Tissue Clearance Research Articles

Sialic acid and PirB are not required for targeting of neural circuits by neurotropic mammalian orthoreovirus.

Serotype 3 (T3) strains of mammalian orthoreovirus (reovirus) spread to the central nervous system to infect the brain and cause lethal encephalitis in newborn mice. Although reovirus targets several regions in the brain, susceptibility to infection is not uniformly distributed. The neuronal subtypes and anatomic sites targeted throughout the brain are not precisely known. Reovirus binds several attachment factors and entry receptors, including sialic acid (SA)-containing glycans and paired immunoglobulin-like receptor B (PirB). While these receptors are not required for infection of some types of neurons, reovirus engagement of these receptors can influence neuronal infection in certain contexts. To identify patterns of T3 neurotropism, we used microbial identification after passive tissue clearance and hybridization chain reaction to stain reovirus-infected cells throughout intact, optically transparent brains of newborn mice. Three-dimensional reconstructions revealed in detail the sites targeted by reovirus throughout the brain volume, including dense infection of the midbrain and hindbrain. Using reovirus mutants incapable of binding SA and mice lacking PirB expression, we found that neither SA nor PirB is required for the infection of various brain regions. However, SA may confer minor differences in infection that vary by region. Collectively, these studies indicate that many regions in the brain of newborn mice are susceptible to reovirus and that patterns of reovirus infection are not dependent on reovirus receptors SA and PirB.IMPORTANCENeurotropic viruses invade the central nervous system (CNS) and target various cell types to cause disease manifestations, such as meningitis, myelitis, or encephalitis. Infections of the CNS are often difficult to treat and can lead to lasting sequelae or death. Mammalian orthoreovirus (reovirus) causes age-dependent lethal encephalitis in many young mammals. Reovirus infects neurons in several different regions of the brain. However, the complete pattern of CNS infection is not understood. We found that reovirus targets almost all regions of the brain and that patterns of tropism are not dependent on receptors sialic acid and paired immunoglobulin-like receptor B. These studies confirm that two known reovirus receptors do not completely explain the cell types infected in brain tissue and establish strategies that can be used to understand complete patterns of viral tropism in an intact brain.

177Lu-labeling of nuclear localization sequence (NLS)-grafted HER2-receptor affine peptide

Tagging of cell permeable nuclear localization sequence (NLS) with receptor targeting peptide vectors is an attractive strategy for selectively targeted translocation of therapeutic cargoes. The present study aimed at grafting nuclear localization sequence (NLS) onto breast cancer targeting rL-A9 peptide. Molecular docking analysis revealed higher binding affinity of the peptide, DOTA-NLS-rL-A9 (−26.1 kJ/mol) towards HER2 receptor in comparison to DOTA-rL-A9 peptide (−22.2 kJ/mol). Confocal microscopy data suggested significantly enhanced cellular internalization of NLS-tagged peptide. The engineered HER2-selective, DOTA-NLS-rL-A9 peptide scaffold was radiolabeled with Lu-177 for intracellular delivery of the theranostic radionuclide into tumor cells. [177Lu]Lu-DOTA-NLS-rL-A9 exhibited significantly enhanced binding affinity (4.58 ± 1.77 nM) towards human breast carcinoma SKBR3 cells and cellular internalization (85 % at 24 h) compared to its original analog, [177Lu]Lu-DOTA-rL-A9. In vivo biodistribution studies showed consistent retention of [177Lu]Lu-DOTA-NLS-rL-A9 in the tumor with negligible washout of radioactivity (∼4.1 % ID/g at 48 h). Prolonged tumor activity with rapid off-target tissue clearance resulted in significantly high tumor-to-background ratios. The radiopeptide, [177Lu]Lu-DOTA-NLS-rL-A9 thus, being precisely confined into HER2-expressing tumor cells and exhibiting favourable pharmacokinetic features is an efficient candidate for further screening.

FAP Radioligand Linker Optimization Improves Tumor Dose and Tumor-to-Healthy Organ Ratios in 4T1 Syngeneic Model.

Fibroblast activation protein (FAP) has attracted considerable attention as a possible target for the radiotherapy of solid tumors. Unfortunately, initial efforts to treat solid tumors with FAP-targeted radionuclides have yielded only modest clinical responses, suggesting that further improvements in the molecular design of FAP-targeted radiopharmaceutical therapies (RPT) are warranted. In this study, we report several advances on the previously described FAP6 radioligand that increase tumor retention and accelerate healthy tissue clearance. Seven FAP6 derivatives with different linkers or albumin binders were synthesized, radiolabeled, and investigated for their effects on binding and cellular uptake. The radioligands were then characterized in 4T1 tumor-bearing Balb/c mice using both single-photon emission computed tomography (SPECT) and ex vivo biodistribution analyses to identify the conjugate with the best tumor retention and tumor-to-healthy organ ratios. The results reveal an optimized FAP6 radioligand that exhibits efficacy and safety properties that potentially justify its translation into the clinic.

P-693 Three-dimensional modeling of follicular microenvironment dynamics in the human adult ovary

Abstract Study question How does the ovarian microenvironment, involving neural fibers, vascular networks, and immune cells, interplay with follicular dynamics in the human adult ovary? Summary answer Neural fibers growing into the ovarian cortex and vascular network expansion were associated with primordial follicle activation and growth. Macrophages associated with antral follicular atresia. What is known already The ovarian follicles, responsible for preserving and supplying oocytes, play a crucial role in fertility, with their fate influenced by the ovarian microenvironment. However, the three-dimensional (3D), irregular, and dynamic structural changes of the ovarian microenvironment are difficult to interpret using two-dimensional analysis. 3D-imaging technologies have been developed to reveal intricate structural and functional relationships particularly in rodents, but these approaches have not been widely adopted for human ovarian research. Study design, size, duration Our study aims to explore the follicular microenvironment dynamics in the human adult ovary. In this work, we optimized whole-mount immunofluorescence using TUBB3, PODXL, PECAM1, ACTA2 and CD68, and conducted 3D imaging on intact adult ovarian tissue, offering a visualization of the 3D structures and dynamic changes within ovarian follicles, neural fibers, vascular networks, and immune cells in human adult ovary at the single-cell level. Participants/materials, setting, methods The human ovarian samples were collected from donors (N = 5) who underwent oophorectomy after testosterone treatment. Part of the intact ovary, including cortex and medulla, was fixed and used for whole-mount immunofluorescence using markers for neural fibers (TUBB3), blood vessels (PODXL, PECAM1, ACTA2), and immune cells (CD68). Subsequently, an enhanced iDiSCO method was employed for tissue clearance. Finally, 3D images were acquired using a lightsheet microscope, followed by image analysis and 3D reconstruction using Imaris software. Main results and the role of chance Our results showed that TUBB3-labeled nerve fibers predominantly distributed in the ovarian cortex, with few presented in the medullary region. Notably, their presence surrounding secondary and antral follicles was limited. We observed a rich distribution of PODXL-labeled capillaries surrounding primordial and primary follicles in the cortical layer of the adult ovary. As follicles progressed to the secondary stage, PECAM1-labeled vasculature contributed to the construction of a vascular network enveloping the secondary follicles and connecting extensively to larger-caliber arteries containing (ACTA2+) smooth muscle cells. In antral follicles, large larger-caliber arteries were also detected, maintaining proximity to the theca cell layer. Interestingly, in cases of follicular atresia, vascular invasion within the follicular cavity was observed. Additionally, clusters of CD68+ macrophages exhibited a scattered distribution around the follicles at various stages, with a significant accumulation in atretic small-antral follicles. Limitations, reasons for caution Our study was conducted on ovaries from a limited number of donors after gender-affirming surgery. Although we aimed to provide insights into the ovarian microenvironment, we were so far unable to obtain ovaries with large antral follicles, ovulatory follicles and the formation of the corpus luteum. Wider implications of the findings The views provided by 3D-image analysis of the human ovary will augment the current knowledge of the complex interactions between the follicles and the ovarian microenvironment. 3D ovarian mapping will also provide a roadmap for generating oocytes in vitro for fertility preservation and ovarian disease modelling. Trial registration number N/A.

Supramolecular self-assembling hydrogel based on imidazole/d-sorbitol deep eutectic solvent for tissue clearance

Embedding and tissue clearing with high refractive index reagents are essential for 3D tissue imaging. However, the current liquid-based clearing condition and dye environment suffer from solvent evaporation and photo-bleaching, causing difficulties in maintain the tissue optical and fluorescent features. In traditional embedding technique, paraffin is highly astigmatic and paraffin-tissue complex is opaque; CLARITY series of gel-type tissue transparency methods usually achieve cross-linking of tissue and gel network first, and then active or passive degreasing by using SDS, TritonX-100 and so on, and finally, the tissue achieves a transparent state by refractive index matching. Based on high permeability of deep eutectic solvents (DES) and the optical transparency potential of polyols, this study developed a DES self-assembled hydrogel-based copolymer to embed mouse tissue for clearing and imaging prepared with imidazole and d-sorbitol. In addition to preserving the high optic transparency of the tissue, the DES hydrogel system is compatible with multiple fluorescence labeling, particularly lipophilic dyes. This DES hydrogel system provides a unique opportunity for simultaneous tissue embedding and clearing. The self-assembled hydrogel achieves 3D imaging at a solid state, and 2D images can be obtained through slicing after 3D imaging of complete organs. Due to its temperature dependent hydrogel properties, the hydrogel-tissue complex achieves gel-sol transition and allows tissue reuse. This transparent and hydrogel condition provides a friendly tissue and cellular environment to facilitate high resolution 3D imaging, preservation, transfer, and sharing among laboratories to investigate the morphologies of interest in experimental and clinical conditions.

The physiological basis of renal nuclear medicine.

Renal physiology underpins renal nuclear medicine, both academic and clinical. Clearance, an important concept in renal physiology, comprises tissue uptake rate of tracer (tissue clearance), disappearance rate from plasma (plasma clearance), appearance rate in urine (urinary clearance) and disappearance rate from tissue. In clinical research, steady-state plasma clearances of para-amino-hippurate and inulin have been widely used to measure renal blood flow (RBF) and glomerular filtration rate (GFR), respectively. Routinely, GFR is measured at non-steady state as plasma clearance of a filtration agent, such as technetium-99m diethylenetriaminepentaacetic acid. Scaled to three-dimensional whole body metrics rather than body surface area, GFR in women is higher than in men but declines faster with age. Age-related decline is predominantly from nephron loss. Tubular function determines parenchymal transit time, which is important in renography, and the route of uptake of technetium-99m dimercaptosuccinic acid, which is via filtration. Resistance to flow is defined according to the pressure-flow relationship but in renography, only transit time can be measured, which, being equal to urine flow divided by collecting system volume, introduces further uncertainty because the volume is also unmeasurable. Tubuloglomerular feedback governs RBF and GFR, is regulated by the macula densa, mediated by adenosine and renin, and can be manipulated with proximal tubular sodium-glucose cotransporter-2 inhibitors. Other determinants of renal haemodynamics include prostaglandins, nitric oxide and dopamine, while protein meal and amino acid infusion are used to measure renal functional reserve. In conclusion, for measuring renal responses to exogenous agents, steady-state para-amino-hippurate and inulin clearances should be replaced with rubidium-82 and gallium-68 EDTA for measuring RBF and GFR.

Ischemia-Reperfusion Injury in Kidney Transplantation: Mechanisms and Potential Therapeutic Targets.

Kidney transplantation offers a longer life expectancy and a better quality of life than dialysis to patients with end-stage kidney disease. Ischemia-reperfusion injury (IRI) is thought to be a cornerstone in delayed or reduced graft function and increases the risk of rejection by triggering the immunogenicity of the organ. IRI is an unavoidable event that happens when the blood supply is temporarily reduced and then restored to an organ. IRI is the result of several biological pathways, such as transcriptional reprogramming, apoptosis and necrosis, innate and adaptive immune responses, and endothelial dysfunction. Tubular cells mostly depend on fatty acid (FA) β-oxidation for energy production since more ATP molecules are yielded per substrate molecule than glucose oxidation. Upon ischemia-reperfusion damage, the innate and adaptive immune system activates to achieve tissue clearance and repair. Several cells, cytokines, enzymes, receptors, and ligands are known to take part in these events. The complement cascade might start even before organ procurement in deceased donors. However, additional experimental and clinical data are required to better understand the pathogenic events that take place during this complex process.

Combining BNCT with carbonic anhydrase inhibition for mesothelioma treatment: Synthesis, in vitro, in vivo studies of ureidosulfamido carboranes

Mesothelioma is a malignant neoplasm of mesothelial cells caused by exposure to asbestos. The average survival time after diagnosis is usually nine/twelve months. A multi-therapeutic approach is therefore required to treat and prevent recurrence. Boronated derivatives containing a carborane cage, a sulfamido group and an ureido functionality (CA-USF) have been designed, synthesised and tested, in order to couple Boron Neutron Capture Therapy (BNCT) and the inhibition of Carbonic Anhydrases (CAs), which are overexpressed in many tumours. In vitro studies showed greater inhibition than the reference drug acetazolamide (AZ). To increase solubility in aqueous media, CA-USFs were used as inclusion complexes of hydroxypropyl β-cyclodextrin (HP-β-CD) in all the inhibition and cell experiments. BNCT experiments carried out on AB22 (murine mesothelioma) cell lines showed a marked inhibition of cell proliferation by CA-USFs, and in one case a complete inhibition of proliferation twenty days after neutron irradiation. Finally, in vivo neutron irradiation experiments on a mouse model of mesothelioma demonstrated the efficiency of combining CA IX inhibition and BNCT treatment. Indeed, a greater reduction in tumour mass was observed in treated mice compared to untreated mice, with a significant higher effect when combined with BNCT. For in vivo experiments CA-USFs were administered as inclusion complexes of higher molecular weight β-CD polymers thus increasing the selective extravasation into tumour tissue and reducing clearance. In this way, boron uptake was maximised and CA-USFs demonstrated to be in vivo well tolerated at a therapeutic dose. The therapeutic strategy herein described could be expanded to other cancers with increased CA IX activity, such as melanoma, glioma, and breast cancer.

Abstract 6028: Preferential tumor-to-normal tissue biodistribution and single-dose efficacy with ABD147, a DLL3-targeted engineered antibody-based radiotherapeutic, in preclinical small cell lung cancer models

Abstract Targeted radiotherapies represent an emerging treatment modality for aggressive cancers with limited treatment options, such as small cell lung cancer (SCLC). ABD147 is a Delta Ligand 3 (DLL3)-targeted antibody conjugate designed to carry and deliver cytotoxic radioactive isotopes to DLL3-expressing tumor cells. DLL3 is commonly expressed on the cell surface of neuroendocrine cancer cells, including SCLC, but has limited and predominantly intracellular expression in non-malignant tissues. The ABD147 antibody specifically binds human DLL3 with high affinity and is internalized by DLL3-expressing cancer cells. To minimize radiation exposure to normal tissue, ABD147 has been engineered to clear quickly from the blood compartment. Using indium-111 to determine ABD147 pharmacokinetics and biodistribution in mice, we demonstrate rapid blood and normal tissue clearance of 111In-ABD147, as designed, following a single intravenous dose administration. However, 111In-ABD147 retains high tumor accumulation (activity concentration of up to 30% ID/g) in xenograft mouse models of SCLC despite low DLL3 surface antigen expression on tumor cells (≤ 3000 copies). 111In-ABD147 shows a favorable tumor-to-normal tissue distribution with predominant liver clearance. Following ABD147 therapeutic radioisotope delivery of actinium-225 or lutetium-177 (225Ac-ABD147, 177Lu-ABD147), we demonstrate single-dose tumor regression and dose-dependent sustained anti-tumor efficacy, corresponding to an extension of survival out to 84 days in multiple SCLC xenograft models. In summary, ABD147 can preferentially deliver radionuclides to DLL3-expressing tumor tissue while substantially reducing the systemic radioactive exposure typical of conventional IgG radioconjugates. Following promising results in preclinical models, including potent anti-tumor activity, 225Ac-ABD147 is now progressing into clinical development. Citation Format: Iva Kulic, Etienne S. Melese, Emma Cummins, Alex Mandel, Raja Viswas, Michael Abrams, Adam Judge. Preferential tumor-to-normal tissue biodistribution and single-dose efficacy with ABD147, a DLL3-targeted engineered antibody-based radiotherapeutic, in preclinical small cell lung cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6028.

VEGF-C prophylaxis favors lymphatic drainage and modulates neuroinflammation in a stroke model.

Meningeal lymphatic vessels (MLVs) promote tissue clearance and immune surveillance in the central nervous system (CNS). Vascular endothelial growth factor-C (VEGF-C) regulates MLV development and maintenance and has therapeutic potential for treating neurological disorders. Herein, we investigated the effects of VEGF-C overexpression on brain fluid drainage and ischemic stroke outcomes in mice. Intracerebrospinal administration of an adeno-associated virus expressing mouse full-length VEGF-C (AAV-mVEGF-C) increased CSF drainage to the deep cervical lymph nodes (dCLNs) by enhancing lymphatic growth and upregulated neuroprotective signaling pathways identified by single nuclei RNA sequencing of brain cells. In a mouse model of ischemic stroke, AAV-mVEGF-C pretreatment reduced stroke injury and ameliorated motor performances in the subacute stage, associated with mitigated microglia-mediated inflammation and increased BDNF signaling in brain cells. Neuroprotective effects of VEGF-C were lost upon cauterization of the dCLN afferent lymphatics and not mimicked by acute post-stroke VEGF-C injection. We conclude that VEGF-C prophylaxis promotes multiple vascular, immune, and neural responses that culminate in a protection against neurological damage in acute ischemic stroke.

Enhancing r1 Relaxivity in GdDOTA-Monoamide Complexes through Polar Group-Mediated Ordering of Second-Sphere Water Molecules.

This study was designed to test whether the single appended phosphonate group in GdDOTA-1AmP is sufficient for catalyzing the exchange of proton from the single inner-sphere water-exchanging molecule. Unlike the other phosphonate derivatives in this series, GdDOTA-1AmP showed a surprisingly smooth increase in r1 relaxivity from 3.0 to 6.3 mM-1 s-1 at 20 MHz as the pH was lowered from 9 to 2.5. In comparison to the bis-, tris-, and tetrakis-phosphonate analogues, which all show a biphasic dependence of r1 with changes in pH, the unique r1 versus pH characteristics of GdDOTA-1AmP are shown to closely parallel deprotonation of the single appended phosphonate group. Although the tissue biodistribution and clearance rates of GdDOTA-1AmP are more favorable than the other more highly charged phosphonate derivatives, the pH dependency of r1 is substantially reduced at magnetic fields typically used for small animal imaging (7 and 9.4T), so the attractiveness of this new molecule for quantitative imaging of tissue pH is diminished. However, this study provides some new insights into the feasibility of designing pH-responsive MRI contrast agents based upon fundamental acid-base prototropic mechanisms.

A combinatorial approach for achieving CNS-selective RNAi.

RNA interference (RNAi) is an endogenous process that can be harnessed using chemically modified small interfering RNAs (siRNAs) to potently modulate gene expression in many tissues. The route of administration and chemical architecture are the primary drivers of oligonucleotide tissue distribution, including siRNAs. Independently of the nature and type, oligonucleotides are eliminated from the body through clearance tissues, where their unintended accumulation may result in undesired gene modulation. Divalent siRNAs (di-siRNAs) administered into the CSF induce robust gene silencing throughout the central nervous system (CNS). Upon clearance from the CSF, they are mainly filtered by the kidneys and liver, with the most functionally significant accumulation occurring in the liver. siRNA- and miRNA-induced silencing can be blocked through substrate inhibition using single-stranded, stabilized oligonucleotides called antagomirs or anti-siRNAs. Using APOE as a model target, we show that undesired di-siRNA-induced silencing in the liver can be mitigated through administration of liver targeting GalNAc-conjugated anti-siRNAs, without impacting CNS activity. Blocking unwanted hepatic APOE silencing achieves fully CNS-selective silencing, essential for potential clinical translation. While we focus on CNS/liver selectivity, coadministration of differentially targeting siRNA and anti-siRNAs can be adapted as a strategy to achieve tissue selectivity in different organ combinations.

The vicious cycle of chronic endometriosis and depression-an immunological and physiological perspective.

Endometriosis is a chronic, estrogen-dependent, proinflammatory disease that can cause various dysfunctions. The main clinical manifestations of endometriosis include chronic pelvic pain and impaired fertility. The disease is characterized by a spectrum of dysfunctions spanning hormonal signaling, inflammation, immune dysregulation, angiogenesis, neurogenic inflammation, epigenetic alterations, and tissue remodeling. Dysregulated hormonal signaling, particularly involving estrogen and progesterone, drives abnormal growth and survival of endometrial-like tissue outside the uterus. Chronic inflammation, marked by immune cell infiltration and inflammatory mediator secretion, perpetuates tissue damage and pain. Altered immune function, impaired ectopic tissue clearance, and dysregulated cytokine production contribute to immune dysregulation. Enhanced angiogenesis promotes lesion growth and survival. Epigenetic modifications influence gene expression patterns, e.g., HSD11B1 gene, affecting disease pathogenesis. Endometriosis related changes and infertility lead to depression in diagnosed women. Depression changes lifestyle and induces physiological and immunological changes. A higher rate of depression and anxiety has been reported in women diagnosed with endometriosis, unleashing physiological, clinical and immune imbalances which further accelerate chronic endometriosis or vice versa. Thus, both endometriosis and depression are concomitantly part of a vicious cycle that enhance disease complications. A multidimensional treatment strategy is needed which can cater for both endometrial disease and depression and anxiety disorders.

Inhaled pulmonary surfactant biomimetic liposomes for reversing idiopathic pulmonary fibrosis through synergistic therapeutic strategy

Idiopathic pulmonary fibrosis (IPF) stands as a highly heterogeneous and deadly lung disease, yet the available treatment options remain limited. Combining myofibroblast inhibition with ROS modulation in damaged AECs offers a comprehensive strategy to halt IPF progression, but delivering drugs separately to these cell types is challenging. Inspired by the successful application of pulmonary surfactant (PS) replacement therapy in lung disease treatment, we have developed PS nano-biomimetic liposomes (PSBs) to utilize its natural transport pathway for targeting AECs while reducing lung tissue clearance. In this collaborative pulmonary drug delivery system, PSBs composed of DPPC/POPG/DPPG/CHO (20:9:5:4) were formulated for inhalation. These PSBs loaded with ROS-scavenger astaxanthin (AST) and anti-fibrosis drug pirfenidone (PFD) were aerosolized for precise quantification and mimicking patient inhalation. Through aerosol inhalation, the lipid membrane of PSBs gradually fused with natural PS, enabling AST delivery to AECs by hitchhiking with PS circulation. Simultaneously, PFD was released within the PS barrier, effectively penetrating lung tissue to exert therapeutic effects. In vivo results have shown that PSBs offer numerous therapeutic advantages in mice with IPF, particularly in terms of lung function recovery. This approach addresses the challenges of drug delivery to specific lung cells and offers potential benefits for IPF patients.

Single-cell transcriptomics identifies new blood cell populations in Drosophila released at the onset of metamorphosis.

Drosophila blood cells called hemocytes form an efficient barrier against infections and tissue damage. During metamorphosis, hemocytes undergo tremendous changes in their shape and behavior, preparing them for tissue clearance. Yet, the diversity and functional plasticity of pupal blood cells have not been explored. Here, we combine single-cell transcriptomics and high-resolution microscopy to dissect the heterogeneity and plasticity of pupal hemocytes. We identified undifferentiated and specified hemocytes with different molecular signatures associated with distinct functions such as antimicrobial, antifungal immune defense, cell adhesion or secretion. Strikingly, we identified a highly migratory and immune-responsive pupal cell population expressing typical markers of the posterior signaling center (PSC), which is known to be an important niche in the larval lymph gland. PSC-like cells become restricted to the abdominal segments and are morphologically very distinct from typical Hemolectin (Hml)-positive plasmatocytes. G-TRACE lineage experiments further suggest that PSC-like cells can transdifferentiate to lamellocytes triggered by parasitoid wasp infestation. In summary, we present the first molecular description of pupal Drosophila blood cells, providing insights into blood cell functional diversification and plasticity during pupal metamorphosis.

Comparison of three 18F-labeled 2-nitroimidazoles for imaging hypoxia in breast cancer xenografts: [18F]FBNA, [18F]FAZA and [18F]FMISO

BackgroundTumour hypoxia is associated with increased metastasis, invasion, poor therapy response and prognosis. Most PET radiotracers developed and used for clinical hypoxia imaging belong to the 2-nitroimidazole family. Recently we have developed novel 2-nitroimidazole-derived PET radiotracer [18F]FBNA (N-(4-[18F]fluoro-benzyl)-2-(2-nitro-1H-imidazol-1-yl)-acet-amide), an 18F-labeled analogue of antiparasitic drug benznidazole. The present study aimed to analyze its radio-pharmacological properties and systematically compare its PET imaging profiles with [18F]FMISO and [18F]FAZA in preclinical triple-negative (MDA-MB231) and estrogen receptor-positive (MCF-7) breast cancer models. MethodsIn vitro cellular uptake experiments were carried out in MDA-MB321 and MCF-7 cells under normoxic and hypoxic conditions. Metabolic stability in vivo was determined in BALB/c mice using radio-TLC analysis. Dynamic PET experiments over 3 h post-injection were performed in MDA-MB231 and MCF-7 tumour-bearing mice. Those PET data were used for kinetic modelling analysis utilizing the reversible two-tissue-compartment model. Autoradiography was carried out in tumour tissue slices and compared to HIF-1α immunohistochemistry. Detailed ex vivo biodistribution was accomplished in BALB/c mice, and this biodistribution data were used for dosimetry calculation. ResultsUnder hypoxic conditions in vitro cellular uptake was elevated in both cell lines, MCF-7 and MDA-MB231, for all three radiotracers. After intravenous injection, [18F]FBNA formed two radiometabolites, resulting in a final fraction of 65 ± 9 % intact [18F]FBNA after 60 min p.i. After 3 h p.i., [18F]FBNA tumour uptake reached SUV values of 0.78 ± 0.01 in MCF-7 and 0.61 ± 0.04 in MDA-MB231 tumours (both n = 3), representing tumour-to-muscle ratios of 2.19 ± 0.04 and 1.98 ± 0.15, respectively. [18F]FMISO resulted in higher tumour uptakes (SUV 1.36 ± 0.04 in MCF-7 and 1.23 ± 0.08 in MDA-MB231 (both n = 4; p < 0.05) than [18F]FAZA (0.66 ± 0.11 in MCF-7 and 0.63 ± 0.14 in MDA-MB231 (both n = 4; n.s.)), representing tumour-to-muscle ratios of 3.24 ± 0.30 and 3.32 ± 0.50 for [18F]FMISO, and 2.92 ± 0.74 and 3.00 ± 0.42 for [18F]FAZA, respectively. While the fraction per time of radiotracer entering the second compartment (k3) was similar within uncertainties for all three radiotracers in MDA-MB231 tumours, it was different in MCF-7 tumours. The ratios k3/(k3 + k2) and K1*k3/(k3 + k2) in MCF-7 tumours were also significantly different, indicating dissimilar fractions of radiotracer bound and trapped intracellularly: K1*k3/(k2 + k3) [18F]FMISO (0.0088 ± 0.001)/min, n = 4; p < 0.001) > [18F]FAZA (0.0052 ± 0.002)/min, n = 4; p < 0.01) > [18F]FBNA (0.003 ± 0.001)/min, n = 3). In contrast, in MDA-MB231 tumours, only K1 was significantly elevated for [18F]FMISO. However, this did not result in significant differences for K1*k3/(k2 + k3) for all three 2-nitroimidazoles in MDA-MB231 tumours. ConclusionNovel 2-nitroimidazole PET radiotracer [18F]FBNA showed uptake into hypoxic breast cancer cells and tumour tissue presumably associated with elevated HIF1-α expression. Systematic comparison of PET imaging performance with [18F]FMISO and [18F]FAZA in different types of preclinical breast cancer models revealed a similar tumour uptake profile for [18F]FBNA with [18F]FAZA and, despite its higher lipophilicity, still a slightly higher muscle tissue clearance compared to [18F]FMISO.

New liposome-radionuclide-chelate combination for tumor targeting and rapid healthy tissue clearance

Radionuclide imaging and therapy are promising methods for controlling systemic cancers; however, their clinical application has been limited by excessive radionuclide accumulation in healthy tissues. To minimize radionuclide accumulation in non-cancerous tissues while ensuring sufficient build up in tumors, we aimed to develop a method that controlled the in vivo dynamics of radionuclides post-administration. To this end, we describe a novel strategy that combines liposomes, a potent carrier system for drug delivery, with unique radionuclide-ligand complexes based on 111In-ethylenedicysteine.Conventional 111In-ligand-complexes-carrying liposomes delivered substantial amounts of radionuclides to tumors; however, they also accumulated in the liver and spleen. In contrast, 111In-ethylenedicysteine-carrying liposomes greatly reduced non-specific accumulation, while being retained selectively at high doses within tumors. Liposomes were rapidly broken down in the liver, releasing encapsulated 111In-ligand complexes. Among the chelates used, only 111In-ethylenedicysteine could escape from the liver and be excreted in the urine. Instead, most liposomes remained intact in tumors, retaining the radionuclide-ligand complexes within them. Therefore, high tumor accumulation was obtained regardless of the type of 111In-ligand complexes in the liposomes. In vivo single photon emission computed tomography/computed tomography imaging with 111In-ethylenedicysteine-carrying liposomes accurately revealed tumor-selective radionuclide retention with little background. Hence, our new strategy could greatly enhance tumor-to-healthy tissue ratios, improve diagnostic imaging, boost therapeutic efficacy, reduce toxicity to healthy tissues, and facilitate radionuclide imaging and therapy.

Effect of sp3/sp2 carbon ratio and hydrodynamic size on the biodistribution kinetics of nanodiamonds in mice via intravenous injection

BackgroundNanodiamonds (NDs) have gained a rapidly growing interest in biomedical applications; however, little is known regarding their biokinetics owing to difficulties in measurements and limited synthesis/purification technologies. In this study, we investigated the distribution kinetics of detonation-synthesized NDs in mice via intravenous injection to evaluate the parameters that determine the behavior of the particles. We prepared two distinctive NDs that controlled the sp3/sp2 carbon ratio and particle size by coating them with serum proteins. The four control samples were intravenously injected into mice, and tissue distribution and clearance were evaluated at 30 min and 1, 7, and 28 days post-injection.ResultsThe sp3/sp2 carbon ratio showed no correlation with the organ distribution of the NDs. However, hydrodynamic size showed an excellent correlation with organ distribution levels: a negative correlation in the liver and positive correlations in the spleen and lungs. Furthermore, the deposition levels of NDs in the lung suggest that particles smaller than 300 nm could avoid lung deposition. Finally, a similar organ distribution pattern was observed in mice injected with carbon black nanoparticles controlled hydrodynamic size.ConclusionsIn conclusion, the tissue distribution of NDs is modulated not by the sp3/sp2 carbon ratio but by the hydrodynamic size, which can provide helpful information for targeting the tissue of NDs. Furthermore, the organ distribution pattern of the NDs may not be specific to NDs but also can apply to other nanoparticles, such as carbon black.

Perivascular clearance of blood proteins after blood-brain barrier disruption in a rat model of microinfarcts.

Microinfarcts result in a transient loss of the blood-brain barrier (BBB) in the ischemic territory. This leads to the extravasation of blood proteins into the brain parenchyma. It is not clear how these proteins are removed. Here we studied the role of perivascular spaces in brain clearance from extravasated blood proteins. Male and female Wistar rats were infused with microspheres of either 15, 25, or 50μm in diameter (n=6 rats per group) via the left carotid artery. We infused either 25,000 microspheres of 15μm, 5500 of 25μm, or 1000 of 50μm. One day later, rats were infused with lectin and hypoxyprobe to label perfused blood vessels and hypoxic areas, respectively. Rats were then euthanized and perfusion-fixed. Brains were excised, sectioned, and analyzed using immunostaining and confocal imaging. Microspheres induced a size-dependent increase in ischemic volume per territory, but the cumulative ischemic volume was similar in all groups. The total volumes of ischemia, hypoxia and infarction affected 1-2% of the left hemisphere. Immunoglobulins (IgG) were present in ischemic brain tissue surrounding lodged microspheres in all groups. In addition, staining for IgG was found in perivascular spaces of blood vessels nearby areas of BBB disruption. About 2/3 of these vessels were arteries, while the remaining 1/3 of these vessels were veins. The subarachnoid space (SAS) of the affected hemisphere stained stronger for IgG than the contralateral hemisphere in all groups: +27%, +44% and +27% respectively. Microspheres of various sizes induce a local loss of BBB integrity, evidenced by parenchymal IgG staining. The presence of IgG in perivascular spaces of both arteries and veins distinct from the ischemic territories suggests that both contribute to the removal of blood proteins. The strong staining for IgG in the SAS of the affected hemisphere suggests that this perivascular route egresses via the CSF. Perivascular spaces therefore play a previously unrecognized role in tissue clearance of fluid and extravasated proteins after BBB disruption induced by microinfarcts.

Spatial characterization of tangle-bearing neurons and ghost tangles in the human inferior temporal gyrus with three-dimensional imaging.

Studies of post-mortem human tissue provide insight into pathological processes, but are inherently limited by practical considerations that limit the scale at which tissue can be examined, and the obvious issue that the tissue reflects only one time point in a continuous disease process. We approached this problem by adapting new tissue clearance techniques to an entire cortical area of human brain, which allows surveillance of hundreds of thousands of neurons throughout the depth of the entire cortical thickness. This approach allows detection of 'rare' events that may be difficult to detect in standard 5 micrometre-thick paraffin sections. For example, it is well established that neurofibrillary tangles begin within a neuron, and ultimately, in at least some instances, persist in the brain even after the neuron has died. These are referred to as 'ghost tangles', a term that appropriately implies their 'difficult to see' ephemeral qualities. We set out to find ghost tangles as one example of the power of the tissue clearance/image analysis techniques to detect rare events, and to learn what happens at the end-point of a tangle's life history. We were able to identify 8103 tau tangles, 132 465 neurons and 299 640 nuclei in tissue samples from three subjects with severe Alzheimer's disease (Braak V-VI) and 4 tau tangles, 200 447 neurons and 462 715 nuclei in tissue samples from three subjects with no significant tau pathology (Braak 0-I). Among these data, we located 57 ghost tangles, which makes them only 0.7% of the total tau tangles observed. We found that ghost tangles are more likely to be found in cortical layers 3 and 5 (49/57), with a select few scattered across other layers 1, 2, 4 and 6. This ability to find rare events, such as ghost tangles, in large enough quantities to statistically test their distribution exemplifies how tissue clearing can be used as a powerful tool for studying selective vulnerability or resilience to pathology across brain regions.