Abstract
BackgroundThe B-subunit of Shiga toxin (STxB) specifically binds to the glycosphingolipid Gb3 that is highly expressed on a number of human tumors and has been shown to target tumor cells in mouse models and ex vivo on primary colon carcinoma specimen.MethodsUsing a novel ex vivo STxB labeling (ESL) method we studied Gb3 expression in cytological specimens of primary human breast tumors from 107 patients, and in synchronous lymph node metastases from 20 patients. Fluorescent STxB was incubated with fine-needle aspiration (FNA) specimens, and Gb3 expression was evaluated by fluorescence microscopy. Furthermore, 11 patient-derived human breast cancer xenografts (HBCx) were evaluated for expression of Gb3 by ESL and FACS. In addition, the biodistribution of fluorescent STxB conjugate was studied after intravenous injection in a Gb3 positive HBCx model.ResultsGb3 expression was detected in 62 of 107 patients (57.9%), mainly in epithelial tumor cells. Gb3 positivity correlated with estrogen receptor expression (p ≤ 0.01), whereas absence of Gb3 expression in primary tumors was correlated with the presence of lymph node metastases (p ≤ 0.03). 65% of lymph node metastases were Gb3 positive and in 40% of tested patients, we observed a statistically significant increase of metastatic Gb3 expression (p ≤ 0.04). Using concordant ESL and flow cytometry analysis, 6 out of 11 HBCx samples were scored positive. Intravenous injections of fluorescent STxB into HBC xenografted mice showed preferential STxB accumulation in epithelial cells and cells with endothelial morphology of the tumor.ConclusionThe enhanced expression of Gb3 in primary breast carcinomas and its lymph node metastases indicate that the development of STxB-based therapeutic strategies is of interest in this pathology. Gb3 expressing HBCx can be used as a model for preclinical studies with STxB conjugates. Finally, the ESL technique on FNA represents a rapid and cost effective method for the stratification of patients in future clinical trials.
Highlights
The B-subunit of Shiga toxin (STxB) binds to the glycosphingolipid Gb3 that is highly expressed on a number of human tumors and has been shown to target tumor cells in mouse models and ex vivo on primary colon carcinoma specimen
In order to evaluate Gb3 expression on epithelial cancer cells, we developed a novel technique for the ex vivo STxB labeling (ESL) in which live cells from dissociated tumors are incubated with STxB prior to microscopical viewing
Gb3 expression is increased in human breast carcinoma compared to normal tissue In order to evaluate global quantity of Gb3 in normal and tumor breast tissue, Gb3 was extracted and quantified receptor (ER), 35/62 (56.5%) for progesterone receptor (PR), and 40/62 (64.5%) for HER2. 5/62 (8.1%) were triple negative (Table 1)
Summary
The B-subunit of Shiga toxin (STxB) binds to the glycosphingolipid Gb3 that is highly expressed on a number of human tumors and has been shown to target tumor cells in mouse models and ex vivo on primary colon carcinoma specimen. Shiga and Shiga-like toxins are produced by Shigella dysenteriae and enterohemorrhagic strains of Escherichia coli. These toxins are composed of two non-covalently attached parts: the enzymatically active A-subunit, and the non-toxic, pentameric B-subunit (STxB) [4]. STxB binds to the sugar moiety of the glycosphingolipid globotriaosylceramide (known as CD77, Gb3, and ceramide trihexoside) in the plasma membrane of target cells, and mediates uptake and intracellular transport of the toxin [5,6]. The A1 fragment irreversibly modifies ribosomal 28S RNA, leading to the inhibition of protein biosynthesis and cell death by apoptosis [5]
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