NITROGEN INDEPENDENCE IDENTIFIES A HIGHLY MALIGNANT POPULATION OF TUMOR STEM CELLS
The present invention is directed to a method for isolating and establishing Growth Factor-Independent (GF-I) Tumor Stem Cells (TSCs) from tumor biopsies or tumor cell lines consisting in culturing cells in serum-free mitogen-free culture medium. The method discloses cell growth in a culture medium, which does neither comprise serum, nor EGF (Epidermal Growth factor) and FGF-2 (Fibroblast Growth Factor), nor both, nor EGF or FGF-2 derivatives with the same mitogenic characteristics of the parent molecules. According to a preferred embodiment, the method is directed to the isolation of Tumor stem cells (TSCs) from glioblastoma multiforme (GBM) or from other brain tumors or brain tumor cell lines. GF-Independent TSCs can be identified and expanded in vitro providing a homogeneous population of multipotent, self-renewing and highly tumorigenic Growth Factor-Independent TSCs, distinguishable from tumor stem cells derived with other methods, grown in parallel, for the above characteristics. The invention also encompasses therapeutic methods based on Tumor Stem Cells isolated as described.
1 . A method for isolating and establishing Growth Factor-Independent (GF-I) Tumor Stem Cells (TSCs) from a tumor biopsy or a tumor cell line consisting essentially in culturing isolated cells in serum-free mitogen-free culture medium.
2 . The method according to claim 1 wherein said culture medium does neither comprise EGF (Epidermal Growth factor) nor FGF-2 (Fibroblast Growth Factor), nor EGF or FGF-2 derivatives.
3 . The method according to claim 2 wherein said culturing is carried out for at least 10 cell passages in said serum free mitogen free culture medium.
4 . The method according to claim 3 wherein said culture medium is a mix of DMEM and F12, further comprising at least: a buffer system, an osmotic regulator, a hormone mixture, a protein carrier or
5 .- 7 . (canceled)
8 . The method according to claim 3 further comprising culturing cells in growth-factor independent conditions for at least 10 culturing passages.
9 . The method according to claim 5 further comprising culturing isolated cells from a tumor biopsy or from a tumor cell line, in parallel, in growth-factor dependent (GF-D) conditions, obtaining proliferation curves from both dependent and independent growth factor conditions for at least 10 culturing passages, comparing said curves, wherein a slower proliferation curve in GF-I is indicative that “bona fide” GF-I TSC have been isolated.
10 .- 12 . (canceled)
13 . The method according to claim 1 wherein said tumor or tumor cell line is human.
14 . The method according to claim 13 wherein said tumor or tumor cell line is from brain, colon, breast and/or pancreas.
15 . The method according to claim 14 wherein said brain tumor is a human anaplastic ganglioglioma (AGG), a glioblastoma multiforme (GBM) or a medulloblastoma.
16 . (canceled)
17 . The method according to claim 8 further comprising sorting and/or selecting for a EGF-R positive cell population either before or after growth in serum-free or mitogen-free cell culture medium.
18 . The method according to claim 1 further comprising a step of transformation with heterologous DNA sequences.
19 . A Growth Factor Independent Tumor Stem Cell (GF-I TSC), prepared according to the method of claim 8 .
20 . A Growth Factor Independent Glioblastoma Tumor Stem Cell (GF-I GBM TSC) obtainable according to the method of claim 9 and characterized by the following surface markers immunologic profile: EGF-R pos (positivity) AC133 neg (negativity) Wnt5a pos (positivity).
21 . A method for screening compounds with anti-tumorigenic activity comprising the method for isolating and establishing Growth Factor-Independent (GF-I) Tumor Stern Cells (TSCs) from a tumor biopsy or a tumor cell line according to claim 1 and further comprising measuring at least one among a cell response selected from the group consisting of a) short-term self-renewing capacity, b) long-term self-renewing capacity, c) proliferation, d) differentiation, e) migration and/or invasion ability of TSCs, f) morphological modifications, and g) gene expression profile, and comparing said cell response to the response of untreated cells or of the same cells grown in Growth Factor Dependent conditions.
22 .- 39 . (canceled)
40 . The method according to claim 8 further comprising adding suitable excipients and/or diluents for preparing an immunogenic composition comprising TSCs.