Anetumab ravtansine: a novel mesothelin-targeting antibody-drug conjugate cures tumors with heterogeneous target expression favored by bystander effect.

Mesothelin is a tumor differentiation antigen frequently overexpressed in tumors such as mesothelioma, ovarian, pancreatic, and lung adenocarcinomas while showing limited expression in nonmalignant tissues. Mesothelin is therefore an attractive target for cancer therapy using antibody-drug conjugates (ADC). This study describes the detailed characterization of anetumab ravtansine, here referred to as BAY 94-9343, a novel ADC consisting of a human anti-mesothelin antibody conjugated to the maytansinoid tubulin inhibitor DM4 via a disulfide-containing linker. Binding properties of the anti-mesothelin antibody were analyzed using surface plasmon resonance, immunohistochemistry, flow cytometry, and fluorescence microscopy. Effects of BAY 94-9343 on cell proliferation were first studied in vitro and subsequently in vivo using subcutaneous, orthotopic, and patient-derived xenograft tumor models. The antibody binds to human mesothelin with high affinity and selectivity, thereby inducing efficient antigen internalization. In vitro, BAY 94-9343 demonstrated potent and selective cytotoxicity of mesothelin-expressing cells with an IC(50) of 0.72 nmol/L, without affecting mesothelin-negative or nonproliferating cells. In vivo, BAY 94-9343 localized specifically to mesothelin-positive tumors and inhibited tumor growth in both subcutaneous and orthotopic xenograft models. In addition, BAY 94-9343 was able to induce a bystander effect on neighboring mesothelin-negative tumor cells. Antitumor efficacy of BAY 94-9343 correlated with the amount of mesothelin expressed and was generally superior to that of standard-of-care regimen resulting in complete tumor eradication in most of the models. BAY 94-9343 is a selective and highly potent ADC, and our data support its development for the treatment of patients with mesothelin-expressing tumors.

Angiopoietin-2 drives lymphatic metastasis of pancreatic cancer.

Lymphatic metastasis constitutes a critical route of disease dissemination, which limits the prognosis of patients with pancreatic ductal adenocarcinoma (PDAC). As lymphangiogenesis has been implicated in stimulation of lymphatic metastasis by vascular endothelial growth factor-C (VEGF-C) and VEGF-D, we studied the effect of the angioregulatory growth factor angiopoietin-2 (Ang-2) on PDAC progression. Ang-2 was found to be expressed in transformed cells of human PDAC specimens, with corresponding Tie-2 receptors present on blood and lymphatic endothelium. In vitro in PDAC cells, Ang-2 was subject to autocrine/paracrine TGF-ß stimulation (2-fold induction, P=0.0106) acting on the -61- to +476-bp element of the human Ang-2 promoter. In turn, Ang-2 regulated the expression of genes involved in cell motility and tumor suppression. Orthotopic PDAC xenografts with forced expression of Ang-2, but not Ang-1, displayed increased blood and lymphatic vessel density, and an enhanced rate of lymphatic metastasis (6.7- to 9.1-fold, P<0.01), which was prevented by sequestration of Ang-2 via coexpression of soluble Tie-2. Notably, elevated circulating Ang-2 in patients with PDAC correlated with the extent of lymphatic metastasis. Furthermore, median survival was reduced from 28.4 to 7.7 mo in patients with circulating Ang-2 ≥ 75th percentile (P=0.0005). These findings indicate that Ang-2 participates in the control of lymphatic metastasis, constitutes a noninvasive prognostic biomarker, and may provide an accessible therapeutic target in PDAC.

D-18F-fluoromethyl tyrosine imaging of bone metastases in a mouse model.

UNLABELLED: The presence and localization of metastatic bone lesions is important for the staging of the disease and subsequent treatment decisions. Detecting tumor cells would have additional value over the current indirect bone scintigraphy method for detecting areas of elevated skeletal metabolic activity. d-(18)F-fluoromethyl tyrosine (d-(18)F-FMT) has recently shown good uptake and fast elimination, resulting in good tumor-to-background ratios. The potential of d-(18)F-FMT for imaging bone metastases has been investigated. METHODS: 786-O/luciferase human renal adenocarcinoma cells were injected intracardially, resulting in the formation of bone metastases in mice. Small-animal PET was performed 51 and 65 d after tumor cell inoculation. RESULTS: d-(18)F-FMT showed specific uptake in the bone metastases, giving excellent images with a little background in the pancreas. All imaged metastases were histologically confirmed. A bone scan with (18)F-fluoride showed elevated skeletal metabolic activity in the areas of osteolytic lesions. CONCLUSION: d-(18)F-FMT is a useful PET tracer for the detection of bone metastases and should be evaluated in the clinical setting.

Characterization of a new renal cell carcinoma bone metastasis mouse model.

Metastatic bone disease caused by renal cell carcinoma (RCC) occurs frequently and becomes more and more prevalent presumably because survival times among patients with disseminated cancers are increasing. Patients with bone metastases from renal cell carcinoma suffer from severe pain, nerve compression syndromes and pathologic fractures. Very little is known about the mechanisms of skeletal metastases of RCC. Thus, to better understand the molecular mechanism of renal cell cancer (RCC) bone metastasis, it is crucial to develop new animal models. We have established a new animal model of RCC metastasis to bone by inoculation of human 786-O/luciferase cells into the left cardiac ventricle of athymic nude mice. The animals developed aggressive osteolytic bone destruction as monitored by radiography and micro-CT-scans with the mean endpoint at 62 +/- 8 days. The extensive bone destruction observed was comparable to the clinical setting and mainly occurred in hind limbs, forelimbs and the spine. The tumors were primarily located within the bone and resulted in destruction of cortical bone. No soft tissue metastases were detected by BLI or histomorphometry. To increase the bone-metastatic potential of the 786-O cell line, an in vivo selection was done yielding a subpopulation causing osteolytic lesions with the mean endpoint of 47 +/- 3 days. The selected subline secreted more proangiogenic factors VEGF and bFGF in vitro compared to the parental cell line suggesting that these tumors are highly vascular. This model provides a reliable reproduction of the clinical situation and therefore, is suitable for designing and evaluating more effective treatments for RCC bone metastasis.

Angiopoietin-2 promotes disease progression of neuroendocrine tumors.

PURPOSE: Inhibition of angiogenesis represents a promising therapeutic strategy in neuroendocrine tumors. Angiopoietin-2 (Ang-2), a ligand of the endothelial tyrosine kinase Tie-2, is emerging as a key regulator of vascular remodeling during tumor angiogenesis. We therefore addressed the expression and biological significance of Ang-2 in human neuroendocrine tumors. EXPERIMENTAL DESIGN: Surgical specimens and serum from neuroendocrine tumor patients were used to determine Ang-2 expression by in situ hybridization or ELISA (circulating Ang-2). Ang-2 biological effects were evaluated following stable transfection into BON human pancreatic neuroendocrine tumor cells. BON clones were grown as orthotopic xenografts in nude mice to determine tumor growth and abdominal metastatic spread. Further analyses included microvessel density, lymphatic vessel density, and nodal invasion. RESULTS: Specimens from pancreatic neuroendocrine tumors and nontransformed pancreatic tissue revealed uniform expression of Ang-2 mRNA in endothelial cells. In contrast, epithelial expression of Ang-2 mRNA occurred exclusively in neuroendocrine tumors. Overexpression of Ang-2 in BON orthotopic xenografts did not affect primary tumor growth, although successful Ang-2 induction was confirmed from elevated serum levels. However, increased microvessel density and enhanced lymphatic metastasis were evident in Ang-2-expressing tumors, indicating a functional role of Ang-2 in experimental neuroendocrine tumors. Consistent with this notion, circulating Ang-2 was significantly elevated in neuroendocrine tumor patients compared with healthy controls. Circulating Ang-2 furthermore correlated with metastatic versus localized disease. The highest Ang-2 concentrations occurred in patients with liver metastasis, and concentrations >or=75th percentile predicted shorter survival (P = 0.0003). CONCLUSION: Induction of Ang-2 in neuroendocrine tumors represents a clinically relevant pathomechanism of disease progression and constitutes an adverse prognostic marker.

High-resolution ultrasound imaging: a novel technique for the noninvasive in vivo analysis of endometriotic lesion and cyst formation in small animal models.

Endometriosis, the presence of endometrial tissue at ectopic sites, is a highly prevalent gynecological disease severely affecting a patient's quality of life. To analyze the mechanisms involved in the disease and to identify new molecular targets for effective therapies, small animal models are an important approach. Herein, we report the first use of high-resolution ultrasound imaging for the in vivo analysis of intraperitoneal endometriotic lesions in mice. This noninvasive technology allows for the repetitive quantitative analysis of growth, cyst development, and adhesion formation of endometriotic lesions with a low intra- and interobserver variability. Moreover, it enables one to easily differentiate between endometrial cysts and stroma. Accordingly, volume measurements of both endometrial cysts and stroma indicated that the initial establishment of endometriotic lesions is associated with enhanced cellular proliferation, followed by a phase of increased secretory activity of endometrial glands. Results of ultrasound analysis correlated well with measurements of lesion volumes by caliper and histology. Importantly, ultrasound imaging could be performed repetitively and noninvasively and reflected best the in vivo situation. The technique could further be demonstrated to successfully monitor the significant inhibition of growth of endometriotic lesions after specific estrogen receptor destabilizator treatment. Thus, high-resolution ultrasound imaging represents an important tool for future preclinical small animal studies, which address the pathophysiology of endometriosis and the development of new treatment strategies.

Comparison of conventional time-intensity curves vs. maximum intensity over time for post-processing of dynamic contrast-enhanced ultrasound.

Our aim was to prospectively compare two post-processing techniques for dynamic contrast-enhanced ultrasound and to evaluate their impact for monitoring antiangiogenic therapy. Thus, mice with epidermoid carcinoma xenografts were examined during administration of polybutylcyanoacrylate-microbubbles using a small animal ultrasound system (40 MHz). Cine loops were acquired and analyzed using time-intensity (TI) and maximum intensity over time (MIOT) curves. Influences of fast (50 microl/2s) vs. slow (50 microl/10s) injection of microbubbles on both types of curves were investigated. Sensitivities of both methods for assessing effects of antiangiogenic treatment (SU11248) were examined. Correlative histological analysis was performed for vessel-density. Mann-Whitney test was used for statistical analysis. Microbubble injection rates significantly influenced upslope, time-to-peak and peak enhancement of conventional TI curves (p<0.05) but had almost no impact on maximum enhancement of MIOT curves (representing relative blood volume). Additionally, maximum enhancement of MIOT curves captured antiangiogenic therapy effects more reliably and earlier (already after 1 day of therapy; p<0.05) than peak enhancement of TI curves. Immunohistochemistry validated the significantly (p<0.01) lower vessel densities in treated tumors and high correlation (R(2)=0.95) between vessel-density and maximum enhancement of MIOT curves was observed. In conclusion, MIOT is less susceptible to variations of the injection's speed. It enables to assess changes of the relative blood volume earlier and with lower standard deviations than conventional TI curves. It can easily be translated into clinical practice and thus may provide a promising tool for cancer therapy monitoring.

Luminescent imaging technology as an opportunity to reduce and refine animal experiments: Light at the end of the tunnel?

In vivo luminescent imaging technology was introduced in experimental life science research several years ago and has rapidly gained wide acceptance. By making use of this technology substantially more information can be gained from animal experiments than was previously possible. The concept of the 3Rs describes the aim to Refine, Reduce and Replace animal models in research. The goal of the present paper is to systematically investigate the impact of luminescent imaging on the 3Rs. In particular, three examples of applications are explained in detail so as to be accessible to the reader unfamiliar with the procedure. The examples are subsequently analysed for and categorised according to their concrete effect on animal welfare as defined by the 3Rs.

Molecular ultrasound imaging of early vascular response in prostate tumors irradiated with carbon ions.

Individualized treatments with combination of radiotherapy and targeted drugs require knowledge about the behavior of molecular targets after irradiation. Angiogenic marker expression has been studied after conventional radiotherapy, but little is known about marker response to charged particles. For the very first time, we used molecular ultrasound imaging to intraindividually track changes in angiogenic marker expression after carbon ion irradiation in experimental tumors. Expression of intercellular adhesion molecule-1 (ICAM-1) and of alpha(v)beta(3)-integrin in subcutaneous AT-1 prostate cancers in rats treated with carbon ions (16 Gy) was studied using molecular ultrasound and immunohistochemistry. For this purpose, cyanoacrylate microbubbles were synthesized and linked to specific ligands. The accumulation of targeted microbubbles in tumors was quantified before and 36 hours after irradiation. In addition, tumor vascularization was analyzed using volumetric Doppler ultrasound. In tumors, the accumulation of targeted microbubbles was significantly higher than in nonspecific ones and could be inhibited competitively. Before irradiation, no difference in binding of alpha(v)beta(3)-integrin-specific or ICAM-1-specific microbubbles was observed in treated and untreated animals. After irradiation, however, treated animals showed a significantly higher binding of alpha(v)beta(3)-integrin-specific microbubbles and an enhanced binding of ICAM-1-specific microbubbles than untreated controls. In both groups, a decrease in vascularization occurred during tumor growth, but no significant difference was observed between irradiated and nonirradiated tumors. In conclusion, carbon ion irradiation upregulates ICAM-1 and alpha(v)beta(3)-integrin expression in tumor neovasculature. Molecular ultrasound can indicate the regulation of these markers and thus may help to identify the optimal drugs and time points in individualized therapy regimens.

Sagopilone inhibits breast cancer bone metastasis and bone destruction due to simultaneous inhibition of both tumor growth and bone resorption.

PURPOSE: Bone metastases have a considerable impact on quality of life in patients with breast and other cancers. Tumors produce osteoclast-activating factors, whereas bone resorption promotes the growth of tumor cells, thus leading to a "vicious cycle" of bone metastasis. Sagopilone, a novel, fully synthetic epothilone, inhibits the growth of breast cancer cells in vitro and in vivo, and here we report its activity in the MDA-MB-231(SA) breast cancer bone metastasis mouse model. EXPERIMENTAL DESIGN: The potency of sagopilone was determined in treatment models simulating the adjuvant (preventive) and metastatic (therapeutic) settings in the clinic. RESULTS: We showed that sagopilone inhibited tumor burden and bone destruction, in addition to reducing tumor-induced cachexia and paraplegia. The reduction in osteolytic lesions, tumor growth in bone, and weight loss was statistically significant in the preventive model compared with the vehicle group. In the therapeutic model, sagopilone treatment significantly lowered the number of activated osteoclasts and significantly reduced the osteolytic lesion area, bone volume loss, and bone resorption compared with vehicle treatment while simultaneously inhibiting tumor burden. An in vitro assay confirmed that sagopilone inhibited osteoclast activation without cytotoxic effects, whereas paclitaxel resulted in lower inhibition and high levels of cytotoxicity. CONCLUSIONS: Sagopilone seems to inhibit the vicious cycle at both the tumor growth and bone resorption stages, suggesting the possibility for substantial benefit in the treatment of patients with breast cancer at risk from bone metastases or with bone lesions already present. Phase II clinical trials with sagopilone in patients with breast cancer are ongoing.

Development of pH-responsive core-shell nanocarriers for delivery of therapeutic and diagnostic agents.

In this paper new dendritic core-shell architectures with pH-labile linkers based on hyperbranched polyglycerol cores and biocompatible poly(ethylene glycol) shells were synthesized which encapsulate the anticancer agent doxorubicin and a dye for near-infrared imaging, an indotricarbocyanine. Acid-sensitive properties of the new nanocarriers and in vitro cytotoxicity of the doxorubicin-nanocarrier are presented as well as preliminary data regarding their toxicity and tumor targeting potential in nude mice.

Vessel fractions in tumor xenografts depicted by flow- or contrast-sensitive three-dimensional high-frequency Doppler ultrasound respond differently to antiangiogenic treatment.

High-frequency volumetric Power Doppler ultrasound (HF-VPDU) captures flow-dependent signals in blood vessels and can be used to assess antiangiogenic therapy effects in rodent tumors. However, the sensitivity is limited to vessels larger than capillaries. Contrast-enhanced HF-VPDU reveals all perfused vessels by assessing stimulated acoustic emissions from disintegrating microbubbles. Thus, we investigated whether flow-sensitive and contrast-enhanced HF-VPDU can depict different vessel fractions and assess their early response to antiangiogenic therapy. Mice with A431 tumors were scanned before and after administration of polybutylcyanoacrylate microbubbles by HF-VPDU. Animals received either antiangiogenic treatment (SU11248) or a control substance and were imaged repeatedly over 9 days. At each time point, tumors were removed for immunohistochemical analysis. During growth of untreated tumors, vascularization decreased correspondingly on flow-sensitive and contrast-enhanced scans. Treated tumors showed a significantly (P < 0.05) stronger decline in vascularization than controls, which was more pronounced in contrast-enhanced scans. Surprisingly, whereas vascularization remained low in contrast-enhanced scans, flow-sensitive ultrasound indicated a reincrease after day 6 with a higher vascularization than the controls at day 9. Histologic evaluation indicated that immature vessels degraded markedly on therapy, whereas large mature vessels on the tumor periphery were more therapy resistant and drew closer due to tumor shrinkage. In conclusion, contrast-enhanced HF-VPDU and flow-sensitive HF-VPDU are both capable of assessing the effects of antiangiogenic therapy. Because contrast-sensitive ultrasound is more sensitive for small immature vessels and flow-sensitive ultrasound mostly captures large vessels at the tumor periphery, the combination of both methods can provide evidence of vascular maturity in tumors.

Dendritic multishell architectures for drug and dye transport.

Here we present the efficiency and versatility of newly developed core-multishell nanoparticles (CMS NPs), to encapsulate and transport the antitumor drugs doxorubicin hydrochloride (Dox), methotrexate (Mtx) and sodium ibandronate (Ibn) as well as dye molecules, i.e., a tetrasulfonated indotricarbocyanine (ITCC) and nile red. Structurally, the CMS NPs are composed of hyperbranched poly(ethylene imine) core functionalized by alkyl diacids connected to monomethyl poly(ethylene glycol). In order to evaluate their transport in aqueous media in vitro, we have used and compared SEC, UV, ITC, and NMR techniques. We observed that the CMS NPs were able to spontaneously encapsulate and transport Dox, Mtx and nile red in both organic and aqueous media as determined by SEC and UV-VIS spectroscopy. For the VIS transparent Ibn Isothermal Titration Calorimetric (ITC) experiments show an exothermic interaction with the CMS NPs. The enthalpic stabilization (DeltaH) upon encapsulation was in the order of approximately 7 kcals/mol which indicates stable interaction between Ibn and nanoparticles. A T(1) inversion recovery NMR experiment was carried out for 31P and 1H nuclei of Ibn and an increment of spin-lattice relaxation time for respective nuclei was observed upon encapsulation. CMS NPs were also found to encapsulate ITCC dye with stoichiometry of 6-8 molecules/nanocarrier. For in vivo imaging studies the dye loaded CMS NPs were injected to F9 teratocarcinoma bearing mice and a strong contrast was observed in the tumor tissues compared to free dye after 6 h of administration.

Differentiation of angiogenic burden in human cancer xenografts using a perfusion-type optical contrast agent (SIDAG).

INTRODUCTION: Use of fluorescence imaging in oncology is evolving rapidly, and nontargeted fluorochromes are currently being investigated for clinical application. Here, we investigated whether the degree of tumour angiogenesis can be assessed in vivo by planar and tomographic methods using the perfusion-type cyanine dye SIDAG (1,1'-bis- [4-sulfobutyl]indotricarbocyanine-5,5'-dicarboxylic acid diglucamide monosodium). METHOD: Mice were xenografted with moderately (MCF7, DU4475) or highly vascularized (HT1080, MDA-MB435) tumours and scanned up to 24 hours after intravenous SIDAG injection using fluorescence reflectance imaging. Contrast-to-noise ratio was calculated for all tumours, and fluorochrome accumulation was quantified using fluorescence-mediated tomography. The vascular volume fraction of the xenografts, serving as a surrogate marker for angiogenesis, was measured using magnetic resonance imaging, and blood vessel profile (BVP) density and vascular endothelial growth factor expression were determined. RESULTS: SIDAG accumulation correlated well with angiogenic burden, with maximum contrast to noise ratio for MDA-MB435 (P < 0.0001), followed by HT1080, MCF7 and DU4475 tumours. Fluorescence-mediated tomography revealed 4.6-fold higher fluorochrome concentrations in MDA-MB435 than in DU4475 tumours (229 +/- 90 nmol/l versus 49 +/- 22 nmol/l; P < 0.05). The vascular volume fraction was 4.5-fold (3.58 +/- 0.9% versus 0.8 +/- 0.53%; P < 0.01), blood vessel profile density 5-fold (399 +/- 36 BVPs/mm2 versus 78 +/- 16 BVPs/mm2) and vascular endothelial growth factor expression 4-fold higher for MDA-MB435 than for DU4475 tumours. CONCLUSION: Our data suggest that perfusion-type cyanine dyes allow assessment of angiogenesis in vivo using planar or tomographic imaging technology. They may thus facilitate characterization of solid tumours.

Pharmacodynamics of streptavidin-coated cyanoacrylate microbubbles designed for molecular ultrasound imaging.

OBJECTIVES: To assess the pharmacodynamic behavior of cyanoacrylate, streptavidin-coated microbubbles (MBs) and to investigate their suitability for molecular ultrasound imaging. MATERIALS AND METHODS: Biodistribution of MBs was analyzed in tumor-bearing mice using gamma-counting, immunohistochemistry, flow cytometry, and ultrasound. Further, vascular endothelial growth factor receptor 2-antibody coupled MBs were used to image tumor neovasculature. RESULTS: After 1 minute >90% of MBs were cleared from the blood and pooled in the lungs, liver, and spleen. Subsequently, within 1 hour a decent reincrease of MB-concentration was observed in the blood. The remaining MBs were removed by liver and spleen macrophages. About 30% of the phagocytosed MBs were intact after 48 hours. Shell fragments were found in the kidneys only. No relevant MB-accumulation was observed in tumors. In contrast, vascular endothelial growth factor receptor 2-specific MBs accumulated significantly within the tumor vasculature (P < 0.05). CONCLUSIONS: The pharmacokinetic behavior of streptavidin-coated cyanoacrylate MBs has been studied. In this context, the low amount of MBs in tumors after >5 minutes is beneficial for specific targeting of angiogenesis.

Molecular profiling of angiogenesis with targeted ultrasound imaging: early assessment of antiangiogenic therapy effects.

Molecular ultrasound is capable of elucidating the expression of angiogenic markers in vivo. However, the capability of the method for volumetric "multitarget quantification" and for the assessment of antiangiogenic therapy response has rather been investigated. Therefore, we generated cyanoacrylate microbubbles linked to vascular endothelial growth factor receptor 2 (VEGFR2) and alphavbeta3 integrin binding ligands and quantified their accumulation in squamous cell carcinoma xenografts (HaCaT-ras-A-5RT3) in mice with the quantitative volumetric ultrasound scanning technique, sensitive particle acoustic quantification. Specificity of VEGFR2 and alphavbeta3 integrin binding microbubbles was shown, and changes in marker expression during matrix metalloproteinase inhibitor treatment were investigated. In tumors, accumulation of targeted microbubbles was significantly higher compared with nonspecific ones and could be inhibited competitively by addition of the free ligand in excess. Also, multimarker imaging could successfully be done during the same imaging session. Molecular ultrasound further indicated a significant increase of VEGFR2 and alphavbeta3 integrin expression during tumor growth and a considerable decrease in both marker densities after matrix metalloproteinase inhibitor treatment. Histologic data suggested that the increasing VEGFR2 and alphavbeta3 integrin concentrations in tumors during growth are related to an up-regulation of its expression by the endothelial cells, whereas its decrease under therapy is more related to the decreasing relative vessel density. In conclusion, targeted ultrasound appears feasible for the longitudinal molecular profiling of tumor angiogenesis and for the sensitive assessment of therapy effects in vivo.

Evaluation of gas-filled microparticles and sonoporation as gene delivery system: feasibility study in rodent tumor models.

PURPOSE: To evaluate the feasibility of gene delivery mediated with diagnostic ultrasound and plasmid DNA (pDNA) encapsulated in gas-filled microparticles (GFMP) in rodent tumor models. MATERIALS AND METHODS: This study was performed according to a protocol approved by the regional animal research committee. The model plasmid UT651 (pUT651) that contained the Escherichia coli LacZ gene for beta-galactosidase was used to demonstrate the feasibility of ultrasound-mediated gene delivery in CC531 liver tumors in rats. In preliminary experiments, a single injection of pUT651-containing GFMP was administered intraarterially (n=4) or intravenously (n=6) with simultaneous sonication (color Doppler mode, maximum mechanical index) of the GFMP passing through the capillaries of the tumors. All animals were sacrificed 2-5 days later, and liver tumors were examined for beta-galactosidase expression histochemically. Subsequently, potential medical usefulness of this delivery system was tested in nude mice bearing Capan-1 tumors (adenocarcinoma of the human pancreas) by using the plasmid RC/CMV-p16 (pRC/CMV-p16), which contains tumor suppressor gene p16. The tumor suppressor gene p16 is deleted in Capan-1 cells. Twenty-five tumor-bearing mice were classified into five groups (four to six mice per group, one treatment group, four control groups) at random. All mice were treated once weekly for 5 weeks with intravenous infusion of p16-containing GFMP or control substances with simultaneous tumor sonication with color Doppler mode ultrasound and maximum mechanical index or without ultrasound treatment. The therapeutic effect of p16 was measured as an increase in tumor volume doubling time. Data were analyzed with analysis of variance. Results were considered significant at the 5% critical level (P < .05). RESULTS: A clear expression of pDNA was found in tumors in rats treated with a combination of pUT651-containing GFMP and ultrasound; relevant controls showed a significantly lower expression of marker gene. The controlled ultrasound-triggered release of pRC/CMV-p16 from GFMP leads to a strong tumor growth inhibition, which is significant (P < .002), compared with that in controls. CONCLUSION: A combination of GFMP and ultrasound provides an effective approach for nonviral gene therapy-based cancer treatment.

Comparison of two tricarbocyanine-based dyes for fluorescence optical imaging.

Optical technologies are evolving in many biomedical areas including the biomedical imaging disciplines. Regarding the absorption properties of physiological molecules in living tissue, the optical window ranging from 700 to 900 nm allows to use fluorescent dyes for novel diagnostic solutions. Here we investigate the potential of two different carbocyanine-based dyes fluorescent in the near infrared as contrast agents for in vivo imaging of subcutaneously grown tumours in laboratory animals. The primary aim was to modify the physicochemical properties of the previously synthesized dye SIDAG to investigate the effect on the in vivo imaging properties.

Molecular imaging of tumor angiogenesis.

Advances in imaging provide new insights into the pathophysiology of many diseases. Established imaging technologies such as MRI, CT, PET, and ultrasound are routinely applied to determine features of tumor blood vessels that distinguish them from normal blood vessels. These techniques yield information on blood flow, blood volume, and vessel permeability. Often, an intravenously injected imaging contrast agent without affinity to a specific target structure is applied to enable detection of malignant lesions. One of the emerging innovations in diagnostic imaging is the evolution of molecular imaging techniques. Molecular imaging is a noninvasive approach to determine the expression of indicative marker molecules of the tumor angiogenesis process. Meanwhile, this approach has been established for all imaging modalities and may further improve sensitivity of diagnostic tumor imaging. Another goal is to provide information with respect to drug treatment monitoring and therapeutic vascular targeting strategies.

Activated signal transducer and activator of transcription 3 (STAT3) supports the malignant phenotype of human pancreatic cancer.

BACKGROUND & AIMS: Constitutive activation of signal transducer and activator of transcription 3 (STAT3) has been implicated in regulation of growth and malignant transformation. We therefore analyzed the expression and biologic significance of STAT3 in human pancreatic cancer cells. METHODS: Expression and activation of STAT3 were investigated by immunohistochemistry and immunoblotting. Functional inactivation of STAT3 was achieved by stable transfection of dominant-negative STAT3 constructs in 2 pancreatic cancer cell lines and confirmed by electrophoretic mobility shift assay and immunoblotting. Cell proliferation and tumorigenicity were evaluated by cell counting, colony formation in soft agar, and xenotransplantation in nude mice. STAT3-dependent cell cycle distribution was monitored by flow cytometry, immunoprecipitation, immunoblotting, and histone H1 and GST-Rb kinase assays. RESULTS: Compared with nontransformed human pancreas, activated STAT3 is overexpressed in ductal carcinoma cells but not in ducts from chronic pancreatitis. Constitutive activation was also observed in all human pancreatic cancer cell lines examined. Functional inactivation of STAT3 resulted in significant inhibition of anchorage-dependent and -independent proliferation in vitro and reduced tumor growth in vivo. Cell cycle analysis showed a delay of G(1)/S-phase progression due to inhibition of cyclin-dependent kinase 2 activity based on increased expression of p21(WAF1) in vitro and in vivo. Blocking of the STAT3 upstream activator Janus kinase 2 by tyrphostin also resulted in growth arrest because of delayed G(1)/S-phase progression and increased expression of p21(WAF1). CONCLUSIONS: On malignant transformation, activated STAT3 promotes cellular proliferation by acceleration of G(1)/S-phase progression and thereby contributes to the malignant phenotype of human pancreatic cancer.