A single cell mRNA atlas of rhabdomyosarcoma at presentation and relapse - identifying foetal developmental targets (co-funded by Children with Cancer UK)

BACKGROUND Following dramatic improvement in the outlook of children suffering from RMS in the 1970s and 1980s, progress has stalled since, in spite of extensive clinical and biological research.

Therefore, it would appear unlikely that the prognosis of children with RMS will improve significantly by variations of “more of the same”. One aspect of RMS biology that remains relatively unexplored is the foetal root of RMS.

RMS is thought to form from foetal mesenchymal cells that differentiate aberrantly along a trajectory of striated muscle cells. We speculate that within these trajectories of aberrant development lie novel therapeutic opportunities.

At its simplest, there may be transcripts that are exclusive to the foetal period, yet persist in RMS and thus represent formidable targets for chemical compounds or immune therapy approaches.

Or, more fundamentally, we may be able to predict what transcriptional states we require, to induce therapeutic maturation of cancer cells.

AIMS Our aim is to build a developmental single cell atlas of rhabdomyosarcoma (RMS) at diagnosis and relapse, in order to identify foetal developmental targets.

We will profile single RMS cell transcriptomes in relation to differentiation of normal foetal cells and to RMS models of maturation, resistance and relapse.

METHODS To build the atlas we will: (i) Profile 250,000 RMS cells obtained from 50 fresh tumour specimens at diagnosis and relapse, using single cell mRNA sequencing (Chromium10x platform), integrated with bulk mRNA and DNA data. (ii) We will validate and spatially locate RMS cell types using multiplexed single molecule FISH and immunohistochemistry. (iii) Our data analyses will aim to elucidate treatment targets, esp. mRNA states of maturing RMS cells.

In in vitro models of RMS we will perform pertubation experiments to: (i) study the transcription underpinning RMS maturation, resistance and metastases (ii) to provide proof-of-principle validation of manipulating developmental trajectories.

HOW THE RESULTS OF THIS RESEARCH WILL BE USED We envision that our results will provide novel mechanistic and therapeutic hypotheses. We may then be able to pursue these in follow-up studies, including dedicated translational efforts.

Moreover, as we will make our data freely available, we will provide what will hopefully be a transformational data resource for the RMS research community.

Finally, our approach to study the foetal roots of RMS may define a blue print for similar enquiries into other childhood tumours.