*2.11. Tumor Reoccurrence Inhibition after Tumor Resection*

To evaluate the tumor reoccurrence inhibition efficacy after tumor resection, the B16F10 melanoma-bearing mice model was established in a similar procedure. After 12 days of melanoma incubation, tumor-bearing C57BL/6 mice with an average tumor volume of around 300–400 mm<sup>3</sup> were chosen for establishing the tumor-resection model. The mice were randomly divided into three groups with 12 mice in each group, including PBS group, free Dox + aCTLA-4 + aPD-1 mixed solution group (Dox + aCTLA-4 + aPD-1) and Dox/aCTLA-4/aPD-1 co-loaded hydrogel group (Dox + aPD-1 + aCTLA-4)@gel. After being anesthetized with 100 µL pentobarbital (1%), each melanoma-bearing mouse was excised ~90% of melanoma, surgically sutured and subsequently treated with subcutaneous injection of the given formulation at the surgical site. The volume of hydrogel and the dosages of drugs were the same as before. The volumes of melanoma and body weights of the animals were recorded at predetermined time points. At day 12, five mice in each group were randomly selected for analysis of immune cells, and the rest of the mice were continuously monitored for evaluation of survival time (*n* = 7).
