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

Abstract

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.

Fig 1. Primary canine inflammatory mammary carcinoma origin of the cell line IPC-366.

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).

Fig 2. Morphology of IPC-366 cells.

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). (Fig. 3C-H).

Fig 3. IPC-366 cells in pellet resin sections.

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 (Fig. 4).

Fig 4. Growth curve of IPC-366 at 30th passage.

Fig 5. Tumors from mice inoculated with IPC-366.

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).

Fig 6. Immunohistochemistry of IPC-366 pellet, paraffin sections.

A) Pancytokeratin (AE1/AE3) (10X); intense immunolabeling and numerous positive cells. B) CK14; intense immunolabeling and numerous positive cells (10X). C) Vimentin; moderate immunolabeling and numerous positive cells (10X). D) p63; negative (20X). E) α-smooth muscle actin, negative cells (10X). F) Estrogen receptor; negative cells (20X). G) Progesterone receptor; negative cells (20X). H) HER-2; negative cells (20X). I) E-Cadherin; intense membranous immunolabeling in numerous cells (20X). J) COX-2; most of the cells are moderately positive; some cells are intensely positive (10X). K) COX-2; some cells strongly positive showing features of malignancy (binucleated cell, arrow) (40X). L) Ki-67 proliferation marker; many nuclei are positive (10X).