An observational pilot study to assess the potential of a microfluidic tissue culture model to predict rectal cancer response to neo-adjuvant therapy

Patel, Rikesh

Medicine
March 2016

Thesis or dissertation


Rights
© 2016 Rikesh Patel. All rights reserved. No part of this publication may be reproduced without the written permission of the copyright holder.
Abstract

Radiotherapy has been reported to induce apoptosis and prevent the proliferation of malignant cells. Complete clinical response to neo-adjuvant long course chemoradiotherapy has been identified in up to 30% of patients with locally advanced rectal cancer. The aim of this study was firstly to maintain rectal cancer biopsies in a viable state within a microfluidic device and subsequently interrogate this ex vivo rectal cancer tissue with radiation and measure changes in morphology and induction of cell death through apoptosis.

Murine colorectal tissue was used for initial optimisation, followed by biopsies from patients with locally advanced rectal cancer taken prior to neo- adjuvant therapy. This tissue was maintained in a biomimetic environment within a bespoke, glass microfluidic device. Subsequently, murine tissue was interrogated with single fractions of radiation (2Gy, 10Gy or 30Gy) to identify suitable doses for delivery to human tissue. Morphology was assessed using H&E staining of the tissue. Effluent from the tissue was collected for subsequent analysis of cell death using a lactate dehydrogenase (LDH) assay and metabolite release using a mass spectrometry-metabolomics approach. Apoptosis was evaluated using the M30 CytoDeath™ monoclonal antibody and terminal deoxynucleotidyl transferase dUTP nick-end labelling (TUNEL) assay to identify DNA fragmentation.

Tissue was successfully maintained for over 70 hours with evidence of viability, as determined by preservation of morphology and increased LDH release after lysis. Rectal cancer biopsies (n = 11 patients) were subsequently interrogated with radiation. Only high doses of radiation (30Gy) delivered to murine colorectal tissue reproducibly induced high levels of LDH release, however architectural losses were seen in all tissue after irradiation regardless of dose. Human tissue was therefore irradiated with 2Gy as an approximation of the dose delivered clinically.

Levels of apoptosis using M30 CytoDeath™ ELISA were not significantly increased in the irradiated groups when compared to control groups. However, using immunohistochemical assessment with M30 CytoDeath™ and TUNEL, significant increases in the irradiated groups were seen (p < 0.05). Evaluation of individual patients using these markers identified several patients with significant rises (p < 0.05) in levels of apoptosis, however there was no correlation with clinical response. Metabolomic analysis identified 28 differentially expressed (p < 0.0001) compounds in effluents collected prior to and after irradiation, however this appeared to be a time-dependent effect, rather than due to irradiation.

This work has demonstrated that the microfluidic device can be used to reliably maintain both ex vivo healthy murine colorectal and human rectal cancer tissue for a sufficient period of time to permit interrogation with radiation. Findings demonstrated that apoptosis and morphological changes are induced by irradiation. Further work is required to correlate findings with clinical outcome, but important progress has been made to allow use of this platform as a predictive tool of response to neo-adjuvant therapy to deliver personalised therapy.

Publisher
Hull York Medical School, The University of Hull and University of York
Supervisor
Greenman, John (Professor of tumour immunology); Hunter, Iain Andrew
Qualification level
Doctoral
Qualification name
MD
Language
English
Extent
5 MB
Identifier
hull:14518
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