psb 10 hydrochloride  (Bio-Techne corporation)

Journal: Journal of Extracellular Vesicles

Article Title: The immunomodulatory ballet of tumour‐derived extracellular vesicles and neutrophils orchestrating the dynamic CD73/PD‐L1 pathway in cancer

doi: 10.1002/jev2.12480

Figure Lengend Snippet: The dynamic modulation of CD73/PD‐L1 axis by pro‐tumour neutrophils. (a) Experimental design of the in vitro modulation in HNC cell lines SCC47, SCC9, PCI52 and FaDu. (b) Immunodetection of PD‐L1, CD73 and CDK6 after treatment with Neutrophils (+NØ), NØ with 10 µg of TEX (+TEX), NØ pre‐treated with CD73 inhibitor followed by TEX incubation (+AMPCP +TEX), NØ pre‐treated with A2 B R antagonist MRS1754 followed by TEX incubation (+MRS +TEX), NØ pre‐treated with A3R antagonist followed by TEX incubation (+PSB10 +TEX) compared to control (HNC cell line = CTRL). Prior to lysis, NØ were washed out to reduce NØ interference in the measurement. Per sample, 10 µg of protein was loaded. β‐actin was used as loading control. HNC cell line PCI52 was used as a representative. Additional cell lines FaDu, SCC9 and SCC47 are shown in Figure . N = 3. (c) Semi‐quantitative analysis of western blots. Protein expression was normalized to the loading control β‐actin. Graphs show mean ± SD and significance values were calculated using multiple two‐way ANOVA. * p < 0.05, *** p < 0.001, **** p < 0.0001. (d) Illustration of the chicken embryo chorioallantoic membrane (CAM) assay design. (e) Immunodetection of PD‐L1, CD73, CDK6 and Cyclin D1 after 7‐day CAM protocol. Groups divided as treatment of HNSCC primary tumour with healthy neutrophil supernatant (PT +NØ), NØ with TEX (+TEX), or NØ pre‐treated with P1R antagonist followed by TEX incubation (+CGS +TEX) compared to control (HNSCC primary tumour = PT). Per sample, 25 µg of protein was loaded. β‐actin was used as loading control. N = 3. Original blots are shown in Figure .

Article Snippet: The following antagonists were used to block ADO signalling: AMPCP (CD73 inhibitor–100 µM, Tocris), CGS15943 (P1 antagonist–0.1 µM, Tocris), PSB36 (A1R antagonist—1 µM, Tocris), SCH442416 (A2 A R antagonist—1 µM, Tocris), MRS1754 (A2 B R antagonist—1 µM, Tocris) and PSB10 hydrochloride (A3R antagonist—1 µM, Tocris, UK).

Techniques: In Vitro, Immunodetection, Incubation, Control, Lysis, Western Blot, Expressing, Membrane, Chick Chorioallantoic Membrane Assay

Journal: Journal of Extracellular Vesicles

Article Title: The immunomodulatory ballet of tumour‐derived extracellular vesicles and neutrophils orchestrating the dynamic CD73/PD‐L1 pathway in cancer

doi: 10.1002/jev2.12480

Figure Lengend Snippet: Endorsement of pro‐tumour potential of TEX‐modulated Neutrophils. Functional assays were performed with HNC cell lines SCC47, SCC9, PCI52 and FaDu with the following treatment groups: control (HNC cell line = CTRL), tumour cells with Neutrophils (+NØ), tumour cells with NØ with 10 µg TEX (+TEX), tumour cells with NØ pre‐treated with CD73 inhibitor followed by TEX incubation (+AMPCP +TEX), tumour cells with NØ pre‐treated with A2 B R antagonist MRS1754 followed by TEX incubation (+MRS +TEX) and tumour cells with NØ pre‐treated with A3R antagonist followed by TEX incubation (+PSB10 +TEX) indicated by distinct symbols and colours. (a) Quantitative analysis of cell proliferation assay by MTS absorbance (OD) with the representative HNSCC cell line SCC47. N = 5. Cell lines SCC9, PCI52 and FaDu are shown in Figure . (b) Quantitative analysis of cell death assay by caspase 3/7 fluorescence as relative fluorescence units (RFU) with the representative HNSCC cell line SCC47. N = 3. (c) Quantitative analysis of spreading assay by area ratio (area 48 h after incubation divided by area right before treatment) with the representative HNSCC cell line SCC47. N = 3. (d) One representative image of spreading assay per group at the beginning of the experiment (0 h) and after 48 h of incubation (48 h), performed with representative HNSCC cell line SCC47. Spreading area is highlighted with a dot line. Scale bar = 0.2 mm. N = 3. (e) Representative images of wound healing assay 0, 5, 10, 12, 15 and 18 h after treatment, performed with SCC47. Scratch is highlighted with a dot line. Scale bar = 0.4 mm. N = 3. Graphs express mean ± SD and significance values were calculated using multiple 2‐way ANOVA. * p < 0.05, ** p < 0.01, *** p < 0.001. For proper comparison, cells from all groups were incubated in the same cell culture media condition. HNSCC, head and neck squamous cell carcinoma.

Article Snippet: The following antagonists were used to block ADO signalling: AMPCP (CD73 inhibitor–100 µM, Tocris), CGS15943 (P1 antagonist–0.1 µM, Tocris), PSB36 (A1R antagonist—1 µM, Tocris), SCH442416 (A2 A R antagonist—1 µM, Tocris), MRS1754 (A2 B R antagonist—1 µM, Tocris) and PSB10 hydrochloride (A3R antagonist—1 µM, Tocris, UK).

Techniques: Functional Assay, Control, Incubation, Proliferation Assay, Fluorescence, Wound Healing Assay, Comparison, Cell Culture

Journal: Journal of Extracellular Vesicles

Article Title: The immunomodulatory ballet of tumour‐derived extracellular vesicles and neutrophils orchestrating the dynamic CD73/PD‐L1 pathway in cancer

doi: 10.1002/jev2.12480

Figure Lengend Snippet: The role of tumour‐derived extracellular vesicles and neutrophils in orchestrating the dynamic CD73/PD‐L1 axis in HNC. (1) Tumour cells secrete TEX carrying immunosuppressive factors, such as PD‐L1 and active ADO producing enzyme, CD73. (2) TEX interact with NØ inducing a pro‐tumour phenotype by increasing expression of CD170, CD73 and PD‐L1. (3) Pro‐tumour NØ secrete immunosuppressive cytokines, recruiting Tregs. (4) Extracellular ADO is increased and upregulates CD73 activity on tumour cells. (5) CD73 expression inhibits Cyclin D1‐CDK4/6, and therefore, increases PD‐L1 expression in tumour cells. (6) By blocking P1R on NØ, the phenotype is modulated: A2 B R acts synergistic to exosome influence and potentiates the pro‐tumour phenotype. A3R inhibits the pro‐tumour phenotype by reducing CD73 and PD‐L1 expression. (7) The reduced ADO and immunosuppressive TME downregulate CD73, which activates Cyclin D1‐CDK4/6 signalling, phosphorylating SPOP, resulting in the degradation of PD‐L1. SPOP, speckle‐type POZ protein; TME, tumour microenvironment.

Article Snippet: The following antagonists were used to block ADO signalling: AMPCP (CD73 inhibitor–100 µM, Tocris), CGS15943 (P1 antagonist–0.1 µM, Tocris), PSB36 (A1R antagonist—1 µM, Tocris), SCH442416 (A2 A R antagonist—1 µM, Tocris), MRS1754 (A2 B R antagonist—1 µM, Tocris) and PSB10 hydrochloride (A3R antagonist—1 µM, Tocris, UK).

Techniques: Derivative Assay, Expressing, Activity Assay, Blocking Assay