Bacterial colonization of tumors drives immune activation and checkpoint blockade efficacy.

The tumor microenvironment (TME), which heavily influences immune checkpoint blockade (ICB) efficacy, is shaped by host-microbe interactions. Endogenous intratumoral bacteria have been implicated in efficacy of ICB, such as anti-PD-1 (aPD-1), however, their role in modulating the TME, and whether these TME interactions are represented in murine tumor models, remains unknown. Using 16S rRNA qPCR, sequencing, and culture-based methods on multiple murine tumor models, we identified intratumoral microbiota in MCA-205 tumors but not in MOC1 tumors, underscoring the role of tumor-intrinsic factors in shaping microbial colonization. In MCA-205 tumors, intratumoral microbiota increased dendritic cell (DC) maturation and CD8+ T cell infiltration and activation, boosting ICB therapeutic efficacy. Specific depletion of only the intratumoral microbiota abrogated these effects. We found these bacteria-dependent effects were not due to canonical TLR signaling, as aPD-1 efficacy was not reduced by blocking TLR signaling through MyD88 depletion or enhanced by adding E. coli into the TME. This suggests that specific microbial species or communities, rather than bacterial colonization alone, are required to modulate antitumor immunity. These results reveal that intratumoral microbiota, when present, can dictate ICB efficacy, highlighting the importance of understanding this variable in murine experiments aiming to optimize cancer immunotherapy.