High Glycolytic Activity Research Articles

High Glycolytic Activity Signature Reveals CCNB2 as a Key Therapeutic Target in Triple-Negative Breast Cancer.

Aerobic glycolysis and the cell cycle are well-established tumor hallmarks. Understanding their relationship could help to unravel the pathogenic mechanisms of breast cancer (BC) and suggest potential new strategies for treatment. Glycolysis-related genes (GRGs) were downloaded from the Reactome database and screened using univariate Cox analysis. The consensus clustering method was employed to identify a glycolytic activity signature (GAS) using the Gene Expression Omnibus (GEO) dataset. A nomogram risk prediction model was constructed using coefficients from univariate Cox analysis. Immune cell infiltration was evaluated using single-sample gene set enrichment analysis (ssGSEA) and the ESTIMATE algorithm. Gene co-expression modules were created using weighted correlation network analysis (WGCNA) to identify hub genes. Gene expression in three BC cell lines was quantified using Quantitative Reverse Transcriptase Polymera (qRT-PCR). Single-cell RNA sequencing (scRNA-seq) data was used to examine the relationship between GAS and hub genes. The sensitivity of different groups to cell cycle-related clinical drugs was also examined. BC with high GAS (HGAS) showed high tumor grade and recurrence rate. HGAS was a prognostic indicator of worse overall survival (OS) in BC patients. HGAS BC showed more abundant immune cells and significantly higher expression of immunomodulators compared to BC with low GAS (LGAS). HGAS BC also showed enhanced cell cycle pathway, with high mRNA and protein expression levels of Cyclin B2 (CCNB2), a key component of the cell cycle pathway. Importantly, scRNA-seq analysis revealed that elevated CCNB2 expression was positively correlated with HGAS in triple-negative BC (TNBC). This was validated in clinical samples from TNBC patients. High expression of CCNB2 was found in three BC cell lines, and was also an indicator of poor prognosis. HGAS BC showed high sensitivity to several cell cycle-related clinical drugs, with 9 of these also showing activity in BC with high CCNB2 expression. HGAS was associated with enhanced cell cycle pathway and immune activity in BC. These results suggest that CCNB2 is a potential key therapeutic target in BC patients.

G2A as a key modulator of carbonyl stress and apoptosis resistance in glucose-loaded cancer cells

Cancer cells exhibit high glycolytic activity, metabolizing glucose as their primary energy substrate. Toxic metabolites produced during glycolysis, such as methylglyoxal, induce carbonyl stress (CS), promoting inflammation and oxidative stress. The elevated glucose metabolism in cancer cells creates this toxic environment. However, little research has focused on the molecules mediating these reactions and stresses, and their role in selecting and enriching apoptosis-resistant cells. This study investigated the impact of constitutively suppressing oxidized lipid receptor G2A (GPR132) expression on the relationship between CS and oxidative stress in glucose-loaded cancer cells. G2A has recently attracted attention as a tumor promoter. However, our study shows that G2A suppression under glucose loading significantly reduces CS and associated oxidative stress, thereby enhancing cancer cell survival. This suggests a new mechanism contrary to conventional thinking, involving the acute induction of glyoxalase 1 (Glo1). G2A may thus play a role in selecting and enriching apoptosis-resistant cell populations under high glucose conditions by regulating Glo1 expression. These findings improve our understanding of the adaptive capacity of cancer cells to glucose toxicity.

Phosphorylation of PFKL regulates metabolic reprogramming in macrophages following pattern recognition receptor activation

Innate immune responses are linked to key metabolic pathways, yet the proximal signaling events that connect these systems remain poorly understood. Here we show that phosphofructokinase 1, liver type (PFKL), a rate-limiting enzyme of glycolysis, is phosphorylated at Ser775 in macrophages following several innate stimuli. This phosphorylation increases the catalytic activity of PFKL, as shown by biochemical assays and glycolysis monitoring in cells expressing phosphorylation-defective PFKL variants. Using a genetic mouse model in which PFKL Ser775 phosphorylation cannot take place, we observe that upon activation, glycolysis in macrophages is lower than in the same cell population of wild-type animals. Consistent with their higher glycolytic activity, wild-type cells have higher levels of HIF1α and IL-1β than PfklS775A/S775A after LPS treatment. In an in vivo inflammation model, PfklS775A/S775A mice show reduced levels of MCP-1 and IL-1β. Our study thus identifies a molecular link between innate immune activation and early induction of glycolysis.

On the Importance of Acidity in Cancer Cells and Therapy.

Cancer cells are associated with high glycolytic activity, which results in acidification of the tumor microenvironment. The occurrence of this stressful condition fosters tumor aggressiveness, with the outcome of invasiveness and metastasis that are linked to a poor clinical prognosis. Acidosis can be both the cause or consequence of alterations in the functions and expressions of transporters involved in intracellular acidity regulation. This review aims to explore the origin of acidity in cancer cells and the various mechanisms existing in tumors to resist, survive, or thrive in the acidic environment. It highlights the difficulties in measuring the intracellular pH evolution that impedes our understanding of the many regulatory and feedback mechanisms. It finally presents the consequences of acidity on tumor development as well as the friend or foe role of acidity in therapy.

Abstract 6205: Identify drugs for metastatic castration-resistance prostate cancer (mCRPC) patients with high glycolytic activity through in silico approach

Abstract Prostate cancer (PC) remains a prominent cause of cancer-related mortality among men in the United States. A significant factor contributing to the mortality of PC patients is the development of resistance to second-generation antiandrogen therapies, particularly androgen receptor-signaling inhibitors (ARSI), which is pronounced in patients with late-stage PC, metastatic castration-resistance prostate cancer (mCRPC). Therefore, there is an urgent need for novel therapeutics beyond AR signaling inhibition for patients with mCRPC. One of the key drivers of resistance to ARSI and a critical feature of CRPC is cancer metabolism reprogramming to elevated glycolysis. However, the current research on finding drugs to impede high-glycolytic tumor growth is hurdled by a resistance raised from metabolic plasticity. The aim of this research is to quickly repurpose existing drugs for inhibiting high-glycolytic mCRPC growth while proactively circumstancing resistance from metabolic plasticity through computational approaches. To do this, we imputed a large number of drug responses in patients with mCRPC using a computational algorithm (OncoPredict), identified drugs that were projected to show higher sensitivity in high-glycolytic tumors, and performed in vitro validation. Furthermore, to maximize drug efficacy, we identified biomarkers for drug candidates. By implementing this computational pipeline, we identified a number of drugs with various mechanisms of action that show higher efficacy in mCRPC with high glycolysis. Furthermore, we successfully validated the efficacy of these nominated drug candidates including an anti-parasitic agent, an HSP90 inhibitor, and a CDK inhibitor. These drug candidates exhibited superior tumor growth inhibition in in vitro models of high glycolysis-induced advanced PC. In conclusion, this study introduces an innovative in silico approach to unveil the existing drugs’ unknown potential for the treatment of patients with mCRPC. Our findings hold promise for development of effective therapeutics for combating this challenging disease. Citation Format: Mei-Chi Su, Adam M. Lee, R. Stephanie Huang. Identify drugs for metastatic castration-resistance prostate cancer (mCRPC) patients with high glycolytic activity through in silico approach [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6205.

Targeting tumor-intrinsic SLC16A3 to enhance anti-PD-1 efficacy via tumor immune microenvironment reprogramming

Immunotherapy, especially immune checkpoint inhibitors, has revolutionized clinical practice within the last decade. However, primary and secondary resistance to immunotherapy is common in patients with diverse types of cancer. It is well-acknowledged that tumor cells can facilitate the formation of immunosuppressive microenvironments via metabolism reprogramming, and lactic acid, the metabolite of glycolysis, is a significant contributor. SLC16A3 (also named as MCT4) is a transporter mediating lactic acid efflux. In this study, we investigated the role of glycolysis in immunotherapy resistance and aimed to improve the immunotherapy effects via Slc16a3 inhibition. Bioinformatical analysis revealed that the expression of glycolysis-related genes correlated with less CD8+ T cell infiltration and increased myeloid-derived suppressor cells (MDSC) enrichment. We found that high glycolytic activity in tumor cells adversely affected the antitumor immune responses and efficacy of immunotherapy and radiotherapy. As the transporter of lactic acid, SLC16A3 is highly expressed in glycolytic B16–F10 (RRID: CVCL_0159) cells, as well as human non-small cell lung carcinoma. We validated that Slc16a3 expression in tumor cells negatively correlated with anti-PD-1 efficiency. Overexpression of Slc16a3 in tumor cells promoted lactic acid production and efflux, and reduced tumor response to anti-PD-1 inhibitors by inhibiting CD8+ T cell function. Genetic and pharmacological inhibition of Slc16a3 dramatically reduced the glycolytic activity and lactic acid production in tumor cells, and ameliorated the immunosuppressive tumor microenvironments (TMEs), leading to boosted antitumor effects via anti-PD-1 blockade. Our study therefore demonstrates that tumor cell-intrinsic SLC16A3 may be a potential target to reverse tumor resistance to immunotherapy.

It's a Trap! Aldolase-Prescribed C4 Deoxyradiofluorination Affords Intracellular Trapping and the Tracing of Fructose Metabolism by PET.

Fructose metabolism has been implicated in various diseases, including metabolic disorders, neurodegenerative disorders, cardiac disorders, and cancer. However, the limited availability of a quantitative imaging radiotracer has hindered its exploration in pathology and diagnostic imaging. Methods: We adopted a molecular design strategy based on the catalytic mechanism of aldolase, a key enzyme in fructolysis. We successfully synthesized a radiodeoxyfluorinated fructose analog, [18F]4-fluoro-4-deoxyfructose ([18F]4-FDF), in high molar activity. Results: Through heavy isotope tracing by mass spectrometry, we demonstrated that C4-deoxyfluorination of fructose led to effective trapping as fluorodeoxysorbitol and fluorodeoxyfructose-1-phosphate invitro, unlike C1- and C6-fluorinated analogs that resulted in fluorolactate accumulation. This observation was consistent invivo, where [18F]6-fluoro-6-deoxyfructose displayed substantial bone uptake due to metabolic processing whereas [18F]4-FDF did not. Importantly, [18F]4-FDF exhibited low uptake in healthy brain and heart tissues, known for their high glycolytic activity and background levels of [18F]FDG uptake. [18F]4-FDF PET/CT allowed for sensitive mapping of neuro- and cardioinflammatory responses to systemic lipopolysaccharide administration. Conclusion: Our study highlights the significance of aldolase-guided C4 radiodeoxyfluorination of fructose in enabling effective radiotracer trapping, overcoming limitations of C1 and C6 radioanalogs toward a clinically viable tool for imaging fructolysis in highly glycolytic tissues.

Comparative responses of flocculating and nonflocculating yeasts to cell density and chemical stress in lactic acid fermentation.

While flocculation has demonstrated its efficacy in enhancing yeast robustness and ethanol production, its potential application for lactic acid fermentation remains largely unexplored. Our study examined the differences between flocculating and nonflocculating Saccharomyces cerevisiae strains in terms of their metabolic dynamics when incorporating an exogenous lactic acid pathway, across varying cell densities and in the presence of lignocellulose-derived byproducts. Comparative gene expression profiles revealed that cultivating a nonflocculant strain at higher cell density yielded a substantial upregulation of genes associated with glycolysis, energy metabolism, and other key pathways, resulting in elevated levels of fermentation products. Meanwhile, the flocculating strain displayed an inherent ability to sustain high glycolytic activity regardless of the cell density. Moreover, our investigation revealed a significant reduction in glycolytic activity under chemical stress, potentially attributable to diminished ATP supply during the energy investment phase. Conversely, the formation of flocs in the flocculating strain conferred protection against toxic chemicals present in the medium, fostering more stable lactic acid production levels. Additionally, the distinct flocculation traits observed between the two examined strains may be attributed to variations in the nucleotide sequences of the flocculin genes and their regulators. This study uncovers the potential of flocculation for enhanced lactic acid production in yeast, offering insights into metabolic mechanisms and potential gene targets for strain improvement.

Metabolic Programming of Hematopoietic Stem Cell Function By Prenatal Folate

Hematopoietic stem cells (HSCs) are programmed by metabolic changes resulting in alterations to HSC function. Here, we discovered that varying prenatal folate status metabolically programs HSCs during development and affects adult HSC self-renewal and engraftment potential into adulthood. Folate status varies greatly in the global population based on nutritional intake, common genetic polymorphisms, and widespread supplementation. Folate-derived one-carbon metabolism (OCM) regulates cellular methylation, de novo nucleotide biosynthesis and mitochondrial metabolism, processes critical to HSC function and establishment. Despite immense therapeutic potential, there is limited understanding of mechanisms driving HSC developmental programming. We hypothesize that varying prenatal folate status affects risk for hematologic dysfunction by developmentally programming HSCs. To test how prenatal folate modulated HSC function, female mice were assigned to one of three experimental diets to model population-wide folate consumption: 0mg/kg (deficient, FD), 2mg/kg (control, FC) and 8mg/kg (supplemented, FS). Profiling of fetal liver (FL) hematopoietic cells revealed that FD diet caused expansion of long term (LT)-HSCs that was propagated across the hematopoietic hierarchy, including myeloid and lymphoid progenitors and mature myeloid cells. In contrast, FS decreased FL cell output. We next determined if these changes were driven by metabolic activity. FD fetal HSPCs exhibited higher mitochondrial membrane potential and lower glycolytic activity, whereas FS fetal HSPCs displayed both higher glycolytic activity and oxygen consumption despite impaired production of downstream hematopoietic cells. Together, these data suggest that HSPC metabolic programming initiates during fetal development and is folate-dose dependent. To determine if prenatal folate programs hematopoiesis into adulthood, we quantified bone marrow (BM) hematopoietic cells in adult offspring after weaning onto standard chow. Surprisingly, FD expanded lymphoid progenitors in adult offspring with no impact on mature lymphoid cell output. In contrast, FS diet expanded myeloid and lymphoid progenitors in the BM as well as downstream mature cells, including monocytes and B-cells. To determine if these effects were HSC cell-intrinsic, we performed competitive transplantation assays of LT-HSCs isolated from adult offspring. FD offspring HSCs showed worse engraftment by chimerism across all peripheral blood (PB) populations compared to FC. In comparison, FS offspring HSCs showed enhanced total cellular output by donor cells/uL across all PB mature lineages except platelets. Secondary whole BM transplantation revealed that FS HSCs retained enhanced total cellular output and superior engraftment compared to FC, whereas FD HSCs demonstrated complete exhaustion. These data indicate that adult HSC function is programmed by prenatal folate status. To investigate molecular alterations driving metabolic programming of adult hematopoiesis by prenatal folate, we performed single-cell RNA sequencing (Scseq) and metabolomics on adult offspring HSPCs. Scseq analysis revealed most differential gene expression (DE) in HSC clusters, with less DE in progenitors. Re-clustering of all HSCs revealed distinct heterogeneity within this population, where both FD and FS HSCs were identified as distinct clusters compared to FC. Metabolomic profiling further suggested that FD HSCs exhibited signs of mitochondrial dysfunction through, 1) accumulating glucose and lactate suggesting reliance on glycolysis and, 2) accumulating succinate and glutamine suggesting complex II impairment and inadequate utilization of mitochondrial metabolism. Surprisingly, the same changes associated with mitochondrial dysfunction in FD HSCs persisted downstream into lymphoid-biased multipotent progenitors. In contrast, limited metabolic changes were observed in FS HSCs, suggesting alternative pathways for sustained programming. These changes indicate that FD and FS offspring have distinct metabolic programming. Together, our data suggest that prenatal folate status programs hematopoietic stem cell function into adulthood and is driven by alterations to metabolic activity. Ongoing studies examine how prenatal folate status affects the adult hematological and immune response to challenge.

Characterization of the Intracellular Acidity Regulation of Brain Tumor Cells and Consequences for Therapeutic Optimization of Temozolomide.

A well-known feature of tumor cells is high glycolytic activity, leading to acidification of the tumor microenvironment through extensive lactate production. This acidosis promotes processes such as metastasis, aggressiveness, and invasiveness, which have been associated with a worse clinical prognosis. Moreover, the function and expression of transporters involved in regulation of intracellular pH might be altered. In this study, the capacity of tumor cells to regulate their intracellular pH when exposed to a range of pH from very acidic to basic was characterized in two glioma cell lines (F98 and U87) using a new recently published method of fluorescence imaging. Our results show that the regulation of acidity in tumors is not the same for the two investigated cell lines; U87 cells are able to reduce their intracellular acidity, whereas F98 cells do not exhibit this property. On the other hand, F98 cells show a higher level of resistance to acidity than U87 cells. Intracellular regulation of acidity appears to be highly cell-dependent, with different mechanisms activated to preserve cell integrity and function. This characterization was performed on 2D monolayer cultures and 3D spheroids. Spatial heterogeneities were exhibited in 3D, suggesting a spatially modulated regulation in this context. Based on the corpus of knowledge available in the literature, we propose plausible mechanisms to interpret our results, together with some new lines of investigation to validate our hypotheses. Our results might have implications on therapy, since the activity of temozolomide is highly pH-dependent. We show that the drug efficiency can be enhanced, depending on the cell type, by manipulating the extracellular pH. Therefore, personalized treatment involving a combination of temozolomide and pH-regulating agents can be considered.

Uncovering the Heterogeneity of Cardiac Lin-KIT+ Cells: A scRNA-seq Study on the Identification of Subpopulations.

The reparative potential of cardiac Lin-KIT+ (KIT) cells is influenced by their population, but identifying their markers is challenging due to changes in phenotype during in vitro culture. Resolving this issue requires uncovering cell heterogeneity and discovering new subpopulations. Single-cell RNA sequencing (scRNA-seq) can identify KIT cell subpopulations, their markers, and signaling pathways. We used 10× genomic scRNA-seq to analyze cardiac-derived cells from adult mice and found 3 primary KIT cell populations: KIT1, characterized by high-KIT expression (KITHI), represents a population of cardiac endothelial cells; KIT2, which has low-KIT expression (KITLO), expresses transcription factors such as KLF4, MYC, and GATA6, as well as genes involved in the regulation of angiogenic cytokines; KIT3, with moderate KIT expression (KITMOD), expresses the cardiac transcription factor MEF2C and mesenchymal cell markers such as ENG. Cell-cell communication network analysis predicted the presence of the 3 KIT clusters as signal senders and receivers, including VEGF, CXCL, and BMP signaling. Metabolic analysis showed that KIT1 has the low activity of glycolysis and oxidative phosphorylation (OXPHOS), KIT2 has high glycolytic activity, and KIT3 has high OXPHOS and fatty acid degradation activity, indicating distinct metabolic adaptations of the 3 KIT populations. Through the systemic infusion of KIT1 cells in a mouse model of myocardial infarction, we observed their involvement in promoting the formation of new micro-vessels. In addition, in vitro spheroid culture experiments demonstrated the cardiac differentiation capacity of KIT2 cells.

Transcriptomic analysis of mesocarp tissue during fruit development of the oil palm revealed specific isozymes related to starch metabolism that control oil yield.

The oil palm (Elaeis guineensis Jacq.) produces a large amount of oil from the fruit. However, increasing the oil production in this fruit is still challenging. A recent study has shown that starch metabolism is essential for oil synthesis in fruit-producing species. Therefore, the transcriptomic analysis by RNA-seq was performed to observe gene expression alteration related to starch metabolism genes throughout the maturity stages of oil palm fruit with different oil yields. Gene expression profiles were examined with three different oil yields group (low, medium, and high) at six fruit development phases (4, 8, 12, 16, 20, and 22 weeks after pollination). We successfully identified and analyzed differentially expressed genes in oil palm mesocarps during development. The results showed that the transcriptome profile for each developmental phase was unique. Sucrose flux to the mesocarp tissue, rapid starch turnover, and high glycolytic activity have been identified as critical factors for oil production in oil palms. For starch metabolism and the glycolytic pathway, we identified specific gene expressions of enzyme isoforms (isozymes) that correlated with oil production, which may determine the oil content. This study provides valuable information for creating new high-oil-yielding palm varieties via breeding programs or genome editing approaches.

Reversal of Lactate and PD-1-mediated Macrophage Immunosuppression Controls Growth of PTEN/p53-deficient Prostate Cancer.

Phosphatase and tensin homolog (PTEN) loss of function occurs in approximately 50% of patients with metastatic castrate-resistant prostate cancer (mCRPC), and is associated with poor prognosis and responsiveness to standard-of-care therapies and immune checkpoint inhibitors. While PTEN loss of function hyperactivates PI3K signaling, combinatorial PI3K/AKT pathway and androgen deprivation therapy (ADT) has demonstrated limited anticancer efficacy in clinical trials. Here, we aimed to elucidate mechanism(s) of resistance to ADT/PI3K-AKT axis blockade, and to develop rational combinatorial strategies to effectively treat this molecular subset of mCRPC. Prostate-specific PTEN/p53-deficient genetically engineered mice (GEM) with established 150-200 mm3 tumors, as assessed by ultrasound, were treated with either ADT (degarelix), PI3K inhibitor (copanlisib), or anti-PD-1 antibody (aPD-1), as single agents or their combinations, and tumors were monitored by MRI and harvested for immune, transcriptomic, and proteomic profiling, or ex vivo co-culture studies. Single-cell RNA sequencing on human mCRPC samples was performed using 10X Genomics platform. Coclinical trials in PTEN/p53-deficient GEM revealed that recruitment of PD-1-expressing tumor-associated macrophages (TAM) thwarts ADT/PI3Ki combination-induced tumor control. The addition of aPD-1 to ADT/PI3Ki combination led to TAM-dependent approximately 3-fold increase in anticancer responses. Mechanistically, decreased lactate production from PI3Ki-treated tumor cells suppressed histone lactylation within TAM, resulting in their anticancer phagocytic activation, which was augmented by ADT/aPD-1 treatment and abrogated by feedback activation of Wnt/β-catenin pathway. Single-cell RNA-sequencing analysis in mCRPC patient biopsy samples revealed a direct correlation between high glycolytic activity and TAM phagocytosis suppression. Immunometabolic strategies that reverse lactate and PD-1-mediated TAM immunosuppression, in combination with ADT, warrant further investigation in patients with PTEN-deficient mCRPC.

Lactate exposure shapes the metabolic and transcriptomic profile of CD8+ T cells.

CD8+ T cells infiltrate virtually every tissue to find and destroy infected or mutated cells. They often traverse varying oxygen levels and nutrient-deprived microenvironments. High glycolytic activity in local tissues can result in significant exposure of cytotoxic T cells to the lactate metabolite. Lactate has been known to act as an immunosuppressor, at least in part due to its association with tissue acidosis. To dissect the role of the lactate anion, independently of pH, we performed phenotypical and metabolic assays, high-throughput RNA sequencing, and mass spectrometry, on primary cultures of murine or human CD8+ T cells exposed to high doses of pH-neutral sodium lactate. The lactate anion is well tolerated by CD8+ T cells in pH neutral conditions. We describe how lactate is taken up by activated CD8+ T cells and can displace glucose as a carbon source. Activation in the presence of sodium lactate significantly alters the CD8+ T cell transcriptome, including the expression key effector differentiation markers such as granzyme B and interferon-gamma. Our studies reveal novel metabolic features of lactate utilization by activated CD8+ T cells, and highlight the importance of lactate in shaping the differentiation and activity of cytotoxic T cells.

Lactic acidosis induces metabolic and phenotypic reprogramming in cholangiocarcinoma cells via the upregulation of thrombospondin-1.

The high glycolytic activity of cancer cells leads to lactic acidosis (LA) in the tumor microenvironment. LA is not merely a consequence of metabolic activities but also has functional roles in metabolic reprogramming and cancer progression. Cholangiocarcinoma (CCA) cells exhibit a high dependency on glycolysis for survival and growth, but the specific effects of LA on cellular characteristics remain unknown. Here, we demonstrate that long-term LA (LLA) reprograms the metabolic phenotype of CCA cells from glycolytic to oxidative and enhances their migratory activity. In CCA cell culture, short-term LA (24 h) showed a growth inhibitory effect, while extended LA exposure for more than 2 weeks (LLA) led to enhanced cell motility. Coincidentally, LLA enhanced the respiratory capacity with an increase in mitochondrial mass. Inhibition of mitochondrial function abolished LLA-induced cell motility, suggesting that metabolic remodeling affects the phenotypic outcomes. RNA-sequencing analysis revealed that LLA upregulated genes associated with cell migration and epithelial-mesenchymal transition (EMT), including thrombospondin-1 (THBS1), which encodes a pro-EMT-secreted protein. Inhibition of THBS1 resulted in the suppression of both LLA-induced cell motility and respiratory capacity. Moreover, high THBS1 expression was associated with poor survival in patients with CCA. Collectively, our study suggests that the increased expression of THBS1 by LLA promotes phenotypic alterations, leading to CCA progression.

Von Willebrand factor: from figurant to main character in the scene of inflammation

von Willebrand factor: from figurant to main character in the scene of inflammation

Immunology of Giant Cell Arteritis.

Giant cell arteritis is an autoimmune disease of medium and large arteries, characterized by granulomatous inflammation of the three-layered vessel wall that results in vaso-occlusion, wall dissection, and aneurysm formation. The immunopathogenesis of giant cell arteritis is an accumulative process in which a prolonged asymptomatic period is followed by uncontrolled innate immunity, a breakdown in self-tolerance, the transition of autoimmunity from the periphery into the vessel wall and, eventually, the progressive evolution of vessel wall inflammation. Each of the steps in pathogenesis corresponds to specific immuno-phenotypes that provide mechanistic insights into how the immune system attacks and damages blood vessels. Clinically evident disease begins with inappropriate activation of myeloid cells triggering the release of hepatic acute phase proteins and inducing extravascular manifestations, such as muscle pains and stiffness diagnosed as polymyalgia rheumatica. Loss of self-tolerance in the adaptive immune system is linked to aberrant signaling in the NOTCH pathway, leading to expansion of NOTCH1+CD4+ T cells and the functional decline of NOTCH4+ T regulatory cells (Checkpoint 1). A defect in the endothelial cell barrier of adventitial vasa vasorum networks marks Checkpoint 2; the invasion of monocytes, macrophages and T cells into the arterial wall. Due to the failure of the immuno-inhibitory PD-1 (programmed cell death protein 1)/PD-L1 (programmed cell death ligand 1) pathway, wall-infiltrating immune cells arrive in a permissive tissues microenvironment, where multiple T cell effector lineages thrive, shift toward high glycolytic activity, and support the development of tissue-damaging macrophages, including multinucleated giant cells (Checkpoint 3). Eventually, the vascular lesions are occupied by self-renewing T cells that provide autonomy to the disease process and limit the therapeutic effectiveness of currently used immunosuppressants. The multi-step process deviating protective to pathogenic immunity offers an array of interception points that provide opportunities for the prevention and therapeutic management of this devastating autoimmune disease.

Metabolic reprogramming of cancer-associated fibroblasts in pancreatic cancer contributes to the intratumor heterogeneity of PET-CT

Intratumor heterogeneity of positron emission tomography-computed tomography (PET-CT) is reflected by variable 18F-fluorodeoxyglucose (FDG) uptake. Increasing evidence has shown that neoplastic and non-neoplastic components can affect the total 18F-FDG uptake in tumors. Cancer-associated fibroblasts (CAFs) is considered as the main non-neoplastic components in tumor microenvironment (TME) of pancreatic cancer. Our study aims to explore the impact of metabolic changes in CAFs on heterogeneity of PET-CT. A total of 126 patients with pancreatic cancer underwent PET-CT and endoscopic ultrasound elastography (EUS-EG) before treatment. High maximum standardized uptake value (SUVmax) from the PET-CT was positively correlated with the EUS-derived strain ratio (SR) and indicated poor prognosis of patients. In addition, single-cell RNA analysis showed that CAV1 affected glycolytic activity and correlated with glycolytic enzyme expression in fibroblasts in pancreatic cancer. We also observed the negative correlation between CAV1 and glycolytic enzyme expression in the tumor stroma by using immunohistochemistry (IHC) assay in the SUVmax-high and SUVmax-low groups of pancreatic cancer patients. Additionally, CAFs with high glycolytic activity contributed to pancreatic cancer cell migration, and blocking CAF glycolysis reversed this process, suggesting that glycolytic CAFs promote malignant biological behavior in pancreatic cancer. In summary, our research demonstrated that the metabolic reprogramming of CAFs affects total 18F-FDG uptake in tumors. Thus, an increase in glycolytic CAFs with decreased CAV1 expression promotes tumor progression, and high SUVmax may be a marker for therapy targeting the neoplastic stroma. Further studies should clarify the underlying mechanisms.

Activated B cells suppress T-cell function through metabolic competition

BackgroundB cells play a pivotal role in regulating the immune response. The induction of B cell-mediated immunosuppressive function requires B cell activating signals. However, the mechanisms by which activated B...

Cross-Talk between Hematopoietic Cells and Fibroblast Subsets Drives Inflammation and Remodelling of the Bone Marrow Microenvironment in Myeloproliferative Neoplasms

Introduction: In a proportion of patients with myeloproliferative neoplasms (MPNs), severe remodelling of the bone marrow microenvironment develops, with myelofibrosis (MF) and osteosclerosis leading to cytopenias and poor survival. Mesenchymal stromal cells (MSCs) have been implicated in MF pathogenesis (Leimkühler, Cell Stem Cell, 2021), including Nestin+ (Arranz, Nature, 2014), Gli1+ (Schneider, Cell Stem Cell, 2017) and Leptin-receptor+ MSCs (Decker, Nat Cell Bio, 2017). However, little is known about the cellular subtypes and transcriptional states of fibroblast subtypes in MF, and the key receptor-ligand (R-L) interactions that lead to their activation. Our aim was to build a fully comprehensive atlas of MF bone marrow and clarify the pathological interactions between MPN cells and their stromal environment to determine the key drivers of inflammation and fibrosis. Methods: We performed single cell RNA sequencing of mononuclear cells, hematopoietic stem/progenitor cells (HSPCs), CD41+ cells and CD45- Lin- Ter119- CD71low/- stromal cells from MF (MPLW515L) mice and controls, as well as CD45- Lin- CD235ab- CD71low/- stromal cells from MPN patients. Targets were validated using a novel human bone marrow organoid platform(Khan et al, bioRxiv, 2022) and by analysis of bone marrow biopsies from MPN patients and controls. Results: More than 75,000 cells were analysed from control and MF mice, capturing 42,319 hematopoietic cells (including HSPCs, myeloid, lymphoid, megakaryocytes) and 34,969 stromal cells, including fibroblasts, MSCs, osteoblasts, chondrocytes, pericytes, arterial and sinusoidal endothelial cells (Fig.1A). Erythroid, neutrophil, megakaryocytes and also eosinophil-basophil-mast (EBM) cells were increased in abundance in MF mice, together with a ~4 - 8 fold expansion of MSCs and a more modest increase in overall abundance of fibroblasts. Extracellular matrix factors were expressed mainly by stromal cells (MSCs, fibroblasts) but also by EBM cells and neutrophils. As reported (Leimkühler, Cell Stem Cell, 2021), expression of hematopoiesis support factors by MSCs was reduced in MF. However, prior studies have not captured fibroblast subtypes. We found that the reduction in niche support from MSCs was compensated by a significant increase in hematopoiesis support factors from fibroblasts and EBM cells. Despite minimal increase in fibroblast cells overall, a subset of inflammatory fibroblasts (iFib) with high metabolic and glycolytic activity, TNFα signaling via NF-κB and TGFβ signaling was dramatically expanded in the MF mice, and also detectable in stromal cells from MPN patients. This atlas enabled detailed study of aberrant cross-talk between hematopoietic and stromal compartments. Overall, receptor-ligand (R-L) interactions were 20% higher in MF mice due to increased signaling from megakaryocytes, EBM, MSC and iFib cells, implicating these four cellular subsets as key mediators of pathogenic cross-talk. R-L interaction analyses predicted increased TNFα signaling (TNFRSF1A → TNF) from EBM to both MSC and iFib, and increased TGFβ signaling (TGFBR2 → TGFB1) between EBM and megakaryocytes to MSC and fibroblast subtypes, including iFib. The expanded subsets of megakaryocytes and EBM cells were noted to express notably high levels of galectins LGALS1 and LGALS3, encoding soluble β-galactoside-binding proteins associated with inflammation and tumorigenesis, and galectin signaling via predicted interacting partners was significantly increased in MF. High LGALS1 correlated with significantly shorter survival in patients with myeloid malignancies in the Cancer Genome Atlas myeloid leukemia cohort. Inhibition of galectin-1 with OTX008 significantly reduced TGFβ-induced collagen 1 and αSMA expression by human bone marrow stromal cells in 2D cultures as well as in human iPSC-derived bone marrow organoids, and bone marrow biopsies from a cohort of MF and non-fibrotic ET/PV patients showed a striking association of galectin-1 with fibrosis (Fig.1B). Conclusions: We present a roadmap of the cellular and molecular landscape of normal and myelofibrotic bone marrow. We identify two cellular subsets (EBM and iFib) whose role as mediators of the inflammatory microenvironment in MF was previously unappreciated, and propose galectin-1 as a novel biomarker and potential mediator of fibrosis. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal