TLR5 is a new reporter for triple-negative breast cancer indicated by radioimmunoimaging and fluorescent staining.

Triple-negative breast cancer (TNBC) is a highly aggressive tumour that lacks marker for targeted diagnosis. Recently, it was reported that toll-like receptor 5 (TLR5) was associated with some kind of tumours, especially in TNBC, but whether it could be used as a non-invasive monitoring target is not fully understood. Here, we established TLR5^- 4T1 cell line with lentivirus-shRNA-TLR5 knock-down transfection (with tag GFP, green fluorescent protein, TLR5^- 4T1) and control TLR5⁺ 4T1 cell line with negative control lentivirus transfection. The effect of TLR5 down-regulation was detected with qPCR and Western blot. ¹²⁵ I-anti-TLR5 mAb and control isotype ¹²⁵ I-IgG were prepared and injected to TLR5^+/- 4T1-bearing mice models, respectively. Whole-body phosphor-autoradiography, fluorescence imaging and biodistribution were performed. Furthermore, ex vivo tumour TLR5 expression was proved through immunohistochemistry staining. We found that ¹²⁵ I-anti-TLR5 mAb could bind to TLR5⁺ 4T1 with high affinity and specificity. Whole-body phosphor-autoradiography after ¹²⁵ I-anti-TLR5 mAb injection showed TLR5⁺ 4T1 tumour images in 24 hours, more clearly in 48 hours. Radioactivities in tumour tissues were positively related with TLR5 expression. Biodistribution assay showed that ¹²⁵ I-anti-TLR5 mAb was mainly metabolized through the liver and kidney, and ¹²⁵ I-anti-TLR5 mAb was much more accumulated in TLR5⁺ 4T1 tumour than TLR5^- 4T1. In vivo fluorescence imaging successfully showed tumour tissues clearly both in TLR5⁺ and TLR5^- 4T1 mice compared with lentivirus untreated 4T1 tumour. Immunohistochemistry staining showed that TLR5 expression in tumours was indeed down-regulated in TLR5^- 4T1 mice. Our results indicated that ¹²⁵ I-antiTLR5 mAb was an ideal agent for non-invasive imaging of TLR5⁺ tumours; TLR5 may be as a novel molecular target for TNBC non-invasive diagnosis. © 2019 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

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