Down-Regulation of DNA Mismatch Repair Enhances Initiation and Growth of Neuroblastoma and Brain Tumour Multicellular Spheroids

Samuel L Collins,Jeremy P Blaydes,C W Keevil,Jeremy S Webb,Rodolphe Hervé,Irina V Lebedeva

doi:10.1371/journal.pone.0028123

Samuel L Collins, Jeremy P Blaydes + Show 4 more

Open Access

https://doi.org/10.1371/journal.pone.0028123

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Journal: PloS one	Publication Date: Dec 1, 2011
Citations: 51	License type: CC BY 4.0

Affiliation: University of Southampton, Cancer Research UK

Abstract

Multicellular tumour spheroid (MCTS) cultures are excellent model systems for simulating the development and microenvironmental conditions of in vivo tumour growth. Many documented cell lines can generate differentiated MCTS when cultured in suspension or in a non-adhesive environment. While physiological and biochemical properties of MCTS have been extensively characterized, insight into the events and conditions responsible for initiation of these structures is lacking. MCTS are formed by only a small subpopulation of cells during surface-associated growth but the processes responsible for this differentiation are poorly understood and have not been previously studied experimentally. Analysis of gene expression within spheroids has provided clues but to date it is not known if the observed differences are a cause or consequence of MCTS growth. One mechanism linked to tumourigenesis in a number of cancers is genetic instability arising from impaired DNA mismatch repair (MMR). This study aimed to determine the role of MMR in MCTS initiation and development. Using surface-associated N2a and CHLA-02-ATRT culture systems we have investigated the impact of impaired MMR on MCTS growth. Analysis of the DNA MMR genes MLH1 and PMS2 revealed both to be significantly down-regulated at the mRNA level compared with non-spheroid-forming cells. By using small interfering RNA (siRNA) against these genes we show that silencing of MLH1 and PMS2 enhances both MCTS initiation and subsequent expansion. This effect was prolonged over several passages following siRNA transfection. Down-regulation of DNA MMR can contribute to tumour initiation and progression in N2a and CHLA-02-ATRT MCTS models. Studies of surface-associated MCTS differentiation may have broader applications in studying events in the initiation of cancer foci.

Highlights

First utilised by Sutherland et al [1,2,3] multicellular tumour spheroid (MCTS) cultures have become ideal model systems for studying many aspects of tumour growth and physiology [1]
Down-regulation of MLH1 and PMS2 in MCTS To determine whether the mismatch repair (MMR) genes MLH1 and PMS2 are differentially expressed in N2a and CHLA-02-ATRT MCTS we carried out reverse transcription PCR (RT-PCR) analyses on separated monolayer and spheroid-derived cells
Our data show that silencing of these two genes results in an increase in the number and size of MCTS that was observed over several subsequent passages following transfection

Summary

Introduction

First utilised by Sutherland et al [1,2,3] multicellular tumour spheroid (MCTS) cultures have become ideal model systems for studying many aspects of tumour growth and physiology [1]. Previous experimental approaches have shown that MCTS are morphologically and characteristically similar to solid tumours in vivo [1,2,4]. They are composed of symmetrically arranged aggregates of cells in discrete proliferating, dormant and necrotic populations and are subject to varying oxygen gradients, nutrient deficiencies and acidosis making them physiologically unique from standard 2-dimensional cultures. The mechanisms governing the differentiation between 2D culture and 3D MCTS are poorly understood and elucidation of these mechanisms may provide new insight into early events in tumourigenesis

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