Metabolic rewiring tunes dermal macrophages in staphylococcal skin infection.

The skin needs to balance tolerance of colonizing microflora with rapid detection of potential pathogens. Flexible response mechanisms would seem most suitable to accommodate the dynamic challenges of effective antimicrobial defense and restoration of tissue homeostasis. Here, we dissected macrophage-intrinsic mechanisms and microenvironmental cues that tune macrophage signaling in localized skin infection with the colonizing and opportunistic pathogen Staphylococcus aureus. Early in skin infection, the cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) produced by γδ T cells and hypoxic conditions within the dermal microenvironment diverted macrophages away from a homeostatic M-CSF- and hypoxia-inducible factor 1α (HIF-1α)-dependent program. This allowed macrophages to be metabolically rewired for maximal inflammatory activity, which requires expression of Irg1 and generation of itaconate, but not HIF-1α. This multifactorial macrophage rewiring program was required for both the timely clearance of bacteria and for the provision of local immune memory. These findings indicate that immunometabolic conditioning allows dermal macrophages to cycle between antimicrobial activity and protection against secondary infections.

Mass Spectrometric Analysis of Cucurbitacins and Dihydrocucurbitacins from the Tuber of Citrullus naudinianus.

The vast pool of structurally and functionally distinct secondary metabolites (i.e., natural products (NPs)) is constantly being expanded, a process also driven by the rapid progress in the development of analytical techniques. Such NPs often show potent biological activities and are therefore prime candidates for drug development and medical applications. The ethyl acetate extract of the tuber of Citrullus naudinianus (C. naudinianus), an African melon with edible fruits and seeds, shows in vitro immunomodulatory activity presumably elicited by cucurbitacins that are known major constituents of this plant. Further potentially immunomodulatory cucurbitacins or cucurbitacin derivatives were assumed to be in the tuber. Given the typically high content of cucurbitacins with similar physicochemical features but often distinct bioactivities, an efficient and reliable separation process is a prerequisite for their detailed characterization and assessment in terms of bioactivity. We therefore developed a detection method to screen and differentiate cucurbitacins via high-performance liquid chromatography/quadrupole-time-of-flight tandem mass spectrometry (HPLC-QTOF-MS/MS). In order to confirm the identification, the fragmentation patterns of two cucurbitacins and one 23,24-dihydrocucurbitacin were also investigated. Six characteristic fragments were identified and three of them were employed for the identification of cucurbitacins and 23,24-dihydrocucurbitacins in the extract. As a result, in addition to eight previously reported cucurbitacins from this plant four distinct 23,24-dihydrocucurbitacins (B, D, E, and I) were putatively identified and newly found in the ethyl acetate extract of the tuber of C. naudinianus. The established methodology enables rapid and efficient LC-MS-based analysis and identification of cucurbitacins and 23,24-dihydrocucurbitacins in plant extracts.

Integrated Metabolomic and Transcriptomic Analysis of Modified Nucleosides for Biomarker Discovery in Clear Cell Renal Cell Carcinoma.

Clear cell renal cell carcinoma (ccRCC) accounts for ~75% of kidney cancers. The biallelic inactivation of the von Hippel-Lindau tumor suppressor gene (VHL) is the truncal driver mutation of most cases of ccRCC. Cancer cells are metabolically reprogrammed and excrete modified nucleosides in larger amounts due to their increased RNA turnover. Modified nucleosides occur in RNAs and cannot be recycled by salvage pathways. Their potential as biomarkers has been demonstrated for breast or pancreatic cancer. To assess their suitability as biomarkers in ccRCC, we used an established murine ccRCC model, harboring Vhl, Trp53 and Rb1 (VPR) knockouts. Cell culture media of this ccRCC model and primary murine proximal tubular epithelial cells (PECs) were investigated by HPLC coupled to triple-quadrupole mass spectrometry using multiple-reaction monitoring. VPR cell lines were significantly distinguishable from PEC cell lines and excreted higher amounts of modified nucleosides such as pseudouridine, 5-methylcytidine or 2'-O-methylcytidine. The method's reliability was confirmed in serum-starved VPR cells. RNA-sequencing revealed the upregulation of specific enzymes responsible for the formation of those modified nucleosides in the ccRCC model. These enzymes included Nsun2, Nsun5, Pus1, Pus7, Naf1 and Fbl. In this study, we identified potential biomarkers for ccRCC for validation in clinical trials.

SCD5 Regulation by VHL Affects Cell Proliferation and Lipid Homeostasis in ccRCC.

Clear cell renal cell carcinoma (ccRCC) is the most common histological subtype of renal cancer, and inactivation of the VHL tumor suppressor gene is found in almost all cases of hereditary and sporadic ccRCCs. CcRCC is associated with the reprogramming of fatty acid metabolism, and stearoyl-CoA desaturases (SCDs) are the main enzymes controlling fatty acid composition in cells. In this study, we report that mRNA and protein expression of the stearoyl-CoA desaturase SCD5 is downregulated in VHL-deficient cell lines. Similarly, in C. elegans vhl-1 mutants, FAT-7/SCD5 activity is repressed, supporting an evolutionary conservation. SCD5 regulation by VHL depends on HIF, and loss of SCD5 promotes cell proliferation and a metabolic shift towards ceramide production. In summary, we identify a novel regulatory function of VHL in relation to SCD5 and fatty acid metabolism, and propose a new mechanism of how loss of VHL may contribute to ccRCC tumor formation and progression.

Mitochondrial Metabolomics of Sym1-Depleted Yeast Cells Revealed Them to Be Lysine Auxotroph.

Metabolomics has expanded from cellular to subcellular level to elucidate subcellular compartmentalization. By applying isolated mitochondria to metabolome analysis, the hallmark of mitochondrial metabolites has been unraveled, showing compartment-specific distribution and regulation of metabolites. This method was employed in this work to study a mitochondrial inner membrane protein Sym1, whose human ortholog MPV17 is related to mitochondria DNA depletion syndrome. Gas chromatography-mass spectrometry-based metabolic profiling was combined with targeted liquid chromatography-mass spectrometry analysis to cover more metabolites. Furthermore, we applied a workflow employing ultra-high performance liquid chromatography-quadrupole time of flight mass spectrometry with a powerful chemometrics platform, focusing on only significantly changed metabolites. This workflow highly reduced the complexity of acquired data without losing metabolites of interest. Consequently, forty-one novel metabolites were identified in addition to the combined method, of which two metabolites, 4-guanidinobutanal and 4-guanidinobutanoate, were identified for the first time in Saccharomyces cerevisiae. With compartment-specific metabolomics, we identified sym1Δ cells as lysine auxotroph. The highly reduced carbamoyl-aspartate and orotic acid indicate a potential role of the mitochondrial inner membrane protein Sym1 in pyrimidine metabolism.

ZBTB18 inhibits SREBP-dependent lipid synthesis by halting CTBPs and LSD1 activity in glioblastoma.

Enhanced fatty acid synthesis is a hallmark of tumors, including glioblastoma. SREBF1/2 regulate the expression of enzymes involved in fatty acid and cholesterol synthesis. Yet, little is known about the precise mechanism regulating SREBP gene expression in glioblastoma. Here, we show that a novel interaction between the co-activator/co-repressor CTBP and the tumor suppressor ZBTB18 regulates the expression of SREBP genes. In line with our findings, metabolic assays and glucose tracing analysis confirm the reduction in several phospholipid species upon ZBTB18 expression. Our study identifies CTBP1/2 and LSD1 as co-activators of SREBP genes and indicates that the functional activity of the CTBP-LSD1 complex is altered by ZBTB18. ZBTB18 binding to the SREBP gene promoters is associated with reduced LSD1 demethylase activity of H3K4me2 and H3K9me2 marks. Concomitantly, the interaction between LSD1, CTBP, and ZNF217 is increased, suggesting that ZBTB18 promotes LSD1 scaffolding function. Our results outline a new epigenetic mechanism enrolled by ZBTB18 and its co-factors to regulate fatty acid synthesis that could be targeted to treat glioblastoma patients.

Novel lectin-based chimeric antigen receptors target Gb3-positive tumour cells.

The link between cancer and aberrant glycosylation has recently become evident. Glycans and their altered forms, known as tumour-associated carbohydrate antigens (TACAs), are diverse, complex and difficult to target therapeutically. Lectins are naturally occurring glycan-binding proteins  that offer a unique opportunity to recognise TACAs. T cells expressing chimeric antigen receptors (CARs) have proven to be a successful immunotherapy against leukaemias, but so far have shown limited success in solid tumours. We developed a panel of lectin-CARs that recognise the glycosphingolipid globotriaosylceramide (Gb3), which is overexpressed in various cancers, such as Burkitt's lymphoma, colorectal, breast and pancreatic. We have selected the following lectins: Shiga toxin's B-subunit from Shigella dysenteriae, LecA from Pseudomonas aeruginosa, and the engineered lectin Mitsuba from Mytilus galloprovincialis as antigen-binding domains and fused them to a well-known second-generation CAR. The Gb3-binding lectin-CARs have demonstrated target-specific cytotoxicity against Burkitt's lymphoma-derived cell lines as well as solid tumour cells from colorectal and triple-negative breast cancer. Our findings reveal the big potential of lectin-based CARs as therapeutical applications to target Gb3 and other TACAs expressed in haematological malignancies and solid tumours.

Retraction notice to "Enhanced AC133-specific CAR T cell therapy induces durable remissions in mice with metastatic small cell lung cancer" [Canc. Lett. 520 (2021) 385-399].

This article has been retracted: please see Elsevier Policy on Article Withdrawal (https://www.elsevier.com/about/our-business/policies/article-withdrawal). This article has been retracted at the request of the Editor-in-Chief. Following the publication of the above article, the Editor was notified that an error occurred in which all images were published with incorrect versions. The Editor has taken the decision that the manuscript is no longer acceptable in its current form, nor with a corrigendum, as the extensive changes to the figures and publication would lead to ambiguity for our readers. We have therefore made the decision to retract this manuscript from Cancer Letters with the possibility of resubmission and republication of the manuscript in its corrected form after peer review.

Enhanced AC133-specific CAR T cell therapy induces durable remissions in mice with metastatic small cell lung cancer.

Metastatic small cell lung cancer (SCLC) is not curable. While SCLC is initially sensitive to chemotherapy, remissions are short-lived. The relapse is induced by chemotherapy-selected tumor stem cells, which express the AC133 epitope of the CD133 stem cell marker. We studied the effectiveness of AC133-specific CAR T cells post-chemotherapy using human primary SCLC and an orthotopic xenograft mouse model. AC133-specific CAR T cells migrated to SCLC tumor lesions, reduced the tumor burden, and prolonged survival in a humanized orthotopic SCLC model, but were not able to entirely eliminate tumors. We identified CD73 and PD-L1 as immune-escape mechanisms and combined PD-1-inhibition and CD73-inhibition with CAR T cell treatment. This triple-immunotherapy induced cures in 25% of the mice, without signs of graft-versus-host disease or bone marrow failure. AC133+ cancer stem cells and PD-L1+CD73+ myeloid cells were detectable in primary human SCLC tissues, suggesting that patients may benefit from the triple-immunotherapy. We conclude that the combination of AC133-specific CAR T cells, anti-PD-1-antibody and CD73-inhibitor specifically eliminates chemo-resistant tumor stem cells, overcomes SCLC-mediated T cell inhibition, and might induce long-term complete remission in an otherwise incurable disease.

BECLIN1 Is Essential for Podocyte Secretory Pathways Mediating VEGF Secretion and Podocyte-Endothelial Crosstalk.

Vascular endothelial growth factor A (VEGFA) secretion from podocytes is crucial for maintaining endothelial integrity within the glomerular filtration barrier. However, until now, the molecular mechanisms underlying podocyte secretory function remained unclear. Through podocyte-specific deletion of BECLIN1 (ATG6 or Becn1), a key protein in autophagy initiation, we identified a major role for this molecule in anterograde Golgi trafficking. The Becn1-deficient podocytes displayed aberrant vesicle formation in the trans-Golgi network (TGN), leading to dramatic vesicle accumulation and complex disrupted patterns of intracellular vesicle trafficking and membrane dynamics. Phenotypically, podocyte-specific deletion of Becn1 resulted in early-onset glomerulosclerosis, which rapidly progressed and dramatically reduced mouse life span. Further, in vivo and in vitro studies clearly showed that VEGFA secretion, and thereby endothelial integrity, greatly depended on BECLIN1 availability and function. Being the first to demonstrate the importance of a secretory pathway for podocyte integrity and function, we identified BECLIN1 as a key component in this complex cellular process. Functionally, by promoting VEGFA secretion, a specific secretory pathway emerged as an essential component for the podocyte-endothelial crosstalk that maintains the glomerular filtration barrier.

Photoaffinity Capture Compounds to Profile the Magic Spot Nucleotide Interactomes.

Magic Spot Nucleotides (MSN) regulate the stringent response, a highly conserved bacterial stress adaptation mechanism, enabling survival under adverse external challenges. In times of antibiotic crisis, a detailed understanding of stringent response is essential, as potentially new targets for pharmacological intervention could be identified. In this study, we delineate the MSN interactome in Escherichia coli and Salmonella typhimurium applying a family of trifunctional photoaffinity capture compounds. We introduce MSN probes covering a diverse phosphorylation pattern, such as pppGpp, ppGpp, and pGpp. Our chemical proteomics approach provides datasets of putative MSN receptors both from cytosolic and membrane fractions that unveil new MSN targets. We find that the activity of the non-Nudix hydrolase ApaH is potently inhibited by pppGpp, which itself is converted to pGpp by ApaH. The capture compounds described herein will be useful to identify MSN interactomes across bacterial species.

Bacterial flavoprotein monooxygenase YxeK salvages toxic S-(2-succino)-adducts via oxygenolytic C-S bond cleavage.

Thiol-containing nucleophiles such as cysteine react spontaneously with the citric acid cycle intermediate fumarate to form S-(2-succino)-adducts. In Bacillus subtilis, a salvaging pathway encoded by the yxe operon has recently been identified for the detoxification and exploitation of these compounds as sulfur sources. This route involves acetylation of S-(2-succino)cysteine to N-acetyl-2-succinocysteine, which is presumably converted to oxaloacetate and N-acetylcysteine, before a final deacetylation step affords cysteine. The critical oxidative cleavage of the C-S bond of N-acetyl-S-(2-succino)cysteine was proposed to depend on the predicted flavoprotein monooxygenase YxeK. Here, we characterize YxeK and verify its role in S-(2-succino)-adduct detoxification and sulfur metabolism. Detailed biochemical and mechanistic investigation of YxeK including 18 O-isotope-labeling experiments, homology modeling, substrate specificity tests, site-directed mutagenesis, and (pre-)steady-state kinetics provides insight into the enzyme's mechanism of action, which may involve a noncanonical flavin-N5-peroxide species for C-S bond oxygenolysis.

Metabolic and Lipidomic Assessment of Kidney Cells Exposed to Nephrotoxic Vancomycin Dosages.

Vancomycin is a glycopeptide antibiotic used against multi-drug resistant gram-positive bacteria such as Staphylococcus aureus (MRSA). Although invaluable against resistant bacteria, vancomycin harbors adverse drug reactions including cytopenia, ototoxicity, as well as nephrotoxicity. Since nephrotoxicity is a rarely occurring side effect, its mechanism is incompletely understood. Only recently, the actual clinically relevant concentration the in kidneys of patients receiving vancomycin was investigated and were found to exceed plasma concentrations by far. We applied these clinically relevant vancomycin concentrations to murine and canine renal epithelial cell lines and assessed metabolic and lipidomic alterations by untargeted and targeted gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry analyses. Despite marked differences in the lipidome, both cell lines increased anabolic glucose reactions, resulting in higher sorbitol and lactate levels. To the best of our knowledge, this is the first endometabolic profiling of kidney cells exposed to clinically relevant vancomycin concentrations. The presented study will provide a valuable dataset to nephrotoxicity researchers and might add to unveiling the nephrotoxic mechanism of vancomycin.

RETRACTED: Enhanced AC133-specific CAR T cell therapy induces durable remissions in mice with metastatic small cell lung cancer.

This article has been retracted: please see Elsevier Policy on Article Withdrawal (https://www.elsevier.com/about/our-business/policies/article-withdrawal). This article has been retracted at the request of the Editor-in-Chief. Following the publication of the above article, the Editor was notified that an error occurred in which all images were published with incorrect versions. The Editor has taken the decision that the manuscript is no longer acceptable in its current form, nor with a corrigendum, as the extensive changes to the figures and publication would lead to ambiguity for our readers. We have therefore made the decision to retract this manuscript from Cancer Letters with the possibility of resubmission and republication of the manuscript in its corrected form after peer review.

The Gb3-enriched CD59/flotillin plasma membrane domain regulates host cell invasion by Pseudomonas aeruginosa.

The opportunistic pathogen Pseudomonas aeruginosa has gained precedence over the years due to its ability to develop resistance to existing antibiotics, thereby necessitating alternative strategies to understand and combat the bacterium. Our previous work identified the interaction between the bacterial lectin LecA and its host cell glycosphingolipid receptor globotriaosylceramide (Gb3) as a crucial step for the engulfment of P. aeruginosa via the lipid zipper mechanism. In this study, we define the LecA-associated host cell membrane domain by pull-down and mass spectrometry analysis. We unraveled a predilection of LecA for binding to saturated, long fatty acyl chain-containing Gb3 species in the extracellular membrane leaflet and an induction of dynamic phosphatidylinositol (3,4,5)-trisphosphate (PIP3) clusters at the intracellular leaflet co-localizing with sites of LecA binding. We found flotillins and the GPI-anchored protein CD59 not only to be an integral part of the LecA-interacting membrane domain, but also majorly influencing bacterial invasion as depletion of either of these host cell proteins resulted in about 50% reduced invasiveness of the P. aeruginosa strain PAO1. In summary, we report that the LecA-Gb3 interaction at the extracellular leaflet induces the formation of a plasma membrane domain enriched in saturated Gb3 species, CD59, PIP3 and flotillin thereby facilitating efficient uptake of PAO1.

A reciprocal regulation of spermidine and autophagy in podocytes maintains the filtration barrier.

Podocyte maintenance and stress resistance are exquisitely based on high basal rates of autophagy making these cells a unique model to unravel mechanisms of autophagy regulation. Polyamines have key cellular functions such as proliferation, nucleic acid biosynthesis and autophagy. Here we test whether endogenous spermidine signaling is a driver of basal and dynamic autophagy in podocytes by using genetic and pharmacologic approaches to interfere with different steps of polyamine metabolism. Translational studies revealed altered spermidine signaling in focal segmental glomerulosclerosis in vivo and in vitro. Exogenous spermidine supplementation emerged as new treatment strategy by successfully activating autophagy in vivo via inhibition of EP300, a protein with an essential role in controlling cell growth, cell division and prompting cells to differentiate to take on specialized functions. Surprisingly, gas chromatography-mass spectroscopy based untargeted metabolomics of wild type and autophagy deficient primary podocytes revealed a positive feedback mechanism whereby autophagy itself maintains polyamine metabolism and spermidine synthesis. The transcription factor MAFB acted as an upstream regulator of polyamine metabolism. Thus, our data highlight a novel positive feedback loop of autophagy and spermidine signaling allowing maintenance of high basal levels of autophagy as a key mechanism to sustain the filtration barrier. Hence, spermidine supplementation may emerge as a new therapeutic to restore autophagy in glomerular disease.

Metabolic Profiling of Early and Late Recurrent Pancreatic Ductal Adenocarcinoma Using Patient-Derived Organoid Cultures.

Pancreatic ductal adenocarcinoma (PDAC) is associated with high mortality and will become the second most common cause of cancer-associated mortality by 2030. The poor prognosis arises from a lack of sensitive biomarkers, limited therapeutic options, and the astonishingly high recurrence rate after surgery of 60-80%. The factors driving this recurrence, however, remain enigmatic. Therefore, we generated patient-derived organoids (PDOs) from early- and late-recurrent PDAC patients. Cellular identity of PDOs was confirmed by qPCR, ddPCR, and IHC analyses. This is the first study investigating the metabolism in PDOs of different, clinically significant PDAC entities by untargeted GC/MS profiling. Partial least square discriminant analysis unveiled global alterations between the two sample groups. We identified nine metabolites to be increased in early recurrent PDOs in comparison to late recurrent PDOs. More than four-times increased were fumarate, malate, glutamate, aspartate, and glutamine. Hence, α-keto acids were elevated in PDO-conditioned medium derived from early recurrent patients. We therefore speculate that an increased anaplerotic metabolism fuels the Krebs-cycle and a corresponding higher accessibility to energy fastens the recurrence in PDAC patients. Therein, a therapeutic intervention could delay PDAC recurrence and prolong survival of affected patients or could serve as biomarker to predict recurrence in the future.

Cells grown in three-dimensional spheroids mirror in vivo metabolic response of epithelial cells.

Metabolism in cells adapts quickly to changes in nutrient availability and cellular differentiation status, including growth conditions in cell culture settings. The last decade saw a vast increase in three-dimensional (3D) cell culture techniques, engendering spheroids and organoids. These methods were established to improve comparability to in vivo situations, differentiation processes and growth modalities. How far spheroids mimic in vivo metabolism, however, remains enigmatic. Here, to our knowledge, we compare for the first time metabolic fingerprints between cells grown as a single layer or as spheroids with freshly isolated in situ tissue. While conventionally grown cells express elevated levels of glycolysis intermediates, amino acids and lipids, these levels were significantly lower in spheroids and freshly isolated primary tissues. Furthermore, spheroids differentiate and start to produce metabolites typical for their tissue of origin. 3D grown cells bear many metabolic similarities to the original tissue, recommending animal testing to be replaced by 3D culture techniques.

Metabolic Adaptation during nab-Paclitaxel Resistance in Pancreatic Cancer Cell Lines.

Pancreatic ductal adenocarcinoma (PDAC) correlates with high mortality and is about to become one of the major reasons for cancer-related mortality in the next decades. One reason for that high mortality is the limited availability of effective chemotherapy as well as the intrinsic or acquired resistance against it. Here, we report the impact of nab-paclitaxel on the cellular metabolome of PDAC cell lines. After establishment of nab-paclitaxel resistant cell lines, comparison of parental and resistant PDAC cell lines by metabolomics and biochemical assessments revealed altered metabolism, enhanced viability and reduced apoptosis. The results unveiled that acute nab-paclitaxel treatment affected primary metabolism to a minor extent. However, acquisition of resistance led to altered metabolites in both cell lines tested. Specifically, aspartic acid and carbamoyl-aspartic acid were differentially abundant, which might indicate an increased de novo pyrimidine synthesis. This pathway has already shown a similar behavior in other cancerous entities and thus might serve in the future as vulnerable target fighting resistance acquisition occurring in common malignancies.

Impact of Diabetic Stress Conditions on Renal Cell Metabolome.

Diabetic kidney disease is a major complication in diabetes mellitus, and the most common reason for end-stage renal disease. Patients suffering from diabetes mellitus encounter glomerular damage by basement membrane thickening, and develop albuminuria. Subsequently, albuminuria can deteriorate the tubular function and impair the renal outcome. The impact of diabetic stress conditions on the metabolome was investigated by untargeted gas chromatography-mass spectrometry (GC-MS) analyses. The results were validated by qPCR analyses. In total, four cell lines were tested, representing the glomerulus, proximal nephron tubule, and collecting duct. Both murine and human cell lines were used. In podocytes, proximal tubular and collecting duct cells, high glucose concentrations led to global metabolic alterations in amino acid metabolism and the polyol pathway. Albumin overload led to the further activation of the latter pathway in human proximal tubular cells. In the proximal tubular cells, aldo-keto reductase was concordantly increased by glucose, and partially increased by albumin overload. Here, the combinatorial impact of two stressful agents in diabetes on the metabolome of kidney cells was investigated, revealing effects of glucose and albumin on polyol metabolism in human proximal tubular cells. This study shows the importance of including highly concentrated albumin in in vitro studies for mimicking diabetic kidney disease.