Sirpa Salo

EXPRESSION OF THE LAMININ γ2 CHAIN IN PANCREATIC ADENOCARCINOMA

Forty‐two pancreatic adenocarcinomas were investigated immunohistochemically and by in situ hybridization for the expression of the laminin γ2 chain. In 41 cases, intracytoplasmic immunoreactivity for the γ2 chain was seen. Positive tumour cells were located especially at the epithelial–stromal interface of the tumour cell islands. In 22 cases, diffuse laminin γ2 chain immunoreactivity could also be seen in stroma and in seven cases, occasional positivity was detected in the neoplastic basement membranes. Signals for laminin γ2 chain mRNA in tumour cells displayed a distribution similar to that observed on immunohistochemistry. There were significantly more cases with less than 20 per cent of laminin γ2 chain‐positive tumour cells in tumours extending to peripancreatic tissues and/or tumours with regional or distant metastases (P=0·029). A corresponding statistical significance could also be noted in the mRNA level (P=0·025). The results show that pancreatic adenocarcinomas display a high activity of laminin γ2 chain synthesis. Tumours with a strong laminin γ2 chain synthesis show a lower invasive and metastatic potential than tumours with a weak or moderate laminin γ2 chain expression.

Altered distribution and synthesis of laminin‐5 (kalinin) in oral lichen planus, epithelial dysplasias and squamous cell carcinomas

Laminin‐5 is a glycoprotein which mediates epithelial cell adhesion to the basement membrane. This study describes the distribution and synthesis of laminin‐5 in oral lichen planus, epithelial dysplasias, squamous cell carcinomas and a lymph node metastasis using immunohistochemistry and in situ hybridization. In normal oral mucosa and lichen planus, immunoreaction to the laminin‐5 was seen as a thin continuous, delicate line in the basement membrane region, although slight irregularities in the thickness and intensity of the immunoreaction could be detected in some cases with lichen planus. In epithelial dysplasias, the laminin‐5 staining was discontinuous and more diffuse compared to lichen planus and normal mucosa. The immunoreaction was generally extracellular, although in some cases with lichen planus and epithelial dysplasia there were a few basal epithelial cells showing cytoplasmic staining. The invasive carcinomas and the lymph node metastasis showed a striking, intense cytoplasmic, staining of the carcinoma cells along the invasive border of the neoplastic islands and in individual infiltrating carcinoma cells. Using in situ hybridization, the laminin‐5 γ2 chain mRNA expression could not be detected in normal oral mucosa whereas, in non‐dysplastic lichen planus and, more strongly, in dysplasias, there was a clear increase in the expression of laminin‐5 mRNA in the basal epithelial cells. The most intensive signal was detected in the invasive front of the oral squamous cell carcinomas and the lymph node metastasis. We conclude that, in oral squamous cell carcinoma, there is altered synthesis and secretion of laminin‐5 mRNA and protein. It is also evident that in dysplastic lesions of oral epithelium the synthesis and distribution of laminin‐5 is abnormal.

A Novel Organotypic Model Mimics the Tumor Microenvironment

Carcinoma cell invasion is traditionally studied in three-dimensional organotypic models composed of type I collagen and fibroblasts. However, carcinoma cell behavior is affected by the various cell types and the extracellular matrix (ECM) in the tumor microenvironment. In this study, a novel organotypic model based on human uterine leiomyoma tissue was established and characterized to create a more authentic environment for carcinoma cells. Human tongue squamous cell carcinoma cells (HSC-3) were cultured on top of either collagen or myoma. Organotypic sections were examined by immunohistochemistry and in situ hybridization. The maximal invasion depth of HSC-3 cells was markedly increased in myomas compared with collagen. In myomas, various cell types and ECM components were present, and the HSC-3 cells only expressed ECM molecules in the myoma model. Organotypic media were analyzed by radioimmunoassay, zymography, or Western blotting. During carcinoma cell invasion, matrix metalloprotease-9 production and collagen degradation were enhanced particularly in the myoma model. To evaluate the general applicability of the myoma model, several oral carcinoma, breast carcinoma, and melanoma cell lines were cultured on myomas and found to invade in highly distinct patterns. We conclude that myoma tissue mimics the native tumor microenvironment better than previous organotypic models and possibly enhances epithelial-to-mesenchymal transition. Thus, the myoma model provides a promising tool for analyzing the behavior of carcinoma cells.

Expression of the laminin γ2 chain in different histological types of lung carcinoma. A study by immunohistochemistry and in situ hybridization

Sixty‐four malignant lung tumours and 12 of their regional lymph node metastases were analysed for expression of the laminin γ2 chain by immunohistochemistry and in situ hybridization. Expression of the laminin γ2 chain was strongest in squamous cell carcinomas, followed by adenocarcinomas and large cell carcinomas. Positive cells, except for large cell carcinomas, were located at the epithelial–stromal interface of tumour clusters. An important exception was small cell lung carcinoma, with only a low level of laminin γ2 chain expression. Apart from tumour type, this may reflect the relatively scanty fibrous stroma in these tumours and supports previous observations that small cell lung carcinoma cells, contrary to other types, lack surface expression of α6β4 integrin, the specific laminin‐5 binding receptor. In frozen sections, immunohistochemistry showed linear basement membranes around tumour clusters in squamous cell carcinomas and adenocarcinomas. This shows that carcinoma cells are capable of heavy deposition of the laminin γ2 chain around tumour clusters and suggests that a laminin γ2 chain‐containing substrate may be of significance for the spread and growth of malignant tumours. Copyright

Chain specificity assignment of monoclonal antibodies to human laminins by using recombinant laminin β1 and γ1 chains

Abstract In the present study, the chain specificity of 16 commonly used monoclonal antibodies to human laminin(s) was analysed by using recombinant laminin β1 and γ1 chains. By ELISA, all antibodies reacted with purified placenta laminin, and most antibodies recognised either recombinant β1 or γ1 chains. Reactivity and chain specificity was confirmed against the recombinant chains in Western blotting under non-reducing conditions, and only a few antibodies were reactive under reducing conditions. Most antibodies were able to immunoprecipitate associated laminin β1/γ1 chains from platelet lysates. Based on these results and data from the literature, a tentative epitope map is presented.

Snail promotes an invasive phenotype in lung carcinoma

BackgroundSnail is a transcriptional factor which is known to influence the epitheliomesenchymal transition (EMT) by regulating adhesion proteins such as E-cadherin and claudins as well as matrix metalloproteases (MMP).MethodsTo evaluate the functional importance of snail, a transciptional factor involved in EMT in lung tumors, we investigated its expression in a large set of lung carcinomas by immunohistochemistry. Expression of snail and effects of snail knockdown was studied in cell lines.ResultsNuclear snail expression was seen in 21% of cases this being strongest in small cell lung carcinomas (SCLC). There was significantly greater snail expression in SCLC compared to squamous cell or adenocarcinoma. Positive snail expression was associated with poor survival in the whole material and separately in squamous cell and adenocarcinomas. In Cox regression analysis, snail expression showed an independent prognostic value in all of these groups. In several cell lines knockdown of snail reduced invasion in both matrigel assay and in the myoma tissue model for invasion. The influence of snail knockdown on claudin expression was cell type specific. Snail knockdown in these cell lines modified the expression of MMP2 and MMP9 but did not influence the activation of these MMPs to any significant degree.ConclusionsThe results show that snail plays an important role in the invasive characteristics of lung carcinoma influencing the survival of the patients. Snail knockdown might thus be one option for targeted molecular therapy in lung cancer. Snail knockdown influenced the expression of claudins individually in a cell-line dependent manner but did not influence MMP expressions or activations to any significant degree.

Molecular crosstalk between cancer cells and tumor microenvironment components suggests potential targets for new therapeutic approaches in mobile tongue cancer

We characterized tumor microenvironment (TME) components of mobile tongue (MT) cancer patients in terms of overall inflammatory infiltrate, focusing on the protumorigenic/anti‐inflammatory phenotypes and on cancer‐associated fibroblasts (CAFs) in order to determine their interrelations and associations with clinical outcomes. In addition, by culturing tongue carcinoma cells (HSC‐3) on a three‐dimensional myoma organotypic model that mimics TME, we attempted to investigate the possible existence of a molecular crosstalk between cancer cells and TME components. Analysis of 64 cases of MT cancer patients revealed that the overall density of the inflammatory infiltrate was inversely correlated to the density of CAFs (P = 0.01), but that the cumulative density of the protumorigenic/anti‐inflammatory phenotypes, including regulatory T cells (Tregs, Foxp3+), tumor‐associated macrophages (TAM2, CD163+), and potentially Tregs‐inducing immune cells (CD80+), was directly correlated with the density of CAFs (P = 0.01). The hazard ratio (HR) for recurrence in a TME rich in CD163+ Foxp3+ CD80+ was 2.9 (95% CI 1.03–8.6, P = 0.043 compared with low in CD163+ Foxp3+ CD80+). The HR for recurrence in a TME rich in CAFs was 4.1 (95% confidence interval [CI] 1.3–12.8, P = 0.012 compared with low in CAFs). In vitro studies showed cancer‐derived exosomes, epithelial–mesenchymal transition process, fibroblast‐to‐CAF‐like cell transdifferentiation, and reciprocal interrelations between different cytokines suggesting the presence of molecular crosstalk between cancer cells and TME components. Collectively, these results highlighted the emerging need of new therapies targeting this crosstalk between the cancer cells and TME components in MT cancer.

Differential expression of basement membrane components in lymphatic tissues

Peripheral lymphoid tissues act as important organs of immunological defense. Characteristic of their architecture is the rich reticular fiber meshwork composed of various extracellular matrix (ECM) molecules with which the stationary non-lymphatic cells stay in intimate contact and form channels through which the lymphatic cells travel. Here we studied the distribution of various laminin (Ln) chains and different types of collagens in human spleen, lymph node, and tonsil to clarify their chain-specific distribution. The most widely distributed proteins in all these organs were Ln chains α5, β1, γ1 and collagen types IV and XVIII, which were present in practically all compartments. Conversely, Ln α1, α2, α4, and type VII collagen showed a more restricted expression pattern. A unique feature was that Ln α3-, β3-, and γ2-chains, which normally are not localized to the vascular wall in non-lymphatic tissues, were present also in capillary basement membranes (BMs) of the follicular structures of lymph node and tonsil and in Ln α1-chain and type VII collagen also in the splenic white pulp. We also found that collagen XVII was exclusively present in the ring fibers of the spleen. The results indicate that BMs of lymphatic tissues contain a variety of macromolecules that probably contribute strongly to immunological events. In addition, capillaries of the lymphoid tissue exhibit a specified BM composition resembling that in epithelial BMs of non-lymphoid tissues.

Laminin-5 gamma2 chain expression facilitates detection of invasive squamous cell carcinoma of the uterine cervix.

Summary:Because it has been suggested that laminin-5 can be used as a sensitive marker for epithelial cell invasion, specimens from patients with invasive squamous cell carcinoma of the uterine cervix with a previous history of preinvasive lesions were evaluated by a newly developed monoclonal antibody directed against the γ2 chain of laminin-5. Thirty-two archival paraffin specimens consisting of the matched preinvasive and invasive lesions from 15 women were evaluated to determine whether γ2 chain laminin-5 staining was present in lesions that progressed to invasive cancer. With the exception of one tumor (a small cell nonkeratinizing squamous carcinoma), all squamous cell carcinomas exhibited positive staining. Five of 17 preinvasive lesions also were immunoreactive for the laminin-5 protein. A blinded histologic reevaluation revealed invasion or lesions suspicious for invasion in four of five preinvasive lesions. Our findings suggest that laminin-5 determined by a monoclonal antibody facilitates the identification of invasive lesions that are difficult or impossible to identify on routinely stained histologic sections.

Antibodies blocking adhesion and matrix binding domains of laminin-332 inhibit tumor growth and metastasis in vivo.

Laminin‐332 (LN‐332), which is essential for epithelial cell adhesion and migration, is up‐regulated in most invasive carcinomas. Association between LN‐332 and carcinoma cell integrins and stroma collagen is thought to be important for tumor growth and metastasis. Here, we show that function blocking LN‐332 antibodies interfering with cellular adhesion and migration in vitro evoke apoptotic pathways. The antibodies also target epithelial tumors in vivo. Antibodies against the cell binding domain of the α3 chain of LN‐332 inhibited tumor growth by up to 68%, and antibodies against the matrix binding domains of the β3 and γ2 chains significantly decreased lung metastases. The LN‐332 antibodies appear to induce tumor cell anoikis and subsequent programmed cell death and reduce migration by interfering with tumor cell matrix interactions.