From phenotypical investigation to RNA-sequencing for gene expression analysis: A workflow for single and pooled rare cells

: Combining phenotypical and molecular characterization of rare cells is challenging due to their scarcity and difficult handling. In oncology, circulating tumor cells (CTCs) are considered among the most important rare cell populations. Their phenotypic and molecular characterization is necessary to define the molecular mechanisms underlying their metastatic potential. Several approaches that require cell fixation make difficult downstream molecular investigations on RNA. Conversely, the DEPArray technology allows phenotypic analysis and handling of both fixed and unfixed cells, enabling a wider range of applications. Here, we describe an experimental workflow that allows the transcriptomic investigation of single and pooled OE33 cells undergone to DEPArray analysis and recovery. In addition, cells were tested at different conditions (unfixed, CellSearch fixative (CSF)- and ethanol (EtOH)-fixed cells). In a forward-looking perspective, this workflow will pave the way for novel strategies to characterize gene expression profiles of rare cells, both single-cell and low-resolution input

Adipocytes and micrornas crosstalk: A key tile in the mosaic of breast cancer microenvironment

Breast cancer (BC) is a disease characterized by a high grade of heterogeneity. Consequently, despite the great achievements obtained in the last decades, most of the current therapeutic regimens still fail. The identification of new molecular mechanisms that will increase the knowledge of all steps of tumor initiation and growth is mandatory in finding new clinical strategies. The BC microenvironment, consisting of endothelial cells, fibroblasts, immune cells and adipocytes, plays an essential role in regulating BC development, and recently it has gained great attention in the scientific community. In particular, adipose tissue is emerging as an important target to investigate among mammary gland components. The mechanisms underlying BC progression driven by adipocytes are predominantly unexplored, especially that involving the switch from normal adipocytes to the so-called cancer-associated adipocytes (CAAs). MicroRNAs (miRNAs), a class of gene expression modulators, have emerged as the regulators of key oncogenes and tumor suppressor genes that affect multiple pathways of the tumor microenvironment and adipose tissue. This review concerns a presentation of the role of adipocytes in breast tissue, and describes the most recent discoveries about the interplay between adipocytes and miRNAs, which collaborate in the arrangement of a pro-inflammatory and cancerous microenvironment, laying the foundations for new concepts in the prevention and treatment of BC

Is GRB 050904 at z=6.3 absorbed by dust?

Claim of dust extinction for this GRB has been debated in the past. We suggest that the discrepant results occur primarily because most of previous studies have not simultaneously investigated the X-ray to near-IR spectral energy distribution of this GRB. The difficulty with this burst is that the X-ray afterglow is dominated by strong flares at early times and is poorly monitored at late times. In addition, the Z band photometry, which is the most sensitive to dust extinction, has been found to be affected by strong systematics. In this paper we carefully re-analyze the Swift/XRT afterglow observations of this GRB, using extensive past studies of X-ray flare properties when computing the X-ray afterglow flux level and exploiting the recent reanalysis of the optical (UV rest frame) data of the same GRB. We extract the X-ray to optical/near-IR afterglow SED for the three epochs where the best spectral coverage is available: 0.47, 1.25, and 3.4 days after the trigger. A spectral power-law model has been fitted to the extracted SEDs. We discuss that no spectral breaks or chromatic temporal breaks are expected in the epochs of interest. To fit any UV rest-frame dust absorption, we tested the Small Magellanic Cloud (SMC) extinction curve, the mean extinction curve (MEC) found for a sample of QSO at $z>4$ and its corresponding attenuation curve, as well as a starburst attenuation curve, and the extinction curve consistent with a supernova dust origin (SN-type). The SMC extinction curve and the SN-type one provide good fit to the data at all epochs, with an average amount of dust absorption at $\lambda_{rest} = 3000 \AA$ of $A_{3000} = 0.25\pm 0.07$ mag. These results indicate that the primeval galaxy at $z = 6.3$ hosting this GRB has already enriched its ISM with dust.Comment: 8 pages, 3 figures, accepted for publication in Astronomy and Astrophysics; figure 1 has been degraded, full resolution version at http://dl.dropbox.com/u/1670579/fig01.p

The rest-frame UV-to-optical spectroscopy of APM 08279+5255 - BAL classification and black hole mass estimates

We present the analysis of the rest-frame optical-to-UV spectrum of APM 08279+5255, a well-known lensed broad absorption line (BAL) quasar at $z = 3.911$. The spectroscopic data are taken with the optical DOLoRes and near-IR NICS instruments at TNG, and include the previously unexplored range between C III] $\lambda$1910 and [O III] $\lambda\lambda$4959,5007. We investigate the possible presence of multiple BALs by computing "balnicity" and absorption indexes (i.e. BI, BI$_0$ and AI) for the transitions Si IV $\lambda$1400, C IV $\lambda$1549, Al III $\lambda$1860 and Mg II $\lambda$2800. No clear evidence for the presence of absorption features is found in addition to the already known, prominent BAL associated to C IV, which supports a high-ionization BAL classification for APM 08279+5255. We also study the properties of the [O III], H$\beta$ and Mg II emission lines. We find that [O III] is intrinsically weak ($F_{\rm [OIII]}/F_{\rm H\beta} \lesssim 0.04$), as it is typically found in luminous quasars with a strongly blueshifted C IV emission line ($\sim$2500 km s$^{-1}$ for APM 08279+5255). We compute the single-epoch black hole mass based on Mg II and H$\beta$ broad emission lines, finding $M_{\rm BH} = (2 \div 3) \times 10^{10}\mu^{-1}$ M$_\odot$, with the magnification factor $\mu$ that can vary between 4 and 100 according to CO and rest-frame UV-to-mid-IR imaging respectively. Using a Mg II equivalent width (EW)-to-Eddington ratio relation, the EW$_{\rm MgII} \sim 27$ \AA\ measured for APM 08279+5255 translates into an Eddington ratio of $\sim$0.4, which is more consistent with $\mu=4$. This magnification factor also provides a value of $M_{\rm BH}$ that is consistent with recent reverberation-mapping measurements derived from C IV and Si IV.Comment: 10 pages, 4 figures, 4 tables, accepted for publication in A&

Gamma Ray Bursts from the early Universe: predictions for present-day and future instruments

Long Gamma Ray Bursts (GRBs) constitute an important tool to study the Universe near and beyond the epoch of reionization. We delineate here the characteristics of an 'ideal' instrument for the search of GRBs at z>6-10. We find that the detection of these objects requires soft band detectors with a high sensitivity and moderately large FOV. In the light of these results, we compare available and planned GRB missions, deriving conservative predictions on the number of high-z GRBs detectable by these instruments along with the maximum accessible redshift. We show that the Swift satellite will be able to detect various GRBs at z>6, and likely at z>10 if the trigger threshold is decreased by a factor of ~2. Furthermore, we find that INTEGRAL and GLAST are not the best tool to detect bursts at z>6: the former being limited by the small FOV, and the latter by its hard energy band and relatively low sensitivity. Finally, future missions (SVOM, EDGE, but in particular EXIST) will provide a good sample of GRBs at z>6 in a few years of operation.Comment: 6 pages, 2 figures, MNRAS in pres

The assembly of "normal" galaxies at z=7 probed by ALMA

We report new deep ALMA observations aimed at investigating the [CII]158um line and continuum emission in three spectroscopically confirmed Lyman Break Galaxies at 6.8<z<7.1, i.e. well within the re-ionization epoch. With Star Formation Rates of SFR ~ 5-15 Msun/yr these systems are much more representative of the high-z galaxy population than other systems targeted in the past by millimeter observations. For the galaxy with the deepest observation we detect [CII] emission at redshift z=7.107, fully consistent with the Lyalpha redshift, but spatially offset by 0.7" (4 kpc) from the optical emission. At the location of the optical emission, tracing both the Lyalpha line and the far-UV continuum, no [CII] emission is detected in any of the three galaxies, with 3sigma upper limits significantly lower than the [CII] emission observed in lower reshift galaxies. These results suggest that molecular clouds in the central parts of primordial galaxies are rapidly disrupted by stellar feedback. As a result, [CII] emission mostly arises from more external accreting/satellite clumps of neutral gas. These findings are in agreement with recent models of galaxy formation. Thermal far-infrared continuum is not detected in any of the three galaxies. However, the upper limits on the infrared-to-UV emission ratio do not exceed those derived in metal- and dust-poor galaxies.Comment: 15 pages, 9 figures, MNRAS in press, replaced with accepted versio

Impressive long-term response with chemo-endocrine therapy in a premenopausal patient with metastatic breast cancer: A case report

RATIONALE: Patients with, or who develop, metastatic breast cancer have a 5-year relative survival of about 25%. Endocrine therapy clearly improves outcomes in patients with estrogen receptor-positive breast cancer. In the metastatic setting, the primary goal of treatment is to maintain long-term disease control with good quality of life. Rarely, exceptional responders achieve durable disease control, and potential cures cannot be ruled out. PATIENT CONCERNS: We report the case of a 39-year-old woman with primary breast cancer and associated synchronous bone metastases, who experienced a disease response of 12 years with hormonal therapy as maintenance after first line chemotherapy, with a good toxicity profile. DIAGNOSIS: The patient was diagnosed with estrogen receptor + human epidermal growth factor receptor 2 (HER2)- metastatic breast cancer with synchronous bone metastases. INTERVENTIONS: This patient was treated with chemotherapy for 6 cycles as a first-line therapy following by endocrine treatment given as a maintenance therapy. OUTCOMES: Our patient experienced a progression-free survival &gt;12 years with an exceptionally good quality of life. LESSONS: Our anecdotal experience highlights the existence of exceptional responders among patients with hormone receptor-positive metastatic breast cancer, who achieve clinical remission and durable disease control with endocrine therapy. Being able to identify these patients could help in the selection of the best treatment option among the many available

The Luminosity Function of Long Gamma-Ray Bursts and their rate at z>6

We compute the luminosity function (LF) and the formation rate of long gamma ray bursts (GRBs) in three different scenarios: i) GRBs follow the cosmic star formation and their LF is constant in time; ii) GRBs follow the cosmic star formation but the LF varies with redshift; iii) GRBs form preferentially in low-metallicity environments. We then test model predictions against the Swift 3-year data, showing that scenario i) is robustly ruled out. Moreover, we show that the number of bright GRBs detected by Swift suggests that GRBs should have experienced some sort of luminosity evolution with redshift, being more luminous in the past. Finally we propose to use the observations of the afterglow spectrum of GRBs at z>5.5 to constrain the reionization history and we applied our method to the case of GRB 050904.Comment: 5 pages, 5 figures, Conference Proceeding for IAU Symposium 255: Low-Metallicity Star Formaion: From the First Stars to Dwarf Galaxie

Case Report: Analysis of Circulating Tumor Cells in a Triple Negative Spindle-Cell Metaplastic Breast Cancer Patient

Circulating tumor cells (CTCs) are a rare population of cells found in the bloodstream and represent key players in the metastatic cascade. Their analysis has proved to provide further core information concerning the tumor. Herein, we aim at investigating CTCs isolated from a 32-year-old patient diagnosed with triple negative spindle-shaped metaplastic breast cancer (MpBC), a rare tumor poorly responsive to therapies and with a dismal prognosis. The molecular analysis performed on the primary tumor failed to underline effective actionable targets to address the therapeutic strategy. Besides the presence of round-shaped CTCs, cells with a spindle shape were present as well, and through molecular analysis, we confirmed their malignant nature. This aspect was coherent with the primary tumor histology, proving that CTCs are released regardless of their morphology. Copy number aberration (CNA) profiling and variant analysis using next-generation sequencing (NGS) showed that these cells did not harbor the alterations exhibited by the primary tumor (PIK3CA G1049A mutation, MYC copy number gain). However, despite the great heterogeneity observed, the amplification of regions involved in metastasis emerged (8q24.22–8q24.23). Our findings support the investigation of CTCs to identify alterations that could have a role in the metastatic process. To the best of our knowledge, this is the first examination of CTCs in an MpBC patient

Single-cell NGS-based analysis of copy number alterations reveals new insights in circulating tumor cells persistence in early-stage breast cancer

Circulating tumor cells (CTCs) are a rare population of cells representing a key player in the metastatic cascade. They are recognized as a validated tool for the identification of patients with a higher risk of relapse, including those diagnosed with breast cancer (BC). However, CTCs are characterized by high levels of heterogeneity that also involve copy number alterations (CNAs), structural variations associated with gene dosage changes. In this study, single CTCs were isolated from the peripheral blood of 11 early-stage BC patients at different time points. A label-free enrichment of CTCs was performed using OncoQuick, and single CTCs were isolated using DEPArray. Libraries were prepared from single CTCs and DNA extracted from matched tumor tissues for a whole-genome low-coverage next-generation sequencing (NGS) analysis using the Ion Torrent S5 System. The analysis of the CNA burden highlighted that CTCs had different degrees of aberration based on the time point and subtype. CTCs were found even six months after surgery and shared CNAs with matched tumor tissue. Tumor-associated CNAs that were recurrent in CTCs were patient-specific, and some alterations involved regions associated with BC and survival (i.e., gains at 1q21-23 and 5p15.33). The enrichment analysis emphasized the involvement of aberrations of terms, associated in particular with interferon (IFN) signaling. Collectively, our findings reveal that these aberrations may contribute to understanding the molecular mechanisms involving CTC-related processes and their survival ability in occult niches, supporting the goal of exploiting their application in patients’ surveillance and follow-up