Joanna Napp

Targeted luminescent near-infrared polymer-nanoprobes for in vivo imaging of tumor hypoxia.

Polystyrene nanoparticles (PS-NPs) were doped with an oxygen-sensitive near-infrared (NIR)-emissive palladium meso-tetraphenylporphyrin and an inert reference dye which are both excitable at 635 nm. The nanosensors were characterized with special emphasis on fundamental parameters such as absolute photoluminescence quantum yield and fluorescence lifetime. The PS-NPs were employed for ratiometric dual-wavelength and lifetime-based photoluminescent oxygen sensing. They were efficiently taken up by cultured murine alveolar macrophages, yielding a characteristic and reversible change in ratiometric response with decreasing oxygen concentration. This correlated with the cellular hypoxic status verified by analysis of hypoxia inducible factor-1α (HIF-1α) accumulation. In addition, the surface of PS-NPs was functionalized with polyethylene glycol (PEG) and the monoclonal antibody herceptin, and their binding to HER2/neu-overexpressing tumor cells was confirmed in vitro. First experiments with tumor-bearing mouse revealed a distinctive ratiometric response within the tumor upon hypoxic condition induced by animal sacrifice. These results demonstrate the potential of these referenced NIR nanosensors for in vitro and in vivo imaging that present a new generation of optical probes for oncology.

Glycosylation of Eag1 (Kv10.1) Potassium Channels INTRACELLULAR TRAFFICKING AND FUNCTIONAL CONSEQUENCES

N-Linked glycosylation is a common post-translational modification of membrane proteins. Here we report that mature Eag1 potassium channels carry sugar moieties linked to asparagines at positions 388 and 406. Asn-388 seems to undergo only core glycosylation, but complex sugars are bound to Asn-406. Correct complex glycosylation is required for proper trafficking of Eag1 to the plasma membrane but is also crucial for the correct function of channels already inserted in the membrane.

Target-specific nanoparticles containing a broad band emissive NIR dye for the sensitive detection and characterization of tumor development

Current optical probes including engineered nanoparticles (NPs) are constructed from near infrared (NIR)-emissive organic dyes with narrow absorption and emission bands and small Stokes shifts prone to aggregation-induced self-quenching. Here, we present the new asymmetric cyanine Itrybe with broad, almost environment-insensitive absorption and emission bands in the diagnostic window, offering a unique flexibility of the choice of excitation and detection wavelengths compared to common NIR dyes. This strongly emissive dye was spectroscopically studied in different solvents and encapsulated into differently sized (15, 25, 100 nm) amino-modified polystyrene NPs (PSNPs) via a one-step staining procedure. As proof-of-concept for its potential for pre-/clinical imaging applications, Itrybe-loaded NPs were surface-functionalized with polyethylene glycol (PEG) and the tumor-targeting antibody Herceptin and their binding specificity to the tumor-specific biomarker HER2 was systematically assessed. Itrybe-loaded NPs display strong fluorescence signals in vitro and in vivo and Herceptin-conjugated NPs bind specifically to HER2 as demonstrated in immunoassays as well as on tumor cells and sections from mouse tumor xenografts in vitro. This demonstrates that our design strategy exploiting broad band-absorbing and -emitting dyes yields versatile and bright NIR probes with a high potential for e.g. the sensitive detection and characterization of tumor development and progression.

Time-domain in vivo near infrared fluorescence imaging for evaluation of matriptase as a potential target for the development of novel, inhibitor-based tumor therapies.

Proteolytic enzymes expressed on the surface of tumor cells, and thus easily accessible to external interventions, represent useful targets for anticancer and antimetastatic therapies. In our study, we thoroughly evaluated matriptase, a trypsin‐like transmembrane serine protease, as potential target for novel inhibitor‐based tumor therapies. We applied time‐domain near infrared fluorescence (NIRF) imaging to characterize expression and activity of matriptase in vivo in an orthotopic AsPC‐1 pancreatic tumor model in nude mice. We show strong and tumor‐specific binding of intravenously injected Cy5.5 labeled antimatriptase antibody (MT‐Ab*Cy5.5) only to primary AsPC‐1 tumors and their metastases over time within living mice, taking into account fluorescence intensities and fluorescence lifetimes of the applied probes. Specific binding of MT‐Ab*Cy5.5 to tumor sites was confirmed by ex vivo NIRF imaging of tumor tissue, NIRF microscopy and by coregistration of the in vivo acquired NIRF intensity maps to anatomical structures visualized by flat‐panel volume computed tomography (fpVCT) in living mice. Moreover, using an activatable synthetic substrate S*DY‐681 we could clearly demonstrate that matriptase is proteolytically active in vitro as well as in vivo in tumor‐bearing mice, and that application of synthetic active‐site inhibitors having high affinity and selectivity toward matriptase can efficiently inhibit its proteolytic activity for at least 24 hr. We thus successfully applied NIRF imaging in combination with fpVCT to characterize matriptase as a promising molecular target for inhibitor‐based cancer therapies.

Semiautomatic landmark-based two-dimensional-three-dimensional image fusion in living mice : correlation of near-infrared fluorescence imaging of Cy5.5-labeled antibodies with flat-panel volume computed tomography

Connecting fluorescence signals with anatomic structures enhances our ability to monitor biologic processes in mice. Here, we present a semiautomated approach to correlate two-dimensional (2D) noninvasive near-infrared fluorescence (NIRF) imaging with three-dimensional (3D), high-resolution, flat-panel volume computed tomography (fpVCT). We developed an algorithm to colocalize fluorescence signals of NIRF-labeled antibodies directed against matriptase and urokinase plasminogen activator receptor (uPAR) to orthotopic carcinomas in mice visualized by fpVCT. For this purpose, mice were anesthetized and fixed on a multimodality animal bed containing fiducial markers filled with iodine-containing contrast agent and fluorescent dye. After intravenous administration of contrast agent and Cy5.5-labeled antibodies, NIRF and fpVCT images were obtained, without repositioning the mice. Binding of Cy5.5-labeled matriptase-specific antibody to pancreatic tumors and Cy5.5-labeled uPAR-specific antibody to mammary carcinomas was assessed by time-domain NIRF imaging measuring the location of fluorescence intensity and its lifetime. In summary, we developed a novel 2D-3D registration technique for image fusion with NIRF imaging and fpVCT to provide complementary information in tumor models on the in vivo association of functional information with anatomic structures. The combination of fpVCT with NIRF imaging will now allow targeted and effective monitoring of preclinical tumor therapies.

High-sensitivity detection of breast tumors in vivo by use of a pH-sensitive near-infrared fluorescence probe.

We investigated the potential of the pH-sensitive dye, CypHer5E, conjugated to Herceptin (pH-Her) for the sensitive detection of breast tumors in mice using noninvasive time-domain near-infrared fluorescence imaging and different methods of data analysis. First, the fluorescence properties of pH-Her were analyzed as function of pH and/or dye-to-protein ratio, and binding specificity was confirmed in cell-based assays. Subsequently, the performance of pH-Her in nude mice bearing orthotopic HER2-positive (KPL-4) and HER2-negative (MDA-MB-231) breast carcinoma xenografts was compared to that of an always-on fluorescent conjugate Alexa Fluor 647-Herceptin (Alexa-Her). Subtraction of autofluorescence and lifetime (LT)-gated image analyses were performed for background fluorescence suppression. In mice bearing HER2-positive tumors, autofluorescence subtraction together with the selective fluorescence enhancement of pH-Her solely in the tumors acidic environment provided high contrast-to-noise ratios (CNRs). This led to an improved sensitivity of tumor detection compared to Alexa-Her. In contrast, LT-gated imaging using LTs determined in model systems did not improve tumor-detection sensitivity in vivo for either probe. In conclusion, pH-Her is suitable for sensitive in vivo monitoring of HER2-expressing breast tumors with imaging in the intensity domain and represents a promising tool for detection of weak fluorescent signals deriving from small tumors or metastases.

Functionalized synchrotron in-line phase-contrast computed tomography: a novel approach for simultaneous quantification of structural alterations and localization of barium-labelled alveolar macrophages within mouse lung samples

This study presents an approach to increase the sensitivity of lung computed tomography (CT) imaging by utilizing in-line phase contrast CT in combination with single-distance phase-retrieval algorithms and a dedicated image-processing regime. As demonstrated here, functional CT imaging can be achieved for the assessment of both structural alterations in asthmatic mouse lung tissue and the accumulation pattern of instilled barium-sulfate-labelled macrophages in comparison with healthy controls.

Preclinical evaluation of near-infrared (NIR) fluorescently labeled cetuximab as a potential tool for fluorescence-guided surgery

The high rate of recurrence in patients with pancreatic ductal adenocarcinoma (PDAC) could be reduced by supporting the surgeons in discriminating healthy from diseased tissues with intraoperative fluorescence‐guidance. Here, we studied the suitability of Cetuximab, a therapeutic monoclonal antibody targeting the human epidermal growth factor receptor (EGFR), near‐infrared (NIR) fluorescently labeled as a new tool for fluorescence‐guided surgery. Distribution and binding of systemically injected Cetuximab Alexa Fluor 647 conjugate (Cetux‐Alexa‐647) and the co‐injected control human IgG Alexa Fluor 750 conjugate (hIgG‐Alexa‐750) was studied over 48 h by NIR fluorescence imaging in mice bearing human orthotopic AsPC‐1 and MIA PaCa‐2 PDAC tumors. Cetux‐Alexa‐647, but not the control hIgG‐Alexa‐750 fluorescence, was specifically detected in vivo in both primary pancreatic tumors with maximum fluorescence intensities at 24 h, and in metastases of AsPC‐1 tumors as small as 1 mm. Lifetime analysis and NIR fluorescence microscopy of tumor sections confirmed the binding specificity of Cetux‐Alexa‐647 to PDAC cells. Comparable results were obtained with Cetuximab conjugated to Alexa Fluor 750 dye (Cetux‐Alexa‐750). Fluorescence‐guided dissection, performed 24 h after injection of Cetuximab conjugated to IRDye 800CW (Cetux‐800CW), enabled a real‐time delineation of AsPC‐1 tumor margins, and small metastases. Odyssey scans revealed that only the vital part of the tumor, but not the necrotic part was stained with Cetux‐800CW. NIR fluorescently labeled Cetuximab may be a promising tool that can be applied for fluorescence‐guided surgery to visualize tumor margins and metastatic sites in order to allow a precise surgical resection.

Targeting of the P2X7 receptor in pancreatic cancer and stellate cells.

The ATP‐gated receptor P2X7 (P2X7R) is involved in regulation of cell survival and has been of interest in cancer field. Pancreatic ductal adenocarcinoma (PDAC) is a deadly cancer and new markers and therapeutic targets are needed. PDAC is characterized by a complex tumour microenvironment, which includes cancer and pancreatic stellate cells (PSCs), and potentially high nucleotide/side turnover. Our aim was to determine P2X7R expression and function in human pancreatic cancer cells in vitro as well as to perform in vivo efficacy study applying P2X7R inhibitor in an orthotopic xenograft mouse model of PDAC. In the in vitro studies we show that human PDAC cells with luciferase gene (PancTu‐1 Luc cells) express high levels of P2X7R protein. Allosteric P2X7R antagonist AZ10606120 inhibited cell proliferation in basal conditions, indicating that P2X7R was tonically active. Extracellular ATP and BzATP, to which the P2X7R is more sensitive, further affected cell survival and confirmed complex functionality of P2X7R. PancTu‐1 Luc migration and invasion was reduced by AZ10606120, and it was stimulated by PSCs, but not by PSCs from P2X7‐/‐ animals. PancTu‐1 Luc cells were orthotopically transplanted into nude mice and tumour growth was followed noninvasively by bioluminescence imaging. AZ10606120‐treated mice showed reduced bioluminescence compared to saline‐treated mice. Immunohistochemical analysis confirmed P2X7R expression in cancer and PSC cells, and in metaplastic/neoplastic acinar and duct structures. PSCs number/activity and collagen deposition was reduced in AZ10606120‐treated tumours.

Tracking of Inhaled Near-Infrared Fluorescent Nanoparticles in Lungs of SKH-1 Mice with Allergic Airway Inflammation

Molecular imaging of inflammatory lung diseases, such as asthma, has been limited to date. The recruitment of innate immune cells to the airways is central to the inflammation process. This study exploits these cells for imaging purposes within the lung, using inhaled polystyrene nanoparticles loaded with the near-infrared fluorescence dye Itrybe (Itrybe-NPs). By means of in vivo and ex vivo fluorescence reflectance imaging of an ovalbumin-based allergic airway inflammation (AAI) model in hairless SKH-1 mice, we show that subsequent to intranasal application of Itrybe-NPs, AAI lungs display fluorescence intensities significantly higher than those in lungs of control mice for at least 24 h. Ex vivo immunofluorescence analysis of lung tissue demonstrates the uptake of Itrybe-NPs predominantly by CD68(+)CD11c(+)ECF-L(+)MHCII(low) cells, identifying them as alveolar M2 macrophages in the peribronchial and alveolar areas. The in vivo results were validated by confocal microscopy, overlapping tile analysis, and flow cytometry, showing an amount of Itrybe-NP-containing macrophages in lungs of AAI mice significantly larger than that in controls. A small percentage of NP-containing cells were identified as dendritic cells. Flow cytometry of tracheobronchial lymph nodes showed that Itrybe-NPs were negligible in lung draining lymph nodes 24 h after inhalation. This imaging approach may advance preclinical monitoring of AAI in vivo over time and aid the investigation of the role that macrophages play during lung inflammation. Furthermore, it allows for tracking of inhaled nanoparticles and can hence be utilized for studies of the fate of potential new nanotherapeutics.