Fig. 3: ICI-resistant RenCa tumors express upregulated Gpnmb expression and immunosuppressive phenotypes.

a Generation of ICI-resistant RenCa cells. RenCa P0 (parental cells) were implanted subcutaneously into BALB/c mice (n = 4) and treated with anti-PD1 or control Ab (shown by blue reverse arrows). After five shots, the largest tumors (shown by red lines) were excised, cultured in vitro, and implanted into new mice and similarly treated (termed P0 to P4). This enrichment was repeated four times. Tumor volume is shown. b P0 to P4 cells were assayed by qRT-PCR for RNA expression of Gpnmb and PDL1 to Gapdh (mean ± SEM, n = 3). c Dot plots of flow-cytometric analysis of P0 and P4 cells fluorescently stained for expression of Gpnmb or PDL1, with mean fluorescence intensity (MFI). d Kinetics of sGpnmb secretion by in vitro cultured P0 and P4 cells. e Growth curves of P0 and P4 subcutaneous tumors in mice (n = 4) treated with anti-PD1 Ab (by closed arrows on the top). Blue arrows show the date of blood drawn. f Blood sGpnmb levels in mice treated with Ab (e) are plotted at varying time points post-implantation of P0 or P4 cells. −3 means 3 days prior to tumor implantation. Untreated P0 and P4 tumors (~1 cm size) are IHC-stained with control IgG (Ctrl), or Ab to CD4, CD8, CD11b, CD31, or tryptase (scale bar, 100 μm, g) and assayed for tumor density of each cell population (h) (mean ± SEM, n = 5); (Ab-stained area μm²/mm² microscopic view), average of nine different views ± SD, from five different tumors. Bone marrows of the same mice were analyzed by qRT-PCR for Gpnmb or PDL1 RNA (i) (mean ± SEM, n = 3). and by flow cytometry for frequencies of different leukocyte populations (j) (mean ± SEM, n = 4). Representative of three separate experiments except (a–c). p values are shown, using Student’s t-test comparing two conditions.