PRSS contributes to cetuximab resistance in colorectal cancer

Tan, Zhaoli; Gao, Lihua; Wang, Yan; Yin, Huihui; Xi, Yongyi; Wu, Xiaojie; Shao, Yong; Qiu, Weiyi; Du, Peng; Shen, Wenlong; Fu, Ling; Jia, Ru; Zhao, Chuanhua; Zhang, Yun; Zhao, Zhihu; Sun, Zhiwei; Chen, Hongxing; Hu, Xianwen; Xu, Jianming; Wang, Youliang

To confirm our in vitro results, which indicated that a combined SPINK1 and cetuximab treatment led to a supra-additive reduction in LoVo tumor growth, we performed a similar xenograft study using HT-29 cells. As expected, both SPINK1/cetuximab and SPINK1/ bevacizumab inhibited tumor growth more than either cetuximab or bevacizumab alone in HT-29 cell-xenografted mice (Fig. 5E ). Similar to the observation in the LoVo cell-xenografted mice ( fig. S6A) , the HT-29 cell-xenografted mice treated with SPINK1 and cetuximab had more significantly decreased pERK levels than those treated with either SPINK1 or cetuximab alone (Fig. 5F ). We also assessed microvascular formation in xenograft tumor sections via staining for CD34 and endomucin. Immunofluorescence and IHC showed more significantly decreased microvascular formation in the HT-29 cell-xenografted mice treated with SPINK1 and bevacizumab than that in mice treated with either SPINK1 or bevacizumab alone ( Fig. 5G and fig. S6B ). CD34 was used to determine the microvascular density. On the basis of CD34 expression, we selected five highmagnification fields of view and counted the number of CD34-positive cells. CD34 expression was localized to microvascular endothelial cells in the xenograft tumor sections. Statistically significant decreases in the number of capillaries and the distribution of the microcirculation were noted in the xenograft tumors treated with SPINK1 and bevacizumab compared with those treated with either SPINK1 or bevacizumab alone (Fig. 5H and fig. S6C ). Consistent with previous reports, we also found that SPINK1 induced cancer cell proliferation (Fig. 5, B and E) . These observations all support the premise that SPINK1 effectively inhibits the trypsin/PRSS1-mediated proteolytic cleavage of mAbs and improves the curative effect of mAbs.