| ObjectiveConstruction of a novel molecular probe targeting S1PR1,99mTc-HYNIC-S1PR1 m Ab,to study the biodistribution characteristics of this nuclide probe in a tumor model with high expression of S1PR1,and to evaluate the feasibility of the probe in SPECT imaging of tumor bearing mice.Methods1.Cell culture,expression level of S1PR1 and blocking experimentSK-HEP-1 cells and MCF-7 cells were cultured in vitro using MEM medium and DMEM medium,respectively,and the expression of S1PR1 of the two cells was detected by flow cytometry and Western blot.Flow cytometry was used to detect the blocking effect of the blocking agent FTY720 at the cell level.2.Tissue S1PR1 expression levelThe lung,liver,spleen,kidney,muscle,and tumor tissues of tumor-bearing mice were removed,and the expression of S1PR1 was analyzed by immunohistochemistry.The expression of S1PR1 in SK-HEP-1 tumor tissue,MCF-7 tumor tissue,and muscle tissue was analyzed by Western blot.3.Antibody labeling,purification and stability testingUsing SHNH as a chelating agent,S1PR1 was first connected and then labeled with 99 m Tc,and a new nuclide probe 99 m Tc-HYNIC-S1PR1 m Ab was finally synthesized.The molecular probe was purified using a PD10 column.The labeling rate and radiochemical purity of the molecular probe were measured before and after purification.The stability test is to determine the radiochemical purity of the molecular probe and the stability at different time points in PBS and fetal bovine serum.4.Cell uptake in vitro99mTc-HYNIC-S1PR1 m Ab was incubated with SK-HEP-1 cells and MCF-7 cells,respectively,and the cell supernatant and cell lysate were collected.Calculate the uptake of the probe by the cells at 30 min,1h,2h,4h,and 6h.In the blocking group,an appropriate amount of FTY720 was added to SK-HEP-1 cells in advance for incubation.5.Establishment of tumor-bearing mouse model,SPECT imaging and biodistributionSK-HEP-1 and MCF-7 tumor-bearing mouse models were constructed,and FTY720(3mg / kg / day)was administered to the tumor-bearing mice in the blocking group one week in advance.Static imaging was performed using SPECT at 3,6,12,18,and 24 h after tail vein injection of 99 m Tc-HYNIC-S1PR1 m Ab.The tumor-bearing mice were sacrificed 24 hours after the probe was injected,and the corresponding organs and tissues were removed.After weighing,the radioactivity count was measured using a γ-ray counter.Results1.Cell S1PR1 expression level and blocking experimentFlow cytometry results showed that the expression rates of S1PR1 in SK-HEP-1cells and MCF-7 cells were 97.9 ± 1.56% and 2.57 ± 0.4%(n> 3),respectively;Western Blot results showed that SK-HEP-1 Cells overexpress the S1PR1,while MCF-7 cells underexpress the S1PR1.Flow cytometry results showed that when the concentration of FTY720 was 0 μM,0.01 μM,0.1 μM,0.5 μM,and 1 μM,the positive rates of S1PR1 receptor in SK-HEP-1 cells were 100%,92.8 ± 2.83%,and 84± 1.41%,62.05 ± 3.89% and 35.6 ± 0.28%,respectively.Indicating that the inhibitory effect of FTY720 on the S1PR1 receptor of SK-HEP-1 cells increased with increasing concentration.2.Tissue S1PR1 expression levelWestern blot confirmed that the S1PR1 of SK-HEP-1 solid tumors is highly expressed,while the expression of S1PR1 in MCF-7 tumor tissues and muscle tissues is very low.Immunohistochemistry confirmed that a small amount of S1PR1 was expressed in vascular endothelial cells in lung,liver,spleen,kidney,muscle and MCF-7 tumor tissues,while SK-HEP-1 tumor tissue highly expressed S1PR1 and was distributed on tumor cell membranes.3.Antibody labeling,purification and stability testingThe labeling rates of 99 m Tc-HYNIC-S1PR1 m Ab was 61.45 ± 9.16%(n> 3).After purification by PD-10 column,the radiochemical purity was 96.7 ± 0.04%(n> 3).This indicates that the molecular probe has high radiochemical purity.At 12 hours,the stability of the molecular probes in PBS and FBS was 67.72 ± 6.06% and 85.49 ±3.48%(n> 3),respectively.4.Cell uptake in vitroThe uptake rate of 99 m Tc-HYNIC-S1PR1 m Ab by SK-HEP-1 cells increased significantly with time.The uptake rate at 30 minutes was 2.08 ± 0.27% and 6h was10.90 ± 0.79%(n> 3).The uptake rate of labeled probes by SK-HEP-1 cells in the blocking group incubating FTY720 4 hours earlier was significantly lower than that in the experimental group,with an uptake rate of 0.62 ± 0.11% at 30 minutes and 4.51 ±0.36% at 6 hours(n> 3).The uptake rate of the labeled probe by the MCF-7 cells in the negative group was similar to that in the blocking group.The uptake rate at 30 minutes was 0.62 ± 0.05% and 6h was 3.96 ± 0.26%(n> 3).This indicates that SK-HEP-1 cells have specific uptake of 99 m Tc-HYNIC-S1PR1 m Ab in vitro.5.SPECT imaging and biodistributionSPECT imaging showed that tumors in the experimental group started to faintly develop at 12 h,and the tumors became clear at 18 h and 24 h.The tumors in the blocking group and the control group had no obvious development.The probe uptake of the tumors in the blocking group and the control group was significantly lower than that in the experimental group at the same time point.Biodistribution results showed that when the 99 m Tc-HYNIC-S1PR1 m Ab was injected into tumor-bearing mice for24 hours,the probes were mainly concentrated in the blood and taken up by the liver,spleen,and kidney.At 24 hours,the tumor uptake value of the experimental group was 5.68±0.14% ID/g(n≥3),which was higher than the tumor uptake values of the blocking group and the control group(3.43±0.02% ID/g,2.87±0.10% ID/g,respectively)(n≥3).At the same time,the tumor/blood and tumor/muscle ratios of tumor-bearing mice in experimental group were higher than those in blocking group and control group.The biodistribution results are consistent with the SPECT imaging.ConclusionSK-HEP-1 cells highly expressed S1PR1,while MCF-7 cells expressed very low.FTY720 showed a good blocking effect in vitro and in vivo.99 m Tc-labeled S1PR1 m Ab has high radiochemical purity and general stability.In vitro and in vivo experiments have shown that 99 m Tc-HYNIC-S1PR1 m Ab has specificity and affinity for SK-HEP-1 cells.Therefore,this method is feasible and effective for targeting cells with high expression of S1PR1,and can be used as a basis to expand the application of this molecular probe.This experiment lays the foundation for further optimization of antibodies and development of monoclonal antibody fragments or mini antibody probes targeting the S1PR1 receptor. |
PDF Full Text Request