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Establishment and characterization of a new triple-negative canine mammary cancer cell line
Tissue and Cell 54 (2018) 10–19
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Establishment and characterization of a new triple-negative canine mammary cancer cell line
T ⁎
Hong Zhanga,1, Shimin Peia,1, Bin Zhoub, Huanan Wangc, Hongchao Dud, Di Zhangd,2, , ⁎ Degui Lind,2, a
Institute of Tropical Agriculture and Forestry, Hainan University, Haikou, Hainan, 570228, China The College of Animal Science and Technology, Zhejiang Agriculture and Forestry University, Hangzhou, Zhejiang, 311300, China c Department of Veterinary Medicine, College of Animal Sciences, Zhejiang University, Hangzhou, Zhejiang, 310058, China d The Clinical Department, College of Veterinary Medicine, China Agricultural University, Beijing, 100193, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Canine mammary cancer Characterization Establishment Epithelial-mesenchymal transition Triple-negative cell line
Canine mammary tumor (CMT) has always been an ideal animal model for human breast cancer (HBC) research, however, there is a lack of various established CMT cell lines corresponding to HBC cell lines. This study was designed to establish a new type of CMT cell line. The primary tumor, CMT-7364, was identified as the intraductal papillary carcinoma, and showed negative immunoreactivity to estrogen receptors (ER), progesterone receptors (PR), and human epidermal growth factor receptor-2 (HER-2) by immunohistochemistry (IHC) analysis. The CMT-7364 cell line from this primary tumor also shows a negative immunoreactivity to ER, PR, and HER-2, and was negative to epithelial cell markers and positive to mesenchymal cell markers by immunocytochemistry (ICC) analysis. This cell line, which has been stably cultured for more than 115 passages, and was characterized by epithelial origin with the expression of the epithelial antigen by ICC analysis and invasion ability by transwell analysis. In vivo, tumor mass and metastases in the lung were found after inoculating the CMT-7364 cells in the nude mice model, and the immune-complete mice model respectively. The tissues from the xenograft tumor were also negative to ER, PR, and HER-2 by IHC analysis. Thus, a novel triple negative canine mammary cancer cell line, CMT-7364, was successfully established, which could be used as a promising model for the research of immunotherapy and Epithelial-Mesenchymal Transition (EMT) mechanism of the triple-negative breast cancer both in canine and human.
1. Introduction The triple-negative breast cancer (TNBC), defined as the absence of hormone receptors (estrogen and progesterone) and negativity for human epidermal growth factor receptor-2 (HER-2), represents approximately 15–20% of breast cancers in human (Giuseppe Palma et al., 2015). Similarly, in a previous study of 241 canine mammary tumors (CMTs), 18.7% CMTs are the triple-negative phenotype which is associated with poor prognosis (Kim et al., 2013). Moreover, spontaneous CMT shares similar epidemiological and biological features with those in human, and these characteristics have been proven to be useful in understanding complex molecular aspects of human tumors (Pinho et al., 2012). Further study of triple-negative CMTs may help to thoroughly comprehend the characteristics of TNBC, and design more
effective therapeutic methods in the future. Compared with other breast cancer subtypes, TNBC is characterized by an aggressive and early pattern of metastases, a relative lack of therapeutic targets, and a poor prognosis (Bosch et al., 2010). It remains a huge challenge to develop the effective treatment of TNBC. While immunotherapies maybe a promising choice for the TNBC treatment, including immune-checkpoint inhibitors, antagonists of immunosuppressive molecules and tumor vaccine. In the laboratory, mouse xenograft model is a useful animal model to explore the immune treatment of TNBC. However, most of the breast cell lines can only be tumorigenic on the immunodeficiency mice which live in abnormally hygienic specific pathogen free (SPF) immunodeficiency barrier facilities. Those mice do not produce the similar immune response with the human to certain diseases until they are raised in the normal
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Correspondence authors. E-mail addresses: [email protected] (D. Zhang), [email protected] (D. Lin). 1 Equal first author (Hong Zhang and Shimin Pei). 2 Equal Corresponding author. https://doi.org/10.1016/j.tice.2018.07.003 Received 17 October 2017; Received in revised form 27 June 2018; Accepted 20 July 2018 Available online 21 July 2018 0040-8166/ © 2018 Elsevier Ltd. All rights reserved.
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Beyotime, Shanghai, China). The growth curve was plotted and the doubling time was calculated from regression equation of the curve with GraphPad Prism 5 software (GraphPad Software, Inc. USA).
environment (Beura et al., 2016). Therefore, it is desired to establish a new TNBC cell line that is tumorigenic on immune-complete mice which can live in the normal environment. In summary, the purpose of our study was to establish a triple-negative CMT cell line which can transplant into immune-complete mice and to determine its characteristics, including morphology, growth potential, protein expression, and metastatic potential.
2.4. Karyotyping For karyotype analysis, cells of passage 60 at the growth phase were treated with 0.05 μg/mL colchicine for 6 h. Then adherent cells were dissociated by 0.25% trypsin and resuspended in 0.075 M hypotonic KCL (preheated to 37 °C) for 30 min at 37 °C. The cell suspension was then fixed in methanol: acetic acid (3:1) and added dropwise to the clean and cold slides. Finally, prepared slides were stained with Giemsa (Beijing Solarbio Science & Technology, China)for 15 min, then chromosome numbers of the cells were counted for 100 metaphase cells. The chromosomes were observed and counted under a Leica microscope (Leica Microsystems GmbH, Wetzlar, Germany) (Tap et al., 1998).
2. Materials and methods 2.1. Establishment and purification of primary cultures Tumor tissues were sterilely surgically excised from the dogs suffered from mammary gland tumors in the China Agricultural University Veterinary Teaching Hospital (CAU-VTH), performed according to CAU-VTH standard operating procedures and with informed owner consent. A part of tissues was used for paraffin embedding to confirm the diagnosis by histology according to Goldschmidt (Goldschmidt et al., 2011). There were 50 primary cultures from 50 distinct female dogs were tried to establish. If there were more than one tumor with one dog, the highest grade malignant one would be further analyzed. Then tumor tissues were washed with Phosphate Buffered Saline (PBS) supplemented with antibiotics (penicillin 100 IU/mL and streptomycin 100 IU/mL), and minced into 1 mm pieces removing blood, fat, and fibro connective tissues. Small pieces were incubated with Dulbecco's Modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS, Gibco, USA) and 0.1% collagenase Ⅱ (Gibco, USA) overnight in a 60-mm (in diameter) tissue culture dish (Costar, Corning Incorporated, USA) at 37 °C in a humidified atmosphere of 5% CO2. The supernatant of digested tissue pieces was transferred into an equal volume of DMEM medium with 10% FBS and antibiotics following completed mixture. The cells were centrifuged at 1000 RPM for 5 min, and resuspended in fresh medium. Then cells were seeded into 60-mm tissue culture dish, containing 3 mL of DMEM with 10% FBS, and maintained at 37 °C in a humidified atmosphere of 5% CO2. After the formation of a complete monolayer in primary culture, 105 cells were transferred into a new flask with fresh DMEM-10% FBS medium. Once the cells cultures were 80–90% confluent, cells were washed three times with PBS, treated with 0.25% trypsin solution (Gibco, USA), and incubated until cells were dislodged from the flask surface, and split in a ratio of 1: 3 in fresh DMEM-10% FBS medium. Also, the cells were sampled and frozen every five passages.
2.5. Invasion assay To assess the invasive capacity of CMT-7364 cells, we utilized Corning transwell chambers (Costar, Corning Incorporated, USA) coated with Matrigel (Becton, Dickinson and Company, USA). Breast cancer cell line MDA-MB-231 cells were used as positive control, the CMT-7364 cells were at passage 65. The experiment was performed in triplicates. Cells were digested with 0.25% trypsin and washed with PBS, then were seeded onto the filters at a concentration of 1 × 104 cells/well in 100 μL DMEM medium without FBS. The lower chambers were filled with 600 μL of medium containing 10% FBS. After 42 h cultured, cells on the topside of the filter were gently removed by scrubbing with a tipped swab. The cells invaded to the lower side of the filter were determined by crystal violet staining for 15 min and were counted in at least 5 randomly chosen fields per well under a microscope. 2.6. Soft agar assay The ability of anchorage-independent growth of the CMT-7364 cell line was determined by growing the cells on semisolid agar at the 65th passage (Sevostianova et al., 2013). A single-cell suspension containing 1000 cells was dispersed in a DMEM medium solution containing 0.6% agar seeded in 35-mm tissue culture dish which was layered a 1.2% agar solution in DMEM supplemented with 20% FBS. The cells were seeded in triplicate. Formation of colonies was determined by inverted microscopy (CKX41, Olympus Corporation, Japan) after 2 weeks incubation.
2.2. Ultrastructure analysis of cells For ultrastructure studies, cells were harvested at passage 61 by centrifugation at 1000 RPM for 5 min, followed by subsequently fixed with 2.5% glutaraldehyde in 0.01 M PBS at 4 °C. Those cells were then post-fixed in 1% OsO4, washed in PBS, dehydrated in graded acetone solutions and embedded in epoxy resin. Silver-to-gold sections were cut with a diamond knife using a Reichert-Jung Ultracut E ultramicrotome (LEICAUC6i, Germany). The ultra-thin sections were cut (400–500 μm), stained with lead citrate and uranyl acetate and examined under a JEOL JEM-1230 EXII transmission electron microscope (JEOL Ltd., Tokyo, Japan) studied under transmission microscope.
2.7. Immunohistochemistry (IHC) For immunohistochemical studies, 3 μm sections were cut from the primary tumor from which CMT-7364 was originated and xenografted tumor. Then they were cut from each specimen and mounted on poly-LLysine-coated slides. Deparaffinized tissues were subjected to heat-induced antigen retrieval at 98 °C for 12 min. After the jars were cooled to room temperature (RT), the slides were covered with 3% hydrogen peroxide to quench endogenous peroxidase activity and then incubation with 5% goat serum at RT for 30 min. Next, the sections were incubated overnight with primary antibodies specific for estrogen receptors (ER), progesterone receptors (PR), HER-2, Cytokeratin-8/18 (CK8/18), Vimentin, E-Cadherin, Transforming growth factor-β1 (TGF-β1) and Ki67 (see Table 1) at 4 °C. The sections were then incubated with secondary antibody (ZSGB-BIO, Beijing, China) for 20 min at 37 °C to detect immunolabelled proteins. Antibody binding was visualized with 3, 3′-diaminobenzidine tetrahydrochloride (DAB kit, ZSGB-BIO, Beijing, China). After a final washing in distilled water, the sections were counterstained with hematoxylin, dehydrated, cleared and mounted.
2.3. Growth studies and doubling time The doubling time of CMT-7364 cell line was determined at passage 54. Cells were diluted to a series of concentration that allowed to easily prepare the standard curve as previously described (Sylvester, 2011). The viable cells were seeded in triplicate in each of 96 well plates in DMEM medium with 10% FBS. Optical Density (OD) was determined with a microplate reader (ELx808 Absorbance Reader, BioTek, USA) using a 450 nm filter. It was determined in triplicate at exactly 24 h intervals for 8 days after 1 h treated with Cell Counting Kit (CCK-8, 11
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instruction. The resulting PCR products were separated by electrophoresis on 1.0% agarose gel, stained with Gelstain (Beijing TransGen Biotech, China), visualized under UV light and documented by photography (JY04S-3C, Beijing Junyi-dongfung Electrophoresis Equipment, China).
Table 1 Technical data of specific antibodies and peroxidase-developing systems for IHC and ICC. Primary Antibody Type
Clone
Manufacturer
Secondary antibody
ER PR HER-2
6F11 Zym5.2 EP3
Goat anti mouse Goat anti rabbit Goat anti rabbit
CK8/18
Zym5.2
Vimentin
V9
E-Cadherin Ki-67
4A2C7 MIB1
Ivitrogen Corporation,USA Ivitrogen Corporation,USA OriGene Technologies, Inc.,USA OriGene Technologies, Inc.,USA OriGene Technologies, Inc.,USA OriGene Technologies, Inc.,USA OriGene Technologies, Inc.,USA Abcam, USA
TGF-β1
Polyclonal
2.11. Statistical analysis The numerical results of invasion cells of different cell lines were expressed as the mean ± standard deviation (Mean ± SD). The results were analyzed by Student’s t-test using SPSS 20 software (Statistical Product and Service Solutions, Chicago, USA). There is the significant difference among different groups when p < 0.05.
Goat anti mouse Goat anti mouse Goat anti mouse Goat anti mouse
3. Result Goat anti rabbit
3.1. Establishment of CMT-7364 cell line from a malignant CMT We initiated 50 primary cultures obtained from female dogs with CMT admitted to the Veterinary Teaching Hospital of China Agricultural University. Out of these primary cultures, a cell line was successfully purified and propagated for more than 115 passages in our laboratory. The tumors were excised from a female dog aged 13 years, and the size of the biggest one was about 6.5 cm × 4 cm × 3.5 cm from which this cell line was originated (Fig. 1A). There was no metastasis in regional lymph node and distant organs. Therefore this mammary gland tumor was diagnosed at stage III according to a modified WHO clinical staging system (Lana et al., 2007). The epithelial cells of the tumor were multilayered with characteristics of malignancy by H＆E staining analysis. It showed moderate tubule formation, nuclear and cellular pleomorphism, and increased numbers of mitotic in the tumor (Fig. 1B and C). Then this mammary gland tumor was cataloged as intraductal papillary carcinoma and histological grade II. It also showed the positive reaction to Ki-67 in the nucleus (Fig. 1D).
For the negative controls, the primary antibody was replaced with PBS (pH 7.2). 2.8. Immunocytochemistry (ICC) An indirect immunofluorescence assay was performed in order to detect the expression of ER, PR, HER-2, CK8/18, Vimentin, E-Cadherin and TGF-β1 in the CMT-7364 cell line at passage 65. The cells growing on 24 well plate were fixed with methanol: acetone(1:1) for 30 min. The cells with primary antibodies were stained for overnight at 4 °C. The next day, cells were washed and stained with FITC–anti-rabbit or FITC–anti-mice IgG (Beijing ComWin Biotech, China) as a secondary antibody for 1 h at RT. The cells were washed and stained with DAPI (BD Transduction Laboratories, Lexington, USA). Then they were examined under a fluorescence microscope (CKX41, Olympus Corporation, Japan).
3.2. The primary tumor shows characters of triple-negative canine mammary cancer
2.9. Transplantation into mice All husbandry practices and experimental operations were performed in compliance with the ARRIVE guidelines and were carried out in accordance with EU Directive 2010/63/EU for animal experiments. All animal experiments were approved by the Institutional Animal Care and Use Committee of China Agricultural University (CAU20150101-2). The 5-week-old female BALB/SCID and BALB/c immune-competent mice (Beijing Vital River Laboratory Animal Technology, China) were used to investigate the tumorigenicity of the CMT-7364 cell line at passage 57. A suspension of 5 × 106 cells in 0.2 mL PBS was transplanted subcutaneously into the left mammary fat pad of mice (n = 6). Mice were observed biweekly. When tumors were detected, their length and width were weekly measured by calipers for 8 weeks. Mice were sacrificed by cervical dislocation when they showed clinical signs such as dyspnoea and weakness or one year after transplantation in cases which were no palpable tumor mass. Mice were euthanized by cervical dislocation and autopsied to detect metastatic lesions in the lungs or other organs. The tumors and organs were removed and fixed in 10% neutral-buffered formalin, and were stained with hematoxylin and eosin (H&E) for histopathologic examination. Tumor volume (V) was calculated according to the following formula (Murai et al., 2012):
We detected the expression of ER, PR, HER-2, CK8/18, Vimentin, ECadherin and TGF-β1 by IHC in the primary canine mammary gland cancer which was the origin of the CMT-7364 cell line. The paraffin sections were negative to ER, PR, and HER-2, but positive to epithelial cell markers (CK8/18 and E-Cadherin) with positive cells localized in the cytomembrane (Fig. 2). The samples had a positive reaction to Vimentin and TGF-β1 in the cytomembrane of epithelial cell (Fig. 2). 3.3. The CMT-7364 cell line shows epithelium-like cell morphology The cells were epithelioid and round- to spindle-shaped under light microscopy (Fig. 3A). The epithelial lineage of CMT-7364 was further confirmed by ultrastructure study of the cells by transmission electron microscopy. Ultrastructural studies revealed that CMT-7364 showed epithelium-like cell morphology with large irregular nuclear outlines, some vacuole structures, mitochondria, numerous free ribosomes, endoplasmic reticulum and intermediate filaments and numerous microvilli exhibited on the cell surface (Fig. 3B). 3.4. Growth studies and doubling time
V = a×b2×1/2(cm3) where a and b are the tumor length and width (in cm), respectively.
Growth studies and population doubling time of the CMT-7364 cell line were determined as Section 2.3. The doubling times of the cell line was calculated to be 21.12 h (Fig. 4).
2.10. Myoplasma detection 3.5. CMT-7364 exhibits abnormalities of chromosomes The mycoplasma DNA was detected by PCR Mycoplasma Test Kit (Hangzhou Hua’an biotechnology, China) as per the manufacturer’s
The CMT-7364 cells showed both numerical and structural 12
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Fig. 1. Primary canine mammary carcinoma. The biggest tumor which the CMT-7364 cell line was derived from was about 6.5 cm × 4.0 cm × 3.5 cm (A). Tumor cells in paraffin sections exhibited marked variation in nuclear size and hyperchromatic nucleus (B and C), often with one or more prominent nucleoli and mitoses (arrow). Tissues from primary tumor were positive to Ki-67 in the nucleus by Immunohistochemical staining.
3.7. CMT-7364 shows high ability of colony
abnormalities of chromosomes by karyotype analysis. Chromosome counts were performed on 100 near-diploid metaphase spreads. The majority of the cells were aneuploid. The chromosome number in the cell line ranged from 65 to 90 while the normal number is 78 in canine (Fig. 5A). Occasionally, the centric fusion of two acrocentric chromosomes resulting in a bi-armed metacentric chromosome was observed. Other structural chromosomal abnormalities (including deletions, gaps, and breaks) were also shown (Fig. 5B).
Large colonies of the CMT-7364 cells were formed (10–20 colonies) at 2 weeks after single cell suspension were planned in 0.3% agar (Fig. 7), MDA-MB-231 cell lines were used as positive control for this experiment which also formed large colonies on agar (data not shown).
3.8. CMT-7364 cell line shows Epithelial-Mesenchymal Transition The expression of ER, PR, HER-2, CK8/18, Vimentin, E-Cadherin and TGF-β1 on the CMT-7364 cells were detected by IHC. The cells were negative to ER, PR, and HER-2, negative to E-Cadherin and weakly positive to CK8/18, but strong positive to Vimentin and TGF-β1 in the cytoplasm (Fig. 8). The result indicated CMT-7364 cell line was a triple-negative canine mammary cancer cell line characterized by the transition from epithelial type to mesenchymal type.
3.6. CMT-7364 has high invasion ability in vitro To examine the invasion capacity of the CMT-7364 cell line, we carried out invasion assay with matrigel coated membrane inserts. The numbers of MDA-MB-231 and CMT-7364 cells that invaded through the basement membrane were 69 ± 6 and 81 ± 21, respectively (Fig. 6). There was no significant difference between them (p = 0.450).
Fig. 2. Immunohistochemical staining of the primary tumor sections. Positive staining was observed as a dark brown color. The tissue in A was the negative control. Tissues from primary tumor were negative to ER (B), PR (C), and HER-2 (D); were positive to CK8/18(E), E-Cadherin (F) and Vimentin (G) with positive cells localized in the cytomembrane and had a positive reaction to TGF-β1 (H) in the cytoplasm. 13
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Fig. 3. Light and electron microscopy of the CMT-7364 cell line. Light microscopy (A) shows the cells were round to spindle-shaped. Ultrastructural studies (B) revealed epitheliumlike cell morphology with large irregular nuclear outlines, some vacuole structures, mitochondria, numerous free ribosomes, endoplasmic reticulum, intermediate filaments and numerous microvilli.
immunophenotype markers of the xenograft tumor was similar with that of the primary tumor (Fig. 10). 3.10. The CMT-7364 cell line is free of mycoplasma contamination CMT-7364 cells were tested for the presence of mycoplasma by a PCR-based method. It had been found to be free of mycoplasma contamination (Fig. 11). 4. Discussion There is no doubt that established breast cancer cell lines are widely used in various studies, particularly in cancer research as in vitro models. They are easily handled and can grow in almost infinite quantities; they exhibit a relatively high degree of homogeneity and is easy to replace from frozen stocks if lost through contamination (Burdall et al., 2003). Spontaneous canine mammary gland cancer is considered as an excellent animal model of human carcinogenesis (Pinho et al., 2012). Those established canine mammary gland cancer cell lines are contributions to deep understand the specific of canine mammary cancer, as well as are likely to have the great impact on improving diagnose, treatment and outcome of human breast cancer patients. In this study, a new triple-negative cell line was successfully established from a primary canine mammary gland cancer. This new canine mammary cell line was aiming for both canine cancer study and human cancer study. Although, there are so many human breast cancer cell lines already, the similarities including epidemiology, biology and
Fig. 4. Population doubling time of CMT-7364 was calculated to be 21.12 h.
3.9. Tumorigenesis and metastasis potentials of the CMT-7364 cells on mice To examine the tumorigenicity of the CMT-7364 cell line, 5 × 106 cells were injected subcutaneously into 5-week-old BALB/c nude and BALB/c immune-competent mice. The growth of tumor was detected in all of the transplanted mice at the transplanted sites (Fig. 9A–D). Tumors occurred necrosis and ulceration (Fig. 9A and C) in some mice and grew rapidly (Fig. 9B and D). In all mice, tumors were histologically confirmed to be carcinomas and high malignant degree; the mitotic index was very high and atypical mitoses were frequently observed (Fig. 9E). All transplanted mice showed metastasis to the regional lymph nodes and lungs (Fig. 9F and G). The immunoreaction to the
Fig. 5. Karyotype analysis. The modal number of chromosome number in the cell line was 78 (A). The biarmed metacentric chromosome, deletions, gaps, and breaks were observed (B). 14
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Fig. 6. The cellular invasion by transwell assay.
Fig. 7. Soft agar assay. The CMT-7364 cells could form large colonies after 2 weeks incubation. It was single cells (A), the colony formed after one week incubation (B) and large colonies formed after 2 weeks incubation (C).
Fig. 8. Immunocytochemistry of CMT-7364 cell line. The cells in A were negative control. The cells were the negative to ER (B), PR (C), HER-2 (D), E-cadherin (F); were weakly positive to CK8/18 (E) and showed the positive reaction to Vimentin (G) and TGF-β1(H) in the cytomembrane. 15
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Fig. 9. Tumorigenesis by xenotransplantation. 5 × 106 cells was injected subcutaneously into mice. Tumors grew rapidly and occurred necrosis and ulceration in some mice (A and C). B and D show the tumor volume of nude and immunocompetent tumor-bearing mice respectively. High mitotic index and atypical mitoses were being frequently observed (E). Necropsy and pathological findings of lungs excised from xenografted mice (F and G).
Fig. 10. Immunohistochemical staining of the xenograft tumor sections. Positive staining was observed as a dark brown color. The tissue in A was the negative control. Tissues from the xenograft tumor were negative to ER (B), PR (C) and HER-2 (D); had a positive reaction to CK8/18(E), E-Cadherin (F) and Vimentin (G) with positive cells localized in the cytomembrane and were positive to TGF-β1 (H) in the cytoplasm.
advance our knowledge on the breast cancer and will translate into better clinical outcomes for both species (Raposo et al., 2017). Therefore, we propose that CMT-7364 has the potential to be a useful cell line for cancer study.
clinical features between dogs and women support the validity of canine mammary cancer as a model for human breast cancer (Liu et al., 2014; Nguyen et al., 2018; Queiroga et al., 2011; Raposo et al., 2017; Vascellari et al., 2016). Further, a natural canine model may help 16
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expression-profiling, including basal-like (BL1 and BL2 of basal or myoepithelial origin), mesenchymal-like (M), mesenchymal stem-like (MSL), luminal androgen receptor expression (LAR), and immunomodulatory (IM) (Lehmann et al., 2011). Whether chemotherapy choices should be different among these subtypes is being debated owing to a majority of TNBC patients do not achieve a pathological complete response (pCR) after chemotherapy (Kalimutho et al., 2015). These challenges have stimulated researchers to identify a more effective therapeutic way for TNBCs patients. It is suggested that immunomodulation may represent a new approach to treat these aggressive breast cancer subtypes as the prognosis of TNBC patients is associated with the level of tumor infiltrating lymphocytes (Loi, 2013). Therefore, the established CMT-7364 cell line that can transplant into immune-competent mice will be a powerful tool to explore immunomodulation and immunotherapy of the triple-negative CMTG in vivo. The CMT-7364 cells possessed the transformed nature by forming large colonies on soft agar (Fig. 7) (Carney et al., 1980). Furthermore, this cell line showed the equal ability of invasion on matrigel compared with MDA-MB-231 cell lines which is a mesenchymal-like cell line that highly expresses genes that makeup components and pathways associated with epithelial-mesenchymal transition (EMT) and cell motility which vastly applies in many works (Lehmann et al., 2011). In addition, all the mice that transplanted with the CMT-7364 cells appeared metastasis to regional lymph nodes (Fig. 9). All these results can prove the CMT-7364 cell line possesses malignant nature and metastatic character in vitro and in vivo. Invasive breast cancer is an important cause of cancer death for both humans and dogs. The classification system classifies human breast cancers into five significant subtypes, including estrogen receptor-positive luminal A and B subtypes, HER-2 overexpression subtype, normal breast-like and basal-like subtype (Sorlie et al., 2001). ER and PR are commonly expressed in breast cancers, but about 15–20% are negative for both ER and PR (Zafrani et al., 2000). Moreover, HER-2 negative or low expression has been seen in about 75–85% of breast cancers (Lal et al., 2005). It is well known that patients with triple-negative breast cancer have an increased likelihood of distant recurrence as well as death within 5 years of diagnosis (Dent et al., 2007). According to the IHC results, the CMT-7364 cell line preserved the immunohistochemical immunophenotype of original canine mammary cancer, i.e. lacked ER, PR, and HER-2 expression, being a novel triple negative cell line that can be used for the research of novel therapeutic targets (Fig. 8). In our study, it is interesting that the expression of biomarker proteins showed different patterns on the primary tumor, the CMT-7364 cell line and mouse xenograft tumors. The CMT-7364 cell line was negative to epithelial cell markers (E-Cadherin and CK 8/18), but positive to mesenchymal cell markers (Vimentin and TGF-β1) (Fig. 8), however, the primary tumor and mouse xenograft tumor were positive to all those markers (Figs. 2 and 10). It may suggest that the cells that we successfully purified from primary tumor are undergoing EMT. During EMT, breast cancer cells shed their differentiated epithelial characteristics, including cell-cell adhesion, polarity and lack of motility, and acquire mesenchymal features, including motility, invasiveness, and stemness (Gao et al., 2016). E-Cadherin is expressed exclusively in all of the mammary epithelial cells and provides a tight connection between epithelial cells which localizes and interacts with components of the adhered junction (Andrews et al., 2012). In the normal canine and human mammary glands, the luminal epithelial cells are characterized by the expression of low molecular weight luminal cytokeratins (CKs), including CK8, CK18 and CK19 (Boecker et al., 2002). High Vimentin and N-cadherin expression are traditional markers currently used to identify cells that have undergone EMT and have been identified as well in circulating tumor cells (Roussos et al., 2010). Vimentin is considered as a regulator to maintain intracellular mechanical homeostasis by mediating cytoskeleton architecture and the
Fig. 11. Mycoplasma detection. There was no mycoplasma contamination of the CMT-7364 cell line. A lane shows the CMT-7364 cells, + lane shows positive control, M lanes shows standard ladder, - lane shows negative control. B lane shows the culture media.
In the past few years, several canine mammary cancer cell lines have been established including CNMp, CHMp, CTBp and CIPp (Uyama et al., 2006), CMT-W1 and CMT-W2 (Krol et al., 2010), DE-E and DE-F (Chang et al., 2010), IPC-366 (Caceres et al., 2015), and FR37-CMT (Raposo et al., 2016). However, none of these cell lines shows tumorigenesis in immune-complete mice. In the present study, we established a new CMGC cell line showed rapid growth in a monolayer fashion whose doubling time was 21.12 h. Ultrastructural features (i.e. the presence of bundles of microfilaments, secretory vesicles, desmosomes, and granular cytoplasm with numerous ribosome) suggested the epithelial origin of this cell line (Fig. 3B). CMT-7364 cells were successfully transplanted into nude and immune-competent mice and resulted in palpable tumors (Fig. 9). Recent studies in human breast cancer and in canine mammary tumors have found that cancer related inflammation has an important role in mammary carcinogenesis, allow cancer cells to survive, proliferate, and disseminate (Carvalho et al., 2016a,b; Karayannopoulou et al., 2017). The lymphocyte infiltration in canine mammary tumors may contribute to that CMT-7364 cells could develop tumors in immune competent mice. The histological findings of the tumors in xenografted mice were similar with those of the original lesions. Thus, the CMT-7364 cell line was thought to have maintained their original histological features and possess tumorigenic properties to immune-competent mice. Data from 21 breast cancer datasets show that TNBCs can be subdivided into seven subclasses based on meta-analysis of gene 17
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balance of cell force generation in EMT (Liu et al., 2015). During tumor progression, upregulation of Vimentin is strongly positive correlated with metastatic spread (Whipple et al., 2008).The overexpression of Vimentin is found to be significantly associated with poor prognosis in TNBC patients (Yamashita et al., 2013),(Karihtala et al., 2013). The TGF-?? shows tumor suppressive effects in the early stages of the breast tumor, however, it increases tumor progression, tumor cell motility, cancer invasiveness, and metastasis in late stages (Syed, 2016; Zarzynska, 2014). This cytokine also participates in EMT and angiogenesis regarded as a metastasis inducer (Miyazono et al., 2012; Parvani et al., 2013). Therefore, the newly established tumourigenic canine mammary cell line, CMT-7364, may be used as a model for the study of the EMT and metastasis in triple-negative canine mammary gland tumors.
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Vimentin, zeb1 and Sip1 are up-regulated in triple-negative and basal-like breast cancers: association with an aggressive tumour phenotype. Breast Cancer Res. Treat. 138, 81–90. Kim, N.H., Lim, H.Y., Im, K.S., Kim, J.H., Sur, J.H., 2013. Identification of triple-negative and basal-like canine mammary carcinomas using four basal markers. J. Comp. Pathol. 148, 298–306. Krol, M., Polanska, J., Pawlowski, K.M., Turowski, P., Skierski, J., Majewska, A., Ugorski, M., Morty, R.E., Motyl, T., 2010. Transcriptomic signature of cell lines isolated from canine mammary adenocarcinoma metastases to lungs. J. Appl. Genet. 51, 37–50. Lal, P., Tan, L.K., Chen, B., 2005. Correlation of HER-2 status with estrogen and progesterone receptors and histologic features in 3,655 invasive breast carcinomas. Am. J. Clin. Pathol. 123, 541–546. Lana, S.E., Rutterman, G.R., Withrow, S.J., 2007. Tumors of the mammary gland. In: Withrow, MacEwen (Eds.), Small Animal Clinical Oncology, 4th ed. Saunders Elsevier, pp. 619–633. Lehmann, B.D., Bauer, J.A., Chen, X., Sanders, M.E., Chakravarthy, A.B., Shyr, Y., Pietenpol, J.A., 2011. Identification of human triple-negative breast cancer subtypes and preclinical models for selection of targeted therapies. J. Clin. Invest. 121, 2750–2767. Liu, D., Xiong, H., Ellis, A.E., Northrup, N.C., Rodriguez, C.O., O’Regan, R.M., Dalton, S., Zhao, S., 2014. Molecular homology and difference between spontaneous canine mammary cancer and human breast cancer. Cancer Res. 74, 5045–5056. Liu, C.Y., Lin, H.H., Tang, M.J., Wang, Y.K., 2015. Vimentin contributes to epithelialmesenchymal transition cancer cell mechanics by mediating cytoskeletal organization and focal adhesion maturation. Oncotarget 6, 15966–15983. Loi, S., 2013. Tumor-infiltrating lymphocytes, breast cancer subtypes and therapeutic efficacy. Oncoimmunology 2, e24720. Miyazono, K., Ehata, S., Koinuma, D., 2012. Tumor-promoting functions of transforming growth factor-beta in progression of cancer. Ups. J. Med. Sci. 117, 143–152. Murai, K., Nakagawa, T., Endo, Y., Kamida, A., Yoshida, K., Mochizuki, M., Nishimura, R., Sasaki, N., 2012. Establishment of a pair of novel cloned tumour cell lines with or without metastatic potential from canine mammary adenocarcinoma. Res. Vet. Sci. 93, 468–472. Nguyen, F., Pena, L., Ibisch, C., Loussouarn, D., Gama, A., Rieder, N., Belousov, A., Campone, M., Abadie, J., 2018. Canine invasive mammary carcinomas as models of human breast cancer. Part 1: natural history and prognostic factors. Breast Cancer Res. Treat. 167, 635–648. Parvani, J.G., Galliher-Beckley, A.J., Schiemann, B.J., Schiemann, W.P., 2013. Targeted inactivation of beta1 integrin induces beta3 integrin switching, which drives breast cancer metastasis by TGF-beta. Mol. Biol. Cell 24, 3449–3459. Pinho, S.S., Carvalho, S., Cabral, J., Reis, C.A., Gartner, F., 2012. Canine tumors: a spontaneous animal model of human carcinogenesis. Transl. Res. 159, 165–172. Queiroga, F.L., Raposo, T., Carvalho, M.I., Prada, J., Pires, I., 2011. Canine mammary tumours as a model to study human breast cancer: most recent findings. In Vivo 25, 455–465. Raposo, L.R., Roma-Rodrigues, C., Faisca, P., Alves, M., Henriques, J., Carvalheiro, M.C., Corvo, M.L., Baptista, P.V., Pombeiro, A.J., Fernandes, A.R., 2016. Immortalization and characterization of a new canine mammary tumour cell line FR37-CMT. Vet. Comp. Oncol. Raposo, T.P., Arias-Pulido, H., Chaher, N., Fiering, S.N., Argyle, D.J., Prada, J., Pires, I., Queiroga, F.L., 2017. Comparative aspects of canine and human inflammatory breast cancer. Semin. Oncol. 44, 288–300. Roussos, E.T., Keckesova, Z., Haley, J.D., Epstein, D.M., Weinberg, R.A., Condeelis, J.S., 2010. AACR special conference on epithelial-mesenchymal transition and cancer progression and treatment. Cancer Res. 70, 7360–7364. Sevostianova, N.N., Linkova, N.S., Polyakova, V.O., Chervyakova, N.A., Kostylev, A.V., Durnova, A.O., Kvetnoy, I.M., Abdulragimov, R.I., Khavinson, V.H., 2013. Immunomodulating effects of Vilon and its analogue in the culture of human and animal thymus cells. Bull. Exp. Biol. Med. 154, 562–565. Sorlie, T., Perou, C.M., Tibshirani, R., Aas, T., Geisler, S., Johnsen, H., Hastie, T., Eisen, M.B., van de Rijn, M., Jeffrey, S.S., Thorsen, T., Quist, H., Matese, J.C., Brown, P.O., Botstein, D., Lonning, P.E., Borresen-Dale, A.L., 2001. Gene expression patterns of breast carcinomas distinguish tumor subclasses with clinical implications. Proc. Natl. Acad. Sci. U. S. A. 98, 10869–10874. Syed, V., 2016. TGF-beta signaling in cancer. J. Cell. Biochem. 117, 1279–1287. Sylvester, P.W., 2011. Optimization of the tetrazolium dye (MTT) colorimetric assay for cellular growth and viability. Methods Mol. Biol. 716, 157–168. Tap, O.T., Rutteman, G.R., Zijlstra, C., de Haan, N.A., Bosma, A.A., 1998. Analysis of chromosome aberrations in a mammary carcinoma cell line from a dog by using canine painting probes. Cytogenet. Cell Genet. 82, 75–79. Uyama, R., Nakagawa, T., Hong, S.H., Mochizuki, M., Nishimura, R., Sasaki, N., 2006. Establishment of four pairs of canine mammary tumour cell lines derived from primary and metastatic origin and their E-cadherin expression. Vet. Comp. Oncol. 4,
5. Conclusion A novel triple negative canine mammary gland cancer cell line, CMT-7364, was established and characterized. The cell line may possess great research significance for future studies on the tumorigenesis and progression of human breast cancer and canine mammary cancer, especially facilitates the immunotherapy explosion and EMT mechanism in triple negative mammary cancer cell line. Conflict of interest statement Authors declare no financial or non-financial competing interests. Acknowledgements This work was supported by the National Natural Science Foundation of China (No. 31572578, No. 31402260, and No.31602218). References Andrews, J.L., Kim, A.C., Hens, J.R., 2012. The role and function of cadherins in the mammary gland. Breast Cancer Res.: BCR 14, 203. Beura, L.K., Hamilton, S.E., Bi, K., Schenkel, J.M., Odumade, O.A., Casey, K.A., Thompson, E.A., Fraser, K.A., Rosato, P.C., Filali-Mouhim, A., Sekaly, R.P., Jenkins, M.K., Vezys, V., Haining, W.N., Jameson, S.C., Masopust, D., 2016. Normalizing the environment recapitulates adult human immune traits in laboratory mice. Nature 532, 512–516. Boecker, W., Moll, R., Dervan, P., Buerger, H., Poremba, C., Diallo, R.I., Herbst, H., Schmidt, A., Lerch, M.M., Buchwalow, I.B., 2002. Usual ductal hyperplasia of the breast is a committed stem (progenitor) cell lesion distinct from atypical ductal hyperplasia and ductal carcinoma in situ. J. Pathol. 198, 458–467. Bosch, A., Eroles, P., Zaragoza, R., Vina, J.R., Lluch, A., 2010. Triple-negative breast cancer: molecular features, pathogenesis, treatment and current lines of research. Cancer Treat. Rev. 36, 206–215. Burdall, S.E., Hanby, A.M., Lansdown, M.R., Speirs, V., 2003. Breast cancer cell lines: friend or foe? Breast Cancer Res.: BCR 5, 89–95. Caceres, S., Pena, L., de Andres, P.J., Illera, M.J., Lopez, M.S., Woodward, W.A., Reuben, J.M., Illera, J.C., 2015. Establishment and characterization of a new cell line of canine inflammatory mammary cancer: IPC-366. PloS One 10, e0122277. Carney, D.N., Gazdar, A.F., Minna, J.D., 1980. Positive correlation between histological tumor involvement and generation of tumor cell colonies in agarose in specimens taken directly from patients with small-cell carcinoma of the lung. Cancer Res. 40, 1820–1823. Carvalho, M.I., Pires, I., Prada, J., Gregorio, H., Lobo, L., Queiroga, F.L., 2016a. Intratumoral FoxP3 expression is associated with angiogenesis and prognosis in malignant canine mammary tumors. Vet. Immunol. Immunopathol. 178, 1–9. Carvalho, M.I., Silva-Carvalho, R., Pires, I., Prada, J., Bianchini, R., Jensen-Jarolim, E., Queiroga, F.L., 2016b. A comparative approach of tumor-associated inflammation in mammary cancer between humans and dogs. Biomed Res. Int. Chang, C.Y., Chiou, P.P., Chen, W.J., Li, Y.H., Yiu, J.C., Cheng, Y.H., Chen, S.D., Lin, C.T., Lai, Y.S., 2010. Assessment of the tumorigenesis and drug susceptibility of three new canine mammary tumor cell lines. Res. Vet. Sci. 88, 285–293. Dent, R., Trudeau, M., Pritchard, K.I., Hanna, W.M., Kahn, H.K., Sawka, C.A., Lickley, L.A., Rawlinson, E., Sun, P., Narod, S.A., 2007. Triple-negative breast cancer: clinical features and patterns of recurrence. Clin. Cancer Res. 13, 4429–4434. Gao, T., Li, J.Z., Lu, Y., Zhang, C.Y., Li, Q., Mao, J., Li, L.H., 2016. The mechanism between epithelial mesenchymal transition in breast cancer and hypoxia microenvironment. Biomed. Pharmacother. 80, 393–405. Giuseppe Palma, G.F., Chirico, Andrea, Esposito, Emanuela, Siani, Claudio, Saturnino,
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S., Aishima, S., Morita, M., Maehara, Y., 2013. Vimentin as a poor prognostic factor for triple-negative breast cancer. J. Cancer Res. Clin. Oncol. 139, 739–746. Zafrani, B., Aubriot, M.H., Mouret, E., De Cremoux, P., De Rycke, Y., Nicolas, A., Boudou, E., Vincent-Salomon, A., Magdelenat, H., Sastre-Garau, X., 2000. High sensitivity and specificity of immunohistochemistry for the detection of hormone receptors in breast carcinoma: comparison with biochemical determination in a prospective study of 793 cases. Histopathology 37, 536–545. Zarzynska, J.M., 2014. Two faces of TGF-beta1 in breast cancer. Mediators Inflamm., 141747 2014.
104–113. Vascellari, M., Capello, K., Carminato, A., Zanardello, C., Baioni, E., Mutinelli, F., 2016. Incidence of mammary tumors in the canine population living in the Veneto region (Northeastern Italy): risk factors and similarities to human breast cancer. Prev. Vet. Med. 126, 183–189. Whipple, R.A., Balzer, E.M., Cho, E.H., Matrone, M.A., Yoon, J.R., Martin, S.S., 2008. Vimentin filaments support extension of tubulin-based microtentacles in detached breast tumor cells. Cancer Res. 68, 5678–5688. Yamashita, N., Tokunaga, E., Kitao, H., Hisamatsu, Y., Taketani, K., Akiyoshi, S., Okada,
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Contents lists available at ScienceDirect
Tissue and Cell journal homepage: www.elsevier.com/locate/tice
Establishment and characterization of a new triple-negative canine mammary cancer cell line
T ⁎
Hong Zhanga,1, Shimin Peia,1, Bin Zhoub, Huanan Wangc, Hongchao Dud, Di Zhangd,2, , ⁎ Degui Lind,2, a
Institute of Tropical Agriculture and Forestry, Hainan University, Haikou, Hainan, 570228, China The College of Animal Science and Technology, Zhejiang Agriculture and Forestry University, Hangzhou, Zhejiang, 311300, China c Department of Veterinary Medicine, College of Animal Sciences, Zhejiang University, Hangzhou, Zhejiang, 310058, China d The Clinical Department, College of Veterinary Medicine, China Agricultural University, Beijing, 100193, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Canine mammary cancer Characterization Establishment Epithelial-mesenchymal transition Triple-negative cell line
Canine mammary tumor (CMT) has always been an ideal animal model for human breast cancer (HBC) research, however, there is a lack of various established CMT cell lines corresponding to HBC cell lines. This study was designed to establish a new type of CMT cell line. The primary tumor, CMT-7364, was identified as the intraductal papillary carcinoma, and showed negative immunoreactivity to estrogen receptors (ER), progesterone receptors (PR), and human epidermal growth factor receptor-2 (HER-2) by immunohistochemistry (IHC) analysis. The CMT-7364 cell line from this primary tumor also shows a negative immunoreactivity to ER, PR, and HER-2, and was negative to epithelial cell markers and positive to mesenchymal cell markers by immunocytochemistry (ICC) analysis. This cell line, which has been stably cultured for more than 115 passages, and was characterized by epithelial origin with the expression of the epithelial antigen by ICC analysis and invasion ability by transwell analysis. In vivo, tumor mass and metastases in the lung were found after inoculating the CMT-7364 cells in the nude mice model, and the immune-complete mice model respectively. The tissues from the xenograft tumor were also negative to ER, PR, and HER-2 by IHC analysis. Thus, a novel triple negative canine mammary cancer cell line, CMT-7364, was successfully established, which could be used as a promising model for the research of immunotherapy and Epithelial-Mesenchymal Transition (EMT) mechanism of the triple-negative breast cancer both in canine and human.
1. Introduction The triple-negative breast cancer (TNBC), defined as the absence of hormone receptors (estrogen and progesterone) and negativity for human epidermal growth factor receptor-2 (HER-2), represents approximately 15–20% of breast cancers in human (Giuseppe Palma et al., 2015). Similarly, in a previous study of 241 canine mammary tumors (CMTs), 18.7% CMTs are the triple-negative phenotype which is associated with poor prognosis (Kim et al., 2013). Moreover, spontaneous CMT shares similar epidemiological and biological features with those in human, and these characteristics have been proven to be useful in understanding complex molecular aspects of human tumors (Pinho et al., 2012). Further study of triple-negative CMTs may help to thoroughly comprehend the characteristics of TNBC, and design more
effective therapeutic methods in the future. Compared with other breast cancer subtypes, TNBC is characterized by an aggressive and early pattern of metastases, a relative lack of therapeutic targets, and a poor prognosis (Bosch et al., 2010). It remains a huge challenge to develop the effective treatment of TNBC. While immunotherapies maybe a promising choice for the TNBC treatment, including immune-checkpoint inhibitors, antagonists of immunosuppressive molecules and tumor vaccine. In the laboratory, mouse xenograft model is a useful animal model to explore the immune treatment of TNBC. However, most of the breast cell lines can only be tumorigenic on the immunodeficiency mice which live in abnormally hygienic specific pathogen free (SPF) immunodeficiency barrier facilities. Those mice do not produce the similar immune response with the human to certain diseases until they are raised in the normal
⁎
Correspondence authors. E-mail addresses: [email protected] (D. Zhang), [email protected] (D. Lin). 1 Equal first author (Hong Zhang and Shimin Pei). 2 Equal Corresponding author. https://doi.org/10.1016/j.tice.2018.07.003 Received 17 October 2017; Received in revised form 27 June 2018; Accepted 20 July 2018 Available online 21 July 2018 0040-8166/ © 2018 Elsevier Ltd. All rights reserved.
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Beyotime, Shanghai, China). The growth curve was plotted and the doubling time was calculated from regression equation of the curve with GraphPad Prism 5 software (GraphPad Software, Inc. USA).
environment (Beura et al., 2016). Therefore, it is desired to establish a new TNBC cell line that is tumorigenic on immune-complete mice which can live in the normal environment. In summary, the purpose of our study was to establish a triple-negative CMT cell line which can transplant into immune-complete mice and to determine its characteristics, including morphology, growth potential, protein expression, and metastatic potential.
2.4. Karyotyping For karyotype analysis, cells of passage 60 at the growth phase were treated with 0.05 μg/mL colchicine for 6 h. Then adherent cells were dissociated by 0.25% trypsin and resuspended in 0.075 M hypotonic KCL (preheated to 37 °C) for 30 min at 37 °C. The cell suspension was then fixed in methanol: acetic acid (3:1) and added dropwise to the clean and cold slides. Finally, prepared slides were stained with Giemsa (Beijing Solarbio Science & Technology, China)for 15 min, then chromosome numbers of the cells were counted for 100 metaphase cells. The chromosomes were observed and counted under a Leica microscope (Leica Microsystems GmbH, Wetzlar, Germany) (Tap et al., 1998).
2. Materials and methods 2.1. Establishment and purification of primary cultures Tumor tissues were sterilely surgically excised from the dogs suffered from mammary gland tumors in the China Agricultural University Veterinary Teaching Hospital (CAU-VTH), performed according to CAU-VTH standard operating procedures and with informed owner consent. A part of tissues was used for paraffin embedding to confirm the diagnosis by histology according to Goldschmidt (Goldschmidt et al., 2011). There were 50 primary cultures from 50 distinct female dogs were tried to establish. If there were more than one tumor with one dog, the highest grade malignant one would be further analyzed. Then tumor tissues were washed with Phosphate Buffered Saline (PBS) supplemented with antibiotics (penicillin 100 IU/mL and streptomycin 100 IU/mL), and minced into 1 mm pieces removing blood, fat, and fibro connective tissues. Small pieces were incubated with Dulbecco's Modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS, Gibco, USA) and 0.1% collagenase Ⅱ (Gibco, USA) overnight in a 60-mm (in diameter) tissue culture dish (Costar, Corning Incorporated, USA) at 37 °C in a humidified atmosphere of 5% CO2. The supernatant of digested tissue pieces was transferred into an equal volume of DMEM medium with 10% FBS and antibiotics following completed mixture. The cells were centrifuged at 1000 RPM for 5 min, and resuspended in fresh medium. Then cells were seeded into 60-mm tissue culture dish, containing 3 mL of DMEM with 10% FBS, and maintained at 37 °C in a humidified atmosphere of 5% CO2. After the formation of a complete monolayer in primary culture, 105 cells were transferred into a new flask with fresh DMEM-10% FBS medium. Once the cells cultures were 80–90% confluent, cells were washed three times with PBS, treated with 0.25% trypsin solution (Gibco, USA), and incubated until cells were dislodged from the flask surface, and split in a ratio of 1: 3 in fresh DMEM-10% FBS medium. Also, the cells were sampled and frozen every five passages.
2.5. Invasion assay To assess the invasive capacity of CMT-7364 cells, we utilized Corning transwell chambers (Costar, Corning Incorporated, USA) coated with Matrigel (Becton, Dickinson and Company, USA). Breast cancer cell line MDA-MB-231 cells were used as positive control, the CMT-7364 cells were at passage 65. The experiment was performed in triplicates. Cells were digested with 0.25% trypsin and washed with PBS, then were seeded onto the filters at a concentration of 1 × 104 cells/well in 100 μL DMEM medium without FBS. The lower chambers were filled with 600 μL of medium containing 10% FBS. After 42 h cultured, cells on the topside of the filter were gently removed by scrubbing with a tipped swab. The cells invaded to the lower side of the filter were determined by crystal violet staining for 15 min and were counted in at least 5 randomly chosen fields per well under a microscope. 2.6. Soft agar assay The ability of anchorage-independent growth of the CMT-7364 cell line was determined by growing the cells on semisolid agar at the 65th passage (Sevostianova et al., 2013). A single-cell suspension containing 1000 cells was dispersed in a DMEM medium solution containing 0.6% agar seeded in 35-mm tissue culture dish which was layered a 1.2% agar solution in DMEM supplemented with 20% FBS. The cells were seeded in triplicate. Formation of colonies was determined by inverted microscopy (CKX41, Olympus Corporation, Japan) after 2 weeks incubation.
2.2. Ultrastructure analysis of cells For ultrastructure studies, cells were harvested at passage 61 by centrifugation at 1000 RPM for 5 min, followed by subsequently fixed with 2.5% glutaraldehyde in 0.01 M PBS at 4 °C. Those cells were then post-fixed in 1% OsO4, washed in PBS, dehydrated in graded acetone solutions and embedded in epoxy resin. Silver-to-gold sections were cut with a diamond knife using a Reichert-Jung Ultracut E ultramicrotome (LEICAUC6i, Germany). The ultra-thin sections were cut (400–500 μm), stained with lead citrate and uranyl acetate and examined under a JEOL JEM-1230 EXII transmission electron microscope (JEOL Ltd., Tokyo, Japan) studied under transmission microscope.
2.7. Immunohistochemistry (IHC) For immunohistochemical studies, 3 μm sections were cut from the primary tumor from which CMT-7364 was originated and xenografted tumor. Then they were cut from each specimen and mounted on poly-LLysine-coated slides. Deparaffinized tissues were subjected to heat-induced antigen retrieval at 98 °C for 12 min. After the jars were cooled to room temperature (RT), the slides were covered with 3% hydrogen peroxide to quench endogenous peroxidase activity and then incubation with 5% goat serum at RT for 30 min. Next, the sections were incubated overnight with primary antibodies specific for estrogen receptors (ER), progesterone receptors (PR), HER-2, Cytokeratin-8/18 (CK8/18), Vimentin, E-Cadherin, Transforming growth factor-β1 (TGF-β1) and Ki67 (see Table 1) at 4 °C. The sections were then incubated with secondary antibody (ZSGB-BIO, Beijing, China) for 20 min at 37 °C to detect immunolabelled proteins. Antibody binding was visualized with 3, 3′-diaminobenzidine tetrahydrochloride (DAB kit, ZSGB-BIO, Beijing, China). After a final washing in distilled water, the sections were counterstained with hematoxylin, dehydrated, cleared and mounted.
2.3. Growth studies and doubling time The doubling time of CMT-7364 cell line was determined at passage 54. Cells were diluted to a series of concentration that allowed to easily prepare the standard curve as previously described (Sylvester, 2011). The viable cells were seeded in triplicate in each of 96 well plates in DMEM medium with 10% FBS. Optical Density (OD) was determined with a microplate reader (ELx808 Absorbance Reader, BioTek, USA) using a 450 nm filter. It was determined in triplicate at exactly 24 h intervals for 8 days after 1 h treated with Cell Counting Kit (CCK-8, 11
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instruction. The resulting PCR products were separated by electrophoresis on 1.0% agarose gel, stained with Gelstain (Beijing TransGen Biotech, China), visualized under UV light and documented by photography (JY04S-3C, Beijing Junyi-dongfung Electrophoresis Equipment, China).
Table 1 Technical data of specific antibodies and peroxidase-developing systems for IHC and ICC. Primary Antibody Type
Clone
Manufacturer
Secondary antibody
ER PR HER-2
6F11 Zym5.2 EP3
Goat anti mouse Goat anti rabbit Goat anti rabbit
CK8/18
Zym5.2
Vimentin
V9
E-Cadherin Ki-67
4A2C7 MIB1
Ivitrogen Corporation,USA Ivitrogen Corporation,USA OriGene Technologies, Inc.,USA OriGene Technologies, Inc.,USA OriGene Technologies, Inc.,USA OriGene Technologies, Inc.,USA OriGene Technologies, Inc.,USA Abcam, USA
TGF-β1
Polyclonal
2.11. Statistical analysis The numerical results of invasion cells of different cell lines were expressed as the mean ± standard deviation (Mean ± SD). The results were analyzed by Student’s t-test using SPSS 20 software (Statistical Product and Service Solutions, Chicago, USA). There is the significant difference among different groups when p < 0.05.
Goat anti mouse Goat anti mouse Goat anti mouse Goat anti mouse
3. Result Goat anti rabbit
3.1. Establishment of CMT-7364 cell line from a malignant CMT We initiated 50 primary cultures obtained from female dogs with CMT admitted to the Veterinary Teaching Hospital of China Agricultural University. Out of these primary cultures, a cell line was successfully purified and propagated for more than 115 passages in our laboratory. The tumors were excised from a female dog aged 13 years, and the size of the biggest one was about 6.5 cm × 4 cm × 3.5 cm from which this cell line was originated (Fig. 1A). There was no metastasis in regional lymph node and distant organs. Therefore this mammary gland tumor was diagnosed at stage III according to a modified WHO clinical staging system (Lana et al., 2007). The epithelial cells of the tumor were multilayered with characteristics of malignancy by H＆E staining analysis. It showed moderate tubule formation, nuclear and cellular pleomorphism, and increased numbers of mitotic in the tumor (Fig. 1B and C). Then this mammary gland tumor was cataloged as intraductal papillary carcinoma and histological grade II. It also showed the positive reaction to Ki-67 in the nucleus (Fig. 1D).
For the negative controls, the primary antibody was replaced with PBS (pH 7.2). 2.8. Immunocytochemistry (ICC) An indirect immunofluorescence assay was performed in order to detect the expression of ER, PR, HER-2, CK8/18, Vimentin, E-Cadherin and TGF-β1 in the CMT-7364 cell line at passage 65. The cells growing on 24 well plate were fixed with methanol: acetone(1:1) for 30 min. The cells with primary antibodies were stained for overnight at 4 °C. The next day, cells were washed and stained with FITC–anti-rabbit or FITC–anti-mice IgG (Beijing ComWin Biotech, China) as a secondary antibody for 1 h at RT. The cells were washed and stained with DAPI (BD Transduction Laboratories, Lexington, USA). Then they were examined under a fluorescence microscope (CKX41, Olympus Corporation, Japan).
3.2. The primary tumor shows characters of triple-negative canine mammary cancer
2.9. Transplantation into mice All husbandry practices and experimental operations were performed in compliance with the ARRIVE guidelines and were carried out in accordance with EU Directive 2010/63/EU for animal experiments. All animal experiments were approved by the Institutional Animal Care and Use Committee of China Agricultural University (CAU20150101-2). The 5-week-old female BALB/SCID and BALB/c immune-competent mice (Beijing Vital River Laboratory Animal Technology, China) were used to investigate the tumorigenicity of the CMT-7364 cell line at passage 57. A suspension of 5 × 106 cells in 0.2 mL PBS was transplanted subcutaneously into the left mammary fat pad of mice (n = 6). Mice were observed biweekly. When tumors were detected, their length and width were weekly measured by calipers for 8 weeks. Mice were sacrificed by cervical dislocation when they showed clinical signs such as dyspnoea and weakness or one year after transplantation in cases which were no palpable tumor mass. Mice were euthanized by cervical dislocation and autopsied to detect metastatic lesions in the lungs or other organs. The tumors and organs were removed and fixed in 10% neutral-buffered formalin, and were stained with hematoxylin and eosin (H&E) for histopathologic examination. Tumor volume (V) was calculated according to the following formula (Murai et al., 2012):
We detected the expression of ER, PR, HER-2, CK8/18, Vimentin, ECadherin and TGF-β1 by IHC in the primary canine mammary gland cancer which was the origin of the CMT-7364 cell line. The paraffin sections were negative to ER, PR, and HER-2, but positive to epithelial cell markers (CK8/18 and E-Cadherin) with positive cells localized in the cytomembrane (Fig. 2). The samples had a positive reaction to Vimentin and TGF-β1 in the cytomembrane of epithelial cell (Fig. 2). 3.3. The CMT-7364 cell line shows epithelium-like cell morphology The cells were epithelioid and round- to spindle-shaped under light microscopy (Fig. 3A). The epithelial lineage of CMT-7364 was further confirmed by ultrastructure study of the cells by transmission electron microscopy. Ultrastructural studies revealed that CMT-7364 showed epithelium-like cell morphology with large irregular nuclear outlines, some vacuole structures, mitochondria, numerous free ribosomes, endoplasmic reticulum and intermediate filaments and numerous microvilli exhibited on the cell surface (Fig. 3B). 3.4. Growth studies and doubling time
V = a×b2×1/2(cm3) where a and b are the tumor length and width (in cm), respectively.
Growth studies and population doubling time of the CMT-7364 cell line were determined as Section 2.3. The doubling times of the cell line was calculated to be 21.12 h (Fig. 4).
2.10. Myoplasma detection 3.5. CMT-7364 exhibits abnormalities of chromosomes The mycoplasma DNA was detected by PCR Mycoplasma Test Kit (Hangzhou Hua’an biotechnology, China) as per the manufacturer’s
The CMT-7364 cells showed both numerical and structural 12
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Fig. 1. Primary canine mammary carcinoma. The biggest tumor which the CMT-7364 cell line was derived from was about 6.5 cm × 4.0 cm × 3.5 cm (A). Tumor cells in paraffin sections exhibited marked variation in nuclear size and hyperchromatic nucleus (B and C), often with one or more prominent nucleoli and mitoses (arrow). Tissues from primary tumor were positive to Ki-67 in the nucleus by Immunohistochemical staining.
3.7. CMT-7364 shows high ability of colony
abnormalities of chromosomes by karyotype analysis. Chromosome counts were performed on 100 near-diploid metaphase spreads. The majority of the cells were aneuploid. The chromosome number in the cell line ranged from 65 to 90 while the normal number is 78 in canine (Fig. 5A). Occasionally, the centric fusion of two acrocentric chromosomes resulting in a bi-armed metacentric chromosome was observed. Other structural chromosomal abnormalities (including deletions, gaps, and breaks) were also shown (Fig. 5B).
Large colonies of the CMT-7364 cells were formed (10–20 colonies) at 2 weeks after single cell suspension were planned in 0.3% agar (Fig. 7), MDA-MB-231 cell lines were used as positive control for this experiment which also formed large colonies on agar (data not shown).
3.8. CMT-7364 cell line shows Epithelial-Mesenchymal Transition The expression of ER, PR, HER-2, CK8/18, Vimentin, E-Cadherin and TGF-β1 on the CMT-7364 cells were detected by IHC. The cells were negative to ER, PR, and HER-2, negative to E-Cadherin and weakly positive to CK8/18, but strong positive to Vimentin and TGF-β1 in the cytoplasm (Fig. 8). The result indicated CMT-7364 cell line was a triple-negative canine mammary cancer cell line characterized by the transition from epithelial type to mesenchymal type.
3.6. CMT-7364 has high invasion ability in vitro To examine the invasion capacity of the CMT-7364 cell line, we carried out invasion assay with matrigel coated membrane inserts. The numbers of MDA-MB-231 and CMT-7364 cells that invaded through the basement membrane were 69 ± 6 and 81 ± 21, respectively (Fig. 6). There was no significant difference between them (p = 0.450).
Fig. 2. Immunohistochemical staining of the primary tumor sections. Positive staining was observed as a dark brown color. The tissue in A was the negative control. Tissues from primary tumor were negative to ER (B), PR (C), and HER-2 (D); were positive to CK8/18(E), E-Cadherin (F) and Vimentin (G) with positive cells localized in the cytomembrane and had a positive reaction to TGF-β1 (H) in the cytoplasm. 13
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Fig. 3. Light and electron microscopy of the CMT-7364 cell line. Light microscopy (A) shows the cells were round to spindle-shaped. Ultrastructural studies (B) revealed epitheliumlike cell morphology with large irregular nuclear outlines, some vacuole structures, mitochondria, numerous free ribosomes, endoplasmic reticulum, intermediate filaments and numerous microvilli.
immunophenotype markers of the xenograft tumor was similar with that of the primary tumor (Fig. 10). 3.10. The CMT-7364 cell line is free of mycoplasma contamination CMT-7364 cells were tested for the presence of mycoplasma by a PCR-based method. It had been found to be free of mycoplasma contamination (Fig. 11). 4. Discussion There is no doubt that established breast cancer cell lines are widely used in various studies, particularly in cancer research as in vitro models. They are easily handled and can grow in almost infinite quantities; they exhibit a relatively high degree of homogeneity and is easy to replace from frozen stocks if lost through contamination (Burdall et al., 2003). Spontaneous canine mammary gland cancer is considered as an excellent animal model of human carcinogenesis (Pinho et al., 2012). Those established canine mammary gland cancer cell lines are contributions to deep understand the specific of canine mammary cancer, as well as are likely to have the great impact on improving diagnose, treatment and outcome of human breast cancer patients. In this study, a new triple-negative cell line was successfully established from a primary canine mammary gland cancer. This new canine mammary cell line was aiming for both canine cancer study and human cancer study. Although, there are so many human breast cancer cell lines already, the similarities including epidemiology, biology and
Fig. 4. Population doubling time of CMT-7364 was calculated to be 21.12 h.
3.9. Tumorigenesis and metastasis potentials of the CMT-7364 cells on mice To examine the tumorigenicity of the CMT-7364 cell line, 5 × 106 cells were injected subcutaneously into 5-week-old BALB/c nude and BALB/c immune-competent mice. The growth of tumor was detected in all of the transplanted mice at the transplanted sites (Fig. 9A–D). Tumors occurred necrosis and ulceration (Fig. 9A and C) in some mice and grew rapidly (Fig. 9B and D). In all mice, tumors were histologically confirmed to be carcinomas and high malignant degree; the mitotic index was very high and atypical mitoses were frequently observed (Fig. 9E). All transplanted mice showed metastasis to the regional lymph nodes and lungs (Fig. 9F and G). The immunoreaction to the
Fig. 5. Karyotype analysis. The modal number of chromosome number in the cell line was 78 (A). The biarmed metacentric chromosome, deletions, gaps, and breaks were observed (B). 14
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Fig. 6. The cellular invasion by transwell assay.
Fig. 7. Soft agar assay. The CMT-7364 cells could form large colonies after 2 weeks incubation. It was single cells (A), the colony formed after one week incubation (B) and large colonies formed after 2 weeks incubation (C).
Fig. 8. Immunocytochemistry of CMT-7364 cell line. The cells in A were negative control. The cells were the negative to ER (B), PR (C), HER-2 (D), E-cadherin (F); were weakly positive to CK8/18 (E) and showed the positive reaction to Vimentin (G) and TGF-β1(H) in the cytomembrane. 15
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Fig. 9. Tumorigenesis by xenotransplantation. 5 × 106 cells was injected subcutaneously into mice. Tumors grew rapidly and occurred necrosis and ulceration in some mice (A and C). B and D show the tumor volume of nude and immunocompetent tumor-bearing mice respectively. High mitotic index and atypical mitoses were being frequently observed (E). Necropsy and pathological findings of lungs excised from xenografted mice (F and G).
Fig. 10. Immunohistochemical staining of the xenograft tumor sections. Positive staining was observed as a dark brown color. The tissue in A was the negative control. Tissues from the xenograft tumor were negative to ER (B), PR (C) and HER-2 (D); had a positive reaction to CK8/18(E), E-Cadherin (F) and Vimentin (G) with positive cells localized in the cytomembrane and were positive to TGF-β1 (H) in the cytoplasm.
advance our knowledge on the breast cancer and will translate into better clinical outcomes for both species (Raposo et al., 2017). Therefore, we propose that CMT-7364 has the potential to be a useful cell line for cancer study.
clinical features between dogs and women support the validity of canine mammary cancer as a model for human breast cancer (Liu et al., 2014; Nguyen et al., 2018; Queiroga et al., 2011; Raposo et al., 2017; Vascellari et al., 2016). Further, a natural canine model may help 16
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expression-profiling, including basal-like (BL1 and BL2 of basal or myoepithelial origin), mesenchymal-like (M), mesenchymal stem-like (MSL), luminal androgen receptor expression (LAR), and immunomodulatory (IM) (Lehmann et al., 2011). Whether chemotherapy choices should be different among these subtypes is being debated owing to a majority of TNBC patients do not achieve a pathological complete response (pCR) after chemotherapy (Kalimutho et al., 2015). These challenges have stimulated researchers to identify a more effective therapeutic way for TNBCs patients. It is suggested that immunomodulation may represent a new approach to treat these aggressive breast cancer subtypes as the prognosis of TNBC patients is associated with the level of tumor infiltrating lymphocytes (Loi, 2013). Therefore, the established CMT-7364 cell line that can transplant into immune-competent mice will be a powerful tool to explore immunomodulation and immunotherapy of the triple-negative CMTG in vivo. The CMT-7364 cells possessed the transformed nature by forming large colonies on soft agar (Fig. 7) (Carney et al., 1980). Furthermore, this cell line showed the equal ability of invasion on matrigel compared with MDA-MB-231 cell lines which is a mesenchymal-like cell line that highly expresses genes that makeup components and pathways associated with epithelial-mesenchymal transition (EMT) and cell motility which vastly applies in many works (Lehmann et al., 2011). In addition, all the mice that transplanted with the CMT-7364 cells appeared metastasis to regional lymph nodes (Fig. 9). All these results can prove the CMT-7364 cell line possesses malignant nature and metastatic character in vitro and in vivo. Invasive breast cancer is an important cause of cancer death for both humans and dogs. The classification system classifies human breast cancers into five significant subtypes, including estrogen receptor-positive luminal A and B subtypes, HER-2 overexpression subtype, normal breast-like and basal-like subtype (Sorlie et al., 2001). ER and PR are commonly expressed in breast cancers, but about 15–20% are negative for both ER and PR (Zafrani et al., 2000). Moreover, HER-2 negative or low expression has been seen in about 75–85% of breast cancers (Lal et al., 2005). It is well known that patients with triple-negative breast cancer have an increased likelihood of distant recurrence as well as death within 5 years of diagnosis (Dent et al., 2007). According to the IHC results, the CMT-7364 cell line preserved the immunohistochemical immunophenotype of original canine mammary cancer, i.e. lacked ER, PR, and HER-2 expression, being a novel triple negative cell line that can be used for the research of novel therapeutic targets (Fig. 8). In our study, it is interesting that the expression of biomarker proteins showed different patterns on the primary tumor, the CMT-7364 cell line and mouse xenograft tumors. The CMT-7364 cell line was negative to epithelial cell markers (E-Cadherin and CK 8/18), but positive to mesenchymal cell markers (Vimentin and TGF-β1) (Fig. 8), however, the primary tumor and mouse xenograft tumor were positive to all those markers (Figs. 2 and 10). It may suggest that the cells that we successfully purified from primary tumor are undergoing EMT. During EMT, breast cancer cells shed their differentiated epithelial characteristics, including cell-cell adhesion, polarity and lack of motility, and acquire mesenchymal features, including motility, invasiveness, and stemness (Gao et al., 2016). E-Cadherin is expressed exclusively in all of the mammary epithelial cells and provides a tight connection between epithelial cells which localizes and interacts with components of the adhered junction (Andrews et al., 2012). In the normal canine and human mammary glands, the luminal epithelial cells are characterized by the expression of low molecular weight luminal cytokeratins (CKs), including CK8, CK18 and CK19 (Boecker et al., 2002). High Vimentin and N-cadherin expression are traditional markers currently used to identify cells that have undergone EMT and have been identified as well in circulating tumor cells (Roussos et al., 2010). Vimentin is considered as a regulator to maintain intracellular mechanical homeostasis by mediating cytoskeleton architecture and the
Fig. 11. Mycoplasma detection. There was no mycoplasma contamination of the CMT-7364 cell line. A lane shows the CMT-7364 cells, + lane shows positive control, M lanes shows standard ladder, - lane shows negative control. B lane shows the culture media.
In the past few years, several canine mammary cancer cell lines have been established including CNMp, CHMp, CTBp and CIPp (Uyama et al., 2006), CMT-W1 and CMT-W2 (Krol et al., 2010), DE-E and DE-F (Chang et al., 2010), IPC-366 (Caceres et al., 2015), and FR37-CMT (Raposo et al., 2016). However, none of these cell lines shows tumorigenesis in immune-complete mice. In the present study, we established a new CMGC cell line showed rapid growth in a monolayer fashion whose doubling time was 21.12 h. Ultrastructural features (i.e. the presence of bundles of microfilaments, secretory vesicles, desmosomes, and granular cytoplasm with numerous ribosome) suggested the epithelial origin of this cell line (Fig. 3B). CMT-7364 cells were successfully transplanted into nude and immune-competent mice and resulted in palpable tumors (Fig. 9). Recent studies in human breast cancer and in canine mammary tumors have found that cancer related inflammation has an important role in mammary carcinogenesis, allow cancer cells to survive, proliferate, and disseminate (Carvalho et al., 2016a,b; Karayannopoulou et al., 2017). The lymphocyte infiltration in canine mammary tumors may contribute to that CMT-7364 cells could develop tumors in immune competent mice. The histological findings of the tumors in xenografted mice were similar with those of the original lesions. Thus, the CMT-7364 cell line was thought to have maintained their original histological features and possess tumorigenic properties to immune-competent mice. Data from 21 breast cancer datasets show that TNBCs can be subdivided into seven subclasses based on meta-analysis of gene 17
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balance of cell force generation in EMT (Liu et al., 2015). During tumor progression, upregulation of Vimentin is strongly positive correlated with metastatic spread (Whipple et al., 2008).The overexpression of Vimentin is found to be significantly associated with poor prognosis in TNBC patients (Yamashita et al., 2013),(Karihtala et al., 2013). The TGF-?? shows tumor suppressive effects in the early stages of the breast tumor, however, it increases tumor progression, tumor cell motility, cancer invasiveness, and metastasis in late stages (Syed, 2016; Zarzynska, 2014). This cytokine also participates in EMT and angiogenesis regarded as a metastasis inducer (Miyazono et al., 2012; Parvani et al., 2013). Therefore, the newly established tumourigenic canine mammary cell line, CMT-7364, may be used as a model for the study of the EMT and metastasis in triple-negative canine mammary gland tumors.
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5. Conclusion A novel triple negative canine mammary gland cancer cell line, CMT-7364, was established and characterized. The cell line may possess great research significance for future studies on the tumorigenesis and progression of human breast cancer and canine mammary cancer, especially facilitates the immunotherapy explosion and EMT mechanism in triple negative mammary cancer cell line. Conflict of interest statement Authors declare no financial or non-financial competing interests. Acknowledgements This work was supported by the National Natural Science Foundation of China (No. 31572578, No. 31402260, and No.31602218). References Andrews, J.L., Kim, A.C., Hens, J.R., 2012. The role and function of cadherins in the mammary gland. Breast Cancer Res.: BCR 14, 203. Beura, L.K., Hamilton, S.E., Bi, K., Schenkel, J.M., Odumade, O.A., Casey, K.A., Thompson, E.A., Fraser, K.A., Rosato, P.C., Filali-Mouhim, A., Sekaly, R.P., Jenkins, M.K., Vezys, V., Haining, W.N., Jameson, S.C., Masopust, D., 2016. Normalizing the environment recapitulates adult human immune traits in laboratory mice. Nature 532, 512–516. Boecker, W., Moll, R., Dervan, P., Buerger, H., Poremba, C., Diallo, R.I., Herbst, H., Schmidt, A., Lerch, M.M., Buchwalow, I.B., 2002. Usual ductal hyperplasia of the breast is a committed stem (progenitor) cell lesion distinct from atypical ductal hyperplasia and ductal carcinoma in situ. J. Pathol. 198, 458–467. Bosch, A., Eroles, P., Zaragoza, R., Vina, J.R., Lluch, A., 2010. Triple-negative breast cancer: molecular features, pathogenesis, treatment and current lines of research. Cancer Treat. Rev. 36, 206–215. Burdall, S.E., Hanby, A.M., Lansdown, M.R., Speirs, V., 2003. Breast cancer cell lines: friend or foe? Breast Cancer Res.: BCR 5, 89–95. Caceres, S., Pena, L., de Andres, P.J., Illera, M.J., Lopez, M.S., Woodward, W.A., Reuben, J.M., Illera, J.C., 2015. Establishment and characterization of a new cell line of canine inflammatory mammary cancer: IPC-366. PloS One 10, e0122277. Carney, D.N., Gazdar, A.F., Minna, J.D., 1980. Positive correlation between histological tumor involvement and generation of tumor cell colonies in agarose in specimens taken directly from patients with small-cell carcinoma of the lung. Cancer Res. 40, 1820–1823. Carvalho, M.I., Pires, I., Prada, J., Gregorio, H., Lobo, L., Queiroga, F.L., 2016a. Intratumoral FoxP3 expression is associated with angiogenesis and prognosis in malignant canine mammary tumors. Vet. Immunol. Immunopathol. 178, 1–9. Carvalho, M.I., Silva-Carvalho, R., Pires, I., Prada, J., Bianchini, R., Jensen-Jarolim, E., Queiroga, F.L., 2016b. A comparative approach of tumor-associated inflammation in mammary cancer between humans and dogs. Biomed Res. Int. Chang, C.Y., Chiou, P.P., Chen, W.J., Li, Y.H., Yiu, J.C., Cheng, Y.H., Chen, S.D., Lin, C.T., Lai, Y.S., 2010. Assessment of the tumorigenesis and drug susceptibility of three new canine mammary tumor cell lines. Res. Vet. Sci. 88, 285–293. Dent, R., Trudeau, M., Pritchard, K.I., Hanna, W.M., Kahn, H.K., Sawka, C.A., Lickley, L.A., Rawlinson, E., Sun, P., Narod, S.A., 2007. Triple-negative breast cancer: clinical features and patterns of recurrence. Clin. Cancer Res. 13, 4429–4434. Gao, T., Li, J.Z., Lu, Y., Zhang, C.Y., Li, Q., Mao, J., Li, L.H., 2016. The mechanism between epithelial mesenchymal transition in breast cancer and hypoxia microenvironment. Biomed. Pharmacother. 80, 393–405. Giuseppe Palma, G.F., Chirico, Andrea, Esposito, Emanuela, Siani, Claudio, Saturnino,
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