Journal: Neoplasia (New York, N.Y.)

Article Title: A Novel Antagonist of the Immune Checkpoint Protein Adenosine A2a Receptor Restores Tumor-Infiltrating Lymphocyte Activity in the Context of the Tumor Microenvironment

doi: 10.1016/j.neo.2017.02.004

Figure Lengend Snippet: PBF-509 is specific for the A2aR. (A) Dose response curves of PBF-509 versus 4 adenosine receptors in transfected CHO (hA1), HeLa (hA2a and hA3), and HEK-293 (hA2b) cells. (B) Representative dose–response curves of PBF-509 in a binding assay against the human A2aR in buffer (median Ki = 12 nM) and in human plasma (median Ki = 32 nM).

Article Snippet: After tumor disaggregation, the cells were stained for Live/Dead NearIR (Life Technologies; L10119), CD3 (Fisher Scientific; BDB563546), CD4 (Fisher Scientific; BDB562970), CD8 (Fisher Scientific; BDB562282), and A2aR (Novus Biologicals; NBP1-39474FR) to determine the percentage of A2aR-positive cells in CD4+ and CD8+ cells.

Techniques: Transfection, Binding Assay, Clinical Proteomics

Journal: Neoplasia (New York, N.Y.)

Article Title: A Novel Antagonist of the Immune Checkpoint Protein Adenosine A2a Receptor Restores Tumor-Infiltrating Lymphocyte Activity in the Context of the Tumor Microenvironment

doi: 10.1016/j.neo.2017.02.004

Figure Lengend Snippet: PBF-509 inhibits accumulation of cAMP. CHO cells transfected with the A2aR were used to determine the concentration of PBF-509 to reduce the concentration of cAMP by 50%.

Article Snippet: After tumor disaggregation, the cells were stained for Live/Dead NearIR (Life Technologies; L10119), CD3 (Fisher Scientific; BDB563546), CD4 (Fisher Scientific; BDB562970), CD8 (Fisher Scientific; BDB562282), and A2aR (Novus Biologicals; NBP1-39474FR) to determine the percentage of A2aR-positive cells in CD4+ and CD8+ cells.

Techniques: Transfection, Concentration Assay

Journal: Neoplasia (New York, N.Y.)

Article Title: A Novel Antagonist of the Immune Checkpoint Protein Adenosine A2a Receptor Restores Tumor-Infiltrating Lymphocyte Activity in the Context of the Tumor Microenvironment

doi: 10.1016/j.neo.2017.02.004

Figure Lengend Snippet: Human primary tumor, CAF, and TIL cell lines express CD73 and A2aR. Tumor (Tm), cancer-associated fibroblast (CAF), and tumor infiltrating lymphocyte (TIL) cell lines were established from 12 resected NSCLC tumors. (A) Tumor cells or CAFs were stained with anti-CD73 and subjected to flow cytometry. Most cells from nearly all of the cell lines (both tumor cells and CAFs) expressed high levels of CD73. (B) Representative experiment of tumor cells stained with anti-CD73 (blue) or isotype control (green). (C) CD4+ or CD8+ TIL cells were stained with anti-A2aR and subjected to flow cytometric analysis showing that high levels of A2aR were being expressed in CD4+ cells, with variable expression in CD8+ cells.

Article Snippet: After tumor disaggregation, the cells were stained for Live/Dead NearIR (Life Technologies; L10119), CD3 (Fisher Scientific; BDB563546), CD4 (Fisher Scientific; BDB562970), CD8 (Fisher Scientific; BDB562282), and A2aR (Novus Biologicals; NBP1-39474FR) to determine the percentage of A2aR-positive cells in CD4+ and CD8+ cells.

Techniques: Staining, Flow Cytometry, Control, Expressing

Journal: Neoplasia (New York, N.Y.)

Article Title: A Novel Antagonist of the Immune Checkpoint Protein Adenosine A2a Receptor Restores Tumor-Infiltrating Lymphocyte Activity in the Context of the Tumor Microenvironment

doi: 10.1016/j.neo.2017.02.004

Figure Lengend Snippet: Anti-PD-L1 and PBF-509 restore immune responsiveness of TILs. Four different resected NSCLC tumors were disaggregated and cultured with or without anti-PD-L1 (aPDL1; 10 mg/ml), anti-PD-1 (aPD1; 10 μg/ml), or the A2aR antagonist PBF-509 (1 μM). After 3 days in culture, the supernatants were assayed for the presence of γ-interferon using an ELISA.

Article Snippet: After tumor disaggregation, the cells were stained for Live/Dead NearIR (Life Technologies; L10119), CD3 (Fisher Scientific; BDB563546), CD4 (Fisher Scientific; BDB562970), CD8 (Fisher Scientific; BDB562282), and A2aR (Novus Biologicals; NBP1-39474FR) to determine the percentage of A2aR-positive cells in CD4+ and CD8+ cells.

Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: Cell biochemistry and function

Article Title: Inhibition of CD73 expression or A2AR blockade reduces MRP1 expression and increases the sensitivity of cervical cancer cells to cisplatin.

doi: 10.1002/cbf.3784

Figure Lengend Snippet: FIGURE 1 Effect of ADO and blockade of ARs on MRP1 expression in CC cells. (A) CaSki and HeLa cells, 1 × 105 of each, were cultured for 48 h with different concentrations of ADO (0, 0.001, 0.01, 0.1, and 1 mM), and MRP1 expression was analyzed by flow cytometry. The mean fluorescence intensity of MRP1 in cells cultured in the absence of ADO was normalized to 1. AF, Autofluorescence. Representative histograms from three independent experiments. (B) MRP1 expression in CaSki and HeLa cells cultured in the presence of 1 mM ADO and presence (+) or absence (−) of 10 μM DPCPX, ZM241385, MRS1754, and MRS1523, specific antagonists of A1R, A2AR, A2BR, and A3R, respectively, which are indicated in parentheses. Representative data from three independent experiments ± SEM. *p < .05, one‐way ANOVA.

Article Snippet: The expression of CD73, MRP1, A1R, A2AR, A2BR, and A3R in CaSki and HeLa cell lines was determined by flow cytometry using the following antibodies: CD73‐PE (PHA550257, BD Pharmingen), anti‐ MRP1 (MRPM5, Abcam), A1R‐AF647 (s‐4235R‐A647, Bioss), A2AR‐ PE (NBP1‐39474PE, Novus Biologicals), A2BR‐AF750 (s‐5900R‐ A750, Bioss), and A3R‐FITC (orb399642, Biorbyt).

Techniques: Expressing, Cell Culture, Flow Cytometry, Fluorescence

Journal: Cell biochemistry and function

Article Title: Inhibition of CD73 expression or A2AR blockade reduces MRP1 expression and increases the sensitivity of cervical cancer cells to cisplatin.

doi: 10.1002/cbf.3784

Figure Lengend Snippet: FIGURE 3 CD73 inhibition or A2AR blockade in CC cells decreases the extrusive capacity of CC cells. (A) CaSki‐WT and CaSki‐pS‐siRNA‐ CD73 cells; (B) CaSki‐WT and CaSki‐ADO + ZM241385 cells; (C) HeLa‐WT and HeLa‐pS‐siRNA‐CD73 cells; and (D) HeLa‐WT and HeLa‐ ADO + ZM241385 cells, 5 × 105 each, were cultured in the presence of different concentrations (0, 10, 20, 40, 60, 80, and 100 μM) of MK‐751, a specific inhibitor of MRP1, and the fluorogenic dye calcein‐AM, a substrate of MRP1, as described in the materials and methods section. The intracellular calcein‐AM content in CaSki‐WT and HeLa‐WT cells in the absence of MK‐751 was normalized to 100%. Representative data from three independent experiments ± SEM. *p < .05; **p < .01; ***p < .001, and ****p < .0001, one‐way ANOVA.

Article Snippet: The expression of CD73, MRP1, A1R, A2AR, A2BR, and A3R in CaSki and HeLa cell lines was determined by flow cytometry using the following antibodies: CD73‐PE (PHA550257, BD Pharmingen), anti‐ MRP1 (MRPM5, Abcam), A1R‐AF647 (s‐4235R‐A647, Bioss), A2AR‐ PE (NBP1‐39474PE, Novus Biologicals), A2BR‐AF750 (s‐5900R‐ A750, Bioss), and A3R‐FITC (orb399642, Biorbyt).

Techniques: Inhibition, Cell Culture

Journal: Cell biochemistry and function

Article Title: Inhibition of CD73 expression or A2AR blockade reduces MRP1 expression and increases the sensitivity of cervical cancer cells to cisplatin.

doi: 10.1002/cbf.3784

Figure Lengend Snippet: FIGURE 4 Effect of CP on the viability of CC cells with downregulated CD73 expression or in which A2AR was blocked. The effect of different concentrations of CP (0, 3.12, 6.25, 12.5, 25, 50, and 100 μM) was evaluated on the viability of (A) parental CaSki and HeLa cells (WT), cells transfected with pSIREN vector (pSIREN), or cells transfected with pSIREN vector containing a siRNA targeting CD73 (pS/siRNA‐CD73); (B) parental CaSki and HeLa cells (WT), treated with 1 mM ADO (ADO) or treated with ADO in the presence of 10 µM A2AR antagonist (ADO + ZM241385); and c) parental CaSki and HeLa cells (WT) or CaSki and HeLa cells treated with 100 µM MK‐751 for 24 h, as described in the materials and methods section. Representative data from three independent experiments. CP IC50 values for each cell type were calculated using GraphPad Prism version 8 using log‐transformed and normalized OD values and CP concentrations (μM) from three experiments. *p < .05; ***p < .001, and ****p < .0001, one‐way ANOVA.

Article Snippet: The expression of CD73, MRP1, A1R, A2AR, A2BR, and A3R in CaSki and HeLa cell lines was determined by flow cytometry using the following antibodies: CD73‐PE (PHA550257, BD Pharmingen), anti‐ MRP1 (MRPM5, Abcam), A1R‐AF647 (s‐4235R‐A647, Bioss), A2AR‐ PE (NBP1‐39474PE, Novus Biologicals), A2BR‐AF750 (s‐5900R‐ A750, Bioss), and A3R‐FITC (orb399642, Biorbyt).

Techniques: Expressing, Transfection, Plasmid Preparation, Transformation Assay