Lymphatic vessel density and function in experimental bladder cancer
© Saban et al; licensee BioMed Central Ltd. 2007
Received: 28 September 2007
Accepted: 29 November 2007
Published: 29 November 2007
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.
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.
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.
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.
De novo lymphangiogenesis influences different pathological courses via modulating tissue fluid homeostasis, macromolecule absorption, and leukocyte transmigration . In addition, lymphatic vessels play a crucial role in a variety of human cancers . Invasion of lymphatic vessels by tumor cells and subsequent development of lymph node metastases significantly influences the prognosis of cancer patients and, therefore, represents an integral part of tumor staging. Increasing knowledge of the tumor's biological significance in lymphatics within the tumors and at the tumor periphery has greatly promoted understanding of tumor access into the lymphatic system by inducing lymphangiogenesis or by co-opting preexisting lymphatics . In contrast, impaired functioning of lymphatic vessels results in lymphedema as observed during breast cancer diagnosis and treatment [3–5].
During cancer progression, a bi-directional communication is established between the tumor microenvironment (TME) and lymphatic vessels. In one direction, the lymphatic vasculature alters TME by draining the interstitial protein-rich exudate fluid (lymph) into the bloodstream. In another direction, inflammation influences the composition and pressure of TME leading to altered lymphatic vessel function.
We choose to study bladder cancer because it represents 2% of all human malignancies. Urothelial carcinoma is one of the most common cancers – it ranks fifth among all cancers in the Western world, and there are 336,000 new cases and 132,000 deaths annually worldwide . In the US alone, the American Cancer Society estimates that 50,040 men and 17,120 women will be diagnosed, and 13,060 men and women will die of cancer of the urinary bladder in 2007 . Although the role of lymphatic vessels during bladder cancer progression is remarkably unknown, invasion of lymphatics during bladder cancer has been reported , whereas in prostate cancer there is a decrease in intratumoral lymphatic vessel density . More recently, Fernandez and collaborators published the first manuscript suggesting the existence of proliferating lymph vessels and, therefore, of lymphangiogenesis in bladder transitional cell carcinoma (TCC), and proposed strong correlation of higher peritumoral LVD with the presence of lymph nodes in clinically localized invasive bladder TCC . However, up to now, no animal model was available for a systematic study of lymphatic vessel density and function during bladder cancer progression.
We previously described that a transgenic mouse (κB-lacZ) with a reporter gene (lacZ) for NF-κB presented constitutive β-galactosidase (β-gal) activity in all lymphatic endothelial cells  and that these mice serve a dual purpose by permitting both visualization of lymphatics and detection of constitutive and inducible NF-κB activity . To study in-depth the role of lymphatic vessels in bladder cancer progression, we generated a double transgenic mouse (SV40-lacZ) by crossing the κB-lacZ mice with a well established model of bladder cancer (UPKII/SV40T) [12, 13]. Here, we demonstrate that these SV40-lacZ mice present an increased lymphatic vessel density during bladder cancer progression.
We also show that a new compound, coined Gd-Cy5.5, which corresponds to a recently described contrast agent (biotin-BSA-Gd-DTPA) [14–16] conjugated to Cy5.5 permits dual imaging by near-infrared fluorescent (NIRF) and MRI. We, therefore, provide proof-of-concept that this contrast agent can be used for determination of lymphatic vessel function during bladder cancer progression.
All experiments were performed according to the "Principles for Research Involving Animals and Human Beings Guidelines" (OUHSC Animal Care & Use Committee protocol # 04–028). Double transgenic mice were obtained by crossing κB-lacZ with UPKII/SV40T mice. The κB-lacZ transgenic model used in this study was first described in 1996  and enriched in the C57Bl/6 background. It was constructed using the promoter of the gene encoding p105, a precursor of the p50 subunit of NF-κB. This promoter contains three NF-κB binding sites in its proximal part driving the expression of lacZ with a nuclear localization sequence. These mice permit the visualization of lymphatic endothelial cells in the urinary bladder and all other tissues examined so far .
A double transgenic mouse model (SV40-lacZ) was generated by crossing the κB-lacZ transgenics with UPKII/SV40T mice. UPKII/SV40T mice present the SV40 large T antigen under the control of a urothelium-specific mouse uroplakin II promoter, and develop specifically bladder cancer [12, 18, 19]. The phenotype of each mouse was confirmed by southern blot analysis of SV40T and PCR for β-galactosidase in tail vein snips. Only double positive mice were used in these experiments. FVB mice were purchased from Jackson Labs, crossed with κB-lacZ, and used as controls for UPKII/SV40T mice.
Lymphatic Vessel Density (LVD)
We follow the "Reporting Recommendations for tumor Marker (REMARK)" guidelines . This consensus report aims to lower the methodological variability of lymphangiogenesis quantification in tissue sections. The recommendations include: 1) double-blinded experiments; 2) the use of multiple IHC stains on serial sections (We used LYVE-1 IHC that has been established in our laboratories  and X-gal staining for β-galactosidase, because the SV40-lacZ enable this stain for visualization of lymphatic vessels ); and 3) the use of a double immunostaining of lymphatic marker and those for cell proliferation. In this context, bladder cross-sections were double stained with LYVE-1 and Ki-67 antibodies.
LVD in bladder whole-mounts
LVD was determined as described before [11, 17]. Eight mice per point were euthanized with sodium pentobarbital (100 mg/kg, i.p.) and tissues were removed rapidly and stained as whole mounts with X-gal. β-galactosidase activity was revealed by X-gal staining at 30°C for 4–6 hours. Whole mounts were examined under a dissecting scope (SMZ 1500, Nikon). All tissues were photographed by a digital camera (DXM1200; Nikon). Exposure times were held constant when acquiring images from different tissues. Afterwards, tissues were washed four times in PBS and post-fixed in 2% paraformaldehyde in PIPES [piperazine diethanesulfonic acid]. Lymphatic vessel density was quantified by morphometric analysis using Neurolucida workstation (MicroBrightField, Inc) , as described (ref ; supplemental material). Morphometric analysis was performed by importing Neurolucida tracings into NeuroExplorer software version 3.70.2 (MicroBrightField, Inc) . The ratio between the area occupied by lymphatics and the total tissue area (μm2) was then determined for each section and statistical analysis was performed using a Wilcoxon's rank sum test. Results are expressed as mean ± SEM. In all cases, a value of p < 0.05 was considered indicative of a significant difference . This method may result in the overestimation of the lymphatic area. The software uses the 3D surface area of lymphatics, estimated from a 2D projection. Also, tissue area was calculated from a 2D projected cross-sectional tissue area (tissue contour).
Primary antibody characteristics
Santa Cruz 
MAC 387 (calprotein Ab-1) monoclonal
Lab Vision 
LVD and image analysis of bladder cross-sections
At least 6 random fields per cross-section were visualized at 20× magnification and used for image analysis that was performed with the NIS-Elements Advanced Research 2.3 imaging software . This software identifies signal by thresholding key intensity values. Further the software permits imposing restrictions to the measurements by excluding false positive signals. Briefly, the number of positive cells expressing a particular antibody was calculated as percent of the region of interest (ROI), as indicated in the individual figure legend. Co-localization of two antibodies was calculated by converting the area occupied by cells positive for the first antibody into a ROI. Then the percent of cells that were positive for the second antibody was calculated within the ROI. Results are expressed as mean ± SEM. In all cases, a value of p < 0.05 was considered indicative of a significant difference .
Mice were fed a low-chlorophyll diet for 2 weeks to reduce auto-fluorescence in the intestinal region  and the abdominal hair was removed. Mice were anesthetized with isoflurane, placed in a heating pad, and received 200 μL of the Gd-Cy5.5 intravenously, and the accumulation of Cy5.5 into the urinary bladder was followed over time. Anesthetized mice were immediately placed on a heating pad inside a FluorChem HD2 (Alpha Innotech, San Leandro, CA) equipped with a Chromalight® multi-wavelength illuminator and a 4-million pixel Cooled camera (F2.8 manual zoon lens and F1.4 fixed lens) coupled to a dedicated computer. The FluorChem cabinet permits continued anesthesia with isoflurane. Images were first acquired and stored with AlphaEase FC® 32-bit software (Alpha Innotech, San Leandro, CA) and subsequently, application of black-and-white and color gradients were performed in Adobe Photoshop® CS3 extended  that permitted the determination of integrated density. For this purpose, an elliptical marquee of fixed size (180 × 180 px) was used to determine the region of interest (ROI) around the luminescence zones corresponding to a bladder area and the count tool was used to determine and record each integrated density. The integrated density corresponded to the sum of the values of the pixels in the ROI, which were equivalent to the product of the area (in pixels) and mean gray value.
All data was acquired using a Bruker 7 Tesla/30 cm horizontal bore magnet. Anatomical scans acquired proved to be useful in elucidating normal and bladder tumor tissue. Bladder and tumor physiology was followed in a time course study using this method at each time point, for each animal. This method is a dual spin-echo technique modified from a MSME (Multi-Slice Multi-Echo) technique designed to yield anatomical T1 and T2 weighted images for each slice position in the slice package. Axial scans acquired had the following parameters: TR = 900 ms, TE = 11.6 ms, slice thickness = 0.75 mm, NA = 4, slice gap = 0.05 mm, FOV = 2.5 cm × 2.5 cm, matrix size = 256 × 256, giving an in-plane resolution of 98 μm × 98 μm. T1-weighted images had an effective TE of 17 ms, and T2-weighted images had an effective TE of 52 ms. Acquired parameter images had TE = 15 ms over a range of 5 TR increments with values = (100, 300, 450, 600, 850, and 1150 ms), slice thickness = 0.75 mm (position of slice varied according to tumor location), FOV = 2.5 cm × 2.5 cm, NA = 2, matrix size = 128 × 128, and an in-plane resolution of 195 μm × 195 μm. Mono-exponential fits were utilized to calculate actual T1 values in post-processing.
Synthesis of biotin-BSA-GdDTPA compound
The macromolecular contrast material, biotin-BSA-GdDTPA, was prepared by the modification of the method of Dafni and collaborators . Bovine serum albumin (BSA, 0.5 g, 8 μmol; Sigma) was dissolved in 0.1 M sodium bicarbonate (7.5 ml, pH 8.5). Sulfo-NHS-Biotin (22.4 mg; 53 μmol; Pierce) was dissolved in double distilled water (DDW, 1.2 ml) and was added to BSA while stirring. The reaction mixture was stirred for 1 hr at 4°C and an additional 2 hrs at room temperature. The dialyzed product in 0.1 M Hepes buffer (pH 8.8) was reacted with diethylene triamine pentaacetic acid anhydride (DTPA, 0.5 g, 1.4 mmol; Sigma) suspended in 2.5 ml of dimethyl sulfoxide (DMSO) at room temperature. DTPA was added in portions and the pH was adjusted immediately after each addition to 8.5 with 5 N NaOH. The reaction mixture was stirred for 2 hrs at 4°C and extensively dialyzed against cold 0.1 M citrate buffer (pH 6.5). Finally, gadolinium (III) chloride (GdCl3, 0.25 g; 0.67 mmol; Sigma) in 2.5 ml 0.1 M sodium acetate buffer (pH 6.0) was added gradually, and the mixture was stirred for 24 hrs at 4°C. The product, biotin-BSA-GdDTPA, was extensively dialyzed against cold citrate buffer (0.1 M, pH 6.5) and then against DDW. The product was lyophilized and stored at 4°C. For Cy5.5 -Gd preparation, 10 mg of dry lyophilized product (biotin-BSA-GdDTPA) was reconstituted in 150 μl of sodium bicarbonate buffer 0.1 M pH 8.8 NaHCO3. Cy5.5 dye (Cy5.5 mono functional reactive dye, Amersham Biosciences) was dissolved in 10–20 μl of DMF and added to the product. The mixture was incubated in the dark at RT for 1 hr while mixing. The final compound biotin-BSA-GdDTPA-Cy5.5 was purified using Zeba 2 ml columns and had a molecular weight of ~82 kDa.
Dextran (500,000 MW)-Cy5.5 (Dex 0003–5) was purchased from Nanocs .
Visualization of bladder lymphatics
Increased lymphatic vessel density (LVD) during mouse BC progression
Bladder cancer-induced lymphangiogenesis
Lymphatic Vessel Function (LVF) by NIRF
For NIRF, a total of 9 SV40-lacZ mice ages 6–11 months were used. Mice were fed a low-chlorophyll diet for 2 weeks to reduce auto-fluorescence in the intestinal region  and the abdominal hair was removed. Mice were anesthetized with isofluorane and received 200 μL Gd-Cy5.5 intravenously (dose of 500 mg/kg), and the accumulation of Cy5.5 into the urinary bladder was followed over time.
Co-localization of Cy5.5 with LYVE-1 positive lymphatics and CD31-positive blood vessels
In particular Figures 6 A–B and 7 A–B indicate the sub-urothelial localization of blood vessels whereas lymphatic vessels are seen in the deeper regions of the mucosa but not in the subepithelial layer. Figures 8 A–D indicate that, at 1140 minutes, a significant amount of Cy5.5 still remains in the bladder parenchyma which may explain the fluorescence observed in Figure 5K.
Image analysis indicates that at all time points the area of the bladder cross-sections occupied by CD31-positive blood vessels was greater than the area occupied by LYVE-1-positive lymphatic vessels (Figure 9). In terms of co-localization of Cy5.5, at 120 minutes, Cy5.5 was found evenly distributed between LYVE-1-positive lymphatics and CD31-positive blood vessels (Figure 9). At 330 minutes a great proportion of Cy5.5 was found within LYVE-positive lymphatic vessels, and at 1140 minutes, most of the Cy5.5 was found within lymphatic vessels (Figures 9). These results provided the basis for subsequent MRI analysis of lymphatic vessel function.
NIRF visualization of abdominal large lymphatic vessel draining the urinary bladder
Although this work is very preliminary, it permitted our laboratory to identify and measure lymphatic function in vivo and in real time. These results indicate that NIRF can be use to study how Cy5.5-conjugate compounds are distributed between the interstitial fluid, blood vessels, and lymphatics. Figure 5K illustrates the kinetics of Gd-Cy5.5 and permitted the follow up of lymphatic vessel function by MRI (see below). In addition, large collecting lymphatics can be identified, the propulsion of lymph within lymphatic vessels can be visualized, and movies can be used to study these dynamics (Figure 11). Finally, NIRF-guided capture permitted us to start observing collecting lymphatics and lymph sacs and, therefore, to further study their structures.
Magnetic Resonance Imaging of mouse urinary bladder cancer
Thanks to a unique rodent-dedicated MRI facility on campus , we were able to meet two major goals regarding the SV40-lacZ mice. The first goal was to detect the presence of BC as early as possible. This permitted a longitudinal study of tumor sizes during cancer progression. The second goal was to determine whether the observed increase in LVD was accompanied by an increased lymphatic function. A total of 7 SV40-lacZ mice and 3 wild type controls (FVB crossed with κB-lacZ mice) entered the study. Out of the 7 SV40-lacZ mice, 5 developed bladder tumors and 1 had to be euthanized.
Dynamic contrast-enhanced MRI and Gd-Cy5.5 for determination of LVF
Our results with SV40-lacZ mice indicate that in the normal bladder, a rich lymphatic vessel network is visible from the adventitia through the detrusor smooth muscle. It is characterized as a vascular network of blind ended, thin-walled capillaries that merge to larger collecting ducts, all positively stained with LYVE-1 antibody. In sharp contrast with the dense blood vessel vascularization of the bladder mucosa, we found that lymphatic vessels are absent of the sub-urothelium being located much deeper in the lamina propria (Figures 6A, 6B, 7A, and 7B) which seems to parallel the clinical findings in the human bladder [35, 36].
Our analysis indicates an increased lymphatic density during cancer progression and a co-localization of Ki-67 with some of LYVE-1-positive lymphatics, suggesting cancer-induced lymphangiogenesis (Figure 4), as it was recently suggested in human bladder cancer [10, 37]. However, the mechanisms for bladder lymphangiogenesis are not clear. In contrast to blood vessel angiogenesis, the mechanisms of lymphangiogenesis in general are still relatively vague . Vascular endothelial growth factor-C (VEGF-C) and VEGF-D have been implicated as specific regulators of lymphangiogenesis [39–43]. Both growth factors mediate their biological activity mainly by VEGF receptor-3 (VEGFR-3, Flt-4) [44, 45]. It remains to be determined whether VEGF-C and VEGF-D along with VEGFR-3 play a role in lymphangiogenesis during bladder cancer development. Another interesting line of research involves LYVE-1-positive tumor associated macrophages  that have indeed been associated with tumor lymphangiogenesis [47, 48]. In the eye, a stepwise mechanism of inflammation-associated de novo lymphangiogenesis involves potential lymphatic progenitor cells  derived from circulation that transmigrate through the connective tissue stroma, presumably in the form of macrophages [49–51], and finally incorporate into the growing lymphatic vessels . Our present findings indicate that in addition to LYVE-1-positive lymphatic endothelial cells, during bladder cancer development, LYVE-1 positive macrophages were found in the bladder detrusor muscle isolated from SV40-lacZ mice. Although this may be only a circumstantial finding, it opens a testable hypothesis on the role of macrophages and other inflammatory cells in bladder lymphangiogenesis. The introduction of SV40-lacZ along with the visualization and quantification techniques described here will permit further investigation on this subject.
It has been proposed that lymphangiogenesis is correlated with tumor metastasis. Increasing knowledge of the tumor's biological significance in lymphatics within the tumors (intratumoral lymphatics, ITLs) and at the tumor periphery (peritumoral lymphatics, PTLs) has greatly promoted understanding of tumor access into the lymphatic system by inducing lymphangiogenesis or by co-opting preexisting lymphatics . Indeed, peritumoral lymphatics have also been associated with both regional metastasis and survival in bladder , lung , breast , and prostate cancer . But the question still remains as to whether pre-existing vessels are sufficient to serve this function, or whether tumor cell dissemination requires de novo lymphatic formation or an increase in lymphatic size. In this regard, Fernandez and collaborators reported that in the human bladder, higher intratumoral LVD correlates significantly with poor histological differentiation, and that higher peritumoral LVD showed a significant association with the presence of lymph node metastasis . Although peritumoral lymphatic vessels contribute to tumor metastasis, opposite views exist as to whether intratumoral lymphatics have any role in tumor metastasis [56, 57]. Padera and collaborators examined functional lymphatics associated with mouse tumors expressing normal or elevated levels of VEGF-C . Although VEGF-C over-expression increased lymphatic surface area in the tumor margin and lymphatic metastasis, these tumors contained no functional lymphatics, as assessed by four independent functional assays and IHC staining . These findings suggest that the functional lymphatics in the tumor margin alone are sufficient for lymphatic metastasis and should be targeted therapeutically . Our MRI results are in agreement with those described by Pandera and collaborators  in the sense that intratumor lymphatic vessels have a reduced function when compared to normal areas of the bladder.
Another question answered by the present work was whether an increase in the number of lymphatic vessels leads to increased function. For this purpose, we followed the strategy recently reviewed by Neeman and collaborators . The contrast agent introduced here was originally described by Dafni and collaborators [14–16], and subsequently by Pathak and collaborators , for visualization of lymphatics and determination of their function. The modification of conjugating biotin-BSA-Gd-DTPA  to Cy5.5 permitted us to use NIRF and MRI to follow the dynamics of the same compound and calculate lymphatic vessel function. The advantage of using NIRF is the faster time for data acquisition. NIRF information permitted us to narrow the number of time points for subsequent MRI studies.
The proposed mechanism of visualization of BSA-Gd-Cy5.5 (~82 kDa) is based on the presence of BSA which prolongs its lifetime in circulation. Initially the probe is confined to blood vessels and it is systemically distributed, and as the time passes it extravasates to the extracellular space in points of increased vascular permeability. Both NIRF and MRI are able to detect when BSA-Gd-cy5.5 starts to accumulate in the extracellular space and when its accumulation reaches a plateau. This was done at the MRI level by following the longitudinal relaxation rates (1/T1) that correlates with increased uptake of the Gd-probe from the blood vessels into the urinary bladder extra vascular space. This phase was called the "early phase" in this manuscript. After 80 minutes of the plateau, a second series of MRI were started and followed the clearance of BSA- Gd-Cy5.5 from the extravascular by the lymphatic vessels. This was supported by ex vivo images which indicated that, at this point, most of the BSA-Gd-Cy5.5 is drained from the tissue by lymphatic vessels.
Although MRI lymphangiography does measure areas of draining and pooling instead of directly evaluating lymphatic vessel function, the delayed enhancement observed at later time points from the Gd-probe may also reflect uptake of Gd-probe in the lymphatics. In addition, this information can not be obtained any other way and clinical studies attest the validity of using lymphangiography for assessment of lymphatic vessel function [60–64]. A step forward in this direction was introduced here by the use of a single probe that can be imaged by MRI and NIRF. The first indicates areas of draining and pooling and the second permitted the temporal association of images with cross-sections indicating the relative distribution of the probe between CD31-positive blood vessels and LYVE-1-positive lymphatic vessels.
As pointed out by Neeman and collaborators , contrast MRI data are typically dominated by vascular permeability, which often masks the relatively slow lymphatic drain. To separate vascular leakage from the lymphatic drain, an avidin chase needs to be introduced [14, 16] which through the rapid clearance of intravenously administered biotin-BSA-Gd-DTPA, allowed them to experimentally track interstitial convection and lymphatic drain in the absence of continuing vascular leakage [14, 16]. Our present MRI results are not corrected for vascular leakage. Therefore, future experiments will take into consideration this point for a more detailed analysis of lymphatic vessel function using the advantage of the presence of biotin in the Gd-Cy5.5 molecule.
Another advantage of NIRF was to permit the collection of tissues exhibiting fluorescence. In this regard, we were able to further pursue the morphology of lymph sacs using dextran-Cy5.5. This will allow us in the near future to determine the contractility of lymph sacs and abdominal lymphatic vessels. The rhythmic activity observed in the abdominal lymphatics draining the urinary bladder is in agreement with recent results that indicated that mesenteric lymphatic vessels have a true pacemaker mechanism [65, 66]. The experiment described in Figures 10, 11, 12 will permit the capture and evaluation of large lymphatic function in health and disease.
The present work introduces a new double transgenic mouse model that permits the visualization of lymphatic vessels during bladder cancer progression, and introduces new technologies for the visualization and quantification of lymphatic vessel density and function by combining NIRF and MRI imaging. The results presented here raise the possibility of the study of lymphatic vessel activity in vivo and in real time and raises the hypothesis regarding the role of lymphangiogenesis during bladder cancer progression. It remains to be determined whether manipulation of lymphatic vessel density and function would alter bladder tumor progression.
magnetic resonance imaging
- and NIRF:
near infrared fluorescence.
This work was supported the Oklahoma Center for the Advancement of Science & Technology (OCAST) – Project HRO4-135S- (MRS), National Institutes of Health grants 5R01 DK066101-02 (RS) and 5R01 DK055828-05 (RS), and Israel Science Foundation (MN).
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