Subcutaneous xenograft study.

To assess the effects of UMCD6 in vivo, we injected SCID beige mice (Charles River) subcutaneously with 5 × 10⁶ luciferase-infected MDA-MB-231 cells. Mice were anesthetized through the i.p. route with a mixture of ketamine (80–120 mg/kg) and xylazine (5–10 mg/kg), and tumor cells were injected into the right flank of each mouse. Tumor growth was monitored by bioluminescence imaging. 26 days after cell implantation, when tumors had reached volumes of at least 100 mm³, 10 mice were randomly allocated to receive an intravenous injection of 1.2 × 10⁷ PBMCs via the tail vein, while the 3 other mice (control group) received PBS.

The following day, mice that had received PBMCs were divided into 2 groups, selected such that the range of tumor sizes was equal in the groups: 5 mice received a single dose of UMCD6 (400 μg/mouse) and 5 mice received an IgG control antibody (400 μg/mouse) i.p. Tumor growth was monitored every other day by bioluminescence imaging. To assess tumor volume via bioluminescence imaging, we i.p. injected mice with 100 μL sterile d-luciferin at 15 mg/mL (Gold Biotechnology) and anesthetized them with 2% isoflurane. Mice were then imaged with a Xenogen IVIS 200 bioluminescence camera following d-luciferin administration, and images were captured after 1 minute of exposure and then quantified using Living Image 2.60.1 software. All images were normalized to the same scale and exposure time.