From Transwells to organ-on-a-chip: design and development of an in vitro 3D model for studying the pancreatic ductal adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) has been recognized as one of the most aggressive and lethal types of cancer worldwide. The absence of specific symptoms in the early stages of the disease, combined with the rapid tumour growth and the limitations of the available imaging techniques, delays the diagnosis of PDAC, revealing it only when an advanced and usually metastasized stage has already been reached. At this point, surgical resection is too risky, and chemotherapy is not totally effective due to the strong resistance of the PDAC to common anti-tumoral drugs, giving patients a five-year survival rate of less than 10%. PDAC onsets from the progressive accumulation of genetic aberrations in the pancreatic ductal cells, that enhance cancer cell survival, proliferation, and immune evasion, while disrupting normal cell-cycle control. Among the altered cells, pancreatic stellate cells (PSCs) play a crucial role in PDAC progression depositing excessive collagen in the extracellular space and generating a dense and hostile tumour microenvironment (TME) characterized by hypoxia and limited drug response. Since, at present, efficient diagnostic tests and therapies to target the PDAC are still lacking, in vitro models have become essential to understand the progression of the disease, providing a reliable base on which perform drug screening studies. The main struggle of these systems is to replicate in vitro the complex mechanisms that happen in vivo, such as the co-presence in the tumour of different cell populations, the complex three-dimensional (3D) organization of the cancerous mass and the interaction between the tumour and its stroma. In this thesis work, we designed an in vitro 3D model to investigate the effects of the TME on the PDAC progression and drug response. Stroma was replicated by loading fibroblasts inside a collagen based hydrogel and a monolayer of pancreatic cells was cultured on a PCL and gelatine electrospun membrane that further improves the overall biomimicry. Preliminary tests performed on transwells inserts confirmed that a 3D environment is essential to mimic the reduced permeability of the tumour’s stroma, limiting the drug targeting. Then, hybrid lipid and polymeric nanoparticles (NPs) formulations were synthesized, showing no cytotoxicity at 0.5 mg/mL concentration for either healthy or tumour cells. From the internalization test, we assessed that even if the nanoparticle itself did not present any specific targeting, cancerous cells internalized more NPs than healthy ones, probably due to their increased metabolic activity. The platform was then miniaturized by designing a custom organ-on-a-chip. The device presents three concentric chambers: an external channel for media perfusion, an intermediate one to host the collagen gel, resembling the stroma, and a central chamber that allows for the insertion of the electrospun membrane, essential to provide a biomimetic environment, improving adhesion of pancreatic cells. The dimensions of the micropillars, placed in between the compartments, were optimized by performing confinement and diffusivity tests, which aimed to assess the non-spreading of the gel in the other channels and, at the same time, sufficient nutrients’ diffusion. The translation of the model into the chip could provide meaningful insights into the PDAC investigation, since the combination of a dynamic environment and the assessed multilayer model provides a high biomimetic platform able to mimic several crucial hallmarks of the in vivo PDAC.