Abstract 2807: Accessing the transcriptome of low-abundance tumor-infiltrating lymphocytes with SPRI beads
Authors: He Aiqing , Ma-Edmonds Manling , Gao Chan , Patel Vishal , Galbraith David , Zhang Yan , Guo Qi , DeSilva Heshani , Dito Gennaro , Truong Amy , Kuppasani Sunil , Ravi Kandasamy , Jabado Omar 
Published: August 2019

Abstract
Background: Multicolor flow cytometry has provided deep characterization of tumor-infiltrating lymphocytes (TILs). Routine fluorescence-activated cell sorting (FACS) methods discard cells after counting, precluding transcriptome analysis, which is key to understanding the activation states of immune cells. FACS-sorted cells pose 3 problems for transcriptomics: low cell count, cell rupture from high-pressure sort process, and inefficient centrifugation and recovery of cells from large collection volumes. SMART-Seq (TaKaRa/Clontech) is a highly sensitive, reverse transcriptase-based method to amplify low-input RNA that is commonly used to generate RNA-Seq libraries. After FACS, cell RNA can be purified by Qiagen RNeasy or nucleic acid binding solid-phase reversible immobilization (SPRI) magnetic beads. Alternatively, cells can be spun down after FACS then directly lysed as input to the SMART-Seq method. We sought to compare different methods and design a high-throughput protocol to capture the transcriptome from FACS-sorted, rare immune cell types.
Methods: A dilution series of 50-5000 human T cells was generated to test efficiency and sensitivity of RNA extraction. We compared RNA extraction yields by column (Qiagen RNeasy Low Input Micro Kit) or SPRI bead (Agencourt RNA-Advanced Cell v2, Beckman Coulter). Direct lysis of cells in SMART-Seq buffer was tested as an alternative to extraction. RNA yields were quantified by High Sensitivity Agilent Bioanalyzer or digital droplet PCR (BioRad). Extracted RNA was reverse transcribed and amplified using SMART-Seq v2 followed by library preparation (Nextera XT DNA kit) and sequencing (NovaSeq Illumina). To obtain TILs, mice were implanted subcutaneously with MC38 tumor cells, then tumors were harvested, disassociated, and FACS sorted to isolate T cells (CD4+, CD8+) and macrophages (yields ranged from 1000-8000 cells).
Results: Direct lysis followed by SMART-Seq was found to work well with diluted T cells; however, the method was sensitive to large buffer volumes and was unreliable for small numbers of pelleted, FACS-sorted cells (&lt;5000). SPRI beads provided higher yields than Qiagen columns and were amenable to plate-based automation. RNA-Seq analysis of a T cell dilution series showed high concordance between SPRI and Qiagen columns, although more genes expressed at very low levels were detected in SPRI-purified samples. We used the SPRI bead method to characterize the transcriptome of TILs from implanted mouse tumors. Expected patterns of immune marker expression by immune cell type were observed.
Conclusions: Use of SPRI beads to extract RNA from FACS-sorted immune cells was sensitive and reproducible, enabling transcriptomic characterization of &lt;5000 cells. Successful profiling of TILs derived from mouse syngeneic tumors suggests that the method can be used to probe activation states of rare immune cell types in the tumor microenvironment.
Citation Format: Aiqing He, Manling Ma-Edmonds, Chan Gao, Vishal Patel, David Galbraith, Yan Zhang, Qi Guo, Heshani DeSilva, Gennaro Dito, Amy Truong, Sunil Kuppasani, Kandasamy Ravi, Omar Jabado. Accessing the transcriptome of low-abundance tumor-infiltrating lymphocytes with SPRI beads [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2807.