Near-infrared fluorescence lymphatic imaging in vascular endothelial growth factor-C overexpressing murine melanoma

: In this study we employ a near-infrared fluorescence lymphatic imaging (NIRFLI) technique to longitudinally image spatial and temporal changes in the lymphatics in mice bearing vascular endothelial growth factor (VEGF)-C overexpressing B16F10 (VEGF-C-B16F10) or mock-transduced B16F10 (mock-B16F10) melanoma tumors. Our NIRFLI data show that ICG-laden lymph accumulates into a VEGF-C-B16F10 tumor compared to mock-B16F10 at 3 days post implantation, presumably due to increased lymphatic vessel permeability. Quantification shows a significantly greater percentage of ICG-perfused area in VEGF-C-B16F10 (7.6 ± 2) as compared to MOCK-B16F10 (1 ± 0.5; p = 0.02), which is also confirmed by quantification of the lymphatic leakage of evans blue dye (optical density at 610nm; VEGF-C-B16F10, 10.5 ± 2; mock-B16F10, 5.1 ± 0.5; p = 0.009); thereafter, lymphatic leakage is visualized only in the peritumoral region. Our imaging data also show that anti-VEGF-C treatment in VEGF-C-B16F10 restores normal lymphatic vessel integrity and reduces dye extravasation. Because NIRFLI technology can be used to non-invasively detect lymphatic changes associated with cancer, it may provide a new diagnostic to assess the lack of lymphatic vessel integrity that promotes lymphovascular invasion and to assess therapies that could through of the lymphatic vasculature.


Introduction
Tumor-associated lymphatic vessel networks undergo significant changes in response to tumor cells, such as lymphatic vessel dilation and leakiness, and sprouting from pre-existing vessels [1,2]. These structural features of tumor lymphatic vessels might make them more susceptible for invasion by malignant cells, resulting in the increased probability of lymphatic metastasis [2]. One of the key lymphangiogenic factors for these changes is vascular endothelial growth factor (VEGF)-C, which has been shown to be critical for the proliferation of lymphatic endothelial cells (LECs) and initial lymphatic vessel sprouting [2].
VEGF-C binds to VEGF receptor (VEGFR)-3, which is predominantly expressed on lymphatic vessels [3]. Overexpression of VEGF-C in cancer cells induces tumor lymphangiogenesis and enhances tumor spread to the regional draining LNs in several mouse models of cancer [4]. Previous studies demonstrate that mice bearing VEGF-C overexpressing tumor show an increase in regional LN metastasis, retrograde lymph flow direction, and an increased number of dilated but functional peri-tumoral lymphatic vessels [5,6]. None of these studies provides longitudinal data showing when and how structural changes of the lymphatics occur in response to VEGF-C overexpressing tumor growth. Moreover, despite the importance of lymphatic vascular permeability in pathophysiological conditions [7], there are limited techniques to image lymphatic leakage due to enhanced permeability in vivo.
agent [8]. In this study, we investigate how VEGF-C impacts the lymphatics imaged by NIRFLI, longitudinally assessing the lymphatics in the hindlimb of mice where VEGF-C overexpressing B16F10 (VEGF-C-B16F10) or mock-transduced B16F10 (MOCK-B16F10) is implanted. Our data demonstrates that dynamic and longitudinal NIRFLI assessment of the lymphatic system may provide a companion diagnostic for therapies that seek to interrupt metastasis through arresting lymphangiogenesis.

Cells and mice
VEGF-C-and mock-B16F10 cells were kindly provided by Dr. Timothy Padera at Massachusetts General Hospital and Harvard Medical School. To transfect VEGF-C-and MOCK-B16F10 cells expressing iRFP gene reporter (iRFP-VEGF-C-B16F10 and iRFP-MOCK-B16F10, respectively), cells were cultured as monolayer in DMEM-F12/10% fetal bovine serum (FBS, BioExpress, Kaysville, UT, USA). At near confluency, the culture was transfected with piRFP plasmid (Addgene, Cambridge, MA, USA) by Lipofectamine 2000 (Invitrogen, Grand Island, NY, USA) as suggested by the manufacturer. Transfected cells were grown under 0.8 mg/ml G418 selection in DMEM-F12/10% FBS growing medium. Transfected cells that survived the antibiotic selection were then sorted through flow cytometry outfitted with 690 nm/730 nm (excitation/emission) wavelengths to obtain the population of high iRFP expressers.
Six to eight week old female C57BL6 mice (Charles River, Wilmington, MA) were housed and fed sterilized pelleted food and sterilized water at the Brown Foundation Institute of Molecular Medicine at the University of Texas Health Science Center -Houston (UTHSC-H). All experiments were performed in accordance with the guidelines of the Institutional Animal Care and Use Committee of UTHSC-H.

Blocking antibody and treatment
A neutralizing rat monoclonal antibody specific for mouse VEGFR-3 (n = 6; mF4-31C1; 800 μg/mouse; ImClone Systems Inc., New York, NY) or control rat IgG (n = 5; 800 μg/mouse; Antibodies incorporated, Davis, CA) was administered at the time of tumor cell injection and every second day.

In vivo fluorescence imaging
Mice were imaged for baseline information with i.d. injection of 10 μl of 645 μΜ of ICG (Akorn, Inc. Buffalo Grove, IL) using 31 gauge needles (BD Ultra-FineTM II Short Needle, Becton and Dickinson Medical, Franklin, NJ). After baseline imaging, iRFP expressing or non-expressing VEGF-C-or mock-B16F10 cells (5 x 10 5 ) in 10 μl PBS were inoculated intradermally into the left hindlimb and thereafter, tumor volume was longitudinally measured using a digital caliper. Tumor volume (mm 3 ) was calculated using the following formula: 0.52 x D1 2 x D2, where D1 and D2 are short and long tumor diameters, respectively. NIRFLI with i.d. injection of 10ul of ICG was performed longitudinally at 3, 7, and 10 days post tumor implantation (p.i.). Therefore, mice were injected four times with ICG (at baseline, and day 3, 7, and 10). In addition, in order to explore whether increased vessel permeability seen with ICG was evident with high MW vascular agents known not to extravasate from intact vasculatures, a subset of mice (n = 2) were injected with 10 μl (10 mg/ml) of FITC-Dextran (2M Da; Sigma) several millimeters proximally away from the ICG injection site at 3 days p.i.. For imaging FITC-Dextran, an Argon-Krypton laser system (50mW, 488nm) was used to illuminate mice. Bandpass (510nm center wavelength) and holographic filters (488nm center wavelength) were used to collect re-emitted fluorescence light and reject the excitation light, respectively. A series of sequential NIRF and FITC-Dextran images were acquired with 200ms exposure time immediately before and for up to 20 min after i.d. injection. NIRF and iRFP images A macrolens to zoom in on anesthetized w

Measure
Two µl of Ev tail 3 days aft injection, anim 1 ml of form (OD610; abso The perce addition, a fix fluorescence fluorescence i

Statistics
Data were p performed wi D'Agostino a was used for comparisons t  owing i.d. injec hown in the hi N), which is co ssels (Fig. 1) Fig. 1(B)) and the fluorescent intensity profiles in the tumor over time ( Fig. 1(I)). We observed this feature in all VEGF-C-B16F10 bearing mice where fluorescent lymphatic vessels pass through the tumor after i.d. injection to the base of the tail. ICG-laden lymph leaked out of lymphatic vessels at the tumor margin and diffused into the tumor as shown in Visualization 1. Magnified fluorescent images showed that VEGF-C-B16F10 draining lymphatic vessels gradually dilated during tumor progression (insets in Figs. 1(B) -1(D)). In contrast, we could not observe extravasation of ICG into mock-B16F10 as seen in VEGF-C-B16F10. ICG-laden lymph drained along the lymphatic vessels in the skin above mock-B16F10 at 3 days p.i. (Fig. 1(F)) and stained around the tumor margin at later time points (Figs. 1(G) and 1(H)). Quantification of the perfused area shows a significant difference between VEGF-C-and MOCK-B16F10 ( Fig. 1(J)). Lymphatic permeability assay showed significant leakage of EBD in VEGF-C-B16F10 as compared to MOCK-B16F10 (Fig. 2(A)), confirming the in vivo imaging data shown in Fig. 1. There was no significant difference in tumor growth rate between VEGF-C-and mock-B16F10 ( Fig. 2(B)).  Molecular weight can be a key factor in extravascular distribution out of the leaky lymphatic vessels. Therefore, we tested if lymphatic vessel leakage as shown from ICG in Fig. 1

Discussio
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GFR-3 treatm
ted if NIRFI c ainage patterns 2], was injecte or cells. NIRF VEGF-C-over to control IgG 4. A. Representativ 10 (red; n = 9 for e ail. The insets sho arison of mock-B sed area in the tum on c system prov o regional dra develop LN m or site by inv genic growth ssel sprouts wi lymphatic ves as been used to ighly disorgan the ear or ta oral injection. nd did not sho non-invasively during VEGFof ICG and FI early stages an to excess VEG vious studies t small molecula to extralumina in is the preval injected ICG hes 67 kDa. Th sculature [11]. erved with ICG -B16F10, but n ment restores n can image the s. To this end ed intraperitone FI data showed rexpression wa (Fig. 4(B)).  [5]. Although FML is useful to understand changes in lymphatic capillaries and cutaneous lymphatic vessels, clinical application is limited owing to the limited penetration depth of light at visible wavelengths and tissue scattering, and the inability to visualize deeper collecting and conducting lymphatic vessels. We show that ICG leakage in VEGF-C expressing tumors occurred at early stages (Fig.  1). When solid tumors grow, interstitial fluid pressure (IFP) is elevated compared with normal tissues due to mechanical stress generated by tumor cell growth [15]. Although we did not measure IFP at 3 days p.i. (as small as 8 mm 3 in tumor volume), previous data showed that IFP in VEGF-C overexpressing tumors is higher than that in normal tissues, but similar to that in control tumors [5]. Therefore, extravasation of ICG-laden lymph in early stage VEGF-C-B16F10 tumors presented in this study may be due to destabilization of the lymphatic vessel wall by tumor-secreted VEGF-C, while anti-VEGFR-3 treatment significantly normalized these vessels.
In conclusion, we demonstrated our ability to image architectural changes of tumorassociated lymphatics in vivo during tumor progression with i.d. injection of ICG. Increasing the permeability of the lymphatic vasculature is one of the hallmarks of cancer and inflammation. Therefore, a better understanding of changes to lymphatic structure and drainage patterns in disease may provide new strategies to improve drug exposure to targets in the lymphatic system and enhance therapeutic utility. Since technology is already used within investigational studies in the clinic to image the lymphatic system longitudinally [16], NIRFLI may also provide information in lymphatic response to anti-VEGF-C and other therapies.

Funding
National Institutes of Health (R21CA159293).