Lymphatic vessel density and function in experimental bladder cancer.
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-kappaB-responsive promoter (kappaB-lacZ) exhibiting constitutive activity of beta-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.


CONCLUSIONS
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.
Department of Physiology, College of Medicine, Oklahoma University Health Sciences Center (OUHSC), Oklahoma City, OK 73104, USA. marcia-saban@ouhsc.edu
Marcia R
MR
Saban
Rheal
R
Towner
Nataliya
N
Smith
Andrew
A
Abbott
Michal
M
Neeman
Carole A
CA
Davis
Cindy
C
Simpson
Julie
J
Maier
Sylvie
S
Mémet
Xue-Ru
XR
Wu
Ricardo
R
Saban
D000818:Animals; D014408:Biomarkers, Tumor / Q000032:analysis*; D002278:Carcinoma in Situ / Q000175:diagnosis* / Q000276:immunology / Q000473:pathology*; D004195:Disease Models, Animal; D006023:Glycoproteins / Q000032:analysis / Q000276:immunology; D007150:Immunohistochemistry; D042583:Lymphangiogenesis; D042601:Lymphatic Vessels / Q000000981:diagnostic imaging* / Q000378:metabolism / Q000473:pathology*; D008220:Lymphography / Q000379:methods; D008279:Magnetic Resonance Imaging; D008565:Membrane Proteins / Q000032:analysis / Q000276:immunology; D026901:Membrane Transport Proteins; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D008856:Microscopy, Fluorescence; D019408:Platelet Endothelial Cell Adhesion Molecule-1 / Q000032:analysis; D019265:Spectroscopy, Near-Infrared; D001743:Urinary Bladder / Q000473:pathology; D001749:Urinary Bladder Neoplasms / Q000175:diagnosis* / Q000276:immunology / Q000473:pathology*; D060170:Uroplakin II
D014408:Biomarkers, Tumor; D006023:Glycoproteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D019408:Platelet Endothelial Cell Adhesion Molecule-1; D060170:Uroplakin II; C554659:Upk2 protein, mouse; C492273:LYVE1 protein, mouse