Liquid biopsy in the diagnosis of HPV DNA in breast lesions

Aim: HPV DNA has never been investigated in nipple discharges (ND) and serum-derived extracellular vesicles, although its presence has been reported in ductal lavage fluids and blood specimens. Materials & methods: We analyzed 50 ND, 22 serum-derived extracellular vesicles as well as 51 pathologic breast tissues for the presence of 16 HPV DNA types. Results: We show that the presence of HPV DNA in the ND is predictive of HPV DNA-positive breast lesions and that HPV DNA is more represented in intraductal papillomas.We also show the presence of HPV DNA in the serum-derived extracellular vesicles. Conclusion: Our data supports the use of liquid biopsy to detect HPV DNA in breast patholog

Density Distribution Maps: A Novel Tool for Subcellular Distribution Analysis and Quantitative Biomedical Imaging

open5noSubcellular spatial location is an essential descriptor of molecules biological function. Presently, super-resolution microscopy techniques enable quantification of subcellular objects distribution in fluorescence images, but they rely on instrumentation, tools and expertise not constituting a default for most of laboratories. We propose a method that allows resolving subcellular structures location by reinforcing each single pixel position with the information from surroundings. Although designed for entry-level laboratory equipment with common resolution powers, our method is independent from imaging device resolution, and thus can benefit also super-resolution microscopy. The approach permits to generate density distribution maps (DDMs) informative of both objects’ absolute location and self-relative displacement, thus practically reducing location uncertainty and increasing the accuracy of signal mapping. This work proves the capability of the DDMs to: (a) improve the informativeness of spatial distributions; (b) empower subcellular molecules distributions analysis; (c) extend their applicability beyond mere spatial object mapping. Finally, the possibility of enhancing or even disclosing latent distributions can concretely speed-up routine, large-scale and follow-up experiments, besides representing a benefit for all spatial distribution studies, independently of the image acquisition resolution. DDMaker, a Software endowed with a user-friendly Graphical User Interface (GUI), is also provided to support users in DDMs creation.openIlaria De Santis; Michele Zanoni; Chiara Arienti; Alessandro Bevilacqua; Anna TeseiIlaria De Santis; Michele Zanoni; Chiara Arienti; Alessandro Bevilacqua; Anna Tese

Specific Biomarkers Are Associated with Docetaxeland Gemcitabine-Resistant NSCLC Cell Lines

AbstractFive-year survival rate for lung cancer is limited to 10% to 15%. Therefore, the identification of novel therapeutic prognostic factors is an urgent requirement. The aim of this study is thus to highlight specific biomarkers in chemoresistant non-small cell lung cancer cell lines. Therefore, we checked—in the control condition as well as after short-term pharmacological treatment with either docetaxel or gemcitabine—the expression of genes such as tumor suppressor genes (CDKN2A, DAPK, FHIT, GSTP1, MGMT, RARβ2, RASSF1A, and TIMP3), genes associated with drug resistance (BRCA1, COX2, ERCC1, IGFBP3, RRM1, and TUBB3), and stemness-related genes (CD133, OCT4, and SLUG) in two cellular models of squamous carcinoma (CAEP) and adenocarcinoma (RAL) of the lung originally established. Their promoter methylation profile was also evaluated. Drug-related genes were upregulated. Cisplatin resistance matched with high levels of BRCA1 and ERCC1 in both cell lines; docetaxel sensitivity of CAEP cells was associated to levels of TUBB3 lower than RAL cells. Although CAEP cells were more sensitive to gemcitabine, both cell lines showed high levels of RRM1. Stemness-related genes were downregulated in the control condition but became upregulated in docetaxel-resistant cells, indicating the selection of a population with stemness features. We did not find an unequivocal correspondence between gene expression and respective DNA promoter methylation status, suggesting the involvement of additional mechanisms of gene expression regulation. These results highlight specific biomarkers consistent with the different responses of the two cell lines to standard pharmacological treatments and indicate specific molecular traits for their chemoresistance

Pro-inflammatory RNA:DNA hybrids are p53 independently boosted by hyperbaric oxygen: a subcellular distribution analysis by automated quantitative imaging

Purpose: RNA:DNA hybrids are co-transcriptional products with acknowledged cytoplasmic pro-inflammatory role as activators of the cGAS-STING pathway. We recently proved them also as radiation-induced senescence messages for the abscopal effect mediation, demonstrating the need for a functional p53 for their production and release in A549 and H1299 tumour cells. However, little is known about their role under different stress conditions, especially in cancer cells. Methods: In this work, we open the investigation making use of automated quantitative imaging to characterize the hybrid subcellular distribution in HeLa cells grown under different oxygen pressures or exposed to different ionizing radiation doses. After cell imaging by confocal fluorescent microscopy, we apply automated imaging methods developed on purpose to quantify hybrid foci and nuclear cluster intensity, regional and local density and dimension. Results: We show that alteration of culture oxygenation increases hybrid cytoplasmic presence, especially when caused by an hyperoxic environment, with evident hybrid gathering at the cell membrane. Ionizing radiations always fail to increase hybrids, in accordance with the absence of functional p53 in HeLa cells. However, dose-dependent effects are still evident and suggest a threshold dose of 7.5 Gy for remarkable hybrid reduction. Conclusion: Together with our previous results, these data demonstrate for the first time that different types of stress can increase hybrid production in cancer cells and by at least two different pathways, one p53-dependent triggerable by ionizing radiations and one p53-independent triggerable by oxidative stress. Together, our findings provide a starting point for understanding hybrid role in tumour stress response

Concerns, challenges and promises of high-content analysis of 3D cellular models

Peer reviewe

Study of molecular mechanisms of pro-apoptotic activity of NCX 4040, a novel nitric oxide-releasing aspirin, in colon cancer cell lines

<p>Abstract</p> <p>Background</p> <p>Despite numerous studies aimed at verifying the antitumor activity of nitric oxide-releasing nonsteroidal antiflammatory drugs (NO-NSAIDs), little is known about the molecular targets responsible for their antineoplastic properties. In the present study, we investigated the mechanisms underlying the cytotoxicity of NCX 4040, a novel NO-aspirin with promising antineoplastic action, in <it>in vitro </it>human colon cancer models.</p> <p>Methods</p> <p>The effect on tumor growth was evaluated in four human colon cancer cell lines (LoVo, LRWZ, WiDr and LoVo Dx) by sulforhodamine B assay, oxidative stress by immunohistochemistry, apoptosis by laddering assay, mitochondrial membrane potential (ΔΨ_m) by flow cytometry, and apoptosis- and chemoresistance-related markers by western-blot and real-time method, respectively. Prostaglandin E₂levels were determined by ELISA.</p> <p>Results</p> <p>NCX 4040 produced a higher cytotoxic effect in all the cell lines than that produced by other NO donors tested. In particular, in LoVo and LRWZ cells, NCX 4040 induced a cytocidal effect and apoptosis through p53 and NAG-1 expression, an early ΔΨ_mcollapse, and a sequential release of cytoplasmatic cytochrome c and caspase -9 and -3 active forms. 8-hydroxyguanine lesions, indicative of oxidative stress, were also observed. Conversely, in WiDr line, the drug caused a cytocidal effect, albeit not through apoptosis, and a concomitant increase in COX-2 activity. In LoVo Dx line, characterized by high levels drug resistance and DNA repair-related markers, only a cytostatic effect was observed, again in concomitance with the increase in COX-2 enzyme activity.</p> <p>Conclusion</p> <p>This study highlights the multiplicity of mechanisms involved in sensitivity or resistance to NCX 4040 and could provide useful indications for tailored therapy by identifying potentially drug-responsive tumors.</p

CometAnalyser : A user-friendly, open-source deep-learning microscopy tool for quantitative comet assay analysis

Comet assay provides an easy solution to estimate DNA damage in single cells through microscopy assessment. It is widely used in the analysis of genotoxic damages induced by radiotherapy or chemotherapeutic agents. DNA damage is quantified at the single-cell level by computing the displacement between the genetic material within the nucleus, typically called ``comet head", and the genetic material in the surrounding part of the cell, considered as the ``comet tail". Today, the number of works based on Comet Assay analyses is really impressive. In this work, besides revising the solutions available to obtain reproducible and reliable quantitative data, we developed an easy-to-use tool named CometAnalyser. It is designed for the analysis of both fluorescent and silver-stained wide-field microscopy images and allows to automatically segment and classify the comets, besides extracting Tail Moment and several other intensity/morphological features for performing statistical analysis. CometAnalyser is an open-source deep-learning tool. It works with Windows, Macintosh, and UNIX-based systems. Source code, standalone versions, user manual, sample images, video tutorial and further documentation are freely available at: https://sourceforge.net/p/cometanalyser. (c) 2022 The Author(s). Published by Elsevier B.V. on behalf of Research Network of Computational and Structural Biotechnology. This is an open access article under the CC BY license (http://creativecommons. org/licenses/by/4.0/).Peer reviewe

Zoledronic acid increases docetaxel cytotoxicity through pMEK and Mcl-1 inhibition in a hormone-sensitive prostate carcinoma cell line

<p>Abstract</p> <p>Background</p> <p>In prostate cancer, the identification of drug combinations that could reduce the tumor cell population and rapidly eradicate hormone-resistant cells potentially present would be a remarkable breakthrough in the treatment of this disease.</p> <p>Methods</p> <p>The study was performed on a hormone-sensitive prostate cancer cell line (LNCaP) grown in normal or hormone-deprived charcoal-stripped (c.s.) medium. Cell viability and apoptosis were assessed by SRB assay and Annexin-V/TUNEL assays, respectively. Activated caspase-3, p21, pMEK and MCL-1 expression levels were detected by western blotting.</p> <p>Results</p> <p>The simultaneous exposure of zoledronic acid [100 μM] and docetaxel [0.01 μM] for 1 h followed by treatment with zoledronic acid for 72, 96 or 120 h produced a high synergistic interaction (R index = 5.1) with a strong decrease in cell viability. This cytotoxic effect was associated with a high induction of apoptosis in both LNCaP and in c.s. LNCaP cells. The induction of apoptosis was paralleled by a decrease in pMEK and Mcl-1 expression.</p> <p>Conclusion</p> <p>The zoledronic acid-docetaxel combination produced a highly significant synergistic effect on the LNCaP cell line grown in normal or hormone-deprived medium, the principal molecular mechanisms involved being apoptosis and decreased pMEK and Mcl-1 expression. This experimentally derived schedule would seem to prevent the selection and amplification of hormone-resistant cell clones and could thus be potentially used alongside standard androgen deprivation therapy in the management of hormone-sensitive prostate carcinoma.</p

In vitro and in vivo evaluation of NCX 4040 cytotoxic activity in human colon cancer cell lines

BACKGROUND: Nitric oxide-releasing nonsteroidal antiinflammatory drugs (NO-NSAIDs) are reported to be safer than NSAIDs because of their lower gastric toxicity. We compared the effect of a novel NO-releasing derivate, NCX 4040, with that of aspirin and its denitrated analog, NCX 4042, in in vitro and in vivo human colon cancer models and investigated the mechanisms of action underlying its antitumor activity. METHODS: In vitro cytotoxicity was evaluated on a panel of colon cancer lines (LoVo, LoVo Dx, WiDr and LRWZ) by sulforhodamine B assay. Cell cycle perturbations and apoptosis were evaluated by flow cytometry. Protein expression was detected by Western blot. In the in vivo experiments, tumor-bearing mice were treated with NCX 4040, five times a week, for six consecutive weeks. RESULTS: In the in vitro studies, aspirin and NCX 4042 did not induce an effect on any of the cell lines, whereas NCX 4040 produced a marked cytostatic dose-related effect, indicating a pivotal role of the -NO(2 )group. Furthermore, in LoVo and LRWZ cell lines, we observed caspase-9 and -3-mediated apoptosis, whereas no apoptotic effect was observed after drug exposure in WiDr or LoVo Dx cell lines. In in vivo studies, both NCX 4040 and its parental compound were administered per os. NCX 4040 induced a 40% reduction in tumor weight. Conversely, aspirin did not influence tumor growth at all. CONCLUSIONS: NCX 4040, but not its parental compound, aspirin, showed an in vitro and in vivo antiproliferative activity, indicating its potential usefulness to treat colon cancer

ReViMS: Software tool for estimating the volumes of 3-D multicellular spheroids imaged using a light sheet fluorescence microscope

Cancer 3-D spheroids are widely used to test drugs and radiotherapy treatments. These 3-D cell clusters range from tens to hundreds of micrometers in size, with shapes that typically differ from a perfect sphere. Change in spheroid volume is one of the most important parameters for evaluating treatment efficacy, and using light sheet fluorescence microscopes (LSFM), optical sections of samples in that size range can be obtained. However, there remains a lack of validated methods for quantifying the volumes of 3-D multicellular aggregates. Here, we present Reconstruction and Visualization from Multiple Sections (ReViMS), an open-source, user-friendly software for automatically segmenting z-stacks of fluorescence images and estimating the volumes of 3-D multicellular spheroids. To assess the precision and accuracy of the volume estimates obtained with ReViMS, we used several cancer spheroids imaged with LSFM. Both the precision and accuracy were >95%, demonstrating the effectiveness of ReViMS