Application of immunohistochemistry and quantitative image analysis to demonstrate in vivo inhibitory activities of PDGF receptor kinase compounds in experimental models of tumor angiogenesis

1499 Although in vitro assays are imperative to evaluate and select compounds for specific molecular targets, experimental in vivo models are necessary for demonstrating efficacy against tumor growth in cancer drug discovery. The most widely employed in vivo tumor models used to determine compound effectiveness against tumor growth is an in vivo animal model where human tumor cells are implanted subcutaneously in immuno-compromised rodents (host), such as nude mice. In this model, human tumor cells are able to orchestrate typical tumor cell behaviors and phenotypes such as cell proliferation and tumor-induced angiogenesis required for solid tumor growth. With the advent of targeted therapeutics against specific molecular target(s), it becomes important to validate and characterize a compound’s intended effect on the molecular target(s) in vivo . In the present study, our objective was to determine the tumor penetration of an inhibitor of platelet-derived growth factor receptor tyrosine kinase (PDGF-RTK) directly in the tumor tissue. The delay of tumor growth was achieved by administering the compound orally in an in vivo animal model of human LoVo colon cancer xenograft. A novel assay was developed to evaluate the anti-PDGF-RTK activity of the inhibitor by determining the phosphorylation status of phospholipase C gamma (PLCγ), a key downstream cellular molecule in the PDGF-RTK pro-mitogenic signaling cascade. We used anti-PLCγ and phospho-specific anti-PLCγ antibodies to immunohistochemically (IHC) detect pan-PLCγ (phosphorylated and unphosphorylated forms) and ph-PLCγ (phosphorylated form), respectively in tumor tissues obtained from in vivo xenograft studies. Image analysis was used to compare the ratio of ph-PLCγ to pan-PLCγ immunolabeling intensities in the tumors from vehicle- (n=14) and compound-treated (n=15) mice. About 63% of the pan-PLCγ detected in serial sections of the tumors in the vehicle group was phosphorylated as compared to 28% in the dosed group. Our data showed significant differences (p in vivo inhibitory activity against PDGF-RTK of the compound, and most importantly, demonstrating activity at the site of the intended molecular target in tumor tissues. This approach using phospho-specific antibodies can be used to evaluate the in vivo cellular activities of other potential inhibitors of PDGF-RTK, while this scheme of IHC and quantitative image analysis can be applied to other targets as well.