Fluorescently labeled Nanobodies (Nbs) provide rapid, specific, and high-contrast molecular imaging capabilities, making them well-suited for applications such as fluorescence-guided surgery. As dye properties can substantially influence tracer performance, this study evaluated three next-generation FNIR-Tag dyes, each conjugated to an anti-EGFR Nb. Procedures. The anti-EGFR Nb 7D12 was conjugated to FNIR-Tag-1.0, FNIR-Tag-766 or FNIR-Tag-804, and characterized in vitro. In vivo imaging and biodistribution studies were performed to assess pharmacokinetics, tumor uptake, tumor-to-background ratios, contrast-to-noise ratios, and renal clearance. All three Nb-based tracers exhibited similar overall pharmacokinetic behavior, characterized by fast tumor accumulation, rapid clearance from blood and non-target tissues, and predominant renal elimination. 7D12–FNIR-Tag-1.0 demonstrated slightly higher tumor uptake, whereas 7D12–FNIR-Tag-766 produced marginally improved tumor-to-background and contrast-to-noise ratios. In contrast, 7D12–FNIR-Tag-804 yielded significantly lower tumor signal intensity, although its imaging contrast remained comparable due to proportionally reduced background fluorescence. Despite differences in dye net charge, no substantial variation in kidney retention was observed over 24 h. Microscopic analysis revealed, however, distinct renal handling: 7D12–FNIR-Tag-766 showed partial endosomal internalization within proximal tubule cells, while 7D12–FNIR-Tag-1.0 remained primarily luminal. FNIR-Tag–labeled Nbs enable effective tumor visualization at 1 h post-injection. Although overall biodistribution was comparable across dyes, differences in tissue uptake, intrinsic brightness, and compatibility with imaging system optics can influence detection sensitivity and should inform dye selection for clinical imaging applications.

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.

Fluorescence molecular imaging (FMI) is becoming a powerful tool to improve surgical precision and diagnostics. Although significant advances have been made in imaging technology and contrast agents, debate continues regarding suitable standards for FMI. Standardizing all FMI aspects is crucial for adoption in clinical procedures and routine practice. Currently, the lack of clear-cut guidelines on FMI study reporting complicates comparison between studies and impedes standardization. This work presents community-driven REFLECT guidelines for FMI study reporting for fluorescence-guided surgery and interventions, covering preclinical, translational, and clinical studies. It aims to improve quality of reporting, enable comparison between studies, and enhance interpretation of results. The resulting structured checklist encompasses all essential details for reporting on the contrast agent, imaging device, imaging protocol, and image processing and analysis methods. Adoption of this framework is encouraged to enhance reproducibility and establish alignment with standardization efforts in the field - thus fostering advancements in FMI.

In vivo fluorescence lifetime (FLT) imaging is an emerging and promising modality with the potential to provide additional biological information compared to fluorescence intensity (FI) imaging. Until now, nearly all studies evaluating the benefits of FLT imaging in vivo have been conducted using cyanine dyes. While these fluorophores are highly successful for conventional in vivo fluorescence imaging due to their unmatched brightness in the NIR-I region (700-900 nm), they might be suboptimal for FLT imaging. Indeed, NIR-I cyanines are characterized by short singlet excited-state lifetime (generally below 1 ns) making it challenging to differentiate them from tissue autofluorescence. Therefore, there is a pressing need for biocompatible fluorophores with longer FLTs. Herein, we report a novel bioconjugatable water-soluble NIR-emissive aza-BODIPY dye with extended FLT values in physiological conditions, compared to cyanine dyes (increase of 100% compared to IRDye® 800CW). The suitability of aza-BODIPY for FLT imaging when conjugated to an antibody was assessed to enhance this technology. Beyond rational design and synthesis of a long-wavelength fluorophore with attractive features for in vivo FLT imaging, this study also highlights for the first time the impact of the conjugation method on the FLT characteristics of the resulting fluorescent antibody conjugates.

Recent studies demonstrated the added-value of fluorescence lifetime (FLT) imaging in tumor identification to intensity-based imaging. The tauCAM is a novel macroscale FLT imaging system that detects the FLT of a fluorophore by measuring fluorescence in the time-domain. FLT images are made simultaneously to fluorescence intensity- and grayscale reflectance images, under ambient illumination. In vivo imaging in subcutaneous tumor bearing-mice revealed that FLT imaging with the tauCAM provides additional information on environmental parameters between tumor and healthy tissue, for tracers sensitive to their physiological environment. This research emphasizes the importance of fluorophore selection for future (pre-) clinical FLT imaging trials.

Macroscale fluorescence lifetime (FLT) imaging is emerging as a promising tool to improve tumor margin delineation and enhance contrast between tumor and healthy tissue during fluorescence-guided surgery. We have been developing the tauCAM, a custom-built time-gated CMOS camera with a high quantum efficiency (QE) in NIR (46%) and a resolution of 128x128 pixels, specifically for this application. The images obtained from the tauCAM are overlaid on high-resolution color images acquired with a dedicated color camera. We demonstrate the capabilities of our camera by performing in vivo studies on subcutaneous mice tumor models administered with nanobody- and antibody-based NIR tumor-targeted fluorescent contrast agents. The results indicate that FLT imaging enhances the contrast between tumor and healthy tissue.

Multimodality reporter gene imaging combines the sensitivity, resolution and translational potential of two or more signals. The approach has not been widely adopted by the animal imaging community, mainly because its utility in this area is unproven. We developed a new complementation-based reporter gene system where the large component of split NanoLuc luciferase (LgBiT) presented on the surface of cells (TM-LgBiT) interacts with a radiotracer consisting of the high-affinity complementary HiBiT peptide labeled with a radionuclide. Radiotracer uptake could be imaged in mice using SPECT/CT and bioluminescence within two hours of implanting reporter-gene-expressing cells. Imaging data were validated by ex vivo biodistribution studies. Following the demonstration of complementation between the TM-LgBiT protein and HiBiT radiotracer, we validated the use of the technology in the highly specific in vivo multimodal imaging of cells. These findings highlight the potential of this new approach to facilitate the advancement of cell and gene therapies from bench to clinic.

A 64 x 64 pixel current-assisted photonic sampler (CAPS) image sensor is designed for real-time fluorescence lifetime (FLT) imaging. Current assistance is used for swift detection and time-gating of the photogenerated carriers within the whole substrate. An additional bias node is used to decrease the dynamic power consumption, and a photogate is applied to the sensor to reduce the carrier transfer time. The sensor is fabricated in a commercial 350-nm CMOS process on a 15- mu m epi-layer, with 30- mu m pixels and a 61% fill factor. The pixel features gating widths below 1 ns subject to a 17-ps standard deviation on the gate width uniformity and a 31-ps standard deviation on its temporal position. The pixel has a quantum efficiency (QE) above 40% for near-infrared (NIR) wavelengths, whereas the peak QE is 61% at 675 nm. The gate's intrinsic decay is observed to be 320 ps, which allows for the FLT imaging of sub-nanosecond lifetime dyes. The real-time FLT imaging capabilities are demonstrated on commercial fluorescence phantoms and in a preclinical experiment.

Intra-operative fluorescence imaging has demonstrated its ability to improve tumor lesion identification. However, the limited tissue penetration of the fluorescent signals hinders the detection of deep-lying or occult lesions. Integrating fluorescence imaging with SPECT and/or intra-operative gamma-probing synergistically combines the deep tissue penetration of gamma rays for tumor localization with the precision of fluorescence imaging for precise tumor resection. In this study, we detail the use of a genetically encoded multifunctional handle, henceforth referred to as a GEM-handle, for the development of fluorescent/radioactive bimodal single-domain antibody (sdAb)-based tracers. A sdAb that targets the urokinase plasminogen activator receptor (uPAR) was engineered to carry a GEM-handle containing a carboxy-terminal hexahistidine-tag and cysteine-tag. A two-step labeling strategy was optimized and applied to site-specifically label IRDye800CW and 99mTc to the sdAb. Bimodal labeling of the sdAbs proved straightforward and successful. 99mTc activity was however restricted to 18.5 MBq per nmol fluorescently-labeled sdAb to prevent radiobleaching of IRDye800CW without impeding SPECT/CT imaging. Subsequently, the in vivo biodistribution and tumor-targeting capacity of the bimodal tracer were evaluated in uPAR-positive tumor-bearing mice using SPECT/CT and fluorescence imaging. The bimodal sdAb showed expected renal background signals due to tracer clearance, along with slightly elevated non-specific liver signals. Four hours post-injection, both SPECT/CT and fluorescent images achieved satisfactory tumor uptake and contrast, with significantly higher values observed for the anti-uPAR bimodal sdAb compared to a control non-targeting sdAb. In conclusion, the GEM-handle is a convenient method for designing and producing bimodal sdAb-based tracers with adequate in vivo characteristics.

Introduction: Surgical resection is one of the main treatment options for several types of cancer, the desired outcome being complete removal of the primary tumor and its local metastases. Any malignant tissue that remains after surgery may lead to relapsing disease, negatively impacting the patient's quality of life and overall survival. Fluorescence imaging in surgical oncology aims to facilitate full resection of solid tumors through the visualization of malignant tissue during surgery, following the administration of a fluorescent contrast agent. An important class of targeting molecules are Nanobodies® (Nbs), small antigen-binding fragments derived from camelid heavy chain only antibodies. When coupled with a fluorophore, Nbs can bind to a specific receptor and demarcate tumor margins through a fluorescence camera, improving the accuracy of surgical intervention. A widely investigated target for fluorescence-guided surgery is the epidermal growth factor receptor (EGFR), which is overexpressed in several types of tumors. Promising results with the fluorescently labeled anti-EGFR Nb 7D12-s775z in murine models motivated a project employing the compound in a pioneering study in dogs with spontaneous cancer. Methods: To determine the safety profile of the study drug, three healthy purpose-bred dogs received an intravenous injection of the tracer at 5.83, 11.66, and 19.47 mg/m2, separated by a 14-day wash-out period. Physical examination and fluorescence imaging were performed at established time points, and the animals were closely monitored between doses. Blood and urine values were analyzed pre- and 24 h post administration. Results: No adverse effects were observed, and blood and urine values stayed within the reference range. Images of the oral mucosa, acquired with a fluorescence imaging device (Fluobeam®), suggest rapid clearance, which was in accordance with previous in vivo studies. Discussion: These are the first results to indicate that 7D12-s775z is well tolerated in dogs and paves the way to conduct clinical trials in canine patients with EGFR-overexpressing spontaneous tumors.

Heptamethine indocyanines are invaluable probes for near-infrared (NIR) imaging. Despite broad use, there are only a few synthetic methods to assemble these molecules, and each has significant limitations. Here, we report the use of pyridinium benzoxazole (PyBox) salts as heptamethine indocyanine precursors. This method is high yielding, simple to implement, and provides access to previously unknown chromophore functionality. We applied this method to create molecules to address two outstanding objectives in NIR fluorescence imaging. First, we used an iterative approach to develop molecules for protein-targeted tumor imaging. When compared to common NIR fluorophores, the optimized probe increases the tumor specificity of monoclonal antibody (mAb) and nanobody conjugates. Second, we developed cyclizing heptamethine indocyanines with the goal of improving cellular uptake and fluorogenic properties. By modifying both the electrophilic and nucleophilic components, we demonstrate that the solvent sensitivity of the ring-open/ring-closed equilibrium can be modified over a wide range. We then show that a chloroalkane derivative of a compound with tuned cyclization properties undergoes particularly efficient no-wash live cell imaging using organelle-targeted HaloTag self-labeling proteins. Overall, the chemistry reported here broadens the scope of accessible chromophore functionality, and, in turn, enables the discovery of NIR probes with promising properties for advanced imaging applications.

PURPOSE:To assess our improved NACA for the detection of tumor necrosis.METHODS:We increased the blood circulation time of our NACA by adding an albumin-binding domain to the molecular structure. We tested the necrosis avidity on dead or alive cultured cells and performed SPECT and fluorescence imaging of both spontaneous and treatment-induced necrosis in murine breast cancer models. We simultaneously recorded [18F]FDG-PET and bioluminescence images for complementary detection of tumor viability.RESULTS:We generated two albumin-binding IRDye800CW derivatives which were labeled with indium-111 with high radiochemical purity. Surprisingly, both albumin-binding NACAs had >10x higher in vitro binding towards dead cells. We selected [111In]3 for in vivo experiments which showed higher dead cell binding in vitro and in vivo stability. The doxorubicin-treated tumors showed increased [111In]3-uptake (1.74 ± 0.08%ID/g after saline treatment, 2.25 ± 0.16%ID/g after doxorubicin treatment, p = 0.044) and decreased [18F]FDG-uptake (3.02 ± 0.51%ID/g after saline treatment, 1.79 ± 0.11%ID/g after doxorubicin treatment, p = 0.040), indicating therapy efficacy. Moreover, we detected increased [111In]3-uptake and tumor necrosis in more rapidly growing EMT6 tumors.CONCLUSIONS:Our albumin-binding NACA based on IRDye800CW facilitates tumor-necrosis imaging for assessment of therapy efficacy and aggressiveness in solid tumors using both fluorescence and SPECT imaging.

Necrosis only occurs in pathological situations and is directly related to disease severity and, therefore, is an important biomarker. Tumor necrosis occurs in most solid tumors due to improperly functioning blood vessels that cannot keep up with the rapid growth, especially in aggressively growing tumors. The amount of necrosis per tumor volume is often correlated to rapid tumor proliferation and can be used as a diagnostic tool. Furthermore, efficient therapy against solid tumors will directly or indirectly lead to necrotic tumor cells, and detection of increased tumor necrosis can be an early marker for therapy efficacy. We propose the application of necrosis avid contrast agents to detect therapy-induced tumor necrosis. Herein, we advance gallium-68-labeled IRDye800CW, a near-infrared fluorescent dye that exhibits excellent necrosis avidity, as a potential PET tracer for in vivo imaging of tumor necrosis. We developed a reliable labeling procedure to prepare [68Ga]Ga-DOTA-PEG4-IRDye800CW ([68Ga]Ga-1) with a radiochemical purity of >96% (radio-HPLC). The prominent dead cell binding of fluorescence and radioactivity from [68Ga]Ga-1 was confirmed with dead and alive cultured 4T1-Luc2 cells. [68Ga]Ga-1 was injected in 4T1-Luc2 tumor-bearing mice, and specific fluorescence and PET signal were observed in the spontaneously developing tumor necrosis. The ip injection of D-luciferin enabled simultaneous bioluminescence imaging of the viable tumor regions. Tumor necrosis binding was confirmed ex vivo by colocalization of fluorescence uptake with TUNEL dead cell staining and radioactivity uptake in dichotomized tumors and frozen tumor sections. Our presented study shows that [68Ga]Ga-1 is a promising PET tracer for the detection of tumor necrosis.

Purpose Current clinical measurements for tumor treatment efficiency rely often on changes in tumor volume measured as shrinkage by CT or MRI, which become apparent after multiple lines of treatment and pose a physical and psychological burden on the patient. Detection of therapy-induced cell death in the tumor can be a fast measure for treatment efficiency. However, there are no reliable clinical tools for detection of tumor necrosis. Previously, we studied the necrosis avidity of cyanine-based fluorescent dyes, which suffered long circulation times before tumor necrosis could be imaged due to low hydrophilicity. We now present the application of radiolabeled 800CW, a commercially available cyanine with high hydrophilicity, to image tumor necrosis in a mouse model. Procedures We conjugated 800CW to DOTA via a PEG linker, for labeling with single-photon emission-computed tomography isotope indium-111, yielding [ 111 In]In-DOTA-PEG 4 -800CW. We then investigated specific [ 111 In]In-DOTA-PEG 4 -800CW uptake by dead cells in vitro , using both fluorescence and radioactivity as detection modalities. Finally, we investigated [ 111 In]In-DOTA-PEG 4 -800CW uptake into necrotic tumor regions of a 4T1 breast tumor model in mice. Results We successfully prepared a precursor and developed a reliable procedure for labeling 800CW with indium-111. We detected specific [ 111 In]In-DOTA-PEG 4 -800CW uptake by dead cells, using both fluorescence and radioactivity. Albeit with a tumor uptake of only 0.37%ID/g at 6 h post injection, we were able to image tumor necrosis with a tumor to background ratio of 7:4. Fluorescence and radioactivity in cryosections from the dissected tumors were colocalized with tumor necrosis, confirmed by TUNEL staining. Conclusions [ 111 In]In-DOTA-PEG 4 -800CW can be used to image tumor necrosis in vitro and in vivo . Further research will elucidate the application of [ 111 In]In-DOTA-PEG 4 -800CW or other radiolabeled hydrophilic cyanines for the detection of necrosis caused by chemotherapy or other anti-cancer therapies. This can provide valuable prognostic information in treatment of solid tumors.

Cancer cells sustain growth by altering their metabolism to accelerated aerobic glycolysis accompanied by increased glucose demand and employ glutamine as additional nutrient source. This metabolic adaptation induces upregulation of glucose transporters GLUT-1 and -3, and simultaneous targeting of both transporters and of glutamine metabolism may offer a promising approach to inhibit cancer cell growth. We describe the discovery of the very potent glucose uptake inhibitor Glutor, which targets glucose transporters GLUT-1, -2, and -3, attenuates glycolytic flux and potently and selectively suppresses growth of a variety of cancer cell lines. Co-treatment of colon cancer cells with Glutor and glutaminase inhibitor CB-839 very potently and synergistically inhibits cancer cell growth. Such a dual inhibition promises to be particularly effective because it targets the metabolic plasticity as well as metabolic rescue mechanisms in cancer cells.