Tumor Model and Melphalan Therapy

All animal studies were conducted according to protocols approved by the Washington University Animal Studies Committee. C57BL/6 KaLwRij mice received a unilateral subcutaneous injection of 10⁶ 5TGM1 cells in the lower flank. All tumor implantation and imaging procedures were conducted under 1%–2% v/v isoflurane/100% O₂ anesthesia.

Tumor-bearing mice were separated into 2 independent studies. The first study consisted of an untreated group (n = 6) and a treated group (n = 7) that received melphalan at weeks 2 (14–18 d) and 3 after tumor implantation, with imaging being performed at week 4 (Fig. 1A). The second study consisted of a group (n = 3) that received melphalan at weeks 3, 4, and 5 after tumor implantation, with imaging being performed immediately before the start of each treatment (Fig. 1B). In both studies, melphalan treatment consisted of a 10 mg/kg intraperitoneal dose in saline (21).

Timeline for treated and untreated groups. Melphalan was administered weekly beginning at either week 2 (A) or week 3 (B) after tumor implantation. Imaging with MRI, ¹⁸F-FDG PET/CT, and ¹⁸F-FDOPA PET/CT was performed on separate, contiguous days. In week 2 group, imaging took place at end of study. In week 3 group, imaging took place before each treatment administration at weeks 3, 4, and 5.