Background: MM and AL are the two most common malignant monoclonal gammopathies. Both diseases result from the accumulation of clonal PCs, but their clinical behavior is significantly different ...suggesting fundamental differences in disease biology. Previous attempts to identify genetic hallmarks that could explain such differences have been unsuccessful. Furthermore, it is unknown if MM and AL arise from the same or different normal PC counterparts.
Aim: To define a transcriptional atlas of the normal PC development in peripheral blood (PB) and bone marrow (BM) for comparison with the transcriptional programs of clonal PCs in MM and AL.
Methods: A total of 93 subjects were studied. In 7 healthy adults (HA), PB PCs were phenotypically sorted according to heavy-chain isotypes (IgG, IgA and IgM). In addition, 5 different BM PCs subsets were isolated based on the differential expression of CD19, CD39, CD81 and CD56, due to their ascribed role in dissecting unique BM PC differentiation states. Clonal PCs from patients with MM (n=38) and AL (n=41) were isolated by FACS according to patient-specific aberrant phenotypes. Due to small numbers of PCs sorted from each subset in HA and clonal PCs in AL patients, we used an RNAseq method optimized for limited cell numbers. Differential expression across all pairwise comparisons between groups was analyzed with Deseq2 R package followed by k-means clustering of genes in R. Single-cell RNAseq (scRNAseq, 10xGenomics) was performed in a total of 35,910 PCs from 3 HA, 2 MM and 2 AL. We used Seurat R package to remove batch effect followed by canonical correlation to perform an integrated analysis of all single PCs from HA, MM and AL subjects.
Results: Principal component analysis of RNAseq data unveiled two major clusters of normal PCs: those in PB and those in BM (with some transcriptional diversity between CD19+ and CD19- PCs), whereas the CD19+CD39+CD81+CD56- BM subset co-localized with PB and CD39- BM PCs (Panel A). Clonal PCs from MM and AL patients clustered together, and both displayed some transcriptional variance related to the spatial location of normal PCs (i.e. PB or BM). In total, 2174 genes were found significantly deregulated after cross-comparing the 10 PC groups (adj.p-value<0.01, logFC>1) and semi-supervised k-means clustering unveiled 8 transcriptional modules (Panel B). Namely, the transition from PB into BM PCs was characterized by genes related to proliferation (clusters 1 & 2), whereas CD39+ and CD39- BM PC subsets differed on the expression of genes associated with proliferation, homing, and metabolism (1, 2, 4 & 6). Thus, CD19+CD39+CD81+CD56- BM PCs emerged as a novel subset that bridges new-born PB with long-lived (CD39-) BM PCs. Interestingly, clonal PCs from MM and AL shared transcriptional programs related to quiescence (5 & 6) with long-lived BM PCs; however, skewing of polyclonal immunoglobulin gene expression (3) and active gene transcription (8) emerged as hallmarks of the neoplastic transformation from normal, long-lived PCs into clonal PCs. That notwithstanding, the later displayed expression levels of the proliferation and homing transcriptional modules (1 & 4) similar to new-born PB and CD39+ BM PCs. Of note, a small transcriptional cluster of genes related to ribosome biogenesis (7) was significantly more expressed in MM than AL. These findings led us to integrate scRNAseq profiles of normal and clonal BM PCs from MM and AL patients, to define PC clusters based on their transcriptional program rather than their normal vs malignant status (Panel C). This strategy unveiled 11 different PC clusters with unequal distribution between groups. Thus, more than half of clonal PCs in MM and AL were assigned to a cluster that is also predominant in normal PCs (1). By contrast, other clusters with a transcriptional program similar to that of new-born PCs (2 & 5) became rarer in MM and AL. Furthermore, a cluster of PCs with an immature-like phenotype (6) was detectable in MM but almost absent in AL.
Conclusions: This is the first integrated analysis of the transcriptional programs of normal PC subsets and clonal PCs in MM and AL, both at the bulk and single-cell levels. Our results unveil shared and exclusive transcriptional states in normal and clonal PCs, together with unique differences between clonal PCs in MM and AL. Thus, we provide here a fundamental resource to understand normal PC development and the cellular origin of both malignant monoclonal gammopathies.
Display omitted
Puig:Takeda: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Research Funding; Celgene: Honoraria, Research Funding. Ocio:Pharmamar: Consultancy; AbbVie: Consultancy; Janssen: Consultancy, Honoraria; Seattle Genetics: Consultancy; BMS: Consultancy; Takeda: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Sanofi: Research Funding; Amgen: Consultancy, Honoraria, Research Funding; Mundipharma: Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Array Pharmaceuticals: Research Funding. Oriol:Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Martinez Lopez:Bristol Myers Squibb: Research Funding, Speakers Bureau; Janssen: Research Funding, Speakers Bureau; Novartis: Research Funding, Speakers Bureau; Celgene: Research Funding, Speakers Bureau. Mateos:Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; GSK: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; GSK: Consultancy, Membership on an entity's Board of Directors or advisory committees; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees. Lahuerta:Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees. San-Miguel:Sanofi: Consultancy; Takeda: Consultancy; Novartis: Consultancy; MSD: Consultancy; Janssen: Consultancy; Celgene: Consultancy; Brystol-Myers Squibb: Consultancy; Amgen: Consultancy; Roche: Membership on an entity's Board of Directors or advisory committees.
Abstract
Checkpoint blockade therapies that aim to reactivate anti-tumor immune responses have revolutionized cancer treatment, resulting in durable responses in a significant proportion of patients ...with advanced disease. Nevertheless, many patients fail to reach long-term clinical benefit, and therefore, a better insight into the mechanisms underlying response to immunotherapy is required to enhance its success rates. Recent findings suggest that immune cell infiltrates in cancer can be highly heterogeneous, potentially contributing to differential therapy outcomes. However, immune cell states have thus far been studied mainly in patients with end-stage melanoma and diverse treatment histories, whereas patients with early-stage metastatic disease are generally treatment naïve, allowing assessment of naturally evolved T cell functionality and immune cell composition. To characterize the heterogeneity in the tumor immune environment within and between patients, we profiled the tumor microenvironment of six stage III melanoma patients by single-cell RNA sequencing, generating an unbiased map of the expression signatures of immune cells as well as tumor and stromal cells. In parallel, we also derived the T cell receptor sequences for single T cells, and used them to determine intra- and inter-tumoral T cell clonality and infer the functionality of clonally expanded T cell populations in the tumor. A series of different cell types and cellular states that have been described previously in end-stage melanoma was also observed in this early-stage metastatic disease, including cytotoxic, exhausted, and regulatory T cells, as well as various myeloid subsets. Notably, despite identical disease stage and treatment background, the composition of the immune cell populations differed considerably between patients. In addition, while analysis of T cell states showed high variability between and within patients, clonally expanded T cells identified within patients predominantly adapted similar cell profiles. Currently we are assessing whether the occurrence of distinct T cell states can be coupled to other aspects of the immune infiltrate. In addition to describing T cell heterogeneity, our data suggest the existence of novel immune cell subsets within the tumor microenvironment. Our findings demonstrate that, using our single-cell RNA-sequencing approach, we can determine the immunological make-up in melanoma during early stages of metastatic disease, in an unbiased manner. Collectively, our approach should be helpful in determining the mechanisms underlying the development and effectiveness of tumor-specific immune responses in metastatic melanoma.
Citation Format: Ido Yofe, Hanjie Li, Anne van der Leun, Lubling Yaniv, Assaf Weiner, Alexander van Akkooi, Amos Tanay, Ton Schumacher, Ido Amit. Dissecting immune cell heterogeneity in human cancer by single-cell RNA-sequencing abstract. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr B54.
Immunogenetics. Chromatin state dynamics during blood formation Lara-Astiaso, David; Weiner, Assaf; Lorenzo-Vivas, Erika ...
Science (American Association for the Advancement of Science),
2014-Aug-22, 20140822, Letnik:
345, Številka:
6199
Journal Article
Recenzirano
Chromatin modifications are crucial for development, yet little is known about their dynamics during differentiation. Hematopoiesis provides a well-defined model to study chromatin state dynamics; ...however, technical limitations impede profiling of homogeneous differentiation intermediates. We developed a high-sensitivity indexing-first chromatin immunoprecipitation approach to profile the dynamics of four chromatin modifications across 16 stages of hematopoietic differentiation. We identify 48,415 enhancer regions and characterize their dynamics. We find that lineage commitment involves de novo establishment of 17,035 lineage-specific enhancers. These enhancer repertoire expansions foreshadow transcriptional programs in differentiated cells. Combining our enhancer catalog with gene expression profiles, we elucidate the transcription factor network controlling chromatin dynamics and lineage specification in hematopoiesis. Together, our results provide a comprehensive model of chromatin dynamics during development.
Minerals are formed by organisms in all of the kingdoms of life. Mineral formation pathways all involve uptake of ions from the environment, transport of ions by cells, sometimes temporary storage, ...and ultimately deposition in or outside of the cells. Even though the details of how all this is achieved vary enormously, all pathways need to respect both the chemical limitations of ion manipulation, as well as the many “housekeeping” roles of ions in cell functioning. Here we provide a chemical perspective on the biological pathways of biomineralization. Our approach is to compare and contrast the ion pathways involving calcium, phosphate, and carbonate in three very different organisms: the enormously abundant unicellular marine coccolithophores, the well investigated sea urchin larval model for single crystal formation, and the complex pathways used by vertebrates to form their bones. The comparison highlights both common and unique processes. Significantly, phosphate is involved in regulating calcium carbonate deposition and carbonate is involved in regulating calcium phosphate deposition. One often overlooked commonality is that, from uptake to deposition, the solutions involved are usually supersaturated. This therefore requires not only avoiding mineral deposition where it is not needed but also exploiting this saturated state to produce unstable mineral precursors that can be conveniently stored, redissolved, and manipulated into diverse shapes and upon deposition transformed into more ordered and hence often functional final deposits.
Many organisms form crystals from transient amorphous precursor phases. In cases where the precursor phases were imaged, they were seen to consist of nanosphere particles. Interestingly, some mature ...biogenic crystals also have a nanosphere particle morphology, but some are characterized by crystallographic faces that are smooth at the nanometer level. There are also biogenic crystals that have both crystallographic faces and nanosphere particle morphology. This highlight presents a working hypothesis, stating that some biomineralization processes involve growth by nanosphere particle accretion, where amorphous nanoparticles are incorporated as such into growing crystals and their morphology is preserved upon crystallization. This process produces biogenic crystals with a nanosphere particle morphology. Other biomineralization processes proceed by ion-by-ion growth, and some cases of biological crystal growth involve both processes. We also identify several biomineralization processes which do not seem to fit this working hypothesis. It is our hope that this highlight will inspire studies that will shed more light on the underlying crystallization mechanisms in biology.
A working hypothesis for the understanding of amorphous-to-crystalline transformations in biogenic skeletal materials formed through transient amorphous precursor phases.
Background Schizophrenia is a neuropsychiatric disorder of a neurodevelopmental origin manifested symptomatically after puberty. Structural neuroimaging studies show that neuroanatomical aberrations ...occur before onset of symptoms, raising a question of whether schizophrenia can be prevented. Treatment with atypical antipsychotic drugs before the development of the full clinical phenotype might reduce the risk of transition to psychosis, but it remains unknown whether neuroanatomical abnormalities can be prevented. We used a neurodevelopmental animal model of schizophrenia to assess the efficacy of the atypical antipsychotic clozapine to prevent neuroanatomical deterioration. Methods Pregnant rats received injection on gestational day 15 with the viral mimic polyriboinosinic-polyribocytidylic acid (PolyI:C) or saline. Structural brain changes in the male offspring were assessed at adolescence and adulthood (35 days and 120 days) with structural neuroimaging. In the second part, male offspring of PolyI:C- and saline-treated dams received daily clozapine (7.5 mg/kg) or saline injection in adolescence (days 34–47) and underwent behavioral testing and imaging at adulthood (from 90 days onward). Results In utero exposure to maternal infection led in the offspring to postpubertal emergence of hallmark structural abnormalities associated with schizophrenia, enlarged ventricles, and smaller hippocampus. These abnormalities were not observed in the offspring of mothers who received PolyI:C that were treated with clozapine in adolescence. This was paralleled by prevention of behavioral abnormalities phenotypic of schizophrenia, attentional deficit, and hypersensitivity to amphetamine. Conclusions This is the first demonstration that pharmacological intervention during adolescence can prevent the emergence of brain structural changes resulting from in-utero insult.
An ugly duckling grows into a swan: Many organisms grow their crystalline mineral phases through the secondary nucleation of nanospheres made of an amorphous precursor phase. Stable amorphous calcium ...carbonate biominerals were used to induce a similar transformation in vitro. The amorphous nanospheres underwent a solid‐phase transformation that resulted in highly ordered calcite crystals composed of aggregated particles (see SEM image).
Many biogenic minerals are composed of aggregated particles at the nanoscale. These minerals usually form through the transformation of amorphous precursors into single crystals inside a privileged ...space controlled by the organism. Here, in vitro experiments aimed at understanding the factors responsible for producing such single crystals with aggregated particle texture are presented. Crystallization is achieved by a two‐step reaction in which amorphous calcium carbonate (ACC) is first precipitated and then transformed into calcite in small volumes of water and in the presence of additives. The additives used are gel‐forming molecules, phosphate ions, and the organic extract from sea urchin embryonic spicules ‐ all are present in various biogenic crystals that grow via the transformation of ACC. Remarkably, this procedure yields faceted single‐crystals of calcite that maintain the nanoparticle texture. The crystals grow predominantly by the accretion of ACC nanoparticles, which subsequently crystallize. Gels and phosphate ions stabilize ACC via a different mechanism than sea urchin spicule macromolecules. It is concluded that the unique nanoparticle texture of biogenic minerals results from formation pathways that may differ from one another, but given the appropriate precursor and micro‐environment, share a common particle accretion mechanism.
Single calcite crystals are grown by amorphous calcium carbonate (ACC) particle‐accretion using a synthetic procedure inspired by biogenic systems. The transformation of solid ACC particles in the presence of certain additives retards the classical dissolution‐precipitation process facilitating growth by a particle‐mediated process. The results provide a mechanistic understanding of biogenic and synthetic single crystal growth.
Cystoliths are amorphous calcium carbonate bodies that form in the leaves of some plant families. Cystoliths are regularly distributed in the epidermis and protrude into the photosynthetic tissue, ...the mesophyll. The photosynthetic pigments generate a steep light gradient in the leaf. Under most illumination regimes the outer mesophyll is light saturated, thus the photosynthetic apparatus is kinetically unable to use the excess light for photochemistry. Here we use micro‐scale modulated fluorometry to demonstrate that light scattered by the cystoliths is distributed from the photosynthetically inefficient upper tissue to the efficient, but light deprived, lower tissue. The results prove that the presence of light scatterers reduces the steep light gradient, thus enabling the leaf to use the incoming light flux more efficiently. MicroCT and electron microscopy confirm that the spatial distribution of the minerals is compatible with their optical function. During the study we encountered large calcium oxalate druses in the same anatomical location as the cystoliths. These druses proved to have similar light scattering functions as the cystoliths. This study shows that certain minerals in the leaves of different plants distribute the light flux more evenly inside the leaf.
Leaf minerals function as internal light scatterers inside leaves. They transfer light from the saturated upper tissue into the light deprived lower tissue. This eases the steep light gradient inside the leaf and improves photosynthetic efficiency on the tissue scale.
PDF
