Acyl-CoA synthetase long-chain family member 4 (ACSL4)-targeting fluorescent probes for precision intraoperative visualization of hepatocellular carcinoma.

Purpose: This study aims to develop and validate a novel ACSL4-targeted fluorescent probe to enhance intraoperative visualization of hepatocellular carcinoma (HCC), emphasizing its binding affinity, specificity, and clinical applicability.

Methods: Transcriptomic sequencing data from TCGA, ICGC, CPTAC, and GSE25097 were analyzed to establish ACSL4 as a viable target for tumor visualization. An ACSL4-specific binding peptide (ABP) was identified using a combination of in vivo and in vitro phage display screening. The peptide was conjugated with IRDye 800CW to create a fluorescent probe (ABP-IRDye 800CW). Binding specificity was assessed using fluorescence imaging and immunoprecipitation assays. Biosafety was evaluated through primary organ histology and blood biochemistry following probe administration.

Results: Surface plasmon resonance analysis demonstrated that ABP exhibited moderate binding affinity to ACSL4 (Kd = 3.767 μM). The probe selectively targeted HCC cells with high ACSL4 expression while sparing normal tissues. In vivo imaging showed the most potent tumor-to-background ratio (TBR) of 4.58 in patient-derived xenograft (PDX) models, correlating with ACSL4 expression. Histological evaluation and blood biochemistry revealed no significant toxicity, with no observable changes in liver or kidney function.

Conclusion: The ACSL4-targeted fluorescent probe holds significant potential for real-time, intraoperative imaging of HCC, paving the way for future clinical applications.