Intravital Microscopy Imaging Research Articles

Recording the wild lives of immune cells.

Intravital microscopic imaging can uncover fundamental aspects of immune cell behavior in real time in both healthy and pathological states. Here, we discuss approaches for single-cell imaging of adaptive and innate immune cells to explore how they migrate, communicate, and mediate regulatory or effector functions in various tissues throughout the body. We further review how intravital single-cell imaging can be used to study drug effects on immune cells.

PO-438 Hyperthermia as neoadjuvant treatment for pancreatic tumours in murine models

IntroductionPancreatic ductal adenocarcinoma (PDAC) is the most common form of pancreatic cancer. The hallmarks of this malignancy, such as it’s deprived vasculature, dense tumour stroma and hypoxic tumour microenvironment are some of the reasons for very poor chemotherapy and radiotherapy treatment efficacy. In these regards, a new combinatory treatment regimen was proposed:1 non-invasive radiofrequency (RF) hyperthermia treatment to increase tumour perfusion, enhance cancer cell death and immune system stimulus;2 chemotherapy regiment with Gemzar and Abraxane.Material and methodsOrthotropic and subcutaneous models of PDAC were induce by injection of PANC-1, AsPc-1 and Pan_O2 cancer cells into Balb/c nude or C57BL/6J mice. Tumour growth was monitored via ultrasound VEVO2100. Tumour perfusion after hyperthermia was monitored via intravital confocal microscopy and ultrasound microbubble contrast imaging. The tumour active vasculature was investigated using Doppler imaging. The chemotherapy with Gemzar and Abraxane was administered in combination with RF hyperthermia. A portable-RF system was used to produce local hyperthermia in the tumour tissue. The device is powered by 200 W and emits 13.56 MHz electric fields. The entire treatment scheme was repeated a total of 6 times at 72 hour time intervals (Q3D × 6 protocol) resulting in 16 days of treatment. The variety of histochemistry staining was performed post mortem to show the influence of treatment on the tumour vasculature and the degree of perfusion and necrosis.Results and discussionsWe showed that RF hyperthermia treatment as a neoadjuvant therapy can be promising for PDAC treatment. However, the effect is still unpredictable and depends on pre-existing tumour physiology before therapy. We established the most effective RF hyperthermia procedure to increase drug perfusion into tumour tissue (41°C for 20–30 min). We studied a variety of chemotherapy dosages to adjust clinical protocols into mouse model. The Doppler ultrasound imaging was found to be a useful tool to investigate vasculature response to treatment.ConclusionThe benefit on Gemzar and Abraxane treatment was confirmed in mice. Although, the effect of treatment was statistically insignificant in comparison to saline treated mice in long-time scale (one year). The benefit of RF hyperthermia was hardly visible in the short-term and completely insignificant during long-term observation. However, increased perfusion in the tumour microenvironment after RF hyperthermia treatment was observed.

A disintegrin and metalloproteinase 15-mediated glycocalyx shedding contributes to vascular leakage during inflammation.

AimsEndothelial hyperpermeability exacerbates multiple organ damage during inflammation or infection. The endothelial glycocalyx, a protective matrix covering the luminal surface of endothelial cells (ECs), undergoes enzymatic shedding during inflammation, contributing to barrier hyperpermeability. A disintegrin and metalloproteinase 15 (ADAM15) is a sheddase capable of cleaving the ectodomains of membrane-bound molecules. Herein, we tested whether and how ADAM15 is involved in glycocalyx shedding and vascular leakage during sepsis.Methods and resultsDextran-150kD exclusion assay revealed lipopolysaccharide (LPS) significantly reduced glycocalyx thickness in mouse cremaster microvessels. Consistently, shedding products of glycocalyx constituents, including CD44 ectodomain, were detected with an increased plasma level after cecal ligation and puncture (CLP)-induced sepsis. The direct effects of CD44 ectodomain on endothelial barrier function were evaluated, which revealed CD44 ectodomain dose-dependently reduced transendothelial electrical resistance (TER) and caused cell–cell adherens junction disorganization. Furthermore, we examined the role of ADAM15 in CD44 cleavage and glycocalyx shedding. An in vitro cleavage assay coupled with liquid chromatography-tandem mass spectrometry confirmed ADAM15 cleaved CD44 at His235-Thr236 bond. In ECs with ADAM15 knockdown, LPS-induced CD44 cleavage and TER reduction were greatly attenuated, whereas, ADAM15 overexpression exacerbated CD44 cleavage and TER response to LPS. Consistently, ADAM15 knockout in mice attenuated CLP-induced increase in plasma CD44. Intravital and electron microscopic images revealed ADAM15 deficiency prevented LPS-induced glycocalyx injury in cremaster and pulmonary microvasculatures. Functionally, ADAM15−/− mice with better-preserved glycocalyx exhibited resistance to LPS-induced vascular leakage, as evidenced by reduced albumin extravasation in pulmonary and mesenteric vessels. Importantly, in intact, functionally vital human lungs, perfusion of LPS induced a significant up-regulation of ADAM15, accompanied by elevated CD44 in the effluent and increased vascular permeability to albumin.ConclusionTogether, our data support the critical role of ADAM15 in mediating vascular barrier dysfunction during inflammation. Its mechanisms of action involve CD44 shedding and endothelial glycocalyx injury.

SPARC preserves endothelial glycocalyx integrity, and protects against adverse cardiac inflammation and injury during viral myocarditis

Myocardial damage as a consequence of cardiotropic viruses leads to a broad variety of clinical presentations and is still a complicated condition to diagnose and treat. Whereas the extracellular matrix protein Secreted Protein Acidic and Rich in Cysteine or SPARC has been implicated in hypertensive and ischemic heart disease by modulating collagen production and cross-linking, its role in cardiac inflammation and endothelial function is yet unknown.Absence of SPARC in mice resulted in increased cardiac inflammation and mortality, and reduced cardiac systolic function upon coxsackievirus-B3 induced myocarditis. Intra-vital microscopic imaging of the microvasculature of the cremaster muscle combined with electron microscopic imaging of the microvasculature of the cardiac muscle uncovered the significance of SPARC in maintaining endothelial glycocalyx integrity and subsequent barrier properties to stop inflammation. Moreover, systemic administration of recombinant SPARC restored the endothelial glycocalyx and consequently reversed the increase in inflammation and mortality observed in SPARC KO mice in response to viral exposure. Reducing the glycocalyx in vivo by systemic administration of hyaluronidase, an enzyme that degrades the endothelial glycocalyx, mimicked the barrier defects found in SPARC KO mice, which could be restored by subsequent administration of recombinant SPARC.In conclusion, the secreted glycoprotein SPARC protects against adverse cardiac inflammation and mortality by improving the glycocalyx function and resulting endothelial barrier function during viral myocarditis.

Real-Time Assessment of Extracellular Vesicles by Intravital Microscopy Imaging

Abstract Extracellular vesicles (EVs), such as exosomes, are essential players in cell-cell communication, which are being considered for developing novel drug delivery applications. Thus, there is much interest in developing reliable methods for tracing EVs in biological settings in real-time. Current standard methods are based on lipophilic fluorescent dyes, though this labeling has not been validated and may result in biased understanding of EVs. Herein, we developed labeling methods directed to faithfully follow the EVs for assessing the fate in biological settings both in vitro and in vivo. Our results showed that by installing fluorescent probes on the surface of EVs by conjugation to the EVs’ amino groups, it is possible to preserve the EVs concentration and stability. Moreover, we found that the EVs labeled by our method were stable in the bloodstream, while the EVs labeled with the conventional lipophilic dyes were immediately removed from circulation, highly accumulating in the lungs. Finally, by using our new labeling method for imaging EVs, we found that EVs from murine breast cancer 4T1 accumulated along the brain capillaries, which correlate with the incidence of brain metastasis from breast tumors.

Ferumoxytol-based Dual-modality Imaging Probe for Detection of Stem Cell Transplant Rejection.

Purpose: Stem cell transplants are an effective approach to repair large bone defects. However, comprehensive techniques to monitor the fate of transplanted stem cells in vivo are lacking. Such strategies would enable corrective interventions at an early stage and greatly benefit the development of more successful tissue regeneration approaches. In this study, we designed and synthesized a dual-modality imaging probe (Feru-AFC) that can simultaneously localize transplanted stem cells and diagnose immune rejection-induced apoptosis at an early stage in vivo.Methods: We used a customized caspase-3 cleavable peptide-dye conjugate to modify the surface of clinically approved ferumoxytol nanoparticles (NPs) to generate the dual-modality imaging probe with fluorescence “light-up” feature. We labeled both mouse mesenchymal stem cells (mMSCs, matched) and pig mesenchymal stem cells (pMSCs, mismatched) with the probe and transplanted the labeled cells with biocompatible scaffold at the calvarial defects in mice. We then employed intravital microscopy (IVM) and magnetic resonance imaging (MRI) to investigate the localization, engraftment, and viability of matched and mismatched stem cells, followed by histological analyses to evaluate the results obtained from in vivo studies.Results: The Feru-AFC NPs showed good cellular uptake efficiency in the presence of lipofectin without cytotoxicity to mMSCs and pMSCs. The fluorescence of Feru-AFC NPs was turned on inside apoptotic cells due to the cleavage of peptide by activated caspase-3 and subsequent release of fluorescence dye molecules. Upon transplantation at the calvarial defects in mice, the intense fluorescence from the cleaved Feru-AFC NPs in apoptotic pMSCs was observed with a concomitant decrease in the overall cell number from days 1 to 6. In contrast, the Feru-AFC NP-treated mMSCs exhibited minimum fluorescence and the cell number also remained similar. Furthermore, in vivo MRI of the Feru-AFC NP-treated mMSC and pMSCs transplants could clearly indicate the localization of matched and mismatched cells, respectively.Conclusions: We successfully developed a dual-modality imaging probe for evaluation of the localization and viability of transplanted stem cells in mouse calvarial defects. Using ferumoxytol NPs as the platform, our Feru-AFC NPs are superparamagnetic and display a fluorescence “light-up” signature upon exposure to activated caspase-3. The results show that the probe is a promising tool for long-term stem cell tracking through MRI and early diagnosis of immune rejection-induced apoptosis through longitudinal fluorescence imaging.

Structural Behavior of the Endothelial Glycocalyx Is Associated With Pathophysiologic Status in Septic Mice: An Integrated Approach to Analyzing the Behavior and Function of the Glycocalyx Using Both Electron and Fluorescence Intravital Microscopy.

The endothelial surface layer (ESL) regulates vascular permeability to maintain fluid homeostasis. The glycocalyx (GCX), which has a complex and fragile ultrastructure, is an important component of the ESL. Abnormalities of the GCX have been hypothesized to trigger pathological hyperpermeability. Here, we report an integrated in vivo analysis of the morphological and functional properties of the GCX in a vital organ. We examined the behavior of the ESL and GCX, using both electron microscopy (EM) and intravital microscopy (IVM). We also compared morphological changes in the ESL of mouse skin in a glycosidase-treated and control group. Combined approaches were also used to examine both morphology and function in a lipopolysaccharide-induced septic model and the pathophysiological features of leukocyte-endothelial interactions and in vivo vascular permeability. Using IVM, we identified an illuminated part of the ESL as the GCX and confirmed our observation using morphological and biochemical means. In septic mice, we found that the GCX was thinner than in nonseptic controls in both an EM image analysis (0.98 ± 2.08 nm vs 70.68 ± 36.36 nm, P< .001) and an IVM image analysis (0.36 ± 0.15 μm vs 1.07 ± 0.39 μm, P< .001). Under septic conditions, syndecan-1, a representative core protein of the GCX, was released into the blood serum at a higher rate in septic animals (7.33 ± 3.46 ng/mL) when compared with controls (below the limit of detection, P< .001). Significant increases in leukocyte-endothelial interactions, defined as the numbers of rolling or firm-sticking leukocytes, and molecular hyperpermeability to the interstitium were also observed after GCX shedding in vivo. Using IVM, we visualized an illuminated part of the ESL layer that was subsequently confirmed as the GCX using EM. Severe sepsis induced morphological degradation of the GCX, accompanied by shedding of the syndecan-1 core protein and an increase in leukocyte-endothelial interactions affecting the vascular permeability. Our in vivo model describes a new approach to deciphering the relationship between structural and functional behaviors of the GCX.

Surgical preparation of rats and mice for intravital microscopic imaging of abdominal organs.

Intravital microscopy is a powerful research tool that can provide insight into cellular and subcellular events that take place in organs in the body. However, meaningful results can only be obtained from animals whose physiology is preserved during the process of microscopy. Here I discuss the importance of preserving the overall state of health of the animal, methods of anesthesia, surgical techniques for intravital microscopy of various abdominal organs, methods to maintain and monitor the physiology of the animal during microscopy and associated peri- and post-operative recovery considerations.

Sphingosine-1-Phosphate Treatment Can Ameliorate Microvascular Leakage Caused by Combined Alcohol Intoxication and Hemorrhagic Shock

Fluid resuscitation following hemorrhagic shock is often problematic, with development of prolonged hypotension and edema. In addition, many trauma patients are also intoxicated, which generally worsens outcomes. We directly investigated how alcohol intoxication impacts hemorrhagic shock and resuscitation-induced microvascular leakage using a rat model with intravital microscopic imaging. We also tested the hypothesis that an endothelial barrier-protective bioactive lipid, sphingosine-1-phosphate (S1P), could ameliorate the microvascular leakage following alcohol intoxication plus hemorrhagic shock and resuscitation. Our results show that alcohol intoxication exacerbated hemorrhagic shock and resuscitation-induced hypotension and microvascular leakage. We next found that S1P effectively could reverse alcohol-induced endothelial barrier dysfunction using both cultured endothelial cell monolayer and in vivo models. Lastly, we observed that S1P administration ameliorated hypotension and microvascular leakage following combined alcohol intoxication and hemorrhagic shock, in a dose-related manner. These findings suggest the viability of using agonists that can improve microvascular barrier function to ameliorate trauma-induced hypotension, offering a novel therapeutic opportunity for potentially improving clinical outcomes in patients with multi-hit injuries.

Using quantitative intravital multiphoton microscopy to dissect hepatic transport in rats.

Hepatic solute transport is a complex process whose disruption is associated with liver disease and drug-induced liver injury. Intravital multiphoton fluorescence excitation microscopy provides the spatial and temporal resolution necessary to characterize hepatic transport at the level of individual hepatocytes in vivo and thus to identify the mechanisms and cellular consequences of cholestasis. Here we present an overview of the use of fluorescence microscopy for studies of hepatic transport in living animals, and describe how we have combined methods of intravital microscopy and digital image analysis to dissect the effects of drugs and pathological conditions on the function of hepatic transporters in vivo.

Computer-aided quantification of microvascular networks: Application to alterations due to pathological angiogenesis in the hamster

Angiogenesis is both a physiological and a pathological process of great complexity, which is difficult to measure objectively and automatically. The hamster cheek pouch (HCP) prepared for intravital-microscopy (IVM) has been used to characterize microvascular functions in many studies and was chosen to investigate microvascular characteristics observed in normal non-infected hamsters as compared to those HCPs parasitized by Trypanosoma cruzi. Images of HCPs captured at IVM were subjected to computer based measurements of angiogenesis and histamine-induced macromolecular (FITC-dextran) leakage with an image segmentation approach that has the capacity to discriminate between fluorescence emitted by macromolecular tracers inside the vasculature and in the extravascular space. We present such an automatic segmentation methodology using known tools from image processing field that, to our knowledge, have not been tested in IVM images. We have compared this methodology with a recently published segmentation strategy based on image intensity thresholding. Our method renders an accurate and robust segmentation of blood vessels for different microvascular scenarios, normal and pathological. Application of the proposed strategy for objective and automatic measurement of angiogenesis detection was explored in detail.

Two-Photon Intravital Fluorescence Lifetime Imaging of the Kidney Reveals Cell-Type Specific Metabolic Signatures.

In the live animal, tissue autofluorescence arises from a number of biologically important metabolites, such as the reduced form of nicotinamide adenine dinucleotide. Because autofluorescence changes with metabolic state, it can be harnessed as a label-free imaging tool with which to study metabolism in vivo Here, we used the combination of intravital two-photon microscopy and frequency-domain fluorescence lifetime imaging microscopy (FLIM) to map cell-specific metabolic signatures in the kidneys of live animals. The FLIM images are analyzed using the phasor approach, which requires no prior knowledge of metabolite species and can provide unbiased metabolic fingerprints for each pixel of the lifetime image. Intravital FLIM revealed the metabolic signatures of S1 and S2 proximal tubules to be distinct and resolvable at the subcellular level. Notably, S1 and distal tubules exhibited similar metabolic profiles despite apparent differences in morphology and autofluorescence emission with traditional two-photon microscopy. Time-lapse imaging revealed dynamic changes in the metabolic profiles of the interstitium, urinary lumen, and glomerulus-areas that are not resolved by traditional intensity-based two-photon microscopy. Finally, using a model of endotoxemia, we present examples of the way in which intravital FLIM can be applied to study kidney diseases and metabolism. In conclusion, intravital FLIM of intrinsic metabolites is a bias-free approach with which to characterize and monitor metabolism in vivo, and offers the unique opportunity to uncover dynamic metabolic changes in living animals with subcellular resolution.

Ultrasound Molecular Imaging of Angiogenesis Using Vascular Endothelial Growth Factor-Conjugated Microbubbles.

Imaging of angiogenesis receptors could provide a sensitive and clinically useful method for detecting neovascularization such as occurs in malignant tumors, and responses to antiangiogenic therapies for such tumors. We tested the hypothesis that microbubbles (MB) tagged with human VEGF121 (MBVEGF) bind to the kinase insert domain receptor (KDR) in vitro and angiogenic endothelium in vivo, and that this specific binding can be imaged on a clinical ultrasound system. In this work, targeted adhesion of MBVEGF was evaluated in vitro using a parallel plate flow system containing adsorbed recombinant human KDR. There was more adhesion of MBVEGF to KDR-coated plates when the amount of VEGF121 on each MB or KDR density on the plate was increased. MBVEGF adhesion to KDR-coated plates decreased with increasing wall shear rate. On intravital microscopic imaging of bFGF-stimulated rat cremaster muscle, there was greater microvascular adhesion of MBVEGF compared to that of isotype IgG-conjugated control MB (MBCTL). To determine if MBVEGF could be used to ultrasonically image angiogenesis, ultrasound imaging was performed in mice bearing squamous cell carcinoma after intravenous injection of MBVEGF. Ultrasound videointensity enhancement in tumor was significantly higher for MBVEGF (17.3 ± 9.7 dB) compared to MBCTL (3.8 ± 4.4 dB, n = 6, p < 0.05). This work demonstrates the feasibility of targeted ultrasound imaging of an angiogenic marker using MBVEGF. This approach offers a noninvasive bedside method for detecting tumor angiogenesis and could be extended to other applications such as molecular monitoring of therapeutic angiogenesis or antiangiogenic therapies in cardiovascular disease or cancer.

Abstract B28: Dynamic interplay between type I collagen signaling and invasion in pancreatic cancer

Abstract The stroma of pancreatic cancer can restrict as well as promote tumor progression. Why the stroma exhibits these paradoxical functions remains unclear. One of the major components of the pancreatic cancer stroma is fibrillar type I collagen. High levels of collagen in primary tumor specimens have been associated with poor survival and resistance to therapy in pancreatic cancer patients. To determine the effects of type I collagen on cancer cell behavior, we performed intravital confocal microscopic imaging in mice. We used mice that expressed GFPtopaz fused to the α2 chain of type I collagen under control of a 3.6kb fragment of the COL1A1 promoter [α2(I)-col-GFP mice]. Live imaging of pancreatic tumors, formed after orthotopic injection of cells derived from the classical KPC mouse models, showed that type I collagen architecture in the tumors was heterogeneous. Strikingly, we observed that invasive cancer cells migrated out of the tumors on linear type I collagen. Linearization of collagen fibers through cross-linking, catalyzed by lysyl oxidases (LOXs), is inversely correlated with metastasis-free survival in breast cancer. However, in our pancreatic cancer model, inhibiting LOX promoted collagen linearization, tumor growth and metastatic spread. Elevated collagen-mediated signaling, through activation of focal adhesion kinase (FAK) and discoidin domain receptor (DDR1), was also observed in the tumors treated with lysyl oxidase inhibitor. Lysyl oxidase inhibition also promoted cell contractility as shown by elevated phosphorylated mysosin light chain 2 (p-MLC2) in vivo and by in collagen contractility assays in vitro. TGF-β signaling is a potent stimulator of collagen secretion and of invasive behavior, and it is aberrantly regulated in pancreatic cancer. We observed that lysyl oxidase inhibitor increased phosphorylated Smad3, a downstream effector of TGF-β signaling. Together, our data suggest that the influence of collagen on tumor behavior depends on its architecture. In areas of linear collagen, we find high cancer cell motility out of the tumors and this correlates with increased FAK and TGF-β signaling activity. The architecture-specific function of collagen could potentially explain the paradoxical role for the stroma in pancreatic cancer progression. Citation Format: Mario A. Shields, Pascal Maguin, Sarah L. Dallas, Mikala Egeblad.{Authors}. Dynamic interplay between type I collagen signaling and invasion in pancreatic cancer. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr B28.

Microglia contributes to plaque growth by cell death due to uptake of amyloid β in the brain of Alzheimer's disease mouse model

Pathological hallmarks of Alzheimer's disease (AD) include extracellularly accumulated amyloid β (Aβ) plaques and intracellular neurofibrillary tangles in the brain. Activated microglia, brain-resident macrophages, are also found surrounding Aβ plaques. The study of the brain of AD mouse models revealed that Aβ plaque formation is completed by the consolidation of newly generated plaque clusters in vicinity of existed plaques. However, the dynamics of Aβ plaque formation, growth and the mechanisms by which microglia contribute to Aβ plaque formation are unknown. In the present study, we confirmed how microglia are involved in Aβ plaque formation and their growth in the brain of 5XFAD mice, the Aβ-overexpressing AD transgenic mouse model, and performed serial intravital two-photon microscopy (TPM) imaging of the brains of 5XFAD mice crossed with macrophage/microglia-specific GFP-expressing CX3CR1GFP/GFP mice. We found that activated microglia surrounding Aβ plaques take up Aβ, which are clusters developed inside activated microglia in vivo and this was followed by microglial cell death. These dying microglia release the accumulated Aβ into the extracellular space, which contributes to Aβ plaque growth. This process was confirmed by live TPM in vivo imaging and flow cytometry. These results suggest that activated microglia can contribute to formation and growth of Aβ plaques by causing microglial cell death in the brain. GLIA 2016;64:2274-2290.

Endothelin-1 Treatment Induces an Experimental Cerebral Malaria–Like Syndrome in C57BL/6 Mice Infected with Plasmodium berghei NK65

Plasmodium berghei ANKA infection of C57BL/6 mice is a widely used model of experimental cerebral malaria (ECM). By contrast, the nonneurotropic P. berghei NK65 (PbN) causes severe malarial disease in C57BL/6 mice but does not cause ECM. Previous studies suggest that endothelin-1 (ET-1) contributes to the pathogenesis of ECM. In this study, we characterize the role of ET-1 on ECM vascular dysfunction. Mice infected with 106 PbN-parasitized red blood cells were treated with either ET-1 or saline from 2 to8 days postinfection (dpi). Plasmodium berghei ANKA-infected mice served as the positive control. ET-1-treated PbN-infected mice exhibited neurological signs, hypothermia, and behavioral alterations characteristic of ECM, dying 4 to 8 dpi. Parasitemia was not affected by ET-1 treatment. Saline-treated PbN-infected mice did not display ECM, surviving until 12 dpi. ET-1-treated PbN-infected mice displayed leukocyte adhesion to the vascular endothelia and petechial hemorrhages throughout the brain at 6 dpi. Intravital microscopic images demonstrated significant brain arteriolar vessel constriction, decreased functional capillary density, and increased blood-brain barrier permeability. These alterations were not present in either ET-1-treated uninfected or saline-treated PbN-infected mice. In summary, ET-1 treatment of PbN-infected mice induced an ECM-like syndrome, causing brain vasoconstriction, adherence of activated leukocytes in the cerebral microvasculature, and blood-brain barrier leakage, indicating that ET-1 is involved in the genesis of brain microvascular alterations thatare the hallmark of ECM.

Tumor Targeting and Pharmacokinetics of a Near-Infrared Fluorescent-Labeled δ-Opioid Receptor Antagonist Agent, Dmt-Tic-Cy5.

Fluorescence molecular imaging can be employed for the development of novel cancer targeting agents. Herein, we investigated the pharmacokinetics (PK) and cellular uptake of Dmt-Tic-Cy5, a delta-opioid receptor (δOR) antagonist-fluorescent dye conjugate, as a tumor-targeting molecular imaging agent. δOR expression is observed normally in the CNS, and pathologically in some tumors, including lung liver and breast cancers. In vitro, in vivo, and ex vivo experiments were conducted to image and quantify the fluorescence signal associated with Dmt-Tic-Cy5 over time using in vitro and intravital fluorescence microscopy and small animal fluorescence imaging of tumor-bearing mice. We observed specific retention of Dmt-Tic-Cy5 in tumors with maximum uptake in δOR-expressing positive tumors at 3 h and observable persistence for >96 h; clearance from δOR nonexpressing negative tumors by 6 h; and systemic clearance from normal organs by 24 h. Live-cell and intravital fluorescence microscopy demonstrated that Dmt-Tic-Cy5 had sustained cell-surface binding lasting at least 24 h with gradual internalization over the initial 6 h following administration. Dmt-Tic-Cy5 is a δOR-targeted agent that exhibits long-lasting and specific signal in δOR-expressing tumors, is rapidly cleared from systemic circulation, and is not retained in non-δOR-expressing tissues. Hence, Dmt-Tic-Cy5 has potential as a fluorescent tumor imaging agent.

Porphyrin Nanodroplets: Sub-micrometer Ultrasound and Photoacoustic Contrast Imaging Agents.

A novel class of all-organic nanoscale porphyrin nanodroplet agents is presented which is suitable for multimodality ultrasound and photoacoustic molecular imaging. Previous multimodality photoacoustic-ultrasound agents are either not organic, or not yet demonstrated to exhibit enhanced accumulation in leaky tumor vasculature, perhaps because of large diameters. In the current study, porphyrin nanodroplets are created with a mean diameter of 185 nm which is small enough to exhibit the enhanced permeability and retention effect. Porphyrin within the nanodroplet shell has strong optical absorption at 705 nm with an estimated molar extinction coefficient >5 × 10(9) m(-1) cm(-1) , allowing both ultrasound and photoacoustic contrast in the same nanoparticle using all organic materials. The potential of nanodroplets is that they may be phase-changed into microbubbles using high pressure ultrasound, providing ultrasound contrast with single-bubble sensitivity. Multispectral photoacoustic imaging allows visualization of nanodroplets when injected intratumorally in an HT1080 tumor in the chorioallantoic membrane of a chicken embryo. Intravital microscopy imaging of Hep3-GFP and HT1080-GFP tumors in chicken embryos determines that nanodroplets accumulated throughout or at the periphery of tumors, suggesting that porphyrin nanodroplets may be useful for enhancing the visualization of tumors with ultrasound and/or photoacoustic imaging.

Formulation and optimization of idarubicin thermosensitive liposomes provides ultrafast triggered release at mild hyperthermia and improves tumor response

Drug delivery through thermosensitive liposomes (TSL) in combination with hyperthermia (HT) has shown great potential. HT can be applied locally forcing TSL to release their content in the heated tumor resulting in high peak concentrations. To perform optimally the drug is ideally released fast (seconds) and taken up rapidly by tumor cells. The aim of this study was to develop a novel thermosensitive liposome formulation of the anthracycline idarubicin (IDA-TSL). The hydrophobicity of idarubicin may improve its release from liposomes and subsequently rapid cellular uptake when combined mild hyperthermia. Here, we investigated a series of parameters to optimize IDA-TSL formulation. The results show that the optimal formulation for IDA-TSL is DPPC/DSPC/DSPE-PEG (6/3.5/0.5mol%), with ammonium EDTA of 6.5 pH as loading buffer and a size of ~85nm. In vitro studies demonstrated minimal leakage of ~20% in FCS at 37°C for 1h, while an ultrafast and complete triggered release of IDA was observed at 42°C. On tumor cells IDA-TSL showed comparable cytotoxicity to free IDA at 42°C, but low cytotoxicity at 37°C. Intravital microscopy imaging demonstrated an efficient in vivo intravascular triggered drug release of IDA-TSL under mild hyperthermia, and a subsequent massive IDA uptake by tumor cells. In animal efficacy studies, IDA-TSL plus mild HT demonstrated prominent tumor growth inhibition and superior survival rate over free IDA with HT or a clinically used Doxil treatment. These results suggest beneficial potential of IDA-TSL combined with local mild HT.

Emerging Applications for Optically Enabled Intravital Microscopic Imaging in Radiobiology

Radiation therapy is an effective cancer treatment used in over 50% of cancer patients. Preclinical research in radiobiology plays a major role in influencing the translation of radiotherapy-based treatment strategies into clinical practice. Studies have demonstrated that various components of tumors and their microenvironments, including vasculature, immune and stem cells, and stromal cells, can influence the response of solid tumors to radiation. Optically enabled imaging techniques used in experimental animal models of cancer offer a unique and powerful way to quantitatively track spatiotemporal changes in these tumor components in vivo at macro-, meso-, and microscopic resolutions following radiotherapy. In this review, we discuss the role of both well-established and emerging intravital microscopy techniques for studying tumors and their microenvironment in vivo, in response to irradiation. The development and application of new animal models, small animal microirradiation technologies, and multimodal optically enabled intravital microscopy techniques are emphasized within the framework of preclinical radiobiology research. We also comment on the potential influence that these newer imaging techniques may have on the clinical translation of new preclinical radiobiology discoveries.