TLR5: A prognostic and monitoring indicator for triple-negative breast cancer

A novel, highly selective biomarker is urgently needed to predict and monitor triple-negative breast cancer (TNBC) because targeting molecules are not currently available. Although associated with various malignant tumors, the role of toll-like receptor 5 (TLR5) in TNBC remains uncertain. We aimed to define the effects of TLR5 in TNBC to determine whether it could serve as a prognostic and monitoring indicator for TNBC. We established TNBC cell line 4T1 with low TLR5 expression (GFP tag; TLR5− 4T1) and with normal TLR5 expression (GFP tag; TLR5+ 4T1) using lentivirus-shRNA-TLR5 knockdown transfection and negative lentivirus transfection, respectively. Detected by western blot and qPCR, we found knockdown of TLR5 resulted in decreased expression of TLR5 and E-cadherin and increased expression of N-cadherin, vimentin, fibronectin, TRAF6, SOX2, and Twist1, which were related to EMT (epithelial–mesenchymal transition). In addition, downregulation of TLR5 increased the invasion and migration of 4T1 cells in vitro, which were investigated by CCK-8 and wound healing, as well as transwell assay and colony formation. Furthermore, the metastatic ability of TLR5− 4T1 cells to the lungs was also increased compared to TLR5+ 4T1 cells in vivo. To verify the effect of TLR5 as a monitor indicator, mice bearing TLR5+ and TLR5− 4T1 tumors injected with 125I-anti-TLR5 mAb or isotype 125I-IgG were assessed by whole body phosphor-autoradiography and fluorescence imaging in vivo. Phosphor-autoradiography of model mice revealed early tumors at 6 days after inoculation with TLR5+ 4T1, but not TLR5− 4T1 cells. Intratumoral accumulation of radioactivity positively correlated with TLR5 expression, and fluorescence imaging in vivo revealed both TLR5+ and TLR5− 4T1 tumors. Our results suggested that downregulation of TLR5 in TNBC increased tumor invasiveness and EMT expression via TRAF6 and SOX2 pathway and TLR5 could serve as a prognostic and monitoring indicator for TLR5-positive tumors.


Introduction
Membrane-bound toll-like receptors (TLRs) play key roles in innate and adaptive immunity. They are primarily expressed in some types of immunocytes and recognize conserved pathogen-associated molecular patterns (PAMPs) [1][2][3][4] . Various members of the TLR family play diversified roles in cancer progression and development 5,6 . For example, positive TLR3 expression indicates a favorable prognosis for patients with neuroblastoma 7 , and TLR4 overexpression is reduced in squamous cell carcinoma of the skin 8 . Unlike other TLRs, TLR5 is not expressed in murine macrophages and conventional dendritic cells 9 , but it is expressed at high levels in some malignancies, such as non-small cell lung cancer 10 and NK cells within breast cancer 11 . Cancer cell proliferation and tumor growth are inhibited by TLR5 signaling [12][13][14] , but the underlying mechanism has not been fully elucidated.
Breast cancer is the most common cancer among women, and it is associated with high mortality rates [15][16][17] . It can be classified into TNBC (triple-negative breast cancer) and non-TNBC subtypes. The TNBC type accounts for 12-24% of all breast cancers and is more aggressive and prone to metastasis than the non-TNBC type. Current clinical management of breast cancer mainly depends on estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) genes as molecular markers and therapeutic and prognostic indicators. However, targeted diagnoses and therapy are not currently feasible for TNBC because it does not express ER, PR, and HER2 18 . Radical surgery is still the first choice of treatment for breast cancer, even though metastasis usually appears within a few years 19 . Hence, the identification of new monitoring molecular markers and clarifying the underlying mechanisms are critical for improved prognosis for patients with TNBC.
Traditional computed tomography (CT) and magnetic resonance imaging (MRI) in tumor diagnosis and staging mainly reveal anatomic changes, and these are usually found at the later stages 20 . Noninvasive nuclear molecular imaging is a whole-body scanning modality that is based on metabolism and abnormal function. It is a combination of anatomical and functional imaging and is more suitable for the detection and quantitation of target molecules in tumor tissues. Presently, F-18-deoxyglucose ( 18 F-FDG) positron emission tomography/computed tomography (PET/CT) is clinically applied to evaluate tumor metabolism 21 . However, 18 F-FDG is not specific, which sometimes results in diagnostic inaccuracy. Hence, identifying more effective molecular targets for tumor prognosis and therapy is critical. Many recent studies have shown that some innate molecules participate in tumor development and prognosis, such as colon cancerassociated transcript2 (CCAT2), metastasis and prognosis 22 and three miRNAs (potential prognostic biomarkers in patients with bladder cancer) 23 . Molecular imaging has rapidly developed and shown great potential in early tumor diagnosis and treatment based on physical and metabolic changes 24 . Radioisotope-labeled target molecules for breast cancer such as 18 F-labeled aptamers of Her2/ErbB2 (human epidermal growth factor receptor 2) 25 , 125 I/ 131 I-labeled anti ICAM-1(intercellular cell adhesion molecule-1) antibody 26 and 89 Zr-Transferrin 27 , have been developed. However, these target molecules are limited by low selection and tumor expression. We developed a 131 I-labeled anti-TLR5 antibody that can indicate allorejection 28 . Here, we aimed to determine whether this radiolabeled antibody could serve as a noninvasive monitoring target for early TNBC using the TNBC cell line, 4T1.

Generation of TLR5 knockdown 4T1 cells
Murine TNBC cell lines 4T1 23 were obtained from the American Type Culture Collection (ATCC). The cells were seeded in 24-well plates (1 × 10 5 /well) and incubated at 37°C in a humidified 5% CO 2 atmosphere overnight. The medium was then replaced with negative control virus or Lentivirus-shRNA TLR5 diluted in fresh medium at a ratio of 1:100, and the cells were incubated at 37°C in a humidified 5% CO 2 atmosphere for 24 h. Finally, the diluted virus was replaced with fresh medium and incubation proceeded under the same conditions for another 48 h. Thereafter, transfection efficiency was calculated. Transfected cells were filtered out using Puromycin (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China).

Western blot and quantitative PCR (qPCR)
Total protein concentrations for western blot were measured using protein assay kits (Beyotime Biotechnology, Shanghai, China). Total protein (20 μg) was loaded onto gel (Bio-Rad Laboratories, Hercules, CA, USA), along with Chameleon Duo ladder protein marker (SMOBIO, Taiwan, R.O.C). Proteins were resolved by electrophoresis at 80 V for 30 min followed by 100 V for 60 min and transferred onto nitrocellulose membranes. Nonspecific binding was blocked with 5% skim milk in blocking buffer for 2 h at room temperature (20°C) and further incubated overnight at 4°C with 1:1000 diluted rabbit anti-mouse TLR5/vimentin/fibronectin/N-cadherin/E-cadherin/TRAF6/SOX2/Twist1 antibodies and 1:10000 diluted rabbit anti-mouse GAPDH antibody. Membranes were washed three times with TBS-Tween 20 (TBS-T), incubated with anti-rabbit IgG antibodies (1:5000) for 2 h at room temperature, then scanned and quantified using a Tanon 4200 imaging system (Tanon Science and Technology Co., Ltd., Shanghai, China).

Flow cytometry
Apoptosis was evaluated by digesting TLR5 + and TLR5 − 4T1 cells with 0.25% trypsin (without EDTA and phenol red), after which, the cells were sedimented by centrifugation (speed in 800g, for 5 min). The cells were washed twice with cold PBS and suspended in 400 μL of 1 × annexin V binding solution at a concentration of 1 × 10 6 /mL. Annexin V-YF647A (5 μL) was gently mixed with 10 μL of PI staining solution, incubated at 4°C for 15 min in darkness, passed through a 200-mesh filter, and immediately analyzed by flow cytometry. Experiments were repeated three times.

Colony formation
TLR5 + and TLR5 − 4T1 cells (1 × 10 3 /well) were seeded in six-well plates and the medium was changed every 3-4 days. The plates were incubated at 37°C in a humidified incubator for 14 days. At the end of the experiment, the cells were washed with PBS, incubated with 0.005% crystal violet for 15 min, then rinsed with PBS. Colony formation rates were calculated as follows: (number of colonies/number of seeded cells) × 100%. Experiments were repeated three times.
Wounding healing assays TLR5 + and TLR5 − 4T1 cells were seeded in six-well plates and incubated overnight to obtain confluent monolayers. Monolayers were scratched using a sterile pipette tip, and wounds were examined after incubation at 37°C in a humidified 5% CO 2 incubator for 24 h. Experiments were repeated three times.

Transwell assays
These assays proceeded in 24-well Transwell units with an 8 μm pore polycarbonate membrane. Matrigel (100 μL; 300 μg/mL) was added to the upper compartment. After overnight starvation, suspended cells were seeded in the upper compartment in serum-free medium, and medium supplemented with 10% FBS as a chemoattractant was placed in the lower compartment. Cells in the upper compartment were removed 24 h later by gentle swabbing. Cells that had migrated to the lower surface of the membrane were stained with crystal violet and counted at 400× magnification in five high power fields. Triplicate samples were tested, and the experiment was repeated three times.

Lung metastasis experiment
Mice were injected with 1 × 10 6 TLR5 + or TLR5 − 4T1 cells via the tail vein, and the mouse lungs were weighed 20 days later and dissected for hematoxylin and eosin (H&E) staining and fluorescence imaging. The experiment was repeated at least three times.

4T1 breast cancer-bearing mice model
All animal studies proceeded in accordance with protocols approved by the Animal Care and Use Committee of Shandong University. Subcutaneous TLR5 + and TLR5 − 4T1 tumor cells were induced in 6-week-old nude male mice, respectively, by injecting their lower left and right flanks with 1 × 10 6 TLR5 + 4T1 and TLR5 − 4T1 cells suspended in 200 μL of PBS. The tumors on mice were monitored every other day. The tumor-bearing mice were assessed by phosphor-autoradiography and fluorescence imaging, and biodistribution was also determined.

Preparation of 125 I-anti-TLR5 mAb and 125 I-IgG
We prepared 125 I-anti-TLR5 and 125 I-IgG as described previously 28 . A 1:2 (v/v) mixture of 0.9% saline and methanol served as an unfolding agent. The stability of 125 I-anti-TLR5 mAb and 125 I-IgG in PBS was evaluated using paper chromatography in PBS and murine serum, then radioactivity was determined using a Gamma counter. Competitive binding was assessed in TLR5 + 4T1 cells (5 × 10 5 /well) seeded in 24-well plates with 0.1-1000 nM anti-TLR5 mAb and 10 nM 125 I-anti-TLR5 mAb and incubated at 37°C for 45 min. The supernatants were discarded, and then the cells were washed twice with iced 1 × PBS containing 0.1% BSA and harvested for radioactivity determination using a Gamma counter.

Dynamic whole-body phosphor-autoradiography
Three days before radiotracers injection, 3% potassium iodide was added to the drinking water of the mice to block iodine uptake by the thyroid gland. Tumor-bearing mice (total n = 50, n = 25 per group, randomly divided) were each injected with 0.37 MBq of 125 I-antiTLR5 mAb (0.38 μg) or 125 I-IgG (0.38 μg) via the tail vein. Wholebody phosphor-autoradiography scanning proceeded on day 6, 8, 10, 12, and 14 after tumor cell inoculation (at 48 h after the 125 I-antiTLR5 or 125 I-IgG injections), respectively. For the blocking group (n = 5, randomly divided), mice were injected with unlabeled anti-TLR5 mAb (100 μg) 30 min before the 125 I-antiTLR5 mAb (0.37 MBq, 0.38 μg) injection. Whole-body phosphorautoradiography scanning proceeded on day 12 after tumor cell inoculation (at 48 h after the 125 I-antiTLR5 injections). Mice anesthetized with sodium pentobarbital (0.6%) were placed supine on a storage phosphor screen plate (back to the plate) and left for 20 min in darkness. Plate was then transferred to a Cyclone Plus scanner (PerkinElmer). Manually drawn rectangular regions of interest (n = 5) within the target area at each time point were semi-quantified. Digital light units (DLU)/mm 2 were obtained using OptiQuant TM image analysis software 5.0 (PerkinElmer). Tumors were individually stripped and imaged.

Fluorescence imaging
The 4T1 cells were transfected with lentivirus-TLR5 knockdown, and the lentivirus expressed green fluorescent protein (GFP) after integration into 4T1 cells. Subcutaneous TLR5 − and TLR5 + 4T1 tumors were induced as described above with TLR5 − 4T1 and TLR5 + 4T1 cells injected into the lower right and left flanks, respectively, and 4T1 cells without Lentivirus transfection were injected into the back. The mice were anesthetized on day 6, 8, 10, 12, and 14 after tumor cell inoculation, respectively, then the skin on the surface of the tumors was peeled, and the mice were placed prone on the imaging plate and photographed using IVIS Spectrum (PerkinElmer).

Biodistribution studies
Three days before injecting the radiotracers, 3% potassium iodide was added to the drinking water of the mice to block iodine uptake by the thyroid gland. Model mice (total n = 30, n = 15 per group, randomly divided) were injected with 125 I-anti-TLR5 mAb or 125 I-IgG (0.37 MBq), then sacrificed, and dissected 24, 48, and 72 h later. Tumors, blood, and major tissues/organs (heart, lung, liver, kidney, spleen, small intestine, and muscle) were harvested and weighed. Samples and primed standards were measured using a Gamma counter. Tissue radioactivity was expressed as the percent injected dose per gram (%ID/g), and the target to non-target (T/NT) ratio was defined as the ratio of radioactivity that accumulated in tumors to that in the contralateral muscle. The experiment was repeated three times.

Statistical analysis
Data were presented as the means ± standard deviation (SD) derived from at least three independent experiments. Student t tests were applied using Graph Pad Prism version 5 (GraphPad Software Inc., La Jolla, CA, USA). Significant differences were considered at *P < 0.05 and **P < 0.01.

TLR5 downregulation prompted epithelial-mesenchymal transition (EMT) in 4T1 cells
We quantified the protein and mRNA expression of TLR5, E-cadherin, N-cadherin, vimentin, fibronectin, TRAF6, SOX2 and the transcription factor, Twist1, by western blot and qPCR to verify the effects of TLR5 on EMT ( Fig. 1a and b, respectively). The protein expression of E-cadherin significantly decreased after TLR5 downregulation, whereas those of vimentin, fibronectin, N-cadherin, TRAF6, SOX2 and Twist1 increased. The trend in mRNA levels was similar. These results suggested that TLR5 downregulation induced EMT increasing via TRAF6 and SOX2 pathway in TNBC.

TLR5 downregulation promoted 4T1 metastasis in vivo
At 21 days after injecting TLR5 + 4T1, TLR5 − 4T1 and non-transfected 4T1 cells via the tail vein, the lungs were stripped from the mice, weighed, and assessed by fluorescence imaging and histological analysis. Figure 3a shows much larger green fluorescence areas indicating metastatic sites in the TLR5 − 4T1 than in the TLR5 + 4T1 group, and green fluorescence was absent in the nontransfected 4T1 group. The lungs were the heaviest, averaging~280 mg and reaching 300 mg, in the TLR5 − 4T1groups, whereas normal lungs weighed approximately 120 mg (Fig. 3b). Differences were quite apparent among the three groups (P < 0.05). Staining with H&E revealed typical tumor tissues changes within the lungs where fluorescence imaging positively. Tumor cells that metastasized to the lungs were closely arranged and had larger nuclei than those in normal lung tissues. The number and size of metastases were significantly smaller in the TLR5 + 4T1 than in the TLR5 − 4T1 group (Fig. 3c).

Successful preparation of 125 I-anti-TLR5 mAb and 125 I-IgG
The radiochemical purity of 125 I-antiTLR5 mAb and 125 I-IgG was both >90%. Both tracers were stable at >95% up to 72 h and relatively stable in serum and normal saline with no detectable significant differences between them ( Fig. 4a and B). Competitive binding analysis using >500fold excess of unlabeled anti-TLR5 mAb almost completely blocked 125 I-anti-TLR5 mAb (<5%) binding, with nonspecific binding for 125 I-IgG being~2% (Fig. 4c).

Dynamic phosphor-autoradiography and fluorescence imaging in vivo
Dynamic phosphor-autoradiography and fluorescence imaging proceeded on day 6, 8, 10, 12, and 14 after the mice were subcutaneously injected with TLR5 + 4T1 and TLR5 − 4T1 cells and at 48 h after injecting them with 125 I-anti-TLR5 mAb (Fig. 5a). Whole-body phosphorautoradiography showed higher and lower radioactivity uptake by TLR5 + 4T1 and TLR5 − 4T1 tumors, respectively, at all time points. Dynamic phosphorautoradiography on day 6 clearly showed TLR5 + 4T1 tumors, whereas TLR5 − 4T1 tumors were unclear. However, fluorescence imaging on day 6 clearly showed the location and boundary of TLR5 − 4T1 tumors, which might be due to the high specificity of 125 I-anti-TLR5 mAb binding. The radioactivity (DLU)/mm 2 was 6.67-fold higher in TLR5 + 4T1 than in TLR5 − 4T1 tumors (141,525 ± 8554 vs. 21,254 ± 2257 cpm). Autoradiography and fluorescence double imaging confirmed that TLR5 was a good reporter for noninvasive monitor and predictor of TNBC. Tumors were not obvious in specifically blocked and 125 I-IgG groups at any checked time point, Representative results of three independent experiments were reported. The data were presented as the means ± SD from three independent experiments, analyzed by Student's t test. *P < 0.05, **P < 0.01.

Fig. 2 TLR5 downregulation enhanced 4T1 tumor cell proliferation, migration and invasiveness in vitro.
Kaplan-Meier plotter online tools were used to identify that the patients suffered from breast cancer with low TLR5 expression had a significantly worse survival rate (a). CCK-8 assay showed that TLR5 − 4T1 cells have higher proliferation ability than TLR5 + 4T1 cells (b).No difference in apoptosis change in TLR5 downregulated 4T1 cells was detected compared with negative control group (c). The colony formation ability of TLR5 − 4T1 cells was significantly increased than TLR5 + 4T1 cells (d).The migration area of TLR5 − 4T1 group was larger than TLR5 + 4T1 group (e). The number of migrated cells was remarkably reduced in TLR5 + 4T1 group than TLR5 − 4T1 group (f). The data were presented as the means ± SD (n = 3) from three independent experiments, analyzed by Student's t test. *P < 0.05, **P < 0.01. Lungs from untreated mice, TLR5 + 4T1 and TLR5 − 4T1 model mice were weighed (b) and H&E staining was performed (c) (n = 3, **P < 0.01). Representative results of three independent experiments were reported. The data were presented as the means ± SD. The data were analyzed by Student's t test. *P < 0.05, **P < 0.01.
suggesting specific 125 I-anti-TLR5 mAb accumulation in TLR5-positive tumors (Fig. 5b). Furthermore, to confirm that tumor fluorescence was GFP-tagged, transfected, virus-derived, and TLR5-specific, we inoculated mice with three types of tumors cells. The 4T1 tumors on the right and left flanks were transfected with lentivirus-TLR5 knockdown and negative-control Lentivirus, respectively, and the 4T1 tumor on the back was not transfected. Figure 5c clearly showed tumors in both flanks, whereas the non-transfected 4T1 tumor appeared black.

Discussion
This study showed that TLR5 downregulation prompted TNBC proliferation, metastasis, and invasion, in vitro The data were presented as the means ± SD. The data were analyzed by Student's t test. *P < 0.05, **P < 0.01. I radiolabeled sodium iodide, h hours **P < 0.01 compared to the TLR5 + 4T1 tumor and the TLR5 − 4T1 tumor in the same mouse. Data are presented as a mean percent injected dose per gram (%ID/g) ± standard deviation (Mean ± SD) of five animals and in vivo. More importantly, we found that TLR5 on TNBC could potentially serve as a reporter for lower metastasis, lower invasion, and better prognosis. Our results revealed a novel target molecule for earlier TNBC detection and prognosis prediction, and this could provide the means for new strategies to monitor and predict TLR5-positive tumors.
Dynamic interactions between tumors and their microenvironments are essential for tumor growth, angiogenesis, and metastasis 29 . High levels of TLR5 were expressed during intestinal epithelial cell-mediated antitumor activity in a mouse xenograft model of human colon cancer 30 . Our findings are consistent with these results. To understand whether the downregulation of TLR5 could promote breast cancer metastasis and invasion, we detected EMT markers and its signal pathway, TRAF6 and SOX2. The EMT represented a fatal transfer of cancer progression 31 . Twist1, a transcription factor, played a key role in cancer development and progression 32 and up-regulated Twist1 induced EMT and Ecadherin repression, indicating that Twist1 promoted metastasis by inducing EMT 33 . Using western bolt and qPCR, we found E-cadherin and TLR5 expression decreased in TLR5 − 4T1 cells, whereas vimentin, fibronectin, N-cadherin and Twist1 expression increased. The metastatic and invasive abilities of TLR5 − 4T1 cells were enhanced. Notably, The expression of TRAF6 and SOX2 increased, and it was reported that elevated TRAF6 expression prompted SOX2 expression 34 , and upregulation of SOX2 prompted tumor metastasis through EMT, in breast cancer and other kind of tumors 35,36 . These results might help to further understand why downregulated TLR5 expression promoted 4T1 cell proliferation, metastasis, and invasion. Our data in vitro showed that TLR5 expressed in breast cancer could serve as a reporter of cancer invasiveness.
Bioprospecting for target molecules represents a promising solution for cancer prognosis 37,38 . TNBC is associated with aggressive tumor behavior and a worse prognosis 39 . However, the early discovery of TNBC is particularly important, since its onset, early metastasis, and aggressive invasiveness is coupled with a lack of targeted therapy [40][41][42] . Therefore, identifying novel targets that closely correlate with TNBC progression is of considerable importance for its monitor and prognosis.
We previously correlated TLR5 expression with allotransplant rejection 28 . The expression of TLR5 in NK cells from patients with breast cancer has recently been discovered 43 , and TLR5 expression could restrain tumor growth and metastasis in vitro and in vivo 44 . Based on these findings, we postulated that TLR5 in TNBC cells might play an important role in the progression of TNBC and serve as a novel target suitable for early TNBC monitor and prediction.
Targeted radioimmunoimaging with monoclonal antibodies against many kinds of cancers has been successful 24 . Radiolabeled 125 I-antiTLR5 mAb might provide a means of visualizing TLR5 expression in vivo. Our data suggested that 125 I-antiTLR5 mAb, which is clinically relevant, can be used in TNBC tumor models.
Our results in vitro indicated that TLR5 in TNBC was closely associated with cell invasion, but whether the same For TLR5 + tumors, representative microscopy images the H&E staining (c) and immunohistochemistry staining for TLR5 (d), black arrow refers to the positive area (brown). n = 5, **p < 0.01. Representative results of five independent experiments were reported. The data were presented as the means ± SD. The data were analyzed by Student's t test. *P < 0.05, **P < 0.01. behavior retained in vivo remains unknown. We therefore established a xeno-subcutaneous tumor model using nude mice, then monitored with an 125 I-labeled anti-TLR5 mAb tracer, which was with high affinity for TLR5 in 4T1 cells in vitro. The distribution in vitro and results of whole-body phosphor-autoradiography in vivo revealed much higher radiotracer retention in TLR5 + 4T1 than in TLR5 − 4T1 tumors at all time points. The 125 I-antiTLR5 mAb could not target TLR5 + 4T1 tumor in group of preinjecting unlabeled anti-TLR5 mAb, and tumors were not visualized by radiography, thus, confirming the specificity of 125 I-antiTLR5 mAb imaging.
Fluorescent reporter gene imaging in vivo has been applied to detect the growth and migration of labeled cells in many kinds of diseases 45 . Here, we monitored tumor locations using a TLR5 knockdown 4T1 cell line labeled with green fluorescent protein. The fluorescence images of virus-transfected 4T1 tumors were much clearer than those of non-transfected 4T1 tumors. Moreover, this method had the advantage of clearly displaying tumor margins. More importantly, we found that locations of fluorescence emission contained more tracer radioactivity in TLR5 + 4T1 tumor, which further confirmed that tumor imaging was TLR5 expression-specific. Additionally, early images showed the TLR5 − 4T1 tumors were larger than TLR5 + 4T1 tumors, which suggested that TLR5 expression might inhibit tumor growth in vivo.
To confirm that TLR5 affects tumor metastasis, we established lung metastasis mouse models by injecting TLR5 + 4T1 and TLR5 − 4T1 cells via tail veins, and evaluated the findings using fluorescence imaging. The results showed much clearer lung tumors in TLR5 − 4T1 group than in TLR5 + 4T1 group, suggesting that TLR5 negatively affected TNBC metastasis.
These data suggested that TLR5 expressed in breast cancer could be considered as a biomarker in vivo for the noninvasive molecular imaging of TNBC. It could also be used to monitor tumor development and metastasis, predict its prognosis, evaluate therapy responses, and as a target for therapy. Low tracer uptake in the lungs and heart, and high uptake in tumors increased image contrast. These cell-specific and favorable non-target clearance features of 125 I-antiTLR5 mAb rendered it a promising radiotracer for TNBC imaging.
This study had some limitations. The molecular weight of labeled antibodies was too high for clinical applications. This might be overcome by reducing extended circulation by selecting antibody fragments, adapters, or other small molecules that specifically bind to TLR5. A radioisotopelabeled TLR5-targeting probe suitable for clinical application, such as iodine 131 or the positron nuclide Fluoro-18, should be further investigated. Hence, additional analyses of TLR5 on breast cancer in vitro and in vivo are required.
In conclusion, the downregulation of TLR5 in TNBC increased tumor invasiveness and EMT expression and promoted TNBC metastasis. Therefore, TLR5 might be useful for monitoring and evaluating TNBC prognosis and serve as a novel target for the early detection of TNBC and other TLR5-positive tumors. The radioisotopelabeled probe, 125 I-antiTLR5 mAb, could potentially serve as an ideal noninvasive monitoring agent for TLR5positive tumors.