Grace Wu

Albumin-Binding Domain Conjugate for Near-Infrared Fluorescence Lymphatic Imaging

PurposeThe aim of this study was to develop and characterize a novel peptide imaging agent for noninvasive near-infrared fluorescence imaging of protein transport by the lymphatics. An imaging agent consisting of a cyclic albumin-binding domain (cABD) peptide, with sequence, Arg-Leu-Ile-Glu-Asp-Ile-Cys-Leu-Pro-Arg-Trp-Gly-Cys-Leu-Trp-Glu-Asp-Asp-Lys, was conjugated to a near-infrared fluorophore, IRDye800CW, allowing for enhanced vascular uptake, retention, and fluorescence imaging.ProcedureCharacterization of the cABD-IRDye800 peptide conjugate was performed using fluorescence spectroscopy to assess optical properties and SDS-PAGE and Biacore binding assays to determine binding affinity and specificity. Fluorescence imaging of normal C57BL/6 mice was conducted to monitor lymphatic uptake and retention.ResultscABD-IRDye800 exhibited approximately six times greater fluorescent yield and greater stability than indocyanine green, an agent previously used in humans to image lymphatic vasculature. The agent exhibited affinity for albumin with IC50 and Kd in the nanomolar range and demonstrated superior retention characteristics within mouse lymphatics when compared with IRDye800CW.ConclusionscABD-IRDye800 has utility for assessing lymphatic function in mouse models of human lymphatic disease and the potential for use in clinical diagnostic imaging of the lymphatic vasculature.

Spatio-temporal changes of lymphatic contractility and drainage patterns following lymphadenectomy in mice

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.

In vivo imaging of orthotopic prostate cancer with far-red gene reporter fluorescence tomography and in vivo and ex vivo validation

Abstract. 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.

Direct visualization of changes of lymphatic function and drainage pathways in lymph node metastasis of B16F10 melanoma using near-infrared fluorescence imaging

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.

In vivo lymphatic imaging of a human inflammatory breast cancer model.

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.

Longitudinal far red gene-reporter imaging of cancer metastasis in preclinical models: a tool for accelerating drug discovery

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.

Dextran sulfate sodium-induced acute colitis impairs dermal lymphatic function in mice.

AIM To investigate whether dermal lymphatic function and architecture are systemically altered in dextran sulfate sodium (DSS)-induced acute colitis. METHODS Balb/c mice were administered 4% DSS in lieu of drinking water ad libitum for 7 d and monitored to assess disease activity including body weight, diarrhea severity, and fecal bleeding. Control mice received standard drinking water with no DSS. Changes in mesenteric lymphatics were assessed following oral administration of a fluorescently-labelled fatty acid analogue, while dermal lymphatic function and architecture was longitudinally characterized using dynamic near-infrared fluorescence (NIRF) imaging following intradermal injection of indocyanine green (ICG) at the base of the tail or to the dorsal aspect of the left paw prior to, 4, and 7 d after DSS administration. We also measured dye clearance rate after injection of Alexa680-bovine serum albumin (BSA). NIRF imaging data was analyzed to reveal lymphatic contractile activity after selecting fixed regions of interest (ROIs) of the same size in fluorescent lymphatic vessels on fluorescence images. The averaged fluorescence intensity within the ROI of each fluorescence image was plotted as a function of imaging time and the lymphatic contraction frequency was computed by assessing the number of fluorescent pulses arriving at a ROI. RESULTS Mice treated with DSS developed acute inflammation with clinical symptoms of loss of body weight, loose feces/watery diarrhea, and fecal blood, all of which were aggravated as disease progressed to 7 d. Histological examination of colons of DSS-treated mice confirmed acute inflammation, characterized by segmental to complete loss of colonic mucosa with an associated chronic inflammatory cell infiltrate that extended into the deeper layers of the wall of the colon, compared to control mice. In situ intravital imaging revealed that mice with acute colitis showed significantly fewer fluorescent mesenteric lymphatic vessels, indicating impaired uptake of a lipid tracer within mesenteric lymphatics. Our in vivo NIRF imaging data demonstrated dilated dermal lymphatic vessels, which were confirmed by immunohistochemical staining of lymphatic vessels, and significantly reduced lymphatic contractile function in the skin of mice with DSS-induced acute colitis. Quantification of the fluorescent intensity remaining in the depot as a function of time showed that there was significantly higher Alexa680-BSA fluorescence in mice with DSS-induced acute colitis compared to pre-treatment with DSS, indicative of impaired lymphatic drainage. CONCLUSION The lymphatics are locally and systemically altered in acute colitis, and functional NIRF imaging is useful for noninvasively monitoring systemic lymphatic changes during inflammation.

Non-invasive imaging of prostate cancer progression in nude mice using iRFP gene reporter

Prostate cancer (PCa) is the second most common cancer in US men. Metastasis is the final step of tumor progression and remains the primary cause of PCa death. Hence preclinical, orthotopic models of PCa metastasis are necessary to develop new therapeutics against metastatic disease. Yet unlike irrelevant subcutaneous tumor models, the deployment of orthotopic models of cancer metastasis in drug research and development is limited by the inability to longitudinally monitor cancer progression/regression in response to administration of experimental pharmaceuticals. Recently, a nearinfrared fluorescent protein (iRFP) was created for deeper imaging [1]. Imaging prostate tumor growth and lymph node metastasis in nude mice therefore becomes possible using this new fluorescent gene reporter. In this study, we first developed an intensified CCD (ICCD)-based iRFP fluorescence imaging device. Then human PCa PC3 cell lines expressing iRFP gene reporter were orthotopically implanted in male Nu/Nu mice at 8-10 weeks old. After 6-10 weeks, in vivo, in situ and ex vivo fluorescence imaging was performed. In vivo iRFP fluorescence imaging showed that the detected fluorescence concentrated at the prostate and became stronger over time, indicating the growth of implanted PCa. Fluorescence was non-invasively detected at locations of prostate-draining lymph nodes as early as 5 weeks post implantation, indicating the metastasis to lymph nodes. In situ and ex vivo fluorescence imaging demonstrated that the detected signals from PCa and lymph nodes were correlated with cancer positive status of tissues as assessed through standard pathology.

Abstract P5-01-04: In vivo lymphatic imaging of a human inflammatory breast cancer model

Background: Inflammatory breast cancer (IBC) is the most aggressive but poorly understood type of breast cancer. IBC constitutes only appoximately 5% of all newly diagnosed breast cancers, yet responsible for 8-10% of breast cancer-related deaths. Unfortunately, there are no definitive molecular or pathological, early diagnostic criteria for IBC. IBC grows in nests or sheets, spreading outward into the skin and eventually distant sites through dermal lymphatic vessels; therefore, one of the key pathological characteristics of IBC is dermal lymphatic invasion by tumor emboli, which can lead to obstruction of the lymphatic drainage possibly causing the clinical inflammatory features of diffuse erythema, rapid breast enlargement and edema as indicated by peau d’orange (orange peel) appearance covering at least a third of the breast surface. However, relatively little is known about the role of lymphatic function in IBC growth and metastasis. The purpose of this study was to non-invasively and longitudinally image changes of lymphatic drainage patterns in mice bearing the triple negative, human IBC SUM149 cells, which were stably transfected with iRFP gene reporter (iRFP-SUM149) in order to better monitor tumor growth and metastasis. Methods: Eight to ten weeks old female non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice were housed and fed special sterilized pelleted food and sterilized water. Two weeks after feeding special diets, mice were subcutaneously injected with iRFP-SUM149 cells in the left hindlimb or orthotopically in the left inguinal mammary fat pad (MFP). Fluorescence images were acquired immediately after and for up to 10 mins after i.d. injection of indocyanine green (ICG) using a custom-built NIRF imaging system. To achieve a greater magnification, a macrolens was used. Results: Our NIRF imaging data showed gradual changes of lymphatic vessels around the tumor. In mice with an orthotopic tumor, ICG accumulation was observed in the distal peritumoral region at up to 3 weeks p.i., indicative of the start of tumor obstruction of normal lymphatic drainage. As tumors grew, the greater extent of lymph flow, but not all, was obstructed by a tumor, resulting in dermal backflow and thus staining more lymphatic capillaries and eventually rerouting of lymphatic drainage was detected due to complete obstruction of lymph flow by a tumor. Similar to an orthotopic model, mice with a s.c. tumor also showed altered lymphatic drainage patterns during tumor growth. Interestingly, extravasation of ICG into iRFP-SUM149 was detected at 3 weeks p.i., due to leaky lymphatic vessels. A similar lymphatic phenotype has also been observed in IBC patients. Previously, we have imaged atypical tortuous lymphatics and altered lymphatic drainage in the arm and affected breast of a patient with IBC using clinical NIRF imaging developed in our team to image as deep as 3-5 cm with a microdose of ICG. Conclusions: Longitudinal, non-invasive imaging of lymphatic functional and architectural changes provides a new method to dynamically monitor lymphatic response to IBC growth and metastasis. Citation Format: Sunkuk Kwon, Germaine Agollah, Grace Wu, Eva M Sevick-Muraca. In vivo lymphatic imaging of a human inflammatory breast cancer model [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P5-01-04.

Abstract 2055: Modulated near-infrared fluorescence light imaging of primary tumor margins, cancer positive lymph nodes, and freshly excised human cancers with imaging agent targeting EpCAM

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA We developed an epithelial cancer-specific, imaging agent labeled for near-infrared florescence (NIRF) imaging together with an intraoperative surgical device for sensitive detection of tumor margins and local metastatic lesions within the surgical field of view. The drug/device combination was tested on an orthotopic model and on freshly resected, human cancerous tissues. To maximize device performance, we illuminated tissue surfaces with modulated near-infrared (NIR) excitation light (785 nm) and collected fluroescent light (830 nm) via using a NIR sensitive image intensifier modulated at with 90 degree phase delays added between 3 consecutively acquired images. These phase-delayed images were collected by a CCD and processed in near-real time to provide images of fluroescence amplitude with significantly higher contrast and without significant reduction in signal to noise ratio as compared to conventional images acquired without modulation. To develop a universal imaging agent for use with the device, a high affinity monoclonal antibody (mAb) against epithelial cell adhesion molecule (EpCAM) was selected from a previously generated panel and labeled with NIRF dye, IRDye800. The labeled mAb was characterized for conserved biological activity. Upon dual-labeling this lead mAb candidate for both PET and NIRF, we showed the accuracy of planar NIRF imaging (96%) to be superior to PET (93%) for detecting cancer positive lymph nodes 24 hours following 40 ug i.v. injection in an orthotopic model of PC3 human metastatic prostate cancer 10-12 weeks post-implantation. Compared to NIRF labeled isotype control mAb, the labeled anti-EpCAM mAb accurately detected primary tumor margins as indicated by both pathology and far-red fluorescent protein reporter, iRFP (excitation 690 nm/ emission 720 nm), that was stably expressed in implanted PC3 cells. Furthermore, modulated light imaging provided greater accuracy in detecting margins compared to the widely used conventional fluorescent collection techniques. Upon immersing freshly excised human squamous cell carcinoma tissues from individual patients in dilute solutions of labeled anti-EpCAM and labeled isotype control and rinsing, we used the device to demonstrate consistent and specific binding of labeled anti-EpCAM mAb to cancer sites. In addition, cancer margins detected by NIRF were pathologically confirmed. When combined with a high-affinity imaging agent targeting an antigen overexpressed on nearly all epithelial cancers, NIRF imaging can be used to accurate detect margins as well as cancer positive LNs. Finally, the modulated NIRF imaging can be deployed for open surgeries as well as endoscopic and robotic surgeries wherein surgical field illumination would otherwise destroy molecular imaging contrast. Citation Format: Banghe Zhu, Barrett Harvey, Melissa Aldrich, Grace Wu, Nathaniel Wilganowski, Holly Robinson, Kenneth Pinkston, Gao Peng, Songlin Zhang, Ron Karni, Eva M. Sevick-Muraca. Modulated near-infrared fluorescence light imaging of primary tumor margins, cancer positive lymph nodes, and freshly excised human cancers with imaging agent targeting EpCAM. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2055. doi:10.1158/1538-7445.AM2014-2055