Near-Infrared Fluorescence Imaging for the Intraoperative Detection of Endometriosis: A Pilot Study

Introduction: Endometriosis surgery is associated with a high risk of reoperation due to an insufficient recognition of endometriotic lesions. Our aim was to explore the role of near-infrared fluorescence (NIRF) imaging for the visualization and identification of endometriotic lesions next to conventional white light (WL) laparoscopy. Materials and methods: Fifteen women scheduled for diagnostic laparoscopy in whom peritoneal endometriosis was suspected were included. Peritoneal exploration was performed in WL, followed by NIRF imaging after ICG administration. Biopsies of all the suspected lesions were taken for histological examination. Subjective evaluations of the equipment and NIRF imaging were also performed. Results: Only 61% (44) of the biopsied lesions contained endometriosis. The positive predictive value (PPV) for the lesions found in WL was 64%. The PPV for the lesions found under NIRF was 69% and the PPV for the lesions found in both modes was 61%. The mean satisfaction of surgeons regarding the surgical procedure and equipment using both imaging modalities was 6.5 (p > 0.05) on a 10 item Likert scale and the mean satisfaction with the quality of the NIRF imaging was 7.4 (p > 0.05). Conclusion: In this study, the additional value of NIRF imaging, although feasible, was found to be limited for the intraoperative detection of endometriotic lesions.

Tracking macrophages in diabetic neuropathy with two-color nanoemulsions for near-infrared fluorescent imaging and microscopy

Background The incidence of diabetes and diabetic peripheral neuropathy continues to rise, and studies have shown that macrophages play an important role in their pathogenesis. To date, macrophage tracking has largely been achieved using genetically-encoded fluorescent proteins. Here we present a novel two-color fluorescently labeled perfluorocarbon nanoemulsion (PFC-NE) designed to monitor phagocytic macrophages in diabetic neuropathy in vitro and in vivo using non-invasive near-infrared fluorescent (NIRF) imaging and fluorescence microscopy. Methods Presented PFC-NEs were formulated with perfluorocarbon oil surrounded by hydrocarbon shell carrying two fluorescent dyes and stabilized with non-ionic surfactants. In vitro assessment of nanoemulsions was performed by measuring fluorescent signal stability, colloidal stability, and macrophage uptake and subsequent viability. The two-color PFC-NE was administered to Leprdb/db and wild-type mice by tail vein injection, and in vivo tracking of the nanoemulsion was performed using both NIRF imaging and confocal microscopy to assess its biodistribution within phagocytic macrophages along the peripheral sensory apparatus of the hindlimb. Results In vitro experiments show two-color PFC-NE demonstrated high fluorescent and colloidal stability, and that it was readily incorporated into RAW 264.7 macrophages. In vivo tracking revealed distribution of the two-color nanoemulsion to macrophages within most tissues of Leprdb/db and wild-type mice which persisted for several weeks, however it did not cross the blood brain barrier. Reduced fluorescence was seen in sciatic nerves of both Leprdb/db and wild-type mice, implying that the nanoemulsion may also have difficulty crossing an intact blood nerve barrier. Additionally, distribution of the nanoemulsion in Leprdb/db mice was reduced in several tissues as compared to wild-type mice. This reduction in biodistribution appears to be caused by the increased number of adipose tissue macrophages in Leprdb/db mice. Conclusions The nanoemulsion in this study has the ability to identify phagocytic macrophages in the Leprdb/db model using both NIRF imaging and fluorescence microscopy. Presented nanoemulsions have the potential for carrying lipophilic drugs and/or fluorescent dyes, and target inflammatory macrophages in diabetes. Therefore, we foresee these agents becoming a useful tool in both imaging inflammation and providing potential treatment in diabetic peripheral neuropathy.

P-L15 Fluorescence guided laparoscopic resection of Colorectal Liver Metastases: Same day administration of indocyanine green with use of SPY- Colour Segmented Fluorescence imaging to identify tumours intra-operatively

Background Near-infrared fluorescent (NIRF) imaging using indocyanine green (ICG) has various applications in minimally invasive surgery. There are a number of techniques in timing and dose administration. Visually different fluorescent enhancement patterns correlate with different pathologies to aid identification of lesions intra-operatively. The aim of this study is to present our experience with utilisation of Colour Segmented Fluorescence ICG mode in laparoscopic liver surgery for colorectal liver metastases. Methods We present a single surgeon (SA) experience with the use of laparoscopic fluorescence guided imaging surgery (L-FGIS). Between November 2020 and July 20201, L-FGIS was used in seven patients with suspected CRLM. ICG was administered intravenously at a dose of 0.2 to 0.3 mg/kg IV 2-3 hours prior to liver surgery. Through use of the SPY Colour Segmented Fluorescence (CSF) imaging mode, the image is scaled as to NIRF fluorescence intensity to allow for the clear identification of the CRLM intra-operatively. Results A total of seven patients (Four males) with median age of 74.3 years (range: 30.5 -86) underwent L-FGIS during the study period. Two out of seven patients underwent re-do liver surgery. The median size of the tumour was 27mm (range: 10-65mm) and median number of tumours were one (range: 1-2). To visualise the tumour and to avoid interference of green background liver, ICG camera was switched to CSF mode. All lesions had signet ring appearance under CSF mode (see figure 1). Except in one patient (necrotic lesion), the histology of resected specimen contained a well to moderately differentiated colorectal adenocarcinoma metastasis. R0 resection was achieved in all patients and median clearance of the tumour was 3mm (range 0.4-10mm). Conclusions In our limited experience ICG administered at least 2-3 hours prior to surgery can identify superficially located colorectal metastases, provided ICG camera is switched to CSF mode. Superficially located lesions are easily identifiable under CSF mode. CSF mode helped us to identify the lesions and to mark the resection margin. The use of ICG is an important advancement in CRLM surgery and further research is needed to optimise image interpretation and correlate with clinical resection outcomes.

Near-Infrared Fluorescence Imaging of Carotid Plaques in an Atherosclerotic Murine Model

Successful imaging of atherosclerosis, one of the leading global causes of death, is crucial for diagnosis and intervention. Near-infrared fluorescence (NIRF) imaging has been widely adopted along with multimodal/hybrid imaging systems for plaque detection. We evaluate two macrophage-targeting fluorescent tracers for NIRF imaging (TLR4-ZW800-1C and Feraheme-Alexa Fluor 750) in an atherosclerotic murine cohort, where the left carotid artery (LCA) is ligated to cause stenosis, and the right carotid artery (RCA) is used as a control. Imaging performed on dissected tissues revealed that both tracers had high uptake in the diseased vessel compared to the control, which was readily visible even at short exposure times. In addition, ZW800-1C’s renal clearance ability and Feraheme’s FDA approval puts these two tracers in line with other NIRF tracers such as ICG. Continued investigation with these tracers using intravascular NIRF imaging and larger animal models is warranted for clinical translation.

Attempt of Real-Time Near-Infrared Fluorescence Imaging Using Indocyanine Green (ICG) in Radical Resection of Gallbladder Cancer: A Case Report

Surgery is the mainstay of treatment for resectable gallbladder cancer. Near-infrared fluorescence (NIRF) imaging using ICG is an innovation in laparoscopic surgery, which can provide real-time navigation during the whole operation. In this article, we present a 56-year older woman with gallbladder cancer, in which we evaluated the applicability of NIRF imaging using ICG for tumor and biliary tree visualization during the operative procedure of gallbladder cancer. The tumor and biliary tree were clearly visualized by utilizing a green fluorescence dye. The patient was successfully operated radical resection of gallbladder cancer under fluorescence laparoscope, without any complications. According to this case, the utilization of ICG based NIRF imaging is feasible and beneficial in identifying tumors and the biliary tree during radical resection. It can assist in the achievement of a negative margin and lymphatic clearance around the biliary tree. However, further studies are needed to corroborate the results of this case.

Intra-operative assessment of the vascularisation of a cross section of the meniscus using near-infrared fluorescence imaging

Purpose The 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). Methods The 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. Results Meniscal 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. Conclusion This 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 evidence IV.

Indocyanine green (ICG) fluorescent cholangiography during laparoscopic cholecystectomy using RUBINA™ technology: preliminary experience in two pediatric surgery centers

Background Recently, we reported the feasibility of indocyanine green (ICG) near-infrared fluorescence (NIRF) imaging to identify extrahepatic biliary anatomy during laparoscopic cholecystectomy (LC) in pediatric patients. This paper aimed to describe the use of a new technology, RUBINA™, to perform intra-operative ICG fluorescent cholangiography (FC) in pediatric LC. Methods During the last year, ICG-FC was performed during LC using the new technology RUBINA™ in two pediatric surgery units. The ICG dosage was 0.35 mg/Kg and the median timing of administration was 15.6 h prior to surgery. Patient baseline, intra-operative details, rate of biliary anatomy identification, utilization ease, and surgical outcomes were assessed. Results Thirteen patients (11 girls), with median age at surgery of 12.9 years, underwent LC using the new RUBINA™ technology. Six patients (46.1%) had associated comorbidities and five (38.5%) were practicing drug therapy. Pre-operative workup included ultrasound (n = 13) and cholangio-MRI (n = 5), excluding biliary and/or vascular anatomical anomalies. One patient needed conversion to open surgery and was excluded from the study. The median operative time was 96.9 min (range 55–180). Technical failure of intra-operative ICG-NIRF visualization occurred in 2/12 patients (16.7%). In the other cases, ICG-NIRF allowed to identify biliary/vascular anatomic anomalies in 4/12 (33.3%), including Moynihan's hump of the right hepatic artery (n = 1), supravescicular bile duct (n = 1), and short cystic duct (n = 2). No allergic or adverse reactions to ICG, post-operative complications, or reoperations were reported. Conclusion Our preliminary experience suggested that the new RUBINA™ technology was very effective to perform ICG-FC during LC in pediatric patients. The advantages of this technology include the possibility to overlay the ICG-NIRF data onto the standard white light image and provide surgeons a constant fluorescence imaging of the target anatomy to assess position of critical biliary structures or presence of anatomical anomalies and safely perform the operation.

Extracellular vesicles-based pre-targeting strategy enables multi-modal imaging of orthotopic colon cancer and image-guided surgery

Backgroud Colon cancer contributes to high mortality rates as the result of incomplete resection in tumor surgery. Multimodal imaging can provide preoperative evaluation and intraoperative image-guiding. As biocompatible nanocarriers, extracellular vesicles hold great promise for multimodal imaging. In this study, we aim to synthesized an extracellular vesicles-based nanoprobe to visualize colon cancer with positron-emission tomography/computed tomography (PET/CT) and near-infrared fluorescence (NIRF) imaging, and investigated its utility in image-guided surgery of colon cancer in animal models. Results Extracellular vesicles were successfully isolated from adipose-derived stem cells (ADSCs), and their membrane vesicles were observed under TEM. DLS detected that the hydrodynamic diameters of the extracellular vesicles were approximately 140 nm and the zeta potential was − 7.93 ± 0.24 mV. Confocal microscopy showed that extracellular vesicles had a strong binding ability to tumor cells. A click chemistry-based pre-targeting strategy was used to achieve PET imaging in vivo. PET images and the biodistribution results showed that the best pre-targeting time was 20 h, and the best imaging time was 2 h after the injection of 68 Ga-L-NETA-DBCO. The NIRF images showed that the tumor had clear images at all time points after administration of nanoparticles and the Tumor/Muscle ratio peaked at 20 h after injection. Our data also showed that both PET/CT and NIRF imaging clearly visualized the orthotopic colon cancer models, providing preoperative evaluation. Under real-time NIRF imaging, the tumor location and tumor boundary could be clearly observed. Conclusions In brief, this novel nanoprobe may be useful for multi-modal imaging of colon cancer and NIRF image-guided surgery. More importantly, this study provides a new possibility for clinical application of extracellular vesicles as nanocarriers. Graphic Abstract

Phase 3, randomized, single-dose, open-label study to investigate the safety and efficacy of pafolacianine sodium injection (OTL38) for intraoperative imaging of folate receptor positive ovarian cancer.

5503 Background: Pafolacianine sodium is under investigation as an adjunct to visual inspection and palpation by providing intra-operative imaging of folate receptor positive (FR+) ovarian cancer. Since complete resection (R0) is the strongest predictor of overall survival, methods to enhance detection of lesions are expected to benefit patient outcomes. Methods: For this phase 3, randomized, multicenter, single dose, open-label pivotal trial (NCT03180307), patients with ovarian cancer who were scheduled to undergo cytoreductive surgery were recruited from 11 sites in the US and Netherlands from March 2018 through April 2020. The study objectives were to confirm efficacy and safety of pafolacianine sodium (0.025 mg/kg i.v., ≥1 h prior to imaging) in combination with intraoperative near-infrared fluorescence (NIRF) imaging to detect additional lesions not detected by palpation and normal white light alone. Results: Pafolacianine sodium was administered to 150 total patients (safety analysis set); 109 patients comprised the full analysis set for efficacy analyses. Patients had primarily serous adenocarcinoma (n = 72; 68.6%) and advanced stage disease (n = 83; 76.1%). In 33% of patients (36 of 109), NIRF imaging with pafolacianine sodium identified additional lesions that were not planned for resection and were not detected by normal white light and palpation ( P < 0.001, 95% CI [0.243, 0.427]). Among patients who underwent interval debulking surgery, the rate was higher, at 39.7% of patients (23 of 58; 95% CI [0.270, 0.534]). At the individual lesion level, the accuracy of pafolacianine sodium with NIRF to detect ovarian cancer is reflected by sensitivity of 83% (95% CI [73.9, 89.4]) and a false positive rate of 32.7% (95% CI [25.6, 40.7]). Investigators reported achieving complete resection (R0) in 62.4% (68 of 109) of patients. Drug-related adverse events (AEs) were reported by 30% of patients (45 out of 150). The most frequently reported drug-related AEs were nausea (18.0%), vomiting (5.3%), and abdominal pain (4.7%). Infusion reactions at the time of the procedure were mostly (96%) mild or moderate in severity; 89% resolved within 24 hours of onset. No drug-related serious AEs or deaths were reported. Conclusions: This phase 3 trial of pafolacianine sodium with NIRF imaging met its primary endpoint, intraoperatively identifying additional cancer not planned for resection in a statistically significant number of patients. Therefore, pafolacianine sodium may offer a novel real-time adjunct to current surgical imaging practice in ovarian cancer surgery. Clinical trial information: NCT03180307.

Targeting Contrast Agents With Peak Near-Infrared-II (NIR-II) Fluorescence Emission for Non-invasive Real-Time Direct Visualization of Thrombosis

Thrombosis within the vasculature arises when pathological factors compromise normal hemostasis. On doing so, arterial thrombosis (AT) and venous thrombosis (VT) can lead to life-threatening cardio-cerebrovascular complications. Unfortunately, the therapeutic window following the onset of AT and VT is insufficient for effective treatment. As such, acute AT is the leading cause of heart attacks and constitutes ∼80% of stroke incidences, while acute VT can lead to fatal therapy complications. Early lesion detection, their accurate identification, and the subsequent appropriate treatment of thrombi can reduce the risk of thrombosis as well as its sequelae. As the success rate of therapy of fresh thrombi is higher than that of old thrombi, detection of the former and accurate identification of lesions as thrombi are of paramount importance. Magnetic resonance imaging, x-ray computed tomography (CT), and ultrasound (US) are the conventional non-invasive imaging modalities used for the detection and identification of AT and VT, but these modalities have the drawback of providing only image-delayed indirect visualization of only late stages of thrombi development. To overcome such limitations, near-infrared (NIR, ca. 700–1,700 nm) fluorescence (NIRF) imaging has been implemented due to its capability of providing non-invasive real-time direct visualization of biological structures and processes. Contrast agents designed for providing real-time direct or indirect visualization of thrombi using NIRF imaging primarily provide peak NIR-I fluorescence emission (ca. 700–1,000 nm), which affords limited tissue penetration depth and suboptimal spatiotemporal resolution. To facilitate the enhancement of the visualization of thrombosis via providing detection of smaller, fresh, and/or deep-seated thrombi in real time, the development of contrast agents with peak NIR-II fluorescence emission (ca. 1000–1,700 nm) has been recently underway. Currently, however, most contrast agents that provide peak NIR-II fluorescence emissions that are purportedly capable of providing direct visualization of thrombi or their resultant occlusions actually afford only the indirect visualization of such because they only provide for the (i) measuring of the surrounding vascular blood flow and/or (ii) simple tracing of the vasculature. These contrast agents do not target thrombi or occlusions. As such, this mini review summarizes the extremely limited number of targeting contrast agents with peak NIR-II fluorescence emission developed for non-invasive real-time direct visualization of thrombosis that have been recently reported.