Probing the Microenvironment of Mammary Tumors Using Multiphoton Microscopy Mazen Sidani & Jeffrey Wyckoff & Chengsen Xue & Jeffrey E. Segall & John Condeelis Published online: 15 November 2006 # Springer Science + Business Media, Inc. 2006 Abstract Advances in optical imaging technologies that allow the subcellular resolution of undissected tissue have begun to offer new clues into the biology of development and disease. For cancer, such advances mean that the primary tumor is no longer a black box and that the disease can be studied throughout the metastatic cascade and not just as an endpoint. In this review we examine the advances in multiphoton imaging technology that have been used to define the microenvironment and its role in delineating the invasion and intravasation steps of metastasis inside living mammary tumors. Results show that the tumor microenvi- ronment is a dynamic place where interactions between tumor cells, macrophages, blood vessels, and extracellular matrix fibers define the metastatic phenotype. Keywords Intravital imaging . GFP . Real-time imaging . Breast cancer . Second harmonic generation Abbreviations ECM extracellular matrix MPIVI multiphoton-based intravital imaging SHG second harmonic generation MMPs matrix metallo-proteases GFP green fluorescent protein CFP cyan fluorescent protein Introduction One out of three cancers diagnosed among US women is due to breast cancer; 212,920 new invasive breast cancer cases and an additional 61,980 in situ breast cancer cases are expected to be diagnosed in 2006. Around 40,970 women are expected to die from breast cancer in 2006 (American Cancer Society, Breast Cancer Facts and Figures 2006). The metastasis of 10–15% of patients with breast cancer can be aggressive and can take between 3 and 10 years to be manifested after the initial diagnosis [1]. The interest in mechanisms behind invasion and metastasis of breast cancer has lead to the use of animal models with tumors that mimic breast cancer in human patients. Animal models employed to study breast cancer have been used for more than 75 years [2], but only recently have these models been improved to employ genetically encoded fluorescent proteins that allow the optical imaging of cells within living primary tumors. The importance of these animal models is that they allow one to recapitulate the different stages of tumor progression, from hyperplasia to late-stage carcinoma, and define the cellular players that lead to tumor metastasis throughout these stages. An important advance in the study of tumor biology is the use of optical imaging techniques in visualizing, at subcellular resolution, the intricate steps of invasion and metastasis in vivo. Early studies were limited by two hurdles: the poor penetration of whole tissue by short wavelengths used for excitation of fluorescence and the J Mammary Gland Biol Neoplasia (2006) 11: 151–163 DOI 10.1007/s10911-006-9021-5 Figures are available in color format in the online version of the article. M. Sidani (*) : J. Wyckoff : C. Xue : J. E. Segall : J. Condeelis (*) Department of Anatomy and Structural Biology, Albert Einstein College of Medicine of Yeshiva University, 1300 Morris Park Avenue, Bronx, NY 10461, USA e-mail: msidani@aecom.yu.edu J. Wyckoff : J. Condeelis Analytical Imaging Facility, Albert Einstein College of Medicine of Yeshiva University, 1300 Morris Park Avenue, Bronx, NY 10461, USA