- Clonogenic High-Throughput Assay for Screening Anti-Cancer Agents and Radiation Modulators
Fast-Track proposals will be accepted.
Number of Anticipated Awards: 3-4
Budget (total costs, per award): Phase I: up to $300,000 for up to 9 months; Phase II: up to $2,000,000 for up to 2 years
PROPOSALS THAT EXCEED THE BUDGET OR PROJECT DURATION LISTED ABOVE MAY NOT BE FUNDED.
The goal of anti-cancer treatment modalities is to eradicate cancer cells via several killing mechanisms that include metabolic death, apoptotic cell death (apoptosis) and reproductive death (clonogenic death). Apoptosis is programmed cell death leading to nuclear DNA fragmentation, mostly assessed by flow cytometry, enzymatic activity or membrane staining (annexin 5). Loss of key metabolic activity such as loss of NAD(P)H-dependent cellular oxidoreductase enzymatic activity can result in non-viable cells. This is mostly assessed by colorimetric methods. Clonogenic death is defined as the loss of ability of a cancer cell to proliferate indefinitely, which can only be assessed accurately by clonogenic assays and considered the gold standard assay to determine efficacy of a given radiation combined therapeutic modality. Colorimetric assay for viability and apoptotic assays measure short-term effects, while clonogenic assays measure long-term effects and integrate all forms of cell death, but these assays are costly and labor-intensive. While high-throughput screening (HTS) systems are available for apoptosis and colorimetric cell viability assessments, there is no HTS technology available for clonogenic assays. Colorimetric / apoptotic assays are more often used in screening than clonogenic assays, but do not directly measure residual cell clonogenic potential. Therefore, there exists an opportunity for using HTS in radiation oncology for screening vast number of drugs or drug combinations to improve the efficacy of radiation treatment, by integrating already developed robotics components, such as automated liquid handlers, centrifuges, incubators, imaging and statistical software as well as an irradiator to assess the effect of radiation in a laboratory. It is well known that cancer recurrence is a common event after treatment and often-attributed to re-population of surviving clones. Thus, evaluating surviving clones becomes a vital test in-vitro to assess treatment efficacy, making colony-forming assays the gold standard. Further, designing an HTS for clonogenic assays will increase its utility to screen for drugs, radiation sensitizers and protectors in vitro.
Chemotherapy is used for both solid and hematologic malignancies. In addition, more than half of US cancer patients undergo radiotherapy alone or in combination with drugs; percentage of which is expected to only increase. Screening that allows for more accurate testing of chemotherapeutic and combinatorial treatments will better focus development to more promising agents and accelerate development of drug and drug-radiotherapy combinations. With expanded global access to radiotherapy and increased utilization rate, pharma and academics will be further incentivized to discover agents with anti-cancer and radiation sensitizing properties. Assays that are adaptable to the incorporation of molecular targeting, imaging, and evaluation of genetically defined cell panels for drug screening and discovery will be required with ongoing precision medicine initiatives. Companies can utilize clonogenic HTS assays to screen for new agents and to test newly identified agents in combination for radiation. Results from this type of screen should improve success in subsequent in vivo model testing and will accelerate clinical translation.
The purpose of this contract solicitation is to: (i) promote stronger academic industry partnerships in radiobiology to develop clonogenic survival-based HTS systems (ii) to exploit recent advances in the technical maturity of HTS technologies and combine them with advances in clonogenic assays, (iii) encourage small businesses to specifically develop HTS systems for screening potential anti-cancer agents based on a clonogenic endpoint, and (iv) integrate relevant technologies. Colony-forming assay survival experiments currently involve the use of several drug and/or drug + radiation doses as well as several plated cell numbers for each cell line and hence the assays are labor and material intensive. Further, developing an HTS system with a clonogenic endpoint will enhance screening/cross validating chemotherapeutic agents as well as radiation effect modulators and combinatorial treatments, while reducing labor and costs.
To apply for this topic, offerors need to design integration of robotic instrumentation, micro-fluidics, thermal and gas control, colony counting microscopic imaging and image analysis. An integrated system may also require the development of “bridging” components and graphic user interfaces. Offerors are required to develop standard operating procedures matched to validated cell lines for use with the integrated system. Offerors must include an integration of microfluidics/culture system with radiation exposure under conditions allowing precise dosimetry, which is critical. Offerors are also required to integrate and adopt software to capture and calculate survival. This solicitation is not intended for development of systems with non-clonogenic endpoints.
Phase I Activities and Expected Deliverables
- Prototype of integrated/customized robotic or automated platform for cell plating, maintaining the temperature and CO2.
- Develop integrated HTS system that couples plating micro-fluidics, irradiation system, microscopy, imaging software and statistical software for estimating cell survival (inactivation radiobiologic estimates) and dose enhancement and modification factors to demonstrate improved efficacy of radiation treatment.
- Integration of localized radiation exposure with precise real-time dosimetry into HTS platform.
Phase II Activities and Expected Deliverables
- Delivery of a prototype system with validated SOPs that are translatable to other laboratories.
- Imaging of colonies and software to capture and calculate the survival.
- Cross-check validation of HTS data with conventional clonogenic assays.
- Defined cell line panels that have been shown to be appropriate for use with the system and the clonogenic endpoint. Validation of representative “hits” using conventional clonogenic assay.
- Software to calculate radiation-inactivation estimates and graphing cell survival curves and calculate dose enhancement factor if done in combination with agents.
- Licensing of individual components for use in the system as needed.