Journal: Signal Transduction and Targeted Therapy

Article Title: Condensate nanovaccine adjuvants augment CD8 + T-Cell-dependent antitumor immunity through mtDNA leakage-triggered cGAS-STING axis activation

doi: 10.1038/s41392-025-02447-w

Figure Lengend Snippet: Evaluation of the in vivo antitumor efficacy and immunological mechanisms of OVA PCD. a Schematic timeline of vaccine and B16-OVA tumor cell inoculation in the C57BL/6 mouse model. The mice were immunized three times via subcutaneous injection at the tail base with OVA PCD, free OVA, or saline. On the seventh day after the final immunization, 5 × 10 5 B16-OVA tumor cells were subcutaneously inoculated into the right thighs of the mice. b Tumor growth curves within 18 days after the subcutaneous inoculation of B16-OVA tumor cells. Compared with that in the free OVA or saline group, tumor growth in the OVA PCD group was significantly inhibited ( n = 5). c Body weight change curves after tumor inoculation. The OVA PCD group showed no significant change in body weight, indicating that there was no apparent systemic toxicity ( n = 4–5). d Individual tumor growth curves for each group after subcutaneous inoculation of B16-OVA cells. e Tumor images from day 18 posttumor inoculation. “x” indicates mice that died on day 18 posttumor inoculation (experimental endpoint). The tumor volume data of this deceased mouse from the statistical analysis for day 18 are included in Fig. 4b. The data in b , c are presented as mean ± s.d. from two independent experiments. Groups were compared via one-way ANOVA with Tukey’s post hoc test. Flow cytometry analysis of the DC maturation rate (CD80 + CD86 + CD11c + ) ( f ) and the percentage of IFN-γ + CD8 + T cells ( g ) in the spleens of mice subjected to different treatments ( n = 3). Representative flow cytometry results ( h ) and quantification ( i ) of OVA antigen peptide-specific CD8 + T cells after ex vivo restimulation with the SIINFEKL peptide (8 μg/mL) ( n = 3). Representative images ( j ) and statistical analysis ( k ) of IFN-γ spot-forming cells in splenocytes after ex vivo restimulation with the SIINFEKL peptide (8 μg/mL) via the ELISPOT assay ( n = 3). l Evaluation of the cytotoxic effect of effector splenocytes (E) on target B16-OVA-GFP cells (T) after coincubation for 24 h at specified ratios, assessing the in vitro tumor cell-targeting cytotoxicity of effector T cells ( n = 3). Statistical analysis data in ( f–l ) are presented as mean ± s.d. from three independent experiments. Groups were compared via one-way ANOVA with Tukey’s post hoc test. Significance levels are indicated as ** p < 0.01, *** p < 0.001, **** p < 0.0001, and ns (not significant)

Article Snippet: The SIINFEKL (OVA 257-264 ) peptide was synthesized by Shanghai Bioengineering Co., Ltd. CpG ODN and the Alhydrogel® adjuvant were purchased from InvivoGen.

Techniques: In Vivo, Injection, Saline, Flow Cytometry, Ex Vivo, Enzyme-linked Immunospot, In Vitro

Journal: Signal Transduction and Targeted Therapy

Article Title: Condensate nanovaccine adjuvants augment CD8 + T-Cell-dependent antitumor immunity through mtDNA leakage-triggered cGAS-STING axis activation

doi: 10.1038/s41392-025-02447-w

Figure Lengend Snippet: Mechanistic research. Representative flow cytometry results ( a ) and quantitative analysis ( b , n = 3) of BMDC maturation. BMDCs were incubated with OVA PCD (10 μg/mL) for 48 h, followed by flow cytometry analysis of DC maturation (CD11c + CD80 + CD86 + ). Representative flow cytometry plots ( c ) and quantitative analysis ( d , n = 3) of MHC II molecule upregulation in BMDCs. BMDCs were incubated with OVA PCD (10 μg/mL) for 48 h, followed by flow cytometry detection of MHC II expression. Quantitative analysis of DC maturation ( e , n = 3) and OVA epitope presentation ( f , n = 3) in lymph nodes. C57BL/6 mice were immunized with two doses (containing an equivalent of 2.5 mg/kg OVA and 0.5 mg/kg CpG ODN). Lymph nodes were harvested 48 h after the final immunization, and single-cell suspensions were prepared for flow cytometry analysis of DC maturation and SIINFEKL-H-2Kb complex presentation on DC surfaces. g Cytokine levels (IL-12 and IFN-β) in supernatants measured by ELISA after BMDCs were incubated with OVA PCD (20 μg/mL) for 24 h ( n = 5). h Representative fluorescence images of mitochondrial colocalization with OVA PCD. Red: mitochondria. Green: FITC-OVA PCD. Scale bar: 15 μm. After 72 h of incubation of OVA PCD with DC2.4 cells, the mitochondria were labeled with a red tracker and observed via confocal microscopy. The white arrows indicate yellow fluorescent signals from the colocalization of FITC-OVA PCD (green) with mitochondria (red). i qPCR analysis of mtDNA leakage in the cytosol after BMDCs were incubated with OVA PCD (20 μg/mL) for 24 h ( n = 5). j cGAMP levels in BMDCs. After treatment with different concentrations of OVA PCD, ELISA was used to detect changes in the cGAMP content in BMDCs ( n = 5). k cGAMP levels in the lymph nodes. C57BL/6 mice ( n = 5) were immunized once a week 2 times. Lymph nodes were harvested 48 h after the final immunization, and single-cell suspensions were prepared for ELISA detection of cGAMP content in lymph node tissues. l Relative mRNA expression levels of type I interferons analyzed by RT‒qPCR after BMDCs were pretreated with different concentrations of the STING-IN-2 inhibitor followed by incubation with OVA PCD (20 μg/mL) for 24 h ( n = 4). All data are expressed as mean ± s.d. from three independent experiments. Unpaired two-tailed Student’s t tests were used for ( g ), and all other statistical analyses were performed via one-way ANOVA with Tukey’s post hoc test. Significance levels are indicated as ** p < 0.01, *** p < 0.001, **** p < 0.0001, and ns (not significant)

Article Snippet: The SIINFEKL (OVA 257-264 ) peptide was synthesized by Shanghai Bioengineering Co., Ltd. CpG ODN and the Alhydrogel® adjuvant were purchased from InvivoGen.

Techniques: Flow Cytometry, Incubation, Expressing, Enzyme-linked Immunosorbent Assay, Fluorescence, Labeling, Confocal Microscopy, Two Tailed Test