Glioblastoma represents an aggressive, immunologically cold primary brain tumor largely unresponsive to conventional immunotherapies. Dissecting patient-specific immunosuppressive cues is limited by current preclinical models. Understanding the functional cross-talk between tumor and immune cells across distinct microenvironmental niches is crucial for advancing personalized immunotherapy. We assessed the immunogenic features of glioblastoma cells and cross-talk with T-cells using bulk and single-cell RNA-seq, multicolor flow cytometry, immunohistochemistry, and functional assays in patient tumors, patient-derived organoids (PDOs), orthotopic xenografts and cell cultures. We developed an advanced 3D co-culture system combining PDOs with human peripheral blood T-cells isolated from glioblastoma patients or healthy donor blood to functionally investigate T-cell behavior in a patient-specific context. T-cell subsets integrated and non-integrated into the PDOs were examined for T-cell activation, exhaustion, and T regulatory cell development in response to tumor-derived signals and hypoxia. Single-cell RNA-seq-based crosstalk predicted direct ligand-receptor and soluble axes between glioblastoma and T-cells. Glioblastoma cells in patient tumors and diverse patient-derived models showed cell surface MHC class I expression across diverse genetic backgrounds and microenvironmental niches, modulable by interferon gamma. Subsets of CD4+ and CD8+ T-cells integrated into 3D PDOs deprived of myeloid component. PDO-integrated T-cells were enriched for T regulatory cells (Treg), and exhibited a heterogeneous spectrum of activation and dysfunction states, with differential expression of PD-1, LAG-3, TIM-3, CD45RO, Granzyme B, TCF1, and TOX. T-cells subjected to soluble factors were largely unaffected and retained capacity to reintegrate PDOs at sequential co-cultures. Hypoxia influenced T-cell phenotype, but did not prevent activation or infiltration within the early stages of co-culture. Our work provides insights into tumor-intrinsic cues of immune evasion in glioblastoma and introduces a scalable patient-relevant ex vivo platform for functional studies. Surface MHC-I was detectable in all assessed glioblastoma models; and was retained in contact with T-cells, indicating that MHC-I loss is not their predominant immune-escape mechanism. Immunosuppression occurred in direct contact with glioblastoma cells, involving both TOX-dependent exhaustion and TOX-independent dysfunction. Advanced co-cultures of glioblastoma PDOs may serve as a powerful tool for functional studies ex vivo, predicting clinically-relevant responses to immunotherapeutic treatments in the future. 1. GBM cells express interferon gamma-responsive cell surface MHC-I. 2. GBM patient-derived organoid-T-cell co-cultures allow for modeling human tumor-immune interactions ex vivo. 3. Direct interaction with GBM cells promotes Treg enrichment and induces a gradient of T-cell activation and dysfunction states, including TOX-driven exhaustion.
Fibroblasts, once viewed as inert producers of extracellular matrix (ECM), are now recognized as dynamic and heterogeneous regulators of tissue identity, homeostasis, and repair. Single-cell and spatial transcriptomic studies have revealed extensive transcriptional and positional diversity among fibroblast populations, demonstrating that they integrate developmental, anatomical, and niche-derived signals to instruct neighboring cells. A particularly striking dimension of this regulatory capacity is their bidirectional engagement with the nervous system. Across diverse organs, fibroblasts engage in reciprocal signaling with neurons and glia, defining what we term neuro-mesenchymal niches: spatially organized microenvironments in which fibroblast-derived neurotrophins, ECM cues, and direct cell-cell contacts shape neuronal growth, survival, and function, while neural inputs, neurotransmitters, neuropeptides, and contact-mediated signals, reciprocally regulate fibroblast identity, proliferation, and activation. In this Review, we synthesize current understanding of the developmental origins, heterogeneity, and canonical functions of fibroblasts, and integrate emerging evidence on fibroblast-neuron crosstalk across the skin, peripheral nerve, lung, musculoskeletal system, intestine and central nervous system (CNS). We further examine how dysregulation of these niches contributes to pathological outcomes including fibrosis, chronic pain, denervation, and neuroinflammation. Conceptualizing fibroblasts as central organizers of neuro-mesenchymal niches provides a unifying framework for dissecting stromal-neural communication and identifies new therapeutic opportunities at the interface of regenerative medicine, neurology, and immunology.
Alzheimer’s disease (AD) exhibits a pronounced sex bias, with women facing disproportionately higher risk and more severe pathology. Postmenopausal estrogen decline is implicated in this vulnerability, yet the molecular mechanisms linking estrogen loss to AD pathogenesis remain incompletely understood. Here, we demonstrate that ovariectomy (OVX) in female 5xFAD mice significantly exacerbates amyloid-β (Aβ) pathology, cognitive deficits, neuroinflammation, and reduces synaptic markers. Pharmacological blockade of estrogen receptor signaling recapitulated these effects, confirming their dependence on estrogen receptor pathways. Single-nucleus RNA sequencing (snRNA-seq) revealed widespread transcriptional reprogramming across brain cell types following estrogen deprivation, with prominent upregulation of the lysosomal protease cathepsin S (Ctss) and the AD risk gene ApoE. Remarkably, partial genetic reduction of CTSS prevented OVX-induced Aβ accumulation, glial activation, and synaptic decline in female 5xFAD mice, establishing CTSS as a critical downstream mediator of estrogen deficiency-driven pathology. Our findings provide mechanistic insight into sex-biased AD vulnerability and identify CTSS as a promising therapeutic target for mitigating AD risk in postmenopausal women.
Focused ultrasound with microbubbles noninvasively and transiently modulates the blood-brain barrier (BBB), enabling precise transvascular solute transport. This procedure minimizes edema and hemorrhage while avoiding long-term physiological and cognitive effects. Currently, this is in clinical trials for Alzheimer’s disease and brain cancers. As with any neurovascular insult, treatment may elicit a sterile inflammatory response (SIR), scaling with increasing mechanical index and microbubble dose. Most studies employ moderate parameters that induce Class II SIR with robust BBB modulation, with reports indicating acute immune activation within 5 min and up to 3 days post-treatment; however, longer-term dynamics remain unclear. Thus, we profiled murine brain transcriptomics for a week post-treatment. Integrative analyses identified a biphasic inflammatory response, characterized by an initial NFκB-driven peak at 3–6 h, followed by an interferon-mediated phase at 48 h that attenuates by a week. This finding reconciles prior inconsistencies, providing insights into establishing therapeutic windows and enhancing neuroinflammatory modulation strategies.
Acute brain injury (ABI) frequently coexists with pulmonary complications, creating bidirectional interactions that can amplify secondary organ injury. This review reframes ABI-associated brain–lung crosstalk as a sequential neuroimmune relay—from generation and peripheral egress of CNS-derived signals, through systemic immune amplification and pulmonary effector injury, to reciprocal lung-to-brain feedback—and explicitly distinguishes directly supported mechanisms from emerging hypotheses. Following ABI, autonomic activation, damage-associated molecular patterns, extracellular vesicles, and peripheral immune responses may contribute to pulmonary endothelial dysfunction, innate immune activation, and alveolar-capillary barrier injury. Glymphatic and lymphatic clearance pathways are discussed as potential routes of CNS-to-systemic molecular transport, but direct evidence linking glymphatic-derived signals to pulmonary injury remains lacking. Conversely, acute pulmonary complications relevant to neurocritical illness—including ALI/ARDS, mechanical ventilation-associated injury, and pulmonary infection—may aggravate the vulnerable brain through systemic inflammation, impaired gas exchange, blood–brain barrier dysfunction, and neuroinflammatory signaling. We further evaluate candidate circulating biomarkers according to their biological source, specificity, and clinical readiness, emphasizing that currently available markers primarily reflect neural injury, endothelial dysfunction, or systemic inflammation rather than brain–lung axis injury itself. Therapeutic strategies targeting inflammasome signaling, autonomic regulation, immune modulation, microbiome-related pathways, and extracellular vesicles are assessed according to their mechanistic evidence and translational stage. By integrating directionality, evidence strength, and translational maturity, this review provides an ABI-centered framework for identifying experimentally testable relay points and priorities for future brain–lung research.
