Abstract 3637: Validation of a workflow to enable massively parallel transcriptome analysis in cryopreserved tissues
Authors: Corney David C. , Han Yang , Qiu Yu , Graham Riley , Fan Yongjun , Mozdzierz Christopher , Latif Haythem , Zhou Ginger 
Published: September 2020

Abstract
Tumor development and growth is a complex process involving interplay between stromal and neoplastic cells. Importantly, neoplastic subclones within tumors often possess different functional properties and metastatic potential, which may be obscured by traditional bulk characterization techniques such as RNA-seq. Such bulk sequencing methods are unable to capture inter-cellular heterogeneity and thus cannot reveal disease-relevant traits occurring within tissues and tumors. These limitations can be overcome by using single-cell RNA-seq (scRNA-seq) and single-cell ATAC-seq (scATAC-seq) methods, which enable a deeper understanding of cell-type specific traits and clonotype density and their contributions to carcinogenesis. However, current single cell techniques rely on obtaining fresh tissues that must be dissociated into single cell suspensions immediately after collection. Delay in processing risks changes in gene expression as well as increased cell mortality. Unfortunately, due to these intractable logistical challenges to carefully prepare single cell suspensions in an OR environment, single cell technologies currently have a modest role in translational and clinical settings despite their potential to revolutionize our understanding and treatment of cancer.
To circumvent these challenges, we present here a cryopreservation workflow to preserve cells harvested from a variety of tissues for single cell sequencing. Following sample collection, tissues are processed through a simple cryopreservation protocol at which point they are amenable to short-term storage to allow batching of samples, as well as transportation to a laboratory for downstream processing. Subsequently, tissues are thawed and subjected to cellular dissociation with optional cell surface marker enrichment resulting in cell or nuclei suspensions suitable for scRNA-seq and scATAC-seq. This approach has been validated for use by analysis of a diverse range of paired fresh and cryopreserved tissues. Cell recovery and cell viability measures are comparable between fresh and cryopreserved across all tested tissues, thereby demonstrating the broad applicability of this approach. Furthermore, profiling by scRNA-seq and scATAC-seq indicates cryopreservation causes only modest effects on gene expression and chromatin accessibility across tested tissue types.
Taken together, this work establishes a workflow to enable analysis of the transcriptome and epigenome in cryopreserved tissues at single cell resolution and make use of precious samples obtained in translational settings.
Citation Format: David C. Corney, Yang Han, Yu Qiu, Riley Graham, Yongjun Fan, Christopher Mozdzierz, Haythem Latif, Ginger Zhou. Validation of a workflow to enable massively parallel transcriptome analysis in cryopreserved tissues [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3637.