Mammosphere Formation Assay Optimization in the Characterization of Cancer Stem Cells of the Primary Breast Tumor

Gizem Uslu; Zehra Seda Ünal Halbutoğulları; Gülçin Gacar; Nihat Zafer Utkan; Yusufhan Yazır

doi:10.31067/acusaglik.1225228

Araştırma Makalesi

BibTex

RIS

Kaynak Göster

Mammosphere Formation Assay Optimization in the Characterization of Cancer Stem Cells of the Primary Breast Tumor

Yıl 2023, Cilt: 14 Sayı: 2, 108 - 118, 17.03.2023

Gizem Uslu Zehra Seda Ünal Halbutoğulları Gülçin Gacar Nihat Zafer Utkan Yusufhan Yazır

https://doi.org/10.31067/acusaglik.1225228

Öz

Background/Purpose: Breast cancer is the most frequently diagnosed cancer and the leading cause of death from cancer among females worldwide. Breast cancer tumors that feature breast cancer stem cells (BCSCs) are known to cause drug resistance and metastasis. Culturing BCSCs from primary tumors as mammospheres is both difficult and costly. Therefore, the ability to form BCSC mammospheres in-vitro has become essential in assessing their characterization. Flow cytometric analysis of surface markers and measurement of aldehyde dehydrogenase (ALDH) activity are among other methods used to evaluate cancer cells' stem cell activity.
Methods:The research material consisted of BCSCs isolated from the tumor tissues collected from two patients with invasive ductal carcinoma breast tumors. Subsequently, several stem cell surface markers, i.e., cluster of differentiation 44 (CD44), CD24, and CD133, were analyzed using flow cytometry during the third passaging of the cells. ALDH assay is performed with negative control verapamil incubated cells. Two mammosphere forming methods, i.e., low attachment and agar-coated wells together with medium seeded in three different cell concentrations, were compared.
Results: CD44+, CD24- and CD133+antibody expressions showed that these cells could be tumor-initiating CSCs. ALDH assay results also indicated that these cells possessed stem cell features. In addition, the results of the mammosphere assay revealed that agar-coated wells at a concentration of 7000 cells/cm2 had more prominent floating features and viable spheres.
Conclusion: The findings of this study supported the hypothesis that agar-coated culture dishes in mammosphere culture would increase the mammosphere formation efficiency (MFE) value and revealed the importance of the number of cells in elucidating the nature of BCSCs.

Anahtar Kelimeler

Breast cancer, mammosphere formation assay, cancer stem cell

Destekleyen Kurum

Kocaeli University Scientific Research Projects Coordination Unit

Proje Numarası

2018/103, 2018/112

Kaynakça

Sung H, Ferlay J, Siegel RL, et al. Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. Ca-a Cancer Journal for Clinicians. 2021;71:209-49. DOI:10.3322/caac.21660
Ellis I, Cornelisse C, Schnitt S, et al. Invasive breast carcinomas. In: Tavassoli FA., Devilee P., eds. WHO Classification of Tumours. Pathology and Genetics of Tumours of the Breast and Female Genital Organs. 3 ed: IARC Press; 2003.
Li CI, Uribe DJ and Daling JR. Clinical characteristics of different histologic types of breast cancer. Brit J Cancer. 2005;93:1046-52. DOI:10.1038/sj.bjc.6602787
Chen XF, Zhang CY, Guo DJ, et al. Distant metastasis and prognostic factors in patients with invasive ductal carcinoma of the breast. Eur J Clin Invest. 2022;52. DOI:10.1111/eci.13704
Lawson JC, Blatch GL and Edkins AL. Cancer stem cells in breast cancer and metastasis. Breast Cancer Res Tr. 2009;118:241-54. DOI:10.1007/s10549-009-0524-9
Gwynne WD, Shakeel MS, Girgis-Gabardo A, et al. The Role of Serotonin in Breast Cancer Stem Cells. Molecules. 2021;26. DOI:10.3390/molecules26113171
Saha T and Lukong KE. Breast Cancer Stem-Like Cells in Drug Resistance: A Review of Mechanisms and Novel Therapeutic Strategies to Overcome Drug Resistance. Front Oncol. 2022;12. DOI:10.3389/fonc.2022.856974
Al-Hajj M, Wicha MS, Benito-Hernandez A, et al. Prospective identification of tumorigenic breast cancer cells. P Natl Acad Sci USA. 2003;100:3983-8. DOI:10.1073/pnas.0530291100
Crabtree JS and Miele L. Breast Cancer Stem Cells. Biomedicines. 2018;6:77. DOI:10.3390/biomedicines6030077
Ponta H, Sherman L and Herrlich PA. CD44: From adhesion molecules to signalling regulators. Nat Rev Mol Cell Bio. 2003;4:33-45. DOI:10.1038/nrm1004
Blick T, Hugo H, Widodo E, et al. Epithelial Mesenchymal Transition Traits in Human Breast Cancer Cell Lines Parallel the CD44(hi/)CD24(lo/-) Stem Cell Phenotype in Human Breast Cancer. J Mammary Gland Biol. 2010;15:235-52. DOI:10.1007/s10911-010-9175-z
Patrawala L, Calhoun T, Schneider-Broussard R, et al. Highly purified CD44(+) prostate cancer cells from xenograft human tumors are enriched in tumorigenic and metastatic progenitor cells. Oncogene. 2006;25:1696-708. DOI:10.1038/sj.onc.1209327
Hou WY, Kong LW, Hou ZP, et al. CD44 is a prognostic biomarker and correlated with immune infiltrates in gastric cancer. Bmc Med Genomics. 2022;15. DOI:10.1186/s12920-022-01383-w
Ciccone V, Terzuoli E, Donnini S, et al. Stemness marker ALDH1A1 promotes tumor angiogenesis via retinoic acid/HIF-1/VEGF signalling in MCF-7 breast cancer cells. J Exp Clin Canc Res. 2018;37. DOI:10.1186/s13046-018-0975-0
De Beca FF, Caetano P, Gerhard R, et al. Cancer stem cells markers CD44, CD24 and ALDH1 in breast cancer special histological types. J Clin Pathol. 2013;66:187-91. DOI:10.1136/jclinpath-2012-201169
Grimshaw MJ, Cooper L, Papazisis K, et al. Mammosphere culture of metastatic breast cancer cells enriches for tumorigenic breast cancer cells. Breast Cancer Res. 2008;10. DOI:10.1186/bcr2106
Shi AP, Fan ZM, Ma KW, et al. Isolation and. characterization of adult mammary stem cells from breast cancer-adjacent tissues. Oncol Lett. 2017;14:2894-902. DOI:10.3892/ol.2017.6485
Lombardo Y, De Giorgio A, Coombes CR, et al. Mammosphere Formation Assay from Human Breast Cancer Tissues and Cell Lines. Jove-J Vis Exp. 2015. DOI:10.3791/52671
Rodriguez-Torres M and Allan AL. Aldehyde dehydrogenase as a marker and functional mediator of metastasis in solid tumors. Clin Exp Metastas. 2016;33:97-113. DOI:10.1007/s10585-015-9755-9
Charafe-Jauffret E, Ginestier C, Iovino F, et al. Breast cancer cell lines contain functional cancer stem cells with metastatic capacity and a distinct molecular signature. Cancer Res. 2009;69:1302-13. DOI:10.1158/0008-5472.CAN-08-2741
Shao J, Fan W, Ma BA, et al. Breast cancer stem cells expressing different stem cell markers exhibit distinct biological characteristics. Mol Med Rep. 2016;14:4991-8. DOI:10.3892/mmr.2016.5899
Lorico A and Rappa G. Phenotypic Heterogeneity of Breast Cancer Stem Cells. J Oncol. 2011;2011. DOI:10.1155/2011/135039
Croker AK, Goodale D, Chu J, et al. High aldehyde dehydrogenase and expression of cancer stem cell markers selects for breast cancer cells with enhanced malignant and metastatic ability. J Cell Mol Med. 2009;13:2236-52. DOI:10.1111/j.1582-4934.2008.00455.x
Brugnoli F, Grassilli S, Al-Qassab Y, et al. CD133 in Breast Cancer Cells: More than a Stem Cell Marker. J Oncol. 2019;2019. DOI:10.1155/2019/7512632
Darvishi B, Boroumandieh S, Majidzadeh-A K, et al. The role of activated leukocyte cell adhesion molecule (ALCAM) in cancer progression, invasion, metastasis and recurrence: A novel cancer stem cell marker and tumor-specific prognostic marker. Exp Mol Pathol. 2020;115. DOI:10.1016/j.yexmp.2020.104443
Xiao M, Wang X, Yan M, et al. A systematic evaluation for the potential translation of CD166-related expression as a cancer biomarker. Expert Rev Mol Diagn. 2016;16:925-32. DOI:10.1080/14737159.2016.1211932
Lu H, Clauser KR, Tam WL, et al. A breast cancer stem cell niche supported by juxtacrine signalling from monocytes and macrophages. Nat Cell Biol. 2014;16:1105-17. DOI:10.1038/ncb3041
Zhang X, Powell K and Li L. Breast Cancer Stem Cells: Biomarkers, Identification and Isolation Methods, Regulating Mechanisms, Cellular Origin, and Beyond. Cancers (Basel). 2020;12. DOI:10.3390/cancers12123765
Sun H, Jia J, Wang X, et al. CD44+/CD24- breast cancer cells isolated from MCF-7 cultures exhibit enhanced angiogenic properties. Clin Transl Oncol. 2013;15:46-54. DOI:10.1007/s12094-012-0891-2
Wang R, Lv Q, Meng W, et al. Comparison of mammosphere formation from breast cancer cell lines and primary breast tumors. J Thorac Dis. 2014;6:829-37. DOI:10.3978/j.issn.2072-1439.2014.03.38