Identification of a Novel TIM-3 Targeting Peptides Probe to Indicate the Immunomodulation of SBRT Combined with Anti-TIM-3 Therapy in HCC.

purposeGiven the escalating global burden and high mortality associated with hepatocellular carcinoma (HCC), immune checkpoint inhibitors (ICIs) have emerged as a critical therapeutic approach. T cell immunoglobulin and mucin-domain containing-3 (TIM-3), an emerging immune checkpoint that is highly expressed in HCC, has been linked to poor prognosis due to its association with exhausted T cells and suppressed immune responses. Anti-TIM-3 therapy may hold potential for activating immunity in HCC patients. Stereotactic body radiotherapy (SBRT), a precise radiation technique, can activate the tumor immunity and modulate IC expression. Therefore, the combination of anti-TIM-3 therapy with SBRT is anticipated to enhance the immune response in HCC. An effective measure to evaluate TIM-3 expression after SBRT for improving the synergistic efficacy is needed. This study aimed to develop a non-invasive tool to monitor TIM-3 expression in HCC and optimize the combination of anti-TIM-3 therapy with SBRT to enhance antitumor efficacy. PROCEDURES: Clinical data and pathological specimens from HCC patients were collected to evaluate TIM-3 expression in tumor tissues via immunohistochemistry (IHC). A 12-amino-acid peptide targeting TIM-3 was screened via phage display technology and subsequently conjugated with a fluorescent moiety to construct a near-infrared fluorescence (NIRF) probe. The probe's targeting capability for TIM-3 imaging and its in vivo biodistribution were evaluated using NIRF imaging. After intravenous administration of the TIM-3-targeted probe to mice, dynamic changes in intratumoral TIM-3 expression under varying radiation doses (0/4/6/8 Gy × 3F) were visualized via longitudinal optical imaging, identifying the optimal radiation regimen for TIM-3 modulation. Splenic cells were isolated for FCM analysis of TIM-3

resultsTIM-3 was highly expressed in human HCC tissues, with its expression level significantly correlated with tumor stage, vascular invasion status, and patient performance status (PS) score (p < 0.0001). TIM-3 targeting peptides probe was constructed successfully. In vivo fluorescence imaging showed significantly higher tumor-specific fluorescence intensity in mice injected with TIM-3-targeted peptide probe compared to those receiving non-specific probe (p < 0.0001), with peak intratumoral probe accumulation observed at 1 h post-injection. Ex vivo organ imaging confirmed predominant probe biodistribution in the liver and kidneys. Radiation dose-response analysis revealed minimal TIM-3 expression in tumors treated with 6 Gy × 3F SBRT (p < 0.0001), accompanied by significant downregulation of splenic TIM-3

conclusionsThis study provides a novel, real-time method to monitor TIM-3 expression in HCC, offering critical insights into SBRT-induced immunomodulation. The combination of SBRT (at the identified optimal dose) with anti-TIM-3 therapy demonstrates enhanced antitumor efficacy, presenting a translational strategy for HCC immunotherapy.