CD133 Positive Embryonal Rhabdomyosarcoma Stem-Like Cell Population Is Enriched in Rhabdospheres

Abstract

Cancer stem cells (CSCs) have been identified in a number of solid tumors, but not yet in rhabdomyosarcoma (RMS), the most frequently occurring soft tissue tumor in childhood. Hence, the aim of this study was to identify and characterize a CSC population in RMS using a functional approach. We found that embryonal rhabdomyosarcoma (eRMS) cell lines can form rhabdomyosarcoma spheres (short rhabdospheres) in stem cell medium containing defined growth factors over several passages. Using an orthotopic xenograft model, we demonstrate that a 100 fold less sphere cells result in faster tumor growth compared to the adherent population suggesting that CSCs were enriched in the sphere population. Furthermore, stem cell genes such as oct4, nanog, c-myc, pax3 and sox2 are significantly upregulated in rhabdospheres which can be differentiated into multiple lineages such as adipocytes, myocytes and neuronal cells. Surprisingly, gene expression profiles indicate that rhabdospheres show more similarities with neuronal than with hematopoietic or mesenchymal stem cells. Analysis of these profiles identified the known CSC marker CD133 as one of the genes upregulated in rhabdospheres, both on RNA and protein levels. CD133+ sorted cells were subsequently shown to be more tumorigenic and more resistant to commonly used chemotherapeutics. Using a tissue microarray (TMA) of eRMS patients, we found that high expression of CD133 correlates with poor overall survival. Hence, CD133 could be a prognostic marker for eRMS. These experiments indicate that a CD133+ CSC population can be enriched from eRMS which might help to develop novel targeted therapies against this pediatric tumor.

Glycogen synthase kinase 3 inhibitors induce the canonical WNT/β-catenin pathway to suppress growth and self-renewal in embryonal rhabdomyosarcoma

“…Sphere and replating assays using RD and 381T cells were completed essentially as previously described (25).…”

“…Sphere-forming assays are widely used as an in vitro measure of tumor cell self-renewal (24) and have been recently used to assess self-renewal in human ERMS cell lines (25). To assess the effect of activating canonical WNT/ β-catenin pathway on the rhabdosphere forming potential, RD and 381T cells were seeded at limiting dilution and treated with BIO and WNT3A.…”

p53 suppresses muscle differentiation at the myogenin step in response to genotoxic stress

“…Next we examined the gene expression of the following factors in RD cells upon p53 activation: MRFs 8,9,14 (MyoD and myogenin), muscle stem cell markers 33,34 (Pax3, Pax7, and cMet), and cancer stem cell-like markers 35 (Oct4, Nanog, Sox2, and CD133). We did not observe significant changes in expression of MyoD, Pax genes, and most of the cancer stem cell-like markers ( Figure 1c and Supplementary Figure 4a).…”

Interfering with Hedgehog Pathway: New Avenues for Targeted Therapy in Rhabdomyosarcoma

“…Importantly, one of the biological limitations of RMS-directed therapies is the possible existence of functionally defined sub-populations of cells within the tumor having increased tumorigenicity, self-renewal and chemoresistance [70]. This is prominent for NRMS where the hierarchical model may be applied and has to be taken into account when developing novel treatment strategies [45,[71][72][73][74]. Indeed, we found that Hh inhibition might provide a promising anti-cancer stem cell (CSC) therapy in NRMS, and therefore multistrategy approaches with bulk-reducing drugs may be more effective in tumor eradication, particularly for high-risk group patients [45].…”