Grantee Research Project Results
Final Report: 3D Breast Tissue Co-Cultures for Screening Mammary Carcinogens
EPA Contract Number: EPD13034Title: 3D Breast Tissue Co-Cultures for Screening Mammary Carcinogens
Investigators: Applegate, Dawn R.
Small Business: RegeneMed, Inc.
EPA Contact: Richards, April
Phase: I
Project Period: May 15, 2013 through November 14, 2013
Project Amount: $80,000
RFA: Small Business Innovation Research (SBIR) - Phase I (2013) RFA Text | Recipients Lists
Research Category: Small Business Innovation Research (SBIR) , SBIR - Innovation in Manufacturing
Description:
Breast cancer is not a disease of individual cells, but principally a failure of cells and tissues to communicate properly. Malignant transformation of breast cells is thought to be a complex multistage process, involving not only immortalization and de-regulated growth, but also modified interactions with surrounding stromal components and systemic factors. While good progress has been made in understanding a number of fundamental mechanisms of cancer progression, a comprehensive pathologically relevant model for the oncogenic transformation of normal human breast cells is still lacking. Rodent models and immortalized cell lines derived from human mammary epithelial cells (HMECs) grown in conventional “two-dimensional” (2D) adherent cultures have yielded a wealth of important information about general pathways of preneoplastic change, but they have failed to recapitulate the cell- and tissue-specific aspects of breast cancer as it generally occurs in patients.
Optimally, understanding the roles that the differentiation, microenvironment and hormonal milieu play in determining the relative susceptibility of human breast cells to malignant transformation, and determining the favored routes of in situ transformation, requires the use of more sophisticated, physiologically relevant in vitro systems. For conventional 2D and single-cell cultures, lack of a complete microenvironment with the cellular milieu, cell-cell and cell-extracellular matrix (ECM) interactions found in vivo may limit the replication of molecular pathways and cell signaling required to accurately model breast cancer development. To meet this need, a physiologically relevant, three-dimensional, human breast tissue co-culture model (3DBC) was developed. The 3DBC was established from all cell types isolated from normal human breast tissue grown in a microporous scaffold that induced the cells to express the physiologic milieu of extracellular matrix (ECM) proteins and growth factors, creating a tissue architecture that sustained organotypic function in vitro. The 3DBC was used to study normal breast tissue function and malignant transformation upon treatment with mammary gland carcinogens.
One communication mechanism that is frequently disrupted in breast cancer involves the hormone estrogen. Despite recognition that exposure to compounds with estrogenic activity can promote breast cancer formation and progression, no good mechanistic explanation presently exists for how this actually happens. Previously, inability to stably culture endocrine receptor positive (ERα[+]) cells from normal human breast impeded mechanistic study of the acquisition of proliferative potential by such cells. The physiologic relevance of the 3DBC was assessed, including maintenance of ERα(+) luminal epithelial cells (LEC). The 3DBC was treated with prototypical mammary gland carcinogens to demonstrate increased co-expression of ERα and proliferative markers such as Ki67 indicating disordered control of cell division, which may represent an important early pathogenic step in the development of breast cancer. This new experimentally manipulable system may enable quick and efficient determination of both genetic and environmental factors that cause deregulated co-expression of ERα and proliferative markers in human breast cells. In this study, the 3DBC were used to assess whether known carcinogens can cause the emergence of proliferative ERα(+) breast cells, and to understand the mechanisms responsible for this preneoplastic change. Knowledge of the mechanisms involved would enable the design of better carcinogen screening methods and prevention measures.
Summary/Accomplishments (Outputs/Outcomes):
Human 3DBC were successfully grown from surgically discarded normal reduction mammoplasty specimens (gram quantities) from anonymous donors. The specimens were mechanical dissociated to yield epithelial organoids and surround stromal cells. Stromal cells were limitedly expanded, with maintenance of cell phenotypes. Parenchyma (organoids) was verified to retain estrogen receptor alpha (ERa) function following cryopreservation. All cell types were grown in RegeneMed’s proprietary 3D co-culture system by a process that induces the cells to express the physiologic milieu of extracellular matrix proteins (ECM) and growth factors to form tissue architecture that sustained organotypic function for the 28-day culture duration (maximum attempted in this short 6-month study).
Maintenance of organotypic function was demonstrated through sustained cell viability and cell and ECM specific markers. Sustained high cell viability was confirmed using an Adenylate Kinase enzyme leakage assay. Luminal epithelial cell (LEC) markers (lactoferrin, Keratin 19 (K19) and ERα) assessed via immunofluorescence and gene expression verified maintenance of ERα(+) LEC through the 28-day culture duration. Persistence of stromal cells and their expression of extracellular matrix proteins, induced by the 3D culture method, were confirmed microscopically.
The 3D breast co-cultures (3DBC) were treated with pharmacologically relevant chronic doses of mammary gland carcinogens 17b-estradiol (E2) and diethylstilbestrol (DES) for up to 21 days of the 28-day culture duration. Dysregulated co-expression of proliferation (Ki67) in ERα(+) LEC induced by the estrogenic compounds would indicate disordered control of cell division, which may represent an important early pathogenic step in the development of breast cancer. Co-localization of ERa and Ki67 was measured by immunofluorescence. Interpretation of proliferative response to the estrogens was complicated by statistical sampling issues encountered in the histological methods used in the immunofluorescence assays. Whole culture assays are being pursued to assess proliferation while histological methods amenable to 3DBC are being developed.
Conclusions:
The 3DBC represents the first system to sustain normal luminal epithelial cell function beyond a few days in culture. Future work will confirm preliminary indications that the cultures can recapitulate oncogenic transformation as it occurs in vivo.
The 3DBC static multiwell bioreactors enables seamless integration into high content and high throughput (HTS/HCS) screening workflow in chemical and pharmaceutical screening programs, which will facilitate rapid adoption of the new product.
RegeneMed has successfully commercialized 3D liver and 3D skin platforms for application to safety and efficacy assessment of compounds for the environmental, chemical, cosmetic, consumer product and pharmaceutical industries. RegeneMed plans to commercialize the 3DBC platform (tissues, reagents, assays and equipment). A commercialized 3DBC platform should enable differentiation of proliferating agents from carcinogens, classification of carcinogenic agents such as genotoxins from non-genotoxins, and environmental risk assessment in life-cycle stages. The 3DBC and associated assays and reagents would serve HTS and HCS activities such as the U.S. Environmental Protection Agency's (EPA) Endocrine Disruptor Screening Program, U.S. Tox21 and ToxCast, international regulatory testing programs for chemicals (e.g. OECD, ICCVAM, ECVAM), basic research on breast cancer etiology including identification of potential breast carcinogens, and breast cancer mechanistic studies and therapeutic development in the research and pharmaceutical communities.
Supplemental Keywords:
breast cancer, estrogen, co-culture, carcinogensThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.