TIPE1 limits virus replication by disrupting PKM2/ HIF-1α/ glycolysis feedback loop.

OBJECTIVE: Virus infection is a major threat to human health and remains a significant cause of death to date. Macrophages are important innate immune cells that exhibit indispensable roles in controlling virus replication. It was recently reported that metabolic adaption determines the functional state of macrophages. Thus, to further unravel the crucial factors involving in metabolic adaption of macrophages might provide the potential candidates for optimizing their anti-viral capabilities. METHODS: RT-PCR, Western blotting, virus plaque assay and HE were used to evaluate the viral load in virus-infected Tipe1M-KO and Tipe1f/f mice or cultured macrophages. RNA sequencing were performed with Tipe1M-KOor Tipe1f/f BMDMs upon virus infection. Extracellular acidification rate (ECAR) was applied for analyzing glycolysis rate in virus-infected BMDMs. Co-immunoprecipitation (Co-IP) assay and LC-MS/MS were used to determine the potential interacting proteins of TIPE1. RESULTS: TIPE1 level was significantly reduced in BMDMs infected with either RNA viruses or DNA virus. Deficiency of Tipe1 in macrophages increased viral load and aggravated tissue damage. Mechanistically, TIPE1 suppressed the glycolytic capacity of macrophages through interacting with PKM2 and promoting its ubiquitination degradation, which in turn decreased HIF1α transcription and viral replication in macrophages. CONCLUSIONS: TIPE1 functions as a novel regulator for metabolic reprogramming and virus infection in macrophages.

VEGFR2 blockade inhibits glioblastoma cell proliferation by enhancing mitochondrial biogenesis.

BACKGROUND: Glioblastoma is an aggressive brain tumor linked to significant angiogenesis and poor prognosis. Anti-angiogenic therapies with vascular endothelial growth factor receptor 2 (VEGFR2) inhibition have been investigated as an alternative glioblastoma treatment. However, little is known about the effect of VEGFR2 blockade on glioblastoma cells per se. METHODS: VEGFR2 expression data in glioma patients were retrieved from the public database TCGA. VEGFR2 intervention was implemented by using its selective inhibitor Ki8751 or shRNA. Mitochondrial biogenesis of glioblastoma cells was assessed by immunofluorescence imaging, mass spectrometry, and western blot analysis. RESULTS: VEGFR2 expression was higher in glioma patients with higher malignancy (grade III and IV). VEGFR2 inhibition hampered glioblastoma cell proliferation and induced cell apoptosis. Mass spectrometry and immunofluorescence imaging showed that the anti-glioblastoma effects of VEGFR2 blockade involved mitochondrial biogenesis, as evidenced by the increases of mitochondrial protein expression, mitochondria mass, mitochondrial oxidative phosphorylation (OXPHOS), and reactive oxygen species (ROS) production, all of which play important roles in tumor cell apoptosis, growth inhibition, cell cycle arrest and cell senescence. Furthermore, VEGFR2 inhibition exaggerated mitochondrial biogenesis by decreased phosphorylation of AKT and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α), which mobilized PGC1α into the nucleus, increased mitochondrial transcription factor A (TFAM) expression, and subsequently enhanced mitochondrial biogenesis. CONCLUSIONS: VEGFR2 blockade inhibits glioblastoma progression via AKT-PGC1α-TFAM-mitochondria biogenesis signaling cascade, suggesting that VEGFR2 intervention might bring additive therapeutic values to anti-glioblastoma therapy.

Dynamic Hybrid Module-Driven NK Cell Stimulation and Release for Tumor Immunotherapy.

Natural killer (NK) cells have become a powerful candidate for adoptive tumor immunotherapy, while their therapeutic efficacy in solid tumors remains unsatisfactory. Here, we developed a hybrid module with an injectable hydrogel and hydroxyapatite (HAp) nanobelts for the controlled delivery of NK cells to enhance the therapy of solid tumors. Surface-functionalized HAp nanobelts modified with agonistic antibodies against NKG2D and 4-1BB and cytokines IL-2 and IL-21 support survival and dynamic activation. Thus, the HAp-modified chitosan (CS) thermos-sensitive hydrogel not only improved the retention of NK cells for more than 20 days in vivo but also increased NK cell function by more than one-fold. The unique architecture of this biomaterial complex protects NK cells from the hostile tumor environment and improves antitumor efficacy. The generation of a transient inflammatory niche for NK cells through a biocompatible hydrogel reservoir may be a conversion pathway to prevent cancer recurrence of resectable tumors.

Urolithin A Hijacks ERK1/2-ULK1 Cascade to Improve CD8+ T Cell Fitness for Antitumor Immunity.

According to the latest evidence, the microbial metabolite Urolithin A (UA), known for its role in promoting cellular health, modulates CD8+ T cell-mediated antitumor activity. However, the direct target protein of UA and its underlying mechanism remains unclear. Here, this research identifies ERK1/2 as the specific target crucial for UA-mediated CD8+ T cell activation. Even at low doses, UA markedly enhances the persistence and effector functions of primary CD8+ cytotoxic T lymphocytes (CTLs) and human chimeric antigen receptor (CAR) T cells both in vitro and in vivo. Mechanistically, UA interacts directly with ERK1/2 kinases, enhancing their activation and subsequently facilitating T cell activation by engaging ULK1. The UA-ERK1/2-ULK1 axis promotes autophagic flux in CD8+ CTLs, enhancing cellular metabolism and maintaining reactive oxygen species (ROS) levels, as evidenced by increased oxygen consumption and extracellular acidification rates. UA-treated CD8+ CTLs also display elevated ATP levels and enhanced spare respiratory capacity. Overall, UA activates ERK1/2, inducing autophagy and metabolic adaptation, showcasing its potential in tumor immunotherapy and interventions for diseases involving ERKs.

Cholesterol: A friend to viruses.

Cholesterol is a key life-sustaining molecule which regulates membrane fluidity and serves as a signaling mediator. Cholesterol homeostasis is closely related to various pathological conditions including tumor, obesity, atherosclerosis, Alzheimer's disease and viral infection. Viral infection disrupts host cholesterol homeostasis, facilitating their own survival. Meanwhile, the host cells strive to reduce cholesterol accessibility to limit viral infection. This review focuses on the regulation of cholesterol metabolism and the role of cholesterol in viral infection, specifically providing an overview of cholesterol as a friend to promote viral entry, replication, assembly, release and immune evasion, which might inspire valuable thinking for pathogenesis and intervention of viral infection.

Cholesterol is a metabolically important molecule. The disruption of cholesterol homeostasis is closely related to various diseases including tumor, atherosclerosis and Alzheimer's disease. Moreover, viral infection is a highly cholesterol-dependent process. Important stages in the life cycle of viruses require the involvement of cholesterol. Viral infection breaks the cholesterol homeostasis in host cells, which is conducive to their own survival. This review aims to characterize the regulation of cholesterol metabolism and the role of cholesterol in viral infection, which would shed new light on the design of antiviral drugs.

Beta-Cell Tipe1 Orchestrates Insulin Secretion and Cell Proliferation by Promoting Gαs/cAMP Signaling via USP5.

Inadequate ß-cell mass and insulin secretion are essential for the development of type 2 diabetes (T2D). TNF-α-induced protein 8-like 1 (Tipe1) plays a crucial role in multiple diseases, however, a specific role in T2D pathogenesis remains largely unexplored. Herein, Tipe1 as a key regulator in T2D, contributing to the maintenance of ß cell homeostasis is identified. The results show that the ß-cell-specific knockout of Tipe1 (termed Ins2-Tipe1BKO) aggravated diabetic phenotypes in db/db mice or in mice with high-fat diet-induced diabetes. Notably, Tipe1 improves ß cell mass and function, a process that depends on Gαs, the α subunit of the G-stimulating protein. Mechanistically, Tipe1 inhibited the K48-linked ubiquitination degradation of Gαs by recruiting the deubiquitinase USP5. Consequently, Gαs or cAMP agonists almost completely restored the dysfunction of ß cells observed in Ins2-Tipe1BKO mice. The findings characterize Tipe1 as a regulator of ß cell function through the Gαs/cAMP pathway, suggesting that Tipe1 may emerge as a novel target for T2D intervention.

Increased Siglec-9/Siglec-9L interactions on NK cells predict poor HCC prognosis and present a targetable checkpoint for immunotherapy.

BACKGROUND & AIMS: Natural killer (NK) cell-based anti-hepatocellular carcinoma (HCC) therapy is an increasingly attractive approach that warrants further study. Siglec-9 interacts with its ligand (Siglec-9L) and restrains NK cell functions, suggesting it is a potential therapeutic target. However, in situ Siglec-9/Siglec-9L interactions in HCC have not been reported, and a relevant interventional strategy is lacking. Herein, we aim to illustrate Siglec-9/Siglec-9L-mediated cell sociology and identify small-molecule inhibitors targeting Siglec-9 that could improve the efficacy of NK cell-based immunotherapy for HCC. METHODS: Multiplexed immunofluorescence staining was performed to analyze the expression pattern of Siglec-7, -9 and their ligands in HCC tissues. Then we conducted docking-based virtual screening combined with bio-layer interferometry assays to identify a potent small-molecule Siglec-9 inhibitor. The therapeutic potential was further evaluated in vitro and in hepatoma-bearing NCG mice. RESULTS: Siglec-9 expression, rather than Siglec-7, was markedly upregulated on tumor-infiltrating NK cells, which correlated significantly with reduced survival of patients with HCC. Moreover, the number of Siglec-9L+ cells neighboring Siglec-9+ NK cells was increased in HCC tissues and was also associated with tumor recurrence and reduced survival, further suggesting that Siglec-9/Siglec-9L interactions are a potential therapeutic target in HCC. In addition, we identified a small-molecule Siglec-9 inhibitor MTX-3937 which inhibited phosphorylation of Siglec-9 and downstream SHP1 and SHP2. Accordingly, MTX-3937 led to considerable improvement in NK cell function. Notably, MTX-3937 enhanced cytotoxicity of both human peripheral and tumor-infiltrating NK cells. Furthermore, transfer of MTX-3937-treated NK92 cells greatly suppressed the growth of hepatoma xenografts in NCG mice. CONCLUSIONS: Our study provides the rationale for HCC treatment by targeting Siglec-9 on NK cells and identifies a promising small-molecule inhibitor against Siglec-9 that enhances NK cell-mediated HCC surveillance. IMPACT AND IMPLICATIONS: Herein, we found that Siglec-9 expression is markedly upregulated on tumor-infiltrating natural killer (TINK) cells and correlates with reduced survival in patients with hepatocellular carcinoma (HCC). Moreover, the number of Siglec-9L+ cells neighboring Siglec-9+ NK cells was increased in HCC tissues and was also associated with tumor recurrence and reduced survival. More importantly, we identified a small-molecule inhibitor targeting Siglec-9 that augments NK cell functions, revealing a novel immunotherapy strategy for liver cancer that warrants further clinical investigation.

Mnox Nanoenzyme Armed CAR-NK Cells Enhance Solid Tumor Immunotherapy by Alleviating the Immunosuppressive Microenvironment.

Adoptively transferred cells usually suffer from exhaustion, limited expansion, and poor infiltration, partially attributing to the complicated immunosuppressive microenvironment of solid tumors. Therefore, it is necessary to explore more effective strategies to improve the poor tumor microenvironment (TME) to efficaciously deliver and support extrinsic effector cells in vivo. Herein, an intelligent biodegradable hollow manganese dioxide nanoparticle (MnOX) that possesses peroxidase activity to catalyze excess H2O2 in the TME to produce oxygen and relieve the hypoxia of solid tumors is developed. MnOX nanoenzymes modified with CD56 antibody could specifically bind CAR-NK (chimeric antigen receptor modified natural killer) cells. It is demonstrated that CAR-NK cells incorporated with MnOX nanoenzymes effectively infiltrate into tumor tissues with an improved TME, which results in superior antitumor activity in solid tumor-bearing mice. The antibody connection between MnOX nanoenzymes and CAR-NK endows the lowest efficient dosage of MnOX. This study features a smart synergistic immunotherapy approach for solid tumors using MnOX nanoenzyme-armed CAR-NK cells, which would provide a valuable tool for immunocyte therapy in solid tumors.

Identification of a small-molecule Tim-3 inhibitor to potentiate T cell-mediated antitumor immunotherapy in preclinical mouse models.

T cell immunoglobulin and mucin-containing molecule 3 (Tim-3), expressed in dysfunctional and exhausted T cells, has been widely acknowledged as a promising immune checkpoint target for tumor immunotherapy. Here, using a strategy combining virtual and functional screening, we identified a compound named ML-T7 that targets the FG-CC' cleft of Tim-3, a highly conserved binding site of phosphatidylserine (PtdSer) and carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1). ML-T7 enhanced the survival and antitumor activity of primary CD8+ cytotoxic T lymphocytes (CTLs) and human chimeric antigen receptor (CAR) T cells and reduced their exhaustion in vitro and in vivo. In addition, ML-T7 promoted NK cells' killing activity and DC antigen-presenting capacity, consistent with the reported activity of Tim-3. ML-T7 strengthened DCs' functions through both Tim-3 and Tim-4, which is consistent with the fact that Tim-4 contains a similar FG-CC' loop. Intraperitoneal dosing of ML-T7 showed comparable tumor inhibitory effects to the Tim-3 blocking antibody. ML-T7 reduced syngeneic tumor progression in both wild-type and Tim-3 humanized mice and alleviated the immunosuppressive microenvironment. Furthermore, combined ML-T7 and anti-PD-1 therapy had greater therapeutic efficacy than monotherapy in mice, supporting further development of ML-T7 for tumor immunotherapy. Our study demonstrates a potential small molecule for selectively blocking Tim-3 and warrants further study.

ZHX2 emerges as a negative regulator of mitochondrial oxidative phosphorylation during acute liver injury.

Mitochondria dysfunction contributes to acute liver injuries, and mitochondrial regulators, such as PGC-1α and MCJ, affect liver regeneration. Therefore, identification of mitochondrial modulators may pave the way for developing therapeutic strategies. Here, ZHX2 is identified as a mitochondrial regulator during acute liver injury. ZHX2 both transcriptionally inhibits expression of several mitochondrial electron transport chain genes and decreases PGC-1α stability, leading to reduction of mitochondrial mass and OXPHOS. Loss of Zhx2 promotes liver recovery by increasing mitochondrial OXPHOS in mice with partial hepatectomy or CCl4-induced liver injury, and inhibition of PGC-1α or electron transport chain abolishes these effects. Notably, ZHX2 expression is higher in liver tissues from patients with drug-induced liver injury and is negatively correlated with mitochondrial mass marker TOM20. Delivery of shRNA targeting Zhx2 effectively protects mice from CCl4-induced liver injury. Together, our data clarify ZHX2 as a negative regulator of mitochondrial OXPHOS and a potential target for developing strategies for improving liver recovery after acute injuries.

Lipid accumulation-mediated histone hypoacetylation drives persistent NK cell dysfunction in anti-tumor immunity.

Hyperlipidemia impairs anti-tumor immune responses and is closely associated with increased human cancer incidence and mortality. However, the underlying mechanisms are not well understood. In the present study, we show that natural killer (NK) cells isolated from high-fat-diet mice or treated with oleic acid (OA) in vitro exhibit sustainable functional defects even after removal from hyperlipidemic milieu. This is accompanied by reduced chromatin accessibility in the promoter region of NK cell effector molecules. Mechanistically, OA exposure blunts P300-mediated c-Myc acetylation and shortens its protein half-life in NK cells, which in turn reduces P300 accumulation and H3K27 acetylation and leads to persistent NK cell dysfunction. NK cells engineered with hyperacetylated c-Myc mutants surmount the suppressive effect of hyperlipidemia and display superior anti-tumor activity. Our findings reveal the persistent dysfunction of NK cells in dyslipidemia milieu and extend engineered NK cells as a promising strategy for tumor immunotherapy.

Transcription factor Zhx2 is a checkpoint that programs macrophage polarization and antitumor response.

Macrophages are usually educated to tumor-associated macrophages (TAMs) in cancer with pro-tumor functions by tumor microenvironment (TME) and TAM reprogramming has been proposed as a potential tumor immunotherapy strategy. We recently demonstrated the critical role of Zinc-fingers and homeoboxes 2 (Zhx2) in macrophages' metabolic programming. However, whether Zhx2 is responsible for macrophage polarization and TAMs reprogramming is largely unknown. Here, we show that Zhx2 controls macrophage polarization under the inflammatory stimulus and TME. Myeloid-specific deletion of Zhx2 suppresses LPS-induced proinflammatory polarization but promotes IL-4 and TME-induced anti-inflammatory and pro-tumoral phenotypes in murine liver tumor models. Factors in TME, especially lactate, markedly decrease the expression of Zhx2 in TAMs, leading to the switch of TAMs to pro-tumor phenotype and consequent cancer progression. Notably, reduced ZHX2 expression in TAM correlates with poor survival of HCC patients. Mechanistic studies reveal that Zhx2 associates with NF-κB p65 and binds to the Irf1 promoter, leading to transcriptional activation of Irf1 in macrophages. Zhx2 functions in maintaining macrophage polarization by regulating Irf1 transcription, which may be a potential target for macrophage-based cancer immunotherapy.

Zhx2 maintains islet ß-cell mass and function by transcriptionally regulating Pax6.

Emerging evidence shows that pancreatic ß-cell function and quality are key determinants in the progression of type 2 diabetes (T2D). The transcription factor zinc finger homeobox 2 (Zhx2) is involved in proliferation and development of multiple cells. However, the exact role of Zhx2 in ß-cells and T2D remains completely unknown. Here, we report that Zhx2 orchestrates ß-cell mass and function by regulating paired box protein pax-6 (Pax6). We found that ß-cell-specific knockout Zhx2 (Zhx2BKO) mice showed a decrease in ß-cell proliferation and glucose homeostasis. Under prediabetic and diabetic conditions, we discovered glucose intolerance in both Zhx2BKO-HFD mice and Zhx2BKO-db/db mice, with reduced ß-cell mass and insulin secretion. Mechanistically, we demonstrated that Zhx2 targeted the Pax6 promoter region (-1740∼-1563; -862∼-559; -251∼+75), enhanced promoter activity. Overall, Zhx2 maintains ß-cell function by transcriptionally regulating Pax6, which provides a therapeutic target for diabetes intervention.

Lipophagy-mediated cholesterol synthesis inhibition is required for the survival of hepatocellular carcinoma under glutamine deprivation.

Glutamine is critical for tumor progression, and restriction of its availability is emerging as a potential therapeutic strategy. The metabolic plasticity of tumor cells helps them adapting to glutamine restriction. However, the role of cholesterol metabolism in this process is relatively unexplored. Here, we reported that glutamine deprivation inhibited cholesterol synthesis in hepatocellular carcinoma (HCC). Reactivation of cholesterol synthesis enhanced glutamine-deprivation-induced cell death of HCC cells, which is partially duo to augmented NADPH depletion and lipid peroxidation. Mechanistically, glutamine deprivation induced lipophagy to transport cholesterol from lipid droplets (LDs) to endoplasmic reticulum (ER), leading to inhibit SREBF2 maturation and cholesterol synthesis, and maintain redox balance for survival. Glutamine deprivation decreased mTORC1 activity to induce lipophagy. Importantly, administration of U18666A, CQ, or shTSC2 viruses further augmented GPNA-induced inhibition of xenograft tumor growth. Clinical data supported that glutamine utilization positively correlated with cholesterol synthesis, which is associated with poor prognosis of HCC patients. Collectively, our study revealed that cholesterol synthesis inhibition is required for the survival of HCC under glutamine-restricted tumor microenvironment.

Depletion and reconstitution of macrophages in mice with vesicular stomatitis virus infection.

Acting as the first line of host defense, macrophages play a central role in antiviral response. Here, we present a protocol for depletion and reconstitution of macrophages in mice with vesicular stomatitis virus (VSV) infection. We describe steps for induction and isolation of peritoneal macrophages from CD45.2+ donor mice, macrophage depletion in CD45.1+ recipient mice, adoptive transfer of CD45.2+ macrophages to CD45.1+ recipient mice, and VSV infection. This protocol highlights the role of exogenous macrophages in antiviral response in vivo. For complete details on the use and execution of this profile, please refer to Wang et al.1.

Carryover Contamination-Controlled Amplicon Sequencing Workflow for Accurate Qualitative and Quantitative Detection of Pathogens: a Case Study on SARS-CoV-2.

Carryover contamination during amplicon sequencing workflow (AMP-Seq) put the accuracy of the high-throughput detection for pathogens at risk. The purpose of this study is to develop a carryover contaminations-controlled AMP-Seq (ccAMP-Seq) workflow to enable accurate qualitative and quantitative detection for pathogens. By using the AMP-Seq workflow to detect SARS-CoV-2, Aerosols, reagents and pipettes were identified as potential sources of contaminations and ccAMP-Seq was then developed. ccAMP-Seq used filter tips and physically isolation of experimental steps to avoid cross contamination, synthetic DNA spike-ins to compete with contaminations and quantify SARS-CoV-2, dUTP/uracil DNA glycosylase system to digest the carryover contaminations, and a new data analysis procedure to remove the sequencing reads from contaminations. Compared to AMP-Seq, the contamination level of ccAMP-Seq was at least 22-folds lower and the detection limit was also about an order of magnitude lower-as low as one copy/reaction. By testing the dilution series of SARS-CoV-2 nucleic acid standard, ccAMP-Seq showed 100% sensitivity and specificity. The high sensitivity of ccAMP-Seq was further confirmed by the detection of SARS-CoV-2 from 62 clinical samples. The consistency between qPCR and ccAMP-Seq was 100% for all the 53 qPCR-positive clinical samples. Seven qPCR-negative clinical samples were found to be positive by ccAMP-Seq, which was confirmed by extra qPCR tests on subsequent samples from the same patients. This study presents a carryover contamination-controlled, accurate qualitative and quantitative amplicon sequencing workflow that addresses the critical problem of pathogen detection for infectious diseases. IMPORTANCE Accuracy, a key indicator of pathogen detection technology, is compromised by carryover contamination in the amplicon sequencing workflow. Taking the detection of SARS-CoV-2 as case, this study presents a new carryover contamination-controlled amplicon sequencing workflow. The new workflow significantly reduces the degree of contamination in the workflow, thereby significantly improving the accuracy and sensitivity of the SARS-CoV-2 detection and empowering the ability of quantitative detection. More importantly, the use of the new workflow is simple and economical. Therefore, the results of this study can be easily applied to other microorganism, which has great significance for improving the detection level of microorganism.

Early life gut microbiota sustains liver-resident natural killer cells maturation via the butyrate-IL-18 axis.

Liver-resident natural killer cells, a unique lymphocyte subset in liver, develop locally and play multifaceted immunological roles. However, the mechanisms for the maintenance of liver-resident natural killer cell homeostasis remain unclear. Here we show that early-life antibiotic treatment blunt functional maturation of liver-resident natural killer cells even at adulthood, which is dependent on the durative microbiota dysbiosis. Mechanistically, early-life antibiotic treatment significantly decreases butyrate level in liver, and subsequently led to defective liver-resident natural killer cell maturation in a cell-extrinsic manner. Specifically, loss of butyrate impairs IL-18 production in Kupffer cells and hepatocytes through acting on the receptor GPR109A. Disrupted IL-18/IL-18R signaling in turn suppresses the mitochondrial activity and the functional maturation of liver-resident natural killer cells. Strikingly, dietary supplementation of experimentally or clinically used Clostridium butyricum restores the impaired liver-resident natural killer cell maturation and function induced by early-life antibiotic treatment. Our findings collectively unmask a regulatory network of gut-liver axis, highlighting the importance of the early-life microbiota in the development of tissue-resident immune cells.

TIM-4 orchestrates mitochondrial homeostasis to promote lung cancer progression via ANXA2/PI3K/AKT/OPA1 axis.

Mitochondrial function and homeostasis are critical to the proliferation of lung cancer cells. T-cell immunoglobulin and mucin domain-containing molecule 4 (TIM-4) promotes the development and progression of lung cancer. However, the role of TIM-4 in mitochondria homeostasis in tumor cells remains completely unknown. In this study, we found that TIM-4 promoted growth and proliferation of lung cancer cells by the oxidative phosphorylation (OXPHOS) pathway. Consistently, inhibition of OXPHOS reversed TIM-4-induced proliferation of lung cancer cells. Notably, TIM-4 promoted mitochondrial fusion via enhancing L-OPA1 protein expression. Mechanistically, TIM-4 regulated protein of L-OPA1 through the PI3K/AKT pathway, and TIM-4 interacted with ANXA2 to promote the activation of PI3K/AKT signaling. Collectively, TIM-4 promotes oxidative phosphorylation of lung cancer cells to accelerate tumor progress via ANXA2/PI3K/AKT/OPA1 axis, which sheds significant new lights on the potential role of TIM-4 in regulating tumor cell metabolism.