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Includes Supplemental Figure S1 and Supplemental Tables S1-S7

Raw cell counts of FISH gene probes for (1) primary breast tumors, (2) metastatic sites, (3) pre- and post-treatment [Hungarian sample], and (4) TNBC cases.

Spatially resolved ambient mass spectrometry imaging methods have gained popularity to characterize cancer sites and their borders using molecular changes in the lipidome. This utility, however, is predicated on metabolic homogeneity at the border, which would create a sharp molecular transition at the morphometric borders. We subjected murine models of human medulloblastoma brain cancer to mass spectrometry imaging, a technique that provides a direct readout of tissue molecular content in a spatially resolved manner. We discovered a distance-dependent gradient of cancer-like lipid molecule profiles in the brain tissue within 1.2 mm of the cancer border, suggesting that a cancer-like state progresses beyond the histologic border, into the healthy tissue. The results were further corroborated using orthogonal liquid chromatography and mass spectrometry (LC-MS) analysis of selected tissue regions subjected to laser capture microdissection. LC-MS/MS analysis for robust identification of the affected molecules implied changes in a number of different lipid classes, some of which are metabolized from the essential docosahexaenoic fatty acid (DHA) present in the interstitial fluid. Metabolic molecular borders are thus not as sharp as morphometric borders, and mass spectrometry imaging can reveal molecular nuances not observed with microscopy. Caution must be exercised in interpreting multimodal imaging results stipulated on a coincidental relationship between metabolic and morphometric borders of cancer, at least within animal models used in preclinical research.

Tumor cells that preferentially enter circulation include the precursors of metastatic cancer. Previously, we characterized circulating tumor cells (CTC) from patients with breast cancer and identified a signature of genomic regions with recurrent copy number gains. Through fluorescence in situ hybridization, we now show that these CTC-associated regions are detected within the matched untreated primary tumors of these patients (21-69%, median 55.5%, n=19). Furthermore, they are more prevalent in the metastases of patients who died from breast cancer after multiple rounds of treatment (70-100%, median 93%, n=41). Diversity indices revealed that higher spatial heterogeneity for these regions within primary tumors is associated with increased dissemination and metastasis. An identified subclone with multiple regions gained (MRG clone) was enriched in a post-treatment primary breast carcinoma as well as multiple metastatic tumors and local breast recurrences obtained at autopsy, indicative of a distinct early subclone with the capability to resist multiple lines of treatment and eventually cause death. Additionally, multiplex immunofluorescence revealed that tumor heterogeneity is significantly associated with the degree of infiltration of B lymphocytes in triple-negative breast cancer, a subtype with a large immune component. Collectively, these data reveal the functional potential of genetic subclones that comprise heterogeneous primary breast carcinomas and are selected for in CTCs and post-treatment breast cancer metastases. In addition, they uncover a relationship between tumor heterogeneity and host immune response in the tumor microenvironment.

Macrophage colony stimulating factor-1 (CSF-1) plays a critical role in maintaining myeloid lineage cells. However, congenital global deficiency of CSF-1 (Csf1

Hematopoietic stem and progenitor cells reside within specialized niches. Colony stimulating factor 1 (CSF1) might serve as a monocyte niche factor given its critical role in regulating survival, proliferation, and differentiation of myeloid lineage cells. However, the role of CSF1 in adult monopoiesis and its functionally important cellular sources remain uncertain. Here, we show that conditional deletion of Csf1 in adulthood regulated monocyte abundance independent of its effects on bone development. Bone histology revealed monocytes reside near sinusoidal endothelial cells (ECs) and leptin receptor (Lepr)-expressing perivascular stromal cells. Concomitantly, targeted in vivo deletion of Csf1 from sinusoidal but not arteriolar ECs selectively reduced Ly6Cnegative monocytes in the bone marrow and blood. Combined depletion of Csf1 from both Lepr-expressing perivascular stromal cells and ECs had additive effects, further decreasing classical Ly6Chigh cells. Thus, different monocyte subsets are supported by distinct cellular sources of CSF1 within a perivascular bone marrow niche.%%%%M.Sc.

The formation of hypoxic microenvironments within solid tumors is known to contribute to radiation resistance, chemotherapy resistance, immune suppression, increased metastasis, and an overall poor prognosis. It is therefore crucial to understand the spatial and molecular mechanisms that contribute to tumor hypoxia formation to improve the efficacy of radiation treatment, develop hypoxia-directed therapies, and increase patient survival. The objective of this study is to present a number of complementary novel methods for quantifying tumor hypoxia and proliferation, especially in relation to the location of perfused blood vessels.Multiplexed immunofluorescence staining can produce whole slide scanned image datasets that are amenable for computational pathology analysis. A standard marker analysis strategy is to take a positive pixel count approach, in which a threshold for positive stain is used to compute a positive area fraction for hypoxia. This work is a reassessment of that approach, utilizing not only cell segmentation but also distance to nearest blood vessel in order to incorporate spatial information into the analysis. We describe a reproducible pipeline for the visualization and quantitative analysis of hypoxia using a vessel distance analysis approach. This methodological pipeline can serve to further elucidate the relationship between vessel distance and microenvironment-linked markers such as hypoxia and proliferation, can help to quantify parameters relating to oxygen consumption and hypoxic tolerance in tissues, as well as potentially serve as a hypothesis generating tool for future studies testing hypoxia-linked markers.

Multiple sclerosis (MS) is characterized by demyelinated and inflammatory lesions in the brain and spinal cord that are highly variable in terms of cellular content. Here, we used imaging mass cytometry (IMC) to enable the simultaneous imaging of 15+ proteins within staged MS lesions. To test the potential for IMC to discriminate between different types of lesions, we selected a case with severe rebound MS disease activity after natalizumab cessation. With post-acquisition analysis pipelines we were able to: (1) Discriminate demyelinating macrophages from the resident microglial pool; (2) Determine which types of lymphocytes reside closest to blood vessels; (3) Identify multiple subsets of T and B cells, and (4) Ascertain dynamics of T cell phenotypes vis-à-vis lesion type and location. We propose that IMC will enable a comprehensive analysis of single-cell phenotypes, their functional states and cell-cell interactions in relation to lesion morphometry and demyelinating activity in MS patients., eLife digest It takes an army of immune cells to defend the body against infection. But sometimes the body’s immune system mistakenly attacks its own cells and chronic inflammatory conditions develop. In multiple sclerosis – also known as “MS” – a horde of immune cells infiltrate the brain and spinal cord, forming lesions which strip nerve cells of their insultation, a protective fatty material called myelin. Nerve cells become damaged, scarred and exposed, and this interferes with messages between the brain and other parts of the body. Advanced imaging techniques have revolutionized the diagnosis of multiple sclerosis by capturing lesions as they develop in the brain and spinal cord. Researchers have also focused their efforts on understanding how immune cells activated in the blood stream invade the central nervous system. To better understand how a mistaken immune response leads to nerve damage in multiple sclerosis, a forensic examination of which immune cells accumulate in brain tissue to form lesions is needed. Standard techniques for analyzing whole tissue samples are however limited by design, capable of detecting only a few cell markers in one section of tissue. Ramaglia et al. have now validated a new imaging technique for looking at an array of cell types in brain tissue in a single sample. The technique – called imaging mass cytometry (or IMC for short) – was used to look at post-mortem brain tissue from a multiple sclerosis patient with an acute form of the illness. The tissue examined had multiple sclerosis lesions present. Different types of immune cells were simultaneously identified and characterized using a panel of antibodies which recognize the signature proteins each immune cell makes when active. The state of the underlying myelin content of the tissue was also characterized. The imaging approach could distinguish between the immune cells of the brain (known as resident microglia) and a type of white blood cell summoned as part of the immune response (infiltrating macrophages). The analysis showed that, in the particular patient examined, microglia are abundant in active lesions in multiple sclerosis; also, different subsets of white blood cells were detected. Measuring how far different immune cells had migrated from nearby blood vessels added insights as to how immune cells move through the brain and which cells may have arrived first. Altogether, Ramaglia et al. have shown that IMC can be used as a discovery tool to gain a deeper understanding of multiple sclerosis lesions and immune cells active in the inflamed brain. Further work will apply this now validated imaging approach to large cohorts of multiple sclerosis patients.

Background: The appropriate location for biopsy procurement relative to an ulcer in active Crohn's disease is unknown. Aim: To explore the relationship between biopsy location, histological disease activity, proinflammatory gene expression and the presence of inflammatory cells. Methods: Fifty-one patients with Crohn's disease and ulcers >0.5 cm diameter in the colon and/or ileum were prospectively enrolled at three centres. Biopsies were obtained from 0 mm, 7 to 8 mm and 21 to 24 mm from the edge of the largest ulcer. Histological activity was blindly assessed with the Global Histological Disease Activity Score, the Robarts Histopathology and Nancy Histological indices. Messenger ribonucleic acid (mRNA) levels for interleukins-6, -8 and -23 (p19 and p40 subunits), CD31 and S100A9 were measured using quantitative polymerase chain reaction. The number of CD3+, CD68+ and myeloperoxidase-positive cells was quantified by immunohistochemistry. Data were analysed using mixed models with location and segment as fixed effects and patients as random effect to account for correlation among segments within a patient. Results: Histological disease activity scores (P

Multiple Sclerosis (MS) is characterized by demyelinated and inflammatory lesions in the brain and spinal cord. Lesions contain immune cells with variable phenotypes and functions. Here we use imaging mass cytometry (IMC) to enable the simultaneous imaging of 15+ proteins within 11 staged MS lesions. Using this approach, we demonstrated that the majority of demyelinating macrophage-like cells in active lesions were derived from the resident microglial pool. Although CD8+ T cells predominantly infiltrated the lesions, CD4+ T cells were also abundant but localized closer to blood vessels. B cells with a predominant switched memory phenotype were enriched across all lesion stages and were found to preferentially infiltrate the tissue as compared to unswitched B cells which localized to the vasculature. We propose that IMC will enable a comprehensive analysis of single-cell phenotypes, their functional states and cell-cell interactions in relation to lesion morphometry and demyelinating activity in the MS brain.

Objective Discoidin domain receptor 1 (DDR1) is a collagen binding receptor tyrosine kinase implicated in atherosclerosis, fibrosis, and cancer. Our previous research showed that DDR1 could regulate smooth muscle cell trans-differentiation, fibrosis and calcification in the vascular system in cardiometabolic disease. This spectrum of activity led us to question whether DDR1 might also regulate adipose tissue fibrosis and remodeling. Methods We have used a diet-induced mouse model of cardiometabolic disease to determine whether DDR1 deletion impacts upon adipose tissue remodeling and metabolic dysfunction. Mice were fed a high fat diet (HFD) for 12 weeks, followed by assessment of glucose and insulin tolerance, respiration via indirect calorimetry, and brown fat activity by FDG-PET. Results Feeding HFD induced DDR1 expression in white adipose tissue, which correlated with adipose tissue expansion and fibrosis. Ddr1−/− mice fed an HFD had improved glucose tolerance, reduced body fat, and increased brown fat activity and energy expenditure compared to Ddr1+/+ littermate controls. HFD-fed DDR1−/− mice also had reduced fibrosis, smaller adipocytes with multilocular lipid droplets, and increased UCP-1 expression characteristic of beige fat formation in subcutaneous adipose tissue. In vitro, studying C3H10T1/2 cells stimulated to differentiate, DDR1 inhibition caused a shift from white to beige adipocyte differentiation, whereas DDR1 expression was increased with TGFβ-mediated pro-fibrotic differentiation. Conclusion This study is the first to identify a role for DDR1 as a driver of adipose tissue fibrosis and suppressor of beneficial beige fat formation., Graphical abstract Image 1, Highlights • DDR1 deletion results in decreased obesity, and increased energy expenditure and brown fat activity. • DDR1 expression was increased in adipose and correlated with obesity and fibrosis. • DDR1 deletion increased UCP-1 expression in brown and white fat in vivo, and in mesenchymal cells in vitro. • In vitro studies suggest that DDR1 suppresses UCP-1 and drives pro-fibrotic differentiation of mesenchymal cells.

Dynamic simulations of the isotropic-nematic phase transformation of liquid crystal droplets under homeotropic anchoring are found to predict chiral symmetry-breaking dynamics. These observations occur when using material parameters for pentyl-cyanobiphenyl (5CB) but not under the single elastic constant approximation of this material, frequently used in simulation. The twisting dynamic process occurs during the relaxation of the domain from an unstable radial texture to a stable uniform texture and involves simultaneous defect loop motion and twisting of the bulk nematic texture., 5 pages, 3 figures, submitted to Physical Review Letters

Polymer-dispersed liquid crystal (PDLC) composites have long been a focus of study for their unique electro-optical properties which have resulted in various applications such as switchable (transparent/translucent) windows. These composites are manufactured using desirable "bottom-up" techniques, such as phase separation of a liquid crystal/polymer mixture, which enable production of PDLC films at very large scales. LC domains within PDLCs are typically spheroidal, as opposed to rectangular for an LCD panel, and thus exhibit substantially different behaviour in the presence of an external field. The fundamental difference between spheroidal and rectangular nematic domains is that the former results in the presence of nanoscale orientational defects in LC order while the latter does not. Progress in the development and optimization of PDLC electro-optical properties has progressed at a relatively slow pace due to this increased complexity. In this work, continuum simulations are performed in order to capture the complex formation and electric field-driven switching dynamics of approximations of PDLC domains. Using a simplified elliptic cylinder (microcapillary) geometry as an approximation of spheroidal PDLC domains, the effects of geometry (aspect ratio), surface anchoring, and external field strength are studied through the use of the Landau--de Gennes model of the nematic LC phase., 22 pages, 9 figures, Physical Review E

A novel microstrip diplexer with low insertion loss, high selectivity and isolation performance is proposed through the combination of two compact filters composed of short-circuit centred stepped impedance resonators and, a side-coupled shorted microstrip line, which are designed for GPS and UWB application, respectively. Due to the intrinsic characteristics and mutual loading effect of the two filters, multiple transmission zeros can be created to improve the out-of-band rejection and isolation of the diplexer. More compact size is obtained without an extra junction matching networks. Simulated results of the fabricated diplexer agree well with measured ones.

A force balance model was developed to predict the effects of particle size, particle size distribution and surface potential on the structure of the filter cake. The model predicts that a stable filter cake is formed at low surface potentials and that the filter cake becomes unstable when the surface potential is larger than 30 mV. The model predicts a minimum porosity and permeability for filter cakes formed at intermediate surface potential. The surface potential that produces the lowest porosity increases with increasing particle size in the system. The specific resistance of the filter cake was a function of particle size and surface potential. The surface potential for the maximum specific resistance in the uniform particle system agreed with reports in the literature.

A topologically-informed method is presented for seeding of hyperstreamlines for visualization of alignment tensor fields. The method is inspired by and applied to visualization of nematic liquid crystal (LC) reorientation dynamics simulations. The method distributes hyperstreamlines along domain boundaries and edges of a nearest-neighbor graph whose vertices are degenerate regions of the alignment tensor field, which correspond to orientational defects in a nematic LC domain. This is accomplished without iteration while conforming to a user-specified spacing between hyperstreamlines and avoids possible failure modes associated with hyperstreamline integration in the vicinity of degeneracies of alignment (orientational defects). It is shown that the presented seeding method enables automated hyperstreamline-based visualization of a broad range of alignment tensor fields which enhances the ability of researchers to interpret these fields and provides an alternative to using glyph-based techniques., Comment: 8 pages, 9 figures

This paper discusses the preferred study methods of Chinese students based on the research findings involving Chinese students studying in the School of Business, Wintec. In order to provide relevant academic delivery, quality education and to maintain sustainability in the international market, New Zealand education institutes have to understand Chinese students background, behaviour and learning difficulties while they are studying in New Zealand.