Arnaud Briat

Preclinical applications of imaging for cancer gene therapy.

Gene therapy is a very attractive strategy in experimental cancer therapy. Ideally, the approach aims to deliver therapeutic genes selectively to cancer cells. However, progress in the improvement of gene therapy formulations has been hampered by difficulties in measuring transgene delivery and in quantifying transgene expression in vivo. In clinical trials, endpoints rely almost exclusively on the analysis of biopsies by molecular and histopathological methods, which provide limited information. Therefore, to ensure the rational development of gene therapy, a crucial issue is the utilisation of technologies for the non-invasive monitoring of spatial and temporal gene expression in vivo upon administration of a gene delivery vector. Such imaging technologies would allow the generation of quantitative information about gene expression and the assessment of cancer gene therapy efficacy. In the past decade, progress has been made in the field of in vivo molecular imaging. This review highlights the various methods currently being developed in preclinical models.

Reduction of renal uptake of 111In-DOTA-labeled and A700-labeled RAFT-RGD during integrin αvβ3 targeting using single photon emission computed tomography and optical imaging

Integrin αvβ3 expression is upregulated during tumor growth and invasion in newly formed endothelial cells in tumor neovasculature and in some tumor cells. A tetrameric RGD‐based peptide, regioselectively addressable functionalized template‐(cyclo‐[RGDfK])4 (RAFT‐RGD), specifically targets integrin αvβ3 in vitro and in vivo. When labeled with indium‐111, the RAFT‐RGD is partially reabsorbed and trapped in the kidneys, limiting its use for further internal targeted radiotherapy and imaging investigations. We studied the effect of Gelofusine on RAFT‐RGD renal retention in tumor‐bearing mice. Mice were imaged using single photon emission computed tomography and optical imaging 1 and 24 h following tracer injection. Distribution of RAFT‐RGD was further investigated by tissue removal and direct counting of the tracer. Kidney sections were analyzed by confocal microscopy. Gelofusine significantly induced a >50% reduction of the renal reabsorption of 111In‐DOTA‐RAFT‐RGD and A700‐RAFT‐RGD, without affecting tumor uptake. Injection of Gelofusine significantly reduced the renal retention of labeled RAFT‐RGD, while increasing the tumor over healthy tissue ratio. These results will lead to the development of future therapeutic approaches. (Cancer Sci 2012; 103: 1105–1110)

Assessment of insulin resistance in fructose-fed rats with 125I-6-deoxy-6-iodo-D-glucose, a new tracer of glucose transport

PurposeInsulin resistance, characterised by an insulin-stimulated glucose transport defect, is an important feature of the pre-diabetic state that has been observed in numerous pathological disorders. The purpose of this study was to assess variations in glucose transport in rats using 125I-6-deoxy-6-iodo-D-glucose (6DIG), a new tracer of glucose transport proposed as an imaging tool to assess insulin resistance in vivo.MethodsTwo protocols were performed, a hyperinsulinaemic–euglycaemic clamp and a normoinsulinaemic–normoglycaemic protocol, in awake control and insulin-resistant fructose-fed rats. The tracer was injected at steady state, and activity in 11 tissues and the blood was assessed ex vivo at several time points. A multicompartmental mathematical model was developed to obtain fractional transfer coefficients of 6DIG from the blood to the organs.ResultsInsulin sensitivity of fructose-fed rats, estimated by the glucose infusion rate, was reduced by 40% compared with control rats. At steady state, 6DIG uptake was significantly stimulated by insulin in insulin-sensitive tissues of control rats (basal versus insulin: diaphragm, p < 0.01; muscle, p < 0.05; heart, p < 0.001), whereas insulin did not stimulate 6DIG uptake in insulin-resistant fructose-fed rats. Moreover, in these tissues, the fractional transfer coefficients of entrance were significantly increased with insulin in control rats (basal vs insulin: diaphragm, p < 0.001; muscle, p < 0.001; heart, p < 0.01) whereas no significant changes were observed in fructose-fed rats.ConclusionThis study sets the stage for the future use of 6DIG as a non-invasive means for the evaluation of insulin resistance by nuclear imaging.

A new transgenic mouse line to image chemically induced p53 activation in vivo

Monitoring p53 transcriptional activity to identify genotoxic damages induced by drugs has been proposed and validated in vitro. However, this methodology is by design limited to the cell line tested. In this study, we have fully validated a luciferase‐based p53‐reporter system in vitro and in vivo. We generated a mouse transgenic line to monitor non‐invasively p53 activation in response to chemically induced DNA damage. Doxorubicin was used as a drug of known toxicity to validate our model. Reporter gene expression was measured using bioluminescence imaging. In females, a weak p53 luciferase activity driven by a p53‐responsive promoter was detectable in the oral cavity region after doxorubicin treatment. In males, the signal increased in the lower abdominal region. Imaging of various organs revealed that the luciferase activity was mainly generated from the testes. Immunohistology demonstrated that the cells in the seminiferous tubules were damaged by the drug and confirmed that they were luciferase and p53 positive. Therefore, these transgenic mice could provide a powerful tool to predict, map and characterize at the organ and cellular levels the toxicity of compounds and help to develop new therapeutic agents in humans. (Cancer Sci 2008; 99: 683–688)

Antibody PEGylation in bioorthogonal pretargeting with trans-cyclooctene/tetrazine cycloaddition: in vitro and in vivo evaluation in colorectal cancer models

Bioorthogonal chemistry represents a challenging approach in pretargeted radioimmunotherapy (PRIT). We focus here on mAb modifications by grafting an increase amount of trans-cyclooctene (TCO) derivatives (0 to 30 equivalents with respect to mAb) bearing different polyethylene glycol (PEG) linkers between mAb and TCO (i.e. PEG0 (1), PEG4 (2) and PEG12 (3)) and assessing their functionality. We used colorectal xenograft (HT29/Ts29.2) and peritoneal carcinomatosis (A431-CEA-Luc/35A7) as tumor cells/mAbs models and fluorescent tetrazines (TZ). MALDI-TOF MS shows that grafting with 2,3 increases significantly the number of TCO per mAb compared with no PEG. In vitro immunofluorescence showed that Ts29.2 and 35A7 labeling intensity is correlated with the number of TCO when using 1,3 while signals reach a maximum at 10 equivalents when using 2. Under 10 equivalents conditions, the capacity of resulting mAbs-1–3 for antigen recognition is similar when reported per grafted TCO and comparable to mAbs without TCO. In vivo, on both models, pretargeting with mAbs-2,3 followed by TZ injection induced a fluorescent signal two times lower than with mAbs-1. These findings suggest that while PEG linkers allow a better accessibility for TCO grafting, it might decrease the number of reactive TCO. In conclusion, mAb-1 represents the best candidate for PRIT.

Tetraspanin 8 (TSPAN 8) as a potential target for radio-immunotherapy of colorectal cancer

Tetraspanin 8 (TSPAN8) overexpression is correlated with poor prognosis in human colorectal cancer (CRC). A murine mAb Ts29.2 specific for human TSPAN8 provided significant efficiency for immunotherapy in CRC pre-clinical models. We therefore evaluate the feasability of targeting TSPAN8 in CRC with radiolabeled Ts29.2. Staining of tissue micro-arrays with Ts29.2 revealed that TSPAN8 espression was restricted to a few human healthy tissues. DOTA-Ts29.2 was radiolabeled with 111In or 177Lu with radiochemical purities >95%, specific activity ranging from 300 to 600 MBq/mg, and radioimmunoreactive fractions >80%. The biodistribution of [111In]DOTA-Ts29.2 in nude mice bearing HT29 or SW480 CRC xenografts showed a high specificity of tumor localization with high tumor/blood ratios (HT29: 4.3; SW480-TSPAN8: 3.9 at 72h and 120h post injection respectively). Tumor-specific absorbed dose calculations for [177Lu]DOTA-Ts29.2 was 1.89 Gy/MBq, establishing the feasibility of using radioimmunotherapy of CRC with this radiolabeled antibody. A significant inhibition of tumor growth in HT29 tumor-bearing mice treated with [177Lu]DOTA-Ts29.2 was observed compared to control groups. Ex vivo experiments revealed specific DNA double strand breaks associated with cell apoptosis in [177Lu]DOTA-Ts29.2 treated tumors compared to controls. Overall, we provide a proof-of-concept for the use of [111In/177Lu]DOTA-Ts29.2 that specifically target in vivo aggressive TSPAN8-positive cells in CRC.