Highlights d Glycolytic index in melanoma negatively correlates with response to anti-PD1 therapy d Blocking lactate transport or knock out of glycolytic genes improves checkpoint therapy d Diclofenac blocks the lactate transporters MCT1 and MCT4 in a COX-independent manner d Inhibition of glycolysis by MCT blockade does not impede T cell function SUMMARY Tumor-derived lactic acid inhibits T and natural killer (NK) cell function and, thereby, tumor immunosurveillance.Here, we report that melanoma patients with high expression of glycolysis-related genes show a worse progression free survival upon anti-PD1 treatment. The non-steroidal anti-inflammatory drug (NSAID) diclofenac lowers lactate secretion of tumor cells and improves anti-PD1-induced T cell killing in vitro. Surprisingly, diclofenac, but not other NSAIDs, turns out to be a potent inhibitor of the lactate transporters monocarboxylate transporter 1 and 4 and diminishes lactate efflux. Notably, T cell activation, viability, and effector functions are preserved under diclofenac treatment and in a low glucose environment in vitro. Diclofenac, but not aspirin, delays tumor growth and improves the efficacy of checkpoint therapy in vivo. Moreover, genetic suppression of glycolysis in tumor cells strongly improves checkpoint therapy. These findings support the rationale for targeting glycolysis in patients with high glycolytic tumors together with checkpoint inhibitors in clinical trials.

Increased glucose consumption distinguishes cancer cells from normal cells and is known as the "Warburg effect" because of increased glycolysis. Lactate dehydrogenase A (LDHA) is a key glycolytic enzyme, a hallmark of aggressive cancers, and believed to be the major enzyme responsible for pyruvate-to-lactate conversion. To elucidate its role in tumor growth, we disrupted both the and genes in two cancer cell lines (human colon adenocarcinoma and murine melanoma cells). Surprisingly, neither nor knockout strongly reduced lactate secretion. In contrast, double knockout (-DKO) fully suppressed LDH activity and lactate secretion. Furthermore, under normoxia, -DKO cells survived the genetic block by shifting their metabolism to oxidative phosphorylation (OXPHOS), entailing a 2-fold reduction in proliferation rates and compared with their WT counterparts. Under hypoxia (1% oxygen), however, suppression completely abolished growth, consistent with the reliance on OXPHOS. Interestingly, activation of the respiratory capacity operated by the-DKO genetic block as well as the resilient growth were not consequences of long-term adaptation. They could be reproduced pharmacologically by treating WT cells with an LDHA/B-specific inhibitor (GNE-140). These findings demonstrate that the Warburg effect is not only based on high LDHA expression, as both and need to be deleted to suppress fermentative glycolysis. Finally, we demonstrate that the Warburg effect is dispensable even in aggressive tumors and that the metabolic shift to OXPHOS caused by / genetic disruptions is responsible for the tumors' escape and growth.

Lectins are a group of diverse proteins which bind to specific configurations of sugar residues. The binding of lectins to sugar residues present in polysaccharides resembles the specific binding of antibodies to antigens (4). Lectins have been widely used to characterize surfaces of eucaryotic cells and polysaccharides. Recently, fluorescently labeled lectins have been applied in the study of biofilm formation and biofilm composition. Excretion of adhesive polymers during attachment of bacterial cells to surfaces has been described using a panel of fluorescent lectins (9,14,20). The formation of biofilms on living and nonliving surfaces has been investigated with lectins (17). Lectins in conjunction with confocal laser scanning microscopy (CLSM) have been valuable tools in the study of the threedimensional structure of biofilms (12,15) or of the composition of extracellular polymeric substances (EPS) involved in accumulation of chlorinated organic compounds (24). Strains of Sphingomonas spp. are known for their interesting catabolic capabilities to degrade a wide variety of environmentally hazardous compounds, including polycyclic aromatics (25), dioxine compounds (6), and chlorinated phenols (3). Theoretically, lectins may be used to study the interaction between Sphingomonas cells and environmental surfaces during biofilm formation or to investigate the interaction of EPS with organic compounds. The common approach has been to deduce the structure or composition of biofilm EPS on the basis of the specific binding of lectins to different sugar residues. In this study, we evaluate the use of lectins for the characterization of Sphingomonas biofilms by investigating the binding of five fluorescent lectins with known specificities to Sphingomonas biofilms and to industrially produced Sphingomonas exopolysaccharides (sphingans) with known molecular structures. MATERIALS AND METHODSBacterial strains and growth conditions. Sphingomonas paucimobilis EPA505 was obtained from J. Mueller (19), and Sphingomonas sp. strain LH128 and Sphingomonas sp. strain LB126 were received from L. Bastiaens (1). All strains were stored in 43% glycerol at Ϫ80°C. The bacteria were grown at room temperature in phosphate minimal medium supplemented with glucose as the sole carbon source (PMMG) containing (in grams/liter) the following: glucose, 2; Na 2 HPO 4 ⅐ 2H 2 O, 0.875; KH 2 PO 4 , 0.1; (NH 4 ) 2 SO 4 , 0.25; MgCl 2 ⅐ 6H 2 O, 0.05; CaCl 2 ⅐ 2H 2 O, 0.015; NaNO 3 , 0.018. The medium was amended with 5 ml of a trace element solution consisting of (in milligrams/liter) the following: Na-EDTA, 800; FeCl 2 , 300; MnCl 2 ⅐ 4H 2 O, 10; CoCl 2 ⅐ 6H 2 O, 4; CuSO 4 , 1; Na 2 MoO 4 ⅐ 2H 2 O, 3; ZnCl 2 , 2; LiCl, 0.5; SnCl 2 ⅐ 2H 2 O, 0.5; H 3 BO 3 , 1; KBr, 2; KI, 2; BaCl 2 , 0.5. Phosphate and glucose were autoclaved separately.Cultivation of biofilms on microscope slides. Single-species biofilms were grown on Cel-Line HTC printed microscope slides with six wells on each slide (Cel-Line Associates, Inc., Newfield, N.J.). The slides were sterili...

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Functional systemic and local immunity is required for effective anti-tumor responses. In addition to an active engagement with cancer cells and tumor stroma, immune cells can be affected and are often found to be dysregulated in cancer patients. The impact of tumors on local and systemic immunity can be assessed using a variety of approaches ranging from low-dimensional analyses that are performed on large patient cohorts to multi-dimensional assays that are technically and logistically challenging and are therefore confined to a limited sample size. Many of these strategies have been established in recent years leading to exciting findings. Not only were analyses of immune cells in tumor patients able to predict the clinical course of the disease and patients’ survival, numerous studies also detected changes in the immune landscape that correlated with responses to novel immunotherapies. This review will provide an overview of established and novel tools for assessing immune cells in tumor patients and will discuss exemplary studies that utilized these techniques to predict patient outcomes.