PhD Candidate The University of Texas Austin Austin, Texas, United States
Introduction: In Pancreatic ductal adenocarcinoma (PDAC), cancer stem cells (CSCs) are thought to drive tumor initiation, progression, and recurrence, likely contributing to one of the highest mortality rates of any malignancy with a 5-year survival rate under 5%. Despite the breadth of resources devoted to developing effective therapeutics, there remains a significant gap in our understanding and ability to target the heterogeneous nature of this deadly malignancy. One key feature of the PDAC tumor microenvironment (TME) is the desmoplastic reaction, comprising a variety of cell types and extracellular matrix (ECM) components that greatly increase tissue stiffness. Researchers have begun to resolve the extracellular cues that shift stem cells (SCs) into a metastatic state. However, the site of tumor initiation is often mechanically distinct from that of the metastatic tumor microenvironment, which may contribute to the failure of current treatments. Our initial results demonstrate cellular “memory” after exposure to certain synthetic matrices. Therefore, we hypothesize that mechanical properties at the primary site drive epigenetic changes dictating pancreatic CSC behavior at the metastatic site.
Materials and
Methods: Stiffening of an alginate-Matrigel hydrogel (MVG, 4%) from normal to PDAC-mimetic stiffnesses of 0.2kPa and 2.0kPa, respectively, was achieved by varying the concentration of the divalent cation crosslinker calcium proportionally with the acidifier glucono-deltalactone (GDL). Storage moduli were confirmed by oscillatory rheometry. The Rheoplus software was used to take frequency sweep measurements from 0.05 to 500 rad/s with 5% initial strain. Pancreatic cancer Mia Paca 2 cells were cultured on 8.0μm transwell inserts coated with our matrix-mimetic alginate gels for 72 hrs before assessment of invasion to the well surface. Cells were also cultured for 4 days on Advanced Biomatrix CytoSoft substrates of 0.2kPa or 2kPa stiffness before exposure to the same of opposite stiffness for an additional 4 days (SoSo: Soft -> Soft, SoSt: Soft -> Stiff, etc). After each assay, cells were analyzed for differential gene and protein expression, 2D migration, and 3D invasion.
Results, Conclusions, and Discussions: Our initial results demonstrate cellular “memory” after exposure to certain synthetic matrices. Therefore, we hypothesize that mechanical properties at the primary site drive epigenetic changes dictating pancreatic CSC behavior at the metastatic site. With our alginate-based model, we have successfully achieved normal- and PDAC-mimetic stiffnesses of 0.2kPa and 2.0kPa. Mia Paca 2 on stiff gels exhibit fibroblast-like morphology, while those on soft gels maintain rounded morphology in cellular aggregates. Preliminary results demonstrate how the initial mechanical environment affects stemness and metastasis at the gene level, for example by the differential expression of EMT-associated genes. This research supports the narrative that TME stiffness can independently drive pancreatic CSC behavior and lays the foundation for further investigation of therapeutic strategies targeting the heterogeneous nature of PDAC.
