ABSTRACT
Objective:To explore the feasibility of pretargeting technique for immunoPET with epidermal growth factor receptor (EGFR) monoclonal antibody in EGFR positive/negative tumor bearing mice.Methods:Cetuximab- Trans-cyclooctene (TCO)was obtained by modifying Cetuximab with TCO- N-hydroxysuccinimide (NHS). 2, 2′-((6-amino-1-(4, 7-bis-(carboxymethyl)-1, 4, 7-triazonan-1-yl)hexan-2-yl)azanediyl)-diacetic acid (L-NETA)was used as a chelating agent to prepare the radioligand 68Ga-L-NETA-tetrazine (Tz), then the labeling rate and in vitro stability of the product were determined. Human basal breast cancer cells MDA-MB-468 (EGFR+ ) and MDA-MB-231 (EGFR-) were cultured in vitro. In vitro experiments were performed to explore the specificity of the probe and the feasibility of pretargeting technique. Nude mice (Balb/c-nu) bearing xenografts of the above two cell lines were established. Cetuximab-TCO (50 μg) was injected into the tumor-bearing mice in advance, then 68Ga-L-NETA-Tz was injected at different time points (48, 36, 24 and 12 h), and pretargeting was realized through " click chemistry" . Small-animal PET imaging and biodistribution were performed to evaluate pharmacokinetic properties and specificity of the probe. The one-way analysis of variance was used to compare the data. Results:The 68Ga-L-NETA-Tz molecular probe was successfully prepared with the labeling yield >95%, and the radiochemical purity was >95% after 2 h. Cetuximab-TCO and 68Ga-L-NETA-Tz were added to MDA-MB-468 cells successively, and the cell uptake rate reached (0.69±0.04)% at 1 h, which demonstrated the feasibility of the pretargeting technique. PET imaging and biodistribution results showed that the best imaging results were obtained in 36 h pre-injection group, in which the tumor uptake was the highest ((0.77±0.05) percentage activity of injection dose per gram of tissue (%ID/g), 1 h) and the tumor/muscle ratio was optimal (4.67±0.46); the tumor uptake in the blocking group, the group without injecting Cetuximab-TCO, and the MDA-MB-231 group were significantly lower ((0.35±0.01), (0.39±0.05), (0.45±0.10) %ID/g; F=15.50, P=0.002). Conclusions:EGFR targeted immunoPET imaging is successfully performed in mouse models of breast cancer by injecting Cetuximab-TCO and 68Ga-L-NETA-Tz successively. It provides an effective method for immunoPET imaging of monoclonal antibodies.
ABSTRACT
Objective:To explore the feasibility and conditions of in vitro and in vivo imaging of triple-negative breast cancer using visible light emitted quantum dots(QDs) as the carrier to target epidermal growth factor receptor (EGFR). Methods:The water-soluble QDs reacted with Cetuximab to synthesize the probe QD-Cetuximab. The morphology, particle size, stability and luminescence properties of the probe were examined. Human breast cancer cells MDA-MB-468 (EGFR+ ) and MDA-MB-453 (EGFR-) were cultured. Cytotoxicity assays, in vitro imaging and fluorescence intensity quantification were performed after cells incubation with QD-Cetuximab and QDs. Eight MDA-MB-468 tumor-bearing mice models were constructed, 100 μl QD-Cetuximab and QDs were injected through the tail vein. In vivo imaging and probe distribution were obtained at different time points. Independent-sample t test was used to analyze the data. Results:QD-Cetuximab had a particle size of (40.34±2.44) nm detected by transmission electron microscope (TEM), a hydrated particle size of (57.85±4.69) nm detected by dynamic light scattering (DLS), and a stable structure. When the concentration of QD-Cetuximab was ≤50 nmol/L, the relative survival rate of cells was more than 90%, and when the concentration exceeded 100 nmol/L, the relative survival rate of cells was reduced to (72.52±4.91)% ( P<0.05). The red fluorescence of MDA-MB-468 incubated with QD-Cetuximab was stronger than that of MDA-MB-468 incubated with QDs and MDA-MB-453 incubated with QD-Cetuximab or QDs. The confocal fluorescent intensity quantitative determination showed that the ratio of QD-Cetuximab group/QDs group was 5.1 (863.36/169.97). Flow cytometry showed that the uptake of QD-Cetuximab and QDs by MDA-MB-468 increased with incremental incubating concentration, and the former was more significantly( t values: 12.25-38.11, all P<0.05). When the incubating concentration was 25, 50, 100, and 200 nmol/L, the quantitative average fluorescent intensity ratio of QD-Cetuximab group/QDs group was 5.4, 6.9, 7.4 and 6.2, respectively. The QD-Cetuximab and QDs probes mainly accumulated in the liver in vivo. The fluorescence emitted by tumor was not obvious under the high fluorescence of liver as a background. However, the fluorescence was visible in the isolated tumor tissue, and the quantitative fluorescence intensity of experimental group and control group were (2.46±0.60)×10 4 and (1.29±0.05)×10 4, respectively ( t=3.392, P=0.015). Conclusions:Cetuximab can increase the targeting ability of QDs and promote cell uptake. Although the isolated tumor imaging results are acceptable, further modification of QDs should be considered to reduce the liver uptake and improving in vivo fluorescence imaging efficiency.