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The cascade of events leading to tumor formation includes induction of a tumor supporting neovasculature, as a primary hallmark of cancer. Developing vasculature is difficult to evaluate in vivo but can be captured using microfluidic chip technology and patient derived cells. Herein, we established an on chip approach to investigate the mechanisms promoting tumor vascularization and vascular targeted therapies via co-culture of cancer spheroids and endothelial cells in a three dimensional environment. Methods: We investigated both, tumor neovascularization and therapy, via co-culture of human derived endothelial cells and adjacently localized metastatic renal cell carcinoma spheroids on a commercially available microfluidic chip system. Metastatic renal cell carcinoma spheroids adjacent to primary vessels model tumor, and induce vessels to sprout neovasculature towards the tumor. We monitored real time changes in vessel formation, probed the interactions of tumor and endothelial cells, and evaluated the role of important effectors in tumor vasculature. In addition to wild type endothelial cells, we evaluated endothelial cells that overexpress Prostate Specific Membrane Antigen (PSMA), that has emerged as a marker of tumor associated neovasculature. We characterized the process of neovascularization on the microfluidic chip stimulated by enhanced culture medium and the investigated metastatic renal cell carcinomas, and assessed endothelial cells responses to vascular targeted therapy with bevacizumab via confocal microscopy imaging. To emphasize the potential clinical relevance of metastatic renal cell carcinomas on chip , we compared therapy with bevacizumab on chip with an in vivo model of the same tumor. Results: Our model permitted real-time, high-resolution observation and assessment of tumor-induced angiogenesis, where endothelial cells sprouted towards the tumor and mimicked a vascular network. Bevacizumab, an antiangiogenic agent, disrupted interactions between vessels and tumors, destroying the vascular network. The on chip approach enabled assessment of endothelial cell biology, vessel's functionality, drug delivery, and molecular expression of PSMA. Conclusion: Observations in the vascularized tumor on chip permitted direct and conclusive quantification of vascular targeted therapies in weeks as opposed to months in a comparable animal model, and bridged the gap between in vitro and in vivo models., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Labeling and tracking existing and emerging cell-based immunotherapies using nuclear imaging is widely used to guide the preclinical phases of development and testing of existing and new emerging off-the-shelf cell-based immunotherapies. In fact, advancing our knowledge about their mechanism of action and limitations could provide preclinical support and justification for moving towards clinical experimentation of newly generated products and expedite their approval by the Food and Drug Administration (FDA).Here we provide the reader with a ready to use protocol describing the labeling methodologies and practical procedures to render different candidate cell therapies in vivo traceable by nuclear-based imaging. The protocol includes sufficient practical details to aid researchers at all career stages and from different fields in familiarizing with the described concepts and incorporating them into their work., (© 2024. The Author(s).)

Background: Male dogs can develop spontaneous prostate cancer, which is similar physiologically to human disease. Recently, Tweedle and coworkers have developed an orthotopic canine prostate model allowing implanted tumors and therapeutic agents to be tested in a more translational large animal model. We used the canine model to evaluate prostate-specific membrane antigen (PSMA)-targeted gold nanoparticles as a theranostic approach for fluorescence (FL) imaging and photodynamic therapy (PDT) of early stage prostate cancer., Methods: Dogs (four in total) were immunosuppressed with a cyclosporine-based immunosuppressant regimen and their prostate glands were injected with Ace-1-hPSMA cells using transabdominal ultrasound (US) guidance. Intraprostatic tumors grew in 4-5 weeks and were monitored by ultrasound (US). When tumors reached an appropriate size, dogs were injected intravenously (iv) with PSMA-targeted nano agents (AuNPs-Pc158) and underwent surgery 24 h later to expose the prostate tumors for FL imaging and PDT. Ex vivo FL imaging and histopathological studies were performed to confirm PDT efficacy., Results: All dogs had tumor growth in the prostate gland as revealed by US. Twenty-four hours after injection of PSMA-targeted nano agents (AuNPs-Pc158), the tumors were imaged using a Curadel FL imaging device. While normal prostate tissue had minimal fluorescent signal, the prostate tumors had significantly increased FL. PDT was activated by irradiating specific fluorescent tumor areas with laser light (672 nm). PDT bleached the FL signal, while fluorescent signals from the other unexposed tumor tissues were unaffected. Histological analysis of tumors and adjacent prostate revealed that PDT damaged the irradiated areas to a depth of 1-2 mms with the presence of necrosis, hemorrhage, secondary inflammation, and occasional focal thrombosis. The nonirradiated areas showed no visible damages by PDT., Conclusion: We have successfully established a PSMA-expressing canine orthotopic prostate tumor model and used the model to evaluate the PSMA-targeted nano agents (AuNPs-Pc158) in the application of FL imaging and PDT. It was demonstrated that the nano agents allowed visualization of the cancer cells and enabled their destruction when they were irradiated with a specific wavelength of light., (© 2023 The Authors. The Prostate published by Wiley Periodicals LLC.)

The cascade of events leading to tumor formation includes induction of a tumor supporting neovasculature as a primary hallmark of cancer. Developing vasculature is difficult to evaluate in vivo but can be captured using microfluidic chip technology and patient derived cells. Herein, we established an on chip approach to investigate the mechanisms promoting tumor vascularization and vascular targeted therapies via co-culture of metastatic renal cell carcinoma spheroids and endothelial cells in a 3D environment. Our model permitted real-time, high-resolution observation and assessment of tumor-induced angiogenesis, where endothelial cells sprout towards the tumor and mimic a vascular network. Bevacizumab, an angiogenic inhibitor, disrupted interactions between vessels and tumors, destroying the vascular network. The on chip approach enabled assessment of endothelial cell biology, vessel's functionality, drug delivery, and molecular expression of PSMA. Finally, observations in the vascularized tumor on chip permitted direct and conclusive quantification of this therapy in weeks as opposed to months in a comparable animal model., Teaser: Vascularized tumor on microfluidic chip provides opportunity to study targeted therapies and improves preclinical drug discovery.

In conventional positron emission tomography (PET), only one radiotracer can be imaged at a time, because all PET isotopes produce the same two 511 keV annihilation photons. Here we describe an image reconstruction method for the simultaneous in vivo imaging of two PET tracers and thereby the independent quantification of two molecular signals. This method of multiplexed PET imaging leverages the 350-700 keV range to maximize the capture of 511 keV annihilation photons and prompt γ-ray emission in the same energy window, hence eliminating the need for energy discrimination during reconstruction or for signal separation beforehand. We used multiplexed PET to track, in mice with subcutaneous tumours, the biodistributions of intravenously injected [ 124 I]I-trametinib and 2-deoxy-2-[ 18 F]fluoro-D-glucose, [ 124 I]I-trametinib and its nanoparticle carrier [ 89 Zr]Zr-ferumoxytol, and the prostate-specific membrane antigen (PSMA) and infused PSMA-targeted chimaeric antigen receptor T cells after the systemic administration of [ 68 Ga]Ga-PSMA-11 and [ 124 I]I. Multiplexed PET provides more information depth, gives new uses to prompt γ-ray-emitting isotopes, reduces radiation burden by omitting the need for an additional computed-tomography scan and can be implemented on preclinical and clinical systems without any modifications in hardware or image acquisition software., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

Background: Extracorporeal membrane oxygenation has a high risk of acute brain injury and resultant mortality. Transcranial Doppler characterizes cerebral hemodynamics in real time, but limited data exist on its interpretation in ECMO. Here, we report TCD mean flow velocity and pulsatility index in a large ECMO population., Methods: This was a prospective cohort study at a tertiary care center. The patients were adults on venoarterial ECMO or venovenous ECMO undergoing TCD studies., Results: A total of 135 patients underwent a total of 237 TCD studies while on VA-ECMO (n = 95, 70.3%) or VV-ECMO (n = 40, 29.6%). MFVs were captured reliably (approximately 90%) and were similar to a published healthy cohort in all vessels except the internal carotid artery. Presence of a recordable PI was strongly associated with ECMO mode (57% in VA vs. 95% in VV, p < 0.001). Absence of TCD pulsatility was associated with intraparenchymal hemorrhage (14.7 vs. 1.6%, p = 0.03) in VA-ECMO patients., Conclusions: Transcranial Doppler analysis in a single-center cohort of VA-ECMO and VV-ECMO patients demonstrates similar MFVs and PIs. Absence of PIs was associated with a higher frequency of intraparenchymal hemorrhage and a composite bleeding event. However, cautious interpretation and external validation is necessary for these findings with a multicenter study with a larger sample size., (© 2022. Springer Science+Business Media, LLC, part of Springer Nature and Neurocritical Care Society.)

Schizophrenia is a major psychiatric disorder that significantly reduces the quality of life. Early treatment is extremely important in order to mitigate the long-term negative effects. In this paper, a machine learning based diagnostics of schizophrenia was designed. Classification models were applied to the event-related potentials (ERPs) of patients and healthy subjects performing the visual cued Go/NoGo task. The sample consisted of 200 adult individuals ranging in age from 18 to 50 years. In order to apply the machine learning models, various features were extracted from the ERPs. The process of feature extraction was parametrized through a special procedure and the parameters of this procedure were selected through a grid-search technique along with the model hyperparameters. Feature extraction was followed by sequential feature selection transformation in order to prevent overfitting and reduce the computational complexity. Various models were trained on the resulting feature set. The best model was support vector machines with a sensitivity and specificity of 91% and 90.8%, respectively.

Cellular immunotherapies have emerged as a successful therapeutic approach to fight a wide range of human diseases, including cancer. However, responses are limited to few patients and tumor types. An in-depth understanding of the complexity and dynamics of cellular immunotherapeutics, including what is behind their success and failure in a patient, the role of other immune cell types and molecular biomarkers in determining a response, is now paramount. As the cellular immunotherapy arsenal expands, whole-body non-invasive molecular imaging can shed a light on their in vivo fate and contribute to the reliable assessment of treatment outcome and prediction of therapeutic response. In this review, we outline the non-invasive strategies that can be tailored toward the molecular imaging of cellular immunotherapies and immune-related components, with a focus on those that have been extensively tested preclinically and are currently under clinical development or have already entered the clinical trial phase. We also provide a critical appraisal on the current role and consolidation of molecular imaging into clinical practice., Competing Interests: Competing interests: Dr Adusumilli declares research funding from ATARA Biotherapeutics, has served on the Scientific Advisory Board or as a consultant to ATARA Biotherapeutics, Bayer, BioArdis, Carisma Therapeutics, Imugene, ImmPACT Bio, and Johnson & Johnson, and has patents, royalties, and intellectual property on mesothelin-targeted CARs and T-cell therapies. Memorial Sloan Kettering Cancer Center has licensed intellectual property related to mesothelin-targeted CARs and T-cell therapies to ATARA Biotherapeutics and has associated financial interests., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Objective: Extracorporeal membrane oxygenation (ECMO) carries a high morbidity of acute brain injury (ABI) with resultant mortality increase. Transcranial Doppler (TCD) allows real-time characterization of regional cerebral hemodynamics, but limited data exist on the interpretation of microembolic signals (MES) in ECMO., Methods: This prospective cohort study was conducted at a single tertiary care center, November 2017 through February 2022, and included all adult patients receiving venoarterial (VA) and venovenous (VV) ECMO undergoing TCD examinations, which all included MES monitoring., Results: Of 145 patients on ECMO who underwent at least 1 TCD examination, 100 (68.9%) patients on VA-ECMO received 187 examinations whereas 45 (31.1%) patients on VV-ECMO received 65 examinations ( P  = .81). MES were observed in 35 (35.0%) patients on VA-ECMO and 2 (4.7%) patients on VV-ECMO ( P  < .001), corresponding to 46 (24.6%) and 2 (3.1%) TCD examinations, respectively. MES were present in 29.4% of patients on VA-ECMO without additional cardiac support, compared with 38.1% with intra-aortic balloon pump and 57.1% with left ventricular assist device, but these differences were not statistically significant ( P  = .39; P  = .20, respectively). Presence or number of MES was not associated with VA-ECMO cannulation mode (23.4% MES presence in peripheral cannulation vs 25.8% in central cannulation, P  = .80). In both VA- and VV-ECMO, MES presence or number was not associated with presence of clot or fibrin in the ECMO circuit or with any studied hemodynamic, laboratory, or ECMO parameters at the time of TCD. ABI occurred in 38% and 31.1% of patients on VA- and VV-ECMO, respectively. In multivariable logistic regression analyses, neither ABI nor a composite outcome of arterial thromboembolic events was associated with presence or number of MES in VA- ECMO., Conclusions: TCD analysis in a large cohort of patients on ECMO demonstrates a significant number of MES, especially in patients on VA-ECMO with intra-aortic balloon pump, and/or left ventricular assist device. However, clinical associations and significance of TCD MES remain unresolved and warrant further correlation with systematic imaging and long-term neurologic follow-up., (© 2022 The Author(s).)

Background: The purpose of the data paper was to introduce into scientific literature the results of scientific work carried out for the third edition of the 'Red Data Book of the Komi Republic'. The article reflects methodological approaches to the formation of a list of rare and in need of protection species and describes the corresponding datasets published in GBIF., New Information: Information about 7,187 occurrences of 438 rare species and infraspecies included in the third edition of the 'Red Data Book of the Komi Republic' have been published., (Svetlana Degteva, Anatoly Bobretsov, Yury Bobrov, Modest Dolgin, Mikhail Dulin, Nickolay Filippov, Nadezhda Goncharova, Janolof Hermansson, Vladimir Kanev, Dmitry Kirillov, Irina Kirillova, Olga Kirsanova, Sergey Kochanov, Alla Kolesnikova, Tatyana Konakova, Andrey Korolev, Denis Kosolapov, Oksana Kulakova, Ekaterina Kulyugina, Olga Loskutova, Elena Melekhina, Oleg Mineev, Yuri Mineev, Vladimir Morozov, Gleb Nakul, Marina Palamarchuk, Elena Patova, Sergej Pestov, Anatoly Petrov, Irina Poletaeva, Vasily Ponomarev, Tatiana Pystina, Yury Rebriev, Roman Romanov, Natalya Selivanova, Anton Shiryaev, Tatyana Shubina, Irina Sterlyagova, Andrey Tatarinov, Boris Teteryuk, Ludmila Teteryuk, Zinaida Ulle, Olga Valuyskikh, Alexander Zakharov, Galina Zheleznova, Aurika Zinovyeva, Yuriy Dubrovskiy, Boris Gruzdev, Anna Ichetkina, Vera Martynenko, Nadezhda Oplesnina, Vera Panova, Irina Romanova, Mikhail Rubtsov, Leonid Rybin, Nataliya Semenova.)

Cell-based therapies have been making great advances toward clinical reality. Despite the increase in trial activity, few therapies have successfully navigated late-phase clinical trials and received market authorization. One possible explanation for this is that additional tools and technologies to enable their development have only recently become available. To support the safety evaluation of cell therapies, the Health and Environmental Sciences Institute Cell Therapy-Tracking, Circulation and Safety Committee, a multisector collaborative committee, polled the attendees of the 2017 International Society for Cell & Gene Therapy conference in London, UK, to understand the gaps and needs that cell therapy developers have encountered regarding safety evaluations in vivo. The goal of the survey was to collect information to inform stakeholders of areas of interest that can help ensure the safe use of cellular therapeutics in the clinic. This review is a response to the cellular imaging interests of those respondents. The authors offer a brief overview of available technologies and then highlight the areas of interest from the survey by describing how imaging technologies can meet those needs. The areas of interest include imaging of cells over time, sensitivity of imaging modalities, ability to quantify cells, imaging cellular survival and differentiation and safety concerns around adding imaging agents to cellular therapy protocols. The Health and Environmental Sciences Institute Cell Therapy-Tracking, Circulation and Safety Committee believes that the ability to understand therapeutic cell fate is vital for determining and understanding cell therapy efficacy and safety and offers this review to aid in those needs. An aim of this article is to share the available imaging technologies with the cell therapy community to demonstrate how these technologies can accomplish unmet needs throughout the translational process and strengthen the understanding of cellular therapeutics., (Copyright © 2021 International Society for Cell & Gene Therapy. Published by Elsevier Inc. All rights reserved.)

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. The use of tumor imaging alone has also proven to be insufficient to interpret response to therapy. To address these limitations, we conducted dual tumor and T-cell imaging by use of a bioluminescent reporter and positron emission tomography in clinically relevant mouse models of pleural mesothelioma and non-small cell lung cancer. We observed that the mode of delivery of T cells (systemic versus regional), T-cell activation status (presence or absence of antigen-expressing tumor), and tumor-antigen expression heterogeneity influence T-cell kinetics. The observations from our study underscore the need to identify and develop a T-cell reporter-in addition to standard parameters of tumor imaging and antitumor efficacy-that can be used for repeat imaging without compromising the efficacy of CAR T cells in vivo ., Competing Interests: P.S.A. has received research funding from ATARA Biotherapeutics and Acea Biosciences; has served on the Scientific Advisory Board or as consultant to ATARA Biotherapeutics, Bayer, Carisma Therapeutics, Imugene, ImmPACT Bio, and Takeda Therapeutics; and has patents, royalties, and intellectual property on MSLN-targeted CARs and other T-cell therapies, which has been licensed to ATARA Biotherapeutics, as well as method for detection of cancer cells using virus, and pending patent applications on T-cell therapies. Memorial Sloan Kettering Cancer Center (MSK) has licensed intellectual property related to mesothelin-targeted CARs and T-cell therapies to ATARA Biotherapeutics, and has associated financial interests., (© 2021 The Authors.)

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., Competing Interests: The authors declare no competing interests., (© 2021 The Author(s).)

Chimeric antigen receptor (CAR)-T cell therapy has generated unprecedented advances in the treatment of hematologic cancers, but readily translatable imaging approaches to visualize the in vivo dynamics of CAR-T cells are lacking. Noninvasive PET imaging is the ideal tool to monitor CAR-T cells. See related article by Simonetta et al., p. 1058 ., (©2020 American Association for Cancer Research.)

Several types of gene- and cell-based therapeutics are now emerging in the cancer immunotherapy, transplantation, and regenerative medicine landscapes. Radionuclear-based imaging can be used as a molecular imaging tool for repetitive and non-invasive visualization as well as in vivo monitoring of therapy success. In this review, we discuss the principles of nuclear-based imaging and provide a comprehensive overview of its application in gene and cell therapy. This review aims to inform investigators in the biomedical field as well as clinicians on the state of the art of nuclear imaging, from probe design to available radiopharmaceuticals and advances of direct (probe-based) and indirect (transgene-based) strategies in both preclinical and clinical settings. Notably, as the nuclear-based imaging toolbox is continuously expanding, it will be increasingly incorporated into the clinical setting where the distribution, targeting, and persistence of a new generation of therapeutics can be imaged and ultimately guide therapeutic decisions., Competing Interests: The authors declare no competing interests., (© 2021 The Authors.)

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. Since the PET/NIRF nanoparticles did not affect cell viability or cytotoxic functionality and enabled long-term whole-body CAR T cell tracking using PET and NIRF in an ovarian peritoneal carcinomatosis model, this platform is a viable imaging technology to be applied in other cancer models., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Background: Treatment of patients with Parkinson disease (PD) using autologous mesenchymal stem cells (MSCs) is a promising method to influence the pathogenesis of the disease. The aim of this study was to assess the immediate results of the introduction of MSCs on the effectiveness of motor and nonmotor symptoms in patients with PD., Methods: MSCs were transplanted to 12 patients with PD through intravenous and tandem (intranasal + intravenous) injections. Effectiveness of the therapy was evaluated 1 and 3 months posttransplantation. Neurological examination of the intensity of motor symptoms was carried out in the morning after a 12 or 24 h break in taking antiparkinsonian drugs, then 1 h after they were taken. The intensity of motor symptoms was assessed with the help of Section III of the Unified PD Rating Scale of the International Society for Movement Disorders (UPDRS). The intensity of nonmotor symptoms was assessed with the help of the following scales: Hamilton Depression Rating Scale, the Pittsburgh Sleep Quality Index, the Epworth Sleepiness Scale, Nonmotor Symptoms Scale, and the 39-item Parkinson's Disease Questionnaire., Results: We found a statistically significant decrease in the severity of motor and nonmotor symptoms in the study group in the posttransplant period., Conclusion: Positive results allow us to consider MSCs transplantation as a disease-modifying therapeutic strategy in PD. However, this method of PD treatment is not a fully understood process, which requires additional studies and a longer follow-up period to monitor the patients' condition posttransplantation., Competing Interests: There are no conflicts of interest., (Copyright: © 2020 Surgical Neurology International.)

CAR T-cell therapy for multiple myeloma (MM) targeting B-cell maturation antigen ( TNFRSF17 ; BCMA) induces high overall response rates; however, relapse occurs commonly. Implicated in relapse is a reservoir of MM if cells lacking sufficient BCMA surface expression (antigen escape). We demonstrate that simultaneous targeting of an additional antigen-here, G protein-coupled receptor class-C group-5 member-D ( GPRC5D )-can prevent BCMA escape-mediated relapse in a model of MM. To identify an optimal approach, we compare subtherapeutic doses of different forms of dual-targeted cellular therapy. These include (1) parallel-produced and pooled mono-targeted CAR T-cells, (2) bicistronic constructs expressing distinct CARs from a single vector, and (3) a dual-scFv "single-stalk" CAR design. When targeting BCMA-negative disease, bicistronic and pooled approaches had the highest efficacy, whereas for dual-antigen-expressing disease, the bicistronic approach was more efficacious than the pooled approach. Mechanistically, expressing two CARs on a single cell enhanced the strength of CAR T-cell/target cell interactions.

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., (© 2020 The Authors.)

Cellular senescence is characterized by stable cell-cycle arrest and a secretory program that modulates the tissue microenvironment 1,2 . Physiologically, senescence serves as a tumour-suppressive mechanism that prevents the expansion of premalignant cells 3,4 and has a beneficial role in wound-healing responses 5,6 . Pathologically, the aberrant accumulation of senescent cells generates an inflammatory milieu that leads to chronic tissue damage and contributes to diseases such as liver and lung fibrosis, atherosclerosis, diabetes and osteoarthritis 1,7 . Accordingly, eliminating senescent cells from damaged tissues in mice ameliorates the symptoms of these pathologies and even promotes longevity 1,2,8-10 . Here we test the therapeutic concept that chimeric antigen receptor (CAR) T cells that target senescent cells can be effective senolytic agents. We identify the urokinase-type plasminogen activator receptor (uPAR) 11 as a cell-surface protein that is broadly induced during senescence and show that uPAR-specific CAR T cells efficiently ablate senescent cells in vitro and in vivo. CAR T cells that target uPAR extend the survival of mice with lung adenocarcinoma that are treated with a senescence-inducing combination of drugs, and restore tissue homeostasis in mice in which liver fibrosis is induced chemically or by diet. These results establish the therapeutic potential of senolytic CAR T cells for senescence-associated diseases.

Bispecific antibodies (BsAb) that engage T cells bind to tumor cells via a tumor-associated antigen and to T cells through surface CD3. BsAbs have promising antitumor properties in vivo Here, we describe the effects of Fc silencing on BsAb-driven T-cell trafficking to solid tumors. We used BsAbs specific for disialoganglioside GD2 or oncoprotein ErbB2 (HER2) and built on the IgG(L)-scFv platform with or without Fc silencing. We studied the kinetics of T-cell infiltration from blood into solid tumor masses when driven by these BsAbs. We also investigated the therapeutic efficacy of these BsAbs in two mouse models: immunodeficient mice xenografted with patient-derived GD2 + neuroblastoma or HER2 + breast cancer, and human CD3ε transgenic mice implanted with a GD2 + murine tumor. BsAbs built with intact Fc domain were unable to drive T cells to tumor, thereby failing to achieve an antitumor effect in mice. T cells became sequestered in lungs by myeloid cells or depleted in circulation. In contrast, when Fc function was silenced by N297A ± K322A mutations, T cells were able to infiltrate into subcutaneous solid tumors, a prerequisite for successful therapy outcome., (©2019 American Association for Cancer Research.)

There remains an urgent need for the noninvasive tracking of transfused chimeric antigen receptor (CAR) T cells to determine their biodistribution, viability, expansion, and antitumor functionality. DOTA antibody reporter 1 (DAbR1) comprises a single-chain fragment of the antilanthanoid-DOTA antibody 2D12.5/G54C fused to the human CD4-transmembrane domain and binds irreversibly to lanthanoid ( S )-2-(4-acrylamidobenzyl)-DOTA (AABD). The aim of this study was to investigate whether DAbR1 can be expressed on lymphocytes and used as a reporter gene as well as a suicide gene for therapy of immune-related adverse effects. Methods: DAbR1 was subcloned together with green fluorescent protein into an SFG-retroviral vector and used to transduce CD3/CD28-activated primary human T cells and second-generation 1928z (CAR) T cells. Cell surface expression of DAbR1 was confirmed by cell uptake studies with radiolabeled AABD. In addition, the feasibility of imaging of DAbR1-positive T cells in vivo after intravenous injection of 86 Y/ 177 Lu-AABD was studied and radiation doses determined. Results: A panel of DAbR1-expressing T cells and CAR T cells exhibited greater than 8-fold increased uptake of 86 Y-AABD in vitro when compared with nontransduced cells. Imaging studies showed 86 Y-AABD was retained by DAbR1-positive T cells while it continuously cleared from normal tissues, allowing for in vivo tracking of intravenously administered CAR T cells. Normal-organ dose estimates were favorable for repeated PET/CT studies. Selective T cell ablation in vivo with 177 Lu-AABD seems feasible for clustered T-cell populations. Conclusion: We have demonstrated for the first time that T cells can be modified with DAbR1, enabling their in vivo tracking via PET and SPECT. The favorable biodistribution and high image contrast observed warrant further studies of this new reporter gene., (© 2018 by the Society of Nuclear Medicine and Molecular Imaging.)

The DNA repair enzyme poly(ADP-ribose) polymerase 1 (PARP-1) is overexpressed in glioblastoma, with overall low expression in healthy brain tissue. Paired with the availability of specific small molecule inhibitors, PARP-1 is a near-ideal target to develop novel radiotherapeutics to induce DNA damage and apoptosis in cancer cells, while sparing healthy brain tissue. Methods: We synthesized an 131 I-labeled PARP-1 therapeutic and investigated its pharmacology in vitro and in vivo. A subcutaneous tumor model was used to quantify retention times and therapeutic efficacy. A potential clinical scenario, intratumoral convection-enhanced delivery, was mimicked using an orthotopic glioblastoma model combined with an implanted osmotic pump system to study local administration of 131 I-PARPi (PARPi is PARP inhibitor). Results: 131 I-PARPi is a 1(2H)-phthalazinone, similar in structure to the Food and Drug Administration-approved PARP inhibitor AZD-2281. In vitro studies have shown that 131 I-PARPi and AZD-2281 share similar pharmacologic profiles. 131 I-PARPi delivered 134.1 cGy/MBq intratumoral injected activity. Doses to nontarget tissues, including liver and kidney, were significantly lower. Radiation damage and cell death in treated tumors were shown by p53 activation in U87-MG cells transfected with a p53-bioluminescent reporter. Treated mice showed significantly longer survival than mice receiving vehicle (29 vs. 22 d, P < 0.005) in a subcutaneous model. Convection-enhanced delivery demonstrated efficient retention of 131 I-PARPi in orthotopic brain tumors, while quickly clearing from healthy brain tissue. Conclusion: Our results demonstrate 131 I-PARPi's high potential as a therapeutic and highlight PARP's relevance as a target for radionuclide therapy. Radiation plays an integral role in brain tumor therapy, and radiolabeled PARP therapeutics could ultimately lead to improvements in the standard of care., (© 2018 by the Society of Nuclear Medicine and Molecular Imaging.)

Prostate-specific membrane antigen (PSMA) or folate hydrolase 1 (FOLH1) is highly expressed on prostate cancer. Its expression correlates inversely with survival and increases with tumor grade. However, the biological role of PSMA has not been explored, and its role in prostate cancer remained elusive. Filling this gap, we demonstrate that in prostate cancer, PSMA initiates signaling upstream of PI3K through G protein-coupled receptors, specifically via the metabotropic glutamate receptor (mGluR). PSMA's carboxypeptidase activity releases glutamate from vitamin B9 and other glutamated substrates, which activate mGluR I. Activated mGluR I subsequently induces activation of phosphoinositide 3-kinase (PI3K) through phosphorylation of p110β independent of PTEN loss. The p110β isoform of PI3K plays a particularly important role in the pathogenesis of prostate cancer, but the origin of its activation was so far unknown. PSMA expression correlated with PI3K-Akt signaling in cells, animal models, and patients. We interrogated the activity of the PSMA-PI3K axis through positron emission tomography and magnetic resonance imaging. Inhibition of PSMA in preclinical models inhibited PI3K signaling and promoted tumor regression. Our data present a novel oncogenic signaling role of PSMA that can be exploited for therapy and interrogated with imaging., (© 2018 Kaittanis et al.)

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.

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.

Tumor hypoxia is a major cause of treatment failure for a variety of malignancies. However, hypoxia also leads to treatment opportunities as demonstrated by the development of compounds that target regions of hypoxia within tumors. Evofosfamide is a hypoxia-activated prodrug that is created by linking the hypoxia-seeking 2-nitroimidazole moiety to the cytotoxic bromo-isophosphoramide mustard (Br-IPM). When evofosfamide is delivered to hypoxic regions of tumors, the DNA cross-linking toxin, Br-IPM, is released leading to cell death. This study assessed the anticancer efficacy of evofosfamide in combination with the Proapoptotic Receptor Agonists (PARAs) dulanermin and drozitumab against human osteosarcoma in vitro and in an intratibial murine model of osteosarcoma. Under hypoxic conditions in vitro, evofosfamide cooperated with dulanermin and drozitumab, resulting in the potentiation of cytotoxicity to osteosarcoma cells. In contrast, under the same conditions, primary human osteoblasts were resistant to treatment. Animals transplanted with osteosarcoma cells directly into their tibiae developed mixed osteosclerotic/osteolytic bone lesions and consequently developed lung metastases 3 weeks post cancer cell transplantation. Tumor burden in the bone was reduced by evofosfamide treatment alone and in combination with drozitumab and prevented osteosarcoma-induced bone destruction while also reducing the growth of pulmonary metastases. These results suggest that evofosfamide may be an attractive therapeutic agent, with strong anticancer activity alone or in combination with either drozitumab or dulanermin against osteosarcoma., (© 2017 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.)

Immunotherapies include various approaches, ranging from stimulating effector mechanisms to counteracting inhibitory and suppressive mechanisms, and creating a forum for discussing the most effective means of advancing these therapies through imaging is the focus of the newly formed Imaging in Cellular and Immune Therapies (ICIT) interest group within the World Molecular Imaging Society. Efforts are being made in the identification and validation of predictive biomarkers for a number of immunotherapies. Without predictive biomarkers, a considerable number of patients may receive treatments that have no chance of offering a benefit. This will reflect poorly on the field of immunotherapy and will yield false hopes in patients while at the same time contributing to significant cost to the healthcare system. This review summarizes the main strategies in cancer immune and cell-based therapies and discusses recent advances in imaging strategies aimed to improve cancer immunotherapy outcomes.

Myeloperoxidase (MPO) and eosinophil peroxidase (EPO) are heme-containing enzymes, well known for their antimicrobial activity, are released in high quantities by infiltrating immune cells in breast cancer. However, the functional importance of their presence within the tumour microenvironment is unclear. We have recently described a new role for peroxidases as key regulators of fibroblast and endothelial cell functionality. In the present study, we investigate for the first time, the ability of peroxidases to promote breast cancer development and progression. Using the 4T1 syngeneic murine orthotopic breast cancer model, we examined whether increased levels of peroxidases in developing mammary tumours influences primary tumour growth and metastasis. We showed that MPO and EPO stimulation increased mammary tumour growth and enhanced lung metastases, effects that were associated with reduced tumour necrosis, increased collagen deposition and neo-vascularisation within the primary tumour. In vitro, peroxidase treatment, robustly stimulated human mammary fibroblast migration and collagen type I and type VI secretion. Mechanistically, peroxidases induced the transcription of pro-tumorigenic and metastatic MMP1, MMP3 and COX-2 genes. Taken together, these findings identify peroxidases as key contributors to cancer progression by augmenting pro-tumorigenic collagen production and angiogenesis. Importantly, this identifies inflammatory peroxidases as therapeutic targets in breast cancer therapy.

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-[ 18 F]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 [ 18 F]FEAU, L-[ 18 F]FEAU, [ 14 C]FMAU, L-[ 18 F]FMAU, and [ 124 I]FIAU. Micro-positron emission tomography/X-ray computed tomography imaging of xenograft-bearing nu/nu mice was conducted with [ 18 F]FEAU, L-[ 18 F]FEAU, L-[ 18 F]FMAU, and [ 124 I]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 [ 18 F]FEAU exhibited the highest sensitivity and selectivity of the human reporters, second only to HSVTK/[ 18 F]FEAU. L-[ 18 F]FEAU biodistribution in mice was on par with [ 18 F]FEAU and L-[ 18 F]FMAU. L-[ 18 F]FMAU uptake in isogenic xenografts was highest for all human reporter genes. However, [ 18 F]FEAU was the most selective of the short half-life reporter probes due to its minimal recognition by human dCK and relative sensitivity, whereas [ 124 I]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.

Bone metastases occur in over 75% of patients with advanced breast cancer and are responsible for high levels of morbidity and mortality. In this study, ex vivo expanded cytotoxic Vγ9Vδ2 T cells isolated from human peripheral blood were tested for their anti-cancer efficacy in combination with zoledronic acid (ZOL), using a mouse model of osteolytic breast cancer. In vitro, expanded Vγ9Vδ2 T cells were cytotoxic against a panel of human breast cancer cell lines, and ZOL pre-treatment further sensitised breast cancer cells to killing by Vγ9Vδ2 T cells. Vγ9Vδ2 T cells adoptively transferred into NOD/SCID mice localised to osteolytic breast cancer lesions in the bone, and multiple infusions of Vγ9Vδ2 T cells reduced tumour growth in the bone. ZOL pre-treatment potentiated the anti-cancer efficacy of Vγ9Vδ2 T cells, with mice showing further reductions in tumour burden. Mice treated with the combination also had reduced tumour burden of secondary pulmonary metastases, and decreased bone degradation. Our data suggests that adoptive transfer of Vγ9Vδ2 T cell in combination with ZOL may prove an effective immunotherapeutic approach for the treatment of breast cancer bone metastases., (Copyright © 2016. Published by Elsevier Ireland Ltd.)

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). PDL-1 expression was detected in Myc-CaP murine prostate tumors growing in immune competent FVB/N and immune-deficient SCID mice. Endogenous CD3 + T cells were restricted from the centers of Myc-CaP tumor nodules growing in FVB/N mice. Following anti-programmed cell death protein 1 (PD-1) treatment, the restriction of CD3 + T cells was reversed, and a tumor-treatment response was observed. Adoptive hPSMA-CAR T cell immunotherapy was enhanced when combined with PD-1 blockade, but the treatment response was of comparatively short duration, suggesting other immune modulation mechanisms exist and restrict CAR T cell targeting, function, and persistence in hPSMA expressing Myc-CaP tumors. Interestingly, an "inverse pattern" of CAR T cell BLI intensity was observed in control and test tumors, which suggests CAR T cells undergo changes leading to a loss of signal and/or number following hPSMA-specific activation. The lower BLI signal intensity in the hPSMA test tumors (compared with controls) is due in part to a decrease in T cell mitochondrial function following T cell activation, which may limit the intensity of the ATP-dependent Luciferin-luciferase bioluminescence signal.

Increased polyamine synthesis is known to play an important role in prostate cancer. We aimed to explore its functional significance in prostate tumor initiation and its link to androgen receptor (AR) signaling. For this purpose, we generated a new cell line derived from normal epithelial prostate cells (RWPE-1) with overexpression of ornithine decarboxylase (ODC) and used it for in vitro and in vivo experiments. We then comprehensively analyzed the expression of the main metabolic enzymes of the polyamine pathway and spermine abundance in 120 well-characterized cases of human prostate cancer and high-grade prostate intraepithelial neoplasia (HGPIN). Herein, we show that the ODC-overexpressing prostate cells underwent malignant transformation, revealing that ODC is sufficient for de novo tumor initiation in 94% of injected mice. This oncogenic capacity was acquired through alteration of critical signaling networks, including AR, EIF2, and mTOR/MAPK. RNA silencing experiments revealed the link between AR signaling and polyamine metabolism. Human prostate cancers consistently demonstrated up-regulation of the main polyamine enzymes analyzed (ODC, polyamine oxidase, and spermine synthase) and reduction of spermine. This phenotype was also dominant in HGPIN, rendering it a new biomarker of malignant transformation. In summary, we report that ODC plays a key role in prostate tumorigenesis and that the polyamine pathway is altered as early as HGPIN., (Copyright © 2016 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

As people age, their performance on tasks requiring cognitive control often declines. Such a decline is frequently explained as either a general or specific decline in cognitive functioning with age. In the context of hypotheses suggesting a general decline, it is often proposed that processing speed generally declines with age. A further hypothesis is that an age-related compensation mechanism is associated with a specific cognitive decline. One prominent theory is the compensation hypothesis, which proposes that deteriorated functions are compensated for by higher performing functions. In this study, we used event-related potentials (ERPs) in the context of a GO/NOGO task to examine the age-related changes observed during cognitive control in a large group of healthy subjects aged between 18 and 84 years. The main question we attempted to answer was whether we could find neurophysiological support for either a general decline in processing speed or a compensation strategy. The subjects performed a relatively demanding cued GO/NOGO task with similar omissions and reaction times across the five age groups. The ERP waves of cognitive control, such as N2, P3cue and CNV, were decomposed into latent components by means of a blind source separation method. Based on this decomposition, it was possible to more precisely delineate the different neurophysiological and psychological processes involved in cognitive control. These data support the processing speed hypothesis because the latencies of all cognitive control ERP components increased with age, by 8 ms per decade for the early components (<200 ms) and by 20 ms per decade for the late components. At the same time, the compensatory hypothesis of aging was also supported, as the amplitudes of the components localized in posterior brain areas decreased with age, while those localized in the prefrontal cortical areas increased with age in order to maintain performance on this simple task at a relatively stable level.

Tumor hypoxia is a major cause of treatment failure for a variety of malignancies. However, hypoxia offers treatment opportunities, exemplified by the development of compounds that target hypoxic regions within tumors. Evofosfamide (TH-302) is a prodrug created by the conjugation of 2-nitroimidazole to bromo-isophosphoramide mustard (Br-IPM). When evofosfamide is delivered to hypoxic regions, the DNA cross-linking effector, Br-IPM, is released. This study assessed the cytotoxic activity of evofosfamide in vitro and its antitumor activity against osteolytic breast cancer either alone or in combination with paclitaxel in vivo. A panel of human breast cancer cell lines were treated with evofosfamide under hypoxia and assessed for cell viability. Osteolytic MDA-MB-231-TXSA cells were transplanted into the mammary fat pad, or into tibiae of mice, allowed to establish and treated with evofosfamide, paclitaxel, or both. Tumor burden was monitored using bioluminescence, and cancer-induced bone destruction was measured using micro-CT. In vitro, evofosfamide was selectively cytotoxic under hypoxic conditions. In vivo evofosfamide was tumor suppressive as a single agent and cooperated with paclitaxel to reduce mammary tumor growth. Breast cancer cells transplanted into the tibiae of mice developed osteolytic lesions. In contrast, treatment with evofosfamide or paclitaxel resulted in a significant delay in tumor growth and an overall reduction in tumor burden in bone, whereas combined treatment resulted in a significantly greater reduction in tumor burden in the tibia of mice. Evofosfamide cooperates with paclitaxel and exhibits potent tumor suppressive activity against breast cancer growth in the mammary gland and in bone., (© 2016 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.)

Intracellular tumor antigens presented on the cell surface in the context of human leukocyte antigen (HLA) molecules have been targeted by T cell-based therapies, but there has been little progress in developing small-molecule drugs or antibodies directed to these antigens. Here we describe a bispecific T-cell engager (BiTE) antibody derived from a T-cell receptor (TCR)-mimic monoclonal antibody (mAb) ESK1, which binds a peptide derived from the intracellular oncoprotein WT1 presented on HLA-A*02:01. Despite the very low density of the complexes at the cell surface, ESK1-BiTE selectively activated and induced proliferation of cytolytic human T cells that killed cells from multiple leukemias and solid tumors in vitro and in mice. We also discovered that in an autologous in vitro setting, ESK1-BiTE induced a robust secondary CD8 T-cell response specific for tumor-associated antigens other than WT1. Our study provides an approach that targets tumor-specific intracellular antigens without using cell therapy and suggests that epitope spreading could contribute to the therapeutic efficacy of this BiTE.

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-β-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., (© 2015 by the Society of Nuclear Medicine and Molecular Imaging, Inc.)

Tumor hypoxia is a major cause of treatment failure for a variety of malignancies. However, tumor hypoxia also offers treatment opportunities, exemplified by the development compounds that target hypoxic regions within tumors. TH-302 is a pro-drug created by the conjugation of 2-nitroimidazole to bromo-isophosphoramide (Br-IPM). When TH-302 is delivered to regions of hypoxia, Br-IPM, the DNA cross linking toxin, is released. In this study we assessed the cytotoxic activity of TH-302 against osteosarcoma cells in vitro and evaluated its anticancer efficacy as a single agent, and in combination with doxorubicin, in an orthotopic mouse model of human osteosarcoma (OS). In vitro, TH-302 was potently cytotoxic to osteosarcoma cells selectively under hypoxic conditions, whereas primary normal human osteoblasts were protected. Animals transplanted with OS cells directly into their tibiae and left untreated developed mixed osteolytic/osteosclerotic bone lesions and subsequently developed lung metastases. TH-302 reduced tumor burden in bone and cooperated with doxorubicin to protect bone from osteosarcoma induced bone destruction, while it also reduced lung metastases. TH-302 may therefore be an attractive therapeutic agent with strong activity as a single agent and in combination with chemotherapy against OS., (Crown Copyright © 2014. Published by Elsevier Ireland Ltd. All rights reserved.)

Background/aim: Drozitumab is a fully human agonistic monoclonal antibody that binds to death receptor DR5 and induces apoptosis. However, drozitumab resistance is a major obstacle limiting anticancer efficacy., Materials and Methods: We examined the potential for the chemotherapeutic agent doxorubicin to overcome resistance against drozitumab-resistant breast cancer cells both in vitro and in vivo., Results: Treatment with doxorubicin increased cell surface expression of DR5, reduced levels of Inhibitors of Apoptosis Proteins (cIAPs) and re-sensitised cells to drozitumab-induced apoptosis. Animals implanted with resistant breast cancer cells into the mammary fat pad and treated with a combination of drozitumab and doxorubicin showed inhibition of tumor growth and a substantial delay in tumor progression compared to untreated controls and mice treated with each agent alone., Conclusion: These results suggest that combination of drozitumab with chemotherapy and agents that modulate IAP levels could potentially be a useful strategy in the treatment of breast cancer., (Copyright© 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.)

Osteoprotegerin (OPG) is a secreted member of the TNF receptor superfamily, which binds to the receptor activator of nuclear factor κB ligand (RANKL) and inhibits osteoclast activity and bone resorption. Systemic administration of recombinant OPG was previously shown to inhibit tumor growth in bone and to prevent cancer-induced osteolysis. In this study, we examined the effect of OPG, when produced locally by breast cancer cells located within bone, using a mouse model of osteolytic breast cancer. MDA-MB-231-TXSA breast cancer cells, tagged with a luciferase reporter gene construct and engineered to overexpress full-length human OPG, were transplanted directly into the tibial marrow cavity of nude mice. Overexpression of OPG by breast cancer cells protected the bone from breast cancer-induced osteolysis and diminished intra-osseous tumor growth but had no effect on extra-skeletal tumor growth. This effect was associated with a significant reduction in the number of osteoclasts that lined the bone surface, resulting in a net increase in bone volume. Despite limiting breast cancer-mediated bone loss, OPG overexpression resulted in a significant increase in the incidence of pulmonary metastasis. Our results demonstrate that inhibition of osteoclastic bone resorption by OPG when secreted locally by tumors in bone may affect the behaviour of cancer cells within the bone microenvironment and their likelihood of spreading and establishing metastasis elsewhere in the body.

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.

Photodynamic therapy (PDT) is an established treatment modality for cancer. ADPM06 is an emerging non-porphyrin PDT agent which has been specifically designed for therapeutic application. Recently, we have demonstrated that ADPM06-PDT is well tolerated in vivo and elicits impressive complete response rates in various models of cancer when a short drug-light interval is applied. Herein, the mechanism of action of ADPM06-PDT in vitro and in vivo is outlined. Using a drug and light combination that reduces the clonogenicity of MDA-MB-231 cells by >90%, we detected a well-orchestrated apoptotic response accompanied by the activation of various caspases in vitro. The generation of reactive oxygen species (ROS) upon photosensitizer irradiation was found to be the key instigator in the observed apoptotic response, with the endoplasmic reticulum (ER) found to be the intracellular site of initial PDT damage, as determined by induction of a rapid ER stress response post-PDT. PDT-induced apoptosis was also found to be independent of p53 tumor suppressor status. A robust therapeutic response in vivo was demonstrated, with a substantial reduction in tumor proliferation observed, as well as a rapid induction of apoptosis and initiation of ER stress, mirroring numerous aspects of the mechanism of action of ADPM06 in vitro. Finally, using a combination of (18) F-labeled 3'-deoxy-3'-fluorothymidine ((18) F-FLT) nuclear and optical imaging, a considerable decrease in tumor proliferation over 24-hr in two models of human cancer was observed. Taken together, this data clearly establishes ADPM06 as an exciting novel PDT agent with significant potential for further translational development., (Copyright © 2011 UICC.)

Osteoprotegerin (OPG) is a secreted member of the tumor necrosis factor (TNF) receptor superfamily that binds to the ligand for receptor activator of nuclear factor κB (RANKL) and inhibits bone resorption. OPG can also bind and inhibit the activity of the TNF-related apoptosis-inducing ligand (Apo2L/TRAIL), raising the possibility that the anticancer efficacy of soluble Apo2L/TRAIL may be abrogated in the bone microenvironment where OPG expression is high. In this study we used a murine model of breast cancer growth in bone to evaluate the efficacy of recombinant soluble Apo2L/TRAIL against intratibial tumors that were engineered to overexpress native full-length human OPG. In vitro, OPG-overexpressing breast cancer cells were protected from Apo2L/TRAIL-induced apoptosis, an effect that was reversed with the addition of soluble RANKL or neutralizing antibodies to OPG. In vivo, mice injected intratibially with cells containing the empty vector developed large osteolytic lesions. In contrast, OPG overexpression preserved the integrity of bone and prevented breast cancer-induced bone destruction. This effect was due primarily to the complete absence of osteoclasts in the tibias of mice inoculated with OPG-transfected cells, confirming the biologic activity of the transfected OPG in vivo. Despite the secretion of supraphysiologic levels of OPG, treatment with Apo2L/TRAIL resulted in strong growth inhibition of both empty vector and OPG-overexpressing intratibial tumors. While Apo2L/TRAIL-induced apoptosis may be abrogated in vitro by OPG overexpression, the in vivo anticancer efficacy of recombinant soluble Apo2L/TRAIL is retained in the bone microenvironment even in the presence of biologically active OPG at supraphysiologic concentrations., (Copyright © 2011 American Society for Bone and Mineral Research.)

Unlabelled: In this article, we describe a series of new human-derived reporter genes based on human deoxycytidine kinase (dCK) suitable for clinical PET., Methods: Native dCK and its mutant reporter genes were tested in vitro and in vivo for their phosphorylation of pyrimidine- and acycloguanosine-based radiotracers including 2'-deoxy-2'-fluoroarabinofuranosylcytosine, 2'-fluoro-2'-deoxyarabinofuranosyl-5-ethyluracil (FEAU), penciclovir, and 9-[4-fluoro-3-(hydroxymethyl)butyl]guanine (FHBG) and clinically applied antiviral and anticancer drugs., Results: Cells transduced with dCK mutant reporter genes showed high in vitro and in vivo uptake of pyrimidine-based radiopharmaceuticals ((18)F-FEAU) comparable to that of herpes simplex virus type-1 thymidine kinase (HSV1-tk)-transduced cells. These mutants did not phosphorylate acycloguanosine-based radiotracers ((18)F-FHBG) or antiviral drugs (ganciclovir). Furthermore, the mutants displayed suicidal activation of clinically used pyrimidine-based prodrugs (cytarabine, gemcitabine)., Conclusion: The mutants of human dCK can be used as pyrimidine-specific PET reporter genes for imaging with (18)F-FEAU during treatment with acycloguanosine-based antiviral drugs. Additionally, the prosuicidal activity of these reporters with pyrimidine-based analogs will allow for the safe elimination of transduced cells.

The lymphatic system is an important pathway for tumor dissemination to the lymph nodes, but to which extent it contributes to the formation of distant metastases remains unknown. We report that induction of lymphangiogenesis by vascular endothelial growth factor-C (VEGF-C) at the secondary site, in the lung, facilitates expansion of already disseminated cancer cells throughout the lung tissue. By using orthotopic spontaneous metastasis models in nude mice, we show that VEGF-C expression by tumor cells altered the pattern of pulmonary metastases from nodular to diffuse and facilitated disease progression. Metastases expressing VEGF-C were tightly associated with the airways, in contrast to the control cells that were scattered in the lung parenchyma, throughout the alveolar region. VEGF-C induced lung lymphangiogenesis and promoted intralymphatic spread of metastases in the lung and formation of tumor emboli in the pulmonary arteries. This pattern of metastasis corresponds to lymphangitic carcinomatosis metastatic phenotype in human cancer patients, an extremely aggressive pattern of pulmonary metastases. In accordance, pulmonary breast cancer metastases from patients which were classified as lymphangitic carcinomatosis showed high levels of VEGF-C expression in cancer cells. These data show that VEGF-C promotes late steps of the metastatic process and identify the VEGF-C/VEGF receptor-3 pathway as the target not only for prevention of metastases, but also for treatment of established metastatic disease.