Question

Biology of Stem Cells:

You injected 1000 Ovarian cancer stem cells into a SCID mouse and a tumor forms. How do you determine tumorigenic potential of the cancer stem cells

Answer 1

The tumorigenic potential of any cells is its ability to form tumors in immunocompromised host. Tumorigenic potential of cancer stem cells can be studied by xenotransplantation of subpopulations of cancer cells into flanks of highly immunosuppressive animals such as NOD/SCID mice. Cancer stem cells have the potential to form all kinds of cells, cells that are tumorigenic and cells that are not. Cancer stem cells phenotypes of self-renewal and pluripotency are acquired by the activation of oncogenic genes or the inactivation of tumor suppressor genes. These cells express CD44, CD117, CD133, CD24 and aldehyde dehydrogenase-1 A1 (ALDH1 or ALDH1A1) markers or a combination of them in ovarian tumors. Several pathways such as Notch, Wnt, Hedgehog, and transforming growth factor-β (TGF-β) regulate self-renewal and maintain ovarian stem cell populations.

A footpad syngeneic melanoma model is normally established to study tumorigenic potential. Briefly, a 10-15, 6-week-old mice are anesthetized. The left hind limb footpad is sterilized with alcohol. The left footpad is injected with 0.05 ml cell suspension containing 1000 ovarian stem cells within 2-minute period. Mice are allowed to be awake and the footpad monitored for tumor development and signs of pain and ulceration twice a week. The control group includes 10-15 mice injected with PBS. The experiment is repeated thrice. The tumors development is assessed and the numbers of mice exhibiting the tumors are counted over a period of atleast a month. The size and weight of the tumor is measured over this period. A graph of mean tumor size vs day will give the tumor kinetics. The mice are sacrificed and the tissue fixed for staining. Haematoxylin and Eosin staining is performed to study the tumor histology, if formed. Immunostaining with proliferation markers such as Ki67 or PCNA and melanoma markers such as Melan-A are performed to analyze the tumor potential. All stem cells that could form the tumor will be positive for these markers. If there is induction of tumors indicates that the cancer stem cells are tumorigenic and can induce tumor formation. The cells can be isolated from the tumors and immunostained for the different markers of ovarian cancer stem cells. If cells exhibiting such markers are present, then the cancer stem cells had self-renewal potential. Expression of markers of different lineages, indicates that they had pluripotency. Pluripotency is ability to differentiate into other cancerous cell types.

Another method to detect pluripotency is by injection of cells into kidney capsules. The pure cell suspension can be injected under mouse kidney capsules of SCID mouse and monitored for tumor formation. If a tumor develops, then the ovarian cells definitely had tumorigenic potential.