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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.
ItemTargeting p21-activated kinases in the treatment of pancreatic cancerWang, Kai ( 2019)Pancreatic ductal adenocarcinoma (PDA) is one of the most lethal malignancies worldwide, with a very poor prognosis and a 5-year survival rate less than 9%. This dismal outcome is largely due to lack of early diagnosis, quick disease progression, high rate of post-surgery recurrence and resistance to conventional therapies. Oncogenic Kras mutation is a well-defined hallmark of pancreatic cancer. It is presented in over 95% cases and leads to constitutively active form of Kras protein. Kras still remains as an undruggable target due to absence of a well-defined drug-binding domain. With the aim to fight against Ras-driven cancers, high priority has been given to the novel therapeutic strategies targeting Ras-dependent signalling. P21-activated kinases (PAKs) are a family of serine/threonine kinases that are important down-stream effectors of multiple small GTPases including Ras, Rac1 and Cdc42. Based on the difference in the structure and sequence, all the six members of PAK family are divided into two groups: group I (PAK1-3) and group II (PAK4-6). PAK1 and PAK4 are the most widely studied members and have been reported to be up-regulated in PDA. PAK1 is situated at the convergence of multiple signalling pathways that are associated with cell proliferation, survival/apoptosis, migration/invasion and cytoskeletal regulation. Immunotherapy is now emerging as a promising treatment in the era of personalised anti-cancer therapeutics. However, it can only bring limited clinical benefits for PDA patients, which is largely attributed to the immunosuppressive tumour microenvironment (TME). The role of PAK1 has not been fully elucidated in pancreatic cancer, especially its involvement in re-programming TME. The work presented in this thesis investigated the role of PAK1 in tumour biology and therapeutic regimens, with a focus on its linkage to stroma modulation and anti-tumour immune response. Firstly, the anti-tumour effect of a potent PAK inhibitor (PF-3758309) was determined on a panel of clinical patient-derived PDA cell lines (TKCC 2.1, TKCC15, TKCC18, TKCC22, TKCC23, TKCC26). PF-3758309 treatment inhibited cell proliferation and sensitized PDA cells to different chemotherapies (fluorouracil, gemcitabine and nab-paclitaxel) with a synergistic effect, which was associated with reduction in PAK1 and PAK4 activity and down-regulation of HIF-1α, α-SMA and palladin in vitro. Combination of PF-3758309 and gemcitabine maximally suppressed tumour growth in vivo and had a comparable or even greater therapeutic efficacy compared to combination of gemcitabine and nab-paclitaxel. As mentioned above, the expression of α-SMA was observed in PDA cells. All-trans retinoid acid (ATRA), a well-known compound that induced quiescence of pancreatic stellate cell (PSC) by decreasing the expression of α-SMA, was utilized to investigate its anti-tumour effect and association with PAK protein and α-SMA in PDA cells. Inhibitory effect of ATRA on PDA cell growth and migration and its synergism with gemcitabine was observed in both wildtype and gemcitabine-resistant PDA cell lines. Expression of PAK1, PAK2, PAK4 and α-SMA was down-regulated by ATRA. Inhibition of PAK1 by shRNA knockdown or PF-3758309 sensitized PDA cells to ATRA. This was the first study to demonstrate the role of PAK proteins in ATRA treatment. The role of PAK1 in tumour immune response was evaluated using an orthotopic mouse model of pancreatic cancer. Inhibition of PAK1 by PF-3758309 or genetic knockout up-regulated intra-tumoural infiltration of CD3+ lymphocytes and splenic CD3+ or CD8+ lymphocytes. Combination of PF-3758309 and gemcitabine synergistically inhibited PDA cell growth in vitro and in vivo. This study not only confirmed the anti-tumour effect of PF-3758309 and its synergism with gemcitabine, but also revealed the potential role of PAK1 in anti-tumour immunity. Finally, the underlying mechanisms of PAK1 in regulating anti-tumour immunity was investigated. Immunohistochemistry was performed on human tissue microarray and KPC (LSL-KrasG12D/+; LSL-Trp53R172H/+; Pdx-1-Cre) mice samples. PAK1 was identified to be a negative prognostic marker and positively correlated with α-SMA expression. Depletion of PAK1 decreased PSC activity, reduced PSC-stimulated PDA cell proliferation and migration and increased intra-tumoural infiltration of CD4+ or CD8+ lymphocytes. Inhibition of PAK1 decreased both intrinsic and PSC-stimulated PD-L1 expression in PDA cells, which could enhance lymphocyte-induced PDA cell death. This was the first study to demonstrate the important role of PAK1 in regulating PSC activity and PD-L1 expression in PDA. In a summary, these studies revealed the importance of PAK signalling in PDA development, the therapeutic value of PAK inhibitors and its synergism with gemcitabine in PDA treatment. Most importantly, PAK1 is emerging as a potential target to enhance anti-tumour immunity and to facilitate the development of novel immunotherapies.