Cell Phenotype Research Articles

APMAT analysis reveals the association between CD8 T cell receptors, cognate antigen, and T cell phenotype and persistence

APMAT analysis reveals the association between CD8 T cell receptors, cognate antigen, and T cell phenotype and persistence

The absolute number of circulating Treg cells is reduced in difficult-to-treat RA patients and is ameliorated by low-dose IL-2

ObjectiveCirculating regulatory T cells (Tregs) are closely related to immune tolerance and maintenance of immune homeostasis. Perhaps, there is a unique immune cell phenotype for difficult-to-treat rheumatoid arthritis (D2T RA). Low-dose interleukin-2 (IL-2) has been considered for the treatment of autoimmune diseases. This study focused on the uniqueness of D2T RA lymphocyte subsets and the feasibility of low-dose IL-2 therapy.MethodsParticipants included 1,042 RA patients who were divided into three groups according to the presence or absence of treatment and their response to treatment in the last 6 months—new group, treated group, and D2T group—and 339 healthy controls (HCs). A total of 381 patients—107, 151, and 123 in each of the three experimental groups—received low-dose IL-2 treatment [0.5 million international units (MIU) per day, subcutaneous injection from day 1 to day 5]. The absolute numbers of peripheral blood lymphocyte subsets were detected by flow cytometry (FCM) and serum cytokine levels were detected by flow cytometry bead array (CBA).ResultsThe absolute number of T, CD4+ T, and Treg cells in the D2T RA group was lower than that in the HC, new, and treated RA groups. Compared with the HC and new RA group, the ratio of Th17/Treg cells in the D2T RA group increased. The new, treated, and D2T RA groups had higher cytokine levels than the HC. The number of Treg cells in RA patients was negatively correlated with the disease activity index. Treg cells in the new, treated, and D2T RA groups could be increased by low-dose IL-2 therapy without any side effects.ConclusionsThe number of lymphocytes and subsets in D2T RA patients was reduced, especially Treg cells, resulting in a shift in the balance of effector T cells/Treg cells toward effector T cells, which is ameliorated by low-dose IL-2 without obvious side effects.

Differences in phenotypes of normal and malignant cells concerning chromosomal fragility and ganglioside expression

The spontaneous chromosomal fragility was tested by light microscopy observation of metaphases from peripheral blood lymphocytes of 8 patients with malignancies and of 8 healthy controls. In the tested patients, a significantly higher frequency of the spontaneous chromosomal fragility was observed compared to the controls, especially in the centromere chromosomal regions. Of particular interest were interactions involving gangliosides, the reduced form of tri-peptide glutathione (GSH) and/or of tumor-suppressor protein HACE1. The average titers of gangliosides and of anti-ganglioside antibodies in extracts from experimental in vitro models of laboratory-incubated cultures of mouse embryonic 3T3 fibroblasts, of mouse malignant myeloma cells, as well as of mixed cultures of both cellular types, were determined after previous passing of each one through GSH-agarose columns about the “selection” of the molecules in each one of the described samples, possessing affinity to GSH. Additionally, the presence and expression of the tumor-suppressor gene HACE-1 in the genome of mouse embryonic stem cells (mESCs) and malignant human cervical carcinoma HeLa cells, both containing an additional copy of this gene, inserted by transfection with appropriate recombinant DNA vectors containing a copy of the tumor-suppressor gene, were tested. The developed experimental in vitro models show specific intermolecular interactions, which could prevent the disease development. Furthermore, a possibility about the production of antibodies/immunoglobulins by non-lymphoid cellular types was shown. Because the antibodies produced in this manner are outside the germinal centers in the specialized lymphoid tissues and organs, the control of their functions by small ions and molecules, such as gangliosides, is important.

Ruxolitinib synergizes with regulatory T cells to improve inflammation but has no added benefits in decreasing albuminuria in SLE

BackgroundUmbilical cord blood (UCB)-derived CD4+CD25+CD127low regulatory T cells (Tregs) can decrease albuminuria and anti-dsDNA IgG in systemic lupus erythematosus (SLE). Ruxolitinib, a JAK/STAT inhibitor, has been shown to improve cutaneous manifestations of SLE. We hypothesize that the addition of ruxolitinib to UCB-Tregs may improve SLE outcomes.MethodsIn vitro cell suppression, phenotype change, IL-10 secretion, and cytokine levels in coculture supernatants were determined to quantify the impact of adding ruxolitinib to UCB-Tregs. A xenogeneic SLE model was utilized to study their in vivo combination.ResultsIn a dose-dependent manner, ruxolitinib addition synergizes with UCB-Tregs to suppress SLE-PBMC proliferation, inhibit CD8+ T cells, and reduce phosphorylation of STAT3/STAT5/AKT in CD8+ T cells. UCB-Treg and ruxolitinib combination also downregulates the soluble form of inflammatory cytokines including IFN-γ, IP-10, TNF-α, IL-6, sCD40L, IL-17A, IL-17F, IL-1α, and LIF in cocultures. The addition of ruxolitinib increases UCB-Treg cell persistence in peripheral blood in vivo and decreases the soluble form of human inflammatory cytokines including IFN-γ, TNF-α, and sCD40L in plasma along with improvement of skin lesions in SLE xenografts. Compared to control, significantly lesser CD3+, CD4+, CD8+, and Ki-67+ infiltrates are observed in the lung and kidney of UCB-Tregs and/or ruxolitinib recipients. No added benefit of addition of ruxolitinib is observed on the significant improvement in the urine albumin/creatinine ratio and the anti-dsDNA IgG levels induced by UCB-Tregs.ConclusionsOur results demonstrate that the addition of ruxolitinib to UCB-Tregs increases UCB-Tregs suppressor function and their persistence in vivo, downregulates systemic inflammation, and controls cutaneous SLE but does not add to UCB-Treg-mediated improvement in renal manifestations.

Immunomodulation by allograft endothelial cells

It is increasingly appreciated that the expression of immunoregulatory molecules within tumors have potential to shape a microenvironment that promotes local immunoevasion and immunoregulation. However, little is known about tissue-intrinsic immunomodulatory mechanisms following transplantation. We propose that differences in the phenotype of microvascular endothelial cells impact the alloantigenicity of the graft and its potential to promote immunoregulation following transplantation. We focus this review on the concept that graft-dependent immunoregulation may evolve post-transplantation, and that it is dependent on the phenotype of select subsets of intragraft endothelial cells. We also discuss evidence that long-term graft survival is critically dependent on adaptive interactions among immune cells and endothelial cells within the transplanted tissue microenvironment.

Patient-Derived Cancer-Associated Fibroblasts Support the Colonization of Tumor Cells in Head and Neck Squamous Cell Carcinoma

Background: The crosstalk between cancer-associated fibroblasts (CAFs) and tumor cells promotes proliferation, tumor relapse, and the acquisition of a partial epithelial-to-mesenchymal (pEMT) phenotype in tumor cells. The aim of this study was to investigate the effects of patient-derived CAFs on tumor cell growth and radioresistance in head and neck squamous cell carcinoma (HNSCC). Methods: CAFs were isolated and cultured in a three-dimensional spheroid formation. SCC-25 tumor cells educated by the CAFs (SCC25-E cells) were subjected to irradiation, and the response of the CAF-stimulated tumor cells to radiotherapy was determined using an MTT assay, a clonogenic assay, and Western blotting. Tumor cell morphological changes and growth dynamics were assessed using 3D holotomographic microscopy and a live video microscope. Results: Patient-derived CAFs significantly increased the growth rate of SCC-25 cells. CAFs drove fibrosis in the tumor microenvironment (TME), functioned as a physical barrier, temporarily stopped tumor growth, and induced the p38 mitogen-activated protein kinase (MAPK) signaling pathway. Viability after irradiation at 4–8 Gy was significantly higher in SCC25-E cells than in the controls (p = 8 × 10–4 or lower). Furthermore, irradiation triggered the pEMT profile in HNSCC cells. Conclusions: CAFs’ education of tumor cells and the induced p38 phosphorylation had no influence on irradiation sensitivity. SCC25-E cultures demonstrated increased tumor cell growth, viability, and stress-induced phospho-p38 activation.

Discovery and in vitro characterization of a human anti-CD36 scFv

IntroductionCD36 is a membrane receptor that participates in the cellular uptake of fatty acids and lipid metabolism. CD36 overexpression favors progression of different pathologies, such as atherosclerosis and cancer. Thus, CD36 targeting has medicinal relevance. Herein, we aimed to identify human anti-CD36 single-chain variable fragment (scFv) with therapeutic potential.MethodsThe semisynthetic ALTHEA Gold Plus Libraries™ were panned using recombinant human CD36. Clone selection was performed by ELISA. Analysis of scFv binding and blocking function was evaluated by flow cytometry in macrophage-like THP-1 cells and hepatocellular carcinoma HepG2 cells. The phenotypic changes induced by CD36 ligands were assessed in vitro by: i) oil red staining, ii) tumorsphere assays, and iii) RT-qPCR.ResultsWe identified an anti-CD36 scFv, called D11, that competes with a commercial anti-CD36 antibody with proven efficacy in disease models. D11 binds to CD36 expressed in the membrane of the cellular models employed and reduces the uptake of CD36 ligands. In macrophage-like THP-1 cells, D11 impaired the acquisition of foam cell phenotype induced by oxLDL, decreasing lipid droplet content and the expression of lipid metabolism genes. Treatment of HepG2 cells with D11 reduced lipid accumulation and the enhanced clonogenicity stimulated by palmitate.ConclusionWe discovered a new fully human scFv that is an effective blocker of CD36. Since D11 reduces the acquisition of pathogenic features induced by CD36 ligands, it could support the generation of therapeutic proteins targeting CD36.

FAM210B activates STAT1/IRF9/IFIT3 axis by upregulating IFN-α/β expression to impede the progression of lung adenocarcinoma

FAM210B (family with sequence similarity 210 member B) is a novel protein that has been linked to tumor development. However, its role and underlying mechanisms in lung adenocarcinoma (LUAD) progression remain largely unexplored. In this study, FAM210B was observed to be down-regulated in LUAD cells. Analyses of public datasets revealed that decreased expression of FAM210B predicts poor survival. Accordingly, in vitro and in vivo studies have confirmed the inhibitory role of FAM210B on the growth and tumor metastasis of LUAD cells. RNA-seq analysis further indicated that FAM210B plays a role in regulating innate immune-related signaling pathways in LUAD cells, particularly involving the production of type I interferon (IFN-α/β). Specifically, FAM210B activates STAT1/IRF9/IFIT3 axis by upregulating IFN-α/β expression, leading to the inhibition of proliferation and migration of LUAD cells. Furthermore, TOM70 (Translocase of outer mitochondrial membrane 70, also named as TOMM70) has been identified as a functional interacting partner of FAM210B in its modulation on the expression of IFN-α/β, as well as the proliferative and metastatic phenotypes of LUAD cells. In conclusion, our study indicates that FAM210B is an important suppressor of cellular viability and mobility during lung cancer progression.

3D Single-Molecule Super-Resolution Imaging of Microfabricated Multiscale Fractal Substrates for Calibration and Cell Imaging.

Microstructures arrayed over a substrate have shown increasing interest due to their ability to provide advanced 3D cellular models, which open up new possibilities for cell culture, proliferation, and differentiation. Still, the mechanisms by which physical cues impact the cell phenotype are not fully understood, hence the necessity to interrogate cell behavior at the highest resolution. However, cell 3D high-resolution optical imaging on such microstructured substrates remains challenging due to their complexity as well as axial calibration issues. In this work, we address this issue by leveraging the geometrical characteristics of fractal-like structures, which serve as axial calibration tools and modulate cell growth. To this end, we use multiscale 3D SiO2 substrates consisting of spatially arrayed octahedral features of a few micrometers to hundreds of nanometers. Through optimizations of both the structures and optical imaging conditions, we demonstrate the potential of these 3D multiscale structures as an alternative to electron microscopy for material imaging but also as calibration tools for 3D super-resolution microscopy. We used their multiscale and known geometry to perform lateral and axial calibrations in 3D single-molecule localization microscopy (SMLM) and assess imaging resolutions. We then utilized these substrates as a platform for high-resolution bioimaging. As a proof of concept, we cultivate human mesenchymal stem cells on these substrates, revealing very different growth patterns compared to flat glass. Specifically, the spatial distribution of cytoskeleton proteins is vastly modified, as we demonstrate with a 3D SMLM assessment.

Cigarette smoke components modulate the MR1-MAIT axis.

Tobacco smoking is prevalent across the world and causes numerous diseases. Cigarette smoke (CS) compromises immunity, yet little is known of the components of CS that impact T cell function. MR1 is a ubiquitous molecule that presents bacterial metabolites to MAIT cells, which are highly abundant in the lungs. Using in silico, cellular, and biochemical approaches, we identified components of CS that bind MR1 and impact MR1 cell surface expression. Compounds, including nicotinaldehyde, phenylpropanoid, and benzaldehyde-related scaffolds, bound within the A' pocket of MR1. CS inhibited MAIT cell activation, ex vivo, via TCR-dependent and TCR-independent mechanisms. Chronic CS exposure altered MAIT cell phenotype and function and attenuated MAIT cell responses to influenza A virus infection in vivo. MR1-deficient mice were partially protected from the development of chronic obstructive pulmonary disease (COPD) features that were associated with CS exposure. Thus, CS can impair MAIT cell function by diverse mechanisms, and potentially contribute to infection susceptibility and disease exacerbations.

CircICMT upregulates and suppresses the malignant behavior of bladder cancer.

Circular RNA (circRNA) is a new type of endogenous single-stranded RNA with a covalently closed circular structure. Increasing evidence shows that circRNA plays an important role in regulating gene expression in tumors. circICMT is a circular RNA produced by the ICMT gene. Currently, the molecular function of circICMT in bladder cancer remains unclear. Differentially expressed circRNAs were identified from RNA sequencing data and circICMT was identified as a new candidate circRNA. qRT-PCR and sanger sequencing were used to detect the expression of circICMT in bladder cancer tissue specimens. Stable cell lines overexpressing and knocking down circICMT were constructed to explore the effect of circICMT on bladder cancer cells. Its biological effects were detected through wound healing experiments, colony formation experiments, CCK-8 experiments and xenogeneic tumorigenesis experiments. This study found that circICMT was significantly upregulated in bladder cancer tissue specimens. Overexpression of circICMT can inhibit cell migration, proliferation and colony formation ability, while knockdown of circICMT promotes the malignant phenotype of bladder cancer cells. Bioinformatics predictions have found that circICMT can bind to a variety of miRNAs and RBPs and may form a complex regulatory network to regulate the progression of bladder cancer. circICMT is significantly highly expressed in bladder cancer, and intervening circICMT expression affects the malignant phenotype of bladder cancer cells in vivo and in vitro, which may provide potential biomarkers and therapeutic targets for the management of bladder cancer.

Spinal AT1R contributes to neuroinflammation and neuropathic pain via NOX2-dependent redox signaling in microglia

Microglia-mediated neuroinflammation demonstrates a crucial act in the progression of neuropathic pain. Oxidative damage induced by reactive oxygen species (ROS) derived from NADPH oxidase (NOX) in microglia drives proinflammatory microglia activation. Recent evidence points to the central renin angiotensin system (RAS) is involved in oxidative stress and neuroinflammation, with the angiotensin converting enzyme/angiotensin II/angiotensin receptor-1 (ACE/Ang II/AT1R) axis promoting inflammation through increased ROS production, counteracted by the ACE2/Ang (1-7)/ Mas receptor (MasR) axis. While interventions targeting spinal AT1R have been shown to alleviate nociceptive hypersensitivity; yet the mechanisms remain elusive. Here, we discovered that spared nerve injury (SNI)-induced mechanical allodynia in rats were associated with M1-like microglia activation, oxidative stress and overactivity of ACE/Ang II/AT1R axis in the spinal cord. Increased AT1R and NOX2 expression were observed in activated dorsal horn microglia following SNI. Blockade of AT1R with losartan potassium (LOP) suppressed NOX2-mediated oxidative stress, and promoted a shift in microglia from the proinflammatory M1 phenotype to the anti-inflammatory M2 phenotype in LPS-treated BV-2 cells. Additionally, NOX2 overexpression triggered the activation of the high-mobility group box 1/ nuclear factor-kappa B (HMGB1/NF-κB) signaling pathway. Intrathecal administration of LOP effectively inhibited SNI-induced NOX2 overactivation in microglia and suppressed the HMGB1/NF-kB pathway, reducing oxidative stress and shifting the microglia polarization from M1 to M2 in the spinal cord, thereby attenuating neuroinflammation and pain hypersensitivity. Collectively, these findings underscore the neuroimmune-modulating effects of spinal AT1R in neuropathic pain, highlighting the regulation of redox homeostasis in microglia via a NOX2 dependent mechanism.

Epithelial stem cells from human small bronchi offer a potential for therapy of idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a fibrosing interstitial pneumonia with restrictive ventilation. Recently, the structural and functional defects of small airways have received attention in the early pathogenesis of IPF. This study aimed to elucidate the characteristics of small airway epithelial dysfunction in patients with IPF and explore novel therapeutic interventions to impede IPF progression by targeting the dysfunctional small airways. Airway trees spanning the proximal-distal axis were harvested from control lungs and explanted lungs with end-stage IPF undergoing transplant. Qualified basal cells (BCs, p63/Krt5/ITGA6/NGFR) were expanded, and their cellular functions, feasibility, safety and efficacy for transplantation therapy in IPF were validated with experiments invitro and mouse model. Single-cell RNA-sequencing was employed to elucidate the underlying mechanisms governing the BCs based therapy. Based upon these evidences, three patients with advanced IPF and small airway dysfunction received autologous-BCs transplantation. Post-transplantation assessments included lung function, exercise capacity and high resolution computed tomography (HRCT) scans were analyzed to quantify the clinical benefits conferred by the BCs transplantation. An overall landscape of senescent phenotype in airway epithelial cells and airway stem/progenitor cells along the proximal-distal axis of the airway tree in IPF were outlined. In contrast to the cells situated in distal airways, BCs located in small bronchi in IPF displayed a non-senescent phenotype, with comparable proliferative, differentiative capabilities, and similar transcriptomic profiles to normal controls. In a mouse model of pulmonary fibrosis, BCs exhibited promising protective efficacy and safety for transplantation therapy. Autologous BCs transplantation in three advanced IPF patients with small airway dysfunction yielded significant clinical improvements in pulmonary function, particularly evidence in lung volume and small airway function. Epithelia of small bronchi in IPF contain functional and expandable basal stem cells, which exert therapeutic benefits via bronchoscopic implantation. Our findings offer a potential for IPF treatment by targeting small airways. National Natural Science Foundation of China (82430001, 81930001, and 81900059), Shanghai Shenkang Hospital Development Center (SHDC2020CR3063B), Department of Science and Technology of Shandong Province (2024HWYQ-058).

Loss of tumor cell surface hepatocyte growth factor activator inhibitor-1 predicts worse prognosis in esophageal squamous cell carcinoma.

Hepatocyte growth factor activator inhibitor-1 (HAI-1) is an epithelial type-1 transmembrane protease inhibitor that regulates the pericellular activities of hepatocyte growth factor activator and type-2 transmembrane serine proteases. It is strongly expressed in the stratified squamous epithelium and functions on the cell surface. We previously reported that the cell surface immunoreactivity of HAI-1 was reduced at the invasion front of oral squamous cell carcinoma. In this study, we investigate the relationship between cell surface HAI-1 (csHAI-1) and prognosis of esophageal squamous cell carcinoma (ESCC) after surgery. The effect of HAI-1 knockdown on cultured ESCC cells was also analyzed in vitro. HAI-1 exhibited distinct cell surface immunoreactivity in normal esophageal epithelium. In contrast, alterations in HAI-1 immunoreactivity were frequent in cancer cells, which exhibited aberrant intracytoplasmic localization and decreased cell surface immunoreactivity. The preservation of csHAI-1 immunoreactivity was a sign of a well-differentiated phenotype of ESCC cells. The decreased csHAI-1 was associated with shorter overall survival (OS) and disease-free survival (DFS) in the patients. In 55 cases of early (T1) ESCC cases, decreased csHAI-1 also predicted poor OS and DFS. The loss of HAI-1 enhanced migration and invasion of ESCC cells in vitro. These results suggest that the decreased cell surface immunoreactivity of HAI-1 is associated with a less differentiated phenotype and worse prognosis in ESCC. The cell surface-localized HAI-1 may serve as a promising marker for predicting recurrence and prognosis of ESCC.

The dominance of old blood, and age-related increase in protein production and noise.

This concise review provides new perspectives on systemic reduction of tissue aging by comparing different strategies, such as heterochronic parabiosis, injections of young blood plasma, neutral blood exchange (NBE) and therapeutic plasma exchange (TPE). Unlike previous literature that primarily discusses the need for young blood factors, we emphasize the potential of diluting age-elevated proteins as the way to re-calibrate systemic proteome to its younger state without donor blood. Furthermore, we introduce modulation of proteome noise, as an important part of understanding tissue aging and as a critical mechanism for tissue rejuvenation. We discuss studies on the dominance of aged systemic milieu in promoting progeric phenotypes in young cells, in vitro, and in multiple tissues of young animals, in vivo. We support our arguments with evidence showing a significant age-related increase in protein synthesis, in noise of newly synthesized proteomes, and in the rapid induction of these aging phenotypes in young muscle by exposure to aged tissue. We summarize the significance of these findings for future research on aging and longevity.

The role and mechanism of triptolide, a potential new DMARD, in the treatment of rheumatoid arthritis.

Triptolide (TP) is the primary pharmacological component of Tripterygium Glycosides (TG), which has anti-inflammatory, antiproliferative, and immunosuppressive properties, among other pharmacological actions, and has excellent potential for developing into a new DMARD. We have reviewed the effects and mechanisms of TP on immunosuppression, inhibiting synovial proliferation, and preventing articular bone destruction in the treatment of rheumatoid arthritis (RA), which is a common disease in the elderly in this paper. We have found that TP has regulatory effects on multiple vital cells in the above-mentioned pathological process of RA, such as monocytes/macrophages, dendritic cells, T cells, fibroblast-like synoviocytes, and osteoclasts. We also found that TP can regulate multiple key signaling pathways such as NF-κB, JAK/STAT, and MAPK through various molecular regulatory mechanisms, achieving regulatory effects on numerous phenotypes of the above-mentioned vital cells.

SMURF1 leads to the β-catenin signaling-mediated progression of esophageal squamous carcinoma by losing PATZ1-induced CCNG2 transcription

Cyclin G2 (CCNG2), a known inhibitor of cell cycle progression, has been identified as a suppressor for the canonical β-catenin pathway. This study explores the impact of CCNG2 on β-catenin activity and malignant characteristics of esophageal squamous cell carcinoma (ESCC) cells, and the mechanism behind CCNG2 dysregulation. In ESCC tissues and cells, CCNG2 was under-expressed and associated with poor clinical outcomes, whereas β-catenin showed an opposite trend. Inducing CCNG2 overexpression in ESCC cells led to a reduction in β-catenin levels, which in turn suppressed proliferation, cell cycle progression, migration, invasion, stemness, and tumorigenesis. Additionally, it enhanced the cytotoxicity and proliferation of T cells in co-culture systems. However, these beneficial effects were negated by the Wnt signaling agonist BML-284. Furthermore, PATZ1 was found as a transcription factor promoting CCNG2 transcription. However, the PATZ1 protein in ESCC cells was degraded by SMURF1. Silencing of SMURF1 restored CCNG2 expression and inhibited β-catenin, thereby suppressing the malignant phenotype of ESCC cells and reducing T cell exhaustion. Yet, these effects were blocked by further silencing of PATZ1. In summary, this research demonstrates that SMURF1 activates β-catenin signaling by suppressing the PATZ1/CCNG2 axis, thereby promoting the progression of ESCC.

Network analyses of brain tumor multiomic data reveal pharmacological opportunities to alter cell state transitions

Glioblastoma Multiforme (GBM) remains a particularly difficult cancer to treat, and survival outcomes remain poor. In addition to the lack of dedicated drug discovery programs for GBM, extensive intratumor heterogeneity and epigenetic plasticity related to cell-state transitions are major roadblocks to successful drug therapy in GBM. To study these phenomenon, publicly available snRNAseq and bulk RNAseq data from patient samples were used to categorize cells from patients into four cell states (i.e., phenotypes), namely: (i) neural progenitor-like (NPC-like), (ii) oligodendrocyte progenitor-like (OPC-like), (iii) astrocyte-like (AC-like), and (iv) mesenchymal-like (MES-like). Patients were subsequently grouped into subpopulations based on which cell-state was the most dominant in their respective tumor. By incorporating phosphoproteomic measurements from the same patients, a protein–protein interaction network (PPIN) was constructed for each cell state. These four-cell state PPINs were pooled to form a single Boolean network that was used for in silico protein knockout simulations to investigate mechanisms that either promote or prevent cell state transitions. Simulation results were input into a boosted tree machine learning model which predicted the cell states or phenotypes of GBM patients from an independent public data source, the Glioma Longitudinal Analysis (GLASS) Consortium. Combining the simulation results and the machine learning predictions, we generated hypotheses for clinically relevant causal mechanisms of cell state transitions. For example, the transcription factor TFAP2A can be seen to promote a transition from the NPC-like to the MES-like state. Such protein nodes and the associated signaling pathways provide potential drug targets that can be further tested in vitro and support cell state-directed (CSD) therapy.

Dose-responsive effects of endothelial cell-sourced exosomes on vascular cell proliferation and phenotype transition.

Endothelial cell-sourced exosomes are potential participants in the process of atherosclerosis, and their function is mainly affected by concentration. By studying the effects of exosome concentrations on vascular cells, atherosclerosis can be better intervened. In this study, exosomes with concentrations of 0, 0.07, 0.35, 1.75 and 8.75μg/mL were set to interact with endothelial cells, macrophages and smooth muscle cells respectively. The results suggested that EC-Exo altered vascular cells' proliferation, migration and nitric oxide release abilities, increasing with EC-Exo concentrate from 0 to 1.75μg/mL and varing with cell types at 8.75μg/mL. The effects of exosome on cells is dose-responsive，and endothelial cells-sourced exosome favors vascular repair within the concentration of 0.35-1.75μg/mL，showing potential for atherosclerosis regulation.

CD8 + T cell proximity analysis of the tumor microenvironment (TME) in patients with pancreatic ductal adenocarcinoma (PDAC) and associated clinical outcomes.

754 Background: Immunosuppressive cells (cancer-associated fibroblasts [CAFs], tumor associated neutrophils [TANs], M2/M1 macrophages) can influence CD8 + T cell infiltration in the PDAC TME. The spatial proximity of these cells to CD8 + T cells and association with clinical outcomes is not well known. We hypothesize the cell distances between CD8 + T cells and these phenotypes: CXC chemokine receptor 4 (CXCR4) + TANs, CAFs, focal adhesion kinase (FAK) + tumor cells, FAK + M2 and M1 macrophages will correlate with survival outcomes. Methods: Twenty-two stage I/II PDAC patients who consented for tissue collection were stratified into two groups: standard responders (n=11) with overall survival (OS) >2 years and poor responders (n=11) with OS ≤ 2 years. Multiplex IHC was performed on specimens using multiple biomarkers (CD8, CXCR4, CD66b, FAK, FAP, CD68, CSF1R, CD163, EPCAM) to identify cell phenotypes. Comprehensive spatial analysis with HALO software was used to measure mean cell distance between CD8 + T cells and other cells. The mean cell-to-cell distance among evaluable specimens was determined by phenotype. Patients were divided into two groups with average cell distance below or above the mean by phenotype. OS of each subgroup was compared using Kaplan-Meier method. Results: Median OS for standard and poor responders was 47.6 months vs 13.1 months (p<.0001), respectively. There was a higher % of CD8 + T cells closer (<50 µm) to CXCR4 + TANs in standard responders (56% vs 28%, p<.0001). There were no significant differences in CD8 + T cell distance to other phenotypes. Longer survival was seen with average cell distance below the mean when comparing CD8 + T cells to FAK + tumor cells (1274 d vs 742 d, p=.306), to CXCR4 + TANs (854 d vs 442 d, p=.947) and to FAP + FAK + CAFs (890.5 d vs 672 d, p=.662). In contrast, longer survival was seen with a distance above the mean comparing CD8 + T cells to M2 (622 d vs 1274 d, p=.732) and to M1 (613 d vs 1397 d, p=.517) macrophages. Conclusions: Our study shows clinical outcomes may be correlated with spatial distance among TME immune cells in PDAC. Longer survival was seen when CD8 + T cells were closer to FAK + tumor cells, CXCR4 + TANs, and CAFs and further away from M2 and M1 macrophages. Larger cohort analyses may better elucidate the prognostic and biological significance of cell distance among TME phenotypes, which may have potential implications for response to immunotherapy.