# Comparison of the procoagulant activity between extracellular vesicles obtained from cellular monolayers and spheroids

## TL;DR

This study compares how different cell culture methods affect the release of blood-clotting factors from cancer cells.

## Contribution

It shows that monolayer cultures release more extracellular vesicles and tissue factor than spheroid cultures.

## Key findings

- Monolayer cells released sevenfold more extracellular vesicles than spheroid cells.
- Monolayer cultures showed 11-fold higher tissue factor antigen release compared to spheroids.
- PAR2-AP activation increased EV and TF release in monolayers but not in most spheroids.

## Abstract

Tissue factor (TF) is the main activator of blood coagulation and is associated with thrombosis and tumor progression. It can be released into the blood circulation incorporated within cancer cell-derived extracellular vesicles (EVs). In this study, we investigated the influence of two-dimensional (monolayer) and three-dimensional (spheroid) tumor cell culture methods, and co-culture with cancer-associated fibroblasts (CAF), on the level of EVs release and the associated TF release and activity. The density of EVs and TF released from spheroids and monolayers of Hs578t human breast cancer and CAF were measured by the concentration of the phosphatidylserine and TF-ELISA. For some experiments, cells were activated using a protease-activated receptor (PAR)-2-activating peptide (PAR2-AP). The concentration and EV’s size were accessed by nanoparticle tracking analysis, and a clotting assay was used to evaluate TF pro-coagulant activity. Hs578t monolayers released sevenfold more EVs, and it was associated with an 11-fold higher TF antigen release than the spheroids cultures. Activation of the cells with a PAR2-AP resulted in a significant increase in the release of EVs and TF from the Hs578t monolayers, but no significant increase was observed in the spheroids, only from half Hs578t, half CAF spheroids. Taken together, our results demonstrate that monolayer cell cultures are capable of releasing more significant amounts of EVs and associated TF than spheroid cultures. Monolayers and spheroids have different behavior when we compare the release of EVs and TF. It is essential to consider it when choosing a cell model to study cancer-associated thrombosis.

Generation of extracellular vesicles (EVs) and release of tissue factor (TF), the initiator of coagulation. This was studied after growing cells in monolayers or more physiological spheroids. Monolayer culture cells were shown to release more EVs, and more TF, suggesting that EV and TF shedding using monolayer-based research is not representative of human pathologies such as cancer.