Fifth European Dirofilaria and Angiostrongylus Days (FiEDAD) 2016

Peer reviewe

Steroid Tumor Environment in Male and Female Mice Model of Canine and Human Inflammatory Breast Cancer

Canine inflammatory mammary cancer (IMC) shares clinical and histopathological characteristics with human inflammatory breast cancer (IBC) and has been proposed as a good model for studying the human disease. The aim of this study was to evaluate the capacity of female and male mice to reproduce IMC and IBC tumors and identify the hormonal tumor environment. To perform the study sixty 6–8-week-old male and female mice were inoculated subcutaneously with a suspension of 106 IPC-366 and SUM149 cells. Tumors and serum were collected and used for hormonal analysis. Results revealed that IPC-366 reproduced tumors in 90% of males inoculated after 2 weeks compared with 100% of females that reproduced tumor at the same time. SUM149 reproduced tumors in 40% of males instead of 80% of females that reproduced tumors after 4 weeks. Both cell lines produce distant metastasis in lungs being higher than the metastatic rates in females. EIA analysis revealed that male tumors had higher T and SO4E1 concentrations compared to female tumors. Serum steroid levels were lower than those found in tumors. In conclusion, IBC and IMC male mouse model is useful as a tool for IBC research and those circulating estrogens and intratumoral hormonal levels are crucial in the development and progression of tumors

Establishment and characterization of a new cell line of canine inflammatory mammary cancer: IPC-366.

Canine inflammatory mammary cancer (IMC) shares epidemiologic, histopathological and clinical characteristics with the disease in humans and has been proposed as a natural model for human inflammatory breast cancer (IBC). The aim of this study was to characterize a new cell line from IMC (IPC-366) for the comparative study of both IMC and IBC. Tumors cells from a female dog with clinical IMC were collected. The cells were grown under adherent conditions. The growth, cytological, ultrastructural and immunohistochemical (IHC) characteristics of IPC-366 were evaluated. Ten female Balb/SCID mice were inoculated with IPC-366 cells to assess their tumorigenicity and metastatic potential. Chromosome aberration test and Karyotype revealed the presence of structural aberration, numerical and neutral rearrangements, demonstrating a chromosomal instability. Microscopic examination of tumor revealed an epithelial morphology with marked anysocytosis. Cytological and histological examination of smears and ultrathin sections by electron microscopy revealed that IPC-366 is formed by highly malignant large round or polygonal cells characterized by marked atypia and prominent nucleoli and frequent multinucleated cells. Some cells had cytoplasmic empty spaces covered by cytoplasmic membrane resembling capillary endothelial cells, a phenomenon that has been related to s vasculogenic mimicry. IHC characterization of IPC-366 was basal-like: epithelial cells (AE1/AE3+, CK14+, vimentin+, actin-, p63-, ER-, PR-, HER-2, E-cadherin, overexpressed COX-2 and high Ki-67 proliferation index (87.15 %). At 2 weeks after inoculating the IPC-366 cells, a tumor mass was found in 100 % of mice. At 4 weeks metastases in lung and lymph nodes were found. Xenograph tumors maintained the original IHC characteristics of the female dog tumor. In summary, the cell line IPC-366 is a fast growing malignant triple negative cell line model of inflammatory mammary carcinoma that can be used for the comparative study of both IMC and IBC

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

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

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