CD73 sorted cell RNA-sequencing and GeoMX sequencing

CD73 marks cells with increased mesenchymal morphologies and phenotypes. To understand the transcriptional differences in CD73+ and - cells, we performed bulk RNA-seq on the CD73 sorted cells. In this study, we investigated if and how transcriptional heterogeneity of EwS cells contributes to heterogeneity of tumor phenotypes in vivo. Digital spatial profiling and human whole transcriptome analysis interrogated transcriptomic heterogeneity in EwS CHLA10 cell line xenografts. Overall design: Bulk RNA-sequencing for CD73 + and - cells in A673 and CHLA10 Ewing sarcoma cell lines GeoMx Digital Spatial Profiling (NanoString) was carried out according to manufacturer instructions. Tumor specimens were generated as follows: subcutaneous injection of 4x10^5 CHLA10 cells in 50% Matrigel, primary tumors collected 54 days post-injection; tail vein injection of 7x10^5 CHLA10 cells injected in PBS, tumors collected 28 days post-injection (NSG immunocompromised mice). Tumors were fixed for 24 hours in 10% non-buffered formalin before paraffin embedding. 3 sections of FFPE tumor tissues (1 from subcutaneous tumor, 2 from 2 different tail vein-injected mice) were sectioned and adhered to a single slide. Whole Human Transcriptome Atlas Digital Spatial Profiling was performed (GeoMx) by Nanostring according to manufacturer protocols for 12 regions of interest (4 per tumor) with NGS readout.