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Open Access Highly Accessed Research article

Lymphatic vessel density and function in experimental bladder cancer

Marcia R Saban1*, Rheal Towner2, Nataliya Smith2, Andrew Abbott2, Michal Neeman3, Carole A Davis1, Cindy Simpson1, Julie Maier4, Sylvie Mémet5, Xue-Ru Wu6 and Ricardo Saban1

Author Affiliations

1 Department of Physiology, College of Medicine, Oklahoma University Health Sciences Center (OUHSC), Oklahoma City, OK 73104, USA

2 Small Animal MRI Core Facility, Free Radical Biology & Aging, Oklahoma Medical Research Foundation (OMRF), Oklahoma City, OK 73104, USA

3 Department of Biological Regulation, Weizmann Institute of Science, Rehovot, 76100, Israel

4 Oklahoma Medical Research Foundation (OMRF), Imaging Core Facility, Oklahoma City, Oklahoma 73104, USA

5 Unité de Mycologie Moléculaire, URA CNRS 3012, Institut Pasteur, 75724 Paris Cedex 15, France

6 Department of Urology, New York University Medical School, New York, NY 10016, USA

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BMC Cancer 2007, 7:219  doi:10.1186/1471-2407-7-219

Published: 29 November 2007

Abstract

Background

The lymphatics form a second circulatory system that drains the extracellular fluid and proteins from the tumor microenvironment, and provides an exclusive environment in which immune cells interact and respond to foreign antigen. Both cancer and inflammation are known to induce lymphangiogenesis. However, little is known about bladder lymphatic vessels and their involvement in cancer formation and progression.

Methods

A double transgenic mouse model was generated by crossing a bladder cancer-induced transgenic, in which SV40 large T antigen was under the control of uroplakin II promoter, with another transgenic mouse harboring a lacZ reporter gene under the control of an NF-κB-responsive promoter (κB-lacZ) exhibiting constitutive activity of β-galactosidase in lymphatic endothelial cells. In this new mouse model (SV40-lacZ), we examined the lymphatic vessel density (LVD) and function (LVF) during bladder cancer progression. LVD was performed in bladder whole mounts and cross-sections by fluorescent immunohistochemistry (IHC) using LYVE-1 antibody. LVF was assessed by real-time in vivo imaging techniques using a contrast agent (biotin-BSA-Gd-DTPA-Cy5.5; Gd-Cy5.5) suitable for both magnetic resonance imaging (MRI) and near infrared fluorescence (NIRF). In addition, IHC of Cy5.5 was used for time-course analysis of co-localization of Gd-Cy5.5 with LYVE-1-positive lymphatics and CD31-positive blood vessels.

Results

SV40-lacZ mice develop bladder cancer and permitted visualization of lymphatics. A significant increase in LVD was found concomitantly with bladder cancer progression. Double labeling of the bladder cross-sections with LYVE-1 and Ki-67 antibodies indicated cancer-induced lymphangiogenesis. MRI detected mouse bladder cancer, as early as 4 months, and permitted to follow tumor sizes during cancer progression. Using Gd-Cy5.5 as a contrast agent for MRI-guided lymphangiography, we determined a possible reduction of lymphatic flow within the tumoral area. In addition, NIRF studies of Gd-Cy5.5 confirmed its temporal distribution between CD31-positive blood vessels and LYVE-1 positive lymphatic vessels.

Conclusion

SV40-lacZ mice permit the visualization of lymphatics during bladder cancer progression. Gd-Cy5.5, as a double contrast agent for NIRF and MRI, permits to quantify delivery, transport rates, and volumes of macromolecular fluid flow through the interstitial-lymphatic continuum. Our results open the path for the study of lymphatic activity in vivo and in real time, and support the role of lymphangiogenesis during bladder cancer progression.