Maxim Moroz

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.

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 Expression of the Human Somatostatin Receptor Subtype-2 Reporter Gene with 68Ga-DOTATOC

The human somatostatin receptor subtype 2 (hSSTr2)–68Ga-DOTATOC reporter system has several attractive features for potential translation to human studies. These include a low expression of hSSTr2 in most organs, a rapid internalized accumulation of 68Ga-DOTATOC in the SSTr2-expressing cells, and a rapid excretion of unbound radioligand by the renal system. We performed a series of in vitro and in vivo validation studies of this reporter system. Methods: A retroviral vector containing a dual reporter, pQCXhSSTr2-IRES-GFP (IRES: internal ribosome entry site; GFP: green fluorescent protein), was constructed and transduced into Jurkat, C6, and U87 cells. Stably transduced reporter cells were characterized in vitro using optical and radiometric methods. Multiple tumor-bearing mice were evaluated with 68Ga-DOTATOC PET studies. Results: The dual-reporter genes were incorporated into all tumor cell lines, and their expression levels were confirmed by fluorescence-activated cell sorting (FACS), GFP visualization, and reverse-transcriptase polymerase chain reaction (RT-PCR) analysis for hSSTr2. In vitro, hSSTr2 cell membrane expression was 36,000, 280,000, and 1,250,000 copies per cell for the SSTR2-transfected Jurkat, U87, and C6 cell lines. Small-animal PET of 68Ga-DOTATOC in tumor-bearing mice demonstrated that the in vivo uptake of this radioligand was directly proportional to the in vitro expression of hSSTr2. The in vivo uptake of 68Ga-DOTATOC, at 2 h after injection, was low in all organs except the kidneys (7.8 percentage of injected dose per gram [%ID/g]) and as high as 15.2 %ID/g in transduced C6 tumors. The corresponding transduced–to–nontransduced tumor uptake ratio was 64, and the tumor-to-muscle uptake ratio was around 500. Conclusion: 68Ga-DOTATOC is an excellent specific ligand for this hSSTr2 reporter system and for hSSTr2 reporter gene PET. Because DOTATOC has undergone extensive clinical testing, this human reporter system has the potential for translation to human studies.

Imaging Colon Cancer Response Following Treatment with AZD1152: A Preclinical Analysis of [18F]Fluoro-2-deoxyglucose and 3′-deoxy-3′-[18F]Fluorothymidine Imaging

Purpose: To determine whether treatment response to the Aurora B kinase inhibitor, AZD1152, could be monitored early in the course of therapy by noninvasive [18F]-labeled fluoro-2-deoxyglucose, [18F]FDG, and/or 3′-deoxy-3′-[18F]fluorothymidine, [18F]FLT, PET imaging. Experimental design: AZD1152-treated and control HCT116 and SW620 xenograft-bearing animals were monitored for tumor size and by [18F]FDG, and [18F]FLT PET imaging. Additional studies assessed the endogenous and exogenous contributions of thymidine synthesis in the two cell lines. Results: Both xenografts showed a significant volume-reduction to AZD1152. In contrast, [18F]FDG uptake did not demonstrate a treatment response. [18F]FLT uptake decreased to less than 20% of control values in AZD1152-treated HCT116 xenografts, whereas [18F]FLT uptake was near background levels in both treated and untreated SW620 xenografts. The EC50 for AZD1152-HQPA was approximately 10 nmol/L in both SW620 and HCT116 cells; in contrast, SW620 cells were much more sensitive to methotrexate (MTX) and 5-Fluorouracil (5FU) than HCT116 cells. Immunoblot analysis demonstrated marginally lower expression of thymidine kinase in SW620 compared with HCT116 cells. The aforementioned results suggest that SW620 xenografts have a higher dependency on the de novo pathway of thymidine utilization than HCT116 xenografts. Conclusions: AZD1152 treatment showed antitumor efficacy in both colon cancer xenografts. Although [18F]FDG PET was inadequate in monitoring treatment response, [18F]FLT PET was very effective in monitoring response in HCT116 xenografts, but not in SW620 xenografts. These observations suggest that de novo thymidine synthesis could be a limitation and confounding factor for [18F]FLT PET imaging and quantification of tumor proliferation, and this may apply to some clinical studies as well. Clin Cancer Res; 17(5); 1099–110. ©2011 AACR.

Comparison of Corticotropin-Releasing Factor, Dexamethasone and Temozolomide: Treatment Efficacy and Toxicity in U87 and C6 Intracranial Gliomas

Purpose/Experimental Design: Treatment of cerebral tumors and peritumoral brain edema remains a clinical challenge and is associated with high morbidity and mortality. Dexamethasone is an effective drug for treating brain edema, but it is associated with well-documented side effects. Corticorelin acetate (Xerecept) or human corticotrophin-releasing factor (hCRF) is a comparatively new drug and has been evaluated in two orthotopic glioma models (U87 and C6), by a direct comparison with dexamethasone and temozolomide. Results: In vitro combination therapy and monotherapy showed a variable response in 6 different glioma cell lines. In vivo studies showed a dose-dependent effect of hCRF (0.03 and 0.1 mg/kg q12h) on survival of U87 intracranial xenograft–bearing animals [median survival: control – 41 days (95% CI 25–61); “low-hCRF” 74.5 days (95% CI 41–88); “high-hCRF” >130 days (95% CI not reached)]. Dexamethasone treatment had no effect on survival, but significant toxicity was observed. A survival benefit was observed with temozolomide and temozolomide + hCRF-treated animals but with significant temozolomide toxicity. C6-bearing animals showed no survival benefit, but there were similar treatment toxicities. The difference in hCRF treatment response between U87 and C6 intracranial gliomas can be explained by a difference in receptor expression. RT-PCR identified CRF2r mRNA in U87 xenografts; no CRF receptors were identified in C6 xenografts. Conclusions: hCRF was more effective than either dexamethasone or temozolomide in the treatment of U87 xenografts, and results included improved prognosis with long-term survivors and only mild toxicity. The therapeutic efficacy of hCRF seems to be dependent on tumor hCRF receptor (CRFr) expression. These results support further clinical assessment of the therapeutic efficacy of hCRF and levels of CRFr expression in different human gliomas. Clin Cancer Res; 17(10); 3282–92. ©2011 AACR.

Adoptively transferred TRAIL + T cells suppress GVHD and augment antitumor activity

Current strategies to suppress graft-versus-host disease (GVHD) also compromise graft-versus-tumor (GVT) responses. Furthermore, most experimental strategies to separate GVHD and GVT responses merely spare GVT function without actually enhancing it. We have previously shown that endogenously expressed TNF-related apoptosis-inducing ligand (TRAIL) is required for optimal GVT activity against certain malignancies in recipients of allogeneic hematopoietic stem cell transplantation (allo-HSCT). In order to model a donor-derived cellular therapy, we genetically engineered T cells to overexpress TRAIL and adoptively transferred donor-type unsorted TRAIL+ T cells into mouse models of allo-HSCT. We found that murine TRAIL+ T cells induced apoptosis of alloreactive T cells, thereby reducing GVHD in a DR5-dependent manner. Furthermore, murine TRAIL+ T cells mediated enhanced in vitro and in vivo antilymphoma GVT response. Moreover, human TRAIL+ T cells mediated enhanced in vitro cytotoxicity against both human leukemia cell lines and against freshly isolated chronic lymphocytic leukemia (CLL) cells. Finally, as a model of off-the-shelf, donor-unrestricted antitumor cellular therapy, in vitro-generated TRAIL+ precursor T cells from third-party donors also mediated enhanced GVT response in the absence of GVHD. These data indicate that TRAIL-overexpressing donor T cells could potentially enhance the curative potential of allo-HSCT by increasing GVT response and suppressing GVHD.

Comparative Analysis of T Cell Imaging with Human Nuclear Reporter Genes

Monitoring genetically altered T cells is an important component of adoptive T cell therapy in patients, and the ability to visualize their trafficking/targeting, proliferation/expansion, and retention/death using highly sensitive reporter systems that do not induce an immunologic response would provide useful information. Therefore, we focused on human reporter gene systems that have the potential for translation to clinical studies. The objective of the in vivo imaging studies was to determine the minimum number of T cells that could be visualized with the different nuclear reporter systems. We determined the imaging sensitivity (lower limit of T cell detection) of each reporter using appropriate radiolabeled probes for PET or SPECT imaging. Methods: Human T cells were transduced with retroviral vectors encoding for the human norepinephrine transporter (hNET), human sodium-iodide symporter (hNIS), a human deoxycytidine kinase double mutant (hdCKDM), and herpes simplex virus type 1 thymidine kinase (hsvTK) reporter genes. After viability and growth were assessed, 105 to 3 × 106 reporter T cells were injected subcutaneously on the shoulder area. The corresponding radiolabeled probe was injected intravenously 30 min later, followed by sequential PET or SPECT imaging. Radioactivity at the T cell injection sites and in the thigh (background) was measured. Results: The viability and growth of experimental cells were unaffected by transduction. The hNET/meta-18F-fluorobenzylguanidine (18F-MFBG) reporter system could detect less than 1 × 105 T cells because of its high uptake in the transduced T cells and low background activity. The hNIS/124I-iodide reporter system could detect approximately 1 × 106 T cells; 124I-iodide uptake at the T cell injection site was time-dependent and associated with high background. The hdCKDM/2′-18F-fluoro-5-ethyl-1-β-d-arabinofuranosyluracil (18F-FEAU) and hsvTK/18F-FEAU reporter systems detected approximately 3 × 105 T cells, respectively. 18F-FEAU was a more efficient probe (higher uptake, lower background) than 124I-1-(2-deoxy-2-fluoro-1-d-arabinofuranosyl)-5-iodouracil for both hdCKDM and hsvTK. Conclusion: A comparison of different reporter gene–reporter probe systems for imaging of T cell number was performed, and the hNET/18F-MFBG PET reporter system was found to be the most sensitive and capable of detecting approximately 35–40 × 103 T cells at the site of T cell injection in the animal model.

Non-invasive molecular imaging and reporter genes

Molecular-genetic imaging in living organisms has become a new field with the exceptional growth over the past 5 years. Modern imaging is based on three technologies: nuclear, magnetic resonance and optical imaging. Most current molecular-genetic imaging strategies are “indirect,” coupling a “reporter gene” with a complimentary “reporter probe.” The reporter transgene usually encodes for an enzyme, receptor or transporter that selectively interacts with a radiolabeled probe and results in accumulation of radioactivity in the transduced cell. In addition, reporter systems based on the expression of fluorescence or bioluminescence proteins are becoming more widely applied in small animal imaging. This review begins with a description of Positron Emission Tomography (PET)-based imaging genes and their complimentary radiolabeled probes that we think will be the first to enter clinical trials. Then we describe other imaging genes, mostly for optical imaging, which have been developed by investigators working with a variety of disease models in mice. Such optical reporters are unlikely to enter the clinic, at least not in the near-term. Reporter gene constructs can be driven by constitutive promoter elements and used to monitor gene therapy vectors and the efficacy of gene targeting and transduction, as well as to monitor adoptive cell-based therapies. Inducible promoters can be used as “sensors” to monitor endogenous cell processes, including specific intracellular molecular-genetic events and the activity of signaling pathways, by regulating the magnitude of reporter gene expression.

Ex Vivo Radiolabeling and In Vivo PET Imaging of T Cells Expressing Nuclear Reporter Genes

Recent advances in T cell-based immunotherapies from bench to bedside have highlighted the need for improved diagnostic imaging of T cell trafficking in vivo and the means to noninvasively investigate failures in treatment response. T cells expressing tumor-associated T cell receptors (TCRs) or engineered with chimeric antigen receptors (CARs) face multiple challenges, including possible influence of genetic engineering on T cell efficacy, inhibitory effects of the tumor microenvironment, tumor checkpoint proteins and on-target, off-tissue toxicities (Kershaw et al., Nat Rev Cancer 13:525-541, 2013; Corrigan-Curay et al., Mol Ther 22:1564-1574, 2014; June et al., Sci Trans Med 7:280-287, 2015; Whiteside et al., Clin Cancer Res 22:1845-1855, 2016; Rosenberg and Restifo, Science 348:62-68, 2015). Positron emission tomography (PET) imaging with nuclear reporter genes is potentially one of the most sensitive and noninvasive methods to quantitatively track and monitor function of adoptively transferred cells in vivo. However, in vivo PET detection of T cells after administration into patients is limited by the degree of tracer accumulation per cell in situ and cell density in target tissues. We describe here a method for ex vivo radiolabeling of T cells, a reliable and robust technique for PET imaging of the kinetics of T cell biodistribution from the time of administration to subsequent localization in targeted tumors and other tissues of the body. This noninvasive technique can provide valuable information to monitor and identify the potential efficacy of adoptive cell therapies.

Abstract 1120: Bioenergetics of T cells in the context of adoptive immunotherapy

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Tumor-specific T cell therapy has been used as an experimental approach for an anti-tumor therapy (Sadelain M 2003, Gattinoni L 2006). Adoptive T cell transfer therapies rely on ex vivo T cell isolation, transduction, activation and expansion of autologous tumor-reactive T cell populations prior to patient administration (Yee C 2002, Dudley ME 2003, Bollard CM 2004). The metabolic changes occurring in modified T-cells are the object of growing interest. For example, it has been shown that quiescent T cells display low energetic and biosynthetic demands, generating ATP through the TCA cycle and OXPHOS (oxidative phosphorylation). Activated T cells undergo conversion from a resting to an active state and ATP production switches from OXPHOS to high glycolysis. Our objective was to assess T cell metabolism and evaluate changes in glycolytic and mitochnodrial function that occur during transduction and expansion, prior to administration of T-cells in adoptive immunotherapy protocols. Methods. Human T-cells were isolated from buffy coat using ficoll separation. 48 hours after phytohemagglutinin (PHA) stimulation, cells were transduced with the retroviral vector bearing chimeric antigen receptor (CAR) targeting PSMA (Maher, Brentjens et al. 2002), and transduction efficacy was assessed by FACS. T-cells further were stimulated by exposure to antigen-presenting cells, APC. The metabolic profiles were determined at different steps of T cell stimulation and transduction. A Seahorse XF96 Analyzer was used to measure glycolysis (extracellular acidification rate, ECAR) and the oxygen consumption rate (OCR), a measure of oxidative phosphorylation (OXPHOS). Results: Glycolysis and oxygen consumption was low in resting non-stimulated cells. Both CAR-transduced and non-transduced T cells increased glycolysis (5-fold), oxygen consumption (3-fold) and ATP-linked OCR following PHA stimulation. No significant metabolic differences were observed between CAR-transduced and non-transduced T cells. High glycolytic activity was maintained by T cells over the 25 day course of T cell transduction and expansion. Mitochondrial function (oxygen consumption) declined during this period. Restimulation by exposure to antigen-presenting cells resulted in a small mitochondrial response in transduced T cells, whereas glycolysis remained high and showed no additional response. Conclusion: Glycolysis (ECAR) remains high, whereas oxygen consumption (OCR and OXPHOS) declines during T cell preparation for adoptive T cell therapy. Whether the specific changes in glucose metabolism and mitochondria respiration can influence cytotoxic function remains to be elucidated. Citation Format: Ekaterina Moroz, Maxim Moroz, Inna Serganova, Juan Zurita, Jason Lee, Nisargbhai Shah, Vladimir Ponomarev, Ronald Blasberg. Bioenergetics of T cells in the context of adoptive immunotherapy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1120. doi:10.1158/1538-7445.AM2014-1120