Toll-like receptor 5 (TLR5) is overexpressed in several cancers and metastases, and presents an enticing target for molecular imaging of primary tumors. In the present study, 131I-anti-TLR5 monoclonal antibody (mAb) was evaluated for its use as a novel radiotracer for imaging hepatocarcinoma in mice bearing H22 tumors. The expression of TLR5 was analyzed by quantitative polymerase chain reaction and immunohistochemistry. The anti-TLR5 mAb and isotype immunoglobulin G (IgG) were radiolabeled with iodine-131 by the Iodogen method. The
Hepatocellular carcinoma (HCC) is the most common tumor that is highly aggressive and has a high recurrence (
It is known that Toll-like receptors (TLRs) play prominent roles in inflammatory responses against pathogen infection. These receptors are primarily expressed on innate immune cells and recognize conserved pathogen-associated molecular patterns (
Previously, much attention has been paid to investigating the role of TLR5 in cancer progression and metastasis (
Due to its anatomical site, the liver is constantly exposed to gut-derived bacterial products, viral infection, alcohol or other products, which may be the reason for chronic liver damage, thus increasing the risk for HCC. Possibly as a consequence of this, TLRs play a key role in liver physiology and pathophysiology, due to their role in the immune system and their significant contribution to several biological processes, including promotion of epithelial regeneration and carcinogenesis (
The main obstacle in the diagnosis of HCC is the low sensitivity for the detection of tumors <2 cm in size. The traditional imaging modalities indicated for small-HCC detection are contrast-enhanced ultrasound and contrast-enhanced magnetic resonance imaging (MRI) that have shown a high false-negative detection rate. Thus, a novel and more sensitive detection method is urgently required for the diagnosis of small HCC without a biopsy. Nuclear molecular imaging is such an emerging and promising science that has been applied in a broad range of clinical diagnoses and therapy. 11C-acetate and 18F-fluorodeoxyglucose (FDG) are complementary tracers in the role of a functional and biochemical probe for detecting both primary and secondary HCC through the degree of tumor cell differentiation. Although increasing evidence has shown that TLR5 plays a prominent role in cancer progression, its expression and role in HCC remain unclassified.
As aforementioned, we hypothesize that TLR5 may be a good biomarker for the detection of HCC, and therefore a radioiodinated anti-TRL5 monoclonal antibody (mAb) was prepared and its tumor-targeting potential was evaluated using the H22 hepatocarcinoma-bearing mice model.
The H22 hepatoma cell line was stored in our laboratory (Institute of Experimental Nuclear Medicine, School of Medicine, Shandong University, Shandong, China). The cells were cultured in Dulbecco’s modified Eagle’s medium (Gibco, Invitrogen Life Technologies, Grand Island, NY, USA) supplemented with 10% (v/v) fetal bovine serum (Gibco), 100 U/ml penicillin and 100 mg/l streptomycin (Beyotime Biotech, Ltd., Shanghai, China) in humidified air containing 5% CO2 at 37°C.
Female BALB/c mice, 6 and 8 weeks of age, were purchased from the Experimental Animal Center of Shandong University (Shangdong, China). The mice were inoculated subcutaneously on the rear flanks with 4×106 H22 cells in 100 μl normal saline. The animals were used for biodistribution and autoradiography experiments when the tumor size reached 6–8 mm in diameter. All experimental protocols described in the present study were under the approval of the Ethics Review Committee for Animal Experimentation of Shandong University (Jinan, China).
The TLR5 mRNA expression level in the H22 tumor-bearing mice was measured using RT-PCR. Briefly, total RNA was extracted in accordance with the manufacturer’s instructions and then reverse transcribed to cDNA using the Gene Amp RNA PCR kit in a DNA thermal cycler (Bio-Rad, Hercules, CA, USA). A non-template control was included in all experiments. Primer sequences were as follows: TLR5 forward, 5′-GCAGGATCATGGCATGTCAAC-3′ and reverse, 5′-AATGGTCAAGTTAGCATACTGGG-3′; GAPDH forward, 5′-AGGCCGGTGCTGAGTATGTC-3′ and reverse, 5′-TGCCTGCTTCACCACCTTCT-3′. The amplification products were separated on an agarose gel (Gene Ltd., Hong Kong, China) and visualized with ethidium bromide. The predicted size for TLR5 and GAPDH was 269 and 530 bp, respectively.
Sections (4 μm thick) cut from the archived paraffin blocks, were attached to slides and deparaffinized with toluene, and gradually dehydrated through a descending alcohol series. To block non-specific binding of the antibodies, the sections were incubated with 2% goat serum in phosphate-buffered saline (PBS; blocking buffer) for 2 h at room temperature. Subsequently, the slides were stained using the rabbit anti-mouse anti-TLR5 mAb (1:200; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) or immunoglobulin G (IgG; Beijing Biosynthesis Biotechnology Co., Ltd., Beijing, China) as the negative control. Positive expression was indicated by brownish-yellow granules in the plasma membrane of hepatoma cells for TLR5. The sections were analyzed using an Olympus DP72 digital camera (Olympus Co., Centre Valley, PA, USA) at a magnification of ×400, and images were captured.
Sodium [131I] iodide was obtained from the China Institute of Atomic Energy (Beijing, China). Anti-TLR5 mAb and control IgG were labeled with 131I-NaI by the Iodogen method. Briefly, 100 μl 0.05 M phosphate buffer (PB) and 22.8 MBq 131I-NaI were added into the prepared Iodogen-coated tubes (Pierce Biotechnology Ltd., Rockford, IL, USA) and then 20 μg of anti-TLR5 mAb or IgG were added respectively. Subsequently, the mixture was incubated at room temperature for 15 min with occasional shaking. The reaction was quenched by incubation with 150 μl 0.05 M PB for 15 min at room temperature. Radiolabeled antibodies were then purified by size-exclusion chromatography using a PD-10 Sephadex G-25 column (GE-Healthcare, Diegem, Belgium). The radiochemical purity was measured by a Wipe Test/Well Counter (Caprac; Capintec, Inc., Ramsey, NJ, USA). The
The H22 tumor-bearing mice were injected via the tail vein with a PBS solution (100 μl) of 131I-anti-TLR5 mAb or 131I-IgG (0.74 MBq) respectively. To block the uptake in the thyroid gland, 5% potassium iodide was fed to the mice for three days before injection. Serial images were performed at 12, 24, 36 and 48 h post-injection. The anesthetized groups of mice (n=4, per group) were placed in the supine position on the storage phosphor screen plate for 15 min. Subsequently, the plate was scanned by the Cyclone Plus Storage Phosphor system (Perkin-Elmer, Waltham, MA, USA) and analyzed using the OptiQuant Acquisition software (Perkin-Elmer).
To validate the imaging studies and further quantify the 131I-mAb uptake, biodistribution studies were performed at various times in the H22 tumor-bearing mice model. The mice were administered 0.37 MBq of 131I-mAb (100 μl) via the lateral tail vein. Subsequently, groups of four mice were sacrificed by cervical dislocation at 12, 24, 36 and 48 h after injection, respectively. Blood was collected and the selected tissues were rapidly harvested, weighed and analyzed for total γ-counts by the Wipe Test/Well Counter. Data were corrected for radioactive decay and the radioactivity values were expressed as percentage of the injected dose [ID (%)] per organ, per gram of tissue and as T/NT (target/non-target) ratio.
Data are expressed as the mean ± standard deviation and P<0.05 was considered to indicate a statistically significant difference. An unpaired two-tailed t-test was used, and statistical analysis was performed using PRIZM SPSS 15.0 software (SPSS, Inc., Chicago, IL, USA).
The expression of TLR5 mRNA in the H22 cell line, H22 xenograft tumor tissue and normal liver tissue are shown in
131I-anti-TLR5 mAb and its control 131I-IgG were successfully radioiodinated. The radiochemical purity of 131I-anti-TLR5 mAb and 131I-IgG were both >95%. The specific activity of 131I-anti-TLR5 mAb and 131I-IgG was 29.56 and 25.43 GBq/μmol, respectively. 131I-anti-TLR5 mAb (
The tissue distributions of radioactivity at 12, 24, 36 and 48 h after injection are illustrated in
As shown in the imaging of autoradiography, it was found that 131I-anti-TLR5 became preferentially accumulated in the xenografted H22 tumor at 24 h (
Novel diagnostic imaging approaches for HCC have been developed during the past decades. Ultrasound scanning is non-invasive and widely used in clinical diagnosis of hepatoma, however the false-negative detection rate is >50% (
TLRs are extraordinarily notable in cancer research due to their role in a number of biological processes, including induction of innate and adaptive immune responses, carcinogenesis and regulation of inflammation. Previously, intense links have emerged between inflammation and the initiation and progression of several cancer types, including stomach, breast, ovary and liver (
Predominantly, TLRs may operate in two ways, which is dependent on the cell type. Cancer cells are more aggressive in response to TLRs activation, whilst immune cells usually respond to TLRs agonist by applying antitumor effects. Higher expression levels of TLR and the structural aberrations that characterize malignant epithelia, including the loss of cell polarity and abnormal intercellular junctions, may allow bacteria and their components to induce TLRs, therefore contributing to the disease progression (
As a pattern recognition receptor, TLR5 can recognize flagellin, which is a component of bacterial flagella. In malignant cells, TLR5 activates inflammatory responses and also induces invasion, migration and chemokine secretion (
Therefore, the present study investigated the expression of TLR5 in the hepatocarcinoma cells. It was found that H22 cells and H22-xenografted tumor tissue exhibited higher levels of TLR5 expression than normal liver tissue, indicating that TLR5 may be a novel biomarker of hepatocarcinoma, although the mechanisms underlying remain far from understood.
The 131I-labeled anti-TLR5 mAb was also evaluated as a specific targeted radiotracer in H22 xenograft-bearing mice models. The biodistribution data showed that 131I-anti-TLR5 mAb had a high tumor uptake and T/NT ratio. In addition, the result of autoradiography showed that the radioactive accumulation in the tumor site became visible from 12 h post injection, and increased continually.
These results showed the potential of 131I-anti-TLR5 mAb as a promising molecular imaging agent for HCC diagnosis and encouraged further investigation. Nevertheless, since TLR5 mAb has a large molecular weight and an immunogenicity that may hinder its application in the clinic, it remains a great challenge to explore a novel small fragment of mAb or a small molecule with improved TLR5 targeting. In addition, to analyze the association between the expression of TLR5 in HCC and clinical stage, and to evaluate its significance in early-stage diagnosis, will be extremely helpful for the prognosis of patients suffering from HCC.
This study was supported by grants from the National Natural Science Foundation of China (81071172) and the Natural Science Foundation of Shandong Province (ZR2010CM025).
Analysis of TRL5 mRNA and protein expression. (A) mRNA expression and (B) relative mRNA expression of TLR5 by reverse transcription polymerase chain reaction in H22 cells, H22 xenograft tumor and normal mice liver tissue. *P<0.05, vs. normal liver tissue. Each bar represents the mean±SD of three experiments. (C) Immunohistochemical staining of H22 xenograft tumor tissue and normal mice liver tissue with TLR5 mAb or isotype IgG. Magnification, ×400. Toll-like receptor 5; mAb, monoclonal antibody; IgG, immunoglobulin G.
Distribution of 131I-anti-TLR5-mAb in the H22 tumor-bearing mice.
Tissue | 12 h | 24 h | 36 h | 48 h |
---|---|---|---|---|
Blood | 6.37±0.48 | 4.26±0.35 | 2.30±0.22 | 1.57±0.20 |
Heart | 2.98±0.16 | 2.36±0.06 | 1.51±0.05 | 0.86±0.04 |
Liver | 6.28±0.51 | 4.64±0.31 | 3.21±0.10 | 2.04±0.21 |
Spleen | 2.83±0.14 | 2.95±0.17 | 1.39±0.04 | 1.22±0.06 |
Kidney | 11.60±0.92 | 9.56±0.12 | 7.23±0.38 | 4.91±0.73 |
Stomach | 1.70±0.27 | 1.04±0.06 | 0.86±0.03 | 0.41±0.02 |
Intestine | 1.69±0.13 | 0.79±0.08 | 1.00±0.04 | 0.64±0.10 |
Bone | 0.87±0.10 | 0.74±0.03 | 0.74±0.11 | 0.34±0.01 |
Muscle | 0.98±0.05 | 0.99±0.03 | 0.48±0.07 | 0.23±0.02 |
Lung | 2.79±0.40 | 1.69±0.10 | 1.08±0.14 | 0.94±0.08 |
Thyroid gland | 1.62±0.04 | 1.24±0.07 | 0.77±0.04 | 0.58±0.01 |
Tumor | 6.81±0.73 | 8.26±0.91 | 4.98±0.17 | 2.17±0.53 |
Data are presented as the mean±SD percentage of the injected dose per gram of tissue of four mice. TLR5, toll-like receptor 5; mAb, monoclonal antibody.
Distribution of 131I-IgG in the H22 tumor-bearing mice.
Tissue | 12 h | 24 h | 36 h | 48 h |
---|---|---|---|---|
Blood | 6.16±0.43 | 3.69±0.12 | 2.09±0.07 | 1.12±0.24 |
Heart | 3.43±0.19 | 2.01±0.09 | 1.40±0.25 | 0.42±0.08 |
Liver | 5.79±0.41 | 4.15±0.07 | 3.73±0.13 | 1.03±0.08 |
Spleen | 2.61±0.12 | 2.15±0.10 | 0.79±0.03 | 0.55±0.12 |
Kidney | 10.51±1.08 | 8.20±0.80 | 5.38±0.33 | 3.71±0.29 |
Stomach | 1.69±0.13 | 0.92±0.13 | 0.74±0.02 | 0.48±0.13 |
Intestine | 1.48±0.25 | 1.02±0.09 | 0.87±0.14 | 0.45±0.11 |
Bone | 1.17±0.19 | 0.95±0.27 | 1.02±0.06 | 0.36±0.05 |
Muscle | 1.21±0.31 | 0.67±0.11 | 0.59±0.08 | 0.32±0.07 |
Lung | 3.01±0.68 | 1.77±0.12 | 1.33±0.12 | 0.62±0.03 |
Thyroid gland | 1.63±0.07 | 0.97±0.05 | 0.80±0.06 | 0.50±0.01 |
Tumor | 3.83±0.26 | 3.27±0.34 | 2.68±0.06 | 1.13±0.18 |
Data are presented as the mean±SD percentage of the injected dose per gram of tissue of four mice. IgG, immunoglobulin G.