NIR-II Fluorescence Surgical Navigation for Gastric Cancer: Systematic Preclinical Validation Toward Clinical Application

Abstract

The precise intraoperative delineation of gastric tumors and their micrometastases remains a major challenge in surgical oncology. To address this, we developed a novel NIR-II fluorescent probe named SPN01 targeting integrin αvβ3, a receptor upregulated in gastric cancer and associated tumor vasculature. SPN01 was constructed by conjugating RGD peptide to a high-performance cyanine fluorophore with net neutral charge. In multiple preclinical models, SPN01 enabled high-contrast visualization of sub-centimeter orthotopic tumors, detection of occult peritoneal micrometastases as small as 0.5 mm, and specific identification of metastatic lymph nodes. Concurrently, near-infrared II (NIR-II, 900–1880 nm) fluorescence guidance facilitated the complete resection of tumors with clear margins, owing to the superior brightness of SPN01 in the NIR-II window. Notably, ex vivo validation using human gastric cancer specimens confirmed target-specific binding. Comprehensive pharmacokinetic and safety studies demonstrated rapid dual-pathway clearance and no obvious toxicity. Following these findings, we conducted a first-in-human Phase I clinical trial involving 24 healthy volunteers, which demonstrated a favorable safety profile for SPN01. These findings support SPN01-assisted NIR-II fluorescence-guided surgery as a promising candidate for clinical translation with the potential to enhance the completeness of gastric cancer resection.

Keywords

gastric cancer / integrin / micrometastasis / NIR-II fluorescence / preclinical / surgical navigation

References

[1] Y. Zhou, T. Huang, H. L. Siu, et al., “IGF2BP3 Functions as a Potential Oncogene and Is a Crucial Target of Mir-34a in Gastric Carcinogenesis,” Molecular Cancer 16 (2017): 77.

[2] J. Guo, A. Xu, X. Sun, et al., “Three-Year Outcomes of the Randomized Phase III SEIPLUS Trial of Extensive Intraoperative Peritoneal Lavage for Locally Advanced Gastric Cancer,” Nature Communications 12 (2021): 6598.

[3] E. P. Weledji, “The Principles of The Surgical Management of Gastric Cancer,” International Journal of Surgery Oncology 2 (2017): e11.

[4] F. Wang, L. Qu, F. Ren, et al., “High-Precision Tumor Resection Down to Few-Cell Level Guided by NIR-Iib Molecular Fluorescence Imaging,” Proceedings of the National Academy of Sciences of the United States of America119 (2022): e2123111119.

[5] Y. X. Ye, C. Y. Wu, L. Q. Chen, S. J. Wu, and K. Ye, “Controversies and Consensus Surrounding Laparoscopic Pylorus-Preserving Gastrectomy for Early Gastric Cancer,” World Journal of Gastrointestinal Surgery 17 (2025): 105295.

[6] Y. Tian, Y. Pang, P.-G. Yang, et al., “Clinical Implications of Micro Lymph Node Metastasis for Patients With Gastric Cancer,” BMC Cancer 23 (2023): 536.

[7] R. D. Houvast, M. Van Duijvenvoorde, K. Thijse, et al., “Selecting Targets for Molecular Imaging of Gastric Cancer: An Immunohistochemical Evaluation,” Molecular Diagnosis & Therapy 29 (2025): 213–227.

[8] X. Liu, J. Cheng, S. Jin, et al., “Mapping 20 Years of Fluorescence-Guided Surgery: A Bibliometric Landscape Analysis,” Med-X 3 (2025): 22.

[9] T. Luo, Z. Xia, Y. Guo, X. Lian, and Y. Lin, “Application of Fluorescent Tracers in Ovarian Cancer Surgical Navigation: A Systematic Review and Meta-Analysis,” World Journal of Surgical Oncology 23 (2025): 319.

[10] A. D. Newton, J. D. Predina, S. Nie, P. S. Low, and S. Singhal, “Intraoperative Fluorescence Imaging in Thoracic Surgery,” Journal of Surgical Oncology 118 (2018): 344–355.

[11] J. Zhou, Z. Tan, B. Sun, Y. Leng, and S. Liu, “Application of Indocyanine Green Fluorescence Imaging in Hepatobiliary Surgery,” International Journal of Surgery 110 (2024): 7948–7961.

[12] F. Ding, J. Feng, X. Zhang, J. Sun, C. Fan, and Z. Ge, “Responsive Optical Probes for Deep-Tissue Imaging: Photoacoustics and Second Near-Infrared Fluorescence,” Advanced Drug Delivery Reviews 173 (2021): 141–163.

[13] J. Huang and K. Pu, “Near-Infrared Fluorescent Molecular Probes for Imaging and Diagnosis of Nephro-Urological Diseases,” Chemical Science 12 (2020): 3379–3392.

[14] Z. Feng, T. Tang, T. Wu, et al., “Perfecting and Extending the Near-Infrared Imaging Window,” Light: Science & Applications 10 (2021): 197.

[15] B. Guo, Z. Feng, D. Hu, et al., “Precise Deciphering of Brain Vasculatures and Microscopic Tumors With Dual NIR-II Fluorescence and Photoacoustic Imaging,” Advanced Materials 31 (2019): e1902504.

[16] X. Fan, Y. Li, Z. Feng, et al., “Nanoprobes-Assisted Multichannel NIR-II Fluorescence Imaging-Guided Resection and Photothermal Ablation of Lymph Nodes,” Advanced Science 8 (2021): 2003972.

[17] Z. Feng, Y. Li, S. Chen, et al., “Engineered NIR-II Fluorophores With Ultralong-Distance Molecular Packing for High-Contrast Deep Lesion Identification,” Nature Communications 14 (2023): 5017.

[18] D. Lin, W. Huang, H. Yang, et al., “Ultrabright NIR-II Nanoparticles for High-Resolution In Vivo Imaging: From Systemic Vasculature Visualization to Pathological Microenvironment Monitoring,” Advanced Materials 38 (2026): E10493.

[19] Z. Hu, C. Fang, B. Li, et al., “First-In-Human Liver-Tumour Surgery Guided by Multispectral Fluorescence Imaging in the Visible and Near-Infrared-I/II Windows,” Nature Biomedical Engineering 4 (2020): 259–271.

[20] R. Li, K. Liu, Q. Hu, et al., “NIR-II AIEgens Nanosystem for Fluorescence and Chemiluminescence Synergistic Imaging-Guided Precise Resection in Osteosarcoma Surgery,” Aggregate 6 (2025): e658.

[21] X. He, Y. Luo, Y. Li, et al., “D-Type Neuropeptide Decorated AIEgen/RENP Hybrid Nanoprobes With Light-Driven ROS Generation Ability for NIR-II Fluorescence Imaging-Guided Through-Skull Photodynamic Therapy of Gliomas,” Aggregate 5 (2024): e396.

[22] W. Ge, B. Gao, S. Li, et al., “Self-Calibrated Identification of Metastatic Lymph Nodes Using Full-NIR-II Tunable Ag₂ Se Quantum Dots Engineered by Short-Chain Phosphines,” Small 21, no. 50 (2025): e10056.

[23] P. Minges, M. Eder, and A. C. Eder, “Dual-Labeled Small Peptides in Cancer Imaging and Fluorescence-Guided Surgery: Progress and Future Perspectives,” Pharmaceuticals 18 (2025): 143.

[24] R. Liu, Y. Xu, K. Xu, and Z. Dai, “Current Trends and Key Considerations in the Clinical Translation of Targeted Fluorescent Probes for Intraoperative Navigation,” Aggregate 2 (2021): e23.

[25] T. M. Lwin, M. A. Turner, S. Amirfakhri, et al., “Rapid Tumor-Labeling Kinetics With a Site-Specific Near-Infrared Anti-CEA Nanobody in a Patient-Derived Orthotopic Xenograft Mouse Model of Colon Cancer,” Journal of Surgical Oncology 124 (2021): 1121–1127.

[26] L. Van Manen, L. De Muynck, V. M. Baart, et al., “Near-Infrared Fluorescence Imaging of Pancreatic Cancer Using a Fluorescently Labelled Anti-CEA Nanobody Probe: A Preclinical Study,” Biomolecules 13 (2023): 618.

[27] M. H. Hwang, J. E. Kim, S. Y. Kim, et al., “Biological Production of an Integrin Αvβ3 Targeting Imaging Probe and Functional Verification,” BioMed Research International 2015 (2015): 681012.

[28] N. Li, S. Qiu, Y. Fang, J. Wu, and Q. Li, “Comparison of Linear vs. Cyclic RGD Pentapeptide Interactions With Integrin αvβ3 by Molecular Dynamics Simulations,” Biology 10 (2021): 688.

[29] B. Bogdanović, D. Fagret, C. Ghezzi, and C. Montemagno, “Integrin Targeting and Beyond: Enhancing Cancer Treatment With Dual-Targeting RGD (Arginine–Glycine–Aspartate) Strategies,” Pharmaceuticals 17 (2024): 1556.

[30] V. S. Lebleu, “Imaging the Tumor Microenvironment,” Cancer Journal 21 (2015): 174–178.

[31] X. Li, S. Ai, X. Lu, S. Liu, and W. Guan, “Nanotechnology-Based Strategies for Gastric Cancer Imaging and Treatment,” RSC Advances 11 (2021): 35392–35407.

[32] Z. Feng, S. Bai, J. Qi, et al., “Biologically Excretable Aggregation-Induced Emission Dots for Visualizing Through the Marmosets Intravitally: Horizons in Future Clinical Nanomedicine,” Advanced Materials 33 (2021): e2008123.

[33] H. Tong, K. Lou, and W. Wang, “Near-Infrared Fluorescent Probes for Imaging of Amyloid Plaques in Alzheimer׳S Disease,” Acta Pharmaceutica Sinica B 5 (2015): 25–33.

[34] Z. Dai, R. Liu, and Y. Xu, “Polyethylene Glycol Modified Zwitterionic Fluorescent Probe, Preparation and Application Thereof,” Chinese Patent no. CN114214061B, 2022.

[35] S. Cho, J. Y. K. Lee, and S. Sheikh, “ 3248 Fluorescence-Guided Neurosurgery With 5-Aminolevulinic-Acid and Second-Window-Indocyanine-Green: A Murine Model and Investigation Into Suitable Cell Lines,” Supplement, Journal of Clinical and Translational Science3 (2019): 105.

[36] A. L. Parker, M. Benguigui, J. Fornetti, et al., “Current Challenges in Metastasis Research and Future Innovation for Clinical Translation,” Clinical & Experimental Metastasis 39 (2022): 263–277.

[37] Z. Y. Zhang and H. Y. Ge, “Micrometastasis in Gastric Cancer,” Cancer Letters 336 (2013): 34–45.

[38] L. Liu, H. Hao, L. Zhao, J. Hu, and B. Si, “Analysis of Survival and Prognosis of 298 Gastric Adenocarcinoma Patients With no Distant Metastasis,” Oncology Letters 14 (2017): 7813–7816.

[39] C. E. S. Hoogstins, Q. R. J. G. Tummers, K. N. Gaarenstroom, et al., “A Novel Tumor-Specific Agent for Intraoperative Near-Infrared Fluorescence Imaging: A Translational Study in Healthy Volunteers and Patients With Ovarian Cancer,” Clinical Cancer Research 22 (2016): 2929–2938.

[40] J. A. Carr, D. Franke, J. R. Caram, et al., “Shortwave Infrared Fluorescence Imaging With the Clinically Approved Near-Infrared Dye Indocyanine Green,” Proceedings of the National Academy of Sciences of the United States of America115 (2018): 4465–4470.

[41] Z. Feng, X. Yu, M. Jiang, et al., “Excretable IR-820 for In Vivo NIR-II Fluorescence Cerebrovascular Imaging and Photothermal Therapy of Subcutaneous Tumor,” Theranostics 9 (2019): 5706–5719.

[42] Q. Wen, Y. Zhang, C. Li, et al., “NIR-II Fluorescent Self-Assembled Peptide Nanochain for Ultrasensitive Detection of Peritoneal Metastasis,” Angewandte Chemie International Edition 58 (2019): 11001–11006.

[43] T. M. Lwin, M. A. Turner, H. Nishino, et al., “Fluorescent Anti-CEA Nanobody for Rapid Tumor-Targeting and Imaging in Mouse Models of Pancreatic Cancer,” Biomolecules 12 (2022): 711.

[44] G. Lu, N. S. van den Berg, B. A. Martin, et al., “Tumour-Specific Fluorescence-Guided Surgery for Pancreatic Cancer Using Panitumumab-Irdye800cw: A Phase 1 Single-Centre, Open-Label, Single-Arm, Dose-Escalation Study,” Lancet Gastroenterology & Hepatology 5 (2020): 753–764.

Rights & permissions

2026 The Author(s). Aggregate published by SCUT, AIEI, and John Wiley & Sons Australia, Ltd.