Establishment and characterization of a HER2-enriched canine mammary cancerous myoepithelial cell line

Abstract

Background: Canine mammary tumors (CMTs) have a poor prognosis, along with tumor recurrence and metastasis. Cell lines are vital in vitro models for CMT research. Many CMT epithelial cell lines were reported. However, canine mammary myoepithelial cells, the contractile component of the canine mammary tissue were overlooked. This study aimed at establishing such a cell line. CMT-1 cell line was obtained from a canine mammary tumor CMT-1 and characterized molecularly through qPCR, western blotting, immunochemistry and immunofluorescence. Its doubling time, cytogenetic analysis and migration rate were evaluated using growth study, karyotype analysis and wound healing assay respectively. To determine its tumorigenesis, xenograft transplantation was performed.

Results: CMT-1 tumor was a complex canine mammary carcinoma that stained negative to estrogen receptors (ER) and progesterone receptors (PR), but positive to human epidermal growth receptor-2 (HER2), defined as HER2-enriched subtype. In this study, a CMT-1 cell line obtained from CMT-1 tumor was immune-positive to vimentin, α-SMA, p63 and negative to E-cadherin (E-cad), indicating CMT-1 cells were myoepithelial cells. It was successfully cultured for more than 50 passages showing the same immunoreactivity to ER, PR, and HER2 as the primary canine tumor. The doubling time of CMT-1 cell line was 26.67 h. The chromosome number of CMT-1 cells ranged from 31 to 64. A potential spontaneous epithelial to mesenchymal transition (EMT) was noticed during cell cultures. Potential EMT-induced CMT-1 cells showed no significance in migration rate compared to the original CMT-1 cells. CMT-1 cells was able to grow on a 3D culture and formed grape-like, solid, and cystic mammospheres at different time period. Inoculation of CMT-1 cells induced a complex HER2-enriched mammary tumor with metastasis in mice.

Conclusions: A canine cancerous HER2-enriched myoepithelial cell line was successfully established and a canine mammosphere developed from myoepithelial cells was documented in this study. We are expecting this novel cell line and its associated mammospheres could be used as a model to elucidate the role of myoepithelial cells in CMT carcinogensis in the future.

Keywords: CMT; EMT; HER2; Mammosphere; Myoepithelium.

Fig. 1

Histopathology of the CMT-1 tumor with H&E and IHC staining. Lower left: Uncapsulated mass composed of proliferative mammary epithelial and myoepithelial cells. Lower right: Nuclei were oval to elongated containing finely stippled scant chromatin and indistinct nucleoli

Fig. 2

Histopathology of the CMT-1 tumor with IHC staining. IHC staining images showed that CMT was ER negative, PR negative, HER2 positive, α-SMA positive, and E-cadherin positive

Fig. 3

Growth studies and doubling time of the CMT-1 cell line. A CMT-1 cells demonstrated moderate anisocytosis and anisokaryosis with the majority of cells having multiple nuclei. B CMT-1 cells (passage 9) showed slight anisocytosis and anisokaryosis with the majority of cells having a single nucleus while CMT-1 (passage 50) became more uniformly round to oval in morphology. C The population doubling time of CMT-1 (passage 50) was calculated to be 26.67 h

Fig. 4

Karyotype analysis of the CMT-1 cell line. A The modal number of chromosome number in CMT-1 cell line was 61. B Chromosomal structural abnormalities such as gaps, breaks and fragments were noted

Fig. 5

Western blotting results of different cell lines and immunofluorescence assay results of CMT-1 cells. A The results of western blotting of MDCK cells, CMT-1 cells (passage 5), CHMp cell line, and CMT-1 cells (passage 50) were ER⁺/PR⁻/HER2⁻, ER⁺/PR⁻/HER2⁻, ER⁺/PR⁻/HER2⁻ and ER⁻/PR⁻/HER2.⁺ respectively. B IFA results of CMT-1 cells were consistent with WB results. The original images of the western blot were shown in the Supplementary file 1

Fig. 6

Molecular analysis of the CMT-1 cell line. A&B The results of western blotting showed CMT-1 cells were positive to α-SMA and p63. C IFA results demonstrated CMT-1 cells had weak reaction to E-cadherin and moderate to strong reaction to vimentin. IFA results shared the same immunoreactivity to α-SMA and p63 for CMT-1 cells. The original images of the western blot were shown in the Supplementary file 1

Fig. 7

Migration assay. A Cell migration ability was tested at 24 and 48 h. The black vertical lines in the images define the open wound areas. B The average migration distance of various preparations is shown. All assays were independently repeated at least three times. ns indicates not significant

Fig. 8

Expression of E-cadherin, and vimentin of CMT-1 cells. A WB for E-cadherin and vimentin. MDCK cells showed weak reaction to E-cadherin while CMT-1 cells (passage 5) strongly expressed E-cadherin. CMT-1 cells (passage 50) lost E-cadherin expression but gained greater vimentin expression. B Relative fold changes in E-cadherin and vimentin expression using qPCR between CMT1-P5 and CMT1-P50. All experiments were independently repeated at least three times. The data are shown as the mean ± SD, n = 3, * p < 0.05, ** p < 0.01, *** p < 0.001). The original images of the western blot were shown in the Supplementary file 1

Fig. 9

Representative bright-field images of different morphologies of CMT-1 mammary organoids. Black arrow: grape-like, Blue arrow: solid, Red arrow: cystic. Scale bar = 50 μm (indicated in the bottom right corner of the image)

Fig. 10

Nude mouse tumorigenicity assay. A The primary tumor appeared as a small solid mass at the injection site. Diffuse to coalescing whitish lesions (white arrow) were noted on the diaphragm. B Tumor volume of the nude mice. The data are shown as the mean ± SD, n = 10, (C) Epithelial cells formed nests and irregular tubules on a spindoloid stromal proliferation in a primary xenograft tumor. Scale bar = 100 μm (indicated in the upper left corner of the image). D Diaphragmatic muscle degeneration and atrophy adjacent to the neoplastic cell infiltrates. Scale bar = 50 μm (indicated in the upper left corner of the image)

Fig. 11

IHC of the xenograft tumor (A) and the metastatic diaphragm (B). Both the xenograft tumor and metastatic diaphragm were negative for ER and PR, but positive for HER2, α-SMA, and E-cadherin. The metastatic diaphragm had stronger staining for HER2 and α-SMA