Saunders, John
(2017)
Ex vivo modelling of oesophago-gastric cancer.
PhD thesis, University of Nottingham.
Abstract
Introduction
The response to neoadjuvant chemotherapy in oesophago-gastric (OG) cancer is only 40%, so over half of the patient’s disease will progress, whilst they also suffer the toxic chemotherapy side-effects. A model to predict chemotherapy response would provide a marked clinical benefit, by enabling personalised treatment of OG cancer.
Methods
Live chemo-naïve tumour biopsies were obtained following informed consent at staging endoscopy, before patients underwent their routine neoadjuvant chemotherapy. Tumour cells from the endoscopic biopsies were expanded, using an in vitro feeder layer system and supplemented medium. With ethics committee approval and under Home Office guidance, these individual patient cancer cells were engrafted into immuno-compromised mice, where they formed representative tumour xenografts. Primary patient tissue, the corresponding individual patient cancer cells and their matching xenografts were analysed using immunohistochemistry, demonstrating that the in vitro and in vivo cells had retained the characteristics of the original patient’s oesophageal adenocarcinoma.
To model the human tumour micro-environment (TME), a three dimensional tumour growth assay (3D-TGA) was developed, whereby the individual patient’s primary tumour cells were grown as 3D cancer cell clusters. This was performed by seeding individual patient’s primary tumour cells within a biological basement membrane extract, rich in extracellular matrix (ECM) components, with and without human mesenchymal stem cells to provide stromal support.
The individual patient cancer clusters in the 3D-TGA were subjected to detailed chemotherapeutic assessment, to quantify their chemo-sensitivity to the standard chemotherapy which was administered to the patient in the clinic. This 3D-TGA predicted chemo-sensitivity was then compared with the patient’s actual clinical chemotherapy response, as measured by the histological tumour regression grade, which directly relates to prognosis. In combination with standard platinum-based chemotherapy, the 3D-TGA was assessed as a platform for evaluating new chemotherapeutics: the novel emerging HDAC inhibitor Panobinostat, and the phosphodiesterase type 5 inhibitor Vardenafil, which has recently been shown to be active against cancer stroma, were evaluated.
Results
Individual patient tumours were grown from primary endoscopic biopsy tissue in over half of samples obtained within a clinically applicable timescale of 2-4 weeks. Incorporating human mesenchymal cells into the 3D-TGA significantly changed the growth and drug resistance profiles (p <0.005). This 3D-TGA chemo-response in the presence of stroma reflected the clinical chemo-sensitivity, with an accurate correlation between the 3D-TGA predicted chemo-resistance and actual clinical response for the patients evaluated.
As well as predicting potential chemo-sensitivity for individual patients, the method allows individual drugs and combinations to be evaluated, trends in chemo-sensitivity between patients to be appraised, and analysis of the effect of the TME on tumour growth and chemotherapy resistance.
Combination with Panobinostat enhanced response and proved efficacious in otherwise chemo-resistant tumours. Addition of PDE5i demonstrated an overall significantly enhanced chemotherapeutic response (p=0.003), and consequently provided efficacy in 60% of the otherwise chemo-resistant tumours.
Discussion
The novel method of growing individual patient OG cancers, using a 3D model with specific components of the tumour micro-environment in particular ECM and mesenchymal cells, provides a clinically-relevant oesophageal cancer model with application for chemo-sensitivity testing. Mesenchymal cells have a significant effect enhancing chemotherapy drug resistance in OG cancer, and this 3D model allowed identification of patients in which stromal targeting using PDE5i provided a significant reduction in chemotherapy drug resistance. In these patients, addition of PDE5i to routine chemotherapy could result in a marked change in the clinical efficacy of their chemotherapy regimen.
The 3D model’s chemo-response accurately reflects individual patients’ clinical chemo-sensitivity and so this research has direct clinical application: if this assay proves to be predictive across a wider patient population, then following clinical trials, it could potentially be used to routinely guide individual patient therapy in the clinic, with administration of tailored chemotherapy for individual patient benefit.
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