In Vitro Establishment of a Genetically Engineered Murine Head and Neck Cancer Cell Line using an Adeno-Associated Virus-Cas9 System

Manu Prasad; Sankar Jagadeeshan; Maurizio Scaltriti; Irit Allon; Moshe Elkabets

doi:10.3791/60410

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Method Article

In Vitro Establishment of a Genetically Engineered Murine Head and Neck Cancer Cell Line using an Adeno-Associated Virus-Cas9 System

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DOI:

10.3791/60410

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January 9th, 2020

Manu Prasad^*¹^,² , Sankar Jagadeeshan^*¹^,² , Maurizio Scaltriti³ , Irit Allon²^,⁴ , Moshe Elkabets¹^,²

¹The Shraga Segal Department of Microbiology, Immunology and Genetics, Ben-Gurion University of the Negev, ²Faculty of Health Sciences, Ben-Gurion University of the Negev, ³Human Oncology & Pathogenesis Program (HOPP), Memorial Sloan Kettering Cancer Center, ⁴Institute of Pathology, Barzilai University Medical Center

^* These authors contributed equally

Summary

Development of murine models with specific genes mutated in head and neck cancer patients is required for understanding of neoplasia. Here, we present a protocol for in vitro transformation of primary murine tongue cells using an adeno-associated virus-Cas9 system to generate murine HNC cell lines with specific genomic alterations.

Abstract

The use of primary normal epithelial cells makes it possible to reproducibly induce genomic alterations required for cellular transformation by introducing specific mutations in oncogenes and tumor suppressor genes, using clustered regulatory interspaced short palindromic repeat (CRISPR)-based genome editing technology in mice. This technology allows us to accurately mimic the genetic changes that occur in human cancers using mice. By genetically transforming murine primary cells, we can better study cancer development, progression, treatment, and diagnosis. In this study, we used Cre-inducible Cas9 mouse tongue epithelial cells to enable genome editing using adeno-associated virus (AAV) in vitro. Specifically, by altering KRAS, p53, and APC in normal tongue epithelial cells, we generated a murine head and neck cancer (HNC) cell line in vitro,which is tumorigenic in syngeneic mice. The method presented here describes in detail how to generate HNC cell lines with specific genomic alterations and explains their suitability for predicting tumor progression in syngeneic mice. We envision that this promising method will be informative and useful to study tumor biology and therapy of HNC.

Introduction

HNC is a common malignancy worldwide¹. Modeling the genesis of HNC neoplasia is currently at a scientific turning point². While many genetic mutations have been identified in HNC²^,³^,⁴ (e.g., TP53, PIK3CA, NOTCH1, FAT1, and RAS) the specific combinations of genomic alterations required in concert to induce HNC remain unclear.

The current use of human HNC cell lines has significantly helped to elucidate the mechanisms associated with pathogenesis and treatment³. However, the stud....