Igg2a control

B16.F10 shoulder allografts were allowed to develop for 5 days before treatment. Mice received 3 intraperitoneal (IP) injections of 10μg/ml anti-C3a (HyCult Biotech, clone: 3/11) or IgG2a control (BioXCell, clone 2A3) 5, 7 and 9 days after tumour induction. Animals were randomised and technicians undertaking procedures were blinded to treatment groups. Animals were sacrificed 6 days (24hs after the first treatment) and 11 days post tumour induction and tumours isolated. Blood was collected by cardiac puncture and kept in capped EDTA tubes at RT before processing. Non-invasive tumour measurements were recorded daily and volumes calculated using the following formula (π/6)(shortest length*longest length)2. Data points from all animals were included unless tumours failed to form following technical issues with injection of cells. For C3aR antagonism, 100μg (5% DMSO in PBS) of the small molecule SB290157 (Sigma) or vehicle control, was injected IP at either day 4 and 6 or day 8 and 10 into B16-F10 tumour bearing mice. Blood and tumour samples were collected 11 days post induction, as previously described.

One-month-old C57Bl/6 or 129/S6 Pkd1^RC/RC mice were treated twice a week for 8 weeks by i.p. injection with 10 mg/kg anti-CD8⁺ depleting antibody or 10 mg/kg IgG2a control (BioXCell, West Lebanon, NH). After 8 weeks of treatment, the animals were killed and evaluated as previously described.