Proteome dynamics during establishment of California grunion (Leuresthes tenuis) cell lines

Establishing continuous cell lines is hindered by limited molecular resolution of culture establishment and the transition to sustained proliferation. Here, proteome changes across passages were quantified in two independently derived California grunion (Leuresthes tenuis) embryonic-derived cell cultures, alongside morphology and growth metrics, to identify proteome dynamics associated with early establishment and subsequent stabilization of continuous proliferation under the standard conditions.

Morphological analysis identified a reproducible transition window centered on passage 4 (P4), coincident with changes in growth trajectories and consolidation toward epithelial-like morphology in three replicate cell lines LtE-1, LtE-2, and LtE3. Two of these replicate lines (LtE1 and LtE2) were analyzed by quantitative cell population proteomics confirming this transition, consistent with establishment-associated selection and/or cell-state change in a mixed early culture. Marker proteins associated with epithelial identity increased in abundance while fibroblast-associated markers declined. Across the transition window and subsequent passages, LtE-1 and LtE-2 shared broad remodeling of biosynthetic, proteostatic, adhesion/ECM, and lipid-related functions, and recurrence-filtered interaction networks highlighted passage-linked module consolidation. However, LtE-1 and LtE-2 differed in their temporal trajectories (transient surges in LtE-1 versus sustained reinforcement in LtE-2). Because early cultures contained mixed morphologies and cell population proteomics integrates across subpopulations, these patterns are presented as proteome dynamics of establishment and candidate biosignatures rather than definitive cell-intrinsic mechanisms.

Passage-resolved cell population proteomics in two replicate California grunion embryonic-derived cell cultures define a baseline of establishment-associated remodeling and identifies candidate biosignatures linked to a reproducible transition window and subsequent stabilization of proliferation. The resulting passage-resolved baseline motivates lineage-resolved validation to distinguish the relative contributions of selection, cell-state transitions, and media adaptation to the observed establishment trajectory.