A method to culture human alveolar rhabdomyosarcoma cell lines as rhabdospheres demonstrates an enrichment in stemness and notch signaling

Abstract

The development of three-dimensional cell culture techniques has allowed cancer researchers to study the stemness properties of cancer cells in in vitro culture. However, a method to grow PAX3-FOXO1 fusion-positive rhabdomyosarcoma (FP-RMS) - an aggressive soft tissue sarcoma of childhood - has to date not been reported, hampering efforts to identify the dysregulated signaling pathways that underlie FP-RMS stemness. Here, we first examine the expression of canonical stem cell markers in human RMS tumors and cell lines. We then describe a method to grow FP-RMS cell lines as rhabdospheres and demonstrate that these spheres are enriched in expression of canonical stemness factors as well as Notch signaling components. Specifically, FP-RMS rhabdospheres have increased expression of SOX2, POU5F1 (OCT4), and NANOG, and several receptors and transcriptional regulators in the Notch signaling pathway. FP-RMS rhabdospheres also exhibit functional stemness characteristics including multipotency, increased tumorigenicity in vivo, and chemoresistance. This method provides a novel practical tool to support research into FP-RMS stemness and chemoresistance signaling mechanisms.

High Frequency of Tumor Propagating Cells in Fusion-Positive Rhabdomyosarcoma

“…Therefore, we hypothesized that FPRMS, contrary to FNRMS but similarly to acute lymphoblastic leukemia and melanoma [48,51], possess a high abundance of cells with tumor initiating potential. Corroborating this hypothesis, expression of the pluripotency genes NANOG, OCT4, and SOX2 was homogeneous in all analyzed monolayer cultures of FPRMS cell lines, as also confirmed by Slemmons et al [49].…”

“…Although we could maintain three out of seven FPRMS lines for five weeks under the new established sphere conditions, the observed increase in sphere numbers as well as the upregulation of the core stem cell genes compared to the monolayer cell culture was very moderate and not consistent for all the analyzed cell lines. Recently, Slemmons et al established other sphere culture conditions able to maintain two FPRMS cell lines in suspension [ 49 ]. Although the spheres were not passaged at clonal level, they showed upregulation of NANOG, OCT4, and SOX2 after serial passaging when compared to the monolayer culture, but not with xenograft derived cells.…”

“…The sphere medium used by Slemmons et al contained eight times more bFGF and four times more EGF than the medium used in our study. Instead of PDGF they added insulin to the neurobasal media supplemented with B27 [ 49 ]. We consciously used the minimal concentration of growth factors allowing passaging of the spheres in order to exclude a media-dependent induced cell plasticity.…”

EHMT1 promotes tumor progression and maintains stemness by regulating ALDH1A1 expression in alveolar rhabdomyosarcoma

“…Fourth, inhibition of ALDH1A1 expression or activity had effects similar to those of EHMT1 depletion. These observations are consistent with the finding that ARMS tumorspheres (rhabdospheres) display stem cell characteristics [29].…”

“…Tumorsphere formation was accomplished by slightly modifying previously described methods [28,29]. In brief, RH41 (25 000 cells per well) or RH30 (20 000 cells per well) cells were grown in neurobasal medium (Gibco, Waltham, MA, USA) enriched with 2X or 1X B27 (Invitrogen), 40 ng/ml bFGF (Invitrogen), 80 ng/ ml EGF (Sigma), and 50 μg/ml insulin (Sigma).…”