Adoptive T cell therapy (ACT) remains limited in solid tumors by poor T cell persistence within the metabolically hostile tumor microenvironment (TME). Although IL-9-producing CD8+ T cells (Tc9) consistently demonstrate superior antitumor efficacy compared with conventional Tc1 cells, the selective pressures that shape their functional advantage remain unclear. Here, we show that effective ACT-mediated tumor control is accompanied by a marked increase in intratumoral extracellular ATP (eATP), representing a common metabolic consequence of tumor cell destruction. Despite comparable ATP accumulation following Tc1 or Tc9 treatment, these subsets exhibit strikingly distinct responses to ATP stress. Tc1 cells are highly susceptible to ATP-induced apoptosis, whereas Tc9 cells display intrinsic resistance, resulting in superior in vivo persistence. Mechanistically, Tc9 cells actively convert ATP signaling into enhanced mitochondrial fitness, characterized by increased oxidative phosphorylation and spare respiratory capacity. ATP exposure further drives Tc9 cells toward a tissue-resident memory (TRM) phenotype through activation of the TGF-β signaling axis. Transcriptomic and molecular analyses reveal that purinergic signaling pathways, including elevated expression of the ATP receptor P2RX7, are intrinsically enriched in Tc9 cells and are further amplified upon ATP stimulation. Collectively, our findings identify extracellular ATP as a metabolic lineage selection signal in ACT, demonstrating that ATP stress preferentially stabilizes metabolically resilient Tc9 cells by linking purinergic sensing to mitochondrial remodeling and TRM programming, thereby providing a conceptual basis for enhancing the persistence and efficacy of engineered T cell therapies in solid tumors.

Digital polymerase chain reaction (dPCR) is a powerful tool for absolute quantification of nucleic acids, offering higher sensitivity than quantitative PCR (qPCR). However, current dPCR platforms struggle to detect gene copy number variations (CNVs) at the single-cell level due to limited droplet control and visualization. To address these challenges, we developed a digital microfluidic (DMF)-based platform that integrates droplet manipulation, sample processing, and nucleic acid amplification into a single, fully controllable workflow. The DMF chip, featuring 16,384 addressable electrodes, enables precise manipulation of nanoliter droplets containing single cells, supporting on-chip cell lysis, droplet merging, and splitting without complex workflows. Using cell lines, we validated the platform’s capability for both qPCR and dPCR, achieving single-cell resolution. To demonstrate clinical relevance, we applied the platform to primary chimeric antigen receptor T (CAR-T) cells and successfully quantified vector copy numbers (VCNs) at the single-cell level, with performance comparable to commercial dPCR systems. This fully integrated, high-throughput platform minimizes sample loss and contamination while offering real-time visualization and scalability. Our DMF-based dPCR system provides a robust tool for single-cell genetic analysis, with broad potential applications in clinical diagnostics, personalized medicine, and research.

Macrophage migration inhibitory factor (MIF), derived from either tumor cells or the tumor microenvironment, promotes multiple myeloma (MM) progression by suppressing T cell-mediated antitumor responses. Here, we demonstrate that MIF drives immune evasion in MM through both TME-mediated and tumor-intrinsic mechanisms. In the TME, MIF promotes the expansion of myeloid-derived suppressor cells (MDSCs) and enhances the expression of immunosuppressive molecules, including CD84, PD-L1, and CD38, thereby augmenting MDSC-mediated immunosuppression and impairing T cell function. In MM cells, MIF suppresses the COPS5/STAT1/NLRC5 signaling axis, resulting in downregulation of the major histocompatibility complex class I (MHC-I) antigen presentation pathway and reduced T cell-mediated cytotoxicity against MM cells. Therapeutically, combined treatment with the MIF inhibitor 4-IPP and dexamethasone delayed MM progression and prolonged survival in a mouse MM model. In summary, our findings reveal MIF as a critical mediator of immune escape in MM and highlight the therapeutic potential of MIF-targeted strategies for MM treatment.

Glioblastoma (GBM) is a highly aggressive form of brain tumor characterized by dysregulated metabolism. Increased fatty acid oxidation (FAO) protects tumor cells from lipid peroxidation-induced cell death, although the precise mechanisms involved remain unclear. Here, we report that loss of TNF receptor-associated factor 3 (TRAF3) in GBM critically regulated lipid peroxidation and tumorigenesis by controlling the oxidation of polyunsaturated fatty acids (PUFAs). TRAF3 was frequently repressed in GBM due to promoter hypermethylation. TRAF3 interacted with enoyl-CoA hydratase 1 (ECH1), an enzyme that catalyzes the isomerization of unsaturated FAs (UFAs) and mediates K63-linked ubiquitination of ECH1 at Lys214. ECH1 ubiquitination impeded TOMM20-dependent mitochondrial translocation of ECH1, which otherwise promoted the oxidation of UFAs, preferentially the PUFAs, and limited lipid peroxidation. Overexpression of TRAF3 enhanced the sensitivity of GBM to ferroptosis and anti-programmed death-ligand 1 (anti-PD-L1) immunotherapy in mice. Thus, the TRAF3/ECH1 axis played a key role in the metabolism of PUFAs and was crucial for lipid peroxidation damage and immune elimination in GBM.

Immune checkpoint blockade (ICB) and chimeric antigen receptor (CAR) T cell therapies have revolutionized cancer immunotherapy, offering significant benefits across various cancers. However, challenges remain, particularly in solid tumors where immunosuppressive tumor microenvironments and T cell exhaustion limit effectiveness. Combining ICB with CAR T cell therapy has shown potential but requires further optimization for effective synergy. Here, the bioinformatic analysis identified that CXCL13 expression is highly elevated in T cells from patients who respond to ICB, indicating its possible role in enhancing T cell antitumor responses. Mouse CAR T cells are engineered to overexpress CXCL13 and observed that these cells displayed reduced exhaustion, increased central memory phenotype, and improved mitochondrial function and proliferation in an AKT-mTOR dependent manner. CXCL13-overexpressing CAR T cells show significantly increased antitumor activity in vivo, particularly when combined with PD-1 inhibition, promoting the expansion and persistence of early exhausted CD8+ CAR T cells. CXCL13 also conferred similar in vitro phenotypic enhancements in human CAR T cells as observed in murine cells. These results indicate that CXCL13 expression improves CAR T cell function and responsiveness to ICB, offering a promising and translationally relevant strategy to optimize CAR T cell therapy for solid tumors in clinical settings.

Multiple myeloma (MM) remains an incurable malignancy characterized by the proliferation of malignant plasma cells and significant dysregulation within the bone marrow microenvironment. Plasmacytoid dendritic cells (pDCs) play a crucial role in the immune landscape of MM, often being co-opted by MM cells to support tumor progression. In this study, we identified all-trans retinoic acid (RA) as a potent modulator of pDC function through a high-throughput screening of 2,000 small-molecule drugs. RA significantly enhances pDCs' capacity to secrete interferon (IFN)-α upon CpG or Resiquimod stimulation, reversing the MM-induced suppression of IFN-α secretion. Mechanistically, RA upregulates Toll-like receptor (TLR)7/9 expression in pDCs, amplifying TLR7/9 agonist-induced IFN-α production and enhancing retinoic acid-inducible gene Ⅰ (RIG-Ⅰ)-like signaling and IFN-stimulated gene expression. In vivo, RA combined with CpG or Resiquimod significantly improves the survival of MM-bearing mice, with even greater pro-survival benefits observed when RA, Resiquimod, and bortezomib are combined. These findings demonstrate that RA rejuvenates pDCs, leading to improved control of MM growth in preclinical models, offering novel insights into developing more effective MM therapies.

Adoptive cell therapy (ACT) relies on durable and functional T cells to mediate tumor clearance. Th9 cells are a metabolically fit CD4+ T cell subset with strong persistence but limited cytotoxicity. Here, we identified endomelipeptide A (EpA), a cyclic peptide isolated from Ganoderma lucidum-associated endophytic fungi, as a potent enhancer of Th9 cell differentiation. EpA promoted a cytotoxic Th9 phenotype with enhanced mitochondrial function and metabolic fitness. Mechanistically, EpA dually targeted ZAP70 and SREBP1, coupling T cell receptor signaling activation with lipid metabolism suppression. EpA-treated Th9 cells mediated robust, CD8+ T cell-dependent tumor control and enhanced the efficacy of human Th9 CAR T cell therapy in vivo. These findings establish EpA as a distinct cyclic peptide that reprograms Th9 cells and provides a potential approach to boost ACT efficacy.

KEY POINTS:Extracellular matrix protein 1 is specifically expressed in glomerular mesangial cells in both human and mice. Loss of extracellular matrix protein 1 leads to excessive mesangial matrix expansion and disrupted glomerular architecture. Extracellular matrix protein 1 binds integrin α 8 β 1 to stabilize mesangial cell adhesion to the mesangial matrix and glomerular basement membrane. BACKGROUND:The mesangium consists of mesangial cells and mesangial matrix that maintain normal glomerular structure and function. The mechanisms by which mesangial cells regulate mesangial matrix dynamics remain poorly understood. This study investigated the role of mesangial cell-derived extracellular matrix protein 1 (ECM1) in regulating mesangial matrix homeostasis and glomerular structure in mice. METHODS:Immunofluorescence and single-nucleus RNA sequencing were used to clarify the ECM1 expression pattern in kidney. Global and tamoxifen-inducible Ecm1 -knockout (KO) mice demonstrated the in vivo function of Ecm1 . Immunoprecipitation, mass spectrometry, and RNA-seq were used to reveal the mechanism by which ECM1 regulates mesangial matrix homeostasis. RESULTS:snRNA-seq and immunofluorescence revealed that ECM1 was specifically expressed in glomerular mesangial cells and downregulated in diabetic kidney disease and IgA nephropathy. Genetic deletion of Ecm1 in mice resulted in mesangial matrix expansion at 4 and 8 weeks of age, whereas early stages appeared unaffected. Similarly, tamoxifen-induced global deletion of Ecm1 in 4-week-old mice led to a trend toward mesangial matrix expansion. Transcriptomic profiling and immunofluorescence confirmed substantial alterations of the mesangial matrix components in Ecm1 -KO kidneys. As a result, histological examination showed profound glomerular abnormalities in Ecm1 -KO mice. Ecm1 deficiency also elevated TGF β 1 expression and downstream signaling, likely contributing to the excessive collagen deposition in glomeruli. Mechanistically, ECM1 interacted with integrin α 8 β 1 to promote mesangial cell-matrix adhesion, and loss of this interaction led to mesangial cell disaggregation and detachment from glomerular basement membrane, thereby promoting glomerular pathology. Notably, adeno-associated virus-mediated ECM1 expression in vivo effectively rescued kidney dysfunction in Ecm1 -KO mice. CONCLUSIONS:ECM1 was highly expressed in glomerular mesangial cells. Loss of Ecm1 led to altered mesangial matrix composition, matrix expansion, and glomerular abnormalities, while restoration of ECM1 expression reversed these defects, demonstrating an essential role of mesangial cell-derived ECM1 in preserving normal mesangial homeostasis and glomerular structure in mice.

Three p -terphenyl metabolites ( 1 - 3 ), three indole-diterpenoids ( 4 - 6 ), an herbicide sesquiterpene ( 7 ), a flavonoid ( 8 ), and five other small molecules containing nitrogen ( 9 - 13 ) were isolated from the medicinal insect ( Peri- planeta americana )-derived endophytic Aspergillus taichungensis SMU01. Their chemical structures were elucidated on the basis of spectroscopic data and quantum chemical computational methods. Biological activity of these isolates in the differentiation of mouse CD4 + T cell subsets was evaluated. Importantly, metabolites 2 targeting JAK-STAT signaling pathway could hold potential benefits in maintaining peripheral immune homeostasis and alleviating the progression of autoimmune diseases.

BACKGROUND:Bendamustine-rituximab (BR) therapy stands out as a promising alternative for elderly patients with diffuse large B-cell lymphoma (DLBCL), demonstrating notable efficacy when conventional regimens pose challenges. Despite its clinical success, the intricate mechanisms underlying BR therapy have remained elusive. METHODS:DLBCL cell lines were used to investigate the mechanism of BR therapy in vitro. RNA-seq and Western blot were used to explore the target pathways of BR therapy. STING was knocked out using Crispr-cas9 and inhibited using H-151 to investigate its role in BR therapy. Bulk RNA-seq and single-cell RNA-seq data from patients were analyzed to investigate the association between STING and pyroptosis pathways, validated using STING downregulated cells. Flow cytometry, transwell experiments and co-culture experiments were performed to investigate the inflammatory phenotype of DLBCL cells after BR treatment and its effect on T-cell recruitment and activation. RESULTS:This study elucidates that BR elicits direct tumoricidal effects by promoting apoptosis and inducing cell cycle arrest. The synergistic impact with rituximab is further potentiated by complement addition, demonstrating the pivotal role of in vivo antibody-dependent cellular cytotoxicity. Moreover, our investigation reveals that, through a cGAS-STING-dependent pathway, prolonged exposure to BR induces pyroptosis in DLBCL cells. Activation of the cGAS-STING pathway by BR therapy triggers the release of inflammatory factors and upregulates major histocompatibility complex molecules, shaping an immunologically hot tumor microenvironment. CONCLUSIONS:This unique dual influence not only directly targets DLBCL cells but also engages the patient's immune system, paving the way for innovative combination therapies. The study provides comprehensive insights into the multifaceted actions of BR in DLBCL, offering a foundation for refined and personalized treatment strategies in elderly patients.

The importance of CD4 + T cells in cancer immunotherapy has gained increasing recognition. Particularly, a specific subset of CD4 + T cells coexpressing the T helper type 1 (Th1) and Th17 markers has demonstrated remarkable antitumor potential. However, the underlying mechanisms governing the differentiation of these cells and their subsequent antitumor responses remain incompletely understood. Single-cell RNA sequencing (scRNA-seq) data reanalysis demonstrated the presence of Th 17 1 cells within tumors. Subsequent trajectory analysis found that these Th 17 1 cells are initially primed under Th17 conditions and then converted into IFN-γ-producing cells. Following the in vivo differentiation trajectory of Th 17 1 cells, we successfully established in vitro Th 17 1 cell culture. Transcriptomic profiling has unveiled a substantial resemblance between in vitro-generated Th 17 1 cells and their tumor-infiltrating counterparts. Th 17 1 cells exhibit more potent antitumor responses than Th1 or Th17 cells. Additionally, Th 17 1chimeric antigen receptor T (CAR-T) cells eradicate solid tumors more efficiently. Importantly, Th 17 1 cells display an early exhaustion phenotype while retaining stemness. Mechanistically, Th 17 1 cells migrate faster and accumulate more in tumors in an extracellular matrix protein 1 (ECM1)-dependent manner. Furthermore, we show that IFN-γ up-regulated IRF7 to promote the type I interferon response network and ECM1 expression but decreased the exhaustion status in Th 17 1 cells. Taken together, our findings position Th 17 1 cells as a great candidate for improving targeted immunotherapies in solid malignancies.

The antitumor effector T helper 1 (Th1) and Th17 cells represent two T cell paradigms: short-lived cytolytic Th1 cells and “stem cell-like” memory Th17 cells. We report that Th9 cells represent a third paradigm—they are less-exhausted, fully cytolytic, and hyperproliferative. Only tumor-specific Th9 cells completely eradicated advanced tumors, maintained a mature effector cell signature with cytolytic activity as strong as Th1 cells, and persisted as long as Th17 cells in vivo. Th9 cells displayed a unique Pu.1-Traf6-NF-κB activation-driven hyperproliferative feature, suggesting a persistence mechanism rather than an antiapoptotic strategy. Th9 antitumor efficacy depended on interleukin-9 and upregulated expression of Eomes and Traf6. Thus, tumor-specific Th9 cells are a more effective CD4+ T cell subset for adoptive cancer therapy.

Background: Rhabdomyosarcoma (RMS) is the most common paediatric soft-tissue sarcoma. Approximately 15-20% of RMS cases arise from the bladder and prostate (B/P). The optimal treatment strategy for B/P RMS remains unclear. Objective: To retrospectively evaluate the applicability of our procedure performed to treat paediatric patients with B/P RMS. Design, setting, and participants: This is a retrospective analysis from a single tertiary referral hospital. From August 2003 to March 2021, 62 children pathologically diagnosed with B/P RMS underwent radical cystectomy and orthotopic detaenial sigmoid neobladder reconstruction in our centre. Surgical procedure: Surgical procedures included laparoscopic radical cystectomy and detaenial sigmoid neobladder reconstruction, which is demonstrated in the accompanying video. Measurements: Demographic, clinical, and follow-up data were collected. Perioperative and long-term oncological and functional outcomes were reported. A logistic regression analysis was also performed. Results and limitations: All surgeries, including three intracorporeal laparoscopic surgeries, were completed successfully. Of the 62 patients, 54 were alive without evidence of disease recurrence or metastasis at the last follow-up. Five of the 14 >12-yr-old boys reported that they experienced erections. Two female patients >12 yr old reported that they menstruated. However, this was a retrospective study conducted at a single centre with limited surgeon experience. Conclusions: Our results confirmed the safety and feasibility of primary orthotopic sigmoid neobladder reconstruction after radical cystectomy for paediatric patients with B/P RMS. Good outcomes in terms of oncological control and functional recovery were achieved. The high histocompatibility and tissue adaptability of children are inspiring. Patient summary: We describe our stepwise technique of radical cystectomy and detaenial sigmoid neobladder reconstruction for paediatric patients with bladder and prostate rhabdomyosarcoma. With this technique, we were able to achieve good functional recovery without compromising cancer control and significantly increasing complications.

Detailed experimental procedures of cell culture and shRNA-mediated knockdown, collection of Serum samples, analysis of published single-cell sequencing data of human cancers,Flow Cytometry, in vitro generation of TAMs,immunofluorescence microscopy,microarray analysis, construction of mouse lymphoma and multiple myeloma model.

Fig. S1, relates to Figure 1: Lipid accumulation in mouse TAMs. Fig. S2, relates to Figure 2: Expression of scavenger receptors in control MΦs and TAMs. Fig. S3, relates to Figure 2: Knockdown of Cd36 decreases lipid accumulation in MФs. Fig. S4, relates to Figure 3: Expression of fatty acid β-oxidation-responsible genes and the glucose metabolism in indicated MΦs. Fig. S5, relates to Figure 5: Ki-67 expression and cell cycle progression in different murine tumor cells cocultured with control MΦs or TAMs. Fig. S6, relates to Figure 6: The phosphorylation of JAK-STAT6 pathway in human MΦs and the production of IL-4 and IL-13 in human and mouse tumor cells. Fig. S7. Clinical significance of CD36 expression in TAMs. Fig. S8., relates to Figure 7: Effect of CD36 deficiency on the infiltration and differentiation of MΦs in vivo.

e16602 Background: Cisplatin-based neoadjuvant chemotherapy is the standard of care in MIBC with improved pathologic response (PaR) and overall survival (OS) compared to radical cystectomy (RC) alone. The efficacy of PD-1 inhibitor in combination with chemotherapy as a new neoadjuvant treatment option for MIBC still needs further confirmation. This study evaluated the efficacy and safety of toripalimab (an anti-PD-1 monoclonal antibody) combined with gemcitabine-cisplatin (GC) as neoadjuvant therapy for patients with MIBC. Methods: Eligible pts with MIBC (cT2-T4a, N≤1, M0) who were candidates for RC were enrolled. Toripalimab (240mg, d1) combined with GC (gemcitabine 1000mg/m 2 d1,d8 plus cisplatin 35mg/m 2 d1,d2) every 21 days, up to 4 cycles. RC was performed within 6 weeks after the last dose treatment. The primary endpoint was PaR (≤ pT1,N0). Secondary endpoints were PFS at 2 years and safety. Results: Between Apr 2021 and Jun 2022, 16 pts (4 pts cT2, 11 pts cT3, 1 pts cT4) were enrolled and received 4 cycles of neoadjuvant therapy, with median age of 63.5 and 75% male. 14 of the 16 pts underwent RC and two declined RC. Among 14 evaluable pts, PaR ≤pT1N0 was achieved in 12 pts (85.71%, 4 were pT0, 8 were pT1/a/is). Survival data are not yet mature. The most common adverse events (AEs) of any grade were anemia (14/16; 87.5%) and nausea/vomiting (11/16; 68.75%). One had Grade≥3 AEs (hemorrhagic shock) while on study that was unrelated to GC or toripalimab. Five pts experienced grade 1-2 immune related AEs, including thyroiditis (3/16, 18.75%) and rash (2/16,12.5%). Conclusions: Neoadjuvant toripalimab combined with GC showed promising safety and efficacy in MIBC with significant pathologic downstaging rates. Clinical trial information: NCT04861584 .

Macrophages activation is crucial in pathogenesis of rheumatic diseases like ankylosing spondylitis (AS). Circular RNAs (circRNAs)-induced macrophage-associated inflammation participates in many autoimmune diseases but remains elusive in AS. Here, we verified increased expression of circIFNGR2 in peripheral blood mononuclear cells from patients with AS and its expression levels were correlated with the AS severity. In vitro assays revealed that circIFNGR2 enhances macrophage proliferation, and regulates M1/M2 macrophage polarization and NF-κB/Akt pathways. We identified that circIFNGR2 promoted the expression of iNOS/TNFα and M1 polarization, and restrained M2 polarization by sponging miR-939. Additionally, the RNA-binding protein, eIF4A3, was found to enhance the production of circIFNGR2. Interestingly, miR-939 attenuated joint damage in collagen-induced arthritis mice, whereas circIFNGR2 reversed this effect. Our findings highlight the pro-inflammatory roles of eIF4A3-induced circIFNGR2 in AS by modulating macrophage-associated inflammation through miR-939.

Supplementary Table 1. Primer sequences used for qPCR and shRNA construction.

Supplementary Table 2. Microarray dataset information