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

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

Attenuation of Experimental Colitis in Glutathione Peroxidase 1 and Catalase Double Knockout Mice through Enhancing Regulatory T Cell Function

<div><p>Reactive oxygen species (ROS) have been implicated in the progression of inflammatory diseases including inflammatory bowel diseases (IBD). Meanwhile, several studies suggested the protective role of ROS in immune-mediated inflammatory diseases, and it was recently reported that dextran sodium sulfate (DSS)-induced colitis was attenuated in mice with an elevated level of ROS due to deficiency of peroxiredoxin II. Regulatory T cells (Tregs) are critical in the prevention of IBD and Treg function was reported to be closely associated with ROS level, but it has been investigated only in lowered levels of ROS so far. In the present study, in order to clarify the relationship between ROS level and Treg function, and their role in the pathogenesis of IBD, we investigated mice with an elevated level of ROS due to deficiency of both glutathione peroxidase (GPx)-1 and catalase (Cat) for the susceptibility of DSS-induced colitis in association with Treg function. The results showed that DSS-induced colitis was attenuated and Tregs were hyperfunctional in GPx1^−/− × Cat^−/− mice. In vivo administration of N-acetylcysteine (NAC) aggravated DSS-induced colitis and decreased Treg function to the level comparable to WT mice. Attenuated Th₁₇ cell differentiation from naïve CD4⁺ cells as well as impaired production of IL-6 and IL-17A by splenocytes upon stimulation suggested anti-inflammatory tendency of GPx1^−/− × Cat^−/− mice. Suppression of Stat3 activation in association with enhancement of indoleamine 2,3-dioxygenase and FoxP3 expression might be involved in the immunosuppressive mechanism of GPx1^−/− × Cat^−/− mice. Taken together, it is implied that ROS level is critical in the regulation of Treg function, and IBD may be attenuated in appropriately elevated levels of ROS.</p></div

Intracellular ROS level was higher in GPx1^−/−× Cat^−/− than WT lymphocytes.

<p>Splenocytes were stained with DC-FDA (5 µM) and simultaneously with anti-CD4-PerCP and anti-CD25-PE (A) or anti-CD11c-PE (B). Then the cells were washed and acquired using FACSCalibur. After stabilization for 30 sec, acquisition was briefly suspended and PMA (100 nM) was added to the tube, and the cells were acquired further for 482 sec. ROS level was expressed as mean fluorescence intensity (MFI) of DC-FDA fluorescence. Data are mean ± SE (n = 12). *, P<0.05.</p

Th₁₇ cell differentiation was mitigated in KO mice.

<p>Naïve CD4⁺ cells isolated from the spleens of WT or KO mice were induced to differentiate into Th₁₇ cells by stimulating in the presence of TGF-β1 and IL-6. KO, GPx1^−/− × Cat^−/−. Data are mean ± SE of six separate experiments.</p

In vivo administration of NAC into KO mice reduced Treg function.

<p>Twenty four hours after the intraperitoneal injection of NAC (10 mg in PBS) or PBS (control), cells were prepared and analyzed for the suppressive function of Tregs in the same way as in Fig. 6. KO, GPx1^−/− × Cat^−/−. Data are mean ± SE of six separate experiments. <i>P</i> value indicates significance of difference between KO Treg and KO Treg-NAC.</p