Discovery of a novel lactate dehydrogenase tetramerization domain using epitope mapping and peptides.

Despite being initially regarded as a metabolic waste product, lactate is now considered to serve as a primary fuel for the tricarboxylic acid cycle in cancer cells. At the core of lactate metabolism, lactate dehydrogenases (LDHs) catalyze the interconversion of lactate to pyruvate and as such represent promising targets in cancer therapy. However, direct inhibition of the LDH active site is challenging from physicochemical and selectivity standpoints. However, LDHs are obligate tetramers. Thus, targeting the LDH tetrameric interface has emerged as an appealing strategy. In this work, we examine a dimeric construct of truncated human LDH to search for new druggable sites. We report the identification and characterization of a new cluster of interactions in the LDH tetrameric interface. Using nanoscale differential scanning fluorimetry, chemical denaturation, and mass photometry, we identified several residues (E62, D65, L71, and F72) essential for LDH tetrameric stability. Moreover, we report a family of peptide ligands based on this cluster of interactions. We next demonstrated these ligands to destabilize tetrameric LDHs through binding to this new tetrameric interface using nanoscale differential scanning fluorimetry, NMR water-ligand observed via gradient spectroscopy, and microscale thermophoresis. Altogether, this work provides new insights on the LDH tetrameric interface as well as valuable pharmacological tools for the development of LDH tetramer disruptors.

Selective HIF stabilization alleviates hepatocellular steatosis and ballooning in a rodent model of 70% liver resection.

BACKGROUND: Small-for-size syndrome (SFSS) looms over patients needing liver resection or living-donor transplantation. Hypoxia has been shown to be crucial for the successful outcome of liver resection in the very early postoperative phase. While poorly acceptable as such in real-world clinical practice, hypoxia responses can still be simulated by pharmacologically raising levels of its transducers, the hypoxia-inducible factors (HIFs). We aimed to assess the potential role of a selective inhibitor of HIF degradation in 70% hepatectomy (70%Hx). METHODS: In a pilot study, we tested the required dose of roxadustat to stabilize liver HIF1α. We then performed 70%Hx in 8-week-old male Lewis rats and administered 25 mg/kg of roxadustat (RXD25) at the end of the procedure. Regeneration was assessed: ki67 and 5-ethynyl-2'-deoxyuridine (EdU) immunofluorescent labeling, and histological parameters. We also assessed liver function via a blood panel and functional gadoxetate-enhanced magnetic resonance imaging (MRI), up to 47 h after the procedure. Metabolic results were analyzed by means of RNA sequencing (RNAseq). RESULTS: Roxadustat effectively increased early HIF1α transactivity. Liver function did not appear to be improved nor liver regeneration to be accelerated by the experimental compound. However, treated livers showed a mitigation in hepatocellular steatosis and ballooning, known markers of cellular stress after liver resection. RNAseq confirmed that roxadustat unexpectedly increases lipid breakdown and cellular respiration. CONCLUSIONS: Selective HIF stabilization did not result in an enhanced liver function after standard liver resection, but it induced interesting metabolic changes that are worth studying for their possible role in extended liver resections and fatty liver diseases.

Metabolic imaging using hyperpolarized 13 C-pyruvate to assess sensitivity to the B-Raf inhibitor vemurafenib in melanoma cells and xenografts.

Nearly all melanoma patients with a BRAF-activating mutation will develop resistance after an initial clinical benefit from BRAF inhibition (BRAFi). The aim of this work is to evaluate whether metabolic imaging using hyperpolarized (HP) 13 C pyruvate can serve as a metabolic marker of early response to BRAFi in melanoma, by exploiting the metabolic effects of BRAFi. Mice bearing human melanoma xenografts were treated with the BRAFi vemurafenib or vehicle. In vivo HP 13 C magnetic resonance spectroscopy was performed at baseline and 24 hours after treatment to evaluate changes in pyruvate-to-lactate conversion. Oxygen partial pressure was measured via electron paramagnetic resonance oximetry. Ex vivo qRT-PCR, immunohistochemistry and WB analysis were performed on tumour samples collected at the same time-points selected for in vivo experiments. Similar approaches were applied to evaluate the effect of BRAFi on sensitive and resistant melanoma cells in vitro, excluding the role of tumour microenvironment. BRAF inhibition induced a significant increase in the HP pyruvate-to-lactate conversion in vivo, followed by a reduction of hypoxia. Conversely, the conversion was inhibited in vitro, which was consistent with BRAFi-mediated impairment of glycolysis. The paradoxical increase of pyruvate-to-lactate conversion in vivo suggests that such conversion is highly influenced by the tumour microenvironment.

Acetate: Friend or foe against breast tumour growth in the context of obesity?

Acetate is reported as a regulator of fat mass but also as lipogenic source for cancer cells. Breast cancer is surrounded by adipose tissue and has been associated with obesity. However, whether acetate contributes to cancer cell metabolism as lipogenic substrate and/or by changing fat storage and eventually obesity-induced breast cancer progression remains unknown. Therefore, we studied the contribution of acetate to breast cancer metabolism and progression. In vitro, we found that acetate is not a bioenergetic substrate under normoxia and did not result in a significant change of growth. However, by using lipidomic approaches, we discovered that acetate changes the lipid profiles of the cells under hypoxia. Moreover, while mice fed a high-fat diet (HFD) developed bigger tumours than their lean counterparts, exogenous acetate supplementation leads to a complete abolishment of fat mass gain without reverting the HFD-induced obesity-driven tumour progression. In conclusion, although acetate protects against diet-induced obesity, our data suggest that it is not affecting HFD-driven tumour progression.

Obesity and triple-negative-breast-cancer: Is apelin a new key target?

Epidemiological studies have shown that obese subjects have an increased risk of developing triple-negative breast cancer (TNBC) and an overall reduced survival. However, the relation between obesity and TNBC remains difficult to understand. We hypothesize that apelin, an adipokine whose levels are increased in obesity, could be a major factor contributing to both tumour growth and metastatization in TNBC obese patients. We observed that development of obesity under high-fat diet in TNBC tumour-bearing mice significantly increased tumour growth. By showing no effect of high-fat diet in obesity-resistant mice, we demonstrated the necessity to develop obesity-related disorders to increase tumour growth. Apelin mRNA expression was also increased in the subcutaneous adipose tissue and tumours of obese mice. We further highlighted that the reproduction of obesity-related levels of apelin in lean mice led to an increased TNBC growth and brain metastases formation. Finally, injections of the apelinergic antagonist F13A to obese mice significantly reduced TNBC growth, suggesting that apelinergic system interference could be an interesting therapeutic strategy in the context of obesity and TNBC.

Combined endogenous MR biomarkers to assess changes in tumor oxygenation induced by an allosteric effector of hemoglobin.

Hypoxia is a crucial factor in cancer therapy, determining prognosis and the effectiveness of treatment. Although efforts are being made to develop methods for assessing tumor hypoxia, no markers of hypoxia are currently used in routine clinical practice. Recently, we showed that the combined endogenous MR biomarkers, R1 and R2 *, which are sensitive to [dissolved O2 ] and [dHb], respectively, were able to detect changes in tumor oxygenation induced by a hyperoxic breathing challenge. In this study, we further validated the ability of the combined MR biomarkers to assess the change in tumor oxygenation induced by an allosteric effector of hemoglobin, myo-inositol trispyrophosphate (ITPP), on rat tumor models. ITPP induced an increase in tumor pO2 , as observed using L-band electron paramagnetic resonance oximetry, as well as an increase in both R1 and R2 * MR parameters. The increase in R1 indicated an increase in [O2 ], whereas the increase in R2 * resulted from an increase in O2 release from blood, inducing an increase in [dHb]. The impact of ITPP was then evaluated on factors that can influence tumor oxygenation, including tumor perfusion, saturation rate of hemoglobin, blood pH and oxygen consumption rate (OCR). ITPP decreased blood [HbO2 ] and significantly increased blood acidity, which is also a factor that right-shifts the oxygen dissociation curve. No change in tumor perfusion was observed after ITPP treatment. Interestingly, ITPP decreased OCR in both tumor cell lines. In conclusion, ITPP increased tumor pO2 via a combined mechanism involving a decrease in OCR and an allosteric effect on hemoglobin that was further enhanced by a decrease in blood pH. MR biomarkers could assess the change in tumor oxygenation induced by ITPP. At the intra-tumoral level, a majority of tumor voxels were responsive to ITPP treatment in both of the models studied.

Combined endogenous MR biomarkers to predict basal tumor oxygenation and response to hyperoxic challenge.

Hypoxia is a common feature of solid tumors, which translates into increased angiogenesis, malignant phenotype cell selection, change in gene expression and greater resistance to radiotherapy and chemotherapy. Therefore, there is a need for markers of hypoxia to stratify patients, in order to personalize treatment to improve therapeutic outcome. However, no modality has yet been validated for the screening of hypoxia in routine clinical practice. Magnetic resonance imaging (MRI) R1 and R2 * relaxation parameters are sensitive to tissue oxygenation: R1 is sensitive to dissolved oxygen and R2 * is sensitive to intravascular deoxyhemoglobin content. Two rat tumor models with distinct levels of hypoxia, 9L-glioma and rhabdomyosarcoma, were imaged for R1 and R2 * under air and carbogen (95% O2 and 5% CO2 ) breathing conditions. It was observed that the basal tumor oxygenation level had an impact on the amplitude of response to carbogen in the vascular compartment (R2 *), but not in the tissue compartment (R1 ). In addition, the change in tissue oxygenation estimated by ΔR1 correlated with the change in vascular oxygenation estimated by ΔR2 *, which is consistent with an increase in oxygen supply generating an elevated tumor pO2 . At the intra-tumoral level, we identified four types of voxel to which a hypoxic feature was attributed (mild hypoxia, severe hypoxia, normoxia and vascular steal), depending on the carbogen-induced change in R1 and R2 * values for each voxel. The results showed that 9L-gliomas present more normoxic fractions, whereas rhabdomyosarcomas present more hypoxic fractions, which is in accordance with a previous study using 18 F-fluoroazomycin arabinoside (18 F-FAZA) and electron paramagnetic resonance (EPR) oximetry. The response of the combined endogenous MRI contrasts to carbogen challenge could be a useful tool to predict different tumor hypoxic fractions.

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.

Exploring the Impact of Irradiation on Glioblastoma Blood-Brain-Barrier Permeability: Insights from Dynamic-Contrast-Enhanced-MRI and Histological Analysis.

(1) Background: Glioblastoma (GB) presents a formidable challenge in neuro-oncology due to its aggressive nature, limited treatment options, and poor prognosis. The blood-brain barrier (BBB) complicates treatment by hindering drug delivery to the tumor site, particularly to the infiltrative cells in the margin of the tumor, which are mainly responsible for tumor recurrence. Innovative strategies are therefore needed to enhance drug delivery in the margins of the tumor. This study explores whether irradiation can enhance BBB permeability by assessing hemodynamic changes and the distribution of contrast agents in the core and the margins of GB tumors. (2) Methods: Mice grafted with U-87MG cells were exposed to increasing irradiation doses. The distribution of contrast agents and hemodynamic parameters was evaluated using both non-invasive magnetic resonance imaging (MRI) techniques with gadolinium-DOTA as a contrast agent and invasive histological analysis with Evans blue, a fluorescent vascular leakage marker. Diffusion-MRI was also used to assess cytotoxic effects. (3) Results: The histological study revealed a complex dose-dependent effect of irradiation on BBB integrity, with increased vascular leakage at 5 Gy but reduced leakage at higher doses (10 and 15 Gy). However, there was no significant increase in the diffusion of Gd-DOTA outside the tumor area by MRI. (4) Conclusions: The increase in BBB permeability could be an interesting approach to enhance drug delivery in glioblastoma margins for low irradiation doses. In this model, DCE-MRI analysis was of limited value in assessing the BBB opening in glioblastoma after irradiation.

Highly Sensitive Detection of Melanin in Melanomas Using Multi-harmonic Low Frequency EPR.

PURPOSE: Low frequency EPR can noninvasively detect endogenous free radical melanin in melanocytic skin lesions and could potentially discriminate between benign atypical nevi and malignant melanoma lesions. We recently succeeded in demonstrating the ability of clinical EPR to noninvasively detect the endogenous melanin free radical in skin lesions of patients. However, the signal-to-noise ratio (SNR) was extremely low warranting further research to boost the sensitivity of detection. In the present study, we assessed the performance of a clinical EPR system with the capability to perform multi-harmonic (MH) analysis for the detection of melanin. PROCEDURES: The sensitivity of MH-EPR was compared with a classical continuous wave (CW)-EPR (1st harmonic) detection in vitro in melanin phantoms, in vivo in melanoma models with cells implanted in the skin, in lymph nodes and having colonized the lungs, and finally on phantoms placed at the surface of human skin. RESULTS: In vitro, we observed an increase in SNR by a factor of 10 in flat melanin phantoms when using MH analysis compared to CW combined with an increase in modulation amplitude. In B16 melanomas having grown in the skin of hairless mice, we observed a boost in sensitivity in vivo similar to that observed in vitro with the capability to detect melanoma cells at an earlier stage of development. MH-EPR was also able to detect non-invasively the melanin signal coming from melanoma cells present in lymph nodes as well as in lungs. We also observed a boost of sensitivity using phantoms of melanin placed at the surface of human skin. CONCLUSIONS: Overall, our results are paving the way for new clinical trials that will use MH clinical EPR for the characterization of pigmented skin lesions.

Sensitive simultaneous measurements of oxygenation and extracellular pH by EPR using a stable monophosphonated trityl radical and lithium phthalocyanine.

The monitoring of acidosis and hypoxia is crucial because both factors promote cancer progression and impact the efficacy of anti-cancer treatments. A phosphonated tetrathiatriarylmethyl (pTAM) has been previously described to monitor both parameters simultaneously, but the sensitivity to tackle subtle changes in oxygenation was limited. Here, we describe an innovative approach combining the pTAM radical and lithium phthalocyanine (LiPc) crystals to provide sensitive simultaneous measurements of extracellular pH (pHe) and pO2. Both parameters can be measured simultaneously as both EPR spectra do not overlap, with a gain in sensitivity to pO2 variations by a factor of 10. This procedure was applied to characterize the impact of carbogen breathing in a breast cancer 4T1 model as a proof-of-concept. No significant change in pHe and pO2 was observed using pTAM alone, while LiPc detected a significant increase in tumor oxygenation. Interestingly, we observed that pTAM systematically overestimated the pO2 compared to LiPc. In addition, we analyzed the impact of an inhibitor (UK-5099) of the mitochondrial pyruvate carrier (MPC) on the tumor microenvironment. In vitro, the exposure of 4T1 cells to UK-5099 for 24 h induced a decrease in pHe and oxygen consumption rate (OCR). In vivo, a significant decrease in tumor pHe was observed in UK-5099-treated mice, while there was no change for mice treated with the vehicle. Despite the change observed in OCR, no significant change in tumor oxygenation was observed after the UK-5099 treatment. This approach is promising for assessing in vivo the effect of treatments targeting tumor metabolism.

Assessment of Hyperosmolar Blood-Brain Barrier Opening in Glioblastoma via Histology with Evans Blue and DCE-MRI.

BACKGROUND: While the blood-brain barrier (BBB) is often compromised in glioblastoma (GB), the perfusion and consequent delivery of drugs are highly heterogeneous. Moreover, the accessibility of drugs is largely impaired in the margins of the tumor and for infiltrating cells at the origin of tumor recurrence. In this work, we evaluate the value of methods to assess hemodynamic changes induced by a hyperosmolar shock in the core and the margins of a tumor in a GB model. METHODS: Osmotic shock was induced with an intracarotid infusion of a hypertonic solution of mannitol in mice grafted with U87-MG cells. The distribution of fluorescent dye (Evans blue) within the brain was assessed via histology. Dynamic contrast-enhanced (DCE)-MRI with an injection of Gadolinium-DOTA as the contrast agent was also used to evaluate the effect on hemodynamic parameters and the diffusion of the contrast agent outside of the tumor area. RESULTS: The histological study revealed that the fluorescent dye diffused much more largely outside of the tumor area after osmotic shock than in control tumors. However, the study of tumor hemodynamic parameters via DCE-MRI did not reveal any change in the permeability of the BBB, whatever the studied MRI parameter. CONCLUSIONS: The use of hypertonic mannitol infusion seems to be a promising method to increase the delivery of compounds in the margins of GB. Nevertheless, the DCE-MRI analysis method using gadolinium-DOTA as a contrast agent seems of limited value for determining the efficacy of opening the BBB in GB after osmotic shock.

Towards Characterization of Skin Melanoma in the Clinic by Electron Paramagnetic Resonance (EPR) Spectroscopy and Imaging of Melanin.

The incidence of melanoma is continuously increasing over time. Melanoma is the most aggressive skin cancer, significantly reducing quality of life and survival rates of patients at advanced stages. Therefore, early diagnosis remains the key to change the prognosis of patients with melanoma. In this context, advanced technologies are under evaluation to increase the accuracy of the diagnostic, to better characterize the lesions and visualize their possible invasiveness in the epidermis. Among the innovative methods, because melanin is paramagnetic, clinical low frequency electron paramagnetic resonance (EPR) that characterizes the melanin content in the lesion has the potential to be an adjunct diagnostic method of melanoma. In this review, we first summarize the challenges faced by dermatologists and oncologists in melanoma diagnostic and management. We also provide a historical perspective on melanin detection with a focus on EPR spectroscopy/imaging of melanomas. We describe key elements that allow EPR to move from in vitro studies to in vivo and finally to patients for melanoma studies. Finally, we provide a critical view on challenges to meet to make EPR operational in the clinic to characterize pigmented lesions.

Mismatch between Bioluminescence Imaging (BLI) and MRI When Evaluating Glioblastoma Growth: Lessons from a Study Where BLI Suggested "Regression" while MRI Showed "Progression".

Orthotopic glioblastoma xenografts are paramount for evaluating the effect of innovative anti-cancer treatments. In longitudinal studies, tumor growth (or regression) of glioblastoma can only be monitored by noninvasive imaging. For this purpose, bioluminescence imaging (BLI) has gained popularity because of its low cost and easy access. In the context of the development of new nanomedicines for treating glioblastoma, we were using luciferase-expressing GL261 cell lines. Incidentally, using BLI in a specific GL261 glioblastoma model with cells expressing both luciferase and the green fluorescent protein (GL261-luc-GFP), we observed an apparent spontaneous regression. By contrast, the magnetic resonance imaging (MRI) analysis revealed that the tumors were actually growing over time. For other models (GL261 expressing only luciferase and U87 expressing both luciferase and GFP), data from BLI and MRI correlated well. We found that the divergence in results coming from different imaging modalities was not due to the tumor localization nor the penetration depth of light but was rather linked to the instability in luciferase expression in the viral construct used for the GL261-luc-GFP model. In conclusion, the use of multi-modality imaging prevents possible errors in tumor growth evaluation, and checking the stability of luciferase expression is mandatory when using BLI as the sole imaging modality.

Quantitative MR estimates of blood oxygenation based on T2*: a numerical study of the impact of model assumptions.

Several MR methods have been proposed over the last decade to obtain quantitative estimates of the tissue blood oxygen saturation (StO2) using a quantification of the blood oxygen level dependent effect. These approaches are all based on mathematical models describing the time evolution of the MR signal in biological tissues in the presence of magnetic field inhomogeneities. Although the experimental results are very encouraging, possible biases induced by the model assumptions have not been extensively studied. In this study, a numerical approach was used to examine the influence on T(2)*, blood volume fraction, and StO2 estimates of possible confounding factors such as water diffusion, intravascular signal, and presence of arterial blood in the voxel. To evaluate the impact of the vessel geometry, straight cylinders and realistic data from two-photon microscopy for microvascular geometry were compared. Our results indicate that the models are sufficiently realistic, based on a good correlation between ground truth and MR estimates of StO2.

Evaluation of Syrosingopine, an MCT Inhibitor, as Potential Modulator of Tumor Metabolism and Extracellular Acidification.

Extracellular acidification has been shown to be an important characteristic of invasive tumors, as it promotes invasion and migration but also resistance to treatments. Targeting transporters involved in the regulation of tumor pH constitutes a promising anti-tumor approach, as it would disrupt cellular pH homeostasis and negatively impact tumor growth. In this study, we evaluated the impact of syrosingopine, an inhibitor of MCT1 and MCT4, as a modulator of tumor metabolism and extracellular acidification in human breast cancer (MDA-MB-231) and pharyngeal squamous cell carcinoma (FaDu) cell models. In both models in vitro, we observed that exposure to syrosingopine led to a decrease in the extracellular acidification rate, intracellular pH, glucose consumption, lactate secretion and tumor cell proliferation with an increase in the number of late apoptotic/necrotic cells. However, in vivo experiments using the MDA-MB-231 model treated with a daily injection of syrosingopine did not reveal any significant change in extracellular pH (pHe) (as measured using CEST-MRI) or primary tumor growth. Overall, our study suggests that targeting MCT could lead to profound changes in tumor cell metabolism and proliferation, and it warrants further research to identify candidates without off-target effects.

Noninvasive detection of the endogenous free radical melanin in human skin melanomas using electron paramagnetic resonance (EPR).

We explored the capability of low-frequency Electron Paramagnetic Resonance (EPR) to noninvasively detect melanin (a stable semiquinone free radical) in the human skin. As previous in vitro studies on biopsies suggested that the EPR signal from melanin was different when measured in skin melanomas or benign nevi, we conducted a prospective first-in-man clinical EPR study in patients with skin lesions suspicious of melanoma. EPR spectra were obtained using a spectrometer operating at 1 GHz, with a surface coil placed over the area of interest. Two clinical studies were carried out: 1) healthy volunteers (n = 45) presenting different skin phototypes; 2) patients (n = 88) with skin lesions suspicious of melanoma (n = 100) requiring surgical resection. EPR data obtained before surgery were compared with histopathology results. The method was not sensitive enough to measure differences in melanin content due to changes in skin pigmentation. In patients, 92% of the spectra were analyzable. The EPR signal of melanin was significantly higher (p < 0.0001) in melanoma lesions (n = 26) than that in benign atypical nevi (n = 62). A trend toward a higher signal intensity (though not significant) was observed in high Breslow depth melanomas (a marker of skin invasion) than in low Breslow lesions. To date, no naturally occurring free radicals have been detected by low-frequency EPR systems adapted for clinical studies. Here, we demonstrated for the first time the ability of this technology to detect an endogenous free radical, opening new avenues for evaluating clinical EPR as a potential aid in the diagnosis of pigmented skin lesions.

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.

Impact of Inhibition of the Mitochondrial Pyruvate Carrier on the Tumor Extracellular pH as Measured by CEST-MRI.

(1) Background: The acidosis of the tumor micro-environment may have profound impact on cancer progression and on the efficacy of treatments. In the present study, we evaluated the impact of a treatment with UK-5099, a mitochondrial pyruvate carrier (MPC) inhibitor on tumor extracellular pH (pHe); (2) Methods: glucose consumption, lactate secretion and extracellular acidification rate (ECAR) were measured in vitro after exposure of cervix cancer SiHa cells and breast cancer 4T1 cells to UK-5099 (10 µM). Mice bearing the 4T1 tumor model were treated daily during four days with UK-5099 (3 mg/kg). The pHe was evaluated in vivo using either chemical exchange saturation transfer (CEST)-MRI with iopamidol as pHe reporter probe or 31P-NMR spectroscopy with 3-aminopropylphosphonate (3-APP). MR protocols were applied before and after 4 days of treatment; (3) Results: glucose consumption, lactate release and ECAR were increased in both cell lines after UK-5099 exposure. CEST-MRI showed a significant decrease in tumor pHe of 0.22 units in UK-5099-treated mice while there was no change over time for mice treated with the vehicle. Parametric images showed a large heterogeneity in response with 16% of voxels shifting to pHe values under 7.0. In contrast, 31P-NMR spectroscopy was unable to detect any significant variation in pHe; (4) Conclusions: MPC inhibition led to a moderate acidification of the extracellular medium in vivo. CEST-MRI provided high resolution parametric images (0.44 µL/voxel) of pHe highlighting the heterogeneity of response within the tumor when exposed to UK-5099.

Multi-compartment metabolomics and metagenomics reveal major hepatic and intestinal disturbances in cancer cachectic mice.

BACKGROUND: Cancer cachexia is a multifactorial syndrome characterized by multiple metabolic dysfunctions. Besides the muscle, other organs such as the liver and the gut microbiota may also contribute to this syndrome. Indeed, the gut microbiota, an important regulator of the host metabolism, is altered in the C26 preclinical model of cancer cachexia. Interventions targeting the gut microbiota have shown benefits, but mechanisms underlying the host-microbiota crosstalk in this context are still poorly understood. METHODS: To explore this crosstalk, we combined proton nuclear magnetic resonance (1 H-NMR) metabolomics in multiple compartments with 16S rDNA sequencing. These analyses were complemented by molecular and biochemical analyses, as well as hepatic transcriptomics. RESULTS: 1 H-NMR revealed major changes between control (CT) and cachectic (C26) mice in the four analysed compartments (i.e. caecal content, portal vein, liver, and vena cava). More specifically, glucose metabolism pathways in the C26 model were altered with a reduction in glycolysis and gluconeogenesis and an activation of the hexosamine pathway, arguing against the existence of a Cori cycle in this model. In parallel, amino acid uptake by the liver, with an up to four-fold accumulation of nine amino acids (q-value <0.05), was mainly used for acute phase response proteins synthesis rather than to fuel the tricarboxylic acid cycle and gluconeogenesis. We also identified a 35% reduction in hepatic carnitine levels (q-value <0.05) and a lower activation of the phosphatidylcholine pathway as potential contributors to the hepatic steatosis present in this model. Our work also reveals a reduction of different beneficial intestinal bacterial activities in cancer cachexia. We found decreased levels of two short-chain fatty acids, acetate and butyrate (72% and 88% reduction in C26 caecal content; q-value <0.001), and a reduction in aromatic amino acid metabolites, which may contribute to the altered intestinal homeostasis in these mice. A member of the Ruminococcaceae family (ASV 2) was identified as the main bacterium responsible for the drop in butyrate. Finally, we report a two-fold intestinal transit acceleration (P-value <0.001) as a key factor shaping the gut microbiota composition and activity in cancer cachexia, which together lead to a faecal loss of proteins and amino acids. CONCLUSIONS: Our work highlights new metabolic pathways potentially involved in cancer cachexia and further supports the interest of exploring the gut microbiota composition and activity, as well as intestinal transit, in cancer patients with and without cachexia.