Grantee Research Project Results
2015 Progress Report: Cancer MAPs: A 3D Organotypic Microfluidic Culture System to Identify Chemicals that Impact Progression and Development of Breast Cancer
EPA Grant Number: R835737C003Subproject: this is subproject number 003 , established and managed by the Center Director under grant R835737
(EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
Center: Human Models for Analysis of Pathways (H MAPs) Center
Center Director: Murphy, William L
Title: Cancer MAPs: A 3D Organotypic Microfluidic Culture System to Identify Chemicals that Impact Progression and Development of Breast Cancer
Investigators: Beebe, David , Murphy, William L
Institution: University of Wisconsin - Madison
EPA Project Officer: Aja, Hayley
Project Period: December 1, 2014 through November 30, 2018 (Extended to November 30, 2019)
Project Period Covered by this Report: December 1, 2014 through November 30,2015
RFA: Organotypic Culture Models for Predictive Toxicology Center (2013) RFA Text | Recipients Lists
Research Category: Chemical Safety for Sustainability
Objective:
To produce a 3D organotypic culture model of ductal breast cancer that is compatible with higher throughput screening and high-content screening approaches to discern toxic effects of chemical substances on breast cancer development and progression. The major goals of this work have not changed since the initiation of this award.
Aim 1. Optimize and automate a microfluidic 3D in vitro ductal breast cancer model to be used for chemical library screening.
Aim 2. Develop an adverse outcome pathway (AOP) based model of estrogen-receptor (ER) mediated invasive ductal carcinoma (IDC) by utilizing quantitative physiological and molecular endpoints to identify key steps between the initiating event (estrogen-receptor ligand binding) and the adverse outcome (IDC) in our microfluidic platform.
Aim 3. Conduct high-throughput screens using chemicals from the ToxCast library to identify chemicals that promote ER-mediated and non ER-mediated IDC.
Progress Summary:
Current Progress in Model Development: Our strategy for this project is to write an ER-mediated IDC AOP, and using this as a way to determine the integral cellular and microenvironmental components of the platform, as well as the critical readouts to be monitored. To increase confidence that our model can identify chemicals that accelerate breast cancer progression, we look to develop a system that can readout multiple endpoints in a single sample at different levels of biological complexity utilizing the AOP to identify what readouts should be considered. We have begun to define each event of the AOP, assessing (1) if the given event can be evaluated in our model, (2) if the key event of the AOP occurs in our system and (3) if the readout is compatible with high-throughput screening. We have completed and submitted a perspective review on this subject.
Significant progress has been made since the initiation of the grant period in platform development. To improve the reproducibility of our platform, we have moved to a new platform design, dubbed LumeNEXT that allows us to create highly reproducible lumen geometries through Extracellular Matrix (ECM) hydrogels with less dependence on the viscosity of the hydrogel materials integrated into the model. This platform expands our capabilities and enables modeling mammary epithelial ducts in natural derived hydrogels, as well as hydrogels synthetically derived in our center’s Synthetic Matrices Core. The LumeNEXT device is easy to modify to allow for compartmentalized co-culture of several types of cells so that we can better model the microenvironment of the human mammary duct.
Currently, the cellular components in our platform include the common cell lines MCF12a, a normal mammary epithelial line, and MCF7, an ER-positive cancerous cell line, in different natural and synthetic ECM hydrogel conditions. We have explored the co-culture of both MCF7s and MCF12as with human mammary fibroblasts (HMFs). In addition to using immortalized cell lines in this model, we’ve also explored the use of induced pluripotent stem cell derived human mammary epithelial cells and primary patient cells. Our experiments have shown that our model supports viability, correct apical/basal polarity, tight junctions, and long term culture of all cell types. We are determining other critical aspects including robustness and estrogen responsiveness in these cell lines, to determine which is the most suitable cell type moving forward. To evaluate the cellular components, we are utilizing immunohistochemical staining to show cell junction (e-cadherin) and apical/basal polarity, cytoskeletal structure, and model viability (calcein AM/ethidium homodimer). The optimization of these staining procedures and imaging processing in ImageJ was not facile because we have found that 2D culture assays/readout protocols do not translate to our 3D model in a straightforward manner. Our model has shown tissue organization with correct polarity, confluence of a monolayer around the ductal geometry, and high viability (>80%) in both MCF12a and MCF7 cell lines via manual scoring. We are working through ImageJ and JEX (an image processing pipeline utilizing ImageJ and R) to develop a robust automated quantification strategy for these analyses.
We also screened a subset of these gels with MCF7 cells that were transfected with a luciferase gene downstream from the estrogen response element promoter (MVLN) and treated them with a dose of 100nM estradiol and vehicle control. Preliminary data suggest that certain combinations of these gels promote higher estrogen receptor activity, however we determined that cell viability on the gels may have confounded results. These results will help guide our decisions in developing the screening platform we will use to screen chemicals from the ToxCast library.
Future Activities:
Once we have completed optimizing culture conditions, we plan to develop and automate other endpoints belonging to our AOP including invasion, tumor growth and extracellular matrix rearrangement. We are also at position in which we plan to develop a 3D model in synthetic matrices and compare its reproducibility to the model in natural hydrogels to increase the robustness of the screening platform.
Journal Articles on this Report : 1 Displayed | Download in RIS Format
Other subproject views: | All 6 publications | 6 publications in selected types | All 6 journal articles |
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Other center views: | All 215 publications | 82 publications in selected types | All 81 journal articles |
Type | Citation | ||
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Morgan MM, Johnson BP, Livingston MK, Schuler LA, Alarid ET, Sung KE, Beebe DJ. Personalized in vitro cancer models to predict therapeutic response:challenges and a framework for improvement. Pharmacology & Therapeutics 2016;165:79-92. |
R835737 (2016) R835737 (2017) R835737C003 (2015) R835737C003 (2016) |
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Supplemental Keywords:
Ductal carcinoma in situ, DCIS, estrogen disrupting chemicals, EDC, mammary duct, microenvironment, microfluidics, stromaProgress and Final Reports:
Original AbstractMain Center Abstract and Reports:
R835737 Human Models for Analysis of Pathways (H MAPs) Center Subprojects under this Center: (EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
R835737C001 Liver MAPs
R835737C002 Brain MAPs
R835737C003 Cancer MAPs: A 3D Organotypic Microfluidic Culture System to
Identify Chemicals that Impact Progression and Development of Breast Cancer
R835737C004 Vascular MAPs: Vascular and Neurovascular Tissue Models
R835737C005 Pathway Analysis Core
The 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.
Project Research Results
6 journal articles for this subproject
Main Center: R835737
215 publications for this center
81 journal articles for this center