Methods of monitoring angiogenesis and metastasis in three dimensional co-cultures
Inventors
Zudaire, Enrique • Cuttitta, Frank • Fang, Changge
Assignees
US Department of Health and Human Services
Publication Number
US-8679836-B2
Publication Date
2014-03-25
Expiration Date
2028-04-01
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Abstract
This disclosure relates to fluorescent cell lines and to the use of such cell lines in monitoring cellular activity, such as angiogenesis. This disclosure further relates to the use of such cell lines in a three-dimensional cell culture to monitor angiogenic and metastatic potential of tumor cells and selecting personalized therapeutics for treatment of cancer.
Core Innovation
This invention provides fluorescent mammalian cell lines that are stably transfected with mammalian expression vectors encoding fluorescent proteins, such as green, yellow, red, and cyan fluorescent proteins. These cell lines enable monitoring of cellular activities such as angiogenesis in vitro. The fluorescent proteins emit signals proportional to the cell number, allowing direct quantification of growth, migration, tubule formation, and cell death without additional staining reagents or chemicals that might interfere with the cellular processes under study.
The invention further discloses methods using these fluorescent cell lines in co-cultures, including three-dimensional (3D) co-cultures comprising layers of neutral polysaccharide polymer gels, solidified gel matrices with dispersed endothelial and tumor cells, and culture medium. These 3D co-cultures simulate in vivo environments to monitor angiogenic and metastatic potentials of tumor cells by detecting endothelial proliferation, tubule formation, and tumor cell angiotropism. This enables assessing patient tumor biopsy samples and screening for personalized anti-angiogenic or anti-metastatic treatments.
The problem addressed is the need for in vitro assays that can accurately model complex biological interactions underlying angiogenesis and metastasis, especially in cancer. Existing methods are limited in simultaneously monitoring multiple cell types, require staining or reagents that interfere with assays, and often do not correlate with in vivo tumor angiogenesis. Moreover, current single drug, non-personalized anti-angiogenesis therapies lack efficacy due to insufficient knowledge of multi-cell interactions and patient-specific angiogenic potential. This invention provides multiplex assays with fluorescent cell lines to overcome these limitations and offers a 3D co-culture model that better mimics in vivo tumor angiogenesis and metastasis for personalized therapeutic selection.
Claims Coverage
The patent includes one independent claim directed to a method for monitoring angiogenic or metastatic potential of tumor cells in a three-dimensional co-culture system, and additional independent claims directed to methods for testing efficacy and selecting personalized treatment using such co-cultures. The main inventive features involve the specific composition and layering of the 3D co-culture and the use of fluorescently labeled cells to detect functional cellular activities that correlate with in vivo tumor behaviors.
Three-dimensional co-culture system with defined layered composition
A 3D co-culture comprising three layers: a first layer of neutral polysaccharide polymer gel (e.g., agarose) contacting the culture dish bottom; a second layer of solidified gel matrix containing dispersed endothelial cells and tumor cells (either a single monoclonal tumor spheroid colony or a tumor biopsy sample); and a third layer comprising culture medium.
Monoclonal tumor spheroid colony production method
Producing monoclonal tumor spheroid colonies by culturing isolated tumor cells suspended in 0.2% agarose with specific supplements over a bottom layer of 1% agarose with FBS and antibiotics, incubating to grow spheroids, harvesting colonies, and resuspending in buffered saline with supplements.
Detection of angiogenesis and metastasis correlating with in vivo tumor behavior
Detecting endothelial cell proliferation, tubule formation, or tumor cell angiotropism in the second layer of the 3D co-culture, wherein these parameters correlate in a tumor-specific manner to in vivo angiogenesis and metastasis.
Use of fluorescent proteins in endothelial and tumor cells
Endothelial cells and tumor cells stably and constitutively express fluorescent proteins with distinguishable emission spectra to facilitate detection and differentiation of cell types in the co-culture.
Inclusion of additional mammalian cell types in the gel matrix
The second layer can include at least one additional mammalian cell type selected from macrophage, mast cell, fibroblast, adipocyte, and pericyte, which may also express fluorescent proteins with emission spectra distinguishable from other cells.
Incorporation of test agents in any layer
At least one test agent, including known or potential inhibitors or promoters of angiogenesis or metastasis, can be included in the first, second, or third layer of the co-culture to assess its effect on cellular activities.
Methods for testing efficacy of anti-angiogenic or anti-metastatic treatments
Using 3D co-cultures with tumor cells derived from the subject and test agents as candidate anticancer treatments to monitor angiogenic or metastatic potential and evaluate treatment efficacy.
Methods for selecting personalized cancer treatments
Preparing multiple 3D co-cultures with tumor and endothelial cells and various test agents, incubating, detecting endothelial proliferation, tubule formation or tumor cell angiotropism, and selecting test agents that decrease these activities relative to controls, thereby identifying personalized effective anti-angiogenic or anti-metastatic therapies.
The claims cover the innovative approach of using a layered 3D co-culture system incorporating fluorescently labeled endothelial and tumor cells for monitoring angiogenic and metastatic activity with correlation to in vivo tumor behavior. This system includes methods for growing monoclonal tumor spheroids, detecting functional cellular responses, including angiotropism, integrating additional stromal cell types, applying test agents within the culture layers, and using the system for evaluating treatment efficacy and selecting personalized therapies.
Stated Advantages
Enables real-time, direct monitoring of cell growth, migration, and tubule formation without staining or additional reagents, simplifying and shortening assay protocols.
Allows simultaneous assessment of multiple histologically different cell lines in co-culture through distinct fluorescent protein emissions.
Provides an in vitro assay that closely simulates in vivo angiogenic and metastatic tumor microenvironments, improving correlation with in vivo tumor behavior.
Facilitates personalized therapeutic screening by testing patient-derived tumor samples with candidate anti-angiogenic and anti-metastatic agents.
Reduces inter-experimental variability associated with staining protocols and enables live cell imaging for morphological analysis over time.
Allows multiplex screening of drug candidates for angiogenic modulation and cytotoxicity in high throughput formats.
Documented Applications
Monitoring cellular activities such as growth, migration, and tubule formation in vitro using fluorescent immortalized mammalian cell lines.
Assessing angiogenic and metastatic potential of tumor cells from cell lines, tumor spheroids, or patient biopsies in 3D co-cultures.
Screening and identifying modulators of angiogenesis and metastasis including inhibitors and stimulators using co-cultures.
Personalizing anti-angiogenic and anti-metastatic cancer therapies by testing patient tumor samples with various test agents in 3D co-cultures to select effective treatments.
High-throughput screening of chemical or biological libraries for compounds that modulate angiogenesis using fluorescent cell line assays.
Studying tumor cell angiotropism as a model for metastasis in 3D in vitro co-cultures.
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