Fluorescent LYVE-1 antibody to image dynamically lymphatic trafficking of cancer cells in vivo

Abstract

Background: The lymphatic system is a major route for cancer cell dissemination, and a potential target for antitumor therapy. Despite ongoing interest in this area of research, the real-time behavior of cancer cells trafficking in the lymphatic system is poorly understood due to lack of appropriate tools to image this process.

Materials and methods: We have used monoclonal-antibody and fluorescence technology to color-code lymphatic vessels and the cancer cells inside them in a living animal. Monoclonal anti-mouse LYVE-1 antibody was conjugated to a green fluorophore and delivered to the lymphatic system of a nude mouse, allowing imaging of mouse lymphatics. Tumor cells engineered to express red fluorescent protein were then imaged traveling within the labeled lymphatics in real time.

Results: AlexaFluor-labeled monoclonal anti-mouse LYVE-1 created a durable signal with clear delineation of lymphatic architecture. The duration of fluorescent signal after conjugated LYVE-1 delivery was far superior to that of fluorescein isothiocyanate-dextran or control fluorophore-conjugated IgG. Tumor cells engineered to express red fluorescent protein delivered to the inguinal lymph node enabled real-time tracking of tumor cell movement within the green fluorescent-labeled lymphatic vessels.

Conclusions: This technology offers a powerful tool for the in vivo study of real-time trafficking of tumor cells within lymphatic vessels, for the deposition of the tumor cells in lymph nodes, as well as for screening of potential antitumor lymphatic therapies.

Figure 1

Fluorescence imaging of in vivo murine lymphatic system of the anterior abdominal wall after injection of AlexaFluor-conjugated LYVE-1 to the inguinal lymph node. The inguinal (a, large arrow) and axillary (a, small arrow) lymph nodes, as well as the connecting lymphatics of the anterior abdominal wall (a-c) reveal durable fluorescent staining after delivery of AlexaFluor-conjugated monoclonal anti-mouse LYVE-1 antibody. Neighboring blood vessels did not stain (b, c).

Figure 2

In vivo time course of fluorescence imaging of mouse lymphatics labeled with a one-time dose of fluorescent-labeled anti-LYVE1 antibody versus control fluorescent-labeled IgG or FITC-dextran. The hours post-delivery are denoted to the left of the images. All scale bars represent 0.1mm. While there is comparable fluorescence within the lymphatics immediately after antibody or FITC delivery (a-c), after four hours the unbound compound has washed away, leaving a strong positive signal only within the LYVE-1 labeled lymphatics (d-f). The small amount of nonspecific binding present within the conjugated IgG lymphatics at 4 hours post delivery (e) was completely gone by 12 hours after delivery (h). After a single administration of conjugated anti-LYVE1 antibody, the fluorescent signal remained present for up to 48 hours in the living animal (p). See the Materials and Methods section for antibody labeling procedures and means of in vitro delivery.

Figure 3

Fluorescent images of nude mouse axillary lymph node (a-c) and mesentery (d) stained with AlexaFluor-labeled anti-LYVE1 monoclonal antibody. Stained lymphatic tissue was imaged using a fluorescent inverted microscope at 10× (d), 20× (c) and 40× (a, b). Fluorescence microscopy showed staining of lymphatic vessels at the lymph node periphery with sparing of the follicles (a-c). Lymphatic vessels showed positive fluorescence while neighboring arteries and veins within the tissue did not stain (d). Binding of conjugated LYVE-1 to the lymphatic endothelium in vivo outlines vessel walls and valves (e, f).

Figure 4

Sequential images of cancer cells traveling through anterior abdominal wall lymphatics. Following administration of conjugated LYVE-1, the pancreatic cancer cell line XPA-1 RFP was injected into the inguinal lymph node. Red fluorescent XPA-1 cells, both individually (small arrows) and in clusters could be seen traveling through the fluorescent LYVE-1 labeled lymphatics. (a-f). RFP labeled cancer cells could also be seen collecting in the axillary lymph node after labeling of the node and lymphatics with green fluorescent LYVE-1 antibody (g).