Citations

Abstract Background & Objectives Patients with cholangiocarcinoma (CCA) commonly progress within months on first-line gemcitabine/cisplatin–based therapy with immune checkpoint inhibition (ICI), consistent with the emergence of treatment-induced adaptive resistance. Cellular senescence and its secretory program (senescence-associated secretory phenotype, SASP) can remodel the tumor immune microenvironment. We hypothesized that gemcitabine/cisplatin induces tumor-cell senescence that promotes immunosuppression and progression and prioritized SASP mediators that are mechanistically linked to this phenotype. Method We quantified senescence and lineage markers in chemotherapy-naïve (n = 18) and gemcitabine/cisplatin treated (n = 23) human cholangiocarcinoma specimens and analyzed single-nucleus RNA sequencing in 9 human CCA tumors. In murine orthotopic CCA models, senescent tumor cells were ablated using p16 or p21 promoter restricted inducible apoptosis (ATTAC), followed by immune profiling by flow cytometry in endpoint cohorts, or tested alone and in combination with gemcitabine/cisplatin-based therapy and immune checkpoint inhibition (ICI) for survival cohorts. Conditioned media from proliferating versus senescent tumor cells were applied to bone marrow-derived macrophages (BMDMs) to assess polarization and suppression on CD8+ T-cell proliferation. SASP candidates were screened by cytokine arrays and Growth/Differentiation Factor 15 (GDF-15) was validated by expression and genetic perturbation. Results Senescent cells were enriched in the tumor compartment in human cholangiocarcinoma and increased after chemotherapy, as assessed by p16, p21and telomere-associated DNA foci (TAF). In mice, ablation of p16+ or p21+ senescent tumor cells reduced tumor burden, prolonged survival, and improved response to gemcitabine/cisplatin-based regimens. Chemotherapy-induced senescence increased immunosuppressive tumor-associated macrophages (TAMs), an effect reversed by senescent tumor cell ablation. Mechanically, GDF-15 was the top enriched secreted factor from senescent human CCA cells, tracked with senescence associated transcriptional programs, and decreased after senescent tumor cell ablation. GDF-15 promoted immunosuppressive macrophage phenotype switching and macrophage-mediated suppression on CD8+ T cell proliferation. In comparison, senescence-restricted GDF-15 knockdown reduced tumor growth and macrophage-mediated immunosuppression. Conclusion Chemotherapy-induced tumor cell senescence promotes an immunosuppressive microenvironment through GDF-15 linked macrophage remodeling. Targeting senescent tumor cells or intercepting senescence associated GDF-15 signaling may improve response to first-line therapy in CCA.

Abstract The trabecular meshwork (TM) and ciliary body (CB) regulate aqueous humor dynamics and intraocular pressure (IOP), and TM/Schlemm’s canal (SC) dysfunction underlies glaucoma. Here, we present a spatially resolved multi-omics atlas of human TM and CB, integrating snRNA-seq, scRNA-seq, and snATAC-seq from over one million cells and nuclei across 112 donors with Xenium spatial transcriptomics. We identified 9 major cell classes and 21 cell types, revealing heterogeneity, including undercharacterized fibroblast and epithelial subpopulations. Spatial mapping supported TM fibroblast zonation and CB epithelial organization. Regulatory analyses identified cell type–specific programs, including OTX/PAX networks in CB epithelium and SMAD3/TGF-β signaling in fibroblasts. Integration with glaucoma loci showed enrichment of non-coding variants in regulatory elements associated with POAG and PACG. Age- and ancestry-associated remodeling revealed divergent fibroblast aging with increased PIEZO1 , suggesting impaired outflow and elevated IOP. Together, this high-resolution atlas links cellular, regulatory, and genetic variation to anterior segment function and glaucoma susceptibility.

Introduction Large-scale production of mammalian cells is pivotal for applications in biotechnology, regenerative medicine, and therapeutic manufacturing. However, current bioreactor technologies face significant technical and economic challenges, including excessive cell aggregation, shear stress-induced cell death, batch-to-batch inconsistencies, and limited scalability. We propose that engineering a cell-friendly microenvironment can enhance culture efficiency. Previously, we developed alginate hydrogel microtubes (AlgTubes) that significantly improved cell density and growth rates; however, AlgTubes lack adhesion sites essential for anchorage-dependent cells and frequently break, causing cell leakage and production inconsistencies. Methods To address these limitations, we reinforced AlgTubes with collagen nanofibers, creating collagen-alginate hybrid hydrogel microtubes (ColAlgTubes). Collagen was integrated to form a dense nanofiber network interwoven with the alginate mesh, with the dual aim of enhancing mechanical properties and providing cell adhesion sites. ColAlgTubes were fabricated to maintain cell mass within a 400 μm diameter to ensure efficient nutrient exchange and waste removal. Results ColAlgTubes successfully formed a reinforced hybrid hydrogel structure in which the collagen nanofiber network improved the mechanical integrity of the alginate matrix while supplying adhesion sites for anchorage-dependent cells. The optimized 400 μm diameter microenvironment supported high cell viability, rapid proliferation, and exceptional yields of 5 × 10 8 cells/mL. Discussion These results demonstrate that ColAlgTubes overcome the key limitations of AlgTubes by combining structural reinforcement with biological functionality. The improved mechanical properties reduce tube breakage and cell leakage, while collagen-derived adhesion sites broaden compatibility to anchorage-dependent cell types. With their scalability, cost-effectiveness, and high efficiency, ColAlgTubes represent a transformative solution for large-scale mammalian cell production across biotechnology, regenerative medicine, and therapeutic manufacturing.

BACKGROUND AND OBJECTIVES: Pediatric large-volume brain arteriovenous malformations (AVMs) carry a substantial lifelong hemorrhage risk, neurological symptoms, and treatment morbidity. Single-session stereotactic radiosurgery (SRS) is often unsuitable due to constraints on dose-volume toxicity. Volume-staged SRS (VS-SRS) enables sequential dosing of large nidus volumes, potentially enhancing safety while maintaining efficacy. Evidence in children remains limited. We aimed to evaluate outcomes of VS-SRS for large AVMs in pediatric patients. METHODS: A multicenter retrospective cohort was assembled from 21 centers, including patients aged younger than 21 years treated with VS-SRS for AVMs >10 cm 3 . Clinical and radiological end points included obliteration, hemorrhage, and permanent symptomatic radiation-induced changes (RIC). RESULTS: A total of 103 patients were included (median age 14 years; IQR, 12-17). The median nidus volume at first stage was 18.2 cm 3 (IQR, 12.3-25.6). Median prescription dose per stage was 17 Gy (IQR, 16-18). The median clinical follow-up from the first stage was 57.5 months (IQR, 25-138). Obliteration occurred in 42 of 103 patients (40.8%), with actuarial rates of 6.9% (95% CI: 2.8-14) at 3 years and 29% (95% CI: 20-39) at 5 years. Hemorrhage occurred in 17 of 103 patients (16.5%) during follow-up, and permanent RIC was observed in 9 of 103 patients (8.7%). CONCLUSION: VS-SRS is a reasonably safe, selected option for pediatric large-volume AVMs when microsurgical or endovascular cure is not feasible or prudent. Delivering ≥17 Gy per stage while limiting each treatment volume to <15 cm 3 supports durable nidus control with acceptable toxicity. VS-SRS represents a key modality in multidisciplinary management of this historically difficult-to-treat population.

Abstract Genome sequencing of the heterogeneous primary mitochondrial disorders (PMD) frequently reveals variants of uncertain significance that require functional tests for diagnosis, and does not identify variants in all patients. We analyzed mitochondrial enzyme assays, blue native polyacrylamide gel electrophoresis (BN-PAGE) with in-gel activity staining, complex I assembly blot, and select protein abundances in fibroblasts of a case series of 204 PMD patients divided into functional classes, in comparison to 51 controls and 53 differential diagnostic conditions. Overall, sensitivity and specificity for respiratory chain enzyme assays were 46% and 93% respectively, for BN-PAGE 40% and 98%, for complex I assembly assay 49% and 99%. The overall sensitivity of all tests was 76%, specificity 93%, with positive predictive value 96% and negative predictive value 67%. Categories with high sensitivity were isolated complex deficiencies, nuclear DNA-encoded mitochondrial protein synthesis defects, co-factor defects, and mitochondrial amino-acyl-tRNA synthetase conditions when aided by protein abundance. Mitochondrial DNA mutations and maintenance disorders showed poor sensitivities. Secondary dysfunctions were rare. A complete battery of functional tests showed strong diagnostic clinical utility in fibroblasts. One sentence summary A combination of four mitochondrial functional tests to identify or confirm suspected primary mitochondrial disease in fibroblasts had good sensitivity and excellent specificity, well beyond what was perceived using enzyme assays only.