Inflammatory bowel disease (IBD) is characterized by chronic inflammation and impaired immune tolerance, for which current therapies provide only partial and transient relief. Here, we introduce PrEXO-a23, a biomimetic nanotherapeutic engineered by fusing regulatory T cell (Treg)-derived exosomes with platelet membrane vesicles and conjugating interleukin-23 (IL-23) antibodies via a matrix metalloproteinase (MMP)-cleavable linker. This design exploits the inherent homing ability of platelets and Tregs, enabling PrEXO-a23 to preferentially accumulate in inflamed colonic tissues in murine IBD models. At the disease site, elevated MMP activity triggers antibody release to inhibit IL-23-mediated inflammation, while exosomal cargo reprograms dendritic cells and promotes Treg expansion, thereby restoring immune tolerance. This dual-action strategy significantly alleviates IBD, prevents complications like intestinal fibrosis and colitis-associated colorectal cancer, and shows p53-dependent efficacy in carcinogenesis prevention. These findings highlight PrEXO-a23 as a promising nanotherapeutic platform for durable immune reprogramming and long-term IBD management.

Raman spectroscopy delivers label-free, non-destructive, single-cell measurements and directly senses intrinsic biochemical signals of nucleic acids, proteins, and lipids. Conventional semen analysis offers limited prognostic power for assisted reproductive technology (ART). We developed a single-cell, label-free Raman workflow integrated with artificial intelligence (AI) to assess sperm functional status and predict outcomes of ART. Spectra from sperm of thirty-one ART patients were processed to extract diagnostically informative bands spanning nucleic acids, proteins, lipids, and glycogen. Spectral analysis resolved two robust spectral subpopulations. The subpopulation exhibiting higher chromatin integrity, more favorable protein conformation, and better membrane lipid dynamics molecular features was associated with better outcomes, higher two-pronuclear (2PN) fertilization rate (76.08% vs 50.17%, P = 0.044), high-quality embryo rate (40.53% vs 17.57%, P = 0.010), and blastocyst formation (59% vs 9%, P < 0.001). Among evaluated classifiers, a gated recurrent unit (GRU) model showed the best predictive performance (accuracy 94%, sensitivity 94%, AUC 0.98). This Raman-AI assay enables objective, non-invasive stratification of sperm quality and clinically relevant prognosis, offering an analytically rigorous tool to guide precise sperm selection in ART.

Cerebrospinal fluid (CSF) is central to neurological diagnostics, yet biomarkers are lacking for many clinical needs. To enable its large-scale proteomic characterization, we developed a high-throughput mass spectrometry workflow quantifying approximately 1,500 proteins per CSF sample across 5,000 individuals, covering a spectrum of neurological disorders. This revealed proteomic alterations associated with blood-CSF barrier impairment, age, and sex, enabling deconvolution of shared and disease-specific signatures. We then focused on multiple sclerosis (MS), using an improved analytical technology that quantified 2,100 proteins per sample. From these data, we derived a 22-protein panel that distinguished MS from related inflammatory diseases and outperformed established markers in challenging cases. A targeted mass spectrometry assay using isotope-labeled standards validated this panel in an independent cohort, offering a clinically compatible format. Additionally, we highlight proteins of therapeutic interest and demonstrate proteome-based staging of individuals along the relapsing-progressive MS spectrum, which correlates with clinical outcomes.

Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer. Although an immune checkpoint blockade can reduce metastasis, its effectiveness is hindered by the immunosuppressive microenvironment in TNBC. EZH2 is overexpressed in TNBC, and patients with high EZH2 expression are associated with poor prognoses. The study developed EIP103 as a first-in-class peptide degrader that targets EZH2 through multivalent, high-affinity interactions and induces conformational destabilization, representing a mechanism distinct from that of the small molecule inhibitor EPZ-6438. The results demonstrated that EIP103 induces immunogenic cell death through lipid peroxidation, resulting in enhanced immune cell infiltration. Additionally, molecular dynamics (MD) simulations and biochemical assays revealed that the peptide EIP103 binds to the SET domain of EZH2, altering its structure and triggering proteasomal degradation via Praja Ring Finger Ubiquitin Ligase 2 (PJA2)-mediated ubiquitination. Harboring both enzymatic inhibition and post-translational regulation properties, EIP103 exerts durable efficacy and activates antitumor immunity, making it a promising therapeutic candidate for TNBC.

We present FoxNovo, a hybrid deep learning-combinatorial framework for de novo sequencing of immunopeptides trained on a large-scale HLA-I immunopeptidomics dataset assembled and reprocessed from public mass spectrometry (MS) repositories. This integration achieved >90% peptide accuracy on the reported benchmarks while enabling repository-scale analysis at ~2,800 spectra per second—more than 100-fold faster than the evaluated beam-search baseline under the reported benchmark conditions. To mimic the heterogeneous spectral quality encountered in experimental MS analyses, we constructed controlled peak-removal stress tests, in which FoxNovo retained higher accuracy than the evaluated methods at different simulation levels. We subsequently re-analyzed 168 million spectra from all collected 4,423 MS raw files in only 18 hours on a single GPU, equivalent to ~245 raw files per hour. This repository-scale application yielded score-filtered canonical and putative ncORF-mapped peptide predictions and recovered 41 of 42 non-canonical HLA-I peptides previously validated by targeted MS. FoxNovo demonstrates the potential of integrating AI with combinatorial decoding for scalable immunopeptidomics. The source code is available at https://github.com/fennomix/fennomix.novo.

Background Preserving dental pulp vitality is crucial for maintaining the physiological function of the tooth. Naringenin (Nar), known for its immunomodulatory properties, has shown pharmacological effects on various inflammatory diseases. This study aimed to investigate the therapeutic potential of Nar in treating Porphyromonas gingivalis lipopolysaccharide (P.g-LPS)-induced pulpitis in a swine model and to elucidate the mechanisms underlying its therapeutic efficacy. Methods Swine premolars with P.g-LPS-induced pulpitis were divided into four groups: sham, hydrogel, iRoot BP PLUS, and Nar hydrogel. Treatment outcomes were evaluated by assessing neutrophil infiltration, dentin-like tissue regeneration, and coronal pulp tissue preservation using histological and immunohistochemical techniques. To further examine potential cellular responses, the effects of Nar were studied in human dental pulp fibroblasts (hDPFs), human peripheral blood-derived neutrophils (hNeu.), and differentiated HL-60 cells (dHL-60). Surface markers were analyzed using fluorescence-activated cell sorting (FACS), cytokine levels were measured by ELISA, and mineralization was assessed using alkaline phosphatase and Alizarin Red S staining. Neutrophil phagocytosis, bactericidal activity, intracellular reactive oxygen species (ROS) levels, and translocation of transcription factor EB (TFEB)-mediated lysosomal activity were evaluated. Statistical analyses included Shapiro-Wilk test and one-way ANOVA or Kruskal-Wallis test with appropriate post hoc comparisons. Results The sham group showed severe pulp tissue necrosis and an intense inflammatory response. Hydrogel alone exhibited limited therapeutic effects. Both Nar hydrogel and iRoot BP PLUS promoted dentin-like tissue formation; however, Nar hydrogel reduced inflammatory infiltration and preserved a greater proportion of coronal pulp tissue. In vitro, Nar reduced inflammatory cytokine secretion from hDPFs and neutrophils and improved P.g-LPS-impaired mineralization capacity in hDPFs. Nar also enhanced the phagocytic and bactericidal activities of dHL-60 cells, accompanied by controlled ROS elevation and increased TFEB-mediated lysosomal activity. Conclusions Nar demonstrates therapeutic potential for pulpitis management by modulating inflammation and promoting dentin-like tissue regeneration. Its efficacy likely stems from the fine-tuning of inflammatory responses. These findings suggest that Nar may represent a potential biological alternative to conventional pulp capping materials for vital pulp therapy.

Solid tumors consist of tumor cells and the tumor microenvironment (TME), a dynamic system comprising various immune cells, mesenchymal cells, cytokines, microvesicles, extracellular matrix, and vascular lymphatic networks. The recruitment of lymphocytes into tumors is essential for effective anti-tumor immunity and successful immunotherapy. The tumor stroma serves as the initial site for effector immune cell infiltration, and the mesenchymal network directly influences immune cell function. Cancer-associated fibroblasts (CAFs) are the largest proportion of TME intrinsic stromal cells, with significant heterogeneity and multiple origins, and perform different biological functions. CAFs can not only induce PD-L1 expression in tumor cells, but also promote PD-L1 expression in TME immune cells through the release of cytokines and vesicles, thereby facilitating tumor immune escape. In this study, we found that in some breast cancer patients, PD-L1 in tumor stroma was abundantly expressed and associated with immunosuppression and poor prognosis, and the distribution of tumor stromal PD-L1 was also closely related to the activation status of CAFs. Furthermore, we performed high-throughput RNA sequencing on PD-L1low and PD-L1high stroma cells, respectively. We found a CAF subset in high PD-L1 expression group, which was characterized by immune exhaustion and hyperactivation. Notably, Regulator of G protein signaling 4 (RGS4) pathways were significantly upregulated in this subset. Furthermore, we confirmed that RGS4 induces fibroblasts to adopt a PD-L1 immunodepleting phenotype and establishes an immune-exhaustive mesenchymal network that modulates anti-tumor immunity. In addition, we validated in humanized murine models that targeting RGS4 significantly enhances the efficacy of immunotherapy in animal models. The spatial heterogeneity of PD-L1 expression in TME is a primary factor contributing to the limited efficacy of PD-L1/PD-1 antibody therapies. Currently, numerous studies aim to enhance the immune response by inhibiting or degrading PD-L1. A comprehensive understanding of the molecular mechanisms governing PD-L1's spatial distribution could fundamentally enhance its clinical application. This study explored the mechanism governing the spatial distribution difference of PD-L1, and provided more possibilities for the development of targeted intervention in CAFs. Huiping Chen, Wenfeng Zeng. RGS4+aSMA+CAF subsets promote tumor immune escape by regulating the formation of immune-exhaustive mesenchymal networks [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5218.

Supplementary figures S1-S8 and tables S1-S6

The majority of low immune-reactive ‘cold’ tumors present an immune-excluded phenotype and it was proposed that stromal fibroblasts together with their collagen products constitute a physical barrier precluding lymphocytic infiltration into tumors. Here, we demonstrated a histopathological structure characterized by stromal fibroblasts embedded with dead lymphocytes in ‘cold’ tumors, named Crab-Nebula sign (CNs), indicating poor patient prognosis. Moreover, we identified a unique fibroblast subset in CNs-enriched structures overexpressing a sulfotransferase which triggers instant activation-induced cell death (AICD) in CTLs and NK cells. Mechanistically, we reveal that sulfonated HSPG receptor on activated immunocytes engaged by sulfonated HSPG inhibit p38-MAPK activation and reduce PI9 expression, leading to Granzyme B release into the cytosol and GSDME cleavage. Furthermore, sulfotransferase-knockout in fibroblasts or blocking sulfonated HSPG receptor in CTLs enhances anti-tumor effects of immune cell therapy in vivo. Collectively, we revealed that stromal fibroblasts construct a toxic biological barrier in ‘cold’ tumors and highlighted the therapeutic potential of disrupting the interaction between stromal fibroblasts and immunocytes in turning ‘immune-cold’ tumors hot. Jianing Chen, Wenfeng Zeng, Penghan Huang, Erwei Song. Tumor stromal fibroblasts constitute a biological immune barrier by inducing instant immune cell death [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5295.

Data-independent acquisition (DIA)-based mass spectrometry is becoming an increasingly popular mass spectrometry acquisition strategy for carrying out quantitative proteomics experiments. Most of the popular DIA search engines make use of in silico generated spectral libraries. However, the generation of high-quality spectral libraries for DIA data analysis remains a challenge, particularly because most such libraries are generated directly from data-dependent acquisition (DDA) data or are from in silico prediction using models trained on DDA data. In this study, we introduce Carafe, a tool that generates high-quality experiment-specific in silico spectral libraries by training deep learning models directly on DIA data. We demonstrate the performance of Carafe on a wide range of DIA datasets, where we observe improved fragment ion intensity prediction and peptide detection relative to existing pretrained DDA models. To make Carafe more accessible to the community, we integrate Carafe into the widely used Skyline tool.

Damage-associated molecular patterns generated by cancer treatment can modulate antitumor immunity, but the underlying mechanisms of this effect are unclear. Here we show that CA-enriched DNA fragments resulting from DNA-damaging chemotherapy in MSH2-low tumors preferentially bind cGAS with strong affinity and form biomolecular condensates by phase separation in the cytoplasm, resulting in antitumor immunity. However, classical CA-poor DNAs released from MSH2-high tumor cells engage AIM2, resulting in immunosuppression by upregulating PD-L1 and IDO. Intratumoral administration of CA-rich DNA fragments enhanced antitumor immunity in syngrafted PyMT tumors. Clinically, CA-rich DNA abundance in breast cancer following chemotherapy was associated with increased tumor-antigen-reactive T cells and better chemotherapeutic responses. Therefore, different tumor DNA fragments can trigger opposing immune responses depending on their preference for differential sensors. This study highlights another mechanistic link between genome instability and immune modulation and the therapeutic potential of CA-rich DNAs to enhance antitumor immunity. DNA damage from chemotherapy can activate cGAS–STING and interferon pathways. Here, the authors show that cGAS–STING signaling is specific to DNA rich in CA repeats, whereas DNA not rich in CA repeats is detected by AIM2, resulting in opposing tumor immune responses.

Spermidine metabolism influences tumor progression and anti-tumor immunity, thereby affecting treatment sensitivity. However, the precise role and therapeutic potential of spermidine in breast cancer remain unclear. Integrated multi-omics analyses (bulk and single-cell RNA sequencing) revealed a significant positive correlation between intratumoral spermidine abundance and immunophenotypic markers of CD8+ T cell infiltration and activation (GZMB+CD8+ T cells). Immunohistochemical and multiplexed immunohistochemistry validation (IHC/mIHC) demonstrated that breast cancer specimens with elevated spermidine production exhibited increased numbers of activated CD8⁺ T cells. Exogenous supplementation with spermidine promoted CD8⁺ T cell activation directly. Furthermore, supplementing spermidine in vivo promoted anti-tumor immune responses and enhanced sensitivity to anti-PD-1 immunotherapy combined with chemotherapy. Our findings indicate that boosting spermidine metabolism is a promising strategy to reinvigorate CD8⁺ T cell function and improve the efficacy of checkpoint blockade immunotherapy.

The clinical benefits of personalized therapeutic tumor vaccines are mainly challenged by the need to identify immunogenic neoantigens promptly, given the rapid pace of tumor mutations. An increasing body of literature addresses the potential of tumor-derived extracellular vesicles (TEVs) as an anti-tumor "cell-free" vaccine due to their substantial presence of neoantigens. However, their immunosuppression and limited presentation efficiency of dendritic cells (DCs) restrict their further application. Here, we have developed a novel tumor-personalized vaccine, termed P-Pev, based on remodeled TEVs by polymeric surfactant polyethylene glycol-phosphatidyleolamine (PEG-PE) and adjuvant monophosphoryl lipid A (MPLA). Our results show that PEG-PE transforms TEVs into micelle-like complexes by disrupting the original structure, facilitating antigens delivery to the cytoplasm, and cross-presentation by DCs. P-Pev particularly prevents the immunosuppressive impacts of TEVs on the ability of DCs to prime CD8+ T cells and eliminates the potency of TEVs to promote lung metastasis through their membrane-bound PD-L1. Finally, the P-Pev effectively induces neoantigen-specific cytotoxic T lymphocytes (CTLs) responses and exhibits excellent therapeutic effects in various murine tumor models. Also, the P-Pev induces neoantigen-specific antibodies, suggesting the involvement of humoral immunity in its anti-tumor effects. More importantly, it has been shown that P-Pev prepared by mutated tumor cells can retard these mutated tumor cell-established syngeneic tumors better than P-Pev prepared by original tumor cells, indicating the feasibility that leverages TEVs to prepare personalized tumor vaccines, and it is synergistically enhanced by PD-1 mAb combination. Collectively, we present a general strategy that offers a streamlined, cost-effective, and time-consuming approach to preparing personalized therapeutic tumor vaccines.

Peripheral nerve damage is intricately linked to the progression of various solid tumors. However, its effect on antitumor immunity and precise underlying mechanisms remain poorly understood. This study aimed to elucidate the effect of peripheral nerve damage and its subsequent immune-modulating effects influence on breast cancer progression. We analyzed nerve injury markers in the TCGA-BRCA database and clinical samples. In vivo experiments were conducted using orthotopic breast cancer models with chemical sympathetic denervation (6-OHDA) or nerve lysate/neurofilament light chain (NFL) treatment, where NFL was identified as a key effector molecule through mass spectrometry screening. The tumor microenvironment was evaluated by flow cytometry, multiplex immunohistochemistry, and single-cell RNA sequencing. In vitro co-culture systems were established to investigate the effects of NFL on macrophages and CD8+ T cells, with transcriptomic profiling revealing that NFL-activated macrophage supernatants induced CD8+ T cell senescence via NF-κB signal pathway activation. Peripheral nerve injury was associated with poor prognosis and immune evasion in breast cancer patients. In mouse models, chemical sympathectomy (6-OHDA) and nerve lysates injection both accelerated tumor growth, suggesting that nerve damage promotes immune escape. Single-cell RNA sequencing (scRNA-seq) further revealed that nerve injury increased tumor-associated macrophages (TAMs) proportion by promoting TAMs proliferation and attracting macrophages. The key effector molecule of nerve lysates neurofilament light chain (NFL) was identified with the TAMs proliferation effect, and intratumoral NFL administration recapitulated the pro-tumor effects of nerve damage and perfomed the same immune-modulating effects as 6-OHDA and nerve lysates. Importantly, NFL-induced TAM enrichment and remodeling promoted CD8+ T cell senescence, as evidenced by transcriptomic analysis showing NF-κB pathway activation and verified with NF-κB inhibitor (BAY 11-7082) in vitro, resulting in breast cancer immune escape. These findings underscore the critical role of peripheral nerve injury in reshaping the interplay between TAMs and antitumor immunity, via NFL-driven NF-κB activation and T cell dysfunction. Suggesting that neuroprotection could serve as a promising strategy to restore anticancer immunosurveillance.

The scale of data generated for mass-spectrometry-based proteomics and modern acquisition strategies poses a challenge to bioinformatic analysis. Search engines need to make optimal use of the data for biological discoveries while remaining statistically rigorous, transparent and performant. Here we present alphaDIA, a modular open-source search framework for data-independent acquisition (DIA) proteomics. We developed a feature-free identification algorithm that performs machine learning directly on the raw signal and is particularly suited for detecting patterns in data produced by time-of-flight instruments. Benchmarking demonstrates competitive identification and quantification performance. While the method supports empirical spectral libraries, we propose a search strategy named DIA transfer learning that uses fully predicted libraries. This entails continuously optimizing a deep neural network for predicting machine-specific and experiment-specific properties, enabling the generic DIA analysis of any post-translational modification. AlphaDIA provides a high performance and accessible framework running locally or in the cloud, opening DIA analysis to the community.

Dear Editor, The microenvironment within a solid tumor is a complex entity comprising cellular components,encompassing tumor cells,fibroblasts,adipocytes,endothelial cells and immune cells,alongside noncellular components such as extracellular matrix(ECM)and tumor interstitial fluid(TIF)(Cox,2021;Di Martino et al.,2021;Giussani et al.,2019;Poltavets et al.,2018).

Abstract Inducing ferroptosis in tumor cells is emerging as a strategy for treating malignancies that are refractory to traditional treatment modalities. However, the consequences of ferroptosis of immune cells in the tumor microenvironment need to be better understood in order to realize the potential of this approach. In this study, we discovered that neutrophils in chemoresistant breast cancer are highly sensitive to ferroptosis. Reduction of the acyltransferase MOAT1 in chemoresistance-associated neutrophils induced phospholipid reprogramming, switching the preference from monounsaturated fatty acids to polyunsaturated fatty acids, which increased their susceptibility to ferroptosis. Ferroptotic neutrophils secreted PGE2, IDO, and oxidized lipids that suppressed the proliferation and cytotoxicity of antitumor CD8+ T cells. Furthermore, neutrophil ferroptosis was closely related to a distinct subset of IL1β+CXCL3+CD4+ (Fer-CD4) T lymphocytes, which were enriched in chemoresistant tumors. Fer-CD4 T cells orchestrated neutrophil ferroptosis by modulating MOAT1 expression via IL1β/IL1R1/NF-κB signaling. Moreover, Fer-CD4 T cells secreted CXCL3, IL8, and S100A9 to replenish the neutrophil pool in the tumor microenvironment. Ferroptotic neutrophils in turn fostered Fer-CD4 T-cell differentiation. In spontaneous tumorigenesis mouse models, targeting IL1β+ CD4+ T cells or IL1R1+ neutrophils broke the cross-talk, restraining neutrophil ferroptosis, enhancing antitumor immunity, and overcoming chemoresistance. Overall, these findings uncover the role of neutrophil ferroptosis in shaping the immune landscape and propose appealing targets for restoring immunosurveillance and chemosensitivity in breast cancer. Significance: In chemoresistant breast cancer, IL1β+CXCL3+CD4+ T cells mediate neutrophil ferroptosis that suppresses antitumor immunity, indicating that interfering with this intercellular cross-talk could be an attractive strategy to reverse chemoresistance.