Protein-based fluorescence imaging is a powerful modality for visualizing diverse biological processes. Biological imaging in the near-infrared (NIR, 800-1000 nm) and shortwave infrared (SWIR, 1000-2000 nm) ranges confers a number of photophysical advantages, but remains a challenge in practice due to the dearth of suitable protein probes in these optical windows. To address this limitation, we sought to develop a general approach integrating computational protein design with organic synthesis for creating long-wavelength fluorescence-activating proteins from scratch. We used this approach to de novo design proteins that specifically bind to synthetic merocyanine dyes, forming Schiff base covalent linkages, which when protonated activate fluorescence with large redshifts in both excitation and emission wavelengths. We describe a designed far-red fluorescence-activating protein, MC7BP34, with a brightness greater than that of existing fluorescent proteins in a similar wavelength range, and an NIR design MC9BP81 with excitation at 892 nm and emission extending into the SWIR range with higher contrast and imaging sensitivity in vivo than the previously developed iRFP720 (excitation 672 nm) owing to the reduced tissue autofluorescence at longer wavelengths. Our results are a substantial step toward genetically encodable probes in the SWIR region, and our approach lays the groundwork for the development of NIR biosensors for specific biological applications.

Modular, tunable linker chemistries that are stable in circulation yet selectively cleaved are needed to realize the therapeutic potential of antibody-drug conjugates (ADCs). Near-infrared (NIR) fluorogenic imaging using norcyanine carbamate (CyBam) probes can quantitatively compare ADC linkers across in vitro and in vivo settings. A series of substituted CyBams modified with phosphoramidates, peptides, and control triggers were conjugated to EGFR-targeting monoclonal antibody (mAb) panitumumab. Imaging in cellular and in vivo settings reveals the potential for novel phosphoramidate linkers, showing selective cellular activation, excellent tumor localization, and reduced liver signal compared to conventional proteolytic linkers. Guided by these imaging results, monomethyl auristatin E (MMAE) conjugates bearing the new linkers were prepared and displayed picomolar potency, receptor-dependent activity, and promising in vivo tumor growth inhibition. In total, these studies demonstrate that quantitative fluorogenic imaging can enable the discovery and prioritization of new ADC linkers.

While radiopharmaceutical therapy (RPT) has become part of the standard-of-care for patients with advanced prostate cancers and neuroendocrine tumors (NETs), cures are elusive and normal tissue toxicity remain a challenge. Chemical groups susceptible to cleavage by enzymes present in tumors, tumor microenvironment or in normal tissues, have the potential to improve the therapeutic index for RPT. Using DOTA-TATE as an example and drawing from strategies used to develop antibody-drug conjugates, we designed, and synthesized, a chemically diverse series of linkers between the chelator (DOTA) and the targeting vector (TATE). Of the 10 agents we tested, two with cleavable linker domains reduced kidney retention compared to DOTA-TATE: the previously reported DOTA-MVK(ε)-TATE, and a novel agent bearing cleavable beta-galactose (β-Gal) unit, DOTA-β-Gal-TATE. In murine models of NETs, positron emission tomography (PET) was used to image yttrium-86 ( 86 Y)-labeled variants and show that, while the 86 Y-DOTA-MVK(ε)-TATE exhibits similar tumor uptake to the parent non-cleavable 86 Y-DOTA-TATE, 86 Y-DOTA-β-Gal-TATE shows enhanced tumor uptake, resulting in up to 10-fold improvement in the tumor-to-kidney ratios compared to 86 Y-DOTA-TATE. In vitro and in vivo studies confirm high efficiency, enzyme-specific cleavage of 86 Y-DOTA-MVK(ε)-TATE and 86 Y-DOTA-β-Gal-TATE, supporting a key role for cleavable linker chemistry in the observed outcomes. RPT studies using actinium-225 ( 225 Ac)-labeled variants confirm that all agents are therapeutically effective and well tolerated. While both cleavable variants exhibit superior local control, overall survival, and more favorable toxicity profile when compared with 225 Ac-DOTA-TATE, 225 Ac-DOTA-β-Gal-TATE demonstrated lower nephrotoxicity. Our findings suggest a potentially generalizable strategy for improving the pharmacokinetics of radiopharmaceutical therapy agents. One Sentence Summary:A β-galactose-cleavable linker reduces kidney toxicity, enhances tumor targeting and therapeutic efficacy in radiopharmaceutical therapy.

An alternative TREN-based bifunctional chelator for 89 Zr was synthesized. Its chemical characterization, in vitro performance, and computational modeling are presented here.

Significance:Pancreatic neuroendocrine neoplasms (PNENs) are an uncommon cancer whose incidence rate has increased dramatically in recent years. Surgery is the only potentially curative treatment, which relies on both preoperative tumor localization and postoperative margin definition using histopathological examination for decision making. If pathology could be automated, valuable time and resources could be saved. Aim:In this study, we investigate the ability of machine learning (ML) with handcrafted features, as well as deep learning, to classify label-free microscopy images of PNENs as a first step toward automated pathology of such tumors. Approach:Patient samples of two different preparation types were imaged, and ML and convolutional neural networks (CNNs) were developed to test the ability of such algorithms to classify PNENs. Results:Our classification algorithms were able to distinguish PNENs from normal tissue with high accuracy using multiphoton microscopy (MPM) images, regardless of sample preparation. Using a combined FFPE and fixed frozen dataset, we achieved an AUC value of 0.793 and an accuracy of 80.6% with ML, and an AUC value of 0.977 and an accuracy of 96.43% using CNNs. Conclusions:Label-free MPM combined with deep learning can provide fast, accurate classification of PNENs. With the ability to assess margins rapidly and potentially automatically, both disease recurrence and the need for resections after initial surgery could be reduced.

Bile duct injury (BDI) is a morbid complication of laparoscopic cholecystectomy due to poor recognition of the anatomy and inadequate visualization of the extra-hepatic biliary ducts. Near-infrared indocyanine green (ICG) is the most commonly used non-invasive option to assist with identification of the extra-hepatic biliary structures. However ICG is limited by its slow onset of action and lack of specificity for the biliary tree. In light of these limitations our team previously reported bile-label 760 (BL-760), a pre-clinical near-infrared dye, as a novel tool for intraoperative identification of biliary structures. This study builds upon our previous work and assesses the intraoperative detection of the extra-hepatic biliary ducts in a swine model of biliary obstruction using intravenously administered BL-760. A survival swine study utilizing BL-760 was performed in two 30 kg female Yorkshire swine. Each swine underwent two surgeries. In the initial surgeries, laparoscopic clipping of an extra-hepatic biliary duct was performed under BL-760 guidance. The cystic duct (CD) was clipped in Swine #1 and the common bile duct (CBD) was clipped in Swine #2. On the third postoperative day, a laparoscopic cholecystectomy was performed in each swine under BL-760 guidance. Target-to-background ratios (TBRs) of the extra-hepatic biliary ducts to the liver were measured using ImageJ. The surgeries were performed without complication. The TBR in the initial surgeries were 2.42 (Swine #1) and 3.22 (Swine #2) for the CBD, without the need for surrounding dissection. In the second surgeries, the gallbladders were clearly inflamed without perforation, and the CBDs were visualized with BL-760 with a TBR of 2.83 (Swine #1) and 2.60 (Swine #2). BL-760 demonstrates high specificity for the biliary tree in an obstructive biliary model. BL-760’s rapid, enhanced visualization has the potential to improve the accuracy of identifying biliary anatomy and enhance cholecystectomy safety.

Near-infrared fluorescent molecular imaging is increasingly being used as a tool to guide intraoperative decision-making. While research into novel fluorescent molecular tracers often prioritizes the targeting moiety, compelling evidence indicates that the choice of fluorophore can substantially influence tracer pharmacokinetics. In this study, tumor-specific Nanobodies were conjugated with four widely used dyes─IRDye800CW, ZW800-1, FNIR-Tag, and s775z-, and a side-by-side comparison of their in vivo biodistribution and tumor targeting was performed. Nanobodies labeled with FNIR-Tag or s775z showed markedly superior results compared to those labeled with IRDye800CW or ZW800-1, demonstrating strong tumor accumulation as early as 1 h postinjection and minimal background signal, particularly in the liver. The effect of increasing dye density on tracer pharmacokinetics was also assessed. Compared with Nbs labeled with a DOL of 1, those with an average of 2 dyes exhibited higher mean fluorescent tumor signals but no improvement in tumor-to-background ratios, owing to increased background signals.

Zirconium-89 (89Zr) bears characteristics that are ideal for positron emission tomography (PET) imaging of long-circulating radiopharmaceuticals. However, the most commonly used chelator, deferoxamine (DFO), suffers from limited in vivo stability, leading to leaching of 89Zr and subsequent bone accumulation that compromises diagnostic accuracy and dosimetric calculations. Here, we report the first application of the enterobactin-inspired chelator TRENTAM and its bifunctional analogue TRENTAM-COOH for chelating 89Zr. Both compounds were synthesized via modular, high-yielding routes and demonstrated efficient radiolabelling with 89Zr under mild conditions. In vitro assays showed that [89Zr]Zr-TRENTAM and [89Zr]Zr-TRENTAM-COOH outperform DFO and TRENHOPO in EDTA and serum stability challenges over 7 days. TRENTAM also exhibited exceptional stability across physiologically relevant pH ranges and resisted transchelation by biologically abundant metal ions. Biodistribution studies in mice demonstrated low nonspecific uptake of [89Zr]Zr-TRENTAM. Furthermore, TRENTAM demonstrated negligible cytotoxicity in cells, supporting its biocompatibility. Collectively, these findings establish TRENTAM as a high-affinity, stable, and versatile chelator for 89Zr, advancing its potential for use in next-generation targeted PET imaging agents and theranostic applications.

Monoclonal antibodies (mAbs) are essential agents for cancer treatment and diagnosis. Advanced optical imaging strategies have the potential to address specific questions regarding their complex in vivo life cycle. This study presents responsive shortwave infrared (SWIR) probes and an associated imaging scheme to assess mAb biodistribution, cellular uptake, and proteolysis. Specifically, we identify a Pegylated benzo-fused norcyanine derivative (Benz-NorCy7) that is activated in acidic environments and can be appended to mAbs without significant changes in optical properties. As a mAb conjugate, this agent shows high tumor specificity in a longitudinal imaging study in a murine model. To enable independent tracking of mAb uptake and lysosomal uptake and retention, a two-color ratiometric imaging strategy was employed using an "always-ON" heptamethine cyanine dye (λex = 785 nm) and the pH-responsive Benz-NorCy7 (λex = 890 nm). To assess proteolytic catabolism, we append a cleavable carbamate to Benz-NorCy7 to create turn-ON probes. These agents facilitate the comparison of two common peptide linkers and provide insights into their in vivo properties. Overall, these studies provide a strategy to assess the fate of protein-based therapeutics using optical imaging.

Photobleaching of fluorescence labels poses a major limitation in single-molecule and super-resolution microscopy. Conventional photostabilization methods, such as oxygen removal and addition of high concentrations of photostabilization additives, often require careful fluorophore selection and can disrupt the biological environment. To address these limitations, we developed a modular and minimally invasive photostabilization approach that utilizes DNA-mediated delivery of a photostabilizer directly to the imaging site. Under lower excitation intensities, the DNA-mediated strategy outperformed solution-based approaches, achieving efficient photostabilization at significantly lower additive concentrations. However, at higher excitation intensities, the stability of a single photostabilizer molecule became the limiting factor. To overcome this and reduce the loss of localizations in DNA-PAINT experiments we have also implemented a recovery scheme where the photostabilizer is continuously replenished at the imaging site. We further extended the approach to cell imaging, demonstrating improved localization rate and precision in 3D-DNA PAINT measurements. DNA-mediated photostabilization offers a promising solution for imaging applications where high additive concentrations are prohibited. Its modularity enables adaptation to various imaging schemes and ultimately expands the repertoire of fluorophores suitable for single-molecule and super-resolution imaging.

Chemical modification of monoclonal antibodies (mAbs) and their fragments gives rise to imaging probes and targeted therapies. Depending on the isotope used, radiolabeled mAbs enable positron emission tomography (PET) and single photon emission computed tomography (SPECT) imaging and can also be applied as cytotoxic therapies. Fluorescent mAb conjugates are used for a range of preclinical applications with clinical utility for intraoperative visualization of tumors. Antibody-drug conjugates (ADCs) enhance the therapeutic efficacy of mAbs and are the topic of extensive clinical development. In all these cases, chemical modifications can significantly affect mAb tumor targeting and clearance. Whole-body imaging techniques provide crucial insights into the in vivo consequences of these changes by directly tracking antibody conjugate distribution and clearance. This review examines in vivo imaging studies that compare “parental” and “modified” mAbs imaged under identical conditions to assess the effects of the cargo itself (e.g. fluorophore, chelator, drug), as well as the chemical conjugation methods. Additionally, we also describe studies that evaluate alternative strategies, including pretargeting, Fc modifications and pre- or co-dosing strategies that seek to tune the biodistribution of a given conjugate. Overall, we highlight the critical role of imaging in characterizing the in vivo performance of mAb conjugates, underscoring how these insights can inform both therapeutic efficacy and toxicity, and enable clinical translation.

BackgroundNoninvasive in vivo cell tracking is valuable in understanding the mechanisms that enhance anti-cancer immunity. We have recently developed a new method called phototruncation-assisted cell tracking (PACT), that uses photoconvertible cell tracking technology to detect in vivo cell migration. This method has the advantages of not requiring genetic engineering of cells and employing tissue-penetrant near-infrared light.MethodsWe applied PACT to monitor the migration of immune cells between a tumour and its tumour-draining lymph node (TDLN) after near-infrared photoimmunotherapy (NIR-PIT).FindingsPACT showed a significant increase in the migration of dendritic cells (DCs) and macrophages from the tumour to the TDLN immediately after NIR-PIT. This migration by NIR-PIT was abrogated by inhibiting the sphingosine-1-phosphate pathway or Gαi signaling. These results were corroborated by intranodal immune cell profiles at two days post-treatment; NIR-PIT significantly induced DC maturation and increased and activated the CD8+ T cell population in the TDLN. Furthermore, PACT revealed that NIR-PIT significantly enhanced the migration of CD8+ T cells from the TDLN to the tumour four days post-treatment, which was consistent with the immunohistochemical assessment of tumour-infiltrating lymphocytes and tumour regression.InterpretationImmune cells dramatically migrated between the tumour and TDLN following NIR-PIT, indicating its potential as an immune-stimulating therapy. Also, PACT is potentially applicable to a wide range of immunological research.FundingThis work was supported by the Intramural Research Program of the National Institutes of Health, National Cancer Institute, Centre for Cancer Research (grant number: ZIA BC011513 and ZIA BC011506).

Pyrrole-containing natural products form a large group of structurally diverse compounds that occur in both terrestrial and marine organisms. In the present study the formation of trideuteromethylated artifacts of pyrrole-containing natural products was investigated, focusing on the discorhabdins. Three deuterated discorhabdins, 1, 3, and 5, were identified to be isolation procedure artifacts caused by the presence of DMSO-d(6) during NMR sample preparation and handling. Three additional semisynthetic derivatives, 7-9, were made during the investigation of the mechanism of formation, which was shown to be driven by trideuteromethyl radicals in the presence of water, methanol, TFA, and traces of iron in the deuterated solvent. Generation of trideuteromethylated artifacts was also confirmed for other classes of pyrrole-containing metabolites, namely, makaluvamines, tambjamines, and dibromotryptamines, which had also been dissolved in DMSO-d(6) during the structure elucidation process. Semisynthetic discorhabdins were assessed for antiproliferative activity against a panel of human tumor cell lines, and 14-trideuteromethyldiscorhabdin L (3) averaged low micromolar potency.

Targeted payload delivery strategies, such as antibody-drug conjugates (ADCs), have emerged as important therapeutics. Although considerable efforts have been made in the areas of antibody engineering and labeling methodology, improving the overall physicochemical properties of the linker/payload combination remains an important challenge. Here we report an approach to create an intrinsically hydrophilic linker domain. We find that benzyl α-ammonium carbamates (BACs) undergo tandem 1,6–1,2-elimination to release secondary amines. Using a fluorogenic hemicyanine as a model payload component, we show that a zwitterionic BAC linker improves labeling efficiency and reduces antibody aggregation when compared to a commonly used para -amino benzyl (PAB) linker as well as a cationic BAC. Cellular and in vivo fluorescence imaging studies demonstrate that the model payload is specifically released in antigen-expressing cells and tumors. The therapeutic potential of the BAC linker strategy was assessed using an MMAE payload, a potent microtubule-disrupting agent frequently used for ADC applications. The BAC-MMAE combination enhances labeling efficiency and cellular toxicity when compared to the routinely used PAB-Val-Cit ADC analogue. Broadly, this strategy provides a general approach to mask payload hydrophobicity and improve the properties of targeted agents.

Colorectal cancer (CRC) is the third most common cancer worldwide. In the United States alone, CRC was responsible for approximately 52,550 deaths in 2023, with an estimated 153,020 new cases. CRC presents with synchronous peritoneal spread in 5-10% of patients, and up to 20-50% of patients with recurrent disease will develop metachronous colorectal cancer peritoneal metastatic (CRC-PM) disease. Eradication of the tumor, tumor margins and microscopic residual disease is paramount, as microscopic residual disease is associated with local recurrences, with 5-year survival rates of less than 35%. The success of resection and reduction of residual disease depends on the accuracy with which cancer cells and normal tissue can be intra-operatively distinguished. Fluorescence Molecular Imaging (IFMI) and tumor-targeted contrast agents represent a promising approach for intraoperative detection and surgical intervention. Proper target selection, the development of scalable imaging agents and enhanced real-time tumor and tumor microenvironment imaging are critical to enabling enhanced surgical resection. LGR5 (leucine-rich repeat-containing G-protein-coupled receptor 5), a colonic crypt stem cell marker and the receptor for the R-spondins (RSPO) in the Wnt signaling pathway, is also expressed on colorectal cancer stem cells (CSC) and on CRC tumors and metastases, suggesting it could be a useful target for imaging of CRC. However, there are numerous diverging reports on the role of LGR5 in CRC therapy and outcomes. Herein, we report on the synthesis and validation of a 37 amino acid RSPO1-mimetic peptide, termed RC18, that was specifically designed to access the R-spondin binding site of LGR5 to potentially be used for interoperative imaging of CRC-PM. The receptor-binding capabilities of the RC18 indicate that direct interactions with LGR5 neither significantly increased LGR5 signaling nor blocked RSPO1 binding and signal transduction, suggesting that the RSPO1-mimetic is functionally inert, making it an attractive contrast agent for intraoperative CRC-PM imaging.

Cyanines are the most commonly used fluorescent probes for in vivo FGS applications, despite challenging properties for in vivo use. To address this, our research focuses on developing new synthetic methods that modify the key polymethine chromophore unit. We have developed probes with rapid and exclusive renal or hepatobiliary clearance for abdominal surgery applications. Additionally, we have extensively examined the role of probe chemistry on tumor targeting of monoclonal antibody (mAb) conjugates. This research has led to the discovery of FNIR-Tag, a molecule with excellent characteristics for in vivo mAb-targeted imaging, including improved labeling properties, reduced hepatic clearance, and enhanced in vivo tumor uptake and signal. Collaborative studies have shown that FNIR-Tag also enhances the in vivo properties of various other targeting agents, including virus-like particles, nanobodies, and peptides. An ongoing challenge in the field is the development of probes that target intracellular structures. To address this, we've recently developed probes capable of reversible cyclization chemistry, enabling efficient intracellular labeling.

Head and neck cancers are the seventh most common cancers worldwide, with squamous cell carcinoma being the most prevalent histologic subtype. Surgical resection is a primary treatment modality for many patients with head and neck squamous cell carcinoma, and accurately identifying tumor boundaries and ensuring sufficient resection margins are critical for optimizing oncologic outcomes. This letter presents an innovative autonomous system for tumor resection (ASTR) and conducts a feasibility study by performing supervised autonomous midline partial glossectomy for pseudotumor with millimeter accuracy. The proposed ASTR system consists of a dual-camera vision system, an electrosurgical instrument, a newly developed vacuum grasping instrument, two 6-DOF manipulators, and a novel autonomous control system. The letter introduces an ontology-based research framework for creating and implementing a complex autonomous surgical workflow, using the glossectomy as a case study. Porcine tongue tissues are used in this study, and marked using color inks and near-infrared fluorescent (NIRF) markers to indicate the pseudotumor. ASTR actively monitors the NIRF markers and gathers spatial and color data from the samples, enabling planning and execution of robot trajectories in accordance with the proposed glossectomy workflow. The system successfully performs six consecutive supervised autonomous pseudotumor resections on porcine specimens. The average surface and depth resection errors measure $0.73\pm 0.60$ $\text{mm}$ and $1.89\pm 0.54$ $\text{mm}$ , respectively, with no positive tumor margins detected in any of the six resections. The resection accuracy is demonstrated to be on par with manual pseudotumor glossectomy performed by an experienced otolaryngologist.