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Updated: Sep 2, 2026
Isolation and Functional Assessment of Human Breast Cancer Stem Cells from Cell and Tissue Samples
Published on: October 2, 2020
Midkine and CA9 Are Potential Cancer Stem Cell Biomarkers in Triple-Negative Breast Tumours
Jorge Choque1, Aparicio Aguilar1,2, Javier Enciso-Benavides3
1Regenerative Medicine Group, Scientific University of the South, Lima, Peru.
Introduction:
There is no specific therapy for triple-negative breast cancer (TNBC), and the recurrence rate is high. Cancer stem cells (CSCs) play an important role in cancer chemoresistance and metastasis, but there is no consensus marker in this type of cancer. The aim of this study was to identify CSC biomarkers in the plasma secretome of TNBC patients.
Methods:
CD44+/CD24- CSCs were isolated from MDA-MB-436 and MDA-MB-231 (ATCC) lines by magnetic immunoselection. The extracellular vesicles (EVs) of CSCs were isolated by size exclusion chromatography (SEC) of conditioned medium (CM) without fetal bovine serum (FBS) and blood plasma from TNBC and healthy women. Tetraspanins CD9 and CD81 identified the EVs, while electron microscopy determined the morphology and tunable resistive pulse sensing (TRPS) established particle size. Luminex technology was used to determine the presence of L1 cell adhesion molecule (L1CAM), carbonic anhydrase IX (CA9), mesothelin, midkine, hepsin, kallikrein-6 (KLK6), transglutaminase 2 (TGM2), aldehyde dehydrogenase 1a1 (ALDH1A1), epithelial cell adhesion molecule (EpCAM), and differentiation cluster 44 (CD44).
Results:
Particles of 0-200 nm in size were more frequent in CSC-MDA-MB-436, while particles of 201-500 nm were more frequent in CSC-MDA-MB-231. In blood plasma, the particle size was 150-250 nm in TNBC patients and 150-300 nm in healthy individuals. Particle concentrations were 1.3 × 1010 in CSCs of the MDA-MB-231 (CSC-MDA-MB-231) cell line and 3.8 × 107 in the MDA-MB-231 cell line (L-MDA-MB-231). The concentration of CSCs in MDA-MB-436 was 2.7 × 108 compared to 9.4 × 107 in L-MDA-MB-436. Midkine and CA9 were present in the EVs of both CSC lines and in the plasma of women with TNBC but absent in the blood plasma of healthy women. Midkine showed better statistical discrimination.
Conclusions:
The size and concentration of EVs are heterogeneous. Midkine and CA9 are produced by CSCs in women with TNBC and may be possible biomarkers of these cells in this tumour type.
