Mixed Cell Populations Research Articles

Abstract 4628: Profiling the tumor microenvironments of triple- and quadruple-negative breast cancers

Abstract Triple-negative breast cancer (TNBC) is a heterogeneous breast cancer subtype that lacks expression of estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2. Approximately 15%-20% of breast cancers are triple-negative. Based on expression of the androgen receptor (AR), TNBC can be categorized as AR-positive (AR+ TNBC) or AR-negative (quadruple-negative breast cancer, QNBC). Studies have reported that the loss of AR expression is associated with poor prognoses. Recent evidence suggests that QNBCs disproportionately affect women of African descent and contributes to poor overall survival. QNBC is a more aggressive disease compared to TNBC, and patients with QNBC currently have no treatment options beyond chemotherapy. Data from interrogating public databases such as TCGA indicates that QNBC is distinct from TNBC in molecular makeup, particularly in immune tumor microenvironment. Currently, QNBC is not considered distinct from the broader TNBC subtype, hence a comprehensive molecular characterization is lacking. We aim to examine differences between the TNBC and QNBC tumor microenvironments that consist of both immune and stromal components. We analyzed RNA expression data from the METABRIC (TNBC= 11, QNBC= 185) and TCGA (TNBC= 29, QNBC= 103) datasets. Based on AR transcript expression, patients with TNBC were stratified into AR-low (considered QNBC) and AR-high (deemed ‘true’ TNBC) groups. The analytical immune tools - CIBERSORTx and xCELL were used to analyze the RNA-Seq data. CIBERSORTx can distinguish 22 mature human hematopoietic populations and provides abundance of member cell types in a mixed cell population, from gene expression data. xCELL is a gene signature-based method that can perform cell type enrichment analysis from gene expression for 64 immune and stromal cell types. Thus, xCell can discriminate between closely related cell types. Using these immune tools, the tumor microenvironments of the TNBC and QNBC groups were compared. Correlations between individual immune and stromal components with patient survival probability were assessed. Significant differences (p-value <0.05) in several immune and stromal cell types were noted when comparing the TNBC and QNBC groups within these datasets. CIBERSORTx and xCELL yielded both unique and common sets of cell types. Notably, significant differences in the stroma score (a composite score of stromal cell types provided by xCELL) were observed between TNBC and QNBC in both the METABRIC and TCGA datasets. These immune tools are based on different methodologies and our results show that they can provide complementary information. Our data suggest that QNBC and TNBC have distinct tumor microenvironments, and that QNBC should be considered as an independent breast cancer subtype that warrants comprehensive characterization. Citation Format: Geetanjali Saini, Manali Rupji, Mahak Bhargava, Sunil Badve, Ritu Aneja. Profiling the tumor microenvironments of triple- and quadruple-negative breast cancers. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4628.

Abstract 186: Cellos: High-throughput deconvolution of 3D organoid dynamics at cellular resolution for cancer pharmacology

Abstract Three-dimensional (3D) culture models, such as organoids, are flexible systems to interrogate cellular growth, multicellular spatial architecture, and morphology in response to drug treatment, including the potential to resolve the individual behaviors and interactions of mixed cancer cell populations. However, new computational methods to segment and analyze 3D models at cellular resolution are needed to realize these possibilities. Here we report Cellos (Cell Organoid Segmentation), an accurate, high throughput image analysis pipeline for 3D organoid and nuclear segmentation analysis. Cellos segments 3D organoids using classical algorithms and segments nuclei using a Stardist-3D convolutional neural network trained on a manually annotated dataset of 3,862 cells from 36 organoids confocally imaged at 5 μm z-resolution. To evaluate the capabilities of Cellos, we then analyzed 74,450 organoids with 1.65 million cells from multiple experiments on triple negative breast cancer organoids containing clonal mixtures with complex cisplatin sensitivity. Cellos was able to accurately distinguish cell ratios in organoids in 96-well plates to within 2.99% and was effective for fluorescently labelled nuclei and independent DAPI stained datasets. Cellos was able to recapitulate traditional luminescence based drug responses by analyzing 3D images, including parallel analysis of multiple cancer clones in the same well. Moreover, Cellos was able to identify organoid and nuclear morphology feature changes associated with treatment. Finally, Cellos enables 3D analysis of cell spatial relationships, which we used to detect ecological affinity between cancer cells, beyond expectations from local cell division or organoid composition. Cellos provides powerful tools to perform high throughput analysis for pharmacological testing and biological investigation of organoids based on 3D imaging. Citation Format: Patience Mukashyaka, Pooja Kumar, Dave Mellert, Shadae Nicholas, Javad Noorbakhsh, Mattia Brugiolo, Olga Anczukow, Edison T. Liu, Jeffrey H. Chuang. Cellos: High-throughput deconvolution of 3D organoid dynamics at cellular resolution for cancer pharmacology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 186.

Use of multi-color flow cytometry for canine immune cell characterization in cancer.

Although immunotherapy is becoming a standard approach of human cancer treatment, only a small but critical fraction of patients responds to the therapy. It is therefore required to determine the sub-populations of patients who will respond to immunotherapies along with developing novel strategies to improve efficacy of anti-tumor immune reactions. Current development of novel immunotherapies relies heavily on mouse models of cancer. These models are important for better understanding of mechanisms behind tumor immune escape and investigation of novel strategies to overcome it. Nevertheless, the murine models do not necessarily represent the complexity of spontaneously occurring cancers in humans. Dogs spontaneously develop a wide range of cancer types with an intact immune system under similar environment and exposure to humans, which can serve as translational models in cancer immunotherapy research. To date though, there is still a relatively limited amount of information regarding immune cell profiles in canine cancers. One possible reason could be that there are hardly any established methods to isolate and simultaneously detect a range of immune cell types in neoplastic tissues. To date only a single manuscript describes characterization of immune cells in canine tumour tissues, concentrating solely on T-cells. Here we describe a protocol for multi-color flow cytometry to distinguish immune cell types in blood, lymph nodes, and neoplastic tissues from dogs with cancer. Our results demonstrate that a 9-color flow cytometry panel enables characterization of different cell subpopulations including myeloid cells. We also show that the panel allows detection of minor/aberrant subsets within a mixed population of cells in various neoplastic samples including blood, lymph node and solid tumors. To our knowledge, this is the first simultaneous immune cell detection panel applicable for solid tumors in dogs. This multi-color flow cytometry panel has the potential to inform future basic research focusing on immune cell functions in translational canine cancer models.

Determination of Intracellular Esterase Activity Using Ratiometric Raman Sensing and Spectral Phasor Analysis

Carboxylesterases (CEs) are a class of enzymes that catalyze the hydrolysis of esters in a variety of endogenous and exogenous molecules. CEs play an important role in drug metabolism, in the onset and progression of disease, and can be harnessed for prodrug activation strategies. As such, the regulation of CEs is an important clinical and pharmaceutical consideration. Here, we report the first ratiometric sensor for CE activity using Raman spectroscopy based on a bisarylbutadiyne scaffold. The sensor was shown to be highly sensitive and specific for CE detection and had low cellular cytotoxicity. In hepatocyte cells, the ratiometric detection of esterase activity was possible, and the result was validated by multimodal imaging with standard viability stains used for fluorescence microscopy within the same cell population. In addition, we show that the detection of localized ultraviolet damage in a mixed cell population was possible using stimulated Raman scattering microscopy coupled with spectral phasor analysis. This sensor demonstrates the practical advantages of low molecular weight sensors that are detected using ratiometric Raman imaging and will have applications in drug discovery and biomedical research.

Hodgkin lymphoma in the bone marrow aspirate of a young patient with human immunodeficiency virus.

Hodgkin lymphoma in the bone marrow aspirate of a young patient with human immunodeficiency virus.

A Simplified and Effective Approach for the Isolation of Small Pluripotent Stem Cells Derived from Human Peripheral Blood

Pluripotent stem cells are key players in regenerative medicine. Embryonic pluripotent stem cells, despite their significant advantages, are associated with limitations such as their inadequate availability and the ethical dilemmas in their isolation and clinical use. The discovery of very small embryonic-like (VSEL) stem cells addressed the aforementioned limitations, but their isolation technique remains a challenge due to their small cell size and their efficiency in isolation. Here, we report a simplified and effective approach for the isolation of small pluripotent stem cells derived from human peripheral blood. Our approach results in a high yield of small blood stem cell (SBSC) population, which expresses pluripotent embryonic markers (e.g., Nanog, SSEA-3) and the Yamanaka factors. Further, a fraction of SBSCs also co-express hematopoietic markers (e.g., CD45 and CD90) and/or mesenchymal markers (e.g., CD29, CD105 and PTH1R), suggesting a mixed stem cell population. Finally, quantitative proteomic profiling reveals that SBSCs contain various stem cell markers (CD9, ITGA6, MAPK1, MTHFD1, STAT3, HSPB1, HSPA4), and Transcription reg complex factors (e.g., STAT5B, PDLIM1, ANXA2, ATF6, CAMK1). In conclusion, we present a novel, simplified and effective isolating process that yields an abundant population of small-sized cells with characteristics of pluripotency from human peripheral blood.

Cover Story: Eur. J. Immunol. 3'23

European Journal of ImmunologyVolume 53, Issue 3 2370031 Cover PictureFree Access Cover Story: Eur. J. Immunol. 3'23 First published: 15 March 2023 https://doi.org/10.1002/eji.202370031AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Graphical Abstract Our cover features an immunofluorescence image of native mouse thymic epithelial cells (TEC) after culture on a polymer substrate identified by Rouse et al. in a novel medium throughput screening assay. The screen was designed to identify synthetic polymers able to support mouse and human TEC in culture. The primary screen employed induced TEC, produced in vitro by reprogramming. This image from the secondary screen shows cells grown on polymer ID287; TEC are identified among a mixed thymic cell population using a pan-cytokeratin antibody (green). The original image was digitally modified for the cover. Volume53, Issue3March 20232370031 RelatedInformation

Packaging cells for lentiviral vectors generated using the cumate and coumermycin gene induction systems and nanowell single-cell cloning

Lentiviral vectors (LVs) are important for cell therapy because of their capacity to stably modify the genome after integration. This study describes a novel and relatively simple approach to generate packaging cells and producer clones for self-inactivating (SIN) LVs pseudotyped with the vesicular stomatitis virus glycoprotein (VSV-G). A novel gene regulation system, based on the combination of the cumate and coumermycin induction systems, was developed to ensure tight control for the expression of cytotoxic packaging elements. To accelerate clone isolation and ensure monoclonality, the packaging genes were transfected simultaneously into human embryonic kidney cells (293SF-3F6) previously engineered with the induction system, and clones were isolated after limiting dilution into nanowell arrays using a robotic cell picking instrument with scanning capability. The method's effectiveness to isolate colonies derived from single cells was demonstrated using mixed populations of cells labeled with two different fluorescent markers. Because the recipient cell line grew in suspension culture, and all the procedures were performed without serum, the resulting clones were readily adaptable to serum-free suspension culture. The best producer clone produced LVs expressing GFP at a titer of 2.3×108 transduction units (TU)/mL in the culture medium under batch mode without concentration.

Cell-Selective Multifunctional Surface Covalent Reconfiguration Using Aptamer-Enabled Proximity Catalytic Labeling.

Cell surface engineering provides access to custom-made cell interfaces with desirable properties and functions. However, cell-selective covalent labeling methods that can simultaneously install multiple molecules with different functions are scarce. Herein, we report an aptamer-enabled proximity catalytic covalent labeling platform for multifunctional surface reconfiguration of target cells in mixed cell populations. By conjugating peroxidase with cell-selective aptamers, the probes formed can selectively bind target cells and catalyze target-cell-localized covalent labeling in situ. The universal applicability of the platform to different phenol-modified functional molecules allows us to perform a variety of manipulations on target cells, including labeling, tracking, assembly regulation, and surface remodeling. In particular, the platform has the ability of multiplexed covalent labeling, which can be used to install two mutually orthogonal click reactive molecules simultaneously on the surface of target cells. We thus achieve "multitasking" in complex multicellular systems: programming and tracking specific cell-cell interactions. We further extend the functional molecules to carbohydrates and perform ultrafast neoglycosylation on target living cells. These newly introduced sugars on the cell membrane can be recognized and remodeled by a glycan-modifying enzyme, thus providing a method package for cell-selective engineering of the glycocalyx.

The impact of spatial correlation on methylation entropy with application to mouse brain methylome

BackgroundWith the advance of bisulfite sequencing technologies, massive amount of methylation data have been generated, which provide unprecedented opportunities to study the epigenetic mechanism and its relationship to other biological processes. A commonly seen feature of the methylation data is the correlation between nearby CpG sites. Although such a spatial correlation was utilized in several epigenetic studies, its interaction to other characteristics of the methylation data has not been fully investigated.ResultsWe filled this research gap from an information theoretic perspective, by exploring the impact of the spatial correlation on the methylation entropy (ME). With the spatial correlation taken into account, we derived the analytical relation between the ME and another key parameter, the methylation probability. By comparing it to the empirical relation between the two corresponding statistics, the observed ME and the mean methylation level, genomic loci under strong epigenetic control can be identified, which may serve as potential markers for cell-type specific methylation. The proposed method was validated by simulation studies, and applied to analyze a published dataset of mouse brain methylome.ConclusionsCompared to other sophisticated methods developed in literature, the proposed method provides a simple but effective way to detect CpG segments under strong epigenetic control (e.g., with bipolar methylation pattern). Findings from this study shed light on the identification of cell-type specific genes/pathways based on methylation data from a mixed cell population.

Harnessing CRISPR-Cas9 as an anti-mycobacterial system

Rapid emergence of drug resistance has posed new challenges to the treatment of mycobacterial infections. As the pace of development of new drugs is slow, alternate treatment approaches are required. Recently, CRISPR-Cas systems have emerged as potential antimicrobials. These sequence-specific nucleases introduce double strand cuts in the target DNA, which if left unrepaired, prove fatal to the host. For most bacteria, homologous recombination repair (HRR) is the only pathway for repair and survival. Mycobacteria is one of the few bacteria which possesses the non-homologous end joining (NHEJ) system in addition to HRR for double strand break repair. To assess the antimicrobial potential of CRISPR-system, Cas9-induced breaks were introduced in the genome of Mycobacterium smegmatis and the survival was studied. While the single strand breaks were efficiently repaired, the organism was unable to repair the double strand breaks efficiently. In a mixed population of antibiotic-resistant and sensitive mycobacterial cells, selectively targeting a factor that confers hygromycin resistance, turned the entire population sensitive to the drug. Further, we demonstrate that the sequence-specific targeting could also be used for curing plasmids from mycobacterium cells. Considering the growing interest in nucleic acid-based therapy to curtail infections and combat antimicrobial resistance, our data shows that CRISPR-systems hold promise for future use as an antimicrobial against drug-resistant mycobacterial infections.

Selective ablation of primary and paracrine senescent cells by targeting iron dyshomeostasis.

Senescent cells can spread the senescent phenotype to other cells by secreting senescence-associated secretory phenotype factors. The resulting paracrine senescent cells make a significant contribution to the burden of senescent cell accumulation with age. Previous efforts made to characterize paracrine senescence are unreliable due to analyses being based on mixed populations of senescent and non-senescent cells. Here, we use dipeptidyl peptidase-4 (DPP4) as a surface maker to isolate senescent cells from mixed populations. Using this technique, we enrich the percentage of paracrine senescence from 40% to 85%. We then use this enriched culture to characterize DPP4+ primary and paracrine senescent cells. We observe ferroptosis dysregulation and ferrous iron accumulation as a common phenomenon in both primary and paracrine senescent cells. Finally, we identify ferroptosis induction and ferrous iron-activatable prodrug as a broad-spectrum senolytic approach to ablate multiple types of primary and paracrine senescent cells.

Abstract A008: An indocyanine green-based liquid biopsy test for circulating tumor cells for hepatoblastoma

Abstract Introduction: Hepatoblastoma (HB) is the most common pediatric primary liver tumor and has the fastest rising incidence of all pediatric solid tumors. Tumor progression is monitored with MRI, chest CT, and serum levels of Alpha fetoprotein. Unfortunately, these assays involve exposing children to radiation and anesthesia and are not always accurate. The goal of this work is to develop and validate a liquid biopsy test for circulating tumor cells (CTCs) with a panel of HB-specific markers to provide a less invasive and more accurate way of assessing disease. Methods: We first explored accumulation of the far-red fluorescent dye indocyanine green (ICG) in widely available cell lines (HepG2, Huh-6, Hep3B, HepRG, 293T, SH-SY5Y, A549), our own HB patient-derived cell line (HB17), and primary fibroblasts. We assessed ICG accumulation with fluorescence microscopy and flow cytometry, including standard flow cytometry on a BD Symphony instrument and imaging flow cytometry on the Amnis ImageStream X MK II instrument. Based on this data, we then developed a new method for quantifying CTC burden from patient whole blood samples using HB-specific markers ICG, Glypican-3 (GPC3), and DAPI with fluorescence microscopy and flow cytometry readouts. We tested this panel with cell lines and non-cancer control blood samples, as well as blood samples from mice harboring intrahepatic patient-derived xenograft (PDX) tumors representing HB. Finally, we used this panel to analyze whole blood samples for CTC burden with a cohort of HB patients and correlated with patient characteristics and outcomes. Results: First, we showed that ICG accumulation is specific to liver cancer cells (HepG2, Huh-6, Hep3B, HB17), compared to non-malignant liver cells (HepRG), non-liver solid tumor cells (SH-SY5Y, A549), and non-malignant cells (293T, primary fibroblasts). In addition, we found that ICG+ liver tumor cells remain positive for at least 96 hours after uptake. We also showed that ICG accumulation can be used to identify HepG2 HB cells in a mixed population of cells down to a dilution of 1:100 (2.7% ICG+ in the HepG2/SH-SY5Y mix versus 1.9% ICG+ in the SH-SY5Y control). Flow cytometry with HepG2 and A549 cell lines with the ICG/GPC3/DAPI panel showed 94.4% of HepG2 cells were triple ICG+/GPC3+/DAPI+ while 0% of A549 cells were triple ICG+/GPC3+/DAPI+. With non-cancer control blood samples, we identified less than 1 ICG+ cell per ml of blood. We showed the presence of triple ICG+/GPC3+/DAPI+ cells in the blood of animals harboring PDX tumors. Most importantly, with a low-risk HB patient sample, we found 2.25 triple ICG+/GPC3+/DAPI+ cells per ml of blood; with a high-risk sample we counted 17.25 triple ICG+/GPC3+/DAPI+ cells per ml of blood. Conclusions: This work shows the translation from the use of ICG to identify HB lesions in patients during surgery to its role in a novel liquid biopsy test for CTCs, which has the real potential to improve the standard of care by offering a more accurate and less invasive means of monitoring disease. Citation Format: Andres F. Espinoza, Roma H. Patel, Bryan W. Armbruster, Pavan Kureti, Saiabhiroop R. Govindu, Sanjeev A. Vasudevan, Sarah E. Woodfield. An indocyanine green-based liquid biopsy test for circulating tumor cells for hepatoblastoma [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr A008.

Spatially Resolved Top-Down Proteomics of Tissue Sections Based on a Microfluidic Nanodroplet Sample Preparation Platform

Conventional proteomic approaches measure the averaged signal from mixed cell populations or bulk tissues, leading to the dilution of signals arising from subpopulations of cells that might serve as important biomarkers. Recent developments in bottom-up proteomics have enabled spatial mapping of cellular heterogeneity in tissue microenvironments. However, bottom-up proteomics cannot unambiguously define and quantify proteoforms, which are intact (i.e., functional) forms of proteins capturing genetic variations, alternatively spliced transcripts and posttranslational modifications. Herein, we described a spatially resolved top-down proteomics (TDP) platform for proteoform identification and quantitation directly from tissue sections. The spatial TDP platform consisted of a nanodroplet processing in one pot for trace samples–based sample preparation system and an laser capture microdissection–based cell isolation system. We improved the nanodroplet processing in one pot for trace samples sample preparation by adding benzonase in the extraction buffer to enhance the coverage of nucleus proteins. Using ∼200 cultured cells as test samples, this approach increased total proteoform identifications from 493 to 700; with newly identified proteoforms primarily corresponding to nuclear proteins. To demonstrate the spatial TDP platform in tissue samples, we analyzed laser capture microdissection–isolated tissue voxels from rat brain cortex and hypothalamus regions. We quantified 509 proteoforms within the union of top-down mass spectrometry–based proteoform identification and characterization and TDPortal identifications to match with features from protein mass extractor. Several proteoforms corresponding to the same gene exhibited mixed abundance profiles between two tissue regions, suggesting potential posttranslational modification–specific spatial distributions. The spatial TDP workflow has prospects for biomarker discovery at proteoform level from small tissue sections.

The Rapid Development of Airway Organoids: A Direct Culture Strategy.

The lung is a complex organ composed of numerous cell types. Exposure to air pollutants, cigarette smoke, bacteria, viruses, and many others may cause injury to the epithelial cells that line the conducting airways and alveoli. Organoids are the 3D self-organising structures grown from stem cells and generated from adult stem and progenitor cells. Lung organoids are fascinating tools to investigate human lung development in vitro. The objective of this study was to establish a rapid method for generating lung organoids with a direct culture strategy. Trachea and lung organoids were derived from mixed cell populations of mice primary airway epithelial cells, fibroblasts, and lung microvascular endothelial cells and directly digested from the whole cell population in the distal lung. The formation of spheres appeared as early as 3days and continued to proliferate until day 5. The generation of trachea and lung organoids self-organised into discrete epithelial structures was formed within less than 10days. We conclude that researchers will be able to examine cellular involvement during organ formation and molecular networks because organoids come in a variety of morphologies and stages of development, and this organoid protocol may be used for modelling lung diseases as a platform for therapeutic purposes and suitable for personalised medicine for respiratory diseases.

Probing Single-Cell Fermentation Fluxes and Exchange Networks via pH-Sensing Hybrid Nanofibers.

The homeostatic control of their environment is an essential task of living cells. It has been hypothesized that, when microenvironmental pH inhomogeneities are induced by high cellular metabolic activity, diffusing protons act as signaling molecules, driving the establishment of exchange networks sustained by the cell-to-cell shuttling of overflow products such as lactate. Despite their fundamental role, the extent and dynamics of such networks is largely unknown due to the lack of methods in single-cell flux analysis. In this study, we provide direct experimental characterization of such exchange networks. We devise a method to quantify single-cell fermentation fluxes over time by integrating high-resolution pH microenvironment sensing via ratiometric nanofibers with constraint-based inverse modeling. We apply our method to cell cultures with mixed populations of cancer cells and fibroblasts. We find that the proton trafficking underlying bulk acidification is strongly heterogeneous, with maximal single-cell fluxes exceeding typical values by up to 3 orders of magnitude. In addition, a crossover in time from a networked phase sustained by densely connected "hubs" (corresponding to cells with high activity) to a sparse phase dominated by isolated dipolar motifs (i.e., by pairwise cell-to-cell exchanges) is uncovered, which parallels the time course of bulk acidification. Our method addresses issues ranging from the homeostatic function of proton exchange to the metabolic coupling of cells with different energetic demands, allowing for real-time noninvasive single-cell metabolic flux analysis.

Clinical dielema-epithelial myoepithelial carcinoma- rare presentation

To describe our experience with a case of epithelial myoepithelial carcinoma that was reported as two different entities and thus proved to be a challenging case.: Retrospective review of histo-pathological reports with detailed work up of final findings and treatment. Four year follow up of the patient shows successful treatment with no recurrence. High index of suspicion and detailed histo-pathological evaluation with IHC testing is needed in tumours presenting with mixed cell population.

Retrospective Evaluation of the Most Frequently Observed Histological Changes in Duodenal and Rectal Mucosal Biopsies in Horses with Recurrent Colic.

Colic, a condition affecting the gastrointestinal tract of horses, manifests as severe pain and may be a life-threatening condition. It is possible to distinguish between an acute, disposable process, as well as recurrent colic symptoms (abdominal pain) caused by an ongoing chronic inflammatory process. This paper presents a retrospective analysis of the histopathological findings of duodenal and rectal samples taken from horses with recurrent colic, with the aim to determine the frequency and extent of inflammation. The samples, i.e., duodenal biopsy (60 samples) and rectal biopsy (17 samples), were taken from 77 horses showing recurrent colic symptoms. Histopathological examination included staining with hematoxylin and eosin. The examination included evaluation of the superficial epithelium, mucosal lamina propria, and submucosa. All samples from the duodenum and rectum showed the presence of leukocyte infiltration in the mucosal lamina propria. The most frequently observed cellular infiltration was a moderate infiltration consisting of lymphocytes and plasma cells in duodenum and mixed populations of plasma cells, lymphocytes, and eosinophilia in the rectum. Mott cells were also noted among the inflammatory infiltrates. More than one-fourth of the horses were found to have shortened intestinal villi. The results presented here showed the involvement of inflammation in the course of recurrent colic, which can be both its cause (by impairing motility and absorption) and its effect (as a result of obstruction or ischemia).

Assessing Spatial Distribution of Multicellular Self-Assembly Enables the Prediction of Phenotypic Heterogeneity in Glioblastoma.

Phenotypic heterogeneity of glioblastomas is a leading determinant of therapeutic resistance and treatment failure. However, functional assessment of the heterogeneity of glioblastomas is lacking. We developed a self-assembly-based assessment system that predicts inter/intracellular heterogeneity and phenotype associations, such as cell proliferation, invasiveness, drug responses, and gene expression profiles. Under physical constraints for cellular interactions, mixed populations of glioblastoma cells are sorted to form a segregated architecture, depending on their preference for binding to cells of the same phenotype. Cells distributed at the periphery exhibit a reduced temozolomide (TMZ) response and are associated with poor patient survival, whereas cells in the core of the aggregates exhibit a significant response to TMZ. Our results suggest that the multicellular self-assembly pattern is indicative of the intertumoral and intra-patient heterogeneity of glioblastomas, and is predictive of the therapeutic response.

A simple transwell-based infection system for obtaining pure populations of VZV-infected cells

Varicella-Zoster virus (VZV) is a human herpesvirus and causes chickenpox and shingles. Research into its molecular virology has been hampered by a lack of methods for generation of high-titre, cell-free infectious virus preparations. VZV propagation and infection in vitro are therefore commonly achieved by co-culture of uninfected ‘target’ cells with infected ‘inoculum’ cells. A major drawback of this approach is that it results in mixed cell populations after infection. To overcome this limitation we developed a transwell-based VZV infection system. Infected inoculum cells and uninfected target cells are spatially separated by a transwell membrane. While cell-cell contact and VZV spread can occur through membrane pores, the two cell populations do not mix. This simple protocol requires no special instrumentation or reagents. We successfully used this system for infection of a range of target cells and obtained pure populations for downstream analyses such as flow cytometry and RT-qPCR. In sum, we developed a broadly applicable approach to study the molecular and cellular biology as well as host-pathogen interactions of VZV.