Comparison of Different Approaches to Single Cell RNA Sequencing of Cancer Associated Fibroblasts

Cancer associated fibroblasts (CAFs) represent a major stromal component of PDAC with both tumor-promoting and restraining properties. Studies have demonstrated functional heterogeneity among different subpopulations of CAFs, highlighting the need to identify specific subpopulations when targeting CAFs. We hereby used two types of processing method, whole tumor or negatively-isolated CAFs, to determine how best to uncover CAF heterogeneity with single-cell RNA sequencing (scRNA-seq). Overall design: Murine orthotopic pancreatic tumors were dissected and collected on day 13 after tumor implantation for scRNA-seq processing. Tumors were mechanically and enzymatically processed with the mouse Miltenyi Tumor Dissociation Kit (Miltenyi Biotec) and gentleMACS Octo Dissociator (Miltenyi Biotec). Samples were then filtered through 100 µm strainer, which was then washed with 10 mL T cell media. Cell suspensions were centrifuged at 1500 RPM for 5 minutes. Cell pellets were resuspended in 10 mL T cell media, filtered through 100 µm strainer, and centrifuged again at 1500 RPM for 5 minutes. For fibroblast enrichment, cells were resuspended with PBS and blocked with anti-mouse Fc antibody (BD Biosciences) in FACS buffer for 10 minutes on ice. Cells were then stained with CD45-AF657 (BioLegend clone 30-F11, 1:20), CD31-AF647 (BioLegend clone 390, 1:20), EPCAM-AF647 (BioLegend, clone G8.8, 1:20), and TER119-AF647 (BioLegend clone TER-119 1:20) for 30 minutes on ice. Single cell suspension was mixed every 10 minutes. Cells were washed with PBS twice, resuspended at 5-10 million cells/mL in T cell media, and filtered through 35 µm flow cytometry tubes (Falcon 352235). Sorting for CD45-, CD31-, EPCAM-, and TER119-negative cells was performed to obtain the fibroblast enriched fraction. Both the unenriched and enriched samples were washed and resuspended to cell concentration of 700-1200 cells/µL in PBS + 0.1% BSA for loading onto 10X Chromium microfluidic chips

癌症相关成纤维细胞（Cancer associated fibroblasts, CAFs）是胰腺导管腺癌（pancreatic ductal adenocarcinoma, PDAC）的主要基质成分之一，兼具促瘤与抑瘤双重特性。已有研究证实不同CAFs亚群间存在功能异质性，这凸显了靶向CAFs时需明确其特异性亚群的必要性。本研究采用两种处理方案：完整肿瘤样本与经负向分选纯化的CAFs，旨在通过单细胞RNA测序（single-cell RNA sequencing, scRNA-seq）最优地解析CAF异质性。
实验整体设计：于肿瘤植入后第13天剖取小鼠原位胰腺肿瘤，用于后续scRNA-seq实验处理。采用小鼠Miltenyi肿瘤解离试剂盒（Miltenyi Biotec）与gentleMACS Octo组织解离仪（Miltenyi Biotec）对肿瘤进行机械与酶学解离。随后将样本通过100 μm细胞筛过滤，并用10 mL T细胞培养基冲洗筛网。细胞悬液以1500转/分钟离心5分钟。沉淀的细胞重悬于10 mL T细胞培养基中，再次通过100 μm细胞筛过滤，并以1500转/分钟离心5分钟。
为富集成纤维细胞，将细胞重悬于磷酸盐缓冲液（PBS）中，于冰上用FACS缓冲液配制的抗小鼠Fc抗体（BD Biosciences）封闭10分钟。随后于冰上用CD45-AF657（BioLegend，克隆号30-F11，稀释比例1:20）、CD31-AF647（BioLegend，克隆号390，稀释比例1:20）、EPCAM-AF647（BioLegend，克隆号G8.8，稀释比例1:20）及TER119-AF647（BioLegend，克隆号TER-119，稀释比例1:20）对细胞进行染色30分钟，每10分钟混匀一次细胞悬液。细胞用PBS洗涤两次后，重悬于T细胞培养基中，调整细胞浓度至5×10^6~1×10^7个/mL，随后通过35 μm流式细胞仪分选管（Falcon 352235）过滤。通过流式分选获取CD45⁻、CD31⁻、EPCAM⁻及TER119⁻的细胞群，以此获得成纤维细胞富集组分。将未富集与富集后的样本均洗涤并重悬于含0.1%牛血清白蛋白（BSA）的PBS中，调整细胞浓度至700~1200个/μL，用于上样至10X Chromium微流控芯片。