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. 2025 May 13;10(1):23.
doi: 10.1186/s41181-025-00345-8.

Evaluation of in vivo and in vitro binding property of a novel candidate PET tracer for CSF1R imaging and comparison with two currently-used CSF1R-PET tracers

Xiyan Rui #  1   2 , Yuzhou Ding #  2 , Nailian Zhang  2 , Xinran Zhao  2 , Chie Seki  3 , Tomoteru Yamasaki  4 , Masayuki Fujinaga  4 , Ming-Rong Zhang  4 , Jun Qian  2   5   6 , Bin Ji  7   8   9   10   11   12   13 , Rong Zhou  14
Affiliations

Evaluation of in vivo and in vitro binding property of a novel candidate PET tracer for CSF1R imaging and comparison with two currently-used CSF1R-PET tracers

Xiyan Rui et al. EJNMMI Radiopharm Chem. .

Abstract

Background: Colony-stimulating factor 1 receptor (CSF1R) is a promising imaging biomarker for neuroinflammation and tumor-associated macrophages. However, existing positron emission tomography (PET) tracers for CSF1R imaging often suffer from limited specificity or sensitivity.

Results: We have performed 11C-labeled radiosynthesis of compound FJRD (3-((2-amino-5-(1-methyl-1H-pyrazol-4-yl)pyridin-3-yl)ethynyl)-N-(4-methoxyphenyl)-4-methylbenzamide), which exhibits excellent affinity for CSF1R, and evaluated its in vivo and in vitro binding properties. PET images of [11C]FJRD show low brain uptake and specific binding in the living organs, except the kidneys in both normal mice and rats. In vitro autoradiographs demonstrate high levels of specific binding in all investigated organs, including the brain, spleen, liver, kidneys and lungs, when self-blocking was used. The addition of CPPC partially blocked in vitro [11C]FJRD binding in these organs, with blocking effects ranging from 9 to 67%. In contrast, the other two CSF1R inhibitors, GW2580 and BLZ945, showed minimal blocking effects, suggesting unignorable off-target binding in these organs. Furthermore, specific binding of [11C]CPPC and [11C]GW2580 was faint in the mouse organs, with [11C]CPPC demonstrating detectable binding only in the spleen.

Conclusions: These results suggest that [11C]FJRD is a potential CSF1R-PET tracer for more sensitive detection of CSF1R, compared to [11C]CPPC and [11C]GW2580. However, the high level off-target binding necessitates further improvements in specificity for CSF1R imaging.

Keywords: Autoradiography; Colony-stimulating factor 1 receptor (CSF1R); Positron emission computed tomography (PET); [11C]CPPC; [11C]o-aminopyridyl alkynyl derivative.

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Conflict of interest statement

Declarations. Ethics approval and consent to participate: The mice and rats in this study were maintained and handled in accordance with the National Research Council’s Guide for the Care and Use of Laboratory Animals, as well as the specific institutional guidelines. The experimental protocols involving animals were thoroughly reviewed and approved by the Animal Ethics Committees of Fudan University and the National Institutes for Quantum Science and Technology. These approvals ensure that all procedures complied with ethical standards for the humane treatment of animals, reflecting the researchers’ commitment to ethical scientific practices and the welfare of the animals involved in the study. Consent for publication: Not applicable. Competing interests: The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Chemical synthesis route of V-2 (FJRD) and V-1 (precursor for radiolabeling): (a) Pd(PPh3)2Cl2, CuI, CsF, Et3N, 6 h, 88.2%; (b) Pd(OAc)2, X-phos, K2CO3, THF, 6 h, 80.0%; (c) LiOH·H2O, 12 h, 94.0%; (d) HATU, DIPEA, 4 h, 79.6%
Fig. 2
Fig. 2
Chemical structures of CSF1R-PET tracers used in the present study and radiosynthesis of [11C]FJRD. (A) The affinities of [11C]FJRD (Xie et al. 2020) [11C]CPPC (Wildt et al. 2021a) [11C]GW2580 (Zhou at al. 2021) and [11C]BLZ945 (Wildt et al. 2021c) for CSF1R from the previous publications. (B) [11C]FJRD was radiosynthesized by one-step reaction of precursor and [11C]NaI
Fig. 3
Fig. 3
Whole body PET imaging with [11C]FJRD in mice. (A) Summation PET images of [11C]FJRD (0–1, 1–2, 12–14 and 20–25 min from left to right) with or without pretreatment of FJRD as indicated. (B) Time-activity curves in the brain, heart, lung, liver, and kidneys. Data are expressed as SUV. -C: Baseline; -B: Preblock
Fig. 4
Fig. 4
PET imaging of [11C]FJRD in rat brain. (A) Summation PET/MRI-fused images of [11C]FJRD (0–90 min) with or without pretreatment of FJRD. (B) Time-activity curves in the striatum (ST), hippocampus (Hip), thalamus (Tha), cerebellum (CB) and whole brain (WB). Data are expressed as SUV. -C: Baseline; -B: Preblock
Fig. 5
Fig. 5
In vitro autoradiography with [11C]FJRD and [11C]CPPC in the peripheral and central organs of healthy mice. Representative in vitro autoradiographs of [11C]FJRD (A) and [11C]CPPC (B) in the various organs from healthy mice in the absence or presence of non-radioactive compounds including FJRD, CPPC, GW2580 and BLZ945 at the concentration of 10 µM as indicated
Fig. 6
Fig. 6
Quantitative analysis for in vitro binding properties of [11C]FJRD and [11C]CPPC. (A) Inhibitory effects of various CSF1R inhibitors on [11C]FJRD in vitro binding. Total binding (TB) is the binding of [11C]FJRD without the addition of any cold compound. N = 3 for each group. Data are expressed as mean ± SD. (B) The proportions of non-specific binding (non-SB), specific binding to CSF1R (CSF1R-SB) and specific binding to off-target (OT-SB) of [11C]FJRD (FJRD) and [11C]CPPC (CPPC) with respective total binding as 100%. Data from Fig. 5
Fig. 7
Fig. 7
Representative in vitro autoradiography with [11C]GW2580 in the peripheral and central organs of healthy mouse in the absence or presence of non-radioactive GW2580 (10 µM)
Fig. 8
Fig. 8
Immunohistochemstry for CSF1R in the normal mouse organs. In vitro autoradiographic image with [11C]CPPC (A) and immunohistochemistry images of CSF1R (B-H) in the tissue sections of the spleen (A-C), brain (D), lung (E), kidney (F), liver (G) and heart (H) from a three-month-old male C57BL/6J mouse. The area enclosed by dotted line in low-power image (B) was shown in high-power image (C) of the spleen. Scale bar: 1 mm in panel B; 100 μm in panel C-H
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