Using picoliter droplet deposition to track clonal competition in adherent and organoid cancer cell cultures

Abstract Clonal growth and competition underlie processes of key relevance in etiology, progression and therapy response across all cancers. Here, we demonstrate a novel experimental approach, based on multi-color, fluorescent tagging of cell nuclei, in combination with picoliter droplet deposition, to study the clonal dynamics in two- and three-dimensional cell cultures. The method allows for the simultaneous visualization and analysis of multiple clones in individual multi-clonal colonies, providing a powerful tool for studying clonal dynamics and identifying clonal populations with distinct characteristics. Results of our experiments validate the utility of the method in studying clonal dynamics in vitro, and reveal differences in key aspects of clonal behavior of different cancer cell lines in monoculture conditions, as well as in co-cultures with stromal fibroblasts

Multimodal profiling of chordoma immunity reveals distinct immune contextures

Background Chordomas are rare cancers from the axial skeleton which present a challenging clinical management with limited treatment options due to their anatomical location. In recent years, a few clinical trials demonstrated that chordomas can respond to immunotherapy. However, an in-depth portrayal of chordoma immunity and its association with clinical parameters is still lacking.Methods We present a comprehensive characterization of immunological features of 76 chordomas through application of a multimodal approach. Transcriptomic profiling of 20 chordomas was performed to inform on the activity of immune-related genes through the immunologic constant of rejection (ICR) signature. Multidimensional immunophenotyping through imaging mass cytometry was applied to provide insights in the different immune contextures of 32 chordomas. T cell infiltration was further evaluated in all 76 patients by means of multispectral immunofluorescence and then associated with clinical parameters through univariate and multivariate Cox proportional hazard models as well as Kaplan-Meier estimates. Moreover, distinct expression patterns of human leukocyte antigen (HLA) class I were assessed by immunohistochemical staining in all 76 patients. Finally, clonal enrichment of the T cell receptor (TCR) was sought through profiling of the variable region of TCRB locus of 24 patients.Results Chordomas generally presented an immune “hot” microenvironment in comparison to other sarcomas, as indicated by the ICR transcriptional signature. We identified two distinct groups of chordomas based on T cell infiltration which were independent from clinical parameters. The highly infiltrated group was further characterized by high dendritic cell infiltration and the presence of multicellular immune aggregates in tumors, whereas low T cell infiltration was associated with lower overall cell densities of immune and stromal cells. Interestingly, patients with higher T cell infiltration displayed a more pronounced clonal enrichment of the TCR repertoire compared with those with low T cell counts. Furthermore, we observed that the majority of chordomas maintained HLA class I expression.Conclusion Our findings shed light on the natural immunity against chordomas through the identification of distinct immune contextures. Understanding their immune landscape could guide the development and application of immunotherapies in a tailored manner, ultimately leading to an improved clinical outcome for patients with chordoma

Multifocality in Sporadic Medullary Thyroid Carcinoma:An International Multicenter Study

Background: Current surgical standard of care in sporadic medullary thyroid carcinoma (sMTC) consists of a minimum of total thyroidectomy with central neck dissection. Some have suggested thyroid lobectomy with isthmusectomy and central neck dissection for patients with sMTC, given their lower frequency of bilateral disease, although this topic has not been thoroughly studied. This study assessed the prevalence of multifocality in sMTC via a large international multi-institutional retrospective review to quantify this prevalence, including the impact of geography, to assess more accurately the risks associated with alternative surgical approaches. Methods: A retrospective chart review of sMTC patients from 11 institutions over 29 years (1983-2011) was undertaken. Data regarding focality, extent of disease, RET germline analysis plus family and clinical history for multiple endocrine neoplasia type 2 (MEN2), and demographic data were collected and analyzed. Results: Patients from four continents and seven countries were included in the sample. Data for 313 patients with documented sMTC were collected. Of these, 81.2% were confirmed with negative RET germline testing, while the remaining 18.8% demonstrated a negative family history and nomanifestations ofMEN2 syndromes other thanMTC. Bilateral disease was identified in 17/306 (5.6%) patients, while multifocal disease was noted in 50/312 (16.0%) sMTC patients. When only accounting for germline negative patients, these rates were not significantly different (5.6% and 17%, respectively). Among them, when disease was unifocal in the ipsilateral lobe and isthmus, bilateral disease was present in 6/212 (2.8%) cases. When disease was multifocal in the ipsilateral lobe or isthmus, then bilateral disease was present in 8/37 (21.6%) cases (p < 0.001). No geographic differences in focality were identified. Conclusions: The 5.6% prevalence of bilateral foci in sMTC suggests that total thyroidectomy should remain the standard of care for initial surgery, as less complete thyroid surgery may fail to address fully the primary site of disease. Whether ipsilateral tumor focality should be an independent factor determining the need for completion thyroidectomy when sMTC is diagnosed after hemithyroidectomy remains to be determined.National Cancer Institute (NCI) National Institutes of Health (NIH) Health and Human Services (HHS)Medical Research CouncilOhio State Univ, Wexner Med Ctr, Dept Otolaryngology Head & Neck Surg, Columbus, OH 43210 USAOhio State Univ, Wexner Med Ctr, Ctr Biostat, Columbus, OH 43210 USAUniv Wisconsin, Sch Med & Publ Hlth, Sect Endocrine Surg, Madison, WI USAUniv Naples Federico II, Dept Clin Med & Surg, Naples, ItalyUniv Fed Sao Paulo, Dept Med, Lab Mol & Translat Endocrinol, Div Endocrinol, Sao Paulo, BrazilUniv Siena, Sect Endocrinol & Metab, Dept Med Surg & Neurol Sci, Siena, ItalyMonash Univ, Alfred Hlth, Dept Endocrinol & Diabet, Melbourne, Vic, AustraliaPierre Oudot Hosp, Dept Endocrinol & Nephrol, Bourgoin Jallieu, FranceUniv Birmingham, Sch Clin & Expt Med, Ctr Endocrinol Diabet & Metab, Inst Biomed Res, Birmingham, W Midlands, EnglandUniv Birmingham, Inst Head & Neck Studies & Educ, Birmingham, W Midlands, EnglandRadboud Univ Nijmegen, Med Ctr, Dept Internal Med, Nijmegen, NetherlandsAlbert Schweitzer Hosp, Dept Internal Med, Dordrecht, NetherlandsUniv Athens, Evgenid Hosp, Thyroid Sect, Unit Endocrinol Diabet & Metab, Athens, GreeceInst Jules Bordet, Dept Med, Brussels, BelgiumUniv Roma La Sapienza, Dipartimento Med Interna, Rome, ItalyVeracyte Inc, 6000 Shoreline Court,Suite 300, San Francisco, CA 94080 USAOhio State Univ, Wexner Med Ctr, Div Endocrinol Diabet & Metab, Columbus, OH 43210 USAOhio State Univ, Wexner Med Ctr, Div Nucl Med, Columbus, OH 43210 USADivision of Endocrinology, Laboratory of Molecular and Translational Endocrinology, Department of Medicine, Universidade Federal de São Paulo (UNIFESP), São Paulo, BrazilNCI NIH HHS: P50 CA168505Medical Research Council: MR/J001414/1Medical Research Council: G0601811Web of Scienc

Double hits in schizophrenia.

The co-occurrence of a copy number variant (CNV) and a functional variant on the other allele may be a relevant genetic mechanism in schizophrenia. We hypothesized that the cumulative burden of such double hits-in particular those composed of a deletion and a coding single-nucleotide variation (SNV)-is increased in patients with schizophrenia. We combined CNV data with coding variants data in 795 patients with schizophrenia and 474 controls. To limit false CNV-detection, only CNVs called by two algorithms were included. CNV-affected genes were subsequently examined for coding SNVs, which we termed "CNV-SNVs." Correcting for total queried sequence, we assessed the CNV-SNV-burden and the combined predicted deleterious effect. We estimated P-values by permutation of the phenotype. We detected 105 CNV-SNVs; 67 in duplicated and 38 in deleted genic sequence. Although the difference in CNV-SNVs rates was not significant, the combined deleteriousness inferred by CNV-SNVs in deleted sequence was almost 4-fold higher in cases compared with controls (nominal P = 0.009). This effect may be driven by a higher number of CNV-SNVs and/or by a higher degree of predicted deleteriousness of CNV-SNVs. No such effect was observed for duplications. We provide early evidence that deletions co-occurring with a functional variant may be relevant, albeit of modest impact, for the genetic etiology of schizophrenia. Large-scale consortium studies are required to validate our findings. Sequence-based analyses would provide the best resolution for detection of CNVs as well as coding variants genome-wide

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field

Processing of nanostructured polymers and advanced polymeric based nanocomposites

The control of the nanostructure and the addition of nanoparticles to polymers have led to structural and functional property enhancements in a number of polymeric systems as a material answer to continuous requirements from advanced industrial sectors. The availability of new nanoparticles with extraordinary properties (i.e. carbon nanotubes, graphenes, but also nanoclays, nanocellulose, metals and ceramics) have determined new and exciting possibilities for a continuous enlargement of polymer markets. However, the potentialities of these new materials are still strongly dependent on the development and scaling-up of reliable processing routes. Therefore, the purpose of this report is to review the main processing approaches for nanostructured polymers and nanocomposites starting with a brief review of available nanoparticles and on their functionalization to promote a better polymer-particle interaction. Regarding processing, the review firstly addresses the bottom-up approaches typically adopted for nanostructured polymers, blends and copolymers. Then, the different technologies required by the top-down processing of thermoplastic and thermosetting polymer matrix systems are reviewed. Finally, the report addresses the recent applications of nanostructured polymers and nanocomposites as matrices of advanced composite materials. In all cases, the main processing approaches and the main structural and functional properties characterizing these materials and their potential and current industrial applications are specifically addressed.We are indebted to the Spanish Ministry of Economy and Competitiveness (MINECO) for their economic support of this research (MAT2013-48059-C2-1-R). LP acknowledges the support of a JAEDoc grant from CSIC cofinanced by FSE. JMK acknowledges the support of the Russian Ministry of Education and Science within State Contract No. 14.Z50.31.0002.Peer Reviewe