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Dihydrouridine (D) is an abundant RNA modification, yet its roles in mammals remain poorly understood due to limited detection methods. We even do not have a comprehensive profile of D site location and modification stoichiometry in tRNA. Here, we introduce Chemical Reduction Assisted Cytosine Incorporation sequencing (CRACI), a highly sensitive, quantitative approach for mapping D at single-base resolution. Using CRACI, we generate the transcriptome-wide maps of D in both cytoplasmic and mitochondrial tRNAs from mammals and plants. We uncover D sites in mitochondrial tRNAs and identify DUS2L as the ‘writer’ protein responsible for human mitochondrial tRNAs. Furthermore, we demonstrate that most D modifications have a limited impact on tRNA stability, except for D20a, which also exhibits cis-regulation of adjacent D20 sites. Application of CRACI to human mRNA reveals that D modifications are present but rare and occur at very low stoichiometry. CRACI thus provides a powerful platform for investigating D biology across species. Dihydrouridine (D) is an abundant RNA modification but its functions are currently unclear. Here, authors develop CRACI, a sensitive method for transcriptome-wide, single-base D mapping, identifying sites across mammalian and plant transcriptomes and identifying DUS2L as a mitochondrial D writer.

Acute myeloid leukemia (AML) is the most common type of leukemia in adults. Despite advances in treatment, the average survival rate after diagnosis is low. AML also alters the patient's immune response further contributing to disease progression. The aim of this study was to analyze the expression of LAG-3, TIM-3 and PD-1 inhibitory checkpoints on NK cells and T cells from newly diagnosed AML patients and its impact in patient survival. NK cells and T cells from AML patients showed a lower expression of TIM-3 compared with healthy donors, whereas no significant differences were observed in the expression of LAG-3. The percentages of cells co-expressing these receptors showed a decrease in the percentage of LAG-3 + TIM-3 + PD-1 − NK and T cells from AML patients in comparison with healthy donors. Remarkably, the survival analysis of AML patients according to the expression of these receptors showed that higher expression of LAG-3 on NK cells and T cells was associated with better survival. In addition, those AML patients showing higher co-expression of LAG-3 and TIM-3 on NK and T cells had better survival (higher than 6 months) than those with lower co-expression of LAG-3 and TIM-3, therefore supporting that the expansion of NK and T cells with lower expression of these checkpoint receptors reflects a dysfunctional state associated with poor prognosis. This study identifies the expression of LAG-3 and TIM-3 checkpoints on NK and T cells as potential biomarkers of AML prognosis, contributing to define those patients that could benefit from checkpoint blockade therapies.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00262-025-04169-y.

IntroductionT cell hematological malignancies are aggressive blood cancers that remain challenging despite various treatments. Current chimeric antigen receptor (CAR)-T and natural killer (NK) therapies show potential but struggle with nonselective elimination during tumor targeting. Since CAR signal strength is determined by the single-chain variable fragment (scFv) and CAR expression levels, fine-tuning these parameters enables selective recognition of malignant cells while preserving normal cells. Here, we aimed to develop optimized CD5 CAR-NK cells (OptiCAR-NK) to achieve potent anti-tumor activity with minimized off-tumor toxicity.MethodsWe engineered CD5 CAR-NK cells with different scFv and CAR expression levels. CAR expression was modulated by single-cell isolation and mRNA transfection to assess activity against both malignant and normal T cells in vitro. Therapeutic efficacy and safety were further validated in xenograft and humanized mouse models.ResultsOptimization of scFv and CAR expression levels (OptiCAR-NK) enabled selective recognition of CD5+ malignant T cells while maintaining strong anti-tumor activity with minimal toxicity. Mechanistic analysis revealed that NK cells’ innate ability to discriminate malignant from normal T cells depends on fine-tuned CAR signal strength and endogenous ligands on target cells.DiscussionOptimized modulation of scFv and CAR expression is crucial for designing a CAR that achieves high anti-cancer efficacy and is safe in normal cells. Our results suggest a promising avenue for optimized CD5 CAR-NK cell therapy to manage T cell malignancies while minimizing off-tumor effects.

AbstractSeveral gene editing tools have entered the clinic, representing varied options for eliminating or correcting mutations. Although gene editing by homologous recombination (HR) can potentially accomplish any type of gene edit (insertions, deletions, and replacements), as the outcome is defined by a recombinant repair template, gene editing enzymes that support efficient HR are rare. ARCUS nucleases, engineered from the homing endonuclease I-CreI, have programmable sequence specificity and support precise, high-frequency transgene insertion. In this study, we demonstrate that the 3′ overhangs that ARCUS nucleases generate when cutting DNA are key to triggering high rates of HR. We show that a single editor can be used to accomplish the full range of currently understood DNA editing approaches, allowing all combinations of single base changes, introducing small, specific deletions, small and large insertions, and the ability to replace large segments of genomic DNA with efficiencies ranging from 60% to 90% in lymphocytes. ARCUS also supports precise, efficient insertion (30%–40%) in noncycling hepatocytes via nonclassical HR pathways. Collectively, this work characterizes a flexible and efficient gene insertion system for potential therapeutic use. Graphical Abstract Graphical Abstract

Immunotherapy has emerged as a promising strategy against cancer, but many patients fail to achieve durable responses. Inefficiency of immunotherapy is often caused by the immunosuppressive tumor microenvironment. Previously, we demonstrated that treatment with an FcαRI-stimulating bi-specific antibody (BsAb), designed to recruit myeloid cells as cytotoxic effector cells, significantly decreased tumor growth in a murine cancer model. Nonetheless, complete tumor eradication was not achieved. In this study, we investigated if co-treatment with the pro-inflammatory cytokine TNF-α enhances the therapeutic efficacy of FcαRI BsAb. Although TNF-α did not affect antibody-dependent cellular phagocytosis (ADCP) of tumor cells, macrophage polarization, or antibody-dependent cellular cytotoxicity (ADCC) by natural killer cells, its combination with FcαRI BsAb increased tumor cell trogocytosis, neutrophil degranulation and tumor cell death. To exploit this synergy, we engineered a TNF-α x FcαRI bi-specific immunocytokine (FcαRI-TNF). Surface plasmon resonance and cellular binding assays demonstrated that FcαRI-TNF retained binding affinities for FcαRI, FcɣRIII, and the tumor-associated antigen EGFR comparable to FcαRI BsAb. Consistent with the combination of TNF-α and FcαRI BsAb, FcαRI-TNF neither influenced macrophage function nor polarization but enhanced neutrophil-mediated tumor killing in vitro. Intravital imaging in a murine MC38-cEGFR tumor model showed that FcαRI-TNF promoted in vivo neutrophil activation and swarming behavior. These findings suggest that FcαRI-TNF represents a promising candidate to improve neutrophil-driven immunotherapy of cancer.

KANSL3 is an epigenetic regulator of hepatocyte metabolism and differentiation, essential for liver development and key to maintaining liver health. Liver disease is a leading cause of mortality worldwide. Emerging evidence highlights the significant role of epigenetic regulation in sustaining liver homeostasis, providing new therapeutic strategies for liver disease. Hepatocyte-specific deletion of the epigenetic regulator KANSL3, a key component of the NSL complex, results in early-onset liver disease marked by biliary hyperplasia and hepatic fibrosis. KANSL3 is essential for regulating hepatocyte transcriptional networks important for hepatic steroid and lipid metabolism through histone acetylation. Moreover, single-cell RNA sequencing demonstrated that the loss of KANSL3 disrupts the differentiation of hepatocytes in vivo. The transcriptional programs necessary for hepatocyte differentiation of ductal and fetal liver organoids were severely compromised in the absence of KANSL3. These findings collectively demonstrate a crucial role of the epigenetic regulator KANSL3 in hepatocyte differentiation in liver development and disease.

PurposeOculocutaneous albinism type 1A (OCA1A) is a rare recessive genetic condition caused by mutations in TYROSINASE (TYR) that results in pigmentation defects of the skin, hair and eyes. This study was performed to understand melanosome biogenesis and maturation defects in an OCA1A in vitro model using retinal pigment epithelium (RPE) derived from TYR knockout human induced pluripotent stem cells (iPSC).MethodsCRISPR-Cas9 was used to knockout the TYR gene in iPSC to generate an isogenic pair. A developmentally guided protocol was used to differentiate the isogenic iPSC pair towards RPE monolayer tissue. Monolayer organization, melanosome formation and maturation were studied using electron microscopy. Loss of TYR protein was studied using Western blot and immuno-fluorescence staining. RPE cellular morphology and junction integrity was studied using immunofluorescence staining and transepithelial resistance measurements.ResultAn isogenic pair comprising of untargeted control and TYR knockout iPSC were successfully differentiated towards RPE monolayer tissue with polygonal cell morphology. TYR knockout RPE exhibited significantly reduced TYR protein, increased presence of immature pre-melanosomes and a complete lack of mature melanosomes. We observed abnormal junctional localization of β-catenin staining pattern, as has been reported previously for albino mouse RPE– and OCA1A patient–derived RPE.ConclusionsDifferentiation of TYR-deficient iPSC toward RPE displayed pigmentation defects and absence of mature melanosomes, whereas melanosome biogenesis was not affected, because pre-melanosomes were still observed. These observations were also similar to what was observed in OCA1A patient–derived RPE monolayer tissue, independently confirming the validity of these previous findings.

BackgroundOvarian cancer (OC) remains the deadliest gynecological cancer, primarily due to late-stage diagnosis and high rates of chemotherapy resistance and recurrence. Lack of representative preclinical models complicate the challenges of discovering effective therapies, especially for platinum-resistant OC. Patient-derived xenograft (PDX) models maintain the genetic characteristics of the original tumor and are ideal for testing candidate therapies in vivo, but their high cost limits their feasibility for high-throughput drug screening. Organoid models mimic the tumor’s 3D structure and preserve intra-tumoral heterogeneity. While organoids established directly from primary patient tumors are the optimal model for personalized drug response studies, the supply of primary tissue is often limited. Patient-derived xenograft tumors can be passaged in mice and provide a renewable source of cancer cells for organoids. This study aimed to determine if PDX-derived organoids (PDXOs) can reflect patient responses to chemotherapy similarly to primary patient-derived organoids (PDOs).Methods3D Organoid models were established from the malignant ascites of five high grade serous ovarian cancer patients: two platinum-sensitive, two platinum-resistant, and one platinum-refractory, along with their matched PDX samples from ascites and solid tumor. Organoid viability after 72-hour treatment with paclitaxel (PTX), carboplatin (CBDCA), or their combination was compared between organoids derived directly from the patient or from the PDX models. The in vitro drug responses of PDXOs and PDOs were then compared to defined patient clinical responses: platinum-sensitive (initial response to standard platinum/paclitaxel therapy lasting > 6 months post-treatment), platinum-resistant (initial response to standard chemotherapy lasting < 6 months), or platinum-refractory (no initial response to standard chemotherapy).ResultsIn drug response assays, PDXOs and PDOs demonstrated similar sensitivity to standard chemotherapy and also reliably reflected patient responses based on the clinical designation of platinum sensitivity. While organoids derived from the ascites were smaller with a denser morphology, their drug response mirrored that of the organoids derived from solid tumor. Platinum-sensitive cases exhibited significant reductions (around 50% reduction) in organoid viability when treated with carboplatin, paclitaxel, or their combination. Platinum-resistant or refractory organoids showed little to no reduction in viability with carboplatin or paclitaxel monotherapy or the combination. Organoids derived from one platinum-resistant case did show a small but significant reduction in viability with single-agent paclitaxel, suggesting that organoid models might predict response to second-line paclitaxel therapy.ConclusionThis study demonstrates that PDXOs respond to drugs similarly to PDOs and confirms that both models effectively mirror patient response to standard chemotherapy. This highlights the potential of PDXOs as renewable models for screening novel therapies and developing personalized strategies in OC.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12885-025-14811-8.

Pulmonary fibrosis (PF) is a progressive disease histologically defined by pathological fibroblasts and epithelial cells. PF lungs contain alveolar type 2 epithelial cells (AT2s) that acquire an aberrant intermediate state phenotype. However, the direct role of these cells in PF and the signals causing them to arise and persist are not fully known. To address this, we harness the SftpcC121G mouse model, where expression of a PF-associated mutation in the AT2-specific surfactant protein C (Sftpc) gene results in AT2 dysfunction and spontaneous lung fibrosis. Here, we identify aberrant intermediate epithelial cells in SftpcC121G lungs that share transcriptional profiles with human PF aberrant basaloid cells and develop a profibrotic interactome with fibroblasts. We develop a sorting method to enrich for these cells, and through ex vivo assays, identify a profibrotic secretome mediated by TGF-β signaling. Coupling this murine model with a newly developed patient-specific induced pluripotent stem cell-derived mutant SFTPC model, we discover that human SFTPC-mutant AT2s express an aberrant basaloid program, and that loss of canonical progenitor signals coupled with TGF-β stimulation causes AT2s to enter this state. We conclude that aberrant intermediate epithelial cells drive pathogenic fibroblast activation, and that reciprocal signaling contributes to their entry into this profibrotic state. The direct contribution of aberrant epithelial cells to lung fibrosis is largely unknown. The authors use murine and human systems to identify how these cells activate fibroblasts, and how reciprocal signals cause them to enter a profibrotic state.

Neoadjuvant chemoimmunotherapy offers promise to improve outcomes for patients with resectable non-small cell lung cancer (NSCLC). Yet not all patients derive treatment benefits, and reliable biomarkers of response are still lacking. We here assess the long-term clinical outcome of neoadjuvant chemotherapy and perioperative anti-PD-L1 inhibition in resectable stage IIIA NSCLC in the SAKK 16/14 trial and provide a comprehensive characterization of anti-tumor immune responses for biomarker-based treatment personalization. We report secondary outcomes of median event-free survival (EFS) of 4.0 years and median overall survival not being reached after a median follow-up of 5.4 years. Computer-aided spatial image analysis emphasizes the importance of CD8+ T cell positioning in tumors, and larger tertiary lymphoid structures in pre-treatment biopsies correlate with improved EFS. Genomic techniques reveal the association of intratumoral TCR diversity with response. Finally, circulating proliferating CD39+ PD-1+ CD8+ T cells and elevated levels of CCL15 post-treatment are seen in patients with sustained therapeutic benefit. NCT02572843. Neoadjuvant immunochemotherapy against NSCLC has been tested in clinical trials. Here, the authors follow up longer-term survival and measure immune cell phenotype changes in a single-arm phase II clinical trial of neoadjuvant immunochemotherapy, indicating association of intratumoural TCR diversity and CD8 T cell positioning.

BACKGROUNDResearch has been increasingly conducted on the connection between mesenchymal stem cell (MSC)-conditioned medium (MSC-CM) and aging. However, most studies have focused on adipose-derived MSC-CM (ADMSC-CM), resulting in a research bias. We hypothesized that umbilical cord-derived MSCs, being younger than adipose-derived MSCs, would be more suitable for overcoming aging-related processes.AIMTo assess the efficacy and safety of umbilical cord-derived MSC-CM (UCMSC-CM) for preventing and treating skin aging.METHODS In vitro and in vivo studies were conducted to compare UCMSC-CM with ADMSC-CM, the most studied active aging-preventive conditioned medium to date. Additionally, the most effective delivery method of UCMSC-CM for aged skin was identified.RESULTSUCMSC-CM had a higher content of effective factors, stimulated higher proliferation of fibroblasts, and strongly inhibited melanin production in B16F1 cells. In aged mice, UCMSC-CM application increased skin thickness, the number of Ki-67-positive cells, and the area of collagen deposition. UCMSC-CM was more effective than ADMSC-CM in preventing and treating skin aging. Additionally, a safety evaluation of UCMSC-CM performed in various animal models indicated that it was safe even when used directly on the skin.CONCLUSIONUCMSC-CM is effective and safe for preventing and treating skin aging.

BackgroundRelapsed and refractory multiple myeloma (RRMM) remains a major clinical challenge, as most patients eventually relapse following standard treatments and are left with limited therapeutic options. Although b-cell maturation antigen (BCMA) CAR-T cell therapy has recently shown remarkable efficacy in select patients, broader implementation is hindered by its reliance on autologous cells, prolonged manufacturing timelines, high costs, and severe immune-related toxicities. These challenges have prompted an urgent demand for safer, more accessible, and rapidly applicable immunotherapeutic alternatives.MethodsCBMC (cord blood mononuclear cells) were cultured with irradiated BMMC (bone marrow mononuclear cells) from RRMM patients in the presence of defined cytokines, aiming to develop a new therapeutic immune cell product for RRMM. Their phenotypic and functional characteristics, including non-MHC-restricted and MHC-restricted cytotoxicity mechanisms, were analyzed using surface marker profiling, cytokine secretion assays, in vitro cytotoxicity assays, functional and blocking assays. Antitumor activity was evaluated in xenograft mouse models using MM.1 S and RPMI-8226 cells.ResultsWe successfully generated CD8+ NKT-like cells through tumor priming, which exhibited potent cytotoxicity and elevated cytokine production against multiple myeloma cell lines and primary RRMM samples. Mechanistically, tumor-priming CD8+ NKT-like cells (TPNC) cytotoxicity was mediated by both non-MHC–restricted pathways involving LFA-1 and DNAM-1, and MHC-restricted, TCR-mediated recognition. TPNC efficiently formed immune synapses, rapidly polarized cytotoxic granules, and engaged in serial killing. In xenograft models, TPNC significantly suppressed tumor progression, prolonged survival, and persisted in circulation without observable toxicity. Based on these findings, we extended the tumor-priming strategy to acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL), successfully generating TPNC with robust cytotoxic activity. In ALL samples, TPNC exhibited cytotoxicity comparable to anti-CD19 CAR-NK cells.ConclusionsTPNC represents a novel cytotoxic lymphocyte product generated through tumor-driven priming. Their dual recognition capacity, functional versatility, and favorable safety profile highlight their potential as a scalable and personalized immunotherapy platform for hematologic malignancies.Supplementary InformationThe online version contains supplementary material available at 10.1186/s40164-025-00707-7.