Journal: bioRxiv
Article Title: MYC and p53 alterations cooperate through VEGF signaling to repress cytotoxic T cell and immunotherapy responses in prostate cancer
doi: 10.1101/2024.07.24.604943
Figure Lengend Snippet: A, Representative IHC staining of prostate tumors from FVB mice transplanted orthotopically with Myc-CaP (Myc) or Myc-p53 KO cells or from C57BL/6 mice transplanted orthotopically with MP , MPten or PtPRb EPO-GEMM-derived cell lines. Arrowheads indicate VEGF positive cells in tumor areas. Scale bars, 50μm. B-C, Quantification of VEGF + cells ( B ) and CD31 + blood vessels ( C ) per field in A (n = 3-4 mice per group). D, Representative co-IF staining for CD8 and VEGFR2 in indicated Myc-CaP or EPO-GEMM cell line-derived transplant prostate tumors. White arrowheads indicate VEGFR2 + CD8 + double positive cells. Scale bars, 50μm. E, Quantification of percentage of CD8 + T cells that are VEGFR2 + from co-IF analysis in D (n = 3 mice per group). F, Schematic of ex vivo tumor-immune co-culture assay using spleen-derived CD8 + T cells and murine prostate cancer cell lines. Created with Biorender.com. G, Flow cytometry analysis of VEGFR2 expression on CD8 + T cells cultured ex vivo with indicated prostate cancer cell lines (n = 3 biological replicates per group) or in the presence of recombinant VEGF (50ng/mL). H-I, Flow cytometry analysis of IFNγ ( H ) and Granzyme B (GZMB) ( I ) expression in CD8 + T cells cultured with indicated prostate cancer cell lines in the presence or absence of a VEGFR2 blocking antibody (DC101; 1µg/mL) (n = 3 biological replicates per group) or recombinant VEGF (50ng/mL). J, Quantification of VEGFR2 staining scores in primary prostate cancer patient samples stratified by MYC staining score into high and low groups (n = 7 samples per group). K, Representative IHC staining in primary prostate cancer patient samples stratified by VEGFR2 staining score into low, intermediate, and high groups. Arrowheads indicate positive staining for immune cells. Scale bars, 50μm. L, Quantification of CD8 + T cell and NKp46 + NK cell numbers in primary prostate cancer patient samples stratified by VEGFR2 staining score in K (n = 4-6 samples per group). M, Representative co-IF staining of CD8 and VEGFR2 expression in primary prostate cancer patient tumors stratified by MYC staining score into high and low groups. Scale bars, 50μm. N, Quantification of percentage of CD8 + T cells that are VEGFR2 + in MYC hi and MYC lo patient prostate tumors in M (n = 5-7 per group). Data represent mean ± SEM. P-values were calculated by two-tailed, unpaired Student’s t-test.
Article Snippet: Some CD8 + T cells from C57BL/6 mice were directly exposed to 50ng/mL recombinant murine VEGF from R&D Systems (493-MV-005/CF) in the absence of tumor cell co-culture as a control condition.
Techniques: Immunohistochemistry, Derivative Assay, Staining, Ex Vivo, Co-culture Assay, Flow Cytometry, Expressing, Cell Culture, Recombinant, Blocking Assay, Two Tailed Test
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