Schisandrin C (JOT-10307) was purchased from Chengdu Pufei De Biotech Co., Ltd. (Chengdu, China). Cisplatin (T1564) was purchased from TargetMol (United States). DMSO (HY-Y0320) was purchased from MedChemExpress (United States). Color Prestained Protein marker (20AB01) was purchased from GenStar (Beijing, China). DMEM Medium (CM10013) and penicillin-streptomycin were purchased from MacGene (Beijing, China). FBS was purchased from VivaCell (Shanghai, China). Mouse 1× Lymphocyte Separation Medium was purchased from Dakewe (Shenzhen, China). The antibodies used include: rabbit monoclonal anti-pIRF3 (GTX86691) was from GeneTex (USA). IRF3 polyclonal antibody (11312-1-AP), and TMEM173/STING polyclonal antibody (19851-1-AP) were from the Proteintech Group (USA). HSP90 antibody (sc-101494) was purchased from Santa Cruz (USA). RBC Lysis Buffer (420301), TruStain FcX™ PLUS (anti-mouse CD16/32) (S17011E, 156603), FITC anti–mouse CD45 (30-F11, 103107), Brilliant Violet 421™ anti-mouse CD3ε (145-2C11, 100335), PE/Cyanine7 anti-mouse CD4 (RM4-5, 100527), PE anti–mouse CD8a (53-6.7, 100708), Zombie Aqua™ Fixable Viability Kit (423101), APC anti-mouse NK1.1 (PK136, 108709) were purchased from BioLegend (United States).

BALB/c and C57BL/6 mice were procured from SPF Biotechnology Co., Ltd. (Beijing, China). All experimental protocols involving animals were performed in strict adherence to the guidelines established by the Animal Care Committee of the Fifth Medical Center of PLA General Hospital.

Before tumor cell injection, the mice, aged between 5 to 6 weeks and closely age-matched, had one of their flanks shaved. Subsequently, tumor cells were administered into the shaved flank. Volume of the tumor was measured every 3 days and computed using the given formula: (length) × (width)²/2. Eventually, the mice were sacrificed on day 21.

The 4T1 cells were cultivated using the RPMI 1640 medium (MacGene). Meanwhile, DMEM medium was employed for growing the MC38 cells. The cells were incubated in a humidified atmosphere with 5% CO₂ at 37°C.

MC38 cells and 4T1 cells were seeded in 96-well plates at a density of 1 × 10⁵ cells/mL, each well containing 100 μL. The following day, these cells underwent treatment with various SC concentrations over 24 h. Cell viability was subsequently assessed with the Cell Counting Kit-8, following the manufacturer’s strict procedures.

Total RNA was isolated using Trizol (Sigma) and synthesized into cDNA using reverse transcription reagents (RT Master Mix for qPCR II, MedChemExpress). Subsequently, the cDNA was amplified through a Quant Studio 6 real-time PCR instrument employing SYBR Green qPCR Master Mix (MedChemExpress). Actin was used to normalize mRNA expression levels. The sequences for the mouse quantitative PCR primers are shown in Table 1.

On the 21st day after the tumor cells were inoculated, the tumors were removed, weighed, mechanically minced, and incubated for 20 min in Collagenase IV (1 mg/mL) and DNase I (40 μg/mL) at 37°C. The cells were then filtered to prepare a single-cell suspension. We enriched lymphocytes using a mouse lymphocyte separation medium. The enriched cells were blocked with anti-CD16/32 antibodies and then stained with surface antibodies at room temperature for 25 min. The antibodies used are listed in the antibody section above. The BD FACSCanto flow cytometry was used to analyze stained cells.

Total RNA was extracted and subjected to quality testing, followed by the synthesis, purification, and identification of double-stranded cDNA using messenger RNA procedures. Subsequently, the construction and sequencing of sample libraries were performed. The high-throughput Illumina HiSeq 4000 sequencing platform was utilized to generate raw data. Sample processing and online sequencing were completed by the Sangon Biotech (Shanghai) Co., Ltd. DESeq2 was used to identify the differentially expressed genes (DEGs) (|log₂FC| ≥ 1, and p ≤ 0.05) between each group. After constructing appropriate gene sets, gene set enrichment analysis (GSEA) analyses were conducted using the Cluster Profiler R package.

Cell lysates were prepared using a loading buffer that includes a radioimmunoprecipitation assay buffer. Following this, the sample mixtures were denatured at a temperature of 105°C for 15 min. Subsequently, equal protein quantities were subjected to 10% SDS-PAGE, followed by transfer onto 0.22 µm PVDF membranes. To minimize non-specific interactions, membranes were blocked with 5% non-fat milk at room temperature for 1 hour. Overnight incubation at 4°C with specific primary antibodies preceded a 1-h room temperature incubation with secondary antibodies. Signal detection was carried out with the enhanced chemiluminescent reagent, supplied by Promega (United States), and analyses were performed subsequently.

Statistical analyses were conducted utilizing the GraphPad Prism 8.0 software. The scrutiny of differences among multiple groups was accomplished through a one-way ANOVA, whereas the assessment of disparities between two individual groups was executed employing an unpaired Student’s t-test. Survival curves were delineated using the Kaplan-Meier estimator, and the comparative analysis of survival rates was performed with the log-rank (Mantel-Cox) test. All experimental data were conveyed as the mean ± SD. p < 0.05 was considered significant.

We first investigated the antitumor effect of SC in 4T1 tumor-bearing mice (Figure 2A). There was no significant difference in body weight between the groups (Figure 2B), suggesting that SC had no severe side effects on mice. Importantly, our results showed that the SC-treated group experienced a significant delay in tumor growth (Figures 2C–E). Tumors in the SC treatment group (30 mg/kg) demonstrated a significant volume reduction compared to the control group after 21 days of treatment. Concurrently, the tumor weight in the SC treatment group significantly decreased. Consistently, photographs of the tumors taken on day 21 clearly illustrated the retardation of tumor growth. To further investigate the therapeutic impact of SC, we conducted a survival analysis. The overall survival was significantly improved in the SC-treated group compared with the vehicle group (Figure 2F). Therefore, our results in the 4T1 murine tumor model strongly suggested that SC could effectively inhibit tumor growth.

Subsequently, we established a mouse model using MC38 colon cancer cells to assess the potential universality of SC’s antitumor effect (Figure 2G). SC significantly inhibited tumor growth in comparison to the control group, without affecting body weight (Figures 2H, I). Tumors were significantly smaller in the SC-treated group compared with the control group (Figures 2J, K). Moreover, mice treated with SC exhibited a significantly higher survival rate (Figure 2L). Therefore, our findings strongly suggested that SC could effectively inhibit tumor growth from both 4T1 and MC38 murine tumor models, with no observed clinical signs of toxicity in mice.

To elucidate the mechanism through which SC mediates the delay in tumor progression, we conducted a comprehensive RNA sequencing analysis of the tumor tissues. Subsequent Principal Component Analysis (PCA) confirmed that the SC treatment significantly altered gene expression profiles (Figure 3A). In comparison with the control group, we identified 206 upregulated and 127 downregulated genes in SC-treated tumor tissues (Figure 3B). A Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis unveiled SC exerted antitumor effects by activating cytokine-cytokine receptor interaction, chemokine signaling pathway and T cell receptor signaling pathway (Figure 3C), affirming the critical role of antitumor immunity in SC’s inhibition of tumor growth. A Gene Ontology (GO) enrichment analysis correlated with KEGG findings, pointing toward immune-related biological processes as the major site of upregulated gene enrichment (Figure 3D).

Motivated by the outcomes of the KEGG and GO analyses, we supplemented these findings with a Gene Set Enrichment Analysis (GSEA) to gain further insights into immune function involvement. GSEA analysis revealed that the SC treatment resulted in a higher degree of innate immune response and response to IFNβ, highlighting the participation of type I IFN response in the antitumor effect (Figures 3E, F). It is widely recognized that type I IFN are the downstream cytokines regulated by the cGAS-STING signaling pathway (Zheng et al., 2020). Moreover, our previous studies have shown that SC activates the cGAS-STING pathway (Zhao et al., 2023), prompting us to focus further on this mechanism.

During tumor progression, the DNA of tumor cells is relatively fragile and prone to leakage, leading to activation of the cGAS-STING signaling pathway (Mackenzie et al., 2017). According to analysis, SC exerted subsequent antitumor effects by enhancing the activation of the cGAS-STING pathway. To verify the enrichment results, we analyzed the mRNA expression of IFNβ, CXCL10, ISG15, and IFIT1 within tumors. Consistently, SC markedly enhanced the transcription of these cytokines in tumors formed by MC38 cells (Figures 3G–J), testifying to type I IFN response’s critical role in SC’s antitumor mechanism. Therefore, the treatment of SC increased the activation of the cGAS-STING pathway, leading to the further expression of type I IFN and various inflammatory cytokines, sparking off an innate immune response.

Evidence from prior research indicates that type I IFN catalyzes the upregulation of several chemokines essential for directing the infiltration of T cells and NK cells into tumor sites (Zheng et al., 2020). Intriguingly, Gene Set Enrichment Analysis (GSEA) disclosed that treatment with SC led to an enriched presence of activated T cells and NK cells (Figures 4A, B), which were pivotal in the elimination of tumor cells. To examine the involvement of T cells and NK cells in the antitumor process of SC, we analyzed the levels of tumor-infiltrating T cells and NK cells in both vector control and SC-treated tumors by flow cytometry (Figures 4C, D). Notably, SC-treated mice exhibited an elevated frequency of CD4⁺ T cells, CD8⁺ T cells, and NK cells within the tumors compared to control (Figures 4E–G). Correspondingly, a marked increase in the infiltration of both CD4⁺ and CD8⁺ T cells, alongside NK cells, was observed in tumors from the SC-treated group (Figures 4H–J). In conclusion, our data suggested that SC effectively inhibited tumor growth by initiating T cell and NK cell-driven immune responses in a type I IFN-dependent manner.

Fascinatingly, recent studies propose that the amalgamation of cGAS-STING pathway enhancers with chemotherapy drugs potentially amplifies antitumor effects (Liu et al., 2023). In addition to the leaked DNA of tumor cells that are susceptible to genotoxic stress during tumor progression, chemotherapy with platinum drugs, such as cisplatin, can also cause DNA damage and activate the cGAS-STING signaling pathway (Grabosch et al., 2019; Glorieux et al., 2022). Therefore, to maximize the antitumor effect of SC, we attempted to verify whether SC could enhance the activity of the cisplatin-induced cGAS-STING pathway to induce type I IFN response in MC38 cells and 4T1 cells, thereby enhancing antitumor immunity against DNA damage caused by chemotherapy and further improving antitumor efficacy.

Before further exploring the regulatory role of SC in the cisplatin-induced cGAS-STING pathway, we initially assessed the cytotoxicity of SC in MC38 cells. SC was applied to MC38 cells for 24 h before assessing cell viability. The results showed that SC exhibited minimal toxicity (Figure 5A), consistent with our previous observations in normal cells (Zhao et al., 2023). Subsequently, we assessed the impact of SC on the cisplatin-induced cGAS-STING pathway in MC38 cells, utilizing a concentration of 15 μM based on previous studies (Zhao et al., 2023). Immunoblotting analyses revealed a notable increase in the protein levels of phosphorylated IRF3 and STING in the combination group of SC and cisplatin, as compared to using cisplatin alone (Figure 5B). In addition, we detected mRNA expression of several ISGs downstream of cGAS-STING in MC38 cells: IFNβ, CXCL10, ISG15, and IFIT1 (Figures 5C–F). Consistent with previous results, SC synergistically enhanced the transcription of these genes induced by cisplatin. Consistent with the results in MC38 cells, SC also enhanced the protein levels of phosphorylated IRF3 and STING as well as the transcription levels of downstream genes such as IFNβ, CXCL10, ISG15, and IFIT1 in 4T1 cells (Figures 5G–L). Collectively, SC boosted the cisplatin-induced type I IFN response in a cGAS-STING dependent way. In MC38 cells and 4T1 cells, SC alone didn't increase the expression of downstream factors related to the cGAS-STING signaling pathway, consistent with our previous findings that SC requires signaling stimulation to promote expression. Conversely, in vivo analyses revealed a distinctive outcome whereby the solitary treatment with SC managed to catalyze the activation of pathway-associated factors. This difference can be attributed to the fragility and proneness to leakage of tumor cell DNA during tumoral progression, thereby precipitating the activation of the cGAS-STING signaling pathway. SC enhances the activation of the cGAS-STING pathway, subsequently exerting its antitumor effects.

Upon establishing that SC augmented the cisplatin-induced activation of the cGAS-STING pathway in vitro, our focus then shifted to examining whether SC could enhance the antitumor efficacy of cisplatin, specifically through the type I IFN response pathway. The efficacy of the antitumor action was evaluated in MC38 tumor-bearing mice that were either unstimulated or stimulated with cisplatin, under conditions both with and without the presence of SC (Figure 6A). Notably, the combined use of SC with cisplatin significantly reduced MC38 tumor growth compared to the individual treatments alone (Figures 6B, C). Complementing this finding, photographs of the tumors clearly illustrated the significant retardation of tumor growth in the group treated with combination therapy (Figure 6D), compared to the groups treated individually with either SC or cisplatin. These findings significantly implied a potent combined effect of SC and cisplatin in successfully inhibiting tumor growth in vivo.

Exposure to cisplatin was observed to result in significant weight loss in the mice. However, this weight loss was significantly ameliorated in the group receiving combined treatment with cisplatin and SC compared to the cisplatin group (Figure 6E). In addition, the survival rate of mice also improved dramatically with the co-administration of SC and cisplatin, compared to cisplatin-only treatment (Figure 6F). These results highlight the potential of SC in reducing side effects and improving the therapeutic effect of cisplatin.

Our earlier research illustrated that SC enhances the cisplatin-induced type I IFN response through the cGAS-STING signaling pathway. Accordingly, the mRNA expression of IFNβ, CXCL10, ISG15, and IFIT1 in the tumor tissues was analyzed. The data revealed that SC markedly enhanced cisplatin-induced gene transcription in the tumors established from MC38 cells (Figures 6G–J), underscoring the potentiated effect of SC on the type I IFN response prompted by cisplatin. Flow cytometry analysis confirmed that the combination therapy of SC and cisplatin significantly resulted in an appreciable increase in the infiltration of CD4⁺ and CD8⁺ T cells, along with NK cells, into the tumors (Figures 7A–H). Collectively, SC amplifies the chemotherapeutic efficacy of cisplatin by bolstering antitumor immunity and enhancing the type I IFN response.