Ex Vivo Near-Infrared Molecular Imaging of Human Upper Urinary Tract Urothelial Carcinoma With a CD47-Based Targeted Tracer

Abstract

Objective: The low detection rate of early and small tumors remains a clinical problem that urgently needs to be solved in the accurate diagnosis and treatment of upper urinary tract urothelial carcinoma (UTUC). The objective of this study is to evaluate the feasibility of CD47 as a target for optical molecular imaging of human UTUC and conduct preliminary ex vivo imaging experiments.

Methods: We firstly analyzed the genome-wide mRNA expression data from Gene Expression Omnibus (GEO). Paraffin-embedded tissue specimens comprising UTUC and normal urothelium were collected. All tissue specimens were used for immunohistochemistry to compare CD47 protein expression in normal and cancer tissue. Meanwhile, 12 patients undergoing radical nephroureterectomy were prospectively included in ex vivo imaging experiments. Freshly isolated upper urinary tract specimens were incubated with anti-CD47-Alexa Fluor 790 and then imaged under white light and near-infrared (NIR) light. Standard histopathologic evaluation was performed, and findings were correlated with CD47-targeted NIR molecular imaging.

Results: The GEO data revealed that CD47 mRNA expression was higher in UTUC specimens than that in paracancer normal tissue. In immunohistochemical analysis, the CD47 protein expression level was higher in both non-muscle-invasive and muscle-invasive (stage ≥T2) UTUCs than that in normal uroepithelium, and the localization of CD47 protein was the tumor cell membrane. In the ex vivo imaging experiments, all patients were pathologically diagnosed with UTUC, and no adverse effects of anti-CD47-Alexa Fluor 790 on the histological structure of the tumor and normal uroepithelium were observed. In the NIR grayscale images, the mean fluorescence intensity of the tumor tissue was significantly higher than that of the adjacent normal background tissue, which greatly improved the visualization of the tumor.

Conclusions: CD47-targeted NIR molecular imaging could be a feasible and powerful strategy for the accurate diagnosis of UTUC. Larger-scale randomized trials are needed.

Keywords: CD47; molecular imaging; near-infrared fluorescence; targeted imaging; upper urinary tract urothelial carcinoma.

Figure 1

Identification and analysis of differentially expressed genes (DEGs) and CD47 mRNA expression. (A) Hierarchical clustering of DEGs, with rows representing genes and columns representing samples. For a gene, red represents a higher expression level, blue represents a lower expression level, and yellow represents the median expression level for all samples. (B) CD47 mRNA expression levels in the upper urinary tract urothelial carcinoma (UTUC) (n = 90) group and normal group (n = 10) (|log2FC| = 1.43, P < 0.001), and P-values were determined using Wilcoxon rank-sum test.

Figure 2

Immunohistochemistry (IHC) for CD47 protein expression and distribution. (A) Qualitative analysis of IHC results revealed that CD47 protein expression was higher in both non-muscle-invasive (n = 21) and muscle-invasive (n = 36) upper urinary tract urothelial carcinomas (UTUCs) than that in normal uroepithelium (n = 20) (P < 0.01), and P-values were determined using unpaired t tests. (B) CD47 was expressed in trace amounts in normal uroepithelium. (C) CD47 was highly expressed in UTUC tissues, CD47-positive cells were widely distributed in the cancer tissue, including on the luminal surface. (D) The localization of CD47 protein was mainly the tumor cell membrane.

Figure 3

Near-infrared (NIR) molecular imaging of freshly isolated human upper urinary tract urothelial carcinoma (UTUC) with anti-CD47-Alexa Fluor 790. Representative white-light, NIR-light, and fusion images with the relevant hematoxylin and eosin-stained (H&E, ×100) photomicrographs for colocalization of anti-CD47-Alexa Fluor 790 binding and histopathology. (A) One upper urinary tract specimen was incubated and imaged successively with IgG control-Alexa Fluor 790 and anti-CD47-Alexa Fluor 790. White- and NIR-light images of normal urothelium and a noninvasive papillary urothelial carcinoma after incubation with the two antibodies are shown. (B) Representative images of renal pelvis, upper ureter, and lower ureter UTUC after incubation with anti-CD47-Alexa Fluor 790. Positive fluorescence at the surgical margin (at the arrow) was seen in the lower ureteral tumor. (C) Quantitative mean fluorescence intensity (MFI) of 12 specimens. MFIs of tumor tissue and normal tissue were 54.67 ± 16.17 and 19.11 ± 6.41 (P < 0.001), respectively. (D) Tumor-to-background ratio (TBR) data of each patient.