Korean Journal of Veterinary Research (대한수의학회지)

The Korean Society of Veterinary Science (대한수의학회)
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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
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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.

Keywords

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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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References

  1. Al-Hajj M, Wicha MS, Benito-Hernandez A, Morrison SJ, Clarke MF. Prospective identification of tumorigenic breast cancer cells. Proc Natl Acad Sci U S A 2003, 100, 3983-3988. https://doi.org/10.1073/pnas.0530291100
  2. Bao S, Wu Q, McLendon RE, Hao Y, Shi Q, Hjelmeland AB, Dewhirst MW, Bigner DD, Rich JN. Glioma stem cells promote radioresistance by preferential activation of the DNA damage response. Nature 2006, 444, 756-760. https://doi.org/10.1038/nature05236
  3. Bapat SA, Mali AM, Koppikar CB, Kurrey NK. Stem and progenitor-like cells contribute to the aggressive behavior of human epithelial ovarian cancer. Cancer Res 2005, 65, 3025-3029. https://doi.org/10.1158/0008-5472.CAN-04-3931
  4. Blacking TM, Waterfall M, Argyle DJ. CD44 is associated with proliferation, rather than a specific cancer stem cell population, in cultured canine cancer cells. Vet Immunol Immunopathol 2011, 141, 46-57. https://doi.org/10.1016/j.vetimm.2011.02.004
  5. Bonnet D, Dick JE. Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell. Nat Med 1997, 3, 730-737. https://doi.org/10.1038/nm0797-730
  6. Chen K, Huang Y, Chen J. Understanding and targeting cancer stem cells: therapeutic implications and challenges. Acta Pharmacol Sin 2013, 34, 732-740. https://doi.org/10.1038/aps.2013.27
  7. Cocola C, Anastasi P, Astigiano S, Piscitelli E, Pelucchi P, Vilardo L, Bertoli G, Beccaglia M, Veronesi MC, Sanzone S, Barbieri O, Reinbold RA, Luvoni GC, Zucchi I. Isolation of canine mammary cells with stem cell properties and tumourinitiating potential. Reprod Domest Anim 2009, 44 (Suppl 2), 214-217. https://doi.org/10.1111/j.1439-0531.2009.01413.x
  8. Dean M, Fojo T, Bates S. Tumour stem cells and drug resistance. Nat Rev Cancer 2005, 5, 275-284. https://doi.org/10.1038/nrc1590
  9. Dobson JM, Samuel S, Milstein H, Rogers K, Wood JLN. Canine neoplasia in the UK: estimates of incidence rates from a population of insured dogs. J Small Anim Pract 2002, 43, 240-246. https://doi.org/10.1111/j.1748-5827.2002.tb00066.x
  10. Dontu G, Abdallah WM, Foley JM, Jackson KW, Clarke MF, Kawamura MJ, Wicha MS. In vitro propagation and transcriptional profiling of human mammary stem/progenitor cells. Genes Dev 2003, 17, 1253-1270. https://doi.org/10.1101/gad.1061803
  11. Else RW, Norval M, Neill WA. The characteristics of a canine mammary carcinoma cell line, REM 134. Br J Cancer 1982, 46, 675-681. https://doi.org/10.1038/bjc.1982.254
  12. Fan X, Liu S, Su F, Pan Q, Lin T. Effective enrichment of prostate cancer stem cells from spheres in a suspension culture system. Urol Oncol 2012, 30, 314-318. https://doi.org/10.1016/j.urolonc.2010.03.019
  13. Geraldes M, Gartner F, Schmitt F. Immunohistochemical study of hormonal receptors and cell proliferation in normal canine mammary glands and spontaneous mammary tumors. Vet Rec 2000, 146, 403-406. https://doi.org/10.1136/vr.146.14.403
  14. Kol A, Arzi B, Athanasiou KA, Farmer DL, Nolta JA, Rebhun RB, Chen X, Griffiths LG, Verstraete FJM, Murphy CJ, Borjesson DL. Companion animals: translational scientist's new best friends. Sci Transl Med 2015, 7, 308ps21. https://doi.org/10.1126/scitranslmed.aaa9116
  15. Korsching E, Packeisen J, Liedtke C, Hungermann D, Wulfing P, van Diest PJ, Brandt B, Boecker W, Buerger H. The origin of vimentin expression in invasive breast cancer: epithelial-mesenchymal transition, myoepithelial histogenesis or histogenesis from progenitor cells with bilinear differentiation potential? J Pathol 2005, 206, 451-457. https://doi.org/10.1002/path.1797
  16. Lee CH, Yu CC, Wang BY, Chang WW. Tumorsphere as an effective in vitro platform for screening anti-cancer stem cell drugs. Oncotarget 2016, 7, 1215-1226.
  17. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the $2^{-{\Delta}{\Delta}Cr}$ method. Methods 2001, 25, 402-408. https://doi.org/10.1006/meth.2001.1262
  18. MacEwen EG. Spontaneous tumors in dogs and cats: models for the study of cancer biology and treatment. Cancer Metastasis Rev 1990, 9, 125-136. https://doi.org/10.1007/BF00046339
  19. Magalhaes GM, Terra EM, de Oliveira Vasconcelos R, de Barros Bandarra M, Moreira PRR, Rosolem MC, Alessi AC. Immunodetection of cells with a CD44+/CD24- phenotype in canine mammary neoplasms. BMC Vet Res 2013, 9, 205. https://doi.org/10.1186/1746-6148-9-205
  20. Michishita M, Akiyoshi R, Yoshimura H, Katsumoto T, Ichikawa H, Ohkusu-Tsukada K, Nakagawa T, Sasaki N, Takahashi K. Characterization of spheres derived from canine mammary gland adenocarcinoma cell lines. Res Vet Sci 2011, 91, 254-260. https://doi.org/10.1016/j.rvsc.2010.11.016
  21. Murai K, Nakagawa T, Endo Y, Kamida A, Yoshida K, Mochizuki M, Nishimura R, Sasaki N. Establishment of a pair of novel cloned tumour cell lines with or without metastatic potential from canine mammary adenocarcinoma. Res Vet Sci 2012, 93, 468-472. https://doi.org/10.1016/j.rvsc.2011.06.012
  22. O'Brien CA, Pollett A, Gallinger S, Dick JE. A human colon cancer cell capable of initiating tumour growth in immunodeficient mice. Nature 2007, 445, 106-110. https://doi.org/10.1038/nature05372
  23. Pang LY, Argyle DJ. Using naturally occurring tumours in dogs and cats to study telomerase and cancer stem cell biology. Biochim Biophys Acta 2009, 1792, 380-391. https://doi.org/10.1016/j.bbadis.2009.02.010
  24. Pang LY, Cervantes-Arias A, Else RW, Argyle DJ. Canine mammary cancer stem cells are radio- and chemo-resistant and exhibit an epithelial-mesenchymal transition phenotype. Cancers (Basel) 2011, 3, 1744-1762. https://doi.org/10.3390/cancers3021744
  25. Pinho SS, Carvalho S, Cabral J, Reis CA, Gartner F. Canine tumors: a spontaneous animal model of human carcinogenesis. Transl Res 2012, 159, 165-172. https://doi.org/10.1016/j.trsl.2011.11.005
  26. Reya T, Morrison SJ, Clarke MF, Weissman IL. Stem cells, cancer, and cancer stem cells. Nature 2001, 414, 105-111. https://doi.org/10.1038/35102167
  27. Ricci-Vitiani L, Lombardi DG, Pilozzi E, Biffoni M, Todaro M, Peschle C, De Maria R. Identification and expansion of human colon-cancer-initiating cells. Nature 2007, 445, 111-115. https://doi.org/10.1038/nature05384
  28. Rutteman GR, Foekens JA, Portengen H, Vos JH, Blankenstein MA, Teske E, Cornelisse CJ, Misdorp W. Expression of epidermal growth factor receptor (EGFR) in nonaffected and timorous mammary tissue of female dogs. Breast Cancer Res Treat 1994, 30, 139-146. https://doi.org/10.1007/BF00666057
  29. Schatton T, Murphy GF, Frank NY, Yamaura K, Waaga-Gasser AM, Gasser M, Zhan Q, Jordan S, Duncan LM, Weishaupt C, Fuhlbrigge RC, Kupper TS, Sayegh MH, Frank MH. Identification of cells initiating human melanomas. Nature 2008, 451, 345-349. https://doi.org/10.1038/nature06489
  30. Sorenmo K. Canine mammary gland tumors. Vet Clin North Am Small Anim Pract 2003, 33, 573-596. https://doi.org/10.1016/S0195-5616(03)00020-2
  31. Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 2006, 126, 663-676. https://doi.org/10.1016/j.cell.2006.07.024
  32. Timmermans-Sprang EPM, Gracanin A, Mol JA. Molecular signaling of progesterone, growth hormone, Wnt, and HER in mammary glands of dogs, rodents, and humans: new treatment target identification. Front Vet Sci 2017, 4, 53.
  33. Torres CG, Olivares A, Stoore C. Simvastatin exhibits antiproliferative effects on spheres derived from canine mammary carcinoma cells. Oncol Rep 2015, 33, 2235-2244. https://doi.org/10.3892/or.2015.3850
  34. Weiswald LB, Bellet D, Dangles-Marie V. Spherical cancer models in tumor biology. Neoplasia 2015, 17, 1-15. https://doi.org/10.1016/j.neo.2014.12.004
  35. Wilson H, Huelsmeyer M, Chun R, Young KM, Friedrichs K, Argyle DJ. Isolation and characterisation of cancer stem cells from canine osteosarcoma. Vet J 2008, 175, 69-75. https://doi.org/10.1016/j.tvjl.2007.07.025
  36. Yang ZF, Ho DW, Ng MN, Lau CK, Yu WC, Ngai P, Chu PWK, Lam CT, Poon RTP, Fan ST. Significance of CD90+ cancer stem cells in human liver cancer. Cancer Cell 2008, 13, 153-166. https://doi.org/10.1016/j.ccr.2008.01.013
  37. Zhu ZW, Chen L, Liu JX, Huang JW, Wu G, Zheng YF, Yao KT. A novel three-dimensional tumorsphere culture system for the efficient and low-cost enrichment of cancer stem cells with natural polymers. Exp Ther Med 2018, 15, 85-92.