Abstract 6197: Improving sample preparation workflows for single cell multiomic analysis of infiltrating leukocytes from solid tumors
Authors: Middlebrook Aaron J. , Huang Wei , Nakamoto Margaret , Shi Xiaoshan , Lo Evelyn , Neuhoefer Patrick , Bornheimer Scott , Ghanekar Smita 
Published: September 2020

Single cell multiomic (scM) methods are powerful tools to characterize patient-specific tumor-immune microenvironment, but current methods of sample handling and preparation can introduce bias. Obtaining non-biased results is particularly important in studies to identify new biomarkers for patient stratification of responders versus non-responders for immunotherapies in oncology. This problem is becoming acute as the number of immune-therapy modalities and combinations in clinical trials and in medical practice continue to expand. Current methodologies used to analyze patient specimens in routine pathology are not adequate to address heterogeneity and complexity of immune cell ecosystem of individuals. SCM analysis of immune cells from tumor microenvironment using technologies that allow high parameter analysis of genes and proteins will allow a comprehensive atlas of immune cells in the tumor tissue, however the quality of single cell sample preparation is critical. Here, we report evaluation of several available solid tumor dissociation methods, including mechanical dissociation by scalpel, BD™ Medimachine System, Miltenyi gentleMACS™ dissociator. Mechanical dissociation was followed by enzymatic dissociation using BD Horizon™ Dri Tumor & Tissue Dissociation Reagent or, in the case of gentleMACS™, the Miltenyi Tumor Dissociation Kit. Metrics evaluated were cell recovery, viability, and proportion of various cell lineage populations using flow cytometry; and expression of 399 mRNA transcripts and 31 proteins using single cell multiomics (BD Rhapsody™, BD Rhapsody™ Immune Response Targeted Panel, and BD™ AbSeq). Tumor tissues were from a mouse a pancreatic (PDAC) tumor model and humanized Patient-Derived Xenograft (PDX; from Onco-hu® mice) models of colon and lung cancer. Method dependent differences in viable cell yield and proportions of immune vs. tumor cells were observed. SCM analysis of FACS sorted CD45+ tumor-infiltrating lymphocytes (TILs) showed differences in gene and protein expression profiles, particularly for expression of rare vs. abundant genes depending on the dissociation method. Our data identify protocols to interrogate tumor immune microenvironment starting from tumor tissue specimen through scM all the way to informatics analysis with consistent results. Similar studies and further optimization are recommended for other tumor types. Consistent adoption of standardized protocols will enable robust findings in research and clinical trials, with the ultimate goal of improved therapies, biomarkers and outcomes.

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Citation Format: Aaron J. Middlebrook, Wei Huang, Margaret Nakamoto, Xiaoshan Shi, Evelyn Lo, Patrick Neuhoefer, Scott Bornheimer, Smita Ghanekar. Improving sample preparation workflows for single cell multiomic analysis of infiltrating leukocytes from solid tumors [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 6197.