Fig. 4

Tumor acidosis increases PD-L1 expression on cancer cells and alleviates the immigration of CD3⁺ T cells. A Representative pH_e maps overlaid on T₂-weighted axial MRI images of MC38^wt tumor-bearing mice injected with iopamidol (i.v.) at day 10 after the cancer cell injection. Mice with intact IFN-γ signaling received either NaHCO₃-enriched water three days prior to cancer cell injection or regular drinking water (Control). Individual images of the tumors are shown in Fig. S7. B Average pH_e across the whole tumor. The NaHCO₃ treatment significantly increased the tumor pH_e measured using acidoCEST MRI (n = 3–4 animals per group; 2 mice were excluded from the quantitative analysis because the measured pH_e-values were out of the calibration range, see Fig. S7, statistics: one-tailed Mann–Whitney test). C The percentage of necrosis in MC38^wt tumors from the NaHCO₃ and Control groups was quantified using H&E staining, as shown in Fig. S8. D Representative H&E staining of MC38^wt tumors isolated from experimental mice on day 18 after control or NaHCO₃ treatment. H&E staining show the well-delimited necrotic area in the MC38^wt tumor of a control treated mouse (center, pink area). In contrast, within the MC38^wt tumor of a NaHCO₃ treated mouse the necrosis is diffuse surrounded by areas of hypoxia reflected by abundant pyknotic cells (hyperchromatic; magnitude: 10x; scalebar: 100 µm). E Representative PD-L1 IHC of a MC38^wt tumor isolated from experimental mice on day 18 after control treatment. (magnitude: 12.5x, scalebar 2 mm; insert: 50x; scalebar 500 µm). F Representative fluorescence microscopy images of PD-L1, the proliferation marker Ki67 in MC38^wt tumors isolated on day 18 after treatment with NaHCO₃ and control groups. G PD-L1 expression was quantified in proliferating and non-proliferating cells from MC38^wt tumor regions. Statistics: two-tailed Mann–Whitney test. H Representative CD3 immunohistochemistry of s.c. MC38^wt tumors derived from wild-type C57BL/6 J (day 18) or IFN-γ^−/− mice (day 17) after the four different treatment conditions. Animals received regular drinking water (control), NaHCO₃ in water (NaHCO₃), anti-PD-L1 mAb or NaHCO₃ & anti-PD-L1 mAb. N = 3 – 4 representative tumors of each experimental group were subjected to CD3 staining. (magnitude: 400x; scalebar: 100 µm)