High-Content and High-Throughput Clonogenic Survival Assay Using Fluorescence Barcoding

Author:

Qian Haibin12,Baglamis Selami2345ORCID,Redeker Fumei12,Raaijman Julia12,Hoebe Ron A.12,Sheraton Vivek M.23456ORCID,Vermeulen Louis2345ORCID,Krawczyk Przemek M.12

Affiliation:

1. Department of Medical Biology, Amsterdam University Medical Centers (Location AMC), Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands

2. Cancer Center Amsterdam, 1081 HV Amsterdam, The Netherlands

3. Laboratory for Experimental Oncology and Radiobiology, Center for Experimental and Molecular Medicine, Amsterdam UMC Location University of Amsterdam, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands

4. Oncode Institute, 3521 AL Utrecht, The Netherlands

5. Amsterdam Gastroenterology Endocrinology Metabolism, 1105 AZ Amsterdam, The Netherlands

6. Institute for Advanced Study, University of Amsterdam, 1012 WX Amsterdam, The Netherlands

Abstract

The Clonogenic Survival Assay (CSA) is a fundamental tool employed to assess cell survival and proliferative potential in cancer research. Despite its importance, CSA faces limitations, primarily its time- and labor-intensive nature and its binary output. To overcome these limitations and enhance CSA’s utility, several approaches have been developed, focusing on increasing the throughput. However, achieving both high-content and high-throughput analyses simultaneously has remained a challenge. In this paper, we introduce LeGO-CSA, an extension of the classical CSA that employs the imaging of cell nuclei barcoded with fluorescent lentiviral gene ontology markers, enabling both high-content and high-throughput analysis. To validate our approach, we contrasted it with results from a classical assay and conducted a proof-of-concept screen of small-molecule inhibitors targeting various pathways relevant to cancer treatment. Notably, our results indicate that the classical CSA may underestimate clonogenicity and unveil intriguing aspects of clonal cell growth. We demonstrate the potential of LeGO-CSA to offer a robust approach for assessing cell survival and proliferation with enhanced precision and throughput, with promising implications for accelerating drug discovery and contributing to a more comprehensive understanding of cellular behavior in cancer.

Funder

Dutch Cancer Society

The New York Stem Cell Foundation, grants from the European Research Council

ZonMw

Publisher

MDPI AG

Subject

Cancer Research,Oncology

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