SPECT and helical CT scans were performed at 24 h p.i. in an effective eradication of tumors upon combination with additional radiotherapy. Conclusion: Our findings demonstrate that M2 macrophage-targeted imaging allows for noninvasively predicting post-chemotherapy tumor relapse and sensitively detecting the metastatic lymph nodes IVIS optical imaging system (Xenogen, Alameda, CA) starting from 10 min after D-luciferin administration (150 mg/kg by intraperitoneal injection). Cyclophosphamide treatment Cyclophosphamide (CTX) treatment started when the 4T1 tumor-bearing mice reached a tumor volume of 100-150 mm3. Mice were separated into 3 groups (n =15 or 20 per group), and were intraperitoneally administered CTX (in phosphate buffered answer; PBS) at a single dose (150 mg/kg, once on day 0) or multiple doses (75 mg/kg, on days 0, 3, 6, 9, 12, and 15), or PBS only (vehicle control). On day 8, three mice from each group were euthanized. Tumors were harvested, slice into 8 m solid frozen sections, and stained for mouse CD206 and F4/80. In the mean time, 5 mice from each group were euthanized, and their tumors were enzymatically digested using a previously explained method 16 to obtain single-cell suspensions for circulation cytometry analysis. On day 32, BRD4 Inhibitor-10 mice from SDF-5 each group (n = 6 or 8 per group) were euthanized. The lungs were filled with 15% India ink via the upper trachea and fixed in Fekete’s answer (100 mL of 70% alcohol, 10 mL of 4% formalin, and 5 mL of glacial acetic acid) for 48 h. Metastatic lesions, which appeared as white nodules around the lung surface, were counted and photographed. In a separate experiment, 4T1 tumor-bearing BALB/c mice were treated with PBS, a single dose of CTX (150 mg/kg, once on day 0), or multiple doses of CTX (75 mg/kg, on days 0, 3, and 6). On day 7, five mice from each group were subjected to CD206-targeted NIRF imaging as explained below. On day 8, three mice from each group were subjected to CD206-targeted SPECT/CT imaging and five mice from each group were subjected BRD4 Inhibitor-10 to a BRD4 Inhibitor-10 biodistribution analysis, respectively, as explained below. Circulation cytometry analysis Single-cell suspensions were incubated with phycoerythrin (PE)-conjugated rat anti-mouse F4/80 (clone BM8; Sungene, Tianjin, China) and fluorescein isothiocyanate (FITC)-conjugated rat anti-mouse CD206 antibodies (clone C068C2; Sungene, Tianjin, China) for 1 h at 4C, then analyzed using a FACSCalibur LSR- circulation cytometer (Becton Dickinson, Germany). Preparation of CD206-targeting probes The CD206-targeting NIRF probe was generated using a previously explained method 16. Briefly, anti-CD206 antibody (CD206, clone C068C2, IgG2a; Biolegend, San Diego, CA) was mixed with Dylight755-N-hydroxysuccinimide (NHS) ester (Pierce, Rockford, IL) in sodium bicarbonate buffer (pH 8.4) at a 1:10 molar ratio. After incubation for 12 h at 4C, the combination was purified using a PD-10 desalting column (GE Healthcare, Piscataway, NJ). The degree of labeling (dye/protein molar ratio) of Dylight755-CD206 (Dye-CD206) was 6:1, as detected using an ultraviolet spectrophotometer (Thermo Fisher Scientific, Waltham, MA). Dylight755-labeled isotype-matched control rat IgG (Dye-IgG) was synthesized as a control using the same method. The CD206-targeting radiotracer was generated by radiolabeling anti-CD206 antibody (100 g) with 185 MBq Na125I using a previously explained method 24. Briefly, 100 g of antibody dissolved in 0.2 M PBS (pH 7.4) was mixed with 185 MBq of Na125I in a vial pre-coated with Iodogen (Sigma, St. Louis, MO). After incubating at room heat for 10 min, the product, 125I-CD206, was purified using a PD-10 desalting column. The radiochemical purity of 125I-CD206 was 98%. 125I-labeled isotype-matched IgG (125I-IgG) was also prepared using the same method. NIRF BRD4 Inhibitor-10 imaging For CD206-targeted NIRF imaging, each 4T1 tumor-bearing mouse was intravenously (i.v.) administered 0.5 nmol of Dye-CD206 or control Dye-IgG. At 4, 24, 48, 72, and 96 h postinjection (p.i.), mice (n = 5 per group) were imaged using a Maestro In-Vivo Imaging System (CRI, Woburn, MA). For each scan, aliquots made up of a.