Immortalization of human primary prostate epithelial cells via CRISPR inactivation of the CDKN2A locus and expression of telomerase

Ziran Zhao,Joseph R. Testa,Henkel Valentine,Jianming Pei,Simone Badal,Yinfei Tan,Xavier Graña,Zemin Liu,Holly Fowle

doi:10.1038/s41391-020-00274-4

Abstract

BackgroundImmortalization of primary prostate epithelial cells (PrEC) with just hTERT expression is particularly inefficient in the absence of DNA tumor viral proteins or p16INK4A knockdown.Materials and methodsHere, we describe the establishment of immortalized normal prostate epithelial cell line models using CRISPR technology to inactivate the CDKN2A locus concomitantly with ectopic expression of an hTERT transgene.ResultsUsing this approach, we have obtained immortal cell clones that exhibit fundamental characteristics of normal cells, including diploid genomes, near normal karyotypes, normal p53 and pRB cell responses, the ability to form non-invasive spheroids, and a non-transformed phenotype. Based on marker expression, these clones are of basal cell origin.ConclusionsUse of this approach resulted in the immortalization of independent clones of PrEC that retained normal characteristics, were stable, and non-transformed. Thus, this approach could be used for the immortalization of normal primary prostate cells. This technique could also be useful for establishing cell lines from prostate tumor tissues of different tumor grades and/or from patients of diverse ethnicities to generate cell line models that facilitate the study of the molecular basis of disease disparity.

Highlights

Prostate cancer is the most common cancer diagnosed in men, accounting for an estimated 20% of new cancer cases and 10% of cancer deaths in US males in 2020 [1]
To help define the minimal relevant genetic alterations needed for prostate cell transformation and 3D organoid aberrant growth, it is necessary to generate a collection of immortal normal prostate cell lines and develop a methodology that can be used for the efficient establishment of primary prostate cancer cells that often fail to establish in culture
Our attempts to establish immortalized primary normal human prostate epithelial cells (PrEC) via ectopic expression of human telomerase reverse transcriptase (hTERT) alone failed to generate any immortal clones following selection. This indicated that the introduction of a single hTERT transgene is very inefficient as a single hit for immortalization of PrEC, supporting why long-term successful stable immortalization of PrEC has been rarely accomplished [8]

Summary

Introduction

Prostate cancer is the most common cancer diagnosed in men, accounting for an estimated 20% of new cancer cases and 10% of cancer deaths in US males in 2020 [1]. The most widely used cell lines, PC-3, DU145, and LNCaP, were established spontaneously and all derived from metastases [4] They have already acquired numerous genomic alterations [5]. To help define the minimal relevant genetic alterations needed for prostate cell transformation and 3D organoid aberrant growth, it is necessary to generate a collection of immortal normal prostate cell lines and develop a methodology that can be used for the efficient establishment of primary prostate cancer cells that often fail to establish in culture. Materials and methods Here, we describe the establishment of immortalized normal prostate epithelial cell line models using CRISPR technology to inactivate the CDKN2A locus concomitantly with ectopic expression of an hTERT transgene. These clones are of basal cell origin

Methods

Results

Conclusion