PyMT-Maclow: A novel, inducible, murine model for determining the role of CD68 positive cells in breast tumor development

CD68+ tumor-associated macrophages (TAMs) are pro-tumorigenic, pro-angiogenic and are associated with decreased survival rates in patients with cancer, including breast cancer. Non-specific models of macrophage ablation reduce the number of TAMs and limit the development of mammary tumors. However, the lack of specificity and side effects associated with these models compromise their reliability. We hypothesized that specific and controlled macrophage depletion would provide precise data on the effects of reducing TAM numbers on tumor development. In this study, the MacLow mouse model of doxycycline-inducible and selective CD68+ macrophage depletion was crossed with the murine mammary tumor virus (MMTV)-Polyoma virus middle T antigen (PyMT) mouse model of spontaneous ductal breast adenocarcinoma to generate the PyMT-MacLow line. In doxycycline-treated PyMT-MacLow mice, macrophage numbers were decreased in areas surrounding tumors by 43%. Reducing the number of macrophages by this level delayed tumor progression, generated less proliferative tumors, decreased the vascularization of carcinomas and down-regulated the expression of many pro-angiogenic genes. These results demonstrate that depleting CD68+ macrophages in an inducible and selective manner delays the development of mammary tumors and that the PyMT-MacLow model is a useful and unique tool for studying the role of TAMs in breast cancer

The genetic architecture of the human cerebral cortex

The cerebral cortex underlies our complex cognitive capabilities, yet little is known about the specific genetic loci that influence human cortical structure. To identify genetic variants that affect cortical structure, we conducted a genome-wide association meta-analysis of brain magnetic resonance imaging data from 51,665 individuals. We analyzed the surface area and average thickness of the whole cortex and 34 regions with known functional specializations. We identified 199 significant loci and found significant enrichment for loci influencing total surface area within regulatory elements that are active during prenatal cortical development, supporting the radial unit hypothesis. Loci that affect regional surface area cluster near genes in Wnt signaling pathways, which influence progenitor expansion and areal identity. Variation in cortical structure is genetically correlated with cognitive function, Parkinson's disease, insomnia, depression, neuroticism, and attention deficit hyperactivity disorder

Genome-wide identification and phenotypic characterization of seizure-associated copy number variations in 741,075 individuals

Copy number variants (CNV) are established risk factors for neurodevelopmental disorders with seizures or epilepsy. With the hypothesis that seizure disorders share genetic risk factors, we pooled CNV data from 10,590 individuals with seizure disorders, 16,109 individuals with clinically validated epilepsy, and 492,324 population controls and identified 25 genome-wide significant loci, 22 of which are novel for seizure disorders, such as deletions at 1p36.33, 1q44, 2p21-p16.3, 3q29, 8p23.3-p23.2, 9p24.3, 10q26.3, 15q11.2, 15q12-q13.1, 16p12.2, 17q21.31, duplications at 2q13, 9q34.3, 16p13.3, 17q12, 19p13.3, 20q13.33, and reciprocal CNVs at 16p11.2, and 22q11.21. Using genetic data from additional 248,751 individuals with 23 neuropsychiatric phenotypes, we explored the pleiotropy of these 25 loci. Finally, in a subset of individuals with epilepsy and detailed clinical data available, we performed phenome-wide association analyses between individual CNVs and clinical annotations categorized through the Human Phenotype Ontology (HPO). For six CNVs, we identified 19 significant associations with specific HPO terms and generated, for all CNVs, phenotype signatures across 17 clinical categories relevant for epileptologists. This is the most comprehensive investigation of CNVs in epilepsy and related seizure disorders, with potential implications for clinical practice

GWAS meta-analysis of over 29,000 people with epilepsy identifies 26 risk loci and subtype-specific genetic architecture

Epilepsy is a highly heritable disorder affecting over 50 million people worldwide, of which about one-third are resistant to current treatments. Here we report a multi-ancestry genome-wide association study including 29,944 cases, stratified into three broad categories and seven subtypes of epilepsy, and 52,538 controls. We identify 26 genome-wide significant loci, 19 of which are specific to genetic generalized epilepsy (GGE). We implicate 29 likely causal genes underlying these 26 loci. SNP-based heritability analyses show that common variants explain between 39.6% and 90% of genetic risk for GGE and its subtypes. Subtype analysis revealed markedly different genetic architectures between focal and generalized epilepsies. Gene-set analyses of GGE signals implicate synaptic processes in both excitatory and inhibitory neurons in the brain. Prioritized candidate genes overlap with monogenic epilepsy genes and with targets of current antiseizure medications. Finally, we leverage our results to identify alternate drugs with predicted efficacy if repurposed for epilepsy treatment

Doxycycline reduces the number of macrophages surrounding mammary tumors in PyMT-MacLow mice.

<p>(<b>A</b>) Sections of mammary tissue were labelled for F4/80 (DAB brown cells) and counterstained with haematoxylin; a representative image at the early carcinoma stage of tumor development is shown for each genotype and treatment. (<b>B</b>) Images captured from slides scanned on an Aperio slide scanner were used to quantify the number of macrophages within (intratumoral) and on the perimeter of mammary tumors (peritumoral). The data was then grouped according to genotype and treatment group and the mean value +/- SD shown. Each individual data point represent the mean values for an individual tumor. Tumor samples were obtained from the following numbers of animals per treatment group the number of tumors analysed is indicated in brackets: PyMT UT = 5(27), PyMT Doxy = 5(33), PyMT-MacLow UT = 7(31), PyMT-MacLow Doxy = 7(39). Significance is from the SPSS nested analysis comparing data from doxycycline treated versus control animals for each tumor and genotype. NS = not significant, **P<0.01. Scalebar = 100 μm. (C) The percentage of animals that had any tumors of hyperplasia and/or Adenoma/mammary intraepithelial neoplasia stages (Adenoma/MIN) was calculated for each genotype and treatment group. A Chi-square test was used to calculate statistical significance.</p

CD68+ and F4/80+ macrophages are analogous populations in PyMT tumors.

<p>Tumors from PyMT mice were enzymatically digested and made into a single cell suspension. Cells were stained with antibodies against the surface markers CD45, CD11b and F4/80. Cells were then fixed, permeabilized and stained with anti-CD68. Fluorescence was measured by flow cytometry and the data was analyzed using Flowjo software. Dot plots shown were generated from CD45⁺ cells. CD68 expression was determined after gating on tumor-associated macrophages (CD45⁺CD11b⁺F4/80⁺), CD11b⁺F4/80^—cells and CD11b^—F4/80^—cells. Representative dot plots are shown from one of four mice analysed.</p

The proliferative capacity of Adenoma/MIN tumors is reduced in macrophage deficient mice.

<p>Mammary sections were labelled with an anti-Ki67 antibody as a marker of proliferation and counterstained with haematoxylin. (<b>A</b>) Images were captured from slides scanned on an Aperio slide scanner and split according to tumor grade, a representative image of an Adeno/MIN tumor (A/M) is shown for each genotype and treatment group. (<b>B</b>) The amount of Ki67 labelling in individual tumor areas (cells stained dark brown with DAB) was quantified and expressed as positivity using the Aperio positive pixel algorithm. Each data point on the graph represents the mean positivity for an individual tumor and the number of animals these tumors were taken from is shown below the x axis on each graph. The mean value ± SD was plotted and a nested analysis carried out in SPSS to compare data from doxycycline treated versus control animals from each tumor grade and genotype. ***P<0.001. Scalebar = 100 μm.</p