Additional file 1: of Local angiotensin II contributes to tumor resistance to checkpoint immunotherapy

Figure S1. The efficacy of knockdown was detected through qRT-PCR and western blotting. The efficacy of knockdown in 4T1 cells (A) and CT26 cells (B). Figure S2. Role of AGT gene-silencing in growth of 4T1 and CT26 cells. AGT-silencing didn't significantly inhibit tumor growth of 4T1 (A) and CT26 cells (B) in NOD/SCID mice. (C) AGT silencing inhibited CT26 growth in BALB/c mice and the depletion of CD8+ T cells reversed this role. Figure S3. Strategy of combined AGT gene-silencing and PD1 blockade. Figure S4. AngII signaling blockage sensitizes tumors to checkpoint immunotherapy. AGT gene-silencing in 4T1 (A) and CT26 cells (B) rendered tumors more sensitive to anti-PD1 immunotherapy. Figure S5. Percentages of CD3+, CD45+, and CD11b+ cells in 4T1 tumors from BALB/c mice treated with different Ang II-receptor blockers. (A), (B), and (C) correspond to Figure 3A, 3E, and 3F. Figure S6. Representative FACS plot of Teffs (CD8+CD44+) and Tregs (CD4+Foxp3+) in AGT-silenced and control 4T1 tumors from BALB/c mice. Bar chart (right) indicated statistic difference (n = 3). Figure S7. α-SMA, CD8 or CD206 positive cells in hypoxic regions of 4T1 tumors. Positive cells were counted in 4 random 400× microscope visions in hypoxic regions of AGT-silenced or control tumors which were from 3 independent mice, (n = 12). Figure S8. The content of TAMs, Mo-MDSCs and G-MDSCs in shRNA-AGT 4T1 tumors. (A-B) Representative FACS plot. (C) Percentages of these populations (n = 3). Figure S9. AGT-silencing triggers an immune-activating cytokine profile in hypoxic 4T1 cells. The levels of 6 cytokinesby ELISA analysis (A). Gene Ontology analysis showed hypoxia induced significantly higher frequencies of cytokines which were associated with 39 biological processes (B, p < 0.01) and 17 signaling pathways (D, left, p < 0.05). The cytokines influenced by AGT-silencing in hypoxia condition were associated with 58 biological processes (C, p < 0.01) and 22 signaling pathways (D, right, p < 0.05). Table S1. Antibodies for Immunofluorescence. (DOCX 48272 kb