Fig 2: SN-38 or metformin significantly suppresses tumor growth in vivo monitored by bioluminescent imaging. (A) Timeline (weekly) of bioluminescent signals in a representative showed growing mouse tumor injected with 5.0×106 ID8-luc2 cells. (B) In the DMSO control group, he bioluminescence level increased significantly at weeks 4 and 5 (*p<0.05, **p<0.005). All scales in photons/second/cm2/steradian. (C) This figure shows flow cytometry analysis of tumor cells treated with controls or two different drugs (DMSO, metformin, or SN-38). Gray color: isotype control; blue color: DMSO-treated tumor cells; green color: metformin-treated tumor cells; and orange color: SN-38-treated tumor cells. The right panel shows elevated FOXO3 expression on each drug-treated tumor cells (n=3, *p<0.05, **p<0.005). (D) Gray color: isotype control; blue color: DMSO-treated tumor cells; green color: metformin-treated tumor cells; and orange color: SN-38-treated tumor cells. The right panel shows decreased PD-L1 expression on each drug-treated tumor cells (n=3, *p<0.05, **p<0.005). (E) This figure shows flow cytometry analysis of CD3 or NKp46 expressing immune cells isolated from mouse tumors. CD3 (T cell marker) or NKp46 (NK cell marker) shows each changed population in the drug-treated tumor. Metformin or SN-38 treated tumor tissues show increased CD3 positive cells or NKp46 positive cells. The right panel shows a significantly increased CD3 or NKp46 positive population in tumor tissues (n=3, *p<0.05, **p<0.005). (F) This figure shows flow cytometry analysis of CD8 or NKp46 expressing immune cells isolated from mouse tumors. CD8 (cytotoxic T cell marker) or NKp46 (NK cell marker) shows each changed population in the drug-treated tumor. Metformin or SN-38 treated tumor tissues show increased CD8 positive cells or NKp46 positive cells. The right panel shows a significantly increased CD8 (cytotoxic T cells) positive population in tumor tissues (n=3, *p<0.05). DMSO, dimethylsulfoxide; NK, natural killer; PD-L1, programmed death-ligand 1.

Fig 3: Preventing ESCRT-dependent cell membrane repair enhanced pyroptosis and augmented antitumor immune response.a Flow cytometry assay of dendritic cell maturation in the lymph nodes after treatments with PBS, aPD-1, VNP@Gel, GD/EI-NP@Gel, VNP-GD@Gel, VNP-GD/EI-NP@Gel and VNP-GD/EI-NP@Gel+aPD-1 (n = 4 mice). b Flow cytometry assay of CD8+ T cell infiltration in the tumor tissue after treatments as mentioned above, n = 4 mice. c Dendritic cell maturation in the lymph nodes after different treatments. Data are shown as mean ± s.d. and analyzed with one way ANOVA followed by Tukey’s multiple comparisons test, n = 4 mice (VNP-GD@Gel vs. VNP-GD/EI-NP@Gel: ***P = 0.0008; VNP-GD@Gel vs. VNP-GD/EI-NP@Gel+aPD-1: ***P = 0.0002; VNP-GD/EI-NP@Gel vs. VNP-GD/EI-NP@Gel+aPD-1: nsP = 0.4116). d The number of CD8+ T cells per mg tumor tissue after different treatments. Data are shown as mean ± s.d. and analyzed with one way ANOVA followed by Tukey’s multiple comparisons test, n = 4 mice (VNP-GD@Gel vs. VNP-GD/EI-NP@Gel: *P = 0.0215; VNP-GD@Gel vs. VNP-GD/EI-NP@Gel+aPD-1: ****P < 0.0001; VNP-GD/EI-NP@Gel vs. VNP-GD/EI-NP@Gel+aPD-1: **P = 0.0042). e The number of Granzyme B+CD8+ T cells per mg tumor tissue after different treatments, n = 4 mice (VNP-GD@Gel vs. VNP-GD/EI-NP@Gel: **P = 0.0065 VNP-GD@Gel vs. VNP-GD/EI-NP@Gel+aPD-1: ****P < 0.0001; VNP-GD/EI-NP@Gel vs. VNP-GD/EI-NP@Gel+aPD-1: **P = 0.0028). HMGB1 (f), IFNγ (g), and TNFα (h) expressions in the tumor tissues after different treatments detected by ELISA. Data are shown as mean ± s.d. and analyzed with one way ANOVA followed by Tukey’s multiple comparisons test, n = 4 mice. (f, VNP-GD@Gel vs. VNP-GD/EI-NP@Gel: **P = 0.0080; VNP-GD@Gel vs. VNP-GD/EI-NP@Gel+aPD-1: **P = 0.0017; VNP-GD/EI-NP@Gel vs. VNP-GD/EI-NP@Gel+aPD-1: nsP = 0.5319. g, VNP-GD@Gel vs. VNP-GD/EI-NP@Gel: *P = 0.0256; VNP-GD@Gel vs. VNP-GD/EI-NP@Gel+aPD-1: ***P = 0.0002; VNP-GD/EI-NP@Gel vs. VNP-GD/EI-NP@Gel+aPD-1: *P = 0.0127. h, VNP-GD@Gel vs. VNP-GD/EI-NP@Gel: **P = 0.0047; VNP-GD@Gel vs. VNP-GD/EI-NP@Gel+aPD-1: ****P < 0.0001; VNP-GD/EI-NP@Gel vs. VNP-GD/EI-NP@Gel+aPD-1: **P = 0.0053.) i Luminex-based quantification of cytokines and chemokines, including IL-1α, IFN-γ, TNF-α, MCP-1, IL-12, IL-1β, IL-6, IL-27, IL-17A, GM-CSF (n = 5 mice). Source data are provided as a Source Data file.

Fig 4: The composition of tumor-associated macrophages varies with treatment modality(A) Immunohistochemical staining with F4/80 shows a dramatic increase in macrophages with radiation therapy. Student’s t test. n = 3 animals per treatment group. Rsssssesults are expressed as mean ± SEM. **p < 0.01. Field: 3,190 × 1,974 μm. Scale bar: 200 μM.(B) UMAP plots of myeloid cells from treated tumors show higher microglia-derived and monocyte-derived macrophages with GDC-0449 and radiation treatment, respectively.(C) RNAscope in situ (micrograph, right) with Siglech (black) and Clec12 (red) probes show the large increase in Clec12a-expressing, monocyte-derived macrophages with radiation that is consistent with scRNA-seq data (UMAP, left). Scale bar: 50 μM.(D) Heatmap of TAMs from irradiated tumors shows clusters 3 and 5 have higher expression of genes associated with migration of the cells from the vasculature and IL-10 signaling.(E) Distribution (UMAP, left) and expression (violin plot, right) of Ly6c2 and Ccr2 show high levels in monocyte-derived clusters that accumulate after radiation.(F) T cell proliferation assay shows Cd11b+Cd45hiLy6G−Ly6chi, but not Cd11b+Cd45hiLy6G−Ly6clo, from irradiated Ccr2WT tumors suppresses proliferation of CD3/CD28-stimulated splenic CD8+ cells. The x axis shows carboxyfluorescein succinimidyl ester (CFSE)-associated fluorescence in CD8+ T cells. Student’s t test. n = 3 (triplicate samples from one mouse). An additional two mice were analyzed to confirm differences in suppression of T cell proliferation between the two cell types. Results are expressed as mean ± SEM. ***p < 0.001.See also Figure S5.

Fig 5: PD-L1 expression is very positively associated with c-Myc or STAT3 expression in patients with various tumors, and a model for the mechanism is proposed. (A–C) Using IF analysis, we determined the protein expression of PD-L1 and c-Myc in TMAs of OvCa (A), invasive BCa (B), and HCC (C) specimens. Each tumor TMA slide was incubated with anti-PD-L1 or anti-c-Myc Abs and followed by specific fluorescent secondary Abs and IF analysis, as described previously. (D–F) We analyzed both PD-L1 versus STAT3 mRNA expression data in human primary ovarian tumor (D), breast tumor (E), and HCC tumor (F) tissues from the Cancer Genome Atlas (TCGA) datasets using cBioPortal.32 PD-L1 expression was positively associated with STAT3 expression in ovarian ad breast tumors. P values between the PD-L1 group versus the STAT3 group were shown. (G and H) We analyzed PD-L1 versus STAT3 and PD-L1 versus FOXO3 mRNA expression data in human primary pediatric neuroblastoma and stomach cancer tissues from TCGA datasets. P values between the tested groups were shown. (I) We analyzed PD-L1 versus FOXO3 mRNA expression data in various human primary tumor tissues (n=2158) from TCGA datasets. P values between the tested groups were shown. (J) A schema depicts a proposed model for the FOXO3-mediated blocking dual c-Myc-PDL1 and STAT3-PD-L1 pathways by SN-38 to promote NK or CD8+ T cell antitumor immunity. Abs, antibodies; BCa, breast cancer; HCC, hepatocellular carcinoma; IF, immunofluorescence; NK, natural killer; OvCa, PD-L1, programmed death-ligand 1; TMA, tissue microarray.