Silvia Valtorta

VEGF-Targeted Therapy Stably Modulates the Glycolytic Phenotype of Tumor Cells

Anti-VEGF therapy perturbs tumor metabolism, severely impairing oxygen, glucose, and ATP levels. In this study, we investigated the effects of anti-VEGF therapy in multiple experimental tumor models that differ in their glycolytic phenotypes to gain insights into optimal modulation of the metabolic features of this therapy. Prolonged treatments induced vascular regression and necrosis in tumor xenograft models, with highly glycolytic tumors becoming treatment resistant more rapidly than poorly glycolytic tumors. By PET imaging, prolonged treatments yielded an increase in both hypoxic and proliferative regions of tumors. A selection for highly glycolytic cells was noted and this metabolic shift was stable and associated with increased tumor aggressiveness and resistance to VEGF blockade in serially transplanted mice. Our results support the hypothesis that the highly glycolytic phenotype of tumor cells studied in xenograft models, either primary or secondary, is a cell-autonomous trait conferring resistance to VEGF blockade. The finding that metabolic traits of tumors can be selected by antiangiogenic therapy suggests insights into the evolutionary dynamics of tumor metabolism.

Comparison of 18F-Fluoroazomycin-Arabinofuranoside and 64Cu-Diacetyl-Bis(N4-Methylthiosemicarbazone) in Preclinical Models of Cancer

Hypoxic regions are present in different types of cancer and are a negative prognostic factor for disease progression and response to therapy. 18F-fluoroazomycin-arabinofuranoside (18F-FAZA) and 64Cu-diacetyl-bis(N4-methylthiosemicarbazone) (64Cu-ATSM) have been widely used to visualize hypoxic regions in preclinical and clinical studies. Although both these radioligands have high signal-to-noise ratios, 64Cu-ATSM may be suitable for use in in vivo imaging and as a radiotherapeutic agent. Despite encouraging results suggesting that it may have a role as a prognostic tracer, 64Cu-ATSM was recently shown to display cell line–dependent kinetics of oxygen-dependent uptake. We set out to evaluate the kinetics of 64Cu-ATSM distribution in different cancer models, using 18F-FAZA as the gold standard. Methods: 18F-FAZA and 64Cu-ATSM uptake were compared ex vivo using dual-tracer autoradiography and in vivo using PET in different xenograft mouse models (FaDu, EMT-6, and PC-3). 18F-FAZA uptake was compared with 64Cu-ATSM uptake in PET studies acquired at early (2 h after injection) and delayed time points (24 h after injection). To evaluate the presence of hypoxia and copper pumps, the tumors from animals submitted to PET were harvested and analyzed by an immunohistochemical technique, using antibodies against carbonic anhydrase IX (CAIX) and copper pumps (Ctr1 and ATP7B). Results: 64Cu-ATSM showed a higher tumor-to-muscle ratio than did 18F-FAZA. In the FaDu mouse model, radioactivity distribution profiles were overlapping irrespective of the hypoxic agent injected or the time of 64Cu acquisition. Conversely, in the EMT-6 and PC-3 models there was little similarity between the early and delayed 64Cu-ATSM images, and both the radiotracers showed a heterogeneous distribution. The microscopic analysis revealed that 18F-FAZA–positive areas were also positive for CAIX immunostaining whereas immunolocalization for copper pumps in the 3 models was not related to radioactivity distribution. Conclusion: The results of this study confirm the cell-dependent distribution and retention kinetics of 64Cu-ATSM and underline the need for proper validation of animal models and PET acquisition protocols before exploration of any new clinical applications.

The p53-p66Shc Apoptotic Pathway is Dispensable for Tumor Suppression whereas the p66Shc-generated Oxidative Stress Initiates Tumorigenesis

Reactive oxygen species (ROS) are regarded as hazardous by-products of mitochondrial respiration. In addition to the respiratory chain, specific ROS-generating systems have evolved. In particular, p66Shc is a mitochondrial redox protein that oxidizes cytochrome c to generate H2O2. Consistently, the deletion of p66Shc in cells and tissue results in reduced levels of ROS and oxidative stress. Taking advantage of the p66Shc knock out (p66KO) mouse model of decreased ROS production, we assessed the role of endogenously-produced ROS in tumorigenesis. Spontaneous tumor incidence was investigated and found unaltered in two different strains, 129Sv and C57Bl/6J, p66KO mice. In addition, papilloma formation upon exposure to ultraviolet radiation (UV) or 7,12-Dimethylbenz(a)anthracene/12-O-tetradecanoylphorbol- 13-acetate (DMBA/TPA) was found to be slightly lower in the absence of p66Shc. The role of p66Shc in tumorigenesis was also investigated in the absence of the tumor suppressor gene p53 (p53KO) by generating p53-p66Shc double knock out (DKO) mice. Notably, DKO mice displayed a significantly increased lifespan compared to p53KO mice. In addition, 2-deoxy-2-(18F)fluoro-D-glucose Positron Emission Tomography ([18F]FDG PET) analysis allowed to determine that disease onset occurred later in life in DKO mice compared to p53KO and that a low percentage of these mice did not develop tumors. Overall, these results indicate that although tumor incidence is not decreased in p66KO mice, p66Shc contributes to tumor initiation, in particular upon activation by carcinogens as well as when p53- mediated tumor suppression mechanisms defect.

Evaluation of the Role of Tumor-Associated Macrophages in an Experimental Model of Peritoneal Carcinomatosis Using 18F-FDG PET

PET is widely used at the clinical and preclinical levels for tumor assessment and evaluation of treatment efficacy. Here, we established and took advantage of a preclinical model of peritoneal carcinomatosis to evaluate the contribution of inflammatory infiltrating macrophages in tumor progression that was followed using 18F-FDG PET. Methods: Groups of mice with peritoneal carcinomatosis were longitudinally evaluated with 18F-FDG PET. Intraperitoneal depletion of macrophages was achieved by an approach (i.e., administration of clodronate encapsulated into liposomes) that proved to be safe and effective. Sham liposomes were used in control animal cohorts. Results: 18F-FDG PET allowed us to detect and monitor peritoneal lesion growth and diffusion. Macrophage-depleted animals showed a substantial reduction in tumor burden paralleled by a decrement in the extent of radioactivity distribution. A significant correlation between lesion dimension and metabolic volume was observed not only in macrophage-depleted but also in sham-treated mice. Conclusion: 18F-FDG PET allowed a noninvasive detection of peritoneal carcinomatosis lesions. Although macrophages play a key role in the early growth and spreading of lesions in the peritoneal cavity, neoplastic cells apparently represent the major player in this system in the uptake of 18F-FDG.

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.

Leukocytes recruited by tumor-derived HMGB1 sustain peritoneal carcinomatosis

ABSTRACT 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.

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.

Human malignant mesothelioma is recapitulated in immunocompetent BALB/c mice injected with murine AB cells

Malignant Mesothelioma is a highly aggressive cancer, which is difficult to diagnose and treat. Here we describe the molecular, cellular and morphological characterization of a syngeneic system consisting of murine AB1, AB12 and AB22 mesothelioma cells injected in immunocompetent BALB/c mice, which allows the study of the interplay of tumor cells with the immune system. Murine mesothelioma cells, like human ones, respond to exogenous High Mobility Group Box 1 protein, a Damage-Associated Molecular Pattern that acts as a chemoattractant for leukocytes and as a proinflammatory mediator. The tumors derived from AB cells are morphologically and histologically similar to human MM tumors, and respond to treatments used for MM patients. Our system largely recapitulates human mesothelioma, and we advocate its use for the study of MM development and treatment.

Divergent in vitro/in vivo responses to drug treatments of highly aggressive NIH-Ras cancer cells: a PET imaging and metabolomics-mass-spectrometry study

Oncogenic K-ras is capable to control tumor growth and progression by rewiring cancer metabolism. In vitro NIH-Ras cells convert glucose to lactate and use glutamine to sustain anabolic processes, but their in vivo environmental adaptation and multiple metabolic pathways activation ability is poorly understood. Here, we show that NIH-Ras cancer cells and tumors are able to coordinate nutrient utilization to support aggressive cell proliferation and survival. Using PET imaging and metabolomics-mass spectrometry, we identified the activation of multiple metabolic pathways such as: glycolysis, autophagy recycling mechanism, glutamine and serine/glycine metabolism, both under physiological and under stress conditions. Finally, differential responses between in vitro and in vivo systems emphasize the advantageous and uncontrolled nature of the in vivo environment, which has a pivotal role in controlling the responses to therapy.

Metabolic Evaluation of Non–Small Cell Lung Cancer Patient–Derived Xenograft Models Using 18F-FDG PET: A Potential Tool for Early Therapy Response

Lung cancer heterogeneity makes response to therapy extremely hard to predict. Patient-derived xenografts (PDXs) are a reliable preclinical model that closely recapitulates the main characteristics of the parental tumors and may represent a useful asset for testing new therapies. Here, using PET imaging, we investigated whether lung cancer PDXs reproduce the metabolic characteristics of the corresponding parental tumors. Methods: We performed longitudinal 18F-FDG PET studies on 9 different PDX groups obtained by implanting primary-cancer fragments harvested from patients into mice. The SUVmax of each PDX was calculated and compared with the SUVmax of the corresponding parental tumor. Results: Tumor growth rate and uptake varied among the different PDXs and confirmed the preservation of individual characteristics. The intragroup reproducibility of PET measurements was good. Furthermore, PDXs from tumors with a higher metabolic rate displayed a rank order of uptake similar to that of the parental tumors. Conclusion: PDXs reproduced the glucose metabolism of the parental tumors and therefore represent a promising preclinical model for the early assessment of therapy efficacy.