Culturing human fusion-positive rhabdomyosarcoma cell lines as rhabdospheres enriches for stemness and Notch signaling

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-positive fusion-positive rhabdomyosarcoma (FP-RMS) - an aggressive soft tissue sarcoma of childhood - has thus far not been achieved, 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. We used microarray to understand the transcriptome profile of rhabdomyosarcoma in different culture condition (adherent, sphere, and xenograft). RNA was isolated from adherent, sphere, xenograft cells using an RNeasy kit (Qiagen) according to the manufacturer’s protocol. Affymetrix U133 Plus 2.0 arrays were used and performed by the Duke University Microarray Facility according to manufacturer’s instruction.