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ItemExploring Treatments to Inhibit tumour recurrence Following Resection of Colorectal Liver MetastasesKastrappis, Georgios Loizou ( 2021)Background: Colorectal cancer (CRC) accounts for 9.2% of all cancer related deaths making it the second most common cause of cancer related death worldwide. The majority of CRC deaths are attributed to metastases, with liver being the most common metastatic site. Currently the best available treatment for colorectal liver metastasis (CRLM) that offers high survival rates and a potential for cure is liver resection surgery. However, only a small fraction of CRLM patients are eligible for surgery. Furthermore, liver resection and the ensuing liver regeneration (LR) upregulate growth factors and cytokines leading to a pro-inflammatory response, creating a favourable environment for any dormant tumours to grow. Thus, liver resected patients experience high tumour recurrence rates. Anti-inflammatory treatments administered perioperatively may reduce tumour recurrence. Previous experimental studies have shown that inhibition of the Renin Angiotensin System (RAS) classical pathway reduces tumour growth and accelerates liver regeneration together with a reduction in inflammation. This study investigates mechanisms by which captopril a RAS inhibitor (RASi) influences the environment of a regenerating liver to reduce inflammation. Additionally, it investigates the potential of a VEGFR-3 specific inhibitor, SAR131675, to inhibit tumour growth and reduce inflammation. Aims: 1) To investigate the effects Captopril, an angiotensin I converting enzyme (ACE) inhibitor, has on pro-inflammatory cytokines during LR. 2) To investigate the effect Captopril has on the global proteome and phosphoproteome of the liver during the early stages of LR 3) To investigate the effect SAR131675, a VEGFR-3 tyrosine kinase inhibitor, has on liver metastases in a CRLM mouse model and determine the likely mechanisms. Methods: Male CBA mice were used for all experiments in this study. For the liver regeneration study a 70% partial hepatectomy mouse model was used. Captopril (750mg/kg) was administered intraperitoneally and given daily starting 4 days before surgery until the endpoint (1 hour, 3 hours, 4 hours, 1 day and 2 days). Serum cytokine (IL-2, IL-6, IL-10, IL-12p70, IL-17A, TNF, IFNgamma and MCP-1) levels were assessed at the 1, 3 and 4 hour timepoints while liver regeneration was assessed at the day 1 and 2 timepoints, by measuring liver to body weight ratio and the liver regeneration rate. In addition, the 4 hour timepoint was used to conduct global proteomic and phosphoproteomic analyses. To investigate the effects SAR131675 had on CRLM and the mechanisms involved a mouse model of CRLM was used where metastases were established via intrasplenic injection of tumour cells. Immunohistochemistry was used for the analysis of proliferation, apoptosis, lymphatic and blood vessel densities, macrophage and T-cell tumour infiltration. Furthermore, FACS analysis was used to investigate changes in immune lymphoid and myeloid cell populations due to SAR131675 treatment. Results: Captopril treatment significantly reduced IL-6 levels in the serum of mice in the early phase of liver regeneration. This result was reinforced by the results of the global proteomic and phospho-proteomic study indicating that Captopril induced changes in a great number of proteins involved in inflammatory pathways in almost every cell process including cell proliferation, apoptosis transcription, translation and stress response. Interestingly, the largest proportion of protein changes were associated with lipid metabolism which is also closely associated with inflammatory pathways. SAR131675 treatment significantly reduced tumour growth in the mouse model of liver metastases. Mechanistically SAR131675 treatment changed the tumour microenvironment and promoted anti-tumour immune responses by modulating the tumour infiltrating immune cell composition; increasing the ratio of T lymphocytes to monocytes and by modifying the T-cell and myeloid cell subtype and activation to that favouring an anti-tumour immune response. Conclusion: Both Captopril and SAR131675 were able to modulate inflammatory pathways creating a microenvironment that is inhibitory towards tumour growth. These treatments have potential to be used in order to reduce tumour recurrence in patients that have undergone liver resection surgery.
ItemReprogramming tumour immune microenvironment of colorectal liver metastases with renin-angiotensin inhibitorsVallejo Ardila, Dora Lucia ( 2021)Background Renin-angiotensin system inhibitors (RASi) have shown anti-tumour effects that may significantly impact the response to current cancer immunotherapies and the prognosis in patients with colorectal liver metastases (CLM). RAS components are expressed by various immune cells and adult hematopoietic cells. The mechanisms by which RASi reprogram the tumour immune microenvironment toward an immunostimulatory milieu involves modulating the function of immune T lymphocytes, myeloid cells and cancer-associated fibroblasts. Experimental Design Liver metastases were established in a mouse model using an autologous colorectal cancer cell line. RASi captopril 750mg/kg or saline was administered to the mice daily via intraperitoneal injection, from day one post-tumour induction to the endpoint, day 15 or 21 post tumour induction. At the endpoint, tumour growth was determined, and lymphocyte and myeloid-derived infiltration and composition in the tumour and liver tissues were analyzed by flow cytometry and immunohistochemistry. At endpoint day 15, livers and tumour tissues were collected, and tissue lysates were prepared and analyzed using liquid chromatography-tandem mass spectrometry proteomic techniques to determine relative changes in protein abundance. The proteomics results were analyzed using open-source software such as MaxQuant and Perseus. A 7-plex OPAL protocol was used to assess the composition and spatial distribution of T cell markers CD3, CD8, FoxP3 and CD103 and the epithelial to mesenchymal transition markers alpha- Smooth muscle actin and E-cadherin. The protocol was manually optimized and validated in two independent cohorts of formalin-fixed, paraffin-embedded CLM patient tissues using well-established antibodies, a single spectral library, negative controls, and biological controls corroborating the staining pattern of immune infiltrates. Results Captopril significantly decreased tumour viability and impaired metastatic growth, increased the infiltration of CD3 T cells into both tissues, and the primary contributing phenotype to this influx is a CD4 and CD8 double-negative T cell subtype, while CD4 T cells decreased and CD8 T cells remained unchanged. Furthermore, clustering and functional enrichment showed that captopril modulates the expression of some of the immunoproteasome subunits, including PSMB8, PSMB10, PSMB5 and PSME1. Also, T lymphocyte phenotypes’ spatial distribution was seen in significantly higher levels in the invasive tumour margin compared to their density within the liver parenchyma or the tumour centre in human CLM. Thus, high CD3 density in the tumour core or high CD8 density in the invasive tumour margin predicts a significantly better overall survival. Moreover, high CD3 T cell density at the adjacent liver parenchyma and a high CD8 density at the tumour core segment influenced patient survival after CLM resection in patients who received RASi antihypertensive medication. In the case of myeloid cells, a significant decline in myeloid cells expressing CD31pos was induced by captopril, while the relative proportion of CD31neg cells was preserved when comparing between the liver and the tumour. RASi significantly reduces a CD31pos monocyte-derived subset F480neg Ly6C intermediate and High, which may have a pro-angiogenic and immunosuppressive function. Conclusion The work of this thesis highlights the mechanism by which RAS inhibitors decreased tumour viability and impaired metastatic growth by effectively restoring the immunogenicity of CLM toward an immunostimulatory milieu.
ItemThe role of the renin-angiotensin system in liver regeneration and colorectal cancer liver metastasesKOH, SHIR LIN ( 2012)Background: Colorectal cancer (CRC) is the second most common cause of cancer related death in Australia with over 4700 deaths reported annually. CRC liver metastasis (CRCLM) contributes to over 70% of the disease mortality. While unresected patients rarely survive beyond 2 years, partial hepatectomy (PH) improves their survival to 25%-60% at 5 years. Blockade of the renin-angiotensin system(RAS) has been shown to enhance liver regeneration and, separately, to inhibit CRCLM. Targeting the RAS may offer a unique synergistic anti-cancer therapy by inhibiting CRCLM tumour growth while simultaneously enhancing liver regeneration following PH. Aim: This study investigated the expression of the RAS during liver regeneration and in CRCLM. The effects of RAS blockade on liver regeneration and CRCLM in the regenerating liver were determined to investigate its potential benefits as a therapeutic avenue for CRCLM patients. Methods: Male CBA mice (10-12 weeks) were used in this study. After 70% partial hepatectomy (PH) alone, captopril (750mg/kg) or saline (control), were administered intraperitoneally on a daily basis until the endpoints (days 1, 2, 4, 6 and 8 post-surgery). A mouse model of CRCLM in the regenerating liver was developed. Mice induced with CRCLM and subjected to 70% PH were treated with captopril (250mg/kg) daily until the endpoints (days 2, 6, 16 and 21). At study endpoints, liver regeneration was assessed by measuring the liver-to-body weight ratio. CRCLM tumour burden (percentage of liver metastases) was calculated using total liver and tumour volumes using quantitative stereology. Liver function tests were performed on mouse serum collected from days 2 and 6. The expression of the RAS components, cell proliferation, apoptosis, hepatic stellate cells (HSC) and liver endothelial cell densities, matrix metalloproteinase (MMP)-9, transforming growth factor (TGF)-β were quantified. Statistical analyses were performed using 2-sample independent T-test, one-way ANOVA with post-hoc analysis, or Kruskal Wallis followed by Mann-Whitney U tests as appropriate (SPSS v.18). P-value of <0.05 was considered statistically significant. Results: Captopril significantly inhibited CRCLM tumour growth and increased tumour cell apoptosis in the regenerating liver at day 21. Captopril also enhanced early liver regeneration and this was associated with an increase in hepatocyte proliferation at 6 hours after PH as well as an increase in HSC density and MMP-9 levels 2 days after PH. The decrease in hepatocyte proliferation at day 2 was transient. By day 4 onwards there was no significant difference between control and treated livers. Captopril also decreased the hepatocyte injury marker, alanine transaminase. The ability of captopril to increase human hepatocyte proliferation was confirmed in vitro. The RAS was expressed in the liver and tumours during liver regeneration and tumour growth phases. Liver and tumour differed in their RAS expression; tumour AT1R expression levels were lower than normal liver, while tumour MasR and AT2R levels were upregulated during cancer progression. Conclusion: This thesis showed a tumour-specific RAS expression which could be targeted to inhibit tumour growth while allowing the liver to regenerate following PH. This is supporting by my findings that RAS blockade with captopril following PH was associated with a reduction in CRCLM tumour growth without impairing liver regeneration. Thus, captopril may offer a new avenue to improve CRCLM patient outcomes by inhibiting tumour growth whilst enhancing the early stage of liver regeneration.