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. 2024 Mar 8;21(4):2034–2042. doi: 10.1021/acs.molpharmaceut.4c00082

KRAS Mutation Detection with (2S,4R)-4-[18F]FGln for Noninvasive PDAC Diagnosis

Song Liu 1, Futao Liu 1, Xingguo Hou 1, Qian Zhang 1, Ya’nan Ren 1, Hua Zhu 1, Zhi Yang 1,*, Xiaoxia Xu 1,*
PMCID: PMC10989612  PMID: 38456403

Abstract

graphic file with name mp4c00082_0007.jpg

Pancreatic ductal adenocarcinoma (PDAC), which has a poor prognosis and nonspecific symptoms and progresses rapidly, is the most common pancreatic cancer type. Inhibitors targeting KRAS G12D and G12C mutations have been pivotal in PDAC treatment. Cancer cells with different KRAS mutations exhibit various degrees of glutamine dependency; in particular, cells with KRAS G12D mutations exhibit increased glutamine uptake. (2S,4R)-4-[18F]FGln has recently been developed for clinical cancer diagnosis and tumor cell metabolism analysis. Thus, we verified the heterogeneity of glutamine dependency in PDAC models with different KRAS mutations by a visual and noninvasive method with (2S,4R)-4-[18F]FGln. Two tumor-bearing mouse models (bearing the KRAS G12D or G12C mutation) were injected with (2S,4R)-4-[18F]FGln, and positron emission tomography (PET) imaging features and biodistribution were observed and analyzed. The SUVmax in the regions of interest (ROI) was significantly higher in PANC-1 (G12D) tumors than in MIA PaCa-2 (G12C) tumors. Biodistribution analysis revealed higher tumor accumulation of (2S,4R)-4-[18F]FGln and other metrics, such as T/M and T/B, in the PANC-1 mouse models compared to those in the MIAPaCa-2 mouse models. In conclusion, PDAC cells with the KRAS G12D and G12C mutations exhibit various degrees of (2S,4R)-4-[18F]FGln uptake, indicating that (2S,4R)-4-[18F]FGln might be applied to detect KRAS G12C and G12D mutations and provide treatment guidance.

Keywords: (2S,4R)-4-[18F]FGln; PET imaging; KRAS mutation

Introduction

In general, glucose metabolism in tumor cells is achieved through glucose transportation and phosphorylation catalyzed by hexokinase. However, another important process regulating tumor growth and reproduction is glutamine hydrolysis.1 This pathway has become a focal point in the diagnosis and treatment of cancer.2 Glutamine is a nonessential amino acid and is the most abundant amino acid in the blood (0.5–1 mM); it enters the cell through membrane transport. The development of the first 18F-labeled positron emission tomography (PET) imaging agent, (2S,4R)-4-[18F]FGln, has been reported; this agent was modified based on glutamine.3,4 There is wide potential for the application of (2S,4R)-4-[18F]FGln in the clinical detection of cancers, such as gliomas and breast cancer, as well as in bone marrow imaging and atherosclerotic inflammation detection.5−9

Pancreatic ductal adenocarcinoma (PDAC) is the main type of pancreatic cancer and is regarded as one of the deadliest cancers. The nonspecific symptoms and rapid progression of PDAC, the lack of early diagnostic methods, and the limited availability of treatment alternatives contribute to the poor prognosis.10 There have been advances in therapeutic methods, including surgical approaches and chemotherapy regimens; however, the prognosis of patents with PDAC has not improved in recent years.11,12 Thus, individualized therapeutic approaches for PDAC guided by precision medicine may be a future trend.12,13

KRAS mutations are detected in more than 90% of the PDAC patients, and mutations in codon 12 are the most common; the most common KRAS mutations in PDAC are KRAS G12D (45%) and KRAS G12 V (35%).14 KRAS mutations lead to the loss of GTPase activity, and the combination of both KRAS and GTP causes the activation of tyrosine kinases, which can activate downstream signaling pathways, such as PI3K, RAF-MEK-ERK, and RAL-GEF. These signaling pathways stimulate cell proliferation and migration, contributing to the occurrence and development of tumors.15 With the development of molecular targeted therapies, some molecular inhibitors targeting G12C mutations have been developed and proven to have antitumor functions; these include AMG510 and MRTX849.16,17 Recently, additional G12D inhibitors, such as MRTX1133, TH-Z827, and TH-Z835, have been identified as exciting candidate drugs for PDAC, and these small molecule inhibitors were confirmed to be effective in targeting KRAS G12D.18,19 Thus, it is essential to estimate the KRAS mutation status in PDAC, especially G12C and G12D mutations, to identify patients who would benefit from molecular targeted therapy.

Basic research on (2S,4R)-4-[18F]FGln has recently been developed to probe tumor cell metabolism, such as the transportation and kinetics of glutamine.3,20,21 In recent years, glutamine metabolism has been reported to be activated by KRAS mutation in nonsmall cell lung cancer (NSCLC), and an effective therapeutic strategy of dual inhibition of the MEK-ERK pathway and glutamine metabolism has been proposed.22 Previous studies have shown that KRAS mutations often lead to glutamine dependency, with different mutations displaying various degrees of reliance on extracellular glutamine.23 Cancer cells bearing the KRAS G12D mutation presented increased glutamine uptake.24 Due to the relationship between KRAS G12D and glutamine, we suppose that (2S,4R)-4-[18F]FGln might be an effective tool for (1) identifying patients with KRAS mutations who may benefit from a KRAS inhibitor and (2) guiding treatment for patients with KRAS mutation.

Experimental Section

General

All the reagents used were commercial products without further purification unless otherwise stated. The PANC-1 cell line and MIA PaCa-2 cell line were purchased from the National Collection of Authenticated Cell Cultures, Shanghai China. Dulbecco’s modified Eagle medium (DMEM) culture and fetal bovine serum were purchased from Shanghai XP Biomed Ltd. Penicillin–streptomycin was purchased from New Cell & Molecular Biotech Co., Ltd. Kunming mice (18–20 g) and Balc/c nude mice (∼4 weeks) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. Micro-PET imaging data were recorded on a Micro PET/CT of PINGSENG Healthcare Inc. (Shanghai, China).

Cell Lines

The PANC-1 cell line and MIA PaCa-2 cell line were cultured in DMEM culture supplemented with 10% fetal bovine serum and 1% penicillin–streptomycin. These cells were all incubated under humidified incubator conditions (37 °C and 5% CO2).

Immunohistochemistry (IHC) Studies

PANC-1 and MIA PaCa-2 tumors were mounted in paraffin, and these tumor sections were dehydrated with xylene and rehydrated with graded alcohol (100%, 95%, 80%, 5 min each). H2O2 (3%) was added for 10 min to block endogenous peroxidase, and then, the sections were washed three times for 5 min with phosphate-buffered saline (PBS). In antigen retrieval, the sections were placed in citrate buffer (pH 6.0) and heated in a microwave (450 W, 10 min). After returning to room temperature, the slides were washed with PBS (for 5 min, three times). For blocking, the sections were incubated with goat serum for 1 h at room temperature. Primary antibody (Abcam, #ab221163) was added to the sections, which were then incubated overnight at 4 °C. After the sections were washed with PBS (5 min, three times), they were incubated with goat antirabbit IgG/HRP polymer at room temperature for 40 min. Then, the sections were washed with PBS and developed with 3′,3-diaminobenzidine (DAB) solution. The sections were counterstained with hematoxylin and washed with water. Hydrochloric acid alcohol differentiation was used to restore the blue color of the slide. Finally, the sections were dehydrated after sealing.

Western Blotting

PANC-1 and MIA PaCa-2 cell lines were harvested with radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime, P0013B), and approximately 15 μg of protein from each cell line was separated via sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE). After transfer, the nitrocellulose membranes were blocked with skim milk (5%) and then incubated with primary anti-KRAS antibody (Sino Biological, 101667-T32) and GAPDH antibody (Proteintech, 10494-1-AP) overnight at 4 °C. After incubation with an HRP-conjugated secondary antibody, KRAS expression and GAPDH expression were detected via enhanced chemiluminescence.

Polymerase Chain Reaction (PCR)

PANC-1 and MIA PaCa-2 tumor tissues and cell lines were placed in PCR tubes, and the required reagents were added. After several mixing and centrifugation steps, the DNA was extracted from the samples. After the PCR system and primer parameters are determined, each thermocycler will be amplified. The sample PCR amplification program included predenaturation, denaturation, annealing, extension, and cooling. Finally, the amplified DNA was evaluated via agarose gel electrophoresis.

Animal Model

PANC-1 xenograft mouse models were established in BALB/c nude mice (4–6 weeks, female). PANC-1 cells were dissociated with 0.25% trysin–ethylenediaminetetraacetic acid (EDTA) and resuspended with PBS in 5 × 106 cells/0.1 mL. BALB/c nude mice were subcutaneously injected with 5 × 106 cells in the right axilla. MIA PaCa-2 mouse models were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. All animal experiments were performed in accordance with the guidelines of the Peking University Institutional Animal Care and Use Committee (EAEC 2022-01).

Radiolabeling and Quality Control of (2S,4R)-4-[18F]FGln

A QMA light cartridge (SepPak) was activated with 10 mL of 0.05 M NaHCO3 and 10 mL of H2O and loaded with 18F (2960–3330 MBq), which was produced by a cyclotron using the 18O(p,n)18F reaction. The 18F was eluted with a phase-transfer catalyst solution (8 mg of 18-crown-6 and 1.44 mg of KHCO3 in 1 mL of acetonitrile and 0.18 mL of water). The solution was evaporated and azeotropically dried twice with 1 mL of acetonitrile with a stream of nitrogen at 90 °C. Then, the 1.7 mg precursor dissolved in 1 mL of acetonitrile was added to the dried 18F. This mixture was reacted at 90 °C for 15 min. Purification processes of the mixture proceeded using an activated Oasis HLB 3 cm3 column; then, the intermediate product was eluted with 0.5 mL of ethanol. The above product was blown to dryness, and a mixture of 500 μL of trifluoroacetic acid (TFA) was added. This reaction mixture was heated at 50 °C for 5 min. The radioproduct (2S,4R)-4-[18F]FGln was diluted with 0.9% NaCl and filtered through a filter, after removing the TFA with a stream of nitrogen. The radiochemical and stereochemical purity was measured by chiral HPLC (Chirex 3126 (d)-penicillamine, 1 mM CuSO4).

Micro-PET Imaging of (2S,4R)-4-[18F]FGln and [18F]FDG

PANC-1 and MIA PaCa-2 xenograft mouse models were intravenously injected with 7.4 MBq of (2S,4R)-4-[18F]FGln. All mice were sedated with isoflurane (1–2%, 1 L/min oxygen) and monitored for respiration and any other signs of distress throughout the PET imaging procedure (SuperNova, PINGSENG Healthcare, China). The imaging scans were conducted at 30, 60, and 120 min after injection. After reconstructing the PET images by Avatar 3, regions of interest (ROI) were drawn on the computed tomography (CT) images and mapped on the PET image over the tumors. The process in [18F]FDG micro-PET imaging was similar to that of (2S,4R)-4-[18F]FGln. However, when [18F]FDG was injected into the mouse models, the animals were fasted for more than 6 h, and the anesthetics were maintained throughout the imaging.

Biodistribution Studies

The biodistribution study of (2S,4R)-4-[18F]FGln was performed in mice bearing tumors (PANC-1 and MIA PaCa-2). These mouse models were divided into four groups (n = 3) and intravenously injected with (2S,4R)-4-[18F]FGln. At 30 min and 1 h post-injection, the mouse models were euthanized. The organs of interest were removed, weighed, and counted with a γ-counter, including the blood, heart, lung, liver, spleen, stomach, muscle, bone, brain, and gastrointestinal organs. Standard samples containing a 1% injected dose of (2S,4R)-4-[18F]FGln were utilized as a standard control for quantification purposes. The results were quantified as the percentage of injected dose per gram of tissue (% ID/g) and expressed as the mean ± standard deviation (SD).

Results

Western Blotting, PCR, and IHC Results

The Western blotting results confirmed that KRAS was expressed in the PDAC cell lines. PCR revealed that the G12D mutation presented G/A (guanine/adenine) peaks, and the G12C mutation was distinguished by substitution by replacing G (guanine) with T (thymine). PANC-1 tumor tissue and cell lines presented the G12D mutation, and MIA PaCa-2 cells presented the G12C mutation. These results are shown in Table 1, Supporting Figures 1, and 2. Immunohistochemistry analysis indicated that the G12D mutation was present in the PANC-1 tumor tissue but not in the MIA PaCa-2 tumor tissue, as shown in Figure 1.

Table 1. PCR Results for PANC-1 and MIA PaCa-2 Tumor and Cell Lines.

samples G12D mutation G12C mutation
PANC-1 tumor tissue set of G/A peaks N
MIA PaCa-2 tumor tissue N Substitution of G with T
PANC-1 cell line set of G/A peaks N
MIA PaCa-2 cell line N Substitution of G with T
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Figure 1.

Figure 1

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IHC images and HE images of PANC-1 and MIA PaCa-2 tumor tissues. (A, C) IHC for PANC-1 and MIA PaCa-2 tumors with an anti-Ras antibody (mutated G12D, ab221163); (B, D) HE for PANC-1 and MIA PaCa-2.

Radiochemical Quality Control of (2S,4R)-4-[18F]FGln

The radiolabeling procedure was conducted in two steps (Figure 2). Radio-high performance liquid chromatography (Radio-HPLC) showed that the radiochemical purity (RCP) of (2S,4R)-4-[18F]FGln was over 90%, and the radiochemical yield was 1–3%.

Figure 2.

Figure 2

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Synthetic scheme of (2S,4R)-4-[18F]FGln (top) and radio-HPLC profiles of [18F]FGln (below) on a chiral column (Chirex 3126 (d)-penicillamine, 1 mM CuSO4 solution, 1 mL/min).

Micro-PET Imaging of (2S,4R)-4-[18F]FGln

In these studies, PET images of PANC-1 and MIA PaCa-2 mouse models injected with (2S,4R)-4-[18F]FGln are shown in Figure 3. (2S,4R)-4-[18F]FGln was rapidly cleared from the intestinal tract, kidneys, and bladder; moreover, this tracer mainly accumulated in the intestinal tract, and slight uptake in the bone was also observed. In 30 min, the accumulation in the intestinal tract was the highest compared with uptake at 60 and 120 min. Tracer uptake in the bone increased with time. In two kinds of mouse models, (2S,4R)-4-[18F]FGln uptake significantly differed in the tumors. In PET imaging, higher uptake was observed in PANC-1 tumors than in MIA PaCa-2 tumors after macroscopic analysis and ROI delineation. The comparison of SUVmax values for (2S,4R)-4-[18F]FGln for PANC-1 and MIA PaCa-2 tumors is shown in Figure 3C (1.03 ± 0.02 vs 0.41 ± 0.02, respectively, at 30 min, 0.92 ± 0.05 vs 0.43 ± 0.03 at 60 min, and 0.62 ± 0.10 vs 0.38 ± 0.02 at 120 min). There was no statistically significant difference in the tumor/muscle ratio (TMR) of PANC-1 compared with MIA PaCa-2.

Figure 3.

Figure 3

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(2S,4R)-4-[18F]FGln PET imaging and analysis of PANC-1 and MIA PaCa-2 tumor-bearing mice. (A, B) PET imaging for PANC-1 and MIA PaCa-2 tumor-bearing mice at 30, 60, and 120 min (white dashed circles indicate the tumors); (C) SUVmax comparison of PANC-1 and MIA PaCa-2 tumors; and (D) comparison of the tumor/muscle ratio (TMR) between PANC-1 and MIA PaCa-2 tumor-bearing mice.

[18F]FDG generally accumulates in the intestinal tract, heart, brain, and bladder and occasionally in brown fat. A comparison of [18F]FDG accumulation between PANC-1 and MIA PaCa-2 tumor-bearing mice is shown in Figure 4. Both PANC-1 and MIA PaCa-2 tumors exhibited a high uptake of [18F]FDG. In PANC-1 tumors, the SUVmax for [18F]FDG was 1.05 ± 0.01 at 30 min, 1.13 ± 0.01 at 60 min, and 1.01 ± 0.06 at 120 min. In MIA PaCa-2 tumors, the SUVmax was 0.66 ± 0.18 at 30 min, 0.77 ± 0.22 at 60 min, and 0.83 ± 0.24 at 120 min. However, there was no significant difference in the accumulation of [18F]FDG between PANC-1 and MIA PaCa-2 tumors between PANC-1 and MIA PaCa-2 TMR tumors.

Figure 4.

Figure 4

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[18F]FDG PET imaging and analysis of PANC-1 and MIA PaCa-2 tumor-bearing mice. (A, B) PET imaging for PANC-1 and MIA PaCa-2 tumor-bearing mice at 30, 60, and 120 min (white dashed circles indicate the tumors); (C) SUVmax comparison of PANC-1 and MIA PaCa-2 tumors; and (D) comparison of the tumor/muscle ratio (TMR) between PANC-1 and MIA PaCa-2 tumor-bearing mice.

Biodistribution Studies of (2S,4R)-4-[18F]FGln

The biodistribution data of PANC-1 and MIA PaCa-2 tumor-bearing mice at 30 and 60 min postinjection of (2S,4R)-4-[18F]FGln and [18F]FDG are presented in Figure 5. In all mouse models, (2S,4R)-4-[18F]FGln exhibited greater uptake in the kidneys, liver, and intestinal tract at 30 and 60 min. Less uptake was observed in the brain, heart, and blood. Uptake of (2S,4R)-4-[18F]FGln decreased rapidly with time in most organs, except the bone. High uptake of [18F]FDG was present in the heart, brain, and intestinal tract. The uptake values of organs postinjection of (2S,4R)-4-[18F]FGln and [18F]FDG are listed in Supporting Tables 1 and 2.

Figure 5.

Figure 5

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Biodistribution of (2S,4R)-4-[18F]FGln and 18F-FDG in PANC-1 and MIA PaCa-2 tumor-bearing mice at different time points postinjection (n = 3, 20 μCi per mouse i.v.).

Comparisons of the two tracers on PANC-1 and MIA PaCa-2 tumors, tumor/blood ratio (T/B), and tumor/muscle ratio (T/M) at 30 and 60 min postinjection are presented in Figure 6. There was a significant difference between PANC-1 and MIA PaCa-2 tumors: the accumulation of (2S,4R)-4-[18F]FGln in PANC-1 tumors was more pronounced than that in MIA PaCa-2 tumors. In the groups injected with (2S,4R)-4-[18F]FGln, the values for the percentage of injected dose per gram of tissue (% ID/g) were 5.24 ± 0.55 vs 2.09 ± 0.27 at 30 min and 4.74 ± 0.63 vs 2.07 ± 0.15 at 60 min in PANC-1 and MIA PaCa-2 tumors, respectively. The values of T/B were 2.57 ± 0.07 vs 1.29 ± 0.24 at 30 min and 3.20 ± 0.42 vs 2.16 ± 0.09 at 60 min in PANC-1 and MIA PaCa-2 tumor-bearing mice. The values of T/M were 1.51 ± 0.07 vs 0.84 ± 0.16 at 30 min and 1.68 ± 0.16 vs 1.19 ± 0.19 at 60 min in PANC-1 and MIA PaCa-2 tumor-bearing mice. In contrast, there was no notable distinction in the [18F]FDG uptake. In this group injected with [18F]FDG, the % ID/g values were 3.66 ± 0.56 vs 3.60 ± 0.99 at 30 min and 2.73 ± 0.74 vs 2.88 ± 0.96 at 60 min in PANC-1 and MIA PaCa-2 tumors, respectively. The values of T/B were 2.94 ± 0.68 vs 4.92 ± 1.22 at 30 min and 5.02 ± 0.51 vs 5.25 ± 1.37 at 60 min in PANC-1 and MIA PaCa-2 tumor-bearing mice. The values of T/M were 1.09 ± 0.16 vs 1.82 ± 1.60 at 30 min and 0.75 ± 0.29 vs 1.07 ± 0.46 at 60 min in PANC-1 and MIA PaCa-2 tumor-bearing mice.

Figure 6.

Figure 6

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Comparisons with PANC-1 and MIA PaCa-2 tumor-bearing mice injected (2S,4R)-4-[18F]FGln and [18F]FDG in terms of the percentage of injected dose per gram of tissue (% ID/g), tumor/blood ratio (TBR), and tumor/muscle ratio (TMR).

Discussion

Glutamine is one of the most crucial nutrients metabolized by numerous cancer cell lines. Once imported into cells, glutamine acts as a carbon source to fuel the tricarboxylic acid cycle and as a nitrogen source for the biosynthesis of nucleotides, nonessential amino acids, and hexosamines.25 It has been established that cancer cells, particularly those harboring oncogenic KRAS, are extremely reliant on glutamine for survival and proliferation.23,26 Moreover, a previous study indicated that KRAS mutations generally result in glutamine dependency, with different mutations manifesting variable degrees of dependency on extracellular glutamine.27

Varshavi et al. reported that compared with WT KRAS, KRAS G12D had a significantly lower concentration of l-alanyl-l-glutamine (fold change of ca. 0.16) but a minor increase in extracellular glutamine (fold change of ca. 1.13), which indicated enhanced glutamine uptake. KRAS G12C, however, displayed no changes in either l-alanyl-l-glutamine or extracellular glutamine compared to WT KRAS, which is interesting, as this specific KRAS mutation is now targeted with drugs in the clinic.28 Additionally, cumulative evidence has demonstrated that KRAS is the predominant mutated isoform in almost all PDAC patients, with its most common oncogenic form encoding KRAS G12D.29

The verification of the mutation by Western blotting, PCR, and IHC experiments confirmed that PANC-1 cell lines and tumor xenografts harbor the KRAS G12D mutation and that MIA PaCa-2 cell lines and tumor xenografts harbor the KRAS G12C mutation. These conclusions have also been proven by other studies.19,30 Hence, this study used KRAS G12D and G12C mutation pancreatic cancer tumor models to explore whether (2S,4R)-4-[18F]FGln PET imaging can noninvasively detect KRAS mutations in tumors, thus guiding clinically individualized clinical treatment.

The radiochemical purity (RCP) of (2S,4R)-4-[18F]FGln was over 90% according to radio-HPLC. This radiotracer (RCP > 90%) was allowed for use in animal experiments in nuclear medicine.31 The presentation of the radiopeak at 22.54 min might be caused by fluorine-18 labeling of glutamic acid analogues due to the labile amide group in (2S,4R)-4-[18F]FGln, which could be easily hydrolyzed to form [18F]-4-fluoro-glutamic acid.3 It should be pointed out that the radiochemical yield (1–3%) was lower than that in previous studies. The main reason was contributed to the lower dosage of the precursor (approximately 1.7 mg).

Compared with that in MIA PaCa-2 tumors, the accumulation of (2S,4R)-4-[18F]FGln was significantly greater in PANC-1 tumors according to both the PET imaging study and the biodistribution study. Furthermore, in noninvasive imaging research, SUVmax values were compared between two kinds of mouse models bearing tumor xenografts. PANC-1 tumors presented increased uptake of (2S,4R)-4-[18F]FGln, which was similar to the absolute accumulation value. However, a comparison of TMR values showed the opposite pattern, which may be due to the partial volume effect of the PET image; namely, the strong absorption of the tracer by the bone in PANC-1 mice led to a false increase in SUVmax in the adjacent muscle when the ROI was delineated. It was reported that the increased uptake in the bone might be due to defluorination of (2S,4R)-4-[18F]FGln or the specific uptake of (2S,4R)-4-[18F]FGln by bone marrow.6 Although the disadvantages of tracer defluorination have been discussed, (2S,4R)-4-[18F]FGln is still useful as an imaging tracer, especially in the context of brain tumors.20,32 Therefore, we further verified the in vivo biodistribution. In terms of biodistribution, the TMR of the PANC-1 and MIA PaCa-2 mouse models was as expected: the TMR of the PANC-1 mouse model was higher than that of the MIA PaCa-2 mouse model, and the effect of bone uptake on the muscle was excluded in this study. Additionally, in the biodistribution studies, the uptake of (2S,4R)-4-[18F]FGln was significantly higher in PANC-1 tumors after comparing the percentage of injected dose per gram of tissue and TBR and TMR values.

One study reported that the oncogenic mutation G12D is more prone to shift the KRAS-GTP conformation to the active state than the G12C mutation. The G12D mutation results in increased exposure of the binding nucleotide site. These signaling pathways stimulate cell proliferation and migration, contributing to the occurrence and development of tumors. Moreover, the G12D mutation results in increased exposure of the binding nucleotide site.33 In addition, a previous report suggested that an increase in the tumor number might be driven by more rapid growth by KRAS G12D than tumors driven by KRAS G12C.34 Thus, there might be a more vigorous metabolism and a higher requirement for carbon and nitrogen sources in the PDAC mouse model with the G12D mutation. As a major source of nitrogen and carbon, glutamine contributes to intracellular biosynthesis, energy metabolism, and redox homeostasis and is essential for cancer cell survival. The liver is regarded as the main site for glutamine metabolism due to glutaminase (GLS2), which is able to convert glutamine to glutamic acid. Therefore, the biodistribution of (2S,4R)4-[18F]FGln was slightly greater in PANC-1 model mice (with G12D mutation) than in MIA PaCa-2 model mice (with G12C mutation), especially in the heart, liver, stomach, and intestinal tract. In addition, the higher metabolic rate in the body accelerates the defluorination of (2S,4R)-4-[18F]FGln. These findings suggested that the PANC-1 model presented higher values in micro-PET imaging. Due to differences in systemic metabolic rates, there may be differences in drug distribution visually in the images shown in this article.

These results proved that (2S,4R)-4-[18F]FGln has the potential to differentiate G12D and G12C mutations via a noninvasive method. In comparison, [18F]FDG significantly accumulated in both PANC-1 and MIA PaCa-2 tumors. Additionally, there were no significant differences in the the TBR and TMR between the two groups of mice. This result indicated that [18F]FDG could not distinguish tumors bearing KRAS G12D and G12C mutations.

Conclusions

In summary, (2S,4R)-4-[18F]FGln exhibited differential accumulation in PDAC cancer cells with KRAS G12D and G12C mutations. As a widely applied radiotracer in diagnosing cancer, (2S,4R)-4-[18F]FGln is expected to be applied in detecting KRAS mutations, which is valuable for guiding molecular targeted therapy and avoiding overtreatment.

Acknowledgments

The authors thank the teams of Prof. Hank F. Kung and Prof. Lin Zhu for synthesis and supply of the precursor. This work was supported by the National Natural Science Foundation of China (82001857), the National Natural Science Foundation of China (82171980), the National Natural Science Foundation of China (22334001), the Beijing Hospitals Authority’s Ascent Plan (DFL20191102), the Science Foundation of Peking University Cancer Hospital (2022-23), and the National Key Research and Development Program of China (No. 2022YFC2406900).

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.molpharmaceut.4c00082.

  • PCR analysis for PANC-1 and MIA PaCa-2 tumor tissues and cell lines, Western blot result for PANC-1 and MIA PaCa-2 cell lines, and comparison of biodistribution results of (2S,4R)4-[18F]FGln and [18F]FDG between PANC-1 and MIA PaCa-2 tumor-bearing mouse models 30 and 60 min postinjection (PDF)

Author Contributions

† S.L. and F.L. were co-first authors and contributed equally to this work, including creating all images in the TOC figure.

The authors declare no competing financial interest.

Supplementary Material

mp4c00082_si_001.pdf (239.5KB, pdf)

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