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Recent reports show that colorectal tumors contain microbiota that are distinct from those that reside in a ‘normal’ colon environment, and that these microbiota can contribute to cancer progression.  Fusobacterium nucleatum  is the most commonly observed species in the colorectal tumor microenvironment and reportedly influences disease progression through numerous mechanisms. However, a detailed understanding of the role of this organism in cancer progression is limited, in part due to challenges in maintaining  F .  nucleatum  viability under standard aerobic cell culture conditions. Herein we describe the development of a 3-dimensional (3D) tumor spheroid model that can harbor and promote the growth of anaerobic bacteria. Bacteria-tumor cell interactions and metabolic crosstalk were extensively studied by measuring the kinetics of bacterial growth, cell morphology and lysis, cancer-related gene expression, and metabolomics. We observed that viable  F .  nucleatum  assembles biofilm-like structures in the tumor spheroid microenvironment, whereas heat-killed  F .  nucleatum  is internalized and sequestered in the cancer cells. Lastly, we use the model to co-culture 28  Fusobacterium  clinical isolates and demonstrate that the model successfully supports co-culture with diverse fusobacterial species. This bacteria-spheroid co-culture model enables mechanistic investigation of the role of anaerobic bacteria in the tumor microenvironment.

Colorectal cancer-associated anaerobic bacteria proliferate in tumor spheroids and alter the microenvironment