Noninvasive evaluation of 18F-FDG/18F-FMISO-based Micro PET in monitoring hepatic metastasis of colorectal cancer

Abstract

This study aimed to explore the application of two radiotracers (¹⁸F-fluorodeoxyglucose (FDG) and ¹⁸F-fluoromisonidazole (FMISO)) in monitoring hepatic metastases of human colorectal cancer (CRC). Mouse models of CRC hepatic metastases were established by implantation of the human CRC cell lines LoVo and HT29 by intrasplenic injection. Wound healing and Transwell assays were performed to examine cell migration and invasion abilities. Radiotracer-based cellular uptake in vitro and micro-positron emission tomography imaging of liver metastases in vivo were performed. The incidence of liver metastases in LoVo-xenografted mice was significantly higher than that in HT29-xenografted ones. The SUVmax/mean values of ¹⁸F-FMISO, but not ¹⁸F-FDG, in LoVo xenografts were significantly greater than in HT29 xenografts. In vitro, LoVo cells exhibited stronger metastatic potential and higher radiotracer uptake than HT29 cells. Mechanistically, the expression of HIF-1α and GLUT-1 in LoVo cells and LoVo tumor tissues was remarkably higher than in HT29 cells and tissues. Linear regression analysis demonstrated correlations between cellular ¹⁸F-FDG/¹⁸F-FMISO uptake and HIF-1α/GLUT-1 expression in vitro, as well as between ¹⁸F-FMISO SUVmax and GLUT-1 expression in vivo. ¹⁸F-FMISO uptake may serve as a potential biomarker for the detection of liver metastases in CRC, whereas its clinical use warrants validation.

Figure 1

Survival curves of the LoVo and HT29 xenograft models. **P < 0.01; n = 20.

Figure 2

(A) ¹⁸F-FDG/¹⁸F-FMISO-based micro-positron emission tomography (PET) images of liver metastases of LoVo and HT29 xenografts 7 weeks after injection with corresponding tumor cells. The gray outline area of the PET images of the mice with no liver metastasis represents the liver area. White arrows indicate the uptake of radiotracers in tumor tissues. (B) The timeline of micro-PET. (C) Quantification of micro-PET regions of interest analysis (ROI) of ¹⁸F-FDG and ¹⁸F-FMISO uptake in xenograft tumor tissues. (D) Demonstration of ROI delineation. *P < 0.05, n = 10 (LoVo) and n = 4 (HT29).

Figure 3

(A) LoVo and HT29 cell monolayers were scratched using pipette tips. The images were acquired at 0, 24, and 48 h after wound formation. Magnification 100×. (B) LoVo and HT29 cells were subjected to a Matrigel extracellular matrix cell invasion assay. The cells were allowed to invade for 72 h. The invading cells were stained with Giemsa and observed under a microscope at a magnification of 40×. (C) Quantification of cell wound closure (%) of LoVo and HT29 cells. (D) Quantification of cell invasion assay. *P < 0.05; n = 3.

Figure 4

Time courses of in vitro cellular uptake of ¹⁸F-FDG/¹⁸F-FMISO in LoVo and HT29 cells. Cells were incubated with ¹⁸F-FDG or ¹⁸F-FMISO for 30, 60, 120, and 240 min. Data are expressed as means ± standard deviation of the percentage of the injected dose (%ID) of ¹⁸F-FDG (A) or ¹⁸F-FMISO (B) per 5 × 10⁵ cells. *P < 0.05; n = 3 (LoVo VS HT29).

Figure 5

Comparison of induction of HIF-1α and GLUT-1 between LoVo and HT29 cells/tumor tissues. (A) Micrographs of immunofluorescence staining for nuclei (blue) and HIF-1α/GLUT-1 (green) in LoVo (upper panel) and HT29 (lower panel) cells (magnification, 200×). (B) and (C) Quantification of HIF-1α and GLUT-1 staining using Live cell Imaging System. (D) Immunohistochemical staining for GLUT-1/HIF-1α (brown) in LoVo (upper panel) and HT29 (lower panel) xenograft tumor tissues (magnification, 200×), respectively. (E) Western blot analyses of HIF-1α and GLUT-1 protein expression in LoVo (right lane) and HT29 (left lane) xenograft tumor tissues. (F) Quantification of immunohistochemical staining. (G) Quantification of Western blot analyses. ***P < 0.0001, **P < 0.001, *P < 0.05, n = 3.

Figure 6

Correlation analyses between various parameters. (A) and (B) correlation of 18F-FDG uptake with GLUT-1 (A) and HIF-1α (B) expression in vitro. (C,D) correlation of ¹⁸F-FMISO uptake with GLUT-1 (C) and HIF-1α (D) expression in vitro. (E) and (F) correlation of in vivo values of ¹⁸F-FMISO SUVmax with GLUT-1 (E) and HIF-1α (F) expression in tumor tissues.