Targeting of the P2X7 receptor in pancreatic cancer and stellate cells

Abstract

The ATP-gated receptor P2X7 (P2X7R) is involved in regulation of cell survival and has been of interest in cancer field. Pancreatic ductal adenocarcinoma (PDAC) is a deadly cancer and new markers and therapeutic targets are needed. PDAC is characterized by a complex tumour microenvironment, which includes cancer and pancreatic stellate cells (PSCs), and potentially high nucleotide/side turnover. Our aim was to determine P2X7R expression and function in human pancreatic cancer cells in vitro as well as to perform in vivo efficacy study applying P2X7R inhibitor in an orthotopic xenograft mouse model of PDAC. In the in vitro studies we show that human PDAC cells with luciferase gene (PancTu-1 Luc cells) express high levels of P2X7R protein. Allosteric P2X7R antagonist AZ10606120 inhibited cell proliferation in basal conditions, indicating that P2X7R was tonically active. Extracellular ATP and BzATP, to which the P2X7R is more sensitive, further affected cell survival and confirmed complex functionality of P2X7R. PancTu-1 Luc migration and invasion was reduced by AZ10606120, and it was stimulated by PSCs, but not by PSCs from P2X7(-/-) animals. PancTu-1 Luc cells were orthotopically transplanted into nude mice and tumour growth was followed noninvasively by bioluminescence imaging. AZ10606120-treated mice showed reduced bioluminescence compared to saline-treated mice. Immunohistochemical analysis confirmed P2X7R expression in cancer and PSC cells, and in metaplastic/neoplastic acinar and duct structures. PSCs number/activity and collagen deposition was reduced in AZ10606120-treated tumours.

Keywords: AZ10606120; P2X7; PDAC; fibrosis; stellate cells.

Figure 1

Orthotopic implantation of PancTu‐1 Luc cells and timeline of AZ10606120 therapy. (a) PancTu‐1 Luc cells injection in the head of pancreas of a nude mouse at day 0. (b) Schematic diagram of the experimental protocol used for the treatment with AZ10606120 and for the bioluminescence analysis. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Figure 2

Expression of P2X7R and cell proliferation with ATP, AZ10606120 and BzATP. (a) Representative gel of P2X7 mRNA expression (284 bp) in HPDE (H) and PancTu‐1 Luc (P) cells (n = 3). (b) Western blot on the whole cell lysates with polyclonal C‐terminal antibody for P2X7R shows the A isoform (70 kDa). β actin (42 kDa) was used as loading control (n = 4). Bar graph shows the level of P2X7R protein as a ratio to β actin. Significant difference in comparison to HPDE cells p < 0.001 (**) is reported. (c) Effect of ATP and ATP plus AZ10606120 on PancTu‐1 Luc cell proliferation was assayed using BrdU incorporation. Black bars show the effect of increasing concentration of exogenous ATP (0 μM, 10 μM, 100 μM and 1 mM). White bars represent the effect of increasing ATP concentration in combination with 10 μM AZ10606120 (P2X7R allosteric inhibitor). (d) Effect of increasing concentrations of BzATP (P2X7R agonist) on BrdU incorporation. Each run was performed in triplicates and the graphs show data from four independent experiments (mean ± SEM). Significant differences p < 0.05 (*, #) and p < 0.001 (**, ##) from the respective control, without exogenous ATP or BzATP (*, **) and with/without inhibitor (#, ##) are indicated. (e) The graph shows PancTu‐1 Luc cell proliferation after transfection with siRNA against P2X7 mRNA (siP2X7), scramble siRNA (siSCR) and siP2X7 plus BzATP (100 μM). Data are represented as mean ± SEM. Significant differences p < 0.001 (**) from the control are indicated. (f) Western blot shows the protein expression of P2X7R in PancTu‐1 Luc cells transfected with siRNA against P2X7R (siP2X7) or scramble Naito‐1 siRNA as control (siSCR).

Figure 3

Effect of AZ10606120, BzATP and mPSCs on PancTu‐1 Luc cells migration. PancTu‐1 Luc cells were grown to confluence in the two‐chamber silicon insert. After removal, phase‐contrast images were taken every hour for 30 hr. (a) Representative images at 0, 15 and 30 hr for control, AZ10606120, BzATP and P2X7R inhibitor plus BzATP. (b) White, black, grey and light grey circles show the percentage of the wound/gap open every hour in control cells (vehicle); cells treated with 10 μM AZ10606120, with 10 μM BzATP and AZ10606120 plus BzATP, respectively. Significant differences p < 0.05 (*) between control and inhibitor/agonist was calculated from the slope of the curves after 11 hr. (c) The graph shows the endpoint, i.e., percentage of wound still open after 30 hr; significant differences p < 0.05 (*) and p ≤ 0.001 (**) are indicated. Four to seven fields of view were analyzed per each experiment. The graphs show data from 3 to 4 independent experiments (mean ± SEM). (d) The graph shows effect of mPSCs from wild‐type and P2X7 knockout (+/−AZ10606120) on PancTu‐1 Luc cells migration. The graph shows data from four independent experiments (mean ± SEM). Significant differences p < 0.001 (**) are indicated.

Figure 4

P2X7R expression in PancTu‐1 Luc in vivo model. (a) Immunofluorescence detection of EGFR in the tumour cells (red), α‐SMA for the pancreatic stellate cells (cyan) and P2X7R (ATTO‐488, green). The staining was performed in two subsequent 2 µm sections. (b–d) Representative pictures of P2X7R expression in PancTu‐1 Luc tumour paraffin sections. The slides were stained with three different P2X7R antibodies: (b) APR‐008 against an epitope in the extracellular loop. (c) APR‐004 against the C‐terminal; (d) PA5‐28020 against the C‐terminal and more specific for human tissues embedded in paraffin. (e) Control of P2X7R antibody (PA5‐28020) in a tumour‐free pancreas of the PDAC mice. In images (b–d) following structures are indicated: Islets of Langerhans (a), ducts (b), PanINs (c) and acinar‐like structures with increased lumen size (arrows). All images are representative of staining carried out on n = 3–6. Images were taken with different magnifications but scale bars are 50 µm on all panels. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Figure 5

Effect of AZ10606120 on tumour growth. (a) In vivo representative bioluminescence images obtained by IVIS Spectrum of PancTu‐1 Luc mice treated either with 0.9% NaCl (upper panel, n = 6) or with AZ10606120 (lower panel, n = 7) at day 10, 17, 23 and 30 after cells implantation. Bioluminescence signals detected 15 min after the i.p. injection of luciferin over tumour areas of the nontreated mice increased faster than in the treated group. The given images and a scale is depicting radiance at day 30 to avoid oversaturation. (b) The graph shows the radiances (photons/second) measured over primary tumours using 2D ROIs at the indicated days after cells implantation normalized to the total flux at day 10 for each mouse (mean ± SD). The black symbols represent the tumour growth of the mice treated with NaCl (n = 4) and the white symbols the tumour growth of the mice treated with AZ10606120 (n = 4). Progression of BLI over time indicated significant difference between NaCl and inhibitor‐treated mice (p < 0.05 (*) slope analysis), and point to point comparison between groups is significant at day 23 and 30 (p < 0.05). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Figure 6

Effect of AZ10606120 on mPSCs and collagen deposition. Representative pictures of Masson Goldner Trichrom (a) and Picrosirius Red (b) staining of treated and untreated PancTu‐1 Luc tumours. (c) The graph shows the difference in collagen deposition (stained area) between the NaCl and AZ10606120 groups. The staining was performed in all tumours (n = 6–7) and eight to nine fields of view were analyzed in each slide (see Methods). Significant difference p < 0.05 (*) is indicated. (d) Representative pictures of AEC reaction and immunofluorescence (e) for pancreatic stellate cells detection, using an antibody against α‐SMA. (f) The graph shows the results of the stained area analysis of the α‐SMA immunofluorescence staining in treated and untreated groups (mean ± SEM). Eight to nine fields of view, per each tumour section, were analyzed. Significant difference p < 0.05 (*) between NaCl and AZ10606120 mice is indicated. Different magnifications were used on right and left images in panels A and D but scale bars are all 50 µm, also in panels B and E. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]