The TNF Family of Ligands and Receptors: Communication Modules in the Immune System and Beyond.

The tumor necrosis factor (TNF) and TNF receptor (TNFR) superfamilies (TNFSF/TNFRSF) include 19 ligands and 29 receptors that play important roles in the modulation of cellular functions. The communication pathways mediated by TNFSF/TNFRSF are essential for numerous developmental, homeostatic, and stimulus-responsive processes in vivo. TNFSF/TNFRSF members regulate cellular differentiation, survival, and programmed death, but their most critical functions pertain to the immune system. Both innate and adaptive immune cells are controlled by TNFSF/TNFRSF members in a manner that is crucial for the coordination of various mechanisms driving either co-stimulation or co-inhibition of the immune response. Dysregulation of these same signaling pathways has been implicated in inflammatory and autoimmune diseases, highlighting the importance of their tight regulation. Investigation of the control of TNFSF/TNFRSF activities has led to the development of therapeutics with the potential to reduce chronic inflammation or promote anti-tumor immunity. The study of TNFSF/TNFRSF proteins has exploded over the last 30 yr, but there remains a need to better understand the fundamental mechanisms underlying the molecular pathways they mediate to design more effective anti-inflammatory and anti-cancer therapies.

Quantification of 782 Plasma Peptides by Multiplexed Targeted Proteomics.

Blood analysis is one of the foundations of clinical diagnostics. In recent years, the analysis of proteins in blood samples by mass spectrometry has taken a jump forward in terms of sensitivity and the number of identified proteins. The recent development of parallel reaction monitoring with parallel accumulation and serial fragmentation (prm-PASEF) combines ion mobility as an additional separation dimension. This increases the proteome coverage while allowing the use of shorter chromatographic gradients. To demonstrate the method's full potential, we used an isotope-labeled synthetic peptide mix of 782 peptides, derived from 579 plasma proteins, spiked into blood plasma samples with a prm-PASEF measurement allowing the quantification of 565 plasma proteins by targeted proteomics. As a less time-consuming alternative to the prm-PASEF method, we describe guided data independent acquisition (dia)-PASEF (g-dia-PASEF) and compare its application to prm-PASEF for measuring blood plasma. To demonstrate both methods' performance in clinical samples, 20 patient plasma samples from a colorectal cancer (CRC) cohort were analyzed. The analysis identified 14 differentially regulated proteins between the CRC patient and control individual plasma samples. This shows the technique's potential for the rapid and unbiased screening of blood proteins, abolishing the need for the preselection of potential biomarker proteins.

Highly Multiplexed Targeted Proteomics Acquisition on a TIMS-QTOF.

Targeted proteomics allows the highly sensitive detection of specific peptides and proteins in complex biological samples. Here, we describe a methodology for targeted peptide quantification using a trapped ion mobility quadrupole time-of-flight mass spectrometer (timsTOF Pro). The prm-PASEF method exploits the multiplexing capability provided by the trapped ion mobility separation, allowing more than 200 peptides to be monitored over a 30 min liquid chromatography separation. Compared to conventional parallel reaction monitoring (PRM), precursor ions are accumulated in the trapped ion mobility spectrometry (TIMS) cells and separated according to their shape and charge before eluting into the quadrupole time-of-flight (QTOF) part of the mass spectrometer. The ion mobility trap allows measuring up to six peptides from a single 100 ms ion mobility separation with the current setup. Using these improved mass spectrometric capabilities, we detected and quantified 216 isotope-labeled synthetic peptides (AQUA peptides) spiked in HeLa human cell extract with limits of quantification of 17.2 amol for some peptides. The acquisition method is highly reproducible between injections and enables accurate quantification in biological samples, as demonstrated by quantifying KRas, NRas, and HRas as well as several Ras mutations in lung and colon cancer cell lines on fast 10 min gradient separations.

In search of definitions: Cancer-associated fibroblasts and their markers.

The tumor microenvironment has been identified as one of the driving factors of tumor progression and invasion. Inside this microenvironment, cancer-associated fibroblasts (CAFs), a type of perpetually activated fibroblasts, have been implicated to have a strong tumor-modulating effect and play a key role in areas such as drug resistance. Identification of CAFs has typically been carried based on the expression of various "CAF markers", such as fibroblast activation protein alpha (FAP) and alpha smooth muscle actin (αSMA), which separates them from the larger pool of fibroblasts present in the body. However, as outlined in this Review, the expression of various commonly used fibroblast markers is extremely heterogeneous and varies strongly between different CAF subpopulations. As such, novel selection methods based on cellular function, as well as further characterizing research, are vital for the standardization of CAF identification in order to improve the cross-applicability of different research studies in the field. The aim of this review is to give a thorough overview of the commonly used fibroblast markers in the field and their various strengths and, more importantly, their weaknesses, as well as to highlight potential future avenues for CAF identification and targeting.

CD95-ligand on peripheral myeloid cells activates Syk kinase to trigger their recruitment to the inflammatory site.

Injury to the central nervous system initiates an uncontrolled inflammatory response that results in both tissue repair and destruction. Here, we showed that, in rodents and humans, injury to the spinal cord triggered surface expression of CD95 ligand (CD95L, FasL) on peripheral blood myeloid cells. CD95L stimulation of CD95 on these cells activated phosphoinositide 3-kinase (PI3K) and metalloproteinase-9 (MMP-9) via recruitment and activation of Syk kinase, ultimately leading to increased migration. Exclusive CD95L deletion in myeloid cells greatly decreased the number of neutrophils and macrophages infiltrating the injured spinal cord or the inflamed peritoneum after thioglycollate injection. Importantly, deletion of myeloid CD95L, but not of CD95 on neural cells, led to functional recovery of spinal injured animals. Our results indicate that CD95L acts on peripheral myeloid cells to induce tissue damage. Thus, neutralization of CD95L should be considered as a means to create a controlled beneficial inflammatory response.

Crosstalk between different family members: IL27 recapitulates IFNγ responses in HCC cells, but is inhibited by IL6-type cytokines.

Interleukin-27 (IL27) is a type-I-cytokine of the IL6/IL12 family predominantly secreted by activated macrophages and dendritic cells. In the liver, IL27 expression was observed to be upregulated in patients with hepatitis B, and sera of hepatocellular carcinoma (HCC) patients contain significantly elevated levels of IL27 compared to healthy controls or patients with hepatitis and/or liver cirrhosis. In this study, we show that IL27 induces STAT1 and STAT3 phosphorylation in 5 HCC lines and 3 different types of non-transformed liver cells. We were especially interested in the relevance of the IL27-induced STAT3 activation in liver cells. Thus, we compared the IL27 responses with those induced by IFNγ (STAT1-dominated response) or IL6-type cytokines (IL6, hyper-IL6 (hy-IL6) or OSM) (STAT3-dominated response) by microarray analysis and find that in HCC cells, IL27 induces an IFNγ-like, STAT1-dependent transcriptional response, but we do not find an effective STAT3-dependent response. Validation experiments corroborate the finding from the microarray evaluation. Interestingly, the availability of STAT1 seems critical in the shaping of the IL27 response, as the siRNA knock-down of STAT1 revealed the ability of IL27 to induce the acute-phase protein γ-fibrinogen, a typical IL6 family characteristic. Moreover, we describe a crosstalk between the signaling of IL6-type cytokines and IL27: responses to the gp130-engaging cytokine IL27 (but not those to IFNs) can be inhibited by IL6-type cytokine pre-stimulation, likely by a SOCS3-mediated mechanism. Thus, IL27 recapitulates IFNγ responses in liver cells, but differs from IFNγ by its sensitivity to SOCS3 inhibition.

A new ALK isoform transported by extracellular vesicles confers drug resistance to melanoma cells.

BACKGROUND: Drug resistance remains an unsolved clinical issue in oncology. Despite promising initial responses obtained with BRAF and MEK kinase inhibitors, resistance to treatment develops within months in virtually all melanoma patients. METHODS: Microarray analyses were performed in BRAF inhibitor-sensitive and resistant cell lines to identify changes in the transcriptome that might play a role in resistance. siRNA approaches and kinase inhibitors were used to assess the involvement of the identified Anaplastic Lymphoma Kinase (ALK) in drug resistance. The capability of extracellular vesicles (EVs) to transfer drug resistant properties was investigated in co-culture assays. RESULTS: Here, we report a new mechanism of acquired drug resistance involving the activation of a novel truncated form of ALK. Knock down or inhibition of ALK re-sensitised resistant cells to BRAF inhibition and induced apoptosis. Interestingly, truncated ALK was also secreted into EVs and we show that EVs were the vehicle for transferring drug resistance. CONCLUSIONS: To our knowledge, this is the first report demonstrating the functional involvement of EVs in melanoma drug resistance by transporting a truncated but functional form of ALK, able to activate the MAPK signalling pathway in target cells. Combined inhibition of ALK and BRAF dramatically reduced tumour growth in vivo. These findings make ALK a promising clinical target in melanoma patients.

Tumor-Initiating Cells: a criTICal review of isolation approaches and new challenges in targeting strategies.

Most cancers contain a subpopulation of highly tumorigenic cells, known as cancer stem cells (CSCs) or tumor-initiating cells (TICs). Targeting TICs may be essential to achieve cure, because of their self-renewal and tumorigenic properties as well as their resistance to conventional therapies. Despite significant advances in TIC biology, their isolation and identification remain largely disputed and incompletely established. In this review, we discuss the latest developments in isolation and culturing approaches of TICs, with focus on colorectal cancer (CRC). We feature recent findings on TIC-relevant signaling pathways and the metabolic identity of TICs, as well as their current clinical implications. Lastly, we highlight the influence of inter- and intra-tumoral heterogeneity on TIC function and targeting approaches.

Loss of Myosin Vb in colorectal cancer is a strong prognostic factor for disease recurrence.

BACKGROUND: Selecting the most beneficial treatment regimens for colorectal cancer (CRC) patients remains challenging due to a lack of prognostic markers. Members of the Myosin family, proteins recognised to have a major role in trafficking and polarisation of cells, have recently been reported to be closely associated with several types of cancer and might thus serve as potential prognostic markers in the context of CRC. METHODS: We used a previously established meta-analysis of publicly available gene expression data to analyse the expression of different members of the Myosin V family, namely MYO5A, 5B, and 5C, in CRC. Using laser-microdissected material as well as tissue microarrays from paired human CRC samples, we validated both RNA and protein expression of Myosin Vb (MYO5B) and its known adapter proteins (RAB8A and RAB25) in an independent patient cohort. Finally, we assessed the prognostic value of both MYO5B and its adapter-coupled combinatorial gene expression signatures. RESULTS: The meta-analysis as well as an independent patient cohort study revealed a methylation-independent loss of MYO5B expression in CRC that matched disease progression. Although MYO5B mutations were identified in a small number of patients, these cannot be solely responsible for the common downregulation observed in CRC patients. Significantly, CRC patients with low MYO5B expression displayed shorter overall, disease-, and metastasis-free survival, a trend that was further reinforced when RAB8A expression was also taken into account. CONCLUSIONS: Our data identify MYO5B as a powerful prognostic biomarker in CRC, especially in early stages (stages I and II), which might help stratifying patients with stage II for adjuvant chemotherapy.

An Organ-on-Chip Platform for Simulating Drug Metabolism Along the Gut-Liver Axis.

The human microbiome significantly influences drug metabolism through the gut-liver axis, leading to modified drug responses and potential toxicity. Due to the complex nature of the human gut environment, the understanding of microbiome-driven impacts on these processes is limited. To address this, a multiorgan-on-a-chip (MOoC) platform that combines the human microbial-crosstalk (HuMiX) gut-on-chip (GoC) and the Dynamic42 liver-on-chip (LoC), mimicking the bidirectional interconnection between the gut and liver known as the gut-liver axis, is introduced. This platform supports the viability and functionality of intestinal and liver cells. In a proof-of-concept study, the metabolism of irinotecan, a widely used colorectal cancer drug, is imitated within the MOoC. Utilizing liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS), irinotecan metabolites are tracked, confirming the platform's ability to represent drug metabolism along the gut-liver axis. Further, using the authors' gut-liver platform, it is shown that the colorectal cancer-associated gut bacterium, Escherichia coli, modifies irinotecan metabolism through the transformation of its inactive metabolite SN-38G into its toxic metabolite SN-38. This platform serves as a robust tool for investigating the intricate interplay between gut microbes and pharmaceuticals, offering a representative alternative to animal models and providing novel drug development strategies.

PKM2 diverts glycolytic flux in dependence on mitochondrial one-carbon cycle.

Modeling tumor metabolism in vitro remains challenging. Here, we used galactose as an in vitro tool compound to mimic glycolytic limitation. In contrast to the established idea that high glycolytic flux reduces pyruvate kinase isozyme M2 (PKM2) activity to support anabolic processes, we have discovered that glycolytic limitation also affects PKM2 activity. Surprisingly, despite limited carbon availability and energetic stress, cells induce a near-complete block of PKM2 to divert carbons toward serine metabolism. Simultaneously, TCA cycle flux is sustained, and oxygen consumption is increased, supported by glutamine. Glutamine not only supports TCA cycle flux but also serine synthesis via distinct mechanisms that are directed through PKM2 inhibition. Finally, deleting mitochondrial one-carbon (1C) cycle reversed the PKM2 block, suggesting a potential formate-dependent crosstalk that coordinates mitochondrial 1C flux and cytosolic glycolysis to support cell survival and proliferation during nutrient-scarce conditions.

Serine metabolism is crucial for cGAS-STING signaling and viral defense control in the gut.

Inflammatory bowel diseases are characterized by the chronic relapsing inflammation of the gastrointestinal tract. While the molecular causality between endoplasmic reticulum (ER) stress and intestinal inflammation is widely accepted, the metabolic consequences of chronic ER stress on the pathophysiology of IBD remain unclear. By using in vitro, in vivo models, and patient datasets, we identified a distinct polarization of the mitochondrial one-carbon metabolism and a fine-tuning of the amino acid uptake in intestinal epithelial cells tailored to support GSH and NADPH metabolism upon ER stress. This metabolic phenotype strongly correlates with IBD severity and therapy response. Mechanistically, we uncover that both chronic ER stress and serine limitation disrupt cGAS-STING signaling, impairing the epithelial response against viral and bacterial infection and fueling experimental enteritis. Consequently, the antioxidant treatment restores STING function and virus control. Collectively, our data highlight the importance of serine metabolism to allow proper cGAS-STING signaling and innate immune responses upon gut inflammation.

Formate promotes invasion and metastasis in reliance on lipid metabolism.

Metabolic rewiring is essential for cancer onset and progression. We previously showed that one-carbon metabolism-dependent formate production often exceeds the anabolic demand of cancer cells, resulting in formate overflow. Furthermore, we showed that increased extracellular formate concentrations promote the in vitro invasiveness of glioblastoma cells. Here, we substantiate these initial observations with ex vivo and in vivo experiments. We also show that exposure to exogeneous formate can prime cancer cells toward a pro-invasive phenotype leading to increased metastasis formation in vivo. Our results suggest that the increased local formate concentration within the tumor microenvironment can be one factor to promote metastases. Additionally, we describe a mechanistic interplay between formate-dependent increased invasiveness and adaptations of lipid metabolism and matrix metalloproteinase activity. Our findings consolidate the role of formate as pro-invasive metabolite and warrant further research to better understand the interplay between formate and lipid metabolism.

A Th17 cell-intrinsic glutathione/mitochondrial-IL-22 axis protects against intestinal inflammation.

Although the intestinal tract is a major site of reactive oxygen species (ROS) generation, the mechanisms by which antioxidant defense in gut T cells contribute to intestinal homeostasis are currently unknown. Here we show, using T cell-specific ablation of the catalytic subunit of glutamate cysteine ligase (Gclc), that the ensuing loss of glutathione (GSH) impairs the production of gut-protective IL-22 by Th17 cells within the lamina propria. Although Gclc ablation does not affect T cell cytokine secretion in the gut of mice at steady-state, infection with C. rodentium increases ROS, inhibits mitochondrial gene expression and mitochondrial function in Gclc-deficient Th17 cells. These mitochondrial deficits affect the PI3K/AKT/mTOR pathway, leading to reduced phosphorylation of the translation repressor 4E-BP1. As a consequence, the initiation of translation is restricted, resulting in decreased protein synthesis of IL-22. Loss of IL-22 results in poor bacterial clearance, enhanced intestinal damage, and high mortality. ROS-scavenging, reconstitution of IL-22 expression or IL-22 supplementation in vivo prevent the appearance of these pathologies. Our results demonstrate the existence of a previously unappreciated role for Th17 cell-intrinsic GSH coupling to promote mitochondrial function, IL-22 translation and signaling. These data reveal an axis that is essential for maintaining the integrity of the intestinal barrier and protecting it from damage caused by gastrointestinal infection.

Formate overflow drives toxic folate trapping in MTHFD1 inhibited cancer cells.

Cancer cells fuel their increased need for nucleotide supply by upregulating one-carbon (1C) metabolism, including the enzymes methylenetetrahydrofolate dehydrogenase-cyclohydrolase 1 and 2 (MTHFD1 and MTHFD2). TH9619 is a potent inhibitor of dehydrogenase and cyclohydrolase activities in both MTHFD1 and MTHFD2, and selectively kills cancer cells. Here, we reveal that, in cells, TH9619 targets nuclear MTHFD2 but does not inhibit mitochondrial MTHFD2. Hence, overflow of formate from mitochondria continues in the presence of TH9619. TH9619 inhibits the activity of MTHFD1 occurring downstream of mitochondrial formate release, leading to the accumulation of 10-formyl-tetrahydrofolate, which we term a 'folate trap'. This results in thymidylate depletion and death of MTHFD2-expressing cancer cells. This previously uncharacterized folate trapping mechanism is exacerbated by physiological hypoxanthine levels that block the de novo purine synthesis pathway, and additionally prevent 10-formyl-tetrahydrofolate consumption for purine synthesis. The folate trapping mechanism described here for TH9619 differs from other MTHFD1/2 inhibitors and antifolates. Thus, our findings uncover an approach to attack cancer and reveal a regulatory mechanism in 1C metabolism.

Evolution of the murine gut resistome following broad-spectrum antibiotic treatment.

The emergence and spread of antimicrobial resistance (AMR) represent an ever-growing healthcare challenge worldwide. Nevertheless, the mechanisms and timescales shaping this resistome remain elusive. Using an antibiotic cocktail administered to a murine model along with a longitudinal sampling strategy, we identify the mechanisms by which gut commensals acquire antimicrobial resistance genes (ARGs) after a single antibiotic course. While most of the resident bacterial populations are depleted due to the treatment, Akkermansia muciniphila and members of the Enterobacteriaceae, Enterococcaceae, and Lactobacillaceae families acquire resistance and remain recalcitrant. We identify specific genes conferring resistance against the antibiotics in the corresponding metagenome-assembled genomes (MAGs) and trace their origins within each genome. Here we show that, while mobile genetic elements (MGEs), including bacteriophages and plasmids, contribute to the spread of ARGs, integrons represent key factors mediating AMR in the antibiotic-treated mice. Our findings suggest that a single course of antibiotics alone may act as the selective sweep driving ARG acquisition and incidence in gut commensals over a single mammalian lifespan.

Cancer-Associated Fibroblast Diversity Shapes Tumor Metabolism in Pancreatic Cancer.

Despite extensive research, the 5-year survival rate of pancreatic cancer (PDAC) patients remains at only 9%. Patients often show poor treatment response, due partly to a highly complex tumor microenvironment (TME). Cancer-associated fibroblast (CAF) heterogeneity is characteristic of the pancreatic TME, where several CAF subpopulations have been identified, such as myofibroblastic CAFs (myCAFs), inflammatory CAFs (iCAFs), and antigen presenting CAFs (apCAFs). In PDAC, cancer cells continuously adapt their metabolism (metabolic switch) to environmental changes in pH, oxygenation, and nutrient availability. Recent advances show that these environmental alterations are all heavily driven by stromal CAFs. CAFs and cancer cells exchange cytokines and metabolites, engaging in a tight bidirectional crosstalk, which promotes tumor aggressiveness and allows constant adaptation to external stress, such as chemotherapy. In this review, we summarize CAF diversity and CAF-mediated metabolic rewiring, in a PDAC-specific context. First, we recapitulate the most recently identified CAF subtypes, focusing on the cell of origin, activation mechanism, species-dependent markers, and functions. Next, we describe in detail the metabolic crosstalk between CAFs and tumor cells. Additionally, we elucidate how CAF-driven paracrine signaling, desmoplasia, and acidosis orchestrate cancer cell metabolism. Finally, we highlight how the CAF/cancer cell crosstalk could pave the way for new therapeutic strategies.

Investigating children's chemical exposome - Description and possible determinants of exposure in the region of Luxembourg based on hair analysis.

The specific physiology and behaviour of children makes them particularly vulnerable to chemical exposure. Specific studies must therefore be conducted to understand the impact of pollution on children's health. Human biomonitoring is a reliable approach for exposure assessment, and hair, allowing the detection of parent chemicals and metabolites, and covering wider time windows than urine and blood is particularly adapted to study chronic exposure. The present study aims at assessing chemical exposure and investigating possible determinants of exposure in children living in Luxembourg. Hair samples were collected from 256 children below 13 y/o and tested for 153 compounds (140 pesticides, 4 PCBs, 7 BDEs and 2 bisphenols). Moreover, anthropometric parameters, information on diet, residence, and presence of pets at home was collected through questionnaires. Correlations, regressions, t-tests, PLS-DA and MANOVAs, were used to investigate exposure patterns. Twenty-nine to 88 (median = 61) compounds were detected per sample. The highest median concentration was observed for BPA (133.6 pg/mg). Twenty-three biomarkers were detected in ≥ 95% of the samples, including 13 in all samples (11 pesticides, BPA and BPS). Exposure was higher at younger ages (R2 = 0.57), and boys were more exposed to non-persistent pesticides than girls. Presence of persistent organic pollutants in most children suggests that exposure is still ongoing. Moreover, diet (e.g. imazalil: 0.33 pg/mg in organic, 1.15 pg/mg in conventional, p-value < 0.001), residence area (e.g. imidacloprid: 0.29 pg/mg in urban, 0.47 pg/mg in countryside, p-value = 0.03), and having pets (e.g. fipronil: 0.32 pg/mg in pets, 0.09 pg/mg in no pets, p-value < 0.001) were identified as determinants of exposure. The present study demonstrates that children are simultaneously exposed to multiple pollutants from different chemical classes, and confirms the suitability of hair to investigate exposure. These results set the basis for further investigations to better understand the determinants of chemical exposure in children.

Mitochondria preserve an autarkic one-carbon cycle to confer growth-independent cancer cell migration and metastasis.

Metastasis is the most common cause of death in cancer patients. Canonical drugs target mainly the proliferative capacity of cancer cells, which leaves slow-proliferating, persistent cancer cells unaffected. Metabolic determinants that contribute to growth-independent functions are still poorly understood. Here we show that antifolate treatment results in an uncoupled and autarkic mitochondrial one-carbon (1C) metabolism during cytosolic 1C metabolism impairment. Interestingly, antifolate dependent growth-arrest does not correlate with decreased migration capacity. Therefore, using methotrexate as a tool compound allows us to disentangle proliferation and migration to profile the metabolic phenotype of migrating cells. We observe that increased serine de novo synthesis (SSP) supports mitochondrial serine catabolism and inhibition of SSP using the competitive PHGDH-inhibitor BI-4916 reduces cancer cell migration. Furthermore, we show that sole inhibition of mitochondrial serine catabolism does not affect primary breast tumor growth but strongly inhibits pulmonary metastasis. We conclude that mitochondrial 1C metabolism, despite being dispensable for proliferative capacities, confers an advantage to cancer cells by supporting their motility potential.

The gut microbial metabolite formate exacerbates colorectal cancer progression.

The gut microbiome is a key player in the immunomodulatory and protumorigenic microenvironment during colorectal cancer (CRC), as different gut-derived bacteria can induce tumour growth. However, the crosstalk between the gut microbiome and the host in relation to tumour cell metabolism remains largely unexplored. Here we show that formate, a metabolite produced by the CRC-associated bacterium Fusobacterium nucleatum, promotes CRC development. We describe molecular signatures linking CRC phenotypes with Fusobacterium abundance. Cocultures of F. nucleatum with patient-derived CRC cells display protumorigenic effects, along with a metabolic shift towards increased formate secretion and cancer glutamine metabolism. We further show that microbiome-derived formate drives CRC tumour invasion by triggering AhR signalling, while increasing cancer stemness. Finally, F. nucleatum or formate treatment in mice leads to increased tumour incidence or size, and Th17 cell expansion, which can favour proinflammatory profiles. Moving beyond observational studies, we identify formate as a gut-derived oncometabolite that is relevant for CRC progression.