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.
Highlights
The cancer stem cell hypothesis suggests that a small subpopulation of cells sharing common characteristics with normal stem cells (SCs) - such as capacity to self renew, potential to differentiate, extensive proliferation in vivo, and resistance to chemotherapeutics - is responsible for tumor development [1] and that tumors are organized hierarchically
Rhabdospheres are enriched with cancer stem-like cells To determine whether RMS cells might contain a subpopulation of Cancer stem cells (CSCs) cells, we attempted to grow embryonal rhabdomyosarcoma cell lines (RD, Rh36 and Ruch2) as rhabdomyosarcoma spheres in stem cell medium (SC-medium)
Compared to the positive control U87MG sphere cultures which showed the highest enrichment over 10 passages, 1300 spheres were counted for RD cultures after 10 passages, while Ruch2 and Rh36 sphere cultures could be enriched albeit to a lesser extent (600 counted spheres) indicating that a subpopulation of cells with self renewal property can be enriched from three different embryonal rhabdomyosarcoma (eRMS) cell lines
Summary
The cancer stem cell hypothesis suggests that a small subpopulation of cells sharing common characteristics with normal stem cells (SCs) - such as capacity to self renew, potential to differentiate, extensive proliferation in vivo, and resistance to chemotherapeutics - is responsible for tumor development [1] and that tumors are organized hierarchically This concept was first established in acute myeloid leukemia (AML) [2] and subsequently in a number of solid tumors such as breast cancer where a CD44+/CD242/low CSC population was identified [3], in brain tumors [4], colon cancer [5], and melanomas [6]. The existence of such a cellular subpopulation most likely has to be established for each tumor type [12]
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