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Disruption of antagonism between SWI/SNF chromatin remodelers and polycomb repressor complexes drives the formation of numerous cancer types. Recently, an inhibitor of the polycomb protein EZH2 was approved for the treatment of a sarcoma mutant in the SWI/SNF subunit SMARCB1, but resistance occurs. Here, we performed CRISPR screens in SMARCB1-mutant rhabdoid tumor cells to identify genetic contributors to SWI/SNF-polycomb antagonism and potential resistance mechanisms. We found that loss of the H3K36 methyltransferase NSD1 caused resistance to EZH2 inhibition. We show that NSD1 antagonizes polycomb via cooperation with SWI/SNF and identify co-occurrence of NSD1 inactivation in SWI/SNF-defective cancers, indicating in vivo relevance. We demonstrate that H3K36me2 itself has an essential role in the activation of polycomb target genes as inhibition of the H3K36me2 demethylase KDM2A restores the efficacy of EZH2 inhibition in SWI/SNF-deficient cells lacking NSD1. Together our data expand the mechanistic understanding of SWI/SNF and polycomb interplay and identify NSD1 as the key for coordinating this transcriptional control., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Adaptation of cellular function with the nutrient environment is essential for survival. Failure to adapt can lead to cell death and/or disease. Indeed, energy metabolism alterations are a major contributing factor for many pathologies, including cancer, cardiovascular disease, and diabetes. In particular, a primary characteristic of cancer cells is altered metabolism that promotes survival and proliferation even in the presence of limited nutrients. Interestingly, recent studies demonstrate that metabolic pathways produce intermediary metabolites that directly influence epigenetic modifications in the genome. Emerging evidence demonstrates that metabolic processes in cancer cells fuel malignant growth, in part, through epigenetic regulation of gene expression programs important for proliferation and adaptive survival. In this review, recent progress toward understanding the relationship of cancer cell metabolism, epigenetic modification, and transcriptional regulation will be discussed. Specifically, the need for adaptive cell metabolism and its modulation in cancer cells will be introduced. Current knowledge on the emerging field of metabolite production and epigenetic modification will also be reviewed. Alterations of DNA (de)methylation, histone modifications, such as (de)methylation and (de)acylation, as well as chromatin remodeling, will be discussed in the context of cancer cell metabolism. Finally, how these epigenetic alterations contribute to cancer cell phenotypes will be summarized. Collectively, these studies reveal that both metabolic and epigenetic pathways in cancer cells are closely linked, representing multiple opportunities to therapeutically target the unique features of malignant growth., (© 2021 Federation of European Biochemical Societies.)

The multiple myeloma (MM) landscape has changed in the last few years, but most patients eventually relapse because current treatment modalities do not target clonogenic stem cells, which are drug-resistant and can self-renew. We hypothesized that side population (SP) cells represent myeloma clonogenic stem cells and, searching for new treatment strategies, analyzed the anti-myeloma activity of natural killer (NK) cells against clonogenic cells. Activated and expanded NK cells (NKAE) products were obtained by co-culturing NK cells from MM patients with K562-mb15-41BBL cell line and characterized by flow cytometry. Functional experiments against MM cells were performed by Eu-TDA release assays and methylcellulose clonogenic assays. Side population was detected by Dye Cycle Violet labeling and then characterized by flow cytometry and RNA-Seq. Self-renewal capacity was tested by clonogenic assays. Sorting of both kind of cells was performed for time-lapse microscopy experiments. SP cells exhibited self-renewal potential and overexpressed genes involved in stem cell metabolism. NK cells from MM patients exhibited dysregulation and had lower anti-tumor potential against clonogenic cells than healthy donors' NK cells. Patients' NK cells were activated and expanded. These cells recovered cytotoxic activity and could specifically destroy clonogenic myeloma cells. They also had a highly cytotoxic phenotype expressing NKG2D receptor. Blocking NKG2D receptor decreased NK cell activity against clonogenic myeloma cells, and activated NK cells were able to destroy SP cells, which expressed NKG2D ligands. SP cells could represent the stem cell compartment in MM. This is the first report describing NK cell activity against myeloma clonogenic cells., (© 2021. The Author(s).)

Polysaccharide hydrogels are promising candidate matrices for recapitulating the characteristics of extracellular matrix (ECM) in breast tumors in terms of their structure and composition. Herein, to obtain an ECM-mimetic matrix, hydroxyethyl chitosan (HECS) hydrogels were prepared through Schiff-base crosslinking reaction using dialdehyde hyaluronic acid as crosslinker. The obtained HECS hydrogels displayed a highly porous structure, a stiffness comparable to that of breast tissue, and a fast water-absorption speed. The amount of crosslinker had great effects on the swelling and rheological behaviors of the HECS hydrogels. Preliminary results from in vitro biological assessments confirmed that MCF-7 cells incubated within HECS hydrogels preferred to grow into three-dimensional spheroids. Importantly, the cells displayed enhanced migrative capability and upregulated expression levels of MMP-2, TGF-β and VEGF in comparison to two-dimension cultured cells. Hence, the HECS hydrogels show great promise as a biomimetic ECM in constructing breast tumor models., (Copyright © 2021 Elsevier B.V. All rights reserved.)

This protocol describes the necessary preparations and procedures to photo-activate Yes-associated protein (YAP) with optoYAP in cancer cell spheroids in 3D collagen matrices. We detail steps for immunofluorescent staining of the resulting YAP-activated HeLa spheroids. In addition, we describe handling of optoYAP on 2D substrates. While this protocol focuses on the use of optoYAP in 3D HeLa cell culture, it can be modified for other cell types. For complete details on the use and execution of this protocol, please refer to Illes et al. (2021)., Competing Interests: The authors declare no competing interests., (© 2021 The Authors.)

Cancer is one of the leading causes of annual deaths worldwide, accounting for nearly 10 million deaths each year. Metastasis, the process by which cancer spreads across the patient's body, is the main cause of death in cancer patients. Because the rising trend observed in statistics of new cancer cases and cancer-related deaths does not allow for an optimistic viewpoint on the future-in relation to this terrible disease-the scientific community has sought methods to enable early detection of cancer and prevent the apparition of metastatic tumors. One such method is known as liquid biopsy, wherein a sample is taken from a bodily fluid and analyzed for the presence of CTCs or other cancer biomarkers (e.g., growth factors). With this objective, interest is growing by year in electrokinetically-driven microfluidics applied for the concentration, capture, filtration, transportation, and characterization of CTCs. Electrokinetic techniques-electrophoresis, dielectrophoresis, electrorotation, and electrothermal and EOF-have great potential for miniaturization and integration with electronic instrumentation for the development of point-of-care devices, which can become a tool for early cancer diagnostics and for the design of personalized therapeutics. In this contribution, we review the state of the art of electrokinetically-driven microfluidics for cancer cells manipulation., (© 2020 Wiley-VCH GmbH.)

Disseminated tumor cells (dTCs) can frequently be detected in the bone marrow (BM) of colorectal cancer (CRC) patients, raising the possibility that the BM serves as a reservoir for metastatic tumor cells. Identification of dTCs in BM aspirates harbors the potential of assessing therapeutic outcome and directing therapy intensity with limited risk and effort. Still, the functional and prognostic relevance of dTCs is not fully established. We have previously shown that CRC cell clones can be traced to the BM of mice carrying patient-derived xenografts. However, cellular interactions, proliferative state and tumorigenicity of dTCs remain largely unknown. Here, we applied a coculture system modeling the microvascular niche and used immunofluorescence imaging of the murine BM to show that primary CRC cells migrate toward endothelial tubes. dTCs in the BM were rare, but detectable in mice with xenografts from most patient samples (8/10) predominantly at perivascular sites. Comparable to primary tumors, a substantial fraction of proliferating dTCs was detected in the BM. However, most dTCs were found as isolated cells, indicating that dividing dTCs rather separate than aggregate to metastatic clones-a phenomenon frequently observed in the microvascular niche model. Clonal tracking identified subsets of self-renewing tumor-initiating cells in the BM that formed tumors out of BM transplants, including one subset that did not drive primary tumor growth. Our results indicate an important role of the perivascular BM niche for CRC cell dissemination and show that dTCs can be a potential source for tumor relapse and tumor heterogeneity., (© 2020 The Authors. International Journal of Cancer published by John Wiley & Sons Ltd on behalf of UICC.)

Metabolic fingerprinting is a strong tool for characterization of biological phenotypes. Classification with machine learning is a critical component in the discrimination of molecular determinants. Cellular activity can be traced using stable isotope labelling of metabolites from which information on cellular pathways may be obtained. Nuclear magnetic resonance (NMR) spectroscopy is, due to its ability to trace labelling in specific atom positions, a method of choice for such metabolic activity measurements. In this study, we used hyperpolarization in the form of dissolution Dynamic Nuclear Polarization (dDNP) NMR to measure signal enhanced isotope labelled metabolites reporting on pathway activity from four different prostate cancer cell lines. The spectra have a high signal-to-noise, with less than 30 signals reporting on 10 metabolic reactions. This allows easy extraction and straightforward interpretation of spectral data. Four metabolite signals selected using a Random Forest algorithm allowed a classification with Support Vector Machines between aggressive and indolent cancer cells with 96.9% accuracy, -corresponding to 31 out of 32 samples. This demonstrates that the information contained in the few features measured with dDNP NMR, is sufficient and robust for performing binary classification based on the metabolic activity of cultured prostate cancer cells., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The metabolic activity of cells is interrelated with cell signaling, functions, and fate. Uncontrolled cancer cell proliferation requires metabolic adaptations. Research focusing on understanding the characteristics of cell metabolism is crucial for the development of novel diagnostic and therapeutic strategies. Here, we describe protocols for the ATP profiling of single cancer cells by fluorescence live-cell imaging. In response to distinct metabolic inhibitions, we record individual mitochondrial ATP dynamics using established Förster resonance energy transfer-based genetically encoded fluorescent ATP probes. For complete details on the use and execution of this protocol, please refer to Depaoli et al. (2018)., Competing Interests: W.F.G. and R.M. are founders of a company, referred to as NGFI GmbH, and are members of its scientific advisory board., (© 2020 The Author(s).)

Exosomes, small-sized extracellular vesicles, carry components of the purinergic pathway. The production by cells of exosomes carrying this pathway remains poorly understood. Here, we asked whether type 1, 2A, or 2B adenosine receptors (A 1 Rs, A 2A Rs, and A 2B Rs, respectively) expressed by producer cells are involved in regulating exosome production. Preglomerular vascular smooth muscle cells (PGVSMCs) were isolated from wildtype, A 1 R -/- , A 2A R -/- , and A 2B R -/- rats, and exosome production was quantified under normal or metabolic stress conditions. Exosome production was also measured in various cancer cells treated with pharmacologic agonists/antagonists of A 1 Rs, A 2A Rs, and A 2B Rs in the presence or absence of metabolic stress or cisplatin. Functional activity of exosomes was determined in Jurkat cell apoptosis assays. In PGVSMCs, A 1 R and A 2A R constrained exosome production under normal conditions (p = 0.0297; p = 0.0409, respectively), and A 1 R, A 2A R, and A 2B R constrained exosome production under metabolic stress conditions. Exosome production from cancer cells was reduced (p = 0.0028) by the selective A 2A R agonist CGS 21680. These exosomes induced higher levels of Jurkat apoptosis than exosomes from untreated cells or cells treated with A 1 R and A 2B R agonists (p = 0.0474). The selective A 2A R antagonist SCH 442416 stimulated exosome production under metabolic stress or cisplatin treatment, whereas the selective A 2B R antagonist MRS 1754 reduced exosome production. Our findings indicate that A 2A Rs suppress exosome release in all cell types examined, whereas effects of A 1 Rs and A 2B Rs are dependent on cell type and conditions. Pharmacologic targeting of cancer with A 2A R antagonists may inadvertently increase exosome production from tumor cells.

Purpose: We investigated the expression of vasopressin receptor 2 and 3 on corticotrophin tumor cells, their role in regulating ACTH secretion, and their potential therapeutic implications., Methods: We retrospectively assessed 52 hospitalized patients with pathologically confirmed ACTH-secreting tumors. The expression of vasopressin receptor 2 and 3 was explored via qualitative and quantitative immunohistochemistry analyses. The role of vasopressin receptors in regulating ACTH secretion was further studied in the AtT-20 cell line., Results: Among 50 cases of pituitary corticotrophin adenoma, 31 were vasopressin receptor 2 positive, 38 were vasopressin receptor 3 positive, and 24 were both vasopressin receptor 2 and 3 positive. Two patients with ectopic ACTH syndrome were vasopressin receptor 3 positive, and one was also vasopressin receptor 2 positive. In 12 patients who underwent bilateral inferior petrosal sinus sampling before surgery, the central ACTH increment ratio after desmopressin stimulation was correlated with vasopressin receptor 2 but not with vasopressin receptor 3 staining intensity. In an in vitro study, the expression of both vasopressin receptor 2 and 3 on AtT-20 cells was confirmed. The vasopressin receptor 2 antagonist Tolvaptan inhibited desmopressin-induced ACTH secretion in a dose-dependent manner., Conclusions: Both vasopressin receptor 2 and 3 are expressed in ACTH-secreting tumors. Vasopressin receptor 2 rather than vasopressin receptor 3 is the primary receptor that seems to mediate the ACTH response in corticotrophin tumors. A vasopressin receptor 2 antagonist can inhibit ACTH secretion induced by desmopressin in AtT-20 cells., Competing Interests: The authors declare that they have no conflicts of interest., (© Georg Thieme Verlag KG Stuttgart · New York.)

microRNAs (miRNAs) are small non-coding RNAs acting as negative regulators of gene expression and involved in tumor progression. We recently showed that inhibition of the pro-metastatic miR-214 and simultaneous overexpression of its downstream player, the anti-metastatic miR-148b, strongly reduced metastasis formation. To explore the therapeutic potential of miR-148b, we generated a conjugated molecule aimed to target miR-148b expression selectively to tumor cells. Precisely, we linked miR-148b to GL21.T, an aptamer able to specifically bind to AXL, an oncogenic tyrosine kinase receptor highly expressed on cancer cells. Axl-148b conjugate was able to inhibit migration and invasion of AXL-positive, but not AXL-negative, cancer cells, demonstrating high efficacy and selectivity in vitro . In parallel, expression of ALCAM and ITGA5, two miR-148b direct targets, was reduced. More importantly, axl-148b chimeric aptamers were able to inhibit formation and growth of 3D-mammospheres, to induce necrosis and apoptosis of treated xenotransplants, as well as to block breast cancer and melanoma dissemination and metastatization in mice. Relevantly, axl aptamer acted as specific delivery tool for miR-148b, but it also actively contributed to inhibit metastasis formation, together with miR-148b. In conclusion, our data show that axl-148b conjugate is able to inhibit tumor progression in an axl- and miR-148b-dependent manner, suggesting its potential development as therapeutic molecule., Competing Interests: Competing Interests: The chimeric aptamer axl-148b has been patented (number 102019000015806)., (© The author(s).)

Podosomes are a singular category of integrin-mediated adhesions important in the processes of cell migration, matrix degradation and cancer cell invasion. Despite a wealth of biochemical studies, the effects of mechanical forces on podosome integrity and dynamics are poorly understood. Here, we show that podosomes are highly sensitive to two groups of physical factors. First, we describe the process of podosome disassembly induced by activation of myosin-IIA filament assembly. Next, we find that podosome integrity and dynamics depends upon membrane tension and can be experimentally perturbed by osmotic swelling and deoxycholate treatment. We have also found that podosomes can be disrupted in a reversible manner by single or cyclic radial stretching of the substratum. We show that disruption of podosomes induced by osmotic swelling is independent of myosin-II filaments. The inhibition of the membrane sculpting protein, dynamin-II, but not clathrin, resulted in activation of myosin-IIA filament formation and disruption of podosomes. The effect of dynamin-II inhibition on podosomes was, however, independent of myosin-II filaments. Moreover, formation of organized arrays of podosomes in response to microtopographic cues (the ridges with triangular profile) was not accompanied by reorganization of myosin-II filaments. Thus, mechanical elements such as myosin-II filaments and factors affecting membrane tension/sculpting independently modulate podosome formation and dynamics, underlying a versatile response of these adhesion structures to intracellular and extracellular cues. This article is part of a discussion meeting issue 'Forces in cancer: interdisciplinary approaches in tumour mechanobiology'.

Aims: Multicellular tumor spheroids (MCTS) produced by different methods vary in forms, sizes, and properties. The aim of this work was to characterize MCTS formed by six pancreatic cell lines on a non-adherent surface., Materials and Methods: Human pancreatic cells were grown in 2D and 3D conditions and compared for the expression of E- and desmosomal cadherins (PCR, confocal microscopy), growth, cell cycling, apoptosis (flow cytometry), and a response to antitumor drugs doxorubicin and gemcitabine (MTT-assay)., Key Findings: Three types of MCTS were identified: BxPC-3, T 3 M 4 formed small number of large and dense spheroids representing type I MCTS; COLO-357 and AsPC-1 generated type II multiple and loose MCTS of different sizes while MiaPaCa-2 and PANC-1 represented type III cultures which grew almost as floating monolayer films. Formation of type I MCTS depended on the simultaneous expression of DSG3 and several DSC proteins; II MCTS expressed solely DSG2-DSC2 but not DSG3, while type III cells either did not express E-cadherin or a pair of DSG and DSC proteins. Cells in type I MCTS but not in types II and III ones quickly became quiescent which correlated with a decrease in the proliferation, increased apoptosis, and a higher resistance to antitumor drugs doxorubicin and gemcitabine., Significance: Taken collectively, pancreatic cells significantly vary in the expression of desmosomal cadherins, resulting in the formation of MCTS with different characteristics. The sensitivity of MCTS to various drugs depends on the type of cells and the method of spheroid preparation used., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Tumorigenesis depends on intricate interactions between genetically altered tumor cells and their surrounding microenvironment. While oncogenic drivers in lung squamous carcinoma (LUSC) have been described, the role of stroma in modulating tissue architecture, particularly cell polarity, remains unclear. Here, we report the establishment of a 3D coculture system of LUSC epithelial cells with cancer-associated fibroblasts (CAFs) and extracellular matrix that together capture key components of the tumor microenvironment (TME). Single LUSC epithelial cells develop into acinar-like structures with 0.02% efficiency, and addition of CAFs provides proper tumor-stromal interactions within an appropriate 3D architectural context. Using this model, we recapitulate key pathological changes during tumorigenesis, from hyperplasia to dysplasia and eventually invasion, in malignant LUSC spheroids that undergo phenotypic switching in response to cell intrinsic and extrinsic changes. Overexpression of SOX2 is sufficient to mediate the transition from hyperplasia to dysplasia in LUSC spheroids, while the presence of CAFs makes them invasive. Unexpectedly, CAFs suppress the activity of high SOX2 levels, restore hyperplasia, and enhance the formation of acinar-like structures. Taken together, these observations suggest that stromal factors can override cell intrinsic oncogenic changes in determining the disease phenotype, thus providing fundamental evidence for the existence of dynamic reciprocity between the nucleus and the TME of LUSC., Competing Interests: Conflict of interest statement: All authors are employees and shareholders of Pfizer., (Copyright © 2018 the Author(s). Published by PNAS.)

Manipulation of cellular functions and structures by introduction of genetic materials inside cells has been one of the most prominent research areas in biomedicine. High-frequency ultrasound acoustic-transfection has recently been developed and confirmed by intracellular delivery of small molecules into HeLa cells at the single-cell level with high cell viability. After we proved the concept underlying the acoustic-transfection technique, treatment conditions for different human cancer cell lines have been intensively investigated to further develop acoustic-transfection as a versatile and adaptable transfection method by satisfying the requirements of high-delivery efficiency and cell membrane permeability with minimal membrane disruption. To determine optimal treatment conditions for different cell lines, we developed a quantitative intracellular delivery score based on delivery efficiency, cell membrane permeability and cell viability after 4 and 20 h of treatment. The intracellular delivery of macromolecules and the simultaneous intracellular delivery of two molecules under optimal treatment conditions were successfully achieved. We found that DNA plasmid was delivered by acoustic-transfection technique into epiblast stem cells, which expressed transient mCherry fluorescence., (Copyright © 2018 World Federation for Ultrasound in Medicine and Biology. Published by Elsevier Inc. All rights reserved.)

Background: Colorectal cancer (CRC) liver metastasis is highly unfavorable for patient outcome and is a leading cause of cancer-related death. Pre-clinical research of CRC liver metastasis predominately utilizes CRC cell lines grown in tissue culture. Here, we demonstrate that CRC liver metastases organoids derived from human specimens recapitulate some aspects of human disease., Methods: Human CRC liver metastases pathological specimens were obtained following patient consent. Tumor disaggregates were plated and organoids were allowed to expand. CRC markers were identified by immunofluorescence. Stem cell genes were analysed by QPCR and flow cytometry. Response to drug therapy was quantified using time-lapse imaging and MATLAB analysis., Results: Organoids showed global expression of the epithelial marker, EpCAM and the adenocarcinoma marker, CEA CAM1. Flow cytometry analysis demonstrated that organoids express the stem cell surface markers CD24 and CD44. Finally, we demonstrated that CRC liver metastases organoids acquire chemotherapy resistance and can be utilized as surrogates for drug testing., Conclusion: These data demonstrate that CRC liver metastases organoids recapitulate some aspects of human disease and may provide an invaluable resource for investigating novel drug therapies, chemotherapy resistance and mechanism of metastasis., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Accumulating studies have linked the disruptions of microRNA-10 (miR-10) to acute myeloid leukemia (AML) with NPM1 mutation. However, miR-10 expression and its clinical implication in AML remain poorly defined. Although a recent report showed high serum level of miR-10a was associated with adverse prognosis in AML, herein, we found bone marrow (BM) miR-10 overexpression was not a prognostic biomarker in AML., Methods: BM miR-10 expression was examined by real-time quantitative PCR in BM mononuclear cells in 115 de novo AML patients and 45 controls., Results: BM miR-10 (miR-10a/b) expression was significantly up-regulated in AML patients, and was positively correlated with each other. Overexpression of miR-10a was associated with lower percentage of BM blasts, whereas miR-10b overexpression tended to correlate with higher percentage of BM blasts. Importantly, miR-10a overexpression was significantly associated with FAB-M3/t(15;17) subtypes and NPM1 mutation, meanwhile, overexpression of miR-10b was correlated with NPM1 and DNMT3A mutations. However, miR-10a/b overexpression was not associated with complete remission rate, and did not have an impact on both leukemia free survival and overall survival time in non-M3 AML patients without NPM1 mutation., Conclusions: BM miR-10 overexpression is associated with genetic events but not affects clinical outcome in AML., (Copyright © 2017 Elsevier GmbH. All rights reserved.)

Specific 3D conditions of cancer cell lines have been optimized over last years, with growing significance of serum-free and xeno-free culture variants. The choice of proper culture media enables cancer stem cells proliferation in primary and stable cell lines. To obtain renal cell cancer stem-like phenotype, we employed media dedicated for mesenchymal cells and adult stem cells. Developed RCC cell line 3D culture system enables effective drug testing, including tyrosine kinase inhibitor anti-cancer cell toxicity. To induce formation of 3D spheroids by RCC cell lines, StemXvivo and NutriStem media must be used. Usage of laminin- or poly-D-lysine coated plates enhances also the formation of spheroids in 3D-promoting media. Seeding is optimal with Caki-1 or ACHN cell lines as well as 786-O or HKCSC cells. Our bio-mimic 3D RCC cell culture model promotes cell viability and stem-related gene expression including E-cadherin, N-cadherin, HIF1, HIF2, VEGF, Sox2, Pax2, and Nestin. 3D spheroid formation ability and spheroid volume increase are disturbed upon drug treatment. Untreated 3D structures reach ~100 μm in diameter at the end of 14-day long experiment. Sorter-based cell cycle analysis and Ki-67 staining should be conducted to verify specific toxicity. We suggest that due to the more complex architecture 3D RCC culture is more relevant to investigate the in vivo-like tumor drug response.

Background: Several tumor entities including brain tumors aberrantly overexpress intermediate conductance Ca2+ activated KCa3.1 K+ channels. These channels contribute significantly to the transformed phenotype of the tumor cells., Method: PubMed was searched in order to summarize our current knowledge on the molecular signaling upstream and downstream and the effector functions of KCa3.1 channel activity in tumor cells in general and in glioblastoma cells in particular. In addition, KCa3.1 expression and function for repair of DNA double strand breaks was determined experimentally in primary glioblastoma cultures in dependence on the abundance of proneural and mesenchymal stem cell markers., Results: By modulating membrane potential, cell volume, Ca2+ signals and the respiratory chain, KCa3.1 channels in both, plasma and inner mitochondrial membrane, have been demonstrated to regulate many cellular processes such as migration and tissue invasion, metastasis, cell cycle progression, oxygen consumption and metabolism, DNA damage response and cell death of cancer cells. Moreover, KCa3.1 channels have been shown to crucially contribute to resistance against radiotherapy. Futhermore, the original in vitro data on KCa3.1 channel expression in subtypes of glioblastoma stem(-like) cells propose KCa3.1 as marker for the mesenchymal subgroup of cancer stem cells and suggest that KCa3.1 contributes to the therapy resistance of mesenchymal glioblastoma stem cells., Conclusion: The data suggest KCa3.1 channel targeting in combination with radiotherapy as promising new tool to eradicate therapy-resistant mesenchymal glioblastoma stem cells., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

The liver's role in metabolism of chemicals makes it an appropriate tissue for toxicity testing. Current testing protocols, such as animal testing and two-dimensional liver cell systems, offer limited resemblance to in vivo liver cell behaviour, in terms of gene expression profiles and metabolic competence; thus, they do not always accurately predict human toxicology. In vitro three-dimensional liver cell models offer an attractive alternative. This study reports on the development of a 3D liver model, using HepG2 cells, by a hanging-drop technique, with a focus on evaluating spheroid growth characteristics and suitability for genotoxicity testing. The cytokinesis-blocked micronucleus assay protocol was adapted to enable micronucleus (MN) detection in the 3D spheroid models. This involved evaluating the difference between hanging vs non-hanging drop positions for dosing of the test agents and comparison of automated Metafer scoring with manual scoring for MN detection in HepG2 spheroids. The initial seeding density, used for all experiments, was 5000 cells/20 μl drop hanging spheroids, harvested on day 4, with >75% cell viability. Albumin secretion (7.8 g/l) and both CYP1A1 and CYP1A2 gene expression were highest in the 3D environment at day 4. Exposure to metabolically activated genotoxicants for 24 h resulted in a 6-fold increase in CYP1A1 enzyme activity (3 μM B[a]P) and a 30-fold increase in CYP1A2 enzyme activity (5 μM PhIP) in 3D hanging spheroids. MN inductions in response to B[a]P or PhIP were 2-fold and 3-fold, respectively, and were greater in 3D hanging spheroids than in 2D format, showing that hanging spheroids are more sensitive to genotoxic agents. HepG2 hanging-drop spheroids are an exciting new alternative system for genotoxicity studies, due to their improved structural and physiological properties, relative to 2D cultures., (Copyright © 2017 Elsevier B.V. All rights reserved.)

In breast cancer research S100A4-positive tumour-associated stromal cells are assumed as primary source of Tenascin C (TNC) in the metastatic environment. Aim of the present study was to isolate and characterize S100A4/TNC positive stromal canine mammary tumour (CMT) cells. Cells grown as scaffold-free spheroids were investigated for S100A4, TNC, and proliferative activity under 1.8% DMSO stimulation by means of Western blot and immunohistochemistry. DMSO is a commonly used drug solvent despite well-known side effects on cells including TNC expression. DMSO did not affect proliferation, but TNC was significantly reduced under DMSO exposure for 7 and 14 days, whereby for S100A4 a reducing effect was only observed after 14 days. Without DMSO, cells stably expressed TNC and S100A4 which makes them suitable to be used in experimental approaches requiring S100A4/TNC expressing CMT stromal cells. Results show that 1.8% DMSO should not be used as solvent for experiments concerning TNC/S100A4 expression., (© 2017 John Wiley & Sons Ltd.)

The sex-determining region Y-related high-mobility group box transcription factor 4 (SOX4) plays a fundamental role during embryogenesis and controls cell fate and differentiation. Recently, increased SOX4 expression has been reported in various cancer types, contributing to the progression and survival of cancer cells. However, the distinct functions and downstream targets of SOX4 remain to be fully elucidated. In this study, we initially found elevated SOX4 expression in melanoma. SOX4 regulates apoptosis and cell cycle arrest, affects glucose consumption and lactate production, and consequently, promotes melanoma cell proliferation. Moreover, we found that SOX4 rewires glucose metabolism by regulating the expression of glucose transporter type 1, hexokinase 2, and lactate dehydrogenase A at the transcriptional level. Mechanistically, SOX4 knockdown reduced activation of acutely transforming retrovirus AKT8 in rodent T-cell lymphoma and mTORC1, leading to an attenuated malignant phenotype. We also identified p70 ribosomal S6 kinase and eukaryotic initiation factor 4E-binding protein 1 as key substrates involved in the regulation of mTORC1 in melanoma cells. In conclusion, our study demonstrates the essential role of SOX4 in melanoma glycolytic metabolism through the acutely transforming retrovirus AKT8 in rodent T-cell lymphoma signaling pathway and highlights its potential as a therapeutic target in melanoma management., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Specific antitumor immunotherapy with autologous dendritic cell vaccines is one of the new approaches of modern medicine. For activation of dendritic cells highly immunogenic antigens are used, however optimal antigens in case of bladder cancer (BC) are still not researched. Cancer-testis antigens (CTA) are the most promising target in the context of creation of antitumor vaccines, because they are distinguished by pronounced immunogenicity, they are detected in different types of tumors and have limited pattern of expression in healthy tissues of grown-up organism. Regarding the level of mutational load, bladder cancer (BC) holds the third position among all malignant growths, which creates particular opportunities for use of immunotherapy in case of this disease. At chromosomal level most times the following cytogenetic anomalies specific for BC are detected: hyperploidies at 3, 7 and 17 chromosomes and deletion of 9p 21 locus. Besides, in the literature there is information about possible monosomy at 2, 3, 6, 8, 13, 14, 17 and frequent loss of Y chromosome in case of BC. Development of personified dendritic cell antitumor vaccines (PDAV) against bladder cancer (BC) is a relevant problem, which covers many aspects, necessary for its standardization. In particular, in case of cultivation of tumor cells under in vitro conditions their transformation goes at higher pace in comparison with in vivo tumor development. Moreover, the article presents the results of the study of molecular-genetic features of BC of tumor cultures in case of long-term cultivation, the level of expression of CTA (MAGE, NY-ESO-1, GAGE, BAGE) by urothelial carcinoma cells (UCC). There has been described the karyotypes of cells of urothelial low differentiated carcinoma of high malignant potential at various passages with prolonged cultivation, as well as the correlation between cytogenetic profile and expression of tumor-specific cancer-testis antigens has been identified. There have been developed two verified cell line cultures of muscle invasive and muscle-non-invasive urothelial carcinoma, that are potentially useful for the producing of tumor-associated vaccines against BC.

Glioblastoma (GBM) represents the most common and aggressive histologic subtype among malignant astrocytoma and is associated with poor outcomes because of heterogeneous tumour cell population including mature non-stem-like cell and immature stem-like cells within the tumour. Thus, it is critical to find new target-specific therapeutic modalities. Protein arginine methyltransferase enzyme 5 (PRMT5) regulates many cellular processes through its methylation activity and its overexpression in GBM is associated with more aggressive disease. Previously, we have shown that silencing of PRMT5 expression in differentiated GBM cell lines results in apoptosis and reduced tumour growth in mice. Here, we report the critical role of PRMT5 in GBM differentiated cells (GBMDC) grown in serum and GBM neurospheres (GBMNS) grown as neurospheres in vitro. Our results uncover a very significant role for PRMT5 in GBMNS self-renewal capacity and proliferation. PRMT5 knockdown in GBMDC led to apoptosis, knockdown in GBMNS led to G1 cell cycle arrest through upregulation of p27 and hypophoshorylation of retinoblastoma protein, leading to senescence. Comparison of impact of PRMT5 on cellular signalling by the Human Phospho-Kinase Array and chromatin immunoprecipitation-PCR revealed that unlike GBMDC, PRMT5 regulates PTEN expression and controls Akt and ERk activity in GBMNS. In vivo transient depletion of PRMT5 decreased intracranial tumour size and growth rate in mice implanted with both primary tumour-derived GBMNS and GBMDC. This is the first study to identify PTEN as a potential downstream target of PRMT5 and PRMT5 is vital to support both mature and immature GBM tumour cell populations., Competing Interests: The authors declare no conflict of interest.

Nanomaterials can be synthesized from a wide range of material systems in numerous morphologies, creating an extremely diverse portfolio. As result of this tunability, these materials are emerging as a new class of nanotherapeutics and imaging agents. One particularly interesting nanomaterial is the gold nanoparticle. Due to its inherent biocompatibility and tunable photothermal behavior, it has made a rapid transition from the lab setting to in vivo testing. In most nanotherapeutic applications, the efficacy of the agent is directly related to the target of interest. However, the optimization of the AuNP size and shape for efficacy in vitro, prior to testing in in vivo models of a disease, has been largely limited to two dimensional monolayers of cells. Two dimensional cell cultures are unable to reproduce conditions experienced by AuNP in the body. In this article, we systematically investigate the effect of different properties of AuNP on the penetration depth into 3D cell spheroids using two-photon microscopy. The 3D spheroids are formed from the HCT116 cell line, a colorectal carcinoma cell line. In addition to studying different sizes and shapes of AuNPs, we also study the effect of an oligo surface chemistry. There is a significant difference between AuNP uptake profiles in the 2D monolayers of cells as compared to the 3D cell spheroids. Additionally, the range of sizes and shapes studied here also exhibit marked differences in uptake penetration depth and efficacy. Finally, our results demonstrate that two-photon microscopy enables quantitative AuNP localization and concentration data to be obtained at the single spheroid level without fluorescent labeling of the AuNP, thus, providing a viable technique for large scale screening of AuNP properties in 3D cell spheroids as compared to tedious and time consuming techniques like electron microscopy., Competing Interests: The authors have declared that no competing interests exist.

Purpose: Human papillomavirus (HPV)-associated oropharyngeal squamous cell carcinoma (OPSCC) carries a distinct clinical behaviour. c-Met oncogene is an important driver for tumour progression and its relationship with HPV in OPSCC was explored in the present study., Experimental Design: Knockdown of HPV oncogene E6 or p53 alone and in combination was performed to examine their effects on c-Met expression by Western blot and quantitative real-time polymerase chain reaction. The effects of c-Met inhibition on cell proliferation, migration, and colony formation were examined in HPV-positive head and neck squamous cell carcinoma (HNSCC) cells. Retrospectively collected OPSCC patient specimens (N = 78) were stained for c-Met by immunohistochemistry and the staining levels were correlated with HPV status and patient outcomes., Results: E6 knockdown decreased c-Met protein and mRNA expression in HPV-positive HNSCC cells, which was partially abolished by the elimination of p53. Reducing c-Met decreased cell proliferation, migration, and colony formation in HPV-positive HNSCC cells. In OPSCC patient samples, high c-Met expression was associated with HPV-positive status (OR = 4.11, 95%CI: 1.16-14.55, P = 0.028) and tumour stage (OR = 0.27, 95%CI: 0.08-0.93, P = 0.039) by multivariable analysis. In T3/T4 stage patients, high c-Met expression was associated with HPV positivity and low p53 levels, supporting an axis of E6-p53-c-Met regulation. Furthermore, high c-Met expression was marginally associated with poor disease-free survival in HPV-positive patients., Conclusions: Our results suggest that c-Met may serve as a novel target for treating HPV-associated OPSCC. The data also demonstrate that HPV E6 upregulates c-Met expression partially through p53 downregulation., Competing Interests: statement All authors declared no conflicts of interest., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Adenosine monophosphate-activated protein kinase (AMPK) acts as a fuel gauge that maintains energy homeostasis in both normal and cancerous cells, and has emerged as a tumor suppressor. The present study aims to delineate the functional relationship between AMPK and transforming growth factor beta (TGF-β). Our results showed that expression of liver kinase B1 (LKB1), an upstream kinase of AMPK, impeded TGF-β-induced Smad phosphorylation and their transcriptional activity in breast cancer cells, whereas knockdown of LKB1 or AMPKα1 subunit by short hairpin RNA (shRNA) enhanced the effect of TGF-β. Furthermore, AMPK activation reduced the promoter activity of TGF-β1. In accordance, type 2 diabetic patients taking metformin displayed a trend of reduction of serum TGF-β1, as compared with those without metformin. A significant reduction of serum TGF-β1 was found in mice after treatment with metformin. These results suggest that AMPK inhibits the transcription of TGF-β1, leading to reduction of its concentration in serum. Finally, metformin suppressed epithelial-to-mesenchymal transition of mammary epithelial cells. Taken together, our study demonstrates that AMPK exerts multiple actions on TGF-β signaling and supports that AMPK can serve as a therapeutic drug target for breast cancer., Competing Interests: Compliance with ethical standards Conflicts of interest None

Unlabelled: Pancreatic ductal adenocarcinoma (PDAC) has a characteristically dense stroma comprised predominantly of cancer-associated fibroblasts (CAF). CAFs promote tumor growth, metastasis, and treatment resistance. This study aimed to investigate the molecular changes and functional consequences associated with chemotherapy treatment of PDAC CAFs. Chemoresistant immortalized CAFs (R-CAF) were generated by continuous incubation in gemcitabine. Gene expression differences between treatment-naïve CAFs (N-CAF) and R-CAFs were compared by array analysis. Functionally, tumor cells (TC) were exposed to N-CAF- or R-CAF-conditioned media and assayed for migration, invasion, and viability in vitro Furthermore, a coinjection (TC and CAF) model was used to compare tumor growth in vivo R-CAFs increased TC viability, migration, and invasion compared with N-CAFs. In vivo, TCs coinjected with R-CAFs grew larger than those accompanied by N-CAFs. Genomic analysis demonstrated that R-CAFs had increased expression of various inflammatory mediators, similar to the previously described senescence-associated secretory phenotype (SASP). In addition, SASP mediators were found to be upregulated in response to short duration treatment with gemcitabine in both immortalized and primary CAFs. Inhibition of stress-associated MAPK signaling (P38 MAPK or JNK) attenuated SASP induction as well as the tumor-supportive functions of chemotherapy-treated CAFs in vitro and in vivo These results identify a negative consequence of chemotherapy on the PDAC microenvironment that could be targeted to improve the efficacy of current therapeutic regimens., Implications: Chemotherapy treatment of pancreatic cancer-associated fibroblasts results in a proinflammatory response driven by stress-associated MAPK signaling that enhances tumor cell growth and invasiveness. Mol Cancer Res; 14(5); 437-47. ©2016 AACR., (©2016 American Association for Cancer Research.)

Mesenchymal stem cells (MSCs) possess some unique features (inherent tumor tropism, anti-inflammatory and immunosuppressive properties) that are not commonly found in conventional anti-cancer agents. These cells are known to secrete a vast array of proteins including growth factors, cytokines, chemokines, extracellular matrix metalloproteinases, and their corresponding inhibitors which exhibit profound effects on the microenvironment. However, the lack of a uniform method for culturing MSCs and their paracrine factors has hindered our understanding of MSC biology. In this chapter, we describe methods for the isolation, in vivo expansion, and phenotypic characterization of MSCs. In addition, methods for the collection and concentration of conditioned medium from these MSCs are described. Using tumor cells that constitutively express fluorescence reporter proteins, the effect of conditioned medium on tumor cell viability can be easily tested in vitro.

Purpose: Conjunctival melanoma (CM) is an ocular surface tumor that can lead to fatal metastases. Patients developing, tumor-associated lymphangiogenesis have a significantly increased risk of metastatic disease, because tumor spread primarily occurs via lymphatic vessels to the draining lymph node. Here, we describe a novel immune-competent mouse model of CM that displays tumor-associated lymphangiogenesis with development of metastatic tumors., Methods: C57BL/6N mice received C57BL/6N-derived dermal melanoma cells (hepatocyte growth factor [HGF] cyclin dependent kinase-4 [Cdk4]+) or B16F10 via subconjunctival injection. A clinical score quantified primary tumor growth and metastases were identified by macroscopic examination of the draining lymph nodes, lung, and spleen. Confirmation of tumors and metastases was achieved by immunohistochemical staining for markers of pigmented cells (tyrosinase related protein-2 [TRP2]) and S-100, and of cell proliferation (Ki67). The intra- and peritumoral CD31+ blood and lymphatic vessel endothelium hyaluronan receptor-1 (LYVE-1)+ lymphatic vessels were quantified immunohistochemically., Results: All mice rapidly developed aggressive TRP2+, S100+, and Ki67+ CM. Metastatic tumors were found in the lymph node (9%) and lung (6%) of HGF-Cdk4(R24C)-treated mice and in the spleen (8%) and lung (17%) of B16F10-treated mice. The amount of peri- and intratumoral blood vessels was significantly increased compared with lymphatic vessels., Conclusions: This CM model in immune-competent animals offers new possibilities to study the pathobiology of tumor growth, invasion, and mechanisms of metastatic tumor spread, and provides a robust model to explore new immune-based and antilymphangiogenic treatment modalities of this malignancy.

The uptake of E -combretastatins, potential prodrugs of the anticancer Z -isomers, into multicellular spheroids has been imaged by intrinsic fluorescence in three dimensions using two-photon excited fluorescence lifetime imaging with 625-nm ultrafast femtosecond laser pulses. Uptake is initially observed at the spheroid periphery but extends to the spheroid core within 30 min. Using agarose gels as a three-dimensional model, the conversion of Z(trans)→E(cis) via two-photon photoisomerization is demonstrated and the location of this photochemical process may be precisely selected within the micron scale in all three dimensions at depths up to almost 2 mm. We discuss these results for enhanced tissue penetration at longer near-infrared wavelengths for cancer therapy and up to three-photon excitation and imaging using 930-nm laser pulses with suitable combretastatin analogs.

A new and simple method was developed to evaluate the distribution of therapeutics in three-dimensional multicellular tumor spheroids (MCTS) by combining serial trypsinization and nanoflow liquid chromatography-tandem mass spectrometry (nLC-MS/MS). This methodology was validated with quantitative measurements of irinotecan and its bioactive metabolite, SN-38, in distinct spatial regions of HCT 116 MCTS. Irinotecan showed a time-dependent permeability into MCTS with most of the drug accumulating in the core after 24 h of treatment. The amount of SN-38 detected was 30 times lower than that of the parent drug, and was more abundant in the outer rim and intermediate regions of MCTS where proliferating cells were present. This method can be used to investigate novel and established drugs. It enables investigation of drug penetration properties and identification of metabolites with spatial specificity in MCTS. The new approach has great value in facilitating the drug evaluation process.

Retinoblastoma (RB) is the most common malignant intraocular childhood tumor. Forty years after their first description, in the present study, we re-characterized seven established retinoblastoma cell lines with regard to their RB1 mutation status, morphology, growth pattern, endogenous apoptosis levels, colony formation efficiency in soft agar and invasiveness and dissemination capacity in chick chorioallantoic membrane (CAM) assays. All RB cell lines predominantly resemble small epithelioid cells with little cytoplasm and large nucleus, which mainly grow in cell clusters, but sometimes form chain-like structures with incident loops or three-dimensional aggregates. We observed different growth rates for the different retinoblastoma cells investigated. RBL-30, RBL-13 and RBL 383 cells grew very slowly, whereas Y-79 cells grew fastest under our culture conditions. Apoptosis rates likewise differed with highest cell death levels in RB 383 and RB 355 and lowest in WERI-Rb1 and RBL-15. Contradicting former reports, six of the seven RB cell lines analyzed were able to form colonies in soft agarose after single cell seeding within 3 weeks of incubation. Upon inoculation of four out of seven RB cell lines on the dorsal CAM, GFP-positive cells were detectable in the ventral CAM and two RB cell lines caused tumor development, indicating their intravasation and dissemination potential. All RB cell lines exhibited the potential to extravasate from the capillary system after intravenous CAM injection. Our study provides valuable new details for future therapy-related retinoblastoma basic research in vitro.

Recombinant human erythropoietin (rHuEPO) is an effective treatment for anaemia but concerns that it causes disease progression in cancer patients by activation of EPO receptors (EPOR) in tumour tissue have been controversial and have restricted its clinical use. Initial clinical studies were flawed because they used polyclonal antibodies, later shown to lack specificity for EPOR. Moreover, multiple isoforms of EPOR caused by differential splicing have been reported in cancer cell lines at the mRNA level but investigations of these variants and their potential impact on tumour progression, have been hampered by lack of suitable antibodies. The EpoCan consortium seeks to promote improved pathological testing of EPOR, leading to safer clinical use of rHuEPO, by producing well characterized EPOR antibodies. Using novel genetic and traditional peptide immunization protocols, we have produced mouse and rat monoclonal antibodies, and show that several of these specifically recognize EPOR by Western blot, immunoprecipitation, immunofluorescence, flow cytometry and immunohistochemistry in cell lines and clinical material. Widespread availability of these antibodies should enable the research community to gain a better understanding of the role of EPOR in cancer, and eventually to distinguish patients who can be treated safely by rHuEPO from those at increased risk from treatment., (© 2014 John Wiley & Sons Ltd.)

Purpose: To address the effect of resveratrol and other red wine polyphenols on cell proliferation, apoptosis and androgen receptor (AR) expression in human prostate cancer LNCaP cells., Materials and Methods: LNCaP cells (5 × 102) were cultured in microtiter plate modules and treated with gallic acid, tannic acid and quercetin (1, 5 and 10 μM), rutin and morin (25, 50 and 75 μM) and resveratrol (5, 10 and 25 μM). To address the extent of proliferation at 24, 48, 72 and 96 hours, a colorimetric immunoassay method was used. An activity caspase 3/7 detection assay was used to disclose apoptosis at 24, 48 and 72 hours. AR mARN levels were determined by real time RT-PCR., Results: All polyphenols studied significantly inhibited (P<.05) cell proliferation compared to control. However, there were moderate differences between them. Resveratrol was the strongest inhibitor at different times and doses. Also, caspase-3 and caspase-7 activity was significantly higher (P<.05) than control in the presence of all the compounds, but the earlier response was achieved by resveratrol. Resveratrol, quercetin and morin were the only nutrients that significantly inhibited AR mRNA expression. Again resveratrol produced the highest inhibition (90-250 times less than control), followed by morin (67-100 times) and quercetin (55-91 times)., Conclusions: All polyphenols studied showed important antiproliferative effects and induced apoptosis when added to LNCaP cells culture. We confirm that resveratrol, morin and quercetin may achieve such effect through reduced expression of AR. The synergistic effects of these compounds and their potential to prevent progression of hormone-dependent prostate cancer merit further study., (Copyright © 2014 AEU. Published by Elsevier Espana. All rights reserved.)

By a proteomics-based approach, we identified an overexpression of fascin in colon adenocarcinoma cells (FPCKpP-3) that developed from nontumorigenic human colonic adenoma cells (FPCK-1-1) and were converted to tumorigenic by foreign-body-induced chronic inflammation in nude mice. Fascin overexpression was also observed in the tumors arising from rat intestinal epithelial cells (IEC 6) converted to tumorigenic in chronic inflammation which was induced in the same manner. Upregulation of fascin expression in FPCK-1-1 cells by transfection with sense fascin cDNA converted the cells tumorigenic, whereas antisense fascin-cDNA-transfected FPCKpP-3 cells reduced fascin expression and lost their tumor-forming ability in vivo. The tumorigenic potential by fascin expression was consistent with their ability to survive and grow in the three-dimensional multicellular spheroids. We found that resistance to anoikis (apoptotic cell death as a consequence of insufficient cell-to-substrate interactions), which is represented by the three-dimensional growth of solid tumors in vivo, was regulated by fascin expression through caspase-dependent apoptotic signals. From these, we demonstrate that fascin is a potent suppressor to caspase-associated anoikis and accelerator of the conversion of colonic adenoma cells into adenocarcinoma cells by chronic inflammation., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Controlling metastatic lesions is an important part of improving cancer prognosis, in addition to controlling the primary lesion. There have been numerous histological studies on primary and metastatic lesions, but little basic research has been performed using cell lines from primary and metastatic lesions belonging to the same patient. In this study, we successfully established a cell line derived from lower gingival carcinoma (WK2) as well as a line derived from secondary cervical lymph node metastasis (WK3F) through primary cultures of tissue from a patient with oral squamous cell carcinoma. We then investigated the biological characteristics of the cancer cell lines from these primary and metastatic lesions and analyzed metastasis-related genes. Comparison of the biological characteristics in vitro showed that WK3F had higher cell proliferation ability and shorter cell doubling time than WK2. WK3F also had increased cell migratory ability and higher invasive and self-replication abilities. Heterotransplantation into nude mice resulted in high tumor formation rates in the tongue and high metastasis rates in the cervical lymph nodes. Changes in WK2 and WK3F gene expression were then comprehensively analyzed using microarrays. Genes with increased expression in WK3F compared to WK2 were extracted when the Z-score was ≥2.0 and the ratio was ≥5.0, while genes with reduced expression in WK3F compared to WK2 were extracted when the Z-score was ≤-2.0 and the ratio was ≤0.2; differences were found in 604 genes. From these, MAGEC1 (88.0-fold), MMP-7 (18.6-fold), SNAI1 (6.6-fold), MACC1 (6.2-fold), and HTRA1 (0.012-fold) were selected as metastasis-related candidate genes. The results suggest that these molecules could be important for clarifying the mechanisms that regulate metastasis and provide new therapeutic targets for inhibiting tumor invasion.

Background: Head and neck squamous cell carcinomas (HNSCCs) have devastating morbidity rates with mortality mainly because of metastasis., Methods: Multiplex enzyme-linked immunosorbent assay (ELISA) to assay a variety of cytokine levels secreted by a panel of stage-specific and anatomic site-specific primary, and recurrent and metastatic University of Michigan-HNSCC cell lines over a 72-hour time course., Results: Conditioned medium from metastatic or recurrent HNSCC showed significantly higher amounts of interleukin (IL)-6, IL-6 receptor, tumor growth factor-beta (TGF-β) and vascular endothelial growth factor (VEGF) than nonmetastatic cells or normal oral keratinocytes. Tumor necrosis factor (TNF) was only secreted by stage IV, metastatic, or recurrence-derived cell lines., Conclusion: The cytokine profile of cultured HNSCC cells suggests that high levels of IL-6 and IL-6R, TGF-β, and VEGF are significantly related with their metastatogenic potential and provide rationale for determining if serum testing for a combination of these 4 soluble factors could be of predictive value for the HNSCC tumor progression and clinical outcome., (Copyright © 2013 Wiley Periodicals, Inc.)

Early and specific diagnosis is critical for treatment of cholangiocarcinoma (CCA). In this study, a carbohydrate antigen-S27 (CA-S27) monoclonal antibody (mAb) was established using pooled CCA tissue-extract as immunogen. The epitope recognized by CA-S27-mAb was a new Lewis-a (Le(a)) associated modification of MUC5AC mucin. A Soybean agglutinin/CA-S27-mAb sandwich ELISA to determine CA-S27 in serum was successfully developed. High level of CA-S27 was detected in serum of CCA patients and could differentiate CCA patients from those of gastro-intestinal cancers, hepatomas, benign hepatobiliary diseases and healthy subjects with high sensitivity (87.5%) and high negative predictive value (90.4%). The level of serum CA-S27 was dramatically reduced after tumor removal, indicating tumor origin of CA-S27. Patients with high serum CA-S27 had significantly shorter survivals than those with low serum CA-S27 regardless of serum MUC5AC levels. Fucosyltransferase-III (FUT3) was shown to be a regulator of CA-S27 expression. Suppression of CA-S27 expression with siRNA-FUT3 or neutralization with CA-S27 mAb significantly reduced growth, adhesion, invasion and migration potentials of CCA cells in vitro. In summary, we demonstrate that serum CA-S27, a novel carbohydrate antigen, has potential as diagnostic and prognostic markers for CCA patients. CA-S27 involves in promoting cell growth, adhesion, migration and invasion of CCA cells., (© 2013 Japanese Cancer Association.)

Purpose: Our group previously noted that HMGB1 release by urothelial carcinoma cells occurs as a consequence of bacillus Calmette-Guérin induced nonapoptotic cell death. Additional studies demonstrated that HMGB1 release in response to bacillus Calmette-Guérin is required for the in vivo tumor response to bacillus Calmette-Guérin. We evaluated the steps required for HMGB1 release by human urothelial carcinoma cells in response to bacillus Calmette-Guérin exposure., Materials and Methods: We used the T24 and 253J human urothelial carcinoma cell lines. HMGB1 concentrations in cell culture supernatant with and without bacillus Calmette-Guérin treatment served as the principal end point to assess the role of potentially involved variables. Specific techniques were used to determine the role of α5β1 antigen receptor cross-linking, TLR signaling, inducible nitric oxide synthase expression/nitric oxide production, p21 expression, and bacillus Calmette-Guérin adherence, internalization and viability., Results: Cross-linking of α5β1 integrin or signaling through TLR2/4 did not contribute to HMGB1 release. Optimal HMGB1 release required bacillus Calmette-Guérin adherence and internalization. Bacillus Calmette-Guérin viability correlated with the magnitude of HMGB1 release. Inhibition of oxidative stress and p21 expression in response to bacillus Calmette-Guérin decreased the magnitude of HMGB1 release., Conclusions: Bacillus Calmette-Guérin induced nonapoptotic cell death and HMGB1 release occur as a consequence of a complex multistep process. Understanding the steps and mechanisms involved in the induced HMGB1 release would provide an opportunity for targeted strategies to improve bacillus Calmette-Guérin treatment efficacy., (Copyright © 2013 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.)

Boronic acid-rich chitosan-poly(N-3-acrylamidophenylboronic acid) nanoparticles (CS-PAPBA NPs) with the tunable size were successfully prepared by polymerizing N-3-acrylamidophenylboronic acid in the presence of chitosan in an aqueous solution. The CS-PAPBA NPs were then functionalized by a tumor-penetrating peptide iRGD and loading doxorubicin (DOX). The interaction between boronic acid groups of hydrophobic PAPBA and the amino groups of hydrophilic chitosan inside the nanoparticles was examined by solid-state NMR measurement. The size and morphology of nanoparticles were characterized by dynamic light scattering and electron microscopy. The cellular uptake, tumor penetration, biodistribution and antitumor activity of the nanoparticles were evaluated by using three-dimensional (3-D) multicellular spheroids (MCs) as the in vitro model and H22 tumor-bearing mice as the in vivo model. It was found that the iRGD-conjugated nanoparticles significantly improved the efficiency of DOX penetration in MCs, compared with free DOX and non-conjugated nanoparticles, resulting in the efficient cell killing in the MCs. In vivo antitumor activity examination indicated that iRGD-conjugated CS-PAPBA nanoparticles promoted the accumulation of nanoparticles in tumor tissue and enhanced their penetration in tumor areas, both of which improved the efficiency of DOX-loaded nanoparticles in restraining tumor growth and prolonging the life time of H22 tumor-bearing mice., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Sustained NF-κB activation is often observed in acute myeloid leukemia (AML); therefore, proteasome inhibition has been proposed to efficiently target AML cells. In this study, we questioned whether leukemic stem cell-enriched CD34(+) cells are sensitive to the proteasome inhibitor bortezomib. Surprisingly, we observed in short-term and long-term culture assays that CD34(-) AML cells were more sensitive to bortezomib treatment compared with the CD34(+) AML cells at a clinical relevant dosage. Cotreatment with the apoptosis-inducing cytokine TRAIL did not enhance cell death in CD34(+) AML cells, in contrast to the effects in AML cell lines. The better survival of CD34(+) AML cells upon bortezomib treatment was due to a persisting NF-κB activity that could be overcome by the IKK inhibitor BMS-345541. This difference in sensitivity might be related to differences in NF-κB activation in AML CD34(+) versus CD34(-) cells, as suggested by a gene expression profiling study. Besides NF-κB, MCL-1 strongly determines the effectiveness of bortezomib. MCL-1 accumulated in CD34(+) AML cells upon bortezomib treatment and inhibition of MCL-1 by shRNA, or Obatoclax, significantly improved the sensitivity of CD34(+) AML cells to bortezomib. These results demonstrate that combining bortezomib with specific NF-κB or MCL-1 inhibitors might potentially target the leukemic stem cells., (Copyright © 2013 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.)

Multiple myeloma (MM) cells typically grow in focal lesions, stimulating osteoclasts that destroy bone and support MM. Osteoclasts and MM cells are hypermetabolic. The coenzyme nicotinamide adenine dinucleotide (NAD(+)) is not only essential for cellular metabolism; it also affects activity of NAD-dependent enzymes, such as PARP-1 and SIRT-1. Nicotinamide phosphoribosyltransferase (NAMPT/PBEF/visfatin, encoded by PBEF1) is a rate-limiting enzyme in NAD(+) biosynthesis from nicotinamide. Coculture of primary MM cells with osteoclasts induced PBEF1 upregulation in both cell types. PBEF1 expression was higher in experimental myelomatous bones than in nonmyelomatous bone and higher in MM patients' plasma cells than in healthy donors' counterparts. APO866 is a specific PBEF1 inhibitor known to deplete cellular NAD(+). APO866 at low nanomolar concentrations inhibited growth of primary MM cells or MM cell lines cultured alone or cocultured with osteoclasts and induced apoptosis in these cells. PBEF1 activity and NAD(+) content were reduced in MM cells by APO866, resulting in lower activity of PARP-1 and SIRT-1. The inhibitory effect of APO866 on MM cell growth was abrogated by supplementation of extracellular NAD(+) or NAM. APO866 inhibited NF-κB activity in osteoclast precursors and suppressed osteoclast formation and activity. PBEF1 knockdown similarly inhibited MM cell growth and osteoclast formation. In the SCID-rab model, APO866 inhibited growth of primary MM and H929 cells and prevented bone disease. These findings indicate that MM cells and osteoclasts are highly sensitive to NAD(+) depletion and that PBEF1 inhibition represents a novel approach to target cellular metabolism and inhibit PARP-1 and bone disease in MM., (Published by Elsevier Inc.)

Cell migration is a key hallmark of malignant cells that contributes to the progression of cancers from a primary, localized mass to an invasive and/or metastatic phenotype. Traditional methods for the evaluation of tumor cell migration in vitro generally employ two-dimensional (2D), homogeneous cultures that do not take into account tumor heterogeneity, three-dimensional (3D) cell-cell contacts between tumor and/or host cells or interactions with extracellular matrix proteins. Here we describe a 3D tumor spheroid-based migration assay which more accurately reflects the solid tumor microenvironment and can accommodate both extracellular matrix and host cell interactions. It is a rapid and highly reproducible 96-well plate-based technique and we demonstrate its utility for the evaluation of therapeutic agents/drugs with anti-migratory properties.

Aim: To isolate and culture human breast cancer stem cells, and characterize their biological properties in vitro., Methods: Fresh tumor tissues of breast cancer patients were collected from surgical rooms. The tissue samples were mechanically sheared and enzyme-digested to get single cell suspensions. Cancer cell surface antigens were analyzed by flow cytometry, and cell growth curve was established by MTT assay; Real-time quantitative PCR (qPCR) was employed to detect the expressions of stem cell-specific genes Sox2 and Nanog in the cancer cells; Immunofluorescence staining was performed to examine the expressions of breast cancer-specific proteins Bcl-2 and progesterone receptor (PR)., Results: Human breast cancer stem cells grew in the form of spheres. These stem cells had a CD44(+);/CD24(-); phenotype as characterized by flow cytometry, expressed high levels of Sox2 and Nanog genes, and were positive for Bcl-2 and PR., Conclusion: Human breast tumors contain CD44(+);/CD24(-); cancer stem cells which are capable of self-renewal and proliferation, and can be long-term cultured and expanded in vitro.

MicroRNAs (miRNAs) are small noncoding RNAs whose function as modulators of gene expression is crucial for the proper control of cell development, differentiation, and homeostasis. The total number and composition of miRNAs expressed per cell at different stages of development varies widely, and the same miRNA may function differently at different stages of development. In this prospective study, we evaluated the function of miR-125b at different developmental stages of glioblastoma cells, such as primary glioblastoma cells and the corresponding stem cells. CD133 is an important surface marker in glioblastoma stem cells. We found that the upregulation of miR-125b had no effects on the invasion of primary glioblastoma CD133-negative cells but that it could inhibit the invasion of corresponding CD133-positive cells; however, the downregulation of miR-125b also had no effects on the invasion of primary glioblastoma CD133-negative cells but promoted the invasion of CD133-positive cells. Further research into the underlying mechanism demonstrated that the effects of miR-125b on the invasion of glioblastoma CD133-positive cells were associated with the alteration of the expression of MMPs (MMP-2 and MMP-9) and corresponding inhibitors (RECK and TIMP3). Our results demonstrate that miR-125b expression plays an essential role in the invasion of glioblastoma CD133-positive cells but not CD133-negative cells. Therefore, miR-125b may represent a novel target for therapy targeting the invasion of glioblastoma stem cells in the future.