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. 2021 Nov 18;6(47):32253–32261. doi: 10.1021/acsomega.1c05073

Gallium-68 Labeling of the Cyclin-Dependent Kinase 4/6 Inhibitors as Positron Emission Tomography Radiotracers for Tumor Imaging

Cheng Liu †,‡,§,∥,⊥, Ziyi Yang †,‡,∥, Mingyu Liu †,‡,∥,⊥, Xiangwei Wang †,‡,∥, Shaoli Song †,‡,§,∥,⊥,*, Xiaoping Xu †,‡,∥,*, Zhongyi Yang †,‡,∥,*
PMCID: PMC8638300  PMID: 34870045

Abstract

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Cyclin-dependent kinase 4 and 6 (CDK4/6) have emerged as interesting therapeutic drug targets with many potential applications in anti-tumors, especially in breast cancer. A novel CDK4/6 kinase-derived positron emission tomography (PET) imaging agent was designed based on palbociclib modified with a chelator DOTA. This new compound with a chelator DOTA-palbociclib was radiolabeled with gallium 68 (68Ga). After labeling, the purity and stability were evaluated, and the blood pharmacokinetics were carried out in normal healthy mice. Human breast cancer MCF-7 (ER+/HER2−) cells were used for in vitro cell uptake tests. PET imaging and ex vivo biodistribution were conducted in MCF-7 tumor-bearing mice. Specific binding of tumors was evaluated by the blocking assay. Furthermore, the uptake of 68Ga-DOTA-palbociclib in tumors was studied by autoradiography of tissue sections followed by immunofluorescence evaluation of CDK4 and CDK6. 68Ga-DOTA-palbociclib was synthesized very simply in a high labeling rate and radiochemical purity in 10 min. The labeling compound showed excellent stability both in vitro and in vivo and exhibited good pharmacokinetics, making it suitable for in vivo imaging. Cell uptake studies display that co-incubation with palbociclib can inhibit cellular uptake of 68Ga-DOTA-palbociclib. In vivo imaging and ex vivo biodistribution in mice bearing MCF-7 tumors both showed obvious radioactive uptake in the tumor and higher tumor-to-muscle ratios, while the tumor radioactivity accumulation was significantly decreased when prior administered with an excess of cold palbociclib, confirming CDK4/6 specific binding of 68Ga-DOTA-palbociclib in vivo. Autoradiography of the avid tumor section showed a high correlation between immunofluorescence with the CDK4/6 positive areas of the tumor, further demonstrating that 68Ga-DOTA-palbociclib specifically targeted CDK4/6 positive tumors. We synthesized 68Ga-DOTA-palbociclib, a new CDK4/6 kinase PET imaging agent, and validated its excellent stability, pharmacokinetics, and specific tumor binding. Based on our primary results, 68Ga-DOTA-palbociclib is a promising imaging agent with the potential to tailor a precise treatment program for CDK4/6 inhibitors.

Introduction

The mammalian cell cycle is a sequence of events essential for cellular reproduction and function, which contains four distinct phases (G1, S, G2, and M).1 Cell cycle control is frequently disrupted in most cancers,2,3 and research on it has become a new attractive target for novel cancer therapeutics. Cyclin-dependent kinase 4 and 6 (CDK4/6) are fundamental drivers of the cell cycle.4 Once activated and after binding with cyclin D, CDK4/6 phosphorylates the retinoblastoma protein (Rb), an event that causes Rb to lose the ability to bind to the E2F family of transcription factors, and thus helps to drive the progression from the G1 to S phase of cells.5 Amplification and overexpression of the CDK4/6 and cyclin D have been found to exist in a variety of malignancies including breast cancer.6,7 As noted above, inhibiting CDK4 and CDK6 prevents cell cycle progression, suppressing tumor development and promoting senescence. Thus, the development of selective CDK4/6 inhibitors has become a novel therapeutic frontier for patients with advanced cancer.

To date, three selective CDK4/6 inhibitors, palbociclib, ribociclib, and abemaciclib, have been approved for the treatment of hormone receptor (HR)-positive, human epidermal growth factor receptor 2 (HER2)-negative breast cancer. A large number of clinical trials have demonstrated that the combination of CDK4/6 inhibitors and endocrine therapy significantly improved clinical outcomes, such as extending the progression-free survival (PFS) and overall survival (OS) in HR-positive metastatic breast cancer.8−11 Despite the prominent benefit of this combination, some patients might show de novo resistance to CDK4/6 inhibitors, and nearly all patients will eventually acquire resistance to these drugs.12,13 Therefore, the early identification of patients who will benefit from CDK4/6 inhibitors is very important. The early prediction will avoid treatment-related toxicity and can lead to improving patient survival by allowing early intensive treatment.

CDK4/6 expression is routinely determined by the invasive method of immunohistochemistry (IHC) at the time of diagnosis of the primary tumor. Furthermore, the role of IHC is limited in analyzing the heterogeneity of CDK4/6 expression in primary tumors and the heterogeneity of CDK4/6 expression between primary and metastatic lesions. In contrast, noninvasive molecular imaging provides a potentially valuable approach for visualization of CDK4/6 positive lesions and quantification of CDK4/6 expression in vivo, including the metastasis that cannot be a biopsy.

Gallium 68 is a metal positron emitter, an isotope used for positron emission tomography (PET) imaging produced by a 68Ge/68Ga generator. 68Ga, with a suitable half-life (68 min) and simple labeling condition, is an ideal radionuclide for developing a new molecular probe based on targeted drugs. With the commercialization and universalization of the 68Ge/68Ga generator, 68Ga-labeled radiopharmaceuticals have displayed a good application prospect in PET imaging and can be seen as a scheme of alternatives to cyclotron-produced PET isotopes, such as 18F.14−16

The goal of the current study was to develop a novel PET tracer (68Ga-DOTA-palbociclib) for imaging of CDK4/6 in vivo. We outline the synthesis and the radiochemical and biological properties of 68Ga-DOTA-palbociclib involved in cellular uptake studies, metabolism, and biodistribution studies and explore its potential for PET imaging. Both in vitro and in vivo studies were performed in MCF7 breast carcinoma cells, and subcutaneous xenografts were derived from this cell line, a molecular subtype of HR+/HER2– breast cancer.

Results

Radiolabeling and Octanol–Water Partition Coefficient

Figure 1 shows the structure and synthesis scheme for 68Ga-DOTA-palbociclib. Using the iTLC-SG, the radiochemical purity (RCP) of 68Ga-DOTA-palbociclib could be obtained in 5 min. The Rf of 68Ga-DOTA-palbociclib was 0.6–0.7, and the Rf of 68GaCl3 was 0–0.1. As displayed in Figure 2, the radiochemical purity of 68Ga-DOTA-palbociclib was >95%. Furthermore, radio-HPLC confirmed the 68Ga-DOTA-palbociclib and its RCP. The retention time of DOTA-palbociclib (UV peak) was 6.18 min, and the retention time of 68Ga-DOTA-palbociclib (radioactive peak) was 6.58 min. The proximity in retention time demonstrated that the Ga-68 had radiolabeled DOTA-palbociclib successfully. The radio-HPLC spectrum confirmed the high RCP of 68Ga-DOTA-palbociclib (>95%). The log Poctanol/water value was −1.64 ± 0.12, revealing that 68Ga-DOTA-palbociclib was more hydrophilic than palbociclib.

Figure 1.

Figure 1

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Structure and synthesis scheme for 68Ga-DOTA-palbociclib.

Figure 2.

Figure 2

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Quality control of 68Ga-DOTA-palbociclib. (A) Typical radio-TLC chromatograms of 68GaCl3 (left) and 68Ga-DOTA-palbociclib (right). (B) Typical HPLC chromatograms of DOTA-palbociclib (left) and 68Ga-DOTA-palbociclib (right).

In Vitro and In Vivo Stability

First of all, the stability of 68Ga-DOTA-palbociclib was measured in PBS (0.1 M pH = 7.4). 68Ga-DOTA-palbociclib was stable in PBS for 3 h, and no free Ga-68 was found by radio-TLC. To better mimic the complex internal environment, fresh mouse plasma was used to evaluate the stability of 68Ga-DOTA-palbociclib. After 3 h of incubation and protein precipitation, the supernatant of plasma was analyzed by radio-HPLC. The result is illustrated in Figure 3A. The peak time of the sample was consistent with the retention time of 68Ga-DOTA-palbociclib, and no other obvious peak was found. These results demonstrated that 68Ga-DOTA-palbociclib remained prototype in plasma for 3 h, and there was not any degradation. All in all, 68Ga-DOTA-palbociclib was very stable in vitro.

Figure 3.

Figure 3

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In vitro and in vivo stability. (A) Typical HPLC chromatograms of DOTA-palbociclib (blue line), 68Ga-DOTA-palbociclib (red line), and 68Ga-DOTA-palbociclib incubation with fresh mouse plasma after 3 h (black line). (B) Typical radio-HPLC chromatograms of 68Ga-DOTA-palbociclib in saline before injection and in the plasma at 5, 30, and 60 min p.i.

Since all the imaging probes will be used in vivo ultimately, it is necessary to determine the in vivo stability of 68Ga-DOTA-palbociclib. After intravenous injection of 68Ga-DOTA-palbociclib, the mice were killed with painless bleeding under anesthesia at 5, 30, and 60 min. The blood was collected and mixed with acetonitrile for protein precipitation. The supernatant was also analyzed by radio-HPLC, and the result is displayed in Figure 3B. There was only one peak in the radioactive chromatograms of the supernatant collected from the blood sample at 5 or 30 min after 68Ga-DOTA-palbociclib injection. Although the analysis result of blood collected at 60 min after injection showed that the radioactivity was very low, no other obvious peak was observed in the HPLC chromatogram. By comparison with the chromatogram of 68Ga-DOTA-palbociclib, it was proven that the peak of the blood sample at 5, 30, or 60 min represented 68Ga-DOTA-palbociclib. As a consequence, it could be considered that 68Ga-DOTA-palbociclib displayed high stability in vivo.

Cell Uptake and Pharmacokinetics

In vitro binding of 68Ga-DOTA-palbociclib to CDK4/6 on MCF-7 cells was investigated using the cell uptake assay. In the presence of an excess of unlabeled palbociclib, cell uptake could be blocked by approximately 86% (Figure 4A, P < 0.001). The radioactivity in mouse blood shows that the 68Ga-DOTA-palbociclib cleared relatively fast in vivo. The distribution phase half-life t1/2α of 68Ga-DOTA-palbociclib was estimated to be 4.30 min, and the clear-phase half-life t1/2β was estimated to be 69.315 min (Figure 4B).

Figure 4.

Figure 4

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Cell uptake and pharmacokinetics. (A) In vitro uptake by MCF-7 cells incubated with 68Ga-DOTA-palbociclib in the absence (red column) or presence (black column) of an excess of unlabeled palbociclib after 1 h (*P < 0.001). (B) Blood pharmacokinetics of 68Ga-DOTA-palbociclib in healthy mice (t1/2α = 4.30 min, t1/2β = 69.31 5 min).

68Ga-DOTA-Palbociclib PET/CT Imaging

The PET/CT images were obtained for 68Ga-DOTA-palbociclib in MCF-7 tumor-bearing mice at 1 h after injection. As shown in Figure 5A, there was a high radioactivity accumulation in the tumor. The specificity of 68Ga-DOTA-palbociclib for CDK4/6 was again confirmed by the blocking experiment. The tumor uptake was almost completely decreased to the background level in the mouse pretreated with excessive cold palbociclib, which was a statistically significant difference compared with that in the unblocked mice (Figure 4B, 0.63 ± 0.05 vs 0.21 ± 0.05 SUV, P = 0.002). Consistent with the above results, there is a significant statistical difference in T/M between the blocked group and the unblocked group (Figure 4C, 3.72 ± 0.47 vs 1.26 ± 0.22, P < 0.001). Equally notable is that the PET images showed obvious abdominal uptake.

Figure 5.

Figure 5

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In vivo small-animal PET/CT images and quantitative analysis parameters. (A) Representative MicroPET/CT images of 68Ga-DOTA-palbociclib in MCF-7 tumor xenografts in vivo. Transverse and coronal images at 1 h after injection (without an excess of palbociclib, left; with an excess of palbociclib 30 min before 68Ga-DOTA-palbociclib, right; red arrows point to tumor tissues). (B) Significance of tumor SUV between 68Ga-DOTA-palbociclib and the blocked group. (C) The ratio of the tumor to the contralateral muscle was analyzed in the 68Ga-DOTA-palbociclib image. *P < 0.05.

Ex Vivo Biodistribution, Autoradiography, and Immunofluorescence

The in vivo tumor targeting and imaging features of 68Ga-DOTA-palbociclib were further evaluated by an ex vivo biodistribution study (Figure 6). There was a high (4.07 ± 0.18% ID/g) accumulation of the radioactivity in the tumors at 1 h post-injection. At the time points of 2 h post-injection, the uptake of the radiotracer in the tumor was slightly decreased (3.75 ± 0.22% ID/g), but there is no significant difference compared with 1 h points (P = 0.193). It is worth noting that high normal tissue radioactivities were observed in the small intestine (11.89 ± 1.58 or 10.84 ± 1.66% ID/g) and kidney (7.72 ± 0.31 or 6.93 ± 0.81% ID/g) at 1 or 2 h after injection. High uptake of the small intestine may be one of the causes for the marked abdominal accumulated in the PET image.

Figure 6.

Figure 6

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68Ga-DOTA-palbociclib ex vivo biodistribution in MCF-7 tumor-bearing mice (n = 3 per group), at 1 h (red bar) and 2 h (blue bar) post-injection or with a pre-injection excess of palbociclib blocking (black bar), here expressed as %ID/g.

To evaluate the specificity of 68Ga-DOTA-palbociclib binding to CDK4/6, a group of MCF-7 tumor-bearing mice (n = 3) was injected with an excess of palbociclib 30 min prior to receiving 68Ga-DOTA-palbociclib (at 1 h point). In the blocking group, the tumor uptake was decreased to 1.67 ± 0.07% ID/g, which was remarkably different from the unblocked group (P = 0.001).

To further investigate the correlation between the protein of CDK4/6 expression and radioactive uptake, we performed 68Ga-DOTA-palbociclib autoradiography and immunofluorescence staining in adjacent sections. As shown in autoradiography images (Figure 7A), the 68Ga-DOTA-palbociclib signal was visibly accumulated in the tumor and consistent with paralleled immunofluorescence for the positive staining of CDK4 and CDK6 in the tumor (Figure 7B). Consequently, we demonstrated that 68Ga-DOTA-palbociclib was specifically bound to CDK4 and CDK6 positive tumors.

Figure 7.

Figure 7

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Autoradiography and immunofluorescence. (A) Autoradiography image showing distribution of radioactivity. (B) Immunofluorescence image showing CDK4 (red), CDK6 (pink), and nuclei (blue).

Discussion

Here, we elaborate on the use of 68Ga-DOTA-palbociclib PET to perform non-invasive imaging of CDK4/6, which is the key regulator of the cell cycle process by regulating the G1-S checkpoint. Palbociclib is the first oral CDK4/6 inhibitor approved by the FDA and currently the most widely used CDK4/6 inhibitor in the world, including China.17 Therefore, the palbociclib analogue was selected as the precursor to synthesize targeted CDK4/6 radiopharmaceutical agents.

In the clinical practice aspect, the application of CDK4/6 inhibitors on breast cancer becomes broader and broader; therefore, it is of great significance to further understand the mechanisms of resistance and potential biomarkers of CDK4/6 inhibitors.18,19 However, it is well known that many potential drug resistance mechanisms have been identified in preclinical studies but cannot be further confirmed in real clinical trials.20−22 In view of this, we synthesized 68Ga-DOTA-palbociclib to monitor the expression of CDK4/6 in tumors or other tissues as a valuable method for predicting which patients will respond to CDK4/6 inhibitor therapy. In addition, we combined multiple molecular imaging technologies, such as 18F-FES23 or 18F-FLT,24 to evaluate the target engagement and early therapeutic efficacy of CDK4/6 inhibitors combined with endocrine therapy. Finally, serial 68Ga-DOTA-palbociclib imaging can be used to study the pharmacokinetics of palbociclib by evaluating the residual availability of CDK4/6 on tumors during palbociclib treatment and therefore has the potential to assist in the design of an optimum therapeutic regimen.

To our knowledge, this is the first report on CDK4/6 imaging using Ga-68 labeling palbociclib analogues. The earliest reports on CDK4 imaging radiotracers were 124I-CKIA and 124I-CKIB.25 Unfortunately, these tracers showed no radioactivity accumulation in vivo xenografts in small-animal PET imaging, and there is only a slight uptake in ex vivo autoradiography. Recently, Song et al.26,27 provided a proof of concept that palbociclib labeled with 99mTc was a potential tracer for CDK4/6 detection by SPECT. Consistent with the results of Gan et al.,27 the blood uptake of 68Ga-DOTA-palbociclib was higher at 1 h after injection, but it was quickly cleared and decreased by about 54% at 2 h after injection (from 3.61 ± 0.53 to 1.69 ± 0.29% ID/g). The most important difference is that the MCF-7 tumor uptake of 68Ga-DOTA-palbociclib was significantly higher than that of blood after 2 h of injection (3.75 ± 0.22% vs 1.69 ± 0.29% ID/g), while the uptake of 99mTc-labeled tracers was still lower than that of blood, indicating that 68Ga-DOTA-palbociclib possessed better imaging to CDK4/6. However, compared with the 18F-CDKi studied by Ramos et al.,28 the tumor-blood ratio of 68Ga-DOTA-palbociclib seems unsatisfactory, and the development of radiotracers with lower blood retention might be the main focus of a subsequent study. Furthermore, PET imaging tracers may improve the characteristics of small lesions due to the higher spatial resolution of clinical PET imaging than SPECT imaging tracers and provide a reliable quantitative method for evaluating the uptake of radiotracers by tumors and normal organs.29 In this context, Ramos et al. synthesized 18F-CDKi to represent the PET imaging tracer to monitor against CDK4/6 kinases.28 Although 18F-CDKi is a promising imaging agent, the application of 18F requires a cyclotron on-site and the cumbersome synthesis method limits it further in a clinical setting in certain aspects. According to the report,30 the binding site of palbociclib to CDK4/6 is the dihydropyrido-pyrimidine and pyridine, and the piperazine is not an active site. Therefore, 99mTc-labeled palbociclib and 18F-CDKi both introduced the nuclide through the piperazine. As a consequence, the nuclide introduced through piperazine did not affect the binding affinity. In the present study, we also conjugated DOTA (1,4,7,10-tetraazacyclododecane-N,N′,N″,N‴-tetraacetic acid) through piperazine to enable the complexation of Ga-68. This chelating system allows 68Ga radiolabeling in high efficiency, and the synthesis method is more convenient and time-saving.31,32

In the present study, the cell uptake experiment showed that the tracer exhibited high levels of uptake by MCF-7 tumor cells, and the uptake can be sharply inhibited by an excess of palbociclib, indicating that CDK4/6 is the key to the specific uptake of 68Ga-DOTA-palbociclib. Taking into account the interesting findings in vitro, a small-animal PET/CT scan was used to further explore the biological properties of the tracer in MCF-7 tumor-bearing mice. From the image, we could find that the radioactivity had significant accumulation in the tumor at 1 h post-injection. Tracer uptake in the tumor decreased by approximately 67% (SUV from 0.63 ± 0.05 to 0.21 ± 0.05) when mice were blocked with an excess of palbociclib, suggesting a CDK4/6-targeting uptake mechanism in vivo.

The results of biodistribution data were also in agreement with in vitro and PET imaging, and tumor accumulation was very high at 1 and 2 h after injection and significantly decreased when mice received an excess of palbociclib prior to the injection of 68Ga-DOTA-palbociclib. The results of autoradiography and immunofluorescence staining further indicated that 68Ga-DOTA-palbociclib was specifically bound to CDK4 and CDK6 positive tumors. Among the normal organs, the small intestine presented the highest uptake of 68Ga-DOTA-palbociclib, which may explain the relatively high concentration of radioactivity in the abdomen of mice in PET imaging. A high concentration of 68Ga-DOTA-palbociclib was also found in the kidneys, suggesting that the urinary system was the main way to clear 68Ga-DOTA-palbociclib. The uptake in the liver was relatively low over the entire time course of the experiments, which will help improve the diagnosis of hepatic disease in clinical practice. In addition, the distribution in other normal tissues was also relatively low and thus helped to improve the detection of target lesions.

Although palbociclib is the first CDK4/6 inhibitor approved by the FDA and occupied the half part of the CDK4/6 inhibitor clinical application, ribociclib and abemaciclib are also widely used in the clinic. In the present study, the CDK4/6 targeted PET probe was based on palbociclib; therefore, it may be efficient in predicting the response after palbociclib treatment. More precisely, it is not clear that 68Ga-DOTA-palbociclib is also effective in ribociclib and abemaciclib assessment. We will find out the answer in a follow-up study. In the future, we will also explore more CDK4/6 targeted probes based on other inhibitors and will modify the structure of DOTA-palbociclib, improving the characteristics of the tracer to reduce the accumulation in nontarget organs, especially in the small intestine.

Conclusions

Our results indicate that 68Ga-DOTA-palbociclib could be straightforwardly radiolabeled in a simple and efficient method and exhibited excellent stability and favorable biological performance in vitro and in vivo. 68Ga-DOTA-palbociclib, as a promising tracer for imaging CDK4/6 with PET, may offer an early assessment of individualized response to CDK4/6 inhibitors in cancer patients.

Materials and Methods

DOTA-palbociclib was synthesized by Chinese Peptide Company (Hangzhou, China). NaAc and hydrochloric acid were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai). Acetonitrile (ACN) and trifluoroacetic acid (TFA) were purchased from J&K Science Co., Ltd. (Shanghai, CHN). Instant thin layer chromatography silica gel (ITLC-SG) was obtained from Agilent Technologies (Folsom, CA, USA). A 68Ge-68Ga generator was obtained from Eckert & Ziegler (Berlin, Germany).

The radioactive pharmaceutical was analyzed by radioactive thin layer chromatography (TLC; Raytest mini-GITA) and high-performance liquid chromatography (HPLC), the latter using an Agilent 1200 system equipped with a flow-through γ-detector (Raytest GABI). A ZORBAX 300 SB-C18 (5 μm, 250 × 4.6 mm) column was used for analysis, and the eluting solvents (1 mL/min) used in HPLC were water (solvent A) and acetonitrile (solvent B) with 0.1% trifluoroacetic acid following the gradient: 0–10 min, 25% solvent B.

68Ga Radiolabeling DOTA-Palbociclib

DOTA-palbociclib (20 μg) was dissolved in 640 μL of NaAc (0.5 M) solution. 68GaCl3 was eluted from a 68Ge-68Ga generator with 0.1 M HCl, and 2 mL of 68GaCl3 (370 MBq) elution was added into the DOTA-palbociclib solution. The final pH of the reaction was adjusted to 4 by HCl, and radiolabeling was performed at 100 °C for 10 min. The product was analyzed by radio-TLC (1 M NH4Ac/CH3OH = 1/1 as the mobile phase and on iTLC-SG) and radio-HPLC.

Measurement of the Octanol–Water Partition Coefficient

The lipophilicity of the radiolabeled palbociclib was measured according to our previous study. Approximately 10 μL of 68Ga-DOTA-palbociclib (74 KBq) was mixed with 0.5 mL of water and 0.5 mL of 1-octanol. After stirring in a vortex mixer for 1 min, the organic and water layers were separated by centrifugation. Samples (100 μL) were taken from each layer, and radioactivity was measured in a γ-counter. The experiment was repeated five times.

In Vitro and In Vivo Stability

The stability of 68Ga-DOTA-palbociclib was evaluated in phosphate buffer saline (PBS; 0.1 M pH = 7.4) and mouse plasma. 68Ga-DOTA-palbociclib (0.2 mL, 18.5 MBq) was incubated with 0.5 mL of PBS at room temperature for 3 h, and then, the mixture was analyzed by radio-TLC to determine the stability of 68Ga-DOTA-palbociclib. Meanwhile, 0.2 mL of 68Ga-DOTA-palbociclib (18.5 MBq) was added into 0.5 mL of fresh mouse plasma and remained at 37 °C. After 3 h of incubation, 0.5 mL of acetonitrile was added into the plasma to precipitate the protein. The supernatant was analyzed using radio-HPLC to determine the degradation of 68Ga-DOTA-palbociclib. Three normal mice were used to evaluate the in vivo stability. 68Ga-DOTA-palbociclib (0.2 mL, 7.4 MBq) was injected into each mouse intravenously. At 5, 30, and 60 min after injection, the mouse was killed by CO2/O2 asphyxiation. Blood was obtained by cardiac puncture and collected in a 2 mL microtube. Acetonitrile (200 μL) was added into a 0.5 mL blood sample for the precipitation of protein. The speed of the centrifugation was 10,000 rpm, and the supernatant was indeed analyzed by HPLC with a radioactive detector.

Cell Culture and Animal Models

MCF-7, a human ER+/HER2– breast cancer cell line, was purchased from Cell Bank, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. The cells were cultured in an RPMI1640 medium with penicillin (100 μg/mL), streptomycin (100 μg/mL), and 10% fetal bovine serum (FBS) in a humidified 5% CO2 atmosphere at 37 °C.

All animal studies were approved by the Fudan University Laboratory Animal Ethics Committee and performed according to the ethical principles of animal experimentation. Female BALB/c nude mice (6–8 weeks old) were purchased from Shanghai Lingchang Inst Biotech (Shanghai, CHN) and housed under the conditions of standardized 12 h light/dark cycle, suitable temperature, and ad libitum to feed and water. Five healthy BALB/c mice without a tumor were used for pharmacokinetic measurement. Three days before MCF-7 cell implantation, all tumor-bearing mice were supplemented with an estradiol pellet (0.72 mg, 90 day release, Innovative Research, USA) embedded in the subcutaneous neck area. Twenty-four BALB/c nude mice received subcutaneous injection of MCF-7 cells (2 × 106) in the right shoulder. The mice were studied when the tumor volume reached approximately 320 ± 100 mm3, and tumor volumes were calculated with the formula (length × width2)/2.

In Vitro Uptake

MCF-7 ER+/HER2– breast cancer cells were seeded in 24-well plates at a density of 2 × 105 per well for 24 h. The next day, cells were incubated with 68Ga-DOTA-palbociclib (0.037 MBq/1 mL per well) at 37 °C for 60 min. When the time is up, cells were first washed three times with phosphate buffer saline (PBS) to remove unbound 68Ga-DOTA-palbociclib. Then, 0.5 mL of NaOH was added, and cell suspensions were collected individually. Finally, radioactivity counts were measured with a γ-counter (SN-697, Shanghai Nuclear Institute Rihuan Photoelectric Instrument Co., Ltd.) at an energy window of 140 ± 19 keV. To determine a specific cell uptake, 68Ga-DOTA-palbociclib was added together with an excess of palbociclib (>1000 molar equivalents, without chelating DOTA) to the MCF-7 cells. After incubation for 1 h, the MCF-7 cell uptake assay was repeated as described above. The experiment was repeated in triplicate. The radioactivity was expressed as counts per minute (CPM).

Pharmacokinetics in Normal Mice

The pharmacokinetics of 68Ga-DOTA-palbociclib was determined in normal mice. Mice (n = 5) were injected via the tail vein with 68Ga-DOTA-palbociclib at a dose of 0.74 MBq/mouse, approximately 200 μL volume. At different time points after injection (1, 3, 5, 7, 10, 15, 30, 60, 120, and 240 min), blood samples were immediately collected by the tail vein using tared capillary tubes. All samples were weighed; radioactivity was measured by a γ-counter and decay-corrected to the time of injection. The pharmacokinetics was analyzed by Prism 5 (Graph-Pad Software) using a two-phase decay least-squares fitting method and expressed as percentage injected dose (%ID)/g.

Small-Animal PET/CT Imaging

For PET/CT imaging, MCF-7 tumor-bearing mice (n = 6) were administered with 68Ga-DOTA-palbociclib (5.55 MBq, 0.2 mL) via tail vein injection. Micro-PET/CT (Inveon, Siemens) scanning was performed at 1 h after radiotracer injection. A blocking study was also performed, and mice (n = 3) were injected with an excess of palbociclib (>1000 molar equivalents) 0.5 h before radiotracer injection. Then, a Micro-PET/CT scan was acquired 1 h later employing the same method. During the scanning, the mice were anesthetized by 1.5% isoflurane gas in oxygen flowing at 0.5 mL/min, and the mice body temperature was maintained by heated air flow under the bed. The PET and CT images were reconstructed using a three-dimensional ordered-subset expectation–maximization (OSEM3D)/maximum algorithm and fused using Inveon Research Workplace software (Siemens Medical Solutions). For data analysis, regions of interest (ROI) were manually drawn around the whole tumor and within quadricep muscles in the fused images. Data were expressed as the mean of standardized uptake value (SUV). The tumor-to-muscle (T/M) ratio was calculated as the ratio of the mean SUV of a tumor to that of contralateral quadricep muscles.

Biodistribution in Tumor-Bearing Mice

For biodistribution studies, MCF-7 tumor-bearing mice were injected with 68Ga-DOTA-palbociclib (0.74 MBq/0.1 mL) via the tail vein. At 1 and 2 h after injection of the radiotracer, the mice (n = 3/per time point) were sacrificed. Tumors and major organs of interest (liver, lung, kidney, spleen, stomach, large intestine, bone, muscle, brain, blood, and heart) were collected, weighed, and measured for their radioactivity by a γ-counter. To confirm the CDK4/6-specific uptake by tumors, a blocking study was performed. In the blocking group, MCF-7 tumor-bearing mice (n = 3) received an injection of an excess of palbociclib (>1000 molar equivalents) 30 min before the injection of 68Ga-DOTA-palbociclib (0.74 MBq/0.1 mL) via the tail vein and sacrificed at 1 h post-injection. The radioactivity uptakes in the organs were expressed as a percentage of the injected radioactive dose per gram of tissue (%ID/g).

Autoradiography and Immunofluorescence

After the γ-counting of tumors from the biodistribution study, tumor tissues were frozen using an optimal cutting temperature (OCT) compound at −80 °C and divided by a cryotome (RM2235, Leica Instruments) into 10 μm-thick slices. Consecutive sections were used for autoradiography and immunofluorescence. The cryosections were placed in an imaging plate for autoradiography. After a 3 h exposure, the plates were scanned with a Fuji Analyzer BAS-5000 (Fuji, Tokyo, Japan). Phosphor imaging plates were read by a Typhoon 9500 IP plate reader (GE Healthcare Life Sciences).

For immunofluorescence techniques, the adjacent slides were fixed with 4% paraformaldehyde in PBS and stained with anti-CDK4 (ab68266, Abcam) and anti-CDK6 antibodies (ab68266, Abcam). The sections were placed in a humidified chamber and incubated overnight at 4 °C. Sections were washed with PBS, and Alexa Fluor-conjugated secondary antibodies (Thermo Fisher Scientific) were incubated for 50 min at room temperature and shielded from direct light. Slides were counterstained with DAPI and mounted in medium for fluorescence (Invitrogen). Sections were observed under an inverted fluorescence microscope (NIKON ECLIPSE CI-S), and images were collected.

Statistical Analysis

All data were expressed as mean ± standard deviation (SD). Differences between cohorts were analyzed with one-way analysis of variance or t test using GraphPad Prism (version 5.01, San Diego, California USA). Results with a P-value less than 0.05 were considered statistically significant.

Acknowledgments

We wish to thank Jianping Zhang and Simin He for excellent technical assistance.

Author Contributions

# C.L. and Z.Y. contributed equally to this work. C.L., Z.Y., and X.X. participated in the experimental and data analysis. C.L. and X.X. wrote the manuscript. C.L., Z.Y., M.L., and X.W. were involved in cell studies and animal imaging and dealt with the tumors. X.X., S.S., and Z.Y. helped in polishing the articles. C.L., X.X., and Z.Y. designed and controlled the quality of the study. All authors have reviewed the manuscript.

This study was funded by the National Key Research and Development Program of China (2020YFA0909003), Shanghai Sailing Program (20YF1408500), Science and Technology Development Fund of Shanghai Pudong New Area (PKJ2020-Y54), Shanghai Committee of Science and Technology Fund (19ZR1411300), and Shanghai Municipal Health Commission (202040269).

The authors declare no competing financial interest.

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