GFP/Luc Reporter Cell Line - LLC

Subcutaneous (s.c.) tumor models are widely utilized in preclinical studies of cancer metastasis. Nevertheless, the dynamics and natural evolution of circulating tumor cells (CTCs) and their clusters (CTCCs) within the peripheral blood of these models remain poorly understood. Here, researchers employed a novel technique known as "Diffuse in vivo Flow Cytometry" (DiFC) to investigate the dissemination of CTCs and CTCCs in a subcutaneous Lewis Lung Carcinoma (LLC) mouse model. Tumors were implanted in the posterior flank of the mice, and DiFC measurements were conducted continuously for up to 31 days post-implantation. At the study endpoint, the mice's lungs were harvested and subjected to bioluminescence imaging (BLI) to assess the extent of pulmonary metastasis. DiFC results revealed that, despite the use of genetically identical LLC cells and consistent tumor implantation sites, the numbers of CTCs and CTCCs exhibited significant heterogeneity across all study mice. The highest count rates recorded by DiFC corresponded to a concentration of 0.1 to 14 CTCs per milliliter of peripheral blood. Overall, CTC counts did not demonstrate a monotonic increase over time and showed only a weak correlation with tumor volume. However, a strong correlation was observed between the numbers of CTCs and CTCCs in the peripheral blood and the extent of pulmonary metastasis. The researchers attributed the observed variability in CTC counts primarily to the growth patterns of the primary tumors.

Here, the researchers utilized LLC cells capable of expressing both luciferase and green fluorescent protein. They first validated in mice-via direct tail vein injection-that GFP/Luc+ LLC cells could be detected using DiFC technology (Figure 1). Figure 1a presents a representative DiFC data trace acquired over a 10-minute period from a control mouse (neither injected with cells nor bearing a tumor). Figure 1b displays a representative DiFC data trace, also acquired over 10 minutes, from a mouse that had received an intravenous (i.v.) tail vein injection of 5 × 10⁵ GFP/Luc LLC cells. Figure 1c illustrates the distribution of peak amplitudes measured during a single 2-hour scanning session. Figure 1d presents a raster plot (event map) depicting the temporal distribution of DiFC detection events following the injection of LLC cells. The cells remained detectable for at least two hours post-injection, at which point the researchers ceased data acquisition. Throughout the entire scanning period, the average DiFC count rate exhibited a continuous downward trend (Figure 1e). The final count rate measured after two hours was approximately 25% of the initial value, implying that the in vivo half-life of these circulating tumor cells (CTCs) is approximately one hour.

Figure 1. Detection of LLC cells in circulation with DiFC. (Fitzgerald J E, et al., 2020)