Human breast tumors override the antiangiogenic effect of stromal thrombospondin-1 in vivo

The antiangiogenic extracellular matrix protein thrombospondin-1 (TSP-1) inhibits tumor growth and metastasis in animals. However, the clinical relevance of such findings are equivocal as increased stromal TSP-1 expression has been associated with either good or poor prognosis. In an effort to obtain a more integrated understanding of the role of TSP-1 in breast cancer, we first used a breast tumorigenesis model in which tumor-associated stromal fibroblasts were engineered to produce high levels of TSP-1. We demonstrate here that stromal TSP-1 delayed human MDA-MB-231/B02 breast tumor growth. However, this delay in MDA-MB-231/B02 tumor growth upon exposure to TSP-1 was associated with an increased vascular endothelial growth factor (VEGF) expression in tumor cells themselves, leading to a tumor growth rate comparable to that of tumors whose fibroblasts did not overproduce TSP-1. Clinical evidence also suggested that primary breast carcinomas have adapted to escape the effects of stromal TSP-1. TSP-1 was found to be expressed in the stroma of human breast carcinomas where, although its level correlated with decreased vascularization, it was unexpectedly associated with a reduction of relapse-free survival. In metastatic axillary lymph nodes, tumor cells expressed high levels of VEGF and TSP-1 expression were no longer associated with a decreased vascularization. Overall, these results suggest that a resistance may develop early in human breast cancers as a result of high in situ exposure to stromal TSP-1, leading to disease progression.     

Inhibition of malignant ascites and growth of human ovarian carcinoma by oral administration of a potent inhibitor of the vascular endothelial growth factor receptor tyrosine kinases

We determined whether inhibition of the catalytic tyrosine kinase activity of the receptors for vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) inhibits the formation of malignant ascites and the progressive growth of human ovarian carcinoma cells implanted into the peritoneal cavity of nude mice. The novel protein tyrosine inhibitor PTK 787 was evaluated in two models of human ovarian cancer: Hey-A8 cells, which express low levels of VEGF/VPF and grow as solid tumor foci on the surface of peritoneal organs, and SKOV3 i.p.1 cells, which express high levels of VEGF/VPF and grow as solid peritoneal tumors and ascites. Treatment of nude mice by daily oral administration of 50 mg/kg PTK 787 was not effective against Hey-A8 tumors. In sharp contrast, it significantly inhibited growth of SKOV3 i.p.1 cells and formation of ascites, significantly increasing survival of mice with the implants. Tumor-induced vascular hyperpermeability in the peritoneum of tumor-bearing mice was inhibited by PTK 787, which accounted for its inhibition of ascites formation. Our results suggest that blockade of the VEGF/VPF receptor may be an efficient strategy to inhibit formation of malignant ascites and growth of VEGF/VPF-dependent human ovarian carcinomas.     

Clinicopathological significance of stromal myofibroblasts in invasive ductal carcinoma of the breast.

The purpose of this study was to investigate the distribution of CD34-positive fibroblasts and alpha-smooth muscle actin (alpha-SMA)-reactive myofibroblasts in the stroma of benign and malignant breast lesions and, secondly, to determine whether the presence of stromal myofibroblasts is associated with some of the clinicopathological characteristics of patients with invasive ductal carcinoma. The presence of stromal CD34-positive fibroblasts and myofibroblasts was investigated (as defined immunohistochemically) in 8 normal breast tissue samples, 58 invasive ductal carcinomas, 9 ductal carcinomas in situ and 16 specimens with benign lesions of the breast (fibroadenomas, ductal hyperplasias). We further studied the correlations between the presence of stromal myofibroblasts with 7 clinicopathological parameters in 58 invasive ductal carcinomas. The results indicated that the stroma of normal breast tissues contained CD34-positive fibroblasts. All benign breast lesions exhibited stromal CD34-positive fibroblasts. In contrast, the stroma of ductal carcinomas showed a complete loss of CD34-positive fibroblasts. alpha-SMA expression in stromal fibroblasts (myofibroblasts) was not detected in normal tissue samples or benign lesions except in 1 case of fibroadenoma, whereas positive myofibroblasts were found in 44.4% of ductal carcinomas in situ and 56.9% of invasive breast carcinomas. Comparison of clinicopathological parameters between invasive ductal carcinomas with and without stromal myofibroblasts revealed significant differences in lymph node metastasis, high histological grade and high microvessel density. These results suggest that CD34 loss and the presence of myofibroblasts favor the diagnosis of breast carcinoma. In invasive ductal carcinoma, the presence of stromal myofibroblasts correlated significantly with pathological parameters associated with a poor prognosis.     

Stromal expression of cancer-associated fibroblast-related molecules,versican and lumican,is strongly associated with worse relapse-free and overall survival times in patients with esophageal squamous cell carcinoma

Cancer-associated fibroblasts (CAFs) in the tumor microenvironment play an essential role in the tumor progression of esophageal squamous cell carcinoma (ESCC). The present study aimed to investigate the expression of CAF-related molecules, versican, periostin and lumican, in cancer stroma, to provide prognostic stratification for patients with ESCC after surgery. A total of 106 patients with ESCC who underwent curative esophagectomy without preoperative chemotherapy or radiotherapy were enrolled. The expression of CAF-related stromal proteins, including versican, periostin and lumican, was examined using immunohistochemistry, and the prognostic value was assessed by Kaplan-Meier survival analysis, and univariate and multivariate Cox regression models. The expression of versican, periostin and lumican was found specifically in the stromal component of ESCC. Kaplan-Meier analysis demonstrated that, compared with a low expression level, a high expression level of versican, periostin or lumican in the cancer stroma was significantly associated with worse relapse-free survival (RFS) and overall survival times in patients with ESCC. The prognostic values of stromal versican and lumican remained significant in a stratified analysis of stage I patients. Moreover, univariate and multivariate analysis revealed that high stromal versican or lumican expression was an independent prognostic factor for RFS in the patients. The present study demonstrated that CAF-related molecules, including versican, periostin and lumican, were expressed in the stroma of ESCC, and that stromal expression of versican and lumican in particular may have clinical utility as a prognostic biomarker for poor RFS in postoperative patients with ESCC.     

Collagenase-3 expression in breast myofibroblasts as a molecular marker of transition of ductal carcinoma in situ lesions to invasive ductal carcinomas

Collagenase-3 (matrix metalloproteinase 13; MMP-13), a protease originally identified in breast carcinoma, is characterized by a potent degrading activity against a wide spectrum of extracellular matrix proteins. The aims of this study were to localize and identify the MMP-13-expressing cells in invasive human breast carcinoma and to evaluate the role of MMP-13 in transition to invasive lesions by studying ductal carcinoma in situ (DCIS). We found expression of MMP-13 in stromal fibroblast-like cells in all 21 invasive ductal carcinomas studied and in 4 of 9 invasive lobular carcinomas. In most carcinomas, expression of MMP-13 was limited to small stromal foci in the tumor area. Combined in situ hybridization and immunohistochemistry showed coexpression of alpha-smooth muscle actin immunoreactivity and MMP-13 mRNA in myofibroblasts. In contrast, cytokeratin-positive cancer cells, alpha-smooth muscle actin-positive vascular smooth muscle cells, CD68-positive macrophages, and CD31-positive endothelial cells were all MMP-13 mRNA negative. In situ hybridization for MMP-13 in 17 DCIS lesions revealed expression in 10 cases. Immunohistochemical analysis of all DCIS cases identified microinvasion in 8 of the 17 lesions. Seven of the eight lesions with microinvasion were MMP-13 positive. Further analysis showed that MMP-13 expression was often associated with the microinvasive events. This particular expression pattern was unique for MMP-13 among other MMPs analyzed, including MMP-2, -11, and -14. We conclude that MMP-13 is primarily expressed by myofibroblasts in human breast carcinoma and that expression in DCIS lesions often is associated with microinvasive events. On the basis of these data, we propose that MMP-13 may play an essential role during transition of DCIS lesions to invasive ductal carcinomas.     

Inhibition of carcinoma cell-derived VEGF reduces inflammatory characteristics in xenograft carcinoma

The stroma of carcinomas shares several characteristics with inflamed tissues including a distorted vasculature, active angiogenesis and macrophage infiltration. In addition, the tumor interstitial fluid pressure (P(IF)) of the stroma is pathologically elevated. We show here that bevacizumab [rhuMab vascular endothelial growth factor (VEGF), Avastin], a monoclonal antibody to VEGF, at a dose of 5 mg/kg modulated inflammation in KAT-4 xenograft human anaplastic thyroid carcinoma tissue. At this dose, bevacizumab reduced the density of macrophages, MHC class II antigen expression by macrophages and IL-1beta mRNA expression. Furthermore, bevacizumab lowered tumor extracellular fluid volume, plasma protein leakage from tumor vessels, the number of CD31-positive structures and tumor P(IF). The tumor plasma volume and the number of alpha-smooth muscle actin-positive vessels, however, remained unchanged. Our data suggest that carcinoma cell-derived VEGF either directly or indirectly participates in maintaining an inflammatory microenvironment in experimental KAT-4 carcinoma. Furthermore, our data indicate that the reduction of inflammation resulting in reduced vascular permeability and decrease in the tumor extracellular fluid volume by bevacizumab contributes to reduced tumor P(IF).     

Study of fibronectin and mRNA in human laryngeal and ectocervical carcinomas by in situ hybridization and image analysis.

The expression of fibronectin (FN) mRNA was studied in histological sections of surgical biopsies from human laryngeal and ectocervical invasive carcinomas of different grading stages by in situ hybridization and image analysis. This approach made it possible to identify the cell types synthesizing FN mRNA in the tissue sections and to compare semi-quantitatively the FN mRNA levels expressed in the different specimens. The carcinoma cells synthesized low levels of FN mRNA, comparable to those detected in control epithelia and connective-tissue fibroblasts. Well-differentiated (G1) laryngeal and ectocervical carcinomas induced the synthesis of FN mRNA--to levels 7 to 13 times higher than in control connective tissues--in the stromal fibroblasts surrounding the tumors. In carcinoma samples analysed, the amount of FN mRNA detected in the stroma decreased in relation to tumor grading (from G1 to G3) and the stromal destruction. FN mRNA was not detectable in the endothelial cells of venules while it was present in large amounts in those surrounding the capillaries present in the stroma. These data indicate that FN, usually observed around carcinomas, is produced by stromal fibroblasts, which are induced to express FN mRNA, presumably in response to diffusible factors produced by the tumor cells, and/or by endothelial cells of the infiltrating capillary vessels. The induction of FN mRNA, inversely proportional to the tumor grading, may be useful in evaluating the invasion potential of the tumor.     

Expression of vascular endothelial growth factor (VEGF)-D and its receptor, VEGF receptor 3, as a prognostic factor in endometrial carcinoma.

PURPOSE: To evaluate the prognostic value of vascular endothelial growth factor (VEGF)-D and VEGF receptor (VEGFR)-3 in endometrial carcinoma. EXPERIMENTAL DESIGN: We assessed the levels of immunoreactivity for VEGF-D and VEGFR-3 in 71 endometrial carcinomas, 14 complex atypical endometrial hyperplasias, and 16 normal endometria by immunohistochemistry. RESULTS: VEGF-D was stained in both tumor cells and adjacent stromal cells. VEGFR-3 was stained in both tumor cells and adjacent endothelial cells. Immunoreactivity for VEGF-D in tumor cells and adjacent stromal cells became significantly stronger as lesions progressed from normal endometrium to advanced carcinoma. Similarly, immunoreactivity for VEGFR-3 in tumor cells and adjacent endothelial cells was significantly greater as lesions progressed from normal endometrium to advanced carcinoma. A strong correlation was found between high levels of VEGF-D immunoreactivity in carcinoma cells and VEGFR-3 in both carcinoma cells and adjacent endothelial cells. Similarly, high levels of VEGF-D immunoreactivity in stromal cells were significantly correlated with those of VEGFR-3 in both carcinoma cells and endothelial cells. High levels of VEGF-D in carcinoma cells and stromal cells, as well as those of VEGFR-3 in carcinoma cells and endothelial cells, were significantly related to myometrial invasion and lymph node metastasis. A strong correlation was found between poor survival and high levels of VEGF-D in both carcinoma cells and stromal cells and between poor survival and high levels of VEGFR-3 in carcinoma cells. Moreover, the high levels of VEGF-D in stromal cells and VEGFR-3 in carcinoma cells were independent prognostic factors in endometrial carcinoma. CONCLUSIONS: The presence of VEGF-D and VEGFR-3 in endometrial carcinoma may predict myometrial invasion and lymph node metastasis and may prospectively identify patients who are at increased risk for poor outcome. In addition, VEGF-D and VEGFR-3 may be promising targets for new therapeutic strategies in endometrial carcinoma.     

Expression of extracellular matrix components versican, chondroitin sulfate, tenascin, and hyaluronan, and their association with disease outcome in node-negative breast cancer.

PURPOSE: The purpose is to determine whether the levels of expression of extracellular matrix components in peritumoral stroma are predictive of disease outcome for women with node-negative breast cancer. EXPERIMENTAL DESIGN: Tumor tissue from 86 patients with node-negative breast cancer was examined by immunohistochemical staining for the expression of versican, chondroitin sulfate (CS), tenascin, and hyaluronan (HA). With the exception of HA, the expression of the extracellular matrix components was measured by video image analysis. Statistical correlation of the immunohistochemical data with clinicopathological characteristics and disease outcome was performed. RESULTS: All of the extracellular matrix components were present in the peritumoral stroma of the entire study cohort. In contrast, immunoreactivity within the cancer cell was observed in 82% of tumors for HA, 12% for CS, and 4% for tenascin; no immunostaining of cancer cells for versican was observed for any of the tumors. Cox regression and Kaplan-Meier analyses indicated that elevated expression of stromal versican predicted increased risk and rate of relapse in this cohort. Elevated expression of tenascin was predictive of increased risk and rate of death only. Although neither CS nor HA were predictive of disease outcome in this cohort, tumor size was predictive of increased risk and rate of both relapse and survival. CONCLUSIONS: Elevated expression within peritumoral stromal matrix of versican and tenascin was predictive of relapse-free and overall survival, respectively, in women with node-negative breast cancer.     

Presence of active gelatinases in endometrial carcinoma and correlation of matrix metalloproteinase expression with increasing tumor grade and invasion

BACKGROUND: The actions of the extracellular-matrix degrading enzymes, matrix metalloproteinases (MMPs), are implicated in tumorigenesis. The cellular localization of MMP-2, MMP-9, membrane type 1 (MT1)-MMP, tissue inhibitors of metalloproteinases (TIMPs) 1-3, and the presence of active gelatinases were investigated in endometrial carcinoma. METHODS: Endometrial carcinomas were grouped according to histologic grade (Grades 1-3), depth of myometrial invasion (0, < 50%, > 50%) and the presence of vascular/lymphatic invasion. Twenty-nine endometrial carcinoma biopsies were investigated immunohistochemically to determine the tissue localization of MMP-2 (gelatinase A), MMP-9 (gelatinase B), MT1-MMP, and TIMPs 1-3. In situ hybridization was performed to localize MMP-2 and MMP-9 mRNA. The presence of active gelatinases was assessed using in situ zymography. RESULTS: Epithelial tumor cells were the main site of MMP-2, MMP-9, and MT1-MMP protein. Variable stromal cell localization was also observed, particularly in areas adjacent to tumor nests. Semiquantitative analysis revealed increases in MMP-9 and MMP-2 but not MT1-MMP staining scores in tumor epithelial cells in the transition from histologic Grade 1 to Grades 2 and 3. Matrix metalloproteinase-9 and MT1-MMP staining scores in tumor cells were significantly associated with the presence of myometrial invasion and vascular/lymphatic invasion, while MMP-2 did not correlate with these factors. In addition, MT1-MMP was co-localized with MMP-2, supporting its role in the activation of proMMP-2. Tumor cells from all histologic grades stained intensely for TIMP-2 and TIMP-3 proteins, while variable stromal staining was observed. In Grade 1 carcinomas TIMP-1 was predominantly immunolocalized to the stromal compartment with variable tumor cell localization being observed in Grades 2 and 3 carcinomas. Matrix metalloproteinase-9 and MMP-2 mRNAs were predominantly observed in tumor epithelial cells as well as in the stroma to varying degrees. In situ zymography revealed active forms of gelatinases at the cellular surface and in association with tumor epithelial cells within endometrial carcinoma tissues. CONCLUSIONS: These data suggest that increasing expression of MMPs and endometrial carcinoma progression are closely related. Active gelatinases are present in endometrial carcinoma, resulting in alterations to the microenvironment that promote tumor invasion and metastasis.     

Spontaneous apoptosis in the intra-ductal component's stroma of breast invasive carcinoma

Spontaneous apoptosis by in situ detection of DNA fragmentation (DNAf) was investigated in breast invasive ductal carcinoma (IDC) frozen samples removed from 61 untreated patients. The incidence of DNAf was low in carcinoma cells and was mainly detected in the stroma. In the stroma at a distance from carcinoma cells, DNAf was inversely related to estradiol plasma level variations (p=0.01), indicating that it probably remained under physiological hormonal regulation. In the stroma adjacent to carcinoma cells, DNAf was correlated to tumor progression parameters such as the presence of a comedo intra ductal carcinoma (DCIS) component (p=0.001) and axillary lymph node metastasis (p=0.002), suggesting that this stromal compartment more probably represented a tumoral component closely associated to epithelial tumor cells. Therefore, the detection of DNAf in the adjacent stroma of breast carcinoma could help to predict progression in non invasive tumors and also in invasive tumors in those patients without lymph node invasion.     

Tenascin expression in primary and recurrent breast carcinomas and the effect of tenascin on breast tumor cell cultures

Tenascin is generally classified as an anti-adhesive protein. Many cells do not adhere to tenascin or if they adhere they do not spread. In this study we analysed the stromal expression of tenascin-C in primary, second primary and recurrent breast carcinomas and the ability of tenascin-C to stimulate the focal adhesion plaques in MDA-MB-435 breast carcinoma cell line. To assess the tenascin-C expression formalin-fixed, paraffin-embedded specimens of 20 specially selected breast carcinomas and their recurrences (14) or a second primary breast cancer of the same patient (6) were examined with immunohistochemical methods. We also studied the effect of tenascin-C on focal adhesion plaques added to MDA-MB-435 breast carcinoma cell line. During a median 2,9-year patient follow up 14 local recurrences and 6-second primary breast carcinomas developed in the 20 patients. In 3 cases a second recurrence occurred. The presence of tenascin in tumor cells, in the proliferating and some normal ducts, near to the tumor cell nests, in the stroma and in ductal carcinoma in situ component of the invasive carcinoma may suggest the role of tenascin played in tumor cell migration. Soluble tenascin added to the cell culture had minimal or no effect on focal adhesion plaques. Tenascin only seems not to be of prognostic value in predicting the local recurrence of breast cancer.     

Analysis of stromal signatures in the tumor microenvironment of ductal carcinoma in situ

Recent advances in the study of the tumor microenvironment have revealed significant interaction between tumor cells and their surrounding stroma in model systems. We have previously shown that two distinct stromal signatures derived from a macrophage (CSF1) response and a fibroblastic (DTF-like) response are present in subsets of invasive breast cancers and show a correlation with clinical outcome [1–3]. In the present study we explore whether these signatures also exist in the stroma of ductal carcinoma in situ (DCIS). We studied the signatures by both gene expression profile analysis of a publically available data set of DCIS and by immunohistochemistry (IHC) on a tissue microarray of DCIS and invasive breast cancer cases. Both the gene expression and immunohistochemical data show that the macrophage response and fibroblast expression signatures are present in the stroma of subsets of DCIS cases. The incidence of the stromal signatures in DCIS is similar to the incidence in invasive breast cancer that we have previously reported. We also find that the macrophage response signature is associated with higher grade DCIS and cases which are ER and PR negative, whereas the fibroblast signature was not associated with any clinicopathologic features in DCIS. A comparison of 115 matched cases of DCIS and invasive breast cancer found a correlation between the type of stromal response in DCIS and invasive ductal carcinoma (IDC) within the same patient for both the macrophage response and the fibroblast stromal signatures (P = 0.03 and 0.08, respectively). This study is a first characterization of these signatures in DCIS. These signatures have significant clinicopathologic associations and tend to be conserved as the tumor progresses from DCIS to invasive breast cancer.     

Syndecan-1 expression by stromal fibroblasts promotes breast carcinoma growth in vivo and stimulates tumor angiogenesis

The induction of the cell surface heparan sulfate proteoglycan syndecan-1 (Sdc1) in stromal fibroblasts is observed in more than 70% of human breast carcinomas. Using a coculture model, we have recently shown that stromal cell-derived Sdc1 stimulates carcinoma cell proliferation in vitro, and that this activity requires Sdc1 glycanation. In the present study, we investigated the effect of stromal cell Sdc1 on breast carcinoma growth in vivo. MDA-MB-231 human breast carcinoma cells were inoculated into the flanks of athymic nude mice either alone, or as mixed suspensions with Sdc1-transfected or mock-transfected 3T3 mouse fibroblasts. The mixed tumors showed an intimate association between carcinoma cells and stromal fibroblasts and histologically closely resembled poorly differentiated human breast carcinomas. The presence of fibroblasts led to significantly accelerated tumor growth, which was further augmented (88% increase) by forced expression of stromal Sdc1. The hyperemic macroscopic appearance of tumors containing Sdc1-positive stromal cells contrasted with pale tumors developing in the presence of mock-transfected fibroblasts, which prompted us to examine tumor microvessels. Stromal Sdc1 expression was associated with a significantly elevated microvessel density (36% increase) and a larger vessel area (153% increase). To evaluate the relevance of this finding in human breast cancer, the relationship between stromal Sdc1 and tumor vascularity was also examined in a tissue array containing 207 human breast carcinoma samples. Similar to the xenografts, stromal Sdc1 expression correlated with both vessel density (P=0.013) and total vessel area (P=0.0026). In conclusion, stromal fibroblast-derived Sdc1 stimulates breast carcinoma growth and angiogenesis in vivo.     

The signaling adapter protein PINCH is up-regulated in the stroma of common cancers,notably at invasive edges

BACKGROUND: PINCH is an LIM (double zinc finger domain) protein that functions as an adapter at a key convergence point for integrin and growth factor signal transduction. Because no information is available regarding its expression in vivo in human tissues, this study evaluated the distribution and abundance of PINCH in patients with breast, prostate, lung, colon, and skin carcinomas. METHODS: A polyclonal antibody was raised to a purified 6-histidine PINCH fusion protein and used to evaluate 74 cases comprising 33 breast carcinomas (21 ductal carcinomas, 6 lobular carcinomas, 4 ductal carcinomas in situ, 2 lobular carcinomas in situ), 22 prostate carcinomas, 5 colon carcinomas, 6 lung carcinomas (3 adenocarcinomas and 3 squamous carcinomas), and 8 skin carcinomas (4 basal cell carcinomas and 4 squamous cell carcinomas) by immunoperoxidase histochemistry of formalin-fixed, paraffin-embedded tissues. Lysates of frozen tissue from the epithelium of two normal breasts and six breast carcinomas were evaluated by immunoblotting. RESULTS: Immunostaining for PINCH was increased in the cytoplasm of fibroblastoid cells in areas of the tumor-associated stroma in all carcinomatous tissues evaluated. The most intense stromal immunostaining for PINCH was noted at invasive edges, particularly in breast carcinomatous tissue. Immunoblotting of lysates from normal breast and breast carcinomatous tissue confirmed that PINCH protein expression was markedly increased in breast carcinomatous tissues. CONCLUSIONS: PINCH is up-regulated in tumor-associated stromal cells, particularly at invasive edges, and may be a marker for stroma manifesting the ability to facilitate invasion. Because of this and because PINCH functions as a "molecular switch" in signal transduction, PINCH may be a new target for drug discovery aimed at the tumor-associated stroma.     

Distribution of laminin and fibronectin isoforms in oral mucosa and oral squamous cell carcinoma

The expression of laminin and fibronectin isoforms varies with cellular maturation and differentiation and these differences may well influence cellular processes such as adhesion and motility. The basement membrane (BM) of fetal oral squamous epithelium contains the laminin chains, alpha2, alpha3, alpha5, beta1, beta2, beta3, gamma1 and gamma2. The BM of adult normal oral squamous epithelium comprises the laminin chains, alpha3, alpha5, beta1, beta3, gamma1 and gamma2. A re-expression of the laminin alpha2 and beta2 chains could be shown in adult hyperproliferative, dysplastic and carcinomatous lesions. In dysplasia and oral squamous cell carcinoma (OSCC), multifocal breaks of the BM are present as indicated by laminin chain antibodies. These breaks correlate to malignancy grade in their extent. Moreover, in the invasion front the alpha3 and gamma2 chain of laminin-5 can immunohistochemically be found outside the BM within the cytoplasm of budding carcinoma cells and in the adjacent stroma. The correlation between the morphological pattern of invasive tumour clusters and a laminin-5 immunostaining in the adjacent stroma may suggest, first, that a laminin-5 deposition outside the BM is an immunohistochemical marker for invasion and second, that OSCC invasion is guided by the laminin-5 matrix. Expression of oncofetal fibronectins (IIICS de novo glycosylated fibronectin and ED-B fibronectin) could be demonstrated throughout the stromal compartment. However, the ED-B fibronectin synthesizing cells (RNA/RNA in situ hybridization) are confined to small stroma areas and to single stroma and inflammatory cells in the invasion front. A correlation of the number of ED-B fibronectin synthesizing cells to malignancy grade could not be seen. ED-B fibronectin mRNA-positive cells seem to be concentrated in areas of fibrous stroma recruitment with a linear alignment of stromal fibro-/myofibroblasts (desmoplasia). Double staining experiments (ED-B fibronectin in situ hybridization and alpha-smooth muscle actin immunohistochemistry) indicated that the stroma myofibroblasts are a preferential source of ED-B fibronectin. In conclusion, in OSCC, a fetal extracellular matrix conversion is demonstrable. Tumour cells (laminin alpha2 and beta2 chain) and recruited stromal myofibroblasts (oncofetal ED-B fibronectin) contribute to the fetal extracellular matrix milieu.     

Expression of cathepsin D, stromelysin-3, and urokinase by reactive stromal cells on breast carcinoma prognosis.

BACKGROUND: Current literature suggests that several proteases act in a cascade to mediate remodeling of the extracellular matrix and favor cancer progression. Others and the authors of this study recently identified cathepsin D, stromelysin-3, and urokinase plasminogen activator (uPA) expression by reactive stromal cells as significant factors of poor prognosis in breast carcinoma. The authors evaluated the joint effect of protease expression on cancer aggressiveness. METHODS: Protease expression was analyzed by immunohistochemistry (cathepsin D) and in situ hybridization (stromelysin-3 and uPA) on formalin fixed paraffin embedded specimens from 557 breast carcinomas without distant metastasis at diagnosis and with an average of 10 years of follow-up. RESULTS: Of the 557 breast carcinomas, 80 (14.3%) expressed all 3 proteases, and 134 (24%) expressed none of them. An adjusted Cox model revealed significantly worse distant metastasis free survival (DMFS) with expression of all three proteases (P < 0.0001). The DMFS of patients whose tumor lacked at least one of the three proteases was similar to that of patients without any protease expression, irrespective of the type or number of proteases missing. CONCLUSIONS: This study suggests that proteases expressed by reactive stromal cells are interdependent and that a breach in the protease pathway may impair breast carcinoma progression.