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

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.

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Immune Cell Modulation of the Extracellular Matrix Contributes to the Pathogenesis of Pancreatic Cancer

Biomolecules ◽

10.3390/biom11060901 ◽

2021 ◽

Vol 11 (6) ◽

pp. 901

Author(s):

Ramiz S. Ahmad ◽

Timothy D. Eubank ◽

Slawomir Lukomski ◽

Brian A. Boone

Keyword(s):

Pancreatic Cancer ◽

Extracellular Matrix ◽

Immune Cells ◽

Immune Cell ◽

Treatment Strategies ◽

Stellate Cells ◽

Pancreatic Stellate Cells ◽

Ductal Adenocarcinoma ◽

Cellular Mechanisms ◽

Novel Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal malignancy with a five-year survival rate of only 9%. PDAC is characterized by a dense, fibrotic stroma composed of extracellular matrix (ECM) proteins. This desmoplastic stroma is a hallmark of PDAC, representing a significant physical barrier that is immunosuppressive and obstructs penetration of cytotoxic chemotherapy agents into the tumor microenvironment (TME). Additionally, dense ECM promotes hypoxia, making tumor cells refractive to radiation therapy and alters their metabolism, thereby supporting proliferation and survival. In this review, we outline the significant contribution of fibrosis to the pathogenesis of pancreatic cancer, with a focus on the cross talk between immune cells and pancreatic stellate cells that contribute to ECM deposition. We emphasize the cellular mechanisms by which neutrophils and macrophages, specifically, modulate the ECM in favor of PDAC-progression. Furthermore, we investigate how activated stellate cells and ECM influence immune cells and promote immunosuppression in PDAC. Finally, we summarize therapeutic strategies that target the stroma and hinder immune cell promotion of fibrogenesis, which have unfortunately led to mixed results. An enhanced understanding of the complex interactions between the pancreatic tumor ECM and immune cells may uncover novel treatment strategies that are desperately needed for this devastating disease.

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ITGA5 inhibition in pancreatic stellate cells attenuates desmoplasia and potentiates efficacy of chemotherapy in pancreatic cancer

Science Advances ◽

10.1126/sciadv.aax2770 ◽

2019 ◽

Vol 5 (9) ◽

pp. eaax2770 ◽

Cited By ~ 7

Author(s):

Praneeth R. Kuninty ◽

Ruchi Bansal ◽

Susanna W. L. De Geus ◽

Deby F. Mardhian ◽

Jonas Schnittert ◽

...

Keyword(s):

Therapeutic Potential ◽

Tumor Stroma ◽

Stellate Cells ◽

Pancreatic Stellate Cells ◽

Ductal Adenocarcinoma ◽

Tumor Penetration ◽

Desmoplastic Stroma ◽

Integrin Α5

Abundant desmoplastic stroma is the hallmark for pancreatic ductal adenocarcinoma (PDAC), which not only aggravates the tumor growth but also prevents tumor penetration of chemotherapy, leading to treatment failure. There is an unmet clinical need to develop therapeutic solutions to the tumor penetration problem. In this study, we investigated the therapeutic potential of integrin α5 (ITGA5) receptor in the PDAC stroma. ITGA5 was overexpressed in the tumor stroma from PDAC patient samples, and overexpression was inversely correlated with overall survival. In vitro, knockdown of ITGA5 inhibited differentiation of human pancreatic stellate cells (hPSCs) and reduced desmoplasia in vivo. Our novel peptidomimetic AV3 against ITGA5 inhibited hPSC activation and enhanced the antitumor effect of gemcitabine in a 3D heterospheroid model. In vivo, AV3 showed a strong reduction of desmoplasia, leading to decompression of blood vasculature, enhanced tumor perfusion, and thereby the efficacy of gemcitabine in co-injection and patient-derived xenograft tumor models.

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Hypoxia Inducible Factor 1 (HIF-1) Recruits Macrophage to Activate Pancreatic Stellate Cells in Pancreatic Ductal Adenocarcinoma

International Journal of Molecular Sciences ◽

10.3390/ijms17060799 ◽

2016 ◽

Vol 17 (6) ◽

pp. 799 ◽

Cited By ~ 29

Author(s):

Na Li ◽

Yang Li ◽

Zengxun Li ◽

Chongbiao Huang ◽

Yanhui Yang ◽

...

Keyword(s):

Pancreatic Ductal Adenocarcinoma ◽

Hypoxia Inducible Factor ◽

Stellate Cells ◽

Pancreatic Stellate Cells ◽

Ductal Adenocarcinoma ◽

Hypoxia Inducible Factor 1

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Proton Pump Inhibitors Reduce Pancreatic Adenocarcinoma Progression by Selectively Targeting H+, K+-ATPases in Pancreatic Cancer and Stellate Cells

Cancers ◽

10.3390/cancers12030640 ◽

2020 ◽

Vol 12 (3) ◽

pp. 640 ◽

Cited By ~ 5

Author(s):

Marco Tozzi ◽

Christiane E. Sørensen ◽

Lara Magni ◽

Nynne M. Christensen ◽

Rayhana Bouazzi ◽

...

Keyword(s):

Pancreatic Cancer ◽

Cell Proliferation ◽

Proton Pump Inhibitors ◽

Proton Pump ◽

Human Cancer ◽

Stellate Cells ◽

In Vivo Studies ◽

Pancreatic Stellate Cells ◽

Ductal Adenocarcinoma ◽

Collagen Secretion

Pancreatic duct cells are equipped with acid/base transporters important for exocrine secretion. Pancreatic ductal adenocarcinoma (PDAC) cells may utilize such transporters to acidify extracellular tumor microenvironment, creating a niche favoring cell proliferation, fibrosis and resistance to chemotherapy—all contributing to the notoriously bad prognosis of this disease. Here, we report that gastric and non-gastric H+, K+-ATPases (coded by ATP4A and ATP12A) are overexpressed in human and murine pancreatic cancer and that we can target them specifically with proton pump inhibitors (PPIs) and potassium-competitive acid blockers (P-CABs) in in vitro models of PDAC. Focusing on pantoprazole, we show that it significantly reduced human cancer cell proliferation by inhibiting cellular H+ extrusion, increasing K+ conductance and promoting cyclin D1-dependent cell cycle arrest and preventing STAT3 activation. Pantoprazole also decreased collagen secretion from pancreatic stellate cells. Importantly, in vivo studies show that pantoprazole treatment of tumor-bearing mice reduced tumor size, fibrosis and expression of angiogenic markers. This work provides the first evidence that H+, K+-ATPases contribute to PDAC progression and that these can be targeted by inhibitors of these pumps, thus proving a promising therapeutic strategy.

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

Cancers ◽

10.3390/cancers12123628 ◽

2020 ◽

Vol 12 (12) ◽

pp. 3628

Author(s):

Manoj Amrutkar ◽

Nils Tore Vethe ◽

Caroline S. Verbeke ◽

Monica Aasrum ◽

Anette Vefferstad Finstadsveen ◽

...

Keyword(s):

Pancreatic Cancer ◽

Cancer Cells ◽

Cell Lines ◽

Cancer Cell ◽

Stellate Cells ◽

Drug Uptake ◽

Pancreatic Stellate Cells ◽

Human Pancreatic Cancer ◽

Pancreatic Cancer Cells ◽

The Impact

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.

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