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Heterogeneous glioblastoma cell cross-talk promotes phenotype alterations and enhanced drug resistance.

ABSTRACT: Glioblastoma multiforme is the most lethal of brain cancer, and it comprises a heterogeneous mixture of functionally distinct cancer cells that affect tumor progression. We examined the U87, U251, and U373 malignant cell lines as in vitro models to determine the impact of cellular cross-talk on their phenotypic alterations in co-cultures. These cells were also studied at the transcriptome level, to define the mechanisms of their observed mutually affected genomic stability, proliferation, invasion and resistance to temozolomide. This is the first direct demonstration of the neural and mesenchymal molecular fingerprints of U87 and U373 cells, respectively. U87-cell conditioned medium lowered the genomic stability of U373 (U251) cells, without affecting cell proliferation. In contrast, upon exposure of U87 cells to U373 (U251) conditioned medium, U87 cells showed increased genomic stability, decreased proliferation rates and increased invasion, due to a plethora of produced cytokines identified in the co-culture media. This cross talk altered the expression 264 genes in U87 cells that are associated with proliferation, inflammation, migration, and adhesion, and 221 genes in U373 cells that are associated with apoptosis, the cell cycle, cell differentiation and migration. Indirect and direct co-culturing of U87 and U373 cells showed mutually opposite effects on temozolomide resistance. In conclusion, definition of transcriptional alterations of distinct glioblastoma cells upon co-culturing provides better understanding of the mechanisms of glioblastoma heterogeneity, which will provide the basis for more informed glioma treatment in the future.

SUBMITTER: Motaln H

PROVIDER: S-EPMC4747385 | biostudies-literature | 2015 Dec

REPOSITORIES: biostudies-literature

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Heterogeneous glioblastoma cell cross-talk promotes phenotype alterations and enhanced drug resistance.

Motaln Helena H Koren Ana A Gruden Kristina K Ramšak Živa Ž Schichor Christian C Lah Tamara T TT

Oncotarget 20151201 38

Glioblastoma multiforme is the most lethal of brain cancer, and it comprises a heterogeneous mixture of functionally distinct cancer cells that affect tumor progression. We examined the U87, U251, and U373 malignant cell lines as in vitro models to determine the impact of cellular cross-talk on their phenotypic alterations in co-cultures. These cells were also studied at the transcriptome level, to define the mechanisms of their observed mutually affected genomic stability, proliferation, invasi ...[more]

PMID: 26517510

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Project description:Despite tremendous research efforts, successful targeting of aberrant tumor metabolism in clinical practice has remained elusive. Tumor heterogeneity and plasticity may play a role in the clinical failure of metabolism-targeting interventions for treating cancer patients. Moreover, compensatory growth-related processes and adaptive responses exhibited by heterogeneous tumor subpopulations to metabolic inhibitors are poorly understood. Here, by using clinically-relevant patient-derived glioblastoma (GBM) cell models, we explore the cross-talk between glycolysis, autophagy, and senescence in maintaining tumor stemness. We found that stem cell-like GBM tumor subpopulations possessed higher basal levels of glycolytic activity and increased expression of several glycolysis-related enzymes including, GLUT1/SLC2A1, PFKP, ALDOA, GAPDH, ENO1, PKM2, and LDH, compared to their non-stem-like counterparts. Importantly, bioinformatics analysis also revealed that the mRNA expression of glycolytic enzymes positively correlates with stemness markers (CD133/PROM1 and SOX2) in patient GBM tumors. While treatment with glycolysis inhibitors induced senescence in stem cell-like GBM tumor subpopulations, as evidenced by increased β-galactosidase staining and upregulation of the cell cycle regulators p21Waf1/Cip1/CDKN1A and p16INK4A/CDKN2A, these cells maintained their aggressive stemness features and failed to undergo apoptotic cell death. Using various techniques including autophagy flux and EGFP-MAP1LC3B+ puncta formation analysis, we determined that inhibition of glycolysis led to the induction of autophagy in stem cell-like GBM tumor subpopulations, but not in their non-stem-like counterparts. Similarly, blocking autophagy in stem cell-like GBM tumor subpopulations induced senescence-associated growth arrest without hampering stemness capacity or inducing apoptosis while reciprocally upregulating glycolytic activity. Combinatorial treatment of stem cell-like GBM tumor subpopulations with autophagy and glycolysis inhibitors blocked the induction of senescence while drastically impairing their stemness capacity which drove cells towards apoptotic cell death. These findings identify a novel and complex compensatory interplay between glycolysis, autophagy, and senescence that helps maintain stemness in heterogeneous GBM tumor subpopulations and provides a survival advantage during metabolic stress.

Project description:Malignant gliomas (glioblastoma multiforme) have a poor prognosis with an average patient survival under current treatment regimens ranging between 12 and 14 months. The tumors are characterized by rapid cell growth, extensive neovascularization, and diffuse cellular infiltration of normal brain structures. We have developed a human glioblastoma xenograft model in nude rats that is characterized by a highly infiltrative non-angiogenic phenotype. Upon serial transplantation this phenotype will develop into a highly angiogenic tumor. Thus, we have developed an animal model where we are able to establish two characteristic tumor phenotypes that define human glioblastoma (i.e. diffuse infiltration and high neovascularization). Here we aimed at identifying potential biomarkers expressed by the non-angiogenic and the angiogenic phenotypes and elucidating the molecular pathways involved in the switch from invasive to angiogenic growth. Focusing on membrane-associated proteins, we profiled protein expression during the progression from an invasive to an angiogenic phenotype by analyzing serially transplanted glioma xenografts in rats. Applying isobaric peptide tagging chemistry (iTRAQ) combined with two-dimensional LC and MALDI-TOF/TOF mass spectrometry, we were able to identify several thousand proteins in membrane-enriched fractions of which 1460 were extracted as quantifiable proteins (isoform- and species-specific and present in more than one sample). Known and novel candidate proteins were identified that characterize the switch from a non-angiogenic to a highly angiogenic phenotype. The robustness of the data was corroborated by extensive bioinformatics analysis and by validation of selected proteins on tissue microarrays from xenograft and clinical gliomas. The data point to enhanced intercellular cross-talk and metabolic activity adopted by tumor cells in the angiogenic compared with the non-angiogenic phenotype. In conclusion, we describe molecular profiles that reflect the change from an invasive to an angiogenic brain tumor phenotype. The identified proteins could be further exploited as biomarkers or therapeutic targets for malignant gliomas.

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Heterogeneous glioblastoma cell cross-talk promotes phenotype alterations and enhanced drug resistance.

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Heterogeneous glioblastoma cell cross-talk promotes phenotype alterations and enhanced drug resistance.

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