Subcutaneous Administration of D-Luciferin is an Effective Alternative to Intraperitoneal Injection in Bioluminescence Imaging of Xenograft Tumors in Nude Mice

Currently, intraperitoneal (IP) injection of D-luciferin is the preferred method of providing substrate for bioluminescence imaging (BLI); however it has a failure rate of 3–10% due to accidental intestinal injection. The present study evaluates the quality of BLI after subcutaneous (SC) injection of D-luciferin and demonstrates the effectiveness of SC injection in anatomically disparate tumor models. Mice bearing luciferase-expressing tumors underwent BLI after SC or IP injection of D-luciferin. The average time to maximal luminescence was 6？min (range 5–9？min) after SC injection and 8？min (range 5–8？min) after IP injection. Within 7 minutes of injection, SC and IP routes yielded similar luminescence in subcutaneous, intracranial, tongue, and lung xenograft tumor models. In a model of combined subcutaneous and intracranial xenografts, SC injection resulted in proportional luminescence at all sites, confirming that preferential delivery of substrate does not occur. While tumors were occasionally not visualized with IP injection, all tumors were visualized reliably with SC injection. Thus, SC injection of D-luciferin is a convenient and effective alternative to IP injection for BLI in nude mice. It may be a preferable approach, particularly for tumors with weaker signals and/or when greater precision is required. 1. Introduction Bioluminescence is a naturally occurring phenomenon in certain species such as the firefly resulting from oxidation of luciferin to oxyluciferin in the presence of molecular oxygen and adenosine triphosphate. This reaction is catalyzed by the enzyme luciferase and results in the emission of light [1]. Bioluminescence imaging (BLI) is an in vivo optical imaging technique that detects light emitted from firefly luciferase-expressing cells by special charge coupled device (CCD) camera [2]. This is an extremely valuable experimental tool in cancer biology [3]; noninvasive whole body BLI allows repeated, real-time, and in vivo monitoring of tumors in experimental animal models, regardless of tumor location [2, 4]. This technique can be used to monitor tumor growth [5, 6], cell trafficking [7], protein-protein interactions [8], and response to treatment, including gene therapy and immunotherapy [9, 10]. Sensitivity of BLI is dependent on various factors including the level of cellular luciferase expression, implantation site of the tumor, and oxygenation and viability of the target tissue [11]. The availability of luciferin at the site of interest is a key element of BLI. D-luciferin can be delivered by intraperitoneal (IP) or