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Tumorsphere formation and cancer stem cell characterization of REM134 canine mammary carcinoma cells

개 REM134 유선종양세포의 sphere 형성을 통한 암 줄기세포 특성 분석

  • Byeon, Jeong Su (Viral Disease Research Division, Animal and Plant Quarantine Agency) ;
  • Lee, Jienny (Viral Disease Research Division, Animal and Plant Quarantine Agency) ;
  • Jeong, Da-Un (Viral Disease Research Division, Animal and Plant Quarantine Agency) ;
  • Gu, Na-Yeon (Viral Disease Research Division, Animal and Plant Quarantine Agency) ;
  • Cho, In-Soo (Viral Disease Research Division, Animal and Plant Quarantine Agency) ;
  • Cha, Sang-Ho (Viral Disease Research Division, Animal and Plant Quarantine Agency)
  • 변정수 (농림축산검역본부 바이러스질병과) ;
  • 이지현 (농림축산검역본부 바이러스질병과) ;
  • 정다운 (농림축산검역본부 바이러스질병과) ;
  • 구나연 (농림축산검역본부 바이러스질병과) ;
  • 조인수 (농림축산검역본부 바이러스질병과) ;
  • 차상호 (농림축산검역본부 바이러스질병과)
  • Received : 2018.07.03
  • Accepted : 2018.09.27
  • Published : 2018.12.31

Abstract

Canine mammary tumors are among the most frequently observed cutaneous tumors in female dogs. Cancer stem cells (CSCs), referred to as tumor-initiating cells, are thought to have properties similar to normal stem cells such as the ability to self-renewal and to differentiate into various cell types. Biological understanding of CSCs and the critical pathways involved in their maintenance are important in research and therapy for mammary tumors. We conducted the present study on sphere formation from REM134 cells by using methylcellulose to produce tumorspheres on a large scale and compared the specific markers of the spheres-formed and plating-cultured REM134 cells. The results revealed that the tumorspheres cultured in methylcellulose had higher seeding density and improved morphology compared to those produced in normal sphere formation medium. Expression levels of stemness markers and CSC-related markers were higher in tumorsphere-forming cells than in plating-cultured cells. Subsequently, we transplanted the tumorsphere-forming and plating-cultured cells into female nude mice to examine their tumorigenic potential. Tumor volume increased rapidly in mice transplanted with tumorsphere-derived cells compared to plating-cultured cells. We observed a novel sphere-forming condition for REM134 cells and showed that REM134 cell tumorspheres can exhibit improved CSC properties.

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Fig. 2. Sphere-forming capacity of REM134 cells. Spheres formed from REM134 cells plated at a density of 2 × 104 cells/well in six well ultra-low attachment plates for different tumorsphere formation mediums: (A) condition 1, (B) condition 2, and (C) condition 3 for 7 days. (D) Representative images and (E) histogram showing the sphere forming capacity of spheres from REM134 cells in condition 2 medium with methylcellulose (Sphere Me) or without methylcellulose (Sphere). Values are mean ± SD of three independent experiments (**p < 0.001). Scale bars = 100 μm (A-D).

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Fig. 1. The morphology of REM134 cells. REM134 cells show an epithelial-like phenotype at the following magnifications (A) 100× and (B) 200×. Scale bars = 100 μm.

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Fig. 3. Flow cytometry analysis of the expression of CD24, CD44, and CD133 in REM134 cells. (A) Expression of the CD44+/CD24− phenotype in plating-cultured and (B) sphereforming cells of REM134 cells. (C) Expression of CD133 in plating-cultured and (D) sphere-forming cells of REM134 cells compared to an isotype control. Values are mean ± SD of three independent experiments.

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Fig. 5. Expression of stem cell-related markers. (A) Expression of surface molecules (CD34, CD44, and CD90), (B) pluripotency markers (Oct4, Sox2, and Nanog), and (C) cancer stem cell markers (CD133, Bmi-1, EGFR, HER-2, PR, and vimentin) in platingcultured and sphere-forming cells from REM134 cells with methylcellulose (Sphere Me) or without methylcellulose (Sphere) by qRT-PCR analysis. β-actin was used as a housekeeping gene. Values are means ± SD of three individual experiments. *p < 0.05, **p < 0.001, compared with the plating-cultured cells.

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Fig. 4. Drug resistance assay of plating-cultured and sphereforming cells from REM134 cells. Cell viability assays in the presence of doxorubicin. Sphere-forming (white dots) and plating-cultured cells (black dots) were treated with different concentrations of doxorubicin for 48 h. Values are means ± SD of three individual experiments. **p < 0.001, compared with the plating-cultured cells.

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Fig. 6. Tumorigenic capacity of REM134 cells. (A) 1 × 105 cells of singly suspended REM134 cells from plating-cultured or sphere-forming cells into injected nude mice. Tumor formation was observed for six weeks after injection. Significant differences were detected within four weeks in sphereforming cells injected mice compared to that in platingcultured cells. Values are means ± SD of three mice. *p < 0.05, compared with the adherent cells. (B) Macroscopic appearances of dissected tumors from plating-cultured cells on the left and sphere-forming cells on the right at six weeks. Scale bars = 12 mm (left), 15 mm (right).

Table 1. Primer sequences

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Acknowledgement

Supported by : 농림축산검역본부

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