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Teratoma formation can be a serious drawback after the therapeutic transplantation of human embryonic stem (hES) cells. Therefore, noninvasive imaging of teratomas could be a valuable tool for monitoring patients undergoing hES cell treatment. Here, we investigated the angiogenic process within teratomas derived from hES cells and now report the first example of using 64Cu-labeled RGD tetramer (64Cu-DOTA-RGD4) for positron emission tomography imaging of teratoma formation by targeting αvβ3 integrin. H9 hES cells (2 × 106), stably expressing firefly luciferase, and enhanced green fluorescence protein (Fluc-eGFP) were injected into adult nude mice (n = 12) s.c. Eight weeks after transplantation, these hES cell grafts evolved into teratomas as confirmed by longitudinal bioluminescence imaging. Under micropositron emission tomography imaging, 2-deoxy-2-[18F]fluoro-D-glucose and 3′-deoxy-3′-[18F]-fluorothymidine both failed to detect hES cell–derived teratomas (0.8 ± 0.5 versus 1.1 ± 0.4 %ID/g, respectively; P = not significant versus background signals). By contrast, 64Cu-DOTA-RGD4 revealed specific and prominent uptake in vascularized teratoma and significantly lower uptake in control tumors (human ovarian carcinoma 2008 cell line), which had low intergrin expression (10.1 ± 3.4 versus 1.4 ± 1.2 %ID/g; P < 0.01). Immunofluorescence staining of CD31 and β3 integrin also supported our in vivo imaging results (P < 0.05). Moreover, we found that the cells dissociated from teratomas showed higher αvβ3 integrin expression than the 2008 cells. In conclusion, by targeting αvβ3 integrin, we successfully showed the ability of 64Cu-DOTA-RGD4 to noninvasively visualize teratoma formation in vivo for the first time.
Human embryonic stem (hES) cells are characterized by their ability for indefinite self-renewal and their potential for pluripotent differentiation. These properties make hES cells an unlimited and ideal cellular source for biomedical research and transplantation (1). However, hES cells as well as preimplantation or early postimplantation embryos often develop into tumors (teratomas) when transplanted into an ectopic location of recipient animals. These teratomas are composed of differentiated somatic tissues from all three germ layers and are a serious drawback after transplantation of hES cells for regenerative purposes (2). Given the importance of ensuring the safety of hES cell transplantation in patients, the ability to noninvasively diagnose cellular misbehavior is a top priority of stem cell research.
Angiogenesis is an invasive process characterized by endothelial cell proliferation, modulation of the extracellular matrix, and cell adhesion/migration. The up-regulation of αvβ3 integrin is known to play a key role in tumor angiogenesis and metastasis (3). Radiolabeled cyclic arginine-glycine-aspartic acid (RGD) peptides can be used for noninvasive imaging of αvβ3 expression and targeted radionuclide therapy (4). Here, we hypothesize that molecular imaging of αvβ3 integrin expression during teratoma angiogenesis might allow the visualization and quantification of angiogenesis levels in teratoma growth. We investigated the angiogenic process within teratomas derived from hES cells and evaluated the feasibility of using 64Cu-labeled RGD tetramer (64Cu-DOTA-RGD4; ref. 4) for positron emission tomography (PET) imaging of teratoma formation by targeting αvβ3 integrin.
A description of procurement of materials used in this study is described in the Supplementary Materials and Methods section.
Growth conditions for the federally approved H9 hES cell line (Wicell) and the human ovarian carcinoma 2008 cell line are described in the Supplementary Methods section.
Subcutaneous xenograft H9 and ovarian cell tumors were formed by injection of 2 × 106 cells into the subcutaneous regions of athymic mice (n = 12). Detailed procedures are described in the Supplementary Materials and Methods section.
Transduction of hES cells with LV-pUB-Fluc-eGFP is described in the Supplementary Materials and Methods section.
Quantitative data were expressed as mean ± SD. Means were compared using one-way ANOVA and the Student’s t test. P values of <0.05 were considered statistically significant.
To assess the effects of Fluc-eGFP double fusion (DF) reporter gene expression on hES cell pluripotency, the modified hES (hES-DF) and control hES cells were subjected to immunohistochemical staining to assess known early stem cell markers (Oct-4, AKP) as shown in Fig. 1A. Cells expressing eGFP were sorted by fluorescence-activated cell sorting and the resultant population exhibited a robust correlation between Fluc expression and cell number (r2 = 0.99; Fig. 1B). The in vitro differentiation assays showed that hES-DF cells expressed endodermic (Albumin), mesodermic (α-MHC), and ectodermic (Ncam1) germ layer markers in a similar manner as control hES cells (Fig. 1C). Furthermore, we observed no significant differences in cell proliferation induced by the DF reporter gene (Fig. 1D) at 24-, 48-, and 72-h time points.
H&E staining revealed a large number of circular vessels of various sizes in teratomas (Fig. 2). To test whether hES cells contributed to the formation of blood vessels, intrateratoma endothelial cells were stained for anti-mouse endothelial marker CD31 (platelet/endothelial cell adhesion molecule; PECAM). We found that most of these cells expressed anti-mouse PECAM, indicating their host-derived origin. This also clearly shows that endothelial cells may contribute to angiogenesis in developing teratomas.
Static microPET scans were performed on the hES cell–derived teratoma model and a control human ovarian carcinoma model (2008 cell line). We performed 18F-FDG and 18F-FLT microPET 8 weeks after the hES cell inoculation (Fig. 3A). Teratomas showed only 0.8 ± 0.5 %ID/g uptake of 18F-FDG and 1.1 ± 0.4 %ID/g uptake of 18F-FLT, compared with 22.1 ± 3.2 %ID/g uptake of 18F-FDG and 25.3 ± 5.1 %ID/g uptake of 18F-FLT in the control tumor. By contrast, vascularized teratoma showed specific and prominent uptake of 64Cu-DOTA-RGD4 (10.1 ± 3.4 %ID/g) compared with control human ovarian carcinoma tumor (1.4 ± 1.2 %ID/g; P < 0.01) at week 8 (Fig. 3B). To confirm 64Cu-DOTA-RGD4 uptake in the teratomas was mediated by binding of the RGD4 peptide to αvβ3 integrin, we conducted a blocking experiment in which a saturating dose of nonradiolabeled RGD multimer (15 mg/kg) was coinjected with 64Cu-DOTA-RGD4 into a second set of animals (n = 5; Supplementary Fig. S1). Uptake of the 64Cu-DOTA-RGD4 tracer was severely diminished in animals that received a blocking dose of the RGD multimer compared with positive controls.
Proliferating endothelial cells express large amounts of αvβ3 and αvβ5 integrins. To determine whether the hES-derived teratomas express β3 integrin, the tissues were double-immunostained with PECAM and β3 integrin (Fig. 3C). A high expression of β3 integrin was observed and colocalized with CD31 staining.
The sigmoid curves for the whole-cell binding assay using 125I-echistatin as a radioligand and unlabeled echistatin as a competitor were obtained by nonlinear regression fitting of the data using GraphPad Prism. The Scatchard transformation and maximum number of binding sites (Fig. 4A–C) were generated from the linear portion of the sigmoid curve (Fig. 4D). The density order for integrin αvβ3 receptors on the cell surface was found to be in the order of differentiated teratoma cells undifferentiated hES cells > control cells (2.31 × 104 receptors per teratoma cell, 4.54 × 103 receptors per hES cell, and 2.13 × 103 receptors per 2008 cell, respectively).
hES cell therapy holds tremendous promise for treating many of the most intractable diseases. However, the risk of teratoma formation is a major obstacle that must be successfully resolved before hES cell–based therapies can be safely conducted in the future. Innovative methods to noninvasively monitor teratoma formation de novo are therefore essential steps to the continuing progress of stem cell therapy. Multimodality approaches have been applied to image teratoma formation in vivo, including magnetic resonance imaging and reporter gene–based optical imaging (6, 7). However, sensitivity (magnetic resonance imaging) or tissue penetration (optical imaging) issues may limit their applications. Due to the high sensitivity and reasonably good spatial/temporal resolution, PET probes are thus highly attractive for real-time and noninvasive monitoring of the location and formation of teratoma. In the present study, we showed that the angiogenic process within hES cell–derived teratomas can be imaged by PET using the 64Cu-labeled RGD tetramer (64Cu-DOTA-RGD4).
FDG-PET has shown utility in distinguishing malignant tumors from benign tumors from various histologic diagnoses, in staging malignant tumors, and in evaluating treatment efficacy in cancer patients (8). The increased glucose metabolism found in malignant tumors compared with benign tissues is a property that can be exploited to distinguish the tissues. Recently, Sugawara and colleagues (9) compared FDG uptake in viable tumors and teratomas. They found that viable tumors showed high FDG uptake but mature teratomas exhibit lower uptake in human patients (9). Our data show a similar pattern in that mature teratomas have relatively low FDG signals. Therefore, FDG may not be suitable for teratoma imaging.
The thymidine analogue FLT has been used for imaging tumor proliferation (10). Thymidine kinase 1 was revealed as the key enzyme responsible for intracellular trapping of 18F-FLT (10–12). Buck and colleagues (13) determined the ability of 18F-FLT to detect manifestation sites of bone and soft tissue tumors, making possible the distinction of malignant versus benign tumors. They found that the 18F-FLT uptake correlated significantly with tumor grading. The signal was 10-fold lower in benign lesions compared with malignant tumor. However, our 18F-FLT results showed low uptake within teratoma, which may limit its use for clinical detection.
Angiogenesis, also called neovascularization, is a fundamental process whereby new blood vessels are formed. Under normal physiologic conditions, angiogenesis is highly regulated and essential for reproduction, embryonic development, and wound healing (14). ES cells are normal embryonal cells that differentiate into many somatic tissues and can give rise to teratomas (2). Bloch and colleagues (15) have shown that the growth of ES cell–derived teratomas is dependent on the expression of β1 integrin. Members of the integrin family are noncovalently associated α/β heterodimers that mediate cell-cell, cell-extracellular matrix, and cell-pathogen interactions. The ligands for the extracellular domain of many integrins are the proteins of the extracellular matrix, which contain a consensus motif with the amino acid sequence RGD (arginine-glycine-aspartate). To test the role of αvβ3 integrin in this process, we analyzed the integrin binding ex vivo and αvβ3 integrin expression in vivo. Immunofluorescence staining of CD31 and β3 integrin showed significantly higher expression compared with the control 2008 cell line. These results revealed that human teratomas expressed high levels of β3 integrin, suggesting a potentially important role of β3 integrin in angiogenesis during teratoma formation. The in vivo bioluminescence and microPET imaging results also confirmed the ex vivo results. Taken together, these findings might prove useful for the treatment of cell misbehavior and neoplasm of ES cells, as neoplasm or formation of a tumor is characterized by angiogenesis.
In summary, this study shows the ability of 64Cu-DOTA-RGD4 to visualize integrin expression during teratoma formation in vivo. Compared with most commonly used PET tracers (18F-FDG and 18F-FLT), 64Cu-DOTA-RGD4 may have superior clinical applicability for monitoring tumorigenicity after hES cell transplantation in the future.
Grant support: R01CA119053 (X. Chen), R21HL091453 (J.C. Wu), R21HL089027 (J.C. Wu), CABCRP141B-0039 (J.C. Wu), and CIRM RS1-00322 (J.C. Wu).
We thank the University of Wisconsin-Madison for 64Cu production and the Stanford cyclotron team for 18F-FDG and 18F-FLT production.
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.