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AbstractMucus is an important component of airway host defenses that acts by enabling the trapping and clearance of infectious materials such as bacteria and viruses. It can be difficult, however, to design experiments that independently determine the extent to which mucus contributes to innate barrier functions in the lung. Here, we provide detailed protocols to collect mucus from human airway epithelial cultures and evaluate how the properties of mucus impact mucociliary transport and protection from viral infection. We include recommended test parameters depending on the specific research question as it relates to respiratory infectious diseases. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Analysis of mucociliary transport and ciliary beat frequency in HAE cultures Basic Protocol 2: Collection of mucus from HAE cultures Basic Protocol 3: Transplantation of mucus to HAE cultures and infection with virus

PurposeEndothelial–pericyte interaction disruption causes vascular dropout and pathological angiogenesis, severely impacting visual function in ocular microvascular diseases. This study examines VEGF receptor 2 (VEGFR2) signaling in endothelial–pericyte interactions, highlighting VEGFR2 as a potential therapeutic target for promoting pericyte coverage and decreasing vascular leakage in diseased retinas.MethodCell–cell interactions with VEGFR2 signaling were assessed using isogenic endothelial cells and pericytes from induced pluripotent stem cells. We investigated changes in VEGFR2 signaling resulting from endothelial–pericyte interactions using quantitative Reverse Transcription PCR, western blot analysis, immunofluorescence staining, migration assays, permeability assays, transendothelial electrical resistance measurements, flow cytometry, and three-dimensional collagen gel vascular networks. We validated VEGFR2 as a therapeutic target via intravitreal injection in the oxygen-induced retinopathy mouse model. Treatment effects were evaluated using western blot analysis, immunofluorescence staining, and an FITC–dextran permeability assay to assess protein expression, pericyte recruitment, and retinal vascular function in response to VEGFR2 modulation.ResultsWe demonstrate that direct endothelial-pericyte contact, mediated by N-cadherin, downregulates phosphorylated VEGFR2 in endothelial cells, thereby enhancing pericyte migration and promoting endothelial cell barrier function. Intravitreal injection of a VEGFR2 inhibitor in mouse models of the developing retina and oxygen-induced retinopathy increased pericyte recruitment and decreased vascular leakage. The VEGFR2 inhibitor further rescued ischemic retinopathy by enhancing vascularization and tissue growth.ConclusionsOur findings uncover a novel mechanism by which VEGFR2 signaling is regulated through endothelial–pericyte interactions, promoting pericyte migration and strengthening endothelial barrier function. These results suggest a pathway that could be harnessed to support the growth of functional and mature microvasculature in ocular microvascular diseases and tissue regeneration overall.

AbstractPeritoneal carcinomatosis is a frequent metastatic condition in gastroesophageal cancer and is associated with poor prognosis and limited therapeutic options. Here, we establish clinically relevant mouse models of peritoneal carcinomatosis to evaluate the efficacy of a second-generation mesothelin-targeted chimeric antigen receptor (CAR) T-cell therapy. Our model recapitulates key clinical features, including ascites, bowel obstruction, and an immunosuppressive tumor microenvironment characterized by tumor-cell programmed death-ligand 1 expression and elevated TGF-β levels in ascites. To overcome T-cell exhaustion, we engineered a CAR T-cell construct (M28z1XXPD1DNR) that incorporates a programmed cell death protein-1 decoy receptor lacking the intracellular signaling domain, which enhances functional persistence. We demonstrate that intraperitoneal (regional) administration of CAR T cells at low doses achieves superior antitumor efficacy, longer survival, and sustained functional persistence, compared with intravenous (systemic) administration. Of note, intraperitoneal treatment also exhibits potency against distant disease sites. These findings provide a strong rationale for clinical translation; we are now conducting a clinical trial (NCT06623396) to evaluate intraperitoneal administration of M28z1XXPD1DNR CAR T cells in patients with gastroesophageal cancer peritoneal carcinomatosis.

AbstractOsteosarcoma 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).Significance: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.

Simple SummaryCancer-associated fibroblasts (CAFs) play a crucial role in the tumor stroma. Our RNA sequencing analysis with 22 CAF and 11 normal fibroblast (NF) samples of non-small-cell carcinoma (NSCLC) revealed specific CAF markers. COL11A1, GREM1, CD36, and GAS6 are highly expressed in CAFs. Both GREM1 and GAS6 showed a strong expression in CAFs from lymph nodes and CAFs from lung specimens relative to NFs. TNC and CXCL2 are prominent in NFs. Differential expression patterns were observed in lymph node and lung specimens. In the co-culture model of CAFs and THP-1 cells, the knockdown of GREM1 or GAS6 in CAFs significantly decreased the M2 marker expression in macrophages. In NSCLC, GREM1 and GAS6 represent potential diagnostic targets for CAFs derived from both primary tumors and metastatic sites. AbstractBackground/Objectives: Cancer-associated fibroblasts (CAFs) play a pivotal role in the tumor microenvironment. We conducted an analysis using RNA sequencing to identify specific markers for CAFs compared to normal fibroblasts (NFs) in non-small-cell carcinoma (NSCLC). Methods: CAFs and NFs were isolated and cultured from tumor tissues (primary tumor or metastatic lymph nodes) and matched non-tumor tissues, respectively. Bulk RNA sequencing was conducted on isolated CAFs and normal fibroblast NFs. Differential expressions, gene set enrichment, and CAF subpopulation prediction analyses were performed. Results: During the study period, 27 CAFs and 12 NFs were isolated and cultured from tumor and non-tumor tissues in patients with treatment-naïve NSCLC. Among them, 22 CAFs and 11 NFs were included in the RNA sequencing analysis. The 22 CAF samples consisted of 12 adenocarcinomas and 10 squamous cell carcinomas (SqCC), with 16 samples from the lungs and 6 samples from the lymph nodes. Notably, COL11A1, GREM1, CD36, and GAS6 showed a higher expression in CAFs than in NFs, whereas TNC and CXCL2 were more abundantly expressed in NFs. CD36 levels were elevated in CAFs from lymph nodes (LN-CAFs) compared with those from lung specimens (Lung-CAFs) and NFs. COL11A1 levels in Lung-CAFs surpassed those in LN-CAFs and NFs. Both GREM1 and GAS6 showed a strong expression in Lung-CAFs and LN-CAFs relative to NFs. CAFs exhibited features of the myofibroblast CAF subpopulation, whereas NFs displayed traits of the antigen-presenting CAF subtype. In the co-culture model of CAFs and THP-1 cells, the knockdown of GREM1 or GAS6 in CAFs significantly decreased the M2 marker expression in macrophages. Conclusions: In NSCLC, GREM1 and GAS6 can be valuable diagnostic targets for CAFs from primary tumors and metastatic sites; they warrant further study.

Simple SummaryThe study explored the complex link between the immune system and bone structure, emphasizing the key role of IFN-γ in preserving skeletal integrity. Targeting the IFN-γ signaling pathway offers a promising way to strengthen bones and tackle cancer-induced bone disorders. It also highlighted the impact of supercharged NK (sNK) cells and probiotic bacteria in preventing tumor growth and spread in humanized-BLT (hu-BLT) mice. When probiotics are given alone or combined with sNK cell infusions, they boost IFN-γ secretion in hu-BLT mice, effectively stopping tumor-driven bone damage. This research points to the potential of probiotics, alone or with sNK cells, as innovative treatments for osteolytic cancers. It stresses the need to understand the mechanisms behind bone-tumor progression and calls for deeper insights into the bone marrow environment to combat cancer-related bone issues effectively. AbstractThis study found a strong link between interferon-gamma (IFN-γ) secretion from immune cells and changes in bone quality in pancreatic tumor-bearing humanized-BLT (hu-BLT) mice. Tumor presence in hu-BLT mice led to bone resorption and reduced IFN-γ production compared to healthy mice. Interestingly, oral supplementation with probiotic bacteria AJ2, either alone or combined with supercharged NK (sNK) cells, inhibited tumor growth and increased IFN-γ levels in tissue compartments and tumor sites. Enhanced IFN-γ secretion was observed in cell cultures from the pancreas, spleen, PBMCs, splenocyte-derived NK cells, and bone marrow of mice treated with sNK cells and AJ2 compared to untreated tumor-bearing mice. Higher IFN-γ levels were associated with improved bone integrity in hu-BLT mice. TRAP staining showed increased osteoclastic activity and bone resorption in untreated tumor mice, in contrast to those treated with sNK and AJ2. This research highlights the role of immune cell-derived IFN-γ in preventing tumor-induced bone loss and improving bone quality, suggesting that probiotics, alone or with immunotherapies, have potential as treatments for osteolytic cancers.

Human induced pluripotent stem cells (iPSCs) are gaining momentum as a powerful starting material in cell therapy. To fully harness their potential, CRISPR technology permits endogenous gene modifications as well as the introduction of advanced features, to increase the immune compatibility of the cells or insert suicide genes for enhancing therapeutic safety, for instance. However, genetic manipulation of iPSCs, in particular the generation of knock-in lines, remains relatively inefficient. Conventional mitigation strategies, such as enriching for positive cells using antibiotic selection or complex instrumentation, may, however, cause conflicts with good manufacturing practice (GMP) requirements. To address this challenge, we have systematically optimized a basic gene editing procedure using both Cas9 and Cas12a-based ribonucleoprotein (RNP) complexes. Based on the sequential delivery of RNPs and donor plasmids as a critical hallmark, this virus-free approach permits knock-ins of full-length transgenes at above 30% efficiency, while readily identifying positive clones through random screening at small scale. We exemplify these advances by creating and characterizing homozygous iPSC lines depleted of HLA class I and carrying an inducible caspase-9 suicide gene. Isolated clones from independent GMP iPSC lines retained genomic integrity, differentiation capability, and functionality of the safety switch in the differentiated state. This improved methodology will form a flexible platform for custom gene editing universally applicable both in basic iPSC research and therapy.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-025-17876-4.

AbstractINTRODUCTIONThis study investigates ancestry‐specific changes in induced pluripotent stem cell (iPSC)‐derived oligodendroglia genomic regulation in Alzheimer's disease (AD), addressing diversity gaps by including African, Amerindian, and European ancestries in the analysis.METHODSWe generated 12 iPSC lines from AD patients and controls with different apolipoprotein E (APOE) genotypes, APOE ε3/ ε3 and APOE ε 4/ ε4, across three ancestries. Lines were differentiated into neural spheroids containing oligodendrocyte lineage cells and analyzed by single‐nucleus RNA sequencing, Assay for Transposase‐Accessible Chromatin with sequencing (ATACseq)APO, and High‐throughput Chromosome Conformation Capture (Hi‐C).RESULTSWe identified ancestry‐specific differences in gene expression and chromatin accessibility of AD genome‐wide association study candidate genes. APOE ε4/ ε4 carriers across all ancestries showed upregulated cholesterol biosynthesis genes with decreased myelination markers. iPSC‐derived oligodendrocytes demonstrated high correlation (R 2 > 0.85) with human brain transcriptomes.DISCUSSIONOur findings highlight the importance of studying diverse ancestries in AD research and suggest early APOE ε4 effects on cholesterol metabolism. The validated iPSC model provides a valuable tool for investigating ancestry‐specific disease mechanisms.Highlights First study comparing iPSC‐derived oligodendroglia across three ancestries. APOE ε4 carriers show upregulated cholesterol synthesis in oligodendroglia.Reduced myelin gene expression observed in APOE ε4/ε4 oligodendroglia.Ancestry‐specific differences found in AD GWAS genes and chromatin states.Novel insights into oligodendrocyte biology relevant to Alzheimer’s disease.

IntroductionBromodomain and Extraterminal (BET) proteins play a crucial role in cellular proliferation and differentiation through the epigenetic regulation of gene transcription. As a result, inhibiting BET family proteins emerges as a promising epigenetic approach for treating various cancers. However, clinical trials have indicated that thrombocytopenia is a dose-limiting toxicity associated with BET inhibition. This study aims to explore the mechanism and clinical pharmacology of BMS-986158-induced thrombocytopenia and to identify biomarkers as tools to identify patients at higher risk, thereby better managing toxicity and improving efficacy.MethodsBlood samples from preclinical rats and clinical trial patients treated with BMS-986158 were collected for transcriptional expression profiling. Target engagement was confirmed by measuring HEXIM1 and monitoring thrombocytopenia following BET inhibition. Genes regulated by GATA1 and associated with thrombopoiesis, including NFE2 and PF4, were investigated. The outcomes of the rat and human studies were compared to identify biomarkers for the early prediction of thrombocytopenia associated with BET inhibition.ResultsTarget engagement was confirmed with dose-dependent responses of HEXIM1 expression and platelet counts. Blood samples from rats treated with BMS-986158 showed dose-dependent downregulation of GATA1, NFE2, and PF4 at 24 h or earlier post-treatment. Similarly, patients’ blood samples collected within 24 h post-treatment with BMS-986158 also showed dose-dependent downregulation of GATA1 and PF4 in all treated groups. Significant downregulation of PF4 and NFE2 genes was found in patients with low platelet counts. A strong correlation between the expression of GATA1 and the genes NFE2 and PF4 was observed in both preclinical and clinical studies.DiscussionThe consistent downregulation of GATA1, NFE2, and PF4 transcription within hours post-BMS-986158 treatment in both preclinical and clinical studies demonstrates that BET inhibitors induce thrombocytopenia by altering GATA1 gene expression and its downstream genes, NFE2 and PF4, which regulate megakaryopoiesis and thrombopoiesis. Early detection of transcriptional changes in blood samples during treatment courses positions NFE2 and PF4 as promising biomarkers for proactively monitoring and mitigating treatment-emergent thrombocytopenia. Graphical abstract Flowchart illustrating the effects of BET inhibitors. BET inhibitor impacts BRD2/BRD4, linked to GATA1 hematopoietic transcription factor. GATA1 affects NFE2 and PF4, influencing thrombopoiesis and leading to thrombocytopenia. Separately, BRD2/BRD4 affects oncogenes, including MYC, related to cancer.

A Cytokine storm is critical in severe dengue, significantly contributing to disrupted endothelial integrity, plasma leakage, and haemorrhage manifestations in affected patients. Various reports have demonstrated that mononuclear phagocytes, including monocytes, dendritic cells, and macrophages, are target cells of DENV infection. They contribute to viral spread into tissues and promote robust inflammatory responses and immunopathology. However, it remains unclear whether the early events of DENV infection play a role in triggering cytokine storms in infected mononuclear phagocytes. To address this knowledge gap, we conducted a comprehensive analysis of the transcriptomic profile of in vitro DENV-2-infected human monocyte-derived macrophages (MDMs) based on the kinetics of viral replication through a standard growth curve. To verify the accuracy of our approach, we used RT-qPCR, ELISA, and transcriptomic data from in vitro DENV-2-infected monocyte-derived dendritic cells (MDDCs) and monocytes obtained from acute dengue patients. RNA-Seq analysis revealed dynamic changes in the transcriptional profile of DENV-2-infected MDMs throughout the viral growth curve. Two waves of differentially expressed genes were observed: the first occurred during the eclipse period of viral replication (3 to 5.5 h.p.i) and was associated with the induction of NF-kB-dependent pro-inflammatory factors, while the second wave at 24 h.p.i coincided with peaks of DENV-2 replication and induction of both NF-kB- and STAT1-dependent pro-inflammatory responses. Additionally, DENV-2 infection promoted the dynamic activation of Toll-like receptors, RIG-like receptors, inflammasomes, and inflammatory pathways, triggering innate pro-inflammatory and antiviral responses. A robust multi-type IFN-dependent antiviral response was also observed at the late stage of infection. A similar transcriptomic profile was found in DENV-2-infected MDDCs and monocyte subsets from acute dengue patients, further confirming the reliability of our in vitro model of DENV-infected MDMs. Together, results suggest that recognizing viral PAMPs during the eclipse period of DENV-2 infection promotes a robust NF-kB-dependent pro-inflammatory response in MDMs. In addition, at later stages of infection, recognizing structural DENV-PAMPs and/or viral replication intermediates induces both NF-kB- and STAT1-dependent pro-inflammatory responses, leading to a cytokine storm. These findings highlight the critical role of monocytes, macrophages, and dendritic cells in detecting DENV infection and triggering a cytokine storm in vitro and in vivo. This suggests that these cell populations could be potential targets for future immunotherapies to modulate the inflammatory response to DENV infection. Author summaryDengue virus (DENV) infection can elicit an excessive and pathological immune response known as a “cytokine storm,” which plays a central role in the pathogenesis of severe dengue, leading to vascular leakage, haemorrhage, and potentially fatal outcomes. Although monocytes, macrophages, and dendritic cells are recognized contributors to this hyperinflammation, the early events that initiate cytokine storm development remain poorly defined. Here, we investigated the temporal dynamics of DENV-2-induced inflammatory responses in human macrophages by analysing transcriptomic changes throughout infection. We identified two distinct waves of immune activation: an early phase (3–5.5 hours post-infection) initiated by viral components before productive replication, and a later, more robust inflammatory response (24 hours post-infection) coinciding with peak viral replication. Both phases engaged canonical immune signalling pathways, including NF-κB and IFN-STAT1, as well as pattern recognition receptors such as Toll-like and RIG-I-like receptors. These transcriptional signatures were recapitulated in DENV-2-infected dendritic cells, and monocytes from dengue patients, underscoring their physiological relevance. Our work reveals how DENV leverages host immune sensing mechanisms to drive pathogenic inflammation. By delineating early molecular triggers, we identify potential therapeutic targets to mitigate cytokine storms and improve clinical outcomes in severe dengue.

IntroductionCOVID-19 was less severe in Sub-Saharan Africa (SSA) compared with Europe and North America. It is unclear whether these differences could be explained immunologically. Here we determined levels of ex vivo SARS-CoV-2 peptide-specific IFN-γ producing cells, and plasma cytokines and chemokines over the first month of COVID-19 diagnosis among Kenyan COVID-19 patients from urban and rural areas.MethodsBetween June 2020 and August 2022, we recruited and longitudinally monitored 188 COVID-19 patients from two regions in Kenya, Nairobi (urban, n = 152) and Kilifi (rural, n = 36), with varying disease severity – severe, mild/moderate, and asymptomatic. IFN-γ secreting cells were enumerated at 0-, 7-, 14- and 28-days post diagnosis by an ex vivo enzyme-linked immunospot (ELISpot) assay following in vitro stimulation of peripheral blood mononuclear cells (PBMCs) with overlapping peptides from several SARS-CoV-2 proteins. A multiplexed binding assay was used to measure levels of 22 plasma cytokines and chemokines.ResultsHigher frequencies of IFN-γ-secreting cells against SARS-CoV-2 spike peptides were observed on the day of diagnosis among asymptomatic compared to patients with severe COVID-19. Higher concentrations of 17 of the 22 cytokines and chemokines measured were positively associated with severe disease, particularly interleukin (IL)-8, IL-18 and IL-1ra (p < 0.0001), while a lower concentration of SDF-1α was associated with severe disease (p < 0.0001). Concentrations of 8 and 16 cytokines and chemokines including IL-18 were higher among Nairobi asymptomatic and mild patients compared to their respective Kilifi counterparts. Conversely, concentrations for SDF-1α were higher in rural Kilifi compared to Nairobi (p = 0.012).ConclusionIn Kenya, as seen elsewhere, pro-inflammatory cytokines and chemokines were associated with severe COVID-19, while an early IFN-γ cellular response to overlapping SARS-CoV-2 spike peptides was associated with reduced risk of disease. Living in urban Nairobi (compared with rural Kilifi) was associated with increased levels of pro-inflammatory cytokines and chemokines.

Neutrophil extracellular traps (NETs) are DNA-protein structures released during a form of programmed neutrophil death known as NETosis. While NETs have been implicated in both tumor inhibition and promotion, their functional role in cancer remains ambiguous. In this study, we compared the NET-forming capacity and functional effects of NETs derived from lung cancer (LC) patients and healthy donors (H). Neutrophils were isolated from peripheral blood and stimulated in vitro with phorbol 12-myristate 13-acetate (PMA) to induce NETosis. Isolated NETs were quantified and assessed for their cytotoxicity against A549 lung cancer cells and their impact on cancer cell migration. Whereas LC neutrophils (LCN) were less cytotoxic to tumor cells than H neutrophils (HN), their NETs maintained similar tumoricidal capacity – 41.6% ± 25.3% (LCN) vs. 46.4% ± 14.5% (HN), ns. Interestingly, we noted a correlation between the amount of NETs and their cytotoxicity to tumor cells. This effect could not be recapitulated with purified genomic DNA, inducing only 3.99% of cytotoxicity to tumor cells, and confirming that intact NETs are required for the anti-tumor activity. LCN displayed an increased frequency of NETosis following PMA stimulation, yet produced significantly fewer NETs per cell – 1569 ± 306 ng (LCN) vs. 2619 ± 313 ng (HN); p = 0.025. Reactive oxygen species (ROS) production was elevated in LC neutrophils, indicating that the NETosis defect was not due to impaired oxidative burst. LCN had increased expression of immunosuppression (PDL-1) as well as exhaustion and aging markers CD62L and CD11b). Only NETs from HN inhibited the migration of A549 tumor cells, whereas those from LCN failed to suppress, and in some cases appeared to enhance, cell motility. Our data suggest that NETs in lung cancer retain anti-tumor cytotoxicity capabilities but lose their anti-migratory capacity, highlighting their dual role in tumor biology and potential as therapeutic targets.