pA20-36 specifically binds primary and metastatic A20 tumor cells. (A) The pA20-36–FITC peptide specifically detects A20 tumor cells ex vivo. Cells (1 × 10⁶) derived from the site of subcutaneously injection (top) or spleen (middle) from A20 B lymphoma–engrafted BALB/c mice at day 12 were incubated with FITC-conjugated pA20-36 or pCNT peptides (10 μg/mL), and phycoerythrin-labeled anti–mouse B220 antibody and analyzed by flow cytometry. Control splenocytes from tumor-free mice were also analyzed (bottom). Numbers in the quadrants indicate the percentage of cells. (B) The pA20-36–FITC peptide detects niches of metastatic A20 tumor cells in the spleen of A20 B lymphoma–engrafted BALB/c mice. Spleens from A20 B lymphoma–bearing mice were stained with pA20-36–FITC, anti–mouse B220 antibody, and the nuclear dye TOPO-3 and analyzed by confocal microscopy. Pictures were captured with a Leica TCS SP2 confocal microscope with a HC PL FLUOTAR 20×/0.50 oil UV objective (NA 0.50) in glycerol and acquired with Leica Confocal Software Version 2.61. Image manipulation was performed with Adobe Photoshop CS. (C) MicroPET images showing the specific retention of [¹⁸F]-pA20-36 in the A20 tumor. BALB/c mice bearing a palpable A20 tumor mass on the flank (5-6 mm in maximal diameter) were injected intravenously with 10 μg (8 MBq peptide) of [¹⁸F]-pA20-36 (left) or [¹⁸F]-pCNT (middle). Mice bearing a palpable 5T33MM tumor mass on the flank were used as control for the specific retention of pA20-36 (right). Images shown are coronal (top) or axial (bottom) sections of static scans of a single mouse for each group collected at 10, 30, and 60 minutes after peptide injection (see also supplemental Videos). Tumors are indicated by white arrows in all cases.