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Aberrant activation of Wnt/β-catenin signaling is proposed as a major molecular mechanism underlying the occurrence and progression of colorectal cancer (CRC). However, the precise mechanisms controlling the accumulation of β-catenin protein in CRC cells remain incompletely understood. Here, we show that TRIM24 is elevated in CRC tissues and partially distributed in the cytoplasm. TRIM24 is phosphorylated at serine 1042 by Aurora kinase B (AURKB), which promotes its cytoplasmic distribution. Subsequently, TRIM24 activates Wnt/β-catenin signaling by facilitating AKT activation through interaction with and ubiquitination of its negative regulator von Hippel-Lindau (VHL), resulting in β-catenin accumulation and enhanced proliferation of CRC cells. Moreover, chemical inhibition of AURKB suppresses tumor growth in subcutaneous mouse model and exhibits particular effectiveness against tumors derived from CRC cells characterized by prominent cytoplasmic TRIM24 distribution. Together, these findings reveal a critical role of TRIM24 in CRC cell proliferation, particularly through activating Wnt/β-catenin signaling. Aberrant activation of Wnt/β-catenin signaling contributes to colorectal cancer (CRC) pathogenesis. Here the authors find that the cytoplasmic distribution of TRIM24 E3 ubiquitin ligase triggers ubiquitination and degradation of von Hippel-Lindau, resulting in the accumulation of β-catenin protein in CRC cells.

Inflammatory bowel disease (IBD) arises from genetic-environmental interactions. Adenosine deaminases acting on RNA 1 (ADAR), an RNA-editing enzyme converting adenosine (A) to inosine (I), is essential for tissue homeostasis. Here we report that intestinal ADAR deficiency contributes to IBD pathogenesis in humans with reduced ADAR expression in patient intestinal crypts. Genetic or pharmacological inhibition of ADAR in mice causes spontaneous ileitis and colitis. Organoid studies show that ADAR loss leads to double-strand RNA (dsRNA) and endogenous retroviruses (ERVs) accumulation, disrupting intestinal homeostasis via melanoma differentiation-associated protein 5 (MDA5)-mediated dsRNA sensing and Janus kinase (JAK)-signal transducer and activator of transcription (STAT) signaling. Editome analyses identify Mda5 as an ADAR target, and edited Mda5 exhibits impaired dsRNA sensing. The human ADAR p.N173S mutation is a loss-of-function variant that fails to rescue IBD in intestinal Adar deficient mice, whereas JAK1/2 inhibitor Ruxolitinib attenuates IBD. We conclude that the ADAR-dsRNA/ERVs-MDA5-JAK/STAT axis is a potential therapeutic target for IBD. Environmental and genetic factors affect the pathogenesis of inflammatory bowel disease (IBD). Here the authors show a loss of function Adenosine deaminases acting on RNA 1 (ADAR) variant that results in accumulation of dsRNA which is sensed by MDA5 and promotes bowel inflammation.

Background/Objectives: Colorectal cancer (CRC) remains a major global health challenge and current therapies are not always effective. In addition, certain immune cell populations, such as myeloid-derived suppressor cells (MDSCs), pose a significant barrier to immune-based treatments. Some phytochemicals, particularly compounds derived from Allium spp. like Propyl-Propane Thiosulfonate (PTSO), have shown strong immunomodulatory potential in digestive disorders. This study aims to investigate the capacity of PTSO to modulate immune responses and affect tumor progression in CRC models, in vitro and in vivo, with a focus on the immune cell populations that comprise the tumor microenvironment. Methods: Human peripheral blood mononuclear cells (hPBMCs) were incubated with PTSO (25 μM for 48 h) and characterized by flow cytometry. These cells (1 × 106) were then injected into NOD scid gamma (NSG) immunodeficient mice, which were simultaneously induced to develop a subcutaneous tumor by injection of HCT116 enriched cancer stem cells (CSCs) colonospheres (60,000 cells/mouse). Results: PTSO reduced MDSC populations, specifically, it significantly reduced monocytic (M-MDSCs, Control: 7.27 ± 0.53% vs. PTSO: 4.70 ± 2.39%; p = 0.0458) and polymorphonuclear (PMN-MDSCs, Control: 5.28 ± 0.99% vs. PTSO: 3.41 ± 1.58%; p = 0.0385) MDSCs. In parallel, PTSO increased T cell subpopulations, particularly interferon gamma (IFNG)-producing cytotoxic CD8+ T cells (Control: 9.52 ± 2.06% vs. PTSO: 15.04 ± 5.01%; p = 0.0685). In the humanized tumor xenograft mouse, the administration of PTSO-pretreated hPBMCs led to a significant reduction in tumor size (Control: 1.43 ± 0.82 cm3 vs. PTSO: 0.44 ± 0.35 cm3; p = 0.0068), accompanied by increased infiltration of CD4+ T lymphocytes and Natural Killer (NK) cells and downregulation of immunosuppressive genes. These effects resulted in a reduction in cancer cell proliferation and invasiveness. Conclusions: The dual effect of PTSO on immune cell populations, reducing immunosuppressive myeloid cells and enhancing effector T lymphocyte and NK cell responses, resulted in an anti-tumor effect, highlighting this bioactive compound as a promising adjuvant in CRC immunotherapy and opening avenues for future research combining immunotherapy with PTSO in alternative models to optimize dosing and enhance translational potential.

Cysteine-rich angiogenic inducer 61 (CYR61) promotes prostate cancer (PCa) cell growth, but its role in disease progression remains unclear. Given its insulin-like growth factor (IGF)-binding domain and the known involvement of insulin-like growth factor-1 (IGF1) in PCa, we investigated the molecular interplay between CYR61 and IGF1. CYR61 was silenced using small interfering RNA (siRNA) in prostate carcinoma 3 (PC3), lymph node carcinoma of the prostate (LNCaP), and androgen receptor (AR)-positive 22Rv1 cells, followed by assessments of their proliferation, viability, colony formation, migration, and signaling pathway activation. CYR61 knockdown significantly reduced cell growth, viability, prostasphere formation, and migration across all three cell lines. Mechanistically, CYR61 silencing inhibited PI3K/AKT signaling but had no effect on MAPK activation. In addition, treatment with recombinant IGF1 induced CYR61 expression in a time-dependent manner, and the inhibition of PI3K/AKT signaling suppressed both CYR61 expression and cell proliferation. These findings suggest that IGF1 promotes PCa progression through CYR61 and that CYR61 may serve as a potential therapeutic target for limiting tumor growth and metastasis.

Aging negatively impacts proteasome activity and/or content, and this impairment contributes to disrupted protein homeostasis and cellular dysfunction. However, little is known about proteasome complex dynamics during aging, particularly in the context of immunosenescence. Indeed, only limited data are available on the immunoproteasome, a specialized variant expressed in immune cells. We establish an in vitro model of monocyte-derived human macrophages that develop a senescence-like phenotype upon long-term culture. Our data demonstrate that immunoproteasome complexes undergo deep structural and functional alterations, with the downregulation of immunosubunit expression at the mRNA and protein level, uncapping of the 20S catalytic particle by the PA28αβ regulator, and loss of activity. Immunosubunits are partly replaced by their constitutive counterparts with a shift toward the building of 19S-capped 20S complexes to maintain proteostasis. Similar proteasome dynamics are found in the lymph nodes of aged C57BL/6 and BTBR mice, the latter of which have a naturally activated immune system. Overall, these findings propose long-term cultures of human monocyte-derived macrophages as a model to study macrophage senescence. They also provide a molecular rationale for immunoproteasome dysfunction with remodeling of the proteasome, indicating that the loss of the PA28αβ regulator is a critical event and a hallmark of immunosenescence. A study on proteasome complex dynamics in an in vitro model of senescent human macrophages and in the lymph nodes of aged mice reveals immunoproteasome remodeling and the loss of PA28αβ regulator capping as hallmarks of immunosenescence.

To explore the relevance of non-malignant B cells (NMBCs) in chronic lymphocytic leukaemia (CLL), 201 blood samples from 79 previously untreated CLL patients receiving chemoimmunotherapy (CIT) in a phase III clinical trial were subjected to mass cytometry analysis using a bespoke panel of 24 antibodies. One memory (CD27+CD38−) and two naïve (CD27−CD38+) CD19+ clusters with a non-malignant phenotype (NMP; CD5−CD43−ROR1−CD20hiCD79bhiCD81+) were identified by FlowSOM and UMAP algorithms. NMP clusters were most prevalent in healthy controls, almost undetectable in untreated and relapsed CLL, and variable following CIT, where they correlated with haematopoietic recovery (naïve NMP clusters) and polyclonal antibody production (memory NMP cluster). Larger NMP cluster size at the early post-treatment timepoint was associated with a significantly longer progression free survival (PFS; HR 0.40 [95% CI: 0.22–0.75]; P = 0.003). Similar findings were obtained when NMBCs were prospectively defined by Boolean gating using the non-redundant features of the NMP clusters. NMBCs measured in this way negatively correlated with MRD and provided complementary prognostic information in patients with persistent MRD. Our findings raise the possibility that re-emergence of circulating NMBCs after treatment reflects clearance of CLL from secondary lymphoid organs, thereby complementing MRD as a biomarker of bone marrow clearance.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-025-16558-5.

Background/Objectives: Untargeted 1H NMR metabolomics is a robust and reproducible approach used to study the metabolism in biological samples, providing unprecedented insight into altered cellular processes associated with human diseases. Metabolomics is increasingly used alongside other techniques to detect an instantaneous altered cellular function, for example, the role of neutrophils in the inflammatory response. However, in some clinical settings, blood samples may be limited, restricting the amount of cellular material available for a metabolomic analysis. In this study, we wanted to establish an optimal 1D 1H NMR metabolomic pipeline for use with human neutrophil samples with low amounts of input material. Methods: We compared the effect of different neutrophil isolation protocols on metabolite profiles. We also compared the effect of the absolute cell counts (100,000 to 5,000,000) on the identities of metabolites that were detected with an increasing number of scans (NS) from 256 to 2048. Results/Conclusions: The variance in the neutrophil profile was equivalent between the isolation methods, and the choice of isolation method did not significantly alter the metabolite profile. The minimum number of cells required for the detection of neutrophil metabolites was 400,000 at an NS of 256 for the spectra acquired with a cryoprobe (700 MHz). Increasing the NS to 2048 increased metabolite detection at the very lowest cell counts (<400,000 neutrophils); however, this was associated with a significant increase in the analysis time, which would be rate-limiting for large studies. The application of a correlation-reliability-score-filtering method to the spectral bins preserved the essential discriminatory features of the PLS-DA models whilst improving the dataset robustness and analytical precision.

Even though the survival and proliferation stages of cancer cells that have newly settled at a metastatic site are the rate-limiting stages and the most promising targets for drugs, there is a lack of models of the earliest stage of metastasis formation. A method for modeling breast cancer liver metastasis is described here: a stage of transition of a differentiated tumor cell into a cell actively proliferating in a three-dimensional (3D) liver spheroid. Opposite to existing heterocellular 3D models of metastases, the protocol allows modeling the initial stage of liver colonization by metastatic cells, the so-called “micrometastases.” The method includes obtaining a line of fluorescent tumor cells, fluorescence-activated sorting of differentiated cells, preparing a single-cell suspension of liver cells, forming a liver spheroid in an agarose mold, inducing the tumor cell dedifferentiation and proliferation using IL-6, and intravital microscopy of spheroids, with subsequent processing and analysis of fluorescent images in the ImageJ software. The performance of the proposed model was demonstrated using microRNA therapeutics. The ability of a combination of microRNAs to suppress the transition of micrometastasis to macrometastasis in the 3D liver spheroid was confirmed by an immunofluorescent assay of spheroid sections and transcriptome analysis. Key features • The method introduces a 3D model of liver micrometastasis formation using differentiated tumor cells.• The 3D spheroid consists of all the main types of normal liver cells and better reproduces the microenvironment.• The method allows one to evaluate the effectiveness of a drug that blocks the transition of micrometastases to macrometastases.• The model is optimal for studying RNA-based therapeutic agents, as well as prodrugs that require metabolism in the liver for activation.

Neuropsychiatric disorders such as schizophrenia (SCZ) and bipolar disorder (BPD) remain challenging to diagnose due to the absence of objective biomarkers, with current assessments relying largely on subjective clinical evaluations. In this study, we present a computational analysis pipeline designed to identify disease-specific electrophysiological signatures from multi-electrode array (MEA) recordings of patient-derived cerebral organoids (COs) and two-dimensional cortical interneuron cultures (2DNs). Using a Support Vector Machine classifier optimized for high-dimensional data, we achieved 95.8% classification accuracy in distinguishing SCZ from control samples in 2DNs under both baseline and post-electrical-stimulation (PES) conditions with the extracted electrophysiological signatures. In COs, classification accuracy improved from 83.3% at baseline to 91.6% following PES, enabling robust separation of control, SCZ, and BPD cohorts. Key discriminative features included channel-specific measures of network activity, with PES significantly enhancing classification performance, particularly for BPD. These results underscore the potential of MEA-based functional phenotyping, coupled with machine learning, to uncover reliable, stimulation-sensitive electrophysiological biomarkers, offering a path toward more objective diagnosis and personalized treatment strategies for neuropsychiatric disorders.

AbstractThe NOTCH pathway regulates cell proliferation, differentiation, and stem cell maintenance. Thus, aberrant NOTCH activation plays a key role in cancer initiation, progression, and chemoresistance. Mutations and amplification of NOTCH pathway genes have been identified in high-grade serous ovarian cancers and are associated with poor clinical outcomes. Among the four NOTCH receptors, NOTCH3 alterations were strongly correlated with poor overall survival. Previously, we identified auranofin, an oral gold salt therapeutic compound, as a novel NOTCH pathway inhibitor that disrupts the DNA binding of RBPJ, the major downstream transcriptional effector of the NOTCH pathway. In this study, we surveyed the response of eight ovarian cancer cell lines to auranofin and found IC50 values ranging from 1.7 to 12 μmol/L, with NOTCH3-negative SKOV3 cells having the highest IC50 value. In NOTCH-dependent OVCAR3 cells, auranofin synergized with cisplatin to enhance cell death. Importantly, auranofin treatment led to a dose-dependent decrease in RBPJ occupancy at the NOTCH-dependent promoters, HES1 and HES4. Furthermore, knocking down NOTCH3 in OVCAR3 cells significantly decreased sensitivity to auranofin, further supporting the notion that NOTCH3 signaling is a major target of auranofin. Moreover, auranofin increased cisplatin efficacy in an OVCAR3-derived xenograft mouse model. Using eight patient-derived cancer organoid models, we found that auranofin increased cisplatin efficacy in killing cancer organoids generated from clinically platinum-sensitive patients but also restored platinum response in a subset of organoid models developed from platinum-resistant patients. These studies underscore the potential of auranofin to improve platinum-based cancer therapy, particularly in NOTCH3-expressing cancers.Significance:NOTCH signaling underlies cancer initiation, progression, and chemoresistance. Our study revealed the potential of auranofin as a NOTCH pathway inhibitor to enhance the efficacy of platinum-based ovarian cancer therapy.

ABSTRACTMemory B cells are critical in kidney transplantation for rapid anti‐HLA antibody production upon re‐exposure, yet pretransplant serum antibodies often remain undetectable despite prior sensitisation. Using R848 stimulation (a TLR7/8 agonist inducing polyclonal B‐cell activation), we tested 45 kidney recipients with past sensitisation events but no circulating antibodies. The assay identified 56 memory B‐cell‐derived HLA antibodies (HLAm) in 8 patients, primarily from pregnancy or transfusion sensitisation, with a 17‐year average latency between exposure and transplantation. This pilot demonstrates that latent memory B cells may reactivate post‐transplant, driving antibody production and adverse outcomes. Our findings advocate integrating memory B‐cell assays into pretransplant risk assessment to improve sensitivity in detecting subclinical HLA sensitisation, refining donor‐recipient matching strategies.

AbstractBackgroundOne in ten Americans carry a lifetime risk of ischemic heart failure, the most severe form of ischemic heart disease. Carrying a nearly 50% five‑year mortality rate, no interventional therapy exists to treat the underlying cause, microvascular malperfusion. In efforts to combat microvascular malperfusion, our group has utilized synergistic application of endothelial progenitor cells (EPCs) and smooth muscle cells (SMCs) to induce angiogenesis in ischemic myocardium.MethodsCells are then embedded into a rapidly manufacturable compressed collagen (CC) patch to provide a biosimilar scaffold ready for transplantation. The performance of the cellular compressed collagen patch was then tested on a rodent acute myocardial infarction model of ischemic heart failure.ResultsBy post‑transplantation Day 28, the cellular CC patch improved left ventricular ejection fraction when compared to an acellular CC patch and control (cellular: 49.1 ± 1.8%; acellular: 38.0 ± 2.6%; control: 39.2 ± 2.1%; ANOVA P = .0006). Cellular CC patch transplantation also induced mature angiogenesis as shown by arteriolar density (cellular: 1084 ± 98 αSMA+vWF+/mm2; acellular: 338 ± 57 αSMA+vWF+/mm2; control: 449 ± 39 αSMA+vWF+/mm2; ANOVA P = .0003) and vascular maturation index (cellular: 0.67 ± 0.04; acellular: 0.48 ± 0.02; and control: 0.46 ± 0.04, P = .001).ConclusionsIn conclusion, transplantation of a rapidly manufacturable EPC‑SMC‑based compressed collagen patch effectively rescues myocardial function by enhancing neovascularization and attenuating post‑infarction myocardial injury. Graphical abstract