Figure 1.

Analysis of TSPAN8 expression in breast cancer cell lines and human tumours. (A) TSPAN8 mRNA expression was tested in different organs in mice using RT‐PCR. Highest expression was detected in the organs of the digestive system – stomach, intestine, and colon – which exhibited 100‐fold higher expression than in the other organs tested (see values on the y‐axis). (B) TSPAN8 expression was examined on RNA (upper panel) and protein levels (bottom panel) in seven breast cancer cell lines; GAPDH and vinculin were used as loading controls for RNA and proteins, respectively. TSPAN8 was strongly expressed only in the MDA‐MB‐361 cells derived from brain metastases. (C) MDA‐MB‐231 cell model, consisting of MDA‐MB‐231 parental cell line, 231‐BR brain‐seeking clone, and BSC60 bone‐seeking clone, was tested for TSPAN8 (upper panel) and TSPAN8 protein (bottom panel) expression. The 231‐Tspan8 cells – parental cell line stably transfected with Tspan8, and MDA‐MB‐361‐expressing endogenous Tspan8 were used as positive controls. Tspan8 revealed strongest expression in the 231‐BR cells. QPCR was performed in technical duplicates and biological triplicates; WB was repeated twice on independent preparations. (D) Immunohistochemistry of human samples for TSPAN8 was performed on paraffin blocks and representative specimens were chosen: on the upper panel, carcinoma in situ shows cytoplasmic localization of TSPAN8; four pairs of primary tumours (pT1, pT2, pT3, pT4) and corresponding metastases (brain M1, brain M2, brain M3, liver M4) originating from the same patient exhibit heterogeneous staining of TSPAN8 in primary tumours and in metastases. Right panel: a brain metastasis with strong membrane and cytoplasmic TSPAN8 staining, a lung metastasis with strong membrane and cytoplasmic staining, and a bone metastasis with cytoplasmic TSPAN8 staining are demonstrated. Arrowheads indicate cytoplasmic TSPAN8 and arrows membrane TSPAN8. Scale bar = 20 μm.