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. 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.

CD73可标记具有间充质形态及表型增强的细胞。为解析CD73阳性与阴性细胞间的转录组差异，本研究对分选得到的CD73阳性、阴性细胞进行了批量RNA测序（bulk RNA-seq）。本研究旨在探究尤因肉瘤（EwS）细胞的转录组异质性是否以及如何在体内推动肿瘤表型异质性的形成。本研究通过数字空间分析（Digital Spatial Profiling, DSP）及人类全转录组分析，对尤因肉瘤CHLA10细胞系异种移植瘤的转录组异质性进行了检测。针对A673与CHLA10两种尤因肉瘤细胞系中的CD73阳性、阴性细胞开展批量RNA测序。GeoMx数字空间分析（NanoString）实验严格按照厂商说明书进行操作。肿瘤标本构建流程如下：将4×10^5个CHLA10细胞与50%基质胶（Matrigel）混合后进行皮下注射，于接种后54天采集原发肿瘤；将7×10^5个CHLA10细胞以磷酸盐缓冲液（PBS）重悬后进行尾静脉注射，于接种后28天采集肿瘤（使用NSG免疫缺陷小鼠）。肿瘤组织经10%非缓冲福尔马林固定24小时后进行石蜡包埋。取3份福尔马林固定石蜡包埋（FFPE）肿瘤组织块（1份来自皮下移植瘤，2份分别来自2只不同尾静脉注射建模小鼠的移植瘤），制备切片并粘附至同一张玻片上。Nanostring公司按照厂商实验方案，针对12个感兴趣区域（每个肿瘤取4个区域）采用下一代测序（NGS）读长，完成了人类全转录组图谱数字空间分析（GeoMx）检测。