Differential Gemcitabine Sensitivity in Primary Human Pancreatic Cancer Cells and Paired Stellate Cells Is Driven by Heterogenous Drug Uptake and Processing

Manoj Amrutkar; Nils Tore Vethe; Caroline S Verbeke; Monica Aasrum; Anette Vefferstad Finstadsveen; Petra Sántha; Ivar P Gladhaug

doi:10.3390/cancers12123628

Differential Gemcitabine Sensitivity in Primary Human Pancreatic Cancer Cells and Paired Stellate Cells Is Driven by Heterogenous Drug Uptake and Processing

Cancers (Basel). 2020 Dec 3;12(12):3628. doi: 10.3390/cancers12123628.

Authors

Manoj Amrutkar^{1

2}, Nils Tore Vethe³, Caroline S Verbeke^{4

5}, Monica Aasrum¹, Anette Vefferstad Finstadsveen⁴, Petra Sántha⁴, Ivar P Gladhaug^{2

6}

Affiliations

¹ Department of Pharmacology, Institute of Clinical Medicine, University of Oslo, P.O. Box 1057 Blindern, 0316 Oslo, Norway.
² Department of Hepato-Pancreato-Biliary Surgery, Institute of Clinical Medicine, University of Oslo, P.O. Box 1171 Blindern, 0318 Oslo, Norway.
³ Department of Pharmacology, Oslo University Hospital Rikshospitalet, P.O. Box 4950 Nydalen, 0424 Oslo, Norway.
⁴ Department of Pathology, Oslo University Hospital Rikshospitalet, P.O. Box 4950 Nydalen, 0424 Oslo, Norway.
⁵ Department of Pathology, Institute of Clinical Medicine, University of Oslo, P.O. Box 1072 Blindern, 0316 Oslo, Norway.
⁶ Department of Hepato-Pancreato-Biliary Surgery, Oslo University Hospital Rikshospitalet, P.O. Box 4950 Nydalen, 0424 Oslo, Norway.

Abstract

Gemcitabine resistance in pancreatic ductal adenocarcinoma (PDAC) is attributed to cancer cell-intrinsic drug processing and the impact of the tumor microenvironment, especially pancreatic stellate cells (PSCs). This study uses human PDAC-derived paired primary cancer cells (PCCs) and PSCs from four different tumors, and the PDAC cell lines BxPC-3, Mia PaCa-2, and Panc-1, to assess the fate of gemcitabine by measuring its cellular uptake, cytotoxicity, and LC-MS/MS-based metabolite analysis. Expression analysis and siRNA-mediated knockdown of key regulators of gemcitabine (hENT1, CDA, DCK, NT5C1A) was performed. Compared to PSCs, both the paired primary PCCs and cancer cell lines showed gemcitabine-induced dose-dependent cytotoxicity, high uptake, as well as high and variable intracellular levels of gemcitabine metabolites. PSCs were gemcitabine-resistant and demonstrated significantly lower drug uptake, which was not influenced by co-culturing with their paired PCCs. Expression of key gemcitabine regulators was variable, but overall strong in the cancer cells and significantly lower or undetectable in PSCs. In cancer cells, hENT1 inhibition significantly downregulated gemcitabine uptake and cytotoxicity, whereas DCK knockdown reduced cytotoxicity. In conclusion, heterogeneity in gemcitabine processing among different pancreatic cancer cells and stellate cells results from the differential expression of molecular regulators which determines the effect of gemcitabine.

Keywords: gemcitabine uptake and metabolism; human pancreatic ductal adenocarcinoma; pancreatic stellate cells and cancer cells; primary cultures.

Abstract

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