Single Cell Profiling of Circulating Tumor Cells: Transcriptional Heterogeneity and Diversity from Breast Cancer Cell Lines

BACKGROUND: To improve cancer therapy, it is critical to target metastasizing cells. Circulating tumor cells (CTCs) are rare cells found in the blood of patients with solid tumors and may play a key role in cancer dissemination. Uncovering CTC phenotypes offers a potential avenue to inform treatment. However, CTC transcriptional profiling is limited by leukocyte contamination; an approach to surmount this problem is single cell analysis. Here we demonstrate feasibility of performing high dimensional single CTC profiling, providing early insight into CTC heterogeneity and allowing comparisons to breast cancer cell lines widely used for drug discovery. METHODOLOGY/PRINCIPAL FINDINGS: We purified CTCs using the MagSweeper, an immunomagnetic enrichment device that isolates live tumor cells from unfractionated blood. CTCs that met stringent criteria for further analysis were obtained from 70% (14/20) of primary and 70% (21/30) of metastatic breast cancer patients; none were captured from patients with non-epithelial cancer (n = 20) or healthy subjects (n = 25). Microfluidic-based single cell transcriptional profiling of 87 cancer-associated and reference genes showed heterogeneity among individual CTCs, separating them into two major subgroups, based on 31 highly expressed genes. In contrast, single cells from seven breast cancer cell lines were tightly clustered together by sample ID and ER status. CTC profiles were distinct from those of cancer cell lines, questioning the suitability of such lines for drug discovery efforts for late stage cancer therapy. CONCLUSIONS/SIGNIFICANCE: For the first time, we directly measured high dimensional gene expression in individual CTCs without the common practice of pooling such cells. Elevated transcript levels of genes associated with metastasis NPTN, S100A4, S100A9, and with epithelial mesenchymal transition: VIM, TGFß1, ZEB2, FOXC1, CXCR4, were striking compared to cell lines. Our findings demonstrate that profiling CTCs on a cell-by-cell basis is possible and may facilitate the application of 'liquid biopsies' to better model drug discovery

Interconnected Microphysiological Systems for Quantitative Biology and Pharmacology Studies

Microphysiological systems (MPSs) are in vitro models that capture facets of in vivo organ function through use of specialized culture microenvironments, including 3D matrices and microperfusion. Here, we report an approach to co-culture multiple different MPSs linked together physiologically on re-useable, open-system microfluidic platforms that are compatible with the quantitative study of a range of compounds, including lipophilic drugs. We describe three different platform designs - "4-way", "7-way", and "10-way" - each accommodating a mixing chamber and up to 4, 7, or 10 MPSs. Platforms accommodate multiple different MPS flow configurations, each with internal re-circulation to enhance molecular exchange, and feature on-board pneumatically-driven pumps with independently programmable flow rates to provide precise control over both intra- and inter-MPS flow partitioning and drug distribution. We first developed a 4-MPS system, showing accurate prediction of secreted liver protein distribution and 2-week maintenance of phenotypic markers. We then developed 7-MPS and 10-MPS platforms, demonstrating reliable, robust operation and maintenance of MPS phenotypic function for 3 weeks (7-way) and 4 weeks (10-way) of continuous interaction, as well as PK analysis of diclofenac metabolism. This study illustrates several generalizable design and operational principles for implementing multi-MPS "physiome-on-a-chip" approaches in drug discovery.United States. Army Research Office (Grant W911NF-12-2-0039

Validating AU Microscopii d with Transit Timing Variations

AU Mic is a young (22 Myr) nearby exoplanetary system that exhibits excess TTVs that cannot be accounted for by the two known transiting planets nor stellar activity. We present the statistical "validation" of the tentative planet AU Mic d (even though there are examples of "confirmed" planets with ambiguous orbital periods). We add 18 new transits and nine midpoint times in an updated TTV analysis to prior work. We perform the joint modeling of transit light curves using EXOFASTv2 and extract the transit midpoint times. Next, we construct an O-C diagram and use Exo-Striker to model the TTVs. We generate TTV log-likelihood periodograms to explore possible solutions for the period of planet d and then follow those up with detailed TTV and RV MCMC modeling and stability tests. We find several candidate periods for AU Mic d, all of which are near resonances with AU Mic b and c of varying order. Based on our model comparisons, the most-favored orbital period of AU Mic d is 12.73596+/-0.00793 days (T_{C,d}=2458340.55781+/-0.11641 BJD), which puts the three planets near a 4:6:9 mean-motion orbital resonance. The mass for d is 1.053+/-0.511 M_E, making this planet Earth-like in mass. If confirmed, AU Mic d would be the first known Earth-mass planet orbiting a young star and would provide a valuable opportunity in probing a young terrestrial planet's atmosphere. Additional TTV observation of the AU Mic system are needed to further constrain the planetary masses, search for possible transits of AU Mic d, and detect possible additional planets beyond AU Mic c.Comment: 89 pages, 35 figures, 34 tables. Redid EXOFASTv2 transit modeling to recover more reasonable stellar posteriors, so redid Exo-Striker TTV modeling for consistency. Despite these changes, the overall results remain unchanged: the 12-7-day case is still the most favored. Submitted to AAS Journals on 2023 Feb 9t

Validating AU Microscopii d with Transit Timing Variations

AU Mic is a young (22 Myr), nearby exoplanetary system that exhibits excess transit timing variations (TTVs) that cannot be accounted for by the two known transiting planets nor stellar activity. We present the statistical “validation” of the tentative planet AU Mic d (even though there are examples of “confirmed” planets with ambiguous orbital periods). We add 18 new transits and nine midpoint times in an updated TTV analysis to prior work. We perform the joint modeling of transit light curves using EXOFASTv2 and extract the transit midpoint times. Next, we construct an O − C diagram and use Exo-Striker to model the TTVs. We generate TTV log-likelihood periodograms to explore possible solutions for d’s period, then follow those up with detailed TTV and radial velocity Markov Chain Monte Carlo modeling and stability tests. We find several candidate periods for AU Mic d, all of which are near resonances with AU Mic b and c of varying order. Based on our model comparisons, the most-favored orbital period of AU Mic d is 12.73596 ± 0.00793 days ( T _C _,d = 2458340.55781 ± 0.11641 BJD), which puts the three planets near 4:6:9 mean-motion resonance. The mass for d is 1.053 ± 0.511 M _⊕ , making this planet Earth-like in mass. If confirmed, AU Mic d would be the first known Earth-mass planet orbiting a young star and would provide a valuable opportunity in probing a young terrestrial planet’s atmosphere. Additional TTV observations of the AU Mic system are needed to further constrain the planetary masses, search for possible transits of AU Mic d, and detect possible additional planets beyond AU Mic c

Haze in Pluto's atmosphere: Results from SOFIA and ground-based observations of the 2015 June 29 Pluto occultation

On UT 29 June 2015, the occultation by Pluto of a bright star (r′ = 11.9) was observed from the Stratospheric Observatory for Infrared Astronomy (SOFIA) and several ground-based stations in New Zealand and Australia. Pre-event astrometry allowed for an in-flight update to the SOFIA team with the result that SOFIA was deep within the central flash zone (~22 km from center). Analysis of the combined data leads to the result that Pluto's middle atmosphere is essentially unchanged from 2011 and 2013 (Person et al. 2013; Bosh et al. 2015); there has been no significant expansion or contraction of the atmosphere. Additionally, our multi-wavelength observations allow us to conclude that a haze component in the atmosphere is required to reproduce the light curves obtained. This haze scenario has implications for understanding the photochemistry of Pluto's atmosphere

The LHS 1678 system : two earth-sized transiting planets and an astrometric companion orbiting an M dwarf near the convective boundary at 20 pc

Funding: The MEarth Team gratefully acknowledges funding from the David and Lucile Packard Fellowship for Science and Engineering (awarded to D.C.). This material is based upon work supported by the National Science Foundation under grants AST-0807690, AST-1109468, AST-1004488 (Alan T. Waterman Award), and AST-1616624, and upon work supported by the National Aeronautics and Space Administration under Grant No. 80NSSC18K0476 issued through the XRP Program. This work is made possible by a grant from the John Templeton Foundation. N. A.-D. acknowledges the support of FONDECYT project 3180063. TD acknowledges support from MIT’s Kavli Institute as a Kavli postdoctoral fellow. KH acknowledges support from STFC grant ST/R000824/1. E.A.G. thanks the LSSTC Data Science Fellowship Program, which is funded by LSSTC, NSF Cybertraining Grant #1829740, the Brinson Foundation, and the Moore Foundation; The material is based upon work supported by NASA under award number 80GSFC21M0002. This work was supported by the lead author’s appointment to the NASA Postdoctoral Program at the Goddard Space Flight Center, administered by Universities Space Research Association under contract with NASAWe present the Transiting Exoplanet Survey Satellite (TESS) discovery of the LHS 1678 (TOI-696) exoplanet system, comprised of two approximately Earth-sized transiting planets and a likely astrometric brown dwarf orbiting a bright (VJ = 12.5, Ks = 8.3) M2 dwarf at 19.9 pc. The two TESS-detected planets are of radius 0.70 ± 0.04 R⊕ and 0.98 ± 0.06 R⊕ in 0.86 day and 3.69 day orbits, respectively. Both planets are validated and characterized via ground-based follow-up observations. High Accuracy Radial Velocity Planet Searcher RV monitoring yields 97.7 percentile mass upper limits of 0.35 M⊕ and 1.4 M⊕ for planets b and c, respectively. The astrometric companion detected by the Cerro Tololo Inter-American Observatory/Small and Moderate Aperture Telescope System 0.9 m has an orbital period on the order of decades and is undetected by other means. Additional ground-based observations constrain the companion to being a high-mass brown dwarf or smaller. Each planet is of unique interest; the inner planet has an ultra-short period, and the outer planet is in the Venus zone. Both are promising targets for atmospheric characterization with the James Webb Space Telescope and mass measurements via extreme-precision radial velocity. A third planet candidate of radius 0.9 ± 0.1 R⊕ in a 4.97 day orbit is also identified in multicycle TESS data for validation in future work. The host star is associated with an observed gap in the lower main sequence of the Hertzsprung–Russell diagram. This gap is tied to the transition from partially to fully convective interiors in M dwarfs, and the effect of the associated stellar astrophysics on exoplanet evolution is currently unknown. The culmination of these system properties makes LHS 1678 a unique, compelling playground for comparative exoplanet science and understanding the formation and evolution of small, short-period exoplanets orbiting low-mass stars.Publisher PDFPeer reviewe

RNA-Seq of untreated wastewater to assess COVID-19 and emerging and endemic viruses for public health surveillance

BackgroundThe COVID-19 pandemic showcased the power of genomic sequencing to tackle the emergence and spread of infectious diseases. However, metagenomic sequencing of total microbial RNAs in wastewater has the potential to assess multiple infectious diseases simultaneously and has yet to be explored.MethodsA retrospective RNA-Seq epidemiological survey of 140 untreated composite wastewater samples was performed across urban (n = 112) and rural (n = 28) areas of Nagpur, Central India. Composite wastewater samples were prepared by pooling 422 individual grab samples collected prospectively from sewer lines of urban municipality zones and open drains of rural areas from 3rd February to 3rd April 2021, during the second COVID-19 wave in India. Samples were pre-processed and total RNA was extracted prior to genomic sequencing.FindingsThis is the first study that has utilised culture and/or probe-independent unbiased RNA-Seq to examine Indian wastewater samples. Our findings reveal the detection of zoonotic viruses including chikungunya, Jingmen tick and rabies viruses, which have not previously been reported in wastewater. SARS-CoV-2 was detectable in 83 locations (59%), with stark abundance variations observed between sampling sites. Hepatitis C virus was the most frequently detected infectious virus, identified in 113 locations and co-occurring 77 times with SARS-CoV-2; and both were more abundantly detected in rural areas than urban zones. Concurrent identification of segmented virus genomic fragments of influenza A virus, norovirus, and rotavirus was observed. Geographical differences were also observed for astrovirus, saffold virus, husavirus, and aichi virus that were more prevalent in urban samples, while the zoonotic viruses chikungunya and rabies, were more abundant in rural environments.InterpretationRNA-Seq can effectively detect multiple infectious diseases simultaneously, facilitating geographical and epidemiological surveys of endemic viruses that could help direct healthcare interventions against emergent and pre-existent infectious diseases as well as cost-effectively and qualitatively characterising the health status of the population over time

The Magellan-TESS Survey I: Survey Description and Mid-Survey Results

One of the most significant revelations from Kepler is that roughly one-third of Sun-like stars host planets which orbit their stars within 100 days and are between the size of Earth and Neptune. How do these super-Earth and sub-Neptune planets form, what are they made of, and do they represent a continuous population or naturally divide into separate groups? Measuring their masses and thus bulk densities can help address these questions of their origin and composition. To that end, we began the Magellan-TESS Survey (MTS), which uses Magellan II/PFS to obtain radial velocity (RV) masses of 30 transiting exoplanets discovered by TESS and develops an analysis framework that connects observed planet distributions to underlying populations. In the past, RV measurements of small planets have been challenging to obtain due to the faintness and low RV semi-amplitudes of most Kepler systems, and challenging to interpret due to the potential biases in the existing ensemble of small planet masses from non-algorithmic decisions for target selection and observation plans. The MTS attempts to minimize these biases by focusing on bright TESS targets and employing a quantitative selection function and multi-year observing strategy. In this paper, we (1) describe the motivation and survey strategy behind the MTS, (2) present our first catalog of planet mass and density constraints for 25 TESS Objects of Interest (TOIs; 20 in our population analysis sample, five that are members of the same systems), and (3) employ a hierarchical Bayesian model to produce preliminary constraints on the mass-radius (M-R) relation. We find qualitative agreement with prior mass-radius relations but some quantitative differences (abridged). The the results of this work can inform more detailed studies of individual systems and offer a framework that can be applied to future RV surveys with the goal of population inferences.Comment: 101 pages (39 of main text and references, the rest an appendix of figures and tables). Submitted to AAS Journal