3D culture system containing gellan gum restores oncogene dependence in ROS1 rearrangements non-small cell lung cancer.

The ROS1 fusion gene has been identified in approximately 1% of non-small cell lung cancer (NSCLC) cases. Several clinical studies have highlighted ROS1 as a promising therapeutic target because crizotinib, a multi-targeted drug against ROS1, ALK, and the MET proto-oncogene, has elicited remarkable responses in ROS1-rearrangements NSCLC. However, acquired resistance mediated by ROS1 kinase domain mutations has been identified and a system to assess ROS1 inhibitors for these resistant mutations is necessary for the promotion of drug development. Publicly available NSCLC cell lines harboring the ROS1 fusion gene are limited to only HCC78 cells carrying SLC34A2-ROS1. This cell line exhibits resistance to ROS1 inhibitors through activation of the EGFR pathway, although the cells were established from ROS1-TKI naïve pleural effusion. Here, we demonstrate that 3D culture with gellan gum can restore the ROS1 oncogene dependence of HCC78 cells by upregulating the expression of the ROS1 fusion gene and reducing the activity of the EGFR pathway. Moreover, we established the HCC78xe3 cell line, a subclone of the HCC78 cell line, by repeated in vitro cultures from tumor xenografts and created xenograft tumors three times using in vitro cultured cells. This eventually enabled us to engraft and stably grow the cells in vivo, and subsequently evaluate various ROS1 inhibitors against HCC78xe3 cells by overexpressing crizotinib-resistant mutations in the ROS1 kinase domain including G2032R and D2033 N. We newly found that lorlatinib, a next generation ROS1/ALK inhibitor, remain the activity against D2033 N mutation. Furthermore, we demonstrated that HCC78xe3 cells expressing SLC34A2-ROS1 G2032R, and D2033 N, but not wild type (WT) cells, were resistant to crizotinib in vivo. Taken together, our data suggested that 3D cultures of HCC78 might reflect the features in patients and this new system will be a useful tool for evaluating ROS1 inhibitors.