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). 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