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The role of the intestinal microbiota in the promotion, progression, and response to therapies is gaining importance, but recent studies confirm the presence of microbiota also in the tumor, thus becoming a component of the tumor microenvironment. There is not much knowledge on the characteristics and mechanisms of action of the tumor resident microbiota, but there are already indications of its involvement in conditioning the response to therapies. In this review, we discuss recent publications on the interaction between microbiota and anticancer treatments, mechanisms of resistance and possible strategies for manipulating the microbiota that could improve treatments in a personalized medicine perspective.

Cancer is a complex disease with a rapid growing incidence and often characterized by a poor prognosis. Although impressive advances have been made in cancer treatments, resistance to therapy remains a critical obstacle for the improvement of patients outcome. Current treatment approaches as chemo-, radio-, and immuno-therapy deeply affect the tumor microenvironment (TME), inducing an extensive selective pressure on cancer cells through the activation of the immune system, the induction of cell death and the release of inflammatory and damage-associated molecular patterns (DAMPS), including nucleosides (adenosine) and nucleotides (ATP and ADP). To survive in this hostile environment, resistant cells engage a variety of mitigation pathways related to metabolism, DNA repair, stemness, inflammation and resistance to apoptosis. In this context, purinergic signaling exerts a pivotal role being involved in mitochondrial function, stemness, inflammation and cancer development. The activity of ATP and adenosine released in the TME depend upon the repertoire of purinergic P2 and adenosine receptors engaged, as well as, by the expression of ectonucleotidases (CD39 and CD73) on tumor, immune and stromal cells. Besides its well established role in the pathogenesis of several tumors and in host–tumor interaction, purinergic signaling has been recently shown to be profoundly involved in the development of therapy resistance. In this review we summarize the current advances on the role of purinergic signaling in response and resistance to anti-cancer therapies, also describing the translational applications of combining conventional anticancer interventions with therapies targeting purinergic signaling.

Three samples of meta-acidic rocks with pre-Alpine metamorphic relicts from the Sesia-Lanzo Zone eclogitic continental crust were investigated using stepwise controlled elemental maps by means of the Quantitative X-ray Maps Analyzer (Q-XRMA). Samples were chosen with the aim of analysing the reacting zones along the boundaries between the pre-Alpine and Alpine mineral phases, which developed in low chemically reactive systems. The quantitative data treatment of the X-ray images was based on a former multivariate statistical analytical stage followed by a sequential phase and sub-phase classification and permitted to isolate and to quantitatively investigate the local paragenetic equilibria. The parageneses thus observed were interpreted as related to the pre-Alpine metamorphic or magmatic stages as well as to local Alpine re-equilibrations. On the basis of electron microprobe analysis, specific compositional ranges were defined in micro-domains of the relict and new paragenetic equilibria. In this way calibrated compositional maps were obtained and used to contour different types of reacting boundaries between adjacent solid solution phases. The pre-Alpine and Alpine mineral parageneses thus obtained allowed to perform geothermobarometry on a statistically meaningful and reliable dataset. In general, metamorphic temperatures cluster at 600–700 °C and 450–550 °C, with lower temperatures referred to a retrograde metamorphic re-equilibration. In all the cases described, pre-Alpine parageneses were overprinted by an Alpine metamorphic mineral assemblage. Pressure-temperature estimates of the Alpine stage averagely range between 420 to 550 °C and 12 to 16.5 kbar. The PT constraints permitted to better define the pre-Alpine metamorphic scenario of the western Austroalpine sectors, as well as to better understand the influence of the pre-Alpine metamorphic inheritance on the forthcoming Alpine tectonic evolution.

Additional file 1: Fig. S1. WTP53 is required to trigger abscopal effects after irradiation. Fig. S2. A549sip53 cells grown as 3D models did not show irradiation-induced SASP; A549 cells grown as monolayer and exposed to IR A549sip53 EVs did not show expression of DNA:RNA hybrid structures; DNA:RNA ORF1 co-localization analysis in A549sip53 after irradiation exposure. Fig. S3. EVs secreted by irradiated A549 induce M1 polarization in a human macrophage cell line. Fig. S4. Analysis of DNA damage and growth rate in A549 and A549sip53 cells exposed to irradiation doses of 10 Gy or 20 Gy.

Additional file 2. Supplementary notes.

OBJECTIVE: High-content screening (HCS) is a powerful approach to discover new drugs and carry out basic biological research. HCS is widely performed in pharmaceutical and biotechnology industries, but always by using 2D cell cultures. In the literature, there are several papers proving that many cell lines behave differently when cultured as monolayer rather than when cultured in 3D. Accordingly, in vitro-built 3D multicellular spheroids are receiving increased interest with evidence showing that the biological response is closer to that of in vivo cells. Today, more and more biological laboratories are using spheroids to test drugs and radiotherapy treatments. However, a well-established and general purpose validated protocol to generate spheroids with characteristics compliant with 3D HCS experiments does not exist. Here we compared several generation methods with the aim of investigating the possibility to generate in a short time hundreds of homogeneous 3D spheroids, suitable for HCS experiments. MATERIALS AND METHODS: We analysed dimension, shape, and homogeneity of Mesenchymal Stromal Cell (MSC) and A549 lung cancer cell spheroids generated by using: (a) a NASA rotatory bioreactor (Synthecon, Houston, USA); (b) several Perfecta3D (3DBiomatrix, Ann Arbor, USA) and GravityPLUS (InSphero, Schlieren, Switzerland) hanging drop plates; (c) magnetic levitation method with magnetic nanoparticle; (d) pellet culture method. To compute the morphological features we used AnaSP (http://sourceforge.net/p/anasp/) and ReVISP (http://sourceforge.net/p/revisp/). RESULTS: Spheroids generated by using magnetic levitation and hanging drop plates are quite small (diameter < 500 µm) and irregular (sphericity index < 0.90). Instead, spheroids generated with the NASA bioreactor are irregular, but characterized by a large dimension. In addition, after waiting few days, the so-called spheroidization time, they become much more spherical. Spheroids generated with the pellet culture method have a large dimension and a spherical shape. As regards the number of spheroids generated, magnetic levitation and NASA bioreactor provide an easy solution to generate a number of spheroids compliant with HCS experiments in a very short time. The pellet culture method depends on the number of chambers in the centrifuge, and more rounds are typically required to generate a sufficient number of spheroids. A high number of hanging drop plates has to be used to generate a sufficient amount of spheroids. To generate the spheroids the NASA bioreactor required two weeks, magnetic levitation and hanging drop plates one week, pellet culture method one day. CONCLUSION: An absolute best solution does not exist to generate spheroids for HCS experiments. Both NASA bioreactor and pellet culture method seem to be a good choice if a number of large spherical spheroids is required. However, due to the short time needed to generate the spheroids, and the absence of a spheroidization time, the pellet culture method seems to be the best solution at one’s disposal.

Three-dimensional (3D) multicellular aggregates, typically known as “spheroids”, are used as in vitro model for several applications, ranging from drug testing in case of cancer spheroids, to regenerative medicine applications for Mesenchymal Stromal Cells (MSC) ones. In particular, MSC spheroids have been demonstrated to improve several MSC features, such as multilineage potential, secretion of proangiogenic and chemotactic factors, and resistance against hypoxic condition. Accordingly they represent an extremely promising tool for many clinical applications. Many approaches have been proposed to generate spheroids and each approach has its own disadvantages. Whichever generation approach is used, a certain degree of shape and volume heterogeneity is always appreciable in the spheroids obtained. Different volumes determine a different viability of the spheroids. In particular, large spheroids (starting from about 500 µm in diameter) are characterized by an external proliferating zone, an internal quiescent zone (caused by limited distribution of oxygen, nutrients and metabolites), and a necrotic core. On the contrary, small spheroids are typically composed by proliferating cells only. It is intuitive understanding that a volume-based preselection is necessary to obtain statistical significant data when the spheroids are used as an in vitro model for drug testing. Less intuitive is understanding that spheroids of same volume, but different shape, are characterized by a different general viability. In this work we proved that a shape-based preselection of the spheroids used as in vitro model, and not only a volume-based preselection, is necessary to obtain statistical significant data from the experiments performed. First of all we generated spheroids of MSC and cells of different cancer lineages by using the well-known pellet culture method simply based on a centrifuge used to collapse in a spherical aggregate single cells contained in a tube. Then, we automatically computed several morphological parameters, including the volume of the spheroids obtained, by using the open-source software AnaSP (freely available at http://sourceforge.net/p/anasp/). We selected two groups of 15 spheroids of similar volume, but different sphericity index, and we compared their metabolic activity as an index of viability. The data obtained by using the luminescence CellTiter-Glo®3D Cell Viability assay (Promega) showed after one week of culture a significantly reduced viability of the group of spherical spheroids with respect to the irregular-shaped group (p

The aim of this thesis is to understand the basic physico/chemical behaviour of novel nitrospiropyran-substituted-(poly)terthiophene materials. Attractive characteristics that distinguish these materials include electrochemical/photochemical-switching processes. Understanding the fundamental behaviour of these new derivatives is the key to taking advantage of these characteristics. The stimuli induced isomerisation between the colourless spiropyran and the highly coloured merocyanine is the key-process studied in this research. Chapter 2 reports the electrochromic behaviour of spiropyran, through irreversible electrooxidative isomerisation in at least two merocyanine isomers. The molecular photophysical processes of the electrochemically-generated polymer are reported and the properties studied. In Chapter 3 a new monomer, which differs by a single methylene group in the linking ester-linker functionality from the previous one, will be introduced. A nitrospiropyran was used as a reference, and a detailed spectroscopy analysis will help in the description of the thermodynamic properties. Chapter 4 highlights how the polymeric molecular switches can bind an important extracellular protein, fibronectin, and explores by means of microscopy the on-demand tuning of the protein adhesion. A new derivative is presented in Chapter 5, wherein a spiropyran moiety is intercalated between two thiophenes. The electrochemical and optical switching of the spiropyran and thiophene groups were investigated. In Chapter 6 three new spiropyran-based derivatives are presented, and routes to multi-modal switching behavior are explored via photochemical, electrochemical and chemical stimuli, such as pH. Their reversibility and their properties are investigated using both spectroscopic and electroanalytical methods. The final chapter outlines future work and initial progress related to the integration of spiropyran-thiophene polymers into photo-electro active portable analytical devices.

The photochemistry and thermodynamics of two terthiophene (TTh) derivatives bearing benzospiropyran (BSP) moieties, 1-(3,3’’-dimethylindoline-6’-nitrobenzospiropyranyl)-2-ethyl 4,4’’-didecyloxy-2,2’:5’,2’’-terthiophene-3’-acetate (BSP-2) and 1-(3,3’’-dimethylindoline-6’-nitrobenzospiropyranyl)-2-10 ethyl 4,4’’-didecyloxy-2,2’:5’,2’’-terthiophene-3’-carboxylate (BSP-3), differing only by a single methylene spacer unit, have been studied. The kinetics of photogeneration of the equivalent merocyanine (MC) isomers (MC-2 and MC-3, respectively), the isomerisation properties of MC-2 and MC-3, and the thermodynamic parameters have been studied in cetonitrile, and compared to the parent, non-TThfunctionalised, benzospiropyran derivative, BSP-1. Despite the close structural similarity of BSP-2 and 15 BSP-3, their physicochemical properties were found to differ significantly; examples include activation energies (Ea(MC-2) = 75.05 KJ mol-1, Ea(MC-3) = 100.39 kJ mol-1) and entropies of activation (S‡ MC-2 = - 43.38 J K-1 mol-1, S‡ MC-3 = 37.78 J K-1 mol-1) for the thermal relaxation from MC to BSP, with the MC-3 value much closer to the unmodified MC-1 value (46.48 J K -1 mol-1) for this latter quantity. The thermal relaxation kinetics and solvatochromic behaviour of the derivatives in a range of solvents of 20 differing polarity (ethanol, dichloromethane, acetone, toluene and diethyl ether) are also presented. Differences in the estimated values of these thermodynamic and kinetic parameters are discussed with reference to the molecular structure of the derivatives.

Adaptive materials have the ability to modify their behaviour or characteristics in response to external stimuli, for example through electrochemical changes, macroscopically in the molecular structure. These materials may have only one form with binding skills whilst others are passive. In principle this material can be switched ‘on’ and ‘off’ using external stimuli [3]. In this field hybrid Polythiophenes substituted with Spiropyrans are an important representative class of conjugated polymers that form environmentally and thermally stable materials and stimulate deep interest for their optoelectronic and reversible properties. An enhancement in the electronic and photonic properties of the materials and the creation of new functions, such as new sensory materials or new biocompatible structures, critically depends on the synthesis of polythiophene [1,4]. This problem can be bypassed with some synthetically procedures that have been developed by our group and published on previous outputs. In this work the new family of hybrid spiropyran-terthiophenes moieties has been fully characterized and analyzed. An important aspect emerged in the study consists is the charge transfer during the processes of activation and deactivation of the material [2]. It has been observed spectroelectrochemically that a consistent charge transfer occurs while the activated surface is stimulated with an external potential. The kinetics of activation and deactivation has been detected at different potentials. This plays a remarkable role in the complete description of the properties of the structure under different conditions, especially during the electrochemical activation of the material in an electrolytical cell. This offers the possibility of inducing dramatic changes to the bulk properties of the active system by electrochemical stimulus. Herein we present the electrochemical and spectrelectrochemical behaviour of our materials. BIBLIOGRAPHY 1. S. Gambhir, K. Wagner, D. L. Officer, Towards functionalised terthiophene-based polymers. Synthetic Metals 154, 2005: 117-120. 2. S. Hammes-Schiffer, A. V.Soudakov. Proton-coupled electron transfer in solution, Proteins and electrochemistry. Journal of Physical Chemistry B, 2008, 14108-14123. 3. A. Radu, S. Scarmagnani, R. Byrne, C. Slater, K. T. Lau, D. Diamond, Photonic modulation of surface properties: a novel concept in chemical sensing. Journal of Physics D: Applied Physics, 40, 2007, 7238-7244. 4. H. Mehenni, L. H. Dao, Synthesis and characterization of novel conducting homopolymers based on β-styryl terthiophene. Canadian Journal of Chemistry, 86, 2008, 1010-1018.

Polythiophenes are an important representative class of conjugated polymers that form some of the most environmentally and thermally stable materials that can be used as electrical conductors, nonlinear optical devices, polymer LEDs, electrochromic windows, sensors, solar cells, polymer electronic interconnects, nanoelectronic and optical devices.1 Gaining control over the structure, properties, and function in polythiophenes continues to make the synthesis of polythiophenes a critical subject in the development of new advanced materials. An enhancement in the electronic and photonic properties of the materials and the creation of new functions, such as new sensory materials, critically depends on the synthesis of the polythiophene.4 This leads to the exciting prospect that the properties of polythiophenes can be selectively engineered through synthesis and assembly. Herein, we show the incorporation of molecular photochromic switches, such as benzospiropyran, into the polythiophene backbone. These are an intriguing class of organic molecules, which allow the control of molecular structure and function with light.2, 3 This offers the possibility of inducing dramatic changes to the bulk properties of a system by photonic irradiation. More importantly, benzospiropyran forms photo- reversible transition metal ion complexes. Herein, we present our work on the electro-chemical and optical properties of these hybrid materials.

Prostate cancer represents the major cause of cancer-related death in men and patients frequently develop drug-resistance and metastatic disease. Most studies focus on hormone-resistance mechanisms related to androgen receptor mutations or to the acquired property of prostate cancer cells to over-activate signaling pathways. Tumor microenvironment plays a critical role in prostate cancer progression. However, the mechanism involving androgen/androgen receptor signaling in cancer associated fibroblasts and consequences for prostate cancer progression still remains elusive. We now report that prostate cancer associated fibroblasts express a transcriptional-incompetent androgen receptor. Upon androgen challenging, the receptor co-localizes with the scaffold protein filamin A in the extra-nuclear compartment of fibroblasts, thus mediating their migration and invasiveness. Cancer-associated fibroblasts move towards epithelial prostate cancer cells in 2D and 3D cultures, thereby inducing an increase of the prostate cancer organoid size. Androgen enhances both these effects through androgen receptor/filamin A complex assembly in cancer-associated fibroblasts. An androgen receptor-derived stapled peptide, which disrupts the androgen receptor/filamin A complex assembly, abolishes the androgen-dependent migration and invasiveness of cancer associated fibroblasts. Notably, the peptide impairs the androgen-induced invasiveness of CAFs in 2D models and reduces the overall tumor area in androgen-treated 3D co-culture. The androgen receptor in association with β1 integrin and membrane type-matrix metalloproteinase 1 activates a protease cascade triggering extracellular matrix remodeling. The peptide also impairs the androgen activation of this cascade. This study offers a potential new marker, the androgen receptor/filamin A complex, and a new therapeutic approach targeting intracellular pathways activated by the androgen/androgen receptor axis in prostate cancer-associated fibroblasts. Such a strategy, alone or in combination with conventional therapies, may allow a more efficient treatment of prostate cancer.

This study aimed to assess the cytotoxic effect of low molecular weight components (LMWC) and conventional silicone oils (SOs) 1000 cSt with different degree of purification (raw, intermediate, and purified) using in vitro cytotoxicity tests. Direct contact cytotoxicity tests were performed in BALB 3T3 and human retinal pigment epithelial cells (ARPE-19) using quantitative and qualitative evaluation according to the ISO 10993-5 (2009) standards. Conventional SOs 1000 cSt in form of raw, intermediate (intermediate product obtained during distillation process), and purified SO (final product after distillation) and a concentrate of LMWC (including siloxane chains with molecular weight up to 1557 g/mol) were directly applied to 100% of cell layer area for 24 h. Cell viability was quantified using 3-(4,5-dimethylthiazole-2-yl)-2,5–28 diphenyltetrazolium bromide (MTT) and neutral red uptake assays in ARPE-19 and BALB3T3, respectively. All tested samples, including the concentrate of LMWC, resulted to be not cytotoxic according to ISO 10993-5 in both qualitative and quantitative evaluations. However, the cellular viability was significantly higher in the intermediate and purified SO compared with the raw SO in ARPE-19 cells. No reduction in cell viability was detected by LMWC. The absence of cytotoxicity was observed for all tested samples in both BALB3T3 and ARPE-19 after 24 h of application. A direct cytotoxic effect is not likely to be involved in the potential complications related to SO and LMWC. Long-term potential adverse effects of SO could be related to the raw material and to different concentrations of LMWC.

A tectonic mixing of thin slivers of calcschist, fine-grained gneisses (metasediments and metagranitoids), quartzite, metabasites and serpentinites occurs all along the western margin of central and southern Sesia-Lanzo Zone (SLZ - Austroalpine Domain) at the boundary with Piemonte Zone (PZ) metaophiolites, and extends from Santanel klippe to Lanzo Massif, over a distance of 50 km. All the rocks belonging to SLZ and PZ together underwent four episodes of deformation, giving rise to a complex regional tectonostratigraphy. The metamorphic mineral assemblages marking successive foliations, detected from the central to the southern part of the SLZ western margin, indicate that various metamorphic conditions dominate in different portions of this tectonic mixing. In the southern sector of this “mixing zone” parageneses marking superposed foliations indicate that rocks of SLZ and PZ experienced an early eclogite facies imprint, followed by reequilibration under blueschist facies conditions, and that they were finally widely retrogressed under greenschist facies conditions, during the last two deformational stages. The strong syn-metamorphic deformation of this rock assemblage leaves, at the moment, the interpretation on its origin open: i) the present day configuration could be entirely due to transposition that occurred in a mantle wedge at the early stages of deformation, up to eclogite facies conditions, during active subduction; ii) a detrital origin of these alternating layers of terrigeneous and carbonaceous rocks may correspond to a primary sequence of an extensionally-thinned continental margin reworked in the Alpine subduction system. Similar kinds of “tectonic mélange” of continental and oceanic crustal slices, with variably serpentinized peridotites, are expected to be generated in an hydrated mantle wedge during ablative subduction as it results by 2D numerical simulations of ocean/continent subduction systems.

The past two decades have seen a shift from cytotoxic drugs to targeted therapy in medical oncology. Although targeted therapeutic agents have shown more impressive clinical efficacy and minimized adverse effects than traditional treatments, drug resistance has become the main limitation to their benefits. Several preclinical in vitro/in vivo models of acquired resistance to targeted agents in clinical practice have been developed mainly by using two strategies: i) genetic manipulation for modeling genotypes of acquired resistance, and ii) in vitro/in vivo selection of resistant models. In the present work, we propose a unifying framework, for investigating the underlying mechanisms responsible for acquired resistance to targeted therapeutic agents, starting from the generation of drug-resistant cellular subclones to the description of silencing procedures used for restoring the sensitivity to the inhibitor. This simple time- and cost-effective approach is widely applicable, and could be easily extended to investigate resistance mechanisms to other targeted therapeutic drugs in different tumor histotypes.