Longitudinal Proteomic Changes in HCT 116 Colon Cancer Spheroids During Growth.

Area of Science:

• Biomedical Engineering
• Proteomics
• Cancer Modeling

Background:

• The FDA Modernization Act 2.0 allows advanced microphysiological systems, like spheroids, for drug testing.
• Spheroids are increasingly used as disease models, but growth duration is often overlooked.
• Proteomics has mainly validated spheroid utility, not tracked longitudinal changes.

Purpose of the Study:

• To investigate temporal proteomic changes in HCT 116 spheroids during 18 days of growth.
• To assess how spheroid growth duration affects their resemblance to avascular tumors.
• To determine the optimal growth phase for spheroid use in preclinical drug development.

Main Methods:

• Data-independent acquisition with gas phase fractionation (DIA-GPF) proteomics was applied.
• HCT 116 spheroids were analyzed every 2 days over 18 days.
• Differential expression analysis and Gene Ontology (GO) term analysis were performed.

Main Results:

• 6,835 proteins were identified across all spheroid samples.
• Day 2 spheroids showed greater similarity to monolayer cells compared to later time points.
• Spheroid maturation involved downregulation of DNA replication and upregulation of glycolysis.

Conclusions:

• Spheroid growth duration is a critical factor for accurate disease modeling.
• Early-stage spheroids (day 2) are more representative of avascular tumors.
• Proteomic analysis provides insights into spheroid temporal dynamics and maturation processes.