Advanced engineering methods for investigating the mechanotransduction mechanisms of pancreatic ductal adenocarcinoma: characterization of substrate stiffness by nanoindentation and of cell traction forces by micropillar arrays

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive disease of the exocrine pancreas with poor prognosis. Its abundant stroma is largely composed of activated pancreatic stellate cells, responsible for the synthesis of great amounts of extracellular matrix (ECM) proteins. The high deposition of ECM components causes an increase in tissue rigidity and interstitial fluid pressure (from ~1 kPa in healthy tissue to ~10-17 kPa in tumoral tissue), while reducing tissue permeability. Thus, treating the PDAC with conventional approaches like chemotherapy and immunotherapy gives poor results. Cancer and stroma cells sense the mechanical cues of the tumor environment through mechanosensitive membrane proteins, prominently ion channels involved in the Ca2+-signaling of cells, and their crucial role in integrating signals makes them good therapeutic target candidates. To understand how ECM stiffness regulates cell mechanotransduction signaling and tumor progression, there is an urgent need for in vitro investigation of the behavior of pancreatic cancer cells when cultured on substrates with tunable mechanical properties. With the purpose of mimicking the stiffness of healthy and tumoral pancreatic environment, two polyacrylamide substrates with low (PAM low) and high (PAM high) stiffness (fabricated at the Laboratory of Cellular and Molecular Angiogenesis at DBIOS, University of Turin), with and without collagen coating, were mechanically characterized. Nanoindentation tests were performed in wet conditions (PBS), at 37°C, and using the PIUMA nanoindenter (Optics11, Netherlands). Through the analysis of the indentation curves, the effective elastic modulus Eeff was quantified. The obtained averaged Eeff values (0.56 ± 0.63 kPa for PAM low without collagen, 1.05 ± 0.76 kPa for PAM low with collagen, 18.79 ± 5.29 kPa for PAM high without collagen and 15.98 ± 5.08 kPa for PAM high with collagen) confirmed the suitability of PAM substrates to replicate the stiffness of healthy and tumoral pancreatic tissue. Furthermore, preliminary biological tests, carried out culturing human pancreatic cancer cells (PANC-1 cell line) on PAM substrates, revealed a higher motility and a reduced Piezo1-mediated Ca2+ signaling in cells seeded on PAM high substrates. Moreover, in order to characterize the cell traction forces developed by PDAC cells depending on the substrate stiffness, the micropillar array method was adopted. When micropillars are used as cell culture substrates, cells attach, spread, and bend the posts, which act as microcantilevers, and their deflections are proportional to the exerted traction forces. Thus, different micropillar arrays were designed for mimicking the mechanical properties of the PAM substrates and were then manufactured in polydimethylsiloxane (PDMS) and characterized at the Laboratory of Micro-Biomechanics of the Hokkaido University (Japan). The evaluation of the spring constant of the fabricated micropillars through geometrical parameter measurement and a finite element method analysis revealed that values were 3-7 times higher than the expected ones. However, preliminary cellular tests were carried out on the fabricated posts. In conclusion, adopting advanced engineering methods, such as nanoindentation and micropillar arrays, combined with biological tests made it possible to investigate the mechano-biological response of PDAC cells to substrates with different stiffnesses, increasing the knowledge in the field of mechanotransduction investigation of PDAC.