The Journal of Histotechnology / Vol. 33, No. 1 / March 2010 15 Address reprint requests to Bin Shi, Merck Research Laboratories, Department of Cancer Research, 770 Sumneytown Pike, WP26-462, West Point, PA 19486. E-mail: bin_shi@merck.com Abstract Antiangiogenic agents such as vascular endothelial growth factor receptor 2 (VEGFR2) inhibitors may prove most effi- cacious in the setting of early disease and in the prevention of dissemination and growth of micrometastases. This hypoth- esis was tested in a metastatic orthotopic rat model of breast cancer with the use of a novel orally bioavailable VEGFR2 kinase inhibitor, MK-0888. Mat B III rat mammary cancer cells were implanted into the mammary fat pads of synge- neic female F344 rats. Primary tumor growth was very aggressive, with micrometastases detected 8 days after cell implantation in ipsilateral axillary and inguinal lymph nodes. Lung metastases were detected 15 days after cell implanta- tion by histological analysis. MK-0888 suppressed primary and metastatic tumor growth and reduced the incidence of metastasis in a dose- and schedule-dependent manner. Inhibitions of primary and metastatic tumor growth, as well as intratumoral antiangiogenesis effects, were detected in situ by immunohistochemical analysis of tumor cells, endothelial cell proliferation, microvascular density, and blood vessel maturity. In the Mat B III rat mammary cancer metastasis model, our results provide further evidence sup- porting the ongoing clinical development of VEGFR2 kinase inhibitors, as well as clinically applicable in situ detection and verification of the inhibitor effect in tumor and metasta- sis biopsies. ( The J Histotechnol 33(1):15–24, 2010) Submitted November 20, 2009; accepted February 8, 2010. Key words: angiogenesis, dual-staining, immunohistochem- istry, metastasis, microvascular density, rat cancer model, VEGF, VEGFR2 Introduction Multifunctional vascular endothelial growth factor (VEGF) is a critical survival factor for endothelial cells (ECs). As the major functional receptor of VEGF, vascular endothelial growth factor receptor 2 (VEGFR2), or kinase insert domain receptor, is specifically expressed by ECs and plays a cru- cial role in tumor angiogenesis (1,2). Solid tumors less than 2 mm in diameter can absorb sufficient oxygen and nutri- ents by passive diffusion. However, for tumors to grow fur- ther, tumor angiogenesis, the process of new blood vessel growth, is required inside and surrounding the tumor. The new blood vessels not only nourish the tumor but also pro- vide a direct route for tumor cells to enter the circulation (3,4) and travel to lymph nodes (LNs), lungs, and other sec- ondary target tissues. Tumor cells that survive in the circu- lation and successfully extravasate from the blood vessels form preangiogenic micrometastases in the new tissue they colonize. The growth of a micrometastasis into a secondary tumor mass depends upon the development and maintenance of blood vessels at the new site. Therefore, angiogenesis is essential for both tumor growth and metastasis. Targeting neovascularization through inhibition of EC proliferation and promotion of EC apoptosis is an attractive antican- cer strategy that has been explored in preclinical models and clinical studies (5–8). Numerous small molecules Immunohistochemical Detection of Antitumor, Antimetastasis, and Antiangiogenesis Effects of a Vascular Endothelial Growth Factor Receptor 2 Kinase Inhibitor in an Orthotopic Breast Cancer Metastasis Model Bin Shi 1,2 , James Hardwick 1 , Brett Connolly 3 , Xianzhi Mao 1 , Rosemary McFall 1 , Susan Hill 4 , Kenneth A. Thomas 1 , Nancy E. Kohl 1 , and Laura Sepp-Lorenzino 1,2 1 Department of Cancer Research, Merck Research Laboratories, Merck & Co., Inc., West Point, PA 2 Department of RNA Therapeutics, Merck Research Laboratories, Merck & Co., Inc., West Point, PA 3 Department of Imaging, Merck Research Laboratories, Merck & Co., Inc., West Point, PA 4 Laboratory Animal Resources, Merck Research Laboratories, Merck & Co., Inc., West Point, PA