Real-Time Monitoring of TIGIT Expression in Hepatocellular Carcinoma for Immunotherapy Guidance

Hepatocellular carcinoma (HCC) is a significant global health concern, ranking as the fifth most common cancer and the second leading cause of cancer-related deaths worldwide. The treatment of advanced HCC has posed challenges due to its inherent immune tolerance mechanisms. Local radiotherapy (RT) has emerged as a promising approach for modulating the immune response against tumors, and one such technique gaining attention is ¹²⁵I seed implantation brachytherapy. This novel form of radiotherapy utilizes ¹²⁵I radioactive seeds, which have demonstrated effectiveness in treating various tumors, including liver cancer. The ¹²⁵I seed brachytherapy approach, characterized by its low-dose-rate radiation, has exhibited a favorable safety profile in HCC treatment. Moreover, it has been increasingly recognized for its ability to inhibit tumor growth and activate antitumor immunity. However, while RT alone can contribute to these immunomodulatory effects, it may also lead to immunosuppression as radiation doses accumulate. Thus, combining RT with other therapeutic strategies, such as immune checkpoint inhibitors (ICIs), offers a promising avenue for enhancing the immunomodulatory potential of these treatments.

T cell immune receptor with immunoglobulin and ITIM domains (TIGIT) emerges as a key player in regulating adaptive and innate immunity, particularly in the context of tumor immune surveillance. TIGIT is primarily expressed on T cells, natural killer cells (NK), and other antigen-presenting cells (APCs), exerting potent immunosuppressive effects by reducing immunostimulatory cytokines production. Notably, in HCC, TIGIT has been identified as a crucial inhibitory immune checkpoint (ICP). RT has been shown to influence the expression of TIGIT in tumors, and the combination of RT with anti-TIGIT therapy has demonstrated promising antitumor effects. Thus, TIGIT represents a valuable marker for assessing the immunoregulation induced by ¹²⁵I seed brachytherapy in HCC.

Advancements in molecular imaging technology have opened new possibilities for dynamically assessing changes in the tumor immune microenvironment in real-time. Near-infrared fluorescence (NIRF) emitters have emerged as powerful tools for real-time imaging due to their excellent tissue penetration and high target-background contrast. NIRF-labeled therapeutic molecules and ICPs have shown great promise in evaluating cancer therapy, offering a safe and accurate means of characterizing the tumor microenvironment. Among the various targeting groups used in NIRF imaging probes, peptides stand out for their low immunogenicity, strong tissue penetration, rapid blood clearance, and ease of production. Phage display technology, which combines the antigen recognition capabilities of recombinant proteins, holds immense potential in developing tumor-specific peptides.

In a new study published in the Frontiers in Oncology Journal led by Dr. Peng Zeng, Dr. Duo Shen, Dr. Wenbin Shu, Dr. Shudan Min, Dr. Min Shu, Dr. Xijuan Yao, Dr. Yong Wang and Dr. Rong Chen from the Medical School at Southeast University developed a novel approach that combines ¹²⁵I seed implantation brachytherapy to regulate TIGIT expression and a 12-amino acid peptide targeting TIGIT for imaging purposes. The peptide is labeled with Cy5 for NIRF imaging, enabling the visualization of immune regulation induced by ¹²⁵I seed brachytherapy. This approach holds significant promise for monitoring the immunoregulation of HCC in real-time and guiding immunotherapeutic strategies.

The authors began by examining the expression of TIGIT protein in human HCC samples collected from different tissue regions and various tumor differentiations. Immunofluorescence staining reveals a marked increase in TIGIT expression in tumor tissue compared to adjacent and normal tissues. Furthermore, TIGIT expression correlates with the degree of malignancy, with poorly differentiated tumors exhibiting the highest expression. Flow cytometry (FCM) analysis corroborates these findings, confirming the significant upregulation of TIGIT on lymphocytes in tumor tissue. The study then proceeds to identify and synthesize a TIGIT-targeted peptide using phage display technology. This process involves multiple rounds of screening, leading to the selection of a 12-amino acid peptide (Po-12) with a high binding affinity for TIGIT. Cy5 fluorophore is added to the peptide to enable NIRF imaging. The synthesized peptide, Po-12-Cy5, demonstrates strong binding affinity to TIGIT-expressing lymphocytes in vitro, as evidenced by FCM and confocal microscopy.

The research team validated the targeting capabilities of Po-12-Cy5 using In vivo experiments involving tumor-bearing mice. The probe showed rapid accumulation in subcutaneous H22 tumors, peaking at 1-hour post-injection, and gradually declining over 7 hours. Ex vivo optical imaging of tumors and organs confirms the specific accumulation of Po-12-Cy5 in H22 tumors, with renal clearance being the primary excretion route. To assess the impact of brachytherapy on TIGIT expression in HCC, the study establishes mouse subcutaneous and orthotopic tumor models and isolates tumor-infiltrating lymphocytes. Confocal microscopy imaging and western blotting (WB) reveal that TIGIT expression is significantly upregulated in the tumor control group but downregulated in the ¹²⁵I seed radiation group, highlighting the potential of brachytherapy to modulate TIGIT expression in HCC.

In conclusion, Dr. Peng Zeng and colleagues addressed a critical challenge in cancer therapy. Traditional assessment methods often fall short in capturing real-time changes in immune molecules, making it difficult to tailor treatments effectively. Molecular imaging technology, particularly NIRF imaging, offers a promising solution by allowing for non-invasive, real-time monitoring of immune events in vivo. The development of the Po-12-Cy5 probe, which specifically targets TIGIT in HCC, represents a significant advancement in this field. Phage display technology facilitated the identification of a TIGIT-targeted peptide with high binding affinity, offering a valuable tool for imaging TIGIT expression in tumors. The probe’s ability to accumulate in TIGIT-expressing lymphocytes, both in vitro and in vivo, underscores its specificity and potential for clinical translation. The study’s findings regarding TIGIT expression patterns in human HCC tissues, where TIGIT levels correlate with tumor differentiation, shed light on the potential clinical relevance of this biomarker. Furthermore, the ability of ¹²⁵I seed brachytherapy to modulate TIGIT expression in HCC presents an intriguing avenue for immunomodulation, supporting the notion that combining radiotherapy with immunotherapies targeting TIGIT could yield promising results. This innovative tool holds promise for enhancing our understanding of the dynamic immune microenvironment in HCC and paves the way for tailored immunotherapeutic strategies in the future.