Global proteomics for routine assessment of in vitro 3D liver models used for drug development

Introduction

3D spheroids of primary human hepatocytes (3D PHH) retain a differentiated phenotype with largely conserved metabolic function and proteomic fingerprint over weeks in culture. As a result, 3D PHH are gaining importance as a model for drug discovery. However, different protocols are applied and comprehensive characterization of 3D PHH phenotypes is typically missing in the studies. One such gap is the quantification of human absorption, distribution, metabolism, and excretion (ADME) proteins across diverse primary human donors and culture conditions. In this regard, global proteomics serve as an indispensable tool for the large scale characterization of ADME protein expression along with detailed description of 3D PHH phenotype.

Aim

To integrate global proteomics analysis in the routine phenotypical assessment of the 3D PHH cultures, which subsequently will improve drug development applicability and mechanistic understanding of these cultures.

Methods

The 3D PHH were cultured for one to three weeks in a chemically defined physiologically relevant medium. Applicability for the drug development studies was demonstrated by ADME protein expression pattern. In brief, bottom-up global proteomics analysis constituted of controlled digestion by LysC and trypsin, peptide separation on an EASY-spray C18-column, and analysis on an Orbitrap Q Exactive HF mass spectrometer operating in a data-dependent mode. Proteins were identified by searching MS and MS/MS data of peptides against a reference human proteome using MaxQuant software. ADMExtract, an in-house developed streamlined workflow for mining of ADME protein was used to summarize the expression pattern of ADME proteins

Results

We demonstrate that 3D PHH proteomes largely vary dependent on the culture conditions like cell culture medium or drug exposure, and these quintessential phenotypic differences are hard to detect with other basic functional culture assessment tests.^1,2 We further confirm quantitative gene expression analysis routinely applied in drug exposure studies does not provide a correct representation of expression of drug metabolizing enzymes and transporters.² To further integrate global proteomics analysis in drug development pipelines, we have optimized the analytical workflow for 3D PHH. We have demonstrated that the amount of initial material and sample preparation method directly affects the understanding of ADME proteins expression.³ Lastly, we have optimized a user-friendly in-house developed streamlined workflow for mining of ADME proteins and simplification of global proteomics analysis for phenotypic description of 3D PHH, that could be used by non-proteomics experts.

Conclusion

Here we demonstrate that global proteomics analysis is indispensable for understanding 3D PHH phenotypic features. It provides a comprehensive understanding of ADME proteins expression and directly reflects conclusions drawn from 3D PHH experiments performed in drug development.