Effects of cilengitide in osteoclast maturation and behavior.

Bone metastasis is a common burden in many types of cancer and has a severe impact on the quality of life in patients. Hence, specific therapeutic strategies inhibiting tumor induced osteolysis are urgently needed. In this study, we aimed to interfere with integrin adhesion receptors, which are central players of the bone resorption process. For this purpose, we used cilengitide, a cyclic RGD peptide, which blocks integrin αVß3 and αVß5-ligand binding. Our results revealed that cilengitide blocked osteoclast maturation in a dose-dependent manner. In detail, pre-osteoclasts treated with cilengitide exhibited reduced cell spreading, cell migration and cell adhesion on RGD-containing matrix proteins, which are ligands of integrin αV. The activation of the most upstream signal transduction molecules of the integrin receptor-initiated pathway, FAK and c-Src, were consistently blocked by cilengitide. First evidence suggests that cilengitide might interfere with metastatic bone disease in vivo and this study describes a potential underlying mechanism of the inhibitory effect of cilengitide on αV-integrin expressing pre-osteoclasts by blocking integrin ligand binding and interfering with osteoclast maturation and cell behavior. In conclusion, our findings suggest that cilengitide, which interferes with αV-integrins on osteoclasts, may represent a novel therapeutic strategy in the treatment of malignant bone disease.

Treatment Efficiency of Free and Nanoparticle-Loaded Mitoxantrone for Magnetic Drug Targeting in Multicellular Tumor Spheroids.

Major problems of cancer treatment using systemic chemotherapy are severe side effects. Magnetic drug targeting (MDT) employing superparamagnetic iron oxide nanoparticles (SPION) loaded with chemotherapeutic agents may overcome this dilemma by increasing drug accumulation in the tumor and reducing toxic side effects in the healthy tissue. For translation of nanomedicine from bench to bedside, nanoparticle-mediated effects have to be studied carefully. In this study, we compare the effect of SPION, unloaded or loaded with the cytotoxic drug mitoxantrone (MTO) with the effect of free MTO, on the viability and proliferation of HT-29 cells within three-dimensional multicellular tumor spheroids. Fluorescence microscopy and flow cytometry showed that both free MTO, as well as SPION-loaded MTO (SPION(MTO)) are able to penetrate into tumor spheroids and thereby kill tumor cells, whereas unloaded SPION did not affect cellular viability. Since SPION(MTO) has herewith proven its effectivity also in complex multicellular tumor structures with its surrounding microenvironment, we conclude that it is a promising candidate for further use in magnetic drug targeting in vivo.

CD98hc (SLC3A2) drives integrin-dependent renal cancer cell behavior.

BACKGROUND: Overexpression of CD98hc (SLC3A2) occurs in a variety of cancers and is suspected to contribute to tumor growth. CD98, a heterodimeric transmembrane protein, physically associates with certain integrin ß subunit cytoplasmic domains via its heavy chain, CD98hc. CD98hc regulates adhesion-induced intracellular signal transduction via integrins, thereby, affecting cell proliferation and clonal expansion. Disruption of CD98hc led to embryonic lethality in mice (E 3.5 and E 9.5) and CD98hc -/- embryonic stem cell transplantation failed to form teratomas, while CD98hc over-expression in somatic cells resulted in anchorage-independent growth. However, it is unclear whether interference with CD98hc expression tumor cell behavior. METHODS: Renal cell cancer cell lines have been used to determine the effect of CD98hc expression on cancer cell behavior using cell adhesion, cell trans-migration and cell spreading assays. Flow cytometric analysis was performed to study the rate of apoptosis after detachment or serum starvation. shRNA-lentiviral constructs were used to stably knockdown or reconstitute full length or mutated CD98hc. The role of CD98 as a promotor of tumorigenesis was evaluated using an in in vivo tumor transplantation animal model. Immunohistochemical analysis was performed to analyze cell proliferation and CD98 expression in tumors. RESULTS: This report shows that CD98hc silencing in clear cell renal cancer cells reverts certain characteristics of tumorigenesis, including cell spreading, migration, proliferation and survival in vitro, and tumor growth in vivo. Acquisition of tumorigenic characteristics in clear cell renal cancer cells occurred through the integrin binding domain of CD98hc. A CD98hc/integrin interaction was required for adhesion-induced sustained FAK phosphorylation and activation of the major downstream signaling pathways PI3k/Akt and MEK/ERK, while overexpression of a constitutive active form of FAK rescued the CD98hc deficiency. CONCLUSIONS: In this study we demonstrate that loss of CD98hc blocks tumorigenic potential in renal cell cancer.

Dextran-coated superparamagnetic iron oxide nanoparticles for magnetic resonance imaging: evaluation of size-dependent imaging properties, storage stability and safety.

BACKGROUND: Rising criticism of currently available contrast agents for magnetic resonance imaging, either due to their side effects or limited possibilities in terms of functional imaging, evoked the need for safer and more versatile agents. We previously demonstrated the suitability of novel dextran-coated superparamagnetic iron oxide nanoparticles (SPIONDex) for biomedical applications in terms of safety and biocompatibility. METHODS: In the present study, we investigated the size-dependent cross-linking process of these particles as well as the size dependency of their imaging properties. For the latter purpose, we adopted a simple and easy-to-perform experiment to estimate the relaxivity of the particles. Furthermore, we performed an extensive analysis of the particles' storage stability under different temperature conditions, showing their superb stability and the lack of any signs of agglomeration or sedimentation during a 12 week period. RESULTS: Independent of their size, SPIONDex displayed no irritation potential in a chick chorioallantoic membrane assay. Cell uptake studies of ultra-small (30 nm) SPIONDex confirmed their internalization by macrophages, but not by non-phagocytic cells. Additionally, complement activation-related pseudoallergy (CARPA) experiments in pigs treated with ultra-small SPIONDex indicated the absence of hypersensitivity reactions. CONCLUSION: These results emphasize the exceptional safety of SPIONDex, setting them apart from the existing SPION-based contrast agents and making them a very promising candidate for further clinical development.

Toxicity of Mitoxantrone-loaded Superparamagnetic Iron Oxide Nanoparticles in a HT-29 Tumour Spheroid Model.

BACKGROUND/AIM: Cancer research is commonly carried out in two-dimensional (2D) cell cultures, which poorly reflect in vivo settings where the growing tumours are exposed to mechanical forces and biochemical gradients. In this study we established a HF-29 colon carcinoma tumor spheroid model to investigate the effect of free mitoxantrone (MTO) and its nanoparticle-bound form (SPION(MTO)) under 3D cell culture conditions. MATERIALS AND METHODS: Tumour spheroids were generated by seeding HT-29 colon carcinoma cells on agarose-coated cell culture wells. Growth of the spheroids was monitored daily by transmission microscopy upon treatment with free MTO, SPION(MTO) or unloaded SPION. RESULTS AND CONCLUSION: Unloaded SPION did not affect the spheroid size compared to untreated controls, while both free MTO and SPION(MTO) inhibited growth of the spheroids in a dose- and time-dependent manner. In comparison to free MTO, the effect of SPION(MTO) on spheroid growth was slightly delayed. Further analyses are necessary to investigate if MTO infiltrates spheroids in its nanoparticle-bound form or whether it is released from SPION before infiltration.

Hypericin-bearing magnetic iron oxide nanoparticles for selective drug delivery in photodynamic therapy.

Combining the concept of magnetic drug targeting and photodynamic therapy is a promising approach for the treatment of cancer. A high selectivity as well as significant fewer side effects can be achieved by this method, since the therapeutic treatment only takes place in the area where accumulation of the particles by an external electromagnet and radiation by a laser system overlap. In this article, a novel hypericin-bearing drug delivery system has been developed by synthesis of superparamagnetic iron oxide nanoparticles (SPIONs) with a hypericin-linked functionalized dextran coating. For that, sterically stabilized dextran-coated SPIONs were produced by coprecipitation and crosslinking with epichlorohydrin to enhance stability. Carboxymethylation of the dextran shell provided a functionalized platform for linking hypericin via glutaraldehyde. Particle sizes obtained by dynamic light scattering were in a range of 55-85 nm, whereas investigation of single magnetite or maghemite particle diameter was performed by transmission electron microscopy and X-ray diffraction and resulted in approximately 4.5-5.0 nm. Surface chemistry of those particles was evaluated by Fourier transform infrared spectroscopy and ζ potential measurements, indicating successful functionalization and dispersal stabilization due to a mixture of steric and electrostatic repulsion. Flow cytometry revealed no toxicity of pure nanoparticles as well as hypericin without exposure to light on Jurkat T-cells, whereas the combination of hypericin, alone or loaded on particles, with light-induced cell death in a concentration and exposure time-dependent manner due to the generation of reactive oxygen species. In conclusion, the combination of SPIONs' targeting abilities with hypericin's phototoxic properties represents a promising approach for merging magnetic drug targeting with photodynamic therapy for the treatment of cancer.

Nanomedical innovation: the SEON-concept for an improved cancer therapy with magnetic nanoparticles.

Nanomedicine offers tremendous opportunities for the development of novel therapeutic and diagnostic tools. During the last decades, extensive knowledge was gained about stabilizing and the coating of nanoparticles, their functionalization for drug binding and drug release and possible strategies for therapies and diagnostics of different diseases. Most recently, more and more emphasis has been placed on nanotoxicology and nanosafety aspects. The section of experimental oncology and nanomedicine developed a concept for translating this knowledge into clinical application of magnetic drug targeting for the treatment of cancer and other diseases using superparamagnetic iron oxide nanoparticles. This approach includes reproducible synthesis, detailed characterization, nanotoxicological testing, evaluation in ex vivo models, preclinical animal studies and production of superparamagnetic iron oxide nanoparticles according to good manufacturing practice regulations.

Flow cytometry for intracellular SPION quantification: specificity and sensitivity in comparison with spectroscopic methods.

Due to their special physicochemical properties, iron nanoparticles offer new promising possibilities for biomedical applications. For bench to bedside translation of super-paramagnetic iron oxide nanoparticles (SPIONs), safety issues have to be comprehensively clarified. To understand concentration-dependent nanoparticle-mediated toxicity, the exact quantification of intracellular SPIONs by reliable methods is of great importance. In the present study, we compared three different SPION quantification methods (ultraviolet spectrophotometry, magnetic particle spectroscopy, atomic adsorption spectroscopy) and discussed the shortcomings and advantages of each method. Moreover, we used those results to evaluate the possibility to use flow cytometric technique to determine the cellular SPION content. For this purpose, we correlated the side scatter data received from flow cytometry with the actual cellular SPION amount. We showed that flow cytometry provides a rapid and reliable method to assess the cellular SPION content. Our data also demonstrate that internalization of iron oxide nanoparticles in human umbilical vein endothelial cells is strongly dependent to the SPION type and results in a dose-dependent increase of toxicity. Thus, treatment with lauric acid-coated SPIONs (SEON(LA)) resulted in a significant increase in the intensity of side scatter and toxicity, whereas SEON(LA) with an additional protein corona formed by bovine serum albumin (SEON(LA-BSA)) and commercially available Rienso(®) particles showed only a minimal increase in both side scatter intensity and cellular toxicity. The increase in side scatter was in accordance with the measurements for SPION content by the atomic adsorption spectroscopy reference method. In summary, our data show that flow cytometry analysis can be used for estimation of uptake of SPIONs by mammalian cells and provides a fast tool for scientists to evaluate the safety of nanoparticle products.

Soluble carcinoembryonic antigen activates endothelial cells and tumor angiogenesis.

Carcinoembryonic antigen (CEA, CD66e, CEACAM-5) is a cell-surface-bound glycoprotein overexpressed and released by many solid tumors that has an autocrine function in cancer cell survival and differentiation. Soluble CEA released by tumors is present in the circulation of patients with cancer, where it is used as a marker for cancer progression, but whether this form of CEA exerts any effects in the tumor microenvironment is unknown. Here, we present evidence that soluble CEA is sufficient to induce proangiogenic endothelial cell behaviors, including adhesion, spreading, proliferation, and migration in vitro and tumor microvascularization in vivo. CEA-induced activation of endothelial cells was dependent on integrin ß-3 signals that activate the focal-adhesion kinase and c-Src kinase and their downstream MAP-ERK kinase/extracellular signal regulated kinase and phosphoinositide 3-kinase/Akt effector pathways. Notably, while interference with VEGF signaling had no effect on CEA-induced endothelial cell activation, downregulation with the CEA receptor in endothelial cells attenuated CEA-induced signaling and tumor angiogenesis. Corroborating these results clinically, we found that tumor microvascularization was higher in patients with colorectal cancer exhibiting higher serum levels of soluble CEA. Together, our results elucidate a novel function for soluble CEA in tumor angiogenesis.

Angiogenesis in cancer: Anti-VEGF escape mechanisms.

It is now widely accepted that tumor-angiogenesis plays a crucial role in tumor growth, tumor propagation and metastasis formation. Among several angiogenic activators, the vascular endothelial growth factor (VEGF) and its receptors represent one of the major inducers of tumor angiogenesis. Thus, this system has become the focus of therapeutic interventions, which led to the approval of the anti-VEGF blocking antibody bevacizumab and the VEGFR-2 pathway inhibitors pazopanib, sorafenib and sunitinib. However, not every cancer patient benefits from such treatment or finally becomes resistant to anti-VEGF approaches; others are suffering from adverse effects. Thus, there is an urgent need for a better understanding of VEGF-independent mechanisms leading to angiogenesis in cancer. This review focuses on anti-VEGF escape mechanisms of tumor cells and its microenvironment.

The urokinase receptor (CD87) represents a central mediator of growth factor-induced endothelial cell migration.

Angiogenesis, the sprouting of blood vessels form pre-existing vasculature after injury or in neoplastic diseases, is initiated by growth factor-induced endothelial cell migration. Recently, the major angiogenic growth factor VEGF165 has become the target of therapeutic interventions. However, this approach has been clinically proven to be of limited efficacy, which might be due to the fact that tumour angiogenesis is not only induced by VEGF, but also by a variety of other growth factors. Thus, the identification of a common downstream mediator of growth-factor-induced endothelial cell migration is mandatory to effectively interfere with (tumour-) angiogenesis. We found that the urokinase-type plasminogen activator (uPA)-system, which affects proteolytic as well as adhesive capacities, represents an essential regulatory mechanism in growth factor-induced endothelial cell migration and invasion. This mechanism was not limited to VEGF165, but mediated pro-angiogenic endothelial cell behaviour induced by various growth factors. Thus, VEGF165, VEGF-E, FGF-2, EGF as well as HGF induced a PI3k-dependent activation of pro-uPA when bound to uPAR, which led to an increase in cell surface fibrinolytic activity. As a consequence, uPAR became internalised and redistributed via LDLR-proteins. Interference with these events led to a reduced migratory response of endothelial cells towards VEGF in vitro as well as endothelial cell invasion in vivo. These data give first evidence that the uPA-system, which represents the only level-of-evidence-1 cancer biomarker system for prognosis and/or prediction in node negative breast cancer, might directly affect (tumour-) angiogenesis.

Targeting of VEGF-dependent transendothelial migration of cancer cells by bevacizumab.

Cancer progression is often associated with the formation of malignant effusions. Vascular endothelial growth factor (VEGF) is a major regulator of vascular permeability and has been implicated as mediator of tumor progression. We examined the production and secretion of VEGF(165) in various primary cancer cells derived from malignant effusions, and the role of exogenous VEGF(165) as a mediator of effusion formation. VEGF(165) was constantly secreted by all cultured tumor cells in an mTOR-dependent manner, as it was inhibited by the mTOR inhibitor rapamycin. Secreted VEGF(165) showed functional activity by inducing endothelial leakiness and tumor cell-transendothelial migration in vitro, effects which could be reverted by the anti-VEGF antibody bevacizumab. Thus, mTOR inhibitors as well as bevacizumab should be considered as potential agents in cancer patients suffering from malignant effusions.