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Gene expression analysis of osteosarcoma tumor-initiating cells (High) vs bulk tumor cells (Low)

ABSTRACT: In the cancer stem cell model a cell hierarchy has been suggested as an explanation for intratumoral heterogeneity, and tumor formation is thought to be driven by this tumor cell subpopulation. The identification of cancer stem cells in osteosarcoma (OS) and the biological processes dysregulated in this cell subpopulation, also known as tumor-initiating cells (TICs), may provide new therapeutic targets. The goal of this study, therefore, was to identify and characterize the gene expression profiles of TICs isolated from human OS cell lines. We analyzed the self-renewal capacity of OS cell lines and primary OS tumors based upon their ability to form sphere-like structures (sarcospheres) under serum-starving conditions. TICs were identify from OS cell lines using the long-term label retention dye PKH26. OS TICs and the bulk of tumor cells were isolated and used to assess their ability to initiate tumor in NOD/SCID mice. Gene expression profiles of OS TICs were obtained from fresh orthotopic tumor samples. We observed that increased sarcosphere efficiency correlated with an enhanced tumorigenic potential in OS. PKH26High cells were shown to constitute OS TICs based upon their capacity to form more sarcospheres, as well as to generate both primary bone tumors and lung metastases efficiently in NOD/SCID mice. Genomic profiling of OS TICs revealed that both bone development and cell migration processes were dysregulated in this tumor cell subpopulation. PKH26 labeling represents a valuable tool to identify OS TICs and gene expression analysis of this tumor cell compartment evidences potential therapeutic targets.

ORGANISM(S): Homo sapiens

PROVIDER: GSE33458 | GEO | 2011/11/04

SECONDARY ACCESSION(S): PRJNA148675

REPOSITORIES: GEO

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Project description:In the cancer stem cell model a cell hierarchy has been suggested as an explanation for intratumoral heterogeneity, and tumor formation is thought to be driven by this tumor cell subpopulation. The identification of cancer stem cells in osteosarcoma (OS) and the biological processes dysregulated in this cell subpopulation, also known as tumor-initiating cells (TICs), may provide new therapeutic targets. The goal of this study, therefore, was to identify and characterize the gene expression profiles of TICs isolated from human OS cell lines. We analyzed the self-renewal capacity of OS cell lines and primary OS tumors based upon their ability to form sphere-like structures (sarcospheres) under serum-starving conditions. TICs were identify from OS cell lines using the long-term label retention dye PKH26. OS TICs and the bulk of tumor cells were isolated and used to assess their ability to initiate tumor in NOD/SCID mice. Gene expression profiles of OS TICs were obtained from fresh orthotopic tumor samples. We observed that increased sarcosphere efficiency correlated with an enhanced tumorigenic potential in OS. PKH26High cells were shown to constitute OS TICs based upon their capacity to form more sarcospheres, as well as to generate both primary bone tumors and lung metastases efficiently in NOD/SCID mice. Genomic profiling of OS TICs revealed that both bone development and cell migration processes were dysregulated in this tumor cell subpopulation. PKH26 labeling represents a valuable tool to identify OS TICs and gene expression analysis of this tumor cell compartment evidences potential therapeutic targets. Gene expression analysis was performed in PKH26Hi cells (TICs) and PKH26Lo cells (non-TICs) obtained from five orthotopic bone tumors generated by each MNNG/HOS and 143B cell lines.The soft tissue component from each bone tumor was dissected, minced and processed. Immediately after FACS, 1.5x10^5 PKH26Hi cells and 7x10^5 PKH26Lo cells from each bone tumor sample were snap frozen and stored at -80°C until RNA processing. The microarray data were first evaluated for quality control using the AffyQCReport library of BioConductor 50.Two 143B PKH26Hi tumor samples were processed together due to insufficient RNA material.

Project description:Background: cancer cells rely on glycolysis as main ATP source (Warbürg effect). Tumor-initiating cells (TICs) are the fraction of cells that give raise and repopulate tumors. TICs are exposed to prolonged periods of oxygen and glucose deprivation (OGD), as they live in a hypoxic niche and they withstand prolonged lack of blood vessels during initial tumorigenesis or metastasis formation (avascular phase). Warbürg effect is energetically inefficient; we hypothesize that TICs might have differential metabolic features. Tumor eradication requires killing TICS; finding such features would have therapeutic implications. Methodology/principal findings: 106 MDA-MB-231 breast-cancer cells (hereafter Wt) were exposed for 5 weeks to 0.2% oxygen and 0.1g/l glucose, recovering 9 clones. Both flow-cytometry (50-fold enrichment in the CD24-/CD44+/CD133+ population) and xenografts in NOD/SCID mice using 100 cells (75% vs. 16% engraftment) suggest that OGD-resistant clones are true TICs (hereafter “TIC clones”). TIC clones showed a 30-fold higher replication and viability (BRDU incorporation, colony assay) than Wt. When exposed to OGD, ATP-production dropped 5-fold in WT, but was maintained in TIC clones by increasing 5-fold the fatty acid and oxygen consumption. These properties were explained by lack of upregulation of HIF-1 alpha and PDK1, as well as an increase in ATP-synthase. Analysis with metabolic inhibitors (2-deoxyglucose, antimycin-A) confirmed glycolysis and mitochondrial respiration as main routes of metabolism for Wt and TIC clones respectively. Metabolomics revealed that glutamine catabolism generated the NADPH required to quench reactive oxygen species generated during mitochondrial respiration in TIC clones. Glutamine deprivation or mitochondrial blockers were able to abrogate the viability of TIC clones. Conclusions/significance: the TIC-fraction of a cancer cell population withstands prolonged OGD by switching from the Warbürg effect to mitochondrial respiration. Targeting this metabolic feature abrogates the survival of TICs.

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Gene expression analysis of osteosarcoma tumor-initiating cells (High) vs bulk tumor cells (Low)

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