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04 FEBRUARY 2012

Brain tumour invasion

Geoffrey Pilkington is Professor of Cellular & Molecular Neuro-oncology at the School of Pharmacy and Biomedical Sciences, University of Portsmouth, and Honorary Professor at King’s College Hospital, London. Professor Pilkington has been involved in brain tumour research since 1971 and has particular expertise in the field of brain tumour cell invasion.

The research assistant supported by the Dr Hadwen Trust grant to work on this project was Samantha Murray BSc MSc.

Invasion of tumour cells into adjacent normal brain tissue is a major biological obstacle to the successful therapy of malignant primary brain tumours. Rogue cells which have violated the healthy brain are either inaccessible to drug treatments or refractory to radiotherapy. The majority of novel therapeutic strategies therefore aim to either prevent invasion or target cells which have already began their assault on the brain.

Animal models of brain tumour invasion exhibit major biological differences from spontaneously occurring human tumours. In particular, they generally grow by expansive, rather than infiltrative growth and are cellularly homogenous rather than heterogeneous as well as having dissimilar growth rates, antigenic expression profiles and vasculature.

Existing three-dimensional models of tumour invasion have used mixed cultures of human and animal cells, and such experiments are usually cultured with fetal calf serum (FCS). This Dr Hadwen Trust-funded project has developed the first “all human” three-dimensional model for the investigation of brain tumour invasion.

Initial work investigated culture of human brain cells under different sera conditions and showed that human serum could be used to supplement in vitro growth of human brain tumours, both as monolayer and spheroid cultures. Human serum modulates expression of glioma cell adhesion, proliferation and differentiation and this is accompanied by both proteomic and genomic changes which drive cellular and molecular pathways underlying disease progression. The use of human serum as a tissue culture supplement better approximates the conditions which prevail in the intact human central nervous system in vivo.

Gene microarrays were used to investigate the effect of different sera supplements on the expression of oncology-related gene expression profiles of brain tumour cells. This data set is still undergoing analysis.

An in vitro model to study brain tumour invasion was developed using exclusively human brain cells. Spheroids consisting of human non-neoplastic glial cells (from epilepsy resections) were cultured juxtaposed to spheroids consisting of cells from human biopsy-derived glioma (glioblastoma multiforme) to produce a 3-D confrontation model.

Spheroids were created using a modified version of the hanging drop method. Combinations of tumour-v-tumour; tumour-v-non-tumour; and non-tumour-v-non-tumour were examined with a range of microscopy techniques. Phase contrast, light, fluorescence, TIRF (total internal reflected fluorescence), live-cell, scanning and transmission electron microscopy was central to this investigation.

Scanning electron micrographs
A. Two spheroids day 2 confrontation have adhered to each other.
B. Magnification of A at a junction between the two spheroids.
C. Magnification of B.

A

B

C

This new all-human in vitro model can be used for live cell imaging and to provide a sound basis for investigation of cellular and molecular features of invading tumour cells as well as facilitating studies on interaction between normal and neoplastic cell populations within the central nervous system. Elucidating the precise mechanisms which underlie invasive behaviour is an essential precursor to the development of successful new therapies.

Pilot studies utilising the new in vitro model are underway to investigate the potential anti-invasive properties of flavonoids. Future adaptations using this system may include studies of cancer stem cell growth and the development of drug resistance, and the investigation of hypoxic environment.

This new model will help to obviate the use of animal-based strategies in the future. Indeed, these findings may lead the way in establishing a trend towards wide-spread use of three-dimensional in vitro real time live cell imaging models for biological studies and drug delivery system evaluation in the field of cancer research which will not only replace animal experiments but will better serve the purpose of answering questions related to the mechanisms underlying brain tumour invasiveness.

Summary

• Normal and neoplastic cells of the human nervous system have been maintained under human serum supplemented conditions, both as monolayer and spheroid cultures.
• Serum conditions influence cell adhesive properties, proliferation and antigenic expression.
• Human neural cells grown in human serum supplements more closely parallel the situation in vivo.
• Serum supplements also affect the expression of various genes of interest, the data from which is still being analysed and the full implications are being investigated.
• Normal brain spheroids and malignant primary brain tumour spheroids have been maintained in confrontation cultures, enabling the evaluation of the interaction between normal and neoplastic cells during the invasion cascade.
• To the best of our knowledge this is the first reproducible all-human in vitro model of tumour invasion to be developed.
• The model is already being used to investigate potential anti-invasive agents.
• This new model will help to replace animal-based research strategies and provide and improved system for investigating the mechanisms underlying brain tumour invasiveness.

Publications

GJ Pilkington & K Parker (2007) Models for CNS Malignancies. In: The Cancer Handbook (2nd Ed). Ed M R Alison. Blackwell Sci. pp 1176-1190. ISBN-13 978-0-470-01852-1

Murray SA, Fry K & Pilkington GJ (2008). Development of three-dimensional all-human, in vitro models for study of the biology of primary and metastatic brain tumours. 109th Meeting of the British Neuropathological Society Institute of Child Health, London, 9-11 January 2008. Neuropathology & Applied Neurobiology 34 Supp1:20

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