# Targeted and Untargeted Proteomics-based Comparison of Adenoviral Infected hCMEC/D3 and hBMEC as a Human Brain Endothelial Cells to Study the OATP2B1 Transporter

## TL;DR

This study compares two human brain endothelial cell lines to evaluate their suitability for studying the OATP2B1 transporter after adenoviral infection.

## Contribution

The study provides a proteomics-based comparison of hCMEC/D3 and hBMEC cell lines for modeling the blood-brain barrier and drug transport.

## Key findings

- hCMEC/D3 showed higher BBB marker expression compared to hBMEC.
- Adenoviral infection increased OATP2B1 levels in hBMEC more effectively than in hCMEC/D3.
- Untargeted proteomics confirmed OATP2B1 upregulation not detected by targeted methods.

## Abstract

The blood–brain barrier (BBB) is essential for central nervous system (CNS) homeostasis by regulating permeability between the bloodstream and brain. This study evaluates the immortalized human brain capillary endothelial cell lines hCMEC/D3 and hBMEC for their use as a brain endothelial cells to investigate the OATP2B1 transporter following adenoviral infection. We assessed the impact of adenoviral-mediated OATP2B1 expression on BBB marker proteins and transporters using targeted and untargeted mass spectrometry-based proteomics. Targeted proteomics identified measurable levels of endothelial markers PECAM1 and CDH5 in hCMEC/D3, whereas these markers were undetectable in hBMEC. Both cell lines exhibited similar Pgp levels, while BCRP was absent in hCMEC/D3. The expression of uptake transporters was also evaluated, revealing comparable levels of GLUT1, ENT1, MCT1 and OAT7 in hCMEC/D3 and hBMEC. Although OATP2B1 levels did not significantly increase post-infection in targeted proteomics, untargeted proteomics confirmed enhanced OATP2B1 expression. Other BBB markers and transporters remained unaffected in both cell lines. Notably, hCMEC/D3 demonstrated a stronger BBB phenotype, indicated by higher expression of BBB markers and transporters, while adenoviral infection was more effective in hBMEC. The differences between targeted and untargeted proteomics underscore the need for diverse methods to verify protein expression levels. This comparative analysis provides insights into the strengths and limitations of hCMEC/D3 and hBMEC for BBB research, particularly regarding drug transport mechanisms.

The online version contains supplementary material available at 10.1007/s12035-025-04807-7.