The human colorectal cancer cell lines LoVo and LS174T were purchased from the Type Culture Collection of the Chinese Academy of Sciences, Shanghai, China. LS174T cell line was derived from the primary lesion of colorectal adenocarcinoma, Dukes' type B; LoVo cell line was derived from the left supraclavicular metastatic site of colorectal adenocarcinoma, Dukes' type C. The LoVo and LS174T cell lines were maintained in Dulbecco’s Modified Eagle Medium (DMEM, Gibco Corporation, USA) supplemented with 10% fetal bovine serum (Hyclone, USA) and 1% penicillin-streptomycin (Beyotime Biotechnology, China). All cells were cultured in a humidified atmosphere containing 5% CO2 air at 37ºC.
Five-week-old female BALA/C nude mice (weight, 16–18 g) were purchased from Animal Laboratory of Cavens Corporate of Changzhou (Changzhou, China). Colorectal cancer liver metastases (CLM) xenograft models were established by injecting LoVo or LS174T cells (2.0 × 107 cells in 0.15 mL of phosphate-buffered saline) into spleen which anesthetized by intraperitoneal injection of 10% chloral hydrate, respectively (n=20). All the animals were housed in an environment with temperature of 22 ± 1 ºC, relative humidity of 50 ± 1% and a light/dark cycle of 12/12 hr. All animal experiments were conducted in compliance with the protocols approved by the Institutional Animal Care and Use Committee (IACUC) of Jiangsu Institute of Nuclear Medicine. The body weight of nude mice was recorded every three days.
18F-AIF-NOTA-E[PEG4-c(RGDfk)]2 (denoted as 18F-RGD) was acquired by Jiangsu Key Laboratory of Molecular Nuclear Medicine, Jiangsu Institute of Nuclear Medicine. Synthesis of 18F-RGD has been described previously . 18F-RGD was a glutamic acid linked dimeric RGD labeled with NOTA-18F-AIF that has been proved to be safe and stable for PET imaging .
Cellular uptake in vitro
LoVo and LS174T cells were maintained in corresponding medium for 24 hours before 18F-RGD cellular uptake. For normal group, the cells, 18F-RGD and buffer (DMEM containing 0.2% bovine serum albumin) were mixed into a glass test tube (2ml). For block group, the cells, 18F-RGD and inhibitor buffer were mixed into a glass test tube (2ml). All the glass test tube were incubated at 37°C for 1 hour. The tubes were divided in three groups: Group O was used as a control tube, composed of 100μl radionuclides and 200μl buffer (DMEM buffer with 0.2% BSA); Group T was used to measure the radionuclide dose, contained 100μl radionuclides; Group X, was composed of 100μl radionuclides, 100μl cell suspension and 100μl corresponding buffer. The cellular uptake was normalized to 5×105cells/tube; each cell line was tested three times at each time point. The radioactivity of each cell line at 1 hour was accurately measured using Automatic Gamma Counter (PerkinElmer, 2480, USA). The cellular uptake ratio was calculated using the following formula: X(cpm)-0(cpm)/T(cpm)%.
Micro-PET Imaging and analysis
18F-RGD PET imaging of LoVo and LS174T-CLM mice models (n=20) were performed seven weeks after implantation. Six-minute static 18F-RGD (About 9.25MBq, 250μCi) was acquired 1 hours (radiopharmaceutical administration time in mice) after injection via tail vein. All the mice were anesthetized with 2% isoflurane in 100% oxygen with a flow rate of 2 L/min prior to imaging. Micro-PET scans were acquired in 3-dimensional mode using an Inveon micro-PET scanner (Siemens Medical Solutions) with an ordered-subset expectation maximization/maximum: matrix, 128×128×159; pixel size, 0.86×0.86×0.8mm; β-value, 1.5, with uniform resolution. PET images were reconstructed and postprocessed using Inveon Acquisition Workplace software (version 2.0, Siemens Preclinical Solutions).
Regions of interests (ROIs) were drawn on images around the entire liver metastasis or primary tumor lesions and normal liver tissue using ASI Pro VM 126.96.36.199 software (Concorde Microsystems, LLC). The maximum/mean standardized uptake value (SUVmax/mean) ratio was calculated as the tumor-to-liver SUVmax/mean (T/L ratio). All Micro-PET imaging procedures were conducted according to protocol approved by the Jiangsu Institute of Nuclear Medicine Animal Care and Use Committee.
The tumor specimens were fixed in 10% formalin for 48 h, paraffin-embedded, and cut into 3 μm-thick sections. Immunohistochemical staining was performed as previously described . Briefly, the slides were incubated with anti-Ki67 (1:100, ab16667, Abcam) or anti-VEGF (1:200, ab46154, Abcam) at 4°C overnight. Next, the slides were incubated with horseradish peroxidase-labeled goat anti-mouse or anti-rabbit secondary antibody (Boster, Wuhan, China) at room temperature followed by counterstaining with hematoxylin. The staining was observed under a BX53 Olympus microscope (Olympus, Japan) at magnification 200×. A brown-yellow staining was defined as positive expression. Ki67 or VEGF protein were quantitated by Image-J software (NIH, Bethesda, MD, USA).
All data were expressed as mean±standard deviation. Statistical analyses were performed using R version 3.6.1 (www.R-project.org). The difference between two groups was assessed using Student’s unpaired t-test. The Fisher exact test was used for comparing differences in liver metastatic potential in vivo. The correlation between the RGD parameters and tumor marker was analyzed using Pearson correlation analysis. The receiver operating characteristic (ROC) curve was used to differentiate LoVo tumor from LS174T tumor. A P value <0.05 was considered statistically significant.