Can TGF-β Differentiate Fibroblasts and Endothelial Cells into CAFs?: A Research Protocol

Abstract

Introduction: Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal forms of cancer in Canada. Tumour metastasis contributes to most of the deaths, a process heavily influenced by cancer-associated fibroblasts (CAFs). While the functions of CAFs have been widely researched, such as their metastasis-promoting secretion of growth factors, their origins remain unclear. This research protocol, therefore, seeks to confirm that PDAC cells are capable of differentiating both fibroblasts and endothelial cells into CAFs by secreting transforming growth-factor beta (TGF-β). Methods: The effects of culturing Hs68 fibroblasts and HMEC-1 endothelial cells in media containing TGF-β will be examined using media supplemented with TGF-β and conditioned media obtained from PANC-1 cells. To confirm these results, TGF-β receptor-inhibited cells will be included also. Proliferation assays, migration assays, RT-qPCR, and western blotting will then be used to determine successful differentiation into CAFs. Results: It is expected that the presence of TGF-β in culture media will lead to the increased proliferation, migration, and presence of CAF cell markers within the cell culture. The inhibited conditions grown in standard media with the added factor are expected to be comparable to their control groups. The same is expected of the inhibited HMEC-1 cells grown in PANC-1 conditioned media, however the Hs68 culture should more closely resemble its uninhibited condition. Discussion: The increased results described above for the uninhibited conditions grown in TGF-β-containing media would indicate the following; that this factor is capable of differentiating Hs68 and HMEC-1 cells into CAFs, and that PANC-1 cells are capable of initiating this change. This would be confirmed by the lack of difference between the inhibited versus control conditions; showing that this secreted factor is indeed responsible for these effects. Conclusion: The results from this protocol will help to solidify fibroblasts and endothelial cells as origins of CAFs, and TGF-β as a CAF-generating factor. By knowing more about their origin, the development of new potential drugs that target the formation of TGF-β is possible. Further directions could include the possibility of in vivo experiments confirming the results of this protocol.