human cd73 elisa kit  (R&D Systems)

Journal: Journal of Extracellular Vesicles

Article Title: CD73 in small extracellular vesicles derived from HNSCC defines tumour‐associated immunosuppression mediated by macrophages in the microenvironment

doi: 10.1002/jev2.12218

Figure Lengend Snippet: CD73 was overexpressed in the tumour cells derived sEVs of HNSCC patients. (a) Schematic of the progress of extracting sEVs from Conditional Reprogramming (CR) cells. (b) The histological and morphological characteristics of CR coculture of keratinocytes and feeder cells from HNSCC and adjacent normal tissues. (c) Electron microscopy images of sEVs from HNSCC. Scale bar, 100 and 200 nm. (d) Nanoparticle tracking analysis (NTA) of sEVs from HNSCC. (e) Immunoblotting for sEVs biomarkers. (f) Quantitative proteome results of cells released sEVs from HNSCC or adjacent normal tissues, shown by venn diagram. (g) The mass spectrum identified CD73 in sEVs derived from HNSCC cells. (h) Representative western blotting for CD73 in sEVs from six pairs of HNSCC samples

Article Snippet: A human CD73 ELISA kit (R&D Systems) and ELISA ancillary reagent kit (R&D Systems) were used to measure the CD73 level in sEVs from the serum.

Techniques: Derivative Assay, Electron Microscopy, Western Blot

Journal: Journal of Extracellular Vesicles

Article Title: CD73 in small extracellular vesicles derived from HNSCC defines tumour‐associated immunosuppression mediated by macrophages in the microenvironment

doi: 10.1002/jev2.12218

Figure Lengend Snippet: CD73 in sEVs was closely contributed to tumour associated macrophages and HNSCC malignant progress. (a) IHC analysis of CD73 expression levels in tissue microarrays, containing 10 normal tissues and 92 HNSCC tissues. Representative immunohistochemistry images of normal tissue, weak positive, modest positive, and strong positive CD73 staining were shown. Scale bar: 200 μm, 40 μm. (b) Statistical analysis about lymph node metastasis (LN metastasis), tumour stage, pathologic stage and overall survival rate with CD73 stain intensity in HNSCC tissues. (c) The correlation of NT5E expression with immune infiltration level in HNSCC investigated in TCGA database based on six deconvolution algorithms. (d) Immunofluorescence staining of CD73 distribution (green) and different resident immune‐associated cell types (red), including macrophages (CD68 + ), CD4 + T cells, CD8 + T cells, Tregs (Foxp3 + ) and CAFs(α‐SMA + ) in tissues from HNSCC patients. Scale bar: 40 μm. (e) The percentage of costaining of CD73 with macrophages, CD4 + T cells, CD8 + T cells, Tregs and CAFs in HNSCC patient tumour samples. (f) NT5E expression ( NT5E high , NT5E low ) as a marker for prediction of overall survival rate in TCGA HNSCC cohort. Data were classified into low macrophage/low M2 macrophage signature (Mac low /M2 low ) and high macrophage/high M2 macrophage signature (Mac high /M2 high ). Log‐rank Mantel‐Cox test was used to assess significance. (g) The association between sensitivity of anti‐PD‐L1 treatment and NT5E expression were studied through the public data of IMvigor210CoreBiologies, lower NT5E group exhibited increased sensitivity to PD‐L1 blockade than higher NT5E group. (h) The percentage of SCC7 Nt5e OE‐GFP ‐derived sEVs CD73‐GFP signal that distributed on macrophages (F4/80 + ), CD4 + T cells, CD8 + T cells, Tregs (Foxp3 + ) in SCC7 tumour‐bearing C3H mice. (i) The SCC7 Nt5e OE‐GFP ‐derived sEVs CD73‐GFP were injected into tumours on C3H mice. After 24 h, fluorescence visualization identified the coexpression of CD73‐GFP in sEVs (green) with immunocytes(red) in tumours. Scale bar: 20 μm. (j) Schematic of sEVs injection through foot pad and its draining lymph node (DLNs). (k) Fluorescence microscopy showed sEVs CD73‐GFP (green) in whole DLNs imaging after 30 or 60 min of sEVs CD73‐GFP (10 μg) injection. Scale bar: 200 μm, 50 μm. (l) Flow cytometry analysis for subpopulation in CD73‐GFP + cells of DLNs (sEVs CD73‐GFP : 25 μg, 24 h). (m) The sEVs SCC7 (25 μg) were injected into foot pads. After 24 h, flow cytometry analyzed the expression of CD73 + or PD‐1 + immunocytes in DLNs. (n) Flow cytometry analysis for percentage of macrophages in DLNs after injecting sEVs (25 μg) derived from SCC7 cells (sEVs SCC7 ), mBMSC cells (sEVs mBMSC ) or mBMSC‐ Nt5e OE cells (sEVs mBMSC‐ Nt5e OE ) every day. DLNs were harvested at different time point. Lymph nodes from untreated mice were used as normal control. CD4 + T: CD4 + T cells, CD8 + T: CD8 + T cells, Mac: Macrophages. Data were analysed by Mann‐Whitney test. (ns, no significant difference, *: p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001)

Article Snippet: A human CD73 ELISA kit (R&D Systems) and ELISA ancillary reagent kit (R&D Systems) were used to measure the CD73 level in sEVs from the serum.

Techniques: Expressing, Immunohistochemistry, Staining, Immunofluorescence, Marker, Derivative Assay, Injection, Fluorescence, Microscopy, Imaging, Flow Cytometry, Control, MANN-WHITNEY

Journal: Journal of Extracellular Vesicles

Article Title: CD73 in small extracellular vesicles derived from HNSCC defines tumour‐associated immunosuppression mediated by macrophages in the microenvironment

doi: 10.1002/jev2.12218

Figure Lengend Snippet: Relationship between CD73 level and clinicopathologic features in HNSCC tumour tissues ( n = 92)

Article Snippet: A human CD73 ELISA kit (R&D Systems) and ELISA ancillary reagent kit (R&D Systems) were used to measure the CD73 level in sEVs from the serum.

Techniques: Expressing, Significance Assay

Journal: Journal of Extracellular Vesicles

Article Title: CD73 in small extracellular vesicles derived from HNSCC defines tumour‐associated immunosuppression mediated by macrophages in the microenvironment

doi: 10.1002/jev2.12218

Figure Lengend Snippet: The effect of CD73 in sEVs derived from HNSCC cells on the function of macrophages. (a) Macrophages were cocultured with DMEM or two HNSCC lines (SCC25, HN6) with or without NT5E/RAB27AKO for 24 h. HNSCC NT5E OE , referred to overexpression of NT5E performed on HNSCC NT5E KO cells. (b) Flow cytometry analysis for percentage of CD73 + macrophages and M2 macrophages (CD163 + CD206 + ) in coculture system. (c–i) The sEVs (50 μg) were cocultured with macrophages (1 × 10 6 ) for 24 h. The sEVs HNSCC derived from two HNSCC lines: SCC25 and HN6. The sEVs hBMSC‐ NT5E OE derived from hBMSC cells with NT5E overexpression. Latrunculin A (Lat A, 30 μM) was used as the inhibitor of sEVs uptaken. (c) Compromised phagocytosis of M2 macrophages treated with or without sEVs or Lat A. The percentage of pHrodo dyes of M2 macrophages was analyzed by flow cytometry. (d) The percentage of CD73 + macrophages and M2 macrophages after macrophages cocultured with sEVs. (e, f) Concentration of IL‐6, IL‐10, TNF‐α, and TGF‐β1 levels in macrophages conditional medium after sEVs education by ELISA. (e)The sEVs were derived from HNSCC cells or HNSCC NT5E KO cells. (f) The sEVs were derived from HNSCC, hBMSC or hBMSC NT5E OE cells with or without Lat A (30 μM). (g) Macrophages were cocultured with anti‐CD73‐FITC labelled sEVs from HNSCC cell lines control or RAB27A KO, CD73‐GFP labelled sEVs from hBMSC treated with or without Lat A were cocultured with macrophages for 1 h, and Laser Scanning Confocal Microscopy was used to analyze the internalization of HNSCC‐derived sEVs into macrophages (Scale bar = 25 μm). (h) Flow cytometry analysis for differential expression of immune check point (PD‐1, PD‐L1, LAG3, CTLA‐4, VISTA) comparing M0 and M2 macrophages which were educated with sEVs from HNSCC cells (h) or hBMSC with or without Lat A (i). Data were analysed by Mann‐Whitney test (ns, no significant difference, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001)

Article Snippet: A human CD73 ELISA kit (R&D Systems) and ELISA ancillary reagent kit (R&D Systems) were used to measure the CD73 level in sEVs from the serum.

Techniques: Derivative Assay, Over Expression, Flow Cytometry, Concentration Assay, Enzyme-linked Immunosorbent Assay, Control, Confocal Microscopy, Quantitative Proteomics, MANN-WHITNEY

Journal: Journal of Extracellular Vesicles

Article Title: CD73 in small extracellular vesicles derived from HNSCC defines tumour‐associated immunosuppression mediated by macrophages in the microenvironment

doi: 10.1002/jev2.12218

Figure Lengend Snippet: Absence of CD73 in sEVs rescues immune suppression and restrains tumour growth in vivo. (a) Schematic of subcutaneous tumorigenesis in vivo experiment, followed with intratumoral injection of sEVs which were collected from SCC7, SCC7‐ Nt5e KO or SCC7‐ Nt5e OE cells grown in vitro. (b) The exhibition of isolated tumours. (c and d) The tumour weight and the time course of tumour growth in grams for 15 days postinjection with SCC7 or SCC7 Rab27a KO cells with or without CD73 in sEVs. Lat A was used as inhibitor of sEVs uptaken. (e) Flow cytometry analysis for infiltration of macrophages and percentage of CD73 + /PD‐1 + macrophages in tumours. (f). Flow cytometry analysis for infiltration of CD8 + T cells and percentage of CD73 + /PD‐1 + CD8 + T cells in tumours. (g) Flow cytometry analysis for infiltration of Tregs and percentage of CD73 + /PD‐1 + Tregs in tumours. (h) Schematic of subcutaneous tumorigenesis followed with intratumoral injection of engineered sEVs from mBMSC or mBMSC Nt5e OE . (i) The exhibition of dissected tumours. (j and k) The tumour weight and tumour growth curve of SCC7 control or SCC7 Rab27a KO with indicated treatment. (l and m) Flow cytometry analysis for infiltration of macrophages and percentage of CD73 + macrophages in tumours. (n) Flow cytometry analysis for infiltration of CD8 + T cells and Tregs in tumours. Data were analysed by Mann‐Whitney test (ns, no significant difference, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001)

Article Snippet: A human CD73 ELISA kit (R&D Systems) and ELISA ancillary reagent kit (R&D Systems) were used to measure the CD73 level in sEVs from the serum.

Techniques: In Vivo, Injection, In Vitro, Isolation, Flow Cytometry, Control, MANN-WHITNEY

Journal: Journal of Extracellular Vesicles

Article Title: CD73 in small extracellular vesicles derived from HNSCC defines tumour‐associated immunosuppression mediated by macrophages in the microenvironment

doi: 10.1002/jev2.12218

Figure Lengend Snippet: CD73 in sEVs regulates the immune functions of TAMs through NF‐κB pathway. (a) Venn diagram showing the differential expressed gene (DEG) of M2 macrophages depending on the regulation by sEVs CD73 or sEVs NT5E KO , the numbers of shared and exclusive genes were exhibited. (b) Bubble plot showing the predicted transcription factors of shared DEGs from AC. The shared DEGs and the signalling pathways which they belonged to were shown in circos gram, the number of genes was represented by node size. (d and e) GSEA analysis of up‐stream events of NF‐κB regulation. IκB phosphorylation and NIK signaling activity was measure after sEVs CD73 (up regulated) or sEVs NT5E KO (down regulated) incubation. (f) M2 macrophages were treated by HNSCC cell lines‐derived sEVs with or without CD73 for 3 h, with or without pretreatment of 100 μM PDTC for 1h. IF was applied for assessing the translocation of p65 in M2 macrophages (Scale bar = 40 μm). (g) Western blot was performed to validate the status of IκBα degradation, IκBα phosphorylation, p65 and phosphorylated p65 in M2 macrophages following treatment with sEVs or sEVs NT5E KO from HNSCC cell lines. (h) The heatmap showed the mRNA levels of IL6 , IL10 , TNFA , TGFB1 , CD274 , CD279 , and LAG3 in M2 macrophages which were stimulated by HNSCC derived sEVs, the expressions were partially downregulated when sEVs NT5E KO were added, and further inhibited when pretreated with PDTC

Article Snippet: A human CD73 ELISA kit (R&D Systems) and ELISA ancillary reagent kit (R&D Systems) were used to measure the CD73 level in sEVs from the serum.

Techniques: Phospho-proteomics, Activity Assay, Incubation, Derivative Assay, Translocation Assay, Western Blot

Journal: Journal of Extracellular Vesicles

Article Title: CD73 in small extracellular vesicles derived from HNSCC defines tumour‐associated immunosuppression mediated by macrophages in the microenvironment

doi: 10.1002/jev2.12218

Figure Lengend Snippet: The sEVs CD73 predicts HNSCC metastasis and targeting sEVs CD73 abolishes immunotherapy resistance. (a) Schematic of the circulating sEVs from HNSCC patients processing. (b) Percentage of CD73 + macrophages in PBMC. (c) The concentration of CD73 in serum sEVs (log2 TPM) detected by ELISA from HNSCC patients. (d) Level of CD73 (log2 TPM) on serum sEVs predicts higher risk of lymph node metastasis and larger tumour size. (e) CD73 indicated as a potential candidate to predict anti‐PD‐1 therapy response for HNSCC patients compared to other existed biomarkers, analysis by TIDE framework ( http://tide.dfci.harvard.edu ), TIDE, tumour immune dysfunction and exclusion. (f) The exhibition of isolated tumours 15 days after injection of SCC7 or SCC7 Rab27a KO cells with or without CD73 absent in sEVs combined with anti‐PD‐1 therapy. (g and h) The tumour weight and tumour growth curve of SCC7 control or SCC7 Rab27a KO with indicated treatment combined with anti‐PD‐1 therapy. (j) Flow cytometry analysis for infiltration of macrophages and percentage of CD73 + macrophages in tumours. (k) Flow cytometry analysis for infiltration of CD8 + T cells and Tregs in tumours. Data were analyzed by Mann–Whitney test (ns, no significant difference, *: p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001)

Article Snippet: A human CD73 ELISA kit (R&D Systems) and ELISA ancillary reagent kit (R&D Systems) were used to measure the CD73 level in sEVs from the serum.

Techniques: Concentration Assay, Enzyme-linked Immunosorbent Assay, Isolation, Injection, Control, Flow Cytometry, MANN-WHITNEY

Journal: Journal of Extracellular Vesicles

Article Title: CD73 in small extracellular vesicles derived from HNSCC defines tumour‐associated immunosuppression mediated by macrophages in the microenvironment

doi: 10.1002/jev2.12218

Figure Lengend Snippet: Relationship between sEVs CD73 in serum and clinicopathologic features ( n = 54)

Article Snippet: A human CD73 ELISA kit (R&D Systems) and ELISA ancillary reagent kit (R&D Systems) were used to measure the CD73 level in sEVs from the serum.

Techniques: Expressing, Significance Assay