Hyperpolarized [1- 13 C]pyruvate NMR spectroscopy reveals transition of tumor energy metabolism in tiny multicellular spheroids

Hyperpolarized [1- 13 C]pyruvate NMR spectroscopy reveals transition of tumor energy metabolism in tiny multicellular spheroids | 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 Hyperpolarized [1- 13 C]pyruvate NMR spectroscopy reveals transition of tumor energy metabolism in tiny multicellular spheroids Yoichi Takakusagi, Kaori Takakusagi, Kaori Inoue, Keita Saito, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5900705/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 02 Jun, 2025 Read the published version in Scientific Reports → Version 1 posted 12 You are reading this latest preprint version Abstract Hyperpolarized (HP) [1- 13 C]pyruvate nuclear magnetic resonance (NMR) spectroscopy was employed to investigate tumor energy metabolism in tiny multicellular spheroids, serving as a model of early-phase tumorigenesis in vivo . A three-dimensional static culture of murine squamous cell carcinoma (SCCVII) cells formed uniform smaller multicellular spheroids (~ 150 µm in diameter), without hypoxic or necrotic cores, yet these spheroids exhibited resistance to anti-tumor drugs. HP [1- 13 C]pyruvate NMR spectroscopy of SCCVII spheroids revealed an increased conversion of pyruvate to lactate compared to monolayer cultures, indicating enhanced aerobic glycolysis in the aggregated cells. Additionally, HP spectroscopy differentiated the degree of aerobic glycolysis in human prostate tumor spheroids―DU145 (~ 120 µm) and PC-3 (~ 230 µm)―as evidenced by the upregulation of genes associated with lactate production and cellular transport. The Lac/Pyr ratio among spheroids correlated with those observed in biopsy samples of corresponding malignant tumors grown in mice. These findings suggest that HP [1- 13 C]pyruvate NMR spectroscopy may serve as a metabolic biomarker for early-phase tumorigenesis in vivo . Biological sciences/Biological techniques/Analytical biochemistry/Biochemical assays Biological sciences/Cancer/Tumour biomarkers Health sciences/Oncology/Cancer/Cancer metabolism Physical sciences/Engineering/Biomedical engineering Hyperpolarization Pyruvate Spheroid SCC Prostate tumor NMR Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction The development of diagnostic systems and technologies for the early detection of tumorigenesis remains a significant challenge. Among the advancements in imaging, 18 F-fluoro-2-deoxyglucose positron emission tomography ( 18 F-FDG-PET) has proven to be a major breakthrough in clinical applications, enabling the detection of tumors with an internal diameter of less than 1 mm 1 . This diagnostic modality relies on the uptake and subsequent phosphorylation of the tracer by actively proliferating tumor cells, leading to its accumulation within these cells. However, while 18 F-FDG-PET provides valuable insights about tumor location, it offers limited information regarding the metabolic profile of the detected tumors, leaving gaps in understanding their biochemical characteristics. Hyperpolarization (HP) is a powerful technique that enhances the sensitivity of nuclear magnetic resonance (NMR) by several orders of magnitude 2 . This advancement enables noninvasive and quantitative assessment of metabolic processes in living systems using functional chemical probes labeled with non-radioactive nuclei, such as 13 C 3 . Unlike imaging modalities that rely on radionuclides as tracers, HP provides real-time information about structural alterations of probes during metabolism, detectable as changes in spin frequency or chemical shifts at specific locations of interest 4 . Additionally, this method allows for the simultaneous detection of multiple metabolites 5 . Among the HP chemical probes developed to date 6 – 8 , 13 C-labeled pyruvate ([1- 13 C]pyruvate) is the most promising. This probe facilitates direct monitoring of aerobic glycolysis ( Fig. 1 ), a process significantly elevated in tumors compared to normal tissues 9 . Furthermore, HP[1- 13 C]pyruvate has been utilized as a noninvasive tool for assessing tumor malignancy and treatment responses in in vivo tumor xenografts and in patients with prostate cancer 10 – 15 . The recent advancements in cell and tissue engineering have enabled the production of biological mimetics for analyzing tissue-like features in living systems, even in vitro . For instance, similar to tumor xenografts grown in mice, three-dimensional spheroid cultures of tumor cells replicate tissue-like characteristics in vitro . These characteristics include static cells in the inner core surrounded by a layer of actively proliferating cells, the development of hypoxic regions or necrotic cores, and reduced sensitivity to chemotherapeutic agents 16 – 18 . The application of spheroids smaller than 1 mm in diameter is particularly relevant for studying early-phase tumorigenesis in vivo . Although extensive biochemical and biomedical research has focused on investigating tumor energy metabolism using spheroidal cultures of tumor cell lines 19 , little attention has been paid to the direct monitoring of metabolic transitions in tiny spheroids 20 – 23 . Indeed, the potential of such smaller spheroids to serve as mimetics of early tumorigenesis remains largely unexplored. If specific physiological or metabolic characteristics of these tiny spheroids could be directly monitored, they might serve as valuable diagnostic biomarkers for detecting early tumorigenesis in vivo 24 . In this study, hyperpolarized [1- 13 C]pyruvate NMR spectroscopy was adapted to directly monitor the conversion of [1- 13 C]pyruvate to [1- 13 C]lactate in tiny multicellular tumor spheroids of murine squamous cell carcinoma (SCC), serving as a model for early tumorigenesis in vivo . The metabolic properties of tumors can vary depending on the cell line and culture format. To evaluate the broader applicability of this approach, additional experiments were conducted using human prostate-specific antigen (PSA)-negative prostate tumor models, which exhibit distinct metabolic characteristics and are under investigation in clinical trials of hyperpolarized [1- 13 C]pyruvate MRI for malignant diagnosis 12 . 2. Results 2.1 Evaluation of the multicellular spheroidal culture of SCCVII cells Multicellular spheroids of murine SCCVII tumor cells were generated using a static culture method with a spheroid culture dish (EZSPHERE™, Iwaki). The growth characteristics of these spheroids were compared to those of SCCVII cells cultured conventionally. Figure 2 A presents a representative microscopic image of SCCVII spheroids and conventionally cultured cells grown in a 10-cm dish over the indicated period. By day 3 after initiating the culture with 5 × 10 4 cells/ml, the conventionally cultured cells became > 90% confluent. In contrast, SCCVII cells cultured in the spheroid dish formed multicellular aggregates at the bottom of the wells by day 7, which then showed a slight reduction in size by day 10 (Fig. 2 B, Fig. S1 C ). The median internal diameter (i.d.) of the multicellular aggregates on day 7 was approximately 150 µm. Histological analysis revealed a radial organization characteristic of typical tumor spheroids. The outer layer of the aggregates consisted of Ki-67-positive proliferating SCCVII cells, whereas Ki-67-negative, non-proliferating cells were present in the internal core (Fig. 2 C). Notably, for most spheroids with an i.d. of approximately 150 µm, no pimonidazole (pimo)-positive hypoxic regions (Fig. 2 C, green) or H&E-stained necrotic areas (Fig. 2 D) were observed. These findings suggest that the SCCVII spheroids closely mimic the structural and physiological features of early-phase in vivo tumors, characterized by limited malignant transformation. The chemosensitivity of SCCVII spheroids was evaluated using an MTS assay. Compared to standard SCCVII cells, the spheroids exhibited resistance to nearly all tested drugs (Table 1 , Fig. S2A, B ). Notably, SCCVII spheroids showed the highest resistance to docetaxel, a mitotic inhibitor. This observation suggests that the multicellular spheroid culture reduces the proliferation of many SCCVII cells, particularly those in the inner core (Fig. 2 C) 25 . Similarly, the spheroids demonstrated strong resistance to the topoisomerase inhibitor irinotecan, moderate resistance to the thymidylate synthase (TS) inhibitor 5-fluorouracil (5-FU), and significant resistance to the multi-targeted receptor tyrosine kinase (RTK) inhibitor sunitinib. These findings can be attributed to tissue-like alterations in the physiological characteristics of cells within the spheroids 16 . Tirapazamine, a hypoxia-activated prodrug known to be potentiated under tumor hypoxia 26 , was also tested. Interestingly, the SCCVII spheroids displayed increased resistance to tirapazamine compared to standard SCCVII cells, indicating the absence of hypoxic regions in the inner core of these tiny spheroids. There was no significant difference in the IC 50 values between monolayer and spheroidal SCCVII cells for the lactate dehydrogenase A (LDHA) inhibitor FX11, as assessed by the MTS assay 27 , 28 . However, the IC 90 value was higher in SCCVII spheroids (Table 1 ), suggesting a subtle but notable increase in aerobic glycolysis. This slight metabolic alteration was more clearly detected using HP [1- 13 C]pyruvate NMR spectroscopy. As shown in Fig. 2 E, the [1- 13 C]lactate signal (182.4 ppm), a metabolite of HP[1- 13 C]pyruvate (170.2 ppm), was significantly elevated in SCCVII spheroids compared to normally cultured SCCVII cells (Fig. 2 F). Both lactate production and the lactate-to-pyruvate ratio (Lac/Pyr) were higher in spheroids than in monolayer cultures (Fig. 2 G, H), indicating enhanced aerobic glycolysis in SCCVII spheroids. Moreover, the conversion rate from pyruvate to lactate was faster in the spheroids than in monolayers (Fig. 2 I). In contrast, mitochondrial activity in SCCVII spheroids was reduced by 51% compared to SCCVII cells from monolayer cultures (Fig. 2 J), suggesting decreased mitochondrial oxidative metabolism. Collectively, these findings demonstrate a significant metabolic shift characteristic of tumor energy metabolism, known as the Warburg effect, in SCCVII spheroids. Notably, this metabolic reprogramming was amplified even in spheroids with a median i.d. of only 150 µm and without evidence of malignant transformation 29 , 30 . Furthermore, HP [1- 13 C]pyruvate NMR spectroscopy enabled the detection of early increase in aerobic glycolysis in smaller tumor cell aggregates, a feature that is otherwise nearly undetectable using conventional methods. This highlights the potential of HP [1- 13 C]pyruvate NMR spectroscopy as a sensitive tool for monitoring early metabolic changes preceding malignant transformation. Table 1. IC 50 and IC 90 values (μM) of anti-tumor drugs for monolayer and spheroid-cultured cells in a 96-well format. Each cell culture was performed at 37°C in an atmosphere containing 5% CO 2 , and treated with various concentrations of drugs for 48 hours. Cell viability was assessed using the MTS assay reagent (Promega). Data were analyzed using a nonlinear regression curve-fitting package in Prism 4 (GraphPad Software Inc.). The drugs tested included: docetaxel, a mitotic inhibitor; irinotecan, a topoisomerase I inhibitor; 5-fluorouracil (5-FU), a thymidylate synthase inhibitor; sunitinib, a multi-receptor tyrosine kinase (RTK) inhibitor; tirapazamine, a hypoxia-activated prodrug; and FX11, a lactate dehydrogenase inhibitor. 2.2 HP [1- 13 C]pyruvate NMR spectroscopy for human prostate tumor spheroids To further validate the applicability of the HP [1- 13 C]pyruvate NMR approach in conjunction with spheroidal culture, analyses were conducted using different human prostate tumor cell lines. Specifically, PSA-negative prostate tumor cells, DU145 and PC-3, both characterized by aggressive metastatic potential and distinct levels of mitochondrial activity ( Fig. S1 B ), were employed 10 , 31 . A suspension of 5 × 10 4 cells/ ml for each cell type was seeded into a 10-cm spheroid culture dish, and the growth of multicellular spheroids was monitored over a 10-day period. DU145 cells formed relatively uniform spheroids, achieving a median i.d. of 88 µm by day 3 and increasing to 114 µm by day 10 (Fig. 3 A). In contrast, PC-3 cells exhibited a heterogeneous growth pattern, forming sparse aggregates with i.d. values ranging from 150 to 300 µm between days 3 and 10 (Fig. 3 B). These differences in growth morphology likely reflect intrinsic variances in cell-cell adhesion and metabolic activity between the two cell lines. Notably, both DU145 and PC-3 spheroids demonstrated a marked resistance to most of the anti-tumor drugs tested, compared to their respective monolayer cultures (Table 1 ). This observation aligns with previous studies suggesting that the three-dimensional architecture of spheroidal cultures confers a tissue-like environment that reduces drug sensitivity, mimicking the in vivo tumor microenvironment 19 , 25 , 32 . To assess the impact of culture format and cell type on the expression levels of genes related to glucose metabolism and pyruvate transport, qRT-PCR was performed. As shown in Fig. 3 C, Ki-67 expression decreased to 90.3 ± 1.1% (n = 3, P < 0.05) in DU145 spheroids, consistent with the increase in quiescent cells observed in SCCVII spheroids (Fig. 2 C). The expression levels of LDHA and ALT1 , both encoding enzymes that utilize pyruvate as a substrate ( Fig. 1 ), were significantly elevated in DU145 spheroids 10 days after culture initiation [ LDHA : 3.5 ± 0.1-fold (n = 3, P < 0.001); ALT1 : 2.8 ± 0.3-fold (n = 3, P < 0.05)]. In contrast, the expression of the glucose transporter GLUT1 remained unchanged between monolayered and spheroidal DU145 cells, likely reflecting the intrinsically aggressive nature of these prostate tumor cells. By contrast, the expression of the monocarboxylate transporter genes MCT1 and MCT4 , which mediate the cellular transport of pyruvate and lactate ( Fig. 1A ), increased significantly in the spheroids [ MCT1 : 2.2 ± 0.2-fold (n = 3, P < 0.001), MCT4 : 1.9 ± 0.5-fold (n = 3, P < 0.01)]. These findings suggest enhanced lactate production from pyruvate, along with increased pyruvate and lactate transport in DU145 spheroids compared to monolayer cultures. Similarly, in PC-3 spheroids, Ki-67 expression was reduced to 62.3 ± 1.6% (n = 3, P < 0.001) 10 days after spheroids formation (Fig. 3 D). Notably, unlike DU145, GLUT1 expression in PC-3 spheroids was significantly upregulated by 2.92 ± 0.1-fold (n = 3, P < 0.001) compared to monolayer PC-3 cells. This finding indicates an increased glucose uptake rate, as GLUT1 expression in monolayer PC-3 cells was only 46% of that observed in monolayer DU145 cells. In addition, the expression levels of LDHA , ALT1 , MCT1 , and MCT4 also increased in PC-3 spheroids after spheroid formation [ LDHA : 1.3 ± 0.1-fold (n = 3, P < 0.001); ALT1 : 1.5 ± 0.1-fold (n = 3, P < 0.05); MCT1 : 1.65 ± 0.04-fold (n = 3, P < 0.001); MCT4 : 2.2 ± 0.4-fold (n = 3, P < 0.05)]. These results indicate that PC-3 spheroids exhibit amplified energy metabolism transition and increased lactate flux compared to monolayer cultures. Following these results, HP [1- 13 C]pyruvate NMR spectroscopy was performed to directly monitor the enhanced lactate signal in both types of spheroid. Consistent with the qRT-PCR findings, HP [1- 13 C]lactate production from HP [1- 13 C]pyruvate significantly increased in DU145 spheroids (Fig. 4 A) compared to monolayer cultures of DU145 (> 90% confluent) (Fig. 4 C). Specifically, the Lac/Pyr ratio in DU145 spheroids was 3.24 × 10 − 3 (n = 3), compared to 0.33 × 10 − 3 (n = 3) in monolayer DU145 cells, representing a 9.8-fold increase (n = 3, P < 0.01, Fig. 4 E). Concurrently, mitochondrial activity decreased to 42% (n = 5–6, P < 0.001) in DU145 spheroids (Fig. 4 E), indicating a pronounced amplification of the Warburg effect. Similarly, an increase in lactate production was noninvasively detected in PC-3 spheroids (Fig. 4 B) compared to monolayer PC-3 cells (Fig. 4 D), though the effect was less pronounced than in DU145 spheroids (Fig. 4 A). The Lac/Pyr ratio in PC-3 spheroids was 2.12 × 10 − 3 (n = 4) compared to 0.23 × 10 − 3 (n = 3) in monolayer PC-3 cells, representing a 9.2-fold increase (n = 3–4, P < 0.01, Fig. 4 F). Interestingly, unlike DU145 spheroids, PC-3 spheroids showed a slight increase in mitochondrial activity compared to monolayer PC-3 cells (Fig. 4 F), consistent with the metabolic profile of docetaxel-resistant PC-3 cell lines 33 . Taken together, these results suggest that HP [1- 13 C]pyruvate NMR spectroscopy can noninvasively detect the enhanced aerobic glycolysis associated with upregulated expression of glycolysis-related genes in tiny spheroids. Moreover, this method effectively distinguishes metabolic differences between cell types, providing a valuable tool for studying the metabolic characteristics of tumor cells in three-dimensional culture systems. Figure 5 presents the Lac/Pyr ratio obtained from multicellular spheroids at their maximum size ( Fig. S1 C ) or biopsy samples from malignant tumors grown on mice ( Fig. S3 ). These values were calculated from HP [1- 13 C]pyruvate NMR experiments, carefully accounting for sample weight and HP probe concentration. The Lac/Pyr ratio varied by cell type, ranking from highest to lowest as DU145, PC-3 and SCCVII, respectively. Notably, the Lac/Pyr ratios derived from biopsy homogenates of tumor xenografts closely correlated with those from in vitro multicellular spheroids of the corresponding cell lines (Fig. 5 ). These findings suggest that the energy metabolism transition observed in tumor xenografts in mice can be partially replicated by in vitro spheroidal culture of their respective cell lines. 3. Discussion 18 F-FDG-PET is widely recognized as a valuable tool for detecting tumors in clinical practice, particularly in the early phases of disease and during post-treatment monitoring. However, the reliance on this imaging technique is accompanied by inherent limitations. Specifically, 18 F-FDG-PET exposures normal tissues and organs to ionizing radiation, which restricts the frequency of its application for individual patients due to safety concerns. In addition, the diagnostic utility of 18 F-FDG-PET has been questioned in certain tumor types. For instance, in canine squamous cell carcinoma, 18 F-FDG-PET failed to consistently reflect the Warburg effect―a hallmark of tumor metabolism―and demonstrated mismatches between 18 F-FDG signal accumulation and metabolically active regions 34 . These discrepancies highlight the need for alternative approaches that provide more direct and detailed insights into tumor energy metabolism. To improve tumor phenotyping and enable the detection of early-phase tumorigenesis in vivo , a deeper understanding of the metabolic transitions specific to tumors, particularly those in small, early-stage lesions, is essential. This need is underscored by the increasing recognition that metabolic shifts, such as the Warburg effect, occur early during tumor development and may serve as valuable biomarkers for early detection. In this study, we directly observed a significant increase in lactate production from pyruvate in tumor cells cultured as spheroids, using HP [1- 13 C]pyruvate NMR spectroscopy. The changes in 13 C chemical shifts allowed for real-time detection of increased HP [1- 13 C]lactate production from HP [1- 13 C]pyruvate in multicellular spheroids. Furthermore, differences in the metabolic transition ( i.e. the Warburg effect) between two human PSA-negative prostate tumor spheroids (DU145 and PC-3) were clearly distinguished using HP [1- 13 C]pyruvate NMR spectroscopy (Fig. 4 A, B). These differences were consistent with variations in the Lac/Pyr ratios derived from biopsy sample homogenates of larger tumors of the respective cell lines grown in mice (Fig. 5 ). These findings have significant implications for both the diagnosis and management of PSA-negative prostate tumors with metastatic potential. Specifically, HP [1- 13 C]pyruvate NMR spectroscopy could serve as a powerful tool for assessing therapeutic response prior to treatment and for developing specific MR imaging biomarkers for PSA-negative prostate tumors, particularly in the early stages of disease or after metastasis in vivo 14 , 35 , 36 . Importantly, our results demonstrated that amplification in aerobic glycolysis is significant even in smaller spheroids (< 300 µm) formed by 3D aggregation or proliferation of tumor cells, despite the absence of tumor vasculature, extracellular matrix, or malignant transformations (Fig. 2 C, D). Tumor progression is characterized by a heterogeneous tumor microenvironment, including pathological microvessel formation driven by vascular growth factors or extracellular matrix-mediated signaling, the development of hypoxic or necrotic regions, disruptions in redox signaling, exessive lactate production, and the resulting acidification of tissue pH, which promotes tissue invasion and metastasis. The relationship between these physiological phenomena is highly complex and varies among tumors. However, as shown in this study, the promotion of aerobic glycolysis appears to precede hypoxia formation, angiogenesis, and other extracellular events associated with tumor malignant progression in vivo . Further investigation is warranted to explore the mechanistic basis of these transitions and their potential as diagnostic or therapeutic targets. In conclusion, this study directly demonstrated the transition of tumor energy metabolism in tiny multicellular spheroids using HP [1- 13 C]pyruvate NMR spectroscopy on an NMR apparatus. This approach enables the investigation of tumor energy metabolism in a widely accessible chemical laboratory setting, without the need for MRI scanners or animal tumor models. Additionally, this method provides a straightforward platform for evaluating the HP lifetime and metabolic functionality of newly developed HP probes in biological environments. Looking forward, the clinical implementation of more advanced polarizers and MRI systems with improved resolution could make it feasible to detect early tumorigenesis or small metastatic tumors using HP metabolic imaging with [1- 13 C]pyruvate or other functional probes. Furthermore, the integration of HP NMR spectroscopy with cellular engineering has the potential to accelerate the development of novel functional HP probes and facilitate in vitro and ex vivo metabolic studies, offering new insights into tumor biology and metabolic reprogramming. 4. Materials and methods 4.1 Chemicals Docetaxel and Irinotecan were purchased from Wako Chemicals Inc. (Tokyo, Japan). Tirapazamine was obtained from LKT Laboratories Inc. (St Paul, MN). Sunitinib was obtained from Selleck Chemicals LLC (Houston, TX). 5-Fluorouracil was purchased from Nacalai Tesque (Kyoto, Japan). FX11 was obtained from Calbiochem (Temecula, CA). The chemical structure of these compounds is shown in Fig. S2A . [1- 13 C]pyruvic acid was purchased from Cambridge Isotope Laboratories, Inc. (Tewksbury, MA). OX063 was obtained from Oxford Instruments Co. Ltd. (Abingdon, UK) and Polarized IVS Co. Ltd. (Lyngby, Denmark). 4.2 Normal and spheroidal cultures of tumor cell lines Murine SCCVII cell line was kindly provided from Dr. O. Inanami, Hokkaido University 37 . The two androgen-independent human prostate cancer cell lines DU145 (RBRC-RCB2143) and PC-3 (RBRC-RCB2145) were obtained from RIKEN Bioresource center (BRC), Japan. All cells were cultured in 10-cm normal culture dishes and maintained in RPMI1640 supplemented with 10% fetal bovine serum and antibiotics. The cells were maintained in a humidified chamber at 37°C containing 5% CO 2 . Static spheroidal cultures of tumor cell lines were conducted using 10-cm or 96-well formatted EZSPHERE™ (IWAKI) plates. Cells (5 × 10 4 /ml) were transferred to a 10-cm dish (10 ml) or 96 well (100 µl) and maintained in a humidified chamber at 37°C containing 5% CO 2 . Digital images of the spheroids in wells were captured at ×4 magnification using a BZ-9000 microscope (Keyence, Osaka, Japan). The internal diameters (i.d.) of the spheroids were measured using ImageJ software ( https://imagej.nih.gov/ij/ ). The morphological figure of each spheroid was captured at ×20 magnification. 4.3 Cell viability assay The viability of cells exposed to chemotherapeutics was assessed using the 96-well aqueous cell proliferation (MTS) assay (Promega, Madison, WI). Cells (5 × 10 3 /100 µl/well) were plated onto a 96-well format microplate and aliquots (100 µl) of various concentrations of drug sample (RPMI 1640–2% DMSO) were added to each well (final level of DMSO 1%). After 48 h incubation at 37°C in an atmosphere containing 5% CO 2 , medium was exchanged to fresh RPMI1640 (100 µl). A 20 µl aliquot of MTS assay reagent was then added and the culture was allowed to continue. Optical density (OD) at 490 nm was measured every 20 min using a Varioskan® Flash spectral scanning reader (Thermo Fisher Scientific, Pittsburgh, PA). The drug concentration resulting in 50% or 90% growth inhibition (IC 50 , IC 90 ) was calculated using a nonlinear regression curve fitting package (sigmoidal dose-response) in Prism 4 (GraphPad Software Inc., La Jolla, CA). For the chemosensitivity test of spheroids, cells (5 × 10 3 /100 µl/well) were plated onto a 96-well format spheroid culture plate (EZSPHERE™, Iwaki) and incubated for the indicated number of days. After exchanging the medium to 100 µl of fresh RPMI1640, a 100 µl aliquot of various concentrations of drug sample (RPMI1640–2% DMSO) was added to each well (final level of DMSO 1%) and the plate was incubated for a further 48 h at 37°C in an atmosphere containing 5% CO 2 . Cell viability was determined as described above. 4.4 qRT-PCR RNA was extracted from human prostate tumor cells (day 4) or spheroids (day 10) using Isogen (Wako Chemicals Inc.). cDNA was synthesized using SuperScript™ III Reverse Transcriptase (Invitrogen, Carlsbad, CA). Quantitative real time PCR (qRT-PCR) was performed in triplicate using TaqMan® Universal Master Mix II (Applied Biosystems, Foster City, CA) with the StepOne™ real time PCR system (Applied Biosystems). Specific primers were used for Ki-67 (gene: MKI67 in GeneCards) (Assay ID in TaqMan™ Assays and Arrays, Thermo Fisher Scientific: Hs01032443_m1), GLUT1 (gene: SLC2A1 ) (Assay ID: Hs00892681_m1), MCT1 (gene: SLC16A1 ) (Assay ID: Hs01560299_m1), MCT4 ( SLC16A4 ) (Assay ID: Hs01006127_m1), LDHA (gene: LDHA ) (Assay ID: Hs01378790_g1), ALT1 (gene: GPT ) (Assay ID: Hs01548671_g1), and β-actin ( ACTB ) (Assay ID: Hs99999903_m1) (Applied Biosystems). 4.5 Hyperpolarization (HP) of [1- 13 C]pyruvate and 13 C NMR measurement In Figs. 4 and 5 and Fig. S3 , the HP of [1- 13 C]pyruvate was conducted using HyperSense™ (Oxford Instruments, Co. Ltd.) according to the manufacturer’s instructions. Briefly, a 30 µl aliquot of [1- 13 C]pyruvic acid (14.2 M) doped with 15 mM OX063 was placed in a sample cup. The sample cup was then set into the bore unit of the HyperSense™ instrument (3.35 T, 1.4 K, 2.8 mbar) using a sample insertion rod. The frozen sample was then polarized for 50–60 min by microwave radiation (100 mW) at 93.96 GHz. During the polarization, the 13 C NMR signal intensity was recorded every 5 min with the built-in solid-state polarimeter using RINMR software (Oxford Instruments, Co. Ltd.). After polarization, the sample was rapidly dissolved in 4.5 ml of dissolution buffer (100 mM Tris, 100 mg/L EDTA) preheated to 10 bar (~ 459 K), which warmed the sample to biological temperature (308 ~ 313 K, pH 7). The resulting solution was received in a 50-ml reservoir and quickly transferred to a 10-mm NMR tube containing biological samples; 1-ml suspension of freshly harvested standard culture cells or spheroids (10 8 cells from 50 of 10-cm culture dishes), or 1 ml of tumor tissue homogenate (2 ml/g tumor)]. After suspending the samples quickly, the NMR tube was placed in a Japan Redox JXI-400Z spectrometer (9.4 T). Collection of the NMR spectra started 30 ~ 35 s after dissolution of HP [1- 13 C]pyruvate. Tetramethylsilane (TMS, 0 ppm) was used as the external standard for 13 C NMR (1- 13 C in pyruvate: 170.2 ppm, alanine: 176.9 ppm, pyruvate・H 2 O: 178.5 ppm, and lactate: 182.4 ppm) ( Fig. 1B ). The spectra were recorded using a flip angle of 5° and time of repetition (TR) of 2.5 sec. Resulting 13 C NMR spectra were processed using JEOL Delta NMR software v5.0.1 (JEOL Ltd., Tokyo, Japan). In Fig. 2 , the HP of [1- 13 C]pyruvate was conducted using SpinAligner (Polarize ApS, Denmark) according to the manufacturer’s instructions. Briefly, a 18 µl aliquot of [1- 13 C]pyruvic acid (14.2 M) doped with 25 mM OX063 was placed in a sample vial. The sample vial was then attached to a fluid path and set into the bore unit of the SpinAligner instrument (6.7 T, 1.25 K, 1.2 mbar). The frozen sample was then polarized for 60–80 min by microwave radiation (22 mW) at 187.770 GHz. During the polarization, the 13 C NMR signal intensity was recorded every 5 min with the built-in solid-state NMR spectrometer controlled by SPINit software (RS 2 D, France.). After polarization, the sample was rapidly dissolved in 3.2 ml of dissolution buffer (40 mM Tris, 50 mM NaCl, 80 mM NaOH, 100 mg/L EDTA) preheated to 12 bar (~ 461 K), which warmed the sample to biological temperature (308 ~ 313 K, pH 7). The resulting solution was received in a 50-ml reservoir, and 50 µL of the HP [1- 13 C]pyruvate solution was quickly transferred to a micro tube containing 550 µL suspension of cultured SCC cells or SCC spheroids. After suspending the samples quickly, the suspension was transferred to a 5-mm NMR tube and the NMR tube was placed in a Spinsolve 60 spectrometer (1.5 T, Magritek, Germany). Acquisition of the NMR spectra was started 30 s after adding HP [1- 13 C]pyruvate to the cells or spheroids. The spectra were recorded using a flip angle of 5° and TR of 2.5 sec. Resulting 13 C NMR spectra were analyzed using MestReNova software v14.2.3 (Mestrelab Research S.L., Spain). 4.6 Histological analysis SCCVII spheroids were collected in 1.5-ml Eppendorf tubes and centrifuged. The pellet was suspended into 900 µl PBS and 100 µl of pimonidazole (1 mM) solution and incubated for 1 h at room temperature. After washing three times with PBS, spheroids were spread onto a glass slide and fixed with 3.7% formalin. After 30 min, the slide glass was washed three times with PBS and nonspecific binding sites on the spheroids were blocked with Protein Block Serum-Free reagent (Dako North America Inc., Carpinteria, CA) at 4°C overnight. After washing three times with PBS, the slides were submerged in a solution containing rabbit anti-Ki-67 antibody (Abcam®; 1:100) and incubated overnight at 4°C. The spheroids were incubated with an Alexa Fluor 546 F(ab’)2 fragment of goat anti-rabbit IgG (H + L) (Invitrogen/Life Sciences, Grand Island, NY; 1:2000) and Hypoxyprobe 4.3.11.3 mouse MAb (hpi; 1:250) for pimonidazole staining (1 h at room temperature). Samples were then mounted with Prolong Gold antifade reagent plus DAPI (Invitrogen). Fluorescence microscopy was performed using a FV1200 IX81 confocal microscope (Olympus, Tokyo, Japan), and images were captured using FLUOVIEW Ver.4.2a imaging software (Olympus). 4.7 Animal experiments All procedures were conducted in full compliance with the ARRIVE guidelines. Animal experiments ( ex vivo ) were performed according to the Institutional Guidance of Kyushu University on Animal Experimentation and this study obtained approval from the animal experiment committee of Kyushu University. Female 7-week old C3H/HeNJcl mice (17–20 g) and athymic BALB/cAJcl-nu/nu mice were purchased from Japan CLEA Japan Inc. (Kawasaki, Japan). A mouse SCCVII tumor was formed by injecting 1 × 10 6 SCCVII cells subcutaneously into the hind leg of C3H mice. Formation of human DU145 and PC-3 solid tumor xenografts were carried out by injecting 1 × 10 6 of each cell type subcutaneously into the hind leg of BALB mice. After tumor growth, the mice were anesthetized with 3% isoflurane (Pfizer Japan Inc., Tokyo, Japan) before being euthanized by exsanguination via transcardial perfusion with PBS, followed by ice-cold 4% paraformaldehyde (PFA) in PBS. Tumor tissues were excised and homogenized in PBS (2 ml/g tumor) prior to analysis by hyperpolarized [1- 13 C]pyruvate spectroscopy. 4.8 Statistical analysis All results were expressed as the mean ± SE. The differences in means of groups were determined by two-tailed Student’s t test. The minimum level of significance was set at P < 0.05. Declarations Acknowledgements We thank to Prof. Shinsuke Sando and Dr. Hiroshi Nonaka (The University of Tokyo) for discussion about this study. We acknowledge Dr. Tatsuya Naganuma (Japan Redox Co. Ltd.) and Mr. Shinya Goto (Oxford Instruments Co. Ltd.) for the technical assistance for the use of HyperSense TM , and Ms. Junko Yamaki (Fukushima Medical University School of Medicine) for assistance with the qRT-PCR experiments. We also thank Ms. Ayumi Koike and Yasuko Shimuta (National Institutes for Quantum Science and Technology) for their technical support for cell experiments. Author contributions Conceived and designed the experiments: YT. Performed the experiments: YT KT KIn KS. Analyzed the data: YT KT KIn KS KIc. Contributed reagents/materials/analysis tools: YT YH KIc. Contributed to the writing of the manuscript: YT. Competing interests The authors declare no competing interests. Funding This work was supported partly by JST FOREST Program [grant number JPMJFR225G (to Y.T.)]; JST CREST [grant number JPMJCR13L4]; MEXT Q-LEAP [grant number JPMXS0120330644 (to Y.T.)]; MEXT Promotion of Development of a Joint Usage/ Research System Project: Coalition of Universities for Research Excellence Program (CURE) [grant number JPMXP1323015488]; JSPS KAKENHI [grant number 23K27561 (to Y.T.), 23K19228 (to K.S.), 24K03317 (to K.I.), 15H03035 (to K.I.) and Research Grant from Fukushima Medical University. The use of HyperSense TM was in part supported by the funding program ‘Creation of Innovation Centers for Advanced Interdisciplinary Research Areas’ from JST. Data availability The datasets generated and/or analyzed during the current study are available upon reasonable request from the corresponding author. The gene sequences used in this study are available in GeneCards ® , the human gene database [https://www.genecards.org/]. 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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-5900705","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":408572355,"identity":"a809024b-f45b-4ad1-b873-07360f2a0738","order_by":0,"name":"Yoichi Takakusagi","email":"data:image/png;base64,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","orcid":"","institution":"National Institutes for Quantum Science and Technology (QST)","correspondingAuthor":true,"prefix":"","firstName":"Yoichi","middleName":"","lastName":"Takakusagi","suffix":""},{"id":408572356,"identity":"9f9b64cd-e649-40a7-9725-7f80773420ed","order_by":1,"name":"Kaori Takakusagi","email":"","orcid":"","institution":"Fukushima Medical University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Kaori","middleName":"","lastName":"Takakusagi","suffix":""},{"id":408572357,"identity":"16a6cfcb-e50d-467a-90f0-2b4a07557a36","order_by":2,"name":"Kaori Inoue","email":"","orcid":"","institution":"Kyushu University","correspondingAuthor":false,"prefix":"","firstName":"Kaori","middleName":"","lastName":"Inoue","suffix":""},{"id":408572358,"identity":"dd54394b-f306-4fee-b9a3-b16acadb68c5","order_by":3,"name":"Keita Saito","email":"","orcid":"","institution":"National Institutes for Quantum Science and Technology (QST)","correspondingAuthor":false,"prefix":"","firstName":"Keita","middleName":"","lastName":"Saito","suffix":""},{"id":408572359,"identity":"7ebe3abc-9d28-4225-bbd5-30fc56868b16","order_by":4,"name":"Yoshimi Homma","email":"","orcid":"","institution":"Fukushima Medical University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Yoshimi","middleName":"","lastName":"Homma","suffix":""},{"id":408572360,"identity":"bfc1bc02-60a5-49d2-8176-ce789fa1b571","order_by":5,"name":"Kazuhiro Ichikawa","email":"","orcid":"","institution":"Nagasaki International University","correspondingAuthor":false,"prefix":"","firstName":"Kazuhiro","middleName":"","lastName":"Ichikawa","suffix":""}],"badges":[],"createdAt":"2025-01-25 09:23:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5900705/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5900705/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-03454-1","type":"published","date":"2025-06-02T15:57:14+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":75163422,"identity":"161e18c8-5800-491e-abdf-fd6f43805119","added_by":"auto","created_at":"2025-01-31 12:52:12","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":776774,"visible":true,"origin":"","legend":"\u003cp\u003eMetabolic pathway and pyruvate cellular transport. \u003cstrong\u003eA\u003c/strong\u003e, Schematic representation of the glucose metabolic pathway and cellular transport of [1-\u003csup\u003e13\u003c/sup\u003eC]pyruvate. Key components include GLUT (glucose transporter), MCT (monocarboxylate transporter), LDH (lactate dehydrogenase), ALT (alanine transaminase), and the TCA (tricarboxylic acid) cycle. \u003cstrong\u003eB\u003c/strong\u003e, The chemical structure of [1-\u003csup\u003e13\u003c/sup\u003eC]pyruvate and its associated metabolites, along with the corresponding \u003csup\u003e13\u003c/sup\u003eC chemical shifts (ppm).\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-5900705/v1/dca4395da258c46e7049b3a8.png"},{"id":75164565,"identity":"3a977cda-78c9-43b1-9a1f-971389e6f9b7","added_by":"auto","created_at":"2025-01-31 13:00:12","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":448976,"visible":true,"origin":"","legend":"\u003cp\u003eCharacterization of murine SCCVII spheroids and detection of aerobic glycolysis using hyperpolarized (HP) [1-\u003csup\u003e13\u003c/sup\u003eC]pyruvate NMR spectroscopy (6.7 T SpinAligner polarizer, 1.5 T Spinsolve 60 benchtop NMR). \u003cstrong\u003eA\u003c/strong\u003e, Microscopic images comparing SCCVII spheroid cultures grown in spheroid culture dishes (EZSPHERE\u003csup\u003eTM\u003c/sup\u003e) and conventional monolayer cultures seeded at 5 × 10\u003csup\u003e4\u003c/sup\u003e cells/ml in 10-cm dishes (Day 0: ×10 magnification; Days 1-7: ×20 magnification). \u003cstrong\u003eB\u003c/strong\u003e, Growth kinetics of SCCVII spheroids, with spheroid size expressed as diameter (mm). Dashed lines indicate standard deviation (S.D.). \u003cstrong\u003eC\u003c/strong\u003e, Immunohistochemical analysis of SCCVII spheroids. Blue: DAPI nuclear stain; Red: Ki-67, indicating proliferating cells; Green: pimonidazole, marking hypoxic regions (\u0026lt;10 mmHg pO\u003csub\u003e2\u003c/sub\u003e). \u003cstrong\u003eD\u003c/strong\u003e, Hematoxylin and Eosin (H\u0026amp;E) staining of SCCVII spheroids, showing no necrotic core at this spheroid size. \u003cstrong\u003eE, F\u003c/strong\u003e, Serial \u003csup\u003e13\u003c/sup\u003eC NMR spectra of SCCVII spheroids (Day 7) (\u003cstrong\u003eE\u003c/strong\u003e) and monolayer cells (\u0026gt;90% confluent, Day 3) (\u003cstrong\u003eF\u003c/strong\u003e) acquired 30 seconds post-reaction with HP [1-\u003csup\u003e13\u003c/sup\u003eC]pyruvate (repetition time: 2.5 s; flip angle: 5°). \u003cstrong\u003eG\u003c/strong\u003e, Lactate signal intensity normalized by the maximum pyruvate signal in spheroids and monolayer cells. \u003cstrong\u003eH\u003c/strong\u003e, Lactate-to-pyruvate (Lac/Pyr) ratio calculated from the area under the curves (AUC) of lactate and pyruvate signals. \u003cstrong\u003eI\u003c/strong\u003e, Time course of Lac/Pyr ratio for monolayer cells and spheroids. \u003cstrong\u003eJ\u003c/strong\u003e, Mitochondrial activity measured by the conversion of MTS to formazan (absorbance at 490 nm). **P \u0026lt; 0.05 (\u003cem\u003en\u003c/em\u003e = 4 for spheroids; \u003cem\u003en\u003c/em\u003e = 6 for monolayer cells); ***P \u0026lt; 0.001 (\u003cem\u003en\u003c/em\u003e = 3).\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-5900705/v1/07c2e3f16ed53e21f02e885c.png"},{"id":75163424,"identity":"9ad740a0-baec-46d2-965a-dc6bf7b95cc9","added_by":"auto","created_at":"2025-01-31 12:52:12","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1580240,"visible":true,"origin":"","legend":"\u003cp\u003eGrowth characteristics of prostate tumor spheroids (DU145 and PC-3) and gene expression levels in monolayer and spheroid cultures. \u003cstrong\u003eA\u003c/strong\u003e, \u003cstrong\u003eB\u003c/strong\u003e, Frequency distribution of spheroid sizes during multicellular culture. Each cell type (DU145 in \u003cstrong\u003eA\u003c/strong\u003e and PC-3 in \u003cstrong\u003eB\u003c/strong\u003e) was seeded at 5 × 10\u003csup\u003e4\u003c/sup\u003e cells/ml (10 ml in a 10-cm dish), and spheroid sizes were measured at the indicated time points. The diameters of approximately 150 spheroids per condition were recorded, and spheroid size is presented as the median diameter (μm). Frequency (%) is shown for spheroid diameters grouped in 10-μm intervals. Black bars in the images represent 50 mm. \u003cstrong\u003eC\u003c/strong\u003e,\u003cstrong\u003e D\u003c/strong\u003e, Gene expression levels in DU145 (\u003cstrong\u003eC\u003c/strong\u003e) and PC-3 (\u003cstrong\u003eD\u003c/strong\u003e) cells measured by qRT-PCR. Samples were collected from monolayer cultures at Day 4 (\u0026gt;90% confluent, Mono) and spheroid cultures at Day 10 (Sphe). Gene expression levels were normalized to b-actin, and the relative expression levels were calculated as ratios to DU145 monolayer cells. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001 (\u003cem\u003en\u003c/em\u003e = 3).\u003c/p\u003e","description":"","filename":"imag3.png","url":"https://assets-eu.researchsquare.com/files/rs-5900705/v1/4279e170bec734c44ff9beb2.png"},{"id":75163426,"identity":"60711818-266e-46f8-a6bd-26eef216a12a","added_by":"auto","created_at":"2025-01-31 12:52:12","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":282028,"visible":true,"origin":"","legend":"\u003cp\u003eEnhanced aerobic glycolysis in tiny prostate tumor spheroids detected by hyperpolarized [1-\u003csup\u003e13\u003c/sup\u003eC]pyruvate NMR spectroscopy (3.35 T HyperSense\u003csup\u003eTM\u003c/sup\u003e polarizer, 9.4 T Japan Redox JXI-400Z spectrometer). \u003cstrong\u003eA\u003c/strong\u003e, \u003cstrong\u003eB\u003c/strong\u003e, Single-scan \u003csup\u003e13\u003c/sup\u003eC NMR spectra of DU145 (\u003cstrong\u003eA\u003c/strong\u003e) and PC-3 (\u003cstrong\u003eB\u003c/strong\u003e) tumor spheroids on Day 10. \u003cstrong\u003eC\u003c/strong\u003e, \u003cstrong\u003eD\u003c/strong\u003e, Single-scan \u003csup\u003e13\u003c/sup\u003eC NMR spectra of DU145 (\u003cstrong\u003eC\u003c/strong\u003e) and PC-3 (\u003cstrong\u003eD\u003c/strong\u003e) tumor cells cultured as monolayers on Day 4. \u003cstrong\u003eE\u003c/strong\u003e, \u003cstrong\u003eF\u003c/strong\u003e, Lac/Pyr ratio and mitochondrial activity in monolayer and spheroid cultures of DU145 (\u003cstrong\u003eE\u003c/strong\u003e) or PC-3 (\u003cstrong\u003eF\u003c/strong\u003e). **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001 (\u003cem\u003en\u003c/em\u003e = 3-4).\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-5900705/v1/6a2ffff56d5ac951a4bd335c.png"},{"id":75163427,"identity":"6ad792fe-935b-4a95-90c3-a0ccd5b7932a","added_by":"auto","created_at":"2025-01-31 12:52:12","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":194553,"visible":true,"origin":"","legend":"\u003cp\u003eCorrelation of the Lac/Pyr ratio between multicellular spheroids at maximum size and biopsy homogenates from each malignant tumors grown in mice. Lac/Pyr ratios were measured in multicellular spheroids and corresponding biopsy homogenates from tumor xenografts of DU145, PC-3, and SCCVII cells. DU145: spheroids on Day 10 (\u003cem\u003en\u003c/em\u003e = 3); biopsy homogenates from tumors of 800 mm\u003csup\u003e3\u003c/sup\u003e (\u003cem\u003en\u003c/em\u003e = 3). PC-3: spheroids on Day 10 (n = 4); biopsy homogenates from tumors of 700 mm\u003csup\u003e3\u003c/sup\u003e (\u003cem\u003en\u003c/em\u003e = 3). SCCVII: spheroids on Day 7 (n = 2); biopsy homogenates from tumors of 1000 mm\u003csup\u003e3\u003c/sup\u003e (\u003cem\u003en\u003c/em\u003e = 4). The dashed line represents the linear regression equation: y = 2.7027x + 0.0065 (r\u003csup\u003e2\u003c/sup\u003e = 0.975).\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-5900705/v1/b23ecf5792cd63a41389fb71.png"},{"id":84242689,"identity":"1383b373-9336-4a25-867a-7acfb9d3f12e","added_by":"auto","created_at":"2025-06-09 16:11:22","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4253383,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5900705/v1/424a8333-ee5f-4484-a2f5-b1e3395506af.pdf"},{"id":75163434,"identity":"da46f8d4-2b02-4576-aea1-0591a73be7d4","added_by":"auto","created_at":"2025-01-31 12:52:13","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":5672551,"visible":true,"origin":"","legend":"","description":"","filename":"2.SFSciRep.docx","url":"https://assets-eu.researchsquare.com/files/rs-5900705/v1/afc014bacebf658c31cbf408.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Hyperpolarized [1- 13 C]pyruvate NMR spectroscopy reveals transition of tumor energy metabolism in tiny multicellular spheroids","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eThe development of diagnostic systems and technologies for the early detection of tumorigenesis remains a significant challenge. Among the advancements in imaging, \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003eF-fluoro-2-deoxyglucose positron emission tomography (\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003eF-FDG-PET) has proven to be a major breakthrough in clinical applications, enabling the detection of tumors with an internal diameter of less than 1 mm\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. This diagnostic modality relies on the uptake and subsequent phosphorylation of the tracer by actively proliferating tumor cells, leading to its accumulation within these cells. However, while \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003eF-FDG-PET provides valuable insights about tumor location, it offers limited information regarding the metabolic profile of the detected tumors, leaving gaps in understanding their biochemical characteristics.\u003c/p\u003e \u003cp\u003eHyperpolarization (HP) is a powerful technique that enhances the sensitivity of nuclear magnetic resonance (NMR) by several orders of magnitude\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. This advancement enables noninvasive and quantitative assessment of metabolic processes in living systems using functional chemical probes labeled with non-radioactive nuclei, such as \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Unlike imaging modalities that rely on radionuclides as tracers, HP provides real-time information about structural alterations of probes during metabolism, detectable as changes in spin frequency or chemical shifts at specific locations of interest\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Additionally, this method allows for the simultaneous detection of multiple metabolites\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Among the HP chemical probes developed to date\u003csup\u003e\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e, \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC-labeled pyruvate ([1-\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC]pyruvate) is the most promising. This probe facilitates direct monitoring of aerobic glycolysis (\u003cb\u003eFig.\u0026nbsp;1\u003c/b\u003e), a process significantly elevated in tumors compared to normal tissues\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Furthermore, HP[1-\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC]pyruvate has been utilized as a noninvasive tool for assessing tumor malignancy and treatment responses in \u003cem\u003ein vivo\u003c/em\u003e tumor xenografts and in patients with prostate cancer\u003csup\u003e\u003cspan additionalcitationids=\"CR11 CR12 CR13 CR14\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe recent advancements in cell and tissue engineering have enabled the production of biological mimetics for analyzing tissue-like features in living systems, even \u003cem\u003ein vitro\u003c/em\u003e. For instance, similar to tumor xenografts grown in mice, three-dimensional spheroid cultures of tumor cells replicate tissue-like characteristics \u003cem\u003ein vitro\u003c/em\u003e. These characteristics include static cells in the inner core surrounded by a layer of actively proliferating cells, the development of hypoxic regions or necrotic cores, and reduced sensitivity to chemotherapeutic agents\u003csup\u003e\u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. The application of spheroids smaller than 1 mm in diameter is particularly relevant for studying early-phase tumorigenesis \u003cem\u003ein vivo\u003c/em\u003e. Although extensive biochemical and biomedical research has focused on investigating tumor energy metabolism using spheroidal cultures of tumor cell lines\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e, little attention has been paid to the direct monitoring of metabolic transitions in tiny spheroids\u003csup\u003e\u003cspan additionalcitationids=\"CR21 CR22\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Indeed, the potential of such smaller spheroids to serve as mimetics of early tumorigenesis remains largely unexplored. If specific physiological or metabolic characteristics of these tiny spheroids could be directly monitored, they might serve as valuable diagnostic biomarkers for detecting early tumorigenesis \u003cem\u003ein vivo\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn this study, hyperpolarized [1-\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC]pyruvate NMR spectroscopy was adapted to directly monitor the conversion of [1-\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC]pyruvate to [1-\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC]lactate in tiny multicellular tumor spheroids of murine squamous cell carcinoma (SCC), serving as a model for early tumorigenesis \u003cem\u003ein vivo\u003c/em\u003e. The metabolic properties of tumors can vary depending on the cell line and culture format. To evaluate the broader applicability of this approach, additional experiments were conducted using human prostate-specific antigen (PSA)-negative prostate tumor models, which exhibit distinct metabolic characteristics and are under investigation in clinical trials of hyperpolarized [1-\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC]pyruvate MRI for malignant diagnosis\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e"},{"header":"2. Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Evaluation of the multicellular spheroidal culture of SCCVII cells\u003c/h2\u003e \u003cp\u003eMulticellular spheroids of murine SCCVII tumor cells were generated using a static culture method with a spheroid culture dish (EZSPHERE\u0026trade;, Iwaki). The growth characteristics of these spheroids were compared to those of SCCVII cells cultured conventionally. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003eA presents a representative microscopic image of SCCVII spheroids and conventionally cultured cells grown in a 10-cm dish over the indicated period. By day 3 after initiating the culture with 5 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells/ml, the conventionally cultured cells became\u0026thinsp;\u0026gt;\u0026thinsp;90% confluent. In contrast, SCCVII cells cultured in the spheroid dish formed multicellular aggregates at the bottom of the wells by day 7, which then showed a slight reduction in size by day 10 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, \u003cb\u003eFig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eC\u003c/b\u003e). The median internal diameter (i.d.) of the multicellular aggregates on day 7 was approximately 150 \u0026micro;m. Histological analysis revealed a radial organization characteristic of typical tumor spheroids. The outer layer of the aggregates consisted of Ki-67-positive proliferating SCCVII cells, whereas Ki-67-negative, non-proliferating cells were present in the internal core (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Notably, for most spheroids with an i.d. of approximately 150 \u0026micro;m, no pimonidazole (pimo)-positive hypoxic regions (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, green) or H\u0026amp;E-stained necrotic areas (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003eD) were observed. These findings suggest that the SCCVII spheroids closely mimic the structural and physiological features of early-phase \u003cem\u003ein vivo\u003c/em\u003e tumors, characterized by limited malignant transformation.\u003c/p\u003e \u003cp\u003eThe chemosensitivity of SCCVII spheroids was evaluated using an MTS assay. Compared to standard SCCVII cells, the spheroids exhibited resistance to nearly all tested drugs (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, \u003cb\u003eFig. S2A, B\u003c/b\u003e). Notably, SCCVII spheroids showed the highest resistance to docetaxel, a mitotic inhibitor. This observation suggests that the multicellular spheroid culture reduces the proliferation of many SCCVII cells, particularly those in the inner core (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003eC)\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Similarly, the spheroids demonstrated strong resistance to the topoisomerase inhibitor irinotecan, moderate resistance to the thymidylate synthase (TS) inhibitor 5-fluorouracil (5-FU), and significant resistance to the multi-targeted receptor tyrosine kinase (RTK) inhibitor sunitinib. These findings can be attributed to tissue-like alterations in the physiological characteristics of cells within the spheroids\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Tirapazamine, a hypoxia-activated prodrug known to be potentiated under tumor hypoxia\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e, was also tested. Interestingly, the SCCVII spheroids displayed increased resistance to tirapazamine compared to standard SCCVII cells, indicating the absence of hypoxic regions in the inner core of these tiny spheroids.\u003c/p\u003e \u003cp\u003eThere was no significant difference in the IC\u003csub\u003e50\u003c/sub\u003e values between monolayer and spheroidal SCCVII cells for the lactate dehydrogenase A (LDHA) inhibitor FX11, as assessed by the MTS assay\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e,\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. However, the IC\u003csub\u003e90\u003c/sub\u003e value was higher in SCCVII spheroids (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), suggesting a subtle but notable increase in aerobic glycolysis. This slight metabolic alteration was more clearly detected using HP [1-\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC]pyruvate NMR spectroscopy. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003eE, the [1-\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC]lactate signal (182.4 ppm), a metabolite of HP[1-\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC]pyruvate (170.2 ppm), was significantly elevated in SCCVII spheroids compared to normally cultured SCCVII cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). Both lactate production and the lactate-to-pyruvate ratio (Lac/Pyr) were higher in spheroids than in monolayer cultures (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003eG, H), indicating enhanced aerobic glycolysis in SCCVII spheroids. Moreover, the conversion rate from pyruvate to lactate was faster in the spheroids than in monolayers (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003eI). In contrast, mitochondrial activity in SCCVII spheroids was reduced by 51% compared to SCCVII cells from monolayer cultures (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003eJ), suggesting decreased mitochondrial oxidative metabolism. Collectively, these findings demonstrate a significant metabolic shift characteristic of tumor energy metabolism, known as the Warburg effect, in SCCVII spheroids. Notably, this metabolic reprogramming was amplified even in spheroids with a median i.d. of only 150 \u0026micro;m and without evidence of malignant transformation\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e,\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. Furthermore, HP [1-\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC]pyruvate NMR spectroscopy enabled the detection of early increase in aerobic glycolysis in smaller tumor cell aggregates, a feature that is otherwise nearly undetectable using conventional methods. This highlights the potential of HP [1-\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC]pyruvate NMR spectroscopy as a sensitive tool for monitoring early metabolic changes preceding malignant transformation.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eTable 1.\u003c/strong\u003e\u0026nbsp; IC\u003csub\u003e50\u003c/sub\u003e and IC\u003csub\u003e90\u003c/sub\u003e values (\u0026mu;M) of anti-tumor drugs for monolayer and spheroid-cultured cells in a 96-well format.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cimg src=\"https://myfiles.space/user_files/127393_c7e80a1c9bb65875/127393_custom_files/img1738327622.png\"\u003e\u003c/strong\u003e\u003cbr\u003e\u003c/p\u003e\n \u003cp\u003eEach cell culture was performed at 37\u0026deg;C in an atmosphere containing 5% CO\u003csub\u003e2\u003c/sub\u003e, and treated with various concentrations of drugs for 48 hours. Cell viability was assessed using the MTS assay reagent (Promega). Data were analyzed using a nonlinear regression curve-fitting package in Prism 4 (GraphPad Software Inc.). The drugs tested included: docetaxel, a mitotic inhibitor; irinotecan, a topoisomerase I inhibitor; 5-fluorouracil (5-FU), a thymidylate synthase inhibitor; sunitinib, a multi-receptor tyrosine kinase (RTK) inhibitor; tirapazamine, a hypoxia-activated prodrug; and FX11, a lactate dehydrogenase inhibitor.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 HP [1-\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC]pyruvate NMR spectroscopy for human prostate tumor spheroids\u003c/h2\u003e \u003cp\u003eTo further validate the applicability of the HP [1-\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC]pyruvate NMR approach in conjunction with spheroidal culture, analyses were conducted using different human prostate tumor cell lines. Specifically, PSA-negative prostate tumor cells, DU145 and PC-3, both characterized by aggressive metastatic potential and distinct levels of mitochondrial activity (\u003cb\u003eFig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eB\u003c/b\u003e), were employed\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. A suspension of 5 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells/ ml for each cell type was seeded into a 10-cm spheroid culture dish, and the growth of multicellular spheroids was monitored over a 10-day period. DU145 cells formed relatively uniform spheroids, achieving a median i.d. of 88 \u0026micro;m by day 3 and increasing to 114 \u0026micro;m by day 10 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). In contrast, PC-3 cells exhibited a heterogeneous growth pattern, forming sparse aggregates with i.d. values ranging from 150 to 300 \u0026micro;m between days 3 and 10 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). These differences in growth morphology likely reflect intrinsic variances in cell-cell adhesion and metabolic activity between the two cell lines. Notably, both DU145 and PC-3 spheroids demonstrated a marked resistance to most of the anti-tumor drugs tested, compared to their respective monolayer cultures (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). This observation aligns with previous studies suggesting that the three-dimensional architecture of spheroidal cultures confers a tissue-like environment that reduces drug sensitivity, mimicking the \u003cem\u003ein vivo\u003c/em\u003e tumor microenvironment\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e,\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eTo assess the impact of culture format and cell type on the expression levels of genes related to glucose metabolism and pyruvate transport, qRT-PCR was performed. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, Ki-67 expression decreased to 90.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1% (n\u0026thinsp;=\u0026thinsp;3, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in DU145 spheroids, consistent with the increase in quiescent cells observed in SCCVII spheroids (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). The expression levels of \u003cem\u003eLDHA\u003c/em\u003e and \u003cem\u003eALT1\u003c/em\u003e, both encoding enzymes that utilize pyruvate as a substrate (\u003cb\u003eFig.\u0026nbsp;1\u003c/b\u003e), were significantly elevated in DU145 spheroids 10 days after culture initiation [\u003cem\u003eLDHA\u003c/em\u003e: 3.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1-fold (n\u0026thinsp;=\u0026thinsp;3, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001); \u003cem\u003eALT1\u003c/em\u003e: 2.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3-fold (n\u0026thinsp;=\u0026thinsp;3, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05)]. In contrast, the expression of the glucose transporter GLUT1 remained unchanged between monolayered and spheroidal DU145 cells, likely reflecting the intrinsically aggressive nature of these prostate tumor cells. By contrast, the expression of the monocarboxylate transporter genes \u003cem\u003eMCT1\u003c/em\u003e and \u003cem\u003eMCT4\u003c/em\u003e, which mediate the cellular transport of pyruvate and lactate (\u003cb\u003eFig.\u0026nbsp;1A\u003c/b\u003e), increased significantly in the spheroids [\u003cem\u003eMCT1\u003c/em\u003e: 2.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2-fold (n\u0026thinsp;=\u0026thinsp;3, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), \u003cem\u003eMCT4\u003c/em\u003e: 1.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5-fold (n\u0026thinsp;=\u0026thinsp;3, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01)]. These findings suggest enhanced lactate production from pyruvate, along with increased pyruvate and lactate transport in DU145 spheroids compared to monolayer cultures.\u003c/p\u003e \u003cp\u003eSimilarly, in PC-3 spheroids, \u003cem\u003eKi-67\u003c/em\u003e expression was reduced to 62.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.6% (n\u0026thinsp;=\u0026thinsp;3, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) 10 days after spheroids formation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). Notably, unlike DU145, \u003cem\u003eGLUT1\u003c/em\u003e expression in PC-3 spheroids was significantly upregulated by 2.92\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1-fold (n\u0026thinsp;=\u0026thinsp;3, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) compared to monolayer PC-3 cells. This finding indicates an increased glucose uptake rate, as \u003cem\u003eGLUT1\u003c/em\u003e expression in monolayer PC-3 cells was only 46% of that observed in monolayer DU145 cells. In addition, the expression levels of \u003cem\u003eLDHA\u003c/em\u003e, \u003cem\u003eALT1\u003c/em\u003e, \u003cem\u003eMCT1\u003c/em\u003e, and \u003cem\u003eMCT4\u003c/em\u003e also increased in PC-3 spheroids after spheroid formation [\u003cem\u003eLDHA\u003c/em\u003e: 1.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1-fold (n\u0026thinsp;=\u0026thinsp;3, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001); \u003cem\u003eALT1\u003c/em\u003e: 1.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1-fold (n\u0026thinsp;=\u0026thinsp;3, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05); \u003cem\u003eMCT1\u003c/em\u003e: 1.65\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04-fold (n\u0026thinsp;=\u0026thinsp;3, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001); \u003cem\u003eMCT4\u003c/em\u003e: 2.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4-fold (n\u0026thinsp;=\u0026thinsp;3, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05)]. These results indicate that PC-3 spheroids exhibit amplified energy metabolism transition and increased lactate flux compared to monolayer cultures.\u003c/p\u003e \u003cp\u003eFollowing these results, HP [1-\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC]pyruvate NMR spectroscopy was performed to directly monitor the enhanced lactate signal in both types of spheroid. Consistent with the qRT-PCR findings, HP [1-\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC]lactate production from HP [1-\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC]pyruvate significantly increased in DU145 spheroids (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eA) compared to monolayer cultures of DU145 (\u0026gt;\u0026thinsp;90% confluent) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Specifically, the Lac/Pyr ratio in DU145 spheroids was 3.24 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e (n\u0026thinsp;=\u0026thinsp;3), compared to 0.33 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e (n\u0026thinsp;=\u0026thinsp;3) in monolayer DU145 cells, representing a 9.8-fold increase (n\u0026thinsp;=\u0026thinsp;3, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). Concurrently, mitochondrial activity decreased to 42% (n\u0026thinsp;=\u0026thinsp;5\u0026ndash;6, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) in DU145 spheroids (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eE), indicating a pronounced amplification of the Warburg effect.\u003c/p\u003e \u003cp\u003eSimilarly, an increase in lactate production was noninvasively detected in PC-3 spheroids (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eB) compared to monolayer PC-3 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eD), though the effect was less pronounced than in DU145 spheroids (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). The Lac/Pyr ratio in PC-3 spheroids was 2.12 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e (n\u0026thinsp;=\u0026thinsp;4) compared to 0.23 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e (n\u0026thinsp;=\u0026thinsp;3) in monolayer PC-3 cells, representing a 9.2-fold increase (n\u0026thinsp;=\u0026thinsp;3\u0026ndash;4, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eF). Interestingly, unlike DU145 spheroids, PC-3 spheroids showed a slight increase in mitochondrial activity compared to monolayer PC-3 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eF), consistent with the metabolic profile of docetaxel-resistant PC-3 cell lines\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eTaken together, these results suggest that HP [1-\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC]pyruvate NMR spectroscopy can noninvasively detect the enhanced aerobic glycolysis associated with upregulated expression of glycolysis-related genes in tiny spheroids. Moreover, this method effectively distinguishes metabolic differences between cell types, providing a valuable tool for studying the metabolic characteristics of tumor cells in three-dimensional culture systems.\u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003e presents the Lac/Pyr ratio obtained from multicellular spheroids at their maximum size (\u003cb\u003eFig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eC\u003c/b\u003e) or biopsy samples from malignant tumors grown on mice (\u003cb\u003eFig. S3\u003c/b\u003e). These values were calculated from HP [1-\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC]pyruvate NMR experiments, carefully accounting for sample weight and HP probe concentration. The Lac/Pyr ratio varied by cell type, ranking from highest to lowest as DU145, PC-3 and SCCVII, respectively. Notably, the Lac/Pyr ratios derived from biopsy homogenates of tumor xenografts closely correlated with those from \u003cem\u003ein vitro\u003c/em\u003e multicellular spheroids of the corresponding cell lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003e). These findings suggest that the energy metabolism transition observed in tumor xenografts in mice can be partially replicated by \u003cem\u003ein vitro\u003c/em\u003e spheroidal culture of their respective cell lines.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Discussion","content":"\u003cp\u003e \u003csup\u003e \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e \u003c/sup\u003eF-FDG-PET is widely recognized as a valuable tool for detecting tumors in clinical practice, particularly in the early phases of disease and during post-treatment monitoring. However, the reliance on this imaging technique is accompanied by inherent limitations. Specifically, \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003eF-FDG-PET exposures normal tissues and organs to ionizing radiation, which restricts the frequency of its application for individual patients due to safety concerns. In addition, the diagnostic utility of \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003eF-FDG-PET has been questioned in certain tumor types. For instance, in canine squamous cell carcinoma, \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003eF-FDG-PET failed to consistently reflect the Warburg effect―a hallmark of tumor metabolism―and demonstrated mismatches between \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003eF-FDG signal accumulation and metabolically active regions\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. These discrepancies highlight the need for alternative approaches that provide more direct and detailed insights into tumor energy metabolism. To improve tumor phenotyping and enable the detection of early-phase tumorigenesis \u003cem\u003ein vivo\u003c/em\u003e, a deeper understanding of the metabolic transitions specific to tumors, particularly those in small, early-stage lesions, is essential. This need is underscored by the increasing recognition that metabolic shifts, such as the Warburg effect, occur early during tumor development and may serve as valuable biomarkers for early detection.\u003c/p\u003e \u003cp\u003eIn this study, we directly observed a significant increase in lactate production from pyruvate in tumor cells cultured as spheroids, using HP [1-\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC]pyruvate NMR spectroscopy. The changes in \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC chemical shifts allowed for real-time detection of increased HP [1-\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC]lactate production from HP [1-\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC]pyruvate in multicellular spheroids. Furthermore, differences in the metabolic transition (\u003cem\u003ei.e.\u003c/em\u003e the Warburg effect) between two human PSA-negative prostate tumor spheroids (DU145 and PC-3) were clearly distinguished using HP [1-\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC]pyruvate NMR spectroscopy (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, B). These differences were consistent with variations in the Lac/Pyr ratios derived from biopsy sample homogenates of larger tumors of the respective cell lines grown in mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003e). These findings have significant implications for both the diagnosis and management of PSA-negative prostate tumors with metastatic potential. Specifically, HP [1-\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC]pyruvate NMR spectroscopy could serve as a powerful tool for assessing therapeutic response prior to treatment and for developing specific MR imaging biomarkers for PSA-negative prostate tumors, particularly in the early stages of disease or after metastasis \u003cem\u003ein vivo\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e,\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eImportantly, our results demonstrated that amplification in aerobic glycolysis is significant even in smaller spheroids (\u0026lt;\u0026thinsp;300 \u0026micro;m) formed by 3D aggregation or proliferation of tumor cells, despite the absence of tumor vasculature, extracellular matrix, or malignant transformations (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, D). Tumor progression is characterized by a heterogeneous tumor microenvironment, including pathological microvessel formation driven by vascular growth factors or extracellular matrix-mediated signaling, the development of hypoxic or necrotic regions, disruptions in redox signaling, exessive lactate production, and the resulting acidification of tissue pH, which promotes tissue invasion and metastasis. The relationship between these physiological phenomena is highly complex and varies among tumors. However, as shown in this study, the promotion of aerobic glycolysis appears to precede hypoxia formation, angiogenesis, and other extracellular events associated with tumor malignant progression \u003cem\u003ein vivo\u003c/em\u003e. Further investigation is warranted to explore the mechanistic basis of these transitions and their potential as diagnostic or therapeutic targets.\u003c/p\u003e \u003cp\u003eIn conclusion, this study directly demonstrated the transition of tumor energy metabolism in tiny multicellular spheroids using HP [1-\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC]pyruvate NMR spectroscopy on an NMR apparatus. This approach enables the investigation of tumor energy metabolism in a widely accessible chemical laboratory setting, without the need for MRI scanners or animal tumor models. Additionally, this method provides a straightforward platform for evaluating the HP lifetime and metabolic functionality of newly developed HP probes in biological environments. Looking forward, the clinical implementation of more advanced polarizers and MRI systems with improved resolution could make it feasible to detect early tumorigenesis or small metastatic tumors using HP metabolic imaging with [1-\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC]pyruvate or other functional probes. Furthermore, the integration of HP NMR spectroscopy with cellular engineering has the potential to accelerate the development of novel functional HP probes and facilitate \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003eex vivo\u003c/em\u003e metabolic studies, offering new insights into tumor biology and metabolic reprogramming.\u003c/p\u003e"},{"header":"4. Materials and methods","content":"\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e4.1 Chemicals\u003c/h2\u003e \u003cp\u003eDocetaxel and Irinotecan were purchased from Wako Chemicals Inc. (Tokyo, Japan). Tirapazamine was obtained from LKT Laboratories Inc. (St Paul, MN). Sunitinib was obtained from Selleck Chemicals LLC (Houston, TX). 5-Fluorouracil was purchased from Nacalai Tesque (Kyoto, Japan). FX11 was obtained from Calbiochem (Temecula, CA). The chemical structure of these compounds is shown in \u003cb\u003eFig. S2A\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e[1-\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC]pyruvic acid was purchased from Cambridge Isotope Laboratories, Inc. (Tewksbury, MA). OX063 was obtained from Oxford Instruments Co. Ltd. (Abingdon, UK) and Polarized IVS Co. Ltd. (Lyngby, Denmark).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e4.2 Normal and spheroidal cultures of tumor cell lines\u003c/h2\u003e \u003cp\u003eMurine SCCVII cell line was kindly provided from Dr. O. Inanami, Hokkaido University\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. The two androgen-independent human prostate cancer cell lines DU145 (RBRC-RCB2143) and PC-3 (RBRC-RCB2145) were obtained from RIKEN Bioresource center (BRC), Japan. All cells were cultured in 10-cm normal culture dishes and maintained in RPMI1640 supplemented with 10% fetal bovine serum and antibiotics. The cells were maintained in a humidified chamber at 37\u0026deg;C containing 5% CO\u003csub\u003e2\u003c/sub\u003e. Static spheroidal cultures of tumor cell lines were conducted using 10-cm or 96-well formatted EZSPHERE\u0026trade; (IWAKI) plates. Cells (5 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e/ml) were transferred to a 10-cm dish (10 ml) or 96 well (100 \u0026micro;l) and maintained in a humidified chamber at 37\u0026deg;C containing 5% CO\u003csub\u003e2\u003c/sub\u003e. Digital images of the spheroids in wells were captured at \u0026times;4 magnification using a BZ-9000 microscope (Keyence, Osaka, Japan). The internal diameters (i.d.) of the spheroids were measured using ImageJ software (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://imagej.nih.gov/ij/\u003c/span\u003e\u003cspan address=\"https://imagej.nih.gov/ij/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The morphological figure of each spheroid was captured at \u0026times;20 magnification.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e4.3 Cell viability assay\u003c/h2\u003e \u003cp\u003eThe viability of cells exposed to chemotherapeutics was assessed using the 96-well aqueous cell proliferation (MTS) assay (Promega, Madison, WI). Cells (5 \u0026times; 10\u003csup\u003e3\u003c/sup\u003e/100 \u0026micro;l/well) were plated onto a 96-well format microplate and aliquots (100 \u0026micro;l) of various concentrations of drug sample (RPMI 1640\u0026ndash;2% DMSO) were added to each well (final level of DMSO 1%). After 48 h incubation at 37\u0026deg;C in an atmosphere containing 5% CO\u003csub\u003e2\u003c/sub\u003e, medium was exchanged to fresh RPMI1640 (100 \u0026micro;l). A 20 \u0026micro;l aliquot of MTS assay reagent was then added and the culture was allowed to continue. Optical density (OD) at 490 nm was measured every 20 min using a Varioskan\u0026reg; Flash spectral scanning reader (Thermo Fisher Scientific, Pittsburgh, PA). The drug concentration resulting in 50% or 90% growth inhibition (IC\u003csub\u003e50\u003c/sub\u003e, IC\u003csub\u003e90\u003c/sub\u003e) was calculated using a nonlinear regression curve fitting package (sigmoidal dose-response) in Prism 4 (GraphPad Software Inc., La Jolla, CA).\u003c/p\u003e \u003cp\u003eFor the chemosensitivity test of spheroids, cells (5 \u0026times; 10\u003csup\u003e3\u003c/sup\u003e/100 \u0026micro;l/well) were plated onto a 96-well format spheroid culture plate (EZSPHERE\u0026trade;, Iwaki) and incubated for the indicated number of days. After exchanging the medium to 100 \u0026micro;l of fresh RPMI1640, a 100 \u0026micro;l aliquot of various concentrations of drug sample (RPMI1640\u0026ndash;2% DMSO) was added to each well (final level of DMSO 1%) and the plate was incubated for a further 48 h at 37\u0026deg;C in an atmosphere containing 5% CO\u003csub\u003e2\u003c/sub\u003e. Cell viability was determined as described above.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e4.4 qRT-PCR\u003c/h2\u003e \u003cp\u003eRNA was extracted from human prostate tumor cells (day 4) or spheroids (day 10) using Isogen (Wako Chemicals Inc.). cDNA was synthesized using SuperScript\u0026trade; III Reverse Transcriptase (Invitrogen, Carlsbad, CA). Quantitative real time PCR (qRT-PCR) was performed in triplicate using TaqMan\u0026reg; Universal Master Mix II (Applied Biosystems, Foster City, CA) with the StepOne\u0026trade; real time PCR system (Applied Biosystems). Specific primers were used for Ki-67 (gene: \u003cem\u003eMKI67\u003c/em\u003e in GeneCards) (Assay ID in TaqMan\u0026trade; Assays and Arrays, Thermo Fisher Scientific: Hs01032443_m1), GLUT1 (gene: \u003cem\u003eSLC2A1\u003c/em\u003e) (Assay ID: Hs00892681_m1), MCT1 (gene: \u003cem\u003eSLC16A1\u003c/em\u003e) (Assay ID: Hs01560299_m1), MCT4 (\u003cem\u003eSLC16A4\u003c/em\u003e) (Assay ID: Hs01006127_m1), LDHA (gene: \u003cem\u003eLDHA\u003c/em\u003e) (Assay ID: Hs01378790_g1), ALT1 (gene: \u003cem\u003eGPT\u003c/em\u003e) (Assay ID: Hs01548671_g1), and β-actin (\u003cem\u003eACTB\u003c/em\u003e) (Assay ID: Hs99999903_m1) (Applied Biosystems).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e4.5 Hyperpolarization (HP) of [1-\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC]pyruvate and \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC NMR measurement\u003c/h2\u003e \u003cp\u003eIn Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003e and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003e and \u003cb\u003eFig. S3\u003c/b\u003e, the HP of [1-\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC]pyruvate was conducted using HyperSense\u0026trade; (Oxford Instruments, Co. Ltd.) according to the manufacturer\u0026rsquo;s instructions. Briefly, a 30 \u0026micro;l aliquot of [1-\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC]pyruvic acid (14.2 M) doped with 15 mM OX063 was placed in a sample cup. The sample cup was then set into the bore unit of the HyperSense\u0026trade; instrument (3.35 T, 1.4 K, 2.8 mbar) using a sample insertion rod. The frozen sample was then polarized for 50\u0026ndash;60 min by microwave radiation (100 mW) at 93.96 GHz. During the polarization, the \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC NMR signal intensity was recorded every 5 min with the built-in solid-state polarimeter using RINMR software (Oxford Instruments, Co. Ltd.). After polarization, the sample was rapidly dissolved in 4.5 ml of dissolution buffer (100 mM Tris, 100 mg/L EDTA) preheated to 10 bar (~\u0026thinsp;459 K), which warmed the sample to biological temperature (308\u0026thinsp;~\u0026thinsp;313 K, pH 7). The resulting solution was received in a 50-ml reservoir and quickly transferred to a 10-mm NMR tube containing biological samples; 1-ml suspension of freshly harvested standard culture cells or spheroids (10\u003csup\u003e8\u003c/sup\u003e cells from 50 of 10-cm culture dishes), or 1 ml of tumor tissue homogenate (2 ml/g tumor)]. After suspending the samples quickly, the NMR tube was placed in a Japan Redox JXI-400Z spectrometer (9.4 T). Collection of the NMR spectra started 30\u0026thinsp;~\u0026thinsp;35 s after dissolution of HP [1-\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC]pyruvate. Tetramethylsilane (TMS, 0 ppm) was used as the external standard for \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC NMR (1-\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC in pyruvate: 170.2 ppm, alanine: 176.9 ppm, pyruvate・H\u003csub\u003e2\u003c/sub\u003eO: 178.5 ppm, and lactate: 182.4 ppm) (\u003cb\u003eFig.\u0026nbsp;1B\u003c/b\u003e). The spectra were recorded using a flip angle of 5\u0026deg; and time of repetition (TR) of 2.5 sec. Resulting \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC NMR spectra were processed using JEOL Delta NMR software v5.0.1 (JEOL Ltd., Tokyo, Japan).\u003c/p\u003e \u003cp\u003eIn Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003e, the HP of [1-\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC]pyruvate was conducted using SpinAligner (Polarize ApS, Denmark) according to the manufacturer\u0026rsquo;s instructions. Briefly, a 18 \u0026micro;l aliquot of [1-\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC]pyruvic acid (14.2 M) doped with 25 mM OX063 was placed in a sample vial. The sample vial was then attached to a fluid path and set into the bore unit of the SpinAligner instrument (6.7 T, 1.25 K, 1.2 mbar). The frozen sample was then polarized for 60\u0026ndash;80 min by microwave radiation (22 mW) at 187.770 GHz. During the polarization, the \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC NMR signal intensity was recorded every 5 min with the built-in solid-state NMR spectrometer controlled by SPINit software (RS\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003eD, France.). After polarization, the sample was rapidly dissolved in 3.2 ml of dissolution buffer (40 mM Tris, 50 mM NaCl, 80 mM NaOH, 100 mg/L EDTA) preheated to 12 bar (~\u0026thinsp;461 K), which warmed the sample to biological temperature (308\u0026thinsp;~\u0026thinsp;313 K, pH 7). The resulting solution was received in a 50-ml reservoir, and 50 \u0026micro;L of the HP [1-\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC]pyruvate solution was quickly transferred to a micro tube containing 550 \u0026micro;L suspension of cultured SCC cells or SCC spheroids. After suspending the samples quickly, the suspension was transferred to a 5-mm NMR tube and the NMR tube was placed in a Spinsolve 60 spectrometer (1.5 T, Magritek, Germany). Acquisition of the NMR spectra was started 30 s after adding HP [1-\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC]pyruvate to the cells or spheroids. The spectra were recorded using a flip angle of 5\u0026deg; and TR of 2.5 sec. Resulting \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC NMR spectra were analyzed using MestReNova software v14.2.3 (Mestrelab Research S.L., Spain).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e4.6 Histological analysis\u003c/h2\u003e \u003cp\u003eSCCVII spheroids were collected in 1.5-ml Eppendorf tubes and centrifuged. The pellet was suspended into 900 \u0026micro;l PBS and 100 \u0026micro;l of pimonidazole (1 mM) solution and incubated for 1 h at room temperature. After washing three times with PBS, spheroids were spread onto a glass slide and fixed with 3.7% formalin. After 30 min, the slide glass was washed three times with PBS and nonspecific binding sites on the spheroids were blocked with Protein Block Serum-Free reagent (Dako North America Inc., Carpinteria, CA) at 4\u0026deg;C overnight. After washing three times with PBS, the slides were submerged in a solution containing rabbit anti-Ki-67 antibody (Abcam\u0026reg;; 1:100) and incubated overnight at 4\u0026deg;C. The spheroids were incubated with an Alexa Fluor 546 F(ab\u0026rsquo;)2 fragment of goat anti-rabbit IgG (H\u0026thinsp;+\u0026thinsp;L) (Invitrogen/Life Sciences, Grand Island, NY; 1:2000) and Hypoxyprobe 4.3.11.3 mouse MAb (hpi; 1:250) for pimonidazole staining (1 h at room temperature). Samples were then mounted with Prolong Gold antifade reagent plus DAPI (Invitrogen). Fluorescence microscopy was performed using a FV1200 IX81 confocal microscope (Olympus, Tokyo, Japan), and images were captured using FLUOVIEW Ver.4.2a imaging software (Olympus).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e4.7 Animal experiments\u003c/h2\u003e \u003cp\u003eAll procedures were conducted in full compliance with the ARRIVE guidelines. Animal experiments (\u003cem\u003eex vivo\u003c/em\u003e) were performed according to the Institutional Guidance of Kyushu University on Animal Experimentation and this study obtained approval from the animal experiment committee of Kyushu University. Female 7-week old C3H/HeNJcl mice (17\u0026ndash;20 g) and athymic BALB/cAJcl-nu/nu mice were purchased from Japan CLEA Japan Inc. (Kawasaki, Japan). A mouse SCCVII tumor was formed by injecting 1 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e SCCVII cells subcutaneously into the hind leg of C3H mice. Formation of human DU145 and PC-3 solid tumor xenografts were carried out by injecting 1 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e of each cell type subcutaneously into the hind leg of BALB mice. After tumor growth, the mice were anesthetized with 3% isoflurane (Pfizer Japan Inc., Tokyo, Japan) before being euthanized by exsanguination via transcardial perfusion with PBS, followed by ice-cold 4% paraformaldehyde (PFA) in PBS. Tumor tissues were excised and homogenized in PBS (2 ml/g tumor) prior to analysis by hyperpolarized [1-\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC]pyruvate spectroscopy.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e4.8 Statistical analysis\u003c/h2\u003e \u003cp\u003eAll results were expressed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SE. The differences in means of groups were determined by two-tailed Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e test. The minimum level of significance was set at \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; We thank to Prof. Shinsuke Sando and Dr. Hiroshi Nonaka (The University of Tokyo) for discussion about this study. We acknowledge Dr. Tatsuya Naganuma (Japan Redox Co. Ltd.) and Mr. Shinya Goto (Oxford Instruments Co. Ltd.) for the technical assistance for the use of HyperSense\u003csup\u003eTM\u003c/sup\u003e, and Ms. Junko Yamaki (Fukushima Medical University School of Medicine) for assistance with the qRT-PCR experiments. We also thank Ms. Ayumi Koike and Yasuko Shimuta (National Institutes for Quantum Science and Technology) for their technical support for cell experiments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceived and designed the experiments: YT. Performed the experiments: YT KT KIn KS. Analyzed the data: YT KT KIn KS KIc. Contributed reagents/materials/analysis tools: YT YH KIc. Contributed to the writing of the manuscript: YT.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported partly by JST FOREST Program [grant number JPMJFR225G (to Y.T.)]; JST CREST [grant number JPMJCR13L4]; MEXT Q-LEAP [grant number JPMXS0120330644 (to Y.T.)]; MEXT Promotion of Development of a Joint Usage/ Research System Project: Coalition of Universities for Research Excellence Program (CURE) [grant number JPMXP1323015488]; JSPS KAKENHI [grant number 23K27561 (to Y.T.), 23K19228 (to K.S.), 24K03317 (to K.I.), 15H03035 (to K.I.) and Research Grant from Fukushima Medical University. The use of HyperSense\u003csup\u003eTM\u003c/sup\u003e was in part supported by the funding program \u0026lsquo;Creation of Innovation Centers for Advanced Interdisciplinary Research Areas\u0026rsquo; from JST.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated and/or analyzed during the current study are available upon reasonable request from the corresponding author. The gene sequences used in this study are available in GeneCards\u003csup\u003e\u0026reg;\u003c/sup\u003e, the human gene database [https://www.genecards.org/].\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBen-Haim, S. \u0026amp; Ell, P. 18F-FDG PET and PET/CT in the evaluation of cancer treatment response. \u003cem\u003eJ. Nucl. Med.\u003c/em\u003e \u003cb\u003e50\u003c/b\u003e, 88\u0026ndash;99 (2009).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eArdenkjaer-Larsen, J. 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Commun.\u003c/em\u003e \u003cb\u003e437\u003c/b\u003e, 420\u0026ndash;425 (2013).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Hyperpolarization, Pyruvate, Spheroid, SCC, Prostate tumor, NMR","lastPublishedDoi":"10.21203/rs.3.rs-5900705/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5900705/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eHyperpolarized (HP) [1-\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC]pyruvate nuclear magnetic resonance (NMR) spectroscopy was employed to investigate tumor energy metabolism in tiny multicellular spheroids, serving as a model of early-phase tumorigenesis \u003cem\u003ein vivo\u003c/em\u003e. A three-dimensional static culture of murine squamous cell carcinoma (SCCVII) cells formed uniform smaller multicellular spheroids (~\u0026thinsp;150 \u0026micro;m in diameter), without hypoxic or necrotic cores, yet these spheroids exhibited resistance to anti-tumor drugs. HP [1-\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC]pyruvate NMR spectroscopy of SCCVII spheroids revealed an increased conversion of pyruvate to lactate compared to monolayer cultures, indicating enhanced aerobic glycolysis in the aggregated cells. Additionally, HP spectroscopy differentiated the degree of aerobic glycolysis in human prostate tumor spheroids―DU145 (~\u0026thinsp;120 \u0026micro;m) and PC-3 (~\u0026thinsp;230 \u0026micro;m)―as evidenced by the upregulation of genes associated with lactate production and cellular transport. The Lac/Pyr ratio among spheroids correlated with those observed in biopsy samples of corresponding malignant tumors grown in mice. These findings suggest that HP [1-\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC]pyruvate NMR spectroscopy may serve as a metabolic biomarker for early-phase tumorigenesis \u003cem\u003ein vivo\u003c/em\u003e.\u003c/p\u003e","manuscriptTitle":"Hyperpolarized [1- 13 C]pyruvate NMR spectroscopy reveals transition of tumor energy metabolism in tiny multicellular spheroids","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-01-31 12:52:07","doi":"10.21203/rs.3.rs-5900705/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-04-02T05:38:42+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-03-31T14:53:28+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-03-30T13:49:42+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"151014602289231675341705204155331169333","date":"2025-03-21T11:15:37+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-02-27T13:47:02+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"36167721036708262517885997202189346650","date":"2025-02-18T14:02:53+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"75936919251900369801103903121217763189","date":"2025-02-18T10:33:36+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-02-18T08:32:27+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-02-18T08:30:07+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-01-31T06:36:59+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-01-29T10:14:21+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-01-25T09:13:16+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d39d9919-9c87-4b33-b973-f526fd95496f","owner":[],"postedDate":"January 31st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":43564996,"name":"Biological sciences/Biological techniques/Analytical biochemistry/Biochemical assays"},{"id":43564997,"name":"Biological sciences/Cancer/Tumour biomarkers"},{"id":43564998,"name":"Health sciences/Oncology/Cancer/Cancer metabolism"},{"id":43564999,"name":"Physical sciences/Engineering/Biomedical engineering"}],"tags":[],"updatedAt":"2025-06-09T16:05:45+00:00","versionOfRecord":{"articleIdentity":"rs-5900705","link":"https://doi.org/10.1038/s41598-025-03454-1","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-06-02 15:57:14","publishedOnDateReadable":"June 2nd, 2025"},"versionCreatedAt":"2025-01-31 12:52:07","video":"","vorDoi":"10.1038/s41598-025-03454-1","vorDoiUrl":"https://doi.org/10.1038/s41598-025-03454-1","workflowStages":[]},"version":"v1","identity":"rs-5900705","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5900705","identity":"rs-5900705","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}