Current Clinical and Pre-Clinical Imaging Approaches to Study the Cancer-Associated Immune System.

In the light of the success and the expected growth of its arsenal, immuno-therapy may become the standard neoadjuvant procedure for many cancers in the near future. However, aspects such as the identity, organization and the activation status of the peri- and intra-tumoral immune cells would represent important elements to weigh in the decision for the appropriate treatment. While important progress in non-invasive imaging of immune cells has been made over the last decades, it falls yet short of entering the clinics, let alone becoming a standard procedure. Here, we provide an overview of the different intra-vital imaging approaches in the clinics and in pre-clinical settings and discuss their benefits and drawbacks for assessing the activity of the immune system, globally and on a cellular level. Stimulated by further research, the future is likely to see many technological advances both on signal detection and emission as well as image specificity and resolution to tackle current hurdles. We anticipate that the ability to precisely determine an immune stage of cancer will capture the attention of the oncologist and will create a change in paradigm for cancer therapy.

The functions of EZH2 in immune cells: Principles for novel immunotherapies.

Enhancer of zeste homolog 2 (EZH2) is aberrantly expressed or mutated in multiple types of cancer cells and plays an oncogenic role in tumorigenesis and development in most cancers. Results from pilot clinical studies have implied that EZH2 inhibitors have therapeutic potential against some cancers. However, the exact mechanisms by which EZH2 plays oncogenic roles and EZH2 inhibition exerts anticancer effects are incompletely understood. To date, the findings of studies focusing on EZH2 and cancer cells have failed to fully explain the observations in preclinical and clinical studies. Therefore, recent studies about the roles of EZH2 in cancers have shifted from cancer cells to immune cells. The human immune system is a complex network comprising multiple subpopulations of immune cells. Immune cells communicate and interact with cancer cells during cancer development and treatment, dictating the fate of cancer cells. Elucidating the roles of EZH2 in immune cells, especially in cancer patients, promises the identification of novel immunotherapeutic strategies or priming of existing immunotherapies against cancer. Hence, we reviewed the studies focusing on the involvement of EZH2 in various immune cells, aiming to provide ideas for immunotherapies targeting EZH2 in immune cells.

In vivo Imaging Technologies to Monitor the Immune System.

The past two decades have brought impressive advancements in immune modulation, particularly with the advent of both cancer immunotherapy and biologic therapeutics for inflammatory conditions. However, the dynamic nature of the immune response often complicates the assessment of therapeutic outcomes. Innovative imaging technologies are designed to bridge this gap and allow non-invasive visualization of immune cell presence and/or function in real time. A variety of anatomical and molecular imaging modalities have been applied for this purpose, with each option providing specific advantages and drawbacks. Anatomical methods including magnetic resonance imaging (MRI), computed tomography (CT), and ultrasound provide sharp tissue resolution, which can be further enhanced with contrast agents, including super paramagnetic ions (for MRI) or nanobubbles (for ultrasound). Conjugation of the contrast material to an antibody allows for specific targeting of a cell population or protein of interest. Protein platforms including antibodies, cytokines, and receptor ligands are also popular choices as molecular imaging agents for positron emission tomography (PET), single-photon emission computerized tomography (SPECT), scintigraphy, and optical imaging. These tracers are tagged with either a radioisotope or fluorescent molecule for detection of the target. During the design process for immune-monitoring imaging tracers, it is important to consider any potential downstream physiologic impact. Antibodies may deplete the target cell population, trigger or inhibit receptor signaling, or neutralize the normal function(s) of soluble proteins. Alternatively, the use of cytokines or other ligands as tracers may stimulate their respective signaling pathways, even in low concentrations. As in vivo immune imaging is still in its infancy, this review aims to describe the modalities and immunologic targets that have thus far been explored, with the goal of promoting and guiding the future development and application of novel imaging technologies.

Immunomodulatory effects and potential clinical applications of dimethyl sulfoxide.

Dimethyl sulfoxide (DMSO) was discovered during the 19th century by the German chemical industry. DMSO comprises a highly polar group and two non-polar domains, which render it soluble in both aqueous solutions and organic solutions. Furthermore, DMSO can penetrate the cell membrane of both the mammalian cells and the non-mammalian cells and prevent freeze-thaw injuries to the cells. Thus, it is frequently used for the cryopreservation of cells and tissues for laboratory and clinical applications. In contrast to this traditional application, DMSO has recently been shown to possess immunomodulatory effects, such as immune enhancement, and anti-inflammatory effects in the innate immunity. In addition, DMSO also affects the adaptive immunity by regulating the expression of transcription factors in immune cells. This review briefly summarizes and highlights the roles and immunomodulatory effects of DMSO on the immune system and reveals the future clinical therapeutic potential of DMSO treatment in cancer, in autoimmune diseases and in chronic inflammatory diseases.

Small-Animal PET/CT Imaging of Local and Systemic Immune Response Using 64Cu-αCD11b.

Current noninvasive imaging methods for monitoring immune response were largely developed for interrogation of the local reaction. This study developed the radiotracer 64Cu-labeled anti-CD11b (64Cu-αCD11b) for longitudinal assessment of local and systemic immune response involving mobilization of CD11b+ myeloid cells by small-animal PET/CT. Methods: Acute or chronic inflammation in the ears of BALB/c mice was induced by 12-o-tetradecanoylphorbol-13-acetate. Acute lung inflammation was induced by intratracheal lipopolysaccharide inoculation. αCD11b was conjugated with p-SCN-Bn-DOTA followed by labeling with 64Cu. PET/CT and biodistribution were evaluated at different times after intravenous injection of 64Cu-αCD11b. Cell populations from bone marrow (BM) and spleen were analyzed by flow cytometry. Results:64Cu-αCD11b was primarily taken up by BM and spleen in control mice. In comparison, 64Cu-αCD11b uptake was significantly reduced in the BM and spleen of CD11b-knockout mice, indicating that 64Cu-αCD11b selectively homed to CD11b+ myeloid cells in vivo. In mice with ear inflammation, for the local inflammatory response, 64Cu-αCD11b PET/CT revealed significantly higher 64Cu-αCD11b uptake in the inflamed ears in the acute inflammation phase than the chronic phase, consistent with markedly increased infiltration of CD11b+ cells into the inflammatory lesions at the acute phase. Moreover, imaging of 64Cu-αCD11b also showed the difference in mouse systemic response for different inflammatory stages. Compared with uptake in control mice, BM 64Cu-αCD11b uptake in mice with ear inflammation was significantly lower in the acute phase and higher in the chronic phase, reflecting an initial mobilization of CD11b+ cells from the BM to the inflammatory foci followed by a compensatory regeneration of CD11b+ myeloid cells in the BM. Similarly, in mice with lung inflammation, 64Cu-αCD11b PET/CT readily detected acute lung inflammation and recruitment of CD11b+ myeloid cells from the BM. Immunohistochemistry staining and flow cytometry results confirmed the noninvasive imaging of PET/CT. Conclusion:64Cu-αCD11b PET/CT successfully tracked ear and pulmonary inflammation in mice and differentiated acute from chronic inflammation at the local and systemic levels. 64Cu-αCD11b PET/CT is a robust quantitative method for imaging of local and systemic immune responses.

DIY: "Do Imaging Yourself" - Conventional microscopes as powerful tools for in vivo investigation.

Intravital imaging has been increasingly employed in cell biology studies and it is becoming one of the most powerful tools for in vivo investigation. Although some protocols can be extremely complex, most intravital imaging procedures can be performed using basic surgery and animal maintenance techniques. More importantly, regular confocal microscopes - the same that are used for imaging immunofluorescence slides - can also acquire high quality intravital images and movies after minor adaptations. Here we propose minimal adaptations in stock microscopes that allow major improvements in different fields of scientific investigation.

In-vivo imaging of tumor-infiltrating immune cells: implications for cancer immunotherapy.

Dynamic interactions between tumor cells and immune cells promote the initiation, progression, metastasis and therapy-resistance of cancer. With respect to immunotherapy, immune cell populations such as cytotoxic CD8+ T-cells, CD56+ NK cells and myeloid phagocytic cells play decisive roles. From an imaging perspective, the immune system displays unique challenges, which have implications for the design and performance of studies. The immune system comprises highly mobile cells that undergo distinct phases of development and activation. These cells circulate through several compartments during their active life span and accumulate in rather limited numbers in cancer lesion, where their effector phenotype further diversifies. Given these features, accurate evaluation of the tumor microenvironment and its cellular components during anti-cancer immunotherapy is challenging. In-vivo imaging currently offers quantitative and sensitive modalities that exploit long-lived tracers to interrogate, e.g. distinct immune cell populations, metabolic phenotypes, specific targets relevant for therapy or critical for their effector function. This review provides a comprehensive overview of current status for in-vivo imaging tumor-infiltrating immune cell populations, focusing on lymphocytes, NK cells and myeloid phagocytic cells, with emphasis on clinical translation.

Harnessing the potential of noninvasive in vivo preclinical imaging of the immune system: challenges and prospects.

Preclinical imaging has become a powerful method for investigation of in vivo processes such as pharmacokinetics of therapeutic substances and visualization of physiologic and pathophysiological mechanisms. These are important aspects to understand diseases and develop strategies to modify their progression with pharmacologic interventions. One promising intervention is the application of specifically tailored nanoscale particles that modulate the immune system to generate a tumor targeting immune response. In this complex interaction between immunomodulatory therapies, the immune system and malignant disease, imaging methods are expected to play a key role on the way to generate new therapeutic strategies. Here, we summarize examples which demonstrate the current potential of imaging methods and develop a perspective on the future value of preclinical imaging of the immune system.

Intravital imaging technology reveals immune system dynamics in vivo.

Fluorescent 'intravital' imaging is a new research technique by which the interior of living tissues and organs (in living bodies, if possible) can be observed, revealing the kinetics of cell and molecular processes in real time. Recent technological innovations in optical equipment and fluorescence imaging techniques have enabled a variety of cellular phenomena in different tissues and organs to be characterized under completely native conditions. This shift from static to dynamic biology constitutes the beginning of a new era in biomedical sciences.

The syndrome of degenerative calcific aortic stenosis: prevalence of multiple pathophysiologic disorders in association with valvular stenosis and their implications.

BACKGROUND: We hypothesized that degenerative calcific aortic stenosis (DCAS) is a syndrome influenced by factors beyond aortic valve stenosis (AS). The aim of this study was to assess how frequently DCAS is complicated by increased vascular load, systolic and/or diastolic left ventricular (LV) dysfunction, and comorbid disorders. METHODS: In 215 consecutive patients > 60 years of age with severe and moderate AS, we analyzed systemic arterial compliance, global hemodynamic load, LV ejection fraction (EF), the presence of diastolic dysfunction, and other valvular or systemic disorders. RESULTS: A total of 164 patients had severe AS and 51 had moderate AS. In patients with severe AS, the prevalence of increased vascular load was 42%; LV systolic and diastolic dysfunction was present in 27% and 42%; other valve diseases in 23%; and comorbid disorders in 82%. In the moderate AS group, abnormal vascular load was found in 52%; LV systolic and diastolic dysfunction was prevalent in 26% and 31%; other valve diseases in 17%; and comorbid disorders in 78% patients. More than half the patients in both groups had symptoms. In both severe and moderate AS groups, the prevalence of increased vascular load and systolic dysfunction was higher in the symptomatic group. CONCLUSION: Considerable number of patients with DCAS have abnormal vascular load, abnormal LV function, and significant coexisting disorders. These could influence the total pathophysiologic burden on the heart and symptom expression. Thus, DCAS should not be considered just as valvular stenosis, but a syndrome of DCAS because of the diagnostic, prognostic, and therapeutic implications of various factors associated with it.

PET imaging of the immune system: immune monitoring at the whole body level.

As newer immunotherapies are developed, the necessity to non-invasively and temporally assess the changes in the immune system will be more important. Currently, a variety of cytokine therapies, vaccines, adoptive cellular therapy, and immunoregulatory antibodies are being advanced in the preclinical and clinical arenas. These developments highlight the necessity to use non-invasive imaging techniques to follow the therapeutic site of action, duration of immune response and the response of the tumor. Positron emission tomography (PET) imaging has emerged as a flexible tool which allows the user to assess multiple aspects of the immune response, including the ability to monitor the primary and secondary immune response, particular effector subpopulations of the immune response, and with novel probes, to more selectively monitor the immune response versus the tumor. This review focuses on the use of PET imaging to monitor the dynamic, multicellular and distinct spatiotemporal aspects of immunotherapy for malignancy.

Imaging immune response in vivo: cytolytic action of genetically altered T cells directed to glioblastoma multiforme.

PURPOSE: Clinical trials have commenced to evaluate the feasibility of targeting malignant gliomas with genetically engineered CTLs delivered directly to the tumor bed in the central nervous system. The objective of this study is to determine a suite of magnetic resonance imaging (MRI) measurements using an orthotopic xenograft murine model that can noninvasively monitor immunologically mediated tumor regression and reactive changes in the surrounding brain parenchyma. EXPERIMENTAL DESIGN: Our preclinical therapeutic platform is based on CTL genetic modification to express a membrane tethered interleukin-13 (IL-13) cytokine chimeric T-cell antigen receptor. This enables selective binding and signal transduction on encountering the glioma-restricted IL-13 alpha2 receptor (IL-13Ralpha2). We used MRI to visualize immune responses following adoptive transfer of IL-13Ralpha2-specific CD8+ CTL clones. RESULTS: Based on MRI measurements, several phases following IL-13Ralpha2-specific T-cell adoptive transfer could be distinguished, all of which correlated well with glioblastoma regression confirmed on histology. The first detectable changes, 24 hours post-treatment, were significantly increased T2 relaxation times and strongly enhanced signal on T1-weighted postcontrast images. In the next phase, the apparent diffusion coefficient was significantly increased at 2 and 3 days post-treatment. In the last phase, at day 3 after IL-13Ralpha2-specific T-cell injection, the volume of hyperintense signal on T1-weighted postcontrast image was significantly decreased, whereas apparent diffusion coefficient remained elevated. CONCLUSIONS: The present study indicates the feasibility of MRI to visualize different phases of immune response when IL-13Ralpha2-specific CTLs are administered directly to the glioma tumor bed. This will further the aim of better predicting clinical outcome following immunotherapy.