ARDF's Annual Open grant program was established to fund research projects that develop alternative methods to advance science and replace or reduce animal use. Proposals are welcome from any nonprofit educational or research institution worldwide, although there is a preference for U.S. applications in order to more quickly advance alternatives here.
Expert reviewers evaluate proposals based on scientific merit and feasibility, and the potential to reduce or replace the use of animals in the near future. Proposals are considered in fields of research, testing, or education. The maximum grant is $40,000, with an average 25% funding rate from 2012 to 2017.
Since 1993, ARDF has provided over $3.25 million in funds for projects in 29 states and 5 countries.
Scientific reviews are coordinated by the Institute for In Vitro Sciences, Gaithersburg, Maryland.
Kim Boekelheide, MD, PhD
Brown University, Providence, RI
Screening estrogenic endocrine disrupting chemicals with human MCF-7 3D microtissues by in vitro pathology
Gargi Ghosh, PhD
University of Michigan, Dearborn, MI
Endocrine disrupting chemicals (EDCs) can adversely affect estrogenic, androgenic, and thyroid hormone sensitive targets. In this project, we develop an innovative screening approach for estrogenic EDCs using human MCF-7 breast cancer cell 3-dimensional (3D) microtissue cultures. Grown in agarose hydrogels, these cells self-assemble into microtissues reminiscent of normal in vivo
human mammary epithelial tissue with lumen-forming glands, up-regulated cell-type specific differentiation markers, and more complex responses to estrogenic stimulation when compared to the same cells grown in 2D [1-3].
This simple and reproducible in vitro
model is amenable to high-throughput confocal imaging analysis, offering a unique opportunity to study morphological and molecular changes associated with exposure to estrogenic EDCs as an indicator of adverse effects over a broad concentration range .
Progress toward the goal of transforming toxicity testing is made by addressing these Specific Aims:
- Specific Aim 1. Optimize the estrogenic MCF-7 3D microtissue platform to screen for proliferation, differentiated morphology, and estrogenic pathway activation endpoints
- Specific Aim 2. Screen a test battery of estrogenic EDCs for their concentration-dependent effects on the MCF-7 3D microtissue endpoints
- Specific Aim 3. Integrate the imaging and molecular endpoints within a computational systems biology framework for the purpose of human safety assessment
The overall goal of this project is to accelerate development of human 3D microtissue platforms as alternatives to animal toxicity testing.
Development of 3D tumor angiogenesis model for pre-clinical drug screening
Matteo Minghetti, PhD
Oklahoma State University, Stillwater, OK
Angiogenesis is a requisite for cancer to grow beyond 1-2 mm3. While many anti-angiogenic agents have been developed, the magnitude of clinical responses and survival benefit from utilization of these inhibitors has been moderate. Identification of new therapeutic interventions and subsequent screening of drug candidates is essential for successful elimination/disruption of vascular networks within tumor.
While animal testing is mandatory for any drug prior to clinical trial, in cancer, a high attrition rate prior to clinical deployment results in utilization of large number of animals for drug evaluation. This is due to lack of physiologically relevant in vitro model capable of high quality triage of experimental compounds.
Proposed here is development of 3D tumor angiogenesis model, which by virtue of recapitulation of many relevant aspects of in vivo tumor vascularization and 96-well plate assay format, will permit discovery and high-throughput screening of new experimental compounds. Selection of only those compounds which displayed optimal efficacy in in vitro model to proceed to in vivo studies will facilitate reducing the usage of animal models significantly. Although, the project involves developing breast cancer angiogenesis model, the platform can be extended to other tumor types and thus will have wide application in drug discovery.
Predicting toxicity in effluent waters using an in vitro model of the fish gill (RTgill-W1)
Thomas Sanderson, PhD
Institut National de la Recherche Scientifique, Laval, QC, Canada
Managing and maintaining water quality is key to human and environmental health. To prevent the contamination of our waters, the Clean Water Act requires major dischargers of effluent wastewater (municipalities, power plants, mining companies, etc.) to perform whole effluent toxicity (WET) tests. WET testing exposes laboratory populations of aquatic organisms, including fish, to effluent samples under controlled conditions in order to estimate the environmental toxicity of those samples. It is estimated that in the United States alone, approximately six million fish are used every year for WET testing.
An alternative to whole animal testing would be highly desirable for ethical reasons and also in practical terms by reducing the cost and time of testing. Previous studies have shown a strong correlation between the concentrations of chemicals lethal to 50% of a fish population (LC50) and the concentrations affecting cellular markers in 50% (EC50) of gill cells (RTgill-W1). We will therefore allow a gill cell culture to inform us of the presence of biologically active compounds in natural waters. The proposed approach, to correlate cell viability in RTgill-W1 in vitro to mortality of fish in vivo, is envisioned to validate the use of RTgill-W1 cells as an alternative to WET testing.
An in vitro human co-culture model of the hormone-dependent breast cancer microenvironment
Aranzazu Villasante, PhD
Columbia University, New York, NY
We propose to develop an innovative coculture system of Hs578T human mammary stromal-like and T47D hormone-dependent breast epithelial tumor cells, in which the interactions between estrogen-dependent T47D and estrogen producing Hs578T cells are expected to be representative in vitro model of the human hormone-dependent mammary tumor microenvironment.
Hs578T cells express aromatase (CYP19) and are capable of producing estrogens in the presence of the adrenocortical androgen precursor dehydroepiandrosterone (DHEA). CYP19 gene expression in Hs578T cells is regulated by the healthy stromal I.4 CYP19 promoter, but the cells can undergo a "promoter-switch" where the normally silent CYP19 promoters PII, I.3 and I.7 become activated, which mimics the in vivo situation in human breast cancer patients. The estrogens produced by Hs578T cells stimulates the proliferation of T47D mammary tumor cells, which in turn secrete inflammatory factors (such as prostaglandin E2) that to further induce CYP19 expression in Hs578T cells. This positive feedback loop is the hallmark of the hormone-dependent breast tumor microenvironment. Using this coculture model, we will evaluate the (inter)cellular signaling pathways involved in the promoter-specific regulation of CYP19 and estrogen-dependent gene expression, and determine potential stimulation or otherwise endocrine disruption of the tumor microenvironment by pesticides and the consequences for tumor cell growth.
Tissue-engineered platform for studies of antiosteolytic and antineoplastic drugs
Ewing's sarcoma (EWS) is the second most aggressive bone cancer affecting children and young adults that frequently metastasizes in bone. Interactions between the tumor and bone cells orchestrate a vicious cycle in which tumor cells induce osteoclast differentiation and activation to cause osteolytic bone resorption.
The lack of predictable models that can recapitulate bone resorption in EWS limits our understanding of how tumor cells regulate osteoclasts activation and how to inhibit the process. Consequently, new experimental models would be transformative to cancer research and new therapeutic options to many patients in need. Here, I propose to generate a living bone-engineered niche with osteoclasts and osteoblasts in vitro in which osteolysis can be assessed; and also to test compounds that inhibit bone resorption in a highly predictive fashion.
The significance of this proposal is that (a) I will develop a protocol for engineering EWS models using animal serum-free media, (b) I will validate the use of tissue-engineered models as predictive systems, and (c) it will provide a bone-engineered-based platform for osteolysis research, in configurations tailored for high-throughput drug screening and in-depth studies of EWS treatments. Together, this novel technology will contribute to reduce the number of animals used for cancer research.