3Din vitromodeling of the exocrine pancreatic unit using tomographic volumetric bioprinting

1. Pancreatic ductal adenocarcinoma (PDAC) is the most frequent type of pancreatic cancer, one of the leading causes of cancer-related deaths worldwide. The first lesions associated with PDAC occur within the functional units of exocrine pancreas. The crosstalk between PDAC cells and stromal cells plays a key role in tumor progression. Thus, i n vitro , fully human models of the pancreatic cancer microenvironment are needed to foster the development of new, more effective therapies. However, it is challenging to make these models anatomically and functionally relevant. Here, we used tomographic volumetric bioprinting, a novel method to fabricate three-dimensional cell-laden constructs, to produce a portion of the complex convoluted exocrine pancreas in vitro . Human fibroblast-laden gelatin methacrylate-based pancreatic models were processed to reassemble the tubuloacinar structures of the exocrine pancreas and, then human pancreatic ductal epithelial (HPDE) cells overexpressing the KRAS oncogene (HPDE-KRAS) were seeded in the acinar lumen to reproduce the pathological exocrine pancreatic tissue. The growth and organization of HPDE cells within the structure was evaluated and the formation of a thin epithelium which covered the acini inner surfaces in a physiological way inside the 3D model was successfully demonstrated. Interestingly, immunofluorescence assays revealed a significantly higher expressions of alpha smooth muscle actin (α-SMA) vs. actin in the fibroblasts co-cultured with cancerous than with wild-type HPDE cells. Moreover, α-SMA expression increased with time, and it was found to be higher in fibroblasts that laid closer to HPDE cells than in those laying deeper into the model. Increased levels of interleukin (IL)-6 were also quantified in supernatants from co-cultures of stromal and HPDE-KRAS cells. These findings correlate with inflamed tumor-associated fibroblast behavior, thus being relevant biomarkers to monitor the early progression of the disease and to target drug efficacy. To our knowledge, this is the first demonstration of a 3D bioprinted portion of pancreas that recapitulates its true 3-dimensional microanatomy, and which shows tumor triggered inflammation.