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 foci formation. To determine whether these results were unique to NSC11 tumors, similar experiments were performed using CD133+ GBMJ1 TSCs. The intracerebral implantation of these cells results in the formation of highly invasive brain tumors with cells expressing GFAP, betaIII tubulin as well as CD133, similar to NSC11 tumors. After irradiation (6 Gy) of ic xenografts, gammaH2AX foci were readily detectable at 0.5h returning to control levels by 6h. These results were then compared to those obtained from GBMJ1 cells maintained under 2 types of in vitro growth conditions. As for the NSC11 model, the dispersal of gammaH2AX foci was more rapid and complete after irradiation of GBMJ1 ic xenografts. In addition, the susceptibility of cells grown orthotopically to radiation-induced gammaH2AX foci was substantially less than for GBMJ1 cells irradiated in vitro. To begin to address the mechanisms responsible for the differences in foci induction, microarray analysis was used to generate gene expression signatures for NSC11 and GBMJ1 cells grown in vivo as ic xenografts and in vitro under stem cell and differentiated conditions. To define the changes in gene expression resulting from in vivo growth, gene expression signatures of the ic xenografts were directly compared to those generated from their corresponding in vitro cultures (CD133+ and differentiated). According to cluster analysis, within each of the GBM models, there were a significant number of genes in CD133+ and differentiated cultures that were commonly affected by in vivo growth and a significant number of genes similarly affected by in vivo growth in both NSC11 and GBMJ1. These results suggest that the effects of the brain microenvironment on gene expression are not tumor specific and may be of a more general nature. The gene expression profiles comparing in vivo to in vitro growth were then evaluated in terms of genes involved in ROS scavenging and anti-oxidant response. Accordingly, gene expression signatures for ic xenografts versus their respective in vitro cultures were interrogated using a list of genes associated with ROS metabolism and anti-oxidant response. Each xenograft contained substantially more commonly up-regulated ROS/ARE genes than their respective in vitro CD133+ and differentiated cultures. These results indicate that a consequence of orthotopic growth of GBM cells is an increased expression of ROS/ARE related genes, which would be consistent with greater anti-oxidant capacity and reduced susceptibility to radiation-induced DSBs. Based on gammaH2AX and 53BP1 foci analyses the data generated in this fiscal year indicate that GBM cells irradiated within orthotopic xenografts have a greater capacity to repair DSBs and are less susceptible to their induction than tumor cells irradiated under in vitro growth conditions. Because DSB induction and repair are critical determinants of radiosensitivity, these results imply that the brain microenvironment contributes to GBM radioresistance. Although the relative decrease in susceptibility of ic xenografts to DSB induction can be linked to modifications in gene expression, the specific mechanisms through which the microenvironment regulates radiation-induced DNA damage and repair, and thus radiosensitivity, remain the subject of future investigations. However, based on these results it appears that for preclinical studies aimed at the identification of targets for GBM radiosensitization and the evaluation of corresponding radiosensitizers it will be necessary to account for the brain microenvironment.
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