Single-cell transcriptomics reveals immune suppression and cell states predictive of patient outcomes in rhabdomyosarcoma

Abstract

Paediatric rhabdomyosarcoma (RMS) is a soft tissue malignancy of mesenchymal origin which is thought to arise as a consequence of derailed myogenic differentiation. Despite intensive treatment regimens, the prognosis for high-risk patients remains dismal. The cellular differentiation states underlying RMS and how these relate to patient outcomes remain largely elusive. Here, we used single-cell mRNA-sequencing to generate a transcriptomic atlas of RMS. Analysis of the RMS tumour niche revealed evidence of an immunosuppressive microenvironment. We also identified an interaction between NECTIN3 and TIGIT, specific to the more aggressive fusion-positive (FP) RMS subtype, as a putative cause of tumour-induced T-cell dysfunction. In malignant RMS cells we defined transcriptional programs reflective of normal myogenic differentiation. Furthermore, we showed that these cellular differentiation states are predictive of patient outcomes in both FP RMS and the more clinically homogenous fusion-negative subtype. Our study reveals the potential of therapies targeting the immune microenvironment of RMS and suggests that assessing tumour differentiation states may enable a more refined risk stratification.

Single cell transcriptomic profiling identifies tumor-acquired and therapy-resistant cell states in pediatric rhabdomyosarcoma

“…Collectively, we detected significant overlap between gene signatures identified across the three datasets indicating that the previously published studies had independently uncovered largely similar dominant RMS cell states. These data are also consistent with recently published findings from DeMartino et al that defined a tripartite cell state landscape in FN-and FP-RMS using a different nomenclature [24].…”

“…The absence of neuronal cells within cell lines may explain why this rare subpopulation was not identified previously by scRNA sequencing analysis by Danielli et al [23], and may indicate that PDXs are the appropriate experimental model for better clarifying the function of neuronal cells in FP-RMS. Patel et al and DeMartino et al also failed to identify these neuronal-pathway enriched tumor cell states, likely reflecting the use of bioinformatic approaches that combined subtypes together for defining cell states [21, 24].…”

“…Moreover, although the FP-progenitor cells do expresses mesenchymal markers, they are transcriptionally distinct from the SkM.Mesen cells found in fetal muscle development and the FN-SkM.Mes-like state discovered here. Indeed, DeMartino et al, also independently identified key differences in mesenchymal-pathway enriched cell states in comparing scRNA sequencing expression of FP– and FN-RMS [24]. In addition, a subset of FP-RMS have tumor cells that have neural-pathway activation that are not found in human muscle development and yet are enriched after chemotherapy.…”

Fusion-negative rhabdomyosarcoma orthotopic tongue xenografts for study of invasion, intravasation and metastasis in live animals

“…We observe heterogeneous cellular phenotypes through IVM including primitive rounded cellular morphologies, elongating mesenchymal morphologies and differentiated striated myofibril-like structures in the tongue tumors. This diversity of in cellular phenotypes is reminiscent of the variations in myogenic differentiation states observed in patient FN-RMS tumors(59). However, also revealed by IVM, these heterogenous populations are diminished in cervical lymph node metastases which are dominated by tumor initiation; (2) rodent specific factors might confound interpretation of results, such as the high levels of growth factors secreted from the salivary glands(57,58); and (3) precise injection technique in the tongue is vital to avoid injuring…”

Complex structural variation is prevalent and highly pathogenic in pediatric solid tumors

“…Of special interest is the ecDNA identified in tumor M911AAA resulting in the rare fusion gene PAX3::WWRT1 (Figure S8). Previously, we found that the transcription profile of this particular tumor clustered with canonical fusion-positive RMS tumors which form a high-risk subgroup [26]. Moreover, for three of these seven fusion-negative RMS with ecDNA/amplicons resulting in oncogene amplification, we found no alterations in cancer driver genes outside of these complex events (Figure 3).…”