Hyperpolarized 13C-Pyruvate to Assess Response to Anti-PD1 Immune Checkpoint Inhibition in YUMMER 1.7 Melanoma Xenografts.

There is currently no consensus to determine which advanced melanoma patients will benefit from immunotherapy, highlighting the critical need to identify early-response biomarkers to immune checkpoint inhibitors. The aim of this work was to evaluate in vivo metabolic spectroscopy using hyperpolarized (HP) 13C-pyruvate and 13C-glucose to assess early response to anti-PD1 therapy in the YUMMER1.7 syngeneic melanoma model. The xenografts showed a significant tumor growth delay when treated with two cycles of an anti-PD1 antibody compared to an isotype control antibody. 13C-MRS was performed in vivo after the injection of hyperpolarized 13C-pyruvate, at baseline and after one cycle of immunotherapy, to evaluate early dynamic changes in 13C-pyruvate-13C-lactate exchange. Furthermore, ex vivo 13C-MRS metabolic tracing experiments were performed after U-13C-glucose injection following one cycle of immunotherapy. A significant decrease in the ratio of HP 13C-lactate to 13C-pyruvate was observed in vivo in comparison with the isotype control group, while there was a lack of change in the levels of 13C lactate and 13C alanine issued from 13C glucose infusion, following ex vivo assessment on resected tumors. Thus, these results suggest that hyperpolarized 13C-pyruvate could be used to assess early response to immune checkpoint inhibitors in melanoma patients.

Manipulation of tumor oxygenation and radiosensitivity through modification of cell respiration. A critical review of approaches and imaging biomarkers for therapeutic guidance.

Tumor hypoxia has long been considered as a detrimental factor for the response to irradiation. In order to improve the sensitivity of tumors cells to radiation therapy, tumor hypoxia may theoretically be alleviated by increasing the oxygen delivery or by decreasing the oxygen consumption by tumor cells. Mathematical modelling suggested that decreasing the oxygen consumption should be more efficient than increasing oxygen delivery in order to alleviate tumor hypoxia. In this paper, we review several promising strategies targeting the mitochondrial respiration for which alleviation of tumor hypoxia and increase in sensitivity to irradiation have been demonstrated. Because the translation of these approaches into the clinical arena requires the use of pharmacodynamics biomarkers able to identify shift in oxygen consumption and tumor oxygenation, we also discuss the relative merits of imaging biomarkers (Positron Emission Tomography and Magnetic Resonance) that may be used for therapeutic guidance. This article is part of a Special Issue entitled Mitochondria in Cancer, edited by Giuseppe Gasparre, Rodrigue Rossignol and Pierre Sonveaux.

17 O MRS assesses the effect of mild hypothermia on oxygen consumption rate in tumors.

Although oxygen consumption is a key factor in metabolic phenotyping, its assessment in tumors remains critical, as current technologies generally display poor specificity. The objectives of this study were to explore the feasibility of direct 17 O nuclear magnetic resonance (NMR) spectroscopy to assess oxygen metabolism in tumors and its modulations. To investigate the impact of hypometabolism induction in the murine fibrosarcoma FSAII tumor model, we monitored the oxygen consumption of normothermic (37°C) and hypothermic (32°C) tumor-bearing mice. Hypothermic animals showed an increase in tumor pO2 (measured by electron paramagnetic resonance oximetry) contrary to normothermic animals. This was related to a decrease in oxygen consumption rate (assessed using 17 O magnetic resonance spectroscopy (MRS) after the inhalation of 17 O2 -enriched gas). This study highlights the ability of direct 17 O MRS to measure oxygen metabolism in tumors and modulations of tumor oxygen consumption rate.

Monitoring Combretastatin A4-induced tumor hypoxia and hemodynamic changes using endogenous MR contrast and DCE-MRI.

PURPOSE: To benchmark MOBILE (Mapping of Oxygen By Imaging Lipid relaxation Enhancement), a recent noninvasive MR method of mapping changes in tumor hypoxia, electron paramagnetic resonance (EPR) oximetry, and dynamic contrast-enhanced MRI (DCE-MRI) as biomarkers of changes in tumor hemodynamics induced by the antivascular agent combretastatin A4 (CA4). METHODS: NT2 and MDA-MB-231 mammary tumors were implanted subcutaneously in FVB/N and nude NMRI mice. Mice received 100 mg/kg of CA4 intraperitoneally 3 hr before imaging. The MOBILE sequence (assessing R1 of lipids) and the DCE sequence (assessing K(trans) hemodynamic parameter), were assessed on different cohorts. pO2 changes were confirmed on matching tumors using EPR oximetry consecutive to the MOBILE sequence. Changes in tumor vasculature were assessed using immunohistology consecutive to DCE-MRI studies. RESULTS: Administration of CA4 induced a significant decrease in lipids R1 (P = 0.0273) on pooled tumor models and a reduction in tumor pO2 measured by EPR oximetry. DCE-MRI also exhibited a significant drop of K(trans) (P < 0.01) that was confirmed by immunohistology. CONCLUSION: MOBILE was identified as a marker to follow a decrease in oxygenation induced by CA4. However, DCE-MRI showed a higher dynamic range to follow changes in tumor hemodynamics induced by CA4.

Impact of Oxygenation Status on 18F-FDG Uptake in Solid Tumors.

The influence of changes in tumor oxygenation (monitored by EPR oximetry) on the uptake of 18F-FDG tracer was evaluated using micro-PET in two different human tumor models. The 18F-FDG uptake was higher in hypoxic tumors compared to tumors that present a pO2 value larger than 10 mmHg.

Combined HP 13C Pyruvate and 13C-Glucose Fluxomic as a Potential Marker of Response to Targeted Therapies in YUMM1.7 Melanoma Xenografts.

A vast majority of BRAF V600E mutated melanoma patients will develop resistance to combined BRAF/MEK inhibition after initial clinical response. Resistance to targeted therapy is described to be accompanied by specific metabolic changes in melanoma. The aim of this work was to evaluate metabolic imaging using 13C-MRS (Magnetic Resonance Spectroscopy) as a marker of response to BRAF/MEK inhibition in a syngeneic melanoma model. Tumor growth was significantly delayed in mice bearing YUMM1.7 melanoma xenografts treated with the BRAF inhibitor vemurafenib, and/or with the MEK inhibitor trametinib, in comparison with the control group. 13C-MRS was performed in vivo after injection of hyperpolarized (HP) 13C-pyruvate, at baseline and 24 h after treatment, to evaluate dynamic changes in pyruvate-lactate exchange. Furthermore, ex vivo 13C-MRS steady state metabolic tracing experiments were performed after U-13C-glucose or 5-13C-glutamine injection, 24 h after treatment. The HP 13C-lactate-to-pyruvate ratio was not modified in response to BRAF/MEK inhibition, whereas the production of 13C-lactate from 13C-glucose was significantly reduced 24 h after treatment with vemurafenib, trametinib, or with the combined inhibitors. Conversely, 13C-glutamine metabolism was not modified in response to BRAF/MEK inhibition. In conclusion, we identified 13C-glucose fluxomic as a potential marker of response to BRAF/MEK inhibition in YUMM1.7 melanoma xenografts.

Targeting the bicarbonate transporter SLC4A4 overcomes immunosuppression and immunotherapy resistance in pancreatic cancer.

Solid tumors are generally characterized by an acidic tumor microenvironment (TME) that favors cancer progression, therapy resistance and immune evasion. By single-cell RNA-sequencing analysis in individuals with pancreatic ductal adenocarcinoma (PDAC), we reveal solute carrier family 4 member 4 (SLC4A4) as the most abundant bicarbonate transporter, predominantly expressed by epithelial ductal cells. Functionally, SLC4A4 inhibition in PDAC cancer cells mitigates the acidosis of the TME due to bicarbonate accumulation in the extracellular space and a decrease in lactate production by cancer cells as the result of reduced glycolysis. In PDAC-bearing mice, genetic or pharmacological SLC4A4 targeting improves T cell-mediated immune response and breaches macrophage-mediated immunosuppression, thus inhibiting tumor growth and metastases. In addition, Slc4a4 targeting in combination with immune checkpoint blockade is able to overcome immunotherapy resistance and prolong survival. Overall, our data propose SLC4A4 as a therapeutic target to unleash an antitumor immune response in PDAC.

Lactate Production Precedes Inflammatory Cell Recruitment in Arthritic Ankles: an Imaging Study.

PURPOSE: Inflammation is involved in many disease processes. However, accurate imaging tools permitting diagnosis and characterization of inflammation are still missing. As inflamed tissues exhibit a high rate of glycolysis, pyruvate metabolism may offer a unique approach to follow the inflammatory response and disease progression. Therefore, the aim of the study was to follow metabolic changes and recruitment of inflammatory cells after onset of inflammation in arthritic ankles using hyperpolarized 1-13C-pyruvate magnetic resonance spectroscopy (MRS) and 19F magnetic resonance imaging (MRI), respectively. PROCEDURE: Experimental rheumatoid arthritis (RA) was induced by intraperitoneal injection of glucose-6-phosphate-isomerase-specific antibodies (GPI) containing serum. To monitor pyruvate metabolism, the transformation of hyperpolarized 1-13C-pyruvate into hyperpolarized 1-13C-lactate was followed using MRS. To track phagocytic immune cell homing, we intravenously injected a perfluorocarbon emulsion 48 h before imaging. The animals were scanned at days 1, 3, or 6 after GPI-serum injection to examine the different stages of arthritic inflammation. Finally, to confirm the pyruvate metabolic activity and the link to inflammatory cell recruitment, we conducted hematoxylin-eosin histopathology and monocarboxylase transporter (MCT-1) immune histochemistry (IHC) of inflamed ankles. RESULTS: Hyperpolarized 1-13C-pyruvate MRS revealed a high rate of lactate production immediately at day 1 after GPI-serum transfer, which remained elevated during the progression of the disease, while 19F-MRI exhibited a gradual recruitment of phagocytic immune cells in arthritic ankles, which correlated well with the course of ankle swelling. Histopathology and IHC revealed that MCT-1 was expressed in regions with inflammatory cell recruitment, confirming the metabolic shift identified in arthritic ankles. CONCLUSIONS: Our study demonstrated the presence of a very early metabolic shift in arthritic joints independent of phagocytic immune cell recruitment. Thus, hyperpolarized 1-13C-pyruvate represents a promising tracer to monitor acute arthritic joint inflammation, even with minor ankle swelling. Furthermore, translated to the clinics, these methods add a detailed characterization of disease status and could substantially support patient stratification and therapy monitoring.

Biomarkers of tumour redox status in response to modulations of glutathione and thioredoxin antioxidant pathways.

The ability of certain cancer cells to maintain a highly reduced intracellular environment is correlated with aggressiveness and drug resistance. Since the glutathione (GSH) and thioredoxin (TRX) systems cooperate to a tight regulation of ROS in cell physiology, and to a stimulation of tumour initiation and progression, modulation of the GSH and TRX pathways are emerging as new potential targets in cancer. In vivo methods to assess changes in tumour redox status are critically needed to assess the relevance of redox-targeted agents. The current study assesses in vitro and in vivo biomarkers of tumour redox status in response to treatments targeting the GSH and TRX pathways, by comparing cytosolic and mitochondrial redox nitroxide electron paramagnetic resonance (EPR) probes, and cross-validation with redox dynamic fluorescent measurement. For that purpose, the effect of the GSH modulator buthionine sulfoximine (BSO) and of the TRX reductase inhibitor auranofin were measured in vitro using both cytosolic and mitochondrial EPR and roGFP probes in breast and cervical cancer cells. In vivo, mice bearing breast or cervical cancer xenografts were treated with the GSH or TRX modulators and monitored using the mito-TEMPO spin probe. Our data highlight the importance of using mitochondria-targeted spin probes to assess changes in tumour redox status induced by redox modulators. Further in vivo validation of the mito-tempo spin probe with alternative in vivo methods should be considered, yet the spin probe used in vivo in xenografts demonstrated sensitivity to the redox status modulators.

2'-deoxy-2'-[18F] fluoro-D-glucose positron emission tomography, diffusion-weighted magnetic resonance imaging, and choline spectroscopy to predict the activity of cetuximab in tumor xenografts derived from patients with squamous cell carcinoma of the head and neck.

We investigated changes on 2'-deoxy-2'-[18F]fluoro-D-glucose positron emission tomography (18FDG-PET), diffusion-weighted magnetic resonance imaging (DW-MRI), and choline spectroscopy as early markers of cetuximab activity in squamous cell carcinoma of the head and neck (SCCHN). SCCHN patient-derived tumor xenografts models were selected based on their cetuximab sensitivity. Three models were resistant to cetuximab and two were sensitive (one was highly sensitive and the other one was moderately sensitive). Cetuximab was infused on day 0 and 7. Maximal standardized uptake values (SUVmax), apparent diffusion coefficient (ADC), and total choline pool were measured at baseline and at day 8. To investigate the possible clinical relevance of our pre-clinical findings, we also studied the SUVmax and ADC modifications induced by cetuximab in five patients. Cetuximab induced a significant decrease in SUVmax and an increase in ADC at day 8 compared to baseline in the most cetuximab-sensitive model but not in the other models. At day 8, in one resistant model, SUVmax was decreased compared to baseline and was significantly lower than the controls. Choline spectroscopy was not able to predict cetuximab activity. The five patients treated with cetuximab had a 18FDG-PET partial response. One patient had a partial response according to RECISTv1.1. Interestingly, this last had also an increase in ADC value above 25%. Our preclinical data support the use of PDTX to investigate imaging techniques to detect early treatment response. Our pre-clinical and clinical data suggest that DW-MRI and 18FDG-PET should be further investigated to predict cetuximab activity.

The Blood Flow Shutdown Induced by Combretastatin A4 Impairs Gemcitabine Delivery in a Mouse Hepatocarcinoma.

In recent clinical studies, vascular disrupting agents (VDAs) are mainly used in combination with chemotherapy. However, an often overlooked concern in treatment combination is the VDA-induced impairment of chemotherapy distribution in the tumor. The work presented here investigated the impact of blood flow shutdown induced by Combretastatin A4 (CA4) on gemcitabine uptake into mouse hepatocarcinoma. At 2 h after CA4 treatment, using DCE-MRI, a significant decrease in the perfusion-relevant parameters Ktrans and Vp were observed in treated group compared with the control group. The blood flow shutdown was indeed confirmed by a histology study. In a third experiment, the total gemcitabine uptake was found to be significantly lower in treated tumors, as assessed in a separate experiment using ex vivo fluorine nuclear magnetic resonance spectroscopy. The amount of active metabolite gemcitabine triphosphate was also lower in treated tumors. In conclusion, the blood flow shutdown induced by VDAs can impact negatively on the delivery of small cytotoxic agents in tumors. The present study outlines the importance of monitoring the tumor vascular function when designing drug combinations.

Inhibition of the pentose phosphate pathway by dichloroacetate unravels a missing link between aerobic glycolysis and cancer cell proliferation.

Glucose fermentation through glycolysis even in the presence of oxygen (Warburg effect) is a common feature of cancer cells increasingly considered as an enticing target in clinical development. This study aimed to analyze the link between metabolism, energy stores and proliferation rates in cancer cells. We found that cell proliferation, evaluated by DNA synthesis quantification, is correlated to glycolytic efficiency in six cancer cell lines as well as in isogenic cancer cell lines. To further investigate the link between glycolysis and proliferation, a pharmacological inhibitor of the pentose phosphate pathway (PPP) was used. We demonstrated that reduction of PPP activity decreases cancer cells proliferation, with a profound effect in Warburg-phenotype cancer cells. The crucial role of the PPP in sustaining cancer cells proliferation was confirmed using siRNAs against glucose-6-phosphate dehydrogenase, the first and rate-limiting enzyme of the PPP. In addition, we found that dichloroacetate (DCA), a new clinically tested compound, induced a switch of glycolytic cancer cells to a more oxidative phenotype and decreased proliferation. By demonstrating that DCA decreased the activity of the PPP, we provide a new mechanism by which DCA controls cancer cells proliferation.

Multi-modality imaging to assess metabolic response to dichloroacetate treatment in tumor models.

Reverting glycolytic metabolism is an attractive strategy for cancer therapy as upregulated glycolysis is a hallmark in various cancers. Dichloroacetate (DCA), long used to treat lactic acidosis in various pathologies, has emerged as a promising anti-cancer drug. By inhibiting the pyruvate dehydrogenase kinase, DCA reactivates the mitochondrial function and decreases the glycolytic flux in tumor cells resulting in cell cycle arrest and apoptosis. We recently documented that DCA was able to induce a metabolic switch preferentially in glycolytic cancer cells, leading to a more oxidative phenotype and decreasing proliferation, while oxidative cells remained less sensitive to DCA treatment. To evaluate the relevance of this observation in vivo, the aim of the present study was to characterize the effect of DCA in glycolytic MDA-MB-231 tumors and in oxidative SiHa tumors using advanced pharmacodynamic metabolic biomarkers. Oxygen consumption, studied by 17O magnetic resonance spectroscopy, glucose uptake, evaluated by 18F-FDG PET and pyruvate transformation into lactate, measured using hyperpolarized 13C-magnetic resonance spectroscopy, were monitored before and 24 hours after DCA treatment in tumor bearing mice. In both tumor models, no clear metabolic shift was observed. Surprisingly, all these imaging parameters concur to the conclusion that both glycolytic tumors and oxidative tumors presented a similar response to DCA. These results highlight a major discordance in metabolic cancer cell bioenergetics between in vitro and in vivo setups, indicating critical role of the local microenvironment in tumor metabolic behaviors.

Multimodality Imaging Identifies Distinct Metabolic Profiles In Vitro and In Vivo.

The study of alterations of tumor metabolism should allow the identification of new targets for innovative anticancer strategies. Metabolic alterations are generally established in vitro, and conclusions are often extrapolated to the in vivo situation without further tumor metabolic phenotyping. To highlight the key role of microenvironment on tumor metabolism, we studied the response of glycolytic and oxidative tumor models to metabolic modulations in vitro and in vivo. MDA-MB-231 and SiHa tumor models, characterized in vitro as glycolytic and oxidative, respectively, were studied. Theoretically, when passing from a hypoxic state to an oxygenated state, a Warburg phenotype should conserve a glycolytic metabolism, whereas an oxidative phenotype should switch from glycolytic to oxidative metabolism (Pasteur effect). This challenge was applied in vitro and in vivo to evaluate the impact of different oxic conditions on glucose metabolism. 18F-fluorodeoxyglucose uptake, lactate production, tumor oxygenation, and metabolic fluxes were monitored in vivo using positron emission tomography, microdialysis, electron paramagnetic resonance imaging, and 13C-hyperpolarizated magnetic resonance spectroscopy, respectively. In vitro, MDA-MB-231 cells were glycolytic under both hypoxic and oxygenated conditions, whereas SiHa cells underwent a metabolic shift after reoxygenation. On the contrary, in vivo, the increase in tumor oxygenation (induced by carbogen challenge) led to a similar metabolic shift in glucose metabolism in both tumor models. The major discordance in metabolic patterns observed in vitro and in vivo highlights that any extrapolation of in vitro metabolic profiling to the in vivo situation should be taken cautiously and that metabolic phenotyping using molecular imaging is mandatory in vivo.

The increase in tumor oxygenation under carbogen breathing induces a decrease in the uptake of [(18)F]-fluoro-deoxy-glucose.

We investigated the impact of oxygenation status (measured by EPR oximetry) on the uptake of (18)F-FDG (measured by PET) in two different tumor models during a carbogen breathing challenge. We observed a significant drop in (18)F-FDG uptake under carbogen breathing that suggests a rapid metabolic adaptation to the oxygen environment.

Qualification of a noninvasive magnetic resonance imaging biomarker to assess tumor oxygenation.

PURPOSE: Although hypoxia has been long recognized as a crucial factor impairing tumor response in many therapeutic schemes, atraumatic and reliable methods of individually quantifying tumor oxygenation are still lacking in day-to-day clinical practice. The aim of this work was to investigate the potentially quantitative properties of our recently described noninvasive magnetic resonance (MR) technique "MOBILE" (mapping of oxygen by imaging lipids relaxation enhancement) and to qualify this endogenous contrast as a tumor hypoxia marker. EXPERIMENTAL DESIGN: The "MOBILE" technique, which assesses the longitudinal MR relaxation rate, R1, of lipid protons, was benchmarked with the parent technique which assesses the global (or water) R1, in response to a hyperoxic challenge (carbogen breathing) and to a hypoxic challenge (combretastatin A4) in MDA-MB-231 xenografts and in NT2 mammary tumors. Electron paramagnetic resonance (EPR) oximetry was used to quantitatively assess the tumor pO2 in matching tumors longitudinally. RESULTS AND CONCLUSION: Our study evidenced that (i) positive and negative changes in tumor oxygenation can be detected using MOBILE; (ii) a change in the R1 of lipids is positively correlated with a change in the tumor pO2 (P = 0.0217, r = 0.5097); (iii) measured lipid R1 values are positively correlated with absolute pO2 values in both tumor models (P = 0.0275, r = 0.3726); and (iv) changes in the R1 of lipids are more sensitive than changes in the global R1. As this technique presents unique translational properties, it seems promising for the individual longitudinal monitoring of tumor oxygenation in a clinical setting.