Extreme Prematurity and Pulmonary Outcomes Program in Saitama: Protocol for a Prospective Multicenter Cohort Study in Japan.

BACKGROUND: Because of the improvements in survival rates for preterm infants, not only the rates of bronchopulmonary dysplasia (BPD) but also those of long-term respiratory complications of premature birth are increasing, resulting in financial and health burdens in developed countries. Thus far, the risk factors of respiratory morbidities in extremely preterm infants remain unknown. Furthermore, the definition and the predictive ability of BPD for long-term respiratory outcomes are yet to be determined. OBJECTIVE: The objective of our study, Extreme Prematurity and Pulmonary Outcomes Program in Saitama, is to develop the diagnostic criteria for BPD and to determine the prognostic factors contributing to the long-term pulmonary outcomes manifesting in extremely preterm infants. METHODS: The Extreme Prematurity and Pulmonary Outcomes Program in Saitama is an observational prospective cohort study performed by a consortium of six neonatal intensive care units (NICUs) in Saitama, Japan. The subjects included in this study are infants (from each clinical center) with gestational ages 22 to 27 weeks. The target is 400 subjects. This study aims to determine the definition of BPD and other perinatal factors that accurately predict the long-term pulmonary outcomes in survivors of extreme prematurity. Moreover, the association between BPD and postprematurity respiratory disease will be investigated using generalized linear models. RESULTS: The protocol and consent forms were evaluated and approved on September 5, 2019, by the Ethics Committee of Saitama Medical Center, Saitama Medical University. Enrollment began on April 1, 2020. It is expected to end on March 31, 2023. The follow-up for 1 year corrected age is expected to continue through the middle of 2024. CONCLUSIONS: The Extreme Prematurity and Pulmonary Outcomes Program in Saitama incorporates aspects of neonatal care in secondary- and tertiary-level NICUs to develop existing research studies on the definition of BPD, objective biomarkers, and outcome measures of respiratory morbidity in extremely preterm infants beyond NICU hospitalization, thereby leading to a novel understanding of the nature and natural history of BPD and potential mechanistic and therapeutic targets in at-risk subjects. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): DERR1-10.2196/22948.

PET Tracer 18F-Fluciclovine Can Detect Histologically Proven Bone Metastatic Lesions: A Preclinical Study in Rat Osteolytic and Osteoblastic Bone Metastasis Models.

18F-Fluciclovine (trans-1-amino-3-18F-fluorocyclobutanecarboxylic acid; anti-18F-FACBC) is a positron emission tomography (PET) tracer for diagnosing cancers (e.g., prostate and breast cancer). The most frequent metastatic organ of these cancers is bone. Fluciclovine-PET can visualize bony lesions in clinical practice; however, such lesions have not been described histologically. Methods: We investigated the potential of 14C-fluciclovine in aiding the visualization of osteolytic and osteoblastic bone metastases (with histological analyses), compared with 3H-2-deoxy-2-fluoro-D-glucose (3H-FDG), 3H-choline chloride (3H-choline), and 99mTc-hydroxymethylene diphosphonate (99mTc-HMDP) by using triple-tracer autoradiography in rat breast cancer osteolytic (on day 12 ± 1 postinjection of MRMT-1) and prostate cancer osteoblastic (on day 20 ± 3 postinjection of AT6.1) metastatic models. Results: The distribution patterns of 14C-fluciclovine, 3H-FDG, and 3H-choline, but not 99mTc-HMDP, were similar in both models, and the lesions where these tracers accumulated were, histologically, typical osteolytic and osteoblastic lesions. 99mTc-HMDP accumulated mostly in osteoblastic lesions. 14C-fluciclovine could visualize the osteolytic lesions as early as day 6 postinjection of MRMT-1. However, differential distributions in 14C-fluciclovine and 3H-FDG existed, based on histological differences: low 14C-fluciclovine and high 3H-FDG accumulation in osteolytic lesions with inflammation. In the osteoblastic metastatic model, visualization of osteoblastic lesions with 14C-fluciclovine was not clear, yet clearer than with 3H-FDG. Although half of the osteoblastic lesions with 14C-fluciclovine accumulation showed negligible 3H-choline accumulation in comparison, they were histologically similar to lesions with marked 14C-fluciclovine and 3H-choline accumulation. Conclusion: These results suggest that fluciclovine-PET can visualize true osteolytic and osteoblastic bone metastatic lesions.

Fasting Enhances the Contrast of Bone Metastatic Lesions in 18F-Fluciclovine-PET: Preclinical Study Using a Rat Model of Mixed Osteolytic/Osteoblastic Bone Metastases.

18F-fluciclovine (trans-1-amino-3-18F-fluorocyclobutanecarboxylic acid) is an amino acid positron emission tomography (PET) tracer used for cancer staging (e.g., prostate and breast). Patients scheduled to undergo amino acid-PET are usually required to fast before PET tracer administration. However, there have been no reports addressing whether fasting improves fluciclovine-PET imaging. In this study, the authors investigated the influence of fasting on fluciclovine-PET using triple-tracer autoradiography with 14C-fluciclovine, [5,6-³H]-2-fluoro-2-deoxy-d-glucose (³H-FDG), and 99mTc-hydroxymethylene diphosphonate (99mTc-HMDP) in a rat breast cancer model of mixed osteolytic/osteoblastic bone metastases in which the animals fasted overnight. Lesion accumulation of each tracer was evaluated using the target-to-background (muscle) ratio. The mean ratios of 14C-fluciclovine in osteolytic lesions were 4.6 ± 0.8 and 2.8 ± 0.6, respectively, with and without fasting, while those for ³H-FDG were 6.9 ± 2.5 and 5.1 ± 2.0, respectively. In the peri-tumor bone formation regions (osteoblastic), where 99mTc-HMDP accumulated, the ratios of 14C-fluciclovine were 4.3 ± 1.4 and 2.4 ± 0.7, respectively, and those of ³H-FDG were 6.2 ± 3.8 and 3.3 ± 2.2, respectively, with and without fasting. These results suggest that fasting before 18F-fluciclovine-PET improves the contrast between osteolytic and osteoblastic bone metastatic lesions and background, as well as 18F-FDG-PET.

Assessment of Amino Acid/Drug Transporters for Renal Transport of [18F]Fluciclovine (anti-[18F]FACBC) in Vitro.

[18F]Fluciclovine (trans-1-amino-3-[18F]fluorocyclobutanecarboxylic acid; anti-[18F]FACBC), a positron emission tomography tracer used for the diagnosis of recurrent prostate cancer, is transported via amino acid transporters (AATs) with high affinity (Km: 97-230 µM). However, the mechanism underlying urinary excretion is unknown. In this study, we investigated the involvement of AATs and drug transporters in renal [18F]fluciclovine reuptake. [14C]Fluciclovine (trans-1-amino-3-fluoro[1-14C]cyclobutanecarboxylic acid) was used because of its long half-life. The involvement of AATs in [14C]fluciclovine transport was measured by apical-to-basal transport using an LLC-PK1 monolayer as model for renal proximal tubules. The contribution of drug transporters herein was assessed using vesicles/cells expressing the drug transporters P-glycoprotein (P-gp), breast cancer resistance protein (BCRP), multidrug resistance-associated protein 4 (MRP4), organic anion transporter 1 (OAT1), organic anion transporter 3 (OAT3) , organic cation transporter 2 (OCT2), organic anion transporting polypeptide 1B1 (OATP1B1), and organic anion transporting polypeptide 1B3 (OATP1B3). The apical-to-basal transport of [14C]fluciclovine was attenuated by l-threonine, the substrate for system alanine-serine-cysteine (ASC) AATs. [14C]Fluciclovine uptake by drug transporter-expressing vesicles/cells was not significantly different from that of control vesicles/cells. Fluciclovine inhibited P-gp, MRP4, OAT1, OCT2, and OATP1B1 (IC50 > 2.95 mM). Therefore, system ASC AATs may be partly involved in the renal reuptake of [18F]fluciclovine. Further, given that [18F]fluciclovine is recognized as an inhibitor with millimolar affinity for the tested drug transporters, slow urinary excretion of [18F]fluciclovine may be mediated by system ASC AATs, but not by drug transporters.

[(14)C]Fluciclovine (alias anti-[(14)C]FACBC) uptake and ASCT2 expression in castration-resistant prostate cancer cells.

INTRODUCTION: trans-1-Amino-3-[(18)F]fluorocyclobutanecarboxylic acid ([(18)F]fluciclovine, also known as anti-[(18)F]FACBC), is a tracer for positron emission tomography (PET) imaging for detection of tumors such as prostate cancer (PCa). Our previous study showed that ASCT2 (Na(+)-dependent amino acid transporter (AAT)) mediates fluciclovine uptake in androgen-dependent PCa cells; its expression is influenced by androgen, a key hormone in the progression of primary PCa and castration-resistant prostate cancer (CRPC). In this study, we investigated the uptake mechanisms and feasibility of [(18)F]fluciclovine for CRPC in the androgen-dependent PCa cell line LNCaP and LNCaP-derivatives LNCaP-SF and LN-REC4. METHODS: LNCaP-SF was established after long-term cultivation of LNCaP in steroid-free conditions, and LN-Pre and LN-REC4 were established from LNCaP inoculated in intact and castrated severe combined immunodeficient mice, respectively. Uptake and competitive inhibition experiments were performed with trans-1-amino-3-fluoro[1-(14)C]cyclobutanecarboxylic acid ([(14)C]fluciclovine) to characterize the involvement of AATs in androgen-dependent PCa (LNCaP and LN-Pre) and CRPC-like (LNCaP-SF and LN-REC4) cell lines. AAT expression was analyzed by Western blotting, and [(14)C]fluciclovine uptake in androgen-dependent PCa and CRPC-like cell lines were investigated in the presence or absence of dihydrotestosterone (DHT). RESULTS: The contribution of Na(+)-dependent AATs to [(14)C]fluciclovine uptake in all cell lines was 88-98%, and [(14)C]fluciclovine uptake was strongly inhibited by L-glutamine and L-serine, the substrates for Na(+)-dependent alanine-serine-cysteine (system ASC) AATs, in the presence of Na(+). DHT enhanced ASCT2 expression in LNCaP, LN-Pre, and LN-REC4, but not in LNCaP-SF, and the responses of ASCT2 expression to DHT correlated with [(14)C]fluciclovine uptake. CONCLUSIONS: System ASC, especially ASCT2, could play a major role in [(14)C]fluciclovine uptake into CRPC-like and androgen-dependent PCa cells, suggesting [(18)F]fluciclovine-PET is applicable to the detection of CRPC as well as androgen-dependent PCa. ADVANCE IN KNOWLEDGE: [(18)F]fluciclovine-PET may be applied for the detection of CRPC. IMPLICATION FOR PATIENT CARE: [(18)F]fluciclovine-PET may permit early intervention for CRPC treatment.

Evaluation of trans-1-amino-3-18F-fluorocyclobutanecarboxylic acid accumulation in low-grade glioma in chemically induced rat models: PET and autoradiography compared with morphological images and histopathological findings.

INTRODUCTION: Magnetic resonance imaging (MRI) can have a problem to delineate diffuse gliomas with an intact blood-brain barrier (BBB) especially when a marked peritumoral edema is present. We evaluated the potential of trans-1-amino-3-(18)F-fluorocyclobutanecarboxylic acid (anti-(18)F-FACBC) positron emission tomography (PET) to delineate the extent of diffuse gliomas by comparing PET findings with autoradiography, in vivo and ex vivo MRI, and histopathology findings. METHODS: Dynamic PET was performed in rats with N-ethyl-N-nitrosourea-induced glioma for 60 min after anti-(18)F-FACBC injection. Contrast-enhanced MRI was performed before or after PET. The PET images were fused with in vivo and ex vivo MR images, and histopathological images for direct comparisons. Autoradiograms were compared with the results of Evans Blue (EB) extravasation (to assess BBB integrity) and hematoxylin-eosin staining. RESULTS: Histopathological examination, including EB extravasation assessment, and enhanced T1-weighted MRI identified several diffuse gliomas with slight BBB disruption, similar to low-grade human gliomas. Anti-(18)F-FACBC uptake was specific and high in the gliomas, irrespective of BBB integrity. Higher anti-(18)F-FACBC uptake corresponded to areas of T2 hyperintensity, independent of gadolinium enhancement. Ex vivo autoradiography also showed high anti-(18)F-FACBC accumulation in tumors lacking EB extravasation and a correlation between anti-(18)F-FACBC accumulation and tumor cell density, but not EB extravasation. CONCLUSIONS: Anti-(18)F-FACBC-PET allowed visualization of gliomas irrespective of BBB integrity. The tumor-to-normal uptake ratio of anti-(18)F-FACBC generally correlated with the relative cell density. Anti-(18)F-FACBC PET combined with MRI shows promise for preoperative glioma delineation. ADVANCES IN KNOWLEDGE: Radiopharmaceuticals that cross the BBB, such as anti-(18)F-FACBC, are taken up by low-grade gliomas with equivocal MRI findings due to an intact BBB. IMPLICATIONS FOR PATIENT CARE: Surgery is the first-line therapy for low-grade gliomas; therefore, delineation of their extent in the presence of an intact BBB is essential to planning surgery that removes the entire neoplasm, which will positively affect long-term survival.

Amino acid PET tracers are reliable markers of treatment responses to single-agent or combination therapies including temozolomide, interferon-ß, and/or bevacizumab for glioblastoma.

INTRODUCTION: We examined whether the amino acid PET tracers, trans-1-amino-3-(18)F-fluorocyclobutanecarboxylic acid (anti-(18)F-FACBC) and (11)C-methyl-l-methionine ((11)C-Met), are suitable for detecting early responses to combination therapies including temozolomide (TMZ), interferon-ß (IFN), and bevacizumab (Bev) in glioblastoma. METHODS: Human glioblastoma U87MG (U87) cells were incubated with low dose TMZ to induce chemoresistance. Both trans-1-amino-3-fluoro-1-(14)C-cyclobutanecarboxylic acid (anti-(14)C-FACBC) and (3)H-methyl-l-methionine ((3)H-Met) uptake were quantified using triple-label accumulation assays to examine the relationship between tracer uptake and proliferation ((3)H-thymidine (TdR) accumulation) in vitro. U87 and U87R (TMZ-resistant subculture) cells were inoculated into the right and left basal ganglia, respectively, of F344/N-rnu rats. The efficacy of single-agent (TMZ, Bev) and combination therapy (TMZ/IFN, TMZ/Bev, TMZ/IFN/Bev) was examined in orthotopic gliomas using MRI, Evans blue extravasation, anti-(14)C-FACBC, and (3)H-Met autoradiography, and MIB-1 immunostaining. RESULTS: TMZ treatment decreased (3)H-TdR accumulation and the volume distribution of anti-(14)C-FACBC and (3)H-Met in U87 but not U87R cells. TMZ/IFN combination therapy significantly decreased these parameters in U87R cells; however, Bev had no additional effect in vitro. In vivo, U87R-derived gliomas were observed as equivocal tumors on MRI and T2-high intensity lesions. Bev treatment, either alone or in combination, markedly decreased U87 enhancing lesions. By contrast, autoradiographic images using anti-(14)C-FACBC and (3)H-Met clearly delineated tumor extent, which spread widely beyond T2-high intensity lesions and enhancing lesions. TMZ therapy significantly decreased tracer accumulation and proliferation of U87- but not U87R-derived tumors. TMZ/IFN combination treatment significantly decreased these parameters in U87R tumors, which were further reduced (in both tumor types) by Bev addition. Tracer uptake correlated with the MIB-1 proliferation index. However, MRI was unsuitable for tumor delineation and assessment of Bev treatment response. CONCLUSIONS: Triple-agent therapy (TMZ/IFN/Bev) was effective against even TMZ-resistant glioblastomas. PET with amino acid tracers provides useful information on the early response of glioblastomas to single-agent and combination therapy.

Anti-1-amino-3-18F-fluorocyclobutane-1-carboxylic acid: physiologic uptake patterns, incidental findings, and variants that may simulate disease.

UNLABELLED: Anti-1-amino-3-(18)F-fluorocyclobutane-1-carboxylic acid ((18)F-FACBC) is a synthetic amino acid analog PET radiotracer undergoing clinical trials for the evaluation of prostate and other cancers. We aimed to describe common physiologic uptake patterns, incidental findings, and variants in patients who had undergone (18)F-FACBC PET. METHODS: Sixteen clinical trials involving 611 (18)F-FACBC studies from 6 centers, which included dosimetry studies on 12 healthy volunteers, were reviewed. Qualitative observations of common physiologic patterns, incidental uptake, and variants that could simulate disease were recorded and compared with similar observations in studies of the healthy volunteers. Quantitative analysis of select data and review of prior published reports and observations were also made. RESULTS: The liver and pancreas demonstrated the most intense uptake. Moderate salivary and pituitary uptake and variable mild to moderate bowel activity were commonly visualized. Moderate bone marrow and mild muscle activity were present on early images, with marrow activity decreasing and muscle activity increasing with time. Brain and lungs demonstrated activity less than blood pool. Though (18)F-FACBC exhibited little renal excretion or bladder uptake during the clinically useful early imaging time window, mild to moderate activity might accumulate in the bladder and interfere with evaluation of adjacent prostate bed and seminal vesicles in 5%-10% of patients. Uptake might also occur from benign processes such as infection, inflammation, prostatic hyperplasia, and metabolically active benign bone lesions such as osteoid osteoma. CONCLUSION: Common physiologic uptake patterns were similar to those noted in healthy volunteers. The activity in organs followed the presence of amino acid transport and metabolism described with other amino acid-based PET radiotracers. As with other PET radiotracers such as (18)F-FDG, focal nonphysiologic uptake may represent incidental malignancy. Uptake due to benign etiologies distinct from physiologic background also occurred and could lead to misinterpretations if the reader is unaware of them.

Accumulation of trans-1-amino-3-[(18)F]fluorocyclobutanecarboxylic acid in prostate cancer due to androgen-induced expression of amino acid transporters.

PURPOSE: Androgens play a crucial role in prostate cancer progression, and trans-1-amino-3-[(18)F]fluorocyclobutanecarboxylic acid (anti-[(18) F]FACBC) are used for visualization of prostate cancer. We examined the effect of androgen on the expression of amino acid transporters related to anti-[(18)F]FACBC transport and uptake of trans-1-amino-3-fluoro-[1-(14)C]cyclobutanecarboxylic acid (anti-[(14)C]FACBC). PROCEDURES: Expression of amino acid transporters and uptake of anti-[(14)C]FACBC in androgen receptor (AR)-positive LNCaP and AR-negative DU145 human prostate cancer cells cultured with/without 5α-dihydrotestosterone (DHT) and the effect of bicalutamide, an AR antagonist, on DHT-associated changes were investigated. RESULTS: DHT stimulated the expression of amino acid transporters ASCT2, SNAT5, 4F2 heavy chain, and LAT3 in LNCaP but not in DU145 cells. Anti-[(14)C]FACBC uptake was enhanced, in a DHT-dependent manner, in LNCaP cells only. CONCLUSIONS: DHT enhanced the expression of ASCT2, the transporter responsible for anti-[(18)F]FACBC uptake, thereby increasing anti-[(14)C]FACBC uptake in AR-positive LNCaP cells. Androgen-mediated induction may contribute to the distinct anti-[(18)F]FACBC accumulation pattern in prostate cancer.

Comparison of trans-1-amino-3-[18F]fluorocyclobutanecarboxylic acid (anti-[18F]FACBC) accumulation in lymph node prostate cancer metastasis and lymphadenitis in rats.

INTRODUCTION: Trans-1-amino-3-[(18)F]fluorocyclobutanecarboxylic acid (anti-[(18)F]FACBC) is a positron emission tomography (PET) tracer used to visualize prostate cancer (PCa). In this study, we investigated the differences in anti-[(18)F]FACBC accumulation between metastatic and inflamed lymph node (LN) lesions. METHODS: A PCa LN metastasis (PLM) model was developed by inoculating a rat PCa cell line, MAT-Ly-Lu-B2, into popliteal LNs of Copenhagen rats. Acute lymphadenitis (AL) was induced by injecting concanavalin A (Con A) into the hind footpad, and chronic lymphadenitis (CL) was induced by daily injection of Con A into the tissues surrounding the popliteal LNs for 2weeks. Main lesions of all animal models were established in lumbar and/or inguinal LNs. Biodistribution and dynamic PET imaging data were acquired after tracer injection. T2-weighted magnetic resonance (MR) images were registered with PET images. RESULTS: In the biodistribution study, the uptake ratios of PLM-to-lymphadenitis in lesional lumbar and inguinal LNs were 0.97-1.57 and 1.47-2.08 at 15 and 60min post-anti-[(18)F]FACBC injection respectively. In PET imaging, the lesional lumbar LNs of CL and PLM, but not of AL, were visualized on anti-[(18)F]FACBC-PET/MR fusion images without disturbance from radioactivity from urine, and　the rank order of anti-[(18)F]FACBC accumulation at 50-60 post-injection in lesional lumbar LNs was PLM>CL>AL. CONCLUSIONS: Anti-[(18)F]FACBC accumulation in LNs with PLM was higher than that in inflamed LNs. ADVANCES IN KNOWLEDGE: The study showed that although low but significant levels of anti-[(18)F]FACBC uptake by chronic inflamed lesions might cause false-positives in anti-[(18)F]FACBC-PET in some PCa patients, uptake of the tracer at acutely inflamed sites was minimal. IMPLICATIONS FOR PATIENT CARE: The findings of this study suggest the potential of Anti-[(18)F]FACBC for distinguishing between tumors and acute inflammation in clinical practice.

Differences in transport mechanisms of trans-1-amino-3-[18F]fluorocyclobutanecarboxylic acid in inflammation, prostate cancer, and glioma cells: comparison with L-[methyl-11C]methionine and 2-deoxy-2-[18F]fluoro-D-glucose.

PURPOSE: We aimed to elucidate trans-1-amino-3-[(18)F]fluorocyclobutanecarboxylic acid (anti-[(18)F]FACBC) uptake mechanisms in inflammatory and tumor cells, in comparison with those of L-[methyl-(11)C]methionine ([(11)C]Met) and 2-deoxy-2-[(18)F]fluoro-D-glucose ([(18)F]FDG). PROCEDURES: Using carbon-14-labeled tracers, in vitro time-course, pH dependence, and competitive inhibition uptake experiments were performed in rat inflammatory (T cells, B cells, granulocytes, macrophages), prostate cancer (MLLB2), and glioma (C6) cells. RESULTS: Anti-[(14)C]FACBC uptake ratios of T/B cells to tumor cells were comparable, while those of granulocytes/macrophages to tumor cells were lower than those for [(14)C]FDG. Over half of anti-[(14)C]FACBC uptake by T/B and tumor cells was mediated by Na(+)-dependent amino acid transporters (system ASC), whereas most [(14)C]Met transport in all cells was mediated by Na(+)-independent carriers (system L). CONCLUSIONS: The low anti-[(18)F]FACBC accumulation in granulocytes/macrophages may be advantageous in discriminating inflamed regions from tumors. The significant anti-[(18)F]FACBC uptake in T/B cells may cause false-positives in some cancer patients who undergo FACBC-positron emission tomography (PET).

Comparative evaluation of transport mechanisms of trans-1-amino-3-[¹8F]fluorocyclobutanecarboxylic acid and L-[methyl-¹¹C]methionine in human glioma cell lines.

Positron emission tomography (PET) with amino acid tracers is useful for the visualization and assessment of therapeutic effects on gliomas. Our purpose is to elucidate the transport mechanisms of trans-1-amino-3-[¹8F]fluorocyclobutanecarboxylic acid (anti-[¹8F]FACBC) and L-[methyl-¹¹C]methionine ([¹¹C]Met) in normal human astrocytes (NHA), low-grade (Hs683, SW1088), and high-grade (U87MG, T98G) human glioma cell lines. Because the short half-lives of fluorine-18 and carbon-11 are inconvenient for in vitro experiments, trans-1-amino-3-fluoro[1-¹4C]cyclobutanecarboxylic acid (anti-[¹4C]FACBC) and L-[methyl-¹4C]methionine ([¹4C]Met) were used instead of the PET tracers. Time-course uptake experiments showed that uptake of anti-[¹4C]FACBC was 1.4-2.6 times higher than that of [¹4C]Met in NHA and low-grade glioma cells, and was almost equal to that of [¹4C]Met in high-grade glioma cells. To identify the amino acid transporters (AATs) involved in the transport of anti-[¹4C]FACBC and [¹4C]Met, we carried out competitive inhibition experiments using synthetic/naturally-occurring amino acids as inhibitors. We found that anti-[¹4C]FACBC uptake in the presence of Na⁺ was strongly inhibited by L-glutamine and L-serine (the substrates for ASC system AATs), whereas L-phenylalanine and 2-amino-bicyclo[2,2,1]heptane-2-carboxylic acid (BCH, the substrates for L system AATs) robustly inhibited Na⁺-independent anti-[¹4C]FACBC uptake. Regardless of Na⁺, [¹4C]Met uptake was inhibited strongly by L-phenylalanine and BCH. Moreover, the exchange transport activity of L-glutamine for anti-[¹4C]FACBC was stronger than that of BCH in the presence of Na⁺, whereas that for [¹4C]Met was almost equal to BCH. These results demonstrate that ASC and L are important transport systems for anti-[¹8F]FACBC uptake, while system L is predominantly involved in [¹¹C]Met transport in human astrocytes and glioma cells.

Trans-1-amino-3-18F-fluorocyclobutanecarboxylic acid (anti-18F-FACBC) is a feasible alternative to 11C-methyl-L-methionine and magnetic resonance imaging for monitoring treatment response in gliomas.

INTRODUCTION: Amino acid PET tracers are promising for visualizing gliomas and evaluating radiochemotherapeutic effects. We compared the glioma detection and early response assessment utility between trans-1-amino-3-fluoro-1-(14)C-cyclobutanecarboxylic acid (anti-(14)C-FACBC) and (3)H-methyl-l-methionine ((3)H-Met) by simultaneously analyzing their uptake by rat gliomas treated with and without temozolomide (TMZ) in vitro and in vivo. METHODS: C6 rat gliomas were incubated with low-dose TMZ to induce chemoresistance. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay demonstrated a significantly greater surviving fraction in the TMZ-resistant subline (C6R) than in drug-naive cells (C6). The anti-(14)C-FACBC and (3)H-Met uptakes were quantified using a triple-label accumulation assay to examine the relationship between tracer uptake and proliferation ((3)H-thymidine (TdR) accumulation rate) in tumor cells. C6 and C6R cells were inoculated into the right and left basal ganglia, respectively, of rats. Efficacy of TMZ against the orthotopic gliomas was analyzed by MRI, Evans blue extravasation, anti-(14)C-FACBC and (3)H-Met autoradiography, and MIB-5 proliferation index. RESULTS: The (3)H-TdR accumulation rate and amino acid tracer (anti-(14)C-FACBC and (3)H-Met) uptake significantly decreased 48 and 72 h, respectively, after TMZ treatment in C6 but not C6R cells. Anti-(14)C-FACBC uptake correlated significantly with (3)H-Met uptake and the (3)H-TdR accumulation rate. In the intracerebral glioma model, anti-(14)C-FACBC and (3)H-Met autoradiography clearly delineated the tumor extent, which spread well beyond the high-T2-intensity and enhancing lesions visible on MRI and Evans blue extravasation. TMZ significantly decreased anti-(14)C-FACBC and (3)H-Met uptake and the MIB-5 index of C6 but not C6R tumors. TMZ inhibited tracer uptake and tumor proliferation before morphological changes on MRI. CONCLUSIONS: Anti-(14)C-FACBC, like (3)H-Met, was more sensitive than post-contrast T1-weighted MRI for detecting tumor extent and early tumor response to TMZ treatment. Anti-(18)F-FACBC should be a sensitive and precise imaging biomarker for tumor extent visualization and response assessment in glioma patients.

Kinetic analyses of trans-1-amino-3-[18F]fluorocyclobutanecarboxylic acid transport in Xenopus laevis oocytes expressing human ASCT2 and SNAT2.

INTRODUCTION: Trans-1-amino-3-[(18)F]fluorocyclobutanecarboxylic acid (anti-[(18)F]FACBC) is a promising amino acid positron emission tomography (PET) radiotracer for visualizing prostate cancer. We previously showed that anti-FACBC is transported by amino acid transporters, especially by alanine-serine-cysteine transporter 2 (ASCT2), which is associated with tumor growth. We studied this affinity to assess the mechanism of anti-FACBC transport in prostate cancer cells. METHODS: Kinetic assays for trans-1-amino-3-fluoro-[1-(14)C]cyclobutanecarboxylic acid ([(14)C]FACBC) were performed in Xenopus laevis oocytes over-expressing either ASCT2 or sodium-coupled neutral amino acid transporter 2 (SNAT2), both of which are highly expressed in prostate cancer cells. We also examined the kinetics of [(14)C]FACBC uptake using mammalian cell lines over-expressing system L amino acid transporter 1 or 2 (LAT1 or LAT2). Results: ASCT2 and SNAT2 transported [14C]FACBC with Michaelis­Menten kinetics Km values of 96.7 ± 45.2 µM and 196.5 ± 19.7 µM, respectively. [correted]. LAT1 and LAT2 transported [(14)C]FACBC with Michaelis-Menten Km values of 230.4 ± 184.5 µM and 738.5 ± 87.6 µM, respectively. CONCLUSIONS: Both ASCT2 and SNAT2 recognize anti-FACBC as a substrate. Anti-FACBC has higher affinity for ASCT2 than for SNAT2, LAT1, or LAT2. The ASCT2-preferential transport of anti-[(18)F]FACBC in cancer cells could be used for more effective prostate cancer imaging.

Transport mechanisms of trans-1-amino-3-fluoro[1-(14)C]cyclobutanecarboxylic acid in prostate cancer cells.

INTRODUCTION: We investigated the mechanisms of trans-1-amino-3-fluoro[1-(14)C]cyclobutanecarboxylic acid (anti-[(14)C]FACBC) transport by human-derived prostate cancer (PCa) cells and normal human prostatic epithelial cells (PrECs). METHODS: Using PCa cells (DU145, PC-3, LNCaP) and PrECs, we performed the following in vitro experiments: time-course, kinetics, competitive inhibition by synthetic/naturally occurring amino acids (AAs), exchange transport with synthetic/naturally occurring AAs and pH-dependency of anti-[(14)C]FACBC uptake. We also examined the amino acid transporter (AAT) expression using flow cytometry. RESULTS: The uptake of anti-[(14)C]FACBC by LNCaP and DU145 cells was higher than that by PC-3 and PrECs. The K(m) values for anti-[(14)C]FACBC were 64.4 and 191.7 µmol/L in the DU145 cells and PrECs, respectively. Total levels of anti-[(14)C]FACBC uptake were positively correlated with the expression level of system ASC in PCa cells. The contributions of Na(+)-dependent AATs to anti-[(14)C]FACBC uptake were greater than those of Na(+)-independent AATs, especially in PCa cells. In the presence of Na(+), glutamine and serine showed the strongest inhibitory effect against anti-[(14)C]FACBC uptake, suggesting that system ASC, especially ASCT2, is an important AAT for anti-[(14)C]FACBC. In contrast, phenylalanine and 2-amino-bicyclo[2,2,1]heptane-2-carboxylic acid, but not N-ethylmaleimide, almost completely inhibited the anti-[(14)C]FACBC uptake in the absence of Na(+), indicating the contribution of LAT1. In the exchange transport experiments, glutamine showed the strongest transstimulation of intracellular anti-[(14)C]FACBC efflux in DU145 cells. Furthermore, the contributions of Na(+)-independent AATs to the uptake of anti-[(14)C]FACBC in DU145 and PrECs were greater under acidic pH conditions than under neutral or alkaline pH conditions. CONCLUSIONS: Total uptake of anti-[(14)C]FACBC by PCa cells correlates with the expression level of system ASC in PCa cells. Furthermore, LAT1 is an important transport system for anti-[(14)C]FACBC uptake, especially in an acidic environment, such as the intra-tumoural environment.

A preliminary study of anti-1-amino-3-18F-fluorocyclobutyl-1-carboxylic acid for the detection of prostate cancer.

UNLABELLED: We evaluated the feasibility of anti-1-amino-3-(18)F-fluorocyclobutyl-1-carboxylic acid (anti-(18)F-FACBC) in diagnosing prostate cancer (PCa), using a rat orthotopic prostate cancer transplantation (OPCT) model. Furthermore, using in vivo experiments, we examined the potential of anti-(18)F-FACBC for differentiating between PCa and inflammation and between PCa and benign prostatic hyperplasia (BPH). METHODS: The OPCT model was developed by transplanting DU145, a human PCa cell line, into the ventral prostate of athymic F344 rats. To develop a dual PCa and inflammation (DPCI) model, MAT-Ly-Lu-B2--a rat PCa cell line--was transplanted subcutaneously into male Copenhagen rats. Streptozotocin was injected into the hind footpad of these rats for inducing popliteal lymphadenitis. For inducing the BPH, normal F344 rats were castrated and injected subcutaneously with testosterone propionate. In biodistribution studies, the rats were injected with anti-(18)F-FACBC or (18)F-FDG and sacrificed at 15 or 60 min after injection. We performed dynamic small-animal PET of the abdominal portion of the OPCT rats for 60 min after the injection of anti-(18)F-FACBC or (18)F-FDG. RESULTS: The biodistribution in the OPCT rats at 60 min after injection showed that the uptake of anti-(18)F-FACBC and (18)F-FDG into the PCa tissue was 1.58 +/- 0.40 %ID/cm(3) (percentage injected dose per cm(3)) and 1.48 +/- 0.90 %ID/cm(3), respectively (P > 0.05). The accumulation of anti-(18)F-FACBC in the urinary bladder at 60 min after injection was 3.09 +/- 1.43 %ID/cm(3), whereas that of (18)F-FDG was 69.31 +/- 16.55 %ID/cm(3) (P < 0.05). Consequently, small-animal imaging with anti-(18)F-FACBC facilitated the visualization of the PCa tissue of the OPCT rats with higher contrast than (18)F-FDG. Furthermore, in comparison with (18)F-FDG, apparently higher ratios of PCa to inflammation and PCa to BPH accumulation of anti-(18)F-FACBC were demonstrated in the animal models. CONCLUSION: FACBC PET is believed to be useful not only for the visualization of human PCa but also for differentiating between PCa and inflammation and between PCa and BHP.