Pancreatic ductal adenocarcinoma (PDAC) is characterised by an abundant desmoplastic reaction driven by pancreatic stellate cells (PSCs). There is accumulating evidence that PSCs influence the malignant phenotype of PDAC. The aim of this study was to analyse the tumour response to radiation treatment in the presence of PSCs and to investigate the cytokine network in the coculture of PSCs and pancreatic cancer cells (PCCs). PSCs were used in coculture with different PCC lines. Clonogenic survival assays of several PCC lines cocultured with PSCs showed decreased radiosensitivity. This effect was abrogated by inhibition of the β1-integrin/FAK signalling pathway. Furthermore, tumour regrowth experiments after irradiation showed that coinjected PSCs were radioprotective for PCCs after single-dose and fractionated irradiation in xenografts. In addition, we examined the expression of 50 proteins in the supernatants of PCCs and PSCs in mono- and coculture conditions. The detected cytokine expression profile of PSCs included many proinflammatory factors. Also, we identified IP-10 as the chemokine with the highest differential upregulation in PSCs by paracrine stimuli from five different PCC lines. Human PDAC with a high stroma component had elevated IP-10 mRNA expression. IP-10 did not stimulate tumour cell growth and migration in our conditions even though several PCCs expressed its cognate receptor CXCR3. Nevertheless, we discovered that in human PDAC samples IP-10 and CXCR3 mRNA levels correlated with the presence of CD3ε, CD4, FoxP3, CTLA4 and CD39 used as surrogate markers for T regulatory cells (Tregs), known to exert an immunosuppressive effect. In conclusion, these data demonstrate that PSCs enhance survival of PCCs to radiation by activating β1-integrin/FAK signalling. Furthermore, the interaction between the tumour stroma in pancreatic cancer may support an immunosuppression by chemoattraction of Tregs following upregulation of IP-10. Further characterisation of the paracrine signalling between PCCs, PSCs and immune cells will improve the understanding of pancreatic cancer biology and could lead to the identification of new targets for multimodal therapy.

In developed countries, breast cancer is the commonest malignancy among women. Understanding the mechanisms involved in breast cancer progression and the influence of the microenvironment on cancer cell proliferation, results in better treatments. This study aimed to optimise breast cancer xenograft rates using a novel chamber developed for tissue engineering purposes. The established tumours were subjected to enzyme digestion, creating a single cell suspension, which was then injected into immunocompromised mice at primary, metastatic and intra-cardiac sites. The resulting tumours in the mammary fat pad (MFP) and bone were compared using species-specific reverse-transcription polymerase chain reaction (RT-PCR) and cDNA microarray, to examine the influence of the microenvironment on gene expression. The achieved xenograft graft rates of 25% were similar to those previously reported. The matrix metalloproteinase family of enzymes (MMPs) degrade extracellular matrix, influencing invasion and migration of malignant cells. RT-PCR results showed that the majority of the MMPs expressed in the cancers were stromal rather than tumour in origin. MT1-MMP, MMP-2 and MMP-11 had significantly higher expression levels in the MFP than in the bone, but MMP-9 was expressed more in the bone than MFP. There was also an up-regulation of stromal production of MT1-MMP and MMP-13 in the MFP in the presence of tumour. This may have significance when considering which MMPs are the most appropriate targets for inhibition during cancer treatment. The most significant of the differentially expressed genes on microarray analysis were trefoil factor 1 (TFF1) and insulin growth-factor binding protein 3 (IGFBP-3), both expressed significantly more in tumours from the MFP than the bone. The thesis presented demonstrates some of the complexities of tumour-stromal interactions and supports Paget’s seed-soil theory, confirming in several ways the variation in gene expression in breast cancer between primary and metastatic sites.

Directed cancer cell migration induced by the environmental signals is a critical process in cancer metastasis. Cancer cells are exposed to complex chemical and mechanical signals stimulating directed migration in the tumor microenvironment, where the physical nature is highly complex. It is still barely understood how cells sense and process the complex environmental signals through the complex intercellular signaling networks to execute the cell responses. This study explores the migratory response of cancer cells under a single and combined signal. The driving hypothesis is that the cell innate capability constraints the signal stimulations physically in inducing directed cell migration. We assess the hypothesis by engineering the microenvironment in the microfluidic platform, exposing a single or combined signal environment. The combined signal environment is established by 1) two different chemoattractants (TGF-β1 and EGF) and 2) the convection-driven signal environment (TGF-β1 and interstitial flow). The results show that the performance of cancer cell directed migration is physically constrained when the environmental stimulation meets the cell’s innate physical limit. We illustrate the results in a physical and quantitative manner. This approach provides a novel insight to understand the cellular process and eventually enables to predict the cellular response under the complex environmental signals. <br>

β-catenin is a multifunctional protein that plays crucial roles in embryonic development, physiological homeostasis, and a wide variety of human cancers. Previously, we showed that in vivo targeted ablation of β-catenin in melanoma-associated fibroblasts after melanoma formation significantly suppressed tumor growth. However, when the expression of β-catenin was ablated in melanoma-associated fibroblasts before tumor initiation, melanoma development was surprisingly accelerated. How stromal β-catenin deficiency leads to opposite biological effects in melanoma progression is not completely understood. Here, we report that β-catenin is indispensable for the activation of primary human stromal fibroblasts and the mediation of fibroblast-melanoma cell interactions. Using coimmunoprecipitation and proximity ligation assays, we identified Yes-associated protein (YAP) as an important β-catenin-interacting partner in stromal fibroblasts. YAP is highly expressed in the nuclei of cancer-associated fibroblasts (CAFs) in both human and murine melanomas. Mechanistic investigation revealed that YAP nuclear translocation is significantly modulated by Wnt/β-catenin activity in fibroblasts. Blocking Wnt/β-catenin signaling in stromal fibroblasts inhibited YAP nuclear translocation. In the absence of YAP, the ability of stromal fibroblasts to remodel the extracellular matrix (ECM) was inhibited, which is consistent with the phenotype observed in cells with β-catenin deficiency. Further studies showed that the expression of ECM proteins and enzymes required for remodeling the ECM was suppressed in stromal fibroblasts after YAP ablation. Collectively, our data provide a new paradigm in which the β-catenin-YAP signaling axis regulates the activation and tumor-promoting function of stromal fibroblasts.

Metastasis is the main cause of death from solid cancer. The dissemination of cancer cells from a primary tumour is a very complex process that involves many steps and cells must overcome many obstacles to colonize distant organs. The tumour microenvironment influences the mode and the dynamics of invasion of cancer cells. Cancer cells have the ability to adapt to distinct environmental conditions in order to stay motile. Invasive cancer cells form membrane protrusions called invadopodia that are able to degrade extracellular matrix. The formation of invadopodia by cancer cells is interconnected to the production of matrix metalloproteases (MMPs). Metastasizing tumour cells use MMPs to break through extracellular matrix barriers and migrate in dense matrix. Both invadopodia formation and MMPs secretion is crucial for the degradation of the extracellular matrix. The most important is the membrane bound MMP-14 (MT1-MMP) and soluble MMP-2 and MMP-9. The invasive structures of tumour cells and the proteolytic enzymes in 2D environment is well described. However, a suitable model of localization and transport of MMPs and connection with invadopodia of tumour cells in 3D environment is still lacking. This diploma thesis focused on the extension of current knowledge of these key MMPs and on the...