Bioluminescence and Fluorescence Imaging for Preclinical Cancer Research
Oncology researchers often rely on traditional mouse disease models using human tumor cells implanted in immunodeficient mice. The most common metric used to assess tumor progression or response to treatment is the physical measurement (via micrometer) of tumor length and width for the calculation of tumor volume. This approach is often hindered by the inherent inaccuracies and variability of hand calculation and is only useful for accessible subcutaneous tumors. The use of anatomical imaging modalities such as magnetic resonance, computed tomography (CT), and ultrasound have improved the measurement of tumor dimensions, however, change in the size of a tumor is an insensitive metric that does not capture the complexity of tumor biology and is relatively slow to change with treatment. The additional assessment of biological changes often relies on terminal histopathology or fluorescence microscopy. In vivo molecular imaging approaches have arisen in response to the need of researchers for improvements in noninvasive methods for understanding tumor biology.
To provide the necessary tools for in vivo molecular imaging, PerkinElmer has instruments for small animal epifluorescence and bioluminescence imaging (IVIS Lumina Series) as well as for deep tissue optical tomography (IVIS Spectrum and FMT 4000). Pairing of these imaging technologies with luciferase-expressing tumor cell lines and near infrared (NIR) in vivo imaging agents allows detection and quantification of changes in biological processes, rather than alterations in morphology. In the study shown in Figure 1, NU/NU mice were implanted orthotopically with 4T1-luc2 tumor (mouse breast adenocarcinoma) cells. Mice with established tumors were injected intravenously with Annexin-Vivo 750 (to detect cell death) and FolateRSense 680 (to detect folate receptor expression) 2 hours and 24 hours prior to imaging, respectively. Mice received an additional injection of D-luciferin 12 to 15 minutes prior to imaging to detect luciferase-expressing tumor cells. Mice were imaged on the IVIS Spectrum CT, first by μCT, then for bioluminescence and fluorescence signal. In a single representative mouse, the μCT image shows the size and location of the tumors as well as some internal anatomical detail. The bioluminescence signal correlates well with the size, shape, and location of the tumors and the signal is found to be proportional to the tumor size. The NIR Annexin-Vivo fluorescence imaging agent reveals a distinctly different pattern of signal attributed to regions of low-level spontaneous cell death, further showing an absence of signal in the necrotic core of the tumor, as expected. FolateRSense shows a third pattern of tumor signal, detecting folate receptor expression attributed to both the tumor and the inflammatory cells in the tumor margin.
In another study, NU/NU mice with established subcutaneous HT-29 tumor xenografts (Figure 2) received either no treatment or a single 170 mg/kg intraperitoneal dose of cyclophosphamide (n = 24 mice per group). Mice were injected 24 hours later with Annexin-Vivo and imaged 2 hours later by FMT 4000. Although the representative control and CY-treated mice had tumors of nearly identical size, the Annexin-Vivo fluorescent signal was greater in the treated mouse as measured by both epifluorescence (2D) and fluorescence tomography (3D) imaging. Assessment of the changes in all 24 treated mice were measured on the tomographic datasets, showing a highly significant increase in fluorescence (p < 0.0002) relative to controls, despite the use of only a single suboptimal dose of the cyclophosphamide. There was no difference in tumor sizes between the two groups at this time point, highlighting the benefit of using molecular imaging agents and not relying on tumor size to assess early therapeutic effects.
The combination of bioluminescence with fluorescence molecular imaging agents provides both tumor burden and specific biological data regarding the status of tumor growth or response to treatment. A variety of PerkinElmer NIR agents, detecting protease activity, integrin expression, cell death, vascular leak, hypoxia and other biological activities, can be used to provide either a deeper understanding of tumor biological changes, or as sensitive tools for early assessment of treatment efficacy, in living animals.
Figure 1. IVIS Spectrum CT imaging of 4T1-luc2 tumors in NU/NU mice by (A) μCT, (B) bioluminescence, (C) NIR imaging of in vivo Annexin-Vivo 750, and (D) NIR imaging of FolateRSense 680.
Figure 2. (A) FMT 4000 imaging of HT-29 tumors in NU/NU mice by 2D epifluorescence and 3D fluorescence tomography. (B) Quantification of tomographic data by FMT 4000 TrueQuant 3.0 in comparison to calculated tumor volumes based on caliper measurements of tumors.