IPC-366 cells in pellet resin sections.

<p>A and B) Semithin sections stained with toluidin blue (40X); some endothelial-like cells are observed (arrows). C to H) Ultraestructure in ultrathin sections. C) Cells with large nuclei and evident nucleoli. Mitosis (arrow). D) Single cell with elongated nucleus, very evident nucleolus and numerous organelles, vacuoles and proteinaceous secretion. E) Tumor degenerated cell with shrunken nucleus and abundant proteinaceous secretion. F) Cytoplasm of a tumor cell containing numerous organelles (mitochondria, endoplasmic reticulum) and a secretory vacuole. G) Tumor cell with three cytoplasmic empty spaces (asterisks) covered by cytoplasmic membrane; progression to a cytoplasmic “lumen” characteristic of endothelial-like cells. Evident nucleolus. H) Binucleate tumor cell with a cytoplasmic central empty space (“lumen”, asterisk) (ELC) resembling a capillary vessel (vasculogenic mimicry). Figs. E and G show different fields from the same grid. At 25^th passage, cells were cryogenic storage (-180°C). Re-cultured thawed cells presented similar growth and morphological characteristics with a viability of 95–99%. The doubling time of IPC-366 cells at 30^th passage was approximately 24 hours and the cells reached a plateau on day 6 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122277#pone.0122277.g004" target="_blank">Fig. 4</a>).</p

Morphology of IPC-366 cells.

<p>A) IPC-366 cells in culture at inverted microscopy (40X). B) IPC-366 pellet; paraffin section, H&E (40X). Highly malignant tumor cells with epithelial morphology; marked anisocytosis and anisokaryosis and evident nucleoli. Endothelial-like cell (ELC) showing cytoplasmic clear space (arrow). IPC-366 ultrastructural features were evaluated by electron microscopy. The cells had abundant cytoplasmic projections, numerous organelles and proteinaceous secretory products. By electron microscopy, the clear cytoplasmic vacuoles seen by optic microscopy, resulted empty spaces lined by cytoplasmic membranes that occasionally joined to form an internal lumen (ELCs, vasculogenic mimicry). (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122277#pone.0122277.g003" target="_blank">Fig. 3C-H</a>).</p

Tumors from mice inoculated with IPC-366.

<p>A) IPC-366 mouse xenografts at four weeks (arrow). B to E: tumor paraffin sections H&E. B) Solid tumor infiltrating the dermis (10X). C) Neoplastic embolus in a superficial dermal lymphatic vessel (arrow) and solid tumor; marked edema in dermis (4X). D) Highly malignant tumor cells with marked anisocytosis and anisokaryosis; evident nucleoli (40X).E) Microvascular channels lined up by neoplastic cells (vasculogenic mimicry, arrows) (20X).</p

Growth curve of IPC-366 at 30th passage.

<p>Growth curve of IPC-366 at 30th passage.</p

Primary canine inflammatory mammary carcinoma origin of the cell line IPC-366.

<p>Tumor paraffin sections, H&E. A (10X) y B (20X). Neoplastic emboli in superficial dermis. Tumor cells exhibit marked anisocitosis and anisokaryosis, and evident large nucleoli. Infiltrating tumor cells (arrow).</p

Technical data of specific antibodies and peroxidase-developing systems for immunohistochemistry.

<p>* Mab = monoclonal antibody, Pab = polyclonal antibody.</p><p>** MAD = Master Diagnostica.</p><p>Technical data of specific antibodies and peroxidase-developing systems for immunohistochemistry.</p

High Serum miR-19a Levels Are Associated with Inflammatory Breast Cancer and Are Predictive of Favorable Clinical Outcome in Patients with Metastatic HER2⁺ Inflammatory Breast Cancer

<div><p>Introduction</p><p>Altered serum microRNA (miRNA) levels may be correlated with a dysregulated expression pattern in parental tumor tissue and reflect the clinical evolution of disease. The overexpression of miR-21, miR-10b, and miR-19a is associated with the acquisition of malignant characteristics (increased tumor cell proliferation, migration, invasion, dissemination, and metastasis); thus, we determined their utility as serum biomarkers for aggressive breast cancer (HER2-overexpressed or -amplified [HER2⁺] and inflammatory breast cancer [IBC]).</p><p>Experimental Design</p><p>In this prospective study, we measured miR-21, miR-10b, and miR-19a levels using quantitative reverse transcriptase-polymerase chain reaction in the serum of 113 breast cancer patients and determined their association with clinicopathologic factors and clinical outcome. Thirty healthy donors with no history of cancer were enrolled as controls.</p><p>Results</p><p>Patients with non-metastatic HER2⁺ breast cancer had higher serum miR-21 median levels than patients with non-metastatic HER2⁻ disease (p = 0.044); whereas patients with metastatic HER2⁺ breast cancer had higher serum miR-10b median levels than patients with metastatic HER2⁻ disease (p = 0.0004). There were no significant differences in serum miR-19a median levels between HER2⁺ and HER2⁻ groups, regardless of the presence of metastases. High serum miR-19a levels were associated with IBC (p = 0.039). Patients with metastatic IBC had significantly higher serum miR-19a median levels than patients with metastatic non-IBC (p = 0.019). Finally, high serum miR-19a levels were associated with longer progression-free survival time (10.3 vs. 3.2 months; p = 0.022) and longer overall survival time (median not reached vs. 11.2 months; p = 0.003) in patients with metastatic HER2⁺ IBC.</p><p>Conclusion</p><p>High levels of miR-21 and miR-10b were present in the serum of patients with non-metastatic and metastatic HER2⁺ breast cancer, respectively. High levels of serum miR-19a may represent a biomarker for IBC that is predictive for favorable clinical outcome in patients with metastatic HER2⁺ IBC.</p></div

The Antihelmintic Drug Pyrvinium Pamoate Targets Aggressive Breast Cancer

<div><p>WNT signaling plays a key role in the self-renewal of tumor initiation cells (TICs). In this study, we used pyrvinium pamoate (PP), an FDA-approved antihelmintic drug that inhibits WNT signaling, to test whether pharmacologic inhibition of WNT signaling can specifically target TICs of aggressive breast cancer cells. SUM-149, an inflammatory breast cancer cell line, and SUM-159, a metaplastic basal-type breast cancer cell line, were used in these studies. We found that PP inhibited primary and secondary mammosphere formation of cancer cells at nanomolar concentrations, at least 10 times less than the dose needed to have a toxic effect on cancer cells. A comparable mammosphere formation IC50 dose to that observed in cancer cell lines was obtained using malignant pleural effusion samples from patients with IBC. A decrease in activity of the TIC surrogate aldehyde dehydrogenase was observed in PP-treated cells, and inhibition of WNT signaling by PP was associated with down-regulation of a panel of markers associated with epithelial-mesenchymal transition. <i>In vivo</i>, intratumoral injection was associated with tumor necrosis, and intraperitoneal injection into mice with tumor xenografts caused significant tumor growth delay and a trend toward decreased lung metastasis. In <i>in vitro</i> mammosphere-based and monolayer-based clonogenic assays, we found that PP radiosensitized cells in monolayer culture but not mammosphere culture. These findings suggest WNT signaling inhibition may be a feasible strategy for targeting aggressive breast cancer. Investigation and modification of the bioavailability and toxicity profile of systemic PP are warranted.</p></div

Inhibitory effect of PP on mammosphere formation.

<p>(A, B) Primary mammosphere formation. SUM-149 (A) and SUM-159 (B) cells were incubated with indicated doses of PP or DMSO in mammosphere formation media for 7 days. PP treatment reduced the number of primary mammospheres in a dose-dependent manner. (C, D) Secondary mammosphere formation. SUM-149 (C) and SUM-159 (D) cells were grown in mammosphere formation media and treated with indicated doses of PP for 4 days. Then primary spheres were trypsinized and seeded as secondary mammospheres. [Add sentence summarizing effect of PP.] The results shown are representative of three independent experiments.</p

Mean threshold cycle values of miR-192± standard deviation and 95% confidence interval in the serum of breast cancer patients and healthy donors.

<p>Unpaired <i>t</i>-test (Mann-Whitney U test).</p><p>SD: standard deviation; CI: confidence interval; Ct: threshold cycle.</p