?(Fig.4e4e). The B7-H3/KIF15 axis confers radioresistance in vitro and in vivo We hypothesized that B7-H3 may contribute to CRC cell radioresistance by controlling KIF15. NF-B pathway. And small interfering RNA (siRNA)-mediated KIF15 silencing or KIF15 blockade by the inhibitor SB743921 abolished the effect of B7-H3 on radioresistance in vitro and in vivo. Similar to B7-H3, we find that the protein expression levels of KIF15, which SMOH showed a positive correlation with B7-H3, was abnormal upregulated in cancer tissues than in adjacent normal tissues and associated with TNM stage. Finally, B7-H3/KIF15 enhanced resistance against irradiation in CRC cells via activating ERK1/2 signaling, a key pathway Triclosan involved in radioresistance in cancer. Our findings reveal an alternative mechanism by which CRC cells can acquire radioresistance via the B7-H3/KIF15/ERK axis. strong class=”kwd-title” Subject terms: Radiotherapy, Colorectal cancer Introduction Colorectal cancer (CRC) is the third most commonly occurring malignancy and accounts for more than 9% of all cancer-related deaths worldwide1. Neoadjuvant radiotherapy/chemoradiotherapy (neoRT/CRT) following surgery has been approved by NCCN and is required for comprehensive therapy for locally advanced stage II and III CRC2. In addition, preoperative chemoradiotherapy combined with surgery could improve Triclosan the locoregional control of CRC. However, the treatment effect of both neoRT/CRT and preoperative chemoradiotherapy is still unsatisfactory because of chemoresistance and radioresistance3. Importantly, approximately 50% of CRC patients experience recurrence and metastasis after radiotherapy4. Therefore, there is an urgent need to understand the mechanisms of radioresistance and identify biomarkers that predict radioresistance in CRC patients. As a type I transmembrane protein, B7-H3 belongs to the important immune checkpoint B7 ligand family, which provides a costimulatory signal for T-cells in the tumor microenvironment and promotes tumor progression and escape5C7. Many studies have suggested that the aberrant Triclosan expression of B7-H3 exists in different cancer types, including pancreatic carcinoma8, esophageal carcinoma9, hepatocellular cancer10, colorectal cancer11, non-small cell lung cancer12 and gastric cancer13. The frequency of B7-H3-positive circulating epithelial tumor cells (CETCs) is significantly higher in breast cancer patients who receive radiotherapy than in patients who do not receive irradiation, suggesting that the upregulation of B7-H3 expression on CETCs could be a possible mechanism of acquired radioresistance in breast cancer patients14. Nevertheless, the functional roles and underlying signaling cascades of B7-H3 associated with radioresistance in CRC have yet to be investigated. Herein, we first demonstrated that the expression of B7-H3 was obviously increased in CRC cells after irradiation. Further in vitro and in vivo functional analyses showed that B7-H3 enhanced resistance against irradiation in CRC cells by upregulating KIF15 expression via NF-B, which activated ERK1/2 signaling, a key pathway involved in radioresistance in cancers15. B7-H3 blockade by 3E8, a specific B7-H3 antibody16, significantly sensitized CRC cells to irradiation in vivo. Moreover, the expression of B7-H3 was positively correlated with KIF15 expression in CRC tissue samples. Overall, our data suggest that B7-H3 contributes to CRC radioresistance and that targeting this molecule may be beneficial for CRC treatment. Materials and methods Cell lines and cell culture NCM460, HCT8, HT29, SW480, SW620, HCT116 and RKO CRC cell lines were purchased from the Chinese Academy of Science Cell Bank and cultured in DMEM and RPMI-1640 medium (BioInd, Beit Haemek, Israel) including 10% fetal bovine serum (FBS, BioInd), 100 U/ml penicillin and 100?mg/ml streptomycin (Gibco, Grand Island, USA) in a humidified atmosphere of 5% CO2 at 37?C. Immunohistochemistry Sections from paraffin-embedded tissues were incubated with a goat anti-human B7-H3 antibody (1:200, R&D Systems, #AF1027), a rabbit anti-human KIF15 antibody (1:2000, Proteintech, #55407-1-AP) or a rabbit anti-human Ki67 antibody (1:500, Abcam, ab15580) overnight at 4?C. This step was followed by staining (45?min at room temperature) with the corresponding HRP-labeled rabbit anti-goat secondary antibody or goat anti-rabbit secondary antibody (Invitrogen). Next, the sections were visualized by staining with 3,3-diaminobenzidine (Biocare Medical, CA, Triclosan USA) and counterstaining with hematoxylin (Sigma). The numbers of Ki67-positive cells and total cells were analyzed using a microscope (Leica, Buffalo Grove, USA). All sections were then reviewed blindly by two experienced pathologists (Dr. Cao and Dr. Zhan). The scoring criteria for B7-H3 and KIF15 immunostaining were based on clinical data and adopted the semiquantitative immunoreactive score (IRS) system17. Briefly, category A (intensity of immunostaining) was scored using the following criteria: 0, negative; 1, weak; 2, moderate; and 3, strong. Category B (percentage of immunoreactive cells) was scored using the following criteria: 1 (0C25%); 2 (26C50%); 3 (51C75%); and 4 (76C100%). Final scores were calculated by multiplying the scores of categories.