Single Cell Suspensions Research Articles

Transcutaneous electrical acustimulation promotes wound healing in mice by modulating signaling molecules and mitochondria function

Previous research has identified a variety of factors that contribute to the development and maintenance of wounds. Concurrently, electroacupuncture has been demonstrated to facilitate wound healing. However, the effects of transcutaneous electrical acustimulation (TEA) on wound healing, as well as its relationship with key factors such as Wnt3a, TGF-β, Akt, c-Myc, VEGF-A, SP1, nitric oxide (NO), and mitochondrial function, remain largely unexplored. We hypothesize that TEA will activate the signaling factors and enhance mitochondrial functions to promote the repair of skin wounds in mice. An in vivo experimental study was conducted utilizing mouse models with skin wounds. The study comprised three groups: a TEA treatment with wound group, a skin wound model group, and a control group. Wound areas were measured by calculating the product of the length and width of each wound using calipers. Single-cell suspensions were prepared by excising the wound and the immediately surrounding tissue. These suspensions were stained with Trypan blue to assess cell viability, with specific probes to measure the rate of reactive oxygen species (ROS) positivity, and with reagents to quantify NO content. Western blotting (WB) was employed to evaluate protein levels associated with tissue changes, while quantitative polymerase chain reaction (qPCR) was used to assess RNA expression levels. Immunofluorescence staining was performed to visualize protein content and other relevant cellular structures within tissue sections. TEA exhibited anti-inflammatory properties and promoted wound healing in mice. Western blot analysis revealed that TEA enhanced the expression of proteins associated with Wnt3a, TGF-β, Akt, c-Myc, VEGF-A, and SP1 during the wound healing process. Immunofluorescence staining of tissue sections indicated that TEA upregulated the expression of COL1A1, MFN1, GRP75, GRP78, GRP75/ROS, GRP78/ROS, ISCU, and UCP1 while downregulating FIS1. Additionally, qPCR results demonstrated that TEA promoted the expression of IL-10 and miRNA205-5p while inhibiting MMP9 levels. TEA modulates various signaling molecules, influences chaperone proteins related to stress recovery responses, along with mitochondrial dynamics and metabolism.Graphical abstract

Protein structure and interactions elucidated with in-cell NMR for different cell cycle phases and in 3D human tissue models

Most of our knowledge of protein structure and function originates from experiments performed with purified proteins resuspended in dilute, buffered solutions. However, most proteins function in crowded intracellular environments with complex compositions. Significant efforts have been made to develop tools to study proteins in their native cellular settings. Among these tools, in-cell NMR spectroscopy has been the sole technique for characterizing proteins in the intracellular space of living cells at atomic resolution and physiological temperature. Nevertheless, due to technological constraints, in-cell NMR studies have been limited to asynchronous single-cell suspensions, precluding obtaining information on protein behavior in different cellular states. In this study, we present a methodology that allows for obtaining an atomically resolved NMR readout of protein structure and interactions in living human cells synchronized in specific cell cycle phases and within 3D models of human tissue. The described approach opens avenues for investigating how protein structure or drug recognition responds to cell-cell communication or changes in intracellular space composition during transitions among cell cycle phases.

Growth factors IGF-1 and KGF and adipose-derived stem cells promote migration and viability of primary human keratinocytes in an in vitro wound model

IntroductionIn the field of plastic surgery, epidermal transplantation is a potential treatment for chronic wounds that results in only minor donor site morbidity. Improving the regenerative capacities of epidermal grafts or single-cell suspensions and therefore accelerating healing processes would be of significant interest.MethodsIn the present study, we analyzed the effects of growth factors and adipose-derived stem cells (ADSCs) on keratinocyte properties. For optimum translation into the clinical setting, primary human keratinocytes and patient-matched ADSCs were isolated and used in an in vitro wound model.ResultsThe keratinocyte migration and viability increased after treatment with the growth factors insulin-like growth factor 1 (IGF-1) and keratinocyte growth factor (KGF). A similar effect was observed with the use of a concentrated ADSC-conditioned medium (ADSC-CM). It was further possible to isolate the keratinocytes in a xenogen-free medium, which is essential for clinical translation. Importantly, a patient-dependent influence on the effects of the growth factors and ADSC-CM was observed.DiscussionThis study provides potential for the improvement of epidermal transplantation in the treatment of chronic wounds using xenogen-free isolated and cultivated keratinocytes, growth factors, and ADSC. Translating these results into clinical application may help accelerate wound healing and shorten the time until patients can return to everyday life.

Role of intraperitoneal paclitaxel in eosinophil activation and recruitment in the peritoneal cavity and anti-tumor effects against peritoneal metastasis from gastric cancer.

471 Background: Recent studies indicate that the efficacy of chemotherapy is significantly influenced by the tumor immune microenvironment. Peritoneal cavity contains many immune cells, however, the relationship between immune response within the peritoneal cavity and tumor response remains poorly understood. Methods: Single cell suspensions were obtained from ascites or peritoneal lavages from 41 patients with PM from GC. In 28 patients, cells were collected both before and after 1-3 courses of IP chemotherapy. These samples were immunestained with mAbs targeting to specific lymphoid or myeloid subsets, and their proportions in CD45(+) leukocytes were analyzed using flowcytometry. Eosinophils were purified by magnetic cell sorting for subsequent RNA-Seq analysis. Results: Among 41 patients with PM, tumor leukocyte ratio (TLR) calculated as CD326(+) tumor cells divided by CD45(+) leukocytes varied from 0.002% to 58.1%. TLR was not correlated with any ratios of immune cells, however, the ratios of CD8(+) T cells, NK cells and CD16(-) CD193(+) eosinophils tended to decrease with the increase in TLR. Conversely, the ratios of CD19(+) B cell, CD14(+) macrophages and CD16(+) neutrophils increased with elevated TLR. Mean survival time of these patients was 17.7 months. None of the leukocyte subsets showed significant association with patient outcome. In 28 patients, the ratios of lymphocytes mostly decreased with significant difference in CD4(+) T cells and CD19(+) B cells after IP treatment. In contrast, the ratio of CD11b(+) myeloid cells increased in peritoneal cavity after IP treatment. Among the myeloid cells, the ratio of CD16(-) CD193(+) eosinophils significantly increased. The change was particularly prominent in in post-treatment negative peritoneal lavage cytology (CY0) patients (M=0.47% vs M=10.0%, p<0.0001), while the difference was not significant in post-treatment positive peritoneal lavage cytology (CY1) patients (M=0.93% vs M=0.97%, p=0.84). Patients with eosinophil ratio of ≥2% after IP chemotherapy in peritoneal cavity had significantly longer overall survival compared to those with lower eosinophil ratios (17.3 mo vs 26.7 mo, p=0.034; HR =0.23[0.06-0.89]). The peritoneal eosinophils after IP treatment exhibited elevated levels of CD11b and CD63 expression compared to circulating eosinophils (p<0.01), while SSC-A levels tended to be lower. Gene ontology pathways revealed enrichment of cytokine-mediated signaling pathway, extracellular matrix organization and response to interferon-gamma in peritoneal eosinophils (p<0.0001). Conclusions: IP-PTX induces eosinophil activation and recruitment in the peritoneal cavity, while also affecting other types of immune cells. These effects may potentially contribute to the anti-tumor response against peritoneal metastases.

Abstract TMP114: Characterizing Anemia’s Impact on Cerebral Immune Response after Intracerebral Hemorrhage

Background: Anemia is a risk factor for worse intracerebral hemorrhage (ICH) outcomes, yet the underlying drivers remain unclear. Though anemia and inflammation are interrelated, it is unknown whether anemia influences inflammatory responses to ICH. We investigated the impact of anemia on inflammatory cell phenotypes seen in murine brains with and without ICH. Methods: Two different models of anemia were generated from 8-week-old, female C57/BL6 mice. The separate cohorts included: 1) chronic anemia model via iron-deficient chow compared to iron replete control diet, and 2) acute anemia model via red blood cell hemolysis using anti-TER119 injection compared to IgG control injection. After confirmation of anemia vs control via modified Drabkin assays, ICH was induced via collagenase injection into the right striatum, and brains and peripheral blood harvested 24 hours after ICH. Separate single cell suspensions were prepared from ICH and non-ICH hemispheres as well as peripheral blood and cells were stained for immunophenotyping using flow cytometry. Similar procedures were performed in mice without ICH. Two-tailed Student's t-tests were performed to compare immune cell populations between anemic vs non-anemic mice. Results: We identified a robust cerebral immune response to ICH in all groups. In the chronic anemia model, infiltrating macrophages and lymphocytes, particularly T cells, were elevated, while helper and gamma-delta T cells were lower in the ICH hemisphere in iron deficient anemic mice compared to iron replete controls (p<0.05). However, in the acute anemia model, only non-parenchymal intracranial macrophages were significantly elevated in the ICH hemisphere in anemic anti-TER119 injected mice compared to IgG injected controls (p<0.05). Notably, we observed a significant increase in infiltrating lymphocytes in the contralateral hemispheres in both acute and chronic models of anemia, compared to non-anemic controls. These findings in the brain were distinct from peripheral blood, where no major differences in immune cell populations were noted. Finally, in anemic mice without ICH, we did not identify a differential cerebral immune response compared to non-anemic controls. Conclusions: Anemia can differentially impact the inflammatory response to ICH in the central nervous system, based on its etiology and chronicity. Further work is required to assess whether anemia modification can abrogate pathologic immune pathways and improve ICH outcomes.

Comparison of methods for the preparation of single-cell suspensions of rat retina and immunophenotyping by flow cytometry

Comparison of methods for the preparation of single-cell suspensions of rat retina and immunophenotyping by flow cytometry

Harnessing cell aggregates for enhanced adeno-associated virus manufacturing: Cultivation strategies and scale-up considerations.

The possibility to produce recombinant adeno-associated virus (rAAV) by adherent HEK293T cells was studied in a stirred tank bioreactor (STR) culture of cell aggregates. A proof-of-concept of rAAV production was successfully demonstrated in a process where single cells were first expanded, then cell aggregates were formed by dilution into a different medium 1 day before triple plasmid transfection was conducted. An alternative approach for the STR inoculation using a seed taken from a high cell density perfusion (HCDP) culture was also investigated. It was, however, found that the spent medium of the HCDP inhibited the transfection of HEK293T cell aggregates, which was confirmed when testing with single-cell suspension culture. The formation of aggregates in shaken multi-well plates was also investigated to develop a screening system using the average power input as a scale-down criterion, which revealed that cell aggregates could be generated in 12-well plates, however with a larger size than in a STR. Taking into account the reported higher rAAV production of adherent cells in comparison with single cells for triple-plasmid transfection, HEK293T cell aggregates can possibly surpass single-cell suspension in space-time rAAV yield. The formation of HEK293T cell aggregates in a STR system offers a promising approach for scaling up and intensifying rAAV production by triple-plasmid transfection, in comparison with traditional 2D scale-up methods.

Protocol for the preparation of zebrafish whole heart cell suspension for single-cell analyses.

Obtaining viable cell suspension that accurately represents the diversity of complex tissues is challenging due to the distinct characteristics of each cell type. Here, we present a protocol for preparing a single-cell suspension of the zebrafish embryonic whole heart, detailing steps for heart extraction, cell dissociation, quantification, and quality assessment. This suspension is compatible with downstream analysis on various single-cell platforms. For details on the use and execution of this protocol, please refer to Abu Nahia etal.1.

Single-cell RNA-seq reveals that granulosa cells are a target of phthalate toxicity in the ovary

Abstract Phthalates are known endocrine-disrupting chemicals and ovarian toxicants that are used widely in consumer products. Phthalates have been shown to exert ovarian toxicity on multiple endpoints, altering transcription of genes responsible for normal ovarian function. However, the molecular mechanisms by which phthalates act on the ovary are not well understood. In this study, we hypothesized that phthalates specifically target granulosa cells within the ovarian follicle. To test our hypothesis, we cultured whole mouse antral follicles for 96 h in the presence of vehicle or 10 µg/ml of a phthalate metabolite mixture. At the end of the culture period, follicles were dissociated into single-cell suspensions and subjected to single-cell RNA-sequencing. We used markers from published studies to identify cell-type clusters, the largest of which were granulosa and theca/stroma cells. We further identified subpopulations of granulosa, theca, and stromal cells and analyzed differentially expressed genes between the phthalate treatment and control. Granulosa cells, specifically mural granulosa cells, had the most differentially expressed genes. Pathway analysis of differentially expressed genes from the overall granulosa cell cluster revealed disruption of cell cycle and mitosis, whereas pathway analysis of the mural granulosa cell subcluster identified terms related to translation, ribosome, and endoplasmic reticulum. Our findings suggest that phthalates have both broad impacts on cell types and specific impacts on cellular subtypes, emphasizing the complexity of phthalate toxicity and highlighting how bulk sequencing can mask effects on vulnerable cell types.

A platinum polymer probe for multiplexed single cell suspension mass cytometry and imaging mass cytometry assays

A platinum polymer probe for multiplexed single cell suspension mass cytometry and imaging mass cytometry assays

Energy Metabolism Dysregulation in Myocardial Infarction: An Integrative Analysis of Ischemic Cardiomyopathy.

Myocardial metabolism is closely related to functional changes after myocardial infarction (MI). This study aimed to present an integrative examination of human ischemic cardiomyopathy. We used both GSE121893 single-cell suspension sequencing and GSE19303 transcription microarray data sets from the GEO database, along with a murine MI model for full-spectrum metabolite detection. Through a systematic investigation that involved differential metabolite identification and functional enrichment analysis, we shed light on the pivotal role of energy metabolism dysregulation in the progression of MI. Our findings revealed an association between the core regulatory genes CDKN1A, FOS, ITGB4, and MAP2K1 and the underlying pathophysiology of the disease. These genes are identified as critical elements in the complex landscape of myocardial ischemic disorder, highlighting novel insights into therapeutic targets and the intricate biological mechanisms involved. This analysis provides a framework for future research on the metabolic alterations associated with MI.

A Thermo-responsive collapse system for controlling heterogeneous cell localization, ratio and interaction for three-dimensional solid tumor modeling.

Cancer immunotherapy using engineered cytotoxic effector cells has demonstrated significant potential. The limited spatial complexity of existing in vitro models, however, poses a challenge to mechanistic studies attempting to approve existing approaches of effector cell-mediated cytotoxicity within a three-dimensional, solid tumor-like environment. To gain additional experimental control, we developed an approach for constructing three-dimensional (3D) culture models using smart polymers that form temperature responsive hydrogels. By embedding cells in these hydrogels, we constructed 3D models to organize multiple cell populations at specified ratios on-demand and gently position them by exploiting the hydrogel phase transition. These systems were amenable to imaging at low- and high-resolution to evaluate cell-to-cell interactions, as well as to dissociation to allow for single cell analyses. We have called this approach "thermal collapse of strata" (TheCOS) and demonstrated its use in creating complex cell assemblies on demand in both layers and spheroids. As an application, we utilized TheCOS to evaluate the impact of directionality of degranulation of natural killer (NK) cell lytic granules. Blocking lytic granule convergence and polarization by inhibiting dynein has been shown to induce bystander killing in single cell suspensions. Using TheCOS we showed that lytic granule dispersion induced by dynein inhibition can be sustained in 3D and results in a multi-directional killing including that of non-triggering bystander cells. By imaging TheCOS experiments, we were able to map a "kill zone" associated with multi-directional degranulation in simulated solid tumor environments. TheCOS should allow for the testing of approaches to alter the mechanics of cytotoxicity as well as to generate a wide-array of human tumor microenvironments to assist in the acceleration of tumor immunotherapy.

Mechanism of miR-130b-3p in relieving airway inflammation in asthma through HMGB1-TLR4-DRP1 axis

Asthma is a chronic inflammatory respiratory disease characterized by recurrent breathing difficulties caused by airway obstruction and hypersensitivity. Although there is diversity in their specific mechanisms, microRNAs (miRNAs) have a significant impact on the development of asthma. Currently, the contribution of miR-130b-3p to asthma remains elusive. The goal of this study was to examine whether miR-130b-3p attenuates house dust mite (HDM)-induced asthma through High-mobility group box protein 1 (HMGB1)/Toll-like receptor 4 (TLR4)/mitochondrial fission protein (DRP1) signaling pathway. We elucidate that miR-130b-3p can bind to the HMGB1 3'UTR, attenuating HMGB1 mRNA and protein levels, and nucleo-cytoplasmic translocation of HMGB1. We observed that miR-130b-3p agomir or HMGB1 CKO attenuated HDM-induced airway inflammation and hyperresponsiveness, and decreased Th2-type cytokines in bronchoalveolar lavage fluid (BALF) and mediastinal lymph nodes. Further, HMGB1 CKO contributes to alleviating Th2 inflammation in AT-II cells (CD45.2−/CD31−/Epcam−+/proSP-C+/MHC-II+) from lung single cell suspensions of asthmatic mice by flow cytometry. Our findings identified miR-130b-3p as a potent regulator in asthma that exerts its anti-inflammatory effects by targeting HMGB1 and the subsequent HMGB1/TLR4/DRP1axis, presenting a prospective novel therapeutic avenue for asthma management.

Tualang Honey Has a Protective Effect Against Photodamage and Skin Cancer: An In Vivo Study.

Ultraviolet (UV) B radiation leads to DNA damage by generating cyclobutane pyrimidine dimers (CPDs). UVB-induced CPDs can also result in immune suppression, which is a major risk factor for non-melanoma skin cancer (NMSC). UVB-induced CPDs are repaired by nucleotide repair mechanisms (NER) mediated by xeroderma pigmentosum complementation group A (XPA). The purpose of this study was to investigate the use of TH as a chemopreventive agent against the development of skin cancer. SKH-1 hairless mice were exposed were fed with TH (0.1% v/v) for two weeks and exposed to a single dose of UVB (180 mJ/cm2). Dorsal skin was harvested 24 h post-UVB exposure for evaluation of DNA damage and repair. Lymph nodes were also harvested to prepare single cell suspension for flow cytometric evaluation. For carcinogenesis experiments, SKH-1 hairless mice were given TH (0.1% v/v) ad libitum and exposed to UVB (180 mJ/cm2) thrice a week for 30 weeks. Feeding SKH-1 hairless mice with TH (0.1% v/v) for two weeks prior to a single dose of UVB (180 mJ/cm2) led to a significant increase in XPA in skin and DNA repair cytokines IL-12 and IL-23 in draining lymph nodes. Furthermore, when subjected to the photocarcinogenesis protocol; mice fed with TH developed significantly fewer tumors in comparison to mice fed on drinking water. Our data demonstrate that TH has a protective effect against UVB-induced DNA damage, immune suppression, and skin cancer. Future studies will further investigate the potential of TH as a preventive treatment for NMSC.

Guidelines for preparation and flow cytometry analysis of human nonlymphoid tissue DC.

This article is part of the Dendritic Cell Guidelines article series, which provides a collection of state-of-the-art protocols for the preparation, phenotype analysis by flow cytometry, generation, fluorescence microscopy, and functional characterization of mouse and human dendritic cells (DC) from lymphoid organs, and various nonlymphoid tissues. Within this article, detailed protocols are presented that allow for the generation of single-cell suspensions from human nonlymphoid tissues including lung, skin, gingiva, intestine as well as from tumors and tumor-draining lymph nodes with a subsequent analysis of dendritic cells by flow cytometry. Further, prepared single-cell suspensions can be subjected to other applications including cellular enrichment procedures, RNA sequencing, functional assays, etc. While all protocols were written by experienced scientists who routinely use them in their work, this article was also peer-reviewed by leading experts and approved by all co-authors, making it an essential resource for basic and clinical DC immunologists.

Electro-deformation spectroscopy: A unified method for simultaneous electrical and mechanical characterization of single cells.

The intrinsic electrical and mechanical properties of cells are not only valuable biophysical markers reflective of physiological conditions but also play important roles in the development and progression of human diseases. Existing single-cell techniques are restricted to assessing either mechanical or electrical properties. We introduce the development of electro-deformation spectroscopy (EDS), namely the frequency-dependent electro-deformation, as a new method for simultaneous electrical and mechanical characterization of individual cells in suspension. To facilitate the practical use of this technology, we developed a testing procedure that evaluates red blood cells (RBCs) directly from whole blood in a simple microfluidic system, employing an electric field magnitude of 34 kV/m over a frequency range of 15 MHz to 100 kHz. The EDS measurement is performed under stationary conditions without special cell stabilization, at a moderate throughput of 50-100 cells per minute. We develop an experimental-computational framework to decouple cell electromechanics by optimizing the most suitable parameters of the relative permittivity of cell membrane, cytoplasm electrical conductivity, and membrane shear modulus. This technique, tested on RBCs from 4 healthy human samples, revealed membrane relative permittivity of 3.6 - 5.8, membrane shear modulus of 2.2 - 2.8 µN/m, and cytoplasm conductivity of 0.47 - 0.81 S/m. EDS analysis identifies the marked intrasample heterogeneity and individual variability in both cellular electrical and mechanical properties. The EDS framework can be readily used to test RBCs across different species, pathological states, and other cell types of similar structures as RBCs. STATEMENT OF SIGNIFICANCE: This work introduces electro-deformation spectroscopy (EDS) as a unified method for simultaneous electrical and mechanical characterization of single cells in suspension. This is the first-of-its-kind technology for such purposes. EDS can be performed in a simple microfluidic system with minimal sample preparation, making it a convenient and powerful tool for label-free, non-invasive single-cell analysis. We validate the applicability of EDS by measuring the intrasample heterogeneity and individual variability based on the electromechanical parameters of interest for human red blood cells. Single-cell EDS has the potential to enable rapid and reliable detection of cellular changes by diseases or drug treatments and provide insights into the fundamental bioelectromechanical mechanisms of cellular adaptation and dysfunction. This work advances the field of single-cell analysis and contributes to the development of biomaterials and biotechnologies based on cellular electromechanics.

MIRD Pamphlet No. 31: MIRDcell V4-Artificial Intelligence Tools to Formulate Optimized Radiopharmaceutical Cocktails for Therapy.

Radiopharmaceutical cocktails have been developed over the years to treat cancer. Cocktails of agents are attractive because 1 radiopharmaceutical is unlikely to have the desired therapeutic effect because of nonuniform uptake by the targeted cells. Therefore, multiple radiopharmaceuticals targeting different receptors on a cell is warranted. However, past implementations invivo have not met with convincing results because of the absence of optimization strategies. Here we present artificial intelligence (AI) tools housed in a new version of our software platform, MIRDcell V4, that optimize a cocktail of radiopharmaceuticals by minimizing the total disintegrations needed to achieve a given surviving fraction (SF) of tumor cells. Methods: AI tools are developed within MIRDcell V4 using an optimizer based on the sequential least-squares programming algorithm. The algorithm determines the molar activities for each drug in the cocktail that minimize the total disintegrations required to achieve a specified SF. Tools are provided for populations of cells that do not cross-irradiate (e.g., circulating or disseminated tumor cells) and for multicellular clusters (e.g., micrometastases). The tools were tested using model data, flow cytometry data for suspensions of single cells labeled with fluorochrome-labeled antibodies, and 3-dimensional spatiotemporal kinetics in spheroids for fluorochrome-loaded liposomes. Results: Experimental binding distributions of 4 211At-antibodies were considered for treating suspensions of MDA-MB-231 human breast cancer cells. A 2-drug combination reduced the number of 211At decays required by a factor of 1.6 relative to the best single antibody. In another study, 2 radiopharmaceuticals radiolabeled with 195mPt were each distributed lognormally in a hypothetical multicellular cluster. Here, the 2-drug combination required 1.7-fold fewer decays than did either drug alone. Finally, 2 225Ac-labeled drugs that provide different radial distributions within a spheroid require about one half of the disintegrations required by the best single agent. Conclusion: The MIRDcell AI tools determine optimized drug combinations and corresponding molar activities needed to achieve a given SF. This approach could be used to analyze a sample of cells obtained from cell culture, animal, or patient to predict the best combination of drugs for maximum therapeutic effect with the least total disintegrations.

Enhancing Scalability and Consistency in Pulsed Electric Field Processing of Microalgae: Integrating Single-Cell Suspension Rheology and Multiphysics Simulation

Emerging extraction technologies such as microsecond pulsed electric field (μsPEF) offer an energy-efficient and gentle method for downstream processing of single cells. However, attaining consistent processing outcomes in continuous PEF systems across different scales is challenging due to variations in cell residence times caused by cell concentration and flow-dependent rheological behavior. This study employed COMSOL Multiphysics® simulations to model rheological changes in high-density heterotrophically grown Auxenochlorella protothecoides microalgae suspensions. The model was designed to precisely estimate residence times for consistent μsPEF treatment and increased processing capacity at high flow rates and cell concentrations. Implementing the simulated residence times into experimental validations achieved consistent treatment outcomes, resulting in 86 ± 4% of cells being permeabilized and increased processing capacity from 21 to 300 mL min-1. Higher cell concentrations led to increased energy input and decreased protein extraction yields, indicating complex underlying permeabilization and diffusion processes during and after treatment. This work advances scalable and consistent μsPEF technology for single-cell downstream processing by applying cell suspension rheology and residence time simulations.

Stabilization of SQLE mRNA by WTAP/FTO/IGF2BP3-dependent manner in HGSOC: implications for metabolism, stemness, and progression

AbstractThe metabolic reprogramming in high-grade serous ovarian carcinoma (HGSOC) affects the tumor stemness, which mediates tumor recurrence and progression. Knowledge of the stemness and metabolic characteristics of HGSOC is insufficient. Squalene epoxidase (SQLE), a key enzyme in cholesterol metabolism, was significantly upregulated in HGSOC samples with a fold change of about 4 in the RNA sequencing analysis. SQLE was positively related to peritoneal metastasis and poor prognosis of HGSOC patients. Functionally, SQLE drove cancer cell proliferation and inhibited apoptosis to accelerate HGSOC growth. SQLE was highly expressed in ALDH+CD133+ FACS-sorted cells derived from HGSOC cells and ovarian cancer stem cells (OCSCs)-enriched tumorspheres. SQLE overexpression resulted in enhanced CSC-like properties, including increased tumorsphere formation and stemness markers expression. In vivo, SQLE not only promoted cell line-derived xenografts growth but extended the OCSCs subpopulation of single-cell suspension. Moreover, non-targeted metabolomics profiling from UPLC-MS/MS system identified 90 differential metabolites responding to SQLE overexpression in HGSOC cells. Among them, the dysfunctional metabolisms of cholesterol and glutathione were involved in the maintenance of HGSOC stemness. Previous studies showed the alteration of N6-Methyladenosine (m6A) modification in HGSOC development. Herein, the m6A modification in the 3’UTR and CDS regions of SQLE mRNA was increased due to upregulated methyltransferases WTAP and downregulated demethylases FTO, which was recognized by m6A-binding proteins IGF2BP3, rather than IGF2BP1 or IGF2BP2, thereby stabilizing the SQLE mRNA. These results suggested that SQLE was a novel potential clinical marker for predicting the HGSOC development and prognosis, as well as a potential therapeutic target of HGSOC.

IMMUNOREACT 9 metachronous rectal cancers have high HLA-ABC expression on healthy epithelium but a lower infiltration of CD3+ T cells than primary lesions

Lynch syndrome is rarely associated with rectal cancer (RC) and thus, metachronous RC has been scarcely investigated. This study aimed to analyze the mucosal immune microenvironment in sporadic and metachronous RC. We analyzed the mucosal immune microenvironment in the 25 metachronous RCs present in the IMMUNOREACT 1 and 2 multicentre observational studies (624 patients). A panel of immune markers was retrospectively investigated at immunohistochemistry: CD3, CD4, CD8, CD8b, Tbet, FoxP3, PD-L1, MSH6, and PMS2 and CD80. Single-cell suspensions were subjected to flow-cytometry to determine the proportion of epithelial cells (pan-cytokeratin) acting as antigen-presenting cells (expressing CD80, CD86, HLA-ABC) and the proportion of activated CD8 + T cells (CD8 + positive for CD28, CD38), inhibitory T cells (CD3 + CTLA-4+) of activated CD4 + T helper cells (CD4 + CD25+) and activated T regulatory cells (CD4 + CD25 + FoxP3+). No mismatch repair gene deficiencies were observed in the patients. The previous history of colorectal adenoma was significantly more frequent in metachronous RC. In healthy epithelial cells, HLA-ABC expression was significantly higher in patients with metachronous RC. In therapy-naïve metachronous RC patients, a significantly lower level of circulating lymphocytes and CD3 + T-cell infiltration in the healthy mucosa surrounding the RC was observed compared to patients with non-metachronous cancer. Our study supports the hypothesis that metachronous RC can occur in a cancerization field in patients with weak systemic and local immune systems. The peculiar site of RC makes the mismatch-repair genes deficiency in metachronous cancer onset less relevant.