The impact of intestinal microbiota‐derived metabolites on cancer and their potential application in tumor immunotherapy

In recent publications,1, 2 the group of Professor Zhu Shu/Pan Wen and the research team of Wang Liangjing/Chen Shujie, respectively investigated two intestinal microbial metabolites: deoxycholic acid (DCA) and indole-3-propionic acid (IPA). DCA acts on the calcium ion channel plasma membrane Ca2+ ATPase (PMCA), inhibits the effector function of CD8+ T cells and consequently promotes colorectal cancer (CRC) growth. IPA activates precursor-exhausted T (Tpex) cells and induces their transformation into effector T (Teff) cells, thereby increasing T-cell infiltration into the tumor tissue and enhancing the efficacy of immunotherapy (Figure 1). Zhu et al. screened 73 small-molecule compounds derived from microbial sources to examine their effects on CD8+ T-cell-mediated cytotoxicity or IFN-γ production, and identified DCA. Subsequent experiments using sodium dodecyl sulfate (SDS) or other chemical inhibitors confirmed that DCA does not lead to cell death. In tumor immunity, CD8+ T cells are crucial in resisting tumors, with cytokines such as IFN-γ and TNF-α playing key roles in killing tumor cells. CD8+ T-cell activation relies on Ca2+ as a second messenger and the cytoplasmic Ca2+ concentration directly affects CD8+ T-cell activation. The authors found that DCA could inhibit cytoplasmic Ca2+ accumulation by monitoring the real-time fluorescence intensity of cytoplasmic Ca2+ in anti-CD3/CD28-activated CD8+ T cells. NFAT2 is an important transcription factor regulating CD8+ T cells.3 According to immunoblotting results, DCA reduced the transcription of this factor. Using NFAT2-luciferase reporter gene assays, the authors observed that PMA/ionomycin stimulation increased the transcriptional activity of NFAT2. Notably, DCA supplementation attenuated this transcriptional activation, indicating that DCA could inhibit the NFAT2-mediated transcription induced by Ca2+ influx. To further study the specific effects of DCA, the authors examined several key observations. First, DCA did not reduce the levels of free calcium ions in cell-free culture medium, indicating that DCA does not chelate calcium ions. Second, the calcium-release activated channel (CRAC) was the main inward pathway. The immunosuppressive impact of DCA on CD8+ T cells persisted despite the presence of the selective CRAC channel blocker, BTP2, indicating that DCA did not affect calcium influx. Third, PMA/ionomycin induces Ca2+ to be released directly from the endoplasmic reticulum without relying on transporter proteins.1, 4 However, DCA inhibits Ca2+ accumulation, indicating that Ca2+ may flow out of the cell membrane in large quantities or into intracellular stores. Therefore, having blocked various channels known to suppress cytoplasmic Ca2+ levels, the authors found that DCA inhibits cytoplasmic Ca2+ accumulation by enhancing PMCA-mediated Ca2+ efflux, and validated this using a PMCA inhibitor. However, research into the precise binding site of DCA on PMCA remains limited. Further research is required to elucidate the mechanism by which DCA boosts PMCA activity. The authors have proposed a possible therapeutic strategy based on the mechanism of DCA. They isolated a bacteriophage from urban sewage that targets the lysis of C. scindens. This bacteriophage effectively reduces the serum DCA concentration, alleviates tumor growth, and enhances the effector function of CD8+ T cells, thereby presenting a potential therapeutic avenue for CRC.1 Additionally, in previous experiments, the use of the inhibitor LaCl3, or interference with shPMCA, inhibited PMCA, similarly alleviating the inhibition of CD8+ T cells. PMCA inhibitors may be used as an adjuvant therapy for those patients with high DCA-producing bacteria. Zhu et al.'s research highlights the enhancement of CD8+ T cell activity as a pivotal aspect of immunotherapy strategies. Wang et al. found that IPA enhances the activity of CD8+ T cells. They conducted in-depth research on the mechanism by which IPA improves tumor responsiveness to immune checkpoint blockade (ICB). These studies provide a conceptual basis for an adjunct approach to cancer treatment. Wang et al. analyzed microbial community differences by dividing mice into poor-responder group and responder group according to αPD-1 therapy. At the species level, Lactobacillus johnsonii exhibited the largest differences. This is consistent with findings in human CRC tissues, where the abundance of this bacterium was negatively correlated with tumor progression. To further investigate the effects of specific substances, the authors grouped mice and treated them with different culture media containing various bacteria. The results showed that only treatment with L. johnsonii-conditioned medium (Lj.CM) had a good therapeutic effect on tumors, comparable to direct oral administration of L. johnsonii. Moreover, the authors found that the levels of tryptophan-related metabolites, especially IPA, a tryptophan-derived metabolite specific to the intestinal microbiota, were increased in the plasma of mice. Surprisingly, only indole-3-lactate (ILA), a precursor substance that can be converted to IPA, was detected in Lj.CM medium, rather than IPA. Subsequent experiments confirmed that the production of IPA requires the cooperation of L. johnsonii and C. sporogenes.2 To determine whether the action of this substance depends on T cells, the authors transferred IPA or L. johnsonii to immunodeficient mice. Both treatments didn't reduce the growth of tumor. Then, IPA-untreated CD8+ T cells or IPA-pretreated CD8+ T cells was administered to the mice. The authors found that the CD8+ T cell group pretreated with IPA showed better tumor suppression compared to the group without IPA, proving that IPA acts through T cells. In subsequent studies using techniques such as single-cell RNA sequencing and single-cell T-cell receptor sequencing, the authors examined the group treated with αPD-1 alone or the IPA group treated with IPA and αPD-1 in combination therapy. The results revealed that IPA diminished naive CD8+ T cell populations, but augmented the population of Tpex cells which characterized by high TCF1 expression, and promoted the conversion of Tpex cells to Teff cells, thereby enhancing the responsiveness to ICB.2 The authors mapped the results of scRNA-seq to those of scATAC-seq and found that IPA activates Tpex cells by modifying the H3K27 acetylation of the Tcf7 super-enhancer region. Based on these results, the authors simulated the tumor microenvironment in CRC tissues. They found that IPA treatment increased CD8+ T cell infiltration and TCF-1 expression in tumors, effectively improving the efficacy of αPD-1 immunotherapy. However, more investigations are required to validate the feasibility of employing IPA as an adjunct immunotherapeutic agent. In conclusion, two research teams discovered the effects of microbial metabolites in the tumor microenvironment by studying the intestinal microbiota. These two studies provided relevant results from different perspectives on tumor immunity. These discoveries offered potential solutions and research directions for promoting the responsiveness of certain tumors to immunotherapy. Mingyan Zhang wrote the manuscript and prepared the figure. Feng Xie provided valuable discussion. Fangfang Zhou approved the final version of the manuscript. All authors have read and approved the final manuscript. This work was supported by the Chinese National Natural Science Funds (92169122, 32125016) and a project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions. The authors declare no conflicts of interest. The authors have nothing to report. The authors have nothing to report.