maleimide, IR700DX–NHS ester and IR700DX–carboxylate injection and the mean radiant efficiency was determined for each of the dyes. ( i ) An SDS‐PAGE gel of mouse blood serum imaged using the IVIS/Spectrum imaging system to visualize the fluorescent dyes’ association with blood proteins. All data are presented as mean ± SD ( n ≥ 3).

Techniques Used: In Vivo, Functional Assay, Injection, Fluorescence, Imaging, Mouse Assay, SDS Page

Expression of EGFR in GBM cell lines and specificity of the Z EGFR:03115 –IR700DX binding to EGFR. ( a ) Varying EGFR expression in the selected cancer cell lines was confirmed by Western blot. The numbers in the brackets represent the relative EGFR expression as determined by densitometric analysis. ( b ) Z EGFR:03115 –IR700DX (30 nM) binding as assessed by flow cytometry in the selected cancer cells with varying EGFR expression and after blocking with 100‐fold excess of unlabeled Z EGFR:03115 . Data are presented as mean ± SEM ( n = 3). ( c – f ) Confocal microscopy images demonstrating target‐specific binding (4°C) and internalization (37°C) of either the Z EGFR:03115 –IR700DX (1 µM), anti‐EGFR‐FITC antibody (25 nM for visualization purposes) or IR700DX alone (1 µM): ( c ) U251 cell lines (1 h incubation time), ( d + e ) U87‐MGvIII or MCF7 cell lines (1–6 h incubation time), ( f ) U87‐MGvIII spheroids (6 h incubation time). Hoechst®33342 (blue) and LysoTracker™Green DND‐26 (green) were used for counterstaining. ( g ) Quantification of fluorescence intensity (median fluorescence intensity) of ∼8 µm slices through U87‐MGvIII spheroids following a 6 h incubation with Z EGFR:03115 –IR700DX (500 nM) or an anti‐EGFR‐FITC antibody (500 nM). Data are presented as mean ± SEM ( n = 3). ( h ) H E, EGFR and Ki67 immunostaining of U87‐MGvIII spheroid (400–500 µm) sections 72 h after seeding.
Figure Legend Snippet: Expression of EGFR in GBM cell lines and specificity of the Z EGFR:03115 –IR700DX binding to EGFR. ( a ) Varying EGFR expression in the selected cancer cell lines was confirmed by Western blot. The numbers in the brackets represent the relative EGFR expression as determined by densitometric analysis. ( b ) Z EGFR:03115 –IR700DX (30 nM) binding as assessed by flow cytometry in the selected cancer cells with varying EGFR expression and after blocking with 100‐fold excess of unlabeled Z EGFR:03115 . Data are presented as mean ± SEM ( n = 3). ( c – f ) Confocal microscopy images demonstrating target‐specific binding (4°C) and internalization (37°C) of either the Z EGFR:03115 –IR700DX (1 µM), anti‐EGFR‐FITC antibody (25 nM for visualization purposes) or IR700DX alone (1 µM): ( c ) U251 cell lines (1 h incubation time), ( d + e ) U87‐MGvIII or MCF7 cell lines (1–6 h incubation time), ( f ) U87‐MGvIII spheroids (6 h incubation time). Hoechst®33342 (blue) and LysoTracker™Green DND‐26 (green) were used for counterstaining. ( g ) Quantification of fluorescence intensity (median fluorescence intensity) of ∼8 µm slices through U87‐MGvIII spheroids following a 6 h incubation with Z EGFR:03115 –IR700DX (500 nM) or an anti‐EGFR‐FITC antibody (500 nM). Data are presented as mean ± SEM ( n = 3). ( h ) H E, EGFR and Ki67 immunostaining of U87‐MGvIII spheroid (400–500 µm) sections 72 h after seeding.

Techniques Used: Expressing, Binding Assay, Western Blot, Flow Cytometry, Cytometry, Blocking Assay, Confocal Microscopy, Incubation, Fluorescence, Immunostaining

Z EGFR:03115 –IR700DX‐mediated PIT causes cellular death selectively in EGFR+ve cells. Decrease in cell viability as assessed by the CellTiter‐Glo® luminescent cell viability assay 24 or 96 h post‐PIT in 2D cells and 3D spheroids, following 6 h incubation with the Z EGFR:03115 –IR700DX and irradiation with a light dose of 8 or 16 J/cm 2 , was confirmed to be dose dependent and receptor mediated. ( a ) U87‐MGvIII cells 24 h post‐PIT. ( b ) MCF7 cells 24 h post‐PIT. ( c , d ) U87‐MGvIII spheroids 24 and 96 h post‐PIT. ( e ) WSz4 spheroids 96 h post‐PIT. Data are presented as mean ± SEM ( n = 3). Statistical significance in comparison to the control group was determined using an unpaired two‐tailed Student's t‐ test with Welch's correction. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 and **** p ≤ 0.0001. [Color figure can be viewed at http://wileyonlinelibrary.com ]
Figure Legend Snippet: Z EGFR:03115 –IR700DX‐mediated PIT causes cellular death selectively in EGFR+ve cells. Decrease in cell viability as assessed by the CellTiter‐Glo® luminescent cell viability assay 24 or 96 h post‐PIT in 2D cells and 3D spheroids, following 6 h incubation with the Z EGFR:03115 –IR700DX and irradiation with a light dose of 8 or 16 J/cm 2 , was confirmed to be dose dependent and receptor mediated. ( a ) U87‐MGvIII cells 24 h post‐PIT. ( b ) MCF7 cells 24 h post‐PIT. ( c , d ) U87‐MGvIII spheroids 24 and 96 h post‐PIT. ( e ) WSz4 spheroids 96 h post‐PIT. Data are presented as mean ± SEM ( n = 3). Statistical significance in comparison to the control group was determined using an unpaired two‐tailed Student's t‐ test with Welch's correction. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 and **** p ≤ 0.0001. [Color figure can be viewed at http://wileyonlinelibrary.com ]

Techniques Used: Cell Viability Assay, Incubation, Irradiation, Two Tailed Test

In vivo Z EGFR:03115 –IR700DX‐mediated PIT studies. ( a ) Fluorescence imaging of mice bearing subcutaneous U87‐MGvIII tumors 1 h after injecting 18 μg of Z EGFR:03115 –IR700DX or IR700DX–maleimide (top row). Subsequently, mice were irradiated with an optical dose of 100 J/cm 2 by a red LED and, immediately after, imaged again (bottom row). ( b ) Tumor growth inhibition of the Z EGFR:03115 –IR700DX‐targeted PIT in U87‐MGvIII tumors after administering three doses of 18 µg of the conjugate and irradiating with 100 J/cm 2 at days 1, 3 and 5 in comparison to control groups. Data are presented as mean ± SD ( n = 6 for each group, ** p ≤ 0.01 as assessed by the Kruskal–Wallis test). ( c ) Visual observation of normal tissue damage in the PDT treated mice, while no skin damage was present in the Z EGFR:03115 –IR700DX PIT mice. These were the appearances seen in all mice. ( d ) H E staining of treated and untreated U87‐MGvIIII tumors (arrows indicate regions of tissue necrosis).
Figure Legend Snippet: In vivo Z EGFR:03115 –IR700DX‐mediated PIT studies. ( a ) Fluorescence imaging of mice bearing subcutaneous U87‐MGvIII tumors 1 h after injecting 18 μg of Z EGFR:03115 –IR700DX or IR700DX–maleimide (top row). Subsequently, mice were irradiated with an optical dose of 100 J/cm 2 by a red LED and, immediately after, imaged again (bottom row). ( b ) Tumor growth inhibition of the Z EGFR:03115 –IR700DX‐targeted PIT in U87‐MGvIII tumors after administering three doses of 18 µg of the conjugate and irradiating with 100 J/cm 2 at days 1, 3 and 5 in comparison to control groups. Data are presented as mean ± SD ( n = 6 for each group, ** p ≤ 0.01 as assessed by the Kruskal–Wallis test). ( c ) Visual observation of normal tissue damage in the PDT treated mice, while no skin damage was present in the Z EGFR:03115 –IR700DX PIT mice. These were the appearances seen in all mice. ( d ) H E staining of treated and untreated U87‐MGvIIII tumors (arrows indicate regions of tissue necrosis).

Techniques Used: In Vivo, Fluorescence, Imaging, Mouse Assay, Irradiation, Inhibition, Staining

Z EGFR:03115 –IR700DX accumulates in U87‐MGvIII orthotopic glioma tumors. ( a ) T 2 ‐weighted MRI images of an intracranial brain tumor model 11 days post‐cell implantation. ( b ) Photographic image of the brain and the corresponding Z EGFR:03115 –IR700DX fluorescent image demonstrates predominant accumulation of the conjugate within the brain tumor mass. ( c ) Transaxial brain histological sections (10μm) containing tumor tissue were obtained for ex vivo analysis immediately after 1 h in vivo image acquisition. Z EGFR:03115 –IR700DX clearly delineated tumor mass from the surrounding normal tissues which correlated well with H E and EGFR staining of the consecutive sections.
Figure Legend Snippet: Z EGFR:03115 –IR700DX accumulates in U87‐MGvIII orthotopic glioma tumors. ( a ) T 2 ‐weighted MRI images of an intracranial brain tumor model 11 days post‐cell implantation. ( b ) Photographic image of the brain and the corresponding Z EGFR:03115 –IR700DX fluorescent image demonstrates predominant accumulation of the conjugate within the brain tumor mass. ( c ) Transaxial brain histological sections (10μm) containing tumor tissue were obtained for ex vivo analysis immediately after 1 h in vivo image acquisition. Z EGFR:03115 –IR700DX clearly delineated tumor mass from the surrounding normal tissues which correlated well with H E and EGFR staining of the consecutive sections.

Techniques Used: Magnetic Resonance Imaging, Ex Vivo, In Vivo, Staining

In vitro morphological changes following affibody‐based PIT. ( a ) Incubation of U87‐MGvIII spheroids with the Z EGFR:03115 –IR700DX for 6 h and irradiation with a red LED (16 J/cm 2 ) induced phototoxic cell death and disintegration of the architectural structure of the spheroid population. ( b ) U87‐MGvIII cells grown as a monolayer culture showed rapid cell swelling and bleb formation (see arrows) as visualized by a phase‐contrast image 1 h post Z EGFR:03115 –IR700DX (red) irradiation with the 639 nm laser on a confocal microscope. ( c ) Following methanol fixation of U87‐MGvIII cells, either treated by PIT or just irradiated, and staining with an anti‐calreticulin‐AlexaFluor488 antibody overnight (4°C), images were acquired by confocal microscopy. ( d ) Cell membrane disruption was monitored by propidium iodide (1 µg/mL) staining. U87‐MGvIII cells irradiated only or treated with Z EGFR:03115 –IR700DX‐based PIT were analyzed by flow cytometry 1 and 24 h post‐treatment. ( e ) Reactive oxygen species production was assessed using the DCFDA cellular ROS detection assay kit using U87‐MGvIII cells treated with affibody‐based PIT (15 min after light exposure). The results were normalized to the control cells. Data are presented as mean ± SEM ( n = 3).
Figure Legend Snippet: In vitro morphological changes following affibody‐based PIT. ( a ) Incubation of U87‐MGvIII spheroids with the Z EGFR:03115 –IR700DX for 6 h and irradiation with a red LED (16 J/cm 2 ) induced phototoxic cell death and disintegration of the architectural structure of the spheroid population. ( b ) U87‐MGvIII cells grown as a monolayer culture showed rapid cell swelling and bleb formation (see arrows) as visualized by a phase‐contrast image 1 h post Z EGFR:03115 –IR700DX (red) irradiation with the 639 nm laser on a confocal microscope. ( c ) Following methanol fixation of U87‐MGvIII cells, either treated by PIT or just irradiated, and staining with an anti‐calreticulin‐AlexaFluor488 antibody overnight (4°C), images were acquired by confocal microscopy. ( d ) Cell membrane disruption was monitored by propidium iodide (1 µg/mL) staining. U87‐MGvIII cells irradiated only or treated with Z EGFR:03115 –IR700DX‐based PIT were analyzed by flow cytometry 1 and 24 h post‐treatment. ( e ) Reactive oxygen species production was assessed using the DCFDA cellular ROS detection assay kit using U87‐MGvIII cells treated with affibody‐based PIT (15 min after light exposure). The results were normalized to the control cells. Data are presented as mean ± SEM ( n = 3).

Techniques Used: In Vitro, Incubation, Irradiation, Microscopy, Staining, Confocal Microscopy, Flow Cytometry, Cytometry, Detection Assay

3) Product Images from "Comparative Evaluation of Radioiodine and Technetium-Labeled DARPin 9_29 for Radionuclide Molecular Imaging of HER2 Expression in Malignant Tumors"

Article Title: Comparative Evaluation of Radioiodine and Technetium-Labeled DARPin 9_29 for Radionuclide Molecular Imaging of HER2 Expression in Malignant Tumors

Journal: Contrast Media & Molecular Imaging

doi: 10.1155/2018/6930425

Representative LigandTracer sensorgrams of [ 125 I]I-DARPin 9_29 (a) and [ 99m Tc]Tc(CO) 3 -DARPin 9_29 (b) binding to HER2-expressing SKOV-3 cells. The association was measured at concentrations of 1, 4, and 8 nM.
Figure Legend Snippet: Representative LigandTracer sensorgrams of [ 125 I]I-DARPin 9_29 (a) and [ 99m Tc]Tc(CO) 3 -DARPin 9_29 (b) binding to HER2-expressing SKOV-3 cells. The association was measured at concentrations of 1, 4, and 8 nM.

Techniques Used: Binding Assay, Expressing

Cellular processing of [ 99m Tc]Tc(CO) 3 -DARPin 9_29 (a, b) and [ 125 I]I-DARPin 9_29 (c, d) by HER2-expressing SKOV-3 (a, c) and BT474 (b, d) cells. Cells were incubated with the conjugates (10 nM) at 37°C. Data are presented as the mean of three samples ± SD.
Figure Legend Snippet: Cellular processing of [ 99m Tc]Tc(CO) 3 -DARPin 9_29 (a, b) and [ 125 I]I-DARPin 9_29 (c, d) by HER2-expressing SKOV-3 (a, c) and BT474 (b, d) cells. Cells were incubated with the conjugates (10 nM) at 37°C. Data are presented as the mean of three samples ± SD.

Techniques Used: Expressing, Incubation

In vitro binding specificity of [ 99m Tc]Tc(CO) 3 -DARPin (a) and [ 125 I]I-DARPin 9_29 (b) to HER2-expressing cells. In the blocked group, receptors were presaturated with a 100-fold excess of unlabeled DARPin. Data are presented as the mean of three samples ± SD.
Figure Legend Snippet: In vitro binding specificity of [ 99m Tc]Tc(CO) 3 -DARPin (a) and [ 125 I]I-DARPin 9_29 (b) to HER2-expressing cells. In the blocked group, receptors were presaturated with a 100-fold excess of unlabeled DARPin. Data are presented as the mean of three samples ± SD.

Techniques Used: In Vitro, Binding Assay, Expressing

Imaging of HER2 expression in SKOV-3 xenografts (maximum intensity projection) using [ 125 I]I-DARPin 9_29 (a) and [ 99m Tc]Tc(CO) 3 -DARPin 9_29 (b). Small-animal SPECT/CT imaging was performed at 6 h after injection.
Figure Legend Snippet: Imaging of HER2 expression in SKOV-3 xenografts (maximum intensity projection) using [ 125 I]I-DARPin 9_29 (a) and [ 99m Tc]Tc(CO) 3 -DARPin 9_29 (b). Small-animal SPECT/CT imaging was performed at 6 h after injection.

Techniques Used: Imaging, Expressing, Single Photon Emission Computed Tomography, Injection

In vitro binding of [ 125 I]I-DARPin 9_29 to SKOV-3 cells after treatment with 100-fold molar excess of different targeting agents. Data are presented as the mean of three samples ± SD.
Figure Legend Snippet: In vitro binding of [ 125 I]I-DARPin 9_29 to SKOV-3 cells after treatment with 100-fold molar excess of different targeting agents. Data are presented as the mean of three samples ± SD.

Techniques Used: In Vitro, Binding Assay

In vivo specificity of HER2 targeting using [ 125 I]I-DARPin 9_29 and [ 99m Tc]Tc(CO) 3 -DARPin 9_29. Uptake of both imaging probes was significantly ( p
Figure Legend Snippet: In vivo specificity of HER2 targeting using [ 125 I]I-DARPin 9_29 and [ 99m Tc]Tc(CO) 3 -DARPin 9_29. Uptake of both imaging probes was significantly ( p

Techniques Used: In Vivo, Imaging

4) Product Images from "Comparative Evaluation of Radioiodine and Technetium-Labeled DARPin 9_29 for Radionuclide Molecular Imaging of HER2 Expression in Malignant Tumors"

Article Title: Comparative Evaluation of Radioiodine and Technetium-Labeled DARPin 9_29 for Radionuclide Molecular Imaging of HER2 Expression in Malignant Tumors

Journal: Contrast Media & Molecular Imaging

doi: 10.1155/2018/6930425

Representative LigandTracer sensorgrams of [ 125 I]I-DARPin 9_29 (a) and [ 99m Tc]Tc(CO) 3 -DARPin 9_29 (b) binding to HER2-expressing SKOV-3 cells. The association was measured at concentrations of 1, 4, and 8 nM.
Figure Legend Snippet: Representative LigandTracer sensorgrams of [ 125 I]I-DARPin 9_29 (a) and [ 99m Tc]Tc(CO) 3 -DARPin 9_29 (b) binding to HER2-expressing SKOV-3 cells. The association was measured at concentrations of 1, 4, and 8 nM.

Techniques Used: Binding Assay, Expressing

Cellular processing of [ 99m Tc]Tc(CO) 3 -DARPin 9_29 (a, b) and [ 125 I]I-DARPin 9_29 (c, d) by HER2-expressing SKOV-3 (a, c) and BT474 (b, d) cells. Cells were incubated with the conjugates (10 nM) at 37°C. Data are presented as the mean of three samples ± SD.
Figure Legend Snippet: Cellular processing of [ 99m Tc]Tc(CO) 3 -DARPin 9_29 (a, b) and [ 125 I]I-DARPin 9_29 (c, d) by HER2-expressing SKOV-3 (a, c) and BT474 (b, d) cells. Cells were incubated with the conjugates (10 nM) at 37°C. Data are presented as the mean of three samples ± SD.

Techniques Used: Expressing, Incubation

Imaging of HER2 expression in SKOV-3 xenografts (maximum intensity projection) using [ 125 I]I-DARPin 9_29 (a) and [ 99m Tc]Tc(CO) 3 -DARPin 9_29 (b). Small-animal SPECT/CT imaging was performed at 6 h after injection.
Figure Legend Snippet: Imaging of HER2 expression in SKOV-3 xenografts (maximum intensity projection) using [ 125 I]I-DARPin 9_29 (a) and [ 99m Tc]Tc(CO) 3 -DARPin 9_29 (b). Small-animal SPECT/CT imaging was performed at 6 h after injection.

Techniques Used: Imaging, Expressing, Single Photon Emission Computed Tomography, Injection

In vitro binding of [ 125 I]I-DARPin 9_29 to SKOV-3 cells after treatment with 100-fold molar excess of different targeting agents. Data are presented as the mean of three samples ± SD.
Figure Legend Snippet: In vitro binding of [ 125 I]I-DARPin 9_29 to SKOV-3 cells after treatment with 100-fold molar excess of different targeting agents. Data are presented as the mean of three samples ± SD.

Techniques Used: In Vitro, Binding Assay

5) Product Images from "Inhibiting HER3-Mediated Tumor Cell Growth with Affibody Molecules Engineered to Low Picomolar Affinity by Position-Directed Error-Prone PCR-Like Diversification"

Article Title: Inhibiting HER3-Mediated Tumor Cell Growth with Affibody Molecules Engineered to Low Picomolar Affinity by Position-Directed Error-Prone PCR-Like Diversification

Journal: PLoS ONE

doi: 10.1371/journal.pone.0062791

Alanine scanning and library design. A. Alanine scanning of Z 05416 . Histogram showing the results from flow-cytometric analysis of the thirteen staphylococcal-displayed mutants. The residues in the Affibody molecule that were mutated are represented on the x-axis, and a ratio of the mean fluorescence intensity (MFI) corresponding to HER3 binding and the MFI corresponding to surface expression level (monitored by HSA binding) is represented on the y-axis. B. Library design. Schematic overview showing the randomized positions in helix 1 and 2, the relative importance of the respective residue for HER3 binding (as determined by alanine scanning), the amino acids in each position in the original HER3 binders, the percentage of the codon corresponding to the original amino acid and the percentage of each of 15 or 16 (depending on the position) mutation codons.
Figure Legend Snippet: Alanine scanning and library design. A. Alanine scanning of Z 05416 . Histogram showing the results from flow-cytometric analysis of the thirteen staphylococcal-displayed mutants. The residues in the Affibody molecule that were mutated are represented on the x-axis, and a ratio of the mean fluorescence intensity (MFI) corresponding to HER3 binding and the MFI corresponding to surface expression level (monitored by HSA binding) is represented on the y-axis. B. Library design. Schematic overview showing the randomized positions in helix 1 and 2, the relative importance of the respective residue for HER3 binding (as determined by alanine scanning), the amino acids in each position in the original HER3 binders, the percentage of the codon corresponding to the original amino acid and the percentage of each of 15 or 16 (depending on the position) mutation codons.

Techniques Used: Flow Cytometry, Fluorescence, Binding Assay, Expressing, Mutagenesis

Characterization of affinity-matured Affibody molecules. A. Biosensor off-rate ranking of ten affinity-matured HER3-binders. Sensorgrams of ten different Affibody molecules injected over immobilized human HER3-Fc (all affinity-matured clones are shown in black, except for Z 08698 and Z 08699 , which are shown in blue and red, respectively). For comparison, the HER3-binder Z 05417 was included in the analysis (grey curve). B. Analysis of refolding capacity and thermal stability of affinity-matured HER3-binders evaluated by SPR and CD. Sensorgrams showing injections of 50 nM Z 08698 and Z 08699 , respectively, before and after heat treatment at 90°C over immobilized human HER3-Fc. C. Variable temperature measurement (VTM) spectra obtained at 221 nm while heating the HER3-specific Affibody molecules Z 08698 and Z 08699 from 20 to 90°C. D. CD spectra of Z 08698 and Z 08699 at wavelengths ranging from 195 to 250 nm at 20°C.
Figure Legend Snippet: Characterization of affinity-matured Affibody molecules. A. Biosensor off-rate ranking of ten affinity-matured HER3-binders. Sensorgrams of ten different Affibody molecules injected over immobilized human HER3-Fc (all affinity-matured clones are shown in black, except for Z 08698 and Z 08699 , which are shown in blue and red, respectively). For comparison, the HER3-binder Z 05417 was included in the analysis (grey curve). B. Analysis of refolding capacity and thermal stability of affinity-matured HER3-binders evaluated by SPR and CD. Sensorgrams showing injections of 50 nM Z 08698 and Z 08699 , respectively, before and after heat treatment at 90°C over immobilized human HER3-Fc. C. Variable temperature measurement (VTM) spectra obtained at 221 nm while heating the HER3-specific Affibody molecules Z 08698 and Z 08699 from 20 to 90°C. D. CD spectra of Z 08698 and Z 08699 at wavelengths ranging from 195 to 250 nm at 20°C.

Techniques Used: Injection, Clone Assay, SPR Assay

Specificity of binding of labeled Affibody molecules to HER3-expressing cells in vitro . Cells were incubated with 1 nM of radiolabeled conjugates for 1 h. For pre-saturation (blocking) of antigens, 0.7 µM unlabeled non-radioactive Affibody molecules was added. Data are presented as mean values of percent of added radioactivity that is cell-bound from three cell dishes and standard deviations. The difference between uptake by non-blocked and blocked cells was statistically significant (p
Figure Legend Snippet: Specificity of binding of labeled Affibody molecules to HER3-expressing cells in vitro . Cells were incubated with 1 nM of radiolabeled conjugates for 1 h. For pre-saturation (blocking) of antigens, 0.7 µM unlabeled non-radioactive Affibody molecules was added. Data are presented as mean values of percent of added radioactivity that is cell-bound from three cell dishes and standard deviations. The difference between uptake by non-blocked and blocked cells was statistically significant (p

Techniques Used: Binding Assay, Labeling, Expressing, In Vitro, Incubation, Blocking Assay, Radioactivity

6) Product Images from "PET imaging of epidermal growth factor receptor expression in tumours using 89Zr-labelled ZEGFR:2377 affibody molecules"

Article Title: PET imaging of epidermal growth factor receptor expression in tumours using 89Zr-labelled ZEGFR:2377 affibody molecules

Journal: International Journal of Oncology

doi: 10.3892/ijo.2016.3369

Specificity of 89 Zr-DFO-ZEGFR:2377 uptake in A431 xenografts and EGFR-expressing organs in mice at 3 h after injection. In the blocked group, receptors were saturated by pre-injection of large excess of non-labelled affibody molecules.
Figure Legend Snippet: Specificity of 89 Zr-DFO-ZEGFR:2377 uptake in A431 xenografts and EGFR-expressing organs in mice at 3 h after injection. In the blocked group, receptors were saturated by pre-injection of large excess of non-labelled affibody molecules.

Techniques Used: Expressing, Mouse Assay, Injection

7) Product Images from "PET imaging of epidermal growth factor receptor expression in tumours using 89Zr-labelled ZEGFR:2377 affibody molecules"

Article Title: PET imaging of epidermal growth factor receptor expression in tumours using 89Zr-labelled ZEGFR:2377 affibody molecules

Journal: International Journal of Oncology

doi: 10.3892/ijo.2016.3369

(A) In vitro specificity of 89 Zr-DFO-ZEGFR:2377 binding to EGFR-expressing A431 cells. (B) Cellular processing of 89 Zr-DFO-ZEGFR:2377 by A431 cells during continuous incubation.
Figure Legend Snippet: (A) In vitro specificity of 89 Zr-DFO-ZEGFR:2377 binding to EGFR-expressing A431 cells. (B) Cellular processing of 89 Zr-DFO-ZEGFR:2377 by A431 cells during continuous incubation.

Techniques Used: In Vitro, Binding Assay, Expressing, Incubation

Specificity of 89 Zr-DFO-ZEGFR:2377 uptake in A431 xenografts and EGFR-expressing organs in mice at 3 h after injection. In the blocked group, receptors were saturated by pre-injection of large excess of non-labelled affibody molecules.
Figure Legend Snippet: Specificity of 89 Zr-DFO-ZEGFR:2377 uptake in A431 xenografts and EGFR-expressing organs in mice at 3 h after injection. In the blocked group, receptors were saturated by pre-injection of large excess of non-labelled affibody molecules.

Techniques Used: Expressing, Mouse Assay, Injection

Representative LignadTracer sensorgram of 89 Zr-DFO-ZEGFR:2377 binding to EGFR-expressing A431 cells. Uptake curves were recorded at 0.33 and 1 nM.
Figure Legend Snippet: Representative LignadTracer sensorgram of 89 Zr-DFO-ZEGFR:2377 binding to EGFR-expressing A431 cells. Uptake curves were recorded at 0.33 and 1 nM.

Techniques Used: Binding Assay, Expressing

8) Product Images from "Near‐infrared photoimmunotherapy targeting EGFR—Shedding new light on glioblastoma treatment"

Article Title: Near‐infrared photoimmunotherapy targeting EGFR—Shedding new light on glioblastoma treatment

Journal: International Journal of Cancer

doi: 10.1002/ijc.31246

Expression of EGFR in GBM cell lines and specificity of the Z EGFR:03115 –IR700DX binding to EGFR. ( a ) Varying EGFR expression in the selected cancer cell lines was confirmed by Western blot. The numbers in the brackets represent the relative EGFR expression as determined by densitometric analysis. ( b ) Z EGFR:03115 –IR700DX (30 nM) binding as assessed by flow cytometry in the selected cancer cells with varying EGFR expression and after blocking with 100‐fold excess of unlabeled Z EGFR:03115 . Data are presented as mean ± SEM ( n = 3). ( c – f ) Confocal microscopy images demonstrating target‐specific binding (4°C) and internalization (37°C) of either the Z EGFR:03115 –IR700DX (1 µM), anti‐EGFR‐FITC antibody (25 nM for visualization purposes) or IR700DX alone (1 µM): ( c ) U251 cell lines (1 h incubation time), ( d + e ) U87‐MGvIII or MCF7 cell lines (1–6 h incubation time), ( f ) U87‐MGvIII spheroids (6 h incubation time). Hoechst®33342 (blue) and LysoTracker™Green DND‐26 (green) were used for counterstaining. ( g ) Quantification of fluorescence intensity (median fluorescence intensity) of ∼8 µm slices through U87‐MGvIII spheroids following a 6 h incubation with Z EGFR:03115 –IR700DX (500 nM) or an anti‐EGFR‐FITC antibody (500 nM). Data are presented as mean ± SEM ( n = 3). ( h ) H E, EGFR and Ki67 immunostaining of U87‐MGvIII spheroid (400–500 µm) sections 72 h after seeding.
Figure Legend Snippet: Expression of EGFR in GBM cell lines and specificity of the Z EGFR:03115 –IR700DX binding to EGFR. ( a ) Varying EGFR expression in the selected cancer cell lines was confirmed by Western blot. The numbers in the brackets represent the relative EGFR expression as determined by densitometric analysis. ( b ) Z EGFR:03115 –IR700DX (30 nM) binding as assessed by flow cytometry in the selected cancer cells with varying EGFR expression and after blocking with 100‐fold excess of unlabeled Z EGFR:03115 . Data are presented as mean ± SEM ( n = 3). ( c – f ) Confocal microscopy images demonstrating target‐specific binding (4°C) and internalization (37°C) of either the Z EGFR:03115 –IR700DX (1 µM), anti‐EGFR‐FITC antibody (25 nM for visualization purposes) or IR700DX alone (1 µM): ( c ) U251 cell lines (1 h incubation time), ( d + e ) U87‐MGvIII or MCF7 cell lines (1–6 h incubation time), ( f ) U87‐MGvIII spheroids (6 h incubation time). Hoechst®33342 (blue) and LysoTracker™Green DND‐26 (green) were used for counterstaining. ( g ) Quantification of fluorescence intensity (median fluorescence intensity) of ∼8 µm slices through U87‐MGvIII spheroids following a 6 h incubation with Z EGFR:03115 –IR700DX (500 nM) or an anti‐EGFR‐FITC antibody (500 nM). Data are presented as mean ± SEM ( n = 3). ( h ) H E, EGFR and Ki67 immunostaining of U87‐MGvIII spheroid (400–500 µm) sections 72 h after seeding.

Techniques Used: Expressing, Binding Assay, Western Blot, Flow Cytometry, Cytometry, Blocking Assay, Confocal Microscopy, Incubation, Fluorescence, Immunostaining

In vivo Z EGFR:03115 –IR700DX‐mediated PIT studies. ( a ) Fluorescence imaging of mice bearing subcutaneous U87‐MGvIII tumors 1 h after injecting 18 μg of Z EGFR:03115 –IR700DX or IR700DX–maleimide (top row). Subsequently, mice were irradiated with an optical dose of 100 J/cm 2 by a red LED and, immediately after, imaged again (bottom row). ( b ) Tumor growth inhibition of the Z EGFR:03115 –IR700DX‐targeted PIT in U87‐MGvIII tumors after administering three doses of 18 µg of the conjugate and irradiating with 100 J/cm 2 at days 1, 3 and 5 in comparison to control groups. Data are presented as mean ± SD ( n = 6 for each group, ** p ≤ 0.01 as assessed by the Kruskal–Wallis test). ( c ) Visual observation of normal tissue damage in the PDT treated mice, while no skin damage was present in the Z EGFR:03115 –IR700DX PIT mice. These were the appearances seen in all mice. ( d ) H E staining of treated and untreated U87‐MGvIIII tumors (arrows indicate regions of tissue necrosis).
Figure Legend Snippet: In vivo Z EGFR:03115 –IR700DX‐mediated PIT studies. ( a ) Fluorescence imaging of mice bearing subcutaneous U87‐MGvIII tumors 1 h after injecting 18 μg of Z EGFR:03115 –IR700DX or IR700DX–maleimide (top row). Subsequently, mice were irradiated with an optical dose of 100 J/cm 2 by a red LED and, immediately after, imaged again (bottom row). ( b ) Tumor growth inhibition of the Z EGFR:03115 –IR700DX‐targeted PIT in U87‐MGvIII tumors after administering three doses of 18 µg of the conjugate and irradiating with 100 J/cm 2 at days 1, 3 and 5 in comparison to control groups. Data are presented as mean ± SD ( n = 6 for each group, ** p ≤ 0.01 as assessed by the Kruskal–Wallis test). ( c ) Visual observation of normal tissue damage in the PDT treated mice, while no skin damage was present in the Z EGFR:03115 –IR700DX PIT mice. These were the appearances seen in all mice. ( d ) H E staining of treated and untreated U87‐MGvIIII tumors (arrows indicate regions of tissue necrosis).

Techniques Used: In Vivo, Fluorescence, Imaging, Mouse Assay, Irradiation, Inhibition, Staining

9) Product Images from "EGFR oligomerization organizes kinase-active dimers into competent signalling platforms"

Article Title: EGFR oligomerization organizes kinase-active dimers into competent signalling platforms

Journal: Nature Communications

doi: 10.1038/ncomms13307

Pairwise EGF separations in three EGFR deletion mutants. ( a ) (left) FLImP distribution (grey) of pairwise EGF separations on CHO cells expressing ΔC-EGFR treated with 4 nM EGF. The distribution is fitted (black line) with a sum of four Rician peaks (colour lines). The number of peaks used was determined using a Bayesian information criterion. The best-fit positions of the peaks and error bars are shown in the inset. The errors in the fit were calculated as described in Supplementary Methods . (right) The FLImP distribution (grey) and the distributions (green or yellow) compiled from the FLImP measurements whose ranges of 69% confidence overlap with the ranges of EGF separations of the expected dimer (12.5±0.3 nm) or tetramer (19.6±0.5 nm), which are indicated by the vertical blue lines. ( b ) Similar to a for C'698 EGFR. ( c ) Similar to a for C'973 EGFR. Cells stably expressed each mutant at an expression level of ∼10 5 copies per cell. ( d ) Similar to a for the wild-type receptor respectively treated with 4 and 100 nM EGF. The numbers of FLImP measurements included in each distribution are 40 (ΔC-EGFR), 44 (c'698-EGFR), 33 (c'973-EGFR), 51 (wild type receptor at 4 nM EGF), and 37 (wild type receptor at 100 nM EGF); the confidence interval for each included FLImP measurement is 6 nm ( d ) or 7 nm ( a – c ). ( e ) Relative populations of the dimers and the tetramers, determined from the FLImP measurements shown in a – d , right hand panels. For each construct, the dimer percentage is estimated by the ratio of the green integral area to the integral area of all FLImP measurements whose 69% confidence overlaps with the dimer-tetramer region (0–20.1 nm). Tetramer populations are calculated in the same way, using the yellow instead of the green integral area. Error bars were calculated by bootstrap-resampling the data 1,000 times with replacement and repeating the analysis.
Figure Legend Snippet: Pairwise EGF separations in three EGFR deletion mutants. ( a ) (left) FLImP distribution (grey) of pairwise EGF separations on CHO cells expressing ΔC-EGFR treated with 4 nM EGF. The distribution is fitted (black line) with a sum of four Rician peaks (colour lines). The number of peaks used was determined using a Bayesian information criterion. The best-fit positions of the peaks and error bars are shown in the inset. The errors in the fit were calculated as described in Supplementary Methods . (right) The FLImP distribution (grey) and the distributions (green or yellow) compiled from the FLImP measurements whose ranges of 69% confidence overlap with the ranges of EGF separations of the expected dimer (12.5±0.3 nm) or tetramer (19.6±0.5 nm), which are indicated by the vertical blue lines. ( b ) Similar to a for C'698 EGFR. ( c ) Similar to a for C'973 EGFR. Cells stably expressed each mutant at an expression level of ∼10 5 copies per cell. ( d ) Similar to a for the wild-type receptor respectively treated with 4 and 100 nM EGF. The numbers of FLImP measurements included in each distribution are 40 (ΔC-EGFR), 44 (c'698-EGFR), 33 (c'973-EGFR), 51 (wild type receptor at 4 nM EGF), and 37 (wild type receptor at 100 nM EGF); the confidence interval for each included FLImP measurement is 6 nm ( d ) or 7 nm ( a – c ). ( e ) Relative populations of the dimers and the tetramers, determined from the FLImP measurements shown in a – d , right hand panels. For each construct, the dimer percentage is estimated by the ratio of the green integral area to the integral area of all FLImP measurements whose 69% confidence overlaps with the dimer-tetramer region (0–20.1 nm). Tetramer populations are calculated in the same way, using the yellow instead of the green integral area. Error bars were calculated by bootstrap-resampling the data 1,000 times with replacement and repeating the analysis.

Techniques Used: Expressing, Stable Transfection, Mutagenesis, Construct

The structural model of EGFR tetramers. ( a ) Key steps in constructing the model of a ligand-bound EGFR tetramer: (1) an initial EGFR dimer model generated using a crystal structure of a HER3 dimer as a template; (2) a face-to-face dimer produced by simulation of the initial dimer model, in which the interaction interface remained unchanged but domains I–III in each monomer departed from the tethered conformation for the conformation seen in the active dimer; (3) domains IV are manually modelled to mimic the conformation of monomers in an active dimer; and (4) a tetramer model constructed by adding two-ligand-bound monomers in back-to-back interactions with the previous dimer. In addition to the ribbons generated using atomic coordinates, cartoon figures are used to illustrate the modeling procedure. ( b ) The site for the face-to-face interaction (purple) and the outline of the largely overlapping EGF binding site at domains I and III. ( c ) A diagram illustrating the open-ended oligomerization scheme for EGFR extracellular domains based on repeating the back-to-back and the face-to-face interactions. ( d ) The full-length structural model of an EGFR tetramer as a dimer of active dimers assembled by the face-to-face interactions. The predicted separation between the N-termini of the two EGF ligands and the average EGF-membrane distance are marked. The coordinates of the model are available in Supplementary Data . ( e ) The arrangement of the two intracellular active kinase dimers in the tetramer model, by which the phosphorylation site Tyr992 (green) of one receptor is positioned in the proximity of the active site (red) of a kinase domain from the neighbouring dimer.
Figure Legend Snippet: The structural model of EGFR tetramers. ( a ) Key steps in constructing the model of a ligand-bound EGFR tetramer: (1) an initial EGFR dimer model generated using a crystal structure of a HER3 dimer as a template; (2) a face-to-face dimer produced by simulation of the initial dimer model, in which the interaction interface remained unchanged but domains I–III in each monomer departed from the tethered conformation for the conformation seen in the active dimer; (3) domains IV are manually modelled to mimic the conformation of monomers in an active dimer; and (4) a tetramer model constructed by adding two-ligand-bound monomers in back-to-back interactions with the previous dimer. In addition to the ribbons generated using atomic coordinates, cartoon figures are used to illustrate the modeling procedure. ( b ) The site for the face-to-face interaction (purple) and the outline of the largely overlapping EGF binding site at domains I and III. ( c ) A diagram illustrating the open-ended oligomerization scheme for EGFR extracellular domains based on repeating the back-to-back and the face-to-face interactions. ( d ) The full-length structural model of an EGFR tetramer as a dimer of active dimers assembled by the face-to-face interactions. The predicted separation between the N-termini of the two EGF ligands and the average EGF-membrane distance are marked. The coordinates of the model are available in Supplementary Data . ( e ) The arrangement of the two intracellular active kinase dimers in the tetramer model, by which the phosphorylation site Tyr992 (green) of one receptor is positioned in the proximity of the active site (red) of a kinase domain from the neighbouring dimer.

Techniques Used: Generated, Produced, Construct, Binding Assay

Pairwise EGF separations and phosphorylation of R647C/V650C. ( a ) FLImP distribution (grey) of pairwise EGF separations on CHO cells expressing the R647C/V650C-EGFR mutant at a level of ∼10 5 copies per cell. The separations whose confidence intervals overlap with the 12.5±0.3 nm (green) or 19.6±0.5 nm (yellow) expected dimer and tetramer interval are shown. The expected intervals are indicated by the vertical blue lines. The distribution includes data from 40 FLImP measurements with confidence intervals
Figure Legend Snippet: Pairwise EGF separations and phosphorylation of R647C/V650C. ( a ) FLImP distribution (grey) of pairwise EGF separations on CHO cells expressing the R647C/V650C-EGFR mutant at a level of ∼10 5 copies per cell. The separations whose confidence intervals overlap with the 12.5±0.3 nm (green) or 19.6±0.5 nm (yellow) expected dimer and tetramer interval are shown. The expected intervals are indicated by the vertical blue lines. The distribution includes data from 40 FLImP measurements with confidence intervals

Techniques Used: Expressing, Mutagenesis

Dependence of EGFR phosphorylation and oligomerization-related structural parameters on ligand concentration. ( a ) Western blot measurement of wild type total EGFR auto-phosphorylation in CHO cells exposed to increasing concentrations of EGF. The monoclonal pan-phosphotyrosine antibody 4G10 was used in the measurements. Data points and standard deviations are derived from the average of three independent measurements (examples western blot images shown in Supplementary Fig. 6c ). ( b ) Similar to Fig. 3e , estimate of the relative population of EGFR dimers. The estimates are based on the wild-type FLImP distributions at varying EGF concentrations ( Fig. 3e and Supplementary Fig. 7 ). Errors are calculated as in Fig. 3 . ( c ) Western blot measurements of phosphorylation of Tyr1173 and Tyr992 in CHO cells exposed to increasing EGF concentrations. Data points and error bars (s.d.) are derived from the average of four independent measurements (examples shown in Supplementary Fig. 6a,b ). For the Tyr992 data, P values were calculated using Student's t -test to determine whether measured phosphorylation at high EGF concentrations was significantly different from the maximum phosphorylation value measured at 100 nM EGF. P values are: 50 nM EGF, P =0.058; 200 nM EGF, P =0.173; 500 nM EGF, P =0.027; 1,000 nM EGF, P =0.014; 2,000 nM EGF, P =0.011; 5,000 nM EGF, P =0.009. ( d ) The DOCA between EGFR-bound EGF molecules and the membrane, derived from FRET measurements shown in Supplementary Fig. 8 . DOCAs were obtained from 1,000 bootstrap data sets (that is, data sets resampled with replacement). The error bars are the standard deviations of the bootstrap means. Simulations of the tetramer ( Fig. 2d and Supplementary Fig. 5c ) and dimer 9 (bottom left inset) predict a DOCA of ∼5 nm for oligomers and ∼7.5 nm for dimers.
Figure Legend Snippet: Dependence of EGFR phosphorylation and oligomerization-related structural parameters on ligand concentration. ( a ) Western blot measurement of wild type total EGFR auto-phosphorylation in CHO cells exposed to increasing concentrations of EGF. The monoclonal pan-phosphotyrosine antibody 4G10 was used in the measurements. Data points and standard deviations are derived from the average of three independent measurements (examples western blot images shown in Supplementary Fig. 6c ). ( b ) Similar to Fig. 3e , estimate of the relative population of EGFR dimers. The estimates are based on the wild-type FLImP distributions at varying EGF concentrations ( Fig. 3e and Supplementary Fig. 7 ). Errors are calculated as in Fig. 3 . ( c ) Western blot measurements of phosphorylation of Tyr1173 and Tyr992 in CHO cells exposed to increasing EGF concentrations. Data points and error bars (s.d.) are derived from the average of four independent measurements (examples shown in Supplementary Fig. 6a,b ). For the Tyr992 data, P values were calculated using Student's t -test to determine whether measured phosphorylation at high EGF concentrations was significantly different from the maximum phosphorylation value measured at 100 nM EGF. P values are: 50 nM EGF, P =0.058; 200 nM EGF, P =0.173; 500 nM EGF, P =0.027; 1,000 nM EGF, P =0.014; 2,000 nM EGF, P =0.011; 5,000 nM EGF, P =0.009. ( d ) The DOCA between EGFR-bound EGF molecules and the membrane, derived from FRET measurements shown in Supplementary Fig. 8 . DOCAs were obtained from 1,000 bootstrap data sets (that is, data sets resampled with replacement). The error bars are the standard deviations of the bootstrap means. Simulations of the tetramer ( Fig. 2d and Supplementary Fig. 5c ) and dimer 9 (bottom left inset) predict a DOCA of ∼5 nm for oligomers and ∼7.5 nm for dimers.

Techniques Used: Concentration Assay, Western Blot, Derivative Assay

FLImP measurement of pairwise EGF separations. ( a ) Cartoon of an EGFR monomer, a two-ligand active dimer, and an EGFR sequence diagram. ( b ) Steps to determine EGF separations using FLImP 15 : (1) TIRF images are collected from intact cells; (2) spots from individual complexes are tracked to derive intensity time courses; and (3) a spot image of a complex containing two fluorophore-conjugated EGF ligands (red dots) features two intensity levels and decays to zero in two bleaching steps; when one fluorophore bleaches, the centroid position shifts. If more than two steps occur, the lowest two are analysed. (4) A global least-squares seven-parameter-fit is used to identify the best intensity, x - y positions and the full-width at half-maximum of the point spread function for each fluorophore, from which their separation is calculated with a precision determined by the localization error; (5) Example systems of a two-ligand dimer and tetramer, a three-ligand tetramer, and a mixture of a dimer and a tetramer. (6) The empirical posterior distributions (or FLImP measurement) of pairwise ligand separations obtained for each example system with their 69% confidence intervals highlighted. The size of the latter depends on the combined localization errors of the two molecules 15 . FLImP measurements with confidence intervals smaller than the required resolution are retained in a histogram, generating a so-called FLImP distribution that is fitted by the sum of a discrete number of Rician peaks ( Supplementary Fig. 3a ). ( c ) FLImP distribution (grey) of CF640R fluorophore-conjugated EGF on CHO cells (∼10 5 copies of wild-type EGFR per cell) treated with 4 nM EGF at 4 °C with chemical fixation, compiled from 30 FLImP measurements with confidence intervals
Figure Legend Snippet: FLImP measurement of pairwise EGF separations. ( a ) Cartoon of an EGFR monomer, a two-ligand active dimer, and an EGFR sequence diagram. ( b ) Steps to determine EGF separations using FLImP 15 : (1) TIRF images are collected from intact cells; (2) spots from individual complexes are tracked to derive intensity time courses; and (3) a spot image of a complex containing two fluorophore-conjugated EGF ligands (red dots) features two intensity levels and decays to zero in two bleaching steps; when one fluorophore bleaches, the centroid position shifts. If more than two steps occur, the lowest two are analysed. (4) A global least-squares seven-parameter-fit is used to identify the best intensity, x - y positions and the full-width at half-maximum of the point spread function for each fluorophore, from which their separation is calculated with a precision determined by the localization error; (5) Example systems of a two-ligand dimer and tetramer, a three-ligand tetramer, and a mixture of a dimer and a tetramer. (6) The empirical posterior distributions (or FLImP measurement) of pairwise ligand separations obtained for each example system with their 69% confidence intervals highlighted. The size of the latter depends on the combined localization errors of the two molecules 15 . FLImP measurements with confidence intervals smaller than the required resolution are retained in a histogram, generating a so-called FLImP distribution that is fitted by the sum of a discrete number of Rician peaks ( Supplementary Fig. 3a ). ( c ) FLImP distribution (grey) of CF640R fluorophore-conjugated EGF on CHO cells (∼10 5 copies of wild-type EGFR per cell) treated with 4 nM EGF at 4 °C with chemical fixation, compiled from 30 FLImP measurements with confidence intervals

Techniques Used: Sequencing

The tethered ectodomain signature in FLImP distributions. ( a ) FLImP distribution (grey) of pairwise separations of fluorophore-conjugated EGF bound to EGFR on CHO cells treated with 4 nM EGF (identical data as shown in Fig. 3d ) and the distribution (yellow) compiled from all FLImP measurements whose 69% confidence interval overlaps with the range of 0–8 nm for the inactive dimers. ( b ) Similar to a , FLImP distribution of cells treated with 4 nM anti-EGFR Affibody. ( c ) Similar to b on cells pre-treated with 200 nM 9G8 nanobody and 4 nM Affibody. The distributions in b , c contain data from 37 and 33 FLImP measurements, respectively. The expected range of separations for dimers is indicated by the vertical blue lines. ( d ) Estimate of the relative population of the inactive dimers as indicated by ratio of the yellow integral area to the corresponding total grey integral area. Errors are calculated as described in Fig. 3 .
Figure Legend Snippet: The tethered ectodomain signature in FLImP distributions. ( a ) FLImP distribution (grey) of pairwise separations of fluorophore-conjugated EGF bound to EGFR on CHO cells treated with 4 nM EGF (identical data as shown in Fig. 3d ) and the distribution (yellow) compiled from all FLImP measurements whose 69% confidence interval overlaps with the range of 0–8 nm for the inactive dimers. ( b ) Similar to a , FLImP distribution of cells treated with 4 nM anti-EGFR Affibody. ( c ) Similar to b on cells pre-treated with 200 nM 9G8 nanobody and 4 nM Affibody. The distributions in b , c contain data from 37 and 33 FLImP measurements, respectively. The expected range of separations for dimers is indicated by the vertical blue lines. ( d ) Estimate of the relative population of the inactive dimers as indicated by ratio of the yellow integral area to the corresponding total grey integral area. Errors are calculated as described in Fig. 3 .

Techniques Used:

10) Product Images from "RAPID COMMUNICATION: Optimizing Glioma Detection using an EGFR-Targeted Fluorescent Affibody"

Article Title: RAPID COMMUNICATION: Optimizing Glioma Detection using an EGFR-Targeted Fluorescent Affibody

Journal: Photochemistry and photobiology

doi: 10.1111/php.13003

Effect of pre-injection of unlabeled cetuximab on contrast tumor-to-normal brain, where the ratio of ABY-029 to cetuximab was varied, as denoted by the three columns, 1:1, 1:2.5 and 1:10, with the assay done at both 1 h and 24 h.
Figure Legend Snippet: Effect of pre-injection of unlabeled cetuximab on contrast tumor-to-normal brain, where the ratio of ABY-029 to cetuximab was varied, as denoted by the three columns, 1:1, 1:2.5 and 1:10, with the assay done at both 1 h and 24 h.

Techniques Used: Injection

Inhibition of ABY-029 binding to EGFR by unlabeled cetuximab was investigated, with bars showing the incubation conditions. Cetuximab bars show the autofluorescence. Shared letters mean no statistical difference (ANOVA, Tukey’s Multiple Comparison Test).
Figure Legend Snippet: Inhibition of ABY-029 binding to EGFR by unlabeled cetuximab was investigated, with bars showing the incubation conditions. Cetuximab bars show the autofluorescence. Shared letters mean no statistical difference (ANOVA, Tukey’s Multiple Comparison Test).

Techniques Used: Inhibition, Binding Assay, Incubation

11) Product Images from "RAPID COMMUNICATION: Optimizing Glioma Detection using an EGFR-Targeted Fluorescent Affibody"

Article Title: RAPID COMMUNICATION: Optimizing Glioma Detection using an EGFR-Targeted Fluorescent Affibody

Journal: Photochemistry and photobiology

doi: 10.1111/php.13003

The observed ABY-029 contrast in tumor-to-normal brain is shown versus administered dose, where each dose of ABY-029 was sampled from multiple animals and tissue slices as shown by the standard deviation indicated by error bars. A logistic function fitting was completed to estimate the observed saturation level of the data at higher concentrations (Adjusted R 2 = 0.93). One outlier in the dose of 245 µg/kg was removed which was 3× standard deviations outside the other data points.
Figure Legend Snippet: The observed ABY-029 contrast in tumor-to-normal brain is shown versus administered dose, where each dose of ABY-029 was sampled from multiple animals and tissue slices as shown by the standard deviation indicated by error bars. A logistic function fitting was completed to estimate the observed saturation level of the data at higher concentrations (Adjusted R 2 = 0.93). One outlier in the dose of 245 µg/kg was removed which was 3× standard deviations outside the other data points.

Techniques Used: Standard Deviation

Effect of pre-injection of unlabeled cetuximab on contrast tumor-to-normal brain, where the ratio of ABY-029 to cetuximab was varied, as denoted by the three columns, 1:1, 1:2.5 and 1:10, with the assay done at both 1 h and 24 h.
Figure Legend Snippet: Effect of pre-injection of unlabeled cetuximab on contrast tumor-to-normal brain, where the ratio of ABY-029 to cetuximab was varied, as denoted by the three columns, 1:1, 1:2.5 and 1:10, with the assay done at both 1 h and 24 h.

Techniques Used: Injection

Tissue slices and histology comparisons show ( a ) ABY-029 fluorescence in the brain slices, with corresponding H E histology slice ( b ) showing the co-registration between ABY-029 fluorescence and tumor, and ( c ) the histogram of fluorescence range heterogeneity from ABY-029 in the tumor region. Doses of ABY-029 from top to bottom: 245 µg/kg, 490 µg/kg, and 1225 µg/kg, respectively.
Figure Legend Snippet: Tissue slices and histology comparisons show ( a ) ABY-029 fluorescence in the brain slices, with corresponding H E histology slice ( b ) showing the co-registration between ABY-029 fluorescence and tumor, and ( c ) the histogram of fluorescence range heterogeneity from ABY-029 in the tumor region. Doses of ABY-029 from top to bottom: 245 µg/kg, 490 µg/kg, and 1225 µg/kg, respectively.

Techniques Used: Fluorescence

Inhibition of ABY-029 binding to EGFR by unlabeled cetuximab was investigated, with bars showing the incubation conditions. Cetuximab bars show the autofluorescence. Shared letters mean no statistical difference (ANOVA, Tukey’s Multiple Comparison Test).
Figure Legend Snippet: Inhibition of ABY-029 binding to EGFR by unlabeled cetuximab was investigated, with bars showing the incubation conditions. Cetuximab bars show the autofluorescence. Shared letters mean no statistical difference (ANOVA, Tukey’s Multiple Comparison Test).

Techniques Used: Inhibition, Binding Assay, Incubation

12) Product Images from "RAPID COMMUNICATION: Optimizing Glioma Detection using an EGFR-Targeted Fluorescent Affibody"

Article Title: RAPID COMMUNICATION: Optimizing Glioma Detection using an EGFR-Targeted Fluorescent Affibody

Journal: Photochemistry and photobiology

doi: 10.1111/php.13003

Effect of pre-injection of unlabeled cetuximab on contrast tumor-to-normal brain, where the ratio of ABY-029 to cetuximab was varied, as denoted by the three columns, 1:1, 1:2.5 and 1:10, with the assay done at both 1 h and 24 h.
Figure Legend Snippet: Effect of pre-injection of unlabeled cetuximab on contrast tumor-to-normal brain, where the ratio of ABY-029 to cetuximab was varied, as denoted by the three columns, 1:1, 1:2.5 and 1:10, with the assay done at both 1 h and 24 h.

Techniques Used: Injection

Inhibition of ABY-029 binding to EGFR by unlabeled cetuximab was investigated, with bars showing the incubation conditions. Cetuximab bars show the autofluorescence. Shared letters mean no statistical difference (ANOVA, Tukey’s Multiple Comparison Test).
Figure Legend Snippet: Inhibition of ABY-029 binding to EGFR by unlabeled cetuximab was investigated, with bars showing the incubation conditions. Cetuximab bars show the autofluorescence. Shared letters mean no statistical difference (ANOVA, Tukey’s Multiple Comparison Test).

Techniques Used: Inhibition, Binding Assay, Incubation

13) Product Images from "Epidermal Growth Factor Receptor in Glioma: Signal Transduction, Neuropathology, Imaging, and Radioresistance 1"

Article Title: Epidermal Growth Factor Receptor in Glioma: Signal Transduction, Neuropathology, Imaging, and Radioresistance 1

Journal: Neoplasia (New York, N.Y.)

doi:

EGFR signaling and NHEJ. The binding of EGF or TGFα to EGFR activates the following pathways: (1) PI3K-Akt-1 pathway, (2) Ras/RAF/MAPK/extracellular signal-regulated (ERK) pathway, and (3) signal transducer and activation of transcription (STAT) pathway (only the PI3K-Akt-1 pathway is shown for simplicity). EGFRvIII, a common deletion mutant that lacks the ligand-binding extracellular domain, is constitutively active and signals preferentially through the PI3K-Akt-1 pathway. In this pathway, activated PI3K phosphorylates phosphatidylinositol-4,5-biphosphate (PIP2) generating phosphatidylinositol-3,4,5-triphosphate (PIP3). PIP3 anchors Akt-1 to the plasma membrane, where it is phosphorylated by mammalian target of rapamycin complex 2 (mTORC2) and 3-phosphoinositide-dependent kinase 1 (PDK1). Activated Akt-1 phosphorylates a variety of downstream targets that enhance proliferation and inhibit cell death. The PTEN tumor suppressor negatively regulates PI3K-Akt-1 signaling by reversing PIP3 back to PIP2. Two models have been proposed to explain the connection between EGFR and NHEJ. In one scenario, wtEGFR translocates into the nucleus in response to IR, interacts with DNA-PKcs (DNA-dependent protein kinase, catalytic subunit), and stimulates its DNA repair activity (I). In another scenario, Akt-1 translocates into the nucleus in response to IR and interacts with DNA-PKcs (II). Phosphorylation of Akt-1 by DNA-PKcs promotes survival (curved arrow) and we hypothesize that reciprocal phosphorylation of DNA-PKcs by Akt-1 might promote DSB repair through NHEJ.
Figure Legend Snippet: EGFR signaling and NHEJ. The binding of EGF or TGFα to EGFR activates the following pathways: (1) PI3K-Akt-1 pathway, (2) Ras/RAF/MAPK/extracellular signal-regulated (ERK) pathway, and (3) signal transducer and activation of transcription (STAT) pathway (only the PI3K-Akt-1 pathway is shown for simplicity). EGFRvIII, a common deletion mutant that lacks the ligand-binding extracellular domain, is constitutively active and signals preferentially through the PI3K-Akt-1 pathway. In this pathway, activated PI3K phosphorylates phosphatidylinositol-4,5-biphosphate (PIP2) generating phosphatidylinositol-3,4,5-triphosphate (PIP3). PIP3 anchors Akt-1 to the plasma membrane, where it is phosphorylated by mammalian target of rapamycin complex 2 (mTORC2) and 3-phosphoinositide-dependent kinase 1 (PDK1). Activated Akt-1 phosphorylates a variety of downstream targets that enhance proliferation and inhibit cell death. The PTEN tumor suppressor negatively regulates PI3K-Akt-1 signaling by reversing PIP3 back to PIP2. Two models have been proposed to explain the connection between EGFR and NHEJ. In one scenario, wtEGFR translocates into the nucleus in response to IR, interacts with DNA-PKcs (DNA-dependent protein kinase, catalytic subunit), and stimulates its DNA repair activity (I). In another scenario, Akt-1 translocates into the nucleus in response to IR and interacts with DNA-PKcs (II). Phosphorylation of Akt-1 by DNA-PKcs promotes survival (curved arrow) and we hypothesize that reciprocal phosphorylation of DNA-PKcs by Akt-1 might promote DSB repair through NHEJ.

Techniques Used: Non-Homologous End Joining, Binding Assay, Activation Assay, Mutagenesis, Ligand Binding Assay, Activity Assay

14) Product Images from "Evaluation of affibody charge modification identified by synthetic consensus design in molecular PET imaging of epidermal growth factor receptor"

Article Title: Evaluation of affibody charge modification identified by synthetic consensus design in molecular PET imaging of epidermal growth factor receptor

Journal: Molecular systems design & engineering

doi: 10.1039/C7ME00095B

Binding characterization. (A) EGFR high A431 cells were mixed with the indicated concentration of affibody (EA26S (blue circles) or EA62S (green squares)). Binding was detected with fluorophore-conjugated anti-His6 antibody via flow cytometry. Equilibrium dissociation constants were calculated assuming a 1:1 binding model. n = 3 measurements per variant. (B) EGFR high A431 cells (white) or EGFR low MCF7 cells (black) were incubated with 500 nM affibody. Binding was detected as in (A). n = 3 measurements per variant.
Figure Legend Snippet: Binding characterization. (A) EGFR high A431 cells were mixed with the indicated concentration of affibody (EA26S (blue circles) or EA62S (green squares)). Binding was detected with fluorophore-conjugated anti-His6 antibody via flow cytometry. Equilibrium dissociation constants were calculated assuming a 1:1 binding model. n = 3 measurements per variant. (B) EGFR high A431 cells (white) or EGFR low MCF7 cells (black) were incubated with 500 nM affibody. Binding was detected as in (A). n = 3 measurements per variant.

Techniques Used: Binding Assay, Concentration Assay, Flow Cytometry, Cytometry, Variant Assay, Incubation

15) Product Images from "Anti-EGFR Affibodies with Site-Specific Photo-Cross-Linker Incorporation Show Both Directed Target-Specific Photoconjugation and Increased Retention in Tumors"

Article Title: Anti-EGFR Affibodies with Site-Specific Photo-Cross-Linker Incorporation Show Both Directed Target-Specific Photoconjugation and Increased Retention in Tumors

Journal: Journal of the American Chemical Society

doi: 10.1021/jacs.8b07601

(A) Denaturing polyacrylamide gel electrophoresis (SDS-PAGE) showing photo-cross-linking products of the affibody as indicated. All samples were treated with PNGase to degloycosylate and visualize EGFR extracellular domain (∼70 kDa) as a distinct band. Each sample contained both an affibody and EGFR fragment unless otherwise indicated. Note that only the N23BP mutant gave a photoproduct, which corresponded to a mass increase of 10 kDa. Ladder proteins are (top to bottom) 100, 75, and 50 kDa. (B) N23BP and EGFR mixed as in (A) or with the equimolar concentration (260 nM) bovine serum albumin (BSA) as indicated and irradiated for the time listed. A band corresponding to the EGFR-affibody conjugate appears at 5, 15, and 30 min (red arrows). Ladder proteins are (top to bottom) 100, 75, and 50 kDa.
Figure Legend Snippet: (A) Denaturing polyacrylamide gel electrophoresis (SDS-PAGE) showing photo-cross-linking products of the affibody as indicated. All samples were treated with PNGase to degloycosylate and visualize EGFR extracellular domain (∼70 kDa) as a distinct band. Each sample contained both an affibody and EGFR fragment unless otherwise indicated. Note that only the N23BP mutant gave a photoproduct, which corresponded to a mass increase of 10 kDa. Ladder proteins are (top to bottom) 100, 75, and 50 kDa. (B) N23BP and EGFR mixed as in (A) or with the equimolar concentration (260 nM) bovine serum albumin (BSA) as indicated and irradiated for the time listed. A band corresponding to the EGFR-affibody conjugate appears at 5, 15, and 30 min (red arrows). Ladder proteins are (top to bottom) 100, 75, and 50 kDa.

Techniques Used: Polyacrylamide Gel Electrophoresis, SDS Page, Mutagenesis, Concentration Assay, Irradiation

Retention of fluorescently labeled affibodies (black, WT; blue, N23BP; red, Z) in 3D tumor spheroids grown from transfected 4T1 cells either (A) induced with 15 μ g/mL cumate or uninduced (B). Additional induced spheroids were irradiated with 365 nm light for 30 min after 3.5 h of incubation and again measured for retention (dashed lines, triangles) and compared to nonirradiated spheroids (solid lines, circles). Concentrations of retained affibodies were calculated by comparing spheroid lysate fluorescence to standard curves prepared for each labeled affibody. (C) Retention of fluorescently labeled affibodies (black, WT; blue, N23BP) with (dotted line, diamonds) or without (dashed line, triangles) addition of EGF.
Figure Legend Snippet: Retention of fluorescently labeled affibodies (black, WT; blue, N23BP; red, Z) in 3D tumor spheroids grown from transfected 4T1 cells either (A) induced with 15 μ g/mL cumate or uninduced (B). Additional induced spheroids were irradiated with 365 nm light for 30 min after 3.5 h of incubation and again measured for retention (dashed lines, triangles) and compared to nonirradiated spheroids (solid lines, circles). Concentrations of retained affibodies were calculated by comparing spheroid lysate fluorescence to standard curves prepared for each labeled affibody. (C) Retention of fluorescently labeled affibodies (black, WT; blue, N23BP) with (dotted line, diamonds) or without (dashed line, triangles) addition of EGF.

Techniques Used: Labeling, Transfection, Irradiation, Incubation, Fluorescence

Overlaid MALDI-MS spectra of affibodies (black, WT; blue, N23BP; red, Z) after conjugation with maleimide-benzophenone. Cysteine-containing affibodies show near complete conjugation with one maleimide-benzophenone, and smaller peaks show a second conjugation, likely with a lysine residue. Labels indicate the expected mass of each affibody.
Figure Legend Snippet: Overlaid MALDI-MS spectra of affibodies (black, WT; blue, N23BP; red, Z) after conjugation with maleimide-benzophenone. Cysteine-containing affibodies show near complete conjugation with one maleimide-benzophenone, and smaller peaks show a second conjugation, likely with a lysine residue. Labels indicate the expected mass of each affibody.

Techniques Used: Mass Spectrometry, Conjugation Assay

Retention of fluorescently labeled affibodies (black, WT; blue, N23BP; red, Z) in either (A) transfected 4T1 cells induced with 15 μ g/mL cumate or (B) MDA-MB-468 cells, each grown as an adherent monolayer of cells. Wells were either irradiated after 3.5 h of incubation for 30 min with 365 nm light (dashed lines, triangles) or kept in the dark (solid lines, circles). Concentrations of retained affibodies were calculated by comparing spheroid lysate fluorescence to standard curves prepared for each labeled affibody.
Figure Legend Snippet: Retention of fluorescently labeled affibodies (black, WT; blue, N23BP; red, Z) in either (A) transfected 4T1 cells induced with 15 μ g/mL cumate or (B) MDA-MB-468 cells, each grown as an adherent monolayer of cells. Wells were either irradiated after 3.5 h of incubation for 30 min with 365 nm light (dashed lines, triangles) or kept in the dark (solid lines, circles). Concentrations of retained affibodies were calculated by comparing spheroid lysate fluorescence to standard curves prepared for each labeled affibody.

Techniques Used: Labeling, Transfection, Multiple Displacement Amplification, Irradiation, Incubation, Fluorescence

(A) Denaturing polyacrylamide gel electrophoresis (SDS-PAGE) showing photo-cross-linking products of either N23BP or WT to EGFR, with or without irradiation at 980 nm. Note that only 980 nm irradiation of N23BP produced a photoproduct significantly different than free EGFR (right lane). Ladder proteins are (top to bottom) 100, 75, and 50 kDa. (B) Retention of fluorescently labeled affibodies (left, N23BP; right, WT) in 3D tumor spheroids grown from transfected 4T1 cells either induced with 15 μ g/mL cumate. Concentrations of retained affibodies were calculated by comparing spheroid lysate fluorescence to standard curves prepared for each labeled affibody. “20 IR” designates that this sample was irradiated at 980 nm after 3 h incubation, then left to grow to a total of 20 h. Lanes without an IR designation were not irradiated.
Figure Legend Snippet: (A) Denaturing polyacrylamide gel electrophoresis (SDS-PAGE) showing photo-cross-linking products of either N23BP or WT to EGFR, with or without irradiation at 980 nm. Note that only 980 nm irradiation of N23BP produced a photoproduct significantly different than free EGFR (right lane). Ladder proteins are (top to bottom) 100, 75, and 50 kDa. (B) Retention of fluorescently labeled affibodies (left, N23BP; right, WT) in 3D tumor spheroids grown from transfected 4T1 cells either induced with 15 μ g/mL cumate. Concentrations of retained affibodies were calculated by comparing spheroid lysate fluorescence to standard curves prepared for each labeled affibody. “20 IR” designates that this sample was irradiated at 980 nm after 3 h incubation, then left to grow to a total of 20 h. Lanes without an IR designation were not irradiated.

Techniques Used: Polyacrylamide Gel Electrophoresis, SDS Page, Irradiation, Produced, Labeling, Transfection, Fluorescence, Incubation

Left: Spheroids were treated with 1 μ M N23BP (top) or WT (bottom) affibody for a total of 4 h (left column) and 20 h (middle and right columns) and additionally irradiated with 365 nm light for 30 min after 3.5 h incubation (right column). These spheroids were sectioned at approximately the same depth and imaged for Rhodamine and GFP distribution. Micrographs show Rhodamine signal within each section normalized by the average GFP intensity. Scale bars are 200 μ m. Right: Five spheroids were grown and irradiated for 1 h with the indicated N23BP affibody concentration in growth media. Affibody containing media was removed, and spheroids were lysed and loaded onto a PAGE gel and probed for affibody conjugates using an anti-T7 antibody. High molecular weight bands around the expected molecular weight of EGFR were observed only when the spheroid-affibody mixtures were irradiated, indicating photo-cross-linking.
Figure Legend Snippet: Left: Spheroids were treated with 1 μ M N23BP (top) or WT (bottom) affibody for a total of 4 h (left column) and 20 h (middle and right columns) and additionally irradiated with 365 nm light for 30 min after 3.5 h incubation (right column). These spheroids were sectioned at approximately the same depth and imaged for Rhodamine and GFP distribution. Micrographs show Rhodamine signal within each section normalized by the average GFP intensity. Scale bars are 200 μ m. Right: Five spheroids were grown and irradiated for 1 h with the indicated N23BP affibody concentration in growth media. Affibody containing media was removed, and spheroids were lysed and loaded onto a PAGE gel and probed for affibody conjugates using an anti-T7 antibody. High molecular weight bands around the expected molecular weight of EGFR were observed only when the spheroid-affibody mixtures were irradiated, indicating photo-cross-linking.

Techniques Used: Irradiation, Incubation, Concentration Assay, Polyacrylamide Gel Electrophoresis, Molecular Weight

16) Product Images from "Cellular Effects of HER3-Specific Affibody Molecules"

Article Title: Cellular Effects of HER3-Specific Affibody Molecules

Journal: PLoS ONE

doi: 10.1371/journal.pone.0040023

Analysis of receptor phosphorylation of MCF-7 and SKBR-3 cells. Histograms showing ELISA absorbance results for detection of: A. Phospho-HER2 in MCF-7 cells, B. Phospho-HER3 in MCF-7 cells, C. Phospho-HER2 in SKBR-3 cells and D. Phospho-HER3 in SKBR-3 cells. Cells were incubated without HRG (grey bars) or with 0.05 nM HRG (black bars), in combination with the Affibody molecules (100 nM) before being lysed and analysed in an ELISA.
Figure Legend Snippet: Analysis of receptor phosphorylation of MCF-7 and SKBR-3 cells. Histograms showing ELISA absorbance results for detection of: A. Phospho-HER2 in MCF-7 cells, B. Phospho-HER3 in MCF-7 cells, C. Phospho-HER2 in SKBR-3 cells and D. Phospho-HER3 in SKBR-3 cells. Cells were incubated without HRG (grey bars) or with 0.05 nM HRG (black bars), in combination with the Affibody molecules (100 nM) before being lysed and analysed in an ELISA.

Techniques Used: Enzyme-linked Immunosorbent Assay, Incubation

Cell binding of HER3-specific Affibody molecules analysed by flow cytometry. Binding of Alexa Fluor® 488-labelled Affibody molecules (150 nM) to: A. MCF-7, B. SKBR-3 and the HER3-negative cell line SKOV-3. “Bl” = blocking with 15 μM of the corresponding, non-labelled Affibody. MCF-7 cells were stained in a separate experiment, whereas SKBR-3 and SKOV-3 were stained simultaneously.
Figure Legend Snippet: Cell binding of HER3-specific Affibody molecules analysed by flow cytometry. Binding of Alexa Fluor® 488-labelled Affibody molecules (150 nM) to: A. MCF-7, B. SKBR-3 and the HER3-negative cell line SKOV-3. “Bl” = blocking with 15 μM of the corresponding, non-labelled Affibody. MCF-7 cells were stained in a separate experiment, whereas SKBR-3 and SKOV-3 were stained simultaneously.

Techniques Used: Binding Assay, Flow Cytometry, Cytometry, Blocking Assay, Staining

Western blot analysis of phosphorylated Akt and Erk upon addition of heregulin and/or HER3-specific Affibody molecules. Phospho-Akt and phospho-Erk detected by western blot of cell lysates from MCF-7 and SKBR-3 cells treated with (+) or without (−) 0.05 nM heregulin (HRG) and 100 nM Affibody molecules (Z). As a control, β-actin was detected to show that the protein concentrations of the different lysates were equivalent.
Figure Legend Snippet: Western blot analysis of phosphorylated Akt and Erk upon addition of heregulin and/or HER3-specific Affibody molecules. Phospho-Akt and phospho-Erk detected by western blot of cell lysates from MCF-7 and SKBR-3 cells treated with (+) or without (−) 0.05 nM heregulin (HRG) and 100 nM Affibody molecules (Z). As a control, β-actin was detected to show that the protein concentrations of the different lysates were equivalent.

Techniques Used: Western Blot

Analysis of cellular growth inhibitory effects of the HER3-specific Affibody molecules. Mean absorbance values at 450 nm ± SD, which is proportional to the number of living cells, is given on the y-axis. A. Proliferation of MCF-7 and SKBR-3 cells grown in a dilution series of HRG. B. Proliferation of MCF-7 and SKBR-3 cells grown in 40 pM HRG and a dilution series of Affibody molecules Z05416, Z05417 or ZTaq. C. Proliferation of cells grown in medium containing 40 nM Affibody molecules, 0.04 nM HRG. Results are compared to unstimulated cells (no Affibody molecules or HRG added).
Figure Legend Snippet: Analysis of cellular growth inhibitory effects of the HER3-specific Affibody molecules. Mean absorbance values at 450 nm ± SD, which is proportional to the number of living cells, is given on the y-axis. A. Proliferation of MCF-7 and SKBR-3 cells grown in a dilution series of HRG. B. Proliferation of MCF-7 and SKBR-3 cells grown in 40 pM HRG and a dilution series of Affibody molecules Z05416, Z05417 or ZTaq. C. Proliferation of cells grown in medium containing 40 nM Affibody molecules, 0.04 nM HRG. Results are compared to unstimulated cells (no Affibody molecules or HRG added).

Techniques Used:

Competitional binding between the natural ligand HRG and Affibody molecules to the HER3-positive breast cancer cell line AU565 visualised by confocal microscopy. AU565 cells were pretreated with Affibody molecules (Z05416, Z05417 or ZTaq) or PBS only (HRG) before addition of HRG in conjugation with a fluorophore. HRG binding to cells is shown in green while nuclear staining by DAPI is shown in blue.
Figure Legend Snippet: Competitional binding between the natural ligand HRG and Affibody molecules to the HER3-positive breast cancer cell line AU565 visualised by confocal microscopy. AU565 cells were pretreated with Affibody molecules (Z05416, Z05417 or ZTaq) or PBS only (HRG) before addition of HRG in conjugation with a fluorophore. HRG binding to cells is shown in green while nuclear staining by DAPI is shown in blue.

Techniques Used: Binding Assay, Confocal Microscopy, Conjugation Assay, Staining

Immunofluorescent staining of human cancer cell lines. Images showing AU565, SKBR-3, MCF-7 and SKOV-3 cells stained with HER3-specific Affibody molecules Z05416 and Z05417, respectively. The polyclonal anti-HER3 antibody A234 and ZTaq were used as positive and negative staining controls, respectively. Affibody molecules and antibodies binding to cells are shown in green while nuclear staining by DAPI is given in blue. AU565 and SKOV-3 images were acquired on the same day using the same detection gain and laser power, enabling comparison between staining intensities. Cell staining of MCF-7 and SKBR-3 was analysed on different days, using different detection gains for optimal image acquisition. Additionally, MCF-7 images were acquired using increased laser power.
Figure Legend Snippet: Immunofluorescent staining of human cancer cell lines. Images showing AU565, SKBR-3, MCF-7 and SKOV-3 cells stained with HER3-specific Affibody molecules Z05416 and Z05417, respectively. The polyclonal anti-HER3 antibody A234 and ZTaq were used as positive and negative staining controls, respectively. Affibody molecules and antibodies binding to cells are shown in green while nuclear staining by DAPI is given in blue. AU565 and SKOV-3 images were acquired on the same day using the same detection gain and laser power, enabling comparison between staining intensities. Cell staining of MCF-7 and SKBR-3 was analysed on different days, using different detection gains for optimal image acquisition. Additionally, MCF-7 images were acquired using increased laser power.

Techniques Used: Staining, Negative Staining, Binding Assay

17) Product Images from "Hydrophobic Fluorescent Probes Introduce Artifacts into Single Molecule Tracking Experiments Due to Non-Specific Binding"

Article Title: Hydrophobic Fluorescent Probes Introduce Artifacts into Single Molecule Tracking Experiments Due to Non-Specific Binding

Journal: PLoS ONE

doi: 10.1371/journal.pone.0074200

Fluorescence intensity measured from confocal microscopy images of T47D cells labeled with 50-conjugated EGFR affibody, and a mixture of 25 nM dye-conjugated affibody and 25 nM unlabeled affibody. Three dyes were selected to cover the range of mobilities (Alexa 488, high mobility; CF 633, moderate mobility; Atto 565, low mobility). Columns represent the median of the distribution of membrane region pixel intensities derived from at least 100 cells. Error bars represent the positions of the 1 st and 3 rd quartile of the distributions.
Figure Legend Snippet: Fluorescence intensity measured from confocal microscopy images of T47D cells labeled with 50-conjugated EGFR affibody, and a mixture of 25 nM dye-conjugated affibody and 25 nM unlabeled affibody. Three dyes were selected to cover the range of mobilities (Alexa 488, high mobility; CF 633, moderate mobility; Atto 565, low mobility). Columns represent the median of the distribution of membrane region pixel intensities derived from at least 100 cells. Error bars represent the positions of the 1 st and 3 rd quartile of the distributions.

Techniques Used: Fluorescence, Confocal Microscopy, Labeling, Derivative Assay

18) Product Images from "DNA-Assembled Core-Satellite Upconverting-Metal–Organic Framework Nanoparticle Superstructures for Efficient Photodynamic Therapy"

Article Title: DNA-Assembled Core-Satellite Upconverting-Metal–Organic Framework Nanoparticle Superstructures for Efficient Photodynamic Therapy

Journal: Small (Weinheim an der Bergstrasse, Germany)

doi: 10.1002/smll.201700504

TEM images of assembled MOF–UCNP superstructures with DNA1-MOF and DNA2-UCNPs using MOF:UCNP ratios of a) 1:1, b) 1:6, and c) 1:12.
Figure Legend Snippet: TEM images of assembled MOF–UCNP superstructures with DNA1-MOF and DNA2-UCNPs using MOF:UCNP ratios of a) 1:1, b) 1:6, and c) 1:12.

Techniques Used: Transmission Electron Microscopy

Confocal luminescence imaging of MDA-MB-468 cells incubated with a) MOF–UCNP affibody , fluorescence core–satellite superstructures, b) MOF–UCNP fluorescence core–satellite superstructures, and c) MOF–UCNP affibody core–satellite superstructures. d) 3D confocal bright field and luminescence images of MDA-MB-468 cells incubated with MOF–UCNP affibody , fluorescence core–satellite superstructures. (All the figures have the same scale bar. Images were collected using a Nikon A1 microscope with a 0.2% setting of a maximum 70 mW laser power.)
Figure Legend Snippet: Confocal luminescence imaging of MDA-MB-468 cells incubated with a) MOF–UCNP affibody , fluorescence core–satellite superstructures, b) MOF–UCNP fluorescence core–satellite superstructures, and c) MOF–UCNP affibody core–satellite superstructures. d) 3D confocal bright field and luminescence images of MDA-MB-468 cells incubated with MOF–UCNP affibody , fluorescence core–satellite superstructures. (All the figures have the same scale bar. Images were collected using a Nikon A1 microscope with a 0.2% setting of a maximum 70 mW laser power.)

Techniques Used: Imaging, Multiple Displacement Amplification, Incubation, Fluorescence, Microscopy

In vitro cell viability of MDA-MB-468 cells incubated with PEGylated MOF NPs and MOF–UCNP core–satellite superstructures at different concentrations for 4 h.
Figure Legend Snippet: In vitro cell viability of MDA-MB-468 cells incubated with PEGylated MOF NPs and MOF–UCNP core–satellite superstructures at different concentrations for 4 h.

Techniques Used: In Vitro, Multiple Displacement Amplification, Incubation

a) Illustration of PCN-224 MOF NPs synthesis. b) Self-assembled MOF–UCNP core–satellite superstructures (left) and random mixtures of MOFs and UCNPs (right) for PDT.
Figure Legend Snippet: a) Illustration of PCN-224 MOF NPs synthesis. b) Self-assembled MOF–UCNP core–satellite superstructures (left) and random mixtures of MOFs and UCNPs (right) for PDT.

Techniques Used:

Photoluminescence spectra of a) DNA assembled MOF–UCNP core–satellite superstructures made at different MOF:UCNP molar ratios. b) Comparison of photoluminescence from MOF NPs alone and with UCNPs (mixed or DNA assembled).
Figure Legend Snippet: Photoluminescence spectra of a) DNA assembled MOF–UCNP core–satellite superstructures made at different MOF:UCNP molar ratios. b) Comparison of photoluminescence from MOF NPs alone and with UCNPs (mixed or DNA assembled).

Techniques Used:

Singlet oxygen generated from MOF NPs, mixtures of MOF NPs and UCNPs, and DNA assembled MOF–UCNP core–satellite superstructures at different MOF:UCNP molar ratios under 980 nm laser irradiation.
Figure Legend Snippet: Singlet oxygen generated from MOF NPs, mixtures of MOF NPs and UCNPs, and DNA assembled MOF–UCNP core–satellite superstructures at different MOF:UCNP molar ratios under 980 nm laser irradiation.

Techniques Used: Generated, Irradiation

Quantitative analysis of MDA-MB-468 cell viabilities with MOF NPs, mixtures of MOF NPs and UCNPs, and DNA assembled MOF–UCNP core–satellite superstructures after 980 nm laser irradiation for 10 and 20 min. Data are averages ± one SD ( n > 3 experimental replicates). * p
Figure Legend Snippet: Quantitative analysis of MDA-MB-468 cell viabilities with MOF NPs, mixtures of MOF NPs and UCNPs, and DNA assembled MOF–UCNP core–satellite superstructures after 980 nm laser irradiation for 10 and 20 min. Data are averages ± one SD ( n > 3 experimental replicates). * p

Techniques Used: Multiple Displacement Amplification, Irradiation

19) Product Images from "Influence of composition of cysteine-containing peptide-based chelators on biodistribution of 99mTc-labeled anti-EGFR affibody molecules"

Article Title: Influence of composition of cysteine-containing peptide-based chelators on biodistribution of 99mTc-labeled anti-EGFR affibody molecules

Journal: Amino Acids

doi: 10.1007/s00726-018-2571-1

In vivo specificity of 99m Tc-ZEGFR conjugates ( a 99m Tc-ZEGFR-GGEC, b 99m Tc-ZEGFR-GEEC and c 99m Tc-ZEGFR-EEEC) in A431 xenografts and EGFR-expressing organs in mice at 6 h after injection. In the blocked group, receptors were saturated by pre-injection of large excess of anti-EGFR antibody cetuximab. The data are presented as the average ( n = 4) and SD
Figure Legend Snippet: In vivo specificity of 99m Tc-ZEGFR conjugates ( a 99m Tc-ZEGFR-GGEC, b 99m Tc-ZEGFR-GEEC and c 99m Tc-ZEGFR-EEEC) in A431 xenografts and EGFR-expressing organs in mice at 6 h after injection. In the blocked group, receptors were saturated by pre-injection of large excess of anti-EGFR antibody cetuximab. The data are presented as the average ( n = 4) and SD

Techniques Used: In Vivo, Expressing, Mouse Assay, Injection

20) Product Images from "RAPID COMMUNICATION: Optimizing Glioma Detection using an EGFR-Targeted Fluorescent Affibody"

Article Title: RAPID COMMUNICATION: Optimizing Glioma Detection using an EGFR-Targeted Fluorescent Affibody

Journal: Photochemistry and photobiology

doi: 10.1111/php.13003

The observed ABY-029 contrast in tumor-to-normal brain is shown versus administered dose, where each dose of ABY-029 was sampled from multiple animals and tissue slices as shown by the standard deviation indicated by error bars. A logistic function fitting was completed to estimate the observed saturation level of the data at higher concentrations (Adjusted R 2 = 0.93). One outlier in the dose of 245 µg/kg was removed which was 3× standard deviations outside the other data points.
Figure Legend Snippet: The observed ABY-029 contrast in tumor-to-normal brain is shown versus administered dose, where each dose of ABY-029 was sampled from multiple animals and tissue slices as shown by the standard deviation indicated by error bars. A logistic function fitting was completed to estimate the observed saturation level of the data at higher concentrations (Adjusted R 2 = 0.93). One outlier in the dose of 245 µg/kg was removed which was 3× standard deviations outside the other data points.

Techniques Used: Standard Deviation

Effect of pre-injection of unlabeled cetuximab on contrast tumor-to-normal brain, where the ratio of ABY-029 to cetuximab was varied, as denoted by the three columns, 1:1, 1:2.5 and 1:10, with the assay done at both 1 h and 24 h.
Figure Legend Snippet: Effect of pre-injection of unlabeled cetuximab on contrast tumor-to-normal brain, where the ratio of ABY-029 to cetuximab was varied, as denoted by the three columns, 1:1, 1:2.5 and 1:10, with the assay done at both 1 h and 24 h.

Techniques Used: Injection

Tissue slices and histology comparisons show ( a ) ABY-029 fluorescence in the brain slices, with corresponding H E histology slice ( b ) showing the co-registration between ABY-029 fluorescence and tumor, and ( c ) the histogram of fluorescence range heterogeneity from ABY-029 in the tumor region. Doses of ABY-029 from top to bottom: 245 µg/kg, 490 µg/kg, and 1225 µg/kg, respectively.
Figure Legend Snippet: Tissue slices and histology comparisons show ( a ) ABY-029 fluorescence in the brain slices, with corresponding H E histology slice ( b ) showing the co-registration between ABY-029 fluorescence and tumor, and ( c ) the histogram of fluorescence range heterogeneity from ABY-029 in the tumor region. Doses of ABY-029 from top to bottom: 245 µg/kg, 490 µg/kg, and 1225 µg/kg, respectively.

Techniques Used: Fluorescence

Inhibition of ABY-029 binding to EGFR by unlabeled cetuximab was investigated, with bars showing the incubation conditions. Cetuximab bars show the autofluorescence. Shared letters mean no statistical difference (ANOVA, Tukey’s Multiple Comparison Test).
Figure Legend Snippet: Inhibition of ABY-029 binding to EGFR by unlabeled cetuximab was investigated, with bars showing the incubation conditions. Cetuximab bars show the autofluorescence. Shared letters mean no statistical difference (ANOVA, Tukey’s Multiple Comparison Test).

Techniques Used: Inhibition, Binding Assay, Incubation

21) Product Images from "Simultaneous in vivo fluorescent markers for perfusion, protoporphyrin metabolism and EGFR expression for optically guided identification of orthotopic glioma"

Article Title: Simultaneous in vivo fluorescent markers for perfusion, protoporphyrin metabolism and EGFR expression for optically guided identification of orthotopic glioma

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

doi: 10.1158/1078-0432.CCR-16-1400

(A) Flow cytometry results showing the red channel log intensity of F98 wt , F98 EGFR , and F98 EGFRvIII cells following incubation with ABY-029, selected using a live/dead assay. (B) Excitation (solid lines) and emission curves (dashed lines) of pure IRDye800CW and of ABY-029, showing the slight red shift caused by conjugation to anti-EGFR Affibody® molecules.
Figure Legend Snippet: (A) Flow cytometry results showing the red channel log intensity of F98 wt , F98 EGFR , and F98 EGFRvIII cells following incubation with ABY-029, selected using a live/dead assay. (B) Excitation (solid lines) and emission curves (dashed lines) of pure IRDye800CW and of ABY-029, showing the slight red shift caused by conjugation to anti-EGFR Affibody® molecules.

Techniques Used: Flow Cytometry, Cytometry, Incubation, Live Dead Assay, Conjugation Assay

22) Product Images from "Benefit of Later-Time-Point PET Imaging of HER3 Expression Using Optimized Radiocobalt-Labeled Affibody Molecules"

Article Title: Benefit of Later-Time-Point PET Imaging of HER3 Expression Using Optimized Radiocobalt-Labeled Affibody Molecules

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms21061972

Receptor quantification and in vitro specificity. ( A ) HER3 expression was quantified for BxPC-3 (n = 2) and DU145 (n = 2) cells by incubation with [57Co]Co-(HE)3-ZHER3-NOTA until saturation. For the in vitro specificity test in ( B ) BxPC-3 and ( C ) DU145 cells, binding to HER3 was inhibited by addition of 50 nM HER3 binding affibody in the blocked groups. Specificity data is presented as the average of three dishes ± SD.
Figure Legend Snippet: Receptor quantification and in vitro specificity. ( A ) HER3 expression was quantified for BxPC-3 (n = 2) and DU145 (n = 2) cells by incubation with [57Co]Co-(HE)3-ZHER3-NOTA until saturation. For the in vitro specificity test in ( B ) BxPC-3 and ( C ) DU145 cells, binding to HER3 was inhibited by addition of 50 nM HER3 binding affibody in the blocked groups. Specificity data is presented as the average of three dishes ± SD.

Techniques Used: In Vitro, Expressing, Incubation, Binding Assay

In vivo specificity. Tumor-bearing female Balb/c nu/nu mice were injected with 2 µg of labeled conjugates or excess amount (70 µg) of non-labeled anti-HER3 affibody molecules. Data presented as the average ± SD of n = 3–4 animals/group. * Indicates significant difference p
Figure Legend Snippet: In vivo specificity. Tumor-bearing female Balb/c nu/nu mice were injected with 2 µg of labeled conjugates or excess amount (70 µg) of non-labeled anti-HER3 affibody molecules. Data presented as the average ± SD of n = 3–4 animals/group. * Indicates significant difference p

Techniques Used: In Vivo, Mouse Assay, Injection, Labeling

Structural overview of the macrocyclic chelators conjugated to the C-terminus of the HER3-targeting affibody molecule (HE) 3 -Z 08698 via a C-terminal cysteine (further denoted (HE) 3 -Z HER3 -X, with X = NOTA (1-(1,3-carboxypropyl)-4,7-carboxymethyl-1,4,7-triazacyclononane), NODAGA (1,4,7-triazacyclononane-N,N′,N″-triacetic acid), DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid), DOTAGA (1,4,7,10-tetraazacyclododecane, 1-(glutaric acid)-4,7,10-triacetic acid)).
Figure Legend Snippet: Structural overview of the macrocyclic chelators conjugated to the C-terminus of the HER3-targeting affibody molecule (HE) 3 -Z 08698 via a C-terminal cysteine (further denoted (HE) 3 -Z HER3 -X, with X = NOTA (1-(1,3-carboxypropyl)-4,7-carboxymethyl-1,4,7-triazacyclononane), NODAGA (1,4,7-triazacyclononane-N,N′,N″-triacetic acid), DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid), DOTAGA (1,4,7,10-tetraazacyclododecane, 1-(glutaric acid)-4,7,10-triacetic acid)).

Techniques Used:

23) Product Images from "Hydrophobic Fluorescent Probes Introduce Artifacts into Single Molecule Tracking Experiments Due to Non-Specific Binding"

Article Title: Hydrophobic Fluorescent Probes Introduce Artifacts into Single Molecule Tracking Experiments Due to Non-Specific Binding

Journal: PLoS ONE

doi: 10.1371/journal.pone.0074200

Mean instantaneous D fit for different anti-EGFR Affibody conjugates. Each datapoint corresponds to mean ± SEM of at least 10 areas acquired from 3 independent samples.
Figure Legend Snippet: Mean instantaneous D fit for different anti-EGFR Affibody conjugates. Each datapoint corresponds to mean ± SEM of at least 10 areas acquired from 3 independent samples.

Techniques Used:

Fluorescence intensity measured from confocal microscopy images of T47D cells labeled with 50-conjugated EGFR affibody, and a mixture of 25 nM dye-conjugated affibody and 25 nM unlabeled affibody. Three dyes were selected to cover the range of mobilities (Alexa 488, high mobility; CF 633, moderate mobility; Atto 565, low mobility). Columns represent the median of the distribution of membrane region pixel intensities derived from at least 100 cells. Error bars represent the positions of the 1 st and 3 rd quartile of the distributions.
Figure Legend Snippet: Fluorescence intensity measured from confocal microscopy images of T47D cells labeled with 50-conjugated EGFR affibody, and a mixture of 25 nM dye-conjugated affibody and 25 nM unlabeled affibody. Three dyes were selected to cover the range of mobilities (Alexa 488, high mobility; CF 633, moderate mobility; Atto 565, low mobility). Columns represent the median of the distribution of membrane region pixel intensities derived from at least 100 cells. Error bars represent the positions of the 1 st and 3 rd quartile of the distributions.

Techniques Used: Fluorescence, Confocal Microscopy, Labeling, Derivative Assay

Effect of logD and charge on affibody conjugate mobility. Plots of mean instantaneous D fit for different anti-EGFR Affibody conjugates vs charge at pH 7.4 ( A ), and logD ( B ). C) Plot of spot density for selected anti-EGFR Affibody conjugates vs charge at logD. Each datapoint corresponds to mean ± SEM of at least 10 independent areas. Lines show linear regression fit to the data, R 2 values indicating goodness of fit. Alexa 555 is not included in this figure as the structure is not published and charge and logD values are unavailable.
Figure Legend Snippet: Effect of logD and charge on affibody conjugate mobility. Plots of mean instantaneous D fit for different anti-EGFR Affibody conjugates vs charge at pH 7.4 ( A ), and logD ( B ). C) Plot of spot density for selected anti-EGFR Affibody conjugates vs charge at logD. Each datapoint corresponds to mean ± SEM of at least 10 independent areas. Lines show linear regression fit to the data, R 2 values indicating goodness of fit. Alexa 555 is not included in this figure as the structure is not published and charge and logD values are unavailable.

Techniques Used:

Analysis of definitely mobile vs immobile or very slow moving spots. A) Mean instantaneous D fit for different anti-EGFR Affibody conjugates, after removing data for spots with D values below 0.1 µm 2 /s. Each datapoint corresponds to mean ± SD of of the tracks contained in at least 10 different areas containing a minimum of 50 different cells. Blue bars indicate dyes excited at 491 nm, green at 561 nm, and red at 638 nm. B) Percentages of spots for each dye with D values below 0.1 µm 2 /s. C) Plot of mean instantaneous D fit for different anti-EGFR Affibody conjugates (calculated from all spots) vs percentage of spots with D values
Figure Legend Snippet: Analysis of definitely mobile vs immobile or very slow moving spots. A) Mean instantaneous D fit for different anti-EGFR Affibody conjugates, after removing data for spots with D values below 0.1 µm 2 /s. Each datapoint corresponds to mean ± SD of of the tracks contained in at least 10 different areas containing a minimum of 50 different cells. Blue bars indicate dyes excited at 491 nm, green at 561 nm, and red at 638 nm. B) Percentages of spots for each dye with D values below 0.1 µm 2 /s. C) Plot of mean instantaneous D fit for different anti-EGFR Affibody conjugates (calculated from all spots) vs percentage of spots with D values

Techniques Used:

24) Product Images from "Hydrophobic Fluorescent Probes Introduce Artifacts into Single Molecule Tracking Experiments Due to Non-Specific Binding"

Article Title: Hydrophobic Fluorescent Probes Introduce Artifacts into Single Molecule Tracking Experiments Due to Non-Specific Binding

Journal: PLoS ONE

doi: 10.1371/journal.pone.0074200

Mean instantaneous D fit for different anti-EGFR Affibody conjugates. Each datapoint corresponds to mean ± SEM of at least 10 areas acquired from 3 independent samples.
Figure Legend Snippet: Mean instantaneous D fit for different anti-EGFR Affibody conjugates. Each datapoint corresponds to mean ± SEM of at least 10 areas acquired from 3 independent samples.

Techniques Used:

Fluorescence intensity measured from confocal microscopy images of T47D cells labeled with 50-conjugated EGFR affibody, and a mixture of 25 nM dye-conjugated affibody and 25 nM unlabeled affibody. Three dyes were selected to cover the range of mobilities (Alexa 488, high mobility; CF 633, moderate mobility; Atto 565, low mobility). Columns represent the median of the distribution of membrane region pixel intensities derived from at least 100 cells. Error bars represent the positions of the 1 st and 3 rd quartile of the distributions.
Figure Legend Snippet: Fluorescence intensity measured from confocal microscopy images of T47D cells labeled with 50-conjugated EGFR affibody, and a mixture of 25 nM dye-conjugated affibody and 25 nM unlabeled affibody. Three dyes were selected to cover the range of mobilities (Alexa 488, high mobility; CF 633, moderate mobility; Atto 565, low mobility). Columns represent the median of the distribution of membrane region pixel intensities derived from at least 100 cells. Error bars represent the positions of the 1 st and 3 rd quartile of the distributions.

Techniques Used: Fluorescence, Confocal Microscopy, Labeling, Derivative Assay

Effect of logD and charge on affibody conjugate mobility. Plots of mean instantaneous D fit for different anti-EGFR Affibody conjugates vs charge at pH 7.4 ( A ), and logD ( B ). C) Plot of spot density for selected anti-EGFR Affibody conjugates vs charge at logD. Each datapoint corresponds to mean ± SEM of at least 10 independent areas. Lines show linear regression fit to the data, R 2 values indicating goodness of fit. Alexa 555 is not included in this figure as the structure is not published and charge and logD values are unavailable.
Figure Legend Snippet: Effect of logD and charge on affibody conjugate mobility. Plots of mean instantaneous D fit for different anti-EGFR Affibody conjugates vs charge at pH 7.4 ( A ), and logD ( B ). C) Plot of spot density for selected anti-EGFR Affibody conjugates vs charge at logD. Each datapoint corresponds to mean ± SEM of at least 10 independent areas. Lines show linear regression fit to the data, R 2 values indicating goodness of fit. Alexa 555 is not included in this figure as the structure is not published and charge and logD values are unavailable.

Techniques Used:

Analysis of definitely mobile vs immobile or very slow moving spots. A) Mean instantaneous D fit for different anti-EGFR Affibody conjugates, after removing data for spots with D values below 0.1 µm 2 /s. Each datapoint corresponds to mean ± SD of of the tracks contained in at least 10 different areas containing a minimum of 50 different cells. Blue bars indicate dyes excited at 491 nm, green at 561 nm, and red at 638 nm. B) Percentages of spots for each dye with D values below 0.1 µm 2 /s. C) Plot of mean instantaneous D fit for different anti-EGFR Affibody conjugates (calculated from all spots) vs percentage of spots with D values
Figure Legend Snippet: Analysis of definitely mobile vs immobile or very slow moving spots. A) Mean instantaneous D fit for different anti-EGFR Affibody conjugates, after removing data for spots with D values below 0.1 µm 2 /s. Each datapoint corresponds to mean ± SD of of the tracks contained in at least 10 different areas containing a minimum of 50 different cells. Blue bars indicate dyes excited at 491 nm, green at 561 nm, and red at 638 nm. B) Percentages of spots for each dye with D values below 0.1 µm 2 /s. C) Plot of mean instantaneous D fit for different anti-EGFR Affibody conjugates (calculated from all spots) vs percentage of spots with D values

Techniques Used:

25) Product Images from "Fluorogen Activating Protein–Affibody Probes: Modular, No-Wash Measurement of Epidermal Growth Factor Receptors"

Article Title: Fluorogen Activating Protein–Affibody Probes: Modular, No-Wash Measurement of Epidermal Growth Factor Receptors

Journal: Bioconjugate Chemistry

doi: 10.1021/bc500525b

Modular capacity of probes for labeling EGFR on the cell surface. (A) Structures of the fluorogens used. The synthetic and analytical details were shown in Supporting Information or described previously. 26 A431 cell labeled with FAP–affibody fusions and various malachite green derivatives were analyzed by flow cytometry (B) and live-cell fluorescence microscopy (C). 5 × 10 5 /mL of cells were labeled with 250 nM of AFA or F followed by incubation with 100 nM of fluorogens for 5 min. Cells were then either analyzed by flow cytometry for mean fluorescence measurement or cell imaging. Scale bar 20 μm.
Figure Legend Snippet: Modular capacity of probes for labeling EGFR on the cell surface. (A) Structures of the fluorogens used. The synthetic and analytical details were shown in Supporting Information or described previously. 26 A431 cell labeled with FAP–affibody fusions and various malachite green derivatives were analyzed by flow cytometry (B) and live-cell fluorescence microscopy (C). 5 × 10 5 /mL of cells were labeled with 250 nM of AFA or F followed by incubation with 100 nM of fluorogens for 5 min. Cells were then either analyzed by flow cytometry for mean fluorescence measurement or cell imaging. Scale bar 20 μm.

Techniques Used: Labeling, Flow Cytometry, Cytometry, Fluorescence, Microscopy, Incubation, Imaging

Characterization of probes binding on A431 cell surface. (A) Dissociation constant analysis of probes on cell surface. 5 × 10 5 /mL quantities of cells were incubated with probes for 1 h at 37 °C followed by 2 μM MG-B-tau for 5 min. Then cells were kept on ice for flow cytometry. The mean fluorescence intensity was corrected with background of cells incubating with F/MG and then normalized to mean fluorescence at 250 nM of probes. (B) Competition assay of nonfluorescent affibody A binding to the cell surface. Cells were labeled with 250 nM AFA or F and a serial dilution of A followed by 100 nM of MG-B-tau added prior to measurement. (C) Detection of receptor activation by Western blots. Starved cells were labeled with 250 nM probes followed by 100 nM of MG-B-tau and then cells were treated with 100 ng/mL EGF. Then cells were lysed for Western blot in order to detect phosphorylated EGFR and total EGFR. (D) Live-cell fluorescence microscopy of A431 cells labeled by various probes. Cells were labeled with 250 nM of probe and 100 nM of MG-B-tau prior to imaging or 100 nM of Cy5 conjugated affibody dimer. Scale bar 20 μm.
Figure Legend Snippet: Characterization of probes binding on A431 cell surface. (A) Dissociation constant analysis of probes on cell surface. 5 × 10 5 /mL quantities of cells were incubated with probes for 1 h at 37 °C followed by 2 μM MG-B-tau for 5 min. Then cells were kept on ice for flow cytometry. The mean fluorescence intensity was corrected with background of cells incubating with F/MG and then normalized to mean fluorescence at 250 nM of probes. (B) Competition assay of nonfluorescent affibody A binding to the cell surface. Cells were labeled with 250 nM AFA or F and a serial dilution of A followed by 100 nM of MG-B-tau added prior to measurement. (C) Detection of receptor activation by Western blots. Starved cells were labeled with 250 nM probes followed by 100 nM of MG-B-tau and then cells were treated with 100 ng/mL EGF. Then cells were lysed for Western blot in order to detect phosphorylated EGFR and total EGFR. (D) Live-cell fluorescence microscopy of A431 cells labeled by various probes. Cells were labeled with 250 nM of probe and 100 nM of MG-B-tau prior to imaging or 100 nM of Cy5 conjugated affibody dimer. Scale bar 20 μm.

Techniques Used: Binding Assay, Incubation, Flow Cytometry, Cytometry, Fluorescence, Competitive Binding Assay, Labeling, Serial Dilution, Activation Assay, Western Blot, Microscopy, Imaging

26) Product Images from "Affibody Modified and Radiolabeled Gold-Iron Oxide Hetero-nanostructures for Tumor PET, Optical and MR Imaging"

Article Title: Affibody Modified and Radiolabeled Gold-Iron Oxide Hetero-nanostructures for Tumor PET, Optical and MR Imaging

Journal: Biomaterials

doi: 10.1016/j.biomaterials.2013.01.014

A: Schematic three dimensional illustration of the Affibody binding domain with conventional single-component IONP (top) and dumbbell Au-IONP (bottom). B: Schematic illustration of Au-IONPs surface functionalization and conjugation with Affibody.
Figure Legend Snippet: A: Schematic three dimensional illustration of the Affibody binding domain with conventional single-component IONP (top) and dumbbell Au-IONP (bottom). B: Schematic illustration of Au-IONPs surface functionalization and conjugation with Affibody.

Techniques Used: Binding Assay, Conjugation Assay

27) Product Images from "Fluorogen Activating Protein–Affibody Probes: Modular, No-Wash Measurement of Epidermal Growth Factor Receptors"

Article Title: Fluorogen Activating Protein–Affibody Probes: Modular, No-Wash Measurement of Epidermal Growth Factor Receptors

Journal: Bioconjugate Chemistry

doi: 10.1021/bc500525b

Modular capacity of probes for labeling EGFR on the cell surface. (A) Structures of the fluorogens used. The synthetic and analytical details were shown in Supporting Information or described previously. 26 A431 cell labeled with FAP–affibody fusions and various malachite green derivatives were analyzed by flow cytometry (B) and live-cell fluorescence microscopy (C). 5 × 10 5 /mL of cells were labeled with 250 nM of AFA or F followed by incubation with 100 nM of fluorogens for 5 min. Cells were then either analyzed by flow cytometry for mean fluorescence measurement or cell imaging. Scale bar 20 μm.
Figure Legend Snippet: Modular capacity of probes for labeling EGFR on the cell surface. (A) Structures of the fluorogens used. The synthetic and analytical details were shown in Supporting Information or described previously. 26 A431 cell labeled with FAP–affibody fusions and various malachite green derivatives were analyzed by flow cytometry (B) and live-cell fluorescence microscopy (C). 5 × 10 5 /mL of cells were labeled with 250 nM of AFA or F followed by incubation with 100 nM of fluorogens for 5 min. Cells were then either analyzed by flow cytometry for mean fluorescence measurement or cell imaging. Scale bar 20 μm.

Techniques Used: Labeling, Flow Cytometry, Cytometry, Fluorescence, Microscopy, Incubation, Imaging

Characterization of probes binding on A431 cell surface. (A) Dissociation constant analysis of probes on cell surface. 5 × 10 5 /mL quantities of cells were incubated with probes for 1 h at 37 °C followed by 2 μM MG-B-tau for 5 min. Then cells were kept on ice for flow cytometry. The mean fluorescence intensity was corrected with background of cells incubating with F/MG and then normalized to mean fluorescence at 250 nM of probes. (B) Competition assay of nonfluorescent affibody A binding to the cell surface. Cells were labeled with 250 nM AFA or F and a serial dilution of A followed by 100 nM of MG-B-tau added prior to measurement. (C) Detection of receptor activation by Western blots. Starved cells were labeled with 250 nM probes followed by 100 nM of MG-B-tau and then cells were treated with 100 ng/mL EGF. Then cells were lysed for Western blot in order to detect phosphorylated EGFR and total EGFR. (D) Live-cell fluorescence microscopy of A431 cells labeled by various probes. Cells were labeled with 250 nM of probe and 100 nM of MG-B-tau prior to imaging or 100 nM of Cy5 conjugated affibody dimer. Scale bar 20 μm.
Figure Legend Snippet: Characterization of probes binding on A431 cell surface. (A) Dissociation constant analysis of probes on cell surface. 5 × 10 5 /mL quantities of cells were incubated with probes for 1 h at 37 °C followed by 2 μM MG-B-tau for 5 min. Then cells were kept on ice for flow cytometry. The mean fluorescence intensity was corrected with background of cells incubating with F/MG and then normalized to mean fluorescence at 250 nM of probes. (B) Competition assay of nonfluorescent affibody A binding to the cell surface. Cells were labeled with 250 nM AFA or F and a serial dilution of A followed by 100 nM of MG-B-tau added prior to measurement. (C) Detection of receptor activation by Western blots. Starved cells were labeled with 250 nM probes followed by 100 nM of MG-B-tau and then cells were treated with 100 ng/mL EGF. Then cells were lysed for Western blot in order to detect phosphorylated EGFR and total EGFR. (D) Live-cell fluorescence microscopy of A431 cells labeled by various probes. Cells were labeled with 250 nM of probe and 100 nM of MG-B-tau prior to imaging or 100 nM of Cy5 conjugated affibody dimer. Scale bar 20 μm.

Techniques Used: Binding Assay, Incubation, Flow Cytometry, Cytometry, Fluorescence, Competitive Binding Assay, Labeling, Serial Dilution, Activation Assay, Western Blot, Microscopy, Imaging

28) Product Images from "Genetic Assembly of Double‐Layered Fluorescent Protein Nanoparticles for Cancer Targeting and Imaging"

Article Title: Genetic Assembly of Double‐Layered Fluorescent Protein Nanoparticles for Cancer Targeting and Imaging

Journal: Advanced Science

doi: 10.1002/advs.201600471

Time‐course analysis of the intracellular fluorescence of MDA‐MB‐468 cancer cells treated with mC‐DL‐HBVC, mC in ‐HBVC, and mC out ‐HBVC, both of which present affibody peptides on their surface. a) Confocal microscopy images of MDA‐MB‐468 cells at predetermined time points after treated with mC‐DL‐HBVC and mC out ‐HBVC. Nuclei were stained with DAPI (blue). b) Time‐course fluorescence intensities from the MDA‐MB‐468 cells of (a). At each time point, the fluorescence intensities were normalized by the respective initial value. Error bars represent standard deviations, n ≥ 7. *: p ‐value
Figure Legend Snippet: Time‐course analysis of the intracellular fluorescence of MDA‐MB‐468 cancer cells treated with mC‐DL‐HBVC, mC in ‐HBVC, and mC out ‐HBVC, both of which present affibody peptides on their surface. a) Confocal microscopy images of MDA‐MB‐468 cells at predetermined time points after treated with mC‐DL‐HBVC and mC out ‐HBVC. Nuclei were stained with DAPI (blue). b) Time‐course fluorescence intensities from the MDA‐MB‐468 cells of (a). At each time point, the fluorescence intensities were normalized by the respective initial value. Error bars represent standard deviations, n ≥ 7. *: p ‐value

Techniques Used: Fluorescence, Multiple Displacement Amplification, Confocal Microscopy, Staining

Cellular uptake of mC‐DL‐HBVC (2 × 10 −9 m ) and mC monomer (200 × 10 −9 m ) with and without affibody peptides by EGFR‐overexpressing cancer cells (MDA‐MB‐468) in vitro at 37 °C. a) Confocal microscopy images of MDA‐MB‐468 cells. Nuclei were counterstained with DAPI (blue). b) Fluorescence intensities from the MDA‐MB‐468 cells of (a). Error bars represent standard deviations, n ≥ 7. *: p ‐value
Figure Legend Snippet: Cellular uptake of mC‐DL‐HBVC (2 × 10 −9 m ) and mC monomer (200 × 10 −9 m ) with and without affibody peptides by EGFR‐overexpressing cancer cells (MDA‐MB‐468) in vitro at 37 °C. a) Confocal microscopy images of MDA‐MB‐468 cells. Nuclei were counterstained with DAPI (blue). b) Fluorescence intensities from the MDA‐MB‐468 cells of (a). Error bars represent standard deviations, n ≥ 7. *: p ‐value

Techniques Used: Multiple Displacement Amplification, In Vitro, Confocal Microscopy, Fluorescence

TEM images and size distributions (determined by DLS) of purified eGFP in ‐HBVC with different linker lengths [ n = a) 0, b) 4, and c) 14], d) mC‐DL‐HBVC, e) mC out ‐HBVC, and f) mC in ‐HBVC. Scale bars represent 50 nm. All of eGFP in ‐HBVC, mC‐DL‐HBVC, mC out ‐HBVC, and mC in ‐HBVC present affibody peptides on their outer surface, as described in Scheme 1 a,c.
Figure Legend Snippet: TEM images and size distributions (determined by DLS) of purified eGFP in ‐HBVC with different linker lengths [ n = a) 0, b) 4, and c) 14], d) mC‐DL‐HBVC, e) mC out ‐HBVC, and f) mC in ‐HBVC. Scale bars represent 50 nm. All of eGFP in ‐HBVC, mC‐DL‐HBVC, mC out ‐HBVC, and mC in ‐HBVC present affibody peptides on their outer surface, as described in Scheme 1 a,c.

Techniques Used: Transmission Electron Microscopy, Purification

29) Product Images from "Near‐infrared photoimmunotherapy targeting EGFR—Shedding new light on glioblastoma treatment"

Article Title: Near‐infrared photoimmunotherapy targeting EGFR—Shedding new light on glioblastoma treatment

Journal: International Journal of Cancer

doi: 10.1002/ijc.31246

In vitro morphological changes following affibody‐based PIT. ( a ) Incubation of U87‐MGvIII spheroids with the Z EGFR:03115 –IR700DX for 6 h and irradiation with a red LED (16 J/cm 2 ) induced phototoxic cell death and disintegration of the architectural structure of the spheroid population. ( b ) U87‐MGvIII cells grown as a monolayer culture showed rapid cell swelling and bleb formation (see arrows) as visualized by a phase‐contrast image 1 h post Z EGFR:03115 –IR700DX (red) irradiation with the 639 nm laser on a confocal microscope. ( c ) Following methanol fixation of U87‐MGvIII cells, either treated by PIT or just irradiated, and staining with an anti‐calreticulin‐AlexaFluor488 antibody overnight (4°C), images were acquired by confocal microscopy. ( d ) Cell membrane disruption was monitored by propidium iodide (1 µg/mL) staining. U87‐MGvIII cells irradiated only or treated with Z EGFR:03115 –IR700DX‐based PIT were analyzed by flow cytometry 1 and 24 h post‐treatment. ( e ) Reactive oxygen species production was assessed using the DCFDA cellular ROS detection assay kit using U87‐MGvIII cells treated with affibody‐based PIT (15 min after light exposure). The results were normalized to the control cells. Data are presented as mean ± SEM ( n = 3).
Figure Legend Snippet: In vitro morphological changes following affibody‐based PIT. ( a ) Incubation of U87‐MGvIII spheroids with the Z EGFR:03115 –IR700DX for 6 h and irradiation with a red LED (16 J/cm 2 ) induced phototoxic cell death and disintegration of the architectural structure of the spheroid population. ( b ) U87‐MGvIII cells grown as a monolayer culture showed rapid cell swelling and bleb formation (see arrows) as visualized by a phase‐contrast image 1 h post Z EGFR:03115 –IR700DX (red) irradiation with the 639 nm laser on a confocal microscope. ( c ) Following methanol fixation of U87‐MGvIII cells, either treated by PIT or just irradiated, and staining with an anti‐calreticulin‐AlexaFluor488 antibody overnight (4°C), images were acquired by confocal microscopy. ( d ) Cell membrane disruption was monitored by propidium iodide (1 µg/mL) staining. U87‐MGvIII cells irradiated only or treated with Z EGFR:03115 –IR700DX‐based PIT were analyzed by flow cytometry 1 and 24 h post‐treatment. ( e ) Reactive oxygen species production was assessed using the DCFDA cellular ROS detection assay kit using U87‐MGvIII cells treated with affibody‐based PIT (15 min after light exposure). The results were normalized to the control cells. Data are presented as mean ± SEM ( n = 3).

Techniques Used: In Vitro, Incubation, Irradiation, Microscopy, Staining, Confocal Microscopy, Flow Cytometry, Cytometry, Detection Assay

30) Product Images from "Anti-EGFR Affibodies with Site-Specific Photo-Cross-Linker Incorporation Show Both Directed Target-Specific Photoconjugation and Increased Retention in Tumors"

Article Title: Anti-EGFR Affibodies with Site-Specific Photo-Cross-Linker Incorporation Show Both Directed Target-Specific Photoconjugation and Increased Retention in Tumors

Journal: Journal of the American Chemical Society

doi: 10.1021/jacs.8b07601

Retention of fluorescently labeled affibodies (black, WT; blue, N23BP; red, Z) in 3D tumor spheroids grown from transfected 4T1 cells either (A) induced with 15 μ g/mL cumate or uninduced (B). Additional induced spheroids were irradiated with 365 nm light for 30 min after 3.5 h of incubation and again measured for retention (dashed lines, triangles) and compared to nonirradiated spheroids (solid lines, circles). Concentrations of retained affibodies were calculated by comparing spheroid lysate fluorescence to standard curves prepared for each labeled affibody. (C) Retention of fluorescently labeled affibodies (black, WT; blue, N23BP) with (dotted line, diamonds) or without (dashed line, triangles) addition of EGF.
Figure Legend Snippet: Retention of fluorescently labeled affibodies (black, WT; blue, N23BP; red, Z) in 3D tumor spheroids grown from transfected 4T1 cells either (A) induced with 15 μ g/mL cumate or uninduced (B). Additional induced spheroids were irradiated with 365 nm light for 30 min after 3.5 h of incubation and again measured for retention (dashed lines, triangles) and compared to nonirradiated spheroids (solid lines, circles). Concentrations of retained affibodies were calculated by comparing spheroid lysate fluorescence to standard curves prepared for each labeled affibody. (C) Retention of fluorescently labeled affibodies (black, WT; blue, N23BP) with (dotted line, diamonds) or without (dashed line, triangles) addition of EGF.

Techniques Used: Labeling, Transfection, Irradiation, Incubation, Fluorescence

Overlaid MALDI-MS spectra of affibodies (black, WT; blue, N23BP; red, Z) after conjugation with maleimide-benzophenone. Cysteine-containing affibodies show near complete conjugation with one maleimide-benzophenone, and smaller peaks show a second conjugation, likely with a lysine residue. Labels indicate the expected mass of each affibody.
Figure Legend Snippet: Overlaid MALDI-MS spectra of affibodies (black, WT; blue, N23BP; red, Z) after conjugation with maleimide-benzophenone. Cysteine-containing affibodies show near complete conjugation with one maleimide-benzophenone, and smaller peaks show a second conjugation, likely with a lysine residue. Labels indicate the expected mass of each affibody.

Techniques Used: Mass Spectrometry, Conjugation Assay

Circular dichroism spectra of both the WT (black) and the N23 mutant conjugated with maleimide-benzophenone (blue). Inset: Melting curves showing the normalized circular dichroism signal at 222 nm versus temperature for both WT (black) and N23BP (blue) affibodies.
Figure Legend Snippet: Circular dichroism spectra of both the WT (black) and the N23 mutant conjugated with maleimide-benzophenone (blue). Inset: Melting curves showing the normalized circular dichroism signal at 222 nm versus temperature for both WT (black) and N23BP (blue) affibodies.

Techniques Used: Mutagenesis

Retention of fluorescently labeled affibodies (black, WT; blue, N23BP; red, Z) in either (A) transfected 4T1 cells induced with 15 μ g/mL cumate or (B) MDA-MB-468 cells, each grown as an adherent monolayer of cells. Wells were either irradiated after 3.5 h of incubation for 30 min with 365 nm light (dashed lines, triangles) or kept in the dark (solid lines, circles). Concentrations of retained affibodies were calculated by comparing spheroid lysate fluorescence to standard curves prepared for each labeled affibody.
Figure Legend Snippet: Retention of fluorescently labeled affibodies (black, WT; blue, N23BP; red, Z) in either (A) transfected 4T1 cells induced with 15 μ g/mL cumate or (B) MDA-MB-468 cells, each grown as an adherent monolayer of cells. Wells were either irradiated after 3.5 h of incubation for 30 min with 365 nm light (dashed lines, triangles) or kept in the dark (solid lines, circles). Concentrations of retained affibodies were calculated by comparing spheroid lysate fluorescence to standard curves prepared for each labeled affibody.

Techniques Used: Labeling, Transfection, Multiple Displacement Amplification, Irradiation, Incubation, Fluorescence

(A) Denaturing polyacrylamide gel electrophoresis (SDS-PAGE) showing photo-cross-linking products of either N23BP or WT to EGFR, with or without irradiation at 980 nm. Note that only 980 nm irradiation of N23BP produced a photoproduct significantly different than free EGFR (right lane). Ladder proteins are (top to bottom) 100, 75, and 50 kDa. (B) Retention of fluorescently labeled affibodies (left, N23BP; right, WT) in 3D tumor spheroids grown from transfected 4T1 cells either induced with 15 μ g/mL cumate. Concentrations of retained affibodies were calculated by comparing spheroid lysate fluorescence to standard curves prepared for each labeled affibody. “20 IR” designates that this sample was irradiated at 980 nm after 3 h incubation, then left to grow to a total of 20 h. Lanes without an IR designation were not irradiated.
Figure Legend Snippet: (A) Denaturing polyacrylamide gel electrophoresis (SDS-PAGE) showing photo-cross-linking products of either N23BP or WT to EGFR, with or without irradiation at 980 nm. Note that only 980 nm irradiation of N23BP produced a photoproduct significantly different than free EGFR (right lane). Ladder proteins are (top to bottom) 100, 75, and 50 kDa. (B) Retention of fluorescently labeled affibodies (left, N23BP; right, WT) in 3D tumor spheroids grown from transfected 4T1 cells either induced with 15 μ g/mL cumate. Concentrations of retained affibodies were calculated by comparing spheroid lysate fluorescence to standard curves prepared for each labeled affibody. “20 IR” designates that this sample was irradiated at 980 nm after 3 h incubation, then left to grow to a total of 20 h. Lanes without an IR designation were not irradiated.

Techniques Used: Polyacrylamide Gel Electrophoresis, SDS Page, Irradiation, Produced, Labeling, Transfection, Fluorescence, Incubation

31) Product Images from "Near‐infrared photoimmunotherapy targeting EGFR—Shedding new light on glioblastoma treatment"

Article Title: Near‐infrared photoimmunotherapy targeting EGFR—Shedding new light on glioblastoma treatment

Journal: International Journal of Cancer

doi: 10.1002/ijc.31246

Expression of EGFR in GBM cell lines and specificity of the Z EGFR:03115 –IR700DX binding to EGFR. ( a ) Varying EGFR expression in the selected cancer cell lines was confirmed by Western blot. The numbers in the brackets represent the relative EGFR expression as determined by densitometric analysis. ( b ) Z EGFR:03115 –IR700DX (30 nM) binding as assessed by flow cytometry in the selected cancer cells with varying EGFR expression and after blocking with 100‐fold excess of unlabeled Z EGFR:03115 . Data are presented as mean ± SEM ( n = 3). ( c – f ) Confocal microscopy images demonstrating target‐specific binding (4°C) and internalization (37°C) of either the Z EGFR:03115 –IR700DX (1 µM), anti‐EGFR‐FITC antibody (25 nM for visualization purposes) or IR700DX alone (1 µM): ( c ) U251 cell lines (1 h incubation time), ( d + e ) U87‐MGvIII or MCF7 cell lines (1–6 h incubation time), ( f ) U87‐MGvIII spheroids (6 h incubation time). Hoechst®33342 (blue) and LysoTracker™Green DND‐26 (green) were used for counterstaining. ( g ) Quantification of fluorescence intensity (median fluorescence intensity) of ∼8 µm slices through U87‐MGvIII spheroids following a 6 h incubation with Z EGFR:03115 –IR700DX (500 nM) or an anti‐EGFR‐FITC antibody (500 nM). Data are presented as mean ± SEM ( n = 3). ( h ) H E, EGFR and Ki67 immunostaining of U87‐MGvIII spheroid (400–500 µm) sections 72 h after seeding.
Figure Legend Snippet: Expression of EGFR in GBM cell lines and specificity of the Z EGFR:03115 –IR700DX binding to EGFR. ( a ) Varying EGFR expression in the selected cancer cell lines was confirmed by Western blot. The numbers in the brackets represent the relative EGFR expression as determined by densitometric analysis. ( b ) Z EGFR:03115 –IR700DX (30 nM) binding as assessed by flow cytometry in the selected cancer cells with varying EGFR expression and after blocking with 100‐fold excess of unlabeled Z EGFR:03115 . Data are presented as mean ± SEM ( n = 3). ( c – f ) Confocal microscopy images demonstrating target‐specific binding (4°C) and internalization (37°C) of either the Z EGFR:03115 –IR700DX (1 µM), anti‐EGFR‐FITC antibody (25 nM for visualization purposes) or IR700DX alone (1 µM): ( c ) U251 cell lines (1 h incubation time), ( d + e ) U87‐MGvIII or MCF7 cell lines (1–6 h incubation time), ( f ) U87‐MGvIII spheroids (6 h incubation time). Hoechst®33342 (blue) and LysoTracker™Green DND‐26 (green) were used for counterstaining. ( g ) Quantification of fluorescence intensity (median fluorescence intensity) of ∼8 µm slices through U87‐MGvIII spheroids following a 6 h incubation with Z EGFR:03115 –IR700DX (500 nM) or an anti‐EGFR‐FITC antibody (500 nM). Data are presented as mean ± SEM ( n = 3). ( h ) H E, EGFR and Ki67 immunostaining of U87‐MGvIII spheroid (400–500 µm) sections 72 h after seeding.

Techniques Used: Expressing, Binding Assay, Western Blot, Flow Cytometry, Cytometry, Blocking Assay, Confocal Microscopy, Incubation, Fluorescence, Immunostaining

In vitro morphological changes following affibody‐based PIT. ( a ) Incubation of U87‐MGvIII spheroids with the Z EGFR:03115 –IR700DX for 6 h and irradiation with a red LED (16 J/cm 2 ) induced phototoxic cell death and disintegration of the architectural structure of the spheroid population. ( b ) U87‐MGvIII cells grown as a monolayer culture showed rapid cell swelling and bleb formation (see arrows) as visualized by a phase‐contrast image 1 h post Z EGFR:03115 –IR700DX (red) irradiation with the 639 nm laser on a confocal microscope. ( c ) Following methanol fixation of U87‐MGvIII cells, either treated by PIT or just irradiated, and staining with an anti‐calreticulin‐AlexaFluor488 antibody overnight (4°C), images were acquired by confocal microscopy. ( d ) Cell membrane disruption was monitored by propidium iodide (1 µg/mL) staining. U87‐MGvIII cells irradiated only or treated with Z EGFR:03115 –IR700DX‐based PIT were analyzed by flow cytometry 1 and 24 h post‐treatment. ( e ) Reactive oxygen species production was assessed using the DCFDA cellular ROS detection assay kit using U87‐MGvIII cells treated with affibody‐based PIT (15 min after light exposure). The results were normalized to the control cells. Data are presented as mean ± SEM ( n = 3).
Figure Legend Snippet: In vitro morphological changes following affibody‐based PIT. ( a ) Incubation of U87‐MGvIII spheroids with the Z EGFR:03115 –IR700DX for 6 h and irradiation with a red LED (16 J/cm 2 ) induced phototoxic cell death and disintegration of the architectural structure of the spheroid population. ( b ) U87‐MGvIII cells grown as a monolayer culture showed rapid cell swelling and bleb formation (see arrows) as visualized by a phase‐contrast image 1 h post Z EGFR:03115 –IR700DX (red) irradiation with the 639 nm laser on a confocal microscope. ( c ) Following methanol fixation of U87‐MGvIII cells, either treated by PIT or just irradiated, and staining with an anti‐calreticulin‐AlexaFluor488 antibody overnight (4°C), images were acquired by confocal microscopy. ( d ) Cell membrane disruption was monitored by propidium iodide (1 µg/mL) staining. U87‐MGvIII cells irradiated only or treated with Z EGFR:03115 –IR700DX‐based PIT were analyzed by flow cytometry 1 and 24 h post‐treatment. ( e ) Reactive oxygen species production was assessed using the DCFDA cellular ROS detection assay kit using U87‐MGvIII cells treated with affibody‐based PIT (15 min after light exposure). The results were normalized to the control cells. Data are presented as mean ± SEM ( n = 3).

Techniques Used: In Vitro, Incubation, Irradiation, Microscopy, Staining, Confocal Microscopy, Flow Cytometry, Cytometry, Detection Assay

32) Product Images from "Near‐infrared photoimmunotherapy targeting EGFR—Shedding new light on glioblastoma treatment"

Article Title: Near‐infrared photoimmunotherapy targeting EGFR—Shedding new light on glioblastoma treatment

Journal: International Journal of Cancer

doi: 10.1002/ijc.31246

Testing Z EGFR:03115 –IR700DX specificity in vivo and studying the effect of functional groups on dye pharmacokinetics. ( a ) The U87‐MGvIII tumor could easily be differentiated as early as 1 h post Z EGFR:03115 –IR700DX (6 µg/mouse) being intravenously injected, whereas minimal tumor uptake was observed when administering the same amount of the non‐specific Z Taq ‐IR700DX. ( b ) Fluorescence imaging of Z EGFR:03115 –IR700DX uptake in excised tissues (1 h post‐injection) and respective tumor‐to‐organ ratios. ( c ) Mean radiant efficiency in U87‐MGvIII tumors 1 h after administering either 6 µg Z EGFR:03115 –IR700DX, 18 µg Z EGFR:03115 –IR700DX or 6 µg of the non‐specific Z Taq ‐IR700DX. ( d ) Tumor‐to‐background ratio comparison when altering the injected dose of Z EGFR:03115 –IR700DX. ( e , f ) Fluorescence intensity and tumor‐to‐background ratio in the U87‐MGvIII tumors over time after 18 µg Z EGFR:03115 –IR700DX. ( g , h ) Fluorescence imaging of mice bearing subcutaneous U87‐MGvIII tumors. Images were acquired 30 min, 1 h or 3 h post IR700DX–maleimide, IR800CW–maleimide, IR700DX–NHS ester and IR700DX–carboxylate injection and the mean radiant efficiency was determined for each of the dyes. ( i ) An SDS‐PAGE gel of mouse blood serum imaged using the IVIS/Spectrum imaging system to visualize the fluorescent dyes’ association with blood proteins. All data are presented as mean ± SD ( n ≥ 3).
Figure Legend Snippet: Testing Z EGFR:03115 –IR700DX specificity in vivo and studying the effect of functional groups on dye pharmacokinetics. ( a ) The U87‐MGvIII tumor could easily be differentiated as early as 1 h post Z EGFR:03115 –IR700DX (6 µg/mouse) being intravenously injected, whereas minimal tumor uptake was observed when administering the same amount of the non‐specific Z Taq ‐IR700DX. ( b ) Fluorescence imaging of Z EGFR:03115 –IR700DX uptake in excised tissues (1 h post‐injection) and respective tumor‐to‐organ ratios. ( c ) Mean radiant efficiency in U87‐MGvIII tumors 1 h after administering either 6 µg Z EGFR:03115 –IR700DX, 18 µg Z EGFR:03115 –IR700DX or 6 µg of the non‐specific Z Taq ‐IR700DX. ( d ) Tumor‐to‐background ratio comparison when altering the injected dose of Z EGFR:03115 –IR700DX. ( e , f ) Fluorescence intensity and tumor‐to‐background ratio in the U87‐MGvIII tumors over time after 18 µg Z EGFR:03115 –IR700DX. ( g , h ) Fluorescence imaging of mice bearing subcutaneous U87‐MGvIII tumors. Images were acquired 30 min, 1 h or 3 h post IR700DX–maleimide, IR800CW–maleimide, IR700DX–NHS ester and IR700DX–carboxylate injection and the mean radiant efficiency was determined for each of the dyes. ( i ) An SDS‐PAGE gel of mouse blood serum imaged using the IVIS/Spectrum imaging system to visualize the fluorescent dyes’ association with blood proteins. All data are presented as mean ± SD ( n ≥ 3).

Techniques Used: In Vivo, Functional Assay, Injection, Fluorescence, Imaging, Mouse Assay, SDS Page

Expression of EGFR in GBM cell lines and specificity of the Z EGFR:03115 –IR700DX binding to EGFR. ( a ) Varying EGFR expression in the selected cancer cell lines was confirmed by Western blot. The numbers in the brackets represent the relative EGFR expression as determined by densitometric analysis. ( b ) Z EGFR:03115 –IR700DX (30 nM) binding as assessed by flow cytometry in the selected cancer cells with varying EGFR expression and after blocking with 100‐fold excess of unlabeled Z EGFR:03115 . Data are presented as mean ± SEM ( n = 3). ( c – f ) Confocal microscopy images demonstrating target‐specific binding (4°C) and internalization (37°C) of either the Z EGFR:03115 –IR700DX (1 µM), anti‐EGFR‐FITC antibody (25 nM for visualization purposes) or IR700DX alone (1 µM): ( c ) U251 cell lines (1 h incubation time), ( d + e ) U87‐MGvIII or MCF7 cell lines (1–6 h incubation time), ( f ) U87‐MGvIII spheroids (6 h incubation time). Hoechst®33342 (blue) and LysoTracker™Green DND‐26 (green) were used for counterstaining. ( g ) Quantification of fluorescence intensity (median fluorescence intensity) of ∼8 µm slices through U87‐MGvIII spheroids following a 6 h incubation with Z EGFR:03115 –IR700DX (500 nM) or an anti‐EGFR‐FITC antibody (500 nM). Data are presented as mean ± SEM ( n = 3). ( h ) H E, EGFR and Ki67 immunostaining of U87‐MGvIII spheroid (400–500 µm) sections 72 h after seeding.
Figure Legend Snippet: Expression of EGFR in GBM cell lines and specificity of the Z EGFR:03115 –IR700DX binding to EGFR. ( a ) Varying EGFR expression in the selected cancer cell lines was confirmed by Western blot. The numbers in the brackets represent the relative EGFR expression as determined by densitometric analysis. ( b ) Z EGFR:03115 –IR700DX (30 nM) binding as assessed by flow cytometry in the selected cancer cells with varying EGFR expression and after blocking with 100‐fold excess of unlabeled Z EGFR:03115 . Data are presented as mean ± SEM ( n = 3). ( c – f ) Confocal microscopy images demonstrating target‐specific binding (4°C) and internalization (37°C) of either the Z EGFR:03115 –IR700DX (1 µM), anti‐EGFR‐FITC antibody (25 nM for visualization purposes) or IR700DX alone (1 µM): ( c ) U251 cell lines (1 h incubation time), ( d + e ) U87‐MGvIII or MCF7 cell lines (1–6 h incubation time), ( f ) U87‐MGvIII spheroids (6 h incubation time). Hoechst®33342 (blue) and LysoTracker™Green DND‐26 (green) were used for counterstaining. ( g ) Quantification of fluorescence intensity (median fluorescence intensity) of ∼8 µm slices through U87‐MGvIII spheroids following a 6 h incubation with Z EGFR:03115 –IR700DX (500 nM) or an anti‐EGFR‐FITC antibody (500 nM). Data are presented as mean ± SEM ( n = 3). ( h ) H E, EGFR and Ki67 immunostaining of U87‐MGvIII spheroid (400–500 µm) sections 72 h after seeding.

Techniques Used: Expressing, Binding Assay, Western Blot, Flow Cytometry, Cytometry, Blocking Assay, Confocal Microscopy, Incubation, Fluorescence, Immunostaining

Z EGFR:03115 –IR700DX‐mediated PIT causes cellular death selectively in EGFR+ve cells. Decrease in cell viability as assessed by the CellTiter‐Glo® luminescent cell viability assay 24 or 96 h post‐PIT in 2D cells and 3D spheroids, following 6 h incubation with the Z EGFR:03115 –IR700DX and irradiation with a light dose of 8 or 16 J/cm 2 , was confirmed to be dose dependent and receptor mediated. ( a ) U87‐MGvIII cells 24 h post‐PIT. ( b ) MCF7 cells 24 h post‐PIT. ( c , d ) U87‐MGvIII spheroids 24 and 96 h post‐PIT. ( e ) WSz4 spheroids 96 h post‐PIT. Data are presented as mean ± SEM ( n = 3). Statistical significance in comparison to the control group was determined using an unpaired two‐tailed Student's t‐ test with Welch's correction. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 and **** p ≤ 0.0001. [Color figure can be viewed at http://wileyonlinelibrary.com ]
Figure Legend Snippet: Z EGFR:03115 –IR700DX‐mediated PIT causes cellular death selectively in EGFR+ve cells. Decrease in cell viability as assessed by the CellTiter‐Glo® luminescent cell viability assay 24 or 96 h post‐PIT in 2D cells and 3D spheroids, following 6 h incubation with the Z EGFR:03115 –IR700DX and irradiation with a light dose of 8 or 16 J/cm 2 , was confirmed to be dose dependent and receptor mediated. ( a ) U87‐MGvIII cells 24 h post‐PIT. ( b ) MCF7 cells 24 h post‐PIT. ( c , d ) U87‐MGvIII spheroids 24 and 96 h post‐PIT. ( e ) WSz4 spheroids 96 h post‐PIT. Data are presented as mean ± SEM ( n = 3). Statistical significance in comparison to the control group was determined using an unpaired two‐tailed Student's t‐ test with Welch's correction. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 and **** p ≤ 0.0001. [Color figure can be viewed at http://wileyonlinelibrary.com ]

Techniques Used: Cell Viability Assay, Incubation, Irradiation, Two Tailed Test

In vivo Z EGFR:03115 –IR700DX‐mediated PIT studies. ( a ) Fluorescence imaging of mice bearing subcutaneous U87‐MGvIII tumors 1 h after injecting 18 μg of Z EGFR:03115 –IR700DX or IR700DX–maleimide (top row). Subsequently, mice were irradiated with an optical dose of 100 J/cm 2 by a red LED and, immediately after, imaged again (bottom row). ( b ) Tumor growth inhibition of the Z EGFR:03115 –IR700DX‐targeted PIT in U87‐MGvIII tumors after administering three doses of 18 µg of the conjugate and irradiating with 100 J/cm 2 at days 1, 3 and 5 in comparison to control groups. Data are presented as mean ± SD ( n = 6 for each group, ** p ≤ 0.01 as assessed by the Kruskal–Wallis test). ( c ) Visual observation of normal tissue damage in the PDT treated mice, while no skin damage was present in the Z EGFR:03115 –IR700DX PIT mice. These were the appearances seen in all mice. ( d ) H E staining of treated and untreated U87‐MGvIIII tumors (arrows indicate regions of tissue necrosis).
Figure Legend Snippet: In vivo Z EGFR:03115 –IR700DX‐mediated PIT studies. ( a ) Fluorescence imaging of mice bearing subcutaneous U87‐MGvIII tumors 1 h after injecting 18 μg of Z EGFR:03115 –IR700DX or IR700DX–maleimide (top row). Subsequently, mice were irradiated with an optical dose of 100 J/cm 2 by a red LED and, immediately after, imaged again (bottom row). ( b ) Tumor growth inhibition of the Z EGFR:03115 –IR700DX‐targeted PIT in U87‐MGvIII tumors after administering three doses of 18 µg of the conjugate and irradiating with 100 J/cm 2 at days 1, 3 and 5 in comparison to control groups. Data are presented as mean ± SD ( n = 6 for each group, ** p ≤ 0.01 as assessed by the Kruskal–Wallis test). ( c ) Visual observation of normal tissue damage in the PDT treated mice, while no skin damage was present in the Z EGFR:03115 –IR700DX PIT mice. These were the appearances seen in all mice. ( d ) H E staining of treated and untreated U87‐MGvIIII tumors (arrows indicate regions of tissue necrosis).

Techniques Used: In Vivo, Fluorescence, Imaging, Mouse Assay, Irradiation, Inhibition, Staining

Z EGFR:03115 –IR700DX accumulates in U87‐MGvIII orthotopic glioma tumors. ( a ) T 2 ‐weighted MRI images of an intracranial brain tumor model 11 days post‐cell implantation. ( b ) Photographic image of the brain and the corresponding Z EGFR:03115 –IR700DX fluorescent image demonstrates predominant accumulation of the conjugate within the brain tumor mass. ( c ) Transaxial brain histological sections (10μm) containing tumor tissue were obtained for ex vivo analysis immediately after 1 h in vivo image acquisition. Z EGFR:03115 –IR700DX clearly delineated tumor mass from the surrounding normal tissues which correlated well with H E and EGFR staining of the consecutive sections.
Figure Legend Snippet: Z EGFR:03115 –IR700DX accumulates in U87‐MGvIII orthotopic glioma tumors. ( a ) T 2 ‐weighted MRI images of an intracranial brain tumor model 11 days post‐cell implantation. ( b ) Photographic image of the brain and the corresponding Z EGFR:03115 –IR700DX fluorescent image demonstrates predominant accumulation of the conjugate within the brain tumor mass. ( c ) Transaxial brain histological sections (10μm) containing tumor tissue were obtained for ex vivo analysis immediately after 1 h in vivo image acquisition. Z EGFR:03115 –IR700DX clearly delineated tumor mass from the surrounding normal tissues which correlated well with H E and EGFR staining of the consecutive sections.

Techniques Used: Magnetic Resonance Imaging, Ex Vivo, In Vivo, Staining

In vitro morphological changes following affibody‐based PIT. ( a ) Incubation of U87‐MGvIII spheroids with the Z EGFR:03115 –IR700DX for 6 h and irradiation with a red LED (16 J/cm 2 ) induced phototoxic cell death and disintegration of the architectural structure of the spheroid population. ( b ) U87‐MGvIII cells grown as a monolayer culture showed rapid cell swelling and bleb formation (see arrows) as visualized by a phase‐contrast image 1 h post Z EGFR:03115 –IR700DX (red) irradiation with the 639 nm laser on a confocal microscope. ( c ) Following methanol fixation of U87‐MGvIII cells, either treated by PIT or just irradiated, and staining with an anti‐calreticulin‐AlexaFluor488 antibody overnight (4°C), images were acquired by confocal microscopy. ( d ) Cell membrane disruption was monitored by propidium iodide (1 µg/mL) staining. U87‐MGvIII cells irradiated only or treated with Z EGFR:03115 –IR700DX‐based PIT were analyzed by flow cytometry 1 and 24 h post‐treatment. ( e ) Reactive oxygen species production was assessed using the DCFDA cellular ROS detection assay kit using U87‐MGvIII cells treated with affibody‐based PIT (15 min after light exposure). The results were normalized to the control cells. Data are presented as mean ± SEM ( n = 3).
Figure Legend Snippet: In vitro morphological changes following affibody‐based PIT. ( a ) Incubation of U87‐MGvIII spheroids with the Z EGFR:03115 –IR700DX for 6 h and irradiation with a red LED (16 J/cm 2 ) induced phototoxic cell death and disintegration of the architectural structure of the spheroid population. ( b ) U87‐MGvIII cells grown as a monolayer culture showed rapid cell swelling and bleb formation (see arrows) as visualized by a phase‐contrast image 1 h post Z EGFR:03115 –IR700DX (red) irradiation with the 639 nm laser on a confocal microscope. ( c ) Following methanol fixation of U87‐MGvIII cells, either treated by PIT or just irradiated, and staining with an anti‐calreticulin‐AlexaFluor488 antibody overnight (4°C), images were acquired by confocal microscopy. ( d ) Cell membrane disruption was monitored by propidium iodide (1 µg/mL) staining. U87‐MGvIII cells irradiated only or treated with Z EGFR:03115 –IR700DX‐based PIT were analyzed by flow cytometry 1 and 24 h post‐treatment. ( e ) Reactive oxygen species production was assessed using the DCFDA cellular ROS detection assay kit using U87‐MGvIII cells treated with affibody‐based PIT (15 min after light exposure). The results were normalized to the control cells. Data are presented as mean ± SEM ( n = 3).

Techniques Used: In Vitro, Incubation, Irradiation, Microscopy, Staining, Confocal Microscopy, Flow Cytometry, Cytometry, Detection Assay

33) Product Images from "Near‐infrared photoimmunotherapy targeting EGFR—Shedding new light on glioblastoma treatment"

Article Title: Near‐infrared photoimmunotherapy targeting EGFR—Shedding new light on glioblastoma treatment

Journal: International Journal of Cancer

doi: 10.1002/ijc.31246

Testing Z EGFR:03115 –IR700DX specificity in vivo and studying the effect of functional groups on dye pharmacokinetics. ( a ) The U87‐MGvIII tumor could easily be differentiated as early as 1 h post Z EGFR:03115 –IR700DX (6 µg/mouse) being intravenously injected, whereas minimal tumor uptake was observed when administering the same amount of the non‐specific Z Taq ‐IR700DX. ( b ) Fluorescence imaging of Z EGFR:03115 –IR700DX uptake in excised tissues (1 h post‐injection) and respective tumor‐to‐organ ratios. ( c ) Mean radiant efficiency in U87‐MGvIII tumors 1 h after administering either 6 µg Z EGFR:03115 –IR700DX, 18 µg Z EGFR:03115 –IR700DX or 6 µg of the non‐specific Z Taq ‐IR700DX. ( d ) Tumor‐to‐background ratio comparison when altering the injected dose of Z EGFR:03115 –IR700DX. ( e , f ) Fluorescence intensity and tumor‐to‐background ratio in the U87‐MGvIII tumors over time after 18 µg Z EGFR:03115 –IR700DX. ( g , h ) Fluorescence imaging of mice bearing subcutaneous U87‐MGvIII tumors. Images were acquired 30 min, 1 h or 3 h post IR700DX–maleimide, IR800CW–maleimide, IR700DX–NHS ester and IR700DX–carboxylate injection and the mean radiant efficiency was determined for each of the dyes. ( i ) An SDS‐PAGE gel of mouse blood serum imaged using the IVIS/Spectrum imaging system to visualize the fluorescent dyes’ association with blood proteins. All data are presented as mean ± SD ( n ≥ 3).
Figure Legend Snippet: Testing Z EGFR:03115 –IR700DX specificity in vivo and studying the effect of functional groups on dye pharmacokinetics. ( a ) The U87‐MGvIII tumor could easily be differentiated as early as 1 h post Z EGFR:03115 –IR700DX (6 µg/mouse) being intravenously injected, whereas minimal tumor uptake was observed when administering the same amount of the non‐specific Z Taq ‐IR700DX. ( b ) Fluorescence imaging of Z EGFR:03115 –IR700DX uptake in excised tissues (1 h post‐injection) and respective tumor‐to‐organ ratios. ( c ) Mean radiant efficiency in U87‐MGvIII tumors 1 h after administering either 6 µg Z EGFR:03115 –IR700DX, 18 µg Z EGFR:03115 –IR700DX or 6 µg of the non‐specific Z Taq ‐IR700DX. ( d ) Tumor‐to‐background ratio comparison when altering the injected dose of Z EGFR:03115 –IR700DX. ( e , f ) Fluorescence intensity and tumor‐to‐background ratio in the U87‐MGvIII tumors over time after 18 µg Z EGFR:03115 –IR700DX. ( g , h ) Fluorescence imaging of mice bearing subcutaneous U87‐MGvIII tumors. Images were acquired 30 min, 1 h or 3 h post IR700DX–maleimide, IR800CW–maleimide, IR700DX–NHS ester and IR700DX–carboxylate injection and the mean radiant efficiency was determined for each of the dyes. ( i ) An SDS‐PAGE gel of mouse blood serum imaged using the IVIS/Spectrum imaging system to visualize the fluorescent dyes’ association with blood proteins. All data are presented as mean ± SD ( n ≥ 3).

Techniques Used: In Vivo, Functional Assay, Injection, Fluorescence, Imaging, Mouse Assay, SDS Page

Expression of EGFR in GBM cell lines and specificity of the Z EGFR:03115 –IR700DX binding to EGFR. ( a ) Varying EGFR expression in the selected cancer cell lines was confirmed by Western blot. The numbers in the brackets represent the relative EGFR expression as determined by densitometric analysis. ( b ) Z EGFR:03115 –IR700DX (30 nM) binding as assessed by flow cytometry in the selected cancer cells with varying EGFR expression and after blocking with 100‐fold excess of unlabeled Z EGFR:03115 . Data are presented as mean ± SEM ( n = 3). ( c – f ) Confocal microscopy images demonstrating target‐specific binding (4°C) and internalization (37°C) of either the Z EGFR:03115 –IR700DX (1 µM), anti‐EGFR‐FITC antibody (25 nM for visualization purposes) or IR700DX alone (1 µM): ( c ) U251 cell lines (1 h incubation time), ( d + e ) U87‐MGvIII or MCF7 cell lines (1–6 h incubation time), ( f ) U87‐MGvIII spheroids (6 h incubation time). Hoechst®33342 (blue) and LysoTracker™Green DND‐26 (green) were used for counterstaining. ( g ) Quantification of fluorescence intensity (median fluorescence intensity) of ∼8 µm slices through U87‐MGvIII spheroids following a 6 h incubation with Z EGFR:03115 –IR700DX (500 nM) or an anti‐EGFR‐FITC antibody (500 nM). Data are presented as mean ± SEM ( n = 3). ( h ) H E, EGFR and Ki67 immunostaining of U87‐MGvIII spheroid (400–500 µm) sections 72 h after seeding.
Figure Legend Snippet: Expression of EGFR in GBM cell lines and specificity of the Z EGFR:03115 –IR700DX binding to EGFR. ( a ) Varying EGFR expression in the selected cancer cell lines was confirmed by Western blot. The numbers in the brackets represent the relative EGFR expression as determined by densitometric analysis. ( b ) Z EGFR:03115 –IR700DX (30 nM) binding as assessed by flow cytometry in the selected cancer cells with varying EGFR expression and after blocking with 100‐fold excess of unlabeled Z EGFR:03115 . Data are presented as mean ± SEM ( n = 3). ( c – f ) Confocal microscopy images demonstrating target‐specific binding (4°C) and internalization (37°C) of either the Z EGFR:03115 –IR700DX (1 µM), anti‐EGFR‐FITC antibody (25 nM for visualization purposes) or IR700DX alone (1 µM): ( c ) U251 cell lines (1 h incubation time), ( d + e ) U87‐MGvIII or MCF7 cell lines (1–6 h incubation time), ( f ) U87‐MGvIII spheroids (6 h incubation time). Hoechst®33342 (blue) and LysoTracker™Green DND‐26 (green) were used for counterstaining. ( g ) Quantification of fluorescence intensity (median fluorescence intensity) of ∼8 µm slices through U87‐MGvIII spheroids following a 6 h incubation with Z EGFR:03115 –IR700DX (500 nM) or an anti‐EGFR‐FITC antibody (500 nM). Data are presented as mean ± SEM ( n = 3). ( h ) H E, EGFR and Ki67 immunostaining of U87‐MGvIII spheroid (400–500 µm) sections 72 h after seeding.

Techniques Used: Expressing, Binding Assay, Western Blot, Flow Cytometry, Cytometry, Blocking Assay, Confocal Microscopy, Incubation, Fluorescence, Immunostaining

Z EGFR:03115 –IR700DX‐mediated PIT causes cellular death selectively in EGFR+ve cells. Decrease in cell viability as assessed by the CellTiter‐Glo® luminescent cell viability assay 24 or 96 h post‐PIT in 2D cells and 3D spheroids, following 6 h incubation with the Z EGFR:03115 –IR700DX and irradiation with a light dose of 8 or 16 J/cm 2 , was confirmed to be dose dependent and receptor mediated. ( a ) U87‐MGvIII cells 24 h post‐PIT. ( b ) MCF7 cells 24 h post‐PIT. ( c , d ) U87‐MGvIII spheroids 24 and 96 h post‐PIT. ( e ) WSz4 spheroids 96 h post‐PIT. Data are presented as mean ± SEM ( n = 3). Statistical significance in comparison to the control group was determined using an unpaired two‐tailed Student's t‐ test with Welch's correction. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 and **** p ≤ 0.0001. [Color figure can be viewed at http://wileyonlinelibrary.com ]
Figure Legend Snippet: Z EGFR:03115 –IR700DX‐mediated PIT causes cellular death selectively in EGFR+ve cells. Decrease in cell viability as assessed by the CellTiter‐Glo® luminescent cell viability assay 24 or 96 h post‐PIT in 2D cells and 3D spheroids, following 6 h incubation with the Z EGFR:03115 –IR700DX and irradiation with a light dose of 8 or 16 J/cm 2 , was confirmed to be dose dependent and receptor mediated. ( a ) U87‐MGvIII cells 24 h post‐PIT. ( b ) MCF7 cells 24 h post‐PIT. ( c , d ) U87‐MGvIII spheroids 24 and 96 h post‐PIT. ( e ) WSz4 spheroids 96 h post‐PIT. Data are presented as mean ± SEM ( n = 3). Statistical significance in comparison to the control group was determined using an unpaired two‐tailed Student's t‐ test with Welch's correction. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 and **** p ≤ 0.0001. [Color figure can be viewed at http://wileyonlinelibrary.com ]

Techniques Used: Cell Viability Assay, Incubation, Irradiation, Two Tailed Test

In vivo Z EGFR:03115 –IR700DX‐mediated PIT studies. ( a ) Fluorescence imaging of mice bearing subcutaneous U87‐MGvIII tumors 1 h after injecting 18 μg of Z EGFR:03115 –IR700DX or IR700DX–maleimide (top row). Subsequently, mice were irradiated with an optical dose of 100 J/cm 2 by a red LED and, immediately after, imaged again (bottom row). ( b ) Tumor growth inhibition of the Z EGFR:03115 –IR700DX‐targeted PIT in U87‐MGvIII tumors after administering three doses of 18 µg of the conjugate and irradiating with 100 J/cm 2 at days 1, 3 and 5 in comparison to control groups. Data are presented as mean ± SD ( n = 6 for each group, ** p ≤ 0.01 as assessed by the Kruskal–Wallis test). ( c ) Visual observation of normal tissue damage in the PDT treated mice, while no skin damage was present in the Z EGFR:03115 –IR700DX PIT mice. These were the appearances seen in all mice. ( d ) H E staining of treated and untreated U87‐MGvIIII tumors (arrows indicate regions of tissue necrosis).
Figure Legend Snippet: In vivo Z EGFR:03115 –IR700DX‐mediated PIT studies. ( a ) Fluorescence imaging of mice bearing subcutaneous U87‐MGvIII tumors 1 h after injecting 18 μg of Z EGFR:03115 –IR700DX or IR700DX–maleimide (top row). Subsequently, mice were irradiated with an optical dose of 100 J/cm 2 by a red LED and, immediately after, imaged again (bottom row). ( b ) Tumor growth inhibition of the Z EGFR:03115 –IR700DX‐targeted PIT in U87‐MGvIII tumors after administering three doses of 18 µg of the conjugate and irradiating with 100 J/cm 2 at days 1, 3 and 5 in comparison to control groups. Data are presented as mean ± SD ( n = 6 for each group, ** p ≤ 0.01 as assessed by the Kruskal–Wallis test). ( c ) Visual observation of normal tissue damage in the PDT treated mice, while no skin damage was present in the Z EGFR:03115 –IR700DX PIT mice. These were the appearances seen in all mice. ( d ) H E staining of treated and untreated U87‐MGvIIII tumors (arrows indicate regions of tissue necrosis).

Techniques Used: In Vivo, Fluorescence, Imaging, Mouse Assay, Irradiation, Inhibition, Staining

Z EGFR:03115 –IR700DX accumulates in U87‐MGvIII orthotopic glioma tumors. ( a ) T 2 ‐weighted MRI images of an intracranial brain tumor model 11 days post‐cell implantation. ( b ) Photographic image of the brain and the corresponding Z EGFR:03115 –IR700DX fluorescent image demonstrates predominant accumulation of the conjugate within the brain tumor mass. ( c ) Transaxial brain histological sections (10μm) containing tumor tissue were obtained for ex vivo analysis immediately after 1 h in vivo image acquisition. Z EGFR:03115 –IR700DX clearly delineated tumor mass from the surrounding normal tissues which correlated well with H E and EGFR staining of the consecutive sections.
Figure Legend Snippet: Z EGFR:03115 –IR700DX accumulates in U87‐MGvIII orthotopic glioma tumors. ( a ) T 2 ‐weighted MRI images of an intracranial brain tumor model 11 days post‐cell implantation. ( b ) Photographic image of the brain and the corresponding Z EGFR:03115 –IR700DX fluorescent image demonstrates predominant accumulation of the conjugate within the brain tumor mass. ( c ) Transaxial brain histological sections (10μm) containing tumor tissue were obtained for ex vivo analysis immediately after 1 h in vivo image acquisition. Z EGFR:03115 –IR700DX clearly delineated tumor mass from the surrounding normal tissues which correlated well with H E and EGFR staining of the consecutive sections.

Techniques Used: Magnetic Resonance Imaging, Ex Vivo, In Vivo, Staining

In vitro morphological changes following affibody‐based PIT. ( a ) Incubation of U87‐MGvIII spheroids with the Z EGFR:03115 –IR700DX for 6 h and irradiation with a red LED (16 J/cm 2 ) induced phototoxic cell death and disintegration of the architectural structure of the spheroid population. ( b ) U87‐MGvIII cells grown as a monolayer culture showed rapid cell swelling and bleb formation (see arrows) as visualized by a phase‐contrast image 1 h post Z EGFR:03115 –IR700DX (red) irradiation with the 639 nm laser on a confocal microscope. ( c ) Following methanol fixation of U87‐MGvIII cells, either treated by PIT or just irradiated, and staining with an anti‐calreticulin‐AlexaFluor488 antibody overnight (4°C), images were acquired by confocal microscopy. ( d ) Cell membrane disruption was monitored by propidium iodide (1 µg/mL) staining. U87‐MGvIII cells irradiated only or treated with Z EGFR:03115 –IR700DX‐based PIT were analyzed by flow cytometry 1 and 24 h post‐treatment. ( e ) Reactive oxygen species production was assessed using the DCFDA cellular ROS detection assay kit using U87‐MGvIII cells treated with affibody‐based PIT (15 min after light exposure). The results were normalized to the control cells. Data are presented as mean ± SEM ( n = 3).
Figure Legend Snippet: In vitro morphological changes following affibody‐based PIT. ( a ) Incubation of U87‐MGvIII spheroids with the Z EGFR:03115 –IR700DX for 6 h and irradiation with a red LED (16 J/cm 2 ) induced phototoxic cell death and disintegration of the architectural structure of the spheroid population. ( b ) U87‐MGvIII cells grown as a monolayer culture showed rapid cell swelling and bleb formation (see arrows) as visualized by a phase‐contrast image 1 h post Z EGFR:03115 –IR700DX (red) irradiation with the 639 nm laser on a confocal microscope. ( c ) Following methanol fixation of U87‐MGvIII cells, either treated by PIT or just irradiated, and staining with an anti‐calreticulin‐AlexaFluor488 antibody overnight (4°C), images were acquired by confocal microscopy. ( d ) Cell membrane disruption was monitored by propidium iodide (1 µg/mL) staining. U87‐MGvIII cells irradiated only or treated with Z EGFR:03115 –IR700DX‐based PIT were analyzed by flow cytometry 1 and 24 h post‐treatment. ( e ) Reactive oxygen species production was assessed using the DCFDA cellular ROS detection assay kit using U87‐MGvIII cells treated with affibody‐based PIT (15 min after light exposure). The results were normalized to the control cells. Data are presented as mean ± SEM ( n = 3).

Techniques Used: In Vitro, Incubation, Irradiation, Microscopy, Staining, Confocal Microscopy, Flow Cytometry, Cytometry, Detection Assay

34) Product Images from "Influence of composition of cysteine-containing peptide-based chelators on biodistribution of 99mTc-labeled anti-EGFR affibody molecules"

Article Title: Influence of composition of cysteine-containing peptide-based chelators on biodistribution of 99mTc-labeled anti-EGFR affibody molecules

Journal: Amino Acids

doi: 10.1007/s00726-018-2571-1

In vivo specificity of 99m Tc-ZEGFR conjugates ( a 99m Tc-ZEGFR-GGEC, b 99m Tc-ZEGFR-GEEC and c 99m Tc-ZEGFR-EEEC) in A431 xenografts and EGFR-expressing organs in mice at 6 h after injection. In the blocked group, receptors were saturated by pre-injection of large excess of anti-EGFR antibody cetuximab. The data are presented as the average ( n = 4) and SD
Figure Legend Snippet: In vivo specificity of 99m Tc-ZEGFR conjugates ( a 99m Tc-ZEGFR-GGEC, b 99m Tc-ZEGFR-GEEC and c 99m Tc-ZEGFR-EEEC) in A431 xenografts and EGFR-expressing organs in mice at 6 h after injection. In the blocked group, receptors were saturated by pre-injection of large excess of anti-EGFR antibody cetuximab. The data are presented as the average ( n = 4) and SD

Techniques Used: In Vivo, Expressing, Mouse Assay, Injection

35) Product Images from "Heptameric Targeting Ligands against EGFR and HER2 with High Stability and Avidity"

Article Title: Heptameric Targeting Ligands against EGFR and HER2 with High Stability and Avidity

Journal: PLoS ONE

doi: 10.1371/journal.pone.0043077

Binding dynamics of monomeric and heptameric targeting ligands by BIAcore analysis. The extracellular domain of (A) EGFR and (B) HER2 receptors were immobilized on the CM5 chip. Different concentrations of monomer or heptamer proteins were injected into the channels. Analyses were performed at room temperature at a flow rate of 20 µl/min.
Figure Legend Snippet: Binding dynamics of monomeric and heptameric targeting ligands by BIAcore analysis. The extracellular domain of (A) EGFR and (B) HER2 receptors were immobilized on the CM5 chip. Different concentrations of monomer or heptamer proteins were injected into the channels. Analyses were performed at room temperature at a flow rate of 20 µl/min.

Techniques Used: Binding Assay, Chromatin Immunoprecipitation, Injection, Flow Cytometry

Analysis of the protease resistance of the monomer and the heptamer by thermolysin. (A) About 5 µg of monomeric and heptameric Z EGFR and (B) monomeric and heptameric Z HER2 targeting ligands were incubated with 100 ng of thermolysin at different temperatures for 20 min. After incubation, reaction was stopped by adding SDS sample buffer and each reaction mixture was separated on a 10% SDS-PAGE to examine protein degradation.
Figure Legend Snippet: Analysis of the protease resistance of the monomer and the heptamer by thermolysin. (A) About 5 µg of monomeric and heptameric Z EGFR and (B) monomeric and heptameric Z HER2 targeting ligands were incubated with 100 ng of thermolysin at different temperatures for 20 min. After incubation, reaction was stopped by adding SDS sample buffer and each reaction mixture was separated on a 10% SDS-PAGE to examine protein degradation.

Techniques Used: Incubation, SDS Page

Co-localization of EEA1 and heptameric targeting ligands. (A) Two different concentrations of the FITC-labeled heptameric Z EGFR targeting ligands were incubated with A431 cells for 2 h at 37°C. (B) FITC labeled heptameric Z HER2 targeting ligands at two concentrations were incubated with SK-OV3 cells for 2 h at 37°C. EEA1 proteins were detected by Alexa 555-conjugated secondary antibody. Top left panels: cell nuclei stained with DAPI (blue); Top right panels: FITC labeled heptamer (green); bottom left panels: EEA1 antibody (red); bottom right panels: merged image of the three stainings.
Figure Legend Snippet: Co-localization of EEA1 and heptameric targeting ligands. (A) Two different concentrations of the FITC-labeled heptameric Z EGFR targeting ligands were incubated with A431 cells for 2 h at 37°C. (B) FITC labeled heptameric Z HER2 targeting ligands at two concentrations were incubated with SK-OV3 cells for 2 h at 37°C. EEA1 proteins were detected by Alexa 555-conjugated secondary antibody. Top left panels: cell nuclei stained with DAPI (blue); Top right panels: FITC labeled heptamer (green); bottom left panels: EEA1 antibody (red); bottom right panels: merged image of the three stainings.

Techniques Used: Labeling, Incubation, Staining

Heat stability assessment of the monomer and the heptamer by circular dichroism analysis. (A) Monomeric and heptameric Z EGFR , (B) monomeric and heptameric Z HER2 targeting ligands, and (C) heptameric core itself were prepared in a 10 mM phosphate buffer, pH 7.4. Temperature was increased from 25°C to 94°C. Spectra were recorded at various temperatures. The ellipticity at 220 nm was used for the analysis.
Figure Legend Snippet: Heat stability assessment of the monomer and the heptamer by circular dichroism analysis. (A) Monomeric and heptameric Z EGFR , (B) monomeric and heptameric Z HER2 targeting ligands, and (C) heptameric core itself were prepared in a 10 mM phosphate buffer, pH 7.4. Temperature was increased from 25°C to 94°C. Spectra were recorded at various temperatures. The ellipticity at 220 nm was used for the analysis.

Techniques Used:

Cell-based surface receptor binding properties of the monomer and heptamer. (A) EGFR-positive A431 cells were grown on coverslips. Different concentration of FITC-labeled monomeric and heptameric Z EGFR ligands was incubated with A431 cells for 30 min at 25°C. (B) HER2-positive SK-OV3 cells were grown on coverslips. FITC-labeled monomeric and heptameric Z HER2 ligands were incubated with SK-OV3 cells for 30 min at 25°C. (C) EGFR-negative Jurkat cells and HER2-low expressing MCF7 cells were grown on coverslips. 100 nM of FITC-labeled monomeric and heptameric ligands were incubated with Jurkat and MCF cells for 30 min at 25°C.
Figure Legend Snippet: Cell-based surface receptor binding properties of the monomer and heptamer. (A) EGFR-positive A431 cells were grown on coverslips. Different concentration of FITC-labeled monomeric and heptameric Z EGFR ligands was incubated with A431 cells for 30 min at 25°C. (B) HER2-positive SK-OV3 cells were grown on coverslips. FITC-labeled monomeric and heptameric Z HER2 ligands were incubated with SK-OV3 cells for 30 min at 25°C. (C) EGFR-negative Jurkat cells and HER2-low expressing MCF7 cells were grown on coverslips. 100 nM of FITC-labeled monomeric and heptameric ligands were incubated with Jurkat and MCF cells for 30 min at 25°C.

Techniques Used: Cell Surface Receptor Assay, Concentration Assay, Labeling, Incubation, Expressing

Native gel separation of monomeric and heptameric targeting ligands. The purified monomeric Z EGFR , heptameric Z EGFR , monomeric Z HER2 and heptameric Z HER2 ligands were separated on an 8% native gel. About 5 µg of the purified monomer or 20 µg heptamer was loaded to the appropriate lane.
Figure Legend Snippet: Native gel separation of monomeric and heptameric targeting ligands. The purified monomeric Z EGFR , heptameric Z EGFR , monomeric Z HER2 and heptameric Z HER2 ligands were separated on an 8% native gel. About 5 µg of the purified monomer or 20 µg heptamer was loaded to the appropriate lane.

Techniques Used: Purification

Cell binding analysis by flow cytometry. (A) 100 nM FITC-monomeric and heptameric Z EGFR ligands were used for labeling of EGFR positive A431 and negative Jurkat cells, and analyzed by flow cytometry. Cells incubated with PBS were served as negative control. (B) 100 nM FITC-monomeric and heptameric Z HER2 ligands were used for labeling of HER2 positive SK-OV3 and HER2 low expressing MCF7 cells, and analyzed by flow cytometry. Cells incubated with PBS were served as negative control.
Figure Legend Snippet: Cell binding analysis by flow cytometry. (A) 100 nM FITC-monomeric and heptameric Z EGFR ligands were used for labeling of EGFR positive A431 and negative Jurkat cells, and analyzed by flow cytometry. Cells incubated with PBS were served as negative control. (B) 100 nM FITC-monomeric and heptameric Z HER2 ligands were used for labeling of HER2 positive SK-OV3 and HER2 low expressing MCF7 cells, and analyzed by flow cytometry. Cells incubated with PBS were served as negative control.

Techniques Used: Binding Assay, Flow Cytometry, Cytometry, Labeling, Incubation, Negative Control, Expressing

SDS-PAGE analysis of the purified monomeric and heptameric targeting ligands. The purified heptameric Z EGFR , monomeric Z EGFR , heptameric Z HER2 , and monomeric Z HER2 ligands were separated on a 10% SDS-PAGE gel. About 5 µg of each protein was applied to each lane.
Figure Legend Snippet: SDS-PAGE analysis of the purified monomeric and heptameric targeting ligands. The purified heptameric Z EGFR , monomeric Z EGFR , heptameric Z HER2 , and monomeric Z HER2 ligands were separated on a 10% SDS-PAGE gel. About 5 µg of each protein was applied to each lane.

Techniques Used: SDS Page, Purification

Determination of the molecular weights of the heptameric targeting ligands by analytical ultracentrifugation analysis. Purified heptameric Z EGFR and heptameric Z HER2 ligands were centrifuged at 10,000 g for 20 h. Absorbances at 280 nm were recorded every two hours.
Figure Legend Snippet: Determination of the molecular weights of the heptameric targeting ligands by analytical ultracentrifugation analysis. Purified heptameric Z EGFR and heptameric Z HER2 ligands were centrifuged at 10,000 g for 20 h. Absorbances at 280 nm were recorded every two hours.

Techniques Used: Purification

36) Product Images from "Hydrophobic Fluorescent Probes Introduce Artifacts into Single Molecule Tracking Experiments Due to Non-Specific Binding"

Article Title: Hydrophobic Fluorescent Probes Introduce Artifacts into Single Molecule Tracking Experiments Due to Non-Specific Binding

Journal: PLoS ONE

doi: 10.1371/journal.pone.0074200

Effect of logD and charge on affibody conjugate mobility. Plots of mean instantaneous D fit for different anti-EGFR Affibody conjugates vs charge at pH 7.4 ( A ), and logD ( B ). C) Plot of spot density for selected anti-EGFR Affibody conjugates vs charge at logD. Each datapoint corresponds to mean ± SEM of at least 10 independent areas. Lines show linear regression fit to the data, R 2 values indicating goodness of fit. Alexa 555 is not included in this figure as the structure is not published and charge and logD values are unavailable.
Figure Legend Snippet: Effect of logD and charge on affibody conjugate mobility. Plots of mean instantaneous D fit for different anti-EGFR Affibody conjugates vs charge at pH 7.4 ( A ), and logD ( B ). C) Plot of spot density for selected anti-EGFR Affibody conjugates vs charge at logD. Each datapoint corresponds to mean ± SEM of at least 10 independent areas. Lines show linear regression fit to the data, R 2 values indicating goodness of fit. Alexa 555 is not included in this figure as the structure is not published and charge and logD values are unavailable.

Techniques Used:

37) Product Images from "Hydrophobic Fluorescent Probes Introduce Artifacts into Single Molecule Tracking Experiments Due to Non-Specific Binding"

Article Title: Hydrophobic Fluorescent Probes Introduce Artifacts into Single Molecule Tracking Experiments Due to Non-Specific Binding

Journal: PLoS ONE

doi: 10.1371/journal.pone.0074200

Mean instantaneous D fit for different anti-EGFR Affibody conjugates. Each datapoint corresponds to mean ± SEM of at least 10 areas acquired from 3 independent samples.
Figure Legend Snippet: Mean instantaneous D fit for different anti-EGFR Affibody conjugates. Each datapoint corresponds to mean ± SEM of at least 10 areas acquired from 3 independent samples.

Techniques Used:

Fluorescence intensity measured from confocal microscopy images of T47D cells labeled with 50-conjugated EGFR affibody, and a mixture of 25 nM dye-conjugated affibody and 25 nM unlabeled affibody. Three dyes were selected to cover the range of mobilities (Alexa 488, high mobility; CF 633, moderate mobility; Atto 565, low mobility). Columns represent the median of the distribution of membrane region pixel intensities derived from at least 100 cells. Error bars represent the positions of the 1 st and 3 rd quartile of the distributions.
Figure Legend Snippet: Fluorescence intensity measured from confocal microscopy images of T47D cells labeled with 50-conjugated EGFR affibody, and a mixture of 25 nM dye-conjugated affibody and 25 nM unlabeled affibody. Three dyes were selected to cover the range of mobilities (Alexa 488, high mobility; CF 633, moderate mobility; Atto 565, low mobility). Columns represent the median of the distribution of membrane region pixel intensities derived from at least 100 cells. Error bars represent the positions of the 1 st and 3 rd quartile of the distributions.

Techniques Used: Fluorescence, Confocal Microscopy, Labeling, Derivative Assay

Effect of logD and charge on affibody conjugate mobility. Plots of mean instantaneous D fit for different anti-EGFR Affibody conjugates vs charge at pH 7.4 ( A ), and logD ( B ). C) Plot of spot density for selected anti-EGFR Affibody conjugates vs charge at logD. Each datapoint corresponds to mean ± SEM of at least 10 independent areas. Lines show linear regression fit to the data, R 2 values indicating goodness of fit. Alexa 555 is not included in this figure as the structure is not published and charge and logD values are unavailable.
Figure Legend Snippet: Effect of logD and charge on affibody conjugate mobility. Plots of mean instantaneous D fit for different anti-EGFR Affibody conjugates vs charge at pH 7.4 ( A ), and logD ( B ). C) Plot of spot density for selected anti-EGFR Affibody conjugates vs charge at logD. Each datapoint corresponds to mean ± SEM of at least 10 independent areas. Lines show linear regression fit to the data, R 2 values indicating goodness of fit. Alexa 555 is not included in this figure as the structure is not published and charge and logD values are unavailable.

Techniques Used:

Analysis of definitely mobile vs immobile or very slow moving spots. A) Mean instantaneous D fit for different anti-EGFR Affibody conjugates, after removing data for spots with D values below 0.1 µm 2 /s. Each datapoint corresponds to mean ± SD of of the tracks contained in at least 10 different areas containing a minimum of 50 different cells. Blue bars indicate dyes excited at 491 nm, green at 561 nm, and red at 638 nm. B) Percentages of spots for each dye with D values below 0.1 µm 2 /s. C) Plot of mean instantaneous D fit for different anti-EGFR Affibody conjugates (calculated from all spots) vs percentage of spots with D values
Figure Legend Snippet: Analysis of definitely mobile vs immobile or very slow moving spots. A) Mean instantaneous D fit for different anti-EGFR Affibody conjugates, after removing data for spots with D values below 0.1 µm 2 /s. Each datapoint corresponds to mean ± SD of of the tracks contained in at least 10 different areas containing a minimum of 50 different cells. Blue bars indicate dyes excited at 491 nm, green at 561 nm, and red at 638 nm. B) Percentages of spots for each dye with D values below 0.1 µm 2 /s. C) Plot of mean instantaneous D fit for different anti-EGFR Affibody conjugates (calculated from all spots) vs percentage of spots with D values

Techniques Used:

38) Product Images from "Influence of composition of cysteine-containing peptide-based chelators on biodistribution of 99mTc-labeled anti-EGFR affibody molecules"

Article Title: Influence of composition of cysteine-containing peptide-based chelators on biodistribution of 99mTc-labeled anti-EGFR affibody molecules

Journal: Amino Acids

doi: 10.1007/s00726-018-2571-1

Biodistribution of 99m Tc-ZEGFR conjugates in BALB/C nu/nu mice bearing EGFR-expressing A431 xenografts at a 6 h and b 24 h after injection. The data are presented as the average ( n = 4) and SD
Figure Legend Snippet: Biodistribution of 99m Tc-ZEGFR conjugates in BALB/C nu/nu mice bearing EGFR-expressing A431 xenografts at a 6 h and b 24 h after injection. The data are presented as the average ( n = 4) and SD

Techniques Used: Mouse Assay, Expressing, Injection

Imaging of EGFR-expressing A431 xenografts in BALB/C nu/nu mice using a 99m Tc-ZEGFR-GGEC, b 99m Tc-ZEGFR-GEEC and c 99m Tc-ZEGFR-EEEC at 24 h after injection. The scales are adjusted to first red pixels in tumors
Figure Legend Snippet: Imaging of EGFR-expressing A431 xenografts in BALB/C nu/nu mice using a 99m Tc-ZEGFR-GGEC, b 99m Tc-ZEGFR-GEEC and c 99m Tc-ZEGFR-EEEC at 24 h after injection. The scales are adjusted to first red pixels in tumors

Techniques Used: Imaging, Expressing, Mouse Assay, Injection

Tumor-to-organ ratios of 99m Tc-ZEGFR conjugates in BALB/C nu/nu mice bearing EGFR-expressing A431 xenografts at a 6 h and b 24 h after injection. The data are presented as the average ( n = 4) and SD
Figure Legend Snippet: Tumor-to-organ ratios of 99m Tc-ZEGFR conjugates in BALB/C nu/nu mice bearing EGFR-expressing A431 xenografts at a 6 h and b 24 h after injection. The data are presented as the average ( n = 4) and SD

Techniques Used: Mouse Assay, Expressing, Injection

Imaging of EGFR-expressing A431 xenografts in BALB/C nu/nu mice using a 99m Tc-ZEGFR-GGEC, b 99m Tc-ZEGFR-GEEC and c 99m Tc-ZEGFR-EEEC at 6 h after injection. The scales are adjusted to first red pixels in tumors
Figure Legend Snippet: Imaging of EGFR-expressing A431 xenografts in BALB/C nu/nu mice using a 99m Tc-ZEGFR-GGEC, b 99m Tc-ZEGFR-GEEC and c 99m Tc-ZEGFR-EEEC at 6 h after injection. The scales are adjusted to first red pixels in tumors

Techniques Used: Imaging, Expressing, Mouse Assay, Injection

Peptide-based chelators used for labeling with 99m Tc. a General structure of the N 3 S chelators formed by a C-terminal cysteine and three adjacent amino acids. X 1 , X 2 and X 3 denote the side chains of the amino acids. b Sequences of EGFR-binding affibody molecules evaluated in this study. The variable amino acids are marked in bold
Figure Legend Snippet: Peptide-based chelators used for labeling with 99m Tc. a General structure of the N 3 S chelators formed by a C-terminal cysteine and three adjacent amino acids. X 1 , X 2 and X 3 denote the side chains of the amino acids. b Sequences of EGFR-binding affibody molecules evaluated in this study. The variable amino acids are marked in bold

Techniques Used: Labeling, Binding Assay

In vitro stability of 99m Tc-ZEGFR conjugates after labeling using different protocols: a 99m Tc-ZEGFR-APKC, b 99m Tc-ZEGFR-GGGC; c 99m Tc-ZEGFR-GGEC, d 99m Tc-ZEGFR-GEEC; e 99m Tc-ZEGFR-EEEC. Protocol B included pre-purification cysteine challenge. Data present the affibody-bound radioactivity after incubation in PBS (red), PBS containing 300-fold molar excess of cysteine (green), PBS containing sodium ascorbate (blue) and PBS containing tin (II) chloride (yellow). The data are presented as average (n ≥ 3) and SD
Figure Legend Snippet: In vitro stability of 99m Tc-ZEGFR conjugates after labeling using different protocols: a 99m Tc-ZEGFR-APKC, b 99m Tc-ZEGFR-GGGC; c 99m Tc-ZEGFR-GGEC, d 99m Tc-ZEGFR-GEEC; e 99m Tc-ZEGFR-EEEC. Protocol B included pre-purification cysteine challenge. Data present the affibody-bound radioactivity after incubation in PBS (red), PBS containing 300-fold molar excess of cysteine (green), PBS containing sodium ascorbate (blue) and PBS containing tin (II) chloride (yellow). The data are presented as average (n ≥ 3) and SD

Techniques Used: In Vitro, Labeling, Purification, Radioactivity, Incubation

In vivo specificity of 99m Tc-ZEGFR conjugates ( a 99m Tc-ZEGFR-GGEC, b 99m Tc-ZEGFR-GEEC and c 99m Tc-ZEGFR-EEEC) in A431 xenografts and EGFR-expressing organs in mice at 6 h after injection. In the blocked group, receptors were saturated by pre-injection of large excess of anti-EGFR antibody cetuximab. The data are presented as the average ( n = 4) and SD
Figure Legend Snippet: In vivo specificity of 99m Tc-ZEGFR conjugates ( a 99m Tc-ZEGFR-GGEC, b 99m Tc-ZEGFR-GEEC and c 99m Tc-ZEGFR-EEEC) in A431 xenografts and EGFR-expressing organs in mice at 6 h after injection. In the blocked group, receptors were saturated by pre-injection of large excess of anti-EGFR antibody cetuximab. The data are presented as the average ( n = 4) and SD

Techniques Used: In Vivo, Expressing, Mouse Assay, Injection

Cellular processing of 99m Tc-labeled ZEGFR ( a 99m Tc-ZEGFR-GGEC, b 99m Tc-ZEGFR-GEEC and c 99m Tc-ZEGFR-EEEC) variants by EGFR-expressing A431 cells. Cells were incubated with 10 nM 99m Tc-labeled ZEGFR conjugate. The data are presented as average ( n = 3) and SD. Error bars were not seen because they were smaller than point symbols
Figure Legend Snippet: Cellular processing of 99m Tc-labeled ZEGFR ( a 99m Tc-ZEGFR-GGEC, b 99m Tc-ZEGFR-GEEC and c 99m Tc-ZEGFR-EEEC) variants by EGFR-expressing A431 cells. Cells were incubated with 10 nM 99m Tc-labeled ZEGFR conjugate. The data are presented as average ( n = 3) and SD. Error bars were not seen because they were smaller than point symbols

Techniques Used: Labeling, Expressing, Incubation

39) Product Images from "Nonimmunogenetic Viral Capsid Carrier with Cancer Targeting Activity"

Article Title: Nonimmunogenetic Viral Capsid Carrier with Cancer Targeting Activity

Journal: Advanced Science

doi: 10.1002/advs.201800494

Genetic presentation of ABPs on the surface of HBVC. A) Schematic illustration of ABP‐HBVC and plasmid expression vector used in E. coli for the biosynthesis of ABP‐HBVC. B) Results of TEM and DLS analyses of purified HBVC (free of ABPs) and ABP‐HBVC.
Figure Legend Snippet: Genetic presentation of ABPs on the surface of HBVC. A) Schematic illustration of ABP‐HBVC and plasmid expression vector used in E. coli for the biosynthesis of ABP‐HBVC. B) Results of TEM and DLS analyses of purified HBVC (free of ABPs) and ABP‐HBVC.

Techniques Used: Plasmid Preparation, Expressing, Transmission Electron Microscopy, Purification

Albumin‐binding activity of ABP‐HBVC. A) Results of ELISA. B) Results of time‐course DLS analysis of the mixture of HBVC (free of ABPs) and human serum. The blue dotted circles represent the DLS peaks for HBVC. C) Results of time‐course DLS analysis of the mixture of ABP‐HBVC and human serum. The red dotted circles and arrows represent the DLS peaks for HSA and agglomerates of HSA‐ABP‐HBVC‐binding complexes, respectively.
Figure Legend Snippet: Albumin‐binding activity of ABP‐HBVC. A) Results of ELISA. B) Results of time‐course DLS analysis of the mixture of HBVC (free of ABPs) and human serum. The blue dotted circles represent the DLS peaks for HBVC. C) Results of time‐course DLS analysis of the mixture of ABP‐HBVC and human serum. The red dotted circles and arrows represent the DLS peaks for HSA and agglomerates of HSA‐ABP‐HBVC‐binding complexes, respectively.

Techniques Used: Binding Assay, Activity Assay, Enzyme-linked Immunosorbent Assay

Concentration of serum IL‐1β in live mice injected with ABP‐HBVC and HBVC (free of ABPs). A) Time schedule of IV injection of PBS (negative control), HBVC (50 µg), and ABP‐HBVC (50 µg) to C57BL/6 mice ( n = 72). B) Results of time‐course ELISA to measure serum IL‐1β in live mice of (A).
Figure Legend Snippet: Concentration of serum IL‐1β in live mice injected with ABP‐HBVC and HBVC (free of ABPs). A) Time schedule of IV injection of PBS (negative control), HBVC (50 µg), and ABP‐HBVC (50 µg) to C57BL/6 mice ( n = 72). B) Results of time‐course ELISA to measure serum IL‐1β in live mice of (A).

Techniques Used: Concentration Assay, Mouse Assay, Injection, IV Injection, Negative Control, Enzyme-linked Immunosorbent Assay

Anti‐HBVC antibody titer in live mice injected with ABP‐HBVC and HBVC (free of ABPs). A) Time schedule of intraperitoneal injection of PBS (negative control), HBVC (50 µg), and ABP‐HBVC (50 µg) to C57BL/6 mice ( n = 9). B) Results of ELISA to measure anti‐HBVC antibody (immunoglobulins except for IgM) titer in live mice of (A).
Figure Legend Snippet: Anti‐HBVC antibody titer in live mice injected with ABP‐HBVC and HBVC (free of ABPs). A) Time schedule of intraperitoneal injection of PBS (negative control), HBVC (50 µg), and ABP‐HBVC (50 µg) to C57BL/6 mice ( n = 9). B) Results of ELISA to measure anti‐HBVC antibody (immunoglobulins except for IgM) titer in live mice of (A).

Techniques Used: Mouse Assay, Injection, Negative Control, Enzyme-linked Immunosorbent Assay

Tumor targeting and biodistribution of ABP‐HBVC in live mice. A) NIR fluorescence images of live mice that were intravenously injected with Cy5.5‐labeled recombinant HBVC particles (HBVC (aff−, ABP−), HBVC (aff+), and ABP‐HBVC). B) Time‐course NIR fluorescence intensity from the tumor in live mice of (A). C) Ex vivo NIR fluorescence images of five major organs and tumor that were excised from live mice of (A) at 48 h after the IV injection.
Figure Legend Snippet: Tumor targeting and biodistribution of ABP‐HBVC in live mice. A) NIR fluorescence images of live mice that were intravenously injected with Cy5.5‐labeled recombinant HBVC particles (HBVC (aff−, ABP−), HBVC (aff+), and ABP‐HBVC). B) Time‐course NIR fluorescence intensity from the tumor in live mice of (A). C) Ex vivo NIR fluorescence images of five major organs and tumor that were excised from live mice of (A) at 48 h after the IV injection.

Techniques Used: Mouse Assay, Fluorescence, Injection, Labeling, Recombinant, Ex Vivo, IV Injection

40) Product Images from "Simultaneous extracellular and intracellular quantification of EGFR using paired-agent imaging in an in ovo tumor model"

Article Title: Simultaneous extracellular and intracellular quantification of EGFR using paired-agent imaging in an in ovo tumor model

Journal: Proceedings of SPIE--the International Society for Optical Engineering

doi: 10.1117/12.2510778

Validation of iPAI compared to Paired-Agent Imaging (PAI). Bottom : iPAI of EGFR intracellular tyrosine kinase domain was performed using a cell-permeable control BODIPY-N-Erlotinib, an extracellular control Alexa Fluor 647 (AF647) and a cell-membrane-permeable targeted TRITC-Erlotinib to produce a spatial map of iPAI binding potential (BP; i.e., receptor concentration). Top: An in ovo avatar white-light image and the PAI agents – extracellular control agent (IR680-Affibody control) and anti-EGFR-IRDye800 agent (ABY-029) – and the corresponding PAI BP map.
Figure Legend Snippet: Validation of iPAI compared to Paired-Agent Imaging (PAI). Bottom : iPAI of EGFR intracellular tyrosine kinase domain was performed using a cell-permeable control BODIPY-N-Erlotinib, an extracellular control Alexa Fluor 647 (AF647) and a cell-membrane-permeable targeted TRITC-Erlotinib to produce a spatial map of iPAI binding potential (BP; i.e., receptor concentration). Top: An in ovo avatar white-light image and the PAI agents – extracellular control agent (IR680-Affibody control) and anti-EGFR-IRDye800 agent (ABY-029) – and the corresponding PAI BP map.

Techniques Used: Imaging, Binding Assay, Concentration Assay, In Ovo

Related Articles

In Vivo:

Article Title: Near‐infrared photoimmunotherapy targeting EGFR—Shedding new light on glioblastoma treatment
Article Snippet: .. The tumor signal intensity after ZEGFR:03115 –IR700DX injection (6 μg) was calculated to be > 6‐fold higher than the signal measured for ZTaq ‐IR700DX, which confirmed the EGFR specificity of the affibody‐based conjugate in vivo (Figs. a and c ). .. Ex vivo fluorescence images demonstrated that amongst the non‐targeted organs, the kidneys and liver exhibited the highest accumulation of the conjugate, producing tumor‐to‐organ ratios of 0.85 ± 0.15 and 0.09 ± 0.02, respectively (Fig. b , Supporting Information Fig. 3a ).

Fluorescence:

Article Title: Imaging of Metastatic Cancer Cells in Sentinel Lymph Nodes using Affibody Probes and Possibility of a Theranostic Approach
Article Snippet: .. NIR Fluorescence Imaging of Cancer Cells in Lymph Nodes Twenty-four hours after injection of the anti-EGFR affibody probe, 14 lymph nodes were excised from four mice and examined. .. NIR fluorescence signals and EGFR expression were analyzed in all 14 lymph nodes by NIR imaging experiments and immunohistochemical studies.

Labeling:

Article Title: Comparative Evaluation of Radioiodine and Technetium-Labeled DARPin 9_29 for Radionuclide Molecular Imaging of HER2 Expression in Malignant Tumors
Article Snippet: .. The LigandTracer measurements demonstrated that both labeled variants bind with similar affinity to SKOV-3 cells, with K D1 of approximately 0.4 nM and K D2 of 8-9 nM. .. Two affinities are often found during LigandTracer measurement of binding to living cells for tracers targeting receptors belonging to HER family [ – ].

Mouse Assay:

Article Title: Imaging of Metastatic Cancer Cells in Sentinel Lymph Nodes using Affibody Probes and Possibility of a Theranostic Approach
Article Snippet: .. NIR Fluorescence Imaging of Cancer Cells in Lymph Nodes Twenty-four hours after injection of the anti-EGFR affibody probe, 14 lymph nodes were excised from four mice and examined. .. NIR fluorescence signals and EGFR expression were analyzed in all 14 lymph nodes by NIR imaging experiments and immunohistochemical studies.

Imaging:

Article Title: Imaging of Metastatic Cancer Cells in Sentinel Lymph Nodes using Affibody Probes and Possibility of a Theranostic Approach
Article Snippet: .. NIR Fluorescence Imaging of Cancer Cells in Lymph Nodes Twenty-four hours after injection of the anti-EGFR affibody probe, 14 lymph nodes were excised from four mice and examined. .. NIR fluorescence signals and EGFR expression were analyzed in all 14 lymph nodes by NIR imaging experiments and immunohistochemical studies.

Spectroscopy:

Article Title: Inhibiting HER3-Mediated Tumor Cell Growth with Affibody Molecules Engineered to Low Picomolar Affinity by Position-Directed Error-Prone PCR-Like Diversification
Article Snippet: .. In addition, the new HER3-specific Affibody molecules demonstrated high thermal stability and were able to refold into their alpha-helical structure after heat-induced denaturation, as demonstrated by biosensor analysis and CD spectroscopy ( ). .. As expected, retained receptor-specific binding to the HER3-receptor on various cancer cell lines in vitro could be shown by measuring the cellular uptake of radionuclide-labeled binders ( ).

Injection:

Article Title: Near‐infrared photoimmunotherapy targeting EGFR—Shedding new light on glioblastoma treatment
Article Snippet: .. The tumor signal intensity after ZEGFR:03115 –IR700DX injection (6 μg) was calculated to be > 6‐fold higher than the signal measured for ZTaq ‐IR700DX, which confirmed the EGFR specificity of the affibody‐based conjugate in vivo (Figs. a and c ). .. Ex vivo fluorescence images demonstrated that amongst the non‐targeted organs, the kidneys and liver exhibited the highest accumulation of the conjugate, producing tumor‐to‐organ ratios of 0.85 ± 0.15 and 0.09 ± 0.02, respectively (Fig. b , Supporting Information Fig. 3a ).

Article Title: Imaging of Metastatic Cancer Cells in Sentinel Lymph Nodes using Affibody Probes and Possibility of a Theranostic Approach
Article Snippet: .. NIR Fluorescence Imaging of Cancer Cells in Lymph Nodes Twenty-four hours after injection of the anti-EGFR affibody probe, 14 lymph nodes were excised from four mice and examined. .. NIR fluorescence signals and EGFR expression were analyzed in all 14 lymph nodes by NIR imaging experiments and immunohistochemical studies.

Binding Assay:

Article Title: Comparative Evaluation of Radioiodine and Technetium-Labeled DARPin 9_29 for Radionuclide Molecular Imaging of HER2 Expression in Malignant Tumors
Article Snippet: .. As shown in , while reduction in binding after treatment with unlabeled DARPin 9_29 was highly significant (p < 0.000001), there was neither significant reduction in binding after receptor saturation using trastuzumab (p > 0.05) nor any reduction in binding after treatment of SKOV-3 cells with cetuximab or bevacizumab. .. Interestingly, treatment of the SKOV-3 cells with ZHER2:342 affibody molecule resulted in small (12%) but significant (p < 0.05) reduction of [125 I]I-DARPin 9_29 binding.

Article Title: Comparative Evaluation of Radioiodine and Technetium-Labeled DARPin 9_29 for Radionuclide Molecular Imaging of HER2 Expression in Malignant Tumors
Article Snippet: .. To elucidate the binding specificity further, binding of [125 I]I-DARPin 9_29 was determined after treatment of SKOV-3 cells with anti-HER2 affibody molecules ZHER2:342 , anti-HER2 antibody trastuzumab, as well as control antibodies anti-EGFR cetuximab and anti-VEGF bevacizumab. .. As shown in , while reduction in binding after treatment with unlabeled DARPin 9_29 was highly significant (p < 0.000001), there was neither significant reduction in binding after receptor saturation using trastuzumab (p > 0.05) nor any reduction in binding after treatment of SKOV-3 cells with cetuximab or bevacizumab.

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  • 90
    Affibody egfr binding affibody molecule
    Specificity of 89 Zr-DFO-ZEGFR:2377 uptake in A431 xenografts and <t>EGFR-expressing</t> organs in mice at 3 h after injection. In the blocked group, receptors were saturated by pre-injection of large excess of non-labelled <t>affibody</t> molecules.
    Egfr Binding Affibody Molecule, supplied by Affibody, used in various techniques. Bioz Stars score: 90/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/egfr binding affibody molecule/product/Affibody
    Average 90 stars, based on 3 article reviews
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    88
    Affibody compact egfr binding affibody zegfr
    Modular capacity of probes for labeling <t>EGFR</t> on the cell surface. (A) Structures of the fluorogens used. The synthetic and analytical details were shown in Supporting Information or described previously. 26 A431 cell labeled with <t>FAP–affibody</t> fusions and various malachite green derivatives were analyzed by flow cytometry (B) and live-cell fluorescence microscopy (C). 5 × 10 5 /mL of cells were labeled with 250 nM of AFA or F followed by incubation with 100 nM of fluorogens for 5 min. Cells were then either analyzed by flow cytometry for mean fluorescence measurement or cell imaging. Scale bar 20 μm.
    Compact Egfr Binding Affibody Zegfr, supplied by Affibody, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/compact egfr binding affibody zegfr/product/Affibody
    Average 88 stars, based on 1 article reviews
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    90
    Affibody egfr binding fibronectin
    The effect of washing on enrichment ratio and recovery of yeast displaying <t>fibronectin</t> domain ligands panned on <t>EGFR</t> mid cells Yeast displaying E6.2.6′, E6.2.6′ N78S and WT′ (affinities indicated) mixed 1:1,000 with non-displaying yeast were panned against MDA-MB-231. The enrichment (A) and yield (B) of binding ligands is presented as the mean ± standard deviation of 3–9 replicates.
    Egfr Binding Fibronectin, supplied by Affibody, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Specificity of 89 Zr-DFO-ZEGFR:2377 uptake in A431 xenografts and EGFR-expressing organs in mice at 3 h after injection. In the blocked group, receptors were saturated by pre-injection of large excess of non-labelled affibody molecules.

    Journal: International Journal of Oncology

    Article Title: PET imaging of epidermal growth factor receptor expression in tumours using 89Zr-labelled ZEGFR:2377 affibody molecules

    doi: 10.3892/ijo.2016.3369

    Figure Lengend Snippet: Specificity of 89 Zr-DFO-ZEGFR:2377 uptake in A431 xenografts and EGFR-expressing organs in mice at 3 h after injection. In the blocked group, receptors were saturated by pre-injection of large excess of non-labelled affibody molecules.

    Article Snippet: In the present study, an EGFR-binding affibody molecule (ZEGFR:2377) was site-specifically conjugated with a deferoxamine (DFO) chelator and labelled under mild conditions (room temperature and neutral pH) with a positron-emitting radionuclide 89 Zr.

    Techniques: Expressing, Mouse Assay, Injection

    Specific binding and uptake of IRDye800CW-labeled Affibody molecules. (A) The protein expression levels of EGFR and HER2 in MDA-MB-231 (MDA231), A431, SKOV3, and SKBR3 cells. Actin served as an internal control. The relative expression levels were calculated

    Journal: Neoplasia (New York, N.Y.)

    Article Title: In Vivo Imaging of Xenograft Tumors Using an Epidermal Growth Factor Receptor-Specific Affibody Molecule Labeled with a Near-infrared Fluorophore 1

    doi:

    Figure Lengend Snippet: Specific binding and uptake of IRDye800CW-labeled Affibody molecules. (A) The protein expression levels of EGFR and HER2 in MDA-MB-231 (MDA231), A431, SKOV3, and SKBR3 cells. Actin served as an internal control. The relative expression levels were calculated

    Article Snippet: Cellular studies of binding, internalization and retention of a radiolabeled EGFR-binding Affibody molecule.

    Techniques: Binding Assay, Labeling, Expressing, Multiple Displacement Amplification

    The effect of EGF and EGFR-specific Affibody (Eaff) on EGFR-mediated phosphorylation of EGFR and ERK1/2 (P44/42 MAPK) proteins. A431 cells were treated with either Eaff or EGF. Two concentrations (5 and 20 nM) for both Eaff (Eaff5 and Eaff20) and EGF

    Journal: Neoplasia (New York, N.Y.)

    Article Title: In Vivo Imaging of Xenograft Tumors Using an Epidermal Growth Factor Receptor-Specific Affibody Molecule Labeled with a Near-infrared Fluorophore 1

    doi:

    Figure Lengend Snippet: The effect of EGF and EGFR-specific Affibody (Eaff) on EGFR-mediated phosphorylation of EGFR and ERK1/2 (P44/42 MAPK) proteins. A431 cells were treated with either Eaff or EGF. Two concentrations (5 and 20 nM) for both Eaff (Eaff5 and Eaff20) and EGF

    Article Snippet: Cellular studies of binding, internalization and retention of a radiolabeled EGFR-binding Affibody molecule.

    Techniques:

    Modular capacity of probes for labeling EGFR on the cell surface. (A) Structures of the fluorogens used. The synthetic and analytical details were shown in Supporting Information or described previously. 26 A431 cell labeled with FAP–affibody fusions and various malachite green derivatives were analyzed by flow cytometry (B) and live-cell fluorescence microscopy (C). 5 × 10 5 /mL of cells were labeled with 250 nM of AFA or F followed by incubation with 100 nM of fluorogens for 5 min. Cells were then either analyzed by flow cytometry for mean fluorescence measurement or cell imaging. Scale bar 20 μm.

    Journal: Bioconjugate Chemistry

    Article Title: Fluorogen Activating Protein–Affibody Probes: Modular, No-Wash Measurement of Epidermal Growth Factor Receptors

    doi: 10.1021/bc500525b

    Figure Lengend Snippet: Modular capacity of probes for labeling EGFR on the cell surface. (A) Structures of the fluorogens used. The synthetic and analytical details were shown in Supporting Information or described previously. 26 A431 cell labeled with FAP–affibody fusions and various malachite green derivatives were analyzed by flow cytometry (B) and live-cell fluorescence microscopy (C). 5 × 10 5 /mL of cells were labeled with 250 nM of AFA or F followed by incubation with 100 nM of fluorogens for 5 min. Cells were then either analyzed by flow cytometry for mean fluorescence measurement or cell imaging. Scale bar 20 μm.

    Article Snippet: A protein domain (FAP dL5** ) that binds to malachite-green (MG) derivatives for fluorescence activation was expressed as a recombinant fusion to one or two copies of the compact EGFR binding affibody ZEGFR:1907 .

    Techniques: Labeling, Flow Cytometry, Cytometry, Fluorescence, Microscopy, Incubation, Imaging

    Characterization of probes binding on A431 cell surface. (A) Dissociation constant analysis of probes on cell surface. 5 × 10 5 /mL quantities of cells were incubated with probes for 1 h at 37 °C followed by 2 μM MG-B-tau for 5 min. Then cells were kept on ice for flow cytometry. The mean fluorescence intensity was corrected with background of cells incubating with F/MG and then normalized to mean fluorescence at 250 nM of probes. (B) Competition assay of nonfluorescent affibody A binding to the cell surface. Cells were labeled with 250 nM AFA or F and a serial dilution of A followed by 100 nM of MG-B-tau added prior to measurement. (C) Detection of receptor activation by Western blots. Starved cells were labeled with 250 nM probes followed by 100 nM of MG-B-tau and then cells were treated with 100 ng/mL EGF. Then cells were lysed for Western blot in order to detect phosphorylated EGFR and total EGFR. (D) Live-cell fluorescence microscopy of A431 cells labeled by various probes. Cells were labeled with 250 nM of probe and 100 nM of MG-B-tau prior to imaging or 100 nM of Cy5 conjugated affibody dimer. Scale bar 20 μm.

    Journal: Bioconjugate Chemistry

    Article Title: Fluorogen Activating Protein–Affibody Probes: Modular, No-Wash Measurement of Epidermal Growth Factor Receptors

    doi: 10.1021/bc500525b

    Figure Lengend Snippet: Characterization of probes binding on A431 cell surface. (A) Dissociation constant analysis of probes on cell surface. 5 × 10 5 /mL quantities of cells were incubated with probes for 1 h at 37 °C followed by 2 μM MG-B-tau for 5 min. Then cells were kept on ice for flow cytometry. The mean fluorescence intensity was corrected with background of cells incubating with F/MG and then normalized to mean fluorescence at 250 nM of probes. (B) Competition assay of nonfluorescent affibody A binding to the cell surface. Cells were labeled with 250 nM AFA or F and a serial dilution of A followed by 100 nM of MG-B-tau added prior to measurement. (C) Detection of receptor activation by Western blots. Starved cells were labeled with 250 nM probes followed by 100 nM of MG-B-tau and then cells were treated with 100 ng/mL EGF. Then cells were lysed for Western blot in order to detect phosphorylated EGFR and total EGFR. (D) Live-cell fluorescence microscopy of A431 cells labeled by various probes. Cells were labeled with 250 nM of probe and 100 nM of MG-B-tau prior to imaging or 100 nM of Cy5 conjugated affibody dimer. Scale bar 20 μm.

    Article Snippet: A protein domain (FAP dL5** ) that binds to malachite-green (MG) derivatives for fluorescence activation was expressed as a recombinant fusion to one or two copies of the compact EGFR binding affibody ZEGFR:1907 .

    Techniques: Binding Assay, Incubation, Flow Cytometry, Cytometry, Fluorescence, Competitive Binding Assay, Labeling, Serial Dilution, Activation Assay, Western Blot, Microscopy, Imaging

    The effect of washing on enrichment ratio and recovery of yeast displaying fibronectin domain ligands panned on EGFR mid cells Yeast displaying E6.2.6′, E6.2.6′ N78S and WT′ (affinities indicated) mixed 1:1,000 with non-displaying yeast were panned against MDA-MB-231. The enrichment (A) and yield (B) of binding ligands is presented as the mean ± standard deviation of 3–9 replicates.

    Journal: Biotechnology and bioengineering

    Article Title: Geometry and expression enhance enrichment of functional yeast-displayed ligands via cell panning

    doi: 10.1002/bit.26001

    Figure Lengend Snippet: The effect of washing on enrichment ratio and recovery of yeast displaying fibronectin domain ligands panned on EGFR mid cells Yeast displaying E6.2.6′, E6.2.6′ N78S and WT′ (affinities indicated) mixed 1:1,000 with non-displaying yeast were panned against MDA-MB-231. The enrichment (A) and yield (B) of binding ligands is presented as the mean ± standard deviation of 3–9 replicates.

    Article Snippet: EGFR-binding fibronectin clone E6.2.6′, affibody clone EA68, and Gp2 clone GαEGFR2.2.3 were tested.

    Techniques: Multiple Displacement Amplification, Binding Assay, Standard Deviation

    The effect of washing and incubation conditions on enrichment ratio and recovery of yeast displaying fibronectin domain ligands panned on EGFR high cells Yeast displaying E6.2.6′, E6.2.6′ N78S, and WT′ (affinities indicated) mixed 1:1,000 with non-displaying yeast were panned against EGFR-expressing MDA-MB-468. The enrichment and yield of binding ligands is presented as the mean ± standard deviation of 3–9 replicates. (A and B) Selections were performed under baseline conditions with the exception of varied number of washing steps. (C and D) Selections were performed with the indicated modulation of incubation conditions.

    Journal: Biotechnology and bioengineering

    Article Title: Geometry and expression enhance enrichment of functional yeast-displayed ligands via cell panning

    doi: 10.1002/bit.26001

    Figure Lengend Snippet: The effect of washing and incubation conditions on enrichment ratio and recovery of yeast displaying fibronectin domain ligands panned on EGFR high cells Yeast displaying E6.2.6′, E6.2.6′ N78S, and WT′ (affinities indicated) mixed 1:1,000 with non-displaying yeast were panned against EGFR-expressing MDA-MB-468. The enrichment and yield of binding ligands is presented as the mean ± standard deviation of 3–9 replicates. (A and B) Selections were performed under baseline conditions with the exception of varied number of washing steps. (C and D) Selections were performed with the indicated modulation of incubation conditions.

    Article Snippet: EGFR-binding fibronectin clone E6.2.6′, affibody clone EA68, and Gp2 clone GαEGFR2.2.3 were tested.

    Techniques: Incubation, Expressing, Multiple Displacement Amplification, Binding Assay, Standard Deviation