Among the routinely applied assays in the Translational Laboratory are in vitro evaluations of compound activity on cells grown in mass culture and in clonogenic assays. A particular focus of attention is the development of an in vitro “area under the concentration x time curve” (AUC) for 50% growth inhibition (GI50), total growth inhibition (TGI), and 50% cell killing (LC50). This information is crucial to apply to emerging pharmacology information from animal experiments, as promising drugs are able to achieve in animals AUCs that at least begin to mimic those capable of affecting the in vitro parameters. Thus, bioassays will be performed that employ drug concentrations deduced from pharmacokinetic studies in animals (AUCmax) and which evaluate whether these achievable concentrations in vivo will relate to growth and target inhibition in vitro. While it is true that animal protein binding and metabolism can confound the predictive value of such information, a clear knowledge of these parameters is crucial in interpreting initial pharmacology information. The Translational Laboratory also conducts in vitro evaluations of additivity and synergy or antagonism of proposed combinations of agents. This information is a necessary prelude to developing combination regimens for application in the clinic, particularly where the agents affect different points in a growth regulatory pathway or complementary pathways sustaining tumorigenesis.
The Translational Laboratory has the capacity to undertake in vivo evaluation of compound activity in tumor models including athymic, severe combined immunodeficient, and immunocompetent models. It is versed in all aspects of molecular target evaluation, including cell cycle analysis, gene expression at the RNA and protein level (realtime PCR analyses, Western blotting, immunohistochemistry), and collaboration with the Biopolymers shared resource of the UMGCC for DNA microarray and the Proteomic shared resource for qualitative protein analysis by 2-D electrophoresis and time-of flight mass spectroscopic analysis. The methodology for the creation of high-density tissue microarrays is also established. The recent acquisition of digitally enabled mobile stage fluorescent microscopic technology allows protein tracking of fluorescently tagged molecules and by immunofluorescence.
The Translational Laboratory develops pharmacodynamic markers of drug effect suitable for a collaborating and clinical routine laboratory to process subsequent clinical specimens. Alternatively, the Translational Laboratory will receive clinically derived specimens and practice assays derived endogenously or received from collaborating, more basic laboratories. One major output of the our work is data for interpretation of clinical trials results, e.g. correlation of an effect of a drug on patient surrogate or tumor cells with achievable pharmacology in early phase trials. A second major output is preliminary pre-clinical data that would support the development of a full fledged clinical trials opportunity.
The Translational Laboratory provides a spectrum of established in vitro proliferation assays that are all automated and run in a multi-well format to enable high-throughput testing of potentially novel and established anticancer agents in cell-based screens. Over 120 permanent tumor and normal cell lines are available, which have been characterized for a large number of currently highly attractive molecular targets. Proliferation assays that can be performed with these cell lines include the MTT assay (methyl tetrazolium assay, detecting mitochondrial function), the SRB assay (measuring total cellular protein, assay used by the NCI 60 cell line screen), and the propidium iodide (determining total DNA content).
The MTT assay is further employed for drug combination studies that are performed based on the fixed IC50 ratio method by Chou and Talalay. For processing the data resulting from combination studies, we have the Calcsyn software package available that enables us to determine combination indices and other relevant parameters with ease.
A unique proliferation assay format that is available through the Translational Laboratory is the human tumor stem cell assay (HTCA). This assay is a soft agar based procedure performed in 24-well plates. The use of a semi-solid matrix enables only the growth pluripotent cell populations/colonies, which are thought to be most relevant for tumor metastasis and development of drug resistance. We have a custom-made tumor colony counter (Microbiology Systems International, Frederick) available to us which allows for automated colony counts and thus rapid processing of a readout that would be very cumbersome if counting by eye is required. Another major advantage to speed is the possibility, to not only grow cancer cell lines in the HTCA, but also to be able to grow single cell suspensions from human tumor xenograft tissues and patient tumor material for tumor stem cell isolation and chemosensitivity testing.
The Translational Laboratory has an IACUC approved umbrella animal use protocol "Animal Models for Studying Tumor Biology and for the Evaluation of Pharmacodynamic and Pharmacokinetic Endpoints of Novel and Experimental Cancer Therapies" approved for three years (#0405001, till May 2008). Any tumor line or drug can be used in the following approved procedures considered key in preclinical anticancer drug development:
Because it is very complicated and difficult to obtain IACUC approval for investigators without prior and documented experience or experienced personnel in the field of animal research, the Translational Laboratory offers a fast and easy option to perform grant-related or contract-related in vivo experiments. New cell lines or drugs only require a notification of the animal care and use office through the submission of an amendment. The latter is mainly concerned with safety information and is assured fast track review and approval within days.
A LABCAT Tumor Tracking & Measurement Software version 8.0 for tumor data acquisition and processing is available which enables electronic, professional raw data documentation, statistical data evaluation as well as high quality graphical data presentation.
Sample collection and processing for PD analyses is the core business of the Translational Laboratory. Services comprise the development of biochemical, molecular or cellular assays that are tailored and validated according the translational research question associated with a clinical trial, the development of standard operating procedures including instructions for research nurses and collection logistics. Assay technologies are first adapted to handling of micro quantities of patient materials by optimizing the procedures with human tumor xenograft and mouse tissues. They are mostly based on immunohistochemistry or immunofluorescence for tracking of target expression at various time points in cytospins or tissue microarrays. Western blotting for qualitative and quantitative evaluation of protein expression and real time PCR for mRNA expression are also frequently used.
The Translational Laboratory has scientific personnel, which is well experienced in protein-protein interaction studies, creation of stably transfected cell lines that express targets of interest and the use of cell biology, proteomic and genomic analyses before and after drug treatment. This expertise is made available to small biotech and pharmaceutical companies that have limited laboratory capacities or lack qualified laboratory personnel.