Introduction: Pancreatic cancer has the lowest survival rate among the common cancers, mainly due to its symptom-free progression and the advanced stage of the disease at the time of diagnosis. The response to current treatment strategies is low, mainly due to the extensive desmoplasia and fibrotic extracellular matrix (ECM), which acts as a barrier against efficient drug delivery.
One distinctive feature of the pancreatic tumor microenvironment (TME) is the extensive remodeling and stiffening of the matrix, mainly due to the excessive production of ECM proteins . 3-dimensional (3D) in vitro platforms proved to be valuable tools in studying the dynamics of TME and its remodeling during disease progression. However, current 3D in vitro models of pancreatic cancer usually overlook the dynamics of matrix remodeling during the autonomous cell organization . This study aimed to evaluate the dynamics of matrix remodeling during the growth and autonomous organization of pancreatic cancer cells in a protease-sensitive and degradable hydrogel matrix based on starPEG-Heparin with defined biochemical and physical properties .
Methodology: The starPEG-Heparin hydrogels were designed to provide adequate initial stiffness according to the published datasets from patient-derived tissues . The incorporation of protease-sensitive peptides provided the dynamic degradability of hydrogels throughout the 3D culture. RGD-peptide sequences were incorporated into the hydrogel matrix, enabling integrin-mediated cellular adhesion. Oscillatory shear measurements and atomic force microscopy (AFM) were used to evaluate the mechanical properties of the hydrogels. Pancreatic cancer cells, together with stromal cells including cancer-associated fibroblasts (CAFs) and immune cells, were grown encapsulated into hydrogels and cultured for 14 days. The dynamics of stiffness remodeling in these multicellular cultures were evaluated by AFM and compared to mono-cultures containing only pancreatic cancer cells.
Results: The initial stiffness of the hydrogel matrices was set to be in a range with the median value of ~15 kPa, by changing the degree of crosslinking. AFM data showed that while hydrogels with mono- and multicellular-cultured cell populations have comparable stiffnesses at the beginning of the culture period, both cell populations contributed to a progressive stiffness reduction after 14 days of culture. Cells organized themselves autonomously within the hydrogels by forming spheroids, followed by the degradation of the hydrogel matrix. However, the size and the number of spheroids in mono-cultures were significantly less than in multicellular cultures. Compared to the initial matrix, the extent of remodeling was significantly less in mono-cultures. Notably, the stiffness of the matrix distant from spheroids in multicellular cultures was significantly lower after 14 days of culture compared to the beginning of the culture period. In contrast, the stiffness in the vicinity of spheroids was considerably larger, suggesting the production and accumulation of newly synthesized ECM.
Conclusions: The spontaneous organization of cancer cells in hydrogels to form cellular aggregates is observed in many in vitro 3D cancer models. Our results suggest that remodeling degradable starPEG-Heparin hydrogels by pancreatic cancer cells is a heterogeneous process. In the presence of stromal cell populations, the intracellular interactions could produce newly formed ECM, profiling the stiffness of cancer tissue.