Vasohibin 2 expression correlates with malignant behavior of pancreatic cancer. We aimed to study the biological effect of VASH2 gene knockdown. Due to the fact, that vasohibin 2 in engaged in tumor microenvironment remodeling, the biological effect of VASH2 gene expression knockdown was evaluated in tumor associated macrophages.
Pancreatic cancer ductal adenocarcinoma cell line (PANC-1) and THP-1 derived M2-typr tumor associated macrophages were used in the study. Uptake of siRNA with tyrosine-modified PEIs as well as lysosomal escape was confirmed by confocal microscopy analysis. Gene knockdown and biological effect was studied by qPCR. Cell survival and proliferation were tested in both cell lines with MTT and WST-8 assay respectively.
Confocal microscopy analysis revealed that complexes of siRNA (siVASH2) and tyrosine-modified PEIs efficiently entered the cells. Moreover, nanoparticles escaped from lysosomes, the same preventing siRNA from early release and degradation. Cytotoxicity studies revealed that macrophages viability after 24-hours treatment with nanoparticles was about 80%. Proliferation rate of pancreatic cancer cells was reduced, depending on the type of polymer used in complex from 40 to 75% after 72-hour incubation. qPCR analysis confirmed reduced level of VASH2 in both cell types. In M2-type tumor associated macrophages decreased level of vasohibin 2 correlated with the decrease in CD206 – marker of M2-type macrophages. In pancreatic cancer cells decrease in E-cadherin and vimentin level was observed when VASH2 was targeted.
Our studies revealed that vasohibin 2 plays an important role in pancreatic cancer progression. It also affects tumor associated macrophages. There is no available vasohibin 2 inhibitor, thus, gene therapy seems to be a promising strategy for VASH2 inhibition. We have chosen tyrosine-modified PEIs as a siRNA carriers. Due to their low toxicity, good biocompatibility and high biological activity they should be considered as a gene carriers in tumor immunooncology gene therapies.
The authors.
National Science Centre, Poland (2020/37/N/NZ5/02359).
All authors have declared no conflicts of interest.