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The high glycolytic activity of multiple myeloma (MM) cells is the rationale for use of Positron Emission Tomography (PET) with 18 F-fluorodeoxyglucose ([ 18 F]FDG) to detect both bone marrow (BM) and extramedullary disease. However, new tracers are actively searched because [ 18 F]FDG-PET has some limitations and there is a portion of MM patients who are negative. Glutamine (Gln) addiction has been recently described as a typical metabolic feature of MM cells. Yet, the possible exploitation of Gln as a PET tracer in MM has never been assessed so far and is investigated in this study in preclinical models. Firstly, we have synthesized enantiopure (2 S ,4 R )-4-fluoroglutamine (4-FGln) and validated it as a Gln transport analogue in human MM cell lines, comparing its uptake with that of 3 H-labelled Gln. We then radiosynthesized [ 18 F]4-FGln, tested its uptake in two different in vivo murine MM models, and checked the effect of Bortezomib, a proteasome inhibitor currently used in the treatment of MM. Both [ 18 F]4-FGln and [ 18 F]FDG clearly identified the spleen as site of MM cell colonization in C57BL/6 mice, challenged with syngeneic Vk12598 cells and assessed by PET. NOD.SCID mice, subcutaneously injected with human MM JJN3 cells, showed high values of both [ 18 F]4-FGln and [ 18 F]FDG uptake. Bortezomib significantly reduced the uptake of both radiopharmaceuticals in comparison with vehicle at post treatment PET. However, a reduction of glutaminolytic, but not of glycolytic, tumor volume was evident in mice showing the highest response to Bortezomib. Our data indicate that [ 18 F](2 S ,4 R )-4-FGln is a new PET tracer in preclinical MM models, yielding a rationale to design studies in MM patients., Competing Interests: NG received research funding and honoraria from Bristol Mayers Squibb, Celgene, Millenium Pharmaceutical, GSK, Takeda, and Janssen Pharmaceutical. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Valtorta, Toscani, Chiu, Sartori, Coliva, Brevi, Taurino, Grioni, Ruffini, Vacondio, Zanardi, Bellone, Moresco, Bussolati and Giuliani.)

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 [ 18 F]FLT (activity of thymidine kinase 1 related to cell proliferation) and [ 18 F]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 [ 18 F]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, [ 18 F]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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Valtorta, Lo Dico, Raccagni, Martelli, Pieri, Rainone, Todde, Zinnhardt, De Bernardi, Coliva, Politi, Viel, Jacobs, Galli, Ottobrini, Vaira and Moresco.)

Hypoxia is a key driver of tumor adaptation promoting tumor progression and resistance to therapy. Hypoxia related pathways might represent attractive targets for the treatment of Glioblastoma Multiforme (GBM), that up to date is characterized by a poor prognosis. Primary aim of this study was to investigate the role of hypoxia and hypoxia-related modifications in the effect of temozolomide (TMZ) given alone or in association with the antidiabetic agent Metformin (MET) or the PI3K/mTOR blocker, BEZ235. The study was conducted in the TMZ responsive U251 and resistant T98 GBM cells. Our results showed that during hypoxia, TMZ plus MET reduced viability of U251 cells affecting also CD133 and CD90 expressing cells. This effect was associated with a reduction of HIF-1α activity, VEGF release and AKT activation. In T98 TMZ-resistant cells, TMZ plus MET exerted similar effects on HIF-1α. However, in this cell line, TMZ plus MET failed to reduce CD133 positive cells and AKT phosphorylation. Nevertheless, the administration of the dual PI3K/mTOR inhibitor BEZ235 potentiated the effect of TMZ plus MET on cell viability, inducing a pro-apoptotic phenotype during hypoxic condition also in T98 cells, suggesting the block of the PI3K/AKT/mTOR pathway as a complementary target to further overcome GBM resistance during hypoxia. In conclusion, we proposed TMZ plus MET as suitable treatment to revert TMZ-resistance also during hypoxia, an effect potentiated by the inhibition of PI3K/mTOR axis.