Rapid Extravasation Research Articles

Precise diagnosis of cardiac‐cerebral vascular diseases with magnetic resonance imaging‐based nanoprobes

AbstractCardiac‐cerebral vascular diseases (CCVDs) are acknowledged as a major threat to public health, leading to more than one‐third of all deaths worldwide. The complex anatomical structure and immune features of blood vessels significantly affect the development of CCVDs, and magnetic resonance angiography (MRA) is one of the main diagnostic approaches for the accurate diagnosis and prognosis of CCVDs. However, MRA suffers from intrinsic problems derived from its blood flow‐dependency, and the clinical Gd‐chelating contrast agents are limited by their rapid vascular extravasation. Over the past decade, spurred on by nanoscience and nanotechnology, numerous contrast agents based on magnetic nanomaterials have been developed to enhance the contrast of MRA, with these including iron oxide nanoparticles, rare earth‐doped nanoparticles, and metal‐organic coordination polymers. The molecular MR imaging of vasculopathy using specific nanoprobes has been explored to obtain a better understanding of the molecular aspects of CCVDs. In this review, the state of the art in MRA nanoprobes is introduced, and recent achievements in the diagnosis of CCVDs using MR imaging are summarized. Additionally, the future prospects and limitations of MRA based on nanoprobes are discussed. The current review provides methodological designs and ideas for subsequent MRA nanoprobes.

Curvature-mediated rapid extravasation and penetration of nanoparticles against interstitial fluid pressure for improved drug delivery

Elevated interstitial fluid pressure (IFP) within pathological tissues (e.g., tumors, obstructed kidneys, and cirrhotic livers) creates a significant hindrance to the transport of nanomedicine, ultimately impairing the therapeutic efficiency. Among these tissues, solid tumors present the most challenging scenario. While several strategies through reducing tumor IFP have been devised to enhance nanoparticle delivery, few approaches focus on modulating the intrinsic properties of nanoparticles to effectively counteract IFP during extravasation and penetration, which are precisely the stages obstructed by elevated IFP. Herein, we propose an innovative solution by engineering nanoparticles with a fusiform shape of high curvature, enabling efficient surmounting of IFP barriers during extravasation and penetration within tumor tissues. Through experimental and theoretical analyses, we demonstrate that the elongated nanoparticles with the highest mean curvature outperform spherical and rod-shaped counterparts against elevated IFP, leading to superior intratumoral accumulation and antitumor efficacy. Super-resolution microscopy and molecular dynamics simulations uncover the underlying mechanisms in which the high curvature contributes to diminished drag force in surmounting high-pressure differentials during extravasation. Simultaneously, the facilitated rotational movement augments the hopping frequency during penetration. This study effectively addresses the limitations posed by high-pressure impediments, uncovers the mutual interactions between the physical properties of NPs and their environment, and presents a promising avenue for advancing cancer treatment through nanomedicine.

Abstract 2577: Analyzing specific and nonspecific tissue uptake of antibodies by SPECT imaging and non-linear compartmental modeling

Abstract Understanding the mechanisms driving specific target engagement versus off-target nonspecific cellular uptake of antibodies can help inform design and/or delivery strategies that maximize therapeutic indices. Receptor binding kinetics, rates of receptor-mediated internalization and nonspecific pinocytosis are often measured through in vitro cell culture models, but there have been fewer efforts to collectively measure these cellular parameters in vivo. Single photon emission computed tomography (SPECT) imaging is a powerful method of estimating these parameters by quantifying both free/bound (intact) and internalized/catabolized antibody using non-residualizing halogen labels and residualizing radiometal-chelates, respectively. Modeling longitudinal in vivo imaging data of non-residualizing antibody can inform receptor occupancy (RO), while residualizing tracer kinetics can provide additional insights into catabolism due to pinocytosis and receptor-mediated internalization in the intracellular spaces. Here, we describe SPECT imaging of tumor-targeting trastuzumab (anti-HER2) and non-targeting anti-glycoprotein D (anti-gD) antibody, each labeled with non-residualizing ([125I]SIB) and residualizing radiometal-chelate (111In-DOTA) radionuclides in HER2+ xenograft-bearing mice. A non-linear compartmental model was fitted to SPECT imaging data to characterize RO of 10 mg/kg of anti-HER2 out to 7 days post dosing as well as to estimate rate constants of pinocytosis and receptor-mediated internalization of anti-HER2 in tumor. Longitudinal SPECT imaging demonstrated statistically significant differences in tumor drug exposure between 125I- and 111In-labeled anti-HER2 relative to anti-gD (p < 0.01). Tracer kinetics of anti-HER2 and associated RO simulations in tumor showed rapid extravasation from blood to tumor with peak RO of 25% at 13 hours post administration followed by a reduction in RO, reaching ~10% by day 7. Simulations for free, bound and catabolized tracer kinetics from our fitted model showed that anti-HER2 in the tumor interstitium was primarily receptor-bound with minimal pinocytosis of unbound anti-HER2 as well as gradual receptor-mediated catabolism, reaching a peak concentration of 113 µg/mL 7 days post dosing. Rate constants of nonspecific pinocytosis and HER2-mediated receptor internalization were estimated to be 0.0145 and 0.036 hours−1, respectively. Our model allowed us to successfully capture anti-HER2 antibody receptor binding and specific and nonspecific internalization over time in vivo. These data and analyses demonstrate the power of SPECT imaging using both non-residualizing and residualizing radiolabels combined with compartmental modeling to better characterize the different biological states (free, bound, and catabolized) of antibody within interstitial and intracellular compartments. Citation Format: Nicholas Cho, Phillip Spinosa, Danielle Mandikian, Geoffrey Del Rosario, Shang-Fan Yu, Jason Ho, Greg Ferl, Charles A. Boswell. Analyzing specific and nonspecific tissue uptake of antibodies by SPECT imaging and non-linear compartmental modeling [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 2577.

Ultrasmall catechol-PEG-anchored ferrite nanoparticles for highly sensitive magnetic resonance angiography.

Highly sensitive iron oxide nanoparticles with stable, safe and efficient surface functionalization, as potential substitutes for gadolinium-based contrast agents (GBCAs) with increasing biosafety concerns, exhibit great potential for high-performance magnetic resonance angiography (MRA). Herein, we developed ultrasmall catechol-PEG-anchored ferrite nanoparticles (PEG-UMFNPs) for highly sensitive MRA. The obtained nanoprobe has a high T1 relaxivity value (7.2 mM-1 s-1) due to its ultrasmall size and Mn doping. It has a suitable hydrodynamic size of 20 nm, which prevents rapid vascular extravasation and renal clearance and prolongs its blood circulation time. In vivo MRA at 3.0 T using the nanoprobe shows that the arteries and veins of rats, even blood vessels as small as 0.32 mm, are distinctly visible, and the contrast enhancement can last for at least 1 h. In addition, due to the outstanding contrast enhancement and long circulation time, the stenosis and recanalization process of the rat's carotid artery can be continuously monitored with a single injection of the nanoprobe. Our study indicates that PEG-UMFNPs are outstanding MR imaging nanoprobes that can be used to diagnose vascular diseases without the biosafety issues of GBCAs.

Hypersensitive MR angiography based on interlocking stratagem for diagnosis of cardiac-cerebral vascular diseases

Magnetic resonance (MR) angiography is one of the main diagnostic approaches for cardiac-cerebral vascular diseases. Nevertheless, the non-contrast-enhanced MR angiography suffers from its intrinsic problems derived from the blood flow-dependency, while the clinical Gd-chelating contrast agents are limited by their rapid vascular extravasation. Herein, we report a hypersensitive MR angiography strategy based on interlocking stratagem of zwitterionic Gd-chelate contrast agents (PAA-Gd). The longitudinal molar relaxivity of PAA-Gd was 4.6-times higher than that of individual Gd-chelates as well as appropriate blood half-life (73.8 min) and low immunogenicity, enabling sophisticated micro-vessels angiography with a resolution at the order of hundred micrometers. A series of animal models of cardiac-cerebrovascular diseases have been built for imaging studies on a 7.0 T MRI scanner, while the clinical translation potential of PAA-Gd has been evaluated on swine on a 3.0 T clinical MRI scanner. The current studies offer a promising strategy for precise diagnosis of vascular diseases.

A lone spike in blood glucose can enhance the thrombo-inflammatory response in cortical venules

How transient hyperglycemia contributes to cerebro-vascular disease has been a challenge to study under controlled physiological conditions. We use amplified, ultrashort laser-pulses to physically disrupt brain-venule endothelium at targeted locations. This vessel disruption is performed in conjunction with transient hyperglycemia from a single injection of metabolically active D-glucose into healthy mice. The observed real-time responses to laser-induced disruption include rapid serum extravasation, platelet aggregation, and neutrophil recruitment. Thrombo-inflammation is pharmacologically ameliorated by a platelet inhibitor, by a scavenger of reactive oxygen species, and by a nitric oxide donor. As a control, vessel thrombo-inflammation is significantly reduced in mice injected with metabolically inert L-glucose. Venules in mice with diabetes show a similar response to laser-induced disruption and damage is reduced by restoration of normo-glycemia. Our approach provides a controlled method to probe synergies between transient metabolic and physical vascular perturbations and can reveal new aspects of brain pathophysiology.

Extravasation of Blood and Blood Toxicity Drives Tubular Injury from RBC Trapping in Ischemic AKI.

Red blood cell (RBC) trapping is common in ischemic acute kidney injury (AKI) and presents as densely packed RBCs that accumulate within and engorge the kidney medullary circulation. In this study, we tested the hypothesis that "RBC trapping directly promotes tubular injury independent of extending ischemia time." Studies were performed on rats. Red blood cell congestion and tubular injury were compared between renal arterial clamping, venous clamping, and venous clamping of blood-free kidneys. Vessels were occluded for either 15 or 45 min with and without reperfusion. We found that RBC trapping in the medullary capillaries occurred rapidly following reperfusion from renal arterial clamping and that this was associated with extravasation of blood from congested vessels, uptake of blood proteins by the tubules, and marked tubular injury. To determine if this injury was due to blood toxicity or an extension of ischemia time, we compared renal venous and arterial clamping without reperfusion. Venous clamping resulted in RBC trapping and marked tubular injury within 45 min of ischemia. Conversely, despite the same ischemia time, RBC trapping and tubular injury were minimal following arterial clamping without reperfusion. Confirming the role of blood toward tubular injury, injury was markedly reduced in blood-free kidneys with venous clamping. Our data demonstrate that RBC trapping results in the rapid extravasation and uptake of blood components by tubular cells, causing toxic tubular injury. Tubular toxicity from extravasation of blood following RBC trapping appears to be a major component of tubular injury in ischemic AKI, which has not previously been recognized.

Selected Articles from This Issue

Nectin-4 is a validated cancer target through ADC enfortumab vedotin. Rigby and colleagues have identified BT8009, a Bicycle® Toxin Conjugate that selectively binds Nectin-4 providing robust activity, and affording similar, or improved, activity over an ADC based on enfortumab vedotin, in several pre-clinical xenograft cancer models. The small, hydrophilic, nature of BT8009 results in a pharmacokinetic profile very different from an ADC, with a high Cmax, rapid extravasation from systemic circulation and rapid renal elimination. These different physical and PK characteristics may provide benefit for tumor penetration and reduced systemic exposure. BT8009 is in an ongoing Phase 1/2 clinical trial.In metastatic castration-resistant prostate cancer (CRPC), altered expression of steroidogenesis enzymes, androgen receptor (AR) overexpression and mutations in AR ligand binding domain lead to increased AR sensitivity against various different steroids. ODM-208 is a highly selective inhibitor of the rate-limiting enzyme of steroidogenesis, CYP11A1. By inhibiting CYP11A1 it is possible to prevent the synthesis of all steroid ligands that could activate AR. ODM-208 leads to rapid, complete, durable, and reversible inhibition of the steroid hormones in multiple preclinical models and in patients with CRPC (NCT03436485). In addition, tumour growth inhibition has been shown in CRPC xenograft model.Scherrer and colleagues investigated the effects of a stimulator of invariant natural killer T-cells, ABX196, on mouse models of B16F10 melanoma and hepatocarcinoma Hepa 1-6. ABX196 combined with anti-PD-1 resulted in increased melanoma and hepatocarcinoma control and survival. In both models, efficacy was associated with a generation of a more advantageous T-effector to Treg cell ratio within the tumor, resulting in the proliferation and accumulation of cells that would otherwise be anergized. ABX196 plus anti-PD-1 antibody may be a novel strategy to overcome the immunosuppressive microenvironment and to produce anti-tumor activity.Human epidermal growth factor receptor type 2 (HER2) is overexpressed in various cancers. There is an urgent need for novel strategies overcoming resistance to HER2-targeted therapies and single-domain antibodies (sdAbs) could offer a useful platform for radioimmunotherapy. Here, a complete preclinical evaluation of 2Rs15d-sdAb radiolabeled with α-particle-emitting 225Ac radionuclide is reported. [225Ac]Ac-DOTA-2Rs15d efficiently targeted HER2pos cells and was effective in treatment of intraperitoneal disseminated tumors, both alone and as an add-on combination with trastuzumab. [225Ac]Ac-DOTA-2Rs15d holds promise for further investigation, and with the recent successful introduction of 225Ac-labeled peptides in the clinic, might also find its way towards clinical translation.

Red Blood Cell Membrane-Coated Ultrasmall NaGdF4 Nanoprobes for High-Resolution 3D Magnetic Resonance Angiography.

Enhanced angiography based on magnetic resonance imaging (MRI) has emerged as a noninvasive, robust, and high-resolution imaging technique for the clinical evaluation of vascular diseases. However, the effects of clinical Gd-chelating contrast agents are unsatisfactory for MRI contrast enhancement owing to their short blood half-life caused by rapid vascular extravasation, especially in microvessels. To address these issues, nanoprobes based on red blood cell membrane-coated ultrasmall NaGdF4 nanoparticles that exhibit much higher longitudinal molar relaxivity (r1) than the clinically used contrast agent gadolinium diethylenetriaminepentaacetic acid have been developed. Furthermore, the appropriate hydrodynamic diameter and stealth nature aid the nanoprobes to reside longer within the blood vessels without extravasation, thereby increasing the contrast between the blood vessels and surrounding tissues. Through probe-enhanced three-dimensional (3D) dynamic contrast-enhanced MR angiography, the main arteries and veins of the mouse were readily discernible, and even tiny vessels with sub-millimeter diameters could be clearly depicted. With this level of outstanding MR angiography performance, the embolization and recanalization processes of the carotid artery can be serially monitored with high imaging resolution using only a single injection. Additionally, the results of clearance studies and the toxicity tests further highlight the safety features of the nanoprobe. To summarize, the nanoprobes used in this study exhibit less extravascular leakage and a longer blood half-life, thus successfully overcoming the defects of the conventional low-molecular-weight Gd-based contrast agents and demonstrating their potential usefulness in enhanced MR angiography.

Thin Layer-Protected Gold Nanoparticles for Targeted Multimodal Imaging with Photoacoustic and CT.

The large size of nanoparticles prevents rapid extravasation from blood vessels and diffusion into tumors. Multimodal imaging uses the physical properties of one modality to validate the results of another. We aim to demonstrate the use of a targeted thin layer-protected ultra-small gold nanoparticles (Au-NPs) to detect cancer in vivo using multimodal imaging with photoacoustic and computed tomography (CT). The thin layer was produced using a mixed thiol-containing short ligands, including MUA, CVVVT-ol, and HS-(CH2)11-PEG4-OH. The gold nanoparticle was labeled with a heterobivalent (HB) peptide ligand that targets overexpression of epidermal growth factor receptors (EGFR) and ErbB2, hereafter HB-Au-NPs. A human xenograft model of esophageal cancer was used for imaging. HB-Au-NPs show spherical morphology, a core diameter of 4.47 ± 0.8 nm on transmission electron microscopy, and a hydrodynamic diameter of 6.41 ± 0.73 nm on dynamic light scattering. Uptake of HB-Au-NPs was observed only in cancer cells that overexpressed EGFR and ErbB2 using photoacoustic microscopy. Photoacoustic images of tumors in vivo showed peak HB-Au-NPs uptake at 8 h post-injection with systemic clearance by ~48 h. Whole-body images using CT validated specific tumor uptake of HB-Au-NPs in vivo. HB-Au-NPs showed good stability and biocompatibility with fast clearance and contrast-enhancing capability for both photoacoustic and CT imaging. A targeted thin layer-protected gold nanoprobe represents a new platform for molecular imaging and shows promise for early detection and staging of cancer.

Isolation of Human Neutrophils from Whole Blood and Buffy Coats.

Neutrophils (PMNs) are the most abundant leukocytes in human circulation, ranging from 40 to 70% of total blood leukocytes. They are the first cells recruited at the site of inflammation via rapid extravasation through vessels. There, neutrophils perform an array of functions to kill invading pathogens and mediate immune signaling. Freshly purified neutrophils from human blood are the model of choice for study, as no cell line fully replicates PMN functions and biology. However, neutrophils are short-lived, terminally differentiated cells and are highly susceptible to activation in response to physical (temperature, centrifugation speed) and biological (endotoxin, chemo- and cytokines) stimuli. Therefore, it is crucial to follow a standardized, reliable, and fast method to obtain pure and non-activated cells. This protocol presents an updated protocol combining density gradient centrifugation, red blood cell (RBC) sedimentation, and RBC lysis to obtain high PMN purity and minimize cell activation. Furthermore, methods to assess neutrophil isolation quality, viability, and purity are also discussed.

Isolation of Human Neutrophils from Whole Blood and Buffy Coats

Neutrophils (PMNs) are the most abundant leukocytes in human circulation, ranging from 40 to 70% of total blood leukocytes. They are the first cells recruited at the site of inflammation via rapid extravasation through vessels. There, neutrophils perform an array of functions to kill invading pathogens and mediate immune signaling. Freshly purified neutrophils from human blood are the model of choice for study, as no cell line fully replicates PMN functions and biology. However, neutrophils are short-lived, terminally differentiated cells and are highly susceptible to activation in response to physical (temperature, centrifugation speed) and biological (endotoxin, chemo- and cytokines) stimuli. Therefore, it is crucial to follow a standardized, reliable, and fast method to obtain pure and non-activated cells. This protocol presents an updated protocol combining density gradient centrifugation, red blood cell (RBC) sedimentation, and RBC lysis to obtain high PMN purity and minimize cell activation. Furthermore, methods to assess neutrophil isolation quality, viability, and purity are also discussed.

ExoSTING, an extracellular vesicle loaded with STING agonists, promotes tumor immune surveillance

Cyclic dinucleotide (CDN) agonists of the STimulator of InterferoN Genes (STING) pathway have shown immune activation and tumor clearance in pre-clinical models. However, CDNs administered intratumorally also promote STING activation leading to direct cytotoxicity of many cell types in the tumor microenvironment (TME), systemic inflammation due to rapid tumor extravasation of the CDN, and immune ablation in the TME. These result in a failure to establish immunological memory. ExoSTING, an engineered extracellular vesicle (EV) exogenously loaded with CDN, enhances the potency of CDN and preferentially activates antigen presenting cells in the TME. Following intratumoral injection, exoSTING was retained within the tumor, enhanced local Th1 responses and recruitment of CD8+ T cells, and generated systemic anti-tumor immunity to the tumor. ExoSTING at therapeutically active doses did not induce systemic inflammatory cytokines, resulting in an enhanced therapeutic window. ExoSTING is a novel, differentiated therapeutic candidate that leverages the natural biology of EVs to enhance the activity of CDNs.

High-speed delivery to neutrophils.

A recent study in Immunity describes that mast cells can degranulate directly into the bloodstream to prime neutrophils for rapid extravasation at sites of tissue inflammation.

Antibody-activated trans-endothelial delivery of mesoporous organosilica nanomedicine augments tumor extravasation and anti-cancer immunotherapy

Tumor vasculature constitutes a formidable hurdle for the efficient delivery of cancer nanomedicine into tumors. The leverage of passive pathway through inter-endothelial gaps in tumor blood vessels might account for limited extravasation of nanomedicine into tumor microenvironment (TME). Herein, Annexin A1 antibody-installed mesoporous organosilica nanoplatforms carrying immunotherapeutics of anti-PD-L1 antibody (aPD-L1) and Indoximod are developed to target at caveolar Annexin-A1 protein of luminal endothelial cells and to trigger the active trans-endothelial transcytosis of nanomedicine mediated by caveolae. Such strategy enables rapid nanomedicine extravasation across tumor endothelium and relatively extensive accumulation in tumor interstitium. aPD-L1 and Indoximod release from aPD/IND@MON-aANN in a reduction-responsive manner and synergistically facilitate the intratumoral infiltration of cytotoxic T lymphocytes and reverse the immunosuppressive TME, thus demonstrating substantial anti-tumor efficacy in subcutaneous 4T1 breast tumors and remarkable anti-metastatic capacity to extend the survival of 4T1 tumor metastasis model. Moreover, aPD/IND@MON-aANN nanomedicine also exhibits distinct superiority over the combination therapy of free drugs to potently attenuate the progression of urethane-induced orthotopic lung cancers. Collectively, aPD/IND@MON-aANN nanoplatforms with boosted delivery efficiency via antibody-activated trans-endothelial pathway and enhanced immunotherapeutic efficacy provides perspectives for the development of cancer nanomedicines.

Abstract 4481: BT5528, an EphA2-targeting Bicycle Toxin Conjugate (BTC): Profound efficacy without bleeding and coagulation abnormalities in animal models

Abstract Ephrin receptor A2 (EphA2) is part of the Ephrin receptor family of cell-cell junction proteins highly overexpressed in several solid tumours, and is associated with poor prognosis. EphA2 has long been a high-value target for cancer therapeutics, but development of an Antibody Drug Conjugate targeting EphA2 (MEDI-547) was stopped in early clinical development after severe adverse events were seen (Annunziata et al, 2013). We have developed a series of Bicycle Toxin Conjugates (BTC®) comprising a constrained bicyclic peptide (Bicycle®) that binds with high affinity and specificity to EphA2, which is covalently linked to a toxin payload via a cleavable linker. The small size of BTCs offers a significant advantage over other targeted cytotoxic approaches such as antibody-drug conjugates due to rapid extravasation, renal clearance and improved tumour penetration. Bicycle binders for EphA2 were identified using a proprietary phage display peptide technology consisting of highly diverse phage libraries of Bicycles, conjugated to cleavable linkers & toxins to form Bicycle Toxin Conjugates (BTCs). We selected the candidate BTC BT5528 from a panel of >75 BTCs, based on in vivo efficacy, tolerability and drug-like properties. BT5528 is effective in EphA2-expressing xenograft models, with complete tumour regression seen from 0.5mg/kg weekly. Expression of EphA2 is high across a range of cancers of high unmet medical need. A good correlation with efficacy was seen across cell- and patient- derived xenograft models including lung, breast, oesophageal, ovarian, prostate, gastric and sarcoma tumours. No significant efficacy is seen in tumours without EphA2 expression. Rapid tumour penetration, renal elimination and significant bystander effect produce efficacy through a “hit & run” approach, with low systemic exposure to conjugate or free toxin. Efficacy is seen with dosing intervals of 1 or 2 weeks, despite a terminal half-life of ~30min for BTCs across species. In simple xenografts (CDX, ~200mm3), efficacy of BT5528 & anti-EphA2 ADC are comparable. In large & more complex models (heterogeneous PDX, >1000mm3), the ADC shows reduced efficacy, while the efficacy of BT5528 is maintained. Clinical development of MEDI-547 was terminated when patients showed profound bleeding & coagulation toxicity, consistent with the nature of toxicology seen in preclinical studies. In contrast, BT5528 shows no sign of bleeding in toxicology studies in rat and NHP and no changes in coagulation parameters or liver enzymes. The EphA2 targeting BTC BT5528 shows potent anti-tumour activity in a range of solid tumour xenograft models without the limiting toxicity observed with previous Antibody Drug Conjugate approaches. IND-enabling studies for BT5528 are currently underway. Citation Format: Gavin S. Bennett, Amy Brown, Gemma Mudd, Johanna Lahdenranta, Nicholas Keen. BT5528, an EphA2-targeting Bicycle Toxin Conjugate (BTC): Profound efficacy without bleeding and coagulation abnormalities in animal models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4481.

An unusual cause of upper gastrointestinal bleeding

A 46-year-old man with stage IV esophageal cancer (Figure 1, left) presented with dysphagia and weight loss. A palliative esophageal stent (ultraflex) was placed to relieve the dysphagia (Figure 1, right) in another hospital for symptom relief. He was administered concurrent chemoradiotherapy after oral intake resumed. On the third hospital day of the fourth chemotherapy course, he experienced sudden-onset large-volume hematemesis and went into shock. After resuscitation, an emergency endoscopy demonstrated bloody content in the esophagus, and the bleeding was uncontrollable. A contrast-enhanced chest computed tomography (CT) was performed (Figure 2). What is the diagnosis? The chest CT demonstrated active contrast extravasation from the aorta (Figure 2, left) into the upper margin of the esophageal stent (Figure 2, right), and aortoesophageal fistula (AEF) was diagnosed. The patient declined surgery because of advanced esophageal cancer. Emergency endovascular repair using an aortic stent was performed to control the bleeding. The patient experienced severe pneumonia after resolution of the bleeding episode, and a tracheoesophageal fistula was found. A week later, he died of multiple organ failure. Esophageal stent for advanced esophageal cancer is aimed at maintaining oral intake to improve the quality of life.1-3 Stent-related major complications, such as perforation and hemorrhage, occur more frequently after radiotherapy and limit its use as a bridge to surgery or before preoperative chemoradiotherapy.1 Patients with AEF may experience mid-thoracic pain, heralded hemorrhage, and then fatal hemorrhage due to rapid blood extravasation from the aorta to the esophagus, so-called Chiari's triad.4 Unlike other causes of upper gastrointestinal bleeding, AEF can be diagnosed using endoscopy in less than 10% of cases. Diagnosis is usually made using CT arteriogram showing active extravasation of blood from the aorta to the esophagus. The treatment options for AEF include definitive surgical aortic replacement, temporary endovascular aortic repair, or further esophageal stenting. The prognosis is poor because most of these patients have contraindications to major surgery due to poor general condition. The authors receive funding from Changhua Christian Hospital 106-CCHIRP-030 for this manuscript. The authors declare no conflict of interest. Yu-Chun Hsu performed the endoscopy and final approval of the manuscript. Chia-Bung Chen provided 3-D reconstruction of the image and final approval of the manuscript. Hsu-Heng Yen was involved in drafting, writing and final approval and drafting of the manuscript.

Glucose transporter 1-mediated vascular translocation of nanomedicines enhances accumulation and efficacy in solid tumors

Nanomedicine modification with ligands directed to receptors on tumor blood vessels has the potential for selectively enhancing nanomedicine accumulation in malignant tissues by overcoming the vascular barrier of tumors. Nevertheless, the development of broadly applicable ligand approaches capable of promoting the transvascular transport of nanomedicines in a wide spectrum of tumors has been elusive so far. By considering the indispensable and persistent glycolytic fueling of tumors, we developed glucose-installed polymeric micelles loading cisplatin (Gluc-CDDP/m) targeting the glucose transporter 1 (GLUT1), which is overexpressed in most tumors and present on vascular endothelial cells, toward improving the delivery efficiency and therapeutic efficacy. The design of the glucose ligands on Gluc-CDDP/m was engineered to control the conjugation via the carbon 6 of the glucose moieties, as well as the ligand density on the poly (ethylene glycol) (PEG) shell of the micelles. The series of micelles was then studied in vitro and in vivo against GLUT1-high human squamous cell carcinoma of the head and neck OSC-19 cells and GLUT1-low human glioblastoma-astrocytoma U87MG cells. Our results showed that precisely tuning the micelles to have glucose ligands on 25% of their PEG chains increased the efficacy against the tumors by significantly enhancing the tumor accumulation, even in GLUT1-low U87MG tumors. The enhancement of the intratumoral levels of these micelles was hindered by concomitant administration of glucose, or the GLUT1 inhibitor STF-31, confirming a GLUT1/glucose-mediated increment of the accumulation. Intravital confocal laser scanning microscopy imaging of tumor tissues further demonstrated the rapid extravasation and penetration of Gluc-CDDP/m in OSC-19 tumors compared to non-targeted CDDP/m. These findings indicate GLUT1-targeting as a promising approach for overcoming the vascular barrier and boosting the delivery of nanomedicine in tumors.

Roles of inflammation, neurogenic inflammation, and neuroinflammation in pain.

Inflammation is the body's response to injury and infection, involving a complex biological response of the somatosensory, immune, autonomic, and vascular systems. Inflammatory mediators such as prostaglandin, proinflammatory cytokines, and chemokines induce pain via direct activation of nociceptors, the primary sensory neurons that detect noxious stimuli. Neurogenic inflammation is triggered by nerve activation and results in neuropeptide release and rapid plasma extravasation and edema, contributing to pain conditions such as headache. Neuroinflammation is a localized inflammation in the peripheral nervous system (PNS) and central nervous system (CNS). A characteristic feature of neuroinflammation is the activation of glial cells in dorsal root ganglia, spinal cord, and brain which leads to the production of proinflammatory cytokines and chemokines in the PNS and CNS that drives peripheral sensitization and central sensitization. Here, we discuss the distinct roles of inflammation, neurogenic inflammation, and neuroinflammation in the regulation of different types of pain conditions, with a special focus on neuroinflammation in postoperative pain and opioid-induced hyperalgesia.

Abstract 5855: Bicycle Drug Conjugates targeting EphA2 for the treatment of solid tumors: Discovery and selection of BT5528

Abstract Ephrin receptor A2 (EphA2) is a member of the Ephrin receptor family of cell-cell junction proteins and is both highly overexpressed in several solid tumors and associated with poor prognosis in patients. Bicycles® are novel therapeutic agents: bicyclic peptides constrained via a chemical scaffold, which confer structural stability leading to high affinity and selectivity usually associated with antibodies. Bicycles can be elaborated to carry payloads to a specific target and their relatively small size (1.5-3 kDa) allows rapid tissue penetration and extravasation. Bicycle binders for EphA2 were identified using a proprietary phage display peptide technology consisting of highly diverse phage libraries of Bicycles. Initial screening identified Bicycle binders with high affinity for EphA2. EphA2 binding Bicycles were conjugated to cytotoxic payload via a cleavable linker to form a Bicycle Toxin Conjugate (BTC) and evaluated for EphA2 dependent cytotoxicity in vitro and in vivo. Efficacy was seen in EphA2-expressing xenograft models, but toxicity was seen at higher doses. These Bicycles were highly plasma protein bound, and relatively lipophilic. Visualization of Bicycle distribution by microPET showed localization to tumor, but with significant distribution to liver. Subsequent screening with a wider range of phage libraries and scaffolds identified Bicycle binders with alternate structures, lower PPB and lipophilicity. Crystal structures of these Bicycles bound to EphA2 enabled rational design to enhance potency and drug-like properties. Visualization of bio-distribution of these optimised Bicycles by microPET showed localization to tumor, with negligible localization to liver and renal clearance. A range of Bicycle Toxin Conjugates were produced by linking bicycle binders to cleavable linkers and toxin payloads. A series of BTCs were developed with DM1 payload, with linkers ranging from uncleavable to unhindered disulfide. Similarly, a series of BTCs were developed with MMAE payload, using uncleavable linker and a variety of cleavable peptidic linkers with different enzyme sensitivity. Binding affinity is maintained to EphA2 protein and cells expressing EphA2. BTC efficacy was evaluated in EphA2-expressing xenograft models, initially the HT-1080 fibrosarcoma model, but also MDA-MB-231 triple-negative breast cancer and NCI-H1975 lung cancer models. Efficacy was seen from 1mg/kg qw and complete regression of tumors from 2 weeks dosing at 2mg/kg qw. No efficacy was seen in xenograft models without EphA2 expression (eg MOLP-8).From >75 BTCs evaluated, BT5528 was chosen as a candidate molecule to progress to in-depth preclinical profiling. The development of Bicycle Toxin Conjugates against EphA2 allows fast and efficient targeting of solid tumors while sparing non-tumor tissues. Citation Format: Gavin Bennett, Philip Huxley, Amy Brown, Gemma Mudd, Katerine van Rietschoten, Silvia Pavan, Liuhong Chen, Sophie M. Watcham, Peter U. Park, Nicholas Keen. Bicycle Drug Conjugates targeting EphA2 for the treatment of solid tumors: Discovery and selection of BT5528 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5855.