Figure 2: Deletion of PRMT7 in melanomas increases immune cell infiltration and increases melanocytic plasticity.
(A-D) Tumors were digested into a single cell suspension and their immune cell composition analyzed. Quantification of Myeloid-Derived Suppressor Cell (MDSC) populations such as Granulocytic MDSC (G-MDSC, F4/80neg) (A), Monocytic MDSC (M-MDSC, F4/80pos) (B), total T cells (C) and non-lymphatic dendritic cells (NLT DC) (D) in sgCTL (black) and sgPRMT7-1 (red) B16.F10 tumors. (A-D) The data represents the mean ± SD and is from two to three independent experiments with a minimum of 3 mice per group. Each dot represents one mouse. Statistical significance was calculated using paired student t test. (*p <0.05; **p <0.01; ns: non-significant).
(E) Representative flow cytometry plots using anti-CD45, anti-CD3 and anti-CD8 antibodies in sgCTL and sgPRMT7-1 B16.F10 tumors.
(F) Quantification graphs from (E) showing frequencies of double positive CD3pos, CD8pos cells (gated on CD45pos cells) in sgCTL (black) and sgPRMT7-1 (red) B16.F10 tumors. Cells were gated as indicated and the relative percentage of cells shown; n=13 animals per group; data from three independent experiments. Statistical significance was calculated using paired student t test (****p <0.0001).
(G) Representative immunofluorescent images of sgCTL and sgPRMT7-1 tumor sections treated with anti-CTLA-4 and anti-PD-1 in vivo and stained with anti-CD8α antibody at 10x magnification. DAPI, 4’,6-diamidino-2-phenylindole, was used to visualize nuclei by Zeiss confocal microscopy. A minimum of 3 biological replicates were used for each experiment.
(H) Immunofluorescence intensity of the CD8 staining done in (G) using ImageJ software. Bar graphs show fluorescence mean intensity ± SEM. Statistical significance was calculated using unpaired student t test (****p <0.0001).
(I) Representative pictures of subcutaneous sgCTL and sgPRMT7-1 derived-melanomas in C57BL/6J mice treated with anti-CTLA-4 and anti-PD-1 (day 21, top) and corresponding representative images of H&E-stained tumor sections (day 21, bottom). Black arrowheads indicate the pigmented areas.
(J) RT-qPCR analysis of Mitf, Gp100 and Melan-A mRNA transcripts in sgCTL, sgPRMT7-1 and sgPRMT7-2 B16.F10 cells. Data are mean ± SD. Data representative for 3 independent experiments. Statistical significance was calculated by unpaired student t test (**p <0.01; ***p <0.001; ****p <0.0001).
(K) Western blot showing the expression of the indicated proteins (PRMT7, MITF, GP100 and Melan-A) in sgCTL, sgPRMT7-1 and sgPRMT7-2 B16.F10 melanoma cells. β-actin was used as the loading control. Molecular mass markers are indicated in the left in kDa. Data are representative of three independent experiments. Cell pellet representative images were shown (bottom; note the black pellet in sgPRMT7 clones).