Microbiota-driven compartment-specific changes in T cell subpopulations associated with bone remodeling regulation in triple-negative breast cancer

Almost 70% of patients diagnosed with advanced mammary tumors are compromised by bone marrow (BM) colonization and skeletal related events. Our group has previously shown that, in response to the triple-negative 4T1 murine breast carcinoma (4T1 BC), RANKL-producing T cells arrive at the BM compartment before tumor cells, inducing a RANKL-dependent bone loss that generates a pre-metastatic niche permissive to bone marrow colonization. Concurrently and independently, the comprehension of the impact of gut microbiota in the regulation of bone remodeling, especially through the modulation of lymphocytes and the RANKL-OPG axis, has substantially advanced. Notably, the microbial-dependent expansion and trafficking of intestinal T cells to the bone marrow has been described as a major component in the gut-bone axis. Recently, intestinal T cells were also implicated in the process of tissue remodeling of the mammary gland, adding interconnections to the role of gut microbiota and immunity to the progression of breast tumors. Given these findings, we investigated the role of the commensal microbiota in the bone progression of 4T1 BC. Using fecal 16S rRNA gene sequencing, we identified that 4T1 BC modifies expressively the intestinal microbiota composition. Antibiotic-induced microbiota depletion decreased the number of metastatic clones in the tumor draining lymph node (tdLN) and in the BM. Regarding bone remodeling, microbiota depletion altered the secretory profile of BM and tdLN cells, reducing their osteoclastogenic activity in vitro. In vivo, morphometric parameters of iliac bone generated by micro-CT showed that the drastic decrease in microbial charge restrained the tumor-induced bone loss. T cell subpopulations in the primary tumor, tdLNs, mesenteric lymph nodes (mLNs), and BM were characterized by flow cytometry and exhibited microbiota-driven, tissue-specific alterations in bone-damaging and osteoprotective phenotypes, highlighting a pathogenic role of microbiota.