Purpose:Intraoperative image guidance may aid in clinical decision-making during surgical treatment of colorectal cancer. We developed the dual-labeled carcinoembryonic antigen–targeting tracer, [111In]In-DTPA-SGM-101, for pre- and intraoperative imaging of colorectal cancer. Subsequently, we investigated the tracer in preclinical biodistribution and multimodal image-guided surgery studies, and assessed the clinical feasibility on patient-derived colorectal cancer samples, paving the way for rapid clinical translation.Experimental Design:SGM-101 was conjugated with p-isothiocyanatobenzyl–diethylenetriaminepentaacetic acid (DTPA) and labeled with Indium-111 (111In). The biodistribution of 3, 10, 30, and 100 μg [111In]In-DTPA-SGM-101 was assessed in a dose escalation study in BALB/c nude mice with subcutaneous LS174T human colonic tumors, followed by a study to determine the optimal timepoint for imaging. Mice with intraperitoneal LS174T tumors underwent micro-SPECT/CT imaging and fluorescence image–guided resection. In a final translational experiment, we incubated freshly resected human tumor specimens with the tracer and assessed the tumor-to-adjacent tissue ratio of both signals.Results:The optimal protein dose of [111In]In-DTPA-SGM-101 was 30 μg (tumor-to-blood ratio, 5.8 ± 1.1) and the optimal timepoint for imaging was 72 hours after injection (tumor-to-blood ratio, 5.1 ± 1.0). In mice with intraperitoneal tumors, [111In]In-DTPA-SGM-101 enabled preoperative SPECT/CT imaging and fluorescence image–guided resection. After incubation of human tumor samples, overall fluorescence and radiosignal intensities were higher in tumor areas compared with adjacent nontumor tissue (P < 0.001).Conclusions:[111In]In-DTPA-SGM-101 showed specific accumulation in colorectal tumors, and enabled micro-SPECT/CT imaging and fluorescence image–guided tumor resection. Thus, [111In]In-DTPA-SGM-101 could be a valuable tool for preoperative SPECT/CT imaging and intraoperative radio-guided localization and fluorescence image–guided resection of colorectal cancer.