Evaluation Of The Effect And Significance Ofdown-regulation Of TLR5 On VEGFR Expression In Triple Negative Breast Cancer Based On Radionuclide Molecular Imaging

BackgroundWith the progress of clinical medical diagnosis and treatment technology,the survival rate of patients with breast cancer has been significantly improved,but so far there are still great difficulties in the diagnosis and treatment of triple negative breast cancer(TNBC).TNBC often occurs in young women under the age of 40.Due to the lack of estrogen receptor(ER),progesterone receptor(PR)and human epidermal growth hormone receptor 2(HER2),it is ineffective to endocrine therapy and targeted therapy for HER2.Clinically,systemic chemotherapy is mainly used.Although other targeted drugs are gradually used,the curative effect is not good due to the lack of a clear treatment target.TNBC has poor histopathological grade,high malignant degree,high metastasis rate in viscera and brain,high drug resistance rate,poor prognosis,early recurrence and high mortality.Early diagnosis and treatment have become the focus that we need to solve urgent ly.As a necessary condition for the sustainable survival and development of tumor cells,especially malignant tumors,angiogenesis plays an important role in tumor growth,invasion,diffusion and metastasis.Studies have shown that vascular endothelial growth factor(VEGF)and its receptor(VEGFR)are highly expressed in TNBC than in non-TNBC.VEGF and VEGFR may become a new therapeutic target,especially in refractory TNBC patients.Previous studies have shown that apatinib can enhances the anti-tumor effect of cisplatin on TNBC by inhibiting VEGFR2;targeted SHP-1 can inhibits the metastasis of TNBC after blocking VEGF paracrine and autocrine.In the treatment of TNBC,anti-angiogenic drugs and immune checkpoint inhibitors have synergistic effects,such as low-dose anti-angiogenic therapy makes breast cancer sensitive to PD-1 blockage,preoperative and postoperative anti-PD-L 1 combined with anti-angiogenic treatment of breast cancer metastasis.These studies show that anti-angiogenic therapy plays an irreplaceable role in TNBC,which is worthy of further exploration.The previous experiments in our laboratory showed that TLR5(Toll-like receptor 5)on TNBC activated can inhibit the growth,invasion and metastasis of TNBC.However,the relationship between TLR5 and VEGFR or whether TLR5 inhibits the growth of TNBC by inhibiting angiogenesis is not clear yet.Early detection,early diagnosis and early treatment of malignant tumors are very important to improve the cure rate and survival rate of patients.Nuclear medicine molecular imaging uses the identity and traceability of radionuclide tracers to recognize diseases at the molecular level.C ompared with the classical imaging diagnosis method,it can visually and non-invasively monitor the changes of molecules in vivo before the change of anatomical structure.In this study,we prepared TLR5 knocked-down TNBC cell line,prepared 125I-anti-TLR5 mAb and 125I-VEGF,established TLR5+and TLR5-4T1 tumor-bearing mice models,performed whole body phosphorus autoradiography and biological distribution study at different time points after injection of 125I-anti-TLR5 mAb/125I-VEGF,to explore the effect and significance of down-regulation of TLR5 on the expression of VEGF in triple-negative breast cancer.Methods1.4T1 cell lines with low expression and normal expression of TLR5 were established by transfecting TLR5-shRNA and negative control lentivirus4T1 cells were plated in 96-well plate overnight.Lentivirus-shRNA-TLR5 and negative control virus were added to the culture medium with a MOI value of 10.After 48 hours of continuous culture,20μg/mL puromycin was added and screened continuously for 7 days.Finally,the bright field and the corresponding fluorescence were captured using the optical microscope to calculate the transfection efficiency.2.Detection of TLR5 and VEGFR expression The expression of TLR5 and VEGFR was detected at mRNA and protein level by RT-PCR and Western Blot,respectively.3.Preparation of the 125I-anti-TLR5 mAb/115I-VEGF/125I-IgG 125I-anti-TLR5 mAb/125I-VEGF/125I-IgG was prepared by using Idogen method,125I-IgG was used as the isotype control.The labeling rate,radiochemical purity and stability of these three tracers were identified.4.Animal models were established6-8-weeks-old female BALB/c mice were subcutaneously injected with 2×106 cells into the right shoulder.The tumor-bearing mice were fed according to the SPF standard,and the tumor size and growth status of the mice were monitored every day.5.Whole-body phosphor autoradiographyOne day before injection of radiotracers,3.5%potassium iodide was added to the drinking water to block the thyroid gland uptake of iodine.The tumor-bearing mice were injected intraperitoneally with 125I-anti-TLR5 mAb/125I-IgG,125I-IgG as a non-specific control.The whole-body dynamic phosphor autoradiography was performed at 24h,48h and 72h after injection of 125I-anti-TLR5 mAb/125I-IgG while 125I-VEGF was 6h,12h,24h,48h to monitor the radioactive tracer concentration of the tumor.6.Biodistribution studies of tumor-bearing miceOne day before injection of radiotracers,3.5%potassium iodide was added to the drinking water to block the thyroid gland uptake of iodine.For the mice injected with 125I-anti-TLR5 mAb/125I-IgG,the biodistribution was prepared at 48 hours after injection.For the mice injected with 125I-VEGF,the biodistribution was prepared at 24 hours after injection.7.Fluorescence imaging4T1 TLR5+/-cells expressed green fluorescent protein(GFP),In order to prevent fluorescence quenching,tumors should be paid attention to avoid light after tumor exfoliation.Then we put them on the imaging board of the living animal imaging system,and selected the appropriate wavelength for fluorescence imaging.8.Autoradiography of tumor in vitroThe mice were killed and the tumor was peeled off.After the tumor was exposed to 30min on the phosphorus screen,the phosphorus screen was placed into the Cyclone Plus phosphorus screen scanning system for image acquisition.9.H&E and immunohistochemical stainingThe isolated tumors were fixed with 4%paraformaldehyde.H&E staining and immunohistochemical staining of TLR5,VEGFR and CD31 were performedResults1.Triple negative breast cancer 4T1 cell line with low TLR5 expression(TLR5-)and normal TLR5 expression TLR5(TLR5+)was constructed successfully,and the transfection efficiency reached almost 100%.The down-regulation of TLR5 expression in 4T1 cells and up-regulation of VEGFR expression was confirmed by RT-PCR and Western Blot at mRNA and protein levels.2.The labeling rate of 125I-anti-TLR5 mAb/125I-VEGF/125I-IgG was 92.5%,94.3%,and 89%,respectively.The radiochemical purity of 125I-anti-TLR5mAb/125I-VEGF/125I-IgG was 97.5%,94.2%,and 97.1%,respectively.The radiochemical purity of 125I-anti-TLR5 mAb/125I-IgG can reach more than 85%in 0.9%NaCl and FBS for 72 h,The radiochemical purity of 125I-VEGF in 0.9%NaCl and FBS can reach more than 85%after 48 hours The stability of radioactive tracers were good.3.The tumor-bearing mice models were successfully established.8-10 days after cell inoculation,the tumor diameter of tumor-bearing mice could reach 8-10mm.4.For 121I-anti-TLR5 mAb,the mouse was clearly visiblly imaged at 24h,however,the radioactivity background was very high,and 1251-anti-TLR5 mAb was mainly concentrated in the abdomen while the uptake of radioactive tracer in the tumor was not high.At 48h,the background was relatively low,and the concentration of radioactive tracer in the tumor increased and reached the highest.The radioactivity in vivo decreased at 72 h.The uptake of 125I-anti-TLR5 mAb in 4T1 TLR5+ tumor was significantly higher than that in TLR5-group.There was no obvious tumor image in 125I-IgG group at any time point suggesting that there was a specific accumulation of 125I-anti-TLR5 mAb in the tumor-bearing mice.For isolated tumour ex vivo imaging,4T1 TLR5+tumours showed much higher uptake of radioactivity than 4T1 TLR5-tumours5.For the whole-body phosphor autoradiography of mice in 125I-VEGF injection group,the results showed that mice image was clearly visible at 6h,the radioactivity background was very high,the 125I-VEGF were mainly accumulated in abdomen,and the uptake of radioactive tracer was not high at the tumor;at 12h,the radioactivity background was higher and the tumor uptake was increased;At 24h,the radioactive background in the body was relatively low and the image of tumor is the most clear.The radioactivity decreased at 48h.The 125I-VEGF uptake of 4T1 TLR5+tumor was significantly lower than that of 4T1 TLR5-group.There was no obvious imaging in the blocking group at any checked time point.The imaging results of tumors ex vivo were consistent with those of whole-body phosphor autoradiography6.For 125I-anti-TLR5 mAb injection group,the results of biodistribution study showed that the radioactivity of liver,spleen and kidney was the highest,suggesting the metabolism of radiotracer was mainly through liver and kidney.The uptake of 125I-anti-TLR5 mAb in 4T1 TLR5+tumor was higher than that in TLR5-,which was statistical differences.The uptake of 125I-anti-TLR5 mAb by tumor was significantly higher than that of 125I-IgG.The difference was statistically significant,which was consistent with the results of imaging.7.For 125I-VEGF injection group,the radioactivity of liver,spleen and kidney was the highest in 24h,and the metabolism of radioactive tracer was mainly through liver and kidney.The results showed that the uptake of 125I-VEGF in 4T1 TLR5+tumor-bearing mice was significantly lower than that in 4T1 TLR5-group,which was consistent with the results of imaging.8.Fluorescence imaging clearly showed the tumor,which further confirmed that the lentivirus with GFP tag was successfully transfected.9.H&E staining showed the characteristics of tumor tissue.immunohistochemistry staining showed that the expression of TLR5 in 4T1 TLR5+tumors was higher than that in 4T1 TLR5-tumors.The average value of VEFGR expression and the MVD of CD31-labeled blood vessel in 4T1 TLR5+tumor was lower than that of 4T1 TLR5-tumor.Conclusion1.In vitro experiments showed that down-regulation of TLR5 expression promoted up-regulation of VEGFR expression in triple-negative breast cancer.2.The prepared 125I-anti-TLR5 mAb/125I-VEGF/125I-IgG probe has good labeling efficiency,radiochemical purity and stability.3.The results of dynamic whole-body phosphor autoradiography imaging were consistent with the results of biodistribution.125I-anti-TLR5 mAb was specifically accumulated in 4T1 TLR5+tumors and the radioactivity uptake in 4T1 TLR5+tumors were higher than that in 4T 1 TLR5-tumors,while the concentration of 125I-VEGF in 4T1 TLR5+tumors was lower than that in 4T1 TLR5-tumors4.Immunohistochemical staining showed that compared with 4T1 TLR5+tumor,the expression of TLR5 decreased,the expression of VEGFR increased,and the MVD value of CD31 staining increased in 4T1 TLR5-tumor.It is further proved that the down-regulation of TLR5 can promote the expression of VEGFR and angiogenesis in triple negative breast cancer.Innovation1.125I-anti-TLR5 mAb/125I-VEGF,was successfully prepared and could specifically bind to TNBC tumor cells,and had specific uptake in the tumor of TNBC tumor-bearing mice.2.Whole-body phosphor autoradiography can dynamically observe the concentration of 125I-anti-TLR5 m Ab/125I-VEGF probe in tumor-bearing mice and realize non-in vasive observation of the effect of TLR5 expression on VEGFR in vivo.3.The expression of TLR5 in triple negative breast cancer is down-regulated,the expression of VEGFR is up-regulated.The effect of TLR5 on the biological behavior of TNBC may be by regulating the expression of VEGFR and angiogenesis.