Standard IHC staining protocols were applied. Briefly, tissue sections were incubated with primary antibodies for 1 hour (or overnight), followed by incubation with the secondary anti-mouse or anti-rabbit for 30 min at 37°C. Slides were analyzed using a Leica DM IRB microscope, and digital images were evaluated with METAMORPH software. For immunohistochemical analysis of lung metastasis, paraffin-embedded samples were cut in 10-μm-thick sections and probed with primary antibodies according to the standard protocols.

An anti-Sema4C immunostaining protocol was specifically set up by the use of BenchMark ULTRA IHC System and UltraView Universal DAB staining kit, according to the following notable steps: dewax at 72°C, EZ Prep 1:10; unmasking for 76 min with ULTRA CC1 (950-224); incubation with primary anti-Sema4C antibody (sc-136445; dilution 1:100) for 48 min at 37°C; detection with amplification kit; and final counterstaining with hematoxylin. In specificity validation experiments, the antibody was blocked by incubation with soluble recombinant Sema4C ectodomain (catalog no. AF6125, R&D Systems).

In the analysis of the breast tumor microarray, a semiquantitative H-score method (55) was applied to classify Sema4C expression levels as low, intermediate, or high. Briefly, we analyzed each section at ×100 microscope magnification and counted the cells characterized by different relative staining intensities (scored as no staining, weak, moderate, or strong staining). Five randomly selected microscopic fields were analyzed for each slide, and the following formula was used to calculate a combined score for each sample in a range from 0 to 300: three times the percentage value of the fraction of strongly stained cells + two times the percentage of moderately stained cells + the percentage of weakly or nonstained cells. On the basis of this global score assigned to individual cases, they were sorted into low, intermediate, or high expression, according to the following ranges: low, combined IHC H-score of 10 to 100; intermediate, combined H-score of 100 to 250; and high, combined H-score of >250.

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