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

Journal: Molecular Metabolism

Article Title: CD73 promotes the immunoregulatory functions of hepatic Tregs through enzymatic and nonenzymatic pathways in MASLD development

doi: 10.1016/j.molmet.2025.102131

Figure Lengend Snippet: CD73 expression on Tregs is increased during MASLD progression . (A–B). The mRNA levels of Nt5e in the livers of the MCD- or CDHFD-fed mice were determined by real-time PCR. (C–D). The Nt5e mRNA levels in hepatocytes or liver MNCs of the MCD- or CDHFD-fed mice were measured by real-time PCR. (E). CD73 expressions on liver CD4 + T, CD8 + T, NK cells, neutrophils, monocytes, and Kupffer cells were determined in NCD- and CDHFD-fed mice. (F). CD73 expressions on blood CD4 + T, CD8 + T, NK, and NKT cells were measured in NCD- and CDHFD-fed mice. (G). CD73 expressions on liver CD4 + T, CD8 + T, NK cells, neutrophils, monocytes, and Kupffer cells were determined in the NCD- and MCD-fed mice. (H). CD73 expressions on blood CD4 + T, CD8 + T, NK, and NKT cells were measured in the MCD-fed mice. (I). Representative flow cytometry plots of CD73 expression on Tregs (CD4 + CD25 + CD127 - ) in the livers or blood of the CDHFD-fed mice. (J–K). Statistical analysis of the percentages of CD73 + Tregs in the liver or blood of the CDHFD- and MCD-fed mice. (L). CD73 concentration in plasma was determined. (M). The ratio of CD73 + Tregs in PBMCs (left) and the concentration of soluble CD73 in plasma (right) from the healthy controls or MASLD patients. n = 5–21 per group. ∗ P < 0.05, ∗∗ P < 0.01.

Article Snippet: FFAs with or without 100 ng/ml recombinant mouse TRAIL protein (1121-TL; R&D) were added to Tregs for 48 h. Tregs were also incubated with 500 ng/ml of recombinant mouse CD73 protein (4488-EN; R&D) or control IgG for 6 h, followed by treatment with 200 μM FFAs and 100 ng/ml of recombinant mouse TRAIL protein for another 48 h. ATP (A6419; Sigma–Aldrich), AMP (A9396; Sigma–Aldrich), or adenosine (ADO) (A4036; Sigma–Aldrich) was separately added to Tregs from WT or Cd73 KO mice for 24 h. For the experiments with inhibitors, Tregs were separately incubated with 10 μM of the p38 inhibitor SB203580 (HY-10256; MCE), 2.5 μM of the AKT inhibitor MK2206 (HY-10358; MCE), 10 μM of the ERK1/2 inhibitor PD98059 (HY-12028; MCE), or 15 μM of the GATA2 inhibitor K-7174 (HY-12743; MCE) for 1 h and then stimulated with 200 μM FFAs for another 48 h.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Flow Cytometry, Concentration Assay

Journal: Molecular Metabolism

Article Title: CD73 promotes the immunoregulatory functions of hepatic Tregs through enzymatic and nonenzymatic pathways in MASLD development

doi: 10.1016/j.molmet.2025.102131

Figure Lengend Snippet: Cd73 knockout decreases intrahepatic Treg survival and immunomodulatory activity in CDHFD-induced MASLD model. (A). Body weight of the NCD- or CDHFD-fed WT or Cd73 knockout mice. (B). Plasma ALT, AST, and TBIL levels were determined. (C). Fasting plasma glucose levels were determined in the NCD- or CDHFD-fed mice. (D). Representative H&E, Oil Red O, and α-SMA staining and quantification of liver histology staining. (E). The relative mRNA expressions of proinflammatory cytokines ( Ifng , Il17 , and Tnfa ) in the liver were measured. (F). Fibrosis-related genes ( Col3a1 , Col1a1 , and Acta2 ) were assessed by real-time PCR. (G). Hydroxyproline levels in liver tissues from the NCD- or CDHFD-fed mice. (H–I). Ratio of Tregs among liver CD4 + T cells and CD45 + cells. (J). Apoptosis (Annexin V, Caspase-3 activity) and proliferation (Ki67, BCL-2) of liver Tregs were detected by flow cytometry. (K). Representative flow cytometry plots and statistical analysis of Granzyme Bpositive Tregs in the liver. (L–N). Proportion of CTLA4 + , IL-10 + , and CD69 + Tregs in each group of mice. n = 5 per group. ∗ P < 0.05, ∗∗ P < 0.01. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: FFAs with or without 100 ng/ml recombinant mouse TRAIL protein (1121-TL; R&D) were added to Tregs for 48 h. Tregs were also incubated with 500 ng/ml of recombinant mouse CD73 protein (4488-EN; R&D) or control IgG for 6 h, followed by treatment with 200 μM FFAs and 100 ng/ml of recombinant mouse TRAIL protein for another 48 h. ATP (A6419; Sigma–Aldrich), AMP (A9396; Sigma–Aldrich), or adenosine (ADO) (A4036; Sigma–Aldrich) was separately added to Tregs from WT or Cd73 KO mice for 24 h. For the experiments with inhibitors, Tregs were separately incubated with 10 μM of the p38 inhibitor SB203580 (HY-10256; MCE), 2.5 μM of the AKT inhibitor MK2206 (HY-10358; MCE), 10 μM of the ERK1/2 inhibitor PD98059 (HY-12028; MCE), or 15 μM of the GATA2 inhibitor K-7174 (HY-12743; MCE) for 1 h and then stimulated with 200 μM FFAs for another 48 h.

Techniques: Knock-Out, Activity Assay, Staining, Real-time Polymerase Chain Reaction, Flow Cytometry

Journal: Molecular Metabolism

Article Title: CD73 promotes the immunoregulatory functions of hepatic Tregs through enzymatic and nonenzymatic pathways in MASLD development

doi: 10.1016/j.molmet.2025.102131

Figure Lengend Snippet: CD73 maintains Treg survival and immunomodulatory activity, protecting against MASLD progression. (A). The Rag1 −/− mice were adoptively transferred with WT mouse-derived CD3 + T cells (W/WT Treg group) or CD3 + T cells without Tregs (W/O Treg group). In the W/ Cd73 KO Treg group, Rag1 −/− mice received CD3 + T cells without Tregs (from WT mice) together with Cd73 KO Tregs (from Cd73 KO mice). The above recipient mice and control Rag1 −/− mice not receiving cell transfer were subsequently fed MCD for 4 weeks. Created in https://BioRender.com . (B–C). Plasma ALT and AST levels were determined. (D). H&E and Oil Red O staining of representative paraffin-embedded liver sections. (E). Quantification of liver H&E and Oil Red O staining. (F). The body weight of mice at a different time. (G). The cytokine levels in plasma were measured by ELISA. (H) Ratio of CD4 + FOXP3 + Tregs in liver CD4 + T cells. (I, J) Ratio of Annexin V + , Tim-3 + , CTLA4 + Tregs in the livers of transplanted Rag1 −/− mice. (K). Ratio of monocytes (CD11b high F4/80 low ) in liver CD45 + Ly6G − cells. (L). The level of TNF-α secreted by monocytes was detected. (M). Ratio of Th1 (IFN-γ + ) or Th17 (IL-17 + ) in CD4 + T cells. n = 3–5 per group. ∗ P < 0.05, ∗∗ P < 0.01. $: A comparison between group Rag1 KO and W/O Treg with P < 0.05; $$: A comparison between group Rag1 KO and W/O Treg with P < 0.01; #: A comparison between group W/O Treg and W/WT Treg with P < 0.05; ##: A comparison between group W/O Treg and W/WT Treg with P < 0.01; &: A comparison between group W/WT Treg and W/ Cd73 KO Treg with P < 0.05. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: FFAs with or without 100 ng/ml recombinant mouse TRAIL protein (1121-TL; R&D) were added to Tregs for 48 h. Tregs were also incubated with 500 ng/ml of recombinant mouse CD73 protein (4488-EN; R&D) or control IgG for 6 h, followed by treatment with 200 μM FFAs and 100 ng/ml of recombinant mouse TRAIL protein for another 48 h. ATP (A6419; Sigma–Aldrich), AMP (A9396; Sigma–Aldrich), or adenosine (ADO) (A4036; Sigma–Aldrich) was separately added to Tregs from WT or Cd73 KO mice for 24 h. For the experiments with inhibitors, Tregs were separately incubated with 10 μM of the p38 inhibitor SB203580 (HY-10256; MCE), 2.5 μM of the AKT inhibitor MK2206 (HY-10358; MCE), 10 μM of the ERK1/2 inhibitor PD98059 (HY-12028; MCE), or 15 μM of the GATA2 inhibitor K-7174 (HY-12743; MCE) for 1 h and then stimulated with 200 μM FFAs for another 48 h.

Techniques: Activity Assay, Derivative Assay, Control, Staining, Enzyme-linked Immunosorbent Assay, Comparison

Journal: Molecular Metabolism

Article Title: CD73 promotes the immunoregulatory functions of hepatic Tregs through enzymatic and nonenzymatic pathways in MASLD development

doi: 10.1016/j.molmet.2025.102131

Figure Lengend Snippet: FFAs increase CD73 expression on Tregs via p38/Gata2 signaling pathway. (A). GSEA of Tregs from MCD-versus NCD-fed mice was performed. (B) After the addition of FFAs for 48 h, the expression of CD73 on Tregs was measured by flow cytometry. (C) Phosphorylated AKT activity in the Tregs after FFAs treatment. (D). MAPK signaling was detected in FFAs-stimulated Tregs by flow cytometry. (E). Tregs isolated from WT mice were preincubated with p38 inhibitor (SB203580), AKT inhibitor (MK2206), or ERK1/2 inhibitor (PD98059) for 1 h followed by FFAs treatment for 48 h, and the CD73 expression was detected by flow cytometry. (F). Transcription factor-binding sites of the Nt5e promoter region were predicted through the JASPAR CORE database and intersected with DEGs of Tregs from MCD- versus NCD-fed mice. (G). Relative mRNA levels of transcription factors ( Runx2, Gata2, Sox9 , and Tcf4 ) were determined by real-time PCR. (H–I). Runx2 and Gata2 expression was detected in Tregs incubated with p38 inhibitor (SB203580) followed by FFAs stimulation. (J). CUT&Tag assays of GATA2 binding sites showed a peak distribution around the Nt5e promoter region in the FFAs-treated Tregs. (K). The enrichment of GATA2 binding sites in the Nt5e promoter region was confirmed by real-time PCR, and the data were normalized to those of IgG. (L). CD73 expression on Tregs with pre-treatment of Gata2 inhibitor (K7174) and subsequent FFA stimulation. n = 4–5 per group. ∗ P < 0.05, ∗∗ P < 0.01.

Article Snippet: FFAs with or without 100 ng/ml recombinant mouse TRAIL protein (1121-TL; R&D) were added to Tregs for 48 h. Tregs were also incubated with 500 ng/ml of recombinant mouse CD73 protein (4488-EN; R&D) or control IgG for 6 h, followed by treatment with 200 μM FFAs and 100 ng/ml of recombinant mouse TRAIL protein for another 48 h. ATP (A6419; Sigma–Aldrich), AMP (A9396; Sigma–Aldrich), or adenosine (ADO) (A4036; Sigma–Aldrich) was separately added to Tregs from WT or Cd73 KO mice for 24 h. For the experiments with inhibitors, Tregs were separately incubated with 10 μM of the p38 inhibitor SB203580 (HY-10256; MCE), 2.5 μM of the AKT inhibitor MK2206 (HY-10358; MCE), 10 μM of the ERK1/2 inhibitor PD98059 (HY-12028; MCE), or 15 μM of the GATA2 inhibitor K-7174 (HY-12743; MCE) for 1 h and then stimulated with 200 μM FFAs for another 48 h.

Techniques: Expressing, Flow Cytometry, Activity Assay, Isolation, Binding Assay, Real-time Polymerase Chain Reaction, Incubation

Journal: Molecular Metabolism

Article Title: CD73 promotes the immunoregulatory functions of hepatic Tregs through enzymatic and nonenzymatic pathways in MASLD development

doi: 10.1016/j.molmet.2025.102131

Figure Lengend Snippet: CD73 protects Tregs from AMP-induced toxicity, while the degradation product adenosine promotes Treg survival and function in the presence of FFAs. (A–C). The plasma concentrations of ATP, AMP, and adenosine (ADO) were measured in the CDHFD- or MCD-fed WT and Cd73 KO mice, as well as the MCD-fed Treg-transplanted Rag1 −/− mice. (D) Relative AMP and ADO levels in the supernatant after the addition of ATP to cultures of WT or Cd73 KO Tregs. (E). Apoptosis of CD4 + T cells without Tregs was detected after ADO treatment with or without FFAs treatment. (F). Apoptosis and TNF-α secretion were measured in ADO-stimulated monocytes with or without FFAs treatment. (G). Apoptosis of ADO-stimulated Tregs was detected by flow cytometry. (H). The expression of immunomodulatory molecules (CTLA4, Perforin, IL-10, Granzyme B, and TGF-β) on WT Tregs were detected after ADO stimulation with or without FFAs treatment. (I–K). Treg apoptosis (Annexin V and 7AAD) and IL-10 expression were compared between WT and Cd73 KO mice after AMP supplement with or without FFAs treatment. (L) The relative mRNA expression of Bcl2, Ctla4, Gzmb, Prf1, Il10 , and Tgfb in Tregs from WT and Cd73 KO mice was determined after AMP supplement with or without FFAs treatment. n = 4–5 per group. ∗ P < 0.05, ∗∗ P < 0.01.

Article Snippet: FFAs with or without 100 ng/ml recombinant mouse TRAIL protein (1121-TL; R&D) were added to Tregs for 48 h. Tregs were also incubated with 500 ng/ml of recombinant mouse CD73 protein (4488-EN; R&D) or control IgG for 6 h, followed by treatment with 200 μM FFAs and 100 ng/ml of recombinant mouse TRAIL protein for another 48 h. ATP (A6419; Sigma–Aldrich), AMP (A9396; Sigma–Aldrich), or adenosine (ADO) (A4036; Sigma–Aldrich) was separately added to Tregs from WT or Cd73 KO mice for 24 h. For the experiments with inhibitors, Tregs were separately incubated with 10 μM of the p38 inhibitor SB203580 (HY-10256; MCE), 2.5 μM of the AKT inhibitor MK2206 (HY-10358; MCE), 10 μM of the ERK1/2 inhibitor PD98059 (HY-12028; MCE), or 15 μM of the GATA2 inhibitor K-7174 (HY-12743; MCE) for 1 h and then stimulated with 200 μM FFAs for another 48 h.

Techniques: Flow Cytometry, Expressing

Journal: Molecular Metabolism

Article Title: CD73 promotes the immunoregulatory functions of hepatic Tregs through enzymatic and nonenzymatic pathways in MASLD development

doi: 10.1016/j.molmet.2025.102131

Figure Lengend Snippet: CD73 inhibits Treg apoptosis by suppressing the TRAIL-DR5 interaction. (A). The relative mRNA expression of Tnfrsf10b (DR5) and Tnfsf10 (TRAIL) in the liver was measured by real-time PCR. (B). Plasma content of TRAIL in the CDHFD- or NCD-fed mice. (C). DR5 expression on liver Tregs. (D). Relative apoptotic levels were compared between CD4 + T cells without Tregs and Tregs followed by FFAs treatment with or without TRAIL. (E). Relative apoptotic levels were compared among CD73 high , CD73 int and CD73 neg Tregs after FFAs treatment with or without TRAIL. (F–G). Relative Annexin V and Caspase-3 activity were measured in the WT and Cd73 KO Tregs treated with FFAs with or without TRAIL. (H). Tregs were blocked with CD73 protein or IgG for 6 h, followed by the addition of FFAs and TRAIL for 48 h. The proportions of Annexin V + Tregs after TRAIL supplement. (I). CD73 on Tregs limits inflammation and maintains homeostasis during MASLD progression (Created in https://BioRender.com ). n = 3–6 per group. ∗ P < 0.05, ∗∗ P < 0.01.

Article Snippet: FFAs with or without 100 ng/ml recombinant mouse TRAIL protein (1121-TL; R&D) were added to Tregs for 48 h. Tregs were also incubated with 500 ng/ml of recombinant mouse CD73 protein (4488-EN; R&D) or control IgG for 6 h, followed by treatment with 200 μM FFAs and 100 ng/ml of recombinant mouse TRAIL protein for another 48 h. ATP (A6419; Sigma–Aldrich), AMP (A9396; Sigma–Aldrich), or adenosine (ADO) (A4036; Sigma–Aldrich) was separately added to Tregs from WT or Cd73 KO mice for 24 h. For the experiments with inhibitors, Tregs were separately incubated with 10 μM of the p38 inhibitor SB203580 (HY-10256; MCE), 2.5 μM of the AKT inhibitor MK2206 (HY-10358; MCE), 10 μM of the ERK1/2 inhibitor PD98059 (HY-12028; MCE), or 15 μM of the GATA2 inhibitor K-7174 (HY-12743; MCE) for 1 h and then stimulated with 200 μM FFAs for another 48 h.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Activity Assay

Journal: Advanced Science

Article Title: Tumor Microenvironment Responsive CD8 + T Cells and Myeloid‐Derived Suppressor Cells to Trigger CD73 Inhibitor AB680‐Based Synergistic Therapy for Pancreatic Cancer

doi: 10.1002/advs.202302498

Figure Lengend Snippet: Human CD73 levels are upregulated in pancreatic cancer cells and correlate with survival. A) The distribution of defined cell clusters comparing three human adjacent/normal pancreas and 16 treatment‐naive PDAC tumors at the time of sample acquisition was shown by UMAP plots in the GSE155698 dataset. B) Dot plot of NT5E expression, the gene encoding CD73, in all identified cell clusters in the GSE155698 dataset C) the distribution of defined cell clusters comparing 11 human adjacent/normal pancreas and 24 treatment‐naive PDAC tumors at the time of sample acquisition was shown by UMAP plots in the CRA001160 dataset. D) Dot plot of NT5E expression, the gene encoding CD73, in all identified cell clusters in the CRA001160 dataset. E) Representative images of IHC staining of CD73 in human adjacent/normal pancreas and PDAC tumors. Scale bar: 500 µm. F) Quantification and statistical analysis of the above IHC staining of CD73 in 17 human adjacent/normal pancreas and 29 PDAC tumors without any treatment. CD73 IHC scores were compared between the two groups using the Mann–Whitney test. *** p < 0.001 G) Correlation analysis of tumor‐infiltrating CD8 + T cell and CD73 IHC scores via Spearman correlation coefficients. H,I) Kaplan–Meier curves showing overall survival (H) and recurrence‐free survival (I) of patients with 29 PDAC stratified by CD73 IHC scores. p ‐values are from log‐rank tests. J) Comparison of CD73 protein levels between 75 normal pancreas and 140 treatment‐naive PDAC tumors in the CTPAC dataset via Student's t ‐test. *** p < 0.001. Data presented as mean ± SD. K) Kaplan–Meier analysis for overall survival in 140 PDAC patients according to CD73 protein level via the log‐rank test.

Article Snippet: Formalin‐fixed, paraffin‐embedded sections of humans and mice were processed and incubated with primary antibodies: CD73 (Proteintech, 12231‐1‐AP), CK19 (Proteintech, 10712‐1‐AP), α‐SMA (Boster, BM0002), CD8 (Abcam, ab209775), granzyme B (Abcam, ab4059), CXCR2 (Proteintech, 19538‐1‐AP), CXCL5 (R&D Systems, AF433), LY6G (Servicebio, GB11229), Ki‐67 (CST, 9129), and cleaved caspase 3 (CST, 9664), followed by biotinylated or fluorescent secondary antibodies.

Techniques: Expressing, Immunohistochemistry, MANN-WHITNEY, Comparison

Journal: Advanced Science

Article Title: Tumor Microenvironment Responsive CD8 + T Cells and Myeloid‐Derived Suppressor Cells to Trigger CD73 Inhibitor AB680‐Based Synergistic Therapy for Pancreatic Cancer

doi: 10.1002/advs.202302498

Figure Lengend Snippet: Overexpression of CD73 in murine tumor cells contributes to tumor growth in vivo. A) Representative images of immunohistochemistry staining of CD73 in murine adjacent/normal pancreas and orthotopic allografts of a KPC cell line. Scale bar: 200 µm. B) UMAP visualization of six identified cell populations and dot plot of Nt5e and Entpd1 expression in these populations in the single‐cell RNA‐sequencing dataset GSE158356 of two orthotopic allografts of a KPC cell line. C) Western blot analysis for CD73 in CD73 overexpressing (OE) and control KPC cells. D–F) Image (D), volumes (E), and weights (F) of the CD73 OE versus Control KPC tumors ( n = 6 samples per group). Tumor volumes and weights were compared between Control and OE groups. Values are means ± SD, * p < 0.05, ** p < 0.01 in a two‐sample t ‐test. G) Quantification and comparison of immunohistochemistry result of CD8 + T cells in the CD73 OE versus Control KPC tumors ( n = 6 samples per group). * p < 0.05, ** p < 0.01 in Student's t ‐test. Data presented as mean ± SD.

Article Snippet: Formalin‐fixed, paraffin‐embedded sections of humans and mice were processed and incubated with primary antibodies: CD73 (Proteintech, 12231‐1‐AP), CK19 (Proteintech, 10712‐1‐AP), α‐SMA (Boster, BM0002), CD8 (Abcam, ab209775), granzyme B (Abcam, ab4059), CXCR2 (Proteintech, 19538‐1‐AP), CXCL5 (R&D Systems, AF433), LY6G (Servicebio, GB11229), Ki‐67 (CST, 9129), and cleaved caspase 3 (CST, 9664), followed by biotinylated or fluorescent secondary antibodies.

Techniques: Over Expression, In Vivo, Immunohistochemistry, Staining, Expressing, RNA Sequencing, Western Blot, Control, Comparison

Journal: Advanced Science

Article Title: Tumor Microenvironment Responsive CD8 + T Cells and Myeloid‐Derived Suppressor Cells to Trigger CD73 Inhibitor AB680‐Based Synergistic Therapy for Pancreatic Cancer

doi: 10.1002/advs.202302498

Figure Lengend Snippet: CD73 inhibition reduces the tumor burden and prolongs survival. A) Schematic showing the schedule of CD73 inhibitor AB680 treatment in KPC subcutaneous transplantation model. B–D) Image (B), volumes (C), and weights (D) of the AB680‐treated versus Control KPC subcutaneous allografts ( n = 6 samples per group). Data presented as mean ± SD, * p < 0.05, ** p < 0.01 in Student's t ‐test. E) Schematic showing the schedule of AB680 treatment in KPC orthotopic transplantation model. F,G) Image (F) and weights (G) of the AB680‐treated versus Control KPC orthotopic allografts ( n = 6 samples per group). Data presented as mean ± SD, * p < 0.05 in Student's t ‐test. H) Kaplan–Meier survival analysis of tumor‐bearing mice untreated or treated with 20 mg k −1 g AB680 via the log‐rank test ( n = 8 samples per group). I) Representative images of H&E staining, Masson's trichrome staining (MTS), and immunohistochemistry staining of CD8, Granzyme B, α‐SMA. Scale bar: 200 µm. J–M) Quantification of CD8 (J), Granzyme B (K), α‐SMA (L), and MTS (M) % positive area in the AB680‐treated versus Control KPC orthotopic allografts ( n = 6 samples per group). Data presented as mean ± SD, * p < 0.05, ** p < 0.01 in Student's t ‐test.

Article Snippet: Formalin‐fixed, paraffin‐embedded sections of humans and mice were processed and incubated with primary antibodies: CD73 (Proteintech, 12231‐1‐AP), CK19 (Proteintech, 10712‐1‐AP), α‐SMA (Boster, BM0002), CD8 (Abcam, ab209775), granzyme B (Abcam, ab4059), CXCR2 (Proteintech, 19538‐1‐AP), CXCL5 (R&D Systems, AF433), LY6G (Servicebio, GB11229), Ki‐67 (CST, 9129), and cleaved caspase 3 (CST, 9664), followed by biotinylated or fluorescent secondary antibodies.

Techniques: Inhibition, Transplantation Assay, Control, Staining, Immunohistochemistry

Journal: Advanced Science

Article Title: Tumor Microenvironment Responsive CD8 + T Cells and Myeloid‐Derived Suppressor Cells to Trigger CD73 Inhibitor AB680‐Based Synergistic Therapy for Pancreatic Cancer

doi: 10.1002/advs.202302498

Figure Lengend Snippet: AB680 regulates CXCL5 released by tumor cells and promotes MDSC accumulation. A) Heatmap of immune‐related differential genes in the RNA‐seq data of control and AB680‐treated tumors ( n = 3 samples per group). B,C) Over‐Representation Analysis B) and Gene Set Enrichment Analysis (C) of the above RNA‐seq data. D) Heatmap of 23 different mouse cytokine expression levels in serums obtained at the time of tumor collection, as shown in Figure using Bio‐Plex Pro Mouse Cytokine 23‐plex immunoassay ( n = 4 samples per group). E) The protein level of CXCL5 in mouse serums from the indicated treatment groups. Data presented as mean ± SD, ** p < 0.01 in Student's t ‐test. F) Dot plot of Cxcl5 expression in the identified populations in the single‐cell RNA‐sequencing dataset GSE158356 of two orthotopic KPC allografts. G,H) Quantification of CXCL5 and CXCR2% positive area in the AB680‐treated versus Control KPC tumor at the endpoint ( n = 6 samples per group). ** p < 0.01, *** p < 0.001 in the Student's t ‐test. Data presented as mean ± SD. I) Representative images of immunohistochemistry staining of CXCL5 and CXCR2 in the KPC tumors from the indicated treatment groups. J,K) The level of adenosine and CXCL5 protein in the AB680‐treated versus Control KPC tumor at the endpoint ( n = 6 samples per group). ** p < 0.01, *** p < 0.001 in Student's t ‐test. Data presented as mean ± SD. L) Schematic representation of the changes in adenosine metabolism after inhibition of CD73 activity by AB680. M) qRT‐PCR analysis of Cxcl5 mRNA expression in the KPC cells with different concentrations of AMP, adenosine, and the adenosine receptor agonist NECA at 0, 10, 20, 50, and 100 µ m ( n = 3). Data presented as mean ± SD. N) The CXCL5 protein concentration in the culture supernatant was evaluated by ELISA in the KPC cells with indicated concentrations of drugs ( n = 3). Data presented as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001 in Student's t ‐test.

Article Snippet: Formalin‐fixed, paraffin‐embedded sections of humans and mice were processed and incubated with primary antibodies: CD73 (Proteintech, 12231‐1‐AP), CK19 (Proteintech, 10712‐1‐AP), α‐SMA (Boster, BM0002), CD8 (Abcam, ab209775), granzyme B (Abcam, ab4059), CXCR2 (Proteintech, 19538‐1‐AP), CXCL5 (R&D Systems, AF433), LY6G (Servicebio, GB11229), Ki‐67 (CST, 9129), and cleaved caspase 3 (CST, 9664), followed by biotinylated or fluorescent secondary antibodies.

Techniques: RNA Sequencing, Control, Expressing, Immunohistochemistry, Staining, Inhibition, Activity Assay, Quantitative RT-PCR, Protein Concentration, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Monitoring and characterizing soluble and membrane-bound ectonucleotidases CD73 and CD39

doi: 10.1101/698373

Figure Lengend Snippet: A. Activity determination of recombinant human 5’-Nucleotidase/CD73 using increasing concentrations of enzyme and 10µM AMP substrate. The reaction was carried out at 23°C for 30min using AMP-Glo Assay System as described in Materials and Methods section. Activity of CD73 is monitored by how much AMP has been consumed in (A), i.e., RLU corresponds to the amount of AMP remaining and thus the activity of the enzyme is reciprocally correlated with RLU (see Schematic 1A); (B) Net RLU after subtracting the control (no-enzyme) from the RLU values at each point of enzyme concentration. The experiment was done in triplicates; results shown are mean ± SD. SD, standard deviation

Article Snippet: Active CD73 enzyme (Recombinant Human 5’-Nucleotidase/CD73 Protein, CF) and active CD39 (His-Tag Human) were from R&D Systems, Minneapolis, MN).

Techniques: Activity Assay, Recombinant, Glo Assay, Concentration Assay, Standard Deviation

Journal: bioRxiv

Article Title: Monitoring and characterizing soluble and membrane-bound ectonucleotidases CD73 and CD39

doi: 10.1101/698373

Figure Lengend Snippet: Time course study using recombinant human 5’-Nucleotidase CD73 protein titration and 25µM AMP for 5,10, and 20 minutes reaction time at 23°C. Activity was determined using AMP-Glo assay. Data are shown as net RLU after subtracting values for no enzyme control from the RLUs values at each point of enzyme concentration. EC 50 represents the amount of enzyme required for 50% maximal activity. Each point represents the average of triplicates; error bars represent SD

Article Snippet: Active CD73 enzyme (Recombinant Human 5’-Nucleotidase/CD73 Protein, CF) and active CD39 (His-Tag Human) were from R&D Systems, Minneapolis, MN).

Techniques: Recombinant, Titration, Activity Assay, Glo Assay, Concentration Assay

Journal: bioRxiv

Article Title: Monitoring and characterizing soluble and membrane-bound ectonucleotidases CD73 and CD39

doi: 10.1101/698373

Figure Lengend Snippet: Determination of Km value for AMP using 0.01ng recombinant human 5’-Nucleotidase/CD73 protein per reaction and varying AMP concentrations for 5min reaction at 23°C followed by AMP-Glo assay protocol. Data shown as net RLU vs. AMP concentration. The experiment was done in triplicates; results shown are mean ± SD.

Article Snippet: Active CD73 enzyme (Recombinant Human 5’-Nucleotidase/CD73 Protein, CF) and active CD39 (His-Tag Human) were from R&D Systems, Minneapolis, MN).

Techniques: Recombinant, Glo Assay, Concentration Assay

Journal: bioRxiv

Article Title: Monitoring and characterizing soluble and membrane-bound ectonucleotidases CD73 and CD39

doi: 10.1101/698373

Figure Lengend Snippet: Reactions contained 0.1 ng of recombinant human 5’-Nucleotidase/CD73 protein and either 5µM or 10µM AMP and different concentrations of inhibitor (AMP-CP). Reactions were carried out for 5min at 23°C and activity was monitored using AMP Glo assay. Experiments were done in triplicates, and results are shown as mean ± SD.

Article Snippet: Active CD73 enzyme (Recombinant Human 5’-Nucleotidase/CD73 Protein, CF) and active CD39 (His-Tag Human) were from R&D Systems, Minneapolis, MN).

Techniques: Recombinant, Activity Assay, Glo Assay

Journal: bioRxiv

Article Title: Monitoring and characterizing soluble and membrane-bound ectonucleotidases CD73 and CD39

doi: 10.1101/698373

Figure Lengend Snippet: Purified CD73 (0.1 ng/Rx) and cN-II (0.6 mU/Rx) were tested for inhibition by various concentrations of AMP-PC using 10µM AMP substrate at 23°C for 5 min (CD73 and 30 min (cN-II). Activities were determined following AMP-Glo protocol. The inhibitor inhibited CD73 activity with IC50 value of 3×10 -7 M with minimal or no inhibition of cN-II. Experiments were done in triplicates; results shown are mean ± SD

Article Snippet: Active CD73 enzyme (Recombinant Human 5’-Nucleotidase/CD73 Protein, CF) and active CD39 (His-Tag Human) were from R&D Systems, Minneapolis, MN).

Techniques: Purification, Inhibition, Activity Assay

Journal: bioRxiv

Article Title: Monitoring and characterizing soluble and membrane-bound ectonucleotidases CD73 and CD39

doi: 10.1101/698373

Figure Lengend Snippet: Five cell lines were evaluated for their CD73 enzyme activity in intact cells using AMP Glo protocol for cells as described in the method section. (A) Time course study of enzyme activity of five cell lines with 10 μM AMP using AMP-Glo for cell bound CD73. T-47D (circle), MDA-MB-231(square), SK-MEL2 (triangle), A375 (reverse triangle), and SK-OV3 (diamond). (B) Determination of abundance of CD73 in membranes of five cell lines using western blotting. Cell lysates (10µg) from each cell line and pure CD73 (0.2µg) as positive control (lane 7) were run on gels and immunoblotted using primary antibodies (anti- NT5E/CD73, Cell Signaling), incubated overnight at 4°C followed by HRP-ECL as described in the method section

Article Snippet: Active CD73 enzyme (Recombinant Human 5’-Nucleotidase/CD73 Protein, CF) and active CD39 (His-Tag Human) were from R&D Systems, Minneapolis, MN).

Techniques: Activity Assay, Western Blot, Positive Control, Incubation

Journal: bioRxiv

Article Title: Monitoring and characterizing soluble and membrane-bound ectonucleotidases CD73 and CD39

doi: 10.1101/698373

Figure Lengend Snippet: Membrane associated CD73 bound MDA-MB-231 activity was determined in presence (open circle) and absence (closed circle) of 50µM AMP-PC. Reactions contained 25k MDA-MB-231 cell per well and incubated at 37°C with 10µM AMP in final 250µl per well. Aliquots of 25µl per sample were withdrawn at each time point and activity was determined following AMP-Glo assay. Each point represents the average of triplicates; the error bars represent the SD.

Article Snippet: Active CD73 enzyme (Recombinant Human 5’-Nucleotidase/CD73 Protein, CF) and active CD39 (His-Tag Human) were from R&D Systems, Minneapolis, MN).

Techniques: Membrane, Activity Assay, Incubation, Glo Assay

Journal: bioRxiv

Article Title: Monitoring and characterizing soluble and membrane-bound ectonucleotidases CD73 and CD39

doi: 10.1101/698373

Figure Lengend Snippet: Three compounds with inhibitory activity against CD73 (AMP-PC, solid circle) and against CD39 (ARL 67156 and POM 1, square and triangle, respectively) were tested using purified CD73 and MDA-MB-231 bound CD73. (A) Purified CD73, 0.1ng per reaction with 1µM AMP for 5 min reaction at 23°C, and (B) cell-based MDA-MB-231, 25K cells per well with 5µM AMP for 90min at 37°C. Each point represents the average of triplicates; the error bars represent the SD

Article Snippet: Active CD73 enzyme (Recombinant Human 5’-Nucleotidase/CD73 Protein, CF) and active CD39 (His-Tag Human) were from R&D Systems, Minneapolis, MN).

Techniques: Activity Assay, Purification

Journal: bioRxiv

Article Title: Monitoring and characterizing soluble and membrane-bound ectonucleotidases CD73 and CD39

doi: 10.1101/698373

Figure Lengend Snippet: Commercially available antibodies against CD73 were tested using cell-based membrane-associated CD73 from MDA-MB-231 following AMP-Glo cell-based assay. (A) Using 9 different commercially available antibodies against CD73 with an antibody against CD39 as a negative control. Antibodies were applied at 10µg/ml per well and incubated with MDA-MB-231 cells for 1, 3, 6, and 16 hrs. Activities are expressed as percentage of control (cells without antibodies). (B) Effect of antibodies concentration of the most effective antibodies (Ab4 and Ab5) and the control CD39 selective antibodies (Ab10) on the activity of membrane bound CD-73. Activities are expressed as percentage of control (cells without antibodies). All antibodies were tested using 25K cells per well cells and incubated at 37°C. Activities were determined in the presence of 10µM AMP substrate for 30min at 37°C. Each point represents the average of triplicates; the error bars represent the SD

Article Snippet: Active CD73 enzyme (Recombinant Human 5’-Nucleotidase/CD73 Protein, CF) and active CD39 (His-Tag Human) were from R&D Systems, Minneapolis, MN).

Techniques: Membrane, Cell Based Assay, Negative Control, Incubation, Concentration Assay, Activity Assay

Journal: bioRxiv

Article Title: Monitoring and characterizing soluble and membrane-bound ectonucleotidases CD73 and CD39

doi: 10.1101/698373

Figure Lengend Snippet: Inhibitors of CD39 and CD73 were incubated with purified CD39 (A, B) and Farage associated enzyme (C, D) and assayed for CD39 activity using ATP (A, C) or ADP (B, D) as substrate. Results show percentage inhibition of CD39 activity using ARL 67156 (circle), POM1 (solid square), and AMP-CP (triangle). Purified CD39 (0.1ng) and 10µM ATP or ADP in each reaction for 30min at 37°C; for cell-based Farage cells (200K cells) were incubated with 10µM ATP or ADP in each reaction for 20min at 37°C. Experiments were carried out in triplicates; results shown are mean ± SD

Article Snippet: Active CD73 enzyme (Recombinant Human 5’-Nucleotidase/CD73 Protein, CF) and active CD39 (His-Tag Human) were from R&D Systems, Minneapolis, MN).

Techniques: Incubation, Purification, Activity Assay, Inhibition

Journal: bioRxiv

Article Title: Monitoring and characterizing soluble and membrane-bound ectonucleotidases CD73 and CD39

doi: 10.1101/698373

Figure Lengend Snippet: Testing the specificity of CD39 inhibitor POM1 (A) and CD73 inhibitor AMP-CP (B) on the activity of different kinases, ATPase, as well as CD39 and CD73. Enzyme tested are protein serine/threonine/tyrosine, lipid, sugar, and inorganic kinases, and K/Na ATPase. Results show that the majority of enzymes are inhibited by POM1 (A), but the more selective CD73 inhibitor AMP-PC) was highly selective for CD73 with minimal inhibition for PKA and no inhibition of the other enzymes. PKA, protein kinase A (solid circle), PKC□ (solid square), Src, protein tyrosine Src kinase (solid triangle), Acka, acetate kinase from Escherichia coli (solid reverse triangle), HK, hexokinase from Saccharomyces cerevisiae (solid circle), PI3□ Kinase, p110□/p85□ (square), K/K ATPase, Adenosine 5’-Triphosphatase from porcine cerebral cortex (circle), CD39 (ATP substrate, triangle), and CD39 (ADP substrate, reverse triangle); for CD73 (triangle) in penal B. Each point represents average of a typical experiment done in triplicates; results shown are mean ± SD.

Article Snippet: Active CD73 enzyme (Recombinant Human 5’-Nucleotidase/CD73 Protein, CF) and active CD39 (His-Tag Human) were from R&D Systems, Minneapolis, MN).

Techniques: Activity Assay, Inhibition

Journal: Scientific Reports

Article Title: Plasma biomarkers for prediction of early tumor recurrence after resection of pancreatic ductal adenocarcinoma

doi: 10.1038/s41598-021-86779-x

Figure Lengend Snippet: Characterization of proteins in pancreatic cancer cell lines (PANC-1, MIA PaCa-2, Capan-2, HPAF-II) and pancreatic stellate cell line (PSC). ( A ) The mRNA levels were determined by qPCR for the genes MAEA, NT5E, AZU1, ATP6AP2, MICA, IFNLR1, CTSO, CDCP1 and TNFRSF12A. The expression level of each gene was normalized against β-actin. Data is presented as mean ± SEM (n = 3). ( B ) Expression of MAEA, NT5E, AZU1, ATP6AP2 and MICA was detected by Western blot analysis. Representative Western blots were selected from three independent experiments. PDI was included as a loading control. Full membrane blots are provided in Supplementary Figure S1. ( C ) Representative immunofluorescence images of PANC-1 cells stained for MAEA, NT5E, AZU1, ATP6AP2 and MICA (red). Nuclei are stained in blue with DAPI. ( D ) Protein concentrations of NT5E, AZU1, ATP6AP2 and MICA in cell culture supernatants were determined by sandwich ELISAs. For values outside the linear range, the value of the smallest standard as minimum or the value of the largest standard as maximum was used for presentation. The mean value ± SEM is given (n = 3 in duplicates). Statistical evaluation was performed by one-way ANOVA + Tukey’s post-hoc test): *p < 0.05; **p < 0.01; ***p < 0.001.

Article Snippet: Levels of respective proteins were quantified with Human MICA ELISA Kit (LS-Bio, LS-F150), Human ATP6AP2 ELISA Kit (Aviva Systems Biology, OKEH04445), Human NT5E ELISA Kit (Biorbyt, orb385324) and Human AZU1 ELISA Kit (Biorbyt, orb390828).

Techniques: Expressing, Western Blot, Control, Membrane, Immunofluorescence, Staining, Cell Culture

Journal: Scientific Reports

Article Title: Plasma biomarkers for prediction of early tumor recurrence after resection of pancreatic ductal adenocarcinoma

doi: 10.1038/s41598-021-86779-x

Figure Lengend Snippet: Gene expression analysis of patient-derived PDAC organoid cultures. The mRNA levels in seven organoid cultures (late recurrence: L1, L2, L3/early recurrence: E1, E2, E3, E4) were determined by qPCR for the genes MAEA, NT5E, AZU1, ATP6AP2, MICA, IFNLR1, CTSO, CDCP1 and TNFRSF12A. A ductal organoid line from a chronic pancreatitis patient (ctrl) served as control. The expression level of each gene was normalized against β-actin. Data is presented as mean ± SEM (n = 3), except AZU1 for E4, L2, L3 (n = 1). For each gene of interest, graphs on the left depict results for individual organoid lines, and graphs on the right a merged analysis including statistics. Statistical evaluation was performed using Student’s t-test: *p < 0.05; **p < 0.01; ***p < 0.001.

Article Snippet: Levels of respective proteins were quantified with Human MICA ELISA Kit (LS-Bio, LS-F150), Human ATP6AP2 ELISA Kit (Aviva Systems Biology, OKEH04445), Human NT5E ELISA Kit (Biorbyt, orb385324) and Human AZU1 ELISA Kit (Biorbyt, orb390828).

Techniques: Gene Expression, Derivative Assay, Control, Expressing

Journal: Scientific Reports

Article Title: Plasma biomarkers for prediction of early tumor recurrence after resection of pancreatic ductal adenocarcinoma

doi: 10.1038/s41598-021-86779-x

Figure Lengend Snippet: Immunohistochemistry staining of PDAC organoid cultures for MAEA, NT5E, AZU1, ATP6AP2 and MICA. The same organoid cultures as in Fig. were analyzed. Brown color represents a positive staining. Staining intensity was semi-quantitatively scored as: 0 (negative), 1 (weak), 2 (moderate), 3 (strong) and is shown in the bottom panels as stacked bar graphs. For representation we selected organoid culture L2 for late recurrence, E2 for early recurrence and the non-tumor chronic pancreatitis organoid line as control.

Article Snippet: Levels of respective proteins were quantified with Human MICA ELISA Kit (LS-Bio, LS-F150), Human ATP6AP2 ELISA Kit (Aviva Systems Biology, OKEH04445), Human NT5E ELISA Kit (Biorbyt, orb385324) and Human AZU1 ELISA Kit (Biorbyt, orb390828).

Techniques: Immunohistochemistry, Staining, Control

Journal: Scientific Reports

Article Title: Plasma biomarkers for prediction of early tumor recurrence after resection of pancreatic ductal adenocarcinoma

doi: 10.1038/s41598-021-86779-x

Figure Lengend Snippet: Summary of characteristics of potential biomarkers indicating early recurrence of PDAC following resection.

Article Snippet: Levels of respective proteins were quantified with Human MICA ELISA Kit (LS-Bio, LS-F150), Human ATP6AP2 ELISA Kit (Aviva Systems Biology, OKEH04445), Human NT5E ELISA Kit (Biorbyt, orb385324) and Human AZU1 ELISA Kit (Biorbyt, orb390828).

Techniques: Activation Assay, Expressing

Journal: Scientific Reports

Article Title: Plasma biomarkers for prediction of early tumor recurrence after resection of pancreatic ductal adenocarcinoma

doi: 10.1038/s41598-021-86779-x

Figure Lengend Snippet:

Article Snippet: Levels of respective proteins were quantified with Human MICA ELISA Kit (LS-Bio, LS-F150), Human ATP6AP2 ELISA Kit (Aviva Systems Biology, OKEH04445), Human NT5E ELISA Kit (Biorbyt, orb385324) and Human AZU1 ELISA Kit (Biorbyt, orb390828).

Techniques:

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: The experiments were divided into seven groups: M0, M2, M2+sEVs HNSCC , M2+sEVs HNSCC‐ NT5E KO , M2+sEVs BMSC , M2+sEVs BMSC‐ NT5E OE , and M2+sEVs BMSC‐ NT5E OE + Lat A (30 μM, MedChemExpress, Monmouth Junction, NJ, USA).

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: The experiments were divided into seven groups: M0, M2, M2+sEVs HNSCC , M2+sEVs HNSCC‐ NT5E KO , M2+sEVs BMSC , M2+sEVs BMSC‐ NT5E OE , and M2+sEVs BMSC‐ NT5E OE + Lat A (30 μM, MedChemExpress, Monmouth Junction, NJ, USA).

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: 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: The experiments were divided into seven groups: M0, M2, M2+sEVs HNSCC , M2+sEVs HNSCC‐ NT5E KO , M2+sEVs BMSC , M2+sEVs BMSC‐ NT5E OE , and M2+sEVs BMSC‐ NT5E OE + Lat A (30 μM, MedChemExpress, Monmouth Junction, NJ, USA).

Techniques: Derivative Assay, Over Expression, Flow Cytometry, Concentration Assay, Enzyme-linked Immunosorbent Assay, Control, Confocal Microscopy, Expressing, 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: The experiments were divided into seven groups: M0, M2, M2+sEVs HNSCC , M2+sEVs HNSCC‐ NT5E KO , M2+sEVs BMSC , M2+sEVs BMSC‐ NT5E OE , and M2+sEVs BMSC‐ NT5E OE + Lat A (30 μM, MedChemExpress, Monmouth Junction, NJ, USA).

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: The experiments were divided into seven groups: M0, M2, M2+sEVs HNSCC , M2+sEVs HNSCC‐ NT5E KO , M2+sEVs BMSC , M2+sEVs BMSC‐ NT5E OE , and M2+sEVs BMSC‐ NT5E OE + Lat A (30 μM, MedChemExpress, Monmouth Junction, NJ, USA).

Techniques: 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: The experiments were divided into seven groups: M0, M2, M2+sEVs HNSCC , M2+sEVs HNSCC‐ NT5E KO , M2+sEVs BMSC , M2+sEVs BMSC‐ NT5E OE , and M2+sEVs BMSC‐ NT5E OE + Lat A (30 μM, MedChemExpress, Monmouth Junction, NJ, USA).

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

Journal: Science Advances

Article Title: Monocytic MDSCs exhibit superior immune suppression via adenosine and depletion of adenosine improves efficacy of immunotherapy

doi: 10.1126/sciadv.adg3736

Figure Lengend Snippet: ( A to C ) CD14 + monocytes from healthy donor peripheral blood mononuclear cells (PBMCs) were cocultured with tumor cells using a Transwell system. Representative dot plots (A), bar graph (B), and histograms (C) showing the expression of CD73 (proportions and MFI) in A549 and A375 tumor cell–induced human M-MDSCs. Data, mean ± SEM of three independent experiments with monocytes from three different donors. ( D and E ) Immune suppressive activity of A549- and A375-induced M-MDSCs. Autologous CFSE-labeled T cells were cultured in the presence of monocytes cultured for 64 hours in the absence of tumor cells (cultured mono) or with monocytes cocultured with A549 or A375 cells (A549-MDSC and A375-MDSC). T cells were activated with anti-CD3/anti-CD28 beads in the absence or presence of the indicated monocytes/MDSCs for 4 days. Representative histograms (D) and bar graph (E) show the percentage of proliferated CFSE-labeled T cells. Data, mean ± SEM of three independent experiments. ( F and G ) Human metastatic lung tumor–associated CD14 + CD3 neg CD16 neg CD68 neg CD15 neg CD206 neg monocytes present in the tumors express higher levels of CD73 when compared to expression levels in the circulating monocytes. Data, mean ± SEM of n = 5 patients. Dot plot in (F) shows CD14 + cells ( x axis) versus CD3, CD16, CD68, CD15, and CD206 markers ( y axis). PE-Cy7, Phycoerythrin-Cyanine 7. ( H ) Tumor and adjacent healthy tissues were resected from the same patients with NSCLC ( n = 9 patients), and adenosine levels were quantified using LC-MS. ( I ) Immunosuppression mediated by human NSCLC-associated CD73 + monocyte cells. CD3 neg CD14 + CD73 + monocyte cells and autologous CD3 + cells were sorted from fresh NSCLC cancer tissues and cocultured for 4 days in the presence of anti-CD3/anti-CD28 Ab. Representative line graph (I) showing the percentage of T cell inhibition. Data, mean ± SEM of cells from four different patients. P values: *** P < 0.0005; ** P < 0.005; * P < 0.05.

Article Snippet: CD14 + cells were then stained with biotinylated antihuman CD73 Ab and streptavidin microbeads, and CD14 + CD73 + cells were separated using AutoMACS ProSeparator; and (ii) murine CD11b + Gr-1 int M-MDSCs were positively selected from PGE 2 -induced BM-MDSCs and from LMCs obtained from naïve, KP1.9-bearing wild-type and Nt5e −/− mice using the murine MDSC isolation kit (Miltenyi Biotec).

Techniques: Expressing, Activity Assay, Labeling, Cell Culture, Liquid Chromatography with Mass Spectroscopy, Inhibition

Journal: Science Advances

Article Title: Monocytic MDSCs exhibit superior immune suppression via adenosine and depletion of adenosine improves efficacy of immunotherapy

doi: 10.1126/sciadv.adg3736

Figure Lengend Snippet: ( A and B ) CD14 + monocytes from healthy donors were either untreated (cultured monocytes) or treated for 48 hours with either GM-CSF (10 ng/ml), GM-CSF and PGE 2 (1 μM), IL-4 (10 ng/ml), or IL-6 (10 ng/ml), and CD73 expression was measured by flow cytometry. Representative dot plots (A) and bar graph (B) show the CD73 expression (%) in the treated cells. Data, mean ± SEM of three independent experiments with monocytes from three different donors. ( C and D ) CD14 + monocytes from healthy donors were treated for 6 days with either GM-CSF, GM-CSF and PGE 2 (PGE 2 -induced M-MDSCs), or GM-CSF and IL-4 [monocyte-derived dendritic cells (mo-DCs)]. Dot plots (C) and bar graph (D) show CD73 expression (%). Data, mean ± SEM of three independent experiments with monocytes from three different donors. ( E ) Indicated cells were added to autologous T cells in the presence of tetanus toxoid (TT; 1.0 μg/ml) for 5 days, and T cell proliferation was measured by incorporation of [3H] thymidine. Data, mean ± SEM. ( F ) Immune suppressive activity (against anti-CD3/anti-CD28 bead–activated T cells) of CD73 + or CD73 neg PGE 2 –M-MDSCs or tumor cell line–induced M-MDSCs. Data, mean ± SEM of three independent experiments. ( G ) Exogenous AMP (10 μM) or AMP and ADA (10 U/ml) were added to fresh medium with CD73 + or CD73 neg M-MDSCs for 6 to 8 hours. Extracellular adenosine was measured in the culture supernatants. Data, mean ± SEM of two independent experiments. ( H ) Untreated or ADA-treated culture supernatants from CD73 + M-MDSCs cultured with AMP were added to T cells, and suppressive activity was measured by incorporation of [3H] thymidine. Data, mean ± SEM of three independent experiments. P values: *** P < 0.0005; ** P < 0.005; * P < 0.05.

Article Snippet: CD14 + cells were then stained with biotinylated antihuman CD73 Ab and streptavidin microbeads, and CD14 + CD73 + cells were separated using AutoMACS ProSeparator; and (ii) murine CD11b + Gr-1 int M-MDSCs were positively selected from PGE 2 -induced BM-MDSCs and from LMCs obtained from naïve, KP1.9-bearing wild-type and Nt5e −/− mice using the murine MDSC isolation kit (Miltenyi Biotec).

Techniques: Cell Culture, Expressing, Flow Cytometry, Derivative Assay, Activity Assay

Journal: Science Advances

Article Title: Monocytic MDSCs exhibit superior immune suppression via adenosine and depletion of adenosine improves efficacy of immunotherapy

doi: 10.1126/sciadv.adg3736

Figure Lengend Snippet: ( A and B ) CD14 + monocytes from healthy donors were treated with either GM-CSF (10 ng/ml), GM-CSF + PGE 2 (1 μM), GM-CSF + EP2 receptor agonist (butaprost; 10 μM), GM-CSF + EP4 receptor agonist (CAY10598; 10 nM), or GM-CSF + IL-4 (10 ng/ml) + EP2 receptor agonist for 6 days. CD73 expression was measured by flow cytometry. Data, mean ± SEM of three independent experiments. ( C ) CD14 + monocytes are treated with either vehicle (Control), PGE 2 (1 μM), or Forskolin (10 μM) for 16 hours, and NF-κB activation was measured by Western blot. ( D ) Bar graphs showing the CD73 and IL-6 mRNA expression in CD14 + monocytes treated as in (C). Data, mean ± SEM. ( E ) Bar graphs showing the CD73 and IL-6 mRNA expression in CD14 + monocytes treated with either vehicle (Control) or D-cAMP (100 μM) for 16 hours. Data, mean ± SEM. ( F ) CD14 + monocytes were treated with either GM-CSF (10 ng/ml), GM-CSF + PGE 2 (1 μM), or GM-CSF + IL-6 (10 ng/ml) for 48 and 72 hours, and activation of Stat3 was assessed by Western blot. ( G ) CD14 + monocytes were treated with either GM-CSF alone, GM-CSF + PGE 2 /vehicle, or GM-CSF + PGE 2 /Stat3 inhibitor (S3I-201; 100 μM) for 48 hours, and Stat3 activation was assessed by Western blot. ( H ) CD14 + monocytes were treated with either GM-CSF alone, GM-CSF + PGE 2 /vehicle, or GM-CSF + PGE 2 /CREB inhibitor (666-15; 2 μM) for 48 hours, and CREB activation was assessed by Western blot. ( I and J ) CD14 + monocytes were treated with either Stat3, CREB, or Stat3/CREB inhibitors as in (G) and (H), and CD39/CD73 expression was measured. Data, mean ± SEM of three independent experiments. P values: *** P < 0.0005; ** P < 0.005; * P < 0.05.

Article Snippet: CD14 + cells were then stained with biotinylated antihuman CD73 Ab and streptavidin microbeads, and CD14 + CD73 + cells were separated using AutoMACS ProSeparator; and (ii) murine CD11b + Gr-1 int M-MDSCs were positively selected from PGE 2 -induced BM-MDSCs and from LMCs obtained from naïve, KP1.9-bearing wild-type and Nt5e −/− mice using the murine MDSC isolation kit (Miltenyi Biotec).

Techniques: Expressing, Flow Cytometry, Control, Activation Assay, Western Blot

Journal: Science Advances

Article Title: Monocytic MDSCs exhibit superior immune suppression via adenosine and depletion of adenosine improves efficacy of immunotherapy

doi: 10.1126/sciadv.adg3736

Figure Lengend Snippet: ( A and B ) Murine BM cells from C57BL/6 mice were cultured with GM-CSF (20 ng/ml) or GM-CSF/PGE 2 (2.6 μM) for 5 days. Accumulation of CD45 + CD11b hi Gr-1 int M-MDSC cells and proportions of CD73 + cells in CD45 + CD11b hi Gr-1 int M-MDSC population are shown in representative contour and dot plots (A) and bar graph (B), respectively. Data, mean ± SEM of three independent experiments. ( C , D , F , and G ) Histograms and bar graphs showing expression of CD73 (C) and (D) and CD39 (F) and (G) in total CD11b hi Gr-1 int BM cells. ( E ) Fresh BM cells or BM cells treated as in (A) were added to splenocytes from OT-I transgenic mice at 1:2, 1:4, and 1:8 ratio in the presence of OVA (250 μg/ml) per well for 4 days. T cell proliferation was measured by [3H] thymidine incorporation. Data, mean ± SEM of three independent experiments. ( H ) CD73 + BM-MDSCs were purified and cultured with AMP in the presence or absence of CD73 inhibitor AMP-CP (10 μM), and supernatants were added to T cells in the presence of anti-CD3 and anti-CD28 antibodies. T cell proliferation was measured by [3H] thymidine incorporation. Data, mean ± SEM of three independent experiments. ( I ) BM cells were cultured with either GM-CSF alone or GM-CSF + PGE 2 for 16, 24, 48, 72, and 96 hours, and Stat3 and CREB activation was assessed by Western blot. β-Actin was used as a loading control. ( J and K ) BM cells are treated with either GM-CSF alone, GM-CSF + PGE 2 /vehicle, or GM-CSF + PGE 2 /Stat3 inhibitor (S3I-201; 100 μM) or GM-CSF + PGE 2 /CREB inhibitor (666-15; 1 μM) for 72 hours and analyzed by flow cytometry. Data, mean ± SEM of two independent experiments. P values: *** P < 0.0005; ** P < 0.005; * P < 0.05.

Article Snippet: CD14 + cells were then stained with biotinylated antihuman CD73 Ab and streptavidin microbeads, and CD14 + CD73 + cells were separated using AutoMACS ProSeparator; and (ii) murine CD11b + Gr-1 int M-MDSCs were positively selected from PGE 2 -induced BM-MDSCs and from LMCs obtained from naïve, KP1.9-bearing wild-type and Nt5e −/− mice using the murine MDSC isolation kit (Miltenyi Biotec).

Techniques: Cell Culture, Expressing, Transgenic Assay, Purification, Activation Assay, Western Blot, Control, Flow Cytometry

Journal: Science Advances

Article Title: Monocytic MDSCs exhibit superior immune suppression via adenosine and depletion of adenosine improves efficacy of immunotherapy

doi: 10.1126/sciadv.adg3736

Figure Lengend Snippet: ( A ) Bar graphs showing PGE 2 levels in culture supernatants of murine B16-F10 melanoma cells, LLC lung cancer cells, and 4T1 breast cancer cells with either PTGES knockdown or scrambled control (4T1 shPTGES , 4T1shScr, inset) as measured by ELISA ( n = 2 independent ELISA expt.). Data, mean ± SEM. ( B to F ) Syngeneic tumors implanted subcutaneously into BALB/c mice (4T1 shPTGES and 4T1shScr cells) and C57BL/6 mice (B16-F10 and LLC cells) ( n = 5 mice per tumor type). Tumors were isolated (800 to 1000 mm 3 ), digested, and stained for CD45, CD11b, Gr-1, and CD73 expression. Contour plots showing the proportions of CD45 + CD11b hi Gr-1 int M-MDSCs (B) and histograms (C) and bar graph (D) showing % CD73 + cells within the CD45 + CD11b hi Gr-1 int M-MDSC population in the implanted tumors. Data, mean ± SEM. (E) Correlation between intratumoral % CD73 + M-MDSCs versus PGE 2 levels produced by the corresponding tumor cells. P = 0.0025, r 2 = 0.9949. (F) Histograms showing CD73 expression in the CD45 + CD11b hi Gr-1 int M-MDSC and CD45 + CD11b hi Gr-1 hi G-MDSC population in the implanted tumors. P values: *** P < 0.0005.

Article Snippet: CD14 + cells were then stained with biotinylated antihuman CD73 Ab and streptavidin microbeads, and CD14 + CD73 + cells were separated using AutoMACS ProSeparator; and (ii) murine CD11b + Gr-1 int M-MDSCs were positively selected from PGE 2 -induced BM-MDSCs and from LMCs obtained from naïve, KP1.9-bearing wild-type and Nt5e −/− mice using the murine MDSC isolation kit (Miltenyi Biotec).

Techniques: Knockdown, Control, Enzyme-linked Immunosorbent Assay, Isolation, Staining, Expressing, Produced

Journal: Science Advances

Article Title: Monocytic MDSCs exhibit superior immune suppression via adenosine and depletion of adenosine improves efficacy of immunotherapy

doi: 10.1126/sciadv.adg3736

Figure Lengend Snippet: ( A to G ) C57BL/6 mice ( n = 20) were injected with KP1.9 cells intravenously. (A) Representative micro-CT images of lungs at day 40. Red arrows, individual tumor. (B) Hematoxylin and eosin–stained section of tumor-bearing mouse lung. Magnification: ×1 and ×8. (C) Bar graph showing PGE 2 levels in the lung tissue measured using mass spectrometry ( n = 4). Data, mean ± SEM. Dot plots (D) and bar graph (E) show % CD73 + cells within the CD45 + CD11b hi Gr-1 int population in naïve and tumor-bearing mouse lungs. (F) CD73 + CD45 + CD11b hi Gr-1 int cells from lung tissue from tumor-bearing and naïve mice ( n = 6 per group) were cocultured with OT-I splenocytes and OVA (250 μg/ml) for 4 days. T cell proliferation was measured using [3H] -thymidine incorporation. Data, mean ± SEM. (G) Culture supernatants from CD73 + M-MDSCs/AMP cultures were added to T cells, and proliferation was measured as above. Data, mean ± SEM of two independent experiments. ( H ) Representative micro-CT images of lungs from wild-type (WT) and Nt5e −/− KP1.9 mice, imaged at day 40. Red arrows, individual tumors. ( I and J ) M-MDSCs from wild-type and Nt5e −/− KP1.9 tumor–bearing mice were cocultured with CFSE-labeled splenocytes from OT1 mice and OVA (250 μg/ml) for 4 days, and CD8 + T proliferation was measured by flow cytometry. Data, mean ± SEM of two independent experiments ( n = 6). ( K and L ) Wild-type or Nt5e −/− mice were injected subcutaneously with KP1.9 cells (1.5 × 10 5 ) mixed with either wild-type or Nt5e −/− BM-derived M-MDSCs or lung tumor–derived M-MDSCs (2 × 10 5 ) ( n = 5 to 7 per group). Bar chart represents tumor volumes. Data, mean ± SEM. P values: *** P < 0.0005; ** P < 0.005; * P < 0.05; ns, not significant.

Article Snippet: CD14 + cells were then stained with biotinylated antihuman CD73 Ab and streptavidin microbeads, and CD14 + CD73 + cells were separated using AutoMACS ProSeparator; and (ii) murine CD11b + Gr-1 int M-MDSCs were positively selected from PGE 2 -induced BM-MDSCs and from LMCs obtained from naïve, KP1.9-bearing wild-type and Nt5e −/− mice using the murine MDSC isolation kit (Miltenyi Biotec).

Techniques: Injection, Micro-CT, Staining, Mass Spectrometry, Labeling, Flow Cytometry, Derivative Assay

Journal: Science Advances

Article Title: Monocytic MDSCs exhibit superior immune suppression via adenosine and depletion of adenosine improves efficacy of immunotherapy

doi: 10.1126/sciadv.adg3736

Figure Lengend Snippet: Tumor-derived PGE 2 initiates a signaling cascade in M-MDSCs resulting in Stat3/CREB-dependent up-regulation of CD73 expression, inducing increased levels of adenosine in the TME culminating in the inhibition of antitumor T cell activity, and thus limits the efficacy of immunotherapy. Depletion of adenosine with PEG-ADA will overcome adenosine-mediated immunosuppression and sensitize tumors to immunotherapy.

Article Snippet: CD14 + cells were then stained with biotinylated antihuman CD73 Ab and streptavidin microbeads, and CD14 + CD73 + cells were separated using AutoMACS ProSeparator; and (ii) murine CD11b + Gr-1 int M-MDSCs were positively selected from PGE 2 -induced BM-MDSCs and from LMCs obtained from naïve, KP1.9-bearing wild-type and Nt5e −/− mice using the murine MDSC isolation kit (Miltenyi Biotec).

Techniques: Derivative Assay, Expressing, Inhibition, Activity Assay

Journal: Human reproduction (Oxford, England)

Article Title: A protein isolated from human oviductal tissue in vitro secretion, identified as human lactoferrin, interacts with spermatozoa and oocytes and modulates gamete interaction.

doi: 10.1093/humrep/det016

Figure Lengend Snippet: Figure 1 (A) Autoradiography of 7.5% SDS-PAGE [35S]-labeled oviductal proteins. Lane 1: proteins (127, 94 and 79 kDa) eluted from the affinity column by high salt as described in Materials and Methods. Lane 2: proteins produced de novo by oviductal tissue in culture. Inset in A: western blot analysis of 5′ nucleotidase in sperm proteins extract, lane 1: oviductal fluid (10 mg protein/lane); lane 2: sperm proteins extract (15 mg protein/lane). St: MW standard. (B) Western blot analysis of LF expression: LF: human milk LF (0.5 mg/ lane). OEH, oviduct epithelial cell homogenate (10 mg protein/ lane). OF, oviductal fluid (10 mg protein/lane). CM, conditioned media from oviductal tissue culture (10 mg protein/lane). HS, human serum (10 mg protein/lane), no signals were observed. IgG: the signal was absent when purified rabbit IgG was used instead of anti-LF antibody to detect LF (0.5 mg/lane). The experiment was repeated three times with different samples and similar results. (C) Expression of LF in OEH at different stages of the menstrual cycle. Proliferative phase: 10 samples from cycle days 7.9+ 0.9. For com- parative purposes, the intensities of LF bands of samples from prolif- erative phase were considered as 100%. Periovulatory phase: mean +SEM % of six samples from cycle days 14.0 +0.2. Secretory phase: mean +SEM % of five samples from cycle days 20.3+0.9. a: P , 0.01; b: P , 0.01.

Article Snippet: As a control of isolation of sperm membrane proteins, 5′ nucleotidase (a sperm membrane enzyme) was assessed in the extracts by 12% sodium dodecyl sulfate–polyacrylamide gel electrophresis (SDS-PAGE) followed by western blot with a specific antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Autoradiography, SDS Page, Labeling, Produced, Western Blot, Expressing

Journal: European journal of pharmacology

Article Title: Anti-inflammatory potency of novel ecto-5'-nucleotidase/CD73 inhibitors in astrocyte culture model of neuroinflammation.

doi: 10.1016/j.ejphar.2023.175943

Figure Lengend Snippet: Fig. 1. Evaluation of the inhibition of CD73 activity by cytosine-based 5’-α,β-methylene diphosphonates MRS4598, MRS4552, and MRS4602. Structural and chemical formulas (A, E, C) and concentration-response curve of MRS4598 (B), MRS4552 (D), and MRS4602 (F). Concentration-response curves were obtained by fitting raw data (percent enzyme inhibition vs [inhibitor concentration]) to the sigmoid curve using a four-parameter logistic regression model. Black symbols represent individual measurements, colored symbols represent mean inhibition (E) at each inhibitor concentration ± SEM from n ≥3 independent measurements.

Article Snippet: After blocking with 5% BSA (Sigma-Aldrich, USA) in Tris-buffer saline/Tween 20 (TBST), the blot was probed overnight at 4 ◦C with the rabbit monoclonal antibodies against CD73 (1:1500 dilution in TBST, Cell Signaling Cat. #13160).

Techniques: Inhibition, Activity Assay, Concentration Assay, Enzyme Inhibition Assay

Journal: European journal of pharmacology

Article Title: Anti-inflammatory potency of novel ecto-5'-nucleotidase/CD73 inhibitors in astrocyte culture model of neuroinflammation.

doi: 10.1016/j.ejphar.2023.175943

Figure Lengend Snippet: Fig. 6. The impact of MRS4598 on CD73 release from astrocyte membranes. A) Representative dot-blot membrane of soluble CD73 detected in culture media, 24 hours after addition of APCP or MRS4598 (both in the range of 1–100 μM). The presence of soluble CD73 was detected by anti-CD73 antibody and chem iluminescence detection. B) Integrated density values determined in ImageJ.

Article Snippet: After blocking with 5% BSA (Sigma-Aldrich, USA) in Tris-buffer saline/Tween 20 (TBST), the blot was probed overnight at 4 ◦C with the rabbit monoclonal antibodies against CD73 (1:1500 dilution in TBST, Cell Signaling Cat. #13160).

Techniques: Dot Blot, Membrane

Journal: European journal of pharmacology

Article Title: Anti-inflammatory potency of novel ecto-5'-nucleotidase/CD73 inhibitors in astrocyte culture model of neuroinflammation.

doi: 10.1016/j.ejphar.2023.175943

Figure Lengend Snippet: Fig. 4. The inhibitory potency of MRS4598 on CD73 activity in astrocytes treated with inflam matory cytokines. A) Representative immunoblot of astrocyte membrane samples isolated from control cultures and cultures treated with TNF-α and IL -1β. Equivalent amounts of native samples or PNGaseF- treated samples (dControl, dTNF-α, and dIL-1β) were resolved on SDS-PAGE, transffered onto PVDF, and immunoblotted with anti-CD73 antibodies. B) CD73 activity in control culture and in cultures treated with TNF-α or IL-1β in the presence (blue bars) or absence (gray bars) of 50 μM MRS4598. Significance inside the graph: # p < 0.05 between cytokine-treated culture and control; * p < 0.05 be tween cultures exposed to MRS4598 and the corre sponding control. C) Concentration-response curve to MRS4598 ranging from 1 to 250 μM in astrocytes treated with 0.1 μg/ml IL -1β. Data were fitted to a sigmoid curve (red lines) with four-parameter sig moid function nonlinear regression analysis using Origin 7.0.

Article Snippet: After blocking with 5% BSA (Sigma-Aldrich, USA) in Tris-buffer saline/Tween 20 (TBST), the blot was probed overnight at 4 ◦C with the rabbit monoclonal antibodies against CD73 (1:1500 dilution in TBST, Cell Signaling Cat. #13160).

Techniques: Activity Assay, Western Blot, Membrane, Isolation, Control, SDS Page, Concentration Assay