Quantification of oxygen tension variation in an in vitro tumor microenvironment model based on co-culture of tumor spheroid and endothelial cells in a microfluidic device

• A microfluidic model replicates the tumor microenvironment with a spheroid and endothelium. • Oxygen-sensing microbeads and FD-FLIM enable oxygen tension mapping for an extended period. • Endothelial cells modulate oxygen microenvironments in proximity to solid tumors. • Quantitative analysis provides novel insights into tumor-endothelium oxygen dynamics. In this study, we developed an in vitro tumor microenvironment model that integrates a tumor spheroid and a monolayer of endothelial cells within a microfluidic device, aiming to replicate the complex conditions of an avascular solid tumor with limited oxygen supply. The tumor spheroid is cultured in a gas-impermeable plastic chamber, which effectively simulates the limited oxygen supply environment of solid tumors, thereby providing a realistic platform for studying tumor behavior and interactions with surrounding cells. To characterize the three-dimensional spatial oxygen tension profiles, we utilized oxygen-sensing microbeads with widefield frequency-domain fluorescence lifetime imaging microscopy (FD-FLIM). The FD-FLIM setup ensures minimal excitation light dosage to reduce photocytotoxicity while providing capabilities for reliable three-dimensional oxygen tension measurements for an extended period. The experimental results show that the oxygen tensions were lowered to approximately 6% in the neighborhood of a colon cancer cell line (HCT116) tumor spheroid. Interestingly, when the tumor spheroid is co-cultured with a monolayer of endothelial cells (human umbilical vein endothelial cell, HUVEC), the lowering of oxygen tension can be alleviated. Our findings highlight the significant interactions between the tumor spheroid and endothelial cells, emphasizing the crucial role of the endothelium in regulating oxygen microenvironments and influencing angiogenesis. Furthermore, the model provides a valuable tool for exploring the dynamics of tumor biology. This capability is particularly beneficial for drug development and preclinical validation, as it allows for the observation of interactions among different cell types and the assessment of stimulations or environmental factors.