Chronic stress disrupts the gut microbiota in patients with cancer; however, how stress-induced microbiota perturbations impact anti-tumor immunity remains unclear. Here, we show that the gut microbiota is required for chronic stress-induced glucocorticoid production, which impairs antigen-specific germinal center B cell responses. In mouse models of colorectal cancer or melanoma, chronic stress promotes translocation of a gut pathobiont, Enterococcus gallinarum (Eg), to tumors. Within tumors, Eg phage DNA induces glucocorticoid production by cancer-associated fibroblasts (CAFs) via TLR9, which suppresses anti-tumor B cell responses through the glucocorticoid receptor. Targeting intratumoral TLR9 or Eg significantly lowers intratumor glucocorticoid levels and reverses the tumor-promoting effects of chronic stress. Extending these findings to human cancer, we identify lytic phages in a Klebsiella pneumoniae isolate from human colorectal tumors that promote tumor growth and detect phage DNA in human brain tumors. Together, our study reveals a chronic stress-induced intratumor phage-CAF-B cell circuit that weakens anti-tumor immunity.

The human microbiota modulates cancer progression through largely unexplored mechanisms. Defining causal pathways is essential for monitoring and fine-tuning the microbiota to improve cancer treatment. Given that amino acid (aa) metabolism is often dysregulated in cancer, we assessed the role of microbiota pathways that modulate intestinal aa levels on colorectal tumor progression in mice. We found that the Bacteroides gene bo-ansB affects tumor responses to dietary asparagine (Asn) by reducing intestinal Asn levels. In mice receiving dietary Asn, bo-ansB promotes tumor progression by altering tumor-infiltrating CD8+ T cells. Mechanistically, bo-ansB depletes Asn in the tumor microenvironment (TME), suppressing the expression of an Asn transporter (SLC1A5) in CD8+ T cells and impairing their stem-like properties and effector functions. In humans, microbiota-encoded genes contributing to aa depletion are associated with colorectal cancer progression. Collectively, these findings reveal nutrient-dependent modulation of anticancer immunity by the gut microbiota and identify diet-microbiota-cancer crosstalk as a potential therapeutic target.

Abstract Introduction Chronic psychological stress is a major driver of cancer progression, yet its effects on anti-tumor B cell immunity remain unclear. Activation of the hypothalamic—pituitary—adrenal axis elevates glucocorticoids that broadly suppress immune responses. Given that the gut microbiota supports germinal center (GC) B cell differentiation, stress-induced dysbiosis may impair B cell—mediated tumor immunity. Methods Mice were subjected to a 21-day chronic unpredictable mild stress (CUMS) regimen. MC38 or B16F10 tumor cells were implanted on day 7, and tumor growth was monitored. Microbiota dependence was tested using broad-spectrum antibiotics, germ-free housing, and monocolonization. Tumor-associated bacteria were isolated and identified by culturomics, 16S rRNA sequencing, and whole-genome analysis to detect prophage activation. CAFs were stimulated with bacterial DNA ± TLR9 antagonist. Interventions included intratumoral TLR9 blockade and localized antibiotic treatment. Human colorectal tumors were analyzed for intratumor corticosterone and GC B cell abundance. Results Chronic stress significantly accelerated tumor growth and suppressed GC B cell responses. These effects were abrogated under antibiotic or germ-free conditions, indicating microbiota dependence. Stress enabled translocation of a gut pathobiont into tumors, where its phage-derived DNA activated TLR9 in cancer-associated fibroblasts, inducing local corticosterone synthesis. This intratumor glucocorticoid suppressed GC B cells and antibody responses. Blockade of the phage—TLR9 pathway restored B cell activity and prevented stress-induced tumor progression. Human colorectal tumors exhibited elevated corticosterone and reduced GC B cells, supporting clinical relevance. Conclusion Chronic stress triggers a microbiota-dependent phage—TLR9—glucocorticoid circuit that suppresses B cell immunity and accelerates tumor growth. Targeting this axis restores anti-tumor B cell function and mitigates stress-driven cancer progression. Funding Source CRI Irvington Fellowship Topic Categories Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)

BACKGROUND:Iron deficiency anemia (IDA) is the most common extra-intestinal complication in inflammatory bowel disease (IBD). The persistence of iron deficiency in patients living with quiescent IBD remains poorly understood. Given the extensive body of research linking IBD pathogenesis to microbiome disruptions, it is hypothesized that alterations in the microbiota or immune responses may drive the persistence of IDA in quiescent Crohn's disease. This study aimed to determine whether changes in the gut microbiota or immune phenotypes contribute to IDA, while uncovering potential mechanisms driving IDA in quiescent disease. METHODS:This cross-sectional, descriptive, and analytical study utilized 141 samples from pediatric Crohn's disease patients with and without iron deficiency as well as healthy controls for initial 16S microbiome analysis and a smaller subset for Shotgun Metagenomics and immunologic analyses. Fecal and peripheral blood samples were obtained from the Jill Roberts Institute Live Cell Bank. RESULTS:While no major differences were observed in the overall gut microbiome composition between pediatric patients with quiescent Crohn's disease, with or without IDA, notable shifts in specific microbial strains were identified. Specifically, levels of Anaerobutyricum soehngenii and Alistipes shahii were significantly altered. Metagenomic analysis revealed an enrichment of pathways related to short-chain fatty acid metabolism and ascorbate degradation, indicative of functional change in these microbes. CONCLUSIONS:This is the first comprehensive microbiome analysis of quiescent pediatric Crohn's disease with concomitant IDA. The findings indicate modest but significant microbial strain-level differences and associated functional pathways, potentially implicating microbiota-mediated mechanisms in the persistence of IDA.

Interleukin (IL-)23 is a major mediator and therapeutic target in chronic inflammatory diseases that also elicits tissue protection in the intestine at homeostasis or following acute infection1-4. However, the mechanisms that shape these beneficial versus pathological outcomes remain poorly understood. To address this gap in knowledge, we performed single-cell RNA sequencing on all IL-23 receptor-expressing cells in the intestine and their acute response to IL-23, revealing a dominance of T cells and group 3 innate lymphoid cells (ILC3s). Unexpectedly, we identified potent upregulation of the immunoregulatory checkpoint molecule cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) on ILC3s. This pathway was activated by gut microbes and IL-23 in a FOXO1- and STAT3-dependent manner. Mice lacking CTLA-4 on ILC3s exhibited reduced regulatory T cells, elevated inflammatory T cells and more-severe intestinal inflammation. IL-23 induction of CTLA-4+ ILC3s was necessary and sufficient to reduce co-stimulatory molecules and increase PD-L1 bioavailability on intestinal myeloid cells. Finally, human ILC3s upregulated CTLA-4 in response to IL-23 or gut inflammation and correlated with immunoregulation in inflammatory bowel disease. These results reveal ILC3-intrinsic CTLA-4 as an essential checkpoint that restrains the pathological outcomes of IL-23, suggesting that disruption of these lymphocytes, which occurs in inflammatory bowel disease5-7, contributes to chronic inflammation.

Group 3 innate lymphoid cells (ILC3s) are abundant in the developing or healthy intestine to critically support tissue homeostasis in response to microbial colonization. However, intestinal ILC3s are reduced during chronic infections, colorectal cancer, or inflammatory bowel disease (IBD), and the mechanisms driving these alterations remain poorly understood. Here we employed RNA sequencing of ILC3s from IBD patients and observed a significant upregulation of RIPK3, the central regulator of necroptosis, during intestinal inflammation. This was modeled in mice where we found that intestinal ILC3s express RIPK3, with conventional (c)ILC3s exhibiting high RIPK3 and low levels of pro-survival genes relative to lymphoid tissue inducer (LTi)-like ILC3s. ILC3-specific RIPK3 is promoted by gut microbiota, further upregulated following enteric infection, and dependent upon IL-23R and STAT3 signaling. However, lineage-specific deletion of RIPK3 revealed a redundant role in ILC3 survival, due to a blockade of RIPK3-mediated necroptosis by caspase 8, which was also activated in response to enteric infection. In contrast, lineage-specific deletion of caspase 8 resulted in loss of cILC3s from the healthy intestine and all ILC3 subsets during enteric infection, which increased pathogen burdens and gut inflammation. This function of caspase 8 required catalytic activity induced by TNF or TL1A and was dispensable if RIPK3 was simultaneously deleted. Caspase 8 activation and cell death were associated with increased Fas on ILC3s, and the Fas-FasL pathway was upregulated by cILC3s during enteric infection, which could restrain the abundance of intestinal ILC3s. Collectively, these data reveal that interpretation of key cytokine signals controls ILC3 survival following microbial challenge, and that an imbalance of these pathways, such as in IBD or across ILC3 subsets, provokes depletion of tissue-protective ILC3s from the inflamed intestine.

Supplemental Figures 1-10. Supplementary figure 1 shows that TLR7 antagonist IRS661 specifically blocks TLR7 expression by human A549 cells Supplementary figure 2 shows that TLR7 agonist induces chemoresistance in murine LL/2 cells Supplementary figure 3 shows the pro-tumoral effect of TLR7 stimulation with increasing doses of chemotherapy Supplementary figure 4 shows that Stimulation of TLR7 favors tumor growth and chemoresistance of murine LL/2 cells while TLR7 blockade with an antagonist inhibits tumor growth Supplementary figure 5 shows that TLR7 stimulation induces myeloid cell recruitment into LL/2 tumors in immunocompetent C57BL/6 mice Supplementary figure 6 shows Determining the optimal cutoff for the cohort of patients not treated with neo-adjuvant chemotherapy Supplementary figure 7 shows Determining the optimal cutoff for the cohort of patients treated with neo-adjuvant chemotherapy Supplementary figure 8 shows that non-responders patients to neo-adjuvant chemotherapy have a poor prognosis Supplementary figure 9 shows the correlation graph for TLR7 expression by tumor cells in metastatic lymph nodes and in primary lung tumor, after treatment with neo-adjuvant chemotherapy Supplementary figure 10 shows that High TLR7 expression on tumor cells is associated with low response to neo-adjuvant chemotherapy

PDF file - 56K, P values corresponding to different cutoff (minimum P value, first, second and third quartile) in CRC and RCC lung metastases. {section sign}P values were corrected by the formula proposed by Altman et al.

PDF file - 83K, Baseline characteristics of 52 patients with RCC lung metastasis. * determined by Heng et al.

PDF file - 123K, CD62L- T cells represent the main T cell population in human lung tumors.

PDF file - 78K, Multivariate Cox proportional hazards analysis for overall survival in NSCLC patients.

PDF file - 139K, Selective presence of CD62L+ T cells in TLS.

PDF file - 380K, Gene expression levels related to immune populations, TLS, Th-orientation, cytotoxicity, T-cell activation, immuno-suppression, inflammation and angiogenesis according to the high and low density of mature DC.

PDF file - 131K, Clinical characteristic of NSCLC patients with DC-Lamp High versus DC-Lamp Low tumors.

PDF file - 65KB, Appendix A5.

PDF file - 95K, Comparison of different methods used for the stratification of patients according to the density of mature DC, stromal CD8+ T cells or tumor nest CD8+ T cells.

PDF file - 191K, Antibodies used in the study.

PDF file - 151K, Expression on tumor-infiltrating T cells of molecules involved in cytotoxicity, activation, and Th1 orientation.