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A new EGFR and c-Met bispecific NIR-II fluorescent probe for visualising colorectal cancer and metastatic lymph nodes
Shangkun Jin1, Changjian Li2, Xiaohua Jia3
1Department of Colorectal Surgery, National Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, 100021, PR China; Department of Colorectal Surgery, Clinical Oncology School of Fujian Medical University, Fujian Cancer Hospital, Fuzhou, 350004, Fujian, PR China; Key Laboratory of Molecular Imaging of Chinese Academy of Sciences, Institute of Automation, Chinese Academy of Sciences, Beijing, 100190, PR China.
Background:
The aim of the study was to increase the specificity and targeting of tumour imaging, targeting molecules that enable the simultaneous recognition and binding of multiple tumour-associated receptors. We constructed a NIR-II fluorescence probe based on a bispecific antibody to epidermal growth factor receptor (EGFR) and cellular mesenchymal-epithelial transition factor (c-Met) for visualising colorectal cancers (CRCs) and metastatic lymph nodes.
Methods:
The expression of EGFR and c-Met in tumour and metastatic lymph node specimens from patients with CRC was examined using immunohistochemistry. The EGFR and c-Met bispecific antibody (Rybrevant) was labelled, and its cell-specific binding ability was assessed using laser confocal microscopy. Subcutaneous CRC and orthotopic tumour models were constructed to evaluate the fluorescence imaging of the probe in vivo. To assess the performance of Rybrevant-IRDye800CW in the differential diagnosis of metastatic lymph nodes, a CRC lymph node metastasis model was constructed using human CRC cells implanted in mouse claw pads. Finally, surgically resected CRC tumours and lymph node specimens were incubated with Rybrevant-IRDye800CW for fluorescence NIR-II imaging to evaluate the efficacy of Rybrevant-IRDye800CW for preclinical visualisation.
Findings:
The combined expression rate of EGFR and c-Met in CRC and metastatic lymph nodes was significantly higher than the single-target expression rate. The bispecific probe Rybrevant-IRDye800CW was successfully synthesised, and its fluorescence signal could be extended up to 1600 nm using NIR-II imaging. Cell incubation experiments showed that the fluorescence intensity of Rybrevant-IRDye800CW was strongly correlated with EGFR and c-Met overexpression of the cells. NIR-II in vivo fluorescence imaging showed that double-positively expressing subcutaneous tumours significantly uptook Rybrevant-IRDye800CW after tail vein injection of the probe, which rapidly accumulated within the tumours in about 6 h. In EGFR and or c-Met blockade assays, subcutaneous tumours showed weaker uptake of Rybrevant-IRDye800CW. Similarly, Rybrevant-IRDye800CW was specifically identified in orthotopic CRC and lymph node metastasis models, with all orthotopic tumours showing high tumour-to-background ratios in NIR-II imaging. In a NIR-II preclinical study, Rybrevant-IRDye800CW could specifically identify fresh human CRC and its metastatic lymph node tissue.
Interpretation:
This study confirmed the complementary EGFR and c-Met expression in CRC and its metastatic lymph nodes. Compared to single-target probes, EGFR and c-Met dual-specific fluorescent probes identified CRC and its metastatic lymph nodes using NIR-II imaging. Thus, NIR-II-guided R0 surgery was performed to resect the CRC and metastatic lymph nodes.
Fundings:
This study was supported by the Beijing Natural Science Foundation (Grant numbers: L222054, 7244517, 4232058, L248026, L232020), National Natural Science Foundation of China (NSFC) (92059207, 92359301, 92259303, 62027901, 81930053, 81227901, U21A20386), CAS Youth Interdisciplinary Team (JCTD-2021-08), and the Fundamental Research Funds for the Central Universities (Grant no. JK2024-2-35-02).
Insights
A novel bispecific fluorescent probe targeting both EGFR and c-Met simultaneously enhances colorectal cancer (CRC) and metastatic lymph node detection using NIR-II imaging for improved surgical outcomes.
Area of Science:
- Oncology
- Medical Imaging
- Molecular Biology
Background:
- Targeting multiple tumor-associated receptors can improve cancer imaging specificity.
- Bispecific antibodies offer simultaneous recognition and binding to multiple targets.
- Epidermal growth factor receptor (EGFR) and cellular mesenchymal-epithelial transition factor (c-Met) are key targets in colorectal cancer (CRC).
Purpose of the Study:
- To develop and evaluate a novel near-infrared II (NIR-II) fluorescence probe for enhanced visualization of colorectal cancers (CRCs) and metastatic lymph nodes.
- To assess the efficacy of a bispecific antibody targeting both EGFR and c-Met for improved tumor imaging specificity and targeting.
- To enable simultaneous recognition and binding of multiple tumor-associated receptors for better diagnostic and surgical guidance.
Main Methods:
- Immunohistochemistry was used to examine EGFR and c-Met expression in CRC tumor and lymph node specimens.
- A bispecific antibody (Rybrevant) targeting EGFR and c-Met was labeled and its cell-specific binding was assessed via laser confocal microscopy.
- In vivo fluorescence imaging was performed using subcutaneous and orthotopic CRC models, and a CRC lymph node metastasis model in mice, followed by NIR-II imaging of resected human tissues.
Main Results:
- Combined EGFR and c-Met expression was significantly higher in CRC and metastatic lymph nodes compared to single-target expression.
- The synthesized bispecific probe Rybrevant-IRDye800CW demonstrated effective fluorescence signal extension up to 1600 nm in NIR-II imaging.
- In vivo and ex vivo imaging showed specific uptake and accumulation of Rybrevant-IRDye800CW in tumors and metastatic lymph nodes, achieving high tumor-to-background ratios.
Conclusions:
- EGFR and c-Met exhibit complementary expression patterns in CRC and its metastatic lymph nodes.
- Dual-specific fluorescent probes targeting both EGFR and c-Met offer superior identification of CRC and metastatic lymph nodes via NIR-II imaging compared to single-target probes.
- NIR-II-guided surgery enabled precise resection of CRC and metastatic lymph nodes, indicating the clinical utility of this dual-targeting approach.
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