Tumorspheres but Not Adherent Cells Derived from Retinoblastoma Tumors Are of Malignant Origin

Wesley S Bond,Mary Y Hurwitz,Patricia Chévez-Barrios,Richard L Hurwitz,Laszlo Perlaky,Patricia Y Akinfenwa,Hiromu Suzuki

doi:10.1371/journal.pone.0063519

Abstract

Verification that cell lines used for cancer research are derived from malignant cells in primary tumors is imperative to avoid invalidation of study results. Retinoblastoma is a childhood ocular tumor that develops from loss of functional retinoblastoma protein (pRb) as a result of genetic or epigenetic changes that affect both alleles of the RB1 gene. These patients contain unique identifiable genetic signatures specifically present in malignant cells. Primary cultures derived from retinoblastoma tumors can be established as non-adherent tumorspheres when grown in defined media or as attached monolayers when grown in serum-containing media. While the RB1 genotypes of tumorspheres match those of the primary tumor, adherent cultures have the germline RB1 genotype. Tumorspheres derived from pRb-negative tumors do not express pRb and express the neuroendocrine tumor markers synaptophysin and microtubule-associated protein 2 (MAP2). Adherent cells are synaptophysin-negative and express pRb, the epithelial cell marker cytokeratin that is expressed in the retinal pigmented epithelium and the vascular endothelial cell marker CD34. While tumorspheres are of malignant origin, our results cast doubt on the assumption that adherent tumor-derived cultures are always valid in vitro models of malignant cells and emphasize the need for validation of primary tumor cultures.

Highlights

Cells derived from primary tumors are commonly used as models for cancer research including for high-throughput genomic and transcriptomic analysis [1] and evaluation of therapeutics for treatment of cancer [2]
Similar to our previously reported observations [14,15], when primary tumor samples were placed in neural stem cell medium with defined supplements, tumorspheres arose in culture (Fig. 1)
Genetic analysis shows that attached cells derived from Rb tumors in serum-containing medium conditions typically are not derived from the malignant cells of the tumor

Summary

Introduction

Cells derived from primary tumors are commonly used as models for cancer research including for high-throughput genomic and transcriptomic analysis [1] and evaluation of therapeutics for treatment of cancer [2]. Primary cultures are not validated genetically and are assumed to be derived from the original malignancy. The formation of tumorspheres commonly requires specific culturing conditions, such as the use of stem cell-optimized media with defined supplements [14]. Culturing tumor cells in serum-containing medium can yield cells with markedly different morphologies and growth characteristics. In a SV40 T-antigen transgenic mouse model of Rb, culturing of tumor cells in medium containing serum typically yields a population of cells with a different phenotype from tumorspheres: an attached monolayer [15]. The true identity of these different primary tumor cultures and definitive knowledge of their origin remain poorly understood

Methods

Results

Conclusion