Nightowl lb981 system

The tumour burden was quantified using whole-body in vivo bioluminescence imaging with a NightOWL LB981 system (Berthold Technologies). Mice were intraperitoneally injected with 2.5 mg of D-luciferin potassium salt (Promega) dissolved in 0.2 mL of PBS. Images were acquired beginning 10 min after D-luciferin injection. Regions of interest (ROIs) were drawn around the whole body. The photons emitted from the ROIs was quantified in units of photons per second using the IndiGO software (Berthold Technologies).

Animal experiments were performed at EPO GmbH, Berlin, in accordance with the UKCCCR guidelines and approved by the responsible local authorities (State Office of Health and Social Affairs, Berlin, Germany). For analysis, 3 × 10⁶ cells of HCT116-LUC-Control or HCT116-LUC-miR-520c were transplanted into the spleens of 8 weeks old severe combined immunodeficiency (SCID) beige mice (randomly assigned to 6 mice per group before transplantation). The mice were imaged once a week in a NightOWL LB 981 system (Berthold Technologies, Germany) by intraperitoneal injection of 150 mg/kg D-luciferin (Biosynth, Switzerland) under temporal anesthesia with isoflurane (Sigma-Aldrich, Germany). Mice were sacrificed 20 days after tumor cell transplantation. Spleens (site of tumor injection) and livers (metastasis target organ) were removed and shock-frozen in liquid nitrogen and cryosections were performed for isolation of genomic DNA (Qiagen, Hilden, Germany) and RNA (TRIzol, Invitrogen). The level of liver metastasis was evaluated by quantifying HMD using PCR with primers provided in Supplementary Table 3. ImageJ was used for color coding of luminescence signal intensity of human cells.

Bioluminescence imaging was performed by using the NightOwl LB 981 system (Berthold Technologies, Bad Wildbad, Germany). For bioluminescence imaging, mice were anesthetized with isofluran (Abbott GmbH, Wiesbaden, Germany) and received 150 mg/kg D-luciferin (Biosynth, Staad, Switzerland) dissolved in sterile PBS i.p. Tumor growth was monitored and semi-quantified by WinLight (Berthold Technologies) and ImageJ 1.48 k (NIH, Bethesda, MD, USA).