Optical Emission Spectroscopy for the Real-Time Identification of Malignant Breast Tissue.

Breast conserving resection with free margins is the gold standard treatment for early breast cancer recommended by guidelines worldwide. Therefore, reliable discrimination between normal and malignant tissue at the resection margins is essential. In this study, normal and abnormal tissue samples from breast cancer patients were characterized ex vivo by optical emission spectroscopy (OES) based on ionized atoms and molecules generated during electrosurgical treatment. The aim of the study was to determine spectroscopic features which are typical for healthy and neoplastic breast tissue allowing for future real-time tissue differentiation and margin assessment during breast cancer surgery. A total of 972 spectra generated by electrosurgical sparking on normal and abnormal tissue were used for support vector classifier (SVC) training. Specific spectroscopic features were selected for the classification of tissues in the included breast cancer patients. The average classification accuracy for all patients was 96.9%. Normal and abnormal breast tissue could be differentiated with a mean sensitivity of 94.8%, a specificity of 99.0%, a positive predictive value (PPV) of 99.1% and a negative predictive value (NPV) of 96.1%. For 66.6% patients all classifications reached 100%. Based on this convincing data, a future clinical application of OES-based tissue differentiation in breast cancer surgery seems to be feasible.

Establishment and Characterization of a Sclerosing Spindle Cell Rhabdomyosarcoma Cell Line with a Complex Genomic Profile.

Sclerosing spindle cell rhabdomyosarcoma (SSRMS) is a rare rhabdomyosarcomas (RMS) subtype. Especially cases bearing a myogenic differentiation 1 (MYOD1) mutation are characterized by a high recurrence and metastasis rate, often leading to a fatal outcome. SSRMS cell lines are valuable in vitro models for studying disease mechanisms and for the preclinical evaluation of new therapeutic approaches. In this study, a cell line established from a primary SSRMS tumor of a 24-year-old female after multimodal chemotherapeutic pretreatment has been characterized in detail, including immunohistochemistry, growth characteristics, cytogenetic analysis, mutation analysis, evaluation of stem cell marker expression, differentiation potential, and tumorigenicity in mice. The cell line which was designated SRH exhibited a complex genomic profile, including several translocations and deletions. Array-comparative genomic hybridization (CGH) revealed an overall predominating loss of gene loci. The mesenchymal tumor origin was underlined by the expression of mesenchymal markers and potential to undergo adipogenic and osteogenic differentiation. Despite myogenic marker expression, terminal myogenic differentiation was inhibited, which might be elicited by the MYOD1 hotspot mutation. In vivo tumorigenicity could be confirmed after subcutaneous injection into NOD/SCID/γcnull mice. Summarized, the SRH cell line is the first adult SSRMS cell line available for preclinical research on this rare RMS subtype.

Interaction of arsenic trioxide and etoposide in Ewing sarcoma cell lines.

Ewing sarcomas (ES) are highly malignant mesenchymal tumors, which most often occur in children and adolescents. The current treatment of choice comprises wide resection in combination with multimodal chemotherapy including etoposide (Eto). Due to the serious side effects associated with common chemotherapeutics and prevalent multidrug resistance in recurrent and metastatic ES, there is a growing demand for alternative strategies and add­on drugs. Previous research has demonstrated efficient cell death induction by Eto in combination with arsenic trioxide (ATO) in ES cell lines. The aim of the present study was to investigate the effect of different temporal sequences of ATO and Eto administration on apoptosis induction and to explore the effect of both drugs on inhibitory glycogen synthase kinase­3ß (GSK3­ß) phosphorylation as well as multidrug transporter gene expression. The intensity of caspase activation was mainly determined by the Eto doses in A673 and TC­71 cells, whereas in RD­ES cells ATO application actively suppressed Eto­induced apoptosis. This coincided with an increase in inhibitory GSK­3ß phosphorylation in ATO­treated RD­ES cells. Inherent mRNA expression of multidrug resistance­associated protein 1 (MRP1) was low in the ES cell lines compared to that observed in the mesenchymal stem cells (MSC), whereas multidrug resistance protein 1 (MDR1) gene expression was considerably increased in the ES cell lines. ATO treatment reduced MRP1 mRNA expression in the A673 and TC­71 cells, while expression was induced in the MSC and RD­ES cells. In contrast, MDR1 mRNA expression was specifically induced by ATO in the A673 and TC­71 cells, reinforcing the expression differences between MSC and the ES cell lines. Although a reliable cell death induction by the combination of ATO and Eto has been previously shown in ES cell lines, the present study showed marked heterogeneity of the ES cell response to ATO and Eto treatment, illustrating the difficulty of prediction of individual treatment outcome in ES.

Onset and Progression of Human Osteoarthritis-Can Growth Factors, Inflammatory Cytokines, or Differential miRNA Expression Concomitantly Induce Proliferation, ECM Degradation, and Inflammation in Articular Cartilage?

Osteoarthritis (OA) is a degenerative whole joint disease, for which no preventative or therapeutic biological interventions are available. This is likely due to the fact that OA pathogenesis includes several signaling pathways, whose interactions remain unclear, especially at disease onset. Early OA is characterized by three key events: a rarely considered early phase of proliferation of cartilage-resident cells, in contrast to well-established increased synthesis, and degradation of extracellular matrix components and inflammation, associated with OA progression. We focused on the question, which of these key events are regulated by growth factors, inflammatory cytokines, and/or miRNA abundance. Collectively, we elucidated a specific sequence of the OA key events that are described best as a very early phase of proliferation of human articular cartilage (AC) cells and concomitant anabolic/catabolic effects that are accompanied by incipient pro-inflammatory effects. Many of the reviewed factors appeared able to induce one or two key events. Only one factor, fibroblast growth factor 2 (FGF2), is capable of concomitantly inducing all key events. Moreover, AC cell proliferation cannot be induced and, in fact, is suppressed by inflammatory signaling, suggesting that inflammatory signaling cannot be the sole inductor of all early OA key events, especially at disease onset.

Chondrosarcoma: A Rare Misfortune in Aging Human Cartilage? The Role of Stem and Progenitor Cells in Proliferation, Malignant Degeneration and Therapeutic Resistance.

Unlike other malignant bone tumors including osteosarcomas and Ewing sarcomas with a peak incidence in adolescents and young adults, conventional and dedifferentiated chondrosarcomas mainly affect people in the 4th to 7th decade of life. To date, the cell type of chondrosarcoma origin is not clearly defined. However, it seems that mesenchymal stem and progenitor cells (MSPC) in the bone marrow facing a pro-proliferative as well as predominantly chondrogenic differentiation milieu, as is implicated in early stage osteoarthritis (OA) at that age, are the source of chondrosarcoma genesis. But how can MSPC become malignant? Indeed, only one person in 1,000,000 will develop a chondrosarcoma, whereas the incidence of OA is a thousandfold higher. This means a rare coincidence of factors allowing escape from senescence and apoptosis together with induction of angiogenesis and migration is needed to generate a chondrosarcoma. At early stages, chondrosarcomas are still assumed to be an intermediate type of tumor which rarely metastasizes. Unfortunately, advanced stages show a pronounced resistance both against chemo- and radiation-therapy and frequently metastasize. In this review, we elucidate signaling pathways involved in the genesis and therapeutic resistance of chondrosarcomas with a focus on MSPC compared to signaling in articular cartilage (AC).

Combined application of arsenic trioxide and lithium chloride augments viability reduction and apoptosis induction in human rhabdomyosarcoma cell lines.

Rhabdomyosarcomas (RMS) are the most prevalent soft tissue sarcomas affecting children and adolescents. Despite intensive treatment consisting of multimodal chemotherapy and surgery RMS patients diagnosed with metastatic disease expect long term survival rates of only 20%. Often multidrug resistance arises upon initial response emphasizing the need for new therapeutic drugs to improve treatment efficiency. Previously, we demonstrated the efficacy of the FDA approved drug arsenic trioxide (ATO) specifically inhibiting viability and clonal growth as well as inducing cell death in human RMS cell lines of different subtypes. In this study, we combined low dose ATO with lithium chloride (LiCl), which is approved as mood stabilizer for the treatment of bipolar disorder, but also inhibits growth and survival of different cancer cell types in pre-clinical research. Indeed, we could show additive effects of LiCl and ATO on viability reduction, decrease of colony formation as well as cell death induction. In the course of this, LiCl induced inhibitory glycogen synthase kinase-3ß (GSK-3ß) serine 9 phosphorylation, whereas glioma associated oncogene family 1 (GLI1) protein expression was particularly reduced by combined ATO and LiCl treatment in RD and RH-30 cell lines, showing high rates of apoptotic cell death. These results imply that combination of ATO with LiCl or another drug targeting GSK-3 is a promising strategy to enforce the treatment efficiency in resistant and recurrent RMS.

Stretching human mesenchymal stromal cells on stiffness-customized collagen type I generates a smooth muscle marker profile without growth factor addition.

Using matrix elasticity and cyclic stretch have been investigated for inducing mesenchymal stromal cell (MSC) differentiation towards the smooth muscle cell (SMC) lineage but not in combination. We hypothesized that combining lineage-specific stiffness with cyclic stretch would result in a significantly increased expression of SMC markers, compared to non-stretched controls. First, we generated dense collagen type I sheets by mechanically compressing collagen hydrogels. Atomic force microscopy revealed a nanoscale stiffness range known to support myogenic differentiation. Further characterization revealed viscoelasticity and stable biomechanical properties under cyclic stretch with >99% viable adherent human MSC. MSCs on collagen sheets demonstrated a significantly increased mRNA but not protein expression of SMC markers, compared to on culture flasks. However, cyclic stretch of MSCs on collagen sheets significantly increased both mRNA and protein expression of α-smooth muscle actin, transgelin, and calponin versus plastic and non-stretched sheets. Thus, lineage-specific stiffness and cyclic stretch can be applied together for inducing MSC differentiation towards SMCs without the addition of recombinant growth factors or other soluble factors. This represents a novel stimulation method for modulating the phenotype of MSCs towards SMCs that could easily be incorporated into currently available methodologies to obtain a more targeted control of MSC phenotype.

Arsenic trioxide potentiates the effectiveness of etoposide in Ewing sarcomas.

Ewing sarcomas (ES) are rare mesenchymal tumours, most commonly diagnosed in children and adolescents. Arsenic trioxide (ATO) has been shown to efficiently and selectively target leukaemic blasts as well as solid tumour cells. Since multidrug resistance often occurs in recurrent and metastatic ES, we tested potential additive effects of ATO in combination with the cytostatic drugs etoposide and doxorubicin. The Ewing sarcoma cell lines A673, RD-ES and SK-N-MC as well as mesenchymal stem cells (MSC) for control were treated with ATO, etoposide and doxorubicin in single and combined application. Viability and proliferation (MTS assay, colony formation, 3D spheroid culture) as well as cell death induction (western blot analysis, flow cytometry) were analysed. In the MTS viability assays ATO treatment significantly reduced the metabolic activity of all three ES cell lines (A673, RD-ES and SK-N-MC) examined. Moreover, all ES cell lines were sensitive to etoposide, whereas MSC remained unaffected by the drug concentrations used. With the exception of ATO in RD-ES cells, all drugs induced apoptosis in the ES cell lines, indicated by caspase-3 and PARP cleavage. Combination of the agents potentiated the reduction of viability as well as the inhibitory effect on clonal growth. In addition, cell death induction was obviously enhanced in RD-ES and SK-N-MC cells by a combination of ATO and etoposide compared to single application. Summarised, the combination of low dose, physiologically easily tolerable ATO with commonly used etoposide and doxorubicin concentrations efficiently and selectively suppressed viability and colony formation in ES cell lines, whereas a combination of ATO and etoposide was favourable for cell death induction. In addition to an increase of the effectiveness of the cytostatic drugs and prevention of potential drug resistance, this approach may also reduce toxicity effects, since the individual doses can be reduced.

Targeting hedgehog signalling by arsenic trioxide reduces cell growth and induces apoptosis in rhabdomyosarcoma.

Rhabdomyosarcomas (RMS) are soft tissue tumours treated with a combination of surgery and chemotherapy. However, mortality rates remain high in case of recurrences and metastatic disease due to drug resistance and failure to undergo apoptosis. Therefore, innovative approaches targeting specific signalling pathways are urgently needed. We analysed the impact of different hedgehog (Hh) pathway inhibitors on growth and survival of six RMS cell lines using MTS assay, colony formation assay, 3D spheroid cultures, flow cytometry and western blotting. Especially the glioma-associated oncogene family (GLI) inhibitor arsenic trioxide (ATO) effectively reduced viability as well as clonal growth and induced cell death in RMS cell lines of embryonal, alveolar and sclerosing, spindle cell subtype, whereas normal skeletal muscle cells were hardly compromised by ATO. Combination of ATO with itraconazole potentiated the reduction of colony formation and spheroid size. These results show that ATO is a promising substance for treatment of relapsed and refractory RMS by directly targeting GLI transcription factors. The combination with itraconazole or other chemotherapeutic drugs has the opportunity to enforce the treatment efficiency of resistant and recurrent RMS.

Predictive biomarker discovery through the parallel integration of clinical trial and functional genomics datasets.

The European Union multi-disciplinary Personalised RNA interference to Enhance the Delivery of Individualised Cytotoxic and Targeted therapeutics (PREDICT) consortium has recently initiated a framework to accelerate the development of predictive biomarkers of individual patient response to anti-cancer agents. The consortium focuses on the identification of reliable predictive biomarkers to approved agents with anti-angiogenic activity for which no reliable predictive biomarkers exist: sunitinib, a multi-targeted tyrosine kinase inhibitor and everolimus, a mammalian target of rapamycin (mTOR) pathway inhibitor. Through the analysis of tumor tissue derived from pre-operative renal cell carcinoma (RCC) clinical trials, the PREDICT consortium will use established and novel methods to integrate comprehensive tumor-derived genomic data with personalized tumor-derived small hairpin RNA and high-throughput small interfering RNA screens to identify and validate functionally important genomic or transcriptomic predictive biomarkers of individual drug response in patients. PREDICT's approach to predictive biomarker discovery differs from conventional associative learning approaches, which can be susceptible to the detection of chance associations that lead to overestimation of true clinical accuracy. These methods will identify molecular pathways important for survival and growth of RCC cells and particular targets suitable for therapeutic development. Importantly, our results may enable individualized treatment of RCC, reducing ineffective therapy in drug-resistant disease, leading to improved quality of life and higher cost efficiency, which in turn should broaden patient access to beneficial therapeutics, thereby enhancing clinical outcome and cancer survival. The consortium will also establish and consolidate a European network providing the technological and clinical platform for large-scale functional genomic biomarker discovery. Here we review our current understanding of molecular mechanisms driving resistance to anti-angiogenesis agents, the current limitations of laboratory and clinical trial strategies and how the PREDICT consortium will endeavor to identify a new generation of predictive biomarkers.

Regulation of p53--insights into a complex process.

The p53 protein is one of the most important tumor suppressor proteins. Normally, the p53 protein is in a latent state. However, when its activity is required, e.g. upon DNA damage, nucleotide depletion or hypoxia, p53 becomes rapidly activated and initiates transcription of pro-apoptotic and cell cycle arrest-inducing target genes. The activity of p53 is regulated both by protein abundance and by post-translational modifications of pre-existing p53 molecules. In the 30 years of p53 research, a plethora of modifications and interaction partners that modulate p53's abundance and activity have been identified and new ones are continuously discovered. This review will summarize our current knowledge on the regulation of p53 abundance and activity.

p53 stabilization in response to DNA damage requires Akt/PKB and DNA-PK.

The p53 protein is one of the major tumor suppressor proteins. In response to DNA damage, p53 is prevented from degradation and accumulates to high levels. Ionizing radiation leads to hypophosphorylation of the p53 ubiquitin ligase Mdm2 at sites where phosphorylation is critical for p53 degradation and to the phosphorylation and activation of Akt/PKB, a kinase that phosphorylates and inhibits GSK-3. GSK-3, which normally phosphorylates Mdm2, is inactivated in response to ionizing radiation. We show that p53 accumulates in lymphoblasts from patients with the hereditary disorder ataxia telangiectasia in response to ionizing radiation despite the absence of a functional ATM kinase. Also, knockdown of ATR did not prevent p53 accumulation in response to ionizing radiation. Instead, p53 stabilization in response to ionizing radiation depended on the inactivation of GSK-3 and the presence of Akt/PKB. Akt/PKB is a target of DNA-PK, a kinase that is activated after ionizing radiation. Correspondingly, down-regulation of DNA-PK prevented phosphorylation of Akt/PKB and GSK-3 after ionizing radiation and strongly reduced the accumulation of p53. We therefore propose a signaling cascade for the regulation of p53 in response to ionizing radiation that involves activation of DNA-PK and Akt/PKB and inactivation of GSK-3 and Mdm2.

Glycogen synthase kinase 3-dependent phosphorylation of Mdm2 regulates p53 abundance.

The Mdm2 oncoprotein regulates abundance and activity of the p53 tumor suppressor protein. For efficient degradation of p53, Mdm2 needs to be phosphorylated at several contiguous residues within the central conserved domain. We show that glycogen synthase kinase 3 (GSK-3) phosphorylated the Mdm2 protein in vitro and in vivo in the central domain. Inhibition of GSK-3 rescued p53 from degradation in an Mdm2-dependent manner while its association with Mdm2 was not affected. Likewise, inhibition of GSK-3 did not alter localization of p53 and Mdm2 or the interaction of Mdm2 and MdmX. Ionizing radiation, which leads to p53 accumulation, directed phosphorylation of GSK-3 at serine 9, which preceded and overlapped with the increase in p53 levels. Moreover, expression of a GSK-3 mutant where serine 9 was replaced with an alanine reduced the accumulation of p53 and induction of its target p21(WAF-1). We therefore conclude that inhibition of GSK-3 contributes to hypophosphorylation of Mdm2 in response to ionizing rays, and in consequence to p53 stabilization.