Flow Cytometric Detection of Newly-formed Breast Cancer Stem Cell-like Cells After Apoptosis Reversal
| Autor: | Hon-Ming Lam, Chun So, Yiyue Xu, Suk Ying Tsang, Ming Chiu Fung |
|---|---|
| Rok vydání: | 2019 |
| Předmět: |
0301 basic medicine
General Chemical Engineering Cell Apoptosis Breast Neoplasms General Biochemistry Genetics and Molecular Biology Flow cytometry 03 medical and health sciences 0302 clinical medicine Breast cancer Cancer stem cell medicine Tumor Cells Cultured Humans Caspase General Immunology and Microbiology biology medicine.diagnostic_test Chemistry General Neuroscience Cell sorting medicine.disease Flow Cytometry 030104 developmental biology medicine.anatomical_structure 030220 oncology & carcinogenesis Caspases Cancer cell biology.protein Cancer research Neoplastic Stem Cells Female |
| Zdroj: | Journal of visualized experiments : JoVE. (143) |
| ISSN: | 1940-087X |
| Popis: | Cancer recurrence has long been studied by oncologists while the underlying mechanisms remain unclear. Recently, we and others found that a phenomenon named apoptosis reversal leads to increased tumorigenicity in various cell models under different stimuli. Previous studies have been focused on tracking this process in vitro and in vivo; however, the isolation of real reversed cells has yet to be achieved, which limits our understanding on the consequences of apoptosis reversal. Here, we take advantage of a Caspase-3/7 Green Detection dye to label cells with activated caspases after apoptotic induction. Cells with positive signals are further sorted out by fluorescence-activated cell sorting (FACS) for recovery. Morphological examination under confocal microscopy helps confirm the apoptotic status before FACS. An increase in tumorigenicity can often be attributed to the elevation in the percentage of cancer stem cell (CSC)-like cells. Also, given the heterogeneity of breast cancer, identifying the origin of these CSC-like cells would be critical to cancer treatment. Thus, we prepare breast non-stem cancer cells before triggering apoptosis, isolating caspase-activated cells and performing the apoptosis reversal procedure. Flow cytometry analysis reveals that breast CSC-like cells re-appear in the reversed group, indicating breast CSC-like cells are transited from breast non-stem cancer cells during apoptosis reversal. In summary, this protocol includes the isolation of apoptotic breast cancer cells and detection of changes in CSC percentage in reversed cells by flow cytometry. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|