A co-culture model to study modulators of tumor immune evasion through scalable arrayed CRISPR-interference screens

Ramiro Martinez; Chiara Finocchiaro; Louis Delhaye; Fien Gysens; Jasper Anckaert; Wim Trypsteen; Maarten Versteven; Eva Lion; Sandra Van Lint; Karim Vermaelen; Eric James de Bony; Pieter Mestdagh

doi:10.3389/fimmu.2024.1444886

A co-culture model to study modulators of tumor immune evasion through scalable arrayed CRISPR-interference screens

Front Immunol. 2024 Oct 21:15:1444886. doi: 10.3389/fimmu.2024.1444886. eCollection 2024.

Authors

Ramiro Martinez^{1

2

3}, Chiara Finocchiaro¹, Louis Delhaye^{1

2

3

4}, Fien Gysens^{1

2

3}, Jasper Anckaert^{1

2

3}, Wim Trypsteen^{1

2

3}, Maarten Versteven⁵, Eva Lion⁵, Sandra Van Lint^{2

3

6}, Karim Vermaelen^{2

3

6}, Eric James de Bony^{1

2

3}, Pieter Mestdagh^{1

2

3}

Affiliations

¹ OncoRNALab, Center for Medical Genetics (CMGG), Ghent University, Ghent, Belgium.
² Cancer Research Institute Ghent (CRIG), Ghent University, Ghent, Belgium.
³ Department of Biomolecular Medicine, Ghent University, Ghent, Belgium.
⁴ Center for Medical Biotechnology, Flanders Institute for Biotechnology - UGENT (VIB-UGENT), Ghent, Belgium.
⁵ Laboratory of Experimental Hematology, Vaccine and Infectious Disease Institute (VAXINFECTIO), Faculty of Medicine and Health Sciences, University of Antwerp, Antwerp, Belgium.
⁶ Tumor Immunology Laboratory, Department of Pulmonary Medicine, Ghent University, Ghent, Belgium.

Abstract

Cancer cells effectively evade immune surveillance, not only through the well-known PD-1/PD-L1 pathway but also via alternative mechanisms that impair patient response to immune checkpoint inhibitors. We present a novel co-culture model that pairs a reporter T-cell line with different melanoma cell lines that have varying immune evasion characteristics. We developed a scalable high-throughput lentiviral arrayed CRISPR interference (CRISPRi) screening protocol to conduct gene perturbations in both T-cells and melanoma cells, enabling the identification of genes that modulate tumor immune evasion. Our study functionally validates the co-culture model system and demonstrates the performance of the CRISPRi-screening protocol by modulating the expression of known regulators of tumor immunity. Together, our work provides a robust framework for future research aimed at systematically exploring mechanisms of tumor immune evasion.

Keywords: CRISPRi; arrayed screens; co-culture; immuno-oncology; long non-coding RNAs.

MeSH terms

CRISPR-Cas Systems*
Cell Line, Tumor
Clustered Regularly Interspaced Short Palindromic Repeats
Coculture Techniques*
Humans
Melanoma* / genetics
Melanoma* / immunology
T-Lymphocytes / immunology
T-Lymphocytes / metabolism
Tumor Escape* / genetics

Grants and funding

The author(s) declare that financial support was received for the research, authorship, and/or publication of this article. This work was supported by the Research Foundation of Flanders (FWO) (grant number: G055022N), the CRIG Young Investigator Proof of Concept Grant (YIPOC), and Flanders Innovation & Entrepreneurship (VLAIO).