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Abstract It is well established that cancer cells acquire energy via the Warburg effect and oxidative phosphorylation. Citrate is considered to play a crucial role in cancer metabolism by virtue of its production in the reverse Krebs cycle from glutamine. Here, we review the evidence that extracellular citrate is one of the key metabolites of the metabolic pathways present in cancer cells. We review the different mechanisms by which pathways involved in keeping redox balance respond to the need of intracellular citrate synthesis under different extracellular metabolic conditions. In this context, we further discuss the hypothesis that extracellular citrate plays a role in switching between oxidative phosphorylation and the Warburg effect while citrate uptake enhances metastatic activities and therapy resistance. We also present the possibility that organs rich in citrate such as the liver, brain and bones might form a perfect niche for the secondary tumour growth and improve survival of colonising cancer cells. Consistently, metabolic support provided by cancer-associated and senescent cells is also discussed. Finally, we highlight evidence on the role of citrate on immune cells and its potential to modulate the biological functions of pro- and anti-tumour immune cells in the tumour microenvironment. Collectively, we review intriguing evidence supporting the potential role of extracellular citrate in the regulation of the overall cancer metabolism and metastatic activity.

Background Solute carrier family 13 member 5 (SLC13A5) is a Na + -coupled citrate co-transporter that mediates entry of extracellular citrate into the cytosol. SLC13A5 inhibition has been proposed as a target for reducing progression of kidney disease. The aim of this study was to leverage the Mendelian randomization paradigm to gain insight into the effects of SLC13A5 inhibition in humans, towards prioritizing and informing clinical development efforts. Methods The primary Mendelian randomization analyses investigated the effect of SLC13A5 inhibition on measures of kidney function, including creatinine and cystatin C-based measures of estimated glomerular filtration rate (creatinine-eGFR and cystatin C-eGFR), blood urea nitrogen (BUN), urine albumin-creatinine ratio (uACR), and risk of chronic kidney disease and microalbuminuria. Secondary analyses included a paired plasma and urine metabolome-wide association study, investigation of secondary traits related to SLC13A5 biology, a phenome-wide association study (PheWAS), and a proteome-wide association study. All analyses were compared to the effect of genetically predicted plasma citrate levels using variants selected from across the genome, and statistical sensitivity analyses robust to the inclusion of pleiotropic variants were also performed. Data were obtained from large-scale genetic consortia and biobanks, with sample sizes ranging from 5023 to 1,320,016 individuals. Results We found evidence of associations between genetically proxied SLC13A5 inhibition and higher creatinine-eGFR ( p  = 0.002), cystatin C-eGFR ( p  = 0.005), and lower BUN ( p  = 3 × 10 −4 ). Statistical sensitivity analyses robust to the inclusion of pleiotropic variants suggested that these effects may be a consequence of higher plasma citrate levels. There was no strong evidence of associations of genetically proxied SLC13A5 inhibition with uACR or risk of CKD or microalbuminuria. Secondary analyses identified evidence of associations with higher plasma calcium levels ( p  = 6 × 10 −13 ) and lower fasting glucose ( p  = 0.02). PheWAS did not identify any safety concerns. Conclusions This Mendelian randomization analysis provides human-centric insight to guide clinical development of an SLC13A5 inhibitor. We identify plasma calcium and citrate as biologically plausible biomarkers of target engagement, and plasma citrate as a potential biomarker of mechanism of action. Our human genetic evidence corroborates evidence from various animal models to support effects of SLC13A5 inhibition on improving kidney function.

Reduced expression of the plasma membrane citrate transporter SLC13A5, also known as INDY, has been linked to increased longevity and mitigated age-related cardiovascular and metabolic diseases. Citrate, a vital component of the tricarboxylic acid cycle, constitutes 1–5% of bone weight, binding to mineral apatite surfaces. Our previous research highlighted osteoblasts’ specialized metabolic pathway facilitated by SLC13A5 regulating citrate uptake, production, and deposition within bones. Disrupting this pathway impairs bone mineralization in young mice. New Mendelian randomization analysis using UK Biobank data indicated that SNPs linked to reduced SLC13A5 function lowered osteoporosis risk. Comparative studies of young (10 weeks) and middle-aged (52 weeks) osteocalcin-cre-driven osteoblast-specific Slc13a5 knockout mice (Slc13a5cKO) showed a sexual dimorphism: while middle-aged females exhibited improved elasticity, middle-aged males demonstrated enhanced bone strength due to reduced SLC13A5 function. These findings suggest reduced SLC13A5 function could attenuate age-related bone fragility, advocating for SLC13A5 inhibition as a potential osteoporosis treatment.

Mammalian INDY (mINDY, NaCT, gene symbol SLC13A5) is a potential target for the treatment of metabolically associated fatty liver disease (MAFLD). This study evaluated the effects of a selective, cross-species active, non-competitive, non-substrate-like inhibitor of NaCT. First, the small molecule inhibitor ETG-5773 was evaluated for citrate and succinate uptake and fatty acid synthesis in cell lines expressing both human NaCT and mouse Nact. Once its suitability was established, the inhibitor was evaluated in a diet-induced obesity (DIO) mouse model. DIO mice treated with 15 mg/kg compound ETG-5773 twice daily for 28 days had reduced body weight, fasting blood glucose, and insulin, and improved glucose tolerance. Liver triglycerides were significantly reduced, and body composition was improved by reducing fat mass, supported by a significant reduction in the expression of genes for lipogenesis such as SREBF1 and SCD1. Most of these effects were also evident after a seven-day treatment with the same dose. Further mechanistic investigation in the seven-day study showed increased plasma β-hydroxybutyrate and activated hepatic adenosine monophosphate-activated protein kinase (AMPK), reflecting findings from Indy (−/−) knockout mice. These results suggest that the inhibitor ETG-5773 blocked citrate uptake mediated by mouse and human NaCT to reduce liver steatosis and body fat and improve glucose regulation, proving the concept of NaCT inhibition as a future liver treatment for MAFLD.

We have recently shown that cancer cells of various origins take up extracellular citrate through the plasma membrane citrate carrier (pmCiC), a specific plasma membrane citrate transporter. Extracellular citrate is required to support cancer cell metabolism, in particular fatty acid synthesis, mitochondrial activity, protein synthesis and histone acetylation. In addition, cancer cells tend to acquire a metastatic phenotype in the presence of extracellular citrate. Our recent study also showed that cancer-associated stromal cells synthesise and release citrate and that this process is controlled by cancer cells. In the present study, we evaluated the expression of pmCiC, fibroblast activation protein-α (FAP) and the angiogenesis marker cluster of differentiation 31 (CD31) in human cancer tissues of different origins. In the cohort studied, we found no correlation between disease stage and the expression of FAP or CD31. However, we have identified a clear correlation between pmCiC expression in cancer cells and cancer-associated stroma with tumour stage. It can be concluded that pmCiC is increased in cancer cells and in cancer-supporting cells in the tumour microenvironment at the later stages of cancer development, particularly at the metastatic sites. Therefore, pmCiC expression has the potential to serve as a prognostic marker, although further studies are needed.

Background: Corneal inflammatory hem- and lymphangiogenesis significantly increase the risk for immune rejection after subsequent allogeneic corneal transplantation. The purpose of this study was to analyze the impact of temporary selective inhibition of lymphangiogenesis after transplantation on graft survival. Methods: Allogeneic transplantation from C57BL/6 mice to BalbC mice was performed as “high-risk” keratoplasty in a prevascularized corneal host bed (suture-induced inflammatory corneal neovascularization). The treatment group received integrin α5β1-blocking small molecules (JSM6427) at the time of transplantation and for two weeks afterwards. Control mice received a vehicle solution. Grafts were evaluated weekly for graft rejection using an opacity score. At the end of the follow-up, immunohistochemical staining of corneal wholemounts for lymphatic vessels as well as CD11b+ immune cells was performed. Results: Temporary postoperative inhibition of lymphangiogenesis by JSM6427 improved the corneal graft survival significantly. At the end of the follow-up, no significant reduction in CD11b+ immunoreactive cells within the graft compared to controls was found. Conclusions: The significant improvement of corneal graft survival by the selective, temporary postoperative inhibition of lymphangiogenesis after keratoplasty using integrin antagonists shows the impact of lymphatic vessels in the early postoperative phase. Retarding lymphatic vessel ingrowth into the graft might be sufficient for the shift to immunological tolerance in the postoperative period, even after high-risk keratoplasty.

Reduced expression of the plasma membrane citrate transporter SLC13A5, also known as INDY, has been linked to increased longevity and mitigated age-related cardiovascular and metabolic diseases. Citrate, a vital component of the tricarboxylic acid cycle, constitutes 1–5% of bone weight, binding to mineral apatite surfaces. Our previous research highlighted osteoblasts’ specialized metabolic pathway facilitated by SLC13A5 regulating citrate uptake, production, and deposition within bones. Disrupting this pathway impairs bone mineralization in young mice. New Mendelian randomization analysis using UK Biobank data indicated that SNPs linked to reduced SLC13A5 function lowered osteoporosis risk. Comparative studies of young (10 weeks) and middle-aged (52 weeks) osteocalcin-cre-driven osteoblast-specific Slc13a5 knockout mice (Slc13a5cKO) showed a sexual dimorphism: while middle-aged females exhibited improved elasticity, middle-aged males demonstrated enhanced bone strength due to reduced SLC13A5 function. These findings suggest reduced SLC13A5 function could attenuate age-related bone fragility, advocating for SLC13A5 inhibition as a potential osteoporosis treatment.