Properties Of Biological Cells Research Articles

Artificial Neural Network-Aided Computational Approach for Mechanophenotyping of Biological Cells Using Atomic Force Microscopy.

The artificial neural network (ANN) based models have shown the potential to provide alternate data-driven solutions in disease diagnostics, cell sorting and overcoming AFM-related limitations. Hertzian model-based prediction of mechanical properties of biological cells, although most widely used, has shown to have limited potential in determining constitutive parameters of cells of uneven shape and nonlinear nature of force-indentation curves in AFM-based cell nano-indentation. We report a new artificial neural network-aided approach, which takes into account, the variation in cell shapes and their effect on the predictions in cell mechanophenotyping. We have developed an artificial neural network (ANN) model which could predict the mechanical properties of biological cells by utilizing the force versus indentation curve of AFM. For cells with 1 μm contact length (platelets), we obtained a recall of 0.97 ± 0.03 and 0.99 ± 0.0 for cells with hyperelastic and linear elastic constitutive properties respectively with a prediction error of less than 10%. Also, for cells with 6-8 μm contact length (red blood cells), we obtained the recall of 0.975 in predicting mechanical properties with less than 15% error. We envisage that the developed technique can be used for better estimation of cells' constitutive parameters by incorporating cell topography into account.

Influence of the physico-chemical bioink composition on the printability and cell biological properties in 3D-bioprinting of a liver tumor cell line.

The selection of a suitable matrix material is crucial for the development of functional, biomimetic tissue and organ models. When these tissue models are fabricated with 3D-bioprinting technology, the requirements do not only include the biological functionality and physico-chemical properties, but also the printability. In our work, we therefore present a detailed study of seven different bioinks with the focus on a functional liver carcinoma model. Agarose, gelatin, collagen and their blends were selected as materials based on their benefits for 3D cell culture and Drop-on-Demand (DoD) bioprinting. The formulations were characterized for their mechanical (G' of 10-350Pa) and rheological (viscosity 2-200Pa*s) properties as well as albumin diffusivity (8-50μm2/s). The cellular behavior was exemplarily shown for HepG2 cells by monitoring viability, proliferation and morphology over 14days, while the printability on a microvalve DoD printer was evaluated by drop volume monitoring in flight (100-250nl), camera imaging of the wetting behavior and microscopy of the effective drop diameter (700µm and more). We did not observe negative effects on cell viability or proliferation, which is due to the very low shear stresses inside the nozzle (200-500Pa). With our method, we could identify the strengths and weaknesses of each material, resulting in a material portfolio. By specifically selecting certain materials or blends, cell migration and possible interaction with other cells can be directed as indicated by the results of our cellular experiments.

Gene Expression Data Points to a Role for Hypoxia in Medulloblastoma Pathogenesis


 
 
 Background:
 Medulloblastoma (MB) is the most common intracranial tumor in children. While molecular classification of MB is well-established, detailing cell origin, biological properties, and biomarkers, little research has been performed concerning the MB tumor microenvironment. Hypoxia is significantly associated with tumor spread, poor prognosis, malignant phenotype, and resistance to radiotherapy and chemotherapy in numerous cancer types. The aim of the present study was to assess a possible role for hypoxia in MB and the potential effect on clinical outcomes.
 Methods:
 We, therefore, performed a systematic review examining the role of hypoxia in MB as well as pediatric brain tumors in general. In vitro studies have identified a role for HIF-1α in chemotherapy resistance, while patient samples suggest hypoxia-induced changes in gene expression as well as proteomic, metabolomic, and lipidomic profiles. Based on this literature review, 55 candidate hypoxia-related genes were identified. The PedcBioPortal for Integrated Childhood Cancer Genomics was used to assess expression differences in pediatric patient samples for these genes of interest.
 Results:
 RNA expression was analyzed for correlation with survival, molecular group, and Chang stage. Expression of DDAH1, HYOU1, MYC, and RBX1 were significantly associated with survival. ANOVA and Ttest with a Bonferroni correction were used to assess for expression differences between groups and Chang stage. Multiple hypoxia candidate genes (ARNT2, BHLHE40, CYP3A5, DDAH1, DDIT4L, EGLN3, MT3, MYC, MYCN, TGFBR2, TP53, VEGFA) were significantly correlated with molecular group. Expression levels of TGFBR3 and GPR37 were associated with Chang stage.
 Conclusion and Potential Impact:
 The results of our systematic review and gene expression analyses support a role for hypoxia in the pathogenesis and potentially the clinical outcomes of children with MB. Future studies comparing gene expression levels at normal oxygen tension (21%) and physiologic oxygen tension (1-3%) will allow us to assess the role of hypoxia in medulloblastoma pathogenesis.
 
 

Characterization of Dental Pulp Stem Cell Populations in the Teeth of Patients With Neurofibromatosis Type 1 - Therapeutic Potential for Bone Tissue Engineering.

Neurofibromas (NF) are the most common benign nerve sheath tumors in the tongue, gingiva, major salivary glands, and jaw bones. Nowadays, tissue engineering is a revolutionary technique for reconstructing tissues. To explore the feasibility of using stem cells derived from NF teeth to treat orofacial bone defects, the differences in cell biological properties between an NF teeth group and Normal teeth group. The intra-dental pulp tissues from each tooth were extracted. The cell survival rates, morphology, proliferation rates, cell activity, and differentiation abilities were contrastively analyzed between the NF teeth group and Normal teeth group. Between the two groups, there were no differences in the primary generation (P0) cells (p>0.05), the cell yield, and the time required for the cells to grow out of the pulp tissue and attach to the culture plate. Furthermore, no differences were found at the first generation (passage) between the two groups in colony formation rate and cell survival rate. The proliferation capacity, cell growth curve, and surface marker expression of dental pulp cells was not altered in the third generation (p>0.05). Dental pulp stem cells from NF teeth were successfully obtained and were not different from normal dental pulp stem cells. Although, clinical research using tissue-engineered bone to repair bone defects is still in its infancy, it will eventually enter the clinic and become a routine means of bone defect reconstruction treatment as related disciplines and technologies develop.

The Study of Mechanical Properties of Biological Cells

Cells are the basic unit of all living organisms. They can realize various specific functions of living organisms such as energy conversion, material transportation, signal transduction, and expression of genetic information. They are the foundation for human research on the life activities of all living organisms. As a line of defense for cells to protect themselves, the cell membrane has certain mechanical properties that can ensure the balance and stability of various normal biochemical reactions inside and outside the cell during its survival, such as cell growth, movement, division, etc. When cells face changes in the environment, their surface mechanical properties will also change to a certain extent. However, the mechanism and manner in which cells respond to environmental influences are still not very clear. Therefore, in order to understand this complex mechanical behavior of cells in living organisms, it is necessary to analyze the mechanical properties of the cell surface.

I.09 Induced pluripotent stem cells for modeling neuromuscular disorders: development of disease-specific assays, live cells functional testing and drug design

Induced pluripotent stem cells (iPSC) represent an innovative, easily obtained and renewable stem cell source. This somatic cell-derived model that can be derived without considerable ethical issues has generated significant enthusiasm for its potential application in basic and translational research or development of new therapeutics. In particular, patient-specific iPS-derived tissues and latterly, organoids offer attractive platform to model a wide range of diseases but also personalized or regenerative medicine. Compared to other tissues, only a limited number of large-scale protocols describe the production of mature skeletal muscle fibers from human iPSCs (hiPSCs). We developed a procedure for simultaneous differentiation of hiPSC into muscle cells and motor neurons, that generates innervated and contractile multinucleated skeletal muscle fibers with sarcomeric organization and formation of neuromuscular junction. Our protocol permits the production of expandable skeletal muscle progenitor cells and mature skeletal muscle fibers that can be maintained and regenerates on the long-term. These in vitro muscle constructs can be used for the exploration of skeletal muscle differentiation for basic research, disease modeling and drug discovery. Our “muscle in a dish” model has been used to successfully model a number of neuromuscular disorders. By combining transcriptomics with artificial intelligence, we were able to design specific pipelines for a comprehensive understanding of the molecular, cell biological and functional properties of hiPSC-derived muscle tissue in diseases. Overall, this strategy opens a wide rage of opportunities for disease modeling, personalized medicine for patients in diagnosis deadlocks, drug design or tissue bioengineering and bioprinting. Induced pluripotent stem cells (iPSC) represent an innovative, easily obtained and renewable stem cell source. This somatic cell-derived model that can be derived without considerable ethical issues has generated significant enthusiasm for its potential application in basic and translational research or development of new therapeutics. In particular, patient-specific iPS-derived tissues and latterly, organoids offer attractive platform to model a wide range of diseases but also personalized or regenerative medicine. Compared to other tissues, only a limited number of large-scale protocols describe the production of mature skeletal muscle fibers from human iPSCs (hiPSCs). We developed a procedure for simultaneous differentiation of hiPSC into muscle cells and motor neurons, that generates innervated and contractile multinucleated skeletal muscle fibers with sarcomeric organization and formation of neuromuscular junction. Our protocol permits the production of expandable skeletal muscle progenitor cells and mature skeletal muscle fibers that can be maintained and regenerates on the long-term. These in vitro muscle constructs can be used for the exploration of skeletal muscle differentiation for basic research, disease modeling and drug discovery. Our “muscle in a dish” model has been used to successfully model a number of neuromuscular disorders. By combining transcriptomics with artificial intelligence, we were able to design specific pipelines for a comprehensive understanding of the molecular, cell biological and functional properties of hiPSC-derived muscle tissue in diseases. Overall, this strategy opens a wide rage of opportunities for disease modeling, personalized medicine for patients in diagnosis deadlocks, drug design or tissue bioengineering and bioprinting.

Global research trends and hotspots on glioma stem cells.

BackgroundGlioma stem cells (GSCs) are a sub-population of cancer stem cells with capacity of self-renewal and differentiation. Accumulated evidence has revealed that GSCs were shown to contribute to gliomagenesis, distant metastasis as well as the resistance to radiotherapy and chemotherapy. As a result, GSCs were regarded as a promising therapeutic target in human glioma. The purpose of our study is to identify current state and hotspots of GSCs research by analyzing scientific publications through bibliometric methods.MethodsAll relevant publications on GSCs during 2003-2021 were extracted from the Science Citation Index Expanded of Web of Science Core Collection (WoSCC), and related information was collected and analyzed using Microsoft Excel 2016, GraphPad Prism 8 and VOSviewer software.ResultsA total of 4990 papers were included. The United States accounted for the largest number of publications (1852), the second average citations per item (ACI) value (67.54) as well as the highest H-index (157). Cancer Research was the most influential journal in this field. The most contributive institution was League of European Research Universities. RICH JN was the author with the most publications (109) and the highest H-index (59). All studies were clustered into 3 groups: “glioma stem cell properties”, “cell biological properties” and “oncology therapy”. The keywords “identification”, “CD133” and “side population” appeared earlier with the smaller average appearing years (AAY), and the keywords”radiotherapy” and “chemotherapy” had the latest AAY. The analysis of top cited articles showed that “temozolomide”, “epithelial-mesenchymal transition”, and “immunotherapy” emerged as new focused issues.ConclusionThere has been a growing number of researches on GSCs. The United States has always been a leading player in this domain. In general, the research focus has gradually shifted from basic cellular biology to the solutions of clinical concerns. “Temozolomide resistance”, “epithelial-mesenchymal transition”, and “immunotherapy” should be given more attention in the future.

High Expression of circ_0001821 Promoted Colorectal Cancer Progression Through miR-600/ISOC1 Axis

It has been reported that circRNAs play an important regulatory role in the progression of colorectal cancer (CRC). However, the molecular role of circ_0001821 in CRC development is unclear. In this study, we aimed to investigate the regulatory role and molecular mechanisms of circ_0001821 in CRC. Reverse transcription-quantitative PCR and western blot assays were used to detect the expression of circ_0001821, miR-600 and isochorismatase domain containing 1 (ISOC1) in CRC tissues as well as its cell lines. Colony formation assay and EDU assay were used to detect the proliferative capacity of cells. Transwell assay was used to assess cell migration and invasion ability. Flow cytometry was used to analyze cell apoptosis. ELISA was used to measure the glycolytic capacity of cells. Dual-luciferase reporter assay and RNA pull-down assay were used to analyze the relationships between circ_0001821, miR-600 and ISOC1. Animal experimentation was used to validate the functional study of circ_0001821 in vivo. Immunohistochemistry (IHC) of Ki67 staining analysis was conducted to assess tumor growth. Circ_0001821 and ISOC1 were significantly increased in CRC tissues and its cell lines, and miR-600 was significantly decreased in CRC tissues and its cell lines. Silencing circ_0001821 inhibited cell proliferation, migration, invasion and glycolytic capacity, while inducing apoptosis. And it could inhibit tumor growth in vivo. Circ_0001821 could act as a sponge for miR-600 to regulate CRC processes. ISOC1 was identified as a downstream regulator of miR-600, also miR-600 could regulate the expression of ISOC1. In addition, circ_0001821 could regulate ISOC1 expression changes through miR-600. Mechanistically, either miR-600 inhibitor or overexpression of ISOC1 could reverse the effects of knockdown of circ_0001821 on cell biological properties. Circ_0001821 regulated the developmental process of CRC through miR-600/ISOC1 axis.

Tetrahydroxyquinone: A Suitable Coating for Ferrofluids Used in Magnetic Hyperthermia

In this work, tetrahydroxyquinone (THQ) was used for the first time to coat iron oxide nanoparticles (IONPs) and to carry out in vitro experiments in magnetic hyperthermia. Synthesis by co-precipitation resulted in spherical IONPs with a core diameter of 13 ± 3 nm and covered by a 0.5 nm thick coat of THQ, which provided them with a reasonably good zeta potential of ζ = −28 ± 2 mV at pH = 7.3, and thus colloidal stability. The magnetic properties of the THQ-coated IONPs are promising: the low coercive field of Hc = 7 Oe, the high magnetic saturation of Ms = 70.5 emu/g and the low blocking temperature of Tb = 273 K indicate superparamagnetic characteristics at room temperature. Additionally, a high specific absorption rate SAR = 135 W/g (at 300 Oe and 530 kHz) was determined. Cell biological experiments using the human cell line HT-29 evidenced negligible cytotoxicity up to 2 mg/mL. Magnetic hyperthermia (MHT) assays demonstrated fast and reliable heating and reduced the metabolic activity of the cells to 42% upon reaching 42 °C within 15 min. The production of ROS by THQ-coated IONPs could not be detected, which may indicate a reduction in the undesired side effects caused by oxidative stress. Considering these good physicochemical and cell biological properties, this ferrofluid is a promising candidate for the initiation of in vivo experiments for cancer treatment by MHT in murine models.

Abstract 3719: Loss of Kmt2c, of the COMPASS-like complex, cooperates with oncogenic signaling in cancer

Abstract In mammals, the histone methyltransferase KMT2C is one of the major regulators of gene expression and is one of the most commonly lost epigenetic regulators across all cancers. Studies in mice demonstrated that Kmt2c is a haploinsufficient tumor suppressor in AML and inoculation of mammary fat pads with breast cancer cells that lack KMT2C had significantly larger tumor growth and reduced survival compared to mice injected with isogenic wild-type cells. Analysis of cBioportal’s curated set of non-redundant cancer patient studies indicates that loss of function KMT2C mutations significantly co-occur with RAS gain of function mutations. Our lab previously discovered that cells expressing oncogenic RAS displayed altered activation of enhancer gene regulator elements. We will test how loss of KMT2C cooperates with oncogenic Ras by: 1) characterizing the impact of KMT2C loss, with or without expression of oncogenic RAS, on biological properties of cells and global chromatin architecture and 2) characterizing how KMT2C loss may cooperate with oncogenic RAS to alter hematopoiesis and the global enhancer landscape in animal models. MEFs expressing heterozygous loss of Kmt2c have a proliferative advantage relative to WT littermate controls. Interestingly, WT MEFs demonstrate enhanced migration in a wound assay, but heterozygotes demonstrate an advantage in a transwell membrane assay. Heterozygotes further display greater EdU incorporation, indicating a greater propensity for entering S-phase and undergoing cell-division. RNA-seq experiments have indicated an upregulation of MAPK & Ras signaling pathway genes in heterozygotes, whilst simultaneously downregulating gene pathways critical for proper extra-cellular matrix organization. Therefore, loss of Kmt2c disrupts biological cell phenotypes, in a manner relatable to the hallmarks of cancer, demonstrating how Kmt2c loss may promote malignancy. Further studies must now be performed to elucidate the molecular mechanisms altered by loss of KMT2C and how they cause changes in cellular phenotypes. Citation Format: Gabriel Prado, Richard L. Bennett, Jonathan D. Licht. Loss of Kmt2c, of the COMPASS-like complex, cooperates with oncogenic signaling in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3719.

Cytochalasin B Modulates Nanomechanical Patterning and Fate in Human Adipose-Derived Stem Cells.

Cytoskeletal proteins provide architectural and signaling cues within cells. They are able to reorganize themselves in response to mechanical forces, converting the stimuli received into specific cellular responses. Thus, the cytoskeleton influences cell shape, proliferation, and even differentiation. In particular, the cytoskeleton affects the fate of mesenchymal stem cells (MSCs), which are highly attractive candidates for cell therapy approaches due to their capacity for self-renewal and multi-lineage differentiation. Cytochalasin B (CB), a cyto-permeable mycotoxin, is able to inhibit the formation of actin microfilaments, resulting in direct effects on cell biological properties. Here, we investigated for the first time the effects of different concentrations of CB (0.1–10 μM) on human adipose-derived stem cells (hASCs) both after 24 h (h) of CB treatment and 24 h after CB wash-out. CB influenced the metabolism, proliferation, and morphology of hASCs in a dose-dependent manner, in association with progressive disorganization of actin microfilaments. Furthermore, the removal of CB highlighted the ability of cells to restore their cytoskeletal organization. Finally, atomic force microscopy (AFM) revealed that cytoskeletal changes induced by CB modulated the viscoelastic properties of hASCs, influencing their stiffness and viscosity, thereby affecting adipogenic fate.

The Effects of a Novel Curcumin Derivative Loaded Long-Circulating Solid Lipid Nanoparticle on the MHCC-97H Liver Cancer Cells and Pharmacokinetic Behavior.

PurposeThe objective of this study was to develop long-circulating solid lipid nanoparticles (LSLN) containing a novel curcumin (CU) derivative (CU1), to improve CU1ʹs pharmacokinetic behavior and its anti-cancer effects in MHCC-97H liver cancer cells.MethodsLSLN loaded with CU1 (CU1-LSLN) was optimized and characterized. The cell biological properties and the anti-cancer mechanism of CU1-LSLN on MHCC-97H cells were evaluated by MTT, flow cytometry, Transwell, and Western blot. CU1-LSLN was further evaluated for pharmacokinetic behavior, biodistribution, and liver toxicity in SD rats.ResultsThe optimized CU1-LSLN formulation showed the ideal particle size (PS), polydispersity index (PDI), zeta potential (ZP), encapsulation efficiency (EE%), and drug loading (DL%) of 122.10 ± 6.63 nm, 0.19 ± 0.02, −36.30 ± 1.25 mV, 94.98 ± 0.90% and 4.53 ± 0.69%, respectively. X-ray powder diffraction (XRD), differential scanning calorimetry (DSC), and Fourier transform infrared spectrometry (FTIR) indicated that CU1 was well encapsulated by LSLN and existed in amorphous form. Storage stability of CU1-LSLN was up to 180 days with a sustained-release of drug over 96 h. The uptake efficiency of CU1-LSLN to MHCC-97H cells was 3.24 and 2.98 times higher than that of CU and CU1 after treatment for 3 h, which helped to enhance the inhibitive effect of CU1-LSLN on the proliferation, migration, and invasion potential of MHCC-97H cells and increased its ability to promote apoptosis. Meanwhile, the expression levels of NF-κB, COX-2, MMP-2, MMP-9, and uPA decreased significantly. In vivo, CU1-LSLN prolonged the retention time of the drug, the area under the curve (AUC) increased significantly (CU: 69.9-fold, CU1: 85.9-fold), and no significant liver toxicity was observed.ConclusionCU1-LSLN is a novel preparation with great potential for treating liver cancer.

Fabrication of highly bioactive zirconia ceramics via grain‐boundary activation for dental implants

AbstractYttria‐stabilized tetragonal zirconia polycrystal (Y‐TZP) ceramics with outstanding mechanical properties and aesthetic origins are expected to be used in dental implant applications. However, tetragonal zirconia ceramics are not bioactive, which affect the osseointegration and reliability as dental implant materials. Herein, in this study, Y‐TZP ceramics were modified by grain‐boundary activation via coating a bioactive glass (BG) sol with different content on the crystal surfaces of zirconia powder and followed by being gelled, dried, granulated, low‐temperature treated, molded and sintered at 1450°C for 3 h in air. The effects of BG content on the morphology, phase compositions, mechanical properties, in vitro mineralization ability and cell biological properties of the bioactivity modified Y‐TZP ceramics were evaluated. The BG additive did not affect the tetragonal–monoclinic phase transformation of ZrO2. However, the addition of BG decreased the flexural strength of the modified Y‐TZP ceramics compared to that of Y‐TZP. The in vitro mineralization results showed that a homogeneous apatite layer was produced on the surface of the Y‐TZP ceramics when they were immersed in the simulated body fluid for 21 days. The cell response results indicated that the bioactive surface modification of Y‐TZP ceramics could promote cell adhesion, propagation and osteogenic differentiation performance. Thus, our research results suggest that the highly bioactive Y‐TZP ceramics could be a potential candidate for dental implant material.

SmartFlareTM is a reliable method for assessing mRNA expression in single neural stem cells.

BACKGROUNDOne of the most challenging tasks of modern biology concerns the real-time tracking and quantification of mRNA expression in living cells. On this matter, a novel platform called SmartFlareTM has taken advantage of fluorophore-linked nanoconstructs for targeting RNA transcripts. Although fluorescence emission does not account for the spatial mRNA distribution, NanoFlare technology has grown a range of theranostic applications starting from detecting biomarkers related to diseases, such as cancer, neurodegenerative pathologies or embryonic developmental disorders.AIMTo investigate the potential of SmartFlareTM in determining time-dependent mRNA expression of prominin 1 (CD133) and octamer-binding transcription factor 4 (OCT4) in single living cells through differentiation.METHODSBrain fragments from the striatum of aborted human fetuses aged 8 wk postconception were processed to obtain neurospheres. For the in vitro differentiation, neurospheres were gently dissociated with Accutase solution. Single cells were resuspended in a basic medium enriched with fetal bovine serum, plated on poly-L-lysine-coated glass coverslips, and grown in a lapse of time from 1 to 4 wk. Live cell mRNA detection was performed using SmartFlareTM probes (CD133, Oct4, Actin, and Scramble). All the samples were incubated at 37 °C for 24 h. For nuclear staining, Hoechst 33342 was added. SmartFlareTM CD133- and OCT4-specific fluorescence signal was assessed using a semiquantitative visual approach, taking into account the fluorescence intensity and the number of labeled cells.RESULTSIn agreement with previous PCR experiments, a unique expression trend was observed for CD133 and OCT4 genes until 7 d in vitro (DIV). Fluorescence resulted in a mixture of diffuse cytoplasmic and spotted-like pattern, also detectable in the contacting neural branches. From 15 to 30 DIV, only few cells showed a scattered fluorescent pattern, in line with the differentiation progression and coherent with mRNA downregulation of these stemness-related genes.CONCLUSIONSmartFlareTM appears to be a reliable, easy-to-handle tool for investigating CD133 and OCT4 expression in a neural stem cell model, preserving cell biological properties in anticipation of downstream experiments.

Impact of LMWH and Specific Factor Xa Inhibitors, Apixaban and Fondaparinux, on Cancer Cell Biology and Procoagulant Properties of Cancer Microenvironment

Background Cancer patients with venous thromboembolism (VTE) or at risk of VTE are treated with antithrombotic agents. Cancer cells express procoagulant properties and induce hypercoagulability in the microenvironment, that could impact the efficiency of the antithrombotic agents.Aims In the present study, we investigated the interaction between antithrombotic agents with pancreatic cancer cells, as well as with their microenvironment. The impact of apixaban, fondaparinux, enoxaparin and tinzaparin on the procoagulant properties of pancreatic cancer cells BXPC3 was examinated. Reciprocally, we also investigated the impact of BXPC3 on the potency of these antithrombotic agents.Methods BXPC3 cells (400 cells/μl) were exposed for 48 hours to apixaban (2 µg/ml), fondaparinux (2 µg/ml), enoxaparin, tinzaparin (2 anti-Xa IU/ml) or NaCL (control). Then, conditioned media (CM) and BXPC3 cells were harvested, separated and put in contact with normal platelet-poor plasma (PPP). Subsequently, thrombin generation (TG) was assessed using Thrombogram-Thrombinoscope® assay (Diagnostica Stago). Cells' viability was also assessed with the MTT assay. Gene expression for Tissue Factor (TF), Vascular Endothelial Growth Factor (VEGF), Thrombospondin 1 (THSB1) was assessed with RT-qPCR at the cells exposed or not to the antithrombotic agents. Expression of TF protein and activity of cancer cells was assessed using ELISA method. Residual anti-Xa activity in CM was measured using specific amidolytic assays for each antithrombotic agent.Results Apixaban, fondaparinux, enoxaparin, and tinzaparin significantly reduced cell viability by 25%, 12%, 14%, and 11% respectively. In the control experiment non treated BXPC3 cells enhanced TG. Pre-treatment of BXPC3 with the antithrombotic agents did not significantly modify their capacity to trigger and enhance TG. Among the studied agents only apixaban resulted in significant decrease of TF mRNA expression. However, protein expression of TF was not significantly modified by any of the antithrombotic agents. VEGF's mRNA expression was significantly decreased by fondaparinux and enoxaparin. THBS1's mRNA expression was significantly increased by apixaban. The concentrations of the anti-Xa activity of fondaparinux, enoxaparin and tinzaparin in the CM obtained at 48h after exposure of cells were reduced by 27%, 48% and 26% respectively as compared to those initially added in the culture medium. In contrast, the concentration of apixaban in the CM did not significantly change. The CM obtained by cells exposed to apixaban, fondaparinux, enoxaparin and tinzaparin inhibited TG by 70%, 30%, 40% and 90% respectively.Conclusion. Antithrombotic agents reduced the viability of BXPC3 cells. Among the studied agents, apixaban had the most pronounced effect on cells' viability. The antithrombotic agents had a potential downregulating effect on the proangiogenetic properties of BXPC3 via the decrease of VEGF gene expression (fondaparinux and enoxaparin) and enhancement of THBS1 gene expression (apixaban). Nevertheless, preincubation of BXPC3 with the antithrombotic agents did not alter the expression of TF protein and their effect on thrombin generation. Moreover, BXPCE exerted a “degradation” effect on LMWH and fondaparinux. Apixaban appeared to escape from this effect of the cancer cells. A significant inhibitory effect on thrombin generation was exerted by the residual concentrations of the antithrombotic agents in the microenvironment of cancer cells. The ensemble of these data highlight for the first time that the presence of antithrombotic agents in cancer cell microenvironment alters the biology of cancer cells and offer a constant antithrombotic effect in the microenvironment. DisclosuresNo relevant conflicts of interest to declare.

Circadian regulation of cancer cell and tumor microenvironment crosstalk.

Circadian rhythms regulate a remarkable variety of physiologic functions in living organisms. Circadian disruption is associated with tumorigenesis and tumor progression through effects on cancer cell biological properties, including proliferation, DNA repair, apoptosis, metabolism, and stemness. Emerging evidence indicates that circadian clocks also play an influential role in the tumor microenvironment (TME). This review outlines recent discoveries on how cancer cell clock components (including circadian clock and clock genes/proteins) regulate TME biology and, reciprocally, how TME clock components affect tumor growth, metastasis, and therapeutic response. An improved understanding of how clock components regulate the symbiosis between cancer cells and the TME will inform the development of novel clock-oriented therapeutic strategies, including immunotherapy.

A Cell's Viscoelasticity Measurement Method Based on the Spheroidization Process of Non-Spherical Shaped Cell.

The mechanical properties of biological cells, especially the elastic modulus and viscosity of cells, have been identified to reflect cell viability and cell states. The existing measuring techniques need additional equipment or operation condition. This paper presents a cell’s viscoelasticity measurement method based on the spheroidization process of non-spherical shaped cell. The viscoelasticity of porcine fetal fibroblast was measured. Firstly, we introduced the process of recording the spheroidization process of porcine fetal fibroblast. Secondly, we built the viscoelastic model for simulating a cell’s spheroidization process. Then, we simulated the spheroidization process of porcine fetal fibroblast and got the simulated spheroidization process. By identifying the parameters in the viscoelastic model, we got the elasticity (500 Pa) and viscosity (10 Pa·s) of porcine fetal fibroblast. The results showed that the magnitude of the elasticity and viscosity were in agreement with those measured by traditional method. To verify the accuracy of the proposed method, we imitated the spheroidization process with silicone oil, a kind of viscous and uniform liquid with determined viscosity. We did the silicone oil’s spheroidization experiment and simulated this process. The simulation results also fitted the experimental results well.

Microfluidic investigation of the effect of graphene oxide on mechanical properties of cell and actin cytoskeleton networks: experimental and theoretical approaches

Biomechanical and morphological analysis of the cells is a novel approach for monitoring the environmental features, drugs, and toxic compounds’ effects on cells. Graphene oxide (GO) has a broad range of medical applications such as tissue engineering and drug delivery. However, the effects of GO nanosheets on biological systems have not been completely understood. In this study, we focused on the biophysical characteristics of cells and their changes resulting from the effect of GO nanosheets. The biophysical properties of the cell population were characterized as follows: cell stiffness was calculated by atomic force microscopy, cell motility and invasive properties were characterized in the microfluidic chip in which the cells are able to visualize cell migration at a single-cell level. Intracellular actin was stained to establish a quantitative picture of the intracellular cytoskeleton. In addition, to understand the molecular interaction of GO nanosheets and actin filaments, coarse-grained (CG) molecular dynamics (MD) simulations were carried out. Our results showed that GO nanosheets can reduce cell stiffness in MCF7 cells and MDA-MB-231 cell lines and highly inhibited cell migration (39.2%) in MCF-7 and (38.6%) in MDA-MB-231 cell lines through the GO nanosheets-mediated disruption of the intracellular cytoskeleton. In the presence of GO nanosheets, the cell migration of both cell lines, as well as the cell stiffness, significantly decreased. Moreover, after GO nanosheets treatment, the cell actin network dramatically changed. The experimental and theoretical approaches established a quantitative picture of changes in these networks. Our results showed the reduction of the order parameter in actin filaments was 23% in the MCF7 cell line and 20.4% in the MDA-MB-231 cell line. The theoretical studies also showed that the GO nanosheet–actin filaments have stable interaction during MD simulation. Moreover, the 2D free energy plot indicated the GO nanosheet can induce conformational changes in actin filaments. Our findings showed that the GO nanosheets can increase the distance of actin-actin subunits from 3.22 to 3.5 nm and in addition disrupt native contacts between two subunits which lead to separate actin subunits from each other in actin filaments. In this study, the biomechanical characteristics were used to explain the effect of GO nanosheets on cells which presents a novel view of how GO nanosheets can affect the biological properties of cells without cell death. These findings have the potential to be applied in different biomedical applications.

Bitter Taste Receptors Are Expressed on Acute Myeloid Leukemia Cells and Their Activation Results in Quiescence Induction and Prevention of Migration

Acute Myeloid Leukemia (AML) is a clonal disease sprouting from a rare population of leukemic stem cells (LSCs). Over the past years, increasing interest is gaining the contribution that cell-extrinsic factors have in AML generation and maintenance. In this context, AML cell ability to sense the changes in the microenvironment is important for different cell functions, including the responsiveness to antineoplastic agents. Bitter taste receptors (T2Rs) are typical G-protein coupled receptors and are normally found on the surface of the tongue. Recent studies showed that T2Rs are widely expressed in various parts of human anatomy and have been shown to be involved in physiology of respiratory system, gastrointestinal tract and endocrine system thus suggesting a wider function in “sensing microenvironment”. So far, very few data about T2Rs expression in cancer cells are available. In particular, their expression and function in AML cells has not been investigated. Our aim was to investigate T2Rs expression and role in regulation of leukemic cell functionality.In the present work, for the first time, we showed that leukemia cell lines OCI-AML3, THP-1, KG1 and AML primary cells expressed several T2R subtypes (Fig.1A). The expression of TAS2R was associated with GPR-related downstream targets, including the beta subunit of gustducin and the PLC beta 2 (Fig.2B). Stimulation of leukemia cell lines with denatonium (DEN), a T2Rs agonist, induced intracellular Ca2+ concentration increase, thus demonstrating T2Rs functionality (Fig. 1C). To investigate whether bitter compound could modulate AML cell biological properties, we performed GEP analysis of DEN treated cells. GEP analysis identified a number of genes significantly modulated by denatonium treatment. Enrichment analysis identify significantly affected biological processes, among which, cell cycle/proliferation, apoptosis, migration/adhesion and DNA repair (Fig.1D). Specifically, leukemic cells stimulated with T2R agonist underwent down-regulation of genes involved in positive regulation of cell proliferation, migration, and cell-cycle. Whereas genes involved in cell adhesion and DNA repair were up-regulated. Functional assays confirmed molecular data. Indeed, depending on the extent of stimulation, TAS2R activation inhibited leukemia cell proliferation (Fig.2A) inducing cell cycle arrest in G0/G1 phase (Fig. 2B) or reduced cell viability inducing apoptosis (Fig. 2C), as demonstrated by caspase cascade activation and mitochondrial stress induction (Fig. 2D-E). Of note, a pronounced inhibitory effect of denatonium on leukemia cells motility, both spontaneous and in response to CXCL-12, was observed (Fig. 2F).Overall, our results indicate that in AML cells the activation of fully functional T2Rs is associated with quiescence induction and prevention of migration. These data may suggest a role for microenvironment “bitter” molecules in regulating leukemia cell functions, including lethal sensitivity to antineoplastic agents. [Display omitted] DisclosuresMartinelli:Pfizer: Consultancy; Celgene: Consultancy; Ariad/Incyte: Consultancy; Amgen: Consultancy; Johnson&Johnson: Consultancy; Roche: Consultancy.

Genetic and biological hallmarks of colorectal cancer.

Colorectal cancer has served as a genetic and biological paradigm for the evolution of solid tumors, and these insights have illuminated early detection, risk stratification, prevention, and treatment principles. Employing the hallmarks of cancer framework, we provide a conceptual framework to understand how genetic alterations in colorectal cancer drive cancer cell biology properties and shape the heterotypic interactions across cells in the tumor microenvironment. This review details research advances pertaining to the genetics and biology of colorectal cancer, emerging concepts gleaned from immune and single-cell profiling, and critical advances and remaining knowledge gaps influencing the development of effective therapies for this cancer that remains a major public health burden.