PhD Scientific Days 2021

MO_II_L: Molecular Sciences II. Lectures

Regulating extracellular vesicle release from pancreatic ductal adenocarcinoma

Text of the abstract

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest cancers. PDAC cells can harbour various mutations that lead to intratumoral cellular heterogeneity. Patient-derived organoids maintain cellular heterogeneity of the original tissues so they are one of the best models of cancers. PDAC cells positive for the Wnt-target CD44 and CD133 show a more aggressive phenotype. A PDAC organoid library highlighted the importance of Wnt proteins in PDAC progression produced by tumor or stromal cells. Extracellular vesicles (EV) are membrane-surrounded particles and represent a novel way of intercellular communication. Although EVs hold a great promise for early cancer diagnostics, however factors modifying their release are not well known.
Since we found that increased Wnt activation and cell proliferation were coupled to a higher EV secretion in intestinal adenomas, here we tested this mechanism in PDAC.
We cultured PDAC patient-derived organoids in Matrigel as 3D matrix. We detected gene expression changes with RT-qPCR, whole-mount immunostaining and flow cytometry. EVs were detected with Nanoparticle Tracking Analysis.
All of our PDAC organoid lines showed heterogeneity for CD44 and CD133, but organoid cells sorted for different expression levels of CD44 and CD133 did not maintain this expression pattern. We collected RNA from organoids after sorting and measured the expression level of genes involved in the epithelial-mesenchymal transition, however no difference was found between CD44low-CD44high and CD133low-CD133high tumor cells. For further investigation of Wnt-signalling, we examined the Wnt dependency and Wnt secretion of organoids. All of our organoids were independent of external Wnt proteins and they expressed PORCN, an enzyme necessary for Wnt secretion. Organoids expressed epithelial Wnt genes but we could not detect the stromal Wnt5a. PORCN inhibitor resulted in a decreased expression of Wnt target genes in organoids, suggesting the responsiveness of PDAC cells for Wnt. Surprisingly, we found no change in the percentage of proliferating cells and EV secretion when blocking Wnt secretion.
Unlike intestinal tumors, we found that Wnt activity, cell proliferation and EV release are uncoupled in PDAC. Furthermore, we found a high plasticity of PDAC tumor cells in their CD44 and CD133 expression pattern.
This study was supported by ÚNKP-20-3-I-SE-4

University and Doctoral School

Semmelweis University, Doctoral School of Molecular Medicine