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Mouse model of lymph node metastasis via afferent lymphatic vessels for development of imaging modalities.

ABSTRACT: Animal studies of lymph node metastasis are constrained by limitations in the techniques available for noninvasive monitoring of the progression of lymph node metastasis, as well as difficulties in the establishment of appropriate animal models. To overcome these challenges, this study has developed a mouse model of inter-lymph-node metastasis via afferent lymphatic vessels for use in the development of imaging modalities. We used 14- to 18-week-old MRL/MpJ-/lpr/lpr (MRL/lpr) mice exhibiting remarkable systemic lymphadenopathy, with proper axillary lymph nodes (proper-ALNs) and subiliac lymph nodes (SiLNs) that are 6 to 12 mm in diameter (similar in size to human lymph nodes). When KM-Luc/GFP malignant fibrous histiocytoma-like cells stably expressing the firefly luciferase gene were injected into the SiLN, metastasis could be detected in the proper-ALN within 3 to 9 days, using in vivo bioluminescence imaging. The metastasis route was found to be via the efferent lymphatic vessels of the SiLN, and metastasis incidence depended on the number of cells injected, the injection duration and the SiLN volume. Three-dimensional contrast-enhanced high-frequency ultrasound imaging showed that the blood vessel volume and density in the metastasized proper-ALN significantly increased at 14 days after tumor cell inoculation into the SiLN. The present metastasis model, with lymph nodes similar in size to those of humans, has potential use in the development of ultrasound imaging with high-precision and high-sensitivity as well as other imaging modalities for the detection of blood vessels in lymph nodes during the progression of metastasis.

SUBMITTER: Li L

PROVIDER: S-EPMC3565997 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundLymph node metastasis constitutes a key event in tumor progression. The molecular control of this process is poorly understood. Heparan sulfate is a linear polysaccharide consisting of unique sulfate-modified disaccharide repeats that allow the glycan to bind a variety of proteins, including chemokines. While some chemokines may drive lymphatic trafficking of tumor cells, the functional and genetic importance of heparan sulfate as a possible mediator of chemokine actions in lymphatic metastasis has not been reported.ResultsWe applied a loss-of-function genetic approach employing lymphatic endothelial conditional mutations in heparan sulfate biosynthesis to study the effects on tumor-lymphatic trafficking and lymph node metastasis. Lymphatic endothelial deficiency in N-deacetylase/N-sulfotransferase-1 (Ndst1), a key enzyme involved in sulfating nascent heparan sulfate chains, resulted in altered lymph node metastasis in tumor-bearing gene targeted mice. This occurred in mice harboring either a pan-endothelial Ndst1 mutation or an inducible lymphatic-endothelial specific mutation in Ndst1. In addition to a marked reduction in tumor metastases to the regional lymph nodes in mutant mice, specific immuno-localization of CCL21, a heparin-binding chemokine known to regulate leukocyte and possibly tumor-cell traffic, showed a marked reduction in its ability to associate with tumor cells in mutant lymph nodes. In vitro modified chemotaxis studies targeting heparan sulfate biosynthesis in lymphatic endothelial cells revealed that heparan sulfate secreted by lymphatic endothelium is required for CCL21-dependent directional migration of murine as well as human lung carcinoma cells toward the targeted lymphatic endothelium. Lymphatic heparan sulfate was also required for binding of CCL21 to its receptor CCR7 on tumor cells as well as the activation of migration signaling pathways in tumor cells exposed to lymphatic conditioned medium. Finally, lymphatic cell-surface heparan sulfate facilitated receptor-dependent binding and concentration of CCL21 on the lymphatic endothelium, thereby serving as a mechanism to generate lymphatic chemokine gradients.ConclusionsThis work demonstrates the genetic importance of host lymphatic heparan sulfate in mediating chemokine dependent tumor-cell traffic in the lymphatic microenvironment. The impact on chemokine dependent lymphatic metastasis may guide novel therapeutic strategies.

Project description:Purpose: Sentinel lymph node biopsy (SLNB) has emerged as the preferred standard procedure in patients with breast cancer, melanoma and other types of cancer. Herein, we developed a method to intra-operatively map SLNs and differentiate tumor metastases within SLNs at the same time, with the aim to provide more accurate and real-time intraoperative guidance. Experimental Design: Hyaluronic acid (HA), a ligand of lymphatic vessel endothelial hyaluronan receptor (LYVE)-1, is employed as a SLN mapping agent after being conjugated with a near-infrared fluorophore (Cy5.5). Different sized HAs (5, 10 and 20K) were tested in normal mice and mice with localized inflammation to optimize LN retention time and signal to background ratio. Cetuximab, an antibody against epidermal growth factor receptor (EGFR), and trastuzumab, an antibody against human epidermal growth factor receptor 2 (HER2), were labeled with near-infrared fluorophore (IRDye800) for detecting metastatic tumors. LN metastasis model was developed by hock injection of firefly luciferase engineered human head neck squamous carcinoma cancer UM-SCC-22B cells or human ovarian cancer SKOV-3 cells. The metastases within LNs were confirmed by bioluminescence imaging (BLI). IRDye800-Antibodies were intravenously administered 24 h before local administration of Cy5.5-HA. Optical imaging was then performed to identify nodal metastases. Results: Binding of HA with LYVE-1 was confirmed by ELISA and fluorescence staining. HA with a size of 10K was chosen based on the favorable migration and retention profile. After sequential administration of IRDye800-antibodies intravenously and Cy5.5-HA locally to a mouse model with LN metastases and fluorescence optical imaging, partially metastasized LNs were successfully distinguished from un-metastasized LNs and fully tumor occupied LNs, based on the different signal patterns. Conclusions: Fluorophore conjugated HA is a potential lymphatic mapping agent for SLNB. Dual-tracer imaging with the combination of lymphatic mapping agents and tumor targeting agents can identify tumor metastases within SLNs, thus may provide accurate and real-time intra-operative guidance to spare the time spent waiting for a biopsy result.

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Mouse model of lymph node metastasis via afferent lymphatic vessels for development of imaging modalities.

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