a740003 p2rx7 antagonist  (Tocris)

Journal: bioRxiv

Article Title: P2RX7 inhibition reduces breast cancer induced osteolytic lesions - implications for bone metastasis

doi: 10.1101/2021.12.31.474644

Figure Lengend Snippet: A) mRNA expression for P2RX7 in the different molecular subtypes of breast cancer. Data analysed from the TCGA and SCAN-B datasets (n=4669). B) mRNA expression of P2RX7 in triple-negative breast cancer (TNBC, n=317) compared to non-TNBC (n=4119). C) Kaplan-Meier plot for distant-metastasis free survival for patients with low (tercile 1) and high (tercile 3) P2RX7 expression. D) P2RX7 agonist BzATP (100μM) mediated calcium response (green) in E0771 murine breast cancer cells, which is inhibited in presence of a specific antagonist A740003 (10μM, red). Peak intensity and the area-under-curve (AUC) for the calcium response in (D) are presented in (E) and (F) respectively. G) P2RX7 mediated pore formation with higher concentrations of BzATP (300μM) assessed by ethidium bromide uptake in E0771 cells (red), which is inhibited by A740003 (green). P2RX7 over-expressing HEK-293 cells are used as a positive control (blue). (H) Analysis of the AUC for these pore-formation responses. I) Effect of P2RX7 antagonist A740003 on E0771 cell proliferation assessed by WST-1 at 72h. J) The effect of A740003 (10μM) on E0771 migration using scratch assay. All in vitro data is presented relative to the untreated controls and are from 3 separate biological repeats. The data has been presented as mean ± SD and analysed using one-way ANOVA. *P<0.05, **P<0.01 and ***P<0.0001.

Article Snippet: Cells were washed with PBS and were incubated with Fluo-4 Direct™ Calcium reagent for 1h at 37°C, with or without 10μM A740003 P2RX7 antagonist (Bio-Techne Ltd, Abingdon, UK).

Techniques: Expressing, Positive Control, Migration, Wound Healing Assay, In Vitro

Journal: bioRxiv

Article Title: P2RX7 inhibition reduces breast cancer induced osteolytic lesions - implications for bone metastasis

doi: 10.1101/2021.12.31.474644

Figure Lengend Snippet: The effect of P2RX7 inhibition on the bone microenvironment. A) μCT analyses of osteolytic lesions in tibias of E0771 tumour bearing C57BL/6J mice treated daily with P2RX7 antagonist (10mg/kg A740003) or vehicle controls (n=5 per group); AMC are non-tumour bearing age-matched controls. B) Representative μCT images of proximal tibias from each group. Morphometric analyses of the proximal tibia was done to characterise the structural changes in bone by measuring C) trabecular bone volume fraction (Tr BV/TV), D) trabecular thickness (Tr.Th), E) trabecular number (Tr.N), and F) cortical bone volume. G) Representative 3D μCT images of the tibial trabecular and cortical bone are shown alongside. Historphometric analyses of the tibial trabecular bone was performed by assessing G) osteoblast number per bone perimeter (N.Ob/B.Pm), H) osteoblast surface per bone perimeter (Ob.Pm/B.Pm), I) osteoclast number per bone perimeter (N.Oc/B.Pm), and J) osteoclast surface per bone perimeter (Oc.Pm/B.Pm). K) Representative images of the tibial sections with TRAP-positive osteoclasts (stars) and osteoblasts (arrows) shown on trabecular bone. Scale Bar = 50μm. The data has been presented as mean ± SD and analysed using unpaired t-test. *P<0.05, **P<0.01 and ***P<0.0001.

Article Snippet: Cells were washed with PBS and were incubated with Fluo-4 Direct™ Calcium reagent for 1h at 37°C, with or without 10μM A740003 P2RX7 antagonist (Bio-Techne Ltd, Abingdon, UK).

Techniques: Inhibition

Journal: bioRxiv

Article Title: P2RX7 inhibition reduces breast cancer induced osteolytic lesions - implications for bone metastasis

doi: 10.1101/2021.12.31.474644

Figure Lengend Snippet: Extracellular vesicles (EVs) from E0771 cells were isolated using size-exclusion columns. A) EVs were enriched in elute fractions 5-8 which were pooled together for subsequent studies. B) size distribution of the isolated EVs as measured by nanoparticle tracking analyses (NTA). C) Western blotting for CD9 and TSG101 as markers for EVs in the cell lysates (CL), EV fractions and post-EV (pEV) fractions. E0771 cells were cultured in hypoxia (1% O 2 ) or normoxia for 8h and EVs isolated subsequently from the conditioned media. D) NTA of the isolated EVs from hypoxic and normoxic cells. E) P2RX7 expression in E0771 cells cultured in normoxic and hypoxic conditions for 8h as assessed by real-time PCR. F) Hif1a and P2RX7 protein expression over time under hypoxic conditions with GAPDH as a loading control. G) The effect of hypoxia on P2RX7 expression was also assessed in primary tumours in vivo . IHC staining for GLUT-1 (as a marker for hypoxia) correlates with P2RX7 in serial sections. H) E0771 cells were treated with varying concentrations of BzATP (0-300μM), and EVs isolated from the media. Western blotting for CD9 and TSG101 shows the effect of P2RX7 activation on EV secretion, with β-Actin serving as a loading control. I) A740003 mediated inhibition of P2RX7 activation by 300μM reduces the EVs (CD9 and TSG101) secreted in the conditioned media. All in vitro data are from 3 separate biological repeats. The data has been presented as mean ± SD and analysed using unpaired t-test. ***P<0.0001.

Article Snippet: Cells were washed with PBS and were incubated with Fluo-4 Direct™ Calcium reagent for 1h at 37°C, with or without 10μM A740003 P2RX7 antagonist (Bio-Techne Ltd, Abingdon, UK).

Techniques: Isolation, Western Blot, Cell Culture, Expressing, Real-time Polymerase Chain Reaction, In Vivo, Immunohistochemistry, Marker, Activation Assay, Inhibition, In Vitro