CMT-1026 was established from a canine solid carcinoma procured after surgical removal of a 5-year-old teddy breed female dog. The neoplasm was clinically and histopathologically identified with a canine mammary tumor (CMT). The mammary glands showed firmness, thickening, warmth and pain. The original CMT was identified as a large solid carcinoma with three small granulitic tumors on the dermal surface. After surgical extraction, the tumor samples were rapidly soaked in phosphate-buffered saline (PBS) with penicillin-streptomycin solution (Gibco, USA). Cancer samples were processed for cell culture and histopathological confirmation.

Neoplasm tissues were ground and washed three times with PBS (Gibco, USA). Cancer samples were disaggregated with collagenase type II (Sigma-Aldrich, V900892) on a rocker under 5% carbon dioxide for 4 h. The disaggregated tissue suspension was placed in a 50 mL centrifuge tube and centrifuged at 1,000 rpm for 5 min, and the collected cells at the bottom of the tube were resuspended in Dulbecco's Modified Eagle Medium Nutrient Mixture Ham's F-12 (DMEM/F12) with 10% fetal bovine serum (FBS) (Gibco, USA) and 1% penicillin-streptomycin solution. The cells were added into 25 cm² culture flasks (Corning, USA) and cultivated at 37°C in a humidified atmosphere containing 5% CO₂. Cells were observed daily by microscopy.

When the cell cultures reached 90% confluence, the cells were incubated with 0.25% trypsin (HyClone, USA) containing EDTA. Secondary cultures were obtained from digested cells, and the cells were placed in new 25 cm² flasks at a density of ~1 × 10⁵ cells/mL. The rest of the digested cells were mixed in DMEM/F12 with 20% FBS and 10% dimethyl sulfoxide (Sigma-Aldrich, USA) and stored at 4°C for 30 min, −20°C for 2 h and −80°C overnight. The cells were sampled and frozen every five passages. The next day, cells were placed in liquid nitrogen. CMT-1026 was considered to be established at passage 30.

CMT-1026 was diluted to different concentrations to provide a normal curve diagram (34). Cells were seeded in triplicate in 96 well plates. After 24 h of cultivation, CMT-1026 cells were treated in triplicate with a Cell Counting Kit for 4 h (CCK-8, Beyotime, Shanghai, China) and then detected at 24 h intervals for 9 days. Then the different concentrations were determined with a 450 nm filter and a microplate reader (ELx808 Absorbance Reader, BioTek, USA). When cell growth was in the exponential phase, the CMT-1026 doubling time was calculated with GraphPad Prism 8 software (GraphPad Software, Inc. USA).

CMT-1026 cells were collected for 24 h and resuspended with 2.5% glutaraldehyde (Panreac, Germany) and 4% paraformaldehyde (Panreac, Germany) solution at 4°C for 16 h. Then cells were added into 1% OsO4 for 1 h, gently washed twice with PBS, treated with graded acetone solutions (30, 50, 70, 80, and 100%) and encapsulated in epoxy resin. Ultra-thin sections were prepared with a Reichert-Jung Ultracut E ultramicrotome (LEICAUC6i, Germany). Lead citrate and uranyl acetate were used to stain the cells, and a JEOL JEM 3000F transmission electron microscope (JEOL Ltd., Tokyo, Japan) was used to obtain photographs.

For karyotype analysis, colchicine (Gibco-Life Technology, USA) was added at 0.06 μg/mL for 6 h before the cell suspension was harvested. Then attached cells were dissociated, and cell suspensions were added into 0.075 M hypotonic KCl for 30 min. The cells were fixed with 1:3 acetic acid:methanol at 4°C overnight. Finally, the prepared cells were stained with Giemsa stain (Beijing Solarbio Science & Technology, China) for 10 min and examined under a microscope (Leica Microsystems GmbH, Germany).

An indirect immunofluorescence experiment was used to monitor the expression of Ki-67 (Thermo Fisher Scientific, 14-5698-82), SOX-2 (Thermo Fisher Scientific, MA1014), Delta-catenin (Thermo Fisher Scientific, MA5-16386) and Claudin-1 (Thermo Fisher Scientific, 374900) in the CMT-1026 cell line. Ki-67 and SOX-2 were diluted to 5 μg/mL; Delta catenin and Claudin-1 were diluted to 1 μg/mL. CMT-1026 cells were seeded in 24 well plates. When cell cultures reached 90% confluence, cells were treated with acetone:methanol (1:1) for 20 min. Primary antibodies were used to stain cells at 4°C for 16 h. Then CMT-1026 cells were washed with PBS and cultured with secondary antibody for 1 h. The secondary antibody was Alexa Fluor 488 goat anti-rabbit IgG (Abcam, ab150077) or FITC goat anti-mouse IgG (Abcam, ab6785), which was diluted to 1 μg/mL. Finally, cells were washed with PBS and incubated with DAPI (BD Transduction Laboratories, USA). A fluorescence microscope (CKX41, Olympus Corporation, Japan) was used to examine the cells.

For IHC assessment, CMT-1026 originating primary cancer and xenograft tumors were prepared in 3 μm samples. Cancer pellets were assessed for the following markers: cytokeratin 5/6 (CK5/6, Invitrogen, 53-9003-82), estrogen receptor (ER, Invitrogen, MA1-12692), progesterone receptor (PR, Invitrogen, PA5-82322), human epidermal receptor-2 (HER-2, Invitrogen, MA5-13105), α-smooth muscle actin (α-SMA, Invitrogen, 14-9760-82), and P63 (Invitrogen, 14-9760-82). All these makers were used at a concentration of 1 μg/mL. Deparaffinized tissues were placed in a PT module with EDTA buffer solution (pH 8.0) (Master Diagnostica, MAD-004072R/D) and subjected to heat-induced antigen retrieval at 95°C for 30 min, then cooled to 60°C. To quench endogenous peroxidase activity, the slides were covered with 3% hydrogen peroxide and incubated with 5% FBS for 1 h. The specimens were incubated with primary antibodies at 4°C for 16 h and with secondary antibody (ZSGB-BIO, Beijing, China) for 1 h at room temperature to allow for the identification of immunolabeled proteins. Subsequently, 3,3′-diaminobenzidine tetrahydrochloride (ZSGB-BIO, China) was used to visualize antibody binding. The samples were washed in distilled water three times, then counterstained with hematoxylin, dehydrated, cleared and mounted. The primary antibody was substituted with PBS for negative control samples. The primary CMT was considered positive for CK5/6, ER, PR, HER-2, SMA, and p63 when more than 10% of the tumor cells were positive (35). ImageJ (National Cancer Institute, USA) was used to quantify the protein in the IHC images.

To evaluate the metastatic ability of CMT-1026 cells, Corning Transwell chambers (Corning Incorporated, USA) were coated with Matrigel (Becton, USA). Cells were digested before being seeded onto the filters at 1 × 10⁵ cells/well in 200 μL DMEM/F12 medium, and 600 μL DMEM/F12 medium containing 10% FBS was mixed in the lower chambers for 48 h. The cells that invaded to the bottom of the chamber were detected after Crystal Violet staining for 5 min and washing with PBS. Five randomly fields per well were chosen to count cells. The experiment was performed in triplicate.

To study the migration of CMT-1026 cells, wound-healing assays were used. In this assay, a scratch is made on a confluent cells with a pipette tip (36, 37). Subsequently, migration was measured in ImageJ software, and the wound healing percentage was calculated.

For murine xenotransplantation models, a cell suspension of 10⁷ in 0.2 mL PBS was inoculated into the left mammary fat pads of ten 5-week-old female Balb/SCID nude mice subcutaneously. The growth of tumors was monitored weekly. When goiters were observed, the width and length of the tumors were regularly monitored with calipers for 8 weeks. When mice exhibited dyspnea and weakness or there were no palpable tumors, they were sacrificed. Neoplasms and organs were collected in 4% paraformaldehyde (pH = 7.4) for histopathological detection. All procedures were conducted according to the Guide for the Care and Use of Laboratory Animals and conformed to the relevant EU Directive.

Mycoplasma DNA was detected with a EZ-PCR Mycoplasma Detection Kit (Biological Industries, Israel) according to the manufacturer's instructions. The PCR products were then separated with 0.8% agarose gel electrophoresis and visualized under UV light (Gel DocTM XR, BIO-RAD, America).

The invaded cells were calculated as the mean ± standard deviation (mean ± SD). The results were analyzed in SPSS 20 software. When the p value was < 0.05, it was regarded as indicating a significant difference among groups.

A canine mammary neoplasm was obtained from the China Agricultural University Veterinary Teaching Hospital. Histological observation of H.E.-stained paraffin sections from the tumor revealed substantial cell hyperplasia and elliptical, and round and polygonal cell shapes. Malignant multinucleated cells frequently had clearly enlarged nuclei. Anisocytosis, anisokaryosis, and elongated eccentric nuclei were observed (Figure 1). The CMT-1026 cells have been continuously sub-cultured for 54 generations.

ER, PR, HER-2, P63, CK5/6, and α-SMA expression was detected through IHC in the primary CMT. The paraffin samples were negative for HER-2, ER and PR, but positive for CK5/6, P63 on the cell membrane and α-SMA in cytolymph (Figure 2). Image J was used to quantify the protein in the IHC images. P63 showed a positive expression rate of 12.89%, CK5/6 showed a positive expression rate of 29.45%, and α-SMA showed a positive expression rate of 23.17%. The neoplasm exhibited characteristics of a triple-negative canine mammary tumor.

CMT-1026 was purified and grown for more than 51 passages. The CMT-1026 cells were observed under light microscopy exhibited an epithelioid and spindle shape (Figure 3A). Diff-Quik staining showed malignant cancer cells with an epithelial morphology, marked anisokaryosis, and multinucleated giant cells (Figure 3B). Furthermore, ultrastructural studies revealed that CMT-1026 cells had large nuclei and apparent nucleoli. The cytoplasm contained multiple organelles, mitochondria, secretory vacuoles, and endoplasmic reticulum. Some vacuole structures were observed near the cell surface (Figure 3C). Degenerated tumor cells showed shrunken nuclei and abundant proteinaceous secretion (Figure 3D). The CMT-1026 doubling time was found to be 23.95 h (Figure 4).

Karyotype analysis was used to determine CMT-1026 cell chromosome numbers. Chromosome numbers were calculated on 80 near-diploid metaphase spreads. Most of the cells had 78 chromosomes, the chromosome number in canines (Figure 5).

The CMT-1026 cells showed strong invasion ability through the basement membrane (63 ± 13) cells, whereas CMT-U27 cells showed an invasion ability of 172 ± 9 cells (Figures 6A,B). As predicted, 48 h after scratching, new growth CMT-1026 cells and CMT-U27 cells covered the scratched area. A wound healing percentage of 80.4% ± 1.22 in CMT-1026 was observed 48 h after the scratch. Notably, in comparison, the wound healing percentage for CMT-U27 was ~78.2% ± 1.43 after the same period of time (Figure 7).

The expression levels of Ki-67, SOX-2, Delta-catenin, and Claudin-1 on CMT-1026 cells were observed by immunofluorescence. All samples were positive for these four antigens in the cytoplasm (Figure 8). The results indicated that the CMT-1026 cell line had a strong ability to metastasize.

CMT-1026 cells were injected into the abdomens of female BALB/c nude mice, and neoplasms grew 2 weeks after inoculation in 100% of the mice (n = 10, length: 0.5–0.8 cm, width: 0.2–0.3 cm). After 8 weeks of monitoring, the xenograft cancers were necrotic and ulcerated (length 0.8–1.3 cm; width 1.1–1.2 cm), and the mice were then euthanized (Figure 9A). Histological examination revealed infiltrating cellular growth in the subcutaneous dermis (Figure 9B). The lung tissue was invaded by glandular epithelial cells (Figure 9C). No clear tumor cell invasion was found in liver tissue, but the overall heteromorphism of liver cells was clear, the nucleoli had increased, and clear nucleoli and necrosis were observed (Figure 9D).

ER, PR, HER-2, P63, CK5/6, and α-SMA expression was detected through IHC in the xenograft tumors. The results were consistent with the original tumor results. The paraffin samples were negative for HER-2, ER, and PR, but positive for CK5/6 and P63 on the cell membrane and for α-SMA in the cytolymph (Figure 10). Image J was used to quantify the protein in the xenograft tumors IHC images. P63 showed a positive expression rate of 38.94%, CK5/6 showed a positive expression rate of 50.01%, and α-SMA showed a positive expression rate of 52.35%. The xenograft tumors exhibited characteristics of triple-negative canine mammary tumors.

CMT-1026 cells were tested with a PCR-based method and found to be free of mycoplasma contamination (Figure 11).