A Novel Near-Infrared Fluorescence Imaging Study Targeting Hepatocellular Carcinoma (HCC)

Hepatocellular carcinoma (HCC) ranks as the sixth most common malignancy and the third leading cause of cancer-related mortality worldwide. Statistics indicate that the postoperative recurrence rate of HCC reaches as high as 80%, with liver fibrosis or cirrhosis forming the pathological basis for over 80% of HCC cases. The high recurrence rate of HCC is closely associated with residual peri-tumoral microlesions, including components of the tumor stroma, after surgery. In current clinical practice, determining if a tumor has been completely excised primarily relies on the surgeon’s experience and preoperative imaging, or on intraoperative frozen section analysis. However, these methods are often limited by insufficient sampling and the heterogeneous expression of tumor markers, making precise and objective intraoperative assessment difficult.

To address this issue, the authors focused on a key component of the HCC tumor microenvironment—cancer-associated fibroblasts (CAFs). CAFs are abundantly present in the tumor stroma and play a role in tumor growth, recurrence, metastasis, and postoperative immunosuppression. Compared to the highly heterogeneous HCC parenchymal cells, CAFs are genetically more stable and less prone to mutation. Fibroblast activation protein (FAP), a principal biomarker of CAFs, is highly expressed in over 80% of HCC stromal samples while exhibiting very low expression in normal tissues. Thus, FAP is a highly specific and stable target for tumor imaging.

To overcome the limitations of existing HCC imaging techniques, this study proposed a novel stromal-targeted imaging strategy. By developing a new near-infrared (NIR) fluorescence probe (ICG-FAP-TATA) that targets FAP-positive CAFs, the study aimed to achieve precise imaging of FAP+ CAFs, facilitate the detection of HCC lesions, and visualize high-risk areas for metastasis, thereby providing potential technical support for precision therapies.

---

Research Team and Context

This study was a collaborative effort by researchers from multiple institutions, including The Fifth Affiliated Hospital of Sun Yat-Sen University, the Institute of Automation (Chinese Academy of Sciences), and the Shanghai Institute of Materia Medica (Chinese Academy of Sciences). First authors include En Lin, Miaomiao Song, and others, with Jian Li, Zhenhua Hu, and Zhen Cheng serving as corresponding authors. The study was published in the European Journal of Nuclear Medicine and Molecular Imaging. The research was received on September 24, 2024, and accepted on January 10, 2025.

---

Research Workflow in Detail

1. Probe Synthesis and Characterization

The research team designed and synthesized a novel FAP-targeting NIR fluorescence probe, ICG-FAP-TATA. The synthesis process involved:

• Modifying a traditional FAP-targeting peptide (FAP-Pep) by replacing it with 1,3,5-Triaacryloyl-Triazinane, creating a stabilized cyclic structure.
• Conjugating the cyclized FAP-TATA with an indocyanine green derivative (ICG-NH2), a near-infrared fluorophore.

The successful synthesis of the probe was confirmed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), with purity exceeding 95% as verified by high-performance liquid chromatography (HPLC). The probe had an average particle diameter of approximately 11.32 nm and demonstrated excellent near-infrared absorption and emission properties.

---

2. In Vitro Validation of the Probe

In vitro experiments were conducted using cell lines with varying levels of FAP expression:

• FAP-negative cells (HCC cell line LM3-Luc and LX2 hepatic stellate cells).
• FAP-high-expressing cells (LX2-hFAP, created by transfecting LX2 cells with the FAP gene).

The probe exhibited significant fluorescence signals in the FAP-high-expressing LX2-hFAP cells but showed substantially weaker signals in FAP-negative controls, confirming its high specificity toward FAP.

---

3. In Vivo Imaging

To evaluate the in vivo imaging capability of ICG-FAP-TATA, the research team used HCC xenograft models:

• Tumor models with varying levels of FAP expression were created, including a high-expression group (1:1 mix of LM3-Luc and LX2), a moderate-expression group, and a low-expression group.
• The probe was administered via tail vein injection. Fluorescence signals rapidly increased in tumor regions, peaking at day 4 and persisting for over 10 days.
• The probe primarily accumulated in tumors, liver, and kidneys. Tumors in the high-FAP-expression group showed significantly higher tumor-to-background ratios (TBR), which correlated strongly with fluorescence intensity (R^2 > 0.8, p < 0.05).

---

4. Ex Vivo Tissue Imaging

Ex vivo tissue imaging was conducted on fresh tissues from both mouse models and human HCC patients:

• Mouse Models: Xenograft tumors and liver tissues were excised and incubated with ICG-FAP-TATA or a control probe. Strong fluorescence signals were observed in tumor regions incubated with ICG-FAP-TATA, with significantly higher TBR values compared to the control probe (p < 0.0001).
• Human HCC Samples: Fresh tumor and adjacent liver tissues from seven HCC patients were analyzed. The probe demonstrated enhanced fluorescence signals in tumor margins and blood flow regions, corresponding to areas of high metastatic risk and aligning with known HCC progression mechanisms.

---

Key Findings and Conclusions

1. High Targeting Specificity and Stability
   The ICG-FAP-TATA probe demonstrated exceptionally high specificity toward FAP+ CAFs and consistent imaging stability across in vivo and ex vivo experiments. In the near-infrared II window (NIR-II, 1000–1700 nm), the probe achieved significantly higher TBR compared to the NIR-I window (700–900 nm), offering superior resolution.

2. Functional Imaging Reflects Tumor Biology
   The probe accurately visualized the expression and distribution of FAP within the tumor microenvironment, correlating strongly with regions of higher metastatic and invasive risk, especially at tumor margins and in blood flow directions. This provides a macroscopic perspective on tumor biology and suggests applications in guiding surgical decision-making.

3. Feasibility Across Fibrotic Liver Conditions
   The probe’s imaging performance remained consistent in healthy and fibrotic liver tissues, demonstrating its robustness across different liver disease backgrounds.

---

Research Significance

This study presents a rapid, intuitive, and precise imaging method for HCC, shifting the focus from tumor parenchymal cells to the more stable stromal CAFs as imaging targets. The research achieves not only accurate lesion localization but also provides valuable insights for identifying high-risk areas during surgery. Furthermore, the ex vivo tissue incubation imaging strategy can potentially be extended to other solid tumors.

The design of ICG-FAP-TATA demonstrates significant potential for translational applications, particularly in intraoperative decision-making, metastatic risk assessment, and tumor progression analysis.