Additional file 1 of High-precision detection and navigation surgery of colorectal cancer micrometastases
Creators: name: Ma, Shengjie
name: Sun, Bin
name: Li, Mengfei
name: Han, Tianyang
name: Yu, Chenlong
name: Wang, Xin
name: Zheng, Xue
name: Li, Shuang
name: Zhu, Shoujun
name: Wang, Quan
Publisher: name: figshare
Year: 2024
Subjects: subject: Biophysics
subject: Physical Sciences not elsewhere classified
subject: Cell Biology
subject: Molecular Biology
subject: Physiology
subject: FOS: Biological sciences
subject: Pharmacology
subject: Biotechnology
subject: Chemical Sciences not elsewhere classified
subject: Biological Sciences not elsewhere classified
subject: Mathematical Sciences not elsewhere classified
subject: Cancer

description: Additional file 1: Scheme S1. Synthesis of IR-FE and IR-FE-N3. Figure S1. Characterization of ICG and BSA@IR-780. a. Schematic diagram of molecular structure of ICG. b. Schematic diagram of the molecular structure for BSA@IR-780. c. Absorption and emission spectra of ICG. d. Absorption and emission spectra of BSA@IR780. Figure S2. The scheme of the NIR-II imaging and operating platform. Created with BioRender.com. Figure S3. Photostability of FE-2PEG and ICG in popliteal and sacral lymph nodes. a. Continuously irradiation of popliteal and sacral lymph nodes under 808 nm laser. The left hind limb was injected with FE-2PEG (600 μM, 25 μL), and the right hind limb was injected with ICG (100 μM, 25 μL) in the footpad. &gt; 1100 nm. 65 mW/cm2. Scale bar: 1 cm. b. The normalized fluorescence intensity curve of (a) (n = 3, data were shown as means ± SD). Figure S4. Photostability of BSA@IR-780 in popliteal and sacral lymph nodes in vivo. a. Continuously irradiation of popliteal and sacral lymph nodes under 808 nm laser. The bilateral hind limbs were injected with BSA@IR-780 (300 μM, 25 μL) in the footpad. &gt; 1100 nm. 65 mW/cm2. Scale bar: 1 cm. b. The normalized fluorescence intensity curve of (a) (n = 3, data were shown as means ± SD). Figure S5. Evaluation of blood circulation time of BSA@IR-780. a. NIR-II imaging of blood ex vivo post-injected of BSA@IR-780 (150 μM, 200 μL) via tail vein at different time points. b. The normalized quantitative curve of fluorescence intensity of blood at different time points (n = 3, data were shown as means ± SD). Figure S6. Representative NIR-II images of mesenteric LNs at different time points. The FE-2PEG was injected into the subserous layer of the cecum (300 μM, 20 μL). The injection site was massaged for 3 min to facilitate the flow of the probe in the lymphatic fluid. Over 1100 nm collection; 65 mW/cm2; Scale bar: 1 mm. Figure S7. The NIR-II imaging of mesenteric LNs at different time points. a. Representative NIR-II images of mesenteric LNs. The probe FE-2PEG was injected into the subserous layer of the cecum (300 μM, 20 μL). The injection site was massaged for 3 min at the start, and then intermittently massaged for 30 seconds before each capture. b. The normalized fluorescent intensity curve of mesenteric LNs (n=5, data were shown as mean ± SD). Over 1100 nm collection; 65 mW/cm2; Scale bar: 1 mm. Figure S8. NIR-II imaging of FE-2PEG and ICG in blood vessels of the hindlimb. a. Representative NIR-II imaging of FE-2PEG and ICG in blood vessels of the hindlimb at different time points. Scale bar: 5 mm. b. Normalized FL intensity in vessels of FE-2PEG and ICG. c. Plot profiles of lines on blood vessels of hindlimb at 2 hours (n = 3, data were shown as means ± SD). Figure S9. The wavelength-dependence NIR-II imaging of FE-2PEG in cerebral vessels. a. Representative images of the cerebral vessels in NIR-II (900 nm, 1000 nm, 1100 nm, 1200 nm, and 1300 nm) windows. Scale bar: 5 mm. b. Fluorescence cross-sectional intensity distribution of cerebral vessels in different NIR-II windows (red line in (a)) (n = 3). The full-width half maxima (FWHM) are 0.81124, 0.80007, 0.7731, 0.71646, and 0.69999 for 900 nm, 1000 nm, 1100 nm, 1200 nm, and 1300 nm, respectively. Figure S10. The wavelength-dependence NIR-II imaging of FE-2PEG in blood vessels of the hindlimb. a. Representative images of blood vessels in the hindlimb in NIR-II (900 nm, 1000 nm, 1100 nm, 1200 nm, and 1300 nm) windows. Scale bar: 5 mm. b. Fluorescence cross-sectional intensity distribution of blood vessels in hindlimb in different NIR-II windows (red line in (a)) (n = 3). The full-width half maxima (FWHM) are 0.86926, 0.77146, 0.74858, 0.65666, and 0.55798 for 900 nm, 1000 nm, 1100 nm, 1200 nm, and 1300 nm, respectively. Figure S11. NIR-II in vivo imaging of BSA@IR-780 in the subcutaneous tumor. a. Representative NIR-II in vivo images of BSA@IR-780 (150 μM, 200 μL) in subcutaneous tumor at different time points. Lateral position. Tail vein injection. Scale bar: 1 cm. b. The T/NT ratio of subcutaneous tumors at different time points in vivo. Figure S12. Evaluation of the biodistribution of FE-2PEG. a. White light and NIR-II imaging of the tumor, organs (heart, liver, spleen, lung, kidney, intestine), and tissues (skin, muscle) at 12h. Scale bar: 1 cm. b. White light and NIR-II imaging of the tumor, organs, and tissues at 24h. Scale bar: 1 cm. c. White light and NIR-II imaging of the tumor, organs, and tissues at 48h. Scale bar: 1 cm. d. Quantitative analysis of fluorescence intensity signals of tumors, organs, and tissues at different time points (n = 3, data were shown as means ± SD). Figure S13. The establishment of the orthotopic mouse model of CRC. a. The preparation of surgical instruments and materials. Materials for surgery include cotton (A), cotton swabs (B), gauze (C), and absorbable sutures (D). Instruments for surgery include bent scissor (E), needle holder (F), straight tweezer (G), bent tweezer (H), and micro-syringe (I). b. The surgical operating platform includes the stereomicroscope (A), operating table (B), monitor (C), and LED lamp (D). c. The cecum was exposed through an abdominal incision. The dotted line shows the cecum. The gauze was used to protect the cecum. d. The CT-26-Luc cells were injected into the subserous membrane of the cecum with the micro-syringe. The red arrow shows tumor cells. e. The incisions in the abdomen were sutured layer by layer with absorbable sutures. The dotted line shows the abdominal incision after the suture. Figure S14. BLI monitoring of the orthotopic CRC model formation. Figure S15. The NIR-II imaging of post-operative orthotopic CRC mice. a. The photograph of post-operative orthotopic CRC mice. b. Representative NIR-II imaging of post-operative orthotopic CRC mice post-injected with FE-2PEG via the tail vein at 24 hours. Scale bar: 1 cm. Figure S16. Dual-NIR-II color testing of FE-2PEG, QDs, and mixture in vitro. a. The photograph of the FE-2PEG, QDs, and mixture solutions. b. The NIR-II fluorescent signals were collected under &gt; 1100, 1100 ~ 1300, and &gt; 1500 nm windows, respectively. Figure S17. NIR-II imaging of ICG in orthotopic CRC in vivo. a. The schematic diagram showed that the orthotopic CRC mouse was injected with ICG (150 μM, 200 μL) via the tail vein. b. BLI showed the successful establishment of the orthotopic CRC model. c. Representative images of orthotopic CRC post-injected ICG at different time points in vivo. Scale bar: 1 cm. d. White light and NIR-II imaging of exposed orthotopic CRC in vivo. &gt; 1100 nm. Exposure time = 100 ms. Scale bar: 5 mm. e. White light and NIR-II imaging of the organs and tumor ex vivo &gt; 1100 nm, exposure time = 100 ms. Scale bar: 1 cm. f. The fluorescence intensity ratio of tumor to feces ex vivo. (n = 3, data were shown as means ± SD). g. H&amp;E staining of the orthotopic tumor. Scale bar: 100 μm (10x); Scale bar: 50 μm (20x). Figure S18. NIR-II imaging of BSA@IR-780 in orthotopic CRC. a. The schematic diagram showed that the orthotopic CRC mouse was injected with BSA@IR-780 (150 μM, 200 μL) via the tail vein. b. BLI showed the successful establishment of the orthotopic CRC model. c. Representative NIR-II images of BSA@IR-780 in orthotopic CRC at different time points with a supine position. Scale bar: 1 cm. d. Quantitative intensity signal analysis of abdominal ROI. e. White light and NIR-II imaging of the exposed orthotopic tumor in vivo. The tumor size is 4.2 mm in length and 4.1 mm in width. Scale bar: 5 mm. f. White light and NIR-II imaging of the isolated tumor and feces ex vivo. Scale bar: 5 mm. g. Plot profiles quantitative analysis of the tumor in vivo (line 1), the isolated feces (line 2), and the tumor ex vivo (line 3). h. White light and NIR-II imaging of isolated organs, feces, and the tumor ex vivo. Scale bar: 1 cm. i. H&amp;E staining of the orthotopic tumor. Scale bar: 100 μm (10x); Scale bar: 50 μm (20x). Figure S19. NIR-II imaging of ICG in orthotopic CRC with lymphatic metastases. a. White light and NIR-II imaging of orthotopic tumor and metastatic LNs before and after laparotomy in vivo. Scale bar: 1 cm. b. White light and NIR-II imaging of intestinal canal, orthotopic tumor, and metastatic LNs ex vivo. Scale bar: 1 cm. c. T/NT ratio of the orthotopic tumor and metastatic LNs in vivo and ex vivo (n = 3, data were shown as means ± SD). Figure S20. NIR-II imaging of BSA@IR-780 of orthotopic CRC with lymphatic metastases. a. White light and NIR-II imaging of primary tumor and metastatic lymph nodes displayed before and after laparotomy in vivo. Scale bar: 1 cm. b. White light and NIR-II imaging of intestinal canal, orthotopic tumor, metastatic LNs, and feces ex vivo. Scale bar: 1 cm. c. T/NT ratio of the orthotopic tumor and metastatic LNs in vivo and ex vivo. Figure S21. BLI of the establishment of CRC peritoneal metastasis model. Figure S22. NIR-II in vivo imaging of FE-2PEG in CRC peritoneal metastasis model. a. Representative NIR-II images of FE-2PEG in peritoneal metastasis mice at different time points. Supine position,tail vein injection, 300 μM, 300 μL. Scale bar: 1 cm. b. Quantitative signal intensity analysis of abdominal ROI. Figure S23. Biological safety of FE-2PEG. a. CCK-8 viabilities of CT-26-Luc cells after incubated with various concentrations of FE-2PEG for 12 h and 24 h; b. Viabilities of L-929 cells after incubation with various concentrations of FE-2PEG for 12 h and 24 h. (n = 6, data were shown as means ± SD). Figure S24. Biological safety of FE-2PEG, ICG, and BSA@IR-780. a. H&amp;E staining of organs (Heart, liver, spleen, lung, and kidney) from mice after intravenous injection of FE-2PEG (300 μM, 300 μL), ICG (150 μM, 200 μL), and BSA@IR-780 (150 μM, 200 μL) in 30 days (n = 3). Scale bar: 100 μm. Figure S25. Biological safety of FE-2PEG, ICG, and BSA@IR780. a. Blood routine examination in 30 days post-injected of FE-2PEG (300 μM, 300 μL), ICG (200 μM, 150 μL), and BSA@IR-780 (200 μM, 150 μL) via tail vein. (n = 3, data were shown as means ± SD). b. Liver and renal function examination in 30 days post injection of FE-2PEG (300 μM, 300 μL), ICG (200 μM, 150 μL), and BSA@IR-780 (200 μM, 150 μL) via tail vein. (n = 3, data were shown as means ± SD). c. Body weight change in two weeks post-injected of FE-2PEG (300 μM, 300 μL), ICG (200 μM, 150 μL), and BSA@IR-780 (200 μM, 150 μL) via tail vein. (n = 3, data were shown as means ± SD).