In very early stages of cancer development, one or a few cells expressing cancer-associated genes appear among a much larger number of surrounding normal cells. To analyze the molecular changes induced by this co-existence, we artificially prepared transformed cells with complete loss of tumor suppressor gene, SCRIB, among normal human embryonic kidney (HEK293T) cells. A cell strain with SCRIB-knockout was successfully constructed by using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 nuclease system and co-cultured with normal cells. By measuring the time-course changes in cell numbers when SCIRB-knockout cells (cancer model) or cells with normal level of SCRIB expression were respectively co-cultured with wild-type normal HEK293T cells, it was shown that the SCRIB-knockout strain was beneficial for proliferation when mixed together with normal cells. Moreover, as a result of proteome analysis on wild-type cells separated from co-culture with SCRIB-knockout cells, a total of 843 proteins were identified, among which 139 proteins were specific. Among the specifically identified proteins, 22 proteins were annotated to be involved in cytoskeletons including microtubule motor activity-associated proteins. It was implied that molecular changes in cytoskeletons occurred in normal cells when co-cultured with SCRIB knockout cells, but the SCRIB knockout might affect proliferation of the transformed cells with SCRIB knockout by defensive or offensive mechanism of surrounding normal cells. (C) 2016, The Society for Biotechnology, Japan. All rights reserved.