Abstract Hypoxia and the development of spatially heterogeneous hypoxic and/or necrotic regions in non-small cell lung cancer (NSCLC) and other solid tumors is associated with chemotherapy and radiation resistance. One mechanism by which this could occur is through modification of the cell-cell interactions (or games) between the resistant and naïve cells within the tumor. Cell-cell interactions can modify the fitness of cells and thereby change the evolutionary dynamics of a tumor. Hypoxia may, in this way, change the game dynamics between cells and directly support maintenance of a population of resistant cells. To understand the mechanism by which hypoxic heterogeneity modulates evolutionary dynamics and therapy response, we combine computational modeling and in vitro experiments under a spatial hypoxia gradient. We utilize in-house game assay protocols to probe the evolutionary games between cell types. The experimental system is adapted from Carmona-Fontaine's MEMIC plate. This system allows us to create stable oxygen and nutrient gradients for 12 evolutionary replicates of NSCLC PC-9 cells. Fluorescence labeled gefitinib-resistant PC-9 cells and naïve parental PC-9 cells are co-cultured in a closed chamber with limited availability to oxygen and nutrient supply at one end. A green hypoxia probe is used to verify the establishment of a hypoxia gradient and a red Annexin V probe monitors apoptosis. Once microenvironmental gradients are established due to cellular consumption of oxygen and nutrients, images of cells are taken every 1.5 hours for 5 days. We carry out multiple experiments in which we vary the initial proportions of parental and resistant cells. Computationally, green and red fluorescence from the images captured by the microscopy system is quantified by an in-house image processing pipeline. We apply a random forest machine learning classifier, illumination correction, and object identification to each image to gather fluorescence counts for each well. These counts are used to compute the background rate of cell death and absolute vs. relative oxygen levels. The subsequent cell counts over time were used to calculate the growth rate (fitness) of the cell types in different environments and therefore the strength of environmental and frequency dependent interactions between the cell types. This framework allows for tightly coupled computational and in vitro experiments aimed at understanding and predicting the clinical implications of hypoxia resistance across a range of tumor types and microenvironments. We have measured the effect of hypoxia upon game interactions between gefinib-resistant and susceptible NSCLC cells. The evolutionary game changes with changing levels of hypoxia–and for the first time we are able to write down a continuous equation representing the game as a function of oxygen. Citation Format: Mina Dinh, Bezhou Feng, Jeff Maltas, Emily Dolson, Masahiro Hitomi, Steph Owen, Jacob Scott. Exploring the effect of hypoxia and spatial interactions on the dynamics between gefitinib resistant and naïve NSCLC cell lines [abstract]. In: Proceedings of the AACR Special Conference on the Evolutionary Dynamics in Carcinogenesis and Response to Therapy; 2022 Mar 14-17. Philadelphia (PA): AACR; Cancer Res 2022;82(10 Suppl):Abstract nr A027.