Steven Machtaler

Molecular Imaging of Inflammation in Inflammatory Bowel Disease with a Clinically Translatable Dual-Selectin–targeted US Contrast Agent: Comparison with FDG PET/CT in a Mouse Model

PURPOSE To develop and test a molecular imaging approach that uses ultrasonography (US) and a clinically translatable dual-targeted (P- and E-selectin) contrast agent (MBSelectin) in the quantification of inflammation at the molecular level and to quantitatively correlate selectin-targeted US with fluorodeoxyglucose (FDG) combined positron emission tomography (PET) and computed tomography (CT) in terms of visualization and quantification of different levels of inflammation in a murine acute colitis model. MATERIALS AND METHODS Animal studies were approved by the Institutional Administrative Panel on Laboratory Animal Care at Stanford University. MBSelectin was developed by covalently binding an analog of the naturally occurring binding ligand P-selectin glycoprotein ligand 1 fused to a human fragment crystallizable(or Fc) domain onto the lipid shell of perfluorobutane and nitrogen-containing MBs. Binding specificity of MBSelectin was assessed in vitro with a flow chamber assay and in vivo with a chemically induced acute colitis murine model. US signal was quantitatively correlated with FDG uptake at PET/CT and histologic grade. Statistical analysis was performed with the Student t test, analysis of variance, and Pearson correlation analysis. RESULTS MBSelectin showed strong attachment to both human and mouse P- and E-selectin compared with MBControl in vitro (P ≤ .002). In vivo, US signal was significantly increased (P < .001) in mice with acute colitis (173.8 arbitrary units [au] ± 134.8 [standard deviation]) compared with control mice (5.0 au ± 4.5). US imaging signal strongly correlated with FDG uptake on PET/CT images (ρ = 0.89, P < .001). Ex vivo analysis enabled confirmation of inflammation in mice with acute colitis and high expression levels of P- and E-selectin in mucosal capillaries (P = .014). CONCLUSION US with MBSelectin specifically enables detection and quantification of inflammation in a murine acute colitis model, leveraging the natural pathway of leukocyte recruitment in inflammatory tissue. US imaging with MBSelectin correlates well with FDG uptake at PET/CT imaging.

Detection of Pancreatic Ductal Adenocarcinoma in Mice by Ultrasound Imaging of Thymocyte Differentiation Antigen 1

BACKGROUND & AIMS Early detection of pancreatic ductal adenocarcinoma (PDAC) allows for surgical resection and increases patient survival times. Imaging agents that bind and amplify the signal of neovascular proteins in neoplasms can be detected by ultrasound, enabling accurate detection of small lesions. We searched for new markers of neovasculature in PDAC and assessed their potential for tumor detection by ultrasound molecular imaging. METHODS Thymocyte differentiation antigen 1 (Thy1) was identified as a specific biomarker of PDAC neovasculature by proteomic analysis. Up-regulation in PDAC was validated by immunohistochemical analysis of pancreatic tissue samples from 28 healthy individuals, 15 with primary chronic pancreatitis tissues, and 196 with PDAC. Binding of Thy1-targeted contrast microbubbles was assessed in cultured cells, in mice with orthotopic PDAC xenograft tumors expressing human Thy1 on the neovasculature, and on the neovasculature of a genetic mouse model of PDAC. RESULTS Based on immunohistochemical analyses, levels of Thy1 were significantly higher in the vascular of human PDAC than chronic pancreatitis (P = .007) or normal tissue samples (P < .0001). In mice, ultrasound imaging accurately detected human Thy1-positive PDAC xenografts, as well as PDACs that express endogenous Thy1 in genetic mouse models of PDAC. CONCLUSIONS We have identified and validated Thy1 as a marker of PDAC that can be detected by ultrasound molecular imaging in mice. The development of a specific imaging agent and identification of Thy1 as a new biomarker could aid in the diagnosis of this cancer and management of patients.

Ultrasound-guided delivery of microRNA loaded nanoparticles into cancer

Ultrasound induced microbubble cavitation can cause enhanced permeability across natural barriers of tumors such as vessel walls or cellular membranes, allowing for enhanced therapeutic delivery into the target tissues. While enhanced delivery of small (<1nm) molecules has been shown at acoustic pressures below 1MPa both in vitro and in vivo, the delivery efficiency of larger (>100nm) therapeutic carriers into cancer remains unclear and may require a higher pressure for sufficient delivery. Enhanced delivery of larger therapeutic carriers such as FDA approved pegylated poly(lactic-co-glycolic acid) nanoparticles (PLGA-PEG-NP) has significant clinical value because these nanoparticles have been shown to protect encapsulated drugs from degradation in the blood circulation and allow for slow and prolonged release of encapsulated drugs at the target location. In this study, various acoustic parameters were investigated to facilitate the successful delivery of two nanocarriers, a fluorescent semiconducting polymer model drug nanoparticle as well as PLGA-PEG-NP into human colon cancer xenografts in mice. We first measured the cavitation dose produced by various acoustic parameters (pressure, pulse length, and pulse repetition frequency) and microbubble concentration in a tissue mimicking phantom. Next, in vivo studies were performed to evaluate the penetration depth of nanocarriers using various acoustic pressures, ranging between 1.7 and 6.9MPa. Finally, a therapeutic microRNA, miR-122, was loaded into PLGA-PEG-NP and the amount of delivered miR-122 was assessed using quantitative RT-PCR. Our results show that acoustic pressures had the strongest effect on cavitation. An increase of the pressure from 0.8 to 6.9MPa resulted in a nearly 50-fold increase in cavitation in phantom experiments. In vivo, as the pressures increased from 1.7 to 6.9MPa, the amount of nanoparticles deposited in cancer xenografts was increased from 4- to 14-fold, and the median penetration depth of extravasated nanoparticles was increased from 1.3-fold to 3-fold, compared to control conditions without ultrasound, as examined on 3D confocal microscopy. When delivering miR-122 loaded PLGA-PEG-NP using optimal acoustic settings with minimum tissue damage, miR-122 delivery into tumors with ultrasound and microbubbles was 7.9-fold higher compared to treatment without ultrasound. This study demonstrates that ultrasound induced microbubble cavitation can be a useful tool for delivery of therapeutic miR loaded nanocarriers into cancer in vivo.

Vascular Endothelial Growth Factor Receptor Type 2–targeted Contrast-enhanced US of Pancreatic Cancer Neovasculature in a Genetically Engineered Mouse Model: Potential for Earlier Detection

PURPOSE To test ultrasonographic (US) imaging with vascular endothelial growth factor receptor type 2 (VEGFR2)-targeted microbubble contrast material for the detection of pancreatic ductal adenocarcinoma (PDAC) in a transgenic mouse model of pancreatic cancer development. MATERIALS AND METHODS Experiments involving animals were approved by the Institutional Administrative Panel on Laboratory Animal Care at Stanford University. Transgenic mice (n = 44; Pdx1-Cre, KRas(G12D), Ink4a(-/-)) that spontaneously develop PDAC starting at 4 weeks of age were imaged by using a dedicated small-animal US system after intravenous injection of 5 × 10(7) clinical-grade VEGFR2-targeted microbubble contrast material. The pancreata in wild-type (WT) mice (n = 64) were scanned as controls. Pancreatic tissue was analyzed ex vivo by means of histologic examination (with hematoxylin-eosin staining) and immunostaining of vascular endothelial cell marker CD31 and VEGFR2. The Wilcoxon rank sum test and linear mixed-effects model were used for statistical analysis. RESULTS VEGFR2-targeted US of PDAC showed significantly higher signal intensities (26.8-fold higher; mean intensity ± standard deviation, 6.7 linear arbitrary units [lau] ± 8.5; P < .001) in transgenic mice compared with normal, control pancreata of WT mice (mean intensity, 0.25 lau ± 0.25). The highest VEGFR2-targeted US signal intensities were observed in smaller tumors, less than 3 mm in diameter (30.8-fold higher than control tissue with mean intensity of 7.7 lau ± 9.3 [P < .001]; and 1.7-fold higher than lesions larger than 3 mm in diameter with mean intensity of 4.6 lau ± 5.8 [P < .024]). Ex vivo quantitative VEGFR2 immunofluorescence demonstrated that VEGFR2 expression was significantly higher in pancreatic tumors (P < .001; mean fluorescent intensity, 499.4 arbitrary units [au] ± 179.1) compared with normal pancreas (mean fluorescent intensity, 232.9 au ± 83.7). CONCLUSION US with clinical-grade VEGFR2-targeted microbubbles allows detection of small foci of PDAC in transgenic mice.

The gap junction protein Cx43 regulates B-lymphocyte spreading and adhesion

The gap junction protein connexin43 (Cx43) is widely expressed in mammalian cells and forms intercellular channels for the transfer of small molecules between adjacent cells, as well as hemichannels that mediate bidirectional transport of molecules between the cell and the surrounding environment. Cx43 regulates cell adhesion and migration in neurons and glioma cells, and we now show that Cx43 influences BCR-, LFA-1- and CXCL12-mediated activation of the Rap1 GTPase. Using shRNA knockdown of Cx43 in WEHI 231 cells, we show that Cx43 is required for sustained Rap1 activation and BCR-mediated spreading. To determine the domains of Cx43 that are important for this effect, Cx43-null J558 μm3 B cells (which express a wild-type IgM BCR) were transfected with wild-type Cx43–GFP or a C-terminal-truncated Cx43 (Cx43ΔT–GFP). Expression of wild-type Cx43–GFP, but not Cx43ΔT–GFP, was sufficient to restore sustained, BCR-mediated Rap1 activation and cell spreading. Cx43, and specifically the C-terminal domain, was also important for LFA-1- and CXCL12-mediated Rap1 activation, spreading and adhesion to an endothelial cell monolayer. These data show that Cx43 has an important and previously unreported role in B-cell processes that are essential to normal B-cell development and immune responses.

The role of the gap junction protein connexin43 in B lymphocyte motility and migration.

The gap junction family of proteins is widely expressed in mammalian cells and form intercellular channels between adjacent cells, as well as hemichannels, for transport of molecules between the cell and the surrounding environment. In addition, gap junction proteins have recently been implicated as important for the regulation of cell adhesion and migration in a variety of cell types. The gap junction protein connexin43 (Cx43) regulates B lymphocyte adhesion, BCR‐ and LFA‐1‐mediated activation of the GTPase Rap1, and cytoskeletal rearrangements resulting in changes to cell shape and membrane spreading. We demonstrate here that the actin cytoskeleton is important for the distribution of Cx43 in the B cell plasma membrane and for other cell processes involving the cytoskeleton. Using shRNA knockdown of Cx43 in B lymphoma cells we show that Cx43 is also necessary for chemokine‐mediated Rap 1 activation, motility, CXCL12‐directed migration, and movement across an endothelial cell monolayer. These results demonstrate that in addition to its role in B cell spreading, Cx43 is an important regulator of B‐cell motility and migration, processes essential for normal B‐cell development and immune responses.

Photoacoustic Tomography Detects Early Vessel Regression and Normalization During Ovarian Tumor Response to the Antiangiogenic Therapy Trebananib

The primary aim of this study was to assess the potential of in vivo photoacoustic tomography for direct functional measurement of ovarian tumor response to antiangiogenic therapy. Methods: In vivo studies were performed with institutional animal care and use committee approval. We used an orthotopic mouse model of ovarian cancer treated with trebananib (n = 9) or vehicle (n = 9). Tumor-bearing mice were randomized into trebananib or vehicle groups at day 10 and dosed on days 12, 15, and 18 after implantation. Photoacoustic tomography and blood draws were performed at day 10 and then 24 h after each drug dose. Tumors were excised for histopathology after the final studies on day 19. Data analysis to test for statistical significance was performed blinded. Results: Blockade of angiopoietin signaling using trebananib resulted in reduced total hemoglobin–weighted photoacoustic signal (n = 9, P = 0.01) and increased oxyhemoglobin-weighted photoacoustic signal (n = 9, P < 0.01). The latter observation indicated normalization of the residual tumor vessels, which was also implied by low levels of angiopoietin 1 in serum biomarker profiling (0.76 ± 0.12 ng/mL). These noninvasive measures reflected a 30% reduction in microvessel density and increased vessel maturation in ex vivo sections. Conclusion: Photoacoustic tomography is able to evaluate both vessel regression and normalization in response to trebananib. Noninvasive imaging data were supported by modulation of serum markers in vitro and ex vivo histopathology.

The role of Ig-α/β in B cell antigen receptor internalization.

The B cell antigen receptor (BCR) is expressed on the surface of B lymphocytes where it can bind antigen then transmit signals which regulate activation, growth, and differentiation. These signals can induce a number of cytoskeletal rearrangements leading to dynamic cellular processes including internalization of the bound antigen which is then processed and presented to T cells on MHC II. The relative importance of regions within the Igα and Igβ cytoplasmic domains has been well studied in terms of signaling but their roles in BCR internalization and trafficking are less clear. We hypothesize that the Igα and Igβ cytoplasmic domains is important for normal internalization and trafficking of the 4 chain BCR. An Igα and Igβ deficient murine lymphoid cell line was used to express mIgM along with a panel of Igα and Igβ mutants in order to compare their internalization and subcellular localization. Here we show that the Igα and Igβ cytoplasmic domains are each sufficient for internalization, though Igα is dominant in this process. We also show that the internalization signal is contained in a region past the first cytoplasmic tyrosine residue of Igα and Igβ, Y176 and Y195 respectively. We also show that a 4 amino acid motif normally contained within the Igα ITAM is sufficient to rescue aberrant internalization. In terms of receptor trafficking, each cytoplasmic domain is sufficient for trafficking to lysosomal compartments but that a normal rate of trafficking likely requires the tandem effects of both Igα and Igβ.

Quantitative Assessment of Inflammation in a Porcine Acute Terminal Ileitis Model: US with a Molecularly Targeted Contrast Agent

PURPOSE To evaluate the feasibility and reproducibility of ultrasonography (US) performed with dual-selectin-targeted contrast agent microbubbles (MBs) for assessment of inflammation in a porcine acute terminal ileitis model, with histologic findings as a reference standard. MATERIALS AND METHODS The study had institutional Animal Care and Use Committee approval. Acute terminal ileitis was established in 19 pigs; four pigs served as control pigs. The ileum was imaged with clinical-grade dual P- and E-selectin-targeted MBs (MBSelectin) at increasing doses (0.5, 1.0, 2.5, 5.0, 10, and 20 × 10(8) MB per kilogram of body weight) and with control nontargeted MBs (MBControl). For reproducibility testing, examinations were repeated twice after the MBSelectin and MBControl injections. After imaging, scanned ileal segments were analyzed ex vivo both for inflammation grade (by using hematoxylin-eosin staining) and for expression of selectins (by using quantitative immunofluorescence analysis). Statistical analysis was performed by using the t test, intraclass correlation coefficients (ICCs), and Spearman correlation analysis. RESULTS Imaging signal increased linearly (P < .001) between a dose of 0.5 and a dose of 5.0 × 10(8) MB/kg and plateaued between a dose of 10 and a dose of 20 × 10(8) MB/kg. Imaging signals were reproducible (ICC = 0.70), and administration of MBSelectin in acute ileitis resulted in a significantly higher (P < .001) imaging signal compared with that in control ileum and MBControl. Ex vivo histologic grades of inflammation correlated well with in vivo US signal (ρ = 0.79), and expression levels of both P-selectin (37.4% ± 14.7 [standard deviation] of vessels positive; P < .001) and E-selectin (31.2% ± 25.7) in vessels in the bowel wall of segments with ileitis were higher than in control ileum (5.1% ± 3.7 for P-selectin and 4.8% ± 2.3 for E-selectin). CONCLUSION Quantitative measurements of inflammation obtained by using dual-selectin-targeted US are reproducible and correlate well with the extent of inflammation at histologic examination in a porcine acute ileitis model as a next step toward clinical translation.

Assessment of Inflammation in an Acute on Chronic Model of Inflammatory Bowel Disease with Ultrasound Molecular Imaging.

Background: Ultrasound (US) molecular imaging has shown promise in assessing inflammation in preclinical, murine models of inflammatory bowel disease. These models, however, initiated acute inflammation on previously normal colons, in contrast to patients where acute exacerbations are often in chronically inflamed regions. In this study, we explored the potential of dual P- and E-selectin targeted US imaging for assessing acute inflammation on a murine quiescent chronic inflammatory background. Methods: Chronic colitis was induced using three cycles of 4% DSS in male FVB mice. Acute inflammation was initiated 2 weeks after the final DSS cycle through rectal administration of 1% TNBS. Mice at different stages of inflammation were imaged using a small animal ultrasound system following i.v. injection of microbubbles targeted to P- and E-selectin. In vivo imaging results were correlated with ex vivo immunofluorescence and histology. Results: Induction of acute inflammation resulted in an increase in the targeted US signal from 5.5 ± 5.1 arbitrary units (a.u.) at day 0 to 61.0 ± 45.2 a.u. (P < 0.0001) at day 1, 36.3 ± 33.1 a.u. at day 3, returning to levels similar to control at day 5. Immunofluorescence showed significant increase in the percentage of P- and E-selectin positive vessels at day 1 (P-selectin: 21.0 ± 7.1% of vessels; P < 0.05; E-selectin: 16.4 ±3.7%; P < 0.05) compared to day 0 (P-selectin: 10.3 ± 5.7%; E-selectin: 7.3 ± 7.0%). Conclusions: Acute inflammation can be accurately measured in a clinically relevant murine model of chronic IBD using ultrasound molecular imaging with a dual P- and E- selectin-targeted contrast agent.