In Vitro/In Vivo PD Assays
Cell Proliferation assay
Colorimetric MTT, MTS or WST-1 assays will be used to measure effect of candidate drug on cancer cell proliferation and cytotoxicity. These assays measure chromogenic products produced by viable mammalian cells from specific substrates.
Activation of caspases initiates apoptosis in mammalian cells. Colorimetric Caspase-3 assays will be used to monitor for apoptosis due to candidate drug treatment. Caspase-3 assay detects formation of chromophore by cleavage from a labeled substrate.
DNA damage assay
Phosphorylated histone H2AX (γH2AX) is a biomarker for double strand breaks (DSB). A chemiluminescent ELISA assay will be utilized to measure the amount of γH2AX in cancer cells or tumor biopsies to monitor DNA damage due to candidate drug.
Migration and invasion of cancer cells into healthy host tissue are fundamental cellular events underlying metastasis. The Boyden Chamber will be utilized to measure the effects of candidate drug on cancer cell migration and invasion. The Cell Migration Assay measures the number of cells traversing a porous membrane, while the Cell Invasion Assay monitors cell movement through extracellular matrices.
In vivo assay to measure growth and metastasis of cancer cells implanted subcutaneously in immunodeficient mice, in presence and absence of candidate drug treatment.
Biomarker assays on tumors from the xenograft model
Apoptosis (Caspase-3 or TUNEL assays) and DNA damage assays (γH2AX assays) performed on primary and metastatic (if there are any) tumors isolated from drug treated and untreated xenograft mouse model. Other assays upon request.
Genetic mouse models for PD assays.
Test drug efficacy in genetic mouse models that develop breast, bone, skin, colon or pancreatic cancers. Monitor biomarker changes in tissues of genetic models treated with candidate drug.