Enhanced Anti-Liver Tumor Efficacy of Chimeric Antigen Receptor-T Cells via SATB1 Modulation

Enhanced Anti-Liver Tumor Efficacy of Chimeric Antigen Receptor-T Cells via SATB1 Modulation | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Enhanced Anti-Liver Tumor Efficacy of Chimeric Antigen Receptor-T Cells via SATB1 Modulation Tongbiao Zhao, Lin Zhang, Chenxi Cheng, Xinyi Bi, Jiani Cao, Xiaoyan Li This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6776099/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 10 Dec, 2025 Read the published version in Cell Death & Disease → Version 1 posted 7 You are reading this latest preprint version Abstract Although Chimeric antigen receptor (CAR) T-cell therapy has achieved remarkable success in treating hematopoietic malignancies, its clinical application in solid tumors is profoundly hindered by persistent T-cell exhaustion within the immunosuppressive tumor microenvironment (TME). Here, we identified SATB1—a genome organizer regulating chromatin architecture—as a key suppressor of CAR-T cell exhaustion. In Glypican-3 (GPC3)-targeted CAR-T cells, SATB1 was significantly downregulated in tumor-infiltrating exhausted populations. SATB1 overexpression not only reduced expression of multiple inhibitory receptors (PD-1, CTLA-4, TIM3 and LAG-3), but also promoted a central memory phenotype, enhancing cytokine production and cytotoxicity against hepatocellular carcinoma (HCC) cells in vitro . In vivo , SATB1-engineered CAR-T cells exhibited superior tumor control and promoted survival, accompanied by reduced exhaustion markers in tumor-infiltrating T cells. These functional improvements are consistent with the reported role of SATB1 in modulating T cell exhaustion, positioning it as a multifunctional enhancer of CAR-T cell fitness. Collectively, our study unveils SATB1 as a multifunctional modulator that simultaneously targets exhaustion and memory differentiation, offering a novel strategy to enhance CAR-T efficacy against solid tumors. Biological sciences/Immunology/Immunotherapy/Immunization Health sciences/Diseases/Cancer Figures Figure 1 Figure 2 Figure 3 Figure 4 1. Introduction Adoptive cellular immunotherapy (ACT) based on functional immune cell transfer holds great promise in treating various malignant diseases, especially cancer(1, 2). Among ACT strategies, chimeric antigen receptor (CAR-T) cell therapy has emerged as an effective clinical strategy for the treatment of several hematopoietic malignancies(3–6). However, its efficacy in solid tumors, including hepatocellular carcinoma (HCC), remains limited due to antigenic heterogeneity, suboptimal infiltration, and immunosuppressive tumor microenvironment (TME)-driven T cell exhaustion(7, 8). Overcoming these challenges is critical for improving the antitumor efficacy of CAR-T cell therapy for solid tumors. T cell exhaustion is a state characterized by diminished proliferation, impaired effector function, transcriptional and epigenetic alterations, and upregulation of inhibitory receptors such as Programmed Death-1 (PD-1), Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4), and Lymphocyte-Activation Gene 3 (LAG-3)(9–13). In CAR-T cells, exhaustion is primarily driven by persistent antigenic stimulation and sustained autoactivation due to CAR structure aggregation(12, 14). Recent efforts to combat T cell exhaustion have focused on modulating transcriptional regulators such as c-Jun(15), NR4A family members(16, 17), BATF (from the AP-1 family)(18, 19), FOXO1(20, 21), Id2(22) and Stat5(23). Genetic modulation of these factors has been shown to improve CD8 + T cell and CAR-T cell function by mitigating exhaustion, enhancing stem-like properties or metabolic adaptability, ultimately leading to tumor regression and extending survival in preclinical models. Moreover, the epigenetic reprogramming underlying T cell exhaustion remains poorly understood, hindering the development of durable solutions. Special AT-Rich Sequence Binding Protein 1 (SATB1) is a critical genome organizer that reprograms chromatin structure and broadly regulates transcriptional profiles to promote tumor cell proliferation(24) and metastasis(25, 26). Beyond its oncogenic functions, predominantly expressed in thymocytes, SATB1 is indispensable for thymocyte development(27, 28) and T cell differentiation(29–31). The absence of SATB1 disrupts thymocyte development, particularly during the double-positive stage(32). Recent studies have highlighted SATB1's role in the anti-tumor function of cytotoxic T lymphocytes (CTL) through its recruitment of the nucleosome remodeling deacetylase (NuRD) complex at genomic regions, thereby regulating PD-1 expression and suggesting its potential role in mitigating T cell exhaustion(33). Additionally, within TME, TGF-β-mediated SATB1 silencing promotes follicular helper T (Tfh) cell differentiation and tertiary lymphoid structures (TLS) formation, further underscoring its multifaceted role in immune regulation and tumor immunity(34). In this study, we identified SATB1 as a key regulator of T cell exhaustion in Glypican-3 (GPC3)-targeting CAR-T cells within hepatocellular carcinoma (HCC) xenograft models. We observed significant downregulation of SATB1 and concomitant upregulation of PD-1 in tumor-infiltrating exhausted CAR-T cells. We hypothesized that SATB1 overexpression could reprogram CAR-T cell epigenetics to resist exhaustion and enhance anti-tumor efficacy in HCC. Overexpression of SATB1 enhanced the immunophenotypic characteristics, improved effector function, and reduced exhaustion levels in CAR-T cells in vitro . Subsequent studies demonstrated that SATB1-overexpressing CAR-T cells exhibited improved resistance to exhaustion and superior immunotherapeutic efficacy in vivo , leading to accelerated tumor eradication and prolonged survival of tumor-bearing mice. These findings suggest that SATB1 modulation represents a promising strategy to enhance the efficacy of CAR-T cells in treating solid tumors. 2. Results 2.1. SATB1 is downregulated in tumor-infiltrating exhausted T cells. Exhausted T cells in TME are characterized by the upregulation of immune checkpoint molecules like PD-1, reduced proliferative capacity, and impaired effector functions(10, 13, 14). To better elucidate the transcriptional profiles of tumor-infiltrating exhausted T cells, we analyzed transcriptional profiles across models. In a B78ChOVA melanoma mouse model (GSE201071), Satb1 mRNA expression was preferentially downregulated in tumor-infiltrated exhausted OT-1 T cells at days 4 and 14 compared to naïve and effector T cells (Figure S1 A)(35). This finding was corroborated in chronically activated T cells from an LCMV Arm5 infection model (GSE88987), where SATB1 expression decreased in exhausted populations (Figure S1 B)(16). To assess clinical relevance, we analyzed HCC patient data (GSE111389) and observed consistent SATB1 downregulation in PD-1 high (PD-1 hi) tumor-infiltrating CD8 + T cells across all six patients (Figure S1 C)(36). Further validation using the Pan-Cancer Human T Cell Atlas (scRNA-seq data) revealed high SATB1 expression in naïve and memory T cells, but marked reduction in exhausted subsets (Figures S1 D and S1E)(37). Collectively, these results implicate SATB1 downregulation as a conserved feature of T cell exhaustion. Engineering CAR-T cells to overexpress SATB1 may represent a promising strategy to mitigate exhaustion and enhance the efficacy of CAR-T cell therapy against solid tumors. 2.2. The potent antitumor activity of GPC3-Targeted CAR-T Cells in Hepatocellular Carcinoma. To investigate the role of SATB1 in exhausted CAR-T cells, we developed a GPC3-targeted CAR-T model. GPC3, a membrane-bound heparan sulfate proteoglycan highly expressed in 70% of HCC patients but absent in normal adult tissues(38), is under clinical evaluation in 11 of 22 ongoing HCC CAR-T trials(39–41). Our analysis confirmed high GPC3 expression in human HCC cell lines Huh7, HepG2, and Hep3B but not in SK-HEP-1 (Figure S2A). Subsequently, second-generation GPC3-CAR-T cells were engineered using an EF1α-promoter lentiviral vector (Figure S2B)(41). Primary human T cells, stimulated with anti-CD3/anti-CD28 Dynabeads and IL-2 achieved 60% transduction efficiency (Figure S2C). These CAR-T cells specifically secreted IFN-γ/IL-2 (Figure S2D) and lysed GPC3 + HCC lines (Huh7, HepG2, Hep3B), but not GPC3 − SK-HEP-1 cells (Figures S2E and S2F), demonstrating their target-dependent specificity and efficacy. To further evaluate the anti-tumor effects of GPC3-CAR-T cells in vivo , we established a cell line-derived xenograft (CDX) model by subcutaneously injecting GFP/Luc + Huh7 into immunodeficient NCG mice. Following tumor engraftment, mice were intravenously infused with either Ctrl-T or CAR-T cells, and the tumor size was measured at the indicated time points (Figure S2G). CAR-T cell treatment induced significant tumor regression and improved survival of tumor-bearing mice compared to Ctrl-T cells (Figures S2H-S2J). However, incomplete regression in some tumors highlighted the need to enhance therapeutic potency—a goal addressed by SATB1 engineering in subsequent studies. 2.3. SATB1 is downregulated in tumor-infiltrating exhausted CAR-T cells. We next asked whether SATB1 downregulation occurs in CAR-T cells within tumors. We analyzed SATB1 expression in human T cells under resting or anti-CD3/CD28-activated conditions. Consistent with prior studies, SATB1 was highly expressed in activated human CD4 + and CD8 + T cells (Figs. 1 A- 1 C)(33). Similarly, anti-CD3/CD28 stimulation significantly increased SATB1 levels in mouse splenic CD3 + T cells (Fig. 1 D). We transferred GPC3-CAR-T cells into GFP/Luc + Huh7 xenograft-bearing mice to detect SATB1 expression in CAR-T cells within tumors (Fig. 1 E). Two weeks post-infusion, tumor-infiltrating CAR-T cells exhibited elevated PD-1 expression (Figs. 1 F and 1 G), resembling endogenous exhausted CD8 + T cells. Strikingly, SATB1 levels were significantly reduced in these cells compared to splenic CAR-T counterparts, corroborating the RNA-seq data (Figs. 1 H and 1 I). These findings suggest that SATB1 downregulation is associated with CAR-T cell exhaustion in the TME, highlighting its potential as a target to enhance functionality. 2.4. SATB1 efficiently enhances CAR-T cell functions in vitro. Considering the downregulation of SATB1 in tumor-infiltrated exhausted CAR-T cells, we investigated whether SATB1 overexpression could enhance CAR-T cell functionality. We co-transduced human T cells with either an empty vector or a SATB1 overexpression vector alongside the CAR construct. Successful SATB1 overexpression in SATB1-CAR-T cells was confirmed at both the mRNA (Fig. 2 A) and protein levels (Figs. 2 B and 2 C). To evaluate the functional impact of SATB1 overexpression, Ctrl-T, SATB1-overexpressing T (SATB1-T), CAR-T, or SATB1-CAR-T cells were co-cultured with HCC cell lines (SK-HEP-1, Huh-7, HepG2, and Hep3B). SATB1-CAR-T cells exhibited enhanced cytokine release compared to conventional CAR-T cells (Fig. 2 D). Consistently, SATB1-CAR-T cells exhibited increased killing efficiency against GFP/Luc + Huh-7, HepG2, and Hep3B cells (Fig. 2 E). These results indicate that SATB1 could enhance the anti-tumor efficacy of CAR-T cells in vitro , highlighting its potential as a strategy to improve the antitumor efficacy of CAR-T cells. 2.5. SATB1 modulates the immunophenotypes of CAR-T cells in vitro . Given the enhanced functionality of SATB1-CAR-T cells, we investigated the influence of SATB1 overexpression on T cell biological properties and immunophenotypic profiles. SATB1-T and SATB1-CAR-T cells exhibited significantly enhanced proliferation over a two-week culture period compared to controls (Figs. 3 A and 3 B). Moreover, SATB1-CAR-T cells exhibited reduced apoptosis levels upon co-culture with GPC3 + Huh7 cells, indicating improved resistance to tumor-induced stress (Figs. 3 C and 3 D). To explore the underlying phenotypic changes, we analyzed T cell differentiation markers CD45RA and CD62L, which classify primary human T cells into four kinds of differentiation subsets: CD45RA + CD62L + naïve T cells (T N ), CD45RA − CD62L + central memory T cells (T CM ), CD45RA − CD62L − effector memory T cells (T EM ), and CD45RA + CD62L − effector memory T cells (T RAEM )(42, 43). Strikingly, SATB1 overexpression drove both CD4 + and CD8 + T cells toward a central memory T cell phenotype (Figs. 3 E and 3 F). This memory reprogramming was further supported by elevated CCR7 expression, a key mediator of T cell homeostasis, lymphoid homing and sustained anti-tumor responses (Figs. 3 G and 3 H). Notably, SATB1 overexpression had no effect on CD4 + or CD8 + T cell subset distribution (Figure S3A) and did not alter regulatory T cell (T reg ) proportions (Figure S3B), suggesting selective modulation of effector/memory subsets without perturbing other immune cells. 2.6. SATB1 attenuates TGF-β-induced Immunosuppression in T Cells. SATB1 has been shown to recruit the nucleosome remodeling deacetylase (NuRD) complex to Pdcd1 regulatory regions, and the loss of Satb1 increases PD-1 expression upon T cell activation(33). Consistent with these mechanism, SATB1-T cells exhibited significantly reduced PD-1 expression compared to controls (Figures S4A and S4B), while other exhaustion markers (CTLA-4, TIM3, LAG-3) remained unchanged (Figure S4C). Transforming growth factor-beta (TGF-β), mainly secreted by immunosuppressive cells and tumor cells, limits CAR-T cell efficacy in solid tumors by suppressing T cell activation, proliferation, migration, and differentiation(44–46). Therapeutic strategies targeting TGF-β signaling have shown considerable promise in preclinical and clinical studies(47–49). In line with previous research, TGF-β1 treatment downregulated SATB1 expression and increased PD-1 levels in control T cells (Figure S4D-S4G). Strikingly, SATB1-T cells resisted this regulation, maintaining significantly higher SATB1 and lower PD-1 expression compared to Ctrl-T cells under TGF-β1 exposure (Figure S4D-S4G). These findings suggest that SATB1 overexpression may counteract TGF-β-driven exhaustion, potentially preserving T cell functionality in immunosuppressive microenvironments. 2.7. SATB1 ameliorates CAR-T cell exhaustion in vitro . To systematically evaluate CAR-T cell exhaustion in vitro , we established a repetitive co-culture model with Huh7 tumor cells (Figure S5A). After three rounds of co-incubation, conventional CAR-T cells showed significant upregulation of exhaustion-associated markers, including PD-1, CTLA-4, TIM3, and LAG-3, confirming successful exhaustion induction (Figure S5B-S5E). Consistent with the suppressive effect of SATB1 on PD-1 under TGF-β exposure (Figure S4), SATB1 overexpression markedly reduced PD-1 expression in both CD4 + and CD8 + CAR-T cells post-co-culture (Figure S5F). Furthermore, SATB1 overexpression SATB1-CAR-T cells also showed attenuated upregulation of CTLA-4, TIM3, and LAG-3 (Figures S5G-S5I). These data collectively suggest that SATB1 overexpression could ameliorate CAR-T cell exhaustion in vitro , potentially preserving their anti-tumor functionality through epigenetic regulation of immune checkpoint molecules. 2.8. SATB1 enhances immunotherapeutic efficacy of CAR-T cells in vivo . To assess the in vivo efficacy of SATB1-CAR-T cells, we established a human liver cancer cell line-derived xenograft (CDX) model (Fig. 4 A). Mice receiving SATB1-CAR-T cells showed superior tumor regression compared to those receiving conventional CAR-T cells (Figs. 4 B and 4 C). Bioluminescence imaging on day 7 confirmed noticeably reduced tumor burden in SATB1-CAR-T-treated mice (Figs. 4 B- 4 D). Flow cytometry analysis revealed nearly 2-fold higher infiltration of human CD3 + T cells in both peripheral blood and tumor tissue of SATB1-CAR-T-treated mice (Figs. 4 E and 4 F). Notably, tumor-infiltrating CD8 + T cells from SATB1-CAR-T group exhibited 50% lower PD-1, CTLA-4, TIM3 and LAG-3 expression compared to controls (Figure S6), aligning with their exhaustion-resistant phenotype in vitro . Importantly, SATB1-CAR-T cell therapy significantly extended the survival of tumor-bearing mice, with over 50% surviving beyond 100 days post-infusion (Fig. 4 G). Collectively, these findings highlight that SATB1 overexpression enhances CAR-T cell persistence and functionality in solid tumors. 3. Discussion The clinical application of CAR-T cells has rapidly expanded, with 23% of 517 registered clinical trials in China focusing on solid tumors, particularly HCC(4, 8, 39, 50). Bioinformatics analyses have revealed a TME enriched with exhausted CD8 + T cells and regulatory CD4 + T regs in HCC, emphasizing the need for engineering exhaustion-resistant CAR-T cells to improve clinical efficacy(37, 51). Comparative analysis of the unique transcriptional programs, epigenetic programs, and metabolic properties of exhausted T cells has identified several T cell exhaustion-related proteins and transcription factors such as CD38(52), Rgs1(53, 54), and Tigit(55). While previous studies have focused on highly expressed genes in exhausted T cells, we identified special AT-rich binding protein SATB1, an epigenetic remodeling factor downregulated in exhausted T cells, as a crucial regulator of CAR-T cell exhaustion. Our study demonstrates that SATB1 overexpression mitigates CAR-T cell exhaustion and improves anti-tumor efficacy, potentially not only through mechanisms involving broadly suppression of inhibitory receptors (PD-1, CTLA-4, TIM3 and LAG-3) (Figure S5, S6) but also promotion of memory-like phenotypes (Figs. 3 E- 3 H) and enhanced resistance to TGF-β-mediated immunosuppression (Figure S4) in the HCC microenvironment. To further explore T cell exhaustion mechanisms, we developed an ex vivo co-culture system for chronic antigen stimulation and rapid T cell exhaustion induction (Figure S5A)(10, 12, 56, 57). In our co-culture system, CAR-T cells exhibited significant upregulation of inhibitory receptors (Figures S5B-S5E). This system, which can be optimized with TGF-β or immunosuppressive cells, provides a platform for screening genes and drugs that enhance CAR-T cell function. SATB1, initially detected at high levels in thymocytes, progenitor cells (such as osteoblasts), and the epidermal basal layer, plays critical roles in embryogenesis, neurogenesis and malignancies(58). It critically regulates hematopoietic development, thymus maturation, and T cell differentiation(27–31). In mature T cells, SATB1 is dynamically regulated by T cell receptor (TCR) signaling and maintains the distinct naive chromatin state within naive CD8 + T cells, also regulates chemokine genes through enhancer-promoter interactions(28, 59–61). SATB1 also contributes to T cell 3D genome homeostasis and immune tolerance(31, 62, 63). Recent studies identified SATB1 as an epigenetic negative regulator of PD-1, mitigating T cell exhaustion(33). Align with its reported role in epigenetic regulation, SATB1 modulation broadly enhance CAR-T fitness, addressing both exhaustion and persistence—a dual challenge that single-pathway interventions may incompletely resolve. Future studies are required to directly link SATB1 overexpression to chromatin remodeling in CAR-T cells. Our study further demonstrates that SATB1 overexpression enhances CAR-T cell functionality without altering T reg proportions (Figure S3B), consistent with its role in maintaining T cell homeostasis(64). Compared to previous work, the expansion of the T CM subset in SATB1-CAR-T cells supports the superior efficacy of T CM in CAR-T therapy, characterized by robust proliferation capacity, prolonged persistence, and reduced exhaustion(19). Since SATB1 deletion causes mitochondrial function impairment and oxidative stress in CD4 + T cells, its overexpression may preserve mitochondrial integrity in TME, thereby sustaining CAR-T cell functionality(65). Furthermore, the precise mechanisms by which SATB1 modulates CAR-T-cell immunophenotypes and the potential of SATB1 to enhance CAR-T cell function in other solid tumors warrants further investigation. In conclusion, our study not only identifies SATB1 as a potential therapeutic target to alleviate CAR-T cell exhaustion, but also provides a novel strategy to enhance CAR-T cell efficacy in solid tumors, particularly HCC. While SATB1 overexpression shows promise, potential side effects such as metabolic stress, autoimmune reactions, and cytokine release syndrome (CRS) require further evaluation. These risks could be mitigated through inducible expression systems and close monitoring of cytokine levels. The development of potent small-molecule drugs targeting SATB1 and integration with immune checkpoint inhibitors or multi-targeted CAR-T cells may further improve therapeutic outcomes. 4. Materials and methods 4.1. Cell lines and culture conditions Hepatocellular carcinoma (HCC) cell lines Huh7, HepG2 (HB-8065, ATCC), Hep3B (BNCC360312, BNCC), SK-HEP-1 (HTB-52, ATCC) and Lentiviral producer cell line 293T (CRL-11268, ATCC) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Vistech), 2 mM GlutaMAX TM -I (Gibco), 1 mM sodium pyruvate (Gibco), 0.1 mM nonessential amino acids (Gibco), 100 µg/mL streptomycin and 100 U/mL penicillin (Gibco). For luciferase-based experiments, the lentivirus of green fluorescent protein and firefly luciferase fusion protein (GFP/Luc) was transduced into tumor cells to produce stable cell lines Huh7-GFP/Luc, HepG2-GFP/Luc, Hep3B-GFP/Luc and SK-HEP-1-GFP/Luc. 4.2. Animals The use of animals for this study was approved by the Institutional Animal Care and Use Committee (IOZ2016004). NCG (NOD/ShiLtJGpt- Prkdc em26Cd52 Il2rg em26Cd22 /Gpt) mice aged 6–12 weeks (Gempharmatech Co., Ltd) were used for human tumor cell line CDX models. 4.3. Generation of CAR constructs To target GPC3, the chimeric antigen receptor (CAR) was designed based on the single-chain variable fragment (scFv) derived from GC33 antibody, which was linked to the CD8 hinge and transmembrane domain, followed by the intracellular domains of 4-1BB and the signaling moiety of the CD3ζ chain. The CAR sequence was synthesized at Sangon Technology (Shanghai, China) and cloned into the pFUW-EF1α-P2A-eGFP lentiviral vector. CAR-T cells were identified by eGFP expression. 4.4. Lentivirus preparation The human SATB1 full sequence was synthesized at GenScript Technology (Shanghai, China) and cloned into the pFUW-EF1α-P2A-mCherry lentiviral backbone to generate gene expression plasmid pFUW-EF1α-SATB1-P2A-mCherry. For CAR and gene vector, the pFUW vectors harboring P2A-eGFP and P2A-mCherry sequences were respectively used as a negative control. We obtained pLenti-CMV-eGFP-linker-Luc-PGK-Puro lentiviral vector (GFP/Luc) from OBiO Technology (Shanghai, China). We obtained Lentiviral particles from 293T-packaging cells by calcium phosphate transfection. The CAR or gene vector plasmid, pMD2.G plasmid and psPAX2 plasmid were transfected into 293T-packaging cells and incubated at 37°C for 12 hours. Then 293T-packaging cells were cultured in fresh medium for 48 hours. The supernatants were collected and filtered through 0.45 µm filter. Lentiviral particles were concentrated by ultracentrifugation at 20 000 rpm for 2 hours at 4°C. The viral granules were resuspended in X-VIVO™ 15 medium and filtered through 0.22 µm filter and stored at -80°C. 4.5. CAR-T cell generation and cell culture For CAR-T cell generation, human peripheral blood mononuclear cells (PBMCs) were obtained from the Biobank of Peking Union Medical College Hospital through Ficoll-Paque PLUS gradient centrifugation (17-1440-02, GE Healthcare). Primary human T cells were negatively selected from PBMCs with a Pan T Cell Isolation Kit (130-096-535, Miltenyi) and were stimulated CD3/CD28 Dynabeads (11161D, ThermoFisher) at the ratio of 1:1. The T cell culture medium was X-VIVO™ 15 medium (04-418Q, Lonza) supplemented with 5% heat-inactivated FBS (Vistech), 1 mM sodium pyruvate (Gibco), 2 mM GlutaMAX TM -I (Gibco) and 100 IU/mL recombinant human IL-2 (200-02, PeproTech). T cells were transduced with control vector, GPC3-CAR and/or SATB1 overexpression lentiviral particles after 24 hours activation and were cultured at a concentration of 10 6 cells/mL in 6-well plates for 2–3 weeks. 4.6. Quantitative real-time PCR Total RNA was extracted from sorted mCherry + Ctrl-T cells and SATB-T cells with an RNAeasy Mini Kit (Qiagen). Total RNA (1 µg) was reverse transcribed into cDNA using a StarScript III All-in-one RT Mix with gDNA Remover (A230-10, GenStar). Quantitative real-time PCR was performed with GoTaq® qPCR Master Mix (Promega) and a QuantStudio™ 6 Flex Real-Time PCR System. All samples were analyzed in duplicate and normalized to GAPDH . The following primers were used: GAPDH -F 5’-GGAGCGAGATCCCTCCAAAAT-3’, GAPDH -R 5’-GGCTGTTGTCATACTTCTCATGG-3’, SATB1 -F 5’-GATCATTTGAACGAGGCAACTCA-3’, SATB1 -R 5’-TGGACCCTTCGGATCACTCA-3. The threshold cycle was determined and the relative gene expression ratio was calculated as follows, fold-change = 2 −ΔΔCt . 4.7. Western blotting and antibodies Whole-cell lysates of T cells were generated by lysing on ice in RIPA buffer for 30 min containing a protease inhibitor cocktail (04693116001, Roche) and 1 mM PMSF (ST506, Beyotime). Equivalent protein quantities (15 µg) of total protein were run in 10% SDS–PAGE gel (Bio-Rad) and transferred to nitrocellulose membranes (Millipore). The membranes were then blocked with 5% non-fat milk in 1× TBST (T1082, Solarbio) and probed with a primary antibody directed against SATB1 (1:1 000, ab109122, Abcam) and β-Actin (1:2500, A5441, Sigma-Aldrich) overnight at 4°C. After incubation with appropriate HRP-conjugated secondary antibody (Beyotime), signals from bound antibodies were detected with a Luminata Forte Western HRP Substrate Kit (WBLUF0100, Millipore) and quantified using Image J Software. 4.8. Flow cytometry analysis and antibodies Flow cytometry was used to detect the expression of cell surface markers. The CAR expression was detected by eGFP, and the SATB1 expression was detected by mCherry. CAR-T cells were stained with Human TruStain FcX™ Antibody (422301), Pacific Blue™ anti-human CD45 (368539), APC anti-human CD3 (300411), Brilliant Violet 421™ anti-human CD4 (357423), APC/Cyanine7 anti-human CD8a (301016), APC anti-human CD45RA (304111), PE anti-human CD62L (304805), Alexa Fluor® 647 anti-human CD279 (PD-1) (329910), APC anti-human CD152 (CTLA-4) (369611), APC anti-human CD366 (TIM3) (364803), Brilliant Violet 421™ anti-human CD223 (LAG-3) (369313) and Alexa Fluor® 647 anti-human FOXP3 (320113) antibodies from Biolegend; and Recombinant APC Anti-Glypican-3 (ab275695), Recombinant Anti-SATB1 (ab109122) antibodies from Abcam; and Goat anti-Rabbit IgG (H + L) Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 555 (A-21428) antibody from Invitrogen. For surface markers detection, T cells were collected, washed with PBS. 1×10 6 T cells were suspended in 100 µL PBS with 1 µL TruStain FcX™ Antibody for 20 minutes at 4°C, then incubated with specific surface antibodies for 30 minutes at 4°C. For intracellular staining, T cells were stained with surface markers first, then were stained with intracellular antibodies by Foxp3/Transcription Factor Staining Buffer Set (00-5523-00, eBioscience) on the basis of the manufacturer’s instructions. The experiment data were acquired using BD FACS AriaIII and were then analyzed using FlowJo software. 4.9. Apoptosis and proliferation assays For apoptosis level detection of T cells, we used Annexin V-APC (40310ES20, Yeasen,) to stain T cells based on the manufacturer’s instructions after T cell membrane protein staining. To test the proliferation level of T cells, the fluorescent dye CFDA SE was used according to the manufacturer’s instructions. For the cell number counting experiment, T cells (5×10 4 ) were seeded in a 48-well plate on day 0 and were passaged at a concentration of 10 6 cells/mL, counted and recorded every two days. 4.10. Cytotoxicity assay We evaluated the cytotoxicity of CAR-T cells by co-cultured with luciferase labeled tumor cells in vitro . One day before the experiment, the anti-CD3/CD28 magnetic beads were removed. The target tumor cells (Huh7-GFP/Luc, HepG2-GFP/Luc, Hep3B-GFP/Luc and SK-HEP-1-GFP/Luc) were seeded in white 96-well plates as a density of 5 000 cells/50 µL each well. At a ratio of 9:1, 3:1, 1:1 or 1:3, the indicated T cells were seeded with target tumor cells for 18 hours. Target tumor cells alone were determined as the maximal luciferase activity. Then D-luciferin, sodium salt (40901ES03, Yeasen) was prepared and added to each well according to the manufacturer’s instructions. The luminescence signal of each well was measured by a PerkinElmer Victor X3 Reader. The specific lysis rate was calculated using the formula: [(Maximal luciferase activity – Experimental Luciferase Activity)/Maximal luciferase activity] × 100. 4.11. Enzyme-linked immunosorbent assay (ELISA) assay To analyze the cytokine secretion by CAR-T cells, one day before the experiment, the anti-CD3/CD28 magnetic beads were removed. CAR-T cells and target tumor cells were seeded at a density of 5 000 cells/50 µL each well at an effector-to-target ratio of 1:1 in 96-well round bottom plates (Nunc) for 24 hours. The supernatants were collected and cytokines IL-2 and interferon (IFN-γ) were measured using ELISA Kits (70-EK102, 70-EK180, Multi-Science) following the manufacturer’s protocol by ELISA plate reader (EL-808, Biotek). The cytokine concentration was quantified by a standard curve. 4.12. In vivo antitumor model To assess the antitumor activity of CAR-T and SATB1-CAR-T cells in vivo , we constructed an HCC cell line-derived CDX model. 5×10 5 Huh7-GFP/Luc cells were subcutaneously (s.c.) injected into NCG mice on the right flank. 4 days after tumor cell implantation, tumor-bearing mice with equal tumor burden were selected and randomized to different treatment groups and 1×10 6 Ctrl-T, SATB1-T, CAR-T or SATB1-CAR-T cells were injected intravenously (i.v.) via the tail vein. Tumor-bearing mice were then intraperitoneally (i.p.) injected with 150 mg/kg D-luciferin at indicated time points and luminescence signals were monitored and counted by an IVIS Spectrum Imaging platform (Caliper, Boston, MA, USA). To assess the survival curves, GraphPad Prism Software was used to record and analyze. 4.13. Statistical analysis All data presented graphically as the mean ± standard deviation (S.D.) was from at least three independent experiments. Each exact n value is stated in the corresponding figure legend. The statistical data was analyzed using paired or unpaired two-tailed Student’s t -test for two-sample comparisons. The log-rank test was performed for comparison of survival curves. All statistical data was analyzed by GraphPad Prism and statistical significance was defined as * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001. Declarations A CKNOWLEDGMENTS CORRESPONDENCE Tongbiao Zhao, The State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, 1 Beichen West Road, Chaoyang District, Beijing, 100101, People's Republic of China. Email: [email protected] . Phone: (+86)-10-64806307. FUNDING INFORMATION This work was supported by grants from the China National Basic Research Program 2022YFA1103601; the Strategic Priority Research Program of the Chinese Academy of Sciences E42GP41107; and the Strategic Collaborative Research Program of the Ferring Institute of Reproductive Medicine Grant No.33. AUTHOR CONTRIBUTIONS L.Z. and T.Z. designed the experiments and wrote the manuscript; L.Z., C.C., X.B., J.C. and X.L. performed the experiments and analyzed the data; T.Z. coordinated and supervised the experiments. CONFLICT OF INTEREST The authors declare no competing interest. DATA AVAILABILITY STATEMENT The data and materials during the current study are available from the corresponding author upon reasonable request. References Liu Q, Li J, Zheng H, Yang S, Hua Y, Huang N, et al. Adoptive cellular immunotherapy for solid neoplasms beyond CAR-T. Mol Cancer. 2023;22(1):28. June CH, O'Connor RS, Kawalekar OU, Ghassemi S, Milone MC. CAR T cell immunotherapy for human cancer. Science. 2018;359(6382):1361-5. Venetis K, Invernizzi M, Sajjadi E, Curigliano G, Fusco N. Cellular immunotherapy in breast cancer: The quest for consistent biomarkers. Cancer Treat Rev. 2020;90:102089. Kirtane K, Elmariah H, Chung CH, Abate-Daga D. Adoptive cellular therapy in solid tumor malignancies: review of the literature and challenges ahead. J Immunother Cancer. 2021;9(7). Cappell KM, Kochenderfer JN. Long-term outcomes following CAR T cell therapy: what we know so far. Nat Rev Clin Oncol. 2023;20(6):359-71. Maude SL, Laetsch TW, Buechner J, Rives S, Boyer M, Bittencourt H, et al. Tisagenlecleucel in Children and Young Adults with B-Cell Lymphoblastic Leukemia. N Engl J Med. 2018;378(5):439-48. Larson RC, Maus MV. Recent advances and discoveries in the mechanisms and functions of CAR T cells. Nat Rev Cancer. 2021;21(3):145-61. Zhang T, Tai Z, Miao F, Zhang X, Li J, Zhu Q, et al. Adoptive cell therapy for solid tumors beyond CAR-T: Current challenges and emerging therapeutic advances. J Control Release. 2024;368:372-96. Zinkernagel RM, Moskophidis D, Kundig T, Oehen S, Pircher H, Hengartner H. Effector T-cell induction and T-cell memory versus peripheral deletion of T cells. Immunol Rev. 1993;133:199-223. Pauken KE, Wherry EJ. Overcoming T cell exhaustion in infection and cancer. Trends Immunol. 2015;36(4):265-76. Wherry EJ, Ha SJ, Kaech SM, Haining WN, Sarkar S, Kalia V, et al. Molecular signature of CD8+ T cell exhaustion during chronic viral infection. Immunity. 2007;27(4):670-84. Wherry EJ, Kurachi M. Molecular and cellular insights into T cell exhaustion. Nat Rev Immunol. 2015;15(8):486-99. Gumber D, Wang LD. Improving CAR-T immunotherapy: Overcoming the challenges of T cell exhaustion. EBioMedicine. 2022;77:103941. Delgoffe GM, Xu C, Mackall CL, Green MR, Gottschalk S, Speiser DE, et al. The role of exhaustion in CAR T cell therapy. Cancer Cell. 2021;39(7):885-8. Lynn RC, Weber EW, Sotillo E, Gennert D, Xu P, Good Z, et al. c-Jun overexpression in CAR T cells induces exhaustion resistance. Nature. 2019;576(7786):293-300. Scott-Browne JP, Lopez-Moyado IF, Trifari S, Wong V, Chavez L, Rao A, et al. Dynamic Changes in Chromatin Accessibility Occur in CD8(+) T Cells Responding to Viral Infection. Immunity. 2016;45(6):1327-40. Chen J, Lopez-Moyado IF, Seo H, Lio CJ, Hempleman LJ, Sekiya T, et al. NR4A transcription factors limit CAR T cell function in solid tumours. Nature. 2019;567(7749):530-4. Quigley M, Pereyra F, Nilsson B, Porichis F, Fonseca C, Eichbaum Q, et al. Transcriptional analysis of HIV-specific CD8+ T cells shows that PD-1 inhibits T cell function by upregulating BATF. Nat Med. 2010;16(10):1147-51. Zhang X, Zhang C, Qiao M, Cheng C, Tang N, Lu S, et al. Depletion of BATF in CAR-T cells enhances antitumor activity by inducing resistance against exhaustion and formation of central memory cells. Cancer Cell. 2022;40(11):1407-22 e7. Chan JD, Scheffler CM, Munoz I, Sek K, Lee JN, Huang YK, et al. FOXO1 enhances CAR T cell stemness, metabolic fitness and efficacy. Nature. 2024;629(8010):201-10. Doan AE, Mueller KP, Chen AY, Rouin GT, Chen Y, Daniel B, et al. FOXO1 is a master regulator of memory programming in CAR T cells. Nature. 2024;629(8010):211-8. Li Y, Han M, Wei H, Huang W, Chen Z, Zhang T, et al. Id2 epigenetically controls CD8(+) T-cell exhaustion by disrupting the assembly of the Tcf3-LSD1 complex. Cell Mol Immunol. 2024;21(3):292-308. Beltra JC, Abdel-Hakeem MS, Manne S, Zhang Z, Huang H, Kurachi M, et al. Stat5 opposes the transcription factor Tox and rewires exhausted CD8(+) T cells toward durable effector-like states during chronic antigen exposure. Immunity. 2023;56(12):2699-718 e11. Naik R, Galande S. SATB family chromatin organizers as master regulators of tumor progression. Oncogene. 2019;38(12):1989-2004. Li QQ, Chen ZQ, Cao XX, Xu JD, Xu JW, Chen YY, et al. Involvement of NF-kappaB/miR-448 regulatory feedback loop in chemotherapy-induced epithelial-mesenchymal transition of breast cancer cells. Cell Death Differ. 2011;18(1):16-25. Han HJ, Russo J, Kohwi Y, Kohwi-Shigematsu T. SATB1 reprogrammes gene expression to promote breast tumour growth and metastasis. Nature. 2008;452(7184):187-93. Yasui D, Miyano M, Cai S, Varga-Weisz P, Kohwi-Shigematsu T. SATB1 targets chromatin remodelling to regulate genes over long distances. Nature. 2002;419(6907):641-5. Patta I, Madhok A, Khare S, Gottimukkala KP, Verma A, Giri S, et al. Dynamic regulation of chromatin organizer SATB1 via TCR-induced alternative promoter switch during T-cell development. Nucleic Acids Res. 2020;48(11):5873-90. Ahlfors H, Limaye A, Elo LL, Tuomela S, Burute M, Gottimukkala KV, et al. SATB1 dictates expression of multiple genes including IL-5 involved in human T helper cell differentiation. Blood. 2010;116(9):1443-53. Nussing S, Miosge LA, Lee K, Olshansky M, Barugahare A, Roots CM, et al. SATB1 ensures appropriate transcriptional programs within naive CD8(+) T cells. Immunol Cell Biol. 2022;100(8):636-52. Trujillo-Ochoa JL, Kazemian M, Afzali B. The role of transcription factors in shaping regulatory T cell identity. Nat Rev Immunol. 2023;23(12):842-56. Alvarez JD, Yasui DH, Niida H, Joh T, Loh DY, Kohwi-Shigematsu T. The MAR-binding protein SATB1 orchestrates temporal and spatial expression of multiple genes during T-cell development. Genes Dev. 2000;14(5):521-35. Stephen TL, Payne KK, Chaurio RA, Allegrezza MJ, Zhu H, Perez-Sanz J, et al. SATB1 Expression Governs Epigenetic Repression of PD-1 in Tumor-Reactive T Cells. Immunity. 2017;46(1):51-64. Chaurio RA, Anadon CM, Lee Costich T, Payne KK, Biswas S, Harro CM, et al. TGF-beta-mediated silencing of genomic organizer SATB1 promotes Tfh cell differentiation and formation of intra-tumoral tertiary lymphoid structures. Immunity. 2022;55(1):115-28 e9. Kersten K, Hu KH, Combes AJ, Samad B, Harwin T, Ray A, et al. Spatiotemporal co-dependency between macrophages and exhausted CD8(+) T cells in cancer. Cancer Cell. 2022;40(6):624-38 e9. Kim HD, Song GW, Park S, Jung MK, Kim MH, Kang HJ, et al. Association Between Expression Level of PD1 by Tumor-Infiltrating CD8(+) T Cells and Features of Hepatocellular Carcinoma. Gastroenterology. 2018;155(6):1936-50 e17. Zheng L, Qin S, Si W, Wang A, Xing B, Gao R, et al. Pan-cancer single-cell landscape of tumor-infiltrating T cells. Science. 2021;374(6574):abe6474. Capurro M, Wanless IR, Sherman M, Deboer G, Shi W, Miyoshi E, et al. Glypican-3: a novel serum and histochemical marker for hepatocellular carcinoma. Gastroenterology. 2003;125(1):89-97. Dal Bo M, De Mattia E, Baboci L, Mezzalira S, Cecchin E, Assaraf YG, et al. New insights into the pharmacological, immunological, and CAR-T-cell approaches in the treatment of hepatocellular carcinoma. Drug Resist Updat. 2020;51:100702. Zhou F, Shang W, Yu X, Tian J. Glypican-3: A promising biomarker for hepatocellular carcinoma diagnosis and treatment. Med Res Rev. 2018;38(2):741-67. Ishiguro T, Sugimoto M, Kinoshita Y, Miyazaki Y, Nakano K, Tsunoda H, et al. Anti-glypican 3 antibody as a potential antitumor agent for human liver cancer. Cancer Res. 2008;68(23):9832-8. Gattinoni L, Lugli E, Ji Y, Pos Z, Paulos CM, Quigley MF, et al. A human memory T cell subset with stem cell-like properties. Nat Med. 2011;17(10):1290-7. Cieri N, Camisa B, Cocchiarella F, Forcato M, Oliveira G, Provasi E, et al. IL-7 and IL-15 instruct the generation of human memory stem T cells from naive precursors. Blood. 2013;121(4):573-84. Batlle E, Massague J. Transforming Growth Factor-beta Signaling in Immunity and Cancer. Immunity. 2019;50(4):924-40. Koehler H, Kofler D, Hombach A, Abken H. CD28 costimulation overcomes transforming growth factor-beta-mediated repression of proliferation of redirected human CD4+ and CD8+ T cells in an antitumor cell attack. Cancer Res. 2007;67(5):2265-73. Golumba-Nagy V, Kuehle J, Hombach AA, Abken H. CD28-zeta CAR T Cells Resist TGF-beta Repression through IL-2 Signaling, Which Can Be Mimicked by an Engineered IL-7 Autocrine Loop. Mol Ther. 2018;26(9):2218-30. Tang N, Cheng C, Zhang X, Qiao M, Li N, Mu W, et al. TGF-beta inhibition via CRISPR promotes the long-term efficacy of CAR T cells against solid tumors. JCI Insight. 2020;5(4). Stuber T, Monjezi R, Wallstabe L, Kuhnemundt J, Nietzer SL, Dandekar G, et al. Inhibition of TGF-beta-receptor signaling augments the antitumor function of ROR1-specific CAR T-cells against triple-negative breast cancer. J Immunother Cancer. 2020;8(1). Hou AJ, Shih RM, Uy BR, Shafer A, Chang ZNL, Comin-Anduix B, et al. IL-13Ralpha2/TGF-beta bispecific CAR-T cells counter TGF-beta-mediated immune suppression and potentiate anti-tumor responses in glioblastoma. Neuro Oncol. 2024. Hu Y, Feng J, Gu T, Wang L, Wang Y, Zhou L, et al. CAR T-cell therapies in China: rapid evolution and a bright future. Lancet Haematol. 2022;9(12):e930-e41. Zhang L, Yu X, Zheng L, Zhang Y, Li Y, Fang Q, et al. Lineage tracking reveals dynamic relationships of T cells in colorectal cancer. Nature. 2018;564(7735):268-72. Huang Y, Shao M, Teng X, Si X, Wu L, Jiang P, et al. Inhibition of CD38 enzymatic activity enhances CAR-T cell immune-therapeutic efficacy by repressing glycolytic metabolism. Cell Rep Med. 2024;5(2):101400. Huang D, Chen X, Zeng X, Lao L, Li J, Xing Y, et al. Targeting regulator of G protein signaling 1 in tumor-specific T cells enhances their trafficking to breast cancer. Nat Immunol. 2021;22(7):865-79. Bai Y, Hu M, Chen Z, Wei J, Du H. Single-Cell Transcriptome Analysis Reveals RGS1 as a New Marker and Promoting Factor for T-Cell Exhaustion in Multiple Cancers. Front Immunol. 2021;12:767070. Jackson Z, Hong C, Schauner R, Dropulic B, Caimi PF, de Lima M, et al. Sequential Single-Cell Transcriptional and Protein Marker Profiling Reveals TIGIT as a Marker of CD19 CAR-T Cell Dysfunction in Patients with Non-Hodgkin Lymphoma. Cancer Discov. 2022;12(8):1886-903. Wherry EJ. T cell exhaustion. Nat Immunol. 2011;12(6):492-9. Chow A, Perica K, Klebanoff CA, Wolchok JD. Clinical implications of T cell exhaustion for cancer immunotherapy. Nat Rev Clin Oncol. 2022;19(12):775-90. Glatzel-Plucinska N, Piotrowska A, Dziegiel P, Podhorska-Okolow M. The Role of SATB1 in Tumour Progression and Metastasis. Int J Mol Sci. 2019;20(17). Gottimukkala KP, Jangid R, Patta I, Sultana DA, Sharma A, Misra-Sen J, et al. Regulation of SATB1 during thymocyte development by TCR signaling. Mol Immunol. 2016;77:34-43. Russ BE, Barugahare A, Dakle P, Tsyganov K, Quon S, Yu B, et al. Active maintenance of CD8(+) T cell naivety through regulation of global genome architecture. Cell Rep. 2023;42(10):113301. Wang B, Bian Q. SATB1 prevents immune cell infiltration by regulating chromatin organization and gene expression of a chemokine gene cluster in T cells. Commun Biol. 2024;7(1):1304. Wang B, Ji L, Bian Q. SATB1 regulates 3D genome architecture in T cells by constraining chromatin interactions surrounding CTCF-binding sites. Cell Rep. 2023;42(4):112323. Gupta PK, Allocco JB, Fraipont JM, McKeague ML, Wang P, Andrade MS, et al. Reduced Satb1 expression predisposes CD4(+) T conventional cells to Treg suppression and promotes transplant survival. Proc Natl Acad Sci U S A. 2022;119(40):e2205062119. Beyer M, Thabet Y, Muller RU, Sadlon T, Classen S, Lahl K, et al. Repression of the genome organizer SATB1 in regulatory T cells is required for suppressive function and inhibition of effector differentiation. Nat Immunol. 2011;12(9):898-907. Kuwabara T, Ishikawa F, Ikeda M, Ide T, Kohwi-Shigematsu T, Tanaka Y, et al. SATB1-dependent mitochondrial ROS production controls TCR signaling in CD4 T cells. Life Sci Alliance. 2021;4(11). Additional Declarations (Not answered) Supplementary Files OriginalDataFiles.pptx Original Data Files orignaldata.zip Dataset 1 SupplementaryFigurelegends.docx SupplementaryFigureS1.png Supplementary Figure S1 SupplementaryFigureS2.png Supplementary Figure S2 SupplementaryFigureS3.png Supplementary Figure S3 SupplementaryFigureS4.png Supplementary Figure S4 SupplementaryFigureS5.png Supplementary Figure S5 SupplementaryFigureS6.png Supplementary Figure S6 Cite Share Download PDF Status: Published Journal Publication published 10 Dec, 2025 Read the published version in Cell Death & Disease → Version 1 posted Editorial decision: revise 12 Aug, 2025 Review # 1 received at journal 25 Jul, 2025 Reviewer # 1 agreed at journal 11 Jul, 2025 Reviewers invited by journal 16 Jun, 2025 Submission checks completed at journal 30 May, 2025 First submitted to journal 29 May, 2025 Editor assigned by journal 29 May, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6776099","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":472204811,"identity":"f6993a92-4b94-4647-bdbe-02a60e3e3c24","order_by":0,"name":"Tongbiao Zhao","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAtUlEQVRIiWNgGAWjYDACCQY2IGnDQ7wOHoiWNNK1HCbBXfbS7c8e/Kg4L8PPf4Dxww8GuzzCtsgcSDfsOXObR7LhALNkD0NyMREOSzgmwdt2m8fgYAODNAPDgcQGwloS2yT/tp3jMTjMwPybSC3JbNK8bQd4DI4xsBFpy51jbNIyZ5J5JHsY2yx7DJIJa2Gf3f5M8k2FnT0//+HDN35U2BHWggQYgYoNSFA/CkbBKBgFowA3AADy/TMmF+mKgAAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0003-1429-5818","institution":"Institute of Zoology Chinese Academy of Sciences","correspondingAuthor":true,"prefix":"","firstName":"Tongbiao","middleName":"","lastName":"Zhao","suffix":""},{"id":472204812,"identity":"debb473e-92df-4d5f-9413-d780a3c7c57c","order_by":1,"name":"Lin Zhang","email":"","orcid":"","institution":"Institute of Zoology Chinese Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Lin","middleName":"","lastName":"Zhang","suffix":""},{"id":472204813,"identity":"65b4487f-69dd-40e2-b27c-fd4f1f2b0af7","order_by":2,"name":"Chenxi Cheng","email":"","orcid":"","institution":"Institute of Zoology Chinese Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Chenxi","middleName":"","lastName":"Cheng","suffix":""},{"id":472204814,"identity":"aafc9741-d7ad-4ba3-a668-5b81e9582e83","order_by":3,"name":"Xinyi Bi","email":"","orcid":"","institution":"Institute of Zoology Chinese Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Xinyi","middleName":"","lastName":"Bi","suffix":""},{"id":472204815,"identity":"07f5cb40-651d-4364-859d-52b8be6b7f6a","order_by":4,"name":"Jiani Cao","email":"","orcid":"","institution":"Institute of Zoology Chinese Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Jiani","middleName":"","lastName":"Cao","suffix":""},{"id":472204816,"identity":"905e40cb-ac4b-4a3a-b867-a52c7f8016be","order_by":5,"name":"Xiaoyan Li","email":"","orcid":"","institution":"Institute of Zoology Chinese Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Xiaoyan","middleName":"","lastName":"Li","suffix":""}],"badges":[],"createdAt":"2025-05-29 11:31:16","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6776099/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6776099/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41419-025-08307-3","type":"published","date":"2025-12-10T05:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":84886708,"identity":"ed5b41be-d1c9-4ba7-820f-4e08663473a1","added_by":"auto","created_at":"2025-06-18 11:51:01","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":310651,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSATB1 is downregulated in tumor-infiltrating CAR-T cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eA\u003c/strong\u003e) SATB1 expression in resting and activated human CD4\u003csup\u003e+\u003c/sup\u003e and CD8\u003csup\u003e+\u003c/sup\u003e T cells was detected by flow cytometry. (\u003cstrong\u003eB\u003c/strong\u003e) Quantification of SATB1 MFI of T cells in (\u003cstrong\u003eA\u003c/strong\u003e). Data was shown as mean ± SD, \u003cem\u003en\u003c/em\u003e=3; ****, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001; Student’s \u003cem\u003et\u003c/em\u003e-test. (\u003cstrong\u003eC\u003c/strong\u003e) Western Blot analysis of SATB1 protein levels in resting and activated human T cells were analyzed by Western Blot. (\u003cstrong\u003eD\u003c/strong\u003e) SATB1 expression in resting and activated mouse T cells was measured by flow cytometry. Data was shown as mean ± SD, \u003cem\u003en\u003c/em\u003e=4; **, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; Student’s \u003cem\u003et\u003c/em\u003e-test. (\u003cstrong\u003eE\u003c/strong\u003e) Schematic of GPC3-CAR-T cell therapy for HCC CDX model. (\u003cstrong\u003eF\u003c/strong\u003e) PD-1 expression on the surface of CD4\u003csup\u003e+\u003c/sup\u003e and CD8\u003csup\u003e+\u003c/sup\u003e CAR-T cells isolated from the spleen and tumor was evaluated by flow cytometry. (\u003cstrong\u003eG\u003c/strong\u003e) Quantification of PD-1 expression in (\u003cstrong\u003eF\u003c/strong\u003e). \u003cem\u003en\u003c/em\u003e=4; **, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; Student’s \u003cem\u003et\u003c/em\u003e-test. (\u003cstrong\u003eH\u003c/strong\u003e) SATB1 expression in spleen- and tumor-derived CD4\u003csup\u003e+\u003c/sup\u003e and CD8\u003csup\u003e+\u003c/sup\u003e CAR-T cells was detected by flow cytometry. (\u003cstrong\u003eI\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003eSATB1 MFI quantification in (\u003cstrong\u003eH\u003c/strong\u003e). \u003cem\u003en\u003c/em\u003e=4; **, \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01; *, \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05; Student’s \u003cem\u003et\u003c/em\u003e-test.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-6776099/v1/43e3f0b82a7df8a27aa5cfb7.png"},{"id":84887511,"identity":"7135a49e-3e64-44ad-adb7-5ec56c1c19f5","added_by":"auto","created_at":"2025-06-18 11:59:01","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":320276,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSATB1 enhances the cytotoxicity of CAR-T cells \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vitro\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Human SATB1 mRNA levels in CAR-T and SATB1-CAR-T cells were quantified by real-time PCR. Data were shown as mean ± SD, \u003cem\u003en\u003c/em\u003e=3; *, \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05; Student’s \u003cem\u003et\u003c/em\u003e-test. (\u003cstrong\u003eB\u003c/strong\u003e) SATB1 protein expression in CAR-T and SATB1-CAR-T cells was assessed by Western blot, with β-actin as the loading control. (\u003cstrong\u003eC\u003c/strong\u003e) Relative SATB1 protein levels in (\u003cstrong\u003eB\u003c/strong\u003e) were quantified and shown as mean ± SD, \u003cem\u003en\u003c/em\u003e=3; *, \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05; Student’s \u003cem\u003et\u003c/em\u003e-test. (\u003cstrong\u003eD\u003c/strong\u003e) IL-2 and IFN-γ secretion by activated CAR-T cells co-cultured with GPC3\u003csup\u003e+\u003c/sup\u003e HCC tumor cells (1:1 E:T\u0026nbsp;ratio, 18 h) was detected. Data are shown as mean ± SD, \u003cem\u003en\u003c/em\u003e=3; **, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; *, \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05; Student’s \u003cem\u003et\u003c/em\u003e-test. (\u003cstrong\u003eE\u003c/strong\u003e) Cytotoxicity of Ctrl-T, SATB1-T, CAR-T, and SATB1-CAR-T cells against GFP/Luc\u003csup\u003e+\u003c/sup\u003e HCC cells was evaluated. Data are shown as mean ± SD, \u003cem\u003en\u003c/em\u003e=3; ***, \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001; *, \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05; Student’s \u003cem\u003et\u003c/em\u003e-test.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-6776099/v1/7e1320215cf935ece77032d0.png"},{"id":84887958,"identity":"d964e36d-9e08-443e-acb4-087e15d37d08","added_by":"auto","created_at":"2025-06-18 12:07:01","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":485558,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOverexpression of SATB1 affects CAR-T cell immunophenotypes \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vitro\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eA\u003c/strong\u003e) CFSE stained Ctrl-T and SATB1-T cells were cultured for 3 days, and proliferation was analyzed by flow cytometry. (\u003cstrong\u003eB\u003c/strong\u003e) SATB1 overexpression promoted CAR-T cell proliferation \u003cem\u003ein vitro\u003c/em\u003e by cell counting analysis. Data were shown as mean ± SD, \u003cem\u003en\u003c/em\u003e=3; *, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; Student’s \u003cem\u003et\u003c/em\u003e-test. (\u003cstrong\u003eC\u003c/strong\u003e) SATB1-CAR-T cells exhibited lower apoptosis levels compared to CAR-T cells after incubation with Huh7 tumor cells (1:1 E:T ratio, 3 days). (\u003cstrong\u003eD\u003c/strong\u003e) Quantification of Annexin V-positive T cells (shown in \u003cstrong\u003eC\u003c/strong\u003e). Data are shown as mean ± SD, \u003cem\u003en\u003c/em\u003e=5; ***, \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001; *, \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05; paired two-tailed \u003cem\u003et\u003c/em\u003e-test. (\u003cstrong\u003eE\u003c/strong\u003e) SATB1 Overexpression increased central memory T cells during \u003cem\u003ein vitro\u003c/em\u003e culture. (\u003cstrong\u003eF\u003c/strong\u003e) Percentages of CD4\u003csup\u003e+\u003c/sup\u003e and CD8\u003csup\u003e+\u003c/sup\u003e T cell subtypes from (\u003cstrong\u003eE\u003c/strong\u003e) were shown as mean ± SD, \u003cem\u003en\u003c/em\u003e=3; *, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; Student’s \u003cem\u003et\u003c/em\u003e-test. (\u003cstrong\u003eG\u003c/strong\u003e) CCR7 expression in CAR-T and SATB1-CAR-T cells was analyzed by flow cytometry. (\u003cstrong\u003eH\u003c/strong\u003e) Statistics of CCR7-positive T cells in (\u003cstrong\u003eG\u003c/strong\u003e). Data are shown as mean ± SD, \u003cem\u003en\u003c/em\u003e=3; *, \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05; Student’s \u003cem\u003et\u003c/em\u003e-test.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-6776099/v1/f2b4a6d737d9e40d4a7b3cd8.png"},{"id":84887514,"identity":"dfdec086-d6b2-44bc-8e70-5c431d4ac20b","added_by":"auto","created_at":"2025-06-18 11:59:01","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":750742,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOverexpression of SATB1 enhances the efficiency of CAR-T cells\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vivo\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Schematic of the human liver cancer CDX model. (\u003cstrong\u003eB\u003c/strong\u003e) Tumor luminescence signals after infusion of Ctrl-T, SATB1-T, CAR-T and SATB1-CAR-T cells in the CDX model. (\u003cstrong\u003eC\u003c/strong\u003e) Quantification of tumor bioluminescence signals at indicated time points from (\u003cstrong\u003eB\u003c/strong\u003e). Ctrl-T: \u003cem\u003en\u003c/em\u003e=4; SATB1-T: \u003cem\u003en\u003c/em\u003e=5; CAR-T: \u003cem\u003en\u003c/em\u003e=6; SATB1-CAR-T: \u003cem\u003en\u003c/em\u003e=7. (\u003cstrong\u003eD\u003c/strong\u003e) Statistics of tumor bioluminescence signals on day 7 after CAR-T and SATB1-CAR-T cells treatment. Data are shown as mean ± SD, CAR-T: \u003cem\u003en\u003c/em\u003e=6; SATB1-CAR-T: \u003cem\u003en\u003c/em\u003e=7; *, \u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05; Student’s \u003cem\u003et\u003c/em\u003e-test. (\u003cstrong\u003eE\u003c/strong\u003e) Survival of peripheral blood human T cells on day 7 after T cell treatment. Data are shown as mean ± SD, Ctrl-T: \u003cem\u003en\u003c/em\u003e=3; SATB1-T: \u003cem\u003en\u003c/em\u003e=3; CAR-T: \u003cem\u003en\u003c/em\u003e=5; SATB1-CAR-T: \u003cem\u003en\u003c/em\u003e=4; *, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; Student’s \u003cem\u003et\u003c/em\u003e-test. (\u003cstrong\u003eF\u003c/strong\u003e) Survival of tumor-infiltrated human T cells after T cell treatment. Data are shown as mean ± SD, CAR-T: \u003cem\u003en\u003c/em\u003e=4; SATB1-CAR-T: \u003cem\u003en\u003c/em\u003e=4; **, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; Student’s \u003cem\u003et\u003c/em\u003e-test. (\u003cstrong\u003eG\u003c/strong\u003e) SATB1-CAR-T cell treatment prolonged the survival of CDX model mice. Ctrl-T: \u003cem\u003en\u003c/em\u003e=4; SATB1-T: \u003cem\u003en\u003c/em\u003e=4; CAR-T: \u003cem\u003en\u003c/em\u003e=8; SATB1-CAR-T: \u003cem\u003en\u003c/em\u003e=8; *, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; log-rank test.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-6776099/v1/de7b1a11d8196ae3e48a5977.png"},{"id":101032616,"identity":"577883d7-4b63-4c93-9690-0123c7f31d2d","added_by":"auto","created_at":"2026-01-24 08:11:02","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2891423,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6776099/v1/d4db1231-0e30-48fd-ae2b-2140aacde3e4.pdf"},{"id":84886722,"identity":"ee4ec3d7-227e-43d5-93ee-6832c0ebaf58","added_by":"auto","created_at":"2025-06-18 11:51:01","extension":"pptx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":4787241,"visible":true,"origin":"","legend":"Original Data Files","description":"","filename":"OriginalDataFiles.pptx","url":"https://assets-eu.researchsquare.com/files/rs-6776099/v1/ae0a0a4ae5b3b4b2c4241c1b.pptx"},{"id":84886716,"identity":"5b13330f-fccf-4006-93f6-54b8fa284cf8","added_by":"auto","created_at":"2025-06-18 11:51:01","extension":"zip","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1802694,"visible":true,"origin":"","legend":"Dataset 1","description":"","filename":"orignaldata.zip","url":"https://assets-eu.researchsquare.com/files/rs-6776099/v1/c4496a8c2ad32ff09c00dddb.zip"},{"id":84886712,"identity":"a35ac7c0-a2b6-4c0b-bdc2-f8b2e490c7c3","added_by":"auto","created_at":"2025-06-18 11:51:01","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":16398,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigurelegends.docx","url":"https://assets-eu.researchsquare.com/files/rs-6776099/v1/d8d09eda29cafd33cf0bbc17.docx"},{"id":84886720,"identity":"6fed3834-ee27-4c58-8f51-1ef514f1661e","added_by":"auto","created_at":"2025-06-18 11:51:01","extension":"png","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":1568511,"visible":true,"origin":"","legend":"Supplementary Figure S1","description":"","filename":"SupplementaryFigureS1.png","url":"https://assets-eu.researchsquare.com/files/rs-6776099/v1/b3e3a3d5e6d1f3fb15ccc37c.png"},{"id":84886718,"identity":"86998015-c0dd-432b-9d8e-b5153f633548","added_by":"auto","created_at":"2025-06-18 11:51:01","extension":"png","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":1192600,"visible":true,"origin":"","legend":"Supplementary Figure S2","description":"","filename":"SupplementaryFigureS2.png","url":"https://assets-eu.researchsquare.com/files/rs-6776099/v1/8c9b3cec2f158f29087b74aa.png"},{"id":84886731,"identity":"e5f24643-3dad-4e64-a449-59b58445851a","added_by":"auto","created_at":"2025-06-18 11:51:01","extension":"png","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":138727,"visible":true,"origin":"","legend":"Supplementary Figure S3","description":"","filename":"SupplementaryFigureS3.png","url":"https://assets-eu.researchsquare.com/files/rs-6776099/v1/67dc0e933df3f9670a6025d0.png"},{"id":84887526,"identity":"dc15fbe0-0682-473a-a677-37ae6c633ab6","added_by":"auto","created_at":"2025-06-18 11:59:02","extension":"png","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":366629,"visible":true,"origin":"","legend":"Supplementary Figure S4","description":"","filename":"SupplementaryFigureS4.png","url":"https://assets-eu.researchsquare.com/files/rs-6776099/v1/3983d5483989a22b211a19e8.png"},{"id":84887516,"identity":"c0a45240-a034-4a75-bb69-78fa64a3511f","added_by":"auto","created_at":"2025-06-18 11:59:01","extension":"png","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":402371,"visible":true,"origin":"","legend":"Supplementary Figure S5","description":"","filename":"SupplementaryFigureS5.png","url":"https://assets-eu.researchsquare.com/files/rs-6776099/v1/d34f1546e076728909a12a93.png"},{"id":84886723,"identity":"b02efb4a-87ba-4fbd-8cc2-9ae77c771851","added_by":"auto","created_at":"2025-06-18 11:51:01","extension":"png","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":276369,"visible":true,"origin":"","legend":"Supplementary Figure S6","description":"","filename":"SupplementaryFigureS6.png","url":"https://assets-eu.researchsquare.com/files/rs-6776099/v1/de0cbb4a18f3c3b39117f420.png"}],"financialInterests":"(Not answered)","formattedTitle":"Enhanced Anti-Liver Tumor Efficacy of Chimeric Antigen Receptor-T Cells via SATB1 Modulation","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eAdoptive cellular immunotherapy (ACT) based on functional immune cell transfer holds great promise in treating various malignant diseases, especially cancer(1, 2). Among ACT strategies, chimeric antigen receptor (CAR-T) cell therapy has emerged as an effective clinical strategy for the treatment of several hematopoietic malignancies(3\u0026ndash;6). However, its efficacy in solid tumors, including hepatocellular carcinoma (HCC), remains limited due to antigenic heterogeneity, suboptimal infiltration, and immunosuppressive tumor microenvironment (TME)-driven T cell exhaustion(7, 8). Overcoming these challenges is critical for improving the antitumor efficacy of CAR-T cell therapy for solid tumors.\u003c/p\u003e \u003cp\u003eT cell exhaustion is a state characterized by diminished proliferation, impaired effector function, transcriptional and epigenetic alterations, and upregulation of inhibitory receptors such as Programmed Death-1 (PD-1), Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4), and Lymphocyte-Activation Gene 3 (LAG-3)(9\u0026ndash;13). In CAR-T cells, exhaustion is primarily driven by persistent antigenic stimulation and sustained autoactivation due to CAR structure aggregation(12, 14). Recent efforts to combat T cell exhaustion have focused on modulating transcriptional regulators such as c-Jun(15), NR4A family members(16, 17), BATF (from the AP-1 family)(18, 19), FOXO1(20, 21), Id2(22) and Stat5(23). Genetic modulation of these factors has been shown to improve CD8\u003csup\u003e+\u003c/sup\u003e T cell and CAR-T cell function by mitigating exhaustion, enhancing stem-like properties or metabolic adaptability, ultimately leading to tumor regression and extending survival in preclinical models. Moreover, the epigenetic reprogramming underlying T cell exhaustion remains poorly understood, hindering the development of durable solutions.\u003c/p\u003e \u003cp\u003eSpecial AT-Rich Sequence Binding Protein 1 (SATB1) is a critical genome organizer that reprograms chromatin structure and broadly regulates transcriptional profiles to promote tumor cell proliferation(24) and metastasis(25, 26). Beyond its oncogenic functions, predominantly expressed in thymocytes, SATB1 is indispensable for thymocyte development(27, 28) and T cell differentiation(29\u0026ndash;31). The absence of SATB1 disrupts thymocyte development, particularly during the double-positive stage(32). Recent studies have highlighted SATB1's role in the anti-tumor function of cytotoxic T lymphocytes (CTL) through its recruitment of the nucleosome remodeling deacetylase (NuRD) complex at genomic regions, thereby regulating PD-1 expression and suggesting its potential role in mitigating T cell exhaustion(33). Additionally, within TME, TGF-β-mediated SATB1 silencing promotes follicular helper T (Tfh) cell differentiation and tertiary lymphoid structures (TLS) formation, further underscoring its multifaceted role in immune regulation and tumor immunity(34).\u003c/p\u003e \u003cp\u003eIn this study, we identified SATB1 as a key regulator of T cell exhaustion in Glypican-3 (GPC3)-targeting CAR-T cells within hepatocellular carcinoma (HCC) xenograft models. We observed significant downregulation of SATB1 and concomitant upregulation of PD-1 in tumor-infiltrating exhausted CAR-T cells. We hypothesized that SATB1 overexpression could reprogram CAR-T cell epigenetics to resist exhaustion and enhance anti-tumor efficacy in HCC. Overexpression of SATB1 enhanced the immunophenotypic characteristics, improved effector function, and reduced exhaustion levels in CAR-T cells \u003cem\u003ein vitro\u003c/em\u003e. Subsequent studies demonstrated that SATB1-overexpressing CAR-T cells exhibited improved resistance to exhaustion and superior immunotherapeutic efficacy \u003cem\u003ein vivo\u003c/em\u003e, leading to accelerated tumor eradication and prolonged survival of tumor-bearing mice. These findings suggest that SATB1 modulation represents a promising strategy to enhance the efficacy of CAR-T cells in treating solid tumors.\u003c/p\u003e"},{"header":"2. Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. \u003cem\u003eSATB1\u003c/em\u003e is downregulated in tumor-infiltrating exhausted T cells.\u003c/h2\u003e \u003cp\u003eExhausted T cells in TME are characterized by the upregulation of immune checkpoint molecules like PD-1, reduced proliferative capacity, and impaired effector functions(10, 13, 14). To better elucidate the transcriptional profiles of tumor-infiltrating exhausted T cells, we analyzed transcriptional profiles across models. In a B78ChOVA melanoma mouse model (GSE201071), \u003cem\u003eSatb1\u003c/em\u003e mRNA expression was preferentially downregulated in tumor-infiltrated exhausted OT-1 T cells at days 4 and 14 compared to na\u0026iuml;ve and effector T cells (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA)(35). This finding was corroborated in chronically activated T cells from an LCMV Arm5 infection model (GSE88987), where SATB1 expression decreased in exhausted populations (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eB)(16).\u003c/p\u003e \u003cp\u003eTo assess clinical relevance, we analyzed HCC patient data (GSE111389) and observed consistent SATB1 downregulation in PD-1 high (PD-1 hi) tumor-infiltrating CD8\u003csup\u003e+\u003c/sup\u003e T cells across all six patients (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eC)(36). Further validation using the Pan-Cancer Human T Cell Atlas (scRNA-seq data) revealed high SATB1 expression in na\u0026iuml;ve and memory T cells, but marked reduction in exhausted subsets (Figures \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eD and S1E)(37). Collectively, these results implicate SATB1 downregulation as a conserved feature of T cell exhaustion. Engineering CAR-T cells to overexpress SATB1 may represent a promising strategy to mitigate exhaustion and enhance the efficacy of CAR-T cell therapy against solid tumors.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. The potent antitumor activity of GPC3-Targeted CAR-T Cells in Hepatocellular Carcinoma.\u003c/h2\u003e \u003cp\u003eTo investigate the role of SATB1 in exhausted CAR-T cells, we developed a GPC3-targeted CAR-T model. GPC3, a membrane-bound heparan sulfate proteoglycan highly expressed in 70% of HCC patients but absent in normal adult tissues(38), is under clinical evaluation in 11 of 22 ongoing HCC CAR-T trials(39\u0026ndash;41). Our analysis confirmed high GPC3 expression in human HCC cell lines Huh7, HepG2, and Hep3B but not in SK-HEP-1 (Figure S2A).\u003c/p\u003e \u003cp\u003eSubsequently, second-generation GPC3-CAR-T cells were engineered using an EF1α-promoter lentiviral vector (Figure S2B)(41). Primary human T cells, stimulated with anti-CD3/anti-CD28 Dynabeads and IL-2 achieved 60% transduction efficiency (Figure S2C). These CAR-T cells specifically secreted IFN-γ/IL-2 (Figure S2D) and lysed GPC3\u003csup\u003e+\u003c/sup\u003e HCC lines (Huh7, HepG2, Hep3B), but not GPC3\u003csup\u003e\u0026minus;\u003c/sup\u003e SK-HEP-1 cells (Figures S2E and S2F), demonstrating their target-dependent specificity and efficacy.\u003c/p\u003e \u003cp\u003eTo further evaluate the anti-tumor effects of GPC3-CAR-T cells \u003cem\u003ein vivo\u003c/em\u003e, we established a cell line-derived xenograft (CDX) model by subcutaneously injecting GFP/Luc\u003csup\u003e+\u003c/sup\u003e Huh7 into immunodeficient NCG mice. Following tumor engraftment, mice were intravenously infused with either Ctrl-T or CAR-T cells, and the tumor size was measured at the indicated time points (Figure S2G). CAR-T cell treatment induced significant tumor regression and improved survival of tumor-bearing mice compared to Ctrl-T cells (Figures S2H-S2J). However, incomplete regression in some tumors highlighted the need to enhance therapeutic potency\u0026mdash;a goal addressed by SATB1 engineering in subsequent studies.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. SATB1 is downregulated in tumor-infiltrating exhausted CAR-T cells.\u003c/h2\u003e \u003cp\u003eWe next asked whether SATB1 downregulation occurs in CAR-T cells within tumors. We analyzed SATB1 expression in human T cells under resting or anti-CD3/CD28-activated conditions. Consistent with prior studies, SATB1 was highly expressed in activated human CD4\u003csup\u003e+\u003c/sup\u003e and CD8\u003csup\u003e+\u003c/sup\u003e T cells (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA-\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC)(33). Similarly, anti-CD3/CD28 stimulation significantly increased SATB1 levels in mouse splenic CD3\u003csup\u003e+\u003c/sup\u003e T cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe transferred GPC3-CAR-T cells into GFP/Luc\u003csup\u003e+\u003c/sup\u003e Huh7 xenograft-bearing mice to detect SATB1 expression in CAR-T cells within tumors (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). Two weeks post-infusion, tumor-infiltrating CAR-T cells exhibited elevated PD-1 expression (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG), resembling endogenous exhausted CD8\u003csup\u003e+\u003c/sup\u003e T cells. Strikingly, SATB1 levels were significantly reduced in these cells compared to splenic CAR-T counterparts, corroborating the RNA-seq data (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI). These findings suggest that SATB1 downregulation is associated with CAR-T cell exhaustion in the TME, highlighting its potential as a target to enhance functionality.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. SATB1 efficiently enhances CAR-T cell functions \u003cem\u003ein vitro.\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eConsidering the downregulation of SATB1 in tumor-infiltrated exhausted CAR-T cells, we investigated whether SATB1 overexpression could enhance CAR-T cell functionality. We co-transduced human T cells with either an empty vector or a SATB1 overexpression vector alongside the CAR construct. Successful SATB1 overexpression in SATB1-CAR-T cells was confirmed at both the mRNA (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA) and protein levels (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo evaluate the functional impact of SATB1 overexpression, Ctrl-T, SATB1-overexpressing T (SATB1-T), CAR-T, or SATB1-CAR-T cells were co-cultured with HCC cell lines (SK-HEP-1, Huh-7, HepG2, and Hep3B). SATB1-CAR-T cells exhibited enhanced cytokine release compared to conventional CAR-T cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). Consistently, SATB1-CAR-T cells exhibited increased killing efficiency against GFP/Luc\u003csup\u003e+\u003c/sup\u003e Huh-7, HepG2, and Hep3B cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). These results indicate that SATB1 could enhance the anti-tumor efficacy of CAR-T cells \u003cem\u003ein vitro\u003c/em\u003e, highlighting its potential as a strategy to improve the antitumor efficacy of CAR-T cells.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. SATB1 modulates the immunophenotypes of CAR-T cells \u003cem\u003ein vitro\u003c/em\u003e.\u003c/h2\u003e \u003cp\u003eGiven the enhanced functionality of SATB1-CAR-T cells, we investigated the influence of SATB1 overexpression on T cell biological properties and immunophenotypic profiles. SATB1-T and SATB1-CAR-T cells exhibited significantly enhanced proliferation over a two-week culture period compared to controls (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Moreover, SATB1-CAR-T cells exhibited reduced apoptosis levels upon co-culture with GPC3\u003csup\u003e+\u003c/sup\u003e Huh7 cells, indicating improved resistance to tumor-induced stress (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo explore the underlying phenotypic changes, we analyzed T cell differentiation markers CD45RA and CD62L, which classify primary human T cells into four kinds of differentiation subsets: CD45RA\u003csup\u003e+\u003c/sup\u003e CD62L\u003csup\u003e+\u003c/sup\u003e na\u0026iuml;ve T cells (T\u003csub\u003eN\u003c/sub\u003e), CD45RA\u003csup\u003e\u0026minus;\u003c/sup\u003e CD62L\u003csup\u003e+\u003c/sup\u003e central memory T cells (T\u003csub\u003eCM\u003c/sub\u003e), CD45RA\u003csup\u003e\u0026minus;\u003c/sup\u003e CD62L\u003csup\u003e\u0026minus;\u003c/sup\u003e effector memory T cells (T\u003csub\u003eEM\u003c/sub\u003e), and CD45RA\u003csup\u003e+\u003c/sup\u003e CD62L\u003csup\u003e\u0026minus;\u003c/sup\u003e effector memory T cells (T\u003csub\u003eRAEM\u003c/sub\u003e)(42, 43). Strikingly, SATB1 overexpression drove both CD4\u003csup\u003e+\u003c/sup\u003e and CD8\u003csup\u003e+\u003c/sup\u003e T cells toward a central memory T cell phenotype (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). This memory reprogramming was further supported by elevated CCR7 expression, a key mediator of T cell homeostasis, lymphoid homing and sustained anti-tumor responses (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH).\u003c/p\u003e \u003cp\u003eNotably, SATB1 overexpression had no effect on CD4\u003csup\u003e+\u003c/sup\u003e or CD8\u003csup\u003e+\u003c/sup\u003e T cell subset distribution (Figure S3A) and did not alter regulatory T cell (T\u003csub\u003ereg\u003c/sub\u003e) proportions (Figure S3B), suggesting selective modulation of effector/memory subsets without perturbing other immune cells.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. SATB1 attenuates TGF-β-induced Immunosuppression in T Cells.\u003c/h2\u003e \u003cp\u003eSATB1 has been shown to recruit the nucleosome remodeling deacetylase (NuRD) complex to \u003cem\u003ePdcd1\u003c/em\u003e regulatory regions, and the loss of \u003cem\u003eSatb1\u003c/em\u003e increases PD-1 expression upon T cell activation(33). Consistent with these mechanism, SATB1-T cells exhibited significantly reduced PD-1 expression compared to controls (Figures S4A and S4B), while other exhaustion markers (CTLA-4, TIM3, LAG-3) remained unchanged (Figure S4C).\u003c/p\u003e \u003cp\u003eTransforming growth factor-beta (TGF-β), mainly secreted by immunosuppressive cells and tumor cells, limits CAR-T cell efficacy in solid tumors by suppressing T cell activation, proliferation, migration, and differentiation(44\u0026ndash;46). Therapeutic strategies targeting TGF-β signaling have shown considerable promise in preclinical and clinical studies(47\u0026ndash;49). In line with previous research, TGF-β1 treatment downregulated SATB1 expression and increased PD-1 levels in control T cells (Figure S4D-S4G). Strikingly, SATB1-T cells resisted this regulation, maintaining significantly higher SATB1 and lower PD-1 expression compared to Ctrl-T cells under TGF-β1 exposure (Figure S4D-S4G). These findings suggest that SATB1 overexpression may counteract TGF-β-driven exhaustion, potentially preserving T cell functionality in immunosuppressive microenvironments.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7. SATB1 ameliorates CAR-T cell exhaustion \u003cem\u003ein vitro\u003c/em\u003e.\u003c/h2\u003e \u003cp\u003eTo systematically evaluate CAR-T cell exhaustion \u003cem\u003ein vitro\u003c/em\u003e, we established a repetitive co-culture model with Huh7 tumor cells (Figure S5A). After three rounds of co-incubation, conventional CAR-T cells showed significant upregulation of exhaustion-associated markers, including PD-1, CTLA-4, TIM3, and LAG-3, confirming successful exhaustion induction (Figure S5B-S5E).\u003c/p\u003e \u003cp\u003eConsistent with the suppressive effect of SATB1 on PD-1 under TGF-β exposure (Figure S4), SATB1 overexpression markedly reduced PD-1 expression in both CD4\u003csup\u003e+\u003c/sup\u003e and CD8\u003csup\u003e+\u003c/sup\u003e CAR-T cells post-co-culture (Figure S5F). Furthermore, SATB1 overexpression SATB1-CAR-T cells also showed attenuated upregulation of CTLA-4, TIM3, and LAG-3 (Figures S5G-S5I). These data collectively suggest that SATB1 overexpression could ameliorate CAR-T cell exhaustion \u003cem\u003ein vitro\u003c/em\u003e, potentially preserving their anti-tumor functionality through epigenetic regulation of immune checkpoint molecules.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8. SATB1 enhances immunotherapeutic efficacy of CAR-T cells \u003cem\u003ein vivo\u003c/em\u003e.\u003c/h2\u003e \u003cp\u003eTo assess the in vivo efficacy of SATB1-CAR-T cells, we established a human liver cancer cell line-derived xenograft (CDX) model (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Mice receiving SATB1-CAR-T cells showed superior tumor regression compared to those receiving conventional CAR-T cells (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Bioluminescence imaging on day 7 confirmed noticeably reduced tumor burden in SATB1-CAR-T-treated mice (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB-\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFlow cytometry analysis revealed nearly 2-fold higher infiltration of human CD3\u003csup\u003e+\u003c/sup\u003e T cells in both peripheral blood and tumor tissue of SATB1-CAR-T-treated mice (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF). Notably, tumor-infiltrating CD8\u003csup\u003e+\u003c/sup\u003e T cells from SATB1-CAR-T group exhibited 50% lower PD-1, CTLA-4, TIM3 and LAG-3 expression compared to controls (Figure S6), aligning with their exhaustion-resistant phenotype \u003cem\u003ein vitro\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eImportantly, SATB1-CAR-T cell therapy significantly extended the survival of tumor-bearing mice, with over 50% surviving beyond 100 days post-infusion (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG). Collectively, these findings highlight that SATB1 overexpression enhances CAR-T cell persistence and functionality in solid tumors.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Discussion","content":"\u003cp\u003eThe clinical application of CAR-T cells has rapidly expanded, with 23% of 517 registered clinical trials in China focusing on solid tumors, particularly HCC(4, 8, 39, 50). Bioinformatics analyses have revealed a TME enriched with exhausted CD8\u003csup\u003e+\u003c/sup\u003e T cells and regulatory CD4\u003csup\u003e+\u003c/sup\u003e T\u003csub\u003eregs\u003c/sub\u003e in HCC, emphasizing the need for engineering exhaustion-resistant CAR-T cells to improve clinical efficacy(37, 51). Comparative analysis of the unique transcriptional programs, epigenetic programs, and metabolic properties of exhausted T cells has identified several T cell exhaustion-related proteins and transcription factors such as CD38(52), Rgs1(53, 54), and Tigit(55). While previous studies have focused on highly expressed genes in exhausted T cells, we identified special AT-rich binding protein SATB1, an epigenetic remodeling factor downregulated in exhausted T cells, as a crucial regulator of CAR-T cell exhaustion. Our study demonstrates that SATB1 overexpression mitigates CAR-T cell exhaustion and improves anti-tumor efficacy, potentially not only through mechanisms involving broadly suppression of inhibitory receptors (PD-1, CTLA-4, TIM3 and LAG-3) (Figure S5, S6) but also promotion of memory-like phenotypes (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE-\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH) and enhanced resistance to TGF-β-mediated immunosuppression (Figure S4) in the HCC microenvironment.\u003c/p\u003e \u003cp\u003eTo further explore T cell exhaustion mechanisms, we developed an \u003cem\u003eex vivo\u003c/em\u003e co-culture system for chronic antigen stimulation and rapid T cell exhaustion induction (Figure S5A)(10, 12, 56, 57). In our co-culture system, CAR-T cells exhibited significant upregulation of inhibitory receptors (Figures S5B-S5E). This system, which can be optimized with TGF-β or immunosuppressive cells, provides a platform for screening genes and drugs that enhance CAR-T cell function.\u003c/p\u003e \u003cp\u003eSATB1, initially detected at high levels in thymocytes, progenitor cells (such as osteoblasts), and the epidermal basal layer, plays critical roles in embryogenesis, neurogenesis and malignancies(58). It critically regulates hematopoietic development, thymus maturation, and T cell differentiation(27\u0026ndash;31). In mature T cells, SATB1 is dynamically regulated by T cell receptor (TCR) signaling and maintains the distinct naive chromatin state within naive CD8\u003csup\u003e+\u003c/sup\u003e T cells, also regulates chemokine genes through enhancer-promoter interactions(28, 59\u0026ndash;61). SATB1 also contributes to T cell 3D genome homeostasis and immune tolerance(31, 62, 63). Recent studies identified SATB1 as an epigenetic negative regulator of PD-1, mitigating T cell exhaustion(33). Align with its reported role in epigenetic regulation, SATB1 modulation broadly enhance CAR-T fitness, addressing both exhaustion and persistence\u0026mdash;a dual challenge that single-pathway interventions may incompletely resolve. Future studies are required to directly link SATB1 overexpression to chromatin remodeling in CAR-T cells. Our study further demonstrates that SATB1 overexpression enhances CAR-T cell functionality without altering T\u003csub\u003ereg\u003c/sub\u003e proportions (Figure S3B), consistent with its role in maintaining T cell homeostasis(64). Compared to previous work, the expansion of the T\u003csub\u003eCM\u003c/sub\u003e subset in SATB1-CAR-T cells supports the superior efficacy of T\u003csub\u003eCM\u003c/sub\u003e in CAR-T therapy, characterized by robust proliferation capacity, prolonged persistence, and reduced exhaustion(19). Since SATB1 deletion causes mitochondrial function impairment and oxidative stress in CD4\u003csup\u003e+\u003c/sup\u003e T cells, its overexpression may preserve mitochondrial integrity in TME, thereby sustaining CAR-T cell functionality(65). Furthermore, the precise mechanisms by which SATB1 modulates CAR-T-cell immunophenotypes and the potential of SATB1 to enhance CAR-T cell function in other solid tumors warrants further investigation.\u003c/p\u003e \u003cp\u003eIn conclusion, our study not only identifies SATB1 as a potential therapeutic target to alleviate CAR-T cell exhaustion, but also provides a novel strategy to enhance CAR-T cell efficacy in solid tumors, particularly HCC. While SATB1 overexpression shows promise, potential side effects such as metabolic stress, autoimmune reactions, and cytokine release syndrome (CRS) require further evaluation. These risks could be mitigated through inducible expression systems and close monitoring of cytokine levels. The development of potent small-molecule drugs targeting SATB1 and integration with immune checkpoint inhibitors or multi-targeted CAR-T cells may further improve therapeutic outcomes.\u003c/p\u003e"},{"header":"4. Materials and methods","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e4.1. Cell lines and culture conditions\u003c/h2\u003e \u003cp\u003eHepatocellular carcinoma (HCC) cell lines Huh7, HepG2 (HB-8065, ATCC), Hep3B (BNCC360312, BNCC), SK-HEP-1 (HTB-52, ATCC) and Lentiviral producer cell line 293T (CRL-11268, ATCC) were cultured in Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium (DMEM) (Gibco) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Vistech), 2 mM GlutaMAX\u003csup\u003eTM\u003c/sup\u003e-I (Gibco), 1 mM sodium pyruvate (Gibco), 0.1 mM nonessential amino acids (Gibco), 100 \u0026micro;g/mL streptomycin and 100 U/mL penicillin (Gibco).\u003c/p\u003e \u003cp\u003eFor luciferase-based experiments, the lentivirus of green fluorescent protein and firefly luciferase fusion protein (GFP/Luc) was transduced into tumor cells to produce stable cell lines Huh7-GFP/Luc, HepG2-GFP/Luc, Hep3B-GFP/Luc and SK-HEP-1-GFP/Luc.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e4.2. Animals\u003c/h2\u003e \u003cp\u003eThe use of animals for this study was approved by the Institutional Animal Care and Use Committee (IOZ2016004). NCG (NOD/ShiLtJGpt-\u003cem\u003ePrkdc\u003c/em\u003e \u003csup\u003eem26Cd52\u003c/sup\u003e\u003cem\u003eIl2rg\u003c/em\u003e \u003csup\u003eem26Cd22\u003c/sup\u003e/Gpt) mice aged 6\u0026ndash;12 weeks (Gempharmatech Co., Ltd) were used for human tumor cell line CDX models.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e4.3. Generation of CAR constructs\u003c/h2\u003e \u003cp\u003eTo target GPC3, the chimeric antigen receptor (CAR) was designed based on the single-chain variable fragment (scFv) derived from GC33 antibody, which was linked to the CD8 hinge and transmembrane domain, followed by the intracellular domains of 4-1BB and the signaling moiety of the CD3ζ chain. The CAR sequence was synthesized at Sangon Technology (Shanghai, China) and cloned into the pFUW-EF1α-P2A-eGFP lentiviral vector. CAR-T cells were identified by eGFP expression.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e4.4. Lentivirus preparation\u003c/h2\u003e \u003cp\u003eThe human SATB1 full sequence was synthesized at GenScript Technology (Shanghai, China) and cloned into the pFUW-EF1α-P2A-mCherry lentiviral backbone to generate gene expression plasmid pFUW-EF1α-SATB1-P2A-mCherry. For CAR and gene vector, the pFUW vectors harboring P2A-eGFP and P2A-mCherry sequences were respectively used as a negative control. We obtained pLenti-CMV-eGFP-linker-Luc-PGK-Puro lentiviral vector (GFP/Luc) from OBiO Technology (Shanghai, China).\u003c/p\u003e \u003cp\u003eWe obtained Lentiviral particles from 293T-packaging cells by calcium phosphate transfection. The CAR or gene vector plasmid, pMD2.G plasmid and psPAX2 plasmid were transfected into 293T-packaging cells and incubated at 37\u0026deg;C for 12 hours. Then 293T-packaging cells were cultured in fresh medium for 48 hours. The supernatants were collected and filtered through 0.45 \u0026micro;m filter. Lentiviral particles were concentrated by ultracentrifugation at 20 000 rpm for 2 hours at 4\u0026deg;C. The viral granules were resuspended in X-VIVO\u0026trade; 15 medium and filtered through 0.22 \u0026micro;m filter and stored at -80\u0026deg;C.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e4.5. CAR-T cell generation and cell culture\u003c/h2\u003e \u003cp\u003eFor CAR-T cell generation, human peripheral blood mononuclear cells (PBMCs) were obtained from the Biobank of Peking Union Medical College Hospital through Ficoll-Paque PLUS gradient centrifugation (17-1440-02, GE Healthcare). Primary human T cells were negatively selected from PBMCs with a Pan T Cell Isolation Kit (130-096-535, Miltenyi) and were stimulated CD3/CD28 Dynabeads (11161D, ThermoFisher) at the ratio of 1:1. The T cell culture medium was X-VIVO\u0026trade; 15 medium (04-418Q, Lonza) supplemented with 5% heat-inactivated FBS (Vistech), 1 mM sodium pyruvate (Gibco), 2 mM GlutaMAX\u003csup\u003eTM\u003c/sup\u003e-I (Gibco) and 100 IU/mL recombinant human IL-2 (200-02, PeproTech). T cells were transduced with control vector, GPC3-CAR and/or SATB1 overexpression lentiviral particles after 24 hours activation and were cultured at a concentration of 10\u003csup\u003e6\u003c/sup\u003e cells/mL in 6-well plates for 2\u0026ndash;3 weeks.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e4.6. Quantitative real-time PCR\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted from sorted mCherry\u003csup\u003e+\u003c/sup\u003e Ctrl-T cells and SATB-T cells with an RNAeasy Mini Kit (Qiagen). Total RNA (1 \u0026micro;g) was reverse transcribed into cDNA using a StarScript III All-in-one RT Mix with gDNA Remover (A230-10, GenStar). Quantitative real-time PCR was performed with GoTaq\u0026reg; qPCR Master Mix (Promega) and a QuantStudio\u0026trade; 6 Flex Real-Time PCR System. All samples were analyzed in duplicate and normalized to \u003cem\u003eGAPDH\u003c/em\u003e. The following primers were used: \u003cem\u003eGAPDH\u003c/em\u003e-F 5\u0026rsquo;-GGAGCGAGATCCCTCCAAAAT-3\u0026rsquo;, \u003cem\u003eGAPDH\u003c/em\u003e-R 5\u0026rsquo;-GGCTGTTGTCATACTTCTCATGG-3\u0026rsquo;, \u003cem\u003eSATB1\u003c/em\u003e-F 5\u0026rsquo;-GATCATTTGAACGAGGCAACTCA-3\u0026rsquo;, \u003cem\u003eSATB1\u003c/em\u003e-R 5\u0026rsquo;-TGGACCCTTCGGATCACTCA-3. The threshold cycle was determined and the relative gene expression ratio was calculated as follows, fold-change\u0026thinsp;=\u0026thinsp;2\u003csup\u003e\u0026minus;ΔΔCt\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e4.7. Western blotting and antibodies\u003c/h2\u003e \u003cp\u003eWhole-cell lysates of T cells were generated by lysing on ice in RIPA buffer for 30 min containing a protease inhibitor cocktail (04693116001, Roche) and 1 mM PMSF (ST506, Beyotime). Equivalent protein quantities (15 \u0026micro;g) of total protein were run in 10% SDS\u0026ndash;PAGE gel (Bio-Rad) and transferred to nitrocellulose membranes (Millipore). The membranes were then blocked with 5% non-fat milk in 1\u0026times; TBST (T1082, Solarbio) and probed with a primary antibody directed against SATB1 (1:1 000, ab109122, Abcam) and β-Actin (1:2500, A5441, Sigma-Aldrich) overnight at 4\u0026deg;C. After incubation with appropriate HRP-conjugated secondary antibody (Beyotime), signals from bound antibodies were detected with a Luminata Forte Western HRP Substrate Kit (WBLUF0100, Millipore) and quantified using Image J Software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e4.8. Flow cytometry analysis and antibodies\u003c/h2\u003e \u003cp\u003eFlow cytometry was used to detect the expression of cell surface markers. The CAR expression was detected by eGFP, and the SATB1 expression was detected by mCherry. CAR-T cells were stained with Human TruStain FcX\u0026trade; Antibody (422301), Pacific Blue\u0026trade; anti-human CD45 (368539), APC anti-human CD3 (300411), Brilliant Violet 421\u0026trade; anti-human CD4 (357423), APC/Cyanine7 anti-human CD8a (301016), APC anti-human CD45RA (304111), PE anti-human CD62L (304805), Alexa Fluor\u0026reg; 647 anti-human CD279 (PD-1) (329910), APC anti-human CD152 (CTLA-4) (369611), APC anti-human CD366 (TIM3) (364803), Brilliant Violet 421\u0026trade; anti-human CD223 (LAG-3) (369313) and Alexa Fluor\u0026reg; 647 anti-human FOXP3 (320113) antibodies from Biolegend; and Recombinant APC Anti-Glypican-3 (ab275695), Recombinant Anti-SATB1 (ab109122) antibodies from Abcam; and Goat anti-Rabbit IgG (H\u0026thinsp;+\u0026thinsp;L) Cross-Adsorbed Secondary Antibody, Alexa Fluor\u0026trade; 555 (A-21428) antibody from Invitrogen.\u003c/p\u003e \u003cp\u003eFor surface markers detection, T cells were collected, washed with PBS. 1\u0026times;10\u003csup\u003e6\u003c/sup\u003e T cells were suspended in 100 \u0026micro;L PBS with 1 \u0026micro;L TruStain FcX\u0026trade; Antibody for 20 minutes at 4\u0026deg;C, then incubated with specific surface antibodies for 30 minutes at 4\u0026deg;C. For intracellular staining, T cells were stained with surface markers first, then were stained with intracellular antibodies by Foxp3/Transcription Factor Staining Buffer Set (00-5523-00, eBioscience) on the basis of the manufacturer\u0026rsquo;s instructions. The experiment data were acquired using BD FACS AriaIII and were then analyzed using FlowJo software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e4.9. Apoptosis and proliferation assays\u003c/h2\u003e \u003cp\u003eFor apoptosis level detection of T cells, we used Annexin V-APC (40310ES20, Yeasen,) to stain T cells based on the manufacturer\u0026rsquo;s instructions after T cell membrane protein staining. To test the proliferation level of T cells, the fluorescent dye CFDA SE was used according to the manufacturer\u0026rsquo;s instructions. For the cell number counting experiment, T cells (5\u0026times;10\u003csup\u003e4\u003c/sup\u003e) were seeded in a 48-well plate on day 0 and were passaged at a concentration of 10\u003csup\u003e6\u003c/sup\u003e cells/mL, counted and recorded every two days.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e4.10. Cytotoxicity assay\u003c/h2\u003e \u003cp\u003eWe evaluated the cytotoxicity of CAR-T cells by co-cultured with luciferase labeled tumor cells \u003cem\u003ein vitro\u003c/em\u003e. One day before the experiment, the anti-CD3/CD28 magnetic beads were removed. The target tumor cells (Huh7-GFP/Luc, HepG2-GFP/Luc, Hep3B-GFP/Luc and SK-HEP-1-GFP/Luc) were seeded in white 96-well plates as a density of 5 000 cells/50 \u0026micro;L each well. At a ratio of 9:1, 3:1, 1:1 or 1:3, the indicated T cells were seeded with target tumor cells for 18 hours. Target tumor cells alone were determined as the maximal luciferase activity. Then D-luciferin, sodium salt (40901ES03, Yeasen) was prepared and added to each well according to the manufacturer\u0026rsquo;s instructions. The luminescence signal of each well was measured by a PerkinElmer Victor X3 Reader. The specific lysis rate was calculated using the formula: [(Maximal luciferase activity \u0026ndash; Experimental Luciferase Activity)/Maximal luciferase activity] \u0026times; 100.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003e4.11. Enzyme-linked immunosorbent assay (ELISA) assay\u003c/h2\u003e \u003cp\u003eTo analyze the cytokine secretion by CAR-T cells, one day before the experiment, the anti-CD3/CD28 magnetic beads were removed. CAR-T cells and target tumor cells were seeded at a density of 5 000 cells/50 \u0026micro;L each well at an effector-to-target ratio of 1:1 in 96-well round bottom plates (Nunc) for 24 hours. The supernatants were collected and cytokines IL-2 and interferon (IFN-γ) were measured using ELISA Kits (70-EK102, 70-EK180, Multi-Science) following the manufacturer\u0026rsquo;s protocol by ELISA plate reader (EL-808, Biotek). The cytokine concentration was quantified by a standard curve.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003e4.12. \u003cem\u003eIn vivo\u003c/em\u003e antitumor model\u003c/h2\u003e \u003cp\u003eTo assess the antitumor activity of CAR-T and SATB1-CAR-T cells \u003cem\u003ein vivo\u003c/em\u003e, we constructed an HCC cell line-derived CDX model. 5\u0026times;10\u003csup\u003e5\u003c/sup\u003e Huh7-GFP/Luc cells were subcutaneously (s.c.) injected into NCG mice on the right flank. 4 days after tumor cell implantation, tumor-bearing mice with equal tumor burden were selected and randomized to different treatment groups and 1\u0026times;10\u003csup\u003e6\u003c/sup\u003e Ctrl-T, SATB1-T, CAR-T or SATB1-CAR-T cells were injected intravenously (i.v.) via the tail vein. Tumor-bearing mice were then intraperitoneally (i.p.) injected with 150 mg/kg D-luciferin at indicated time points and luminescence signals were monitored and counted by an IVIS Spectrum Imaging platform (Caliper, Boston, MA, USA). To assess the survival curves, GraphPad Prism Software was used to record and analyze.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec25\" class=\"Section2\"\u003e \u003ch2\u003e4.13. Statistical analysis\u003c/h2\u003e \u003cp\u003eAll data presented graphically as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (S.D.) was from at least three independent experiments. Each exact \u003cem\u003en\u003c/em\u003e value is stated in the corresponding figure legend. The statistical data was analyzed using paired or unpaired two-tailed Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e-test for two-sample comparisons. The log-rank test was performed for comparison of survival curves. All statistical data was analyzed by GraphPad Prism and statistical significance was defined as *\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, ***\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, and ****\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e\u003cstrong\u003eCKNOWLEDGMENTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCORRESPONDENCE\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTongbiao Zhao, The State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, 1 Beichen West Road, Chaoyang District, Beijing, 100101, People\u0026apos;s Republic of China. Email: [email protected]. Phone: (+86)-10-64806307.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFUNDING INFORMATION\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by grants from the China National Basic Research Program 2022YFA1103601; the Strategic Priority Research Program of the Chinese Academy of Sciences E42GP41107; and the Strategic Collaborative Research Program of the Ferring Institute of Reproductive Medicine Grant No.33.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAUTHOR CONTRIBUTIONS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eL.Z. and T.Z. designed the experiments and wrote the manuscript; L.Z., C.C., X.B., J.C. and X.L. performed the experiments and analyzed the data; T.Z. coordinated and supervised the experiments.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCONFLICT OF INTEREST\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDATA AVAILABILITY STATEMENT\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data and materials during the current study are available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eLiu Q, Li J, Zheng H, Yang S, Hua Y, Huang N, et al. Adoptive cellular immunotherapy for solid neoplasms beyond CAR-T. Mol Cancer. 2023;22(1):28.\u003c/li\u003e\n\u003cli\u003eJune CH, O\u0026apos;Connor RS, Kawalekar OU, Ghassemi S, Milone MC. CAR T cell immunotherapy for human cancer. Science. 2018;359(6382):1361-5.\u003c/li\u003e\n\u003cli\u003eVenetis K, Invernizzi M, Sajjadi E, Curigliano G, Fusco N. Cellular immunotherapy in breast cancer: The quest for consistent biomarkers. Cancer Treat Rev. 2020;90:102089.\u003c/li\u003e\n\u003cli\u003eKirtane K, Elmariah H, Chung CH, Abate-Daga D. Adoptive cellular therapy in solid tumor malignancies: review of the literature and challenges ahead. J Immunother Cancer. 2021;9(7).\u003c/li\u003e\n\u003cli\u003eCappell KM, Kochenderfer JN. Long-term outcomes following CAR T cell therapy: what we know so far. Nat Rev Clin Oncol. 2023;20(6):359-71.\u003c/li\u003e\n\u003cli\u003eMaude SL, Laetsch TW, Buechner J, Rives S, Boyer M, Bittencourt H, et al. Tisagenlecleucel in Children and Young Adults with B-Cell Lymphoblastic Leukemia. N Engl J Med. 2018;378(5):439-48.\u003c/li\u003e\n\u003cli\u003eLarson RC, Maus MV. Recent advances and discoveries in the mechanisms and functions of CAR T cells. Nat Rev Cancer. 2021;21(3):145-61.\u003c/li\u003e\n\u003cli\u003eZhang T, Tai Z, Miao F, Zhang X, Li J, Zhu Q, et al. Adoptive cell therapy for solid tumors beyond CAR-T: Current challenges and emerging therapeutic advances. J Control Release. 2024;368:372-96.\u003c/li\u003e\n\u003cli\u003eZinkernagel RM, Moskophidis D, Kundig T, Oehen S, Pircher H, Hengartner H. Effector T-cell induction and T-cell memory versus peripheral deletion of T cells. Immunol Rev. 1993;133:199-223.\u003c/li\u003e\n\u003cli\u003ePauken KE, Wherry EJ. Overcoming T cell exhaustion in infection and cancer. Trends Immunol. 2015;36(4):265-76.\u003c/li\u003e\n\u003cli\u003eWherry EJ, Ha SJ, Kaech SM, Haining WN, Sarkar S, Kalia V, et al. Molecular signature of CD8+ T cell exhaustion during chronic viral infection. Immunity. 2007;27(4):670-84.\u003c/li\u003e\n\u003cli\u003eWherry EJ, Kurachi M. Molecular and cellular insights into T cell exhaustion. Nat Rev Immunol. 2015;15(8):486-99.\u003c/li\u003e\n\u003cli\u003eGumber D, Wang LD. Improving CAR-T immunotherapy: Overcoming the challenges of T cell exhaustion. EBioMedicine. 2022;77:103941.\u003c/li\u003e\n\u003cli\u003eDelgoffe GM, Xu C, Mackall CL, Green MR, Gottschalk S, Speiser DE, et al. The role of exhaustion in CAR T cell therapy. Cancer Cell. 2021;39(7):885-8.\u003c/li\u003e\n\u003cli\u003eLynn RC, Weber EW, Sotillo E, Gennert D, Xu P, Good Z, et al. c-Jun overexpression in CAR T cells induces exhaustion resistance. Nature. 2019;576(7786):293-300.\u003c/li\u003e\n\u003cli\u003eScott-Browne JP, Lopez-Moyado IF, Trifari S, Wong V, Chavez L, Rao A, et al. Dynamic Changes in Chromatin Accessibility Occur in CD8(+) T Cells Responding to Viral Infection. Immunity. 2016;45(6):1327-40.\u003c/li\u003e\n\u003cli\u003eChen J, Lopez-Moyado IF, Seo H, Lio CJ, Hempleman LJ, Sekiya T, et al. NR4A transcription factors limit CAR T cell function in solid tumours. Nature. 2019;567(7749):530-4.\u003c/li\u003e\n\u003cli\u003eQuigley M, Pereyra F, Nilsson B, Porichis F, Fonseca C, Eichbaum Q, et al. Transcriptional analysis of HIV-specific CD8+ T cells shows that PD-1 inhibits T cell function by upregulating BATF. Nat Med. 2010;16(10):1147-51.\u003c/li\u003e\n\u003cli\u003eZhang X, Zhang C, Qiao M, Cheng C, Tang N, Lu S, et al. Depletion of BATF in CAR-T cells enhances antitumor activity by inducing resistance against exhaustion and formation of central memory cells. Cancer Cell. 2022;40(11):1407-22 e7.\u003c/li\u003e\n\u003cli\u003eChan JD, Scheffler CM, Munoz I, Sek K, Lee JN, Huang YK, et al. FOXO1 enhances CAR T cell stemness, metabolic fitness and efficacy. Nature. 2024;629(8010):201-10.\u003c/li\u003e\n\u003cli\u003eDoan AE, Mueller KP, Chen AY, Rouin GT, Chen Y, Daniel B, et al. FOXO1 is a master regulator of memory programming in CAR T cells. Nature. 2024;629(8010):211-8.\u003c/li\u003e\n\u003cli\u003eLi Y, Han M, Wei H, Huang W, Chen Z, Zhang T, et al. Id2 epigenetically controls CD8(+) T-cell exhaustion by disrupting the assembly of the Tcf3-LSD1 complex. Cell Mol Immunol. 2024;21(3):292-308.\u003c/li\u003e\n\u003cli\u003eBeltra JC, Abdel-Hakeem MS, Manne S, Zhang Z, Huang H, Kurachi M, et al. Stat5 opposes the transcription factor Tox and rewires exhausted CD8(+) T cells toward durable effector-like states during chronic antigen exposure. Immunity. 2023;56(12):2699-718 e11.\u003c/li\u003e\n\u003cli\u003eNaik R, Galande S. SATB family chromatin organizers as master regulators of tumor progression. Oncogene. 2019;38(12):1989-2004.\u003c/li\u003e\n\u003cli\u003eLi QQ, Chen ZQ, Cao XX, Xu JD, Xu JW, Chen YY, et al. Involvement of NF-kappaB/miR-448 regulatory feedback loop in chemotherapy-induced epithelial-mesenchymal transition of breast cancer cells. Cell Death Differ. 2011;18(1):16-25.\u003c/li\u003e\n\u003cli\u003eHan HJ, Russo J, Kohwi Y, Kohwi-Shigematsu T. SATB1 reprogrammes gene expression to promote breast tumour growth and metastasis. Nature. 2008;452(7184):187-93.\u003c/li\u003e\n\u003cli\u003eYasui D, Miyano M, Cai S, Varga-Weisz P, Kohwi-Shigematsu T. SATB1 targets chromatin remodelling to regulate genes over long distances. Nature. 2002;419(6907):641-5.\u003c/li\u003e\n\u003cli\u003ePatta I, Madhok A, Khare S, Gottimukkala KP, Verma A, Giri S, et al. Dynamic regulation of chromatin organizer SATB1 via TCR-induced alternative promoter switch during T-cell development. Nucleic Acids Res. 2020;48(11):5873-90.\u003c/li\u003e\n\u003cli\u003eAhlfors H, Limaye A, Elo LL, Tuomela S, Burute M, Gottimukkala KV, et al. SATB1 dictates expression of multiple genes including IL-5 involved in human T helper cell differentiation. Blood. 2010;116(9):1443-53.\u003c/li\u003e\n\u003cli\u003eNussing S, Miosge LA, Lee K, Olshansky M, Barugahare A, Roots CM, et al. SATB1 ensures appropriate transcriptional programs within naive CD8(+) T cells. Immunol Cell Biol. 2022;100(8):636-52.\u003c/li\u003e\n\u003cli\u003eTrujillo-Ochoa JL, Kazemian M, Afzali B. The role of transcription factors in shaping regulatory T cell identity. Nat Rev Immunol. 2023;23(12):842-56.\u003c/li\u003e\n\u003cli\u003eAlvarez JD, Yasui DH, Niida H, Joh T, Loh DY, Kohwi-Shigematsu T. The MAR-binding protein SATB1 orchestrates temporal and spatial expression of multiple genes during T-cell development. Genes Dev. 2000;14(5):521-35.\u003c/li\u003e\n\u003cli\u003eStephen TL, Payne KK, Chaurio RA, Allegrezza MJ, Zhu H, Perez-Sanz J, et al. SATB1 Expression Governs Epigenetic Repression of PD-1 in Tumor-Reactive T Cells. Immunity. 2017;46(1):51-64.\u003c/li\u003e\n\u003cli\u003eChaurio RA, Anadon CM, Lee Costich T, Payne KK, Biswas S, Harro CM, et al. TGF-beta-mediated silencing of genomic organizer SATB1 promotes Tfh cell differentiation and formation of intra-tumoral tertiary lymphoid structures. Immunity. 2022;55(1):115-28 e9.\u003c/li\u003e\n\u003cli\u003eKersten K, Hu KH, Combes AJ, Samad B, Harwin T, Ray A, et al. Spatiotemporal co-dependency between macrophages and exhausted CD8(+) T cells in cancer. Cancer Cell. 2022;40(6):624-38 e9.\u003c/li\u003e\n\u003cli\u003eKim HD, Song GW, Park S, Jung MK, Kim MH, Kang HJ, et al. Association Between Expression Level of PD1 by Tumor-Infiltrating CD8(+) T Cells and Features of Hepatocellular Carcinoma. Gastroenterology. 2018;155(6):1936-50 e17.\u003c/li\u003e\n\u003cli\u003eZheng L, Qin S, Si W, Wang A, Xing B, Gao R, et al. Pan-cancer single-cell landscape of tumor-infiltrating T cells. Science. 2021;374(6574):abe6474.\u003c/li\u003e\n\u003cli\u003eCapurro M, Wanless IR, Sherman M, Deboer G, Shi W, Miyoshi E, et al. Glypican-3: a novel serum and histochemical marker for hepatocellular carcinoma. Gastroenterology. 2003;125(1):89-97.\u003c/li\u003e\n\u003cli\u003eDal Bo M, De Mattia E, Baboci L, Mezzalira S, Cecchin E, Assaraf YG, et al. New insights into the pharmacological, immunological, and CAR-T-cell approaches in the treatment of hepatocellular carcinoma. Drug Resist Updat. 2020;51:100702.\u003c/li\u003e\n\u003cli\u003eZhou F, Shang W, Yu X, Tian J. Glypican-3: A promising biomarker for hepatocellular carcinoma diagnosis and treatment. Med Res Rev. 2018;38(2):741-67.\u003c/li\u003e\n\u003cli\u003eIshiguro T, Sugimoto M, Kinoshita Y, Miyazaki Y, Nakano K, Tsunoda H, et al. Anti-glypican 3 antibody as a potential antitumor agent for human liver cancer. Cancer Res. 2008;68(23):9832-8.\u003c/li\u003e\n\u003cli\u003eGattinoni L, Lugli E, Ji Y, Pos Z, Paulos CM, Quigley MF, et al. A human memory T cell subset with stem cell-like properties. Nat Med. 2011;17(10):1290-7.\u003c/li\u003e\n\u003cli\u003eCieri N, Camisa B, Cocchiarella F, Forcato M, Oliveira G, Provasi E, et al. IL-7 and IL-15 instruct the generation of human memory stem T cells from naive precursors. Blood. 2013;121(4):573-84.\u003c/li\u003e\n\u003cli\u003eBatlle E, Massague J. Transforming Growth Factor-beta Signaling in Immunity and Cancer. Immunity. 2019;50(4):924-40.\u003c/li\u003e\n\u003cli\u003eKoehler H, Kofler D, Hombach A, Abken H. CD28 costimulation overcomes transforming growth factor-beta-mediated repression of proliferation of redirected human CD4+ and CD8+ T cells in an antitumor cell attack. Cancer Res. 2007;67(5):2265-73.\u003c/li\u003e\n\u003cli\u003eGolumba-Nagy V, Kuehle J, Hombach AA, Abken H. CD28-zeta CAR T Cells Resist TGF-beta Repression through IL-2 Signaling, Which Can Be Mimicked by an Engineered IL-7 Autocrine Loop. Mol Ther. 2018;26(9):2218-30.\u003c/li\u003e\n\u003cli\u003eTang N, Cheng C, Zhang X, Qiao M, Li N, Mu W, et al. TGF-beta inhibition via CRISPR promotes the long-term efficacy of CAR T cells against solid tumors. JCI Insight. 2020;5(4).\u003c/li\u003e\n\u003cli\u003eStuber T, Monjezi R, Wallstabe L, Kuhnemundt J, Nietzer SL, Dandekar G, et al. Inhibition of TGF-beta-receptor signaling augments the antitumor function of ROR1-specific CAR T-cells against triple-negative breast cancer. J Immunother Cancer. 2020;8(1).\u003c/li\u003e\n\u003cli\u003eHou AJ, Shih RM, Uy BR, Shafer A, Chang ZNL, Comin-Anduix B, et al. IL-13Ralpha2/TGF-beta bispecific CAR-T cells counter TGF-beta-mediated immune suppression and potentiate anti-tumor responses in glioblastoma. Neuro Oncol. 2024.\u003c/li\u003e\n\u003cli\u003eHu Y, Feng J, Gu T, Wang L, Wang Y, Zhou L, et al. CAR T-cell therapies in China: rapid evolution and a bright future. Lancet Haematol. 2022;9(12):e930-e41.\u003c/li\u003e\n\u003cli\u003eZhang L, Yu X, Zheng L, Zhang Y, Li Y, Fang Q, et al. Lineage tracking reveals dynamic relationships of T cells in colorectal cancer. Nature. 2018;564(7735):268-72.\u003c/li\u003e\n\u003cli\u003eHuang Y, Shao M, Teng X, Si X, Wu L, Jiang P, et al. Inhibition of CD38 enzymatic activity enhances CAR-T cell immune-therapeutic efficacy by repressing glycolytic metabolism. Cell Rep Med. 2024;5(2):101400.\u003c/li\u003e\n\u003cli\u003eHuang D, Chen X, Zeng X, Lao L, Li J, Xing Y, et al. Targeting regulator of G protein signaling 1 in tumor-specific T cells enhances their trafficking to breast cancer. Nat Immunol. 2021;22(7):865-79.\u003c/li\u003e\n\u003cli\u003eBai Y, Hu M, Chen Z, Wei J, Du H. Single-Cell Transcriptome Analysis Reveals RGS1 as a New Marker and Promoting Factor for T-Cell Exhaustion in Multiple Cancers. Front Immunol. 2021;12:767070.\u003c/li\u003e\n\u003cli\u003eJackson Z, Hong C, Schauner R, Dropulic B, Caimi PF, de Lima M, et al. Sequential Single-Cell Transcriptional and Protein Marker Profiling Reveals TIGIT as a Marker of CD19 CAR-T Cell Dysfunction in Patients with Non-Hodgkin Lymphoma. Cancer Discov. 2022;12(8):1886-903.\u003c/li\u003e\n\u003cli\u003eWherry EJ. T cell exhaustion. Nat Immunol. 2011;12(6):492-9.\u003c/li\u003e\n\u003cli\u003eChow A, Perica K, Klebanoff CA, Wolchok JD. Clinical implications of T cell exhaustion for cancer immunotherapy. Nat Rev Clin Oncol. 2022;19(12):775-90.\u003c/li\u003e\n\u003cli\u003eGlatzel-Plucinska N, Piotrowska A, Dziegiel P, Podhorska-Okolow M. The Role of SATB1 in Tumour Progression and Metastasis. Int J Mol Sci. 2019;20(17).\u003c/li\u003e\n\u003cli\u003eGottimukkala KP, Jangid R, Patta I, Sultana DA, Sharma A, Misra-Sen J, et al. Regulation of SATB1 during thymocyte development by TCR signaling. Mol Immunol. 2016;77:34-43.\u003c/li\u003e\n\u003cli\u003eRuss BE, Barugahare A, Dakle P, Tsyganov K, Quon S, Yu B, et al. Active maintenance of CD8(+) T cell naivety through regulation of global genome architecture. Cell Rep. 2023;42(10):113301.\u003c/li\u003e\n\u003cli\u003eWang B, Bian Q. SATB1 prevents immune cell infiltration by regulating chromatin organization and gene expression of a chemokine gene cluster in T cells. Commun Biol. 2024;7(1):1304.\u003c/li\u003e\n\u003cli\u003eWang B, Ji L, Bian Q. SATB1 regulates 3D genome architecture in T cells by constraining chromatin interactions surrounding CTCF-binding sites. Cell Rep. 2023;42(4):112323.\u003c/li\u003e\n\u003cli\u003eGupta PK, Allocco JB, Fraipont JM, McKeague ML, Wang P, Andrade MS, et al. Reduced Satb1 expression predisposes CD4(+) T conventional cells to Treg suppression and promotes transplant survival. Proc Natl Acad Sci U S A. 2022;119(40):e2205062119.\u003c/li\u003e\n\u003cli\u003eBeyer M, Thabet Y, Muller RU, Sadlon T, Classen S, Lahl K, et al. Repression of the genome organizer SATB1 in regulatory T cells is required for suppressive function and inhibition of effector differentiation. Nat Immunol. 2011;12(9):898-907.\u003c/li\u003e\n\u003cli\u003eKuwabara T, Ishikawa F, Ikeda M, Ide T, Kohwi-Shigematsu T, Tanaka Y, et al. SATB1-dependent mitochondrial ROS production controls TCR signaling in CD4 T cells. Life Sci Alliance. 2021;4(11).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cell-death-and-disease","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"cddis","sideBox":"Learn more about [Cell Death \u0026 Disease](http://www.nature.com/cddis/)","snPcode":"41419","submissionUrl":"https://mts-cddis.nature.com/cgi-bin/main.plex","title":"Cell Death \u0026 Disease","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-6776099/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6776099/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAlthough Chimeric antigen receptor (CAR) T-cell therapy has achieved remarkable success in treating hematopoietic malignancies, its clinical application in solid tumors is profoundly hindered by persistent T-cell exhaustion within the immunosuppressive tumor microenvironment (TME). Here, we identified SATB1\u0026mdash;a genome organizer regulating chromatin architecture\u0026mdash;as a key suppressor of CAR-T cell exhaustion. In Glypican-3 (GPC3)-targeted CAR-T cells, SATB1 was significantly downregulated in tumor-infiltrating exhausted populations. SATB1 overexpression not only reduced expression of multiple inhibitory receptors (PD-1, CTLA-4, TIM3 and LAG-3), but also promoted a central memory phenotype, enhancing cytokine production and cytotoxicity against hepatocellular carcinoma (HCC) cells \u003cem\u003ein vitro\u003c/em\u003e. \u003cem\u003eIn vivo\u003c/em\u003e, SATB1-engineered CAR-T cells exhibited superior tumor control and promoted survival, accompanied by reduced exhaustion markers in tumor-infiltrating T cells. These functional improvements are consistent with the reported role of SATB1 in modulating T cell exhaustion, positioning it as a multifunctional enhancer of CAR-T cell fitness. Collectively, our study unveils SATB1 as a multifunctional modulator that simultaneously targets exhaustion and memory differentiation, offering a novel strategy to enhance CAR-T efficacy against solid tumors.\u003c/p\u003e","manuscriptTitle":"Enhanced Anti-Liver Tumor Efficacy of Chimeric Antigen Receptor-T Cells via SATB1 Modulation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-18 11:50:56","doi":"10.21203/rs.3.rs-6776099/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"revise","date":"2025-08-12T13:49:14+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"This content is not available.","date":"2025-07-25T19:21:30+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2025-07-11T09:50:40+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2025-06-16T21:54:26+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-05-30T10:49:20+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cell Death \u0026 Disease","date":"2025-05-29T11:29:31+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-05-29T11:29:31+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cell-death-and-disease","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"cddis","sideBox":"Learn more about [Cell Death \u0026 Disease](http://www.nature.com/cddis/)","snPcode":"41419","submissionUrl":"https://mts-cddis.nature.com/cgi-bin/main.plex","title":"Cell Death \u0026 Disease","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"543f38ba-7b52-44fc-9660-7556caaa99bd","owner":[],"postedDate":"June 18th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":50147082,"name":"Biological sciences/Immunology/Immunotherapy/Immunization"},{"id":50147083,"name":"Health sciences/Diseases/Cancer"}],"tags":[],"updatedAt":"2026-01-24T08:10:55+00:00","versionOfRecord":{"articleIdentity":"rs-6776099","link":"https://doi.org/10.1038/s41419-025-08307-3","journal":{"identity":"cell-death-and-disease","isVorOnly":false,"title":"Cell Death \u0026 Disease"},"publishedOn":"2025-12-10 05:00:00","publishedOnDateReadable":"December 10th, 2025"},"versionCreatedAt":"2025-06-18 11:50:56","video":"","vorDoi":"10.1038/s41419-025-08307-3","vorDoiUrl":"https://doi.org/10.1038/s41419-025-08307-3","workflowStages":[]},"version":"v1","identity":"rs-6776099","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6776099","identity":"rs-6776099","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}