Multilevel Proteomic Profiling of Colorectal Adenocarcinoma Caco‑2 Cell Differentiation to Characterize an Intestinal Epithelial Model

Emergent advancements on the role of the intestinal microbiome
for human health and disease necessitate well-defined intestinal cellular
models to study and rapidly assess host, microbiome, and drug interactions.
Differentiated Caco-2 cell line is commonly utilized as an epithelial
model for drug permeability studies and has more recently been utilized
for investigating host–microbiome interactions. However, its
suitability to study such interactions remains to be characterized.
Here, we employed multilevel proteomics to demonstrate that both spontaneous
and butyrate-induced Caco-2 differentiations displayed similar protein
and pathway changes, including the downregulation of proteins related
to translation and proliferation and upregulation of functions implicated
in host–microbiome interactions, such as cell adhesion, tight
junction, extracellular vesicles, and responses to stimuli. Lysine
acetylomics revealed that histone protein acetylation levels were
decreased along with cell differentiation, while the acetylation in
proteins associated with mitochondrial functions was increased. This
study also demonstrates that, compared to spontaneous differentiation
methods, butyrate-containing medium accelerates Caco-2 differentiation,
with earlier upregulation of proteins related to host–microbiome
interactions, suggesting its superiority for assay development using
this intestinal model. Altogether, this multiomics study emphasizes
the controlled progression of Caco-2 differentiation toward a specialized
intestinal epithelial-like cell and establishes its suitability for
investigating the host–microbiome interactions.