Functional analysis of GNA15 role in PDAC cells utilizing an in vitro biomaterial based 3D model.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy with a five-year survival rate of approximately 13%. Its poor prognosis is primarily due to rapid infiltrative growth and early metastatic dissemination. This study investigated the role of the Gα15 protein, encoded by the GNA15 gene, which is ectopically expressed in pancreatic ductal adenocarcinoma (PDAC) by transformed cells. Abnormal expression of GNA15 has been associated with poor prognosis. Given the critical role of the extracellular matrix (ECM) composition and mechanical properties in PDAC progression, transcriptomic data revealing that COL1A1 expression is modulated by GNA15 prompted further investigation. To explore GNA15’s influence on PDAC invasiveness, we developed a 3D biomaterial-based in vitro model composed of collagen type I (Col1) and hyaluronic acid (HA), two major components of the PDAC tumor microenvironment (TME). This model was designed to replicate key biophysical features of the TME and to assess differences in invasive behaviour between PANC-1 wild-type (WT) cells and GNA15-knockout clones, specifically clone 2 (Cl2) and clone 20 (Cl20). Immunofluorescence analysis on 2D and 3D cell culture confirmed that GNA15-knockout dramatically reduces COL1A1 expression. However, no significant impact was observed on the ability of PANC-1 to aggregate in diluted Matrigel. Among three hydrogels with distinct viscoelastic profiles, we found that only the hydrogel formulation with the highest viscous loss modulus supported PANC-1 invasion in Col1-HA matrix. Under this settings, WT invasive properties resulted comparable to GNA15-knockout clones. We conclude that neither GNA15 signaling, nor COL1A1 autocrine production are essential to allow PANC-1 cells to penetrate ECM.