Abstract 589: Long-term acclimation of target cell lines to tumor microenvironment culture condition provides mechanistic insights into cell therapy effectiveness

The tumor microenvironment (TME) has several characteristics that distinguish it from normal tissue, including elevated interstitial fluid pressures and hypoxia. To study the effects of TME culture conditions, commonly used target cell lines were serially passaged under hyperbaric and hypoxic conditions for a period of eight weeks. Conventionally expanded parental cell lines (normoxia) were compared against TME-acclimated tumor cell lines (TACTLs; hyperbaric and hypoxic), using RNAseq, ATACseq, and flow cytometry analysis. Commonly used target cell lines for cell therapy development were selected for TME acclimation (SKOV3, A549, JEKO1, and NALM6). TACTLs were generated using an AVATAR system, with culture conditions set at 1% O2 and 2.0 PSI, and serially passaged twice a week for a minimum of 8 weeks. Cell doubling times were measured weekly and compared against parental lines maintained under normoxic culture conditions in a conventional CO2 incubator. In-depth characterization was performed on TME-Acclimated Tumor Cell Lines (TACTLs), utilizing a multi-omic approach. Differential gene expression analysis was performed using RNAseq datasets, and surface biomarkers/targets used for cell therapy development were assessed via nanostring and flow cytometry. Growth kinetics and cell doubling times of TACTLs were initially inhibited during the first two weeks of culture under TME conditions, but eventually reached parity with their normoxia maintained parental cell lines at 6 to 8 weeks, signaling a successful adaptation of the tumor lines to low oxygen and hyperbaric conditions. RNAseq analysis revealed upregulation in glycolytic pathways and epithelial-to-mesenchymal signaling, accompanied by altered metabolic profiles. Surface target expression showed increased expression of checkpoint ligands, such as PD-L1. Cell therapy targets, such as ROR1 were also upregulated in A549 and JEKO1 cell lines passaged under TME. Preliminary drug screening experiments were conducted using checkpoint inhibitors and CAR-T candidates on TACTLs, revealing significant changes in half maximal inhibitory concentration (IC50) when compared to parental tumor lines maintained under normoxia. In summary, acclimation of tumor cell lines to TME culture conditions can provide unique mechanistic insights that can facilitate drug development efforts. Future studies will incorporate TACTLs for CDX tumor models with the goal of identifying cell therapies that work effectively in the tumor mi