MRI of ASCT2-mediated amino acid uptake in xenograft tumor models

MRI of ASCT2-mediated amino acid uptake in xenograft tumor models | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article MRI of ASCT2-mediated amino acid uptake in xenograft tumor models Behnaz Ghaemi, Balaji Krishnamachary, Natalie Dillman, Kirsten N. Bains Williams, and 10 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7367339/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 9 You are reading this latest preprint version Abstract We investigated alanine as a magnetic resonance imaging (MRI) biomarker for alanine-serine-cysteine transporter 2 (ASCT2), the primary transporter for glutamine in cancer. Alanine exhibited higher contrast using the chemical exchange saturation transfer (CEST) method than other major ASCT2 substrates. Upon bolus injection of alanine (6 mmole/kg), CEST MRI enhancement in vivo was higher in SLC1A5 -overexpressing Pa20c pancreatic tumors than naïve tumors (p < 0.0001). Enhancement was more pronounced in LNCaP prostate tumors than DU-145 prostate tumors in vivo (p < 0.0001). The temporal dynamics of alanine-weighted CEST (ALAwCEST) signal enhancement depended on the volume of the tumor, which histological analysis revealed to be due to alterations in the expression and distribution of ASCT2 and CD31-positive blood vessels. Mass spectrometric imaging of 13 C-labeled metabolites confirmed differences in alanine uptake and metabolism in prostate tumors. We demonstrated the feasibility of ALAwCEST imaging for reporting ASCT2-mediated uptake in multiple human cancer models. Health sciences/Biomarkers Biological sciences/Cancer Health sciences/Oncology Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Cellular glutamine dependence is an attractive target for cancer imaging and therapy. Glutamine is actively transported into the cell, upon which it is metabolized to serve as a nitrogen and carbon source for biosynthesis of amino acids and nucleosides and as a supplemental energy source 1 . Glutamine transporters have been bi-directionally linked to the activation of oncogenes: Mdm2 2 , which has been shown to enhance the expression of these transporters, and Myc 3 , which is activated by them. Alanine-serine-cysteine transporter 2 (ASCT2; gene: SLC1A5 ), the primary transporter for glutamine import, is associated with poor prognosis for many cancers 4 – 8 and their therapies 9 . At the clinical stage, several glutamine antagonists that include ASCT2 inhibition as their mode of action have been tested, though none beyond phase I trials 10 . In preclinical stages, multiple inhibitors have been developed to target this transporter 11 , 12 . Assays for measuring the transport capabilities of ASCT2 ex vivo are not yet commercially available. Histopathological grading of its expression is a current clinical metric for ASCT2. For prostate cancer, higher ASCT2 expression corresponds with cancer recurrence 9 . Still, expression level is not proportional to cell uptake by ASCT2, unless glycosylated to translocate from the cytoplasm to the plasma membrane 13 . In addition, functionality of ASCT2 is sodium-dependent and alterations in the transporters responsible for regulating sodium levels have been reported in multiple cancers 14 . If a suitable MRI-detectable substrate could be found, in vivo MRI may allow evaluation of ASCT2-mediated transport and report on the tumor environment. We hypothesized that natural substrates could serve as chemical exchange saturation transfer (CEST) magnetic resonance imaging (MRI) agents for evaluating ASCT2 activity. CEST MRI has the advantage of detecting low-concentration molecules (mM level) without modification or the need for specialized equipment through the presence of water-exchanging protons, for instance in their amine groups 15 . In this report we investigated the potential utility of several natural amine-containing substrates as CEST imaging biomarkers for ASCT2 activity, namely: alanine, asparagine, glutamine, serine, and threonine. Next, we measured CEST contrast upon intravenous injection of alanine in mice bearing Pa20c pancreatic tumors engineered to express different levels of ASCT2. Afterwards, we evaluated alanine-weighted CEST (ALAwCEST) contrast in vivo for DU-145 and LNCaP prostate tumors that differentially express ASCT2. Subsequently, we correlated ALAwCEST enhancement to ASCT2 expression in these tumors. Lastly, we correlated ALAwCEST enhancement to the uptake and metabolism of 13 C-labeled alanine using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging. Results CEST MRI of Major ASCT2 Substrates Initially, we assessed the potential of naturally occurring CEST MRI agents to report on ASCT2 activity. We screened 5 natural amino acids that are major substrates of ASCT2—glutamine, alanine, serine, threonine, and asparagine 16 —in PBS solution (20 mM, pH of 7.3, T = 37°C) for amineCEST signal generation (Fig. 1 ). They were also compared to ACBC (1-aminocyclobutane carboxylic acid), the nonfluorinated synthetic amino acid from which [18F]-fluciclovine ([18F]-FACBC) is derived. Notably, glutamine 17 , asparagine 17 and ACBC 18 can also be transported by the LAT1 amino acid exchanger. Of them, alanine generated the highest signal, followed by glutamine and serine, and then ACBC. Threonine and asparagine generated minimal signal compared to PBS alone. Thus, we continued investigating alanine based on its high CEST signal, as well as its relatively good clinical safety profile 19 compared to the other ASCT2 substrates. Next, we evaluated the sensitivity of ALAwCEST MRI to ASCT2 activity using a genetically-engineered cancer model. We characterized ASCT2 expression in naïve and SLC1A5 -overexpressing Pa20c patient-derived pancreatic xenograft tumors (Fig. 2 ) and confirmed that SLC1A5 -overexpression in this cell line increased ASCT2 protein levels in its tumors. Then, we compared their CEST signal enhancement at the + 3.1 ppm frequency upon intravenous alanine injection (6 mmol/kg). As shown in Fig. 2 c, CEST signal remained close to baseline levels over a period of 60 min following infusion in naïve Pa20c tumors (n = 3), but amine-weighted CEST (i.e. ALAwCEST) rose steadily in Pa20c tumors engineered to express higher levels of ASCT2 (n = 4). Their CEST enhancement significantly differed (p < 0.0001). Alanine-Weighted CEST MRI of Prostate Tumor Models Subsequently, we assessed the potential of ALAwCEST to differentiate naïve prostate tumor models with different levels of ASCT2 expression and glutamine uptake. We characterized ASCT2 expression (Fig. S1 ) in prostatic cancer cell lines DU-145 and LNCaP using fluorescent microscopy. The average intensity of ASCT2 in LNCaP was 11% higher than that of DU-145 (p = 0.0203). Since cellular uptake by ASCT2 requires translocation of the transporter to the plasma membrane, we investigated the intensity across the cell area. A line profile revealed expression at the cellular border was higher than that in the cytoplasm for LNCaP cells. It also revealed that the cell line had higher fluorescent intensity compared to DU-145 across the cellular space. Since ALAwCEST signal could be dependent on downstream enzymatic conversion, we also quantified the expression of alanine transaminase (ALT2) and glutamate dehydrogenase 1 (GDH1) in these cells (Fig. S2). The average intensity of ALT2 and GDH1 was 58% (p = 0.0078) and 25% (p = 0.0190) higher in LNCaP than DU-145, respectively. Furthermore, since hypoxia is a common feature of prostate tumors, we also evaluated changes in their expression after short-term (24 h) exposure to hypoxia (1%). Within that time frame we found ASCT2 expression was significantly lower (5%: p = 0.0108) in LNCaP cells exposed to hypoxic conditions compared to LNCaP cells exposed to normoxic conditions. We then characterized changes in the CEST signal of their tumor spheres (100 µL cell volume) when exposed to alanine (400 µL of 10 mM; Fig. S3). CEST signal in LNCaP-containing phantoms was higher than DU-145-containing phantoms (p = 0.0090) and the alanine only control phantoms (p = 0.0021) after 4 hours of exposure (210 minutes after the initial acquisition). ALAwCEST enhancement of DU-145 containing phantoms was also higher than alanine only control phantoms (p = 0.0012). Subsequently, we compared ALAwCEST signal enhancement between LNCaP (n = 8) and DU-145 (n = 9) tumors. We observed necrotic cores in some of the larger DU-145 tumors (> 630 mm 3 , similar to others 20 ) and excluded those mice from further analysis. In dynamic CEST scans, CEST enhancement was higher in LNCaP tumors than DU-145 tumors (p < 0.0001). In static CEST scans, acquired before and after the dynamic ones, CEST contrast at the + 3.0 to + 3.2 ppm frequency range and the − 3.2 to -3.0 ppm frequency range did not significantly change upon administration of alanine. We then stratified tumors by volume. ALAwCEST enhancement profiles differed visually in LNCaP tumors with volumes less than 600 mm 3 (n = 5) and greater than 630 mm 3 (n = 3) when alanine was administered (Fig. S4). For smaller tumors ( 630 mm 3 ), CEST signal steadily increased during the scan session. CEST signal dynamics between these two size classes significantly differed (p = 0.04). CEST enhancement profiles differed visually in DU-145 tumors with volumes less than 165 mm 3 (n = 5) and greater than 310 mm 3 (n = 4) when alanine was administered (Fig. S5). ALAwCEST signal at the + 3.1 ppm frequency was higher than baseline for smaller tumors ( 310 mm 3 ). Their CEST signal dynamics significantly differed (p < 0.0001). CEST enhancement at the end of the dynamic scan negatively correlated (ρ = -0.7579, p = 0.02) with tumor volume. CEST signal enhancement amongst the volume-stratified tumor groups were then compared (Fig. 3 ). At early times (~ 15 minutes after alanine injection), signal at the + 3.1 ppm frequency was significantly (0.008 < p < 0.043) higher in smaller LNCaP tumors ( 310 mm 3 ). Towards the end of the scan session, both smaller (< 600 mm 3 : 0.0261 < p 630 mm 3 : 0.0148 < p 310 mm 3 ). Fluorescent Imaging of ASCT2 Content in Prostate Tumors Next, we characterized ASCT2 and CD31 expression in larger and smaller LNCaP and DU-145 tumors (Fig. 4 , Fig. S6). Fluorescent intensity of ASCT2 was highest in smaller DU-145 tumors. Levels were comparable in the periphery of smaller LNCaP tumors, but were much lower in the center, reminiscent of the ring of bright CEST signal observed in Fig. 3 . ASCT2 staining intensity in the periphery of larger DU-145 tumors was comparable to that in larger LNCaP tumors, but in the center of larger DU-145 tumors, staining levels were low and heterogenous proximal to necrotic (acellular) lesions. Endothelial (CD31) levels were generally comparable, in line with what was reported in the literature for these two tumors 21 – 24 . Still, CD31 staining was lower in the center of smaller LNCaP and larger DU-145 tumors. In the former, CD31 staining patterns were similar to ASCT2 staining patterns, i.e. ring-like. In the latter, CD31 staining was also heterogenous, but localized independently of ASCT2 staining. MALDI Mass Spectrometry Imaging of Prostate Tumor Uptake and Metabolism of Alanine To demonstrate that the source of the CEST MRI signal enhancement was the uptake of alanine, we compared the presence of 13 C3-labeled alanine and its metabolites in tumors ex vivo using isotopomer analysis of MALDI mass spectrometry images (Fig. 5 ). The presence of 13 C3-alanine (p = 0.0329) and its immediately downstream metabolic product 13 C3-pyruvate (generated intracellularly by alanine transaminase: p = 0.0357) was higher in LNCaP tumors than in DU-145 tumors. In contrast, the presence of 13 C3-lactate, a product of the CEST agent entering the glycolytic pathway, was similar (p = 0.0877) amongst the tumors. Discussion Although best known for transporting glutamine, the neutral amino acid transporter ASCT2 has multiple, naturally-occurring substrates 16 , 25 – 27 , i.e. alanine, asparagine, glycine, leucine, methionine, serine, threonine, and valine. All of these substrates can be metabolized via their respective transaminases 28 . We investigated the utility of CEST MRI to detect and image ASCT2-mediated transport using some of these unmodified, naturally occurring substrates. We then selected alanine from this pool of candidate agents in part due to its readiness for translation. Alanine is a well-recognized substrate for ASCT2 that has been injected intravenously at a dose of 6 mmole/kg 29 into humans for other applications without symptoms. Its suitability as an ASCT2 agent for cancer imaging has already been demonstrated with PET imaging in preclinical models using a fluorinated version of the metabolite 30 . In addition, 13 C-labeled alanine 31 has also been injected in humans and is readily hyperpolarized 32 , permitting spectroscopic imaging. Most recently, its T2 relaxation was leveraged to permit imaging of a glioblastoma model (U87MG) using MRI 33 . The relatively low toxicity of alanine in humans permitted us to use high doses for intravenous administration in our experiments, which should be translatable to the clinic. Its relatively high safety profile 19 has the added advantage of enabling the repetition of these scans, which is beneficial for monitoring therapeutic efficacy in cancer management. We elected to investigate alanine-mediated CEST enhancement in multiple tumor xenograft models. The use of genetically-engineered pancreatic tumor models enabled us to establish the feasibility of alanine to report ASCT2 activity. The use of the DU-145 and LNCaP prostate cancer models enabled us to establish the sensitivity. Their relative expression (protein) of ASCT2 is well-established 9 , 34 , 35 and the uptake of ASCT2 substrates in LNCaP cells is reported to be nearly 1.5 times faster than that of DU-145 cells 36 , 37 . Still, compared to other cancers, prostate cancer expression (mRNA) is quite low, ranking fourth from last according to a systematic review 38 . This combination of attributes permitted us to examine the utility of dynamic ALAwCEST MRI at the lower limits of ASCT2 for cancer applications. When naturally occurring molecules are used, CEST enhancement is dependent on not only the pH and concentration of the agents in tissue compartments, but also on the concentration of their metabolized products when the latter contain the same exchangeable proton groups 39 – 44 . In the tumor microenvironment intracellular pH becomes more alkaline, reducing CEST contrast for amine groups. On the other hand, pH in the interstitium (extracellular, extravascular space) acidifies, increasing amine proton contrast 45 – 49 . Reported values of intracellular and extracellular pH in prostate cancer models range from 7.1 to 7.6 50–52 and from 6.4 to 6.8 53–55 , respectively. In larger (> 310 mm 3 ) DU-145 tumors the directionality of CEST enhancement was slightly negative (Fig. S5), which could result from the lower ASCT2 levels observed in histological images compared to the smaller tumors (Fig. 4 , Fig. S6). Since hypoxia is often featured in larger 56 – 58 , more advanced 59 tumors, particularly prostate cancer 60 , we investigated its impact on their expression in DU-145 and LNCaP cells. We did not observe significant alterations in ASCT2 expression (protein) in DU-145 cells when cultured in hypoxic conditions in vitro , but the same conditions decreased ASCT2 expression in LNCaP cells (Fig. S2) and increased ASCT2 expression (mRNA, protein) in pancreatic cancer cell lines 61 . Still, decreased ASCT2 expression and/or function may occur under longer-term exposure. Alternatively, low glutamine availability—which is associated with less vascularized tumors such as the larger DU-145 tumors in this report (Fig. 4 )—has been shown to decrease ASCT2 expression in liver 62 and gastric 63 cancer cell lines. In larger LNCaP tumors (cutoff between 600–630 mm 3 ), a decreased vascular fraction also explains the size-dependence of CEST profiles for LNCaP tumors as the initial peak seen in smaller tumors was not present in larger tumors. Peaks within the first 30 minutes has been observed with other CEST agents delivered at these doses 41 . Additionally, early-stage blood vessel marker CD31 revealed larger LNCaP tumors were less vascularized than smaller ones (Fig. 4 , Fig. S6). MALDI mass spectrometry imaging was used in our studies to validate the uptake of our CEST agents (Fig. 5 ). MALDI imaging confirmed the presence of 13 C-labeled alanine in tumor tissue. However, similar to in vivo imaging techniques, mass spectrometry imaging is unable to differentiate the compartment, i.e. vascular, extracellular and intracellular spaces, of these metabolites unless they are known not to be transportable. To minimize the contribution of the vascular and extracellular compartments to this measurement, we extracted the tumors after multiple half-lives (< 15 minutes 64 , 65 ). Furthermore, as alanine is metabolized inside the cell, we compared differences in the presence of its 13 C-labeled metabolic products to indirectly confirm the occurrence of cellular uptake. These findings warrant more extensive in vivo and ex vivo metabolic studies to establish the fate of this imaging agent and improve the interpretation of its images. There are several limitations to our studies. Firstly, we did not image pH in vivo or ex vivo to evaluate its impact on our measurement of ASCT2 function. Still, several MRI and magnetic resonance spectroscopy methods to measure pH have been translated to human use 66 that can be used to evaluate its contribution. Secondly, we did not image tumor vascular function to evaluate its impact on CEST enhancement and MALDI images. However, CD31 staining (Fig. S6) revealed vessel density in DU-145 and LNCaP tumors was comparable. Furthermore, the blood clearance half-lives of small molecules such as alanine in rodent blood are less than 15 minutes 64 , 65 ; therefore, the contribution of the vascular fraction to the CEST signal would be expectedly minimal by the end of the scan session. Still, we recognize that the measurement and consideration of these confounding factors would improve the interpretation of ASCT2 imaging in the clinic. Gadolinium-enhanced MRI is already a feature of cancer patient care used to evaluate the vascular profile of tumors. These evaluations would complement the visual information provided by ALAwCEST enhancement. In conclusion, we evaluated alanine for imaging ASCT2-mediated glutamine transport. ALAwCEST MRI detected its uptake without modification via the water-exchanging protons of its amine group. Dynamic CEST imaging of alanine was able to differentiate Pa20c-naïve and Pa20c-ASCT2 pancreatic tumor models as well as DU-145 and LNCaP prostate tumor models. The direction and magnitude of dynamic ALAwCEST enhancement when alanine was administered was sensitive to size-dependent differences in the ASCT2 content, particularly for DU-145 tumors. Lastly, as alanine has been intravenously injected into humans for other applications at a dose (6 mmole/kg) that should allow ALAwCEST MRI detection, ASCT2 imaging using this metabolite warrants further optimization and development as its use as a noninvasive, natural imaging agent is readily translatable. Methods Prostate cell line culture . For monolayer cultures, the prostatic cancer lines DU-145 (ATCC, 1 x 10 4 ) and LNCaP (ATCC, 2 x 10 5 ) were seeded onto 6-well plates and cultured in RPMI media (Gibco, A10491-01) supplemented with fetal bovine serum (10%; Gibco, 25140-079) and penicillin-streptomycin (1%; Gibco, 15140-122). For spheroid culture, DU-145 cells (1.5 x 10 3 ) and LNCaP (1.5 x 10 4 ) cells were seeded onto low-attachment 6-well plates (Corning Costar®) and cultured in DMEM/F12 media (Gibco, 10565-018) supplemented with 1% penicillin-streptomycin, B27 (2%; Gibco 17504-044), insulin (3 µg/ml; Sigma, I2643) epidermal growth factor (20 ng/mL; Gibco, PHG0311), and knock-out serum (3%; Gibco, 10828-010) until a 100 µL volume of cells was attained. Pancreatic cell line generation and culture . The Pa20c (Panc198/RRID: CVCL_E285, male) human pancreatic cancer cell line is from a primary pancreatic tumor characterized previously in a published work 67 that was kindly provided by Dr. Anirban Maitra (MD Anderson Cancer Center). To generate ASCT2-overexpressing Pa20c cells, a 1.62 kb open reading frame (ORF) of human solute carrier family 1 member 5 (SLC1A5) transcript variant 1 (ASCT2) was spliced into a lentiviral vector (LV-231-SLC1A5-eGFP) purchased from GeneCopoeia (Cat. No. EX-A6442-Lv231, Rockville, MD) and selected using puromycin. Virions for expressing the ASCT2 gene were produced by co-transfecting 293T cells with 12 µg of lentiviral construct, 6 µg of packaging plasmid pCMVΔR8.2 DVPR (VPR deleted) and 1.5 µg of pCMV-VSVg plasmid using lipofectamine 2000 (Invitrogen, Carlsbad, CA). Supernatant containing the virions were collected 48 h after transfection and gently centrifuged to remove cell debris. Pa20c cells were transfected daily with fresh lentivirus in serum-free media with 1µL/mL of polybrene (Sigma-Aldrich) for 1 week. These cells were cultured in DMEM (Sigma-Aldrich) with 10% FBS, 25 mmol/L glucose, and 4 mmol/L glutamine. Immunocytochemistry . Prostatic cancer cell lines were cultured in standard conditions until approximately 50% confluency. One set of each cell line was left to incubate 24 hours at 37°C in standard incubating conditions (20% oxygen). The other set was placed for the same amount of time in an incubator set at 1% oxygen, which was reported by multiple groups to induce changes in protein expression in these cell lines 68 – 70 . Afterwards, cells were fixed using paraformaldehyde (4%, Sigma) for 10 minutes, permeabilized with Triton-X (0.1%) for 30 minutes, stained overnight with rabbit anti-ASCT2 (Cell Signaling Technology, cat. no. 5345; 1:100), rabbit anti-ALT2 (ProteinTech, 16757-1-AP; 1:400), or goat anti-GDH1 (Invitrogen, PA5-19267; 1:400), and then stained for 1 hr using the following secondary antibodies: donkey anti-goat AF647 (1:200) or anti-rabbit AF488 (1:200) antibodies. Data shown is representative of 4–5 independent characterizations. Histology . Tumors were fixed with paraformaldehyde (2%) via cardiac perfusion and frozen (-80°C) until use. Upon cryosectioning the tissues to 14 µm thick slices, they were permeabilized with Triton-X (0.1%) and stained overnight with rat anti-CD31 (Novus Biologicals, NB600-1475, 1:200) and with rabbit anti-ASCT2 (1:100). Donkey anti-rat AF488 and donkey anti-rabbit AF594 were used as secondary antibodies. Western blot . Pa20c-naïve and Pa20c-ASCT2 cells were lysed for whole-cell protein extraction using RIPA (Radio Immuno Precipitation Assay) buffer (Sigma-Aldrich) following the manufacturer’s instructions. Protein (100 µg total) was resolved on a 4–15% gradient SDS-PAGE gel. Proteins were transferred to a nitrocellulose membrane and then incubated with the rabbit anti-ASCT2 antibody overnight. Mouse anti-GAPDH monoclonal antibody (Sigma, 1:5000) was used as loading control. After washing, the membrane was incubated with a horseradish peroxidase-conjugated donkey anti-rabbit or anti-mouse secondary antibody (GE healthcare, 1:2000) for 1 hour. Immunoblots were developed using SuperSignal™ West Pico PLUS Chemiluminescent Substrate kit (Thermo Fisher) following the manufacturer’s instructions. MALDI mass spectrometry imaging . Tumors were embedded in M1 media (Thermo Fisher), cryosectioned at a 30 µm slice thickness and placed on indium tin oxide-coated glass slides (Delta Technologies). Tissue sections were warmed to room temperature (RT) in a vacuum desiccator for 10 mins prior to spraying. 1,5-diaminonaphthalene (10 mg/mL) in 70% acetonitrile with 0.1% trifluoroacetic acid was applied using an HTX M5 sprayer (HTX Technologies) with the following parameters: 30-degree nozzle temperature, 4 passes, 0.1 ml/min flow rate, 1200 mm/min velocity, 2.5 mm track spacing criss-cross spray pattern, 10 psi pressure, and 2 L/min gas flow rate. MALDI imaging was performed in reflectron-negative mode at a 100-micron pixel and raster size with 200 laser shots per pixel using a Bruker RapifleX MALDI TOF/TOF instrument. Metabolite phantom preparation and MRI . Alanine (Sigma, PHR1110), 1-aminocyclobutane carboxylic acid (ACBC; Sigma, 652369), asparagine (Sigma, A4159), glutamine (Sigma, G3126), serine (Sigma, S4311), or threonine (Sigma, T8441) were dissolved separately in phosphate buffered saline (PBS). Solutions (20 mM) were titrated to a pH of 7.3 ± 0.03 and placed into 5 mm NMR tubes (Wilmad, WG-1000-7) for MR imaging at 37°C. Phantom images were acquired using a Bruker 9.4T vertical bore spectrometer and a 20 mm coil. Amine CEST maps were generated from MTR asym spectra. Scan parameters were TR/TE = 8000/5.58 ms, RARE factor = 32, NA = 1, repetitions = 1, B 1 ​= ​3.6 ​µT, saturation length ​(block pulse) = ​4 ​s, frequency sweep from − 8 to + 8 ​ppm with 0.1 ​ppm increments, matrix size ​= ​76 x 76, slice thickness = 2 mm, and field of view = 20 × 20 mm 2 . Spectral shifts due to B 0 inhomogeneity were corrected for using the water saturation shift referencing (WASSR) method 71 , 72 . Cell phantom preparation and MRI . The cell-rich layer consisted of prostate cancer spheres placed on top of a layer of agarose (1% in PBS) in 5 mm NMR tubes at a height of 5 mm (~ 100 µL in volume), confirmed using a caliper. The supernatant-rich layer consisted of PBS (pH of ~ 7.3; 400 µL) with bovine serum albumin (BSA; 1%) and alanine (10 mM). Phantoms without cells that contained PBS with BSA with or without alanine served as controls. Phantom images of the cell-rich and supernatant-rich layers were acquired in alternation at 37°C using a Bruker 11.7T vertical bore spectrometer and a 20 mm coil. CEST enhancement maps were generated from Z-spectra. Scan parameters were TR/TE = 10000/4.53 ms, RARE factor = 32, NA = 1, repetitions = 1, B 1 ​= ​3.6 ​µT, saturation length ​= ​3 ​s, frequency sweep from − 8 to + 8 ​ppm with 0.4 ​ppm increments, matrix size ​= ​64 x 64, slice thickness = 2 mm, and field of view = 20 × 20 mm 2 . B 0 inhomogeneity was corrected using the WASSR method 71 . Tumor induction and MRI . Animal work performed was approved by the institutional ACUC. DU-145 (n = 9, 1–2 x 10 6 ) or LNCaP (n = 8, 2–3 x 10 6 ) prostatic cancer cells were subcutaneously injected into the flank of male, 6–10 week old Rag2 mice (Jackson Laboratory, #8449). Pa20c-naïve (n = 3, 1–3 x 10 6 ) and Pa20c-ASCT2 (n = 4, 1–3 x 10 6 ) pancreatic cancer cell lines were subcutaneously injected into the flank of male 6–10 week old NCI Scid NCr mice (Charles River, #561). Mice were imaged 1–2 months after tumor induction using a Bruker 11.7T horizontal bore spectrometer and an 8-element phased-array surface coil. Animals were anesthetized with isoflurane (1–2%). Before and after dynamic CEST imaging, static CEST imaging was performed using a frequency sweep from − 8 to + 8 ​ppm with 0.2 ​ppm increments with a scan time of 14 minutes. Dynamic CEST images were collected at the + 3.1 ppm amine proton frequency prior to alanine injection (6 mmol/kg in bolus) to establish a baseline and over 60 minutes after agent injection. Dynamic CEST enhancement ΔS(t)/S 0 was quantified by subtracting signal S/S 0 at time t from the average signal obtained prior to injection. S 0 (signal at + 40 ppm) was collected at the beginning and end of the scan (n = 5 each) and then linearly fitted over time to correct for drift. Static ALAwCEST enhancement was quantified from Z-spectra (ΔS/S 0 ) and from MTR asym spectra (ΔΔS/S 0 ) by subtracting the signal in the ± 3.0 to ± 3.2 ppm range after injection from the signal before injection. CEST MRI scan parameters for in vivo images were TR/TE = 10000/3.49 ms, RARE factor = 32, NA = 1, repetitions = 1, B 1 ​= 3.6 ​µT, saturation length ​= ​3 ​s, matrix size ​= ​48 x 48, slice thickness = 1.5 mm, and field of view = 30 × 30 mm 2 . For static CEST maps and spectra, B 0 inhomogeneity was corrected using the WASSR method as before 71 . Tumor size (mm 3 ) was calculated as the sum of the cross-sectional areas of T2-weighted images multiplied by the slice thickness. Statistics . Student’s t-tests, paired or unpaired, and two-way ANOVAs were performed as appropriate. Spearman’s rank was used for correlations. Significance was defined as p < 0.05. Histograms of the intensity frequencies were normalized (by pixel number) and averaged. Line profiles are from representative regions as outlined in the figure. Declarations Acknowledgements This study was funded by NIH: K01 EB030612 (A.M.T.), Patrick C. Walsh Prostate Cancer Research Fund (A.M.T.), R21 EB035705 (A.M.T.), P50 CA272391 (A.M.T.), P41 EB024495 (P.C.M.v.Z., M.T.M., M.G.P.), and R01 EB030376 (J.W.M.B.). The funder played no role in study design, data collection, analysis and interpretation of data, or the writing of this manuscript. MALDI imaging experiments were performed in the Johns Hopkins Applied Imaging Mass Spectrometry (AIMS) Core facility. MRI experiments were performed in the Johns Hopkins NMR service facility and in the F.M. Kirby Research Center for Functional Brain Imaging at Kennedy Krieger Institute preclinical facility. Author contributions B.G – investigation (prostate models). B.K. – methodology and investigation (pancreatic models). N.D. – investigation and formal analysis (MALDI). K.N.B.W. – investigation (cell culture, staining, and imaging). Y.H. – investigation (tissue staining and imaging). Y.L. – investigation (animal imaging). M.-F.P.V. – investigation (pancreatic models). M.G.P – supervision (imaging validation); funding acquisition. C.M.T – methodology, supervision, and resources (MALDI). Z.M.B. – supervision and resources (pancreatic models). J.W.M.B. – resources (prostate models); writing (review and editing); funding acquisition. M.T.M. – methodology, supervision, and resources (imaging sequences and phantoms); funding acquisition. P.C.M.v.Z. – supervision and resources; writing (review and editing); funding acquisition. A.M.T. – conceptualization, investigation, formal analysis, and visualization; supervision (prostate models); writing (original); funding acquisition. All authors read and approved the final manuscript. Competing interests The authors declare no financial or non-financial competing interests. Code availability MATLAB codes for performing WASSR for B 0 correction, regions of interest analysis, and CEST map generation can be found at our website: godzilla.kennedykrieger.org/CEST/, https://www.kennedykrieger.org/kirby-research-center/software-databases. Data availability The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. References Choi, Y. K. & Park, K. G. Targeting Glutamine Metabolism for Cancer Treatment. Biomol Ther (Seoul) 26 , 19-28 (2018). https://doi.org:10.4062/biomolther.2017.178 Riscal, R. et al. Chromatin-Bound MDM2 Regulates Serine Metabolism and Redox Homeostasis Independently of p53. Molecular Cell 62 , 890-902 (2016). https://doi.org:10.1016/j.molcel.2016.04.033 Yue, M., Jiang, J., Gao, P., Liu, H. & Qing, G. Oncogenic MYC Activates a Feedforward Regulatory Loop Promoting Essential Amino Acid Metabolism and Tumorigenesis. Cell Reports 21 , 3819-3832 (2017). https://doi.org:10.1016/j.celrep.2017.12.002 Roux, C. et al. Endogenous glutamine decrease is associated with pancreatic cancer progression. Oncotarget 8 (2017). Sun, H.-W. et al. GLUT1 and ASCT2 as Predictors for Prognosis of Hepatocellular Carcinoma. PLOS ONE 11 , e0168907 (2016). https://doi.org:10.1371/journal.pone.0168907 Shimizu, K. et al. ASC amino-acid transporter 2 (ASCT2) as a novel prognostic marker in non-small cell lung cancer. British Journal of Cancer 110 , 2030-2039 (2014). https://doi.org:10.1038/bjc.2014.88 Luo, Y. et al. ASCT2 overexpression is associated with poor survival of OSCC patients and ASCT2 knockdown inhibited growth of glutamine-addicted OSCC cells. Cancer Medicine 9 , 3489-3499 (2020). https://doi.org:doi.org/10.1002/cam4.2965 Bernhardt, S. et al. Proteomic profiling of breast cancer metabolism identifies SHMT2 and ASCT2 as prognostic factors. Breast Cancer Research 19 , 112 (2017). https://doi.org:10.1186/s13058-017-0905-7 Wang, Q. et al. Targeting ASCT2-mediated glutamine uptake blocks prostate cancer growth and tumour development. The Journal of Pathology 236 , 278-289 (2015). https://doi.org:doi.org/10.1002/path.4518 Shen, Y.-A. et al. Inhibition of glutaminolysis in combination with other therapies to improve cancer treatment. Current Opinion in Chemical Biology 62 , 64-81 (2021). https://doi.org:https://doi.org/10.1016/j.cbpa.2021.01.006 Choi, Y.-K. & Park, K.-G. Targeting Glutamine Metabolism for Cancer Treatment. Biomolecules & Therapeutics 26 , 19-28 (2018). https://doi.org:10.4062/biomolther.2017.178 Feng, Y. et al. Identification and Characterization of IMD-0354 as a Glutamine Carrier Protein Inhibitor in Melanoma. Molecular Cancer Therapeutics 20 , 816-832 (2021). https://doi.org:10.1158/1535-7163.Mct-20-0354 Console, L., Scalise, M., Tarmakova, Z., Coe, I. R. & Indiveri, C. N-linked Glycosylation of human SLC1A5 (ASCT2) transporter is critical for trafficking to membrane. Biochimica et Biophysica Acta (BBA) - Molecular Cell Research 1853 , 1636-1645 (2015). https://doi.org:doi.org/10.1016/j.bbamcr.2015.03.017 Leslie, T. K. et al. Sodium homeostasis in the tumour microenvironment. Biochimica et Biophysica Acta (BBA) - Reviews on Cancer 1872 , 188304 (2019). https://doi.org:doi.org/10.1016/j.bbcan.2019.07.001 van Zijl, P. C. M. & Yadav, N. N. Chemical exchange saturation transfer (CEST): What is in a name and what isn't? Magnetic Resonance in Medicine 65 , 927-948 (2011). https://doi.org:https://doi.org/10.1002/mrm.22761 Utsunomiya-Tate, N., Endou, H. & Kanai, Y. Cloning and Functional Characterization of a System ASC-like Na+-dependent Neutral Amino Acid Transporter*. Journal of Biological Chemistry 271 , 14883-14890 (1996). https://doi.org:https://doi.org/10.1074/jbc.271.25.14883 Yanagida, O. et al. Human L-type amino acid transporter 1 (LAT1): characterization of function and expression in tumor cell lines. Biochimica et Biophysica Acta (BBA) - Biomembranes 1514 , 291-302 (2001). https://doi.org:https://doi.org/10.1016/S0005-2736(01)00384-4 Goodman, M. M., Yu, W. & Jarkas, N. Synthesis and biological properties of radiohalogenated α,α-disubstituted amino acids for PET and SPECT imaging of amino acid transporters (AATs). Journal of Labelled Compounds and Radiopharmaceuticals 61 , 272-290 (2018). https://doi.org:https://doi.org/10.1002/jlcr.3584 Garlick, P. J. The Nature of Human Hazards Associated with Excessive Intake of Amino Acids. The Journal of Nutrition 134 , 1633S-1639S (2004). https://doi.org:10.1093/jn/134.6.1633S Tan, Z. et al. Saturation transfer properties of tumour xenografts derived from prostate cancer cell lines 22Rv1 and DU145. Scientific Reports 10 , 21315 (2020). https://doi.org:10.1038/s41598-020-78353-8 Mussawy, H. et al. The bone microenvironment promotes tumor growth and tissue perfusion compared with striated muscle in a preclinical model of prostate cancer in vivo. BMC Cancer 18 , 979 (2018). https://doi.org:10.1186/s12885-018-4905-5 Adeno-Associated Virus 2-Mediated Intratumoral Prostate Cancer Gene Therapy: Long-Term Maspin Expression Efficiently Suppresses Tumor Growth. Human Gene Therapy 16 , 699-710 (2005). https://doi.org:10.1089/hum.2005.16.699 Li, Y., Zhong, W., Zhu, M., Li, M. & Yang, Z. miR-185 inhibits prostate cancer angiogenesis induced by the nodal/ALK4 pathway. BMC Urology 20 , 49 (2020). https://doi.org:10.1186/s12894-020-00617-2 Kai, L. et al. Targeting prostate cancer angiogenesis through metastasis-associated protein 1 (MTA1). The Prostate 71 , 268-280 (2011). https://doi.org:https://doi.org/10.1002/pros.21240 Scopelliti, A. J., Font, J., Vandenberg, R. J., Boudker, O. & Ryan, R. M. Structural characterisation reveals insights into substrate recognition by the glutamine transporter ASCT2/SLC1A5. Nature Communications 9 , 38 (2018). https://doi.org:10.1038/s41467-017-02444-w Yao, D. et al. A Novel System A Isoform Mediating Na+/Neutral Amino Acid Cotransport *. Journal of Biological Chemistry 275 , 22790-22797 (2000). https://doi.org:10.1074/jbc.M002965200 Foster, A. C. et al. D-Serine Is a Substrate for Neutral Amino Acid Transporters ASCT1/SLC1A4 and ASCT2/SLC1A5, and Is Transported by Both Subtypes in Rat Hippocampal Astrocyte Cultures. PLOS ONE 11 , e0156551 (2016). https://doi.org:10.1371/journal.pone.0156551 Vettore, L., Westbrook, R. L. & Tennant, D. A. New aspects of amino acid metabolism in cancer. British Journal of Cancer 122 , 150-156 (2020). https://doi.org:10.1038/s41416-019-0620-5 Fernandes, J. & Blom, W. The intravenous L-alanine tolerance test as a means for investigating gluconeogenesis. Metabolism - Clinical and Experimental 23 , 1149-1156 (1974). https://doi.org:10.1016/0026-0495(74)90031-6 Liu, H. et al. 18F-Alanine Derivative Serves as an ASCT2 Marker for Cancer Imaging. Molecular Pharmaceutics 15 , 947-954 (2018). https://doi.org:10.1021/acs.molpharmaceut.7b00884 Battezzati, A., Haisch, M., Brillon, D. J. & Matthews, D. E. Splanchnic utilization of enteral alanine in humans. Metabolism - Clinical and Experimental 48 , 915-921 (1999). https://doi.org:10.1016/S0026-0495(99)90229-9 Ji, X. et al. Transportable hyperpolarized metabolites. Nature Communications 8 , 13975 (2017). https://doi.org:10.1038/ncomms13975 Yang, S.-H. et al. MRI measurement of alanine uptake in a mouse xenograft model of U-87 MG glioblastoma. Magnetic Resonance Imaging 93 , 189-194 (2022). https://doi.org:https://doi.org/10.1016/j.mri.2022.08.015 Cardoso, H. J. et al. Glutaminolysis is a metabolic route essential for survival and growth of prostate cancer cells and a target of 5α-dihydrotestosterone regulation. Cellular Oncology 44 , 385-403 (2021). https://doi.org:10.1007/s13402-020-00575-9 Hodo, Y. et al. Imaging the uptake and metabolism of glutamine in prostate tumor models using CEST MRI. npj Imaging 3 , 34 (2025). https://doi.org:10.1038/s44303-025-00100-3 Okudaira, H. et al. Accumulation of Trans-1-Amino-3-[18F]Fluorocyclobutanecarboxylic Acid in Prostate Cancer due to Androgen-Induced Expression of Amino Acid Transporters. Molecular Imaging and Biology 16 , 756-764 (2014). https://doi.org:10.1007/s11307-014-0756-x Oka, S. et al. Transport mechanisms of trans-1-amino-3-fluoro[1-14C]cyclobutanecarboxylic acid in prostate cancer cells. Nuclear Medicine and Biology 39 , 109-119 (2012). https://doi.org:doi.org/10.1016/j.nucmedbio.2011.06.008 Liu, Y. et al. The role of ASCT2 in cancer: A review. European Journal of Pharmacology 837 , 81-87 (2018). https://doi.org:doi.org/10.1016/j.ejphar.2018.07.007 Rivlin, M. & Navon, G. CEST MRI of 3-O-methyl-D-glucose on different breast cancer models. Magnetic Resonance in Medicine 79 , 1061-1069 (2018). https://doi.org:doi.org/10.1002/mrm.26752 Anemone, A. et al. In vitro and in vivo comparison of MRI chemical exchange saturation transfer (CEST) properties between native glucose and 3-O-Methyl-D-glucose in a murine tumor model. NMR in Biomedicine 34 , e4602 (2021). https://doi.org:https://doi.org/10.1002/nbm.4602 Rivlin, M., Horev, J., Tsarfaty, I. & Navon, G. Molecular imaging of tumors and metastases using chemical exchange saturation transfer (CEST) MRI. Scientific Reports 3 , 3045 (2013). https://doi.org:10.1038/srep03045 Walker-Samuel, S. et al. In vivo imaging of glucose uptake and metabolism in tumors. Nature Medicine 19 , 1067-1072 (2013). https://doi.org:10.1038/nm.3252 Nasrallah, F. A., Pagès, G., Kuchel, P. W., Golay, X. & Chuang, K.-H. Imaging Brain Deoxyglucose Uptake and Metabolism by Glucocest MRI. Journal of Cerebral Blood Flow & Metabolism 33 , 1270-1278 (2013). https://doi.org:10.1038/jcbfm.2013.79 Knutsson, L., Xu, X., van Zijl, P. C. M. & Chan, K. W. Y. Imaging of sugar-based contrast agents using their hydroxyl proton exchange properties. NMR in Biomedicine n/a , e4784 https://doi.org:https://doi.org/10.1002/nbm.4784 Ellingson, B. M. et al. pH-weighted molecular MRI in human traumatic brain injury (TBI) using amine proton chemical exchange saturation transfer echoplanar imaging (CEST EPI). NeuroImage: Clinical 22 , 101736 (2019). https://doi.org:https://doi.org/10.1016/j.nicl.2019.101736 Harris, R. J. et al. pH-weighted molecular imaging of gliomas using amine chemical exchange saturation transfer MRI. Neuro-Oncology 17 , 1514-1524 (2015). https://doi.org:10.1093/neuonc/nov106 Wermter, F. C., Bock, C. & Dreher, W. Investigating GluCEST and its specificity for pH mapping at low temperatures. NMR in Biomedicine 28 , 1507-1517 (2015). https://doi.org:https://doi.org/10.1002/nbm.3416 Yao, J., Wang, C. & Ellingson, B. M. Influence of phosphate concentration on amine, amide, and hydroxyl CEST contrast. Magnetic Resonance in Medicine 85 , 1062-1078 (2021). https://doi.org:doi.org/10.1002/mrm.28481 Cho, N. S. et al. Amine-weighted chemical exchange saturation transfer magnetic resonance imaging in brain tumors. NMR in Biomedicine 36 , e4785 (2023). https://doi.org:https://doi.org/10.1002/nbm.4785 Lee, Z.-W. et al. Intracellular Hyper-Acidification Potentiated by Hydrogen Sulfide Mediates Invasive and Therapy Resistant Cancer Cell Death. Frontiers in Pharmacology 8 (2017). https://doi.org:10.3389/fphar.2017.00763 Wilson, L. T. et al. A new class of ratiometric small molecule intracellular pH sensors for Raman microscopy. Analyst 145 , 5289-5298 (2020). https://doi.org:10.1039/D0AN00865F Furuya, Y., Lundmo, P., Short, A. D., Gill, D. L. & Isaacs, J. T. The role of calcium, pH, and cell proliferation in the programmed (apoptotic) death of androgen-independent prostatic cancer cells induced by thapsigargin. Cancer Res 54 , 6167-6175 (1994). Vāvere, A. L. et al. A Novel Technology for the Imaging of Acidic Prostate Tumors by Positron Emission Tomography. Cancer Research 69 , 4510-4516 (2009). https://doi.org:10.1158/0008-5472.Can-08-3781 Ibrahim-Hashim, A. et al. Systemic Buffers Inhibit Carcinogenesis in TRAMP Mice. Journal of Urology 188 , 624-631 (2012). https://doi.org:doi:10.1016/j.juro.2012.03.113 Korenchan, D. E. et al. Hyperpolarized in vivo pH imaging reveals grade-dependent acidification in prostate cancer. Oncotarget 10 (2019). https://doi.org:10.18632/oncotarget.27225 Dunst, J. et al. Tumor Volume and Tumor Hypoxia in Head andNeck Cancers. Strahlentherapie und Onkologie 179 , 521-526 (2003). https://doi.org:10.1007/s00066-003-1066-4 Gaustad, J.-V., Simonsen, T. G., Andersen, L. M. K. & Rofstad, E. K. Vascular abnormalities and development of hypoxia in microscopic melanoma xenografts. Journal of Translational Medicine 15 , 241 (2017). https://doi.org:10.1186/s12967-017-1347-9 Kis, A. et al. In Vivo Imaging of Hypoxia and Neoangiogenesis in Experimental Syngeneic Hepatocellular Carcinoma Tumor Model Using Positron Emission Tomography. BioMed Research International 2020 , 4952372 (2020). https://doi.org:10.1155/2020/4952372 Kiraga, Ł. et al. Changes in hypoxia level of CT26 tumors during various stages of development and comparing different methods of hypoxia determination. PLOS ONE 13 , e0206706 (2018). https://doi.org:10.1371/journal.pone.0206706 Vaupel, P., Kallinowski, F. & Okunieff, P. Blood Flow, Oxygen and Nutrient Supply, and Metabolic Microenvironment of Human Tumors: A Review1. Cancer Research 49 , 6449-6465 (1989). Yoo, H. C. et al. A Variant of SLC1A5 Is a Mitochondrial Glutamine Transporter for Metabolic Reprogramming in Cancer Cells. Cell Metabolism 31 , 267-283.e212 (2020). https://doi.org:https://doi.org/10.1016/j.cmet.2019.11.020 BUNGARD, Claire I. & McGIVAN, John D. Glutamine availability up-regulates expression of the amino acid transporter protein ASCT2 in HepG2 cells and stimulates the ASCT2 promoter. Biochemical Journal 382 , 27-32 (2004). https://doi.org:10.1042/bj20040487 Ma, H. et al. Inhibition of Glutamine Uptake Improves the Efficacy of Cetuximab on Gastric Cancer. Integrative Cancer Therapies 20 , 15347354211045349 (2021). https://doi.org:10.1177/15347354211045349 Kee, A. J., Smith, R. C., Gross, A. S., Madsen, D. C. & Rowe, B. The effect of dipeptide structure on dipeptide and amino acid clearance in rats. Metabolism 43 , 1373-1378 (1994). https://doi.org:doi: 10.1016/0026-0495(94)90030-2 Qi, Z. et al. Serial determination of glomerular filtration rate in conscious mice using FITC-inulin clearance. American Journal of Physiology-Renal Physiology 286 , F590-F596 (2004). https://doi.org:10.1152/ajprenal.00324.2003 Kim, H., Krishnamurthy, L. C. & Sun, P. Z. Brain pH Imaging and its Applications. Neuroscience 474 , 51-62 (2021). https://doi.org:doi.org/10.1016/j.neuroscience.2021.01.026 Jones, S. et al. Core Signaling Pathways in Human Pancreatic Cancers Revealed by Global Genomic Analyses. Science 321 , 1801-1806 (2008). https://doi.org:doi:10.1126/science.1164368 Geng, H. et al. Interplay between hypoxia and androgen controls a metabolic switch conferring resistance to androgen/AR-targeted therapy. Nature Communications 9 , 4972 (2018). https://doi.org:10.1038/s41467-018-07411-7 Ravenna, L. et al. Distinct Phenotypes of Human Prostate Cancer Cells Associate with Different Adaptation to Hypoxia and Pro-Inflammatory Gene Expression. PLOS ONE 9 , e96250 (2014). https://doi.org:10.1371/journal.pone.0096250 Ma, Y. et al. Prostate Cancer Cell Lines under Hypoxia Exhibit Greater Stem-Like Properties. PLOS ONE 6 , e29170 (2011). https://doi.org:10.1371/journal.pone.0029170 Thomas, A. M., Xu, J., Calabresi, P. A., van Zijl, P. C. M. & Bulte, J. W. M. Monitoring diffuse injury during disease progression in experimental autoimmune encephalomyelitis with on resonance variable delay multiple pulse (onVDMP) CEST MRI. NeuroImage 204 , 116245 (2020). https://doi.org:doi.org/10.1016/j.neuroimage.2019.116245 Kim, M., Gillen, J., Landman, B. A., Zhou, J. & van Zijl, P. C. M. Water saturation shift referencing (WASSR) for chemical exchange saturation transfer (CEST) experiments. Magnetic Resonance in Medicine 61 , 1441-1450 (2009). https://doi.org:https://doi.org/10.1002/mrm.21873 Additional Declarations No competing interests reported. Supplementary Files ASCT2SI.docx Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 27 Oct, 2025 Reviews received at journal 24 Oct, 2025 Reviews received at journal 15 Oct, 2025 Reviewers agreed at journal 12 Oct, 2025 Reviewers agreed at journal 12 Oct, 2025 Reviewers agreed at journal 11 Oct, 2025 Reviewers invited by journal 10 Oct, 2025 Submission checks completed at journal 02 Sep, 2025 First submitted to journal 02 Sep, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7367339","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":532365938,"identity":"963b1a48-0f1b-4b8f-8cf8-684ef79f8327","order_by":0,"name":"Behnaz Ghaemi","email":"","orcid":"","institution":"Johns Hopkins University","correspondingAuthor":false,"prefix":"","firstName":"Behnaz","middleName":"","lastName":"Ghaemi","suffix":""},{"id":532365939,"identity":"ed08dc7f-a909-4e37-9d36-a04da738bfaa","order_by":1,"name":"Balaji Krishnamachary","email":"","orcid":"","institution":"Johns Hopkins University","correspondingAuthor":false,"prefix":"","firstName":"Balaji","middleName":"","lastName":"Krishnamachary","suffix":""},{"id":532365940,"identity":"6d34f396-af77-4c59-93e3-aab0a1ce98a4","order_by":2,"name":"Natalie Dillman","email":"","orcid":"","institution":"Johns Hopkins University","correspondingAuthor":false,"prefix":"","firstName":"Natalie","middleName":"","lastName":"Dillman","suffix":""},{"id":532365941,"identity":"aa30888c-8f65-4e28-8fb8-5c3ac9eb4baf","order_by":3,"name":"Kirsten N. Bains Williams","email":"","orcid":"","institution":"Johns Hopkins University","correspondingAuthor":false,"prefix":"","firstName":"Kirsten","middleName":"N. Bains","lastName":"Williams","suffix":""},{"id":532365942,"identity":"500a5e01-d852-4f8d-9179-6e5d937b7b3d","order_by":4,"name":"Yuki Hodo","email":"","orcid":"","institution":"Johns Hopkins University","correspondingAuthor":false,"prefix":"","firstName":"Yuki","middleName":"","lastName":"Hodo","suffix":""},{"id":532365943,"identity":"1cafc4ce-ea2f-4798-ba52-9a2e69ecfde8","order_by":5,"name":"Yuguo Li","email":"","orcid":"","institution":"Kennedy Krieger Institute","correspondingAuthor":false,"prefix":"","firstName":"Yuguo","middleName":"","lastName":"Li","suffix":""},{"id":532365944,"identity":"c3dbaa9b-b815-4645-a5d7-3ff125a6de23","order_by":6,"name":"Marie-France Penet Vidaver","email":"","orcid":"","institution":"Johns Hopkins University","correspondingAuthor":false,"prefix":"","firstName":"Marie-France","middleName":"Penet","lastName":"Vidaver","suffix":""},{"id":532365945,"identity":"09d8237c-5f35-4560-a4a8-f335dd308df9","order_by":7,"name":"Martin G. Pomper","email":"","orcid":"","institution":"University of Texas Southwestern Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Martin","middleName":"G.","lastName":"Pomper","suffix":""},{"id":532365946,"identity":"17c7e39d-af12-462d-a4de-8b30dd95e191","order_by":8,"name":"Caitlin M. Tressler","email":"","orcid":"","institution":"Johns Hopkins University","correspondingAuthor":false,"prefix":"","firstName":"Caitlin","middleName":"M.","lastName":"Tressler","suffix":""},{"id":532365947,"identity":"4ccb6c4e-e33b-47f4-aa77-35e0d04562af","order_by":9,"name":"Zaver M. Bhujwalla","email":"","orcid":"","institution":"Johns Hopkins University","correspondingAuthor":false,"prefix":"","firstName":"Zaver","middleName":"M.","lastName":"Bhujwalla","suffix":""},{"id":532365948,"identity":"da55eef1-0cd6-4a83-a9f6-f92fa0915999","order_by":10,"name":"Michael T. McMahon","email":"","orcid":"","institution":"Kennedy Krieger Institute","correspondingAuthor":false,"prefix":"","firstName":"Michael","middleName":"T.","lastName":"McMahon","suffix":""},{"id":532365949,"identity":"382c8dd2-e6c8-4730-90fe-ff46034eadd5","order_by":11,"name":"Jeff W.M. Bulte","email":"","orcid":"","institution":"Johns Hopkins University","correspondingAuthor":false,"prefix":"","firstName":"Jeff","middleName":"W.M.","lastName":"Bulte","suffix":""},{"id":532365950,"identity":"ae4ce2e8-08d8-4e66-adb5-5e0bf12b8db2","order_by":12,"name":"Peter C.M. Zijl","email":"","orcid":"","institution":"Johns Hopkins University","correspondingAuthor":false,"prefix":"","firstName":"Peter","middleName":"C.M.","lastName":"Zijl","suffix":""},{"id":532365951,"identity":"22c378b4-a525-4643-9d5c-652d19b092dc","order_by":13,"name":"Aline M. Thomas","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAvklEQVRIiWNgGAWjYFACHiA2kODhZzgD4jEToYMNpKXAQk6ygTQtHyqMDQ7wEKmFf37vwUc3DCQSNx88e0yCocI6sYGQFoljfMnGOUAt2w6cS5NgOJNOWAvDMR4zaYiWM2YSjG2HCWuRh2nZ3ADS8o8ILQZQLcYGDCAtDURoMTyWYwzyi5zEgTPGFgnH0o0JapE7fMbwcc6fOh7+GWcMb3yosZYlqAUBJA4wMCQQrxwE+EkwfhSMglEwCkYWAADF5zyxIyPo1AAAAABJRU5ErkJggg==","orcid":"","institution":"Johns Hopkins University","correspondingAuthor":true,"prefix":"","firstName":"Aline","middleName":"M.","lastName":"Thomas","suffix":""}],"badges":[],"createdAt":"2025-08-13 17:53:17","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7367339/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7367339/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":94398621,"identity":"822f9621-5bd2-400e-9917-c232ae27d767","added_by":"auto","created_at":"2025-10-27 13:57:09","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":5557113,"visible":true,"origin":"","legend":"","description":"","filename":"ASCT2Final082725.docx","url":"https://assets-eu.researchsquare.com/files/rs-7367339/v1/184f15151e3d78155fbc2d35.docx"},{"id":94398091,"identity":"34475d27-8684-4d13-a5c1-ce8793843149","added_by":"auto","created_at":"2025-10-27 13:56:58","extension":"json","order_by":1,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":14510,"visible":true,"origin":"","legend":"","description":"","filename":"3a511fd12ab04b8dadf90e03f598f15a.json","url":"https://assets-eu.researchsquare.com/files/rs-7367339/v1/ad0d5326a68cc836b05a4d0d.json"},{"id":94397536,"identity":"ea8e2f39-211c-4623-945a-8807566227e9","added_by":"auto","created_at":"2025-10-27 13:56:42","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":3400113,"visible":true,"origin":"","legend":"","description":"","filename":"ASCT2SI.docx","url":"https://assets-eu.researchsquare.com/files/rs-7367339/v1/c88da8fe9a7d1268398ddf41.docx"},{"id":94396437,"identity":"590c473e-d121-41c7-a854-4f7932ab075b","added_by":"auto","created_at":"2025-10-27 13:56:00","extension":"xml","order_by":3,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":167240,"visible":true,"origin":"","legend":"","description":"","filename":"3a511fd12ab04b8dadf90e03f598f15a1enriched.xml","url":"https://assets-eu.researchsquare.com/files/rs-7367339/v1/89f303c3c8187a8b20fe3960.xml"},{"id":94398401,"identity":"e1176c0c-e3cd-4d99-b198-74fe28cf8668","added_by":"auto","created_at":"2025-10-27 13:57:04","extension":"png","order_by":4,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":55229,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7367339/v1/f07237843cfa911983419607.png"},{"id":94398991,"identity":"f4392b9e-ee56-410b-9a19-81def7137bf6","added_by":"auto","created_at":"2025-10-27 13:57:18","extension":"png","order_by":5,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":519231,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage10.png","url":"https://assets-eu.researchsquare.com/files/rs-7367339/v1/4d12f3f41a4cd6cc1e36192b.png"},{"id":94398175,"identity":"7cdb0267-0a88-4273-8683-e5d0a16a32ba","added_by":"auto","created_at":"2025-10-27 13:57:00","extension":"png","order_by":6,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1022824,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage11.png","url":"https://assets-eu.researchsquare.com/files/rs-7367339/v1/ec88449e6939f50a6bf4f450.png"},{"id":94489233,"identity":"5f4be3e2-3ce5-4dd0-ba98-0ba202781927","added_by":"auto","created_at":"2025-10-27 17:03:55","extension":"png","order_by":7,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":290090,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7367339/v1/98e83e571b4621f3eb36db5a.png"},{"id":94398075,"identity":"1714b578-ad40-4de1-989c-97deeb7e78b2","added_by":"auto","created_at":"2025-10-27 13:56:58","extension":"png","order_by":8,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":607891,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7367339/v1/ba0b0eb9669dd1571ce8b29d.png"},{"id":94398734,"identity":"d145051f-76c1-4d55-a8b3-96b831743376","added_by":"auto","created_at":"2025-10-27 13:57:12","extension":"png","order_by":9,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1017851,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7367339/v1/0d53b9ce105679a13d3ef909.png"},{"id":94398591,"identity":"58fca3f7-ce5c-4663-96c8-bb43cfaf0237","added_by":"auto","created_at":"2025-10-27 13:57:08","extension":"png","order_by":10,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":80028,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7367339/v1/93f87b78b4c102f74f257f41.png"},{"id":94397028,"identity":"9c96cb88-87cb-4a90-9866-ff5e67492fe6","added_by":"auto","created_at":"2025-10-27 13:56:25","extension":"png","order_by":11,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":226434,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-7367339/v1/47290e16faf042515ebbbed8.png"},{"id":94397275,"identity":"ea1152b1-ee21-490d-b211-065ee9e947c4","added_by":"auto","created_at":"2025-10-27 13:56:35","extension":"png","order_by":12,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":952546,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-7367339/v1/d2f23ba114fea9089ff50cbb.png"},{"id":94398627,"identity":"e77da3e2-b88f-477c-9db8-acc7e0c4faed","added_by":"auto","created_at":"2025-10-27 13:57:09","extension":"png","order_by":13,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":173181,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-7367339/v1/886eaff7902a7c84eec6955a.png"},{"id":94397246,"identity":"a7c4d997-6623-4411-afbf-e7bdd822e884","added_by":"auto","created_at":"2025-10-27 13:56:34","extension":"png","order_by":14,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":486574,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-7367339/v1/aa595dde9d705d5f8e8168a0.png"},{"id":94397823,"identity":"aa61fe27-0b2e-4b8e-b288-f069c4737417","added_by":"auto","created_at":"2025-10-27 13:56:49","extension":"png","order_by":15,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":13376,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7367339/v1/681fef68ceb3d96930ea709f.png"},{"id":94489657,"identity":"054f2ff3-abde-4adb-abde-8bcf2a4d71c3","added_by":"auto","created_at":"2025-10-27 17:05:32","extension":"png","order_by":16,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":54802,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage10.png","url":"https://assets-eu.researchsquare.com/files/rs-7367339/v1/5e7068b9dd8082a03005c8b1.png"},{"id":94399193,"identity":"7a37654e-181b-4932-b8bf-e6214e81fde7","added_by":"auto","created_at":"2025-10-27 13:57:23","extension":"png","order_by":17,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":187032,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage11.png","url":"https://assets-eu.researchsquare.com/files/rs-7367339/v1/ea8bd1079a871d8c855f2922.png"},{"id":94398446,"identity":"19887768-69f2-422c-b981-df03a257efa0","added_by":"auto","created_at":"2025-10-27 13:57:05","extension":"png","order_by":18,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":39260,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7367339/v1/9b8c338df4b3eed5a8e35c51.png"},{"id":94397436,"identity":"64dd4a84-6710-4a9d-b3d8-15637180cf23","added_by":"auto","created_at":"2025-10-27 13:56:40","extension":"png","order_by":19,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":82802,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7367339/v1/fd3f04518d3736cbc15d0ecf.png"},{"id":94397030,"identity":"836d1319-9c27-4cf5-87e2-86015ddae2cc","added_by":"auto","created_at":"2025-10-27 13:56:25","extension":"png","order_by":20,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":147368,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7367339/v1/be068135451bb32be1100361.png"},{"id":94397315,"identity":"495fc1a9-9d12-40d0-947a-6768f5180316","added_by":"auto","created_at":"2025-10-27 13:56:37","extension":"png","order_by":21,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":21299,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7367339/v1/5a96db742144783ea86184a1.png"},{"id":94397847,"identity":"f46d14f3-cd5e-4b92-a3e5-155186d7d7d5","added_by":"auto","created_at":"2025-10-27 13:56:51","extension":"png","order_by":22,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":35623,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-7367339/v1/aba4f2ee45219812db0facc3.png"},{"id":94396450,"identity":"70feba51-dd88-49de-82dd-0d7f9754105c","added_by":"auto","created_at":"2025-10-27 13:56:00","extension":"png","order_by":23,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":117642,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-7367339/v1/11be93e189b7664566458a0c.png"},{"id":94396831,"identity":"0f8f852a-f759-4d3b-ad62-9ef1e0d6bd44","added_by":"auto","created_at":"2025-10-27 13:56:17","extension":"png","order_by":24,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":38309,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-7367339/v1/ef45749dced78a652829e329.png"},{"id":94398631,"identity":"ff82d1ec-0d68-4220-9863-7d66ccb6f076","added_by":"auto","created_at":"2025-10-27 13:57:09","extension":"png","order_by":25,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":55377,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-7367339/v1/df6ace9ca6a4ae5db625d196.png"},{"id":94398742,"identity":"9a1d3b00-cfe6-4bc6-8de3-a6a5f452e0e1","added_by":"auto","created_at":"2025-10-27 13:57:12","extension":"xml","order_by":26,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":165901,"visible":true,"origin":"","legend":"","description":"","filename":"3a511fd12ab04b8dadf90e03f598f15a1structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7367339/v1/dfcfbb4ae190f6e3e51c728f.xml"},{"id":94397428,"identity":"fdbbc293-e1f8-4eb2-8a2c-504c1765f184","added_by":"auto","created_at":"2025-10-27 13:56:39","extension":"html","order_by":27,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":181918,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7367339/v1/e5ae0ca52b997f0ed75c3f7d.html"},{"id":94398982,"identity":"a458fad9-fdad-4580-8697-31d225be6fe8","added_by":"auto","created_at":"2025-10-27 13:57:18","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":43473,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCEST imaging ASCT2 substrates.\u003c/strong\u003e (\u003cstrong\u003eA\u003c/strong\u003e) Chemical structure and (\u003cstrong\u003eB\u003c/strong\u003e) CEST signal at +3.1 ppm and 37 °C of major, natural ASCT2 substrates compared to the synthetic ASCT2 substrate ACBC (from which fluciclovine is derived). Phantoms consisted of 20 mM solutions of these metabolites in PBS titrated to a pH of 7.3. ACBC = 1-aminocyclobutane carboxylate. Ala = alanine. Asn = asparagine. Gln = glutamine. Ser = serine. Thr = threonine.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7367339/v1/1b9d76b6406afce83d7565dd.png"},{"id":94489225,"identity":"8aa5d555-15eb-4969-b313-2898d7a79637","added_by":"auto","created_at":"2025-10-27 17:03:51","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":152088,"visible":true,"origin":"","legend":"\u003cp\u003eDynamic CEST enhancement in genetically-engineered pancreatic tumors upon alanine injection. (A) Western blot of ASCT2 expression levels in naïve and genetically-engineered Pa20c tumors. (B) Visualization of CEST enhancement at the +3.1 ppm frequency over the course of the scan upon administration of alanine. (C) Quantification of ALAwCEST enhancement over the course of the scan in naïve and ASCT2-overexpressing Pa20c tumors. * = p \u0026lt; 0.05. n = 4 for the +ASCT2 group and n = 3 for the naïve group.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7367339/v1/c45c706561a42de845eecabc.png"},{"id":94398111,"identity":"182df100-78e1-48a1-bcdc-adf7312d6f21","added_by":"auto","created_at":"2025-10-27 13:56:59","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":208064,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDynamic CEST enhancement in prostate tumors upon alanine injection.\u003c/strong\u003e (\u003cstrong\u003eA\u003c/strong\u003e)\u0026nbsp; Visualization of CEST enhancement at the +3.1 ppm frequency over the course of the scan. (\u003cstrong\u003eB\u003c/strong\u003e) Quantification of CEST enhancement over the course of the scan in LNCaP tumors and DU-145 tumors. * = p \u0026lt; 0.05 of larger DU-145 tumors (\u0026gt;310 mm\u003csup\u003e3\u003c/sup\u003e) compared to the smaller LNCaP tumors (\u0026lt; 600mm\u003csup\u003e3\u003c/sup\u003e). # = p \u0026lt; 0.05 of larger LNCaP (\u0026gt;630 mm\u003csup\u003e3\u003c/sup\u003e) compared to the larger DU-14 5 (\u0026gt; 310 mm\u003csup\u003e3\u003c/sup\u003e) tumors. ^ = p \u0026lt; 0.05 of smaller LNCaP (\u0026lt;600 mm\u003csup\u003e3\u003c/sup\u003e) compared to the larger DU-14 5 (\u0026gt; 310 mm\u003csup\u003e3\u003c/sup\u003e) tumors. n = 5 and n = 3 for LNCaP tumors that were \u0026lt; 600 mm\u003csup\u003e3\u003c/sup\u003e and \u0026gt; 630 mm\u003csup\u003e3\u003c/sup\u003e, respectively. n = 5 and n = 4 for DU-145 tumors that were \u0026lt; 165 mm\u003csup\u003e3\u003c/sup\u003e and \u0026gt; 310 mm\u003csup\u003e3\u003c/sup\u003e, respectively. Individual enhancement curves are in supplemental figures 4 and 5.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7367339/v1/47563c9705a9a3bc90b2d9f7.png"},{"id":94397814,"identity":"988e926b-3f1c-420c-a888-ccfa6d8d6b45","added_by":"auto","created_at":"2025-10-27 13:56:49","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":392609,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExpression of ASCT2 and CD31 in prostate tumors.\u003c/strong\u003e Visualization of ASCT2 (green) and CD31 (red) expression in (left) smaller and (right) larger DU-145 and LNCaP at the (top) center region or (bottom) periphery of the tumor. DAPI staining (blue) of nucleic structures is shown for reference. White squares outline the location of the magnified images above.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7367339/v1/24c7f500c346dc038c8cd512.png"},{"id":94396255,"identity":"fe3267bd-66dd-4958-afa2-0f11a89d34d0","added_by":"auto","created_at":"2025-10-27 13:55:55","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":67952,"visible":true,"origin":"","legend":"\u003cp\u003eMALDI imaging of \u003csup\u003e13\u003c/sup\u003eC3-alanine and its downstream metabolic products. (A) Schematic of the carbon (pink) flow from the imaging agent to its metabolic products prior to tricarboxylic acid (TCA) cycling. (B) Visualization and (C) quantification of \u003csup\u003e13\u003c/sup\u003eC-labeled metabolites in LNCaP and DU-145 tumors. Metabolites: Ala=alanine, Lac=lactate, and Pyr=pyruvate. Proteins: αKGDH=alpha-ketoglutarate dehydrogenase, ALT=alanine transaminase, ASCT2=alanine serine cysteine transporter 2, GDH=glutamate dehydrogenase, GLS=glutaminase, GS=glutamine synthetase, IDH=isocitrate dehydrogenase, and LDH=lactate dehydrogenase. * = p \u0026lt; 0.05. Mean ± s.e.m. n = 8 for the LNCaP group and n = 6 for the DU-145 group\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7367339/v1/c23cc82e79623ff55f6085d5.png"},{"id":94491278,"identity":"2e7e0014-c85a-427f-9113-3bac34088b53","added_by":"auto","created_at":"2025-10-27 17:24:13","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1817708,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7367339/v1/da756e12-0a64-4d2c-aad9-a605120e0975.pdf"},{"id":94397432,"identity":"8e335a9a-b2ff-427e-967f-8526bffb3395","added_by":"auto","created_at":"2025-10-27 13:56:40","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":3400113,"visible":true,"origin":"","legend":"","description":"","filename":"ASCT2SI.docx","url":"https://assets-eu.researchsquare.com/files/rs-7367339/v1/87936dafd0baa0c27f48f11d.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"MRI of ASCT2-mediated amino acid uptake in xenograft tumor models","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCellular glutamine dependence is an attractive target for cancer imaging and therapy. Glutamine is actively transported into the cell, upon which it is metabolized to serve as a nitrogen and carbon source for biosynthesis of amino acids and nucleosides and as a supplemental energy source\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Glutamine transporters have been bi-directionally linked to the activation of oncogenes: Mdm2\u003csup\u003e2\u003c/sup\u003e, which has been shown to enhance the expression of these transporters, and Myc\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e, which is activated by them. Alanine-serine-cysteine transporter 2 (ASCT2; gene: \u003cem\u003eSLC1A5\u003c/em\u003e), the primary transporter for glutamine import, is associated with poor prognosis for many cancers\u003csup\u003e\u003cspan additionalcitationids=\"CR5 CR6 CR7\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e and their therapies\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. At the clinical stage, several glutamine antagonists that include ASCT2 inhibition as their mode of action have been tested, though none beyond phase I trials\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. In preclinical stages, multiple inhibitors have been developed to target this transporter\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Assays for measuring the transport capabilities of ASCT2 \u003cem\u003eex vivo\u003c/em\u003e are not yet commercially available.\u003c/p\u003e\u003cp\u003eHistopathological grading of its expression is a current clinical metric for ASCT2. For prostate cancer, higher ASCT2 expression corresponds with cancer recurrence\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Still, expression level is not proportional to cell uptake by ASCT2, unless glycosylated to translocate from the cytoplasm to the plasma membrane\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. In addition, functionality of ASCT2 is sodium-dependent and alterations in the transporters responsible for regulating sodium levels have been reported in multiple cancers\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. If a suitable MRI-detectable substrate could be found, \u003cem\u003ein vivo\u003c/em\u003e MRI may allow evaluation of ASCT2-mediated transport and report on the tumor environment.\u003c/p\u003e\u003cp\u003eWe hypothesized that natural substrates could serve as chemical exchange saturation transfer (CEST) magnetic resonance imaging (MRI) agents for evaluating ASCT2 activity. CEST MRI has the advantage of detecting low-concentration molecules (mM level) without modification or the need for specialized equipment through the presence of water-exchanging protons, for instance in their amine groups\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. In this report we investigated the potential utility of several natural amine-containing substrates as CEST imaging biomarkers for ASCT2 activity, namely: alanine, asparagine, glutamine, serine, and threonine. Next, we measured CEST contrast upon intravenous injection of alanine in mice bearing Pa20c pancreatic tumors engineered to express different levels of ASCT2. Afterwards, we evaluated alanine-weighted CEST (ALAwCEST) contrast \u003cem\u003ein vivo\u003c/em\u003e for DU-145 and LNCaP prostate tumors that differentially express ASCT2. Subsequently, we correlated ALAwCEST enhancement to ASCT2 expression in these tumors. Lastly, we correlated ALAwCEST enhancement to the uptake and metabolism of \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC-labeled alanine using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eCEST MRI of Major ASCT2 Substrates\u003c/h2\u003e\u003cp\u003eInitially, we assessed the potential of naturally occurring CEST MRI agents to report on ASCT2 activity. We screened 5 natural amino acids that are major substrates of ASCT2\u0026mdash;glutamine, alanine, serine, threonine, and asparagine\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e\u0026mdash;in PBS solution (20 mM, pH of 7.3, T\u0026thinsp;=\u0026thinsp;37\u0026deg;C) for amineCEST signal generation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). They were also compared to ACBC (1-aminocyclobutane carboxylic acid), the nonfluorinated synthetic amino acid from which [18F]-fluciclovine ([18F]-FACBC) is derived. Notably, glutamine\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e, asparagine\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e and ACBC\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e can also be transported by the LAT1 amino acid exchanger. Of them, alanine generated the highest signal, followed by glutamine and serine, and then ACBC. Threonine and asparagine generated minimal signal compared to PBS alone. Thus, we continued investigating alanine based on its high CEST signal, as well as its relatively good clinical safety profile\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e compared to the other ASCT2 substrates. Next, we evaluated the sensitivity of ALAwCEST MRI to ASCT2 activity using a genetically-engineered cancer model. We characterized ASCT2 expression in na\u0026iuml;ve and \u003cem\u003eSLC1A5\u003c/em\u003e-overexpressing Pa20c patient-derived pancreatic xenograft tumors (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) and confirmed that \u003cem\u003eSLC1A5\u003c/em\u003e-overexpression in this cell line increased ASCT2 protein levels in its tumors. Then, we compared their CEST signal enhancement at the +\u0026thinsp;3.1 ppm frequency upon intravenous alanine injection (6 mmol/kg). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec, CEST signal remained close to baseline levels over a period of 60 min following infusion in na\u0026iuml;ve Pa20c tumors (n\u0026thinsp;=\u0026thinsp;3), but amine-weighted CEST (i.e. ALAwCEST) rose steadily in Pa20c tumors engineered to express higher levels of ASCT2 (n\u0026thinsp;=\u0026thinsp;4). Their CEST enhancement significantly differed (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eAlanine-Weighted CEST MRI of Prostate Tumor Models\u003c/h3\u003e\n\u003cp\u003eSubsequently, we assessed the potential of ALAwCEST to differentiate na\u0026iuml;ve prostate tumor models with different levels of ASCT2 expression and glutamine uptake. We characterized ASCT2 expression (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e) in prostatic cancer cell lines DU-145 and LNCaP using fluorescent microscopy. The average intensity of ASCT2 in LNCaP was 11% higher than that of DU-145 (p\u0026thinsp;=\u0026thinsp;0.0203). Since cellular uptake by ASCT2 requires translocation of the transporter to the plasma membrane, we investigated the intensity across the cell area. A line profile revealed expression at the cellular border was higher than that in the cytoplasm for LNCaP cells. It also revealed that the cell line had higher fluorescent intensity compared to DU-145 across the cellular space. Since ALAwCEST signal could be dependent on downstream enzymatic conversion, we also quantified the expression of alanine transaminase (ALT2) and glutamate dehydrogenase 1 (GDH1) in these cells (Fig. S2). The average intensity of ALT2 and GDH1 was 58% (p\u0026thinsp;=\u0026thinsp;0.0078) and 25% (p\u0026thinsp;=\u0026thinsp;0.0190) higher in LNCaP than DU-145, respectively. Furthermore, since hypoxia is a common feature of prostate tumors, we also evaluated changes in their expression after short-term (24 h) exposure to hypoxia (1%). Within that time frame we found ASCT2 expression was significantly lower (5%: p\u0026thinsp;=\u0026thinsp;0.0108) in LNCaP cells exposed to hypoxic conditions compared to LNCaP cells exposed to normoxic conditions. We then characterized changes in the CEST signal of their tumor spheres (100 \u0026micro;L cell volume) when exposed to alanine (400 \u0026micro;L of 10 mM; Fig. S3). CEST signal in LNCaP-containing phantoms was higher than DU-145-containing phantoms (p\u0026thinsp;=\u0026thinsp;0.0090) and the alanine only control phantoms (p\u0026thinsp;=\u0026thinsp;0.0021) after 4 hours of exposure (210 minutes after the initial acquisition). ALAwCEST enhancement of DU-145 containing phantoms was also higher than alanine only control phantoms (p\u0026thinsp;=\u0026thinsp;0.0012).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eSubsequently, we compared ALAwCEST signal enhancement between LNCaP (n\u0026thinsp;=\u0026thinsp;8) and DU-145 (n\u0026thinsp;=\u0026thinsp;9) tumors. We observed necrotic cores in some of the larger DU-145 tumors (\u0026gt;\u0026thinsp;630 mm\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e, similar to others\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e) and excluded those mice from further analysis. In dynamic CEST scans, CEST enhancement was higher in LNCaP tumors than DU-145 tumors (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). In static CEST scans, acquired before and after the dynamic ones, CEST contrast at the +\u0026thinsp;3.0 to +\u0026thinsp;3.2 ppm frequency range and the \u0026minus;\u0026thinsp;3.2 to -3.0 ppm frequency range did not significantly change upon administration of alanine. We then stratified tumors by volume. ALAwCEST enhancement profiles differed visually in LNCaP tumors with volumes less than 600 mm\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e (n\u0026thinsp;=\u0026thinsp;5) and greater than 630 mm\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e (n\u0026thinsp;=\u0026thinsp;3) when alanine was administered (Fig. S4). For smaller tumors (\u0026lt;\u0026thinsp;600 mm\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e), CEST signal reached a peak approximately 15 minutes after agent injection, returned to baseline approximately 45 minutes after agent injection, and then plateaued. For larger tumors (\u0026gt;\u0026thinsp;630 mm\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e), CEST signal steadily increased during the scan session. CEST signal dynamics between these two size classes significantly differed (p\u0026thinsp;=\u0026thinsp;0.04). CEST enhancement profiles differed visually in DU-145 tumors with volumes less than 165 mm\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e (n\u0026thinsp;=\u0026thinsp;5) and greater than 310 mm\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e (n\u0026thinsp;=\u0026thinsp;4) when alanine was administered (Fig. S5). ALAwCEST signal at the +\u0026thinsp;3.1 ppm frequency was higher than baseline for smaller tumors (\u0026lt;\u0026thinsp;165 mm\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e), but lower than baseline for larger tumors (\u0026gt;\u0026thinsp;310 mm\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e). Their CEST signal dynamics significantly differed (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). CEST enhancement at the end of the dynamic scan negatively correlated (ρ = -0.7579, p\u0026thinsp;=\u0026thinsp;0.02) with tumor volume. CEST signal enhancement amongst the volume-stratified tumor groups were then compared (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003e). At early times (~\u0026thinsp;15 minutes after alanine injection), signal at the +\u0026thinsp;3.1 ppm frequency was significantly (0.008\u0026thinsp;\u0026lt;\u0026thinsp;p\u0026thinsp;\u0026lt;\u0026thinsp;0.043) higher in smaller LNCaP tumors (\u0026lt;\u0026thinsp;600 mm\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e) compared to larger DU-145 tumors (\u0026gt;\u0026thinsp;310 mm\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e). Towards the end of the scan session, both smaller (\u0026lt;\u0026thinsp;600 mm\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e: 0.0261\u0026thinsp;\u0026lt;\u0026thinsp;p\u0026thinsp;\u0026lt;\u0026thinsp;0.0427) and larger (\u0026gt;\u0026thinsp;630 mm\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e: 0.0148\u0026thinsp;\u0026lt;\u0026thinsp;p\u0026thinsp;\u0026lt;\u0026thinsp;0.0497) LNCaP tumor groups had significantly higher CEST enhancement than larger DU-145 tumors (\u0026gt;\u0026thinsp;310 mm\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003eFluorescent Imaging of ASCT2 Content in Prostate Tumors\u003c/h3\u003e\n\u003cp\u003eNext, we characterized ASCT2 and CD31 expression in larger and smaller LNCaP and DU-145 tumors (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e4\u003c/span\u003e, Fig. S6). Fluorescent intensity of ASCT2 was highest in smaller DU-145 tumors. Levels were comparable in the periphery of smaller LNCaP tumors, but were much lower in the center, reminiscent of the ring of bright CEST signal observed in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003e. ASCT2 staining intensity in the periphery of larger DU-145 tumors was comparable to that in larger LNCaP tumors, but in the center of larger DU-145 tumors, staining levels were low and heterogenous proximal to necrotic (acellular) lesions. Endothelial (CD31) levels were generally comparable, in line with what was reported in the literature for these two tumors\u003csup\u003e\u003cspan additionalcitationids=\"CR22 CR23\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Still, CD31 staining was lower in the center of smaller LNCaP and larger DU-145 tumors. In the former, CD31 staining patterns were similar to ASCT2 staining patterns, i.e. ring-like. In the latter, CD31 staining was also heterogenous, but localized independently of ASCT2 staining.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003eMALDI Mass Spectrometry Imaging of Prostate Tumor Uptake and Metabolism of Alanine\u003c/h3\u003e\n\u003cp\u003eTo demonstrate that the source of the CEST MRI signal enhancement was the uptake of alanine, we compared the presence of \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC3-labeled alanine and its metabolites in tumors \u003cem\u003eex vivo\u003c/em\u003e using isotopomer analysis of MALDI mass spectrometry images (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e5\u003c/span\u003e). The presence of \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC3-alanine (p\u0026thinsp;=\u0026thinsp;0.0329) and its immediately downstream metabolic product \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC3-pyruvate (generated intracellularly by alanine transaminase: p\u0026thinsp;=\u0026thinsp;0.0357) was higher in LNCaP tumors than in DU-145 tumors. In contrast, the presence of \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC3-lactate, a product of the CEST agent entering the glycolytic pathway, was similar (p\u0026thinsp;=\u0026thinsp;0.0877) amongst the tumors.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eAlthough best known for transporting glutamine, the neutral amino acid transporter ASCT2 has multiple, naturally-occurring substrates\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan additionalcitationids=\"CR26\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e–\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e, i.e. alanine, asparagine, glycine, leucine, methionine, serine, threonine, and valine. All of these substrates can be metabolized via their respective transaminases\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. We investigated the utility of CEST MRI to detect and image ASCT2-mediated transport using some of these unmodified, naturally occurring substrates. We then selected alanine from this pool of candidate agents in part due to its readiness for translation. Alanine is a well-recognized substrate for ASCT2 that has been injected intravenously at a dose of 6 mmole/kg\u003csup\u003e29\u003c/sup\u003e into humans for other applications without symptoms. Its suitability as an ASCT2 agent for cancer imaging has already been demonstrated with PET imaging in preclinical models using a fluorinated version of the metabolite\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. In addition, \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC-labeled alanine\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e has also been injected in humans and is readily hyperpolarized\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e, permitting spectroscopic imaging. Most recently, its T2 relaxation was leveraged to permit imaging of a glioblastoma model (U87MG) using MRI\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. The relatively low toxicity of alanine in humans permitted us to use high doses for intravenous administration in our experiments, which should be translatable to the clinic. Its relatively high safety profile\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e has the added advantage of enabling the repetition of these scans, which is beneficial for monitoring therapeutic efficacy in cancer management. We elected to investigate alanine-mediated CEST enhancement in multiple tumor xenograft models. The use of genetically-engineered pancreatic tumor models enabled us to establish the feasibility of alanine to report ASCT2 activity. The use of the DU-145 and LNCaP prostate cancer models enabled us to establish the sensitivity. Their relative expression (protein) of ASCT2 is well-established\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e,\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e and the uptake of ASCT2 substrates in LNCaP cells is reported to be nearly 1.5 times faster than that of DU-145 cells\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e,\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. Still, compared to other cancers, prostate cancer expression (mRNA) is quite low, ranking fourth from last according to a systematic review\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. This combination of attributes permitted us to examine the utility of dynamic ALAwCEST MRI at the lower limits of ASCT2 for cancer applications.\u003c/p\u003e\u003cp\u003eWhen naturally occurring molecules are used, CEST enhancement is dependent on not only the pH and concentration of the agents in tissue compartments, but also on the concentration of their metabolized products when the latter contain the same exchangeable proton groups\u003csup\u003e\u003cspan additionalcitationids=\"CR40 CR41 CR42 CR43\" citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e–\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. In the tumor microenvironment intracellular pH becomes more alkaline, reducing CEST contrast for amine groups. On the other hand, pH in the interstitium (extracellular, extravascular space) acidifies, increasing amine proton contrast\u003csup\u003e\u003cspan additionalcitationids=\"CR46 CR47 CR48\" citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e–\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. Reported values of intracellular and extracellular pH in prostate cancer models range from 7.1 to 7.6\u003csup\u003e50–52\u003c/sup\u003e and from 6.4 to 6.8\u003csup\u003e53–55\u003c/sup\u003e, respectively. In larger (\u0026gt; 310 mm\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e) DU-145 tumors the directionality of CEST enhancement was slightly negative (Fig. S5), which could result from the lower ASCT2 levels observed in histological images compared to the smaller tumors (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e4\u003c/span\u003e, Fig. S6). Since hypoxia is often featured in larger\u003csup\u003e\u003cspan additionalcitationids=\"CR57\" citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e–\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e, more advanced\u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e tumors, particularly prostate cancer\u003csup\u003e\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e, we investigated its impact on their expression in DU-145 and LNCaP cells. We did not observe significant alterations in ASCT2 expression (protein) in DU-145 cells when cultured in hypoxic conditions \u003cem\u003ein vitro\u003c/em\u003e, but the same conditions decreased ASCT2 expression in LNCaP cells (Fig. S2) and increased ASCT2 expression (mRNA, protein) in pancreatic cancer cell lines\u003csup\u003e\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e. Still, decreased ASCT2 expression and/or function may occur under longer-term exposure. Alternatively, low glutamine availability—which is associated with less vascularized tumors such as the larger DU-145 tumors in this report (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e4\u003c/span\u003e)—has been shown to decrease ASCT2 expression in liver\u003csup\u003e\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u003c/sup\u003e and gastric\u003csup\u003e\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e\u003c/sup\u003e cancer cell lines. In larger LNCaP tumors (cutoff between 600–630 mm\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e), a decreased vascular fraction also explains the size-dependence of CEST profiles for LNCaP tumors as the initial peak seen in smaller tumors was not present in larger tumors. Peaks within the first 30 minutes has been observed with other CEST agents delivered at these doses\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. Additionally, early-stage blood vessel marker CD31 revealed larger LNCaP tumors were less vascularized than smaller ones (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e4\u003c/span\u003e, Fig. S6).\u003c/p\u003e\u003cp\u003eMALDI mass spectrometry imaging was used in our studies to validate the uptake of our CEST agents (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e5\u003c/span\u003e). MALDI imaging confirmed the presence of \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC-labeled alanine in tumor tissue. However, similar to \u003cem\u003ein vivo\u003c/em\u003e imaging techniques, mass spectrometry imaging is unable to differentiate the compartment, i.e. vascular, extracellular and intracellular spaces, of these metabolites unless they are known not to be transportable. To minimize the contribution of the vascular and extracellular compartments to this measurement, we extracted the tumors after multiple half-lives (\u0026lt; 15 minutes\u003csup\u003e\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e,\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e\u003c/sup\u003e). Furthermore, as alanine is metabolized inside the cell, we compared differences in the presence of its \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC-labeled metabolic products to indirectly confirm the occurrence of cellular uptake. These findings warrant more extensive \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003eex vivo\u003c/em\u003e metabolic studies to establish the fate of this imaging agent and improve the interpretation of its images.\u003c/p\u003e\u003cp\u003eThere are several limitations to our studies. Firstly, we did not image pH \u003cem\u003ein vivo\u003c/em\u003e or \u003cem\u003eex vivo\u003c/em\u003e to evaluate its impact on our measurement of ASCT2 function. Still, several MRI and magnetic resonance spectroscopy methods to measure pH have been translated to human use\u003csup\u003e\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e\u003c/sup\u003e that can be used to evaluate its contribution. Secondly, we did not image tumor vascular function to evaluate its impact on CEST enhancement and MALDI images. However, CD31 staining (Fig. S6) revealed vessel density in DU-145 and LNCaP tumors was comparable. Furthermore, the blood clearance half-lives of small molecules such as alanine in rodent blood are less than 15 minutes\u003csup\u003e\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e,\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e\u003c/sup\u003e; therefore, the contribution of the vascular fraction to the CEST signal would be expectedly minimal by the end of the scan session. Still, we recognize that the measurement and consideration of these confounding factors would improve the interpretation of ASCT2 imaging in the clinic. Gadolinium-enhanced MRI is already a feature of cancer patient care used to evaluate the vascular profile of tumors. These evaluations would complement the visual information provided by ALAwCEST enhancement.\u003c/p\u003e\u003cp\u003eIn conclusion, we evaluated alanine for imaging ASCT2-mediated glutamine transport. ALAwCEST MRI detected its uptake without modification via the water-exchanging protons of its amine group. Dynamic CEST imaging of alanine was able to differentiate Pa20c-naïve and Pa20c-ASCT2 pancreatic tumor models as well as DU-145 and LNCaP prostate tumor models. The direction and magnitude of dynamic ALAwCEST enhancement when alanine was administered was sensitive to size-dependent differences in the ASCT2 content, particularly for DU-145 tumors. Lastly, as alanine has been intravenously injected into humans for other applications at a dose (6 mmole/kg) that should allow ALAwCEST MRI detection, ASCT2 imaging using this metabolite warrants further optimization and development as its use as a noninvasive, natural imaging agent is readily translatable.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cb\u003eProstate cell line culture\u003c/b\u003e. For monolayer cultures, the prostatic cancer lines DU-145 (ATCC, 1 x 10\u003csup\u003e4\u003c/sup\u003e) and LNCaP (ATCC, 2 x 10\u003csup\u003e5\u003c/sup\u003e) were seeded onto 6-well plates and cultured in RPMI media (Gibco, A10491-01) supplemented with fetal bovine serum (10%; Gibco, 25140-079) and penicillin-streptomycin (1%; Gibco, 15140-122). For spheroid culture, DU-145 cells (1.5 x 10\u003csup\u003e3\u003c/sup\u003e) and LNCaP (1.5 x 10\u003csup\u003e4\u003c/sup\u003e) cells were seeded onto low-attachment 6-well plates (Corning Costar®) and cultured in DMEM/F12 media (Gibco, 10565-018) supplemented with 1% penicillin-streptomycin, B27 (2%; Gibco 17504-044), insulin (3 µg/ml; Sigma, I2643) epidermal growth factor (20 ng/mL; Gibco, PHG0311), and knock-out serum (3%; Gibco, 10828-010) until a 100 µL volume of cells was attained.\u003c/p\u003e\u003cp\u003e\u003cb\u003ePancreatic cell line generation and culture\u003c/b\u003e. The Pa20c (Panc198/RRID: CVCL_E285, male) human pancreatic cancer cell line is from a primary pancreatic tumor characterized previously in a published work\u003csup\u003e\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e\u003c/sup\u003e that was kindly provided by Dr. Anirban Maitra (MD Anderson Cancer Center). To generate ASCT2-overexpressing Pa20c cells, a 1.62 kb open reading frame (ORF) of human solute carrier family 1 member 5 (SLC1A5) transcript variant 1 (ASCT2) was spliced into a lentiviral vector (LV-231-SLC1A5-eGFP) purchased from GeneCopoeia (Cat. No. EX-A6442-Lv231, Rockville, MD) and selected using puromycin. Virions for expressing the ASCT2 gene were produced by co-transfecting 293T cells with 12 µg of lentiviral construct, 6 µg of packaging plasmid pCMVΔR8.2 DVPR (VPR deleted) and 1.5 µg of pCMV-VSVg plasmid using lipofectamine 2000 (Invitrogen, Carlsbad, CA). Supernatant containing the virions were collected 48 h after transfection and gently centrifuged to remove cell debris. Pa20c cells were transfected daily with fresh lentivirus in serum-free media with 1µL/mL of polybrene (Sigma-Aldrich) for 1 week. These cells were cultured in DMEM (Sigma-Aldrich) with 10% FBS, 25 mmol/L glucose, and 4 mmol/L glutamine.\u003c/p\u003e\u003cp\u003e\u003cb\u003eImmunocytochemistry\u003c/b\u003e. Prostatic cancer cell lines were cultured in standard conditions until approximately 50% confluency. One set of each cell line was left to incubate 24 hours at 37°C in standard incubating conditions (20% oxygen). The other set was placed for the same amount of time in an incubator set at 1% oxygen, which was reported by multiple groups to induce changes in protein expression in these cell lines\u003csup\u003e\u003cspan additionalcitationids=\"CR69\" citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e–\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e\u003c/sup\u003e. Afterwards, cells were fixed using paraformaldehyde (4%, Sigma) for 10 minutes, permeabilized with Triton-X (0.1%) for 30 minutes, stained overnight with rabbit anti-ASCT2 (Cell Signaling Technology, cat. no. 5345; 1:100), rabbit anti-ALT2 (ProteinTech, 16757-1-AP; 1:400), or goat anti-GDH1 (Invitrogen, PA5-19267; 1:400), and then stained for 1 hr using the following secondary antibodies: donkey anti-goat AF647 (1:200) or anti-rabbit AF488 (1:200) antibodies. Data shown is representative of 4–5 independent characterizations.\u003c/p\u003e\u003cp\u003e\u003cb\u003eHistology\u003c/b\u003e. Tumors were fixed with paraformaldehyde (2%) via cardiac perfusion and frozen (-80°C) until use. Upon cryosectioning the tissues to 14 µm thick slices, they were permeabilized with Triton-X (0.1%) and stained overnight with rat anti-CD31 (Novus Biologicals, NB600-1475, 1:200) and with rabbit anti-ASCT2 (1:100). Donkey anti-rat AF488 and donkey anti-rabbit AF594 were used as secondary antibodies.\u003c/p\u003e\u003cp\u003e\u003cb\u003eWestern blot\u003c/b\u003e. Pa20c-naïve and Pa20c-ASCT2 cells were lysed for whole-cell protein extraction using RIPA (Radio Immuno Precipitation Assay) buffer (Sigma-Aldrich) following the manufacturer’s instructions. Protein (100 µg total) was resolved on a 4–15% gradient SDS-PAGE gel. Proteins were transferred to a nitrocellulose membrane and then incubated with the rabbit anti-ASCT2 antibody overnight. Mouse anti-GAPDH monoclonal antibody (Sigma, 1:5000) was used as loading control. After washing, the membrane was incubated with a horseradish peroxidase-conjugated donkey anti-rabbit or anti-mouse secondary antibody (GE healthcare, 1:2000) for 1 hour. Immunoblots were developed using SuperSignal™ West Pico PLUS Chemiluminescent Substrate kit (Thermo Fisher) following the manufacturer’s instructions.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMALDI mass spectrometry imaging\u003c/b\u003e. Tumors were embedded in M1 media (Thermo Fisher), cryosectioned at a 30 µm slice thickness and placed on indium tin oxide-coated glass slides (Delta Technologies). Tissue sections were warmed to room temperature (RT) in a vacuum desiccator for 10 mins prior to spraying. 1,5-diaminonaphthalene (10 mg/mL) in 70% acetonitrile with 0.1% trifluoroacetic acid was applied using an HTX M5 sprayer (HTX Technologies) with the following parameters: 30-degree nozzle temperature, 4 passes, 0.1 ml/min flow rate, 1200 mm/min velocity, 2.5 mm track spacing criss-cross spray pattern, 10 psi pressure, and 2 L/min gas flow rate. MALDI imaging was performed in reflectron-negative mode at a 100-micron pixel and raster size with 200 laser shots per pixel using a Bruker RapifleX MALDI TOF/TOF instrument.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMetabolite phantom preparation and MRI\u003c/b\u003e. Alanine (Sigma, PHR1110), 1-aminocyclobutane carboxylic acid (ACBC; Sigma, 652369), asparagine (Sigma, A4159), glutamine (Sigma, G3126), serine (Sigma, S4311), or threonine (Sigma, T8441) were dissolved separately in phosphate buffered saline (PBS). Solutions (20 mM) were titrated to a pH of 7.3 ± 0.03 and placed into 5 mm NMR tubes (Wilmad, WG-1000-7) for MR imaging at 37°C. Phantom images were acquired using a Bruker 9.4T vertical bore spectrometer and a 20 mm coil. Amine CEST maps were generated from MTR\u003csub\u003easym\u003c/sub\u003e spectra. Scan parameters were TR/TE = 8000/5.58 ms, RARE factor = 32, NA = 1, repetitions = 1, B\u003csub\u003e1\u003c/sub\u003e ​= ​3.6 ​µT, saturation length ​(block pulse) = ​4 ​s, frequency sweep from − 8 to + 8 ​ppm with 0.1 ​ppm increments, matrix size ​= ​76 x 76, slice thickness = 2 mm, and field of view = 20 × 20 mm\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Spectral shifts due to B\u003csub\u003e0\u003c/sub\u003e inhomogeneity were corrected for using the water saturation shift referencing (WASSR) method\u003csup\u003e\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e,\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003e\u003cb\u003eCell phantom preparation and MRI\u003c/b\u003e. The cell-rich layer consisted of prostate cancer spheres placed on top of a layer of agarose (1% in PBS) in 5 mm NMR tubes at a height of 5 mm (~ 100 µL in volume), confirmed using a caliper. The supernatant-rich layer consisted of PBS (pH of ~ 7.3; 400 µL) with bovine serum albumin (BSA; 1%) and alanine (10 mM). Phantoms without cells that contained PBS with BSA with or without alanine served as controls. Phantom images of the cell-rich and supernatant-rich layers were acquired in alternation at 37°C using a Bruker 11.7T vertical bore spectrometer and a 20 mm coil. CEST enhancement maps were generated from Z-spectra. Scan parameters were TR/TE = 10000/4.53 ms, RARE factor = 32, NA = 1, repetitions = 1, B\u003csub\u003e1\u003c/sub\u003e ​= ​3.6 ​µT, saturation length ​= ​3 ​s, frequency sweep from − 8 to + 8 ​ppm with 0.4 ​ppm increments, matrix size ​= ​64 x 64, slice thickness = 2 mm, and field of view = 20 × 20 mm\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. B\u003csub\u003e0\u003c/sub\u003e inhomogeneity was corrected using the WASSR method\u003csup\u003e\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003e\u003cb\u003eTumor induction and MRI\u003c/b\u003e. Animal work performed was approved by the institutional ACUC. DU-145 (n = 9, 1–2 x 10\u003csup\u003e6\u003c/sup\u003e) or LNCaP (n = 8, 2–3 x 10\u003csup\u003e6\u003c/sup\u003e) prostatic cancer cells were subcutaneously injected into the flank of male, 6–10 week old Rag2 mice (Jackson Laboratory, #8449). Pa20c-naïve (n = 3, 1–3 x 10\u003csup\u003e6\u003c/sup\u003e) and Pa20c-ASCT2 (n = 4, 1–3 x 10\u003csup\u003e6\u003c/sup\u003e) pancreatic cancer cell lines were subcutaneously injected into the flank of male 6–10 week old NCI Scid NCr mice (Charles River, #561). Mice were imaged 1–2 months after tumor induction using a Bruker 11.7T horizontal bore spectrometer and an 8-element phased-array surface coil. Animals were anesthetized with isoflurane (1–2%). Before and after dynamic CEST imaging, static CEST imaging was performed using a frequency sweep from − 8 to + 8 ​ppm with 0.2 ​ppm increments with a scan time of 14 minutes. Dynamic CEST images were collected at the + 3.1 ppm amine proton frequency prior to alanine injection (6 mmol/kg in bolus) to establish a baseline and over 60 minutes after agent injection. Dynamic CEST enhancement ΔS(t)/S\u003csub\u003e0\u003c/sub\u003e was quantified by subtracting signal S/S\u003csub\u003e0\u003c/sub\u003e at time t from the average signal obtained prior to injection. S\u003csub\u003e0\u003c/sub\u003e (signal at + 40 ppm) was collected at the beginning and end of the scan (n = 5 each) and then linearly fitted over time to correct for drift. Static ALAwCEST enhancement was quantified from Z-spectra (ΔS/S\u003csub\u003e0\u003c/sub\u003e) and from MTR\u003csub\u003easym\u003c/sub\u003e spectra (ΔΔS/S\u003csub\u003e0\u003c/sub\u003e) by subtracting the signal in the ± 3.0 to ± 3.2 ppm range after injection from the signal before injection. CEST MRI scan parameters for \u003cem\u003ein vivo\u003c/em\u003e images were TR/TE = 10000/3.49 ms, RARE factor = 32, NA = 1, repetitions = 1, B\u003csub\u003e1\u003c/sub\u003e ​= 3.6 ​µT, saturation length ​= ​3 ​s, matrix size ​= ​48 x 48, slice thickness = 1.5 mm, and field of view = 30 × 30 mm\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. For static CEST maps and spectra, B\u003csub\u003e0\u003c/sub\u003e inhomogeneity was corrected using the WASSR method as before\u003csup\u003e\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e\u003c/sup\u003e. \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eTumor size\u003c/span\u003e (mm\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e) was calculated as the sum of the cross-sectional areas of T2-weighted images multiplied by the slice thickness.\u003c/p\u003e\u003cp\u003e\u003cb\u003eStatistics\u003c/b\u003e. Student’s t-tests, paired or unpaired, and two-way ANOVAs were performed as appropriate. Spearman’s rank was used for correlations. Significance was defined as p \u0026lt; 0.05. Histograms of the intensity frequencies were normalized (by pixel number) and averaged. Line profiles are from representative regions as outlined in the figure.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was funded by NIH: K01 EB030612 (A.M.T.), Patrick C. Walsh Prostate Cancer Research Fund (A.M.T.), R21 EB035705 (A.M.T.), P50 CA272391 (A.M.T.), P41 EB024495 (P.C.M.v.Z., M.T.M., M.G.P.), and R01 EB030376 (J.W.M.B.). The funder played no role in study design, data collection, analysis and interpretation of data, or the writing of this manuscript. MALDI imaging experiments were performed in the Johns Hopkins Applied Imaging Mass Spectrometry (AIMS) Core facility. MRI experiments were performed in the Johns Hopkins NMR service facility and in the F.M. Kirby Research Center for Functional Brain Imaging at Kennedy Krieger Institute preclinical facility.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eB.G \u0026ndash; investigation (prostate models). B.K. \u0026ndash; methodology and investigation (pancreatic models). N.D. \u0026ndash; investigation and formal analysis (MALDI). K.N.B.W. \u0026ndash; investigation (cell culture, staining, and imaging). Y.H. \u0026ndash; investigation (tissue staining and imaging). Y.L. \u0026ndash; investigation (animal imaging). M.-F.P.V. \u0026ndash; investigation (pancreatic models). M.G.P \u0026ndash; supervision (imaging validation); funding acquisition. C.M.T \u0026ndash; methodology, supervision, and resources (MALDI). Z.M.B. \u0026ndash; supervision and resources (pancreatic models). J.W.M.B. \u0026ndash; resources (prostate models); writing (review and editing); funding acquisition. M.T.M. \u0026ndash; methodology, supervision, and resources (imaging sequences and phantoms); funding acquisition. P.C.M.v.Z. \u0026ndash; supervision and resources; writing (review and editing); funding acquisition. A.M.T. \u0026ndash; conceptualization, investigation, formal analysis, and visualization; supervision (prostate models); writing (original); funding acquisition.\u003c/p\u003e\n\u003cp\u003eAll authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no financial or non-financial competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCode availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMATLAB codes for performing WASSR for B\u003csub\u003e0\u003c/sub\u003e correction, regions of interest analysis, and CEST map generation can be found at our website: godzilla.kennedykrieger.org/CEST/, https://www.kennedykrieger.org/kirby-research-center/software-databases.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eChoi, Y. K. \u0026amp; Park, K. G. Targeting Glutamine Metabolism for Cancer Treatment. \u003cem\u003eBiomol Ther (Seoul)\u003c/em\u003e \u003cstrong\u003e26\u003c/strong\u003e, 19-28 (2018). https://doi.org:10.4062/biomolther.2017.178\u003c/li\u003e\n \u003cli\u003eRiscal, R.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e Chromatin-Bound MDM2 Regulates Serine Metabolism and Redox Homeostasis Independently of p53. \u003cem\u003eMolecular Cell\u003c/em\u003e \u003cstrong\u003e62\u003c/strong\u003e, 890-902 (2016). https://doi.org:10.1016/j.molcel.2016.04.033\u003c/li\u003e\n \u003cli\u003eYue, M., Jiang, J., Gao, P., Liu, H. \u0026amp; Qing, G. Oncogenic MYC Activates a Feedforward Regulatory Loop Promoting Essential Amino Acid Metabolism and Tumorigenesis. \u003cem\u003eCell Reports\u003c/em\u003e \u003cstrong\u003e21\u003c/strong\u003e, 3819-3832 (2017). https://doi.org:10.1016/j.celrep.2017.12.002\u003c/li\u003e\n \u003cli\u003eRoux, C.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e Endogenous glutamine decrease is associated with pancreatic cancer progression. \u003cem\u003eOncotarget\u003c/em\u003e \u003cstrong\u003e8\u003c/strong\u003e (2017).\u003c/li\u003e\n \u003cli\u003eSun, H.-W.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e GLUT1 and ASCT2 as Predictors for Prognosis of Hepatocellular Carcinoma. \u003cem\u003ePLOS ONE\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e, e0168907 (2016). https://doi.org:10.1371/journal.pone.0168907\u003c/li\u003e\n \u003cli\u003eShimizu, K.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e ASC amino-acid transporter 2 (ASCT2) as a novel prognostic marker in non-small cell lung cancer. \u003cem\u003eBritish Journal of Cancer\u003c/em\u003e \u003cstrong\u003e110\u003c/strong\u003e, 2030-2039 (2014). https://doi.org:10.1038/bjc.2014.88\u003c/li\u003e\n \u003cli\u003eLuo, Y.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e ASCT2 overexpression is associated with poor survival of OSCC patients and ASCT2 knockdown inhibited growth of glutamine-addicted OSCC cells. \u003cem\u003eCancer Medicine\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e, 3489-3499 (2020). https://doi.org:doi.org/10.1002/cam4.2965\u003c/li\u003e\n \u003cli\u003eBernhardt, S.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e Proteomic profiling of breast cancer metabolism identifies SHMT2 and ASCT2 as prognostic factors. \u003cem\u003eBreast Cancer Research\u003c/em\u003e \u003cstrong\u003e19\u003c/strong\u003e, 112 (2017). https://doi.org:10.1186/s13058-017-0905-7\u003c/li\u003e\n \u003cli\u003eWang, Q.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e Targeting ASCT2-mediated glutamine uptake blocks prostate cancer growth and tumour development. \u003cem\u003eThe Journal of Pathology\u003c/em\u003e \u003cstrong\u003e236\u003c/strong\u003e, 278-289 (2015). https://doi.org:doi.org/10.1002/path.4518\u003c/li\u003e\n \u003cli\u003eShen, Y.-A.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e Inhibition of glutaminolysis in combination with other therapies to improve cancer treatment. \u003cem\u003eCurrent Opinion in Chemical Biology\u003c/em\u003e \u003cstrong\u003e62\u003c/strong\u003e, 64-81 (2021). https://doi.org:https://doi.org/10.1016/j.cbpa.2021.01.006\u003c/li\u003e\n \u003cli\u003eChoi, Y.-K. \u0026amp; Park, K.-G. Targeting Glutamine Metabolism for Cancer Treatment. \u003cem\u003eBiomolecules \u0026amp; Therapeutics\u003c/em\u003e \u003cstrong\u003e26\u003c/strong\u003e, 19-28 (2018). https://doi.org:10.4062/biomolther.2017.178\u003c/li\u003e\n \u003cli\u003eFeng, Y.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e Identification and Characterization of IMD-0354 as a Glutamine Carrier Protein Inhibitor in Melanoma. \u003cem\u003eMolecular Cancer Therapeutics\u003c/em\u003e \u003cstrong\u003e20\u003c/strong\u003e, 816-832 (2021). https://doi.org:10.1158/1535-7163.Mct-20-0354\u003c/li\u003e\n \u003cli\u003eConsole, L., Scalise, M., Tarmakova, Z., Coe, I. R. \u0026amp; Indiveri, C. N-linked Glycosylation of human SLC1A5 (ASCT2) transporter is critical for trafficking to membrane. \u003cem\u003eBiochimica et Biophysica Acta (BBA) - Molecular Cell Research\u003c/em\u003e \u003cstrong\u003e1853\u003c/strong\u003e, 1636-1645 (2015). https://doi.org:doi.org/10.1016/j.bbamcr.2015.03.017\u003c/li\u003e\n \u003cli\u003eLeslie, T. K.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e Sodium homeostasis in the tumour microenvironment. \u003cem\u003eBiochimica et Biophysica Acta (BBA) - Reviews on Cancer\u003c/em\u003e \u003cstrong\u003e1872\u003c/strong\u003e, 188304 (2019). https://doi.org:doi.org/10.1016/j.bbcan.2019.07.001\u003c/li\u003e\n \u003cli\u003evan Zijl, P. C. M. \u0026amp; Yadav, N. N. Chemical exchange saturation transfer (CEST): What is in a name and what isn\u0026apos;t? \u003cem\u003eMagnetic Resonance in Medicine\u003c/em\u003e \u003cstrong\u003e65\u003c/strong\u003e, 927-948 (2011). https://doi.org:https://doi.org/10.1002/mrm.22761\u003c/li\u003e\n \u003cli\u003eUtsunomiya-Tate, N., Endou, H. \u0026amp; Kanai, Y. Cloning and Functional Characterization of a System ASC-like Na+-dependent Neutral Amino Acid Transporter*. \u003cem\u003eJournal of Biological Chemistry\u003c/em\u003e \u003cstrong\u003e271\u003c/strong\u003e, 14883-14890 (1996). https://doi.org:https://doi.org/10.1074/jbc.271.25.14883\u003c/li\u003e\n \u003cli\u003eYanagida, O.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e Human L-type amino acid transporter 1 (LAT1): characterization of function and expression in tumor cell lines. \u003cem\u003eBiochimica et Biophysica Acta (BBA) - Biomembranes\u003c/em\u003e \u003cstrong\u003e1514\u003c/strong\u003e, 291-302 (2001). https://doi.org:https://doi.org/10.1016/S0005-2736(01)00384-4\u003c/li\u003e\n \u003cli\u003eGoodman, M. M., Yu, W. \u0026amp; Jarkas, N. Synthesis and biological properties of radiohalogenated \u0026alpha;,\u0026alpha;-disubstituted amino acids for PET and SPECT imaging of amino acid transporters (AATs). \u003cem\u003eJournal of Labelled Compounds and Radiopharmaceuticals\u003c/em\u003e \u003cstrong\u003e61\u003c/strong\u003e, 272-290 (2018). https://doi.org:https://doi.org/10.1002/jlcr.3584\u003c/li\u003e\n \u003cli\u003eGarlick, P. J. The Nature of Human Hazards Associated with Excessive Intake of Amino Acids. \u003cem\u003eThe Journal of Nutrition\u003c/em\u003e \u003cstrong\u003e134\u003c/strong\u003e, 1633S-1639S (2004). https://doi.org:10.1093/jn/134.6.1633S\u003c/li\u003e\n \u003cli\u003eTan, Z.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e Saturation transfer properties of tumour xenografts derived from prostate cancer cell lines 22Rv1 and DU145. \u003cem\u003eScientific Reports\u003c/em\u003e \u003cstrong\u003e10\u003c/strong\u003e, 21315 (2020). https://doi.org:10.1038/s41598-020-78353-8\u003c/li\u003e\n \u003cli\u003eMussawy, H.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e The bone microenvironment promotes tumor growth and tissue perfusion compared with striated muscle in a preclinical model of prostate cancer in vivo. \u003cem\u003eBMC Cancer\u003c/em\u003e \u003cstrong\u003e18\u003c/strong\u003e, 979 (2018). https://doi.org:10.1186/s12885-018-4905-5\u003c/li\u003e\n \u003cli\u003eAdeno-Associated Virus 2-Mediated Intratumoral Prostate Cancer Gene Therapy: Long-Term Maspin Expression Efficiently Suppresses Tumor Growth. \u003cem\u003eHuman Gene Therapy\u003c/em\u003e \u003cstrong\u003e16\u003c/strong\u003e, 699-710 (2005). https://doi.org:10.1089/hum.2005.16.699\u003c/li\u003e\n \u003cli\u003eLi, Y., Zhong, W., Zhu, M., Li, M. \u0026amp; Yang, Z. miR-185 inhibits prostate cancer angiogenesis induced by the nodal/ALK4 pathway. \u003cem\u003eBMC Urology\u003c/em\u003e \u003cstrong\u003e20\u003c/strong\u003e, 49 (2020). https://doi.org:10.1186/s12894-020-00617-2\u003c/li\u003e\n \u003cli\u003eKai, L.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e Targeting prostate cancer angiogenesis through metastasis-associated protein 1 (MTA1). \u003cem\u003eThe Prostate\u003c/em\u003e \u003cstrong\u003e71\u003c/strong\u003e, 268-280 (2011). https://doi.org:https://doi.org/10.1002/pros.21240\u003c/li\u003e\n \u003cli\u003eScopelliti, A. J., Font, J., Vandenberg, R. J., Boudker, O. \u0026amp; Ryan, R. M. Structural characterisation reveals insights into substrate recognition by the glutamine transporter ASCT2/SLC1A5. \u003cem\u003eNature Communications\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e, 38 (2018). https://doi.org:10.1038/s41467-017-02444-w\u003c/li\u003e\n \u003cli\u003eYao, D.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e A Novel System A Isoform Mediating Na\u0026lt;sup\u0026gt;+\u0026lt;/sup\u0026gt;/Neutral Amino Acid Cotransport *. \u003cem\u003eJournal of Biological Chemistry\u003c/em\u003e \u003cstrong\u003e275\u003c/strong\u003e, 22790-22797 (2000). https://doi.org:10.1074/jbc.M002965200\u003c/li\u003e\n \u003cli\u003eFoster, A. C.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e D-Serine Is a Substrate for Neutral Amino Acid Transporters ASCT1/SLC1A4 and ASCT2/SLC1A5, and Is Transported by Both Subtypes in Rat Hippocampal Astrocyte Cultures. \u003cem\u003ePLOS ONE\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e, e0156551 (2016). https://doi.org:10.1371/journal.pone.0156551\u003c/li\u003e\n \u003cli\u003eVettore, L., Westbrook, R. L. \u0026amp; Tennant, D. A. New aspects of amino acid metabolism in cancer. \u003cem\u003eBritish Journal of Cancer\u003c/em\u003e \u003cstrong\u003e122\u003c/strong\u003e, 150-156 (2020). https://doi.org:10.1038/s41416-019-0620-5\u003c/li\u003e\n \u003cli\u003eFernandes, J. \u0026amp; Blom, W. The intravenous L-alanine tolerance test as a means for investigating gluconeogenesis. \u003cem\u003eMetabolism - Clinical and Experimental\u003c/em\u003e \u003cstrong\u003e23\u003c/strong\u003e, 1149-1156 (1974). https://doi.org:10.1016/0026-0495(74)90031-6\u003c/li\u003e\n \u003cli\u003eLiu, H.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e 18F-Alanine Derivative Serves as an ASCT2 Marker for Cancer Imaging. \u003cem\u003eMolecular Pharmaceutics\u003c/em\u003e \u003cstrong\u003e15\u003c/strong\u003e, 947-954 (2018). https://doi.org:10.1021/acs.molpharmaceut.7b00884\u003c/li\u003e\n \u003cli\u003eBattezzati, A., Haisch, M., Brillon, D. J. \u0026amp; Matthews, D. E. Splanchnic utilization of enteral alanine in humans. \u003cem\u003eMetabolism - Clinical and Experimental\u003c/em\u003e \u003cstrong\u003e48\u003c/strong\u003e, 915-921 (1999). https://doi.org:10.1016/S0026-0495(99)90229-9\u003c/li\u003e\n \u003cli\u003eJi, X.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e Transportable hyperpolarized metabolites. \u003cem\u003eNature Communications\u003c/em\u003e \u003cstrong\u003e8\u003c/strong\u003e, 13975 (2017). https://doi.org:10.1038/ncomms13975\u003c/li\u003e\n \u003cli\u003eYang, S.-H.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e MRI measurement of alanine uptake in a mouse xenograft model of U-87 MG glioblastoma. \u003cem\u003eMagnetic Resonance Imaging\u003c/em\u003e \u003cstrong\u003e93\u003c/strong\u003e, 189-194 (2022). https://doi.org:https://doi.org/10.1016/j.mri.2022.08.015\u003c/li\u003e\n \u003cli\u003eCardoso, H. J.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e Glutaminolysis is a metabolic route essential for survival and growth of prostate cancer cells and a target of 5\u0026alpha;-dihydrotestosterone regulation. \u003cem\u003eCellular Oncology\u003c/em\u003e \u003cstrong\u003e44\u003c/strong\u003e, 385-403 (2021). https://doi.org:10.1007/s13402-020-00575-9\u003c/li\u003e\n \u003cli\u003eHodo, Y.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e Imaging the uptake and metabolism of glutamine in prostate tumor models using CEST MRI. \u003cem\u003enpj Imaging\u003c/em\u003e \u003cstrong\u003e3\u003c/strong\u003e, 34 (2025). https://doi.org:10.1038/s44303-025-00100-3\u003c/li\u003e\n \u003cli\u003eOkudaira, H.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e Accumulation of Trans-1-Amino-3-[18F]Fluorocyclobutanecarboxylic Acid in Prostate Cancer due to Androgen-Induced Expression of Amino Acid Transporters. \u003cem\u003eMolecular Imaging and Biology\u003c/em\u003e \u003cstrong\u003e16\u003c/strong\u003e, 756-764 (2014). https://doi.org:10.1007/s11307-014-0756-x\u003c/li\u003e\n \u003cli\u003eOka, S.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e Transport mechanisms of trans-1-amino-3-fluoro[1-14C]cyclobutanecarboxylic acid in prostate cancer cells. \u003cem\u003eNuclear Medicine and Biology\u003c/em\u003e \u003cstrong\u003e39\u003c/strong\u003e, 109-119 (2012). https://doi.org:doi.org/10.1016/j.nucmedbio.2011.06.008\u003c/li\u003e\n \u003cli\u003eLiu, Y.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e The role of ASCT2 in cancer: A review. \u003cem\u003eEuropean Journal of Pharmacology\u003c/em\u003e \u003cstrong\u003e837\u003c/strong\u003e, 81-87 (2018). https://doi.org:doi.org/10.1016/j.ejphar.2018.07.007\u003c/li\u003e\n \u003cli\u003eRivlin, M. \u0026amp; Navon, G. CEST MRI of 3-O-methyl-D-glucose on different breast cancer models. \u003cem\u003eMagnetic Resonance in Medicine\u003c/em\u003e \u003cstrong\u003e79\u003c/strong\u003e, 1061-1069 (2018). https://doi.org:doi.org/10.1002/mrm.26752\u003c/li\u003e\n \u003cli\u003eAnemone, A.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e In vitro and in vivo comparison of MRI chemical exchange saturation transfer (CEST) properties between native glucose and 3-O-Methyl-D-glucose in a murine tumor model. \u003cem\u003eNMR in Biomedicine\u003c/em\u003e \u003cstrong\u003e34\u003c/strong\u003e, e4602 (2021). https://doi.org:https://doi.org/10.1002/nbm.4602\u003c/li\u003e\n \u003cli\u003eRivlin, M., Horev, J., Tsarfaty, I. \u0026amp; Navon, G. Molecular imaging of tumors and metastases using chemical exchange saturation transfer (CEST) MRI. \u003cem\u003eScientific Reports\u003c/em\u003e \u003cstrong\u003e3\u003c/strong\u003e, 3045 (2013). https://doi.org:10.1038/srep03045\u003c/li\u003e\n \u003cli\u003eWalker-Samuel, S.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e In vivo imaging of glucose uptake and metabolism in tumors. \u003cem\u003eNature Medicine\u003c/em\u003e \u003cstrong\u003e19\u003c/strong\u003e, 1067-1072 (2013). https://doi.org:10.1038/nm.3252\u003c/li\u003e\n \u003cli\u003eNasrallah, F. A., Pag\u0026egrave;s, G., Kuchel, P. W., Golay, X. \u0026amp; Chuang, K.-H. Imaging Brain Deoxyglucose Uptake and Metabolism by Glucocest MRI. \u003cem\u003eJournal of Cerebral Blood Flow \u0026amp; Metabolism\u003c/em\u003e \u003cstrong\u003e33\u003c/strong\u003e, 1270-1278 (2013). https://doi.org:10.1038/jcbfm.2013.79\u003c/li\u003e\n \u003cli\u003eKnutsson, L., Xu, X., van Zijl, P. C. M. \u0026amp; Chan, K. W. Y. Imaging of sugar-based contrast agents using their hydroxyl proton exchange properties. \u003cem\u003eNMR in Biomedicine\u003c/em\u003e \u003cstrong\u003en/a\u003c/strong\u003e, e4784 https://doi.org:https://doi.org/10.1002/nbm.4784\u003c/li\u003e\n \u003cli\u003eEllingson, B. M.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e pH-weighted molecular MRI in human traumatic brain injury (TBI) using amine proton chemical exchange saturation transfer echoplanar imaging (CEST EPI). \u003cem\u003eNeuroImage: Clinical\u003c/em\u003e \u003cstrong\u003e22\u003c/strong\u003e, 101736 (2019). https://doi.org:https://doi.org/10.1016/j.nicl.2019.101736\u003c/li\u003e\n \u003cli\u003eHarris, R. J.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e pH-weighted molecular imaging of gliomas using amine chemical exchange saturation transfer MRI. \u003cem\u003eNeuro-Oncology\u003c/em\u003e \u003cstrong\u003e17\u003c/strong\u003e, 1514-1524 (2015). https://doi.org:10.1093/neuonc/nov106\u003c/li\u003e\n \u003cli\u003eWermter, F. C., Bock, C. \u0026amp; Dreher, W. Investigating GluCEST and its specificity for pH mapping at low temperatures. \u003cem\u003eNMR in Biomedicine\u003c/em\u003e \u003cstrong\u003e28\u003c/strong\u003e, 1507-1517 (2015). https://doi.org:https://doi.org/10.1002/nbm.3416\u003c/li\u003e\n \u003cli\u003eYao, J., Wang, C. \u0026amp; Ellingson, B. M. Influence of phosphate concentration on amine, amide, and hydroxyl CEST contrast. \u003cem\u003eMagnetic Resonance in Medicine\u003c/em\u003e \u003cstrong\u003e85\u003c/strong\u003e, 1062-1078 (2021). https://doi.org:doi.org/10.1002/mrm.28481\u003c/li\u003e\n \u003cli\u003eCho, N. S.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e Amine-weighted chemical exchange saturation transfer magnetic resonance imaging in brain tumors. \u003cem\u003eNMR in Biomedicine\u003c/em\u003e \u003cstrong\u003e36\u003c/strong\u003e, e4785 (2023). https://doi.org:https://doi.org/10.1002/nbm.4785\u003c/li\u003e\n \u003cli\u003eLee, Z.-W.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e Intracellular Hyper-Acidification Potentiated by Hydrogen Sulfide Mediates Invasive and Therapy Resistant Cancer Cell Death. \u003cem\u003eFrontiers in Pharmacology\u003c/em\u003e \u003cstrong\u003e8\u003c/strong\u003e (2017). https://doi.org:10.3389/fphar.2017.00763\u003c/li\u003e\n \u003cli\u003eWilson, L. T.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e A new class of ratiometric small molecule intracellular pH sensors for Raman microscopy. \u003cem\u003eAnalyst\u003c/em\u003e \u003cstrong\u003e145\u003c/strong\u003e, 5289-5298 (2020). https://doi.org:10.1039/D0AN00865F\u003c/li\u003e\n \u003cli\u003eFuruya, Y., Lundmo, P., Short, A. D., Gill, D. L. \u0026amp; Isaacs, J. T. The role of calcium, pH, and cell proliferation in the programmed (apoptotic) death of androgen-independent prostatic cancer cells induced by thapsigargin. \u003cem\u003eCancer Res\u003c/em\u003e \u003cstrong\u003e54\u003c/strong\u003e, 6167-6175 (1994).\u003c/li\u003e\n \u003cli\u003eVāvere, A. L.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e A Novel Technology for the Imaging of Acidic Prostate Tumors by Positron Emission Tomography. \u003cem\u003eCancer Research\u003c/em\u003e \u003cstrong\u003e69\u003c/strong\u003e, 4510-4516 (2009). https://doi.org:10.1158/0008-5472.Can-08-3781\u003c/li\u003e\n \u003cli\u003eIbrahim-Hashim, A.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e Systemic Buffers Inhibit Carcinogenesis in TRAMP Mice. \u003cem\u003eJournal of Urology\u003c/em\u003e \u003cstrong\u003e188\u003c/strong\u003e, 624-631 (2012). https://doi.org:doi:10.1016/j.juro.2012.03.113\u003c/li\u003e\n \u003cli\u003eKorenchan, D. E.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e Hyperpolarized in vivo pH imaging reveals grade-dependent acidification in prostate cancer. \u003cem\u003eOncotarget\u003c/em\u003e \u003cstrong\u003e10\u003c/strong\u003e (2019). https://doi.org:10.18632/oncotarget.27225\u003c/li\u003e\n \u003cli\u003eDunst, J.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e Tumor Volume and Tumor Hypoxia in Head andNeck Cancers. \u003cem\u003eStrahlentherapie und Onkologie\u003c/em\u003e \u003cstrong\u003e179\u003c/strong\u003e, 521-526 (2003). https://doi.org:10.1007/s00066-003-1066-4\u003c/li\u003e\n \u003cli\u003eGaustad, J.-V., Simonsen, T. G., Andersen, L. M. K. \u0026amp; Rofstad, E. K. Vascular abnormalities and development of hypoxia in microscopic melanoma xenografts. \u003cem\u003eJournal of Translational Medicine\u003c/em\u003e \u003cstrong\u003e15\u003c/strong\u003e, 241 (2017). https://doi.org:10.1186/s12967-017-1347-9\u003c/li\u003e\n \u003cli\u003eKis, A.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e \u0026lt;i\u0026gt;In Vivo\u0026lt;/i\u0026gt; Imaging of Hypoxia and Neoangiogenesis in Experimental Syngeneic Hepatocellular Carcinoma Tumor Model Using Positron Emission Tomography. \u003cem\u003eBioMed Research International\u003c/em\u003e \u003cstrong\u003e2020\u003c/strong\u003e, 4952372 (2020). https://doi.org:10.1155/2020/4952372\u003c/li\u003e\n \u003cli\u003eKiraga, Ł.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e Changes in hypoxia level of CT26 tumors during various stages of development and comparing different methods of hypoxia determination. \u003cem\u003ePLOS ONE\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e, e0206706 (2018). https://doi.org:10.1371/journal.pone.0206706\u003c/li\u003e\n \u003cli\u003eVaupel, P., Kallinowski, F. \u0026amp; Okunieff, P. Blood Flow, Oxygen and Nutrient Supply, and Metabolic Microenvironment of Human Tumors: A Review1. \u003cem\u003eCancer Research\u003c/em\u003e \u003cstrong\u003e49\u003c/strong\u003e, 6449-6465 (1989).\u003c/li\u003e\n \u003cli\u003eYoo, H. C.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e A Variant of SLC1A5 Is a Mitochondrial Glutamine Transporter for Metabolic Reprogramming in Cancer Cells. \u003cem\u003eCell Metabolism\u003c/em\u003e \u003cstrong\u003e31\u003c/strong\u003e, 267-283.e212 (2020). https://doi.org:https://doi.org/10.1016/j.cmet.2019.11.020\u003c/li\u003e\n \u003cli\u003eBUNGARD, Claire I. \u0026amp; McGIVAN, John D. Glutamine availability up-regulates expression of the amino acid transporter protein ASCT2 in HepG2 cells and stimulates the ASCT2 promoter. \u003cem\u003eBiochemical Journal\u003c/em\u003e \u003cstrong\u003e382\u003c/strong\u003e, 27-32 (2004). https://doi.org:10.1042/bj20040487\u003c/li\u003e\n \u003cli\u003eMa, H.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e Inhibition of Glutamine Uptake Improves the Efficacy of Cetuximab on Gastric Cancer. \u003cem\u003eIntegrative Cancer Therapies\u003c/em\u003e \u003cstrong\u003e20\u003c/strong\u003e, 15347354211045349 (2021). https://doi.org:10.1177/15347354211045349\u003c/li\u003e\n \u003cli\u003eKee, A. J., Smith, R. C., Gross, A. S., Madsen, D. C. \u0026amp; Rowe, B. The effect of dipeptide structure on dipeptide and amino acid clearance in rats. \u003cem\u003eMetabolism\u003c/em\u003e \u003cstrong\u003e43\u003c/strong\u003e, 1373-1378 (1994). https://doi.org:doi: 10.1016/0026-0495(94)90030-2\u003c/li\u003e\n \u003cli\u003eQi, Z.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e Serial determination of glomerular filtration rate in conscious mice using FITC-inulin clearance. \u003cem\u003eAmerican Journal of Physiology-Renal Physiology\u003c/em\u003e \u003cstrong\u003e286\u003c/strong\u003e, F590-F596 (2004). https://doi.org:10.1152/ajprenal.00324.2003\u003c/li\u003e\n \u003cli\u003eKim, H., Krishnamurthy, L. C. \u0026amp; Sun, P. Z. Brain pH Imaging and its Applications. \u003cem\u003eNeuroscience\u003c/em\u003e \u003cstrong\u003e474\u003c/strong\u003e, 51-62 (2021). https://doi.org:doi.org/10.1016/j.neuroscience.2021.01.026\u003c/li\u003e\n \u003cli\u003eJones, S.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e Core Signaling Pathways in Human Pancreatic Cancers Revealed by Global Genomic Analyses. \u003cem\u003eScience\u003c/em\u003e \u003cstrong\u003e321\u003c/strong\u003e, 1801-1806 (2008). https://doi.org:doi:10.1126/science.1164368\u003c/li\u003e\n \u003cli\u003eGeng, H.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e Interplay between hypoxia and androgen controls a metabolic switch conferring resistance to androgen/AR-targeted therapy. \u003cem\u003eNature Communications\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e, 4972 (2018). https://doi.org:10.1038/s41467-018-07411-7\u003c/li\u003e\n \u003cli\u003eRavenna, L.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e Distinct Phenotypes of Human Prostate Cancer Cells Associate with Different Adaptation to Hypoxia and Pro-Inflammatory Gene Expression. \u003cem\u003ePLOS ONE\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e, e96250 (2014). https://doi.org:10.1371/journal.pone.0096250\u003c/li\u003e\n \u003cli\u003eMa, Y.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e Prostate Cancer Cell Lines under Hypoxia Exhibit Greater Stem-Like Properties. \u003cem\u003ePLOS ONE\u003c/em\u003e \u003cstrong\u003e6\u003c/strong\u003e, e29170 (2011). https://doi.org:10.1371/journal.pone.0029170\u003c/li\u003e\n \u003cli\u003eThomas, A. M., Xu, J., Calabresi, P. A., van Zijl, P. C. M. \u0026amp; Bulte, J. W. M. Monitoring diffuse injury during disease progression in experimental autoimmune encephalomyelitis with on resonance variable delay multiple pulse (onVDMP) CEST MRI. \u003cem\u003eNeuroImage\u003c/em\u003e \u003cstrong\u003e204\u003c/strong\u003e, 116245 (2020). https://doi.org:doi.org/10.1016/j.neuroimage.2019.116245\u003c/li\u003e\n \u003cli\u003eKim, M., Gillen, J., Landman, B. A., Zhou, J. \u0026amp; van Zijl, P. C. M. Water saturation shift referencing (WASSR) for chemical exchange saturation transfer (CEST) experiments. \u003cem\u003eMagnetic Resonance in Medicine\u003c/em\u003e \u003cstrong\u003e61\u003c/strong\u003e, 1441-1450 (2009). https://doi.org:https://doi.org/10.1002/mrm.21873\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"npj-imaging","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [npj Imaging](https://www.nature.com/npjimaging)","snPcode":"44303","submissionUrl":"https://submission.springernature.com/new-submission/44303/3","title":"npj Imaging","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"NPJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-7367339/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7367339/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eWe investigated alanine as a magnetic resonance imaging (MRI) biomarker for alanine-serine-cysteine transporter 2 (ASCT2), the primary transporter for glutamine in cancer. Alanine exhibited higher contrast using the chemical exchange saturation transfer (CEST) method than other major ASCT2 substrates. Upon bolus injection of alanine (6 mmole/kg), CEST MRI enhancement \u003cem\u003ein vivo\u003c/em\u003e was higher in \u003cem\u003eSLC1A5\u003c/em\u003e-overexpressing Pa20c pancreatic tumors than na\u0026iuml;ve tumors (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). Enhancement was more pronounced in LNCaP prostate tumors than DU-145 prostate tumors \u003cem\u003ein vivo\u003c/em\u003e (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). The temporal dynamics of alanine-weighted CEST (ALAwCEST) signal enhancement depended on the volume of the tumor, which histological analysis revealed to be due to alterations in the expression and distribution of ASCT2 and CD31-positive blood vessels. Mass spectrometric imaging of \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC-labeled metabolites confirmed differences in alanine uptake and metabolism in prostate tumors. We demonstrated the feasibility of ALAwCEST imaging for reporting ASCT2-mediated uptake in multiple human cancer models.\u003c/p\u003e","manuscriptTitle":"MRI of ASCT2-mediated amino acid uptake in xenograft tumor models","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-26 00:39:54","doi":"10.21203/rs.3.rs-7367339/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-10-27T13:42:22+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-24T09:58:51+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-15T12:04:42+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"288681377220256905600852692788938675258","date":"2025-10-12T15:10:23+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"185086655933968530776885099614766013593","date":"2025-10-12T14:01:03+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"81948513977611450166137803143649925212","date":"2025-10-11T08:49:58+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-10-10T13:55:38+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-09-02T18:09:53+00:00","index":"","fulltext":""},{"type":"submitted","content":"npj Imaging","date":"2025-09-02T18:05:17+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"npj-imaging","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [npj Imaging](https://www.nature.com/npjimaging)","snPcode":"44303","submissionUrl":"https://submission.springernature.com/new-submission/44303/3","title":"npj Imaging","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"NPJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"7e3cca1a-c1a4-4186-8d09-2001067f1804","owner":[],"postedDate":"October 26th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":56588578,"name":"Health sciences/Biomarkers"},{"id":56588579,"name":"Biological sciences/Cancer"},{"id":56588580,"name":"Health sciences/Oncology"}],"tags":[],"updatedAt":"2026-04-30T19:11:33+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-26 00:39:54","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7367339","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7367339","identity":"rs-7367339","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}