Molecular Discovery and Evaluation

 


Molecular Discovery and Evaluation Shared Resource


V. Jo Davisson, Ph.D. MDE Director

Professor of Medicinal Chemistry & Molecular Pharmacology
Laboratory for Chemical Biology & Drug Development
BindleyBioscienceCenterat PurdueDiscoveryPark
1203 W. State St.
West Lafayette, IN  47907-2057
davisson@purdue.edu
 


 

General Information and Purpose:  

The Molecular Discovery and Evaluation Shared Resource (MDE) provides services and capabilities through the Purdue University Center for Cancer Research to accelerate translation of early discoveries, new molecular agents, and enhancement technologies for improved therapies in oncology. The primary focus is to conduct pre-clinical evaluation to annotate and/or define the potency and efficacy of candidates for therapeutic development, test the specificity of cancer diagnostics agents, or develop critical new tools for cancer research and oncology.

The Shared Resource will assist investigations to:

  • Provide cost-effective standardized services through automated in vitro cellular analysis;
  • Provide cost-effective standardized services for in vivo animal xenograft testing;
  • Implement new cell-based assays for evaluation of therapeutic targets or pathways;
  • Develop additional capabilities and services for in vitro and in vivo evaluation.

 

The MDE offers a variety of flexible and customizable services for in vitro testing and in vivo mouse models to assess the efficacy of anti-cancer drugs on human and murine tumors. Our facility staff will consult and work with investigators to select an appropriate study design including high throughput in vitro cell-based testing, in vivo model determination, maximum tolerated dose, drug vehicle selection, drug routes and administration schedules, and evaluation of tumor response.

Additional experimental design expertise including statistical analysis http://www.stat.purdue.edu/scs/, histopathology http://www.vet.purdue.edu/mdru/phpl.html, and related features are coordinated with other Shared Resources on campus.

 

CURRENT SERVICES


In vitro cell-based evaluations

Human Tumor Cell Lines: The primary focus of the in vitro evaluation is on advancing early discovery to evaluate therapeutic potential through chemical-genetic cancer biology approaches in the MDE Shared Resource. The in vitro testing uses and maintains authenticated (DNA fingerprinting) human tumor cell lines panels and assures quality control (cell passage numbers and mycoplasma-free) for both in vivo and in vitro assay formats. The available oncogene mutations and marker gene expression patterns are used to annotate the specific cell lines and guide assay design and data interpretation. Validation assays of expression for specific targets or pathways can be conducted for project applications. Each of the cell lines is adapted to appropriate media for testing and storage.


Current Human Tumor Cell Lines (updated periodically):

 

A549, H1299 (NSCLC) PC3, LNCaP (prostate)
PaCa2 (pancreatic) HepG2 (hepatoma)
SH-SY5Y (neuroblastoma) DG75 (lymphoblast)
HL60, K562 (AML, CML) Breast cell panel (9 lines)
HCT116 (colorectal)  



 

Cancer Cell Growth and Inhibition (CCGI) Assay: The current assays include a simple primary screen against a panel of human tumor-derived cancer cell line panels representing a diversity of genotypes. This screening service is designed for rapid turn-around, robotic assay implemented in 96-well format for testing in parallel multiple agents at varied dosing options against multiple cell lines. The current basic assay uses 3-day growth MTT end-point for monitoring effects in a dose-response format. Five core cell lines are offered as a baseline to evaluate tumor cell growth inhibition (GI50), total inhibition of growth (TGI) and death (LC50), by necrotic or cellular apoptotic events.
 

Basic Cell Panel Screen: The baseline profiling tumor cell lines used in the assay are A549 (non small cell lung), HCT116 (colorectal), MCF7 (breast), PaCa2 (pancreatic), and PC3 (prostate). Additional cell lines can be tested when the expression and pathway data gives a better indication of tumor types likely to be affected. These cell lines have been adapted to automated cell culture protocols on the same economical media (RPMI-1640); the growth characteristics are standardized for plating in the parallel screening assays.
 

Secondary Assays: Additional tumor cell line panels are available to incorporate into assay panels. Alternative end-point parameters are available in secondary assay formats and include cell cycle analysis. Other functional dye-based or molecular marker assays for specific pathways or target proteins are also options for incorporation into secondary assays.
 

Charge units: Each plate represents a separate cell line and charges are calculated upon a per plates basis. Samples can be tested in dose steps of 2, 4, or 10 per plate. For compound submission, a minimum of 100 µL of sample at 10 mM concentration in cell culture-grade DMSO is preferred. Sample numbers of up to 6 can be handled by direct submission in suitable tubes or vials; larger numbers of samples require plate formatting set up.
 

Implementation and Development projects: The Shared Resource also develops and implements new services or conducts special projects to meet the needs of cancer-focused research projects. Assays using alternative tumor cell lines or models are also available upon request. Cellular models and/or assays developed in investigator projects can be adapted to the parallel screening format by the MDE and offered as a service. Additional high throughput cell-based assays including multi-parameter readouts are under development.


In Vivo Evaluations:

General Information and Models: The current primary in vivo testing involves exposure of mice bearing tumor xenografts to evaluate pre-clinical candidate anti-tumor agents, delivery or diagnostic tools, or otherwise assess biological models. Commercially available immune-competent or immune-compromised mouse strain can be used to tailor experimental design for specific projects. Animals are purchased and housed in Purdue’s AAALAC-approved Animal Holding Facility and allowed to acclimate for one week before experimental use. Approved animal protocols are also available for several standardized models and evaluations. Current models make use of human cell lines MDA-MB-231 (breast), MCF7 (breast) PaCa2 (pancreatic), HCT116 (colorectal), A549 (lung), LNCaP (prostate), PC3 (prostate). Other investigator-supplied cell lines will also be possible to meet the needs of a project. A few select mouse tumor cell lines are also available for studies with specific needs.


Xenogenic Animal Assays (XAA) Services: the following services can be provided with unit costs associated with each.

  • In vitro cytotoxicity testing (see CCGI section)
    • As needed, prepare animal protocol amendment for PACUC approval prior to study initiation
      • In vivo maximum tolerated dose, acute and sub-acute toxicity studies

        -Xenogeneic, allogeneic, or syngeneic setting

        -Immunocompromised or immunocompetent mice

        -Heterotopic or orthotopic tumor implantation

        -Route of drug administration

        -Power analysis for group size

        -Proper controls

        -Randomization

        • In vivo project design and model selection
        • Tumor implantation, drug administration, tumor measurement
          • Tissue collection/preservation; deliver solid tissue to histopathology (http://www.vet.purdue.edu/mdru/phpl.html )
          • Statistical assessment of therapeutic efficacy of treatment
          • Untreated tumor repository for routine models
             

Charge units: The variation in experimental design is accommodated by a menu selection to allow for appropriate costs calculation per project. All PCCR members receive a discounted rate, but due to the variety of models and drug regimens requested, the specific costs of each study will be calculated once the design is finalized.

Implementation and Development projects: The Shared Resource also develops and implements new services or conducts special projects to meet the needs of cancer-focused research projects. Assays using alternative tumor cell lines or animal models are also available upon request through development projects. Efforts to incorporate image-based observation of agents or impact on local tumors are in process. 

Although the Molecular Discovery and Shared Resource operates under an approved PACUC blanket protocol for drug discovery in animals, unique features of each individual study may require an amendment before initiation.  The review process can take up to one month before approval.  Additionally, depending on the vendor, particular strains of mice can be on back order. Therefore, once an in vivo study design is finalized, it may take several weeks to months before it can be initiated.
 


Client Request for Services and Consultation:


Contact is made through the Molecular Discovery and Evaluation Shared Resource Website for assignment of a user access code and a LIMS based tracking number.
http://bbc-lims.bbc.purdue.edu:8080/lablink

Once the user account is initiated, each investigator/project will submit a request form to the MDE for in vitro cell-based testing, in vivo testing, or both. This request is initiated by filling out an appropriate order form for each particular assay. There is required technical information to fully define assays. Consultation with the MDE is available to most accurately describe the requested services.

To initiate a request for services, the principal investigator should submit one of the completed Molecular Discovery and Evaluation Shared Resource Request Forms. An initial meeting will be arranged to determine the appropriate study design and budget development.