Biomimetic platforms for modeling in vitro the functional unit of the exocrine pancreas

Pancreatic ductal adenocarcinoma (PDAC), commonly known as pancreatic cancer, is the most frequent type of exocrine pancreas tumors and one of the leading causes of cancer-related death worldwide with a five-year survival rate around 10%. The main reason that leads to consider PDAC a notably aggressive disease concerns the rapid cancer’s evolution without clear symptoms in the early stages, resulting in a late diagnosis and poor clinical prognosis. In addition, the unique bioarchitecture of the pancreatic tumor microenvironment (TME) weakens the effectiveness of the current treatments that, despite the advances in the discovery of new therapeutic strategies, result insufficient to treat this particularly aggressive pathology. Current research is focusing on better understanding the key mechanisms involved in pancreatic cancer through creating effective in vitro models that can be used to recapitulate the cancer evolution. Indeed, in vitro models represent an important alternative for animal experiments and powerful tools for biomedical research, drug discovery, diagnostics, and regenerative medicine. 2D models, cancer-on-a-chip platforms, multicellular spheroids, organoids and 3D biofabricated constructs (scaffolds or hydrogel-based models) are the currently available bioengineered models mimicking the pancreatic TME. However, although recent studies have shown the possibility of modeling the PDAC microenvironment in vitro, the crosstalk between tumor and the surrounding stromal cells remains extremely challenging to be reproduced and monitored in effective models. Moreover, only a few novel studies in literature focus on the development of biomimetic platforms reproducing the microanatomy (in terms of 3D architecture and cellular composition) of the exocrine pancreas and lack to resemble the native compartmentalized architecture of tumor microenvironment that is widely recognized to affect cell functionality and cancer-cell response to therapeutics. Specifically, the gland complex geometry has been reproduced in simplified ways, which result in low reproducibility, throughput, and shape fidelity. My PhD research focuses on replicating the functional unit of the exocrine pancreas in vitro, where the earliest lesions of pancreatic cancer manifest. The objective of this work is to create advanced bioengineered platforms that mimic the intricate processes involved in the initial stages of tumor development. To achieve this goal, different biofabrication strategies were explored to obtain in vitro models which can be classified as two-dimensional (2D), two-and-a-half-dimensional (2.5D) and three-dimensional (3D). Each one, although being a simplified model of the in vivo conditions, represented an important step in the process toward the development of a valuable and effective human model for the study of pancreatic cancer. Moreover, all the here designed and fabricated models constitute, for different reasons, innovative approaches that go beyond the state-of-the-art in cancer research. • The 2D model, composed by transwell inserts including a polycaprolactone/gelatin (PCL/Gel) electrospun membrane, allowed to preliminary study the reciprocal influence of different cell types (i.e., stromal and epithelial cells) and it was able to replicate the highest cytokines release and changes in cell morphology by fibroblasts co-cultured with epithelial cells overexpressing the KRAS oncogene which are also reported in vivo. • The information acquired using this simplified model were then transferred to a more complex 2.5D model, represented by a multilayer PDAC-on-chip system. This microfluidic device was designed to incorporate PDAC cells and a stromal cell-laden type I collagen hydrogel in the top and bottom layers, respectively. The use of a nanofibrous and biomimetic electrospun membrane inside the chip represents an innovation since it allowed to compartmentalize the microfluidic device and thus separate the cancer component from the stromal tissue. In this way, the effect of the inflammation stimuli on stromal cells was studied in a controlled and specific way. This 2.5D model permitted to perform tests (e.g., evaluation of cell resistance to chemotherapy) in a fast, medium-throughput and accessible manner. • The 3D models were obtained at different length scales by using distinct additive manufacturing approaches: fused deposition modeling (FDM), melt electrowriting (MEW) and volumetric bioprinting (VBP). These techniques allowed to design and develop innovative biomimetic 3D constructs that fully replicate the complex microanatomy of the human pancreatic gland, which was not yet been accurately reproduced in the currently available in vitro models. Specifically, these engineered platforms intrinsically provide the morphological cues that cells experience in vivo and can reproduce the inflammatory cascade typical of pancreatic cancer from the very early stages. The layer-by-layer techniques used in this thesis project allowed to obtain macro- and microscale models replicating the half-structure of the complex gland morphology. Specifically, the FDM model was used to preliminary assess the feasibility of reproducing the glandular structure by using a layer-by-layer approach and to monitor the fibroblasts viability on polycaprolactone printed structures over several weeks. Nevertheless, melt electrowriting (MEW) permitted to achieve better resolutions of the printed structures, that have dimensions about four times smaller than those of FDM constructs. These studies led us to proceed with the implementation of co-culture conditions only in MEW scaffolds. The biomimicry of this model was demonstrated in terms of (i) capability to recreate the compartmentalization of stroma and epithelium found in PDAC microenvironment and (ii) ability to mirror the fibroblasts inflammation process occurring during pathology development. The cutting-edge technique of volumetric bioprinting was here adopted to develop a 3D in vitro model at the microscale, resembling the physiological “closed” structure typical of the pancreatic gland. In particular, a gelatin metacrylate (GelMA) hydrogel was ad hoc prepared and loaded with human fibroblasts to mimic the stromal compartment. Healthy or KRAS-mutated human pancreatic ductal epithelial cells were then introduced inside the construct’s cavity to reproduce the exocrine tissue that evolves to neoplastic lesions during pancreatic carcinogenesis. The ability of VBP model in recapitulating the tumor-stroma interplay occurring in pancreatic cancer while also accurately reproducing the microanatomy of the exocrine gland was proved. In summary, the in vitro models devised in this PhD study offer compelling and potent instruments for developing novel diagnostic methodologies and for the rigorous screening and evaluation of therapeutical agents. Consequently, they stand as pivotal assets in advancing our understanding of the intricate mechanisms underlying PDAC and in pioneering innovative therapeutic approaches to fight pancreatic cancer.