Structured Review

Agilent technologies egfr
Immunophenotype of ZJU-0327, ZJU-0725 and ZJU-1127 cells in sections from original and xenografted tumors. On the sections of ZJU-0725 and ZJU-1127, strongly positive for ERα, <t>Her2,</t> <t>EGFR,</t> P120 and E-cadherin in original tumors and Her2, EGFR, P120 and E-cadherin in the ZJU-0725 xenografted tumors and Her2, P120 and E-cadherin in the ZJU-0725 xenografted tumors; positive for ki67 in original tumors and ERα and Ki67 in the ZJU-0725 xenografted tumors and ERα, Ki67 and EGFR in the ZJU-1127 xenografted tumors. But negative for PR and ck5/6 in both original and xenografted tumors. On the sections of ZJU-0327, strongly staining for ERα, PR, Her2, P120 and E-cadherin in the original tumors and Her2, P120 and E-cadherin; positive for Ki67 in the original tumors and ERα, ki67 and EGFR. But negative for ck5/6 and EGFR in the original tumors and PR and ck5/6 in the xenografted
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Images

1) Product Images from "Establishment and characterization of three stable Basal/HER2-positive breast cancer cell lines derived from Chinese breast carcinoma with identical missense mutations in the DNA-binding domain of TP53"

Article Title: Establishment and characterization of three stable Basal/HER2-positive breast cancer cell lines derived from Chinese breast carcinoma with identical missense mutations in the DNA-binding domain of TP53

Journal: Cancer Cell International

doi: 10.1186/s12935-018-0617-9

Immunophenotype of ZJU-0327, ZJU-0725 and ZJU-1127 cells in sections from original and xenografted tumors. On the sections of ZJU-0725 and ZJU-1127, strongly positive for ERα, Her2, EGFR, P120 and E-cadherin in original tumors and Her2, EGFR, P120 and E-cadherin in the ZJU-0725 xenografted tumors and Her2, P120 and E-cadherin in the ZJU-0725 xenografted tumors; positive for ki67 in original tumors and ERα and Ki67 in the ZJU-0725 xenografted tumors and ERα, Ki67 and EGFR in the ZJU-1127 xenografted tumors. But negative for PR and ck5/6 in both original and xenografted tumors. On the sections of ZJU-0327, strongly staining for ERα, PR, Her2, P120 and E-cadherin in the original tumors and Her2, P120 and E-cadherin; positive for Ki67 in the original tumors and ERα, ki67 and EGFR. But negative for ck5/6 and EGFR in the original tumors and PR and ck5/6 in the xenografted
Figure Legend Snippet: Immunophenotype of ZJU-0327, ZJU-0725 and ZJU-1127 cells in sections from original and xenografted tumors. On the sections of ZJU-0725 and ZJU-1127, strongly positive for ERα, Her2, EGFR, P120 and E-cadherin in original tumors and Her2, EGFR, P120 and E-cadherin in the ZJU-0725 xenografted tumors and Her2, P120 and E-cadherin in the ZJU-0725 xenografted tumors; positive for ki67 in original tumors and ERα and Ki67 in the ZJU-0725 xenografted tumors and ERα, Ki67 and EGFR in the ZJU-1127 xenografted tumors. But negative for PR and ck5/6 in both original and xenografted tumors. On the sections of ZJU-0327, strongly staining for ERα, PR, Her2, P120 and E-cadherin in the original tumors and Her2, P120 and E-cadherin; positive for Ki67 in the original tumors and ERα, ki67 and EGFR. But negative for ck5/6 and EGFR in the original tumors and PR and ck5/6 in the xenografted

Techniques Used: Staining

2) Product Images from "Immunohistochemical Classification of Primary and Secondary Glioblastomas"

Article Title: Immunohistochemical Classification of Primary and Secondary Glioblastomas

Journal: Korean Journal of Pathology

doi: 10.4132/KoreanJPathol.2013.47.6.541

Characteristic immunohistochemical expression of epidermal growth factor receptor (EGFR), p53, and isocitrate dehydrogenase 1 (IDH-1) in primary glioblastoma (A-C) and secondary glioblastoma (D-F). EGFR (A, D), p53 (B, E), and IDH-1 (C, F).
Figure Legend Snippet: Characteristic immunohistochemical expression of epidermal growth factor receptor (EGFR), p53, and isocitrate dehydrogenase 1 (IDH-1) in primary glioblastoma (A-C) and secondary glioblastoma (D-F). EGFR (A, D), p53 (B, E), and IDH-1 (C, F).

Techniques Used: Immunohistochemistry, Expressing

3) Product Images from "Establishment and characterization of three stable Basal/HER2-positive breast cancer cell lines derived from Chinese breast carcinoma with identical missense mutations in the DNA-binding domain of TP53"

Article Title: Establishment and characterization of three stable Basal/HER2-positive breast cancer cell lines derived from Chinese breast carcinoma with identical missense mutations in the DNA-binding domain of TP53

Journal: Cancer Cell International

doi: 10.1186/s12935-018-0617-9

Immunophenotype of ZJU-0327, ZJU-0725 and ZJU-1127 cells in sections from original and xenografted tumors. On the sections of ZJU-0725 and ZJU-1127, strongly positive for ERα, Her2, EGFR, P120 and E-cadherin in original tumors and Her2, EGFR, P120 and E-cadherin in the ZJU-0725 xenografted tumors and Her2, P120 and E-cadherin in the ZJU-0725 xenografted tumors; positive for ki67 in original tumors and ERα and Ki67 in the ZJU-0725 xenografted tumors and ERα, Ki67 and EGFR in the ZJU-1127 xenografted tumors. But negative for PR and ck5/6 in both original and xenografted tumors. On the sections of ZJU-0327, strongly staining for ERα, PR, Her2, P120 and E-cadherin in the original tumors and Her2, P120 and E-cadherin; positive for Ki67 in the original tumors and ERα, ki67 and EGFR. But negative for ck5/6 and EGFR in the original tumors and PR and ck5/6 in the xenografted
Figure Legend Snippet: Immunophenotype of ZJU-0327, ZJU-0725 and ZJU-1127 cells in sections from original and xenografted tumors. On the sections of ZJU-0725 and ZJU-1127, strongly positive for ERα, Her2, EGFR, P120 and E-cadherin in original tumors and Her2, EGFR, P120 and E-cadherin in the ZJU-0725 xenografted tumors and Her2, P120 and E-cadherin in the ZJU-0725 xenografted tumors; positive for ki67 in original tumors and ERα and Ki67 in the ZJU-0725 xenografted tumors and ERα, Ki67 and EGFR in the ZJU-1127 xenografted tumors. But negative for PR and ck5/6 in both original and xenografted tumors. On the sections of ZJU-0327, strongly staining for ERα, PR, Her2, P120 and E-cadherin in the original tumors and Her2, P120 and E-cadherin; positive for Ki67 in the original tumors and ERα, ki67 and EGFR. But negative for ck5/6 and EGFR in the original tumors and PR and ck5/6 in the xenografted

Techniques Used: Staining

4) Product Images from "Radioresistance of human glioma spheroids and expression of HSP70, p53 and EGFr"

Article Title: Radioresistance of human glioma spheroids and expression of HSP70, p53 and EGFr

Journal: Radiation Oncology (London, England)

doi: 10.1186/1748-717X-6-156

p53, Hsp70 and EGFr immunohistochemical expression in UGBM1, U-87MG and MO59J spheroids following irradiation . Immunostaining score was calculated for non-treated (control) or irradiated (5 Gy) as determined 24 h (left column) and 6 days (right column) after treatment. Arbitrary scoring system was calculated as described in Methods section. Data was plotted as the mean ± SD of three different experiments. *Significantly different from control (p
Figure Legend Snippet: p53, Hsp70 and EGFr immunohistochemical expression in UGBM1, U-87MG and MO59J spheroids following irradiation . Immunostaining score was calculated for non-treated (control) or irradiated (5 Gy) as determined 24 h (left column) and 6 days (right column) after treatment. Arbitrary scoring system was calculated as described in Methods section. Data was plotted as the mean ± SD of three different experiments. *Significantly different from control (p

Techniques Used: Immunohistochemistry, Expressing, Irradiation, Immunostaining

Representative photomicrographs of the p53 content evaluated by immunohistochemistry in UGBM1 human GBM spheroids (A); Hsp 70 content following 5 Gy irradiation in MO59J spheroids (B); EGFr contents in no-irradiated (control) (C) and 5 Gy irradiated MO59J spheroids (D) . EGFr antibody clone H11, recognizes wild-type EGFr and the deleted mutant form (EGFrvIII). Original magnification × 400.
Figure Legend Snippet: Representative photomicrographs of the p53 content evaluated by immunohistochemistry in UGBM1 human GBM spheroids (A); Hsp 70 content following 5 Gy irradiation in MO59J spheroids (B); EGFr contents in no-irradiated (control) (C) and 5 Gy irradiated MO59J spheroids (D) . EGFr antibody clone H11, recognizes wild-type EGFr and the deleted mutant form (EGFrvIII). Original magnification × 400.

Techniques Used: Immunohistochemistry, Irradiation, Mutagenesis

5) Product Images from "A comprehensive morphological study for basal-like breast carcinomas with comparison to nonbasal-like carcinomas"

Article Title: A comprehensive morphological study for basal-like breast carcinomas with comparison to nonbasal-like carcinomas

Journal: Diagnostic Pathology

doi: 10.1186/1746-1596-7-145

Immunohistochemical staining of BLBCs (All photographs were taken at x100 magnification). A . CK 5/6. B . EGFR. C . CK14. D . Vimentin.
Figure Legend Snippet: Immunohistochemical staining of BLBCs (All photographs were taken at x100 magnification). A . CK 5/6. B . EGFR. C . CK14. D . Vimentin.

Techniques Used: Immunohistochemistry, Staining

6) Product Images from "Phosphorylated AKT1 is associated with poor prognosis in esophageal squamous cell carcinoma"

Article Title: Phosphorylated AKT1 is associated with poor prognosis in esophageal squamous cell carcinoma

Journal: Journal of Experimental & Clinical Cancer Research : CR

doi: 10.1186/s13046-015-0212-z

Representative findings on immunohistochemical staining for the tested biomarkers (original magnification × 200): EGFR, phosphorylated (p)-EGFR, AKT1, p-AKT1, AKT2, p-AKT2, ERK1, ERK2, p-ERK1/2, STAT3, and p-STAT3
Figure Legend Snippet: Representative findings on immunohistochemical staining for the tested biomarkers (original magnification × 200): EGFR, phosphorylated (p)-EGFR, AKT1, p-AKT1, AKT2, p-AKT2, ERK1, ERK2, p-ERK1/2, STAT3, and p-STAT3

Techniques Used: Immunohistochemistry, Staining

7) Product Images from "Safety and efficacy of afatinib as add-on to standard therapy of gemcitabine/cisplatin in chemotherapy-naive patients with advanced biliary tract cancer: an open-label, phase I trial with an extensive biomarker program"

Article Title: Safety and efficacy of afatinib as add-on to standard therapy of gemcitabine/cisplatin in chemotherapy-naive patients with advanced biliary tract cancer: an open-label, phase I trial with an extensive biomarker program

Journal: BMC Cancer

doi: 10.1186/s12885-018-5223-7

Immunohistochemical staining, a ) negative control, positive staining of b ) EGFR (3+), c ) HER2neu (1+), d ) HER3 (2+) and e ) HER4 (only sporadic)
Figure Legend Snippet: Immunohistochemical staining, a ) negative control, positive staining of b ) EGFR (3+), c ) HER2neu (1+), d ) HER3 (2+) and e ) HER4 (only sporadic)

Techniques Used: Immunohistochemistry, Staining, Negative Control

8) Product Images from "Differences in biological features of gastric dysplasia, indefinite dysplasia, reactive hyperplasia and discriminant analysis of these lesions"

Article Title: Differences in biological features of gastric dysplasia, indefinite dysplasia, reactive hyperplasia and discriminant analysis of these lesions

Journal:

doi: 10.3748/wjg.v11.i23.3595

MUC5AC, MUC6, APC, EGFR, and p53 expressions in different gastric lesions.
Figure Legend Snippet: MUC5AC, MUC6, APC, EGFR, and p53 expressions in different gastric lesions.

Techniques Used:

9) Product Images from "Selective Modulation of Hedgehog/GLI Target Gene Expression by Epidermal Growth Factor Signaling in Human Keratinocytes †"

Article Title: Selective Modulation of Hedgehog/GLI Target Gene Expression by Epidermal Growth Factor Signaling in Human Keratinocytes †

Journal:

doi: 10.1128/MCB.02317-05

Coexpression of IL1R2, EGFR, and p-ERK in the ORS of human anagen hair follicles. (A) IL1R2 protein is localized to the ORS of human hair follicles below the sebaceous gland. Inset shows intense IL1R2 staining in the ORS at higher magnification. (B) EGFR
Figure Legend Snippet: Coexpression of IL1R2, EGFR, and p-ERK in the ORS of human anagen hair follicles. (A) IL1R2 protein is localized to the ORS of human hair follicles below the sebaceous gland. Inset shows intense IL1R2 staining in the ORS at higher magnification. (B) EGFR

Techniques Used: Staining

10) Product Images from "Clinical variables serve as prognostic factors in a model for survival from glioblastoma multiforme: an observational study of a cohort of consecutive non-selected patients from a single institution"

Article Title: Clinical variables serve as prognostic factors in a model for survival from glioblastoma multiforme: an observational study of a cohort of consecutive non-selected patients from a single institution

Journal: BMC Cancer

doi: 10.1186/1471-2407-13-402

Examples of IHC stainings. p53, EGFR, and MGMT expression by IHC scored semiquantitatively on the scale:
Figure Legend Snippet: Examples of IHC stainings. p53, EGFR, and MGMT expression by IHC scored semiquantitatively on the scale:

Techniques Used: Immunohistochemistry, Expressing

11) Product Images from "Co-Expression of Cox-2, C-Met and ?-catenin in Cells Forming Invasive front of Gallbladder Cancer"

Article Title: Co-Expression of Cox-2, C-Met and ?-catenin in Cells Forming Invasive front of Gallbladder Cancer

Journal:

doi: 10.4143/crt.2005.37.3.171

Representative immunostaining for EGFR, c-erbB2, Cox-2, c-Met and β-catenin in gallbladder cancer. A-B) Cancer cells showed selective membrane staining for EGFR (A, ×200, scale bar 100 µm) and c-erbB2 (B, ×400, scale bar
Figure Legend Snippet: Representative immunostaining for EGFR, c-erbB2, Cox-2, c-Met and β-catenin in gallbladder cancer. A-B) Cancer cells showed selective membrane staining for EGFR (A, ×200, scale bar 100 µm) and c-erbB2 (B, ×400, scale bar

Techniques Used: Immunostaining, Staining

12) Product Images from "Dual targeting of glioblastoma with chimeric antigen receptor-engineered natural killer cells overcomes heterogeneity of target antigen expression and enhances antitumor activity and survival"

Article Title: Dual targeting of glioblastoma with chimeric antigen receptor-engineered natural killer cells overcomes heterogeneity of target antigen expression and enhances antitumor activity and survival

Journal: Oncoimmunology

doi: 10.1080/2162402X.2015.1119354

Antitumor activity of CAR NK cells against orthotopic LNT-229/EGFR and LNT-229/EGFRvIII GBM xenografts. (A) LNT-229/EGFR cells were stereotactically injected into the right striatum of NSG mice. Seven days later, the animals were treated by intratumoral injection of parental NK-92, EGFR-specific NK-92/R1, EGFRvIII-specific NK-92/MR1-1, or dual-specific NK-92/225 cells once per week for 12 weeks (n = 6). Control mice received injection medium. Tumor growth was monitored by MRI. Tumor development in representative animals from each group at day 53 is shown. (B) Symptom-free survival of the mice from the experiment described in (A). (C) LNT-229/EGFRvIII cells were stereotactically injected into the right striatum of NSG mice. Seven days later, the animals were treated as described above with NK-92 (n = 6), NK-92/R1 (n = 6), NK-92/MR1-1 (n = 5), or NK-92/225 (n = 6) cells once per week for 8 weeks. Control mice received injection medium (n = 6). Tumor development in representative animals from each group at day 53 is shown. (D) Symptom-free survival of the mice from the experiment described in (C). *** p ≤ 0.001; ** p ≤ 0.01; ns, p > 0.05.
Figure Legend Snippet: Antitumor activity of CAR NK cells against orthotopic LNT-229/EGFR and LNT-229/EGFRvIII GBM xenografts. (A) LNT-229/EGFR cells were stereotactically injected into the right striatum of NSG mice. Seven days later, the animals were treated by intratumoral injection of parental NK-92, EGFR-specific NK-92/R1, EGFRvIII-specific NK-92/MR1-1, or dual-specific NK-92/225 cells once per week for 12 weeks (n = 6). Control mice received injection medium. Tumor growth was monitored by MRI. Tumor development in representative animals from each group at day 53 is shown. (B) Symptom-free survival of the mice from the experiment described in (A). (C) LNT-229/EGFRvIII cells were stereotactically injected into the right striatum of NSG mice. Seven days later, the animals were treated as described above with NK-92 (n = 6), NK-92/R1 (n = 6), NK-92/MR1-1 (n = 5), or NK-92/225 (n = 6) cells once per week for 8 weeks. Control mice received injection medium (n = 6). Tumor development in representative animals from each group at day 53 is shown. (D) Symptom-free survival of the mice from the experiment described in (C). *** p ≤ 0.001; ** p ≤ 0.01; ns, p > 0.05.

Techniques Used: Activity Assay, Injection, Mouse Assay, Magnetic Resonance Imaging

Generation of CAR NK cells. (A) Lentiviral transfer plasmids pS-R1.28.z-IEW, pS-MR1-1.28.z-IEW and pS-225.28.z-IEW encoding under control of the Spleen Focus Forming Virus promoter (SFFV) CARs consisting of an immunoglobulin heavy chain signal peptide (SP), scFv fragments derived from EGFR-specific antibody R1, EGFRvIII-specific MR1-1, or 225 recognizing EGFR and EGFRvIII, followed by a Myc-tag (M), CD8α hinge region (CD8α), transmembrane and intracellular domains of CD28, and the intracellular domain of CD3ζ. Enhanced green fluorescent protein (EGFP) cDNA separated from the CAR sequence by an internal ribosome entry site (IRES) served as a marker. (B) CAR surface expression on NK-92/R1, NK-92/MR1-1 and NK-92/225 single cell clones was determined by flow cytometry with Myc-tag-specific antibody (open areas). Isotype antibody (filled areas) and parental NK-92 cells served as controls. (C) Binding of recombinant EGFR-Fc protein to the surface of CAR NK cells was measured by flow cytometry (open areas). CAR NK cells only treated with secondary antibody (filled areas) and parental NK-92 cells served as controls. MFI: mean fluorescence intensity (geometric mean).
Figure Legend Snippet: Generation of CAR NK cells. (A) Lentiviral transfer plasmids pS-R1.28.z-IEW, pS-MR1-1.28.z-IEW and pS-225.28.z-IEW encoding under control of the Spleen Focus Forming Virus promoter (SFFV) CARs consisting of an immunoglobulin heavy chain signal peptide (SP), scFv fragments derived from EGFR-specific antibody R1, EGFRvIII-specific MR1-1, or 225 recognizing EGFR and EGFRvIII, followed by a Myc-tag (M), CD8α hinge region (CD8α), transmembrane and intracellular domains of CD28, and the intracellular domain of CD3ζ. Enhanced green fluorescent protein (EGFP) cDNA separated from the CAR sequence by an internal ribosome entry site (IRES) served as a marker. (B) CAR surface expression on NK-92/R1, NK-92/MR1-1 and NK-92/225 single cell clones was determined by flow cytometry with Myc-tag-specific antibody (open areas). Isotype antibody (filled areas) and parental NK-92 cells served as controls. (C) Binding of recombinant EGFR-Fc protein to the surface of CAR NK cells was measured by flow cytometry (open areas). CAR NK cells only treated with secondary antibody (filled areas) and parental NK-92 cells served as controls. MFI: mean fluorescence intensity (geometric mean).

Techniques Used: Derivative Assay, Sequencing, Marker, Expressing, Clone Assay, Flow Cytometry, Cytometry, Binding Assay, Recombinant, Fluorescence

Cytotoxicity of CAR NK cells against EGFR- and EGFRvIII-expressing LNT-229 cells. (A) EGFR (170 kDa) and EGFRvIII (140 kDa) were detected in cell lysates of LNT-229 GBM cells ectopically overexpressing full-length EGFR (LNT-229/EGFR) or mutant EGFRvIII (LNT-229/EGFRvIII) by immunoblotting with an EGFR-specific antibody binding to both receptors. Parental LNT-229 served as control. (B) Cytotoxicity of CAR NK cells against LNT-229/EGFR, LNT-229/EGFRvIII and parental LNT-229 cells was investigated after co-incubation of effector and target cells for 2 h at different E/T ratios. Parental NK-92 were included as control. Mean values ± SEM are shown; n = 3. (C) Conjugate formation between CAR NK cells and LNT-229/EGFR and LNT-229/EGFRvIII cells was investigated by confocal microscopy. Tumor (T) and EGFP-positive CAR NK (N; green) cells were co-incubated for 1 h, fixed, permeabilized and stained for perforin (red) to identify cytotoxic granules. Cell nuclei were labeled with DAPI (blue). Parental NK-92 and NK-92/225.TM cells expressing a CAR without intracellular signaling domains were included as controls. Scale bar: 10 µm.
Figure Legend Snippet: Cytotoxicity of CAR NK cells against EGFR- and EGFRvIII-expressing LNT-229 cells. (A) EGFR (170 kDa) and EGFRvIII (140 kDa) were detected in cell lysates of LNT-229 GBM cells ectopically overexpressing full-length EGFR (LNT-229/EGFR) or mutant EGFRvIII (LNT-229/EGFRvIII) by immunoblotting with an EGFR-specific antibody binding to both receptors. Parental LNT-229 served as control. (B) Cytotoxicity of CAR NK cells against LNT-229/EGFR, LNT-229/EGFRvIII and parental LNT-229 cells was investigated after co-incubation of effector and target cells for 2 h at different E/T ratios. Parental NK-92 were included as control. Mean values ± SEM are shown; n = 3. (C) Conjugate formation between CAR NK cells and LNT-229/EGFR and LNT-229/EGFRvIII cells was investigated by confocal microscopy. Tumor (T) and EGFP-positive CAR NK (N; green) cells were co-incubated for 1 h, fixed, permeabilized and stained for perforin (red) to identify cytotoxic granules. Cell nuclei were labeled with DAPI (blue). Parental NK-92 and NK-92/225.TM cells expressing a CAR without intracellular signaling domains were included as controls. Scale bar: 10 µm.

Techniques Used: Expressing, Mutagenesis, Binding Assay, Incubation, Confocal Microscopy, Staining, Labeling

Antitumor activity of CAR NK cells against mixed LNT-229/EGFR and LNT-229/EGFRvIII GBM xenografts. (A) LNT-229/EGFR and LNT-229/EGFRvIII cells were mixed at a 1:1 ratio before stereotactic injection of the cells into the right striatum of NSG mice. Seven days later, the animals were treated by intratumoral injection of parental NK-92, EGFR-specific NK-92/R1, EGFRvIII-specific NK-92/MR1-1, dual-specific NK-92/225, or a 1:1 mixture of NK-92/R1 and NK-92/MR1-1 cells once per week for 8 weeks (n = 6). Control mice received injection medium. Tumor development in representative animals from each group at day 54 is shown. (B) Symptom-free survival of the mice from the experiment described in (A). *** p ≤ 0.001; * p ≤ 0.05; ns, p > 0.05. (C) Sections of tumors from individual animals of each treatment group sacrificed at the indicated time point were stained with EGFR- or EGFRvIII-specific antibodies. NK cells present in tumor tissues were detected with CD45-specific antibody. Scale bar: 300 µm.
Figure Legend Snippet: Antitumor activity of CAR NK cells against mixed LNT-229/EGFR and LNT-229/EGFRvIII GBM xenografts. (A) LNT-229/EGFR and LNT-229/EGFRvIII cells were mixed at a 1:1 ratio before stereotactic injection of the cells into the right striatum of NSG mice. Seven days later, the animals were treated by intratumoral injection of parental NK-92, EGFR-specific NK-92/R1, EGFRvIII-specific NK-92/MR1-1, dual-specific NK-92/225, or a 1:1 mixture of NK-92/R1 and NK-92/MR1-1 cells once per week for 8 weeks (n = 6). Control mice received injection medium. Tumor development in representative animals from each group at day 54 is shown. (B) Symptom-free survival of the mice from the experiment described in (A). *** p ≤ 0.001; * p ≤ 0.05; ns, p > 0.05. (C) Sections of tumors from individual animals of each treatment group sacrificed at the indicated time point were stained with EGFR- or EGFRvIII-specific antibodies. NK cells present in tumor tissues were detected with CD45-specific antibody. Scale bar: 300 µm.

Techniques Used: Activity Assay, Injection, Mouse Assay, Staining

13) Product Images from "Distinct gene expression profiles in ovarian cancer linked to Lynch syndrome"

Article Title: Distinct gene expression profiles in ovarian cancer linked to Lynch syndrome

Journal: Familial Cancer

doi: 10.1007/s10689-014-9728-1

Immunohistochemical stainings for p-mTOR, EGFR and PTEN in ×40 magnification with Lynch syndrome-associated tumors presented on the top row and sporadic tumors on the bottom row. The left and middle columns show positive ( top row ) and negative ( bottom row ) p-mTOR and EGFR expression in tumor cells respectively. The right column shows negative PTEN expression in tumor cells but retained staining in surrounding tissue ( top row ) and positive PTEN expression in tumor cells and surrounding tissue ( bottom row )
Figure Legend Snippet: Immunohistochemical stainings for p-mTOR, EGFR and PTEN in ×40 magnification with Lynch syndrome-associated tumors presented on the top row and sporadic tumors on the bottom row. The left and middle columns show positive ( top row ) and negative ( bottom row ) p-mTOR and EGFR expression in tumor cells respectively. The right column shows negative PTEN expression in tumor cells but retained staining in surrounding tissue ( top row ) and positive PTEN expression in tumor cells and surrounding tissue ( bottom row )

Techniques Used: Immunohistochemistry, Expressing, Staining

14) Product Images from "Multiparameter Analysis, including EMT Markers, on Negatively Enriched Blood Samples from Patients with Squamous Cell Carcinoma of the Head and Neck"

Article Title: Multiparameter Analysis, including EMT Markers, on Negatively Enriched Blood Samples from Patients with Squamous Cell Carcinoma of the Head and Neck

Journal: PLoS ONE

doi: 10.1371/journal.pone.0042048

Representative, confocal images of a cytospin of an enriched peripheral blood sample from a SCCHN patient. This is the same patient sample used to create the cytospin presented in Figure 4 . However, for this specific, four color staining, the anti-EGFR antibody was replaced with anti-CD44. The cells highlighted with a red arrow are positive for all four markers, while cells highlighted with yellow arrows are either negative, or very weak for CK, vimentin, and CD44.
Figure Legend Snippet: Representative, confocal images of a cytospin of an enriched peripheral blood sample from a SCCHN patient. This is the same patient sample used to create the cytospin presented in Figure 4 . However, for this specific, four color staining, the anti-EGFR antibody was replaced with anti-CD44. The cells highlighted with a red arrow are positive for all four markers, while cells highlighted with yellow arrows are either negative, or very weak for CK, vimentin, and CD44.

Techniques Used: Staining

Representative, confocal images of a cytospin of an enriched peripheral blood sample from a 27 year old, HPV positive, SCCHN patient. Sample taken in the OR, 214 cytokeratin positive cells per ml of blood were detected, and she had a reoccurrence 2 months after surgery. For this specific four color staining, the cell structure/protein targeted is listed in each image. The cells highlighted with a red arrow are positive for all four markers, yellow are negative for CK, weak or negative for vimentin and EGFR, and white is negative for all but nuclei. Green arrows highlight cells that have a granulocytic like nuclei.
Figure Legend Snippet: Representative, confocal images of a cytospin of an enriched peripheral blood sample from a 27 year old, HPV positive, SCCHN patient. Sample taken in the OR, 214 cytokeratin positive cells per ml of blood were detected, and she had a reoccurrence 2 months after surgery. For this specific four color staining, the cell structure/protein targeted is listed in each image. The cells highlighted with a red arrow are positive for all four markers, yellow are negative for CK, weak or negative for vimentin and EGFR, and white is negative for all but nuclei. Green arrows highlight cells that have a granulocytic like nuclei.

Techniques Used: Staining

Representative set of confocal images of a cytospin of stained normal blood samples and SSC4 cell lines. The cell structure/protein targeted is listed in each image, the type of cells listed on right. Note, the second row of images presents cells stained for EGFR, while the third row shows cells stained for CD44.
Figure Legend Snippet: Representative set of confocal images of a cytospin of stained normal blood samples and SSC4 cell lines. The cell structure/protein targeted is listed in each image, the type of cells listed on right. Note, the second row of images presents cells stained for EGFR, while the third row shows cells stained for CD44.

Techniques Used: Staining

15) Product Images from "Combining the Multi-Targeted Tyrosine Kinase Inhibitor Vandetanib with the Anti-Estrogen Fulvestrant Enhances its Anti-tumor Effect in Non-Small Cell Lung Cancer"

Article Title: Combining the Multi-Targeted Tyrosine Kinase Inhibitor Vandetanib with the Anti-Estrogen Fulvestrant Enhances its Anti-tumor Effect in Non-Small Cell Lung Cancer

Journal: Journal of Thoracic Oncology

doi: 10.1097/JTO.0b013e31824177ea

Protein expression in NSCLC cell lines and VEGF secretion. A . Western blot analysis for VEGFR-2, VEGFR-3, EGFR, RET and actin in whole cell lysates (50 μg) from NSCLC cells 201T, 273T and A549. Cell line controls were specific for each protein
Figure Legend Snippet: Protein expression in NSCLC cell lines and VEGF secretion. A . Western blot analysis for VEGFR-2, VEGFR-3, EGFR, RET and actin in whole cell lysates (50 μg) from NSCLC cells 201T, 273T and A549. Cell line controls were specific for each protein

Techniques Used: Expressing, Western Blot

16) Product Images from "Randomized Phase II Trial of Erlotinib Versus Temozolomide or Carmustine in Recurrent Glioblastoma: EORTC Brain Tumor Group Study 26034"

Article Title: Randomized Phase II Trial of Erlotinib Versus Temozolomide or Carmustine in Recurrent Glioblastoma: EORTC Brain Tumor Group Study 26034

Journal: Journal of Clinical Oncology

doi: 10.1200/JCO.2008.17.5984

Correlation between progression-free survival and (A) epidermal growth factor receptor (EGFR) amplification in the erlotinib arm; (B) EGFR amplification in the control arm; (C) the presence of EGFRvIII mutant in the erlotinib arm; (D) EGFRvIII mutant
Figure Legend Snippet: Correlation between progression-free survival and (A) epidermal growth factor receptor (EGFR) amplification in the erlotinib arm; (B) EGFR amplification in the control arm; (C) the presence of EGFRvIII mutant in the erlotinib arm; (D) EGFRvIII mutant

Techniques Used: Amplification, Mutagenesis

17) Product Images from "ErbB/HER receptor activation and preclinical efficacy of lapatinib in vestibular schwannoma †"

Article Title: ErbB/HER receptor activation and preclinical efficacy of lapatinib in vestibular schwannoma †

Journal: Neuro-Oncology

doi: 10.1093/neuonc/noq012

Immunohistochemistry of VS specimens. Representative staining of VS for EGFR (A), ErbB2 (B), phospho-ERK1/2 (C), and survivin (D) is shown. Antigens are visualized by brown membranous and/or nuclear staining.
Figure Legend Snippet: Immunohistochemistry of VS specimens. Representative staining of VS for EGFR (A), ErbB2 (B), phospho-ERK1/2 (C), and survivin (D) is shown. Antigens are visualized by brown membranous and/or nuclear staining.

Techniques Used: Immunohistochemistry, Staining

18) Product Images from "Optical molecular imaging can differentiate metastatic from benign lymph nodes in head and neck cancer"

Article Title: Optical molecular imaging can differentiate metastatic from benign lymph nodes in head and neck cancer

Journal: Nature Communications

doi: 10.1038/s41467-019-13076-7

Panitumumab-IRDye800CW Distribution and Correlation to Immunohistochemistry. a Hematoxylin and eosin (H E) staining, near-infrared fluorescence image and EGFR, cytokeratin 5/6, CD68 and CD31 immunohistochemistry results of a 4 µm cut section of representative true positive, false positive, false negative and true negative LNs. Dotted line: tumor region. Scale bars = 5 mm. b Representative microscopic fluorescence images of panitumumab-IRDye800CW (IRDye800CW) and EGFR immunohistochemistry in macro-metastasis (upper row), micro-metastasis (middle row) and uninvolved LN tissue (bottom row). Scale bars = 100 µm. EGFR epidermal growth factor receptor, DAPI 4′,6-diamidino-2-phenylindole (cell nucleus staining), LN lymph node
Figure Legend Snippet: Panitumumab-IRDye800CW Distribution and Correlation to Immunohistochemistry. a Hematoxylin and eosin (H E) staining, near-infrared fluorescence image and EGFR, cytokeratin 5/6, CD68 and CD31 immunohistochemistry results of a 4 µm cut section of representative true positive, false positive, false negative and true negative LNs. Dotted line: tumor region. Scale bars = 5 mm. b Representative microscopic fluorescence images of panitumumab-IRDye800CW (IRDye800CW) and EGFR immunohistochemistry in macro-metastasis (upper row), micro-metastasis (middle row) and uninvolved LN tissue (bottom row). Scale bars = 100 µm. EGFR epidermal growth factor receptor, DAPI 4′,6-diamidino-2-phenylindole (cell nucleus staining), LN lymph node

Techniques Used: Immunohistochemistry, Staining, Fluorescence

19) Product Images from "Immunohistochemical Analysis of Phosphotyrosine Signal Transducer and Activator of Transcription 3 and Epidermal Growth Factor Receptor Autocrine Signaling Pathways in Head and Neck Cancers and Metastatic Lymph Nodes"

Article Title: Immunohistochemical Analysis of Phosphotyrosine Signal Transducer and Activator of Transcription 3 and Epidermal Growth Factor Receptor Autocrine Signaling Pathways in Head and Neck Cancers and Metastatic Lymph Nodes

Journal:

doi: 10.1158/1078-0432.CCR-07-1543

Relationship between TGF-α and EGFR protein levels in 55 SCCHNs (Spearman correlation = 0.51; P
Figure Legend Snippet: Relationship between TGF-α and EGFR protein levels in 55 SCCHNs (Spearman correlation = 0.51; P

Techniques Used:

STAT3-mediated TGF-α/EGFR signaling is detected in metastatic cervical lymph nodes
Figure Legend Snippet: STAT3-mediated TGF-α/EGFR signaling is detected in metastatic cervical lymph nodes

Techniques Used:

Representative immunostaining of tumors and paired cervical metastases. Tumors from primary tumor ( top ) and paired metastatic lymph nodes ( bottom ) were stained for ( A ) GPRR, ( B ) TGF-α, ( C ) EGFR, and ( D ) pSTAT3 (original magnification, ×100).
Figure Legend Snippet: Representative immunostaining of tumors and paired cervical metastases. Tumors from primary tumor ( top ) and paired metastatic lymph nodes ( bottom ) were stained for ( A ) GPRR, ( B ) TGF-α, ( C ) EGFR, and ( D ) pSTAT3 (original magnification, ×100).

Techniques Used: Immunostaining, Staining

20) Product Images from "Stratification of Prognosis of Triple-Negative Breast Cancer Patients Using Combinatorial Biomarkers"

Article Title: Stratification of Prognosis of Triple-Negative Breast Cancer Patients Using Combinatorial Biomarkers

Journal: PLoS ONE

doi: 10.1371/journal.pone.0149661

Kaplan Meier curves of disease free survival of risk groups for TNBC patients. The patients were stratified into different risk groups. Two low basal (EGFR≤15% and CK5/6≤50%) risk groups: group 1 (n = 20) with low expressions/values of EGFR (≤15%), CK5/6 (≤50%), Ki-67 (≤50%), pT (≤ 3), and pN (≤ 1), and group 2 (n = 30) with low expressions of EGFR and CK5/6, and any high expressions/values of Ki-67, pT, and pN. Two high basal risk groups: group 3 (n = 106) with single high basal expression (EGFR > 15% or CK5/6 > 50%), and group 4 (n = 36) with double high basal expressions (EGFR > 15% and CK5/6 > 50%).
Figure Legend Snippet: Kaplan Meier curves of disease free survival of risk groups for TNBC patients. The patients were stratified into different risk groups. Two low basal (EGFR≤15% and CK5/6≤50%) risk groups: group 1 (n = 20) with low expressions/values of EGFR (≤15%), CK5/6 (≤50%), Ki-67 (≤50%), pT (≤ 3), and pN (≤ 1), and group 2 (n = 30) with low expressions of EGFR and CK5/6, and any high expressions/values of Ki-67, pT, and pN. Two high basal risk groups: group 3 (n = 106) with single high basal expression (EGFR > 15% or CK5/6 > 50%), and group 4 (n = 36) with double high basal expressions (EGFR > 15% and CK5/6 > 50%).

Techniques Used: Expressing

Immunoreactivity of 69-yr-old patient with T1bN0M0 TNBC. (A) Hematoxylin and Eosin (H E), (B) EGFR ( > 70%), (C) CK5/6 ( > 70%), (D) ER, (E) PR, (F) HER2, (G) Ki-67 ( > 60%), and (H) P53 ( > 90%). The patient was undergone BCT, had 3.8 event free survival and 22.8 months overall survival.
Figure Legend Snippet: Immunoreactivity of 69-yr-old patient with T1bN0M0 TNBC. (A) Hematoxylin and Eosin (H E), (B) EGFR ( > 70%), (C) CK5/6 ( > 70%), (D) ER, (E) PR, (F) HER2, (G) Ki-67 ( > 60%), and (H) P53 ( > 90%). The patient was undergone BCT, had 3.8 event free survival and 22.8 months overall survival.

Techniques Used:

Kaplan Meier curves of disease free survival for basal biomarkers. (a) EGFR at cut-off level 15% with log-rank p = 0.0016, (b) time-dependent ROC analysis of EGFR with AUC = 0.723, where cutoff point 15% (red circled), (c) CK5/6 at cut-off level 50% with log-rank p = 0.0066, and (d) the significance of survival difference at different cutoff values for EGFR and CK5/6. The most significant cutoff values were red-circled with 15% for EGFR and 50% for CK5/6.
Figure Legend Snippet: Kaplan Meier curves of disease free survival for basal biomarkers. (a) EGFR at cut-off level 15% with log-rank p = 0.0016, (b) time-dependent ROC analysis of EGFR with AUC = 0.723, where cutoff point 15% (red circled), (c) CK5/6 at cut-off level 50% with log-rank p = 0.0066, and (d) the significance of survival difference at different cutoff values for EGFR and CK5/6. The most significant cutoff values were red-circled with 15% for EGFR and 50% for CK5/6.

Techniques Used:

21) Product Images from "Dermcidin exerts its oncogenic effects in breast cancer via modulation of ERBB signaling"

Article Title: Dermcidin exerts its oncogenic effects in breast cancer via modulation of ERBB signaling

Journal: BMC Cancer

doi: 10.1186/s12885-015-1022-6

The effect of DCD levels on global gene expression profiles. A , Heatmap depicting relatedness of gene expression profiles of control and DCD shRNA expressing cells. Hierarchical clustering was applied to Microarray data and selected portions of the clustering heat map are shown here. Each row represents a probe and the official symbol of gene is shown. Red and green indicate high and low gene expression levels, respectively. B , Gene ontology biological process categories highly represented in DCD shRNA expressing cells. Categories with an enrichment score > 2 using the DAVID Functional Annotation Tool are plotted. C , Development_ERBB signaling canonical pathway map generated by MetaCore. Blue thermometers in Betacellulin, amphiregulin, EGFR and c-Myc indicate fold-change in expression levels in each of the three different DCD shRNA expressing cell pools compared to control pLKO. See Additional file 5 for network legend.
Figure Legend Snippet: The effect of DCD levels on global gene expression profiles. A , Heatmap depicting relatedness of gene expression profiles of control and DCD shRNA expressing cells. Hierarchical clustering was applied to Microarray data and selected portions of the clustering heat map are shown here. Each row represents a probe and the official symbol of gene is shown. Red and green indicate high and low gene expression levels, respectively. B , Gene ontology biological process categories highly represented in DCD shRNA expressing cells. Categories with an enrichment score > 2 using the DAVID Functional Annotation Tool are plotted. C , Development_ERBB signaling canonical pathway map generated by MetaCore. Blue thermometers in Betacellulin, amphiregulin, EGFR and c-Myc indicate fold-change in expression levels in each of the three different DCD shRNA expressing cell pools compared to control pLKO. See Additional file 5 for network legend.

Techniques Used: Expressing, shRNA, Microarray, Functional Assay, Generated

Signaling pathways modulated by DCD. A , Expression of ERBB family of receptors and their ligands (B) in control and DCD shRNA-expressing cells. Y-axis indicates mean ± SD mRNA levels (normalized to HPRT) relative to pLKO controls. C , Proliferation-inducing activity of highly purified recombinant human DCD in MDA-MB-361 based on BrdU incorporation assay. Columns indicate mean of absorbance units, bars, SD. Real time PCR analyses of EGFR (D) , C-myc (E) , ERBB ligands (F) and ERBB receptors (G) at the indicated doses of rhDCD. Each sample was analyzed in three independent experiments performed in duplicates. * and ** denotes P
Figure Legend Snippet: Signaling pathways modulated by DCD. A , Expression of ERBB family of receptors and their ligands (B) in control and DCD shRNA-expressing cells. Y-axis indicates mean ± SD mRNA levels (normalized to HPRT) relative to pLKO controls. C , Proliferation-inducing activity of highly purified recombinant human DCD in MDA-MB-361 based on BrdU incorporation assay. Columns indicate mean of absorbance units, bars, SD. Real time PCR analyses of EGFR (D) , C-myc (E) , ERBB ligands (F) and ERBB receptors (G) at the indicated doses of rhDCD. Each sample was analyzed in three independent experiments performed in duplicates. * and ** denotes P

Techniques Used: Expressing, shRNA, Activity Assay, Purification, Recombinant, Multiple Displacement Amplification, BrdU Incorporation Assay, Real-time Polymerase Chain Reaction

22) Product Images from "Prognostic value of expression of molecular markers in adenoid cystic cancer of the salivary glands compared with lymph node metastasis: a retrospective study"

Article Title: Prognostic value of expression of molecular markers in adenoid cystic cancer of the salivary glands compared with lymph node metastasis: a retrospective study

Journal: World Journal of Surgical Oncology

doi: 10.1186/1477-7819-10-266

Immunohistochemical staining of adenoid cystic carcinomas of the salivary gland. C-kit (CD 117) expression of weakly (1+, A ), moderately (2+, B ), and strongly positive (3+, C ) cases; EGFR (epidermal growth factor receptor) expression of weakly (D) and moderately positive (E) cases; VEGF (vascular endothelial growth factor) expression of weakly (F) , moderately (G) , and strongly positive (H) cases. Expressions of c-kit (membranous/cytoplasmic), EGFR (membranous), and VEGF (cytoplasmic) were scored as follows: 0, reactivity in
Figure Legend Snippet: Immunohistochemical staining of adenoid cystic carcinomas of the salivary gland. C-kit (CD 117) expression of weakly (1+, A ), moderately (2+, B ), and strongly positive (3+, C ) cases; EGFR (epidermal growth factor receptor) expression of weakly (D) and moderately positive (E) cases; VEGF (vascular endothelial growth factor) expression of weakly (F) , moderately (G) , and strongly positive (H) cases. Expressions of c-kit (membranous/cytoplasmic), EGFR (membranous), and VEGF (cytoplasmic) were scored as follows: 0, reactivity in

Techniques Used: Immunohistochemistry, Staining, Expressing

23) Product Images from "The Prognostic Value of Ki-67, p53, EGFR, 1p36, 9p21, 10q23, and 17p13 in Skull Base Chordomas"

Article Title: The Prognostic Value of Ki-67, p53, EGFR, 1p36, 9p21, 10q23, and 17p13 in Skull Base Chordomas

Journal: Archives of pathology & laboratory medicine

doi: 10.1043/2009-0380-OA.1

Histologic and immunohistochemical characterization of skull base chordomas. H E stains showed both conventional (A, B) and chondroid (C) histologic subtypes of chordomas in the cohort. 32% had a prominent solid component (B), 32% showed elevated Ki67 PI (D), and 44% showed increased p53 accumulation (E). EGFR expression, in contrast, was seen in only 8% of cases (F).
Figure Legend Snippet: Histologic and immunohistochemical characterization of skull base chordomas. H E stains showed both conventional (A, B) and chondroid (C) histologic subtypes of chordomas in the cohort. 32% had a prominent solid component (B), 32% showed elevated Ki67 PI (D), and 44% showed increased p53 accumulation (E). EGFR expression, in contrast, was seen in only 8% of cases (F).

Techniques Used: Immunohistochemistry, Expressing

24) Product Images from "Frequent activation of EGFR in advanced chordomas"

Article Title: Frequent activation of EGFR in advanced chordomas

Journal: Clinical Sarcoma Research

doi: 10.1186/2045-3329-1-4

Representative images from phospho-RTK (left panel) and phospho-kinase (right panel) arrays from chordoma cases 18, 17b, 20 and 21 . The EGFR and EPHB2 TK are frequently activated and downstream RTK signaling intermediates are activated consistently in chordomas. Each kinase is spotted in duplicate. The pairs of dots in each corner are positive controls. Each pair of the most positive kinase dots is denoted by a numeral, with the identity of the corresponding kinases listed as follows: 1) EGFR, 2) CSF1R, 3) MSPR, 4) PDGFRB, 5) FGFR3, 6) EPHB2, 7) HER2, 8) TOR, 9) AKT, 10) TP53, 11) RSK1/2/3, 12) S6K, 13) CREB, 14) YES, 15) MSK1/2, 16) RSK1/2, 17) eNOS.
Figure Legend Snippet: Representative images from phospho-RTK (left panel) and phospho-kinase (right panel) arrays from chordoma cases 18, 17b, 20 and 21 . The EGFR and EPHB2 TK are frequently activated and downstream RTK signaling intermediates are activated consistently in chordomas. Each kinase is spotted in duplicate. The pairs of dots in each corner are positive controls. Each pair of the most positive kinase dots is denoted by a numeral, with the identity of the corresponding kinases listed as follows: 1) EGFR, 2) CSF1R, 3) MSPR, 4) PDGFRB, 5) FGFR3, 6) EPHB2, 7) HER2, 8) TOR, 9) AKT, 10) TP53, 11) RSK1/2/3, 12) S6K, 13) CREB, 14) YES, 15) MSK1/2, 16) RSK1/2, 17) eNOS.

Techniques Used:

25) Product Images from "Identification of EGFR expression status association with metastatic lymph node density (ND) by expression microarray analysis of advanced gastric cancer"

Article Title: Identification of EGFR expression status association with metastatic lymph node density (ND) by expression microarray analysis of advanced gastric cancer

Journal: Cancer Medicine

doi: 10.1002/cam4.311

High EGFR expression is a strongly associated with high ND. (A) Microscopic analysis of cell membrane EGFR immunohistochemical staining. EGFR expression was graded using a 3-point scale, where 1+ = light staining of more than 10% of the specimens, 2+ = moderate staining of more than 10% and less than or equal to 30% of the specimens, and 3+ = strong staining of more than 30% of the specimens. (B) Statistical analysis of EGFR expression using Student's t -test. EGFR 2+/3+ expression group included more patients with high ND than the EGFR 1+ group. The most suitable ND cutoff level was deemed ND of 35%. (c) Kaplan–Meier curves indicate that EGFR 2+/3+ expression was significantly associated with poor outcome in patients with 13th JGCA stage II/III disease ( P = 0.039). (D) ND ≥35 was significantly associated with poor outcome ( P = 0.0012).
Figure Legend Snippet: High EGFR expression is a strongly associated with high ND. (A) Microscopic analysis of cell membrane EGFR immunohistochemical staining. EGFR expression was graded using a 3-point scale, where 1+ = light staining of more than 10% of the specimens, 2+ = moderate staining of more than 10% and less than or equal to 30% of the specimens, and 3+ = strong staining of more than 30% of the specimens. (B) Statistical analysis of EGFR expression using Student's t -test. EGFR 2+/3+ expression group included more patients with high ND than the EGFR 1+ group. The most suitable ND cutoff level was deemed ND of 35%. (c) Kaplan–Meier curves indicate that EGFR 2+/3+ expression was significantly associated with poor outcome in patients with 13th JGCA stage II/III disease ( P = 0.039). (D) ND ≥35 was significantly associated with poor outcome ( P = 0.0012).

Techniques Used: Expressing, Immunohistochemistry, Staining

26) Product Images from "Stratification of Prognosis of Triple-Negative Breast Cancer Patients Using Combinatorial Biomarkers"

Article Title: Stratification of Prognosis of Triple-Negative Breast Cancer Patients Using Combinatorial Biomarkers

Journal: PLoS ONE

doi: 10.1371/journal.pone.0149661

Kaplan Meier curves of disease free survival of risk groups for TNBC patients. The patients were stratified into different risk groups. Two low basal (EGFR≤15% and CK5/6≤50%) risk groups: group 1 (n = 20) with low expressions/values of EGFR (≤15%), CK5/6 (≤50%), Ki-67 (≤50%), pT (≤ 3), and pN (≤ 1), and group 2 (n = 30) with low expressions of EGFR and CK5/6, and any high expressions/values of Ki-67, pT, and pN. Two high basal risk groups: group 3 (n = 106) with single high basal expression (EGFR > 15% or CK5/6 > 50%), and group 4 (n = 36) with double high basal expressions (EGFR > 15% and CK5/6 > 50%).
Figure Legend Snippet: Kaplan Meier curves of disease free survival of risk groups for TNBC patients. The patients were stratified into different risk groups. Two low basal (EGFR≤15% and CK5/6≤50%) risk groups: group 1 (n = 20) with low expressions/values of EGFR (≤15%), CK5/6 (≤50%), Ki-67 (≤50%), pT (≤ 3), and pN (≤ 1), and group 2 (n = 30) with low expressions of EGFR and CK5/6, and any high expressions/values of Ki-67, pT, and pN. Two high basal risk groups: group 3 (n = 106) with single high basal expression (EGFR > 15% or CK5/6 > 50%), and group 4 (n = 36) with double high basal expressions (EGFR > 15% and CK5/6 > 50%).

Techniques Used: Expressing

Immunoreactivity of 69-yr-old patient with T1bN0M0 TNBC. (A) Hematoxylin and Eosin (H E), (B) EGFR ( > 70%), (C) CK5/6 ( > 70%), (D) ER, (E) PR, (F) HER2, (G) Ki-67 ( > 60%), and (H) P53 ( > 90%). The patient was undergone BCT, had 3.8 event free survival and 22.8 months overall survival.
Figure Legend Snippet: Immunoreactivity of 69-yr-old patient with T1bN0M0 TNBC. (A) Hematoxylin and Eosin (H E), (B) EGFR ( > 70%), (C) CK5/6 ( > 70%), (D) ER, (E) PR, (F) HER2, (G) Ki-67 ( > 60%), and (H) P53 ( > 90%). The patient was undergone BCT, had 3.8 event free survival and 22.8 months overall survival.

Techniques Used:

Kaplan Meier curves of disease free survival for basal biomarkers. (a) EGFR at cut-off level 15% with log-rank p = 0.0016, (b) time-dependent ROC analysis of EGFR with AUC = 0.723, where cutoff point 15% (red circled), (c) CK5/6 at cut-off level 50% with log-rank p = 0.0066, and (d) the significance of survival difference at different cutoff values for EGFR and CK5/6. The most significant cutoff values were red-circled with 15% for EGFR and 50% for CK5/6.
Figure Legend Snippet: Kaplan Meier curves of disease free survival for basal biomarkers. (a) EGFR at cut-off level 15% with log-rank p = 0.0016, (b) time-dependent ROC analysis of EGFR with AUC = 0.723, where cutoff point 15% (red circled), (c) CK5/6 at cut-off level 50% with log-rank p = 0.0066, and (d) the significance of survival difference at different cutoff values for EGFR and CK5/6. The most significant cutoff values were red-circled with 15% for EGFR and 50% for CK5/6.

Techniques Used:

27) Product Images from "Safety and efficacy of afatinib as add-on to standard therapy of gemcitabine/cisplatin in chemotherapy-naive patients with advanced biliary tract cancer: an open-label, phase I trial with an extensive biomarker program"

Article Title: Safety and efficacy of afatinib as add-on to standard therapy of gemcitabine/cisplatin in chemotherapy-naive patients with advanced biliary tract cancer: an open-label, phase I trial with an extensive biomarker program

Journal: BMC Cancer

doi: 10.1186/s12885-018-5223-7

Immunohistochemical staining, a ) negative control, positive staining of b ) EGFR (3+), c ) HER2neu (1+), d ) HER3 (2+) and e ) HER4 (only sporadic)
Figure Legend Snippet: Immunohistochemical staining, a ) negative control, positive staining of b ) EGFR (3+), c ) HER2neu (1+), d ) HER3 (2+) and e ) HER4 (only sporadic)

Techniques Used: Immunohistochemistry, Staining, Negative Control

Related Articles

Staining:

Article Title: Establishment and characterization of three stable Basal/HER2-positive breast cancer cell lines derived from Chinese breast carcinoma with identical missense mutations in the DNA-binding domain of TP53
Article Snippet: .. The specimen were cut into 4-μm-thick sections, that were mounted on poly- l -lysine-coated slides, dried overnight at 37 °C, deparaffinized in xylene, rehydrated through a graded series of alcohol, and stained with H & E. Immunohistochemical analysis was performed using a standard avidin–biotin technique after a microwave antigen retrieval step in which slides were heated in a pressure cooker containing 10 mM sodium citrate (pH 6.5) for 10 min. Endogenous peroxidase was deactivated by treatment with 3% H2 O2 for 5 min, blocked with 10% normal goat serum for 30 min at room temperature, and probed overnight at 4 °C with primary antibodies against ER-α, PR, HER2, E-cadherin, CK5/6, EGFR, p120, and Ki-67 (Dako, Carpinteria, CA, USA). .. The following day, slides were incubated with poly-HRP secondary antibodies for 1 h in the dark at room temperature.

Article Title: Phosphorylated AKT1 is associated with poor prognosis in esophageal squamous cell carcinoma
Article Snippet: .. IHC staining was performed on 4-μm paraffin-embedded sections from TMA blocks by the standard Envision method using a panel of antibodies: EGFR (113, dilution 1:50; Dako), AKT1 (C73H10, dilution 3 μg/ml; Cell Signaling), AKT2 (302501, dilution 25 μg/ml; R & D), ERK1 (Y72, dilution 1:100; Abcam), ERK2 (E460, dilution 1:250; Abcam), STAT3 (E121-21, dilution 1:50; Abcam), phosphorylated-EGFR (p-EGFR) (Tyr1068) (EP774Y, dilution 1:250; Abcam), phosphorylated-AKT1 (p-AKT1) (Ser473) (EP2109Y, dilution 1:100; Abcam), phosphorylated-AKT2 (p-AKT2) (Ser474) (D3H2, dilution 1:100; Cell Signaling), phosphorylated-ERK1/2 (p-ERK1/2) (MAPK-YT, dilution 1:100; Abcam), and phosphorylated- STAT3 (p-STAT3) (EP2147Y, dilution 1:250; Abcam) (Fig. ). .. IHC scoring A modified semiquantitative method H-score was used to evaluate IHC staining [ , ].

Article Title: Establishment and characterization of three stable Basal/HER2-positive breast cancer cell lines derived from Chinese breast carcinoma with identical missense mutations in the DNA-binding domain of TP53
Article Snippet: .. The specimen were cut into 4-μm-thick sections, that were mounted on poly-l -lysine-coated slides, dried overnight at 37 °C, deparaffinized in xylene, rehydrated through a graded series of alcohol, and stained with H & E. Immunohistochemical analysis was performed using a standard avidin–biotin technique after a microwave antigen retrieval step in which slides were heated in a pressure cooker containing 10 mM sodium citrate (pH 6.5) for 10 min. Endogenous peroxidase was deactivated by treatment with 3% H2 O2 for 5 min, blocked with 10% normal goat serum for 30 min at room temperature, and probed overnight at 4 °C with primary antibodies against ER-α, PR, HER2, E-cadherin, CK5/6, EGFR, p120, and Ki-67 (Dako, Carpinteria, CA, USA). .. The following day, slides were incubated with poly-HRP secondary antibodies for 1 h in the dark at room temperature.

Immunohistochemistry:

Article Title: Establishment and characterization of three stable Basal/HER2-positive breast cancer cell lines derived from Chinese breast carcinoma with identical missense mutations in the DNA-binding domain of TP53
Article Snippet: .. The specimen were cut into 4-μm-thick sections, that were mounted on poly- l -lysine-coated slides, dried overnight at 37 °C, deparaffinized in xylene, rehydrated through a graded series of alcohol, and stained with H & E. Immunohistochemical analysis was performed using a standard avidin–biotin technique after a microwave antigen retrieval step in which slides were heated in a pressure cooker containing 10 mM sodium citrate (pH 6.5) for 10 min. Endogenous peroxidase was deactivated by treatment with 3% H2 O2 for 5 min, blocked with 10% normal goat serum for 30 min at room temperature, and probed overnight at 4 °C with primary antibodies against ER-α, PR, HER2, E-cadherin, CK5/6, EGFR, p120, and Ki-67 (Dako, Carpinteria, CA, USA). .. The following day, slides were incubated with poly-HRP secondary antibodies for 1 h in the dark at room temperature.

Article Title: Phosphorylated AKT1 is associated with poor prognosis in esophageal squamous cell carcinoma
Article Snippet: .. IHC staining was performed on 4-μm paraffin-embedded sections from TMA blocks by the standard Envision method using a panel of antibodies: EGFR (113, dilution 1:50; Dako), AKT1 (C73H10, dilution 3 μg/ml; Cell Signaling), AKT2 (302501, dilution 25 μg/ml; R & D), ERK1 (Y72, dilution 1:100; Abcam), ERK2 (E460, dilution 1:250; Abcam), STAT3 (E121-21, dilution 1:50; Abcam), phosphorylated-EGFR (p-EGFR) (Tyr1068) (EP774Y, dilution 1:250; Abcam), phosphorylated-AKT1 (p-AKT1) (Ser473) (EP2109Y, dilution 1:100; Abcam), phosphorylated-AKT2 (p-AKT2) (Ser474) (D3H2, dilution 1:100; Cell Signaling), phosphorylated-ERK1/2 (p-ERK1/2) (MAPK-YT, dilution 1:100; Abcam), and phosphorylated- STAT3 (p-STAT3) (EP2147Y, dilution 1:250; Abcam) (Fig. ). .. IHC scoring A modified semiquantitative method H-score was used to evaluate IHC staining [ , ].

Article Title: Establishment and characterization of three stable Basal/HER2-positive breast cancer cell lines derived from Chinese breast carcinoma with identical missense mutations in the DNA-binding domain of TP53
Article Snippet: .. The specimen were cut into 4-μm-thick sections, that were mounted on poly-l -lysine-coated slides, dried overnight at 37 °C, deparaffinized in xylene, rehydrated through a graded series of alcohol, and stained with H & E. Immunohistochemical analysis was performed using a standard avidin–biotin technique after a microwave antigen retrieval step in which slides were heated in a pressure cooker containing 10 mM sodium citrate (pH 6.5) for 10 min. Endogenous peroxidase was deactivated by treatment with 3% H2 O2 for 5 min, blocked with 10% normal goat serum for 30 min at room temperature, and probed overnight at 4 °C with primary antibodies against ER-α, PR, HER2, E-cadherin, CK5/6, EGFR, p120, and Ki-67 (Dako, Carpinteria, CA, USA). .. The following day, slides were incubated with poly-HRP secondary antibodies for 1 h in the dark at room temperature.

Avidin-Biotin Assay:

Article Title: Establishment and characterization of three stable Basal/HER2-positive breast cancer cell lines derived from Chinese breast carcinoma with identical missense mutations in the DNA-binding domain of TP53
Article Snippet: .. The specimen were cut into 4-μm-thick sections, that were mounted on poly- l -lysine-coated slides, dried overnight at 37 °C, deparaffinized in xylene, rehydrated through a graded series of alcohol, and stained with H & E. Immunohistochemical analysis was performed using a standard avidin–biotin technique after a microwave antigen retrieval step in which slides were heated in a pressure cooker containing 10 mM sodium citrate (pH 6.5) for 10 min. Endogenous peroxidase was deactivated by treatment with 3% H2 O2 for 5 min, blocked with 10% normal goat serum for 30 min at room temperature, and probed overnight at 4 °C with primary antibodies against ER-α, PR, HER2, E-cadherin, CK5/6, EGFR, p120, and Ki-67 (Dako, Carpinteria, CA, USA). .. The following day, slides were incubated with poly-HRP secondary antibodies for 1 h in the dark at room temperature.

Article Title: Establishment and characterization of three stable Basal/HER2-positive breast cancer cell lines derived from Chinese breast carcinoma with identical missense mutations in the DNA-binding domain of TP53
Article Snippet: .. The specimen were cut into 4-μm-thick sections, that were mounted on poly-l -lysine-coated slides, dried overnight at 37 °C, deparaffinized in xylene, rehydrated through a graded series of alcohol, and stained with H & E. Immunohistochemical analysis was performed using a standard avidin–biotin technique after a microwave antigen retrieval step in which slides were heated in a pressure cooker containing 10 mM sodium citrate (pH 6.5) for 10 min. Endogenous peroxidase was deactivated by treatment with 3% H2 O2 for 5 min, blocked with 10% normal goat serum for 30 min at room temperature, and probed overnight at 4 °C with primary antibodies against ER-α, PR, HER2, E-cadherin, CK5/6, EGFR, p120, and Ki-67 (Dako, Carpinteria, CA, USA). .. The following day, slides were incubated with poly-HRP secondary antibodies for 1 h in the dark at room temperature.

Incubation:

Article Title: Radioresistance of human glioma spheroids and expression of HSP70, p53 and EGFr
Article Snippet: .. The respective primary antibodies p53, Hsp70, EGFr and phospho-Akt (Dako, Carpinteria, CA, USA) were applied, and the slides incubated for 30 min at 37°C and overnight at 4°C in a humidity chamber. .. Subsequently, slides were incubated with biotinylated secondary antibody (Vector, CA, USA) for 30 min. After incubation with VECTASTAIN® ABC Reagent for 30 min, peroxidase activity was developed with DAB Substrate-Chromogen System (Merck, WS, NJ, USA) identifying bound antibody.

Article Title: A comprehensive morphological study for basal-like breast carcinomas with comparison to nonbasal-like carcinomas
Article Snippet: .. Sections were incubated with primary antibody solutions for CK5/6 (monoclonal mouse anti-human, clone D5/16 B4, Dako, Denmark), EGFR (monoclonal mouse anti-human, cloneE30, Dako), CK14 (monoclonal mouse anti-human, clone SPM 263, Spring bioscience, CA, USA) and vimentin (monoclonal mouse anti-human, cloneV9, Dako, Denmark) at a dilution of 1:100 with PBS for 1 hour at room temperature. .. After washing with PBS, they were incubated with secondary antibody (multispecies ultra streptavidine detection system-HRP, Zymed, Massachusetts, USA) and streptavidin-biotin complex (Zymed, Massachusetts, USA) for 20 minutes at room temperature.

Article Title: Immunohistochemical Classification of Primary and Secondary Glioblastomas
Article Snippet: .. Sections were then incubated with combinations of EGFR (1:150, Dako, Camarillo, CA, USA), p53 (1:1,000, Dako, Glostrup, Denmark), and/or IDH-1 (1:100, DIANOVA, Hamburg, Germany) antibodies. .. Immunohistochemical stains for EGFR were graded as follows: 0 (no cell stained), 1+ ( < 5% tumor cells stained), 2+ (5-50% cells stained), and 3+ ( > 50% cells stained).

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  • 88
    Agilent technologies relative egfr
    <t>HGF/MET</t> pathway expression and activation in recurrent/metastatic HNSCC. a Representative pictures showing a range of <t>EGFR,</t> MET, p-MET, and HGF expression levels observed by IHC in human HNSCC. b MET identification in FFPE tissue samples by SISH. Left panel detail of a MET -amplified case (clusters of black dots are seen in the nuclei of tumoral cells, representing several copies of the MET locus; as opposed to just two red dots per cell, corresponding to the centromeric region on chromosome 7). Right panel non-amplified sample. c Protein expression levels and gene amplification in the complete series. Case numbers are ordered from test (#1–33) to controls (#34–57), and a dashed line has been drawn in between the two groups. The color intensity of the boxes is indicative of abundance level in each column, either protein level by IHC (EGRF, MET, p-MET, HGF) or gene amplification level by SISH ( MET gene). Expression levels are indicated in a color gradient, from white (minimum) to red (maximum), with missing data in gray
    Relative Egfr, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Agilent technologies α egfr tm
    Development of the novel nb-based <t>α-EGFR</t> TM. (A) Two α-EGFR TM constructs (A I, α-EGFR TM (eu); A II, α-EGFR TM (pro)) were cloned for expression either in CHO cells (α-EGFR TM (eu)) or in E. coli (α-EGFR TM (pro)). As schematically shown, both nb-based α-EGFR TM constructs consist of the open reading frame encoding the EGFR-specific nb. For binding to the UniCAR the E5B9-tag is fused to the C-terminus. Furthermore, both TMs are tagged with 6xhis residues at the C-terminus for protein purification and detection. To enable eukaryotic expression, the α-EGFR TM (eu) construct additionally contains an N-terminal signal peptide (SP). To facilitate the interaction of UniCAR T cells with the TM the E5B9 tag was N- and C-terminally flanked with a glycine (4x)-serine (1x) linker (G 4 S). (B) The elution fraction of the purified α-EGFR TM (eu) (lane 1) and α-EGFR TM (pro) (lane 2) was separated via SDS-PAGE and subsequently stained with Coomassie brilliant blue G-250 (BI) or transferred onto a nitrocellulose membrane for detection of the purified α-EGFR TM (eu) (lane 1) and α-EGFR TM (pro) (lane 2) via its C-terminal his-tag (BII). M, molecular weight marker. (C) Both TMs were further analyzed by size exclusion HPLC using either 15 µL of the α-EGFR TM (eu) (10 µg) or 7.5 µL of the α-EGFR TM (pro) (15 µg).
    α Egfr Tm, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Agilent technologies anti human egfr polyclonal antibodies
    Comparison of frequency shifts observed when (A) 1 ng/mL and (B) 4 ng/mL of PAb peptide were administered to a freshly prepared <t>EGFR</t> ECD conjugated PEMS (empty squares) and a regenerated PEMS (filled squares). For both concentrations, the regenerated PEMS yields a frequency shift that was similar to the freshly prepared PEMS with the recovery rate described in text. (C) Comparison of frequency shifts resulting from the binding of 4 ng/mL of specific PAb peptide anti-EGFR <t>polyclonal</t> antibodies (filled squares) and 4 ng/mL of non-binding, non-reactive polyclonal antibodies (empty squares) to a regenerated EGFR ECD conjugated PEMS. The injection of non-binding antibody yielded a minimal frequency shift, suggesting that non-specific binding was not a major factor.
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    80
    biolegend egfr quantification
    Killing of <t>EGFR</t> low -expressing tumor cells by UniCAR T cells redirected by αEGFR TMs. The estimated number of EGFR molecules per A431 or MDA-MB-435S tumor cell was evaluated using the <t>QIFIKIT</t> ® ( A ). MDA-MB-435S tumor cells were stained with αEGFR mAb and detected via goat-α-mouse IgG-Pacific Blue TM to determine EGFR surface expression by flow cytometry ( B ). Binding of indicated αEGFR TMs to the MDA-MB-435S target cell line was analyzed by staining and flow cytometry ( B ). Therefore, cells were incubated with αEGFR TM, the αE5B9 mAb and goat-α-mouse IgG-Pacific Blue TM ( B ). As a negative control, cells were stained with the detection Ab alone (white graphs) ( B ). Percentage (%) and median fluorescence intensity (MFI) of positively stained cells were shown ( B ). In a standard chromium release cytotoxicity assay, EGFR low -expressing MDA-MB-435S tumor cells were incubated with T cells genetically modified to express EGFP (vector control), non-signaling UniCAR stop or signaling UniCAR CD28/ζ in the presence or absence of indicated αEGFR TMs at an effector to target cell ratio of 5:1 ( C ). Mean specific lysis and SEM are shown for two individual donors in the presence of Hu scFv αEGFR TM and three individual donors in the presence of nb or Mu scFv αEGFR TM ( C ). In addition, MDA-MB-435S tumor cells were co-cultured together with UniCAR CD28/ζ T cells and increasing concentrations of the respective αEGFR TMs under the same conditions ( D ). Mean specific lysis and SEM are shown. Statistical analysis was performed by two-way ANOVA with Tukey’s multiple comparison test ( C ) or paired t -test for each specific concentration ( D ). ( # p
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    HGF/MET pathway expression and activation in recurrent/metastatic HNSCC. a Representative pictures showing a range of EGFR, MET, p-MET, and HGF expression levels observed by IHC in human HNSCC. b MET identification in FFPE tissue samples by SISH. Left panel detail of a MET -amplified case (clusters of black dots are seen in the nuclei of tumoral cells, representing several copies of the MET locus; as opposed to just two red dots per cell, corresponding to the centromeric region on chromosome 7). Right panel non-amplified sample. c Protein expression levels and gene amplification in the complete series. Case numbers are ordered from test (#1–33) to controls (#34–57), and a dashed line has been drawn in between the two groups. The color intensity of the boxes is indicative of abundance level in each column, either protein level by IHC (EGRF, MET, p-MET, HGF) or gene amplification level by SISH ( MET gene). Expression levels are indicated in a color gradient, from white (minimum) to red (maximum), with missing data in gray

    Journal: Journal of Translational Medicine

    Article Title: Activation of MET pathway predicts poor outcome to cetuximab in patients with recurrent or metastatic head and neck cancer

    doi: 10.1186/s12967-015-0633-7

    Figure Lengend Snippet: HGF/MET pathway expression and activation in recurrent/metastatic HNSCC. a Representative pictures showing a range of EGFR, MET, p-MET, and HGF expression levels observed by IHC in human HNSCC. b MET identification in FFPE tissue samples by SISH. Left panel detail of a MET -amplified case (clusters of black dots are seen in the nuclei of tumoral cells, representing several copies of the MET locus; as opposed to just two red dots per cell, corresponding to the centromeric region on chromosome 7). Right panel non-amplified sample. c Protein expression levels and gene amplification in the complete series. Case numbers are ordered from test (#1–33) to controls (#34–57), and a dashed line has been drawn in between the two groups. The color intensity of the boxes is indicative of abundance level in each column, either protein level by IHC (EGRF, MET, p-MET, HGF) or gene amplification level by SISH ( MET gene). Expression levels are indicated in a color gradient, from white (minimum) to red (maximum), with missing data in gray

    Article Snippet: Relative EGFR and HGF expression ratios were calculated using the Pfaffl method [ ] relative to the calibrator sample (MVP Human Breast Total RNA, Agilent Technologies, USA).

    Techniques: Expressing, Activation Assay, Immunohistochemistry, Formalin-fixed Paraffin-Embedded, Amplification

    Development of the novel nb-based α-EGFR TM. (A) Two α-EGFR TM constructs (A I, α-EGFR TM (eu); A II, α-EGFR TM (pro)) were cloned for expression either in CHO cells (α-EGFR TM (eu)) or in E. coli (α-EGFR TM (pro)). As schematically shown, both nb-based α-EGFR TM constructs consist of the open reading frame encoding the EGFR-specific nb. For binding to the UniCAR the E5B9-tag is fused to the C-terminus. Furthermore, both TMs are tagged with 6xhis residues at the C-terminus for protein purification and detection. To enable eukaryotic expression, the α-EGFR TM (eu) construct additionally contains an N-terminal signal peptide (SP). To facilitate the interaction of UniCAR T cells with the TM the E5B9 tag was N- and C-terminally flanked with a glycine (4x)-serine (1x) linker (G 4 S). (B) The elution fraction of the purified α-EGFR TM (eu) (lane 1) and α-EGFR TM (pro) (lane 2) was separated via SDS-PAGE and subsequently stained with Coomassie brilliant blue G-250 (BI) or transferred onto a nitrocellulose membrane for detection of the purified α-EGFR TM (eu) (lane 1) and α-EGFR TM (pro) (lane 2) via its C-terminal his-tag (BII). M, molecular weight marker. (C) Both TMs were further analyzed by size exclusion HPLC using either 15 µL of the α-EGFR TM (eu) (10 µg) or 7.5 µL of the α-EGFR TM (pro) (15 µg).

    Journal: Oncoimmunology

    Article Title: A novel nanobody-based target module for retargeting of T lymphocytes to EGFR-expressing cancer cells via the modular UniCAR platform

    doi: 10.1080/2162402X.2017.1287246

    Figure Lengend Snippet: Development of the novel nb-based α-EGFR TM. (A) Two α-EGFR TM constructs (A I, α-EGFR TM (eu); A II, α-EGFR TM (pro)) were cloned for expression either in CHO cells (α-EGFR TM (eu)) or in E. coli (α-EGFR TM (pro)). As schematically shown, both nb-based α-EGFR TM constructs consist of the open reading frame encoding the EGFR-specific nb. For binding to the UniCAR the E5B9-tag is fused to the C-terminus. Furthermore, both TMs are tagged with 6xhis residues at the C-terminus for protein purification and detection. To enable eukaryotic expression, the α-EGFR TM (eu) construct additionally contains an N-terminal signal peptide (SP). To facilitate the interaction of UniCAR T cells with the TM the E5B9 tag was N- and C-terminally flanked with a glycine (4x)-serine (1x) linker (G 4 S). (B) The elution fraction of the purified α-EGFR TM (eu) (lane 1) and α-EGFR TM (pro) (lane 2) was separated via SDS-PAGE and subsequently stained with Coomassie brilliant blue G-250 (BI) or transferred onto a nitrocellulose membrane for detection of the purified α-EGFR TM (eu) (lane 1) and α-EGFR TM (pro) (lane 2) via its C-terminal his-tag (BII). M, molecular weight marker. (C) Both TMs were further analyzed by size exclusion HPLC using either 15 µL of the α-EGFR TM (eu) (10 µg) or 7.5 µL of the α-EGFR TM (pro) (15 µg).

    Article Snippet: For size exclusion high-performance liquid chromatography (SE-HPLC) analysis 10-15 µL samples containing the respective α-EGFR TM were applied to a size exclusion column (Agilent Bio SEC-3 (3 µm, 150 A, 7.8 × 300 mm2 ) from Agilent Technologies, Böblingen, Germany).

    Techniques: Construct, Clone Assay, Expressing, Binding Assay, Protein Purification, Purification, SDS Page, Staining, Molecular Weight, Marker, High Performance Liquid Chromatography

    Biodistribution of 64 Cu- and 68 Ga-radiolabeled α-EGFR TM (pro). After conjugation of the α-EGFR TM (pro) with NODAGA the resulting α-EGFR TM (pro) (NODAGA) 1.2 was radiolabeled with either 64 Cu or 68 Ga. The biodistribution of the [ 64 Cu]Cu-α-EGFR TM (pro) (NODAGA) 1.2 complex is shown in (A and C). The biodistribution of the [ 68 Ga]Ga-α-EGFR TM (pro) (NODAGA) 1.2 complex is shown in (B and D). The biodistribution is given as percentage of the total activity of the injected dose (%ID) and the activity concentration (SUV) based on four A431-Luc tumor-bearing mice. (E) Target to background ratios including tumor to muscle-, and tumor to blood ratios.

    Journal: Oncoimmunology

    Article Title: A novel nanobody-based target module for retargeting of T lymphocytes to EGFR-expressing cancer cells via the modular UniCAR platform

    doi: 10.1080/2162402X.2017.1287246

    Figure Lengend Snippet: Biodistribution of 64 Cu- and 68 Ga-radiolabeled α-EGFR TM (pro). After conjugation of the α-EGFR TM (pro) with NODAGA the resulting α-EGFR TM (pro) (NODAGA) 1.2 was radiolabeled with either 64 Cu or 68 Ga. The biodistribution of the [ 64 Cu]Cu-α-EGFR TM (pro) (NODAGA) 1.2 complex is shown in (A and C). The biodistribution of the [ 68 Ga]Ga-α-EGFR TM (pro) (NODAGA) 1.2 complex is shown in (B and D). The biodistribution is given as percentage of the total activity of the injected dose (%ID) and the activity concentration (SUV) based on four A431-Luc tumor-bearing mice. (E) Target to background ratios including tumor to muscle-, and tumor to blood ratios.

    Article Snippet: For size exclusion high-performance liquid chromatography (SE-HPLC) analysis 10-15 µL samples containing the respective α-EGFR TM were applied to a size exclusion column (Agilent Bio SEC-3 (3 µm, 150 A, 7.8 × 300 mm2 ) from Agilent Technologies, Böblingen, Germany).

    Techniques: Conjugation Assay, Activity Assay, Injection, Concentration Assay, Mouse Assay

    Estimation of range of working concentrations and EC 50 values for the novel α-EGFR TMs. For a direct comparison of the capability to mediate a UniCAR-dependent lysis of EGFR-positive tumor cells A431 cells and UniCAR 28/ζ T cells were incubated with increasing amounts of (A) the α-EGFR TM (eu) or (B) the α-EGFR TM (pro). Chromium release was measured after 48 h of incubation. Data shown represent mean specific lysis and SD for 6 independent donors (*** p

    Journal: Oncoimmunology

    Article Title: A novel nanobody-based target module for retargeting of T lymphocytes to EGFR-expressing cancer cells via the modular UniCAR platform

    doi: 10.1080/2162402X.2017.1287246

    Figure Lengend Snippet: Estimation of range of working concentrations and EC 50 values for the novel α-EGFR TMs. For a direct comparison of the capability to mediate a UniCAR-dependent lysis of EGFR-positive tumor cells A431 cells and UniCAR 28/ζ T cells were incubated with increasing amounts of (A) the α-EGFR TM (eu) or (B) the α-EGFR TM (pro). Chromium release was measured after 48 h of incubation. Data shown represent mean specific lysis and SD for 6 independent donors (*** p

    Article Snippet: For size exclusion high-performance liquid chromatography (SE-HPLC) analysis 10-15 µL samples containing the respective α-EGFR TM were applied to a size exclusion column (Agilent Bio SEC-3 (3 µm, 150 A, 7.8 × 300 mm2 ) from Agilent Technologies, Böblingen, Germany).

    Techniques: Lysis, Incubation

    Time-activity curves (TAC) of regions of interest (ROI). The TAC curves are derived from PET studies of four A431-Luc tumor-bearing mice. The data points were collected over 20 h to 35 h after injection of the [ 64 Cu]Cu-α-EGFR TM (pro) (NODAGA) 1.2 . ( A ) TAC of the ROI over the heart representing primarily the blood activity concentration (SUV mean ) supporting the fast elimination of the TM with half-lifes of 4.2 min and 23.2 min of the fast and slow distribution and elimination phase, respectively. (B) TAC of the tumor activity concentration with a maximum at 22 min p.i. and the clearance with half-life of 3.7 h. (C) TAC of the total activity (%ID) in the tumor with 3.2% ID at the maximum. (D), (E) TAC of the tumor ratios to muscle and blood, respectively, showing the increasing image contrast up to 2 h. (F) accumulation of the 64 Cu-activity in the kidneys with a maximum of 46.4% ID after 2 h.

    Journal: Oncoimmunology

    Article Title: A novel nanobody-based target module for retargeting of T lymphocytes to EGFR-expressing cancer cells via the modular UniCAR platform

    doi: 10.1080/2162402X.2017.1287246

    Figure Lengend Snippet: Time-activity curves (TAC) of regions of interest (ROI). The TAC curves are derived from PET studies of four A431-Luc tumor-bearing mice. The data points were collected over 20 h to 35 h after injection of the [ 64 Cu]Cu-α-EGFR TM (pro) (NODAGA) 1.2 . ( A ) TAC of the ROI over the heart representing primarily the blood activity concentration (SUV mean ) supporting the fast elimination of the TM with half-lifes of 4.2 min and 23.2 min of the fast and slow distribution and elimination phase, respectively. (B) TAC of the tumor activity concentration with a maximum at 22 min p.i. and the clearance with half-life of 3.7 h. (C) TAC of the total activity (%ID) in the tumor with 3.2% ID at the maximum. (D), (E) TAC of the tumor ratios to muscle and blood, respectively, showing the increasing image contrast up to 2 h. (F) accumulation of the 64 Cu-activity in the kidneys with a maximum of 46.4% ID after 2 h.

    Article Snippet: For size exclusion high-performance liquid chromatography (SE-HPLC) analysis 10-15 µL samples containing the respective α-EGFR TM were applied to a size exclusion column (Agilent Bio SEC-3 (3 µm, 150 A, 7.8 × 300 mm2 ) from Agilent Technologies, Böblingen, Germany).

    Techniques: Activity Assay, Derivative Assay, Positron Emission Tomography, Mouse Assay, Injection, Concentration Assay

    Estimation of the K d values of the novel α-EGFR TMs. Increasing amounts of the α-EGFR TM (eu) (left panel) or α-EGFR TM (pro) (right panel) were used for staining of A431 cells. Binding was detected via the E5B9-tag using an α-E5B9 mAb and a PE-conjugated α-mouse-IgG mAb. The respective K d value was calculated from the resulting binding curve (see also Materials and methods ).

    Journal: Oncoimmunology

    Article Title: A novel nanobody-based target module for retargeting of T lymphocytes to EGFR-expressing cancer cells via the modular UniCAR platform

    doi: 10.1080/2162402X.2017.1287246

    Figure Lengend Snippet: Estimation of the K d values of the novel α-EGFR TMs. Increasing amounts of the α-EGFR TM (eu) (left panel) or α-EGFR TM (pro) (right panel) were used for staining of A431 cells. Binding was detected via the E5B9-tag using an α-E5B9 mAb and a PE-conjugated α-mouse-IgG mAb. The respective K d value was calculated from the resulting binding curve (see also Materials and methods ).

    Article Snippet: For size exclusion high-performance liquid chromatography (SE-HPLC) analysis 10-15 µL samples containing the respective α-EGFR TM were applied to a size exclusion column (Agilent Bio SEC-3 (3 µm, 150 A, 7.8 × 300 mm2 ) from Agilent Technologies, Böblingen, Germany).

    Techniques: Staining, Binding Assay

    Cytokine release from EGFR-redirected UniCAR T cells as estimated by either (A) a multiplex assay or (B) ELISA. (A and B) A431 cells were incubated in the presence of genetically engineered UniCAR 28/ζ T cells either in the absence (UniCAR CD28/ζ + A431, white bars) or presence of 50 nM of the α-EGFR TM (eu) (gray bars) or α-EGFR TM (pro) (black bars) at an e:t ratio of 5:1 for 48 h. Cytokines secreted into cell culture supernatants were estimated for three individual donors. As controls UniCAR T cells were co-cultivated with A431 cells in the absence of 50 nM TM as indicated in ( A ). As additional controls T cells were engrafted with the vector control or the UniCAR Stop construct and incubated in the presence of 50 nM TM as indicated in (B). ( A ) Using the MACSPlex Cytokine 12 Kit, variable amounts of the cytokines GM-CSF, IFNγ, IL-2, IL-4, IL-9, and TNF-α were detected (x, not detectable, n.d. not defined) but not the other cytokines IFN-α , IL-5, IL-6, IL-9, IL-10, IL-17A (Data not shown). (B) The concentration of selected cytokines including IFNγ (left), IL-2 (middle) and TNF (right) in co-culture supernatants was also estimated by ELISA. Mean cytokine concentrations and SD of triplicates for three independent donors are shown (x, not detectable).

    Journal: Oncoimmunology

    Article Title: A novel nanobody-based target module for retargeting of T lymphocytes to EGFR-expressing cancer cells via the modular UniCAR platform

    doi: 10.1080/2162402X.2017.1287246

    Figure Lengend Snippet: Cytokine release from EGFR-redirected UniCAR T cells as estimated by either (A) a multiplex assay or (B) ELISA. (A and B) A431 cells were incubated in the presence of genetically engineered UniCAR 28/ζ T cells either in the absence (UniCAR CD28/ζ + A431, white bars) or presence of 50 nM of the α-EGFR TM (eu) (gray bars) or α-EGFR TM (pro) (black bars) at an e:t ratio of 5:1 for 48 h. Cytokines secreted into cell culture supernatants were estimated for three individual donors. As controls UniCAR T cells were co-cultivated with A431 cells in the absence of 50 nM TM as indicated in ( A ). As additional controls T cells were engrafted with the vector control or the UniCAR Stop construct and incubated in the presence of 50 nM TM as indicated in (B). ( A ) Using the MACSPlex Cytokine 12 Kit, variable amounts of the cytokines GM-CSF, IFNγ, IL-2, IL-4, IL-9, and TNF-α were detected (x, not detectable, n.d. not defined) but not the other cytokines IFN-α , IL-5, IL-6, IL-9, IL-10, IL-17A (Data not shown). (B) The concentration of selected cytokines including IFNγ (left), IL-2 (middle) and TNF (right) in co-culture supernatants was also estimated by ELISA. Mean cytokine concentrations and SD of triplicates for three independent donors are shown (x, not detectable).

    Article Snippet: For size exclusion high-performance liquid chromatography (SE-HPLC) analysis 10-15 µL samples containing the respective α-EGFR TM were applied to a size exclusion column (Agilent Bio SEC-3 (3 µm, 150 A, 7.8 × 300 mm2 ) from Agilent Technologies, Böblingen, Germany).

    Techniques: Multiplex Assay, Enzyme-linked Immunosorbent Assay, Incubation, Cell Culture, Plasmid Preparation, Construct, Concentration Assay, Co-Culture Assay

    Evidence for disassembly of UniCAR-TM complexes in vivo . ( A ) Small animal PET/CT scans of mice taken 5 or 90 min after s.c. injection of [ 64 Cu]Cu-α-EGFR (pro) TM (NODAGA) 1.2 (left panel) or the TM pre-incubated with A431 cells (right panel) (ki, kidneys, li, liver, s.c. injected material). ( B ) Dynamic PET analysis of either free TM (TM) or TM pre-incubated with A431 cells (TM+A431) or TM pre-incubated with UniCAR T cells (TM+UniCAR) or TM pre-incubated with A431 cells and UniCAR T cells (TM+A431+UniCAR). ( C ) Based on the elimination curves half-lifes for free and complexed TMs were calculated.

    Journal: Oncoimmunology

    Article Title: A novel nanobody-based target module for retargeting of T lymphocytes to EGFR-expressing cancer cells via the modular UniCAR platform

    doi: 10.1080/2162402X.2017.1287246

    Figure Lengend Snippet: Evidence for disassembly of UniCAR-TM complexes in vivo . ( A ) Small animal PET/CT scans of mice taken 5 or 90 min after s.c. injection of [ 64 Cu]Cu-α-EGFR (pro) TM (NODAGA) 1.2 (left panel) or the TM pre-incubated with A431 cells (right panel) (ki, kidneys, li, liver, s.c. injected material). ( B ) Dynamic PET analysis of either free TM (TM) or TM pre-incubated with A431 cells (TM+A431) or TM pre-incubated with UniCAR T cells (TM+UniCAR) or TM pre-incubated with A431 cells and UniCAR T cells (TM+A431+UniCAR). ( C ) Based on the elimination curves half-lifes for free and complexed TMs were calculated.

    Article Snippet: For size exclusion high-performance liquid chromatography (SE-HPLC) analysis 10-15 µL samples containing the respective α-EGFR TM were applied to a size exclusion column (Agilent Bio SEC-3 (3 µm, 150 A, 7.8 × 300 mm2 ) from Agilent Technologies, Böblingen, Germany).

    Techniques: In Vivo, Positron Emission Tomography, Mouse Assay, Injection, Incubation

    Binding of the novel TMs to EGFR-expressing tumor cells. To analyze binding properties of the α-EGFR TMs, A431 and FaDu cells were stained with the respective TM (20 ng/μL). The specific binding was detected via the E5B9-tag using an α-E5B9 mAb and a PE-conjugated α-mouse-IgG mAb. As positive control, cells were labeled with mAb α-EGFR/PE-Cy7. The histograms show cells stained with either the control Ab (dark gray graphs, upper panel) or the α-EGFR TM (eu) (light gray graphs, lower panel) or α-EGFR TM (pro) (dark gray graphs, lower panel) and their respective controls (transparent graphs). The numbers represent the percentage of antigen-positive cells and mean fluorescence intensity (MFI) of stained cells.

    Journal: Oncoimmunology

    Article Title: A novel nanobody-based target module for retargeting of T lymphocytes to EGFR-expressing cancer cells via the modular UniCAR platform

    doi: 10.1080/2162402X.2017.1287246

    Figure Lengend Snippet: Binding of the novel TMs to EGFR-expressing tumor cells. To analyze binding properties of the α-EGFR TMs, A431 and FaDu cells were stained with the respective TM (20 ng/μL). The specific binding was detected via the E5B9-tag using an α-E5B9 mAb and a PE-conjugated α-mouse-IgG mAb. As positive control, cells were labeled with mAb α-EGFR/PE-Cy7. The histograms show cells stained with either the control Ab (dark gray graphs, upper panel) or the α-EGFR TM (eu) (light gray graphs, lower panel) or α-EGFR TM (pro) (dark gray graphs, lower panel) and their respective controls (transparent graphs). The numbers represent the percentage of antigen-positive cells and mean fluorescence intensity (MFI) of stained cells.

    Article Snippet: For size exclusion high-performance liquid chromatography (SE-HPLC) analysis 10-15 µL samples containing the respective α-EGFR TM were applied to a size exclusion column (Agilent Bio SEC-3 (3 µm, 150 A, 7.8 × 300 mm2 ) from Agilent Technologies, Böblingen, Germany).

    Techniques: Binding Assay, Expressing, Staining, Positive Control, Labeling, Fluorescence

    Evidence for disassembly of UniCAR-TM complexes in vitro . To analyze the stability of UniCAR-TM complexes T cells engrafted with the UniCAR CD28/ζ construct were pre-incubated with 50 nM α-EGFR TM (eu) (A) or α-EGFR TM (pro) (B) and washed several times as indicated before co-cultivation with A431 target cells. After washing standard chromium release assays were performed at an e:t ratio of 5:1. As controls non pre-incubated T cells were cultivated with A431 cells in the presence or absence of the respective TM. After 48 h chromium release assays were performed. As negative controls served T cells transduced with the vector control or the UniCAR Stop construct. Mean specific lysis and SD for three independent T cell donors are shown (* p

    Journal: Oncoimmunology

    Article Title: A novel nanobody-based target module for retargeting of T lymphocytes to EGFR-expressing cancer cells via the modular UniCAR platform

    doi: 10.1080/2162402X.2017.1287246

    Figure Lengend Snippet: Evidence for disassembly of UniCAR-TM complexes in vitro . To analyze the stability of UniCAR-TM complexes T cells engrafted with the UniCAR CD28/ζ construct were pre-incubated with 50 nM α-EGFR TM (eu) (A) or α-EGFR TM (pro) (B) and washed several times as indicated before co-cultivation with A431 target cells. After washing standard chromium release assays were performed at an e:t ratio of 5:1. As controls non pre-incubated T cells were cultivated with A431 cells in the presence or absence of the respective TM. After 48 h chromium release assays were performed. As negative controls served T cells transduced with the vector control or the UniCAR Stop construct. Mean specific lysis and SD for three independent T cell donors are shown (* p

    Article Snippet: For size exclusion high-performance liquid chromatography (SE-HPLC) analysis 10-15 µL samples containing the respective α-EGFR TM were applied to a size exclusion column (Agilent Bio SEC-3 (3 µm, 150 A, 7.8 × 300 mm2 ) from Agilent Technologies, Böblingen, Germany).

    Techniques: In Vitro, Construct, Incubation, Transduction, Plasmid Preparation, Lysis

    Retargeting of EGFR-positive tumor cells in experimental mice. A431 cells were transduced to express firefly luciferase resulting in A431-Luc cells. Per mouse, 1.5×10 6 A431-Luc cells were mixed with 1.5×10 6 UniCAR 28/ζ T cells and 100 µg of the α-EGFR TM (pro). As “untreated” control served 1.5×10 6 A431-Luc cells mixed with 1.5×10 6 UniCAR 28/ζ T cells without any TM. The respective mixture (100 µL) was injected subcutaneously into SCID/beige mice resulting in two groups of animals representing untreated (group A) or treated (group B) mice. Luminescence imaging of anesthetized mice was performed 10 min after i.p. injection of 200 µL of D-luciferin potassium salt (15 mg/mL) starting at day one (D1), and followed at day 2 (D2), and day 8 (D8).

    Journal: Oncoimmunology

    Article Title: A novel nanobody-based target module for retargeting of T lymphocytes to EGFR-expressing cancer cells via the modular UniCAR platform

    doi: 10.1080/2162402X.2017.1287246

    Figure Lengend Snippet: Retargeting of EGFR-positive tumor cells in experimental mice. A431 cells were transduced to express firefly luciferase resulting in A431-Luc cells. Per mouse, 1.5×10 6 A431-Luc cells were mixed with 1.5×10 6 UniCAR 28/ζ T cells and 100 µg of the α-EGFR TM (pro). As “untreated” control served 1.5×10 6 A431-Luc cells mixed with 1.5×10 6 UniCAR 28/ζ T cells without any TM. The respective mixture (100 µL) was injected subcutaneously into SCID/beige mice resulting in two groups of animals representing untreated (group A) or treated (group B) mice. Luminescence imaging of anesthetized mice was performed 10 min after i.p. injection of 200 µL of D-luciferin potassium salt (15 mg/mL) starting at day one (D1), and followed at day 2 (D2), and day 8 (D8).

    Article Snippet: For size exclusion high-performance liquid chromatography (SE-HPLC) analysis 10-15 µL samples containing the respective α-EGFR TM were applied to a size exclusion column (Agilent Bio SEC-3 (3 µm, 150 A, 7.8 × 300 mm2 ) from Agilent Technologies, Böblingen, Germany).

    Techniques: Mouse Assay, Luciferase, Injection, Imaging

    Target-specific lysis of EGFR-positive tumor cells via the novel UniCAR system. In standard chromium release assays (A) A431 or (B) FaDu cells were incubated with T cells engrafted with either the vector control encoding the EGFP marker protein (vector control), the UniCAR Stop construct lacking intracellular signaling domains (UniCAR Stop) or the α-E5B9 signaling construct (UniCAR 28/ζ). The A431 and FaDu cells were cultivated with the respective genetically engineered T cells in the presence or absence of 50 nM α-EGFR TM (eu) (A I, B I) or α-EGFR TM (pro) (A II, B II) for 48 h. Mean specific lysis and SD for seven (A431 and FaDu, e:t ratio of 5:1) or three (A431, e:t ratio of 1:1) independent T cell donors are shown (** p

    Journal: Oncoimmunology

    Article Title: A novel nanobody-based target module for retargeting of T lymphocytes to EGFR-expressing cancer cells via the modular UniCAR platform

    doi: 10.1080/2162402X.2017.1287246

    Figure Lengend Snippet: Target-specific lysis of EGFR-positive tumor cells via the novel UniCAR system. In standard chromium release assays (A) A431 or (B) FaDu cells were incubated with T cells engrafted with either the vector control encoding the EGFP marker protein (vector control), the UniCAR Stop construct lacking intracellular signaling domains (UniCAR Stop) or the α-E5B9 signaling construct (UniCAR 28/ζ). The A431 and FaDu cells were cultivated with the respective genetically engineered T cells in the presence or absence of 50 nM α-EGFR TM (eu) (A I, B I) or α-EGFR TM (pro) (A II, B II) for 48 h. Mean specific lysis and SD for seven (A431 and FaDu, e:t ratio of 5:1) or three (A431, e:t ratio of 1:1) independent T cell donors are shown (** p

    Article Snippet: For size exclusion high-performance liquid chromatography (SE-HPLC) analysis 10-15 µL samples containing the respective α-EGFR TM were applied to a size exclusion column (Agilent Bio SEC-3 (3 µm, 150 A, 7.8 × 300 mm2 ) from Agilent Technologies, Böblingen, Germany).

    Techniques: Lysis, Incubation, Plasmid Preparation, Marker, Construct

    MALDI-TOF MS analysis (A) and SDS-PAGE followed by autoradiography (B) of 64 Cu-radiolabeled α-EGFR TM (pro). (A) A mixture of non-modified and NODAGA modified TMs were analyzed with MALDI-TOF MS. The molecular mass of the unmodified α-EGFR TM (pro) was determined with 16676.611 (m/z). The mean of the molecular mass of the NODAGA conjugated TMs was estimated with 17486.367 (m/z), which yields an average of 1.2 NODAGA chelator groups per molecule of the α-EGFR TM (pro) (NODAGA) 1.2 . (B) After SDS-PAGE, the autoradiogram of the [ 64 Cu]Cu-α-EGFR TM (pro) (NODAGA) 1.2 shows a main fraction with a molecular mass of approximately 18 kDa.

    Journal: Oncoimmunology

    Article Title: A novel nanobody-based target module for retargeting of T lymphocytes to EGFR-expressing cancer cells via the modular UniCAR platform

    doi: 10.1080/2162402X.2017.1287246

    Figure Lengend Snippet: MALDI-TOF MS analysis (A) and SDS-PAGE followed by autoradiography (B) of 64 Cu-radiolabeled α-EGFR TM (pro). (A) A mixture of non-modified and NODAGA modified TMs were analyzed with MALDI-TOF MS. The molecular mass of the unmodified α-EGFR TM (pro) was determined with 16676.611 (m/z). The mean of the molecular mass of the NODAGA conjugated TMs was estimated with 17486.367 (m/z), which yields an average of 1.2 NODAGA chelator groups per molecule of the α-EGFR TM (pro) (NODAGA) 1.2 . (B) After SDS-PAGE, the autoradiogram of the [ 64 Cu]Cu-α-EGFR TM (pro) (NODAGA) 1.2 shows a main fraction with a molecular mass of approximately 18 kDa.

    Article Snippet: For size exclusion high-performance liquid chromatography (SE-HPLC) analysis 10-15 µL samples containing the respective α-EGFR TM were applied to a size exclusion column (Agilent Bio SEC-3 (3 µm, 150 A, 7.8 × 300 mm2 ) from Agilent Technologies, Böblingen, Germany).

    Techniques: Mass Spectrometry, SDS Page, Autoradiography, Modification

    Comparison of frequency shifts observed when (A) 1 ng/mL and (B) 4 ng/mL of PAb peptide were administered to a freshly prepared EGFR ECD conjugated PEMS (empty squares) and a regenerated PEMS (filled squares). For both concentrations, the regenerated PEMS yields a frequency shift that was similar to the freshly prepared PEMS with the recovery rate described in text. (C) Comparison of frequency shifts resulting from the binding of 4 ng/mL of specific PAb peptide anti-EGFR polyclonal antibodies (filled squares) and 4 ng/mL of non-binding, non-reactive polyclonal antibodies (empty squares) to a regenerated EGFR ECD conjugated PEMS. The injection of non-binding antibody yielded a minimal frequency shift, suggesting that non-specific binding was not a major factor.

    Journal: Sensors (Basel, Switzerland)

    Article Title: A Rapid Method to Regenerate Piezoelectric Microcantilever Sensors (PEMS)

    doi: 10.3390/s110505520

    Figure Lengend Snippet: Comparison of frequency shifts observed when (A) 1 ng/mL and (B) 4 ng/mL of PAb peptide were administered to a freshly prepared EGFR ECD conjugated PEMS (empty squares) and a regenerated PEMS (filled squares). For both concentrations, the regenerated PEMS yields a frequency shift that was similar to the freshly prepared PEMS with the recovery rate described in text. (C) Comparison of frequency shifts resulting from the binding of 4 ng/mL of specific PAb peptide anti-EGFR polyclonal antibodies (filled squares) and 4 ng/mL of non-binding, non-reactive polyclonal antibodies (empty squares) to a regenerated EGFR ECD conjugated PEMS. The injection of non-binding antibody yielded a minimal frequency shift, suggesting that non-specific binding was not a major factor.

    Article Snippet: Once a stable baseline was obtained for a period of at least 20 min, anti-human EGFR polyclonal antibodies diluted in PBS were injected into the system and the frequency shift was measured for 90 min with an electrical impedance analyzer (Agilent 4294A, Agilent).

    Techniques: Binding Assay, Injection

    Killing of EGFR low -expressing tumor cells by UniCAR T cells redirected by αEGFR TMs. The estimated number of EGFR molecules per A431 or MDA-MB-435S tumor cell was evaluated using the QIFIKIT ® ( A ). MDA-MB-435S tumor cells were stained with αEGFR mAb and detected via goat-α-mouse IgG-Pacific Blue TM to determine EGFR surface expression by flow cytometry ( B ). Binding of indicated αEGFR TMs to the MDA-MB-435S target cell line was analyzed by staining and flow cytometry ( B ). Therefore, cells were incubated with αEGFR TM, the αE5B9 mAb and goat-α-mouse IgG-Pacific Blue TM ( B ). As a negative control, cells were stained with the detection Ab alone (white graphs) ( B ). Percentage (%) and median fluorescence intensity (MFI) of positively stained cells were shown ( B ). In a standard chromium release cytotoxicity assay, EGFR low -expressing MDA-MB-435S tumor cells were incubated with T cells genetically modified to express EGFP (vector control), non-signaling UniCAR stop or signaling UniCAR CD28/ζ in the presence or absence of indicated αEGFR TMs at an effector to target cell ratio of 5:1 ( C ). Mean specific lysis and SEM are shown for two individual donors in the presence of Hu scFv αEGFR TM and three individual donors in the presence of nb or Mu scFv αEGFR TM ( C ). In addition, MDA-MB-435S tumor cells were co-cultured together with UniCAR CD28/ζ T cells and increasing concentrations of the respective αEGFR TMs under the same conditions ( D ). Mean specific lysis and SEM are shown. Statistical analysis was performed by two-way ANOVA with Tukey’s multiple comparison test ( C ) or paired t -test for each specific concentration ( D ). ( # p

    Journal: OncoTargets and therapy

    Article Title: Highly Efficient Targeting of EGFR-Expressing Tumor Cells with UniCAR T Cells via Target Modules Based on Cetuximab®

    doi: 10.2147/OTT.S245169

    Figure Lengend Snippet: Killing of EGFR low -expressing tumor cells by UniCAR T cells redirected by αEGFR TMs. The estimated number of EGFR molecules per A431 or MDA-MB-435S tumor cell was evaluated using the QIFIKIT ® ( A ). MDA-MB-435S tumor cells were stained with αEGFR mAb and detected via goat-α-mouse IgG-Pacific Blue TM to determine EGFR surface expression by flow cytometry ( B ). Binding of indicated αEGFR TMs to the MDA-MB-435S target cell line was analyzed by staining and flow cytometry ( B ). Therefore, cells were incubated with αEGFR TM, the αE5B9 mAb and goat-α-mouse IgG-Pacific Blue TM ( B ). As a negative control, cells were stained with the detection Ab alone (white graphs) ( B ). Percentage (%) and median fluorescence intensity (MFI) of positively stained cells were shown ( B ). In a standard chromium release cytotoxicity assay, EGFR low -expressing MDA-MB-435S tumor cells were incubated with T cells genetically modified to express EGFP (vector control), non-signaling UniCAR stop or signaling UniCAR CD28/ζ in the presence or absence of indicated αEGFR TMs at an effector to target cell ratio of 5:1 ( C ). Mean specific lysis and SEM are shown for two individual donors in the presence of Hu scFv αEGFR TM and three individual donors in the presence of nb or Mu scFv αEGFR TM ( C ). In addition, MDA-MB-435S tumor cells were co-cultured together with UniCAR CD28/ζ T cells and increasing concentrations of the respective αEGFR TMs under the same conditions ( D ). Mean specific lysis and SEM are shown. Statistical analysis was performed by two-way ANOVA with Tukey’s multiple comparison test ( C ) or paired t -test for each specific concentration ( D ). ( # p

    Article Snippet: EGFR quantification was performed using the QIFIKIT® (Agilent Technologies, Böblingen, Germany) following the manufacturer’s instructions.

    Techniques: Expressing, Multiple Displacement Amplification, Staining, Flow Cytometry, Binding Assay, Incubation, Negative Control, Fluorescence, Cytotoxicity Assay, Genetically Modified, Plasmid Preparation, Lysis, Cell Culture, Concentration Assay