# 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.

## Key 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.