Three-dimensional in vitro model of human preneoplastic breast disease
AssigneeWayne State University
InventorsMalathy P. V. Shekhar, Larry Tait
Life of this patent
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Abstract
A three-dimensional in vitro culture system comprising breast epithelial cells, endothelial cells and breast fibroblasts supports growth and functional differentiation of preneoplastic breast epithelial cells and endothelial cells and recapitulates estrogen induced in vivo effects on angiogenesis and proliferative potential of breast epithelial tissue. This model permits the generation of functional vascular networks and local proliferative ductal alveolar outgrowths with invasive potential. Both these processes are augmented by estrogen. This culture model is used to evaluate agents for their effects, whether stimulatory or inhibitory, on preneoplastic breast disease and its progression to cancer.
Claims33
A method for assaying a test agent for its activity in inhibiting molecular or cellular changes characteristic of preneoplastic breast disease in a three-dimensional in vitro culture system that serves as a model for preneoplastic breast disease, said method comprising:
- (a) providing said three-dimensional culture system, which comprise a co-culture of
- (i) preneoplastic breast epithelial cells,
- (ii) endothelial cells and
- (iii) breast fibroblasts on a reconstituted basement membrane in the presence of medium containing
- (1) effective concentrations of growth factors that act on said epithelial and endothelial cells, and
- (2) effective concentrations of an estrogen such that said cells undergo morphogenesis that results in the formation of a multicellular three dimensional network of branching ductal alveolar units in culture within about 3-7 days,
- (b) adding said test agent to said culture system for an interval sufficient for said agent to act upon said preneoplastic breast epithelial cells, said endothelial cells or said fibroblasts
- (c) to a parallel control culture, adding a negative control agent for said interval, which negative control agent does not prevent or inhibit said progression,
- (d) examining the cultures of (b) and (c), above, for
- i. formation of said branching ductal alveolar units by morphology;
- or
- ii. proliferation of cells;
- or
- iii. the presence in the culture medium of one or more secreted products of said cells
- wherein prevention or inhibition of said ductal alveolar unit formation, said proliferation or generation of said secreted products in cultures of (a) compared to (b), is indicative that said agent has said inhibitory activity.
A method for testing an agent for its activity as an endothelial cell-specific or epithelial cell-specific factor active in promoting ductal-alveolar morphogenesis, angiogenesis and progression of preneoplastic breast epithelial cells to a malignant phenotype, comprising:
- (a) providing a three-dimensional in vitro culture system that serves as a model for the development and progression of preneoplastic breast disease, said system comprising a co-culture of
- (i) preneoplastic breast epithelial cells,
- (ii) endothelial cells and
- (iii) breast fibroblasts on a reconstituted basement membrane in the presence of medium containing
- (1) effective concentrations of growth factors that act on said epithelial and endothelial cells, and,
- (2) effective concentrations of an estrogen such that said cells undergo morphogenesis that results in the formation of a multicellular three dimensional network of branching ductal alveolar units in culture within about 3-7 days.
- (b) adding said test agent to said culture system for an interval sufficient for said agent to act upon said preneoplastic breast epithelial cells or said endothelial cells;
- (c) to a parallel control culture, adding a negative control agent for said interval, which negative control agent does not promote ductal-alveolar morphogenesis, angiogenesis or progression of preneoplastic breast epithelial cell to a malignant phenotype;
- (d) examining the cultures of (b) and (c), above, for
- i. formation of said branching ductal alveolar units by morphology;
- or
- ii. cellular changes corresponding to angiogenesis;
- or
- iii. progression of preneoplastic breast epithelial cells to a malignant phenotype,
- wherein promoting of said ductal alveolar unit formation, said cellular changes corresponding to angiogenesis, or said progression in cultures of (a) compared to (b), is indicative that said agent has said activity.
Dependencies are inferred from the claim text.