(68c) Engineered Matrices Reveal Stiffness-Mediated Chemoresistance in Pancreatic Cancer Organoids

Pancreatic ductal adenocarcinoma (PDAC) is the most prevalent form of pancreatic cancer, exhibiting a 5-year survival rate of <10% and high resistance to chemotherapy. Frontline chemotherapeutics to treat PDAC are often ineffective due to acquired resistance. Understanding the mechanisms of how PDAC tumors develop and retain chemoresistance is critical to advancing impactful treatment strategies. Clinically, PDAC chemoresistance has been correlated with extreme fibrotic remodeling of the extracellular matrix (ECM). The PDAC ECM is characterized by its high stiffness and dense stroma, which worsens throughout disease progression. These clinical observations led us to hypothesize that the PDAC ECM may drive chemoresistance through direct cell-ECM signaling. To test this hypothesis, we developed a 3D in vitro model of patient-specific PDAC organoids grown within a tunable engineered matrix. Specifically, our family of engineered matrices is comprised of hyaluronan (HA) and elastin-like protein (termed HELP) and can be tuned to model the increased stiffness and hyaluronan content of the native tumor microenvironment. After performing mechanical measurements on freshly resected human tissues, we created a range of HELP matrices that mimic the high stiffness of PDAC tumors and the low stiffness of the healthy pancreas, while maintaining similar matrix biochemistry. These HELP matrices supported the robust culture of PDAC organoids from three patients to date. We observed that PDAC organoids grown in stiffer HELP matrices have resistance to gemcitabine drug treatment and increased function of drug efflux transporters (including ABCG2). Mechanistically, our work shows that CD44-HA interactions in high stiffness matrices lead to ABCG2 overexpression. We validated this through CD44 knockout studies (CRISPR/Cas9 gene editing) and efflux pump activity studies (flow cytometry). Interestingly, integrin signaling was not required for this phenotypic effect. Overall, our data strongly suggest PDAC organoids are mechanosensitive through the CD44 receptor and that mechanosignaling can play a direct role in driving PDAC drug sensitivity. Furthermore, we demonstrate the usefulness of tunable, engineered hydrogel platforms to elucidate how ECM properties influence patient-specific disease pathophysiology. Our ongoing work aims to add more complexity to this engineered tissue model by producing a co-culture of PDAC organoids with fibroblasts in both low and highs stiffness matrices. Human pancreatic stellate cells (hPaSCs) are stromal fibroblasts that have contribute significantly to the PDAC microenvironment by secreting ECM and biochemical factors. Morphological differences are observed in both cell types in low vs. high stiffness matrix co-cultures, alluding to stiffness-mediated changes in crosstalk between patient-derived PDAC organoids and hPaSCS.