Inna Serganova

Molecular Imaging of Temporal Dynamics and Spatial Heterogeneity of Hypoxia-Inducible Factor-1 Signal Transduction Activity in Tumors in Living Mice

Tumor hypoxia is a spatially and temporally heterogeneous phenomenon, which results from several tumor and host tissue-specific processes. To study the dynamics and spatial heterogeneity of hypoxia-inducible factor-1 (HIF-1)-specific transcriptional activity in tumors, we used repetitive noninvasive positron emission tomography (PET) imaging of hypoxia-induced HIF-1 transcriptional activity in tumors in living mice. This approach uses a novel retroviral vector bearing a HIF-1–inducible “sensor” reporter gene (HSV1-tk/GFP fusion) and a constitutively expressed “beacon” reporter gene (DsRed2/XPRT). C6 glioma cells transduced with this multireporter system revealed dose-dependent patterns in temporal dynamics of HIF-1 transcriptional activity induced by either CoCl2 or decreased atmospheric oxygen concentration. Multicellular spheroids of C6 reporter cells developed a hypoxic core when >350 μm in diameter. 18F-2′-fluoro-2′deoxy-1β-D-arabionofuranosyl-5-ethyl-uracil (FEAU) PET revealed spatial heterogeneity of HIF-1 transcriptional activity in reporter xenografts in mice as a function of size or ischemia-reperfusion injury. With increasing tumor diameter (>3 mm), a marked increase in HIF-1 transcriptional activity was observed in the core regions of tumors. Even a moderate ischemia-reperfusion injury in small C6 tumors caused a rapid induction of HIF-1 transcriptional activity, which persisted for a long time because of the inability of C6 tumors to rapidly compensate acute changes in tumor microcirculation.

A Human-Derived Reporter Gene for Noninvasive Imaging in Humans: Mitochondrial Thymidine Kinase Type 2

A human-derived intrinsically nonimmunogenic reporter gene was tested for PET imaging of different molecular–genetic processes for potential clinical use. Methods: The human mitochondrial thymidine kinase type 2 (hTK2) reporter gene truncated at the N terminus (ΔhTK2), alone or fused with green fluorescent protein (GFP), was used for preclinical evaluation in a mouse model. The levels of enzymatic activity of ΔhTK2 and ΔhTK2 GFP proteins were assessed using radiotracer accumulation and prodrug activation assays in vitro and in subcutaneous tumors grown from the corresponding cell lines in nude mice. Kinetic analyses of 124I-2′-fluoro-2′-deoxy-1-β-d-β-arabinofuranosyl-5-iodouracil (FIAU), 18F-2′-fluoro-2′-deoxy-1-β-d-β-arabinofuranosyl-5-ethyluracil (FEAU), or 18F-9-(4-18F-fluoro-3-hydroxymethylbutyl)guanine (FHBG) uptake in tumors and biodistribution studies were performed. Results: ΔhTK2 was successfully expressed in the cytoplasm of transduced cells. A new anti-hTK2 monoclonal antibody 8G2 was developed. The levels of FIAU and FEAU accumulation in cells expressing ΔhTK2 and ΔhTK2 GFP were at least 10-fold higher than in wild-type cells in vitro and about 6 times higher in vivo. We determined that FEAU is a more specific reporter substrate for ΔhTK2 than FIAU, whereas FHBG is not phosphorylated by this enzyme. In addition, we showed that ΔhTK2 transduced cells can be eliminated by treatment with d-arabinofuranosyl-cytosine. Conclusion: We have tested a human-derived reporter gene that is likely to be nonimmunogenic and potentially allows for long-term monitoring of different molecular–genetic processes by nuclear imaging techniques in humans. Using 124I-FIAU, 18F-FIAU, or 18F-FEAU, it should be possible to image ΔhTK2 reporter gene expression with PET in preclinical and clinical studies.

Real-Time Imaging of HIF-1α Stabilization and Degradation

HIF-1α is overexpressed in many human cancers compared to normal tissues due to the interaction of a multiplicity of factors and pathways that reflect specific genetic alterations and extracellular stimuli. We developed two HIF-1α chimeric reporter systems, HIF-1α/FLuc and HIF-1α(ΔODDD)/FLuc, to investigate the tightly controlled level of HIF-1α protein in normal (NIH3T3 and HEK293) and glioma (U87) cells. These reporter systems provided an opportunity to investigate the degradation of HIF-1α in different cell lines, both in culture and in xenografts. Using immunofluorescence microscopy, we observed different patterns of subcellular localization of HIF-1α/FLuc fusion protein between normal cells and cancer cells; similar differences were observed for HIF-1α in non-transduced, wild-type cells. A dynamic cytoplasmic-nuclear exchange of the fusion protein and HIF-1α was observed in NIH3T3 and HEK293 cells under different conditions (normoxia, CoCl2 treatment and hypoxia). In contrast, U87 cells showed a more persistent nuclear localization pattern that was less affected by different growing conditions. Employing a kinetic model for protein degradation, we were able to distinguish two components of HIF-1α/FLuc protein degradation and quantify the half-life of HIF-1α fusion proteins. The rapid clearance component (t1/2 ∼4–6 min) was abolished by the hypoxia-mimetic CoCl2, MG132 treatment and deletion of ODD domain, and reflects the oxygen/VHL-dependent degradation pathway. The slow clearance component (t1/2 ∼200 min) is consistent with other unidentified non-oxygen/VHL-dependent degradation pathways. Overall, the continuous bioluminescence readout of HIF-1α/FLuc stabilization in vitro and in vivo will facilitate the development and validation of therapeutics that affect the stability and accumulation of HIF-1α.

Imaging hNET Reporter Gene Expression with 124I-MIBG

The norepinephrine transporter (NET) has recently been suggested as a useful reporter gene. We have extended this effort by constructing an internal ribosomal entry site (IRES)-linked hNET-green fluorescent protein (GFP) hybrid reporter gene for both nuclear and optical imaging. Methods: A retroviral vector pQCXhNET-IRES-GFP was constructed and used to generate several reporter cell lines and xenografts. Transduced cells were sorted by fluorescence-activated cell sorting based on GFP expression and used for both in vitro and in vivo imaging studies. Results: The transduced reporter cells accumulated 123I- or 124I-labeled metaiodobenzylguanidine (MIBG) to high levels compared with the wild-type parent cell lines. Differences in MIBG accumulation between cell lines were primarily due to differences in influx (K1) rather than efflux (k2). The estimated MIBG distribution volumes (Vd) for transduced Jurkat, C6, and COS-7 cells were 572 ± 13, 754 ± 25, and 1,556 ± 38 mL/g, respectively. A correlation between radiotracer accumulation (K1) and GFP fluorescence intensity was also demonstrated. Sequential imaging studies of mice bearing pQCXhNET-IRES-GFP transduced and wild-type C6 xenografts demonstrated several advantages of 124I-MIBG small-animal PET compared with 123I-MIBG γ-camera/SPECT. This was primarily due to the longer half-life of 124I and to the retention and slow clearance (half-time, 63 ± 6 h) of MIBG from transduced xenografts compared with that from wild-type xenografts (half-time, 12 ± 1 h) and other organs (half-time, 2.6–21 h). Very high radioactivity ratios were observed at later imaging times; at 73 h after 124I-MIBG injection, the C6/hNET-IRES-GFP xenograft-to-muscle ratio was 293 ± 48 whereas the C6 xenograft-to-muscle ratio was 0.71 ± 0.19. Conclusion: These studies demonstrate the potential for a wider application of hNET reporter imaging and the future translation to patient studies using radiopharmaceuticals that are currently available for both SPECT and PET.

Metabolic imaging: a link between lactate dehydrogenase A, lactate, and tumor phenotype.

Purpose: We compared the metabolic profiles and the association between LDH-A expression and lactate production in two isogenic murine breast cancer cell lines and tumors (67NR and 4T1). These cell lines were derived from a single mammary tumor and have different growth and metabolic phenotypes. Experimental Design: LDH-A expression, lactate concentration, glucose utilization, and oxygen consumption were measured in cells, and the potential relationship between tumor lactate levels [measured by magnetic resonance spectroscopic imaging (MRSI)] and tumor glucose utilization [measured by [18F]2-deoxy-2-fluoro-d-glucose positron emission tomography ([18F]FDG-PET)] was assessed in orthotopic breast tumors derived from these cell lines. Results: We show a substantial difference in LDH-A expression between 67NR and 4T1 cells under normoxia and hypoxia. We also show that small orthotopic 4T1 tumors generate 10-fold more lactate than corresponding 67NR tumors. The high lactate levels in small primary 4T1 tumors are associated with intense pimonidazole staining (a hypoxia indicator). Less-intense hypoxia staining was observed in the larger 67NR tumors and is consistent with the gradual increase and plateau of lactate concentration in enlarging 67NR tumors. Conclusions: Lactate-MRSI has a greater dynamic range than [18F]FDG-PET and may be a more sensitive measure with which to evaluate the aggressive and metastatic potential of primary breast tumors. Clin Cancer Res; 17(19); 6250–61. ©2011 AACR.

Noninvasive Molecular Imaging Using Reporter Genes

Noninvasive reporter gene imaging is a component of molecular imaging. Reporter imaging can provide noninvasive assessments of endogenous biologic processes in living subjects and can be performed using different imaging modalities. This review will focus on radionuclide-based reporter gene imaging as developed and applied in preclinical and clinical studies. Examples of different reporter systems are presented, with a focus on human reporter systems. Selected applications are discussed, including adoptive cell therapies, gene and oncoviral therapies, oncogenesis, signal pathway monitoring, and imaging drug treatment. Molecular imaging, and noninvasive reporter gene imaging in particular, are making important contributions to our understanding of disease development, progression, and treatment in our current era of molecular medicine and individualized patient care.

A preclinical model for noninvasive imaging of hypoxia-induced gene expression; comparison with an exogenous marker of tumor hypoxia.

PurposeHypoxia is associated with tumor aggressiveness and is an important cause of resistance to radiation therapy and chemotherapy. Assays of tumor hypoxia could provide selection tools for hypoxia-modifying treatments. The purpose of this study was to develop and characterize a rodent tumor model with a reporter gene construct that would be transactivated by the hypoxia-inducible molecular switch, i.e., the upregulation of HIF-1.MethodsThe reporter gene construct is the herpes simplex virus 1-thymidine kinase (HSV1-tk) fused with the enhanced green fluorescent protein (eGFP) under the regulation of an artificial hypoxia-responsive enhancer/promoter. In this model, tumor hypoxia would up-regulate HIF-1, and through the hypoxia-responsive promoter transactivate the HSV1-tkeGFP fusion gene. The expression of this reporter gene can be assessed with the 124I-labeled reporter substrate 2′-fluoro-2′-deoxy-1-β-d-arabinofuranosyl-5-iodouracil (124I-FIAU), which is phosphorylated by the HSV1-tk enzyme and trapped in the hypoxic cells. Animal positron emission tomography (microPET) and phosphor plate imaging (PPI) were used in this study to visualize the trapped 124I-FIAU, providing a distribution of the hypoxia-induced molecular events. The distribution of 124I-FIAU was also compared with that of an exogenous hypoxic cell marker, 18F-fluoromisonidazole (FMISO).ResultsOur results showed that 124I-FIAU microPET imaging of the hypoxia-induced reporter gene expression is feasible, and that the intratumoral distributions of 124I-FIAU and 18F-FMISO are similar. In tumor sections, detailed radioactivity distributions were obtained with PPI which also showed similarity between 124I-FIAU and 18F-FMISO.ConclusionThis reporter system is sufficiently sensitive to detect hypoxia-induced transcriptional activation by noninvasive imaging and might provide a valuable tool in studying tumor hypoxia and in validating existing and future exogenous markers for tumor hypoxia.

Multimodality imaging of TGFβ signaling in breast cancer metastases

The skeleton is a preferred site for breast cancer metastasis. We have developed a multimodality imaging approach to monitor the transforming growth factor β (TGFβ) signaling pathway in bone metastases, sequentially over time in the same animal. As model systems, two MDA‐MB‐231 breast cancer cells lines with different metastatic tropisms, SCP2 and SCP3, were transduced with constitutive and TGFβ‐ inducible reporter genes and were tested in vitro and in living animals. The sites and expansion of metastases were visualized by bioluminescence imaging using a constitutive firefly luciferase reporter, while TGFP signaling in metastases was monitored by microPET imaging of HSV1‐TK/GFP expression with [18F]FEAU and by a more sensitive and cost‐effective bioluminescence reporter, based on nonsecreted Gaussia luciferase. Concurrent and sequential imaging of metastases in the same animals provided insight into the location and progression of metastases, and the timing and course of TGFP signaling. The anticipated and newly observed differences in the imaging of tumors from two related cell lines have demonstrated that TGFβ signal transduction pathway activity can be noninvasively imaged with high sensitivity and reproducibility, thereby providing the opportunity for an assessment of novel treatments that target TGFβ signaling.— Serganova, I.,Moroz, E., Vider, J., Gogiberidze, G., Moroz, M., Pillarsetty, N., Doubrovin, M., Minn, A., Thaler, H. T., Massague, J., Gelovani, J., Blasberg, R. Multimodality imaging of TGFβ signaling in breast cancer metastases. FASEB J. 23, 2662–2672 (2009)

Imaging a Genetically Engineered Oncolytic Vaccinia Virus (GLV-1h99) Using a Human Norepinephrine Transporter Reporter Gene

Purpose: Oncolytic viral therapy continues to be investigated for the treatment of cancer, and future studies in patients would benefit greatly from a noninvasive modality for assessing virus dissemination, targeting, and persistence. The purpose of this study was to determine if a genetically modified vaccinia virus, GLV-1h99, containing a human norepinephrine transporter (hNET) reporter gene, could be sequentially monitored by [123I]metaiodobenzylguanidine (MIBG) γ-camera and [124I]MIBG positron emission tomography (PET) imaging. Experimental Design: GLV-1h99 was tested in human malignant mesothelioma and pancreatic cancer cell lines for cytotoxicity, expression of the hNET protein using immunoblot analysis, and [123I]MIBG uptake in cell culture assays. In vivo [123I]MIBG γ-camera and serial [124I]MIBG PET imaging was done in MSTO-211H orthotopic pleural mesothelioma tumors. Results: GLV-1h99 successfully infected and provided dose-dependent levels of transgene hNET expression in human malignant mesothelioma and pancreatic cancer cells. The time course of [123I]MIBG accumulation showed a peak of radiotracer uptake at 48 hours after virus infection in vitro. In vivo hNET expression in MSTO-211H pleural tumors could be imaged by [123I]MIBG scintigraphy and [124I]MIBG PET 48 and 72 hours after GLV-1h99 virus administration. Histologic analysis confirmed the presence of GLV-1h99 in tumors. Conclusion: GLV-1h99 shows high mesothelioma tumor cell infectivity and cytotoxic efficacy. The feasibility of imaging virus-targeted tumor using the hNET reporter system with [123I]MIBG γ-camera and [124I]MIBG PET was shown in an orthotopic pleural mesothelioma tumor model. The inclusion of human reporter genes into recombinant oncolytic viruses enhances the potential for translation to clinical monitoring of oncolytic viral therapy.

hNIS-IRES-eGFP Dual Reporter Gene Imaging

The human and rodent sodium iodide symporters (NIS) have recently been cloned and are being investigated as potential therapeutic and reporter genes. We have extended this effort by constructing an internal ribosomal entry site (IRES)-linked human NIS (hNIS)-enhanced green fluorescent protein (eGFP) hybrid reporter gene for both nuclear and optical imaging. A self-inactivating retroviral vector, termed pQCNIG, containing hNIS-IRES-eGFP dual reporter gene, driven by a constitutive CMV promoter, was constructed and used to generate RG2-pQCNIG cells and RG2-pQCNIG tumors. 131I-iodide and 99mTcO4-pertechnetate accumulation studies plus fluorescence microscopy and intensity assays were performed in vitro, and gamma camera imaging studies in RG2-pQCNIG and RG2 tumor-bearing athymic rats were performed. RG2-pQCNIG cells expressed high levels of hNIS protein and showed high intensity of eGFP fluorescence compared with RG2 wild-type cells. RG2-pQCNIG cells accumulated Na131I and 99mTcO4– to a 50:1 and a 170:1 tissue/medium ratio at 10 min, compared with 0.8:1.2 tissue/medium ratio in wild-type RG2 cells. A significant correlation between radiotracer accumulation and eGFP fluorescence intensity was demonstrated. RG2-pQCNIG and RG2 tumors were readily differentiated by in vivo gamma camera imaging; radiotracer uptake increased in RG2-pQCNIG but declined in RG2 tumors over the 50-min imaging period. Stomach and thyroid were the major organs of radionuclide accumulation. The IRES-linked hNIS-eGFP dual reporter gene is functional and stable in transduced RG2-pQCNIG cells. Optical and nuclear imaging of tumors produced from these cell lines provides the opportunity to monitor tumor growth and response to therapy. These studies indicate the potential for a wider application of hNIS reporter imaging and translation into patient studies using radioisotopes that are currently available for human use for both SPECT and PET imaging.