Expression of CXCL1 in human endothelial cells induces angiogenesis through the CXCR2 receptor and the ERK1/2 and EGF pathways.

Endothelial cell growth and proliferation are critical for angiogenesis; thus, greater insight into the regulation of pathological angiogenesis is greatly needed. Previous studies have reported on chemokine (C-X-C motif) ligand 1 (CXCL1) expression in epithelial cells and that secretion of CXCL1 from these epithelial cells induces angiogenesis. However, limited reports have demonstrated CXCL1 expression in endothelial cells. In this report, we present data that expand on the role of CXCL1 in human endothelial cells inducing angiogenesis. Specifically, CXCL1 is expressed and secreted from human endothelial cells. Interference of CXCL1 function using neutralizing antibodies resulted in a reduction in endothelial cell migration and viability/proliferation, the latter associated with a decrease in levels of cyclin D and cdk4. In vitro studies revealed that CXCL1 influenced neoangiogenesis through the regulation of epidermal growth factor and ERK1/2. In a xenograft angiogenesis model, interference of CXCL1 function resulted in inhibition of angiogenesis. A better understanding of the role of CXCL1 in the interactions between the endothelial and epithelial components will provide insight into how human tissues use CXCL1 to survive and thrive in a hostile environment.

Multiplex protein signature for the detection of bladder cancer in voided urine samples.

PURPOSE: Accurate urine assays for bladder cancer detection would benefit patients and health care systems. Through extensive genomic and proteomic profiling of urine components we previously identified a panel of 8 biomarkers that can facilitate the detection of bladder cancer in voided urine samples. In this study we confirmed this diagnostic molecular signature in a diverse multicenter cohort. MATERIALS AND METHODS: We performed a case-control, phase II study in which we analyzed voided urine from 102 subjects with bladder cancer and 206 with varying urological disorders. The urinary concentration of 8 biomarkers (IL-8, MMP-9 and 10, PAI-1, VEGF, ANG, CA9 and APOE) was assessed by enzyme-linked immunosorbent assay. Diagnostic performance of the panel of tested biomarkers was evaluated using ROCs and descriptive statistical values, eg sensitivity and specificity. RESULTS: Seven of the 8 urine biomarkers were increased in subjects with bladder cancer relative to those without bladder cancer. The 7 biomarkers were assessed in a new model, which had an AUROC of 0.88 (95% CI 0.84-0.93), and 74% sensitivity and 90% specificity. In contrast, the sensitivity of voided urine cytology and the UroVysion® cytogenetic test in this cohort was 39% and 54%, respectively. Study limitations include analysis performed on banked urine samples and the lack of voided urine cytology and cytogenetic test data on controls. CONCLUSIONS: The study provides further evidence that the reported panel of diagnostic biomarkers can reliably achieve the noninvasive detection of bladder cancer with higher sensitivity than currently available urine based assays.

Chemokine (C-X-C) ligand 1 (CXCL1) protein expression is increased in aggressive bladder cancers.

BACKGROUND: Chemokines, including chemokine (C-X-C motif) ligand 1 (CXCL1), may regulate tumor epithelial-stromal interactions that facilitate tumor growth and invasion. Studies have linked CXCL1 expression to gastric, colon and skin cancers, but limited studies to date have described CXCL1 protein expression in human bladder cancer (BCa). METHODS: CXCL1 protein expression was examined in 152 bladder tissue specimens (142 BCa) by immunohistochemical staining. The expression of CXCL1 was scored by assigning a combined score based on the proportion of cells staining and intensity of staining. CXCL1 expression patterns were correlated with clinicopathological features and follow-up data. RESULTS: CXCL1 protein expression was present in cancerous tissues, but was entirely absent in benign tissue. CXCL1 combined immunostaining score was significantly higher in high-grade tumors relative to low-grade tumors (p = 0.012). Similarly, CXCL1 combined immunostaining score was higher in high stage tumors (T2-T4) than in low stage tumors (Ta-T1) (p < 0.0001). An increase in the combined immunostaining score of CXCL1 was also associated with reduced disease-specific survival. CONCLUSION: To date, this is the largest study describing increased CXCL1 protein expression in more aggressive phenotypes in human BCa. Further studies are warranted to define the role CXCL1 plays in bladder carcinogenesis and progression.

Diagnostic potential of urinary α1-antitrypsin and apolipoprotein E in the detection of bladder cancer.

PURPOSE: The ability to reliably diagnose bladder cancer in voided urine samples would be a major advance. Using high throughput technologies, we identified a panel of bladder cancer associated biomarkers with potential clinical usefulness. In this study we tested 4 potential biomarkers for the noninvasive detection of bladder cancer. MATERIALS AND METHODS: We examined voided urine specimens from 124 patients, including 63 newly diagnosed with bladder cancer and 61 controls. Concentrations of proteins were assessed by enzyme-linked immunosorbent assay, including α1-antitrypsin, apolipoprotein E, osteopontin and pentraxin 3. Data were compared to the results of urinary cytology and the BTA Trak® enzyme-linked immunosorbent assay based bladder cancer detection assay. We used the AUC of ROC curves to compare the usefulness of each biomarker to detect bladder cancer. RESULTS: Urinary levels of α1-antitrypsin, apolipoprotein E and bladder tumor antigen were significantly increased in subjects with bladder cancer. α1-Antitrypsin (AUC 0.9087, 95% CI 0.8555-0.9619) and apolipoprotein E (AUC 0.8987, 95% CI 0.8449-0.9525) were the most accurate biomarkers. The combination of α1-antitrypsin and apolipoprotein E (AUC 0.9399) achieved 91% sensitivity, 89% specificity, and a positive and negative predictive value of 89% and 90%, respectively. Multivariate regression analysis highlighted only apolipoprotein E as an independent predictor of bladder cancer (OR 24.9, 95% CI 4.22-146.7, p = 0.0004). CONCLUSIONS: Alone or in combination, α1-antitrypsin and apolipoprotein E show promise for the noninvasive detection of bladder cancer (OR 24.9, 95% CI 4.22-146.7, p = 0.0004). Larger, prospective studies including more low grade, low stage tumors are needed to confirm these results.

IL-8 as a urinary biomarker for the detection of bladder cancer.

BACKGROUND: Current urine-based assays for bladder cancer (BCa) diagnosis lack accuracy, so the search for improved biomarkers continues. Through genomic and proteomic profiling of urine, we have identified a panel of biomarkers associated with the presence of BCa. In this study, we evaluated the utility of three of these biomarkers, interleukin 8 (IL-8), Matrix metallopeptidase 9 (MMP-9) and Syndecan in the diagnosis of BCa through urinalysis. METHODS: Voided urines from 127 subjects, cancer subjects (n = 64), non-cancer subjects (n = 63) were analyzed. The protein concentrations of IL-8, MMP-9, and Syndecan were assessed by enzyme-linked immunosorbent assay (ELISA). Data were also compared to a commercial ELISA-based BCa detection assay (BTA-Trak©) and urinary cytology. We used the area under the curve of a receiver operating characteristic (AUROC) to compare the performance of each biomarker. RESULTS: Urinary protein concentrations of IL-8, MMP-9 and BTA were significantly elevated in BCa subjects. Of the experimental markers compared to BTA-Trak©, IL-8 was the most prominent marker (AUC; 0.79; 95% confidence interval [CI], 0.72-0.86). Multivariate regression analysis revealed that only IL-8 (OR; 1.51; 95% CI, 1.16-1.97, p = 0.002) was an independent factor for the detection of BCa. CONCLUSIONS: These results suggest that the measurement of IL-8 in voided urinary samples may have utility for urine-based detection of BCa. These findings need to be confirmed in a larger, prospective cohort.

Derivation of molecular signatures for breast cancer recurrence prediction using a two-way validation approach.

Previous studies have demonstrated the potential value of gene expression signatures in assessing the risk of post-surgical breast cancer recurrence, however, many of these predictive models have been derived using simple computational algorithms and validated internally or using one-way validation on a single dataset. We have recently developed a new feature selection algorithm that overcomes some limitations inherent to high-dimensional data analysis. In this study, we applied this algorithm to two publicly available gene expression datasets obtained from over 400 patients with breast cancer to investigate whether we could derive more accurate prognostic signatures and reveal common predictive factors across independent datasets. We compared the performance of three advanced computational algorithms using a robust two-way validation method, where one dataset was used for training and to establish a prediction model that was then blindly tested on the other dataset. The experiment was then repeated in the reverse direction. Analyses identified prognostic signatures that while comprised of only 10-13 genes, significantly outperformed previously reported signatures for breast cancer evaluation. The cross-validation approach revealed CEGP1 and PRAME as major candidates for breast cancer biomarker development.

Urinary proteomic profiling for diagnostic bladder cancer biomarkers.

The ability to detect and monitor bladder cancer in noninvasively obtained urine samples is a major goal. While a number of protein biomarkers have been identified and commercially developed, none have greatly improved the accuracy of sample evaluation over invasive cystoscopy. The ongoing development of high-throughput proteomic profiling technologies will facilitate the identification of molecular signatures that are associated with bladder disease. The appropriate use of these approaches has the potential to provide efficient biomarkers for the early detection and monitoring of recurrent bladder cancer. Identification of disease-associated proteins will also advance our knowledge of tumor biology, which, in turn, will enable development of targeted therapeutics aimed at reducing morbidity from bladder cancer. In this article, we focus on the accumulating proteomic signatures of urine in health and disease, and discuss expected future developments in this field of research.

VEGF induces expression of Bcl-2 and multiple signaling factors in microvascular endothelial cells in a prostate cancer model.

PURPOSE: We have previously demonstrated that prostate tumors that highly express Bcl-2 are not only more tumorigenic, but also more angiogenic than low Bcl-2 expressing tumors. Observed increased rates of angiogenesis are likely due to the secretion of multiple factors from the tumor cells. EXPERIMENTAL DESIGN: Human endothelial cells were subjected to exogenous VEGF or conditioned media from PC-3 cells and assayed by several in vitro systems to better characterize the effects of tumor microenvironment on endothelial cells. RESULTS: VEGF stimulation increased Bcl-2 expression in human microvascular endothelial cells (HMVECs), at least partially through stabilization of Bcl-2 mRNA transcripts, and protected these cells from apoptosis. These effects were mimicked by treatment of HMVECs with conditioned media from cultured PC-3 prostate tumor cells manipulated to overexpress Bcl-2. Through the use of kinase inhibitors and molecular profiling, several distinct pathways were implicated in the regulation of Bcl-2 in HMVECs, including those involving PI3K/AKT, PKC, mTOR, STAT-1, and IL-8, factors associated with tumor survival and growth. CONCLUSIONS: This study identifies molecular elements of a link between Bcl-2 expression in distinct cell types within a tumor and reaffirms that strategies designed to target Bcl-2 are desirable as they might enhance treatment response through dual effects.

Breast tumor metastasis: analysis via proteomic profiling.

The ability to predict the metastatic behavior of a patient's cancer, as well as to detect and eradicate such recurrences, remain major clinical challenges in oncology. While many potential molecular biomarkers have been identified and tested previously, none have greatly improved the accuracy of specimen evaluation over routine histopathological criteria and, to date, they predict individual outcomes poorly. The ongoing development of high-throughput proteomic profiling technologies is opening new avenues for the investigation of cancer and, through application in tissue-based studies and animal models, will facilitate the identification of molecular signatures that are associated with breast tumor cell phenotype. The appropriate use of these approaches has the potential to provide efficient biomarkers, and to improve our knowledge of tumor biology. This, in turn, will enable the development of targeted therapeutics aimed at ameliorating the lethal dissemination of breast cancer. In this review, we focus on the accumulating proteomic signatures of breast tumor progression, particularly those that correlate with the occurrence of distant metastases, and discuss some of the expected future developments in the field.

Differential expression of ribosomal proteins in a human metastasis model identified by coupling 2-D liquid chromatography and mass spectrometry.

BACKGROUND: Proteomic profiling of an experimental tumor metastasis model has the potential to identify gene products that can influence this fatal phenotype of tumor cells. In this study, we focused on the notoriously difficult to assess ribosomal protein component of a pair of cell lines which originate from the same tumor but have opposite metastatic capabilities. MATERIALS AND METHODS: Cell lysate proteins were separated using a two-dimensional liquid chromatographic system directly coupled to an ESI-TOF mass spectrometer for accurate intact protein MW analysis. Characterization of distinct post-translational modifications and sequence variation within several ribosomal proteins was obtained using monolithic capillary LC/MS/MS, MALDI-MS and -MS/MS. RESULTS: The combination of these techniques enabled the identification of 45 unique ribosomal proteins, several of which were differentially expressed in metastatic M4A4 cells. CONCLUSION: The described proteomic profiling approach enables the identification of phenotype-associated ribosomal proteins for subsequent functional analyses and disease biomarker development.

The RhoGAP protein DLC-1 functions as a metastasis suppressor in breast cancer cells.

The identification of molecular signatures characteristic of tumor cells that are capable of metastatic spread is required for the development of therapeutic interventions to abrogate this lethal process. To facilitate this, we have previously characterized an experimental system in which the role of candidate metastasis-related genes can be screened and tested. Monoclonal cell lines M4A4 and NM2C5 are spontaneously occurring sublines of the MDA-MB-435 cell breast tumor cell line that exhibit phenotypic differences in growth, invasion, and metastatic efficiency in athymic mice. In this study, transcriptional profiles of these cell lines were created using oligonucleotide microarrays representing over 12,000 genes. Intensity modeling and hierarchical clustering analysis identified a 171-gene expression signature that correlated with metastatic phenotype and highlighted several GTPase signaling components. Restoration of one of these GTPases, deleted in liver cancer-1 (DLC-1), in metastatic M4A4 cells to levels observed in the nonmetastatic NM2C5 cell line resulted in the inhibition of migration and invasion in vitro and a significant reduction in the ability of these cells to form pulmonary metastases in athymic mice. These studies show the utility of expression profiling, in an appropriate experimental system, to identify genetic determinants of metastatic sufficiency. The finding that DLC-1 can act as a metastasis-suppressor gene supports an influential role for GTPase signaling in tumor progression.

Urinary mRNA biomarker panel for the detection of urothelial carcinoma.

The early detection of bladder cancer is important as the disease has a high rate of recurrence and progression. The development of accurate, non-invasive urinary assays would greatly facilitate detection. In previous studies, we have reported the discovery and initial validation of mRNA biomarkers that may be applicable in this context. In this study, we evaluated the diagnostic performance of proposed molecular signatures in an independent cohort.Forty-four mRNA transcripts were monitored blindly in urine samples obtained from a cohort of 196 subjects with known bladder disease status (89 with active BCa) using quantitative real-time PCR (RT-PCR). Statistical analyses defined associations of individual biomarkers with clinical data and the performance of predictive multivariate models was assessed using ROC curves. The majority of the candidate mRNA targets were confirmed as being associated with the presence of BCa over other clinical variables. Multivariate models identified an optimal 18-gene diagnostic signature that predicted the presence of BCa with a sensitivity of 85% and a specificity of 88% (AUC 0.935). Analysis of mRNA signatures in naturally micturated urine samples can provide valuable information for the evaluation of patients under investigation for BCa. Additional refinement and validation of promising multi-target signatures will support the development of accurate assays for the non-invasive detection and monitoring of BCa.

A microRNA biomarker panel for the non-invasive detection of bladder cancer.

The development of accurate, non-invasive urinary assays for bladder cancer would greatly facilitate the detection and management of a disease that has a high rate of recurrence and progression. In this study, we employed a discovery and validation strategy to identify microRNA signatures that can perform as a non-invasive bladder cancer diagnostic assay. Expression profiling of 754 human microRNAs (TaqMan low density arrays) was performed on naturally voided urine samples from a cohort of 85 subjects of known bladder disease status (27 with active BCa). A panel of 46 microRNAs significantly associated with bladder cancer were subsequently monitored in an independent cohort of 121 subjects (61 with active BCa) using quantitative real-time PCR (RT-PCR). Multivariable modeling identified a 25-target diagnostic signature that predicted the presence of BCa with an estimated sensitivity of 87% at a specificity of 100% (AUC 0.982). With additional validation, the monitoring of a urinary microRNA biomarker panel could facilitate the non-invasive evaluation of patients under investigation for BCa.

Telomerase reverse transcriptase promoter regulation during myogenic differentiation of human RD rhabdomyosarcoma cells.

During terminal differentiation of human and murine cells, telomerase activity and parallel transcription of telomerase reverse transcriptase (hTERT) are inhibited. In this study, we used in vitro and in vivo analyses to determine the role of hTERT promoter elements and associated factors during differentiation-induced inhibition of telomerase expression in RD, a human rhabdomyosarcoma cell line. Assay of telomerase enzyme activity, hTERT mRNA, and reporter gene assays confirmed that the hTERT promoter was silenced during 12-O-tetradecanoylphorbol-13-acetate-induced myogenic differentiation of telomerase-positive RD cells. Promoter deletion and mutation analyses revealed that two E-boxes and an AP-2 site present in a 320-bp region of the promoter were essential for the transcriptional activity of the hTERT gene. Electrophoretic mobility shift assays identified several factors that interact with this region of DNA, including the muscle-specific transcription factors Myf5, Myf6, and myogenin and the ubiquitously expressed factors Sp1 and AP-2. Ectopic expression of the E-box binding factors c-Myc and Mad did influence promoter activity in these cells; indeed, the presence of endogenous c-Myc protein was altered after differentiation. Our findings suggest that the acute regulation of hTERT transcription is primarily controlled by E-box elements, which bind a series of factors during the phased phenotypic changes occurring during the differentiation of RD human muscle cells.

Molecular cytogenetic analysis of a human breast metastasis model: identification of phenotype-specific chromosomal rearrangements.

We have previously characterized an experimental system in which the role of candidate metastasis-related genes can be screened and tested. Monoclonal cell lines M4A4 and NM2C5 originated from the MDA-MB-435 breast tumor cell line but have opposite metastatic capabilities in vivo. To investigate gross genetic changes present in this model, we performed a detailed molecular cytogenetic evaluation of the parental cell line, the M4A4 and NM2C5 cell lines, and related clones of metastatic phenotype. Using a combination of spectral karyotyping (SKY), G-banding, and fluorescence in situ hybridization (FISH), we were able to resolve the identity of all common marker chromosomes present in MDA-MB-435 cells, and to define several chromosomal changes, which were specific to each monoclonal cell line. Twenty identical structural and numerical chromosomal aberrations, including trisomies of chromosomes 2 and 5 as well as t(1;7), t(1;10), t(8;11), t(8;15), and t(20;21), were present in all cell lines. The majority of translocations were relatively simple non-reciprocal rearrangements, most frequently involving chromosomes 19, 1, 6, and 20. Chromosomal gains of 1, 7q, 8q, and 20q are common alterations in breast cancer. The metastatic M4A4 cell line contained numerous unique chromosomal aberrations, of which an abnormal banding region on chromosome 22, abr(22), was the best clone-specific identifier. Conversely, the t(12;15)(q22;q26.1) was found exclusively in the non-metastatic NM2C5 cell line. The integration of these karyotypic data with other cytogenetic and genomic databases will enhance our ability to identify genes that play critical roles in cancer development and progression.

Contrasting expression of thrombospondin-1 and osteopontin correlates with absence or presence of metastatic phenotype in an isogenic model of spontaneous human breast cancer metastasis.

Knowledge of the molecular mechanisms involved in metastatic spread is needed to facilitate advances in prognostic evaluation for individual patients and in the design of therapeutic interventions to inhibit the process. In an effort to establish a methodological framework for analysis of molecules and mechanisms involved in this complex multistep process, we have developed a well defined experimental system, in which the role of candidate genes can be screened and tested. By serial dilution cloning of the MDA-MB-435 breast tumor cell line and screening by orthotopic implantation into the mammary fat pad of athymic mice, we have derived a pair of breast tumor cell lines (M-4A4 and NM-2C5) that originate from the same breast tumor but have diametrically opposite metastatic capabilities. In 74% of inoculated athymic mice, clone M-4A4 metastasized consistently to the lungs, mimicking a major dissemination route of human breast cancer. Conversely, although equally tumorigenic, clone NM-2C5 did not metastasize to any distal site. We have confirmed that the cell lines originate from a single genetic source by spectral karyotyping and evaluated the expression of a number of proteins previously implicated in cellular transformation and metastasis. The ability of M-4A4 to metastasize was not associated with increased angiogenesis, as measured by immunohistochemical microvessel density analysis. However, RNA and protein analyses revealed that two secreted proteins were differentially expressed: osteopontin expression was increased approximately 30-fold in clone M-4A4 and thrombospondin-1 expression was increased approximately 15-fold in clone NM-2C5. These cell lines constitute a stable and accessible model for the identification of genes involved in the multistep process of breast tumor metastasis. Manipulation of candidate genes in these cells will permit evaluation of their functional significance in the geometric progression of breast cancer.

Prolonged dormancy and site-specific growth potential of cancer cells spontaneously disseminated from nonmetastatic breast tumors as revealed by labeling with green fluorescent protein.

This study used an isogenic pair of metastatic (M4A4) and nonmetastatic (NM2C5), green fluorescent protein-labeled human breast cancer cell lines derived from the same patient and inoculated into the mammary glands of nude mice to investigate the dissemination patterns and fate of cells that escaped spontaneously from the resulting tumors. After tumors appeared, fluorescing single tumor cells were regularly seen in the lungs, even in animals inoculated with NM2C5, which fails to form secondary tumors in other organs. The sensitivity of the technique confirmed the continuing presence of scattered NM2C5 cells after primary tumor resection, although they formed no metastases by 6 months. These self-disseminated human tumor cells were retrievable from the tissues and were still viable and malignant, manifested by indefinite proliferation in vitro and green fluorescence and local tumorigenicity in vivo. Therefore, these scattered tumor cells were still immortal but rendered indefinitely quiescent by the microenvironmental conditions in the lung tissue. This is the first unequivocal demonstration of spontaneous distant dissemination of human cancer cells by undisturbed nonmetastatic tumors and comprises a valuable system for the analysis of tumor dormancy. In contrast, although many of the cells disseminating from M4A4 tumors grew into fluorescing metastases in the lungs, others remained solitary and quiescent. Therefore, even in a clonally derived cell population with metastatic properties, many cells do not, or cannot, mobilize the organ-specific growth properties needed to generate metastases. This experimental approach, by using self-disseminating, green fluorescent protein-labeled, sister cell lines of opposing metastatic phenotypes, opens new avenues for investigating topics of clinical relevance, including tumor cell dormancy, anatomical distribution of metastases, and host factors influencing the metastatic process.

The interrelationship between DRIM gene expression and cytogenetic and phenotypic characteristics in human breast tumor cell lines.

BACKGROUND: In order to facilitate the identification of genes involved in the metastatic phenotype we have previously developed a pair of cell lines from the human breast carcinoma cell line MDA-MB-435, which have diametrically opposite metastatic potential in athymic mice. Differential display analysis of this model previously identified a novel gene, DRIM (down regulated in metastasis), the decreased expression of which correlated with metastatic capability. DRIM encodes a protein comprising 2785 amino acids with significant homology to a protein in yeast and C. elegans, but little else is currently known about its function or pattern of expression. In a detailed analysis of the DRIM gene locus we quantitatively evaluated gene dosage and the expression of DRIM transcripts in a panel of breast cell lines of known metastatic phenotype. RESULTS: Fluorescent in situ hybridization (FISH) analyses mapped a single DRIM gene locus to human chromosome 12q23~24, a region of conserved synteny to mouse chromosome 10. We confirmed higher expression of DRIM mRNA in the non-metastatic MDA-MB-435 clone NM2C5, relative to its metastatic counterpart M4A4, but this appeared to be due to the presence of an extra copy of the DRIM gene in the cell line's genome. The other non-metastatic cell lines in the series (T47D MCF-7, SK-BR-3 and ZR-75-1) contained either 3 or 4 chromosomal copies of DRIM gene. However, the expression level of DRIM mRNA in M4A4 was found to be 2-4 fold higher than in unrelated breast cells of non-metastatic phenotype. CONCLUSIONS: Whilst DRIM expression is decreased in metastatic M4A4 cells relative to its non-metastatic isogenic counterpart, neither DRIM gene dosage nor DRIM mRNA levels correlated with metastatic propensity in a series of human breast tumor cell lines examined. Collectively, these findings indicate that the expression pattern of the DRIM gene in relation to the pathogenesis of breast tumor metastasis is more complex than previously recognized.

Elevated osteopontin and thrombospondin expression identifies malignant human breast carcinoma but is not indicative of metastatic status.

BACKGROUND: Our previous characterization of a human breast tumor metastasis model identified several candidate metastasis genes. The expression of osteopontin (OPN) correlated with the metastatic phenotype, whereas thrombospondin-1 (TSP-1) and tyrosinase-related protein-1 (TYRP-1) correlated with the nonmetastatic phenotype of independent MDA-MB-435 cell lines implanted orthotopically into athymic mice. The aim of the present study was to examine the cellular distribution of these molecules in human breast tissue and to determine whether the relative expression level of these three genes is associated with human breast tumor metastasis. METHODS: Sixty-eight fresh, frozen specimens including 31 primary infiltrating ductal carcinomas, 22 nodal metastases, 10 fibroadenomas, and five normal breast tissues were evaluated for OPN expression, TSP-1 expression and TYRP-1 expression. Immunohistochemistry was performed to monitor the cellular distribution and to qualitatively assess expression. Quantitative analysis was achieved by enrichment of breast epithelial cells using laser-capture microdissection and subsequent real-time, quantitative PCR. RESULTS: The epithelial components of the breast tissue were the source of OPN and TSP-1 expression, whereas TYRP-1 was present in both the epithelial and stromal components. Both OPN and TSP-1 expression were significantly higher in malignant epithelial sources over normal and benign epithelial sources, but no difference in expression levels was evident between primary tumors with or without metastases, nor between primary and metastatic carcinomas. CONCLUSION: Elevated expression of OPN and TSP-1 may play a role in the pathogenesis of breast cancer. The multiplex analysis of these molecules may enhance our ability to diagnose and/or prognosticate human breast malignancy.