A human lung tumor microenvironment interactome identifies clinically relevant cell-type cross-talk.

BackgroundTumors comprise a complex microenvironment of interacting malignant and stromal cell types. Much of our understanding of the tumor microenvironment comes from in vitro studies isolating the interactions between malignant cells and a single stromal cell type, often along a single pathway.ResultTo develop a deeper understanding of the interactions between cells within human lung tumors, we perform RNA-seq profiling of flow-sorted malignant cells, endothelial cells, immune cells, fibroblasts, and bulk cells from freshly resected human primary non-small-cell lung tumors. We map the cell-specific differential expression of prognostically associated secreted factors and cell surface genes, and computationally reconstruct cross-talk between these cell types to generate a novel resource called the Lung Tumor Microenvironment Interactome (LTMI). Using this resource, we identify and validate a prognostically unfavorable influence of Gremlin-1 production by fibroblasts on proliferation of malignant lung adenocarcinoma cells. We also find a prognostically favorable association between infiltration of mast cells and less aggressive tumor cell behavior.ConclusionThese results illustrate the utility of the LTMI as a resource for generating hypotheses concerning tumor-microenvironment interactions that may have prognostic and therapeutic relevance

High prevalence of vitamin D deficiency in Cambodian women:A common deficiency in a sunny country

Recent studies have shown that in spite of being generally close to the equator; vitamin D deficiency is common in South East Asian countries. In order to quantify micronutrient status for women and children in Cambodia; a nationally-representative survey was conducted in 2014 linked to the Cambodian Demographic Health Survey. The countrywide median of 25(OH) D was, respectively, 64.9 and 91.1 nmol/L for mothers and children. Based on The Endocrine Society cutoffs (>50<75 nmol/L = insufficiency; <= 50 nmol/L = deficiency); 64.6% of mothers and 34.8% of their children had plasma vitamin D concentrations indicating insufficiency or deficiency. For deficiency alone, 29% of the mothers were found to be vitamin D deficient, but only 13.4% of children. Children who live in urban areas had a 43% higher rate of vitamin D insufficiency versus those who live in rural areas (OR; 1.434; 95% CI: 1.007; 2.041). However, such differences were not observed in their mothers. The high prevalence of vitamin D deficiency is likely in part due to lifestyle choices, including sun avoidance, increasingly predominant indoor work, and covered transport. These survey findings support the need for a broader national Cambodian study incorporating testing of adult men, adolescents and the elderly, and encompassing other parameters such as skeletal health. However, the data presented in this study already show significant deficiencies which need to be addressed and we discuss the benefit of establishing nationally-mandated food fortification programs to enhance the intake of vitamin D

Molecular Characterization of Cryptosporidium Species and Giardia duodenalis from Symptomatic Cambodian Children

Background: In a prospective study, 498 single faecal samples from children aged under 16 years attending an outpatient clinic in the Angkor Hospital for Children, northwest Cambodia, were examined for Cryptosporidium oocysts and Giardia cysts using microscopy and molecular assays. Methodology/Principal Findings: Cryptosporidium oocysts were detected in 2.2% (11/498) of samples using microscopy and in 7.7% (38/498) with molecular tests. Giardia duodenalis cysts were detected in 18.9% (94/498) by microscopy and 27.7% (138/498) by molecular tests; 82% of the positive samples (by either method) were from children aged 1–10 years. Cryptosporidium hominis was the most common species of Cryptosporidium, detected in 13 (34.2%) samples, followed by Cryptosporidium meleagridis in 9 (23.7%), Cryptosporidium parvum in 8 (21.1%), Cryptosporidium canis in 5 (13.2%), and Cryptosporidium suis and Cryptosporidium ubiquitum in one sample each. Cryptosporidium hominis and C. parvum positive samples were subtyped by sequencing the GP60 gene: C. hominis IaA16R6 and C. parvum IIeA7G1 were the most abundant subtypes. Giardia duodenalis was typed using a multiplex real-time PCR targeting assemblages A and B. Assemblage B (106; 76.8% of all Giardia positive samples) was most common followed by A (12.3%) and mixed infections (5.1%). Risk factors associated with Cryptosporidium were malnutrition (AOR 9.63, 95% CI 1.67–55.46), chronic medical diagnoses (AOR 4.51, 95% CI 1.79–11.34) and the presence of birds in the household (AOR 2.99, 95% CI 1.16–7.73); specifically C. hominis (p = 0.03) and C. meleagridis (p<0.001) were associated with the presence of birds. The use of soap was protective against Giardia infection (OR 0.74, 95% CI 0.58–0.95). Conclusions/Significance: This is the first report to describe the different Cryptosporidium species and subtypes and Giardia duodenalis assemblages in Cambodian children. The variety of Cryptosporidium species detected indicates both anthroponotic and zoonotic transmission in this population. Interventions to improve sanitation, increase hand washing after defecation and before preparing food and promote drinking boiled water may reduce the burden of these two parasites

Broken replication forks trigger heritable DNA breaks in the terminus of a circular chromosome

<p><u>(A) Circular map of the <i>E</i>. <i>coli</i> chromosome</u>: <i>oriC</i>, <i>dif</i> and <i>terD</i> to <i>terB</i> sites are indicated. Numbers refer to the chromosome coordinates (in kb) of MG1655. (<u>B) Linear map of the terminus region:</u> chromosome coordinates are shown increasing from left to right, as in the marker frequency panels (see Figure 1C for example), therefore in the opposite direction to the circular map. In addition to <i>dif</i> and <i>ter</i> sites, the positions of the <i>parS</i>_pMT1 sites used for microscopy experiments are indicated. (<u>C) MFA analysis of terminus DNA loss in the <i>recB</i> mutant</u>: sequence read frequencies of exponential phase cells normalized to the total number of reads were calculated for each strain. Ratios of normalized reads in isogenic wild-type and <i>recB</i> mutant are plotted against chromosomal coordinates (in kb). The profile ratio of the terminus region is enlarged and the profile of the corresponding entire chromosomes is shown in inset. Original normalized profiles used to calculate ratios are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007256#pgen.1007256.s005" target="_blank">S1 Fig</a>. The position of <i>dif</i> is indicated by a red arrow. The <i>ter</i> sites that arrest clockwise forks (<i>terC</i>, <i>terB</i>, green arrow) and counter-clockwise forks (<i>terA</i>, <i>terD</i>, blue arrow) are shown. <u>(D) Schematic representation of focus loss in the <i>recB</i> mutant:</u> Time-lapse microscopy experiments showed that loss of a focus in the <i>recB</i> mutant occurs concomitantly with cell division in one of two daughter cells, and that the cell that keeps the focus then generates a focus-less cell at each generation. The percentage of initial events was calculated as the percentage of cell divisions that generate a focus-less cell, not counting the following generations. In this schematic representation, two initial events occurred (generations #2 and #7) out of 9 generations, and focus loss at generation #2 is heritable. Panels shown in this figure were previously published in [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007256#pgen.1007256.ref019" target="_blank">19</a>] and are reproduced here to introduce the phenomenon.</p

Antiglycation and antioxidant properties of Momordica charantia

The accumulation of advanced glycation endproducts (AGEs) and oxidative stress underlie the pathogenesis of diabetic complications. In many developing countries, diabetes treatment is unaffordable, and plants such as bitter gourd (or bitter melon; Momordica charantia) are used as traditional remedies because they exhibit hypoglycaemic properties. This study compared the antiglycation and antioxidant properties of aqueous extracts of M. charantia pulp (MCP), flesh (MCF) and charantin in vitro. Lysozyme was mixed with methylglyoxal and 0–15 mg/ml of M. charantia extracts in a pH 7.4 buffer and incubated at 37°C for 3 days. Crosslinked AGEs were assessed using gel electrophoresis, and the carboxymethyllysine (CML) content was analyzed by enzyme-linked immunosorbent assays. The antioxidant activities of the extracts were evaluated using assays to assess DPPH (1,1-diphenyl-2-picryl-hydrazyl) and hydroxyl radical scavenging activities, metal-chelating activity and reducing power of the extracts. The phenolic, flavonol and flavonoid content of the extracts were also determined. All extracts inhibited the formation of crosslinked AGEs and CML in a dose-dependent manner, with MCF being the most potent. The antioxidant activity of MCF was higher than that of MCP, but MCP showed the highest metal-chelating activity. MCF had the highest phenolic and flavonoid contents, whereas MCP had the highest flavonol content. M. charantia has hypoglycaemic effects, but this study shows that M. charantia extracts are also capable of preventing AGE formation in vitro. This activity may be due to the antioxidant properties, particularly the total phenolic content of the extracts. Thus, the use of M. charantia deserves more attention, as it may not only reduce hyperglycaemia but also protect against the build-up of tissue AGEs and reduce oxidative stress in patients with diabetes

The evolution of lung cancer and impact of subclonal selection in TRACERx

Lung cancer is the leading cause of cancer-associated mortality worldwide. Here we analysed 1,644 tumour regions sampled at surgery or during follow-up from the first 421 patients with non-small cell lung cancer prospectively enrolled into the TRACERx study. This project aims to decipher lung cancer evolution and address the primary study endpoint: determining the relationship between intratumour heterogeneity and clinical outcome. In lung adenocarcinoma, mutations in 22 out of 40 common cancer genes were under significant subclonal selection, including classical tumour initiators such as TP53 and KRAS. We defined evolutionary dependencies between drivers, mutational processes and whole genome doubling (WGD) events. Despite patients having a history of smoking, 8% of lung adenocarcinomas lacked evidence of tobacco-induced mutagenesis. These tumours also had similar detection rates for EGFR mutations and for RET, ROS1, ALK and MET oncogenic isoforms compared with tumours in never-smokers, which suggests that they have a similar aetiology and pathogenesis. Large subclonal expansions were associated with positive subclonal selection. Patients with tumours harbouring recent subclonal expansions, on the terminus of a phylogenetic branch, had significantly shorter disease-free survival. Subclonal WGD was detected in 19% of tumours, and 10% of tumours harboured multiple subclonal WGDs in parallel. Subclonal, but not truncal, WGD was associated with shorter disease-free survival. Copy number heterogeneity was associated with extrathoracic relapse within 1 year after surgery. These data demonstrate the importance of clonal expansion, WGD and copy number instability in determining the timing and patterns of relapse in non-small cell lung cancer and provide a comprehensive clinical cancer evolutionary data resource

Genomic–transcriptomic evolution in lung cancer and metastasis

Intratumour heterogeneity (ITH) fuels lung cancer evolution, which leads to immune evasion and resistance to therapy. Here, using paired whole-exome and RNA sequencing data, we investigate intratumour transcriptomic diversity in 354 non-small cell lung cancer tumours from 347 out of the first 421 patients prospectively recruited into the TRACERx study. Analyses of 947 tumour regions, representing both primary and metastatic disease, alongside 96 tumour-adjacent normal tissue samples implicate the transcriptome as a major source of phenotypic variation. Gene expression levels and ITH relate to patterns of positive and negative selection during tumour evolution. We observe frequent copy number-independent allele-specific expression that is linked to epigenomic dysfunction. Allele-specific expression can also result in genomic–transcriptomic parallel evolution, which converges on cancer gene disruption. We extract signatures of RNA single-base substitutions and link their aetiology to the activity of the RNA-editing enzymes ADAR and APOBEC3A, thereby revealing otherwise undetected ongoing APOBEC activity in tumours. Characterizing the transcriptomes of primary–metastatic tumour pairs, we combine multiple machine-learning approaches that leverage genomic and transcriptomic variables to link metastasis-seeding potential to the evolutionary context of mutations and increased proliferation within primary tumour regions. These results highlight the interplay between the genome and transcriptome in influencing ITH, lung cancer evolution and metastasis

The evolution of non-small cell lung cancer metastases in TRACERx

Metastatic disease is responsible for the majority of cancer-related deaths. We report the longitudinal evolutionary analysis of 126 non-small cell lung cancer (NSCLC) tumours from 421 prospectively recruited patients in TRACERx who developed metastatic disease, compared with a control cohort of 144 non-metastatic tumours. In 25% of cases, metastases diverged early, before the last clonal sweep in the primary tumour, and early divergence was enriched for patients who were smokers at the time of initial diagnosis. Simulations suggested that early metastatic divergence more frequently occurred at smaller tumour diameters (less than 8 mm). Single-region primary tumour sampling resulted in 83% of late divergence cases being misclassified as early, highlighting the importance of extensive primary tumour sampling. Polyclonal dissemination, which was associated with extrathoracic disease recurrence, was found in 32% of cases. Primary lymph node disease contributed to metastatic relapse in less than 20% of cases, representing a hallmark of metastatic potential rather than a route to subsequent recurrences/disease progression. Metastasis-seeding subclones exhibited subclonal expansions within primary tumours, probably reflecting positive selection. Our findings highlight the importance of selection in metastatic clone evolution within untreated primary tumours, the distinction between monoclonal versus polyclonal seeding in dictating site of recurrence, the limitations of current radiological screening approaches for early diverging tumours and the need to develop strategies to target metastasis-seeding subclones before relapse