The Role of Stellate Cells in Pancreatic Ductal Adenocarcinoma: Targeting Perspectives.
ABSTRACT: Pancreatic ductal adenocarcinoma (PDAC) is a gastrointestinal malignancy with a dismal clinical outcome. Accumulating evidence suggests that activated pancreatic stellate cells (PSCs), the major producers of extracellular matrix (ECM), drive the severe stromal/desmoplastic reaction in PDAC. Furthermore, the crosstalk among PSCs, pancreatic cancer cells (PCCs) as well as other stroma cells can establish a growth-supportive tumor microenvironment (TME) of PDAC, thereby enhancing tumor growth, metastasis, and chemoresistance via various pathways. Recently, targeting stroma has emerged as a promising strategy for PDAC therapy, and several novel strategies have been proposed. The aim of our study is to give a profound review of the role of PSCs in PDAC progression and recent advances in stroma-targeting strategies.
Project description:Pancreatic ductal adenocarcinoma (PDAC) is characterized by an abundant desmoplastic reaction driven by pancreatic stellate cells (PSCs) that contributes to tumor progression. Here we sought to characterize the interactions between pancreatic cancer cells (PCCs) and PSCs that affect the inflammatory and immune response in pancreatic tumors. Conditioned media from mono- and cocultures of PSCs and PCCs were assayed for expression of cytokines and growth factors. IP-10/CXCL10 was the most highly induced chemokine in coculture of PSCs and PCCs. Its expression was induced in the PSCs by PCCs. IP-10 was elevated in human PDAC specimens, and positively correlated with high stroma content. Furthermore, gene expression of IP-10 and its receptor CXCR3 were significantly associated with the intratumoral presence of regulatory T cells (Tregs). In an independent cohort of 48 patients with resectable pancreatic ductal adenocarcinoma, high IP-10 expression levels correlated with decreased median overall survival. Finally, IP-10 stimulated the ex vivo recruitment of CXCR3+ effector T cells as well as CXCR3+ Tregs derived from patients with PDAC. Our findings suggest that, in pancreatic cancer, CXCR3+ Tregs can be recruited by IP-10 expressed by PSCs in the tumor stroma, leading to immunosuppressive and tumor-promoting effects.
Project description:Pancreatic ductal adenocarcinoma (PDAC) is characterized by a strong desmoplastic reaction where the stromal compartment often accounts for more than half of the tumor volume. Pancreatic stellate cells (PSC) are a central mediator of desmoplasia. There is increasing evidence that desmoplasia is contributing to the poor therapeutic response of PDAC. We show that PSCs promote radioprotection and stimulate proliferation in pancreatic cancer cells (PCC) in direct coculture. Our in vivo studies show PSC-dependent radioprotection in response to a single dose and to fractionated radiation. Abrogating ?1-integrin signaling abolishes the PSC-mediated radioprotection in PCCs. Furthermore, this effect is independent of PI3K (phosphoinositide 3-kinase) but dependent on FAK. Taken together, we show for the first time that PSCs promote radioprotection of PCCs in a ?1-integrin-dependent manner.
Project description:Activation of pancreatic stellate cells (PSCs) initiates pancreatic fibrosis in chronic pancreatitis and furnishes a niche that enhances the malignancy of pancreatic cancer cells (PCCs) in pancreatic ductal adenocarcinoma (PDAC). Resveratrol (RSV), a natural polyphenol, exhibits potent antioxidant and anticancer effects. However, whether and how RSV influences the biological properties of activated PSCs and the effects of these changes on tumor remain unknown. In the present study, we found that RSV impeded hydrogen peroxide-driven reactive oxygen species- (ROS-) induced activation, invasion, migration, and glycolysis of PSCs. In addition, miR-21 expression in activated PSCs was downregulated after RSV treatment, whereas the PTEN protein level increased. miR-21 silencing attenuated ROS-induced activation, invasion, migration, and glycolysis of PSCs, whereas the overexpression of miR-21 rescued the responses of PSCs treated with RSV. Moreover, RSV or N-acetyl-L-cysteine (NAC) administration or miR-21 knockdown in PSCs reduced the invasion and migration of PCCs in coculture, and the effects of RSV were partly reversed by miR-21 upregulation. Collectively, RSV inhibits PCC invasion and migration through suppression of ROS/miR-21-mediated activation and glycolysis in PSCs. Therefore, targeting miR-21-mediated glycolysis by RSV in tumor stroma may serve as a new strategy for clinical PDAC prevention or treatment.
Project description:Pancreatic ductal adenocarcinoma (PDAC) is characterized by an extremely poor prognosis, and its treatment remains a challenge. As the existing in vitro experimental models offer only a limited resemblance to human PDAC, there is a strong need for additional research tools to better understand PDAC tumor biology, particularly the impact of the tumor stroma. Here, we report for the first time the establishment and characterization of human PDAC-derived paired primary monolayer cultures of (epithelial) cancer cells (PCCs) and mesenchymal stellate cells (PSCs) derived from the same tumor by the outgrowth method. Characterization of cell morphology, cytostructural, and functional profiles and proteomics-based secretome analysis were performed. All PCCs harbored KRAS and TP53 mutations, and expressed cytokeratin 19, ki-67, and p53, while the expression of EpCAM and vimentin was variable. All PSCs expressed ?-smooth muscle actin (?-SMA) and vimentin. PCCs showed a significantly higher growth rate and proliferation than PSCs. Secretome analysis confirmed the distinct nature of PCCs as compared to PSCs and allowed identification of potential molecular regulators of PSC-conditioned medium (PSC-CM)-induced migration of PCCs. Paired primary cultures of PCCs and PSCs derived from the same tumor specimen represent a novel experimental model for basic research in PDAC tumor biology.
Project description:Pancreatic ductal adenocarcinoma (PDAC) has a dismal prognosis largely owing to inefficient diagnosis and tenacious drug resistance. Activation of pancreatic stellate cells (PSCs) and consequent development of dense stroma are prominent features accounting for this aggressive biology<sup>1,2</sup>. The reciprocal interplay between PSCs and pancreatic cancer cells (PCCs) not only enhances tumour progression and metastasis but also sustains their own activation, facilitating a vicious cycle to exacerbate tumorigenesis and drug resistance<sup>3-7</sup>. Furthermore, PSC activation occurs very early during PDAC tumorigenesis<sup>8-10</sup>, and activated PSCs comprise a substantial fraction of the tumour mass, providing a rich source of readily detectable factors. Therefore, we hypothesized that the communication between PSCs and PCCs could be an exploitable target to develop effective strategies for PDAC therapy and diagnosis. Here, starting with a systematic proteomic investigation of secreted disease mediators and underlying molecular mechanisms, we reveal that leukaemia inhibitory factor (LIF) is a key paracrine factor from activated PSCs acting on cancer cells. Both pharmacologic LIF blockade and genetic Lifr deletion markedly slow tumour progression and augment the efficacy of chemotherapy to prolong survival of PDAC mouse models, mainly by modulating cancer cell differentiation and epithelial-mesenchymal transition status. Moreover, in both mouse models and human PDAC, aberrant production of LIF in the pancreas is restricted to pathological conditions and correlates with PDAC pathogenesis, and changes in the levels of circulating LIF correlate well with tumour response to therapy. Collectively, these findings reveal a function of LIF in PDAC tumorigenesis, and suggest its translational potential as an attractive therapeutic target and circulating marker. Our studies underscore how a better understanding of cell-cell communication within the tumour microenvironment can suggest novel strategies for cancer therapy.
Project description:BACKGROUND:Gemcitabine remains a cornerstone in chemotherapy of pancreatic ductal adenocarcinoma (PDAC) despite suboptimal clinical effects that are partly due to the development of chemoresistance. Pancreatic stellate cells (PSCs) of the tumor stroma are known to interact with pancreatic cancer cells (PCCs) and influence the progression of PDAC through a complex network of signaling molecules that involve extracellular matrix (ECM) proteins. To understand tumor-stroma interactions regulating chemosensitivity, the role of PSC-secreted fibronectin (FN) in the development of gemcitabine resistance in PDAC was examined. METHODS:PSC cultures obtained from ten different human PDAC tumors were co-cultured with PCC lines (AsPC-1, BxPC-3, Capan-2, HPAF-II, MIA PaCa-2, PANC-1 and SW-1990) either directly, or indirectly via incubation with PSC-conditioned medium (PSC-CM). Gemcitabine dose response cytotoxicity was determined using MTT based cell viability assays. Protein expression was assessed by western blotting and immunofluorescence. PSC-CM secretome analysis was performed by proteomics-based LC-MS/MS, and FN content in PSC-CM was determined with ELISA. Radiolabeled gemcitabine was used to determine the capacity of PCCs to uptake the drug. RESULTS:In both direct and indirect co-culture, PSCs induced varying degrees of resistance to the cytotoxic effects of gemcitabine among all cancer cell lines examined. A variable degree of increased phosphorylation of ERK1/2 was observed across all PCC lines upon incubation with PSC-CM, while activation of AKT was not detected. Secretome analysis of PSC-CM identified 796 different proteins, including several ECM-related proteins such as FN and collagens. Soluble FN content in PSC-CM was detected in the range 175-350?ng/ml. Neither FN nor PSC-CM showed any effect on PCC uptake capacity of gemcitabine. PCCs grown on FN-coated surface displayed higher resistance to gemcitabine compared to cells grown on non-coated surface. Furthermore, a FN inhibitor, synthetic Arg-Gly-Asp-Ser (RGDS) peptide significantly inhibited PSC-CM-induced chemoresistance in PCCs via downregulation of ERK1/2 phosphorylation. CONCLUSIONS:The findings of this study suggest that FN secreted by PSCs in the ECM plays a key role in the development of resistance to gemcitabine via activation of ERK1/2. FN-blocking agents added to gemcitabine-based chemotherapy might counteract chemoresistance in PDAC and provide better clinical outcomes.
Project description:<h4>Background</h4>Pancreatic ductal adenocarcinoma (PDAC) is a highly desmoplastic tumor with a dismal prognosis for most patients. Fibrosis and inflammation are hallmarks of tumor desmoplasia. We have previously demonstrated that preventing the activation of pancreatic stellate cells (PSCs) and alleviating desmoplasia are beneficial strategies in treating PDAC. Metformin is a widely used glucose-lowering drug. It is also frequently prescribed to diabetic pancreatic cancer patients and has been shown to associate with a better outcome. However, the underlying mechanisms of this benefit remain unclear. Metformin has been found to modulate the activity of stellate cells in other disease settings. In this study, we examine the effect of metformin on PSC activity, fibrosis and inflammation in PDACs.<h4>Methods/results</h4>In overweight, diabetic PDAC patients and pre-clinical mouse models, treatment with metformin reduced levels of tumor extracellular matrix (ECM) components, in particular hyaluronan (HA). In vitro, we found that metformin reduced TGF-ß signaling and the production of HA and collagen-I in cultured PSCs. Furthermore, we found that metformin alleviates tumor inflammation by reducing the expression of inflammatory cytokines including IL-1? as well as infiltration and M2 polarization of tumor-associated macrophages (TAMs) in vitro and in vivo. These effects on macrophages in vitro appear to be associated with a modulation of the AMPK/STAT3 pathway by metformin. Finally, we found in our preclinical models that the alleviation of desmoplasia by metformin was associated with a reduction in ECM remodeling, epithelial-to-mesenchymal transition (EMT) and ultimately systemic metastasis.<h4>Conclusion</h4>Metformin alleviates the fibro-inflammatory microenvironment in obese/diabetic individuals with pancreatic cancer by reprogramming PSCs and TAMs, which correlates with reduced disease progression. Metformin should be tested/explored as part of the treatment strategy in overweight diabetic PDAC patients.
Project description: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.
Project description:Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive cancers with a low response to treatment and a five-year survival rate below 5%. The ineffectiveness of treatment is partly because of an immunosuppressive tumor microenvironment, which comprises tumor-supportive pancreatic stellate cells (PSCs). Therefore, new therapeutic strategies are needed to tackle both the immunosuppressive PSC and pancreatic cancer cells (PCCs). Recently, physical cold atmospheric plasma consisting of reactive oxygen and nitrogen species has emerged as a novel treatment option for cancer. In this study, we investigated the cytotoxicity of plasma-treated phosphate-buffered saline (pPBS) using three PSC lines and four PCC lines and examined the immunogenicity of the induced cell death. We observed a decrease in the viability of PSC and PCC after pPBS treatment, with a higher efficacy in the latter. Two PCC lines expressed and released damage-associated molecular patterns characteristic of the induction of immunogenic cell death (ICD). In addition, pPBS-treated PCC were highly phagocytosed by dendritic cells (DCs), resulting in the maturation of DC. This indicates the high potential of pPBS to trigger ICD. In contrast, pPBS induced no ICD in PSC. In general, pPBS treatment of PCCs and PSCs created a more immunostimulatory secretion profile (higher TNF-? and IFN-?, lower TGF-?) in coculture with DC. Altogether, these data show that plasma treatment via pPBS has the potential to induce ICD in PCCs and to reduce the immunosuppressive tumor microenvironment created by PSCs. Therefore, these data provide a strong experimental basis for further in vivo validation, which might potentially open the way for more successful combination strategies with immunotherapy for PDAC.
Project description:Pancreatic ductal adenocarcinoma is characterised by a dense desmoplastic stroma composed of stromal cells and extracellular matrix (ECM). This barrier severely impairs drug delivery and penetration. Activated pancreatic stellate cells (PSCs) play a key role in establishing this unique pathological obstacle, but also offer a potential target for anti-tumour therapy. Here, we construct a tumour microenvironment-responsive nanosystem, based on PEGylated polyethylenimine-coated gold nanoparticles, and utilise it to co-deliver all-trans retinoic acid (ATRA, an inducer of PSC quiescence) and siRNA targeting heat shock protein 47 (HSP47, a collagen-specific molecular chaperone) to re-educate PSCs. The nanosystem simultaneously induces PSC quiescence and inhibits ECM hyperplasia, thereby promoting drug delivery to pancreatic tumours and significantly enhancing the anti-tumour efficacy of chemotherapeutics. Our combination strategy to restore homoeostatic stromal function by targeting activated PSCs represents a promising approach to improving the efficacy of chemotherapy and other therapeutic modalities in a wide range of stroma-rich tumours.