2.1 Synthesis of PET tracers

The radiotracer ¹⁸F-FDG was manufactured via a fully automated FDG MX module (GE Medical Systems, Germany). The radiochemical purity of ¹⁸F-FDG was > 98%.

An automated TRACERlab FX_F-N radiosynthetic module (GE Medical Systems, USA) was used to prepare ¹⁸F-PBR06 as previously reported^[16]. Further, ¹⁸F-PBR06 was synthesized using a novel tosylate precursor as described previously^[17]. The synthetic procedures of ¹⁸F-PBR06 are shown in Fig. 1a. Radiosynthesis of ¹⁸F-PBR06 using the new tosylated precursor provided similar radiochemical purity, higher specific activity, and higher radiochemical yield in comparison with radiosynthesis using bromine precursor^[17]. The decay corrected radiochemical yields of ¹⁸F-PBR06 were 30–40% (end of synthesis, EOS), and the radiochemical purity of ¹⁸F-PBR06 was greater than 99%.

Figure 1Full-screen View

(Color online) (a) Synthetic procedures of ¹⁸F-PBR06 using a novel tosylate precursor. (b) Radio-HPLC analysis of ¹⁸F-PBR06. (c) Flow chart of the animal study design. Models of inflammation and lung cancer (n = 3 each) were used for ¹⁸F-FDG Micro-PET/CT imaging, followed by ¹⁸F-PBR06 Micro-PET/CT imaging 3 days later. Subsequently, the animals were sacrificed for immunofluorescence analysis.

2.2 Animal models

The animal study protocol was in accordance with the principles and procedures outlined in the Guide for the Care and Use of Laboratory Animals and approved by Zhongshan Hospital. FVB/N (FVB) and athymic nude mice (both female, 6–8 weeks) were purchased from Peking University Laboratory Animal Center (Beijing, China). Each athymic nude moue was inoculated with 2 × 10⁶ A549 lung cancer tumor cells to establish the lung cancer model. When the A549 tumor volumes in athymic nude mice reached 0.5 cm³, the FVB mice were injected with 20 µL turpentine into the muscles of their left hind legs to establish the inflammation model. In total, 14 animals were used in the whole study. Three mice each of inflammation and lung cancer models were used for ¹⁸F-FDG Micro-PET/CT imaging and ¹⁸F-PBR06 Micro-PET/CT imaging. Subsequently, the animals were sacrificed for immunofluorescence analysis (Fig. 1c). Further, an additional four mice each of inflammation and lung cancer models were used for the biodistribution study. All mice were maintained in a temperature-controlled environment under a 12 h light/dark cycle with free access to food and water.

2.3 Biodistribution studies for ¹⁸F-PBR06

The inflammation and lung cancer models were intravenously injected with 5.56 MBq of ¹⁸F-PBR06 (n = 4) and sacrificed 120 min later by ethyl ether anesthesia. Blood was collected from the heart, and normal tissues (kidneys, brain, liver, spleen, intestine, lung, stomach, pancreas, and muscles of the right hind leg), inflamed muscles, and A549 tumors were excised. All samples were weighed, and the radioactivity was measured by a GC-1200 gamma counter (HYMI-1403-015), followed by decay correction. Tracer accumulations of a Three-dimensional Region of Interest (3D-ROI) for a volume were calculated as follows: 3D-ROI = (radioactivity of organs/radioactivity of injected drugs)/quality of organs. The results were expressed as the percentage of injected dose per gram of tissue (% ID/g).

2.4 Micro-PET/CT imaging

A Siemens Inveon Micro-PET/CT scanner (Siemens Preclinical Solutions, Knoxville, TN, USA) was used for Micro-PET/CT scans and imaging analysis. For the inflammation models, 10 min PET images and 5 min CT images were acquired on day 4 post-injection of turpentine oil at 1 h following the 5.56 MBq ¹⁸F-FDG injection via the tail vein. Furthermore, 1.5 h dynamic PET images and 5 min CT images were obtained on day 7 post-injection of turpentine oil following the 5.56 MBq ¹⁸F-PBR06 injection via the tail vein. For the lung cancer models, 10 min PET imaging and 5 min CT images was performed at 1 h following the 5.56 MBq ¹⁸F-FDG injection via the tail vein on day 25 post-inoculation. Subsequently, 1.5 h dynamic PET imaging and 5 min CT images were performed following the 5.56 MBq ¹⁸F-PBR06 injection via the tail vein on day 28. The data were reconstructed using a 3-D OSEM algorithm and segmented attenuation. 3D-ROIs were placed on the inflamed lesions or tumor tissues, and a threshold of 40% of the maximum uptake was used for the final ROI drawing.

2.5 Immunofluorescence

The inflammatory regions on day 7 after turpentine injection and lung cancer models on day 28 after inoculation into mice were excised and embedded in paraffin. The inflamed muscle slices and tumor tissue sections were incubated with rabbit anti-CD68 antibody (1:100, Abcam, USA) for 1 h, followed by Cy3-conjugated anti-rabbit secondary antibodies (1:200, Jackson ImmunoResearch Laboratories) for 30 min in the dark at room temperature. After PBS washes, the slices were mounted with VECTASHIELD^® mounting medium containing DAPI and coverslipped before visualization by epifluorescence microscopy (IX-81, Olympus).

2.6 Statistical analysis

The data are expressed as mean and standard deviation (SD). P-values < 0.05 were considered statistically significant.

3.1 Biodistribution of ¹⁸F-PBR06

The biodistribution data of ¹⁸F-PBR06 in the inflammation and tumor models achieved at 120 min post-injection are presented in Fig. 2a. ¹⁸F-PBR06 displayed higher uptake in the inflamed lesions (4.79 ± 0.48), whereas relatively lower uptake in the lung tumor tissues (1.55 ± 0.29) (P < 0.001) was found. Considering that accumulation in the brain, spleen, lung, blood, kidneys, right hind leg muscles, and intestines of the inflammation models was higher than the lung cancer models (all P < 0.05), we used the target-to-nontarget ratios at 120 min post-injection in the animal models to explore the potential of ¹⁸F-PBR06 to differentiate the inflamed lesions from lung tumor tissues.

Figure 2.Full-screen View

(Color online) (a) Biodistribution of ¹⁸F-PBR06 at 2 h in inflammation and lung cancer models. (b) Parameters of target-to-nontarget at 2 h after injection in inflammation and lung cancer models.

The ratio of inflammation-to-blood (I/B) was 3.53 ± 0.26, and the ratio of tumor-to-blood (T/B) was 1.77 ± 0.35 (P < 0.001). The ratio of inflammation-to-muscle (I/M) was 2.33 ± 0.64, and the ratio of tumor-to-muscle (T/M) was 1.45 ± 0.14 (P = 0.036). The results revealed that ¹⁸F-PBR06 had relatively high uptake in the inflammatory regions, and accumulation of ¹⁸F-PBR06 in the lung tumor tissues was lower than in the inflammatory regions.

3.2 Micro-PET/CT imaging

The time activity curves obtained from the dynamic Micro-PET/CT imaging of ¹⁸F-PBR06 are shown in Fig. 3, indicating that ¹⁸F-PBR06 uptake in the inflamed muscles remained higher than normal muscles during the 90 min of dynamic Micro-PET/CT imaging (All P < 0.05). Moreover, ¹⁸F-PBR06 uptake in the inflamed muscles remained higher than tumor tissues. Based on the biodistribution results, it further confirmed the specificity of ¹⁸F-PBR06 for TSPO evaluation.

Figure 3.Full-screen View

(a) Time activity curves (TACs) of inflamed muscles during 90 min ¹⁸F-PBR06 dynamic Micro-PET/CT imaging. (b) TACs of lung tumor tissues during 90 min ¹⁸F-PBR06 dynamic Micro-PET/CT imaging.

Fig. 4 shows the Micro-PET/CT images of both tracers obtained from the inflammation and lung cancer models. All images were viewed from the top. ¹⁸F-FDG showed high uptake in both inflamed muscles and lung tumor tissues. However, ¹⁸F-PBR06 showed high uptake in inflamed muscles, but low uptake in lung tumor tissues. The results revealed that, compared to ¹⁸F-FDG, ¹⁸F-PBR06 showed superior specificity for inflammation.

Figure 4Full-screen View

(Color online) ¹⁸F-PBR06 and ¹⁸F-FDG images of an inflammation model and lung cancer model. a, d, g, and j show coronal views; b, e, h, and k show transverse views; and c, f, i, and l show sagittal views. ¹⁸F-FDG accumulation showed high uptake during inflammation (g, h, and i). ¹⁸F-FDG accumulation showed high uptake in A549 tumor tissues (j, k, and l). ¹⁸F-PBR06 accumulation showed high uptake during inflammation (a, b, and c). ¹⁸F-PBR06 accumulation showed low uptake in A549 tumor tissues (d, e, and f).

3.3 Immunofluorescence

Immunofluorescence staining revealed macrophages in all specimens of the inflammation and lung cancer models. As seen in Fig. 5, macrophages in the inflammatory regions showed a higher fluorescence signal than the lung tumor tissue. These results revealed that the number of macrophages in the inflammatory regions was significantly higher than the lung tumor tissue. Therefore, the amount of TSPO in the inflammatory regions was also significantly higher than the lung tumor tissue. These results were consistent with the biodistribution and Micro-PET/CT imaging results.

Figure 5Full-screen View

(Color online) Immunofluorescence of a specimen excised from an inflammation model and lung cancer model. A specimen from an inflamed muscle (a, b, c) and A549 tumor (d, e, f) (all samples were stained with CD68⁺ and magnified × 200). The nuclei were stained blue, and TSPO was stained green. A, D are merged images, and the fluorescence signal of TSPO in inflamed muscle was much higher than the A549 tumor.

3.4 Statistical analysis

The time activity curves obtained from the dynamic Micro-PET/CT imaging of ¹⁸F-PBR06 revealed higher uptake in the inflamed muscles than lung tumor tissues during the 90 min imaging (all P < 0.05). Biodistribution demonstrated that the parameters of inflammation-to-blood in ¹⁸F-PBR06 were significantly higher than those of tumor-to-blood (P < 0.001). Additionally, the parameters of inflammation-to-muscle in ¹⁸F-PBR06 were higher than those of tumor-to-muscle (P = 0.036).

4. Discussion

¹⁸F-FDG showed increased accumulation in the inflamed muscles and lung tumor tissues in this study, which was in agreement with previous reports, wherein ¹⁸F-FDG has high sensitivity but low specificity^[5]. However, ¹⁸F-PBR06 exhibited high uptake during inflammation, but low uptake by the lung tumor tissues. Thus, ¹⁸F-PBR06 can be considered as a higher specificity inflammation imaging tracer compared to ¹⁸F-FDG. Furthermore, ¹⁸F-PBR06 may have potential to differentiate between lung cancer and inflammation.

¹⁸F-FDG has been used for diagnosis of inflammation and detection of infectious loci^[18-20]. In the experimental inflammatory muscles, ¹⁸F-FDG uptake was significantly higher than uptake in general muscle tissues. However, ¹⁸F-FDG accumulated not only in the inflamed lesions, but also in a variety of tumors, including lung tumors^[21,22]. Furthermore, ¹⁸F-FDG leads to high local uptake in inflammation and recurrent tumors for evaluation of changes in lesions after radio- or chemotherapy. Therefore, the inflammatory changes and recurrent tumors could not be differentiated from inflammation by ¹⁸F-FDG PET/CT imaging^[23,24].

¹⁸F-PBR06 is a novel radioligand that can characterize inflammation by measuring TSPO receptors; thus, ¹⁸F-PBR06 PET/CT imaging can be a new tool for characterization of inflammation. Studies of some of the TSPO-specific tracers have been reported in neuroinflammatory diseases^[25,26] and inflammatory diseases of the peripheral tissues^[27,28]. In our study, we selected ¹⁸F-PBR06 for its increased binding affinity, higher signal-to-noise ratio, and low lipophilicity, and it has been used in human studies.

In the current study, we used turpentine oil-induced inflammatory models to investigate the feasibility of ¹⁸F-PBR06 PET/CT imaging in diagnosing inflammatory diseases. Thus, we selected day 7 after turpentine oil injection as the window for the macrophage infiltration in the inflammatory regions. In the acute inflammation phase, the cell population within the inflammatory regions primarily included lymphocytes and neutrophils, and the chronic inflammation phase mainly displayed monocytes and macrophages^[29]. The turpentine oil would not be cleared easily, and hence, on day 3–6 post-turpentine oil injection, the inflammatory reaction would become chronic inflammation^[30]. Therefore, ¹⁸F-PBR06 uptake would reach a high level on day 7, and combined with the immunofluorescent images, we demonstrated that ¹⁸F-PBR06 PET/CT imaging reflected macrophage infiltration during chronic inflammation. Therefore, the findings of the current study may indicate the feasibility of using ¹⁸F-PBR06 PET/CT imaging for assessing inflammation via macrophage infiltration.

Compared to ¹⁸F-FDG, ¹⁸F-PBR06 exhibited lower uptake in lung tumor tissues, thereby designating ¹⁸F-PBR06 as a specific tracer to characterize inflammation. In addition, ¹⁸F-PBR06 showed low uptake in lung tumor tissues (1.55 ± 0.29), but high uptake during inflammation (4.79 ± 0.48). Previous reports showed elevated TSPO levels within some specific tumors, such as ovary, breast, colon, and prostate cancer^[31]. However, in this study, lung tumor tissues exhibited significantly low uptake of ¹⁸F-PBR06 (T/B = 1.77 ± 0.35, T/M = 1.45 ± 0.14) as compared to the inflamed muscles (I/B = 3.53 ± 0.26, I/M=2.33 ± 0.64). In ¹⁸F-PBR06 Micro-PET/CT imaging, the lung tumor tissues showed lower uptake than the inflamed muscles (Fig. 4). Combined with the immunofluorescence results, the number of macrophages and the amount of TSPO in the inflammatory regions were significantly higher than the lung tumor tissues. There would also be macrophage infiltration in pulmonary inflammation nodules^[32], suggesting that ¹⁸F-PBR06 PET/CT imaging may allow for diagnosis of pulmonary inflammation nodules. Therefore, ¹⁸F-PBR06 may be utilized to differentiate between peripheral lung cancer and inflammation nodules, thus enhancing accuracy in detection of pulmonary nodules.

Nevertheless, the present study had some limitations. For better comparisons of the data, this experiment could be improved by using mice bearing both inflammation and tumors in the same tissue. ¹⁸F-PBR06 showed increased accumulation in the lung that was higher than uptake in the inflamed muscles; however, a previous study reported that ¹⁸F-PBR06 showed very low uptake by the lung in the biodistribution of humans^[12]. Moreover, a previous study reported that a TSPO-specific tracer might be a useful tool for evaluating lung inflammation^[28]. Turpentine was used to induce inflammation in this study; however, turpentine inhalation might lead to pulmonary inflammation and empyema^[33]. Therefore, pulmonary inflammation induced by turpentine inhalation might be the reason why ¹⁸F-PBR06 showed increased accumulation in the lung in our study. Taken together, further verification is essential to evaluate the usefulness of ¹⁸F-PBR06 in differentiating peripheral lung cancer and inflammation nodules.