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Assessing Cell Trafficking By Noninvasive Imaging Techniques: Applications in Experimental Tumor Immunology

Posted on: Friday, 17 March 2006, 06:00 CST

By Ottobrini, L; Lucignani, G; Clerici, M; Rescigno, M

Tracer methods are increasingly being exploited to examine the trafficking patterns of cells transferred into recipient models of diseases, to optimize immune cell therapies, and to assess cancer gene therapy and vaccines in various cancer models. In animal cancer models, noninvasive monitoring by imaging tumor response could significantly facilitate the development of immune cell therapies against cancer. Currently, ex vivo lymphocyte labeling is primarily done by direct labeling. Major advances in cell labeling procedures have led to the use of reporter constructs to assess gene expression in vivo. With this novel technique, the reporter gene marks the cell with a specific protein that distinguishes the cell and its cellular progeny from other cells after migration, homing and mitosis. Several in vivo imaging procedures, including positron emission tomography, single photon emission tomography and magnetic resonance imaging, have been rescaled for studies in small animais. Other methods initially used for in vitro bioluminescence and fluorescence studies have also been refined for in vivo studies. When combined, these methods allow to assess cell trafficking in a noninvasive fashion, beyond lymphocyte response to inflammation, including metastatic diffusion and stem cell transplantation.

KEY WORDS: Neoplasms * Immunology * Medical imaging.

Radiotracer methods to detect inflammatory cell trafficking in response to infectious pathogens have been exploited for almost thirty years.1,2 The original concepts embedded in seminal research work on blood elements are now being applied to examine the trafficking patterns of cells transferred into recipient models of diseases. Since the cells responsible for immune response are highly mobile and can travel throughout the body in response to a variety of tissue and disease-specific signals, various approaches to monitoring their migration and homing have been pursued with two main aims: to study inflammation and infection3 and to detect graft rejection by monitoring the accumulation of immune cells at the transplanted organ.4-8

A better understanding of the trafficking patterns of immune cells has also become crucial for optimizing immune cell therapies. For example immune cell trafficking is imaged to assess cancer gene therapy and vaccines in various cancer models.9 In a recent study, de Vries et al. labeled dendritic cells from the blood of melanoma patients by using dextran-coated super paramagnetic iron oxide particles (SPIO) and indium-Ill oxine to monitor cellular therapy.10 In addition, hematopoietic stem cells and mature immune cells have been imaged trafficking through the lymphatic system and other tissues in small animals.11 12

The development of immune cell therapies against cancer may he significantly facilitated by imaging techniques for dynamic noninvasive monitoring of tumor response to such therapies in animal models of cancer.

Labeling of immunocompetent cells

Ex vivo labeling of lymphocytes has been primarily done by direct techniques based on radionuclide labeling with single photon emitting radiopharmaceuticals, including technetium-99m hexamethylpropylene amine oxime (^sup 99m^Tc-HMPAO ) and Indium-111 oxine.14-16 This strategy has been complemented by labeling with positron emitters.17, 18 Botti et al.17 used ^sup 99m^Tc-HMPAO, indium-Ill oxine and [^sup 18^F]2-fluoro-2-deoxy-D-glucose (^sup 18^F-FDG) to follow the migratory pattern of activated lymphocytes. Paik et al., improved the ^sup 18^F-FDG labeling of monocytes by incubating the cells in the presence of insulin.18 ^sup 64^Cu- pyruvaldehyde-bis(N4-methylthiosemicarbazone) (^sup 64^CuPTSM) has also been used to label ex vivo glioma cells and lymphocytes. The cells were labeled ex vivo, and their distribution was monitored using a dedicated positron emission tomograph in live animals.19 While cell trafficking can be monitored using ex vivo labeling of cells, where the radionuclide-labeled molecules are retained nonspecifically in the cells, this approach does not allow long- term monitoring of cell viability and proliferation in the body since the radiolabel may be lost owing to apoptosis or mitosis. This problem is particularly relevant for stem cell and immunocompetent cell monitoring during therapy, because, once introduced into the host, the cells need to be followed in their evolution, homing, transformation for several months. So new methods and technologies to assess the migration, survival, and function of antigen-specific T cells in vivo have been sought to elucidate migratory pathways of immune cells within the body and related migration patterns, chronology and immune response.

Reporter gene technology

Major advances in cell labeling procedures derived from recent developments in molecular biology have led to the use of reporter constructs to assess gene expression in vivo.20, 21 The reporter gene marks the cell with a specifically encoded protein that distinguishes the cell and its cellular progeny from other cells after migration, homing and mitosis. Reporter probes based on radionuclide or magnetic resonance imaging (MRI) or optical imaging technologies are able to bind or act as a substrate for the reporter protein (a receptor or an enzyme), thus allowing the detection of reporter gene-labeled cells. Introducing genetically encoded reporter genes into cells makes noninvasive, sequential imaging of cell trafficking possible in vivo. Methods to image genetically encoded reporters need a reporter gene (a gene that encodes an easily detectable protein that directly or indirectly generates a signal), a regulatory element governing reporter gene activity and a device for noninvasive detection of the signal that the reporter gene produces inside or on the surface of an intact cell or organism. The reporter may be an enzyme which catalyses a light- producing reaction or an acceptor which binds a radionuclide tracer with high affinity.

Technological developments and applications

Several in vivo imaging instruments currently used in human studies, e.g. positron emission tomography (PET), single photon emission tomography (SPET) and MRI, have been rescaled for studies in small animals, while other methods initially used for in vitro bioluminescence and fluorescence studies have also been refined for in vivo studies. In animal models of various types of cancer, cells labeled with reporter genes for magnetic resonance, emission tomography and optical imaging 22, 23 are now being increasingly used to assess cell trafficking in a noninvasive fashion, beyond lymphocyte response to inflammation, including metastatic diffusion and stem cell transplantation. These methods have also made it possible to monitor the viability, function and proliferation of labeled cells administered to host recipients and then to monitor cell migration in vivo. Optical imaging is based on fluorescence or bioluminescence. The major limitation to optical reporters is signal absorption and scattering as it passes through the tissues. As nearly 90% of the signal is attenuated per centimeter of tissue depth, the amount of photons detected may not be sufficient to assess signals arising from the innermost organs of even such small animals as the mouse. To overcome this, research has focused on generating reporters that can emit signals at wavelengths above 600 nm. Green fluorescent protein (GFP) and luciferase mutant variants have been created to shift emission closer to infrared wavelengths. Light-stimulated fluorescence of GFP is hampered by higher tissue scattering and absorption than that of bioluminescent reporters. This limits the use of GFP to very small organisms. Bioluminescent reporters such as luciferase generate photons in the presence of an appropriate enzymatic substrate. At present, optical imaging can be achieved with relatively low-cost instrumentation endowed with good sensitivity and temporal resolution. An alternative to optical imaging is PET and SPET technology, by which reporter expression is localized by radiotracers which bind specifically to the reporter protein or the enzyme itself. So far, the best reporters developed for PETbased imaging of small animals are the dopamine D2 receptor and the viral thymidine kinase. Efficient radiotracers are available for both. By combining the radionuclide approach to computed tomography (CT), three dimensional images can be generated with a resolution not yet achievable with optical imaging. Using HSVl- sr39tk as a reporter gene in adoptively transferred lymphocytes, Dubey et al.24 showed that T-cell antitumor response can be quantified using microPET. Similarly, Koehne et al.25 showed, by using microPET, that Epstein-Barr virus (EBV)-specific T lymphocytes marked with HSVl-tk migrate and accumulate in EBV^sup +^ tumors in mice. The researchers marked tumor-specific T lymphocytes ex vivo with retrovirally delivered HSVl-TK as the PET reporter gene and then used either 9-(4-[18F]-fluoro-3-hydroxymethylbutyl) guanine (FHBG) 24 or iodine labeled 2'-fluoro-2'-deoxy-l-beta-D- arabinofuranosyl-5-iodouracil (FIAU) 25 as the PET reporter probe. By monitoring the course and the extent of antigen-dependent tumor localization, investigators can determine the effect of a wide variety of modulatory factors on cellular the\rapy. These two papers are the first examples of applying noninvasive PET reporter gene imaging to analyze the time-dependent accumulation of antigen- specific T cells at a tumor site. The use of distinct cell systems emphasizes the potential of noninvasive imaging techniques to enhance our understanding of immune cell trafficking.26 Labeling cells with MR contrast agents permits the monitoring of transgenic expression 27 as well as cell migration and trafficking in vivo by MRI.28 MRI has been used for in vivo tracking of progenitor cells labeled with SPIO.29 Using magnetic resonance imaging and iron oxide nanoparticles, Kircher et al.30 repeatedly tracked adoptively transferred CD8+ cytotoxic T lymphocytes within tumors in mice.

In a slightly different approach to assess tumor cell trafficking, neoplastic cells can be transfected with a reporter gene and then implanted into recipient nude mice. The labeled tumor cells can then be tracked to follow local tumor growth and regression and metastatic diffusion, and to monitor the effects of therapeutic interventions.31-34 Another promising approach applied to imaging technology in live animals combines animal engineering and cell labeling by reporter genes. Direct labeling of bone marrow- derived cells is limited by the half-life and quantity of the labeling isotope. Reporter gene technology overcomes this limitation and allows a time-extended monitoring of stem cell engraftment. Recently, Cao et al. reported luciferase bioluminescence imaging of hematopoietic stem cells following transplantation into irradiated mice.35 Donor stem cells were derived from either a luciferase or a luciferase/GFP transgenic mouse bred to express the reporter gene(s) in every cell line. After systemic administration, the sites and kinetics of hematopoietic stem cell engraftment were detected by repeated optical imaging. In this perspective, a CD4^sup +^ T-cell subset mouse could be created by collecting the CD4^sup +^ T-cell subset from the bone marrow of a mouse engineered to ubiquitously express a reporter gene. The transplantation of the harvested T- cell subset into the marrow of an identical but reporter-negative mouse could originate a mouse in which CD4^sup +^ T-cells express the reporter gene but no other cell line in the mouse expressing it. This leads to the possibility of monitoring CD4^sup +^ T-cell behavior in the presence of a transplanted or a spontaneously growing tumor. Yu et al.36 demonstrated the real-time visualization of localization, survival and replication of engineered bacteria and vaccinia viruses in implanted tumors and their metastases in live animals. They hypothesized that a small number of microorganisms may enter the tumor through leaky vasculature, thus escaping the host's immunosurveillance. and find a protected environment in the tumor tissue. These systems may be applied to detect tumors and metastases and to develop tumor-specific gene therapy protocols. In another approach, Ponomarev et al.37 monitored TCR-mediated T-cell activation. Jurkat cells were transduced with a retroviral vector encoding a HSVl-TK-GFP fusion protein whose expression was driven by a NFAT-sensitive promoter. When stimulated in vivo by either tetradecanoyl phorbol acetate and ionomycin or anti-CD3, the cells expressed the HSVl-TK-GFP reporter. Subcutaneous xenografts of these tumors in nu/nu mice (which have no mature thymocytes) were relatively devoid of signal following racliolabeled FIAU injection. Following intravenous administration of anti-CD3 and anti-CD28 antibodies, however, a signal was easily revealed by the PET scanner. This is the first example of the use of PET reporter genes in the noninvasive measurement of T-cell activation in a live animal.37

Imaging of activation molecules

It would be of particular benefit to tumor immunology to image cells expressing specific activation molecules. For example, T-cell activation is induced by the presence of a peptide/major histocompatibility molecule complex (MHC) in the presence of co- stimulatory molecules on the surface of antigen-presenting cells (APC). The B7/CD28 family of co-stimulatory molecules plays a pivotal role in the development of optimal T-cell immunity as cross- linking of CD28 on the surface of T lymphocytes activates these cells.38,39 B7-1 (CD80) and B7-2 (CD86) are expressed on APC and engage CD28 or CTLA-4 with different affinity. The binding of these molecules is associated with different functional outcomes as triggering of CD28 has a stimulatory effect, whereas binding of CTLA- 4 is associated with dampening of T-cell activation.40,41 These interactions also play an important role in regulating T-helper differentiation. Hence, the interaction of CD80 or CD86 molecules with CD28 elicits TH2 response, whereas their interaction with CTLA- 4 inhibits TH2 response.40, 41 Besides B7-1 and B7-2, several other new members of the B7 family of costimulatory molecules have been described, among which B7-H1 and B7-H3 are emergent players. B7-H1 is constitutively present on monocytes and may be induced on activated T cells.42 B7-H1 co-stimulation strongly up-regulates IL- 10 production and reduces T-cell proliferation but has no effect on the generation of IL-2 and IL-4.43, 44 Several studies indicate a negative regulatory role for B7-H1 in T-cell responses. Liver, kidney and carcinoma cells express a high density of B7-H1 on their surfaces.45, 46 In this way, tumorspecific T lymphocytes recognize tumor antigens in the context of B7-H1. Their interaction promotes the secretion of IL-10, resulting in anergy and apoptosis of effector cytotoxic T lymphocytes (CTL).45, 46 Thus, the expression of B7-H1 on neoplastic cells actively inhibits immune response and favors neoplastic growth. Chemo- and radiotherapy are associated with a decreased percentage of circulating B7-H1-expressing cells; however, it is still impossible to quantify this marker's expression on the tumoral mass. Recent data have demonstrated that increased B7- H1 expression is also associated with other chronic conditions, including diabetes and HIV infection,47, 48 thus making this marker a key candidate for determining the prognosis of a number of human diseases. As the percentage of immune cells circulating in the blood is minimal (about 3%), it is clear that the development of a By-Hi- specific radiolabeled molecule could greatly enhance our diagnostic and prognostic ability for a wide range of diseases.

Conclusion and future outlook

The prospects for noninvasive reporter gene imaging as a prognostic and a therapeutic tool to analyze the time-dependent accumulation of antigen-specific T cells at a tumor site are promising. The ex vivo expansion of tumor epitopes-specific lymphocytes for radiolabeling would allow the production of T-cell lines of tumor-specific lymphocytes for in vivo detection of micrometastases. Loading these lymphocytes with toxins or apoptosis- inducing agents could create "magic bullets" targeted precisely at the tumoral cells to be eliminated. These exciting advances could be at hand once the technical problems have been solved.

The in vivo sequential monitoring of T-lymphocyte trafficking holds tremendous potential for the advancement of cellular immunotherapy in cancer. Moreover, multiple imaging modalities combined into one hybrid instrument to generate tomographic images having the high anatomic resolution of MRI or CT and the exquisite specificity and the flexibility of functional and molecular imaging that characterizes optical and radionuclide imaging will further improve the potential of this approach to cancer immunology. Advances in new radioactive, optical and MRI probes have been remarkable in recent times. However, new probes need to be developed for labeling specific reporterexpressing cells which can obtain information about the behavior of different cell populations. Immune cells genetically modified to express molecules may help to increase tumor localization of effector cells.49 The use of multiple constructs containing both the therapeutic and the reporter genes could help to evaluate the actual delivery of the therapeutic gene itself. This goal can be achieved by constructing fusion constructs to link the two genes or by bicistronic vectors. Another future possibility is to deliver two genes on two different vectors but each with the same promoter. The use of an inducible promoter driving the expression of the therapeutic and the reporter genes allows controlled gene regulation. Moreover, animals engineered to either constitutively or inclucibly express reporter gene(s) in all cells and tissues could serve as universal cell donors for cell transfer experiments in animal models of cancer.%-S3 A combined application of imaging systems, probes and animal engineering techniques will make it possible to study the dynamics of specific cell populations in transgenic animals. These developments represent extraordinary opportunities for applying imaging procedures and cell biology techniques to experimental tumor immunology.

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L. OTTOBRINI1, G. LLICIGNANI1, 2, M. CLERICI3, M. RESCIGNO4

1 Institute of Radiological Sciences,

University of Milan, Milan, Italy

2 Unit of Molecular Imaging

Division of Radiation Therapy

European Institute of Oncology, Milan, Italy

3 Chair of Immunology DISP LITA Vialba

University of Milan, Milan, Italy

4 Department of Experimental Oncology

European Institute of Oncology, Milan, Italy

Address reprint requests to: G. Lucignani, Unit of Molecular Imaging Division of Radiation Therapy, European Institute of Oncology, Via Ripamonti 435. 20141 Milan, Italy. E-mail: giovanni.lucignani@unimi.it

Copyright Edizioni Minerva Medica Dec 2005

Source: Quarterly Journal of Nuclear Medicine, The

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