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Targeted Molecular Imaging of Antigen OC183B2 in Ovarian Cancers Using MR Molecular Probes

Posted Nov 30, 2010 Updated Apr 7, 2023
By Guangnan Quan MD , Xiangke Du MD PhD , Tianlong Huo MD , Xubin Li MD , Zhengmao Wei MD , Heng Cui MD PhD , Xiaohong Chang MD , Yexia Cheng MD , Xue Ye MD , Hongyan Cheng MD
9 min read

Rationale and Objectives

This study was designed to develop a novel magnetic resonance (MR) probe for the antigen OC183B2 in ovarian cancer cells and investigate its imaging features in vitro and in vivo.

Materials and Methods

Molecular probes were achieved through ultrasmall superparamagnetic iron oxide nanoparticles (USPIOs) conjugated to ovarian cancer monoclonal antibodies 183B2 (OCMab183B2) using a chemical method. In the control group, USPIOs were coupled with murine immunoglobulin G (mIgG) and conjugated the same way. Native polyacrylamide gel electrophoresis was used to evaluate the conjugation reaction. The cytotoxicity of the probe was measured using the methyl thiazolyl tetrazolium assay, and its cell-labeling efficiency was evaluated by Prussian blue staining. In vitro cell MR imaging was performed to evaluate the targeting of the probe to the cells. After that, the OCMab183B2 USPIOs and mIgG USPIOs were injected intravenously into nude mice implanted with ovarian cancer xenograft tumors, respectively. T2-weighted imaging and T2 mapping were then performed on a 3.0-T MR imaging system equipped with an animal birdcage coil at different times. Finally, the nude mice were sacrificed for histologic examination to confirm the imaging results.

Results

Native polyacrylamide gel electrophoresis displayed an optimal conjugation of USPIOs to OCMab183B2 and mIgG. Various blue-staining particles were found in the cells labeled with the molecular probe at different iron concentrations, and the density of particles was positively related to the iron concentration. Its labeling rate was 96.06%, which was higher than that of USPIOs (62.5%) at the same iron concentration (20 μg/mL). The methyl thiazolyl tetrazolium assay showed that there was no difference in cellular bioactivity between OCMab183B2 USPIO–labeled and nonlabeled cells ( P > .05). In vitro cell MR imaging showed that there was an obvious decrease in signal intensity for the probe-labeled cells compared to mIgG USPIO–labeled cells. For in vivo MR imaging, distinct changes of signal intensities and T2 values of ovarian cancers were detected after the injection of OCMab183B2 USPIOs compared to mIgG USPIOs. The histologic analysis showed that iron depositions were visualized in the experimental group but not in the control group.

Conclusion

OCMab183B2 USPIO conjugates have the potential to be useful as OC183B2-targeted MR imaging agents for the early detection of ovarian cancers.

Ovarian cancer accounts for 4% of all cancers in women and is the leading cause of death from gynecologic malignancies. Early detection and accurate diagnosis are crucial for its successful treatment . Magnetic resonance (MR) imaging has emerged as an important contributor to its diagnosis, but current conventional MR imaging has several limitations in early diagnosis due to its imaging mechanism, which is based on anatomic changes . Recent developments in molecular imaging have provided new views for cancer imaging and treatment. This new imaging technology focuses on molecular changes of diseases and enables MR imaging to detect these changes at “predisease states.” In the process of MR molecular imaging, an important step is to develop a targeted imaging probe, which is made of MR contrast agents, mostly including superparamagnetic iron oxide nanoparticles or ultrasmall superparamagnetic iron oxide nanoparticles (USPIOs), and various ligands or antibodies. This imaging strategy relies on targeted imaging of biomarker molecules coupled with imaging probes guided by ligands or antibodies that can recognize and interact with target molecules .

In our previous studies, we have discovered an ovarian cancer antigen 183B2 (OC183B2), which is expressed on both cytoplasm and cytomembrane of SKOV-3 cells, one kind of epithelial ovarian carcinoma cells, and we successfully obtained its monoclonal antibody (OCMab183B2) from mouse ascites . The murine monoclonal antibody OCMab183B2 was raised against human epithelial ovarian adenocarcinoma-associated antigen by the fusion of murine myeloma cells with spleen lymphocytes from BALB/c nude mice immunized with soluble antigens prepared from ovarian papillary serous cyst adenocarcinoma. The monoclonal antibody was stable after culture, and its subclass was immunoglobulin G. In the current study, we aimed to develop novel MR molecular probe OCMab183B2 USPIO targeting to the antigen OC183B2 and then investigate its imaging features in vitro and in vivo.

Materials and methods

Cell Culture

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Immunocytochemistry

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Conjugation of Antibody with Biocompatible Magnetic Nanoparticles

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Native Polyacrylamide Gel Electrophoresis

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Labeling of OC183B2 USPIOs and USPIOs

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Methyl Thiazolyl Tetrazolium (MTT) Assay

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In Vitro MR Imaging

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Animals and Tumor Models

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In Vivo MR Imaging

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Statistical Analysis

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Results

Cell Culture

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Expression of the Antigen OC183B2 in SKOV-3 Cells

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Figure 1, The OC183B2 tumor antigen was detected by immunocytochemical staining of ovarian carcinoma SKOV-3 cells with monoclonal antibody directed to OC183B2. (a) Tumor cells demonstrated positive staining for antigen OC183B2 on cell surface membrane as well as cytoplasm (Streptavidin- perosidase (SP); magnification, 400×). (b) Negative control was the staining of tumor cells with secondary antibody but without primary monoclonal antibody (SP; magnification, 400×).

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Native Polyacrylamide Gel Electrophoresis

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Figure 2, The gel stained by Coomassie blue after electrophoresis. Lanes A, B, C, and D were filled with OCMab183B2 ultrasmall superparamagnetic iron oxide nanoparticle (USPIO) conjugates, a mixture of USPIOs and OCMab183B2, USPIO nanocrystals, and OCMab183B2, respectively. There was no difference between lanes B and D but a remarkable difference between lanes A and D.

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Comparing the Labeling Ability Between OC183B2 USPIOs and USPIOs

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Figure 3, Prussian blue staining of labeled cells with different iron concentration of probes and ultrasmall superparamagnetic iron oxide nanoparticles (USPIOs) (magnification, 100×). (a) Probe at an iron concentration of 40 μg/mL. (b) Probe at an iron concentration of 20 μg/mL. (c) USPIOs at an iron concentration of 20 μg/mL. (d) Blank control.

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MTT Results

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Figure 4, Methyl thiazolyl tetrazolium (MTT) assay of labeled cells with OCMab183B2 ultrasmall superparamagnetic iron oxide nanoparticles (USPIOs) at different iron concentrations. There was no significant difference of optical density (OD) values between labeled and unlabeled cells, demonstrating that OCMab183B2 USPIOs are safe to the SKOV-3 cells.

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In Vitro MR Imaging

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Figure 5, In vitro T2-weighted magnetic resonance imaging of SKOV-3 cells. (a) Unlabelled cells. (b) Labeled cells with ultrasmall superparamagnetic iron oxide nanoparticles (USPIOs). (c) Labeled cells with murine immunoglobulin G USPIOs. (d) Labeled cells with OCMab183B2 USPIOs. Cells cultured with OCMab183B2 USPIOs showed much lower T2 signal intensity than other cells.

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In Vivo MR Imaging

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Figure 6, T2-weighted imaging of the tumor at different times after the injection of OCMab183B2 ultrasmall superparamagnetic iron oxide nanoparticle (USPIO) conjugates. The signal intensity of the tumor slowly decreased from 5 minutes to 4 hours and did not change remarkably from 4 to 12 hours after injection. The signal intensity reached the minimum point 24 hours after injection and then began to increase. Forty-eight hours after injection, there was no remarkable difference compared to the initial state.

Figure 7, The changes of T2 values of the tumors after the injection of OCMab183B2 ultrasmall superparamagnetic iron oxide nanoparticles (USPIOs) and murine immunoglobulin G (mIgG) USPIOs. A different trend existed in both groups. For OCMab183B2 USPIOs, the T2 value slowly decreased and reached the minimum 48 hours after injection. For mIgG USPIOs, the value first decreased from 5 minutes to 4 hours and then began to increase until 48 hours after injection. The T2 values of tumors at 24 hours after injection in the experimental and control groups indicated a significant difference.

Figure 8, T2-weighted imaging of the tumor at different times after the injection of murine immunoglobulin G (mIgG) ultrasmall superparamagnetic iron oxide nanoparticles (USPIOs). The signal intensity of the tumor slowly decreased from 5 minutes to 2 hours and then began to revert to its initial state after 48 hours of injection.

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Histologic Analysis

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Figure 9, Photomicrography of Prussian blue staining of tumors 48 hours after injection. Blue particles deposited in tumors after the injection of OCMab183B2 ultrasmall superparamagnetic iron oxide nanoparticles (USPIOs) (a) , while no blue particles in tumors after the injection of murine immunoglobulin G USPIOs (b) (magnification, 400×).

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Discussion

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Conclusions

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Acknowledgments

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Academic Radiology, Volume 17, Issue 12
Journals General Radiology
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