A CRISPR/Cas9-Based Assay for High-Throughput Studies of Cancer-Induced Innervation

Sapthala Loku Galappaththi; Brenna Katz; Patrick H Howze 4th; Gregory Hoover; Simon Grelet

doi:10.3390/cancers15072026

A CRISPR/Cas9-Based Assay for High-Throughput Studies of Cancer-Induced Innervation

Cancers (Basel). 2023 Mar 29;15(7):2026. doi: 10.3390/cancers15072026.

Authors

Sapthala Loku Galappaththi^{1

2}, Brenna Katz^{1

2}, Patrick H Howze 4th^{1

2}, Gregory Hoover^{1

2}, Simon Grelet^{1

2}

Affiliations

¹ Department of Biochemistry and Molecular Biology, College of Medicine, University of South Alabama, Mobile, AL 36688, USA.
² Mitchell Cancer Institute, The University of South Alabama, Mobile, AL 36604, USA.

Abstract

The aggressive nature of certain cancers and their adverse effects on patient outcomes have been linked to cancer innervation, where neurons infiltrate and differentiate within the cancer stroma. Recently we demonstrated how cancer plasticity and TGFβ signaling could promote breast cancer innervation that is associated with increased cancer aggressivity. Despite the promising potential of cancer innervation as a target for anti-cancer therapies, there is currently a significant lack of effective methods to study cancer-induced neuronal differentiation, hindering the development of high-throughput approaches for identifying new targets or pharmacological inhibitors against cancer innervation. To overcome this challenge, we used CRISPR-based endogenous labeling of the neuronal marker β3-tubulin in neuronal precursors to investigate cancer-induced neuronal differentiation in nerve-cancer cocultures and provide a tool that allows for better standardization and reproducibility of studies about cancer-induced innervation. Our approach demonstrated that β3-tubulin gene editing did not affect neuronal behavior and enabled accurate reporting of cancer-induced neuronal differentiation dynamics in high-throughput settings, which makes this approach suitable for screening large cohorts of cells or testing various biological contexts. In a more context-based approach, by combining this method with a cell model of breast cancer epithelial-mesenchymal transition, we revealed the role of cancer cell plasticity in promoting neuronal differentiation, suggesting that cancer innervation represents an underexplored path for epithelial-mesenchymal transition-mediated cancer aggressivity.

Keywords: axonogenesis; cancer innervation; cancer plasticity; epithelial-mesenchymal transition; neurogenesis; neuronal differentiation; β3-tubulin.

Grants and funding

This research was supported by startup funds from the Department of Biochemistry and Molecular Biology, Frederick P. Whiddon College of Medicine, University of South Alabama, Mitchell Cancer Institute; the Mitchell Endowment and the Patricia Cobb Rodgers Endowment from the Frederick P. Whiddon College of Medicine, University of South Alabama, Mitchell Cancer Institute. The authors gratefully acknowledge the resources and services provided by the Bioimaging Core Facility and the Flow Cytometry Shared Resource Laboratories, both core research facilities supported by the University of South Alabama P. Whiddon College of Medicine.