Enhanced Detection of Early Pulmonary Fibrosis Disease Using 68Ga-FAPI-LM3 PET

Abstract

Early detection of pulmonary fibrosis is a critical yet insufficiently met clinical necessity. This study evaluated the effectiveness of FAPI-LM3, a ⁶⁸Ga-radiolabeled heterobivalent molecular probe that targets fibroblast activating protein (FAP) and somatostatin receptor 2 (SSTR2), in the early detection of pulmonary fibrosis, leveraging its potential for early disease identification. A bleomycin-induced early pulmonary fibrosis model was established in C57BL/6 mice for 7 days. FAP and SSTR2 expression levels were quantitatively assessed in human idiopathic pulmonary fibrosis lung tissue samples and bleomycin-treated mouse lung tissues by using western blotting, real-time quantitative PCR (RT-qPCR), and immunofluorescence techniques. The diagnostic performance of FAPI-LM3 was investigated by synthesizing monomeric radiotracers ⁶⁸Ga-FAPI-46 and ⁶⁸Ga-DOTA-LM3 alongside the heterobivalent probe ⁶⁸Ga-FAPI-LM3. These imaging radiopharmaceuticals were used in small-animal PET to compare their uptake in fibrotic and normal lung tissues. Results indicated significant upregulation of FAP and SSTR2 at both RNA and protein levels in fibrotic lung tissues compared with that in normal controls. PET imaging demonstrated significantly enhanced uptake of the ⁶⁸Ga-FAPI-LM3 probe in fibrotic lung tissues, with superior visual effects compared to monomeric tracers. At 60 min postinjection, early stage fibrotic tissues (day 7) demonstrated low-to-medium uptake of monomeric probes, including ⁶⁸Ga-DOTA-LM3 (0.45 ± 0.04% ID/g) and ⁶⁸Ga-FAPI-46 (0.78 ± 0.09% ID/g), whereas the uptake of the heterobivalent probe ⁶⁸Ga-FAPI-LM3 (1.90 ± 0.10% ID/g) was significantly higher in fibrotic lesions than in normal lung tissue. Blockade experiments confirmed the specificity of ⁶⁸Ga-FAPI-LM3 uptake, which was attributed to synergistic targeting of FAP and SSTR2. This study demonstrates the potential of ⁶⁸Ga-FAPI-LM3 for early pulmonary fibrosis detection via molecular imaging, offering significant benefits over monomeric tracers ⁶⁸Ga-FAPI-46 and ⁶⁸Ga-DOTA-LM3. This strategy offers new possibilities for noninvasive and precise early detection of pulmonary fibrosis.

Keywords: FAPI-LM3; fibroblast-activating protein; molecular imaging; pulmonary fibrosis; somatostatin receptor 2.

Figure 1

Chemical structure and in vivo stability of ⁶⁸Ga-FAPI-LM3. (a) Chemical structure of ⁶⁸Ga-FAPI-LM3. (b–d) Assessment of ⁶⁸Ga-FAPI-LM3, including radiochemical purity (b) and in vivo stability in blood (c) and urine (d) through radio-high-performance liquid chromatography analysis.

Figure 2

Elevated expression of FAP and SSTR2 in fibrotic lung tissue. (a) Comparison of FAP and SSTR2 expression between 40 IPF and 8 normal tissue samples from GSE53845 (compared to the control group, **p < 0.01, ****p < 0.0001.) (b) Measurement of FAP and SSTR2 mRNA concentrations in lung tissue from control mice and bleomycin-treated mice 7 days after instillation (n = 3). p values were calculated using Student’s t test (compared to the control group, ***p < 0.001.) (c) Western blotting was performed for FAP and SSTR2 in mouse lung tissues treated with saline solution and bleomycin (n = 3). β-actin served as a housekeeping standard. (d) Representative hematoxylin and eosin (H&E) stained images of mouse lung sections treated with saline solution and bleomycin are presented. Scale bar: 50 μm. (e) Representative immunofluorescence images of FAP and SSTR2 from mouse lung sections treated with saline solution and bleomycin are presented. Scale bar: 25 μm.

Figure 3

Detection of FAP with ⁶⁸Ga-FAPI-46 PET/CT in mice. PET/CT images and volume of interest (VOI) quantification data for ⁶⁸Ga-FAPI-46 uptake were acquired in both bleomycin-treated and control mice, measured at 30 and 60 min postinjection (n = 3, **p < 0.01).

Figure 4

Detection of SSTR2 with ⁶⁸Ga-DOTA-LM3 PET/CT in mice. PET/CT images and VOI quantification data for ⁶⁸Ga-DOTA-LM3 uptake were acquired in both bleomycin-treated and control mice, measured at 30 and 60 min postinjection (n = 3, ***p < 0.001).

Figure 5

Detection of FAP and SSTR2 with ⁶⁸Ga-FAPI-LM3 PET/CT in mice. PET/CT images and VOI quantification data for ⁶⁸Ga-FAPI-LM3 uptake were acquired in both bleomycin-treated and control mice, measured at 30 and 60 min postinjection (n = 3, ***p < 0.001).

Figure 6

Representative PET imaging and VOI quantification data of ⁶⁸Ga-FAPI-LM3 and blocking with FAPI-46, DOTA-LM3, or FAPI-46 plus DOTA-LM3 (double block) in mice at 60 min postinjection (n = 3, **p < 0.01, ***p < 0.001).