Tumor associated macrophages in breast cancer progression: implications and clinical relevance.

Macrophages represent an immune cell population characterized by high plasticity and a range of properties and functions. Their activation status and specific phenotype are highly associated with their localization and the environmental cues they receive. The roles of macrophages in cancer development are diverse. Despite their antitumor effects at early stages of the disease, their presence in the tumor microenvironment (TME) has been linked to tumor promotion upon disease establishment. Tumor associated macrophages (TAMs) are key components of breast cancer TME and they have been associated with poor clinical outcomes. High TAM densities were found to correlate with tumor progression, increased metastatic potential and poor prognosis. Interestingly, considerably higher levels of TAMs were found in patients with triple negative breast cancer (TNBC)-the most aggressive type of breast cancer-compared to other types. The present review summarizes recent findings regarding the distinct TAM subsets in the TME and TAM involvement in breast cancer progression and metastasis. It highlights the constant interplay between TAMs and breast cancer cells and its major contribution to the progression of the disease, including such aspects as, polarization of macrophages toward a tumor promoting phenotype, induction of epithelial to mesenchymal transition (EMT) in cancer cells and enhancement of cancer stem cell properties. Further, we discuss the clinical relevance of these findings, focusing on how a better delineation of TAM involvement in breast cancer metastasis will facilitate the selection of more efficient treatment options.

Markers of Dermal Fibroblast Subpopulations for Viable Cell Isolation via Cell Sorting: A Comprehensive Review.

Fibroblasts are among the most abundant cell types in the human body, playing crucial roles in numerous physiological processes, including the structural maintenance of the dermis, production of extracellular matrix components, and mediation of inflammatory responses. Despite their importance, fibroblasts remain one of the least characterized cell populations. The advent of single-cell analysis techniques, particularly single-cell RNA sequencing (scRNA-seq) and fluorescence-activated cell sorting (FACS), has enabled detailed investigations into fibroblast biology. In this study, we present an extensive analysis of fibroblast surface markers suitable for cell sorting and subsequent functional studies. We reviewed over three thousand research articles describing fibroblast populations and their markers, characterizing and comparing subtypes based on their surface markers, as well as their intra- and extracellular proteins. Our detailed analysis identified a variety of distinct fibroblast subpopulations, each with unique markers, characteristics dependent on their location, and the physiological or pathophysiological environment. These findings underscore the diversity of fibroblasts as a cellular population and could lead to the development of novel diagnostic and therapeutic tools.

Mechanical force-activated CD109 on periodontal ligament stem cells governs osteogenesis and osteoclast to promote alveolar bone remodeling.

Mechanical force-mediated bone remodeling is crucial for various physiological and pathological processes involving multiple factors, including stem cells and the immune response. However, it remains unclear how stem cells respond to mechanical stimuli to modulate the immune microenvironment and subsequent bone remodeling. Here, we found that mechanical force induced increased expression of CD109 on periodontal ligament stem cells (PDLSCs) in vitro and in periodontal tissues from the force-induced tooth movement rat model in vivo, accompanied by activated alveolar bone remodeling. Under mechanical force stimulation, CD109 suppressed the osteogenesis capacity of PDLSCs through the JAK/STAT3 signaling pathway, whereas it promoted PDLSC-induced osteoclast formation and M1 macrophage polarization through paracrine. Moreover, inhibition of CD109 in vivo by lentivirus-shRNA injection increased the osteogenic activity and bone density in periodontal tissues. On the contrary, it led to decreased osteoclast numbers and pro-inflammatory factor secretion in periodontal tissues and reduced tooth movement. Mechanistically, mechanical force-enhanced CD109 expression via the repression of miR-340-5p. Our findings uncover a CD109-mediated mechanical force response machinery on PDLSCs, which contributes to regulating the immune microenvironment and alveolar bone remodeling during tooth movement.

Inter-laboratory multiplex bead-based surface protein profiling of MSC-derived EV preparations identifies MSC-EV surface marker signatures.

Mesenchymal stromal cells (MSCs) are promising regenerative therapeutics that primarily exert their effects through secreted extracellular vesicles (EVs). These EVs - being small and non-living - are easier to handle and possess advantages over cellular products. Consequently, the therapeutic potential of MSC-EVs is increasingly investigated. However, due to variations in MSC-EV manufacturing strategies, MSC-EV products should be considered as highly diverse. Moreover, the diverse array of EV characterisation technologies used for MSC-EV characterisation further complicates reliable interlaboratory comparisons of published data. Consequently, this study aimed to establish a common method that can easily be used by various MSC-EV researchers to characterise MSC-EV preparations to facilitate interlaboratory comparisons. To this end, we conducted a comprehensive inter-laboratory assessment using a novel multiplex bead-based EV flow cytometry assay panel. This assessment involved 11 different MSC-EV products from five laboratories with varying MSC sources, culture conditions, and EV preparation methods. Through this assay panel covering a range of mostly MSC-related markers, we identified a set of cell surface markers consistently positive (CD44, CD73 and CD105) or negative (CD11b, CD45 and CD197) on EVs of all explored MSC-EV preparations. Hierarchical clustering analysis revealed distinct surface marker profiles associated with specific preparation processes and laboratory conditions. We propose CD73, CD105 and CD44 as robust positive markers for minimally identifying MSC-derived EVs and CD11b, CD14, CD19, CD45 and CD79 as reliable negative markers. Additionally, we highlight the influence of culture medium components, particularly human platelet lysate, on EV surface marker profiles, underscoring the influence of culture conditions on resulting EV products. This standardisable approach for MSC-EV surface marker profiling offers a tool for routine characterisation of manufactured EV products in pre-clinical and clinical research, enhances the quality control of MSC-EV preparations, and hopefully paves the way for higher consistency and reproducibility in the emerging therapeutic MSC-EV field.

Identification of cell surface markers for acute myeloid leukemia prognosis based on multi-model analysis.

Given the extremely high inter-patient heterogeneity of acute myeloid leukemia (AML), the identification of biomarkers for prognostic assessment and therapeutic guidance is critical. Cell surface markers (CSMs) have been shown to play an important role in AML leukemogenesis and progression. In the current study, we evaluated the prognostic potential of all human CSMs in 130 AML patients from The Cancer Genome Atlas (TCGA) based on differential gene expression analysis and univariable Cox proportional hazards regression analysis. By using multi-model analysis, including Adaptive LASSO regression, LASSO regression, and Elastic Net, we constructed a 9-CSMs prognostic model for risk stratification of the AML patients. The predictive value of the 9-CSMs risk score was further validated at the transcriptome and proteome levels. Multivariable Cox regression analysis showed that the risk score was an independent prognostic factor for the AML patients. The AML patients with high 9-CSMs risk scores had a shorter overall and event-free survival time than those with low scores. Notably, single-cell RNA-sequencing analysis indicated that patients with high 9-CSMs risk scores exhibited chemotherapy resistance. Furthermore, PI3K inhibitors were identified as potential treatments for these high-risk patients. In conclusion, we constructed a 9-CSMs prognostic model that served as an independent prognostic factor for the survival of AML patients and held the potential for guiding drug therapy.

Metformin as an Enhancer for the Treatment of Chemoresistant CD34+ Acute Myeloid Leukemia Cells.

Acute myeloid leukemia is the second most frequent type of leukemia in adults. Due to a high risk of development of chemoresistance to first-line chemotherapy, the survival rate of patients in a 5-year period is below 30%. One of the reasons is that the AML population is heterogeneous, with cell populations partly composed of very primitive CD34+CD38- hematopoietic stem/progenitor cells, which are often resistant to chemotherapy. First-line treatment with cytarabine and idarubicin fails to inhibit the proliferation of CD34+CD38- cells. In this study, we investigated Metformin's effect with or without first-line conventional chemotherapy, or with other drugs like venetoclax and S63845, on primitive and undifferentiated CD34+ AML cells in order to explore the potential of Metformin or S63845 to serve as adjuvant therapy for AML. We found that first-line conventional chemotherapy treatment inhibited the growth of cells and arrested the cells in the S phase of the cell cycle; however, metformin affected the accumulation of cells in the G2/M phase. We observed that CD34+ KG1a cells respond better to lower doses of cytarabine or idarubicin in combination with metformin. Also, we determined that treatment with cytarabine, venetoclax, and S63845 downregulated the strong tendency of CD34+ KG1a cells to form cell aggregates in culture due to the downregulation of leukemic stem cell markers like CD34 and CD44, as well as adhesion markers. Also, we found that idarubicin slightly upregulated myeloid differentiation markers, CD11b and CD14. Treatment with cytarabine, idarubicin, venetoclax, metformin, and S63845 upregulated some cell surface markers like HLA-DR expression, and metformin upregulated CD9, CD31, and CD105 cell surface marker expression. In conclusion, we believe that metformin has the potential to be used as an adjuvant in the treatment of resistant-to-first-line-chemotherapy AML cells. Also, we believe that the results of our study will stimulate further research and the potential use of changes in the expression of cell surface markers in the development of new therapeutic strategies.

Phenotypic and Functional Characterization of Memory CD4+ and CD8+ T Cells After Antigenic Stimulation.

The encounter of T cells with the antigen through the interaction of T cell receptors with peptides and major histocompatibility complex (MHC) molecules on the surface of antigen-presenting cells (APCs) can generate effector response and memory T cells. Memory T cells developed following infections or vaccination may persist, leading to the generation of a specific immune response upon reexposure to the same pathogen through rapid clonal proliferation and activation of effector functions. T cell memory subsets can be identified based on the expression of several membrane markers such as CCR7, CD27, and CD45RA. Using fluorescent antibodies against these markers and a flow cytometer, it is possible to perform immunophenotyping via the analysis of cell surface expression of proteins by different subpopulations such as the subsets of naïve, effector, and memory T cells as well as via the analysis of functional markers that further characterize each sample. Intracellular cytokine staining allows for the evaluation of intracellular proteins expressed in T cells in response to antigenic stimulation. This chapter presents the phenotypic and functional characterization of memory T cells after antigenic stimulation, detailing the procedures for identifying intracellular and surface protein markers. Herein, we review and present a reproducible standardized protocol using antibodies for specific markers and applying flow cytometry.

Recent developments in targeting breast cancer stem cells (BCSCs): a descriptive review of therapeutic strategies and emerging therapies.

Despite recent advancements in the diagnosis and treatment of breast cancer (BC), patient outcomes in terms of survival, recurrence, and disease progression remain suboptimal. A significant factor contributing to these challenges is the cellular heterogeneity within BC, particularly the presence of breast cancer stem cells (BCSCs). These cells are thought to serve as the clonogenic nexus for new tumor growth, owing to their hierarchical organization within the tumor. This descriptive review focuses on the evolving strategies to target BCSCs, which have become a pivotal aspect of therapeutic development. We explore a variety of approaches, including targeting specific tumor surface markers (CD133 and CD44), transporters, heat shock proteins, and critical signaling pathways like Notch, Akt, Hedgehog, KLF4, and Wnt/ß-catenin. Additionally, we discuss the modulation of the tumor microenvironment through the CXCR-12/CXCR4 axis, manipulation of pH levels, and targeting hypoxia-inducible factors, vascular endothelial growth factor, and CXCR1/2 receptors. Further, this review focuses on the roles of microRNA expression, strategies to induce apoptosis and differentiation in BCSCs, dietary interventions, dendritic cell vaccination, oncolytic viruses, nanotechnology, immunotherapy, and gene therapy. We particularly focused on studies reporting identification of BCSCs, their unique properties and the efficacy of various therapeutic modalities in targeting these cells. By dissecting these approaches, we aim to provide insights into the complex landscape of BC treatment and the potential pathways for improving patient outcomes through targeted BCSC therapies.

Immune system-related plasma extracellular vesicles in healthy aging.

Objectives: To identify age-related plasma extracellular vehicle (EVs) phenotypes in healthy adults. Methods: EV proteomics by high-resolution mass spectrometry to evaluate EV protein stability and discover age-associated EV proteins (n=4 with 4 serial freeze-thaws each); validation by high-resolution flow cytometry and EV cytokine quantification by multiplex ELISA (n=28 healthy donors, aged 18-83 years); quantification of WI-38 fibroblast cell proliferation response to co-culture with PKH67-labeled young and old plasma EVs. The EV samples from these plasma specimens were previously characterized for bilayer structure, intra-vesicle mitochondria and cytokines, and hematopoietic cell-related surface markers. Results: Compared with matched exo-EVs (EV-depleted supernatants), endo-EVs (EV-associated) had higher mean TNF-α and IL-27, lower mean IL-6, IL-11, IFN-γ, and IL-17A/F, and similar mean IL-1ß, IL-21, and IL-22 concentrations. Some endo-EV and exo-EV cytokine concentrations were correlated, including TNF-α, IL-27, IL-6, IL-1ß, and IFN-γ, but not IL-11, IL-17A/F, IL-21 or IL-22. Endo-EV IFN-γ and exo-EV IL-17A/F and IL-21 declined with age. By proteomics and confirmed by flow cytometry, we identified age-associated decline of fibrinogen (FGA, FGB and FGG) in EVs. Age-related EV proteins indicated predominant origins in the liver and innate immune system. WI-38 cells (>95%) internalized similar amounts of young and old plasma EVs, but cells that internalized PKH67-EVs, particularly young EVs, underwent significantly greater cell proliferation. Conclusion: Endo-EV and exo-EV cytokines function as different biomarkers. The observed healthy aging EV phenotype reflected a downregulation of EV fibrinogen subpopulations consistent with the absence of a pro-coagulant and pro-inflammatory condition common with age-related disease.

[Circulating populations of CD4+ CD25+ CD127- regulatory t lymphocytes in peripheral blood of allergic asthmatic children]. / Determinación de poblaciones circulantes en sangre periférica de linfocitos T, reguladores CD4+ CD25+ CD127-, en niños asmáticos alérgicos.

OBJECTIVE: To carry out a preliminary analysis on the Treg lymphocyte counts present in the peripheral blood of allergic asthmatic children from the city of Cartagena, Colombia, compared to healthy controls. METHODS: We compared cytometry counts of ten asthmatic patients (age 7-16 years) and seven healthy controls (6-12 years), recruited in the city of Cartagena. Peripheral blood samples were stained using Cytek's 14-color cFluor Immunoprofiling kit (Cytek® cFluor® Immunoprofiling Kit 14 Color RUO kit), and analyzed on a Northern Lights™ spectral cytometer (Cytek® Biosciences, Fremont, CA, USA), to read 50.000 events per sample. The data obtained were analyzed in SpectroFlo® and FlowJo. The study was approved by the ethics committee of the University of Cartagena (SGR, Grant BPIN2020000100405). RESULTS: The frequency of CD3+, CD4+, CD25+, CD127- Tregs was 11% of all CD4+ T cells, with a range of minimum 8,1% and maximum 17,7%. There was no significant difference in the proportion of Tregs between allergic asthmatic patients and healthy controls (P = 0,2). CONCLUSIONS: With this preliminary sample size, no significant differences were found in the Treg lymphocyte population between allergic asthmatic patients and healthy controls. The 14-color multiplexed panel is a useful tool not only to count CD3+ and CD4+ populations, but also to obtain the percentage of regulatory T cells using cell surface markers.

OBJETIVO: Realizar un análisis preliminar sobre los conteos de linfocitos Tregs presentes en sangre periférica de niños asmáticos alérgicos de la ciudad de Cartagena, comparado con controles sanos. MÉTODOS: Se compararon los conteos de citometría de diez pacientes asmáticos (entre 7 y16 años) y siete controles sanos (entre 6 y12 años), reclutados en la ciudad de Cartagena. La muestra de sangre periférica fue teñida empleando el kit de inmunofenotipo multiplexado de 14 colores de Cytek (Cytek® cFluor® Immunoprofiling Kit 14 Color), y analizada en un citómetro espectral Northern Lights™ (Cytek® Biosciences, Fremont, CA, USA), a lectura de 50.000 eventos por muestra. Los datos obtenidos fueron analizados en SpectroFlo® y FlowJo. El estudio fue aprobado por el Comité de Ética de la Universidad de Cartagena. RESULTADOS: El panel de tinción funcionó apropiadamente y dentro de los parámetros apropiados. Se obtuvo un promedio de células Tregs CD3+, CD4+, CD25+ y CD127- del 11% de todos los CD4+ en las muestras estudiadas, con un rango de mínimo de 8,1% y un máximo de 17,7%. No hubo diferencias significativas en la proporción de linfocitos Tregs entre los pacientes asmáticos alérgicos y los controles sanos (P = 0.2). CONCLUSIONES: Con este tamaño de muestra preliminar, no se encontraron diferencias significativas en la población de linfocitos Tregs entre los pacientes asmáticos alérgicos y los controles sanos. El panel multiplexado de 14 colores es una herramienta útil no solo para derivar las poblaciones CD3+ y CD4+, sino también para obtener el porcentaje de células T reguladoras empleando marcadores de superficie celular.

Lectin: A Molecular Tool in Cancer Diagnosis and Therapy with Special Reference to Reproductive Cancers.

The prevalence of cancer deaths globally and domestically is higher especially due to the deferment of diagnosis and lack of facilities for women's reproductive cancers. The present review focussed to explore the application of lectins in cancer theranostics. Though there is cancer diagnostic and treatment available there is no promising early diagnostic tool and effective treatment available for the cancer which is the major concern. Lectins are cellulose-binding proteins that are strongly determined in saccharide groups of glycans, glycopeptides, or glycolipids. In the concomitance of events in cells, carbohydrates, and proteins, lectins play an important role. Lectins bind superiorly to the cancer cell membrane and their receptors induce the cytotoxic effect, which results in caspase-mediated cell death, and prohibits tumour development. Lectin snuffing also reveals polyamine stocks and impedes the growth of cancerous cells. They affect the cell cycle by non-apoptotic aggregation, seizure of the cell cycle phase G2, M, and the mediation of caspases. It can also adversely affect the action of telomerase and hinder vascularisation. They promote immunomodulation and adversely limit protein synthesis. Their easy availability and its characteristics support its use in cancer diagnosis and therapy, despite their small corollary effects. Future investigations recommend focussing more on the key applications of lectin by reducing its concurrent effects and carrying out more in-vitro investigations. However, the use of lectin formulations for cancer theranostics is a new area in cancer detection and treatment. In this review, plant lectin appears to be a potential target for cancer research in the fields of diagnosis and theranostics.

Enriching new transplantable RGC-like cells from retinal organoids for RGC replacement therapy.

Optic neuropathies, such as glaucoma, are due to progressive retinal ganglion cells (RGCs) degeneration, result in irreversible vision loss. The promising RGCs replacement therapy for restoring vision are impeded by insufficient RGC-like cells sources. The present work was enriched one new type RGC-like cells using two surface markers CD184 and CD171 from human induced pluripotent stem cells (hiPSCs) by FACS sorting firstly. These new kind cells have well proliferation ability and possessed passage tolerance in vitro 2D or 3D spheroids culture, which kept expressing Pax6, Brn3b and ßIII-Tubulin and so on. The transplanted CD184+CD171+ RGC-like cells could survive and integrate into the normal and optic nerve crush (ONC) mice retina, especially they were more inclined to across the optic nerve head and extend to the damaged optic nerve. These data support the feasible application for cell replacement therapy in RGC degenerative diseases, as well as help to develop new commercial cells sorting reagents and establish good manufacturing practice (GMP) grade RGC-like donor cells for further clinical application.

Capture of the Mouse Organ Membrane Proteome Specificity in Peptidisc Libraries.

Membrane proteins, particularly those on the cell surface, play pivotal roles in diverse physiological processes, and their dysfunction is linked to a broad spectrum of diseases. Despite being crucial biomarkers and therapeutic drug targets, their low abundance and hydrophobic nature pose challenges in isolation and quantification, especially when extracted from tissues and organs. To overcome these hurdles, we developed the membrane-mimicking peptidisc, enabling the isolation of the membrane proteome in a water-soluble library conducive to swift identification through liquid chromatography with tandem mass spectrometry. This study applies the method across five mice organs, capturing between 200 and 450 plasma membrane proteins in each case. More than just membrane protein identification, the peptidisc is used to estimate the relative abundance across organs, linking cell-surface protein molecular functions to organ biological roles, thereby contributing to the ongoing discourse on organ specificity. This contribution holds substantial potential for unveiling new avenues in the exploration of biomarkers and downstream applications involving knowledge of the organ cell-surface proteome.

Innate lymphoid cells (ILCs) in teleosts against data on ILCs in humans.

It is assumed that cells corresponding to innate lymphoid cells (ILCs) in humans, in addition to lymphoid tissue inducer cells (LTi), are also found in teleosts. In this systematic group of organisms, however, they are a poorly understood cell population. In contrast to the data on ILCs in humans, which also remain incomplete despite advanced research, in teleosts, these cells require much more attention. ILCs in teleosts have been presented as cells that may be evolutionary precursors of NK cells or ILCs identified in mammals, including humans. It is a highly heterogeneous group of cells in both humans and fish and their properties, as revealed by studies in humans, are most likely to remain strictly dependent on the location of these cells and the physiological state of the individual from which they originate. They form a bridge between innate and adaptive immunity. The premise of this paper is to review the current knowledge of ILCs in teleosts, taking into account data on similar cells in humans. A review of the knowledge concerning these particular cells, elements of innate immunity mechanisms as equivalent to, or perhaps dominant over, adaptive immunity mechanisms in teleosts, as presented, may inspire the need for further research.

Regulation of the migration of colorectal cancer stem cells via the TLR4/MyD88 signaling pathway by the novel surface marker CD14 following LPS stimulation.

Cell surface markers are most widely used in the study of cancer stem cells (CSCs). However, cell surface markers that are safely and stably expressed in CSCs have yet to be identified. Colonic CSCs express leukocyte CD14. CD14 binding to the ligand lipopolysaccharide (LPS) is involved in the inflammatory response via the Toll-like receptor 4 (TLR4)/myeloid differentiation factor 88 (MyD88) signaling pathway. TLR4 and MyD88 have been reported to promote the proliferation, metastasis and tumorigenicity of colon cancer cells, which is consistent with the characteristics of CSCs. In the present study, the proposed experimental method to detect cell proliferation, metastasis and tumorigenesis was used to confirm that, under LPS stimulation, CD14 promoted the proliferation, migration and tumorigenesis of colonic CSCs via the TLR4/MyD88 signaling pathway. Cell Counting Kit-8 and 5-ethynyl-2'-deoxyuridine assays were used to assess the proliferation and migration of the cells. Colony formation and nude mouse xenograft assays were used to assess the capacity of cells to form tumors. Using western blotting and reverse transcription-quantitative PCR, the mRNA and protein levels of CD14, TLR4 and MyD88 were examined. It was confirmed that CD14 promoted the proliferation, metastasis and tumorigenesis of colon CSCs in response to LPS stimulation via the TLR4/MyD88 signaling pathway, and CD14+ colon cancer cells were successfully isolated and sorted. According to the results of proliferation assay, it was determined that CD14 regulated the LPS-induced proliferation of colon CSCs. CD14, TLR4 and MyD88 protein and mRNA expression was upregulated in colon CSCs in response to LPS stimulation. This indicates a potential novel target for colon CSC-related studies.

Expression of stem cell markers as predictors of therapeutic response in metastatic prostate cancer patients.

BACKGROUND: Cancer stem cells (CSCs) have been implicated in prostate cancer (PCA) progression and therapeutic resistance. This study aimed to compare the expression levels of CSC CD (CD 44, CD 133, and CD 24) markers in treatment-naive patients with metastatic PCA before and after treatment. METHODS: The study included 60 treatment-naïve patients with metastatic PCA who received androgen deprivation therapy (ADT) alone (n = 30) and ADT plus chemotherapy (n = 30). The level of CD44, CD133, and CD24 were obtained by flow cytometric analysis before and after treatment. Baseline characteristics were also assessed, including age, pretreatment testosterone levels, and pretreatment prostate-specific antigen (PSA) levels. RESULTS: The baseline characteristics analysis showed no significant difference in pre-treatment testosterone levels between the ADT+ chemotherapy and ADT-alone groups. In the flow cytometric analysis, no significant difference was observed in pre-treatment CD44+ and CD133+ levels between the 2 treatment groups, although a trend towards higher pretreatment CD24- levels was observed in the ADT+ chemotherapy group. After treatment, significant reductions in testosterone and PSA levels were observed in both treatment arms. The ADT+ chemotherapy group showed a greater reduction in CD44+ and CD133+ levels compared to the ADT-alone group. Bioinformatic analysis using the UALCAN TCGA database also showed a similar trend of CD 44, CD 24, and CD 133 gene expression patterns. CONCLUSION: Combination therapy involving chemotherapy and ADT appears to have a greater impact on suppressing CSCs compared to ADT alone. These findings highlight the potential of targeting CSCs as a prognostic and predictive marker therapeutic strategy in metastatic PCA.

Identification of Surface Markers and Functional Characterization of Myeloid Derived Suppressor Cell-Like Adherent Cells.

Myeloid-derived suppressor cell (MDSC)-like adherent cells (MLACs) are a recently identified CD11b+ F4/80- myeloid cell subset that can infiltrate tumors early in development and promote their growth. Because of these functions, MLACs play an important role in establishing an immunosuppressive tumor microenvironment (TME). However, the lack of MLAC-specific markers has hampered further characterization of this cell type. This study identifies the gene signature of MLACs by analyzing RNA-sequencing (RNA-seq) and public single-cell RNA-seq data, revealing that MLACs are an independent cell population that are distinct from other intratumoral myeloid cells. After combining proteome analysis of membrane proteins with RNA-seq data, H2-Ab1 and CD11c are indicated as marker proteins that can support the isolation of MLAC subsets from CD11b+ F4/80- myeloid cells by fluorescence-activated cell sorting. The CD11b+ F4/80- H2-Ab1+ and CD11b+ F4/80- CD11c+ MLAC subsets represent approximately half of the MLAC population that is isolated based on their adhesion properties and possess gene signatures and functional properties similar to those of the MLAC population. Additionally, membrane proteome analysis suggests that MLACs express highly heterogeneous surface proteins. This study facilitates an integrated understanding of heterogeneous intratumoral myeloid cells, as well as the molecular and cellular details of the development of an immunosuppressive TME.

The effect of pegbovigrastim administration on the nonspecific immunity of calves.

BACKGROUND: Prevention of diseases in the early rearing of calves is important, particularly because disease occurrence most often requires antimicrobial administration but reduction of their use in animals is a priority. Pegbovigrastim is known for its use as an immunoregulator in cows and heifers, but the effect of its administration on calves has not been fully investigated. OBJECTIVES: Investigate whether administration of pegbovigrastim effectively stimulates nonspecific immunity in healthy calves. ANIMALS: Eleven clinically healthy 5-week-old calves. METHODS: Prospective observational study. Calves were randomly allocated to an experimental or control groups to receive pegbovigrastim or the same volume of phosphate-buffered saline twice over a 7-day period. To evaluate nonspecific immunity, the numbers of total leukocytes and cells in the appropriate cell fractions were determined. Cytometric analyses were carried out to identify cells expressing CD11b and to evaluate the phagocytic and oxidative burst activities of granulocytes and monocytes. Myeloperoxidase (MPO) and selected cytokines were assayed using ELISA. RESULTS: Pegbovigrastim significantly increased the number of total leukocytes and of cells in all of the examined subsets (P < .05). The phagocytic activity of leukocytes expressed as mean fluorescence intensity was significantly potentiated after pegbovigrastim administration (P < .05). The cytokine response was modulated by pegbovigrastim administration toward anti-inflammatory activity. CONCLUSIONS AND CLINICAL IMPORTANCE: Pegbovigrastim effectively stimulated nonspecific immunity in clinically healthy calves, which in the long term could make the prevention of diseases during early rearing possible by strengthening the immune defense mechanisms of the host.

Nanoprojectile Secondary Ion Mass Spectrometry Enables Multiplexed Analysis of Individual Hepatic Extracellular Vesicles.

Extracellular vesicles (EVs) are nanoscale lipid bilayer particles secreted by cells. EVs may carry markers of the tissue of origin and its disease state, which makes them incredibly promising for disease diagnosis and surveillance. While the armamentarium of EV analysis technologies is rapidly expanding, there remains a strong need for multiparametric analysis with single EV resolution. Nanoprojectile (NP) secondary ion mass spectrometry (NP-SIMS) relies on bombarding a substrate of interest with individual gold NPs resolved in time and space. Each projectile creates an impact crater of 10-20 nm in diameter while molecules emitted from each impact are mass analyzed and recorded as individual mass spectra. We demonstrate the utility of NP-SIMS for statistical analysis of single EVs derived from normal liver cells (hepatocytes) and liver cancer cells. EVs were captured on antibody (Ab)-functionalized gold substrate and then labeled with Abs carrying lanthanide (Ln) MS tags (Ab@Ln). These tags targeted four markers selected for identifying all EVs, and specific to hepatocytes or liver cancer. NP-SIMS was used to detect Ab@Ln-tags colocalized on the same EV and to construct scatter plots of surface marker expression for thousands of EVs with the capability of categorizing individual EVs. Additionally, NP-SIMS revealed information about the chemical nanoenvironment where targeted moieties colocalized. Our approach allowed analysis of population heterogeneity with single EV resolution and distinguishing between hepatocyte and liver cancer EVs based on surface marker expression. NP-SIMS holds considerable promise for multiplexed analysis of single EVs and may become a valuable tool for identifying and validating EV biomarkers of cancer and other diseases.

Depolarized Mitochondrial Membrane Potential and Elevated Calcium in Platelets of Sickle Cell Disease.

Hemolysis, a crucial feature of Sickle cell disease (SCD), is a key player for cellular activation leading to various complications including thrombosis. In response to hemolysis, platelets get activated and release components that are necessary for further platelet activation and aggregation. Thus, it is believed that platelets contribute to the development of thrombotic complications. Platelets in SCD are expected to be affected due to common cause of hemolysis. To measure the surface markers of platelets including P-Selectin, Phosphatidyl Serine and integrin αIIbß3 in SCD patients and healthy controls in order to understand the status of the platelets in SCD. To measure the surface markers of activated platelets using flow cytometry. Since mitochondria and calcium play an important role in cellular functions, the mitochondrial membrane potential and calcium content of platelets in SCD were also evaluated using flow cytometry. In the present study, we have observed significant increase of calcium level in SCD platelets. Further, the loss of mitochondrial membrane potential in SCD platelets was found to be significantly higher when compared to platelets of healthy controls. Though the surface markers of activated platelets in SCD remain unchanged, increased level of calcium and mitochondrial membrane potential loss suggest that the platelets in SCD are more prone to become activated. In order to understand the status of the platelets in SCD, apart from the surface markers, it is also important to assess the calcium levels and mitochondrial membrane potential of platelets.