Cordyceps militaris Induces Immunogenic Cell Death and Enhances Antitumor Immunogenic Response in Breast Cancer.

Xingguo Quan,Tae Hyun Kim,Anbok Lee,Beom Seok Kwak,Ji-Young Lee,Jin Hee Park,Saegwang Park

doi:10.1155/2020/9053274

Xingguo Quan, Tae Hyun Kim + Show 5 more

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https://doi.org/10.1155/2020/9053274

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Abstract

Cordyceps militaris has been widely used as a traditional medicine in East Asia. Its effects against breast cancer have been reported previously. However, whether C. militaris-induced breast cancer cell death is immunogenic remains unelucidated. This study aimed to determine whether ethanolic extracts of C. militaris (CM-EE) could induce immunogenic cell death (ICD) in breast cancer immunotherapy to improve the efficacy of immune checkpoint inhibitors. Human and mouse breast cancer cells were treated with various concentrations of CM-EE for 72 h, and cytotoxicity was measured using the sulforhodamine B assay. Flow cytometry was used to assess cell death with annexin V/7-AAD staining and measure the surface exposure of damage-associated molecular pattern (DAMP) molecules including calreticulin, HSP70, and HSP90. Western blot for cleaved poly (ADP-ribose) polymerase (PARP) was used to confirm apoptotic cell death. The immunogenicity of CM-EE-induced dead cells was evaluated using the CFSE dilution assay. CM-EE reduced the viability of human (MCF7, MDA-MB-231, HS578T, and SKBR3) and mouse (4T1-neu-HA, TUBO-HA, and TUBO-P2J-HA) breast cancer cells. The IC50 was 25–50 µg/ml in human breast cancer cells and 10–50 µg/ml in mouse breast cancer cells at 72 h. CM-EE-treated breast cancer cells were positively stained by annexin V, cleaved PARP, and cleaved caspase 3/7 which were increased upon CM-EE treatment. Surface exposure of DAMP molecules was increased in dose- and time-dependent manners. The CFSE dilution assay revealed that dendritic cells fed with CM-EE-treated breast cancer cells successfully stimulated tumor-specific T cell proliferation without inhibiting DC function and T cell proliferation. The expression of PD-L1 mRNA and protein level was increased in dose-dependent manners. In addition, CM-EE also potentiated the cytotoxic activity of tumor-specific T cells. CM-EE can induce immunogenic and apoptotic cell death in breast cancer cells, and it is a good candidate for cancer immunotherapy and may improve the efficacy of immune checkpoint inhibitors.

Highlights

Immune checkpoint inhibitors (ICIs), such as programmed cell death 1 (PD-1)/programmed cell death ligand 1 (PD-L1) and cytotoxic T-lymphocyte antigen-4 (CTLA-4) inhibitors, have shown promising clinical outcomes over the past several years
To investigate whether the cell death type induced by CM-EE is apoptosis or necrosis, Annexin V and 7-AAD staining were conducted. e cells were treated with different concentrations (MDA-MB-231 and MCF-7: 25 or 50 μg/ml, 4T1-neu-HA: 10 or 25 μg/ml, and TUBO-HA: 50 or 200 μg/ml) of CM-EE for 72 h and stained with APC-Annexin V and 7-AAD. e stained cells were analyzed by flow cytometry
In other human breast cells (MCF-7 cells and TUBO-HA cells), we found that, after CM treatment, the surface exposure of CRT, HSP70, and HSP90 gradually increased as the drug concentration increased compared with the untreated control (Supplementary Figures 1(a) and 1(b)). erefore, human and mouse breast cancer cells treated with CM-EE were exposed to Immunogenic cell death (ICD) markers on the cell surface

Summary

Introduction

Immune checkpoint inhibitors (ICIs), such as programmed cell death 1 (PD-1)/programmed cell death ligand 1 (PD-L1) and cytotoxic T-lymphocyte antigen-4 (CTLA-4) inhibitors, have shown promising clinical outcomes over the past several years. It exhibits antitumor activity by inhibiting tumor cells from evading immunosurveillance and reinvigorating antitumor immune response [1]. Ere is difference among the various subtypes of malignancies, and the response rate of ICIs alone for advanced breast cancer is low [3,4,5]. Several efforts have been made to overcome the low response rate of ICIs alone in patients with breast cancer. Tumor cells exposed to ICD inducers undergo apoptosis; simultaneously, damage-associated molecular patterns (DAMPs) such as calreticulin (CRT), adenosine triphosphate (ATP), high mobility group box 1 (HMGB1), and heat shock protein (HSP) and 90 are exposed or released on the cell surface [7, 8]. ese DAMPs activate dendritic cells (DCs) and stimulate the presentation of tumor antigens to T cells [9, 10], thereby inhibiting tumor cell growth

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