Propionic Acid-Based PET Imaging of the Fatty Acid Synthesis Pathway in Prostate Cancer

The aim of this study was to evaluate the potential value of 2-[ 18 F]uoropropionic acid ([ 18 F]FPA) for PET imaging of prostate cancer (PCa) and to conrm the correlation between [ 18 F]FPA accumulation and fatty acid synthase (FASN) levels in PCa models. The results of the rst [ 18 F]FPA PET study of a PCa patient are reported.


Background
Prostate cancer (PCa) is the most common malignant tumor and the second leading cause of death in the United States [1,2]. Over the past 20 years, the incidence has increased, while PCa mortality has decreased in most countries due to early diagnosis and effective treatment [3,4]. However, unfortunately, the incidence and mortality have been increasing in China in recent years, partly due to rapid socioeconomic development, and most PCas are found in advanced stages [5][6][7]. Accurate diagnosis and staging of PCa using traditional imaging methods, including ultrasound, computer tomography (CT) and magnetic resonance imaging (MRI), remains a challenge in daily clinical practice [8][9][10]. Positron emission tomography (PET) imaging of cancer with [ 18 F] uoro-2-deoxy-d-glucose ([ 18 F]FDG) is based on a fundamental premise: glucose metabolism in malignant tissues exceeds that in normal tissues [11,12].
However, glucose metabolism tends to be low unless PCa has a high Gleason score, thus seriously limiting the ability of [ 18 F]FDG-PET to detect PCa [13,14]. Additionally, the heterogeneity of glucose metabolism in cancer severely hampers the application of [ 18 F]FDG in the diagnosis of PCa due to the different rates of growth, aggressiveness and metastasis [15,16].
Fatty acids are the other principal energy source for tumor cells besides glucose and are essential constituents of membrane phospholipids and important substrates for the energy metabolism of malignant tumor cells (Fig. 1) [17][18][19]. PCa cells are characterized by the predominant uptake of fatty acids over glucose [20,21], and lipid metabolizing enzymes are upregulated in the initial stage of PCa and throughout tumor progression [22][23][24]. Fatty acid synthase (FASN) is a key cytosolic multienzyme complex that catalyzes the de novo synthesis of long-chain fatty acids (Fig. 1). FASN has been found to be overexpressed in multiple human cancers but shows low expression in normal tissues [22][23][24], and its expression is signi cantly associated with poor prognosis, tumor grade and local invasion. Several studies have demonstrated that FASN, a potential malignant tumor marker, may be a potential antitumor target [25][26][27][28]. Moreover, FASN inhibition can effectively reduce fatty acid synthesis, leading to suppression of cell proliferation and adhesion, migration, and invasion [29][30][31][32]. Orlistat is an irreversible inhibitor of FASN via the thioesterase domain of the enzyme and shows potent anticancer effects in malignant tumors [29][30][31][32].
Lipid metabolism, in particular the de novo synthesis of fatty acids, is an essential cellular process that converts nutrients into metabolic intermediates and is vitally important for cell proliferation [33][34].
Propionic acid is a short-chain fatty acid participating in lipid metabolism and is an energy-providing substrate for heart and tumor cells (Fig. 1 [36][37][38].
Given the nutrient dependence of PCa on fatty acids, we then hypothesized that the uptake mechanism of

Chemicals and equipment
All reagents used in the synthesis were commercial products applied without further puri cation unless otherwise indicated. All chemical reagents and equipment were purchased from commercial sources.  [36][37][38].
Animal models and Small-animal PET imaging Mice were purchased from the Laboratory Animal Center of Sun Yat-sen University (Guangzhou, China) and housed 5-6 animals per cage under standard laboratory conditions. LNCaP, PC-3 and DU145 cells (1×10 7 cells) were injected subcutaneously into BALB/c nude mice (weighing 18-22 g, male). PET-CT imaging was performed when the tumors grew to approximately 5-10 mm in diameter [36][37][38]. The experiments were approved by the Institutional Animal Care and Utilization Committee (IACUU) of the First A liated Hospital, Sun Yat-sen University (approval no. 2016058). Protocol for Small-animal PET imaging were performed as described previously [36][37][38].

Small-animal PET assessment of FASN inhibition
Twenty-four hours after PET imaging with [ 18 F]FPA, the same mice (n=3) received intraperitoneal injections of 240 mg/kg orlistat dissolved in DMSO (<2% DMSO). Sixty minutes after injection of the orlistat, the mice were again injected intravenously with 3.7-5.5 MBq (100-150 μCi, 0.1 mL of saline) of [ 18 F]FPA, and the mice then underwent PET scanning again following the same protocol.

Patient preparation and imaging protocol
The Institutional Review Board of the First A liated Hospital, Sun Yat-sen University approved this human study (Approval No. 2019168). Informed consent was obtained before imaging. The patient fasted for at least 6 hours before 18 F-FDG and [ 18 F]FPA was injected intravenously. The start of PET/CT acquisition was 60 min after 18 F-FDG or [ 18 F]FPA injection (3.7 MBq/kg body weight). Imaging was performed with a uMI 780 128-slice CT apparatus (Shanghai United Imaging Healthcare, China) in the supine position. The CT scan (5 mm slice thickness) from the base of the skull to the inferior border of the pelvis was acquired according to standardized protocol (120 kV and 180 mA). The CT scan was followed immediately by a PET scan (6-7 bed positions, 2 minutes per bed position) acquired in threedimensional mode. Attenuation was corrected using the CT images.

Statistical analysis
Data are represented as the mean ± SEM unless speci ed otherwise. Graphs were plotted, and statistical analyses were performed using Prism software (version 6, GraphPad). All data are expressed as the mean±standard deviation (SD). The statistical analysis was performed with SPSS software version 20.0 (SPSS, Inc.) for Windows (Microsoft). Comparisons between conditions were performed by using unpaired, 2-tailed Student's t-tests. P<0.05 was considered statistically signi cant.

Results
Propionic acid-based tracers show favorable characteristics for in vivo PET imaging of PCa  (Fig. 3). In the livers of the LNCaP and PC-3 xenograft tumor-bearing mice, the radioactivity uptake slightly decreased after treatment with orlistat (P<0.05). In the normal tissues of those tumor-bearing mice, including the brain and muscle, the radioactivity uptake showed no marked reduction after treatment with orlistat (P>0.05).

The [ 18 F]FPA tracer shows a high tumor-to-background uptake ratio in human PCa with progression
The results showed no obvious abnormal [ 18 F]FDG uptake in the regions of the prostate (Fig. 4A).
However, abnormal [ 18 F]FPA avidity was observed in the tumor regions in the prostate (Fig. 4C). Furthermore, this patient's tumor demonstrated a slightly low signal in the T 2 W image (Fig. 4I) and mild contrast enhancement on MRI (Fig. 4H), and no obvious abnormal signals were observed in the T 1 W image on MRI (Fig. 4G). The MRI results were misdiagnosed as hyperplasia of the prostate gland in the preoperative examination. The nal pathological con rmation was PCa in the right medial prostate ( In addition, in ammatory lymph nodes in the hilum and mediastinum showed signi cant [ 18 F]FDG uptake, but slight [ 18 F]FPA uptake was found in those lymph nodes (Fig. S6). Moreover, benign tumors of the right parotid gland showed signi cant [ 18 F]FDG uptake but slight [ 18 F]FPA uptake (Fig. S7).

Discussion
Highly proliferative tumor cells depend on glucose, amino acids and fatty acids as the principal energy sources, and these cells reprogram their metabolism of nutrients through glycolysis, glutaminolysis and fatty acid synthesis, which is vital for cancer survival and growth [39][40][41]. Although [ 18 F]FDG PET is commonly used to measure glucose metabolism in various tumors, it is ineffective for PCa due to the lack of glycolysis [13,14]. Upregulation and increased activity of fatty acid synthase occurs throughout PCa carcinogenesis and usually correlates with a worse prognosis. A growing body of evidence on altered tumor fatty acid synthesis has shown the critical role of FASN in tumorigenesis and progression [22][23][24]. FASN is a potential PET imaging agent and antitumor therapeutic target for PCa. We aimed to develop a means of noninvasively assessing fatty acid energy metabolism in PCa and to transform it into a clinically useful imaging modality, which will promote early clinical diagnosis and evaluation of the therapeutic effects and prognosis of PCa.
PET imaging analogs of fatty acids such as [ 11 C]AC and [ 18 F]FAC have been used to image human PCa, but some factors limit their clinical application [42][43][44][45] provide additional information about the metabolic mechanism that is relevant to propionic acid [35][36][37][38].
Indeed, [ 18 F]FPA PET imaging takes advantage of fatty acid dependence in cancer and may serve as a valuable tool to assess fatty acid accumulation in PCa in vivo.
Our previous research has shown that [ 18 F]FPA uptake in liver cancer could be mainly mediated by FASN [37,38], which is minimally expressed in normal tissue but strongly increased in malignant tumors. The  F]FPA accumulation was noted in the bones of animal models and humans, demonstrating that the probe was stable in vivo and that no de uorination occurred. More importantly, the relatively low background uptake in most normal tissues indicated that [ 18 F]FPA is a potential broad-spectrum PET imaging agent for tumors.
A limitation of this study is that [ 18 F]FPA shows a high background level in the blood pool of animal models and humans, probably due to the involvement of [ 18 F]FPA in plasma protein binding. Another limitation is that only one PCa subject was assessed.

Conclusions
Our data presented here support the assumption that propionic acid-based PET imaging of the fatty acid synthesis pathway through [ 18

Availability of data and materials
Most of the data generated or analysed during the present study are included in this published article.

Ethics approval and consent to participate
The animal experiments were approved by the Institutional Animal Care and Utilization Committee of the First A liated Hospital, Sun Yat-sen University. The Institutional Review Board of the First A liated Hospital, Sun Yat-sen University approved this human study.

Consent for publication
All Authors have seen and approved the manuscript and consent publication.

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