*2.10. In Vivo Antitumor Tests*

B16F10 cells (1 <sup>×</sup> <sup>10</sup><sup>6</sup> ) were injected subcutaneously into each C57BL/6 mouse to establish a B16F10-melanoma bearing mice model to evaluate the antitumor efficiency in vivo. The tumor-bearing mice were randomly divided into 7 groups one week after inoculation of melanoma cells (*n* = 5), as follows: PBS-treated group (denoted as PBS), free aCTLA-4 + aPD-1 mixed solution group (aCTLA-4 + aPD-1), aCTLA-4/aPD-1 co-loaded hydrogel group (aCTLA-4 + aPD-1)@gel, Dox solution group (Dox), Dox-loaded hydrogel group (Dox@gel), free Dox + aCTLA-4 + aPD-1 solution group (Dox + aCTLA-4 + aPD-1), and Dox/aCTLA-4/aPD-1 co-loaded hydrogel group (Dox + aCTLA-4 + aPD-1)@gel. The volume of paratumorally injected solution in each group was 50 µL. The dosages of Dox, aCTLA-4 and aPD-1 were controlled as 4 mg/kg, 1 mg/kg and 2 mg/kg, respectively, in the formulations containing each ingredient. The average melanoma volume and body weight were recorded and calculated in each group at predetermined time points. The melanoma volume was calculated according to the following equation: V (mm<sup>3</sup> ) = L <sup>×</sup> <sup>W</sup>2/2, where L (mm) means the length of melanoma and W (mm) represents the width of melanoma. The mice were euthanized when the volume of tumor was higher than 1500 mm<sup>3</sup> , except those that died naturally. The survival time for all the treated groups was investigated in a separate study (*n* = 7).
