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<p>Gastric cancer (GC) is a prevalent malignancy worldwide with high morbidity and mortality. Its insidious onset and lack of sensitive early biomarkers impede routine screening, so most patients present advanced or metastatic disease at first diagnosis, leading to poor prognosis and scarce curative options. Surgery, chemotherapy and targeted therapy serve as standard GC treatments, however their efficacy is restricted by metastasis, adverse reactions and drug resistance, which drives the rapid development of immune checkpoint inhibitor (ICI)-based immunotherapy. Although monoclonal antibodies targeting programmed cell death protein 1/programmed cell death 1 ligand 1 (PD-1/PD-L1) resulted in significant survival benefits for GC patients, some patients exhibited an ineffective response. Our previous study has shown that Weitiao No. 3 (WD-3) enhanced the efficacy of anti-PD-1 immunotherapy by regulating intestinal flora in gastric cancer (GC) mice, in this study, huPBMC-NOG-dKO mice and MGC803 cells were used for in vivo and in vitro functional validation to explore the regulatory mechanism of WD-3 in GC immunotherapy. 16&#xa0;S rRNA sequencing analysis of mouse fecal samples demonstrated that WD-3 significantly enriched the intestinal abundance of <i>Lachnospiraceae</i> and <i>Ruminococcaceae</i> in GC model mice. Targeted serum metabolomics further verified that WD-3 markedly elevated serum isobutyric acid level in GC mice. Combined with HE staining and tumor growth monitoring, both the intestinal flora mixture consisting of <i>Lachnospiraceae</i> and <i>Ruminococcaceae</i> and isobutyric acid prominently enhanced the anti-tumor efficacy of PD-L1 inhibitors in GC mice. Mechanistically, RNA sequencing and qRT-PCR assays identified <i>ACOX2</i> as the most significantly up-regulated gene in isobutyric acid-treated MGC803 cells. Functional experiments using CCK-8 assay and flow cytometry revealed that isobutyric acid inhibited the viability and induced apoptosis of MGC803 cells, and such phenotypic changes were effectively rescued by <i>ACOX2</i> knockdown. Furthermore, results from the CytoTox 96 cytotoxicity assay and ELISA revealed that combined treatment with isobutyric acid and PD-L1 inhibitor elevated the levels of LDH, IL-2, TNF-α, IFN-γ, Perforin 1 and GzmB, while suppressing IL-8, PD-1 and TIM-3 expression in CD8⁺ T cells co-cultured with MGC803 cells. Notably, all these regulatory effects were abrogated when MGC803 cells were transfected with si-ACOX2. These results suggested that WD-3 modulated intestinal flora to elevate isobutyric acid level, thereby enhancing the inhibitory effect of PD-L1 inhibitor on tumor growth and boosting the anti-tumor activity of CD8⁺ T cells by up-regulating ACOX2 expression in GC. These findings may provide novel targets for improving anti-PD-L1 therapy against GC.</p>

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Weitiao No. 3 increased isobutyric acid to enhance the effect of PD-L1 inhibitor on tumor growth and anti-tumor effect of CD8+T cells in gastric cancer

  • 期刊论文

  • 发表日期：

    2026年7月15日

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  • Hengzhou Zhu,
  • Xiaodan Zhu,
  • Xiaona Huang,
  • Yiyang Qian,
  • Jiahui Li,
  • Jiaying Zhang,
  • Dong Niu,
  • Chunhui Jin

摘要

Gastric cancer (GC) is a prevalent malignancy worldwide with high morbidity and mortality. Its insidious onset and lack of sensitive early biomarkers impede routine screening, so most patients present advanced or metastatic disease at first diagnosis, leading to poor prognosis and scarce curative options. Surgery, chemotherapy and targeted therapy serve as standard GC treatments, however their efficacy is restricted by metastasis, adverse reactions and drug resistance, which drives the rapid development of immune checkpoint inhibitor (ICI)-based immunotherapy. Although monoclonal antibodies targeting programmed cell death protein 1/programmed cell death 1 ligand 1 (PD-1/PD-L1) resulted in significant survival benefits for GC patients, some patients exhibited an ineffective response. Our previous study has shown that Weitiao No. 3 (WD-3) enhanced the efficacy of anti-PD-1 immunotherapy by regulating intestinal flora in gastric cancer (GC) mice, in this study, huPBMC-NOG-dKO mice and MGC803 cells were used for in vivo and in vitro functional validation to explore the regulatory mechanism of WD-3 in GC immunotherapy. 16 S rRNA sequencing analysis of mouse fecal samples demonstrated that WD-3 significantly enriched the intestinal abundance of Lachnospiraceae and Ruminococcaceae in GC model mice. Targeted serum metabolomics further verified that WD-3 markedly elevated serum isobutyric acid level in GC mice. Combined with HE staining and tumor growth monitoring, both the intestinal flora mixture consisting of Lachnospiraceae and Ruminococcaceae and isobutyric acid prominently enhanced the anti-tumor efficacy of PD-L1 inhibitors in GC mice. Mechanistically, RNA sequencing and qRT-PCR assays identified ACOX2 as the most significantly up-regulated gene in isobutyric acid-treated MGC803 cells. Functional experiments using CCK-8 assay and flow cytometry revealed that isobutyric acid inhibited the viability and induced apoptosis of MGC803 cells, and such phenotypic changes were effectively rescued by ACOX2 knockdown. Furthermore, results from the CytoTox 96 cytotoxicity assay and ELISA revealed that combined treatment with isobutyric acid and PD-L1 inhibitor elevated the levels of LDH, IL-2, TNF-α, IFN-γ, Perforin 1 and GzmB, while suppressing IL-8, PD-1 and TIM-3 expression in CD8⁺ T cells co-cultured with MGC803 cells. Notably, all these regulatory effects were abrogated when MGC803 cells were transfected with si-ACOX2. These results suggested that WD-3 modulated intestinal flora to elevate isobutyric acid level, thereby enhancing the inhibitory effect of PD-L1 inhibitor on tumor growth and boosting the anti-tumor activity of CD8⁺ T cells by up-regulating ACOX2 expression in GC. These findings may provide novel targets for improving anti-PD-L1 therapy against GC.