Cytosolic Bacteria in Tumor Cells Promote Immune Evasion during Breast Cancer Metastatic Recurrence

Intratumor microbiota represents an understudied yet influential component of the cancer ecosystem, impacting the physical, chemical and immunological tumor microenvironment. However, the interplay between intratumor microbiota and the tumor-immune network as well as its physiological significance remain poorly understood. In this study, we investigated the long-term developmental process of metastatic seeds from PyMT breast tumor, and identified the key role of intratumor bacteria for efficient metastasis outgrowth after tumor cell colonization in distant organ in an immune-mediated process. Cytosolic bacteria, the dominant form of intratumor bacteria in PyMT tumors, promote metastatic tumor cell immune evasion through activating tumor cell intrinsic cGAS-STING-Il17b response pathway, ultimately inducing neutrophil into a protumor state to suppress cytotoxic T cells. Intriguingly, the upregulation of Il17b is dependent on the intracellular invasion of bacteria and accumulation of cytosolic dsDNA. The extracellular bacteria can not activate the STING pathway but induce neutrophils into an anti-tumor state instead. Under the physiological condition, the eradication of intracellular bacteria effectively abrogates metastatic immune suppression and prevents post-surgical metastatic recurrence, akin to the effect of introducing extracellular bacteria. Our findings underscore the critical role of intratumor bacteria in modulating the immune landscape of recurrent metastatic niches, and emphasize the bacteria-host interaction mode as a novel key factor for immune regulation. Overall design: For FVB mouse experiment, lungs were harvested from PBS(injected PBS), Bacteria(injected bacteria), woBac(injected tumor cell co-cultured with bacteria which showed negative bacteria signal) , withBac(injected tumor cell co-cultured with bacteria which showed positive bacteria signal). For germ free mouse experiment, lungs were harvested from WT(wild type), Control(injected tumor cells co-cultured with bacteria) and ATBx(injected tumor cell co-cultured with bac but use Antibiotics treated) mice at the end of point of experiment. Then lungs were dissected into single cells. Live cells were sorted by FACS to do 10X scRNA-seq. For RNA seq experiment, tumor cells co-cultured with live or dead bacteria. Then tumor cell containing live or dead bacteria were sorted by FACS to do bulk RNA-seq.