2.14. In Vivo Tumour Treatment Studies

Animal experiments were performed in the Experimental Animal Center of Fudan University (Shanghai, China). All experimental protocols involving animals were approved by the Institutional Animal Care and Use Committee of Fudan University, and the agreement number was 20171143A083. Male C57BL/6 mice (aged 6-8 weeks) were purchased from Shanghai Lingchang BioTech Co., Ltd.

Intralesional injections of PBS, RB, the RB-based mDC vaccine and the RB-iDC vaccine were performed as follows. A total of 1 × 10⁶ LLCs were subcutaneously inoculated into the right flank of the C57BL/6 mice (five to six per group). Before the intralesional injection of RB/DCs, we selected mice with round tumours. When the tumour diameter was 4-5 mm (day 0), 1 mM RB (dissolved in PBS) was administered by intralesional injection, and the injection volume was equal to half the volume of the tumour (0.5 ml/cm³). For immunization of the animals, the iDCs or mature DCs pulsed with the RB-treated LLC lysates were washed three times in cold PBS and resuspended in PBS at a final concentration of 1 × 10⁷ cells/ml. DC suspension (100 μl) was subcutaneously injected into syngeneic C57BL/6 mice. PBS injection was used as the control. The RB-iDC vaccine strategy involved pretreating the tumour with intralesional RB for 10 min and then performing an intralesional injection of 1 × 10⁶ iDCs. Tumour sizes were measured with digital callipers and approximated by multiplying the measured dimensions. Tumour sizes were surveyed every day after the tumours appeared, and the mice were sacrificed when their tumour diameter was more than 10-15 mm. Tumour volumes based on the measurements with the digital callipers were calculated by the following equation: V = 0.5 × (d_long × d_short × d_short).