Phenotypic Heterogeneity and Plasticity of Cancer Cell Migration in a Pancreatic Tumor Three-Dimensional Culture Model.
ABSTRACT: Invasive cancer cell migration is a key feature of metastatic human pancreatic ductal adenocarcinoma (PDAC), yet the underlying mechanisms remain poorly understood. Here, we investigated modes of cancer cell invasion using two pancreatic cancer cell lines with differential epithelial-mesenchymal status, PANC-1 and BxPC-3, under 3D culture conditions. Multicellular tumor spheroids (TSs) were grown in a collagen matrix co-cultured with pancreatic stellate cells (PSCs) using microchannel chips. PANC-1 cells showed individual migration from TSs via invadopodium formation. BxPC-3 cells showed plasticity between collective and individual migration in either mesenchymal mode, with filopodium-like protrusions, or blebby amoeboid mode. These two cell lines showed significantly different patterns of extracellular matrix (ECM) remodeling, with MMP-dependent degradation in a limited area of ECM around invadopodia for PANC-1 cells, or MMP-independent extensive deformation of ECM for BxPC-3 cells. Cancer cell migration out of the collagen channel significantly increased by PSCs and directional cancer cell migration was mediated by fibronectin deposited by PSCs. Our results highlight the phenotypic heterogeneity and plasticity of PDAC cell migration and ECM remodeling under 3D culture conditions. This 3D co-culture model of pancreatic cancer cells and PSCs offers a useful tool for studying cancer cell migration and ECM remodeling to identify and develop potential molecular targets and anti-cancer agents against human PDAC.
Project description:BACKGROUND:Pancreatic ductal adenocarcinoma (PDAC) is a stroma-rich carcinoma, and pancreatic stellate cells (PSCs) are a major component of this dense stroma. PSCs play significant roles in metastatic progression and chemoresistance through cross-talk with cancer cells. Preclinical in vitro tumor model of invasive phenotype should incorporate three-dimensional (3D) culture of cancer cells and PSCs in extracellular matrix (ECM) for clinical relevance and predictability. METHODS:PANC-1 cells were cultured as tumor spheroids (TSs) using our previously developed minipillar chips, and co-cultured with PSCs, both embedded in collagen gels. Effects of PSC co-culture on ECM fiber network, invasive migration of cancer cells, and expression of epithelial-mesenchymal transition (EMT)-related proteins were examined. Conditioned media was also analyzed for secreted factors involved in cancer cell-PSC interactions. Inhibitory effect on cancer cell invasion was compared between gemcitabine and paclitaxel at an equitoxic concentration in PANC-1 TSs co-cultured with PSCs. RESULTS:Co-culture condition was optimized for the growth of TSs, activation of PSCs, and their interaction. Increase in cancer cell invasion via ECM remodeling, invadopodia formation and EMT, as well as drug resistance was recapitulated in the TS-PSC co-culture, and appeared to be mediated by cancer cell-PSC interaction via multiple secreted factors, including IL-6, IL-8, IGF-1, EGF, TIMP-1, uPA, PAI-1, and TSP-1. Compared to gemcitabine, paclitaxel showed a greater anti-invasive activity, which was attributed to suppresion of invadopodia formation in cancer cells as well as to PSC-specific cytotoxicity abrogating its paracrine signaling. CONCLUSIONS:Here, we established 3D co-culture of TSs of PANC-1 cells and PSCs using minipillar histochips as a novel tumoroid model of PDAC. Our results indicate usefulness of the present co-culture model and multiplex quantitative analysis method not only in studying the role of PSCs and their interactions with tumor cell towards metastatic progression, but also in the drug evaluation of stroma-targeting drugs.
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:Galectin-1 has previously been shown to be strongly expressed in activated pancreatic stellate cells (PSCs) and promote the development and metastasis of pancreatic ductal adenocarcinoma (PDAC). However, the molecular mechanisms by which Galectin-1 promotes the malignant behavior of pancreatic cancer cells remain unclear. In this study, we examined the effects of Galectin-1 knockdown or overexpression in PSCs co-cultured with pancreatic cancer (PANC-1) cells. Immunohistochemical analysis showed expression of epithelial-mesenchymal transition (EMT) markers and MMP9 were positively associated with the expression of Galectin-1 in 66 human PDAC tissues. In addition, our in vitro studies showed PSC-derived Galectin-1 promoted the proliferation, invasion, and survival (anti-apoptotic effects) of PANC-1 cells. We also showed PSC-derived Galectin-1 induced EMT of PANC-1 cells and activated the NF-?B pathway in vitro. Our mixed (PSCs and PANC-1 cells) mouse orthotopic xenograft model indicated that overexpression of Galectin-1 in PSCs significantly promoted the proliferation, growth, invasion, and liver metastasis of the transplanted tumor. Moreover, Galectin-1 overexpression in PSCs was strongly associated with increased expression of EMT markers in both the orthotopic xenograft tumor in the pancreas and in metastatic lesions of naked mice. We conclude that PSC-derived Galectin-1 promotes the malignant behavior of PDAC by inducing EMT via activation of the NF-?B pathway. Our results suggest that targeting Galectin-1 in PSCs could represent a promising therapeutic strategy for PDAC progression and metastasis.
Project description:Background:The nucleation-promoting factor cortactin is expressed and promotes tumor progression and metastasis in various cancers. However, little is known about the biological role of cortactin in the progression of pancreatic ductal adenocarcinoma (PDAC). Methods:Cortactin and phosphorylated cortactin (Y421) were investigated immunohistochemically in 66 PDAC tumor specimens. To examine the functional role of cortactin in PDAC, we modulated cortactin expression by establishing two cortactin knockout cell lines (Panc-1 and BxPC-3) with CRISPR/Cas9 technique. Cortactin knockout was verified by immunoblotting and immunofluorescence microscopy and functional effects were determined by cell migration and invasion assays. A proteomic screening approach was performed to elucidate potential binding partners of cortactin. Results:Immunohistochemically, we observed higher cortactin expression and Tyr421-phosphorylation in PDAC metastases compared to primary tumor tissues. In PDAC cell lines Panc-1 and BxPC-3, knockdown of cortactin impaired migration and invasion, while cell proliferation was not affected. Three-dimensional spheroid culturing as a model for collective cell migration enhanced cortactin expression and Tyr421-phosphorylation. The activation of cortactin as well as the migratory capacity of PDAC cells could significantly be reduced by dasatinib, a Src family kinase inhibitor. Finally, we identified gelsolin as a novel protein interaction partner of cortactin in PDAC. Conclusion:Our data provides evidence that cohesive cell migration induces cortactin expression and phosphorylation as a prerequisite for the gain of an invasive, pro-migratory phenotype in PDAC that can effectively be targeted with dasatinib.
Project description:SS1P is an antimesothelin recombinant immunotoxin (RIT). Pancreatic ductal adenocarcinoma (PDAC) cell lines are resistant to SS1P, despite high mesothelin expression. The aim of this study is to examine whether combining SS1P and BH3-mimetic ABT-737 induces cell death in a panel of PDAC cell lines. ABT-737 binds and neutralizes several antiapoptotic BCL2 family proteins, but has a low affinity for the short-lived MCL1 and BCL2A1. SS1P inhibits protein synthesis, which has shown to downregulate MCL1. PDAC cell lines KLM-1, BxPc-3, and Panc 3.014 were resistant to SS1P or ABT-737 alone. Combining both compounds led to a significant increase in cell death. After 48 hours of treatment, cell death was observed in 92% of KLM-1, 55% of BxPc-3, and 23% of Panc 3.014 cells. Panc 3.014 had the highest number of mesothelin-binding sites (92×10(3)), followed by KLM-1 (58×10(3)) and BxPc-3 (3×10(3)). ABT-737 had no effect on SS1P internalization, but enhanced SS1P-induced protein synthesis inhibition significantly in KLM-1, to a lesser extent in BxPc-3, and very little in Panc 3.014. SS1P alone or in combination with ABT-737 downregulated MCL1 in KLM-1 and BxPc-3, but not in Panc 3.014. Similar observations were made for BCL2A1, which had the highest levels in Panc 3.014. Compared with KLM-1, Panc 3.014, and BxPc-3 also had lower proapoptotic BAK and a trend toward higher MCL1. Proapoptotic BAX was similar in KLM-1 and BxPc-3, but lower in Panc 3.014. In conclusion, combining SS1P with ABT-737 overcomes SS1P-resistance in PDAC, although to a variable extent. The efficacy of the combination is mainly associated with the RIT-associated inhibition of protein synthesis and the ability to downregulate MCL1 and BCL2A1, while levels of other key apoptotic proteins may also be important. Our data support the combination of an RIT and a BH3-mimetic, and identify factors that potentially limit the efficacy of such therapeutic approach.
Project description:Pancreatic ductal adenocarcinoma (PDAC) is characterized by an acidic and fibrotic stroma. The extracellular matrix (ECM) causing the fibrosis is primarily formed by pancreatic stellate cells (PSCs). The effects of the altered biomechanics and pH landscape in the pathogenesis of PDAC, however, are poorly understood. Mechanotransduction in cells has been linked to the function of mechanosensitive ion channels such as Piezo1. Here, we tested whether this channel plays crucial roles in transducing mechanical signals in the acidic PDAC microenvironment. We performed immunofluorescence, Ca2+ influx and intracellular pH measurements in PSCs and complemented them by live-cell imaging migration experiments in order to assess the function of Piezo1 channels in PSCs. We evaluated whether Piezo1 responds to changes of extracellular and/or intracellular pH in the pathophysiological range (pH 6.6 and pH 6.9, respectively). We validated our results using Piezo1-transfected HEK293 cells as a model system. Indeed, acidification of the intracellular space severely inhibits Piezo1-mediated Ca2+ influx into PSCs. In addition, stimulation of Piezo1 channels with its activator Yoda1 accelerates migration of PSCs on a two-dimensional ECM as well as in a 3D setting. Furthermore, Yoda1-activated PSCs transmit more force to the surrounding ECM under physiological pH, as revealed by measuring the dislocation of microbeads embedded in the surrounding matrix. This is paralleled by an enhanced phosphorylation of myosin light chain isoform 9 after Piezo1 stimulation. Intriguingly, upon acidification, Piezo1 activation leads to the initiation of cell death and disruption of PSC spheroids. In summary, stimulating Piezo1 activates PSCs by inducing Ca2+ influx which in turn alters the cytoskeletal architecture. This results in increased cellular motility and ECM traction, which can be useful for the cells to invade the surroundings and to detach from the tissue. However, in the presence of an acidic extracellular pH, although net Ca2+ influx is reduced, Piezo1 activation leads to severe cell stress also limiting cellular viability. In conclusion, our results indicate a strong interdependence between environmental pH, the mechanical output of PSCs and stromal mechanics, which promotes early local invasion of PDAC cells.
Project description:Baicalein, a flavone ingredient of Scutellaria baicalensis Georgi, is a promising anti-cancer agent. However, its potential anti-pancreatic cancer effects and the underlying mechanisms are still unclear. In this study, we showed that Baicalein not only induced apoptosis, but also suppressed proliferation, migration and invasion of two pancreatic cancer cell lines BxPC-3 and PANC-1 in a dose- and time-dependent manner. Notably, Baicalein exhibited low toxicity to normal human liver or kidney cells. We further discovered that Baicalein suppressed BxPC-3 and PANC-1 cell proliferation and invasion through targeting the expression of NEDD9, a Cas scaffolding protein, to decrease Akt and ERK activities. Especially, Baicalein decreased Akt phosphorylation at T-308 via lowering NEDD9-dependent PDK1 expression. Overexpression of NEDD9 effectively rescued proliferation and invasion of BxPC-3 and PANC-1 cells dampened by Baicalein. Taken together, our findings suggest that Baicalein is a potent remedy applied to pancreatic cancer treatment in the future.
Project description: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 <i>via</i> 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:BACKGROUND:Pancreatic ductal adenocarcinoma (PDAC) is presently one of the cancers with the worst survival rates and least effective treatments. Moreover, total deaths due to PDAC are predicted to increase in the next 15 years. Therefore, novel insights into basic mechanism of PDAC development and therapies are needed. PDAC is characterized by a complex microenvironment, in which cancer and stromal cells release different molecules, such as ATP. ATP can be transported and/or exocytosed from active cancer cells and released from dying cells in the necrotic core of the cancer. We hypothesized that one of the ATP receptors, the P2X7 receptor (P2X7R) could be an important player in PDAC behaviour. METHODS:We determined the expression (real time PCR and Western blot) and localization (immunofluorescence) of P2X7R in human PDAC cell lines (AsPC-1, BxPC-3, Capan-1, MiaPaCa-2, Panc-1) and a "normal" human pancreatic duct epithelial cell line (HPDE). The function of P2X7R in proliferation (BrdU assay), migration (wound assay) and invasion (Boyden chamber with matrigel) was characterized. Furthermore, we studied P2X7R-dependent pore formation (YoPro-1 assay) and cell death (caspase and annexin V / propidium iodide assays). RESULTS:We found higher expression of P2X7R protein in PDAC compared to HPDE cells. P2X7R had notable disparate effects on PDAC survival. Firstly, high concentrations of ATP or the specific P2X7R agonist, BzATP, had cytotoxic effects in all cell lines, and cell death was mediated by necrosis. Moreover, the P2X7R-pore antagonist, A438079, prevented ATP-induced pore formation and cell death. Second, in basal conditions and with low concentrations of ATP/BzATP, the P2X7R allosteric inhibitor AZ10606120 reduced proliferation in all PDAC cell lines. P2X7R also affected other key characteristics of cancer cell behavior. AZ10606120 reduced cell migration and invasion in PDAC cell lines compared to that of untreated/vehicle-treated control cells, and stimulation with sub-millimolar concentrations of ATP or BzATP substantially increased cell invasion. CONCLUSIONS:PDAC cell lines overexpress P2X7R and the receptor plays crucial roles in cell survival, migration and invasion. Therefore, we propose that drugs targeting P2X7R could be exploited in therapy of pancreatic cancer.