Fig 2: Evaluation of SPIN-mediated sono-immunotherapy in Panc02 tumor mouse model.a Schedule for the establishment of primary and distant tumors, triple systemic injection of SPINs (0.2 mL, 0.6 mg/mL) via tail vein, US irradiation (1.0 MHz, 1.2 W/cm2, 50% duty cycle, 10 min), and analysis of immune responses. b, c Relative tumor volumes of primary (b) and distant (c) tumors of Panc02 tumor-bearing C57BL/6 mice (n = 6) after systemic injection of saline, free-drug mixture (on day 0, 3, and 6, 4 mg/kg body weight for NLG919 and aPD-L1), or SPIND2 (0.2 mL, 0.6 mg/mL) with or without US irradiation (1.0 MHz, 1.2 W/cm2, 50% duty cycle, 10 min). SPIND2 + US versus drug + US: P < 0.0001 for primary tumors (b); SPIND2 + US versus drug: P < 0.0001 for distant tumors (c). d Survival curves of Panc02 tumor-bearing C57BL/6 mice (n = 10) receiving different treatments as indicated. e Schematic illustration of treatment of rechallenged tumor mouse models using SPINs. f Growths of rechallenged tumors in Panc02 tumor-bearing mice after injection of saline or SPIND2 (0.2 mL, 0.6 mg/mL) with US irradiation (1.0 MHz, 1.2 W/cm2, 50% duty cycle, 10 min) (n = 5). Saline versus SPIND2 + US: P < 0.0001. g The survival curves of Panc02 tumor-bearing C57BL/6 mice after different treatments followed by tumor rechallenge (n = 10). h Flow cytometry analysis of populations of effector memory T cells in the spleen of Panc02 tumor-bearing C57BL/6 mice after different treatments followed by tumor rechallenge (n = 4). Saline versus SPIND2 + US: P = 0.0059. i Differentially expressed gene numbers in tumor tissues of mice after different treatments. j Relative expression of Carl, Hmgb1-ps1, Hmgb1-ps2, Cd80, Cd86, Cd40, Pdcd1, Cd3e, Cd8a, Ifng, Gzmb, Cxcl1, Cxcl2, Cxcl9, Cxcl10, Cxcl11, Ccl4, Ccl5, Il1b, Il2, Il6, Il7, Il15, Ido1, and Cd274 in tumors of Panc02 tumor-bearing mice after different treatments (the experiment was repeated independently five times with similar results). k Unsupervised hierarchical clustering of relative gene expression in tumors of Panc02 tumor-bearing C57BL/6 mice after different treatments (n = 5). Data are presented as mean values ± SD. Statistical significance was calculated via two-tailed Student’s t test; **P < 0.01, ***P < 0.001. Source data are provided as a Source Data file.

Fig 3: LIA induced reduced systemic toxicity and enhanced immune responses in vivo.a Fluorescence images of the 4T1 tumour-bearing mice at 3, 6, 9, 12, 24 h post intravenous administration of free Ce6 and LIA (Ce6 3 mg/kg), 2 mice per group. b Fluorescence images of major organs (heart, liver, spleen, lung, kidney) and tumour tissues were collected at 24 h post injection, 2 mice per group. c, d ELISA analysis of TNF-α (c) and IL-6 (d) concentrations in serum collected from mice 24 h after different treatments (PBS, LIA, LIA+L, CpG and LPS). e, f Images of spleens (e) and weight of spleens (f) from each group 14 days after treatment with different formulations. g, h The tumour-bearing mice were i.v. injected with PBS, free Ce6, DP-Ce6 and LIA at an equivalent Ce6 dose of 3 mg/kg, then NIR light was used to irradiate (665 nm, 0.15 W/cm2, for 30 min) the tumour site at 4 h post injection. The treatments were performed in triplicates every other day. DC maturation was analysed 72 h after the final treatment (gated on CD11C+ cells). Representative flow cytometry (g) and statistical analysis (h) of CD80+ CD86+ DCs in LNs of mice after different treatments. i, j Representative flow cytometry (g) and statistical analysis (h) of CD8+ T cells in spleens of mice 7 days after different treatments. Data in c, d, f, h, j are presented as mean ± s.d. (n = 3). Statistically significant differences between groups were identified by one-way ANOVA. ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05.

Fig 4: ALDH2 regulates macrophage polarization and inflammation through the cGAS-STING signaling pathway. A, B Pro-inflammatory macrophages (CD68+CD86+) were measured by flow cytometry in human PBMCs treated with oxLDL with or without RU.521 (n = 3). C, D Anti-inflammatory macrophages (CD68+CD163+) were measured by flow cytometry in human PBMCs treated with IL-4/IL-13 with or without RU.521 (n = 3). E, F Pro-inflammatory macrophages (F4/80+CD86+) were measured by flow cytometry in BMDMs of WT and ALDH2-KO mice treated with oxLDL with or without RU.521 (n = 3). G, H Anti-inflammatory macrophages (F4/80+CD163+) were measured by flow cytometry in BMDMs treated with IL-4/IL-13 with or without RU.521 (n = 3). I ALDH2-KO BMDMs and WT BMDMs treated with oxLDL with or without RU.521. RT-qPCR of iNOS, CXCL-10, IL-6, and TNF-α in BMDMs (n = 3–4). J ALDH2-KO BMDMs and WT BMDMs treated with IL-4/IL-13 with or without RU.521. RT-qPCR of TGF-β (n = 8) and IL-10 (n = 6) in BMDMs. K Western blot of iNOS in ALDH2-KO BMDMs and WT BMDMs (n = 4). Data are presented as the mean ± SD. One-way ANOVA was used for the analysis in (B, D, F, H, I). Brown-Forsythe and Welch ANOVA tests followed by Dunnett's T3 multiple comparisons test was used for the analysis in (J). Western blot bands were detected under the same conditions (K). P < 0.05 was considered significant.

Fig 5: Macrophage phenotype regulation based on NM-MS. (A) Flow cytometry of DiR-positive macrophages. (B) Flow cytometry and (C) histograms of CD86+ and CD206+ macrophages (gated by F4/80). (D) Immunofluorescence and (E) semiquantitative analysis of iNOS and CD206 in macrophages cocultured with fiber scaffolds. (F) Western blotting and (G) semiquantitative analysis of iNOS and CD206 protein expression. (H) Real-time OCRs of macrophages and (I) semiquantitative analysis of basal respiration, maximal respiration, and ATP production (n = 3, error bars, means ± SD; all analyses were performed using one-way ANOVA with Tukey’s post hoc test *P < 0.05 and **P < 0.01).