Activation of the GPR35 pathway drives angiogenesis in the tumour microenvironment

Objective Primary sclerosing cholangitis (PSC) is in 70% of cases associated with inflammatory bowel disease. The hypermorphic T108M variant of the orphan G protein-coupled receptor GPR35 increases risk for PSC and ulcerative colitis (UC), conditions strongly predisposing for inflammation-associated liver and colon cancer. Lack of GPR35 reduces tumour numbers in mouse models of spontaneous and colitis associated cancer. The tumour microenvironment substantially determines tumour growth, and tumour-associated macrophages are crucial for neovascularisation. We aim to understand the role of the GPR35 pathway in the tumour microenvironment of spontaneous and colitis-associated colon cancers. Design Mice lacking GPR35 on their macrophages underwent models of spontaneous colon cancer or colitis-associated cancer. The role of tumour-associated macrophages was then assessed in biochemical and functional assays. Results Here, we show that GPR35 on macrophages is a potent amplifier of tumour growth by stimulating neoangiogenesis and tumour tissue remodelling. Deletion of Gpr35 in macrophages profoundly reduces tumour growth in inflammation-associated and spontaneous tumour models caused by mutant tumour suppressor adenomatous polyposis coli. Neoangiogenesis and matrix metalloproteinase activity is promoted by GPR35 via Na/K-ATPase-dependent ion pumping and Src activation, and is selectively inhibited by a GPR35-specific pepducin. Supernatants from human inducible-pluripotent-stem-cell derived macrophages carrying the UC and PSC risk variant stimulate tube formation by enhancing the release of angiogenic factors. Conclusions Activation of the GPR35 pathway promotes tumour growth via two separate routes, by directly augmenting proliferation in epithelial cells that express the receptor, and by coordinating macrophages’ ability to create a tumour-permissive environment.

layers were collected, washed and plated in RPMI/10% FBS containing 100ng/mL of GM-CSF (Miltenyi Biotech). RNA was extracted after 6 days in culture.

CRISPR/Cas9 editing of human iPSC line
The rs3749171 mutation in the human GPR35 gene was generated by a single base substitution (C>T) using CRISPR/Cas9-induced homology directed repair in the Kolf2 human iPSC line as previously reported. (10) iPSC culture and macrophage differentiation KOLF-2 human induced pluripotent stem cells were maintained in mTeSR-E8 medium (Stemcell Technologies) on Vitronectin (rhVTN-N) coated plates (Gibco #A14700). These cells were then differentiated to monocytes using the STEMdiff Monocyte Kit (StemCell Technologies) as per the manufacturer's instructions. After 14 days, macrophage precursors were harvested from the supernatant and plated on standard tissue culture plates in RPMI 1640, 10% FBS and 1% L-glutamine supplemented with 100 ng/mL M-CSF. After 7 days, mature macrophages were used for assays.

AOM/DSS model of colitis associated cancer
Six-to 8-week-old mice were injected intraperitoneally with AOM (12.5 mg/kg) (Sigma-Aldrich). Colitis was induced by two cycles of 2.5% DSS (MP Biomedicals) in drinking water for 5 days, followed by a 16-day tap water period. The final DSS cycle (2%) was administered for 4 days, followed by a 10-day tap water period. Tumour count and tumour area were determined microscopically (2-4× magnification) after 61 days. Tissues were paraffin embedded or frozen for further analysis.
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Transendothelial migration
H2-11 cells were grown to confluence on 5-m-pore size 24-well transwell inserts, which separated the upper and the lower chambers. Bone marrow derived monocytes, stained with calcein-AM were resuspended in RPMI 1640, 0.5% bovine serum and migrated towards macrophage conditioned media for 4h. Fluorescence intensity was then measured in the lower wells, and after washing on the transwell inserts to determine adhesion.
Tube formation assay 48 well plates were coated with 100 μl Geltrex (Thermo Fisher) at 4°C, incubated in a humidified incubator (37°C, 5% CO2) for 30 min. 20,000 freshly passaged calcein-AM stained H2-11 or SVEC cells (passage 2-5 were plated on Geltrex-coated plates. Supernatants from BMDM or iPSC/derived macrophages were added to the endothelial cells and endothelial tube morphogenesis was carried out in the presence or absence of ouabain (100µM) or pNaKtide (1µM). Endothelial tube formation was observed after 6 h and endothelial tubes were photographed. Quantification of tube formation was performed in a blinded manner by counting total tubes per five 40x fields and quantified for tube length and branch complexity using NIH ImageJ software.
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ELISA assays
CXCL-1, VEGF, MMP-2, MMP-9 and IL-1 from mouse tumour tissue or macrophage supernatants were measured by standard ELISA technique. Quantikine ELISA assays were bought from R&D systems and cytokines assessed adhering to the protocols.

MPO assays
Tumour tissue was homogenised and MPO was measured and calculated adhering to the manufacturer's protocol (Sigma Aldrich).

Western blotting
Cells or tissue were lysed in RIPA buffer and equal amounts of lysates were separated by SDS-polyacrylamide gel electrophoresis. After blotting onto Hybond P membranes (GE Healthcare), blots were blocked with 5% milk, and primary antibody was added at 4°C overnight. The protein was then detected by using an HRP-conjugated secondary antibody and visualized with LumiGLO (Cell Signaling Technology).

Immunofluorescence
Frozen sections (5 μm) of murine colon tissue from AOM/DSS model were fixed with 100% acetone at -20°C for 20 minutes and then rinsed with PBS containing 0.05% Tween (PBS-T).

Gelatine zymography
Cell lysates or supernatants were resolved in 12% polyacrylamide gels containing (1 mg/ml) gelatine A (Sigma). After electrophoresis, gels were washed three times in in regeneration buffer (20 min) followed by incubation in developing buffer (18 h at 37C) and stained with 0.25% Coomassie blue and then destained.

RNA extraction and reverse transcription quantitative polymerase chain reaction
RNA was isolated using the RNeasy Mini Kit (QIAGEN). RNA was then reverse-transcribed and SYBR Green (Eurogentec) quantitative polymerase chain reaction was performed using Mx3000 (Agilent Technologies). Target gene expression is expressed as ratio to housekeeping gene expression.