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PET Imaging of 18F-(2S,4R)4-fluoroglutamine Accumulation in Breast Cancer: from Xenografts to Patients Fei Liu, Xiaoxia Xu, Hua Zhu, Yan Zhang, Jianhua Yang, Lifang Zhang, Nan Li, Lin Zhu, Hank F. Kung, and Zhi Yang Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.8b00430 • Publication Date (Web): 09 Jul 2018 Downloaded from http://pubs.acs.org on July 9, 2018

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Molecular Pharmaceutics

PET Imaging of 18F-(2S,4R)4-fluoroglutamine Accumulation in Breast Cancer: from Xenografts to Patients Fei Liu1,#, Xiaoxia Xu1,#, Hua Zhu1,#, Yan Zhang2, Jianhua Yang1, Lifang Zhang2, Nan Li1, Lin Zhu2, Hank F. Kung3,4,*, Zhi Yang1,* 1

Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education/Beijing),

Department of Nuclear Medicine, Peking University Cancer Hospital & Institute, Beijing 100142, China 2

Key Laboratory of Radiopharmaceuticals, Ministry of Education, College of Chemistry, Beijing

Normal University, Beijing 100875, China 3

Department of Radiology, University of Pennsylvania, Philadelphia, PA 19104, USA

4

Beijing Institute for Brain Disorders, Capital Medical University, Beijing 100069, China

#

These authors contributed equally to this article.

ABSTRACT Sustaining the growth of tumor cells requires extra energy and metabolic building blocks. In addition to consuming glucose, glutamine may play the role as an alternative source of nutrient for growth and survival. We aim to characterize, a glutamine analog,

18

F-(2S,4R)4-fluoroglutamine

(18F-(2S,4R)4-FGln), as an imaging agent for interrogating the role of glutamine from in vitro study of tumor cells to clinical manifestation in breast cancer patients. Purity was measured by radio-high performance liquid chromatography (Radio-HPLC), and the stability after production was evaluated in phosphate buffer saline (PBS), saline, mouse and human serum buffers. The presence of Myc expression in MCF-7 and U87 cells was conducted using qPCR. In vitro cell uptake of 18

F-(2S,4R)4-FGln in MCF-7 and U87 cells was directly compared with

(18F-FDG). In vivo biodistribution and micro-PET imaging of BALB/c nude mice were performed. PET/CT imaging of

18

18

18

F-fluorodeoxyglucose

F-(2S,4R)4-FGln in MCF-7 bearing

F-(2S,4R)4-FGln was compared with

18

F-FDG in the same group of breast cancer patients (n = 10). We successfully synthesized

18

F-(2S,4R)4-FGln with a high radiochemical purity (>98 %), and the radiochemical purity was

unchanged in PBS and saline buffers during 2 hr incubation. In vitro cell uptake studies of 18

F-(2S,4R)4-FGln displayed a rapid and higher uptake in MCF-7 and U87 cells as compared with

18

F-FDG. Biodistribution and micro-PET images showed excellent tumor accumulation of

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Page 2 of 23

18

F-(2S,4R)4-FGln in MCF-7 implanted mice tumor model. In a preliminary clinical study,

18

F-(2S,4R)4-FGln/PET detected more lesions in breast cancer patients than

18

F-FDG/PET (90 % vs.

80 %). Additionally, in one patients with breast lobular carcinoma there were higher lesion mean standardized uptake value (SUVmean) and maximum standardized uptake value (SUVmax) values for 18

F-(2S,4R)4-FGln than those obtained by 18F-FDG as determined by PET imaging. 18F-(2S,4R)4-FGln

may be a useful glutamine-targeting metabolic probe for noninvasive imaging of breast cancer. KEYWORDS: glutamine; PET; breast cancer; metabolic imaging; Myc

INTRODUCTION Cancer is a form of uncontrolled growth of cells that can appear in many parts of human body. Each year there are 14 million new cases of cancer occurred in the world. Cancer is now the leading cause of death in China. And lung cancer and breast cancer are the most common types of cancers among men and women, occurring at a rate of 23 % and 17 % of all cancer cases, respectively 1. Development of new diagnostic method for detecting and monitoring tumor growth is highly important. Imaging tumor metabolism using 18F-fluorodeoxyglucose (18F-FDG) in conjunction with positron emission tomography (PET) is the most commonly used nuclear medicine imaging technique. A widespread use of 18F-FDG/PET is closely associated to “Warburg effect” in tumor cells. The “Warburg effect” indicates that tumor cells take up more glucose for energy production. Even in aerobic environment, tumor cells convert glucose mainly to lactic acid, and the lower the pathological differentiation degrees of the tumors are, the more energy the tumors need trials there were false-positive and false-negative reported for

18

2, 3

. However, in clinical

F-FDG/PET due to individual

differences of tumors, which lead to erroneous diagnosis of the diseases

4, 5

. There are three main

reasons leading to the incorrect diagnosis of patients with breast cancer. Firstly, as PET can only detect lesions of 6-10 mm, and false-negative diagnosis may exist in smaller tumors. Secondly, 18F-FDG/PET imaging can’t distinguish between benign breast tumors and inflammation. Thirdly, higher muscle uptake and elevated blood glucose levels may cause false-negative results

6, 7

. Due to the high

prevalence of breast cancer cases and the importance of correct diagnosis, there is an urgent need for developing new probes for improving accuracy of detection using PET imaging. Recently, glutamine, a nonessential amino acid, is found to play a key role in tumor proliferation 8, 9

. It may serve as a substrate for supporting growth and proliferation. In fact, most tissues of mammals

can synthesize glutamine, but under a stressed situation such as development of tumor, the cells need more glutamine for energy supplying. The glutamine consumption rate in tumors cells such as human uterine carcinoma cells, human breast cancer cells and human non-small cell lung cancer cells is


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10-times more than that in normal cells

8, 10

. Glutamine metabolism is considered to be another

important metabolic pathway in tumor cells above and beyond the “Warburg effect” on glycolysis. Molecular imaging especially for glutamine metabolism may provide a new opportunity for diagnosis of glutamine-addicted tumors. Glutamine transporters (ASCT2; LAT1; SNAT) are responsible for moving this amino acid across the cell membrane. Glutamine provides nitrogen for macromolecules biosynthesis. On the other hand it is converted into glutamate by glutaminase, which is then converted by glutamate dehydrogenase to α-ketoglutarate, subsequently. Through which it enters the tricarboxylic acid (TCA) cycle and provides energy for the cellular metabolism. Tumor cells metabolism studies have shown that activation of specific signaling pathways, such as the up-regulation of Myc gene, can 11-13

promote the intake of glutamine in cancer cells

. Myc gene proliferation is the most common

activity observed in tumors, which can promote the progress of lymphoma, neuroblastoma and small cell lung cancer

14

. Moreover, Myc gene can activate transcription of glutamine transporters,

up-regulate the activity of glutaminase and promote glutamine converted into glutamate 15, 16. Many tumors exhibit various degrees of dependence on glutamine. Multiple evidences suggested that non-small cell lung cancer cells showed a range of glutamine dependency, and 15 out of 39 cell lines (46 %) showed high glutamine dependency 17. Son J et al., reported that glutamine could support the growth of pancreatic cancer

18

. Shajahan-Haq et al., has reported that Myc gene regulated the

glutamine uptake in breast cancer 19. Among all tumor types, breast cancer is of the highest morbidity in women. However, clinical application of using

18

F-FDG/PET to detect breast cancer showed less

sensitivity as compared to other types of cancers 20, 21. (18F-(2S,4R)4-FGln), as reported previously

8, 22

18

F-(2S,4R)4-fluoroglutamine

, was a novel radiotracer for metabolism of glutamine

in cancers and may guide therapies based on glutamine-metabolism. The cell uptake of 18

F-(2S,4R)4-FGln in 9L and SF188 cell lines was higher than that of

18

F-FDG, and it showed high

tumor uptake in the corresponding 2 tumor models. This new glutamine probe may be useful for studying tumor metabolism in breast cancer cells. It is generally accepted that glutaminolysis is an alternative metabolic pathway in supporting cancer growth. Various invasive cancers under stressful microenvironments may adopt it to overcome the limitations of nutrients and oxidative potential

8, 10

. It was previously reported by Zhou et al

23

that

triple negative breast cancer (TNBC; cancer cells that do not express the genes for estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2) are especially predisposed to utilize glutamine for survival and growth due to reprogramming of gene expression). 18

F-(2S,4R)4-FGln/PET imaging of xenografted TNBC tumor model in mice exhibited high

glutaminase activity 23. Therefore, they suggested that 18F-(2S,4R)4-FGln may be useful for evaluation



of drugs targeting glutamine metabolism

8, 23

Page 4 of 23

. It was reported that the inhibition of glutaminase may

increase the intracellular glutamine pool in MCF-7 mice tumor model 23. However, TNBC accounts for only 15 %-20 % of breast cancer, we chose the estrogen receptor positive cells (MCF-7 breast cancer cells) as a tumor cell model for further study. Previously, Dunphy et al., has demonstrated that clinically aggressive high-grade gliomas and TNBC tumors displayed high

18

F-(2S,4R)4-FGln uptake.

But the less-aggressive low-grade gliomas and receptor-positive tumors showed no 18F-(2S,4R)4-FGln uptake. Except in one case that brain metastases in one patient with receptor-positive breast cancer with HER2 overexpression (often implicated in glutamine-addiction). Aggressive TNBC is reported to be a “glutamine-addicted” cancer

9, 24, 25

. Therefore, 18F-(2S,4R)4-FGln/PET imaging might have prognostic

value for detecting metabolically aggressive tumor. Reported herein is the evaluation of

18

F-(2S,4R)4-FGln as a probe to study glutamine addicted

breast cancer. Results of radio-synthesis, quality control, characterization of

18

F-(2S,4R)4-FGln in

MCF-7 tumor cells and clinical assessment of the probe in breast cancer patients. Clinical images of 18

18

F-(2S,4R)4-FGln/PET were compared with that of

F-FDG/PET in breast cancer patients to further

confirm its clinical application.

MATERIALS AND METHODS Materials, Cell Culture and Animal Model. Detailed information of the materials, cell culture and animal model is provided in the Supplemental Materials. Radio-synthesis and Quality Control of the Radiotracer. The radio-synthesis and stability of 18

F-(2S,4R)4-FGln are provided in the Supplemental Materials. Real-time Quantitative PCR (qPCR). The real-time quantitative PCR (qPCR) study was

conducted to evaluate the relationship between Myc gene and MCF-7 and U87 cells (Supplemental Materials). Cell Uptake Studies. In vitro cell uptake of

18

F-(2S,4R)4-FGln compared with

18

F-FDG was

performed in MCF-7 and U87 cells (Supplemental Materials). Pharmacokinetics and Biodistribution Studies. Healthy BALB/c mice (18-20 g) were used to conduct the pharmacokinetics study. 3.7 MBq of

18

F-(2S,4R)4-FGln was intravenously injected into

each mouse (n = 5), at the set intervals (2, 5, 10, 15, 30, 45, 60, 90 and 120 min), 10 µL of blood taken from tail vein was acquired, weighed and counted using a gamma counter and the result was determined as percentage of injected dose per gram organ/tissue (% ID/g), and the parameters of pharmacokinetics was analyzed using the Graphpad Prism software (Graphpad Software, California, CA, USA). Biodistribution studies were conducted in healthy BALB/c and BALB/c nude mice bearing


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MCF-7 tumors, respectively. The MCF-7 tumor bearing models were prepared by subcutaneously injecting 2 × 106 cells into the right subaxillary region of the BALB/c nude mice. After about 2 weeks, the tumor diameter could reach 0.6-0.8 mm ready for biodistribution studies. The mice were injected intravenously with 1.11 MBq (0.03 mCi) of the radiotracer to evaluate its biodistribution in tumor and normal organs. After predetermined periods (2, 30, 60, 120 and 180 min for BALB/c and 30, 120 and 180 min for MCF-7 BALB/c nude mice), the mice were sacrificed under anesthesia, blood, tumor tissues and major organs were excised, collected, weighed. The radiotracer activity of the organs and tissues were measured using the gamma counter and the result was calculated as % ID/g. Data of the pharmacokinetics and biodistribution studies were expressed as mean ± SEM (n = 3). Both experiments were repeated for three times. Micro-PET Imaging Studies in Nude Mice. Micro-PET imaging studies was conducted using BALB/c nude mice bearing MCF-7 breast cancer cells with a PET system (SuperArgus, Sedecal, Spain). After the tumor reached about 0.6-0.8 mm in diameter, the mice were intravenously injected (14.8 MBq, 0.4 mCi, of

18

F-(2S,4R)4-FGln). A series of static scans were acquired at 1 and 2 h

post-injection under the anesthesia of 2 % isoflurane. After the scans, the mice were moved out from the scan bed and given enough food and water. The images were reconstructed using the three-dimensional ordered subsets expectation maximum (OSEM) algorithm without correction for attenuation. Region of interests (ROIs) was drawn over the region of tumor, heart, kidney and muscle, and the tumor-to-blood (T/B) and tumor-to-muscle (T/M) ratios were obtained using the ASI Pro 5.2.4.0 software. The experiments were repeated for three times at each time point. PET Imaging Studies in Patients. Patients who were suspected to have breast cancer were recruited from Peking University Cancer Hospital & Institute (Exclusion criteria were those who were pregnant or lactating, who had severe liver and kidney dysfunction, or those who couldn’t keep lying in the scan bed for more than 1 h). A total of ten patients had signed the informed consents before enrolled in the study. The study was approved by the Peking University Cancer Hospital & Institute Ethics Committee (Permission No. 2017KT38). Table 1 provides the details of patients’ information. Each patient underwent

18

F-(2S,4R)4-FGln/PET and

18

F-FDG/PET imaging studies within an interval of

more than 1 d but less than 7 d. On the day of the examination, the patient was fasted for more than 6 h. Each patient received 3.7 MBq/kg of 18F-(2S,4R)4-FGln or 18F-FDG intravenously. A series of 10 min static PET/CT images of

18

F-FDG were obtained at 1 h using the Siemens Biograph mCT Flow 64

scanner (Erlangen, Germany). Dynamic images of 18F-(2S,4R)4-FGln were acquired during the first 1 h post-injection. The length of PET/CT imaging was from vertex to the first 1/3-thigh. Images were reconstructed using OSEM. PET and CT images were fused using the Fusion Viewer software provided



Page 6 of 23

by the manufacturer. Two experienced nuclear medical doctors read the PET/CT images. ROIs were drawn manually over the region of tumor regions. Results were calculated as mean standardized uptake value (SUVmean) and maximum standardized uptake value (SUVmax). Hematoxylin-eosin and Immunohistochemistry Staining. Patients who were suspected of breast cancer by PET/CT examination were subsequently diagnosed by core needle biopsy (CNB). Detailed information is provided in Supplemental Materials. Statistical Analysis. Quantitative data in the study were expressed as mean ± SEM. Mean values were determined using Student t test and one-way ANOVA analysis. A P value of less than 0.05 was considered to be statistically significant.

RESULTS Radio-synthesis and Characterization of 18F-(2S,4R)4-FGln. Detailed results of radio-synthesis, quality control and stability of 18F-(2S,4R)4-FGln are provided in the Supplemental Materials. Real-time Quantitative PCR. To evaluate the relationship between Myc gene and MCF-7 and U87 cells, the qPCR study was conducted. And the mRNA ratios of Myc/GAPDH for MCF-7 and U87 was 5.17 ± 0.16 and 6.45 ± 0.05, respectively (Figure S3). Since up-regulation of Myc gene may stimulate glutaminolysis and render cells addicted to glutamine. Results of the analysis showed the expression level of Myc gene was significantly higher in MCF-7 cells and U87 cells, which suggests the potential of using 18F-(2S,4R)4-FGln to study the glutamine-addiction in MCF-7 and U87 cells. No qPCR measurements were performed for biopsy samples from breast cancer patients. In Vitro Cell Uptake Studies. In vitro cell uptake of

18

F-(2S,4R)4-FGln showed a higher uptake

than that of 18F-FDG in MCF-7 and U87 cells between 5 min to 2 h incubation time periods (Figure 1A and 1C), and the uptake of 18F-(2S,4R)4-FGln in MCF-7 cells was faster than that of

18

F-FDG during

the first 30 min of incubation. For MCF-7 cells, the uptake of 18F-(2S,4R)4-FGln reached 6.38 ± 0.55, 8.51 ± 0.21 and 8.89 ± 0.45 percentage of collected count to total added count (% AD/106 cells) at 30, 60 and 120 min, respectively, while the uptake of

18

F-FDG was 2.01 ± 0.15, 3.11 ± 0.44 and 3.95 ±

0.71 % AD/106 cells, respectively (Figure 1A). While for U87 cells, the uptake of

18

F-(2S,4R)4-FGln

reached 1.51 ± 0.13, 1.69 ± 0.08 and 2.77 ± 0.17 % AD/106 cells at 30, 60 and 120 min, respectively, and the uptake of 18F-FDG was 1.18 ± 0.02, 1.48 ± 0.06 and 1.77 ± 0.27 % AD/106 cells, respectively (Figure 1C). 18

L-glutamine (0.5 mM) significantly blocked the uptake. The uptake of

F-(2S,4R)4-FGln in MCF-7 cells reduced to 1.03 ± 0.04 % AD/106 cells (30 min) and 0.78 ± 0.07 %

AD/106 cells (60 min) after 0.5 mM of cold L-glutamine was added (Figure 1B). The uptake of 18

F-(2S,4R)4-FGln in U87 cells reduced to 0.35 ± 0.02 % AD/106 cells (30 min) and 0.51 ± 0.01 %

AD/106 cells (60 min) after 0.5 mM of cold L-glutamine was added (Figure 1D). And MCF-7 was


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selected as a model for further evaluation of

18

F-(2S,4R)4-FGln by studying biodistribution and

micro-PET imaging in implanted nude mice.

Figure 1. (A and C) In vitro cell uptake of

18

F-(2S,4R)4-FGln and

18

F-FDG in MCF-7 and U87 cells.

(B and D) Blocking studies of 18F-(2S,4R)4-FGln in MCF-7 and U87 cells. Uptake was expressed as % AD/106 cells. Data were represented as mean ± SEM. Pharmacokinetics and Biodistribution of radioactivity in the blood after tail-injection of

18 18

F-(2S,4R)4-FGln in BALB/c mice. Profile of

F-(2S,4R)4-FGln in BALB/c mice was delineated

using Graphpad Prism software (Figure 2). The profile fits well into a two-compartment pharmacokinetics formula presented as follows: Ct (% ID/g) = SpanFast*exp (-αt) + SpanSlow*exp (-βt), where SpanFast and SpanSlow are the y intercepts in the model, α and β are the fast and slow rate constants, expressed in reciprocal of the time axis. In this model the equation of

18

F-(2S,4R)4-FGln in

BALB/c mice was Ct (% ID/g) = 5.45*exp (-0.48t) + 1.57*exp (-0.02t), and the half life was 1.5 and 43.7 h for distribution and elimination phase, respectively. Biodistribution of 18F-(2S,4R)4-FGln in BALB/c and BALB/c nude mice was depicted in Figure 3. As shown in Figure 3A, rapid accumulation was achieved in kidney (15.62 % ID/g), intestine tissues (13.21 and 10.03 % ID/g and bone (7.75 % ID/g) at 2 min, and the radiotracer was quickly cleared through kidney by 30 min, rapid and high uptake was observed with bone tissue. Brain uptake was low and consistent during the 3 h (1.20 % ID/g at 2 min and 0.96 % ID/g at 3 h). And for biodistribution in MCF-7 xenografts in BALB/c nude mice, tumor uptake of 18F-(2S,4R)4-FGln could reach 4.83 % ID/g



at 30 min and 6.29 % ID/g at 60 min, the MCF-7 tumor/blood and tumor/muscle ratio reached from 1.94 and 1.33 at 30 min to 6.37 and 3.91 at 60 min. The biodistribution results also presented high uptake in pancreases (25.76 % ID/g at 30 min) and bone (13.61 % ID/g at 30 min) tissues, and the other organs showed similar uptake with normal BALB/c mice (Figure 3B). 5

4

% ID/g

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 8 of 23

3

2

1

0 0

20

40

60

80

100

120

140

Time (Min)

Figure 2. Blood radioactivity profile of 18F-(2S,4R)4-FGln. Data were expressed as mean ± SEM.

Figure 3. Biodistribution of

18

F-(2S,4R)4-FGln in BALB/c mice (A) and BALB/c nude mice bearing

MCF-7 tumors (B). Data were expressed as mean ± SEM. Micro-PET Imaging Studies in Nude Mice. Micro-PET imaging of 18F-(2S,4R)4-FGln in BALB/c nude mice bearing MCF-7 tumor cells was shown in Figure 4A. The uptake in kidneys decreased rapidly from 60 min to 120 min, indicating the radiotracer was mainly excreted through the kidneys. Rapid excretion from kidney was in accordance with the biodistribution results (Figure 3). Well-defined tumor accumulation of

18

F-(2S,4R)4-FGln was detected from 60 min to 120 min, with

high contrast with surrounding background. The T/B and T/M ratios of the injected mice were shown in Figure 4B, at 1 h and 2 h post-injection, the T/B ratios were 1.25 ± 0.02 and 1.51 ± 0.02, and the T/M ratios were 2.06 ± 0.04 and 2.49 ± 0.08.


Page 9 of 23 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 4. (A) Micro-PET imaging of 18F-(2S,4R)4-FGln (coronal slide) in BALB/c nude mice bearing MCF-7 tumors at 1 and 2 h post-injection. (B) Tumor-to-blood (T/B) and tumor-to-muscle (T/M) ratios of the radiotracer. The white arrows depicted the tumors. PET Imaging Studies in Patients. As in our study, the product of

18

F-(2S,4R)4-FGln was

colorless liquid with a pH value of 6.2-7.0, and there were no remains of acetonitrile and ethanol in the final product. The radiolabeling purity of 18F-(2S,4R)4-FGln was over 98 % and can reach the standard of evaluation of radiopharmaceuticals. The final product was dissolved in 0.9 % saline and filtered using a 0.20 µm Millipore filter. And according to the Provisions for Preparation of Positron Radiopharmaceuticals in Medical Institutions, the product of

18

F-(2S,4R)4-FGln could be used for

further clinical experiments. Among the ten breast cancer patients enrolled in the study, nine primary breast cancer lesions and three 18

metastasized

axillary

lymph

nodes

lesions

were

accurately

diagnosed

with

F-(2S,4R)4-FGln/PET while only eight primary breast cancer lesions and three metastasized axillary

lymph nodes lesions with 18F-FDG/PET (Table 1). All suspected breast cancer lesions and lymph nodes were confirmed by core needle biopsy. Results of immunohistochemical staining assay were included in Table 2. All breast cancer detected by 18F-(2S,4R)4-FGln/PET could clearly be separated from the surrounding breast tissues. Boundaries delineated by

18

F-(2S,4R)4-FGln/PET scan showed a good

correspondence with the matched CT images. Additionally, the SUVmax and SUVmean of 18

F-(2S,4R)4-FGln/PET for breast cancer lesions showed excellent results, which were 4.69±1.87,

3.87±1.62 respectively. The representative PET/CT images of the two radiotracers in a 57-year-old female patient were depicted in Figure 5. The SUVmax of 18F-(2S,4R)4-FGln reached 4.55, which was much higher than that of

18

F-FDG (1.60) for the lesion. And the SUVmean were 3.00 ± 0.68 and 1.20 ± 0.30 for

18

F-(2S,4R)4-FGln and

18

18

F-(2S,4R)4-FGln and 18F-FDG was 7.05 ± 0.05 and 3.38 ± 0.18, respectively.

F-FDG, respectively. The mean lesion to normal breast ratio for



Page 10 of 23

Figure 5. PET/CT imaging of 18F-(2S,4R)4-FGln (A-B), 18F-FDG (C-D) and whole body MIP imaging of 18F-(2S,4R)4-FGln (E) at 1 h in a 57-year-old female patient. The red arrows depicted the tumors. Table 1. Patient Information No.

Age(y)/Sex

1

57/F

2

50/F

3

40/F

4

54/F

5

46/F

6

52/F

7

48/F

8

40/F

9

51/F

10

49/F

Pathological Type

Tumor Avidity for 18 F-(2S,4R)4-F Gln†

SUVmax/S UVmean

Treatment*

lobular carcinoma ductal carcinoma ductal carcinoma ductal carcinoma ductal carcinoma metastasized lymph node metastasized lymph node ductal carcinoma metastasized lymph node ductal carcinoma ductal carcinoma mucinous carcinoma ductal carcinoma

Yes

4.49/3.00

Yes

SUVmax/SUV

NA

Tumor Avidity for 18 F-FD G No

2.75/1.69

NA

Yes

4.31/3.09

Yes

3.91/3.43

NA

Yes

5.20/3.78

Yes

8.89/7.53

NA

Yes

10.41/10.06

Yes

4.78/4.00

NA

Yes

6.39/4.04

Yes

5.67/5.21

NA

Yes

6.60/4.05

Yes

3.42/3.13

NA

Yes

3.57/3.16

Yes

4.94/4.16

NA

Yes

4.70/3.45

Yes

3.12/3.00

NA

Yes

3.9/2.45

Yes

3.45/3.11

NA

Yes

5.11/3.66

Yes

5.59/4.69

NA

Yes

6.89/4.57

No

1.83/1.84

NA

No

2.18/2.08

Yes

6.25/5.22

NA

Yes

5.62/4.07


mean

2.45/2.07

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Note: NA = not applicable or data not available. *Any treatment less than 4 weeks before 18F-(2S,4R)4-FGln PET. †

Avid tumors demonstrated tracer uptake and retention greater than blood pool and background tissues at

18

F-(2S,4R)4-FGln/PET.

Table 2. Results of Immunohistochemical Staining Assay in 10 patients No.

Immunohistochemistry ER

PR

HER2

Ki67

P120

1

95 %

5%

1+

20 %

1+

2

90 %

90 %

2+

20 %

NA

3

95 %

5%

2+

30 %

1+

4

95 %

95 %

1+

15 %

NA

5

90 %

70 %

3+

40 %

NA

6

80 %

80 %

1+

20 %

NA

7

75 %

75 %

1+

14 %

NA

8

75 %

75 %

1+

10 %

NA

9

95 %

95 %

1+

5%

NA

10

90 %

95 %

-

70 %

NA

Note: NA = not applicable or data not available. ER: genes for estrogen receptor. PR: progesterone receptor. HER2: human epidermal growth factor receptor 2. Ki67: nuclear-associated antigen. P120: P120 caterin.

Hematoxylin-eosin and Immunohistochemistry Staining. The represented hematoxylin-eosin (HE) and immunohistochemistry (IHC) results of the 57-year-old female patient were shown in Figure 6. Based on the pathological analysis the patient was diagnosed as invasive lobular breast carcinoma. The HE staining illustrated a typical invasive adipose growth of the tumor cells, the ER expression result showed 95 % of the tumor cells was positive, the PR expression showed 5 % positive, the HER2 expression was 1+ with faint staining of over 10 % of the tumor cells, and the positive staining of P120 catenin (P120) depicted a strong cytoplasmic staining. The nuclear-associated antigen (Ki67) index of the tissues was +20 %.

Figure 6. Immunoprofile of ER and P120 of the 57-year-old female patient. (A) HE staining depiceted



Page 12 of 23

typical tumor invasive histology. (B) ER staining showed diffuse strong expression. (C) P120 showed strong cytoplasmic staining. Original magnification × 200.

DISCUSSION In this study, we evaluated the characteristics of

18

F-(2S,4R)4-FGln uptake in tumor cells and

explored its advantages as a PET imaging probe in glutamine addicted breast cancer cells. We synthesized the radiotracer with a high radiochemical purity, which fulfilled the requirements for a clinically suitable dose (Figure S1). As Myc gene can upregulate the metabolism of glutamine for energy requirement and building block production, breast cancer with upregulated Myc expression may particularly dependent on glutamine

19, 26

. The qPCR result of Myc/GAPDH ratio showed a high Myc gene level in MCF-7 and

U87 cells (Figure S3). The cell uptake of

18

F-(2S,4R)4-FGln was significantly higher than that of

18

F-FDG in MCF-7 cells and the in vivo cell blocking experiment validated the specificity of the

18

F-(2S,4R)4-FGln probe in MCF-7 breast cancer cells (Figure 1). Therefore, this cell line was selected

as tumor model for biodistribution and micro-PET imaging studies. The main reason for inconsistency between cell uptake and Myc mRNA levels in MCF-7 and U87 cells is that the glutamine uptake in tumor cells is not only related to Myc mRNA levels but to other factors such as amino acid transporters (such as sodium-neutral amino acid transporters (SNAT), alanine, serine, cysteine-preferring transporter 2 (ASCT2) and large neutral amino acid transporter 1 (LAT1)) and glutaminase, but Myc mRNA levels is a core influencing factor 8. The in vivo specificity of

18

F-(2S,4R)4-FGln in MCF-7 tumor model was demonstrated by

biodistribution and micro-PET imaging (Figures 3 and 4). The biodistribution of

18

F-(2S,4R)4-FGln

showed a rapid tumor accumulation at 30 min post-injection in BALB/C nude mice bearing MCF-7 tumors, and the uptake consistently increased at 1 h after injection. The results confirmed that the MCF-7 tumor displayed excellent uptake as visualized by micro-PET/CT imaging. Among the ten patients enrolled in the study, PET images showed nine patients could be correctly diagnosed with 18

F-(2S,4R)4-FGln/PET, while only eight patients were diagnosed by

patient (No. 9) was diagnosed negative with both

18

18

F-FDG/PET. However, one

F-(2S,4R)4-FGln/PET and

18

F-FDG/PET. This is

mainly because breast mucinous cancer has a low tumor cell density and a high concentration of mucin, so there is lower

18

F-(2S,4R)4-FGln and

18

F-FDG uptake on pure mucinous carcinomas 27. Moreover,

PET imaging study on one breast cancer patient showed a much higher tumor accumulation with 18

F-(2S,4R)4-FGln than that of 18F-FDG (Figure 5). And the patient（No. 1）was finally identified as

invasive lobular breast carcinoma by CNB. Therefore, results indicated that

18

F-(2S,4R)4-FGln/PET

may have an advantage in the diagnosis of breast lobular carcinoma than 18F-FDG/PET.


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Recent reports suggested that proliferation of tumor cells (including glioma, sarcoma, acute myeloid leukemia and other tumors) associated with mutations of isocitrate dehydrogenase (IDH1 and IDH2), two enzymes within the Krebs Cycle, may lead to a reversal of this cycle promoting the consumption of glutamine

28, 29

. Further studies of genetic mutations on glutamine-addiction in

sustaining different types of tumor growth, particularly in breast cancer, may improve our 18

understanding of using

F-(2S,4R)4-FGln/PET imaging for the diagnosis and treatment of cancer

patients.

CONCLUSION In conclusion, we have successfully synthesized a glutamine analog, 18F-(2S,4R)4-FGln, as a new metabolic probe for imaging breast cancer. Preliminary study of patients showed superior diagnostic properties than

18

18

F-(2S,4R)4-FGln in breast cancer

F-FDG. The results support the possibility of

using 18F-(2S,4R)4-FGln/PET for diagnosis of glutamine-addicted breast cancer.

ASSOCIATED CONTENT Supporting information. Quality control; Myc gene expression levels

AUTHOR INFORMATION Corresponding Authors * Email: [email protected] * Email: [email protected] ORCID Hank F. Kung: 0000-0003-3254-8049 Zhi Yang: 0000-0003-2084-5193 Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS The study was financially supported by the National Natural Science Foundation of China (81371592, 81401467, 81501519, 81571705), the Beijing Natural Science Foundation (7154188, 7162041) and the Beijing Municipal Commission of Health and Family Planning (215 backbone project).

ABBREVIATIONS 18

F-(2S,4R)4-FGln:

18

F-(2S,4R)4-fluoroglutamine; Radio-HPLC: radio-high performance liquid

chromatography; PBS: phosphate buffer saline;

18

F-FDG:

18

F-fluorodeoxyglucose; PET: positron

emission tomography; TCA: tricarboxylic acid; TNBC: triple negative breast cancer; ER: estrogen



receptor; PR: progesterone receptor; HER2: human epidermal growth factor receptor 2; qPCR: Real-time Quantitative PCR; OSEM: ordered subsets expectation maximum; ROIs: region of interests; T/B: tumor-to-blood; T/M: tumor-to-muscle; CNB: core needle biopsy; HE: hematoxylin-eosin; IHC: immunohistochemistry; Ki67: nuclear-associated antigen; IDH: isocitrate dehydrogenase.

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Figure 1. (A and C) In vitro cell uptake of 18F-(2S,4R)4-FGln and 18F-FDG in MCF-7 and U87 cells. (B and D) Blocking studies of 18F-(2S,4R)4-FGln in MCF-7 and U87 cells. Uptake was expressed as % AD/106 cells. Data were represented as mean ± SEM.

129x90mm (300 x 300 DPI)

ACS Paragon Plus Environment

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Figure 2. Blood radioactivity profile of 18F-(2S,4R)4-FGln. Data were expressed as mean ± SEM.

112x98mm (300 x 300 DPI)



Figure 3. Biodistribution of 18F-(2S,4R)4-FGln in BALB/c mice (A) and BALB/c nude mice bearing MCF-7 tumors (B). Data were expressed as mean ± SEM.

129x43mm (300 x 300 DPI)


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Figure 4. (A) Micro-PET imaging of 18F-(2S,4R)4-FGln (coronal slide) in BALB/c nude mice bearing MCF-7 tumors at 1 and 2 h post-injection. (B) Tumor-to-blood (T/B) and tumor-to-muscle (T/M) ratios of the radiotracer. The white arrows depicted the tumors. 129x43mm (300 x 300 DPI)



Figure 5. PET/CT imaging of 18F-(2S,4R)4-FGln (A-B), 18F-FDG (C-D) and whole body MIP imaging of 18F(2S,4R)4-FGln (E) at 1 h in a 57-year-old female patient. The red arrows depicted the tumors.

209x119mm (300 x 300 DPI)


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Figure 6. Immunoprofile of ER and P120 of the 57-year-old female patient. (A) HE staining depiceted typical tumor invasive histology. (B) ER staining showed diffuse strong expression. (C) P120 showed strong cytoplasmic staining. Original magnification × 200.

129x34mm (300 x 300 DPI)


PET Imaging of 18F-(2S,4R)4-fluoroglutamine ... - ACS Publications

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