Invasive Ductal Carcinoma Cells Research Articles

Breast cancer proteomics by laser capture microdissection, sample pooling, 54‐cm IPG IEF, and differential iodine radioisotope detection

The presence of progesterone receptor (PR) in estrogen receptor (ER)-positive breast cancer is associated with a good prognosis, and indicates that tumors are likely to respond to tamoxifen. However, ER+/PR- tumors respond less well. To reveal the potential molecular mechanism of this phenomenon, we sought to identify differential protein abundances between invasive ductal carcinoma cells from cryopreserved ER+/PR+ and ER+/PR- mammary tumor specimens. Because current proteomics methods are hampered in the examination of most primary human tumor samples by the extreme tissue heterogeneity, we used laser capture microdissection (LCM) to isolate tumor cells and developed a sample pooling strategy to analyze small sample protein lysates. Proteins from LCM-harvested tumors were pooled into four sub-pools from each condition of three tumors/sub-pool, and proteins from respective paired sub-pools were co-electrophoresed by 2-DE using 54-cm IEF over pH 4-9. Abundance ratios were accurately quantified by a differential multiplex radioactive ProteoTope method at low attomole levels ( approximately 3.6 microg protein per labeling reaction, <180 ng per multiplex protein sample per 54-cm gel). Applying this approach, differentially displayed proteins were identified by MS using comigrating non-radioactively labeled tumor proteins. They include decreased cytochrome b5 and transgelin, and more abundant CRABP-II, cyclophilin A, Neudesin, and hemoglobin in ER+/PR+ tumors versus ER+/PR- providing a possible explanation for differential susceptibility against tamoxifen as a result of deregulated cytochrome b5-dependent metabolism. This study demonstrates the potential of ProteoTope and LCM to enable extremely sensitive and precise differential analyses from well-defined primary clinical specimen.

Id-1 overexpression in invasive ductal carcinoma cells is significantly associated with intratumoral microvessel density in ER-negative/node-positive breast cancer

The aim of this study is to investigate the possible role of inhibitor of DNA binding (Id-1) overexpression in human breast cancer. We examined Id-1 expression by immunohistochemistry in 263 human breast cancers, 15 in situ lesions and 248 invasive cancers to investigate the relationship between its expression and various clinicopathological factors. Id-1 expression was significantly higher in invasive ductal carcinoma than in in situ ductal carcinoma or other invasive cancer subtypes ( P=0.029 and 0.006, respectively). We also examined the association between Id-1 expression and tumor angiogenesis by measuring microvessel densities (MVD). Regarding the endothelial cells of microvessels showed negative or very weak Id-1 expression, Id-1 overexpression was found to be significantly related to MVD ( P=0.014). Furthermore, Id-1 overexpression was found to be significantly associated with higher MVD in the ER-negative and node-involved subgroups of breast cancer ( P=0.040 and 0.046, respectively). These data indicate that Id-1 overexpression is significantly associated with tumor angiogenesis, especially in the ER-negative and node-positive subtypes of invasive breast cancer. Thus, Id-1 presents a possible therapeutic antitumor target molecule in ER-negative and node-positive breast cancer.

Localisation of COX-2 protein is different in breast ductal carcinoma and adjacent non-tumour ductal epithelium

A number of findings suggest that cyclooxygenase-2 (COX-2) is overexpressed in breast tumours. However, there is a lack of consensus in the literature regarding the pattern of expression of this protein in invasive breast ductal carcinoma and in the adjacent non-tumour ductal epithelium. This study compares the expression of COX-2 mRNA and protein in breast ductal carcinoma relative to non-tumour breast tissue. We analysed the expression of COX-2 mRNA by quantitative PCR, and COX-2 protein by immunohistochemistry in invasive ductal carcinoma as well as in non-tumour adjacent ductal epithelium from 34 breast biopsies diagnosed as being invasive ductal carcinoma. As control, we analysed expression of COX-2 protein by immunohistochemistry in surgically-resected benign breast lesions. Our results show that COX-2 mRNA and protein are overexpressed in non-tumour ductal epithelium compared with invasive ductal carcinoma. However, the pattern of the protein expression is different in tumour and non-tumour tissue: COX-2 protein is expressed predominantly in the membrane of the non-tumour ductal epithelium (including in benign breast lesions) while, in invasive ductal carcinoma cells, it is localised in the cytoplasm. The non-tumour ductal epithelium adjacent to invasive ductal carcinoma shows a higher COX-2 expression than does the invasive ductal carcinoma. However, the different localisation of the immunohistochemically-detected protein suggests a possible post-translational regulation of the protein.

Cathepsin D expression in the tumor cells and juxta-tumoral stromal cells of T1 invasive ductal carcinoma, Nos

Cathepsin D expression in the tumor cells and juxta-tumoral stromal cells of T1 invasive ductal carcinoma, Nos

Cystatin m: a novel candidate tumor suppressor gene for breast cancer.

The contribution of pericellular proteolysis to tumor progression is well documented. To better understand protease biology and facilitate clinical translation, specific proteolytic systems need to be better defined. In particular, the precise role of endogenous protease inhibitors still needs to be deciphered. We reported previously that cystatin M, a potent endogenous inhibitor of lysosomal cysteine proteases, significantly suppressed in vitro cell proliferation, migration, and Matrigel invasion. Here, we show that scid mice orthotopically implanted with breast cancer cells expressing cystatin M show significantly delayed primary tumor growth and lower metastatic burden in the lungs and liver when compared with mice implanted with mock controls. The incidence of metastasis, however, appeared to be unaltered between the cystatin M group and the control group. Experimental metastasis assays suggest that cystatin M suppressed tumor cell proliferation at the secondary site. By using laser capture microdissection and quantitative reverse transcription-polymerase chain reaction, we found consistent expression of cystatin M in normal human breast epithelial cells, whereas expression was decreased by 86% in invasive ductal carcinoma (IDC) cells of stage I to IV patients. Complete loss of expression of cystatin M was observed in two of three IDCs from stage IV patients. Immunohistochemical studies confirmed that expression of cystatin M in IDCs was partially or completely lost. We propose cystatin M as a novel candidate tumor suppressor gene for breast cancer.

Role of cytokeratin expression in differential diagnosis of intraductal proliferative lesions of breast

To evaluate the expression of cytokeratins in intraductal proliferative lesions of breast, including usual ductal hyperplasia (UDH), atypical ductal hyperplasia (ADH), ductal carcinoma-in-situ (DCIS) and its role in differential diagnosis. Ninety two cases of paraffin-embedded lesional breast tissue, 30 cases of frozen samples, cell cultures of hyperplastic ductal cells and 2 invasive ductal carcinoma cell lines (T47D and MCF-7) were used for this study. Immunohistochemistry was performed using EnVision method for 34betaE12, CK8 and CK14. The percentage of 34betaE12-positivity in paraffin-embedded samples of UDH, ADH, DCIS and invasive ductal carcinoma (IDC) was found to be 95.2%, 33.3%, 19.2% and 12.5% respectively. In frozen tissues, all UDH cases and 55% of IDC cases expressed 34betaE12. The primary UDH cell cultures and T47D cell line were also 34betaE12-positive, whereas MCF7 cell line showed negative staining. The expression rate of CK8 and CK14 in UDH was also different from that in ADH and DCIS. 34betaE12 can be useful in differential diagnosis of intraductal proliferative lesions of the breast. However application of this cytokeratin stain in intraoperative frozen sections is relatively limited. The expression patterns of CK8 and CK14 are also helpful in the differential diagnosis of similar lesions.

The Myofibroblast Is the Predominant Plasminogen Activator Inhibitor-1-Expressing Cell Type in Human Breast Carcinomas

The tumor level of plasminogen activator inhibitor-1 (PAI-1) is an informative biochemical marker of a poor prognosis in several cancer types. However, the tumor biological functions of PAI-1 and the identity of PAI-1-expressing cells are controversial. With the aim of immunohistochemically localizing PAI-1 in formalin-fixed, paraffin-embedded invasive ductal breast carcinoma samples, we raised new polyclonal antibodies against PAI-1 from different expression systems. The antibodies were affinity purified by absorption on immobilized preparations of PAI-1 different from those used for immunization. The specificity of the antibodies was ensured by immunoblotting analysis. In immunohistochemistry, the staining pattern obtained with the antibodies showed a good correlation with the PAI-1 mRNA expression pattern. In all 25 cases analyzed, PAI-1 immunoreactivity was predominantly localized in fibroblast-like cells. Double-immunofluorescence analyses showed co-expression of PAI-1 and alpha-smooth muscle actin in these cells, suggesting that they are myofibroblasts. PAI-1 was also seen in some myoepithelial cells surrounding occasional foci of ductal carcinoma in situ (9 of 25), some endothelial cells (8 of 25), some cancer cells (3 of 25), and some mast cells (6 of 25). In conclusion, we have provided a robust immunohistochemical procedure for detection of PAI-1 and shown that the majority of the PAI-1-expressing cells in invasive ductal breast carcinomas are myofibroblasts.

Immunohistochemical demonstration of the expression of CYP2E1 in human breast tumour and non-tumour tissues

Environmental chemicals are one of the risk factors in breast cancer genesis. Cytochrome P450 2E1 (CYP2E1), a member of multigene superfamily of enzymes, plays a major role in the activation of some of these chemicals such as tobacco-derived N-nitrosamines. Using immunohistochemistry, the cellular distribution and the level of expression of CYP2E1 was assessed in breast tumour and surrounding tumour free (control) breast tissue in 25 pairs of samples obtained from females with infiltrating ductal carcinoma. Cells staining with the CYP2E1 antibody showed only cytoplasmic positivity with varying intensities in 100% (25/25) of tumours and 96% (24/25) of normal breast tissues. Cytoplasmic staining was present in invasive ductal carcinoma cells but not in tumour stroma. CYP2E1 was detected in normal epithelial, myoepithelial and to a lesser degree in some of the non-epithelial cells (e.g. endothelial cells). No positive staining was detected in other non-epithelial cells such as fibroblasts in normal breast tissues. When CYP2E1 staining was assessed semiquantitatively the mean staining score values of CYP2E1 was found to be significantly higher in tumours compared to that of normal breast tissues. These results show that CYP2E1 protein is expressed in both tumour and normal breast tissue with an increased expression in breast tumours.

Stromal expression of CD10 in invasive breast carcinoma: a new predictor of clinical outcome.

CD10 is a cell surface neutral endopeptidase that is not consistently expressed in the stromal cells of the normal breast. Expression of CD10 is sometimes observed in the stromal cells of invasive ductal carcinoma, but its clinical significance has never been studied. Immunohistochemical examination of CD10 was performed in 123 cases of breast cancer. The median follow-up period of all patients was 8.0 years, and univariate and multivariate analyses were performed to evaluate the prognostic significance of stromal CD10 expression. There was no staining in the stromal cells of 13 non-invasive ductal carcinomas or normal breast tissue. Of 110 invasive ductal carcinomas, 20 cases (18%) in which more than 10% of the stromal cells stained positive throughout the cancer tissue were judged "positive". The frequency of positive stromal staining was significantly higher in the cases with axillary lymph-node metastasis ( P=0.038), but there were no correlations between stromal CD10 expression and age, tumor size, histologic grade, or clinical stage. The patients whose tumors contained CD10-positive stromal cells had a shorter metastasis-free interval ( P=0.0008). Univariate analysis demonstrated that patients with lymph-node metastasis also had a significantly shorter metastasis-free interval (metastasis vs no metastasis; P=0.0318). In the multivariate analysis, only CD10 remained a significant predictor for time to recurrence ( P=0.0059), and CD10 was the single significant prognostic factor for overall survival in the univariate analysis ( P=0.0021). These results suggest that stromal expression of CD10 in breast cancer is an important novel prognostic factor.

Availability of CD10 Immunohistochemistry as a Marker of Breast Myoepithelial Cells on Paraffin Sections

CD10, also called common acute lymphoblastic leukemia antigen (CALLA), was recently found to be expressed in nonhematopoietic tissues. Although CD10 was also identified in human breast myoepithelial cells, its availability of immunohistochemistry on paraffin sections has not been examined so far. In the present study, we demonstrated CD10 immunohistochemically on paraffin sections of both normal and pathological breast tissues, comparing its staining patterns to those of smooth muscle actin (SMA), which is now commonly used to highlight myoepithelium. CD10 was consistently positive in normal breast myoepithelial cells. CD10 also clearly highlighted myoepithelial cells in intraductal papilloma, adenosis, ductal hyperplasia, fibroadenoma, and phyllodes tumor as well as SMA did. In atypical ductal hyperplasia and ductal carcinoma in situ, continuous, discontinuous, and totally negative stainings of both CD10 and SMA were noted, depending on foci of neoplastic cell nests. However, both stainings clearly demonstrated myoepithelial cells of cancerized acini, being useful in differentiating lobular cancerization from microinvasion. Because SMA was also positive in normal vessels and spindle-shaped stromal cells, CD10, which was negative in vessels, was useful in differentiating myoepithelial cells from thin vascular wall in intracystic lesions with delicate papillae. Although background staining of spindle-shaped stromal cells was also noted in CD10, the positive cell number was less, and the signal was weaker than that of SMA. The absence of myoepithelial cells in invasive ductal carcinomas was more clearly highlighted by CD10 than SMA. We concluded that CD10 could be another useful marker of breast myoepithelial cells on paraffin sections. Combination of CD10 and SMA will provide more sophisticated information about presence or absence of myoepithelial cells in confusing breast lesions.

High-Performance Liquid Chromatography Analysis of Lysozyme in Wine

<h3>Abstract</h3> Invasive lobular carcinoma (ILC) is the second most common subtype of breast cancer following invasive ductal carcinoma (IDC) and characterized by the loss of E-cadherin-mediated adherens junctions. Despite displaying unique histological and clinical features, ILC still remains a chronically understudied disease with limited knowledge on the available laboratory research models. To this end, herein we report a comprehensive 2D and 3D phenotypic characterization of four Estrogen Receptor-positive human ILC cell lines - MDA-MB-134, SUM44, MDA-MB-330 and BCK4. Compared to the IDC cell lines MCF7, T47D and MDA-MB-231, ultra-low attachment culture conditions revealed a remarkable anchorage-independence ability that was unique to the ILC cells, a feature not evident in soft agar gels. 3D Collagen I and Matrigel culture indicated a generally loose morphology for the ILC cell lines, which exhibited differing preferences for adhesion to ECM proteins in 2D. Furthermore, ILC cells had limited migration and invasion ability in wound-scratch and transwell assays with the exception of haptotaxis to Collagen I. Transcriptional comparison of the cell lines confirmed the decreased cell proliferation and E-cadherin-mediated intercellular junctions in ILC, while uncovering the induction of novel pathways related to cyclic nucleotide phosphodiesterase activity, ion channels, drug metabolism and alternative cell adhesion molecules such as N-cadherin, some of which were also differentially regulated in ILC versus IDC tumors. Altogether, these studies will serve as an invaluable resource for the breast cancer research community and facilitate further functional discoveries towards understanding ILC, identifying novel drug targets and ultimately improving the outcome of patients with ILC. <h3>Authors’ Contributions</h3> <b><i>Conception and design:</i></b> N. Tasdemir, NE. Davidson, S. Oesterreich <b><i>Development of methodology:</i></b> N. Tasdemir, L. Zhu, GC. Tseng, S. Oesterreich <b><i>Acquisition of data (performed experiments, processed data, etc.):</i></b> N. Tasdemir, E. Bossart, Z. Li, Z. Li <b><i>Analysis and interpretation of data (e.g. biological interpretation, statistical analysis, computational analysis):</i></b> N. Tasdemir, Z. Li, KM. Levine, NE. Davidson, S. Oesterreich <b><i>Writing, review and/or revision of the manuscript:</i></b> N. Tasdemir, Z. Li, KM. Levine, BM. Jacobson, GC. Tseng, NE. Davidson, S. Oesterreich <b><i>Study supervision:</i></b> NE. Davidson and S. Oesterreich

Immunohistochemical studies on expression of human vascular smooth muscle myosin heavy chain isoforms in normal mammary glands, benign mammary disorders and mammary carcinomas.

The expression of myosin in normal and diseased mammary glands of 199 Japanese women was evaluated immunohistochemically by the avidin-biotin peroxidase complex method using antibodies to three human smooth muscle myosin heavy chain isoforms derived from the vascular smooth muscle: myosin SM1 is expressed consistently from fetal stage to adulthood, myosin SM2 appears only in well-differentiated smooth muscle after birth, and myosin SMemb is more abundant in embryonic aortas. SM1 was expressed in myoepithelial cells of normal mammary glands and fibrocystic diseases and in myoepithelial-like tumor cells in the basal layer of fibroadenomas and phyllodes tumors. SM2 was expressed only in the myoepithelial cells of mammary glands in breastfeeding women. SMemb was expressed more intensely in the cytoplasm of luminal epithelial cells in larger fibroadenomas (P< 0.01), or in the cytoplasm of carcinoma cells in invasive ductal carcinomas with metastasized lymph nodes (P< 0.001) and in those of higher histological grade (P<0.0001). Multivariate logistic analysis showed a significant correlation only between the expression of SMemb and histological grade (P< 0.0001), which is a prognostic factor of mammary carcinomas. These findings suggested the possible prognostic value of SMemb.

Evidence of ED-B + fibronectin synthesis in human tissues by non-radioactive RNA in situ hybridization. Investigations on carcinoma (oral squamous cell and breast carcinoma), chronic inflammation (rheumatoid synovitis) and fibromatosis (Morbus Dupuytren)

The splicing variant of fibronectin containing the ED-B domain (oncofoetal fibronectin) occurs in foetal tissues, reparative processes, organ fibrosis and in tumour tissues. Consequently, a supportive effect of ED-B+ fibronectin for tissue remodelling and tumour progression is assumed. A non-radioactive RNA-RNA in situ hybridization protocol for the investigation of ED-B+ fibronectin synthesis applicable in human tissues is introduced. The ED-B+ fibronectin synthesis was investigated in human disease processes, for which the occurrence of ED-B+ fibronectin is well demonstrated by immunohistochemistry (rheumatoid arthritis, oral squamous cell carcinoma, invasive ductal carcinoma of the breast and nodular palmar fibromatosis). The ED-B+ fibronectin synthesis could be shown in lining cells and in endothelial cells of synovial villi in rheumatoid arthritis, in stromal cells of oral squamous cell carcinoma and invasive ductal carcinoma and in fibro-/myofibroblasts in the proliferative and early involutional phase of nodular palmar fibromatosis. By means of double labelling (alpha-smooth muscle actin immunostaining - ED-B+ fibronectin in situ hybridization), the ED-B+ fibronectin synthesis could be shown to be a typical feature of myofibroblasts. In contrast to the often diffuse ED-B+ fibronectin immunostaining, only a few synthetically active stromal cells were observed focally accentuated within the tumour, which were interpreted as hot spots of tumour-stroma interaction.

Detection of novel genes that are up-regulated (Di12) or down-regulated (T1A12) with disease progression in breast cancer.

Carcinogenesis is a multistep process and it is believed that seven to ten alterations are needed to convert a normal cell into a maligant one. Thus, identification of these specific genetic lesions and their role in the progression of cancer will lead to more effective methods for early diagnosis, as well as provide targets for treatment and therapeutic intervention. To isolate a number of genes that are differentially expressed in human breast carcinomas we used subtractive hybridization and differential display cloning techniques. By using these methods we obtained 952 clones, characterized 288 clones by restriction mapping, and 105 by Northern blotting and DNA sequencing. Twenty-four clones were found to be previously unidentified, unique genes. Full-length complementary (c)DNA was isolated from clone T1A12 and Di12, and further characterized by computer data search. Polyclonal antibodies were generated against N- and/or C-terminal peptides of the predicted T1A12 and Di12 amino acid sequences and immunohistochemistry was used to delineate gene function in breast cancers. We found that T1A12 is a novel member of the insulin-like growth factor binding protein family and that its expression decreases with disease progression. In 60 primary breast tissues examined, strong T1A12-specific immunoperoxidase staining was observed in luminal epithelial cells of normal lobules or ducts, and in blood vessels. Moderate protein expression was detected in hyperplastic and ductal carcinoma in situ (DCIS) cells, but no staining was found in invasive ductal carcinoma (IDC) cells. On the contrary, by using the same primary breast specimen Di12, a gene which shares no sequence homology with any protein, was found to be overexpressed in IDC cells but was not detectable in normal breast tissues. Specific strong Di12 staining was seen in the cytoplasm, with a slight increase in perinuclear regions of less well differentiated cells similar to those present in ductal carcinomas with poor prognosis. Both the T1A12 and Di12 genes, studied alone or in combination, could prove valuable for understanding the biology of breast cancer, detecting early and late stage disease, and for selecting appropriate treatments.

Expression of Beta-1 Integrins on Tumor Cells of Invasive Ductal Breast Carcinoma

The integrins are transmembrane alfabeta heterodimers mediating cell-cell as well as cell-extracellular matrix interactions. The present study was designed to analyse the expression of beta-1 integrins on cryostat sections of invasive ductal carcinomas not otherwise specified by avidin-biotin complex immunoperoxidase technique, and to compare it with the morphometric prognostic index (MPI). The results show that the expression of beta-1 integrins is heterogeneous in the tumors. This heterogeneity was observed in quantitative and qualitative staining pattern. There was an absent expression of beta-1 integrins in 22 out of 55 tumors while 33 showed staining, weak on 23 cases and strong on 10 infiltrative ductal carcinomas. Statistical analysis pointed to some correlation of beta-1 integrins with some morphometric parameters. Low or absent expression of beta-1 integrins correlated significantly with tumors exceeding 2 cm (p < 0.0245). Moreover, a larger proportion of tumors with positive lymph nodes showed absence of beta-1 expression compared with negative lymph node, and this was also statistically significant (p < 0.0076). Correlation between mitotic activity index and staining intensity for beta-1 integrins was not found (p < 0.372). When tumors with different beta-1 expression were subdivided according to MPI values into two groups, one group with a low-risk, < 0.6, and second with a high risk, > 0.6, concordance in prognostic value was shown between MPI and beta-1 expression (p < 0.0193). These results support the idea that loss of beta-1 integrins correlates with the invasive and metastatic potential of tumor cells.