�����ƽ��

Persistent infection with high-risk human papillomavirus (HR-HPV) is the primary cause of cervical cancer. Co-infection with HIV-1 increases the risk of cervical cancer progression 6-fold, despite adherence to antiretroviral therapy (ART). While chronic HIV-1 infection is known to cause inflammation, the paracrine effects of HIV-1-infected immune cells on cervical signaling remain unclear. We performed transcriptomics on cervical swabs from Kenyan women stratified by HIV-1 and cancer status, which revealed HIV-1 infection drove cancer-like gene expression in non-cancerous cervical cells. In parallel, global abundance proteomics and phosphoproteomics of cervical cancer cells exposed to HIV-1-infected human primary CD4+ T cell secretomes revealed altered MAPK, PI3K-AKT, cell cycle, and beta-catenin pathways. Concordantly, IRS1 was upregulated in both patient cervical samples and cultured cells. Our findings suggest HIV-1 dysregulates cervical cell signaling via paracrine mechanisms to phenocopy PIK3CA-activating mutations through IRS1-PI3K-AKT pathway activation. Our findings highlight IRS1 and the PI3K pathway as a potential therapeutic target for cervical cancer in women living with HIV-1.

Animal and human stem cell-derived three-dimensional models to study physio-pathological brain functioning are becoming a gold standard for in vitro electrophysiology, as they enable the recapitulation of complex network properties by accounting for spatial architectural features that better reflect in vivo conditions than simpler 2D models. Standard planar multielectrode arrays (MEAs), typically providing tens of recording electrodes, are commonly used to record activity from 2D neuronal cultures. However, when adapted for use with 3D models, planar 2D MEAs showed limited effectiveness. The main issues are limited specimen adhesion to the chip, a low number of sensing elements, inability to retrieve signals from within the tissue, and reduced perfusion and vitality of the tissue in contact with sensors. To overcome these limitations, a new generation of microchip-based 3D high-density MEAs (3D HD-MEA) has been developed and validated in recent years. This technological advancement has improved the sensing capabilities and the vitality of 3D models, providing a tool tailored to maximize their potential. Here, we present an optimized protocol for neural network activity recordings in 3D models (including acute slices, brain spheroids, and organoids) from various brain regions using 3D HD-MEAs. First, we summarize the critical steps for 1) obtaining viable acute slices from the mouse cerebellum, cortico-hippocampal circuit, and prefrontal cortex, 2) establishing efficient coupling of the slices with the chip, and 3) performing recordings and analyses. We then describe the main procedures required to obtain human and animal brain spheroids and neural organoids, as well as standardized routines to perform effective recordings and analyses. For each section, we highlight the crucial steps, identify tips for specific applications, and propose troubleshooting procedures. For example, the same type of preparation (e.g., acute slices) requires different adjustments when working with different brain areas. The specific information provided here is intended to assist researchers in their daily efforts to obtain efficient and reproducible functional recordings from 3D models by using the cutting-edge technique of 3D HD-MEA. Key features • Comprehensive all-in-one guide covering the complete workflow for acquiring electrophysiological data from brain slices, neural region-specific organoids, and brain spheroids. • Intuitive, step-by-step protocol for brain slice preparation, enriched with practical tips and expert recommendations to ensure high-quality tissue viability. • Detailed instructions for optimal use of 3D HD-MEA technology, including proper handling of the sample holder for recordings from brain slices, neural organoids, and spheroids. • In-depth guidance on BrainWave6 software, providing clear procedures for data acquisition, signal detection, and advanced electrophysiological analysis across all sample types.

Glioblastoma (GBM) is the most aggressive primary malignant brain tumor, and while chimeric antigen receptor-T (CAR-T) cell therapy has shown promise, its efficacy remains limited by antigen heterogeneity and immune escape. Here, we investigated the expression of B7 homolog 3 (B7-H3), epidermal growth factor receptor (EGFR), and interleukin-13 receptor alpha 2 (IL-13RA2) in GBM tissues and cell lines. Although all three antigens were highly expressed, sustained exposure to B7-H3 CAR-T cells led to significant B7-H3 downregulation but concurrent EGFR upregulation, revealing a potential immune escape mechanism. To address this heterogeneity, we engineered T cells to express an anti-B7-H3 CAR and secrete an EGFR-targeting bispecific T-cell engager (EGFR-BsTe). These B7-H3-CAR-T-EGFR-BsTe cells exerted dual functionality: direct B7-H3-dependent cytotoxicity and recruitment of unmodified T cells via secreted EGFR-BsTe to eliminate EGFR-expressing tumor cells. Notably, EGFR-BsTe secretion promoted CAR-T cell proliferation and effector differentiation. In orthotopic GBM xenograft models, including mixed tumors with heterogeneous antigen expression, B7-H3-CAR-T-EGFR-BsTe cells demonstrated superior antitumor activity and prolonged survival compared to conventional B7-H3 CAR-T cells. Quantitative analysis revealed that EGFR-BsTe secretion abrogated EGFR upregulation and enhanced B7-H3 downregulation in a target-dependent manner; however, efficacy was diminished when the CAR-Target (B7-H3) was absent on a substantial fraction of tumor cells. Our findings suggest that arming B7-H3 CAR-T cells with EGFR-targeting bispecific engagers represents a promising strategy to overcome antigen heterogeneity and improve therapeutic outcomes for GBM patients.

Macrophages are pivotal immune cells due to their phagocytic capabilities, yet the impact of aging on macrophage phagocytosis remains poorly understood. Using comprehensive in vitro and in vivo phagocytic assays, we demonstrate significantly reduced phagocytic activity in monocyte-derived macrophages from aged humans and mice compared to young counterparts. RNA-seq analysis revealed upregulated expression of extracellular matrix protein genes, particularly collagens, in aged macrophages; manipulation of COL1A1 expression can significantly affect phagocytosis. Protein interaction assay identified binding between collagen and actin filaments, which inhibits F-actin turnover and consequently impairs phagocytic function. Also, we found that mitochondrial ROS is the driving force of collagen overproduction and MitoTEMPO rejuvenates macrophage phagocytosis via restoring actin dynamics. In a mouse model, MitoTEMPO significantly boosted the phagocytosis of peritoneal macrophages against bacteria. These findings highlight the fundamental role of mitochondrial redox balance and collagen production in controlling macrophage phagocytic function, identifying them as targetable mechanisms for promoting healthy immune aging.

Microglia are the brain's resident immune cells, essential for homeostasis and implicated in common neurodegenerative diseases like Alzheimer's and Parkinson's disease (PD), where their early activation and sustained inflammatory mediator release contribute to neuronal loss. However, their role in rare disorders is unclear. β-propeller protein-associated neurodegeneration (BPAN), caused by WDR45 mutations, shares key features with PD, including iron accumulation and dopaminergic neuron loss, but the impact of microglia and mutant WDR45 in BPAN pathophysiology remains unexplored. To address this, we established the first induced pluripotent stem stell (iPSC)-derived microglia model from BPAN patients. Parallel targeted transcriptomic and secretomic profiling revealed a shift from a homeostatic microglial toward a stress-adapted and transcriptionally reprogrammed state characterized by selective remodeling of immune signaling pathways and dysregulation of autophagy and cellular stress responses. Complementary secretomic analysis identified reduced secretion of lysosomal enzymes alongside increased shedding of immune-associated surface proteins, indicating altered lysosomal trafficking and remodeling of microglial immune signaling. These findings identify a distinct microglial phenotype in BPAN and implicate microglial dysfunction as a potential contributor to disease mechanisms, highlighting new avenues for therapeutic strategies targeting neuroimmune pathways.

Platelet shortages and limited storage stability restrict global platelet transfusion capacity, highlighting the need for efficient in vitro platelet production systems. This study establishes a simple, non-genetic and cost-effective system for efficient in vitro platelet-like particle (PLP) production from human hematopoietic stem/progenitor cells (HSPCs) using a Phase-specific modulation of Hippo-YAP/TAZ signaling modulation. Temporal control of Hippo-YAP/TAZ signaling by lysophosphatidic acid (LPA), an activator of YAP/TAZ activity, significantly enhanced megakaryocyte differentiation, expansion and PLP production, resulting in an approximately 15-fold increase in PLP yield at the end of the procedure. Furthermore, LPA extended the expansion period of HSPC-derived megakaryocytes up to 8 days, resulting in a greater than 20-fold increase in the number of HSPC-derived CD41+ megakaryocytes. Moreover, replacement of expensive commercial recombinant human thrombopoietin (C-rhTPO), one of the major cost-driving components in in vitro PLP production, with recombinant human thrombopoietin produced in Escherichia coli (W-rhTPO) further improved the cost-effectiveness of the procedure. In conclusion, this study demonstrates that temporally controlled Hippo-YAP/TAZ signaling, together with affordable cytokine supplementation, provides a robust and GMP-compatible platform for large-scale PLP manufacturing for future clinical applications. We believe that this system will enable scalable PLP generation, even in resource-constrained settings, to increase human platelet supply for many life-saving therapies in the future.

Natural killer (NK) cells recognize and eliminate malignant cells through multiple receptor-ligand interactions. To uncover genetic determinants of the susceptibility of myeloid leukemia cells to NK cell cytotoxicity, we analyzed several genome-wide CRISPR screens. Among recurrent hits, the BRCA1-associated protein 1 (BAP1) gene emerged as a key factor protecting K562 leukemic cells from NK cell-mediated killing. Using BAP1 knockout (KO) models, we found that loss of BAP1 alone did not alter NK cell sensitivity. However, upon interferon-γ (IFN-γ) stimulation, BAP1 KO K562 cells exhibited reduced HLA class I induction, triggered enhanced NK cell degranulation, and showed increased sensitivity to NK cell-mediated cytotoxicity compared with wild-type cells. Further experiments revealed that BAP1-deficient cells displayed reduced expression of the IFN-γ receptor 1 (IFN-γ-R1). BAP1 knockdown across multiple myeloid leukemia cell lines selectively decreased HLA-E and IFN-γ-R1 expression in ASXL1-mutant backgrounds. These findings suggest that BAP1 may contribute to the regulation of IFN-γ responsiveness and immune evasion in myeloid leukemia.

Multiple sclerosis (MS) is a chronic CNS disease characterized by demyelinated lesions. Gray matter lesions (GMLs) are associated with neurodegeneration and clinical disability, and normal appearing gray matter (NAGM) also shows neuropathology. The extracellular matrix (ECM) supports neuronal health, with perineuronal nets (PNNs) regulating synaptic stability. Microglia remodel the ECM by secreting matrix metalloproteinases, while ECM composition affects microglia behavior. We examined ECM composition in decellularized human control gray matter, NAGM and GML slices and investigated its effects on microglia. Label-free quantitative mass spectrometry of decellularized control gray matter and NAGM revealed differential abundances in PNN ECM components and condition-specific synaptic and basement membrane ECM components. ECM composition was similar between decellularized GMLs and perilesional gray matter. Microglia introduced to decellularized NAGM and GML slices lost IBA1 expression in half the cells, while some remaining IBA1+ microglia acquired proinflammatory marker inducible nitric oxide synthase (iNOS). These findings suggest ECM alterations in NAGM influencing microglial characteristics potentially contributing to MS pathology.

The SIRPα-CD47 "don't eat me" checkpoint axis plays a critical role in shaping antitumor activities of macrophages within the tumor microenvironment (TME). However, targeting this axis with anti-CD47 antibodies to enhance antitumor responses in clinical trials has been challenging. Here, we demonstrated that SIRPA-knockout (KO) iPSC-derived macrophages (iMacs) exhibit superior antitumor activity against various CD47-expressing tumors in vitro when combined with cancer-targeted monoclonal antibodies (mAbs) or chimeric antigen receptors (CARs). Moreover, SIRPA-KO protected macrophages from mAb- and CAR-driven hypophagia, enabling efficient tumoricidal effects even after serial tumor exposures. Retention of phagocytic activities in SIRPA-KO iMacs was associated with heightened surface expression of Fc receptors and GD2-CAR compared to their SIRPA-expressing counterparts. Despite the powerful impact of SIRPA-KO on iMac antitumor activities in vitro, only modest efficacy was observed in human xenograft mouse models of SK-OV3 ovarian carcinoma and CHLA-163 neuroblastoma treated with mAb or CAR-iMac therapy, indicating further engineering or combinatorial therapeutic strategies are needed for potent in vivo antitumor efficacy. Together, these findings identify SIRPα as a regulator in macrophage hypophagia and underscore the advantages of using SIRPA-KO macrophage therapeutic strategies to modulate the SIRPα-CD47 checkpoint to unleash macrophage antitumor activity within the TME.

Colorectal cancer is frequently driven by hyperactivation of Wnt/β-catenin signaling, which also contributes to reduced responsiveness to chemotherapy. However, how aberrant Wnt/β-catenin signaling enables colorectal cancer cells to tolerate chemotherapy-induced DNA damage remains elusive. Identifying actionable downstream effectors of this pathway may provide a more selective strategy to improve chemotherapy response while avoiding the toxicity associated with global Wnt inhibition. Here we show that TELO2-interacting protein 1 (TTI1) is a direct transcriptional target of β-catenin/TCF in colorectal cancer. TTI1 expression correlates with β-catenin abundance and is elevated in tumors from patients with poor response to neoadjuvant chemotherapy. Mechanistically, TTI1 maintains the integrity of the TELO2-TTI1-TTI2 complex and stabilizes the DNA damage response kinases ATM and ATR. Genetic depletion of TTI1 destabilizes ATM and ATR, attenuates DNA damage signaling, impairs double-strand break repair, and sensitizes colorectal cancer cells to 5-fluorouracil and oxaliplatin. Conversely, restoration of TTI1 or ATM/ATR re-establishes DNA damage responses and chemo-insensitivity. Pharmacological suppression of the TTT complex by piperlongumine mimics TTI1 loss and enhances the anti-tumor activity of chemotherapy in cell lines, xenografts, Apc-mutant patient-derived organoids and Apcmin/+ colonic adenomas. These findings define a β-catenin-TTI1-ATM/ATR axis that links Wnt/β-catenin activation to DNA damage tolerance and chemotherapy response in colorectal cancer. Targeting TTI1 is therefore a promising approach to improve chemotherapy efficacy in Wnt/β-catenin-activated colorectal cancer.

Dampening neutrophil-driven inflammation in the airways remains a challenge in treating cystic fibrosis (CF) lung disease. Myeloperoxidase (MPO) is a neutrophilic enzyme that produces reactive oxygen species and is highly concentrated in CF airways. Greater MPO concentrations have previously been correlated with increased mucus plugging in bronchiectasis, suggesting that MPO could impair mucociliary transport. As such, we evaluated the impact of MPO treatment on barrier integrity, mucin production, mucus viscoelasticity and mucociliary transport in fully differentiated human airway epithelial cultures at ionic conditions reflective of healthy and CF-affected airways. Using live-cell imaging and particle velocimetry, we found that MPO inhibits mucociliary transport in vitro at CF-like and normal airway conditions. The impairment of mucus clearance by MPO was similar to that by neutrophil elastase, another neutrophilic granular enzyme that damages the host tissues and impairs airway clearance. We also found that subsequent treatment with the reducing agent, N-acetyl cysteine, could alleviate MPO-mediated mucociliary dysfunction through disulfide bond cleavage. These findings identify MPO as a therapeutic target to resolve deficits in airway clearance function in CF and related muco-obstructive lung diseases.

Nutraceuticals are increasingly investigated for their capacity to modulate oxidative and inflammatory stress, yet preclinical testing still relies largely on immortalized cell lines or animal models that poorly recapitulate human epithelial complexity. To address this gap, we developed an integrated platform based on patient-derived colon organoids generated from non-tumoral mucosa and maintained under proliferative or differentiation conditions to model distinct epithelial states. The system combines millifluidic measurement of individual organoid mass, density, and diameter with bulk RNA sequencing and digital PCR profiling to enable multiparametric characterization. Transcriptional analysis revealed state-specific gene programs and shifts in epithelial and immune-related pathways, while biophysical measurements captured structural remodeling. In this pilot validation, a defined oxidative insult followed by nutraceutical treatment elicited coordinated transcriptional and phenotypic responses. This integrated approach provides a scalable and physiologically relevant framework for functional nutraceutical profiling and mechanistic studies of epithelial stress responses.