Microbiota-mediated CHI3L1 modulation as a novel mechanism for breast cancer progression

Over the past decade, infectious agents, including bacteria, have come under scrutiny for contributing to almost 16% of all tumors. Indeed, emerging data report a microbial presence in many cancer types, with breast cancer accumulating the most diverse bacterial population. Pathogen defense mechanisms, such as CHI3L1, contribute to bacterial elimination; however, CHI3L1 is overexpressed in several cancer types and correlates with poor prognosis and shorter survival. However, the connection between intratumoral bacteria and CHI3L1 and how they contribute to cancer growth has not been fully characterized. To investigate if microbiota and CHI3L1 are connected, female BALB/c mice harboring 4T1-Luc cells were exposed to a broad-spectrum antibiotic mix to deplete the microbiota and to evaluate tumor growth and circulating CHI3L1 levels. 9 and 14 days after tumor inoculation, feces were collected and bacterial DNA isolated for 16S rRNA sequencing. Tumors were plated to identify intratumoral bacteria. To assess whether a specific bacterial strain could modulate CHI3L1 expression, we incubated 4T1 cells with secretome from isolated bacteria, which contain metabolites and extracellular factors. Isolated bacteria that could upregulate CHI3L1 expression were then used to infect 4T1 cells prior to subcutaneous injection in the mammary fat pad of BALB/c mice to evaluate if bacterial infection changes tumor growth rate. In the 4T1 mouse breast cancer model, a broad-spectrum antibiotic treatment was able to: deplete host microbiota, abrogate tumor growth, and reduce CHI3L1 levels in the sera and in CD45+ immune infiltrating cells in tumor and colon. When 4T1 cells were exposed to bacterial supernatants in vitro, we observed that CHI3L1 was upregulated by a specific strain of E. coli. To simulate the effect of intratumoral microbiota, we infected 4T1 cells with either the isolated E. coli or S. aureus (which did not induce any CHI3L1 modulation in vitro) prior to subcutaneous injection. Strikingly, E. coli infection increased tumor progression with respect to the control S. aureus infection (p<0.01). Moreover, we observed an increase in CHI3L1 produced by tumor immune infiltrates in E. coli- and S. aureus infected 4T1, suggesting that CHI3L1 produced by the immune cells is not sufficient to promote tumor progression. At last, we performed a silencing on CHI3L1 in 4T1 cells and we infected them with E. coli. We observed an increase in infection rate and a consequent increase of tumor growth, meaning that CHI3L1 protects tumor cells from infection and reduces bacteria-induced cancer progression. Furthermore, we observed a reduction of tumor immune infiltrates indicating that bacteria could contribute to tumor progression, modulating the immune cell recruitment. Overall, this work sheds light on the mechanisms through which intratumoral microbiota can promote breast cancer growth. We showed that the microbiota leads to increased production of CHI3L1 which can be detrimental to the tumor but also beneficial as it allows controlling the growth of potentially dangerous intratumoral microbial strains. It remains to be established how the microbiota drives CHI3L1 production and how the latter restrains bacterial infection. Future work is needed to address these questions.