Cristina Martelli

In vivo imaging of immune cell trafficking in cancer

Tumour establishment, progression and regression can be studied in vivo using an array of imaging techniques ranging from MRI to nuclear-based and optical techniques that highlight the intrinsic behaviour of different cell populations in the physiological context. Clinical in vivo imaging techniques and preclinical specific approaches have been used to study, both at the macroscopic and microscopic level, tumour cells, their proliferation, metastasisation, death and interaction with the environment and with the immune system. Fluorescent, radioactive or paramagnetic markers were used in direct protocols to label the specific cell population and reporter genes were used for genetic, indirect labelling protocols to track the fate of a given cell subpopulation in vivo. Different protocols have been proposed to in vivo study the interaction between immune cells and tumours by different imaging techniques (intravital and whole-body imaging). In particular in this review we report several examples dealing with dendritic cells, T lymphocytes and macrophages specifically labelled for different imaging procedures both for the study of their physiological function and in the context of anti-neoplastic immunotherapies in the attempt to exploit imaging-derived information to improve and optimise anti-neoplastic immune-based treatments.

Cellular magnetic resonance with iron oxide nanoparticles: long-term persistence of SPIO signal in the CNS after transplanted cell death

AIM To study the specificity of cellular MRI based on superparamagnetic iron oxide particles (SPIOs), especially within the CNS. MATERIALS & METHODS A microglial cell line was engineered for the expression of a suicide gene, the receptor of diphtheria toxin (DT), and two reporter genes, green fluorescent protein and luciferase, in order to induce, in a controlled manner, cell death and test it through bioluminescence. SPIO-labeled DT-sensitive and control DT-insensitive cells were transplanted into the brains of mice, which underwent serial MRI and bioluminescence studies before and up to 90 days after DT-induced cell death. RESULTS No variations in SPIO signal voids were detected along longitudinal monitoring in brain hemispheres transplanted with DT-sensitive cells. Ex vivo analyses showed persistence of iron nanoparticle deposits at transplantation sites. CONCLUSION Due to the long-term persistence of signal after transplanted cell death, caution is advised when SPIOs are employed for cell tracking.

In vivo imaging of lymph node migration of MNP- and (111)In-labeled dendritic cells in a transgenic mouse model of breast cancer (MMTV-Ras).

PurposeThe authors present a protocol for the in vivo evaluation, using different imaging techniques, of lymph node (LN) homing of tumor-specific dendritic cells (DCs) in a murine breast cancer model.ProceduresBone marrow DCs were labeled with paramagnetic nanoparticles (MNPs) or 111In-oxine. Antigen loading was performed using tumor lysate. Mature DCs were injected into the footpads of transgenic tumor-bearing mice (MMTV-Ras) and DC migration was tracked by magnetic resonance imaging (MRI) and single-photon emission computed tomography (SPECT). Ex vivo analyses were performed to validate the imaging data.ResultsDC labeling, both with MNPs and with 111In-oxine, did not affect DC phenotype or functionality. MRI and SPECT allowed the detection of iron and 111In in both axillary and popliteal LNs. Immunohistochemistry and γ-counting revealed the presence of DCs in LNs.ConclusionsMRI and SPECT imaging, by allowing in vivo dynamic monitoring of DC migration, could further the development and optimization of efficient anti-cancer vaccines.

MiR675-5p Acts on HIF-1α to Sustain Hypoxic Responses: A New Therapeutic Strategy for Glioma.

Hypoxia is a common feature in solid tumours. In glioma, it is considered the major driving force for tumour angiogenesis and correlates with enhanced resistance to conventional therapies, increased invasiveness and a poor prognosis for patients. Here we describe, for the first time, that miR675-5p, embedded in hypoxia-induced long non-coding RNA H19, plays a mandatory role in establishing a hypoxic response and in promoting hypoxia-mediated angiogenesis. We demonstrated, in vitro and in vivo, that miR675-5p over expression in normoxia is sufficient to induce a hypoxic moreover, miR675-5p depletion in low oxygen conditions, drastically abolishes hypoxic responses including angiogenesis. In addition, our data indicate an interaction of miR675-5p, HIF-1α mRNA and the RNA Binding Protein HuR in hypoxia-induced responses. We suggest the modulation of miR675-5p as a new therapeutic option to promote or abolish hypoxia induced angiogenesis.

Validation of an engineered cell model for in vitro and in vivo HIF-1α evaluation by different imaging modalities.

PurposeThe aim of this study was to characterize a cell-based model for the molecular study of hypoxia-inducible factor (HIF)-1α activity, in the context of hypoxia, by means of different imaging techniques.ProceduresEngineered U251-HRE glioma cells were used to analyze the molecular mechanisms underlying HIF-1α activity in vitro in relation to luciferase expression. The same cells were orthotopically implanted in mice to evaluate tumor progression and hypoxia induction by bioluminescence imaging, fluorescence imaging, positron emission tomography (PET), and magnetic resonance imaging (MRI).ResultsIn vitro analyses highlighted the relationship between HIF-1α and luciferase activity in hypoxic conditions and after pharmacological treatments in U251-HRE cells. Through in vivo studies, it was possible to assess hypoxia establishment in relation to tumor growth by optical imaging, PET and MRI.ConclusionsThe findings of this study indicate that the U251-HRE orthotopic murine model can be used to reliably evaluate processes modulating HIF-1α activity, using both molecular and preclinical non-invasive imaging techniques.

Metformin and temozolomide, a synergic option to overcome resistance in glioblastoma multiforme models

Glioblastoma multiforme (GBM) is the most aggressive primary brain tumor with poor survival. Cytoreduction in association with radiotherapy and temozolomide (TMZ) is the standard therapy, but response is heterogeneous and life expectancy is limited. The combined use of chemotherapeutic agents with drugs targeting cell metabolism is becoming an interesting therapeutic option for cancer treatment. Here, we found that metformin (MET) enhances TMZ effect on TMZ-sensitive cell line (U251) and overcomes TMZ-resistance in T98G GBM cell line. In particular, combined-treatment modulated apoptosis by increasing Bax/Bcl-2 ratio, and reduced Reactive Oxygen Species (ROS) production. We also observed that MET associated with TMZ was able to reduce the expression of glioma stem cells (GSC) marker CD90 particularly in T98G cells but not that of CD133. In vivo experiments showed that combined treatment with TMZ and MET significantly slowed down growth of TMZ-resistant tumors but did not affect overall survival of TMZ-sensitive tumor bearing mice. In conclusion, our results showed that metformin is able to enhance TMZ effect in TMZ-resistant cell line suggesting its potential use in TMZ refractory GBM patients. However, the lack of effect on a GBM malignancy marker like CD133 requires further evaluation since it might influence response duration.

Immunological Characterization of Whole Tumour Lysate-Loaded Dendritic Cells for Cancer Immunotherapy

Introduction Dendritic cells play a key role as initiators of T-cell responses, and even if tumour antigen-loaded dendritic cells can induce anti-tumour responses, their efficacy has been questioned, suggesting a need to enhance immunization strategies. Matherials & Methods We focused on the characterization of bone marrow-derived dendritic cells pulsed with whole tumour lysate (TAA-DC), as a source of known and unknown antigens, in a mouse model of breast cancer (MMTV-Ras). Dendritic cells were evaluated for antigen uptake and for the expression of MHC class I/II and costimulatory molecules and markers associated with maturation. Results Results showed that antigen-loaded dendritic cells are characterized by a phenotypically semi-mature/mature profile and by the upregulation of genes involved in antigen presentation and T-cell priming. Activated dendritic cells stimulated T-cell proliferation and induced the production of high concentrations of IL-12p70 and IFN-γ but only low levels of IL-10, indicating their ability to elicit a TH1-immune response. Furthermore, administration of Antigen loaded-Dendritic Cells in MMTV-Ras mice evoked a strong anti-tumour response in vivo as demonstrated by a general activation of immunocompetent cells and the release of TH1 cytokines. Conclusion Data herein could be useful in the design of antitumoral DC-based therapies, showing a specific activation of immune system against breast cancer.

Optical imaging probes in oncology

Cancer is a complex disease, characterized by alteration of different physiological molecular processes and cellular features. Keeping this in mind, the possibility of early identification and detection of specific tumor biomarkers by non-invasive approaches could improve early diagnosis and patient management. Different molecular imaging procedures provide powerful tools for detection and non-invasive characterization of oncological lesions. Clinical studies are mainly based on the use of computed tomography, nuclear-based imaging techniques and magnetic resonance imaging. Preclinical imaging in small animal models entails the use of dedicated instruments, and beyond the already cited imaging techniques, it includes also optical imaging studies. Optical imaging strategies are based on the use of luminescent or fluorescent reporter genes or injectable fluorescent or luminescent probes that provide the possibility to study tumor features even by means of fluorescence and luminescence imaging. Currently, most of these probes are used only in animal models, but the possibility of applying some of them also in the clinics is under evaluation. The importance of tumor imaging, the ease of use of optical imaging instruments, the commercial availability of a wide range of probes as well as the continuous description of newly developed probes, demonstrate the significance of these applications. The aim of this review is providing a complete description of the possible optical imaging procedures available for the non-invasive assessment of tumor features in oncological murine models. In particular, the characteristics of both commercially available and newly developed probes will be outlined and discussed.

Identification of imaging biomarkers for the assessment of tumour response to different treatments in a preclinical glioma model

PurposeHypoxia-inducible factor 1α (HIF-1α) activity is one of the major players in hypoxia-mediated glioma progression and resistance to therapies, and therefore the focus of this study was the evaluation of HIF-1α modulation in relation to tumour response with the purpose of identifying imaging biomarkers able to document tumour response to treatment in a murine glioma model.MethodsU251-HRE-mCherry cells expressing Luciferase under the control of a hypoxia responsive element (HRE) and mCherry under the control of a constitutive promoter were used to assess HIF-1α activity and cell survival after treatment, both in vitro and in vivo, by optical, MRI and positron emission tomography imaging.ResultsThis cell model can be used to monitor HIF-1α activity after treatment with different drugs modulating transduction pathways involved in its regulation. After temozolomide (TMZ) treatment, HIF-1α activity is early reduced, preceding cell cytotoxicity. Optical imaging allowed monitoring of this process in vivo, and carbonic anhydrase IX (CAIX) expression was identified as a translatable non-invasive biomarker with potential clinical significance. A preliminary in vitro evaluation showed that reduction of HIF-1α activity after TMZ treatment was comparable to the effect of an Hsp90 inhibitor, opening the way for further elucidation of its mechanism of action.ConclusionThe results of this study suggest that the U251-HRE-mCherry cell model can be used for the monitoring of HIF-1α activity through luciferase and CAIX expression. These cells can become a useful tool for the assessment and improvement of new targeted tracers for potential theranostic procedures.

Development of a bicistronic vector for multimodality imaging of estrogen receptor activity in a breast cancer model: preliminary application

PurposeThe aim of this study was to develop a cellular model for the concurrent imaging of reporter genes expression by using positron emission tomography (PET) and bioluminescence imaging (BLI) for the assessment of estrogen receptor activity in vivo in a breast cancer model.MethodsTwo reporters were chosen: a mutated form of the dopaminergic D2 receptor (D2R80A) for PET imaging, and the Firefly Luciferase for BLI. The presence of an IRES sequence between the two reporters ensured the coordinated expression driven by the same regulatory sequence containing an estrogen responsive element (ERE). To prevent chromatin effects on reporter expression, the construct was flanked by insulator sequences (Matrix Attachment Region, MAR).ResultsIn vitro studies showed that the vector was efficient in coordinating the expression of the two genes. Moreover, stably transfected cells implanted in recipient animals maintained their capacity to express the reporters and react to systemic treatments permitting the in vivo study of ERs activity by PET and BLI imaging. In vitro expression analysis after long-term treatments showed different behaviour of the two reporter proteins in monitoring estrogen-dependent transcription outlining the importance of multi-reporter systems. With this model, PET and BLI can be applied to the concurrent evaluation of gene expression induced by estrogen and its analogues by using a bicistronic construct.ConclusionThe combined features of rapid, sensitive, sequential BLI and tomographic and quantitative PET imaging will allow the use of this strategy for the in vivo evaluation of molecular processes also for pharmacodynamic studies.