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Treatment of Hepatocellular Carcinoma by Intratumoral Injection of 125I-AA98 mAb and Its Efficacy Assessments by Molecular Imaging

Objective: To investigate the therapeutic efficacy of intratumoral injection of 125I-AA98 mAb for hepatocellular carcinoma (HCC) and its therapy efficacy assessment by 99mTc-HYNIC-duramycin and 99mTc-HYNIC-3PRGD2 SPECT/CT imaging. Methods: HCC xenograft tumor mice models were injected intratumorally with a single dose of normal saline, 10 microcurie (μCi) 125I-AA98 mAb, free 125I, AA98 mAb, 80 μCi 125I-AA98 mAb, and 200 μCi 125I-AA98 mAb. 99mTc-HYNIC-duramycin and 99mTc-HYNIC-3PRGD2 micro-SPECT/CT imaging were performed on days 3 and 7, respectively. The T/M ratio for each imaging was compared with the corresponding immunohistochemical staining at each time point. The relative tumor inhibition rates were documented. Results: In terms of apoptosis, the 200 μCi group demonstrated the highest apoptotic index (11.8 ± 3.8%), and its T/M ratio achieved by 99mTc-HYNIC-duramycin imaging on day 3 was higher than that of the normal saline group, 80 μCi group, 10 μCi group and free 125I group on day 3, respectively (all P < 0.05). On day 3, there was a markedly positive correlation between T/M ratio from 99mTc-HYNIC-duramycin imaging and apoptotic index by TUNEL staining (r = 0.6981; P < 0.05). Moreover, the 200 μCi group showed the lowest T/M ratio on 99mTc-HYNIC-3PRGD2 imaging (1.0 ± 0.5) on day 7 (all P < 0.05) comparing to other groups. The T/M ratio on day 7 was not correlated with integrin ανβ3 staining (P > 0.05). The relative inhibitory rates of tumor on day 14 in the AA98 mAb, 10 μCi, 80 μCi, free 125I, and 200 μCi groups were 26.3, 55.3, 60.5, 66.3, and 69.5%, respectively. Conclusion: 125I-AA98 mAb showed more effective apoptosis induced ability for CD146 high expression Hep G2 HCC cells and hold the potential for HCC treatment. Moreover, 99mTc-HYNIC-Duramycin (apoptosis-targeted) imaging and 99mTc-HYNIC-3PRGD2 (angiogenesis-targeted) imaging are reliable non-invasive methods to evaluate the efficacy of targeted treatment of HCC.


INTRODUCTION
Hepatocellular carcinoma (HCC) is the most common primary malignant tumor of the liver in adults (Torre et al., 2015). The majority of patients diagnosed with advanced HCC and failed to meet the criteria for resection or transplantation (Thomas et al., 2010). Although locoregional therapies including transarterial chemoembolization, cryotherapy, and radiofrequency or microwave ablation can reduce tumor burden while preserving liver function, the prognosis of advanced HCC patients still remains dismal. Alternatively, intratumoral injection of radiopharmaceuticals can achieve a high therapeutic concentration at targeted sites with an extended period of time and avoid systemic exposure to radiation and the results are promising (Tian et al., 1996;Wang et al., 1998;Junfeng et al., 2000;Chi et al., 2014).
It is widely accepted that angiogenesis is essential for tumor growth and invasion (Folkman, 1971). HCC is one of the malignant hypervascular solid tumors, and inhibition of angiogenesis is one of the important methods to treat HCC. CD146 has emerged as a biomarker for angiogenesis (Zheng et al., 2009;Wang and Yan, 2013;Nomikou et al., 2015), and it has been identified as an attractive target for imaging and therapy in HCC (Thomann et al., 2014;Hernandez et al., 2016). Furthermore, high expression of CD146 predicted poor prognosis in HCC patients (Jiang et al., 2016). AA98 monoclonal antibody (mAb) is a promising mAb against CD146 by inhibition of angiogenesis and tumor growth, but it does not induce apoptosis in vitro (Yan et al., 2003). Anti-angiogenic mono-therapy is a powerful tool to inhibit tumor growth, but, to date, any available anti-angiogenic mono-therapies targeting cancer do not satisfy the expectation of "starving the tumor." One of the solutions to enhance this anti-angiogenic efficacy is to label the anti-angiogenic agents with therapeutic radionuclides, and this strategy is supported by previous reports (Tijink et al., 2006;Fujiwara et al., 2014;Park et al., 2017;Ehlerding et al., 2018). Iodine-125 ( 125 I) is a long-lived radioisotope with a half-life of 59.5 days and a short-range emitter (Cunningham et al., 1998). Its decay can produce a highly localized deposition of dose by short-range Auger electrons plus X ray and Gamma ray. 125 I can cause non-repairable damage to double strand DNA and then induce tumor cell death with favorable results by several possible mechanisms including apoptosis hypothesis (Hofer and Hughes, 1971;Bagshawe et al., 1991;Cunningham et al., 1998).
Molecular imaging plays a critical role in monitoring tumor response to targeted therapy. 99m Tc is an easily accessible diagnostic radionuclide using 99 Mo/ 99m Tc generator system, and it has a very attractive nuclear property for molecular imaging. The main targets for apoptosis are the Abbreviations: AI, apoptosis index; DAPI, 4 ′ , 6-diamidino-2-phenylindole dihydrochloride; HCC, hepatocellular carcinoma; HE, haematoxylin and eosin; HYNIC, mAb, monoclonal antibody; IOD, integrated optical density; 125 I, Iodine-125; PBS, phosphate buffer solution; PE, phosphatidylethanolamine; PS, phosphatidylserine; RCP, radiochemical purity; T/M, tumor-to-contralateral muscle tissues; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling; VOI, volume of interest; 3PRGD2, 3PEG4-cRGD dimer. two common cell membrane aminophospholipids, such as the richest phosphatidylserine (PS) and the second richest phosphatidylethanolamine (PE). PE can be recognized and bound with high affinity by duramycin with a 19-amino acid peptide during cell apoptosis and necrosis (Zhao, 2011). Moreover, the stable, fast clearing, and highly specific 99m Tc-HYNIC-duramycin can be applied to detect tumor cell death and monitor tumor response to treatment (Elvas et al., 2015;Hu et al., 2018). Integrin α ν β 3 is an attractive molecular target for its highly restricted expression in tumor angiogenesis and metastasis. The 99m Tc-HYNIC-3PRGD2 (3PEG4-cRGD dimer) with a safe, highly stable, rapid blood clearance, and easily available kit formulation has the potential for non-invasive integrin α ν β 3 -targeted imaging and monitoring treatment efficacy of antiangiogenesis (Wang et al., 2009;Jia et al., 2011).
In the present study, AA98 mAb was labeled with 125 I to treat HCC xenograft by intratumoral injection. In the following, apoptosis-targeted imaging with 99m Tc-HYNIC-duramycin and angiogenesis-targeted imaging with 99m Tc-HYNIC-3PRGD2 were employed to evaluate treatment efficacy.

Preparation of 99m Tc-Labeled Probes for Tumor Apoptosis and Angiogenesis
A single-step kit containing 15 µg hydrazinonicotinamide (HYNIC)-duramycin (Molecular Targeting Technologies, Inc., USA) was labeled by Na 99m TcO 4 as previously reported (Zhao et al., 2008;Hu et al., 2018). In brief, 0.5 mL freshly prepared sodium pertechnetate ( 99m Tc-pertechnetate) with specific activity of 30 millicurie (mCi), which was obtained from Mo-99/Tc-99m generator (GMS Pharmaceutical Co., Ltd., Shanghai, China), was added to the kit vial. The vial was heated at 80 • C around 20 min and then cooled down to room temperature. The RCP of the 99m Tc-HYNIC-duramycin (always beyond 95%) was analyzed by Radio-TLC.
A single-step kit containing 20 µg of the HYNIC-3PRGD2 conjugate was labeled by Na 99m TcO 4 as previously reported (Wang et al., 2009). Simply put, 1.0 mL of Na 99m TcO 4 solution (20 mCi) in saline was added into the kit vial and heated at 100 • C around 20 min. After heating, the vial was cooled down to room temperature. The RCP of the 99m Tc-HYNIC-3PRGD2 was >95%.

Cell Lines
The human HCC cell line Hep G2 was obtained from the Cell Bank, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai. Hep G2 cells were cultured in DMEM medium (Gibco) containing 1% penicillin-streptomycin (100 U/mL penicillin; 100 µg/mL streptomycin) and 10% fetal bovine serum at 37 • C with 5% CO 2 . All cells were passaged and collected with trypsin-EDTA solution (0.05% trypsin; 0.02% EDTA).

Animal Care
All animal studies were performed under a protocol approved by the Institutional Animal Care and Use Committee of Zhongshan Hospital, Fudan University. All mice were acclimatized to laboratory conditions (individually ventilated cages, 20 • C, 12/12 h light/dark cycle, and ad libitum to a standard diet) around 1 week prior to studies.

In vivo Micro-SPECT/CT Imaging
Prior to micro-SPECT/CT imaging, mice were placed into a mice anesthesia chamber with 4% isoflurane mixed with 2 L/min O 2 for several minutes to induce deep anesthesia using a VETEQUIP V-1 animal anesthesia system (VetEquip Inc., USA), then mice were transferred to exam table and maintained with 1.5% isoflurane mixed with 0.4 L/min O 2 using the same anesthesia system. The Nano SPECT/CT scanner (Bioscan, USA) was employed to perform in vivo micro-SPECT/CT imaging with the settings (Tan et al., 2017). All 3D OSEM images were reconstructed with a HiSPECT algorithm.
Tumor responses in the tumors were evaluated with 99m Tc-HYNIC-duramycin and 99m Tc-HYNIC-3PRGD2. Before treatment (day 0), baseline micro-SPECT/CT imaging was performed in each of imaging groups using the two molecular probes. After intratumoral injections, micro-SPECT/CT imaging targeting tumor apoptosis was performed using 99m Tc-HYNICduramycin on day 3, and targeting tumor angiogenesis using 99m Tc-HYNIC-3PRGD2 on day 7.

Data Analysis
All micro-SPECT/CT images were displayed with Invivo Scope software (Version 1.43, Bioscan, USA). An appropriate 3D volume of interest (VOI) was placed in the tumor with maximum molecular probe uptake included and necrotic area spared, and the contralateral muscle area was also measured. The radioactivity of tumor or contralateral muscle (in µCi/mm 3 ) was reported by the software for each VOI. The tumor-tocontralateral muscle tissues ratio (T/M ratio) was calculated for each tumor. For assessment of tumor apoptosis and angiogenesis, the T/M ratio of each of the six groups after intratumoral injection was compared with that of the corresponding baseline imaging, and multiple comparisons of the T/M ratio were also conducted among the six groups after intratumoral injection.

Therapeutic Efficacy Study
Tumor size (length and width in mm) and body weight (gram) were measured every other day for 14 days after injection. Tumor volume (mm 3 ) was calculated following the formula (Inaba et al., 1989): Volume = [length × (width) 2 ]/2. The relative tumor inhibition rate was used to assess the antitumor efficacy of the different treatments as follows: Where V 14 is the tumor volume on Day 14, V 0 is the pretreatment tumor volume before injection (Day 0), T represents each of the five treatment groups, and C means the control group. When body weight loss was >20%, or tumor volume was larger than 2 cm 3 , mice were sacrificed and excluded to calculate the relative tumor inhibition rate.

Histiopathological and Immunohistochemistry
After imaging or treatment, all mice were sacrificed, and all tumors were harvested. Adjacent 4 µm thick paraffinembedded sections of all tumor samples were stained with Haematoxylin and Eosin (HE) and terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) fluorescence or integrin α ν β 3 fluorescence. In brief, all tumors were fixed in 4% paraformaldehyde in PBS, dehydrated, and embedded in paraffin, then sliced in the maximal section of the tumor. According to the instruction of the in situ cell death detection kit (Roche), TUNEL fluorescence staining was performed to detect and quantify tumor cell apoptosis.

Pathological and Immunohistochemistry Analysis
The percentage of tumor necrotic area was calculated as: the area of tumor necrosis/the area of total tumor tissue (drawn manually) on HE sections using Case Viewer (3dhistech Ltd., Hungary). Apoptosis index (AI) was determined by calculating the percentage of apoptotic nuclei with green TUNEL fluorescence staining over total nuclei using Quant Center software (3dhistech Ltd., Hungary). Image-Pro Plus 6.0 software (Media Cybernetics, USA) was used to assess the integrin α ν β 3 red fluorescence intensity by measuring the integrated optical density per field of view (IOD/mm 2 ) of the images with an equivalent area.

Statistics
The numerous data were expressed as mean ± standard deviation. The results were analyzed usinig the software package (IBM SPSS version 22.0 for Mac OS, IBM, USA). A P < 0.05 was considered to indicate statistically significant difference.

RESULTS
The labeling rate of the 125 I-AA98 mAb molecular probe was over 95%. Both the radiochemical purity and the radiochemical yield were 95.8 ± 0.5% (Figure S1).

Apoptosis Imaging
In terms of T/M ratio, 99m Tc-HYNIC-Duramycin imaging showed that the baseline scans had no statistically significant difference (P > 0.05, Figure 1). Three days after treatment, the T/M ratio in each group was higher than that before treatment. The T/M ratio of the 10 µCi group, 80 µCi group, free 125 I group, and 200 µCi group on day 3 was higher than that on the corresponding baseline scans (P = 0.0011, P = 0.0002, P = 0.0332, and P = 0.0218, respectively). There was statistically significant difference among all the 6 groups on day 3 (P =  0.0066). In terms of the degree of apoptosis, the 200 µCi group demonstrated the highest apoptotic index (11.8 ± 3.8%), and its T/M ratio on day 3 was higher than that of the normal saline, 10 µCi and free 125 I groups, respectively (P = 0.0016, P = 0.0029, and P = 0.0413, respectively). The T/M ratio in 80 µCi group on day 3 was higher than that in 10 µCi and normal groups (P = 0.0021 and P = 0.0043). In terms of T/M ratio on day 3, there was also a markedly significant difference between 10 µCi group and normal group (P = 0.0375). There were no significant differences among the remaining comparative studies (all P > 0.05). There was a markedly positive correlation (r = 0.6981; P = 0.0013) between T/M ratio and apoptotic index by TUNEL fluorescence staining (Figure 2) on day 3.

Angiogenesis Imaging
In terms of T/M ratio, 99m Tc-HYNIC-3PRGD2 imaging showed there was no statistically significant difference among the baseline scans (P > 0.05) (Figure 3). Seven days after treatment, the T/M ratio was significantly lower than that before treatment, and there had no statistically significant difference among all groups (P = 0.0001). There was marked difference between the baseline scan and the repeated scan on day 7 in 200 µCi group, 80 µCi group, and AA98 mAb group, respectively (all P < 0.05). The 200 µCi group showed the lowest T/M ratio (1.0 ± 0.5) compared with the other five groups on day 7 (all P < 0.05). The T/M ratio in 80 µCi group and AA98 mAb group on day 7 were both higher than that in 10 µCi group and saline control group on day 7 (all P< 0.05). The T/M ratio of angiogenesis-targeted imaging on day 7 was not correlated with IOD/mm 2 by integrin α ν β 3 immunofluorescence staining (Figure 4) (P = 0.1020).

Therapeutic Efficacy
As shown in Figure 5, compared with the control group, the relative inhibitory rates of tumor in AA98 mAb group, 10 µCi group, 80 µCi group, 125 I group, and 200 µCi group on day 14 were 26.3%, 55.3%, 60.5%, 66.3%, and 69.5%, respectively. Fourteen days after intratumoral injection, there is no markedly statistical difference in terms of tumor volume growth rate among all the six groups (P > 0.05). No significant difference of the percentage of tumor necrotic area was noted among the six groups (P > 0.05).

DISCUSSION
CD146 is one of adhesion molecules involving in angiogenesis (St Croix et al., 2000;Chan et al., 2005), and it also plays an essential role in tumor progression. Recently, overexpression of CD146 was found to promote migration and invasion of HCC cells and predict poor prognosis in HCC patients (Jiang et al., 2016). A novel anti-CD146 mAb AA98 has been revealed that it can block angiogenesis both in vitro and in vivo, and it alone can effectively inhibit tumor growth of HCC xenografts via intraperitoneal injections (Yan et al., 2003). In clinical settings, the usual way of treating inoperable HCC is transcatheter arterial chemoembolization (TACE), which is not easily accessible for HCC xenograft mice model. Alternatively, intratumoral injection is preferred to mimic TACE as a way of drug delivery. AA98 mAb, as an angiogenesis inhibitor, can block the formation of new blood vessel, rather than directly damage existing blood vessel to result in secondary tumor cell apoptosis (Siemann and Horsman, 2009). Previous report also showed AA98 mAb could not directly induce cell apoptosis in vitro (Yan et al., 2003). However, 125 I can delivery longer short-range internal radiation over time to induce tumor cell apoptosis. One of the possible molecular mechanisms of tumor cell apoptosis that 125 I may upregulate expression of p53 to downregulate vascular endothelial growth factor and then decrease microvessel density . We were motivated to try the intratumoral injection of 125 I-AA98 mAb to target CD146 with AA98 mAb and then to induce HCC cell apoptosis by 125 I with its potential mechanisms (Cunningham et al., 1998;Ma et al., 2014).
In our experiments, AA98 mAb was labeled with a high specific activity of Na 125 I solution by Iodogen method to yield >95% of RCP. Compared with the baseline T/M ratio of in vivo 99m Tc-HYNIC-Duramycin imaging before intratumoral injection, the T/M ratio of the four groups containing 125 I revealed marked increase of HCC cell apoptosis after 3-day treatment (P < 0.05), but not in AA98 mAb group (P > 0.05). Noticeable, 125 I-AA98 mAb for targeting CD146 might more efficiently induce apoptosis than free 125 I. The central HCC tumor apoptosis with peripheral normal HCC cells among all groups was verified by TUNEL staining. For in vivo 99m Tc-HYNIC-Duramycin imaging on day 3, the T/M ratios of the 3 kinds of molecular probe groups (10 µCi group, 80 µCi group, and 200 µCi group) were significantly different from that of the control group (P < 0.05), but not the free 125 I group (P > 0.05). Compared with the control group before treatment, although there was slight increase tendency of 99m Tc-HYNIC-Duramycin uptake within tumor in the 3 kinds of molecular probe groups on day 3 (P > 0.05), the inherently spontaneous HCC tumor apoptosis in the three kinds of molecular probe groups was ruled out.
We also confirmed that 125 I low-energy radionuclide which resulting a highly localized irradiation can kill HCC cells via apoptosis-mediated cell death. According to dosage of 125 I, there is a tendency that higher dosage may induce higher apoptosis. Moreover, the 200 µCi group alone had the highest apoptotic index and markedly different from the five other groups (all P < 0.05). It means that higher dosage of 125 I-AA98 mAb will cause more serious apoptosis, and the 200 and 80 µCi groups may superior to the free 125 I group. Finally, our results revealed that the T/M ratio of in vivo 99m Tc-HYNIC-Duramycin imaging had a positive correlation with apoptotic index by TUNEL fluorescence staining. It indicates that in vivo 99m Tc-HYNIC-Duramycin imaging can provide a potential tool to evaluate tumor cell apoptosis.
AA98 mAb plays an essential role in inhibiting early angiogenic processes, which control the formation of secondary and tertiary vessel branches (Yan et al., 2003). Tumor neoangiogenesis was able to be identified by overexpression of the integrin α ν β 3 receptor too, which can be visualized by radionuclide-labeled RGD analogs, such as the molecular probe 99m Tc-HYNIC-3PRGD2 (Jin et al., 2016). We found that the groups containing 80 µg of AA98 mAb or more (i.e., 200 µCi group, 80 µCi group, and AA98 mAb group) showed marked difference between the baseline scan on day 0 and the repeated scan on day 7 (all P < 0.05). This finding indicates that AA98 mAb of ≥ 80 µg may be the optimal single dosage to achieve response detected on 99m Tc-HYNIC-3PRGD2 SPECT/CT imaging during 7-day therapy. These results were consistent with previous report (Yan et al., 2003). However, our study found that only 200 µCi group was superior to the other five groups (P < 0.05), although the T/M ratio of in vivo 99m Tc-HYNIC-3PRGD2 SPECT/CT imaging on day 7 was not correlated with IOD/mm 2 by integrin α ν β 3 immunofluorescence staining (P > 0.05). The fixed dosage of AA98 mAb may be one of the reasons to limit the anti-angiogenetic effects. Further work is needed to apply multiple injections for treatment to achieve better response.
In addition, our data also showed that there is a tendency of inhibiting growth of HCC xenografts using 125I-AA98 mAb for the first time, although there is no marked significant difference among the five treatment groups (P > 0.05). We noted that the relative inhibitory rate of tumor in the 125 I group was very well (66.3%). However, the average necrotic area in the 125 I group was just higher than the in the control group and lower than that in the other four groups. It indicates that the percentage of the tumor tissue in the 125 I group was only lower than that in the control group. This is the first point that we thought the 125 I group did not share the best anti-tumor effect. Second, the average apoptotic index in the 125 I group on day 3 also showed it is inferior to the three groups containing 125 I-AA98 mAb. Third, the evaluation of the SPECT/CT imaging in terms of antiapoptosis and anti-angiogenesis demonstrated that the tumormuscle rates did not indicate the 125 I group was the best one. Based on our study, we think our further research may focus on comparing the 125 I-AA98 mAb with free 125 I, optimal multiple injections and optimal injection interval to confirm the definite efficacy.
In conclusion, compared with free 125 I and unlabeled AA98 mAb, it's evident that 125 I-AA98 mAb showed more effective apoptosis induced ability for CD146 high expression Hep G2 HCC cells. Moreover, 99m Tc-HYNIC-Duramycin (apoptosistargeted) imaging and 99m Tc-HYNIC-3PRGD2 (angiogenesistargeted) imaging are reliable non-invasive methods to evaluate the efficacy of targeted treatment of HCC.

DATA AVAILABILITY STATEMENT
The datasets generated for this study are available on request to the corresponding author.

ETHICS STATEMENT
The animal study was reviewed and approved by the Animal Care Committee of Fudan University.

AUTHOR CONTRIBUTIONS
JZ, PH, and DC were involved in the study data analysis and edited the main manuscript text. LQ, HZ, ZF, and JZ contributed to animal models. ZS and DC helped in synthesizing the probe used in this study. HT and WM deal with the pathology. JZ, PH, DC, and HS conceived and designed as well as controlled the quality of this study.

ACKNOWLEDGMENTS
We thank Prof. Xiyun Yan and Prof. Minmin Liang (National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences) who kindly provided AA98 mAb to us for free. A single-step kit of the HYNIC-3PRGD2 conjugate was kindly provided by Prof. Fan Wang (Medical Isotopes Research Center, Peking University, Beijing, China). We want to thank for the technical supports from Dr. Yuxia Liu and Dr. Xiaobei Zheng (Institute of Applied Physics, Chinese Academy of Sciences). We thank for the imaging support from Prof. Yingjian Zhang and Mr. Jianping Zhang (Center for Biomedical Imaging, Fudan University and Department of Nuclear Medicine, Shanghai Cancer Center, Fudan University).