Serial Multitracer PET/CT for Characterization of the Inflammation–Fibrosis Axis in Liver Injury

As the global burden of liver disease is increasing, the interplay between inflammation and fibrosis is emerging as a promising target for antifibrotic hepatic therapies. Such advanced interventions will require biomarker guidance, which is an opportunity for molecular imaging. We tested the feasibility of multiple radiopharmaceuticals to characterize disease progression and regression in a mouse model of fibrotic liver injury. Methods: C57BL/6N mice (n = 31) received intraperitoneally injected carbon tetrachloride (CCl4) along with serial PET/CT at baseline, in the prefibrotic inflammatory phase (3 wk of CCl4), in the fibrosis phase (6 wk of CCl4), and in the regression phase (3 wk after termination of CCl4). PET/CT included the metabolic marker [18F]FDG; the leukocyte marker [68Ga]Ga-pentixafor (chemokine [C-X-C motif] receptor 4 ligand); 2 fibroblast activation protein (FAP) markers, [68Ga]Ga-MHLL2 and [68Ga]Ga-FAP inhibitor (FAPI)–46; and the αvβ6-integrin ligand [68Ga]Ga-trivehexin. Correlative measurements included serum biomarkers, in vitro cell assays, and tissue analyses. Results: CCl4 administration led to elevation of liver transaminases, which was reversible in the regression phase. This was associated with gross morphologic changes, histologic macrophage–monocyte infiltration, and increased collagen deposition. Hepatic [18F]FDG uptake increased in the prefibrosis phase (P = 0.005) and the fibrosis phase (P = 0.049) compared with baseline. [68Ga]Ga-pentixafor reached its peak in the prefibrosis phase (P = 0.003), consistent with the abundance of CD68-positive cells observed upon histology. Although αvβ6-integrin, as a marker of profibrotic activity, was elevated as expected in the fibrosis phase (P = 0.038), we found a surprising reduction of uptake of both FAP tracers in the prefibrosis phase ([68Ga]Ga-MHLL2, P = 0.001; [68Ga]Ga-FAPI-46, P = 0.001) and the fibrosis phase ([68Ga]Ga-MHLL2, P < 0.001; [68Ga]Ga-FAPI-46, P = 0.001), despite histologic proof of FAP-positive stellate cells. In vitro cell assays showed a fast cellular efflux of FAP tracers, potentially limiting specific accumulation in FAP-expressing injured tissue. Conclusion: Activation of the inflammation–fibrosis axis by liver injury is associated with stimulated hepatic glucose utilization. Chemokine receptor 4 targeting specifically identifies hepatic tissue inflammation, whereas αvβ6-integrin identifies fibrotic activity. This supports the feasibility of targeted molecular imaging to characterize hepatic response to injury and provides a foundation for image guidance of antifibrotic therapies. The value of FAP ligands was limited in our model and requires further investigation.