Tumor microenvironment-like conditions alter pancreatic cancer cell metabolism and behavior

The tumor microenvironment is complex and dynamic, characterized by poor vascularization, limited nutrient availability, hypoxia, and an acidic pH. This environment plays a critical role in driving cancer progression. However, standard cell culture conditions used to study cancer cell biology in vitro fail to replicate the in vivo environment of tumors. Recently, "physiological" cell culture media that closely resemble human plasma have been developed (e.g., Plasmax, HPLM), along with more frequent adoption of physiological oxygen conditions (1%-8% O₂). Nonetheless, further refinement of tumor-specific culture conditions may be needed. In this study, we describe the development of a tumor microenvironment medium (TMEM) based on murine pancreatic ductal adenocarcinoma (PDAC) tumor interstitial fluid. Using RNA-sequencing, we show that murine PDAC cells (KPCY) cultured in tumor-like conditions (TMEM, pH 7.0, 1.5% O₂) exhibit profound differences in gene expression compared with plasma-like conditions (mouse plasma medium, pH 7.4, 5% O₂). Specifically, the expression of genes and pathways associated with cell migration, biosynthesis, angiogenesis, and epithelial-to-mesenchymal transition were altered, suggesting tumor-like conditions promote metastatic phenotypes and metabolic remodeling. Using functional assays to validate RNA-seq data, we confirmed increased motility at 1.5% O₂/TMEM, despite reduced cell proliferation. Moreover, a hallmark shift to glycolytic metabolism was identified via measurement of glucose uptake/lactate production and mitochondrial respiration. Taken together, these findings demonstrate that growth in 1.5% O₂/TMEM alters several biological responses in ways relevant to cancer biology, and more closely models hallmark cancerous phenotypes in culture. This highlights the importance of establishing tumor microenvironment-like conditions in standard cancer research. <b>NEW & NOTEWORTHY</b> Standard cell culture conditions do not replicate the complex tumor microenvironment experienced by cells in vivo. Although currently available plasma-like media are superior to traditional supraphysiological media, they fail to model tumor-like conditions. Using RNA-seq analysis and functional metabolic and migratory assays, we show that tumor microenvironment medium (TMEM), used with representative tumor hypoxia, better models cancerous phenotypes in culture. This emphasizes the critical importance of accurately modeling the tumor microenvironment in cancer research.