<h3>Purpose/Objective(s)</h3> Glioblastoma, historically viewed as a single disease entity, is now known to be composed of various subtypes. Here, we present our work evaluating the effects of chemoradiotherapy on cellular biophysical processes using a novel murine model of glioblastoma developed by our group. <h3>Materials/Methods</h3> Primary tumor cell lines were established from a murine glioma model generated from a synthetic DNA transposon system delivered oncogenic drivers SV40-LgTA and either NRASV12 or PDGFB. We have previously shown these cell lines, termed NRAS and PDGF, respectively, recapitulate the mesenchymal and proneural tumor subtypes. Treatment was administered with temozolomide (TMZ) to a maximum of 100µM, single fraction radiation therapy (RT) to a maximum of 10 Gy, or both. Cells were stained with Hoechst dye, imaged with an epifluorescence microscope with 10X objective lens, and counted with an image segmentation algorithm daily for 4 days to estimate cell survival and proliferation. On post-treatment day 4, surviving cells were plated on Matrigel coated 9.3kPa polyacrylamide gel and imaged with an inverted, transmitted light microscope with 10X objective lens every 5 minutes for 4 hours. Single cell mean squared displacement (MSD) was measured from time lapse images via an image segmentation algorithm. The random motility coefficient (µ) was calculated by the relationship MSD(t) = 4µt, where t is time. Data was compared via the Kruskal-Wallis test with Bonferroni correction for multiple comparisons when appropriate. <h3>Results</h3> There was no significant difference in cellular death or proliferation between treatment and control arms for the PDGF cell lines. In the NRAS cell lines, TMZ alone did not induce cell death while RT (P < 0.01) and combined modality therapy (P < 0.01) yielded cellular killing in a dose dependent manner. Furthermore, RT-treated NRAS cells proliferated more slowly than non-treated cells (0.05 vs. 0.20 hr-1, P=0.04). Control NRAS cell lines migrated faster than their PDGF counterparts at baseline (3.82 vs. 0.73 µm2/hr, P<1E-5). Within each cell line, the use of combined modality therapy significantly reduced cellular motility (PDGF: 0.73 vs. 0.21 µm2/hr, P=0.008; NRAS: 0.65 vs. 3.82 µm2/hr, P<1E-5). RT monotherapy additionally reduced motility in NRAS cell lines (1.07 vs. 3.82 µm2/hr, P<1E-5) while no difference was observed in PDGF cells. TMZ monotherapy did not affect motility within either cell line. <h3>Conclusion</h3> We evaluated biophysical properties of a novel murine glioma model utilizing oncogenic drivers overexpressing NRASV12 or PDGFB which we have previously shown recapitulates the mesenchymal and proneural subtypes of human glioblastoma, respectively. Here, we observed phenotypic differences in cellular migration following RT and TMZ regimens which could have subtype-specific ramifications for radiotherapy dose and target volume delineation.