Fluorescent-Labeled Tilmanocept for Sentinel Lymph Node Identification in Rectal Cancer during Robotic Surgery: A Preclinical Study in the Porcine Model

Fluorescent-Labeled Tilmanocept for Sentinel Lymph Node Identification in Rectal Cancer during Robotic Surgery: A Preclinical Study in the Porcine Model | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Fluorescent-Labeled Tilmanocept for Sentinel Lymph Node Identification in Rectal Cancer during Robotic Surgery: A Preclinical Study in the Porcine Model Ryotaro Ogawa, Junichiro Kawamura, Edward T. Ashworth, Soo Bin Park, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6212314/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 27 Oct, 2025 Read the published version in Scientific Reports → Version 1 posted 11 You are reading this latest preprint version Abstract The management of lateral pelvic lymph nodes in locally advanced lower rectal cancer remains controversial. The sentinel lymph node (SLN) mapping can potentially enhance patient selection for lateral pelvic lymph node dissection. The current mapping agent, indocyanine green dye cannot be externally imaged prior to surgery and is not retained after entering the SLN. This study evaluated the ability of tilmanocept, a receptor-specific SLN mapping agent, to provide preoperative PET cross-sectional imaging and sustained intraoperative fluorescence images during rectal cancer surgery. Tilmanocept was labeled with gallium-68, technetium-99m, and a near-infrared fluorophore. Four pigs were studied. Tilmanocept was injected into the submucosal layer of the rectal wall followed one hour later by PET/CT images of the pelvis, which identified ten SLNs, one of which was a pudendal artery regional SLN. Approximately forty-five hours after administration, SLN dissection was guided by fluorescence imaging during robotic surgery. The tenth SLN was excised by open surgery. The radioactive and fluorescent intensities of all ten excised SLNs were significantly higher than the non-SLNs. The ability of dual-labeled tilmanocept to provide preoperative SLN imaging can potentially reduce morbidity and operation time by identifying patients who will not require lateral pelvic lymph node dissections. Biological sciences/Biological techniques Biological sciences/Cancer Rectal cancer Lateral pelvic lymph node Sentinel lymph node Tilmanocept Robotic surgery fluorescent navigation surgery Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 INTRODUCTION Increasing attention has been focused on treatment strategies for lateral pelvic lymph nodes (LPLN) in locally advanced lower rectal cancer. Total mesorectal excision (TME) is the standard procedure in rectal cancer surgery worldwide. TME has been shown to control local recurrence and improve survival [ 1 ] . Recurrence in the lateral pelvic lymph nodes, which are not included in the TME procedure, remains an issue. Treatment of the lateral pelvic lymph nodes is essential to control local recurrence [ 2 ] further and improve survival in patients with rectal cancer. Treatment strategies for locally advanced lower rectal cancer differ between Western countries and Japan, and a consensus has not yet been reached. Chemoradiation therapy (CRT) and TME are used in Europe and the United States [ 3 , 4 ] . In Japan, lateral pelvic lymph node dissection (LPLND) has been widely used as a standard procedure in addition to TME [ 5 ] . In some patients at risk of LPLN disease, both treatments may be inadequate for the lateral pelvic lymph nodes although CRT or LPLND provide better local control than TME alone [ 3 ] . The treatment strategy is shifting to CRT with TME and LPLND for high-risk LPLN-positive cases [ 6 ] . In recent years, preoperative treatments such as total neoadjuvant therapy have become more common worldwide, and the number of CRT-treated cases is also increasing in Japan. In some patients with enlarged clinical metastatic-positive lateral pelvic lymph nodes, CRT alone may be insufficient treatment to eradicate metastasis of lateral pelvic lymph nodes [ 7 – 9 ] , necessitating the addition of LPLND. However, LPLND is a complex procedure that requires preserving the autonomic nervous system to prevent sexual dysfunction and dysuria, significantly prolonging operative time and increasing blood loss [ 10 , 11 ] . Therefore, it is crucial to identify eligible patients so that LPLND does not become overtreatment. In general, size cut off criteria such as 5 − 10mm are commonly used for the preoperative evaluation of lateral pelvic lymph nodes [ 9 , 12 , 13 ] although size, shape, intensity and FDG-PET uptake are considered [ 14 ] . However, there are no gold standard criteria for preoperative evaluation of metastatic lymph nodes. To give the best possible patient care, there is a clinical need to develop a new, more reliable diagnostic method. Today, the sentinel lymph node concept is clinically applied in breast cancer and melanoma using Tc-99m-tilmanocept, various radiolabeled colloids, and dyes [ 15 – 18 ] . SPECT/CT scans of Tc-99m-tilmanocept enable cross-sectional imaging for SLN mapping of head and neck cancer [ 19 ] . In colorectal cancers, navigation surgery in which ICG is administered near the tumor to visualize the lymphatic flow and the dominant lymph nodes is also performed [ 20 – 22 ] . Tc-99m-tilmanocept was approved by the United States FDA for sentinel lymph node mapping of solid tumors in 2013. Fluorescent-labeled tilmanocept [ 23 ] and radiolabeled-tilmanocept [ 24 ] exhibit sub-nanomolar affinity for CD206, a surface receptor of macrophages [ 25 ] and dendritic cells [ 26 ] . We have reported the use of multimodal tilmanocept to identify sentinel nodes in animal models of the prostate [ 27 ] , bladder wall [ 28 ] , endometrium [ 29 ] , and oral cavity [ 30 ] . A gallium-68 label, the radioactive mode, enabled pre-operative imaging with a PET/CT scanner, and a near-infrared label, the fluorescence mode, enabled intra-operative imaging during robotic surgery. When the SLN concept is applied to rectal cancer with complex lymphatic-systems, it may help selecting patients for LPLND [ 31 ] and avoid unnecessary LPLND. Fluorescent-guided surgery with preoperative imaging could substantially reduce morbidity and operation time. In this study, we investigated whether a CD206-specific multimodal tilmanocept could identify sentinel lymph node locations using PET/CT preoperative imaging and intraoperatively under fluorescence guidance using the Firefly (Intuitive, Sunnyvale, CA) camera system. RESULTS Preoperative PET/CT Imaging Preoperative PET/CT imaging identified a total of 10 lymph nodes from four pigs. The mean number of lymph nodes was 2.5 (range 1–4). The maximum standardized uptake value (SUVmax) was 95 − 1,902 (Table 1 ) and that of injection site was 51,470 − 93,275. PET/CT positive lymph nodes were located in intra-mesenteric or ipsilateral lateral pelvic lymph nodes (Table 1 ). Two of four pigs showed mesenteric and lateral pelvic lymph nodes due to both vertical and parallel lymph flow, and in some cases, images of lymphatic flow migrating through the lymphatic vessels could be visualized. The three panels of Fig. 1 are representative PET/CT cross-sections from Pig#3. The injection site is shown in Fig. 1 a. Two ipsilateral sentinel lymph nodes (Figs. 1 b & 1 c) along the right internal iliac artery had SUVmax values of 1,902 and 835, respectively. Pre-operative PET/CT imaging of Pig #4 exhibited a right Pudendal artery regional sentinel lymph node; coronal and transaxial cross-sections are displayed in Figs. 2a and 2b, respectively. Table 1 Comparison of Tilmanocept accumulation in sentinel lymph nodes and visualization by fluorescence Case # Injection site SLN location PET/CT SUV Da Vinci Firefly Fluobeam Max (IU) Tc-99m %ID (%) 1 Left Lt. External 104 Y 4,848 1.73 Lt. Internal 535 Y 4,064 1.99 Mesenteric 409/396* Y 3,232 0.85 Mesenteric Y 2,976 0.42 2 Left Lt. Internal 95 Y 9,840 2.25 3 Right Rt. Internal 835 Y 11,200 2.80 Rt. Internal 1,902 Y 9,120 1.49 4 Right Rt. pudendal 419 ** 6,016 4.19 Mesenteric 318* Y 4,384 1.81 Mesenteric Y 3,952 1.43 *: indistinguishable, **: Out of Firefly detection range Preoperative PET/CT images of Pig#3 at 1 hour after tilmanocept injection. Gallium-68-labeled Timanocept was injected into the lateral wall just above the dentate line. Tilmanocept accumulated in the ipsilateral lymph node of the injection site. a) right injection site (arrowhead) b) right internal iliac regional lymph node, SUVmax = 1,902 (white arrow). c) right internal iliac regional lymph node, SUVmax = 835 (black arrow). Intraoperative Firefly Fluorescence Imaging Identifies Sentinel Lymph Nodes Surgery was performed using the DaVinci Xi Firefly camera system with preoperative PET/CT imaging as the guide. The mean number of lymph nodes per animal resected was 24.25 ± 5.68, with 2.5 ± 1.29 SLNs following the 10% rule. All PET/CT-positive regional lymph nodes were excised. The Firefly system identified nine of the 10 PET/CT-positive lymph nodes. The one lymph node that could not be detected via the intra-operative camera was the pudendal artery regional lymph node of Pig #4, which was outside the abdominal cavity and therefore not within the field-of-view of the FireFly camera. This pudendal artery regional lymph node in Pig#4 was removed by open surgery because the space between the sacrum and the iliac bone was too narrow and deep to be approached by the DaVinci Xi . Figure 3 displays the representative Firefly system images of Pig#3. Figure 3 a is a bright field image, the arrow points to a sentinel lymph node at the right internal iliac artery. The Firefly mode (Fig. 3 b) revealed fluorescence of the tilmanocept-positive lymph node, which was more clearly displayed by the sensitive mode (Fig. 3 c). Ex Vivo Fluorescence Imaging All SLNs identified by the Firefly system were also detectable using Fluobeam800 at an exposure time of 10 ms (Table 1 ). Figure 4 is the brightfield (Fig. 4 a) and fluorescence images (Fig. 4 b) from Pig #3. The optical imager demonstrated intra-nodal fluorescence within two right internal lymph nodes. These two lymph nodes were consistent with the SLNs identified on pre-operative imaging and ex vivo assay of technetium-99m radioactivity. Ex Vivo Radioactivity Counting The radioactive activity results for Tc-99m indicated that the mean %ID ± SD for the ten sentinel lymph node was 1.90 ± 1.05, which was significantly (p < 0.01) different than mean value (0.01 ± 0.02) of the non-sentinel lymph nodes (Fig. 5 a). The maximum fluorescent intensity for SLNs and NSLNs were 5,963.2 ± 2,985.0 and 553.9 ± 199.2, respectively, indicating a statistically significant difference (p < 0.01) (Fig. 5 b). DISCUSSION In this study, we successfully demonstrated the ability of multimodal tilmanocept to preoperatively image and intra-operatively map sentinel lymph nodes. By combining preoperative PET/CT imaging with intraoperative fluorescent navigation, we demonstrated significant potential for improving patient selection for LPLND. Avid binding to CD206 allows tilmanocept to remain detectable in lymph nodes for at least 45 hours post-injection, enabling precise SLN identification with the intra-operative Firefly camera system. The goal of this innovative approach is to reduce unnecessary LPLND procedures, thereby decreasing operative time and minimizing the risk of complications. In the lower rectum, the lymphatic flow becomes more complex, as it approaches the anus. There are two major lymphatic pathways: the upstream and the lateral direction. Upstream lymphatic flow runs along the superior rectal artery and inferior mesenteric artery within the mesorectum. The lateral lymphatic flow, on the other hand, drains into the basin of lateral pelvic lymph nodes, particularly around the internal iliac artery and obturator artery. This lateral lymphatic flow is responsible for lateral pelvic lymph node metastasis. Currently, there are no clear imaging diagnostic criteria for lymph nodes suspected of metastasis [ 32 ] , leading to a lack of established treatment guidelines. Patients with enlarged or borderline-metastatic lateral pelvic lymph nodes are typically treated with a combination of CRT and/or LPLND. However, some patients who undergo LPLND are over-treated [ 6 , 9 ] . Therefore, it is crucial to stratify the patients effectively to ensure that only those who truly need LPLND receive it, especially after CRT, thereby avoiding unnecessary invasive treatments. SLN biopsy is one of the approaches for stratification. The usefulness of ICG-guided sentinel node biopsy in assessing the metastatic status in lateral pelvic lymph nodes has been reported [ 20 , 33 ] . In these reports, sentinel lymph nodes without metastasis in the lateral pelvic region indicate the absence of metastasis in other non-sentinel pelvic lymph nodes. Moreover, evaluating sentinel lymph nodes may contribute to the detection of metastatic lymph nodes, including micrometastasis [ 34 , 35 ] . However, it should be noted that widespread lymph node metastasis or replacement with cancer cells in lymph nodes inhibits staining with blue dye or ICG [ 36 , 37 ] . Based on the above, patients without obvious lymph node metastasis or metastatic border line lymph nodes, such as 5-10mm or less, will be considered as suitable candidates for SLN mapping. The advantage of tilmanocept lies in its versatile labeling capabilities. It can be labeled with gallium-68 for PET/CT imaging or technetium-99m for SPECT/CT imaging. In addition, tilmanocept can also be conjugated with a fluorescent dye such as IRDye800CW , enabling near-infrared imaging during surgery [ 21 ] as well as preoperative imaging. Preoperative lymph node mapping provides anatomical information and potentially detects more SLNs [ 38 ] . This permits preoperative planning of the surgery and reduction of the time required for intra-operative SLNs mappings [ 38 ] . This fluorescence navigation is effective only for superficial areas up to a depth of 10mm, with limited fluorescent penetration [ 39 ] . Identifying the fluorescence of lymph nodes located deeper than 10mm, such as those in the lateral pelvic lymph node and mesorectum, remains challenging. In human rectal cancer, lateral pelvic lymph node metastases are located more frequently in the iliac artery region and obturator artery region [ 40 ] . These regions are sites deeply embedded within the pelvic cavity with thick fat, and can only be detected via external imaging, such as PET/CT or SPECT/CT. The fluorescence of lateral pelvic sentinel lymph nodes in deep region may not be visible from the dissection surface after TME. In such cases, fluorescence should be detected by dissecting through the fat, but vessels, arteries, and the ureter duct are contained in this fat. Random excision to detect fluorescence may cause morbidity and take more time. If there is preoperative lymph node site information, the sentinel node can be accessed safely and accurately. If pre-operative SPECT/CT imaging does not detect a SLN, unnecessary dissection maneuvers can be omitted, which may lead to a lower incidence of complications related to LPLND. One example of the benefit of preoperative lymph node mapping was seen in a pudendal artery regional lymph node in case 4. All SLNs except the pudendal artery regional lymph node could be detected by fluorescence due to less fat. However, the pudendal artery regional SLN existed in the distal pudendal artery region between the sacral and iliac bone spaces. The Da Vinci camera was unable to identify fluorescence due to being physically inaccessible. This lymph node is considered a site of systemic metastasis and is not typically resected in clinical practice. In this preclinical study, the purpose was to confirm the consistency between preoperative PET-CT images and intra- and post-operative fluorescent images. The surgery was transferred to an open approach and the sentinel lymph node was detected by the fluorescent camera and confirmed by Tc-99m activity. This combination of preoperative mapping and intraoperative fluorescent navigation suggests that SLNs can be accurately and efficiently identified and excised. Tilmanocept can reside in lymph nodes for up to 72 hours after injection [ 29 ] ; this study demonstrated a fluorescent signal within sentinel lymph nodes at 45 hours post-administration. The extended retention of tilmanocept in sentinel lymph nodes allows surgeries to be performed the day after imaging, significantly reducing radiation exposure to the staff, and insuring a bright fluorescent emission for detection by the Firefly camera. We envision the following implementation of a technetium and fluorescent version of tilmanocept for sentinel lymph node mapping of rectal cancer. The bi-modal tilmanocept would be administered within the submucosa of the rectal wall near the tumor. One hour later, a 20-minute SPECT/CT of the subject’s pelvis would be acquired. A radiologist would examine the resulting attenuation-corrected cross-sectional images and identify sentinel lymph nodes based on SUVs and the 10%-rule. After imaging, the operation could be performed up to 72 hours after administration of the fluorescent-tilmanocept. Prior to excision, fluorescent-tilmanocept within each sentinel lymph node would be visualized by the fluorescence imaging subsystem of the surgical robot. Each fluorescent lateral pelvic lymph node packet would be submitted for histologic examinate as a sentinel lymph node. The strengths of this study are 1) the use of an animal model to demonstrate the rapid and sustained accumulation of radiolabeled-fluorescent-tilmanocept, 2) a demonstration of the potential significance of pre-operative detection of sentinel lymph nodes invisible field of the surgical robot, and 3) a demonstration of fluorescent detection of bi-modal tilmanocept by a standard robotic surgical system. The limitation of this study is that it is an examination of a limited number of pigs. We only injected tilmanocept into the submucosa of the lateral wall just above the dentate line, not the anterior and posterior sides and proximal rectum. In addition, there may be differences in lymphatic flow between humans and pigs due to different anatomy. CONCLUSION This study demonstrated the ability of pre-operative imaging and intra-operative detection of radiolabeled fluorescent-tilmanocept to efficiently identify sentinel lymph nodes. Sentinel lymph node identification of lateral pelvic lymph nodes using dual-labeled tilmanocept may provide an alternative strategy to the dissection of lateral lymph nodes for all tumors below the peritoneal reflection. PET/CT or SPECT/CT pre-operative sentinel lymph node imaging can potentially reduce morbidity and surgical time by identifying patients who will not require lateral pelvic lymph node dissections. METHODS Experimental design We designed a non-survival study using four female Yorkshire pigs (Premier BioSource, Ramona CA) to evaluate a clinical protocol for sentinel lymph node mapping of patients with rectal cancer. The animals ranged in age from 85 to 90 days and weighed between 31 and 32 kg. The primary goal of this study was a demonstration of bi-modal tilmanocept for pre-operative cross-sectional imaging and intra-operative sentinel lymph node mapping many days after administration. We labeled tilmanocept with a near-infrared fluorescent dye ( IRDye800CW ) and two radioisotopes: gallium-68, and technetium-99m. Gallium-68 is a positron emission isotope with a 68-minute half-life, which enabled pre-operative PET imaging of tilmanocept for in vivo sentinel lymph node mapping and surgical planning. Technetium-99m is a gamma-emitting isotope, with a 6-hour half-life, which provides a measurement of lymph node accumulation, and therefore, an independent identification of SLN status. Animal Model and Reagents The protocol for animal transfer and experiments was approved by the University of California, San Diego Institutional Animal Care and Use Committee. All animal procedures were performed in accordance with 1) the Guide for the Care and Use of Laboratory Animals as published by the National Research Council, 2) Association for Assessment and Accreditation of Laboratory Animal Care regulations, and 3) ARRIVE guidelines. A total of four pigs female were studied. During transportation to the imaging or surgical facility, each pig was sedated by intramuscular injection of an acepromazine/buprenorphine (0.10–0.05 and 0.01 mg/kg) cocktail. All procedures were performed under general anesthesia(propofol i.v., 2 mg induction, 10 mg/kg/hr maintenance). No animals were excluded from the study. Tilmanocept was obtained from Navidea Biopharmaceuticals (Dublin, OH), and IRDye800CW -NHS-ester was purchased from LICOR Biosciences (Lincoln, NB). Fluorescent-labeled tilmanocept was prepared by covalent attachment of IRDye800CW to tilmanocept using a previously described method [ 41 ] . IRDye800CW -tilmanocept was radiolabeled with technetium-99m and gallium-68 as previously reported [ 42 ] . The radiochemical yield (RCY) and fluorescent purity were measured by ITLC using Whatman31 as the stationary phase and acetone as the mobile phase. The RCYs and fluorescent purity of all preparations exceeded over 98%. PET/CT Imaging The pigs were positioned within the gantry of a uMI550 PET-CT scanner (United Imaging, Houston TX) so that the injection of 0.1ml of 68 Ga- 99m Tc-labeled IRDye800CW -tilmaoncept (1.5 nmol Tilmanocept, 〜5 MBq 68 Ga、30 MBq 99m Tc) could be performed above the dentate line on the rectal side wall. Scout CT images were taken for correct positioning followed by a CT scan for attenuation correction. Whole-body PET-CT images were acquired at approximately 60 minutes post-injection and consisted of two bed positions spanning from the rectum to the lower chest of each animal. Each bed position was acquired for 2 minutes. CT attenuation correction was applied, and the reconstructed PET-CT fusion images were viewed, and SUV measurements were made with the Visage 7 software (San Diego, CA). After recovery from anesthesia, each pig was returned to the vivarium. Robotic-assisted surgery and Firefly imaging Within 2 days of PET/CT imaging, each pig was transferred to the UCSD Center for the Future of Surgery. The pigs were anesthetized and set on the bed in the supine position. A camera port was placed on the midline, one on the left abdomen, and two on the right. The uterus was hung to the abdominal wall, and lymph node dissection was started. Lymph nodes around the external and internal iliac arteries were dissected from the internal and external iliac artery bifurcation toward the anal side. The lateral pelvic lymph nodes of both sides were excised completely. In addition, paraaortic lymph node near inferior mesenteric artery and pudendal artery regional lymph node were sampled. Finally, the mesorectum was dissected, and the mesorectum lymph nodes were excised from around the rectum. Intraoperatively, the Firefly camera system was used to check the fluorescence of each lymph node during the resection. We used both Firefly and sensitive mode to detect fluorescent-positive lymph nodes. We used the information from the PET/CT as a guide to confirm the location of the SLNs. Radioactivity and fluorescence measurements The dissected lymph nodes were individually separated from the bulk of lymph nodes embedded in fat tissue. Each lymph node was placed in a plastic scintillation vial and measured for technetium-99m radioactivity (100–200 keV energy window) in an autowell gamma counter ( Wizard2 , PerkinElmer, Waltham, MA) with a counting standard, which consisted of a known amount of injectate of a known dilution. The percent-of-injected dose ( %ID ) was calculated using the counting standard. All lymph nodes were imaged with a hand-held fluorescence imaging system ( Fluobeam800 , Fluoptics, Grenoble, France). Separated lymph nodes images were acquired with a 10-ms exposure time. The fluorescence images were analyzed using ImageJ (NIH, Bethesda MD); ROIs were drawn around each lymph node and the maximum gray luminance value was obtained within each ROI. We followed the “10% rule” to identify sentinel lymph nodes [ 43 ] . According to this rule, lymph nodes that accumulated more than 10% of the highest radioactivity in technetium-99m measurement qualified as sentinel lymph nodes. Statistical Analysis Statistical analysis was performed with JMP® Pro 16.0.0 . Percent-of-injected dose and fluorescent intensity were assessed by the two-tailed Student’s t-test. We considered a P-value less than 0.05 to be statistically significant. Declarations Disclosure David Vera is the inventor of tilmanocept Acknowledgments Authors would like to thank Prof. Shouichiro Saito and Dr. Sawa Onouchi, Laboratory of Veterinary Anatomy, University of Gifu, for their porcine anatomy consultation. Author contributions Conception and design: RO, DRV Development of methodology: RO JK, CKH, DRV Acquisition of data: RO, JK, ETA, SBP, CKH, DVR Analysis and interpretation of data: RO, JK, ETA, SBP, MK, CKH, DVR Writing, review, and/or revision of the manuscript: RO, JK, ETA, SBP, MK, CKH, DRV Study supervision: DRV Corresponding author: RO Competing interests David Vera is the inventor of timanocept. The remaining authors declare no competing interests. Data availability statement The data that support the findings of this study are available from the corresponding author, RO, upon reasonable request. References Dayal, S., Battersby, N. & Cecil, T. Evolution of Surgical Treatment for Rectal Cancer: a Review. J. Gastrointest. Surg. 21 , 1166–1173 (2017). Kusters, M. et al. Patterns of local recurrence in rectal cancer; a study of the Dutch TME trial. Eur. J. Surg. Oncol. 36 , 470–476 (2010). Kusters, M. et al. 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Biol. 42 , 917–922 (2015). Albertini, J. J. et al. Intraoperative radio-lympho-scintigraphy improves sentinel lymph node identification for patients with melanoma. Ann. Surg. 223 , 217–224 (1996). Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 27 Oct, 2025 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 22 Jul, 2025 Reviews received at journal 16 Jul, 2025 Reviews received at journal 08 Jul, 2025 Reviewers agreed at journal 06 Jul, 2025 Reviewers agreed at journal 28 Jun, 2025 Reviewers agreed at journal 06 Apr, 2025 Reviewers invited by journal 04 Apr, 2025 Editor assigned by journal 04 Apr, 2025 Editor invited by journal 25 Mar, 2025 Submission checks completed at journal 24 Mar, 2025 First submitted to journal 12 Mar, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6212314","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":439472614,"identity":"5824025c-1692-4cde-988e-eb2f320fa68b","order_by":0,"name":"Ryotaro Ogawa","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABC0lEQVRIiWNgGAWjYBADOSA2YGwwYGBsYAYLsBHUYky6lsQGsBYGBhDGD8xnJB9+8bPNLn1te/MGxhkFd2T72xkYP/xg4MvDpUXmRlqaZW9bcu62M8cKGDcYPDOecZiBWbKHga0YlxYJiRwzA95tzLnbbuQYMD4wOJzYcJiBQRrol0RcLgRpMfy7rT7d7P4biJb5QFt+E9Bi/Jh32+EEsxs8BkCHHU7ccJiBDb8tPM/SmGX/HTfcdiat4OAMg8PGGw8ztln2GODxC3vy4Y9vzlTLmx0/vPFhz5/DsvPOHz5840fFMZwhxiCQwCYBYx+AUKCoMTiWgFML/wHmD9jEa3BrGQWjYBSMgpEGANYVXOjMOcnDAAAAAElFTkSuQmCC","orcid":"","institution":"University of California, San Diego","correspondingAuthor":true,"prefix":"","firstName":"Ryotaro","middleName":"","lastName":"Ogawa","suffix":""},{"id":439472615,"identity":"6fa91fbc-32df-4f34-9e8c-706b3fd747bd","order_by":1,"name":"Junichiro Kawamura","email":"","orcid":"","institution":"Kindai University Faculty of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Junichiro","middleName":"","lastName":"Kawamura","suffix":""},{"id":439472616,"identity":"69fc067a-05fe-4ced-ba4f-bb782a9ba3cf","order_by":2,"name":"Edward T. Ashworth","email":"","orcid":"","institution":"University of California, San Diego","correspondingAuthor":false,"prefix":"","firstName":"Edward","middleName":"T.","lastName":"Ashworth","suffix":""},{"id":439472617,"identity":"4e3dad4a-b944-42f8-9bca-00a5f65ca214","order_by":3,"name":"Soo Bin Park","email":"","orcid":"","institution":"University of California, San Diego","correspondingAuthor":false,"prefix":"","firstName":"Soo","middleName":"Bin","lastName":"Park","suffix":""},{"id":439472618,"identity":"f829b454-8052-4bfd-849f-532ecf0e74d5","order_by":4,"name":"David J Hall","email":"","orcid":"","institution":"University of California, San Diego","correspondingAuthor":false,"prefix":"","firstName":"David","middleName":"J","lastName":"Hall","suffix":""},{"id":439472619,"identity":"da3bcff3-1a88-4096-ab15-e5d7123902c1","order_by":5,"name":"Masatoshi Kudo","email":"","orcid":"","institution":"Kindai University Faculty of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Masatoshi","middleName":"","lastName":"Kudo","suffix":""},{"id":439472620,"identity":"2e7c2317-9b91-4685-9c90-006d0e982dfc","order_by":6,"name":"Carl K. Hoh","email":"","orcid":"","institution":"University of California, San Diego","correspondingAuthor":false,"prefix":"","firstName":"Carl","middleName":"K.","lastName":"Hoh","suffix":""},{"id":439472621,"identity":"cc4042b9-d5fe-4489-b658-961859c7ebf7","order_by":7,"name":"David R. Vera","email":"","orcid":"","institution":"University of California, San Diego","correspondingAuthor":false,"prefix":"","firstName":"David","middleName":"R.","lastName":"Vera","suffix":""}],"badges":[],"createdAt":"2025-03-12 13:08:20","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6212314/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6212314/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-21160-w","type":"published","date":"2025-10-27T15:58:08+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":82122070,"identity":"eebcaa68-934a-4b1b-9cb1-4497cb5db548","added_by":"auto","created_at":"2025-05-07 03:26:37","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1914176,"visible":true,"origin":"","legend":"\u003cp\u003ePreoperative PET/CT images of Pig#3 at 1 hour after tilmanocept injection. Gallium-68-labeled Timanocept was injected into the lateral wall just above the dentate line. Tilmanocept accumulated in the ipsilateral lymph node of the injection site. a) right injection site (arrowhead) b) right internal iliac regional lymph node, SUVmax = 1,902 (white arrow). c) right internal iliac regional lymph node, SUVmax = 835 (black arrow).\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-6212314/v1/78e9d80d6cdbe8854b49be2d.png"},{"id":82120702,"identity":"646a7833-c5f7-42f7-8c5b-e1cc5a94541f","added_by":"auto","created_at":"2025-05-07 03:18:37","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1734261,"visible":true,"origin":"","legend":"\u003cp\u003ePreoperative PET/CT images of Pig#4 at 1 hour after tilmanocept injection. Gallium-68-labeled Tilmanocept was injected into the lateral wall just above the dentate line. Tilmanocept accumulated in the right pudendal artery regional lymph node (arrow): a) coronal view (SUV = 419), and b) transaxial view. Note short distance to pelvic bone.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-6212314/v1/763eb5b18c9ab71c80501084.png"},{"id":82120718,"identity":"0da58428-2d1b-40e8-9a54-ccaa8e7f6911","added_by":"auto","created_at":"2025-05-07 03:18:38","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":5561333,"visible":true,"origin":"","legend":"\u003cp\u003eIntraoperative Pig#3 images of the around the right internal iliac artery with the following camera modes were acquired approximately 45 hours after injection of \u003cem\u003eIRDye800CW\u003c/em\u003e-tilmanocept. Sentinel Lymph nodes along the right internal iliac artery (arrows) were visualized using a near-infrared camera. a) brightfield image b) standard overlay image c) \u003cem\u003esensitive\u003c/em\u003e mode image\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-6212314/v1/1541649498d008a4bb4067d0.png"},{"id":82122077,"identity":"bc0e99b7-39b8-4f51-8e9b-f9d0a03cea8f","added_by":"auto","created_at":"2025-05-07 03:26:38","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2937933,"visible":true,"origin":"","legend":"\u003cp\u003eEx vivo fluorescence imaging of Pig#3 with a hand-held camera were acquired for 10 ms exposure time. Dissected specimens of each lymph node from each regional lymph node: a) bright field, b) optical image demonstrated intra-nodal fluorescence within the right internal lymph nodes.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-6212314/v1/0051c04e7aca4fd0682440d7.png"},{"id":82122071,"identity":"81f2aa3c-ecd9-4c01-8064-eb5fa8c0d30a","added_by":"auto","created_at":"2025-05-07 03:26:37","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":342691,"visible":true,"origin":"","legend":"\u003cp\u003eTilmanocept accumulation by sentinel lymph node was significantly (P \u0026lt; 0.01) higher (mean +/- SD) than non-sentinel lymph nodes (mean +/- SD): a) percent-of-injection dose (%ID), b) maximum fluorescent intensity (IU).\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-6212314/v1/829f5245c52507b92f57f988.png"},{"id":95040451,"identity":"737b482a-c1bc-474b-8aed-dd1d96e4bc01","added_by":"auto","created_at":"2025-11-03 16:08:57","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":12356465,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6212314/v1/31522f46-92c5-4ff4-99f2-6adad16a1ce1.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Fluorescent-Labeled Tilmanocept for Sentinel Lymph Node Identification in Rectal Cancer during Robotic Surgery: A Preclinical Study in the Porcine Model","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eIncreasing attention has been focused on treatment strategies for lateral pelvic lymph nodes (LPLN) in locally advanced lower rectal cancer. Total mesorectal excision (TME) is the standard procedure in rectal cancer surgery worldwide. TME has been shown to control local recurrence and improve survival\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e. Recurrence in the lateral pelvic lymph nodes, which are not included in the TME procedure, remains an issue. Treatment of the lateral pelvic lymph nodes is essential to control local recurrence\u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e further and improve survival in patients with rectal cancer. Treatment strategies for locally advanced lower rectal cancer differ between Western countries and Japan, and a consensus has not yet been reached. Chemoradiation therapy (CRT) and TME are used in Europe and the United States\u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e. In Japan, lateral pelvic lymph node dissection (LPLND) has been widely used as a standard procedure in addition to TME\u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e. In some patients at risk of LPLN disease, both treatments may be inadequate for the lateral pelvic lymph nodes although CRT or LPLND provide better local control than TME alone\u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e. The treatment strategy is shifting to CRT with TME and LPLND for high-risk LPLN-positive cases\u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn recent years, preoperative treatments such as total neoadjuvant therapy have become more common worldwide, and the number of CRT-treated cases is also increasing in Japan. In some patients with enlarged clinical metastatic-positive lateral pelvic lymph nodes, CRT alone may be insufficient treatment to eradicate metastasis of lateral pelvic lymph nodes\u003csup\u003e[\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e, necessitating the addition of LPLND. However, LPLND is a complex procedure that requires preserving the autonomic nervous system to prevent sexual dysfunction and dysuria, significantly prolonging operative time and increasing blood loss\u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e. Therefore, it is crucial to identify eligible patients so that LPLND does not become overtreatment. In general, size cut off criteria such as 5\u0026thinsp;\u0026minus;\u0026thinsp;10mm are commonly used for the preoperative evaluation of lateral pelvic lymph nodes\u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e although size, shape, intensity and FDG-PET uptake are considered\u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e. However, there are no gold standard criteria for preoperative evaluation of metastatic lymph nodes. To give the best possible patient care, there is a clinical need to develop a new, more reliable diagnostic method.\u003c/p\u003e \u003cp\u003eToday, the sentinel lymph node concept is clinically applied in breast cancer and melanoma using Tc-99m-tilmanocept, various radiolabeled colloids, and dyes\u003csup\u003e[\u003cspan additionalcitationids=\"CR16 CR17\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e. SPECT/CT scans of Tc-99m-tilmanocept enable cross-sectional imaging for SLN mapping of head and neck cancer\u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e. In colorectal cancers, navigation surgery in which ICG is administered near the tumor to visualize the lymphatic flow and the dominant lymph nodes is also performed\u003csup\u003e[\u003cspan additionalcitationids=\"CR21\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e. Tc-99m-tilmanocept was approved by the United States FDA for sentinel lymph node mapping of solid tumors in 2013.\u003c/p\u003e \u003cp\u003eFluorescent-labeled tilmanocept\u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e and radiolabeled-tilmanocept\u003csup\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e exhibit sub-nanomolar affinity for CD206, a surface receptor of macrophages\u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e and dendritic cells\u003csup\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e. We have reported the use of multimodal tilmanocept to identify sentinel nodes in animal models of the prostate\u003csup\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e, bladder wall\u003csup\u003e[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e, endometrium\u003csup\u003e[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e, and oral cavity\u003csup\u003e[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e. A gallium-68 label, the radioactive mode, enabled pre-operative imaging with a PET/CT scanner, and a near-infrared label, the fluorescence mode, enabled intra-operative imaging during robotic surgery.\u003c/p\u003e \u003cp\u003eWhen the SLN concept is applied to rectal cancer with complex lymphatic-systems, it may help selecting patients for LPLND\u003csup\u003e[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/sup\u003e and avoid unnecessary LPLND. Fluorescent-guided surgery with preoperative imaging could substantially reduce morbidity and operation time. In this study, we investigated whether a CD206-specific multimodal tilmanocept could identify sentinel lymph node locations using PET/CT preoperative imaging and intraoperatively under fluorescence guidance using the \u003cem\u003eFirefly\u003c/em\u003e (Intuitive, Sunnyvale, CA) camera system.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003ePreoperative PET/CT Imaging\u003c/h2\u003e\n \u003cp\u003ePreoperative PET/CT imaging identified a total of 10 lymph nodes from four pigs. The mean number of lymph nodes was 2.5 (range 1\u0026ndash;4). The maximum standardized uptake value (SUVmax) was 95\u0026thinsp;\u0026minus;\u0026thinsp;1,902 (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e) and that of injection site was 51,470\u0026thinsp;\u0026minus;\u0026thinsp;93,275. PET/CT positive lymph nodes were located in intra-mesenteric or ipsilateral lateral pelvic lymph nodes (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). Two of four pigs showed mesenteric and lateral pelvic lymph nodes due to both vertical and parallel lymph flow, and in some cases, images of lymphatic flow migrating through the lymphatic vessels could be visualized. The three panels of Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e are representative PET/CT cross-sections from Pig#3. The injection site is shown in Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ea. Two ipsilateral sentinel lymph nodes (Figs. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eb \u0026amp; \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ec) along the right internal iliac artery had SUVmax values of 1,902 and 835, respectively. Pre-operative PET/CT imaging of Pig #4 exhibited a right Pudendal artery regional sentinel lymph node; coronal and transaxial cross-sections are displayed in Figs. 2a and 2b, respectively. \u0026nbsp;\u003c/p\u003e\n \u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eComparison of Tilmanocept accumulation in sentinel lymph nodes and visualization by fluorescence\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eCase\u003c/p\u003e\n \u003cp\u003e#\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eInjection\u003c/p\u003e\n \u003cp\u003esite\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSLN location\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePET/CT\u003c/p\u003e\n \u003cp\u003eSUV\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eDa Vinci\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003e\u003cem\u003eFirefly\u003c/em\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eFluobeam\u003c/p\u003e\n \u003cp\u003eMax (IU)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eTc-99m %ID\u003c/p\u003e\n \u003cp\u003e(%)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"4\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"4\"\u003e\n \u003cp\u003eLeft\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLt. External\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e104\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eY\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e4,848\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.73\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLt. Internal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e535\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eY\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e4,064\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.99\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMesenteric\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003e409/396*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eY\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3,232\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.85\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMesenteric\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eY\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2,976\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.42\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLeft\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLt. Internal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e95\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eY\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e9,840\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.25\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eRight\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eRt. Internal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e835\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eY\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e11,200\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.80\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eRt. Internal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1,902\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eY\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e9,120\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.49\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003eRight\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eRt. pudendal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e419\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e**\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e6,016\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e4.19\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMesenteric\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003e318*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eY\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e4,384\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.81\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMesenteric\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eY\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3,952\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.43\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"7\"\u003e*: indistinguishable, **: Out of Firefly detection range\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"7\"\u003ePreoperative PET/CT images of Pig#3 at 1 hour after tilmanocept injection. Gallium-68-labeled Timanocept was injected into the lateral wall just above the dentate line. Tilmanocept accumulated in the ipsilateral lymph node of the injection site. a) right injection site (arrowhead) b) right internal iliac regional lymph node, SUVmax\u0026thinsp;=\u0026thinsp;1,902 (white arrow). c) right internal iliac regional lymph node, SUVmax\u0026thinsp;=\u0026thinsp;835 (black arrow).\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n \u003cp\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eIntraoperative\u003c/strong\u003e \u003cstrong\u003eFirefly\u003c/strong\u003e \u003cstrong\u003eFluorescence Imaging Identifies Sentinel Lymph Nodes\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eSurgery was performed using the \u003cem\u003eDaVinci Xi Firefly\u003c/em\u003e camera system with preoperative PET/CT imaging as the guide. The mean number of lymph nodes per animal resected was 24.25\u0026thinsp;\u0026plusmn;\u0026thinsp;5.68, with 2.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.29 SLNs following the 10% rule. All PET/CT-positive regional lymph nodes were excised. The \u003cem\u003eFirefly\u003c/em\u003e system identified nine of the 10 PET/CT-positive lymph nodes. The one lymph node that could not be detected via the intra-operative camera was the pudendal artery regional lymph node of Pig #4, which was outside the abdominal cavity and therefore not within the field-of-view of the \u003cem\u003eFireFly\u003c/em\u003e camera. This pudendal artery regional lymph node in Pig#4 was removed by open surgery because the space between the sacrum and the iliac bone was too narrow and deep to be approached by the \u003cem\u003eDaVinci Xi\u003c/em\u003e.\u003c/p\u003e\n \u003cp\u003eFigure \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e displays the representative \u003cem\u003eFirefly\u003c/em\u003e system images of Pig#3. Figure \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ea is a bright field image, the arrow points to a sentinel lymph node at the right internal iliac artery. The \u003cem\u003eFirefly\u003c/em\u003e mode (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eb) revealed fluorescence of the tilmanocept-positive lymph node, which was more clearly displayed by the \u003cem\u003esensitive\u003c/em\u003e mode (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ec).\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eEx Vivo Fluorescence Imaging\u003c/h3\u003e\n\u003cp\u003eAll SLNs identified by the \u003cem\u003eFirefly\u003c/em\u003e system were also detectable using \u003cem\u003eFluobeam800\u003c/em\u003e at an exposure time of 10 ms (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). Figure \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e is the brightfield (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ea) and fluorescence images (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eb) from Pig #3. The optical imager demonstrated intra-nodal fluorescence within two right internal lymph nodes. These two lymph nodes were consistent with the SLNs identified on pre-operative imaging and ex vivo assay of technetium-99m radioactivity.\u003c/p\u003e\n\u003ch3\u003eEx Vivo Radioactivity Counting\u003c/h3\u003e\n\u003cp\u003eThe radioactive activity results for Tc-99m indicated that the mean %ID\u0026thinsp;\u0026plusmn;\u0026thinsp;SD for the ten sentinel lymph node was 1.90\u0026thinsp;\u0026plusmn;\u0026thinsp;1.05, which was significantly (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) different than mean value (0.01\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02) of the non-sentinel lymph nodes (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ea). The maximum fluorescent intensity for SLNs and NSLNs were 5,963.2\u0026thinsp;\u0026plusmn;\u0026thinsp;2,985.0 and 553.9\u0026thinsp;\u0026plusmn;\u0026thinsp;199.2, respectively, indicating a statistically significant difference (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eb).\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eIn this study, we successfully demonstrated the ability of multimodal tilmanocept to preoperatively image and intra-operatively map sentinel lymph nodes. By combining preoperative PET/CT imaging with intraoperative fluorescent navigation, we demonstrated significant potential for improving patient selection for LPLND. Avid binding to CD206 allows tilmanocept to remain detectable in lymph nodes for at least 45 hours post-injection, enabling precise SLN identification with the intra-operative \u003cem\u003eFirefly\u003c/em\u003e camera system. The goal of this innovative approach is to reduce unnecessary LPLND procedures, thereby decreasing operative time and minimizing the risk of complications.\u003c/p\u003e \u003cp\u003eIn the lower rectum, the lymphatic flow becomes more complex, as it approaches the anus. There are two major lymphatic pathways: the upstream and the lateral direction. Upstream lymphatic flow runs along the superior rectal artery and inferior mesenteric artery within the mesorectum. The lateral lymphatic flow, on the other hand, drains into the basin of lateral pelvic lymph nodes, particularly around the internal iliac artery and obturator artery. This lateral lymphatic flow is responsible for lateral pelvic lymph node metastasis. Currently, there are no clear imaging diagnostic criteria for lymph nodes suspected of metastasis\u003csup\u003e[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/sup\u003e, leading to a lack of established treatment guidelines. Patients with enlarged or borderline-metastatic lateral pelvic lymph nodes are typically treated with a combination of CRT and/or LPLND. However, some patients who undergo LPLND are over-treated\u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e. Therefore, it is crucial to stratify the patients effectively to ensure that only those who truly need LPLND receive it, especially after CRT, thereby avoiding unnecessary invasive treatments.\u003c/p\u003e \u003cp\u003eSLN biopsy is one of the approaches for stratification. The usefulness of ICG-guided sentinel node biopsy in assessing the metastatic status in lateral pelvic lymph nodes has been reported\u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/sup\u003e. In these reports, sentinel lymph nodes without metastasis in the lateral pelvic region indicate the absence of metastasis in other non-sentinel pelvic lymph nodes. Moreover, evaluating sentinel lymph nodes may contribute to the detection of metastatic lymph nodes, including micrometastasis\u003csup\u003e[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]\u003c/sup\u003e. However, it should be noted that widespread lymph node metastasis or replacement with cancer cells in lymph nodes inhibits staining with blue dye or ICG\u003csup\u003e[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]\u003c/sup\u003e. Based on the above, patients without obvious lymph node metastasis or metastatic border line lymph nodes, such as 5-10mm or less, will be considered as suitable candidates for SLN mapping.\u003c/p\u003e \u003cp\u003eThe advantage of tilmanocept lies in its versatile labeling capabilities. It can be labeled with gallium-68 for PET/CT imaging or technetium-99m for SPECT/CT imaging. In addition, tilmanocept can also be conjugated with a fluorescent dye such as \u003cem\u003eIRDye800CW\u003c/em\u003e, enabling near-infrared imaging during surgery\u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e as well as preoperative imaging. Preoperative lymph node mapping provides anatomical information and potentially detects more SLNs\u003csup\u003e[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]\u003c/sup\u003e. This permits preoperative planning of the surgery and reduction of the time required for intra-operative SLNs mappings\u003csup\u003e[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThis fluorescence navigation is effective only for superficial areas up to a depth of 10mm, with limited fluorescent penetration\u003csup\u003e[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]\u003c/sup\u003e. Identifying the fluorescence of lymph nodes located deeper than 10mm, such as those in the lateral pelvic lymph node and mesorectum, remains challenging. In human rectal cancer, lateral pelvic lymph node metastases are located more frequently in the iliac artery region and obturator artery region\u003csup\u003e[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]\u003c/sup\u003e. These regions are sites deeply embedded within the pelvic cavity with thick fat, and can only be detected via external imaging, such as PET/CT or SPECT/CT.\u003c/p\u003e \u003cp\u003eThe fluorescence of lateral pelvic sentinel lymph nodes in deep region may not be visible from the dissection surface after TME. In such cases, fluorescence should be detected by dissecting through the fat, but vessels, arteries, and the ureter duct are contained in this fat. Random excision to detect fluorescence may cause morbidity and take more time. If there is preoperative lymph node site information, the sentinel node can be accessed safely and accurately. If pre-operative SPECT/CT imaging does not detect a SLN, unnecessary dissection maneuvers can be omitted, which may lead to a lower incidence of complications related to LPLND.\u003c/p\u003e \u003cp\u003eOne example of the benefit of preoperative lymph node mapping was seen in a pudendal artery regional lymph node in case 4. All SLNs except the pudendal artery regional lymph node could be detected by fluorescence due to less fat. However, the pudendal artery regional SLN existed in the distal pudendal artery region between the sacral and iliac bone spaces. The \u003cem\u003eDa Vinci\u003c/em\u003e camera was unable to identify fluorescence due to being physically inaccessible. This lymph node is considered a site of systemic metastasis and is not typically resected in clinical practice. In this preclinical study, the purpose was to confirm the consistency between preoperative PET-CT images and intra- and post-operative fluorescent images. The surgery was transferred to an open approach and the sentinel lymph node was detected by the fluorescent camera and confirmed by Tc-99m activity. This combination of preoperative mapping and intraoperative fluorescent navigation suggests that SLNs can be accurately and efficiently identified and excised.\u003c/p\u003e \u003cp\u003eTilmanocept can reside in lymph nodes for up to 72 hours after injection\u003csup\u003e[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e; this study demonstrated a fluorescent signal within sentinel lymph nodes at 45 hours post-administration. The extended retention of tilmanocept in sentinel lymph nodes allows surgeries to be performed the day after imaging, significantly reducing radiation exposure to the staff, and insuring a bright fluorescent emission for detection by the Firefly camera.\u003c/p\u003e \u003cp\u003eWe envision the following implementation of a technetium and fluorescent version of tilmanocept for sentinel lymph node mapping of rectal cancer. The bi-modal tilmanocept would be administered within the submucosa of the rectal wall near the tumor. One hour later, a 20-minute SPECT/CT of the subject\u0026rsquo;s pelvis would be acquired. A radiologist would examine the resulting attenuation-corrected cross-sectional images and identify sentinel lymph nodes based on SUVs and the 10%-rule. After imaging, the operation could be performed up to 72 hours after administration of the fluorescent-tilmanocept. Prior to excision, fluorescent-tilmanocept within each sentinel lymph node would be visualized by the fluorescence imaging subsystem of the surgical robot. Each fluorescent lateral pelvic lymph node packet would be submitted for histologic examinate as a sentinel lymph node.\u003c/p\u003e \u003cp\u003eThe strengths of this study are 1) the use of an animal model to demonstrate the rapid and sustained accumulation of radiolabeled-fluorescent-tilmanocept, 2) a demonstration of the potential significance of pre-operative detection of sentinel lymph nodes invisible field of the surgical robot, and 3) a demonstration of fluorescent detection of bi-modal tilmanocept by a standard robotic surgical system. The limitation of this study is that it is an examination of a limited number of pigs. We only injected tilmanocept into the submucosa of the lateral wall just above the dentate line, not the anterior and posterior sides and proximal rectum. In addition, there may be differences in lymphatic flow between humans and pigs due to different anatomy.\u003c/p\u003e"},{"header":"CONCLUSION","content":"\u003cp\u003eThis study demonstrated the ability of pre-operative imaging and intra-operative detection of radiolabeled fluorescent-tilmanocept to efficiently identify sentinel lymph nodes. Sentinel lymph node identification of lateral pelvic lymph nodes using dual-labeled tilmanocept may provide an alternative strategy to the dissection of lateral lymph nodes for all tumors below the peritoneal reflection. PET/CT or SPECT/CT pre-operative sentinel lymph node imaging can potentially reduce morbidity and surgical time by identifying patients who will not require lateral pelvic lymph node dissections.\u003c/p\u003e"},{"header":"METHODS","content":"\u003cdiv id=\"Sec9\" class=\"Section3\"\u003e\n \u003ch2\u003eExperimental design\u003c/h2\u003e\n \u003cp\u003eWe designed a non-survival study using four female Yorkshire pigs (Premier BioSource, Ramona CA) to evaluate a clinical protocol for sentinel lymph node mapping of patients with rectal cancer. The animals ranged in age from 85 to 90 days and weighed between 31 and 32 kg. The primary goal of this study was a demonstration of bi-modal tilmanocept for pre-operative cross-sectional imaging and intra-operative sentinel lymph node mapping many days after administration. We labeled tilmanocept with a near-infrared fluorescent dye (\u003cem\u003eIRDye800CW\u003c/em\u003e) and two radioisotopes: gallium-68, and technetium-99m. Gallium-68 is a positron emission isotope with a 68-minute half-life, which enabled pre-operative PET imaging of tilmanocept for in vivo sentinel lymph node mapping and surgical planning. Technetium-99m is a gamma-emitting isotope, with a 6-hour half-life, which provides a measurement of lymph node accumulation, and therefore, an independent identification of SLN status.\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eAnimal Model and Reagents\u003c/h3\u003e\n\u003cp\u003eThe protocol for animal transfer and experiments was approved by the University of California, San Diego Institutional Animal Care and Use Committee. All animal procedures were performed in accordance with 1) the Guide for the Care and Use of Laboratory Animals as published by the National Research Council, 2) Association for Assessment and Accreditation of Laboratory Animal Care regulations, and 3) ARRIVE guidelines. A total of four pigs female were studied. During transportation to the imaging or surgical facility, each pig was sedated by intramuscular injection of an acepromazine/buprenorphine (0.10\u0026ndash;0.05 and 0.01 mg/kg) cocktail. All procedures were performed under general anesthesia(propofol i.v., 2 mg induction, 10 mg/kg/hr maintenance). No animals were excluded from the study.\u003c/p\u003e\n\u003cp\u003eTilmanocept was obtained from Navidea Biopharmaceuticals (Dublin, OH), and \u003cem\u003eIRDye800CW\u003c/em\u003e-NHS-ester was purchased from LICOR Biosciences (Lincoln, NB). Fluorescent-labeled tilmanocept was prepared by covalent attachment of \u003cem\u003eIRDye800CW\u003c/em\u003e to tilmanocept using a previously described method \u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e41\u003c/span\u003e]\u003c/sup\u003e. \u003cem\u003eIRDye800CW\u003c/em\u003e-tilmanocept was radiolabeled with technetium-99m and gallium-68 as previously reported\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e42\u003c/span\u003e]\u003c/sup\u003e. The radiochemical yield (RCY) and fluorescent purity were measured by ITLC using \u003cem\u003eWhatman31\u003c/em\u003e as the stationary phase and acetone as the mobile phase. The RCYs and fluorescent purity of all preparations exceeded over 98%.\u003c/p\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003ch2\u003ePET/CT Imaging\u003c/h2\u003e\n \u003cp\u003eThe pigs were positioned within the gantry of a \u003cem\u003euMI550\u003c/em\u003e PET-CT scanner (United Imaging, Houston TX) so that the injection of 0.1ml of \u003csup\u003e68\u003c/sup\u003eGa-\u003csup\u003e99m\u003c/sup\u003eTc-labeled \u003cem\u003eIRDye800CW\u003c/em\u003e-tilmaoncept (1.5 nmol Tilmanocept, 〜5 MBq \u003csup\u003e68\u003c/sup\u003eGa、30 MBq \u003csup\u003e99m\u003c/sup\u003eTc) could be performed above the dentate line on the rectal side wall. Scout CT images were taken for correct positioning followed by a CT scan for attenuation correction. Whole-body PET-CT images were acquired at approximately 60 minutes post-injection and consisted of two bed positions spanning from the rectum to the lower chest of each animal. Each bed position was acquired for 2 minutes. CT attenuation correction was applied, and the reconstructed PET-CT fusion images were viewed, and SUV measurements were made with the \u003cem\u003eVisage 7\u003c/em\u003e software (San Diego, CA). After recovery from anesthesia, each pig was returned to the vivarium.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eRobotic-assisted surgery and\u003c/strong\u003e \u003cstrong\u003eFirefly\u003c/strong\u003e \u003cstrong\u003eimaging\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eWithin 2 days of PET/CT imaging, each pig was transferred to the UCSD Center for the Future of Surgery. The pigs were anesthetized and set on the bed in the supine position. A camera port was placed on the midline, one on the left abdomen, and two on the right. The uterus was hung to the abdominal wall, and lymph node dissection was started. Lymph nodes around the external and internal iliac arteries were dissected from the internal and external iliac artery bifurcation toward the anal side. The lateral pelvic lymph nodes of both sides were excised completely. In addition, paraaortic lymph node near inferior mesenteric artery and pudendal artery regional lymph node were sampled. Finally, the mesorectum was dissected, and the mesorectum lymph nodes were excised from around the rectum. Intraoperatively, the \u003cem\u003eFirefly\u003c/em\u003e camera system was used to check the fluorescence of each lymph node during the resection. We used both \u003cem\u003eFirefly\u003c/em\u003e and \u003cem\u003esensitive\u003c/em\u003e mode to detect fluorescent-positive lymph nodes. We used the information from the PET/CT as a guide to confirm the location of the SLNs.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n \u003ch2\u003eRadioactivity and fluorescence measurements\u003c/h2\u003e\n \u003cp\u003eThe dissected lymph nodes were individually separated from the bulk of lymph nodes embedded in fat tissue. Each lymph node was placed in a plastic scintillation vial and measured for technetium-99m radioactivity (100\u0026ndash;200 keV energy window) in an autowell gamma counter (\u003cem\u003eWizard2\u003c/em\u003e, PerkinElmer, Waltham, MA) with a counting standard, which consisted of a known amount of injectate of a known dilution. The percent-of-injected dose (\u003cem\u003e%ID\u003c/em\u003e) was calculated using the counting standard. All lymph nodes were imaged with a hand-held fluorescence imaging system (\u003cem\u003eFluobeam800\u003c/em\u003e, Fluoptics, Grenoble, France). Separated lymph nodes images were acquired with a 10-ms exposure time. The fluorescence images were analyzed using \u003cem\u003eImageJ\u003c/em\u003e (NIH, Bethesda MD); ROIs were drawn around each lymph node and the maximum gray luminance value was obtained within each ROI. We followed the \u0026ldquo;10% rule\u0026rdquo; to identify sentinel lymph nodes \u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e43\u003c/span\u003e]\u003c/sup\u003e. According to this rule, lymph nodes that accumulated more than 10% of the highest radioactivity in technetium-99m measurement qualified as sentinel lymph nodes.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n \u003ch2\u003eStatistical Analysis\u003c/h2\u003e\n \u003cp\u003eStatistical analysis was performed with JMP\u0026reg; \u003cem\u003ePro 16.0.0\u003c/em\u003e. Percent-of-injected dose and fluorescent intensity were assessed by the two-tailed Student\u0026rsquo;s t-test. We considered a P-value less than 0.05 to be statistically significant.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003eDisclosure\u003c/p\u003e\n\u003cp\u003eDavid Vera is the inventor of tilmanocept\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eAcknowledgments \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAuthors would like to thank Prof.\u0026nbsp;Shouichiro Saito and\u0026nbsp;Dr. Sawa Onouchi, Laboratory of Veterinary Anatomy, University of Gifu, for their porcine anatomy consultation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConception and design: RO, DRV\u003c/p\u003e\n\u003cp\u003eDevelopment of methodology: RO JK, CKH, DRV\u003c/p\u003e\n\u003cp\u003eAcquisition of data: RO, JK, ETA, SBP, CKH, DVR\u003c/p\u003e\n\u003cp\u003eAnalysis and interpretation of data: RO, JK, ETA, SBP, MK, CKH, DVR\u003c/p\u003e\n\u003cp\u003eWriting, review, and/or revision of the manuscript: RO, JK, ETA, SBP, MK, CKH, DRV\u003c/p\u003e\n\u003cp\u003eStudy supervision: DRV\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorresponding author:\u003c/strong\u003e RO\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDavid Vera is the inventor of timanocept. The remaining authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data that support the findings of this study are available from the corresponding author, RO, upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eDayal, S., Battersby, N. \u0026amp; Cecil, T. Evolution of Surgical Treatment for Rectal Cancer: a Review. \u003cem\u003eJ. Gastrointest. Surg.\u003c/em\u003e \u003cb\u003e21\u003c/b\u003e, 1166\u0026ndash;1173 (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKusters, M. et al. Patterns of local recurrence in rectal cancer; a study of the Dutch TME trial. \u003cem\u003eEur. J. Surg. Oncol.\u003c/em\u003e \u003cb\u003e36\u003c/b\u003e, 470\u0026ndash;476 (2010).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKusters, M. et al. 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Surg.\u003c/em\u003e \u003cb\u003e223\u003c/b\u003e, 217\u0026ndash;224 (1996).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Rectal cancer, Lateral pelvic lymph node, Sentinel lymph node, Tilmanocept, Robotic surgery, fluorescent navigation surgery","lastPublishedDoi":"10.21203/rs.3.rs-6212314/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6212314/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe management of lateral pelvic lymph nodes in locally advanced lower rectal cancer remains controversial. The sentinel lymph node (SLN) mapping can potentially enhance patient selection for lateral pelvic lymph node dissection. The current mapping agent, indocyanine green dye cannot be externally imaged prior to surgery and is not retained after entering the SLN. This study evaluated the ability of tilmanocept, a receptor-specific SLN mapping agent, to provide preoperative PET cross-sectional imaging and sustained intraoperative fluorescence images during rectal cancer surgery. Tilmanocept was labeled with gallium-68, technetium-99m, and a near-infrared fluorophore. Four pigs were studied. Tilmanocept was injected into the submucosal layer of the rectal wall followed one hour later by PET/CT images of the pelvis, which identified ten SLNs, one of which was a pudendal artery regional SLN. Approximately forty-five hours after administration, SLN dissection was guided by fluorescence imaging during robotic surgery. The tenth SLN was excised by open surgery. The radioactive and fluorescent intensities of all ten excised SLNs were significantly higher than the non-SLNs. The ability of dual-labeled tilmanocept to provide preoperative SLN imaging can potentially reduce morbidity and operation time by identifying patients who will not require lateral pelvic lymph node dissections.\u003c/p\u003e","manuscriptTitle":"Fluorescent-Labeled Tilmanocept for Sentinel Lymph Node Identification in Rectal Cancer during Robotic Surgery: A Preclinical Study in the Porcine Model","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-07 03:18:33","doi":"10.21203/rs.3.rs-6212314/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-07-23T03:11:31+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-16T09:28:31+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-08T23:09:03+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"152801605289601691288211477756377861184","date":"2025-07-06T09:07:27+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"132633160041253180363175895033820942280","date":"2025-06-28T18:43:57+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"306144992833888280558199360195825415714","date":"2025-04-06T11:47:01+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-04-04T10:58:25+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-04-04T10:55:19+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-03-25T07:39:08+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-03-24T06:18:56+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-03-12T13:03:14+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"ae955fc8-fd50-44d3-98dc-9e052a3d9bc5","owner":[],"postedDate":"May 7th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":46785124,"name":"Biological sciences/Biological techniques"},{"id":46785125,"name":"Biological sciences/Cancer"}],"tags":[],"updatedAt":"2025-11-03T16:05:10+00:00","versionOfRecord":{"articleIdentity":"rs-6212314","link":"https://doi.org/10.1038/s41598-025-21160-w","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-10-27 15:58:08","publishedOnDateReadable":"October 27th, 2025"},"versionCreatedAt":"2025-05-07 03:18:33","video":"","vorDoi":"10.1038/s41598-025-21160-w","vorDoiUrl":"https://doi.org/10.1038/s41598-025-21160-w","workflowStages":[]},"version":"v1","identity":"rs-6212314","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6212314","identity":"rs-6212314","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}