The importance of the hemovascular circulatory system has been long recognized to play a critical role in cancer progression.11,15
Halting angiogenesis, or disrupting the process governing the formation and reorganization of the tumor neovasculature that provides nutrients for tumor expansion, is an emerging therapeutic strategy in cancer treatment.9,19
However more recently, there has been increasing attention on the role of the lymphatic vasculature and disrupting the process of new lymphatic vessel formation (termed lymphangiogenesis) to arrest meta-static disease.32,50
The emerging focus upon targeting lymphangiogenesis as a therapeutic strategy to arrest progressive cancer may not be surprising given the long standing clinical practice of lymph node (LN) dissection for (i) cancer staging based upon presence of cancer cells in resected LNs and (ii) regional control of disease associated with removal of potentially cancer-positive LNs and disruption of the lymphatic pathways involved in metastatic dissemination. Histological studies indicate that lymphangiogenesis may proceed prior to metastasis providing a cancer dissemination route.45
For example, histological studies of resected human tissues have correlated an increased number or density of intra-and/or peri-tumoral lymphatic vessels with metastasis. 7,42
Dilation or increase in the cross-sectional area of intra-tumoral lymphatics has also been shown to be associated with metastatic melanoma. In addition, lymph sinus remodeling/dilation along with LN lymphangiogenesis in sentinel LNs (SLNs) and increased lymph flow to tumor-draining LNs have been shown to occur prior to LN metastasis in preclinical models.16,40
Therefore, non-invasive imaging of changes in lymphatic function and remodeling may allow early identification of metastatic potential and lymphatic involvement. Yet the diagnostic tools to evaluate the in vivo
status of tumor-induced lymphangiogenesis and to monitor the consequence of pharmacological interventions on the lymphatics remains lacking, due in part to the lack of imaging techniques than can accommodate the unique structure and function of the lymphatic circulatory system.
Compared to the hemovascular system, the lymphatic system is a poorly understood, unidirectional circulatory system comprised of initial lymphatic capillaries that take up fluid, macromolecules, cellular debris, and foreign contaminants from interstitial spaces. From the capillary plexus, lymph fluid is conducted via
lymphatic vessels and trunks, through LNs for immune presentation and ultimately returns lymph fluid to the blood at the subclavian vein. In contrast to the hemovascular system, the lymphatics have no central pumping organ but instead, the lymphatic vessels are comprised of subunits called lymphangions that are bounded by valves and lined by smooth muscle cells. Lymph is actively “pumped” or propelled through the lymphangions via
orchestrated peristaltic contractions and sequential closing/opening of valves. Additionally, hydrodynamic pressure gradients generated in surrounding tissues by extrinsic factors, such as skeletal muscle contractions, produce passive lymph flow without active lymphatic pumping. Given its diversity of function, the lymphatics have been implicated in a spectrum of diseases including cancer metastasis41,50
and lymphedema, a disease which afflicts many cancer survivors in the U.S. (for review see Cormier et al
Because lymph is typically translucent with relatively low concentrations of cells and particulate matter, there is little endogenous contrast available for conventional imaging of lymphatic vessels using X-ray, magnetic resonance imaging (MRI), and ultrasound techniques. Diagnostic lymphoscintigraphy is the only accepted method to image lymphatic function using 99mc-Technetium radiocolloid as a radiotracer administered subcutaneously in 1–5 cc volumes or intradermally in ~0.1 cc volumes. Intradermal administration results in efficient uptake of the radiocolloid by initial lymphatic capillaries and transport through lymphatic vessels to draining LNs. Upon decay, radiocolloid emits high energy gamma photons that efficiently propagate through tissues and are collected by a gamma camera for planar gamma scintigraphy. Planar gamma scintigraphy is performed over several minutes to identify tumor draining SLNs, visualize major lymphatic vessels, and diagnose lymphatic obstruction resulting in lymphedema.59
Dynamic lymphoscintigraphy captures the advancing radiocolloid front transiting the lymphatics using a series of gamma images, acquired with exposure times as short as 20 s, to discriminate SLNs from those LNs that are secondary or drain the SLN.25
However, lymphoscintigraphy suffers from several significant drawbacks for assessing tumor lymphangiogenesis including: (i) limited photon count rate and sensitivity, (ii) finite radiocolloid size and comparatively long integration times that prevent direct imaging of contractile lymphatic function or active lymphatic pumping, and (iii) poor spatial resolution preventing visualization of small lymphatic neovasculatures that could characterize tumor lymphangiogenesis.
Opportunities for direct lymphangiography, similar to X-ray and magnetic resonance angiography, are limited due to the difficulties of cannulating lymphatic vessels and the transport of large volumes of viscous contrast agents.4
Recent advancements in MRI contrast agents and faster imaging systems have enabled Liu et al
to image anatomic and functional characteristics of diseased lymphatics using indirect lymphangiography in which contrast agent is injected intradermally, typically in the webs of digits. In their studies, interdigit injections of approximately 3 mL of gadobenate dimeglumine (GD) enabled MRI imaging of lymphatic vasculature and measurement of the transit speed of contrast moving from site of injection towards the draining LN basins in the legs of subjects with lymphedema. Yet the MRI approach failed to image lymphatic structure or movement of GD in healthy lymphatics owing, most likely, to significantly faster transit rates in the healthy as opposed to diseased lymphatics and the consequential lack of accumulated contrast agent required for MRI.
Over the past several years near-infrared fluorescence (NIRF) imaging has emerged as a non-invasive modality for in vivo
lymphatic imaging in both animals22,23,35,48,55
NIRF imaging has been used intraoperatively to map the SLN and guide its surgical resection in cancer patients,12,21,36,52
assess lymphatic structure and contractile function in health and disease,34,43,44,47,57,58
and evaluate response to lymphedema therapy.3,30,54
Herein we review the physics and instrumentation of NIRF imaging, briefly review its clinical application in the literature, and present our latest, NIRF images of longitudinal changes of the lymphatics in a preclinical model of progressing melanoma and snap shots in time of the lymphatics in melanoma patients.