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Establishment and characterization of a canine sebaceous epithelial cell line derived from an eyelid mass.

ABSTRACT: Little is known about the pathological roles of sebaceous glands in canine skin diseases, as most examinations have been conducted with cultured human sebaceous epithelial cell lines. To our knowledge, there is no available canine sebaceous epithelial cell line. The purpose of this study was to establish a canine sebaceous epithelial cell line and characterize it. An eyelid mass in a dog was surgically resected for treatment, and it was histologically diagnosed as sebaceous epithelioma. Collected tissue was conducted for culture, and the growing epithelial-like cells were passaged. The cells showed continuous proliferation for over 6 months. After 40 passages, the cells were named CMG-1. Lipid droplets in the cytoplasm of CMG-1 cells were confirmed by Oil Red O staining. As reported in studies with human sebaceous epithelial cell lines, lipogenesis in CMG-1 cells was promoted by linoleic acid, whereas transforming growth factor-? (TGF-?) suppressed it. Additionally, real-time PCR revealed that the expression levels of chemokines and cytokines, including CC chemokine ligand (CCL)-2, CCL-20, CXCL-10, Tumor necrosis factor-? (TNF-?), Interleukin (IL)-1?, IL-1?, and IL-8, were significantly increased in CMG-1 cells following treatment with lipopolysaccharide. In conclusion, we successfully established a new canine sebaceous epithelial cell line. Our data indicated that lipogenesis and inflammatory responses were quantitatively evaluable in this cell line. CMG-1 cells could be useful for the pathological analysis of sebaceous gland diseases in dogs.

SUBMITTER: Matsuda A

PROVIDER: S-EPMC7719885 | biostudies-literature | 2020 Nov

REPOSITORIES: biostudies-literature

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Establishment and characterization of a canine sebaceous epithelial cell line derived from an eyelid mass.

Matsuda Akira A Mitsui Ikki I Shimizu Yuki Y Kanda Teppei T Ohnishi Akihiro A Miyabe Masahiro M Itoh Yoshiki Y

The Journal of veterinary medical science 20200911 11

Little is known about the pathological roles of sebaceous glands in canine skin diseases, as most examinations have been conducted with cultured human sebaceous epithelial cell lines. To our knowledge, there is no available canine sebaceous epithelial cell line. The purpose of this study was to establish a canine sebaceous epithelial cell line and characterize it. An eyelid mass in a dog was surgically resected for treatment, and it was histologically diagnosed as sebaceous epithelioma. Collecte ...[more]

PMID: 32921644

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Project description:PurposeEyelid sebaceous carcinoma (SeC) is the third most frequent eyelid malignancy worldwide and is relatively prevalent in Asian patients. An eyelid SeC cell line model is necessary for experimental research to explore the etiology and pathogenesis of eyelid SeC. This study established and characterized an eyelid SeC cell line with a TP53 mutation that might be useful for analyzing potential treatment options for eyelid SeC.MethodsThe eyelid SeC cell line SHNPH-SeC was obtained from a patient with eyelid SeC at Shanghai Ninth People's Hospital (SHNPH), Shanghai JiaoTong University School of Medicine. Immunofluorescence staining was employed to detect the origination and proliferation activity. Short tandem repeat (STR) profiling was performed for verification. Chromosome analysis was implemented to investigate chromosome aberrations. Whole exome sequencing (WES) was used to discover genomic mutations. Cell proliferation assays were performed to identify sensitivity to mitomycin-C (MMC) and 5-fluorouracil (5-FU).ResultsSHNPH-SeC cells were successively subcultured for more than 100 passages and demonstrated rapid proliferation and migration. Karyotype analysis revealed abundant chromosome aberrations, and WES revealed SeC-related mutations in TP53, KMT2C, and ERBB2. An in vivo tumor model was successfully established in NOD/SCID mice. Biomarkers of eyelid SeC, including cytokeratin 5 (CK5), epithelial membrane antigen (EMA), adipophilin, p53, and Ki-67, were detected in SHNPH-SeC cells, original tumors, and xenografts. MMC and 5-FU inhibited the proliferation and migration of SHNPH-SeC cells, and SHNPH-SeC cells presented a greater drug response than non-TP53-mutated SeC cells.ConclusionsThe newly established eyelid SeC cell line SHNPH-SeC demonstrates mutation in TP53, the most commonly mutated gene in SeC. It presents SeC properties and malignant characteristics that may facilitate the investigation of cellular behaviors and molecular mechanisms of SeC to explore promising therapeutic strategies.

Project description:BackgroundCanine 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.ResultsCMT-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.ConclusionsA 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.

Project description:Background and purposeCanine mammary tumors are the most common tumor disease of female dogs, and adjuvant chemotherapy often results in multi-drug resistance. Currently, the mechanisms underlying the development of tumor multi-drug resistance are unclear. The translation of research applications that can be used to effectively overcome tumor resistance is similarly hampered. Therefore, it is urgent to construct multi-drug resistance models of canine mammary tumors that can be used for research, to explore the mechanisms and means of overcoming resistance.Materials and methodsIn this study, the canine triple negative breast cancer cell line CMT-7364 was induced to develop multidrug resistance using doxorubicin by high-dose drug pulse method. The drug resistance and the expression of drug transport pumps of the cells was verified by CCK8 assay, immunoblotting, qPCR and immunofluorescence. Next, we used scratch assay and Transwell invasion assay to compare the migration and invasion abilities of the two cell lines and examined the expression of EMT-related proteins in both using immunoblotting. The differences of transcriptome between parental and drug-resistant cell lines were detected by RNA-seq sequencing. Finally, mouse xenograft models of drug-resistant and parental cell lines were constructed to evaluate the tumorigenic ability.ResultsAfter more than 50 generations of continuous passages stimulated by high-dose drug pulse method, the morphology of drug-resistant cell line CMT-7364/R tended to be mesenchymal-like and heterogeneous under light microscopy compared with the parental cell line CMT-7364/S, and developed resistance to doxorubicin and other commonly used chemotherapeutic drugs. In CMT-7364/R, BCRP was expressed at higher levels at both transcriptional and protein levels, while P-glycoprotein was not significantly different. Secondly, the migration and invasion ability of CMT-7364/R was significantly enhanced, with decreased expression of E-cadherin and increased expression of vimentin and mucin 1-N terminus. Finally, mouse xenograft models were constructed, while there was no significant difference in the volume of masses formed at 21 days.ConclusionIn summary, by using the canine mammary tumor cell line CMT-7364/S as the parental cell line, we successfully constructed a multidrug-resistant CMT-7364/R with high-dose drug pulse methods. Compared to its parental cell line, CMT-7364/R has decreased growth rate, overexpression of BCRP and increased migration and invasion ability due to EMT. The results of this study showed that CMT-7364/R might serve as a model for future studies on tumor drug resistance.

Dataset Information

Establishment and characterization of a canine sebaceous epithelial cell line derived from an eyelid mass.

Publications

Establishment and characterization of a canine sebaceous epithelial cell line derived from an eyelid mass.

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