We performed comparative analysis for them, which involved developing two HT-29 colorectal cancer models: as monolayers in a conventional incubator and as 3D spheroids under physioxia, in the presence/absence of gamma-interferon.

On each model, a viability assay was performed after 3 days incubation time with inhibitors, followed by large-scale proteome profiling analysis on 2000 targets from each cell lysates.

We established and implemented a physiologically relevant 3D Colorectal cancer cell-based assay. To answer questions related to variations at transcription and protein level, our methodology required long-term imaging at controlled temperature and gas levels (CO₂ and O₂), on both 3D cellular models and patient-derived samples. Impact of an anti-cancerous drug was investigated on colon cell lines (HT-29, HCT116 and LS174T) in conventional 2D monolayer and 3D spheroid cultures, in hypoxia or normoxia. Methods used were metastatic invasion assay, gene expression analysis by RT-PCR on hypoxic response and oncopathway genes, and large-scale protein profiling to detect proteomic levels of metalloproteinases and surface proteins known to be involved in colorectal cancer invasion.

To select the best production conditions in HEK 293 EBNA cells, we first performed a small volume expression study to define optimal production time and ratio of DNA/transfection reagent. Moreover, we tested addition to the cell culture medium of various supplements (amino acids, inorganic salts, lipids…), aiming at boosting the expression level by varying medium composition. On identification of the best production conditions, several buffer compositions were screened at purification level to improve stability and solubility of the protein of interest. Different buffers, detergents, sugars, as well as α-helix stabilizers, and many more, were tested in a 96-well format for high-throughput.

Tebubio lab has developed a plasmid template for the mRNA production, optimized for efficient transcription and translation in mammalian cells. By using Andy Fluor-UTP (refs. C410, C412, C414, C416, C418), it is possible to obtain fluorescent mRNA detectable at green, orange and red wavelengths. As a proof of concept, we have produced eGFP mRNA modified with Andy Fluor 647-X-UTP, transfected it in HEK293 cells and detected the mRNA delivery in the cells as well as the eGFP expression by fluorescent microscopy.