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Updated: May 11, 2026
Mammosphere Assay Reveals Api5-Induced Stemness in Non-Tumorigenic Breast Epithelial Cell Lines
Published on: February 24, 2026
Assessment of potential anti-cancer stem cell activity of marine algal compounds using an in vitro mammosphere assay
Jo-Anne de la Mare1, Jason N Sterrenberg, Mugdha G Sukhthankar
1The Biomedical Biotechnology Research Unit (BioBRU), Department of Biochemistry, Microbiology and Biotechnology, Rhodes University, P, O, Box 94, Grahamstown, 6140, South Africa. a.edkins@ru.ac.za.
Background:
The cancer stem cell (CSC) theory proposes that tumours arise from and are sustained by a subpopulation of cells with both cancer and stem cell properties. One of the key hallmarks of CSCs is the ability to grow anchorage-independently under serum-free culture conditions resulting in the formation of tumourspheres. It has further been reported that these cells are resistant to traditional chemotherapeutic agents.
Methods:
In this study, the tumoursphere assay was validated in MCF-7 cells and used to screen novel marine algal compounds for potential anti-cancer stem cell (CSC) activity in vitro.
Results:
MCF-7 breast cancer cells were observed to generate tumourspheres or mammospheres after 3-5 days growth in anchorage-independent conditions and an apparent enrichment in potential CSCs was observed by an increase in the proportion of CD44high/CD24low marker-bearing cells and Oct4 expression compared to those in the bulk population grown in regular adherent conditions. Using this assay, a set of algal metabolites was screened for the ability to inhibit mammosphere development as a measure of potential anti-CSC activity. We report that the polyhalogenated monoterpene stereoisomers RU017 and RU018 isolated from the red alga Plocamium cornutum, both of which displayed no cytotoxicity against either adherent MCF-7 breast cancer or MCF-12A non-transformed breast epithelial cells, were able to prevent MCF-7 mammosphere formation in vitro. On the other hand, neither the brown algal carotenoid fucoxanthin nor the chemotherapeutic paclitaxel, both of which were toxic to adherent MCF-7 and MCF-12A cells, were able to inhibit mammosphere formation. In fact, pre-treatment with paclitaxel appeared to enhance mammosphere formation and development, a finding which is consistent with the reported resistance of CSCs to traditional chemotherapeutic agents.
Conclusion:
Due to the proposed clinical significance of CSC in terms of tumour initiation and metastasis, the identification of agents able to inhibit this subpopulation has clinical significance.
