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I131 reinforces antitumor activity of metuximab by reversing epithelial–mesenchymal transition via VEGFR‐2 signaling in hepatocellular carcinoma

Abstract

CD147 is highly expressed in hepatocellular carcinoma (HCC) and associated with the invasion and metastasis of HCC. The efficacy of I131‐metuximab (I131‐mab), a newly developed agent that targets CD147, as a radio‐immunotherapy for local HCC, has been validated in clinical practice. However, the synergistic anticancer activity and molecular mechanism of different conjugated components within I131‐mab remain unclear. In this study, the cytological experiments proved that I131‐mab inhibited the proliferation and invasion of HCC cells. Mechanically, this inhibition effect was mainly mediated by the antibody component part of I131‐mab, which could reverse the epithelial–mesenchymal transition of HCC cells partially by suppressing the phosphorylation of VEGFR‐2. The inhibitory effect of I131 on HCC cell proliferation and invasion is limited, whereas, when combined with metuximab, I131 significantly enhanced the sensitivity of HCC cells to CD147‐mab and consequently reinforced the anticancer effects of CD147‐mab, suggesting that the two components of I131‐mab exerted synergistic anti‐HCC capability. Furthermore, the experiments using SMMC‐7721 human HCC xenografts in athymic nude mice showed that I131‐mab and CD147‐mab significantly inhibited the growth of xenograft tumors and that I131‐mab was more effective than CD147‐mab. In conclusion, our results elucidated the mechanism underlying the anti‐HCC effects of I131‐mab and provided a theoretical foundation for the clinical application of I131‐mab.

1 INTRODUCTION

Hepatocellular carcinoma (HCC) is a common malignant tumor that is commonly treated with surgery, minimally invasive treatment and transcatheter arterial chemoembolization (TACE) (Li & Yeo, 2017; Ramaswami et al., 2016). However, postoperative recurrence and metastasis remain important factors that affect the survival of patients with HCC (Grandhi et al., 2016; Yagci, Cetin, & Ercin, 2017). In particular, in HCC treated with chemotherapy or TACE, the residual cancer cells are found to be more invasive and metastatic (Su, 2016; Wan et al., 2016; Yu, Park, Park, & Yoon, 2016). Thus, further studying the genetic and biological properties of HCC is important to identify specific molecular markers of HCC recurrence and metastasis, develop new anti‐HCC agents and ultimately improve the quality of life of HCC patients.

Immunotherapy is an important approach of comprehensive cancer therapy. Specifically, HCC cells strongly express CD147 antigen, which is related to the invasion and metastasis of HCC (Chen et al., 2016). Thus, CD147, a highly glycosylated transmembrane protein and a member of the immunoglobulin superfamily, is an effective target for HCC immunotherapy. CD147 expression is up‐regulated in many tumors and is especially high in HCC tissue and HCC cell lines, where it is expressed in up to 75%. Conversely, it is not expressed in normal hepatic tissue and normal hepatic cell lines (Dai et al., 2009; Gou et al., 2009). Moreover, the over‐expression of CD147 can induce the expression and secretion of matrix metalloproteinase‐2 (MMP‐2) and matrix metalloproteinase‐9 (MMP‐9), which degrade extracellular matrix (ECM) and promote tumor invasion and metastasis by interfering with mesenchymal cells (Cui et al., 2012; Wang et al., 2010). To target CD147, a highly specific monoclonal antibody, HAB18 or metuximab, was developed and conjugated to the radioisotope I131. The resultant I131‐metuximab (I131‐mab) is a radio‐immunotherapy injection that was officially approved by the CFDA in 2007, being under the trade name Licartin for the treatment of local HCC (Ma & Wang, 2015; Wu, Shen, Xia, & Yang, 2016). I131‐mab exhibits several advantages, such as its specificity, rapid response and lethality to cancer cells. The preliminary results of a phase IV clinical trial of TACE combined with I131‐mab for the treatment of advanced HCC at eight Chinese institutions showed that I131‐mab is a safe and effective agent for the treatment of advanced HCC (Xu et al., 2007) and can effectively delay HCC relapse after hepatic transplantation (Wu et al., 2010, 2012). Moreover, the combination of TACE and I131‐mab can significantly prolong progression‐free survival and the overall survival of patients with advanced HCC according to the Barcelona clinical staging (BCLC) (Li et al., 2009). Among the 167 patients with HCC in this study, the 1‐year survival rate was significantly higher for patients receiving the combination treatment than that of patients only receiving the TACE treatment. The extrahepatic metastasis incidence in the combined treatment group was 2.94%, which was significantly lower than the 8.57% rate in the TACE group. This difference suggests that I131‐mab can reduce the extrahepatic metastasis incidence in patients after TACE treatment.

The molecular mechanisms underlying the acceleration effect of CD147 on HCC invasion and metastasis and the synergistic inhibition effect of I131‐mab on HCC progression have been reported (Bian et al., 2014; Gou et al., 2016). Specifically, CD147 can activate the transforming growth factor‐β (TGF‐β) signaling pathway to promote the expression and secretion of MMPs in tumor cells and mesenchymal cells, which induces the epithelial–mesenchymal transition (EMT) in cancer cells and contributes to tumor invasion and metastasis (Ru, Wu, Chen, & Bian, 2015; Xu et al., 2007). Growing evidence shows that EMT plays an important role in the molecular mechanisms underlying HCC occurrence and development. During the process of EMT, cells of epithelial origin transform into mesenchymal cells under specific physiological and pathological conditions, and this transition is accompanied by a series of phenotypic and behavioral changes within cells (Jayachandran, Dhungel, & Steel, 2016). EMT is not only involved in embryonic development but also plays an important role in the pathology of many diseases, such as fibrosis and cancer (Nieto, Huang, Jackson, & Thiery, 2016). In cancer cells, EMT can decrease cell adhesion and increase cell motility, and these changes promote invasion, metastasis and the formation of a new tumor (Giannelli, Koudelkova, Dituri, & Mikulits, 2016; Polyak & Weinberg, 2009). I131‐mab specifically binds to CD147, which is located on the surface of HCC cells, to inhibit TGF‐β signaling and MMP secretion. This inhibition consequently interrupts EMT process and suppresses the invasion and metastasis of cancer cells.

The efficacy of TACE in combination with I131‐mab has been confirmed, and the mechanisms underlying this effect have also been partially elucidated. However, multiple factors are involved in these mechanisms. Do the mechanisms interact? Is the effect synergistic? In addition to the I131‐mab‐mediated inhibition of TGF‐β signaling to interrupt EMT, are other signaling pathways involved in this process? Furthermore, studies are needed to answer these questions. The work described herein shows that I131‐mab can reverse the EMT of HCC cells, and metuximab, a component of I131‐mab, plays an important role in the reversion effect. I131 can increase sensitivity of HCC cells to metuximab to synergistically enhance the efficacy of treatment. Moreover, the mechanism by which I131‐mab reverses the EMT of HCC cells is related to VEGFR‐2 signaling. Overall, this work showed that I131‐mab may interrupt EMT of HCC by inhibiting VEGFR‐2 signaling to prevent HCC metastasis.

2 RESULTS

2.1 Inhibition of HCC cell proliferation by I131‐mab

Western blotting was used to measure the expression levels of CD147 in a variety of cell lines, and CD147 was expressed in HepG2, Hep3B, SMMC‐7721, MHCC97H and MHCC97L cells, with HepG2, Hep3B and MHCC97H cells expressing higher levels of CD147. MHCC97L cells expressed the lowest level of CD147, and WRL‐68 cells were negative for the expression of CD147 (Figure 1a). Based on these results, we selected CD147‐negative WRL‐68 cells to investigate the cytotoxicity of I131‐mab and CD147 antibody (CD147‐mab). CD147‐mab did not affect cell viability, whereas I131‐mab exerted a dose‐dependent effect on cell viability. The IC20 and IC50 values of I131‐mab on WRL‐68 cells were 8.09 μCi/100 μl and 12.69 μCi/100 μl, respectively (Figure 1b). Because WRL‐68 cells did not express CD147 and were not inhibited by CD147‐mab, the cytotoxicity of I131‐mab was attributed to the conjugated I131. Because the unit of measurement of I131‐mab and I131 was μCi and the unit of measurement of CD147‐mab is μM, therefore, to establish the concentration equivalence relation between I131‐mab and CD147‐mab, we regarded that the concentrations of I131‐mab corresponding to IC20 and IC50 could be served as the reference concentrations of I131 and consequently calculated the molar concentrations of CD147‐mab to be 5.62 μM/100 μl and 8.82 μM/100 μl, respectively.

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Inhibition of HCC cell proliferation by I131‐mab. (a) Experimental cell lines were seeded in a 24‐well plate (1 × 105 cells per well) and cultured for 48 hr. Western blotting was used to measure CD147 expression in the harvested cells. GAPDH was used as the internal control. (b) WRL‐68 cells were seeded in 96‐well plate (1 × 104 cells/100 μl per well) and cultured for 24 hr. I131‐mab and CD147‐mab were diluted in serum‐free medium. The cells were treated with 0, 1.0, 5.0, 10.0 and 15.0 μCi (per 100 μl) I131‐mab and 0, 2.5, 5.0, 7.5 and 10.0 μM (per 100 μl) CD147‐mab. After 2 hr of incubation, the medium was replaced with medium supplemented with 10% FBS (100 μl/well). After 48 hr of incubation, MTT reagent was added, and the cells were incubated overnight. The absorbance was measured at 490 nm, and cell viability curves were plotted. (c) HCC cells were seeded in a 96‐well plate (1 × 104 cells/100 μl per well) and cultured for 24 hr. I131‐mab (8.09 μCi/100 μl), I131 (12.69 μCi/100 μl) and CD147‐mab (5.62 μM/100 μl or 8.82 μM/100 μl) were used to treat cells to examine the cell visibility as described above. Compared with the parental cell group (upper panel) or I131 group (lower panel): *< .05, **< .01, and ***< .001

The tested cells were treated with I131‐mab, I131 (both agents administered at doses of 8.09 μCi/100 μl or 12.69 μCi/100 μl) and CD147‐mab (5.62 μM/100 μl and 8.82 μM/100 μl corresponding to the concentration of I131‐mab) to examine their effects on the proliferation of HCC cells. The results showed that I131‐mab was significantly more cytotoxic to all HCC cell lines than CD147‐mab or I131 alone, and specifically, viability was lowest in HepG2, Hep3B and MHCC97H cells, and doses of 8.09 μCi/100 μl and 12.69 μCi/100 μl resulted in viabilities lower than 30% and 10%, respectively. In contrast, I131‐mab was weakly cytotoxic to MHCC97L, resulting in viabilities exceeding 50% at doses of 8.09 μCi/100 μl and 12.69 μCi/100 μl. Moreover, CD147‐mab was significantly more cytotoxic to HepG2, Hep3B, SMMC‐7721 and MHCC97H cells than I131, whereas the effects of I131 and CD147‐mab on MHCC97L cells did not significantly differ (Figure 1c).

2.2 Inhibition of HCC cell invasion by I131‐mab

To verify the effect of I131‐mab on HCC cell invasion, a Transwell device was used to assess cell invasion. Both I131‐mab and CD147‐mab significantly inhibited the invasion of the HCC cell lines. Specifically, I131‐mab inhibited HepG2, Hep3B, SMMC‐7721, MHCC97H and MHCC97L cell invasion more effectively than CD147‐mab, whereas the effects of CD147‐mab on the invasion of Hep3B and MHCC97L did not significantly differ from the control group. These results suggested that inhibition of HCC cells by I131‐mab or CD147‐mab was closely associated with CD147 expression. I131 alone exerted a limited effect on HCC cell invasion, and this effect only differed between the treated and control groups for MHCC97L cells (Figure 2).

image
Inhibition of HCC invasion by I131‐mab. The upper Transwell chambers were coated with 50 μl of 1:6 diluted Matrigel (BD Biosciences, San Jose, USA), and the lower chambers were filled with 500 μl of 10% fetal bovine serum, and the Transwell chambers were placed in a well of a 24‐well plate. HCC cells were seeded into the upper chamber of the Transwell (1 × 105 cells/well) and cultured for 24 hr; the cells were treated with I131‐mab, I131 (8.09 μCi/100 μl) and CD147‐mab (5.62 μM/100 μl) were used to treat cells for 48 hr. The Transwell chambers were collected, and the cells were stained with 0.1% crystal violet for 20 min. Positive cells were counted. Compared with the control group: *< .05, **< .01 and ***< .001

2.3 I131‐mab reversed HCC cell EMT via VEGFR‐2 signaling

To elucidate the mechanism by which I131‐mab inhibits HCC cells, we compared some markers related to EMT in HCC cells treated with I131‐mab, I131 and CD147‐mab. E‐cadherin expression was significantly increased, whereas the expression levels of N‐cadherin and vimentin were decreased in all HCC cells treated with I131‐mab and CD147‐mab. Conversely, I131 did not affect the expression levels of these proteins (Figure 3a). These results suggested that I131‐mab reversed HCC cell EMT via the CD147 antibody in the conjugated molecules but not via I131.

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Impact of I131‐mab on EMT and VEGFR‐2 expression in HCC cells. (a) HCC cells were seeded in 24‐well plates (1 × 105 cells/100 μl/well) and cultured for 24 hr. I131‐mab and CD147‐mab were diluted in serum‐free medium. The cells were then treated with I131‐mab, I131 (8.09 μCi/10 0 μl) or CD147‐mab (5.62 μM/100 μl) for 48 hr. The cells were harvested, total protein was extracted, and the protein concentration was measured. Western blotting was used to measure the expression of E‐cadherin, N‐cadherin and vimentin. GAPDH was used as a loading control. Compared with the parental cell group: *< .05, **< .01 and ***< .001. (b) Cells were cultured and treated as described above. Western blotting was used to measure the expression of VEGFR‐1, p‐VEGFR‐1, VEGFR‐2 and p‐VEGFR‐2. GAPDH was used as a loading control. Compared with the parental cell group: **< .01 and ***< .001

VEGFR expression was further studied in HCC cells treated with I131‐mab, CD147‐mab and I131. The results showed that VEGFR‐2 phosphorylation (p‐VEGFR‐2) significantly decreased in HCC cells treated with I131‐mab and CD147‐mab, whereas the levels of VEGFR‐1, p‐VEGFR‐1 and VEGFR‐2 did not change. I131 alone did not affect the expression levels of these proteins in HCC cells (Figure 3b). These results suggest that I131‐mab reversed EMT by inhibiting the phosphorylation of VEGFR‐2 in HCC cells.

2.4 I131 increases the sensitivity of HCC cells to the cytotoxicity of CD147‐mab

By MTT assay, we found that I131 did not inhibit the proliferation of all HCC cells at doses <8.09 μCi/100 μl, whereas the cytotoxicity of 5.62 μM/100 μl CD147‐mab (concentration corresponding to the I131 dose) varied among HCC cell lines. We cotreated cells with I131 (8.09 μCi/100 μl) and CD147‐mab (5.62 μM/100 μl) to compared the effect of this treatment to that of CD147‐mab alone. We found that the viability of all HCC cell lines significantly decreased in response to the combined treatment (Figure 4a). The results of cell invasion capability were coincident with the MTT assay (Figure 4b,c). The results suggested that I131 increases the sensitivity of HCC cells to CD147‐mab and consequently enhances the cytotoxicity of this agent.

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I131 enhanced the inhibition of CD147‐mab on HCC cells. (a) HCC cells were seeded in 96‐well plates (1 × 104 cells/100 μl/well) and cultured for 24 hr. I131 (8.09 μCi/100 μl) and CD147‐mab (5.62 μM/100 μl) were combined to treat cells. The control group was treated with the same dose of I131 alone or CD147‐mab alone. The cell viabilities were carried out as described in Figure 1b. Compared with the parental cell group: #< .05 and ##< .01; compared with the parental cell group, the I131 group or the CD147‐mab group: ***< .001. (b) The transwell assay was carried out as described in Figure 2 to compare the capability of HCC cell invasion after treated with I131, CD147‐mab or combination of them. (c) The invasive cells were counted within five high‐magnification fields by three persons. Compared with the parental cell group: **< .01 and ***< .001

2.5 Inhibition of SMMC‐7721 human hepatoma xenografts by I131‐mab

The inhibition of HCC by I131‐mab and the molecular mechanisms underlying this inhibition were verified in nude mice harboring SMMC‐7721 human hepatoma xenografts. Treating these xenografts with I131‐mab injection markedly inhibited tumor growth speed; specifically, the tumor volume was significantly smaller in the I131‐mab treatment group than that in the control group 7 days after the first injection, and this difference increased over time. The efficacy of CD147‐mab group was evident 14 days after the start of treatment, whereas no noticeable effects were observed in the I131 treatment group, even at the end of the observation period (Figure 5a). After 21 days of treatment, the tumor volume in the control group exceeded the standard limit, and the observation was ended. The tumors were dissected and weighed, and the tumor weight was lowest in the I131‐mab group, followed by the CD147‐mab group. There is no difference in tumor weight between the I131 group and the control group (Figure 5b). Tissue sections were obtained from the hepatoma xenografts, and immunohistochemistry staining was used to measure E‐cadherin and p‐VEGFR‐2 expression. The results showed that E‐cadherin expression was up‐regulated, whereas p‐VEGFR‐2 expression was down‐regulated in the I131‐mab and CD147‐mab groups (Figure 5c).

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Inhibition of SMMC‐7721 human hepatoma xenografts by I131‐mab. (a) SMMC‐7721 cell suspension (5 × 105 cells/100 μl per mouse) was injected subcutaneously into the right flanks of the nude mice. Tumors formed 12 days after inoculation. The mice were then randomly divided into four groups of five mice each (the I131‐mab group, CD147‐mab group, I131 group and control group). The injections were carried out every other day for 5 times. Each mouse in the I131 and I131‐mab groups received 12.69 μCi/100 μl I131 or I131‐mab, respectively, whereas each mouse in the CD147‐mab group received 8.82 μM/100 μl CD147‐mab. The control group mice were injected with saline (100 μl per mouse per injection). After treatment, tumor size was measured weekly, and the tumor volume was calculated with the following formula: “maximum diameter × minimum diameter2 × 0.5.” Compared with the control group: *< .05, **< .01 and ***< .001. (b) At the end of observation, the mice were killed, and the tumor specimens were harvested and weighed. Compared with the control group: **< .01 and ***< .001; (c) sections of hepatoma xenografts were obtained. The expression levels of E‐cadherin and p‐VEGFR‐2 were measured by immunohistochemical staining. Stained cells were counted in five high‐magnification fields, and the percentages of positive cells were determined. Compared with the control group: *< .05 and ***< .001

3 DISCUSSION

EMT has been observed in many human tumors, and extracellular signaling molecules that induce EMT in tumor cells include MMP‐2, MMP‐3, MMP‐9, type I or III collagen, hepatocyte growth factor (HGF), epidermal growth factor (EGF), fibroblast growth factor (FGF), TGF‐β and tumor necrosis factor‐α (TNF‐α). EMT also modulates the activity of multiple signaling pathways, such as TGF‐β, Wnt, PDGF, Notch, Hedgehog, Akt, PI3K, NF‐κB and Ras, via the Snail family of zinc finger transcription factors (Snail1, Snail2 and Snail3) and the helix‐loop‐helix structure of transcription factors (Twist, ZEB 1 and ZEB2/SIP1) to down‐regulate the epithelial markers E‐cadherin and keratin or up‐regulate the mesenchymal markers N‐cadherin and vimentin (Hanna & Shevde, 2016; Lee & Kong, 2016; Moustakas & Heldin, 2016; Xu, Yang, & Lu, 2015; Zhang, Tian, & Xing, 2016). EMT increases the malignancy of cancer cells because cells lose apical–basal polarity and cell junctions and acquire a migratory mesenchymal phenotype. These cells then invade the lymphatic or vascular system and spread to different sites or organs to grow and form metastatic tumors (Jayachandran et al., 2016; Kölbl, Jeschke, & Andergassen, 2016).

TACE is an important treatment for HCC but has been clinically associated with increases in distant metastasis due to residual cancer cells in many patients with HCC (Xue et al., 2016). This phenomenon has greatly compromised the long‐term efficacy of TACE. Moreover, TACE can induce tissue hypoxia, which down‐regulates E‐cadherin and up‐regulates of N‐cadherin in residual cancer cells. Thus, hypoxia may mediate the negative effects of TACE by inducing EMT in cancer cells (Fransvea, Angelotti, Antonaci, & Giannelli, 2008; Xue et al., 2015). EMT enhances the invasion and metastasis of cancer cells, reduces the sensitivity or increases the resistance of HCC to chemotherapeutic agents (Bae et al., 2014; Nishida, Kitano, Sakurai, & Kudo, 2015) and contributes to the immune escape of HCC cells (Chockley & Keshamouni, 2016; Ye et al., 2016).

The studies of the relationship between EMT and the metastasis of HCC have provided an effective target for HCC treatment. LY2109761, a TGF‐β 1 receptor kinase inhibitor, inhibits EMT to reduce vascular invasion and metastasis in HCC cells (Baldassarre et al., 2012). Blocking the effects of upstream loop regulatory factors of EMT, such as inhibition of NF‐κB/miR‐448, can improve the response of cancer cells to chemotherapeutic agents (Li et al., 2011). CD147, under the regulation of slug at transcriptional level, can promote EMT process in HCC via TGF‐β signaling (Wu et al., 2011). Furthermore, hypoxia caused by TACE can up‐regulate CD147 expression in HCC cells (Gou et al., 2016), and the over‐expression of CD147 enhances EMT in cancer cells by activating TGF‐β signaling. Thus, I131‐mab, which targets CD147, may specifically inhibit EMT to attenuate tumor development and metastasis. In nude mouse HCC models, I131‐mab effectively inhibited the growth and metastasis of HCC and inhibited MMPs and VEGF expression in the para‐tumor microenvironment (Wu et al., 2011). We conducted a prospective controlled clinical study in which the combination of TACE and I131‐mab was used to treat intermediate‐stage HCC (BCLC staging). Specifically, 68 patients with intermediate‐stage HCC were included in the combined treatment group, and 70 patients were included in the TACE alone group. The median survival time was 26.7 months in the combined treatment group but only 20.6 months in the control group, and the overall survival in the combined treatment group was significantly better than that in the control group (= .038). The median time to progression was 18.6 months in the combined treatment group and superior to only 12.5 months in the control group (= .046). In particular, the extrahepatic metastasis rate was 2.94% in the combined treatment group and 8.57% in the TACE group. These results suggested that I131‐mab reduces the risk of extrahepatic metastasis after TACE treatment (Wu et al., 2012).

In this work, the cytological experiments proved that I131‐mab inhibits the proliferation and invasion of HCC cells, and this inhibition is closely related to the level of CD147 expression. HCC cells expressing high levels of CD147 are more sensitive to I131‐mab. The inhibition effect of I131‐mab on HCC cells was mostly attributed to the CD147 antibody within the conjugated molecule. Although the inhibition of HCC cell proliferation and invasion by I131 alone is limited, when combined with CD147‐mab, I131 significantly enhanced the sensitivity of cancer cells to CD147‐mab and consequently enhanced the cytotoxicity of anticancer antibodies. This finding suggested that the two components of I131‐mab synergistically inhibited HCC. Both I131‐mab and CD147‐mab can reverse the EMT of HCC cells partially by inhibiting the phosphorylation of VEGFR‐2 and then reduce the capabilities of proliferation and metastasis of HCC cells. The experiments using SMMC‐7721 human hepatoma xenografts in athymic nude mice also proved that I131‐mab and CD147‐mab significantly inhibited xenograft tumors, and I131‐mab was more effective than CD147‐mab in this inhibition. The synergistic effect of conjugated I131‐mab was attributed to I131 because I131 alone did not significantly inhibit the proliferation and invasion of HCC. Our results elucidated the mechanism underlying the antiproliferative and antimetastatic effects of I131‐mab and provided a theoretical foundation for the clinical application of I131‐mab.

4 EXPERIMENTAL PROCEDURES

4.1 Cells and reagents

Human hepatoma cell lines (HepG2, Hep3B, SMMC‐7721, MHCC97H and MHCC97L) and normal hepatic cells (WRL‐68) were provided by Cell Bank, Institute of Cell Biochemistry, Chinese Academy of Sciences Shanghai Institutes for Biological Sciences (Shanghai, China). The cells were cultured according to the manufacturer's protocol. I131‐mab was developed by the Chengdu Huasun Group Inc., Ltd. (Chengdu, China). CD147‐mab and Sodium Iodide [131] Capsules (I131) were purchased from Abcam Trading Company Ltd. (Shanghai, China) and Atom‐Hitech Co., Ltd. (Beijing, China), respectively.

4.2 Cell proliferation assay

A tetrazolium colorimetric assay (MTT assay) was used to assess the effects of I131‐mab, CD147‐mab and I131 on the proliferation of HCC and normal cells. Briefly, cells were harvested during the logarithmic growth phase and seeded in 96‐well plates at 1 × 104 cells/100 μl/well, and then cultured for 24 hr; serum‐free culture medium was used to dilute I131‐mab, and cells were treated with various concentrations of this agent, with eight duplicates per concentration. After incubated for 2 hr, the medium was then replaced with medium containing 10% serum (100 μl/well). After 48 hr of culture, the medium was replaced with 0.1 M PBS solution (100 μl/well), and MTT labeling reagent (10 μl/well for a final concentration of 0.5 mg/ml; Roche Diagnostics GmbH, Shanghai, China) was then added. After 4 hr of incubation, the solubilization solution (10% SDS in 0.01 mol/L HCl, 100 μl/well) was added for overnight, the absorbance was measured at 490 nm, and the values were plotted to assess cell viability and calculate the IC20 and IC50 of each concentration of I131‐mab and its controls.

4.3 Cell invasion assay

The upper Transwell chambers (8 μm pore size, Corning, Tewksbury, USA) were coated with 50 μl of 1:6 diluted Matrigel (BD Biosciences, San Jose, USA), and the lower chambers were filled with 500 μl of 10% fetal bovine serum. The Transwell chambers were placed in a well of a 24‐well plate. Harvested cells were seeded into the upper Transwell chamber at 1 × 105 cells/well and cultured for 24 hr; I131‐mab was diluted in serum‐free culture medium and added to the wells for a variety of final concentrations. The cells were incubated for 2 hr, and the medium was replaced with medium containing 10% serum (100 μl/well) after 48 hr of incubation. The Transwell inserts were removed, and the cells were stained with 0.1% crystal violet for 20 min before being counted under a microscope; three random fields were photographed (200× magnification).

4.4 Immunoblotting

The above‐studied cells were seeded in a 24‐well plate at 1 × 105 cells/well and cultured for 24 hr. I131‐mab was diluted in serum‐free culture medium and added to the culture wells at various concentrations (based on the protocol). These cells were cultured for 2 hr. Medium containing 10% serum (100 μl/well) was used to replace the previous medium. After 48 hr of incubation, the cells were harvested. Western blotting was used to measure the expression of the target proteins. The antibodies used in the experiments are listed below: rabbit anti‐CD147 (Abcam Trading Company Ltd., Shanghai, China); mouse anti‐E‐cadherin, mouse anti‐N‐cadherin, mouse antivimentin, rabbit anti‐VEGF‐C antibodies (Cell Signaling Technology, Danvers, MA, USA); and mouse anti‐VEGFR‐1, mouse anti‐VEGFR‐2, rabbit anti‐phospho‐VEGFR‐1 and rabbit anti‐p‐VEGFR‐2 (Cell Applications Inc., CA, USA).

4.5 Human hepatoma xenografts in nude mice

A total of 25 healthy, purebred BALB/C male mice aged 4 weeks were purchased from the SLAC Experimental Animal Centre of the Chinese Academy of Sciences (Shanghai, China). SMMC‐7721 cells were harvested during the logarithmic growth phase, and cell suspension was prepared. The cells (5 × 105 cells in 100 μl per mouse) were subcutaneously injected into the right flanks of nude mice, and tumors formed 12 days after inoculation. The three mice with the largest tumors and the two mice with the smallest tumors were excluded. The remaining 20 mice were randomly divided into four groups (the I131‐mab group, the CD147‐mab group, the I131 group and the control group). Animals in the I131 and I131‐mab groups received injections of I131 or I131‐mab at multiple sites (dose: 12.69 μCi/100 μl per mouse); animals in the CD147‐mab group received CD147‐mab at a dose of 8.82 μM/100 μl per mouse. The animals were injected every other day for a total of five injections. The control mice were injected with saline (100 μl per mouse per injection). After treatment, tumor size was measured weekly, and the following formula was used to calculate tumor volume: “maximum diameter × minimum diameter2 × 0.5.” The experiment was terminated immediately when the mean tumor volume exceed 2,000 mm3 in any group, as defined by the Animal Ethics Committee of the Second Military Medical University. At the end of observation, the mice were killed with over‐doses anesthesia. The tumor specimens were harvested and weighed, and the tumor tissue was fixed in 10% neutral buffered formalin, embedded in paraffin, sectioned and immunohistochemically stained.

4.6 Immunohistochemistry

The sections of paraffin‐embedded SMMC‐7721 hepatoma xenografts were subjected to streptavidin–peroxidase immunohistochemistry to detect the expression of E‐cadherin and p‐VEGFR‐2. The antibodies used for staining were the same as those used for Western blotting. The percentages of positive cells in all sections were determined by counting the cells in five high‐magnification fields.

4.7 Statistical analysis

The cytological experiments were repeated three times independently, and a total of five animals were included in each group. The data are expressed as the mean ± SD. An analysis of variance (ANOVA) and a t test were used for the statistical analysis with the PASW Statistics 18 software. p value less than .05 was considered significant.

ACKNOWLEDGMENTS

This work was supported by the National Natural Science Foundation of China (81301302; 81773251; 81702735), and the Science and Technology Commission of Shanghai Municipality (15411966100).

    AUTHOR CONTRIBUTIONS

    L.W., B.S., X.L. and C.L. conceived the experiments. H.Q., L.C. and C.S. conducted the experiments. L.W., B.S., X.L., Y.Y. and F.S. analyzed the results. All authors reviewed the manuscript.

    The information comes from:
    https://onlinelibrary.wiley.com/doi/full/10.1111/gtc.12545


    Iodine Symporter Targeting with 124I/131I Theranostics

    Abstract

    Theranostics, a modern approach combining therapeutics and diagnostics, is among the most promising concepts in nuclear medicine for optimizing and individualizing treatments for many cancer entities. Theranostics has been used in clinical routines in nuclear medicine for more than 60 y—as 131I for diagnostic and therapeutic purposes in thyroid diseases. In this minireview, we provide a survey of the use of 2 different radioiodine isotopes for targeting the sodium–iodine symporter in thyroid cancer and nonthyroidal neoplasms as well as a brief summary of theranostics for neuroendocrine neoplasms and metastatic castration-refractory prostate cancer. In particular, we discuss the role of 124I-based dosimetry in targeting of the sodium–iodine symporter and describe the clinical application of 124I dosimetry in a patient who had radioiodine-refractory thyroid cancer and who underwent a redifferentiation treatment with the mitogen-activated extracellular signal–related kinase kinase inhibitor trametinib.

    Theranostics is a diagnostic approach coupled with treatment modalities in a personalized fashion to improve therapeutic effects and reduce treatment toxicities (1). This term was initially coined by John Funkhouser in a 1998 press release in connection with personalized treatment. Translating theranostics to the field of nuclear medicine means labeling of a compound with different radionuclides for both diagnostic and therapeutic purposes for a specific target. Ideally, the radionuclides used for both diagnostic and therapeutic purposes are derived from the same element, but for chemical and physical reasons this is often not practicable. In this mini review, we provide a brief survey of 2 theranostic approaches for neuroendocrine tumors and prostate cancer, with a focus on targeting of the sodium–iodine symporter (NIS) in thyroidal disorders, and we discuss issues regarding 124I and 131I targeting of the NIS in extrathyroidal disorders.

    CLINICAL USE OF THERANOSTIC AGENTS OTHER THAN IODINE

    Neuroendocrine neoplasms and prostate cancer are currently the most prominent targets of nonthyroidal theranostic agents (24). Because of a lack of efficient treatment options, metastatic, well-differentiated neuroendocrine neoplasms are challenging tumors. These tumors express somatostatin receptors, which can be imaged by PET with, for instance, 68Ga-labeled DOTATATE, DOTATOC, or DOTANOC (5). PET enables in vivo quantification of the tumoral expression level and of the background uptake level in the surrounding tissues or organs at risk. For treatment, the same compound can be labeled with a β-particle emitter (177Lu or 90Y) to target metastatic sites. The higher the level of expression in the tumor (and, thus, the higher the tumor-to-background ratio), the lower the radiation-related toxicity effects in the surrounding tissues.

    Regarding toxicity, protection of the kidneys is important, because a large portion of the injected amount of a radiolabeled compound is eliminated via renal excretion. To address this issue, patients receive an infusion of an amino acid cocktail during treatment to saturate renal reabsorption mechanisms (6). The efficacy of this approach was recently shown in a prospective multicenter trial (2).

    Another promising theranostic approach is being used for metastatic castration-resistant prostate cancer. The target in this challenging disease is the prostate-specific membrane antigen (PSMA), which is expressed at high levels, particularly in recurrent prostate cancer; additionally, expression is not lost with dedifferentiation, making it an ideal target. PSMA-targeting molecules can be labeled with different positron emitters, such as 124I, 18F, 64Cu, or 68Ga (3,7). At present, PSMA ligands are more frequently labeled with 68Ga for PET imaging. For therapeutic purposes, they are usually labeled with the β-particle emitter 177Lu. However, there has even been a report on the labeling of a PSMA ligand with the α-particle emitter 225Ac for the treatment of patients whose disease progressed after 177Lu-PSMA ligand treatment (8).

    THERANOSTICS WITH IODINE

    Radioiodine treatment (using 131I) has been the main pillar of nuclear medicine for more than 60 y. 131I not only is a β-particle emitter but also has penetrating γ-radiation, which makes this tracer trackable in vivo through imaging. However, 131I is not the ideal tracer for quantitative imaging purposes because of poor spatial resolution and quantification capacity using SPECT (Table 1). With the increasing availability of PET scanners during the last 15 y, 124I became the tracer of first choice for the imaging of thyroid disorders, mainly in patients with high-risk and recurrent thyroid cancer (9,10). The properties of 124I and 131I are juxtaposed in Table 1, demonstrating the superiority of 124I for imaging. Moreover, 124I allows more reliable dosimetry, the 2 main pillars of which are shown in Figure 1.

    TABLE 1

    Physical Half-Lives and Qualitative Comparison of Common Radioiodine Isotopes for Imaging in Theranostics

    FIGURE 1.
    FIGURE 1.

    Simplified illustration of 2 main pillars of 124I dosimetry concept. LDpA = lesion-absorbed dose per administered activity; MTA = maximum tolerable activity.

    Most patients typically undergo several radioiodine treatments during their disease history, and each additional radioiodine treatment increases the risk of radiation-associated detrimental effects. To reduce or at least estimate the risks, as well as to increase the efficacy of radioiodine treatment, an individual assessment of absorbed radiation doses to the tumors and the organs at risk is crucial. According to Maxon et al., an absorbed dose of 85 Gy or higher is associated with an 80%–90% likelihood of a therapy response in lymph node metastases (Table 2) (11). The absorbed dose thresholds for other metastatic tissues (and thyroid remnants), derived from current 124I PET dosimetry studies, are also shown in Table 2 (12,13). Of note, a response to radioiodine is already expected for absorbed doses exceeding 20 Gy (11,12). Moreover, the bone marrow is often the dose-limiting organ in the application of high therapy activities.

    TABLE 2

    Relationship Between Absorbed Dose Thresholds and Associated Complete Response Rates for Metastases and Thyroid Remnants

    Pretherapy blood dosimetry has been developed to estimate the toxicity of radioiodine with the aim of avoiding possible life-threatening, radiation-induced bone marrow suppression (Fig. 1). In this organ-at-risk dosimetry approach, the maximum tolerable 131I activity that can be safely administered without producing toxic effects is calculated. Through collection of blood samples and determination of external whole-body counts over a period of 4 d or longer, the maximum tolerable 131I activity can be estimated using an absorbed dose limit of 2 Gy to blood (as a surrogate for bone marrow toxicity) (1416). The key quantities for estimating the absorbed dose to the tumor are mass (or volume), the initial uptake value (for instance, at 24 h), and the effective 131I half-life. The mass can be estimated from CT or 124I PET using sophisticated threshold-based segmentation algorithms. Determination of the 24-h uptake—mainly mediated through the NIS—and the predicted effective 131I half-life requires serial 124I PET/CT scans (17). Pretherapy dosimetry results enable the selection of an optimized therapeutic activity—that is, the activity achieving a high tumor dose (such as 85 Gy for lymph node metastases) while maintaining a dose less than the 2-Gy limit to blood (Fig. 1). Thus, 124I dosimetry is suitable for individual therapy assessment.

    IODINE METABOLISM

    A better understanding of 124I PET dosimetry can be gained through an examination of iodine metabolism (Fig. 2). In brief, iodide is transported actively into the cell via the NIS, which is located in the basolateral membrane of thyrocytes. Next, this iodide enters the colloid on the apical membrane, located on the opposite side, via pendrin or other unspecified channels (18). In the colloid, iodide is oxidized, is bound to thyroglobulin (via the enzyme thyroid peroxidase), and either remains in the colloid or exits the cell as the end product—the thyroid hormone triiodothyronine or tetraiodothyronine. The active transport of iodide into the cell via the NIS is correlated with the level of expression of this symporter and can be estimated in vivo through 124I PET imaging. The effective half-life—that is, the decrease in accumulated radioiodine uptake over time—cannot be quantified on the basis of a single PET scan. Therefore, serial PET scans over time are needed to estimate the kinetics of radioiodine. Even though the precision of the quantification of radioiodine accumulation over time increases with the number of PET scans, these scans are limited in clinical settings for time and economic reasons. Therefore, a 2-time-point model that reasonably balances precision and effort or cost in clinical settings has been developed (19).

    FIGURE 2.
    FIGURE 2.

    Thyroid follicle showing iodine uptake and residence. Expression of NIS is essential for iodide uptake. Iodide is transported via pendrin to colloid in which iodide is bound to thyroglobulin. The latter is crucial to increasing average time on site of radioiodide in the follicle, which is associated with an increased absorbed radiation dose. Nonthyroidal cells expressing NIS lack this storing mechanism. DIT = diiodotyrosine; MIT = monoiodotyrosine; TSH = thyroid-stimulating hormone; TSH-R = TSH receptor. The function of TSH is to stimulate NIS expression.

    An example is provided in Figure 3, which shows 124I PET/CT images of a thyroid cancer patient and describes the lesions along with their predicted absorbed doses. 124I PET was capable of quantifying the increase in NIS expression in this patient with radioiodine-refractory thyroid cancer after redifferentiation treatment with trametinib, a mitogen-activated extracellular signal–related kinase kinase inhibitor. This is the first report of trametinib treatment of a patient with radioiodine-refractory thyroid cancer. Basically, as shown by the 124I PET results, the effective half-lives of the lesions remained similar (Fig. 4). Because of previous experience with mitogen-activated extracellular signal–related kinase kinase inhibition in this setting, we expected an increase in NIS expression without a significant increase in the effective half-lives, as shown in Figure 4 (17). The effects in our patient with radioiodine-refractory thyroid cancer were in line with this expectation. The 124I PET results revealed a 10-fold increase in iodide uptake with a nonsignificant change in the effective half-life.

    FIGURE 3.
    FIGURE 3.

    PET/CT images of 69-y-old patient with follicular thyroid carcinoma diagnosed in 1999. Patient underwent surgery and many treatments with radioiodine and experienced disease progression involving bone and lymph node metastases. Patient had undergone tyrosine kinase inhibitor treatment with sorafenib and lenvatinib but discontinued treatment because of disease progression. Patient was introduced to our hospital for redifferentiation therapy. After confirmation of BRAF-WT mutation status using archival tumor tissue, patient underwent pretreatment lesion dosimetry under thyrotropin stimulation with recombined human thyrotropin (A and C). Target lesions showed absorbed doses of 1–10 Gy/GBq. After 4 wk of trametinib treatment, 124I PET lesion dosimetry revealed absorbed doses of 10–322 Gy/GBq for most metastases (B and D). Blood dosimetry estimated maximum tolerable activity of 7 GBq. Patient was treated with 6 GBq of 131I. This example demonstrates importance of in vivo dosimetry in estimating redifferentiation effects and evaluating radioiodine treatment of nonthyroidal tumors.

    FIGURE 4.
    FIGURE 4.

    Predicted 131I uptake curve derived from 124I PET–based lesion dosimetry before (A) and after (B) redifferentiation. Lines were calculated using 2-point approach (19), and symbols represent measured PET-derived uptake values. Uptake values before differentiation were lower by factor of 10, demonstrating similar effective half-lives but 10-fold-lower 24-h uptake per gram.

    These findings were most likely due to the fact that in such tumors, mitogen-activated extracellular signal–related kinase kinase inhibition alone is not sufficient to reestablish the polarity of the cells and, thus, reshape a functioning colloidal structure. The latter is crucial for proper binding of iodide to thyroglobulin, resulting in an increased effective half-life. Nevertheless, the increased level of NIS expression alone was sufficient to increase the estimated absorbed dose significantly. The dosimetry results showed that the patient should have been treated with 17 GBq of 131I. However, the decision about a treatment must also be based on the blood dosimetry results, which limited the amount of treatment activity to 7 GBq of 131I. Even though there is no single clinical study analyzing the predictive value of pretherapeutic 124I PET dosimetry, the common consensus is that 124I PET dosimetry contributes significantly to pretherapeutic absorbed dose estimations. The example provided here not only illustrates the potential of 124I PET dosimetry but also underlines the importance of applying this dosimetry approach to redifferentiation treatments in clinical routines (20).

    THERANOSTICS WITH IODINE IN NONTHYROIDAL CANCER

    The simplicity and efficacy of radioiodine for the imaging and treatment of thyroid cancer patients attracted many research groups to investigate NIS expression in nonthyroidal tumor entities with the aim of treating these entities with radioiodine as well (21,22). In this context, some research groups investigated the efficacy of transfection of the NIS gene to tumor cells to make them targets for radioiodine. Table 3 shows the results of a study in which NIS expression in nonthyroidal tumors was investigated. Investigating radioiodine accumulation in nonthyroidal tissue requires an appreciation of the lack of a colloidal structure in the tumor cells and, thus, the absence of an ability to metabolize iodine and the consequent negative impact on the effective half-life of iodine. Given these circumstances, the level of expression of the NIS in nonthyroidal tumor cells should compensate for these shortcomings to achieve a significant absorbed dose. This goal is challenging and is probably the main reason why no clinical data showing the efficacy of radioiodine in nonthyroidal tumors have yet been published.

    TABLE 3

    Extrathyroidal Tissues Expressing NIS

    Another issue is the presence of thyroid in patients with nonthyroidal cancer. Because these patients have a functioning thyroid, the applied radioiodine will be actively transported into thyroid cells; therefore, the amount of radioiodine delivered to the targeted tumor cells will be reduced. More important than this reduced efficacy is unintended radiation damage to thyroid cells. However, thyroid uptake can be reduced through the coapplication of triiodothyronine (or tetraiodothyronine) and methimazole. Triiodothyronine downregulates the thyrotropin level (through a feedback loop), resulting in a reduction in NIS expression and, consecutively, a reduction in iodine uptake. Methimazole inhibits the enzyme thyroperoxidase, which catalyzes the binding of iodine to thyroglobulin and, thus, reduces the effective half-life of iodine (23).

    IODINE AND BREAST CANCER

    Breast cancer was one of the first nonthyroidal tumor entities in which NIS expression was convincingly shown (by messenger RNA levels and immunohistochemical staining). Therefore, many groups proposed radioiodine treatment for patients with breast cancer expressing the NIS (24,25). However, the in vivo imaging of NIS expression (131I or 99mTc scans) in the tumor cells did not correlate with NIS expression shown by messenger RNA levels and immunohistochemical staining. This apparently contradictory result is more likely to be due to the fact that the increased expression of the NIS on messenger RNA and protein levels is not translated into a functioning NIS located on the basolateral membrane. The latter is crucial for proper functioning of this symporter.

    There are few reports on the imaging of increased NIS expression in breast cancer xenografts and in brain metastases of breast cancer in mice. Kelkar et al. discussed a potential treatment for patients (25). However, no data analyzing this hypothesis in clinical settings have yet been published. The main shortcoming of the published in vivo data is that the role of tumor dosimetry—that is, an estimation of the absorbed doses delivered to tumors—was not analyzed. As described earlier, quantifying the uptake, effective half-life, and tumor mass is crucial for calculating the doses delivered to tumors; these data, in turn, predict the response to radioiodine.

    Figure 5 shows an estimation of the model-based absorbed doses for a spheric tumor as a function of 24-h 131I uptake per gram and at various effective half-lives. As shown in Figure 4, the 24-h 131I uptake per gram was approximately 0.16%/g after redifferentiation, and the effective half-life was estimated to be 1 d. As shown in Figure 5, the estimated absorbed dose was about 10 Gy/GBq, a value that was similar to the calculated one. This approach may suggest the extent to which the iodide uptake must be increased to achieve a tumoricidal absorbed dose.

    FIGURE 5.
    FIGURE 5.

    Model-based relationship between absorbed dose and actual 24-h 131I uptake per gram of tissue for 1-mL spheric lesion at different effective half-lives (in days) (shown close to straight lines); values within parentheses are estimated slopes (in Gy/GBq per unit percentage uptake per gram) for assessing absorbed doses beyond axis scale limit. Uptake curves decreased monoexponentially using the respective effective half-lives. For volumes ranging from 0.1 to 5 mL, absolute percentage absorbed dose deviations from 1-mL volume were less than or equal to 5%. Nonlinear relationship between slope and half-life resulted from extrapolation from 24-h uptake value to zero time point.

    CONCLUSION

    Theranostics with the matched pair 124I/131I in high-risk or progressive thyroid cancer enables an individualized dosimetry approach to delivering high absorbed doses to tumors and reducing radiation-related toxicity primarily to bone marrow. The target dose delivered through 131I depends on iodine uptake and effective half-life. In this context, NIS expression is critical for iodine uptake and a colloidal configuration with polarized thyrocytes for the effective half-life of radioiodine. Therefore, applying radioiodine isotopes to nonthyroidal tumor cells remains challenging, but individualized dosimetry at least facilitates proper analysis of the expected effectiveness.

    DISCLOSURE

    No potential conflict of interest relevant to this article was reported.

    The information comes from:
    http://jnm.snmjournals.org/content/58/Supplement_2/34S.long


    Studies on 177Lu-labeled methylene diphosphonate as potential bone-seeking radiopharmaceutical for bone pain palliation.

    Abstract

    OBJECTIVE:
    (99m)Tc-MDP (technetium-99(m)-labeled methylene diphosphonate) has been widely used as a radiopharmaceutical for bone scintigraphy in cases of metastatic bone disease. (177)Lu is presently considered as an excellent radionuclide for developing bone pain palliation agents. No study on preparing a complex of (177)Lu with MDP has been reported yet. Based on these facts, it was hypothesized that a bone-seeking (177)Lu-MDP (lutetium-177-labeled MDP) radiopharmaceutical could be developed as an agent for palliative radiotherapy of bone pain due to skeletal metastases. Biodistribution studies after intravenous injection of (177)Lu-MDP complex in rats may yield important information to assess its potential for clinical use as a bone pain palliation agent for the treatment of bone metastases.

    METHODS:
    (177)Lu was produced by irradiating natural Lu(2)O(3) (10 mg) target at a thermal flux ∼ 8.0 × 10(13) n/cm(2) per second for 12 h in the swimming pool-type reactor.(177)Lu was labeled with MDP by adding nearly 37 MBq (1.0 mCi) of (177)LuCl(3) to a vial containing 10 mg MDP. The radiochemical purity and labeling efficiencies were determined by thin layer chromatography. Labeling of (177)Lu with MDP was optimized, and one sample was subjected to high-performance liquid chromatography (HPLC) analysis. Twelve Sprague-Dawley rats were injected with 18.5 MBq (0.5 mCi). (177)Lu-MDP in a volume of 0.1 ml was injected intravenously and then sacrificed at 2 min, 1 h, 2 h and 22 h (three rats at each time point) after injection. Samples of various organs were separated, weighed and measured for radioactivity and expressed as percent uptake of injected dose per gram. Bioevaluation studies with rats under gamma-camera were also performed to verify the results.

    RESULTS:
    The quality control using thin layer chromatography has shown >99% radiochemical purity of (177)Lu-MDP complex. Chromatography with Whatman 3MM paper showed maximum labeling at pH = 6, incubation time = 30 min, and ligand/metal ratio = 60:1. HPLC analysis showed 1.35 ± 0.05 min retention time of (177)Lu-MDP complex. No decrease in labeling was observed at higher temperatures, and the stability of the complex was found adequate. Biodistribution studies of (177)Lu-MDP revealed high skeletal uptake, i.e., 31.29 ± 1.27% of the injected dose at 22 h post injection. Gamma-camera images of (177)Lu-MDP in Sprague-Dawley rats also showed high skeletal uptake and verified the results.


    CONCLUSION:
    The study demonstrated that MDP could be labeled with (177)Lu with high radiochemical yields (>99%). The in vitro stability of the complex was found adequate. Biodistribution studies in Sprague-Dawley rats indicated selective bone accumulation, relatively low uptake in soft tissue (except liver) and higher skeletal uptake, suggesting that it may be useful as a bone pain palliation agent for the treatment of bone metastases.

    Copyright © 2011 Elsevier Inc. All rights reserved.

    The information comes from:
    https://www.ncbi.nlm.nih.gov/pubmed/21492790