Defining an Optimized Workflow for Enriching and Analyzing Residual Tumor Populations Using Intracellular Markers

TL;DR

This paper presents a new workflow to enrich and analyze residual tumor cells using intracellular markers, improving the study of treatment-resistant populations in B-cell lymphoma.

Contribution

A novel workflow combining glyoxal fixation and the cellenONE platform for intracellular marker-based tumor cell enrichment is introduced.

Findings

Glyoxal fixation preserves RNA quality and antibody expression better than other fixatives.

BCL2high populations were effectively enriched and analyzed using the cellenONE platform.

Transcriptomic profiles of fixed and live cells showed high concordance, highlighting B-cell biology.

Abstract

Tumor relapse is well recognized to arise from treatment-resistant residual populations. Strategies enriching such populations for in-depth downstream analyses focus on tumor-specific surface markers; however, enrichment using intracellular biomarkers remains challenging. Using B-cell lymphoma as an exemplar, we demonstrate feasibility to enrich B-cell lymphoma 2 (BCL2)high populations, a surrogate marker for t(14;18)+ lymphomas, for use in downstream applications. Different fixation protocols were assessed for impact on antibody expression and RNA integrity; glyoxal fixation demonstrated superior results regarding minimal effects on surface and intracellular expression, and RNA quality, compared with alternative fixatives evaluated. Furthermore, t(14;18)+ B cells were effectively detected using intracellular BCL2 overexpression to facilitate tumor cell enrichment. Tumor cell populations were enriched using the cellenONE F1.4 single-cell sorting platform, which detected and dispensed BCL2high-expressing cells directly into library preparation reagents for transcriptome analyses. Sorted glyoxal-fixed cells generated good quality sequencing libraries, with high concordance between live and fixed single-cell transcriptomic profiles, discriminating cell populations predominantly on B-cell biology. Overall, we successfully developed a proof-of-concept workflow employing a robust cell preparation protocol for intracellular markers combined with cell enrichment using the cellenONE platform, providing an alternative to droplet-based technologies when cellular input is low or requires prior enrichment to detect rare populations. This workflow has wider prognostic and therapeutic potential to study residual cells in a pan-cancer setting.

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