Activated anti-tumor immunity of senescent B16-F10 cells in vitro.

(A) Images of senescence-associated β-galactosidase staining of B16-F10 cells (top panel scale bar: 200 μm; bottom panel scale bar: 50 μm). (B) The concentrations of HMGB1 and ATP in the supernatant of untreated cells and STCs. (C)The percentage of CD11c+ MHCII+ DCs after treatment with untreated cells and STCs. (D) The percentage of CD3+ CD8+, CD8+ Granzyme B+, and CD8+ IFN-γ+ T cells after treatment with untreated cells and STCs. (E) The percentage of CD3-NK1.1+, NK1.1+ Granzyme B+, and NK1.1+ IFN-γ+ cells after treatment with untreated cells and STCs. The concentrations of (F) Perforin and (G) Granzyme B in the supernatant of untreated cells and STCs (n=4). Statistical significance was assessed by unpaired Student’s t-test. Data were presented as mean ± SD. Statistical significance was determined as *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Immunization with senescent B16-F10 cells promoted anticancer immunity.

(A) Schedule of STCs treatment in a recurrent melanoma model. Created with BioRender.com. (B) The average tumor volume change of mice treated with different groups (n=5). (C) The tumor weight of the first tumor (n=5). (D) The tumor weight of recurrent tumor (n=4). (E) The percentage of CD3+ CD4+ and CD3+ CD8+ T cells within tumors treated with different groups (n=5). (F) The percentage of CD4+ and CD8+ T cells within spleens of mice treated with different groups (n=3). (G) The percentage of CD3- NK1.1+ and NK1.1+ Granzyme B+ cells within spleens of mice treated with different groups (n=3). Statistical significance was assessed by one-way ANOVA and Tukey multiple comparisons tests. Data were presented as mean ± SD. Statistical significance was determined as *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Celecoxib promoted the DCs recruitment.

(A–B) COX2 expression in senescent and normal B16-F10 cells with or without celecoxib treatment detected by western blotting and statistical analysis. (C) PGE2 concentrations in the supernatant of different treatments. (D) Schematic diagram of Transwell coculture system. Created with BioRender.com. (E) The quantitative analysis of migrated DCs after different treatments. Statistical significance was assessed by one-way ANOVA and Tukey multiple comparisons tests. Data were presented as mean ± SD (n=3). Statistical significance was determined as ns (not significant, p > 0.05), **p < 0.01, ***p < 0.001, ****p < 0.0001.

Preparation and characterization of STCs+CLX-Lipo@Gel.

(A) Schematic diagram of the preparation process for STCs+CLX-Lipo@Gel. Created with BioRender.com. (B) Particle size and (C) zeta potential of CLX-Lipo. (D) Transmission Electron Microscopy image of CLX-Lipo (scale bar: 100 nm). (E) Photograph of STCs+CLX-Lipo@Gel post-gelatinization. (F) Scanning Electron Microscopy (SEM) image of STCs+CLX-Lipo@Gel. (G) Rheological evaluation of STCs+CLX-Lipo@Gel over time. (H) Quantitative analysis of cell viability for STCs encapsulated within chitosan hydrogel and (I) representative fluorescence images for Live/Dead staining (green: Calcein-AM; red: PI; scale bar: 40 μm). Statistical significance was assessed by one-way ANOVA and Tukey multiple comparisons tests. Data were presented as mean ± SD (n=3). Statistical significance was determined as ns (not significant, p > 0.05), *p < 0.05, **p < 0.01.

STCs+CLX-Lipo@Gel promoted maturation of DCs and activation of CD8+ T cells.

(A) Schematic diagram of coculture of STCs+CLX-Lipo@Gel and BMDCs. (B) Flow cytometry analysis and (C) quantitative analysis of the percentage of CD11c+ MHCII+ cells after various treatments. (D) Flow cytometry analysis and (E) quantitative analysis of the percentage of cDC1 in matured DCs after various treatments. (F) Schematic diagram of coculture of STCs+CLX-Lipo@Gel and splenic lymphocytes. The percentage of (G) CD8+ T cells, (H) Granzyme B+, and (I) IFN-γ+ in CD8+ T cells after various treatments. (J) Flow cytometry histograms depicting CMFDA fluorescence intensity of CD8+ cells. (K) Quantitative analysis of the percentage of proliferative CD8+ T cells after various treatments. Statistical significance was assessed by one-way ANOVA and Tukey multiple comparisons tests. Data were presented as mean ± SD (n=3). Statistical significance was determined as, **p < 0.01, ***p < 0.001, ****p < 0.0001.

STCs+CLX-Lipo@Gel activated NK cells and restored the functionality of the NK-DC axis.

(A) Schematic diagram of coculture of STCs+CLX-Lipo@Gel and NK cells. The (B) NKG2D+ and (C) NKp46+ cell percentages in NK cells following various treatments. (D) Quantitative analysis of the percentage of proliferative NK cells after various treatments. (E) The ability of NK cell-mediated killing B16-F10 cells after various treatments detected by CCK8 assay. The concentrations of (F) CCL5, (G) XCL1, and (H) PGE2 in the supernatant of NK cells after various treatments. (I) Schematic diagram of Transwell coculture system. Created with BioRender.com. (J) The quantitative analysis and (K) representative images (scale bar: 200 μm) of migrated DCs after different treatments. Statistical significance was assessed by one-way ANOVA and Tukey multiple comparisons tests. Data were presented as mean ± SD (n=3). Statistical significance was determined as ns (not significant, p > 0.05), *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

In vivo biodistribution, prophylactic and immunoregulatory effects of STCs+CLX-Lipo@Gel.

(A) Representative IVIS images at different time points of DiR-labeled STCs encapsulated in chitosan hydrogel and (B) semi-quantitative analysis of fluorescent signal (n=3). (C) Schedule of various treatments in a melanoma challenge model. Created with BioRender.com. (D) Tumor-free rate of different groups on the 7th day. (E) The average tumor growth curve of different groups. (F) The individual tumor growth of each group. (G) The percentage of CD3- NK1.1+ cells in peripheral blood after various treatments. The percentage of (H) NKG2D+ and (I) NKp46+ cells in NK cells in peripheral blood after various treatments. The percentage of (J) CD4+ and (K) CD8+ effector memory T cells (TEMs) in spleens after various treatments. Statistical significance was assessed by one-way ANOVA and Tukey multiple comparisons tests. Data were presented as mean ± SD (n=5). Statistical significance was determined as *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Antitumor and immune-stimulating effects of STCs+CLX-Lipo@Gel in the subcutaneous melanoma model.

(A) Schedule of various treatments in subcutaneous melanoma model. Created with BioRender.com. (B) Tumor growth curve, (C) tumor image, and (D) tumor weight of different groups. (E) The percentage of matured DCs (CD11c+ MHCII+ and CD11c+ CD80+ CD86+) and cDC1 in peritumoral lymph nodes after various treatments. (F) The percentage of CD4+ T cells in tumor tissues after various treatments. (G) The percentage of CD8+ T cells and activated CD8+ T cells (CD8+ Granzyme B+) proliferative CD8+ T cells (CD8+ Ki67+) in tumor tissues after various treatments. (H) The percentage of NK cells (CD3- NK1.1+), and activated NK cells (NK1.1+ NKG2D+, NK1.1+ NKp46+, and NK1.1+ Granzyme B+) in tumor tissues after various treatments. Statistical significance was assessed by one-way ANOVA and Tukey multiple comparisons tests. Data were presented as mean ± SD (n=5). Statistical significance was determined as *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Antitumor effect of STCs+CLX-Lipo@Gel in various tumor models.

(A) Schedule of various treatments in melanoma brain metastasis model. Created with BioRender.com. (B) Survival rate and (C) median survival of mice during the treatment period (n=12). (D) Schedule of various treatments in an orthotopic pancreatic cancer model. Created with BioRender.com. (E) Tumor image of pancreatic tumors after different treatments (n=8). (F) Representative IVIS images of pancreatic tumors at different time points after different treatments and (G) semi-quantitative analysis of bioluminescence (n=3). (H) Pancreatic tumor weight of different groups (n=8). Statistical significance was assessed by one-way ANOVA and Tukey multiple comparisons tests. Data were presented as mean ± SD. Statistical significance was determined as ***p < 0.001, ****p < 0.0001.

Senescent B16-F10 cells characterization.

(A) mRNA expression levels of Cdkn1α, IL1α, CCL2, CXCL2, and MMP9 of senescent B16-F10 cells; (B) The concentrations of IL-6 and CXCL1 in the supernatant of senescent B16-F10 cells. Statistical significance was assessed by unpaired Student’s t-test. Data were presented as mean ± SD. Statistical significance was determined as **p < 0.01, ***p < 0.001, ****p < 0.0001.

Alterations in immune-related cytokines and NK cell-activated receptor ligands at the gene expression level.

(A) mRNA expression levels of chemokines CCL2 and CCL5 in STCs; (B) mRNA expression levels of NKG2D ligands ULBP-1 and RAE-1, and DNAM-1 ligand CD155 in STCs; (C) mRNA expression levels of immune-related cytokines IFN-γ, IL-2, IL-12, and IL-15. Statistical significance was assessed by unpaired Student’s t-test. Data were presented as mean ± SD. Statistical significance was determined as *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Tumor image of the first tumor (n=5).

The percentage of (A) activated CD8+ T cells (CD8+ Granzyme B+), proliferative CD8+ T cells (CD8+ Ki67+), (B) matured DCs (CD11c+ MHCII+ and CD11c+ CD80+ CD86+) in tumor tissues after different treatments.

Statistical significance was assessed by one-way ANOVA and Tukey multiple comparisons tests. Data were presented as mean ± SD. Statistical significance was determined as ns (not significant, p > 0.05), *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Flow cytometry histograms illustrating fluorescence intensity of (A) NKG2D+, (B) NKp46+, and (C) proliferative NK cells.

Tumor inhibition rate of different groups.

Statistical significance was assessed by one-way ANOVA and Tukey multiple comparisons tests. Data were presented as mean ± SD. Statistical significance was determined as ns (not significant, p > 0.05), *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Biosafety evaluation of STCs+CLX-Lipo@Gel.

(A) Body weight changes during different treatments; (B) Organ coefficients after different treatments; (C) Histological examination of the major organs in each group; (D) Tumorigenic potential of senescent B16-F10 cells. Imaging was conducted on the 42nd day post-subcutaneous administration of varying quantities of senescent B16-F10 cells. Data were presented as mean ± SD (n=5).

Raw data of western blots in Figure 3A.

Drug encapsulation efficiency and drug-loading capacity of CLX-Lipo.

The gelation time of CS@Gel at different concentrations of gelating agent.

Primer sequences for qPCR analysis.