Near-infrared fluorescence-labeled anti-PD-L1-mAb for tumor imaging in human colorectal cancer xenografted mice

Abstract

The expression of programmed death ligand-1 (PD-L1) in tumor has been used as a biomarker to predict the anti-PD-L1 immunotherapy response. To develop a noninvasive imaging technique to monitor the dynamic changes in PD-L1 expression in colorectal cancer (CRC), we labeled an anti-PD-L1 monoclonal antibody with near-infrared (NIR) dye and tested the ability of the NIR-PD-L1-mAb probe to monitor the PD-L1 expression in CRC-xenografted mice by performing optical imaging. Consistent with the expression levels of PD-L1 protein in three CRC cell lines in vitro by flow cytometry and Western blot analyses, our in vivo imaging showed the highest fluorescence signal of the xenografted tumors in mice bearing SW620 CRC cells, followed by tumors derived from SW480 and HCT8 cell lines. We detected the highest fluorescent intensity of the tumor at 120 hours after injection of NIR-PD-L1-mAb. The highest fluorescence intensity was seen in the tumor, followed by the spleen and the liver in SW620 xenografted mice. In SW480 and HCT8 xenografted mice, however, the highest fluorescent signals were detected in the spleen, followed by the liver and the tumor. Our findings indicate that SW620 cells express a higher level of PD-L1, and the NIR-PD-L1-mAb binding to PD-L1 on the surface of CRC cells was specific. The technique was safe and could provide valuable information on PD-L1 expression of the tumor for development of a therapeutic strategy of personized targeted immunotherapies as well as treatment response of patients with CRC.

Keywords: colorectal cancer; immunotherapy; noninvasive imaging; optical imaging; programmed death ligand-1/programmed death ligand-1 checkpoint.

Figure 1

NIR‐PD‐L1‐mAb has higher affinity binding to PD‐L1 in SW620 cells than SW480 and HCT8 cell lines. Binding affinity of anti‐PD‐L1 monoclonal antibody to the surface PD‐L1 was analyzed by FACS. A, Colorized histograms represent anti‐human PD‐L1 antibody binding to SW620, SW480 and HCT8 cell lines as indicated. B, Quantification of fluorescent intensity of SW620, SW480, and HCT8 cell lines detected by FACS. Data are expressed as the mean ± standard deviation and the significance of the value is indicated by asterisk, ***P   0.001 (n = 3). NIR, near‐infrared; PD‐L1, programmed death ligand‐1

Figure 2

Comparison of PD‐L1 protein expression levels between SW620, SW480, and HCT8 cells in vitro. A, PD‐L1 expression in SW620, SW480, and HCT8 cells measured by the Western blot. B, Quantification of Western blot analyses. Data are expressed as the mean ± standard deviation and the significance of the value is indicated by an asterisk, ***P   0.001, **P   0.01 (n = 3). PD‐L1, programmed death ligand‐1

Figure 3

NIR‐PD‐L1‐mAb specifically binds to PD‐L1 in human colorectal cancer xenografted mice. A, Optical images in SW620, SW480, and HCT8 grafted mice at different time‐points. B, Quantitative fluorescent intensity of optical imaging regions of interest (ROIs) of tumor to background at 24, 48, 72, and 120 hours, respectively after injection of NIR‐PD‐L1‐mAb (n = 3). Data are expressed as the mean ± standard deviation of the fluorescence intensity ratio of tumor to background. The significance of the value is indicated by an asterisk, ***P   0.001. NIR, near‐infrared; PD‐L1, programmed death ligand‐1

Figure 4

Ex vivo optical biodistribution at 120 hours after injection of NIR‐PD‐L1‐mAb in SW620, SW480, and HCT8 xenograft models. A, B, Optical biodistribution image and corresponding quantitative data of SW620 grafted mice. C, D, Optical biodistribution image and corresponding quantitative data of SW480 grafted mice. E, F, Optical biodistribution image and corresponding quantitative data of HCT8 grafted mice. G, Histograms of biodistribution fluorescent accumulation in SW620, SW480, and HCT8 grafted mice, respectively. Data are expressed as the mean ± standard deviation of the fluorescent intensity ratio of tumor to background. The significance of the value is indicated by an asterisk, ***P   0.001 (n = 3) by one‐way ANOVA. ANOVA, analysis of variance; NIR, near‐infrared; PD‐L1, programmed death ligand‐1