Microbiome derived lactate promotes immunosuppression and radiation resistance

The cervical microbiome plays a pivotal role in shaping vaginal health and influencing tumor prognosis. Tumor associated microbes secrete bioactive metabolites that modulate both tumor behavior and its surrounding microenvironment. Our lab previously identified a species of L-Lactate producing Lactobacillus (LAB), Lactobacillus iners ( L.   iners ), to be associated with poor chemoradiation response. In vitro experiments revealed that conditioned media from L.   iners enhances radiation resistance in cervical cancer cells-a phenomenon replicated by direct lactate supplementation. This study aims to characterize the impact of microbiome-derived lactate on the tumor microenvironment. Hypothesis: We propose that L-lactate produced by cancer-derived LAB supports tumor progression by fostering an immunosuppressive niche. Methods: LAB strains were isolated via targeted culture from cervical, vaginal, vulvar, and oral cavity cancers. These isolates were used in co-culture systems and to generate cell-free bacterial conditioned media (CFS) to assess their capacity to interact with cancer cells and promote tumor-supportive conditions. For initial studies, we focused on cervical cancer isolates. To evaluate LAB-derived lactate's role in radiation resistance, we performed cell viability assays (CyQUANT) on HeLa, SiHa, and CaSki cervical cancer lines exposed to CFS from patient-derived LAB and radiation. Lactate production was quantified using the Diazyme D-/L-Lactate rapid test kit, enabling precise source attribution between bacterial and tumor origins. We developed a syngeneic lactate exposure model using mEER (mouse oral line) xenografts in C57BL/6 mice, administering weekly intratumoral lactate injections. The tumors were radiated after the tumors reached ~75mm 3 . To evaluate the immune landscape, multiplex immunofluorescence using the Lunaphore COMET microfluidic platform was performed on mEER tumors. Results: Cervical cancer cells exhibited increased radiation resistance when co-cultured with L-lactate-producing LAB, the isoform preferentially metabolized by human cells. LAB emerged as the dominant source of lactate in co-culture, confirming its role in driving lactate accumulation within the tumor microenvironment. In vivo, lactate-treated tumors grew significantly larger post-radiation (mean volume 48 mm³ vs. 74 mm³, p  < 0.0001). Immune profiling revealed elevated T-cell infiltration, with a skew toward regulatory T cells and reduced CD8⁺ populations. The lactate-rich microenvironment showed marked upregulation of immune checkpoint proteins CTLA4 and PD-L1, suggesting functional exhaustion of infiltrating immune cells.