Establishment and characterization of a multi-drug resistant cell line for canine mammary tumors

Abstract

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.

Keywords: Breast Cancer Resistance Protein (BCRP or ABCG2); canine mammary tumor (CMT); chemotherapy; mRNA-sequencing (mRNA-seq); multidrug resistance (MDR).

Figure 1

Sensitivity of drug-resistant cell lines and parental cell line to doxorubicin. Relative cell viability was determined by CCK8 assay. The cells were treated with doxorubicin for 48h. Cells treated with vehicle serve as a blank control. All experiments were conducted in triplicate and data were expressed as the mean ± SD (n = 3).

Figure 2

Comparison of cell morphology of drug-resistant cell lines and their parent cell lines under optical microscopy. Notice that after continuous high-dose drug pulse, the cell boundaries of CMT-7364/R are not clear and the refractivity is weak. The scale bar in the figure is 10 μm.

Figure 3

Growth curves and population doubling times of drug-resistant cell lines and their parent cell lines.The growth curve was measured at 24-h intervals for 7 consecutive days. Population doubling time (PDT) of CMT-7364/R was 40.17 ± 0.3700 h, while the PDT of its parental cell line was 32.23 ± 0.2773 h. Data were presented as the mean ± SD of three independent experiments. ****p < 0.0001.

Figure 4

Distribution of cell cycles of drug-resistant cell lines and their parent cell lines under the action of doxorubicin at different concentrations. (A) Cell cycle distribution was analyzed by flow cytometry after 24 h treatment with 5 or 10 μM doxorubicin. (B) Statistical analysis of the cell cycle distribution showed that CMT-7364/R is more resistant to doxorubicin-induced cell cycle block. Data were presented as the mean ± SD of three independent experiments. ^**p < 0.01, ^****p < 0.0001.

Figure 5

Uptake of doxorubicin by drug-resistant cell lines and their parent cell lines. Both type of cells were treated with 5 μM DOX for 12 h. Subsequently, observations were made using a confocal microscope. The red fluorescence received by the mCherry channel was emitted by the excitation of doxorubicin by a 587 nm laser.

Figure 6

The IC₅₀ of various clinically commonly used chemotherapy drugs for drug-resistant cell lines and their parent cell lines. DOX, doxorubicin; 5-Fu, 5-fluorouracil; PTX, Paclitaxel; DDP, Cisplatin. Data were presented as the mean ± SD of three independent experiments. ****p < 0.0001.

Figure 7

Expression level of P-gp and BCRP in drug-resistant cell lines and their parent cell lines. (A) Western Blot and semi-quantitative analysis. (B) RT-qPCR. The results showed that the transcript and protein expression levels of BCRP were significantly higher in CMT-7364/R compared to CMT-7364/S. GAPDH was used as an internal control. Data were presented as the mean ± SD of three independent experiments. ****p < 0.0001.

Figure 8

Immunofluorescence images of P-gp and BCRP in drug-resistant cell lines and parental cell lines. The green fluorescence shown by FITC is the target protein, and the blue fluorescence shown by DAPI is used for localization to the nucleus. S, CMT-7364/S; R, CMT-7364/R. The protein expression of BCRP was significantly elevated in drug-resistant cell lines compared to parental cell lines.

Figure 9

Migration of drug-resistant cell lines and their parental cell lines and percentage of scratch healing area. After 24 h and 48 h, the healing area of the scratches were measured, and after 48 h, the percentage of healed scratches in the CMT-7364/R group was 82.30 ± 1.054, while that in the CMT-7364/S group was 73.30 ± 1.600. Data were presented as the mean ± SD of three independent experiments. **p < 0.01.

Figure 10

Number of cells in which drug-resistant cell lines and their parent cell lines cross the stromal membrane. The pore size of the Transwell chamber used in this experiment is 8.0 μm. After 24 h, the number of cells invading the lower chamber was 700 ± 54 and 426 ± 38 cells in the CMT-7364/R and parental cell line groups, respectively. Data were presented as the mean ± SD of three independent experiments. *p < 0.05.

Figure 11

Expression of EMT-related proteins in drug-resistant cell lines and their parent cell lines. The results showed that the expression level of E-cadherin was reduced in the resistant cell line compared to the parental cell line, while the expression levels of MUC1-N and vimentin were increased, consistent with the characteristics of EMT. GAPDH was used as an internal control. Data were presented as the mean ± SD of three independent experiments. ****p < 0.0001, “ns” stands for not significant.

Figure 12

Comparison of transcriptome expression profiles of drug-resistant cell lines and parental cell lines. (A) GO analysis shows that biological functions related to cell adhesion capacity are elevated in drug-resistant cell lines compared to parental cell lines. (B) KEGG analysis showed that the MAPK pathway was activated in drug-resistant cells compared to parental cells.

Figure 13

Mouse xenograft models. (A) The appearance of masses on the 10th day after inoculation of tumor cells. Notice that the mass formed by CMT-7364/S shows limited growth, clear boundaries and intact epidermis while the tumor formed by CMT-7364/R has a variable appearance and often ulcers. (B) The appearance of masses at the end of the study. (C) Volume growth curve of masses formed by drug-resistant cell lines and their parent cell lines. (D) The final volume of the masses. (E) The histopathological image of CMT-7364/S with scale bar of 50 μm. (F) The histopathological image of CMT-7364/R with scale bar of 50 μm. Data were presented as the mean ± SD of three independent experiments. “ns” stands for not significant.