039*     .852     .099     .005** negative 75 78   66 26   73 80   94 59   positive 15 32   21 87   16 31   18 29   D represents the diameter of tumor, LN represents lymph node status, N represents the amoumt of lymph node excised, grade means the histological grade, and stage means the clinical stage. *P < 0.05, **P < 0.01 In IHC staining, 77% of tumor cells were CXCR4 positive in the cytoplasm, including high and low CXCR4 expression (Figure Poziotinib purchase 1A2). Meanwhile, 73% were positive in the nucleus (Figure 1A2). The amounts of CCR7 (Figure 1B2) and EGFR (Figure 1E2) were

detected in 82% and 66% of tumor cells, respectively, in the cytoplasm and/or membrane. Furthermore, 50% of ER, 49.5% of PR, and 23.5% of HER-2/neu were observed to be positive. Figure 1 IHC staining see more for biomarkers. IHC staining for CXCR4, CXCL12, CCR7, CCL21 and EGFR. PT pertains to primary tumor, while LNMT Adriamycin stands for lymph node metastasis tumor. Rows correspond to the designated chemokine or receptor. The first column represents staining of negative expression in primary breast cancer with the indicated antibody. The second column indicates positive expression in primary breast cancer, and the third column shows positive expression in lymph node metastasis

cancer. Both PT and LNMT columns in each row are obtained from the same patient while the negative column is not. In the CXCR4 row, A2 and A3 exhibit high expression in both cytoplasm and nucleus. CCR7, CXCL12, and CCL21 all exhibit positive reaction in the cytoplasm. In the EGFR row, E2 and E3 indicate that EGFR is expressed mainly Glycogen branching enzyme in the membrane. However,

a number of tumor cells appear to be positive in the cytoplasm as well (Panels A-E, ×200). Association of CXCR4, CCR7, and EGFR with lymph node metastasis The immunoreactivity of CXCR4 was observed in the cytoplasm and/or nucleus of tumor cells. Cytoplasmic reactivity of CXCR4 correlated positively with lymph node metastasis of breast cancer (P < 0.001), but not with the amount of involved lymph nodes. Nuclear reactivity was not observed to be correlated with any pathologic parameters. Meanwhile, CCR7 was positively expressed in the cytoplasm, and the activity was significantly correlated with lymph node metastasis (P < 0.001). Similarly, associations among the lymph node status, histological grade, and EGFR expression were observed in this study (Table 1). To verify the important effect of CXCR4 and CCR7 in metastasis, CXCR4, CCR7, and EGFR expression in primary breast cancer were compared with that in lymph node metastasis tumor. It was observed that CXCR4 and CCR7 expression in metastasis tumor was even higher, although no significant distinction was evident. More importantly, their respective ligands, CXCL12 and CCL21, exhibited significant differences in expression between primary tumor and lymph node metastasis tumor (P = 0.016 and P = 0.004; Table 2).