Abstract
Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children. Fusion-positive RMS (FPRMS), expressing the PAX3/7-FOXO1, has a worse prognosis compared to the more common fusion-negative RMS (FNRMS). Although several studies reported hierarchical organization for FNRMS with the identification of cancer stem cells, the cellular organization of FPRMS is not yet clear. In this study we investigated the expression of key stem cell markers, developed a sphere assay, and investigated the seven most common FPRMS cell lines for subpopulations of tumor propagating cancer stem-like cells, also called cancer stem cells (CSCs). Moreover, loss- and gain-of-functions of the stem cell genes SOX2, OCT4, and NANOG were investigated in the same cells. Single-cell clonal analysis was performed in vitro as well as in vivo. We found that no stable CSC subpopulation could be enriched in FPRMS. Unlike depletion of PAX3-FOXO1, neither overexpression nor siRNA-mediated downregulation of SOX2, OCT4, and NANOG affected physiology of RMS cells. Every single subclone-derived cell clone initiated tumor growth in mice, despite displaying considerable heterogeneity in gene expression. FPRMS appears to contain a high frequency of tumor propagating stem-like cells, which could explain their higher propensity for metastasis and relapse. Their dependency on PAX3-FOXO1 activity reinforces the importance of the fusion protein as the key therapeutic target.
Highlights
To understand tumor origin, maintenance, progression and heterogeneity, two models have been postulated in the past: the stochastic or clonal evolution model and the hierarchical or cancer stem cell model [1]
Cells were seeded at clonal dilution (1 cell/μL) into Petri dishes and cultured in sphere medium consisting of DMEM/HAM’s F12, 20% knockout Serum Replacement (KSR) (GIBCO), 2 mM L-glutamine, 100 U/mL penicillin/streptomycin (P/S) either supplemented with 10 ng/mL bFGF or with 10 ng/mL bFGF, EGF, and PDGF (R&D Systems)
To understand whether Fusionpositive RMS (FPRMS) possess a subpopulation of Cancer stem cells (CSCs), we developed a medium able to maintain the seven common FPRMS cell lines (RH4, RH30, RMS13, RH3, RH5, RH41, CW9019) in suspension, allowing the formation of spheres
Summary
Maintenance, progression and heterogeneity, two models have been postulated in the past: the stochastic or clonal evolution model and the hierarchical or cancer stem cell model [1]. For the development of more effective cancer therapies, it is of fundamental importance to understand the extent of heterogeneity and associated hierarchy within a tumor. Cancer stem cells (CSCs) possess the ability for unlimited self-renewal and to generate the heterogeneous and differentiated cell lineages that form the tumor bulk [2]. Because of these unique characteristics, CSCs are called tumor initiating cells or tumor propagating cells. Based on phenotypic traits such as cell surface markers and on functional properties such as sphere formation, quiescence, and drug resistance, CSCs can nowadays be isolated from a number of primary tumors or cell lines and can be maintained and enriched in vitro, facilitating their molecular characterization
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