Engineered matrices reveal stiffness-mediated chemoresistance in pancreatic cancer organoids

Introduction: Pancreatic ductal adenocarcinoma (PDAC) is the most prevalent form of pancreatic cancer, with 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.

In this work, we engineer a defined and tunable 3D matrix that mimics key biochemical and mechanical properties of the in vivo PDAC ECM and supports long-term culture and passaging of human patient-derived PDAC organoids in vitro. Using our engineered matrix, we identify previously untested causal relationships between PDAC chemoresistance and ECM signaling, which opens new directions of drug development for effective patient therapy.

Materials and

Methods: The in vivo PDAC ECM is characterized by the increased deposition of several matrix components including fibronectin and hyaluronan (HA). Increased matrix deposition also correlated with increased tumour stiffness (~2900 Pa) of freshly-resected human samples compared to normal pancreas tissue (~900 Pa). To test the hypothesis that PDAC ECM may drive chemoresistance through direct cell-ECM signaling, 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. Elastin-like protein (ELP) is a recombinant protein consisting of repeating elastin and fibronectin-derived sequences that include an integrin-binding RGD peptide. 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. Stiffness is tuned in this system by changing the polymer concentration and chemical crosslink density between benzaldehyde and hydrazine groups on the HA and ELP, respectively. These HELP matrices supported the robust culture of PDAC organoids from three patients to date. PDAC response to chemotherapy over time within soft vs. stiff HELP matrices was quantified through immunostaining, Western blot, flow cytometry, mRNA expression and inhibition studies.

Results, Conclusions, and Discussions: We observed that PDAC organoids grown in stiffer HELP matrices led to acquired resistance to gemcitabine drug treatment in three different patient cultures. Sustained exposure to stiffer matrices also led to increased function of drug efflux transporters (including ABCG2). Interestingly, long-term drug efflux inhibition (Ko143) only exacerbated chemoresistance in our model by inducing broad mRNA- and protein-level upregulation of efflux transporters. Our work suggests that increased expression of drug efflux transporters is mediated by CD44 receptor interactions with HA. 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. This drug resistant phenotype was also reversible following transfer of PDAC organoids from high to low stiffness matrices, suggesting combination therapies of anticancer agents and mechanotransduction inhibitors may be beneficial for overcoming chemoresistance. 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 high stiffness matrices. Human pancreatic stellate cells (hPaSCs) are stromal fibroblasts that contribute significantly to the PDAC microenvironment by secreting ECM and other 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.

Acknowledgements (Optional): This work was supported by funding from the National Institutes of Health (R01 EB027171 to S.C.H.; R01CA2515143, U01CA217851, and U54CA224081 to C.J.K.; U01CA217851 and DP1CA238296 to C.C.), Stand Up to Cancer and Cancer Research UK (C.J.K.), and the National Science Foundation (CBET 2033302 to S.C.H.). Flow cytometry data was collected on BD FACS Symphony A5 in the Shared FACS Facility obtained using NIH S10 Shared Instrument Grant (1S10OD026831-01). Part of this work was performed at the Stanford Nano Shared Facilities (SNSF), supported by the National Science Foundation under award ECCS-2026822.