Serum diluent

Snap frozen primary tumor and LN samples were mechanically disrupted and treated by lysis solution (Bio-Rad). Protein concentration in lysates was determined using Bradford assay (Bio-Rad). After sonication, samples were centrifuged at 4,500 g for 6 min at 4 °C. Protein concentration in the supernatant was determined using Bradford assay and adjusted to 1000 μg/ml using serum diluent (both Bio-Rad). Amounts of inflammatory factors in tissue lysates (obtained from tissues from mice treated for 5 days and sacrificed 5 days later) were measured by multiplex technology (Bio-Rad).Transforming growth factor (TGF)-β1 was measured using a single plex kit (Millipore) according to manufacturer's protocol.

Mouse plasma was prepared through centrifugation of EDTA-treated peripheral blood at 1000× g for 10 min at 4 °C. Supernatant was collected and filtered using a 0.22 µm filter and stored at −20 °C. Tumor samples were snap frozen in liquid nitrogen and stored at −80 °C. For the lysis of tumor samples, they were treated with cell lysis kit (Bio-Rad Laboratories, Inc., Hercules, CA, USA) according to the manufacturer’s instructions. Protein amount in lysates and plasma was measured by Pierce BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA). Protein concentration in lysates was adjusted to 1 mg/mL with serum diluent (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Chemokine levels in plasma and tumor tissue were analyzed by multiplex technology (Bio-Rad Laboratories, Inc., Hercules, CA, USA) the Pro Mouse Chemokine 31-plex kit according to the manufacturer’s instruction. Acquisition and data analysis were performed by bio-plex Manager™.