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High-grade gliomas (HGGs) are aggressive brain tumors with limited treatment options and poor survival outcomes. Variants including isocitrate dehydrogenase (IDH)-wildtype, IDH-mutant, and histone 3 lysine to methionine substitution (H3K27M)-mutant subtypes demonstrate considerable tumor heterogeneity at the genetic, cellular, and microenvironmental levels. This presents a major barrier to the development of reliable models that recapitulate tumor heterogeneity, allowing for the development of effective therapies. Glioma tumor organoids (GTOs) have emerged as a promising model, offering a balance between biological relevance and practical scalability for precision medicine. In this study, we present a refined methodology for generating three-dimensional, multiregional, patient-derived GTOs across a spectrum of glioma subtypes (including primary and recurrent tumors) while preserving the transcriptomic and phenotypic heterogeneity of their source tumors. We demonstrate the feasibility of a high-throughput drug-screening platform to nominate multi-drug regimens, finding marked variability in drug response, not only between patients and tumor types, but also across regions within the tumor. These findings underscore the critical impact of spatial heterogeneity on therapeutic sensitivity and suggest that multiregional sampling is critical for adequate glioma model development and drug discovery. Finally, regional differential drug responses suggest that multi-agent drug therapy may provide better comprehensive oncologic control and highlight the potential of multiregional GTOs as a clinically actionable tool for personalized treatment strategies in HGG.

BackgroundNeuroendocrine prostate cancer (NEPC) is an aggressive subtype of prostate cancer with limited therapeutic options. Although cisplatin is recommended as a first-line treatment, its clinical efficacy is hindered by the rapid development of drug resistance, highlighting the urgent need for effective strategies to overcome cisplatin resistance.MethodsWe established a NEPC mouse allograft model and performed RNA sequencing to identify genes associated with cisplatin resistance. The role of CCL5 in tumor-stromal crosstalk was investigated using immunofluorescence, ELISA assays, co-culture assays, and CCL5 knockout mice. Mechanistic studies were conducted to explore CCL5/CCR5-mediated signaling pathways. The therapeutic efficacy of cisplatin combined with maraviroc, an FDA-approved CCR5 antagonist, was evaluated in vitro using NEPC cell lines and patient-derived organoids, and in vivo using NEPC mouse models.ResultsHere, we identify a tumor-stromal interaction mediated by the CCL5/CCR5 axis that drives cisplatin resistance in NEPC. Cisplatin-induced DNA damage promotes a cGAS-STING–dependent senescence program in cancer-associated fibroblasts (CAFs), resulting in the secretion of CCL5, a key senescence-associated secretory phenotype factor. CCL5 from CAFs binds to CCR5 on tumor cells, promoting the formation of a CCR5/β-arrestin1/p85 complex that activates the PI3K/AKT pathway. This activation enhances DNA repair, protecting tumor cells from cisplatin-induced apoptosis. Pharmacologic inhibition of the CCL5/CCR5 pathway using maraviroc, an FDA-approved CCR5 antagonist, sensitizes NEPC cells to cisplatin treatment and significantly prolongs survival in NEPC mouse models.ConclusionsOur findings identify the CCL5/CCR5 axis as a key mediator of tumor-stromal crosstalk driving cisplatin resistance in NEPC. Mechanistically, CAF-derived CCL5 activates AKT signaling in tumor cells by promoting the formation of the CCR5/β-arrestin1/p85 complex. Targeting this pathway with maraviroc in combination with cisplatin offers a promising therapeutic strategy for overcoming drug resistance in NEPC.Graphical Abstract Supplementary InformationThe online version contains supplementary material available at 10.1186/s13046-025-03552-y.

Human NK cells exhibit functional heterogeneity, yet the clinical conditions and cellular processes driving this diversity remain insufficiently understood. Here, we report that NK cell diversity emerges during peripheral development, influenced by the divergence of adaptive NK cells from various stages of canonical NK cell maturation during the resolution phase of severe COVID-19. Using scRNA-seq, we analyzed blood NK cells from patients with severe COVID-19 (acute phase and post-intensive care), individuals vaccinated with the SARS-CoV-2 mRNA vaccine (BNT162b2), and healthy donors. The frequencies of immature CD56bright and proliferating NK cells increased following BNT162b2 vaccination. In contrast, the frequency of adaptive CD56dim cells was markedly elevated in patients recovering from severe COVID-19, alongside clonal expansion and enhanced mitochondrial oxidative phosphorylation. Trajectory analysis revealed a bifurcation in peripheral NK cell development, with CD56bright cells diverging into canonical and adaptive CD56dim subsets during the course of severe COVID-19. Notably, adaptive NK differentiation exhibited transcriptional and signaling profiles analogous to those of T-cell activation. Thus, NK cell diversity is shaped by the induction of an intrinsic adaptive program. These findings uncover the mechanisms underlying NK cell heterogeneity and have implications for medical applications, including the development of immunotherapies that leverage adaptive NK cell functions.

Osteosarcoma (OS) is a relatively rare bone malignancy that primarily affects children and young adults and is associated with significant morbidity and mortality. Cisplatin is a mainstay of treatment, but its efficacy is limited by off-target toxicities. Immunotherapy is not effective due to a poor antigenic tumor microenvironment. Here, we address these challenges by using manufactured M1 macrophage-derived vesicles (MVs) loaded with cisplatin. Human blood and mouse RAW 264.7 M1 macrophages were used to prepare empty (E-MVs) and cisplatin-loaded MVs (C-MVs). Human OS cell lines were used in vitro and in a tibia xenograft mouse model to evaluate the anti-cancer and immune-stimulating abilities of MVs. C-MVs had lower IC50s but equivalent DNA damage in OS cell lines when compared with free cisplatin. E-MVs and C-MVs were observed to accumulate in the tumor in OS tumor-bearing mice. C-MVs significantly reduced tumor burden and prolonged survival in a mouse model of OS. Animals dosed with free cisplatin experienced weight loss and renal and hepatic toxicity, while equivalent doses of C-MVs did not cause these effects. In addition, both E-MVs and C-MVs showed immunomodulation of the tumor microenvironment with a significant increase in the M1/M2 macrophages ratio (7-fold and 22-fold, respectively) and increased levels of TNF-α in serum (1.8-fold and 2.1-fold, respectively) compared to control mice. Collectively, these experiments support further development of C-MVs for the treatment of OS.

While single-cell RNA sequencing has advanced our understanding of cell fate, identifying molecular hallmarks of potency—a cell’s ability to differentiate into other cell types—remains a challenge. Here we introduce CytoTRACE 2, an interpretable deep learning framework for predicting absolute developmental potential from single-cell RNA sequencing data. Across diverse platforms and tissues, CytoTRACE 2 outperformed previous methods in predicting developmental hierarchies, enabling detailed mapping of single-cell differentiation landscapes and expanding insights into cell potency. CytoTRACE 2 is an interpretable deep learning framework that leverages single-cell RNA sequencing data to predict absolute developmental potential across datasets.

BCL6 limits CD8 T-cell responses to tumors by inhibiting Glut3 expression. Conditional deletion of Bcl6 in activated CD8 T cells or pharmacological inhibition of BCL6 in mice represses tumor growth. T cells are one of the most powerful weapons to fight cancer; however, T-cell exhaustion and dysfunction restrict their long-lasting function in antitumor immunity. B-cell lymphoma 6 (BCL6) has many functions in CD8 T cells; however, it is unclear how it regulates the effector function and exhaustion of CD8 cells. Overall, a low level of BCL6 mRNA in human cancer samples is associated with better outcomes, but high expression of BCL6 is specifically observed in cytotoxic CD8 T cells. We found that BCL6 deficiency in activated CD8 T cells enhanced tumor repression in multiple mouse models. More IL-2–expressing CD8 T cells and reduced proportions of exhausted or dysfunctional CD8 T cells were detected within tumors when Bcl6 was knocked out upon T-cell activation. Glycolysis was promoted, and GLUT3 expression was derepressed in BCL6-deficient CD8 T cells. The BCL6 inhibitor Fx1 promoted antitumor immunity in a T cell–dependent manner. These findings suggest a novel pathway to restore effector function of CD8 T cells by changing their energy use pathways to facilitate long-term tumor resistance. Graphical Abstract

Recent advances in single-cell technologies have provided limited insight into the role of protein phosphorylation in T-cell fate and function. Dysregulated protein phosphorylation is associated with adverse clinical outcomes, emphasizing the need for reliable methods to unravel the complexities of T-cell signal transduction and disease-related alterations. While flow cytometry is widely used, it is constrained by spectral overlap, limiting the number of protein targets for simultaneous analysis. To overcome this, we present a robust protocol for whole blood T-cell stimulation and subsequent analysis using mass cytometry by time-of-flight (CyTOF). CyTOF minimizes spillover into adjacent channels by employing highly pure, stable, heavy metal–conjugated antibodies for protein detection. This protocol offers a high-dimensional approach for phenotypic and phospho-protein characterization of key signaling pathways, including JAK/STAT, MAPK, PI3K/mTOR, PKC, and NF-κB. A key feature is the T-cell stimulation reagent, which mimics endogenous activation by engaging the T-cell receptor (TCR)/CD3 complex and providing co-stimulation via an anti-CD28 antibody. Further, we enhance reproducibility and enable batch processing through the implementation of the Prot1/Thaw-Lyse system for immediate cryopreservation of stimulated blood samples. By employing CyTOF, this method permits the simultaneous analysis of 31 protein targets with single-cell resolution, minimizing spillover and providing superior specificity, sensitivity, and resolution over flow cytometric methods. This approach facilitates the robust assessment of TCR activation and its effect on bystander populations, which has been challenging with spectral flow cytometry due to the limited availability of methanol-resistant fluorophores. This protocol is a precise and reproducible method for elucidating the downstream effects of T-cell stimulation and immune status, with significant potential for clinical applications, including the assessment of T-cell-targeted therapies. Key features • Mass cytometry (CyTOF) analysis: A "vein-to-tube" protocol for high-dimensional profiling of key signaling pathways (JAK/STAT, MAPK, PI3K/mTOR, PKC, NF-κB) in human whole blood.• Cryopreservation for batch processing: Enables long-term storage and standardized batch processing of whole blood samples by implementing immediate cryopreservation via the Prot1/Thaw-Lyse system.• Assessment of early T-cell activation: This workflow involves a rapid, 15-min stimulation length using a proprietary T-cell activator that provides TCR engagement and co-stimulation.• Expertise required: Intended for users experienced in surface and intracellular antibody staining techniques for cytometric applications.

Human induced pluripotent stem cell (hiPSC)-derived motor neurons (MNs) provide a critical source for the study of motor neuron diseases (MNDs), which has been hindered by the lack of appropriate disease models for many years. Although many spinal MN differentiation protocols have been established by mimicking in vivo neurogenesis using extrinsic signaling molecules, substantial variations in the duration and efficiency persist due to inconsistencies in concentrations, timing, and delivery methods of these molecules. Here, we present an efficient monolayer culture differentiation strategy that enables the generation of enriched CHAT+ spinal MNs (sMNs) in 18 days and functional sMNs exhibiting extensive network activities, as confirmed by multielectrode array (MEA), within 28 days. Therefore, this optimized MN differentiation protocol facilitates the production of mature sMNs for MND research, high-throughput drug screening, and potential cell replacement therapies. Key features • This protocol provides a rapid and simple monolayer culture strategy for differentiating hiPSCs into sMNs in 18 days. • Early administration of the Notch inhibitor Compound E accelerates the generation of hiPSC-sMNs 10 days in advance compared to a previous protocol. • This protocol uses neural stem cells (NSCs) and MNPs as an intermediate to generate functionally mature sMNs.

Computational trajectory analysis is a key computational task for inferring differentiation trees from this single-cell data. An open challenge is the prediction of complex and multi-branching trees from multimodal data. To address these challenges, we present PHLOWER (decomposition of the Hodge Laplacian for inferring trajectories from flows of cell differentiation), which leverages the harmonic component of the Hodge decomposition on simplicial complexes to infer trajectory embeddings from single-cell multimodal data. These natural representations of cell differentiation facilitate the estimation of their underlying differentiation trees. We evaluate PHLOWER through benchmarking with multi-branching differentiation trees and using kidney organoid multimodal and spatial single-cell data. These demonstrate the power of PHLOWER in both the inference of complex trees and the identification of transcription factors regulating off-target cells in kidney organoids. Thus, PHLOWER enables inference of complex branching trajectories and prediction of transcriptional regulators by leveraging multimodal data. PHLOWER leverages single-cell multimodal data to infer complex, multi-branching cell differentiation trajectories.

NK cells are innate lymphoid cells that can acquire a memory-like phenotype in vitro when stimulated with IL-12, IL-15, and IL-18. These cytokine-induced memory-like (CIML) NK cells exhibit prolonged lifespan and increased cytotoxicity, making them ideal for immunotherapy. This study characterizes two CIML NK cell subsets based on CD16 expression. NK cells were isolated from the peripheral blood of healthy donors and stimulated overnight to induce a memory-like phenotype. After seven days, we analyzed the phenotype and degranulation potential of CD16−/CD56 + and CD16+/CD56 + cells. The subsets were purified by fluorescence-activated cell sorting (FACS) and examined using high-throughput multiplexed quantitative proteomics. CD16 − cells showed higher levels of activating receptors, increased Granulysin expression, and lower inhibitory receptor expression compared to CD16 + cells. Functionally, CD16 − cells exhibited greater degranulation capacity, as determined by CD107a/b expression, when co-incubated with K562 and melanoma cells. Proteomic profiling identified 35 differentially expressed proteins out of 4,750, with 22 downregulated and 13 upregulated in the CD16 − subset. Key proteins included Granzyme family proteins, NCAM1, CALM1, CD247, and Fc receptors. This study provides a detailed characterization of CIML NK cells based on CD16 expression. Our findings highlight the molecular and functional diversity of CIML NK cells and may guide improved cancer immunotherapy strategies.

IntroductionTumor-associated macrophages (TAMs) promote immunosuppression, hindering immune checkpoint blockade and immunotherapy efficacy. To overcome this, we developed a novel multifunctional nanovaccine based on hepatitis B core virus-like particles (HBc VLP) to synergistically remodel the immunosuppressive tumor microenvironment through integrated TAM reprogramming and B7-H3 checkpoint blockade.MethodsThe core VLP co-displayed tumor antigen peptide MAGE-A10 and TAM-targeting peptide M2pep via fusion expression. Immunostimulatory CpG oligodeoxynucleotide 1826 (CpG) was encapsulated within VLP. Anti-B7-H3 antibody (αB7-H3) and polyethylene glycol (PEG) were chemically conjugated to the surface for checkpoint blockade and prolonged circulation, forming CpG@VLP-αB7-H3-PEG.ResultsStructural characterization using transmission electron microscopy and dynamic light scattering confirmed the hollow spherical self-assembly of VLP. Nanovaccines efficiently targeted TAMs in vitro and in vivo. Following CpG encapsulation (5.60 µg/mg), the nanovaccine reprogrammed M2-like TAMs into an M1-like phenotype. This was achieved by elevating the M1/M2 ratios of CD86/CD206 and MHC II/CD206 to 15.50-fold and 3.11-fold, respectively, as determined by flow cytometry. Further conjugation of αB7-H3 (250 µg/mg) significantly enhanced T-cell activation in TAM-T cell co-culture assays. In B16-F10 melanoma-bearing mice, reprogrammed iNOS+ M1-like macrophages triggered robust antitumor immunity, achieving a tumor inhibition rate of 63.47%. These macrophages also function as antigen-presenting cells and increase the proportion of tumor-infiltrating Granzyme B+CD8+ T cells. αB7-H3 conjugation further boosted infiltrating immune cells, M1-like macrophages, activated CD69+CD4+/CD8+ T cells, and cytotoxic T lymphocytes. PEGylation amplified systemic tumor-specific immunity and increased tumor inhibition by 80.12%.ConclusionThis HBc VLP-based nanovaccine constitutes a pioneering multifunctional platform designed to overcome TAM-mediated immunosuppression through synergistic integration of three modalities: antigen presentation, TAM phenotype reprogramming, and B7-H3 checkpoint blockade. To the best of our knowledge, this is the first nanovaccine architecture to enable coordinated immunomodulation. Its modular design supports the clinical translation of solid tumors and personalized immunotherapy. Graphical Abstract

Immune checkpoint inhibitors (ICIs) extend survival in many patients with cancer but are ineffective in patients without pre-existing immunity1–9. Although personalized mRNA cancer vaccines sensitize tumours to ICIs by directing immune attacks against preselected antigens, personalized vaccines are limited by complex and time-intensive manufacturing processes10–14. Here we show that mRNA vaccines targeting SARS-CoV-2 also sensitize tumours to ICIs. In preclinical models, SARS-CoV-2 mRNA vaccines led to a substantial increase in type I interferon, enabling innate immune cells to prime CD8+ T cells that target tumour-associated antigens. Concomitant ICI treatment is required for maximal efficacy in immunologically cold tumours, which respond by increasing PD-L1 expression. Similar correlates of vaccination response are found in humans, including increases in type I interferon, myeloid–lymphoid activation in healthy volunteers and PD-L1 expression on tumours. Moreover, receipt of SARS-CoV-2 mRNA vaccines within 100 days of initiating ICI is associated with significantly improved median and three-year overall survival in multiple large retrospective cohorts. This benefit is similar among patients with immunologically cold tumours. Together, these results demonstrate that clinically available mRNA vaccines targeting non-tumour-related antigens are potent immune modulators capable of sensitizing tumours to ICIs. mRNA vaccines targeting SARS-CoV-2 also sensitize tumours to immune checkpoint inhibitors.