The concentration of the cleaved suPAR forms in pre- and postoperative plasma samples improves the prediction of survival in colorectal cancer: A nationwide multicenter validation and discovery study.

BACKGROUND AND OBJECTIVES: The aim was to evaluate the prognostic biomarker potential of the soluble urokinase-type plasminogen activator receptor (suPAR) in plasma samples collected pre- and postoperatively from patients resected for colorectal cancer (CRC). METHODS: Patients with CRC were recruited prospectively at six centers from 2006 to 2008. Preoperative plasma samples were available from 494 patients and from 328 of these patients at 6 months postoperatively. Determinations of intact soluble uPAR (suPAR) suPAR(I-III) and the cleaved forms suPAR(I-III) + (II-III) and uPAR(I) were performed. Clinical data were retrieved retrospectively. RESULTS: In a multivariable model based on preoperative plasma samples suPAR(I-III) + (II-III) and uPAR(I) showed an independent statistically significant association to long term survival. When including the change in biomarker level between the pre- and postoperatively samples the hazard ratios were 3.06 (95% confidence interval [CI], 1.78-5.28; P < .0001) and 2.24 (95% CI, 1.59-3.16; P < .0001) for suPAR(I-III) + (II-III) and uPAR(I), respectively. A one-unit decrease in biomarker levels between the pre- and postoperative levels resulted in a 55% and 34% reduction in the risk estimate of death for suPAR(I-III) + (II-III) and uPAR(I), respectively. CONCLUSION: This study validates previously findings regarding the prognostic significance of suPAR in preoperative samples. The inclusion of postoperative samples added further prognostic information.

International consensus guidelines for scoring the histopathological growth patterns of liver metastasis.

BACKGROUND: Liver metastases present with distinct histopathological growth patterns (HGPs), including the desmoplastic, pushing and replacement HGPs and two rarer HGPs. The HGPs are defined owing to the distinct interface between the cancer cells and the adjacent normal liver parenchyma that is present in each pattern and can be scored from standard haematoxylin-and-eosin-stained (H&E) tissue sections. The current study provides consensus guidelines for scoring these HGPs. METHODS: Guidelines for defining the HGPs were established by a large international team. To assess the validity of these guidelines, 12 independent observers scored a set of 159 liver metastases and interobserver variability was measured. In an independent cohort of 374 patients with colorectal liver metastases (CRCLM), the impact of HGPs on overall survival after hepatectomy was determined. RESULTS: Good-to-excellent correlations (intraclass correlation coefficient >0.5) with the gold standard were obtained for the assessment of the replacement HGP and desmoplastic HGP. Overall survival was significantly superior in the desmoplastic HGP subgroup compared with the replacement or pushing HGP subgroup (P=0.006). CONCLUSIONS: The current guidelines allow for reproducible determination of liver metastasis HGPs. As HGPs impact overall survival after surgery for CRCLM, they may serve as a novel biomarker for individualised therapies.

Microvessel density and endothelial cell proliferation levels in colorectal liver metastases from patients given neo-adjuvant cytotoxic chemotherapy and bevacizumab.

The treatment of patients with colorectal liver metastasis has improved significantly and first line therapy is often combined chemotherapy and bevacizumab, although it is unknown who responds to this regimen. Colorectal liver metastases grow in different histological growth patterns showing differences in angiogenesis. To identify possible response markers, histological markers of angiogenesis were assessed. Patients who underwent resection of colorectal liver metastasis at Rigshospitalet, Copenhagen, Denmark from 2007 to 2011 were included (n = 254) including untreated and patients treated with chemotherapy or chemotherapy plus bevacizumab. The resected liver metastases were characterised with respect to growth pattern, endothelial and tumour cell proliferation as well as microvessel density and tumour regression. Tumour regression grade of liver metastases differed significantly between untreated/chemotherapy treated patients in comparison to chemotherapy plus bevacizumab treated patients (both p < 0.0001). Microvessel density was decreased in liver metastases from patients treated with bevacizumab in comparison to those from untreated/chemotherapy-treated patients (p = 0.006/p = 0.002). Tumour cell proliferation assessed by Ki67 expression correlated to a shorter recurrence free survival in the total patient cohort. In conclusion, liver metastases from patients treated with neo-adjuvant chemotherapy and bevacizumab had significantly lower microvessel densities and tumour regression grades when compared to liver metastases from untreated or chemotherapy treated patients. This may indicate that bevacizumab treatment results in altered vascular biology and tumour viability, with possible tumour reducing effect.

Tissue inhibitor of matrix metalloproteinase-1 expression in colorectal cancer liver metastases is associated with vascular structures.

Metastatic growth by colorectal cancer cells in the liver requires the ability of the cancer cells to interact with the new microenvironment. This interaction results in three histological growth patterns of liver metastases: desmoplastic, pushing, and replacement. In primary colorectal cancer several proteases, involved in the degradation of extracellular matrix components, are up-regulated. In liver metastases, their expression is growth pattern dependent. Tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) is a strong prognostic marker in plasma from colorectal cancer patients, with significant higher levels in patients with metastatic disease. We therefore wanted to determine the expression pattern of TIMP-1 in primary colorectal cancers and their matching liver metastases. TIMP-1 mRNA was primarily seen in α-smooth-muscle actin (α-SMA)-positive cells. In all primary tumors and liver metastases with desmoplastic growth pattern, TIMP-1 mRNA was primarily found in α-SMA-positive myofibroblasts located at the invasive front. Some α-SMA-positive cells with TIMP-1 mRNA were located adjacent to CD34-positive endothelial cells, identifying them as pericytes. This indicates that TIMP-1 in primary tumors and liver metastases with desmoplastic growth pattern has dual functions; being an MMP-inhibitor at the cancer periphery and involved in tumor-induced angiogenesis in the pericytes. In the liver metastases with pushing or replacement growth patterns, TIMP-1 was primarily expressed by activated hepatic stellate cells at the metastasis/liver parenchyma interface. These cells were located adjacent to CD34-positive endothelial cells, suggesting a function in tumor-induced angiogenesis. We therefore conclude that TIMP-1 expression is growth pattern dependent in colorectal cancer liver metastases.

ADAM12 expression is upregulated in cancer cells upon radiation and constitutes a prognostic factor in rectal cancer patients following radiotherapy.

Radiotherapy is one of the most common cancer treatments, yet, some patients require high doses to respond. Therefore, the development of new strategies leans toward personalizing therapy to avoid unnecessary burden on cancer patients. This approach prevents the administration of ineffective treatments or uses combination strategies to increase the sensitivity of cancer cells. ADAM12 has been shown to be upregulated in many cancers and correlate with poor survival and chemoresistance, thus making it a potential candidate responsible for radioresistance. Here, we show that ADAM12 expression is upregulated in response to irradiation in both mouse and human cancer cells in vitro, as well as in tumor tissues from rectal cancer patients. Interestingly, the expression of ADAM12 following radiotherapy correlates with the initial disease stage and predicts the response of rectal cancer patients to the treatment. While we found no cell-autonomous effects of ADAM12 on the response of colon cancer cells to irradiation in vitro, depletion of ADAM12 expression markedly reduced the tumor growth of irradiated cancer cells when subcutaneously transplanted in syngeneic mice. Interestingly, loss of cancer cell-derived ADAM12 expression increased the number of CD31+FAP- cells in murine tumors. Moreover, conditioned medium from ADAM12-/- colon cancer cells led to increased tube formation when added to endothelial cell cultures. Thus, it is tempting to speculate that altered tumor vascularity may be implicated in the observed effect of ADAM12 on response to radiotherapy in rectal cancer. We conclude that ADAM12 represents a promising prognostic factor for stratification of rectal cancer patients receiving radiotherapy and suggest that targeting ADAM12 in combination with radiotherapy could potentially improve the treatment response.

Concomitant lack of MMP9 and uPA disturbs physiological tissue remodeling.

Urokinase-type plasminogen activator (uPA) and matrix metalloproteinase-9 (MMP9, gelatinase B) have separately been recognized to play important roles in various tissue remodeling processes. In this study, we demonstrate that deficiency for MMP9 in combination with ablation of either uPA- or tissue-type plasminogen activator (tPA)-catalyzed plasminogen activation is critical to accomplish normal gestation in mice. Gestation was also affected by simultaneous lack of MMP9 and the uPA receptor (uPAR). Interestingly, uPA-deficiency additionally exacerbated the effect of MMP9-deficiency on bone growth and an additive effect caused by combined lack in MMP9 and uPA was observed during healing of cutaneous wounds. By comparison, MMP9-deficiency combined with absence of either tPA or uPAR resulted in no significant effect on wound healing, indicating that the role of uPA during wound healing is independent of uPAR, when MMP9 is absent. Notably, compensatory upregulation of uPA activity was seen in wounds from MMP9-deficient mice. Taken together, these studies reveal essential functional dependency between MMP9 and uPA during gestation and tissue repair.

Expression of C4.4A in precursor lesions of pulmonary adenocarcinoma and squamous cell carcinoma.

The protein C4.4A, a structural homologue of the urokinase-type plasminogen activator receptor, is a potential new biomarker in non-small cell lung cancer, with high levels of expression recently shown to correlate to poor survival of adenocarcinoma patients. In this study, C4.4A immunoreactivity in precursor lesions of lung squamous cell carcinoma and adenocarcinoma was investigated by stainings with a specific anti-C4.4A antibody. In the transformation from normal bronchial epithelium to squamous cell carcinoma, C4.4A was weakly expressed in basal cell hyperplasia but dramatically increased in squamous metaplasia. This was confined to the cell membrane and sustained in dysplasia, carcinoma in situ, and the invasive carcinoma. The induction of C4.4A already at the stage of hyperplasia could indicate that it is a marker of very early squamous differentiation, which aligns well with our earlier finding that C4.4A expression levels do not provide prognostic information on the survival of squamous cell carcinoma patients. In the progression from normal alveolar epithelium to peripheral adenocarcinoma, we observed an unexpected, distinct cytoplasmic staining for C4.4A in a fraction of atypical adenomatous hyperplasias, while most bronchioloalveolar carcinomas were negative. Likewise, only a fraction of the invasive adenocarcinomas was positive for C4.4A. With a view to the prognostic impact of C4.4A in adenocarcinoma patients, this finding might suggest that C4.4A could be an early biomarker for a possibly more malignant subtype of this disease.

Prognosis in adenocarcinomas of lower oesophagus, gastro-oesophageal junction and cardia evaluated by uPAR-immunohistochemistry.

Adenocarcinomas of lower oesophagus, gastro-oesophageal junction and cardia in humans are highly invasive tumours with poor prognosis. The localisation of urokinase-type plasminogen activator receptor (uPAR) was determined in 66 patients; 60 with adenocarcinomas and six cases with Barrett's oesophagus. uPAR was expressed in nearly all cases of invasive adenocarcinomas by populations of cancer cells, macrophages and myofibroblasts at both the invasion front and the tumour core. In areas with high-grade dysplasia or with Barrett's metaplasia adjacent to the tumour tissue, no uPAR-immunoreactivity was found. High local expression of uPAR, therefore, appears to be a characteristic marker for invasive behaviour in this tumour, suggesting that uPAR's contribution to matrix degradation during invasive growth is a late event in carcinogenesis. Using a scoring system for semiquantitative estimation of uPAR-positivity on immmunohistochemically stained specimens, a significant association was found between poor overall survival and high uPAR-score for cancer cells in the tumour core and for macrophages peripherally at the tumour invasion zone. In multivariate analysis, these two uPAR-scores were confirmed as highly significant prognostic parameters independent of Tumour, Node, Metastasis (TNM)-stage and World Health Organization (WHO) classification. The proteolytic action of these malignant and nonmalignant accessory cells thus seemed to follow two main patterns: one dominated by uPAR positive cancer cells and one by uPAR-positive macrophages. Scoring of uPAR-positivity might be a useful parameter for onset of invasion and prognosis in these adenocarcinomas.

Urokinase plasminogen activator receptor on invasive cancer cells: a prognostic factor in distal gastric adenocarcinoma.

Gastric cancer is the second cancer causing death worldwide. The five-year survival for this malignancy is below 25% and few parameters have shown an impact on the prognosis of the disease. The receptor for urokinase plasminogen activator (uPAR) is involved in extracellular matrix degradation by mediating cell surface associated plasminogen activation, and its presence on gastric cancer cells is linked to micrometastasis and poor prognosis. Using immunohistochemistry, the prognostic significance of uPAR was evaluated in tissue samples from a retrospective series of 95 gastric cancer patients. uPAR was expressed by neoplastic cells, macrophages, myofibroblasts and neutrophils in both intestinal and diffuse subtypes. No association was demonstrated between the expression of uPAR on cancer cells and histological subtype (p = 0.64) or TNM stage (p = 0.75). Univariate analysis revealed a significant association between the expression of uPAR on tumor cells in the peripheral invasion zone and overall survival of gastric cancer patients (HR = 2.16; 95% CI: 1.13-4.14; p = 0.02). Multivariate analysis showed that uPAR immunoreactivity in cancer cells at the invasive front is an independent prognostic factor for overall survival in gastric cancer (HR = 2.39; 95% CI: 1.22-4.69; p = 0.011). In consequence, scoring of uPAR-positive cancer cells may be a direct measure for the invasive potential of gastric adenocarcinomas.

Circulating Forms of Urokinase-Type Plasminogen Activator Receptor in Plasma Can Predict Recurrence and Survival in Patients with Urothelial Carcinoma of the Bladder.

Urothelial carcinoma of the bladder is a highly aggressive disease characterised by a very heterogeneous clinical outcome. Despite cystectomy, patients still have a high recurrence risk and shortened survival. Urokinase-type plasminogen activator receptor (uPAR) is present in tumour tissue specimens from patients with urothelial carcinoma. The different uPAR forms in blood are strong prognostic markers in other cancer types. We investigate the presence of different uPAR forms in tumour tissue and test the hypothesis that preoperative plasma levels of the uPAR forms predict recurrence free survival, cancer specific survival, and overall survival in patients treated with cystectomy for urothelial carcinoma. Using Western blotting we analyse neoplasia and adjacent benign-appearing urothelium from randomly selected patients for the presence of intact and cleaved uPAR forms. Prospectively collected preoperative plasma samples from 107 patients who underwent radical cystectomy for urothelial carcinoma are analysed. The different uPAR forms are measured by time-resolved fluorescence immunoassays. uPAR in tumour tissue from patients with urothelial carcinoma is demonstrated in both an intact and cleaved form. The different uPAR forms in plasma are all significantly associated with both recurrence free survival, cancer specific survival, and overall survival, high concentrations predicting short survival. uPAR (I) has the strongest association with a HR of 2.56 for overall survival. In the multivariable survival analysis uPAR (I) is significantly associated with cancer specific survival and overall survival.

Urokinase plasminogen activator receptor is expressed in invasive cells in gastric carcinomas from high- and low-risk countries.

Gastric cancer is the second cancer causing death worldwide. Both incidence and mortality rates vary according to geographical regions. The receptor for urokinase plasminogen activator (uPAR) is involved in extracellular matrix degradation by mediating cell surface associated plasminogen activation, and its presence on gastric cancer cells is linked to micro-metastasis and poor prognosis. Immunohistochemical analyses of a set of 44 gastric cancer lesions from Costa Rica showed expression of uPAR in cancer cells in both intestinal subtype (14 of 27) and diffuse subtype (10 of 17). We compared the expression pattern of uPAR in gastric cancers from a high-risk country (Costa Rica) with a low-risk country (Norway). We found uPAR on gastric cancer cells in 24 of 44 cases (54%) from Costa Rica and in 13 of 23 cases (56%) from Norway. uPAR was seen in macrophages and neutrophils in all cases. We also examined the nonneoplastic mucosa and found that uPAR was more frequently seen in epithelial cells located at the luminal edge of the crypts in cases with Helicobacter pylori infection than in similar epithelial cells in noninfected mucosa (p = 0.033; chi(2) = 4.54). In conclusion, the expression of uPAR in cancer cells in more than half of the gastric cancer cases suggests that their uPAR-positivity do not contribute to explain the different mortality rates between the 2 countries, however, the actual prevalence of uPAR-positive cancer cells in the gastric cancers may still provide prognostic information.

Purification and characterization of recombinant full-length and protease domain of murine MMP-9 expressed in Drosophila S2 cells.

Matrix metalloproteinase-9 (MMP-9) is a 92-kDa soluble pro-enzyme implicated in pathological events including cancer invasion. It is therefore an attractive target for therapeutic intervention studies in mouse models. Development of inhibitors requires sufficient amounts of correctly folded murine MMP-9. Constructs encoding zymogens of full-length murine MMP-9 and a version lacking the O-glycosylated linker region and hemopexin domains were therefore generated and expressed in stably transfected Drosophila S2 insect cells. After 7 days of induction the expression levels of the full-length and truncated versions were 5 mg/l and 2 mg/l, respectively. The products were >95% pure after gelatin Sepharose chromatography and possessed proteolytic activity when analyzed by gelatin zymography. Using the purified full-length murine MMP-9 we raised polyclonal antibodies by immunizations of rabbits. These antibodies specifically identified pro-MMP-9 in incisional skin wound extracts from mice when used for Western blotting. Immunohistochemical analysis of paraffin embedded skin wounds from mice showed that MMP-9 protein was localized at the leading-edge keratinocytes in front of the migrating epidermal layer. No immunoreactivity was observed when the antibody was probed against skin wound material from MMP-9 deficient mice. In conclusion, we have generated and purified two proteolytically active recombinant murine MMP-9 protein constructs, which are critical reagents for future cancer drug discovery studies.

Helicobacter pylori Colonization Drives Urokinase Receptor (uPAR) Expression in Murine Gastric Epithelium During Early Pathogenesis.

(1) Background: Persistent Helicobacter pylori infection is the most important risk factor for gastric cancer. The urokinase receptor (uPAR) is upregulated in lesions harboring cancer invasion and inflammation. Circumstantial evidence tends to correlate H. pylori colonization with increased uPAR expression in the human gastric epithelium, but a direct causative link has not yet been established in vivo; (2) Methods: In a mouse model of H. pylori-induced gastritis, we investigated the temporal emergence of uPAR protein expression in the gastric mucosa in response to H. pylori (SS1 strain) infection; (3) Results: We observed intense uPAR immunoreactivity in foveolar epithelial cells of the gastric corpus due to de novo synthesis, compared to non-infected animals. This uPAR induction represents a very early response, but it increases progressively over time as do infiltrating immune cells. Eradication of H. pylori infection by antimicrobial therapy causes a regression of uPAR expression to its physiological baseline levels. Suppression of the inflammatory response by prostaglandin E2 treatment attenuates uPAR expression. Notwithstanding this relationship, H. pylori does induce uPAR expression in vitro in co-cultures with gastric cancer cell lines; (4) Conclusions: We showed that persistent H. pylori colonization is a necessary event for the emergence of a relatively high uPAR protein expression in murine gastric epithelial cells.

Activation of Adenosine Monophosphate-Activated Protein Kinase Reduces the Onset of Diet-Induced Hepatocellular Carcinoma in Mice.

The worldwide obesity and type 2 diabetes epidemics have led to an increase in nonalcoholic fatty liver disease (NAFLD). NAFLD covers a spectrum of hepatic pathologies ranging from simple steatosis to nonalcoholic steatohepatitis, characterized by fibrosis and hepatic inflammation. Nonalcoholic steatohepatitis predisposes to the onset of hepatocellular carcinoma (HCC). Here, we characterized the effect of a pharmacological activator of the intracellular energy sensor adenosine monophosphate-activated protein kinase (AMPK) on NAFLD progression in a mouse model. The compound stimulated fat oxidation by activating AMPK in both liver and skeletal muscle, as revealed by indirect calorimetry. This translated into an ameliorated hepatic steatosis and reduced fibrosis progression in mice fed a diet high in fat, cholesterol, and fructose for 20 weeks. Feeding mice this diet for 80 weeks caused the onset of HCC. The administration of the AMPK activator for 12 weeks significantly reduced tumor incidence and size. Conclusion: Pharmacological activation of AMPK reduces NAFLD progression to HCC in preclinical models.

Two distinct expression patterns of urokinase, urokinase receptor and plasminogen activator inhibitor-1 in colon cancer liver metastases.

Metastatic growth and invasion by colon cancer cells in the liver requires the ability of the cancer cells to interact with the new tissue environment. Plasmin(ogen) is activated on cell surfaces by urokinase-type PA (uPA), and is regulated by uPAR and plasminogen activator inhibitor-1 (PAI-1). To compare the expression patterns of uPA, uPAR and PAI-1 in colon cancer with that in their liver metastases, we analysed matched samples from 14 patients. In all 14 primary colon cancers, we found upregulation of uPAR, uPA mRNA and PAI-1 in primarily stromal cells at the invasive front. In 5 of the 14 liver metastases, we found intense expression of uPAR, uPA-mRNA and PAI-1 in primarily stromal cells at the metastases periphery, and in an expression pattern similar to that found in the primary tumours. In the remaining 9 liver metastases, uPAR and uPA-mRNA were only seen associated with the presence of necrosis within the liver metastases. In addition, PAI-1-immunoreactivity was in all liver metastases seen in hepatocytes at the metastases periphery. Interestingly, the former 5 liver metastases positive for uPAR, uPA mRNA and PAI-1 at the metastasis periphery all had a predominantly desmoplastic reaction, whereas 8 of the remaining 9 showed direct contact between the cancer cells and the liver parenchyma. We conclude that there are 2 distinct patterns of expression of uPAR, uPA and PAI-1 in colon cancer liver metastases and that these correlate closely with 2 morphological growth patterns. These findings may have implication for the treatment of patients with metastatic disease.

Expression of cancer stem cell markers is prognostic in metastatic gastroesophageal adenocarcinoma.

Gastroesophageal adenocarcinoma is a common and highly lethal malignancy. Cancer stem cells (CSCs) have a key role in the development and progression of metastatic disease. While expression of CSC markers CD44, CD133 and aldehyde dehydrogenase 1 (ALDH1) in locoregional gastroesophageal cancer is known to be associated with poorer clinical outcomes, the significance of CSC marker expression in distal metastatic disease is unknown. We investigated the clinicopathological and prognostic associations of the CSC markers, CD44, CD133, and ALDH1, on metastatic deposits from gastroesophageal adenocarcinomas, and evaluated the association of CSC expression with urokinase-type plasminogen activator receptor (uPAR) expression. Of the 36 patients included in the study, 16 (44%) were positive for CD44, 13 (36%) were positive for CD133, and 26 (72%) were positive for ALDH1. CD44 expression was significantly associated with poorer overall survival (OS) in univariate [hazard ratio (HR) 2.9, 95% confidence interval (CI) 1.3-6.9, p=0.008] and multivariate analyses (HR 2.5, 95%CI 1.1-6.2, p=0.04). ALDH1 expression was significantly associated with poorer OS in univariate (HR 2.4, 95% CI 1.01-5.7, p=0.04) analysis but was not significant in multivariate analysis. Both CD44 and ALDH1 expression were significantly associated with uPAR expression. We found no association between CD133 expression and OS. CD44 expression on metastatic disease from gastroesophageal adenocarcinomas is an independent prognostic marker associated with poorer OS. These results expand current evidence to support the role of CSCs as biomarkers in metastatic gastroesophageal cancer.

Altered expression of the urokinase receptor homologue, C4.4A, in invasive areas of human esophageal squamous cell carcinoma.

C4.4A is a glycolipid-anchored membrane protein with structural homology to the urokinase-type plasminogen activator receptor (uPAR). Although C4.4A was identified as a metastasis-associated protein little is known about its actual expression and possible function in malignant disease. In the present study, we have therefore analyzed the expression of C4.4A in 14 esophageal squamous cell carcinomas (ESCC). Normal squamous esophageal epithelium shows a strong cell surface associated C4.4A expression in the suprabasal layers, whereas basal cells are negative. Upon transition to dysplasia and carcinoma in situ the expression of C4.4A is abruptly and coordinately weakened. Double immunofluorescence staining of normal and dysplastic tissue showed that C4.4A colocalizes with the epithelial cell surface marker E-cadherin in the suprabasal cells and has a complementary expression pattern compared to the proliferation marker Ki-67. A prominent, but frequently intracellular, C4.4A expression reappeared in tumor cells located at the invasive front and local lymph node metastases. Because C4.4A was reported previously to be a putative laminin-5 (LN5) ligand, and both proteins are expressed by invasive tumor cells, we analyzed the possible coexpression of C4.4A and the gamma 2-chain of LN5 (LN5-gamma 2). Although these proteins are indeed expressed by either neighboring cancer cells or in a few cases even coexpressed by the same cells in the tumor front and metastases, we found no evidence for a general colocalization in the extracellular compartment by confocal microscopy. In conclusion, C4.4A is expressed during invasion and metastasis of human ESCC and may thus provide a new histological marker in this disease.

Crohn's disease but not chronic ulcerative colitis induces the expression of PAI-1 in enteric neurons.

OBJECTIVES: Chronic inflammation of the intestinal wall is the common characteristic of Crohn's disease and ulcerative colitis; disorders, which in some cases can be difficult to distinguish. The inflammation also affects the local neuronal plexuses of the enteric nervous system. It is known that plasminogen activator inhibitor-1 (PAI-1) and urokinase receptor (uPAR) are upregulated in neurons after experimental peripheral nerve injury and have been linked to nerve regeneration. METHODS: The expression of PAI-1 and uPAR in neuronal cells in lesions of the gastrointestinal tract was analyzed by immunohistochemical techniques. RESULTS: PAI-1 was found in a subset of neurons primarily located in the submucosal plexus of the small and large intestine in 24 of 28 cases (86%) with Crohn's disease, but in none of 17 cases with chronic ulcerative colitis and other severe inflammatory conditions in the intestinal wall. The PAI-1 was seen in the perikarya of the neurons and a few proximal axons, whereas nerves were negative. uPAR was seen in nerves in all types of lesion varying from 21% to 88% of the cases, most frequent in colon adenocarcinomas. No uPAR-positive nerves were detected in normal colon. CONCLUSIONS: PAI-1-positive neurons in inflammatory bowel disease are linked to chronic inflammation in Crohn's disease, implying PAI-1 as a potential parameter for the differential diagnosis between Crohn's disease and ulcerative colitis. The findings also suggest that PAI-1 in neurons is related to pain and that both PAI-1 and uPAR are involved in neuronal repair in the inflamed tissue.

Discrimination of different forms of the murine urokinase plasminogen activator receptor on the cell surface using monoclonal antibodies.

The urokinase plasminogen activator receptor (uPAR) is a versatile three-domain GPI-anchored protein, which binds urokinase plasminogen activator (uPA) and thereby focalises plasminogen activation on the cell surface. Generation of a proteolytic potential is essential in both normal physiological and pathological extracellular tissue remodelling processes. uPA can also cleave uPAR, resulting in liberation of the amino-terminal domain I, which encompasses binding sites for both uPA and the adhesion molecule, vitronectin. In order to localise the different uPAR forms on the plasma membrane of murine monocyte macrophage-like P388D.1 cells, we have now generated and characterised two high-affinity murine mAbs, mR3 and mR4, raised against murine uPAR. mR3 was found to recognise an epitope located in domain I of uPAR. Surface plasmon resonance analyses and cell binding studies revealed that this mAb was able to bind preformed complexes of murine pro-uPA and murine uPAR. In contrast, mR4 recognises domains II-III in uPAR and does not bind preformed pro-uPA-uPAR complexes in similar analyses. Immunofluorescence microscopy of P388D.1 cells revealed that mR3 stained the cells equally well in the presence or absence of saturation with the amino-terminal fragment of uPA, ATF. However, the signal intensity obtained using another uPAR domain I specific mAb, mR1, was significantly reduced upon ATF saturation. Furthermore, when adding ATF, mR4 selectively stained the cleaved receptor. Applying these newly generated mAbs, we additionally demonstrated that cleaved and intact uPAR was evenly distributed on the surface of these cells.

MMP-9 is differentially expressed in primary human colorectal adenocarcinomas and their metastases.

Matrix metalloproteinase-9 (MMP-9) is up-regulated in macrophages in various human cancer types. In human colon cancer, MMP-9 is expressed in a macrophage subpopulation located at the tumor edge, indicating a specific induction of MMP-9 in macrophages in direct association with cancer invasion. To test whether MMP-9 is also induced in tumor edge macrophages in metastases from colorectal adenocarcinomas, we have compared the expression pattern of MMP-9 in primary colorectal adenocarcinomas (n = 15) with that in liver metastases (n = 15) and local lymph node metastases (n = 7) from the same patients by in situ hybridization and immunohistochemistry. In all the colorectal adenocarcinomas, the expression of MMP-9 mRNA and immunoreactivity in macrophages was located at the invasive front. In contrast, only 3 of the 15 liver metastases had MMP-9 mRNA and immunoreactivity at the periphery, and this expression was confined to small foci of macrophages located either among lymphocytes or in a dense desmoplastic stroma. Expression of MMP-9 mRNA and immunoreactivity was in all liver metastases seen in macrophages located in the lumen of malignant glandular structures and in central necrotic tissue. In all the 7 lymph node metastases, MMP-9 mRNA and immunoreactivity was seen in macrophages located in the stromal tissue surrounding the metastases. We conclude that MMP-9 is not up-regulated in tumor edge macrophages in liver metastases like in their primary tumor and local lymph node metastases, suggesting that disseminating colorectal cancer cells can adopt alternative proteolytic mechanisms for invasion depending on the local microenvironment.