Effects of chemotherapy on colorectal liver metastases

© 2012 Dr. Linh My NguyenBackground: Colorectal cancer (CRC) is the fourth most frequently occurring cancer in the world. Despite optimum surgical endeavours, many patients will develop disease recurrence. Treatments available for patients who do not qualify for surgical resection are limited and mainly consist of chemotherapy or radiotherapy. Recent innovative options focus on selective targeting of the tumour blood supply, as a means of achieving greater tumour destruction or slowing overall tumour progression. Two main classes of drugs have been used both clinically and experimentally: the angioinhibitory agents (AIA) that inhibit the formation of new vessels, and the vascular disruptive agents (VDA) that target endothelial cells in immature tumour vessels, causing vessel collapse, tumour hypoxia and death. Treatment by VDAs are characterised by rapid and extensive destruction of tumour limited only by the persistence of a viable rim of tumour cells in the periphery, which subsequently leads to recurrence. The VDA OXi4503 is one of the most potent VDAs being tested, and our own research has demonstrated a rapid onset of microvascular thrombosis leading to tumour necrosis in excess of 90% of the tumour. Despite this efficacy, complete tumour eradication was not achieved as a thin rim of viable tumour invariably survived in the periphery giving rise to recurrence. Understanding the mechanisms that enable tumour to survive in the periphery could lead to formulation of drug combinations for total tumour eradication. Based on the finding that only tumour cells in the periphery survive the VDA treatment this study tests the following hypotheses: • Morphological and molecular differences in the tumour contribute to drug resistance in the tumour periphery (Chapter 4). • Treatment with OXi4503 promotes molecular and morphological changes in the residual tumour rendering the tumour resistant to cytotoxic treatments (Chapter 5 and Chapter 6). • Combination treatment using AIA (Sunitinib) and VDA (OXi4503) may produce complete destruction of colorectal liver tumours and hence improve treatment outcomes (Chapter 7). Experimental Design: Using a murine colorectal liver metastases model, inherent morphological and molecular differences within the periphery and the centre of the tumour that may account for differences in resistance to OXi4503 treatment were investigated. H&E staining and immunostaining were used to examine spatial differences in vessel maturity and stability, accumulation of immune cells, the expression of proangiogenic factors/receptors (HIF-1α, VEGF and ATR1) and the expression of epithelial to mesenchymal transition (EMT) markers (ZEB1, vimentin, E-cadherin and β-catenin) between the periphery and the centre of established tumours. The effects of single dose OXi4503 treatment on tumour vessels, cell kinetics, changes in growth factors (HIF-1α, VEGF and ATR1) and EMT markers (ZEB1, vimentin, E-cadherin and β-catenin) were also investigated by H&E and immunohistochemical staining, western blotting and RT-PCR techniques. A combination study testing the combined efficacy of OXi4503 and Sunitinib (AIA) was conducted and the effects were investigated by macroscopic stereology and immunohistochemistry. Results: In this study, significant differences were found between the tumour periphery and the central regions of mouse colorectal liver metastases, including association of the periphery with mature vessels, higher accumulation of immune cells, increased growth factor expression, minimal levels of hypoxia and increased EMT evidence. OXi4503 treatment resulted in collapse of tumour vessels in the tumour centre; however in the periphery the vasculature remained patent. Similarly, tumour apoptosis and proliferation were differentially modulated between centre and periphery after treatment. Significant increases in hypoxia and up-regulation of the pro-angiogenic growth factors HIF-1α, VEGF, and AT1R were detected within the viable periphery of the tumour. Simultaneously there was a significant down-regulation of E-cadherin, relocation and nuclear accumulation of β -catenin and up-regulation of ZEB1 and vimentin. These changes are strongly suggestive of EMT occurring in the surviving tumour in the periphery. This is the first direct evidence of in vivo EMT occurring almost immediately following treatment and involving all the surviving tumour cells. The data presented demonstrated a possible mechanism employed by tumour cells to evade drug treatment and metastasize, and may be targeted for more effective clinical outcomes. Sunitinib/OXi4503 combination treatment produced significantly lower tumour burden compared to either treatment alone. However, tumour kinetic studies revealed higher tumour proliferation compared to tumour apoptosis and the evidence of widespread EMT, suggesting the combination treatment in this model may successfully delay tumour regrowth but disease recurrence seems likely. Conclusion: The molecular and morphological differences seen between the periphery and the bulk of the tumour may account for the observed differential resistance to OXi4503 treatment. Growth factor and EMT changes following OXi4503 treatment further contribute to tumour resistance by providing escape mechanisms, re-vascularisation and tumour regrowth. Combination treatment targeting two different aspects of the tumour vasculature while more effective than either treatment alone, did not achieve complete tumour eradication. One exciting finding in this study is the observation that all surviving tumour cells undergo EMT in all three treatments holding the promise that EMT inhibitors in combination with VDAs or chemotherapies may result in total tumour eradication