Pancreatic Tumor Cell Invasion Research Articles

Ectopic expression of DOCK8 regulates lysosome-mediated pancreatic tumor cell invasion

Amplified lysosome activity is a hallmark of pancreatic ductal adenocarcinoma (PDAC) orchestrated by oncogenic KRAS that mediates tumor growth and metastasis, though the mechanisms underlying this phenomenon remain unclear. Using comparative proteomics, we found that oncogenic KRAS significantly enriches levels of the guanine nucleotide exchange factor (GEF) dedicator of cytokinesis 8 (DOCK8) on lysosomes. Surprisingly, DOCK8 is aberrantly expressed in a subset of PDAC, where it promotes cell invasion invitro and invivo. DOCK8 associates with lysosomes and regulates lysosomal morphology and motility, with loss of DOCK8 leading to increased lysosome size. DOCK8 promotes actin polymerization at the surface of lysosomes while also increasing the proteolytic activity of the lysosomal protease cathepsin B. Critically, depletion of DOCK8 significantly reduces cathepsin-dependent extracellular matrix degradation and impairs the invasive capacity of PDAC cells. These findings implicate ectopic expression of DOCK8 as a key driver of KRAS-driven lysosomal regulation and invasion in pancreatic cancer cells.

Multiple roles for cholinergic signaling in pancreatic diseases.

Cholinergic nerves are widely distributed throughout the human body and participate in various physiological activities, including sensory, motor, and visceral activities, through cholinergic signaling. Cholinergic signaling plays an important role in pancreatic exocrine secretion. A large number of studies have found that cholinergic signaling overstimulates pancreatic acinar cells through muscarinic receptors, participates in the onset of pancreatic diseases such as acute pancreatitis and chronic pancreatitis, and can also inhibit the progression of pancreatic cancer. However, cholinergic signaling plays a role in reducing pain and inflammation through nicotinic receptors, but enhances the proliferation and invasion of pancreatic tumor cells. This review focuses on the progression of cholinergic signaling and pancreatic diseases in recent years and reveals the role of cholinergic signaling in pancreatic diseases.

Effect of obestatin on the migration and invasion of pancreatic tumor cells.

Effect of obestatin on the migration and invasion of pancreatic tumor cells.

Abstract 1237: Down-regulation of chemokine receptor CXCR4 by gambogic acid suppresses metastasis of pancreatic cancer in an orthotopic mouse model

Abstract More than 90% of patients die of pancreatic cancer due to metastasis. Among the cytokines, CXCL12 and its CXC chemokine receptor 4 (CXCR4) are known to play a role in invasion and metastasis in many types of cancer. Thus, agents that can interrupt the CXCR4/CXCL12 signaling cascade have the potential to suppress cancer metastasis. In the present study, we demonstrate that gambogic acid (GA), a xanthone from Gamboge hanburyi, down-regulated CXCR4 expression in pancreatic cancer cells in a dose- and time-dependent manner. We found that the decrease in CXCR4 by GA was not specific to cell type since its expression was abrogated in multiple myeloma cell lines and in colorectal, head and neck, prostate, and breast cancer cell lines. Furthermore, down-regulation of CXCR4 was not due to proteolytic degradation since proteasome inhibitors had no effect. Instead, the down-regulation of CXCR4 was due to transcriptional regulation, as indicated by the corresponding down-regulation of mRNA expression. This xanthone also inhibited the invasion of pancreatic tumor cells in vitro. Down-regulation of CXCR4 and prevention of cell invasion by the xanthone were both found to be mediated through the reactive oxygen species (ROS) as N-acetylcysteine, a ROS quencher, abolished these effects. When examined in an orthotopic nude mouse model of human pancreatic cancer, intraperitoneal administration of GA suppressed both tumor growth and metastasis. Immunohistochemical analysis of tumor tissue showed that besides decreasing the level of CXCR4, GA also reduced the expression of VEGF and MMP-9, which are also linked to invasion and metastasis. Thus, our results demonstrate gambogic acid’s potential in suppressing tumor invasion and metastasis through down-modulation of CXCR4/CXCL12 signaling pathways. Citation Format: Sahdeo Prasad, Sanjay Srivastava. Down-regulation of chemokine receptor CXCR4 by gambogic acid suppresses metastasis of pancreatic cancer in an orthotopic mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1237.

Abstract B22: Necuparanib inhibits pancreatic cancer progression and invasion in a 3D tumor and stromal cell co-culture system

Abstract Pancreatic cancer is one of the most aggressive types of cancer, with only about 5% of patients surviving 5 years past the initial diagnosis. Despite advances with new chemotherapy combinations, overall survival outcomes are still dismal due to the cancer's complex pathology. Novel multi-targeted agents or therapeutic combinations able to disrupt the aggressive fibrotic microenvironment and restrict tumor proliferation are a current focus in the field. Necuparanib (formerly M402) is a heparan sulfate-like molecule that binds and inhibits multiple heparin-binding growth factors, chemokines, and adhesion molecules in cancer progression and metastasis and as such has been shown to modulate multiple pathways in tumors. In order to further explore how necuparanib affects the tumor and its microenvironment, a 3-dimensional (3D) culture system mimicking pancreatic cancer was developed containing a co-culture of human pancreatic tumor cells and pancreatic stellate cells, the most abundant stromal cell in the pancreas. Morphological and histological assessment of the 3D tumor spheroids demonstrated that necuparanib inhibits a) pancreatic tumor cell growth, b) pancreatic tumor cell invasion, and c) pancreatic stellate cell growth. Necuparanib's activity in these assays was shown to be dose-dependent. Moreover, using a negative control for necuparanib engineered to lack the ability to bind growth factors, we established that necuparanib's activity is growth factor binding dependent. The ability to inhibit both tumor and stromal cells demonstrates that necuparanib is a very promising multi-targeting agent for difficult-to-treat cancers such as desmoplastic pancreatic tumors. These studies aid in our understanding of the key biological targets and pathways responsible for necuparanib's anti-tumor effects and support the current Phase 2 safety and efficacy study being performed in patients with metastatic pancreatic cancer. Citation Format: Amanda MacDonald, Michelle Priess, Jennifer Curran, Silva Krause. Necuparanib inhibits pancreatic cancer progression and invasion in a 3D tumor and stromal cell co-culture system. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr B22.

Polyethylenimine-coated SPION exhibits potential intrinsic anti-metastatic properties inhibiting migration and invasion of pancreatic tumor cells

Due to its aggressive behavior, pancreatic cancer is one of the principal causes of cancer-related deaths. The highly metastatic potential of pancreatic tumor cells demands the development of more effective anti-metastatic approaches for this disease. Although polyethylenimine-coated superparamagnetic iron oxide nanoparticles (PEI-coated SPIONs) have been studied for their utility as transfection agents, little is known of their effect on tumor cell biology. Here we demonstrated that PEI-coated SPIONs have potent inhibitory effects on pancreatic tumor cell migration/invasion, through inhibition of Src kinase and decreased expression of MT1-MMP and MMP2 metalloproteinases. When treated with PEI-coated SPIONs, the pancreatic tumor cell line Pan02 showed reduced invadosome density and thus, a decrease in their ability to invade through basement membrane. These nanoparticles temporarily downmodulated microRNA-21, thereby upregulating the cell migration inhibitors PTEN, PDCD4 and Sprouty-1. PEI-coated SPIONs thus show intrinsic, possibly anti-metastatic properties for modulating pancreatic tumor cell migration machinery, which indicates their potential as anti-metastatic agents for treatment of pancreatic cancer.

Abstract 5499: Necuparanib affects tumor progression and invasion in a 3D co-culture system of pancreatic cancer cells and stellate cells

Abstract Pancreatic cancer is one of the most aggressive and deadliest cancers, with only about 5% of patients surviving 5 years past the initial diagnosis. In over 80% of patients the cancer has already spread to distant organs at the time of diagnosis, making it difficult to treat. Most alarmingly, pancreatic cancer is the only cancer for which deaths are predicted to increase over the next few years. Despite advances with new chemotherapy combinations, overall survival outcomes are still dismal and require novel therapeutic approaches. Necuparanib (formerly M402) is a heparan sulfate-like molecule that binds and inhibits multiple heparin-binding growth factors, chemokines, adhesion molecules and enzymes involved in cancer progression and metastasis and as such modulates multiple elements of the tumor microenvironment. In order to further explore the mechanism of action for epithelial-stromal interactions affected by necuparanib, a 3-dimensional culture system mimicking pancreatic cancer was developed containing a co-culture of pancreatic tumor cells and pancreatic stellate cells, the most abundant stromal cell in the pancreas. In this culture system, pancreatic cancer cells form a tumor spheroid embedded in a matrix that is a mixture of type I collagen and basement membrane extract and contains pancreatic stellate cells. Notably, pancreatic cancer cells required the co-culture with pancreatic stellate cells to become invasive in vitro. Conditioned medium derived from pancreatic stellate cells could not mimic this effect indicating that soluble factors are not sufficient to cause invasion into surrounding matrix. Necuparanib inhibited the invasive behavior in a dose-dependent manner. The effect of pancreatic stellate cells on tumor cell invasion and the effect of necuparanib on stromal cells were further explored in this culture system. We showed an effect of necuparanib on proliferation, migration and invasion of pancreatic tumor cells. These studies aid in our understanding of the key biological targets and pathways responsible for necuparanib's anti-tumor effects and support biomarker discovery in the ongoing Phase 2 efficacy study in metastatic pancreatic cancer. Citation Format: Silva Krause, Amanda Weyers, Katie Loveluck, Birgit Schultes. Necuparanib affects tumor progression and invasion in a 3D co-culture system of pancreatic cancer cells and stellate cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5499. doi:10.1158/1538-7445.AM2015-5499

Ral and Rheb GTPase Activating Proteins Integrate mTOR and GTPase Signaling in Aging, Autophagy, and Tumor Cell Invasion

Diverse environmental cues converge on and are integrated by the mTOR signaling network to control cellular growth and homeostasis. The mammalian Tsc1-Tsc2 GTPase activating protein (GAP) heterodimer is a critical negative regulator of Rheb and mTOR activation. The RalGAPα-RalGAPβ heterodimer shares sequence and structural similarity with Tsc1-Tsc2. Unexpectedly, we observed that C.elegans expresses orthologs for the Rheb andRalA/B GTPases and for RalGAPα/β, but not Tsc1/2. This prompted our investigation to determine whether RalGAPs additionally modulate mTOR signaling. We determined that C.elegans RalGAP loss decreased lifespan, consistent with a Tsc-likefunction. Additionally, RalGAP suppression in mammalian cells caused RalB-selective activation and Sec5- and exocyst-dependent engagement of mTORC1 and suppression of autophagy. Unexpectedly, we also found that Tsc1-Tsc2 loss activated RalA/B independently of Rheb-mTOR signaling. Finally, RalGAP suppression caused mTORC1-dependent pancreatic tumor cell invasion. Our findings identify an unexpected crosstalk and integration of the Ral and mTOR signaling networks.

4 - Catalytically Inactive Extracellular Superoxide Dismutase Reduces Pancreatic Tumor Cell Invasion

4 - Catalytically Inactive Extracellular Superoxide Dismutase Reduces Pancreatic Tumor Cell Invasion

Protein Kinase A Modulates Transforming Growth Factor-β Signaling through a Direct Interaction with Smad4 Protein

Transforming growth factor β (TGFβ) signaling normally functions to regulate embryonic development and cellular homeostasis. It is increasingly recognized that TGFβ signaling is regulated by cross-talk with other signaling pathways. We previously reported that TGFβ activates protein kinase A (PKA) independent of cAMP through an interaction of an activated Smad3-Smad4 complex and the regulatory subunit of the PKA holoenzyme (PKA-R). Here we define the interaction domains of Smad4 and PKA-R and the functional consequences of this interaction. Using a series of Smad4 and PKA-R truncation mutants, we identified amino acids 290-300 of the Smad4 linker region as critical for the specific interaction of Smad4 and PKA-R. Co-immunoprecipitation assays showed that the B cAMP binding domain of PKA-R was sufficient for interaction with Smad4. Targeting of B domain regions conserved among all PKA-R isoforms and exposed on the molecular surface demonstrated that amino acids 281-285 and 320-329 were required for complex formation with Smad4. Interactions of these specific regions of Smad4 and PKA-R were necessary for TGFβ-mediated increases in PKA activity, CREB (cAMP-response element-binding protein) phosphorylation, induction of p21, and growth inhibition. Moreover, this Smad4-PKA interaction was required for TGFβ-induced epithelial mesenchymal transition, invasion of pancreatic tumor cells, and regulation of tumor growth in vivo.

Abstract 502: Schwann cells promote tumor cell invasion through regulation of the laminin receptorA6B1

Abstract Tumor cell migration and invasion of nerves, a process known as perineural nerve invasion (PNI), is a major metastatic route for neurotropic cancers including prostate and pancreatic cancer. The laminin family of extracellular matrix glycoproteins derived from myelinating Schwann cells coat nerve axons and play a prominent role in the development and maintenance of the peripheral nervous system. Tumor cells undergoing PNI utilize the laminin rich nerves as they migrate and invade during metastasis. Evidence has shown that the A6B1 laminin receptor promotes tumor cell migration and invasion following cleavage of the receptor to form the variant A6pB1 by the serine protease urokinase plasminogen activator (uPA). Here we demonstrate Schwann cell regulation of A6pB1 formation on prostate and pancreatic tumor cell lines. Conditioned medium from the S16 and S16Y cell lines, which are immortalized Schwann cells of the myelinating and non-myelinating phenotype, respectively was provided to the prostate and pancreatic tumor cell lines. It was observed that S16Y conditioned medium inhibited A6 conversion to A6p while the myelinating S16 cells increased cleavage of the receptor in DU-145, CF-PAC1 and PC-3 cancer cells. This effect was reversible as replenishing standard media conditions following a 24 hour treatment with S16 or S16Y media brought integrin cleavage levels back to steady state levels by 24 hours. Further, conditioned medium from S16 cells significantly increased pancreatic tumor cell invasion while S16Y conditioned medium decreased invasion in a modified Boyden chamber Matrigel assay. The increased invasion mediated by myelinating Schwann cells was dependent on A6 since a function blocking antibody targeting A6 inhibited invasion. Taken together these results suggest that myelinating Schwann cells (S16Y) in the pancreatic and prostatic nerve environment promote A6B1 and A6pB1 receptor dependent tumor cell invasion during PNI. (Supported in part by NIH CA23074 and CA159406 and HHMI Grant 52005889) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 502. doi:1538-7445.AM2012-502

Quiescin Sulfhydryl Oxidase 1 Promotes Invasion of Pancreatic Tumor Cells Mediated by Matrix Metalloproteinases

Quiescin sulfhydryl oxidase 1 (QSOX1) oxidizes sulfhydryl groups to form disulfide bonds in proteins. We previously mapped a peptide in plasma from pancreatic ductal adenocarcinoma (PDA) patients back to an overexpressed QSOX1 parent protein. In addition to overexpression in pancreatic cancer cell lines, 29 of 37 patients diagnosed with PDA expressed QSOX1 protein in tumor cells, but QSOX1 was not detected in normal adjacent tissues or in a transformed, but nontumorigenic cell line. To begin to evaluate the advantage QSOX1 might provide to tumors, we suppressed QSOX1 protein expression using short hairpin (sh) RNA in two pancreatic cancer cell lines. Growth, cell cycle, apoptosis, invasion, and matrix metalloproteinase (MMP) activity were evaluated. QSOX1 shRNA suppressed both short and long isoforms of the protein, showing a significant effect on cell growth, cell cycle, and apoptosis. However, QSOX1 shRNA dramatically inhibited the abilities of BxPC-3 and Panc-1 pancreatic tumor cells to invade through Matrigel in a modified Boyden chamber assay. Mechanistically, gelatin zymography indicated that QSOX1 plays an important role in activation of MMP-2 and MMP-9. Taken together, our results suggest that the mechanism of QSOX1-mediated tumor cell invasion is by activation of MMP-2 and MMP-9.

Consensus transcriptome signature of perineural invasion in pancreatic carcinoma

Perineural invasion, the growth of tumor cells along nerves, is a key feature of pancreatic cancer. The cardinal symptom of pancreatic cancer, abdominal pain often radiating to the back, as well as the high frequency of local tumor recurrence following resection are both attributed to the unique ability of pancreatic tumor cells to invade the neuronal system. The molecular mechanisms underlying the neuroaffinity of pancreatic tumors are not completely understood. In this study, we developed a novel method to monitor ex vivo perineural invasion into surgically resected rat vagal nerves by different human pancreatic tumor cell lines. Genome-wide transcriptional analyses were employed to identify the consensus set of genes differentially regulated in all highly nerve-invasive (nerve invasion passage 3) versus less invasive (nerve invasion passage 0) pancreatic tumor cells. The critical involvement of kinesin family member 14 (KIF14) and Rho-GDP dissociation inhibitor beta (ARHGDIbeta) in perineural invasion was confirmed on RNA and protein levels in human pancreatic tumor specimens. We found significant up-regulation of KIF14 and ARHGDIbeta mRNA levels in patients with pancreatic cancer, and both proteins were differentially expressed in tumor cells invading the perineural niche of pancreatic cancer patients as detected by immunohistochemistry. Moreover, functional knockdown of KIF14 and ARHGDIbeta using small interfering RNA resulted in altered basal and/or perineural invasion of pancreatic tumor cells. Our work provides novel insights into the molecular determinants of perineural invasion in pancreatic cancer. The established nerve invasion model and the consensus signature of perineural invasion could be instrumental in the identification of novel therapeutic targets of pancreatic cancer as exemplified by KIF14 and ARHGDIbeta.

Fibroblasts stimulate pancreatic tumor cell invasion by up regulating MMP-9 activity

MMP-9 activity is determined by a balance between the production of MMP-9 and tissue inhibitor of metalloproteinases-2 (TIMP-2). We have previously shown that increased MMP-9 activity stimulates pancreatic cancer cell invasion and that MMP-9 localizes to stromal fibroblasts in pancreatic cancer. We now hypothesize that fibroblasts stimulate tumor cell invasion by up regulating MMP-9 activity. Methods. MMP-9 and TIMP-2 expression in PANC-1 cancer cells, fibroblasts, and PANC-1/fibroblast cocultures were determined by ELISA and Western blot. The effect of fibroblasts on PANC-1 cancer cell invasion was evaluated using a Boyden Chamber invasion assay with and without neutralizing antibodies (Ab) against MMP-9 and TIMP-2. Statistical significance was determined by ANOVA and Student’s unpaired t-test. Results. There was no significant difference in MMP-9 production among PANC-1 cancer cells, fibroblasts, and PANC-1/fibroblast cocultures ( P = 0.25). Fibroblasts produced seven times higher amounts of TIMP-2 than PANC-1 cells (28.01 ± 1.21 versus 4.03 ± 0.01 ng/ml, P < 0.02). When cocultured with PANC-1 cells, overall TIMP-2 expression was reduced to 4.65 ± 0.23 ( P < 0.02) as demonstrated by ELISA and confirmed by Western blot (see Figure 1). Fibroblasts induced a 3-fold increase in PANC-1 tumor cell invasion. Fibroblast-induced PANC-1 tumor cell invasion was inhibited by an anti-MMP-9 Ab and up regulated by an anti-TIMP-2 Ab (see Figure 2, P < 0.02 compared to fibroblasts alone). Conclusion. Fibroblasts co-cultured with pancreatic cancer cells decrease overall TIMP-2 expression. The resultant increase in MMP-9 activity promotes tumor cell invasion.

Invasiveness and MMP Expression in Pancreatic Carcinoma

Background. Previous investigations have suggested that expression of matrix metalloproteinases (MMPs) may be related to increased invasiveness of various tumors. This study evaluated a possible relation between pancreatic tumor cell invasiveness and MMPs.Methods. A Matrigel invasion assay was performed with pancreatic tumor cell line SUIT-2 and its sublines S2-007, S2-013, S2-020, and S2-028. The degree of invasiveness of stimulated and unstimulated cell lines was correlated with MMP gene expression measured by RT-PCR and MMP protein product measured by gelatin zymography. Cell lines were stimulated by 12-O-tetradecanoylphorbol-13-acetate (TPA), concanavalin (Con-A), and polymerized collagen type I gel (Vitrogen).Results. For SUIT-2, S2-007, S2-013, S2-020, and S2-028, 3.2, 1.0, 4.1, 6.4, and 0.4%, respectively, of the cells invaded the Matrigel membrane. TPA, Con-A, and Vitrogen resulted in the up-regulation of MMP-2 in S2-020. TPA and Vitrogen resulted in up-regulation of MMP-9 in each of the cell lines, while Con-A could up-regulate MMP-9 expression only in SUIT-2. There was no constitutive expression of either MMP-2 or MMP-9 in SUIT-2 or its sublines. There was a positive relationship between Matrigel invasiveness and up-regulation of MMP-2 and MMP-9 expression.Conclusions. These data suggest that, while MMP-2 and MMP-9 are not constitutively expressed in pancreatic carcinoma cell lines, they may be up-regulated by TPA, Con-A, and Vitrogen. Since MMP-2 and MMP-9 expression correlated with degree of tumor cell invasiveness, the ability to up-regulate MMP-2 and MMP-9 expression may play a role in facilitating pancreatic tumor cell invasion.

Role of MT-MMPs and MMP-2 in pancreatic cancer progression.

Activation of matrix metalloproteinase-2 (MMP-2) by the membrane-type matrix metalloproteinases (MT-MMPs) has been associated with tumor progression. In the present study, we examined the role of MMP-2 and its activators MT1-MMP, MT2-MMP and MT3-MMP in pancreatic tumor cell invasion and the development of the desmoplastic reaction characteristic of pancreatic cancer tissues. Northern blot analyses revealed that transcript levels of MT1-MMP and MT2-MMP, but not MT3-MMP, were enhanced in pancreatic cancer tissues (n = 18) compared with both chronic pancreatitis (n = 9) and healthy pancreas (n = 9). A good correlation was found between MT1-MMP and both MMP-2 expression (p < 0.01) and activity in pancreatic cancer tissues. In addition, expression and activation of MMP-2 were strongly associated with the extent of the desmoplastic reaction in pancreatic cancer tissues. Invasion assays showed a good correlation between MMP-2 expression and activity and the invasive potential of pancreatic cancer cell lines. In cell lines with high levels of MMP-2 expression and activity, the MMP inhibitor Batimastat led to significant reduction of the number of invading cells. Our results suggest that MT1-MMP is involved in the progression of pancreatic cancer via activation of MMP-2. MMP-2 itself plays an important role in tumor cell invasion and appears to be associated with the development of the characteristic desmoplastic reaction in pancreatic cancer.

The Effect of Thrombospondin-1 and TGF-β1 on Pancreatic Cancer Cell Invasion

We have previously shown that thrombospondin-1 (TSP-1) and TGF-β1 upregulate the urokinase plasminogen activator (uPA) and its receptor (uPAR) and promote tumor cell invasion in breast cancer. To date, the effect of TSP-1 and TGF-β1 on the plasminogen/plasmin system in gastrointestinal epithelial malignancies has not been investigated. In this study, we determined the effect of TSP-1 and TGF-β1 on uPA and uPAR expression and on tumor cell invasion in pancreatic cancer. ASPC1 human pancreatic adenocarcinoma cells were incubated for 48 h on cell-conditioned media (CCM) either alone (Control) or with the addition of either TSP-1 (40 μg/ml) or TGF-β1 (5 ng/ml). uPA and uPAR expression were determined by ELISA. ASPC1 cell invasion was determined in a modified Boyden chamber type I collagen invasion assay. The upper chamber was treated with CCM either alone (Control) or with the addition of anti-uPA (10 μg/ml) or anti-uPAR (10 μg/ml). The lower chamber was treated with CCM either alone (Control) or with the addition of either TSP-1 (40 μg/ml) or TGF-β1 (5 ng/ml). TSP-1 and TGF-β1 induced a twofold increase on uPAR expression but only a slight increase on total uPA. Tumor cell invasion was upregulated 3.5 to 4.5-fold by TSP-1 and TGF-β1, respectively. Anti-uPA and anti-uPAR antibodies completely blocked the TSP-1 and TGF-β1-mediated pancreatic tumor cell invasion. We conclude that TSP-1 and TGF-β1 mediate pancreatic tumor cell invasion through upregulation of the plasminogen/plasmin system.