Differential Gemcitabine Sensitivity in Primary Human Pancreatic Cancer Cells and Paired Stellate Cells Is Driven by Heterogenous Drug Uptake and Processing
Abstract
:Simple Summary
Abstract
1. Introduction
2. Results
2.1. Human PDAC-Derived Primary PCCs Are Chemosensitive Whereas PSCs Are Resistant to Gemcitabine
2.2. Gemcitabine Uptake Is Minimal in PSCs and Significantly Higher in PCCs
2.3. High Variability in the Fate of Gemcitabine Following Its Uptake in Both PCCs and PSCs
2.4. Expression Analysis of Key Determinants of Gemcitabine Uptake and Metabolism
2.5. Gemcitabine-Induced Cytotoxicity in Pancreatic Cancer Cells Is Primarily Determined by hENT1 and DCK Expression
2.6. Correlation between Gemcitabine Chemosensitivity and Its Intracellular Metabolites or Expression of Key Regulator Proteins
3. Discussion
4. Material and Methods
4.1. Reagents
4.2. Cell Culture and Patient Information
4.3. Chemosensitivity
4.4. Gemcitabine Uptake
4.5. Western Blot Analysis
4.6. LC-MS/MS Analysis
4.7. Histology, Immunohistochemistry and Immunofluorescence
4.8. Transient Transfections (RNAi Interference)
4.9. Statistical Analysis
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Conflicts of Interest
Abbreviations
CDA | Cytidine deaminase |
DCK | Deoxycytidine kinase |
DCTD | Deoxycytidylate deaminase |
dFdC | 2′,2′-Difluoro deoxycytidine |
dFdCDP | 2′,2′-Difluoro deoxycytidine- 5′-diphosphate |
dFdCTP | 2′,2′-Difluoro deoxycytidine-5′-triphosphate |
dFdU | 2′,2′-Difluorodeoxyuridine |
hENT1 | Human equilibrative nucleoside transporter 1 |
LC-MS/MS | Liquid chromatography tandem mass spectrometry |
MTT | 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide |
NBMPR | S-(4-Nitrobenzyl)-6-thioinosine |
NTC | Negative transfection control |
NT5C1A | 5′-Nucleotidase cytosolic 1A |
PC | Pancreatic cancer |
PCC | Pancreatic cancer cell |
PDAC | Pancreatic ductal adenocarcinoma |
PSC | Pancreatic stellate cell |
PSC-CM | PSC-conditioned medium |
TME | Tumor microenvironment |
References
- Kamisawa, T.; Wood, L.D.; Itoi, T.; Takaori, K. Pancreatic cancer. Lancet 2016, 388, 73–85. [Google Scholar] [CrossRef]
- Kleeff, J.; Korc, M.; Apte, M.; La Vecchia, C.; Johnson, C.D.; Biankin, A.V.; Neale, R.E.; Tempero, M.; Tuveson, D.A.; Hruban, R.H.; et al. Pancreatic cancer. Nat. Rev. Dis. Primers 2016, 2, 16022. [Google Scholar] [CrossRef] [PubMed]
- Labori, K.J.; Katz, M.H.; Tzeng, C.W.; Bjørnbeth, B.A.; Cvancarova, M.; Edwin, B.; Kure, E.H.; Eide, T.J.; Dueland, S.; Buanes, T.; et al. Impact of early disease progression and surgical complications on adjuvant chemotherapy completion rates and survival in patients undergoing the surgery first approach for resectable pancreatic ductal adenocarcinoma—A population-based cohort study. Acta Oncol. 2016, 55, 265–277. [Google Scholar] [CrossRef] [PubMed]
- Burris, H.A.; Moore, M.J.; Andersen, J.; Green, M.R.; Rothenberg, M.L.; Modiano, M.R.; Cripps, M.C.; Portenoy, R.K.; Storniolo, A.M.; Tarassoff, P.; et al. Improvements in survival and clinical benefit with gemcitabine as first-line therapy for patients with advanced pancreas cancer: A randomized trial. J. Clin. Oncol. 1997, 15, 2403–2413. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Wong, A.; Soo, R.A.; Yong, W.-P.; Innocenti, F. Clinical pharmacology and pharmacogenetics of gemcitabine. Drug Metab. Rev. 2009, 41, 77–88. [Google Scholar] [CrossRef]
- Conroy, T.; Hammel, P.; Hebbar, M.; Ben Abdelghani, M.; Wei, A.C.; Raoul, J.L.; Chone, L.; Francois, E.; Artru, P.; Biagi, J.J.; et al. Folfirinox or gemcitabine as adjuvant therapy for pancreatic cancer. N. Engl. J. Med. 2018, 379, 2395–2406. [Google Scholar] [CrossRef]
- Conroy, T.; Desseigne, F.; Ychou, M.; Bouché, O.; Guimbaud, R.; Bécouarn, Y.; Adenis, A.; Raoul, J.L.; Gourgou-Bourgade, S.; de la Fouchardière, C.; et al. Folfirinox versus gemcitabine for metastatic pancreatic cancer. N. Engl. J. Med. 2011, 364, 1817–1825. [Google Scholar] [CrossRef] [Green Version]
- Von Hoff, D.D.; Ervin, T.; Arena, F.P.; Chiorean, E.G.; Infante, J.; Moore, M.; Seay, T.; Tjulandin, S.A.; Ma, W.W.; Saleh, M.N.; et al. Increased survival in pancreatic cancer with nab-paclitaxel plus gemcitabine. N. Engl. J. Med. 2013, 369, 1691–1703. [Google Scholar] [CrossRef] [Green Version]
- Amrutkar, M.; Gladhaug, I.P. Pancreatic cancer chemoresistance to gemcitabine. Cancers 2017, 9, 157. [Google Scholar] [CrossRef] [Green Version]
- Kim, M.P.; Gallick, G.E. Gemcitabine resistance in pancreatic cancer: Picking the key players. Clin. Cancer Res. Off. J. Am. Assoc. Cancer Res. 2008, 14, 1284–1285. [Google Scholar] [CrossRef] [Green Version]
- Neesse, A.; Michl, P.; Frese, K.K.; Feig, C.; Cook, N.; Jacobetz, M.A.; Lolkema, M.P.; Buchholz, M.; Olive, K.P.; Gress, T.M.; et al. Stromal biology and therapy in pancreatic cancer. Gut 2011, 60, 861–868. [Google Scholar] [CrossRef]
- Kadaba, R.; Birke, H.; Wang, J.; Hooper, S.; Andl, C.D.; Di Maggio, F.; Soylu, E.; Ghallab, M.; Bor, D.; Froeling, F.E.; et al. Imbalance of desmoplastic stromal cell numbers drives aggressive cancer processes. J. Pathol. 2013, 230, 107–117. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Pothula, S.P.; Xu, Z.; Goldstein, D.; Pirola, R.C.; Wilson, J.S.; Apte, M.V. Key role of pancreatic stellate cells in pancreatic cancer. Cancer Lett. 2016, 381, 194–200. [Google Scholar] [CrossRef] [PubMed]
- McCarroll, J.A.; Naim, S.; Sharbeen, G.; Russia, N.; Lee, J.; Kavallaris, M.; Goldstein, D.; Phillips, P.A. Role of pancreatic stellate cells in chemoresistance in pancreatic cancer. Front. Physiol. 2014, 5, 141. [Google Scholar] [CrossRef] [Green Version]
- Amrutkar, M.; Aasrum, M.; Verbeke, C.S.; Gladhaug, I.P. Secretion of fibronectin by human pancreatic stellate cells promotes chemoresistance to gemcitabine in pancreatic cancer cells. BMC Cancer 2019, 19, 596. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Dalin, S.; Sullivan, M.R.; Lau, A.N.; Grauman-Boss, B.; Mueller, H.S.; Kreidl, E.; Fenoglio, S.; Luengo, A.; Lees, J.A.; Vander Heiden, M.G.; et al. Deoxycytidine release from pancreatic stellate cells promotes gemcitabine resistance. Cancer Res. 2019, 79, 5723–5733. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Halbrook, C.J.; Pontious, C.; Kovalenko, I.; Lapienyte, L.; Dreyer, S.; Lee, H.J.; Thurston, G.; Zhang, Y.; Lazarus, J.; Sajjakulnukit, P.; et al. Macrophage-released pyrimidines inhibit gemcitabine therapy in pancreatic cancer. Cell Metab. 2019, 29, 1390–1399.e1396. [Google Scholar] [CrossRef] [PubMed]
- Principe, D.R.; Narbutis, M.; Kumar, S.; Park, A.; Viswakarma, N.; Dorman, M.J.; Kamath, S.D.; Grippo, P.J.; Fishel, M.L.; Hwang, R.F.; et al. Long-term gemcitabine treatment reshapes the pancreatic tumor microenvironment and sensitizes murine carcinoma to combination immunotherapy. Cancer Res. 2020, 80, 3101–3115. [Google Scholar] [CrossRef] [Green Version]
- Hessmann, E.; Patzak, M.S.; Klein, L.; Chen, N.; Kari, V.; Ramu, I.; Bapiro, T.E.; Frese, K.K.; Gopinathan, A.; Richards, F.M.; et al. Fibroblast drug scavenging increases intratumoural gemcitabine accumulation in murine pancreas cancer. Gut 2018, 67, 497–507. [Google Scholar] [CrossRef]
- Patzak, M.S.; Kari, V.; Patil, S.; Hamdan, F.H.; Goetze, R.G.; Brunner, M.; Gaedcke, J.; Kitz, J.; Jodrell, D.I.; Richards, F.M.; et al. Cytosolic 5′-nucleotidase 1a is overexpressed in pancreatic cancer and mediates gemcitabine resistance by reducing intracellular gemcitabine metabolites. EBioMedicine 2019, 40, 394–405. [Google Scholar] [CrossRef] [Green Version]
- Maity, G.; Ghosh, A.; Gupta, V.; Haque, I.; Sarkar, S.; Das, A.; Dhar, K.; Bhavanasi, S.; Gunewardena, S.S.; Von Hoff, D.D.; et al. Cyr61/ccn1 regulates dck and ctgf and causes gemcitabine-resistant phenotype in pancreatic ductal adenocarcinoma. Mol. Cancer Ther. 2019, 18, 788–800. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Mini, E.; Nobili, S.; Caciagli, B.; Landini, I.; Mazzei, T. Cellular pharmacology of gemcitabine. Ann. Oncol. 2006, 17 (Suppl. 5), v7–v12. [Google Scholar] [CrossRef]
- Bjanes, T.K.; Jordheim, L.P.; Schjott, J.; Kamceva, T.; Cros-Perrial, E.; Langer, A.; Ruiz de Garibay, G.; Kotopoulis, S.; McCormack, E.; Riedel, B. Intracellular cytidine deaminase regulates gemcitabine metabolism in pancreatic cancer cell lines. Drug Metab. Dispos. 2020, 48, 153–158. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Frese, K.K.; Neesse, A.; Cook, N.; Bapiro, T.E.; Lolkema, M.P.; Jodrell, D.I.; Tuveson, D.A. Nab-paclitaxel potentiates gemcitabine activity by reducing cytidine deaminase levels in a mouse model of pancreatic cancer. Cancer Discov. 2012, 2, 260–269. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Maréchal, R.; Bachet, J.B.; Mackey, J.R.; Dalban, C.; Demetter, P.; Graham, K.; Couvelard, A.; Svrcek, M.; Bardier-Dupas, A.; Hammel, P.; et al. Levels of gemcitabine transport and metabolism proteins predict survival times of patients treated with gemcitabine for pancreatic adenocarcinoma. Gastroenterology 2012, 143, 664–674.e666. [Google Scholar] [CrossRef] [PubMed]
- Greenhalf, W.; Ghaneh, P.; Neoptolemos, J.P.; Palmer, D.H.; Cox, T.F.; Lamb, R.F.; Garner, E.; Campbell, F.; Mackey, J.R.; Costello, E.; et al. Pancreatic cancer hent1 expression and survival from gemcitabine in patients from the espac-3 trial. J. Natl. Cancer Inst. 2014, 106, djt347. [Google Scholar] [CrossRef]
- Weizman, N.; Krelin, Y.; Shabtay-Orbach, A.; Amit, M.; Binenbaum, Y.; Wong, R.J.; Gil, Z. Macrophages mediate gemcitabine resistance of pancreatic adenocarcinoma by upregulating cytidine deaminase. Oncogene 2014, 33, 3812–3819. [Google Scholar] [CrossRef] [Green Version]
- Deer, E.L.; González-Hernández, J.; Coursen, J.D.; Shea, J.E.; Ngatia, J.; Scaife, C.L.; Firpo, M.A.; Mulvihill, S.J. Phenotype and genotype of pancreatic cancer cell lines. Pancreas 2010, 39, 425–435. [Google Scholar] [CrossRef] [Green Version]
- Cros, J.; Raffenne, J.; Couvelard, A.; Pote, N. Tumor heterogeneity in pancreatic adenocarcinoma. Pathobiology 2018, 85, 64–71. [Google Scholar] [CrossRef]
- Ku, J.L.; Yoon, K.A.; Kim, W.H.; Jang, Y.; Suh, K.S.; Kim, S.W.; Park, Y.H.; Park, J.G. Establishment and characterization of four human pancreatic carcinoma cell lines. Genetic alterations in the tgfbr2 gene but not in the madh4 gene. Cell Tissue Res. 2002, 308, 205–214. [Google Scholar] [CrossRef]
- Rückert, F.; Aust, D.; Böhme, I.; Werner, K.; Brandt, A.; Diamandis, E.P.; Krautz, C.; Hering, S.; Saeger, H.D.; Grützmann, R.; et al. Five primary human pancreatic adenocarcinoma cell lines established by the outgrowth method. J. Surg. Res. 2012, 172, 29–39. [Google Scholar] [CrossRef] [PubMed]
- Kim, M.J.; Kim, M.S.; Kim, S.J.; An, S.; Park, J.; Park, H.; Lee, J.H.; Song, K.B.; Hwang, D.W.; Chang, S.; et al. Establishment and characterization of 6 novel patient-derived primary pancreatic ductal adenocarcinoma cell lines from korean pancreatic cancer patients. Cancer Cell Int. 2017, 17, 47. [Google Scholar] [CrossRef] [PubMed]
- Lenggenhager, D.; Amrutkar, M.; Sántha, P.; Aasrum, M.; Löhr, J.M.; Gladhaug, I.P.; Verbeke, C.S. Commonly used pancreatic stellate cell cultures differ phenotypically and in their interactions with pancreatic cancer cells. Cells 2019, 8, 23. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Amrutkar, M.; Larsen, E.K.; Aasrum, M.; Finstadsveen, A.V.; Andresen, P.A.; Verbeke, C.S.; Gladhaug, I.P. Establishment and characterization of paired primary cultures of human pancreatic cancer cells and stellate cells derived from the same tumor. Cells 2020, 9, 227. [Google Scholar] [CrossRef] [Green Version]
- Mackey, J.R.; Mani, R.S.; Selner, M.; Mowles, D.; Young, J.D.; Belt, J.A.; Crawford, C.R.; Cass, C.E. Functional nucleoside transporters are required for gemcitabine influx and manifestation of toxicity in cancer cell lines. Cancer Res. 1998, 58, 4349–4357. [Google Scholar]
- Nakano, Y.; Tanno, S.; Koizumi, K.; Nishikawa, T.; Nakamura, K.; Minoguchi, M.; Izawa, T.; Mizukami, Y.; Okumura, T.; Kohgo, Y. Gemcitabine chemoresistance and molecular markers associated with gemcitabine transport and metabolism in human pancreatic cancer cells. Br. J. Cancer 2007, 96, 457–463. [Google Scholar] [CrossRef]
- Kurata, N.; Fujita, H.; Ohuchida, K.; Mizumoto, K.; Mahawithitwong, P.; Sakai, H.; Onimaru, M.; Manabe, T.; Ohtsuka, T.; Tanaka, M. Predicting the chemosensitivity of pancreatic cancer cells by quantifying the expression levels of genes associated with the metabolism of gemcitabine and 5-fluorouracil. Int J. Oncol. 2011, 39, 473–482. [Google Scholar]
- Wang, W.Q.; Liu, L.; Xu, J.Z.; Yu, X.J. Reflections on depletion of tumor stroma in pancreatic cancer. Biochim. Biophys. Acta Rev. Cancer 2019, 1871, 267–272. [Google Scholar] [CrossRef]
- De Sousa Cavalcante, L.; Monteiro, G. Gemcitabine: Metabolism and molecular mechanisms of action, sensitivity and chemoresistance in pancreatic cancer. Eur. J. Pharmacol. 2014, 741, 8–16. [Google Scholar] [CrossRef]
- Saiki, Y.; Yoshino, Y.; Fujimura, H.; Manabe, T.; Kudo, Y.; Shimada, M.; Mano, N.; Nakano, T.; Lee, Y.; Shimizu, S.; et al. Dck is frequently inactivated in acquired gemcitabine-resistant human cancer cells. Biochem. Biophys. Res. Commun. 2012, 421, 98–104. [Google Scholar] [CrossRef]
- Costantino, C.L.; Witkiewicz, A.K.; Kuwano, Y.; Cozzitorto, J.A.; Kennedy, E.P.; Dasgupta, A.; Keen, J.C.; Yeo, C.J.; Gorospe, M.; Brody, J.R. The role of hur in gemcitabine efficacy in pancreatic cancer: Hur up-regulates the expression of the gemcitabine metabolizing enzyme deoxycytidine kinase. Cancer Res. 2009, 69, 4567–4572. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Lamba, J.K.; Crews, K.; Pounds, S.; Schuetz, E.G.; Gresham, J.; Gandhi, V.; Plunkett, W.; Rubnitz, J.; Ribeiro, R. Pharmacogenetics of deoxycytidine kinase: Identification and characterization of novel genetic variants. J. Pharmacol. Exp. Ther. 2007, 323, 935–945. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Kong, K.; Guo, M.; Liu, Y.; Zheng, J. Progress in animal models of pancreatic ductal adenocarcinoma. J. Cancer 2020, 11, 1555–1567. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Krempley, B.D.; Yu, K.H. Preclinical models of pancreatic ductal adenocarcinoma. Chin. Clin. Oncol. 2017, 6, 25. [Google Scholar] [CrossRef]
- Awaji, M.; Singh, R.K. Cancer-associated fibroblasts’ functional heterogeneity in pancreatic ductal adenocarcinoma. Cancers 2019, 11, 290. [Google Scholar] [CrossRef] [Green Version]
- Öhlund, D.; Handly-Santana, A.; Biffi, G.; Elyada, E.; Almeida, A.S.; Ponz-Sarvise, M.; Corbo, V.; Oni, T.E.; Hearn, S.A.; Lee, E.J.; et al. Distinct populations of inflammatory fibroblasts and myofibroblasts in pancreatic cancer. J. Exp. Med. 2017, 214, 579–596. [Google Scholar] [CrossRef]
- Huelsken, J.; Hanahan, D. A subset of cancer-associated fibroblasts determines therapy resistance. Cell 2018, 172, 643–644. [Google Scholar] [CrossRef] [Green Version]
- Pereira, B.A.; Vennin, C.; Papanicolaou, M.; Chambers, C.R.; Herrmann, D.; Morton, J.P.; Cox, T.R.; Timpson, P. Caf subpopulations: A new reservoir of stromal targets in pancreatic cancer. Trends Cancer 2019, 5, 724–741. [Google Scholar] [CrossRef] [Green Version]
- Bachem, M.G.; Schneider, E.; Gross, H.; Weidenbach, H.; Schmid, R.M.; Menke, A.; Siech, M.; Beger, H.; Grunert, A.; Adler, G. Identification, culture, and characterization of pancreatic stellate cells in rats and humans. Gastroenterology 1998, 115, 421–432. [Google Scholar] [CrossRef]
- Bapiro, T.E.; Richards, F.M.; Goldgraben, M.A.; Olive, K.P.; Madhu, B.; Frese, K.K.; Cook, N.; Jacobetz, M.A.; Smith, D.M.; Tuveson, D.A.; et al. A novel method for quantification of gemcitabine and its metabolites 2′,2′-difluorodeoxyuridine and gemcitabine triphosphate in tumour tissue by lc-ms/ms: Comparison with (19)f nmr spectroscopy. Cancer Chemother. Pharmacol. 2011, 68, 1243–1253. [Google Scholar] [CrossRef] [Green Version]
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. |
© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
Share and Cite
Amrutkar, M.; Vethe, N.T.; Verbeke, C.S.; Aasrum, M.; Finstadsveen, A.V.; Sántha, P.; Gladhaug, I.P. Differential Gemcitabine Sensitivity in Primary Human Pancreatic Cancer Cells and Paired Stellate Cells Is Driven by Heterogenous Drug Uptake and Processing. Cancers 2020, 12, 3628. https://doi.org/10.3390/cancers12123628
Amrutkar M, Vethe NT, Verbeke CS, Aasrum M, Finstadsveen AV, Sántha P, Gladhaug IP. Differential Gemcitabine Sensitivity in Primary Human Pancreatic Cancer Cells and Paired Stellate Cells Is Driven by Heterogenous Drug Uptake and Processing. Cancers. 2020; 12(12):3628. https://doi.org/10.3390/cancers12123628
Chicago/Turabian StyleAmrutkar, Manoj, Nils Tore Vethe, Caroline S. Verbeke, Monica Aasrum, Anette Vefferstad Finstadsveen, Petra Sántha, and Ivar P. Gladhaug. 2020. "Differential Gemcitabine Sensitivity in Primary Human Pancreatic Cancer Cells and Paired Stellate Cells Is Driven by Heterogenous Drug Uptake and Processing" Cancers 12, no. 12: 3628. https://doi.org/10.3390/cancers12123628