Colony stimulating factor-1 producing endothelial cells and mesenchymal stromal cells maintain monocytes within a perivascular bone marrow niche.

Macrophage colony stimulating factor-1 (CSF-1) plays a critical role in maintaining myeloid lineage cells. However, congenital global deficiency of CSF-1 (Csf1op/op) causes severe musculoskeletal defects that may indirectly affect hematopoiesis. Indeed, we show here that osteolineage-derived Csf1 prevented developmental abnormalities but had no effect on monopoiesis in adulthood. However, ubiquitous deletion of Csf1 conditionally in adulthood decreased monocyte survival, differentiation, and migration, independent of its effects on bone development. Bone histology revealed that monocytes reside near sinusoidal endothelial cells (ECs) and leptin receptor (Lepr)-expressing perivascular mesenchymal stromal cells (MSCs). Targeted deletion of Csf1 from sinusoidal ECs selectively reduced Ly6C- monocytes, whereas combined depletion of Csf1 from ECs and MSCs further decreased Ly6Chi cells. Moreover, EC-derived CSF-1 facilitated recovery of Ly6C- monocytes and protected mice from weight loss following induction of polymicrobial sepsis. Thus, monocytes are supported by distinct cellular sources of CSF-1 within a perivascular BM niche.

Mass Spectrometry Imaging Reveals a Gradient of Cancer-like Metabolic States in the Vicinity of Cancer Not Seen in Morphometric Margins from Microscopy.

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.

Formation and field-driven dynamics of nematic spheroids.

Unlike the canonical application of liquid crystals (LCs), LC displays, emerging technologies based on LC materials are increasingly leveraging the presence of nanoscale defects. The inherent nanoscale characteristics of LC defects present both significant opportunities as well as barriers for the application of this fascinating class of materials. Simulation-based approaches to the study of the effects of confinement and interface anchoring conditions on LC domains has resulted in significant progress over the past decade, where simulations are now able to access experimentally-relevant length scales while simultaneously capturing nanoscale defect structures. In this work, continuum simulations were performed in order to study the dynamics of micron-scale nematic LC spheroids of varying shape. Nematic spheroids are one of the simplest inherently defect-containing LC structures and are relevant to polymer-dispersed LC-based "smart" window technology. Simulation results include nematic phase formation and external field-switching dynamics of nematic spheroids ranging in shape from oblate to prolate. Results include both qualitative and quantitative insight into the complex coupling of nanoscale defect dynamics and structure transitions to micron-scale reorientation. Dynamic mechanisms are presented and related to structural transitions in LC defects present in the nematic domain. Domain-averaged metrics including order parameters and response times are determined for a range of experimentally-accessible electric field strengths. These results have both fundamental and technological relevance, in that increased understanding of LC dynamics in the presence of defects is a key barrier to continued advancement in the field.

Deep Learning Accurately Quantifies Plasma Cell Percentages on CD138-Stained Bone Marrow Samples.

The diagnosis of plasma cell neoplasms requires accurate, and ideally precise, percentages. This plasma cell percentage is often determined by visual estimation of CD138-stained bone marrow biopsies and clot sections. While not necessarily inaccurate, estimates are by definition imprecise. For this study, we hypothesized that deep learning can be used to improve precision. We trained a semantic segmentation-based convolutional neural network (CNN) using annotations of CD138+ and CD138- cells provided by one pathologist on small image patches of bone marrow and validated the CNN on an independent test set of image patches using annotations from two pathologists and a non-deep learning commercial software. On validation, we found that the intraclass correlation coefficients for plasma cell percentages between the CNN and pathologist #1, a non-deep learning commercial software and pathologist #1, and pathologists #1 and #2 were 0.975, 0.892, and 0.994, respectively. The overall results show that CNN labels were almost as accurate as pathologist labels at a cell-by-cell level. Once satisfied with performance, we scaled-up the CNN to evaluate whole slide images (WSIs), and deployed the system as a workflow friendly web application to measure plasma cell percentages using snapshots taken from microscope cameras.

Heterogeneity of Circulating Tumor Cell-Associated Genomic Gains in Breast Cancer and Its Association with the Host Immune Response.

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 FISH, we now show that these CTC-associated regions are detected within the matched untreated primary tumors of these patients (21% to 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% to 100%, median 93%, samples 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 posttreatment 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. In addition, 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 posttreatment breast cancer metastases. In addition, they uncover a relationship between tumor heterogeneity and host immune response in the tumor microenvironment. SIGNIFICANCE: As breast cancers progress, they become more heterogeneous for multiple regions amplified in circulating tumor cells, and intratumoral spatial heterogeneity is associated with the immune landscape.

Discoidin domain receptor 1-deletion ameliorates fibrosis and promotes adipose tissue beiging, brown fat activity, and increased metabolic rate in a mouse model of cardiometabolic disease.

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 TGFß-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.

Quantitative Visualization of Hypoxia and Proliferation Gradients Within Histological Tissue Sections.

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 in multiplexed immunofluorescence images, especially in relation to the location of perfused blood vessels. 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.

Multiplexed imaging of immune cells in staged multiple sclerosis lesions by mass cytometry.

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.

Evaluation of optimal biopsy location for assessment of histological activity, transcriptomic and immunohistochemical analyses in patients with active Crohn's disease.

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 < 0.0001), proinflammatory gene expression levels (P < 0.005) and numbers of myeloperoxidase-positive cells (P < 0.0001) were highest in biopsies from the ulcer edge in the colon and ileum, with decreasing gradients observed with distance from the edge (P < 0.05). No differences between colonic and ileal samples were detected for the parameters measured at any location. CONCLUSIONS: Biopsies from the ulcer edge in patients with Crohn's disease yielded the greatest histological disease activity and mRNA levels and had similar readouts in the colon and ileum. Research is needed to confirm this conclusion for other measures.

A Topologically-Informed Hyperstreamline Seeding Method for Alignment Tensor Fields.

A topologically-informed hyperstreamline seeding method is presented for visualization of alignment tensor fields. The method is inspired by and applied to visualization of nematic liquid crystal (LC) orientation 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 in 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.

Field-driven dynamics of nematic microcapillaries.

Polymer-dispersed liquid-crystal (PDLC) composites long have been a focus of study for their unique electro-optical properties which have resulted in various applications such as switchable (transparent or 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 behavior 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.