Balb c athymic nude mice

We examined the effect of miR-375-3p in vivo growth. SAS-L1 cells (1.0 × 10⁶) were seeded in a 100 mm dish (Corning Life Sciences) and treated with 0.3% Lipofectamine RNAiMAX and 20 nM of a miRIDIAN microRNA human hsa-miR-375 mimic or a miRID-IAN microRNA mimic negative control #1 (Dharmacon, Horizon Discovery). After 24 h of reverse transfection, the cells were harvested and injected subcutaneously at two sites in the flanks of 5-week-old male Balb/c athymic nude mice (CLEA Japan, Tokyo, Japan) at a density of 1.5 × 10⁶ treated cells per 150 μL of plain DMEM. Tumor diameter was measured every three days starting one week after injection using digital calipers, and tumor volume (mm³) was calculated using the following formula: length × width × height × 0.523. Twenty-two days after injection, the xenografts were dissected and we measured the tumor weight by using an electronic balance (ATX224, Shimadzu, Kyoto, Japan).

The model was slightly modified according to previous studies [54 (link)]. All animal studies were performed in compliance with the guidelines of the Institute for Laboratory Animal Research, National Cancer Center Research Institute (Number: T19-010, 11 June 2019). Five-week-old female Balb/c athymic nude mice (CLEA Japan, Shizuoka, Japan) were anesthetized by exposure to 3% isoflurane via injection and subjected to in vivo imaging. MDA-MB-231-D3H2LN cells (2 × 10⁶) and 1 × 10⁶ stimulated PBMCs in 50 µL of PBS were mixed with 50 µL of Matrigel (Corning, Merck Sigma, MA, USA), and then injected subcutaneously into the fat pad of the mice. Two days later, 30 µg of Exo-424 or Exo, in 100 µL of PBS was injected intratumorally every other day 4 times in total. The tumor mass was evaluated by measuring the bioluminescence with the IVIS Spectrum (Xenogen, Caliper, New York, USA) according to the manufacturer’s instructions. The data were analyzed using LIVINGIMAGE software (version 2.50, Xenogen, Caliper, New York, NY, USA).