Abstract
Bioluminescence imaging (BLI) has shown its appeal as a sensitive technique for in vivo whole body optical imaging. However, the development of injectable tumor-specific near-infrared fluorescent (NIRF) probes makes fluorescence imaging (FLI) a promising alternative to BLI in situations where BLI cannot be used or is unwanted (e.g., spontaneous transgenic tumor models, or syngeneic mice to study immune effects).In this study, we addressed the questions whether it is possible to detect tumor progression using FLI with appropriate sensitivity and how FLI correlates with BLI measurements. In addition, we explored the possibility to simultaneously detect multiple tumor characteristics by dual-wavelength FLI (∼700 and ∼800 nm) in combination with spectral unmixing. Using a luciferase-expressing 4T1-luc2 mouse breast cancer model and combinations of activatable and targeting NIRF probes, we showed that the activatable NIRF probes (ProSense680 and MMPSense680) and the targeting NIRF probes (IRDye 800CW 2-DG and IRDye 800CW EGF) were either activated by or bound to 4T1-luc2 cells. In vivo, we implanted 4T1-luc2 cells orthotopically in nude mice and were able to follow tumor progression longitudinally both by BLI and dual-wavelength FLI. We were able to reveal different probe signals within the tumor, which co-localized with immuno-staining. Moreover, we observed a linear correlation between the internal BLI signals and the FLI signals obtained from the NIRF probes. Finally, we could detect pulmonary metastases both by BLI and FLI and confirmed their presence histologically.Taken together, these data suggest that dual-wavelength FLI is a feasible approach to simultaneously detect different features of one tumor and to follow tumor progression with appropriate specificity and sensitivity. This study may open up new perspectives for the detection of tumors and metastases in various experimental models and could also have clinical applications, such as image-guided surgery.
Highlights
Both bioluminescence imaging (BLI) and fluorescence imaging (FLI) are widely used optical modalities for non-invasive detection of tumor progression in small animals
Cell culture The mouse mammary gland cancer cell line, 4T1-luc2, expressing a codon-optimized luciferase gene was obtained from Caliper Life Sciences (Hopkinton, MA). This cell line has been shown to spontaneously produce highly metastatic tumors which can metastasize to the lung, liver and lymph nodes while the primary tumor is growing in situ [2,29,30]
Similar results were found with MMPSense680 and IRDye 800CW EGF
Summary
Both bioluminescence imaging (BLI) and fluorescence imaging (FLI) are widely used optical modalities for non-invasive detection of tumor progression in small animals. BLI is appealing for in vivo whole body imaging because of its exceptional sensitivity and almost negligible bioluminescent background [1,2,3]. Under certain circumstances, BLI is not applicable. BLI cannot be employed in tumor models lacking expression of bioluminescent reporters, such as in spontaneous- or chemicalinduced cancer models. The introduction of a foreign reporter protein, such as luciferase, may induce an immune response directed against the reporter protein itself in syngeneic mouse models of cancer [4,5,6,7,8]. For clinical applications, such as image-guided surgery [9], BLI is not applicable as it requires genetic modification of the targeted cells
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