Reduced sulfatide content in deferoxamine-induced senescent HepG2 cells

Iron chelators, such as deferoxamine, exert an anticancer effect by altering the activity of biomolecules critical for regulation of the cell cycle, cell metabolism, and apoptotic processes. Thus, iron chelators are sometimes used in combination with radio- and/or chemotherapy in the treatment of cancer. The possibility that deferoxamine could induce a program of senescence similar to radio- and/or chemotherapy, fostering adaptation in the treatment of cancer cells, is not fully understood. Using established biochemical techniques, biomarkers linked to lipid composition, and coherent anti-Stokes Raman scattering microscopy, we demonstrated that hepatocellular carcinoma-derived HepG2 cells survive after deferoxamine treatment, acquiring phenotypic traits and representative hallmarks of senescent cells. The results support the view that deferoxamine acts in HepG2 cells to produce oxidative stress-induced senescence by triggering sequential mitochondrial and lysosomal dysfunction accompanied by autophagy blockade. We also focused on the lipidome of senescent cells after deferoxamine treatment. Using mass spectrometry, we found that the deferoxamine-induced senescent cells presented marked remodeling of the phosphoinositol, sulfatide, and cardiolipin profiles, which all play a central role in cell signaling cascades, intracellular membrane trafficking, and mitochondria functions. Detection of alterations in glycosphingolipid sulfate species suggested modifications in ceramide generation, and turnover is frequently described in cancer cell survival and resistance to chemotherapy. Blockade of ceramide generation may explain autophagic default, resistance to apoptosis, and the onset of senescence

Deep ensemble learning and transfer learning methods for classification of senescent cells from nonlinear optical microscopy images

The success of chemotherapy and radiotherapy anti-cancer treatments can result in tumor suppression or senescence induction. Senescence was previously considered a favorable therapeutic outcome, until recent advancements in oncology research evidenced senescence as one of the culprits of cancer recurrence. Its detection requires multiple assays, and nonlinear optical (NLO) microscopy provides a solution for fast, non-invasive, and label-free detection of therapy-induced senescent cells. Here, we develop several deep learning architectures to perform binary classification between senescent and proliferating human cancer cells using NLO microscopy images and we compare their performances. As a result of our work, we demonstrate that the most performing approach is the one based on an ensemble classifier, that uses seven different pre-trained classification networks, taken from literature, with the addition of fully connected layers on top of their architectures. This approach achieves a classification accuracy of over 90%, showing the possibility of building an automatic, unbiased senescent cells image classifier starting from multimodal NLO microscopy data. Our results open the way to a deeper investigation of senescence classification via deep learning techniques with a potential application in clinical diagnosis

Impact of biospecimens handling on biomarker research in breast cancer

<p>Abstract</p> <p>Background</p> <p>Gene expression profiling is moving from the research setting to the practical clinical use.</p> <p>Gene signatures able to correctly identify high risk breast cancer patients as well as to predict response to treatment are currently under intense investigation. While technical issues dealing with RNA preparation, choice of array platforms, statistical analytical tools are taken into account, the tissue collection process is seldom considered.</p> <p>The time elapsed between surgical tissue removal and freezing of samples for biological characterizations is rarely well defined and/or recorded even for recently stored samples, despite the publications of standard operating procedures for biological sample collection for tissue banks.</p> <p>Methods</p> <p>Breast cancer samples from 11 patients were collected immediately after surgical removal and subdivided into aliquots. One was immediately frozen and the others were maintained at room temperature for respectively 2, 6 and 24 hrs. RNA was extracted and gene expression profile was determined using cDNA arrays. Phosphoprotein profiles were studied in parallel.</p> <p>Results</p> <p>Delayed freezing affected the RNA quality only in 3 samples, which were not subjected to gene profiling. In the 8 breast cancer cases with apparently intact RNA also in sample aliquots frozen at delayed times, 461 genes were modulated simply as a function of freezing timing. Some of these genes were included in gene signatures biologically and clinically relevant for breast cancer. Delayed freezing also affected detection of phosphoproteins, whose pattern may be crucial for clinical decision on target-directed drugs.</p> <p>Conclusion</p> <p>Time elapsed between surgery and freezing of samples appears to have a strong impact and should be considered as a mandatory variable to control for clinical implications of inadequate tissue handling.</p

Noninvasive morpho-molecular imaging reveals early therapy-induced senescence in human cancer cells

Anticancer therapy screening in vitro identifies additional treatments and improves clinical outcomes. Systematically, although most tested cells respond to cues with apoptosis, an appreciable portion enters a senescent state, a critical condition potentially driving tumor resistance and relapse. Conventional screening protocols would strongly benefit from prompt identification and monitoring of therapy-induced senescent (TIS) cells in their native form. We combined complementary all-optical, label-free, and quantitative microscopy techniques, based on coherent Raman scattering, multiphoton absorption, and interferometry, to explore the early onset and progression of this phenotype, which has been understudied in unperturbed conditions. We identified TIS manifestations as early as 24 hours following treatment, consisting of substantial mitochondrial rearrangement and increase of volume and dry mass, followed by accumulation of lipid vesicles starting at 72 hours. This work holds the potential to affect anticancer treatment research, by offering a label-free, rapid, and accurate method to identify initial TIS in tumor cells

Full-Spectrum CARS Microscopy of Cells and Tissues with Ultrashort White-Light Continuum Pulses

Coherent anti-StokesRaman scattering (CARS) microscopyis an emergingnonlinear vibrational imaging technique that delivers label-free chemicalmaps of cells and tissues. In narrowband CARS, two spatiotemporallysuperimposed picosecond pulses, pump and Stokes, illuminate the sampleto interrogate a single vibrational mode. Broadband CARS (BCARS) combinesnarrowband pump pulses with broadband Stokes pulses to record broadvibrational spectra. Despite recent technological advancements, BCARSmicroscopes still struggle to image biological samples over the entireRaman-active region (400-3100 cm(-1)). Here,we demonstrate a robust BCARS platform that answers this need. Oursystem is based on a femtosecond ytterbium laser at a 1035 nm wavelengthand a 2 MHz repetition rate, which delivers high-energy pulses usedto produce broadband Stokes pulses by white-light continuum generationin a bulk YAG crystal. Combining such pulses, pre-compressed to sub-20fs duration, with narrowband pump pulses, we generate a CARS signalwith a high (&lt;9 cm(-1)) spectral resolution inthe whole Raman-active window, exploiting both the two-color and three-colorexcitation mechanisms. Aided by an innovative post-processing pipeline,our microscope allows us to perform high-speed (approximate to 1 ms pixeldwell time) imaging over a large field of view, identifying the mainchemical compounds in cancer cells and discriminating tumorous fromhealthy regions in liver slices of mouse models, paving the way forapplications in histopathological settings

Is Wilms Tumor a Candidate Neoplasia for Treatment with WNT/β-Catenin Pathway Modulators?-A Report from the Renal Tumors Biology-Driven Drug Development Workshop

The European Network for Cancer Research in Children and Adolescents (ENCCA) consortium organized a workshop in Rome, in June 2012, on “Renal Tumor Biology Driven Drug Development” to discuss the current knowledge in pediatric renal cancers and to recommend directions for further research. Wilms tumor (WT) is the most common renal tumor of childhood and represents a success of pediatric oncology, with cure rates of over 85% of cases. However, a substantial minority (~25%) responds poorly to current therapies and requires ‘high risk’ treatment or relapse. Moreover, the successfully treated majority are vulnerable to the late effects of treatment, with nearly one quarter reporting severe chronic health conditions by 25 years of follow up. Main purposes of this meeting were: to advance our understanding on the molecular drivers in WT, their heterogeneity and interdependencies; to provide updates on the clinic-pathologic associations with biomarkers; to identify eligible populations for targeted drugs; and to model opportunities to use preclinical model systems and prioritize targeted agents for early phase clinical trials. At least three different pathways are involved in WT; this review represents the outcome of the workshop discussion on the WNT/β-catenin pathway in Wilms tumorigenesis

Activated Leukocyte Cell Adhesion Molecule Expression and Shedding in Thyroid Tumors

Activated leukocyte cell adhesion molecule (ALCAM, CD166) is expressed in various tissues, cancers, and cancer-initiating cells. Alterations in expression of ALCAM have been reported in several human tumors, and cell adhesion functions have been proposed to explain its association with cancer. Here we documented high levels of ALCAM expression in human thyroid tumors and cell lines. Through proteomic characterization of ALCAM expression in the human papillary thyroid carcinoma cell line TPC-1, we identified the presence of a full-length membrane-associated isoform in cell lysate and of soluble ALCAM isoforms in conditioned medium. This finding is consistent with proteolytically shed ALCAM ectodomains. Nonspecific agents, such as phorbol myristate acetate (PMA) or ionomycin, provoked increased ectodomain shedding. Epidermal growth factor receptor stimulation also enhanced ALCAM secretion through an ADAM17/TACE-dependent pathway. ADAM17/TACE was expressed in the TPC-1 cell line, and ADAM17/TACE silencing by specific small interfering RNAs reduced ALCAM shedding. In addition, the CGS27023A inhibitor of ADAM17/TACE function reduced ALCAM release in a dose-dependent manner and inhibited cell migration in a wound-healing assay. We also provide evidence for the existence of novel O-glycosylated forms and of a novel 60-kDa soluble form of ALCAM, which is particularly abundant following cell stimulation by PMA. ALCAM expression in papillary and medullary thyroid cancer specimens and in the surrounding non-tumoral component was studied by western blot and immunohistochemistry, with results demonstrating that tumor cells overexpress ALCAM. These findings strongly suggest the possibility that ALCAM may have an important role in thyroid tumor biology

Dasatinib reduces FAK phosphorylation increasing the effects of RPI-1 inhibition in a RET/PTC1-expressing cell line

BACKGROUND: TPC-1 is a papillary thyroid carcinoma (PTC)-derived cell line that spontaneously expresses the oncogene RET/PTC1. TPC-1 treated with the RET/PTC1 inhibitor RPI-1 displayed a cytostatic and reversible inhibition of cell proliferation and a strong activation of focal adhesion kinase (FAK). As dasatinib inhibition of Src results in reduction of FAK activation, we evaluated the effects of TPC-1 treatment with dasatinib in combination with RPI-1. RESULTS: Dasatinib (100 nM) strongly reduced TPC-1 proliferation and induced marked changes in TPC-1 morphology. Cells appeared smaller and more contracted, with decreased cell spreading, due to the inhibition of phosphorylation of important cytoskeletal proteins (p130(CAS), Crk, and paxillin) by dasatinib. The combination of RPI-1 with dasatinib demonstrated enhanced effects on cell proliferation (more than 80% reduction) and on the phosphotyrosine protein profile. In particular, RPI-1 reduced the phosphorylation of RET, MET, DCDB2, CTND1, and PLCγ, while dasatinib acted on the phosphorylation of EGFR, EPHA2, and DOK1. Moreover, dasatinib completely abrogated the phosphorylation of FAK at all tyrosine sites (Y576, Y577, Y861, Y925) with the exception of the autoactivation site (Y397). Notably, the pharmacological treatments induced an overexpression of integrin β1 (ITB1) that was correlated with a mild enhancement in phosphorylation of ERK1/2 and STAT3, known for their roles in prevention of apoptosis and in increase of proliferation and survival. A reduction in Akt, p38 and JNK1/2 activation was observed. CONCLUSIONS: All data demonstrate that the combination of the two drugs effectively reduced cell proliferation (by more than 80%), significantly decreased Tyr phosphorylation of almost all phosphorylable proteins, and altered the morphology of the cells, supporting high cytostatic effects. Following the combined treatment, cell survival pathways appeared to be mediated by STAT3 and ERK activities resulting from integrin clustering and FAK autophosphorylation. EphA2 may also contribute, at least in part, to integrin and FAK activation. In conclusion, these data implicate ITB1 and EphA2 as promising therapeutic targets in PTC