This study aimed to develop a novel combined immune score (CIS)-based model assessing prognosis in triple-negative breast cancer (TNBC).The expression of eight immune markers  was assessed with immunohistochemistry on the tumor cells (TCs) and immune cells (ICs) of 227 TNBC cases, respectively, and subsequently associated with selected clinicopathological parameters and survival. Data retrieved from The Cancer Genome Atlas (TCGA) were further examined to validate our findings.p = 0.040, p = 0.020, and p = 0.020, respectively). In TCs, OX40 positive expression was associated with a shorter PFS (p = 0.025). Subsequently, the TNBC patients were classified into high and low combined immune score groups (CIS-H and CIS-L), based on the expression levels of a selection of biomarkers in TCs (TCIS-H or TCIS-L) and ICs (ICIS-H or ICIS-L). The TCIS-H group was significantly associated with a longer PFS (p < 0.001). Furthermore, the ICIS-H group was additionally associated with a longer PFS (p < 0.001) and overall survival , at significant levels. In the multivariate analysis, both TCIS-H and ICIS-H groups were identified as independent predictors of favorable PFS . ICIS-H was also shown to be an independent predictor of favorable OS (p = 0.003). The analysis of the mRNA expression data from TCGA also validated our findings regarding TNBC.All immune markers were often expressed in TCs and ICs, except for PD-1 which was not expressed in TCs. In ICs, the expression of all immune markers was positively correlated between one another, except between PD-L1 and OX40, also TIM3 and OX40. In ICs, PD-1, PD-L1, and OX40L positive expression was associated with a longer progression-free survival (PFS; Our novel TCIS and ICIS exhibited a significant prognostic value in TNBC. Additional research would be needed to strengthen our findings and identify the most efficient prognostic and predictive biomarkers for TNBC patients.The online version contains supplementary material available at 10.1186/s13058-023-01710-8. Immune checkpoint inhibitors (ICIs), which block checkpoint molecules such as the cytotoxic T lymphocyte antigen-4 (CTLA-4), programmed death 1 (PD-1) and programmed death ligand 1 (PD-L1), have become a standard therapeutic option across several types of malignant tumors .Many recent studies have shown that triple-negative breast cancer (TNBC) is highly immunogenic, exhibiting a better response to ICIs than other molecular subtypes of breast cancer , 4. LateT cell-mediated immunity includes multiple sequential steps. The ultimate amplitude and quality of the response, which is initiated through the antigen recognition of ligand–receptor interactions, is regulated by a balance between co-stimulatory and inhibitory signals. Examples of co-stimulatory molecules include the OX40 and OX40L, which are expressed by T cells and antigen-presenting cells. Co-inhibitory signaling occurs through interactions between receptors, such as the TIM-3 expressed on T cells, and their ligands expressed on antigen-presenting cells and other cells of the microenvironment . Along wRecent state-of-the-art technologies, including single-cell RNA sequencing and multiplex immunohistochemistry, have revealed the complexity of heterogenous tumor-infiltrating immune cells influencing the response to immunotherapy . TherefoAlong with the therapeutic aspects of ICIs, the expression of immune checkpoints has additionally been associated with the prognosis of various cancer types –16. The The purpose of this study was to identify novel checkpoints and define aggressive immune-phenotype subgroups in TNBC. We selected eight immune checkpoint targets previously shown to exhibit clinical significance in various cancers –27 and eA total number of 306 TNBC patients who received surgical resection after being diagnosed with invasive ductal carcinoma at Seoul National University Hospital between 2003 and 2006 were included in this study. Histologic grading was based on the Nottingham grading system . PatientAll enrolled TNBC cases had previously been subjected to estrogen receptor (ER) and progesterone receptor (PR) immunohistochemical analysis, also to Human epidermal growth factor receptor 2 (HER2) immunohistochemical and/or in situ hybridization testing, and had been evaluated based on the 13th St. Galen International Breast Cancer Conference and ASCO/CAP guidelines , 32, 33.Immunohistochemical staining was performed on 227 TNBC cases, arranged in tissue microarrays consisting of 2 mm cores from the aforementioned cases . A 4-μm section from each block was subjected to immunohistochemistry, using the Benchmark automatic immunostaining device . The following eight primary antibodies were used: PD-1 (PDCD1) , PD-L1 (B7-H1) , PD-L2 (B7-DC) , IDO (IDO1) , TIM3 (HAVCR2) , OX40 (TNFRSF4) , OX40L (TNFSF4) , and B7-H2 (ICOSLG) . Interpretation of all eight biomarkers was based on both intensity and proportion of the positively stained TCs and ICs; the latter included lymphocytes, macrophages, dendritic cells, and granulocytes . For eacTo validate our findings, the TCGA breast cancer dataset was used to investigate the mRNA expression of the ICI-related markers exhibiting prognostic relevance in our immunohistochemical analysis. The expression of each biomarker was considered as positive or negative, using the 25th percentile as a cut-off. We also classified all breast cancer and TNBC cases into two groups, based on the gene expression levels of a combination of immune markers, similar to our immunohistochemical analysis. Kaplan–Meier curves and log-rank tests were used to determine whether there was a significant difference in survival. The TCGA data was obtained from cBioPortal and the survival analysis was processed using the R software, version 4.1.0 .p < 0.05. To construct each CIS, we only selected immune markers exhibiting a P-value of less than 0.1 (p < 0.1) in the PFS Kaplan–Meier analysis   vs. TCIS-L, 70.7% (mean 110.8 months); p = 0.012; HR for ICIS = 0.303; 95% CI 0.150–0.615; p = 0.001]. ICIS-H was also shown to be an independent factor predicting a lower risk of death compared with ICIS-L .As both TCIS and ICIS exhibited prognostic significance, we performed univariate and multivariable analysis along with other clinicopathological variables, using a Cox proportional hazard model, to determine whether TCIS and/or ICIS were independent prognostic predictors Table ; Fig. 5.p < 0.001) and 5-year OS (p = 0.0018)  and 5-year OS (p < 0.001)  Fig. a, b. In 01) Fig. c, d, bei01) Fig. : Fig. S4In this study, we propose two combined immune score systems, the TCIS and ICIS, as novel prognostic models in TNBC. Eight immune markers of already established or potential prognostic or predictive value, including PD-L1, were evaluated , 38, 39.Even though the expression of PD-L1 is a crucial predictive factor for the treatment with PD-1/L1 blockade, many recent studies emphasize the importance of deciphering the complex interaction among several types of immune cells and tumor cells to overcome the low rate of therapeutic response. In the present study, we enrolled eight immune checkpoint-related markers and suggested two CIS predicting prognosis in TNBC. Both TCIS and ICIS were identified as independent prognostic factors of PFS in our multivariate analysis. More specifically, the TCIS-H and ICIS-H groups were linked with a longer PFS, while the ICIS-H group was additionally associated with a longer OS. These results reflect that the CIS models may depict more comprehensively the complex immunologic characteristics of TNBC and predict a patient’s prognosis more robustly than the expression of a single checkpoint. Besides PD-1/PD-L1, several other immune checkpoints have recently been under investigation for their potential therapeutic value in TNBC, and any combination of regimens, possibly together with the PD-1/PD-L1 blockade approach, might be more efficacious than a single ICI . TherefoIn this study, we defined the positive group when the staining was present in greater than or equal to 1% of both TCs and ICs, based on already published PD-L1 scoring formulas 36, 50, 51). The combined positive score (CPS) using the 22C3 clone is currently the only approved PD-L1 scoring algorithm in TNBC 0, 51. Th, whereasBy analyzing eight immune markers in TNBC with immunohistochemistry, we highlighted the significance of testing multiple biomarkers and providing combined immune scores. Both TCIS and ICIS exhibited a significant prognostic value in TNBC patients in our study. However, further studies would be needed to identify and implement the most efficient prognostic and predictive biomarkers of immunotherapy in the management of TNBC patients.Additional file 1. Table S1. Expression of the tested immune-related markers in both tumor and immune cells, and its clinicopathological significance.Additional file 2. Table S2. Prognostic role of the expression of each immune marker in both tumor and immune cells.Additional file 3. Fig. S1. Expression of each immune marker in tumor cells and its prognostic significance. Kaplan Meier graph for progression-free survival (PFS) and overall survival (OS). (A and B) in PD-L1; (C and D) in PD-L2; (E and F) in IDO; (G and H) in TIM3; (I and J) in OX40; (K and L) in OX40L and (M and N) in B7-H2.Additional file 4. Fig. S2. Expression of each immune marker in immune cells and its prognostic significance. Kaplan Meier graph for progression-free survival (PFS) and overall survival (OS). (A and B) in PD-1; (C and D) in PD-L1; (E and F) in PD-L2; (G and H) in IDO; (I and J) in TIM3; (K and L) in OX40; (M and N) in OX40L and (O and P) in B7-H2.Additional file 5. Fig. S3. Grouped by combined immune scores (CIS). In tumor cells combined immune score (TCIS), 169 cases were classified as tumor cells combined immune score-high (TCIS-H) and 58 cases as tumor cells combined immune score-low (TCIS-L). In immune cells combined immune score (ICIS), 167 cases were classified as immune cells combined immune score-high (ICIS-H) and 60 cases as immune cells combined immune score-low (ICIS-L).Additional file 6. Fig. S4. The progression-free survival (PFS) and overall survival (OS) of tumor cells combined immune score (TCIS) groups, according to the combination of mRNA expression levels of PD-L1, B7-H2, and OX40 in hormone receptor-positive (HRP) and HER2-positive (HER2) breast cancer from TCGA database. (A) PFS in HRP breast cancer, (B) OS in HRP breast cancer, (C) PFS in HER2-positive breast cancer, and (D) OS in HER2-positive breast cancer. Abbreviations: TCGA, The Cancer Genome Atlas; HRP, hormone receptor positive breast cancer; HER2, HER2 positive breast cancer.


CAR T cell therapy has been successfully used in the treatment of hematological malignancies, and the strategy that deletion of inhibitory receptor on the CAR T cell surface, such as PD-1, greatly enhance the antitumor effects. Here, we describe a one-step electroporation for the co-transfection of Cas9:sgRNA and CAR plasmids on primary T cells to demonstrate the effect of SHP-1 deletion in CAR T cells. By using PiggyBac Transposase system, we can achieve more than 90% of T cells express CAR gene and nearly 60% SHP-1 knockout efficiency in T cells. We show that knockout of SHP-1 in CD133 CAR T cells resulted in significantly improve the cytolysis effect on CD133 positive glioma cell lines. We further demonstrate that the enhanced antitumor efficacy of SHP-1 deletion is due to the increased release of TNF-α, IL-2 and IFN-γ in vitro. Finally, we evaluated the biosafety of Cas9 genome editing and did not find any insertions of Cas9 and obvious editing in off-target sites in CAR T cells. These data provide an approach for achieving both intracellular inhibitory molecule, SHP-1 deletion and CD133 CAR gene over-expression in human T cells. And SHP-1 could be a new potential target for adoptive CAR T cells immunotherapy.The online version contains supplementary material available at 10.1186/s40164-023-00450-x. PTPN6 gene, is a widely expressed inhibitory protein tyrosine phosphatase and is the most studied phosphatase in T cells [SHP-1, encoded by the  T cells . Specifi T cells , 4, whic T cells , 6. Here T cells . Next, twww.crispr.mit.edu) based on the sgRNA sequence targeting SHP-1  transplantation. The first day of CAR T cell injection was defined as Day1, and CAR T cells were injected at Day1, Day8, Day11 and Day18 respectively (Fig. In conclusion, we describe a one-step electroporation for CRISPR/Cas9 editing in CAR T cells and further demonstrate the role of the intracellular molecule SHP-1 in CAR T cell therapy against glioma.Below is the link to the electronic supplementary material.Supplementary Material 1


Breast cancer (BC) is the most common malignant disease worldwide. Although the survival rate is improved in recent years, the prognosis is still bleak once recurrence and metastasis occur. It is vital to investigate more efficient biomarkers for predicting the metastasis and relapse of BC. DYNLT1 has been reported that participating in the progression of multiple cancers. However, there is still a lack of study about the correlation between DYNLT1 and BC.In this study, we evaluated and validated the expression pattern and prognostic implication of DYNLT1 in BC with multiple public cohorts and BC tumor microarrays (TMAs) of paraffin-embedded tissues collected from the Affiliated Hospital of Jining Medical University. The response biomarkers for immune therapy, such as tumor mutational burden (TMB), between different DYNLT1 expression level BC samples were investigated using data from the TCGA-BRCA cohort utilizing public online tools. In addition, colony formation and transwell assay were conducted to verify the effects of DYNLT1 in BC cell line proliferation and invasion.The results demonstrated that DYNLT1 overexpressed in BC and predicted poor relapse-free survival in our own BC TMA cohort. In addition, DYNLT1 induced BC development by promoting MDA-MB-231 cell proliferation migration, and metastasis.Altogether, our findings proposed that DYNLT1 could be a diagnostic and prognostic indicator in BC. Breast carcinoma (BC) is the most common cancer in women and ranks the second leading cause of tumor-related death in women in the United States and in China , 2. AccoIn this study, we identified that dynein light chain tctex type 1 (DYNLT1), a component of the cytoplasmic dynein 1 complex, may predict the prognosis of BC. DYNLT1 is responsible for the intracellular retrograde motility of vesicles and organelles along microtubules, binding to transport cargo, and is involved in apical cargo transport. It is reported that DYNLT1 plays an important role in many biological functions and diseases, such as Huntington's disease , fertiliDYNLT1 between BC and normal breast tissues from multiple public cohorts and validated the results at the protein level by immunohistochemistry (IHC) staining for DYNLT1 in 68 BC samples along with paired 55 adjacent normal breast specimens collected from the Affiliated Hospital of Jining Medical University. In addition, we observed that DYNLT1 affected the migratory and colony-forming abilities of BC cells in vitro. Furthermore, in vivo experiment was conducted to verify that DYNLT1 knockdown suppressed tumor growth and abolished distant metastasis. Therefore, we proposed that DYNLT1 may have the potential to become a promising diagnostic indicator and prognostic predictor of BC patients.In this study, we evaluated the mRNA expression of https://www.ncbi.nlm.nih.gov/geo) for evaluating the mRNA expression of DYNLT1 between BC and normal breast tissues.A total of four Gene Expression Omnibus (GEO) cohorts  –17 were http://gepia2.cancer-pku.cn/#index) database (DYNLT1 in pan cancers using data from The Cancer Genome Atlas (TCGA). In addition, GEPIA 2.0 was also utilized to evaluate the prognostic implication of DYNLT1 in pan cancers.The GEPIA 2.0 (database  was utilhttp://bcgenex.centregauducheau.fr/BC-GEM/GEM-Accueil.php?js=1) database  database (DYNLT1 in cancer.The PrognoScan (database  was utilhttps://www.string-db.org/) database  and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses by R software with the “clusterProfiler” package . Terms whttp://camoip.net/) database  database  database was utilized to evaluate the correlation of DYNLT1 expression with 14 functional states of single BC cells . All participants provided written informed consent.2.MDA-MB-231 and HEK 293T cells were cultured in the DMEM medium (Gibco) with 10% fetal bovine serum (FBS) (Gibco) and 1% penicillin–streptomycin (Gibco) at 37°C with 5% COCells were lysed by denatured buffer and quantified by the Pierce BCA protein assay (Thermo Scientific). The whole cell lysate protein was separated by SDS-PAGE, transferred to NC membranes (Millipore), blocked by no-fat milk, and then detected by primary antibody DYNLT1  and HRP-conjugated secondary antibody (Sigma), followed by being exposed to enhanced chemiluminescence (Vazyme). β-actin  was used as a loading control.Lentivirus was collected to infect MDA-MB-231 cells. The primer sequences are shown as follows: DYNLT1-sh1-F: CCGGGAGGCTATAGAAAGCGCAATTCTCGAGAATTGCGCTTTCTATAGCCTCTTTTTG, DYNLT1-sh1-R: AATTCAAAAAGAGGCTATAGAAAGCGCAATTCTCGAGAATTGCGCTTTCTATAGCCTC; DYNLT1-sh2-F: CCGGCCACAAATGTAGTAGAACAAACTCGAGTTTGTTCTACTACATTTGTGGTTTTTTG, DYNLT1-sh2-R: AATTCAAAAAACCACAAATGTAGTAGAACAAACTCGAGTTTGTTCTACTACATTTGTGG.Annealing and ligation of the DYNLT1-knockdown shRNA were performed and inserted into the enzyme cut pLKO.1. The shDYNLT1 plasmids were then transinfected into HEK293T cells along with psPAX and pMD2.0G. Next, Immunohistochemistry (IHC) staining for DYNLT1  was operated by the standard IHC protocol as described earlier . The IHC2 for 3 weeks. Colonies were fixed with 10% formalin and then stained with 0.1% crystal violet.MDA-MB-231 cells were seeded at a density of 200 cells per well in six-well plates. Single cells were cultured in DMEM with 10% FBS at 37°C with 5% COLentivirus-infected stable cells were seeded into 96-well plates and cultured in 10% FBS DMEM . Then, the cells were collected at different points in time, and the cell number in each well was counted by the CCK-8 reagent. The absorbance at 450 nm was employed to determine the number of viable cells.4 cells per well were seeded into the upper chamber for the migration assays, while 5 × 104 cells per well were seeded into the upper chamber after matrigel was coagulated at 37°C for the invasion assays with serum-free medium, and the bottom of the chamber contained the DMEM medium with 10% FBS. Cells were fixed by 10% formalin and stained by 0.1% crystal violet after migration for 24 h. Migrated BC cells' pictures were captured by an inverted light microscope at × 100 magnification, and three random fields were counted.The migration assays and invasion assays were performed using a transwell chamber (Corning). A total of 2 × 106 cells per mouse). Tumor volume was measured every 7 days and calculated according to the formula as follows: volume = 0.5 × tumor length × width × width. Mice were generally sacrificed when tumors became necrotic or their volume reached 1,500 mm3, recorded as death for the survival curve. The lung and the liver of dead mice were excised and fixed in formalin. Paraffin-embedded lungs were systematically sectioned and stained with hematoxylin and eosin (H&E) staining, and images were captured by Leica Aperio CS2.Female SCID mice (6 weeks old) were purchased from the Shanghai Model organism. SCID mice were injected in the right lower breast fat pad with MDA-MB-231 cells knockdown DYNLT1 or vector shRNA control  risk factor for the prognosis of BC, MESO, and LIHC  at the single cell level of BC by the CancerSEA database  mutational ratio, a high proportion of TMB, high neoantigen loads, high TILs regional fraction, and low TGF-beta response predict well overcome in ICB therapy for patients with cancer , 32. In database . We foundatabase , TILs redatabase , and lowdatabase  compareddatabase  and TMB database  was showdatabase .in vitro experiments were conducted. First, MDA-MB-231-shDYNLT1 and MDA-MB-231-sh scramble (NC) stable cell lines were constructed successfully by lentiviral transduction  female mice. Knockdown of DYNLT1 led to smaller tumor volume . The patients/participants provided their written informed consent to participate in this study. The animal study was reviewed and approved by the Ethics Committee of Affiliated Hospital of Jining Medical University, JNNL-202-DW-02.SM, GJ, CJ, and BX performed the experiments and wrote the manuscript. LihZ, RZ, HD, XY, LinZ, XP, and HZ collected the BC sample and performed the experiments. LijZ, LW, and TZ supervised the project and provided funds for the whole project. All authors approved the final manuscript.


Paediatric-type diffuse High-Grade Gliomas (PDHGG) are highly heterogeneous tumours which include distinct cell sub-populations co-existing within the same tumour mass. We have previously shown that primary patient-derived and optical barcoded single-cell-derived clones function as interconnected networks. Here, we investigated the role of exosomes as a route for inter-clonal communication mediating PDHGG migration and invasion.A comprehensive characterisation of seven optical barcoded single-cell-derived clones obtained from two patient-derived cell lines was performed. These analyses highlighted extensive intra-tumour heterogeneity in terms of genetic and transcriptional profiles between clones as well as marked phenotypic differences including distinctive motility patterns. Live single-cell tracking analysis of 3D migration and invasion assays showed that the single-cell-derived clones display a higher speed and longer travelled distance when in co-culture compared to mono-culture conditions. To determine the role of exosomes in PDHGG inter-clonal cross-talks, we isolated exosomes released by different clones and characterised them in terms of marker expression, size and concentration. We demonstrated that exosomes are actively internalized by the cells and that the inhibition of their biogenesis, using the phospholipase inhibitor GW4689, significantly reduced the cell motility in mono-culture and more prominently when the cells from the clones were in co-culture. Analysis of the exosomal miRNAs, performed with a miRNome PCR panel, identified clone-specific miRNAs and a set of miRNA target genes involved in the regulation of cell motility/invasion/migration. These genes were found differentially expressed in co-culture versus mono-culture conditions and their expression levels were significantly modulated upon inhibition of exosome biogenesis.In conclusion, our study highlights for the first time a key role for exosomes in the inter-clonal communication in PDHGG and suggests that interfering with the exosome biogenesis pathway may be a valuable strategy to inhibit cell motility and dissemination for these specific diseases.The online version contains supplementary material available at 10.1186/s13578-023-01166-5. Paediatric-type diffuse high-grade gliomas (PDHGG) are a family of highly aggressive and heterogeneous tumours of the central nervous system that arise in children and young adults and for which there is still no effective treatment , 2. BiolA significant degree of genetic and phenotypic, inter- and intra-tumour heterogeneity, at both spatial and temporal levels, has been described for PDHGG, representing one of the most challenging aspects in the effort of developing effective therapeutic strategies for these diseases –10. The Intercellular communication is essential for the coordination of several functions in multicellular systems particularly important for cancer progression. This complex process mainly takes place through the active transfer of molecules from one cell to another. Cells communicate with each other via direct cell–cell contact, such as juxtracrine signalling through the gap junctions , 13 and In this context, exosomes, extracellular vesicles of 40–160 nm in diameter secreted by most cells, have been shown to play crucial roles in mediating intercellular communication , 20. TheThe role of exosomes in the aggressive behaviour of PDHGG has not been explored yet. Herein, we fully characterised optical barcoded single-cell-derived clones from two PDHGG patient-derived cell lines and investigated the role of the exosomes, with a focus on the exosome-contained miRNA (exo-miRNA), in mediating the indirect cell–cell communication and in sustaining the glioma network.To investigate the cellular and molecular mechanisms involved in inter-clonal communication in PDHGG, we generated and characterized the phenotypic features of single-cell-derived clones from patient-derived primary cell lines. To this purpose, we have generated the optical barcoded (OB) clones from multifluorescent PDHGG-WT (OPBG-GBM002) and a DMG-H3K27-altered (OPBG-DIPG002) patient-derived cell lines, using the multifluorescent marking technology as previously described . The OB Individual clones displayed different cell morphologies Fig. A, as exeFurthermore, the clones exhibited overall different growth rates, also when compared to the bulk cell line from which they were derived Fig. F, G. Thee.g. H3F3A, TP53, ATRX), the clones showed several shared mutations, not identified in the bulk and in the patient tumour tissue , and clone-specific mutations  , we performed gene set enrichment analysis (GSEA) for the 1C5 and 2B4 clones derived from OPBG-DIPG002 and for 5E2 and 1D3, the two most phenotypically different clones derived from OPBG-GBM002. Interestingly, for the OPBG-DIPG002, 2B4 showed enrichment in genes associated with positive regulation of cell migration and extracellular matrix organization when compared to 1C5  analysis was performed at high depth using a custom-designed targeted panel. In addition to the common mutations . Interestingly, 2B4 also seemed to advantage of the co-culture condition with the 1C5 clone, as it significantly showed higher speed and accumulated distance when compared to its mono-culture  from conditioned medium (CM) of the OPBG-DIPG002 and OPBG-GBM002 bulk cell lines and their corresponding derived clones. The total EV protein content was quantified, showing a relatively variable protein amount between the bulk and the clones Fig. A. WesterAltogether, these results confirmed that we successfully isolated, from our cell cultures, EVs corresponding to exosomes.donor” clone, were added to the culture of the “recipient” clone, and vice versa. After 24 h, internalized fluorescent green signals were visualized in the cytoplasm of the cells , we used a 384 miRNome PCR panel. Following the analysis, we did not identify differentially expressed exo-miRNAs between the paired clones, 1C5 and 2B4 for OPBG-DIPG002 and 5E2 and 1D3 for OPBG-GBM002. However, we found that the expression of some of the exo-miRNAs was clone specific. The miR-200c-3p was found exclusively expressed in the exosomes isolated from the clone 1C5, while miR-887-3p, miR-885-5p, and miR-582-5p were found exclusively expressed in the exosomes secreted by the clone 2B4, both OPBG-DIPG002 derived clones . SMase converts sphingomyelin into ceramide, a rigid lipid element essential for the generation and release of exosomes. GW4869 has been used in different studies that proved its activity in blocking the exosome-mediated transfer of key regulators of oncogenic processes ).RNA were extracted from exosomes using Plasma/Serum Circulating and Exosomal RNA Purification Mini Kit (Slurry Format) (Norgen) following the manufacturer’s instruction. The quantification and quality evaluation was performed by using the Agilent 2100 Bioanalyzer RNA Pico assay (Agilent technologies) following the manufacturer’s instructions. Total RNA from exosomes was mixed with artificial RNAs (RNA spike-ins) used as controls, and the mixture was reverse transcribed at 42 °C for 60 min using the miRCURY LNA™ Universal RT cDNA Synthesis Kit (Qiagen) following the manufacturer’s instruction. The expression of each miRNA (including spike-ins) was evaluated by miRNome PCR Panel  by quantitative PCR (q-PCR) (QuantStudiohttps://mirtarbase.cuhk.edu.cn/~miRTarBase/miRTarBase_2022/php/index.php) . Networks have been drafted using Cytoscape . Obtained tryptic peptides (2 µg) were separated by liquid chromatography (HPLC) using an Ultimate 3000 RSLCnano System device (Thermofisher Scientific). A 90 min gradient, ramping from 5 to 25% aqueous solution of ACN with 0.1% formic acid, was used to elute peptides onto a PepMap RSLC C18 column  working at a flow rate of 250 nL/min. Peptides were eluted in the EasySpray source of an Orbitrap Fusion mass spectrometer (Thermo Scientific) and analysed by a data-dependent acquisition (DDA) method. The Orbitrap analyzer was used for full-MS scans and MS2 scans using 120000 and 15000 resolutions respectively (250–1500 m/z scan range). Fragments were obtained by high energy collision-induced dissociation (HCD) on precursor ions setting the dynamic exclusion at 30 s after a single scan. Two technical replicates from 3 biological replicates were acquired for each sample.Protein identification and Label-Free Quantitation (LFQ) analysis were performed with the Proteome Discoverer 2.5 software (Thermo Fisher Scientific), using the SwissProt database restricted to Homo Sapiens (2022_1). Two miss cleavages were allowed, cysteine carbamidomethylation and oxidation of methionine were set as fixed and variable modifications respectively. Mass error tolerance was set to 10 ppm for precursor ions and 0.02 Da for fragment ions. The INFERYS rescoring was applied before the Percolator algorithm for false discovery rate (FDR) calculation with a cut-off of 0.01. All proteins identified with less than two peptides or no unique peptides were filtered out. Quantification was performed by determining the protein intensity ratio between mono-culture vs co-culture conditions and using the summed abundance of both unique and razor peptides, normalizing on the total peptide amount. The protein ratio was calculated with the “pairwise ratio-based” algorithm using a t-test. Up- or down-regulated proteins were selected by setting the log2 fold change value and the p-value thresholds to 0.66 and 0.05 respectively  and statistical analysis was performed with GraphPad Prism 8.4 software. For the phenotypic characterization, statistical significance was evaluated using One-way ANOVA multiple comparisons, while for 3D live imaging experiments, statistical significance was evaluated with Two-way ANOVA Multiple Comparison Tests and Paired t-tests. For exosome uptake and exosome education experiments, statistical significance was evaluated with the Two-way ANOVA Multiple Comparison Test. For q-PCR, statistical analyses were performed on 2Additional file 1: Figure S1. Overall Survival analysis of HGG patients using TCGA gene expression data. (A-B) The patient cohort was divided into two groups based on the median expression of OPBG-DIPG002 2B4 (A) and OPBG-GBM002 5E2 (B) specific gene signatures. The red curve represents patients with higher expression of this signature, while the blue curve represents patients with lower expression. Figure S2. Motility of single-cell-derived clones in mono and co-culture condition. Individual clones derived from OPBG-DIPG002 (A-D) and OPBG-GBM002 (G) were cultured either alone or co-cultured at an equal cell/cell ratio. Representative brightfield images are shown for the invasion (A) and migration (D-G) assays after 96 hours. The extent of invasion and migration is marked with coloured segmentation: co-culture (light blue); mono-cultures of 1C5 and 5E2 (fuchsia); mono-cultures of 2B4 and 1D3 (yellow). The total area covered by invading and migrating cells was quantified and normalised by the NS area determined at the time t0 (B-E-H). Scale bar = 500μm. Representative fluorescent images of OPBG-DIPG002 single-cell-derived clones 1C5 (Venus) and 2B4 (m-Orange2) 3D invasion (A) and migration (D) and of OPBG-GBM002 single-cell-derived clones 5E2 (Venus) and 1D3 (m-Orange2) migration (G) are shown as mono-culture and as co-culture (overlay of Venus and m-Orange2). Fluorescent images were acquired on a Leica TCS AOBS-SP8X confocal microscope. The percentage of each clone in the invasion (C) and migration (F-I) at 96 hours was analysed with Harmony software. Data are mean ± SD, n = 3. (****) p<0.0001; (***) p<0.001; (**) p<0.01; (*) p<0.05. Figure S3. The exosomal education of single-cell-derived clones does not affect their migratory/invasive phenotype. Exosomal education of the clones was performed. Exosomes isolated from donor clones were used to educate the recipient clone (10 μg/mL of media) every day, over 7 days. The effect on migration (A left panel and B) and invasion (A right panel) was quantified as the total area in the field of view covered by migrating and invading cells was normalized to the size of the NS at t0. Automated image acquisition was performed on a Celigo imaging cytometer. Scale bar = 500μm. Data were derived, and representative images were obtained from n=3 independent experiments. Data are mean ± SD, n = 3. Figure S4. Effect of the inhibition of exosome biogenesis on cell viability. (A) Effect of exosome biogenesis inhibition by GW4869 on the viability of the cells for the clones derived from OPBG-DIPG002 (left panel) and OPBG-GBM002 (right panel) cell lines. Cells were treated with GW4869 or vehicle control. Cell viability was measured with CellTiter Glo assay and dose-response curves are shown. Results are mean ± SD; n = 3. Figure S5. Analysis of exosome biogenesis inhibition upon GW4869 treatment on single-cell-derived clones. (A and C) Plots showing the size distribution of the nanoparticles resulting from the NanoSight particle-tracking analysis performed with the exosomes obtained from OPBG-DIPG002 and OPBG-GBM002 derived-clones CM in the presence or absence of 10μM and 20μM of GW4869 respectively or DMSO as vehicle control . (B and D) Determination of the exosomal protein concentration. The quantification of the total exosomal protein obtained from OPBG-DIPG002 and OPBG-GBM002 derived-clones in untreated and treated with GW4869 conditions, is shown (n=3). (E) Determination of the exosomal RNA concentration. The quantification of total exosomal RNA obtained from OPBG-DIPG002 2B4 clone, in the presence or absence of 10μM of GW4869. Figure S6. Effect of the inhibition of exosome biogenesis on migration of single-cell-derived clones. (A and B) The mean accumulated distance, mean displacement, and speed of OPBG-DIPG002 (A) and OPBG-GBM002 (B) clone migrating cells in mono- and co-culture are shown. Cells were treated with 10μM and 20μM GW4869 or vehicle control, for 96 hours. Analysis was performed with Harmony software. Data are mean ± SD, n = 3. (****) p<0.0001; (***) p<0.001; (**) p<0.01; (*) p<0.05. Figure S7. Effect of the combination GW4869+exosome treatment on migration of single-cell-derived clones. OPBG-DIPG002 1C5 and 2B4 single-cell-derived clones were treated with GW4869 (10μM) or vehicle control (CTRL), in the presence or absence of exosomes (10 μg/mL of media) isolated from donor clones. The extent of migration is marked with coloured segmentation (white). The effect on migration (A) was quantified as the total area in the field of view covered by migrating cells, normalized to the size of the NS at T0. Automated image acquisition was performed on a Celigo imaging cytometer. Scale bar = 200μm. Data were derived, and representative images were obtained from n=2 independent experiments. Data are mean ± SD. (****) p<0.0001; (***) p<0.001; (**) p<0.01; (*) p<0.05. Figure S8. Proteomic analysis of single-cell-derived clones. Volcano plot of the mono-culture/co-culture ratio values measured in the label-free quantitative analysis performed on single-cell-derived clones of OPBG-DIPG002 (A and B) and OPBG-GBM002 (C and D). The red and green boxes include significant up- and down-regulated proteins respectively, based on the thresholds set for abundance ratio and p-value. Identified proteins related to the miRNA-predicted target genes are highlighted with sky-blue dots and tagged with the UniProt entry name.Additional file 2: Gene signatures associated with OPBG-DIPG002 2B4 derived-clone and OPBGGBM0025E2 derived-clone. Additional file 3:  Summary of the proteomic Label-Free Quantitation (LFQ) analysis performed for theclones 2B4, 5E2, 1C5, and 1D3 based on the comparison of mono versus coculture conditions.


Stimulator of interferon genes (STING) is an ER-localized transmembrane protein and the receptor for 2',3'-cyclic guanosine monophosphate–adenosine monophosphate (cGAMP), which is a second messenger produced by cGAMP synthase (cGAS), a cytosolic double-stranded DNA sensor. The cGAS-STING pathway plays a critical role in the innate immune response to infection of a variety of DNA pathogens through the induction of the type I interferons. Pharmacological activation of STING is a promising therapeutic strategy for cancer, thus the development of potent and selective STING agonists has been pursued. Here we report that mouse STING can be activated by phenylarsine oxide (PAO), a membrane permeable trivalent arsenic compound that preferentially reacts with thiol group of cysteine residue (Cys). The activation of STING with PAO does not require cGAS or cGAMP. Mass spectrometric analysis of the peptides generated by trypsin and chymotrypsin digestion of STING identifies several PAO adducts, suggesting that PAO covalently binds to STING. Screening of STING variants with single Cys to serine residues (Ser) reveals that Cys88 and Cys291 are critical to the response to PAO. STING activation with PAO, as with cGAMP, requires the ER-to-Golgi traffic and palmitoylation of STING. Our results identify a non-nucleotide STING agonist that does not target the cGAMP-binding pocket, and demonstrate that Cys of STING can be a novel target for the development of STING agonist. STING is an ER-localized transmembrane protein essential for control of infections of DNA viruses and tumor immune surveillance . After iPhosphatidylinositol-4-phosphate (PI4P) is the most abundant phosphoinositide in cells . InitialIn the present study, we show that PAO binds covalently to STING at the ER and induces the translocation of STING to the Golgi, where STING activates TBK1 and IRF3 in a STING palmitoylation-dependent manner. Our work identifies a novel STING agonist that does not target the cGAMP-binding pocket, and demonstrates that Cys of STING can be a novel target for the development of STING agonist.–/–Sting (SKO) MEFs  induced the phosphorylation of STING, TBK1, and IRF3 in mouse embryonic fibroblasts (MEFs), but not KO) MEFs a. To visKO) MEFs b. Most it manner c. PAO ext manner d. Of nott manner . Given tCxcl10, Ifnb, and Il6 in CKO MEFs  MEFs a, PAO inCKO MEFs b. These CKO MEFs c, PAO, nCKO MEFs d. These N-acetylcysteine (NAC), suppressed PAO-induced phosphorylation of STING, TBK1, and IRF3 (87CLGCPLR94) including Cys88 and Cys91 after trypsin digestion, which was successfully identified with mass spectrometry (87CLGCPIHCMAMILLSSYFYFLQNTADIYLSWMFGLLVLYK127), which appeared not suitable for mass spectrometric detection.As PAO makes a covalent bond with Cys of some proteins, PAO may also bind to Cys of STING to exert its effect. Indeed, the treatment of cells with a reducing agent and IRF3 , suggestWe mutated His94 to Arg94 in mouse STING and used the resultant variant (STING H94R) for the following analyses. Mouse STING H94R was confirmed to have the same reactivity as WT STING to PAO, regarding the generation of the phosphorylation of STING, TBK1, and IRF3 a and thein situ. The resulting peptides were subjected to liquid chromatography-mass spectrometry (LC-MS) analysis, and two PAO-peptide adducts were identified with a MASCOT score higher than 50 using MASCOT cross-linking analysis  also indicated to be modified covalently with PAO. We thus examined if which Cys modification(s) with PAO was essential for the STING activation. To address this, we generated STING variants with single Cys to Ser substitution. We found that STING C88S and STING C291S, but not the other variants  did not translocate to the Golgi . These rCxcl10 d, furtheCxcl10 .i.e., MEFs stably expressing STING vs. HEK293T cells overexpressing STING.Of note, we found that STING C147S and STING C205S appeared to lose the typical ER localization without stimulation and partially accumulated around the perinuclear area, with some co-localization with a Golgi protein GM130 . Thus, CCxcl10  a, b. TheCxcl10 . These rSurf4 is a protein that circulates between the ER and the Golgi d. The weSeveral studies have demonstrated that STING activity is tightly associated with cellular redox status. Treatment of cells with hydrogen peroxide generated intermolecular disulfide bonds between STING, leading to the formation of inactive STING oligomers , an ER-resident protein, was suggested to act as a tether of STING to the ER , rabbit anti-phospho-TBK1 , rabbit anti-IRF3 , rabbit anti-phospho-IRF3  ; rabbit anti-TBK1  (Abcam); mouse anti-α-tubulin  (Sigma-Aldrich); Goat Anti-Rabbit IgG (H + L) Mouse/Human ads-HRP  and Goat Anti-Mouse IgG (H + L) Human ads-HRP  (Southern Biotech); sheep anti-TGN38  (Bio-Rad); mouse anti-GM130  (BD Biosciences).The following reagents were purchased from the manufacturers as noted: DMXAA ; nocodazole ; 2-bromopalmitate ; 2',3'-cGAMP (InvivoGen); D-ceramide-C6 , L-ceramide-C6 ; H-151 was provided by Carna Biosciences, Inc.Sting–/– mice at E13.5 and immortalized with SV40 Large T antigen. MEFs were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and penicillin/streptomycin/glutamine (PSG) in a 5% CO2 incubator. MEFs that stably express tagged proteins were established using retrovirus. Plat-E cells were transfected with pMXs vectors, and the medium that contains the retrovirus was collected. MEFs were incubated with the medium and then selected with puromycin (2 μg mL–1).MEFs were obtained from embryos of WT or N-terminal EGFP- or FLAG-tagged mouse STING (NM_028261) was introduced into pMXs-IPuro. STING variants were generated by site-directed mutagenesis.Cells were fixed with 4% paraformaldehyde (PFA) in PBS at room temperature for 15 min, permeabilized with 0.1% Triton X-100 in PBS at room temperature for 5 min. After blocking with 3% BSA in PBS, cells were incubated with primary antibodies. After washing with PBS three times, cells were then incubated with the secondary antibody at room temperature for 60 min, washed, and mounted with ProLong™ Glass Antifade Mountant .Ifnb; sense primer) and 5'-GGCAGTGTAACTCTTCTGCAT-3' ; 5'-AGTGCTGCCGTCATTTTCTGCCTC-3'  and 5'-GCAGGATAGGCTCGCAGGGATGATT-3' ; 5'-TAGTCCTTCCTACCCCAATTTC-3'  and 5'-TTGGTCCTTAGCCACTCCTTC-3' ; 5'-AGGTCGGTGTGAACGGATTTG-3'  and 5'-TGTAGACCATGTAGTTGAGGTCA-3' . Target gene expression was normalized on the basis of Gapdh content.Total RNA was extracted from cells using Isogen II (Nippongene) or Superprep II (TOYOBO), and reverse-transcribed using ReverTraAce qPCR RT Master Mix with gDNA Remover (TOYOBO). Quantitative real-time PCR (qRT-PCR) was performed using KOD SYBR qPCR (TOYOBO) and LightCycler 96 (Roche). The sequences of the primers were as follows; 5'-CAGCTCCAAGAAAGGACGAAC-3' . The cell lysates were centrifuged at 15,000 rpm for 15 min at 4°C, and the resultant supernatants were pre-cleared with Ig-Accept Protein G  at 4°C for 1 h. The lysates were then incubated for 2 h at 4°C with anti-FLAG M2 affinity Gel. The beads were washed four times with immunoprecipitation wash buffer (50 mM HEPES-NaOH (pH 7.2), 150 mM NaCl, 0.1% Triton X-100) and eluted with 2 × Laemmli sample buffer. The immunoprecipitated proteins were separated with SDS-PAGE and transferred to PVDF membrane, then analyzed by western blot.Cells were washed with ice-cold PBS and scraped in immunoprecipitation buffer composed of 50 mM HEPES-NaOH (pH 7.2), 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, protease inhibitor cocktail   and phosphatase inhibitors . These membranes were incubated with primary antibodies, followed by secondary antibodies conjugated to peroxidase. The proteins were visualized by enhanced chemiluminescence using Fusion SOLO.7S.EDGE (Vilber-Lourmat).–1. The separation was performed with 0.1% formic acid as solvent A and 100% acetonitrile containing 0.1% formic acid as solvent B, with a linear gradient of 40 min with solvent B rising from 0% to 80%. The separated peptides were analyzed using an online coupled Q Exactive Mass Spectrometer (Thermo Fisher Scientific) using the data-dependent Top 10 method. The acquired data were processed using MASCOT 2.8 (Matrix Science) and Proteome Discoverer 2.4 (Thermo Fisher Scientific). The MASCOT search with crosslinking analysis was conducted as follows: Database, in-house database-containing STING sequence; type of search, MS/MS ion; enzyme, trypchymo (trypsin + chymotrypsin); fixed modification, none; variable modifications, oxidation (M), dehydro (C), phospho (ST), palmitoyl (C) and crosslinking with PAO or disulfide; mass values, monoisotopic; peptide mass tolerance, ±15 ppm; fragment mass tolerance, ±30 mmu; max missed cleavages, 4; instrument type, ESI-TRAP.Purified GFP nanobodies from bacteria were incubated with GST-Accept resin  at room temperature for 2 h. Cells were lysed with IP buffer (50 mM HEPES-NaOH (pH 7.2), 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, protease inhibitors, and phosphatase inhibitors). The lysates were centrifugated at 15,000 rpm for 10 min at 4°C, and the resultant supernatants were incubated overnight at 4°C with the resin. The beads were washed six times with immunoprecipitation wash buffer (50 mM HEPES-NaOH (pH 7.2), 150 mM NaCl, 1% Triton X-100), and boiled at 95°C for 5 min with elution buffer (50 mM HEPES-NaOH (pH 7.2), 150 mM NaCl, 5 mM EDTA, 1% Triton X-100). Eluted proteins were applied to SDS-PAGE under non-reducing conditions. The gel was stained with CBB and the band that corresponds to STING was excised. The gel pieces were digested with trypsin  and chymotrypsin  at 37°C for 12 h. The resulting peptides were separated using an Easy nLC 1000 (Thermo Fisher Scientific) equipped with a nano-ESI spray column  at a flow rate of 300 nl minFor quantification of imaging data of multiple cells, individual cells were segmented using ROI. Pearson’s correlation coefficient was then quantified by Coloc 2 in Fiji plugin with ROI data.post hoc test.Error bars displayed in the bar plots throughout this study represent s.e.m. unless otherwise indicated and were calculated from triplicate samples. In box-and-whisker plots, the box bounds the interquartile range (IQR) divided by the median, and whiskers extend to a maximum of 1.5 × IQR beyond the box. The corresponding data points are overlayed on the plots. Statistical significance was determined with one-way ANOVA followed by Tukey-Kramer The data sets generated and/or analyzed during the present study are available from the corresponding author on reasonable request.K.Matsumoto designed and performed the experiments, analyzed the data, interpreted the results, and wrote the paper; S.N. performed experiments with PAO; H.A. discussed the results; T.Toyama and Y.S. designed the experiments with NAC. T.S. and N.D. performed the proteomics analysis; K.Mukai and T.Taguchi designed the experiments, interpreted the results, and wrote the paper.The authors declare no competing financial interests.


In recent years, there has been an intensive development of portable ultralow-field magnetic resonance imaging (MRI) for low-cost, shielding-free, and point-of-care applications. However, its quality is poor and scan time is long. We propose a fast acquisition and deep learning reconstruction framework to accelerate brain MRI at 0.055 tesla. The acquisition consists of a single average three-dimensional (3D) encoding with 2D partial Fourier sampling, reducing the scan time of T1- and T2-weighted imaging protocols to 2.5 and 3.2 minutes, respectively. The 3D deep learning leverages the homogeneous brain anatomy available in high-field human brain data to enhance image quality, reduce artifacts and noise, and improve spatial resolution to synthetic 1.5-mm isotropic resolution. Our method successfully overcomes low-signal barrier, reconstructing fine anatomical structures that are reproducible within subjects and consistent across two protocols. It enables fast and quality whole-brain MRI at 0.055 tesla, with potential for widespread biomedical applications. Large-scale 3T MRI data enables fast whole-brain scanning at 0.055T via deep learning super-resolution and image reconstruction. Magnetic resonance imaging (MRI) is a highly valuable imaging modality in modern health care due to its noninvasive, nonionizing, fundamentally three-dimensional (3D), quantitative, and multiparametric nature  strengths (<0.1 T) for portable and point-of-care clinical applications  at ULF compared to that at high field undermines its clinical value and hinders its widespread clinical adoption  and high-resolution 3D target image data (1.5-mm isotropic resolution and high SNR). We used 1248 and 1182 pairs of such 3D training data for the T1W and T2W models, respectively. These were synthesized from publicly available human brain 3 T MRI data from the HCP Consortium . They were scanned using the 0.055 T MRI head scanner with scan time of 2.5 and 3.2 min for T1W and T2W data, respectively. The acquired data were reconstructed using both the proposed PF-SR method and the traditional non-DL method for direct comparison. The volunteers were also scanned using a standard clinical 3 T MRI scanner with 1.5-mm isotropic resolution for comparison.Typical reconstruction results of experimental ULF T1W and T2W data from one healthy volunteer are shown in Experimental inter-exam reproducibility test on T1W and T2W imaging was performed to evaluate the reconstruction consistency of PF-SR from the same volunteer with different head positions during two different exam sessions . Here, PIn this study, we achieved fast and high-quality 3D whole-brain MRI at 0.055 T through an integrated PF sampling acquisition and DL reconstruction framework. Fast 3D imaging was realized by single-NEX acquisition with 2D PF sampling of a fraction of 0.7 along each of the two PE directions. This scheme shortened the scan time of the 3-mm isotropic ULF T1W and T2W whole-brain imaging protocols to 2.5 and 3.2 min, respectively. The 3D DL PF-SR reconstruction model was trained on synthetic ULF data simulated from large-scale 3 T human brain MRI data. We demonstrated its effective reduction of PF-related blurring and ringing artifacts, noise, and enhancement of spatial resolution to synthesized or synthetic 1.5-mm isotropic resolution in both synthetic and experimental ULF results.MR signal at ULF, e.g., 0.055 T, is approximately three orders of magnitude weaker than that at 3 T  isotropic 3D convolution layers, (ii) multiscale feature extraction, and (iii) channel and spatial attention. All convolution layers and the synthetic ULF model training data are isotropic 3D to maximally facilitate the extraction of the 3D brain structural features from the large-scale 3 T human brain data. Small kernel size at the top-scale level of multiscale feature extraction enables the model to learn local features, e.g., blurring artifact, while the middle to the bottom-scale level allows the extraction of semiglobal feature, e.g., ringing artifact, by increasing the receptive field of convolution layer  as shown in fig. S4. The lowest and least fluctuating training loss is achieved when the model is trained on 100% training sample size. The increase in training sample size allows the model to extract more general 3D brain structural features. In addition, it reduces the bias from individual training data, resulting in more stable model training. On the basis of the performance on synthetic and experimental ULF data , we anticipate that expanding the training sample size by including other high-field brain data may help to improve the PF-SR reconstruction. Doing so would entail complex dataset-specific preprocessing steps and more model training time.We recognize that the model is, to a certain extent, specific to the contrast on which it is trained. This is expected because 3D brain image features depend on both brain anatomical structures and image contrasts. In this study, we train two PF-SR models separately and independently for T1W and T2W data. It is plausible to train single model for both T1W and T2W data although it may compromise the reconstruction performance. We also observe that the model is specific to the orientation of the 3D brain data although this limitation does not really compromise its usefulness in practice.B0 field strength in ULF compared to high-field    synthetic ULF data simulated from 3 T data and (ii) experimental ULF data. The proposed DL method, namely, PF-SR, was compared to a non-DL method, which consisted of 2D iterative POCS  were recruited. Written informed consent was obtained from all volunteers in this study with the approval of the institutional review board. All recruited volunteers were scanned by the 0.055 T MRI head scanner developed in our laboratory (


Mutations in SNCA, encoding for α-synuclein, are a known cause of familial PD, and the G51D mutation causes a particularly aggressive form of the condition. CRISPR/Cas9 technology was used to introduce the G51D mutation into the endogenous rat SNCA gene. SNCAG51D/+ and SNCAG51D/G51D rats were born in Mendelian ratios and did not exhibit any severe behavourial defects. L-3,4-dihydroxy-6-18F-fluorophenylalanine (18F-DOPA) positron emission tomography (PET) imaging was used to investigate this novel rat model. Wild-type (WT), SNCAG51D/+ and SNCAG51D/G51D rats were characterized over the course of ageing  using 18F-DOPA PET imaging and kinetic modelling. We measured the influx rate constant (iK) and effective distribution volume ratio (EDVR) of 18F-DOPA in the striatum relative to the cerebellum in WT, SNCAG51D/+ and SNCAG51D/G51D rats. A significant reduction in EDVR was observed in SNCAG51D/G51D rats at 16 months of age indicative of increased dopamine turnover. Furthermore, we observed a significant asymmetry in EDVR between the left and right striatum in aged SNCAG51D/G51D rats. The increased and asymmetric dopamine turnover observed in the striatum of aged SNCAG51D/G51D rats reflects one aspect of prodromal PD, and suggests the presence of compensatory mechanisms. SNCAG51D rats represent a novel genetic model of PD, and kinetic modelling of 18F-DOPA PET data has identified a highly relevant early disease phenotype.Parkinson’s disease (PD) is a neurodegenerative condition with several major hallmarks, including loss of  The conreleased . The patreleased . Cases o atrophy .L-3,4-dihydroxy-6-18F-fluorophenylalanine (18F-DOPA)  to produce 18F-DA, which is incorporated into synaptic vesicles and then released into the synaptic cleft following neuronal stimulation (iK) of 18F-DOPA is significantly decreased in the caudate and putamen (EDVR) of 18F-DOPA can be calculated  lesion rat model of PD has been demonstrated  positive neurons in the striatum when compared to controls, and this occurs when the condition has significantly progressed . However8F-DOPA) . 18F-DOPmulation . In pati putamen . Dopaminlculated . A decrese in Ki . An incres in Ki . This haes in Ki . The ratnstrated . Furtherel of PD . The MPTroxylase . PET imasing age .SNCA construct with two PD mutations (A30P and A53T) under control of the rat TH promoter, and the major deficits reported in this model were olfactory   serotonergic deficit leading to anxiety-like phenotypes prior to the onset of motor deficits  . Transgecontrols . Furthercontrols . A subsedeficits .SNCA exon 3 to produce a novel SNCAG51D rat model of PD. Striatal dopamine metabolism and turnover were investigated in SNCAG51D/+ and SNCAG51D/G51D rats over the course of ageing using 18F-DOPA PET imaging and kinetic modelling.To date the majority of genetic rat models of PD have been generated by the random insertion of a transgene or the knock-out of a PD-related gene . Replica; RRID:Addgene_41824) and humanized Cas9 nuclease vector . A 80-nt ssDNA mutant donor oligonucleotide with the desired 2-bp mutation (GGA to GAT) was synthesized. The sequence of the ssDNA donor is: 5′-CAATTCTTTTTTTAGGTTCCAAAACTAAGGAGGGAGTCGTTCATGatGTGACAACAGGTAAGCTCTGTTGTCTTTTATCC-3′. The gRNA, Cas9 mRNA, and ssDNA donor oligonucleotide were co-injected into male pronuclei of F344/Stm rat zygotes. Eleven (11) founder rats were screened by Sanger sequencing. Five (5) founders had mutations in exon 3 of SNCA, and one founder had the desired GGA to GAT (G51D) mutation (BspHI restriction site (SNCA exon 3 PCR (forward primer 5′-TGGTGGCTGTTTGTCTTCTG-3′ and reverse primer 5′-TCCTCTGAAGACAATGGCTTTT-3′), a BspHI restriction enzyme digest, and agarose gel electrophoresis .The breeding and maintenance of wild-type (WT), 18F-DOPA was produced using a multi-step nucleophilic fluorination pathway, and used the TRACERlab MX synthesiser and cassette with the nucleophilic precursor to 18F-DOPA , with the final product formulated in PBS , then 10 mg/kg Entacapone  were injected intravenously to prevent the peripheral metabolism of 18F-DOPA by AADC and catechol-O-methyltransferase (COMT), respectively  and used for attenuation correction of PET data.Images were obtained using the nanoScan PET/CT scanner , with PET/CT data acquired using Nucline™ v2.01 acquisition software. Prior to radiotracer injection a scout view CT image was acquired. Dynamic PET imaging commenced upon injection of The PET data that was reconstructed and extended from the olfactory bulbs to the caudal border of the heart. Reconstruction was 3D, dynamic and used the TeraTomo3D reconstruction method. Coincidence mode was 1–3, voxel size 0.4 mm × 0.4 mm × 0.4 mm, used 4 iterations and 6 subsets, and was corrected for scatter, attenuation and randoms. Data was reconstructed into frames comprising 6 frames of 30 s, 3 frames of 60 s, 2 frames of 120 s and 22 frames of 300 s. All PET imaging data is freely accessible.18F-DOPA injected (MBq)/weight of the rat (kg)].Images were analyzed using PMOD 3.409 software (PMOD Technologies LLC) and a hand-drawn template. Volumes of interest (VOIs) comprised the left and right striatum, whole striatum, and the cerebellum which was a reference region for non-specific uptake. The same VOI template was used for all rats and were only moved into position over the respective anatomical areas. PET images for display purposes and to aid with VOI drawing were obtained by averaging over 0–120 min of emission data then a 1 mm × 1 mm × 1 mm Gaussian filter was applied. Standardized uptake value (SUV) images were produced using rat body weight and the activity injected. Similarly, SUV TACs were calculated as follow SUV (g/mL) = activity concentration in the target VOI (kBq/mL)/[decay corrected amount of iK of 18F-DOPA in the striatum and the distribution volume ratio (DVR) of 18F-DOPA in the striatum relative to the cerebellum, respectively  of 18F-DOPA which involved subtracting the TAC (kBq/mL) for the cerebellum from the TACs for the striatum before analysis /EDVR contralateral , quantile-quantile plots of the iK, DVR, and EDVR data-sets indicated the data is normally distributed.GT) to GAT, encoding for aspartic acid. The rat SNCA Gly-51 codon is GGA, which requires a 2-nucleotide mutation to produce the Asp-51 codon GAT. Cas9 mRNA, gRNA and ssDNA donor oligonucleotide with GAT codon were co-injected into F344/Stm rat zygotes. Eleven founder rats were screened by Sanger sequencing and 5 F0 rats had mutations in SNCA, but only one founder had the desired GGA to GAT (G51D) mutation in exon 3 of SNCA  that mutates the Gly-51 codon  NU-4 mouse image quality (IQ) phantom , and since EDVR is estimated to be the inverse of effective dopamine turnover   .EDVR of 18F-DOPA showed decreased mean EDVR in the left striatum when compared to the right in 5 out of 6 groups of rats, with greatest asymmetry in 16 month old SNCAG51D/G51D rats  can be found in the article/The animal study was reviewed and approved by UK Home Office under project licence PC6C08D7D.SNCAG51D rat model. KD (University of Edinburgh) established genotyping of SNCAG51D rats, maintained the rat colony, and conducted preliminary studies. TW and CL (University of Edinburgh) synthesized the 18F-DOPA radiotracer, and CA-C (University of Edinburgh) provided assistance during in vivo18F-DOPA PET imaging experiments. VM and AT designed and conducted PET experiments and data analysis. VM and TK wrote the manuscript and AT edited the manuscript. All authors contributed to the article and approved the submitted version.VM, AT, and TK devised the study. TM (Kyoto University) generated the This work was funded by a Carnegie Trust PhD Scholarship to VM, Parkinson’s UK Senior Fellowship to TK, and a Wellcome Trust ISSF award to TK and AT.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


However, the regulatory mechanism underlying the release of Epstein-Barr virus, a human oncovirus, remains unclear. We demonstrate that disruption of BBLF1, a tegument component, suppressed viral release and resulted in the accumulation of viral particles on the inner side of the vesicular membrane. Organelle separation revealed the accumulation of infectious viruses in fractions containing vesicles derived from the TGN and late endosomes. Deficiency of an acidic amino acid cluster in BBLF1 reduced viral secretion. Moreover, truncational deletion of the C-terminal region of BBLF1 increased infectious virus production. These findings suggest that BBLF1 regulates the viral release pathway and reveal a new aspect of tegument protein function.Enveloped viruses undergo a complex multistep process of assembly, maturation, and release into the extracellular space utilizing host secretory machinery. Several studies of the herpesvirus subfamily have shown that secretory vesicles derived from the IMPORTANCE Several viruses have been linked to the development of cancer in humans. Epstein-Barr virus (EBV), the first identified human oncovirus, causes a wide range of cancers. Accumulating literature has demonstrated the role of viral reactivation in tumorigenesis. Elucidating the functions of viral lytic genes induced by reactivation, and the mechanisms of lytic infection, is essential to understanding pathogenesis. Progeny viral particles synthesized during lytic infection are released outside the cell after the assembly, maturation, and release steps, leading to further infection. Through functional analysis using BBLF1-knockout viruses, we demonstrated that BBLF1 promotes viral release. The acidic amino acid cluster in BBLF1 was also important for viral release. Conversely, mutants lacking the C terminus exhibited more efficient virus production, suggesting that BBLF1 is involved in the fine-tuning of progeny release during the EBV life cycle. Herpesviridae (–Epstein-Barr virus (EBV) is a double-stranded DNA virus belonging to the family sviridae . EBV infsviridae –5. Approviridae –, 7.in vitro by several chemical and biological stimuli, including 12-O-tetradecanoylphorbol-13-acetate, transforming growth factor β, histone deacetylase inhibitors, and anti-human IgG  are attached to nucleocapsids in the nucleus. The nucleocapsids then exit the nucleus and migrate to the cytoplasm through the nuclear membrane via an envelopment and de-envelopment process. In the cytoplasm, the nucleocapsids bud into special vesicles derived from the trans-Golgi network (TGN), early endosome (EE), or autophagosome, where they acquire additional tegument proteins, the viral envelope, and glycoproteins  and subcellular fractionation analysis showed that BBLF1 knockout led to an accumulation of virions in secretory vesicles. Through mutagenesis of BBLF1, we found that optimal shedding of virions required a cluster of acidic amino acids but did not require posttranslational modifications, such as myristoylation and palmitoylation of the protein. By contrast, truncation of the C-terminal region of BBLF1 increased infectious virus production, suggesting that the C terminus of BBLF1 regulates its production function.O-tetradecanoylphorbol-13-acetate, calcium ionophore, and sodium butyrate were harvested sequentially. BBLF1 expression was detected 36 h after lytic reactivation, similar to the L-stage lytic protein BALF4  in lytically induced Akata, HEK293-EBV, and AGS-EBV cells through immunoblotting . To obsein BALF4 . When trin BALF4 . Knockouin BALF4 , suppresnd BALF4 , suggestMGALWSLCRRRVNSIGDVDG-HA tag) was below the detection limit, indicating that it is extremely unstable  using the EBV bacterial artificial chromosome (BAC) system with a homologous recombination technique. A BBLF1-deficient virus (dBBLF1) carrying a stop codon (TGA) was generated from the wild type (WT), and a revertant virus (dBBLF1/R) was constructed from dBBLF1 . Becauseunstable . These dTo examine the impact of BBLF1 knockout on infectious virus production, we infected EBV-negative cells with virion derived from the culture medium (cell-free virions) and cells (cell-associated virions) separately and monitored viral titers as outlined in To investigate the effect of BBLF1 knockout on infectivity, we measured the cell-free viral DNA level by quantitative PCR (qPCR) and then measured viral titer in the presence of an equal quantity of cell-free viral DNA, as outlined in We further investigated whether exogenous supply of BBLF1 to dBBLF1 could restore the reduction in virion release during the lytic cycle. Exogenous expression of BBLF1 significantly increased virion release to a level comparable to WT and dBBLF1/R cells  and B. OTo determine where BBLF1-knockout viral particles accumulate, we observed lytically induced HEK293-EBV WT and dBBLF1 cells by electron microscopy. Viral particles or virus-like particles accompanying vesicles were observed in both types of cells  to H; hoBBLF1 is a myristoylated and palmitoylated protein, and it has two acidic clusters of amino acids, NDE (amino acids 28 to 31) and SDE (amino acids 58 to 65) . In the To further examine whether those posttranslational modifications and acidic clusters affected viral release, we performed a complementation assay using cells infected with dBBLF1  and E. TTo extend the previous results obtained using the B95-8 strain of EBV and HEK293 cells, we aimed to prepare a BBLF1-knockout EBV of the Akata strain using the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system. However, knockout of the BBLF1 gene through gene editing was difficult because the BBLF1 gene has an N-terminal overlap with the BBLF1-adjacent BGLF5 gene, which limits the region available for editing. In addition, the expression of the adjacent BGLF3, BGLF3.5, BGLF4, and BGLF5 genes is regulated by a bicistronic or polycistronic translation mechanism, indicating that these proteins are translated from a common mRNA , 45. TheTo validate the finding that C-terminal truncation of BBLF1 resulted in increased production of both cell-free and cell-associated progeny virions, we conducted transcomplementation assays using HEK293-EBV-dBBLF1 cells, which were prepared as shown in Lastly, we tested HEK293-EBV WT but not HEK293-EBV-dBBLF1 cells. Overexpression of BBLF1-WT in cells infected with WT EBV significantly decreased total progeny production, suggesting that excessive BBLF1 is disadvantageous for EBV reproduction  showed increased efficiency of infectious virus production compared to that of the WT, providing the first example of a tegument protein partially suppressing viral production.The role of HSV-1 UL11, a homolog of BBLF1, in viral replication is well understood: UL11 knockout resulted in marked reduction of extracellular virion levels . The accThe mechanism through which BBLF1 facilitates the transport-to-release process remains unclear. Alphaherpesviruses bud on TGN- or endosome-derived vesicular membranes expressing viral glycoproteins , 49, 50.BBLF1 is required for virus maturation, but its overexpression can potentially be harmful to virus. Notably, the C terminus of BBLF1 might have an autoinhibitory effect . CertainIn conclusion, we performed initial functional analysis of BBLF1 using BBLF1-knockout viruses and found that BBLF1 contributes to the viral release process. Furthermore, we found that an acidic amino acid cluster enhances the efficiency of viral release. This is the first report showing that the CTD of BBLF1 suppresses viral production, suggesting that the BBLF1 protein plays dual roles. The molecular mechanism through which BBLF1 suppresses viral production remains unclear, and BBLF1 may fine-tune the secretion of progeny virions.ggacggggaattctacccatacgatg) and reverse (tagaattccccgtccacgtcgcctat), dMyr forward (AGAatggCTgccctctggtctctttg) and reverse (gagggcAGccatTCTAGAGGGCCCG), dPal forward (tctcttAgccgacgacgagtcaac) and reverse (tcgtcggcTaagagaccagagggcac), dNDE forward (atgCctatgCggCgtttaacctggagactac) and reverse (aacGccGcatagGcattatacaggttgatta), dSDE forward number 1 (tatgCtgCggCctctgaaaatgatgaattg) and reverse number 1 (agagGccGcaGcatattcgagcccctcgtt), dSDE forward number 2 (tctgCaaatgCtgCattgctgtttttgccaaat) and reverse number 2 (caatGcaGcatttGcagagGccGcaGcatattc), and CTD forward (AAACCAACGAATTCTACCCATACGATGTTCCAG) and reverse (TAGAATTCGTTGGTTTCCCCGCAGGCCCTCCCT). To edit BBLF1 of the Akata strain, Cas9-encoded pX459 (Addgene) carrying a BBLF1-targeting guide RNA was generated through cloning of the following annealed oligonucleotides into the BbsI site of the pX459 vector. The three pairs of oligonucleotides were annealed and inserted into the pX459 vector as follows: sgBBLF1 number 1 forward (CACCCAACCTGTATAATGACTATG) and reverse (AAACCATAGTCATTATACAGGTTG), sgBBLF1 number 2 forward (CACCCTATTAGCTTAGTAGTCTCC) and reverse (AAACGGAGACTACTAAGCTAATAG), and sgBBLF1 number 3 forward (CACCATCATATTCGAGCCCCTCGT) and reverse (AAACACGAGGGGCTCGAATATGAT). The amplified PCR product was introduced into competent E. coli DH5 (Nippon Gene), and clonal cells carrying the plasmid with the optimal sequence were grown for plasmid isolation. The plasmid was isolated using the Plasmid midikit (Qiagen).The preparation of the pBZLF1, pBBLF1, and pBDLF4 expression vectors has been described previously , 55, 56.O-tetradecanoylphorbol-13-acetate (Sigma), calcium ionophore (Sigma), sodium butyrate (Wako), and goat anti-human IgG (Jackson Immuno Research Inc.). Phosphonoacetic acid (PAA) (Sigma) was used to inhibit viral DNA replication. Monoclonal antibodies against glyceraldehyde 3-phosphate dehydrogenase (GAPDH), tubulin, HA-tag, calnexin, GM130, Rab5, Rab9, Rab11, and TGN38 were purchased from Cell Signaling Technology. An antibody against CD63 (Ts63) was obtained from Thermo. Antibodies against BZLF1, BMRF1, BALF4, BDLF4, and BALF2 were used as described previously (The following reagents were used for activation of EBV-infected cells: 12-eviously . RabbitsO-tetradecanoylphorbol-13-acetate (20 ng/mL); A, calcium ionophore (1 μM); B, sodium butyrate (5 mM)] for reactivation and PAA (400 μg/mL) to inhibit viral DNA replication.HEK293, HEK293T, and HEK293 cells carrying EBV-BAC as an episome (HEK293-EBV) were cultured in Dulbecco's modified Eagle’s medium (DMEM) (Sigma) supplemented with 100 U/mL penicillin-streptomycin (Gibco) and 10% fetal bovine serum (FBS). Akata EBV-positive and EBV-negative cells, EBV-infected AGS cells (AGS-EBV), and B95-8 cells were cultured in RPMI 1640 medium (Sigma) supplemented with 100 U/mL penicillin-streptomycin and 10% FBS. EBV-infected cell lines including B95-8, Akata-EBV, AGS-EBV, and HEK293-EBV were lytically reactivated for viral production and further analysis. Culture supernatants and cells were generally harvested at 48 to 72 h after lytic reactivation. AGS-EBV cells maintained in 400 μg/mL G418 were transfected with the pBZLF1 vector using the Neon electroporation system (Thermo). HEK293-EBV cells were propagated in DMEM containing 150 μg/mL hygromycin B (TaKaRa) and reactivated via pBZLF1 transfection using Lipofectamine 2000 (Thermo). Goat anti-human IgG was used to reactivate EBV-positive Akata cells maintained in 750 μg/mL G418. B95-8 cells were treated with TAB [T, 12-HEK293T cells were transfected with the designated plasmids using Lipofectamine 2000 (Thermo) for assessment of protein expression. Alternatively, in some cases, EBV-carrying clonal cells were lytically reactivated through targeted methods and harvested at the indicated time points for immunoblotting. To prepare a solubilized protein sample, harvested cells were washed in phosphate-buffered saline (PBS) and lysed in lysis sample buffer . Equal amounts of proteins were resolved through electrophoresis on a sodium dodecyl sulfate-polyacrylamide gel , transferred onto polyvinylidene difluoride membranes (Millipore), reacted with designated primary antibodies, and then reacted with horseradish peroxidase (HRP)-linked anti-rabbit or anti-mouse IgG  as secondary antibodies. Proteins were visualized through reaction with an HRP substrate, namely, Forte (Merck) or Chemi-Lumi One Ultra . Luminescent signals were detected and analyzed using the LuminoGraph II EM imaging system (ATTO).Escherichia coli. During the homologous recombination of EBV-BAC in E. coli, a cassette of neomycin resistance and streptomycin sensitivity (Neos/Str) genes was inserted into the BBLF1 sequence, resulting in intermediate-1; this was later replaced with the BBLF1-nonsense sequence, producing BBLF1-deficient EBV-BAC (dBBLF1). Similarly, after the generation of intermediate-2 with the Neo/St cassette from dBBLF1, a revertant EBV-BAC was constructed that carries the same sequence as WT BBLF1 (dBBLF1/R). PCR products were purified using a PCR product extraction kit (Qiagen), and EBV-BAC was extracted with the NucleoBond BAC 100 kit (TaKaRa). A Gene Pulser III (Bio-Rad) instrument was used for transfection of EBV-BAC and PCR products. The extracted EBV-BAC was digested using EcoRI or BamHI restriction enzymes and visualized through 0.6% agarose gel electrophoresis. Sequencing was conducted to confirm that the targeted manipulation of the EBV-BAC was successful. The isolated EBV-BAC was transfected into HEK293 cells using Lipofectamine 2000 and selected using hygromycin B for 2 weeks. Finally, hygromycin-resistant and green fluorescent protein (GFP)-positive cell colonies were selected and HEK293-EBV clonal lines were established. Individual PCR products were obtained through PCRs, using the rpsL-neo vector (Gene Bridges) as a template and the following primers: for BBLF1 Neo/St, 5′- ATGGGTGCCCTCTGGTCTCTTTGCCGACGACGAGTCAACTCCATAGGCGACGTGGACGGGggcctggtgatgatggcgggatc-3′ and 5′-TCGGCCGCTATTAGCTTAGTAGTCTCCAGGTTAAACTCCTCATAGTCATTATACAGGTTGtcagaagaactcgtcaagaagg-3′. To construct BBLF1 containing an antisense sequence, a vector carrying a nonsense mutation in BBLF1 was first constructed through inverse PCR using the following primers: 5′-ggacgggTgaataatcaacctgtata-3′ and 5′-attattcAcccgtccacgtcgcctat-3′. The PCR product was amplified using a vector.BBLF1-deficient EBV was generated from EBV-BAC, a BAC containing the cloned EBV genome, following the homologous recombination technique . The EBVThe generation of EBV mutants through CRISPR/Cas9-based editing has been described previously . Briefly6 EBV-negative Akata cells were cocultured with 1 mL viral medium using a tube rotator for 2.5 h, after which the medium was replaced with fresh medium and incubation was continued. At 48 h postinfection, cells were fixed with 4% paraformaldehyde, and the frequency of GFP-positive cells was examined by flow cytometry .Reactivated HEK293-EBV or Akata-EBV cells were harvested after a specified incubation period along with the spent culture medium and centrifuged at low speed; the supernatant contained cell-free virions, while cell-associated virions were obtained from the cell pellets after further processing. The cell pellets were frozen, thawed, and resuspended in medium, and cell debris was then removed from the medium through high-speed centrifugation to obtain cell-associated virions. For collection of total virions including both the cell-free and cell-associated fractions, harvested cells and spent media were not initially separated; instead, they were frozen, thawed, vortexed, and centrifuged, after which the supernatant was collected. To determine viral titer, 1 × 10The number of virus particles released into culture medium from lytically induced cells was quantified by enumerating viral genome copies as reported previously . Briefly4, followed by treatment with 1% uranyl acetate. For dehydration, the cells were incubated in a series of ethanol solutions with concentrations increasing gradually from 70% to 100%. This was followed by the embedding of cells in resin, thin sectioning, mounting of the slices on a specimen grid, and staining with uranyl acetate and lead citrate. Transmission electron microscopy (TEM) images were captured using a JEOL JEM-1400 PLUS transmission electron microscope.HEK293-EBV cells were lytically reactivated, harvested after 3 days, washed in PBS, and fixed with 2% glutaraldehyde. Secondary fixation was conducted with 1% OsO7) were harvested, resuspended in homogenization medium , and homogenized on ice with a Potter-Elvehjem grinder. To obtain the cytoplasmic fraction, the cell suspension was centrifuged at 1,000 × g, and the pellet was removed as the nuclear fraction. A continuous 5 to 50% (wt/vol) iodixanol (OptiPrep) gradient was prepared using Gradient Master (BioComp). Next, the cytosolic fraction was poured into the tube as a top layer. The gradient tubes were ultracentrifuged in an Optima XPN-80 (Beckman) centrifuge at 48,000 × g for 18 h. After ultracentrifugation, the gradient solution was collected from top to bottom as 12 fractions. For infectious viral titer measurement, fractions were frozen and thawed, and cell debris was then removed through centrifugation. After Akata cells were infected, each fraction was adjusted to a 1-mL volume through the addition of RPMI medium. Lysis sample buffer was added to the fractioned sample, followed by immunoblotting with the designated antibodies to check for the presence of cellular organelle markers. Fractionated samples were placed on hydrophilized grids (Nisshin EM), reacted with 1% uranyl acetate for 10 s, dried, and examined under an electron microscope.Reactivated HEK293-EBV cells (2.5 × 10


The vitamin D receptor (VDR) has a low level of expression in the keratinocytes of patients with psoriasis and plays a role in the development of the disease. Furthermore, the crosstalk between macrophages and psoriatic keratinocytes-derived exosomes is critical for psoriasis progression. However, the effects of VDR-deficient keratinocytes-derived exosomes (Exos-shVDR) on macrophages and their underlying mechanisms remain largely unknown.VDR-deficient keratinocytes were constructed by infecting HaCaT cells with a VDR-targeting lentivirus, mimicking the VDR-deficient state observed in psoriatic keratinocytes. Exosomes were characterized using transmission electron microscopy, nanoparticle tracking analysis, and Western blot. The effect of Exos-shVDR on macrophage proliferation, apoptosis, and M1/M2 polarization was assessed using cell counting kit-8 assay (CCK-8), flow cytometer, real-time quantitative polymerasechain reaction (RT-qPCR), and enzyme-linked immunosorbent assay (ELISA). The mechanism underlying the effect of Exos-shVDR on macrophage function was elucidated through data mining, bioinformatics, RT-qPCR, and rescue experiments.Our results revealed that both Exos-shVDR and Exos-shNC exhibited typical exosome characteristics, including a hemispheroid shape with a concave side and particle size ranging from 50 to 100 nm. The levels of expression of VDR were significantly lower in Exos-shVDR than in Exos-shNC. Functional experiments demonstrated that Exos-shVDR significantly promoted macrophage proliferation and polarization towards the M1 phenotype while inhibiting macrophage apoptosis. Moreover, miR-4505 was highly expressed in the skin tissue of patients with psoriasis. Its overexpression significantly increased macrophage proliferation and polarization towards M1 and inhibited apoptosis. Furthermore, the effects of Exos-shVDR on macrophage function occur through miR-4505.Exos-shVDR exacerbates macrophage proliferation, promotes polarization towards the M1 phenotype, and inhibits macrophage apoptosis by increasing the levels of miR-4505. These results indicate that modulation of macrophage function is a potential strategy for developing new drugs for the treatment of psoriasis. Psoriasis is a prevalent chronic inflammatory skin disease and has a high relapse rate. Its incidence is approximately 0.4% in China and 2–3% in Europe and America . PsoriasRecently, macrophage polarization was shown to play a crucial role in the development of inflammation . MacrophUnder the influence of various factors, macrophages differentiate into the M1 phenotype, secreting pro-inflammatory factors such as interleukin (IL)-1β, inducible nitric oxide synthase (iNOS), and tumor necrosis factor-α (TNF-α), which contribute to the initiation and aggravation of the inflammatory response. Conversely, M2 macrophages mainly release anti-inflammatory factors such as IL-10, transforming growth factor-β (TGF-β), and arginase 1 (Arg1), inhibiting the inflammatory response .Emerging evidence revealed that polarization of macrophages towards the M1 phenotype contributes to psoriasis by activating inflammatory responses and releasing a multitude of pro-inflammatory factors. In summary, the polarization state of macrophages plays an important role in the pathogenesis of psoriasis. Modulating the differentiation of macrophages towards the M1 phenotype is considered beneficial for the treatment of psoriasis, as it can help reduce inflammation and mitigate the symptoms associated with the condition.Exosomes, small extracellular vesicles involved in intercellular communication, play a role in carrying various molecules, including microRNAs (miRNAs) . Psoriat2D3] (The vitamin D receptor (VDR), a nucleophilic protein belonging to the nuclear receptor superfamily, mediates the biological effects of the active form of vitamin D3, 1,25-dihydroxy vitamin D3  . In pati2D3] . Further2D3] . Additio2D3] .However, it is still unclear whether VDR-deficient keratinocytes-derived exosomes (Exos-shVDR) affect the biological functions of macrophages. We aimed to provide an answer to this question and offer a foundation for exploring new diagnostic and therapeutic targets for psoriasis.Herein, we discovered that Exos-shVDR promoted macrophage proliferation and their polarization towards the M1 phenotype, and inhibited their apoptosis, thereby promoting the development of psoriasis. Mechanistically, these effects of Exos-shVDR were mediated through miR-4505.2. THP-1 cells were differentiated into macrophages by treating them with 100 ng/mL phorbol 12-myristate 13-acetate  for 48 h prior to exosome uptake.The HaCaT (iCell-h066) and THP-1 human monocytic leukemia  cell lines were purchased from iCell Bioscience Inc. . The cells were cultured in Dulbecco’s modified Eagle medium (DMEM)  supplemented with 10% fetal bovine serum  and 100 U/mL penicillin/streptomycin  in an incubator at 37 °C with 5% CO™  following the manufacturer’s instructions. VDR shRNAs were transfected into keratinocytes using Lipofectamine 8000™  for 24 h to achieve VDR deficiency, as confirmed by reverse transcription quantitative polymerase chain reaction (RT-qPCR). Additionally, the miR-4505 inhibitor was transfected into exosomes using Exo-Fect™ Exosome Transfection Kit . The transfection efficiency was verified by RT-qPCR. The utilized sequences of shRNA, mimics, and inhibitors are provided below:The short hairpin RNA (shRNA) targeting VDR , miR-4505 mimics, miR-4505 inhibitor, and their negative controls  were obtained from RiboBio Corporation . Transient transfection was performed using Lipofectamine 8000shVDR#1: CCGGTCGAAGTGTTTGGCAATGAGATCTCGAGATCTCATTGCCAAACACTTCGTTTTTGAATT;shVDR#2: CCGGTCCTCCAGTTCGTGTGAATGATCTCGAGATCATTCACACGAACTGGAGGTTTTTGAATT;shVDR#3: CCGGTGTCATCATGTTGCGCTCCAATCTCGAGATTGGAGCGCAACATGATGACTTTTTGAATT;scramble: CCGGTCCTAAGGTTAAGTCGCCCTCGCTCGAGCGAGGGCGACTTAACCTTAGGTTTTTGAATT;miR-4505 mimics,Sense: 5′-AGGCUGGGCUGGGACGGA-3′, Antisense: 5′- CGUCCCAGCCCAGCCUUU -3′;NC mimics,Sense: 5′- UUGUACUACACAAAAGUACUG-3′, Antisense: 5′-GUACUUUUGUGUAGUACAAUU-3′;miR-4505 inhibitor: TCCGUCCCAGCCCAGCCT;NC inhibitor: CAGUACUUUUGUGUAGUACAA.™ bicinchoninic acid (BCA) protein assay . The characterization of exosomes was assessed through transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and assessment of the protein expression of exosomal markers . For TEM, the exosomes were mixed with 10 μL uranyl acetate for 1 min and photographed using a transmission electron microscope  after a brief drying period. NTA was performed using a particle size analyzer  to track the particle size distribution and concentration of the exosomes. Additionally, the protein expression of exosomal markers was measured by Western blot.The exosomes were isolated from HaCaT cells using Total Exosome Isolation Reagent  following the manufacturer’s instructions. Briefly, the culture supernatants of HaCaT cells stably transfected with sh-VDR or sh-NC were collected and centrifuged at 2,000 g for 20 min to remove cells and debris. The resulting supernatants were filtered through a 0.22 μm filtering membrane to remove residual impurities and collected in an ultrafiltration cube. An equal volume of Total Exosome Isolation Reagent  was added to the ultrafiltration cube. Finally, the mixture in the ultrafiltration cube was centrifuged at 10,000 g for 1 h, and the exosomes were collected from the sediment. All operating procedures were carried out at 4 °C. The concentration of exosomes was determined using the PierceThe indicated exosomes were labeled using the PKH26 Green Fluorescent Cell Linker Kit . Briefly, the exosomes were cultured with diluent C and PKH26 dye for 5 min, and then the unincorporated dye was removed using exosome spin columns. The macrophages were incubated in a medium containing PKH26-labeled exosomes. The treated macrophages were stained with 4′,6-diamidino-2-phenylindole (DAPI) and photographed using a fluorescence microscope .−ΔΔCt method, and all of the RT-qPCR reactions were performed in triplicate. The primers used in the present study were the following:Total RNA was extracted using Trizol reagent  and reverse transcribed into cDNA using PrimeScript™ RT reagent kit and gDNA Eraser . RT-qPCR amplification was performed on an ABI7500 quantitative PCR instrument using the SYBR Green qPCR Mix . U6 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were used as housekeeping genes for miRNAs and other mRNAs, respectively. Relative expression levels of miRNAs and mRNAs were calculated by the 2VDR:F: 5′- GTGAGCTGAGATCGTGCCGTTA -3′,R: 5′- GGTCCTGTCCTGGTCCACTTCT -3′;GAPDH,F: 5′- AAGTATGACAACAGCCTCAAG -3′,R: 5′- TCCACGATACCAAAGTTGTC -3′;miR-4505:F: 5′- TTATCTTTAGGCTGGGCTGG -3′,R: 5′-GTCGTATCCAGTGCGTGTC -3′,RT: 5′- GTCGTATCCAGTGCGTGTCGTGGAGTCGGCAATTGCACTGGATACGACGACGGA -3′;miR-4507:F: 5′- AACTAAACTGGGTTGGGCTGG -3′,R: 5′-GTCGTATCCAGTGCGTGTC -3′,RT: 5′- GTCGTATCCAGTGCGTGTCGTGGAGTCGGCAATTGCACTGGATACGACGCTGGG -3′;miR-4563:F: 5′- CGTGGAGTTAAGGGTTGCT -3′,R: 5′-GTCGTATCCAGTGCGTGTC-3′,RT: 5′- GTCGTATCCAGTGCGTGTCGTGGAGTCGGCAATTGCACTGGATACGACTGGAGA -3′.U6:F: 5′- CTCGCTTCGGCAGCACATATACT -3′,R: 5′- ACGCTTCACGAATTTGCGTGTC-3′,RT: 5′- AAAATATGGAACGCTTCACGAATTTG -3′.Total proteins from cells or exosomes were isolated using RIPA lysis buffer  and quantified using a BCA detection kit  according to the manufacturer’s instructions. Proteins with suitable concentrations were separated using sodium dodecyl-sulfate polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes . The membranes were incubated overnight at 4 °C with the following primary antibodies: anti-CD63 , anti-TSG101 , anti-VDR , and anti-GAPDH , after blocking with 5% blocking solution. Subsequently, the membranes were incubated with a secondary antibody  at room temperature for 2 h. Protein bands and gray values were visualized and quantified using a gel imaging system  and Image J software , respectively.The viability of THP-1 cells was assessed using the CCK-8 kit  following the manufacturer’s instructions. Briefly, the treated macrophages were inoculated in a 96-well plate. CCK-8 solution was added to each well and incubated in the dark for 2 h. The absorbance values at 450 nm were measured using a spectrophotometer .Flow cytometry was used to evaluate cell apoptosis and macrophage polarization. For cell apoptosis analysis, the treated cells were incubated with annexin V conjugated with fluorescein isothiocyanate (FITC) and propidium iodide (PI)  at room temperature in the dark for 20 min. For the macrophage polarization, the treated cells were fixed with 1% paraformaldehyde (PFA) overnight at 4 °C and then incubated with anti-CD86  and anti-CD206  antibodies for 30 min at 20 °C.Following incubations, the cells were analyzed by flow cytometry .GSE115293). The dataset included four psoriasis skin samples and four normal skin samples. Differential expression analysis of miRNAs between patients with psoriasis and without (controls) was performed using the “edgeR” package with the following filter criteria: |log | > 1.5 and adj. P levels < 0.05. Volcano plots were generated to illustrate the differentially expressed miRNAs.The miRNAs expression data of patients with or without psoriasis were obtained from the Gene Expression Omnibus (GEO) database . The Student’s t-test and one-way analysis of variance (ANOVA) were used to compare the results and evaluate the differences between groups. A To silence VDR expression in HaCaT cells, three shRNAs targeting VDR  and negative control (shNC) were constructed and transfected into HaCaT cells. The expression levels of VDR were detected after transfection by RT-qPCR and Western blot. The three shVDRs significantly suppressed the expression of VDR in HaCaT cells. Among them, shVDR#1 (shVDR) demonstrated the most significant inhibitory effect and was selected for the subsequent experiments .Subsequently, HaCaT cells stably expressing shVDR and shNC were constructed by lentiviral transduction of shVDR and shNC, respectively. The resulting exosomes, designated as Exos-shVDR and Exos-shNC, were extracted using Total Exosome Isolation reagent. The exosomes were identified by Western blot, TEM, and NTA.Western blot analysis confirmed the enhanced expression of exosome-specific markers (CD63 and TSG101) in both Exos-shVDR and Exos-shNC, while nearly no expression was observed in the supernatant of HaCaT cells with stable transfected shVDR or shNC treated with exosome inhibitor GW4869 .The morphological characteristics of exosomes were evaluated by TEM, revealing that both Exos-shVDR and Exos-shNC displayed a typical double membrane structure with a particle size of 100 nm . AdditioFurthermore, the expression levels of VDR in Exos-shNC and Exos-shVDR were measured by RT-qPCR and Western blot to confirm VDR silencing in exosomes derived from HaCaT cells with stable shVDR transfection. The expression of VDR was significantly lower in Exos-shVDR than in Exos-shNC . Taken tTo explore the function of Exos-shVDR in macrophages, THP-1 cells were differentiated into macrophages using 100 ng/mL PMA for 48 h. Subsequently, they were co-cultured with PKH26-labeled Exos-shVDR or PKH26-labeled Exos-shNC. The results demonstrated the uptake of both Exos-shVDR and Exos-shNC by macrophages after 24 h of co-culture .Following treatment of PMA-induced macrophages with Exos-shVDR or Exos-shNC for 24 h, cell proliferation and apoptosis were assessed using CCK-8, Western blot, and flow cytometry, as well as by assessing the activity of caspase-3. The CCK-8 assay revealed that Exos-shVDR significantly enhanced macrophage proliferation compared to Exos-shNC . Flow cyTo evaluate the effect of Exos-shVDR on macrophage M1/M2 polarization, we used flow cytometry, RT-qPCR, and ELISA to quantify the expression levels of the markers of macrophage polarization in macrophages treated with Exos-shVDR or Exos-shNC for 24 h. Exos-shVDR significantly promoted the expression levels of the M1 polarization marker CD86, while it had no significant effect on the expression levels of the M2 polarization marker CD206 .Consistent with the result of flow cytometry, RT-PCR analysis revealed that Exos-shVDR significantly increased the mRNA expression levels of M1 polarization markers , while it had no significant effect on the mRNA expression of the M2 polarization marker IL-10 . FurtherGSE115293) associated with psoriasis from the GEO database and performed differential expression profiling of miRNA.Evidence indicates that miRNAs carried by exosomes play a role in various diseases including cancer , stroke A volcano plot revealed 26 dysregulated miRNAs in psoriatic skin tissue compared to normal skin tissue, with 12 upregulated and 14 downregulated miRNAs . Among tTo further explore the function of miR-4505 in macrophages, macrophages were transfected with miR-4505 mimic or NC mimic for 24 h, followed by RT-qPCR, CCK-8 assay, Western blot, caspase-3 activity assessment, and flow cytometry. The level of expression of miR-4505 was significantly increased in macrophages transfected with miR-4505 mimic compared to those transfected with NC mimic . CCK-8 aTo further explore the impact of miR-4505 on macrophage polarization, RT-qPCR and ELISA were used to quantify the levels of expression of macrophage polarization-related markers. RT-PCR revealed that the upregulation of miR-4505 significantly increased the mRNA expression levels of M1 polarization markers , while did not affect the mRNA expression of M2 polarization marker IL-10 . Similar™ Exosome Transfection kit. The results demonstrated that the expression level of miR-4505 in Exos-shVDR transfected with miR-4505 inhibitor (Exos-shVDR-miR-4505 inhibitor) was significantly lower than that in Exos-shVDR transfected with NC inhibitor (Exos-shVDR-NC inhibitor), indicating effective inhibition of miR-4505 in Exos-shVDR transfected with miR-4505 inhibitor 2D3 . VDR is 2D3 . Further2D3  and auto2D3 . HowevermiRNAs are small noncoding RNAs ranging in length from 18 to 24 nucleotides and playing a crucial role in the occurrence and development of various diseases, including psoriasis . Thus, aRecently, exosomal miRNAs have been identified, and extensively studied for their biological functions upon transfer to recipient cells. In gastric cancer, macrophage-derived exosomal transfer has been associated with doxorubicin resistance . FurtherBased on GEO data mining and experimental verification, we demonstrated that miR-4505 was highly expressed in the keratinocytes of patients with psoriasis. Furthermore, we also observed significantly higher levels of expression of miR-4505 in exosomes derived from VDR knockdown HaCaT cells compared to control HaCaT cells. VDR is a ligand-activated transcription factor belonging to the nuclear receptor superfamily. Previous studies have shown that VDR/retinoid X receptor can modulate the expression of miR-22, miR-296-3p, and miR-498 by regulating the maturation of pre-miRNAs upon binding to VDR response element . In addiin vitro experiments.Our findings revealed that miR-4505 promoted macrophage proliferation and their polarization towards M1 phenotype, while inhibiting macrophage apoptosis. Furthermore, we demonstrated that exosomes derived from VDR knockdown keratinocytes exert their effects through miR-4505. Our results indicate that miR-4505 might be a new potential therapeutic target for psoriasis. However, this study also has certain limitations: (1) the downstream mechanism of miR-4505 regulation in macrophage polarization has not been elucidated; (2) the underlying mechanism of VDR knockdown-induced miR-4505 overexpression has not been explored; (3) the results of this study have yet to be validated in animal models as they were limited to Our study demonstrated that Exos-shVDR promoted macrophage proliferation and polarization towards the M1 phenotype while inhibiting macrophage apoptosis. Mechanistically, these effects occurred due to the VDR knockdown-induced increase in the level of expression of miR-4505 in the exosomes secreted by keratinocytes. This study provides a foundation for further exploration of novel diagnostic and therapeutic targets for psoriasis.10.7717/peerj.15798/supp-1Supplemental Information 1Click here for additional data file.