Abstract The survival of GBM cells critically depends on receptor tyrosine kinases like fibroblast growth factor receptors (FGFR) and cell adhesion molecules like discoidin domain receptor 1 (DDR1), both of which represent potential therapeutic targets. Although adaptive responses to pharmacological inhibitors are known, therapeutic adaptation is often hampered due to a lack of underlying mechanisms. Based on the radiochemosensitizing potential of DDR1 monoinhibition, we investigated the adaptive response induced by DDR1 inactivation using a broad-spectrum kinome analysis (PAMgene technology). Since FGFRs have been identified as the most potently induced tyrosine receptors, we investigated the efficacy of concurrent pharmacological FGFR/DDR1 inhibition and observed increased cytotoxicity and radiochemosensitization in a series of human gliomasphere models. Subsequent evaluation of single and concurrent FGFR and DDR1 inhibition in orthotopic GBM mouse models treated with chemoradiotherapy revealed a significant reduction in the efficacy of simultaneous FGFR/DDR1 inhibition compared to FGFR or DDR1 monotherapy. Mechanistically, RNA sequencing analyses demonstrated that strong activation of EGFR/MAPK signaling with concurrent FGFR/DDR1 inactivation greatly reduced the efficacy of chemoradiotherapy compared to monotherapies. In summary, our data clearly demonstrate that DDR1 monotherapy, in particular, effectively complements chemoradiotherapy in GBM, whereas concurrent FGFR/DDR1 inhibition appears harmful and requires further investigation.

IntroductionCirculating tumor cells (CTCs) and L1 cell adhesion molecule (L1CAM) are associated with breast cancer (BC) metastasis. This study investigated their potential as predictive biomarkers for lymph node metastasis in early-stage invasive breast cancer (ESIBC).MethodsNinety-three ESIBC patients were enrolled. CTC phenotypes and L1CAM expression were detected in preoperative blood samples using the CanPatrol® CTC system and RNA-ISH. Associations with clinicopathological variables were analyzed.ResultsCTCs were detected in 79.6% of patients. Hybrid CTCs (H-CTCs) and L1CAM-positive CTCs were significantly correlated with lymph node metastasis and Ki-67 expression. A nomogram integrating H-CTCs, L1CAM, and Ki-67 predicted metastatic risk with excellent accuracy (AUC = 0.98).DiscussionH-CTCs and L1CAM-positive CTCs serve as potential blood-based biomarkers for evaluating metastatic risk in BC.ConclusionThe combined detection of H-CTCs and L1CAM enhances preoperative prediction of lymph node metastasis and provides new insights into BC metastasis mechanisms.

Primary familial brain calcification (PFBC) is a dominantly or recessively inherited neurodegenerative disease characterized by bilateral basal ganglia calcifications. Patients affected by PFBC present with diverse motor and nonmotor symptoms. Mutations in seven genes (SLC20A2, XPR1, PDGFB, PDGFRB, MYORG, NAA60, and JAM2) are associated with PFBC. PFBC genes encode proteins that comprise inorganic phosphate transporters, growth factor and its receptor, a cell adhesion molecule, and enzymes. It remains to be determined whether these proteins interact within a single disrupted pathway or whether mutations affect distinct pathways in the same cell type. Although vessel calcification is a diagnostic criterion of PFBC, its causal role in neurodegeneration needs to be established. This review provides an overview of PFBC genes, including animal models that have yielded insights into the underlying pathophysiologic mechanisms, such as the role of specific cell types in the progression of vascular calcification.

The aim of the study was to evaluate the prospects/likelihood/feasibility of in vitro endothelial-mesenchymal transition (endothelial-mesenchymal transition, EndMT) studies in primary human umbilical vein endothelial cell cultures (human umbilical vein endothelial cells, HUVEC) using specific concentrations and time of TGF1 expression. Material and methods. The study was conducted on primary human umbilical vein endothelial cell culture HUVEC passage 3. Primary HUVEC cultures were cultured in ECGM culture medium for 3 days until a monolayer was formed, after which the nutrient medium was changed to serum-free DMEM F-12 medium for 3 hours. Then, HUVEC culture cells were incubated with recombinant human TGF-1 (10 ng/ml) at 37C for 72 hours. At the end of TGF-1 exposure, the relative amounts of proteins characteristic of EndMT are obtained in cell lysates using Western blotting techniques: endothelial markers - cell adhesion molecules and platelets to endothelial cells-1 PECAM-1 (CD31) and von Willebrand factor (von Willebrand factor, vWF), as well as mesenic markers - fibronectin (fibronectin, FN), vimentin. (vimentin, VIM). Results. The study found that exposure of the liver to TGF-1 at a dose of 10 ng / ml for 3 days on the primary culture of HUVEC led to a significant decrease in the amount of CD31 by 45.4% and vWF by 34.3% compared to the control, confirmed by the services for confirming the loss of characteristic endothelial cell markers (r 0.05). Exposure of primary HUVEC culture to TGF-1 for 72 h significantly increased the relative amount of FN by 43.3% and VIM by 50.7% relative to the control (p0.05), indicating the formation of endothelial cells with mesenchymal characteristics. Conclusions. Exposure of primary HUVEC culture to human recombinant TGF-1 in patients at 10 ng/ml for 3 days allows for reliable induction of EndMT in vitro, which is a statistically significant decrease in the relative amount of endothelial markers EndMT CD31 and vWF and a relative increase in the amount of mesenchymal markers FN and VIM in cultured cell lysates.

Objective The aim of this study is to see the association of P-selectin, E-selectin, Vascular Cell Adhesion Molecule-1 (VCAM-1) and Intercellular Adhesion Molecule-1 (ICAM-1) with Vaso-occlusive crisis (VOC) in sickle cell disease patients. Methods In this longitudinal study, a total of 140 SCD patients admitted into a Government Medical College, Jabalpur, were recruited. Plasma levels of soluble P-selectin, E-Selectin, ICAM-1, and VCAM-1 were measured at three different time points, that is, (i) at the time of admission (n = 140), (ii) at discharge (n = 139), and (iii) during steady state (n = 118). Results The most common presenting symptoms at the time of VOC were severe joint pain (65.7%) and back pain (49.3%). All the cell adhesion molecule levels were similar in males and females except sVCAM-1. The plasma P-selectin, E-Selectin, ICAM-1 and VCAM-1 levels were significantly higher during crisis than at the discharge and steady state. No statistically significant association of any of the CAM levels with the severity of pain was observed. High plasma VCAM-1 levels (≥1676 ng/mL) were associated with longer duration of hospital stay. Conclusions Significant association of soluble CAMs with VOC in SCD suggests that soluble CAMs play a crucial role in the pathophysiology of the VOC.

Despite intensive, multimodal therapy, only half of children diagnosed with high-risk neuroblastoma will survive five years, and survivors harbor significant short- and long-term treatment-related co-morbidities. Although monoclonal antibody therapy targeting GD2 has improved outcomes, GD2-directed immunotherapy remains one of the only FDA-approved immunotherapies for pediatric cancer, and therapy is toxic due to GD2 expression on pain fibers. Thus, there is a critical need to uncover new immunotherapy targets in neuroblastoma. ALCAM is a cell adhesion molecule that promotes tumor growth in a variety of cancers and is highly expressed in neuroblastoma. We generated three inducible CRISPRi cell lines to deplete ALCAM and elucidate its role in neuroblastoma. Depletion of ALCAM reduced cell growth, reduced Ki-67 staining, and increased cleaved PARP. To determine the mechanism of ALCAM overexpression, we used ChIP-sequencing to show MYCN oncoprotein binding at the ALCAM promoter. We generated luciferase reporters from the ALCAM promoter and a putative upstream (10kb) enhancer, which we defined using Promoter-based Capture-C. Treatment with the MYC(N)/MAX dimerization inhibitor MYCi975 reduced ALCAM expression by immunoblotting and luciferase signal from the ALCAM promoter. We validated the activity of the upstream enhancer and uncovered an AP-1 binding motif that is critical for enhancer activity. Finally, as ALCAM is expressed in several normal tissues, we investigated an ALCAM-targeted conditionally activated antibody drug conjugate (ADC), CX-2009 (praluzatamab ravtansine), which delayed tumor growth in two out of three PDX models. Together, these findings credential ALCAM as an immunotherapeutic target in neuroblastoma.

Proteins linked to heritable coronary heart disease (CHD) could uncover new pathophysiological mechanisms of atherosclerosis. We report on the protein profile associated with a family history of early-onset CHD and whether the relation between proteins and coronary atherosclerotic burden differs according to family history status, as well as inferences from Mendelian randomization. Data on coronary atherosclerotic burden from computed tomography angiography and Olink proteomics were retrieved for 4521 subjects, free of known CHD, from the SCAPIS (Swedish Cardiopulmonary Bioimage Study). Records of myocardial infarction and coronary revascularization therapies in any parent or sibling of subjects were retrieved from national registers. Linear associations between family history and proteins were adjusted for age, sex, and study site. Statistical interactions between proteins and family history for the association between proteins and the coronary atherosclerotic burden were also studied. Mendelian randomization for causal associations between proteins and CHD was performed with GWAS summary data from UKB-PPP (UK Biobank Pharma Proteomics Project), CARDIoGRAMplusC4D, and FinnGen. Of 4251 subjects, family history of early-onset CHD was present in 9.5%. Thirty-eight proteins, with biological features of inflammation, lipid metabolism, and vascular function, were associated with family history using a false discovery rate of 0.05. The strongest associations were observed for follistatin and cathepsin D, neither of which was attenuated by adjusting for cardiovascular risk factors. Eighteen proteins were statistical interactors with family history in the association between each protein and the coronary atherosclerotic burden, most notably the LDL (low-density lipoprotein) receptor, transferrin receptor protein 1, and PECAM1 (platelet endothelial cell adhesion molecule 1). In 2-sample Mendelian randomization, a novel association was found for follistatin and myocardial infarction, and previous associations for PCSK9 (proprotein convertase subtilisin/kexin type 9) and PECAM1 were repeated. These findings highlight new potential mechanisms for heritable and general atherosclerosis.

Background: Sex-based differences often influence the therapeutic efficacy and safety of medications. Semen Cuscutae is a traditional tonic botanical drug with sex-specific characteristics, traditionally indicated for conditions such as impotence (exclusive to males) and restless fetus (exclusive to pregnant females). However, most existing studies have focused on a single sex. Objective: To evaluate the sex-specific biological effects of Semen Cuscutae in rats and explore its molecular mechanisms, with the aim of uncovering its pharmacological characteristics through a multiomics approach. Methods: A traditional aqueous extract of Semen Cuscutae (SCA) was used as the experimental material. Forty adult Sprague-Dawley rats (equal numbers of males and females) were randomly divided into 4 groups: male control, male SCA treatment (240 mg/kg), female control, and female SCA treatment (240 mg/kg), with 10 rats in each group. The biological effects were comprehensively evaluated using a combination of open field test, biochemical analyses, proteomics, and gut microbiota profiling. Results: As a tonic botanical drug, SCA appeared to directly affect the mental and behavioral state of rats. It significantly altered the time spent by rats in the center area during the open field test, showing a sex-dependent reversal of behaviors. Proteomic analysis of brain tissue identified 624 differentially expressed proteins across the groups, with 10 key differentially expressed protein related to sex differences, including fibroblast growth factor receptor 3, transcription elongation factor A protein-like 1, 40S ribosomal protein S25, neural cell adhesion molecule, and anion exchange protein 2 (SLC4A2). Enrichment analysis revealed that in male rats, SCA upregulated proteins involved in biological processes such as ribosome function and energy derivation, supporting protein synthesis and enhancing energy supply, showing an overall gain effect. In contrast, in female rats, SCA downregulated proteins associated with processes such as positive regulation of target of rapamycin signaling and vesicle transport, suggesting suppression of neuronal signaling and material transport, indicative of a shift toward a more restrained physiological state. Furthermore, SCA reduced gut microbiota diversity in female rats but increased it in males, including the abundance of Akkermansia , which may serve as a crucial mediator. Conclusion: Overall, the biological effects of SCA differ significantly between male and female rats, with evidence suggesting greater health benefits in males. These findings help elucidate the scientific basis of its traditional applications and provide guidance for the precise application of SCA as a functional health food.

Any strategy that can selectively and persistently lower the brain levels of the cellular prion protein (PrPC) is expected to extend survival in prion diseases. Recent advances in the virus-mediated delivery of gene therapies prompted us to explore if a recombinant adeno-associated virus (rAAV) vector delivering a CRISPR-Cas-based gene editor can be devised that induces a functional knockout of the prion gene. Whereas the eventual objective is to assess the therapeutic potency of an optimized vector in prion-infected mice, in this proof-of-concept study, we evaluated tools and methods that are suited to achieve this goal. The result of these efforts is a first-generation all-in-one rAAV vector that codes for a prion gene-specific guide RNA and a small Cas9 endonuclease, whose expression is controlled by a truncated neural cell adhesion molecule 1 (NCAM1) promoter that is active in PrPC expressing cells. We also constructed a second rAAV vector coding for a prion gene-specific ‘traffic light reporter’ (TLR). The TLR can be used to monitor prion gene-editing efficacy by coding for red and green fluorescent proteins separated by a segment of the prion gene that is targeted by the gene editor. For the purification of AAVs, we adopted a robust and scalable rAAV vector assembly pipeline and undertook proof-of-concept prion gene editing experiments in human cells and mice, which to date yielded prion gene editing rates of approximately 20% and 5%, respectively. Finally, we compared brain distributions of rAAV vectors following intrathalamic versus retro-orbital injection, and selected the 9P31 capsid for future studies based on a 7.5-fold higher heterologous gene expression level as compared to the PHP.eB capsid.

BackgroundMore and more Research has shown that Hashimoto’s thyroiditis (HT) is significantly associated with recurrent miscarriage (RM), but the specific mechanism is not yet clear. This study aimed to identify the key shared biomarkers between HT and RM using bioinformatics methods, reveal the potential molecular mechanisms they were involved in and the characteristics of the immune microenvironment, and provide new theoretical basis and potential diagnostic and therapeutic targets for the association between these two diseases.MethodsThis study adopted an integrated transcriptomic analysis strategy. First, the HT thyroid tissue dataset (GSE138198) and RM endometrial tissue datasets (GSE165004 and GSE26787) were obtained from the Gene Expression Omnibus (GEO) database. Differentially expressed genes (DEGs) were screened, and the intersection was taken to identify key genes. Further verification was conducted through the protein-protein interaction (PPI) network to screen candidate biomarkers. Subsequently, the final biomarkers were determined through consistency verification of expression levels. Gene Set Enrichment Analysis (GSEA) was used to reveal biomarker-related pathways, and the ssGSEA algorithm was applied to quantify immune cell infiltration for analyzing its association with the immune microenvironment. Finally, targeted drugs were predicted via molecular docking, and experimental verification was performed using an HT animal model.ResultsCFL1 and TRAPPC1 were identified as biomarkers, and their expression levels were up-regulated in disease groups. A nomogram with superior diagnostic performance was constructed to predict the occurrence of RM. In the GSE138198 dataset, biomarkers CFL1 and TRAPPC1 were found to be enriched in multiple pathways, like “graft-versus-host disease”, “autoimmune thyroid disease”, and “antigen processing and presentation”. In the GSE165004 dataset, biomarkers were enriched in multiple pathways, like “ribosome”, “Huntington’s disease”, and “cell adhesion molecules (CAMs)”. Additionally, the abundance of infiltration of monocytes and eosinophils infiltration showed significant differences between HT and RM patients (p < 0.05). Biomarkers showed significant positive correlations with monocytes and eosinophils in HT and RM, respectively. Moreover, artenimol and S-palmitoyl-L-cysteine might be potential therapeutic drugs for HT and RM.ConclusionCFL1 and TRAPPC1 were found to be common biomarkers for HT and RM in this study. These genes were thoroughly investigated and analyzed, yielding novel insights for both fundamental experimental research and early clinical diagnosis and treatment of disease.

L1 cell adhesion molecule (L1CAM) is a relevant target in pediatric tumors, including rhabdomyosarcoma (RMS). We validated the recombinant antibody ABCD_AN179, derived from the CE7 hybridoma, for specific detection of L1CAM by flow cytometry. Using CRISPR/Cas9-engineered L1CAM-knockout RMS cell lines (RD, Rh30), ABCD_AN179 showed clear staining of wild-type cells but no signal in knockouts, confirming high specificity. These results demonstrate that ABCD_AN179 specifically detects human L1CAM by flow cytometry and represents a validated recombinant antibody that supports research and translational studies.

Background: The goal of tissue regeneration is to restore structure and function. In adult regenerative wound healing, dermal fibroblasts exhibit multipotency and can be reprogrammed into lineages such as hair follicle cells and adipocytes, etc. Neural cell adhesion molecule 1 (NCAM1), a membrane-bound adhesion protein expressed in dermal fibroblasts, plays a key role in cell-cell and cell-matrix interactions and has been implicated in fate transitions during tissue remodeling. NCAM1 is absent in normal endothelium but is aberrantly expressed in tumor-derived endothelial cells, where it promotes capillary morphogenesis. These observations suggest that NCAM1+ fibroblasts may represent a progenitor-like state more capable of endothelial conversion than NCAM1- cells. While angiogenesis is the main vascularization process in adult wounds, vasculogenesis from progenitors like fibroblasts may also contribute. Fibroblast-to-endothelial reprogramming has been demonstrated in vitro using defined reprogramming factors, but its in vivo evidence remains to be investigated. We hypothesized that NCAM1+ dermal fibroblasts give rise to a subset of regenerated endothelial cells. Aims: (1) Characterize vasculature formation during regenerative wound healing (2) Identify progenitor populations and reprogramming cues, including transcription factors and adhesion molecules Method: Using a wound-induced hair neogenesis (WIHN) mouse model, we performed scRNA sequencing and RNA velocity analyses on post-wounding day 14. Wholemount immunostaining assessed vascular morphology and cell identity. NCAM1-CreERT2×ROSA26 reporter mice for lineage tracing. Results: The regenerated wound bed showed two contrasting vasculature patterns: disrupted, discontinuous vessels in the regenerating wound center and organized vasculature in the wound margin. scRNA-seq and velocity analyses indicated PECAM1+ cells may originate from NCAM1+ fibroblast-like cells that also expressed fibroblast-to-endothelial cells reprogramming factors such as FOXO1, TAL1 and SOX17. Wholemount staining revealed individual cells and a fraction of endothelial cells co-expressing PECAM1, PDGFRα, and NCAM1 in the capillary-like vasculature in wound dermis. Conclusion: Our findings support vasculogenesis during regenerative wound healing, with NCAM1+ fibroblasts contributing to endothelial cell populations. These results provide new insights into fibroblast-endothelial plasticity and vascular regeneration in adult tissue regeneration.

Background: Atrial fibrosis induces atrial fibrillation (AF) and increases the risk of stroke. In our previous study, we demonstrated that tumor endothelial marker 1 (TEM1/CD248), a transmembrane protein only expressed in mesenchymal cells during embryonic development, is re-expressed in atrial cardiac fibroblasts (CFs) of AF patients (pts) and TEM1 modulates CF cellular behaviors. We further explored the role of TEM1 in atrial inflammation, a key driver of atrial fibrosis. Methods and Results: Data are presented as mean ± standard error. Left atrial (LA) appendages were collected from 30 AF pts (mean age 64.0±1.6 yrs; 76.7% male) undergoing cardiac surgery. Western blot analysis and real-time quantitative polymerase chain reaction (RT-qPCR) were used to assess expression levels of TEM1 and pro-inflammatory mediators in the LA tissues. Pearson correlation analyses of western blot revealed significantly positive correlations between TEM1 expression with interleukin (IL)-6 (r=0.53), IL-1β (r=0.52), intercellular adhesion molecule 1 (ICAM1) (r=0.54), and vascular cell adhesion molecule 1 (VCAM1) (r=0.69, all p&lt;0.01). Multivariate regression analysis confirmed these associations were independent of age, sex, hypertension, diabetes, and dyslipidemia. RT-qPCR also shows positive correlations of relative TEM1 expression level with IL-1β (r=0.99), tumor necrosis factor (TNF)-α (r=0.99), ICAM1 (r=0.76), VCAM1 (r=0.69) and the macrophage marker CD68 (r=0.68, all p&lt;0.001). Immunohistochemical staining with F4/80 demonstrated increased macrophage infiltration in the LA tissues of AF pts compared to normal control (US Biomax). Osmotic mini-pumps delivering angiotensin II (Ang II, 1000 ng/kg/min for 4 wks) were implanted in mice to induce atrial fibrosis. Ang II significantly increased atrial macrophage infiltration in wild-type (WT) mice (n=3), which was markedly attenuated in TEM1-deficient transgenic mice (n=3) (F4/80-positive/total area [%]: 0.07±0.01 [WT+saline] vs. 1.62±0.19 [WT+Ang II] vs. 0.53 ± 0.03 [TEM1-deficient+Ang II], p&lt;0.001). Sirius Red staining showed significantly reduced atrial fibrosis in TEM1-deficient mice compared to WT mice after Ang II infusion. Conclusions: TEM1 expression in atrial CFs promotes atrial inflammation and contributes to atrial fibrosis.

Endometriosis is a common disease among women of childbearing age, and endoplasmic reticulum stress (ERS), a response involved in regulating protein homeostasis, has been linked to its pathogenesis. To identify ERS-related hub genes, this study sequentially employed differential expression analysis, weighted gene co-expression network analysis (WGCNA), protein–protein interaction (PPI) network construction, and three machine learning algorithms. These methods led to the identification of four hub genes: Von Willebrand factor (VWF), vascular cell adhesion molecule 1 (VCAM1), endothelial PAS domain protein 1 (EPAS1), and coagulation factor VIII (F8). Unsupervised cluster analysis was conducted to categorize samples into ERS clusters, and the CIBERSORT algorithm was used to calculate immune infiltration scores, revealing two stable clusters. Cluster B was defined as “immune-enriched” with significantly higher immune scores, while Cluster A was “less immune-enriched”. Functional enrichment analysis of differentially expressed genes (DEGs) between the clusters highlighted cell adhesion and regulation of immune cell activation as key to cluster-specific phenotypes. A diagnostic model built with the four hub genes showed robust utility via validation curves, confirming their clinical relevance. DEGs from each cluster were screened in the Connectivity Map database to identify cluster-specific therapeutic agents. RT-qPCR and immunohistochemistry (IHC) validated that both mRNA and protein levels of the four hub genes were elevated in endometriosis tissues, supporting the bioinformatics findings. Overall, this study links ERS-related hub genes to endometriosis subtyping, immune infiltration, and diagnostics, providing a basis for personalized treatments and a potential clinical tool.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-025-22400-9.

Afferent lymphatic vessels (LVs) are present in most vascularized tissues and exert important immune and drainage functions, yet human afferent LVs remain poorly studied. Performing single-cell RNA sequencing of lymphatic endothelial cells (LECs) from human skin and subcutaneous adipose tissue, we identified various LEC subsets, including two valve LEC populations located on the upstream and downstream sides of the valve leaflets. The cell adhesion molecule CD24 emerged as a specific marker of upper valve leaflet LECs in human skin and contributed to lymphatic valve development in murine mesentery. Three-dimensional imaging further revealed several unique features of the human dermal lymphatic network, including a high proportion of LYVE-1+ pre-collecting vessels containing intraluminal valves, virtually no collectors, and absence of lymphatic muscle cell coverage. Moreover, LECs in blind-ended capillaries and around valves in pre-collectors displayed mixed junctional and morphological phenotypes. These findings reveal key differences between human and murine dermal afferent lymphatics and provide a deeper understanding of human lymphatic-related (patho)physiological processes.

Investigation of predictive markers of disease complications in children with sickle cell disease: Preliminary findings from a multicenter prospective inception cohort study

Primary results from Equator, a Phase 3 double-blind, randomized placebo-controlled study evaluating itolizumab in combination with corticosteroids as initial treatment of acute graft-versus-host disease.

Enhanced VLA4-dependent endothelial cell adhesion underpins the higher efficacy of CAR-iNKT over CAR-T cells in limiting leptomeningeal acute leukemia

Mechanism of acquired resistance to histone deacetylase inhibitor, romidepsin, in myeloid leukemia associated with down syndrome

CD44v6 promotes anti-apoptotic phenotypes in AML via an SPP1-mediated autocrine activation of the MAPK-dusp pathway