Supplementary Materials Physique S1 HCT116 colon cancer cells express GPR55 mRNA. SHP2 IN-1 significant effect on the migration of LPI induced migratory responses of GPR55 overexpressing HCT116 in the Transwell migration assay (= 6C8; one\way ANOVA; Tukey’s post hoc test). (B) Incubation with Rock inhibitor H\1152 (10 nM) had no effect on the adhesion of na?ve HCT116 cells onto a HUVEC cell monolayer (= 6; t\test; not significant). Physique S4 PCR amplification of GPR55 transcripts. Gel showing bands of amplicons of passages 4 (p4) and 7 (p7) from HCT116 cancer cells and passage 6 (p6) of HCT116\CMVp\Luc cancer cells (HCT116\Luc). HCT116 as well as HCT116\CMVp\Luc cancer cells express GPR55 transcripts. Amplicons were electrophoresed in 1% agarose gel and SHP2 IN-1 stained with ethidiumbromide. GPR55 pcDNA3.1 plasmid (10 ng; Kargl assay of liver metastasis were performed. The GPR55 antagonist CID16020046, cannabidiol, a putative GPR55 antagonist and GPR55 siRNA were used to block GPR55 activity in HCT116 colon cancer cells. Key Results HCT116 cells showed a significant decrease in adhesion to endothelial cells and in migration after blockade with CID16020046 or cannabidiol. The inhibitory effects of CID16020046 or cannabidiol were averted by GPR55 siRNA knock down in cancer cells. The integrity of endothelial cell monolayers was increased after pretreatment of HCT116 cells with the antagonists or after GPR55 siRNA knockdown while pretreatment with lysophosphatidylinositol SHP2 IN-1 (LPI), the endogenous ligand of GPR55, decreased integrity of the monolayers. LPI also induced migration in GPR55 overexpressing HCT116 cells that was blocked by GPR55 antagonists. In a mouse model of metastasis, the arrest of HCT116 cancer cells in the liver was reduced after treatment with CID16020046 or cannabidiol. Increased levels of LPI (18:0) were found in colon cancer patients when compared with healthy individuals. Conclusions and Implications GPR55 is SHP2 IN-1 usually involved in the migratory behaviour of colon carcinoma cells and may serve as a pharmacological target for the prevention of metastasis. ? 2015 The British Pharmacological Society AbbreviationsCBDcannabidiolCMVcytomegalovirusGPR55G\protein coupled receptor 55LPAlysophosphatidic acidLPIlysophosphatidylinositolMEKmitogen\activated protein kinase kinaseNFATnuclear factor of activated T\cellsROCKRho\associated coiled\coil made up of protein kinase 1 Tables of Links assays exhibited that GPR55 is usually involved in adhesion and migration of colon cancer cells. Using an model of tumour cell metastasis, we show that after intrasplenic injection of HCT116\CMVp\Luc colon cancer cells, the arrest of cells is usually reduced in liver tissue of mice treated with CID16020046 or cannabidiol. We also detected increased LPI (18:0) content in the blood of colon cancer patients when compared with healthy donors. This study provides evidence that GPR55 is usually involved in the metastatic behaviour of colon cancer cells. Methods Cell culture and drugs Colon cancer cells (HCT116, HT\29 and SW480) were purchased from Interlab ANGPT2 Cell Line Collection, Genoa, Italy; HCT\CMVp\Luc cells were kindly provided by Dr Antje Siegert, EPO, Berlin, Germany. Overexpression of human 3xHA\GPR55 or vector alone (pcDNA3.1) in HCT116 cells was performed as previously described using Lipofectamine 2000 (Kargl non\invasive monitoring system (Kent Scientific, Torrington, CT, USA). Three and a half hours after the injection, the left lobe of the liver was removed, rinsed in PBS, blotted and weighed and quickly transferred into lysis buffer [25?mM TRISphosphate (pH?7.8), 10% glycerol, 1% Triton\X\100, 1?mgmL?1 BSA, 2?mM EGTA and 2?mM DTT]. After sonication and centrifugation, 100?L of supernatant was added to assay reagent (reaction buffer, 1?mM luciferin, 2?mM ATP). Reaction buffer consisted of 25?mM glycylglycine, 15?mM MgSO4, 4?mM EDTA, 15?mM K2PO4 (pH?7.8), 1?mM DTT and 1?mM CoA. After 1?min, luminescence was measured for 5?s at 562?nm at a TopCounter (Top Count NXT; Packard Instrument Company, Meriden, CT, USA). Luminescence values were normalized to liver wt and expressed as relative light units. Human blood samples Blood samples were provided as part of the project (http://www.oncotrack.eu/) by the General Hospital Graz West, St John of God Hospital Graz, Graz, Austria, and by the Institute of Experimental and Clinical Pharmacology, Medical University of Graz, Austria. Blood was collected from colon cancer patients and healthy individuals (= 7), drawn into heparin\made up of plasma separation tubes (Greiner\Bio\One, Austria) and centrifuged within 2?h at 1600 x?for 10?min. Plasma was then transferred into cryotubes without disturbing the buffy coat layer. The specimens were stored at ?80C until use. Written informed consent was obtained from all patients. Ethical approval was granted by the ethics committee of the Medical University of Graz and confirmed by the ethics committee of the St John of God Hospital Graz (23\015 ex 10/11 and 17\291 ex 05/06). LC\MS of LPI Lipid.