Abstract 2797: Fusobacterium nucleatum antigens drive tumor-associated macrophage-like cells in an engineered oral squamous cell carcinoma on-a-chip

Abstract Oral squamous cell carcinoma (OSCC) is the sixth most prevalent cancer, representing 90% of all head and neck cancers. Oral cancers are expected to cause 11000 deaths in the US in 2023. OSCC comprises a complex microenvironment involving a 3D collagenous matrix, innate and adaptative immune cells, fibroblasts, and opportunistic microorganisms. Fusobacterium nucleatum, for instance, is related to OSCC development in different aspects, such as inducing epithelial-to-mesenchymal transition and coordinating the immunological response in the tumor microenvironment (TME). Although some advances were achieved in understanding the OSCC TME, there is an urge to develop advanced in vitro models to investigate the immunosuppressive role of opportunistic microorganisms in OSCCs. Offering a promising avenue for studying OSCCs, organs-on-a-chip serve as microphysiological platforms that closely emulate the complexities of biological tissues within the highly controllable microfluidic, cellular communication, and extracellular matrix. Here, we developed an OSCC on-a-chip to understand the immunomodulatory profile of F. nucleatum on macrophages in the TME. We cultivated oral squamous carcinoma cells in 3D in a collagen matrix (2. 5 mg/mL) in a microfluidic device that has a central chamber (1mm in width) and two lateral channels connected by triangular pillars (space between pillars of 115 µm). Our tested groups were collagen matrix containing OSCC (5. 105 cells/mL) in the presence or absence of heat-killed antigens (1. 106 CFUs/mL) from F. nucleatum (ATCC 23726). F. nucleatum cells were integrated with collagen gels and oral squamous carcinoma cells for three days. On the fourth day, we added 2. 103 THP-1 derived macrophages (differentiated with PMA 100 ng/mL) in the lateral channel to evaluate their chemoattraction to the 3D matrix in the presence or absence of F. nucleatum for 48 hours. We noticed a significantly higher number of macrophages invading the 3D matrices that had heat-killed antigens when compared to the groups containing just OSSCs. By immunofluorescence, we stained the cells with markers for M1 (CD86) and M2 (CD163) and observed a significantly lower number of CD86 and a higher number of CD163-positive cells in the F. nucleatum stimulated group. When cytokines and chemokines were measured from these chips by Luminex assay, we found that the presence of heat-killed bacteria upregulated TNF-α, MCP-1, IL-6, VEGF, and CCL5 significantly, showing that the presence of F. nucleatum might stimulate tumor-associated macrophages-like cells. These results enable us to understand how the early interactions between opportunistic microorganisms and the immune system modulate the suppression responses in the tumor microenvironment. Citation Format: Mauricio G. Sousa, Avathamsa Athirasala, May Anny A. Fraga, Dustin Higashi, Justin Merrit, Luiz Bertassoni. Fusobacterium nucleatum antigens drive tumor-associated macrophage-like cells in an engineered oral squamous cell carcinoma on-a-chip abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts) ; 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84 (6Suppl): Abstract nr 2797.