Magnetic resonance spectroscopy ex vivo: A short historical review.

Over the last half century, there have been several periods during which magnetic resonance spectroscopy (MRS) has been used ex vivo, for a variety of reasons, on samples such as microorganisms, cells, animal or human tissue, tissue extracts or biological fluids. These studies began in the days before the acronym MRS had been invented, when all such methods were still called nuclear magnetic resonance (NMR), and have extended to the present day. I will describe the historical development of NMR methods used ex vivo, their influences on the development of MRS in vivo, and their longer-term uses. All the interpretations will be personal, based on what I saw, or discussed with colleagues at the time.

Survival Pathways of HIF-Deficient Tumour Cells: TCA Inhibition, Peroxisomal Fatty Acid Oxidation Activation and an AMPK-PGC-1α Hypoxia Sensor.

The HIF-1 and HIF-2 (HIF1/2) hypoxia responses are frequently upregulated in cancers, and HIF1/2 inhibitors are being developed as anticancer drugs. How could cancers resist anti-HIF1/2 therapy? We studied metabolic and molecular adaptations of HIF-1ß-deficient Hepa-1c4, a hepatoma model lacking HIF1/2 signalling, which mimics a cancer treated by a totally effective anti-HIF1/2 agent. [1,2-13C2]-D-glucose metabolism was measured by SiDMAP metabolic profiling, gene expression by TaqMan, and metabolite concentrations by 1H MRS. HIF-1ß-deficient Hepa-1c4 responded to hypoxia by increasing glucose uptake and lactate production. They showed higher glutamate, pyruvate dehydrogenase, citrate shuttle, and malonyl-CoA fluxes than normal Hepa-1 cells, whereas pyruvate carboxylase, TCA, and anaplerotic fluxes decreased. Hypoxic HIF-1ß-deficient Hepa-1c4 cells increased expression of PGC-1α, phospho-p38 MAPK, and PPARα, suggesting AMPK pathway activation to survive hypoxia. They had higher intracellular acetate, and secreted more H2O2, suggesting increased peroxisomal fatty acid ß-oxidation. Simultaneously increased fatty acid synthesis and degradation would have "wasted" ATP in Hepa-1c4 cells, thus raising the [AMP]:[ATP] ratio, and further contributing to the upregulation of the AMPK pathway. Since these tumour cells can proliferate without the HIF-1/2 pathways, combinations of HIF1/2 inhibitors with PGC-1α or AMPK inhibitors should be explored.

HIF-1-Independent Mechanisms Regulating Metabolic Adaptation in Hypoxic Cancer Cells.

In solid tumours, cancer cells exist within hypoxic microenvironments, and their metabolic adaptation to this hypoxia is driven by HIF-1 transcription factor, which is overexpressed in a broad range of human cancers. HIF inhibitors are under pre-clinical investigation and clinical trials, but there is evidence that hypoxic cancer cells can adapt metabolically to HIF-1 inhibition, which would provide a potential route for drug resistance. Here, we review accumulating evidence of such adaptions in carbohydrate and creatine metabolism and other HIF-1-independent mechanisms that might allow cancers to survive hypoxia despite anti-HIF-1 therapy. These include pathways in glucose, glutamine, and lipid metabolism; epigenetic mechanisms; post-translational protein modifications; spatial reorganization of enzymes; signalling pathways such as Myc, PI3K-Akt, 2-hyxdroxyglutarate and AMP-activated protein kinase (AMPK); and activation of the HIF-2 pathway. All of these should be investigated in future work on hypoxia bypass mechanisms in anti-HIF-1 cancer therapy. In principle, agents targeted toward HIF-1ß rather than HIF-1α might be advantageous, as both HIF-1 and HIF-2 require HIF-1ß for activation. However, HIF-1ß is also the aryl hydrocarbon nuclear transporter (ARNT), which has functions in many tissues, so off-target effects should be expected. In general, cancer therapy by HIF inhibition will need careful attention to potential resistance mechanisms.

Beyond factor H: The impact of genetic-risk variants for age-related macular degeneration on circulating factor-H-like 1 and factor-H-related protein concentrations.

Age-related macular degeneration (AMD) is a leading cause of vision loss; there is strong genetic susceptibility at the complement factor H (CFH) locus. This locus encodes a series of complement regulators: factor H (FH), a splice variant factor-H-like 1 (FHL-1), and five factor-H-related proteins (FHR-1 to FHR-5), all involved in the regulation of complement factor C3b turnover. Little is known about how AMD-associated variants at this locus might influence FHL-1 and FHR protein concentrations. We have used a bespoke targeted mass-spectrometry assay to measure the circulating concentrations of all seven complement regulators and demonstrated elevated concentrations in 352 advanced AMD-affected individuals for all FHR proteins (FHR-1, p = 2.4 × 10-10; FHR-2, p = 6.0 × 10-10; FHR-3, p = 1.5 × 10-5; FHR-4, p = 1.3 × 10-3; FHR-5, p = 1.9 × 10-4) and FHL-1 (p = 4.9 × 10-4) when these individuals were compared to 252 controls, whereas no difference was seen for FH (p = 0.94). Genome-wide association analyses in controls revealed genome-wide-significant signals at the CFH locus for all five FHR proteins, and univariate Mendelian-randomization analyses strongly supported the association of FHR-1, FHR-2, FHR-4, and FHR-5 with AMD susceptibility. These findings provide a strong biochemical explanation for how genetically driven alterations in circulating FHR proteins could be major drivers of AMD and highlight the need for research into FHR protein modulation as a viable therapeutic avenue for AMD.

ApcMin/+ tumours and normal mouse small intestines show linear metabolite concentration and DNA cytosine hydroxymethylation gradients from pylorus to colon.

Topographical variations of metabolite concentrations have been reported in the duodenum, jejunum and ileum of the small intestine, and in human intestinal tumours from those regions, but there are no published metabolite concentrations measurements correlated with linear position in the mouse small intestine or intestinal tumours. Since DNA methylation dynamics are influenced by metabolite concentrations, they too could show linear anatomical variation. We measured metabolites by HR-MAS 1H NMR spectroscopy and DNA cytosine modifications by LC/MS, in normal small intestines of C57BL/6J wild-type mice, and in normal and tumour samples from ApcMin/+ mice. Wild-type mouse intestines showed approximately linear, negative concentration gradations from the pylorus (i.e. the junction with the stomach) of alanine, choline compounds, creatine, leucine and valine. ApcMin/+ mouse tumours showed negative choline and valine gradients, but a positive glycine gradient. 5-Hydroxymethylcytosine showed a positive gradient in the tumours. The linear gradients we found along the length of the mouse small intestine and in tumours contrast with previous reports of discrete concentration changes in the duodenum, jejunum and ileum. To our knowledge, this is also the first report of a systematic measurement of global levels of DNA cytosine modification in wild-type and ApcMin/+ mouse small intestine.

How and Why Are Cancers Acidic? Carbonic Anhydrase IX and the Homeostatic Control of Tumour Extracellular pH.

The acidic tumour microenvironment is now recognized as a tumour phenotype that drives cancer somatic evolution and disease progression, causing cancer cells to become more invasive and to metastasise. This property of solid tumours reflects a complex interplay between cellular carbon metabolism and acid removal that is mediated by cell membrane carbonic anhydrases and various transport proteins, interstitial fluid buffering, and abnormal tumour-associated vessels. In the past two decades, a convergence of advances in the experimental and mathematical modelling of human cancers, as well as non-invasive pH-imaging techniques, has yielded new insights into the physiological mechanisms that govern tumour extracellular pH (pHe). In this review, we examine the mechanisms by which solid tumours maintain a low pHe, with a focus on carbonic anhydrase IX (CAIX), a cancer-associated cell surface enzyme. We also review the accumulating evidence that suggest a role for CAIX as a biological pH-stat by which solid tumours stabilize their pHe. Finally, we highlight the prospects for the clinical translation of CAIX-targeted therapies in oncology.

Intravitreal Pharmacokinetic Study of the Antiangiogenic Glycoprotein Opticin.

Opticin is an endogenous vitreous glycoprotein that may have therapeutic potential as it has been shown that supranormal concentrations suppress preretinal neovascularization. Herein we investigated the pharmacokinetics of opticin following intravitreal injection in rabbits. To measure simultaneously concentrations of human and rabbit opticin, a selected reaction monitoring mass spectrometry assay was developed. The mean concentration of endogenous rabbit opticin in 7 uninjected eyes was measured and found to be 19.2 nM or 0.62 µg/mL. When the vitreous was separated by centrifugation into a supernatant and collagen-containing pellet, 94% of the rabbit opticin was in the supernatant. Intravitreal injection of human opticin (40 µg) into both eyes of rabbits was followed by enucleation at 5, 24, and 72 h and 7, 14, and 28 days postinjection (n = 6 at each time point) and measurement of vitreous human and rabbit opticin concentrations in the supernatant and collagen-containing pellet following centrifugation. The volume of distribution of human opticin was calculated to be 3.31 mL, and the vitreous half-life was 4.2 days. Assuming that rabbit and human opticin are cleared from rabbit vitreous at the same rate, opticin is secreted into the vitreous at a rate of 0.14 µg/day. We conclude that intravitreally injected opticin has a vitreous half-life that is similar to currently available antiangiogenic therapeutics. While opticin was first identified bound to vitreous collagen fibrils, here we demonstrate that >90% of endogenous opticin is not bound to collagen. Endogenous opticin is secreted by the nonpigmented ciliary epithelium into the rabbit vitreous at a remarkably high rate, and the turnover in vitreous is approximately 15% per day.

A Combined Chemical Derivatization/Mass Spectrometric Method for the Enhanced Detection and Relative Quantification of Protein Ubiquitination.

Mass spectrometry (MS) is a sensitive analytical technique with wide application across the sciences including for the detection of peptides and proteins in biological analysis. Ubiquitinated (Ub) proteins are typically analyzed by proteolytic digestion and subsequent chromatographic separation followed by MS detection of the resulting isopeptides. Here we describe a novel method which enables enhanced detection of this important posttranslational modification (PTM) by use of a simple chemical labeling strategy prior to Data-Independent Acquisition (DIA) using a SWATH-based acquisition approach on a suitable Quadrupole-Time-Of-Flight (Q-TOF) mass spectrometer.

Carbonic anhydrase IX is a pH-stat that sets an acidic tumour extracellular pH in vivo.

BACKGROUND: Tumour carbonic anhydrase IX (CAIX), a hypoxia-inducible tumour-associated cell surface enzyme, is thought to acidify the tumour microenvironment by hydrating CO2 to form protons and bicarbonate, but there is no definitive evidence for this in solid tumours in vivo. METHODS: We used 1H magnetic resonance spectroscopic imaging (MRSI) of the extracellular pH probe imidazolyl succinic acid (ISUCA) to measure and spatially map extracellular pH in HCT116 tumours transfected to express CAIX and empty vector controls in SCID mice. We also measured intracellular pH in situ with 31P MRS and measured lactate in freeze-clamped tumours. RESULTS: CAIX-expressing tumours had 0.15 pH-unit lower median extracellular pH than control tumours (pH 6.71 tumour vs pH 6.86 control, P = 0.01). Importantly, CAIX expression imposed an upper limit for tumour extracellular pH at 6.93. Despite the increased lactate concentration in CAIX-expressing tumours, 31P MRS showed no difference in intracellular pH, suggesting that CAIX acidifies only the tumour extracellular space. CONCLUSIONS: CAIX acidifies the tumour microenvironment, and also provides an extracellular pH control mechanism. We propose that CAIX thus acts as an extracellular pH-stat, maintaining an acidic tumour extracellular pH that is tolerated by cancer cells and favours invasion and metastasis.

Thymidine Metabolism as a Confounding Factor for 3'-Deoxy-3'-18F-Fluorothymidine Uptake After Therapy in a Colorectal Cancer Model.

Noninvasive monitoring of tumor therapy response helps in developing personalized treatment strategies. Here, we performed sequential PET and diffusion-weighted MRI to evaluate changes induced by a FOLFOX-like combination chemotherapy in colorectal cancer xenografts, to identify the cellular and molecular determinants of these imaging biomarkers. Methods: Tumor-bearing CD1 nude mice, engrafted with FOLFOX-sensitive Colo205 colorectal cancer xenografts, were treated with FOLFOX (5-fluorouracil, leucovorin, and oxaliplatin) weekly. On days 1, 2, 6, 9, and 13 of therapy, tumors were assessed by in vivo imaging and ex vivo analyses. In addition, HCT116 xenografts, which did not respond to the FOLFOX treatment, were imaged on day 1 of therapy. Results: In Colo205 xenografts, FOLFOX induced a profound increase in uptake of the proliferation PET tracer 3'-deoxy-3'-18F-fluorothymidine (18F-FLT) accompanied by increases in markers for proliferation (Ki-67, thymidine kinase 1) and for activated DNA damage response (γH2AX), whereas the effect on cell death was minimal. Because tracer uptake was unaltered in the HCT116 model, these changes appear to be specific for tumor response. Conclusion: We demonstrated that 18F-FLT PET can noninvasively monitor cancer treatment-induced molecular alterations, including thymidine metabolism and DNA damage response. The cellular or imaging changes may not, however, be directly related to therapy response as assessed by volumetric measurements.

3'-Deoxy-3'-[18F]Fluorothymidine Uptake Is Related to Thymidine Phosphorylase Expression in Various Experimental Tumor Models.

PURPOSE: We recently reported that high thymidine phosphorylase (TP) expression is accompanied by low tumor thymidine concentration and high 3'-deoxy-3'-[18F]fluorothymidine ([18F]FLT) uptake in four untreated lung cancer xenografts. Here, we investigated whether this relationship also holds true for a broader range of tumor models. PROCEDURES: Lysates from n = 15 different tumor models originating from n = 6 institutions were tested for TP and thymidylate synthase (TS) expression using western blots. Results were correlated to [18F]FLT accumulation in the tumors as determined by positron emission tomography (PET) measurements in the different institutions and to previously published thymidine concentrations. RESULTS: Expression of TP correlated positively with [18F]FLT SUVmax (ρ = 0.549, P < 0.05). Furthermore, tumors with high TP levels possessed lower levels of thymidine (ρ = - 0.939, P < 0.001). CONCLUSIONS: In a broad range of tumors, [18F]FLT uptake as measured by PET is substantially influenced by TP expression and tumor thymidine concentrations. These data strengthen the role of TP as factor confounding [18F]FLT uptake.

Fumarate Hydratase Loss Causes Combined Respiratory Chain Defects.

Fumarate hydratase (FH) is an enzyme of the tricarboxylic acid (TCA) cycle mutated in hereditary and sporadic cancers. Despite recent advances in understanding its role in tumorigenesis, the effects of FH loss on mitochondrial metabolism are still unclear. Here, we used mouse and human cell lines to assess mitochondrial function of FH-deficient cells. We found that human and mouse FH-deficient cells exhibit decreased respiration, accompanied by a varying degree of dysfunction of respiratory chain (RC) complex I and II. Moreover, we show that fumarate induces succination of key components of the iron-sulfur cluster biogenesis family of proteins, leading to defects in the biogenesis of iron-sulfur clusters that affect complex I function. We also demonstrate that suppression of complex II activity is caused by product inhibition due to fumarate accumulation. Overall, our work provides evidence that the loss of a single TCA cycle enzyme is sufficient to cause combined RC activity dysfunction.

Exploration of human brain tumour metabolism using pairwise metabolite-metabolite correlation analysis (MMCA) of HR-MAS 1H NMR spectra.

METHODS: We quantified 378 HRMAS 1H NMR spectra of human brain tumours (132 glioblastomas, 101 astrocytomas, 75 meningiomas, 37 oligodendrogliomas and 33 metastases) from the eTumour database and looked for metabolic interactions by metabolite-metabolite correlation analysis (MMCA). RESULTS: All tumour types showed remarkably similar metabolic correlations. Lactate correlated positively with alanine, glutamate with glutamine; creatine + phosphocreatine (tCr) correlated positively with lactate, alanine and choline + phosphocholine + glycerophosphocholine (tCho), and tCho correlated positively with lactate; fatty acids correlated negatively with lactate, glutamate + glutamine (tGlut), tCr and tCho. Oligodendrogliomas had fewer correlations but they still fitted that pattern. CONCLUSIONS: Possible explanations include (i) glycolytic tumour cells (the Warburg effect) generating pyruvate which is converted to lactate, alanine, glutamate and then glutamine; (ii) an association between elevated glycolysis and increased choline turnover in membranes; (iii) an increase in the tCr pool to facilitate phosphocreatine-driven glutamate uptake; (iv) lipid signals come from cytosolic lipid droplets in necrotic or pre-necrotic tumour tissue that has lower concentrations of anabolic and catabolic metabolites. Additional metabolite exchanges with host cells may also be involved. If tumours co-opt a standard set of biochemical mechanisms to grow in the brain, then drugs might be developed to disrupt those mechanisms.

The action of ß-hydroxybutyrate on the growth, metabolism and global histone H3 acetylation of spontaneous mouse mammary tumours: evidence of a ß-hydroxybutyrate paradox.

BACKGROUND: Ketone bodies have both metabolic and epigenetic roles in cancer. In several studies, they showed an anti-cancer effect via inhibition of histone deacetylases; however, other studies observed faster tumour growth. The related molecule butyrate also inhibits growth of some cancer cells and accelerates it in others. This "butyrate paradox" is thought to be due to butyrate mediating histone acetylation and thus inhibiting cell proliferation in cancers that preferentially utilise glucose (the Warburg effect); whereas in cells that oxidise butyrate as a fuel, it fails to reach inhibitory concentrations and can stimulate growth. METHODS: We treated transgenic mice bearing spontaneous MMTV-NEU-NT mammary tumours with the ketone body ß-hydroxybutyrate (ß-OHB) and monitored tumour growth, metabolite concentrations and histone acetylation. In a cell line derived from these tumours, we also measured uptake of ß-OHB and glucose, and lactate production, in the absence and presence of ß-OHB. RESULTS: ß-OHB administration accelerated growth of MMTV-NEU-NT tumours, and their metabolic profile showed significant increases in ATP, glutamine, serine and choline-related metabolites. The ß-OHB concentration within the treated tumours, 0.46 ± 0.05 µmol/g, had no effect on histone acetylation as shown by western blots. Cultured tumour cells incubated with 0.5 mM ß-OHB showed ß-OHB uptake that would be equivalent to 54% of glycolytic ATP phosphorylation and no significant change in glucose consumption or lactate production. CONCLUSIONS: These results suggest that a ß-OHB paradox may occur in these mammary tumours in a manner analogous to the butyrate paradox. At low ß-OHB concentrations (<1 mM, as observed in our tumour model post-treatment), and in the absence of a Warburg effect, ß-OHB is consumed and thus acts as an oxidative energy source and not as an epigenetic factor. This would explain the increase in tumour growth after treatment, the metabolic profiles and the absence of an effect on histone H3 acetylation.

Quantitative and textural analysis of magnetization transfer and diffusion images in the early detection of brain metastases.

PURPOSE: The sensitivity of the magnetization transfer ratio (MTR) and apparent diffusion coefficient (ADC) for early detection of brain metastases was investigated in mice and humans. METHODS: Mice underwent MRI twice weekly for up to 31 d following intracardiac injection of the brain-homing breast cancer cell line MDA-MB231-BR. Patients with small cell lung cancer underwent quarterly MRI for 1 year. MTR and ADC were measured in regions of metastasis and matched contralateral tissue at the final time point and in registered regions at earlier time points. Texture analysis and linear discriminant analysis were performed to detect metastasis-containing slices. RESULTS: Compared with contralateral tissue, mouse metastases had significantly lower MTR and higher ADC at the final time point. Some lesions were visible at earlier time points on the MTR and ADC maps: 24% of these were not visible on corresponding T2 -weighted images. Texture analysis using the MTR maps showed 100% specificity and 98% sensitivity for metastasis at the final time point, with 77% sensitivity 2-4 d earlier and 46% 5-8 d earlier. Only 2 of 16 patients developed metastases, and their penultimate scans were normal. CONCLUSIONS: Some brain metastases may be detected earlier on MTR than conventional T2 ; however, the small gain is unlikely to justify "predictive" MRI. Magn Reson Med 77:1987-1995, 2017. © 2016 The Authors Magnetic Resonance in Medicine published by Wiley Periodicals, Inc. on behalf of International Society for Magnetic Resonance in Medicine. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

Assessment of early treatment response to neoadjuvant chemotherapy in breast cancer using non-mono-exponential diffusion models: a feasibility study comparing the baseline and mid-treatment MRI examinations.

OBJECTIVES: To assess the feasibility of the mono-exponential, bi-exponential and stretched-exponential models in evaluating response of breast tumours to neoadjuvant chemotherapy (NACT) at 3 T. METHODS: Thirty-six female patients (median age 53, range 32-75 years) with invasive breast cancer undergoing NACT were enrolled for diffusion-weighted MRI (DW-MRI) prior to the start of treatment. For assessment of early response, changes in parameters were evaluated on mid-treatment MRI in 22 patients. DW-MRI was performed using eight b values (0, 30, 60, 90, 120, 300, 600, 900 s/mm2). Apparent diffusion coefficient (ADC), tissue diffusion coefficient (D t), vascular fraction (ƒ), distributed diffusion coefficient (DDC) and alpha (α) parameters were derived. Then t tests compared the baseline and changes in parameters between response groups. Repeatability was assessed at inter- and intraobserver levels. RESULTS: All patients underwent baseline MRI whereas 22 lesions were available at mid-treatment. At pretreatment, mean diffusion coefficients demonstrated significant differences between groups (p < 0.05). At mid-treatment, percentage increase in ADC and DDC showed significant differences between responders (49 % and 43 %) and non-responders (21 % and 32 %) (p = 0.03, p = 0.04). Overall, stretched-exponential parameters showed excellent repeatability. CONCLUSION: DW-MRI is sensitive to baseline and early treatment changes in breast cancer using non-mono-exponential models, and the stretched-exponential model can potentially monitor such changes. KEY POINTS: • Baseline diffusion coefficients demonstrated significant differences between complete pathological responders and non-responders. • Increase in ADC and DDC at mid-treatment can discriminate responders and non-responders. • The ƒ fraction at mid-treatment decreased in responders whereas increased in non-responders. • The mono- and stretched-exponential models showed excellent inter- and intrarater repeatability. • Treatment effects can potentially be assessed by non-mono-exponential diffusion models.

Response Monitoring with [18F]FLT PET and Diffusion-Weighted MRI After Cytotoxic 5-FU Treatment in an Experimental Rat Model for Colorectal Liver Metastases.

PURPOSE: The aim of the study was to investigate the potential of diffusion-weighted magnetic resonance imaging (DW-MRI) and 3'-dexoy-3'-[18F]fluorothymidine ([18F]FLT) positron emission tomography (PET) as early biomarkers of treatment response of 5-fluorouracil (5-FU) in a syngeneic rat model of colorectal cancer liver metastases. PROCEDURES: Wag/Rij rats with intrahepatic syngeneic CC531 tumors were treated with 5-FU (15, 30, or 60 mg/kg in weekly intervals). Before treatment and at days 1, 3, 7, and 14 after treatment rats underwent DW-MRI and [18F]FLT PET. Tumors were analyzed immunohistochemically for Ki67, TK1, and ENT1 expression. RESULTS: 5-FU inhibited the growth of CC531 tumors in a dose-dependent manner. Immunohistochemical analysis did not show significant changes in Ki67, TK1, and ENT1 expression. However, [18F]FLT SUVmean and SUVmax were significantly increased at days 4 and 7 after treatment with 5-FU (60 mg/kg) and returned to baseline at day 14 (SUVmax at days -1, 4, 7, and 14 was 1.1 ± 0.1, 2.3 ± 0.5, 2.3 ± 0.6, and 1.5 ± 0.4, respectively). No changes in [18F]FLT uptake were observed in the nontreated animals. Furthermore, the apparent diffusion coefficient (ADCmean) did not change in 5-FU-treated rats compared to untreated rats. CONCLUSION: This study suggests that 5-FU treatment induces a flare in [18F]FLT uptake of responsive CC531 tumors in the liver, while the ADCmean did not change significantly. Future studies in larger groups are warranted to further investigate whether [18F]FLT PET can discriminate between disease progression and treatment response.

Imaging biomarker roadmap for cancer studies.

Imaging biomarkers (IBs) are integral to the routine management of patients with cancer. IBs used daily in oncology include clinical TNM stage, objective response and left ventricular ejection fraction. Other CT, MRI, PET and ultrasonography biomarkers are used extensively in cancer research and drug development. New IBs need to be established either as useful tools for testing research hypotheses in clinical trials and research studies, or as clinical decision-making tools for use in healthcare, by crossing 'translational gaps' through validation and qualification. Important differences exist between IBs and biospecimen-derived biomarkers and, therefore, the development of IBs requires a tailored 'roadmap'. Recognizing this need, Cancer Research UK (CRUK) and the European Organisation for Research and Treatment of Cancer (EORTC) assembled experts to review, debate and summarize the challenges of IB validation and qualification. This consensus group has produced 14 key recommendations for accelerating the clinical translation of IBs, which highlight the role of parallel (rather than sequential) tracks of technical (assay) validation, biological/clinical validation and assessment of cost-effectiveness; the need for IB standardization and accreditation systems; the need to continually revisit IB precision; an alternative framework for biological/clinical validation of IBs; and the essential requirements for multicentre studies to qualify IBs for clinical use.

S-2-hydroxyglutarate regulates CD8+ T-lymphocyte fate.

R-2-hydroxyglutarate accumulates to millimolar levels in cancer cells with gain-of-function isocitrate dehydrogenase 1/2 mutations. These levels of R-2-hydroxyglutarate affect 2-oxoglutarate-dependent dioxygenases. Both metabolite enantiomers, R- and S-2-hydroxyglutarate, are detectible in healthy individuals, yet their physiological function remains elusive. Here we show that 2-hydroxyglutarate accumulates in mouse CD8+ T cells in response to T-cell receptor triggering, and accumulates to millimolar levels in physiological oxygen conditions through a hypoxia-inducible factor 1-alpha (HIF-1α)-dependent mechanism. S-2-hydroxyglutarate predominates over R-2-hydroxyglutarate in activated T cells, and we demonstrate alterations in markers of CD8+ T-cell differentiation in response to this metabolite. Modulation of histone and DNA demethylation, as well as HIF-1α stability, mediate these effects. S-2-hydroxyglutarate treatment greatly enhances the in vivo proliferation, persistence and anti-tumour capacity of adoptively transferred CD8+ T cells. Thus, S-2-hydroxyglutarate acts as an immunometabolite that links environmental context, through a metabolic-epigenetic axis, to immune fate and function.