In this paper, we showed that Cerenkov radiation (CR) escaping from the surface of small living animals injected with (18)F-FDG can be detected with optical imaging techniques. (18)F decays by emitting positrons with a maximum energy of 0.635 MeV; such positrons, when travelling into tissues faster than the speed of light in the same medium, are responsible of CR emission. A detailed model of the CR spectrum considering the positron energy spectrum was developed in order to quantify the amount of light emission. The results presented in this work were obtained using a commercial optical imager equipped with charged coupled detectors (CCD). Our data open the door to optical imaging (OI) in vivo of the glucose metabolism, at least in pre-clinical research. We found that the heart and bladder can be clearly identified in the animal body reflecting the accumulation of the (18)F-FDG. Moreover, we describe two different methods based on the spectral analysis of the CR that can be used to estimate the depth of the source inside the animal. We conclude that (18)F-FDG can be employed as it is as a bimodal tracer for positron emission tomography (PET) and OI techniques. Our results are encouraging, suggesting that it could be possible to apply the proposed approach not only to beta(+) but also to pure beta(-) emitters.

Cerenkov luminescence imaging is an emerging optical preclinical modality based on the detection of Cerenkov radiation induced by beta particles when traveling though biological tissues with a velocity greater than the speed of light. We present the first human Cerenkography obtained by detecting Cerenkov radiation escaping the thyroid gland of a patient treated for hyperthyroidism. The Cerenkov light was detected using an electron multiplied charge coupled device and a conventional C-mount lens. The system set-up has been tested by using a slab of ex vivo tissue equal to a 1 cm slice of chicken breast in order to simulate optical photons attenuation. We then imaged for 2 min the head and neck region of a patient treated orally 24 h before with 550 MBq of I-131. Co-registration between photographic and Cerenkov images showed a good localization of the Cerenkov light within the thyroid region. In conclusion, we showed that it is possible to obtain a planar image of Cerenkov photons escaping from a human tissue. Cerenkography is a potential novel medical tool to image superficial organs of patients treated with beta minus radiopharmaceuticals and can be extended to the imaging of beta plus emitters.

Extracellular vesicles (EVs) play a major role in cell-to-cell communication in physiological and pathological conditions, and their manipulation may represent a promising therapeutic strategy. Microglia, the parenchymal mononuclear phagocytes of the brain, modulate neighboring cells also through the release of EVs. The production of custom EVs filled with desired molecules, possibly targeted to make their uptake cell specific, and their administration in biological fluids may represent a valid approach for drug delivery. We engineered a murine microglia cell line, BV-2, to release EVs overexpressing the endogenous "eat me" signal Lactadherin (Mfg-e8) on the surface to target phagocytes and containing the anti-inflammatory cytokine IL-4. A single injection of 107 IL-4+Mfg-e8+ EVs into the cisterna magna modulated established neuroinflammation and significantly reduced clinical signs in the mouse model of multiple sclerosis, experimental autoimmune encephalomyelitis (EAE). Injected IL-4+Mfg-e8+ EVs target mainly phagocytes (i.e., macrophages and microglia) surrounding liquoral spaces, and their cargo promote the upregulation of anti-inflammatory markers chitinase 3-like 3 (ym1) and arginase-1 (arg1), significantly reducing tissue damage. Engineered EVs may represent a biological drug delivery tool able to deliver multiple functional molecules simultaneously to treat neuroinflammatory diseases.

Quite recently Cerenkov luminescence imaging (CLI) has been introduced as a novel pre-clinical imaging for the in vivo imaging of small animals such as mice. The CLI method is based on the detection of Cerenkov radiation (CR) generated by beta particles as they travel into the animal tissues with an energy such that Cerenkov emission condition is satisfied. This paper describes an image reconstruction method called multi spectral diffuse Cerenkov luminescence tomography (msCLT) in order to obtain 3D images from the detection of CR. The multispectral approach is based on a set of 2D planar images acquired using a number of narrow bandpass filters, and the distinctive information content at each wavelength is used in the 3D image reconstruction process. The proposed msCLT method was tested both in vitro and in vivo using ³²P-ATP and all the images were acquired by using the IVIS 200 small animal optical imager (Caliper Life Sciences, Alameda USA). Source depth estimation and spatial resolution measurements were performed using a small capillary source placed between several slices of chicken breast. The theoretical Cerenkov emission spectrum and optical properties of chicken breast were used in the modelling of photon propagation. In vivo imaging was performed by injecting control nude mice with 10 MBq of ³²P-ATP and the 3D tracer bio-distribution was reconstructed. Whole body MRI was acquired to provide an anatomical localization of the Cerenkov emission. The spatial resolution obtained from the msCLT reconstructed images of the capillary source showed that the FWHM is about 1.5 mm for a 6 mm depth. Co-registered MRI images showed that the Cerenkov emission regions matches fairly well with anatomical regions, such as the brain, heart and abdomen. Ex vivo imaging of the different organs such as intestine, brain, heart and ribs further confirms these findings. We conclude that in vivo 3D bio-distribution of a pure beta-minus emitting radiopharmaceutical such as ³²P-ATP can be obtained using the msCLT reconstruction approach.

Transfer of T-cell receptors (TCRs) specific for tumor-associated antigens is a promising approach for cancer immunotherapy. We developed the TCR gene editing technology that is based on the knockout of the endogenous TCR α and β genes, followed by the introduction of tumor-specific TCR genes, and that proved safer and more effective than conventional TCR gene transfer. Although successful, complete editing requires extensive cell manipulation and 4 transduction procedures. Here we propose a novel and clinically feasible TCR “single editing” (SE) approach, based on the disruption of the endogenous TCR α chain only, followed by the transfer of genes encoding for a tumor-specific TCR. We validated SE with the clinical grade HLA-A2 restricted NY-ESO-1_157-165–specific TCR. SE allowed the rapid production of high numbers of tumor-specific T cells, with optimal TCR expression and preferential stem memory and central memory phenotype. Similarly to unedited T cells redirected by TCR gene transfer (TCR transferred [TR]), SE T cells efficiently killed NY-ESO-1^pos targets; however, although TR cells proved highly alloreactive, SE cells showed a favorable safety profile. Accordingly, when infused in NSG mice previously engrafted with myeloma, SE cells mediated tumor rejection without inducing xenogeneic graft-versus-host disease, thus resulting in significantly higher survival than that observed in mice treated with TR cells. Overall, single TCR gene editing represents a clinically feasible approach that is able to increase the safety and efficacy of cancer adoptive immunotherapy. Key Points TCR SE is a clinically feasible approach to rapidly produce highly performing and specific tumor reactive T cells. NY-ESO-1 TCR SE T cells kill multiple myeloma in the absence of off-target reactivity including alloreactivity.