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Hepatocellular Carcinoma Cells Carrying a Multimodality Reporter Gene for Fluorescence, Bioluminescence, and Magnetic Resonance Imaging In Vitro and In Vivo

Posted Oct 31, 2016 Updated Apr 10, 2023
By Xiaoxiao Qin M.D. , Xiaojun Hu M.D. , Chun Wu M.D. Ph.D. , Mingyue Cai M.D. , Zhengran Li M.D. Ph.D. , Lina Zhang M.D. , Liteng Lin M.D. , Wensou Huang M.D. , Kangshun Zhu M.D. Ph.D.
10 min read

Rationale and Objectives

The study aimed to evaluate the feasibility of imaging or tracking hepatocellular carcinoma cells by modifying these cells to carry a multimodality reporter gene, enabling fluorescence, bioluminescence, and magnetic resonance imaging (MRI) in vitro and in vivo.

Materials and Methods

HepG2 cells were labeled with the enhanced green fluorescent protein (EGFP)/luciferase2/ferritin—the multimodality reporter gene (labeled HepG2 cells). The labeled and unlabeled HepG2 cells were cultured in vitro and then injected subcutaneously into mice as a hepatoma model in vivo. The expressions of EGFP, luciferase2, and ferritin in HepG2 cell suspensions and hepatoma model were investigated using fluorescence, bioluminescence, and MRI.

Results

Individual HepG2 cells expressing EGFP were identified under blue laser excitation. The linear coefficient between the optical signal intensity of luciferase2 and the number of labeled cells was 0.993. MRI was used to distinguish the T2* signal of 2 × 10 7 cells/mL between the labeled (6.67 ± 1.88 ms) and unlabeled cells (10.66 ± 2.22 ms) ( P = 0.034). In vivo, individual HepG2 cells expressing EGFP in frozen sections were observed. Labeled cells expressing luciferase2 have been detected since the second day after injection, and the bioluminescence increased with the tumor size. The T2* signal was significantly different between the labeled (6.04 ± 1.60 ms) and unlabeled cells (17.06 ± 2.17 ms) ( P < 0.001).

Conclusions

A multimodality reporter gene consisting of EGFP, luciferase2, and ferritin was successfully integrated into the HepG2 cell genome via a lentiviral vector and was highly expressed in the daughter cells. These cells could be detected by fluorescence, bioluminescence, and MRI in vitro and in vivo.

Introduction

Hepatocellular carcinoma (HCC) is one of the most common forms of cancer and is the fifth most prevalent cause of tumor-related death globally . The absolute number of new HCC cases continues to increase, and HCC is projected to remain as the largest cancer burden for the next several decades. Although advancements in gene diagnosis and treatment modalities have helped improve the survival and prognosis of HCC patients, HCC diagnosis predominantly depends on imaging studies such as computed tomography and magnetic resonance imaging (MRI), limiting the early diagnosis of the disease . Several biomarkers have been proposed , but no available theory can explain how these malignant cells are capable of evading immune clearance or when and where they prefer to reside and metastasize. This limited knowledge has hindered our understanding of the pathologic changes caused by HCC and our identification of potential therapeutic targets. Thus, tracing or imaging HCC cells in vitro and in vivo at the molecular level is a challenge for the treatment of HCC.

Gene imaging , which is a distinct imaging from exogenous tracers, can be visualized stably in cells, enabling the precise localization of labeled cells. Both optical imaging and MRI facilitate the noninvasive tracking of transplanted cells in living organisms. Due to its advantages in terms of sensitivity and specificity, bioluminescent reporters are among the most widely used, as they can provide qualitative and quantitative information about the cells . However, due to photo attenuation and light scattering within deep tissues, bioluminescence imaging (BLI) lacks tomographic resolution and limits its application to small animals or superficial areas . Ferritin has been used as a T2*-weighted MRI probe to dynamically track cells in vivo because of the low-intensity signals generated from T2*-weighted MRI . In contrast to optical imaging, MRI displays the advantages of unlimited depth penetration, high spatial resolution, and multiple imaging parameters; however, MRI cannot distinguish whether or not the transplanted cells are surviving . Apparently, the combination of these two imaging tools may overcome their disadvantages while benefiting from each of their advantages to provide improved cell tracking in vivo . Moreover, because the expression of enhanced green fluorescent protein (EGFP) can be detected in single cells, the sensitivity of EGFP enables its function as a sorting and qualitative marker for cell tracing. Therefore, the expression of reporter genes that can be used for several imaging modalities in targeted cells would have complementary advantages in sensitivity and specificity and would provide a complete and precise description of the biological processes in these cells.

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Materials and Methods

Instruments and Reagents

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Plasmid Construction

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Culture and Transfection of HepG2 Cells

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Transwell Migration/Invasion Assay

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Fluorescence Analysis of EGFP for In Vitro Targeting of Labeled Cells

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Bioluminescence Analysis of Luciferase2 In Vitro

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Prussian Blue Iron Staining to Detect the Fe3+ Content in Transfected Cells

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MRI Analysis of Ferritin In Vitro

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Nude Mouse Hepatoma Model

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Fluorescence Analysis of EGFP for In Vivo Targeting of Labeled Cells

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Bioluminescence Analysis of Luciferase2 In Vivo

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MRI Analysis of Ferritin In Vivo

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Statistical Methods

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Results

Plasmid Construction and Verification and Lentivirus Packaging

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Figure 1, (a) Schematic diagram of the recombinant plasmid. (b) At 72 hours after transfecting 293FT cells with the recombinant plasmid, the cells had grown to confluence (left), and green fluorescence emission was detected in some of the cells under blue laser excitation (right), indicating that the recombinant plasmid was successfully transferred to some of the 293FT cells (×40; scale bar: 200 µm). (c) At 72 hours after lentiviral transfection of HepG2 cells. At a multiplicity of infection of 50, the HepG2 cells were in a good state, as no mass scarring of necrotic cells occurred (left). Green fluorescence emission was observed under blue laser excitation on the same field of view (right) (×100; scale bar: 100 µm). (d,e) The number of labeled HepG2 cells (left) that succeeded in migrating across the micro-pores of the base membrane was 23.60 ± 1.21 per well, similar to that of the unlabeled cells (right) (24.20 ± 1.16 per well) (×200; scale bar: 100 µm); this difference was not significant ( P > 0.05). The cells in both groups were epithelial-like, extending few processes.

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Culture and Transfection of HepG2 Cells

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Cancer Cell Migration

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Fluorescence Analysis of EGFP Imaging In Vitro

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Figure 2, (a) Green fluorescence emission was observed in individual labeled HepG2 cells under blue laser excitation, and enhanced green fluorescent protein (EGFP) was predominantly expressed in the cytoplasm (left); nuclei counterstained with DAPI are shown in blue (middle), and the fused image of EGFP fluorescence and nuclear staining (right) (×630; scale bar: 50 µm). (b) With the reduction in the cell number, the photon count decreased, revealing a linear correlation between the number of cells and the bioluminescence signal (r = 0.993). (c) Prussian blue staining showed extensive blue staining in the labeled HepG2 cells, whereas the blue granules in the unlabeled HepG2 cells were fewer and were more scattered (×200; scale bar: 100 µm). (d) Comparison of the normalized T2* signal intensity between the labeled (left) and unlabeled HepG2 cells (middle). The average T2* value of the four fields of labeled HepG2 cells was 6.67 ± 1.88 ms, which was significantly lower than that of the unlabeled cells (10.66 ± 2.22 ms) ( P = 0.034) (right).

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Bioluminescence Analysis of Luciferase2 In Vitro

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Prussian Blue Iron Staining for Iron Assessment In Vitro

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MRI for Ferritin In Vitro

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Fluorescence Analysis of EGFP Imaging In Vivo

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Figure 3, (a) A frozen tumor section 14 days after labeled HepG2 cell transplant. Green fluorescence emission was detected in the cytoplasm of individual labeled HepG2 cells under blue laser excitation (left), nuclei counterstained with DAPI (middle), and the fused image of enhanced green fluorescent protein fluorescence and nuclear staining (right) (×400; scale bar: 50 µm). (b) The first, second, third, seventh, and fourteenth day after transplant, the bioluminescence signal was detected using an acquisition time of 5 seconds. Until the 14th day, the signal increased as the tumor size increased. (c) At 14 days after injection, the T2* value of the tumors originating from the labeled cells was 6.04 ± 1.60 ms, which was significantly lower than that from the unlabeled cells (17.06 ± 2.17 ms) ( P < 0.001). This result indicated that the labeled HepG2 cells (ROI 1) transported and integrated more Fe 3+ than the unlabeled HepG2 cells (ROI 2). ROI, region of interest.

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Bioluminescence Analysis of Luciferase2 in Vivo

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MRI for Ferritin In Vivo

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Discussion

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Acknowledgments

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Academic Radiology, Volume 23, Issue 11
Journals General Radiology
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