Abstract
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.
Highlights
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
Since F. nucleatum is consistently found to be enriched in the colorectal tumor microenvironment (TME), we hypothesized that co-culturing anaerobic F. nucleatum with tumor spheroids may provide a niche for maintaining F. nucleatum viability outside the anaerobic chamber (Fig. 1A)
When testing the Bacteria-spheroid co-cultures (BSCCs) model with a different anaerobic species, Faecalibacterium prausnitzii, which is not commonly associated with CRC7,34, no viable bacteria could be recovered at any timepoint for any tumor spheroid size (Supplementary Fig. 1), suggesting that this niche environment cannot universally support the growth and viability of all anaerobic microbes
Summary
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. Recent reports have demonstrated a potential role for enhancing cancer cell proliferation[13,14], modulating tumor immunity[15,16], regulating autophagy[17], and influencing metastasis[10,18,19] Despite these compelling observations, a mechanistic understanding of the role for this organism in cancer progression is limited, in part due to challenges in maintaining the viability of F. nucleatum under standard aerobic human cell culture conditions. While these studies have demonstrated important interactions between the surface components of F. nucleatum and both epithelial and immune cells, they did not reveal any specific effects due to viable F. nucleatum, or characterize any host-microbe metabolite crosstalk This is a challenging problem in microbiome research and is beginning to be addressed by the development of engineered models to co-culture host cells with anaerobic bacteria. We leveraged the 3D nature of these tumor spheroids to study the effects of co-culturing viable F. nucleatum with epithelial cells; including, gene expression, metabolomics, and their morphology
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