Chemotherapy resistance and stemness in mitotically quiescent human breast cancer cells identified by fluorescent dye retention

Metastatic recurrence in breast cancer is a major cause of mortality and often occurs many years after removal of the primary tumour. This process is driven by the reactivation of disseminated tumour cells that are characterised by mitotic quiescence and chemotherapeutic resistance. The ability to reliably isolate and characterise this cancer cell population is critical to enable development of novel therapeutic strategies for prevention of breast cancer recurrence. Here we describe the identification and characterisation of a sub-population of slow-cycling tumour cells in the MCF-7 and MDA-MB-231 human breast cancer cell lines based on their ability to retain the lipophilic fluorescent dye Vybrant® DiD for up to six passages in culture. Vybrant® DiD-retaining (DiD+) cells displayed significantly increased aldehyde dehydrogenase activity and exhibited significantly reduced sensitivity to chemotherapeutic agents compared to their rapidly dividing, Vybrant® DiD-negative (DiD-) counterparts. In addition, DiD+ cells were exclusively capable of initiating population re-growth following withdrawal of chemotherapy. The DiD+ population displayed only partial overlap with the CD44+CD24-/low cell surface protein marker signature widely used to identify breast cancer stem cells, but was enriched for CD44+CD24+ cells. Real-time qPCR profiling revealed differential expression of epithelial-to-mesenchymal transition and stemness genes between DiD+ and DiD- populations. This is the first demonstration that both MCF-7 and MDA-MB-231 human breast cancer lines contain a latent therapy-resistant population of slow-cycling cells capable of initiating population regrowth post-chemotherapy. Our data support that label-retaining cells can serve as a model for identification of molecular mechanisms driving tumour cell quiescence and de novo chemoresistance and that further characterisation of this prospective tumour-reinitiating population could yield novel therapeutic targets for elimination of the cells responsible for breast cancer recurrence

Transcriptomic profiling reveals novel candidate genes and signalling programs in breast cancer quiescence and dormancy

Metastatic recurrence, the major cause of breast cancer mortality, is driven by reactivation of dormant disseminated tumour cells that are defined by mitotic quiescence and chemoresistance. The molecular mechanisms underpinning mitotic quiescence in cancer are poorly understood, severely limiting the development of novel therapies for removal of residual, metastasis-initiating tumour cells. Here, we present a molecular portrait of the quiescent breast cancer cell transcriptome across the four main breast cancer sub-types (luminal, HER2-enriched, basal-like and claudin-low) and identify a novel quiescence-associated 22-gene signature using an established lipophilic-dye (Vybrant® DiD) retention model and whole-transcriptomic profiling (mRNA-Seq). Using functional association network analysis, we elucidate the molecular interactors of these signature genes. We then go on to demonstrate that our novel 22-gene signature strongly correlates with low tumoural proliferative activity, and with dormant disease and late metastatic recurrence (≥5 years after primary tumour diagnosis) in metastatic breast cancer in multiple clinical cohorts. These genes may govern the formation and persistence of disseminated tumour cell populations responsible for breast cancer recurrence, and therefore represent prospective novel candidates to inform future development of therapeutic strategies to target disseminated tumour cells in breast cancer, eliminate minimal residual disease and prevent metastatic recurrence

Anti-angiogenic drugs: direct anti-cancer agents with mitochondrial mechanisms of action

Components of the mitochondrial electron transport chain have recently gained much interest as potential therapeutic targets. Since mitochondria are essential for the supply of energy that is required for both angiogenic and tumourigenic activity, targeting the mitochondria represents a promising potential therapeutic approach for treating cancer. Here we investigate the established anti-angiogenesis drugs combretastatin A4, thalidomide, OGT 2115 and tranilast that we hypothesise are able to exert a direct anti-cancer effect in the absence of vasculature by targeting the mitochondria. Drug cytotoxicity was measured using the MTT assay. Mitochondrial function was measured in intact isolated mitochondria using polarography, fluorimetry and enzymatic assays to measure mitochondrial oxygen consumption, membrane potential and complex I–IV activities respectively. Combretastatin A4, OGT 2115 and tranilast were both shown to decrease mitochondrial oxygen consumption. OGT 2115 and tranilast decreased mitochondrial membrane potential and reduced complex I activity while combretastatin A4 and thalidomide did not. OGT 2115 inhibited mitochondrial complex II–III activity while combretastatin A4, thalidomide and tranilast did not. Combretastatin A4, thalidomide and OGT 2115 induced bi-phasic concentration-dependent increases and decreases in mitochondrial complex IV activity while tranilast had no evident effect. These data demonstrate that combretastatin A4, thalidomide, OGT 2115 and tranilast are all mitochondrial modulators. OGT 2115 and tranilast are both mitochondrial inhibitors capable of eliciting concentration-dependent reductions in cell viability by decreasing mitochondrial membrane potential and oxygen consumption

Verification of molecular subtyping of bladder cancer in the GUSTO clinical trial

The GUSTO clinical trial (Gene expression subtypes of Urothelial carcinoma: Stratified Treatment and Oncological outcomes) uses molecular subtypes to guide neoadjuvant therapies in participants with muscle‐invasive bladder cancer (MIBC). Before commencing the GUSTO trial, we needed to determine the reliability of a commercial subtyping platform (Decipher Bladder; Veracyte) when performed in an external trial laboratory as this has not been done previously. Here, we report our pre‐trial verification of the TCGA molecular subtyping model using gene expression profiling. Formalin‐fixed paraffin‐embedded tissue blocks of MIBC were used for gene expression subtyping by gene expression microarrays. Intra‐ and inter‐laboratory technical reproducibilities, together with quality control of laboratory and bioinformatics processes, were assessed. Eighteen samples underwent analysis. RNA of sufficient quality and quantity was successfully extracted from all samples. All subtypes were represented in the cohort. Each sample was subtyped twice in our laboratory and once in a separate reference laboratory. No clinically significant discordance in subtype occurred between intra‐ or inter‐laboratory replicates. Examination of sample histopathology showed variability of morphological appearances within and between subtypes. Overall, these results show that molecular subtyping by gene expression profiling is reproducible, robust and suitable for use in the GUSTO clinical trial

Search for direct production of charginos and neutralinos in events with three leptons and missing transverse momentum in √s = 7 TeV pp collisions with the ATLAS detector

A search for the direct production of charginos and neutralinos in final states with three electrons or muons and missing transverse momentum is presented. The analysis is based on 4.7 fb−1 of proton–proton collision data delivered by the Large Hadron Collider and recorded with the ATLAS detector. Observations are consistent with Standard Model expectations in three signal regions that are either depleted or enriched in Z-boson decays. Upper limits at 95% confidence level are set in R-parity conserving phenomenological minimal supersymmetric models and in simplified models, significantly extending previous results

Jet size dependence of single jet suppression in lead-lead collisions at sqrt(s(NN)) = 2.76 TeV with the ATLAS detector at the LHC

Measurements of inclusive jet suppression in heavy ion collisions at the LHC provide direct sensitivity to the physics of jet quenching. In a sample of lead-lead collisions at sqrt(s) = 2.76 TeV corresponding to an integrated luminosity of approximately 7 inverse microbarns, ATLAS has measured jets with a calorimeter over the pseudorapidity interval |eta| < 2.1 and over the transverse momentum range 38 < pT < 210 GeV. Jets were reconstructed using the anti-kt algorithm with values for the distance parameter that determines the nominal jet radius of R = 0.2, 0.3, 0.4 and 0.5. The centrality dependence of the jet yield is characterized by the jet "central-to-peripheral ratio," Rcp. Jet production is found to be suppressed by approximately a factor of two in the 10% most central collisions relative to peripheral collisions. Rcp varies smoothly with centrality as characterized by the number of participating nucleons. The observed suppression is only weakly dependent on jet radius and transverse momentum. These results provide the first direct measurement of inclusive jet suppression in heavy ion collisions and complement previous measurements of dijet transverse energy imbalance at the LHC.Comment: 15 pages plus author list (30 pages total), 8 figures, 2 tables, submitted to Physics Letters B. All figures including auxiliary figures are available at http://atlas.web.cern.ch/Atlas/GROUPS/PHYSICS/PAPERS/HION-2011-02

The performance of the jet trigger for the ATLAS detector during 2011 data taking

The performance of the jet trigger for the ATLAS detector at the LHC during the 2011 data taking period is described. During 2011 the LHC provided proton–proton collisions with a centre-of-mass energy of 7 TeV and heavy ion collisions with a 2.76 TeV per nucleon–nucleon collision energy. The ATLAS trigger is a three level system designed to reduce the rate of events from the 40 MHz nominal maximum bunch crossing rate to the approximate 400 Hz which can be written to offline storage. The ATLAS jet trigger is the primary means for the online selection of events containing jets. Events are accepted by the trigger if they contain one or more jets above some transverse energy threshold. During 2011 data taking the jet trigger was fully efficient for jets with transverse energy above 25 GeV for triggers seeded randomly at Level 1. For triggers which require a jet to be identified at each of the three trigger levels, full efficiency is reached for offline jets with transverse energy above 60 GeV. Jets reconstructed in the final trigger level and corresponding to offline jets with transverse energy greater than 60 GeV, are reconstructed with a resolution in transverse energy with respect to offline jets, of better than 4 % in the central region and better than 2.5 % in the forward direction

Search for strong gravity in multijet final states produced in pp collisions at √s=13 TeV using the ATLAS detector at the LHC

A search is conducted for new physics in multijet final states using 3.6 inverse femtobarns of data from proton-proton collisions at √s = 13TeV taken at the CERN Large Hadron Collider with the ATLAS detector. Events are selected containing at least three jets with scalar sum of jet transverse momenta (HT) greater than 1TeV. No excess is seen at large HT and limits are presented on new physics: models which produce final states containing at least three jets and having cross sections larger than 1.6 fb with HT > 5.8 TeV are excluded. Limits are also given in terms of new physics models of strong gravity that hypothesize additional space-time dimensions

Measurement of the correlation between flow harmonics of different order in lead-lead collisions at √sNN = 2.76 TeV with the ATLAS detector

Correlations between the elliptic or triangular flow coefficients vm (m=2 or 3) and other flow harmonics vn (n=2 to 5) are measured using √sNN=2.76 TeV Pb+Pb collision data collected in 2010 by the ATLAS experiment at the LHC, corresponding to an integrated luminosity of 7 μb−1. The vm−vn correlations are measured in midrapidity as a function of centrality, and, for events within the same centrality interval, as a function of event ellipticity or triangularity defined in a forward rapidity region. For events within the same centrality interval, v3 is found to be anticorrelated with v2 and this anticorrelation is consistent with similar anticorrelations between the corresponding eccentricities, ε2 and ε3. However, it is observed that v4 increases strongly with v2, and v5 increases strongly with both v2 and v3. The trend and strength of the vm−vn correlations for n=4 and 5 are found to disagree with εm−εn correlations predicted by initial-geometry models. Instead, these correlations are found to be consistent with the combined effects of a linear contribution to vn and a nonlinear term that is a function of v22 or of v2v3, as predicted by hydrodynamic models. A simple two-component fit is used to separate these two contributions. The extracted linear and nonlinear contributions to v4 and v5 are found to be consistent with previously measured event-plane correlations

Search for H→γγ produced in association with top quarks and constraints on the Yukawa coupling between the top quark and the Higgs boson using data taken at 7 TeV and 8 TeV with the ATLAS detector

A search is performed for Higgs bosons produced in association with top quarks using the diphoton decay mode of the Higgs boson. Selection requirements are optimized separately for leptonic and fully hadronic final states from the top quark decays. The dataset used corresponds to an integrated luminosity of 4.5 fb−14.5 fb−1 of proton–proton collisions at a center-of-mass energy of 7 TeV and 20.3 fb−1 at 8 TeV recorded by the ATLAS detector at the CERN Large Hadron Collider. No significant excess over the background prediction is observed and upper limits are set on the tt¯H production cross section. The observed exclusion upper limit at 95% confidence level is 6.7 times the predicted Standard Model cross section value. In addition, limits are set on the strength of the Yukawa coupling between the top quark and the Higgs boson, taking into account the dependence of the tt¯H and tH cross sections as well as the H→γγ branching fraction on the Yukawa coupling. Lower and upper limits at 95% confidence level are set at −1.3 and +8.0 times the Yukawa coupling strength in the Standard Model