Divergent evolution of temozolomide resistance in glioblastoma stem cells is reflected in extracellular vesicles and coupled with radiosensitization

Abstract

Background: Glioblastoma (GBM) is almost invariably fatal due to failure of standard therapy. The relapse of GBM following surgery, radiation, and systemic temozolomide (TMZ) is attributed to the ability of glioma stem cells (GSCs) to survive, evolve, and repopulate the tumor mass, events on which therapy exerts a poorly understood influence.

Methods: Here we explore the molecular and cellular evolution of TMZ resistance as it emerges in vivo (xenograft models) in a series of human GSCs with either proneural (PN) or mesenchymal (MES) molecular characteristics.

Results: We observed that the initial response of GSC-initiated intracranial xenografts to TMZ is eventually replaced by refractory growth pattern. Individual tumors derived from the same isogenic GSC line expressed divergent and complex profiles of TMZ resistance markers, with a minor representation of O6-methylguanine DNA methyltransferase (MGMT) upregulation. In several independent TMZ-resistant tumors originating from MES GSCs we observed a consistent diminution of mesenchymal features, which persisted in cell culture and correlated with increased expression of Nestin, decline in transglutaminase 2 and sensitivity to radiation. The corresponding mRNA expression profiles reflective of TMZ resistance and stem cell phenotype were recapitulated in the transcriptome of exosome-like extracellular vesicles (EVs) released by GSCs into the culture medium.

Conclusions: Intrinsic changes in the tumor-initiating cell compartment may include loss of subtype characteristics and reciprocal alterations in sensitivity to chemo- and radiation therapy. These observations suggest that exploiting therapy-induced changes in the GSC phenotype and alternating cycles of therapy may be explored to improve GBM outcomes.

Keywords: exosomes; glioma stem cells; molecular subtypes; radiation; temozolomide resistance.

Fig. 1

Phenotypic and molecular heterogeneity of glioma stem cells (GSCs). (A) Morphology of mesenchymal (MES) and proneural (PN) GSCs in sphere culture—phase contrast microscopy, under 10x objective. (B) Proteomes of MES and PN cell lines. The main validated subtype-specific proteins are indicated (arrows). (C) Differentially expressed proteins extracted from the proteome of MES and PN GSCs. (D) Western blot validation of differentially expressed marker proteins in MES and PN GSC lines.

Fig. 2

Differential responses of GSC-initiated intracranial glioblastoma xenografts to TMZ. (A) Experimental design—tumor regression was induced by initial dose of TMZ (120 mg/kg), which was repeated at relapse of individual tumors (red dots) until response was no longer observed. (B, C) Survival curves of mice harboring xenografts initiated by either MES-1123 (B) or PN-528 (C) GSCs. Cells were injected at 1 × 10⁴ cells per inoculum into the brains of NSG mice. Untreated control (discontinuous line; N = 5) or treated with TMZ (continuous line; N = 5 for MES, N = 4 for PN) are shown. (D–G) Heterogeneity in the natural history of the disease in individual mice harboring GSC-initiated GBMs. Clinical response to progression in individual mice was measured by weight changes. Numerical designations in panels D–G indicate the respective individual xenografts with either TMZ sensitive (S) or resistant (R) phenotype (see text). **P < 0.01.

Fig. 3

Secondary GSCs represent a stable and cell intrinsic TMZ resistance phenotype, coupled with a loss of mesenchymal characteristics. (A, B) TMZ resistance of GSCs is maintained in sphere culture (MTS viability assay, see text). MTS assay after TMZ treatment in 2 SGSC lines (1123IC8R, 1123IC7R) from TMZ-unresponsive tumors and 2 SGSC lines from control tumors (1123IC12S, 1123IC13S) (N = 3; 100% = no TMZ). ****P < 0.0001. (C–E) TGM2 downregulation and Nestin induction in TMZ resistant phenotype were observed in vitro after isolation of SGSCs from xenograft tumors, at the mRNA level tested by qPCR (C, D) (1 = average mRNA expression in TMZ-sensitive cell lines), and at the protein level by western blot (E). ****P < 0.0001; **P < 0.01 (N = 3). (F–H) Immunostaining of mesenchymal control (PT1123ICxS) and TMZ-treated (PT1123ICxR) intracranial tumors confirmed the variability in protein expression, a trend toward a decrease in mesenchymal markers TGM2 and CD44, and an upregulation of Nestin expression after acquisition of resistance to TMZ (objective 20x).

Fig. 4

Heterogeneous expression of genes associated with TMZ resistance among secondary GSCs isolated from individual mice following chemotherapy in vivo. Profiles of mRNA among clonally related SGSCs isolated from PN (528) or MES (1123) brain tumors: control (S), TMZ resistant (R) (as in Fig. 2). Markers for which differences were significant are shown as bar graphs (right panel), in MES (A) and PN (B) cell lines (1 = average mRNA expression in TMZ-sensitive cell lines). The heatmap (left panel) represents the global profile of all markers of resistance tested. ****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05 (N = 3).

Fig. 5

Reflection of molecular changes associated with TMZ resistance in GSC-derived EVs. (A) TMZ effects on cell viability (MTS assay). (B, C) Nanoparticle tracking analysis of EVs produced by SGSC lines in the absence (B) or presence of 100 μM TMZ (C). (D, E) Expression of transcripts implicated in TMZ resistance in GSCs and in their EVs. (D) Quantitative PCR analysis reveals changes in EV-associated TGM2, MGMT, and/or Nestin (1 = average mRNA expression in TMZ-sensitive cell lines). (E) The comparison of heatmaps between cell and EV profiles shows a great similarity. *P < 0.05 (N = 2).

Fig. 6

Elevated sensitivity of TMZ-resistant GSC lines to radiation. Individual (A) and averaged (B) relative (%) changes in surviving TMZ-sensitive (1123ICS, white bars) and TMZ-resistant (1123ICR, black bars) cells after exposure to 0, 2, 4, 6, and 8 Gy of radiation therapy in vitro. TMZ-resistant cell lines exhibit greater cytotoxic impact. (C–E) Impact of radiation (2 Gy) on tumor forming potential of TMZ-sensitive and -resistant GSCs. Pre-irradiated cells were injected into NSG mice. (C) Tumor volume after 26 days of growth. (D, E) Tumor growth curves of TMZ-sensitive (D) or TMZ-resistant (E) GSC lines treated (grey symbols) or not (white or black symbols) with radiation. ****P < 0.0001; **P < 0.01; *P < 0.05 (N = 3).