Search

The global incidence of human papillomavirus (HPV) associated head and neck carcinoma is on the rise, in response to this a tetravalent therapeutic vaccine named Qβ-HPVag was developed. This vaccine, utilizing virus-like particles (VLPs) loaded with toll-like receptor ligands and chemically coupled to four HPV16-derived peptides, demonstrated strong anti-tumor effects in a murine head and neck cancer model. Qβ-HPVag impeded tumor progression, increased infiltration of HPV-specific T cells, and significantly improved survival. The vaccine`s efficacy was associated with immune repolarization in the tumor microenvironment, characterized by expanded activated dendritic cell subsets (cDC1, cDC2, DC3). Notably, mice responding to treatment exhibited a higher percentage of migratory DC3 cells expressing CCR7. These findings suggest promising prospects for optimized VLP-based vaccines in treating HPV-associated head and neck cancer., Competing Interests: M.O.M. and M.F.B. are founders of DeepVax GmbH. Additionally, M.O.M., D.E.S., and M.F.B. hold shares in DeepVax GmbH, a company focused on the development of cancer immunotherapy., (© 2024 The Author(s).)

The role of the lymphatics in the clearance of cerebrospinal fluid (CSF) from the brain has been implicated in multiple neurodegenerative conditions. In premature infants, intraventricular hemorrhage causes increased CSF production and, if clearance is impeded, hydrocephalus and severe developmental disabilities can result. In this work, we developed and deployed near-infrared fluorescence (NIRF) tomography and imaging to assess CSF ventricular dynamics and extracranial outflow in similarly sized, intact non-human primates (NHP) following microdose of indocyanine green (ICG) administered to the right lateral ventricle. Fluorescence optical tomography measurements were made by delivering ~10 mW of 785 nm light to the scalp by sequential illumination of 8 fiber optics and imaging the 830 nm emission light collected from 22 fibers using a gallium arsenide intensified, charge coupled device. Acquisition times were 16 seconds. Image reconstruction used the diffusion approximation and hard-priors obtained from MRI to enable dynamic mapping of ICG-laden CSF ventricular dynamics and drainage into the subarachnoid space (SAS) of NHPs. Subsequent, planar NIRF imaging of the scalp confirmed extracranial efflux into SAS and abdominal imaging showed ICG clearance through the hepatobiliary system. Necropsy confirmed imaging results and showed that deep cervical lymph nodes were the routes of extracranial CSF egress. The results confirm the ability to use trace doses of ICG to monitor ventricular CSF dynamics and extracranial outflow in NHP. The techniques may also be feasible for similarly-sized infants and children who may suffer impairment of CSF outflow due to intraventricular hemorrhage.

The lymphatics play important roles in chronic diseases/conditions that comprise the bulk of healthcare worldwide. Yet the ability to routinely image and diagnose lymphatic dysfunction, using commonly available clinical imaging modalities, has been lacking and as a result, the development of effective treatment strategies suffers. Nearly two decades ago, investigational near-infrared fluorescence lymphatic imaging and ICG lymphography were developed as routine diagnostic for clinically evaluating, quantifying, and treating lymphatic dysfunction in cancer-related and primary lymphedema, chronic venous disease, and more recently, autoimmune and neurodegenerative disorders. In this review, we provide an overview of what these non-invasive technologies have taught us about lymphatic (dys) function and anatomy in human studies and in corollary animal studies of human disease. We summarize by commenting on new impactful clinical frontiers in lymphatic science that remain to be facilitated by imaging., Competing Interests: ES-M, JR, and CF have financial interests in a University of Texas Health Science Center start-up, Lymphatic Science, which seeks to commercialize near-infrared fluorescence lymphatic imaging devices and the approaches described herein. The conflict of interest on the part of ES-M and JR is managed by the University Research Conflict of Interest Committee., (Copyright © 2023 Sevick-Muraca, Fife and Rasmussen.)

Breast cancer-related lymphedema (BCRL) occurs in ~ 40% of patients after axillary lymph node dissection (ALND), radiation therapy (RT), or chemotherapy. First-line palliative treatment utilizes compression garments and specialized massage. Reparative microsurgeries have emerged as a second-line treatment, yet both compression and surgical therapy are most effective at early stages of LE development. Identifying patients at the highest risk for BCRL would allow earlier, more effective treatment. Perometric arm volume measurements, near-infrared fluorescent lymphatic imaging (NIRF-LI) data, and blood were collected between 2016 and 2021 for 40 study subjects undergoing treatment for breast cancer. Plasma samples were evaluated using MILLIPLEX human cytokine/chemokine panels at pre-ALND and at 12 months post-RT. A Mann-Whitney t -test showed that G-CSF, GM-CSF, IFN-2α, IL-10, IL-12p40, IL-15, IL-17A, IL-1β, IL-2, IL-3, IL-6, and MIP-1β were significantly higher at pre-ALND in those presenting with BCRL at 12 months post-RT. MIP-1β and IL-6 were significantly higher at pre-ALND in those who developed dermal backflow, but no BCRL, at 12 months post-RT. Plasma IL-15, IL-3, and MIP-1β were elevated at 12 months after RT in those with clinical BCRL. These findings establish BCRL as a perpetual inflammatory disorder, and suggest the use of plasma cytokine/chemokine levels to predict those at highest risk.

Objective: Lipedema is an inflammatory subcutaneous adipose tissue disease that develops in women and may progress to lipolymphedema, a condition similar to lymphedema, in which lymphatic dysfunction results in irresolvable edema. Because it has been shown that dilated lymphatic vessels, impaired pumping, and dermal backflow are associated with presymptomatic, cancer-acquired lymphedema, this study sought to understand whether these abnormal lymphatic characteristics also characterize early stages of lipedema prior to lipolymphedema development., Methods: In a pilot study of 20 individuals with Stage I or II lipedema who had not progressed to lipolymphedema, lymphatic vessel anatomy and function in upper and lower extremities were assessed by near-infrared fluorescence lymphatic imaging and compared with that of a control population of similar age and BMI., Results: These studies showed that, although lower extremity lymphatic vessels were dilated and showed intravascular pooling, the propulsion rates significantly exceeded those of control individuals. Upper extremity lymphatics of individuals with lipedema were unremarkable. In contrast to individuals with lymphedema, individuals with Stage I and II lipedema did not exhibit dermal backflow., Conclusions: These results suggest that, despite the confusion in the diagnoses between lymphedema and lipedema, their etiologies differ, with lipedema associated with lymphatic vessel dilation but not lymphatic dysfunction., (© 2022 The Authors. Obesity published by Wiley Periodicals LLC on behalf of The Obesity Society (TOS).)

An infusion of checkpoint blockade immunotherapy (CBI) has revolutionized cancer treatments for some patients, but the majority of patients experience disappointing responses. Because adaptive immune responses are mounted by the concentrated assembly of antigens, immune cells, and mediators in the secluded and protective environment of draining lymph nodes (dLNs), we hypothesize that lymphatic delivery of CBI (αCTLA-4 and αPD-1) to tumor dLNs (tdLNs) improves anti-tumor responses over intravenous (i.v.) administration, and that vaccination against tumor associated antigen (TAA) further enhances these responses. Mono- and combination CBI were administered i.v. or through image-guided intradermal (i.d.) injection to reach tdLNs in vaccinated and unvaccinated animals bearing either primary or orthotopically metastasizing B16F10 melanoma. Vaccination and boost against TAA, Melan-A, was accomplished with virus-like particles (VLP) directed to tdLNs followed by VLP boost after CBI administration. Lymphatic delivery of CBIs reduced primary tumor size and metastatic tumor burden, alleviated the pro-tumorigenic immune environment, and improved survival over systemic administration of CBIs. Animals receiving CBIs lymphatically exhibited significantly enhanced survival over those receiving therapies administered partially or completely through systemic routes. By combining vaccination and CBI for effective T-cell priming in the protected environment of dLNs, anti-tumor responses may be improved.

Cutaneous tuberculosis secondary to skin inoculation of Mycobacterium tuberculosis is uncommon but it can occur in the health care settings. Herein, we report an unusual case of primary cutaneous tuberculosis of the thumb following a needlestick injury. The infection progressed with a necrotic granuloma, lymphatic dysfunction as visualized by near-infrared fluorescence lymphatic imaging, and the development of an axillary web syndrome., (© The Author(s) 2021. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

Background: Chylothorax is a rare complication of pediatric cardiac operations that occurs more frequently in children with Noonan syndrome, a genetic disorder associated with cardiac defects and lymphatic anomalies., Case Presentation: We report a case of postoperative chylothorax in a 6-month-old infant with Noonan syndrome where multimodality lymphatic imaging guided management was followed. Drainage patterns of the lymphatic capillaries in the lower and upper extremities were visualized during near-infrared fluorescence lymphatic imaging (NIRFLI). Dynamic magnetic resonance lymphangiography (MRL) further identified the site of leakage in the thoracic duct and subsequently guided surgical intervention., Conclusions: Application of multimodality imaging allows for greater individualization of treatment and should be considered in patients with complex cases such as those with syndromes associated with a higher incidence of chylothorax. IRB Number: HSC-MS-13-0754, December 10, 2013.

Although Blood Oxygenation Level Dependent (BOLD) functional MRI (fMRI) is widely used to examine brain function in adults, the need for general anesthesia limits its practical utility in infants and small children. Functional Near-Infrared Spectroscopy - Diffuse Optical Tomography (fNIRS-DOT) imaging promises to be an alternative brain network imaging technique. Yet current versions of continuous-wave fNIRS-DOT systems are restricted to the cortical surface measurements and do not probe deep structures that are frequently injured especially in premature infants. Herein we report a transcranial near infrared optical imaging system, called Cap-based Transcranial Optical Tomography (CTOT) able to image whole brain hemodynamic activity with 3 seconds of data acquisition time. We show the system is capable of whole brain oxygenation mapping in an awake child, and that tomographically reconstructed static CTOT-derived oxy- and deoxygenated blood volumes are spatially correlated with the time-averaged BOLD fMRI volumes. By removing time bottlenecks in the current system, dynamic CTOT mapping should be possible, which would then enable evaluation of functional connectivity in awake infants.

Breast-cancer-acquired lymphedema is routinely diagnosed from the appearance of irreversible swelling that occurs as a result of lymphatic dysfunction. Yet in head and neck cancer survivors, lymphatic dysfunction may not always result in clinically overt swelling, but instead contribute to debilitating functional outcomes. In this review, we describe how cancer metastasis, lymph node dissection, and radiation therapy alter lymphatic function, as visualized by near-infrared fluorescence lymphatic imaging. Using custom gallium arsenide (GaAs)-intensified systems capable of detecting trace amounts of indocyanine green administered repeatedly as lymphatic contrast for longitudinal clinical imaging, we show that lymphatic dysfunction occurs with cancer progression and treatment and is an early, sub-clinical indicator of cancer-acquired lymphedema. We show that early treatment of lymphedema can restore lymphatic function in breast cancer and head and neck cancer patients and survivors. The compilation of these studies provides insights to the critical role that the lymphatics and the immune system play in the etiology of lymphedema and associated co-morbidities.

Recently, fluorescence imaging using shortwave infrared light (SWIR, 1,000-2,000 nm) has been proposed as having advantage over conventional near-infrared fluorescence (NIRF) imaging due to the reduced tissue scattering, negligible autofluorescence, comparable tissue absorption, and the discovery that indocyanine green (ICG), used clinically as a NIRF contrast agent, also has fluorescence emission in SWIR regime. Images of ICG in small animals acquired by commercial Si-based and InGaAs-based imaging cameras have been qualitatively compared, however the lack of working standards to quantify performance of these imaging systems limits quantitative comparison. Without quantification using a traceable in vitro test, clinical adoption of rapidly evolving advances in both NIRF and SWIR imaging devices will become limited. In this work, we developed an ICG based fluorescent solid working standard calibrated with SI units (mW [Formula: see text]cm [Formula: see text]sr -1 ) for quantification of measurement sensitivity of Si, GaAs-intensified Si, and InGaAs based camera systems, their signal-to-noise ratio (SNR), and contrast in non-clinical tests. In addition, we present small animal and large animal imaging with ICG for qualitative comparison of the same SWIR fluorescence and NIRF imaging systems. Results suggest that SWIR fluorescence imaging of ICG may have superior resolution in small animal imaging compared to NIRF imaging, but lack of measurement sensitivity, SNR, contrast, as well as water absorption limits deep penetration in large animals.

Evidence overwhelmingly suggests that the lymphatics play a critical role in the clearance of cerebrospinal fluid (CSF) from the cranial space. Impairment of CSF outflow into the lymphatics is associated with a number of pathological conditions including spaceflight-associated neuro-ocular syndrome (SANS), a problem that limits long-duration spaceflight. We used near-infrared fluorescence lymphatic imaging (NIRFLI) to dynamically visualize the deep lymphatic drainage pathways shared by CSF outflow and disrupted during head-down tilt (HDT), a method used to mimic the cephalad fluid shift that occurs in microgravity. After validating CSF clearance into the lymph nodes of the neck in swine, a pilot study was conducted in human volunteers to evaluate the effect of gravity on the flow of lymph through these deep cervical lymphatics. Injected into the palatine tonsils, ICG was imaged draining into deep jugular lymphatic vessels and subsequent cervical lymph nodes. NIRFLI was performed under HDT, sitting, and supine positions. NIRFLI shows that lymphatic drainage through pathways shared by CSF outflow are dependent upon gravity and are impaired under short-term HDT. In addition, lymphatic contractile rates were evaluated from NIRFLI following intradermal ICG injections of the lower extremities. Lymphatic contractile activity in the legs was slowed in the gravity neutral, supine position, but increased under the influence of gravity regardless of whether its force direction opposed (sitting) or favored (HDT) lymphatic flow toward the heart. These studies evidence the role of a lymphatic contribution in SANS., (© 2020 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.)

Rationale : Cytotoxic T-lymphocyte-associated antigen 4 (CTLA - 4) is a co-inhibitory checkpoint receptor that is expressed by naïve T-cells in lymph nodes (LNs) to inhibit activation against "self" antigens (Ags). In cancer, anti-CTLA-4 blocks inhibitory action, enabling robust activation of T-cells against tumor Ags presented in tumor draining LNs (TDLNs) . However, anti-CTLA-4 is administered intravenously with limited exposure within TDLNs and immune related adverse events (irAEs) are associated with over-stimulation of the immune system. Methods : Herein, we first deliver anti-CTLA-4 in an orthotopic mammary carcinoma murine model using a nanotopographical microneedle-array device to compare its anti-tumor response to that from systemic administration. Additionally, to demonstrate the feasibility of lymphatic delivery in humans using the device, we use near-infrared fluorescence imaging to image delivery of ICG to LNs. Results : Our data show that lymphatic infusion results in more effective tumor growth inhibition, arrest of metastases, increased tumor infiltrating lymphocytes and complete responses when compared to conventional systemic administration. In clinical studies, we demonstrate for the first time that nanotopographic infusion can deliver ICG through the lymphatics directly to the axilla and inguinal LNs of healthy human volunteers. Conclusion : Taken together, these results suggest that regional delivery using a nanotopography-based microneedle array could revolutionize checkpoint blockade immunotherapy by reducing systemic drug exposure and maximizing drug delivery to TDLNs where tumor Ags present. Future work is needed to determine whether lymphatic delivery of anti-CTLA-4 can alleviate irAEs that occur with systemic dosing., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Adeno-associated virus (AAV) has emerged as a promising gene delivery vector because of its non-pathogenicity, simple structure and genome, and low immunogenicity compared to other viruses. However, its adoption as a safe and effective delivery vector for certain diseases relies on altering its tropism to deliver transgenes to desired cell populations. To this end, we have developed a protease-activatable AAV vector, named provector, that responds to elevated extracellular protease activity commonly found in diseased tissue microenvironments. The AAV9-based provector is initially inactive, but then it can be switched on by matrix metalloproteinases (MMP)-2 and -9. Cryo-electron microscopy and image reconstruction reveal that the provector capsid is structurally similar to that of AAV9, with a flexible peptide insertion at the top of the 3-fold protrusions. In an in vivo model of myocardial infarction (MI), the provector is able to deliver transgenes site specifically to high-MMP-activity regions of the damaged heart, with concomitant decreased delivery to many off-target organs, including the liver. The AAV provector may be useful in the future for enhanced delivery of transgenes to sites of cardiac damage., (Copyright © 2019 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)

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.

The relationship between lymphatic and venous malformations in Klippel-Trénaunay syndrome is difficult to assess. Herein the authors describe near-infrared fluorescence lymphatic imaging to assess the lymphatics of a subject with a large port-wine stain and right leg edema. Although lymphatic vessels in the medial, affected knee appeared dilated and perhaps tortuous, no definitive abnormal lymphatic pooling or propulsion was observed. The lymphatics in the affected limb were well defined but less numerous than in the contralateral limb, and active, contractile function was observed in all vessels. As demonstrated, near-infrared fluorescence lymphatic imaging enables the clinical assessment of lymphatics in lymphovenous malformations., (Copyright © 2017 Society for Vascular Surgery. Published by Elsevier Inc. All rights reserved.)

Near-infrared fluorescence imaging (NIRFI) and far-red fluorescence imaging (FRFI) were used to investigate effects of depilation-induced skin pigmentation and diet-induced background fluorescence on fluorescent signal amplitude and lymphatic contraction frequency in C57BL6 mice. Far-red fluorescent signal amplitude, but not frequency, was affected by diet-induced fluorescence, which was removed by feeding the mice an alfalfa-free diet, and skin pigmentation further impacted the amplitude measurement. NIRFI showed minimal background fluorescence; however, skin pigmentation reduced the amplitude of fluorescent signal changes. Therefore, these effects should be taken into account when imaging mice with different states of skin pigmentation and diet-induced background fluorescence in vivo.

Background: Evidence suggests lymphatic function mediates local rheumatoid arthritis (RA) flares. Yet biologics that target the immune system are dosed systemically via the subcutaneous (SC) administration route, thereby inefficiently reaching local lymphatic compartments. Nanotopography has previously been shown to disrupt tight cellular junctions, potentially enhancing local lymphatic delivery and potentially improving overall therapeutic efficacy., Method: We first characterized nanotopography (SOFUSA™) delivery of an anti-TNF drug, etanercept, by comparing pharmacokinetic profiles to those obtained by conventional SC, intravenous (IV), and intradermal (ID) routes of administration, and assessed uptake of radiolabeled etanercept in draining lymph nodes (LNs) in single dosing studies. We then compared etanercept efficacy in a progressive rat model of collagen-induced arthritis (CIA), administered systemically via SC route of administration; via the regional lymphatics through ID delivery; or through a nanotopography (SOFUSA™) device at 10, 12, and 14 days post CIA induction. Measurements of hind limb swelling and near-infrared fluorescence (NIRF) imaging of afferent lymph pumping function and reflux were conducted on days 11, 13, and 18 post CIA induction and compared to untreated CIA animals. Univariate and multivariate analysis of variance were used to compare the group differences for percentage swelling and lymphatic contractile activity., Results: Even though all three modes of administration delivered an equal amount of etanercept, SOFUSA™ delivery resulted in increased lymphatic pumping and significantly reduced swelling as compared to untreated, ID, and SC groups. Pharmacokinetic profiles in serum and LN uptake studies showed that using the nanotopography device resulted in the greatest uptake and retention in draining LNs., Conclusions: Locoregional lymphatic delivery of biologics that target the immune system may have more favorable pharmacodynamics than SC or IV administration. Nanotopography may provide a more efficient method for delivery of anti-TNF drugs to reverse impairment of lymphatic function and reduce swelling associated with RA flares.

Objective: Currently, lymphedema (LED) is typically diagnosed clinically on the basis of a patient's history and characteristic physical findings. Whereas the diagnosis of LED is sometimes confirmed by lymphoscintigraphy (LSG), the technique is limited in both its ability to identify disease and to guide therapy. Recent advancements provide opportunities for new imaging techniques not only to assist in the diagnosis of LED, based on anatomic changes, but also to assess contractile function and to guide therapeutic intervention. The purpose of this contribution was to review these imaging techniques., Methods: Literature for each technique is reviewed and discussed, and the evidence for each of these new diagnostic techniques was assessed on several criteria to determine if each could (1) establish whether disease is present, (2) determine the severity of the disease process, (3) define the pathophysiologic mechanism of the disease process, (4) demonstrate whether intervention is possible as well as what type, and (5) objectively grade the response to therapy., Results: LSG is currently the standard test to confirm LED. Duplex ultrasound (DUS) is a simple, readily available noninvasive test that can identify LED by specific tissue characteristics as well as the response to therapy. Magnetic resonance imaging and computed tomography scans similarly demonstrate the alterations in epidermal and subcutaneous tissue, but the latter can also detect obstructing neoplasms as a cause of secondary LED. Moreover, magnetic resonance lymphangiography details lymphatic vessels and nodes and their function. Newer fluorescence imaging techniques provide opportunities to image lymphatic anatomy and function. Visible microlymphangiography by fluorescein sodium is limited by tissue light absorption to imaging depths of 200 μm. Near-infrared fluorescence lymphatic imaging, a newer test using intradermal injection of indocyanine green, can penetrate several centimeters of tissue and can visualize the initial and conducting lymphatics, the lymph node basins, and the active function of lymphangions (the key module) in exquisite detail., Conclusions: The availability and the noninvasive nature of DUS should make this modality the first choice for establishing the diagnosis of LED based on tissue changes. Further studies comparing DUS with LSG, however, are needed. The costs of magnetic resonance imaging and computed tomography limit their adoption as a means to regularly assess the lymphatics. Whereas lymphatic truncal anatomy and transit times can be delineated by the older technique of LSG, near-infrared fluorescence lymphatic imaging is rapid, highly sensitive, and repeatable and provides exquisite detail for lymphatic vessel anatomy and function of the lymphangions as well as the response to therapy., (Copyright © 2016 Society for Vascular Surgery. Published by Elsevier Inc. All rights reserved.)

Mechanical stress is pervasive in egress routes of malignancy, yet the intrinsic effects of force on tumour cells remain poorly understood. Here, we demonstrate that frictional force characteristic of flow in the lymphatics stimulates YAP1 to drive cancer cell migration; whereas intensities of fluid wall shear stress (WSS) typical of venous or arterial flow inhibit taxis. YAP1, but not TAZ, is strictly required for WSS-enhanced cell movement, as blockade of YAP1, TEAD1-4 or the YAP1-TEAD interaction reduces cellular velocity to levels observed without flow. Silencing of TEAD phenocopies loss of YAP1, implicating transcriptional transactivation function in mediating force-enhanced cell migration. WSS dictates expression of a network of YAP1 effectors with executive roles in invasion, chemotaxis and adhesion downstream of the ROCK-LIMK-cofilin signalling axis. Altogether, these data implicate YAP1 as a fluid mechanosensor that functions to regulate genes that promote metastasis.

To date, no emerging preclinical or clinical near-infrared fluorescence (NIRF) imaging devices for noninvasive and/or surgical guidance have their performances validated on working standards with SI units of radiance that enable comparison or quantitative quality assurance. In this work, we developed and deployed a methodology to calibrate a stable, solid phantom for emission radiance with International System of Units (SI) units of mW ·sr(-1) ·cm(-2) for use in characterizing the measurement sensitivity of ICCD and IsCMOS detection, signal-to-noise ratio, and contrast. In addition, at calibrated radiances, we assess transverse and lateral resolution of ICCD and IsCMOS camera systems. The methodology allowed demonstration of superior SNR of the ICCD over the IsCMOS technology and superior resolution of the IsCMOS over the ICCD approach. Contrast depended upon the camera settings (binning and integration time) and gain of intensifier. Finally, because the architecture of CMOS and CCD camera systems results in vastly different performance, we comment on the utility of these technologies for small animal imaging as well as clinical applications for noninvasive and surgical guidance.

Background: Recent advancements in near-infrared fluorescence lymphatic imaging (NIRFLI) technology provide opportunities for non-invasive, real-time assessment of lymphatic contribution in the etiology and treatment of ulcers. The objective of this study was to assess lymphatics in subjects with venous leg ulcers using NIRFLI and to assess lymphatic impact of a single session of sequential pneumatic compression (SPC)., Methods: Following intradermal microdoses of indocyanine green (ICG) as a lymphatic contrast agent, NIRFLI was used in a pilot study to image the lymphatics of 12 subjects with active venous leg ulcers (Clinical, Etiologic, Anatomic, and Pathophysiologic [CEAP] C6). The lymphatics were imaged before and after a single session of SPC to assess impact on lymphatic function., Results: Baseline imaging showed impaired lymphatic function and bilateral dermal backflow in all subjects with chronic venous insufficiency, even those without ulcer formation in the contralateral limb (C0 and C4 disease). SPC therapy caused proximal movement of ICG away from the active wound in 9 of 12 subjects, as indicated by newly recruited functional lymphatic vessels, emptying of distal lymphatic vessels, or proximal movement of extravascular fluid. Subjects with the longest duration of active ulcers had few visible lymphatic vessels, and proximal movement of ICG was not detected after SPC therapy., Conclusions: This study provides visible confirmation of lymphatic dysfunction at an early stage in the etiology of venous ulcer formation and demonstrates the potential therapeutic mechanism of SPC therapy in removing excess fluid. The ability of SPC therapy to restore fluid balance through proximal movement of lymph and interstitial fluid may explain its value in hastening venous ulcer healing. Anatomical differences between the lymphatics of longstanding and more recent venous ulcers may have important therapeutic implications., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Although lower extremity edema/lymphedema can result from venous and/or lymphatic abnormalities, effective treatment depends upon understanding their relative contributions to the condition. Herein we use near-infrared fluorescence lymphatic imaging in a 16 year-old female diagnosed with unilateral lymphedema of the right leg and previously treated with left iliac vein stenting in an attempt to alleviate lymphedema. The imaging shows that abnormal lymphatic anatomy, rather than venous occlusion, was likely responsible for unilateral swelling., Competing Interests: Conflict of Interest CEF, JCR and EMS are listed as inventors on patents related to near-infrared fluorescence lymphatic imaging. JCR has received fees for consulting from NIRF Imaging, Inc., a UTHSCH start-up company seeking to commercialize the imaging technology. JCR and EMS may receive future financial benefit from NIRF Imaging, Inc. The other authors declare no potential conflict of interest.

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.

The performance evaluation of a variety of small animal tomography measurement approaches and algorithms for recovery of fluorescent absorption cross section has not been conducted. Herein, we employed an intensified CCD system installed in a commercial small animal CT (Computed Tomography) scanner to compare image reconstructions from time-independent, continuous wave (CW) measurements and from time-dependent, frequency domain (FD) measurements in a series of physical phantoms specifically designed for evaluation. Comparisons were performed as a function of (1) number of projections, (2) the level of preprocessing filters used to improve the signal-to-noise ratio (SNR), (3) endogenous heterogeneity of optical properties, as well as in the cases of (4) two fluorescent targets and (5) a mouse-shaped phantom. Assessment of quantitative recovery of fluorescence absorption cross section was performed using a fully parallel, regularization-free, linear reconstruction algorithm with diffusion approximation (DA) and high order simplified spherical harmonics ( SPN) approximation to the radiative transport equation (RTE). The results show that while FD measurements may result in superior image reconstructions over CW measurements, data acquisition times are significantly longer, necessitating further development of multiple detector/source configurations, improved data read-out rates, and detector technology. FD measurements with SP3 reconstructions enabled better quantitative recovery of fluorescent target strength, but required increased computational expense. Despite the developed parallel reconstruction framework being able to achieve more than 60 times speed increase over sequential implementation, further development in faster parallel acceleration strategies for near-real time and real-time image recovery and more precise forward solution is necessary.

Objective: The combination of both nuclear and fluorescent reporters provides unique opportunities for noninvasive nuclear imaging with subsequent fluorescence image-guided resection and pathology. Our objective was to synthesize and optimize a dual-labeled trastuzumab-based imaging agent that can be used to validate an optical imaging agent with potential use in identifying tumor metastases in human epidermal growth factor receptor 2 (HER2) positive breast cancer patients., Methods: [(111)In]-DTPA-trastuzumab-IRDye 800 was synthesized by a three-step procedure. Purity, stability, immunoreactivity, internalization and biodistribution were explored in HER2+ SKBR-3 cells. Biodistribution of [(111)In]-DTPA-trastuzumab-IRDye 800 was performed in a SKBR-3 xenograft model., Results: [(111)In]-DTPA-trastuzumab-IRDye 800 demonstrated high purity by both chemical and fluorometric determinations. Both flow cytometry and the Lindmo assay demonstrated a high binding affinity of [(111)In]-DTPA-trastuzumab-IRDye 800 to HER2-overexpressing cells. The dual-labeled conjugate was stable in PBS, but not in serum after 24 h at 37 °C. Larger molecules (>150 kD) were seen after a 24 h-incubation in human serum. Biodistribution studies revealed tumor-specific accumulation of [(111)In]-DTPA-trastuzumab-IRDye 800 in SKBR-3 tumors, and tumor uptakes at 24 and 48 h were (12.42±1.72)% and (9.96±1.05)%, respectively, following intravenous administration. The tumor-to-muscle ratio was 9.13±1.68 at 24 h, and increased to 12.79±2.13 at 48 h. Liver and kidney showed marked uptake of the dual-labeled imaging agent., Conclusions: [(111)In]-DTPA-trastuzumab-IRDye 800 is an effective diagnostic biomarker that can be used to validate dual-labeled, molecularly targeted imaging agents and can allow these agents to be translated into clinical practice for identifying HER2+ lesions.

The lymphatic vasculature plays a critical role in a number of disease conditions of increasing prevalence, such as autoimmune disorders, obesity, blood vascular diseases, and cancer metastases. Yet, unlike the blood vasculature, the tools available to interrogate the molecular basis of lymphatic dysfunction/disease have been lacking. More recently, investigators have reported that dysregulation of the PI3K pathway is involved in syndromic human diseases that involve abnormal lymphatic vasculatures, but there have been few compelling results that show the direct association of this molecular pathway with lymphatic dysfunction in humans. Using near-infrared fluorescence lymphatic imaging (NIRFLI) to phenotype and next generation sequencing (NGS) for unbiased genetic discovery in a family with non-syndromic lymphatic disease, we discovered a rare, novel mutation in INPPL1 that encodes the protein SHIP2, which is a negative regulator of the PI3K pathway, to be associated with lymphatic dysfunction in the family. In vitro interrogation shows that SHIP2 is directly associated with impairment of normal lymphatic endothelial cell (LEC) behavior and that SHIP2 associates with receptors that are associated in lymphedema, implicating its direct involvement in the lymphatic vasculature.

Background: Inflammatory breast cancer (IBC) remains the most aggressive type of breast cancer with the greatest potential for metastasis and as a result, the highest mortality rate. IBC cells invade and metastasize through dermal lymphatic vessels; however, it is unknown how lymphatic drainage patterns change during IBC growth and metastasis. Herein, we non-invasively and longitudinally imaged lymphatics in an animal model of IBC using near-infrared fluorescence (NIRF) imaging., Materials and Methods: Mice were imaged in vivo prior to, and up to 11 weeks after subcutaneous or orthotopic inoculation of human IBC SUM149 cells, which were stably transfected with infrared fluorescence protein (iRFP) gene reporter (SUM149-iRFP), following intradermal (i.d.) injection of indocyanine green (ICG)., Results: Fluorescence images showed well-defined lymphatic vessels prior to SUM149-iRFP inoculation. However, altered lymphatic drainage patterns including rerouting of lymphatic drainage were detected in mice with SUM149-iRFP, due to lymphatic obstruction of normal lymphatic drainages caused by tumor growth. In addition, we observed tortuous lymphatic vessels and extravasation of ICG-laden lymph in mice with SUM149-iRFP. We also observed increased and dilated fluorescent lymphatic vessels in the tumor periphery, which was confirmed by ex vivo immunohistochemical staining of lymphatic vessels., Conclusions: Our pre-clinical studies demonstrate that non-invasive NIRF imaging can provide a method to assess changes in lymphatic drainage patterns during IBC growth and metastasis.

Objective: Investigational, near-infrared fluorescence (NIRF) lymphatic imaging was used to assess lymphatic architecture and contractile function in participants diagnosed with Dercum's disease, a rare, poorly understood disorder characterized by painful lipomas in subcutaneous adipose tissues., Methods: After informed consent and as part of an FDA-approved feasibility study to evaluate lymphatics in diseases in which their contribution has been implicated, three women diagnosed with Dercum's disease and four control subjects were imaged. Each participant received multiple intradermal and subcutaneous injections of indocyanine green (ICG, total dose ≤400 µg) in arms, legs, and/or trunk. Immediately after injection, ICG was taken up by the lymphatics and NIRF imaging was conducted., Results: The lymphatics in the participants with Dercum's disease were intact and dilated, yet sluggishly propelled lymph when compared to control lymphatics. Palpation of regions containing fluorescent lymphatic pathways revealed tender, fibrotic, tubular structures within the subcutaneous adipose tissue that were associated with painful nodules, and, in some cases, masses of fluorescent tissue indicating that some lipomas may represent tertiary lymphoid tissues., Conclusions: These data support the hypothesis that Dercum's disease may be a lymphovascular disorder and suggest a possible association between abnormal adipose tissue deposition and abnormal lymphatic structure and function., (Copyright © 2014 The Obesity Society.)

Objective: To investigate the redirection of lymphatic drainage post-lymphadenectomy using non-invasive near-infrared fluorescence (NIRF) imaging, and to subsequently assess impact on metastasis., Background: Cancer-acquired lymphedema arises from dysfunctional fluid transport after lymphadenectomy performed for staging and to disrupt drainage pathways for regional control of disease. However, little is known about the normal regenerative processes of the lymphatics in response to lymphadenectomy and how these responses can be accelerated, delayed, or can impact metastasis., Methods: Changes in lymphatic "pumping" function and drainage patterns were non-invasively and longitudinally imaged using NIRF lymphatic imaging after popliteal lymphadenectomy in mice. In a cohort of mice, B16F10 melanoma was inoculated on the dorsal aspect of the paw 27 days after lymphadenectomy to assess how drainage patterns affect metastasis., Results: NIRF imaging demonstrates that, although lymphatic function and drainage patterns change significantly in early response to popliteal lymph node (PLN) removal in mice, these changes are transient and regress dramatically due to a high regenerative capacity of the lymphatics and co-opting of collateral lymphatic pathways around the site of obstruction. Metastases followed the pattern of collateral pathways and could be detected proximal to the site of lymphadenectomy., Conclusions: Both lymphatic vessel regeneration and co-opting of contralateral vessels occur following lymphadenectomy, with contractile function restored within 13 days, providing a basis for preclinical and clinical investigations to hasten lymphatic repair and restore contractile lymphatic function after surgery to prevent cancer-acquired lymphedema. Patterns of cancer metastasis after lymphadenectomy were altered, consistent with patterns of re-directed lymphatic drainage.

Passive protection, the administration of antibodies to prevent infection, has garnered significant interest in recent years as a potential prophylactic countermeasure to decrease the prevalence of hospital-acquired infections. Pili, polymerized protein structures covalently anchored to the peptidoglycan wall of many Gram-positive pathogens, are ideal targets for antibody intervention, given their importance in establishing infection and their accessibility to antibody interactions. In this work, we demonstrated that a monoclonal antibody to the major component of Enterococcus faecalis pili, EbpC, labels polymerized pilus structures, diminishes biofilm formation, and significantly prevents the establishment of a rat endocarditis infection. The effectiveness of this anti-EbpC monoclonal provides strong evidence in support of its potential as a preventative. In addition, after radiolabeling, this monoclonal identified the site of enterococcal infection, providing a rare example of molecularly specific imaging of an established bacterial infection and demonstrating the versatility of this agent for use in future diagnostic and therapeutic applications.

The lymphatic circulatory system has diverse functions in lipid absorption, fluid homeostasis, and immune surveillance and responds dynamically when presented with infection, inflammation, altered hemodynamics, and cancer. Visualization of these dynamic processes in human disease and animal models of disease is key to understanding the contributory role of the lymphatic circulatory system in disease and to devising effective therapeutic strategies. Longitudinal, non-destructive, and repeated imaging is necessary to expand our understanding of disease progression and regression in basic science and clinical investigations. Herein we summarize recent advances in in vivo lymphatic imaging employing magnetic resonance, computed tomography, lymphoscintigraphy, and emerging optical techniques with respect to their contributory roles in both basic science and clinical research investigations.

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.

Non-invasive injectable cellular therapeutic strategies based on sustained delivery of physiological levels of BMP-2 for spinal fusion are emerging as promising alternatives, which could provide sufficient fusion without the associated surgical risks. However, these injectable therapies are dependent on bone formation occurring only at the specific target region. In this study, we developed and deployed fluorescence gene reporter tomography (FGRT) to provide information on in vivo cell localization and viability. This information is sought to confirm the ideal placement of the materials with respect to the area where early bone reaction is required, ultimately providing three dimensional data about the future fusion. However, because almost all conventional fluorescence gene reporters require visible excitation wavelengths, current in vivo imaging of fluorescent proteins is limited by high tissue absorption and confounding autofluorescence. We previously administered fibroblasts engineered to produce BMP-2, but is difficult to determine 3-D information of placement prior to bone formation. Herein we used the far-red fluorescence gene reporter, IFP1.4 to report the position and viability of fibroblasts and developed 3-D tomography to provide placement information. A custom small animal, far-red fluorescence tomography system integrated into a commercial CT scanner was used to assess IFP1.4 fluorescence and to demark 3-D placement of encapsulated fibroblasts with respect to the vertebrae and early bone formation as assessed from CT. The results from three experiments showed that the placement of the materials within the spine could be detected. This work shows that in vivo fluorescence gene reporter tomography of cell-based gene therapy is feasible and could help guide cell-based therapies in preclinical models.

The response of the lymphatic system to inflammatory insult and infection is not completely understood. Using a near-infrared fluorescence (NIRF) imaging system to noninvasively document propulsive function, we noted the short-term cessation of murine lymphatic propulsion as early as 4h following LPS injection. Notably, the effects were systemic, displaying bilateral lymphatic pumping cessation after a unilateral insult. Furthermore, IL-1β, TNF-α, and IL-6, cytokines that were found to be elevated in serum during lymphatic pumping cessation, were shown separately to acutely and systemically decrease lymphatic pulsing frequency and velocity following intradermal administration. Surprisingly, marked lymphatic vessel dilation and leakiness were noted in limbs contralateral to IL-1β intradermal administration, but not in ipsilateral limbs. The effects of IL-1β on lymphatic pumping were abated by pre-treatment with an inhibitor of inducible nitric oxide synthase, L-NIL (N-iminoethyl-L-lysine). The results suggest that lymphatic propulsion is systemically impaired within 4h of acute inflammatory insult, and that some cytokines are major effectors of lymphatic pumping cessation through nitric oxide-mediated mechanisms. These findings may help in understanding the actions of cytokines as mediators of lymphatic function in inflammatory and infectious states., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Fluorescence gene reporters have recently become available for excitation at far-red wavelengths, enabling opportunities for small animal in vivo gene reporter fluorescence tomography (GRFT). We employed multiple projections of the far-red fluorescence gene reporters IFP1.4 and iRFP, excited by a point source in transillumination geometry in order to reconstruct the location of orthotopically implanted human prostate cancer (PC3), which stably expresses the reporter. Reconstruction was performed using a linear radiative-transfer-based regularization-free tomographic method. Positron emission tomography (PET) imaging of a radiolabeled antibody-based agent that targeted epithelial cell adhesion molecule overexpressed on PC3 cells was used to confirm in vivo GRFT results. Validation of GRFT results was also conducted from ex vivo fluorescence imaging of resected prostate tumor. In addition, in mice with large primary prostate tumors, a combination of GRFT and PET showed that the radiolabeled antibody did not penetrate the tumor, consistent with known tumor transport limitations of large (∼150  kDa) molecules. These results represent the first tomography of a living animal using far-red gene reporters.

During acute inflammation, functioning lymphatics are believed to reduce edema and to provide a transiting route for immune cells, but the extent at which the dermal lymphatic remodeling impacts lymphatic transport or the factors regulating these changes remains unclear. Herein we quantify the increase in lymphatic endothelial cells (LECs) and examine the expression of pro-angiogenenic and lymphangiogenic factors during acute cutaneous hypersensitivity (CHS). We found that LECs actively proliferate during CHS but that this proliferation does not affect the lymphatic vessel density. Instead, lymphatic remodeling is accompanied by lymphatic vessel leakiness and lower ejection of lymph fluid, which is observed only in the proximal lymphatic vessel draining the inflamed area. LECs and the immune cells release growth factors and cytokines during inflammation, which impact the lymphatic microenvironment and function. We identified that FGF-2, PLGF-2, HGF, EGF, and KC/CXCL17 are differentially expressed within tissues during acute CHS, but both VEGF-C and VEGF-D levels do not significantly change. Our results indicate that VEGF-C and VEGF-D are not the only players and other factors may be responsible for the LECs proliferation and altered lymphatic function in acute CHS.

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.

Mutations in gene RASA1 have been historically associated with capillary malformation-arteriovenous malformation, but sporadic reports of lymphatic involvement have yet to be investigated in detail. To investigate the impact of RASA1 mutations in the lymphatic system, we performed investigational near-infrared fluorescence lymphatic imaging and confirmatory radiographic lymphangiography in a Parkes-Weber syndrome (PKWS) patient with suspected RASA1 mutations and correlated the lymphatic abnormalities against that imaged in an inducible Rasa1 knockout mouse. Whole-exome sequencing (WES) analysis and validation by Sanger sequencing of DNA from the patient and unaffected biological parents enabled us to identify an early-frameshift deletion in RASA1 that was shared with the father, who possessed a capillary stain but otherwise no overt disease phenotype. Abnormal lymphatic vasculature was imaged in both affected and unaffected legs of the PKWS subject that transported injected indocyanine green dye to the inguinal lymph node and drained atypically into the abdomen and into dermal lymphocele-like vesicles on the groin. Dermal lymphatic hyperplasia and dilated vessels were observed in Rasa1-deficient mice, with subsequent development of chylous ascites. WES analyses did not identify potential gene modifiers that could explain the variability of penetrance between father and son. Nonetheless, we conclude that the RASA1 mutation is responsible for the aberrant lymphatic architecture and functional abnormalities, as visualized in the PKWS subject and in the animal model. Our unique method to combine investigatory near-infrared fluorescence lymphatic imaging and WES for accurate phenoptyping and unbiased genotyping allows the study of molecular mechanisms of lymphatic involvement of hemovascular disorders.

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.

Isotropic scattering coefficient measurements were made of monodisperse polystyrene lattices of two different diameters of 144 nm and 223 nm and at volume fractions ranging from 0.15 to 0.22, using frequency domain photon migration measurements at wavelengths of 660, 685, 785 and 828 nm. The isotropic scattering coefficient measurements were shown to be sensitive to the changing ionic strength (0.5-4 mM, NaCl equiv.) of the dispersions exhibiting hindered scattering owing to structure at the lowest ionic strength values. Monte Carlo simulations and numerical solution of the Ornstein Zernike equations were used to compute isotropic scattering coefficients for comparison to measured values. The interaction potential was modeled as a hard sphere Yukawa potential and the Hypernetted Chain closure was used to solve the OZ equation. Effective particle charges were found after renormalization of the bare particle charge and used to predict the isotropic scattering coefficient. The model data were found to follow similar trends as experimental measurements. The refractive index of the particles has found to be an important factor for predicting experimental isotropic scattering coefficient values., (Published by Elsevier Inc.)

Unlabelled: The proliferation of most carcinomas is associated with an overexpression of epithelial cell adhesion molecule (EpCAM), a 40-kDa type I transmembrane protein found on epithelial cells yet absent from other cell types. The absence of EpCAM in normal lymphatics makes it an attractive marker for studying lymph node (LN) metastases of carcinomas to improve LN staging accuracy. Herein, we developed and quantitatively compared dual-labeled monoclonal antibodies (mAbs) of varying affinities against EpCAM for both noninvasive and intraoperative detection of metastatic LNs in prostate cancer., Methods: A panel of hybridoma-derived anti-EpCAM mAbs was generated and screened. Two high-affinity candidate mAbs with specificity for nonoverlapping epitopes on the EpCAM extracellular domain were chosen for further evaluation. After conjugation with DOTA for (64)Cu radiolabeling and IRDye 800CW as a fluorophore, dual-labeled specific or isotype control mAb was administered intravenously to male nu/nu mice at 10-12 wk after orthotopic implantation of DsRed-expressing PC3 cells. Within 18-24 h, noninvasive small-animal PET/CT and in vivo, in situ, and ex vivo DsRed reporter gene and near-infrared fluorescence (NIRF) imaging were performed to detect primary tumors and metastatic LNs. Using DsRed fluorescence as the true indicator of cancer-positive tissue, we performed receiver operating characteristic curve analyses of percentage injected dose per gram measured from quantitative small-animal PET/CT and fluorescence intensity measured from semiquantitative NIRF imaging for each LN examined to compare mAb sensitivity and specificity., Results: mAbs 7 and 153 generated in-house were found to have higher affinity than commercial mAb 9601. Accuracy, as a function of sensitivity and specificity, for the detection of cancer-positive LNs during in vivo small-animal PET/CT was highest for mAbs 7 (87.0%) and 153 (78.0%) and significantly greater (P < 0.001) than random chance (50.0%). Rates for mAb 9601 (60.7%) and control mAb 69 (27.0%) were not significantly different from chance. Similarly, mAb 7 had significant detection accuracy by NIRF imaging (96.0%, P < 0.001)., Conclusion: mAbs 7 and 153 are attractive, high-affinity candidates for further multimodal imaging agent optimization aimed at enhancing sensitivity and specificity for detection of metastatic LNs in prostate cancer. Fully quantitative NIRF imaging is needed for comprehensive analyses of NIRF-labeled agent accuracy for intraoperative guidance.

The lymphatic system plays an important role in maintaining the fluid homeostasis between the blood vascular and interstitial tissue compartment and there is recent evidence that its transport capabilities may regulate blood pressure in salt-induced hypertension. Yet, there is little known how the lymphatic contractile function and architecture responds to dietary salt-intake. Thus, we longitudinally characterized lymphatic contractile function and vessel remodeling noninvasively using dynamic near-infrared fluorescence imaging in animal models of salt-induced hypertension. The lymphatics of mice and rats were imaged following intradermal injection of indocyanine green to the ear tip or the base of the tail before and during two weeks of either a high salt diet (HSD) or normal chow. Our noninvasive imaging data demonstrated dilated lymphatic vessels in the skin of mice and rats on a HSD as compared to their baseline levels. In addition, our dynamic imaging results showed increased lymphatic contraction frequency in HSD-fed mice and rats. Lymphatic contractile function and vessel remodeling occurs in response to salt-induced hypertension suggesting a possible role for the lymphatics in the regulation of vascular blood pressure.

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.