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.

Imaging CAR T-cell kinetics in solid tumors: Translational implications.

Success in solid tumor chimeric antigen receptor (CAR) T-cell therapy requires overcoming several barriers, including lung sequestration, inefficient accumulation within the tumor, and target-antigen heterogeneity. Understanding CAR T-cell kinetics can assist in the interpretation of therapy response and limitations and thereby facilitate developing successful strategies to treat solid tumors. As T-cell therapy response varies across metastatic sites, the assessment of CAR T-cell kinetics by peripheral blood analysis or a single-site tumor biopsy is inadequate for interpretation of therapy response.

Introducing a new reporter gene, membrane-anchored 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 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.

A dual-modal PET/near infrared fluorescent nanotag for long-term immune cell tracking.

Adoptive cell transfer of targeted chimeric antigen receptor (CAR) T cells has emerged as a highly promising cancer therapy. The pharmacodynamic action or CAR T cells is closely related to their pharmacokinetic profile; because of this as well as the risk of non-specific action, it is important to monitor their biodistribution and fate following infusion. To this end, we developed a dual-modal PET/near infrared fluorescent (NIRF) nanoparticle-based imaging agent for non-genomic labeling of human CAR T cells.

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.

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.

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.

Enhancement of PSMA-Directed CAR Adoptive Immunotherapy by PD-1/PD-L1 Blockade.

Chimeric antigen receptor (CAR) T cell therapy in hematologic malignancies has shown remarkable responses, but the same level of success has not been observed in solid tumors. A new prostate cancer model (Myc-CaP:PSMA(+)) and a second-generation anti-hPSMA human CAR T cells expressing a Click Beetle Red luciferase reporter) were used to study hPSMA targeting and assess CAR T cell trafficking and persistence by bioluminescence imaging (BLI). We investigated the antitumor efficacy of human CAR T cells targeting human prostate-specific membrane antigen (hPSMA), in the presence and absence of the target antigen; first alone and then combined with a monoclonal antibody targeting the human programmed death receptor 1 (anti-hPD1 mAb).

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-[F]FEAU.

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.

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).

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.

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.

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.

Multimodality imaging of TGFbeta 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 beta (TGFbeta) 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 TGFbeta-inducible reporter genes and were tested in vitro and in living animals.

3'-deoxy-3'-[18F]fluorothymidine positron emission tomography is a sensitive method for imaging the response of BRAF-dependent tumors to MEK inhibition.

Activating mutations of BRAF occur in approximately 7% of all human tumors and in the majority of melanomas. These tumors are very sensitive to pharmacologic inhibition of mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK), which causes loss of D-cyclin expression, hypophosphorylation of Rb, and G(1) arrest. Growth arrest is followed by differentiation or senescence and, in a subset of BRAF mutant tumors, by apoptosis.

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.