Immunohistochemical Assessment of Tumor Markers

Immunohistochemical staining and analysis was performed as previously published^{[30 (link)-32 (link)]}. Briefly, Ki67 proliferation indices were determined by counting the number of Ki67 positive tumor cells in 10 individual 40x fields and dividing that number by the total number of tumor cells. The data are expressed as the mean ± S.E.M. of two independent experiments. Expression indices of RRM1, RRM2, hENT1, hCNT1, CDA, dCK, ALDH1, CD133, and CXCR4 were calculated by assigning a staining intensity of 0, 1, 2, or 3 to each specimen, and multiplying this intensity by the percent of tumor cells expressing the protein of interest^{[33 (link)]}. Overall scores ranged from 0 to 300. Data were derived from photomicrographs taken with Olympus BH-2 microscope with DP71 camera and DPS-BSW v3.1 software. Primary antibodies were obtained from multiple sources: Ki67 (ab92742, abcam, Cambridge, MA, USA), RRM1 (NBP2-49415, Novus Biologicals, Littleton, CO, USA), RRM2 (ab57653, abcam), hENT1 (SAB5500117, Sigma-Aldrich, St. Louis, MO, USA), hCNT1 (NBP2-30857, Novus Biologicals), CDA (ab82346, abcam), dCK (sc393099, Santa Cruz, Dallas, TX, USA), ALDH1 (sc166362, Santa Cruz), CD133 (CS#86781T, Cell Signaling, Danvers, MA, USA), CXCR4 (TA305935, Origene, Rockville, MD, USA).