Introduction: Pancreatic cancer is a devastating malignancy, and treatment options are very limited . Progression of the disease and resistance to therapy are mediated by the tumour microenvironment (TME), which is composed of excessive amounts of extracellular matrix proteins, as well as stromal and immune cells, acting as a physical barrier for drug delivery [1-3]. Cell-secreted factors, such as kallikrein-related peptidases (KLKs), are important players in the TME . Elevated expression of KLK6 is associated with poor survival rates in pancreatic cancer, making it an attractive target for alternative treatment strategies . There is a lack of pre-clinical models of pancreatic cancer that reconstruct different elements of the TME to study disease progression and response to treatment. To address this limitation, and to explore the tumour-biological role of KLK6, we developed a TME model using a protease-sensitive star-shaped poly(ethylene glycol) (star-PEG)-heparin hydrogel system in which the mechanical and chemical properties are independently controlled .
Methodology: To mimic the extracellular components of pancreatic cancer, hydrogels were formed by covalently crosslinking protease-sensitive four-arm starPEG with maleimide-functionalized heparin. To allow integrin-mediated cell functions, hydrogels were functionalized with RGD peptides. Hydrogels were globally and locally characterized regarding their mechanical properties by shear rheometry and atomic force microscopy. To increase the complexity of our hydrogel model, and to include the cellular component of tumour tissues, human pancreatic cancer cells, together with cancer-associated fibroblasts and myeloid cells were grown encapsulated in hydrogels for 14 days. Cell viability was assessed by live/dead staining, and the metabolic activity was measured using the PrestoBlue assay. To study the role of KLK6, a CRISPR/Cas9 knockout (KO) approach has been applied.
Results: In order to mimic the stiffness of pancreatic tumour tissues, hydrogels with different mechanical properties ranging from ~4-15 kPa were achieved by varying the crosslinking degree between PEG and heparin. These results are consistent with reported data for patient-derived tissues  and were confirmed with our own, unpublished patient cohort. Our multicellular 3D cultures had a high viability and were metabolically active over the analysed timeframe. To test the clinical value of our TME model, the response towards treatment with different chemotherapeutics including gemcitabine and nab-paclitaxel, as well as stromal-targeting agents are assessed. Our CRISPR/Cas9 approach resulted in a successful KO of KLK6 gene expression in human pancreatic cancer cells. The functional consequences of this KO were analysed using our multicellular 3D cultures and an orthotopic xenograft approach.
Conclusion: Bioengineered starPEG-heparin hydrogels are a powerful 3D model to mimic the mechanical, chemical and multicellular characteristics of human tissues in the lab. Our future studies will now determine their potential as pre-clinical platforms for drug screening. Therefore, we are collecting patient-derived tumour specimens that will be included in our model to strengthen their clinical relevance.
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