Engineered matrices reveal stiffness-mediated chemoresistance in patient-derived pancreatic cancer organoids.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by its fibrotic and stiff extracellular matrix. However, how the altered cell/extracellular-matrix signalling contributes to the PDAC tumour phenotype has been difficult to dissect. Here we design and engineer matrices that recapitulate the key hallmarks of the PDAC tumour extracellular matrix to address this knowledge gap. We show that patient-derived PDAC organoids from three patients develop resistance to several clinically relevant chemotherapies when cultured within high-stiffness matrices mechanically matched to in vivo tumours. Using genetic barcoding, we find that while matrix-specific clonal selection occurs, cellular heterogeneity is not the main driver of chemoresistance. Instead, matrix-induced chemoresistance occurs within a stiff environment due to the increased expression of drug efflux transporters mediated by CD44 receptor interactions with hyaluronan. Moreover, PDAC chemoresistance is reversible following transfer from high- to low-stiffness matrices, suggesting that targeting the fibrotic extracellular matrix may sensitize chemoresistant tumours. Overall, our findings support the potential of engineered matrices and patient-derived organoids for elucidating extracellular matrix contributions to human disease pathophysiology.

Cell Microencapsulation Within Engineered Hyaluronan Elastin-Like Protein (HELP) Hydrogels.

Three-dimensional cell encapsulation has rendered itself a staple in the tissue engineering field. Using recombinantly engineered, biopolymer-based hydrogels to encapsulate cells is especially promising due to the enhanced control and tunability it affords. Here, we describe in detail the synthesis of our hyaluronan (i.e., hyaluronic acid) and elastin-like protein (HELP) hydrogel system. In addition to validating the efficacy of our synthetic process, we also demonstrate the modularity of the HELP system. Finally, we show that cells can be encapsulated within HELP gels over a range of stiffnesses, exhibit strong viability, and respond to stiffness cues. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Elastin-like protein modification with hydrazine Basic Protocol 2: Nuclear magnetic resonance quantification of elastin-like protein modification with hydrazine Basic Protocol 3: Hyaluronic acid-benzaldehyde synthesis Basic Protocol 4: Nuclear magnetic resonance quantification of hyaluronic acid-benzaldehyde Basic Protocol 5: 3D cell encapsulation in hyaluronan elastin-like protein gels.