Eelco F. J. Meijer

Lymph node metastases can invade local blood vessels, exit the node, and colonize distant organs in mice

An alternate route for metastatic cells Metastatic tumor cells are thought to reach distant organs by traveling through the blood circulation or the lymphatic system. Two studies of mouse models now suggest a hybrid route for tumor cell dissemination. Pereira et al. and Brown et al. used distinct methodologies to monitor the fate of tumor cells in lymph nodes. They found that tumor cells could invade local blood vessels within a node, exit the node by entering the blood circulation, then go on to colonize the lung. Whether this dissemination route occurs in cancer patients is unknown; the answer could potentially change the way that affected lymph nodes are treated in cancer. Science, this issue p. 1403, p. 1408 In mice, tumor cells can metastasize to distant organs by entering blood vessels within the local lymph node. Lymph node metastases in cancer patients are associated with tumor aggressiveness, poorer prognoses, and the recommendation for systemic therapy. Whether cancer cells in lymph nodes can seed distant metastases has been a subject of considerable debate. We studied mice implanted with cancer cells (mammary carcinoma, squamous cell carcinoma, or melanoma) expressing the photoconvertible protein Dendra2. This technology allowed us to selectively photoconvert metastatic cells in the lymph node and trace their fate. We found that a fraction of these cells invaded lymph node blood vessels, entered the blood circulation, and colonized the lung. Thus, in mouse models, lymph node metastases can be a source of cancer cells for distant metastases. Whether this mode of dissemination occurs in cancer patients remains to be determined.

The Lymphatic System in Disease Processes and Cancer Progression

Advances in our understanding of the structure and function of the lymphatic system have made it possible to identify its role in a variety of disease processes. Because it is involved not only in fluid homeostasis but also in immune cell trafficking, the lymphatic system can mediate and ultimately alter immune responses. Our rapidly increasing knowledge of the molecular control of the lymphatic system will inevitably lead to new and effective therapies for patients with lymphatic dysfunction. In this review, we discuss the molecular and physiological control of lymphatic vessel function and explore how the lymphatic system contributes to many disease processes, including cancer and lymphedema.

In vivo label-free measurement of lymph flow velocity and volumetric flow rates using Doppler optical coherence tomography.

Direct in vivo imaging of lymph flow is key to understanding lymphatic system function in normal and disease states. Optical microscopy techniques provide the resolution required for these measurements, but existing optical techniques for measuring lymph flow require complex protocols and provide limited temporal resolution. Here, we describe a Doppler optical coherence tomography platform that allows direct, label-free quantification of lymph velocity and volumetric flow rates. We overcome the challenge of very low scattering by employing a Doppler algorithm that operates on low signal-to-noise measurements. We show that this technique can measure lymph velocity at sufficiently high temporal resolution to resolve the dynamic pulsatile flow in collecting lymphatic vessels.

Simultaneous measurements of lymphatic vessel contraction, flow and valve dynamics in multiple lymphangions using optical coherence tomography

Lymphatic dysfunction is involved in many diseases including lymphedema, hypertension, autoimmune responses, graft rejection, atherosclerosis, microbial infections, cancer and cancer metastasis. Expanding our knowledge of lymphatic system function can lead to a better understanding of these disease processes and improve treatment options. Here, optical coherence tomography (OCT) methods were used to reveal intraluminal valve dynamics in 3 dimensions, and measure lymph flow and vessel contraction simultaneously in 3 neighboring lymphangions of the afferent collecting lymphatic vessels to the popliteal lymph node in mice. Flow measurements were based on Doppler OCT techniques in combination with exogenous lymph labeling by Intralipid. Through these imaging methods, it is possible to study lymphatic function and pumping more comprehensively. These capabilities can lead to a better understanding of the regulation and dysregulation of lymphatic vessels in health and disease. The image depicts the dynamic measurements of lymphatic valves, lymphatic vessels cross-sectional area and lymph velocity simultaneously measured in vivo with optical coherence tomography.

Murine chronic lymph node window for longitudinal intravital lymph node imaging

Chronic imaging windows in mice have been developed to allow intravital microscopy of many different organs and have proven to be of paramount importance in advancing our knowledge of normal and disease processes. A model system that allows long-term intravital imaging of lymph nodes would facilitate the study of cell behavior in lymph nodes during the generation of immune responses in a variety of disease settings and during the formation of metastatic lesions in cancer-bearing mice. We describe a chronic lymph node window (CLNW) surgical preparation that allows intravital imaging of the inguinal lymph node in mice. The CLNW is custom-made from titanium and incorporates a standard coverslip. It allows stable longitudinal imaging without the need for serial surgeries while preserving lymph node blood and lymph flow. We also describe how to build and use an imaging stage specifically designed for the CLNW to prevent (large) rotational changes as well as respiratory movement during imaging. The entire procedure takes approximately half an hour per mouse, and subsequently allows for longitudinal intravital imaging of the murine lymph node and surrounding structures for up to 14 d. Small-animal surgery experience is required to successfully carry out the protocol.

Methicillin-resistant Staphylococcus aureus causes sustained collecting lymphatic vessel dysfunction

Impairment in lymph flow caused by methicillin-resistant Staphylococcus aureus toxins persists after skin and soft tissue infections clear. Lymphatics limp along after MRSA Lymphedema is associated with skin and soft tissue infections, and both can be recurring, causing continual suffering in affected patients. To better understand the relationship between bacterial infections and lymphedema, Jones et al. used intravital imaging to examine the lymphatics of mice infected with MRSA. They observed lymphatic muscle cell death, which led to prolonged dysfunction months after the bacteria had been cleared. In vitro experiments with human cells indicated that bacterial toxins were responsible for damaging the lymphatic muscle cells, suggesting that the toxins could be targeted in patients to interrupt this brutal cycle. Methicillin-resistant Staphylococcus aureus (MRSA) is a major cause of morbidity and mortality worldwide and is a frequent cause of skin and soft tissue infections (SSTIs). Lymphedema—fluid accumulation in tissue caused by impaired lymphatic vessel function—is a strong risk factor for SSTIs. SSTIs also frequently recur in patients and sometimes lead to acquired lymphedema. However, the mechanism of how SSTIs can be both the consequence and the cause of lymphatic vessel dysfunction is not known. Intravital imaging in mice revealed an acute reduction in both lymphatic vessel contractility and lymph flow after localized MRSA infection. Moreover, chronic lymphatic impairment is observed long after MRSA is cleared and inflammation is resolved. Associated with decreased collecting lymphatic vessel function was the loss and disorganization of lymphatic muscle cells (LMCs), which are critical for lymphatic contraction. In vitro, incubation with MRSA-conditioned supernatant led to LMC death. Proteomic analysis identified several accessory gene regulator (agr)–controlled MRSA exotoxins that contribute to LMC death. Infection with agr mutant MRSA resulted in sustained lymphatic function compared to animals infected with wild-type MRSA. Our findings suggest that agr is a promising target to preserve lymphatic vessel function and promote immunity during SSTIs.

Lymph node effective vascular permeability and chemotherapy uptake

Lymph node metastases are a poor prognostic factor. Additionally, responses of lymph node metastasis to therapy can be different from the primary tumor. Investigating the physiologic lymph node blood vasculature might give insight into the ability of systemic drugs to penetrate the lymph node, and thus into the differential effect of therapy between lymph node metastasis and primary tumors. Here, we measured effective vascular permeability of lymph node blood vessels and attempted to increase chemotherapy penetration by increasing effective vascular permeability.

Lymphatic function measurements influenced by contrast agent volume and body position

Several imaging modalities have been used to assess lymphatic function, including fluorescence microscopy, near-infrared fluorescence (NIRF) imaging, and Doppler optical coherence tomography (DOCT). They vary in how the mouse is positioned, the invasiveness of the experimental setup, and the volume of contrast agent injected. Here, we present how each of these experimental parameters affects functional measurements of collecting lymphatic vessels. First, fluorescence microscopy showed that supine mice have a statistically lower contraction frequency compared with mice sitting upright. To assess the effect of different injection volumes on these endpoints, mice were injected with 4, 10, or 20 μl of dye. The lowest frequencies were observed after 20-μl injections. Interestingly, lymph-flow DOCT revealed that although there was lower contraction frequency in mice injected with 20 μl versus 4 μl, mice showed a higher volumetric flow with a 20-μl injection. This indicates that contraction frequency alone is not sufficient to understand lymphatic transport. Finally, NIRF revealed that removing the skin reduced contraction frequency. Therefore, this study reveals how sensitive these techniques are to mouse position, removal of skin, and dye volume. Care should be taken when comparing results obtained under different experimental conditions.

Depolarization signatures map gold nanorods within biological tissue

Owing to their electromagnetic properties, tunability and biocompatibility, gold nanorods (GNRs) are being investigated as multifunctional probes for a range of biomedical applications. However, detection beyond the reach of traditional fluorescence and two-photon approaches and quantitation of their concentration in biological tissue remain challenging tasks in microscopy. Here we show how the size and aspect ratio that impart GNRs with their plasmonic properties also make them a source of entropy. We report on how depolarization can be exploited as a strategy to visualize GNR diffusion and distribution in biologically relevant scenarios ex vivo, in vitro and in vivo. We identify a deterministic relation between depolarization and nanoparticle concentration. As a result, some of the most stringent experimental conditions can be relaxed, and susceptibility to artefacts is reduced, enabling microscopic and macroscopic applications.

Measuring Vascular Permeability In Vivo.

Over the past decades, in vivo vascular permeability measurements have provided significant insight into vascular functions in physiological and pathophysiological conditions such as the response to pro- and anti-angiogenic signaling, abnormality of tumor vasculature and its normalization, and delivery and efficacy of therapeutic agents. Different approaches for vascular permeability measurements have been established. Here, we describe and discuss a conventional 2D imaging method to measure vascular permeability, which was originally documented by Gerlowski and Jain in 1986 (Microvasc Res 31:288-305, 1986) and further developed by Yuan et al. in the early 1990s (Microvasc Res 45:269-289, 1993; Cancer Res 54:352-3356, 1994), and our recently developed 3D imaging method, which advances the approach originally described by Brown et al. in 2001 (Nat Med 7:864-868, 2001).