Antibiotics treatment impairs the growth of murine triple negative mammary tumors by altering the tumor associated microbiota

Triple-negative breast cancer (TNBC) is the most aggressive BC subtype and a recent candidate for immunotherapy. Gut microbiota influences tumor growth and immunotherapy response. Other tissues host their own microbiota, that changing in tumor favours immune suppressive cells recruitment. In lung, we identified the targeting of local microbiota by antibiotic aerosol a strategy to promote an increased anti-tumor immunity. BALB/C mice were mammary fat pad transplanted with 5x104 4T1 TNBC cells and received aerosolized or oral Vancomycin /Neomycin, or Ampicillin, or Clindamicin, or saline starting 24 h later. The tumor immune microenvironment was analyzed by Real Time PCR and FACS; the tumor microbiome by 16S rRNA metagenomic analysis. Aerosolized Vanco/Neo decreased spontaneous lung metastases in mice implanted with 4T1 cells (p=0,0193) and impaired mammary tumor growth (p<0,0001) by inducing an M1 local immunity. This effect depend on antibiotics systemic bioavailability, since oral Vanco/Neo, that has minimal access to the bloodstream at intestinal barrier, did not affect mammary tumor. In contrast, antibiotics that cross the intestinal epithelium, Amp or Clinda, impaired mammary tumor by aerosol and also orally (p<0,0001). CD3+ T cells and F4/80+ macrophages resulted expanded in tumors, suggesting that antibiotics affect mammary tumor growth by modulating the local microbiota and in turn the immune microenvironment. Accordingly, in untreated versus Amp-treated tumors we found increased level of pathogen-recognition receptors (PRRs) and a different representation of 8 bacterial taxa, as Staphylococcus genus, over-represented in untreated tumors, and Micrococcaceae genus, over-represented in Amp-treated tumors, by 16S rRNA metagenomic analysis. In vivo peritumoral transfer of Micrococcus luteus and Clostridium perfringens strains, that we isolated from Amp-treated tumors, significantly reduced the mammary tumor growth (p<0,0001 and p=0.0006), while no effect was induced by Staphilococcus epidermidis, isolated from untreated tumors. Our findings support a role of the tumor associated microbiota on the growth of mammary tumor suggesting that its manipulation might create an immune microenvironement favourable to respond to immunotherapy in TNBC patients.