RTRB-24SYMBIOSIS BETWEEN GBM CELL SUBPOPULATIONS, WITH OR WITHOUT EGFR AMPLIFICATION, IS A NEW MECHANISM OF TUMOR RESISTANCE TO RADIATION THERAPY

Abstract

BACKGROUND: EGFR amplification in a tumor cell subpopulation was frequently found of GBMs, through multiple copies of chromosome 7 fragment known as EGFR double minute. Intra-tumoral heterogeneity is thought to confer a survival advantage to tumor, and may be the basis for the observed resistance to therapeutic interventions such as irradiation - a frontline therapy for malignant gliomas. However, direct evidence supporting this hypothesis is currently limited. We have established two syngeneic primary cultures (51A and 51B) of a recurrent GBM with high level of EGFR amplification, under neural sphere (NS) and serum adherent (SA) and culture conditions for more than three months or eight passages, respectively. EGFR-FISH analysis showed 77% cells in 51A carrying EGFR double minute, while absence of such cell subpopulation in 51B. In comparison to 51B, 51A showed higher levels of invasiveness and cell doubling time. METHODS: To study symbiosis between GBM cell subpopulations with or without EGFR amplification in response to radiation, we firstly compared their differential response to radiation on growth/survival, cellular bioenergetics and redox, and cell invasiveness. Then using matrigel invasion assay, we analyzed the effect of radiated 51A to un-radiated 51B cells on their invasiveness, after 1-week culture with serum-supplied conditioned medium of 51A (0 or 0.5GyX10d). RESULTS: Both mitochondrial and glycolytic respirations were increased in 51A but decreased in 51B, 1 week post a 2-Gy dose of irradiation. Reactive oxygen species (ROS) level was increased in 51A, which was not shown in 51B, even after a 5-Gy dose of radiation. Consistently, 51A was more radio-resistance compared to 51B. Interestingly, 51B cells cultured for 1 week in conditioned medium of radiated 51A cells doubled their invasion activity. CONCLUSIONS: There exists a symbiotic relationship between GBM cell subpopulations, with or without EGFR amplification, which supports overall tumor growth/expansion under radiation therapy.