A Preliminary Study On The Imaging Of Human Breast Cancer MCF7 By Fluorescent Probe Targeting Mucin UMUC1

Breast cancer has the highest morbidity among all malignant tumors in female. Hence, early diagnosis and treatment is of prime importance for patients with breast cancers. Molecular imaging aims to detect alterations in the expression of disease-associated molecules and to discriminate cell types at molecular level using imaging techniques. Thus, molecular imaging is invaluable in early diagnosis and treatment of diseases. Mucinl (MUC1) is a membrane protein widely expressed in epithelia. In breast cancer cells, the glycosylation level of MUC1 greatly decreases, whereas the expression level of MUC1 markedly increases and loses the polarity of distribution. Therefore, targeting underglycosylated MUC1 (uMUC) might be helpful for the early diagnosis, prognosis evaluation and treatment of breast cancers. However, it remains incompletely understood whether fluorescent probes that target uMUC1 could be used in the imaging diagnosis of breast cancers in clinical practice.To investigate the effectiveness of the fluorescent probe that targets uMUC1 in imaging breast cancer cells, we used anti-uMUC1 antibody (VU4H5) to construct the probe. To verify the binding of this antibody to uMUC1(+) breast cancer cell line, MCF7 cells, we incubated MCF7 with antibodies and then performed flow cytometry. We observed that the fluorescence density of MCF7 cells incubated with anti-uMUCl antibody was significantly higher than that of MCF7 cells incubated with normal IgG, which indicates that anti-uMUC1 antibody specifically bind to MCF7 cells. Likewise, we incubated another uMUC1(+) breast cancer cell line, MDA-MB-468 cells, with anti-uMUC1 antibody. The results showed that anti-uMUC1 antibody also specifically bind to MDA-MB-468 cells. Hence, anti-uMUC1 antibody (VU4H5) effectively binds to uMUC1(+) breast cancer cell lines.Cell lines used for in vivo evaluation of fluorescent probes should be imaging-detectable and traceable. Green fluorescent protein (GFP), a frequently used fluorescent tag protein, can be detected by imaging equipments. Therefore, to get traceable MCF7 cells, we stably integrated GFP into MCF cells. First, we infected MCF7 cells with hU6-MCS-Ubiquitin-eGFP-IRES-puromycin lentivirus. The results showed that the infection had the highest efficiency when the multiple of infection (MOI) was 50 and 5μg/mL Polybrene was used. Next, we screened the lentivirus-integrated MCF7 cells with 3μg/mL puromycin. After 2 weeks of continuous passaging and screening, MCF7-GFP cell line, which stably expresses GFP, was obtained. Proliferation curve, wound-healing test and propidium iodide (PI) staining followed by flow cytometry showed that the proliferation rate and cell cycle distribution of MCF7-GFP cells were not significantly different from MCF7 control cells. In addition, we utilized optical imaging system (Xenogen, IVIS 300) to examine the fluorescence of MCF7-GFP cells. The imaging experiment confirmed that green fluorescence was detectable in MCF7-GFP cells. Thus, we successfully constructed a MCF7-GFP cell line that stably expresses GFP and we showed that the proliferative properties of this cell line are comparable with normal MCF7 cells.In consideration that lentivirus randomly integrates exogenous genes into the host genome, and therefore may interfere with host gene expression, we next examined uMUC1 expression in MCF7-GFP cells. Through western blot and flow cytometry, we observed that uMUC1 expression in MCF7-GFP cells was lower than that of wild type MCF7 cells. Nevertheless, flow cytometry results showed that anti-uMUC1 antibody still effectively bound to MCF7-GFP cells. These results indicate that, although MCF7-GFP cells have a lower expression level of uMUC1 than normal MCF7 cells, MCF7-GFP cells can still be used in the study of uMUCl-targeted probe imaging.After the construction of MCF7-GFP cell line, we investigate the effectiveness and safety of uMUC1-targeted fluorescent probe binding to MCF7-GFP cells. We coupled IRDye800 NHS Ester, which contains NHS Ester active groups, with anti-uMUC1 antibody to construct the anti-uMUC1-IRDye800 probe. Using optical imaging system, we next evaluated the binding effectiveness of anti-uMUC1-IRDye800 probe to MCF7-GFP. We observed that the probe effectively bound to MCF7-GFP cells in the concentration of 10μg/mL after incubation at 37℃ for 30 min. And further increase of the incubation time did not significantly change the binding efficiency. The binding specificity of the anti-uMUC1-IRDye800 probe was verified using IRDye800 carboxylate as a negative control. The fluorescence density of MCF7-GFP cells incubated with 10μg/mL anti-uMUC1-IRDye800 probe was significantly higher than that of cells incubated with IRDye800 carboxylate in the same concentration, and the fluorescence density gradually increased with the increase in the concentration of anti-uMUC1-IRDye800 probe. By contrast, the fluorescence density of MCF7-GFP cells incubated with IRDye800 carboxylate did not increase with the increase in the concentration of the fluorescent dye. These results confirmed the binding specificity of the anti-uMUC1-IRDye800 probe to MCF7-GFP cells. Next, to investigate the toxicity of the anti-uMUC1-IRDye800 probe to MCF7-GFP cells, we performed MTT assays. The results showed that, only when the concentration was higher than 50μg/mL, the anti-uMUC1-IRDye800 probe significantly impaired MCF7-GFP cell survival. The above results indicate that anti-uMUC1-IRDye800 probe specifically binds to MCF7-GFP cells without causing significant toxicity in effective concentration.To summarize, in the present study, we constructed MCF7-GFP cell line stably expressing GFP, and preliminarily imaged MCF7-GFP cells using an anti-uMUCl (VU4H5)-IRDye800 probe. Our work adds a cell line to the toolkit for further in vivo breast cancer imaging research, and lays the preliminary experimental foundation for the utilization of uMUCl-targeted fluorescent probes in clinical breast cancer imaging.