Additional file 1 of Fibronectin-targeting and metalloproteinase-activatable smart imaging probe for fluorescence imaging and image-guided surgery of breast cancer
Creators: name: Cheng, Zhongquan
name: Jin, Yushen
name: Li, Jiaqian
name: Shi, Guangyuan
name: Yu, Leyi
name: Shao, Bing
name: Tian, Jie
name: Du, Yang
name: Yuan, Zhu
Publisher: name: figshare
Year: 2023
Subjects: Space Science
Medicine
Cell Biology
Biotechnology
Biological Sciences not elsewhere classified
Cancer

description: Additional file 1: Fig. S1 IHC staining of MMP-9 in orthotopic breast cancer and normal breast tissues. High levels of MMP-9 expression were detected by IHC staining in orthotopic breast lesions from 4T1 tumor-bearing mice (scale bar: 50 μm). Relative lower expression levels of MMP-9 were detected in normal breast tissues (scale bar: 50 μm). Fig. S2 IHC staining of fibronectin in orthotopic breast cancer and normal breast tissues. High levels of fibronectin expression were detected by IHC staining in orthotopic lesions from 4T1 tumor-bearing mice (scale bar: 50 μm). Relative lower expression levels of fibronectin were detected in normal breast tissues (scale bar: 50 μm). Fig. S3 IHC staining of MMP-9 in lung metastasis and normal lung tissues. High levels of MMP-9 expression were detected by IHC staining in metastatic lung lesions from 4T1 tumor-bearing mice (scale bar: 50 μm). Relative lower expression levels of MMP-9 were detected in normal lung tissues (scale bar: 50 μm). Fig. S4 IHC staining of fibronectin in lung metastasis and normal lung tissues. High levels of fibronectin expression were detected by IHC staining in metastatic lung lesions from 4T1 tumor-bearing mice (scale bar: 50 μm). Relative lower expression levels of fibronectin were detected in normal lung tissues (scale bar: 50 μm). Fig. S5 Examination of MMP-9 expression on murine 4T1 breast cancer. The expression level of MMP-9 in 4T1 breast tumor and normal breast tissues examined by Western Blot analysis. Densitometric analysis of expression level of MMP-9 normalized to that of GAPDH. Data represented as the mean ± SD, n=3, **p&lt;0.01. Fig. S6 Examination of fibronectin expression on murine 4T1 breast cancer. The expression level of fibronectin in 4T1 breast tumor and normal murine breast tissues examined by Western Blot analysis. Densitometric analysis of expression level fibronectin normalized to that of GAPDH. Data represented as the mean ± SD, n=3, **p&lt;0.01. Fig. S7 HR-MS spectra of Sulfo Cy5.5-GK8-QSY21. Fig. S8 HR-MS spectra of CREKA-GK8-QC. Fig. S9 HR-MS spectra of CERAK-GK8-QC. Fig. S10 IF staining of subcutaneous breast tumors. Analysis of MMP-9 or fibronectin expression and distribution of targeted CREKA-GK8-Cy5.5 and control CERAK-GK8-QC in 4T1 subcutaneous breast tumor sections, respectively. DAPI was used for staining nuclei. Pseudo-colors in the confocal images are assigned as follows: red, Cy5.5; green, MMP-9 or fibronectin; and blue, nucleus. Scale bar 50 µm. Fig. S11 IF staining of lung metastasis. Analysis of MMP-9 or fibronectin expression and distribution of targeted CREKA-GK8-Cy5.5 and control CERAK-GK8-QC in metastatic lung sections, respectively. DAPI was used for staining nuclei. Pseudo-colors in the confocal images are assigned as follows: red, Cy5.5; green, MMP-9 or fibronectin; and blue, nucleus. Scale bar 50 µm. Fig. S12 The cytotoxicity assays of different concentrations of fluorescence imaging probe CREKA-GK8-QC (0, 20, 40, 80, 100, and 200 μg/ml) were measured using CCK-8 with murine 4T1 cells. Fig. S13 Ex vivo fluorescence intensities of tumors and major organs at 48 hours post-injection of CREKA-GK8-QC or CERAK-GK8-QC. Data represented as the mean ± SD, n=3, *p&lt;0.05. Fig. S14 BLI imaging of subcutaneous 4T1 breast tumors. BLI of subcutaneous 4T1 tumor bearing mice which was applied to detect the location of tumors. Fig. S15 In vivo fluorescence imaging of subcutaneous 4T1 breast tumors. In vivo fluorescence images of subcutaneous breast cancer bearing mice injected with CREKA-GK8-QC or CERAK-GK8-QC (n=3) at different time points. The dotted black circles indicated the locations of orthotopic breast tumors. Fig. S16 In vivo TBR at different time points. Quantification of in vivo fluorescence imaging TBR. Data represented as the mean ± SD, n=3, **p&lt;0.01. Fig. S17 Ex vivo fluorescence imaging of subcutaneous 4T1 breast tumors and major organs. Ex vivo fluorescence imaging of tumors and major organs, including liver, kidney, heart, spleen, lung, and intestine, at 48 hours post-injection of CREKA-GK8-QC or CERAK-GK8-QC. Fig. S18 Fluorescence intensities of subcutaneous 4T1 breast tumors and major organs. Ex vivo fluorescence intensities of tumors and major organs at 48 hours post-injection of CREKA-GK8-QC or CERAK-GK8-QC. Data represented as the mean ± SD, n=3, *p&lt;0.05. Fig. S19 BLI imaging of metastatic lung lesions. BLI of lung metastasis in 4T1 tumor bearing mice which was applied to detect the location of metastatic lesions. Fig. S20 In vivo fluorescence imaging of metastatic lung lesions. In vivo fluorescence images of metastatic breast cancer bearing mice injected with CREKA-GK8-QC or CERAK-GK8-QC (n=3) at different time points. The dotted black circles indicated the locations of metastatic lesions. Fig. S21 Ex vivo BLI and fluorescence images of metastatic lung lesions 4T1 tumor bearing mice at 24 h post injection of CREKA-GK8-QC or CERAK-GK8-QC. Fig. S22 Ex vivo fluorescence intensities of metastatic lungs and major organs at 24 hours post-injection of CREKA-GK8-QC or CERAK-GK8-QC. Data represented as the mean ± SD, n=3, ***p&lt;0.001. Fig. S23 Histologic analysis of major organs. HE staining images of major organs (heart, liver, spleen, lung, kidney, and spleen) from different groups. Scale bars: 50 μm. Fig. S24 Liver and kidney function analysis. Liver function, including a. ALT, b. AST, and c. ALP and kidney function, including d. Scr and e. BUN, were all analyzed from different groups. Fig. S25 H&amp;E staining was utilized to confirm the tumor residuals. Scale bar 50 µm. Inset: enlarged images of the metastasis sections (scale bar 10 µm). Table. S1 Local tumor recurrence after surgery.