Content area
Urea cycle (UC) dysfunction drives tumorigenesis and poor prognosis, yet its role in tumor–stroma crosstalk is unclear. Here we show that colorectal cancer (CRC) cells reprogram UC metabolism in cancer-associated fibroblasts (CAFs) via CRC-derived exosomes. Reprogrammed CAFs support CRC cell growth by providing UC metabolites, especially arginine (Arg). Depriving CRC cells of Arg halts their growth and simultaneously increases their reliance on putrescine while up-regulating ornithine decarboxylase (ODC), the polyamine-biosynthesis gatekeeper. Our study illustrates the UC metabolic interaction between CAFs and CRC cells and demonstrates the potential therapeutic utility of Arg restriction and ODC blockade combination treatment for colorectal cancer.
Introduction
Cancer-associated fibroblasts (CAFs) represent one of the most prevalent stromal components in the tumor microenvironment (TME) and play a critical role in cancer progression [1]. Research has demonstrated that CAF accumulation correlates with decreased patient survival in colorectal cancer (CRC) [2]. Mounting evidence indicates that CAFs undergo metabolic reprogramming to facilitate cancer cell growth and metastatic spread [3]. Studies have shown that CAFs secrete energy-rich metabolites, including pyruvate and lactate, to sustain neighboring cancer cells [4]. Additionally, CAFs undergo lipidome reprogramming and, through metabolite-mediated crosstalk, enhance CRC cell migration [5]. Recent findings demonstrate that metabolically altered CAFs increase de novo fatty-acid biosynthesis and significantly enhance colorectal-cancer cell motility through secreted lipid mediators [6]. These observations have prompted renewed mechanistic investigation of CAF metabolic reprogramming within the TME.
Urea cycle (UC) dysregulation in numerous malignancies leads to enhanced anabolic synthesis of macromolecules essential for tumor proliferation and growth by optimizing nitrogen and carbon utilization [7, 8]. Research indicates significant activation of urea cycle metabolism during colorectal tumorigenesis [9]. Arginine (Arg), an intermediate of UC, influences multiple aspects of tumor metabolism, including the synthesis of polyamines, nitric oxide, nucleotides, proline, and glutamate [8, 10]. Studies reveal that various tumor types depend on extracellular Arg to maintain essential biological processes [11], prompting investigation into Arg's role in tumor-stroma crosstalk.
Polyamines, which are arginine-derived catabolites, interact with DNA, RNA, proteins, and phospholipids, thereby promoting nucleic-acid and protein synthesis to regulate cell growth and differentiation [12]. These compounds show ubiquitous dysregulation in cancer, and their accumulation proves essential for tumor progression [13]. Consequently, targeting polyamine metabolism represents a promising anticancer strategy. Ornithine decarboxylase (ODC) serves as the rate-limiting enzyme in polyamine biosynthesis [14]. Research indicates significant ODC upregulation in various cancers, including CRC [15]. Polyamine blockade therapy combines inhibition of polyamine biosynthesis with concurrent suppression of polyamine transport [16]. These insights have led to the development of selective ODC inhibitors. DFMO, an irreversible ODC inactivator, stands as the primary polyamine-targeted agent under evaluation in clinical trials [17, 18].
Here, we delineate a previously unknown UC metabolic axis between CRC cells and CAFs. CRC-derived exosomes reprogram CAFs to secrete UC intermediates—particularly arginine—sustaining adjacent tumor cells. Concomitant arginine depletion and ODC inhibition synergistically suppress CRC proliferation. Our findings establish a metabolic symbiosis within the CRC microenvironment and nominate a readily translatable combination therapy.
Here, we delineate a previously unknown UC metabolic axis between CRC cells and CAFs. CRC-derived exosomes reprogram CAFs to secrete UC intermediates—particularly arginine—sustaining adjacent tumor cells. Concomitant arginine depletion and ODC inhibition synergistically suppress CRC proliferation. Our findings establish a metabolic symbiosis within the CRC microenvironment and nominate a readily translatable combination therapy.
Methods
Cell culture
All of the cell lines were generously gifted by Prof. Mong-Hong Lee (The Sixth Affiliated Hospital of Sun Yat-sen University, Guang Zhou, China). HCT116 (RRID: CVCL_0291) and HT29 (RRID: CVCL_0320) cells were cultured in Dulbecco's modified Eagle's medium (DMEM), DLD1 (RRID: CVCL_0248) and HCT8 (RRID: CVCL_2478) cells were cultured in RPMI 1640. All medium were supplemented with 10% fetal bovine serum (FBS) and 1 × penicillin/streptomycin. Arg deprivation: Complete removal of arginine in the environment through exogenous means (such as ADI-PEG20, ARG-free medium) to bring its concentration close to zero. Arg restriction: Reduce the concentration of arginine in the environment to sub physiological levels (such as in Low-Arg media), but do not completely eliminate it. For Arg-deprived cultures (denoted as “Arg-”), cells were cultured in Arg-free DMEM (Meilunbio, MA0545) supplemented with 10% Dialyzed FBS (Biological Industries, 04–001) and 1 × penicillin/streptomycin. Low-Arg DMEM (denoted as “Low-Arg”) was made from a source stock containing all components of Arg-free DMEM (Meilunbio, MA0545) with arginine (SIGMA, A1270000, 50 mM). CRC cells cultured with nucleotide (Sigma-Aldrich; D7295) in control and Arg-free medium. All cultures were maintained in a humidified tissue culture incubator at 37 °C and 5% CO2. All cell lines were regularly checked for mycoplasma contamination, and no mycoplasma contamination was found.
Exosomes isolation and characterization
Exosomes were isolated from the colorectal cancer (CRC)-derived conditioned medium (CM) through ultracentrifugation. CRC cells (HCT116 and HCT8) were cultured in DMEM medium containing 10% FBS that had been cleared of exosomes through ultracentrifugation at 100,000 × g for 16 h at 4 °C. Conditioned media were harvested after 48 h and subjected to sequential centrifugation steps: 300 × g for 10 min at 4 °C, followed by 2,000 × g for 10 min at 4 °C, and then 10,000 g for 30 min at 4 °C. The supernatants underwent ultracentrifugation at 100,000 × g for 70 min at 4 °C. The exosomes were washed with PBS and underwent a second ultracentrifugation at 100,000 × g for 70 min at 4 °C before being resuspended in PBS. The concentration of exosomes was determined using the BCA Protein Assay kit (Thermo Scientific, 23,225). For cell treatment, 1 μg of exosomes were added to 1 × 105 CAFs. The characterization of exosomes was conducted at Obio Technology (shanghai) Corp., Ltd. Exosomes were visualized using transmission electron microscopy (TEM). The density and size of exosomes were analyzed through nanosight particle tracking analysis (NTA).
Isolation and culture of CAFs
To isolate CAFs, human CRC tissues were obtained from three distinct patient donors at the Sixth Affiliated Hospital of Sun Yat-sen University in Guang Zhou, China (Approval ID: 2020ZSLYEC-225). The primary CAFs were dissociated using Tumor Dissociation Kit (Miltenyi Biotec, Germany). Human CRC tissues were cut from surgical specimen and minced, transferred into the gentleMACS C Tube containing the enzyme mix (4.7 mL of DMEM, 200 μL Enzyme H, 100 μL Enzyme R and 25 μL Enzyme A). The C tube was tightly closed and connected upside down onto the sleeve of the gentleMACS Dissociator. The tissues were dissociated by running the gentleMACS program ‘h_tumor_01’ and incubated for 30 min at 37 °C under continuous rotation. The dissociation and incubation were repeated three times. The resulting cell suspension was collected through a 40 µm cell strainer and centrifuged at 500 × g for 5 min. After aspirating the supernatant completely, the remaining cells were resuspended and cultured in DMEM/F-12 with 10% FBS and 1 × penicillin/streptomycin. After 3 days, the cells were trypsinized with 0.25% trypsin–EDTA and seed with FBS-free DMEM/F-12 followed by removing nonadherent cell after 30 min. Cell passage was repeated three times.
Metabolomics
To obtain conditioned medium (CM) from cancer-associated fibroblasts (CAFs), the cells were treated with PBS or HCT116-derived exosomes for 24 h, followed by culturing in fresh FBS-free DMEM for 48 h after PBS washing. CM from HCT116 cells was generated by culturing HCT116 cells in FBS-free DMEM for 48 h. For CM sample collection, at least 10 mL of cell culture supernatant was collected per sample, centrifuged at 1,000 × g at 4 °C for 5 min. The supernatant was collected, passed through 0.22 μm filters, and stored at − 80 °C. For cell samples, after removing the medium, cells were washed twice in pre-cooled PBS before adding 1 mL pre-cooled methanol: acetonitrile: water (2:2:1, v/v/v). All cells in the culture dish were gently scraped off using a cell scraper and collected in 1.5 mL centrifuge tubes. The samples were frozen in liquid nitrogen and stored at − 80 °C.
Targeted metabolic analysis was performed using LC–MS/MS at Shanghai Applied Protein Technology Co. Ltd. Samples were separated using an Agilent 1290 chromatography system. The mobile phases consisted of A (25 mM ammonium formate + 0.08% FA aqueous solution) and B (0.1% FA acetonitrile). Samples were placed in an automatic sampler at 4 °C, with a column temperature of 40 °C, flow rate of 0.25 μL/min, and injection volume of 2 μL. The liquid phase gradients were as follows: 0–12 min, B changed linearly from 90 to 70%; 12–18 min, B changed linearly from 70 to 50%; 18–25 min, B changed linearly from 50 to 40%; 30–30.1 min, B changed linearly from 40 to 90%; 30.1–37 min, B remained at 90%. Samples were analyzed using a 5500 QTRAP mass spectrometer in positive mode. The ESI source conditions were: Source Temperature 500 °C, Ion Source Gas1(Gas1): 40, Ion Source Gas2(Gas2): 40, Curtain Gas (CUR): 30, Ion Spray Voltage Floating (ISVF) 5500 V. MRM mode was used to detect the ion pairs to be measured.
Multiquant software was used to obtain chromatographic peak area and retention time. The retention time was corrected, and metabolites were identified using standard samples of amino acids and their derivatives.
Lentiviral production and transduction
Lentiviral pLenti-TRE-EGFP-EF1-rtTA3-IRES-Puro-WPRE vectors encoding dox-inducible shRNA targeting SLC7A1 (Sequences of shRNA: 5′-GCTGAGGATGGACTGCTATTT-3′), and Lentiviral pLKD-U69-EF1a-LUC-F2A-Puro vector encoding shRNA targeting RAB27A (Sequences of shRNA: 5′-GCTGCCAATGGGACAAACATA-3′) were obtained from OBiO Technology (Shanghai, China). Lentiviruses were produced in 293 T cells using the Lenti-Pac HIV Expression Packaging Kit (GeneCopoeia, LT001) according to the manufacturer’s instructions.
For the generation of RAB27A knock-down cell lines and SLC7A1 dox-inducible knock-down cell lines, HCT116 cells were transduced with corresponding lentivirus and 5 μg/mL polybrene (Sigma, TR-1003) for 24 h. Forty-eight hours after transduction, HCT116 cells were selected with 1 μg/mL puromycin (Selleck Chemicals, S7417) for 7 days to establish stably expressing cell lines. For puromycin concentration selection, HCT116 cells were exposed to puromycin (0.25, 0.5, 1, 1.5, 2 μg/mL) for 48 h. Working concentration is the lowest concentration that kills all cells. The knock-down efficiency was verified by immunoblotting analysis.
RNA interference and dox-inducible shRNA
For RNA interference, ODC and negative control siRNA (RiboBio, SIGS0007322) were purchased commercially (siODC#1: AGAGGATTATCTATGCAAA, siODC#2: GCATGTATCTGCTTGATAT). We transfected them into HCT116 cells using Lipofectamine RNAiMAX Transfection (Invitrogen, 13778075) Reagent according to the manufacturer’s instructions. The efficiency of transfection was verified by western blotting.
Dox-inducible shSLC7A1 cell models in HCT116 cells were generated according to the protocol described above. In the dox-inducible shSLC7A1 experiments, 1 μg/mL of doxycycline (Selleck Chemicals, S5159) was added and refreshed every 2 days to induce shRNA expression.
L-arginine detection
CAFs treated with PBS or HCT116-derived exosomes for 24 h in six-well plates. After washing by PBS, CAFs were cultured in fresh DMEM with 10% dialyzed FBS for 48 h. HCT116-TRE-shSLC7A1 cells were cultured in DMEM with 10% dialyzed FBS for 48, 24 and 0 h in six-well plates. L-arginine concentrations in cell culture supernatant were measured using L-Arginine Assay Kit (Abcam, ab241028) according to the manufacturer’s instructions. After pre-treating the cell medium sample with cleanup mix, 100 μL of medium was taken up and centrifuged at 13,000 × g for 10 min at 4 °C. 40 μL of resulting sample was used for the assay. Following completion of the kit’s protocol, absorbance was measured at 450 nm using a microplate reader. L-arginine concentrations was calculated by the given kit formula. L-arginine uptake was determined by subtracting 24 and 48 h from the 0 h time point.
Cell viability, clonogenic assay, and co-culture assays
Cell viability was detected using Cell Counting Kit-8 (CCK-8) Kit (Dojindo, CK04) according to the manufacturer’s instructions. Absorbance was measured at 450 nm using a microplate reader. For clonogenic assays, 1,500 cells per well were seeded in triplicate into 6-well plates and allowed to adhere overnight. Cells were then cultured under different conditions as indicated in the figures. After 10 ~ 14 days, the cells were fixed with 4% formaldehyde, stained with Crystal Violet solution (Beyotime, C0121) according to the manufacturer’s instructions, and photographed using a digital camera. Colonies were counted using ImageJ software. For co-culture assays, the transwell noncontact co-culture system (6-well and 24-well) was constructed using polyethylene Terephthalate hanging cell culture inserts with a pore size of 0.4 µm (Merck, MCHT06H48 and MCHT24H48). CAFs were plated into the upper, and HCT116 cells or HCT8 cells were plated into the lower chambers, the cells were co-cultured in DMEM with 1% FBS or arginine-free DMEM with 1% dialysis FBS. For the collection and treatment of CM, CM from CAFs was generated by culturing CAFs using DMEM with 1% FBS for 48 h, and HCT116 cells were treated with 75% CM and 25% fresh DMEM with 1% FBS. CM from CAFs in arginine deprivation was generated by culturing CAF using arginine-free DMEM with 1% dialysis FBS for 48 h, and HCT116 cells and HCT8 cells were treated with 75% CM and 25% fresh arginine-free DMEM with 1% dialysis FBS. All CM was centrifuged at 1,000 × g at 4 °C for 5 min and passed through 0.22 μm filters.
Immunoblotting
Proteins from cells were extracted using M-PER (Thermo Scientific, 78501) containing 1 × protease inhibitor cocktail (Topscience, C0004). The BCA Protein Assay Kit (Thermo Scientific, 23225) was used to determine protein concentrations. Protein extracts were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) in 120 V for 1 h and then transferred to polyvinylidene fluoride (PVDF) membranes (Roche, 3010040001). The membranes were blocked by 5% milk for 1 h at RT, and incubated with primary antibodies at 4 °C overnight. The primary antibodies used were: ODC (0.5 µg/mL, Abcam, ab193338), CPS1 (1:2000, Sino Biological, 203902-T44), OTC (1:1000, Abcam, ab203859), ASS (1:1000, Sino Biological, 200448-T44), ASL (1:1000, Sino Biological, 200445-T44), ARG (1:1000, Sino Biological, 310345-T44), SLC7A1 (1:1000, ABclonal, A14784), beta Actin (1:10000, Arigo, ARG65683) and GAPDH (1:10000, Arigo, ARG65680). After washing for three times, the membranes were incubated with secondary antibodies for 1 h at RT. The secondary antibodies used were: Goat anti-Rabbit IgG (HRP) (1:10000, Arigo, ARG65351) or Goat anti-Mouse IgG (HRP) (1:10000, Arigo, ARG65350). After washing for three times, immunoblotting images were captured using a ChemiDoc XRS + system (Bio-Rad).
Real-time PCR
Total RNA was extracted using TRIzol (Invitrogen, 15596026) according to the manufacturer’s instructions. cDNA was produced from 2 μg of RNA using oligo (dT) (Invitrogen, AM5730G) and RevertAid Reverse Transcriptase (Thermo Scientific, EP0441) according to the manufacturer’s instructions. qPCR was performed using an Applied Biosystems 7500 Fast Sequence Detection System with primers and SYBR Green Master Mix (TIANGEN, FP205). The amplification conditions were 95 °C for 5 min, followed by 40 cycles of amplification at 95 °C for 10 s, 60 °C for 20 s, 72 °C for 20 s. Data were analyzed using the ΔΔCT method with β-actin as a housekeeping gene. The primer sequences used are listed in Supplementary Table 1. All primers were purchased from BGI Genomics.
RNA-sequencing analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis
Exosomes isolated from HCT116 cells were added to CAFs for 24 h with three replicates. After washing by PBS, total RNA of CAFs was extracted using TRIzol according to the manufacturer’s instructions and then sent to the Wuhan Genomics Institution (BGI) for RNA-sequencing analysis. The sequencing reads were mapped to the reference genomes (hg38/GRCh38) using HISAT2 software. Fragments per kilobase of transcript per million (FPKM) for each cell line were log2 transformed. DESeq2 was used for differential gene expression analysis. KEGG pathway enrichment analysis was performed with cluster Profiler package in R Studio software (v.4.2.1, R Studio, Inc. Boston, USA), and p < 0.05 was considered statistically significant.
Statistical analysis
Data analysis was conducted using appropriate statistical methods within GraphPad Prism software. Statistical comparisons between groups employed student's t-test (for two groups), one-way ANOVA (for multiple groups), or two-way ANOVA where applicable. All data are presented as mean ± SD from a minimum of three biological replicates, unless otherwise specified in the figure legend, with corresponding P-values provided in the figure legend. Statistical significance was established at P < 0.05.
Results
CAFs promote CRC growth in an exosome-dependent manner
To investigate the communication interaction between CAFs and CRC cells, we analyzed the cell growth of CRC cells across different culture models. Although conditioned medium from CAFs demonstrated a mild pro-proliferative effect, CAFs further enhanced the cell growth of HCT116 cells when present in the same environment, independent of direct cell–cell contact (Fig. 1A). Given that tumor-secreted exosomes function as essential mediators of intercellular communication between tumor cells and stromal cells in the TME [19], we hypothesized and subsequently confirmed that CRC-derived exosomes coordinate CAFs-mediated tumor promotion. Initially, we isolated exosomes from HCT116 cells' conditioned media. Immunoblotting analysis revealed the expression of exosomal markers CD63 and Alix, while GM130 and β-actin were absent (Fig. S1A). Electron microscopy and NanoSight particle tracking analysis confirmed the double-layer membrane structure and size of the exosomes (Fig. S1B, C). Subsequently, HCT116 cells were co-cultured with CAFs in the transwell system. The cell number of HCT116 cells increased significantly when co-cultured with CAFs and exhibited further enhancement upon addition of HCT116-derived exosomes (Fig. 1B). However, exosomes alone did not influence HCT116 cell growth (Fig. 1B). Furthermore, the enhanced cell growth observed during co-culture with CAFs was substantially reduced by treatment with an exosome-release inhibitor dimethyl amiloride (DMA) (Fig. 1B).
[See PDF for image]
Fig. 1
CAFs promote CRC growth in an exosome-dependent manner. A, B Representative immunofluorescence images of HCT116 cells expressing GFP (Green) (A) and HCT116 cells stained with DAPI (Blue) (B). Scale bar, 100 μm. Cell numbers were quantitated by ImageJ. The values are expressed as the mean ± SD of the average cell numbers of three randomly selected fields relative to the control group. C Western blot analysis of Rab27a in HCT116 cells transfected with shNC or shRab27a. Representative western blots from three independent experiments are shown. D Relative cell viability of HCT116-shNC cells and HCT116-shRab27a cells co-cultured with or without CAFs. n = 3 per group. Data for (A), (B), and (D) represent one of three independent experiments each contain three replicates. Data represent the mean ± SD
The small GTPase Rab27a has been established as critically necessary for exosome secretion [20]. Our findings demonstrated that Rab27a knockdown significantly diminished the capacity of CAFs to promote CRC cell (HCT116) growth in vitro (Fig. 1C, D). These results suggest that exosomes derived from colorectal cancer cells play a crucial role in enabling CAFs to promote CRC cell growth.
Exosomes derived from CRC reprogram the metabolism characteristic of CAFs
The mechanism by which CAFs acquire enhanced cancer-promoting capabilities after exosome induction represents a key scientific question requiring investigation. To examine the effects of CRC-derived exosomes on CAFs, RNA sequencing (RNA-seq) analysis was performed on primary CAFs treated with HCT116-secreted exosomes (HCT116 Exo) or vehicle control. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed upregulation of ECM receptor interaction and focal adhesion in exosome-treated CAFs, indicating CAF activation (Fig. 2A). Notably, the differentially expressed genes showed enrichment in multiple metabolic pathways, suggesting that CAFs undergo metabolic reprogramming following exposure to CRC-derived exosomes (Fig. 2A).
[See PDF for image]
Fig. 2
CAFs treated with CRC-derived exosomes undergo urea cycle metabolic reprogramming. A KEGG pathway enrichment analysis of differentially expressed genes between HCT116 Exo treated CAFs and untreated CAFs. HCT116 Exo represents HCT116-derived exosomes. B Schematic of the metabolomic experiments depicted in (C). C Relative metabolite levels expressed as log₂(Fold Change) in CAFs + Exo CM (medium from CAFs treated with HCT116-derived exosome), CAFs CM (medium from CAFs), Coculturing CM (CAFs + Exo CM was added to HCT116 cells, and then medium was collected), HCT116 cells treated with CAFs + Exo CM and HCT116 cells treated with HCT116 CM. n = 4 per group. D Schematic depicting the urea cycle and putrescine biosynthesis. E qPCR analysis assessing expression of the indicated genes in cells treated with or without HCT116- derived exosomes. n = 3 per group. F Western blot analysis of indicated proteins in cells treated with or without HCT116- derived exosomes. Representative western blots from three independent experiments are shown. Data for (E) represent one of three independent experiments each contain three replicates. Two-sided unpaired t-tests was used in (C and E). *p < 0.05, **p < 0.01. All data are shown as the mean ± SD
Metabolomic profiling was conducted to identify CAF-secreted metabolites absorbed by HCT116 cells (Fig. 2B). Specifically, we sought molecules that were significantly increased in CAFs + Exo CM (and therefore secreted by exosome-treated CAFs), including Arg, Orn and Creatinine; significantly decreased in the CAFs + Exo CM after contact with HCT116 cells (removed by HCT116 cells); significantly increased in HCT116 cells treated with the CAFs + Exo CM (taken up by HCT116 cells). Through targeted metabolomics quantifying 24 metabolites, including Arg and Orn, only Orn exhibited this distinct pattern. In addition, we observed that Arg, an amino acid critically involved in multiple cellular processes including the synthesis of nitric oxide (NO), polyamines and nucleotides [21], was secreted by CAFs treated with exosomes and removed by HCT116 cells, but significantly increased Arg was not detected in HCT116 cells treated with CAFs + Exo CM (Fig. 2C). Arg and Orn, UC intermediates, function as established oncometabolites supporting tumor growth and survival (Fig. 2D) [22]. Further examination of key UC pathway genes revealed that CRC-derived exosomes significantly increased arginine succinate synthase (ASS) and ODC expression in CAFs (Fig. 2E, F). These findings demonstrate that CRC-secreted exosomes induce UC reprogramming in CAFs, suggesting that CAF support for colorectal cancer cells is likely mediated through urea cycle metabolites.
Reprogrammed CAFs secreted Arg to assist CRC cell growth
The experimental data demonstrated that CRC cells utilize Arg and Orn secreted by CAFs for survival. To evaluate whether exogenous UC metabolites are essential for CRC growth, several experiments were conducted. Initially, CRC cells were cultured in Arg- medium. The results indicated that cell viability decreased significantly across all 4 CRC cell lines tested when Arg was absent (Fig. 3A). Subsequently, the addition of exogenous Arg fully restored the decreased cell viability of CRC cells cultured in Arg- medium (Fig. 3B). Additionally, exogenous Arg enhanced the clonogenic capacity of arginine-deprived (Arg-)deprived CRC cells in a dose-dependent manner (Fig. 3C, D). These data indicate that Arg is essential for CRC cell growth and establish the fundamental validity of our specific experimental model system.
[See PDF for image]
Fig. 3
Reprogrammed CAFs support CRC cell growth through Arg secretion. A Relative cell viability of CRC cells cultured in control and Arg-free medium. Arg- represents Arg-free. n = 3 per group. B Relative cell viability of HCT116 cells (left) and HCT8 cells (right) cultured in Arg-free medium in the presence or absence of 500 μM arginine (Arg) or 500 μM ornithine (Orn). n = 3 per group. C, D Colony formation assay of HCT116 cells (C) and HCT8 cells (D) cultured in medium containing increasing amounts of arginine (0, 50, 100, 200, 500 and 1,000 μM). n = 3 per group. E Arginine levels in conditioned medium (CM) from untreated CAFs or HCT116-derived exosomes treated CAFs. n = 3 per group. CAF (Exo) represents HCT116-derived exosomes treated CAFs. F Relative cell viability of HCT116 cells (left) and HCT8 cells (right) co-cultured with CAFs in the absence or presence of HCT116-derived exosomes in Arg-free medium. n = 3 per group. G Relative cell viability of HCT116 cells (left) and HCT8 cells (right) grown in Arg-free medium (black line), CM from CRC cells cultured in Arg-free medium (green line), CM from CAFs (blue line) or CRC-derived exosomes treated CAFs (orange line) cultured in Arg-free medium. n = 3 per group. Two-sided unpaired t-tests was used in (A). One-way ANOVA was used in (B, E and F). Two-way ANOVA was used in (G). All data represent one of three independent experiments each contain three replicates. Data represent the mean ± SD.*p < 0.05, **p < 0.01, ***p < 0.001, n.s., not significant. All data are shown as the mean ± SD
Based on the observation that CRC cells depend on Arg for survival, we investigated whether CAFs secrete Arg to support CRC growth in the TME. Analysis of Arg levels in the medium of CAFs treated with tumor-derived exosomes revealed that exosomes enhanced Arg secretion by CAFs (Fig. 3E). Moreover, co-culture with CAFs partially reversed the reduced cell viability of CRC cells caused by Arg deprivation, and this effect was amplified when CAFs were pretreated with CRC-derived exosomes (Fig. 3F). Furthermore, CAFs promoted the growth of CRC cells in Arg- medium; notably, exosome-pretreated CAFs exhibited a significantly enhanced capacity to promote the proliferation of CRC cells under the same conditions (Fig. 3G). Together, these observations support our hypothesis that exosome-reprogrammed CAFs nourish CRC cells with Arg, leading to sustained cell growth. However, the cellular uptake of arginine depends on specific transporters located on the cell membrane. We hypothesize that targeting these arginine transporters may effectively disrupt the arginine-mediated support provided by CAFs to colorectal cancer cells.
Inhibiting SLC7A1 represses CRC cell growth by suppressing Arg uptake
Arginine transport across cell membranes occurs through solute carrier (SLC) transporters [23]. Transcriptome analysis of clinical CRC samples from The Cancer Genome Atlas (TCGA) database revealed that three SLC transporters—SLC7A1, SLC7A6, and SLC3A2—were upregulated in tumor tissues compared to normal tissues (Fig. 4A). Analysis of transcription levels of these genes was conducted in HCT116 cells and NCM460 cells (human colon mucosal epithelial cells). Among 19 transporters identified as L-arginine transporters [24, 25], including SLC7A1, SLC7A2, SLC7A4, SLC7A6, SLC7A7, and SLC3A2, the data demonstrated that SLC7A1 expression was elevated in HCT116 cells compared to normal cells, with further enhancement under arginine deprivation (Fig. 4B). Our findings reveal that, upon arginine deprivation, cells up-regulate SLC7A1 as the principal transporter to compensate for the insufficient extracellular supply and restore intracellular arginine homeostasis. In addition, Kaplan–Meier analysis revealed that high SLC7A1 expression in CRC tissues correlated with poor survival (Fig. 4C). As SLC7A1 functions as an arginine transporter, depletion of SLC7A1 resulted in reduced arginine consumption in the medium (Fig. 4D, E), indicating decreased arginine uptake by HCT116 cells.
[See PDF for image]
Fig. 4
CRC cells rely on SLC7A1-mediated Arg uptake to ensure growth. A Box plot comparing transcript levels of arginine transporters in tumor tissue (T) and normal colon tissue(N). Data were generated from TCGA datasets. n (T) = 456, n (N) = 41. B qPCR analysis assessing mRNA expression of Arg transporter in NCM460 cells, HCT116 cells and Arg-deprived HCT116 cells. n = 3 per group. C Kaplan–Meier survival curves of patients with colon cancer based on SLC7A1 expression. Data were generated from Gene Expression Omnibus (GEO) (series GSE12945 (left) and series GSE24551 (right). D Western blot analysis of SLC7A1 in cells transfected with doxycycline-inducible shSLC7A1 in the absence or presence of Dox (doxycycline). Representative western blots from three independent experiments are shown. E Measurement of L-arginine uptake from the medium of HCT116-TRE-shSLC7A1 in the absence (Dox-) or presence (Dox +) of 1 μg/mL doxycycline for 24 or 48 h. n = 3 per group. F Relative cell viability of HCT116-TRE-shSLC7A1 cells cultured in control and Arg-free medium in the absence or presence of 1 μg/mL doxycycline. n = 3 per group. G HCT116-TRE-shSLC7A1 cells were cultured for 48 h in control or Arg-free medium, and co-cultured ± HCT116-derived exosomes treated CAFs in the absence or presence of 1 μg/mL doxycycline. Relative cell viability was determined by CCK-8 assay. n = 3 per group. H Relative cell viability of HCT116 cells treated with increasing concentrations of NEM (0, 10, 20 and 50 μM). n = 3 per group. I Relative cell viability of HCT116 cells cultured in control and Arg-free medium in the absence or presence of NEM (20 μM). n = 3 per group. Data for (B), (E), (F), (G), (H), and (I) represent one of three independent experiments each contain three replicates. Two-sided unpaired t-tests was used in (a). One-way ANOVA was used in (B, F, G, H and I). Two-way ANOVA was used in (e). *p < 0.05, **p < 0.01, ***p < 0.001, n.s., not significant. All data are shown as the mean ± SD
Building upon previous research demonstrating arginine's essential role in CRC growth, the study investigated whether SLC7A1 knockdown could affect CRC cell viability by limiting arginine uptake. The data demonstrated that SLC7A1 knockdown significantly reduced cell viability of CRC cells in complete medium, while showing no substantial effect on cell viability in arginine-deficient medium (Fig. 4F). In contrast, SLC7A6 and SLC3A2 demonstrated limited capacity for arginine transport (Fig. S3A, B). Notably, SLC7A1 knockdown inhibited the growth-promoting effect induced by exosome-reprogrammed CAFs in HCT116 cells cultured in arginine-deficient medium (Fig. 4G). Additionally, treatment of HCT116 cells with SLC7A1 inhibitor N-ethylmaleimide (NEM) decreased cell viability in a dose-dependent manner (Fig. 4H). However, NEM cannot synergize with Arg deprivation to inhibit CRC cell viability (Fig. 4I). These findings identify SLC7A1 as a crucial arginine importer in CRC cells and suggest its potential as a therapeutic target for CRC treatment. The subsequent investigation will examine the mechanism through which arginine influences colorectal cancer cell growth.
Putrescine plays an important role in cell growth arrest induced by Arg deprivation
To investigate the effects of Arg on tumor cell growth, CRC cells were supplemented with three major Arg metabolites in low-Arg medium. Nitric oxide (NO), a versatile signaling molecule, has been reported to stimulate cancer cell proliferation at lower concentrations [26, 27]. The addition of NO-donor (SNAP) to Arg-restricted CRC cells did not restore cell growth (Fig. 5A). Although nucleotide supplementation enhanced cell viability of CRC cells in complete medium, its growth-promoting effect was significantly diminished in arginine-restricted CRC cells (Fig. 5B). Importantly, supplementation with putrescine (Put), the precursor for higher polyamine biosynthesis, partially restored cell growth in Arg-restricted CRC cells without affecting the growth of cells in complete medium (Fig. 5C).
[See PDF for image]
Fig. 5
Putrescine contributes to the cell growth inhibition triggered by Arg deprivation. A–C Relative cell viability of CRC cells cultured with SNAP (A), nucleotide B or putrescine (Put) (C) in control and low-Arg medium. n = 3 per group. D Western blot analysis of the indicated proteins in in HCT116 cells (left) and HCT8 cells (right) cultured in control or Arg-free medium. E Western blot analysis of ODC in CRC cells cultured in control and Arg-free conditions after putrescine treatment for 48 h. F CRC cells were cultured in control and Arg-free medium, and co-cultured with CAFs in the absence or the presence of CRC-derived exosomes. ODC levels were monitored by western blot. G Western blot analysis of ODC and SLC7A1 in HCT116-TRE-shSLC7A1 cells treated with or without doxycycline. H Western blot analysis of ODC in CRC cells treated with increasing concentrations of NEM. Data for (A), (B), and (C) represent one of three independent experiments each contain three replicates. One-way ANOVA was used in (A–C). *p < 0.05, **p < 0.01, ***p < 0.001, n.s., not significant. All data are shown as the mean ± SD. In (D, E F, G, H), representative western blots are shown from three independent experiments
To confirm the metabolic changes induced by Arg deprivation, protein levels of UC enzymes and ODC were analyzed. The data demonstrated that Arg deprivation significantly increased ODC expression in both cell lines (Fig. 5D). Previous research has shown that ODC levels and activity are modified in response to intracellular free polyamine levels [28]. Put supplementation decreased ODC protein levels in CRC cells cultured in both complete and Arg-depleted medium (Fig. 5E), suggesting decreased putrescine levels. Significantly, co-culturing CRC cells with exosome-treated CAFs reduced ODC protein levels in Arg-depleted medium (Fig. 5F). Conversely, SLC7A1 knockdown increased ODC protein levels in CRC cells cultured in Arg replete conditions (Fig. 5G). The SLC7A1 inhibitor NEM similarly increased ODC expression in both cell lines (Fig. 5H). These findings indicate that Put contributes to the inhibition of cell viability triggered by Arg deprivation. Since arginine is an upstream metabolite of Put, combining arginine restriction with ODC inhibition may represent a promising therapeutic strategy. Further research will examine the efficacy of this combined approach in suppressing colorectal cancer cell growth.
Combined Arg deprivation and ODC inhibitor effectively suppresses CRC cell growth
Ornithine derived from Arg generates pro-tumorigenic polyamines through ODC upregulation [29]. RNA-seq data from TCGA demonstrated elevated ODC expression in CRC tumors (Fig. 6A). The data indicated that ODC knockdown diminished cell viability in CRC cells (Fig. 6B). Additionally, treatment with the ODC inhibitor DFMO decreased cell viability in a dose-dependent manner (Fig. 6C). Furthermore, supplementation with Put restored the viability of CRC cells treated with ODC-targeting siRNA or DFMO (Fig. 6D), indicating that ODC-mediated maintenance of the Put pool is essential for CRC cell survival.
[See PDF for image]
Fig. 6
Combination treatment with Arg deprivation and ODC inhibitor suppresses CRC cell growth. A Box plot comparing transcript levels of ODC in tumor tissue (T) and normal colon tissue(N). Data were generated from TCGA datasets. n (T) = 456, n (N) = 41. B HCT116 cells were transfected with ODC siRNAs for 48 h. Western blot was used to detect ODC expression. CCK-8 assay was used to determine cell viability. n = 3 per group. Representative western blots from three independent experiments are shown. C Relative cell viability of HCT116 cells treated with increasing concentrations of DMFO. n = 3 per group. D HCT116 cells treated with control siRNA, ODC siRNA or DFMO (500 μM) were cultured in medium containing increasing amounts of putrescine (0, 100 and 500 μM). Cell viability was determined with CCK-8. n = 3 per group. E HCT116 cells were cultured in control or low-Arg medium, colony formation assay was performed after treatment with control siRNA or ODC siRNAs. ODC knockdown efficiency was monitored by western blot. Representative images of the colonies are given. n = 3 per group. Representative western blots from three independent experiments are shown. F HCT116 cells were cultured in control or low-Arg medium, colony formation assay was performed after treatment with vehicle or DFMO. Representative images of the colonies are given. n = 3 per group. G Colony formation assay of HCT116-TRE-shSLC7A1 cells treated with vehicle (0 μM) or DFMO in the absence or the presence of doxycycline (Dox). n = 3 per group. Representative images of the colonies are given. H Colony formation assay of HCT116 cells treated with DMFO (0, 100 and 500 μM), NEM (20 μM) or a combination. n = 3 per group. I A proposed model of the UC metabolic interaction between CRC and CAFs. Representative images of the colonies are given. Data for (C), (D), (E), (F), (G), and (H) represent one of three independent experiments each contain three replicates. Two-sided unpaired t-tests was used in (A, B). One-way ANOVA was used in (D–H). *p < 0.05, **p < 0.01, ***p < 0.001, n.s., not significant. All data are shown as the mean ± SD
Given that Arg deprivation led to ODC upregulation, the effects of combined Arg restriction and ODC inhibition on CRC cells were examined. The data demonstrated that both ODC knockdown and the ODC inhibitor DFMO induced more pronounced inhibition of clonogenic capacity in CRC cells under Arg-restricted conditions compared to complete-medium conditions (Fig. 6E, F). Following the identification of SLC7A1 as a crucial arginine transporter in CRC cells, the effect of combined DFMO treatment and SLC7A1 inhibition on CRC cell growth was investigated. DFMO treatment showed enhanced efficacy in SLC7A1-knockdown cells (Fig. 6G). Similarly, the combination of SLC7A1 inhibitor NEM and DFMO produced stronger inhibition of CRC cell growth compared to individual treatments (Fig. 6H). These findings suggest that combining Arg-depleting agents or Arg restriction with ODC inhibitor represents a potentially more effective therapeutic approach.
Discussion
The metabolic interaction between cancer cells and the tumor microenvironment represents a fundamental characteristic of cancer metabolism [30]. CAFs adjacent to tumor cells modify their metabolism to provide nutrients. However, the precise mechanisms by which tumor cells direct CAFs to support tumor growth remain incompletely understood. Tumor-derived exosomes serve as critical mediators of progression and metastasis [31]. This study demonstrates that CRC cells reprogram CAF metabolism through exosomes to obtain essential nutrients for growth and survival, warranting further investigation into exosome-mediated communication and metabolic interactions between CRC and CAFs.
Modified nutrient metabolism in the tumor stroma has emerged as a crucial aspect of cancer-associated metabolic reprogramming [32]. Studies have shown that in certain tumors, CAFs undergo aerobic glycolysis and release energy-rich pyruvate and lactate to sustain neighboring cancer cells [33, 34]. Through integrated transcriptomic and metabolomic analysis, this study reveals that CRC-secreted exosomes reprogram CAF metabolism, significantly increasing UC metabolites Arg and Orn. Subsequently, CRC cells utilize these UC metabolites released by reprogrammed CAFs to maintain Put synthesis for cellular growth (Fig. 6I). This discovery of previously unrecognized UC metabolic crosstalk between CRC cells and CAFs illuminates a novel role of tumor stroma in promoting CRC progression.
The critical importance of UC metabolites in cancer initiation and progression has been targeted for therapeutic intervention [35, 36]. The depletion of exogenous Arg has been developed as an antitumor therapy [37]. Pegylated arginine deiminase (ADI-PEG20), a bioengineered mycoplasmal enzyme that converts arginine into citrulline and ammonia, has demonstrated favorable tolerability and achieved partial responses in hepatocellular carcinoma and melanoma [38, 39]. Alternative therapeutic approaches involve inhibiting Arg transport into cancer cells, which occurs through multiple plasma membrane transporters with distinct tissue distributions [40]. This study demonstrates that CRC cells depend on SLC7A1 for adequate Arg uptake, and SLC7A1 inhibition diminishes CRC cell growth, suggesting its potential as a therapeutic target for CRC treatment.
Beyond its direct effects on cancer cell proliferation, the modulation of arginine metabolism profoundly influences the tumor immune microenvironment. Arginine is critical for T-cell function, proliferation, and antitumor activity [41]. While systemic arginine depletion can impair T-cell responses, our strategy leverages CAFs for a more localized restriction, the effect of which on intra-tumoral immunity remains to be fully elucidated. Furthermore, the addition of DFMO, which targets downstream polyamine synthesis, could potentially modulate the function of immunosuppressive cells like MDSCs that rely on polyamines [5]. Therefore, the interplay between this metabolic combination therapy and anti-tumor immunity presents a fascinating duality: it may suppress immune function through arginine starvation, or it could potentially reprogram the immunosuppressive milieu by disrupting polyamine metabolism. Future studies should employe immunocompetent mouse models will be crucial to dissect these complex immunometabolic interactions.
The complex tumor microenvironment and metabolic adaptability present challenges for metabolite deprivation monotherapy. For instance, a phase III randomized study of ADI-PEG20 in advanced HCC patients failed to show significant overall survival benefits [42]. While DFMO has demonstrated efficacy in clinical trials for high-risk neuroblastoma [43], extended administration may lead to gradual drug resistance in tumor cells, potentially compromising treatment outcomes. This study reveals that Arg deprivation monotherapy increases ODC expression, potentially directing ornithine away from the UC toward pro-tumorigenic polyamine biosynthesis. Consequently, elevated ODC levels may affect the survival of Arg-deprived cells, creating a specific vulnerability. Therefore, combining Arg-depleting agents or dietary Arg restriction with ODC inhibition may provide a more effective therapeutic approach (Fig. 6I).
While our metabolomic profiling revealed that CAFs secrete multiple metabolites in addition to arginine and ornithine (Fig. 2C), including putrescine, creatine, etc. Our functional studies consistently identified arginine depletion as the principal mechanism mediating the observed growth suppression. Nevertheless, the potential contribution of other CAFs-derived metabolites warrants further investigation through more comprehensive flux analyses.
Multiple plasma-membrane transporters facilitate arginine uptake, with varying tissue distributions [24, 25]. Their abundance and apparent redundancy limit their appeal as therapeutic targets [44]. Alternative approaches are therefore needed to prevent cancer cells from utilizing arginine. SLC7A1 suppression significantly decreased CRC arginine uptake from the TME (Fig. 4F). TCGA data and quantitative PCR suggest possible compensatory arginine transport through SLC7A6 or SLC3A2, though functional confirmation remains pending.
The study presents three primary limitations. First, it lacks in vivo validation, as no xenograft experiments were conducted to verify the synergistic antitumor effects of combining arginine depletion with ODC inhibition or to assess systemic toxicity and pharmacokinetics. Second, while functional data indicate that arginine depletion reduces polyamine availability, direct quantification of intracellular putrescine under these conditions was not performed. Third, currently, no other clinically approved ODC inhibitor matches DFMO in terms of representativeness or validation. The experiments relied exclusively on DFMO; the absence of an additional, structurally distinct ODC inhibitor prevents definitive attribution of the observed effects to ODC blockade. Therefore, the conclusions remain limited to in vitro observations, with future research priorities including in vivo validation, direct putrescine quantification, and the incorporation of alternative ODC inhibitors to establish translational relevance.
Overall, this study reveals a previously unrecognized metabolic crosstalk between CRC cells and CAFs, which depends on tumor-derived exosomes. Furthermore, these findings demonstrate the metabolic flexibility of CRC to fulfill nutritional requirements through exogenous uptake of Arg in the TME, and support the advancement of combined Arg depletion and ODC blockade into clinical applications.
Author contributions
Rui Wang: Conceptualization, supervision, resources, investigation, writing-original draft and writing–review and editing. Guopeng Pan: Investigation, writing-original draft and writing–review and editing. Zexiong Cheng: Investigation. Yiwei Wang: Investigation. Changxi Wu: Investigation. Fang Wang: Investigation. Qing Li: Investigation. Xiangyu Wang: Investigation. Yuequan Zeng: Investigation. Yuqin Li: Investigation. Kai Li: Resources. Xi Lin: Resources. Fan Xing: Resources. Youwei Huang: Resources, methodology. Jun Liu: Resources, methodology.
Funding
This work was supported by grants from the project supported by the youth Fund of the National Natural Science Foundation of China (grant no. 62301245 to R. Wang), the Guangdong Basic and Applied Basic Research Foundation (grant no. 2022A1515110674 to R. Wang), the National Natural Science Foundation of China (grant no. 82173829 to F. Xing) and the youth Fund of the National Natural Science Foundation of China (Grant no. 82403011 to Y. Huang).
Data availability
The data are available from the corresponding author upon reasonable request. The datasets generated and/or analysed during the current study are available in the GEO database repository (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc = GSE285472, https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc = GSE12945 and https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc = GSE24551). The data (Fig. 4A and Fig. 6A) were obtained from The Cancer Genome Atlas (TCGA) Research Network (https://www.cancer.gov/tcga).
Declarations
Ethics approval and consent to participate
Informed consent has been obtained from the tissue donor, and the human experiment plan complies with the Declaration of Helsinki. Approval was granted by the Ethics Committee of Sun Yat-sen University Sixth Affiliated Hospital (October 28th, 2020, No. 2020ZSLYEC-225).
Competing interests
The authors declare no competing interests.
Abbreviations
Arginine
Arginine-deprived
Arginine succinate synthase
Cancer-associated fibroblasts
Conditioned medium
Colorectal cancer
Difluoromethylornithine
Dimethyl amiloride
Exosomes
HCT116-secreted exosomes
Low- arginine
Kyoto Encyclopedia of Genes and Genomes
N-ethylmaleimide
Nitric oxide
Ornithine decarboxylase
Ornithine
Putrescine
RNA sequencing
Solute carrier
Transcriptome analysis of clinical CRC samples from The Cancer Genome Atlas
Tumor microenvironment
Urea cycle
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
References
1. Chen, X; Song, E. Turning foes to friends: targeting cancer-associated fibroblasts. Nat Rev Drug Discov; 2019; 18,
2. Isella, C; Terrasi, A; Bellomo, SE et al. Stromal contribution to the colorectal cancer transcriptome. Nat Genet; 2015; 47,
3. Gentric, G; Mechta-Grigoriou, F. Tumor cells and cancer-associated fibroblasts: an updated metabolic perspective. Cancers (Basel); 2021; [DOI: https://dx.doi.org/10.3390/cancers13030399] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/33499022]
4. Cirri, P; Chiarugi, PJC. Cancer-associated-fibroblasts and tumour cells: a diabolic liaison driving cancer progression. Cancer Metastasis Rev; 2012; 31,
5. Ye, C; Geng, Z; Dominguez, D et al. Targeting ornithine decarboxylase by α-difluoromethylornithine inhibits tumor growth by impairing myeloid-derived suppressor cells. J Immunol; 2016; 196,
6. Gong, J; Lin, Y; Zhang, H et al. Reprogramming of lipid metabolism in cancer-associated fibroblasts potentiates migration of colorectal cancer cells. Cell Death Dis; 2020; 11,
7. Lee, JS; Adler, L; Karathia, H et al. Urea cycle dysregulation generates clinically relevant genomic and biochemical signatures. Cell; 2018; 174,
8. Keshet, R; Szlosarek, P; Carracedo, A et al. Rewiring urea cycle metabolism in cancer to support anabolism. Nat Rev Cancer; 2018; 18,
9. Chen, HY; Tong, TY; Lu, SY et al. Urea cycle activation triggered by host-microbiota maladaptation driving colorectal tumorigenesis. Cell Metab; 2023; 35,
10. Feun, L; You, M; Wu, C et al. Arginine deprivation as a targeted therapy for cancer. Curr Pharm Des; 2008; 14,
11. Dillon, BJ; Prieto, VG; Curley, SA et al. Incidence and distribution of argininosuccinate synthetase deficiency in human cancers - a method for identifying cancers sensitive to arginine deprivation. Cancer; 2004; 100,
12. Casero, RA; Marton, LJ. Targeting polyamine metabolism and function in cancer and other hyperproliferative diseases. Nat Rev Drug Discov; 2007; 6,
13. Murray-Stewart, TR; Woster, PM; Casero, RA. Targeting polyamine metabolism for cancer therapy and prevention. Biochem J; 2016; 473, pp. 2937-2953. [DOI: https://dx.doi.org/10.1042/BCJ20160383] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/27679855]
14. Gerner, EW; Meyskens, FL. Polyamines and cancer: old molecules, new understanding. Nat Rev Cancer; 2004; 4,
15. Hu, HY; Liu, XX; Jiang, CY et al. Ornithine decarboxylase gene is overexpressed in colorectal carcinoma. World J Gastroenterol; 2005; 11,
16. Casero, RA; Murray Stewart, T; Pegg, AE. Polyamine metabolism and cancer: treatments, challenges and opportunities. Nat Rev Cancer; 2018; 18,
17. Meyskens, FL; Mclaren, CE; Pelot, D et al. Difluoromethylornithine plus sulindac for the prevention of sporadic colorectal adenomas: a randomized placebo-controlled, double-blind trial. Cancer Prev Res; 2008; 1,
18. Lewis, EC; Kraveka, JM; Ferguson, W et al. A subset analysis of a phase II trial evaluating the use of DFMO as maintenance therapy for high-risk neuroblastoma. Int J Cancer; 2020; 147,
19. Tkach, M; Théry, C. Communication by extracellular vesicles: where we are and where we need to go. Cell; 2016; 164,
20. Ostrowski, M; Carmo, NB; Krumeich, S et al. Rab27a and Rab27b control different steps of the exosome secretion pathway. Nat Cell Biol; 2010; 12,
21. Hajaj, E; Sciacovelli, M; Frezza, C et al. The context-specific roles of urea cycle enzymes in tumorigenesis. Mol Cell; 2021; 81,
22. Matos, A; Carvalho, M; Bicho, M et al. Arginine and arginases modulate metabolism, tumor microenvironment and prostate cancer progression. Nutrients; 2021; [DOI: https://dx.doi.org/10.3390/nu13124503] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/34960055][PubMedCentral: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8704013]
23. Fotiadis, D; Kanai, Y; Palacin, M. The SLC3 and SLC7 families of amino acid transporters. Mol Aspects Med; 2013; 34,
24. Banjarnahor, S; Rodionov, RN; König, J et al. Transport of l-arginine related cardiovascular risk markers. J Clin Med; 2020; [DOI: https://dx.doi.org/10.3390/jcm9123975] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/33302555][PubMedCentral: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7764698]
25. Surrer, DB; Fromm, MF; Maas, R et al. L-arginine and cardioactive arginine derivatives as substrates and inhibitors of human and mouse NaCT/Nact. Metabolites; 2022; [DOI: https://dx.doi.org/10.3390/metabo12040273] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/35448460][PubMedCentral: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9026504]
26. Pervin, S; Singh, R; Hernandez, E et al. Nitric oxide in physiologic concentrations targets the translational machinery to increase the proliferation of human breast cancer cells: involvement of mammalian target of Rapamycin/eIF4E pathway. Cancer Res; 2007; 67,
27. Coneski, PN; Schoenfisch, MH. Nitric oxide release: part III. measurement and reporting. Chem Soc Rev; 2012; 41,
28. Perez-Leal, O; Merali, S. Regulation of polyamine metabolism by translational control. Amino Acids; 2012; 42,
29. Sk, LAM; Pong, UK; Li, YY et al. Inhibition of ornithine decarboxylase 1 facilitates pegylated arginase treatment in lung adenocarcinoma xenograft models. Oncol Rep; 2018; 40,
30. Pavlova, NN; Thompson, CB. The emerging hallmarks of cancer metabolism. Cell Metab; 2016; 23,
31. Becker, A; Thakur, BK; Weiss, JM et al. Extracellular vesicles in cancer: cell-to-cell mediators of metastasis. Cancer Cell; 2016; 30,
32. Yan, W; Wu, X; Zhou, W et al. Cancer-cell-secreted exosomal miR-105 promotes tumour growth through the MYC-dependent metabolic reprogramming of stromal cells. Nat Cell Biol; 2018; 20,
33. Lisanti, MP; Martinez-Outschoorn, UE; Sotgia, F. Oncogenes induce the cancer-associated fibroblast phenotype: metabolic symbiosis and “fibroblast addiction” are new therapeutic targets for drug discovery. Cell Cycle; 2013; 12,
34. Zhang, D; Wang, Y; Shi, Z et al. Metabolic reprogramming of cancer-associated fibroblasts by IDH3 alpha downregulation. Cell Rep; 2015; 10,
35. Vynnytska, BO; Mayevska, OM; Kurlishchuk, YV et al. Canavanine augments proapoptotic effects of arginine deprivation in cultured human cancer cells. Anticancer Drugs; 2011; 22,
36. Hinrichs, CN; Ingargiola, M; Kaeubler, T et al. Arginine Deprivation Therapy: Putative Strategy to Eradicate Glioblastoma Cells by Radiosensitization. Mol Cancer Ther; 2018; 17,
37. Fultang, L; Vardon, A; De Santo, C et al. Molecular basis and current strategies of therapeutic arginine depletion for cancer. Int J Cancer; 2016; 139,
38. Izzo, F; Marra, P; Beneduce, G et al. Pegylated arginine deiminase treatment of patients with unresectable hepatocellular carcinoma: results from phase I/II studies. J Clin Oncol; 2004; 22,
39. Ascierto, PA; Scala, S; Castello, G et al. Pegylated arginine deiminase treatment of patients with metastatic melanoma: results from phase I and II studies. J Clin Oncol; 2005; 23,
40. Closs, EI; Simon, A; Vékony, N et al. Plasma membrane transporters for arginine. J Nutr; 2004; 134,
41. Geiger, R; Rieckmann, JC; Wolf, T et al. L-arginine modulates T cell metabolism and enhances survival and anti-tumor activity. Cell; 2016; 167,
42. Abou-Alfa, GK; Qin, S; Ryoo, BY et al. Phase III randomized study of second line ADI-PEG 20 plus best supportive care versus placebo plus best supportive care in patients with advanced hepatocellular carcinoma. Ann Oncol; 2018; 29,
43. Oesterheld, J; Ferguson, W; Kraveka, JM et al. Eflornithine as postimmunotherapy maintenance in high-risk neuroblastoma: externally controlled, propensity score-matched survival outcome comparisons. J Clin Oncol; 2024; 42,
44. Werner, A; Pieh, D; Echchannaoui, H et al. Cationic amino acid transporter-1-mediated arginine uptake is essential for chronic lymphocytic leukemia cell proliferation and viability. Front Oncol; 2019; [DOI: https://dx.doi.org/10.3389/fonc.2019.01268] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/31824848][PubMedCentral: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6879669]
© The Author(s) 2025. This work is published under http://creativecommons.org/licenses/by-nc-nd/4.0/ (the “License”). Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.