Fig. 1

Identification of GC metastasis-suppressing genes using CRISPR KO screening. A Schematic of the in vivo genome-wide CRISPR KO screen using a peritoneal dissemination model. B KO library coverage rates based on amplicon next-generation sequencing (NGS) analysis of input samples from three independent experiments. C Peritoneal dissemination rates in syngeneic mice intraperitoneally injected with KO library and control S1M cells. Necropsy was performed either upon showing clinical signs of malignancy or after 105 days post-injection. D Kaplan–Meier survival analysis of metastasis-free survival percentage in syngeneic mice injected with KO library and control S1M cells. The data represent the combined results of three independent screenings of sets. P value, Log-rank test was performed to compare the metastasis-free survival. E Representative gross images at the point of necropsy of syngeneic mice after intraperitoneal injection with control and KO library S1M cells. Bar = 1 cm. F Representative H&E-stained images of metastatic foci developed at peritoneum and diaphragm of syngeneic mice intraperitoneally transplanted with KO library S1M cells. Bar = 200 μm. G Representative Ki-67 immunohistochemical images of metastatic foci in the peritoneum from syngeneic mice intraperitoneally transplanted with KO library S1M cells. Bar = (top) 200 μm, (bottom) 50 µm. H Enriched gRNA-targeted genes were analyzed using TA cloning with Sanger sequencing (set 1) and amplicon NGS analyses (sets 2 and 3). Each color in pie charts represents a specific gene targeted by the gRNA and shows the relative read counts obtained from each mouse with metastasis. Genes with read counts less than 5% of the total are represented in the gray area. I Incidence rates of metastasis after intraperitoneal injection with individual candidate genes-KO cells into syngeneic mice. P value, Fisher's exact test; compared with the results of control cells. In all experiments, Student's t-test was used to obtain the p-value for statistical analysis, unless otherwise specified