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Glioblastoma (GBM) is a highly aggressive tumor of the brain. Despite the efforts, response to current therapies is poor and 2-years survival rate ranging from 6-12%. Here, we evaluated the preclinical efficacy of Metformin (MET) as add-on therapy to Temozolomide (TMZ) and the ability of [18F]FLT (activity of thymidine kinase 1 related to cell proliferation) and [18F]VC701 (translocator protein, TSPO) Positron Emission Tomography (PET) radiotracers to predict tumor response to therapy. Indeed, TSPO is expressed on the outer mitochondrial membrane of activated microglia/macrophages, tumor cells, astrocytes and endothelial cells. TMZ-sensitive (Gli36ΔEGFR-1 and L0627) or -resistant (Gli36ΔEGFR-2) GBM cell lines representative of classical molecular subtype were tested in vitro and in vivo in orthotopic mouse models. Our results indicate that in vitro, MET increased the efficacy of TMZ on TMZ-sensitive and on TMZ-resistant cells by deregulating the balance between pro-survival (bcl2) and pro-apoptotic (bax/bad) Bcl-family members and promoting early apoptosis in both Gli36ΔEGFR-1 and Gli36ΔEGFR-2 cells. In vivo, MET add-on significantly extended the median survival of tumor-bearing mice compared to TMZ-treated ones and reduced the rate of recurrence in the TMZ-sensitive models. PET studies with the cell proliferation radiopharmaceutical [18F]FLT performed at early time during treatment were able to distinguish responder from non-responder to TMZ but not to predict the duration of the effect. On the contrary, [18F]VC701 uptake was reduced only in mice treated with MET plus TMZ and levels of uptake negatively correlated with animals’ survival. Overall, our data showed that MET addition improved TMZ efficacy in GBM preclinical models representative of classical molecular subtype increasing survival time and reducing tumor relapsing rate. Finally, results from PET imaging suggest that the reduction of cell proliferation represents a common mechanism of TMZ and combined treatment, whereas only the last was able to reduce TSPO. This reduction was associated with the duration of treatment response. TSPO-ligand may be used as a complementary molecular imaging marker to predict tumor microenvironment related treatment effects.

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.

Purpose: In tumour, the imbalance between oxygen supply and demand leads to hypoxia, which represents a negative prognostic factor associated with aggressive tumour phenotype and therapy resistance. This review provides an overview of the use of positron emitter-labelled radiopharmaceutical used to image hypoxia in preclinical models of cancer. Methods: A critical and comprehensive PubMed search was performed identifying articles related to PET imaging for hypoxia assessment in preclinical setting from January 2007 up to January 2017. Results: We have considered and described a total of 54 original articles, exploring tumour-associated hypoxia in preclinical models. Results underlined the potential application together with the advantages and pitfalls of the use of PET in preclinical research. Multi-target imaging allowed to better define the relationship between hypoxia and other biological hallmarks of tumour; imaging of hypoxia was proved as a useful tool for lesions stratification and response prediction to radiotherapy; however, cutoff indexes were identified in few studies. Hypoxia PET showed remarkable tracer delivery limitations in the study of vascular disrupting agents but suggested the potential use of PET as a marker of response or resistance to anti-angiogenics. Finally, the effect of anaesthesia on tracer kinetics and tumour oxygenation as well as perfusion dependency in tracer uptake should be carefully evaluated to avoid artefactual results. Conclusions: Preclinical studies highlight the advantages and the limitations of the available hypoxia-radiotracers and their potential usefulness for the evaluation of treatments outcome and radiotherapy planning.

PURPOSE: Lung cancer heterogeneity makes response to therapy extremely hard to predict. Patient-derived xenografts (PDXs) represent a reliable preclinical model that closely recapitulates the main characteristics of the primary tumor and could represent a useful asset to test new therapies. Here, using PET imaging, we verify how lung cancer PDXs reproduce the metabolic features of the corresponding primary tumors. METHODS: We performed longitudinal [18F]FDG-PET studies on nine different PDXs, obtained by implants of primary cancer fragments harvested from patients. Max [18F]FDG uptake values of the lesion for each group were calculated and compared to corresponding patient's uptake. RESULTS: Different PDXs showed variable tumor growth rate and [18F]FDG uptake confirming the preservation of individual characteristics. A good intra-group reproducibility of PET measurements was observed. Furthermore, the subgroup of PDXs originating from primary tumors with higher metabolic rate displayed a rank order of [18F]FDG uptake similar to that of patients' original SUV. CONCLUSION: PDXs reproduced the original glucose metabolism of primary lesions and represent therefore a promising preclinical model also for the early assessment of therapy efficacy.

Rationale Pathological response to neo-adjuvant chemotherapy (NAC) represents a commonly used predictor of survival in triple negative breast cancer (TNBC) and the need to identify markers that predict response to NAC is constantly increasing. Aim of this study was to evaluate the potential usefulness of PET imaging with [18F]FDG and [18F]FLT for the discrimination of TNBC responders to Paclitaxel (PTX) therapy compared to the response assessed by an adapted Response Evaluation Criteria In Solid Tumors (RECIST) criteria based on tumor volume (Tumor Volume Response). Methods Nu/nu mice bearing TNBC lesions of different size were evaluated with [18F]FDG and [18F]FLT PET before and after PTX treatment. SUVmax, Metabolic Tumor Volume (MTV) and Total Lesion Glycolysis (TLG) and Proliferation (TLP) were assessed using a graph-based random walk algorithm. Results We found that in our TNBC model the variation of [18F]FDG and [18F]FLT SUVmax similarly defined tumor response to therapy and that SUVmax variation represented the most accurate parameter. Response evaluation using Tumor Volume Response (TVR) showed that the effectiveness of NAC with PTX was completely independent from lesions size at baseline. Conclusions Our study provided interesting results in terms of sensitivity and specificity of PET in TNBC, revealing the similar performances of [18F]FDG and [18F]FLT in the identification of responders to Paclitaxel.

The factors that determine whether disseminated transformed cells in vivo yield neoplastic lesions have only been partially identified. We established an ad hoc model of peritoneal carcinomatosis by injecting colon carcinoma cells in mice. Tumor cells recruit inflammatory leukocytes, mostly macrophages, and generate neoplastic peritoneal lesions. Phagocyte depletion via clodronate treatment reduces neoplastic growth. Colon carcinoma cells release a prototypic damage-associated molecular pattern (DAMP)/alarmin, High Mobility Group Box1 (HMGB1), which attracts leukocytes. Exogenous HMGB1 accelerates leukocyte recruitment, macrophage infiltration, tumor growth and vascularization. Lentiviral-based HMGB1 knockdown or pharmacological interference with its extracellular impair macrophage recruitment and tumor growth. Our findings provide a preclinical proof of principle that strategies based on preventing HMGB1-driven recruitment of leukocytes could be used for treating peritoneal carcinomatosis.

Purpose: Hypoxia-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. Methods: U251-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. Results: This 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. Conclusion: The 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.

Introduction The prognosis of malignant gliomas remains largely unsatisfactory for the intrinsic characteristics of the pathology and for the delayed diagnosis. Multimodal imaging based on PET and MRI may assess the dynamics of disease onset and progression allowing the validation of preclinical models of glioblastoma multiforme (GBM). The aim of this study was the characterization of a syngeneic rat model of GBM using combined in vivo imaging and immunohistochemistry. Methods Four groups of Fischer rats were implanted in a subcortical region with increasing concentration of rat glioma F98 cells and weekly monitored with Gd-MR, [ 18 F]FDG- and [ 18 F]FAZA-PET starting one week after surgery. Different targets were evaluated on post mortem brain specimens using immunohistochemistry: VEGF, GFAP, HIF-1α, Ki-67 and nestin. Results Imaging results indicated that tumor onset but not progression was related to the number of F98 cells. Hypoxic regions identified with [ 18 F]FAZA and high-glucose metabolism regions recognized with [ 18 F]FDG were located respectively in the core and in external areas of the tumor, with partial overlap and remodeling during disease progression. Histological and immunohistochemical analysis confirmed PET/MRI results and revealed that our model resumes biological characteristics of human GBM. IHC and PET studies showed that necrotic regions, defined on the basis of [ 18 F]FDG uptake reduction, may include hypoxic clusters of vital tumor tissue identified with [ 18 F]FAZA. This last information is particularly relevant for the identification of the target volume during image- guided radiotherapy . Conclusions In conclusion, the combined use of PET and MRI allows in vivo monitoring of the biological modification of F98 lesions during tumor progression.