A nano-platform combats the "attack" and "defense" of cytoskeleton to block cascading tumor metastasis.

The cytoskeleton facilitates tumor cells invasion into the bloodstream via vasculogenic mimicry (VM) for "attack", and protects cells against external threats through cytoskeletal remodeling and tunneling nanotubes (TNTs) for "defense". However, the existing strategies involving cytoskeleton are not sufficient to eliminate tumor metastasis due to mitochondrial energy supply, both within tumor cells and from outside microenvironment. Here, considering the close relationship between cytoskeleton and mitochondria both in location and function, we construct a nano-platform that combats the "attack" and "defense" of cytoskeleton in the cascading metastasis. The nano-platform is composed of KFCsk@LIP and KTMito@LIP for the cytoskeletal collapse and mitochondrial dysfunction. KFCsk@LIP prevents the initiation and circulation of cascading tumor metastasis, but arouses limited suppression in tumor cell proliferation. KTMito@LIP impairs mitochondria to trigger apoptosis and impede energy supply both from inside and outside, leading to an amplified effect for metastasis suppression. Further mechanisms studies reveal that the formation of VM and TNTs are seriously obstructed. Both in situ and circulating tumor cells are disabled. Subsequently, the broken metastasis cascade results in a remarkable anti-metastasis effect. Collectively, based on the nano-platform, the cytoskeletal collapse with synchronous mitochondrial dysfunction provides a potential therapeutic strategy for cascading tumor metastasis suppression.

Inhibition of ATR opposes glioblastoma invasion through disruption of cytoskeletal networks and integrin internalization via macropinocytosis.

BACKGROUND: Glioblastomas have highly infiltrative growth patterns that contribute to recurrence and poor survival. Despite infiltration being a critical therapeutic target, no clinically useful therapies exist that counter glioblastoma invasion. Here, we report that inhibition of ataxia telangiectasia and Rad 3 related kinase (ATR) reduces invasion of glioblastoma cells through dysregulation of cytoskeletal networks and subsequent integrin trafficking. METHODS: Glioblastoma motility and invasion were assessed in vitro and in vivo in response to ATR inhibition (ATRi) and ATR overexpression using time-lapse microscopy, two orthotopic glioblastoma models, and intravital imaging. Disruption to cytoskeleton networks and endocytic processing were investigated via high-throughput, super-resolution and intravital imaging. RESULTS: High ATR expression was associated with significantly poorer survival in clinical datasets while histological, protein expression, and spatial transcriptomics using glioblastoma tumor specimens revealed higher ATR expression at infiltrative margins. Pharmacological inhibition with two different compounds and RNAi targeting of ATR opposed the invasion of glioblastoma, whereas overexpression of ATR drove migration. Subsequent investigation revealed that cytoskeletal dysregulation reduced macropinocytotic internalization of integrins at growth-cone-like structures, resulting in a tumor microtube retraction defect. The biological relevance and translational potential of these findings were confirmed using two orthotopic in vivo models of glioblastoma and intravital imaging. CONCLUSIONS: We demonstrate a novel role for ATR in determining invasion in glioblastoma cells and propose that pharmacological targeting of ATR could have far-reaching clinical benefits beyond radiosensitization.

JNK Inhibition Modulates the Cytoskeleton, Hypoxia, and Neurogenesis on the Protein Level in Glioblastoma Cells and Astrocytes: An Immunofluorescence Study.

AIM: To evaluate the effects of c-Jun N-terminal kinase (JNK) inhibition and signal blocking on hypoxia (hypoxia-inducible factor 1-alpha (HIF-1α)), differentiation and neurogenesis (bone morphogenetic protein (BMP4)), and the cytoskeleton (F-actin) in glioblastoma multiforme cells (GBMCs). MATERIAL AND METHODS: We evaluated the differences between GBMCs and astrocytes in terms of the abovementioned parameters and assessed them with the aim of studying human GBMCs (U-87 MG) and astrocytes (SVG p12). The cells were exposed to different doses of the JNK inhibitor, SP600125, for 24, 48, and 72 hours. HIF-1α, BMP4, and F-actin expressions were evaluated using immunofluorescence image analysis. RESULTS: The half-maximal inhibitory concentration value for SP600125 was determined to be 10 µM at 24 hours of exposure. After SP600125 administration, elevated levels of HIF-1α and BMP4 were detected in GBMCs and astrocytes. F-actin level only increased in GBMCs after SP600125 administration. CONCLUSION: JNKs are important for cell proliferation, differentiation, survival, and death; thus, research on JNKs has become important for the treatment of many human diseases, especially brain tumors, Parkinson's disease, and Alzheimer's disease. The results of this study involving immunofluorescence techniques should be investigated and supported by studies that involve comprehensive molecular techniques.

HPV disrupt the cytoskeleton in oral squamous cell carcinomas from non-oropharyngeal sites via the E-cadherin/Mena/SMA pathway.

In this paper, we aimed to evaluate the mechanism of actin cytoskeleton disruption, in oral squamous cell carcinoma (OSCC). A total of 43 patients with surgically resected OSCCs located in non-oropharyngeal regions were randomly selected. The expression of E-cadherin, ß-catenin, smooth muscle actin (SMA), Mena, maspin, V-set and immunoglobulin domain containing 1 (VSIG1), ß human chorionic gonadotropin (ßhCG), and angiotensin-converting enzyme (ACE) was assessed via immunohistochemistry (IHC) and evaluated in association with the prevalence of high-risk human papillomavirus (HPV). Mena positivity (n = 30; 69.77%) was more frequent in poorly differentiated OSCC of the tongue and lips with high-risk HPV viral DNA and a lymph node ratio (LNR) ≤ 2.5. Loss of E-cadherin was more prevalent among poorly differentiated stage pT4N1 tumors with an LNR ≤ 2.5 and perineural invasion. These cases were classified as SMA-high tumors. Independent negative prognostic factors included high Mena expression, loss of E-cadherin, high SMA expression, and the presence of high-risk HPV. No VSIG1 positivity was observed. In conclusion, in non-oropharyngeal OSCC, cytoskeleton activity might be driven by the Mena/E-cadherin/SMA axis, reflecting active epithelial-mesenchymal interaction. High Mena intensity is an indicator of poorly differentiated carcinomas with high-risk HPV and unfavorable prognosis.

Long-chain perfluoroalkyl carboxylates induce cytoskeletal abnormalities and activate epithelial-mesenchymal transition in both renal cell carcinoma 3D cultures and Caki-1 xenografted mouse model.

Exposure to perfluorooctanoate (PFOA; a type of perfluoroalkyl carboxylates [PFACs]) may be correlated with the incidence of kidney cancer in individuals exposed to high levels of PFOA. However, mechanistic studies on the influence of PFACs on renal cell carcinoma (RCC) development are lacking. We explored the effects of five types of PFACs on RCC using in vitro and in vivo models to fill this knowledge gap and provide information for environmental/usage regulations. Using 2D/3D cultures of Caki-1 cells, a human clear cell RCC line, we examined the effects of short-chain (SC) PFACs and long-chain (LC) PFACs on RCC physio/pathological markers, including the cytoskeleton, epithelial-mesenchymal transition (EMT)-related proteins, and Na+/K+-ATPase. We also administered three different PFACs orally to mice harboring Caki-1 xenografts to assess the impact of these compounds on engrafted RCC in vivo. Compared with the effects of SCPFACs, mice with Caki-1 xenografts treated with LCPFACs showed increased EMT-related protein expression and exhibited liver toxicity. Therefore, LCPFACs induced EMT, influencing cancer metastasis activity, and displayed higher toxicity in vivo compared with SCPFACs. These findings improve our understanding of the effects of PFACs on RCC development and their corresponding in vivo toxicity, which is crucial for regulating these substances to protect public health.

Long Noncoding RNA Cytoskeleton Regulator RNA Suppresses Apoptosis in Hepatoma Cells by Modulating the miR-125a-5p/HS1-Associated Protein X-1 Axis to Induce Caspase-9 Inactivation.

Background/Aims: The involvement of long noncoding RNAs in the carcinogenesis of hepatocellular carcinoma (HCC) has been well documented by substantial evidence. However, whether cytoskeleton regulator RNA (CYTOR) could affect the progression of HCC remains unclear. Methods: The relative expression of CYTOR, miR-125a-5p and HS1-associated protein X-1 (HAX-1) mRNA in HCC cells were determined via quantitative real-time polymerase chain reaction. The viability of treated HCC cells was measured by Cell Counting Kit-8 assay. Cell apoptosis was estimated by flow cytometry analysis, assessment of caspase-9 activity and terminal deoxynucleotidyl transferase dUTP nick-end labeling staining, and Western blot of apoptosis-related proteins. The interplay between CYTOR or HAX-1 and miR-125a-5p was validated by dual-luciferase reporter assay. Results: CYTOR was upregulated and miR-125a-5p was downregulated in HCC cells. CYTOR silencing inhibited cell proliferation and promoted cell apoptosis in HepG2 and SMMC-7721 cells. miR-125a-5p was sponged and negatively regulated by CYTOR, and HAX-1 was directly targeted and negatively modulated by miR-125a-5p. Overexpression of miR-125a-5p enhanced the repressive effects of CYTOR knockdown on HCC cells, and knockdown of HAX-1 enhanced the inhibitory effects of miR-125a-5p mimics on HCC cells. Conclusions: CYTOR silencing facilitates HCC cell apoptosis in vitro via the miR-125a-5p/HAX-1 axis.

Novel long noncoding RNA LINC02820 augments TNF signaling pathway to remodel cytoskeleton and potentiate metastasis in esophageal squamous cell carcinoma.

Esophageal squamous cell carcinoma (ESCC) is one of the most common malignant tumors in China. However, there are no targets to treat ESCC because the molecular mechanism behind the cancer is still unclear. Here, we found a novel long noncoding RNA LINC02820 was upregulated in ESCC and associated with the ESCC clinicopathological stage. Through a series of functional experiments, we observed that LINC02820 only promoted the migration and invasion capabilities of ESCC cell lines. Mechanically, we found that LINC02820 may affect the cytoskeletal remodeling, interact with splice factor 3B subunit 3 (SF3B3), and cooperate with TNFα to amplify the NF-κB signaling pathway, which can lead to ESCC metastasis. Overall, our findings revealed that LINC02820 is a potential biomarker and therapeutic target for the diagnosis and treatment of ESCC.

Oxidative stress disrupts the cytoskeleton of spinal motor neurons.

BACKGROUND AND AIM: Traumatic spinal cord injury (SCI) is a common and devastating central nervous disease, the treatment of which faces many challenges to the medical community and society as a whole. Treatment measures based on oxidative stress of spinal motor neurons during SCI are expected to help restore biological functions of neurons under injury conditions. However, to date, there are no systematic reports regarding oxidative stress on spinal motor neuron injury. Our aim is to better understand and explain the influences and mechanisms of oxidative stress on spinal motor neurons during SCI. METHODS: We first exposed VSC4.1 motor neurons to hydrogen peroxide (H2 O2 ) and evaluated the effects on cell viability, morphology, cycling, and apoptosis, with an emphasis on the changes to the cytoskeleton and the effect of N-acetyl-l-cysteine (NAC) on these changes. Then, we investigated the effects of NAC on these cytoskeletal changes in vitro and in vivo. RESULTS: We found that H2 O2 caused severe damage to the normal cytoskeleton, leading to a reduction in neurite length and number, rearrangement of the actin cytoskeleton, and disorder of the microtubules and neurofilaments in VSC4.1. Importantly, NAC attenuated the oxidative damage of spinal motor neurons in vitro and in vivo, promoting the recovery of hindlimb motor ability in mice with SCI at the early stage of injury. CONCLUSION: This study shows that oxidative stress plays an important role in the cytoskeleton destruction of spinal motor neurons in SCI, and treatment of SCI on this basis is a promising strategy. These findings will help to elucidate the role of oxidative stress in spinal motor neuron injury in SCI and provide references for further research into the study of the pathology and underlying mechanism of SCI.

Rho GTPase-activating protein 35 suppresses gastric cancer metastasis by regulating cytoskeleton reorganization and epithelial-to-mesenchymal transition.

Cytoskeletal reorganization and epithelial-to-mesenchymal transition (EMT) are key processes and typical characteristics of metastatic cancer cells. Rho GTPase­activating protein 35 (ARHGAP35) is a GTPase-activating protein, which has a significant effect on cell motility. However, the particular function of ARHGAP35 in gastric cancer (GC) remains unknown. In the present study, the role of ARHGAP35 in GC was investigated by in vitro loss-of-function and gain-of-function experiments. Cytoskeletal reorganization in GC cells was evaluated using immunofluorescence staining and the protein expression levels of key molecules and active RhoA were detected by western blot analysis. Additionally, the clinical evaluation of proteins in human GC tissues was assessed by immunohistochemistry. The results showed that ARHGAP35, a tumor suppressor, was downregulated in GC tissues and its decreased expression was associated with the metastatic status of GC. Additionally, Transwell and wound healing assays demonstrated that ARHGAP35 knockdown promoted cell motility in vitro. However, the above effects were abrogated following ectopic ARHGAP35 expression. Furthermore, ARHGAP35 could affect cytoskeletal reorganization via directly regulating RhoA activation. In addition, ARHGAP35 upregulated E-cadherin and attenuated EMT in GC cells. Both ARHGAP35 and E-cadherin were associated with overall survival in patients with GC, while their combination allowed for an even greater capacity for distinguishing GC patients with different prognosis. Overall, the results of the current study suggested that ARHGAP35 could directly regulate cell morphology and motility via affecting cytoskeletal reorganization and EMT via targeting RhoA and E-cadherin, respectively. Targeting the ARHGAP35/RhoA/E-cadherin pathway could be a potential approach for treating GC.

Betulonic Acid, as One of the Active Components of the Celastrus orbiculatus Extract, Inhibits the Invasion and Metastasis of Gastric Cancer Cells by Mediating Cytoskeleton Rearrangement In Vitro.

Gastric cancer is a type of malignant tumor that seriously threatens human life and health. Invasion and metastasis present difficulties in the treatment of gastric cancer, and the remodeling of the tumor cytoskeleton plays an important role in mediating the ability of tumor cells to achieve invasion and metastasis. Previous experimental results suggest that Celastrus orbiculatus extract can regulate cytoskeletal remodeling in gastric cancer, but the active component has not been determined. Betulonic acid, as an effective component of COE, inhibits the invasion and metastasis of gastric cancer cells by regulating cytoskeletal remodeling in vitro; its specific mechanisms have been studied here. After betulonic acid was dissolved, it was diluted to various working concentrations in RPMI-1640 medium and added to AGS, HGC-27 and GES-1 cell lines. Cell viability was assessed by CCK-8 and colony formation assays. Cytoskeleton staining was used to detect changes in cytoskeleton morphology. Functional assays including wound healing assays and transwell assays were used to detect the invasion and migration of cells. The effect of betulonic acid on cell invasion and migration was clearly and precisely observed by high-content imaging technology. Western blotting was used to detect the regulation of matrix metalloproteinase-related proteins and epithelial-mesenchymal transformation-related proteins. We found that betulonic acid inhibited the migration and invasion of gastric cancer cells. Therefore, betulonic acid inhibits the invasion and metastasis of gastric cancer cells by mediating cytoskeletal remodeling and regulating epithelial mesenchymal transformation.

Phenotypic and Functional Transformation in Smooth Muscle Cells Derived from a Superficial Thrombophlebitis-affected Vein Wall.

BACKGROUND: Superficial thrombophlebitis (ST) is a frequent pathology, but its exact incidence remains to be determined. This study tested the hypothesis whether relationships exist among smooth muscle cells (SMCs) derived from ST, varicose great saphenous veins (VGSVs), and normal great saphenous veins (GSVs). METHODS: Forty-one samples of ST, VGSVs, and GSVs were collected. SMCs were isolated and cultured. Proliferation, migration, adhesion, and senescence in SMCs from the three vein walls were compared by various methods. Bax, Bcl-2, caspase-3, matrix metalloproteinase-2 (MMP-2), MMP-9, tissue inhibitor of metalloproteinase-1 (TIMP-1), and TIMP-2 messenger RNA (mRNA) and protein expressions were detected by fluorescence quantitative PCR and Western blot. RESULTS: An obvious decrease in cytoskeletal filaments was observed in thrombophlebitic vascular smooth muscle cells (TVSMCs). The quantity of proliferation, migration, adhesion, and senescence in TVSMCs was significantly higher than in varicose vascular smooth muscle cells and normal vascular smooth muscle cells (NVSMCs) (all P < 0.05). Bax and caspase-3 mRNA and protein expression were decreased, while Bcl-2 mRNA and protein expression were increased in the TVSMCs compared with the varicose vascular smooth muscle cells and the NVSMCs (all P < 0.05). MMP-2, MMP-9, TIMP-1, and TIMP-2 mRNA and protein expression were significantly increased in the TVSMCs compared with the VVGSVs and the NVSMCs (all P < 0.05). CONCLUSION: SMCs derived from ST are more dedifferentiated and demonstrate increased cell proliferation, migration, adhesion, and senescence, as well as obviously decreased cytoskeletal filaments. These results suggest that the phenotypic and functional differences could be related to the presence of atrophic and hypertrophic vein segments during the disease course among SMCs derived from ST, VGSVs, and GSVs.

Giant obscurin regulates migration and metastasis via RhoA-dependent cytoskeletal remodeling in pancreatic cancer.

Obscurins, encoded by the OBSCN gene, are giant cytoskeletal proteins with structural and regulatory roles. Large scale omics analyses reveal that OBSCN is highly mutated across different types of cancer, exhibiting a 5-8% mutation frequency in pancreatic cancer. Yet, the functional role of OBSCN in pancreatic cancer progression and metastasis has to be delineated. We herein show that giant obscurins are highly expressed in normal pancreatic tissues, but their levels are markedly reduced in pancreatic ductal adenocarcinomas. Silencing of giant obscurins in non-tumorigenic Human Pancreatic Ductal Epithelial (HPDE) cells and obscurin-expressing Panc5.04 pancreatic cancer cells induces an elongated, spindle-like morphology and faster cell migration via cytoskeletal remodeling. Specifically, depletion of giant obscurins downregulates RhoA activity, which in turn results in reduced focal adhesion density, increased microtubule growth rate and faster actin dynamics. Although OBSCN knockdown is not sufficient to induce de novo tumorigenesis, it potentiates tumor growth in a subcutaneous implantation model and exacerbates metastasis in a hemispleen murine model of pancreatic cancer metastasis, thereby shortening survival. Collectively, these findings reveal a critical role of giant obscurins as tumor suppressors in normal pancreatic epithelium whose loss of function induces RhoA-dependent cytoskeletal remodeling, and promotes cell migration, tumor growth and metastasis.

Dickkopf-related protein 1/cytoskeleton-associated protein 4 signaling activation by Helicobacter pylori-induced activator protein-1 promotes gastric tumorigenesis via the PI3K/AKT/mTOR pathway.

BACKGROUND: Gastric cancer (GC) is a common malignant tumor with high incidence and mortality rates globally, especially in East Asian countries. Helicobacter pylori (H. pylori) infection is a significant and independent risk factor for GC. However, its underlying mechanism of action is not fully understood. Dickkopf-related protein (DKK) 1 is a Wnt signaling antagonist, and cytoskeleton-associated protein (CKAP) 4 is a newly identified DKK1 receptor. Recent studies found that the binding of DKK1 to CAKP4 mediated the procancer signaling of DKK1 inde-pendent of Wnt signaling. We hypothesize that H. pylori-induced activation of DKK1/CKAP4 signaling contributes to the initiation and progression of GC. AIM: To investigate the interaction of H. pylori infection, DKK1 and CAKP4 in GC, as well as the underlying molecular mechanisms. METHODS: RNA sequencing was used to identify differentially expressed genes (DEGs) between H. pylori-infected and uninfected primary GC cells. Gain- and loss-of-function experiments were performed to verify the H. pylori-induced upregulation of activator protein-1 (AP-1) in GC cells. A dual-luciferase reporter assay and co-immunoprecipitation were used to determine the binding of AP-1 to the DKK1 promoter and DKK1 to CKAP4. Western blotting and immunohistochemistry detected the expression of DKK1, CKAP4, and phos-phatidylinositol 3-kinase (PI3K) pathway-related proteins in GC cells and tissues. Functional experiments and tumorigenicity in nude mice detected malignant behavior of GC cells in vitro and in vivo. RESULTS: We identified 32 DEGs between primary GC cells with and without H. pylori infection, including JUN, fos-like antigen-1 (FOSL1), and DKK1, and confirmed that the three proteins and CKAP4 were highly expressed in H. pylori-infected GC cells, H. pylori-infected gerbil gastric tissues, and human GC tissues. JUN and FOSL1 form AP-1 to transcriptionally activate DKK1 expression by binding to the DKK1 promoter. Activated DKK1 bound to CKAP4, but not the most common Wnt coreceptor low-density lipoprotein receptor-related protein 5/6, to promote GC cell growth, colony formation, migration, invasion, and xenograft tumor growth in nude mice. All these effects were driven by activation of the PI3K/AKT/mammalian target of rapamycin (mTOR) pathway. Targeting the PI3K signaling pathway by LY294002 inhibited DKK1-mediated CKAP4/PI3K signaling activity and the malignant behavior of GC cells. CONCLUSION: H. pylori induces JUN and FOSL1 expression to form AP-1, which transcriptionally activates DKK1. Binding of DKK1 to KAKP4 contributes to gastric tumorigenesis via the PI3K/AKT/mTOR pathway.

Cytoskeleton Reorganization in EndMT-The Role in Cancer and Fibrotic Diseases.

Chronic inflammation promotes endothelial plasticity, leading to the development of several diseases, including fibrosis and cancer in numerous organs. The basis of those processes is a phenomenon called the endothelial-mesenchymal transition (EndMT), which results in the delamination of tightly connected endothelial cells that acquire a mesenchymal phenotype. EndMT-derived cells, known as the myofibroblasts or cancer-associated fibroblasts (CAFs), are characterized by the loss of cell-cell junctions, loss of endothelial markers, and gain in mesenchymal ones. As a result, the endothelium ceases its primary ability to maintain patent and functional capillaries and induce new blood vessels. At the same time, it acquires the migration and invasion potential typical of mesenchymal cells. The observed modulation of cell shape, increasedcell movement, and invasion abilities are connected with cytoskeleton reorganization. This paper focuses on the review of current knowledge about the molecular pathways involved in the modulation of each cytoskeleton element (microfilaments, microtubule, and intermediate filaments) during EndMT and their role as the potential targets for cancer and fibrosis treatment.

Mechanosensitive channel inhibition attenuates TGFß2-induced actin cytoskeletal remodeling and reactivity in mouse optic nerve head astrocytes.

Astrocytes within the optic nerve head undergo actin cytoskeletal rearrangement early in glaucoma, which coincides with astrocyte reactivity and extracellular matrix (ECM) deposition. Elevated transforming growth factor beta 2 (TGFß2) levels within astrocytes have been described in glaucoma, and TGFß signaling induces actin cytoskeletal remodeling and ECM deposition in many tissues. A key mechanism by which astrocytes sense and respond to external stimuli is via mechanosensitive ion channels. Here, we tested the hypothesis that inhibition of mechanosensitive channels will attenuate TGFß2-mediated optic nerve head astrocyte actin cytoskeletal remodeling, reactivity, and ECM deposition. Primary optic nerve head astrocytes were isolated from C57BL/6J mice and cell purity was confirmed by immunostaining. Astrocytes were treated with vehicle control, TGFß2 (5 ng/ml), GsMTx4 (a mechanosensitive channel inhibitor; 500 nM), or TGFß2 (5 ng/ml) + GsMTx4 (500 nM) for 48 h. FITC-phalloidin staining was used to assess the formation of f-actin stress fibers and to quantify the presence of crosslinked actin networks (CLANs). Cell reactivity was determined by immunostaining and immunoblotting for GFAP. Levels of fibronectin and collagen IV deposition were also quantified. Primary optic nerve head astrocytes were positive for the astrocyte marker GFAP and negative for markers for microglia (F4/80) and oligodendrocytes (OSP1). Significantly increased %CLAN-positive cells were observed after 48-h treatment with TGFß2 vs. control in a dose-dependent manner. Co-treatment with GsMTx4 significantly decreased %CLAN-positive cells vs. TGFß2 treatment and the presence of f-actin stress fibers. TGFß2 treatment significantly increased GFAP, fibronectin, and collagen IV levels, and GsMTx4 co-treatment ameliorated GFAP immunoreactivity. Our data suggest inhibition of mechanosensitive channel activity as a potential therapeutic strategy to modulate actin cytoskeletal remodeling within the optic nerve head in glaucoma.

NK Cells in a Tug-of-War With Cancer: The Roles of Transcription Factors and Cytoskeleton.

Natural killer (NK) cells are innate immune cells which play a key role in shaping the immune response against cancer. Initially hailed for their potential to recognise and eliminate tumour cells, their application has been greatly hindered by the immunosuppressive tumour microenvironment (TME) which suppresses NK functions (e.g., cytotoxicity). This dysfunctional state that is accompanied by phenotypic changes such as upregulation of inhibitory receptors and downregulation of activating receptors, forms the basis of what many researchers have referred to as 'exhausted' NK cells. However, there is no consensus on whether these phenotypes are sufficient to define an exhausted state of the NK cell. While recent advances in checkpoint inhibition appear to show promise in early-stage pre-clinical studies, much remains to be fully explored and understood in the context of the TME. The TME is where the NK cells are subjected to interaction with various cell types and soluble factors, which could exert an inhibitory effect on NK cytotoxicity. In this review, we provide an overview of the general markers of NK cell exhaustion viz, the surface activating and inhibitory receptors. We also highlight the potential role of T-box transcription factors in characterising such a dysfunctional state and discuss the often-overlooked mechanism of cell cytoskeletal dynamics in regulating NK cell function. These aspects may further contribute to NK exhaustion or NK revival in cancer and may open new avenues to explore cancer treatment strategies.

Functional, proteomic and phenotypic in vitro studies evidence podocyte injury after chronic exposure to heparin.

Unfractionated heparin (UFH) is a widely used anticoagulant that possess numerous properties including anti-inflammatory, anti-viral, anti-angiogenesis, and anti-metastatic effects. The effect of this drug was evaluated on the podocyte, an important actor of the glomerular filtration. Using a functional approach, we demonstrate that heparin treatment leads to a functional podocyte perturbation characterized by the increase of podocyte monolayer permeability. This effect is enhanced with time of exposure. Proteomic study reveals that heparin down regulate focal adhesion and cytoskeletal protein expressions as well as the synthesis of glomerular basement membrane components. This study clearly demonstrates that UFH may affect podocyte function by altering cytoskeleton organization, cell-cell contacts and cell attachment.

Survey of cancer cell anatomy in nonadhesive confinement reveals a role for filamin-A and fascin-1 in leader bleb-based migration.

Cancer cells migrating in confined microenvironments exhibit plasticity of migration modes. Confinement of contractile cells in a nonadhesive environment drives "leader bleb-based migration" (LBBM), morphologically characterized by a long bleb that points in the direction of movement separated from a cell body by a contractile neck. Although cells undergoing LBBM have been visualized within tumors, the organization of organelles and actin regulatory proteins mediating LBBM is unknown. We analyzed the localization of fluorescent organelle-specific markers and actin-associated proteins in human melanoma and osteosarcoma cells undergoing LBBM. We found that organelles from the endolysosomal, secretory, and metabolic systems as well as the vimentin and microtubule cytoskeletons localized primarily in the cell body, with some endoplasmic reticulum, microtubules, and mitochondria extending into the leader bleb. Overexpression of fluorescently tagged actin regulatory proteins showed that actin assembly factors localized toward the leader bleb tip, contractility regulators and cross-linkers in the cell body cortex and neck, and cross-linkers additionally throughout the leader bleb. Quantitative analysis showed that excess filamin-A and fascin-1 increased migration speed and persistence, while their depletion by small interfering RNA indicates a requirement in promoting cortical tension and pressure to drive LBBM. This indicates a critical role of specific actin crosslinkers in LBBM.

Inflammasome-Induced Osmotic Pressure and the Mechanical Mechanisms Underlying Astrocytic Swelling and Membrane Blebbing in Pyroptosis.

Cell swelling and membrane blebbing are characteristic of pyroptosis. In the present study, we explored the role of intracellular tension activity in the deformation of pyroptotic astrocytes. Protein nanoparticle-induced osmotic pressure (PN-OP) was found to be involved in cell swelling and membrane blebbing in pyroptotic astrocytes, and was associated closely with inflammasome production and cytoskeleton depolymerization. However, accumulation of protein nanoparticles seemed not to be absolutely required for pyroptotic permeabilization in response to cytoskeleton depolymerization. Gasdermin D activation was observed to be involved in modification of typical pyroptotic features through inflammasome-induced OP upregulation and calcium increment. Blockage of nonselective ion pores can inhibit permeabilization, but not inflammasome production and ion influx in pyroptotic astrocytes. The results suggested that the inflammasomes, as protein nanoparticles, are involved in PN-OP upregulation and control the typical features of pyroptotic astrocytes.

Glucocorticoid-mediated induction of caveolin-1 disrupts cytoskeletal organization, inhibits cell migration and re-epithelialization of non-healing wounds.

Although impaired keratinocyte migration is a recognized hallmark of chronic wounds, the molecular mechanisms underpinning impaired cell movement are poorly understood. Here, we demonstrate that both diabetic foot ulcers (DFUs) and venous leg ulcers (VLUs) exhibit global deregulation of cytoskeletal organization in genomic comparison to normal skin and acute wounds. Interestingly, we found that DFUs and VLUs exhibited downregulation of ArhGAP35, which serves both as an inactivator of RhoA and as a glucocorticoid repressor. Since chronic wounds exhibit elevated levels of cortisol and caveolin-1 (Cav1), we posited that observed elevation of Cav1 expression may contribute to impaired actin-cytoskeletal signaling, manifesting in aberrant keratinocyte migration. We showed that Cav1 indeed antagonizes ArhGAP35, resulting in increased activation of RhoA and diminished activation of Cdc42, which can be rescued by Cav1 disruption. Furthermore, we demonstrate that both inducible keratinocyte specific Cav1 knockout mice, and MßCD treated diabetic mice, exhibit accelerated wound closure. Taken together, our findings provide a previously unreported mechanism by which Cav1-mediated cytoskeletal organization prevents wound closure in patients with chronic wounds.