TLR5 Targeting Nuclide/Fluorescence Molecular Imaging And Related Mechanism For Triple-negative Breast Cancer

BackgroundBreast cancer is the most common cancer in female worldwide,which can be classified into two groups,triple-negative breast cancer(TNBC)and non-TNBC according to whether there is estrogen receptor(ER),progesterone receptor(PR)and human epidermal growth factor receptor 2(HER2)expression.TNBC is more aggressive and is prone to early metastasis than non-TNBC.At present,targeted therapy for breast cancer mainly relies on ER,PR and HER2 expressed by cancer cells,while triple-negative breast cancer does not express these three types of receptors,so it lacks molecular targets.It seriously affects the early diagnosis,treatment and prognosis of TNBC.Nowadays,the commonly used non-invasive methods for diagnosing TNBC in clinic mainly include imaging examination and serum tumor marker detection,which have obvious limitations in terms of low specificity and low practicability.Therefore,there is an urgent need to search for highly selective and highly specific molecular markers for TNBC,to provide a theoretical and experimental basis for early diagnosis,monitoring and targeted therapy of TNBC.Nuclear medicine molecular imaging is a non-invasive imaging method based on metabolic and functional abnormalities.In terms of tumor diagnosis and staging,compared with traditional imaging methods CT and MRI,nuclear medicine molecular imaging is more suitable for detecting and quantifying tumor targeting molecules.18F-FDG(F-18-deoxyglucose)PET/CT(positron emission tomography/computed tomography)have been widely used in clinical practice.However,18F-FDG is a non-specifically binding probe and,in some cases,cannot be clearly diagnosed.Therefore,finding better molecular targets for tumor diagnosis and treatment is critical to the clinic.The research on molecular imaging is progressing rapidly,and its imaging is based on the changes of function and metabolism,so it shows great potential and good application prospect in the early diagnosis and treatment of tumors.Many researches showed that innate molecules were participated in tumor developments and prognosis,such as CCAT2(a kind of long non-coding RNA,metastasis and prognosis)and three miRNAs(potential prognostic biomarker in patients with bladder cancer).Toll-like receptor(TLR)is a membrane-bound protein that is primarily expressed in immune cells,such as monocytes/macrophages,vascular endothelial cells,etc.,and can also be expressed in other types of cells,such as cancer cells.The TLR family(TLR1-13)recognizes different pathogen-associated molecular patterns(PAMP).As a member of TLR family,TLR5 is known to specifically recognize flagellin,which is the only protein ligand in the TLR family.Recent studies have found that TLR5 is highly expressed in a variety of tumors,such as ovarian,gastric,and colon cancer,and that TLR5 pathway activation significantly inhibited tumor growth.More importantly,research has found that TLR5 was highly expressed in TNBC and is closely related to cancer progression.Considering that TLR5 expression is related to cancer progression,we hypothesized that TLR5 may be a new biomarker for monitoring TNBC,so we chose the TNBC cell line to study whether it might be a target molecule for non-invasive monitoring of TNBC and the underlying mechanism.In this paper,we chose TNBC,which is difficult to diagnose at early stage and with high incidence,as a study object.We prepared radioprobe 125I-anti-TLR5 mAb to study whether TLR5 could be as a reporter for monitoring triple-negative breast cancer and the underlying mechanism,evaluating by radionuclide imaging and fluorescent imaging.We successfully constructed TLR5-4T1 with decreased TLR5 expression and TLR5+4T1 cells with normal TL5 expression.Then we compared the two newly constructed cells in proliferation,invasion,metastasis,and apoptosis both in vivo and in vitro.A new radionuclide molecular probe 125I-anti-TLR5 mAb was prepared and identified.The specificity of its binding to 4T1 cells was studied and compared with control 125I-IgG.Meanwhile,we used radioimmunoimaging and fluorescent imaging in co-determining the location of tumors.This paper aims to find a highly targeted molecule for the monitoring of TNBC and provides a new strategy for the early non-invasive diagnosis of TNBC.Part One TLR5 as a reporter for monitoring triple-negative breast cancer evaluating by radioimmunoimaging and fluorescent imagingMethods1.TLR5-and TLR5+4T1 cell lines were constructed by lentiviral shRNA transfection2.Preparation of 125I-anti-TLR5 mAb and 125I-IgGThe probes,125I-anti-TLR5 mAb and 125I-IgG,were prepared using lodogen method.PD-10 gel column was used for eluting and purifying.We calculated the labeled rate and specific activity of the two probes.Radiochemical purity rate was evaluated using paper chromatography method.125I-anti-TLR5 mAb or 125I-IgG was mixed with saline and serum,respectively.The stability was measured by paper chromatography at room temperature at 24h,48h and 72h.3.Detection of the expression of TLR5 in TLR5-and TLR5+4T1 cells with qPCR and Western blotTLR5-and TLR5+4T1 cells were routinely cultured aseptically.qPCR was used to detect the TLR5 mRNA level;Western blot was used to analyze the TLR5 protein level.4.Binding assay of 125I-anti-TLR5 mAb in vitroTLR5-and TLR5+4T1 cells were respectively seeded in 24-well plates,5 × 105 cells per well,and 125I-anti-TLR5 mAb was added according to a concentration(1?30nM).After 2 hours,the cells were washed with PBS and measured the cell radioactivity count.The differences between the affinity dissociation constant(Kd value)and the maximum amount of receptor binding(Bmax)of 125I-anti-TLR5 mAb for TLR5-and TLR5+4T1 cells were compared and analyzed.5.Binding blocking assay of probes in vitroTLR5+4T1 cells(2 × 105/well)were seeded on 24-well plates,and the quantitative 125I-anti-TLR5 mAb or 125I-IgG and unlabeled anti-TLR5 mAb(0.1,1,10,100,1000 nmol/L).After 45 min,washed with PBS,measuring the cpm,and analyzed the B/B0 of 125I-anti-TLR5 mAb and 125I-IgG.6.Construction of animal model and imaging6-week-old male nude mice were inoculated with TLR5-4T1 cells(1 ×107)on the left shoulder and TLR5+4T1 cells(1 × 107)on the right shoulder.125I-anti-TLR5 mAb or 125I-IgG(24h pre-block thyroid with 3%Nal)were injected through the tail vein at the 4,6,8,10,and 12 days after the inoculation,respectively.Whole body phosphor screen imaging was performed at 24,48,and 72 h after 125I-anti-TLR5(0.37 MBq)or 125I-IgG(0.37 MBq)injection,and fluorescence imaging was performed at 72 h after probes were injected.For the anti-TLR5 mAb blocking group,100 ?g of unlabeled anti-TLR5 mAb was injected through the tail vein 30 minutes before 125I-anti-TLR5 mAb injection.7.Biodistribution studyAfter the whole body phosphor screen imaging and fluorescence imaging were finished,5 mice were randomly selected and the main organs(heart,liver,lung,kidney,thyroid,spleen,bone),tumors and contra-lateral muscle tissues were weighed and radioactivity was measured(cpm),along with injected dose(standard source).Calculating the%ID/g.8.H&E staining and immunohistochemical staining of tumor tissueAfter whole body phosphor screen imaging,tumor tissues were isolated.4%paraformaldehyde was used to preserve tumor tissues and paraffin sections were prepared,H&E staining was performed,and anti-TLR5 antibody was used for immunohistochemistry staining,observe and photograph under a microscope,and calculate the positive expression rate of TLR5.Results1.TLR5-4T1 cell lines with down-regulated TLR5 expression and TLR5+4T1 cell lines with normal TLR5 expression were successfully constructed.2.The radionuclide probes,125I-anti-TLR5 mAb and 125I-IgG,were successfully prepared,with labeling rate was 94.55%and 92.14%,respectively.The specific radioactivity was 925.25MBq/mg and 915.58 MBq/mg,respectively.The radiochemical purity was respectively 95.44%and 92.47%.Both 125I-anti-TLR5 mAb and 125I-IgG have good stability as determined by paper chromatography,and they remain above 95%by 72h.3.The binding assay showed that the Kd values of TLR5-and TLR5+4T1 cells for 125I-anti-TLR5 mAb were 6.463nmol/L and 6.069nmol/L,respectively.The affinity of TLR5+4T1 cells was slightly higher than that of TLR5-4T1 cells;The Bmax values of TLR5-4T1 and TLR5+4T1 cells were respectively 2811 cpm/5 X 105 cells and 6245 cpm/5 × 1 05 cells.The Bmax of TLR5+4T1 cells was significantly higher than TLR5-4T1 cells.4.Competitive binding experiments showed that unlabeled anti-TLR5 mAb can specifically block the binding of TLR5+4T1 cells to 125I-anti-TLR5 mAb,and the amount of unlabeled anti-TLR5 mAb is positively proportional to the blocking rate.However,unlabeled anti-TLR5 mAb failed to block the binding of TLR5+4T1 cells to 125I-IgG.5.From the sixth day of tumor cell inoculation,TLR5+4T1 tumors can be displayed by phosphor screen autoradiography,and by the 14th day,the imaging becomes much clearer.While TLR5-4T1 tumor was not displayed clearly until the 14th day.Fluorescence imaging demonstrated the change of TLR5-and TLR5+4T1 tumors over time.In addition,the 125I-IgG group and the unlabeled anti-TLR5 mAb block group could not clearly show tumors' location at all checked time point.6.The biodistribution results revealed 125I-anti-TLR5mAb was mainly metabolized by liver and kidney,and the T/NT ratio of TLR5+4T1 tumor(8.413 ± 0.5270)was significantly higher than that of TLR5-4T1 tumor(2.489± 0.1541).In the unlabeled anti-TLR5 mAb blocking group,the T/NT ratio of TLR5+4T1 tumors was only 1.57 ± 0.13.In the 125I-IgG group,the T/NT ratio of TLR5+4T1 tumors was only 2.023±0.2149.7.Tumor histopathological examination showed a typical tumor tissue feature.The cell positive rate of TLR5+4T1 tumor in immunohistochemical staining was significantly higher than that of TLR5-4T1 tumor,which was consistent with the expression of TLR5 in two kinds of breast cancer cell lines measured in vitro.Part Two The effects of TLR5 on the invasion and metastasis for triple-negative breast cancer and molecular mechanismMethods1.TLR5-and TLR5+ 4T1 cell lines were constructed as described in the Part One2.CCK-8 analysis was used to evaluate TLR5-and TLR5+ 4T1 cells proliferation abilityTLR5-and TLR5+4T1 cells were plated in 96-well plates with 2 × 103 cells in each well.After cultivating overnight,the absorbance at 450 nm was measured at 0,6,24,36,and 48h with a microplate reader.3.Flow cytometry was used to analyze the differences in apoptosis of TLR5-and TLR5+4T1 cellsTLR5-and TLR5+4T1 cells were digested with 0.25%trypsin without EDTA and phenol red,centrifuged,and the cells were treated with an apoptosis detection kit according to the instructions.After filtration,apoptosis was detected by flow cytometry.4.Colony formation assay was used to analyze the tumorigenicity of TLR5-and TLR5+4T1 cellsTLR5-and TLR5+4T1 cells were seeded in 6-well plates,1 ×103 cells per well,and the medium was changed every three days for 14 days.The medium was discarded on the 15th day,washed with PBS,then stained with 0.005%crystal violet staining solution for 20 minutes,and washed with PBS.5.Wound healing assay was used to analyze the migration ability of TLR5-and TLR5+4T1 cellsTLR5-and TLR5+4T1 cells were seeded in 6-well plates with 5 × 105 cells per well.After overnight,streak with 10 ?L pipette tips,wash with PBS,take pictures for Oh,and change to serum-free medium.After 24h,wash with PBS and take pictures for 24h.6.Transwell experiments were used to analyze the difference in invasion ability between TLR5-and TLR5+4T1 cellsThe upper chamber of Transwell was plated with 100 ?L of 300 pg/mL matrigel.After the gel was solidified,the upper chamber was inoculated with 8 × 1 03 TLR5-or TLR5+4T1 cells.The upper chamber was serum-free medium.The lower chamber uses normal medium.After 24 hours,wipe the matrigel and cells inside the cell with a cotton swab,fix with methanol,and stain with crystal violet,observe and take a picture under a microscope.7.Pulmonary metastasis experiments were performed to analyze the differences in metastatic capacity of TLR5-and TLR5+ 4T1 cells in vivoSix-week-old male nude mice were injected with TLR5-and TLR5+4T1 cells through the tail vein,respectively,with 1 × 106 cells per mouse.After 3 weeks,mice were sacrificed and intact lungs were dissected.After weighing,fluorescence imaging was performed.8.Tumor-bearing mouse models were constructed and the tumor size was analyzed6-week-old male nude mice were inoculated with TLR5-4T1 cells(1 ×107)on the left shoulder and TLR5+4T1 cells(1 × 107)on the right shoulder.Tumors were weighed and measured in size from the day 6 to the day 14(once every another day).9.qPCR and Western blot were performed to verify the signal pathway changes of EMT due to down-regulation of TLR5The relationship between TLR5 down-regulation and EMT pathway was detected by qPCR and Western blot at the mRNA level and protein level.Results1.The absorbance of TLR5-4T1 cells was higher than that of TLR5+4T1 cells at all time points in the CCK-8 assay,which suggested that TLR5-4T 1 cells had stronger proliferation ability than TLR5+4T1 cells.In the clone formation experiment,the colony formation rate of TLR5-4T1 cells was significantly higher than that of TLR5+4T1 cells,which suggested TLR5-4T1 cells had stronger tumorigenicity ability than TLR5+4T1 cells.Wound healing experiments proved that the migration area of TLR5-4T1 cells was larger than that of TLR5+4T1 cells.The results of the Transwell experiment showed that the number of TLR5-4T1 cells successfully transmembrane was significantly larger than TLR5+4T1 cells.TLR5-4T1 cells had stronger proliferation,invasion,metastasis,and tumorigenicity than TLR5+4T1 cells.However,there was no significant difference in apoptosis.2.In vivo,TLR5-4T1 cells caused much more lung metastases,and their metastatic capacity was stronger than TLR5+4T1 cells.3.In vivo,the weight and volume of TLR5-4T1 tumors were higher than TLR5+4T1 tumors at all time points.4.TLR5 down-regulation promoted the EMT process.After down-regulation of TLR5,the expression of E-cadherin decreased and the expression of N-cadherin,vimentin,fibronectin,TWIST1,TRAF6,and SOX2 increased,both in mRNA and protein levels.After TLR5 expression decreased,the EMT process accelerated and the related E-cadherin expression decreased,and the expression of N-cadherin,vimentin,and fibronectin increased;the expression of EMT-related signal molecules TWIST1,TRAF6,and SOX2 also increased significantly;TLR5 was negatively correlated with TRAF6 expression,while TRAF6 and SOX2 expression were positively correlated,and increased SOX2 expression accelerated the EMT process.The result suggested that down-regulation of TLR5 can promote EMT process through the TRAF6-SOX2 pathway.Conclusion1.TLR5-and TLR5+4T1 cell lines were successfully established.2.125I-anti-TLR5 mAb can specifically bind to 4T1 cells in vitro.Binding of 4T1 cells with 125I-anti-TLR5 mAb was positively related to TLR5 expression.3.The biodistribution of TLR5+and TLR5-4T1 tumor mice model was consistent with the results of whole body dynamic imaging.For mice models injected 125I-anti TLR5 mAb,the tumor radioactivity count was positively related to TLR5 expression.Through whole-body phosphor screen imaging,TLR5+4T1 tumor can be seen early on the sixth day after tumor bearing,while the TLR5-4T1 tumor cannot be observed until the 14th day.4.Down-regulation of TLR5 expression promoted the EMT process,enhanced the proliferation,invasion,and metastatic ability of 4T1 cells.Fluorescence imaging and H&E staining further confirmed that the lung metastasis ability of 4T1 cells was significantly enhanced with down-regulated TLR5 expression.TLR5 regulated the activity of 4T1 cells mainly through the TLR5-TRAF6-SOX2 pathway.Innovation1.TLR5-4T1 cell line with down-regulated TLR5 expression and GFP tag was constructed for the first time.2.The first to use radionuclide probe 125I-anti-TLR5 mAb for tumor imaging,and it was found that 125I-anti-TLR5 mAb can specifically bind 4T1 cells both in vitro and in vivo.125I-anti-TLR5 can be specifically accumulated in TLR5 expression-positive tumor regions in tumor-bearing mouse models.So TLR5 has potential clinical application prospects as a new molecular target for triple-negative breast cancer.3.This study found that down-regulation of TLR5 expression affected the biological characteristics of triple-negative breast cancer cells and found a relation between TLR5 and the EMT process and revealed potential signaling pathways.