Differential gene expressions of cancer cells cultured in an oxygen-limiting, micro-metabolic rewiring (μMeRe) device over their normoxia control

Cancer metabolism plays an essential role in therapeutic resistance, where significant inter- and intra-tumoral heterogeneity exists. Hypoxia is a prominent driver of metabolic rewiring behaviors and drug responses. Recapitulating the hypoxic landscape in tumor microenvironment thus offers unique insights into heterogeneity in metabolic rewiring and therapeutic responses, to inform better treatment strategies. We developed a micro-metabolic rewiring (μMeRe) device that provides the scalability and resolution needed to characterize the metabolic rewiring behaviors of different cancer cells in the context of hypoxic solid tumors. Our device generates hypoxia through cellular metabolism without external gas controls, enabling the characterization of cell-specific intrinsic ability to drive hypoxia and undergo metabolic rewiring. We assessed the changes in the transcriptomic profile of different cancer cells in the μMeRe device compared to their normoxia counterpart through bulk RNA-seq. Our data confirms heterogeneity and commonality of rewiring behaviors of diverse cancer cell types. To examine the heterogeneous metabolic rewiring characteristics of different types of cancer cells, we cultured 8 human cancer cell lines originating from 4 different organs (ovary: OAW42, SKOV3, prostate: LNCaP, PC3, lung: A549, PC9, Breast: MCF7, T47D) in μMeRe devices and their normoxia counterparts (serving as control). Cancer cells were injected into the μMeRe device, where they formed a 2D circular micropattern sandwiched between two oxygen diffusion barriers (polycarbonate at the top and glass at the bottom). A natural oxygen gradient was established across the cancer micropattern due to cell-specific consumption and limited lateral diffusion of oxygen across the monolayer. Cancer cells were also injected into the normoxia device sharing the the same dimensions as the µMeRe device except without the PC oxygen diffusion barrier, thus with unobstructed oxygen supply. All µMeRe and corresponding normoxia devices were disassembled after 24 hours of culture to expose the micropatterns for cancer cell extraction, RNA isolation, and RNA-seq. Transcriptome-wide pairwise gene expression analysis was performed between the µMeRe device and its normoxia counterpart for each cancer cell line to identify differentially expressed genes (DEGs) in the oxygen limited microenvironment.