Tumor‐targeting Salmonella typhimurium A1‐R arrests a doxorubicin‐resistant PDGFRA‐amplified patient‐derived orthotopic xenograft mouse model of pleomorphic liposarcoma

Pleomorphic liposarcoma (PLPS) is a recalcitrant soft‐tissue sarcoma (STS) subtype in need of transformative therapy. We have previously established a patient‐derived orthotopic xenograft (PDOX) model, of PLPS with PDGFRA amplification, using surgical orthotopic implantation. In the current study, the PLPS PDOX model was randomized into 3 groups of 7 mice each: untreated control; doxorubicin (DOX)‐treated; and treated with Salmonella typhimurium A1‐R (S. typhimurium A1‐R) expressing green fluorescent protein (GFP). Tumor volume and body weight were monitored during the treatment period. The PLPS PDOX was resistant to DOX. In contrast, the PLPS PDOX was highly sensitive to S. typhimurium A1‐R. There was no significant body‐weight loss among these 3 groups. Fluorescence imaging demonstrated that S. typhimurium A1‐R‐GFP was very effective to target the PLPS PDOX tumor. The current study demonstrates that a PLPS PDOX, resistant to first‐line therapy DOX, was highly sensitive to tumor targeting S. typhimurium A1‐R.     

In‐vivo Tumor detection using diffusion reflection measurements of targeted gold nanorods – a quantitative study

The ability to quantitatively and non‐invasively detect nanoparticles has important implications on their development as an in‐vivo cancer diagnostic tool. The Diffusion Reflection (DR) method is a simple, non‐invasive imaging technique which has been proven useful for the investigation of tissue's optical parameters. In this study, Monte Carlo (MC) simulations, tissue‐like phantom experiments and in‐vivo measurements of the reflected light intensity from tumor bearing mice are presented. Following intravenous injection of antibody conjugated poly (ethylene glycol)‐coated (PEGylated) gold nanorods (GNR) to tumor‐bearing mice, accumulation of GNR in the tumor was clearly detected by the DR profile of the tumor. The ability of DR measurements to quantitate in‐vivo the concentration of the GNR in the tumor was demonstrated and validated with Flame Atomic Absorption spectroscopy results. With GNR as absorbing contrast agents, DR has important potential applications in the image guided therapy of superficial tumors such as head and neck cancer, breast cancer and melanoma. (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

A transgenic red fluorescent protein‐expressing nude mouse for color‐coded imaging of the tumor microenvironment

The tumor microenvironment (TME) is critical for tumor growth and progression. We have previously developed color‐coded imaging of the TME using a green fluorescent protein (GFP) transgenic nude mouse as a host. However, most donor sources of cell types appropriate for study in the TME are from mice expressing GFP. Therefore, a nude mouse expressing red fluorescent protein (RFP) would be an appropriate host for transplantation of GFP‐expressing stromal cells as well as double‐labeled cancer cells expressing GFP in the nucleus and RFP in the cytoplasm, thereby creating a three‐color imaging model of the TME. The RFP nude mouse was obtained by crossing non‐transgenic nude mice with the transgenic C57/B6 mouse in which the β‐actin promoter drives RFP (DsRed2) expression in essentially all tissues. In crosses between nu/nu RFP male mice and nu/+ RFP female mice, the embryos fluoresced red. Approximately 50% of the offspring of these mice were RFP nude mice. In the RFP nude mouse, the organs all brightly expressed RFP, including the heart, lungs, spleen, pancreas, esophagus, stomach, duodenum, the male and female reproductive systems; brain and spinal cord; and the circulatory system, including the heart, and major arteries and veins. The skinned skeleton highly expressed RFP. The bone marrow and spleen cells were also RFP positive. GFP‐expressing human cancer cell lines, including HCT‐116‐GFP colon cancer and MDA‐MB‐435‐GFP breast cancer were orthotopically transplanted to the transgenic RFP nude mice. These human tumors grew extensively in the transgenic RFP nude mouse. Dual‐color fluorescence imaging enabled visualization of human tumor–host interaction. The RFP nude mouse model should greatly expand our knowledge of the TME. J. Cell. Biochem. 106: 279–284, 2009. © 2008 Wiley‐Liss, Inc.     

Effect of the Interleukin‐6 (−174) G/C Promoter Polymorphism on Adiponectin and Insulin Sensitivity

We investigated the relation among the interleukin (IL)‐6 (?174) G/C promoter polymorphism, adipose tissue gene expression of IL6, circulating adiponectin, and systemic insulin sensitivity. Eighty‐five Swedish male subjects who had participated in our previous prediabetic phenotype characterization study were genotyped for the IL6 (?174) G/C polymorphism. Subcutaneous adipose tissue gene expression of IL6 and adiponectin was measured in 44 subjects. The IL6 (?174) G allele carriers had higher fasting plasma insulin levels (C/C, 7.8 ± 1.1; G/C, 9.0 ± 0.6; G/G, 10.5 ± 1.0 mU/L) and higher homeostasis model assessment for insulin resistance (C/C, 1.6 ± 0.2; G/C, 1.9 ± 0.1; G/G, 2.2 ± 0.2) compared with subjects with the C/C genotype. The circulating adiponectin levels were lower in the G allele carriers (C/C, 7.93 ± 0.45; G/C, 7.05 ± 0.44; G/G, 7.02 ± 0.46 μg/mL), whereas the IL‐6 levels did not differ among the three genotypes. Adipose tissue IL6 gene expression was significantly higher in the G allele carriers compared with the subjects homozygous for the C allele (C/C, 0.29 ± 0.15; G/C, 0.84 ± 0.29; G/G, 0.62 ± 0.35). Our results suggest that IL6 (?174) G/C polymorphism is associated with insulin resistance and increased adipose tissue IL6 gene expression, which can impair adiponectin production.     

Feasibility of photoacoustic evaluations on dual‐thermal treatment of ex vivo bladder tumors

A variety of thermal therapeutic methods have been investigated to treat bladder tumors but often cause bowel injury and bladder wall perforation due to high treatment dosage and limited clinical margins. The objective of the current study is to develop a dual‐thermal modality to deeply coagulate the bladder tumors at low thermal dosage and to evaluate therapeutic outcomes with high contrast photoacoustic imaging (PAI). High intensity focused ultrasound (HIFU) is combined with 532 nm laser light to enhance therapeutic depth during thermal treatments on artificial tumor‐injected bladder tissue ex vivo. PAI is employed to identify the margins of the tumors pre‐ and post‐treatments. The dual‐thermal modality achieves 3‐ and 1.8‐fold higher transient temperature changes and 2.2‐ and 1.5‐fold deeper tissue denaturation than laser and HIFU, respectively. PAI vividly identifies the position of the injected tumor and entails approximately 7.9 times higher image contrast from the coagulated tumor as that from the untreated tumor. Spectroscopic analysis exhibits that both 740 nm and 760 nm attains the maximum photoacoustic amplitudes from the treated areas. The proposed PAI‐guided dual‐thermal treatments (laser and HIFU) treatments can be a feasible therapeutic modality to treat bladder tumors in a controlled and efficient manner.

     

Transdifferentiation of human gingival mesenchymal stem cells into functional keratinocytes by Acalypha indica in three‐dimensional microenvironment

Gingival tissue is reportedly a promising, easily accessible, abundant resource of mesenchymal stem cells (MSC) for use in various tissue engineering strategies. Human gingival MSC (HGMSCs) were successfully isolated from gingival tissue and characterized. To analyze in a two‐dimensional form, HGMSCs were cultured with basal medium and induced with 25 µg/ml of Acalypha indica. Quantitative real‐time polymerase chain reaction (qPCR) and western blot analysis showed the presence of keratinocyte‐specific markers, including cytokeratin‐5 and involucrin. To further assess its capability for stratification akin to human keratinocytes, HGMSCs were encapsulated in a HyStem^®‐HP Cell Culture Scaffold Kit and cultured in the presence of A. indica. Calcein AM staining indicated that the HyStem^®‐HP Scaffold Kit has excellent biocompatibility. Immunofluorescence and qPCR analysis revealed the presence of keratinocyte‐specific markers. The study concluded that the three‐dimensional microenvironment is a novel method for inducing epidermal differentiation of HGMSCs to engineer epidermal substitutes with the help of A. indica, which provides an alternative strategy for skin tissue engineering.     

Droplet aerodynamics,cellular uptake,and efficacy of a nebulizable corticosteroid nanosuspension are superior to a micronized dosage form

Inhaled corticosteroids are considered to be an effective prophylactic against the morbid symptoms of several lung diseases, but scope remains for improvement in drug delivery technology to benefit bioavailability and treatment compliance. To ascertain whether dosage form might influence bioavailability, the emission characteristics and efficacy of a nanoparticulate budesonide formulation (Nanagel®) were compared with those of a proprietary micronized suspension (Pulmicort®) when delivered as a nebulized aerosol to human airway epithelial cells in a culture model. Having the visual appearance of a clear solution, Nanagel® was delivered by both jet and vibrating mesh nebulizers as an increased fine particle fraction and with a smaller mass median aerodynamic diameter (MMAD) compared to the micronized suspension. Quantitative high performance liquid chromatography (HPLC) analysis of cultured epithelia one hour after treatment with Nanagel® revealed a significantly greater cellular accumulation of budesonide when compared with Pulmicort® for an equivalent dose, a differential which persisted 24 and 48 h later. A quantitative in vitro assay measuring the activity of enzymes involved in superoxide production revealed that stressed HaCaT cells (a long‐lived, spontaneously immortalized human keratinocyte line) treated with Nanagel® continued to show significantly greater attenuation of inflammatory response compared with Pulmicort®‐treated cells 24 h after the application of an equivalent budesonide dose. The present in vitro findings suggest that formulation of inhalable drugs such as budesonide as aerosolized nanoparticulate, rather than microparticulate, suspensions can enhance bioavailability with concomitant improvements in efficacy. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

Efficacy of a micro‐encapsulated formulation compared with a sticky barrier for excluding ants from citrus canopies

Inesfly IGR FITO^® is an insecticidal paint containing chlorpyrifos and pyriproxyfen incorporated in a micro‐encapsulated formulation that confers the advantage of releasing active ingredients slowly. In this study, a 15‐cm band of Inesfly IGR FITO^® was painted around citrus trunks. The efficacy of this paint and a sticky barrier to exclude ants from foraging in citrus trees was evaluated in two citrus orchards during the season in two different ant communities, one dominated by Lasius grandis and the other by Linepithema humile. Field results demonstrated that a single application of Inesfly IGR FITO^® at the beginning of the season was highly effective in excluding ants from canopies throughout the season. Inesfly IGR FITO^® provides an efficient and more economical alternative than current ant exclusion strategies used in many perennial crops. Further studies should be performed to determine the effects of this strategy on other pests and on beneficial arthropods in citrus.     

Cytotoxic Efficacy of Photodynamic Therapy in Osteosarcoma Cells In Vitro

In recent years, there has been the difficulty in finding more effective therapies against cancer with less systemic side effects. Therefore Photodynamic Therapy is a novel approach for a more tumor selective treatment.Photodynamic Therapy (PDT) that makes use of a nontoxic photosensitizer (PS), which, upon activation with light of a specific wavelength in the presence of oxygen, generates oxygen radicals that elicit a cytotoxic response¹. Despite its approval almost twenty years ago by the FDA, PDT is nowadays only used to treat a limited number of cancer types (skin, bladder) and nononcological diseases (psoriasis, actinic keratosis)².The major advantage of the use of PDT is the ability to perform a local treatment, which prevents systemic side effects. Moreover, it allows the treatment of tumors at delicate sites (e.g. around nerves or blood vessels). Here, an intraoperative application of PDT is considered in osteosarcoma (OS), a tumor of the bone, to target primary tumor satellites left behind in tumor surrounding tissue after surgical tumor resection. The treatment aims at decreasing the number of recurrences and at reducing the risk for (postoperative) metastasis.In the present study, we present in vitro PDT procedures to establish the optimal PDT settings for effective treatment of widely used OS cell lines that are used to reproduce the human disease in well established intratibial OS mouse models. The uptake of the PS mTHPC was examined with a spectrophotometer and phototoxicity was provoked with laser light excitation of mTHPC at 652 nm to induce cell death assessed with a WST-1 assay and by the counting of surviving cells. The established techniques enable us to define the optimal PDT settings for future studies in animal models. They are an easy and quick tool for the evaluation of the efficacy of PDT in vitro before an application in vivo.     

Rhodobacter sphaeroides,a novel tumor‐targeting bacteria that emits natural near‐infrared fluorescence

Several optical imaging techniques have been used to monitor bacterial tropisms for cancer. Most such techniques require genetic engineering of the bacteria to express optical reporter genes. This study investigated a novel tumor‐targeting strain of bacteria, Rhodobacter sphaeroides 2.4.1 (R. sphaeroides), which naturally emits near‐infrared fluorescence, thereby facilitating the visualization of bacterial tropisms for cancer. To determine the penetration depth of bacterial fluorescence, various numbers of cells (from 10⁸ to 10¹⁰ CFU) of R. sphaeroides and two types of Escherichia coli, which stably express green fluorescent protein (GFP) or red fluorescent protein (RFP), were injected s.c. or i.m. into mice. Bacterial tropism for cancer was determined after i.v. injection of R. sphaeroides (10⁸ CFU) into mice implanted s.c. with eight types of tumors. The intensity of the fluorescence signal in deep tissue (muscle) from R. sphaeroides was much stronger than from E. coli‐expressing GFP or RFP. The near‐infrared fluorescence signal from R. sphaeroides was visualized clearly in all types of human or murine tumors via accumulation of bacteria. Analyses of C‐reactive protein and procalcitonin concentrations and body weights indicated that i.v. injection of R. sphaeroides does not induce serious systemic immune reactions. This study suggests that R. sphaeroides could be used as a tumor‐targeting microorganism for the selective delivery of drugs to tumor tissues without eliciting a systemic immune reaction and for visualizing tumors.     

Imaging directed photothermolysis through two‐photon absorption demonstrated on mouse skin – a potential novel tool for highly targeted skin treatment

One‐photon absorption based traditional laser treatment may not necessarily be selective at the microscopic level, thus could result in un‐intended tissue damage. Our objective is to test whether two‐photon absorption (TPA) could provide highly targeted tissue alteration of specific region of interest without damaging surrounding tissues. TPA based laser treatments (785 nm, 140 fs pulse width, 90 MHz) were performed on ex vivo mouse skin using different average power levels and irradiation times. Reflectance confocal microscopy (RCM) and combined second‐harmonic‐generation (SHG) and two‐photon fluorescence (TPF) imaging channels were used to image before, during, and after each laser treatment. The skin was fixed, sectioned and H & E stained after each experiment for histological assessment of tissue alterations and for comparison with the non‐invasive imaging assessments. Localized destruction of dermal fibers was observed without discernible epidermal damage on both RCM and SHG + TPF images for all the experiments. RCM and SHG + TPF images correlated well with conventional histological examination. This work demonstrated that TPA‐based light treatment provides highly localized intradermal tissue alteration. With further studies on optimizing laser treatment parameters, this two‐photon absorption photothermolysis method could potentially be applied in clinical dermatology. (© 2014 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Dynamic cultivation of human mesenchymal stem cells in a rotating bed bioreactor system based on the Z®RP platform

Because the regeneration of large bone defects is limited by quantitative restrictions and risks of infections, the development of bioartificial bone substitutes is of great importance. To obtain a three‐dimensional functional tissue‐like graft, static cultivation is inexpedient due to limitations in cell density, nutrition and oxygen support. Dynamic cultivation in a bioreactor system can overcome these restrictions and furthermore provide the possibility to control the environment with regard to pH, oxygen content, and temperature. In this study, a three‐dimensional bone construct was engineered by the use of dynamic bioreactor technology. Human adipose tissue derived mesenchymal stem cells were cultivated on a macroporous zirconium dioxide based ceramic disc called Sponceram®. Furthermore, hydroxyapatite coated Sponceram® was used. The cells were cultivated under dynamic conditions and compared with statically cultivated cells. The differentiation into osteoblasts was initiated by osteogenic supplements. Cellular proliferation during static and dynamic cultivation was compared measuring glucose and lactate concentration. The differentiation process was analysed determining AP‐expression and using different specific staining methods. Our results demonstrate much higher proliferation rates during dynamic conditions in the bioreactor system compared to static cultivation measured by glucose consumption and lactate production. Cell densities on the scaffolds indicated higher proliferation on native Sponceram® compared to hydroxyapatite coated Sponceram®. With this study, we present an excellent method to enhance cellular proliferation and bone lineage specific growth of tissue like structures comprising fibrous (collagen) and globular (mineral) extracellular components. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Perfusion‐based fluorescence imaging method delineates diverse organs and identifies multifocal tumors using generic near‐infrared molecular probes

Rapid detection of multifocal cancer without the use of complex imaging schemes will improve treatment outcomes. In this study, dynamic fluorescence imaging was used to harness differences in the perfusion kinetics of near‐infrared (NIR) fluorescent dyes to visualize structural characteristics of different tissues. Using the hydrophobic nontumor‐selective NIR dye cypate, and the hydrophilic dye LS288, a high tumor‐to‐background contrast was achieved, allowing the delineation of diverse tissue types while maintaining short imaging times. By clustering tissue types with similar perfusion properties, the dynamic fluorescence imaging method identified secondary tumor locations when only the primary tumor position was known, with a respective sensitivity and specificity of 0.97 and 0.75 for cypate, and 0.85 and 0.81 for LS288. Histological analysis suggests that the vasculature in the connective tissue that directly surrounds the tumor was a major factor for tumor identification through perfusion imaging. Although the hydrophobic dye showed higher specificity than the hydrophilic probe, use of other dyes with different physical and biological properties could further improve the accuracy of the dynamic imaging platform to identify multifocal tumors for potential use in real‐time intraoperative procedures.      

Development of the transgenic cyan fluorescent protein (CFP)‐expressing nude mouse for “Technicolor” cancer imaging

A major goal for in vivo biology is to develop models which can express multiple colors of fluorescent proteins in order to image many processes simultaneously in real time. Towards this goal, the cyan fluorescent protein (CFP) nude mouse was developed by crossing non‐transgenic nude mice with the transgenic CK/ECFP mouse in which the β‐actin promoter drives expression of CFP in almost all tissues. In crosses between nu/nu CFP male mice and nu/+ CFP female mice, approximately 50% of the embryos fluoresced blue. In the CFP nude mice, the pancreas and reproductive organs displayed the strongest fluorescent signals of all internal organs which vary in intensity. Orthotopic implantation of XPA‐1 human pancreatic cancer cells expressing red fluorescent protein (RFP); or green fluorescent protein (GFP) in the nucleus and RFP in the cytoplasm, was performed in female nude CFP mice. Color‐coded fluorescence imaging of these human pancreatic cancer cells implanted into the bright blue fluorescent pancreas of the CFP nude mouse afforded novel insight into the interaction of the pancreatic tumor and the normal pancreas, in particular the strong desmoplastic reaction of the tumor. The naturally enhanced blue fluorescence of the pancreas in the CFP mouse serves as an ideal background for color‐coded imaging of the interaction of implanted cancer cells and the host. The CFP nude mouse will provide unique understanding of the critical interplay between the cancer cells and their microenvironment. J. Cell. Biochem. 107: 328–334, 2009. © 2009 Wiley‐Liss, Inc.     

MYC‐driven inhibition of the glutamate‐cysteine ligase promotes glutathione depletion in liver cancer

How MYC reprograms metabolism in primary tumors remains poorly understood. Using integrated gene expression and metabolite profiling, we identify six pathways that are coordinately deregulated in primary MYC‐driven liver tumors: glutathione metabolism; glycine, serine, and threonine metabolism; aminoacyl‐tRNA biosynthesis; cysteine and methionine metabolism; ABC transporters; and mineral absorption. We then focus our attention on glutathione (GSH) and glutathione disulfide (GSSG), as they are markedly decreased in MYC‐driven tumors. We find that fewer glutamine‐derived carbons are incorporated into GSH in tumor tissue relative to non‐tumor tissue. Expression of GCLC, the rate‐limiting enzyme of GSH synthesis, is attenuated by the MYC‐induced microRNA miR‐18a. Inhibition of miR‐18a in vivo leads to increased GCLC protein expression and GSH abundance in tumor tissue. Finally, MYC‐driven liver tumors exhibit increased sensitivity to acute oxidative stress. In summary, MYC‐dependent attenuation of GCLC by miR‐18a contributes to GSH depletion in vivo, and low GSH corresponds with increased sensitivity to oxidative stress in tumors. Our results identify new metabolic pathways deregulated in primary MYC tumors and implicate a role for MYC in regulating a major antioxidant pathway downstream of glutamine.     

Stability evaluation of temoporfin-loaded liposomal gels for topical application

Temoporfin (mTHPC) is a potent second-generation synthetic photosensitizer. Topical delivery of mTHPC is of great interest for the photodynamic therapy of psoriasis and superficial skin cancer lesions. The aim of this study was to evaluate the stability of hydrophilic gels containing mTHPC-loaded liposomes. Two different mTHPC-loaded liposome dispersions, composed of 15 % (w/w) nonhydrogenated soybean lecithin of different phosphatidylcholine content, were prepared and incorporated (2:1 w/w) into hydrogels of different carbomer concentrations (1.5, 2.25, and 3%; w/w). Obtained liposomal hydrogels, containing 0.15% (w/w) mTHPC, 10% (w/w) phospholipids, and 0, 0.5, or 1% (w/w) carbomer, were analyzed for flow properties, liposome particle size, and polydispersity index (PDI), pH value, and mTHPC content after their preparation and at predetermined time intervals during 6 months of storage at 4 and 23°C. All hydrogels showed, during the whole period of investigation, adequate characteristics for topical application (i.e., they revealed shear-thinning plastic flow behavior). Rheological parameters, particle size, and PDI of liposomes in hydrogels, mTHPC content, and pH value did not show remarkable changes during the storage of gels, which could make them unacceptable for topical use. The obtained results indicated physical and chemical stability of liposomal gels containing mTHPC during 6 months of storage at both temperatures.     

Enhanced human tumor cell transplantability in a new congenic immunodeficient mouse; KSN-BNX

We introduced two mutant genes (beige; bg that induces the deficiency of natural killer (NK) activity andxid that decreases the production of immunoglobulin) into KSN nude mice with high reproductive performances. We produced KSNbg/bg(nu/nu) (KSN-bg), KSN-xid/xid(nu/nn) (KSN-xid), KSNxid/xid;bg/bg(nu/nu) (KSN-BNX) and KSN-nu/+ (KS) mice by backcross (cross-intercross method). All strains showed as high a reproductivity rate as the parental KSN mice. KSN-xid and KSN-BNX mice had a reduced percentage of B220 positive cells in the spleens compared to KSN and KSN-bg mice, but they showed increased percentages, of Thy-1 and asialo GM1 positive cells. The serum immunoglobulin concentrations of KSN-BNX were as low as KSN-xid. Both KSN-bg and KSN-BNX mice showed deficient NK activity in spleens, whereas KSN-xid mice showed an elevated NK activity. Compared to nude mice, the growth of both human tumor cell TCO-1 and BxPc-3 transplanted subcutaneously was enhanced in KSN-BNX mice. However Panc-1 cells that was rejected in nude mice was not accepted in KSN-BNX mice. Liver metastasis of human pancreatic tumor cells; Capan-1, BxPc-3 and MIAPaCa-2 were studied. No significant difference was observed in the percentage of metastasis formed mice between nude and KSN-BNX mice.     

Toxicity and localization studies of a potential photodynamic therapy agent in Drosophila

Photodynamic therapy utilizes light, a photosensitizer, and molecular oxygen as a treatment modality for a variety of cancers. We have recently combined ruthenium(II) polypyridyl groups with a zinc(II) centered porphyrin as a new photosensitizer for the treatment of melanoma. In‐vitro studies have indicated that this photosensitizer is toxic to melanoma cells when irradiated with low energy light; however, it is nontoxic to normal cells under similar conditions. To determine the toxicity and cell viability of this compound in‐vivo we present, herein, a study using Drosophila melanogaster. In the absence of light, the new photosensitizer shows no discernible effects to fly larvae at various concentrations of compound and stages of larval development. When the larvae were fed the photosensitizer it was observed, by fluorescence microscopy, that the compound passes through the cell membrane and localizes in the cytosol at lower concentrations and the nucleus at slightly higher concentrations indicating that the compound is not immediately metabolized. genesis 52:309–314, 2014. © 2014 Wiley Periodicals, Inc.     

H‐Cadherin expression reduces invasion of malignant melanoma

Melanocytic behavior, survival, and proliferation are regulated through a complex system of cell–cell adhesion molecules. Pathologic changes leading to development of malignant melanoma, upset the delicate homeostatic balance between melanocytes and keratinocytes and can lead to altered expression of cell–cell adhesion and cell–cell communication molecules. Malignant transformation of melanocytes frequently coincides with loss of E‐cadherin expression. We now show loss of another member of the superfamily of classical cadherins, H‐cadherin (CDH13), which may be involved in the development of malignant melanoma. The provided data show that H‐cadherin expression is lost in nearly 80% of the analyzed melanoma cell lines. Knockdown of H‐cadherin using siRNA increases invasive capacity in melanocytes. Functional assays show that the re‐expression of H‐cadherin decreases migration and invasion capacity, as well as anchorage‐independent growth in comparison to control melanoma cells. Furthermore, melanoma cells, which re‐express H‐cadherin via stable transfection show a reduction in rate of tumor growth in a nu/nu mouse tumor model in comparison to the parental control transfected cell lines. Our study presents for the first time the down‐regulation of H‐cadherin in malignant melanomas and its possible functional relevance in maintenance healthy skin architecture.     

MALDI Mass Spectrometry Imaging of Early‐ and Late‐Stage Serous Ovarian Cancer Tissue Reveals Stage‐Specific N‐Glycans

Epithelial ovarian cancer is one of the most fatal gynecological malignancies in adult women. As studies on protein N‐glycosylation have extensively reported aberrant patterns in the ovarian cancer tumor microenvironment, obtaining spatial information will uncover tumor‐specific N‐glycan alterations in ovarian cancer development and progression. matrix‐assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) is employed to investigate N‐glycan distribution on formalin‐fixed paraffin‐embedded ovarian cancer tissue sections from early‐ and late‐stage patients. Tumor‐specific N‐glycans are identified and structurally characterized by porous graphitized carbon‐liquid chromatography‐electrospray ionization‐tandem mass spectrometry (PGC‐LC‐ESI‐MS/MS), and then assigned to high‐resolution images obtained from MALDI‐MSI. Spatial distribution of 14 N‐glycans is obtained by MALDI‐MSI and 42 N‐glycans (including structural and compositional isomers) identified and structurally characterized by LC‐MS. The spatial distribution of oligomannose, complex neutral, bisecting, and sialylated N‐glycan families are localized to the tumor regions of late‐stage ovarian cancer patients relative to early‐stage patients. Potential N‐glycan diagnostic markers that emerge include the oligomannose structure, (Hex)₆ + (Man)₃(GlcNAc)₂, and the complex neutral structure, (Hex)₂ (HexNAc)₂ (Deoxyhexose)₁ + (Man)₃(GlcNAc)₂. The distribution of these markers is evaluated using a tissue microarray of early‐ and late‐stage patients.