A Novel Radioimmune 99mTc-Labeled Tracer for Imaging Sphingosine 1-Phosphate Receptor 1 in Tumor Xenografts: An In Vitro and In Vivo Study

Sphingosine-1-phosphate (S1P) is a phospholipid that regulates pleiotropic biological activities and exerts extracellular functions by binding to five specific G-protein-coupled receptors, S1P receptors (S1PR) 1–5. When activated by S1P, S1PR promote the proliferation and invasion of tumor cells by inducing the formation of new blood vessels. We developed and assessed a new monoclonal antibody imaging probe 99mTc-HYNIC-S1PR1mAb, to explore the feasibility of targeting the S1PR1 in vitro and in vivo. S1PR1mAb was prepared and followed by technetium-99m labeling with succinimidyl 6-hydraziniumnicotinate hydrochloride. Cell uptake and blocking studies were performed to investigate the binding specificity of 99mTc-HYNIC-S1PR1mAb in vitro. 99mTc-HYNIC-S1P1mAb was also tested in vivo in mice xenografted with SK-HEP-1 (high-expression of S1PR1) and MCF-7 (low-expression of S1PR1) using single-photon emission-computed tomography (SPECT). Ex vivo gamma counting of tissues from tumor-bearing mice was used to evaluate 99mTc-HYNIC-S1PR1mAb biodistribution. The biodistribution study results showed significantly higher uptake in SK-HEP-1 tumors than in MCF-7 tumors (P < 0.001). Reduced uptake of 99mTc-HYNIC-S1PR1mAb in SK-HEP-1 was observed in tumor-bearing nude mice pretreated with fingolimod, which binds competitively to the receptors, especially S1PR1. 99mTc-HYNIC-S1PR1mAb can be synthesized and specifically targeted to S1PR1 in vitro and in vivo, allowing S1PR1 expression assessment with SPECT imaging.


INTRODUCTION
Sphingosine-1 phosphate (S1P) is a bioactive sphingolipid metabolite that plays an important role in the maturation and homeostasis of the vascular system and in the transport of immune cells (1). S1P signaling pathways are involved in multiple biological processes, including tumor immune regulation, angiogenesis, tumor growth, tumor migration and invasion (2). which could be abnormal in breast cancer, liver cancer, melanoma, papillary thyroid cancer, pancreatic cancer, and prostate cancer (3). S1P can bind to specific G protein-coupled receptors on the cell membrane surface to generate downstream signals that regulate physiological processes. The export of S1P is facilitated by the transporter protein Spinster homolog 2 or the ATP-binding cassette transporter protein family (4,5). There are five known S1P receptor subtypes (S1PR1-5). S1PR1 is involved in, among many processes, promotion of proliferation and invasion of tumor cells and the formation of new blood vessels (6,7). Studies have shown that S1PR1 is highly expressed in colorectal cancer and that the up-regulation of S1PR1 expression is closely related to deep infiltration of cancer cells and liver metastasis. Multivariate survival analysis has shown that S1PR1 expression levels can be used as an independent prognostic indicator of colorectal cancer (8). Among ER-positive breast cancer patients on tamoxifen therapy, patients with high S1PR1 expression had a higher treatment recurrence rate than those with low expression. Therefore, the expression of S1PR1 is promising as a biomarker to predict the drug resistance of tumors, to allow for more effective treatment (9). Further studies have indicated elevated expression of S1PR1 in bladder cancer tissue. S1PR1 stimulates bladder cancer cells to secrete transforming growth factor (TGF)-b and IL-6, thereby inducing the aggregation of regulatory T-cells (Tregs) (10). These studies demonstrate that S1PR1 is a promising cancer biomarker that may play a role in the prognosis of certain tumors.
Some studies have used S1PR1 as a therapeutic target for tumors. The combination of S1PR1 antagonist and chemotherapy drugs can be used as a new strategy for tumor treatment (11). In addition, for the S1PR1 signal is necessary in vascular stability, the loss of S1PR1 function will produce disordered and non-functioning blood vessels and increase the permeability of the blood vessels. Inhibition of S1PR1 can destroy tumor blood vessels in xenograft tumor models and ultimately inhibit tumor growth (12). In vivo studies have shown that S1PR1 inhibitors can reduce the vascular stability of Lewis lung cancer and inhibit angiogenesis and tumor growth (13). On the other hand, the vascular endothelial growth factor (VEGF) can promote tumor angiogenesis and tumor growth (14), and simultaneous inhibition of S1PR1 and VEGFR can maximize the effect of anti-angiogenesis therapy and may become an effective treatment strategy for the treatment of renal cell carcinoma and other types of tumors (15).
In view of the important role of S1PR1 in the occurrence, development and metastasis of tumors, noninvasive monitoring of the expression of S1PR1 in malignant tumors has important clinical significance in formulating tumor treatment plans, monitoring treatment effects, and evaluating prognosis. In some recent studies, Tu's and Haufe's groups have designed and synthesized a series of S1PRs, including specific carbon-11and fluorine-18-labeled radiotracers highly targeted to S1PR1 (16)(17)(18)(19)(20)(21). They have shown promising cell membrane binding assays in vitro towards S1PRs. Some in vivo evaluation of these analogs was accomplished. Single-photon emission computed tomography (SPECT) radioactive probes targeting S1PR1 are rarely known (22,23). In this study, the S1PR1-targeted monoclonal antibody (S1PR1mAb), which can specifically target the S1PR1 protein on the cell surface, was designed and synthesized. The antibody was radiolabeled with technetium-99m using succinimidyl 6-hydraziniumnicotinate hydrochloride (SHNH, Solulink, Inc., San Diego, CA, USA). The targeting efficiency and pharmacokinetics of 99m Tc-labeled S1PR1mAb were then assessed to explore its potential clinical value in both in vitro and in vivo studies.

Antibody Preparation
We searched the human S1PR1 receptor protein sequence in the protein database of the National Center for Biotechnology Information (NCBI). The production and purification of the murine monoclonal antibody was done by ChinaPeptides Co., Ltd.

Cell Culture
The human hepatocellular carcinoma cell line SK-HEP-1 (high expression of S1PR1) and the human breast carcinoma cell line MCF-7 (low expression of S1PR1) were purchased from the Type Culture Collection of the Chinese Academy of Sciences, Shanghai, China (CAT# TCHu109 and TCHu74). SK-HEP-1 cells were cultured in Minimum Essential Medium (MEM, Gibco, USA) and MCF-7 cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM, Gibco, USA). Both medias were supplemented with 10% fetal bovine serum (FBS, HyClone, USA), 100 U/ml penicillin and 100 mg/mL streptomycin (Solarbio, Shanghai, China). The cultures were maintained at 37°C in 5% CO 2 incubators.

Western Blot Analysis
When SK-HEP-1 and MCF-7 cells grew to a density of about 80%, we collected them and added cell lysate buffer (Beyotime, Shanghai, China) and isolated the protein. The protein concentration was determined using a bicinchoninic acid (BCA) protein assay kit (Boster, Wuhan, China). After SDS-PAGE electrophoresis, proteins were transferred to PVDF membrane. The membrane was incubated with the anti-S1PR1 antibody (Abcam, ab233386) for one night and then an antirabbit secondary antibody (Sanjian, Tianjin, China, LK2001) for 2 h. Images were processed with Image J software.
The labeled compound was then purified on a PD-10 column (General Electric, Milwaukee WI, USA). The radiolabeled compound was analyzed by instant thin layer chromatography (ITLC) to calculate its radiolabeling efficiency, radiochemical purity, and in vitro stability (3, 6, 9, and 12 h in FBS, n = 4 per group). Fifty percent acetonitrile and 0.01M PBS were used as the developing solvent system.

Tumor Models
All animal experiments were performed in accordance with a protocol approved by the Institutional Animal Care and Use Committee of Tongji Medical College, Huazhong University of Science and Technology. Female BALB/c nude mice (3-4 weeks old, weighing 15.0-17.0g) were obtained from the Huafukang Bio-Technology Company (Beijing, China). SK-HEP-1 or MCF-7 cells in the logarithmic growth phase were inoculated subcutaneously into the right axilla with 1 × 10 6 cells per mouse. The mice were used as models for in vivo SPECT imaging and biodistribution studies when the tumor size reached 0.5-1.0 cm.
For biodistribution, the SK-HEP-1 and MCF-7 tumorbearing nude mice were injected with 29.6 MBq (29.6 MBq, 0.8 mCi) of 99m Tc-HYNIC-S1PR1mAb by tail vein injection with the blocking group of SK-HEP-1 xenografted mice having received intragastric fingolimod (3 mg/kg/d) for 1 week prior. After the mice were sacrificed by cervical dislocation, blood, brain, lung, heart, liver, spleen, kidney, stomach, small intestine, large intestine, muscle, bone, tail and tumor tissue were taken out and measured using an automatic gamma counter after washing and weighing. After attenuation correction, the percentage of injected dose per gram of tissue (%ID/g) was calculated for each tissue type and the radioactivity count ratio of tumor-to-blood (T/B) and tumor-to-muscle (T/M) of each tumor-bearing mouse were calculated.

Tissue S1PR1 Expression Levels
For immunohistochemistry, SK-HEP-1 and MCF-7 tumorbearing nude mice were sacrificed by cervical dislocation after SPECT imaging. Lung, liver, spleen, kidney, muscle and tumor tissues were removed and fixed with 4% paraformaldehyde. Subsequent tissue staining was performed by Biossci Biotechnology (Hubei, China). For western blotting, appropriate amounts of SK-HEP-1 and MCF-7 tumor tissues were rinsed with PBS 2-3 times, and then minced with scissors. Tissues were lysed in radioimmune precipitation buffer supplemented with a protease inhibitor mixture for 30 min at 4°C, and centrifuged for 10 min (4°C, 10,000 rpm). The supernatant was considered to comprise the total protein extracts, and then followed the remaining steps with the western blot of the cell proteins.

Statistical Analysis
Statistical analysis was performed using Statistical Package for the Social Sciences (SPSS) software (version 22.0, SPSS Inc., Chicago, IL, USA). All data are expressed as mean ± standard deviation (SD). Differences were considered statistically significant when P values were < 0.05.

S1PR1 Expression In Vitro
Western blotting was used to analyze the expression of S1PR1 in SK-HEP-1 and MCF-7 cells. The results demonstrated that SK-HEP-1 cells overexpressed S1PR1, while MCF-7 cells rarely expressed S1PR1 ( Figure 1A and Supplementary Figures 2-4).

SPECT Imaging
SPECT imaging results of SK-HEP-1 tumor model mice at 2, 12, 18, and 24 h are shown in Figure 2A. After injection of 99m Tc-HYNIC-S1PR1mAb, the uptake in SK-HEP-1 tumor-bearing nude mice was mainly in the liver. The uptake at the tumor site gradually increased with time, with tumors becoming clear at 18 h and 24 h. The SK-HEP-1 tumor xenografts in the blocking SK-HEP-1 tumor model group ( Figure 2B) and in the MCF-7 xenografts ( Figure 2C) in the control group were close to the background level at 2 h, and no obvious further increase in uptake occurred. At 24 h, the uptake of the imaging agent by tumors in the blocking group and the control group was significantly lower than that in the experimental group.

Biodistribution Studies
To further explore the metabolism and specificity of 99m Tc-HYNIC-S1PR1mAb in vivo, biodistribution studies were performed on tumor-bearing nude mice in the SK-HEP-1 xenografted mice group, the blocking group (SK-HEP-1 xenografted mice treated with FTY720) and the MCF-7 xenografted mice. The results are shown in Figure 3 and Table 1. When 99m Tc-HYNIC-S1PR1mAb was injected into the SK-HEP-1 xenografted mice group via the tail vein for 24 h, the probes were mainly concentrated in the blood and taken up by the liver, spleen and kidneys, indicated that the blood pool activity was slow to clear. The uptake values of the xenografted nude mice in blood, liver, spleen and kidney were 14.06 ± 0.75%ID/g, 11.89 ± 0.67%ID/g, 8.06 ± 0.54%ID/g, 6.79 ± 0.81%ID/g, respectively (n = 4). At 24 h after the injection of 99m Tc-HYNIC-S1PR1mAb, the SK-HEP-1 tumor uptake was 5.53 ± 0.32%ID/g, which was higher than the tumor uptake values of the tumor-bearing nude mice in the blocking group (P = 0.001) and MCF-7 xenografted mice group (3.64 ± 0.39%ID/g, 2.81 ± 0.21%ID/g, respectively)(P < 0.001). At the same time, the tumor/ blood and tumor/muscle ratios of the tumor-bearing nude mice in the experimental group were higher than those in the blocking group and the control group. The biodistribution results were consistent with the SPECT imaging results.

Tissue S1PR1 Expression
Western blotting confirmed that S1PR1 is highly expressed in SK-HEP-1 solid tumors, while the expression of S1PR1 in muscle  tissues from nude mice in the experimental group and MCF-7 tumor tissues is very low ( Figure 4A). Immunohistochemistry staining 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 the cell membranes of SK-HEP-1 tumors highly expressed S1PR1 (Figures 4B-I).

DISCUSSION
We developed a molecular probe, 99m Tc-HYNIC-S1PR1mAb, that can bind to S1PR1 both in vitro and in vivo. The uptake rate of 99m Tc-HYNIC-S1PR1mAb in tumor cells was related to the expression of S1PR1 and could be specifically blocked. The specificity of 99m Tc-HYNIC-S1PR1mAb to S1PR1 was verified at the cellular level. The uptake rate of 99m Tc-HYNIC-S1PR1mAb in SK-HEP-1 cells (S1PR1 high expression) was more than 2-fold greater than in MCF-7 (S1PR1 low expression) or blocking groups. SPECT imaging and biodistribution results show that tumors with high expression of S1PR1 can specifically take up 99m Tc-HYNIC-S1PR1mAb. The tumor uptake in the blocking group and the MCF-7 xenografted mice group were close to the background level at 2 h and were significantly lower than in the SK-HEP-1 group at 2 h. The results of SPECT imaging were consistent with the biodistribution study results. At 24 h, the tumor uptake value of the SK-HEP-1 group was 5.53 ± 0.32% ID/g, which was higher than both blocking group (3.64 ± 0.39% ID/g, P = 0.003) and MCF-7 group (2.81 ± 0.21% ID/g, P = 0.000), and indicated that the molecular probe had targeting potential and specificity for tumors with high S1PR1 expression. The tumor/blood ratio and tumor/muscle ratio of the SK-HEP-1 group were 0.39 ± 0.01 and 5.99 ± 1.38, and the tumor/muscle ratio was more than twice that of the blocking group (2.84 ± 0.52, Frontiers in Immunology | www.frontiersin.org August 2021 | Volume 12 | Article 660842 P = 0.020) and the MCF-7 group (2.38 ± 0.07, P = 0.011). The results indicated that 99m Tc-HYNIC-S1PR1mAb has a good targeting ability to S1PR1 and could be used as an imaging tracer for S1PR1. SPECT imaging showed that 99m Tc-HYNIC-S1PR1mAb was mainly distributed in the liver, and the tumor in the experimental group was not obvious at 2 h. Subsequently, the imaging agent at the tumor site gradually concentrated with time, and the tumor was seen vaguely at 12 h, and the tumor was clearly seen at 18 h and 24 h. 99m Tc-HYNIC-S1PR1mAb is retained in the blood and non-target organs, especially in the liver, spleen and kidneys. However, the immunohistochemistry result of tissues showed that S1PR1 only had a mild expression in the liver. The high uptake of the tracer might be related to the metabolism of 99m Tc-HYNIC-S1PR1mAb through the liver, which is similar to the results of other experiments using monoclonal antibodies as radioactive probes (24)(25)(26). Antigen-antibody complexes will be non-specifically taken up by the reticuloendothelial system of the liver and spleen, resulting in a concentration of radioactivity. This retention characteristic has a great impact on the evaluation of liver tumors. The high activity in the kidney can be attributed to the renal clearance of radioactive degradation products (27), so it is necessary to further improve the stability of molecular probes in vivo. In addition, the FTY720 may have anti-cancer effects, so the reducing reduction of antibodies may be due to the decrease in tumor, rather than decreased S1PR1. However, in our studies, we usually did not collect the whole tumor tissues in biodistribution to analyze. The valid data of 24 cases (complete tumor was collected), including 12 cases in the experimental group and 12 cases in the blocking group showed that the difference in tumor volume between the two groups was not statistically significant (P>0.05). FTY720 does have the potential to kill tumors, but this is not reflected in our data, probably because our sample size is limited. However, we will explore more blocking conditions or try different blockers (such as the blocking of the antibody itself) in future experiments. S1PR1 has played an important role in tumor, inflammation, immune system, vascular regulation, etc. (11)(12)(13). It could be a key biomarker in immune responses, inflammation, and in the prognosis of certain tumors (4,28). Current research focuses on the synthesis of small molecule radioactive probes binding to S1PRs for PET scanning. For example, 11 C-TZ3321 can detect the up-regulation of S1PR1 in inflammatory blood vessels, and the expression of S1PR1 is higher in neointimal hyperplasia, so 11 C-TZ3321 may realize the detection of early inflammation or can be used in the diagnosis and prognosis of atherosclerosis (29,30). To extend the imaging time, some studies have used 18 Flabeled S1PR1 radioligands. After the molecular probes traversed the blood-brain barrier, an in vivo autoradiographic study of the  All data were expressed as mean ± SD. The uptake in each tissue was expressed as the percentage of injected dose per gram of tissue (%ID/g) (n=4).
neuroinflammatory response in mice was performed, and S1PR1 imaging in primates was achieved with satisfactory results (31,32). Despite the challenges and drawbacks, newer 99m Tc-based S1PR1-targeted tracers should also be studied, for SPECT imaging is widely used. Although the use of radiolabeled monoclonal antibodies can achieve a good target-to-background ratio, the longer circulating half-life is the main limitation of using them as molecular imaging probes (33). For example, the tumor in this experiment was clearly visible at 24 hours, but the image quality obtained after 4 half-lives of the nuclide is not good enough. Longer-lived isotopes may be a better choice, such as indium-111. To optimize the target-to-background ratio, pretargeting techniques may be used in the future. The monoclonal antibody and the small molecule radioligand are injected successively. When the radioligand is injected, the antibody has accumulated in the tumor and most unbound antibody has already been removed from the bloodstream. Pretargeting techniques can significantly reduce the uptake of tracer in non-target tissues, which is beneficial to achieve the purpose of radioimmunoimaging using short half-life nuclides or large molecular weight monoclonal antibodies (34,35).
In addition, to optimize the imaging results, small molecule probes aimed at the same target can be selected, such as small molecule radioligands, targeting peptides, Fab fragments (36). Targeting peptides have a shorter circulating half-life, better permeability, lower immunogenicity, and are easily chemically modified, so they may be more suitable for scientific research and clinical imaging (37)(38)(39)(40). Li used 99m Tc-labeled HER2-targeted peptides for experiments, and the results showed that the maximum absorption value of the was observed 30 min after injection of the imaging agent, and the highest tumor/organ radioactive uptake ratio was observed within 1 h (36). Fab fragments retain the same specificity and affinity activity as monoclonal antibodies. Compared with intact antibodies, Fab fragments have a smaller molecular weight and faster metabolism in the body. The lack of Fc fragments can reduce non-specific uptake. Clear tumor visualization and high tumor/ background signal ratio can be obtained in a short time (33,41). In general, the reduction in molecular weight optimizes the imaging performance of the probe, and small molecule probes can be combined with positron nuclides with a short half-life to perform rapid PET imaging. The sensitivity and spatial resolution of PET are higher than that of SPECT, which can provide more accurate information for the study of S1PR1 and the diagnosis and treatment related to S1PR1. This will be one of the development directions of radioimmunoimaging in the future.

CONCLUSION
We developed a molecular probe, 99m Tc-HYNIC-S1PR1mAb, that can target to S1PR1. Both in vitro and in vivo experiments showed that 99m Tc-HYNIC-S1PR1mAb have good specificity and affinity. Further studies could be performed to optimize the probe for better pharmacokinetics in vivo.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding authors.

ETHICS STATEMENT
The animal study was reviewed and approved by Institutional Animal Care and Use Committee of Tongji Medical College, Huazhong University of Science and Technology.

AUTHOR CONTRIBUTIONS
XX and XL designed the research. MY, YG, HJ, YJ, PQ, and WW performed research. MY analysed the data. MY and XX wrote the paper. YG, YZ, and XL reviewed the paper. All authors contributed to the article and approved the submitted version.