Michael Doubrovin

Imaging transcriptional regulation of p53-dependent genes with positron emission tomography in vivo

A noninvasive method for molecular imaging of the activity of different signal transduction pathways and the expression of different genes in vivo would be of considerable value. It would aid in understanding the role specific genes and signal transduction pathways have in various diseases, and could elucidate temporal dynamics and regulation at different stages of disease and during various therapeutic interventions. We developed and assessed a method for monitoring the transcriptional activation of endogenous genes by positron-emission tomography (PET) imaging. The HSV1-tk/GFP (TKGFP) dual reporter gene was used to monitor transcriptional activation of p53-dependent genes. A retrovirus bearing the Cis-p53/TKGFP reporter system was constructed in which the TKGFP reporter gene was placed under control of an artificial cis-acting p53-specific enhancer. U87 glioma and SaOS-2 osteosarcoma cells were transduced with this retrovirus and used to establish xenografts in rats. We demonstrated that DNA damage-induced up-regulation of p53 transcriptional activity correlated with the expression of p53-dependent downstream genes, such as p21, in U87 (wild-type p53), but not in SaOS-2 osteosarcoma (p53 −/−) cells. We showed that PET, with [124I]FIAU (2′-fluoro-2′-deoxy-1-β-d-arabinofuranosyl-5-[124I]iodouracil) and the Cis-p53TKGFP reporter system, is sufficiently sensitive to image the transcriptional regulation of genes in the p53 signal transduction pathway. These imaging results were confirmed by independent measurements of p53 activity and the expression levels of downstream genes (e.g., p21) by using conventional molecular-biological assays. PET imaging of p53 transcriptional activity in tumor xenografts by using the Cis-p53TKGFP reporter system may be useful in assessing novel therapeutic approaches.

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.

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.

Positron emission tomography (PET) imaging of tumor-localized Salmonella expressing HSV1-TK

In order to noninvasively detect Salmonella delivery vectors within tumors, we used a genetically modified Salmonella, VNP20009, that expresses the herpes simplex thymidine kinase (HSV1-tk) reporter gene. VNP20009-TK were able to selectively localize within murine tumor models and to effectively sequester a radiolabeled nucleoside analogue, 2′-fluoro-1-β-D-arabino-furanosyl-5-iodo-uracil (FIAU). A quantitative relationship between the level of radioactivity accumulated and the number of bacteria in tumor and different tissues was demonstrated. The in vivo accumulation of [14C]FIAU measured in tissue sample homogenates and sections were related to Salmonella number and to immunohistochemical bacterial staining, respectively. Quantitative autoradiography (QAR) revealed the relative intensity of [14C]FIAU accumulation in a tumor cross-section, demonstrating that the peripheral region of the tumor was significantly less active than internal regions. [124I]FIAU positron emission tomography (PET) and subsequent tissue radioactivity and bacterial concentration measurements were compared. A log–log relationship was found, and the PET images could identify multiple tumor sites. The ability to noninvasively detect Salmonella vectors by PET imaging has the potential to be conducted in a clinical setting, and could aid in development of these vectors by demonstrating the efficiency and duration of targeting as well as indicating the locations of tumors.

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 transgene activity in vivo.

The successful translation of gene therapy for clinical application will require the assessment of transgene activity as a measure of the biological function of a therapeutic transgene. Although current imaging permits the noninvasive detection of transgene expression, the critical need for quantitative imaging of the action of the expressed transgene has not been met. In vivo magnetic resonance spectroscopic imaging (MRSI) was applied to quantitatively delineate both the concentration and activity of a cytosine deaminase-uracil phosphoribosyltransferase (CD-UPRT) fusion enzyme expressed from a transgene. MRSI enabled the generation of anatomically accurate maps of the intratumoral heterogeneity in fusion enzyme activity. We observed an excellent association between the CD-UPRT concentration and activity and the percentage of CD-UPRT(+) cells. Moreover, the regional levels of UPRT activity, as measured by imaging, correlated well with the biological affect of the enzyme. This study presents a translational imaging paradigm for precise, in vivo measurements of transgene activity with potential applications in both preclinical and clinical settings.

In vivo 5‐fluorouracil and fluoronucleotide T1 relaxation time measurements using the variable nutation angle method

19Fluorine NMRS has the potential to enable noninvasive predictions of tumor response to 5‐fluorouracil (5FU) therapy based on tumor pharmacokinetics. Knowledge of the T1s of 5FU and its fluoronucleotide anabolites (FNuc) is required for quantitative spectral analysis and selection of optimal pulse parameters. We used the variable nutation angle (VNA) method to determine T1s of 5FU and FNuc in subcutaneous Walker 256 rat mammary carcinosarcoma tumors transfected with a cytosine deaminase/uracil phosphoribosyltransferase fusion gene. We calibrated in vivo NAs using methoxydifluoroacetate to ensure the accuracy of these measurements. The T1s were calculated based on signal intensities acquired with NAs of 20°, 35°, 45°, 60°, and 75°. The acquisition order of these NAs was shuffled to reduce the effect of signal variations. The determined T1s for 5FU and FNuc (2.3 ± 0.1 s and 1.3 ± 0.1 s, respectively) represent the first reported in vivo measurements for these metabolites in tumor. Magn Reson Med 52:169–173, 2004.

Imaging Genes for Viral and Adoptive Therapies

The concept of using gene therapy for cancer treatment was initially met with enthusiasm. The possibility of replacing or altering damaged genes, the introduction of suicide genes into cancer cells, and the alteration of cell function as a consequence of exogenous gene expression were advocated. Therapeutic genes can be transferred to patients through a variety of vehicles. These include retroviruses, herpes viruses, adenoviruses, adeno-associated viruses, lentiviruses, baculoviruses, liposomes, bacterial hosts, naked DNA, DNA precipitates, and protein-DNA conjugates (Table 1) (1,2). However, the practical application of gene therapy to treat cancer has been somewhat disappointing so far. Major obstacles remain, including the inability to target appropriate tissues and deliver therapeutic genes to a sufficient number of target cells, the inability to monitor the level of expression of the therapeutic gene, the loss of therapeutic gene expression over time, and the inability to correlate the level and duration of gene expression with therapeutic outcome.

258. Multi-Modality Non-Invasive Imaging of NK-92 Cells Trafficking and Tumor Targeting in Mice

Objective. The studies of NK cell based therapies often have to address the question of the NK cells trafficking, homing, tumor targeting and therapeutic effect. We tested feasibility of applying a multi-modality imaging approach for assessment of therapeutic NK-92 cells trafficking and tumor targeting in mice model.