Grantee Research Project Results
2017 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, 2016 through November 30,2017
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 effects of chemicals on breast cancer development and progression.
Aim 1: Optimize and automate our microfluidic 3D in vitro ductal breast cancer model to be used for chemical library screening.
Aim 2: Develop an AOP based model of estrogen-receptor (ER) mediated invasive ductal carcinoma (IDC).
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:
Since our last update, we concluded experiments evaluating the impact of stromal cells on ER-driven responses, optimized extracellular matrix (ECM) conditions, finalized our chemical testing system, and began chemical testing. We previously reported that we were investigating the influence of mammary fibroblasts and preadipocytes on response to 17β-estradiol (E2) as well as testosterone (T) due to their regulation of estrogen receptor (ER) protein levels and production of aromatase, respectively. We have completed these studies, and found that immortalized human mammary fibroblasts increased the severity and sped the onset of E2-induced ductal hyperplasia by reducing apoptosis, which reduced cell loss in the system and increased cell density. Interestingly, ER protein expression was similar in E2-treated monocultures compared to E2-treated co-cultures, while maximal ER transactivation was significantly higher in the E2-treated co-cultures. A manuscript detailing these findings is under review in Scientific Reports. Experiments evaluating the role of pre-adipocytes on estrogenic response used adipose-derived mesenchymal stem cells (ADMSCs), which are primary human cells that can be differentiated into adipocytes. We previously reported that exposure to T induced ER-driven responses when MCF7-derived ducts were co-cultured with ADMSCs, and speculated that these responses were relevant to aromatase. We have evaluated this hypothesis by exposing our system to T alone, along with a mixture of T and aromatase inhibitor anastrazole. As expected, anastrazole inhibited T-induced ER transactivation and proliferation in a concentration dependent manner.
In addition to optimizing stromal cell components, we have explored choices for the ECM hydrogel. We hypothesized that utilizing a synthetically designed poly(ethylene) glycol (PEG) based hydrogel in the model would provide higher reliability in cellular responses to E2. To test this hypothesis we designed two PEG hydrogels, each with the same stiffness, pore size, and cellular cleavability as the synthetic counter to animal derived collagen 1 and Matrigel. The PEG hydrogels differ in the concentration of RGD sequence (cyclic conformation) incorporated into the matrix to promote cell adhesion and were chosen based on several discovery based cell adhesion screens. We seeded MCF7 cells on top of the four different hydrogels in a 2D cell-on-gel format then exposed each condition to five concentrations of E2 for 48 hours. At the end of the culture period, ERE activity, cell redox potential, and proliferation were measured. We then assessed the variability in measurements across five independent experiments, with 3 technical replicates at each E2 concentration. Preliminary results indicate that the synthetic hydrogel tissue models have a lower standard deviation in ERE activity compared to animal derived tissue model counterparts. Surprisingly, the standard deviations of both PEG and collagen 1 tissue models were not significantly different. However, the tissue model that included collagen 1 and Matrigel resulted in a much higher and significantly different standard deviation in calculated EC50s, indicating that Matrigel, at least when mixed in collagen 1, contributes to a much higher amount of error.
We finalized our platform using the findings described in this report as well as by using the guidelines defined by the AOP that was discussed in our previous report, and have thus completed Aims 1 and 2. Our final chemical testing platform consists of a MCF7-derived duct surrounded by a 4.5 mg/mL collagen matrix containing ADMSCs. Next, we confirmed that the MCF7-derived duct model is responsive to weak ER agonists by exposing the system to five chemicals with varying levels of estrogenic activity (in order of estrogenic activity): E2, estrone, genistein, bisphenol-A, and fenarimol. Exposure to all test chemicals increased ER transactivation as well as proliferation. We also exposed our model to ER antagonists fulvestrant and tamoxifen, which led to a decrease in E2-induced ER transactivation and proliferation. Altogether, these studies validate the MCF7-derived duct model as a suitable platform to evaluate the effects of ER agonists as well as ER antagonists on breast cancer progression.
Future Activities:
For future activities, we will evaluate a set of test chemicals in the finalized model defined above, thus completing Aim 3. 20 chemicals will be evaluated at five different concentrations alone and in combination with 1 nM testosterone. 1 nM of testosterone was chosen because the primary source of estrogen in post-menopausal women occurs locally through aromatase metabolizing T to E2, and 1 nM is a physiologically relevant concentration; therefore, exposing our system to T will flag compounds that antagonize ER as well as modulate aromatase levels. Chemical hits will be compared back to the ToxCast database as well as previously published in vitro and in vivo studies. Our future efforts will also include developing a protocol to isolate ER+ breast epithelial cells from primary breast tissue, to replace the immortalized MCF7 cells used in the model.
Journal Articles on this Report : 2 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, Livingston MK, Warrick JW, Stanek EM, Alarid ET, Beebe DJ, Johnson BP. Mammary fibroblasts reduce apoptosis and speed estrogen-induced hyperplasia in an organotypic MCF7-derived duct model. Scientific Reports 2018;8(1):7139. |
R835737 (2017) R835737C003 (2017) R835737C003 (Final) |
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Stallcop LE, Alvarez-Garcia YR, Reyes-Ramos AM, Ramos-Cruz KP, Morgan MM, Shi Y, Li L, Beebe DJ, Domenech M, Warrick JW. Razor-printed sticker microdevices for cell-based applications. Lab on a Chip 2018;18(3):451-462. |
R835737 (2017) R835737C003 (2017) R835737C003 (Final) |
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Progress 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