Stromal accumulation of chondroitin sulphate in mammary tumours of dogs.

To contribute to the investigation of the composition of the extracellular matrix in epithelial tumours, mammary gland tissues of dogs (including tumours, hyperplasias and normal tissue as well as metastatic lesions in lymph nodes and lung) were studied histochemically and immunohistochemically for distribution of sulphated glycosaminoglycans (s-GAGs). The formaline-fixed tissue was stained by alcian blue at pH 5.8, using the 'critical electrolyte concentration' to study the degree of sulphation of s-GAGs. s-GAGs were characterized by degradation with enzymes and nitrous acid and by immunohistochemistry with two anti-chondroitin sulphate monoclonal antibodies. The light microscopic investigation of s-GAG deposits revealed a limited number of patterns of their distribution. The main s-GAGs found in the mammary gland tumours of dogs and in metastatic lesions were chondroitin sulphate (CS) and heparin/heparan sulphate (HEP/HS). CS accumulated in diffuse structures between epithelial cells as well as around clusters of tumour cells. The latter pattern, possibly representing a mesenchymal reaction to the tumour, was present in 74% of the tumours, and in 67% of these, highly sulphated CS was present. A diffuse accumulation of CS was present almost exclusively in complex and mixed tumours; because of the expression of the 3B3 epitope for CS in immature cartilage the spindle cells of complex tumours are argued to be the precursors of the cartilage in mixed tumours. HEP/HS was stored mainly in mast cells that were found in increased numbers in hyperplasias and tumours. By pretreatment of microscopic slides with chondroitinase AC or ABC immunostaining of fibronectin could be made possible in areas in which CS was abundantly present, suggesting that CS may mask fibronectin epitopes. It is concluded that CS with different degrees of sulphation is the most important s-GAG in the extracellular matrix of mammary tumours of dogs. CS and other s-GAGs accumulate at different sites and may have a different pathogenetic significance.     

Expression and degeneration of tenascin-C in human lung cancers.

Tenascin-C is an extracellular matrix glycoprotein produced in response to epithelial-mesenchymal interactions during organogenesis and tissue remodelling. It has therefore been proposed as a stromal marker for epithelial malignancy. To test this hypothesis, 30 human lung cancers, presenting a variety of clinicopathological features, and six specimens of normal tissue were examined by Western and Northern blotting of tenascin-C protein and mRNA. The results obtained were: (1) elevated tenascin-C expression was detected in all 30 cases by Western blotting, with mRNA increase in 22 of them; (2) mRNA for a large isoform of tenascin-C, including an alternatively spliced sequence, was expressed in lung cancer tissues but not in normal lungs; and (3) metastasis to lymph nodes was frequently found in cases whose tenascin-C was degraded into small fragments. These results suggest that tenascin-C degradation can be used as a marker for metastatic potential of a tumour.     

Extracellular matrix regulates steady-state mRNA levels of the proliferation associated protein Ki-67 in endometrial cancer cells

We investigated whether components of the extracellular matrix have the potential to regulate the proliferative activity of endometrial adenocarcinoma cells. Culturing of cells on the reconstituted basement membrane matrigel™ down-regulated the steady-state mRNA levels of the proliferation associated protein, Ki-67, in the endometrial adenocarcinoma cell lines HEC 1B(L) and Ishikawa after 48–96 h of culture on the matrix substrate. Proliferation of Ishikawa was stimulated again if cells were cultured on matrigel and challenged by proteins representing functional domains of tenascin-C, a mesenchymal glycoprotein. The fibronectin-type-III-like repeats 6–8 of tenascin-C were found to be the most potent. In summary, evidence is provided that components of both epithelial and stromal extracellular matrices can function as regulators of cell growth.     

Tumour-associated tenascin-C isoforms promote breast cancer cell invasion and growth by matrix metalloproteinase-dependent and independent mechanisms

Introduction  

The stromal microenvironment has a profound influence on tumour cell behaviour. In tumours, the extracellular matrix (ECM) composition differs from normal tissue and allows novel interactions to influence tumour cell function. The ECM protein tenascin-C (TNC) is frequently up-regulated in breast cancer and we have previously identified two novel isoforms – one containing exon 16 (TNC-16) and one containing exons 14 plus 16 (TNC-14/16).     

Tenascin in human neoplasia

Tenascin (TN), a recently characterized extracellular matrix protein, largely confined to the process with the development of embryo in areas of epithelial-mesenchymal interactions, developing neural tissues, bone and cartilage where there are morphogenetic movements and tissue patterning, has a highly restricted expression in adult tissues. The function(s) and molecular mechanisms of enhanced expression of TN in neoplastic lesions, however, remain unclear. Tissue specimens of human neoplasia obtained from 1050 cases of biopsy or surgery and their representative fetal and normal adult tissues were evaluated for immunoreactivity of TN using seven different clones of monoclonal antibodies in a three stage streptavidin biotin immunoperoxidase method. The normal epithelial lined structures such as the mucous membrane and skin had a thin delicate immunoreactive band just beneath the basement membrane at the epithelial mesenchymal interface. Hyperplastic or dysplastic lesions of the epithelium, such as leukoplakia of the oral mucosa, senile keratosis and intraepithelial carcinoma of skin showed an enhanced expression of TN extending down into the connective tissue where the degree of enhanced expression correlated with increasing hyperplasia or dysplasia. The squamous cell carcinoma and adenocarcinoma of different organs, both at the primary site and in lymph node metastasis, showed a widespread stromal immunoreactivity and the expression was affected by infiltration of inflammatory cells, fibrous tissue reaction and tumor cell differentiation. There was heterogeneity in the expression of TN in benign epithelial neoplasms, squamous cell carcinoma and adenocarcinoma which varied in different areas of the same tumor specimen ranging from no reaction to intensely reactive areas. The undifferentiated carcinomas often had low expression in the stroma. The non-epithelial or mesenchymal tumors such as chondrogenic, osteogenic, myogenic, neurogenic, vascular and other miscellaneous neoplasms also had an enhanced expression of TN. When the immunoreactivity of seven different monoclonal antibodies to detect TN were compared, the pattern of expression of TN with different monoclonal antibody in majority of the cases were identical and equivocal, but there were slight differences in the intensity of staining. Using a single monoclonal antibody nearly 94% of the cases of human neoplasia showed an enhanced immunoreaction for TN. In conclusion, the results of the present study when combined with our previous in vitro study (Shrestha et al, Eur J Cancer Oral Oncol, in press) suggest that the enhanced expression of TN in vivo in a wide variety of neoplastic lesions of both epithelial and non-epithelial in origin may affect tumor growth, differentiation, vascularity, cellular adhesion, invasion and metastasis.     

Tumor-targeting properties of novel antibodies specific to the large isoform of tenascin-C.

BACKGROUND: The targeted delivery of bioactive molecules with antibodies specific to tumor-associated antigens represents a promising strategy for improving the efficacy of tumor therapy. The large isoform of tenascin-C, an abundant glycoprotein of the tumor extracellular matrix, is strongly overexpressed in adult tissue undergoing tissue remodeling, including wound healing and neoplasia, and has been implicated in a variety of different cancers while being virtually undetectable in most normal adult tissues. EXPERIMENTAL DESIGN: We have used antibody phage technology to generate good-quality human recombinant antibodies (F16 and P12) specific to the alternatively spliced domains A1 and D of the large isoform of tenascin-C. The tumor-targeting properties of F16 and P12 were assessed by biodistribution studies in tumor xenografts using the antibodies in small immunoprotein (SIP) format. RESULTS: SIP(F16) selectively accumulated at the tumor site with 4.5%ID/g at 24 hours in the U87 glioblastoma model but was rapidly cleared from other organs (tumor-to-organ ratios, approximately 10:1). The accumulation of SIP(P12) in the tumor was lower compared with SIP(F16) and persistent levels of radioactivity were observed in the intestine. CONCLUSIONS: These data suggest that the F16 antibody, specific to domain A1 of tenascin-C, is a promising building block for the development of antibody-based pharmaceuticals in view of its excellent tumor-targeting performance and the strong expression of the antigen in a variety of primary and metastatic tumors.     

Inhibition of tenascin-C expression in mammary epithelial cells by thyroid hormone

Multiple data suggest a relationship between thyroid hormone (triiodothyronine (T3)) and carcinogenesis. Studies on breast cancer have been inconclusive, suggesting contradictory effects of thyroid status and diseases. Recently, we reported that expression of the extracellular matrix glycoprotein tenascin-C is modulated by T3 during rat brain development. Because tenascin-C has been reported to have growth-, motility-, and angiogenic-promoting activities and to become upregulated during tumorigenesis in breast carcinoma and stromal cells, we analyzed the effects of T3 on tenascin-C expression in mammary epithelial cells. In this study, we showed that tenascin-C RNA expression was inhibited by T3 in normal un-transformed EpH4 mouse mammary epithelial cells expressing appropriate receptors. T3's action appeared to be due to a decreased half-life of the tenascin-C mRNA, with a maximum effect (85% at 100 nM) 48 h after addition. T3 also downregulated tenascin-C in the human mammary tumor cell line SKBR-3, which expresses endogenous thyroid receptors. Immunoprecipitation experiments confirmed that tenascin-C protein content was also decreased by T3 in EpH4 cells (70% reduction at 100 nM). Dexamethasone had a similar inhibitory effect (70% at 100 nM), whereas estradiol, the antiestrogen ICI 164,384, progesterone, and all-trans retinoic acid did not alter tenascin-C expression. Our data demonstrate an inhibitory action of T3 on tenascin-C expression in mammary epithelial cells that may play a role in the physiological regulation of this gene and in neoplastic processes.     

Differential gene expression in liposarcoma, lipoma, and adipose tissue

Malignant transformation is thought to be associated with changes in the expression of a number of genes, and this alteration in gene expression is felt to be critical to the development of the malignant phenotype. Sarcomas represent a diverse group of tumors derived from cells of mesenchymal origin. Marked heterogeneity exists in the biological behavior of sarcomas, even within histologic subtypes of sarcomas. In an effort to better understand the biology of liposarcomas, gene expression in normal adipose tissue, lipomas, and liposarcomas was examined using the Affymetrix® microarray technology. Differences in gene expression were quantified as the fold change in gene expression among the sample sets. Differences in gene expression among normal adipose tissue, lipomas, and liposarcomas were observed. In addition, genes expressed uniquely in liposarcoma among these and 18 other tissue sample sets were identified. Gene sets were devised that allowed the separation of liposarcomas from other samples, and most normal adipose tissue from most lipomas using the Eisen clustering software “Cluster.” We conclude that differences in gene expression can be identified among different tumors derived from the adipocyte series. Such differences in gene expression may help differentiate among subtypes of sarcomas, and may also yield clues to the pathophysiology of this heterogeneous group of tumors.     

Basal lamina redevelopment in tumours metastatic to brain:an immunoperoxidase study using an antibody to type-IV collagen

The basal lamina in a variety of metastatic tumours in brain was assessed with an antibody to type-IV collagen and the indirect immunoperoxidase technique. The antibody was raised in rabbits against type-IV collagen isolated from human placental tissue. Basal lamina redevelopment was demonstrated around individual melanoma cells, between melanoma cells and cerebral parenchyma, and at the tumour-stroma interface in both metastatic melanoma and metastatic carcinoma. At the periphery of metastatic carcinomatous deposits, no basal lamina was observed between tumour cells and the adjacent cerebral parenchyma. Basal lamina staining other than that of cerebral vessels was absent in deposits of poorly differentiated tumours which were unaccompanied by the development of a tumour stroma. It is concluded that metastatic tumours retain the ability to produce basal lamina and, in the case of metastatic epithelial tumours, the redevelopment of basal lamina is dependent on interaction with mesenchymal tissue. The stromal dependency of basal lamina formation by metastatic epithelial tumours suggests the reactivation of a control mechanism acting in normal tissue. Although basal lamina formation in metastatic melanoma can occur in the absence of mesenchymal tissue, there may be some interaction between tumour cells and stroma in the redevelopment of basal lamina at the tumour-stroma interface.     

Monoclonal antibodies to human colorectal epithelium: markers for differentiation and tumour characterization

A panel of monoclonal antibodies (MAbs) has been obtained, directed against determinants of normal human colorectal epithelium. BALB/c mice were immunized and boosted with mucosal scrapings and cell membrane preparations from normal large intestine. In one case boosting was also performed with HT29 colon carcinoma cells. Hybridoma supernatants were screened immunohistochemically on frozen sections of normal colorectal epithelium, leading to the selection of 12 MAbs which recognized determinants of the major epithelial cell lineages. These antibodies fall into two groups: group 1 antibodies react with mucus constituents, with or without other cell components; group 2 antibodies react only with non-mucus components of cells. The normal tissue distribution of the antibody panel has been characterized immunohistochemically. Three of the mucus-reactive group-1 antibodies, PR.4D4, PR.5D5 and PR.3A5, and also PR.1A3 of group 2, have a very restricted distribution. In all 4 cases, their reactivity outside the gastrointestinal tract is mainly confined to tracheal epithelium. A series of benign and malignant colorectal tumours has been studied with the antibody panel. The antibodies of group 1, and PR.1A3 from group 2, show a marked heterogeneity in their reactions with malignant cells and seem to be defining patterns of functional differentiation, independently of standard histological criteria. The reactivity of 6 colorectal carcinoma cell lines has also been assessed with the antibodies. The group-1 antibodies and PR.1A3 identify those cell lines which have retained some capacity for differentiation.     

TGF-α can act as morphogen and/or mitogen in a colon-cancer cell line

Transforming growth factor alpha (TGF-α) has multifunctional biological effects on a variety of mesenchymal and epithelial cells. It is a potent mitogen for a number of normal and transformed cell types, regulates extracellular matrix (ECM) production and promotes breast, kidney and lung morphogenesis. To clarify the role of ECM proteins in the morphogenetic and mitogenic effects of TGF-α, we have used a human colon carcinoma cell line (SW1222) which expresses EGF receptor. Here we show, that TGF-α at 1 ng/ml increases the proliferation of SW1222 cells, but only when they are cultured on plastic rather than collagen-coated plates. Higher concentrations of TGF-α (10ng/ml) did not increase cell proliferation but significantly enhanced the crypt-like glandular differentiation when cells were grown in 3-dimensional collagen gel (p = 0.027). These effects were accompanied by increased expression of α₂β₁ and α₃β₁ integrin molecules, which are receptors for extracellular matrix proteins, and by a statistically significant increase in binding of SW1222 cells to type-1 collagen. The effects of TGF-α both on binding to type-1 collagen and on morphological differentiation in 3-dimensional collagen gel were inhibited by monoclonal antibodies recognizing the α₂β₁ integrin. These data indicate that the morphogenetic or mitogenic activities of TGF-α are critically dependent on cellular interactions with extracellular matrix proteins and are primarily mediated by the α₂β₁ integrin receptor. Inappropriate expression of this growth factor, seen in tumours whose cell-matrix interactions are greatly impaired, could have deleterious effects on the maintenance of normal tissue architecture and growth control.     

Streptozotocin-induced renal tumours in rats.

Forty-six separate renal tumours developed in 36/80 Wistar male rats given a single i.v. dose of streptozotocin (25 mg/kg body wt) to induce diabetes mellitus. Fourteen of the tumours were epithelial in type, 8 were wholly mesenchymal and 24 were largely mesenchymal but also contained epithelial elements. The purely epithelial tumours correspond to the renal adenomas and adenocarcinomas seen in man. The mesenchymal tumours were composed either of undifferentiated spindle cells or of a mixutre of poorly differentiated mesenchyme and epithelial glands. Microscopically, the mixed tumours resembled the nephroblastomas seen in man; both elements appeared to be malignant, but in the absence of metastases this remains unproven. The management of the diabetic state did not influence the incidence of tumours, but insulin appeared to enhance tumour growth.     

Laminin and fibronectin in rectal adenocarcinoma: relationship to tumour grade, stage and metastasis

Using an immunoperoxidase procedure, we have examined the distribution of laminin and fibronectin in normal human large intestinal mucosa and in 50 cases of rectal adenocarcinoma for which extensive clinical follow up was available. In normal tissue, laminin staining was largely restricted to basement membranes, including that underlying the epithelial cells, whereas fibronectin was found in both basement membranes and surrounding connective tissue. In rectal carcinomas, basement membrane-like staining for laminin associated with tumour cells was found in only 27 out of the 50 cases studied. Statistical analysis showed that the presence of laminin-containing basement membranes was correlated with low histological grade (well-differentiated tumours), but not with stage (progression through the bowel wall and the development of lymph node metastases) and, in a highly significant way, with a reduced incidence of distant metastases and increased patient survival. Although fibronectin was found in tumour cell basement membranes where these were present, it was also found in the stroma of all 50 tumours. There was no apparent correlation between the presence of stromal fibronectin and grade, stage or development of metastases. Finally, attention is drawn to some of the technical difficulties in detecting basement membrane antigens in formalin-fixed tissue, the material most frequently available for retrospective study.     

Production and characterization of monoclonal antibodies showing a different spectrum of reactivity to human breast tissue

We have generated three hybridoma-producing monoclonal antibodies (MAs) that show a different spectrum of reactivity to human mammary tissues. Two of these antibodies, 1F10B4 and 1F10G2, recognize a cytoplasmic determinant highly expressed in most of the primary and metastatic breast carcinomas studied, and weakly (or not at all) in normal breast and nonbreast tissues. 3C6F9 detected a surface determinant common to both normal and neoplastic mammary epithelium. Five hundred hybridomas were obtained from the fusion of NS-1 myeloma cells with spleen cells of mice hyperimmunized with the well-characterized human breast carcinoma cell line BT-20. After the initial screenings and clonings, three monoclonal antibodies (1F10B4, 1F10G2, and 3C6F9) showing a restricted range of reactivity were selected for further investigation. These three antibodies recognized a panel of neoplastic mammary cell lines; however, the degree of reactivity could not be correlated to any of the various characteristics of these epithelial cell lines. Moreover, immunofluorescence analysis of acetone-fixed cryostat section showed that 1F10B4 and 1F10G2 recognize the vast majority of the 37 primary and metastatic breast cancers tested, binding strongly to 47% and 67% of them respectively. Only one of the primary carcinomas was not recognized by 1F10B4. On the other hand, these two MAs reacted weakly or not at all with normal breast and nonbreast tissues showing only few focal reactivities with the luminal pole of some ducts of the breast; very weak staining in renal tubular epithelial cells, in few keratinocytes and epithelial cells lining some sebaceous glands in the skin; and a moderate staining in biliary ducts of the liver. All mesenchymal structures including smooth and striated muscle tissues, lymph nodes, and connective tissue were negative. On the other hand, 3C6F9 recognized a more limited number of human mammary tumors and reacted with normal ductal epithelium in the breast and with nonbreast tissues. Because of their wide spectrum of reactivity with breast cancer cells and restricted recognition of normal mammary tissues, their cytoplasmic localization, and their heterogeneous distribution within a single neoplasm, 1F10B4 and 1F10G2 are now being used to characterize antigenic phenotypes of tumor-associated antigens in retrospective studies performed on conventional formalin-fixed, paraffin-embedded human mammary carcinomas.     

An antigen associated with mesenchyme in human tumours that cross-reacts with brain glycoprotein.

Anti-NSA3 antiserum was found to react with many kinds of benign and malignant tumours, as well as foetal skin and intestinal extracts. The corresponding antigens isolated from nervous tissue, benign breast adenoma, and a fibrosarcoma were compared. Immunoprecipitation cannot distinguish between these antigens, and their amino-acid contents were comparable. However, immuno-absorption identified an antigenic determinant that was confined to nervous tissue. Indirect immunofluorescence further confirmed the validity of the concept of a nervous form vs a mesenchymal form of the antigen. Furthermore, immunofluorescence enabled the localization of the antigen found in non-nervous tissue to mesenchyme (mesenchyme-associated antigen: MAA), whether the mesenchymal tissue be normal (foetal organs), tumoral (fibrosarcoma) or reactional (connective-tissue stroma of epithelial tumours).     

Extracellular matrix components in breast carcinomas

A malignant process interferes with the normal 'programme' of extracellular matrix biosynthesis and can modify extensively the structure and composition of the matrix. This effect appears to be attributable to several processes such as direct production of some selected matrix macromolecules by malignant cells or indirectly by the production of factors by malignant cells interfering with the regulation of normal matrix production. Other possibilities may also exist, such as the direct action of an environmental carcinogen on otherwise normal mesenchymal cells. The result is a more or less profound modification of tissue structure and composition with possible feedback effects on the malignant process. Some examples will be discussed such as elastin production by some tumours as well as the biosynthesis of some other selected matrix macromolecules as tenascin and osteopontin by breast tumours. Although the detailed mechanisms of these specific matrix productions is not yet completely elucidated, the rapidly increasing knowledge on the regulation of specific matrix production process and deranged matrix production might represent a new area of crosstalk between cancer research and matrix biology.     

Endogenous tenascin-C enhances glioblastoma invasion with reactive change of surrounding brain tissue

Tenascin-C is an extracellular matrix glycoprotein implicated in embryogenesis, wound healing and tumor progression. We previously revealed that tenascin-C expression is correlated with the prognosis of patients with glioblastoma. However, the exact role of endogenous tenascin-C in regulation of glioblastoma proliferation and invasion remains to be established. We show here that endogenous tenascin-C facilitates glioblastoma invasion, followed by reactive change of the surrounding brain tissue. Although shRNA-mediated knockdown of endogenous tenascin-C does not affect proliferation of glioblastoma cells, it abolishes cell migration on a two-dimensional substrate and tumor invasion with brain tissue changes in a xenograft model. The tyrosine phosphorylation of focal adhesion kinase, a cytoplasmic tyrosine kinase that associates with integrins, was decreased in tenascin-C-knockdown cells. In the analysis of clinical samples, tenascin-C expression correlates with the volume of peritumoral reactive change detected by magnetic resonance imaging. Interestingly, glioblastoma cells with high tenascin-C expression infiltrate brain tissue in an autocrine manner. Our results suggest that endogenous tenascin-C contributes the invasive nature of glioblastoma and the compositional change of brain tissue, which renders tenascin-C as a prime candidate for anti-invasion therapy for glioblastoma. ( Cancer Sci 2009)     

Comparison of IgG diffusion and extracellular matrix composition in rhabdomyosarcomas grown in mice versus in vitro as spheroids reveals the role of host stromal cells

The tumour extracellular matrix acts as a barrier to the delivery of therapeutic agents. To test the hypothesis that extracellular matrix composition governs the penetration rate of macromolecules in tumour tissue, we measured the diffusion coefficient of nonspecific IgG in three rhabdomyosarcoma subclones growing as multicellular spheroids in vitro or as subcutaneous tumours in dorsal windows in vivo. In subcutaneous tumours, the diffusion coefficient decreased with increasing content of collagen and sulphated glycosaminoglycans. When grown as multicellular spheroids, no differences in either extracellular matrix composition or diffusion coefficient were found. Comparison of in vitro vs in vivo results suggests an over-riding role of host stromal cells in extracellular matrix production subjected to modulation by tumour cells. Penetration of therapeutic macromolecules through tumour extracellular matrix might thus be largely determined by the host organ. Hence, caution must be exercised in extrapolating drug penetrability from spheroids and multilayer cellular sandwiches consisting of only tumour cells to tumours in vivo.     

Incidental liposarcomas identified during hernia repair operations.

BACKGROUND AND OBJECTIVES: Since the inguinal region communicates with the retroperitoneum, both retroperitoneal as well as de novo spermatic cord liposarcomas may be detected during hernia repair operations. We assessed the incidence of liposarcomas presenting at hernia repair in our hospital. METHODS: We performed a clerical review of pathology reports on adult tissue accessioned during hernia repair operations and reviewed operating room logs to obtain information concerning the total number of hernia repair operations (since some operations afford no accessioned tissue). RESULTS: Between 1992 and 1997, 1,736 adult hernia repair specimens were accessioned from approximately 2,000 operations. Among these, 22% had an associated cord lipoma; 2 cases were well-differentiated liposarcomas. These were from males aged 56 and 64 years in contrast to the mean age of 35 years for cord lipoma and measured 13 and 10 cm, whereas the mean size for cord lipomas was 5.5 cm. One of the liposarcomas had radiographic evidence of extension from a retroperitoneal lesion; the other appeared confined to the groin. On surgical exploration, the lesion was restricted to the spermatic cord region in both cases despite the suggestion of retroperitoneal extension/involvement in one. CONCLUSIONS: Incidental liposarcomas identified during hernia operations are rare (<0.1% at our institution) but their presence merits histologic evaluation of adipose tissue from these cases. However, if efforts to contain costs are implemented and histologic review of such tissue is deemed generally unrewarding, large (>10 cm) fatty masses from this area should still be sampled.