Role of c-MET Inhibitors in Overcoming Drug Resistance in Spheroid Models of Primary Human Pancreatic Cancer and Stellate Cells.

Omidreza Firuzi,Rainer Heuchel,Godefridus J Peters,Ilaria Carnevale,Amir Avan,Thomas Schmidt,Pei Pei Che,Elisa Giovannetti,Mark Buijs,Matthias Löhr,Stefano Coppola,Giulia Mantini,Btissame El Hassouni,Luciano Saso,Niccola Funel

doi:10.3390/cancers11050638

Abstract

Pancreatic stellate cells (PSCs) are a key component of tumor microenvironment in pancreatic ductal adenocarcinoma (PDAC) and contribute to drug resistance. c-MET receptor tyrosine kinase activation plays an important role in tumorigenesis in different cancers including PDAC. In this study, effects of PSC conditioned medium (PCM) on c-MET phosphorylation (by immunocytochemistry enzyme-linked immunosorbent assay (ELISA)) and drug response (by sulforhodamine B assay) were investigated in five primary PDAC cells. In novel 3D-spheroid co-cultures of cyan fluorescence protein (CFP)-firefly luciferase (Fluc)-expressing primary human PDAC cells and green fluorescence protein (GFP)-expressing immortalized PSCs, PDAC cell growth and chemosensitivity were examined by luciferase assay, while spheroids’ architecture was evaluated by confocal microscopy. The highest phospho-c-MET expression was detected in PDAC5 and its subclone sorted for “stage specific embryonic antigen-4” (PDAC5 (SSEA4)). PCM of cells pre-incubated with PDAC conditioned medium, containing increased hepatocyte growth factor (HGF) levels, made PDAC cells significantly more resistant to gemcitabine, but not to c-MET inhibitors. Hetero-spheroids containing both PSCs and PDAC5 (SSEA4) cells were more resistant to gemcitabine compared to PDAC5 (SSEA4) homo-spheroids. However, c-MET inhibitors (tivantinib, PHA-665752 and crizotinib) were equally effective in both spheroid models. Experiments with primary human PSCs confirmed the main findings. In conclusion, we developed spheroid models to evaluate PSC–PDAC reciprocal interaction, unraveling c-MET inhibition as an important therapeutic option against drug resistant PDAC.

Highlights

With a five-year survival rate of only 8%, pancreatic ductal adenocarcinoma (PDAC) has the worst prognosis among major malignancies [1,2]
We successfully developed a 3D hetero-spheroid model consisting of the same primary PDAC cells and pancreatic stellate cell (PSC), in which we showed that the presence of PSCs significantly enhances the growth and gemcitabine resistance of PDAC5 (SSEA4) cells
PDAC cell lines have been orthotopically co-injected with PSCs in mice and it has been observed that tumor size and progression was significantly higher compared to mice that had been injected with PDAC cells alone [20,39]

Summary

Introduction

With a five-year survival rate of only 8%, pancreatic ductal adenocarcinoma (PDAC) has the worst prognosis among major malignancies [1,2]. One of the main causes for this dismal prognosis, which has only slightly changed in the past 50 years, is the inherent resistance of PDAC to available therapies. This prompts the re-evaluation of peculiar aspects of PDAC that may contribute to its chemoresistance and lead to almost invariable therapeutic failure [2,3,4]. The stroma, a collective term for extracellular matrix (ECM) and non-tumor cells, constitutes a major part of the tumor mass in PDAC and recent evidence has shown that in addition to serving as a physical barrier to drug delivery, it promotes tumor growth and metastasis [8,9,10]. There has been some debate initially on whether stroma restrains or supports PDAC cells [11], recent evidence clearly demonstrates the supportive role of stromal cells on PDAC growth and chemoresistance and suggests the usefulness of developing new stroma-targeted therapies [12,13,14]

Methods

Findings

Discussion

Conclusion