Supplementary Figures S1-S9. Figure S1. Functional evaluation of the GpNLuc LumiFluor. Figure S2. In vitro characterization of FACS sorted mouse Em-Myc lymphoma and human non-small cell lung cancer (NSCLC) cells. Figure S3. Temporal development of A549 NSCLC xenograft tumors expressing GpNLuc or GpNLuc and the tumor suppressor LKB1. Figure S4. Detection of spontaneous lymph node metastasis from primary lung tumors. Figure S5. In vitro and in vivo characterization of independently derived E?-Myc lymphoma cells stably expressing GpNLuc. Figure S6. Bioluminescence imaging of E?-Myc lymphoma-GpNLuc allografts. Figure S7. A, in vivo dose-response kinetics of GpNLuc signal strength. Figure S8. Quantification of in vivo bioluminescence imaging and confirmation ex vivo. Figure S9. Flow cytometric analysis of inducibly expressed fluorescent proteins in stable lymphoma cell lines.
ARTICLE ABSTRACT
Fluorescent proteins are widely used to study molecular and cellular events, yet this traditionally relies on delivery of excitation light, which can trigger autofluorescence, photoxicity, and photobleaching, impairing their use in vivo. Accordingly, chemiluminescent light sources such as those generated by luciferases have emerged, as they do not require excitation light. However, current luciferase reporters lack the brightness needed to visualize events in deep tissues. We report the creation of chimeric eGFP-NanoLuc (GpNLuc) and LSSmOrange-NanoLuc (OgNLuc) fusion reporter proteins coined LumiFluors, which combine the benefits of eGFP or LSSmOrange fluorescent proteins with the bright, glow-type bioluminescent light generated by an enhanced small luciferase subunit (NanoLuc) of the deep-sea shrimp Oplophorus gracilirostris. The intramolecular bioluminescence resonance energy transfer that occurs between NanoLuc and the fused fluorophore generates the brightest bioluminescent signal known to date, including improved intensity, sensitivity, and durable spectral properties, thereby dramatically reducing image acquisition times and permitting highly sensitive in vivo imaging. Notably, the self-illuminating and bifunctional nature of these LumiFluor reporters enables greatly improved spatiotemporal monitoring of very small numbers of tumor cells via in vivo optical imaging and also allows the isolation and analyses of single cells by flow cytometry. Thus, LumiFluor reporters are inexpensive, robust, noninvasive tools that allow for markedly improved in vivo optical imaging of tumorigenic processes. Cancer Res; 75(23); 5023–33. ©2015 AACR.
