Airborne respiratory viruses, such as coronaviruses and influenza, pose major threats to public health and the economy, as highlighted by the COVID-19 pandemic. Preclinical research is hindered by models that poorly mimic human tissue structure and function, often relying on immortalized cell lines and low-throughput animal studies. This limits accurate prediction of disease mechanisms, drug effects, and target suitability. Here, we report a custom-engineered, passive-flow, high-containment chip for culturing human primary bronchial epithelial cells (hPBECs) at air-liquid interface (ALI) on a large-area membrane. The dual-chamber microfluidic chip, separated by a horizontal support membrane, is enclosed in a 35 mm sealed Petri dish, enabling safe use in standard incubators without leakage or biosafety concerns. The platform supports high-resolution in-situ imaging, apical viral infection, and retrieval of cells and secretions (e.g., mucus, viral lysate) for molecular analysis. We demonstrate robust infection and replication of human coronavirus NL63 (HCoV-NL63) in differentiated hPBECs cultured up to 4 weeks at ALI. Epithelial differentiation was confirmed by immunofluorescence (e.g., ciliated cells), and infection kinetics were monitored by RT-qPCR over 7 days. The interferon-based immune response showed increased activity, with upregulation of viral response pathways (e.g., replication, inflammation, immunoregulation), and consistent activation across donors (e.g., ISG15, IFIT1). Collectively, we present a reproducible, small-scale chip model that enables high-containment in vitro studies of respiratory viruses and their effects on human airway epithelia.
© 2025 The Authors. Published by Elsevier Ltd.

Several FDA-approved anti-PD-L1 (programmed cell death ligand-1) monoclonal antibodies (mAbs) are used to treat cancer. While these mAbs primarily target and intercept PD-L1:PD-1 inhibitory signaling in T-cells, the Fc-domains of these mAbs are distinct, and the unique cellular cascades triggered by differing Fc-domains of PD-L1 mAbs have not been directly investigated. In this study, we compared the innate immune effects of two widely used anti-PD-L1 IgG1 mAbs which bear distinct Fc-domains, avelumab (native-Fc) and durvalumab (mutated-Fc), using two-cell and three-cell co-culture systems containing Natural Killer cells (NK-cells), dendritic cells (DCs) and various tumor cell lines of multiple cancer origins. We show a robust enhancement in NK-cell effector function, DC maturation, reciprocal NK:DC crosstalk and DC editing that is unique to avelumab treatment using multiple functional immune assays. By transcriptomic analysis, we show for the first time pivotal differences in gene sets involved in NK-cell effector function, DC maturation, immunoregulatory interactions, and cytokine production between innate immune cells treated with avelumab versus durvalumab. Furthermore, we report several previously unknown Fc-receptor-associated biological pathways uniquely triggered by avelumab. Our findings elucidate novel mechanisms of Fc-dependent actions of PD-L1 mAbs which may inform their use in future clinical trials.

Patient-derived organoids (PDOs) have emerged as powerful tools in personalized medicine applicable to both non-malignant conditions and to cancer, where they are increasingly used for personalized drug screening and precision treatment strategies in part due to their ability to replicate tumor heterogeneity. They also serve as study model systems to understand disease mechanisms, pathways, and the impact of ex vivo exposures. We present a detailed step-by-step protocol for generating organoids from normal crypts, polyps, and tumors, including methods for tissue processing, crypt isolation, culture establishment, and the transition from basolateral to apical-out polarity for co-culture and exposure-based studies. The protocol also includes immunofluorescence staining procedures for cellular characterization and quality control measures. Our standardized approach successfully generates organoids from diverse colorectal tissues with high efficiency and reproducibility. This comprehensive guide addresses common technical challenges and provides troubleshooting strategies to improve success rates across different sample types. We believe that this resource will enhance reproducibility in organoid research and expand their utility in translational applications, particularly for personalized medicine approaches in colorectal cancer.

Recurrent acute anterior uveitis is a frequent extra-articular manifestation of the axial spondyloarthropathies (AxSpA): chronic inflammatory diseases affecting the spine, enthesis, peripheral joints, skin, and gastrointestinal tract. Pathology in AxSpA has been associated with local tissue-resident populations of IL-23 responsive lymphoid cells. Here we characterize a population of ocular T cell defined by CD3+CD4-CD8-CD69+γδTCR+IL-23R+ that reside within the anterior uvea as an ocular entheseal analogue of the mouse eye. Localized cytokine expression demonstrates that uveal IL-23R+ IL-17A-producing cells are both necessary and sufficient to drive uveitis in response to IL-23. This T cell population is also present in humans, occupying extravascular tissues of the anterior uveal compartment. Consistent with the concept of IL-23 as a unifying mediator in AxSpA, we present evidence that IL-23 can also act locally on tissue resident T cells in the anterior compartment of the eye at sites analogous to the enthesis to drive ocular inflammation.

Osteosarcoma is the most common primary malignant bone tumor and predominantly affects adolescents and young adults. It is the third most common cause of cancer-related deaths among 9 to 24 year olds. Despite aggressive chemotherapeutic and surgical therapies, the survival rate is only 25% for patients with detectable lung metastases at diagnosis and only 70% in patients who present without detectable lung metastases. The poor prognosis is due to growth of metastases irrespective of whether they are initially large enough to detect clinically. It is therefore necessary to develop new methods to target the growth of lung micrometastases. An NCI panel of FDA-approved oncology drugs was therefore screened using three highly metastatic human osteosarcoma cell lines. To more closely approximate in vivo micrometastases, the screen used a three-dimensional multicellular in vitro osteosarcoma spheroid (sarcosphere) model. Among 13 hits from the initial screen, we identified the histone deacetylase inhibitor (HDI) romidepsin as the most promising inhibitor in secondary screens comparing effects on sarcospheres with clinically achievable levels and to effects on non-transformed cells. Romidepsin potency was evident with and without standard-of-care chemotherapeutics (MAP: methotrexate, adriamycin, and cisplatin) at romidepsin concentrations that are clinically achievable and did not affect non-transformed cells. Romidepsin also substantially outperformed the other three FDA-approved HDIs and eight HDIs in clinical trials. The effects of romidepsin were a transient cell cycle block at G2/M and cell death. Importantly, sarcospheres derived from ∼30% of human and 50% of canine patient samples responded to romidepsin at clinically tolerable concentrations (ED50s <70 nmol/L).
Our unbiased sarcosphere-based drug screen identified romidepsin as a promising candidate to repurpose for human and canine patients with metastatic osteosarcoma. This screening strategy allowed us to identify romidepsin-sensitive and -resistant patients. Sarcosphere-based screening may therefore be useful to identify patients most likely to respond clinically to romidepsin or other drugs.
©2025 The Authors; Published by the American Association for Cancer Research.