Journal: Communications Biology

Article Title: Apoptosis-inducing anti-HER2 agents operate through oligomerization-induced receptor immobilization

doi: 10.1038/s42003-021-02253-4

Figure Lengend Snippet: a Monovalent, monoparatopic DARPins (9.26 binding to extracellular subdomain I, vermillion; G3 binding to extracellular subdomain IV, yellow) and design of bipDARPin 6L1G (right), connecting 9.26 and G3 through a short linker. b Ligand-activated (e.g., by heregulin, mint) HER2:HER3 (sky blue, blue) heterodimers are able to stimulate proliferation and survival. c , d Bivalent intermolecular binding of bipDARPins may induce the formation of ( c ) inactive dimers or ( d ) oligomerization into daisy chains. In both cases, the kinase domains are separated and thus inactive. HRG heregulin, TM transmembrane helix, KD kinase domain.

Article Snippet: For specific kinase inhibition, cells were pre-treated in complete medium for ~3 h with 12.5 µM HER2-specific kinase inhibitor (Selleckchem, cat. no. S2752, an analog of ARRY-380) or dimethyl sulfoxide.

Techniques: Binding Assay

Journal: Communications Biology

Article Title: Apoptosis-inducing anti-HER2 agents operate through oligomerization-induced receptor immobilization

doi: 10.1038/s42003-021-02253-4

Figure Lengend Snippet: a Comparison of the size of HER2 complexes after treatment with the bipDARPin 6L1G (32 kDa) with those obtained with growth factor heregulin (HRG, 27 kDa), monovalent DARPins (9.26 and G3, ~12 kDa each) or a bivalent, monoparatopic DARPin (GL4G, ~30 kDa). Primary antibodies against HER2 and DARPins were used. b Crosslinked HER2 complexes upon treatment with antibodies trastuzumab (TZB, ~145 kDa) and pertuzumab (PZB, ~145 kDa), detected by primary antibodies against HER2 and human IgG.

Article Snippet: For specific kinase inhibition, cells were pre-treated in complete medium for ~3 h with 12.5 µM HER2-specific kinase inhibitor (Selleckchem, cat. no. S2752, an analog of ARRY-380) or dimethyl sulfoxide.

Techniques:

Journal: Communications Biology

Article Title: Apoptosis-inducing anti-HER2 agents operate through oligomerization-induced receptor immobilization

doi: 10.1038/s42003-021-02253-4

Figure Lengend Snippet: a , b Confocal images from a representative FRAP experiment of HT-HER2 labeled with Alexa Fluor 660, by photobleaching either a in the equatorial (eq.) or b basal (bs.) membrane plane. The arrow indicates the location of the bleach spot (2 µm diameter). c , d FRAP curves (bleach-corrected, normalized mean fluorescence intensities in the bleached ROI after subtraction of residual intensity at t = 0) for various DARPin and antibody treatments and controls. c Massive reduction of mobility upon treatment with DARPin 6L1G. Data from the basal membrane are shown in addition to those obtained by regular measurements in the equatorial plane. d Slight reduction in mobility is seen upon treatment with the therapeutic mAbs PZB and TZB individually but complete immobilization by their combination (PZB + TZB). Untr., untreated.

Article Snippet: For specific kinase inhibition, cells were pre-treated in complete medium for ~3 h with 12.5 µM HER2-specific kinase inhibitor (Selleckchem, cat. no. S2752, an analog of ARRY-380) or dimethyl sulfoxide.

Techniques: Labeling, Fluorescence

Journal: Communications Biology

Article Title: Apoptosis-inducing anti-HER2 agents operate through oligomerization-induced receptor immobilization

doi: 10.1038/s42003-021-02253-4

Figure Lengend Snippet: a Individual FRAP curves (no averaging) before and after addition of DARPin 6L1G, colored according to the starting time relative to DARPin addition, show immediate immobilization. b HER2 immobilization is similar after 5 min and 6 h of treatment. c FRAP experiments in cells treated with Latrunculin B in the absence of DARPins (LatB), and cells treated with LatB before (LatB, 6L1G) or after (6L1G, LatB) bipDARPin addition indicate that cytoskeleton interactions are not directly involved in HER2 immobilization upon DARPin treatment. d Pre-treatment with a HER2-specific HER2 kinase inhibitor (Selleckchem S2752, an analog of ARRY 380) does not affect immobilization, suggesting that immobilization is independent of HER2 kinase activation. e HER2 immobilization occurs at a wide range of expression levels, as indicated by FRAP measurements on HER2-overexpressing BT-474 cells and non-induced HEK cells stably transfected with HaloTagged HER2. Note that the sensitivity of the FRAP method limits the cell lines that can be investigated to those with at least intermediate expression levels (Supplementary Table and Supplementary Fig. ). Data for untreated cells (Untr.) and 6L1G in c are identical to those shown in Fig. .

Article Snippet: For specific kinase inhibition, cells were pre-treated in complete medium for ~3 h with 12.5 µM HER2-specific kinase inhibitor (Selleckchem, cat. no. S2752, an analog of ARRY-380) or dimethyl sulfoxide.

Techniques: Activation Assay, Expressing, Stable Transfection, Transfection

Journal: Communications Biology

Article Title: Apoptosis-inducing anti-HER2 agents operate through oligomerization-induced receptor immobilization

doi: 10.1038/s42003-021-02253-4

Figure Lengend Snippet: a Dramatic loss of mobility upon treatment with bipDARPin 6L1G compared to untreated cells (Untr.). b Concentration dependence of the mobility reduction as illustrated by the relative fluorescence recovery after 48 s. c Homo-bivalent, monoparatopic DARPins 6L46 and GL4G reduce HER2 mobility only slightly, while a mixture of both (GL4G + 6L46) fully immobilizes HER2 through crosslinking. d Similarly, moderate mobility changes are observed upon treatment with therapeutic mAbs PZB and TZB but almost complete immobilization by their combination (PZB + TZB).

Article Snippet: For specific kinase inhibition, cells were pre-treated in complete medium for ~3 h with 12.5 µM HER2-specific kinase inhibitor (Selleckchem, cat. no. S2752, an analog of ARRY-380) or dimethyl sulfoxide.

Techniques: Concentration Assay, Fluorescence

Journal: Communications Biology

Article Title: Apoptosis-inducing anti-HER2 agents operate through oligomerization-induced receptor immobilization

doi: 10.1038/s42003-021-02253-4

Figure Lengend Snippet: a Representative SMLM super-resolution images rendered from 500 consecutive frames (localization precision ~20 nm) with localization densities encoded as gray values. Scale bar: 10 µm. Magnified center regions are shown as insets (scale bar: 2 µm). b Particle densities in the DY-649 channel observed in different single-molecule experiments. c , d Comparison of the immobile fraction identified by spatiotemporal clustering analysis ( c ) and the diffusion coefficient within the mobile fraction ( d ) for HER2 for different treatments. e , f Colocalized HER2 molecules in the mobile ( e ) and the immobile fraction ( f ) quantified by dual-color co-tracking. g Correlation of the total fraction of co-localized molecules with the immobile fraction and linear regression (R 2 = 0.97, Pearson’s r = 0.99, p = 3.5 × 10 −5 ). Different colors (cyan, control or monovalent; red, homo-bivalent; green, biparatopic) in b – g indicate the three distinct groups of agents as referenced in the main text. The DARPins are shown schematically in Supplementary Fig. . Significance values for b – f are provided in Supplementary Fig. .

Article Snippet: For specific kinase inhibition, cells were pre-treated in complete medium for ~3 h with 12.5 µM HER2-specific kinase inhibitor (Selleckchem, cat. no. S2752, an analog of ARRY-380) or dimethyl sulfoxide.

Techniques: Diffusion-based Assay

Journal: Communications Biology

Article Title: Apoptosis-inducing anti-HER2 agents operate through oligomerization-induced receptor immobilization

doi: 10.1038/s42003-021-02253-4

Figure Lengend Snippet: a , b , c Transient arrest events (highlighted in red) within individual HER2 trajectories (top) determined from the confinement index calculated along a representative trajectory (bottom) upon treatment with OL4G ( a ), GL4G ( b ), and 6L1G ( c ). The threshold defining transient arrest events is indicated by the red line. d Identified fraction of confined particles. e Probability of transitioning into state of arrested diffusion. f Probability of mobilization out of arrested diffusion. Different colors in d – f indicate the three distinct groups of agents as described in Fig. and referenced in the main text. The DARPins are shown schematically in Supplementary Fig. . Significance values for d – f are provided in Supplementary Fig. .

Article Snippet: For specific kinase inhibition, cells were pre-treated in complete medium for ~3 h with 12.5 µM HER2-specific kinase inhibitor (Selleckchem, cat. no. S2752, an analog of ARRY-380) or dimethyl sulfoxide.

Techniques: Diffusion-based Assay

Journal: Communications Biology

Article Title: Apoptosis-inducing anti-HER2 agents operate through oligomerization-induced receptor immobilization

doi: 10.1038/s42003-021-02253-4

Figure Lengend Snippet: a Characteristic diffusion modes of HER2 monomers (cyan), dimers (red), and oligomers (green) induced by different agents are due to differential propensity of partitioning into membrane nanodomains. Schematic trajectories and transiently forming nanodomains integrated over time. Trajectory time resolution: ~5 ms; hop diffusion inflicted by the membrane skeleton omitted for clarity. Scale bar: 50 nm. b Efficient inhibition of HER2 by bipDARPins caused by arrest in an inactive conformation upon formation of daisy chains and by strongly enhanced partitioning of HER2 daisy chains into membrane nanodomains. Scale bar: 20 nm.

Article Snippet: For specific kinase inhibition, cells were pre-treated in complete medium for ~3 h with 12.5 µM HER2-specific kinase inhibitor (Selleckchem, cat. no. S2752, an analog of ARRY-380) or dimethyl sulfoxide.

Techniques: Diffusion-based Assay, Inhibition

Journal: Molecular cancer research : MCR

Article Title: Let-7 Status is Crucial for PARP1 Expression in HER2-overexpressing Breast Tumors

doi: 10.1158/1541-7786.MCR-16-0287-T

Figure Lengend Snippet: (A, B) Cells were transfected with HER2 or control (CON) siRNA for 72 hours and then analyzed by Western blot analysis for HER2, PARP1, and PARP2 protein levels. β-actin was used as a loading control. MDA-MB-231 cells that (C) transiently or (D) stably express the HER2 vector (231 HER2) as compared to cells expressing the control plasmid (231 NEO) were subjected to Western blot analysis for HER2, PARP1, PARP2, and β-actin. Data shown are representative immmunoblots from one of two independent experiments.

Article Snippet: The following are the primary antibodies used in this study: anti-human HER2 (2165S; Cell Signaling, Danvers, MA, USA), PARP1 (9532S; Cell Signaling, Danvers, MA, USA), PARP2 (ab176330; Abcam, Cambridge, MA, USA), c-Myc (5605S; Cell Signaling, Danvers, MA, USA), and β-actin HRP (sc-47778 HRP; Santa Cruz, Dallas, TX, USA).

Techniques: Transfection, Western Blot, Stable Transfection, Plasmid Preparation, Expressing

Journal: Molecular cancer research : MCR

Article Title: Let-7 Status is Crucial for PARP1 Expression in HER2-overexpressing Breast Tumors

doi: 10.1158/1541-7786.MCR-16-0287-T

Figure Lengend Snippet: Expression of the Let-7 miRNA family is altered after HER2 overexpression in breast cancer cells lines.

Article Snippet: The following are the primary antibodies used in this study: anti-human HER2 (2165S; Cell Signaling, Danvers, MA, USA), PARP1 (9532S; Cell Signaling, Danvers, MA, USA), PARP2 (ab176330; Abcam, Cambridge, MA, USA), c-Myc (5605S; Cell Signaling, Danvers, MA, USA), and β-actin HRP (sc-47778 HRP; Santa Cruz, Dallas, TX, USA).

Techniques: Expressing, Over Expression

Journal: Molecular cancer research : MCR

Article Title: Let-7 Status is Crucial for PARP1 Expression in HER2-overexpressing Breast Tumors

doi: 10.1158/1541-7786.MCR-16-0287-T

Figure Lengend Snippet: Ten nanograms of isolated miRNA from 70% confluent (A) 231 NEO and 231 HER2 or (B) MCF7, BT-474, and SKBR3 human breast cancer cells were reverse transcribed to cDNA and then analyzed by qRT-PCR for let-7a, let-7i, and U6 expression. The figures are representative from (A, B) one of two independent experiments performed in triplicate. A t-test or one-way ANOVA test was performed. (C) Western blot analysis of HER2, PARP1, PARP2 and β-actin levels in untreated 231 NEO, 231 HER2, MCF7, BT-474, and SKBR3 cell lines. **p<0.01, ***p<0.001 and ****p<0.0001.

Article Snippet: The following are the primary antibodies used in this study: anti-human HER2 (2165S; Cell Signaling, Danvers, MA, USA), PARP1 (9532S; Cell Signaling, Danvers, MA, USA), PARP2 (ab176330; Abcam, Cambridge, MA, USA), c-Myc (5605S; Cell Signaling, Danvers, MA, USA), and β-actin HRP (sc-47778 HRP; Santa Cruz, Dallas, TX, USA).

Techniques: Isolation, Quantitative RT-PCR, Expressing, Western Blot

Journal: Molecular cancer research : MCR

Article Title: Let-7 Status is Crucial for PARP1 Expression in HER2-overexpressing Breast Tumors

doi: 10.1158/1541-7786.MCR-16-0287-T

Figure Lengend Snippet: (A–C) BT-474, SKBR3 and 231 HER2 cells were transiently transfected with a let-7a miRNA (hsa-let-7a-5p) or its neg control miRNA (negative control). Following transfection, cells were analyzed by Western blot analysis for PARP-1, PARP-2, and β-actin. (D) The 231 NEO cell line was transiently transfected with an anti-let-7a miRNA (let-7a antagomiR) or a neg control miRNA (negative control). Following transfection, cells were analyzed by Western blot analysis for PARP-1, PARP-2, and β-actin. Results shown are from one of two independent experiments performed duplicate.

Article Snippet: The following are the primary antibodies used in this study: anti-human HER2 (2165S; Cell Signaling, Danvers, MA, USA), PARP1 (9532S; Cell Signaling, Danvers, MA, USA), PARP2 (ab176330; Abcam, Cambridge, MA, USA), c-Myc (5605S; Cell Signaling, Danvers, MA, USA), and β-actin HRP (sc-47778 HRP; Santa Cruz, Dallas, TX, USA).

Techniques: Transfection, Negative Control, Western Blot

Journal: Molecular cancer research : MCR

Article Title: Let-7 Status is Crucial for PARP1 Expression in HER2-overexpressing Breast Tumors

doi: 10.1158/1541-7786.MCR-16-0287-T

Figure Lengend Snippet: (A) The PARP1 3’UTR was aligned with the seed sequence of the let-7a miRNA. (B, C) BT-474 and 231 HER2 cells were co-transfected with a let-7a miRNA or its neg control miRNA and a plasmid containing a firefly luciferase gene and the 3’UTR of PARP1 or a control 3’UTR. (D) 231 NEO cell line was co-transfected with an anti-let-7a miRNA or its neg control miRNA and a plasmid containing a firefly luciferase gene and the 3’UTR of PARP1 or a control 3’UTR. Luciferase activity was normalized to cells co-transfected with the respective neg control miRNA and the plasmid containing the control 3’UTR. The fold change values were calculated back to the cells co-transfected with the respective neg control miRNA and the plasmid containing the 3’UTR of PARP1. Results shown are from one of two independent experiments performed in quadruplicate. *p<0.05, **p<0.01

Article Snippet: The following are the primary antibodies used in this study: anti-human HER2 (2165S; Cell Signaling, Danvers, MA, USA), PARP1 (9532S; Cell Signaling, Danvers, MA, USA), PARP2 (ab176330; Abcam, Cambridge, MA, USA), c-Myc (5605S; Cell Signaling, Danvers, MA, USA), and β-actin HRP (sc-47778 HRP; Santa Cruz, Dallas, TX, USA).

Techniques: Sequencing, Transfection, Plasmid Preparation, Luciferase, Activity Assay

Journal: Molecular cancer research : MCR

Article Title: Let-7 Status is Crucial for PARP1 Expression in HER2-overexpressing Breast Tumors

doi: 10.1158/1541-7786.MCR-16-0287-T

Figure Lengend Snippet: (A) Let-7a levels were measured by qRT-PCR analysis in HER2+ and HER2− breast cancer specimens. U6 was used to normalize data. (B) Let-7a gene and PARP1 protein expression levels were correlated in 17 UAB HER2− and HER2+ breast cancer patients (rs=−0.6937, p=0.0020). ***p<0.001

Article Snippet: The following are the primary antibodies used in this study: anti-human HER2 (2165S; Cell Signaling, Danvers, MA, USA), PARP1 (9532S; Cell Signaling, Danvers, MA, USA), PARP2 (ab176330; Abcam, Cambridge, MA, USA), c-Myc (5605S; Cell Signaling, Danvers, MA, USA), and β-actin HRP (sc-47778 HRP; Santa Cruz, Dallas, TX, USA).

Techniques: Quantitative RT-PCR, Expressing

Journal: Molecular cancer research : MCR

Article Title: Let-7 Status is Crucial for PARP1 Expression in HER2-overexpressing Breast Tumors

doi: 10.1158/1541-7786.MCR-16-0287-T

Figure Lengend Snippet: (A) BT-474 and (B) 231 HER2 cells were transiently transfected with a let-7a miRNA or its negative control. Forty-eight hours after transfection, the cells were treated with DMSO or 10µM ABT-888 for 96 hours and then analyzed by a cellular proliferation assay. Results shown are from one of two independent experiments performed in quadruplicate. *p<0.05, **p<0.01, and ***p<0.001

Article Snippet: The following are the primary antibodies used in this study: anti-human HER2 (2165S; Cell Signaling, Danvers, MA, USA), PARP1 (9532S; Cell Signaling, Danvers, MA, USA), PARP2 (ab176330; Abcam, Cambridge, MA, USA), c-Myc (5605S; Cell Signaling, Danvers, MA, USA), and β-actin HRP (sc-47778 HRP; Santa Cruz, Dallas, TX, USA).

Techniques: Transfection, Negative Control, Proliferation Assay

Journal: Cell Death & Disease

Article Title: Increased expression of EHF via gene amplification contributes to the activation of HER family signaling and associates with poor survival in gastric cancer

doi: 10.1038/cddis.2016.346

Figure Lengend Snippet: EHF regulates the expression of HER receptors and the activities of their downstream signaling pathways in gastric cancer cells. ( a ) qRT-PCR assay was used to evaluate mRNA expression of EHF and HER receptors in primary gastric cancers ( n =30). Linear regression analysis was performed to assess the correlations between them. 18S rRNA was used as a normalized control. ( b ) qRT-PCR assay was performed to investigate the effect of EHF knockdown on the expression of HER receptors. Expression levels of these genes were normalized with 18S rRNA levels. Data were presented as mean±S.E. ( c ) The effect of EHF depletion on the expression of HER2-4 was determined in the indicated cells by western blot analysis. GAPDH was used as loading control. ( d ) Cells transfected with si-EHF-979 or si-NC were lysed and lysates were subjected to western blot analysis. The antibodies against phospho-Erk (p-Erk), total Erk (t-Erk), phospho-Akt (p-Akt) and total Akt (t-Akt) were used to determine the effect of EHF knockdown on the activities of the MAPK/Erk and PI3K/Akt cascades. GAPDH was used as a loading control

Article Snippet: The membranes were blocked for 2 h in 5% bovine serum albumin (BSA) in 1 × TBS-T (0.5% Tween-20) and incubated with the indicated primary antibodies, including anti-EHF (Abcam, Inc), anti-total-Erk1/2 (Abcam, Inc), anti-phospho-Erk1/2 (Epitomics, Inc), anti-phospho-AktSer473 (Bioworld Technology, co, Ltd), anti-total-Akt (Bioworld Technology, co, Ltd), anti-HER2 (Sino Biological, Inc), anti-HER3 (Sino Biological, Inc), anti-HER4 (Sino Biological, Inc), anti-E-cadherin (Epitomics, Inc), anti-Vimentin (Epitomics, Inc) and anti-GAPDH (Abgent, Inc).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Transfection

Journal: Cell Death & Disease

Article Title: Increased expression of EHF via gene amplification contributes to the activation of HER family signaling and associates with poor survival in gastric cancer

doi: 10.1038/cddis.2016.346

Figure Lengend Snippet: EHF is identified as a new HER2 transcription factor and the modulator of HER3 and HER4 in gastric cancer. ( a ) BGC823 cells were transiently transfected with pGL3-Basic or luciferase reporter constructs containing various lengths of the promoter region of HER2 gene, as indicated (F1: −607/+11; F2: −175/+11; F3: −607/−175) (left panels). Cotransfection with empty vector was used as a control. The ratio of the Luc/Renilla activity is shown as means±S.E. of three independent assays (right panels). ( b ) The luciferase reporter gene assay was performed to evaluate the effect of EHF knockdown on promoter activity of HER2 in BGC823 cells. The ratio of the Luc/Renilla activity is shown as means±S.E. of three independent assays. ( c ) HEK293T cells were cotransfected pGL3-HER2-Luc-F1 and various amounts of pcDNA3.1(-)A-EHF or empty vector, respectively. Promoter activities of HER2 were measured by luciferase reporter gene assays. All the ratio of the Luc/Renilla activity is shown as means±S.E. of three independent assays. ( d ) Putative promoter regions of HER2 (−607/+11), HER3 (−997/+440) and HER4 (−697/+306) were inserted into the pGL3-Basic to construct the luciferase reporter plasmid pGL3-HER2-Luc, pGL3-HER3-Luc and pGL3-HER4-Luc (upper panels). P1-P7 represent the regions analyzed by ChIP assays for HER2 , HER3 and HER4 , respectively. BGC823 cells were transiently transfected with pcDNA3.1/myc-His(-)A-EHF or empty vector, and were subjected to ChIP-qRT-PCR assays using anti-Myc tag antibody. Flod enrichment was shown as means±S.E. of three independent assays (lower panels). ( e ) EMSA assay was performed to confirm the interaction between EHF and HER2 promoter. Shown are specific DNA-binding of in vitro translated EHF protein to an oligonucleotide sequence (SH2) containing ETS responsive element (GAGGAA) from the HER2 promoter. Unlabeled mutated probes contain specific mutations in the GGAA ETS core or flanking nucleotides of core sequence, as indicated by MT1 and MT2. Unlabeled wild-type (WT) and mutated (MT1 or MT2) competitor probes were added at 100-fold molar excess. Statistically significant differences were indicated: * P <0.05; ** P <0.01; *** P <0.001

Article Snippet: The membranes were blocked for 2 h in 5% bovine serum albumin (BSA) in 1 × TBS-T (0.5% Tween-20) and incubated with the indicated primary antibodies, including anti-EHF (Abcam, Inc), anti-total-Erk1/2 (Abcam, Inc), anti-phospho-Erk1/2 (Epitomics, Inc), anti-phospho-AktSer473 (Bioworld Technology, co, Ltd), anti-total-Akt (Bioworld Technology, co, Ltd), anti-HER2 (Sino Biological, Inc), anti-HER3 (Sino Biological, Inc), anti-HER4 (Sino Biological, Inc), anti-E-cadherin (Epitomics, Inc), anti-Vimentin (Epitomics, Inc) and anti-GAPDH (Abgent, Inc).

Techniques: Transfection, Luciferase, Construct, Cotransfection, Plasmid Preparation, Activity Assay, Reporter Gene Assay, Quantitative RT-PCR, Binding Assay, In Vitro, Sequencing

Journal: Cell Death & Disease

Article Title: Increased expression of EHF via gene amplification contributes to the activation of HER family signaling and associates with poor survival in gastric cancer

doi: 10.1038/cddis.2016.346

Figure Lengend Snippet: HER2 depletion attenuates proliferation-promoting effect of EHF in gastric cancer cells. Inhibitory effect of HER2 depletion on cell proliferation in GES-1 and MGC803 cells overexpressing EHF were evaluated by MTT assay. The data were presented as mean±S.E. Statistically significant differences were indicated: * P <0.05; ** P <0.01; *** P <0.001

Article Snippet: The membranes were blocked for 2 h in 5% bovine serum albumin (BSA) in 1 × TBS-T (0.5% Tween-20) and incubated with the indicated primary antibodies, including anti-EHF (Abcam, Inc), anti-total-Erk1/2 (Abcam, Inc), anti-phospho-Erk1/2 (Epitomics, Inc), anti-phospho-AktSer473 (Bioworld Technology, co, Ltd), anti-total-Akt (Bioworld Technology, co, Ltd), anti-HER2 (Sino Biological, Inc), anti-HER3 (Sino Biological, Inc), anti-HER4 (Sino Biological, Inc), anti-E-cadherin (Epitomics, Inc), anti-Vimentin (Epitomics, Inc) and anti-GAPDH (Abgent, Inc).

Techniques: MTT Assay

Journal: bioRxiv

Article Title: Imaging-guided fine tuning of CAR synapse dynamics and T cell triggering

doi: 10.1101/2021.08.18.456686

Figure Lengend Snippet: A. Fixed anti-HER2 CAR T cell showing maximum intensity projection of CAR, TCR, CD45 and overlay imaged by lattice light-sheet. Bottom row shows zoomed view of region with one microvillus occupied by TCR but not CAR (red arrow), and one occupied by both CAR and TCR (yellow arrow). Top scale bar = 2 µm. Bottom scale bar = 0.7 µm. Human CD8+ T cell expressing anti-HER2 CAR (mut4D5) was labelled with antibodies to MYC (CAR)-Alexa488, TCR (OKT3)-APC, and CD45-Alexa594. B. Schematic showing the cell boundary as mapped to radial intensity profile by angle (left) and pixel intensity (right). C. Pixel intensity (gray line) and binned intensity (black line), defined by taking a moving average of 10 pixels, here showing one z-slice for CAR-mEmerald. Dashed line indicates 3 standard deviations (+3SD) above mean pixel intensity. P indicates binned intensity peaks above +3SD threshold (patches). D. Number of patches/z-slice for each receptor was defined by the number of excursions of binned intensity above the +3SD threshold. T test was performed on data compiled from multiple slices of 3 cells (n = 17 slices per group). Error bar represents standard deviation (s.d.). E. Anti-HER2 CAR T cell interacting with target HER2+ SKBR3 cell (left: side view slice with SKBR3 location marked by dashed yellow line, scale bar = 2 µm; center: zoomed on white box region at 18, 85 seconds later; right: en face synapse surface, scale bar = 4 µm), showing CAR enrichment at projections into the synapse (white arrows). Human CD8+ T cell expressing anti-HER2 CAR (4D5) was labelled with antibodies to MYC (CAR)-Alexa488 and CD45-Alexa647. F. CAR T cell synapse imaged by TIRF showing CAR-mEmerald, microvillar projections (as seen by holes in QD605 signal), and overlay. Human CD8+ T cell expressing anti-HER2 CAR (mut4D5) interacting with lipid bilayer loaded with 625ng HER2 + ICAM + QD605. Scale bar is 2 µm. G. line scan from F shows anti-correlation of CAR-mEmerald and QD605, indicating enrichment of CARs within microvillar contacts.

Article Snippet: After rinsing, protein mixes containing 63 ng recombinant human ICAM-1 (Abcam #AB151393) and 6.25-625 ng biotinylated HER2 (ACROBiosystems #HE2-H822R) or 6 ng pMHC in 2% BSA were injected into each well. pMHC was provided by the NIH Tetramer Facility.

Techniques: Expressing, Standard Deviation

Journal: bioRxiv

Article Title: Imaging-guided fine tuning of CAR synapse dynamics and T cell triggering

doi: 10.1101/2021.08.18.456686

Figure Lengend Snippet: A. Experiments were performed comparing CARs with a high-affinity scFv against HER2-based off trastuzumab (4D5, K D = 0.0523nM, red) and a lower affinity scFv made by substitution of three amino acids (see Supplemental Table 1, mut4D5, K D = 3.63nM, blue). B. Bilayers were loaded with fluorescent quantum dots (QD605) with a height of 16nm. locations where the cell makes close contact with the bilayer (<16nm) are visualized as holes in QD605 signal due to their size-based exclusion. QD605 signal is shown for a low-affinity CAR T cell interacting with a lipid bilayer loaded with 6.25 ng HER2. Outlined contact is shown in C. Scale bar is 3 µm. C. QD605 signal across 5 time points are shown for the same field of view, each on low HER2 bilayers (6.25 ng/well). Top: Microvillus from low-affinity CAR T cell moves out of view. Bottom: Microvillus from high-affinity CAR T cell remains across time points. White scale bar = 1 µm. D. CAR-occupied close contact persistence times (blue, red) and CAR-negative close contact persistence times (black) are shown. All CAR:HER2 interactions tested result in CAR-occupied microvillar contact stabilization above background CAR-negative contacts (black, dotted line). Persistence time is further increased in interactions of high-affinity CAR (red), even at lowest HER2 densities on the bilayer. For low-affinity CAR (blue), only high levels of HER2 yield similar persistence times to high-affinity CAR. Data is shown for at least 11 cells per condition across four experiments (n = 84, 13, 13, 11, 15, 17, 15 cells per group from left to right, respectively). E. low-affinity CAR was retrovirally expressed in primary mouse OT-I CD8+ T cells. Receptor-occupied microvillar persistence times are shown for OT-I:Sl8 (green), low-affinity CAR:low HER2 (light blue), and low-affinity CAR:High HER2 (dark blue) interactions. All cognate interactions are stabilized above background receptor-negative contacts (black, dotted line). CAR:High HER2 persistence is hyper-stable relative to TCR:pMHC stabilization. Data is shown for at least 10 cells per condition across three experiments (n = 36, 16, 10, 10 cells per group from left to right, respectively). F. Dimers (filled dots) and monomers (open dots) are compared on high HER2 bilayers (625ng/well). Only monomeric low-affinity CAR regains natural persistence time of TCR:pMHC contacts (green dashed line). All receptor-occupied contacts are stabilized above non-cognate antigen interactions (black, dotted line). Data is shown for at least 5 cells per condition across three experiments (n = 32, 7, 7, 5, 13 cells per group from left to right, respectively). All error bars represent s.d. and analyses shown are unpaired t tests.

Article Snippet: After rinsing, protein mixes containing 63 ng recombinant human ICAM-1 (Abcam #AB151393) and 6.25-625 ng biotinylated HER2 (ACROBiosystems #HE2-H822R) or 6 ng pMHC in 2% BSA were injected into each well. pMHC was provided by the NIH Tetramer Facility.

Techniques:

Journal: bioRxiv

Article Title: Imaging-guided fine tuning of CAR synapse dynamics and T cell triggering

doi: 10.1101/2021.08.18.456686

Figure Lengend Snippet: A. TIRF imaging CAR-mEmerald is shown for synapses with low-or high-affinity CAR interacting with low or high HER2-loaded bilayers. Time point shown is 93.6 seconds following the initiation of imaging, which began as synapses were starting to form. Yellow line spans CAR microclusters in synapse, indicating diameter quantified in B. B. Diameter across CAR microclusters in synapse (yellow lines in A). For dimeric CARs, only low affinity:low HER2 synapses result in accumulation of CAR microclusters at the center, indicated by lower diameter. Monomerization of CARs improves centralization on high HER2, especially in the low-affinity CAR. Data is shown for at least 7 cells pooled from a minimum two independent experiments per condition (n = 13, 13, 20, 20, 13, 7 cells per group from left to right, respectively). C. Spots with tracking were assigned to CAR microclusters in Imaris, and a random 10 tracks are shown in flower plots for each condition. Top: limited mobility is apparent for dimeric CARs on high-HER2 loaded bilayers. Bottom: On low-HER2 bilayers, low-affinity CAR microclusters show increased mobility. D. Quantification of the average displacement of all CAR microclusters for a given cell (n = 3 cells per group). All error bars represent s.d. and analyses shown are unpaired t tests.

Article Snippet: After rinsing, protein mixes containing 63 ng recombinant human ICAM-1 (Abcam #AB151393) and 6.25-625 ng biotinylated HER2 (ACROBiosystems #HE2-H822R) or 6 ng pMHC in 2% BSA were injected into each well. pMHC was provided by the NIH Tetramer Facility.

Techniques: Imaging

Journal: bioRxiv

Article Title: Imaging-guided fine tuning of CAR synapse dynamics and T cell triggering

doi: 10.1101/2021.08.18.456686

Figure Lengend Snippet: A. low HER2-expressing MCF7 (open circle) and high HER2-expressing SKBR3 (solid circle) cell lines were used to assess differences between HER2 levels in cell-cell interactions in vitro. B. Early activation is similar across all CAR+ conditions with low and high HER2 at 18 hours following co-incubation, as seen by anti-CD69-BUV395 positivity. Box and whiskers plot error bars showing minimum and maximum values (n = 12 samples per group pooled from four independent experiments and two donors). C. Proliferation is induced across all CAR+ conditions with low and high HER2, as seen by VPD dilution at 96 hours following co-incubation. line marks VPD dilution indicating at least 3 replication cycles (quantified in D). D. Percentage of cells that have undergone at least 3 replication cycles. Replicates from 2 independent experiments of different donors are pooled (n = 6). Error bars represent s.d. Dashed lines indicate individual donor trends. E. Percentage of anti-Ki67-PE-eFluor 610 positive cells is similar across all CAR+ conditions with low and high HER2. Replicates from 2 independent experiments of different donors are pooled (n = 6). Error bars represent s.d. F. Intracellular staining with anti-IFN-y-APC at 18hrs following co-incubation. low-affinity CAR induces greater IFN-y than high-affinity, and only monomeric low-affinity CAR, which has overall lower magnitude of response, shows sensitivity to antigen density. Box and whiskers plot error bars showing minimum and maximum values (n = 12 samples per group pooled from four independent experiments and two donors). G. T cells were co-cultured with MC38s expressing low-or high-HER2 for 24 hours in normoxia (20% oxygen) and then separated into normoxic or hypoxic (1.5% oxygen) cultures for an additional six days. Under hypoxic conditions, high-affinity CAR interactions with high HER2 upregulate TOX, while PD-1 expression is upregulated in high HER2 interactions for both high and low-affinity CARs. Box and whiskers plot error bars showing minimum and maximum values (n = 9 samples pooled from 3 independent experiments). All analyses shown are unpaired t tests.

Article Snippet: After rinsing, protein mixes containing 63 ng recombinant human ICAM-1 (Abcam #AB151393) and 6.25-625 ng biotinylated HER2 (ACROBiosystems #HE2-H822R) or 6 ng pMHC in 2% BSA were injected into each well. pMHC was provided by the NIH Tetramer Facility.

Techniques: Expressing, In Vitro, Activation Assay, Incubation, Staining, Cell Culture