Longitudinal Proteomic Changes in HCT 116 Colon Cancer Spheroids During Growth

The
FDA Modernization Act 2.0 permits data from advanced microphysiological
systems, such as spheroids, to be used as a testbed for drug candidates
entering phase 1 clinical trials. Despite their increasing adoption,
spheroids of varying growth durations are often used interchangeably
as disease models. While transcriptomic studies have been employed
to monitor spheroids over time, proteomics has primarily been used
to validate their utility as model systems and assess drug responses
rather than for longitudinal studies. Here, we apply data independent
acquisition with gas phase fractionation (DIA-GPF) proteomics to investigate
temporal changes in HCT 116 spheroids every 2 days throughout 18 days
of growth, identifying 6,835 proteins across all samples. Differential
expression analysis reveals that day 2 spheroids more closely resemble
monolayer cells than spheroids cultured for extended periods. Gene
ontology (GO) term analysis of differentially expressed proteins indicates
that relative to monolayer cells DNA replication is downregulated,
while glycolysis is upregulated during spheroid maturation. Parallel
reaction monitoring (PRM) experiments targeting thymidylate synthase
and fructose-bisphosphate aldolase C validate the initial proteomic
findings and corroborate the trends observed in the GO term analysis.
These results highlight the importance of growth duration when spheroids
are used as a model for avascular tumors.