Dorsal Skinfold Window Chamber Research Articles

MEK Inhibitors, Novel Anti-Adhesive Molecules, Reverse Sickle Red Blood Cell Adhesion and Vaso-Occlusion in Vivo

In sickle cell disease (SCD), painful vaso-occlusive crises and end-organ damage are caused by obstruction of the vessels due in large part to sickle erythrocyte (SSRBC) adherence to both the endothelium and leukocytes. Despite the contribution of SSRBC adhesiveness to vaso-occlusion, we lack treatments that specifically target intracellular signaling mechanisms critical for SSRBC cell-cell interactions. We have previously shown that the mitogen-activated protein (MAP) kinase ERK1/2 is aberrantly activated in SSRBCs. ERK1/2 signaling activation is critical for SSRBC interactions with the vascular endothelium and leukocytes. We have now evaluated the anti-adhesive properties of RDEA119, an inhibitor of MEK1/2, the kinase that activates the MAP kinase ERK1/2, in in vitro adhesion assays and in vivo in both nude mouse and humanized models of vaso-occlusion analyzed by intravital microscopy. We used RDEA119 because this dryg has previously been developed for human use. We found that RDEA119 caused a dose-dependent inhibition of human SSRBC adhesion to cultured TNFα-activated human micro-vascular endothelial cells (HMVECs) in vitro with a half-maximum inhibitory concentration (IC50) < 2 nmol/L. This suggests that RDEA119 would be effective at very low doses in reducing SSRBC adhesion and vaso-occlusion in vivo. To determine if RDEA119 prevents vaso-occlusion in nude mice, animals were injected with TNFα followed by infusion of RDEA119 (or placebo control), and then human SSRBCs. Intravital microscopy observations of enflamed vessels visible through dorsal skin-fold window chamber implants immediately after human SSRBC infusion showed persistent human SSRBC adhesion to both small and large vessels, with mean diameter of 29±3.5 μm, and vaso-occlusion in placebo-treated nude mice for at least 2 h, promoting blood stasis. However, treatment of nude mice with either 0.2 mg/kg or 2 mg/kg RDEA119 markedly reduced human SSRBC adhesion and mitigated vaso-occlusion. While human SSRBC adhered to 68.5±4.6% of total vessels recorded in placebo-treated animals, SSRBCs occupied only 33 ± 6.6% (p<0.0001) and 25 ± 3.7% (p<0.0001) of vessels in 0.2 and 2 mg/kg RDEA119-treated nude mice, respectively. As a result, normal blood flow was restored in 95±3.5% and 76±6.2% of vessels in 0.2 and 2 mg/kg RDEA119-treated animals, respectively, compared to 43±5.8% of vessels with normal blood flow in placebo-treated mice (p<0.001). Nude mice treated with 0.2 mg/kg RDEA119 also exhibited reduced human SSRBC trapping in the lungs, liver and spleen (p<0.0001), and an increased number of human SSRBCs remaining in circulation. RDEA119 was also administered to nude mice after onset of vaso-occlusion for a more clinically relevant outcome. Infusion of a single dose of 1 mg/kg RDEA119 to nude mice reduced human SSRBC adhesion and vaso-occlusion within 5 min of RDEA119 administration, and adhesion progressively decreased thereafter compared to SSRBC adhesion and vaso-occlusion in these same vessel segments prior to drug administration (p<0.0001). RDEA119 led to a 72% reduction in the percentage of vessels occupied by adherent human SSRBCs (p<0.001) and improvement in blood flow in 89±2.4% of vessels (p<0.001). Similar therapeutic benefits were obtained when RDEA119 was administered after the inflammatory trigger of TNFα to induce vaso-occlusion in transgenic sickle mice. RDEA119 resulted in a significant decrease in adherent sickle cells in enflamed vessels of sickle mice treated with only one dose of RDEA119 at 0.1, 0.05 or 0.025 mg/kg (p<0.001). RDEA119 at 0.1, 0.05 or 0.025 mg/kg also significantly reduced adhesion of leukocytes to the vascular endothelium compared with placebo-treated sickle mice (p<0.001). The effect of RDEA119 on both sickle RBCs and leukocyte adhesion was rapid, because a reduction in adhesion of the cells was already detectable within the first 15 minutes after drug infusion, and adhesion further decreased thereafter. In contrast, adherence of leukocytes increased progressively over time in placebo-treated sickle mice. Treatment of sickle mice with RDEA119 led to improved microcirculatory blood flow in enflamed vessels visible through the dorsal skin-fold window chamber and improved survival. These results suggest that RDEA119 may represent a valuable novel therapeutic intervention for acute pain crises that should be further evaluated in humans with SCD. Disclosures No relevant conflicts of interest to declare.

Microvascular effects of the low molecular weight heparins in a colorectal xenograft model: an intravital microscopy study

BackgroundLow molecular weight heparins (LMWHs) are the cornerstone of the prevention of thromboembolic events in surgical patients. Recent clinical data suggest that LMWHs might improve survival of cancer patients, independent of their anticoagulant effect. The anti-cancer mechanism of LMWHs is incompletely understood, but may include effects on tumor angiogenesis. We assessed the effects of LMWHs on tumor angiogenesis and microcirculation in a mouse colorectal xenograft model using in vivo microscopy in window chambers. MethodsHT29 human colorectal cancers were implanted in dorsal skinfold window chambers in athymic mice. Animals (n = 8 per group) were treated with 200 IU of nadroparin, enoxaparin, or saline for 8 d. Repeated intravital fluorescence microscopy was performed to calculate functional microcirculatory parameters as follows: number (N) and length (L) of microvessels, vascular area fraction (AF), and red blood cell velocity. Microvessel density, microvessel fractal dimension, and pericyte coverage were assessed histologically. ResultsActive angiogenesis was observed in control animals, resulting in a significant increase in N, L, and AF. In nadroparin- and enoxaparin-treated animals, however, AF did not change significantly over time and N and L remained significantly lower than untreated animals on day 7. Compared with control animals, nadroparin- and enoxaparin-treated animals showed a significantly lower microvessel density, but a higher pericyte coverage index, indicating a more mature microvessel network. ConclusionsThe LMWHs nadroparin and enoxaparin inhibit tumor angiogenesis and result in microvessel normalization in this in vivo observed colorectal xenograft model.

Abstract 5377: Assessing the chemotherapeutic response of nanoliposomal topotecan in conjunction with radiotherapy on tumor and its microenvironment

Abstract Topotecan (TPT), a highly active, water soluble analogue of camptothecin, is currently being used in the treatment of various localized cancers from different tissue origin including the breast. Topotecan is classically known for its ability to inhibit topoisomerase I activity as well as accumulation of HIF-1α to block upregulation of pro-angiogenic factors. Despite its known anticancer and antiangiogenic properties the clinical use of TPT is limited owing to its rapid clearance and hematological toxicity. Liposomal encapsulation of TPT can potentially increase its efficacy by shielding it from systemic clearance, allowing greater uptake and extended tissue exposure in solid tumors. We hypothesize that tumor cell biology and treatment response would be more informative when done in the presence of stromal components, like endothelial cells. To that end we have developed a 3D in-vitro/ in-vivo murine model of Triple Negative Breast Cancer where 4T1-GFP tumor cells are co-cultured with their stromal component (2H11 murine endothelial cells) in ‘hanging drops’ of medium. In the present study we have determined the effective dose of radiation and TPT for 4T1 tumor and 2H11 endothelial cells by cell viability and clonogenic assays. Based on the observed toxicity treatment response of actively loaded liposomal topotecan (ALLTs) and free drug with or without radiation exposure (3Gy) in tumor-endothelial spheroids was determined by monitoring the spheroid size and quantitation of apoptotic changes observed. 100nm DSPC and m-PEG DSPE (95:5 mol: mol) nanoliposomes were prepared in ammonium besylate and then TPT was actively loaded by pH gradient method. Tumor-endothelial spheroids grown in hanging drops, were subsequently transferred to cell repellent plates for high throughput in-vitro analysis. Our studies indicate that endothelial cells are more sensitive to TPT treatment (IC50 of 0.13µM) than tumor cells (IC50=63µM). Combinatorial treatment with topotecan loaded nanoliposome (ALLT) and radiation of tumor endothelial spheroid reveals a decrease in spheroid size and severe disruption of the peripheral layers with time. ALLTs based chemotherapy in conjunction with radiation was thus observed to be more efficacious in disrupting the integrity, more specifically the extracellular matrix of tumor-endothelial spheroids. Unlike the free drug which gets hydrolyzed into its relatively inactive carboxylate form, we expect that the ability of ALLTs for extended release of active TPT (more than 24 hours) in a metronomic fashion, may contribute to the therapeutic efficacy of its combined treatment with radiation. Efforts are underway to implant these spheroids in mammary fat pad and dorsal skin fold window chamber and study the effect of ALLTs in combination with radiation on tumor growth and neovascularization and metastasis. Supported by NCI grants R25CA153954 and CA173609 Citation Format: Amar Jyoti, Pallavi Sethi, Kyle Fugit, Ulrich Langner, William St. Clair, Ronald C. McGarry, Bradley D. Anderson, Meenakshi Upreti. Assessing the chemotherapeutic response of nanoliposomal topotecan in conjunction with radiotherapy on tumor and its microenvironment. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5377. doi:10.1158/1538-7445.AM2014-5377

Abstract 4589: Impact of tumor microenvironment on tumor growth, metastasis and response to combination therapy via microenvironment-responsive dual drug-loaded nanoparticles and radiation

Abstract Several studies including our own have made it increasingly clear that genetic alterations of tumor cells alone are not the sole driving force behind tumor development but that tumor initiation, growth, progression and response to combinatorial treatments are rather intimately controlled by the microenvironment. Improved knowledge of cancer biology and investigation of the complex functional interrelation between the cellular and noncellular compartments of the tumor microenvironment have provided an ideal platform for the evolution of novel cancer nanotherapies. In the present study we have developed an in-vitro method to co-culture color coded triple negative breast carcinoma cells and cell types that exist in the tumor/tumor microenvironment in a “hanging drop” of medium to the size of an avascular microtissue (∼800 µm). Changes in the fluorescence intensity of different cell types and spheroid/microtissue sizing is being utilized as a method for high throughput therapeutic evaluation of the combination therapy using nanoparticles. When implanted orthotopically and in the dorsal skin fold window chambers in nude mice they facilitate the imaging of neovascularization and understanding response to therapy. We have also observed that radiation (3Gy) can significantly enhances expression of Galectin-1 in tumor vasculature associated endothelial cells which can serve as a specific ligand for the 33 amino acid antiangiogenic, anginex peptide. Thus Galectin-1 is a promising candidate for actively targeting irradiated tumors with anginex conjugated nanoparticles. The objective of this study is to establish a novel strategy for radiation therapy-amplified delivery of dual drugs for targeting the microenvironment-responsive endothelial component of the tumor vasculature. Use of radiation-guided anginex bound nanoparticles for delivery and controlled release of dual drugs (Arsenic-cisplatin) are expected to simulate metronomic therapy and preferentially target the tumor endothelial cells at the irradiated tumor site via tumor stroma enriched Galectin-1 protein. Our molecular studies indicate differential vulnerability for arsenic and cisplatin in cell types constituting the tumor/tumor microenvironment. This is the first study to understand a novel combinatorial nanotherapeutic system in an in-vitro/ in-vivo breast tumor model which incorporates characteristics of the tumor microenvironment and is thus expected to better predict treatment response in patients. Supported by NCI grant CA173609 and (CNPP) U01CA151461 Citation Format: Pallavi Sethi, Amar Jyoti, Elden Swindell, Ulrich W. Langner, William H. St. Clair, Ronald C. McGarry, Thomas V. O'Halloran, Meenakshi Upreti. Impact of tumor microenvironment on tumor growth, metastasis and response to combination therapy via microenvironment-responsive dual drug-loaded nanoparticles and radiation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4589. doi:10.1158/1538-7445.AM2014-4589

Evaluation of doxorubicin-loaded pH-sensitive polymeric micelle release from tumor blood vessels and anticancer efficacy using a dorsal skin-fold window chamber model.

To evaluation the doxorubicin (DOX)-loaded pH-sensitive polymeric micelle release from tumor blood vessels into tumor interstitium using an animal vessel visibility model, the so-called dorsal skin-fold window chamber model. DOX-loaded pH-sensitive polyHis-b-PEG micelles and DOX-loaded pH-insensitive PLLA-b-PEG micelles were prepared. The uptake of the micelles by MDA-MB-231 breast cancer cells in vitro and in vivo was examined using flow cytometry. The pharmacokinetic parameters of the micelles were determined in SD rats after intravenous injection of a DOX dose (6 mg/kg). The release of the micelles from tumor vasculature and the antitumor efficacy were evaluated in MDA-MB-231 breast cancer xenografted in nude mice using a dorsal skin-fold window chamber. The effective elimination half-life t1/2 of the pH-sensitive, pH-insensitive polymeric micelles and DOX-PBS in rats were 11.3 h, 9.4 h, and 2.1 h, respectively. Intravital microscopy in MDA-MB-231 breast cancer xenografted in nude mice showed that the pH-sensitive polymeric micelles rapidly extravasated from the tumor blood vessels, and DOX carried by the pH-sensitive micelles was preferentially released at the tumor site as compared to the pH-insensitive polymeric micelles. Furthermore, the pH-sensitive polymeric micelles exhibited significant greater efficacy in inhibition of tumor growth in the nude mice. When DOX is loaded into pH-sensitive polymeric micelles, the acidity in tumor interstitium causes the destabilization of the micelles and triggers drug release, resulting in high local concentrations within the tumor, thus more effectively inhibiting the tumor growth in vivo.

Optimization of the dorsal skinfold window chamber model and multi-parametric characterization of tumor-associated vasculature.

The dorsal skinfold window chamber (DSWC) model is a unique tool that enables analysis of various aspects of tumor biology and therapeutic response. Although the protocol for the murine DSWC model is standardized, certain tumors fail to grow or require a particular environment to promote growth. Given such limitations, we optimized the DSWC model for a slow-growing tumor that regresses spontaneously in the standard protocol. We further characterized the vascular network in the tumor model compared with that of non-tumor-bearing mice and observed significant differences in multiple parameters related to vascular structure and function.

Limitations of the dorsal skinfold window chamber model in evaluating anti-angiogenic therapy during early phase of angiogenesis.

BackgroundAngiogenesis is an essential process during tumor development and growth. The murine dorsal skinfold window chamber model has been used for the study of both tumor microvasculature and other vascular diseases, including the study of anti-angiogenic agents in cancer therapy. Hyperspectral imaging of oxygen status of the microvasculature has not been widely used to evaluate response to inhibition of angiogenesis in early tumor cell induced vascular development. This study demonstrates the use of two different classes of anti-angiogenic agents, one targeting the Vascular Endothelial Growth Factor (VEGF) pathway involved with vessel sprouting and the other targeting the Angiopoietin/Tie2 pathway involved in vascular destabilization. Studies evaluated the tumor microvascular response to anti-angiogenic inhibitors in the highly angiogenic renal cell carcinoma induced angiogenesis model.MethodsHuman renal cell carcinoma, Caki-2 cells, were implanted in the murine skinfold window chamber. Mice were treated with either VEGF pathway targeted small molecule inhibitor Sunitinib (100 mg/kg) or with an anti-Ang-2 monoclonal antibody (10 mg/kg) beginning the day of window chamber surgery and tumor cell implantation. Hyperspectral imaging of hemoglobin saturation was used to evaluate both the development and oxygenation of the tumor microvasculature. Tumor volume over time was also assessed over an 11-day period post surgery.ResultsThe window chamber model was useful to demonstrate the inhibition of angiogenesis using the VEGF pathway targeted agent Sunitinib. Results show impairment of tumor microvascular development, reduced oxygenation of tumor-associated vasculature and impairment of tumor volume growth compared to control. On the other hand, this model failed to demonstrate the anti-angiogenic effect of the Ang-2 targeted agent. Follow up experiments suggest that the initial surgery of the window chamber model may interfere with such an agent thus skewing the actual effects on angiogenesis.ConclusionsResults show that this model has great potential to evaluate anti-VEGF, or comparable, targeted agents; however the mere protocol of the window chamber model interferes with proper evaluation of Ang-2 targeted agents. The limitations of this in vivo model in evaluating the response of tumor vasculature to anti-angiogenic agents are discussed.

Multimodality pH imaging in a mouse dorsal skin fold window chamber model.

Upregulate levels of expression and activity of membrane H+ ion pumps in cancer cells drives the extracellular pH (pHe,) to values lower than normal. Furthermore, disregulated pH is indicative of the changes in glycolytic metabolism in tumor cells and has been shown to facilitate extracellular tissue remodeling during metastasis Therefore, measurement of pHe could be a useful cancer biomarker for diagnostic and therapy monitoring evaluation. Multimodality in-vivo imaging of pHe in tumorous tissue in a mouse dorsal skin fold window chamber (DSFWC) model is described. A custom-made plastic window chamber structure was developed that is compatible with both imaging optical and MR imaging modalities and provides a model system for continuous study of the same tissue microenvironment on multiple imaging platforms over a 3-week period. For optical imaging of pHe, SNARF-1 carboxylic acid is injected intravenously into a SCID mouse with an implanted tumor. A ratiometric measurement of the fluorescence signal captured on a confocal microscope reveals the pHe of the tissue visible within the window chamber. This imaging method was used in a preliminary study to evaluate sodium bicarbonate as a potential drug treatment to reverse tissue acidosis. For MR imaging of pHe the chemical exchange saturation transfer (CEST) was used as an alternative way of measuring pHe in a DSFWC model. ULTRAVIST®, a FDA approved x-ray/CT contrast agent has been shown to have a CEST effect that is pH dependent. A ratiometric analysis of water saturation at 5.6 and 4.2 ppm chemical shift provides a means to estimate the local pHe.

Selection of Polyhydroxybutyrate-Co-Valerate (PHBV) as An Improved Polymer Choice in FTY720 Loaded Nanofibers for Islet Transplant

Introduction: These studies aim to determine a biodegradeable polymer that can be used within a tissue engineering device that does not reduce the viability of islets to the same degree as poly(lactic-co-glycolic acid) (PLAGA), but can still be successfully electrospun and loaded with FTY720. Drug release profile and in vivo immune response support the new polymer choice. Methods: Nanofibers are electrospun resulting in scaffolds consisting of several types of polymers, including equal blends by weight with polycaprolactone (PCL), with and without FTY720. Images are obtained from dorsal skinfold window chambers where a FTY720 loaded implant and an unloaded implant are placed in the same animal immediately following window surgery. Microvessel metrics are quantified with a MATLAB program. Islets are stained for viability (Propidium Iodide and Fluorescein Diacetate). Release samples are analyzed with Liquid Chromatography-Mass Spectrometry (LC-MS) following lipid extraction. Hematoxylin and Eosin stained slides are obtained from paraffin embedded tissue samples. Results: Human islets tested with PLAGA/PCL nanofibers displayed a decrease in viability compared to the media only controls (68% PLAGA/PCL verses 78% media only). Other polymer types polyhydroxybutyrate (PHB) and polyhydroxybutyrate-co-valerate (PHBV) performed better in this assay (75% and 79% respectively). LC-MS assisted in vitro release data indicate that a burst release (>10% loaded drug within 48 hours) is followed by a steady release resulting in 25% of the loaded FTY720 released by 2 weeks. Water droplet absorption time into nanofiber scaffolds indicate differences when FTY720 is added (< 5 sec FTY720 Loaded verses >3 min Unloaded scaffold, until complete wetting) likely related to the amphiphilic character of FTY720. Image analysis of window chambers with dual implants, indicate that after an initial 3 day time period a preferential blood flow in the area surrounding the FTY720 loaded implant exists as measured by both volume of blood in the tissue and length of visible blood vessels. Preliminary histology from subcutaneous implant studies indicate that the PLAGA/PCL fiber scaffolds contain more inflammatory cells and are characterized by tertiary lymphoid tissue development. In contrast, the PHBV/PCL fibers elicit an immune response, but do not result in formation of lymphoid tissue. Conclusion: The monomers of PLAGA are acidic. A decrease in culture media pH may contribute to a loss in islet viability with PLAGA fibers, which is less of an issue with the slower degrading PHBV. FTY720 releases from the fibers at a rate which can drive microvessel remodeling following injury perhaps by the recruitment of microvessel support cells. A depot of FTY720 is maintained however that will release for an extended period of time. Water droplet absorption combined with the burst release suggest that some of the FTY720 is locating near the surface of the fibers. PHBV scaffolds improved viability of islets in culture over PLAGA/PCL scaffolds. Preliminary in vivo studies demonstrate that PHBV/PCL fibers instigate less of an immune response than PLAGA/PCL fibers. In conclusion, PHBV has shown promise as a replacement polymer for PLAGA in FTY720 loaded nanofiber scaffolds for islet transplant.

QUANTIFYING NANOPARTICLE TRANSPORT IN VIVO USING HYPERSPECTRAL IMAGING WITH A DORSAL SKINFOLD WINDOW CHAMBER.

We have developed a noninvasive imaging method to quantify in vivo drug delivery pharmacokinetics without the need for blood or tissue collection to determine drug concentration. By combining the techniques of hyperspectral imaging and a dorsal skinfold window chamber, this method enabled the real-time monitoring of vascular transport and tissue deposition of nanoparticles labeled with near-infrared (NIR) dye. Using this imaging method, we quantified the delivery pharmacokinetics of the native high-density lipoprotein (HDL) and epidermal growth factor receptor (EGFR)-targeted HDL nanoparticles and demonstrated these HDLs had long circulation time in blood stream (half-life >12 h). These HDL nanoparticles could efficiently carry cargo DiR-BOA to extravasate from blood vessels, diffuse through extracellular matrix, and penetrate and be retained in the tumor site. The EGFR targeting specificity of EGFR-targeted HDL (EGFR-specific peptide conjugated HDL) was also visualized in vivo by competitive inhibition with excess EGFR-specific peptide. In summary, this imaging technology may help point the way toward the development of novel imaging-based pharmacokinetic assays for preclinical drugs and evaluation of drug delivery efficiency, providing a dynamic window into the development and application of novel drug delivery systems.

Intravital microscopy at the single vessel level brings new insights of vascular modification mechanisms induced by electropermeabilization

Electroporation/electropermeabilization, i.e. the result of the application of electric pulses to tissues, is a physical method for delivery of exogenous molecules into cells. It is effective particularly for compounds with limited transmembrane transport. In vivo, electropermeabilization facilitates the delivery of chemotherapeutic drugs into tumor cells that is the basic mechanism of the antitumor effectiveness of electrochemotherapy. This therapy has also blood flow modifying effects in tissues. The aim of our present study was to understand and explain the effects of electropermeabilization on the dynamics (vasomotricity, permeability and recovery) of subcutaneous blood vessels towards different size of molecules. These features were measured in C57Bl/6 mice via a dorsal skin fold window chamber, using fluorescently labeled dextrans of different sizes, intravital fluorescence microscopy imaging and specific image analysis. Application of electric pulses on the skin in vivo resulted in a rapid increase in vascular permeability that gradually recovered to basal levels at different times post-treatment, depending on dextran size. Simultaneously, the immediate constriction of the blood vessels occurred that was more pronounced for arterioles compared to venules. This vasoconstriction of arterioles results in a transient “vascular lock”.The increased permeability of small vessels walls whatever the dextran size associated with delayed perfusion explains the improved delivery of the intravenous injected molecules (i.e. drugs, gene delivery) into the tissues induced by electropermeabilization in vivo.

Arteriolar and Venular Remodeling Are Differentially Regulated by Bone Marrow-Derived Cell-Specific CX3CR1 and CCR2 Expression

The chemokine receptors CCR2 and CX3CR1 are critical for the recruitment of “inflammatory” and “resident” monocytes, respectively, subpopulations that differentially affect vascular remodeling in atherosclerosis. Here, we tested the hypothesis that bone marrow-derived cell (BMC)-specific CCR2 and CX3CR1 differentially control venular and arteriolar remodeling. Venular and arteriolar lumenal remodeling were observed by intravital microscopy in mice with either CCR2 or CX3CR1 deficient BMCs after implantation of a dorsal skinfold window chamber, a model in which arterioles and venules lumenally enlarge in wild-type (WT) mice. Arteriolar remodeling was abolished in mice with either CCR2 or CX3CR1-deficient BMCs. In contrast, the loss of CX3CR1 from BMCs, but not CCR2, significantly reduced small venule remodeling compared to WT controls. We conclude that microvascular remodeling is differentially regulated by BMC-expressed chemokine receptors. Both CCR2 and CX3CR1 regulate arteriole growth; however, only BMC-expressed CX3CR1 impacts small venule growth. These findings may provide a basis for additional investigations aimed at determining how patterns of monocyte subpopulation recruitment spatially influence microvascular remodeling.

Microcirculation and NO-CO Studies of a Natural Extracellular Hemoglobin Developed for an Oxygen Therapeutic Carrier

Extracellular soluble hemoglobins (Hbs) have long been studied for their possible use as safe and effective alternatives to blood transfusion. While remarkable progress has been made in the use of cell-free Hb as artificial oxygen carrier, significant problems remain, including susceptibility to oxidative inactivation and propensity to induce vasoconstriction. Hemarina-M101 is a natural giant extracellular hemoglobin (3600 kDa) derived from marine invertebrate (polychaete annelid). Hemarina-M101 is a biopolymer composed of 156 globins and 44 non-globin linker chains and formulated in a product called HEMOXYCarrier®. Prior work has shown Hemarina-M101 to possess unique anti-oxidant activity and a high oxygen affinity. Topload experiment with this product into rats did not revealed any effect on heart rate (HR) and mean arterial pressure (MAP). A pilot study with the hamster dorsal skinfold window chamber model showed absence of microvascular vasoconstriction and no significant impact on mean arterial blood pressure. In vitro nitric oxide (NO) and carbon monoxide (CO) reaction kinetics measurements show that Hemarina-M101 has different binding rates as compared to human Hb. These results revealed for the first time that the presence of this marine hemoglobin appears to have no vasoactivity at the microvascular level in comparison to others hemoglobin based oxygen carriers (HBOCs) developed so far and merits further investigation.

Abstract 4644: A novel strategy for targeted drug delivery to the tumor vasculature by radiation-induced receptor expression on endothelial cells

Abstract One of the primary goals of a successful cancer treatment regimen is to deliver an effective combination of radiation and/or drugs to tumors while minimizing damage to normal tissues. Many anti-angiogenic agents, while not able to control tumor growth, possess the ability to selectively target the location and process of tumor blood vessel formation. Conversely many chemotherapy agents are highly cytotoxic and lack selective targeting ability thus decreasing the therapeutic ratio. We are investigating a new drug delivery strategy exploiting the tumor endothelium “stimulated” by ionizing radiation to preferentially target and deliver a nanoparticulate formulation of arsenic trioxide (ATO) encapsulated in liposomal vesicles or “nanobins” to the irradiated tumor tissue. The targeting is via the 33 amino acid anti-angiogenic peptide, anginex. The identification of galectin-1 as the receptor for anginex expressed on activated endothelial cells involved in tumor angiogenesis has revealed a solid basis for this therapeutic rationale. Unlike the antiangiogenic agent Avastin, which is an antibody that targets the vascular endothelial growth factor (VEGF) released by the tumor cells into the microenvironment, this peptide binds to galectin-1, a tumor endothelial cell specific antigen that is expressed in solid tumors. We have made the novel discovery that galectin-1 expression is further upregulated in the tumor after radiation exposure, particularly on the endothelial cell surface. Exposure of murine SCK breast tumors to a clinical radiation dose of 2Gy induced a substantial average increase of 141 +/− 49% in anginex uptake as assessed by [18]-F-labeled anginex biodistribution. These results suggest that anginex may be an effective targeting molecule for image and radiation-guided therapy of solid tumors. To assist in delineating the exact mechanisms for our radiation-guided drug delivery strategy, we have developed a system to grow 3D tumor cell-endothelial cell spheroids and have observed increased galectin-1 expression upon radiation exposure. We are using these spheroidal cultures to implant tumors in dorsal skin fold window chambers for intravital wavelength imaging of drug delivery to tumor tissue before and after radiation exposure. Understanding how anginex uptake selectively increases in endothelial cells after irradiation and its nexus to radiation-sensitivity and drug delivery is our current focus. Further studies are underway to characterize the in vivo targeting of arsenic trioxide chemotherapy via anginex-conjugated ‘nanobins’ in combination with radiation exposure and assess the therapeutic potential in our solid tumor model. Supported by NCI grant CA107160 and the Central Arkansas Radiation Therapy Institute. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4644. doi:1538-7445.AM2012-4644

Abstract 4338: Monitoring radiation response in tumor vasculature using intravital photoacoustic imaging in a murine window chamber model in vivo

Abstract VisualSonics has recently developed a preclinical photoacoustic (PA) imaging system called the VevoLAZR that combines the sensitivity of optical imaging and the high resolution of micro-ultrasound. The system incorporates a 40 MHz (centre frequency) ultrasound transducer linear array probe (LZ550) and a tuneable 680-970 nm nanosecond pulsed-laser. We used this system to study in vivo changes in tumor oxygen saturation and haemoglobin density caused by exposure to radiation therapy (RT). For this, DsRed-Me180 human cervical tumors were grown in a nude mouse dorsal skinfold window chamber model until they reached 2.5 mm in diameter. Specifically, we investigated the system's sensitivity and dynamic range to measure relative changes in oxygen saturation in tumor and surrounding healthy tissues 10 days after treatment. Tumors (∼2.5 mm diameter) were focally irradiated with a single dose of 30 Gy using a small animal microirradiator (XRAD225, Precision XRay Inc., North Branford, CT). To measure the dynamic range and stability of our setup for measuring oxygen saturation in vivo, we altered the anesthetised animal's inhaled oxygen from 100% to 7% for 1 min during PA imaging. This test showed that blood oxygen saturation in the healthy dorsal skinfold tissue decreased from 82% to 8% and confirmed the linearity of the measurement technique. Furthermore, we compared vascular morphology obtained by photoacoustic imaging and intravital fluorescent microscopy using FITC-Dextran (2 MDa, injected 20 mins prior). This comparison showed good correlation and confirms that PA imaging can provide important structural information of vascularity. Photoacoustic imaging was performed before and 10 days after irradiation to assess changes in tumour volume, relative blood oxygen saturation, relative tissue oxygen saturation, and relative hemoglobin density. Ten days after irradiation, PA imaging showed that the tumour volume increased from 5.7 to 14.2 mm3, relative blood oxygen saturation decreased from 75.3 to 48.2%, relative tissue oxygen saturation decreased from 40.7 to 0.1%, and hemoglobin density decreased from 18457 to 3253 a.u. These data illustrate the capability of PA imaging to simultaneously measure multiple radiobiological response metrics from a single imaging scan. Pilot results demonstrate: i) the compatibility of the VisualSonics small animal VevoLAZR photoacoustic imaging system with intravital murine tumor models, ii) the sensitivity of the system to detect RT-induced changes in tumor vascular oxygen saturation non-invasively, in real time and in vivo, and iii) a new preclinical application of PA imaging for longitudinal monitoring of tumor response to RT in vivo. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4338. doi:1538-7445.AM2012-4338

Estimation of Minimum Doses for Optimized Quantum Dot Contrast‐Enhanced Vascular Imaging In Vivo

Quantum dot (QD) contrast-enhanced molecular imaging has potential for early cancer detection and image guided treatment, but there is a lack of quantitative image contrast data to determine optimum QD administered doses, affecting the feasibility, risk and cost of such procedures, especially in vivo. Vascular fluorescence contrast-enhanced imaging is performed on nude mice bearing dorsal skinfold window chambers, injected with 4 different QD solutions emitting in the visible and near infrared. Linear relationships are observed among the vascular contrast, injected contrast agent volume, and QD concentration in blood. Due primarily to differential light absorption by blood, the vasculature is optimally visualized when exciting in the 435-480 nm region in 81% of the cases (89 out of 110 regions of interest in 22 window chambers). The threshold dose, defined here as the quantity of injected nanoparticles required to yield a vascular target-to-autofluorescence ratio of 2, varies from 10.6 to 0.15 pmol g(-1) depending on the QD emission wavelength. The wavelength optimization maximum and broadband gain, defined as the ratio of threshold doses estimated for optimal and suboptimal (worst wavelength or broadband) spectral illumination, has average values of 4.5 and 1.9, respectively. This study demonstrates, for the first time, optimized QD imaging in vivo. It also proposes and validates a theoretical framework for QD dose estimation and quantifies the effects of blood absorption, QD emission wavelength, and vessel diameter relative to the threshold dose.

Error analysis of ratiometric imaging of extracellular pH in a window chamber model

Ratiometric fluorescence-imaging technique is commonly used to measure extracellular pH in tumors and surrounding tissue within a dorsal skin-fold window chamber. Using a pH-sensitive fluorophore such as carboxy SNARF-1 one can measure pH distributions with high precision. However, it is often observed that the measured pH is lower than expected, with a bias that varies from one image to another. A comprehensive analysis of possible error sources is presented. These error sources include photon noise, estimator bias, instrument errors, temperature, and calibration errors from biological factors.

Tumor-Endothelial Cell Three-dimensional Spheroids: New Aspects to Enhance Radiation and Drug Therapeutics

Classic cancer research for several decades has focused on understanding the biology of tumor cells in vitro. However, extending these findings to in vivo settings has been impeded owing to limited insights on the impact of microenvironment on tumor cells. We hypothesized that tumor cell biology and treatment response would be more informative when done in the presence of stromal components, like endothelial cells, which exist in the tumor microenvironment. To that end, we have developed a system to grow three-dimensional cultures of GFP-4T1 mouse mammary tumor and 2H11 murine endothelial cells in hanging drops of medium in vitro. The presence of 2H11 endothelial cells in these three-dimensional cocultures was found to sensitize 4T1-GFP tumor cells to chemotherapy (Taxol) and, at the same time, protect cells from ionizing radiation. These spheroidal cultures can also be implanted into the dorsal skinfold window chamber of mice for fluorescence imaging of vascularization and disease progression/treatment response. We observed rapid neovascularization of the tumor-endothelial spheroids in comparison to tumor spheroids grown in nude mice. Molecular analysis revealed pronounced up-regulation of several proangiogenic factors in the tumor tissue derived from the tumor-endothelial spheroids compared with tumor-only spheroids. Furthermore, the rate of tumor growth from tumor-endothelial spheroids in mice was faster than the tumor cell-only spheroids, resulting in greater metastasis to the lung. This three-dimensional coculture model presents an improved way to investigate more pertinent aspects of the therapeutic potential for radiation and/or chemotherapy alone and in combination with antiangiogenic agents.

FTY720-loaded poly(DL-lactide-co-glycolide) Electrospun Scaffold Significantly Increases Microvessel Density over 7 Days in Streptozotocin-Induced Diabetic C57b16/J Mice: Preliminary Results

Background Nanofiber scaffolds could improve islet transplant success by physically mimicking the shape of extracellular matrix and by acting as a drug-delivery vehicle. Scaffolds implanted in alternate transplant sites must be prevascularized or very quickly vascularized following transplantation to prevent hypoxia-induced islet necrosis. The local release of the S1P prodrug FTY720 induces diameter enlargement and increases in length density. The objective of this preliminary study was to evaluate length and diameter differences between diabetic and nondiabetic animals implanted with FTY720-containing electrospun scaffolds using intravital imaging of dorsal skinfold window chambers. Methods Electrospun mats of randomly oriented fibers we created from polymer solutions of PLAGA (50:50 LA:GA) with and without FTY720 loaded at a ratio of 1:200 (FTY720:PLAGA by wt). The implanted fiber mats were 4 mm in diameter and ∼0.2 mm thick. Increases in length density and vessel diameter were assessed by automated analysis of images over 7 days in RAVE, a Matlab program. Results Image analysis of repeated measures of microvessel metrics demonstrated a significant increase in the length density from day 0 to day 7 in the moderately diabetic animals of this preliminary study ( P < .05). Furthermore, significant differences in length density at day 0 and day 3 were found between recently STZ-induced moderately diabetic and nondiabetic animals in response to FTY720 local release ( P < .05, Student t test). Conclusions Driving the islet revascularization process using local release of factors, such as FTY720, from biodegradable polymers makes an attractive system for the improvement of islet transplant success. Preliminary study results suggest that a recently induced moderately diabetic state may potentiate the mechanism by which local release of FTY720 from polymer fibers increases length density of microvessels. Therefore, local release of S1P receptor–targeted drugs is under further investigation for improvement of transplanted islet function.

Exogenous intravascular nitric oxide enhances ventricular function after hemodilution with plasma expander

Aims This study evaluated the hypothesis that exogenous nitric oxide (NO) supplementation during acute hemodilution with plasma expander (PE) provides beneficial effects on cardiac function. Main methods Acute hemodilution in golden Syrian hamsters was induced by a 40% of blood volume exchange with dextran 70 kDa. Intravascular NO supplementation after hemodilution was accomplished with a NO donor, diethylenetriamine NONOate (DETA NONOate). The test group was treated with DETA NONOate, while the control group received only vehicle. Left ventricular cardiac function was studied using pressure–volume measurements obtained with a miniaturized conductance catheter. Key findings Cardiac output increased to 122 ± 5% and 107 ± 1% of the baseline in the group treated with NO donor and the vehicle group, respectively. Stroke work per stroke volume (SW/SV) after hemodilution reduced to 90% of the baseline and the NO donor significantly reduced SW/SV compared to the vehicle. The minimum rate of pressure change (dP/dt min) was significantly lower in animals treated with the NO donor compared to vehicle treated animals. Systemic vascular resistance (SVR) decreased to 62 ± 5% of the baseline in the NO donor group whereas the vehicle group SVR decreased to 83 ± 5% of the baseline. Using intravital microscopy analysis of microvessel in the dorsal skinfold window chamber, we established that the NO donor group induced significant vasodilation compared to the vehicle group. Significance NO supplementation in an acute hemodilution with PE has beneficial effects on cardiac performance. However, the NO supplementation effects with a NO donor are dose-independent and short-lasting.