In Situ Immunofluorescence Imaging of Vital Human Pancreatic Tissue Using Fiber-Optic Microscopy.

Purpose: Surgical resection is the only curative option for pancreatic carcinoma, but disease-free and overall survival times after surgery are limited due to early tumor recurrence, most often originating from local microscopic tumor residues (R1 resection). The intraoperative identification of microscopic tumor residues within the resection margin in situ could improve surgical performance. The aim of this study was to evaluate the effectiveness of fiber-optic microscopy for detecting microscopic residues in vital pancreatic cancer tissues. Experimental Design. Fresh whole-mount human pancreatic tissues, histological tissue slides, cell culture, and chorioallantoic membrane xenografts were analyzed. Specimens were stained with selected fluorophore-conjugated antibodies and studied using conventional wide-field and self-designed multicolor fiber-optic fluorescence microscopy instruments. Results: Whole-mount vital human tissues and xenografts were stained and imaged using an in situ immunofluorescence protocol. Fiber-optic microscopy enabled the detection of epitope-based fluorescence in vital whole-mount tissue using fluorophore-conjugated antibodies and enabled visualization of microvascular, epithelial, and malignant tumor cells. Among the selected antigen-antibody pairs, antibody clones WM59, AY13, and 9C4 were the most promising for fiber-optic imaging in human tissue samples and for endothelial, tumor and epithelial cell detection. Conclusions: Fresh dissected whole-mount tissue can be stained using direct exposure to selected antibody clones. Several antibody clones were identified that provided excellent immunofluorescence imaging of labeled structures, such as endothelial, epithelial, or EGFR-expressing cells. The combination of in situ immunofluorescence staining and fiber-optic microscopy visualizes structures in vital tissues and could be proposed as an useful tool for the in situ identification of residual tumor mass in patients with a high operative risk for incomplete resection.

Clinical Research in Renal Transplantation: A Bibliometric Perspective on a Half-century of Innovation and Progress.

BACKGROUND: Groundbreaking biomedical research has transformed renal transplantation (RT) into a widespread clinical procedure that represents the mainstay of treatment for end-stage kidney failure today. Here, we aimed to provide a comprehensive bibliometric perspective on the last half-century of innovation in clinical RT. METHODS: The Web of Science Core Collection was used for a comprehensive screening yielding 123 303 research items during a 50-y period (January 1973-October 2022). The final data set of the 200 most-cited articles was selected on the basis of a citation-based strategy aiming to minimize bias. RESULTS: Studies on clinical and immunological outcomes (n = 63 and 48), registry-based epi research (n = 38), and randomized controlled trials (n = 35) dominated the data set. Lead US authors have signed 110 of 200 articles. The overall level of evidence was high, with 84% of level1 and -2 reports. Highest numbers of these articles were published in New England Journal of Medicine , Transplantation , and American Journal of Transplantation. Increasing trend was observed in the number of female authors in the postmillennial era (26% versus 7%). CONCLUSIONS: This study highlights important trends in RT research of the past half-century. This bibliometric perspective identifies the most intensively researched areas and shift of research interests over time; however, it also describes important imbalances in distribution of academic prolificacy based on topic, geographical aspects, and gender.

DWH24: a new antibody for fluorescence-based cell death analysis.

Antibodies have gained considerable importance in laboratory and clinical settings. Currently, antibodies are extensively employed for the diagnosis and treatment of several human diseases. Herein, using targeted and cell immunisation approaches, we developed and characterised an antibody clone, DWH24. We found that DWH24 is an IgMκtype antibody that enables excellent visualisation and quantification of dead cells using immunofluorescence, fluorescence microscopy, and flow cytometry. This property was proved by the spontaneous cell death of several tumour cell lines and stimulated T cells, as well as after chemo- and photodynamic therapy. Unlike conventional apoptosis and cell death markers, DWH24 binding occurred in a Ca2+- and protein-independent manner and enabled live imaging of cell death progress, as shown using time-lapse microscopy. The binding specificity of DWH24 was analysed using a human proteome microarray, which revealed a complex response profile with very high spot intensities against various proteins, such as tropomyosin variants and FAM131C. Accordingly, DWH24 can be employed as a suitable tool for the cost-effective and universal analysis of cell death using fluorescence imaging and flow cytometry.

Low efficiency of leucocyte plugging-based drug delivery to cancer in mice.

Cells of the immune system were proposed for use as Trojan horse for tumour-specific drug delivery. The efficacy of such cell-based drug delivery depends on the site-specific cell homing. This present study was aimed to investigate the potential of leucocytes for intratumoural site-specific enrichment using a locoregional application route in experimental liver tumours. Human neutrophils were isolated from peripheral blood and directly labelled with calcein AM or loaded with doxorubicin. The neutrophil loading and release of doxorubicin and the migration and adhesion to ICAM-1 were analysed in vitro. Macrophages were isolated and activated in vitro. Leucocyte plugging and the distribution pattern in the liver microvasculature were studied ex vivo, and the efficacy of leucocyte plugging in tumour blood vessels was analysed in vivo after superselective intra-arterial injection in mouse liver tumour models. Neutrophils were characterised by the high dose-dependent uptake and rapid release of doxorubicin. Doxorubicin loading did not affect neutrophil migration function. Neutrophil plugging in liver microvasculature was very high (> 90%), both after ex vivo perfusion and after injection in vivo. However, neutrophils as well as activated macrophages plugged insufficiently in tumour blood vessels and passed through the tumour microvasculture with a very low sequestration rate in vivo. Neutrophils possess several properties to function as potentially effective drug carriers; however, the tumour site-specific drug delivery after selective locoregional injection was observed to be insufficient owing to low intratumoural microvascular plugging.

YAP-induced Ccl2 expression is associated with a switch in hepatic macrophage identity and vascular remodelling in liver cancer.

BACKGROUND & AIM: The development of hepatocellular carcinoma (HCC) is associated with the formation of communication networks leading to the recruitment of disease-modifying macrophages. However, how oncogenes in tumour cells control paracrine communication is not fully understood. METHODS: Transgenic mice with liver-specific expression of the constitutively active yes-associated protein (YAPS127A ) or an orthotopic implantation model served as tumour models. FACS-sorted F4/80+ /CD11bdim /CD146- /retinoid- macrophages from healthy and tumour-bearing livers were used for transcriptomic profiling. Expression data of 242 human HCCs and a tissue microarray consisting of 91 HCCs and seven liver tissues were analyzed. RESULTS: Screening of primary tumour cells expressing YAPS127A identified CC chemokine ligand 2 (Ccl2) as a macrophage chemoattractant, whose expression was regulated in a YAP/TEA domain family member 4 (TEAD4)-dependent manner. Ccl2 expression was associated with a loss of Kupffer cells (KCs) and an increase in immature macrophages (Mɸimm ) in hepatocarcinogenesis. Recruited Mɸimm were characterized by a lack of functional polarization (M0 signature) and high expression of the Ccl2 receptors C-C motif chemokine receptor 2 (Ccr2), C-X3-C motif chemokine receptor 1 (Cx3cr1) and pro-angiogenic platelet-derived growth factors (Pdgfa/Pdgfb). Mɸimm formed cellular clusters in the perivascular space, which correlated with vascular morphometric changes indicative for angiogenesis. In human HCCs, the M0 signature served as an identifier for poor clinical outcome and CCL2 correlated with YAP expression and vascular network formation. CONCLUSIONS: In conclusion, YAP/TEAD4-regulated Ccl2 associates with perivascular recruitment of unpolarized Mɸimm and may contribute to a proangiogenic microenvironment in liver cancer.

Collagen Organization Does Not Influence T-Cell Distribution in Stroma of Human Pancreatic Cancer.

The dominant intrastromal T-cell infiltration in pancreatic cancer is mainly caused by the contact guidance through the excessive desmoplastic reaction and could represent one of the obstacles to an effective immune response in this tumor type. This study analyzed the collagen organization in normal and malignant pancreatic tissues as well as its influence on T-cell distribution in pancreatic cancer. Human pancreatic tissue was analyzed using immunofluorescence staining and multiphoton and SHG microscopy supported by multistep image processing. The influence of collagen alignment on activated T-cells was studied using 3D matrices and time-lapse microscopy. It was found that the stroma of malignant and normal pancreatic tissues was characterized by complex individual organization. T-cells were heterogeneously distributed in pancreatic cancer and there was no relationship between T-cell distribution and collagen organization. There was a difference in the angular orientation of collagen alignment in the peritumoral and tumor-cell-distant stroma regions in the pancreatic ductal adenocarcinoma tissue, but there was no correlation in the T-cell densities between these regions. The grade of collagen alignment did not influence the directionality of T-cell migration in the 3D collagen matrix. It can be concluded that differences in collagen organization do not change the spatial orientation of T-cell migration or influence stromal T-cell distribution in human pancreatic cancer. The results of the present study do not support the rationale of remodeling of stroma collagen organization for improvement of T-cell-tumor cell contact in pancreatic ductal adenocarcinoma.

YAP Orchestrates Heterotypic Endothelial Cell Communication via HGF/c-MET Signaling in Liver Tumorigenesis.

The oncogene yes-associated protein (YAP) controls liver tumor initiation and progression via cell extrinsic functions by creating a tumor-supporting environment in conjunction with cell autonomous mechanisms. However, how YAP controls organization of the microenvironment and in particular the vascular niche, which contributes to liver disease and hepatocarcinogenesis, is poorly understood. To investigate heterotypic cell communication, we dissected murine and human liver endothelial cell (EC) populations into liver sinusoidal endothelial cells (LSEC) and continuous endothelial cells (CEC) through histomorphological and molecular characterization. In YAPS127A-induced tumorigenesis, a gradual replacement of LSECs by CECs was associated with dynamic changes in the expression of genes involved in paracrine communication. The formation of new communication hubs connecting CECs and LSECs included the hepatocyte growth factor (Hgf)/c-Met signaling pathway. In hepatocytes and tumor cells, YAP/TEA domain transcription factor 4 (TEAD4)-dependent transcriptional induction of osteopontin (Opn) stimulated c-Met expression in EC with CEC phenotype, which sensitized these cells to the promigratory effects of LSEC-derived Hgf. In human hepatocellular carcinoma, the presence of a migration-associated tip-cell signature correlated with poor clinical outcome and the loss of LSEC marker gene expression. The occurrence of c-MET-expressing CECs in human liver cancer samples was confirmed at the single-cell level. In summary, YAP-dependent changes of the liver vascular niche comprise the formation of heterologous communication hubs in which tumor cell-derived factors modify the cross-talk between LSECs and CECs via the HGF/c-MET axis. SIGNIFICANCE: YAP-dependent changes of the liver vascular niche comprise the formation of heterologous communication hubs in which tumor cell-derived factors modify the cross-talk between EC subpopulations. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/24/5502/F1.large.jpg.

Fluorescence-guided fiber-optic micronavigation using microscopic identification of vascular boundary of liver segment and tumors.

Background: The exact identification of tumor boundaries and related liver segments is especially important for liver tumor surgery. This study aimed to evaluate a new approach for vascular boundary assessment and surgical navigation based on fiber-optic microscopy and microvascular fluorescence labeling. Methods: Antibody clones with fast binding ability were identified and selected using immunofluorescence. We evaluated the endothelial capture efficacy for an anti-mouse CD31 antibody labeled with different fluorophores and different degrees of labeling ex vivo. Segment boundary identification and navigation potential using endothelial capture were explored by two different fiber-optic microscopy systems. Finally, microvasculature labeling and fiber-optic microscopy were used to identify and treat microscopic liver tumors in vivo. Results: The following monoclonal antibodies were selected: anti-mouse CD31 (clone 390), anti-mouse CD54 (YN1/1.7.4), anti-human CD31 (WM59), and anti-human CD54 (HA58). These clones showed fast binding to endothelial cells and had long half-lives. The fluorophore choice and the degree of antibody labeling did not significantly affect capture efficacy in an isolated liver perfusion model. The microvascular system was clearly identified with wide-field fiber-optic microscopy after labeling the endothelium with low doses of specific antibodies, and the specifically labeled liver segment could be microscopically dissected. High antibody doses were required for confocal laser endomicroscopy. After microscopically identifying the vascular margin in vivo, tumor thermoablation strongly reduced tumor size or totally eliminated tumors. Conclusions: We demonstrated that vascular boundaries of liver tumors and locally perfused liver segments were accurately identified and surgical micronavigation was facilitated with fiber-optic microscopy and selected endothelium-specific antibodies.

In Vitro Characterization of a Novel Type of Radiopaque Doxorubicin-Loaded Microsphere.

PURPOSE: To evaluate and compare the material characteristics of a novel type of radiopaque doxorubicin-loaded microsphere (V-100) with radiopaque and non-radiopaque doxorubicin-loaded microspheres. MATERIALS AND METHODS: The prototype V-100 featuring inherent radiopacity and three available commercial controls (DC-Bead-LUMI™-70-150, Embozene-Tandem™-100 and DC-Bead™-M1) were analyzed before and after doxorubicin loading (37.5 mg doxorubicin/1 ml microspheres) in suspension with aqua and/or aqua/iodixanol-320. Study goals included inherent radiopacity [e.g., using conventional computed tomography (CT)], doxorubicin loading efficacy, morphology using light and fluorescence microscopy, size distribution using laser diffraction/light scattering, time-in-suspension, rheological properties using rheometer analysis, and microsphere stability observed over a period of 5 days after doxorubicin loading. RESULTS: V-100 showed good inherent radiopacity without adverse imaging artifacts. Under conventional CT, the quantitative radiopacity was as follows: 480.4 ± 2.9HU for V-100, 2432.7 ± 3.2HU for DC-Bead-LUMI™-70-150, 118.1 ± 3.0HU for Embozene-Tandem™-100, and 19.8 ± 1.5HU for DC-Bead™-M1. All of the types of microspheres showed a similar loading efficiency (> 98%) after 24 h; however, there were slower doxorubicin loading velocities for the radiopaque microspheres. The doxorubicin-loaded V-100 and Embozene-Tandem™-100 showed typical narrow-sized distributions. In aqua/iodixanol-320 suspension, doxorubicin-loaded V-100 showed the best suspension features and ideal deformability and elasticity characteristics. Similar to other microspheres, doxorubicin-loaded V-100 was very stable and storable for at least 5 days. CONCLUSION: V-100 is a promising novel type of radiopaque doxorubicin-loaded microsphere. Compared with the controls, V-100 shows good inherent radiopacity without adverse imaging artifacts and with comparable doxorubicin loading efficacy. Further advantages of V-100 include narrow-sized distribution and excellent suspension, rheology, and stability features.

Distinct Origin of Claudin7 in Early Tumor Endosomes Affects Exosome Assembly.

Microvesicles are the body's most powerful intercellular communication system and cancer-initiating cell microvesicles (CIC-TEX) reprogram Non-CIC towards fortified malignancy. Claudin7, a CIC-biomarker in gastrointestinal tumors, is recovered in TEX. Recent evidence suggesting individual cells delivering distinct microvesicles became of particular interest for claudin7, which is part of tight junctions (TJ) and glycolipid-enriched membrane domains (GEM), GEM-located claudin7 is palmitoylated. This offered the unique possibility of exploring the contribution of a CIC marker and its origin from distinct membrane domains on CIC-TEX biogenesis and activities. Proteome and miRNA analysis of wild-type, claudin7-knockdown and a rescue with claudin7 harboring a mutated palmitoylation site (mP) of a rat pancreatic and a human colon cancer line uncovered significant, only partly overlapping contributions of palmitoylated and non-palmitoylated claudin7 to TEX composition. Palmitoylated claudin7 facilitates GEM-integrated plasma membrane and associated signaling molecule recruitment; non-palmitoylated claudin7 supports recruitment of trafficking components, proteins engaged in fatty acid metabolism and TJ proteins into TEX. Claudin7mP also assists TEX recovery of selected miRNA. Thus, distinctly located claudin7 affects CIC-TEX composition and TJ-derived cld7 might play a unique role in equipping CIC-TEX with transporters and lipid metabolism-regulating molecules, awareness of distinct TEX populations being crucial facing therapeutic translation.

Endothelial capture using antibodies and nanoparticles in human tissues: Antigen identification and liver segment imaging.

BACKGROUND: The unique phenomenon of endothelial antibody capture (endocapt) leads to site-specific accumulation of antibodies on the endothelium after its locoregional injection. The potential of this phenomenon has already been demonstrated in animal models. In the present study, the translational potential of several human endothelium-specific antibodies for their use in the endocapt-based approach was analysed. METHODS: The binding of different endothelium-surface specific monoclonal antibody clones was analysed in human tissue and in endothelial cells using image-based immunofluorescence and the determination of half-maximal effective concentration (EC50). The potential of endocapt-based locoregional application of antibodies or antibody-coated liposomes was analysed ex vivo using isolated mouse liver perfusion and in vivo using superselective injection in tumour models. RESULTS: Eight out of ten antibody clones were assigned to the group of "fast binding antibodies". Different antibody clones showed various binding kinetics to the same endothelial marker whereas the binding kinetics of single antibody clones was independent from the tissue type. Anti-CD49e, anti-CD31, anti-CD34 and anti-CD102 antibodies showed the lowest EC50 of antibody binding concentration and constant results in EC50 determination of antibody binding to cells and human tissue. Experimental studies using anti-mouse CD49e antibody and coated immunoliposomes confirmed their effective capture by endothelial cells in vitro and in vivo by which fluorescent liver segment labelling was achieved. CONCLUSIONS: Our findings identify the high potential of several human antibody clones, especially anti-CD49e, -CD31, -CD34 and -CD102, for endocapt technology. We also propose important translational implications of these antibodies for image-guided liver surgery and for use of nanoparticles/immunoliposomes. Toxicological studies are indispensable for further translational development of new antibodies for endocapt. STATEMENT OF SIGNIFICANCE: The phenomenon of endothelial antibody capture (endocapt) leads to site-specific accumulation of antibodies on the endothelium after its locoregional injection. This phenomenon broadly prevents systemic circulation of the antibody or antibody-drug conjugates. In the present study, our findings identify several human antibody clones promising for endothelial capture technology. This study provided the experimental demonstration of liver segment labelling ex vivo using isolated mouse liver perfusion and in vivo using superselective injection in tumor models. In addition, this study proposed the important translational implications of selected antibodies for image-guided liver surgery and for use of nanoparticles/immunoliposomes.

Therapeutic Targeting Cancer-Initiating Cell Markers by Exosome miRNA: Efficacy and Functional Consequences Exemplified for claudin7 and EpCAM.

AIM: Transfer of exosomes (Exo) miRNA was described interfering with tumor progression. We here explored for claudin7 (cld7) and EpCAM (EpC), cancer-initiating-cell markers in colorectal and pancreatic cancer, the efficacy of Exo loading with miRNA and miRNA transfer. METHODS: Exo were collected from nontransformed mouse (NIH3T3) and rat lung fibroblasts (rFb), which were transfected with Tspan8 cDNA (NIH3T3-Tspan8, rFb-Tspan8). Exo were loaded by electroporation with miRNA. The transfer of Exo-miRNA was evaluated in vitro and in vivo in a rat pancreatic (ASML) and a human colon (SW948) cancer line. RESULTS: NIH3T3-Tspan8- or rFb-Tspan8-Exo were efficiently loaded with cld7- or EpC-miRNA. Exo targeting in vivo was strongly improved by tailoring with Tspan8. Exo-miRNA transfer into tumor targets promoted cld7, respectively, EpC downregulation by 33%-60%. Cld7 silencing was accompanied by reduced expression of additional cancer-initiating cell markers and NOTCH. EpC silencing reduced vimentin, N-cadherin, and Nanog expression. The Exo-miRNA transfer affected anchorage-independent growth, motility, and invasion. CONCLUSIONS: Exo are efficiently loaded with miRNA, miRNA-delivery being supported by Exo tailoring. Partial cld7 and EpC silencing by Exo miRNA affects metastasis-promoting tumor cell activities. The findings suggest miRNA loading of tailored Exo as an easy approachable and efficient adjuvant therapy.

Microsurgical Technique of Locoregional Injection into the Hepatic Artery in Tumor-Bearing Mice.

BACKGROUND: Intraarterial injection into the hepatic artery represents an important route for locoregional administration for the treatment of hepatic tumors. In the present work, we describe microsurgical methodology for injection into the hepatic artery in mice. The technique was recently used for analysis of the phenomenon of endothelial capture in liver tumors. METHODS: Two different models of hepatic tumors in C57BL/6 mice were used. Tumors were induced by intrahepatic cell inoculation. The preferential blood supply of tumors was studied using blocking of bioavailability of nontumoral endothelial epitope and the subsequent injection of fluorescent endothelium-specific antibody. The selective intraarterial injection of labeled antibody was performed in tumor-bearing mice. The procedure addressed variations of vascular anatomy of the hepatic artery in mice and used direct intraarterial injection with dispensable catheterization. RESULTS: Both experimental tumor models showed preferential blood supply from the hepatic artery. The technique of hepatic arterial injection was adapted and performed according to two major anatomic variations of the hepatic artery. Using this technique, the selective enrichment of labeled antibody to tumor and liver blood vessels, which were perfused during the first intravascular passage, was demonstrated. CONCLUSIONS: The experimental hepatic arterial injection in mice is a feasible but demanding microsurgical procedure. The choice of subsequent operation steps is dependent on the vascular anatomy of the hepatic artery which has two major variations in mice.

Phenomenon of Endothelial Antibody Capture: Principles and Potential for Locoregional Targeting of Hepatic Tumors.

The systemic drug circulation represents a source of adverse effects during tumor targeting. We studied the binding efficacy of endothelium-specific antibodies after a very short contact with an antigen target, along with assessing the intravascular capture and targeting potential of these antibodies after locoregional injection. Fast-binding anti-CD 146 (clone ME-9F1) and anti-CD31 (clone 390) antibodies were selected based on histological analysis of their binding activity. The efficacy of antibody capture by hepatic endothelium under different conditions was analyzed using an isolated liver perfusion model. The local enrichment of R-phycoerythrin and 125 I-conjugated antibody was studied in vivo in two hepatic tumor models using biodistribution, scintigraphic imaging, and fluorescence microscopy. Upon injection into the tumor-feeding artery, the antibody was immediately captured in the microvasculature during the first passage. At doses not exceeding the saturation level of endothelial epitopes, the capture efficacy was almost 90%. We showed that the efficacy of endothelial capture is controlled by factors such as antibody affinity, number of binding sites on the endothelium, and microvascular flow rate. The targeting potential of endothelial capture was experimentally proven in vivo using scintigraphic imaging and biodistribution analysis after locoregional intra-arterial injection of 125 I-labeled antibodies in hepatic tumor models. Conclusion: The unique phenomenon of endothelial capture can broadly prevent systemic circulation of the antibody or antibody-drug conjugates applied by intravascular injection and may have specific relevance for targeting of hepatic tumors.

Liver segment imaging using monocyte sequestration: a potential tool for fluorescence-guided liver surgery.

Background: The accurate determination of liver segment anatomy is essential to perform liver resection without complications and to ensure long-term outcomes after this operation. There are several perioperative methods for segment identification and surgical navigation, such as intraoperative ultrasound, indigo carmine and near-infrared imaging with indocyanine green. The present study experimentally analyzed the usefulness of monocyte sequestration for liver segment labeling and imaging. Methods: Human monocytes were isolated from peripheral blood and directly or indirectly labeled with calcein or IRDye 800CW. Potential toxicity, labeling stability, and adhesion to ICAM-1 were analyzed in vitro. Monocyte sequestration in the liver microvasculature and liver segment labeling and boundary demarcation were studied using isolated mouse and pig liver perfusion and via intraportal injection in mouse liver tumor models. Results: The highest monocyte labeling efficiency was achieved using direct labeling with IRDye 800CW. Labeling was stable and did not influence cell viability. The labeled monocytes were highly sequestrated in the liver microvasculature, both after ex vivo perfusion and after injection in vivo , resulting in excellent labeling of selected liver segments and strong segment boundary demarcation. In contrast to results to a normal liver, monocyte sequestration was very low in tumor-associated blood vessels. Conclusions: The present experimental study shows that sequestration of labeled monocytes after superselective application demarcates the selected liver segment. These results illustrate potential of this technique for surgical navigation during liver surgery.

Electrochemical Effects after Transarterial Chemoembolization in Combination with Percutaneous Irreversible Electroporation: Observations in an Acute Porcine Liver Model.

PURPOSE: To evaluate the effects of combined use of transarterial chemoembolization and irreversible electroporation (IRE) for focal tissue ablation in an acute porcine liver model. MATERIALS AND METHODS: Two established interventional techniques were combined: IRE with zones of irreversible and reversible electroporation and chemoembolization with microspheres, iodized oil, and doxorubicin. IRE was performed before chemoembolization in two pigs (pigs 1 and 2; IRE/chemoembolization group), chemoembolization was performed before IRE in two pigs (pigs 3 and 4; chemoembolization/IRE group), and only IRE was performed in two pigs (pigs 5 and 6). Five study groups were defined: IRE/chemoembolization (pigs 1 and 2), chemoembolization/IRE (pigs 3 and 4), IRE only (pigs 5 and 6), chemoembolization only (tissue outside the IRE zones in pigs 1-4), and control (untreated liver tissue outside the IRE zones in pigs 5 and 6). Animals were euthanized 2 hours after intervention. Size and shape of IRE zones on contrast-enhanced computed tomography, cell death on light microscopy, and doxorubicin tissue concentrations on chromatography and fluorescence microscopy were analyzed. RESULTS: Size and shape of IRE zones were not significantly different (eg, P = .067 for volume). A histologic marker for irreversible cell death was positive in IRE/chemoembolization, chemoembolization/IRE, and IRE groups only in the macroscopically visible IRE zones. Doxorubicin tissue concentrations were not significantly different (P = .873). However, in the reversible electroporation (RE) zones, broad areas with intense intranuclear doxorubicin accumulation were observed in IRE/chemoembolization but not in chemoembolization/IRE and chemoembolization groups. CONCLUSIONS: IRE before chemoembolization enhances the intranuclear accumulation of doxorubicin in the RE zone.

Impact of wall shear stress and ligand avidity on binding of anti-CD146-coated nanoparticles to murine tumor endothelium under flow.

The endothelial phenotype of tumor blood vessels differs from the liver and forms an important base for endothelium-specific targeting by antibody-coated nanoparticles. Although differences of shear stress and ligand avidity can modulate the nanoparticle binding to endothelium, these mechanisms are still poorly studied. This study analyzed the binding of antibody-coated nanoparticles to tumor and liver endothelium under controlled flow conditions and verified this binding in tumor models in vivo. Binding of anti-CD146-coated nanoparticles, but not of antibody was significantly reduced under increased wall shear stress and the degree of nanoparticle binding correlated with the avidity of the coating. The intravascular wall shear stress favors nanoparticle binding at the site of higher avidity of endothelial epitope which additionally promotes the selectivity to tumor endothelium. After intravenous application in vivo, pegylated self-coated nanoparticles showed specific binding to tumor endothelium, whereas the nanoparticle binding to the liver endothelium was very low. This study provides a rationale that selective binding of mAb-coated nanoparticles to tumor endothelium is achieved by two factors: higher expression of endothelial epitope and higher nanoparticle shearing from liver endothelium. The combination of endothelial marker targeting and the use of shear stress-controlled nanoparticle capture can be used for selective intratumoral drug delivery.

Aspirin counteracts cancer stem cell features, desmoplasia and gemcitabine resistance in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDA) is characterized by an extremely poor prognosis. An inflammatory microenvironment triggers the pronounced desmoplasia, the selection of cancer stem-like cells (CSCs) and therapy resistance. The anti-inflammatory drug aspirin is suggested to lower the risk for PDA and to improve the treatment, although available results are conflicting and the effect of aspirin to CSC characteristics and desmoplasia in PDA has not yet been investigated. We characterized the influence of aspirin on CSC features, stromal reactions and gemcitabine resistance. Four established and 3 primary PDA cell lines, non-malignant cells, 3 patient tumor-derived CSC-enriched spheroidal cultures and tissues from patients who did or did not receive aspirin before surgery were analyzed using MTT assays, flow cytometry, colony and spheroid formation assays, Western blot analysis, antibody protein arrays, electrophoretic mobility shift assays (EMSAs), immunohistochemistry and in vivo xenotransplantation. Aspirin significantly induced apoptosis and reduced the viability, self-renewal potential, and expression of proteins involved in inflammation and stem cell signaling. Aspirin also reduced the growth and invasion of tumors in vivo, and it significantly prolonged the survival of mice with orthotopic pancreatic xenografts in combination with gemcitabine. This was associated with a decreased expression of markers for progression, inflammation and desmoplasia. These findings were confirmed in tissue samples obtained from patients who had or had not taken aspirin before surgery. Importantly, aspirin sensitized cells that were resistant to gemcitabine and thereby enhanced the therapeutic efficacy. Aspirin showed no obvious toxic effects on normal cells, chick embryos or mice. These results highlight aspirin as an effective, inexpensive and well-tolerated co-treatment to target inflammation, desmoplasia and CSC features PDA.

Selective targeting of liver cancer with the endothelial marker CD146.

Hepatocellular carcinomas are well-vascularized tumors; the endothelial cells in these tumors have a specific phenotype. Our aim was to develop a new approach for tumor-specific drug delivery with monoclonal antibody targeting of endothelial ligands. CD146, a molecule expressed on the endothelial surface of hepatocellular carcinoma, was identified as a promising candidate for targeting. In the present study, endothelial cells immediately captured circulating anti-CD146 (ME-9F1) antibody, while antibody binding in tumors was significantly higher than in hepatic endothelium. Macroscopically, after intravenous injection, there were no differences in the mean accumulation of anti-CD146 antibody in tumor compared to liver tissue , due to a compensating higher blood vessel density in the liver tissue. Additional blockade of nontumoral epitopes and intra-arterial administration, improved selective antibody capture in the tumor microvasculature and largely prevented antibody distribution in the lung and liver. The potential practical use of this approach was demonstrated by imaging of radionuclide-labeled ME-9F1 antibody, which showed excellent tumor-selective uptake. Our results provide a promising principle for the use of endothelial markers for intratumoral drug delivery. Tumor endothelium-based access might offer new opportunities for the imaging and therapy of hepatocellular carcinoma and other liver malignancies.

Anti-inflammatory functions of protein C require RAGE and ICAM-1 in a stimulus-dependent manner.

By binding ß 2-integrins both ICAM-1 and the receptor for advanced glycation end products (RAGE) mediate leukocyte recruitment in a stimulus-dependent manner. Using different inflammatory mouse models we investigated how RAGE and ICAM-1 are involved in anti-inflammatory functions of protein C (PC; Ceprotin, 100 U/kg). We found that, depending on the stimulus, RAGE and ICAM-1 are cooperatively involved in PC-induced inhibition of leukocyte recruitment in cremaster models of inflammation. During short-term proinflammatory stimulation (trauma, fMLP, and CXCL1), ICAM-1 is more important for mediation of anti-inflammatory effects of PC, whereas RAGE plays a major role after longer proinflammatory stimulation (TNF α ). In contrast to WT and Icam-1(-/-) mice, PC had no effect on bronchoalveolar neutrophil emigration in RAGE(-/-) mice during LPS-induced acute lung injury, suggesting that RAGE critically mediates PC effects during acute lung inflammation. In parallel, PC treatment effectively blocked leukocyte recruitment and improved survival of WT mice and Icam-1-deficient mice in LPS-induced endotoxemia, but failed to do so in RAGE-deficient mice. Exploring underlying mechanisms, we found that PC is capable of downregulating intracellular RAGE and extracellular ICAM-1 in endothelial cells. Taken together, our data show that RAGE and ICAM-1 are required for the anti-inflammatory functions of PC.