ZIA BC 011372 (ZIA) | |||
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Title | Microenvironmental regulation of glioblastoma radiosensitivity | ||
Institution | NCI, Bethesda, MD | ||
Principal Investigator | Tofilon, Philip | NCI Program Director | N/A |
Cancer Activity | N/A | Division | CCR |
Funded Amount | $515,574 | Project Dates | null - null |
Fiscal Year | 2018 | Project Type | Intramural |
Research Topics w/ Percent Relevance | Cancer Types w/ Percent Relevance | ||
Cancer (100.0%) |
Brain (100.0%) Nervous System (100.0%) |
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Research Type | |||
Localized Therapies - Discovery and Development Combinations of Localized and Systemic Therapies |
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Abstract | |||
In this fiscal year we began studies aimed at determining whether intracerebral (ic) growth of GBM stem like cells (TSCs) influences their gene expression patterns and defined the influence of orthotopic growth on radiation-induced DNA double strand breaks (DSBs) and their repair. For these studies we initially focused on ic xenografts initiated from the CD133+ NSC11 GBM TSC line. The intracerebral implantation of CD133+ NSC11 cells into nu/nu mice results in the formation of a highly invasive brain tumor containing cells expressing GFAP, betaIII tubulin and CD133. Thus, these data indicate that the implanted CD133+ NSC11 cells proliferate and undergo differentiation along both glial and neuronal pathways to form an invasive, phenotypically heterogeneous tumor, i.e., recapitulating GBM in situ. As an indicator of radiation-induced DSBs we used gammaH2AX nuclear foci. Mice bearing NSC11 intracerebral tumors were irradiated (6 Gy) and gammaH2AX foci quantified in individual nuclei using confocal microscopy and a stacking procedure that allowed for evaluation of whole nuclei in the z-direction. The maximum number of gammaH2AX foci was reached at 0.5h after irradiation followed by a rapid decline at 1h with a further reduction by 6h; at 24h there was no significant difference as compared to control. gammaH2AX foci levels were also evaluated in NSC11 cells grown in vitro. For this analysis two in vitro growth conditions were evaluated: CD133+ NSC11 cells maintained in stem cell medium (TSCs) and CD133+ NSC11 cells that had been exposed to FBS for 10 days (differentiated), which results in the loss of CD133 expression and differentiation along astroglial and neuronal pathways. Use of both in vitro growth conditions thus approximates the phenotypic heterogeneity and cell cycle phase distribution of the ic xenografts. Comparison of the time courses for in vivo and in vitro growth conditions indicates that for cells irradiated as ic xenografts the maximum gammaH2AX foci dispersal rate occurred between 0.5 and 1h post-irradiation, whereas for both in vitro models foci dispersal was relatively steady over the 24h evaluation period. These data suggest that repair of radiation-induced DSBs was considerably more rapid in tumor cells irradiated under orthotopic conditions. Comparison of the initial time courses revealed that 6 Gy delivered to ic xenografts resulted in a similar level of gammaH2AX foci as induced by 2 Gy in vitro. To better illustrate this difference in susceptibility to radiation-induced gammaH2AX foci the dose response at 0.5h for each growth condition was determined. With respect to in vitro growth conditions, the differentiated cultures contained fewer radiation-induced gammaH2AX foci at each dose tested than the actively cycling CD133+ cultures, perhaps due to the differences in cell cycle phase distribution. However, the NSC11 cells grown orthotopically, were substantially less susceptible to gammaH2AX induction than both in vitro culture models. The same experiments were performed using 53BP1 foci, which provide an independent measure of radiation-induced DSBs. After irradiation of CD133+ NSC11 initiated xenografts the number of 53BP1 foci reached a maximum at 0.5h followed by a rapid decline at 1h returning to control levels by 6h. Irradiation of the NSC11 cells in vitro resulted in 53BP1 foci induction and dispersal similar to that detected for gammaH2AX. As compared to ic xenografts, the dispersal of 53BP1 foci after irradiation under both in vitro growth conditions was considerably slower, remaining significantly above control levels at 6h. Consistent with the gammaH2AX data, these results suggest that the repair of DSBs is more rapid in NSC11 cells grown orthotopically. In addition, the number of 53BP1 foci induced in both in vitro models at 0.5h after 2 Gy was similar to that induced by 6 Gy for cells within the ic xenografts. Thus, tumor cells grown orthotopically were less susceptible to radiation-induced 53BP1 |