Fig. 2: DOX upregulates antigen-presenting molecules in human GBM cells.

a Representative multiplex immunofluorescence images illustrating SOX2⁺, HLA-ABC⁺, and HLA-DR⁺ cells in pre-treatment and on-treatment GBM samples. b, c Dot plot showing the cell density of SOX2⁺ HLA-ABC⁺ (b) and SOX2⁺ HLA-DR⁺ (c) cells in pre-treatment and on-treatment GBM samples. n = 4 paired GBM samples. A mixed effects model was constructed considering DOX+aPD-1 treatment as a fixed effect and patients as a random effect influencing SOX2⁺ HLA-ABC⁺ and SOX2⁺ HLA-DR⁺ cell densities. P values were obtained by a chi-squared test of the likelihood ratio test of the full model including DOX+aPD-1 treatment as a fixed effect against the model without the fixed effect. d Schematic of the flow cytometry experiment and gating strategy performed to assess the effect of different DOX concentrations on the expression of antigen-presenting molecules in the PDX cell lines, GBM6 and GBM63. e, f (left) Bar plots showing the expression of HLA-ABC and HLA-DR assessed as MFI values in GBM6 (e) and GBM63 (f). n = 3 biological replicates per condition. (right) Representative histograms showing the expression of HLA-ABC and HLA-DR in control and 0.15-0.6 μM DOX-treated cells. Histograms are representative data from three biological replicates. g Experimental setup of CD8⁺ T cells isolated from OT-1 mice co-cultured with GL261-OVA cells treated with varying concentrations of DOX to assess T cell activation and proliferation. h (left) Scatter plot displaying CD8⁺ T cell proliferation after co-culture with GL261-OVA cells, untreated (red dots) or treated with 0.3 µM DOX (blue dots). (right) Bar graph showing the CD8⁺ T cell Proliferation Index for untreated and DOX-treated GL261-OVA cells at different concentrations (0, 0.1, 0.3, and 0.6 µM). The Proliferation Index is calculated based on the fluorescence intensity decay of BV421, indicating the division of T cells. n = 3 biological replicates per condition. P values were obtained by one-way ANOVA with post hoc Dunnett’s multiple comparisons test in e, f and h. Source data are provided as a Source Data file. Data are presented as mean ± SEM in e, f and h.