The role of EGFR double minutes in modulating the response of malignant gliomas to radiotherapy

Abstract

EGFR amplification in cells having double minute chromosomes (DM) is commonly found in glioblastoma multiforme (GBM); however, how much it contributes to the current failure to treat GBM successfully is unknown. We studied two syngeneic primary cultures derived from a GBM with and without cells carrying DM, for their differential molecular and metabolic profiles, in vivo growth patterns, and responses to irradiation (IR). Each cell line has a distinct molecular profile consistent with an invasive “go” (with DM) or angiogenic “grow” phenotype (without DM) demonstrated in vitro and in intracranial xenograft models. Cells with DM were relatively radio-resistant and used higher glycolytic respiration and lower oxidative phosphorylation in comparison to cells without them. The DM-containing cell was able to restore tumor heterogeneity by mis-segregation of the DM-chromosomes, giving rise to cell subpopulations without them. As a response to IR, DM-containing cells switched their respiration from glycolic metabolism to oxidative phosphorylation and shifted molecular profiles towards that of cells without DM. Irradiated cells with DM showed the capacity to alter their extracellular microenvironment to not only promote invasiveness of the surrounding cells, regardless of DM status, but also to create a pro-angiogenic tumor microenvironment. IR of cells without DM was found primarily to increase extracellular MMP2 activity. Overall, our data suggest that the DM-containing cells of GBM are responsible for tumor recurrence due to their high invasiveness and radio-resistance and the mis-segregation of their DM chromosomes, to give rise to fast-growing cells lacking DM chromosomes.

Dual antivascular function of human fibulin‐3 variant, a potential new drug discovery strategy for glioblastoma

“…Immunohistochemistry of glial fibrillary acidic protein (GFAP) revealed negative staining of GFAP in central tumor mass of both PBS and ZR30‐treated xenografts. Because 51A cells express GFAP, whereas 51B cells do not, GFAP‐negative tumor mass is consistent with our prior findings that 51B formed tumor masses in syngeneic GBM i.c. xenograft models of 51A and 51B (9:1).…”

“…As shown previously, i.c. implantation of a mixture of 100 000 cells of 51A and 51B (9:1) formed i.c.…”

“…whereas 51B cells do not,19 GFAP-negative tumor mass is consistent with our prior findings that 51B formed tumor masses in syngeneic Real-time PCR primer sequences and amplicon melting temperature…”

“…Cells used in this study were validated to be free of mycoplasma by analyzing culture medium with MycoAlert PLUS Mycoplasma from Lonza and/or PCR‐based Venor GeM Mycoplasma Detection Kit (Sigma‐Aldrich). Identification of mutations in TP53 , PTEN , IDH1 , and IDH2 and DNA copy number variation of PTEN and EGFR were carried out using cDNA and DNA samples of GBM primary cultures, following PCR‐based mutation assay with sequencing and comparative quantitative PCR, respectively, as described previously . The genetic identity profiles of 16 short tandem repeats were determined based on DNA samples by IDEXX RADIL, with results shown in Table .…”

“…Human GBM-derived syngeneic primary cultures used in this study were established in the Brain Tumor Research Laboratory, UC Irvine, following methods detailed previously. 19 In brief, single cells of GBM specimens were equally divided into 2 or 3 portions to culture in 2 (namely SA and NS) or 3 (namely SA, NS, and EC) conditions. In the SA condition, cells were cultured with DMEM/F12 medium supplemented with 10% FBS in collagen-coated (initial culture) or regular culture dishes (subsequent passages).…”