Background: Fibroblast activation protein (FAP) is commonly expressed in activated stromal fibroblasts in various epithelial tumours. Recently, ⁶⁸Ga-FAPI-04 has been used for tumour imaging in positron emission tomography/computed tomography (PET/CT). This study aimed to compare the diagnostic performances of ⁶⁸Ga-FAPI-04 PET/CT and ¹⁸F-FDG PET/CT in hepatocellular carcinoma (HCC), and to assess factors associated with ⁶⁸Ga-FAPI-04 uptake in HCC.Materials and Methods: Twenty-nine patients with suspiciously HCC who received both ¹⁸F-FDG and ⁶⁸Ga-FAPI-04 PET/CT were included in this retrospective study. The results were interpreted by two experienced nuclear medicine physicians independently. The maximum and mean standardized uptake values (SUV_max and SUV_mean) were measured in the lesions and liver background, respectively. The tumour-to-background ratio (TBR) was then calculated as lesion’s SUV_max divided by background SUV_mean.Results: A total of 35 intrahepatic lesions in 25 patients with HCC were finally involved in the statistical analysis. ⁶⁸Ga-FAPI-04 PET/CT showed a higher sensitivity than ¹⁸F-FDG PET/CT in detecting intrahepatic HCC lesions (85.7% vs. 57.1%, P = 0.002), including in small (≤ 2 cm in diameter; 68.8% vs. 18.8%, P = 0.008) and well- or moderately-differentiated (83.3% vs. 33.3%, P = 0.031) tumors. SUV_max was comparable between ⁶⁸Ga-FAPI-04 and ¹⁸F-FDG (6.96 ± 5.01 vs. 5.89 ± 3.38, P > 0.05), but the TBR was significantly higher in the ⁶⁸Ga-FAPI-04 group compared with the ¹⁸F-FDG group (11.90 ± 8.35 vs. 3.14 ± 1.59, P < 0.001). SUV_max and the TBR in ⁶⁸Ga-FAPI-04 positive lesions were associated with tumour size (both P < 0.05), but not the remaining clinical and pathological features (all P > 0.05).Conclusions: ⁶⁸Ga-FAPI-04 PET/CT is more sensitive than ¹⁸F-FDG PET/CT in detecting HCC lesions, and ⁶⁸Ga-FAPI-04 uptake is correlated mainly with tumour size.

Immune checkpoint inhibitors, particularly PD-1/PD-L1 blockades, have been approved for unresectable hepatocellular carcinoma (HCC). However, high resistance rates still limit their efficacy, highlighting the urgent need to understand the underlying mechanisms and develop strategies for overcoming the resistance. In this study, we demonstrate that HCC with high MER proto-oncogene tyrosine kinase (MerTK) expression exhibits anti-PD-1/PD-L1 resistance in two syngeneic mouse models and in patients who received anti-PD-1/PD-L1 therapy. Mechanistically, MerTK renders HCC resistant to anti-PD-1/PD-L1 by limiting ferroptosis with the upregulation of SLC7A11 via the ERK/SP1 pathway and facilitating the development of an immunosuppressive tumor microenvironment (TME) with the recruitment of myeloid-derived suppressor cells (MDSCs). Sitravatinib, an inhibitor of MerTK, sensitizes resistant HCC to anti-PD-L1 therapy by promoting tumor ferroptosis and decreasing MDSC infiltration into the TME. In conclusion, we find that MerTK could serve as a predictive biomarker for patient stratification and as a promising target to overcome anti-PD-1/PD-L1 resistance in HCC.

Background: Neutrophil extracellular traps (NETs) are net like extracellular structure formed by neutrophils in response to certain stimulation. It works as inflammatory regulator and metastasis promoter in cancer. Mitochondrial-(mt)DNA is a circular, mitochondria derived double strain molecule, which is involved in NETs formation. Its role in NETs induced inflammatory alteration in hepatocellular carcinoma (HCC) remained unexplored.

Background: Immune escape is an important feature of hepatocellular carcinoma (HCC). The overall response rate of immune checkpoint inhibitors (ICIs) in HCC is still limited. Revealing the immune regulation mechanisms and finding new immune targets are expected to further improve the efficacy of immunotherapy. Our study aims to use CRISPR screening mice models to identify potential targets that play a critical role in HCC immune evasion and further explore their value in improving immunotherapy. Methods: We performed CRISPR screening in two mice models with different immune backgrounds (C57BL/6 and NPG mice) and identified the immunosuppressive gene Gsk3a as a candidate for further investigation. Flow cytometry was used to analyze the impact of Gsk3a on immune cell infiltration and T-cell function. RNA sequencing was used to identify the changes in neutrophil gene expression induced by Gsk3a and alterations in downstream molecules. The therapeutic value of the combination of Gsk3a inhibitors and anti-programmed cell death protein-1 (PD-1) antibody was also explored. Results: Gsk3a, as an immune inhibitory target, significantly promoted tumor growth in immunocompetent mice rather than immune-deficient mice. Gsk3a inhibited cytotoxic T lymphocytes (CTLs) function by inducing neutrophil chemotaxis. Gsk3a promoted self-chemotaxis of neutrophil expression profiles and neutrophil extracellular traps (NETs) formation to block T-cell activity through leucine-rich α-2-glycoprotein 1 (LRG1). A significant synergistic effect was observed when Gsk3a inhibitor was in combination with anti-PD-1 antibody. Conclusions: We identified a potential HCC immune evasion target, Gsk3a, through CRISPR screening. Gsk3a induces neutrophil recruitment and NETs formation through the intermediate molecule LRG1, leading to the inhibition of CTLs function. Targeting Gsk3a can enhance CTLs function and improve the efficacy of ICIs.