Fluorophore-conjugated 4-1BB antibody enables early detection of T-cell responses in inflammatory arthritis via NIRF imaging

BackgroundActivated T cells play a pivotal role in rheumatoid arthritis (RA) pathogenesis, and imaging of activated T cells may provide a non-invasive tool for RA detection. Here, we first developed an optical probe targeting human inducible T cell co-stimulator (ICOS) and tested its capacity in RA diagnosis by capturing ICOS+ activated T cells in vivo in a humanized mouse model.MethodsThe humanized arthritis model, Human peripheral blood mononuclear cells- adjuvant induced arthritis (HuPBMC-AIA) was established, and flow cytometry and immunofluorescence were employed to determine ICOS expression in huPBMC-AIA model. Anti-human ICOS monoclonal antibody (mAb) was conjugated to Cy7 via NHS ester amine reaction. A cell uptake study was used to confirm the specificity of Cy7-ICOS mAb to activated T cells. 4-view near-infrared fluorescence (NIRF) imaging study was performed to test Cy7-ICOS mAb in detecting RA in vivo.FindingsICOS was confirmed as an indicator of RA pathogenesis via RNA-seq, flow cytometry and immunofluorescence data. An in-vitro cellular uptake study validated the specificity of Cy7-ICOS mAb to activated T cells. Cy7-ICOS mAb could detect ICOS+ activated T cells in vivo through 4-view NIRF imaging. The receiver operating characteristic (ROC) curve created based on NIRF imaging quantification could distinguish the huPBMC-AIA group from the control group at all time points imaged.ConclusionIn this study, we first developed an optical imaging probe targeting human ICOS, Cy7-ICOS mAb. The 4-view NIRF imaging with Cy7-ICOS mAb could detect pathogenic ICOS+ activated T cells with high sensitivity and specificity in vivo, which indicated the great potential of this imaging probe in RA early diagnosis.

PurposeThe goal of this study was to develop an imaging probe—IRDye-680RD-OX40 mAb—that can be used for noninvasive imaging and optical imaging of rheumatoid arthritis (RA). OX40/OX40 ligand (OX40L) interactions have been shown to exert potent costimulatory effects on T cell activation. Detectable change in T cell activation profiles was observed in early RA.MethodsOX40 expression pattern was analyzed by flow cytometry. N-hydroxysuccinimide (NHS) esters are used to label proteins selectively on free amino groups of OX40 monoclonal antibody (mAb). Characterization of IRDye-680RD-OX40 mAb was measured and a fluorescence spectrum gathered. Cell binding assay was also performed between activated and naïve murine T cells. Longitudinal near-infrared fluorescence (NIRF) imaging of the probe was performed on day 8, day 9, day 10, and day 11 of adjuvant-induced arthritis (AIA) mouse model. Paw thickness and body weight were compared between the OX40 mAb and IgG injection groups.ResultsNIRF imaging with IRDye-680RD-OX40 mAb revealed strong OX40-positive responses with high specificity. Flow analysis showed that OX40 was specifically expressed on the surface of T cells in RP and spleen of AIA model. The AIA group was significantly differentiated from the control group at all time points with imaging monitoring. The region of interest (ROI) was in line with ex vivo imaging and biodistribution study. This study highlights the potential utility of the OX40 NIRF imaging as a new strategy for RA prediction and T cell monitoring.ConclusionThe results provide evidence that IRDye-680RD-OX40 mAb detects organized T cells activation in early RA. The optical probe was capable of detection of RA pathogenesis. It identified transcriptional responses to RA that mediate its immune functions. Thus, it may be an ideal probe for RA imaging.Graphical

Introduction: Near-infrared fluorescence (NIRF) imaging is an emerging technology with several important applications in oncologic surgery. Using fluorescent dyes and dedicated camera systems, real-time intraoperative imaging is provided to guide surgical procedures and allow for instant decision-making. NIRF imaging has the potential to improve the Sentinel Node Biopsy (SNB) procedure for staging of oral cavity cancer by facilitating intraoperative visual identification of the sentinel lymph node (SN). Also, NIRF imaging may be used in the search of head and neck unknown primaries (CUP) in combination with transoral robotic surgery (TORS). The feasibility of NIRF imaging for intraoperative SN detection was investigated in a series of oral cavity cancer patients. In addition, our initial experience of using NIRF imaging during TORS for detection of oropharyngeal cancer will also be reported (ongoing study). Methods: (1) A prospective study of patients with primary oral squamous cell carcinoma (OSCC) planned for tumor resection and SNB. Thirty patients were injected peritumorally with a bimodal tracer (ICG-99mTc-Nanocoll) followed by lymphoscintigraphy (LSG) and SPECT/CT to define the SNs and their anatomical location preoperatively. SNs were detected intraoperatively with a hand-held gamma-probe and a hand-held NIRF camera. (2) In a minor prospective series of patients with oropharyngeal cancer undergoing TORS Indocyanin Green (ICG) was systematically injected preoperatively. The aim was to guide primary tumor detection using the firefly NIRF modality incorporated in the Da Vinci Si robotic system. Results: (1) In the SNB study, 29 of 30 subjects (97%), all preoperatively defined SNs could be identified intraoperatively using a combination of radioactive and fluorescence guidance. Eleven of 94 SNs (12%) could only be identified in vivo using NIRF imaging and the majority of those were located in level 1 close to the primary tumor. (2) In the pilot ICG TORS study, the additional use of ICG for primary tumor identification was varying. Conclusions: Intraoperative NIRF imaging as a tool to guide cancer surgery seems promising and further exploration of this novel technology is warranted. A combined fluorescent and radioactive tracer for SNB is feasible and the additional use of NIRF imaging may improve the accuracy of SN identification in oral cancer patients. Intraoperative fluorescence guidance seems of particular value, when SNs are located in close proximity to the injection site. Combined TORS and NIRF imaging showed with a varying quality the localization of the oropharyngeal cancer. The combined procedure may have a potential role in the diagnostic algorithm in head and neck CUP patients facilitating the detection of the primary tumor. Citation Format: Christian von Buchwald, Anders Christensen, Niclas Rubek, Karina Juhl, Birgitte Charabi, Jann Mortensen, Katalin Kiss, Andreas Kjær. Real-time near-infrared fluorescence tracer imaging to guide sentinel node biopsy and tumor detection in head and neck cancer [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; April 23-25, 2017; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(23_Suppl):Abstract nr IA13.

Background: Near-infrared (NIR) fluorescence imaging using indocyanine green (ICG) is a promising technique to obtain real-time assessment of the extent and number of colorectal liver metastases during liver surgery. The current study aims to optimize dosage and timing of ICG administration. Materials and methods: The Mini-FLARE imaging system was used for real-time identification of liver tumors in 18 rats. Liver tumors were measured at 24, 48, 72 or 96 hours after administration of 0.04, 0.08, or 0.16 mg ICG (0.13, 0.26 or 0.53 mg / kg, respectively). Guided by these results, intraoperative identification of liver metastases was performed in 12 patients undergoing liver resection. NIR fluorescence imaging was performed 24 or 48 hours after administration of 10 or 20 mg ICG (0.13 or 0.26 mg / kg, respectively). After intraoperative imaging, resection specimens were sliced to examine internal fluorescent patterns using the Mini-FLARE imaging system. Subsequently, frozen tissue sections were measured for fluorescence using the Nuance multispectral imager. Results: Using NIR fluorescence imaging and ICG, all colorectal liver metastases (N = 34), could be identified in all rats. Average tumor-to-liver (TLR) ratio over all groups was 3.0 ± 1.2. Liver signal was lower in the 72 h time group compared to other time points, resulting in a significantly higher TLR. ICG dose did not significantly influence TLR, but a trend was found favoring the 0.08 mg dose group. Clinically, during intraoperative NIR fluorescence imaging, all superficially located metastases (< 1 cm beneath liver capsule) were identified (N = 18). Average TLR was 11.1 ± 5.1 and no significant differences between time-points or doses were found. Liver signal was comparable to pre-injection signal at 24 to 48 hours post-injection, eliminating the need to test other time-points. In all patients, a fluorescent rim around the tumor was found, as described in earlier studies. Using fluorescence microscopy, this clear fluorescent rim was localized in stromal tissue in the transition area between tumor and normal liver tissue in all liver metastases. In this area, multiple cell types that are involved in tissue inflammation (e.g. granulocytes, lymphocytes) were found. In two patients, additional small (2 – 8 mm) metastases were identified using NIR fluorescence that were otherwise missed preoperatively and intraoperatively using only visual inspection and ultrasound. Conclusions: This study demonstrates that colorectal cancer liver metastases can be clearly identified during surgery using ICG and the Mini-FLARE imaging system. NIR fluorescence imaging has the potential to improve intraoperative detection of in particular small and superficially located liver metastases and can therefore be seen as an addition to the conventional imaging modalities. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4140. doi:10.1158/1538-7445.AM2011-4140

The intraoperative distinction between normal and abnormal pituitary tissue is crucial during pituitary adenoma surgery to obtain a complete tumor resection while preserving endocrine function. Near-infrared (NIR) fluorescence imaging is a technique to intraoperatively visualize tumors by using indocyanine green (ICG), a contrast agent allowing visualization of differences in tissue vascularization. Although NIR fluorescence imaging has been described in pituitary surgery, it has, in contrast to other surgical areas, never become widely used. To evaluate NIR fluorescence imaging in pituitary surgery, both qualitatively and quantitatively, and to assess the additional value of resecting adenoma tissue under NIR fluorescence guidance. We included 10 patients planned to undergo transnasal transsphenoidal selective adenomectomy. Patients received multiple intravenous administrations of 5 mg ICG, up to a maximum of 15 mg per patient. Endoscopic NIR fluorescence imaging was performed at multiple points in time. The NIR fluorescent signal in both the adenoma and pituitary gland was obtained, and the fluorescence contrast ratio was assessed. Four patients had Cushing disease, 1 had acromegaly, and 1 had a prolactinoma. Four patients had a nonfunctioning macroadenoma. In 9 of 10 patients with a histologically proven pituitary adenoma, the normal pituitary gland showed a stronger fluorescent signal than the adenoma. A fluorescence contrast ratio of normal pituitary gland to adenoma of 1.5 ± 0.2 was obtained. In 2 patients; adenoma resection was actually performed under NIR fluorescence guidance instead of under white light. NIR fluorescence imaging can easily and safely be implemented in pituitary surgery. The timing of ICG administration is important for optimal results and warrants further study. It appears that injection of ICG can best be postponed until some part of the normal pituitary gland is identified. Subsequent repeated low-dose ICG administrations improved the distinction between adenoma and gland.

The main aims of this study are to investigate the clinical application value of using indocyanine green fluorescence imaging for ensuring complete resection of tumour tissue during hepatectomy and to evaluate the diagnostic efficacy of near-infrared (NIR) fluorescence imaging system using indocyanine green in hepatectomy. After undergoing liver resection at the Affiliated Hospital of Southwest Medical University from July 2017 to May 2018, 35 eligible patients were included in this study. The liver surface and resection margin were intraoperatively assessed by intraoperative ultrasonography and NIR fluorescence imaging, after intravenous administration of indocyanine green (0.5 mg/kg) 72-96 h prior to surgery. The intraoperative observations were compared with the pathological findings in the liver. In the 35 patients, a total of 53 lesions were found, of which 42 were malignant lesions. The analysis results showed that the sensitivity and accuracy of detection using NIR fluorescence imaging were significantly higher than with intraoperative ultrasonography (P < 0.05). However, there was no difference between contrast-enhanced helical computed tomography and NIR fluorescence imaging in finding lesions (P > 0.05). In addition, 11 new suspicious lesions were detected only by NIR fluorescence imaging in the liver surface and resection margin during surgery, four of which were hepatocellular carcinoma. We also detected four vein tumour thrombi using the NIR fluorescence navigation system. The NIR fluorescence navigation system enables the identification of small tumours, residual cancer tissues in resection margin and venous tumour embolies in real time and enhances the accuracy and integrity of liver resection.

ObjectiveIn ovarian cancer, two of the most important prognostic factors for survival are completeness of staging and completeness of cytoreductive surgery. Therefore, intra-operative visualization of tumor lesions is of great importance. Preclinical data already demonstrated tumor visualization in a mouse-model using near-infrared (NIR) fluorescence imaging and indocyanine green (ICG) as a result of enhanced permeability and retention (EPR). The aim of this study was to determine feasibility of intraoperative ovarian cancer metastases imaging using NIR fluorescence imaging and ICG in a clinical setting.MethodsTen patients suspected of ovarian cancer scheduled for staging or cytoreductive surgery were included. Patients received 20 mg ICG intravenously after opening the abdominal cavity. The mini-FLARE NIR fluorescence imaging system was used to detect NIR fluorescent lesions.Results6 out of 10 patients had malignant disease of the ovary or fallopian tube, of which 2 had metastatic disease outside the pelvis. Eight metastatic lesions were detected in these 2 patients, which were all NIR fluorescent. However, 13 non-malignant lesions were also NIR fluorescent, resulting in a false-positive rate of 62%. There was no significant difference in tumor-to-background ratio between malignant and benign lesions (2.0 vs 2.0; P=0.99).ConclusionsThis is the first clinical trial demonstrating intraoperative detection of ovarian cancer metastases using NIR fluorescence imaging and ICG. Despite detection of all malignant lesions, a high false-positive rate was observed. Therefore, NIR fluorescence imaging using ICG based on the EPR effect is not satisfactory for the detection of ovarian cancer metastases. The need for tumor-specific intraoperative agents remains.Trial RegistrationISRCTN Registry ISRCTN16945066

Long-term follow-up after near-infrared fluorescence-guided resection of colorectal liver metastases: A retrospective multicenter analysis

Objectives: The early diagnosis and treatment of rheumatoid arthritis (RA) is important to reduce joint destruction. Many of the current imaging techniques have disadvantages, such as the need for contrast agents and interpretation by specialists. Fluorescence imaging is an emerging technique that overcomes some of these problems. The aim of this study was to determine whether near-infrared (NIR) fluorescence imaging of indocyanine green (ICG)-lactosomes can detect joint inflammation in a mouse model of RA.Methods: Control and arthritic SKG/Jcl mice were injected with ICG alone or ICG-lactosomes and examined by NIR fluorescence imaging. Arthritis severity was assessed macroscopically and histopathologically.Results: ICG fluorescence was detected in the liver soon after injection and then decreased over the next several hours. ICG was not detected in the joints of control or arthritic mice. In contrast, ICG-lactosomes remained in mice for at least 48 h and accumulated specifically at inflamed joints. ICG-lactosome fluorescence was higher in arthritic versus normal joints at all times examined and was maximal at 24 h after injection.Conclusions: NIR fluorescence imaging of ICG-lactosomes detects arthritic joints in a mouse model of RA. ICG-lactosomes may preferentially localize to inflamed joints via enhanced permeability and retention.

Detection of Cancer Metastases with a Dual-labeled Near-Infrared/Position Emission Tomography Imaging Agent

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 available. In this study, we non-invasively imaged changes of lymphatic function and drainage patterns using dynamic 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 foot for baseline data and until 21 days post-implant (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). In addition, intravital color images using a stereomicroscope were acquired after a skin incision. Then, the PLNs and inguinal LNs (ILNs) were harvested for ex vivo NIRF and intravital color imaging. We observed significant reduction of lymphatic contractility from 7 days p.i. until 21 days p.i.. Altered lymphatic drainage patterns were detected at 21 days p.i., due to lymphatic obstruction of normal lymphatic drainages caused by extensive tumor invasion of draining LNs. Magnified fluorescent images of tumor-draining lymphatic vessels showed numerous fluorescent lymphatic vessels at day 21 p.i. as compared to well-defined fluorescent vessels from a baseline image. Our CT data showed enlarged tumor-draining PLNs at 21 days p.i. and intravital images confirmed the PLN harbored melanoma cells. Overlay of white light and fluorescent images of dissected LNs showed strong ICG fluorescence in the ILN and to less extent in the PLN on the tumor bearing side, indicating significantly reduced lymph flow to the PLN due to blockage of normal lymph flow by tumor in the PLN. 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. Citation Format: Sunkuk Kwon, Germaine Agollah, Grace Wu, Sevick Sevick-Muraca. Direct visualization of changes of lymphatic function and drainage pathways in lymph node metastasis of B16F10 melanoma using near-infrared fluorescence imaging. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr A43.

Near-infrared (NIR) fluorescence imaging using indocyanine green (ICG) has the potential to improve the sentinel lymph node (SLN) procedure by facilitating percutaneous and intraoperative identification of lymphatic channels and SLNs. Previous studies suggested that a dose of 0.62mg (1.6mL of 0.5mM) ICG is optimal for SLN mapping in breast cancer. The aim of this study was to evaluate the diagnostic accuracy of NIR fluorescence for SLN mapping in breast cancer patients when used in conjunction with conventional techniques. Study subjects were 95 breast cancer patients planning to undergo SLN procedure at either the Dana-Farber/Harvard Cancer Center (Boston, MA, USA) or the Leiden University Medical Center (Leiden, the Netherlands) between July 2010 and January 2013. Subjects underwent the standard-of-care SLN procedure at each institution using (99)Technetium-colloid in all subjects and patent blue in 27 (28%) of the subjects. NIR fluorescence-guided SLN detection was performed using the Mini-FLARE imaging system. SLN identification was successful in 94 of 95 subjects (99%) using NIR fluorescence imaging or a combination of both NIR fluorescence imaging and radioactive guidance. In 2 of 95 subjects, radioactive guidance was necessary for initial in vivo identification of SLNs. In 1 of 95 subjects, NIR fluorescence was necessary for initial in vivo identification of SLNs. A total of 177 SLNs (mean 1.9, range 1-5) were resected: 100% NIR fluorescent, 88% radioactive, and 78% (of 40 nodes) blue. In 2 of 95 subjects (2.1%), SLNs-containing macrometastases were found only by NIR fluorescence, and in one patient this led to upstaging to N1. This study demonstrates the safe and accurate application of NIR fluorescence imaging for the identification of SLNs in breast cancer patients, but calls into question what technique should be used as the gold standard in future studies.

PurposeThe purpose of this study was to assess whether the vascularisation of the meniscus could be visualised intra-operatively using near-infrared fluorescence (NIRF) imaging with indocyanine green (ICG) in patients undergoing total knee arthroplasty (TKA).MethodsThe anterior horn (i.e., Cooper classification: zones C and D) of the meniscus that was least affected (i.e., least degenerative) was removed during TKA surgery in ten patients to obtain a cross section of the inside of the meniscus. Thereafter, 10 mg of ICG was injected intravenously, and vascularisation of the cross section of the meniscus was assessed using the Quest spectrum NIRF camera system. We calculated the percentage of patients in whom vascularisation was observed intra-operatively using NIRF imaging compared to immunohistochemistry.ResultsMeniscal vascularisation using NIRF imaging was observed in six out of eight (75%) patients in whom vascularisation was demonstrated with immunohistochemistry. The median extent of vascularisation was 13% (interquartile range (IQR) 3–28%) using NIRF imaging and 15% (IQR 11–23%) using immunohistochemistry.ConclusionThis study shows the potential of NIRF imaging to visualise vascularisation of the meniscus, as vascularisation was observed in six out of eight patients with histologically proven meniscal vascularisation.Level of evidenceIV.

Intraoperative assessment of myocardial perfusion using near-infrared fluorescence and indocyanine green: A literature review.

Magnetic resonance imaging (MRI) is one of the best imaging modalities for noninvasive cancer detection but MRI does not have enough sensitivity to delineate tumor margins during surgery. Moreover, since most surgical tools contain metal substances, image-guided surgery is hard to perform with a MR machine using magnets. Also, MR imaging is too slow for real-time guided-surgery. On the other hand, near infrared fluorescence (NIRF) imaging has recently received great interest for in vivo imaging due to its high signal-to-noise ratios and short image-acquisition times. NIRF imaging can be used to delineate tumor margins during surgery, but current NIRF imaging cannot provide the penetration depth to detect early-stage cancer inside body. Thus, we have developed dual-modality in vivo imaging for MRI detection of tumors and NIRF-guided surgery using multi-component nanoparticles. NIRF dye (cyanine 5.5, Cy5.5), conjugated glycol chitosan nanoparticles (HGC) exhibited excellent tumor targeting ability with NIRF imaging. Superparamagnetic iron oxide (SPIO) nanoparticles as a MR contrast agent were loaded into the nanoparticles, resulting in SPIO-HGC-Cy5.5 nanoparticles. SPIO-HGC-Cy5.5 nanoparticles were characterized and evaluated in mice by both NIRF and MR imaging. Our results indicate SPIO-HGC-Cy5.5 nanoparticles have the potential for dual-modality in vivo imaging with MRI detection of tumors and NIRF-guided surgery.