Defining an Optimized Workflow for Enriching and Analyzing Residual Tumor Populations Using Intracellular Markers.
J Mol Diagn
; 26(4): 245-256, 2024 04.
Article
in En
| MEDLINE
| ID: mdl-38280422
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.
Full text:
1
Database:
MEDLINE
Main subject:
RNA
/
Neoplasm Recurrence, Local
Type of study:
Guideline
Limits:
Humans
Language:
En
Journal:
J Mol Diagn
Journal subject:
BIOLOGIA MOLECULAR
Year:
2024
Type:
Article