Development of a rat model of lymphedema and the implantation of a collagen-based medical device for therapeutic intervention.

Secondary lymphedema is a common condition among cancer survivors, and treatment strategies to prevent or treat lymphedema are in high demand. The development of novel strategies to diagnose or treat lymphedema would benefit from a robust experimental animal model of secondary lymphedema. The purpose of this methods paper is to describe and summarize our experience in developing and characterizing a rat hindlimb model of lymphedema. Here we describe a protocol to induce secondary lymphedema that takes advantage of micro computed tomography imaging for limb volume measurements and visualization of lymph drainage with near infrared imaging. To demonstrate the utility of this preclinical model for studying the therapeutic benefit of novel devices, we apply this animal model to test the efficacy of a biomaterials-based implantable medical device.

Introducing a new reporter gene, membrane-anchored Cypridina luciferase, for multiplex bioluminescence imaging.

Bioluminescence reporter gene imaging is a robust, high-throughput imaging modality that is useful for tracking cells and monitoring biological processes, both in cell culture and in small animals. We introduced and characterized a novel bioluminescence reporter-membrane-anchored Cypridina luciferase (maCLuc)-paired with a unique vargulin substrate. This luciferase-substrate pair has no cross-reactivity with established d-luciferin- or coelenterazine-based luciferase reporters. We compare maCLuc with several established luciferase-based reporter systems (firefly, click beetle, Renilla, and Gaussia luciferases), using both in vitro and in vivo models. We demonstrate the different imaging characteristics of these reporter systems, which allow for multiplexed-luciferase imaging of 3 and 4 separate targets concurrently in the same animal within 24 h. The imaging paradigms described here can be directly applied for simultaneous in vivo monitoring of multiple cell populations, the activity of selected signal transduction pathways, or a combination of both constitutive and inducible reporter imaging.

Lactate Dehydrogenase A Depletion Alters MyC-CaP Tumor Metabolism, Microenvironment, and CAR T Cell Therapy.

To enhance human prostate-specific membrane antigen (hPSMA)-specific chimeric antigen receptor (CAR) T cell therapy in a hPSMA+ MyC-CaP tumor model, we studied and imaged the effect of lactate dehydrogenase A (LDH-A) depletion on the tumor microenvironment (TME) and tumor progression. Effective LDH-A short hairpin RNA (shRNA) knockdown (KD) was achieved in MyC-CaP:hPSMA+ Renilla luciferase (RLuc)-internal ribosome entry site (IRES)-GFP tumor cells, and changes in tumor cell metabolism and in the TME were monitored. LDH-A downregulation significantly inhibited cell proliferation and subcutaneous tumor growth compared to control cells and tumors. However, total tumor lactate concentration did not differ significantly between LDH-A knockdown and control tumors, reflecting the lower vascularity, blood flow, and clearance of lactate from LDH-A knockdown tumors. Comparing treatment responses of MyC-CaP tumors with LDH-A depletion and/or anti-hPSMA CAR T cells showed that the dominant effect on tumor growth was LDH-A depletion. With anti-hPSMA CAR T cell treatment, tumor growth was significantly slower when combined with tumor LDH-A depletion and compared to control tumor growth (p < 0.0001). The lack of a complete tumor response in our animal model can be explained in part by (1) the lower activity of human CAR T cells against hPSMA-expressing murine tumors in a murine host, and (2) a loss of hPSMA antigen from the tumor cell surface in progressive generations of tumor cells.

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.

Imaging T Cell Dynamics and Function Using PET and Human Nuclear Reporter Genes.

Adoptive cell transfer immunotherapy has demonstrated significant promise in the treatment of cancer, with long-term, durable responses. T cells expressing T cell receptors (TCRs) that recognize tumor antigens, or engineered with chimeric antigen receptors (CARs) can recognize and eliminate tumor cells even in advanced disease. Positron emission tomography (PET) imaging with nuclear reporter genes, a noninvasive method to track and monitor function of engineered cells in vivo, allows quantitative, longitudinal monitoring of these cells, including their expansion/contraction, migration, retention at target and off-target sites, and biological state. As an additional advantage, some reporter genes also exhibit "suicide potential" permitting the safe elimination of adoptively transferred T cells in instances of adverse reaction to therapy. Here, we describe the production of human nuclear reporter gene-expressing T cells and noninvasive PET imaging to monitor their cell fate in vivo.

Comparative Analysis of Human Nucleoside Kinase-Based Reporter Systems for PET Imaging.

PURPOSE: Radionuclide-based reporter gene imaging has the sensitivity to monitor gene- and cell-based therapies in human subjects. Potential immunogenicity of current viral transgenes warrants development of human-based reporter systems. We compared human nucleoside kinase reporters to a panel of nucleoside analogs of FEAU, FMAU, and FIAU, including the first in vivo assessment of L-[18F]FEAU. PROCEDURES: Human isogenic U87 cell lines were transduced to express different human reporter genes including dCK-R104M/D133A (dCKDM), dCK-R104Q/D133N (dCKep16A), dCK-A100V/R104M/D133A (dCK3M), and TK2-N93D/L109F (TK2DM), and wild-type dCK (dCK) and herpes simplex virus type-1 (HSVTK) reporter gene as references. In vitro cell uptake assays were performed with [18F]FEAU, L-[18F]FEAU, [14C]FMAU, L-[18F]FMAU, and [124I]FIAU. Micro-positron emission tomography/X-ray computed tomography imaging of xenograft-bearing nu/nu mice was conducted with [18F]FEAU, L-[18F]FEAU, L-[18F]FMAU, and [124I]FIAU on consecutive days. A cell viability assay was also performed to assess sensitivities to gemcitabine and bromovinyldeoxyuridine (BVdU). RESULTS: In vitro, dCKep16A and dCKDM with [18F]FEAU exhibited the highest sensitivity and selectivity of the human reporters, second only to HSVTK/[18F]FEAU. L-[18F]FEAU biodistribution in mice was on par with [18F]FEAU and L-[18F]FMAU. L-[18F]FMAU uptake in isogenic xenografts was highest for all human reporter genes. However, [18F]FEAU was the most selective of the short half-life reporter probes due to its minimal recognition by human dCK and relative sensitivity, whereas [124I]FIAU permitted imaging at a later time point, improving signal-to-background ratio. Of the human reporter genes, dCKep16A consistently outperformed the other tested reporters. Reporter genes of interest increased potency to the nucleoside analog prodrugs gemcitabine and BVdU. CONCLUSIONS: We demonstrate that human nucleoside kinase reporter systems vary significantly in their sensitivity and selectivity for in vivo imaging. The sufficiently high signal-to-background ratios and enhanced suicide gene potential support clinical translation.

Comparative Analysis of T Cell Imaging with Human Nuclear Reporter Genes.

UNLABELLED: 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, 10(5) to 3 × 10(6) 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-(18)F-fluorobenzylguanidine ((18)F-MFBG) reporter system could detect less than 1 × 10(5) T cells because of its high uptake in the transduced T cells and low background activity. The hNIS/(124)I-iodide reporter system could detect approximately 1 × 10(6) T cells; (124)I-iodide uptake at the T cell injection site was time-dependent and associated with high background. The hdCKDM/2'-(18)F-fluoro-5-ethyl-1-ß-d-arabinofuranosyluracil ((18)F-FEAU) and hsvTK/(18)F-FEAU reporter systems detected approximately 3 × 10(5) T cells, respectively. (18)F-FEAU was a more efficient probe (higher uptake, lower background) than (124)I-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/(18)F-MFBG PET reporter system was found to be the most sensitive and capable of detecting approximately 35-40 × 10(3) T cells at the site of T cell injection in the animal model.

Comparison of corticotropin-releasing factor, dexamethasone, and temozolomide: treatment efficacy and toxicity in U87 and C6 intracranial gliomas.

UNLABELLED: 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.

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 [(18)F]-labeled fluoro-2-deoxyglucose, [(18)F]FDG, and/or 3'-deoxy-3'-[(18)F]fluorothymidine, [(18)F]FLT, PET imaging. EXPERIMENTAL DESIGN: AZD1152-treated and control HCT116 and SW620 xenograft-bearing animals were monitored for tumor size and by [(18)F]FDG, and [(18)F]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, [(18)F]FDG uptake did not demonstrate a treatment response. [(18)F]FLT uptake decreased to less than 20% of control values in AZD1152-treated HCT116 xenografts, whereas [(18)F]FLT uptake was near background levels in both treated and untreated SW620 xenografts. The EC(50) 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 [(18)F]FDG PET was inadequate in monitoring treatment response, [(18)F]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 [(18)F]FLT PET imaging and quantification of tumor proliferation, and this may apply to some clinical studies as well.

Imaging expression of the human somatostatin receptor subtype-2 reporter gene with 68Ga-DOTATOC.

UNLABELLED: 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 hNET reporter gene expression with 124I-MIBG.

UNLABELLED: 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 (123)I- or (124)I-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 (K(1)) rather than efflux (k(2)). The estimated MIBG distribution volumes (V(d)) 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 (K(1)) 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 (124)I-MIBG small-animal PET compared with (123)I-MIBG gamma-camera/SPECT. This was primarily due to the longer half-life of (124)I 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 (124)I-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.