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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 arrest invasion through normalization of the lymphatic vasculature., Competing Interests: The authors declare that there are no conflicts of interest related to this article.

In this short communication, we demonstrate for the first time, the use of far red fluorescent gene reporter, iRFP to longitudinally and non-invasively track the in vivo process of lymphatic metastases from an orthotopic site of mammary implantation through lymphatic vessels and to draining lymph nodes. Potentially useful to accelerate cancer drug discovery as an in vivo screening tool to monitor the pharmacological arrest of metastasis, we show that the custom as well as commercial small animal imaging devices have adequate performance to detect the gene reporter in stably expressing metastatic cancer cells.

One limitation of fluorescence molecular imaging that can limit clinical implementation and hamper small animal imaging is the inability to eliminate ambient light. Herein, we demonstrate the ability to conduct rapid non-invasive, far-red and near-infrared fluorescence imaging in living animals and a phantom under ambient light conditions using a modulated image intensified CCD (ICCD) and a laser diode operated in homodyne detection. By mapping AC amplitude from three planar images at varying phase delays, we show improvement in target-to-background ratios (TBR) and reasonable signal-to-noise ratios (SNR) over continuous wave measurements. The rapid approach can be used to accurately collect fluorescence in situations where ambient light cannot be spectrally conditioned or controlled, such as in the case of fluorescent molecular image-guided surgery.

As part of a proof-of-concept study for future delivery of targeted near-infrared fluorescent (NIRF) tracers, we sought to assess the delivery of micrograms of indocyanine green to all the axillary lymph nodes following intraparenchymal breast injections and intradermal arm injections in 20 subjects with advanced breast carcinoma and undergoing complete axillary lymph node dissection. Lymphatic vessels and nodes were assessed in vivo. Ex vivo images demonstrated that 87% of excised lymph nodes, including 81% of tumor-positive lymph nodes, were fluorescent. Future clinical studies using microdose amounts of tumor-targeting NIRF contrast agents may demonstrate improved surgical intervention with reduced morbidity.

The lymphatic system provides an initial route for cancer cell dissemination in many cancers including melanoma. However, it is largely unknown how the lymphatic system changes during tumor progression due in part to the lack of imaging techniques currently available. In this study, we non-invasively imaged changes of lymphatic function and drainage patterns using near-infrared fluorescence (NIRF) imaging. Dynamic NIRF imaging following intradermal injection of indocyanine green (ICG) was conducted in C57BL/6 mice prior to inoculation of B16F10 murine melanoma cells to the dorsal aspect of the left hindpaw for baseline data or directly to the popliteal lymph node (PLN) and until 21 days post-implantation (p.i.). A series of acquired fluorescent images were quantified to measure lymphatic contractile function. Computed tomography (CT) was also performed to measure the volume of tumor-draining lymph nodes (LNs). We observed significant reduction of lymphatic contractility from 7 days p.i. until 21 days p.i.. Altered lymphatic drainage patterns were also detected at 21 days p.i. in mice with tumor in the paw and at 11 days p.i. in mice with tumor in the PLN, due to lymphatic obstruction of normal lymphatic drainages caused by extensive tumor invasion of draining LNs. Since lymphatic function and architecture were progressively altered during tumor growth and metastasis, non-invasive NIRF imaging may provide a new method to stage disease. In addition, this novel technique can be used as a diagnostic method to non-invasively assess lymphatic response as mechanism of therapeutic action.

Despite the development of a large number of promising candidates, few contrast agents for established medical imaging modalities have successfully been translated over the past decade. The emergence of new imaging contrast agents that employ biomedical optics is further complicated by the relative infancy of the field and the lack of approved imaging devices compared to more established clinical modalities such as nuclear medicine. Herein, we propose a navigational approach (as opposed to a fixed "roadmap") for translation of optical imaging agents that is (i) proposed through consensus by four academic research programs that are part of the cooperative U54 NCI Network for Translational Research, (ii) developed through early experiences for translating optical imaging agents in order to meet distinctly varied needs in cancer diagnostics, and (iii) adaptable to the rapidly changing environment of academic medicine. We describe the pathways by which optical imaging agents are synthesized, qualified, and validated for preclinical testing, and ultimately translated for "first-in-humans" studies using investigational optical imaging devices. By identifying and adopting consensus approaches for seemingly disparate optical imaging modalities and clinical indications, we seek to establish a systematic method for navigating the ever-changing "roadmap" to most efficiently arrive at the destination of clinical adoption and improved outcome and survivorship for cancer patients.

ALFIA (Automated Lymphatic Function Imaging Analysis), an algorithm providing quantitative analysis of investigational near-infrared fluorescence lymphatic images, is described. Images from nine human subjects were analyzed for apparent lymphatic propagation velocities and propulsion periods using manual analysis and ALFIA. While lymphatic propulsion was more easily detected using ALFIA than with manual analysis, statistical analyses indicate no significant difference in the apparent lymphatic velocities although ALFIA tended to calculate longer propulsion periods. With the base ALFIA algorithms validated, further automation can now proceed to provide a clinically relevant analytic tool for quantitatively assessing lymphatic function in humans.

Current treatment of unilateral breast cancer-related lymphedema (BCRL) is only directed to the afflicted arm. Near-infrared fluorescent imaging (NIRF) of arm lymphatic vessel architecture and function in BCRL and control subjects revealed a trend of increased lymphatic abnormalities in both the afflicted and unafflicted arms with increasing time after lymphedema onset. These pilot results show that BCRL may progress to affect the clinically "normal" arm, and suggest that cancer-related lymphedema may become a systemic, rather than local, malady. These findings support further study to understand the etiology of cancer-related lymphedema and lead to better diagnostics and therapeutics directed to the systemic lymphatic system.

Medical imaging is an invaluable tool for diagnosis, surgical guidance, and assessment of treatment efficacy. The Network for Translational Research (NTR) for Optical Imaging consists of four research groups working to "bridge the gap" between lab discovery and clinical use of fluorescence- and photoacoustic-based imaging devices used with imaging biomarkers. While the groups are using different modalities, all the groups face similar challenges when attempting to validate these systems for FDA approval and, ultimately, clinical use. Validation steps taken, as well as future needs, are described here. The group hopes to provide translational validation guidance for itself, as well as other researchers.

We demonstrate the ability to non-invasively and quantitatively image lymphatic architecture and contractile function using dynamic near-infrared (NIR) fluorescence imaging with injection of indocyanine green in normal and transgenic mice. Unlike normal mice, which showed well defined lymphatic drainage patterns and orthograde propagation of contraction waves, we observed tortuous and mispatterned lymphatic vessels and persistent retrograde lymph flow in mice with deficiency in Prox1, a transcription factor essential for lymphatic vascular development. NIR fluorescence imaging provides a method for quantifying lymphatic function for future studies designed to discern differences in lymphatic function in murine models of human lymphatic disease.

Lymphedema affects up to 50% of all breast cancer survivors. Management with pneumatic compression devices (PCDs) is controversial, owing to the lack of methods to directly assess benefit. This pilot study employed an investigational, near-infrared (NIR) fluorescence imaging technique to evaluate lymphatic response to PCD therapy in normal control and breast cancer-related lymphedema (BCRL) subjects. Lymphatic propulsion rate, apparent lymph velocity, and lymphatic vessel recruitment were measured before, during, and after advanced PCD therapy. Lymphatic function improved in all control subjects and all asymptomatic arms of BCRL subjects. Lymphatic function improved in 4 of 6 BCRL affected arms, improvement defined as proximal movement of dye after therapy. NIR fluorescence lymphatic imaging may be useful to directly evaluate lymphatic response to therapy. These results suggest that PCDs can stimulate lymphatic function and may be an effective method to manage BCRL, warranting future clinical trials.