Multi-Phenotypic subtyping of circulating tumor cells using sequential fluorescent quenching and restaining

Area of Science:

• Biomedical Science
• Cancer Research
• Molecular Diagnostics

Background:

• Tissue biopsies using formalin-fixed paraffin-embedded blocks allow extensive proteomic and genomic subtyping of cancer specimens.
• Blood-based biopsies (BBBs) currently offer limited clinical utility, primarily for enumerating circulating tumor cells (CTCs) due to restrictions on the number of detectable fluorescent markers.

Purpose of the Study:

• To develop a simple, inexpensive method to increase the number of subtyping biomarkers detectable on individual CTCs.
• To enhance the clinical utility of BBBs by enabling in-depth interrogation of CTCs.

Main Methods:

• A novel technique for quenching specific fluorophores on stained CTCs.
• Sequential restaining of CTCs with additional biomarkers after quenching.
• Proof-of-principle analysis using CTC, immunosuppression, and stem cell panels.

Main Results:

• Demonstrated a method to sequentially subtype individual fluorescently stained patient CTCs.
• Successfully applied CTC, immunosuppression, and stem cell panels for multi-marker analysis.
• Showcased the ability to analyze multiple clinically applicable immunomarkers from a single CTC sample.

Conclusions:

• The developed method provides a simple and universal technique for analyzing multiple immunomarkers from BBBs.
• This approach significantly expands the potential of CTC analysis for cancer subtyping and clinical decision-making.
• Enhanced CTC subtyping via sequential staining promises to revolutionize the diagnostic and prognostic capabilities of blood-based biopsies.