Breast cancer remains a leading cause of female cancer related mortality worldwide. Loss of genomic stability and dysregulation of cellular metabolism are well-recognized features of breast cancer, presenting an opportunity to study the drivers of breast cancer progression and resistance to chemotherapy. The overarching goal of this work is to perform combined analysis of DNA damage repair and cellular metabolism in response to olaparib treatment in a panel of breast cancer cell lines. By applying a combined untargeted metabolomics and molecular biology approach, our findings show dysregulation of amino acid metabolism and metabolic reprogramming from glycolysis to amino acid utilization to be a common feature in all breast cancer cell lines examined, some of which are consistent with findings from the analysis of clinical breast cancer tumours. Functional assessment of genetic alterations offers the scope to design new prognostic tools and inform the design of new chemotherapies or drug combinations.Metabolic reprogramming and genomic instability are key hallmarks of cancer, the combined analysis of which has gained recent popularity. Given the emerging evidence indicating the role of oncometabolites in DNA damage repair and its routine use in breast cancer treatment, it is timely to fingerprint the impact of olaparib treatment in cellular metabolism. Here, we report the biomolecular response of breast cancer cell lines with DNA damage repair defects to olaparib exposure. Following evaluation of olaparib sensitivity in breast cancer cell lines, we immunoprobed DNA double strand break foci and evaluated changes in cellular metabolism at various olaparib treatment doses using untargeted mass spectrometry-based metabolomics analysis. Following identification of altered features, we performed pathway enrichment analysis to measure key metabolic changes occurring in response to olaparib treatment. We show a cell-line-dependent response to olaparib exposure, and an increased susceptibility to DNA damage foci accumulation in triple-negative breast cancer cell lines. Metabolic changes in response to olaparib treatment were cell-line and dose-dependent, where we predominantly observed metabolic reprogramming of glutamine-derived amino acids and lipids metabolism. Our work demonstrates the effectiveness of combining molecular biology and metabolomics studies for the comprehensive characterisation of cell lines with different genetic profiles. Follow-on studies are needed to map the baseline metabolism of breast cancer cells and their unique response to drug treatment. Fused with genomic and transcriptomics data, such readout can be used to identify key oncometabolites and inform the rationale for the design of novel drugs or chemotherapy combinations. In a bid to develop new therapies against various cancer types, and genomic instability, its underpinning mechanisms and contribution to tumorigenesis have been extensively investigated over the past few decades. Genomic instability, a well-known contributor to cancer, presents a therapeutic vulnerability that can be targeted in the development of novel chemotherapy agents .To maintain their genomic integrity, cells are equipped with a range of DNA damage repair (DDR) pathways and responses to counteract DNA lesions formed in response to endogenous and exogenous insults . HereditPARP inhibitors, as a class of DDR inhibitors, block the activity of PARP enzymes involved in DNA damage repair, therefore leading to accumulation of DNA double-strand breaks that gives rise to genomic instability and subsequent apoptosis . SeveralWhile PARP inhibitors present a therapeutic opportunity for targeting DDR defects in breast and ovarian cancers, emerging evidence has shown a role for oncometabolites—small molecule intermediates of cellular metabolism—in determining the response to these chemotherapies. The biology of oncometabolites and their role in modulating DDR has been increasingly studied over the past few years, guiding new combination therapies and novel biological targets for drug discovery .Metabolic reprogramming—a key feature of all cancers —gives riDDR mechanisms induce cellular metabolic changes through interference with purine and pyrimidine biosynthetic pathways, amino acid metabolism, protein biosynthesis, and energy metabolism, impacting several metabolic routes . MediatoDNA repair and regulation of metabolism is critical for maintaining homeostasis in normal human cells. However, the extensive dysregulation and aberrant function of both these pathways promotes tumorigenesis. Until recently, DNA repair and metabolic pathways have routinely been researched as distinct fields within their own right, but emerging research evidence an intrinsic inter-dependency between these pathways. Here, we report the differential cellular response of breast cancer cell line models with different mutational signatures see  and DDR v/v FBS , 1% v/v non-essential amino acids (NEAA) and 1% v/v penicillin-streptomycin (Invitrogen). Corresponding cell line origins, hormone receptor status and mutational profiles are included in v/v FBS and 1% v/v penicillin-streptomycin. All cell lines were maintained at 37 °C in a pre-humidified atmosphere containing 5% v/v CO2 and used within ten passages for the purposes of this work (passage 2–10). Olaparib  was prepared as a 100 mM stock solution in DMSO, aliquoted, and stored at −20 °C until use. γH2AX, p53BP1 primary antibodies , were used for foci immunostaining alongside the Alexa Fluor® 488-conjugated secondary antibody .All cell lines used in this study were purchased from the vendor and maintained in accordance with manufacturer instructions. All cell culture reagents were obtained from Gibco . MCF7  and MDA-MB-231 cells  were purchased and maintained in Dulbecco’s Modified Eagle Medium  supplemented with 10% v/v CO2. Treatment media were replaced every three days with treatment medium. Following a seven-day incubation, cell viability was measured using CellTiter 96® Aqueous Non-Radioactive Cell Proliferation Assay  -5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) reagent. The resultant absorbance at 490 nm was measured using a GM3500 Glomax® Explorer Multimode Microplate Reader (Promega).MCF-7, MDA-MB-231, and HCC1937 cells undergoing exponential growth were seeded at a density of 4,000 cells/well in 96 well plates and incubated overnight to facilitate cell attachment. On the following day, cells were exposed to either blank growth medium (control) or growth medium containing different concentrations of olaparib (treatment medium) ranging from 0.01–500 µM for seven days at 37 °C and 5% 50) was determined using a linear regression model. Statistical analysis was performed using GraphPad Prism . Three independent biological replicates (five wells per treatment concentration) were performed for each cell line.Growth curves represent percentage cell growth following treatment with different concentrations of olaparib and are plotted as a semi-log dose–response curve. The half maximal inhibitory concentration  for seven days. Briefly, cell monolayers were fixed in chilled 4% w/v formaldehyde containing 2% w/v sucrose in PBS, followed by fixation in ice-cold methanol (100% v/v). Subsequently, cells were permeabilized in 0.25% v/v Triton X-100 in PBS, blocked with 5% v/v goat serum/5% w/v BSA, immunoprobed with either a primary rabbit anti-γH2AX antibody (RRID:AB_420030) (1:1000) or primary rabbit anti-P53BP1 (1:200) antibody  overnight at 4 °C. Cell monolayers were treated with goat, anti-rabbit Alexa Fluor® 488 conjugated secondary antibody and counterstained with DAPI. Image acquisition was carried out using an Invitrogen EVOS Auto Imaging System (AMAFD1000-Thermo Fisher Scientific) with a minimum of 100 cells imaged per treatment condition. Resultant foci images were analyzed in Cell Profiler (v.4.2.1.) using a modified version of the speckle counting pipeline.Foci immunodetection for γH2AX and p53BP1 was performed in both control (growth medium) and for cells treated with olaparib . Following exposure to olaparib, the growth medium was aspirated from each well, centrifuged to remove cell debris, and stored at −80 °C. Next, treated cells were washed with pre-chilled PBS, with the metabolites quenched and extracted in a final volume of 1.5 mL pre-chilled (−80 °C) mixed solvent (Methanol:Acetonitrile:Water = 50:30:20). Resultant cell pellets were collected, and submerged in liquid nitrogen, vortexed, and sonicated for 3 min in an ice-water bath. This procedure was performed in triplicate. Resultant extracts were centrifuged at 13,000× g for 10 min at 4 °C and the pellets were retained for protein quantification using the Bradford assay. The resultant supernatant was collected and dried with a Speed vac centrifuge (Savant-SPD121P). Dried metabolite pellets were reconstituted in Acetonitrile:Water (50:50) at volumes normalized to the relative protein content. Quality control (QC) samples were prepared by pooling samples across all control and treatment groups. Solvent blank and QC samples were inserted in analytical batch after every five samples to assess the stability of detecting system.MCF-7, MDA-MB-231, and HCC1937 cells were seeded at a density of 2 × 10Metabolite separation was performed on a binary Thermo Vanquish ultra-high performance liquid chromatography system, where 5 µL of reconstituted cellular extract was injected on to a Thermo Accucore HILIC column . The temperature of the column oven was maintained at 35 °C, while the autosampler temperature was set at 5 °C. For chromatographic separation, a consistent flow rate of 500 µL/min was used where the mobile phase in positive heated electrospray ionisation mode (HESI+) was composed of buffer A  and buffer B  . Likewism/z with a MS1 resolution of 60,000. Subsequent MS/MS scans were processed with a resolution of 15,000. High-purity nitrogen was used as nebulising and as the collision gas for higher energy collisional dissociation. Further details are included in A high-resolution Exploris 240-Orbitrap mass spectrometer (Thermo Fisher Scientific) was used to perform full scan and fragmentation analyses. Global operating parameters were set as follows: spray voltages of 3900 V in HESI+ mode, and 2700 V in HESI-mode. The temperature of the transfer tube was set as 320 °C with a vaporizer temperature of 300 °C. Sheath, aux gas, and sheath gas flow rates were set at 40, 10, and 1 Arb, respectively. Data-dependent acquisitions (DDA) were performed using the following parameters: full scan range was 70–1050 TM software 4.2 were imported into Compound DiscovererTM 3.2 software where the “Untargeted Metabolomics with Statistics Detect Unknowns with ID Using Online Databases and mzLogic” feature was selected , a HMDB identification code was assigned to each selected metabolite. A joint pathway analysis was performed by integrating the genes relative to each cell line . For cell viability and immunofluorescence quantification data, the Shapiro normality test was performed . Peak areas were log10 transformed and p values were calculated for the sample group by analysis of variance (ANOVA) test. A p value < 0.05 and fold-change of 1.5 was deemed to be statistically significant.All data are presented as mean ± standard deviation  and a non-triple-negative (MCF-7) cell line.50) of 10 µM and 14 µM, respectively. However, in the case of HCC1937 cells, a higher concentration of olaparib was required to achieve the same reduction in cell viability (150 µM), indicating a lower efficacy of response to olaparib in this cell line.Our results show that exposure to olaparib caused a reduction in cell viability in all cell lines in a dose-dependent manner . We obse10, IC25, and IC50—determined from MTS assays) promotes the accumulation of DNA double strand breaks (DSBs) in MCF-7, MDA-MB-231, and HCC1937 cell lines. Key markers for DNA DSB formation include phosphorylated histone H2 variant H2AX (γH2AX) [PARP inhibition induced by olaparib exposure results in the accumulation of DNA damage in cells by compromising their DDR mechanisms. Therefore, we next investigated the extent to which olaparib exposure at various doses (IC (γH2AX) . To measBased on our results, p53BP1 and γH2AX foci levels increased in a dose-dependent manner in both MCF7 and MDA-MB-231 cells in response to ascending doses of olaparib a,b,d,e. To comprehensively measure the extent of variation induced by olaparib exposure in MCF-7, MDA-MB-231, and HCC1937 cell lines, we profiled their metabolome using an in-house untargeted liquid chromatography-mass spectrometry-based metabolomics pipeline . After d50 olaparib treatment dose across all cell lines , suggesting a strong reliance of breast cancer cell metabolism on amino acids under baseline conditions (control samples). Upon olaparib exposure, the same pathways (amino acid metabolism) were among the most significantly altered across all cell lines, while fatty acid (butanoate metabolism) and vitamin B6 metabolism were only significantly perturbed in MCF-7 cells.Next, we explored individual metabolites that were associated with significantly altered metabolic pathways in response to olaparib exposure and evaluated relative changes in their levels between control and treatment samples. These results are presented through a heatmap clustering analysis . A correMultiple amino acids  were found to be depleted following olaparib exposure (relative to control) in all cell lines examined. Arginine and proline metabolism were significantly depleted by olaparib exposure, with depletion of their derived polyamines detected in all cell lines examined. Conversely, catabolic products of arginine and proline metabolism  were enriched. Elevated levels of serine were observed in MCF7 and MDA-MB-231 cells, while depletion of serine levels was seen in HCC1937 cells.Alpha-ketoglutarate (α-KG-glutamine-derived intermediate of the TCA cycle) was enriched in MCF7 and depleted in MDA-MB-231 and HCC1937 cells. A negative correlation was observed between α-KG and glutamine levels, and a positive correlation between α-KG, and citric and fumaric acid (TCA cycle intermediates). Aspartate (a TCA cycle product) accumulated in the KRAS-mutant MDA-MB-231 cells, while aspartate depletion was observed in MCF7 and HCC1937 cells. Glucose levels were significantly elevated relative to control samples in HCC1937 cells. Asparagine (a byproduct of aspartate) was absent in MDA-MB-231 cells, while its enrichment was detected in MCF7 and HCC1937 cells. In parallel, accumulation of AMP was observed in both MCF7 and HCC1937 cell lines, while it was absent in MDA-MB-231 cells, and enrichment of PPi was detected in all cell lines examined following olaparib exposure.In the case of lipid metabolism, we observed a global depletion of phosphocholines (PC) and phosphoethanolamines (PE) in all cell lines following olaparib treatment. Acylcarnitine levels varied across the cell lines, with an overall enrichment of long (C14–C21) and very-long chain acylcarnitines (>C22) in all cell lines treated with olaparib. Moreover, we observed enriched alpha-linoleic acid (a polyunsaturated fatty acid-PUFA) levels in MCF7 and MDA-MB-231 cells, which was absent in HCC1937 cells.Compared to non-treated cells, elevated levels of glucose were detected in all cell lines studied following olaparib treatment, while downregulation of most nucleobases was observed. Finally, NAD+ downregulation was detected in all cell lines treated with olaparib.An overview of the metabolic features altered in response to olaparib exposure is given in PARP inhibitors have shown promising results in the treatment of metastatic breast cancers harboring germline BRCA1/2 mutations ,23. Rece50—150 µM). These findings are in agreement with previous reports of HCC1937 resistance to PARP inhibition, where the identification of predictive biomarkers of response to PARP inhibitor treatments was recommended beyond BRCA1/2 status [In this study we analyzed the sensitivity of two triple-negative (MDA-MB-231 and HCC1937) and MCF-7  cell lines to olaparib PARP inhibition (PARP1/2). The rationale for selecting these cell lines was to explore how their different genetic profiles see  define t2 status .Our analysis of γH2AX and p53BP1 DNA DSB immunolabelled foci  showed aGenetic biomarkers are routinely used in the clinical stratification of breast cancers and predicting treatment-emergent resistance . While gOur goal in the present study was to apply combined analysis of DNA damage foci formation with global untargeted mass-spectrometry based metabolomics to map the metabolic changes occurring following exposure to olaparib. We examined the baseline differences in cellular metabolism across the cell line panel and extended this evaluation to examine cell-line-dependent response to olaparib treatment. Under baseline cell culture conditions, we found overlapping metabolic features  occurring across all three breast cancer cell lines studies, and metabolic signatures that were unique to specific cell lines  .10 treatment concentration, accompanied by a decrease in NAD+ levels at ascending concentrations of olaparib. Reduced levels of NAD+ were also detected in the MDA-MB-231 and HCC1937 cells at all treatment concentrations. Recent studies have shown that in TNBC cells, olaparib enhances the signaling pathways of other NAD+-dependent deacetylase  [Our analysis of metabolites significantly altered in response to olaparib treatment correlate with reports from Bhute et al., where metabolic markers of PARP inhibition were reported as changes in amino acid metabolism , downregulation of osmolyte levels , phosphocreatine, lactate, and pyruvate in MCF7 cells . We repoirtuins) ,30. ThesGlutamine, a precursor for protein, nucleotide, and lipid biosynthesis, is a fundamental amino acid in breast cancer cell metabolism, playing a pivotal role in providing anaplerotic intermediates for the tricarboxylic acid (TCA) cycle . PreviouGlutamine is also a source of nitrogen groups for the synthesis of nucleobases and nucleotides, either directly or through a process involving the transamination of glutamate and the TCA cycle-derived oxaloacetate that generates aspartate ,39,40. OAmong the pathways of aspartate utilization, asparagine is converted through the enzyme asparagine synthetase (ASNS). The reaction requires glutamine as a substrate and consumption of adenosine triphosphate (ATP) to produce adenosine monophosphate (AMP) and pyrophosphate (PPi). Physiological levels of asparagine occur at levels of <0.05 mM in human plasma . Cancer Beyond asparagine synthesis, aspartate amidation through ASNS presents a source of amino building blocks for the synthesis of arginine in the urea cycle, which is in turn responsible for the synthesis of polyamines catalyzed by ornithine decarboxylase (ODC). Polyamine accumulation has previously been correlated with the increased proliferation of both hormone-dependent and independent breast cancer cells , and wasGlutathione (GSH) is involved in the protection against ROS and regulation of intracellular redox homeostasis. Elevated GSH levels have previously been reported in TNBC compared to luminal breast cancers, suggesting the relevance of GSH to our observations of lower sensitivity to olaparib in TNBC cell lines ,52.Lipids mediate various cellular biological functions, including energy storage, cell membrane structural composition and signal transduction, the increased biosynthesis of which is a marker of metabolic rewiring observed in malignant breast cancers ,54. Our Future targeted metabolomics studies using additional TNBC cell lines and clinical tumor clinical specimens are required to validate our observations. Validation of our findings could define prognostic biomarkers that will aid evaluation of patient prognosis in the clinical setting and enable the implementation of precision medicine in the management of breast cancer.Our data show differential sensitivity of breast cancer cell lines to olaparib treatment that was dose-dependent and demonstrated the increased sensitivity of TNBC cells to DNA damage foci accumulation. The application of metabolomics to the study of breast cancer remains in its infancy, with only a handful of studies reporting combined metabolomics and phenotypic analyses. Data acquired from metabolomics analysis can be validated against routine molecular biology and phenotypic assays, providing a powerful platform for biomarker detection or the discovery of novel actionable pathways for drug development.Our results show that fingerprinting the metabolic profile of cells can be a powerful tool for uncovering potential oncometabolites or mechanisms giving rise to chemoresistance. Findings from such studies may provide potential additional actionable targets for modulating response to drug treatment or the design of new drug combinations that will overall enhance DNA damage efficacy, ultimately improving patient response to radiotherapy and adjuvant chemotherapy.


Klebsiella pneumoniae (K. pneumoniae) colonizes the human gut and is a causative factor of pyogenic liver abscess (PLA). Retrospective studies conducted on K. pneumoniae PLA patients revealed subsequent CRC development in later years of their life with increasing prevalence of these strains harbouring polyketide synthase (PKS) genes. To our knowledge there are no known studies directly implicating K. pneumoniae with CRC to date. Our aims are to characterize K. pneumoniae isolates from CRC patients and investigate its effects on cell proliferation in vitro. K. pneumoniae isolates were characterized by screening virulence genes including polyketide synthase (PKS), biofilm assay, antibiotic susceptibility, and string test to determine hypervirulent (hvKp) strains. Solubilised antigens of selected K. pneumoniae isolates were co-cultured with primary colon cell lines and CRC cell lines (Stage I-IV) for 48 h. The enhancement of proliferation was measured through MTT and ECIS assay. Twenty-five percent of K. pneumoniae isolates were PKS-positive out of which 50% were hvKp strains. The majority of the isolates were from the more virulent serotype of K1 (30%) and K2 (50%). PKS-positive K. pneumoniae isolates did not possess genes to confer carbapenem resistance but instead were more highly associated with siderophore genes  and allantoin metabolism genes . Cell proliferation in primary colon, SW1116 (Stage I), and SW480 (Stage II) CRC cell lines were enhanced when co-cultured with PKS-positive K. pneumoniae antigens. ECIS revealed enhanced cell proliferation upon recurrent antigen exposure. This demonstrates the possible role that PKS-positive K. pneumoniae has in exacerbating CRC progression. Klebsiella pneumoniae (K. pneumoniae) is a Gram-negative bacterium of the Enterobacteriaceae family that colonizes human mucosal surfaces such as the nasopharynx and the gastrointestinal tract (GIT) [K. pneumoniae has been an aetiological agent in a number of diseases such as hospital acquired infections, bacteraemia, endocarditis, pneumonia, as well as pyogenic liver abscess (PLA) [K. pneumoniae infections worldwide has risen dramatically due to the emergence of multi-drug resistant (MDR) as well as extremely drug resistant (XDR) strains [K. pneumoniae strains (hvKp) among Asian countries have also caused a peak in PLA cases being reported, especially in Taiwan and China, where K. pneumoniae has exceeded Escherichia coli (E. coli) as the most common pathogen isolated from PLA patients [K. pneumoniae (cKp) due to its increased virulence and ability to cause more severe infections compared to the latter. The hvKp strains are notorious for their biofilm forming properties as well as efficient iron uptake systems and siderophore production. These are some of the virulence factors that enhance the survival of K. pneumoniae within the host through evasion of host immune defences and its ability to sequester iron, allowing it to persist within the host. Furthermore, K. pneumoniae is known to harbour the polyketide synthase (PKS) island within its genome that produces colibactin genotoxin. This phenomenon has also been observed in E. coli, where reports demonstrated colibactin genotoxin’s ability to cleave host DNA double strands bringing about cell cycle arrest as well as DNA damage and mutations which initiates tumorigenesis [K. pneumoniae PLA patients had approximately three times higher risk of developing colorectal cancer (CRC) years after a PLA infection compared to non-K. pneumoniae PLA patients [K. pneumoniae metabolism that enter the portal venous system. [K. pneumoniae and antibiotic treatment was implemented. Furthermore, upon treating the patient with empiric antibiotic therapy (sulbenicillin 8 g/day and metronidazole 2 g/day), there was a reduction in gas accumulation due to the inhibited reproduction of K. pneumoniae. The same patient was then diagnosed with colon cancer (TNM staging: T4bN2bM0) after a few days.ct (GIT) ,2. K. pnss (PLA) ,4. In re strains . The lacpatients . Increasigenesis . This ispatients ,10,11,12 system. . The bloK. pneumoniae is a common colonizer of the GIT, especially the colon, and possibly enters the hepatic portal veins which receive blood from the GIT through the network of mesenteric veins. The inferior mesenteric vein transports blood from the colon and rectum to the hepatic portal vein which sends it to the liver before being transported back to the heart. It could also be postulated that K. pneumoniae enters the liver through this similar pathway to cause PLA.K. pneumoniae isolated from PLA patients, there are still gaps and chinks in the armour that directly link K. pneumoniae with CRC without PLA being a precursor condition. Therefore, this study aims to characterize the virulence genes as well as the prevalence of PKS-positive K. pneumoniae isolates obtained from CRC patients attending University Malaya Medical Centre (UMMC) and to determine their role in CRC progression in vitro.Although numerous studies have performed characterization of K. pneumoniae were isolated from faecal, tumour, and tissue biopsy specimens of CRC patients and healthy individuals attending UMMC upon obtaining ethical approval from UMMC Medical Ethics Committee based on the Declaration of Helsinki 1975 (Ethics Reference Number: 943.18). In this study, 43 CRC patients and 72 healthy individuals attending UMMC for routine colonoscopy screening and who had colon or rectal resection between March 2014 to August 2015 consented to participate. All clinical specimens were processed, isolated, and cultured in the Department of Medical Microbiology, Faculty of Medicine, University of Malaya. In order to isolate pure colonies of K. pneumoniae, the specimens were homogenized and cultured in Luria Bertani broth overnight and streaked onto MacConkey Agar. All mucoidal colonies were subjected for identification of K. pneumoniae isolates, performed using Bruker Biotyper MALDI-TOF MS system according to manufacturer’s instructions. Additionally, species identification was further confirmed through 16s rRNA gene sequencing. Pure colony K. pneumoniae cultures were preserved in 30% glycerol stocks and stored in −80 °C until further use.® CCL-233), SW 480 (ATCC ® CCL-228), HT 29 (ATCC ® HTB-38), HCT 116 (ATCC ® CCL-247) were used as stage I-IV derived cell lines respectively. All cell lines were grown in RPMI 1640 and supplemented with 10% Fetal Bovine Serum (FBS) and 1% each of Penicillin-Streptomycin, L-Glutamine, and Amphotericin B at 37 °C with 5% CO2.A total of 4 human CRC cell lines derived from various CRC stages (Stage I to IV) as well as 1 primary colon cell line were used in this study. Human primary colon cell  was used as a non-CRC in vitro model. SW 1116 (ATCC K. pneumoniae DNA were extracted according to manufacturer’s protocol using the Qiagen DNeasy Blood & Tissue Kit. The purity and concentration of extracted DNA were measured and quantified using the IMPLEN NanoPhotometer. The DNA purity of 1.8–2.0 (A260/A280 ratio) was used for subsequent experiments. Extracted DNA that was not utilized immediately were stored in aliquots at −20 °C. The positive and negative controls used were K. pneumoniae ATCC 13883 extracted DNA and nuclease free water respectively.K. pneumoniae virulence genes which included PKS colibactin gene, K. pneumoniae serotypes (K1(K1-magA), K2 (K2wzy), K5 (K5wzx), K20 (wzyK20), K54 (wzxK54), K57 (wzyK57)) as well as Carbapenem genes (OXA-48 (blaOXA-48), NDM-1 (blaNDM-1), KPC (blaKPC), IMP (blaIMP), and VIM (blaVIM)). Previously published primers that were used in this study along with the length of the expected PCR product are listed in mrkD1, mrkD2, fimH, kpn), Enterobactin siderophores , Ferric aerobactin receptor , Aerobactin siderophore, Yersiniabactin siderophore , Salmochelin catecholate receptor , Klebsiella iron uptake systems , Genotoxins(Colibactin (clbB), Haemolysin (hlyA), Cytotoxic necrotizing factor (cnf)), Capsule synthesis (K2 serotype capsule synthesis (k2A), Fucose capsule synthesis (wcaG), Lipid polysaccharide (LPS) synthesis (wabG), outer membrane lipoprotein (ycfM), Uridine diphosphategalacturonate 4-epimerase (uge), Regulator of mucoid phenotype A , urease synthesis (ureA), and Allantoin metabolism . The cycling conditions for the detection of PKS colibactin gene were initial denaturation at 95 °C for 10 min, denaturation at 94 °C for 45 s, annealing at 54 °C for 45 s, and extension at 72 °C for 1 min. This was repeated for 30 cycles. The amplified PCR products were separated and visualized through gel electrophoresis at 120 V for 2 h in 1.8% (wt/vol) agarose gel containing SYBR Safe DNA Gel Stain (Invitrogen). Cycling conditions used for the multiplex PCR along with the primer grouping will be made available upon request.A total of 41 primer sets were used to screen and detect K. pneumoniae isolates and 1 K. pneumoniae ATCC 13883 strain were screened against a panel of 29 antibiotics from 16 antimicrobial categories using Kirby-Bauer disc diffusion method and broth microdilution methods were based on previously established protocol by Wiegand et al. and Hudzicki et al. [A total of 40 clinical i et al. ,15 accori et al.  antibiotK. pneumoniae isolates were inoculated in 5 mL of trypticase soy broth for 24 h at 37 °C. The inoculum was adjusted to 0.5 MacFarland standard . 200 µL of the adjusted inoculum were seeded into wells of the 96-well flat bottom plates and incubated at 37 °C for 24 h. Inoculum were carefully removed, and the plates were washed three times in sterile phosphate buffered saline (PBS). The adherent layer of bacteria was heat-fixed for 20 min at 60 °C. An amount of 125 µL of 0.1% crystal violet was added to the wells and incubated at room temperature for 15 min. The plates were rinsed with sterile distilled water 3 times and excess water and dye were blotted using paper towels. An amount of 200 µL of 33% glacial acetic acid was added to the wells and incubated at room temperature for 1 h. The absorbance was measured at 570 nm and the biofilm-producing classification was determined based on the values below:This assay was performed according to previously established protocols by Singh et al. and Emery et al. ,19 with cNegative control = DOaAbsorbance value of each strain = DOa ≤ DOcNon-adherent = DOc < DOa ≤ 2xDOcWeakly adherent = DOc < DOa ≤ 4xDOcModerately adherent = 2xDOc < DOaStrongly adherent = 4xDOK. pneumoniae isolates were grown on nutrient agar plates overnight at 37 °C. A sterile inoculating loop was used to touch the surface of a single K. pneumoniae colony and to stretch the colonies by raising the inoculating loop to produce a mucoviscous string. The test was considered positive if the isolates were able to produce a mucoviscous string of > 5 mm in length.K. pneumoniae isolates were grown separately in 200 mL of Luria Bertani (LB) broth in a conical flask at 37 °C for 18 h with shaking at 180 rpm. After incubation, the grown inoculum was transferred to 50 mL conical tubes and centrifuged at 18,000× g for 30 min at 4 °C. The supernatant was discarded, and the bacterial cell pellet was washed twice in cold sterile PBS and resuspended in sterile ice-cold PBS at a concentration of 1 g wet weight per ml. The suspension was sonicated to rupture the bacterial cell wall for 15 min at 4 °C with 40 s burst at 50% power setting. The sonicated suspension was then briefly vortexed at top speed and allowed to stand overnight at 4 °C to allow complete solubilisation of the K. pneumoniae bacterial antigens. The suspension was sequentially filtered through 0.45 µm and 0.20 µm syringe filters. An amount of 10 µL of the filtered solubilised antigen was spread onto nutrient agar and incubated at 37 °C overnight to determine no viable bacterial cells were present. The whole bacterial antigens extracted are protein in nature and will be used as such. Further purification and determination of the antigen properties were not performed in this study. From here on, the K. pneumoniae solubilized antigens will be referred to as bacterial antigens. The protein concentration was estimated using the Bradford’s method and the extracts were stored in aliquots at −20 °C until further use.Single colonies of 3/mL). The cells were incubated at 37 °C with 5% CO2 overnight to allow the cells to adhere. MTT assay was performed to determine the appropriate concentration of bacterial antigen to be used (data not shown). After incubation, 10 µL of prepared bacterial antigen was added to the well at a concentration of 500 µg/mL . The optimal concentration of bacterial antigen to be used was determined through MTT assay. 10 µL of EGF at a final well concentration of 20 ng/mL and 10 µL of sterile PBS were used as positive and negative controls respectively. All conditions were in triplicates. The K. pnuemoniae bacterial antigens used were obtained from 4 main isolate groups, namely PKS-positive of CRC, PKS-positive of healthy, PKS-negative of CRC, and PKS-negative of healthy origin. The cells were then incubated for 48 h at 37 °C with 5% CO2. To measure the enhancement of cell proliferation, 10 µL of 5 mg/mL  MTT dye were added to all wells and incubated for 4 h at 37 °C with 5% CO2. After incubation, the supernatant was carefully aspirated so as to not disturb the cell monolayer and 100 µL of DMSO were added to the wells and kept on a rocking platform for 10 min to solubilise the MTT dye. The absorbance was read on a microtitre plate reader at 570 nm. The enhancement of cell proliferation was read after normalizing the values obtained from the negative control wells.Human primary and CRC cell lines were seeded into wells of 96-well flat bottom plates at a density of 500 cells/100 µL per well . The impedance was measured using the ECIS model Zθ (Z Theta) . The cells were incubated for a period of 6 days at 37 °C with 5% COp value less than 0.05 was considered to be statistically significant.The IBM Statistical Package for Social Science (SPSS) software version (26.0) was used for statistical analysis. A Pearson chi-square was used to find the correlation between the relationship of PKS-positive and hypervirulent isolates against the screened virulence genes. A K. pneumoniae. Out of the 40 clinical isolates, 25% (10/40) of the isolates harboured the PKS colibactin genotoxin gene and a K-antigen capsular serotyping screening revealed that 14/40 of the isolates could be assigned to the six most prevalent serotypes  of K. pneumoniae. The most virulent serotypes that have been associated with hypervirulence are the K1 and K2 serotype. Screening of the ten PKS-positive isolates showed that three (30%) of these isolates belonged to the K1 serotype, five (50%) were K2, one (10%) isolate was K5 and the remaining one isolate did not belong to the any of the serotypes screened. On the other hand, all three of the PKS-positive K1 serotype isolates were obtained from clinical specimens of CRC patients and the remaining seven PKS-positive isolates were obtained from healthy individuals attending UMMC for routine medical examinations. There was no statistical difference observed among the PKS-positive K. pneumoniae strains isolated from CRC and healthy individuals. Further assessment of other virulence genes is vital to understand if other virulence factors play a role in increasing the pathogenicity of PKS-positive K. pneumoniae. The presence of virulence genes was determined solely based on a PCR and sequencing of biosynthetic gene clusters will be required to determine the functionality of the virulence genes. K. pneumoniae isolates.After screening 43 CRC patients and 72 healthy individuals, only 40 individuals  harboured K. pneumoniae sequesters iron from host bound proteins through the secretion of siderophore molecules which are high-affinity iron-chelating agents. The four groups of siderophores are aerobactin, enterobactin, salmochelin, and yersiniabactin, which all play different roles in establishing an infection within the host. In this study, PKS-positive isolates harboured genes responsible for the production of aerobactin, enterobactin, and yersiniabactin siderophores. A key notable difference observed was the lack of the salmochelin  siderophore gene among PKS-positive isolates from CRC patients and healthy individuals as well as lack of iron uptake systems  among PKS-positive isolates from healthy individuals. In addition to that, PKS-negative isolates predominantly possessed genes conferring production of enterobactin siderophore. Similarly, the salmochelin siderophore was also found in approximately 17% of PKS-negative isolates from CRC patients and 11% of PKS-negative isolates from healthy individuals. Iron is an important element that is required for bacterial survival and pathogenesis. Although, iron is abundantly found within the host, most of the iron are bound to host proteins such as haemoglobin and transferrin. To overcome this, K. pneumoniae hypervirulent strains. All the PKS-positive isolates from CRC patients had the rmpA gene. Additionally, the cytotoxic necrotizing factor-1 (cnf-1) works in concert with the colibactin gene to modulate the cell cycle that contribute to tumorigenesis and hyperproliferation. This gene was also found in all PKS-positive isolates of CRC origin. However, the cnf-1 gene was not present in any of the PKS-positive strains isolated from healthy individuals and therefore it would be interesting to observe how it affects the enhancement of cell proliferation. Interestingly, the haemolysin gene (hly) was found in a low abundance of 14% and 8% among PKS-positive from healthy individuals and PKS-negative isolates from CRC patients, respectively. The two fundamental genes that are involved in colonization and infection for K. pneumoniae are the uge and urea gene. The uge gene plays a role in the initial colonization and infection of the gastrointestinal mucosa whereas the urea gene aids in driving the persistence of infection by overcoming the gastrointestinal stress through the utilisation of nitrogen for protein synthesis. The regulator mucoid phenotype gene (rmpA) is responsible for enhancing extracellular capsule synthesis resulting in increased mucoviscosity of the isolates. The presence of the rmpA gene is also one of the virulence determinants for Fimbriae and adhesins are vital for the attachment and colonization on the colonic mucosa. The kpn gene which is responsible for type 1 fimbriae-like adhesin is lacking among PKS-positive isolates from CRC patients. However, it is found in 100%, 50%, and 61% of PKS-positive , PKS-negative (CRC), and PKS-negative  isolates respectively. None of the isolates possessed the mrkD-1 gene which is a type 3 fimbriae but was compensated by the presence of other adhesins and fimbriae. Escherichia coli (E. coli) can only use allantoin as a source of nitrogen. However, K. pneumoniae has the ability to metabolize allantoin and utilize it as a source of carbon and nitrogen. This allows it to persist within the host for a prolonged time and survive under stressful conditions. Allantoin metabolism is vital for the utilisation of allantoin as a main source of nitrogen and carbon. It is also an important hallmark feature to determine the severity of infection. wabG, ycfM, entB, entB1, fimH, iutA, iutA1, iucB, rmpA1, rmpA2, kfuBC, kfu, allS, and allS2 (having a correlation significance at 0.01 level). We also observed that the presence of the pks gene is correlated with the hypervirulent K. pneumoniae strains as previously described. Additionally, these Pks-positive hypervirulent K. pneumoniae isolates possess a high correlation between the ycfM, ybtS, ybtA, (yersiniabactin) iutA, iutA1, aerobactin, iucB, (aerobactin) rmpA1, rmpA2 (regulator of mucoid), and k2A (K2 capsule) genes.A correlation analysis was performed to determine the relationship of PKS-positive and hypervirulent isolates against the screened virulence genes . The anaAntibiotic resistance is a worldwide threat, particularly in clinical settings where growing cases of MDR, XDR, and Pandrug resistance (PDR) infections are being reported. With the rise of hvKp strains being reported in Asian countries it is important to contain the spread of these isolates and prevent the emergence of MDR-hvKp strains prevailing. An isolate is considered to be MDR when it is non-susceptible to ≥1 agent in ≥3 antimicrobial categories. An XDR isolate is defined as non-susceptible to ≥1 agent in all but ≤2 categories and a PDR isolate is non-susceptible to all antimicrobial agents .K. pneumoniae isolates and one K. pneumoniae ATCC 13883 strain, seven clinical isolates were identified as MDR strains, all of which were PKS-negative. PKS-positive K. pneumoniae isolates were only resistant to the penicillin group of antibiotics (ampicillin (6/10) and amoxycillin (10/10)). On the other hand, PKS-negative K. pneumoniae conferred antibiotic resistance to amoxycillin (30/30), ampicillin (27/30), chloramphenicol (6/30), tetracycline (4/30), trimethoprim-sulfamethoxazole (4/30), doxycycline (3/30), levofloxacin (3/30), tigecycline (2/30), amikacin (1/30), amoxicillin-clavulanic acid (1/30), and norfloxacin (1/30). AST results are tabulated in After screening fourty clinical blaOXA-48 gene. Of these strains, two out of three were isolated from healthy individuals and one strain was isolated from a CRC patient. The summary of isolates harbouring carbapenem resistance are shown in Additionally, none of the PKS-positive isolates carried the genes that confer resistance towards carbapenems. However, three PKS-negative isolates carried the K. pneumoniae biofilm-forming capabilities are a cause of the majority of indwelling catheter infections among patients. In this study, 18 out of the 40 isolates screened were strong biofilm producers. Among this group, five isolates were PKS-positive strains and out of which three isolates were obtained from CRC patients. There were six moderate biofilm producers with two PKS-positive isolates, one of which came from a CRC patient. Four isolates were weak biofilm producers having only one PKS-positive isolate obtained from a healthy individual. The remaining 12 out of the 40 isolates screened were non-biofilm producers out of which two PKS -positive isolates were obtained from healthy individuals. K. pneumoniae isolates.K. pneumoniae that the strain is considered to be a hvKp strain [K. pneumoniae isolates screened, 16 (40%) produced a positive string test. In these 16 isolates, 10 isolates (62.5%) were considered hvKp based on the abovementioned definition. The further breakdown of the hvKp isolates is described in K. pneumoniae in CRC is similar to the healthy group, with 34.8% and 34.7% prevalence, respectively  wells. The enhancement of cell proliferation was expressed as the percentage (%) of cell proliferation. Generally, K. pneumoniae antigens were able to enhance the proliferation of human primary and CRC cell lines in vitro.However, PKS-positive isolates isolated from CRC patients had the highest enhancement of cell proliferation across all cell lines except for SW1116 (stage I), where the PKS-positive isolate from healthy patients had a slight edge. The highest enhancement of cell proliferation was observed in the SW480 (stage II) cell line. The enhancement of cell proliferation trend showed a bell-shaped curve that progressed from the human primary cell line and peaked at SW480 (stage II) after which the enhancement of cell proliferation decreased. Additionally, PKS-positive isolates showed better enhancement of cell proliferation compared to PKS-negative isolates, irrespective of isolate origin. There is some plausibility that the presence of the PKS colibactin gene along with co-virulence factors could play a role in enhancing the progression rate of CRC, especially in the normal and early stages of CRC (as observed in the primary and CRC cell line of stage I and II).K. pneumoniae antigens. In this section only, results of the cell lines exposed to PKS +ve CRC and PKS -ve CRC antigens were shown as it had the best enhancement of its group. The enhancement of cell proliferation was seen in all stages of CRC cell lines. Interestingly, the enhancement was more pronounced upon the second exposure of K. pneumoniae antigens. Recurrent exposure to K. pneumoniae antigens simulates the persistent exposure of the colonic mucosa to bacterial toxins within the colonic microenvironment. The enhancement of cell proliferation due to recurrent infection by K. pneumoniae antigens demonstrates the effect that chronic K. pneumoniae infection has on cell proliferation and possibly initiation of tumorigenesis. PKS +ve CRC had a higher cell proliferation rate at the early stages of CRC  whereas PKS-ve CRC antigens enhanced cell proliferation at advanced CRC stages (HT29 and HCT116). These findings could potentially illustrate the role of PKS +ve K. pneumoniae as a driver bacterium involved in the initiation of tumorigenesis and PKS-ve K. pneumoniae as the passenger bacterium that progresses the tumorigenesis into more advanced stages of CRC.The ECIS array aids in detecting an increase in cell proliferation by measuring the impedance of the cell when an alternating current is applied to it. As the cell proliferates and covers the electrodes at the bottom of the well, the impedance increases. K. pneumoniae infections are a huge threat and burden to the healthcare systems as they are the leading cause of nosocomial infections. The emergence of hvKp strains further complicates the treatment and prognosis of a patient. In this study, there was an equal distribution of hvKp strains from PKS-positive and PKS-negative K. pneumoniae isolates as well as from CRC and healthy individual origin. Although hvKp strains are generally sensitive to most antibiotics, it is still more difficult to treat compared to cKp infections due to the arsenal of virulence factors that hvKp isolates possess [OXA-48 gene. Globally, the K1 and K2 serotype are the most prevalent serotypes of the hvKp strain and PKS-positive K. pneumoniae isolates. This could possibly explain the higher rate of antibiotic susceptibility and lack of antibiotic resistance genes observed among PKS-positive strains in this study. The K1 and K2 serotype are known to have increased capsule production that confers the hypermucoviscous variant. This characteristic presents a physical barrier that prevents the horizontal gene transfer of antibiotic resistance genes to occur [K. pneumoniae isolated from PLA patients is rampant and should be addressed. possess . We obseto occur . Howeverto occur ,25. Therto occur . The incK. pneumoniae, three of which were isolated from CRC patients. Moreover, the characterization of isolates based on virulence genes also showed that these three isolates possessed 22/28 of the virulence genes screened in addition to being a strong biofilm producer and belonging to the K1 serotype. These features aid in increasing their virulence, thus making them strong pathogenic bacterium with the ability to cause life threatening and debilitating diseases within the host.There has been plenty of controversy surrounding the definition of hvKp strains through string test . Hence, K. pneumoniae isolates is the abundance of virulence genes present within the PKS-positive K. pneumoniae genome. PKS-positive isolates had a greater abundance of genes responsible for siderophore production, namely aerobactin, enterobactin, and yersiniabactin, compared to PKS-negative isolates that were more associated with only enterobactin siderophore. The ability to produce more than one group of siderophore increases the organism’s ability to sequester iron more efficiently from the host environment, thus enhancing its survival within the host. A similar observation was made by Khaertynov et al. [rmpA-positive isolates.The key observable difference between the PKS-positive and PKS-negative v et al. , where aK. pneumoniae isolates that harbored a single type of siderophore, particularly enterobactin, had an increased virulence compared to isolates that harbored all four types of siderophore production genes. Similarly, PKS-negative isolates in this study primarily had genes for enterobactin siderophore production only, which translated to having a higher antibiotic resistance thus producing MDR strains. However, this does not diminish the pathogenicity of PKS-positive strains as they are primarily implicated in invasive and persistent infections as well as their increased ability to cause severe infections such as PLA, brain abscess, ophthalmitis, and bacteraemia that results in sepsis [A study by Chen et al.  demonstrn sepsis .allS, allS2) was more expressed in PKS-positive strains. Allantoin metabolism is a vital process that allows an organism to utilize allantoin as a source of carbon and nitrogen. Chou et al. [K. pneumoniae was able to utilize not only carbon but nitrogen as a source of energy under both aerobic and anaerobic conditions. This shows the ability of PKS-positive isolates to sustain and persist under stressful conditions and environments such as the tumour microenvironment within a CRC patient. The results of the virulence gene detection are listed in Additionally, the genes coding for allantoin metabolism  that may induce inflammation resulting in increased cell proliferation. This indicates that the colibactin toxin production along with its counterpart virulence genes seen in the PKS-positive isolates has a better ability to enhance CRC progression compared to PKS-negative isolates. The systematic review conducted by Strakova et al. [K. pneumoniae as a driver bacterium inducing mutations in CRC driver genes. As observed in K. pneumoniae antigens, with the highest proliferation occurring in SW 480 (stage II) CRC-derived cell lines. Contrarily, CRC cell lines exposed to PKS-negative K. pneumoniae antigens had a greater enhancement of cell proliferation at advanced CRC stages (stage III-IV). The findings from this study could possibly indicate the driver–passenger relationship between PKS-positive and PKS-negative K pneumoniae, respectively. To further explore this notion, an ECIS assay was carried out to substantiate these findings.To further understand if other virulence factors and the origin of isolate may play a synergistic role in PKS-positive isolates, we co-cultured human primary and CRC cell lines derived from stages I-IV with a et al.  discusseK. pneumoniae antigens. Repeated exposure of bacterial antigens increased the enhancement of cell proliferation significantly after the second antigen introduction. This could be due to the medium renewal as well. However, as seen in K. pneumoniae as a driver bacterium in the initiation and progression of CRC. Another interesting pattern observed here was the ability of PKS-ve CRC-exposed cells to sustain proliferation over longer periods of time compared to PKS+ve CRC-exposed cells. Although we may not see spikes in impedance of cells exposed to PKS-ve CRC cells, they are able to demonstrate a gradual increase in proliferation which will be useful in sustaining the progression of CRC cell proliferation. This is supportive of the notion that PKS-ve K. pneumoniae is a passenger bacterium.The ECIS array was used to confirm the enhancement of CRC cell growth upon exposure to K. pneumoniae isolates in possibly enhancing CRC progression in vitro. However, a more in-depth molecular study should be performed to understand the various aspects affected by virulence gene expression during tumorigenesis. Our observation suggests that the presence of the PKS gene and its counterpart virulence genes do play a role in enhancing CRC progression as summarized in K. pneumoniae strains is vital to dampen the progression of the tumor while improving the management of CRC patients.To our knowledge, this is the first known study to highlight the effect of PKS-positive


The majority of current cancer therapies are aimed at reducing tumour growth, but there is lack of viable pharmacological options to reduce the formation of metastasis. This is a paradox, since more than 90% of cancer deaths are attributable to metastatic progression. Integrin alpha9 (ITGA9) has been previously described as playing an essential role in metastasis; however, little is known about the mechanism that links this protein to this process, being one of the less studied integrins. We have now deciphered the importance of ITGA9 in metastasis and provide evidence demonstrating its essentiality for metastatic dissemination in rhabdomyosarcoma and neuroblastoma. However, the most translational advance of this study is to reveal, for the first time, the possibility of reducing metastasis by pharmacological inhibition of ITGA9 with a synthetic peptide simulating a key interaction domain of ADAM proteins, in experimental metastasis models, not only in childhood cancers but also in a breast cancer model.The online version contains supplementary material available at 10.1007/s00018-022-04557-y. Metastasis is the main cause of cancer-associated mortality and one of the most indicative prognostic factors for cancer patients –4. This The Integrin alpha9 (ITGA9) protein is composed of the α9 itself and a β1 subunit. Unlike other integrins, ITGA9 does not depend on the Arg-Gly-Asp (RGD) sequence for ligand recognition  and bindOne of the most remarkable families of ITGA9 interactors is the ADAM family, which has some members highly expressed in many types of cancer and can regulate cell–cell adhesion or cell-ECM interaction through its binding to adhesion molecules such as integrins and syndecans . Overall2 and were periodically tested for mycoplasma by PCR and authenticated by short tandem repeat profiling.RMS cell lines RH30 and RD were cultured in Minimum Essential Medium with Earle’s Salts (Biowest), NB cell lines BE(2)-C and CHLA90 were grown in Iscove’s Modified Dulbecco’s Medium (Thermo Fisher Scientific) and BC cell lines MDA-MB-231 and MDA-MB-468 were cultured in Dulbecco’s Modified Eagle Medium (Thermo Fisher Scientific). All media were supplemented with 10% FBS (Sigma), 1 × non-essential amino acids, 100U/ml penicillin and 0.1 mg/ml streptomycin . All cell lines were maintained at 37 °C in a controlled atmosphere of 5% CO5 target cells were seeded in 60 mm dishes and incubated together overnight. Positively transduced cells were selected with puromycin . Four different shRNAs were tested and the ones that reduced ITGA9 expression more efficiently were selected for further experiments. Rescue experiments to overexpress ITGA9 were performed using pcDNA3.1(+) vector (GenScript Biotech), carrying ITGA9 complete cDNA (NM_002207.3). Cells were transiently transfected with 2 μg of each construct using Lipofectamine (Thermo Fisher) and protein analyses were performed after 24 h.Genetic ITGA9 downregulation was performed by shRNA cloned into the lentiviral vector pGIPZ . Lentiviruses were produced in HEK293T cells and 2 × 10At 80%-confluence, cells were washed with PBS and scraped in RIPA protein lysis buffer (Thermo Fisher Scientific) supplemented with protease (Roche) and phosphatase inhibitors (Sigma-Aldrich). Cell lysates were incubated for 5 min at 95 °C and after centrifugation at 13000 rpm for 15 min at 4 °C, cell debris was discarded. Supernatant protein concentration was quantified by the DC Protein Assay (Bio-Rad) according to manufacturer’s instructions, and 20-40 µg of total protein was separated on 8–12% SDS-PAGE gels and transferred onto PVDF membranes . After blocking with 5% BSA (Sigma-Aldrich) for 1 h, membranes were incubated overnight at 4 °C with the following primary antibodies: anti-ITGA9 , anti-FAK , anti-pFAK at Tyr397  and anti-actin . Finally, membranes were incubated with the corresponding HRP-conjugated secondary antibodies for 1 h at room temperature and immunoreactive bands were visualized with ECL Prime reagent .5 cells resuspended in serum-free medium were seeded in the upper chamber of a Matrigel-coated Transwell (Corning), and the lower chamber was filled with serum-supplemented medium. Cells were incubated at 37 °C for 24 h and fixed with 4% paraformaldehyde (Sigma-Aldrich). After removal of the Matrigel, the cells that migrated to the lower surface of the membrane were stained with Hoechst-33342  and were counted under the microscope. Three independent wells were counted for each condition. To assess RA08’s effects on cell invasion (and RGD peptide), cells were previously treated for 48 h with different concentrations , which were also present in the medium during the cell invasion assay.1 × 10Cell proliferation was measured using crystal violet staining. Cells were seeded at low-density in 96-well plates, and a different initial number of cells were used for each cell line to ensure lineal growth throughout the assay. Media was changed every 48 h, including RA08 (or RGD peptide) treatment . Cells were allowed to proliferate for 5 days and were washed with PBS, stained with 0.5% crystal violet (Sigma-Aldrich) and washed thoroughly with distilled water. Plates were dried overnight and crystals were dissolved with 15% acetic acid (Carl Roth). Finally, absorbance was measured at 590 nm using an Epoch microplate spectrophotometer (Biotek).Anchorage-independent cell growth was measured by WST-1 (Sigma-Aldrich), following the manufacturer’s instructions. Cells were seeded at low density in polyHEMA-coated dishes (Santa Cruz Biotechnology) that prevented cell attachment to the plate and were allowed to grow for 48 h. At the end of the assay, cell spheres were disaggregated with accutase (Fisher Scientific) and WST-1 reagent was added to the medium. Three hours later, absorbance was measured at 440 and 690 nm using an Epoch microplate spectrophotometer (Biotek).6 RD cells, 1.25 × 105 MDA-MB-468 cells, and 1 × 104 and 2 × 104 BE(2)-C cells for the shRNA and RA08 models, respectively). To test RA08 effects, mice were split into three groups of 10 mice each and treated with the vehicle  or two different RA08 doses by subcutaneous injection. The first RA08 administration was performed two hours before tail vein injection and then three times per week throughout the experiment. Mice were monitored and their body weight measured twice per week. Ethical endpoint criteria were based on: acute weight loss , clear detection of metastases (lumps or swelling) or poor general appearance of the animal. Metastatic foci were found in post-mortem analysis of mice fulfilling at least one of these end-point criteria. After euthanasia, all organs were assessed and macroscopic metastases were detected. Then, tissue samples were collected, fixed in formalin (Sigma-Aldrich) and paraffin-embedded. In the case of RMS models, most of the metastases were found in lungs, ovary and suprarenal gland, while in the NB models the majority of metastases were hepatic. For the BC model, lung sections were deparaffinized and rehydrated for incubation with anti-GATA3 antibody . All procedures were approved by the Ethics Committee of Animal Experimentation of the Vall d’Hebron Research Institute (CEEA) and were in line with EU directive 2010/63/EU.Tumour cells were intravenously injected through the tail vein of five-week-old SCID-beige mice (Charles River Laboratories) housed in specific pathogen-free conditions. Cell number was adjusted according to the cell line’s ability to generate metastases  was purified in a semipreparative system equipped with a NW50 Merck column filled with 10 µm of kromasil silica, and the pure fractions analysed in an analytical RP-HPLC were lyophilized. The ion-exchange step was carried out to obtain the peptide with the acetate counterion. The final acetate compound was recovered via filtering and then lyophilized. (13.4 g were obtained.) RA08 was characterized by mass spectrometry by ESI–MS spectrometry (MW = 1806 a.m.u.).RA08 was synthesized by solid-phase peptide synthesis following the Fmoc/tBu strategy with 4-Methylbenzhydrylamine (p-MBHA) resin at the 30 mmol scale. Fmoc-AM-OH linker (2 eq.), HOBT (2 eq.) and DIPCDI (2 eq.) were used for attaching the linker to the resin. Then, synthesis proceeded with the removal of the Fmoc group by treatment with a solution of 20% piperidine in DMF (twice for 5 min and twice for 10 min). The peptide-resin was washed 5 times with DMF, filtered and the amino acids were coupled. Couplings were performed by using Fmoc-AA-OH:DIPCDI:HOBt (3 eq: 3 eq: 3 eq) in DMF (Fmoc-Msa-OH was coupled using 1.5 eq). All these coupling reactions were left to react for 40–60 min. The completion of each coupling reaction was controlled with a ninhydrin test (or a Chloranil test). After completion of the peptide sequence, the peptidyl-resin was washed with DMF, MeOH and Et2O and dried. 92.6 g of peptidyl-resin were obtained. Then, the peptidyl-resin was left to react for 2 h in a TFA mixture (TFA:TIO:TIS:H2O (85:5:5:5) ) at room temperature to cleave the peptide from the resin. The resin was washed with TFA and EtThe binding of RA08 to ITGA9 was assessed using the method of the cellular thermal shift assay (CETSA) as previously described . BrieflyLZerD protein docking suite [−1). The ADAM-12/ITGA9 binding affinity was also calculated with the same web server. All analyses were performed from protein structure predicted by homology-modelling server SWISS-MODEL [In silico analysis of RA08 binding to ITGA9 was performed using the platform ng suite  to preding suite  and calcSS-MODEL .t test or one-way ANOVA , considering *p < 0.05, **p < 0.01 and ***p < 0.001. Log-rank test was used for survival analysis.At least three independent replicates were performed for each experiment and analysis was performed using GraphPad Prism Software. Error bars in graphical data represent mean ± SD. Statistical significance was determined by Student’s ITGA9 expression was downregulated in cells stably transduced with two specific shRNAs sh#1 and sh#2) both very effective in reducing the amount of protein compared with empty vector-transduced cells (CTRL). Western blots -C, were selected to perform non-adherent cell growth assays and subsequent in vivo studies. Cells overexpressing sh#1 and sh#2 seeded in non-adherent conditions showed a reduction in cell proliferation after 48 h  a higher ITGA9 expression was observed in relapsed patients when compared with the subgroup without relapse  derived from the disintegrin domain of the native sequence of ADAM-12 (-Arg-Asp-Ser-Ser-Asn-Ser-Cys-Asp-Leu-Pro-Glu-Phe-). RA08 has been modified at the N-terminus with the inclusion of an unnatural amino acid , together with other modifications such as the substitution of Cys by Met, the substitution of Phe by 3-mesitylalanine , and the incorporation of two Lys at the N- and C-termini of the sequence. All these modifications aim to increase its stability in plasma and improve its pharmacological properties for inhibiting the ITGA9-ADAM interaction, with the ultimate aim of impairing cancer cell invasion and metastasis. Additionally, the incorporation of two further Lys residues, one at the N-terminus and another at the C-terminus, was performed to improve the peptide sequence’s solubility when formulated in physiological saline  in RMS and NB cell lines. In this case, since it has been previously suggested that ITGA9 played a role in breast cancer (BC) progression, BC cell lines were also included to test the possibility of the compound in this neoplasia. The results showed a remarkable reduction in FAK phosphorylation in RA08-treated cells in a dose-dependent manner, whereas total FAK and ITGA9 levels were unaltered Fig. b to e.p value: 0.016), observable from 55ºC onward, thereby suggesting that RA08 binds ITGA9 and protects it against thermic degradation  between beta-propeller domain for ITGA9 and the domain of ADAM12 that contains the peptide that inspired RA08. For more information and interactive format of the 3D structure pbd files, see Supplementary File 1 (RA08/ITGA9) and 2 (ADAM12/ITGA9).Additionally, the in silico analysis revealed also a high probability of binding between RA08 and ITGA9 (on its beta-propeller domain) with a binding affinity prediction value of − 13.4 Kcal molTreatment with RA08 produced a significant reduction in cell invasiveness in the majority of RMS, NB and BC cell lines, with RA08 concentrations ranging from 100 to 1000 nM. The effects of RA08 were tested in two cell lines for each of the cancers included in this work Fig. a. In RMSSimilarly, treatment with RA08 reduced cell growth when cells were cultured in anchorage-independent conditions, concurring with the previous results obtained with the genetic depletion of ITGA9. Thus, in the 3 cell lines analysed, a reduction of ~ 30% in anchorage-independent cell growth was observed in the presence of RA08 at 1000 nM Fig. b.With the aim of evaluating the therapeutic potential of RA08 in vivo murine metastatic experimental models were established by tail vein injection. In the RMS model Fig. a, all coLikewise, the NB model Fig. c revealeFinally, the BC model showed a diffuse pattern of invading cells and it was not possible to quantify the number of macroscopic lesions. In this case, with the aim of evaluating metastatic foci in mouse lungs, immunohistochemical staining of GATA3 was used as a marker of tumour cells. Although the quantification procedure was different due to the diffuse nature of the metastatic cells, the results also showed a reduction in metastatic foci to half for the two doses analysed (1 mg/kg and 2 mg/kg) Fig. e and f.It is well known that the onset of metastasis very often involves crossing a boundary beyond which the probability of cure becomes clearly lower. The fact that most of cancer-related deaths are attributed to metastases, but, unfortunately, treatments for their blockade have not yet been established, is a tremendous paradox. Probably, the more advanced studies in this direction were focused on inhibiting angiogenesis , 37, matA noticeable variable of metastasis is its temporal course, which strongly depends on cancer type. For instance, in small cell lung cancer and pancreatic cancer, metastatic disease often appears at the time of (or shortly after) the initial diagnosis, whereas many patients with breast and prostate cancers, among many others, tend to show metastasis after a prolonged period of time . These oMYCN-amplified in NB) are completely independent of ITGA9 levels. Conversely, in the case of patients classified as fusion-negative in RMS and non-MYCN amplified in NB, the outcomes showed dependency on ITGA9 levels. Consequently, a blocking of its function may hinder the development of metastasis in these specific subtypes. Interestingly, fusion-negative RMS and non-MYCN-amplified NB are considered to have better prognosis compared with less favourable subgroups of patients; however, unresolved questions persist regarding the optimal management for these groups of patients given that some cases can still relapse and/or metastasize. The fact that, in RMS and NB, ITGA9 can discriminate a subgroup of patients with worse prognosis should be considered for subsequent studies, as this new set of patients with worst prognosis would be candidates for putative ITGA9-based anti-metastatic therapy or, at least, for an intensification of treatment owing to their putative propensity to metastasize. Two BC cell lines were included in this part of the study to test the possible applicability of the compound to other neoplasia. In these cell lines, variability was also observed between the 2 cell lines analysed, with the cell line MDA-MB-468 showing significant sensitivity to the compound whilst in the other (MDA-MB-231), effects were only observed at the highest dose (1000 nM). Both cell lines correspond to triple negative BC, but relative ITGA9 expression is notably higher in MDA-MB-468 , thereby suggesting that the expression level may correlate with sensitivity to treatment with RA08.The experiments herein provided revealed a strong dependency on ITGA9 in several steps of metastasis, even though the experimental tail vein models provided skip initial steps of the process and, therefore, ITGA9 role may be even more extensive. The RGD peptide used as a reference promoted similar effects on cell invasiveness. However, the strong concomitant reduction observed in cell growth pointed to a higher toxicity and to a less-specific activity, probably caused by the wide range of integrins and ligands potentially affected by RGD blocking. This reinforces our approach and suggest a greater specificity of ITGA9 blocking as well as lower toxicity. Interestingly, a strong variability in the response to the inhibition of ITGA9 among different cell lines was detected. Particularly, RD and BE(2)-C cell lines were clearly more sensitive than CHLA90 (slightly sensitive), or RH30 (moderately sensitive). These differences observed among cell lines pointed to a diversity of response probably caused by the molecular background of each cell line, ITGA9 basal expression  and the histology of the tumours from which were derived. Interestingly, the patient’s gene expression data presented , although clear effects upon reduction of metastasis were found in RMS, NB and BC in vivo models. Beyond the effects of RA08 on metastasis, the low toxicity of the compound makes it particularly interesting, since a putative therapy for metastasis prevention is expected to be long-term, probably spanning several months and, therefore, its possible future implantation is going to be enormously facilitated if the compounds used show a complete absence of associated non-desirable side effects.Taken together, our results indicate an important role of ITGA9 in the establishment of metastasis and present RA08 as a new compound able to prevent its origin in RMS and NB models, with potential applicability in a wide range of tumour types. In addition, the prognostic value of ITGA9 expression is also depicted, emphasizing the potential of this protein as prognostic factor in some subsets of patients.ITGA9 is expressed in RMS, NB and BC cell lines. Representative immunoblot showing ITGA9 protein levels in a RMS, b NB and c BC cell lines (PDF 845 KB)Comparative gene expression analysis of alpha integrins in RMS by its molecular subtype (fusion-positive (FP+) vs fusion-negative (FN-)). Scatter dot plots representing the gene expression for each sample. Lines and error bars represent the mean ± CI 95%. No data was available for ITGA3, ITGA11, ITGAD and ITGAX. Statistical significance  was obtained after two-tailed t-test (PDF 71 KB)Comparative gene expression analysis of alpha integrins in RMS FP+ by its state . Scatter dot plots representing the gene expression for each sample. Lines and error bars represent the mean ± CI 95%. No data was available for ITGA3, ITGA11, ITGAD and ITGAX. Statistical significance  was obtained after one-tailed t-test (PDF 46 KB)Comparative gene expression analysis of alpha integrins in RMS FN- by its state . Scatter dot plots representing the gene expression for each sample. Lines and error bars represent the mean ± CI 95%. No data was available for ITGA3, ITGA11, ITGAD and ITGAX. Statistical significance (*p < 0.05) was obtained after one tailed t-test (PDF 54 KB)Gene expression analysis of alpha integrins in NB MYCN amplified compared to MYCN non-amplified. Scatter dot plots representing the gene expression for each sample. Lines and error bars represent the mean ± CI 95%. Statistical significance  was obtained after two-tailed t-test (PDF 155 KB)Gene expression analysis of alpha integrins in relapsed NB MYCN non-amplified compared to non-relapsed. Scatter dot plots representing the gene expression for each sample. Lines and error bars represent the mean ± CI 95%. No data was available for ITGA11 and ITGAD. Statistical significance (*p < 0.05) was obtained after one-tailed t-test (PDF 57 KB)Relapse-free survival curves of NB MYCN non-amplified patients considering each alpha integrin expression. For each alpha integrin analysed, samples (n = 102) were separated in high or low expression considering the median gene expression. Statistical significance was obtained after Log-rank test to compare survival distributions (PDF 158 KB)Supplementary file8 (PDF 110 KB)Relapse-free survival curves of NB MYCN non-amplified patients considering each alpha integrin expression. For each alpha integrin analysed, samples (n = 102) were separated in high or low expression (PDF 11810 KB)Treatment with RGD peptide significantly reduces cell invasiveness and cell growth. a. Relative cell invasiveness and cell proliferation after treatment with increasing concentrations of RGD peptide  in RMS (RD and RH30), NB (BE(2)-C and CHLA90) and BC (MDA-MB-468 and MDA-MB-231). Bars represent cell invasion (significance indicated with asterisks), whereas cell proliferation is represented by dots and lines (significance indicated with hashes). Representative images of the Transwell-invasion assays, including control cells and cells treated with 1000nM are also shown below each plot. All measurements were taken in triplicates. All values are expressed in percentages and referred to those of the control condition. Statistical significance  was obtained after one-way ANOVA test. b. Molecular structure, name and chemical formula of the RGD peptide used for this experiment (PDF 5713 KB)File containing the 3D structure of RA08 and ITGA9 predicted interaction from LZerD protein docking suite platform (PBD 655 KB)File containing the 3D structure of ADAM12 and ITGA9 predicted interaction from LZerD protein docking suite platform (PBD 1174 KB)Below is the link to the electronic supplementary material.


Merkel cell carcinoma (MCC) is a rare but highly aggressive skin cancer. Despite important progress, overall understanding of the events that drive MCC carcinogenesis remains incomplete. We discovered that the plasma membrane citrate transporter (pmCiC) is upregulated in Merkel cell carcinoma cell lines. Cancer cells import extracellular citrate via pmCiC to support their metabolism, which is critical to support proliferation and metastatic spread. In this study, we show that inhibition of pmCiC can decrease the growth rate of Merkel cell carcinoma cell lines. Targeting pmCiC and thereby the tumor metabolism should be considered further as a potential anti-cancer therapy.+-gluconate) blocks this citrate-induced proliferation. Furthermore, the 3D in vivo Chick Chorioallantoic Membrane (CAM) model showed that the application of Na+-gluconate also decreases Merkel cell carcinoma growth. Based on our results, we conclude that pmCiC and extracellular citrate uptake should be considered further as a potential novel target for the treatment of Merkel cell carcinoma.Merkel cell carcinoma (MCC) is a rare but highly aggressive tumor of the skin with a poor prognosis. The factors driving this cancer must be better understood in order to discover novel targets for more effective therapies. In the search for targets, we followed our interest in citrate as a central and critical metabolite linked to fatty acid synthesis in cancer development. A key to citrate uptake in cancer cells is the high expression of the plasma membrane citrate transporter (pmCiC), which is upregulated in the different adenocarcinoma types tested so far. In this study, we show that the pmCiC is also highly expressed in Merkel cell carcinoma cell lines by western blot and human tissues by immunohistochemistry staining. In the presence of extracellular citrate, MCC cells show an increased proliferation rate in vitro; a specific pmCiC inhibitor (Na Merkel cell carcinoma (MCC) is an aggressive tumor of the skin with a rising incidence  that preAfter the diagnosis of MCC, a complete excision (1–2cm margin) and adjuvant radiation is normally performed. Sentinel lymph node mapping and biopsy are recommended together with stage imaging , or positron emission tomography (PET)) . Around Membrane transport proteins are involved in the movement of ions and small molecules across biological membranes. Many different membrane transporters have been shown to facilitate cancer development, progression, and drug resistance . A recenIn this study, we have conducted experiments to explore the hypothesis that pmCiC is important for MCC tumors by first measuring expression in MCC cell lines and human tumor biopsies. We then tested the effects of extracellular citrate on MCC proliferation in vitro and applied gluconate-based pmCiC inhibition to these in vitro and in vivo systems to show whether the tumor promoting effects of citrate can be inhibited.We first tested whether characterized MCC cell lines express pmCiC in vitro. All four MCC cell lines  showed high expression of pmCiC by Western blotting A.Next, we utilized immunohistochemical staining to detect pmCiC expression in formalin-fixed and paraffin-embedded primary MCC tissue sections. These results showed that 26 of the 28 samples stained positive for pmCiC (93%), where 2 samples showed no pmCiC expression (−), 9 showed weak/partial expression (+/−), 9 showed high expression (+), and 8 had very high expression (++) B. In totp = 0.00036; 72 h: p = 0.00007; t-test with citrate 48 h: control p = 0.0017; gluconate p = 0.0008; citrate + gluconate p = 0.0007). After adding 100 µM Na+-gluconate to inhibit pmCiC-mediated citrate transport into cells, the proliferative effects of citrate were abrogated. Notably, Na+-gluconate treatment alone had no effect on PeTa MCC cell proliferation .To investigate the effect of citrate on MCC cells, we used a dialyzed serum that does not contain citrate in experimental cultures of MS-1 cells. For this experiment, one group of cultures was supplemented with 200 µM citrate and the proliferation rate in both groups was measured after 24, 48, and 72 h using the CyQUANT Cell Proliferation Assay. There was a significant difference at 48 and 72 h between cells treated with 200 µM Na+-citrate versus cultures without added citrate,  decreased tumor growth when treated with Na+-gluconate externally for 5 days compared to tumors treated with NaCl (+-gluconate (concentration 500 mg/kg/d) or NaCl was applied every 24 h, as previously described (n = 20) [+-gluconate , as well as for WaGa . These results suggest not only a direct effect of gluconate on proliferation, but also on angiogenesis.Bhat et al. showed that the Chick Chorioallantoic Membrane (CAM) model works well for in vivo testing of MCC tumor cell development . In our ith NaCl B. Na+-gl(n = 20) . Consistluconate C and macol group D. This e+-gluconate as a specific pmCiC inhibitor. Growth inhibition was most probably due to cell cycle inhibition, rather than induction of apoptosis. We validated these findings using the 3D in vivo CAM model, where Na+-gluconate lowered MCC proliferation and showed antiangiogenic effects on tumor-surrounding vessels. Therefore, our study supports further research to therapeutically target the pmCiC in human MCC.In our present study, we primarily provide evidence that citrate and pmCiC expression may play a role in the biology of human MCC. PmCiC is expressed in 93% of MCC tissue samples and in 4 out of 4 tested cell lines. In vitro, we could detect an effect of extracellular citrate on the proliferation rate of PeTa and MS-1 cell lines. This effect could be blocked in PeTa cells by adding NaPmCiC expression in MCC human tissue was present in most of the specimens studied. Due to the incomplete clinical patient history available, it is difficult to make a firm conclusion about the potential correlation of staining intensity with disease outcome. However, our data strongly suggests that pmCiC is expressed in most patients with metastatic disease, which is consistent with our hypothesis that extracellular citrate uptake plays an important role in supporting metastatic behavior in vivo . From a Citrate is the primary substrate in fatty acid synthesis, which is a metabolic hallmark of cancer . By exte+-gluconate as a specific pmCiC blocker [+-gluconate treatment correlates with the lower MCC tumor cell proliferation rate when deprived of citrate [Blocking of citrate uptake by cancer cells using Na blocker  reduced  blocker . The cle citrate . Glucona citrate ,24. Mech citrate .2O2 for 10 min, samples were washed once with bi-distilled water, heated in HIER Citrate Buffer pH6  at 90 °C for 20 min, and cooled for 20 min. Samples were blocked using the blocking solution of the ZytoChem Plus HRP-kit Rabbit/Mouse (Zytomed) for 10 min.Paraffin-embedded sections were dewaxed using xylol  and then rehydrated by washing twice with absolute ethanol, twice with 96% ethanol, and twice with 70% ethanol. After blocking endogenous peroxidases using 3% HSections were labeled at 4 °C overnight using a rabbit monoclonal antibody against pmCiC  and a rabbit monoclonal antibody against Ki67 . After washing with DPBS, sections were incubated with biotinylated secondary anti-rabbit (HRP060-RB) antibodies for 30 min, washed in DPBS, and incubated with streptavidin HRP conjugate for 20 min . After washing with DPBS, slides were stained using AEC+ High Sensitivity Substrate Chromogen Ready-to-Use , counterstained using hematoxylin , and mounted using Aquatex (Merck). Tissues were collected from patients of the department of dermatology at the university hospital of Regensburg and correlated with clinical outcome. Ethics permission No. 22-2834-104.+- gluconate , and dialyzed serum . The following antibodies were used: pmCiC specific antibodies  and Ki67 . Experimental media consisted of glucose free RPMI-1640 , 10% dialyzed serum, 2 mM glutamine, 0.25 g/L glucose, ±200 µM citrate, and ±100 µM Na+-gluconate, unless otherwise stated. The incubation time varied between 24 h, 48 h, 72 h, and 5 weeks, as specified. Western blots were analyzed by measurement of the pixel density using ImageJ software , University of Wisconsin, Madison, WI, USA). The CyQUANT Direct Cell Proliferation Assay  was used to measure cell proliferation. Cells were counted with LUNA-FL™ Automated Fluorescence Cell Counter . Western blots were analyzed by measurement of the pixel density using ImageJ software.Cell lines from primary Merkel cell carcinoma (PeTa), as well as from metastatic disease , were grown in RPMI-1640 medium with 1% penicillin/streptomycin, 1% L-glutamine, and 10% fetal bovine serum. Although cell lines were not further authenticated, they were grown at low passage numbers from original sources and were kept typically in culture for only 2 months. Cells were tested and confirmed to be mycoplasma free. The following chemicals were used: citric acid and Na6 Merkel cell carcinoma cells (WaGa and MKL-1), daily treatment with either Na+-gluconate (500 mg/kg/d) or NaCl 0.9% was performed (n = 20). Pictures were taken and tumors were removed after 5 days. The size of tumors was measured and the surrounding vessels were counted for all tumors.The Chick Chorioallantoic Membrane (CAM) model was performed as described before ,26,27. Tt-test was performed and significance was assumed for p < 0.05.Statistics were performed using GraphPad by Dotmatics. A In conclusion, extracellular citrate uptake via the pmCiC is a potential novel therapeutic target in MCC tumors that highly expresses this critical citrate transporter, especially for cancers that tend to be more aggressive or metastatic. The exact mechanisms that play a role in this effect will require further investigation.