Journal: PLoS ONE

Article Title: Abrogation of Junctional Adhesion Molecule-A Expression Induces Cell Apoptosis and Reduces Breast Cancer Progression

doi: 10.1371/journal.pone.0021242

Figure Lengend Snippet: A) MMTV-PyVmT/JAMA−/− mice had longer disease-free time compared to JAM-A+/+ mice; B) 13 week-old MMTV-PyVmT/JAM-A−/− mice present reduced primary tumor growth. Bars are means ± SEM. The lung metastases ratio (the percentage of mice with metastases out of the total number of mice with tumors) was higher in MMTV-PyVmT/JAM-A+/+ compared to JAM-A−/− mice but did not reach statistical significance (p = 0.091); C) Correlation between JAM-A expression, tumor size and lung metastases: The population was classified for tumor size in two groups on the basis of the median weight of tumors (3.55 g): big tumors (above median) and small tumors (below median), each composed of 41 mice; the population was then classified by expression of JAM-A (31 JAM-A −/− mice and 51 JAM-A +/+ mice) and by incidence of metastases (55 mice did not develop metastases and 27 did). C.1) Venn diagram of Big/Small (above or below median weight) tumors and metastases. There is a significant correlation between size of tumor and development of metastases (overlap between these two sets is 23 mice while the expectation from the null hypothesis would be 13.5, leading to a significant positive correlation by Exact Fisher Test with p<10 −5 ). C.2) MMTV-PyVmT/JAM-A+/+ or JAM-A−/− and Big/Small tumors. Expression of JAM-A positively correlates with tumor size (out of the 51 JAM-A +/+ mice, 41 show big tumors and 30 are in the overlap of the two sets; significance of correlation p<0.034). C.3) MMTV-PyVmT/JAM-A+/+ or JAM-A−/− and metastases. The incidence of metastases is not significantly affected by JAM-A expression if balanced with tumor size. There is no significant correlation between expression of JAM-A (51 mice) and metastases (27 mice out of 82 mice), the overlap is 18 as shown in the Venn Diagram and the deviation from the expected 16.8 is not statistically significant (p = 0.368). All correlations are referred to a population of 82 animals, p Values are calculated by Exact Fisher Test, null values are the expectations for independent events.Tables on the right side of panel C summarize the results.

Article Snippet: Large-scale TMA of 444 patient breast cancer case–control cohort studies were performed using human JAM-A antibody (R&D, Minneapolis, MN) as described .

Techniques: Expressing

Journal: PLoS ONE

Article Title: Abrogation of Junctional Adhesion Molecule-A Expression Induces Cell Apoptosis and Reduces Breast Cancer Progression

doi: 10.1371/journal.pone.0021242

Figure Lengend Snippet: A) Histological examination shows lower cell proliferation and higher apoptosis in MMTV-PyVmT/JAM-A−/− as compared to JAM-A+/+ tumors. Right panels show quantification of Ki-67 or TUNEL positive cells. Data are means +/− SEM *p<0.05 by unpaired Student's t test. Scale bar: 100 µm. B) The growth curve of 4T1 cells in the presence of a control (Ctrl) antibody (Ab) or a JAM-A neutralizing Ab (BV11) was measured in the presence (left upper panel) or absence of fetal bovine serum (FBS) (left lower panel). Under starving conditions, upon exposure to a JAM-A blocking Ab the total cell number was significantly reduced while the number of TUNEL-positive cells was increased. Values are means ± SEM of at least 4 replicates from 3 independent experiments. *p<0.05, **p<0.01 by unpaired Student-t test.

Article Snippet: Large-scale TMA of 444 patient breast cancer case–control cohort studies were performed using human JAM-A antibody (R&D, Minneapolis, MN) as described .

Techniques: TUNEL Assay, Blocking Assay

Journal: PLoS ONE

Article Title: Abrogation of Junctional Adhesion Molecule-A Expression Induces Cell Apoptosis and Reduces Breast Cancer Progression

doi: 10.1371/journal.pone.0021242

Figure Lengend Snippet: A) Phase-contrast microscopy of cultured mammary tumor cells derived from MMTV-PyVmT/JAM-A+/+ and JAM-A−/− tumors. JAM-A+/+ cells formed confluent islands with epithelioid morphology and tight intercellular junctions. In contrast, JAM-A−/− cells form loosely organized islands and frequently grow as single cells with spindle shape morphology. Scale bar; 200 µm; B) Immunofluorescence staining of markers of adherens junctions (E-cadherin, β-catenin) and tight junctions (ZO-1, cingulin). While JAM-A+/+ tumor cells present a continuous junctional staining of E-cadherin, β-catenin, ZO-1 and cingulin at intercellular contacts, in JAM-A−/− cells the distribution of these markers was reduced or discontinuous (arrowhead). Scale bar; 15 µm; C) The effect of BV11 JAM-A blocking Ab on Ca 2+ switch assay. 4T1 cells were incubated with EGTA for 60 min to disrupt E-cadherin junctional localization and Ca 2+ was then added back to the cells to restore E-cadherin staining. In the presence of a JAM-A blocking Ab BV11 E-cadherin staining was partially inhibited and still largely incomplete up to 120 min. Scale bar; 20 µm; D) 4T1cell migration was significantly increased by a JAM-A blocking Ab (BV11). Upper panels report phase contrast microscopy of a typical experiment. Scale bar; 500 µm. Lower panels show a quantification of the distance of migration of the front. Values are means ± SEM of 4 replicates from a typical experiment out of 3 performed. **p<0.01 by unpaired Student-t test.

Article Snippet: Large-scale TMA of 444 patient breast cancer case–control cohort studies were performed using human JAM-A antibody (R&D, Minneapolis, MN) as described .

Techniques: Microscopy, Cell Culture, Derivative Assay, Immunofluorescence, Staining, Blocking Assay, Incubation, Migration

Journal: PLoS ONE

Article Title: Abrogation of Junctional Adhesion Molecule-A Expression Induces Cell Apoptosis and Reduces Breast Cancer Progression

doi: 10.1371/journal.pone.0021242

Figure Lengend Snippet: A) MMTV-PyVmT/JAM-A+/+ tumor sections show decreased JAM-A and E-cadherin expression level at the invasive edge as compared to tumor nodules and mammary duct. Scale bar; 100 µm; B) In tissue sections, TUNEL positive tumor cells were concentrated at the invasive edge of the tumor. Scale bar; 100 µm; C) In the wound assay (see ) cultured MMTV-PyVmT/JAM-A−/− cells show strong TUNEL staining both at the migrating front and in the distal areas while JAM-A+/+ cells presented TUNEL staining only at the leading edge. Quantification is reported at the right panel, data are means +/− SEM of 3 experiments performed in quadruplicates. **p<0.01 by unpaired Student-t test. Scale bar; 300 µm; D) Western blot analysis of JAM-A and ZO-1 expression in 4T1 cells in sparse, sub-confluent, and confluent conditions. Quantification is reported in the right panels. The results are representative of 3 independent experiments.

Article Snippet: Large-scale TMA of 444 patient breast cancer case–control cohort studies were performed using human JAM-A antibody (R&D, Minneapolis, MN) as described .

Techniques: Expressing, TUNEL Assay, Cell Culture, Staining, Western Blot

Journal: PLoS ONE

Article Title: Abrogation of Junctional Adhesion Molecule-A Expression Induces Cell Apoptosis and Reduces Breast Cancer Progression

doi: 10.1371/journal.pone.0021242

Figure Lengend Snippet: A) A total 444 human breast cancer were examined. Scores were evaluated on the level of JAM-A expression at cell membrane. Tumors were divided in JAM-A negative when no JAM-A was detected and JAM-A positive when JAM-A staining, either complete or incomplete, was present on the membrane of more than 10% of tumor cells. Scale bar; 200 µm.

Article Snippet: Large-scale TMA of 444 patient breast cancer case–control cohort studies were performed using human JAM-A antibody (R&D, Minneapolis, MN) as described .

Techniques: Expressing, Staining