Martin Illemann

Stromal cells associated with early invasive foci in human mammary ductal carcinoma in situ coexpress urokinase and urokinase receptor

The transition from ductal carcinoma in situ (DCIS) of the breast to invasive ductal carcinoma is facilitated by proteolytic degradation of basement membrane. The transition can be identified as microinvasive foci in a small proportion of DCIS lesions. We have previously found that MMP‐13 is frequently expressed in such foci. To establish whether plasmin‐directed proteolysis is likely to be involved in early invasion, we have here studied the expression of urokinase plasminogen activator (uPA) and its receptor (uPAR) in human DCIS lesions with and without microinvasion. uPA mRNA was detected in periductal stromal cells in all of 9 DCIS lesions with microinvasion and in 2 of 9 DCIS lesions without microinvasion by in situ hybridization. The uPA mRNA signal was seen in numerous stromal cells in microinvasive areas together with MMP‐13 mRNA expressing cells. Double immunofluorescence analyses, using emission fingerprinting, showed that the uPA expressing stromal cells included both myofibroblasts and macrophages. The early invasive carcinoma cells were negative for uPA. uPAR immunoreactivity was focally upregulated in periductal stromal cells in all of the 9 DCIS lesions with microinvasion and in only 2 of the 9 DCIS lesions without microinvasion. uPAR was seen in both macrophages and myofibroblasts in microinvasive areas, and it was evident that uPA and uPAR colocalized in both fibroblast‐like cells and macrophage‐like cells. We conclude that periductal macrophages and myofibroblasts are strongly involved in the initial steps of breast cancer invasion by focally upregulating the expression of the plasminogen activation system and MMP‐13.

The Multifaceted Role of the Microenvironment in Liver Metastasis: Biology and Clinical Implications

The liver is host to many metastatic cancers, particularly colorectal cancer, for which the last 2 decades have seen major advances in diagnosis and treatment. The liver is a vital organ, and the extent of its involvement with metastatic disease is a major determinant of survival. Metastatic cells arriving in the liver via the bloodstream encounter the microenvironment of the hepatic sinusoid. The interactions of the tumor cells with hepatic sinusoidal and extrasinusoidal cells (endothelial, Kupffer, stellate, and inflammatory cells) determine their fate. The sinusoidal cells can have a dual role, sometimes fatal to the tumor cells but also facilitatory to their survival and growth. Adhesion molecules participate in these interactions and may affect their outcome. Bone marrow-derived cells and chemokines also play a part in the early battle for survival of the metastases. Once the tumor cells have arrested and survived the initial onslaught, tumors can grow within the liver in 3 distinct patterns, reflecting differing host responses, mechanisms of vascularization, and proteolytic activity. This review aims to present current knowledge of the interactions between the host liver cells and the invading metastases that has implications for the clinical course of the disease and the response to treatment.

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. (Mol Cancer Res 2006;4(5):293–302)

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.

Localization of tissue inhibitor of metalloproteinases 1 (TIMP‐1) in human colorectal adenoma and adenocarcinoma

Tissue inhibitor of matrix metalloproteases 1 (TIMP‐1) inhibits the proteolytic activity of matrix metalloproteases and hereby prevents cancer invasion. However, TIMP‐1 also possesses other functions such as inhibition of apoptosis, induction of malignant transformation and stimulation of cell‐growth. We have previously demonstrated that TIMP‐1 is elevated in blood from colorectal cancer patients and that high TIMP‐1 levels predict poor prognosis. To clarify the role of TIMP‐1 in colorectal tumorigenesis, the expression pattern of TIMP‐1 in benign and malignant colorectal tumors was studied. In all of 24 cases of colorectal adenocarcinoma TIMP‐1 mRNA was detected by in situ hybridization. In all cases TIMP‐1 expression was found in fibroblast‐like cells located at the invasive front but was seen only sporadically in normal mucosa. No TIMP‐1 mRNA was seen in any of the cases in benign or malignant epithelial cells, in vascular cells or smooth muscle cells. Comparison of sections processed for TIMP‐1 in situ hybridization with sections immunohistochemically stained with antibodies against TIMP‐1 showed good correlation between TIMP‐1 mRNA and immunoreactivity. Combining TIMP‐1 in situ hybridization with immunohistochemical staining for α‐smooth muscle actin or CD68 showed TIMP‐1 mRNA in myofibroblasts but not in macrophages. TIMP‐1 mRNA was detected in 2 of 7 adenomatous polyps in the adenoma area: in both cases associated with focal stromal inflammation at the epithelial‐stromal interface. In conclusion, TIMP‐1 expression is a rare event in benign human colon tissue but is highly expressed by myofibroblasts in association with invading colon cancer cells.

uPAR as anti-cancer target: evaluation of biomarker potential, histological localization, and antibody-based therapy.

Degradation of proteins in the extracellular matrix is crucial for the multistep process of cancer invasion and metastasis. Compelling evidence has demonstrated the urokinase receptor (uPAR) and its cognate ligand, the urokinase plasminogen activator (uPA), to play critical roles in the concerted action of several proteolytic systems in generation of a high proteolytic potential required for tissue remodeling processes. uPAR is additionally cleaved by uPA on the cell surface, liberating domain I, resulting in abrogated pericellular proteolysis. The expression of both uPAR and uPA is significantly up-regulated during cancer progression and is primarily confined to the tumor-associated stromal compartment. Furthermore, both uPAR and uPA have proven to be prognostic markers in several types of cancer; high levels indicating poor survival. The cleaved forms of uPAR are also prognostic markers, and a potential diagnostic and predictive impact of the different uPAR forms has been reported. Hence, pericellular proteolysis seems to be a suitable target for anti-cancer therapy and numerous approaches have been pursued. Targeting of this process may be achieved by preventing the binding of uPA to uPAR on the cell surface and/or by direct inhibition of the catalytic activity of uPA. Both strategies have been pursued and inhibition of these functions has shown effect in xenogenic cancer models. Pericellular proteolysis has also been inhibited in vivo in mouse models of wound healing and hepatic fibrinolysis using mouse monoclonal antibodies (mAbs) against mouse uPA or uPAR. These reagents will target uPA and uPAR in both stromal cells and cancer cells, and their therapeutic potential can now be assessed in syngenic mouse cancer models.

Leading-Edge Myofibroblasts in Human Colon Cancer Express Plasminogen Activator Inhibitor-1

Plasminogen activator inhibitor-1 (PAI-1) is up-regulated strongly in various cancer tissues, including colon cancer tissue. Highly specific rabbit polyclonal antibodies against PAI-1 were used for immunohistochemical localization of PAI-1 in 12 invasive colorectal adenocarcinomas. PAI-1 immunoreactivity was observed in endothelial cells of some vessels located in the submucosa and in several fibroblast-like cells located at the invasive front. No PAI-1 immunoreactivity was seen in cancer cells in any of the 12 cases. Double immunofluorescence using the PAI-1 antibodies together with antibodies against alpha-smooth muscle actin for myofibroblast/smooth muscle cells and CD34 for endothelial cells showed that more than 80% of the PAI-1-positive fibroblast-like cells in all 12 cases were myofibroblasts. In 4 of 12 cases, a few of the PAI-1-positive fibroblast-like cells in the invasive front were CD34+. We conclude that the majority of PAI-1-positive fibroblast-like cells located at the leading edge of the invasive colon cancers are myofibroblasts.

Neutrophil gelatinase-associated lipocalin (NGAL/Lcn2) is upregulated in gastric mucosa infected with Helicobacter pylori.

Helicobacter pylori infection is one of the most significant risk factors for gastric cancer. The infection is established early in life and persists lifelong leading to a sustained chronic inflammation. Iron is essential for most living organisms. Bacteria use several mechanisms to acquire iron from their hosts, including the synthesis of the potent iron chelators known as siderophores. Hosts cells may express the siderophore-binding protein neutrophil gelatinase-associated lipocalin (NGAL/lipocalin-2 (Lcn2)) in response to infection, thus preventing bacterial iron uptake. We have characterized here the pattern of expression of NGAL/Lcn2 in gastric mucosa (45 non-neoplastic and 38 neoplastic tissue samples) and explored the connection between NGAL/Lcn2 expression and H. pylori infection. Immunohistochemical analysis showed high NGAL/Lcn2 expression in normal and gastritis-affected mucosa compared to low expression in intestinal metaplasia, dysplasia, and gastric cancer. In normal and gastritis-affected mucosa (n = 36 tissue samples), NGAL/Lcn2 was more frequently seen in epithelial cells located at the neck and base of the glands in H. pylori-positive cases than in similar epithelial cells of noninfected cases (Fisher’s exact test, p = 0.04). In conclusion, the high expression of NGAL/Lcn2 in normal and gastritis-affected mucosa infected with H. pylori suggests that NGAL/Lcn2 is upregulated locally in response to this bacterial infection. It is discussed whether this may have a causal relation to the development of gastric 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 γ2‐chain of LN5 (LN5‐γ2). 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.

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.