Near-Infrared Fluorescent Probes Targeting LAG-3 for Guiding Immunomodulation and Efficacy Monitoring of Stereotactic Body Radiotherapy in Liver Cancer.

PURPOSE: Lymphocyte activation gene 3 (LAG-3) is an attractive biomarker for immunotherapy in hepatocellular carcinoma (HCC) due to its high expression. Based on our previously identified LAG-3 peptide, we developed a LAG-3 targeted peptide probe named Cqy-12-Cy5 for monitoring the dynamic regulation of LAG-3 by stereotactic body radiotherapy (SBRT), thereby providing a strategy to enhance the immunotherapeutic efficacy for HCC.

METHODS

Immunohistochemistry (IHC) was used to detect the expression of LAG-3 in HCC tissues and its correlation with clinicopathological characteristics. An optical fluorescent probe (Cqy-12-Cy5) was constructed, and its in vivo targeting ability and metabolic profile were evaluated via near-infrared fluorescent (NIRF). Flow cytometry (FCM) was employed to assess the apoptosis of H22 cells after radiation, aiming to screen the optimal radiation dose. Furthermore, the regulatory effect of SBRT on LAG-3 expression in HCC was detected, along with changes in the proportion of CD8+ T cells, TNF-α and IFN-γ following SBRT. Dynamic observations of tumor growth and LAG-3-targeted fluorescent expression after SBRT were performed using magnetic resonance imaging (MRI) and NIRF.

RESULTS

LAG-3 is highly expressed in HCC and is closely related to tumor progression. The fluorescent uptake of the Cqy-12-Cy5 in the tumor region reached a peak at 1 hour after tail vein injection. A radiation dose of 6 Gy was identified as the optimal dose. SBRT could reduce the size of the tumor, promote CD8+ T cell infiltration and increase the secretion of TNF-α and IFN-γ.

CONCLUSION

The successfully constructed LAG-3-targeting NIRF probe enables non-invasive, real-time monitoring of tumor growth and LAG-3 expression. SBRT can inhibit tumor growth, modulate LAG-3 expression, promote T cell infiltration and cytokine secretion in HCC. The Cqy-12-Cy5 can dynamically monitor the modulation of LAG-3 by SBRT, providing a strategy for guiding the timing of combination therapy and promoting synergistic immunotherapeutic efficacy.