Journal: bioRxiv

Article Title: A novel, RAS-independent role for NF1 in microtubular dynamics and damage repair dictates sensitivity to T-DM1 in HER2-positive breast cancer

doi: 10.1101/2023.12.06.569572

Figure Lengend Snippet: a-c CETSA for DM1 in BT-474 WT and BT-474 KO cells. a experimental design. Cells are harvested and incubated in suspension with DM1 for 2h and then exposed to increasing temperatures as indicated b western blot for β-tubulin of cell extracts from BT-474 WT and BT-474 KO cells . c. Quantification of band intensities from CETSA western blots (n = 3 independent experiments), normalised on baseline signal at 57°C. Ribbons indicate SD. We used 2-way ANOVA separately on WT and KO to test for significance of temperature and drug. We observed that temperature was significant both in the WT and in the KO case (pvalue=2.05x10 - and 1.79 x10 -9 , respectively). However, the addition of DM1 was significant only in the KO case (pvalue=4.62 x10 -3 , 0.78 in the WT case). d. Representative kymographs of microtubules grown in vitro with or without NF1 at indicated concentration and 1μM DM1 from GMPCPP-stabilized seeds and followed by TIRF microscopy e. NF1 increases the fraction of microtubules initiating elongation in the presence of 1μM DM1. The percentage for each condition was calculated as the number of GMPCPP seeds elongating divided by the total number of seeds present in the acquired ROI. f. Quantification of growth rate of microtubules grown with 1μM DM1 and with 100nM or 300nM NF1. g. Quantification of depolymerization rate of microtubules grown with or without 100nM NF1 and with or without 1μM DM1. Data are means ± SEM. Kruskal-wallis test with Dunn post-hoc test was used to assess significance. h. Single frames of time-lapse videos of taxol-stabilized rhodamine-labeled microtubules incubated with 1μM DM1 i. Taxol-stabilized rhodamine-labeled microtubules were first incubated without Taxol and tubulin for 1.5 min and subsequently with 5 mM HiLyte Fluor-488-labeled tubulin with 100nM NF1. After 10 min, the residual free green tubulin was washed out with the wash buffer supplemented with 25% glycerol to prevent microtubule depolymerization, in order to better visualize incorporation of green tubulin. Kymograph showing green tubulin incorporation sites into Rhodamine-labelled microtubule lattices (magenta). Arrows indicate complete repair. j. reconstruction of microtubule tip trajectories after monitoring of microtubule elongation in BT-474 WT and BT-474 KO cells transiently transfected with an EB3-GFP-expressing vector and monitored by live cell imaging for 1 minute with acquisitions every 4 seconds. Microtubule tip fates were reconstructed by the TrackMate ImageJ plugin. k quantification of tip mean speed. At least 1000 events from at least 3 cells were calculated per condition. 2-way ANOVA with Tukey’s HSD. **** p < 0.001. l-m immunofluorescence for acK40 alpha tubulin in HCC1954 WT and HCC1954 KO . m quantification of mean intensity normalized by area occupied by cell cytoplasm. 4 fields of view per condition

Article Snippet: Cells were blocked with 5% BSA in PBS for 30 minutes and then incubated with primary antibody anti-HER2 Alexa Fluor-488 conjugate (#FAB1129G-025, Biotechne) diluted in 1% BSA in PBS for 1 hour at 4° C in agitation.

Techniques: Incubation, Suspension, Western Blot, In Vitro, Concentration Assay, Microscopy, Labeling, Transfection, Expressing, Plasmid Preparation, Live Cell Imaging, Immunofluorescence

Journal: bioRxiv

Article Title: Treatment with anti-HER2 chimeric antigen receptor tumor-infiltrating lymphocytes (CAR-TILs) is safe and associated with antitumor efficacy in mice and companion dogs

doi: 10.1101/2022.09.11.507449

Figure Lengend Snippet: mRNA encoding anti-HER2 CAR was electroporated into TILs from five cutaneous melanomas (MM1, MM3, MM4, MM5, and MM6) and one uveal melanoma (UM22) to produce CAR-TILs. CAR expression was detected by HER2-biotin binding at 5-16 hours post-transfection (a). MM1 TILs and CAR-TILs were co-cultured with the parental or HER2 KO 92-1 uveal melanoma cell line for 4-6 hours, followed by flow cytometry to measure degranulation (CD107a expression) (b) or IFN-γ-secreting cells (c). TILs and CAR-TILs not co-cultured with 92-1 cells (TILs alone; TA) were used as controls in b-c. Alternatively, cells were co-cultured for 48 h, and the supernatant was collected for analysis of secreted IFN-γ by ELISA (d). 92-1 cells not co-cultured with TILs (no TILs) were used as a control. Data are presented as mean ± standard deviation (SD) of duplicates. The experiments were performed twice, and representative results are shown.

Article Snippet: For analysis of HER2 protein binding capacity of CAR-TILs, 100.000 cells were incubated with 1 μg biotinylated HER2 protein (Abcam) for 30 min at 4 °C, followed by incubation with an allophycocyanin-conjugated streptavidin antibody (Jackson Immuno Research) for 25 min at 4 °C, and flow cytometry analysis was performed using an Accuri C6 flow cytometer (BD) equipped with the BD Accuri C6 software.

Techniques: Expressing, Binding Assay, Transfection, Cell Culture, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: bioRxiv

Article Title: Treatment with anti-HER2 chimeric antigen receptor tumor-infiltrating lymphocytes (CAR-TILs) is safe and associated with antitumor efficacy in mice and companion dogs

doi: 10.1101/2022.09.11.507449

Figure Lengend Snippet: Flow cytometry data showing HER2 CAR expression after HER2 CAR mRNA electroporation by detecting bound biotinylated HER2 protein. Representative plots for samples MM1, MM3, MM4, MM5, MM6 and UM22.

Article Snippet: For analysis of HER2 protein binding capacity of CAR-TILs, 100.000 cells were incubated with 1 μg biotinylated HER2 protein (Abcam) for 30 min at 4 °C, followed by incubation with an allophycocyanin-conjugated streptavidin antibody (Jackson Immuno Research) for 25 min at 4 °C, and flow cytometry analysis was performed using an Accuri C6 flow cytometer (BD) equipped with the BD Accuri C6 software.

Techniques: Flow Cytometry, Expressing, Electroporation

Journal: bioRxiv

Article Title: Treatment with anti-HER2 chimeric antigen receptor tumor-infiltrating lymphocytes (CAR-TILs) is safe and associated with antitumor efficacy in mice and companion dogs

doi: 10.1101/2022.09.11.507449

Figure Lengend Snippet: (a-b) TILs and CAR-TILs from MM1 were co-cultured with the parental or HER2 KO 92-1 uveal melanoma cell line for 24 hours, followed by cleaved caspase 3 (CC3) detection in the tumor cells using flow cytometry (a). Additionally, TILs and CAR-TILs from MM5 were co-cultured with autologous cancer cells for 24 hours, followed by cleaved CC3 detection using flow cytometry (b). Cancer cells not treated with TILs were used as a control (no TILs). (c-g) Viability of autologous cancer cells was measured by luciferase signal detected after 48 hours co-culture with increasing doses of TILs and CAR-TILs from UM22 (c), MM3 (d), MM4 (e), MM5 (f) and MM6 (g) in the indicated ratios (TILs:cancer cells). Data is presented as mean with SD of triplicates. Asterisks represent p-values of difference between similar doses of TILs and CAR-TILs. The experiments were performed twice, and representative data from one experiment is shown.

Article Snippet: For analysis of HER2 protein binding capacity of CAR-TILs, 100.000 cells were incubated with 1 μg biotinylated HER2 protein (Abcam) for 30 min at 4 °C, followed by incubation with an allophycocyanin-conjugated streptavidin antibody (Jackson Immuno Research) for 25 min at 4 °C, and flow cytometry analysis was performed using an Accuri C6 flow cytometer (BD) equipped with the BD Accuri C6 software.

Techniques: Cell Culture, Flow Cytometry, Luciferase, Co-Culture Assay

Journal: bioRxiv

Article Title: Treatment with anti-HER2 chimeric antigen receptor tumor-infiltrating lymphocytes (CAR-TILs) is safe and associated with antitumor efficacy in mice and companion dogs

doi: 10.1101/2022.09.11.507449

Figure Lengend Snippet: HER2 expression detected by qPCR (a) and immunohistochemistry (b) in tumors from four companion dogs (dogs 1-4). Canine cell lines D17.os and CF41.mg and canine PBMC were used as controls in a. (c) Expression of human melanoma marker Melan-a in tumors from dogs 3 and 4 with melanoma. (d) Classification of canine tumor biopsies from dogs 3 and 4 based on gene expression relative to TCGA cohort of > 10000 human tumor samples from 32 cancer types. The similarities between canine melanoma and samples in TCGA were visualized using tSNE dimensionality reduction. SKCM: melanoma cases. (e-f) TILs and mRNA electroporated CAR-TILs from dog 3 were co-cultured with the canine cell lines CF41.mg (e) and D17.os (f) for 24 h. The experiment was performed twice and representative data (n=3, data ± SD) from one experiment are shown. (g) Photograph of Dog 3 with metastatic oral malignant melanoma enrolled in the FIDO trial evaluating the safety of CAR-TIL treatment in companion dogs.

Article Snippet: For analysis of HER2 protein binding capacity of CAR-TILs, 100.000 cells were incubated with 1 μg biotinylated HER2 protein (Abcam) for 30 min at 4 °C, followed by incubation with an allophycocyanin-conjugated streptavidin antibody (Jackson Immuno Research) for 25 min at 4 °C, and flow cytometry analysis was performed using an Accuri C6 flow cytometer (BD) equipped with the BD Accuri C6 software.

Techniques: Expressing, Immunohistochemistry, Marker, Cell Culture