Fig 2: P2RY12 modulates pancreatic cancer cell migration and proliferation. (A) Migration assay using trans well chambers was performed with human pancreatic BxPC3 cells without treatment or stimulated with ADP (20 µM) or in presence of ticagrelor (10 µM) or treated with ADP in presence of ticagrelor at the same concentration as before. Graph represents the Mean +/- SEM for each condition normalized to control condition. (B) Proliferation assay of BxPC3 cells was tested using a commercially available BrdU kit. Cells were incubated without treatment or stimulated with ADP (20 µM) or in the presence of ticagrelor (10 µM) or treated with ADP in presence of ticagrelor. Graph represents the Mean +/- SEM for each condition normalized to control condition. (C) Using a holotomography microscope, we analyzed the changes in BxPC3 cell morphology such as Mean Cells Extent Variation, Mean Cell Compactness Variation, and Mean total dry mass Variation. These three parameters used to detect the apoptosis were analyzed in BxPC3 cell without treatment (Control; Full Black circle), BxPC3 cells stimulated with ADP at 20 µM (Open gray circle) or treated with Ticagrelor (10 µM) (Full square), and BxPC3 treated with ADP (20 µM) in the presence of ticagrelor (10 µM) (full triangle). The graphs represent the Mean of three experiments for each condition. (D) Migration assay of mouse pancreatic Panc02 cells. Cells were analyzed without treatment (control) or stimulated with ADP (20 µM) or in presence of ticagrelor (10 µM) or treated with ADP in presence of ticagrelor at the same concentration as before. Graph represents the Mean +/- SEM for each condition normalized to the control condition. (E) Panc02 cells were incubated without stimulation (control) or stimulated with ADP (20 µM) or in the presence of ticagrelor (10 µM) or treated with ADP in the presence of ticagrelor. Graph represents the Mean +/- EM for each condition normalized to the control condition. Statistical analysis was performed with Shapiro-Wilk and T-tests ****P < 0.0001, ***P < 0.001; **P < 0.01. All the experiments were performed in triplicate.

Fig 3: Flow cytometry analysis identified the presence of microglia in iPSC-derived MCOs. The MCOs cultured for 35 and 45 days were subjected to flow cytometry analysis with CD11b and P2RY12. Isotype controls of the corresponding antibodies were used to determine the baseline expression of microglia marker. (A) Representative side scatter (SSC) images of one MCO. (B) Diagram graph showing the average percentage of positive cells per MCO. Data represent mean ± SE of three MCOs per group. The **P < 0.01 indicates the significant difference from corresponding marker CD11b at day 35. The ns indicates no significance statistically as compared with corresponding day 35 in each marker.

Fig 4: P2RY12 mRNA and protein expression in different cancer cell lines. (A) RT-PCR showing the P2RY12 mRNA in human and mouse pancreatic adenocarcinoma cell lines. (B) RT-PCR showing the P2RY1 and P2RY12 RNA in human colorectal adenocarcinoma cell line HT29. (C) Relative P2RY12 mRNA (delta CT) in different human and mouse cancer cell lines compared to platelets. (D) Mean Fluorescence of the P2RY12 protein in different pancreatic cancer cell lines BXPC3 and Panc02 as well as colorectal cancer cell line HT29. (E) P2RY12 protein expression normalized to the total cells in the samples analyzed with flow cytometry to show the relative population expression. Statistical test: Shapiro-Wilk and T-tests, **P < 0.001; ns, not- significant.

Fig 5: Isolated mouse microglia express macrophage markers PU.1 and Iba1, and the microglial-specific marker P2RY12Representative fluorescent images showing mouse microglial cultures are positive for the nuclear microglial marker PU.1, and cytoplasmic protein Iba1 (A). Microglia adopt morphologies ranging from rod-like to ramified. Scale bar, 50 μm. P2RY12 expression by isolated mouse microglia and pericytes via flow cytometry showing microglial purity (B).