ABSTRACT
Tumor behavior is intricately dependent on the oncogenic properties of cancer cells and their multi-cellular interactions. To understand these dependencies within the wider microenvironment, we studied over 270,000 single-cell transcriptomes and 100 microdissected whole exomes from 12 patients with kidney tumors, prior to validation using spatial transcriptomics. Tissues were sampled from multiple regions of the tumor core, the tumor-normal interface, normal surrounding tissues, and peripheral blood. We find that the tissue-type location of CD8+ T cell clonotypes largely defines their exhaustion state with intra-tumoral spatial heterogeneity that is not well explained by somatic heterogeneity. De novo mutation calling from single-cell RNA-sequencing data allows us to broadly infer the clonality of stromal cells and lineage-trace myeloid cell development. We report six conserved meta-programs that distinguish tumor cell function, and find an epithelial-mesenchymal transition meta-program highly enriched at the tumor-normal interface that co-localizes with IL1B-expressing macrophages, offering a potential therapeutic target.
Subject(s)
Carcinoma, Renal Cell , Kidney Neoplasms , Humans , Transcriptome , Gene Expression Profiling , Carcinoma, Renal Cell/genetics , Kidney Neoplasms/genetics , Epithelial-Mesenchymal Transition , Tumor Microenvironment/genetics , Single-Cell AnalysisABSTRACT
The rates and patterns of somatic mutation in normal tissues are largely unknown outside of humans1-7. Comparative analyses can shed light on the diversity of mutagenesis across species, and on long-standing hypotheses about the evolution of somatic mutation rates and their role in cancer and ageing. Here we performed whole-genome sequencing of 208 intestinal crypts from 56 individuals to study the landscape of somatic mutation across 16 mammalian species. We found that somatic mutagenesis was dominated by seemingly endogenous mutational processes in all species, including 5-methylcytosine deamination and oxidative damage. With some differences, mutational signatures in other species resembled those described in humans8, although the relative contribution of each signature varied across species. Notably, the somatic mutation rate per year varied greatly across species and exhibited a strong inverse relationship with species lifespan, with no other life-history trait studied showing a comparable association. Despite widely different life histories among the species we examined-including variation of around 30-fold in lifespan and around 40,000-fold in body mass-the somatic mutation burden at the end of lifespan varied only by a factor of around 3. These data unveil common mutational processes across mammals, and suggest that somatic mutation rates are evolutionarily constrained and may be a contributing factor in ageing.
Subject(s)
Longevity , Mutation Rate , Animals , Humans , Longevity/genetics , Mammals/genetics , Mutagenesis/genetics , MutationABSTRACT
Over the course of an individual's lifetime, normal human cells accumulate mutations1. Here we compare the mutational landscape in 29 cell types from the soma and germline using multiple samples from the same individuals. Two ubiquitous mutational signatures, SBS1 and SBS5/40, accounted for the majority of acquired mutations in most cell types, but their absolute and relative contributions varied substantially. SBS18, which potentially reflects oxidative damage2, and several additional signatures attributed to exogenous and endogenous exposures contributed mutations to subsets of cell types. The rate of mutation was lowest in spermatogonia, the stem cells from which sperm are generated and from which most genetic variation in the human population is thought to originate. This was due to low rates of ubiquitous mutational processes and may be partially attributable to a low rate of cell division in basal spermatogonia. These results highlight similarities and differences in the maintenance of the germline and soma.
Subject(s)
Germ Cells/metabolism , Germ-Line Mutation , Mutation Rate , Organ Specificity/genetics , Aged , Clone Cells/metabolism , Female , Health , Humans , Male , Microdissection , Middle Aged , Oxidative Stress , Spermatogonia/metabolismABSTRACT
Somatic mutations accumulate in healthy tissues as we age, giving rise to cancer and potentially contributing to ageing. To study somatic mutations in non-neoplastic tissues, we developed a series of protocols to sequence the genomes of small populations of cells isolated from histological sections. Here, we describe a complete workflow that combines laser-capture microdissection (LCM) with low-input genome sequencing, while circumventing the use of whole-genome amplification (WGA). The protocol is subdivided broadly into four steps: tissue processing, LCM, low-input library generation and mutation calling and filtering. The tissue processing and LCM steps are provided as general guidelines that might require tailoring based on the specific requirements of the study at hand. Our protocol for low-input library generation uses enzymatic rather than acoustic fragmentation to generate WGA-free whole-genome libraries. Finally, the mutation calling and filtering strategy has been adapted from previously published protocols to account for artifacts introduced via library creation. To date, we have used this workflow to perform targeted and whole-genome sequencing of small populations of cells (typically 100-1,000 cells) in thousands of microbiopsies from a wide range of human tissues. The low-input DNA protocol is designed to be compatible with liquid handling platforms and make use of equipment and expertise standard to any core sequencing facility. However, obtaining low-input DNA material via LCM requires specialized equipment and expertise. The entire protocol from tissue reception through whole-genome library generation can be accomplished in as little as 1 week, although 2-3 weeks would be a more typical turnaround time.
Subject(s)
Laser Capture Microdissection/methods , Sequence Analysis, DNA/methods , Whole Genome Sequencing/methods , DNA/genetics , Gene Expression Profiling/methods , Gene Library , High-Throughput Nucleotide Sequencing/methods , Humans , Mutation/genetics , WorkflowABSTRACT
Many aquatic environments are at risk for oil contamination and alkanes are one of the primary constituents of oil. The alkane hydroxylase (AlkB) is a common enzyme used by microorganisms to initiate the process of alkane-degradation. While many aspects of alkane bioremediation have been studied, the diversity and evolution of genes involved in hydrocarbon degradation from environmental settings is relatively understudied. The majority of work done to-date has focused on the marine environment. Here we sought to better understand the phylogenetic diversity of alkB genes across marine and freshwater settings using culture-independent methods. We hypothesized that there would be distinct phylogenetic diversity of alkB genes in freshwater relative to the marine environment. Our results confirm that alkB has distinct variants based on environment while our diversity analyses demonstrate that freshwater and marine alkB communities have unique responses to oil amendments. Our results also demonstrate that in the marine environment, depth is a key factor impacting diversity of alkB genes.
Subject(s)
Bacteria , Cytochrome P-450 CYP4A/genetics , Genes, Bacterial/genetics , Genetic Variation , Phylogeny , Atlantic Ocean , Bacteria/classification , Bacteria/enzymology , Bacteria/genetics , Cytochrome P-450 CYP4A/metabolism , Great Lakes Region , Petroleum/metabolism , Salinity , Water MicrobiologyABSTRACT
PREMISE: Cryptic species are evolutionarily distinct lineages lacking distinguishing morphological traits. Hidden diversity may be lurking in widespread species whose distributions cross phylogeographic barriers. This study investigates molecular and morphological variation in the widely distributed Caulanthus lasiophyllus (Brassicaceae) in comparison to its closest relatives. METHODS: Fifty-two individuals of C. lasiophyllus from across the species' range were sequenced for the nuclear ribosomal internal transcribed spacer region (ITS) and the chloroplast trnL-F region. A subset of these samples were examined for the chloroplast ndhF gene. All 52 individuals were scored for 13 morphological traits, as well as monthly and annual climate conditions at the collection locality. Morphological and molecular results are compared with the closest relatives-C. anceps and C. flavescens-in the "Guillenia Clade." To test for polyploidy, genome size estimates were made for four populations. RESULTS: Caulanthus lasiophyllus consists of two distinct lineages separated by eight ITS differences-eight times more variation than what distinguishes C. anceps and C. flavescens. Fewer variable sites were detected in trnL-F and ndhF regions, yet these data are consistent with the ITS results. The two lineages of C. lasiophyllus are geographically and climatically distinct; yet morphologically overlapping. Their genome sizes are not consistently different. CONCLUSIONS: Two cryptic species within C. lasiophyllus are distinguished at the molecular, geographic, and climatic scales. They have similar genome sizes and are morphologically broadly overlapping, but an ephemeral basal leaf character may help distinguish the species.
Subject(s)
DNA, Chloroplast , Mustard Plant , Base Sequence , California , Genetic Variation , Phylogeny , Sequence Analysis, DNAABSTRACT
The extent of somatic mutation and clonal selection in the human bladder remains unknown. We sequenced 2097 bladder microbiopsies from 20 individuals using targeted (n = 1914 microbiopsies), whole-exome (n = 655), and whole-genome (n = 88) sequencing. We found widespread positive selection in 17 genes. Chromatin remodeling genes were frequently mutated, whereas mutations were absent in several major bladder cancer genes. There was extensive interindividual variation in selection, with different driver genes dominating the clonal landscape across individuals. Mutational signatures were heterogeneous across clones and individuals, which suggests differential exposure to mutagens in the urine. Evidence of APOBEC mutagenesis was found in 22% of the microbiopsies. Sequencing multiple microbiopsies from five patients with bladder cancer enabled comparisons with cancer-free individuals and across histological features. This study reveals a rich landscape of mutational processes and selection in normal urothelium with large heterogeneity across clones and individuals.
Subject(s)
Genes, Neoplasm , Mutagenesis , Selection, Genetic , Urinary Bladder Neoplasms/genetics , Urinary Bladder/pathology , Urothelium/pathology , APOBEC Deaminases/genetics , Adult , Aged , Biopsy , Chromatin Assembly and Disassembly/genetics , Female , Humans , Male , Middle Aged , Mutagens/analysis , MutationABSTRACT
We conducted a global characterization of the microbial communities of shipping ports to serve as a novel system to investigate microbial biogeography. The community structures of port microbes from marine and freshwater habitats house relatively similar phyla, despite spanning large spatial scales. As part of this project, we collected 1,218 surface water samples from 604 locations across eight countries and three continents to catalogue a total of 20 shipping ports distributed across the East and West Coast of the United States, Europe, and Asia to represent the largest study of port-associated microbial communities to date. Here, we demonstrated the utility of machine learning to leverage this robust system to characterize microbial biogeography by identifying trends in biodiversity across broad spatial scales. We found that for geographic locations sharing similar environmental conditions, subpopulations from the dominant phyla of these habitats (Actinobacteria, Bacteroidetes, Cyanobacteria, and Proteobacteria) can be used to differentiate 20 geographic locations distributed globally. These results suggest that despite the overwhelming diversity within microbial communities, members of the most abundant and ubiquitous microbial groups in the system can be used to differentiate a geospatial location across global spatial scales. Our study provides insight into how microbes are dispersed spatially and robust methods whereby we can interrogate microbial biogeography.IMPORTANCE Microbes are ubiquitous throughout the world and are highly diverse. Characterizing the extent of variation in the microbial diversity across large geographic spatial scales is a challenge yet can reveal a lot about what biogeography can tell us about microbial populations and their behavior. Machine learning approaches have been used mostly to examine the human microbiome and, to some extent, microbial communities from the environment. Here, we display how supervised machine learning approaches can be useful to understand microbial biodiversity and biogeography using microbes from globally distributed shipping ports. Our findings indicate that the members of globally dominant phyla are important for differentiating locations, which reduces the reliance on rare taxa to probe geography. Further, this study displays how global biogeographic patterning of aquatic microbial communities (and other systems) can be assessed through populations of the highly abundant and ubiquitous taxa that dominant the system.
Subject(s)
Actinobacteria/classification , Bacteroidetes/classification , Biodiversity , Cyanobacteria/classification , Proteobacteria/classification , Water Microbiology , Asia , DNA, Bacterial/genetics , Europe , Fresh Water/microbiology , Machine Learning , Microbiota , Phylogeography , RNA, Ribosomal, 16S/genetics , Seawater/microbiology , Ships , United StatesABSTRACT
In the past, ballast water has been a key vector in the ship-mediated dispersal of invasive species. Here, we evaluate the potential for port microorganisms to enter and colonize the hull and bilge water of ships. Due to the small size and ubiquitous nature of bacteria, they also have the potential to be spread through hull fouling and bilge water discharge. The goal of this study was to identify the extent to which the boat microbial community is shaped by the microbial community in the port water where the boat spends most of its time. Here, we compared the microbial communities of the hull and bilge compartments of 20 boats to those of the port water in 20 different ports in five regions around the world. We found that there was a significant difference in microbial diversity between boat and port microbial communities. Despite these differences, we found that Cyanobacteria were present at high abundances in the bilge water of most vessels. Due to the limited light in the bilge, the presence of Cyanobacteria suggests that port microorganisms can enter the bilge. Using source-tracking software, we found that, on average, 40% of the bilge and 52% of the hull microbial communities were derived from water. These findings suggest that the bilge of a vessel contains a diverse microbial community that is influenced by the port microbial community and has the potential to serve as an underappreciated vector for dispersal of life.IMPORTANCE Invasive species have been a worldwide problem for many years. However, the potential for microorganisms to become invasive is relatively underexplored. As the tools to study bacterial communities become more affordable, we are able to perform large-scale studies and examine bacterial communities in higher resolution than was previously practical. This study looked at the potential for bacteria to colonize both boat surfaces and bilge water. We describe the bacterial communities on boats in 20 shipping ports in five regions around the world, describing how these microorganisms were similar to microorganisms found in port water. This suggests that the water influences the bacterial community of a boat and that microorganisms living on a boat could be moved from place to place when the boat travels.
Subject(s)
Microbiota , Ships , Water Microbiology , Water/chemistry , Biodiversity , Cyanobacteria , Introduced Species , Microbiota/genetics , RNA, Ribosomal, 16S/geneticsABSTRACT
Many freshwater environments experience dramatic seasonal changes with some systems remaining ice-covered for most of the winter. Freshwater systems are also highly sensitive to environmental change. However, little is known about changes in microbial abundance and community composition during lake ice formation and times of persistent ice cover. The goal of this study is to characterize temporal dynamics of microbial communities during ice formation and persistent ice cover. Samples were collected in triplicate, five days per week from surface water in the Keweenaw Waterway between November and April. Environmental conditions along with microbial abundance and microbial community composition was determined. Distinct community composition was found between ice-free and ice-covered time periods with significantly different community composition between months. The microbial community underwent dramatic shifts in microbial abundance and diversity during the transitions into and out of ice cover. The richness of the microbial community increased during times of ice cover. Relatives of microbes involved in nitrogen cycling bloomed during times of ice cover as sequences related to known nitrifying taxa were significantly enriched during ice cover. These results help to elucidate how microbial abundance and diversity change over drastic seasonal transitions and how ice cover may affect microbial abundance and diversity.
Subject(s)
Freezing , Ice Cover/microbiology , Lakes/microbiology , Microbiota , Water Microbiology , Archaea/genetics , Bacteria/genetics , Base Sequence , DNA, Archaeal/genetics , DNA, Archaeal/isolation & purification , DNA, Bacterial/genetics , DNA, Bacterial/isolation & purification , Genetic Variation , Michigan , Phylogeny , Polymerase Chain Reaction , RNA, Ribosomal, 16S/genetics , SeasonsABSTRACT
Pathological complete response (pCR) is an accurate predictor of good outcome following neoadjuvant chemotherapy (NAC) for locally advanced breast cancer. The presence of circulating-tumor DNA (ctDNA) has recently been reported to be strongly predictive of poor outcome in similar patient groups. We monitored ctDNA levels from 10 women undergoing NAC for locally advanced breast cancer using a patient-specific, hybrid-capture sequencing technique sensitive to the level of one altered allele in 10,000. Plasma was collected prior to the start of NAC, prior to each infusion of NAC, and during follow-up for between 350 and 1150 d after the start of NAC. Prior to the start of NAC, ctDNA was detectable in 3/3 triple negative, 3/3 HER2+, and 2/4 HER2-, ER+ breast cancer patients. Total cell-free DNA levels were considerably higher when patients were on NAC than at other times. ctDNA dynamics during NAC showed that patients with pCR experienced rapid declines in ctDNA levels, whereas patients without pCR typically showed evidence of residual ctDNA after initiation of treatment. Intriguingly, two of three patients that showed marked increases in ctDNA while on NAC experienced rapid recurrences (<2 yr following start of NAC). The third patient that had increases in ctDNA levels while on NAC had low-grade ER+ disease and showed residual ctDNA after surgery, which became undetectable after local radiation. Taken together, these results demonstrate the ability of our approach to sensitively serially monitor ctDNA during NAC, and identifies a need to further investigate the possibility of stratifying patients who need additional treatment or identify therapies that are ineffective.
Subject(s)
Breast Neoplasms/therapy , Circulating Tumor DNA/genetics , Neoadjuvant Therapy/methods , Sequence Analysis, DNA/methods , Adult , Aged , Breast Neoplasms/genetics , Female , Humans , Mastectomy , Middle Aged , Precision Medicine , Treatment OutcomeABSTRACT
The development of new circulating-tumor DNA (ctDNA) analysis techniques has led to an explosion of studies demonstrating exciting clinical applications. Non-invasive genotyping can characterize mutations of interest without the need for an invasive biopsy. Serial ctDNA monitoring can assess response to treatment, and potentially identify mechanisms of resistance. Perhaps most excitingly, sensitive ctDNA analysis methods allow for detection of minimally residual disease, predicting recurrence months before clinical presentation. In this review, we highlight several recent, key studies in the ctDNA field and discuss future advances which would further improve patient care.
Subject(s)
Biomarkers, Tumor/blood , Circulating Tumor DNA/genetics , Early Detection of Cancer , Neoplasms/blood , Circulating Tumor DNA/isolation & purification , Genotype , Humans , Mutation , Neoplasms/genetics , Neoplasms/pathologyABSTRACT
The identification of the molecular drivers of cancer by sequencing is the backbone of precision medicine and the basis of personalized therapy; however, biopsies of primary tumors provide only a snapshot of the evolution of the disease and may miss potential therapeutic targets, especially in the metastatic setting. A liquid biopsy, in the form of cell-free DNA (cfDNA) sequencing, has the potential to capture the inter- and intra-tumoral heterogeneity present in metastatic disease, and, through serial blood draws, track the evolution of the tumor genome. In order to determine the clinical utility of cfDNA sequencing we performed whole-exome sequencing on cfDNA and tumor DNA from two patients with metastatic disease; only minor modifications to our sequencing and analysis pipelines were required for sequencing and mutation calling of cfDNA. The first patient had metastatic sarcoma and 47 of 48 mutations present in the primary tumor were also found in the cell-free DNA. The second patient had metastatic breast cancer and sequencing identified an ESR1 mutation in the cfDNA and metastatic site, but not in the primary tumor. This likely explains tumor progression on Anastrozole. Significant heterogeneity between the primary and metastatic tumors, with cfDNA reflecting the metastases, suggested separation from the primary lesion early in tumor evolution. This is best illustrated by an activating PIK3CA mutation (H1047R) which was clonal in the primary tumor, but completely absent from either the metastasis or cfDNA. Here we show that cfDNA sequencing supplies clinically actionable information with minimal risks compared to metastatic biopsies. This study demonstrates the utility of whole-exome sequencing of cell-free DNA from patients with metastatic disease. cfDNA sequencing identified an ESR1 mutation, potentially explaining a patient's resistance to aromatase inhibition, and gave insight into how metastatic lesions differ from the primary tumor.
Subject(s)
DNA, Neoplasm/genetics , Estrogen Receptor alpha/genetics , Exome , Mutation, Missense , Neoplasm Proteins/genetics , Phosphatidylinositol 3-Kinases/genetics , Sarcoma/genetics , Amino Acid Substitution , Class I Phosphatidylinositol 3-Kinases , DNA, Neoplasm/blood , Female , Humans , Middle Aged , Neoplasm Metastasis , Sarcoma/blood , Sarcoma/pathologyABSTRACT
BACKGROUND: Circulating biomarkers are urgently needed in hepatocellular carcinoma (HCC). The aims of this study were to determine the feasibility of detecting and isolating circulating tumor cells (CTCs) in HCC patients using enrichment for epithelial cell adhesion molecule (EpCAM) expression, to examine their prognostic value, and to explore CTC-based DNA sequencing in metastatic HCC patients compared to a control cohort with non-malignant liver diseases (NMLD). METHODS: Whole blood was obtained from patients with metastatic HCC or NMLD. CTCs were enumerated by CellSearch then purified by immunomagnetic EpCAM enrichment and fluorescence-activated cell sorting. Targeted ion semiconductor sequencing was performed on whole genome-amplified DNA from CTCs, tumor specimens, and peripheral blood mononuclear cells (PBMC) when available. RESULTS: Twenty HCC and 10 NMLD patients enrolled. CTCs ≥ 2/7.5 mL were detected in 7/20 (35%, 95% confidence interval: 12%, 60%) HCC and 0/9 eligible NMLD (p = 0.04). CTCs ≥ 1/7.5 mL was associated with alpha-fetoprotein ≥ 400 ng/mL (p = 0.008) and vascular invasion (p = 0.009). Sequencing of CTC DNA identified characteristic HCC mutations. The proportion with ≥ 100x coverage depth was lower in CTCs (43%) than tumor or PBMC (87%) (p < 0.025). Low frequency variants were higher in CTCs (p < 0.001). CONCLUSIONS: CTCs are detectable by EpCAM enrichment in metastatic HCC, without confounding false positive background from NMLD. CTC detection was associated with poor prognostic factors. Sequencing of CTC DNA identified known HCC mutations but more low-frequency variants and lower coverage depth than FFPE or PBMC.
Subject(s)
Antigens, Neoplasm/genetics , Carcinoma, Hepatocellular/genetics , Cell Adhesion Molecules/genetics , Liver Neoplasms/genetics , Neoplastic Cells, Circulating , Aged , Aged, 80 and over , Antigens, Neoplasm/blood , Carcinoma, Hepatocellular/blood , Carcinoma, Hepatocellular/pathology , Cell Adhesion Molecules/blood , Epithelial Cell Adhesion Molecule , Epithelial-Mesenchymal Transition/genetics , Female , High-Throughput Nucleotide Sequencing , Humans , Kaplan-Meier Estimate , Liver Diseases/blood , Liver Diseases/genetics , Liver Diseases/pathology , Liver Neoplasms/blood , Liver Neoplasms/pathology , Male , Middle Aged , Neoplasm Metastasis , Polymorphism, Single Nucleotide , PrognosisABSTRACT
Cities have developed into the hotspots of human economic activity. From the appearance of the first cities in the Neolithic to 21st century metropolis their impact on the environment has always been apparent. With more people living in cities than in rural environments now it becomes crucial to understand these environmental impacts. With the immergence of megacities in the 20th century and their continued growth in both, population and economic power, the environmental impact has reached the global scale. In this paper we examine megacity impacts on atmospheric composition and climate. We present basic concepts, discuss various definitions of footprints, summarize research on megacity impacts and assess the impact of megacity emissions on air quality and on the climate at the regional to global scale. The intention and ambition of this paper is to give a comprehensive but brief overview of the science with regard to megacities and the environment.