Cementum and enamel surface mimicry influences soft tissue cell behavior.

AIMS: To test whether titanium surface roughness disparity might be used to specifically guide the behavior of gingiva fibroblasts and keratinocytes, thereby improving the quality of soft tissue (ST) integration around abutments. METHODS: Titanium discs resembling the roughness of enamel (M) or cementum (MA) were created with normal or increased hydrophilicity and used as substrates for human fibroblasts and keratinocytes. Adhesion and proliferation assays were performed to assess cell-type specific responses upon encountering the different surfaces. Additionally, immunofluorescence and qPCR analyses were performed to study more in depth the behavior of fibroblasts and keratinocytes on MA and M surfaces, respectively. RESULTS: While enamel-like M surfaces supported adhesion, growth and a normal differentiation potential of keratinocytes, cementum-emulating MA surfaces specifically impaired the growth of keratinocytes. Vice versa, MA surfaces sustained regular adhesion and proliferation of fibroblasts. Yet, a more intimate adhesion between fibroblasts and titanium was achieved by an increased hydrophilicity of MA surfaces, which was associated with an increased expression of elastin. CONCLUSION: The optimal titanium implant abutment might be achieved by a bimodal roughness design, mimicking the roughness of enamel (M) and cementum with increased hydrophilicity (hMA), respectively. These surfaces can selectively elicit cell responses favoring proper ST barrier by impairing epithelial downgrowth and promoting firm adhesion of fibroblasts.

Reflecting the human lip in vitro: Cleft lip skin and mucosa keratinocytes keep their identities.

OBJECTIVES: Cell models have shown great promise as tools for research, potentially providing intriguing alternatives to animal models. However, the original tissue characteristics must be maintained in culture, a fact that is often assumed, but seldom assessed. We aimed to follow the retention of the original tissue identities of cleft lip-derived skin and mucosa keratinocytes in vitro. METHODS: Cleft lip-derived keratinocytes were isolated from discarded tissue along the cleft margins during cheiloplasty. Cell identities were assessed by immunohistochemistry and quantitative real-time PCR for tissue-specific markers and compared with native lip tissue. Moreover, keratinocytes were regularly analyzed for the retention of the original tissue characteristics by the aforementioned methods as well as by differentiation assays. RESULTS: The various anatomical zones of the human lip could be distinguished using a panel of differentiation and functional-based markers. Using these markers, retention of the original tissue identities could be followed and confirmed in the corresponding primary keratinocytes in culture. CONCLUSIONS: Our findings promote patient-derived cells retaining their original identities as astonishing and clinically relevant in vitro tools. Such cells allow a better molecular understanding of various lip-associated pathologies as well as their modeling in vitro, including but not restricted to orofacial clefts.

Phosphorylated S6 as an immunohistochemical biomarker of vulvar intraepithelial neoplasia.

As life expectancy lengthens, cases of non-viral-associated vulvar squamous cell carcinoma and its precursor lesion, so-called differentiated vulvar intraepithelial neoplasia (VIN), continue to increase in frequency. Differentiated VIN often is difficult to recognize and failure to detect it before invasion results in morbidity and mortality. Thus, identification of a reliable biomarker for this type of lesion would be of great clinical benefit. Our recent studies have identified activation (ser235/236 phosphorylation) of ribosomal protein S6 (p-S6) in basal epithelial cells as an event that precedes and accompanies laminin γ(2) overexpression in most preinvasive oral dysplasias. To test this as a potential biomarker of vulvar dysplasia, we immunostained seven differentiated VINs and nine papillomavirus-related 'classic' VINs, most of which were associated with carcinoma, for p-S6. All carcinomas, all differentiated VINs, and most classic VINs contained regions of p-S6 staining in the basal layer, whereas basal and parabasal cells of normal vulvar epithelium and hyperplastic and inflamed lesions lacking cellular atypia were p-S6 negative. Laminin γ(2) was expressed in a subset of VINs, always occurring within basal p-S6 positive regions, as we had found previously for oral dysplasias. Lichen sclerosus is considered a potential precursor of vulvar carcinoma. Two lichen sclerosus lesions of patients with a concurrent carcinoma and one of six lichen sclerosus lesions without atypia or known concurrent carcinoma were basal p-S6 positive. In summary, there is a distinct difference in p-S6 basal cell layer staining between benign and neoplastic vulvar squamous epithelium, with consistent staining of differentiated VIN and of some lichen sclerosus lesions. These results support further studies to assess the potential of p-S6 as a biomarker to identify vulvar lesions at risk of progressing to invasive cancer.

MAPK/ERK-dependent translation factor hyperactivation and dysregulated laminin γ2 expression in oral dysplasia and squamous cell carcinoma.

Lesions displaying a variety of dysplastic changes precede invasive oral and epidermal squamous cell carcinoma (SCC); however, there are no histopathological criteria for either confirming or staging premalignancy. SCCs and dysplasias frequently contain cells that abnormally express the γ2 subunit of laminin-332. We developed cell culture models to investigate γ2 dysregulation. Normal human keratinocytes displayed density-dependent repression of γ2, whereas premalignant keratinocytes and SCC cells overexpressed γ2 and secreted laminin assembly intermediates. Neoplastic cells had hyperactive EGFR/MAPK(ERK) signaling coordinate with overexpressed γ2, and EGFR and MEK inhibitors normalized γ2 expression. Keratinocytes engineered to express HPV16 E6 or activated mutant HRAS, cRAF1, or MEK1 lost density repression of γ2 and shared with neoplastic cells signaling abnormalities downstream of ERK, including increased phosphorylation of S6 and eIF4 translation factors. Notably, qPCR results revealed that γ2 overexpression was not accompanied by increased γ2 mRNA levels, consistent with ERK-dependent, eIF4B-mediated translation initiation of the stem-looped, 5'-untranslated region of γ2 mRNA in neoplastic cells. Inhibitors of MEK, but not of TORC1/2, blocked S6 and eIF4B phosphorylation and γ2 overexpression. Immunostaining of oral dysplasias identified γ2 overexpression occurring within fields of basal cells that had elevated p-S6 levels. These results reveal a causal relationship between ERK-dependent translation factor activation and laminin γ2 dysregulation and identify new markers of preinvasive neoplastic change during progression to SCC.

Tenascin-W is a better cancer biomarker than tenascin-C for most human solid tumors.

BACKGROUND: Tenascins are large glycoproteins found in the extracellular matrix of many embryonic and adult tissues. Tenascin-C is a well-studied biomarker known for its high overexpression in the stroma of most solid cancers. Tenascin-W, the least studied member of the family, is highly expressed in the stroma of colon and breast tumors and in gliomas, but not in the corresponding normal tissues. Other solid tumors have not been analyzed. The present study was undertaken to determine whether tenascin-W could serve as a cancer-specific extracellular matrix protein in a broad range of solid tumors. METHODS: We analyzed the expression of tenascin-W and tenascin-C by immunoblotting and by immunohistochemistry on multiple frozen tissue microarrays of carcinomas of the pancreas, kidney and lung as well as melanomas and compared them to healthy tissues. RESULTS: From all healthy adult organs tested, only liver and spleen showed detectable levels of tenascin-W, suggesting that tenascin-W is absent from most human adult organs under normal, non-pathological conditions. In contrast, tenascin-W was detectable in the majority of melanomas and their metastases, as well as in pancreas, kidney, and lung carcinomas. Comparing lung tumor samples and matching control tissues for each patient revealed a clear overexpression of tenascin-W in tumor tissues. Although the number of samples examined is too small to draw statistically significant conclusions, there seems to be a tendency for increased tenascin-W expression in higher grade tumors. Interestingly, in most tumor types, tenascin-W is also expressed in close proximity to blood vessels, as shown by CD31 co-staining of the samples. CONCLUSIONS: The present study extends the tumor biomarker potential of tenascin-W to a broad range of solid tumors and shows its accessibility from the blood stream for potential therapeutic strategies.

Immunomarkers in gynecologic cytology: the search for the ideal 'biomolecular Papanicolaou test'.

Harnessing the knowledge we have gained on the cell cycle disruption caused by human papillomaviruses (HPV) will likely lead to improved screening modalities for cervical cancer and its precursors. An easily applied biomarker that has high specificity and sensitivity would represent an attractive alternative or complement to cytology and HPV testing. To date, a number of promising markers have been investigated. These include p16(INK4A), MIB-1, BD-ProEx C, and L1. Newer possibilities involve a variety of gene products associated with aberrations of chromosome 3q, such as telomerase, p63, and PIK3CA, as well the combination of biomarkers such as p16(INK4A) and MIB-1 in the same assay. Although none of them has yet been incorporated into screening algorithms or found its way into routine practice, their performance characteristics remain a focus of current investigations. This review summarizes what we know and where we hope to go in translating basic pathobiology into clinical practice.

Revisiting the Tenascins: Exploitable as Cancer Targets?

For their full manifestation, tumors require support from the surrounding tumor microenvironment (TME), which includes a specific extracellular matrix (ECM), vasculature, and a variety of non-malignant host cells. Together, these components form a tumor-permissive niche that significantly differs from physiological conditions. While the TME helps to promote tumor progression, its special composition also provides potential targets for anti-cancer therapy. Targeting tumor-specific ECM molecules and stromal cells or disrupting aberrant mesenchyme-cancer communications might normalize the TME and improve cancer treatment outcome. The tenascins are a family of large, multifunctional extracellular glycoproteins consisting of four members. Although each have been described to be expressed in the ECM surrounding cancer cells, tenascin-C and tenascin-W are currently the most promising candidates for exploitability and clinical use as they are highly expressed in various tumor stroma with relatively low abundance in healthy tissues. Here, we review what is known about expression of all four tenascin family members in tumors, followed by a more thorough discussion on tenascin-C and tenascin-W focusing on their oncogenic functions and their potential as diagnostic and/or targetable molecules for anti-cancer treatment purposes.

Discovery and characterization of heterogeneous and multipotent fibroblast populations isolated from excised cleft lip tissue.

BACKGROUND: Regularly discarded lip tissue obtained from corrective surgeries to close the cleft lip represents an easily accessible and rich source for the isolation of primary fibroblasts. Primary fibroblasts have been described to show compelling similarities to mesenchymal stem cells (MSCs). Hence, cleft lip and palate (CLP) lip-derived fibroblasts could be thought as an intriguing cell source for personalized regenerative therapies in CLP-affected patients. METHODS: Initially, we thoroughly characterized the fibroblastic nature of the lip-derived mesenchymal outgrowths by molecular and functional assays. Next, we compared their phenotype and genotype to that of bone marrow-mesenchymal stem cells (BM-MSCs) and of human lung-derived fibroblasts WI38, by assessing their morphology, surface marker expression, trilineage differentiation potential, colony-forming (CFU) capacity, and immunomodulation property. Finally, to better decipher the heterogeneity of our CLP cultures, we performed a single cell clonal analysis and tested expanded clones for surface marker expression, as well as osteogenic and CFU potential. RESULTS: We identified intriguingly similar phenotypic and genotypic properties between CLP lip fibroblasts and BM-MSCs, which makes them distinct from WI38. Furthermore, our own data in combination with the complex anatomy of the lip tissue indicated heterogeneity in our CLP cultures. Using a clonal analysis, we discovered single cell-derived clones with increased levels of the MSC markers CD106 and CD146 and clones with variabilities in their commitment to differentiate into bone-forming cells and in their potential to form single cell-derived colonies. However, we were not able to gain clones possessing superior MSC-like capacities when compared to the heterogeneous parental CLP population. Additionally, all clones could still generate contractile forces and retained robust levels of the fibroblast specific marker FSP1, which was not detectable in BM-MSCs. CONCLUSIONS: Our results suggest that we isolate heterogeneous populations of fibroblasts from discarded CLP lip tissue, which show a prominently multipotent character in their entirety avoiding the need for elaborate subpopulation selections in vitro. These findings suggest that CLP lip fibroblasts might be a novel potential cell source for personalized regenerative medicine of clinical benefit for CLP patients.

Consistent downregulation of the cleft lip/palate-associated genes IRF6 and GRHL3 in carcinomas.

Interferon Regulatory Factor 6 (IRF6) and Grainyhead Like Transcription Factor 3 (GRHL3) are transcription factors that orchestrate gene regulatory networks required for the balance between keratinocyte differentiation and proliferation. Absence of either protein results in the lack of a normal stratified epidermis with keratinocytes failing to stop proliferating and to terminally differentiate. Numerous pathological variants within IRF6 and GRHL3 have been identified in orofacial cleft-affected individuals and expression of the two transcription factors has been found to be often dysregulated in cancers. However, whether orofacial cleft-associated IRF6 and GRHL3 variants in patients might also affect their cancer risk later in life, is not clear yet. The fact that the role of IRF6 and GRHL3 in cancer remains controversial makes this question even more challenging. Some studies identified IRF6 and GRHL3 as oncogenes, while others could attribute tumor suppressive functions to them. Trying to solve this apparent conundrum, we herein aimed to characterize IRF6 and GRHL3 function in various types of carcinomas. We screened multiple cancer and normal cell lines for their expression, and subsequently proceeded with functional assays in cancer cell lines. Our data uncovered consistent downregulation of IRF6 and GRHL3 in all types of carcinomas analyzed. Reduced levels of IRF6 and GRHL3 were found to be associated with several tumorigenic properties, such as enhanced cell proliferation, epithelial mesenchymal transition, migration and reduced differentiation capacity. Based on our findings, IRF6 and GRHL3 can be considered as tumor suppressor genes in various carcinomas, which makes them potential common etiological factors for cancer and CLP in a fraction of CLP-affected patients.

Tenascin-W is a specific marker of glioma-associated blood vessels and stimulates angiogenesis in vitro.

The microenvironment hosting a tumor actively participates in regulating tumor cell proliferation, migration, and invasion. Among the extracellular matrix proteins enriched in the stroma of carcinomas are the tenascin family members tenascin-C and tenascin-W. Whereas tenascin-C overexpression in gliomas is known to correlate with poor prognosis, the status of tenascin-W in brain tumors has not been investigated so far. In the present study, we analyzed protein levels of tenascin-W in 38 human gliomas and found expression of tenascin-W in 80% of the tumor samples, whereas no tenascin-W could be detected in control, nontumoral brain tissues. Double immunohistochemical staining of tenascin-W and von Willebrand factor revealed that tenascin-W is localized around blood vessels, exclusively in tumor samples. In vitro, the presence of tenascin-W increased the proportion of elongated human umbilical vein endothelial cells (HUVECs) and augmented the mean speed of cell migration. Furthermore, tenascin-W triggered sprouting of HUVEC spheroids to a similar extent as the proangiogenic factor tenascin-C. In conclusion, our study identifies tenascin-W as a candidate biomarker for brain tumor angiogenesis that could be used as a molecular target for therapy irrespective of the glioma subtype.-Martina, E., Degen, M., Rüegg, C., Merlo, A., Lino, M. M., Chiquet-Ehrismann, R., Brellier, F. Tenascin-W is a specific marker of glioma-associated blood vessels and stimulates angiogenesis in vitro.

Lack of IRF6 Disrupts Human Epithelial Homeostasis by Altering Colony Morphology, Migration Pattern, and Differentiation Potential of Keratinocytes.

Variants within the gene encoding for the transcription factor Interferon Regulatory Factor 6 (IRF6) are associated with syndromic and non-syndromic Cleft Lip/Palate (CLP) cases. IRF6 plays a vital role in the regulation of the proliferation/differentiation balance in keratinocytes and is involved in wound healing and migration. Since a fraction of CLP patients undergoing corrective cleft surgery experience wound healing complications, IRF6 represents an interesting candidate gene linking the two processes. However, Irf6 function has been mainly studied in mice and knowledge on IRF6 in human cells remains sparse. Here, we aimed to elucidate the role of IRF6 in human postnatal skin- and oral mucosa-derived keratinocytes. To do so, we applied CRISPR/Cas9 to ablate IRF6 in two TERT-immortalized keratinocyte cultures, which we used as model cell lines. We show that IRF6 controls the appearance of single cells and colonies, with the latter being less cohesive in its absence. Consequently, IRF6 knockout keratinocytes often moved as single cells instead of a collective epithelial sheet migration but maintained their epithelial character. Lack of IRF6 triggered severe keratinocyte differentiation defects, which were already apparent in the stratum spinosum and extended to the stratum corneum in 3D organotypic skin cultures, while it did not alter their growth rate. Finally, proteomics revealed that most of the differentially expressed proteins in the absence of IRF6 could be associated with differentiation, cell-cell adhesion as well as immune response. Our data expand the knowledge on IRF6 in human postnatal keratinocytes, which will help to better understand IRF6-related pathologies.

A Living Cell Repository of the Cranio-/Orofacial Region to Advance Research and Promote Personalized Medicine.

The prevalence of congenital anomalies in newborns is estimated to be as high as 6%, many of which involving the cranio-/orofacial region. Such malformations, including several syndromes, are usually identified prenatally, at birth, or rarely later in life. The lack of clinically relevant human cell models of these often very rare conditions, the societal pressure to avoid the use of animal models and the fact that the biological mechanisms between rodents and human are not necessarily identical, makes studying cranio-/orofacial anomalies challenging. To overcome these limitations, we are developing a living cell repository of healthy and diseased cells derived from the cranio-/orofacial region. Ultimately, we aim to make patient-derived cells, which retain the molecular and genetic characteristics of the original anomaly or disease in vitro, available for the scientific community. We report our efforts in establishing a human living cell bank derived from the cranio-/orofacial region of otherwise discarded tissue samples, detail our strategy, processes and quality checks. Such specific cell models have a great potential for discovery and translational research and might lead to a better understanding and management of craniofacial anomalies for the benefit of all affected individuals.

Tenascin-W: Discovery, Evolution, and Future Prospects.

Of the four tenascins found in bony fish and tetrapods, tenascin-W is the least understood. It was first discovered in the zebrafish and later in mouse, where it was mistakenly named tenascin-N. Tenascin-W is expressed primarily in developing and mature bone, in a subset of stem cell niches, and in the stroma of many solid tumors. Phylogenetic studies show that it is the most recent tenascin to evolve, appearing first in bony fishes. Its expression in bone and the timing of its evolutionary appearance should direct future studies to its role in bone formation, in stem cell niches, and in the treatment and detection of cancer.

A Novel Van der Woude Syndrome-Causing IRF6 Variant Is Subject to Incomplete Non-sense-Mediated mRNA Decay Affecting the Phenotype of Keratinocytes.

Van der Woude syndrome (VWS) is a genetic syndrome that leads to typical phenotypic traits, including lower lip pits and cleft lip/palate (CLP). The majority of VWS-affected patients harbor a pathogenic variant in the gene encoding for the transcription factor interferon regulatory factor 6 (IRF6), a crucial regulator of orofacial development, epidermal differentiation and tissue repair. However, most of the underlying mechanisms leading from pathogenic IRF6 gene variants to phenotypes observed in VWS remain poorly understood and elusive. The availability of one VWS individual within our cohort of CLP patients allowed us to identify a novel VWS-causing IRF6 variant and to functionally characterize it. Using VWS patient-derived keratinocytes, we reveal that most of the mutated IRF6_VWS transcripts are subject to a non-sense-mediated mRNA decay mechanism, resulting in IRF6 haploinsufficiency. While moderate levels of IRF6_VWS remain detectable in the VWS keratinocytes, our data illustrate that the IRF6_VWS protein, which lacks part of its protein-binding domain and its whole C-terminus, is noticeably less stable than its wild-type counterpart. Still, it maintains transcription factor function. As we report and characterize a so far undescribed VWS-causing IRF6 variant, our results shed light on the physiological as well as pathological role of IRF6 in keratinocytes. This acquired knowledge is essential for a better understanding of the molecular mechanisms leading to VWS and CLP.

Tenascin-W Is a Novel Stromal Marker in Biliary Tract Cancers.

Extrahepatic cancers of the biliary system are typically asymptomatic until after metastasis, which contributes to their poor prognosis. Here we examined intrahepatic cholangiocarcinomas (n = 8), carcinomas of perihilar bile ducts (n = 7), carcinomas of the gallbladder (n = 11) and hepatic metastasis from carcinomas of the gallbladder (n = 4) for the expression of the extracellular matrix glycoproteins tenascin-C and tenascin-W. Anti-tenascin-C and anti-tenascin-W immunoreactivity was found in all biliary tract tumors examined. Unlike tenascin-C, tenascin-W was not detected in normal hepatobiliary tissue. Tenascin-W was also expressed by the cholangiocarcinoma-derived cell line Huh-28. However, co-culture of Huh-28 cells with immortalized bone marrow-derived stromal cells was necessary for the formation and organization of tenascin-W fibrils in vitro. Our results indicate that tenascin-W may be a novel marker of hepatobiliary tumor stroma, and its absence from many normal tissues suggests that it may be a potential target for biotherapies.

Transforming growth factor beta type 1 (TGF-ß) and hypoxia-inducible factor 1 (HIF-1) transcription complex as master regulators of the immunosuppressive protein galectin-9 expression in human cancer and embryonic cells.

Galectin-9 is one of the key proteins employed by a variety of human malignancies to suppress anti-cancer activities of cytotoxic lymphoid cells and thus escape immune surveillance. Human cancer cells in most cases express higher levels of galectin-9 compared to non-transformed cells. However, the biochemical mechanisms underlying this phenomenon remain unclear. Here we report for the first time that in human cancer as well as embryonic cells, the transcription factors hypoxia-inducible factor 1 (HIF-1) and activator protein 1 (AP-1) are involved in upregulation of transforming growth factor beta 1 (TGF-ß1) expression, leading to activation of the transcription factor Smad3 through autocrine action. This process triggers upregulation of galectin-9 expression in both malignant (mainly in breast and colorectal cancer as well as acute myeloid leukaemia (AML)) and embryonic cells. The effect, however, was not observed in mature non-transformed human cells. TGF-ß1-activated Smad3 therefore displays differential behaviour in human cancer and embryonic vs non-malignant cells. This study uncovered a self-supporting biochemical mechanism underlying high levels of galectin-9 expression operated by the human cancer and embryonic cells employed in our investigations. Our results suggest the possibility of using the TGF-ß1 signalling pathway as a potential highly efficient target for cancer immunotherapy.

Tenascin-W is a novel marker for activated tumor stroma in low-grade human breast cancer and influences cell behavior.

This is the first report about human tenascin-W, the fourth and final member of the extracellular matrix protein family of tenascins. Sixty-three human breast tumor extracts were analyzed by Western blotting for the presence of tenascin-W and compared with tenascin-C, an established marker of tumor stroma. Interestingly, we found tenascin-W expression in the majority of the tumor tissues, but no detectable expression in the normal mammary parenchyma. Eighty-one percent of the breast tumor samples were tenascin-W positive and 86% showed expression of tenascin-C. However, tenascin-W and tenascin-C amounts varied greatly between tumors and some contained either tenascin-W or tenascin-C exclusively, indicating independent mechanisms regulating their expression. Although there was no difference between high- or low-grade tumors with respect to the presence of tenascin-C, tenascin-W was more prominent in low-grade tumors. For 42 of the breast cancer tissues, a frozen tumor microarray was available to confirm the Western blot data by immunohistochemistry. Similar to tenascin-C, tenascin-W was detected in the tumor stroma. Fibroblasts adhered to tenascin-W in a beta(1) integrin-dependent manner and spread with a distinctive morphology under conditions where they remained round on tenascin-C. CHOB2 cells expressing alpha(v)beta(1) or alpha4beta(1) integrins were able to spread on tenascin-W. Furthermore, addition of tenascin-W to the culture medium increased migration of breast cancer cells toward a fibronectin substratum in vitro. These data imply that tenascin-W expression in the activated tumor stroma facilitates tumorigenesis by supporting the migratory behavior of breast cancer cells.

The Expression and Possible Functions of Tenascin-W During Development and Disease.

Tenascins are a family of multifunctional glycoproteins found in the extracellular matrix of chordates. Two of the tenascins, tenascin-C and tenascin-W, form hexabrachions. In this review, we describe the discovery and domain architecture of tenascin-W, its evolution and patterns of expression during embryogenesis and in tumors, and its effects on cells in culture. In avian and mammalian embryos tenascin-W is primarily expressed at sites of osteogenesis, and in the adult tenascin-W is abundant in certain stem cell niches. In primary cultures of osteoblasts tenascin-W promotes cell migration, the formation of mineralized foci and increases alkaline phosphatase activity. Tenascin-W is also prominent in many solid tumors, yet it is missing from the extracellular matrix of most adult tissues. This makes it a potential candidate for use as a marker of tumor stroma and a target for anti-cancer therapies.

Tenascin-W, a new marker of cancer stroma, is elevated in sera of colon and breast cancer patients.

Tenascins are extracellular matrix proteins present during the development of organisms as well as in pathological conditions. Tenascin-W, the fourth and last member of the tenascin family remains the least well-characterized one. Our study aimed to evaluate the potential significance of tenascin-W as cancer biomarker by monitoring its presence in the serum of colorectal and breast cancer patients and its expression in colorectal tumor tissues. To measure serum tenascin-W levels, a sensitive sandwich-ELISA was established. Mean tenascin-W concentration in sera of patients with nonmetastatic colorectal cancer at time of diagnosis was highly increased compared to that of healthy volunteers. A similar tendency was observed for tenascin-C in the same patient cohort. However, the increase was much more striking for tenascin-W. We also detected elevated tenascin-W levels in sera of breast cancer patients. Furthermore, we could show a prominent expression of tenascin-W in extracts from colorectal tumor tissues by immunoblot analysis, whereas tenascin-W was not detectable in the corresponding normal colon mucosa. To confirm the western blot results, we performed immunohistochemistry of frozen sections of the same patients as well as of an additional, independently chosen collection of colorectal cancer tissues. In all cases, similarly to tenascin-C, tenascin-W was detected in the tumor stroma. Our results reveal a clear association between elevated levels of tenascin-W and the presence of cancer. These results warrant further studies to evaluate the potential value of serum and tissue tenascin-W levels as diagnostic, prognostic or monitoring biomarker in colorectal, breast and possibly other solid cancers.

Effects of tenascin-W on osteoblasts in vitro.

Tenascin-W is a glycoprotein secreted into the extracellular matrix of developing bones. Here, we have examined possible roles for tenascin-W in osteogenesis. Purified recombinant tenascin-W, like tenascin-C, increases the number of mineralized foci in primary cultures of avian osteoblasts and increases alkaline phosphatase activity in vitro. In addition, tenascin-W in solution promotes the migration of primary osteoblasts across fibronectin-coated filters. The sixth fibronectin type III domain of chicken tenascin-W contains a phylogenetically conserved KGD motif that is predicted to be available to integrin binding. To determine whether this motif is potentially functional, we have cultured osteoblasts on KGD-containing peptides and control peptides. Osteoblasts cultured on peptides with the KGD motif acquire a multipolar phenotype with pseudopods tipped with actin-rich ruffles, which is similar to the morphology of osteoblasts cultured on recombinant tenascin-W. Moreover, the KGD peptides, but not the control peptides, promote proliferation in cultured osteoblasts but not alkaline phosphatase activity or migration. Finally, explanted embryonic frontal bones are significantly thicker when cultured in the presence of tenascin-W, suggesting that tenascin-W can accelerate the formation of new bone in a complex multicellular environment.