# Comparative effects of a TLR7/8 agonist and doxorubicin on immune checkpoint modulation and apoptosis in 4T1 breast cancer cells

## TL;DR

This study compares how a TLR7/8 agonist and doxorubicin affect breast cancer cells, focusing on cell death and immune checkpoint gene expression.

## Contribution

The novel comparison of TLR7/8 agonist and doxorubicin effects on apoptosis and immune checkpoint modulation in 4T1 breast cancer cells.

## Key findings

- R848 and doxorubicin both reduced 4T1 cell viability and induced apoptosis.
- Doxorubicin significantly increased PD-L1 and PVR checkpoint gene expression.
- R848 induced early apoptosis without boosting immune checkpoint ligands.

## Abstract

Breast cancer is still one of the challenges of medical science, despite fast and remarkable advancements in diagnosis and new therapy approaches. This study aims to explore the individual effects of TLR7/8 agonists and to compare their cytotoxic properties with those of the chemotherapy drug doxorubicin on 4T1 breast cancer cells, as well as to evaluate their role in the modulation of immune checkpoint molecules.

4T1 cancer cells were treated with the TLR7/8 agonist R848 and doxorubicin for a duration of 48 h. First, the viability of the cells was assessed using the MTT method. The relative expression of mRNA for Gal-9, PD-L1, and PVR was analyzed with HPRT serving as a housekeeping gene. Finally, an apoptosis test was conducted to evaluate the cytotoxic effects of R848 and doxorubicin on 4T1 cells. And the wound healing assay was completed in order to assess the migratory potential of 4T1 cells after treatment with R848 and doxorubicin.

The expression of the PVR and Gal-9 genes in the group treated with R848 showed a decrease compared to the control group, although this change was not statistically significant. In contrast, the expression of PD-L1 and PVR in the group treated with doxorubicin increased significantly when compared to both the control group and the R848 treated group. Additionally, the total apoptosis rate in both the R848 and doxorubicin treatment groups was significantly higher than that in the control group. After 24 h post-scratch, control 4T1 cells showed about 50 % wound closure, while R848 reduced it to 35 %. Doxorubicin lowered migration to under 10 %. By 48 h, control and R848 nearly closed the wound, unlike the DOX group.

R848 and doxorubicin possess anti-proliferative and pro-apoptotic effects on 4T1 cells, doxorubicin effectively induces cell death and boosts immune checkpoint gene expression, while R848 fosters early apoptosis without raising checkpoint ligand expression.

•R848 (TLR7/8 agonist) and doxorubicin decreased the viability of 4T1 cells.•Doxorubicin greatly elevated the expression of PD-L1 and PVR checkpoint genes.•R848 induced early apoptosis without increasing immune checkpoint ligands.•R848 and doxorubicin agents induced apoptosis whereas doxorubicin revealed stronger late-apoptotic activities.

R848 (TLR7/8 agonist) and doxorubicin decreased the viability of 4T1 cells.

Doxorubicin greatly elevated the expression of PD-L1 and PVR checkpoint genes.

R848 induced early apoptosis without increasing immune checkpoint ligands.

R848 and doxorubicin agents induced apoptosis whereas doxorubicin revealed stronger late-apoptotic activities.