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Updated: Aug 2, 2025
The Use of Mouse Mammary Tumor Cells in an In Vitro Invasion Assay as a Measure of Oncogenic Cell Behavior
Published on: June 12, 2019
Establishment and characterization of a multi-drug resistant cell line for canine mammary tumors
Chaoyu Zhou1, Zixiang Lin1, Xinqiu Li1
1Department of Veterinary Clinical Science, College of Veterinary Medicine, China Agricultural University, Beijing, China.
Background And Purpose:
Canine 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 Methods:
In 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.
Results:
After 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.
Conclusion:
In 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.
Insights
Researchers developed a multidrug-resistant canine mammary tumor cell line (CMT-7364/R) to study chemotherapy resistance. This new model exhibits increased drug resistance, BCRP overexpression, and enhanced migration, aiding future research into overcoming tumor resistance.
Area of Science:
- Veterinary Oncology
- Cancer Biology
- Drug Resistance Mechanisms
Background:
- Canine mammary tumors are common in female dogs, with adjuvant chemotherapy often failing due to multi-drug resistance.
- The mechanisms driving tumor multi-drug resistance in dogs are not well understood, hindering the development of effective treatments.
- There is an urgent need for reliable canine mammary tumor multi-drug resistance models for research.
Purpose of the Study:
- To construct a multidrug-resistant canine mammary tumor cell line for research purposes.
- To investigate the mechanisms underlying multidrug resistance in canine mammary tumors.
- To provide a model for exploring strategies to overcome tumor resistance.
Main Methods:
- Induced multidrug resistance in the CMT-7364 canine triple-negative breast cancer cell line using doxorubicin and a high-dose drug pulse method.
- Verified drug resistance and expression of drug transport pumps via CCK8 assay, immunoblotting, qPCR, and immunofluorescence.
- Assessed cell migration and invasion using scratch and Transwell assays, and analyzed epithelial-mesenchymal transition (EMT) markers.
- Performed RNA-sequencing to compare transcriptomes and constructed mouse xenograft models to evaluate tumorigenic potential.
Main Results:
- The developed drug-resistant cell line (CMT-7364/R) showed mesenchymal-like morphology and resistance to doxorubicin and other chemotherapeutics after continuous passaging.
- CMT-7364/R cells exhibited significantly enhanced migration and invasion abilities, with decreased E-cadherin and increased vimentin and mucin 1-N terminus expression, indicative of EMT.
- BCRP (breast cancer resistance protein) was overexpressed at both transcriptional and protein levels in CMT-7364/R cells, while P-glycoprotein levels were not significantly altered.
- Mouse xenograft models showed no significant difference in tumor volume at 21 days between parental and drug-resistant cell lines.
Conclusions:
- Successfully established a multidrug-resistant canine mammary tumor cell line (CMT-7364/R) using a high-dose drug pulse method.
- The CMT-7364/R model displays decreased growth rate, BCRP overexpression, and increased migration/invasion due to EMT, compared to the parental cell line.
- This CMT-7364/R cell line serves as a valuable model for future research into canine tumor drug resistance mechanisms and therapeutic strategies.
