C57BL/6N WT mice were injected subcutaneously into the right flank with 5 × 105 syngeneic B16-D5-HER2 cells suspended in phenol red-free DMEM (without additives). Mice bearing palpable B16-HER2 tumors (average volume of 20–30 mm3) received intra-tumoral injection of 3 × 108 TU of HER2-targeted and shielded HCAdVs, coding for the indicated payloads, on days 11, 13, 15, and 18 after tumor challenge. The tumor volume was calculated according to the formula: V = ½[D (mm) × d2 (mm)], with D and d being the longest and shortest tumor diameter, respectively. Bodyweight and physiological score, including appearance, behavior, and respiration, were monitored during the whole experiment to detect signs of toxicity.

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