Intelligent image-based deformation-assisted cell sorting with molecular specificity.

Although label-free cell sorting is desirable for providing pristine cells for further analysis or use, current approaches lack molecular specificity and speed. Here, we combine real-time fluorescence and deformability cytometry with sorting based on standing surface acoustic waves and transfer molecular specificity to image-based sorting using an efficient deep neural network. In addition to general performance, we demonstrate the utility of this method by sorting neutrophils from whole blood without labels.

Real-time fluorescence and deformability cytometry.

The throughput of cell mechanical characterization has recently approached that of conventional flow cytometers. However, this very sensitive, label-free approach still lacks the specificity of molecular markers. Here we developed an approach that combines real-time 1D-imaging fluorescence and deformability cytometry in one instrument (RT-FDC), thus opening many new research avenues. We demonstrated its utility by using subcellular fluorescence localization to identify mitotic cells and test for mechanical changes in those cells in an RNA interference screen.

Initiation of acute graft-versus-host disease by angiogenesis.

The inhibition of inflammation-associated angiogenesis ameliorates inflammatory diseases by reducing the recruitment of tissue-infiltrating leukocytes. However, it is not known if angiogenesis has an active role during the initiation of inflammation or if it is merely a secondary effect occurring in response to stimuli by tissue-infiltrating leukocytes. Here, we show that angiogenesis precedes leukocyte infiltration in experimental models of inflammatory bowel disease and acute graft-versus-host disease (GVHD). We found that angiogenesis occurred as early as day+2 after allogeneic transplantation mainly in GVHD typical target organs skin, liver, and intestines, whereas no angiogenic changes appeared due to conditioning or syngeneic transplantation. The initiation phase of angiogenesis was not associated with classical endothelial cell (EC) activation signs, such as Vegfa/VEGFR1+2 upregulation or increased adhesion molecule expression. During early GVHD at day+2, we found significant metabolic and cytoskeleton changes in target organ ECs in gene array and proteomic analyses. These modifications have significant functional consequences as indicated by profoundly higher deformation in real-time deformability cytometry. Our results demonstrate that metabolic changes trigger alterations in cell mechanics, leading to enhanced migratory and proliferative potential of ECs during the initiation of inflammation. Our study adds evidence to the hypothesis that angiogenesis is involved in the initiation of tissue inflammation during GVHD.

Effects of rigosertib on the osteo-hematopoietic niche in myelodysplastic syndromes.

Rigosertib is a novel multi-kinase inhibitor, which has clinical activity towards leukemic progenitor cells of patients with high-risk myelodysplastic syndromes (MDS) after failure or progression on hypomethylating agents. Since the bone marrow microenvironment plays an important role in MDS pathogenesis, we investigated the impact of rigosertib on cellular compartments within the osteo-hematopoietic niche. Healthy C57BL/6J mice treated with rigosertib for 3 weeks showed a mild suppression of hematopoiesis (hemoglobin and red blood cells, both - 16%, p < 0.01; white blood cells, - 34%, p < 0.05; platelets, - 38%, p < 0.05), whereas there was no difference in the number of hematopoietic stem cells in the bone marrow. Trabecular bone mass of the spine was reduced by rigosertib (- 16%, p = 0.05). This was accompanied by a lower trabecular number and thickness (- 6% and - 10%, respectively, p < 0.05), partly explained by the increase in osteoclast number and surface (p < 0.01). Milder effects of rigosertib on bone mass were detected in an MDS mouse model system (NHD13). However, rigosertib did not further aggravate MDS-associated cytopenia in NHD13 mice. Finally, we tested the effects of rigosertib on human mesenchymal stromal cells (MSC) in vitro and demonstrated reduced cell viability at nanomolar concentrations. Deterioration of the hematopoietic supportive capacity of MDS-MSC after rigosertib pretreatment demonstrated by decreased number of colony-forming units, especially in the monocytic lineage, further supports the idea of disturbed crosstalk within the osteo-hematopoietic niche mediated by rigosertib. Thus, rigosertib exerts inhibitory effects on the stromal components of the osteo-hematopoietic niche which may explain the dissociation between anti-leukemic activity and the absence of hematological improvement.

Real-time deformability cytometry: on-the-fly cell mechanical phenotyping.

We introduce real-time deformability cytometry (RT-DC) for continuous cell mechanical characterization of large populations (>100,000 cells) with analysis rates greater than 100 cells/s. RT-DC is sensitive to cytoskeletal alterations and can distinguish cell-cycle phases, track stem cell differentiation into distinct lineages and identify cell populations in whole blood by their mechanical fingerprints. This technique adds a new marker-free dimension to flow cytometry with diverse applications in biology, biotechnology and medicine.

Aromatic Hydrocarbon Receptor Suppresses Prostate Cancer Bone Metastasis Cells-Induced Vasculogenesis of Endothelial Progenitor Cells under Hypoxia.

BACKGROUND/AIMS: Hypoxia leads to the development of neovascularization in solid tumor by regulating VEGF expression. Aromatic hydrocarbon receptor (AHR), a receptor for dioxin-like compounds, functions as a transcription factor through dimerization with hypoxia-inducible factors 1ß (HIF-1ß) and inhibits the secretion of vascular endothelial growth factor (VEGF). The purpose of this study was to explore whether AHR can suppress hypoxia-induced VEGF production in prostate bone metastasis cells and repress neovascularization in endothelial progenitor cells (EPCs), and, if so, through what mechanisms. METHODS: PC-3 or LNCaP cells induced angiogenesis was detected by Matrigel-based tube formation assay, mRNA expression levels was measured by qRT-PCR, VEGF secretion level was determined by ELISA assay, respectively. RESULTS: AHR activation inhibits hypoxia-induced adhesiveness and vasculogenesis of EPCs induced by PC-3 or LNCaP cells under hypoxia. Moreover, AHR activation suppressed hypoxia-induced VEGF production in PC-3 and LNCaP cells (48 ± 14% in PC-3, p = 0.000; 41 ± 14% in LNCaP, p = 0.000) by attenuating HIF-1α and HIF-1ß level that in turn diminished the angiogenic ability of EPCs in vitro. Furthermore, we found the mRNA level of hypoxia-inducible factors 1α (HIF-1α) (1.54 ± 0.13 fold in PC-3, p = 0.002, 1.62 ± 0.12 fold in LNCaP, p = 0.001) and HIF-1ß (1.67 ± 0.23 fold in PC-3, p = 0.007; 1.75 ± 0.26 fold in LNCaP, p=0.008) were upregulated in prostate cancer bone metastasis PC-3 and LNCaP cell lines in response to hypoxia, and revealed that the regulation of VEGF by HIF-1α and HIF-1ß was possibly mediated by the activation of phosphatidylinositol 3-kinase pathway. CONCLUSION: By providing a mechanistic insight into the modulation of neovascularization by AHR ligand, we suggest that AHR ligand has a strong potential of being a new therapeutic agent with applications in the field of bone metastatic prostate cancer.

Icariin promotes angiogenic differentiation and prevents oxidative stress-induced autophagy in endothelial progenitor cells.

Reduced tissue levels of endothelial progenitor cells (EPCs) and functional impairment of endothelium are frequently observed in patients with diabetes and cardiovascular disease. The vascular endothelium is specifically sensitive to oxidative stress, and this is one of the mechanisms that causes widespread endothelial dysfunction in most cardiovascular diseases and disorders. Hence attention has increasingly been paid to enhance mobilization and differentiation of EPCs for therapeutic purposes. The aim of this study was to investigate whether Icariin, a natural bioactive component known from traditional Chinese Medicine, can induce angiogenic differentiation and inhibit oxidative stress-induced cell dysfunction in bone marrow-derived EPCs (BM-EPCs), and, if so, through what mechanisms. We observed that treatment of BM-EPCs with Icariin significantly promoted cell migration and capillary tube formation, substantially abrogated hydrogen peroxide (H2 O2 )-induced apoptotic and autophagic programmed cell death that was linked to the reduced intracellular reactive oxygen species levels and restored mitochondrial membrane potential. Icariin downregulated endothelial nitric oxide synthase 3, as well as nicotinamide-adenine dinucleotide phosphate-oxidase expression upon H2 O2 induction. These antiapoptotic and antiautophagic effects of Icariin are possibly mediated by restoring the loss of mammalian target of rapamycin /p70S6K/4EBP1 phosphorylation as well as attenuation of ATF2 and ERK1/2 protein levels after H2 O2 treatment. In summary, favorable modulation of the angiogenesis and redox states in BM-EPCs make Icariin a promising proangiogenic agent both enhancing vasculogenesis and protecting against endothelial dysfunction.

Decoding Single Cell Morphology in Osteotropic Breast Cancer Cells for Dissecting Their Migratory, Molecular and Biophysical Heterogeneity.

Breast cancer is a heterogeneous disease and the mechanistic framework for differential osteotropism among intrinsic breast cancer subtypes is unknown. Hypothesizing that cell morphology could be an integrated readout for the functional state of a cancer cell, we established a catalogue of the migratory, molecular and biophysical traits of MDA-MB-231 breast cancer cells, compared it with two enhanced bone-seeking derivative cell lines and integrated these findings with single cell morphology profiles. Such knowledge could be essential for predicting metastatic capacities in breast cancer. High-resolution microscopy revealed a heterogeneous and specific spectrum of single cell morphologies in bone-seeking cells, which correlated with differential migration and stiffness. While parental MDA-MB-231 cells showed long and dynamic membrane protrusions and were enriched in motile cells with continuous and mesenchymal cell migration, bone-seeking cells appeared with discontinuous mesenchymal or amoeboid-like migration. Although non-responsive to CXCL12, bone-seeking cells responded to epidermal growth factor with a morphotype shift and differential expression of genes controlling cell shape and directional migration. Hence, single cell morphology encodes the molecular, migratory and biophysical architecture of breast cancer cells and is specifically altered among osteotropic phenotypes. Quantitative morpho-profiling could aid in dissecting breast cancer heterogeneity and in refining clinically relevant intrinsic breast cancer subtypes.

Machine learning assisted real-time deformability cytometry of CD34+ cells allows to identify patients with myelodysplastic syndromes.

Diagnosis of myelodysplastic syndrome (MDS) mainly relies on a manual assessment of the peripheral blood and bone marrow cell morphology. The WHO guidelines suggest a visual screening of 200 to 500 cells which inevitably turns the assessor blind to rare cell populations and leads to low reproducibility. Moreover, the human eye is not suited to detect shifts of cellular properties of entire populations. Hence, quantitative image analysis could improve the accuracy and reproducibility of MDS diagnosis. We used real-time deformability cytometry (RT-DC) to measure bone marrow biopsy samples of MDS patients and age-matched healthy individuals. RT-DC is a high-throughput (1000 cells/s) imaging flow cytometer capable of recording morphological and mechanical properties of single cells. Properties of single cells were quantified using automated image analysis, and machine learning was employed to discover morpho-mechanical patterns in thousands of individual cells that allow to distinguish healthy vs. MDS samples. We found that distribution properties of cell sizes differ between healthy and MDS, with MDS showing a narrower distribution of cell sizes. Furthermore, we found a strong correlation between the mechanical properties of cells and the number of disease-determining mutations, inaccessible with current diagnostic approaches. Hence, machine-learning assisted RT-DC could be a promising tool to automate sample analysis to assist experts during diagnosis or provide a scalable solution for MDS diagnosis to regions lacking sufficient medical experts.

Real-Time Deformability Cytometry Detects Leukocyte Stiffening After Gadolinium-Based Contrast Agent Exposure.

OBJECTIVES: Reports on gadolinium (Gd) retention in soft tissues after administration of Gd-based contrast agents (GBCAs) raise concerns about Gd-induced changes in the biophysical properties of cells and tissues. Here, we investigate if clinical GBCAs of both classes of linear and macrocyclic structure cause changes in the mechanical properties of leukocytes in human blood samples. MATERIAL AND METHODS: Real-time deformability cytometry was applied to human blood samples from 6 donors. The samples were treated with 1 mM gadoteric acid (Dotarem), gadopentetic acid (Magnevist), gadobutrol (Gadovist), or Gd trichloride at 37°C for 1 hour to mimic clinical doses of GBCAs and exposure times. Leukocyte subtypes-lymphocytes, monocytes, and neutrophils-were identified based on their size and brightness and analyzed for deformability, which is inversely correlated with cellular stiffness. RESULTS: We observed significant stiffening (3%-13%, P < 0.01) of all investigated leukocyte subtypes, which was most pronounced for lymphocytes, followed by neutrophils and monocytes, and the effects were independent of the charge and steric structure of the GBCA applied. In contrast, no changes in cell size and brightness were observed, suggesting that deformability and cell stiffness measured by real-time deformability cytometry are sensitive to changes in the physical phenotypes of leukocytes after GBCA exposure. CONCLUSIONS: Real-time deformability cytometry might provide a quantitative blood marker for critical changes in the physical properties of blood cells in patients undergoing GBCA-enhanced magnetic resonance imaging.

AIDeveloper: Deep Learning Image Classification in Life Science and Beyond.

Artificial intelligence (AI)-based image analysis has increased drastically in recent years. However, all applications use individual solutions, highly specialized for a particular task. Here, an easy-to-use, adaptable, and open source software, called AIDeveloper (AID) to train neural nets (NN) for image classification without the need for programming is presented. AID provides a variety of NN-architectures, allowing to apply trained models on new data, obtain performance metrics, and export final models to different formats. AID is benchmarked on large image datasets (CIFAR-10 and Fashion-MNIST). Furthermore, models are trained to distinguish areas of differentiated stem cells in images of cell culture. A conventional blood cell count and a blood count obtained using an NN are compared, trained on >1.2 million images, and demonstrated how AID can be used for label-free classification of B- and T-cells. All models are generated by non-programmers on generic computers, allowing for an interdisciplinary use.

Analysis of Biomechanical Properties of Hematopoietic Stem and Progenitor Cells Using Real-Time Fluorescence and Deformability Cytometry.

Stem cell mechanics, determined predominantly by the cell's cytoskeleton, plays an important role in different biological processes such as stem cell differentiation or migration. Several methods to measure mechanical properties of cells are currently available, but most of them are limited in the ability to screen large heterogeneous populations in a robust and efficient manner-a feature required for successful translational applications. With real-time fluorescence and deformability cytometry (RT-FDC), mechanical properties of cells in suspension can be screened continuously at rates of up to 1,000 cells/s-similar to conventional flow cytometers-which makes it a suitable method not only for basic research but also for a clinical setting. In parallel to mechanical characterization, RT-FDC allows to measure specific molecular markers using standard fluorescence labeling. In this chapter, we provide a detailed protocol for the characterization of hematopoietic stem and progenitor cells (HSPCs) in heterogeneous mobilized peripheral blood using RT-FDC and present a specific morpho-rheological fingerprint of HSPCs that allows to distinguish them from all other blood cell types.

Erratum: Spheroid Culture of Mesenchymal Stromal Cells Results in Morphorheological Properties Appropriate for Improved Microcirculation.

[This corrects the article DOI: 10.1002/advs.201802104.].

Spheroid Culture of Mesenchymal Stromal Cells Results in Morphorheological Properties Appropriate for Improved Microcirculation.

Human bone marrow mesenchymal stromal cells (MSCs) are used in clinical trials for the treatment of systemic inflammatory diseases due to their regenerative and immunomodulatory properties. However, intravenous administration of MSCs is hampered by cell trapping within the pulmonary capillary networks. Here, it is hypothesized that traditional 2D plastic-adherent cell expansion fails to result in appropriate morphorheological properties required for successful cell circulation. To address this issue, a method to culture MSCs in nonadherent 3D spheroids (mesenspheres) is adapted. The biological properties of mesensphere-cultured MSCs remain identical to conventional 2D cultures. However, morphorheological analyses reveal a smaller size and lower stiffness of mesensphere-derived MSCs compared to plastic-adherent MSCs, measured using real-time deformability cytometry and atomic force microscopy. These properties result in an increased ability to pass through microconstrictions in an ex vivo microcirculation assay. This ability is confirmed in vivo by comparison of cell accumulation in various organ capillary networks after intravenous injection of both types of MSCs in mouse. The findings generally identify cellular morphorheological properties as attractive targets for improving microcirculation and specifically suggest mesensphere culture as a promising approach for optimized MSC-based therapies.

Targeting Mechanoresponsive Proteins in Pancreatic Cancer: 4-Hydroxyacetophenone Blocks Dissemination and Invasion by Activating MYH14.

Metastasis is complex, involving multiple genetic, epigenetic, biochemical, and physical changes in the cancer cell and its microenvironment. Cells with metastatic potential are often characterized by altered cellular contractility and deformability, lending them the flexibility to disseminate and navigate through different microenvironments. We demonstrate that mechanoresponsiveness is a hallmark of pancreatic cancer cells. Key mechanoresponsive proteins, those that accumulate in response to mechanical stress, specifically nonmuscle myosin IIA (MYH9) and IIC (MYH14), α-actinin 4, and filamin B, were highly expressed in pancreatic cancer as compared with healthy ductal epithelia. Their less responsive sister paralogs-myosin IIB (MYH10), α-actinin 1, and filamin A-had lower expression differential or disappeared with cancer progression. We demonstrate that proteins whose cellular contributions are often overlooked because of their low abundance can have profound impact on cell architecture, behavior, and mechanics. Here, the low abundant protein MYH14 promoted metastatic behavior and could be exploited with 4-hydroxyacetophenone (4-HAP), which increased MYH14 assembly, stiffening cells. As a result, 4-HAP decreased dissemination, induced cortical actin belts in spheroids, and slowed retrograde actin flow. 4-HAP also reduced liver metastases in human pancreatic cancer-bearing nude mice. Thus, increasing MYH14 assembly overwhelms the ability of cells to polarize and invade, suggesting targeting the mechanoresponsive proteins of the actin cytoskeleton as a new strategy to improve the survival of patients with pancreatic cancer. SIGNIFICANCE: This study demonstrates that mechanoresponsive proteins become upregulated with pancreatic cancer progression and that this system of proteins can be pharmacologically targeted to inhibit the metastatic potential of pancreatic cancer cells.

Abnormal localization and accumulation of FLT3-ITD, a mutant receptor tyrosine kinase involved in leukemogenesis.

Aberrant subcellular localization of mutant transmembrane receptors is increasingly acknowledged as a possible mechanism for an altered signaling quality leading to transformation. There is evidence that mutated receptor tyrosine kinases of subclass III, for example the platelet-derived growth factor receptor (PDGFR) and KIT-protein, are aberrantly localized in human cancers. In order to further analyze this phenomenon, we investigated the localization of FLT3, a subclass III receptor tyrosine kinase frequently mutated in leukemia. By immunofluorescence staining and confocal laser scanning microscopy we found that in retrovirally transduced COS7 cells, wild type FLT3 receptor protein is localized primarily at the cell surface. In contrast, a mutant FLT3 receptor protein with an internal tandem duplication (ITD) accumulates in a perinuclear region and is not detectable at the plasma membrane. Surprisingly, and in contrast to previously published data, intracellular FLT3-ITD accumulation could neither be detected in the endoplasmic reticulum (ER) nor in the Golgi apparatus. Furthermore, transient overexpression per se leads to accumulation of wild type FLT3 receptor protein in the ER in addition to surface localization, probably due to inefficient intracellular transport by the overloaded sorting machinery of the secretory pathway. Based on our data and the immature glycosylation pattern of FLT3-ITD, we speculate that the mutant protein resides most probably in an unidentified compartment of the secretory pathway between the ER and the Golgi apparatus.

Detection of human disease conditions by single-cell morpho-rheological phenotyping of blood.

Blood is arguably the most important bodily fluid and its analysis provides crucial health status information. A first routine measure to narrow down diagnosis in clinical practice is the differential blood count, determining the frequency of all major blood cells. What is lacking to advance initial blood diagnostics is an unbiased and quick functional assessment of blood that can narrow down the diagnosis and generate specific hypotheses. To address this need, we introduce the continuous, cell-by-cell morpho-rheological (MORE) analysis of diluted whole blood, without labeling, enrichment or separation, at rates of 1000 cells/sec. In a drop of blood we can identify all major blood cells and characterize their pathological changes in several disease conditions in vitro and in patient samples. This approach takes previous results of mechanical studies on specifically isolated blood cells to the level of application directly in blood and adds a functional dimension to conventional blood analysis.

Bone marrow niche-mimetics modulate HSPC function via integrin signaling.

The bone marrow (BM) microenvironment provides critical physical cues for hematopoietic stem and progenitor cell (HSPC) maintenance and fate decision mediated by cell-matrix interactions. However, the mechanisms underlying matrix communication and signal transduction are less well understood. Contrary, stem cell culture is mainly facilitated in suspension cultures. Here, we used bone marrow-mimetic decellularized extracellular matrix (ECM) scaffolds derived from mesenchymal stromal cells (MSCs) to study HSPC-ECM interaction. Seeding freshly isolated HSPCs adherent (AT) and non-adherent (SN) cells were found. We detected enhanced expansion and active migration of AT-cells mediated by ECM incorporated stromal derived factor one. Probing cell mechanics, AT-cells displayed naïve cell deformation compared to SN-cells indicating physical recognition of ECM material properties by focal adhesion. Integrin αIIb (CD41), αV (CD51) and ß3 (CD61) were found to be induced. Signaling focal contacts via ITGß3 were identified to facilitate cell adhesion, migration and mediate ECM-physical cues to modulate HSPC function.

Large-scale gene expression profiling data of bone marrow stromal cells from osteoarthritic donors.

This data article contains data related to the research article entitled, "in vitro characterization of bone marrow stromal cells from osteoarthritic donors" [1]. Osteoarthritis (OA) represents the main indication for total joint arthroplasty and is one of the most frequent degenerative joint disorders. However, the exact etiology of OA remains unknown. Bone marrow stromal cells (BMSCs) can be easily isolated from bone marrow aspirates and provide an excellent source of progenitor cells. The data shows the identification of pivotal genes and pathways involved in osteoarthritis by comparing gene expression patterns of BMSCs from osteoarthritic versus healthy donors using an array-based approach.

In vitro characterization of bone marrow stromal cells from osteoarthritic donors.

BMSCs, also known as bone marrow-derived mesenchymal stem cells, provide an excellent source of progenitor cells for regenerative therapy. To assess whether osteoarthritis (OA) affects the regenerative potential of BMSCs we compared the proliferation and differentiation potential as well as the surface marker expression profile of OA- versus control BMSCs. BMSCs were isolated from bone marrow aspirates of n=14 patients with advanced-stage idiopathic hip OA (67±6years) and n=15 healthy individuals (61±4years). Proliferation was quantified by total DNA content and colony-forming-units of fibroblastsmax (CFU-F) assay. Differentiation assays included immunohistology, cell-specific alkaline phosphatase (ALP) activity, and osteogenic, chondrogenic as well as adipogenic marker gene qRT-PCR. Expression of BMSC-associated surface markers was analyzed using flow cytometry. No significant intergroup differences were observed concerning the proliferation potential, cell-specific ALP activity as well as adipogenic and osteogenic differentiation marker gene expressions. Interestingly, SOX9 gene expression levels were significantly increased in OA-BMSCs after 14days of chondrogenic stimulation (p<0.01). The surface markers CD73, CD90 and STRO-1 were elevated in relation to CD14, CD34 and CD45 in both groups (p<0.0001). Notably, OA-BMSCs showed significantly increased CD90 (p<0.01) and decreased CD166 (p<0.001) levels. Overall, the in vitro characteristics of BMSCs are not markedly influenced by OA. However, increased SOX9 and CD90 as well as reduced CD166 expression levels in OA-BMSCs warrant further investigation. These data will help to further understand the role of BMSC in OA and facilitate the application of autologous cell-based strategies for musculoskeletal tissue regeneration in OA patients.