Pre- and post-chemotherapy plasma vascular endothelial growth factor (VEGF) were measured. Tumor expression of tissue VEGF was characterized by tissue microarray. Methods for these assays have been described [20 (link)]. Also, serum total testosterone levels were obtained prior to and after neoadjuvant chemotherapy.
Immune infiltration was examined via immunohistochemistry with a panel of 8 markers in primary prostatic tumors and adjacent normal tissue using a tissue microarray (TMA) of 50 patients treated neoadjuvantly with docetaxel and mitoxantrone. Heat mediated antigen retrieval was performed with citrate buffer (BioGenex, Fremont, CA), with the exception of retrieval prior to CD4 staining, which was achieved using 10 mM Tris/1 mM EDTA. Staining was performed with the Ultravision Detection System (Thermo Scientific, Waltham, MA), followed by counterstaining with methyl green. Antibodies CD3, CD4, CD8, CD15, and CD163 were from Thermo Scientific, CD68 (clone PG-M1) and CD20 were from AbCAM (Cambridge, UK), and FoxP3 was from eBioscience (San Diego, CA). Slides were digitally scanned with an Aperio ScanScope CT Slide Scanner at 40x objective and individual TMA cores were analyzed separately using TMALab (Aperio, Buffalo Grove, IL) and the nuclear stain detection algorithm to quantify the number of positive cells within each core. Data is displayed as cell density (cells/mm2).
Immune infiltration was examined via immunohistochemistry with a panel of 8 markers in primary prostatic tumors and adjacent normal tissue using a tissue microarray (TMA) of 50 patients treated neoadjuvantly with docetaxel and mitoxantrone. Heat mediated antigen retrieval was performed with citrate buffer (BioGenex, Fremont, CA), with the exception of retrieval prior to CD4 staining, which was achieved using 10 mM Tris/1 mM EDTA. Staining was performed with the Ultravision Detection System (Thermo Scientific, Waltham, MA), followed by counterstaining with methyl green. Antibodies CD3, CD4, CD8, CD15, and CD163 were from Thermo Scientific, CD68 (clone PG-M1) and CD20 were from AbCAM (Cambridge, UK), and FoxP3 was from eBioscience (San Diego, CA). Slides were digitally scanned with an Aperio ScanScope CT Slide Scanner at 40x objective and individual TMA cores were analyzed separately using TMALab (Aperio, Buffalo Grove, IL) and the nuclear stain detection algorithm to quantify the number of positive cells within each core. Data is displayed as cell density (cells/mm2).
