Chemically augmented malaria sporozoites display an altered immunogenic profile.

Despite promising results in malaria-naïve individuals, whole sporozoite (SPZ) vaccine efficacy in malaria-endemic settings has been suboptimal. Vaccine hypo-responsiveness due to previous malaria exposure has been posited as responsible, indicating the need for SPZ vaccines of increased immunogenicity. To this end, we here demonstrate a proof-of-concept for altering SPZ immunogenicity, where supramolecular chemistry enables chemical augmentation of the parasite surface with a TLR7 agonist-based adjuvant (SPZ-SAS(CL307)). In vitro, SPZ-SAS(CL307) remained well recognized by immune cells and induced a 35-fold increase in the production of pro-inflammatory IL-6 (p < 0.001). More promisingly, immunization of mice with SPZ-SAS(CL307) yielded improved SPZ-specific IFN-γ production in liver-derived NK cells (percentage IFN-γ+ cells 11.1 ± 1.8 vs. 9.4 ± 1.5%, p < 0.05), CD4+ T cells (4.7 ± 4.3 vs. 1.8 ± 0.7%, p < 0.05) and CD8+ T cells (3.6 ± 1.4 vs. 2.5 ± 0.9%, p < 0.05). These findings demonstrate the potential of using chemical augmentation strategies to enhance the immunogenicity of SPZ-based malaria vaccines.

A Truncated 14-Amino-Acid Myelin Protein-Zero-Targeting Peptide for Fluorescence-Guided Nerve-Preserving Surgery.

BACKGROUND: The occurrence of accidental nerve damage during surgery and the increasing application of image guidance during head-and-neck surgery have highlighted the need for molecular targeted nerve-sparing interventions. The implementation of such interventions relies on the availability of nerve-specific tracers. In this paper, we describe the development of a truncated peptide that has an optimized affinity for protein zero (P0), the most abundant protein in myelin. METHODS AND MATERIALS: Further C- and N-terminal truncation was performed on the lead peptide Cy5-P0101-125. The resulting nine Cy5-labelled peptides were characterized based on their photophysical properties, P0 affinity, and in vitro staining. These characterizations were combined with evaluation of the crystal structure of P0, which resulted in the selection of the optimized tracer Cy5-P0112-125. A near-infrared Cy7-functionalized derivative (Cy7-P0112-125) was used to perform an initial evaluation of fluorescence-guided surgery in a porcine model. RESULTS: Methodological truncation of the 26-amino-acid lead compound Cy5-P0101-125 resulted in a size reduction of 53.8% for the optimized peptide Cy5-P0112-125. The peptide design and the 1.5-fold affinity gain obtained after truncation could be linked to interactions observed in the crystal structure of the extracellular portion of P0. The near-infrared analogue Cy7-P0112-125 supported nerve illumination during fluorescence-guided surgery in the head-and-neck region in a porcine model. CONCLUSIONS: Methodological truncation yielded a second-generation P0-specific peptide. Initial surgical evaluation suggests that the peptide can support molecular targeted nerve imaging.

Comparison of two hybrid sentinel node tracers: indocyanine green (ICG)-99mTc-nanocolloid vs. ICG-99mTc-nanoscan from a nuclear medicine and surgical perspective.

BACKGROUND: Lymph node (LN) metastasis is a relevant predictor for survival in patients with a.o. penile cancer (PeCa), malignant melanoma. The sentinel node (SN) procedure comprises targeted resection of the first tumour-draining SNs. Here, the hybrid tracer indocyanine green (ICG)-99mTc-nanocolloid has been used for several years to combine optical and nuclear detection. Recently, the resource of the nanocolloid precursor stopped production and the precursor was replaced by a different but chemically comparable colloid, nanoscan. Our aim was to study the performance of ICG-99mTc-nanoscan compared to ICG-99mTc-nanocolloid from a nuclear and surgical perspective. METHODS: Twenty-four patients with either PeCa or head-and-neck (H&N) melanoma and scheduled for a SN procedure were included. The initial group (n = 11) received ICG-99mTc-nanocolloid until no longer available; the second group (n = 13) received ICG-99mTc-nanoscan. Tracer uptake was assessed on lymphoscintigraphy and single-photon emission (SPECT). Intraoperatively, SNs were identified using gamma tracing and fluorescence imaging. Ex vivo (back-table) measurements were conducted to quantify the fluorescence emissions. Chemical analysis was performed to compare the ICG assembly on both precursors. RESULTS: The mean tracer uptake in the SNs was similar for ICG-99mTc-nanocolloid (2.2 ± 4.3%ID) and ICG-99mTc-nanoscan (1.8 ± 2.6%ID; p = 0.68). 3 SNs (interquartile range (IQR) 3-4) were detected on lymphoscintigraphy in PeCa patients receiving ICG-99mTc-nanoscan compared to 2 SNs (IQR 2-3) in PeCa patients receiving ICG-99mTc-nanocolloid (p = 0.045), no differences were observed in H&N patients. Back-table measurements of resected SNs revealed a lower total fluorescence intensity in the ICG-99mTc-nanoscan group (24*109 arbitrary units (A.U) IQR 1.6*109-14*109 in the ICG-99mTc-nanocolloid group versus 4.6*109 A.U. IQR 2.4*109-42*109 in the ICG-99mTc-nanoscan group, p = 0.0054). This was consistent with a larger degree of "stacked" ICG observed in the nanoscan formulation. No tracer-related adverse events were reported. CONCLUSIONS: Based on this retrospective analysis, we can conclude that ICG-99mTc-nanoscan has similar capacity for SN identification as ICG-99mTc-nanocolloid and can safely be implemented in SN procedures.

Chemically Enhanced Immunogenicity of Bacteria by Supramolecular Functionalization with an Adjuvant.

Many pathogens blunt immune responses because they lack immunogenic structural features, which typically results in disease. Here, we show evidence suggesting that pathogen immunogenicity can be chemically enhanced. Using supramolecular host-guest chemistry, we complexed onto the surface of a poorly immunogenic bacterium (Staphylococcus aureus) a TLR7 agonist-based adjuvant. "Adjuvanted" bacteria were readily recognized by macrophages and induced a more pro-inflammatory immunophenotype. Future applications of this concept could yield treatment modalities that bolster the immune system's response to pathogenic microbes.

Feasibility of fluorescence imaging at microdosing using a hybrid PSMA tracer during robot-assisted radical prostatectomy in a large animal model.

BACKGROUND: With the rise of prostate-specific membrane antigen (PSMA) radioguided surgery, which is performed using a microdosing regime, demand for visual target confirmation via fluorescence guidance is growing. While proven very effective for radiotracers, microdosing approaches the detection limit for fluorescence imaging. Thus, utility will be highly dependent on the tracer performance, the sensitivity of the fluorescence camera used, and the degree of background signal. Using a porcine model the ability to perform robot-assisted radical prostatectomy under fluorescence guidance using the bimodal or rather hybrid PSMA tracer (99mTc-EuK-(SO3)Cy5-mas3) was studied, while employing the tracer in a microdosing regime. This was followed by ex vivo evaluation in surgical specimens obtained from prostate cancer patients. RESULTS: T50% blood and T50% urine were reached at 85 min and 390 min, in, respectively, blood and urine. Surgical fluorescence imaging allowed visualization of the prostate gland based on the basal PSMA-expression in porcine prostate. Together, in vivo visualization of the prostate and urinary excretion suggests at least an interval of > 7 h between tracer administration and surgery. Confocal microscopy of excised tissues confirmed tracer uptake in kidney and prostate, which was confirmed with PSMA IHC. No fluorescence was detected in other excised tissues. Tumor identification based on ex vivo fluorescence imaging of human prostate cancer specimens correlated with PSMA IHC. CONCLUSION: Intraoperative PSMA-mediated fluorescence imaging with a microdosing approach was shown to be feasible. Furthermore, EuK-(SO3)Cy5-mas3 allowed tumor identification in human prostate samples, underlining the translational potential of this novel tracer. Trial registration Approval for use of biological material for research purposes was provided by the Translational Research Board of the Netherlands Cancer Institute-Antoni van Leeuwenhoek hospital (NKI-AvL) under reference IRBm19-273 (22/10/2019).

Advancing intraoperative magnetic tracing using 3D freehand magnetic particle imaging.

PURPOSE: Sentinel lymph node biopsy is a routine procedure for nodal staging in penile cancer. Most commonly, this procedure is guided by radioactive tracers, providing various forms of preoperative and intraoperative guidance. This is further extended with fluorescence imaging using hybrid radioactive-fluorescence tracers. Alternatively, a magnetic-based approach has become available using superparamagnetic iron-oxide nanoparticles (SPIONs). This study investigates a novel freehand magnetic particle imaging and navigation modality (fhMPI) for intraoperative localization, along with a hybrid approach, combining magnetic and fluorescence guidance. MATERIALS AND METHODS: The fhMPI set-up was built with a surgical navigation device, optical tracking system and magnetometer probe. A dedicated reconstruction software based on a look-up-table method was used to reconstruct a superficial 3D volume of the SPION distribution in tissue. For fluorescence guidance, indocyanine green (ICG) was added to the SPIONs. The fhMPI modality was characterized in phantoms, ex vivo human skin and in vivo porcine surgery. RESULTS: Phantom and human skin explants illustrated that the current fhMPI modality had a sensitivity of 2.2 × 10-2 mg/mL SPIONs, a resolving power of at least 7 mm and a depth penetration up to 1.5 cm. Evaluation during porcine surgery showed that fhMPI allowed for an augmented reality image overlay of the tracer distribution in tissue, as well as 3D virtual navigation. Besides, using the hybrid approach, fluorescence imaging provided a visual confirmation of localized nodes. CONCLUSION: fhMPI is feasible in vivo, providing 3D imaging and navigation for magnetic nanoparticles in the operating room, expanding the guidance possibilities during magnetic sentinel lymph node procedures. Furthermore, the integration of ICG provides the ability to visually refine and confirm correct localization. Further clinical evaluation should verify these findings in human patients as well.

Intraoperative visualization of nerves using a myelin protein-zero specific fluorescent tracer.

BACKGROUND: Surgically induced nerve damage is a common but debilitating side effect in oncological surgery. With the aim to use fluorescence guidance to enable nerve-sparing interventions in future surgery, a fluorescent tracer was developed that specifically targets myelin protein zero (P0). RESULTS: Truncated homotypic P0 protein-based peptide sequences were C-terminally functionalized with the far-red cyanine dye Cy5. The lead compound Cy5-P0101-125 was selected after initial solubility, (photo)physical and in vitro evaluation (including P0-blocking experiments). Cy5-P0101-125 (KD = 105 ± 17 nM) allowed in vitro and ex vivo P0-related staining. Furthermore, Cy5-P0101-125  enabled in vivo fluorescence imaging of the Sciatic nerve in mice after local intravenous (i.v.) administration and showed compatibility with a clinical fluorescence laparoscope during evaluation in a porcine model undergoing robot-assisted surgery. Biodistribution data revealed that i.v. administered [111In]In-DTPA-P0101-125 does not enter the central nervous system (CNS). CONCLUSION: P0101-125 has proven to be a potent nerve-specific agent that is able to target P0/myelin under in vitro, ex vivo, and in vivo conditions without posing a threat for CNS-related toxicity.

The Design and Preclinical Evaluation of a Single-Label Bimodal Nanobody Tracer for Image-Guided Surgery.

Intraoperative guidance using targeted fluorescent tracers can potentially provide surgeons with real-time feedback on the presence of tumor tissue in resection margins. To overcome the limited depth penetration of fluorescent light, combining fluorescence with SPECT/CT imaging and/or gamma-ray tracing has been proposed. Here, we describe the design and preclinical validation of a novel bimodal nanobody-tracer, labeled using a "multifunctional single attachment point" (MSAP) label, integrating a Cy5 fluorophore and a diethylenetriaminepentaacetic acid (DTPA) chelator into a single structure. After conjugation of the bimodal MSAP to primary amines of the anti-HER2 nanobody 2Rs15d and 111In-labeling of DTPA, the tracer's characteristics were evaluated in vitro. Subsequently, its biodistribution and tumor targeting were assessed by SPECT/CT and fluorescence imaging over 24 h. Finally, the tracer's ability to identify small, disseminated tumor lesions was investigated in mice bearing HER2-overexpressing SKOV3.IP1 peritoneal lesions. [111In]In-MSAP.2Rs15d retained its affinity following conjugation and remained stable for 24 h. In vivo SPECT/CT and fluorescence images showed specific uptake in HER2-overexpressing tumors with low background. High tumor-to-muscle ratios were obtained at 1h p.i. and remained 19-fold on SPECT/CT and 3-fold on fluorescence images over 24 h. In the intraperitoneally disseminated model, the tracer allowed detection of larger lesions via nuclear imaging, while fluorescence enabled accurate removal of submillimeter lesions. Bimodal nuclear/fluorescent nanobody-tracers can thus be conveniently designed by conjugation of a single-molecule MSAP-reagent carrying a fluorophore and chelator for radioactive labeling. Such tracers hold promise for clinical applications.

Cyclodextrin/Adamantane-Mediated Targeting of Inoculated Bacteria in Mice.

Cyclodextrin (CD)-based host-guest interactions with adamantane (Ad) have demonstrated use for functionalizing living cells in vitro. The next step in this supramolecular functionalization approach is to explore the concept to deliver chemical cargo to living cells in vivo, e.g., inoculated bacteria, in order to study their dissemination. We validated this concept in two rodent Staphylococcus aureus models. Bacteria (1 × 108 viable S. aureus) were inoculated by (1) intramuscular injection or (2) intrasplenic injection followed by dissemination throughout the liver. The bacteria were prefunctionalized with 99mTc-UBI29-41-Ad2 (primary vector), which allowed us to both determine the bacterial load and create an in vivo target for the secondary host-vector (24 h post-inoculation). The secondary vector, i.e., chemical cargo delivery system, made use of a 111In-Cy50.5CD9PIBMA39 polymer that was administered intravenously. Bacteria-specific cargo delivery as a result of vector complexation was evaluated by dual-isotope SPECT imaging and biodistribution studies (111In), and by fluorescence (Cy5); these evaluations were performed 4 h post-injection of the secondary vector. Mice inoculated with nonfunctionalized S. aureus and mice without an infection served as controls. Dual-isotope SPECT imaging demonstrated that 111In-Cy50.5CD9PIBMA39 colocalized with 99mTc-UBI29-41-Ad2-labeled bacteria in both muscle and liver. In inoculated muscle, a 2-fold higher uptake level (3.2 ± 1.0%ID/g) was noted compared to inoculation with nonfunctionalized bacteria (1.9 ± 0.4%ID/g), and a 16-fold higher uptake level compared to noninfected muscle (0.2 ± 0.1%ID/g). The hepatic accumulation of the host-vector was nearly 10-fold higher (27.1 ± 11.1%ID/g) compared to the noninfected control (2.7 ± 0.3%ID/g; p < 0.05). Fluorescence imaging of the secondary vector corroborated SPECT-imaging and biodistribution findings. We have demonstrated that supramolecular host-guest complexation can be harnessed to achieve an in vivo cargo delivery strategy, using two different bacterial models in soft tissue and liver. This proof-of-principle study paves a path toward developing innovative drug delivery concepts via cell functionalization techniques.

The helminth glycoprotein omega-1 improves metabolic homeostasis in obese mice through type 2 immunity-independent inhibition of food intake.

Type 2 immunity plays an essential role in the maintenance of metabolic homeostasis and its disruption during obesity promotes meta-inflammation and insulin resistance. Infection with the helminth parasite Schistosoma mansoni and treatment with its soluble egg antigens (SEA) induce a type 2 immune response in metabolic organs and improve insulin sensitivity and glucose tolerance in obese mice, yet, a causal relationship remains unproven. Here, we investigated the effects and underlying mechanisms of the T2 ribonuclease omega-1 (ω1), one of the major S mansoni immunomodulatory glycoproteins, on metabolic homeostasis. We show that treatment of obese mice with plant-produced recombinant ω1, harboring similar glycan motifs as present on the native molecule, decreased body fat mass, and improved systemic insulin sensitivity and glucose tolerance in a time- and dose-dependent manner. This effect was associated with an increase in white adipose tissue (WAT) type 2 T helper cells, eosinophils, and alternatively activated macrophages, without affecting type 2 innate lymphoid cells. In contrast to SEA, the metabolic effects of ω1 were still observed in obese STAT6-deficient mice with impaired type 2 immunity, indicating that its metabolic effects are independent of the type 2 immune response. Instead, we found that ω1 inhibited food intake, without affecting locomotor activity, WAT thermogenic capacity or whole-body energy expenditure, an effect also occurring in leptin receptor-deficient obese and hyperphagic db/db mice. Altogether, we demonstrate that while the helminth glycoprotein ω1 can induce type 2 immunity, it improves whole-body metabolic homeostasis in obese mice by inhibiting food intake via a STAT6-independent mechanism.

Multi-wavelength fluorescence imaging with a da Vinci Firefly-a technical look behind the scenes.

The field of fluorescence-guided surgery builds on colored fluorescent tracers that have become available for different clinical applications. Combined use of complementary fluorescent emissions can allow visualization of different anatomical structures (e.g. tumor, lymphatics and nerves) in the same patient. With the aim to assess the requirements for multi-color fluorescence guidance under in vivo conditions, we thoroughly characterized two FDA-approved laparoscopic Firefly camera systems available on the da Vinci Si or da Vinci Xi surgical robot. In this process, we studied the cameras' performance with respect to the photophysical properties of the FDA-approved dyes Fluorescein and ICG. Our findings indicate that multi-wavelength fluorescence imaging of Fluorescein and ICG is possible using clinical-grade fluorescence laparoscopes, but critical factors for success include the photophysical dye properties, imaging system performance and the amount of accumulated dye. When comparing the camera performance, the Xi system provided more effective excitation (adaptions in the light source) and higher detection sensitivity (chip-on-a-tip and/or enhanced image processing) for both Fluorescein and ICG. Both systems can readily be used for multi-wavelength fluorescence imaging of Fluorescein and ICG under clinically relevant conditions. With that, another step has been made towards the routine implementation of multi-wavelength image-guided surgery concepts.

Multi-Wavelength Fluorescence in Image-Guided Surgery, Clinical Feasibility and Future Perspectives.

With the rise of fluorescence-guided surgery, it has become evident that different types of fluorescence signals can provide value in the surgical setting. Hereby a different range of targets have been pursued in a great variety of surgical indications. One of the future challenges lies in combining complementary fluorescent readouts during one and the same surgical procedure, so-called multi-wavelength fluorescence guidance. In this review we summarize the current clinical state-of-the-art in multi-wavelength fluorescence guidance, basic technical concepts, possible future extensions of existing clinical indications and impact that the technology can bring to clinical care.

Fluorescence background quenching as a means to increase Signal to Background ratio - a proof of concept during Nerve Imaging.

Introduction: Adequate signal to background ratios are critical for the implementation of fluorescence-guided surgery technologies. While local tracer administrations help to reduce the chance of systemic side effects, reduced spatial migration and non-specific tracer diffusion can impair the discrimination between the tissue of interest and the background. To combat background signals associated with local tracer administration, we explored a pretargeting concept aimed at quenching non-specific fluorescence signals. The efficacy of this concept was evaluated in an in vivo neuronal tracing set-up. Methods: Neuronal tracing was achieved using a wheat germ agglutinin (WGA) lectin. functionalized with an azide-containing Cy5 dye (N3-Cy5-WGA). A Cy7 quencher dye (Cy7-DBCO) was subsequently used to yield Cy7-Cy5-WGA, a compound wherein the Cy5 emission is quenched by Förster resonance energy transfer to Cy7. The photophysical properties of N3-Cy5-WGA and Cy7-Cy5-WGA were evaluated together with deactivation kinetics in situ, in vitro (Schwannoma cell culture), ex vivo (muscle tissue from mice; used for dose optimization), and in vivo (nervus ischiadicus in THY-1 YFP mice).Results:In situ, conjugation of Cy7-DBCO to N3-Cy5-WGA resulted in >90% reduction of the Cy5 fluorescence signal intensity at 30 minutes after addition of the quencher. In cells, pretargeting with the N3-Cy5-WGA lectin yielded membranous staining, which could efficiently be deactivated by Cy7-DBCO over the course of 30 minutes (91% Cy5 signal decrease). In ex vivo muscle tissue, administration of Cy7-DBCO at the site where N3-Cy5-WGA was injected induced 80-90% quenching of the Cy5-related signal after 10-20 minutes, while the Cy7-related signal remained stable over time. In vivo,Cy7-DBCO effectively quenched the non-specific background signal up to 73% within 5 minutes, resulting in a 50% increase in the signal-to-background ratio between the nerve and injection site. Conclusion: The presented pretargeted fluorescence-quenching technology allowed fast and effective reduction of the background signal at the injection site, while preserving in vivo nerve visualization. While this proof-of-principle study was focused on imaging of nerves using a fluorescent WGA-lectin, the same concept could in the future also apply to applications such as sentinel node imaging.

A Supramolecular Platform Technology for Bacterial Cell Surface Modification.

In an era of antimicrobial resistance, a better understanding of the interaction between bacteria and the sentinel immune system is needed to discover new therapeutic targets for combating bacterial infectious disease. Sentinel immune cells such as macrophages phagocytose intact bacteria and thereby initiate ensuing immune responses. The bacterial surface composition is a key element that determines the macrophage signaling. To study the role of the bacterial cell surface composition in immune recognition, we developed a platform technology for altering bacterial surfaces in a controlled manner with versatile chemical scaffolds. We show that these scaffolds are efficiently loaded onto both Gram-positive and -negative bacteria and that their presence does not impair the capacity of monocyte-derived macrophages to phagocytose bacteria and subsequently signal to other components of the immune system. We believe this technology thus presents a useful tool to study the role of bacterial cell surface composition in disease etiology and potentially in novel interventions utilizing intact bacteria for vaccination.

Hybrid Tracers Based on Cyanine Backbones Targeting Prostate-Specific Membrane Antigen: Tuning Pharmacokinetic Properties and Exploring Dye-Protein Interaction.

Prostate cancer surgery is currently being revolutionized by the use of prostate-specific membrane antigen (PSMA)-targeted radiotracers, for example, 99mTc-labeled PSMA tracer analogs for radioguided surgery. The purpose of this study was to develop a second-generation 99mTc-labeled PSMA-targeted tracer incorporating a fluorescent dye. Methods: Several PSMA-targeted hybrid tracers were synthesized: glutamic acid-urea-lysine (EuK)-Cy5-mas3, EuK-(SO3)Cy5-mas3, EuK-Cy5(SO3)-mas3, EuK-(Ar)Cy5-mas3, and EuK-Cy5(Ar)-mas3; the Cy5 dye acts as a functional backbone between the EuK targeting vector and the 2-mercaptoacetyl-seryl-seryl-seryl (mas3) chelate to study the dye's interaction with PSMA's amphipathic entrance funnel. The compounds were evaluated for their photophysical and chemical properties and PSMA affinity. After radiolabeling with 99mTc, we performed in vivo SPECT imaging, biodistribution, and fluorescence imaging on BALB/c nude mice with orthotopically transplanted PC346C tumors. Results: The dye composition influenced the photophysical properties (brightness range 0.3-1.5 × 104 M-1 × cm-1), plasma protein interactions (range 85.0% ± 2.3%-90.7% ± 1.3% bound to serum, range 76% ± 0%-89% ± 6% stability in serum), PSMA affinity (half-maximal inhibitory concentration [IC50] range 19.2 ± 5.8-175.3 ± 61.1 nM) and in vivo characteristics (tumor-to-prostate and tumor-to-muscle ratios range 0.02 ± 0.00-154.73 ± 28.48 and 0.46 ± 0.28-5,157.50 ± 949.17, respectively; renal, splenic, and salivary retention). Even though all tracer analogs allowed tumor identification with SPECT and fluorescence imaging, 99mTc-EuK-(SO3)Cy5-mas3 had the most promising properties (e.g., half-maximal inhibitory concentration, 19.2 ± 5.8, tumor-to-muscle ratio, 5,157.50 ± 949.17). Conclusion: Our findings demonstrate the intrinsic integration of a fluorophore in the pharmacophore in PSMA-targeted small-molecule tracers. In this design, having 1 sulfonate on the indole moiety adjacent to EuK (99mTc-EuK-(SO3)Cy5-mas3) yielded the most promising tracer candidate for imaging of PSMA.

Size and affinity kinetics of nanobodies influence targeting and penetration of solid tumours.

A compound's intratumoural distribution is an important determinant for the effectiveness of molecular therapy or imaging. Antibodies (Abs), though often used in the design of targeted compounds, struggle to achieve a homogenous distribution due to their large size and bivalent binding mechanism. In contrast, smaller compounds like nanobodies (Nbs) are expected to distribute more homogenously, though this has yet to be demonstrated in vivo at the microscopic level. We propose an intravital approach to evaluate the intratumoural distribution of different fluorescently labeled monomeric and dimeric Nb tracers and compare this with a monoclonal antibody (mAb). Monomeric and dimeric formats of the anti-HER2 (2Rb17c and 2Rb17c-2Rb17c) and control (R3B23 and R3B23-R3B23) Nb, as well as the dimeric monovalent Nb 2Rb17c-R3B23 were generated and fluorescently labeled with a Cy5 fluorophore. The mAb trastuzumab-Cy5 was also prepared. Whole-body biodistribution of all constructs was investigated in mice bearing subcutaneous xenografts (HER2+ SKOV3) using in vivo epi-fluorescence imaging. Next, for intravital experiments, GFP-expressing SKOV3 cells were grown under dorsal window chambers on athymic nude mice (n = 3/group), and imaged under a fluorescence stereo microscope immediately after intravenous injection of the tracers. Consecutive fluorescence images within the tumour were acquired over the initial 20 min after injection and later, single images were taken at 1, 3 and 24 h post-injection. Additionally, two-photon microscopy was used to investigate the colocalization of GFP (tumour cells) and Cy5 fluorescence (tracers) at higher resolution. Whole-body images showed rapid renal clearance of all Nbs, and fast tumour targeting for the specific Nbs. Specific tumour uptake of the mAb could only be clearly distinguished from background after several hours. Intravital imaging revealed that monomeric Nb tracers accumulated rapidly and distributed homogenously in the tumour mere minutes after intravenous injection. The dimeric compounds initially achieved lower fluorescence intensities than the monomeric. Furthermore, whereas the HER2-specific dimeric bivalent compound remained closely associated to the blood vessels over 24 h, the HER2-specific dimeric monovalent tracer achieved a more homogenous tumour distribution from 1 h post-injection onwards. Non-specific tracers were not retained in the tumour. Trastuzumab had the most heterogenous intratumoural distribution of all evaluated compounds, while -due to the long blood retention- achieving the highest overall tumour uptake at 24 h post-injection. In conclusion, monomeric Nbs very quickly and homogenously distribute through tumour tissue, at a rate significantly greater than dimeric Nbs and mAbs. This underlines the potential of monomeric Nb tracers and therapeutics in molecular imaging and targeted therapies.

Image-Guided Surgery: Are We Getting the Most Out of Small-Molecule Prostate-Specific-Membrane-Antigen-Targeted Tracers?

Expressed on virtually all prostate cancers and their metastases, the transmembrane protein prostate-specific membrane antigen (PSMA) provides a valuable target for the imaging of prostate cancer. Not only does PSMA provide a target for noninvasive diagnostic imaging, e.g., PSMA-positron emission tomography (PSMA-PET), it can also be used to guide surgical resections of PSMA-positive lesions. The latter characteristic has led to the development of a plethora of PSMA-targeted tracers, i.e., radiolabeled, fluorescent, or hybrid. With image-guided surgery applications in mind, this review discusses these compounds based on clinical need. Here, the focus is on the chemical aspects (e.g., imaging label, spacer moiety, and targeting vector) and their impact on in vitro and in vivo tracer characteristics (e.g., affinity, tumor uptake, and clearance pattern).

Click Chemistry in the Design and Production of Hybrid Tracers.

Hybrid tracers containing both fluorescent and radioactive imaging labels have demonstrated clinical potential during sentinel lymph node procedures. To combine these two labels on a single targeting vector that allows tumor-targeted imaging, end-labeling strategies are often applied. For αvß3-integrin-targeting hybrid tracers, providing an excellent model for evaluating tracer development strategies, end-labeling-based synthesis provides a rather cumbersome synthesis strategy. Hence, the aim of this study was to investigate the use of heterobifunctional cyanine dyes in a click-chemistry-based synthesis strategy for RGD-based hybrid tracers. The triazole-based hybrid tracers DTPA.DBCO.N 3 (SO 3 )-Cy5-c[RGDyK] and DTPA.BCN.N 3 (SO 3 )-Cy5-c[RGDyK] were obtained in fewer steps than DTPA-Lys(Cy5(SO 3 )methyl)-Cys-c[RGDyK] and had partition coefficients of log P (o/w) = -2.55 ± 0.10, -1.45 ± 0.03, and -2.67 ± 0.12, respectively. Both tracers were chemically stable, and the brightnesses of DTPA.DBCO.N 3 (SO 3 )-Cy5-c[RGDyK] and DTPA.BCN.N 3 (SO 3 )-Cy5-c[RGDyK] were, respectively, 23 × 103 and 40 × 103 M-1 cm-1; lower than that of the reference tracer DTPA-Lys(Cy5(SO 3 )methyl)-Cys-c[RGDyK] (50 × 103 M-1 cm-1). Assessment of serum protein binding revealed no statistically significant difference (44 ± 2 and 40 ± 2% bound for DTPA.DBCO.N 3 (SO 3 )-Cy5-c[RGDyK] and DTPA.BCN.N 3 (SO 3 )-Cy5-c[RGDyK], respectively; 36 ± 5% bound for DTPA-Lys(Cy5(SO 3 )methyl)-Cys-c[RGDyK]; p > 0.05). DTPA.DBCO.N 3 (SO 3 )-Cy5-c[RGDyK] (K D = 17.5 ± 6.0) had a statistically significantly higher affinity than the reference compound DTPA-Lys(Cy5(SO 3 )methyl)-Cys-c[RGDyK] (K D = 30.3 ± 5.7; p < 0.0001), but DTPA.BCN.N 3 (SO 3 )-Cy5-c[RGDyK] had a statistically significantly lower affinity (K D = 76.5 ± 18.3 nM; p < 0.0001). Both [ 111 In]DTPA.DBCO.N 3 (SO 3 )-Cy5-c[RGDyK] and [ 111 In]DTPA.BCN.N 3 (SO 3 )-Cy5-c[RGDyK] enabled in vivo visualization of the 4T1 tumor via fluorescence and single-photon emission computed tomography (SPECT) imaging. Biodistribution data (% ID/g) revealed a significant increase in nonspecific uptake in the kidney, liver, and muscle for both [ 111 In]DTPA.DBCO.N 3 (SO 3 )-Cy5-c[RGDyK] and [ 111 In]DTPA.BCN.N 3 (SO 3 )-Cy5-c[RGDyK]. As a result of the higher background activity, the tumor-to-background ratio of the click-labeled RGD analogues was twofold lower compared to the end-labeled reference compound. The use of click chemistry labeling did not yield a pronounced negative effect on serum protein binding, in vitro stability, and receptor affinity; and tumors could still be visualized using SPECT and fluorescence imaging. However, quantitative in vivo biodistribution data suggest that the triazole and strained cyclooctyne moieties associated with this type of click chemistry negatively influence the pharmacokinetics of RGD peptides. Nevertheless, the design might still hold promise for other targets/targeting moieties.

High-resolution imaging and single-cell analysis via laser ablation-inductively coupled plasma-mass spectrometry for the determination of membranous receptor expression levels in breast cancer cell lines using receptor-specific hybrid tracers.

This work evaluates the possibility of placement of high-resolution imaging and single-cell analysis via laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) within precision medicine by assessing the suitability of LA-ICP-MS as a micro-analytical technique for the localization and quantification of membranous receptors in heterogeneous cell samples that express both the membrane-bound receptors C-X-C chemokine receptor type 4 (CXCR4) and epidermal growth factor receptor (EGFR). Staining of the breast cancer cell lines MDA-MB-231 X4 and MDA-MB-468 was achieved using receptor-specific hybrid tracers, containing both a fluorophore and a DTPA single-lanthanide chelate. Prior to LA-ICP-MS imaging, fluorescence confocal microscopy (FCM) imaging was performed to localize the receptors, hereby enabling direct comparison. Based on the different expression levels of CXCR4 and EGFR, a distinction could be made between the cell lines using both imaging modalities. Furthermore, FCM and LA-ICP-MS demonstrated complementary characteristics, as a more distinct discrimination could be made between both cell lines based on the EGFR-targeting hybrid tracer via LA-ICP-MS, due to the intrinsic CXCR4-related green fluorescent protein (GFP) signal present in the MDA-MB-231 X4 cells. Employing state-of-the-art LA-ICP-MS instrumentation in bidirectional area scanning mode for sub-cellular imaging of MDA-MB-231 X4 cells enabled the specific binding of the CXCR4-targeting hybrid tracer to the cell membrane to be clearly demonstrated. The stretching of cells over the glass substrate led to a considerably higher signal response for pixels at the cell edges, relative to the more central pixels. The determination of the expression levels of CXCR4 and EGFR for the MDA-MB-468 cell line was performed using LA-ICP-MS single-cell analysis (sc-LA-ICP-MS) and external calibration, based on the quantitative ablation of Ho-spiked dried gelatin droplet standards. Additionally, a second calibration approach was applied based on spot ablation of highly homogeneous dried gelatin gels in combination with the determination of the ablated volume using atomic force microscopy (AFM) and yielded results which were in good agreement with the expression levels determined via flow cytometry (FC) and mass cytometry (MC). Hybrid tracers enable a direct comparison between (i) FCM and LA-ICP-MS imaging for the evaluation of the microscopic binding pattern and between (ii) FC, MC and sc-LA-ICP-MS for the quantification of receptor expression levels in single cells.

Multimodal Tracking of Controlled Staphylococcus aureus Infections in Mice.

There is a need to develop diagnostic and analytical tools that allow noninvasive monitoring of bacterial growth and dissemination in vivo. For such cell-tracking studies to hold translational value to controlled human infections, in which volunteers are experimentally colonized, they should not require genetic modification, and they should allow tracking over a number of replication cycles. To gauge if an antimicrobial peptide tracer, 99mTc-UBI29-41-Cy5, which contains both a fluorescent and a radioactive moiety, could be used for such in vivo bacterial tracking, we performed longitudinal imaging of a thigh-muscle infection with 99mTc-UBI29-41-Cy5-labeled Staphylococcus aureus. Mice were imaged using SPECT and fluorescence-imaging modalities at various intervals during a 28 h period. Biodistribution analyses were performed to quantitate radioactivity in the abscess and other tissues. SPECT and fluorescence imaging in mice showed clear retention of the 99mTc-UBI29-41-Cy5-labeled bacteria following inoculation in the thigh muscle. Despite bacterial replication, the signal intensity in the abscess only modestly decreased within a 28 h period: 52% of the total injected radioactivity per gram of tissue (%ID/g) at 4 h postinfection (pi) versus 44%ID/g at 28 h pi (15% decrease). After inoculation, a portion of the bacteria disseminated from the abscess, and S. aureus cultures were obtained from radioactive urine samples. Bacterial staining with 99mTc-UBI29-41-Cy5 allowed noninvasive bacterial-cell tracking during a 28 h period. Given the versatility of the presented bacterial-tracking method, we believe that this concept could pave the way for precise imaging capabilities during controlled-human-infection studies.