Pa5 17245

Expression of CDCP1 was analyzed by IHC on consecutive 2-μm formalin-fixed, paraffin-embedded (FFPE) tumor sections, using rabbit polyclonal anti-CDCP1 (1:50) (PA5-17245, Thermo Fisher Scientific), which is directed against the C-terminus of CDCP1, after antigen retrieval, which as performed by heating the sections for 5 min at 96°C in 10 mM citrate buffer, pH 6.0. Immunoreactions were visualized using streptavidin-biotin-peroxidase (Dako, Agilent Technology, Santa Clara, CA), 3,3′-diaminobenzidine (DAB; brown signal) (Dako) as the chromogen, and the sections were counterstained with hematoxylin. Images were acquired on an ECLIPSE TE2000-S inverted microscope (Nikon Instruments, Melville, NY) at 20× and 40× magnification.
The reactivity of anti-CDCP1 in the TNBC specimens was considered to be positive when ≥ 10% of tumor cells showed membrane staining. This cutoff was chosen, based on distribution analysis of the percentage of CDCP1-positive cells by IHC in each tumor section. No tumors had < 10% CDCP1-positive cells in our series, and cases with different percentages of CDCP1-positive cells were likewise distributed in a 10–100% interval. By explorative Kaplan-Meyer analysis of DFS in our cases—stratified as negative, ≥ 10% and < 50%, or ≥ 50% for CDCP1 expression, both CDCP1-positive groups had a worse and superimposable DFS compared with CDCP1-negative cases (data not shown).

In the biochemical analyses, we used rabbit polyclonal antibody against CDCP1 (Merck Millipore); rabbit polyclonal phospho-CDCP1 (Tyr734) (Cell Signaling); mouse monoclonal anti-Src, clone GD11 (Merck Millipore); rabbit polyclonal phospho-Src family (Tyr416) (Cell Signaling); rabbit polyclonal anti-PKCδ (Cell Signaling); rabbit polyclonal phospho-PKCδ (Tyr311) (Cell Signaling); mouse monoclonal anti-vinculin, clone hVIN-1 (Sigma); anti-rabbit or -mouse IgG (GE Healthcare) as the secondary antibody; and peroxidase-linked mouse monoclonal anti-actin (Sigma-Aldrich). In the IHC analyses, we used CDCP1 polyclonal antibody PA5-17245 (Thermo Fisher Scientific).

Expression of CDCP1 and PDGFRβ was analyzed by IHC in consecutive 2-μm formalin-fixed, paraffin-embedded (FFPE) tumor sections, using rabbit polyclonal anti-CDCP1 (1:50) (PA5–17245, Thermo Fisher Scientific) and rabbit anti-human PDGFRβ (1:200) (Y92, Abcam), respectively. Antigen retrieval was performed by heating the sections for 5 min at 96 °C in 10 mM citrate buffer, pH 6.0. Staining was visualized using streptavidin-biotin-peroxidase (Dako, Agilent Technology, Santa Clara, CA) and 3,3′-diaminobenzidine (DAB; brown signal) (Dako), and the sections were counterstained with hematoxylin. Images were acquired by ECLIPSE TE2000-S inverted microscope (Nikon Instruments, Melville, NY) at 20X and 40X magnification. The reactivity of anti-CDCP1 and anti-PDGFRβ was considered to be positive per Turdo 2016 and D’Ippolito 2016 [3 (link), 25 (link)]. Specifically, based on the intensity of PDGFRβ staining in neoplastic cells, we assigned tumors a score of 0 (absence of signal) or 1 (weak to strong cytoplasmic signal and membrane signal). Reactivity of polyclonal anti-CDCP1 was defined as positive when ≥10% of tumor cells showed membrane staining.