Introduction

Cancer immunotherapy, a cutting-edge approach, activates the anti-tumor immune responses to regulate tumor growth and progression1,2,3. This treatment is garnering increasing attention because of its capacity to selectively target particular tumors while minimizing systemic toxicity4. Several forms of cancer immunotherapy, including monoclonal antibodies, cancer vaccines, adoptive immune-cell immunotherapies, immune-checkpoint inhibitors, and chimeric antigen receptor (CAR-T) therapy, are approved by the US Food and Drug Administration (FDA)5. Yet, the effectiveness of cancer immunotherapy is constrained by the immunosuppressive nature and heterogeneity of the tumor microenvironment, leading to diminished treatment efficacy6,7. Hence, there is a pressing demand for efficient strategies to modulate this intricate immunosuppressive microenvironment and augment the potency of cancer immunotherapy.

Natural products, derived from living organisms, have long been used in traditional medicine for their therapeutic properties8. Combining them with cancer immunotherapy aims for maximal efficacy with minimal adverse effects. They remodel the tumor microenvironment, thwarting immune evasion5,9. Studies reveal their potential to bolster therapies like adoptive immune-cell therapy, cancer vaccines, and immune checkpoint inhibitors5,9,10. For example, a nano-drug pairing curcumin with an anti-PD-1 antibody demonstrated anti-tumor effects in colon cancer mice11. A polyvalent vaccine with a saponin-based immunoadjuvant showed moderate immunogenicity in epithelial ovarian cancer patients12. In a phase II study, combining Pembrolizumab (PD-1 blocker) and curcumin stabilized health-related quality of life in uterine and cervical cancer patients, correlating with increased peripheral T-cells in responders13. This combined approach improves cancer treatment effectiveness and enhances patient quality of life9.

Cordyceps militaris, a traditional medicine in East Asian countries like China, Japan, and Korea for over 3000 years14,15, showcases a diverse array of biological characteristics, delivering substantial health advantages such as anti-oxidative, anti-tumor, anti-inflammatory activities, and immunomodulatory properties16. Extracts from C. militaris is rich in active compounds, including cordycepin, cordycepic acid, adenosine, carotenoids, proteins, essential amino acids, minerals, vitamins, and polysaccharides, which underlie their medicinal attributes17,18. The immunomodulatory potential of C. militaris extracts has been extensively explored in vitro and in vivo models. For instance, the water extract of C. militaris (CME) enhanced ovalbumin (OVA) presentation through both MHC class I and class II pathways in dendritic cells (DCs)19. Oral administration of ethanol extract of C. militaris (CM) in immunocompromised mice led to significant increases in splenocyte proliferation, NK activity and the production of Th1 cytokines including IL-2, IL-12, IFN-γ and TNF-α, compared to immunosuppressed control mice17). Moreover, when cordycepin, the major bioactive constituent of C. militaris, was combined with Natural Killer (NK)-92 cells, a marked enhancement in cell death was observed in the cholangiocarcinoma (CCA) cell line, KKU-213A, compared to treatment with NK cells alone. This study underscored cordycepin’s ability to sensitize KKU-213A cells, prompting them to express TRAIL, thereby augmenting the effectiveness of NK-92 cells in inducing cell death20. Collectively, C. militaris extract and cordycepin demonstrate the capacity to enhance immune responses and sensitize cancer cells to immune cell cytotoxicity. These findings imply that C. militaris extract and cordycepin hold promise as agents to enhance the efficacy of cancer immunotherapy.

This study investigated the effects of cordycepin and C. militaris ethanolic extract (Cm-EE) on cancer sensitization and immunomodulation in breast cancer (BC) and hepatocellular carcinoma (HCC), the most prevalent cancers among females and males in Thailand, respectively, contributing to high global mortality rates. These cancers are notorious for their dismal prognoses, often attributed to drug resistance, frequent recurrence, and metastasis, all influenced by their immunosuppressive milieu 21,22. The enhancement of immunotherapy with natural products holds promise for the development of novel therapeutics for BC and HCC. Thus, we investigated the effects of cordycepin and Cm-EE on human effector immune cells’ ability to eliminate BC and HCC cells. Our findings reveal that cordycepin and Cm-EE can modify the expression of surface molecules such as NKG2D ligand, HLA-ABC, FasR, and DR4 in BC and HCC cells, suggesting their potential to sensitize cancer cells by altering surface molecules, thereby increasing susceptibility to immune cell-mediated killing. Moreover, cordycepin was found to induce the expression of NKG2D receptor and Trail on CD3-negative effector immune cells, particularly on natural killer (NK) cells, while Cm-EE triggered cytokine production, indicating their immunomodulatory effects. Co-culturing cancer cells with effector immune cells in the presence of cordycepin or Cm-EE resulted in increased cytotoxicity of effector immune cells against BC and HCC cell lines.

Results

Analysis of pentostatin, adenosine, and cordycepin in Cm-EE

Ultra-performance liquid chromatography (UPLC) was employed to identify the bioactive compounds, including pentostatin, adenosine and cordycepin, within Cm-EE. The UPLC chromatograms displaying the retention times of these bioactive substances in Cm-EE (Fig. 1b) were compared to standard references of pentostatin, adenosine, and cordycepin (Fig. 1a). The retention times of pentostatin, adenosine, and cordycepin in Cm-EE were determined to be 2.54, 3.70, and 4.67 min, respectively (Fig. 1b). Correspondingly, the retention times for the pentostatin standard, adenosine standard, and cordycepin standard were 2.60, 3.72, and 4.72 min, respectively (Fig. 1a). The quantities of pentostatin, adenosine and cordycepin were calculated relative to the standard references, revealing that Cm-EE contained 10.98 ± 1.80 mg of pentostatin, 9.58 ± 1.67 mg of adenosine, and 21.50 ± 2.31 mg of cordycepin per 1 gram of the extract, as summarized in Fig. 1c. Consequently, cordycepin was included in all experiments as a positive bioactive compound, given its status as the major component found in the Cm-EE.

Fig. 1
figure 1

Ultra-performance liquid chromatography (UPLC) chromatograms of standard compounds and C. militaris ethanolic extract (Cm-EE). (a) standard solutions of 25 µg/mL pentostatin, 20 µg/mL adenosine, and 25 µg/mL cordycepin, and (b) 1 mg/mL Cm-EE. (c) Identification of bioactive compounds in Cm-EE. Data are presented as the mean ± standard error of the mean (SEM) (N = 3).

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Evaluation of cordycepin and Cm-EE cytotoxicity via cell viability assay

The cytotoxic effects of cordycepin and Cm-EE on breast cancer cell lines (BC; MCF-7 and MDA-MB-231), hepatocellular carcinoma cell lines (HCC; Huh-7 and SNU-449) as well as immune cells (PBMCs and non-adherent cells), were assessed using PrestoBlue cell viability reagent. Cells were exposed to varying concentrations of standard cordycepin (0–1,000 µM) or Cm-EE (0–1,000 µg/mL) for 24 h. Subsequently, cell viability was measured and expressed as a percentage relative to untreated controls. As depicted in Fig. 2, revealed that both cordycepin and Cm-EE decreased cell viability in a dose-dependent manner. Cordycepin demonstrated notable cytotoxicity against Huh-7 cells (IC50 = 367.90 ± 4.44 µM), while proving to non-toxic to SNU-449 cells and non-adherent immune cells (IC50 > 1,000 µM). Notably, Huh-7 cells exhibited the highest sensitivity to Cm-EE, with an IC50 of 82.23 ± 3.31 µg/mL. Consequently, sub-lethal doses (100 µM cordycepin and 100 µg/mL Cm-EE) were selected for subsequent experiments involving cancer cells and immune cells.

Fig. 2
figure 2

Cell viability of cancer cell lines and immune cells following treatment with standard cordycepin, or C. militaris ethanolic extract (Cm-EE). Cells were exposed to cordycepin (0–1,000 µM) (a) or Cm-EE (0–1,000 µg/mL) (b) for 24 h. Cytotoxicity was assessed using PrestoBlue Cell Viability Reagent. The percentage of cell viability and inhibitory concentration (IC50) were analyzed using GraphPad Prism version 8 (N = 3).

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Impact of cordycepin and Cm-EE on sensitizing cancer cells

Previous research has indicated that cordycepin treatment can enhance the expression of death receptors and ligands in cancer cells20. To investigate these effects, the expression of ligands such as NKG2D ligand (MICA/B) and HLA-ABC, as well as death receptors including FasR and death receptor 4/5 (DR4 and DR5), on the cell surface of cancer cells was evaluated using flow cytometry following a 24-hour treatment with either 100 µM of cordycepin or different concentrations of Cm-EE (25, 50, 100 µg/mL). As shown in Fig. 3b to 3e, the results demonstrated a significant increase in the percentages of NKG2D ligand-positive cells in various cancer cell lines, including MCF-7 (24.70 ± 2.20%), MDA-MB-231 (49.97 ± 7.22%), Huh-7 (74.70 ± 5.81%), and SNU-449 (94.70± 0.71%), after treatment with Cm-EE at 100 µg/mL compared to the control group (set as 100%) (p < 0.01). Conversely, the proportion of HLA-ABC-positive cells notably decreased in MCF-7 (74.03 ± 2.63%), Huh-7 (82.40 ± 3.71%), and SNU-449 (81.40 ± 5.10%) after treatment with 100 µM of standard cordycepin compared to cells treated with the media control (p < 0.05) (Fig. 3f, 3h and 3i). Furthermore, FasR expression exhibited a dose-dependent increase in HCC cells, particularly Huh-7 and SNU-449, upon treatment with Cm-EE at 100 µg/mL, compared to the diluent control (p < 0.05) as depicted in Fig. 3l and 3m. The percentages of FasR-positive cells for Huh-7 and SNU-449 were 88.77 ± 2.44% and 47.50 ± 12.58%, respectively. Similarly, treatment with Cm-EE at 100 µg/mL significantly increased the expression of DR4 in Huh-7 (92.97 ± 4.14%) and SNU-449 (97.10 ± 1.59%) compared to the control group treated with a diluent (p < 0.01) as shown in Fig. 3p and 3q. The representative data showing the impact of cordycepin and Cm-EE on modulating NKG2D ligand, HLA-ABC, FasR, and DR4 expression are provided in Supplementary Fig. S1 online. Following treatment with cordycepin or Cm-EE, no change was observed in the expression of DR5 between the groups (Supplementary Fig. S2 online). In conclusion, these findings suggest that Cm-EE may enhance the expression of NKG2D ligand and death receptors (FasR and DR4) in cancer cells, while cordycepin appears to reduce the expression of HLA-ABC, potentially enhancing the cytotoxicity of effector immune cells against various cancer cell lines.

Fig. 3
figure 3

The impact of cordycepin (CD) and Cm-EE on modulating NKG2D ligand, HLA-ABC, FasR, and DR4 expression. Cancer cells were exposed with 100 µM cordycepin (CD) and Cm-EE (25, 50, and 100 µg/mL) for 24 h. Gating strategy was used to identify cancer cell population based on forward scatter (FSC) and side scatter (SSC) properties. These cell populations were further gated by using specific fluorescence dye-conjugated monoclonal antibodies (a). The percentages of NKG2D ligand (b-e) and HLA-ABC (f-i), and death receptor expression such as FasR (j-m), and DR4 (n-q) were analyzed by flow cytometry. Data are presented as the mean ± SEM. Statistically significant values (*p < 0.05, **p < 0.01, and *** p < 0.001) were analyzed by one-way ANOVA (N = 3).

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Impact of cordycepin and Cm-EE on NKG2D receptor and Trail expression in effector immune cells

The interaction between NKG2D ligand or death receptor on cancer cells and immunoreceptors like NKG2D or Trail on effector immune cells can potentially trigger the cytolytic function of these immune cells, leading to the elimination of cancerous cells. Therefore, we investigated the expression levels of NKG2D receptor and Trail on effector immune cells (both CD3 positive (CD3+) and CD3 negative (CD3-) cells) following treatment with either 100 µM cordycepin or 25, 50, and 100 µg/mL of Cm-EE using flow cytometry. The results indicated that the percentages of positive cells for KIRs and FasL in both CD3 + and CD3- effector immune cells did not exhibit significant differences between the untreated control group and the groups treated with cordycepin or Cm-EE (Supplementary Fig. S3 online). However, cordycepin treatment significantly increased the expression levels or mean fluorescence intensity (MFI) of NKG2D (1,496 ± 38.93) (Fig. 4b) and Trail (1,290 ± 62.22) (Fig. 4f) on CD3- effector immune cells (p < 0.05), as well as the percentages of NKG2D + CD3- (74.76 ± 2.84%) (Fig. 4c) and Trail + CD3- cells (89.14 ± 1.04%) (Fig. 4g) compared to the control group (p < 0.05). In contrast, treatment with Cm-EE did not alter the expressions of NKG2D immunoreceptors and Trail on effector immune cells. These findings suggest that cordycepin may exert immunomodulatory effects by influencing the expression of the NKG2D receptor and Trail on CD3-effector immune cells specifically on natural killer (NK) cells.

Fig. 4
figure 4

Changes of cell surface expression levels of NKG2D receptor and Trail on effector immune cells following treatment with cordycepin (CD) and Cm-EE. Non-adherent cells were exposed to 100 µM cordycepin (CD) or 25, 50, and 100 µg/mL Cm-EE for 24 h. Gating strategy was used to identify non-adherent cell population based on forward scatter (FSC) and side scatter (SSC) properties. These cell populations were further gated by using specific fluorescence dye-conjugated monoclonal antibodies (a). Analysis of mean fluorescence intensity (b, d, f, and h) and percentages (c, e, g, and i) of NKG2D receptor and Trail expression levels on effector immune cells (CD3 + and CD3- cells) was conducted using flow cytometry. Data are presented as the mean ± SEM. Statistically significant values (*p < 0.05 and **p < 0.01) were determined by one-way ANOVA (N = 3).

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Impact of cordycepin and Cm-EE on cytokine production from effector immune cells

Previously, cordycepin and cordyceps extracts have demonstrated the ability to modulate cytokine production17. In this study, the production of cytokines and mediators in the culture supernatant following treatment of effector immune cells with cordycepin (100 µM) and Cm-EE (100 µg/mL) was assessed using Cytokine Bead Array (CBA) (Fig. 5). The results indicated that Cm-EE significantly increased the fold changes in the production of IL-2 (2.033 ± 0.42), IL-6 (19.11 ± 5.46), and IL-10 (16.51 ± 6.86) compared to the diluent control group (p < 0.05). Notably, the level of TNF-α production after treatment with Cm-EE (2.010 ± 0.49) was significantly higher than the cordycepin treatment condition (0.8833 ± 0.077) (p < 0.05) (Fig. 5a and Supplement Table S1 online). However, the cytotoxicity mediators (Fig. 5b and Supplement Table S2 online), including soluble Fas, soluble FasL, granzyme A and B (GrA and GrB), perforin, and granulysin, which play a crucial role in the cytotoxicity of T and NK cells, did not exhibit significant changes compared to the control group (p > 0.05). Furthermore, the subsets of immune cells including CD4 (CD3 + CD4+), CD8 (CD3 + CD8+), NK(CD3-CD56+) and B (CD3-CD19+) cells after treatment with cordycepin (100 µM) or Cm-EE (100 µg/mL) for 24 h were determined by flow cytometry using cell surface staining. As shown in Fig. 5c and Supplementary Fig. S4 online, the results revealed no significant differences in the percentages of CD4, CD8, NK, and B cells between the groups treated with cordycepin or Cm-EE and the untreated control group. These findings suggest that Cm-EE, but not cordycepin, exhibited an immunomodulatory effect by influencing the production of both inflammatory and anti-inflammatory cytokines.

Fig. 5
figure 5

Changes in cytokines, cytotoxic mediators, and subpopulations of effector immune cells following treatment with cordycepin (CD) and Cm-EE. Non-adherent cells were exposed to 100 µM of cordycepin (CD) or 100 µg/mL Cm-EE for 24 h. Culture supernatants were collected for determination of cytokine and cytotoxic mediator production by using cytokine bead array. The fold changes in cytokine (a) and cytotoxic mediator (b) production from the cordycepin or Cm-EE treatments were compared to the appropriate control. Subpopulations of treated immune cells (c), including CD4 (CD3 + and CD4+), CD8 (CD3 + and CD8+), NK cells (CD3- and CD56+), and B cells (CD3- and CD19+) were characterized using specific fluorescence dye-conjugated monoclonal antibodies. Data are presented as the mean ± SEM. Statistically significant values (*p < 0.05) were determined by Student’s t-test (N = 3).

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Enhanced cytotoxicity of immune cells against BC and HCC with cordycepin or Cm-EE pretreatment and combination treatments

Cordycepin and Cm-EE possess the capability to modulate both cancer cells and effector immune cells, thereby enhancing the vulnerability of cancer cells to immune-mediated eradication. Thus, we investigated the effect of cordycepin and Cm-EE on the cytotoxicity of effector immune cells against various cancer cell lines (MCF-7, MDA-MB-231, Huh-7, and SNU-449) using a killing assay with crystal violet staining. The treatment conditions were categorized into four groups as follows: (1) effector immune cells alone co-culture with target cells (at the E: T ratio 20:1), (2) pretreated effector immune cells with 100 µM of cordycepin or 100 µg/mL of Cm-EE before coculturing with target cells, (3) pretreated cancer cells with 100 µM of cordycepin or 100 µg/mL of Cm-EE before coculturing with effector immune cells, (4) combination treatment with 100 µM of cordycepin or 100 µg/mL of Cm-EE, effector immune cells, and target cells. After 24 h of incubation, the remaining target cells were assessed for percentages of cell viability using crystal violet staining.

As depicted in Fig. 6a, the percentages of cell viability of MCF-7 cells significantly decreased in response to pretreatment of cancer cell with cordycepin (61.27 ± 4.14%) and combination treatment with cordycepin (58.50 ± 4.14) compared to the medium control (100%) (p < 0.001). Notably, the cell viability of MCF-7 cells in the combination treatment with cordycepin was significantly reduced compared to immune cell alone (89.42 ± 2.03%) or pretreatment of immune cell with cordycepin (87.78 ± 6.14%) (p < 0.05). Similarly, the cell viability of MCF-7 cells significantly decreased in response to pretreatment of effector immune cells with Cm-EE (35.80 ± 4.40%), and combination treatment with Cm-EE (41.62 ± 3.55%) compared to treatment with immune cells alone (89.42 ± 2.03%) (p < 0.001) as shown in Fig. 6b. For MDA-MB-231 cells, the cell viability of cancer cells significantly decreased in response to pretreatment of effector immune cells with cordycepin (77.89 ± 2.92%) or Cm-EE (72.81 ± 3.43%), pretreatment of cancer cells with cordycepin (73.00 ± 1.33%) or Cm-EE (69.17 ± 4.61%), and combined treatment with cordycepin (76.17 ± 1.36%) compared to treatment with immune cells alone (95.20 ± 3.70%) (p < 0.01) as shown in Fig. 6c and 6d.

Similar to the findings observed in MCF-7 cells, the viability of Huh-7 cells notably decreased in response to combination treatment with cordycepin (27.66 ± 0.78%) or Cm-EE (21.92 ± 0.85%), showing a significant reduction compared to the viability of immune cell alone (86.52 ± 0.41%), pretreatment of Huh-7 cells with either cordycepin (48.28 ± 0.15%) or Cm-EE (28.87 ± 2.15%), as well as immune cells pretreated with cordycepin (81.87 ± 1.61%) or Cm-EE (60.32 ± 2.16%) (p < 0.01) as shown in Fig. 6e and 6f. Moreover, the cell viability of SNU-449 significantly decreased in response to pretreatment of immune cells with cordycepin (82.83 ± 3.01%) or Cm-EE (84.08 ± 3.04%), pretreatment of cancer cells with cordycepin (66.48 ± 0.83%) or Cm-EE (86.59 ± 2.81%), and combination treatment with cordycepin (73.79 ± 6.15%) or Cm-EE (79.92 ± 2.58%), compared to treatment with immune cells alone (117.0 ± 3.68%) (p < 0.01) as shown in Fig. 6g and 6h. In summary, cordycepin and Cm-EE possess the potential to heighten the susceptibility of breast cancer and hepatocellular carcinoma cells to immune cell-mediated destruction. Furthermore, they can influence the activity of immune cells, ultimately augmenting the cytotoxicity of effector immune cells through pretreatment and combined therapeutic approaches.

Fig. 6
figure 6

The impact of cordycepin and Cm-EE on the cytotoxicity of effector immune cells against various cancer cell lines. Coculture experiments were conducted under four conditions: (1) immune cell alone, (2) immune cells pretreated with 100 µM cordycepin or 100 µg/mL Cm-EE, (3) cancer cells pretreated with 100 µM cordycepin or 100 µg/mL Cm-EE, and (4) combination treatment of effector immune cells with 100 µM cordycepin or 100 µg/mL Cm-EE for 24 h. The remaining cancer cells including MCF-7 (a and b), MDA-MB-231 (c and d), Huh-7 (e and f), and SNU-449 (g and h) were assessed by crystal violet staining. Data are presented as the mean ± SEM. Statistically significant values (*p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001) were compared with the media or diluent group and analyzed by one-way ANOVA. Statistically significant values (#p < 0.05, ##p < 0.01, ###p < 0.001, and ####p < 0.0001) were compared with the immune cell alone condition and analyzed by one-way ANOVA (N = 3).

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Enhanced cytotoxicity of effector cells against mCherry red fluorescence protein-expressing cancer cells via combination treatment with cordycepin or Cm-EE

To ascertain the influence of cordycepin or Cm-EE on the cytotoxicity of effector immune cells, a co-culture experiment was conducted employing MCF-7, MDA-MB-231, Huh-7, and SNU-449 cell lines, all of which express mCherry red fluorescent protein. Cancer cells expressing mCherry red fluorescent protein were co-cultured with effector immune cells at the E:T ratio 20:1, while subjected to a combination treatment of either 100 µM cordycepin or 100 µg/mL Cm-EE for 24 h. Subsequently, the surviving cells were examined using a fluorescent microscope. As depicted in Fig. 7a, the results revealed a notable reduction in red fluorescent protein-expressing cancer cells (MCF-7, MDA-MB-231, Huh-7, and SNU-449 cell lines) following the combination treatment of cordycepin or Cm-EE compared to immune cell treatment alone. The percentages of cell viability of MCF-7, MDA-MB-231, Huh-7, and SNU-449 cell lines after cordycepin combination treatment were 25.59 ± 2.56 (p < 0.05), 37.74 ± 3.52 (p < 0.01), 9.020 ± 3.04 (p < 0.001), and 75.80 ± 8.22, respectively. For Cm-EE combination treatment, the percentages of cell viability of MCF-7, MDA-MB-231, Huh-7, and SNU-449 cell lines were 9.34 ± 1.72 (p < 0.01), 56.26 ± 7.14 (p < 0.05), 13.62 ± 5.31(p < 0.001), and 43.94 ± 11.50 (p < 0.05), respectively. In comparison, the percentages of cell viability for MCF-7, MDA-MB-231, Huh-7, and SNU-449 cell lines after co-culture with effector immune cells alone were 56.80 ± 10.62, 79.98 ± 1.71, 73.08 ± 11.88, and 87.21 ± 11.42, respectively (Fig. 7b). These findings underscore the ability of cordycepin and Cm-EE to enhance the cytotoxicity of effector immune cells against breast cancer (BC) and hepatocellular carcinoma (HCC) cells expressing mCherry red fluorescent protein.

Fig. 7
figure 7

The impact of cordycepin and Cm-EE on the cytotoxicity of effector immune cells against various cancer cell lines. Red-fluorescent protein-expressing MCF-7, MDA-MB-231, Huh-7, and SNU-449 cell lines were co-culture with effector immune cells at an effector (E) to target (T) ratio of 20:1 in the presence of 100 µM cordycepin or 100 µg/mL Cm-EE for 24 h. The remaining cancer cells were visualized under a fluorescence microscope (a). Data are presented as the mean ± SEM (b). Statistically significant values (**p < 0.01, and ****p < 0.0001) were compared with the medium control group and analyzed by one-way ANOVA. Statistically significant values ($$$p < 0.001, and $$$$p < 0.0001) were compared with the diluent control group and analyzed by one-way ANOVA. Statistically significant values (#p < 0.05, ##p < 0.01, and ####p < 0.0001) were compared with the immune cell alone condition and analyzed by one-way ANOVA (N = 3).

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Discussion

Recent advancements in cancer therapy, particularly in immunotherapy, continue to revolutionize treatment approaches. Understanding the complex interactions between the immune system and cancer cells has led to the development of novel therapeutic strategies, including combinations of anticancer and immunomodulatory compounds. Natural compounds have emerged as promising and safe options for cancer therapy, offering notable antitumor effects and immunomodulatory properties capable of reversing cancer progression. C. militaris, a medicinal mushroom with a long-standing history of traditional medicinal applications, has been extensively researched and documented for its biological and therapeutic properties23. Cordycepin, a key component found in C. militaris, has received significant research focus due to its recognized medicinal benefits and potential as a nutraceutical24. This investigation assessed the ex vivo effects of cordycepin and the C. militaris ethanolic extract (Cm-EE) on cancer sensitization and immunomodulation, with the aim of enhancing the cytotoxicity of effector immune cells against diverse cancer cell lines.

We utilized UPLC to identify bioactive compounds, namely cordycepin, adenosine, and pentostatin, within the Cm-EE. The results revealed higher levels of cordycepin (21.50 ± 2.31 mg/g extract) compared to pentostatin (10.98 ± 1.80 mg/g extract) and adenosine (9.58 ± 1.97 mg/g extract), as depicted in Fig. 1. Previous studies using HPLC analysis of ethanolic extract from C. militaris fruiting bodies cultivated in Chiang Mai, Thailand, reported cordycepin content of 32.08 ± 0.10 mg/g extract and adenosine content of 3.78 ± 0.05 mg/g extract. The aqueous extract exhibited cordycepin content of 23.98 ± 0.39 mg/g extract and adenosine content of 2.86 ± 0.01 mg/g extract25. Furthermore, methanol extracts of C. militaris fruiting bodies contained cordycepin and adenosine at levels of 2.65 ± 0.02 and 2.45 ± 0.03 mg/g, respectively26. These findings suggest that varying quantities of these compounds may result from factors such as the solvent ratio, temperature, duration, and frequency of extraction. Notably, cordycepin and adenosine have been investigated for their potential health benefits, including immunomodulatory and anti-cancer effects27,28. Pentostatin has been shown to effectively inhibit the adenosine deaminase (ADA) enzyme, thus preventing the conversion of cordycepin to 3’-deoxyinosine29. Co-administration of cordycepin with pentostatin maintained cordycepin levels in rat plasma30, suggesting pentostatin’s role in stabilizing cordycepin in both the samples and Cm-EE.

The anti-tumor activity of natural killer (NK) cells is governed by the delicate balance between activating and inhibitory signals. The NKG2D ligand, also referred to as MICA/B, engages with the immunoreceptor NKG2D, crucially bolstering natural killer (NK) cell-mediated cytolytic activity against cancer cells. Primarily expressed in liver, lung, breast, and colon cancers, this NKG2D ligand plays a pivotal role in promoting NK cell responses. Retinoic acid serves as a regulator of MICA/B expression on human hepatocellular carcinoma, facilitating the elimination of cancer cells by NK cells via the MICA/B-NKG2D interaction31. Receptors such as killer cell immunoglobulin-like receptors (KIR) and lectin-like NKG2A recognize MHC class I (ABC), triggering inhibitory signals in NK cells32. Additionally, numerous studies have demonstrated that many cancer cells develop resistance to Fas/Trail-mediated apoptosis by downregulating the expression of FasR/FasL, Trail1/DR4, and Trail2/DR533. Consequently, targeting this resistance by modulating the expression of these proteins has been proposed to enhance NK function. Various natural compounds such as curcumin or phytochemicals like 7R-acetylmelodorinol have been identified for their ability to sensitize cancer cells by restoring the expression of these death ligands and receptors, ultimately inducing apoptosis in cancer cells34,35. The study conducted by Panwong  et al. (2021) reported that cordycepin could enhance NK-92 cytotoxicity, resulting in the elimination of CCA cells20. Similarly, our study demonstrated that Cm-EE increased the expression of NKG2D ligand on the cell surface of MCF-7, MDA-MB-231, Huh-7, and SNU-449 cell lines, as depicted in Fig. 3. Additionally, Cm-EE specifically upregulated the expression of FasR and DR4 on the cell surface of Huh-7 and SNU-449 cells. Conversely, cordycepin decreased the expression of HLA-ABC, thereby collectively amplifying the cytotoxicity of effector immune cells against MCF-7, Huh-7 and SNU-449 cells.

The upregulated expression of NKG2D ligand on cancer cells can be influenced by various stressors, including DNA damage, heat shock, and oxidative stress32. Transcriptional mechanisms regulating the expression of death receptors contribute to the activation of signaling pathways such as MEK/ERK/AP-1 or NF-κB36,37. These findings suggest that Cm-EE may play a potential role in sensitizing cancer cells through these mechanisms. Moreover, cordycepin has been shown to decrease the expression of HLA-ABC on cancer cells through epigenetic modifications or transcriptional regulation38. However, the heterogeneity of cancer cell types can influence the response to cordycepin and Cm-EE. Overall, cordycepin and Cm-EE have the potential to enhance the sensitivity of cancer cells to immune cell killing by modulating the expression of NKG2D ligand, HLA-ABC, as well as death receptors such as FasR and DR4.

The immunomodulation properties of Cm-EE and cordycepin, influencing the anti-tumor activity of effector immune cells, are critical and may enhance NK function. To evaluate this, we examined the expression of immune receptors (NKG2D and KIRs) and death ligands (FasL and Trail) on the surface of effector immune cells, including CD3-positive cells (CD4 + and CD8+) and CD3-negative cells (NK and B cells), following treatment with cordycepin or Cm-EE using flow cytometric analysis. As depicted in Fig. 4, treatment with 100 µM of cordycepin for 24 h significantly increased the mean fluorescence intensity (MFI) and percentages of NKG2D + and Trail + on CD3-negative cells, specifically NK cells. compared to controls (p < 0.05), suggesting engagement with their respective ligands or receptors for immune cell-mediated cancer cell killing. Based on our results, particularly the increase in NKG2D expression, future research should focus on NK cells, as their activity appears to be enhanced by cordycepin treatment. To better understand, long-term and in vivo studies are needed to evaluate how cordycepin and Cm-EE affect NK cell function and cancer progression, including their impact on NK cell-mediated tumor control and overall survival.

Exposure of effector immune cells to 100 µg/mL of Cm-EE for 24 h significantly augmented the production of IL-2, IL-6, and IL-10 cytokines compared to controls, as shown in Fig. 5a, indicating Cm-EE promotion of cytokine production. Conversely, levels of cytotoxicity mediators such as sFas, sFasL, GrA, GrB, perforin, and granulysin did not exhibit significant changes compared to controls, as demonstrated in Fig. 5b. This underscores a significant finding: the cytotoxicity of effector immune cells may not rely solely on cytotoxic mediators but rather be regulated through indirect mediators such as cytokines. IL-2, a cytokine promoting proliferation and activation of T cells, B-cells, and NK cells, stimulates the activation of cytotoxic lymphocytes. Additionally, IL-6 plays crucial roles in inflammation, while IL-10, an anti-inflammatory cytokine, contributes to maintaining a balanced inflammatory response39. The induction of these cytokines by Cm-EE collectively reflects its modulation of immune responses. These findings suggest that both cordycepin and Cm-EE possess potent immunomodulatory effects, albeit likely through different pathways.

Pretreating both effector immune cells and cancer cells with cordycepin or Cm-EE, followed by co-culture, resulted in increased cytotoxicity of effector immune cells, as depicted in Figs. 6 and 7. This enhancement was notably evident compared to conditions where only effector immune cells were utilized. The increase was more pronounced in Huh-7 cells, particularly in pretreated with Cm-EE and under combination treatment conditions (Fig. 6f). The strong effect of Cm-EE, with an IC50 of 82.23 ± 3.31 µg/mL (Fig. 2), suggests that the observed effects are primarily attributed to Cm-EE treatment. It was a significant difference between the pretreated and combination groups, which might be due to the high sensitivity of Huh-7 cells to the extracts. The genetic background and characteristics of Huh-7 cells may contribute to this sensitivity. Huh-7 cells are well-differentiated hepatocyte-derived carcinoma cells that contain a point mutation (Y220C) in the DNA-binding domain of the p53 protein. This mutation results in an abnormally stable p53 protein with impaired transcriptional activity40, A previous study found that the small compound tetrahydroisoquinoline (KG13) selectively interacts with the cysteine in p53 mutant (Y220C) resulting restoring wild-type thermal stability and gene activation in Huh-7 cells. The interaction leads to significant tumor regression and cell death41. Likewise, we hypothesize that cordycepin and Cm-EE may bind the mutated p53, potentially increasing the sensitivity of Huh-7 cells to treatment.

Taken together, these findings underscore the potential of both cordycepin and Cm-EE to augment the immune system’s ability to target and eliminate cancer cells, presenting a promising avenue to enhance the efficacy of cancer immunotherapy against various cancer cell lines, including breast cancer and hepatocellular carcinoma. The mechanism underlying the heightened cytotoxicity of effector immune cells may be attributed to the sensitization of cancer cells, rendering them more susceptible to killing by effector immune cells, as well as the immunomodulatory effects of cordycepin or Cm-EE. Cordycepin emerged as the primary bioactive compound in Cm-EE, demonstrating immunomodulation activity that enhances NK cytotoxicity. Nevertheless, we cannot overlook the potential contributions of other substances in Cm-EE. Alongside cordycepin, UPLC analysis identified adenosine and pentostatin. Adenosine has been previously implicated in immunomodulation, impacting the maturation, migration, and effector functions of NK cells42. Additionally, pentostatin, found in Cm-EE, holds promise in preventing cordycepin degradation by adenosine deaminase, potentially prolonging cordycepin’s half-life in Cordyceps crude extract. Consequently, the crude extract may offer advantages over individual synthetic compounds. Notably, quantitative analysis of bioactive compounds in crude extract could serve as quality control, mitigating lot-to-lot variation risks in mass production. Collectively, our findings highlight the potential of Cm-EE as an alternative option for treating breast cancer (BC) and hepatocellular carcinoma (HCC).

Conclusion

We present findings on the cancer sensitization and immunomodulatory effects of cordycepin and the C. militaris ethanolic extract (Cm-EE) in breast cancer (BC) and hepatocellular carcinoma (HCC) models. Our experiments revealed that cordycepin and Cm-EE modulate the expression of NKG2D ligand, HLA-ABC, and death receptors (FasR and DR4) in BC and HCC cell lines. Cordycepin exhibited an immunomodulatory effect by upregulating the expression of NKG2D and Trail in CD3-negative cells especially NK cells, while Cm-EE induced cytokine production (IL-2, IL-6, and IL-10). Furthermore, under combination treatment conditions, cordycepin and Cm-EE enhanced the cytotoxicity of effector immune cells against BC and HCC cells. These findings underscore the potential of cordycepin and Cm-EE as promising sensitizers and immunomodulators, warranting further investigation and development for potential combination with cellular immunotherapy to manage tumor development and progression. Nevertheless, comprehensive in vivo studies and clinical trials are necessary to confirm their effectiveness and safety, as well as to better understand the interactions between immune cells and between immune cells and tumor cells.

Materials and methods

Cell cultures

Human cancer cell lines (MCF-7 and MDA-MB-231 for breast cancer, and Huh-7, and SNU-449 for hepatocellular carcinoma) were sourced from the American Type Culture Collection (ATCC, Manassas, VA, USA). These cells were maintained in a complete medium comprising DMEM/F-12 (Gibco, Thermo Fisher Scientific, Inc., MA, USA), supplemented with 10% fetal bovine serum (FBS; Gibco) and 1x antibiotic-antimycotic (Gibco), and cultured at 37 °C in a humidified atmosphere with 5% CO2. Upon reaching 80–90% confluency, sub-culturing was carried out using the standard trypsinization method.

Ethical approval

The study was conducted under the Declaration of Helsinki, and the project proposal and protocol for collecting blood samples from healthy volunteers was approved by the Human Ethics Committee of the University of Phayao, Phayao, Thailand, all volunteers provided written informed consent. The project identification code is HREC-UP-HSST 1.3/013/67.

Peripheral blood mononuclear cell (PBMC) isolation

Following the manufacturer’s guidelines, peripheral blood mononuclear cells (PBMCs) from healthy donors were isolated through gradient density centrifugation using Lymphocyte Separation Medium (LSM) (Corning, MA, USA). Initially, 20 mL of heparinized blood was obtained, mixed with 20 mL of sterile 1xPBS, and gently blended. The diluted blood was then cautiously layered onto LSM and centrifuged at 500 ×g at 20 °C for 20 min. Subsequently, PBMCs were harvested and treated with red blood cell (RBC) lysis buffer to eliminate any contaminating RBCs. After centrifugation at 500 ×g at 20 °C for 10 min, the supernatant was removed. The PBMC pellet underwent washing with serum-free AIM V medium (Gibco), followed by centrifugation at 500 × g, 20 °C for 10 min, with subsequent removal of the supernatant. PBMCs were resuspended in serum-free AIM V medium and plated in a 6-well plate for 2 h at 37 °C. The resulting non-adherent cells, representing effector immune cells, were then collected and frozen until needed.

Preparation of C. militaris ethanolic extract (Cm-EE)

To obtain the C. militaris ethanolic extract (Cm-EE), fifty grams of dried fruiting bodies of C. militaris (AAVA Group Co., Ltd., Bangkok, Thailand) were combined with 500 mL of 95% ethanol, maintaining a solid-liquid ratio of 1:10 (weight/volume; w/v). The mixture was then allowed to incubate at room temperature for 72 h. Following incubation, the supernatant was separated and filtered using Whatman filter paper no. 1 via vacuum filtration. Subsequently, the ethanol solvent was removed, and the solution was freeze-dried with a rotary evaporator. The Cm-EE was stored at -20 °C.

Ultra-performance liquid chromatography (UPLC)

The concentrations of cordycepin, adenosine and pentostatin present in Cm-EE were determined utilizing the Accela™ UPLC system (Thermo Fisher Scientific, USA). Standard samples, consisting of 25 µg/mL cordycepin, 20 µg/mL adenosine, and 25 µg/mL pentostatin (all from Sigma-Aldrich, Saint Louis, USA), along with 1 mg/ mL Cm-EE, were filtrated using a 0.45 µM membrane filter and then injected into the UPLC system for a duration of 10 min. Chromatographic analysis was conducted using Hypersil™ BDS C18 Columns (150 mm × 4.6 mm ID, 5 μm particle size) with a mobile phase composed of acetonitrile, ethanol, and 10 mM ammonium acetate in water (pH 7.6, in a ratio of 5:5:90), at a flow rate of 1.0 mL per minute at 30 °C. Detection was performed using a UV photodiode array detector at wavelengths of 256 nm (for cordycepin and adenosine) and 280 nm (for pentostatin). The bioactive compound profiles present in the Cm-EE were analyzed and compared to available standards.

Cell viability assay

Cancer cells (5 × 103 cells/well) and immune cells (1 × 105 cells/well) were seeded into a 96-well plate and allowed to adhere overnight. Subsequently, cells were exposed to varying concentrations of cordycepin (0–1,000 µM) or Cm-EE (0–1,000 µg/mL) and then incubated at 37 °C in a humidified atmosphere with 5% CO2 for 24 h. Following the treatment period, PrestoBlue cell viability reagent (Thermo Fisher Scientific) was introduced into each well and further incubated at 37 °C in a humidified atmosphere with 5% CO2 for 4–16 h. The spectrophotometer measured absorbance at 570 nm and 600 nm (as a reference wavelength). The half-maximal inhibitory concentration (IC50) was determined using GraphPad Prism software 8. Cell viability percentage was calculated using the formula: % Cell viability = [(OD570-OD600) treated cells/(OD570-OD600) untreated cells] × 100.

Cell surface staining assay

Human cancer cell lines were seeded at a density of 1 × 105 cells/well in a 12-well plate and subsequently exposed to cordycepin (100 µM) or Cm-EE (25, 50, 100 µg/mL) for 24 h. Following treatment, cells were collected and stained with monoclonal fluorescent-conjugated antibodies: anti‑DR4‑PE (Clone: DJR1), anti‑DR5‑PE (Clone: DJR2-4(7–8)), anti‑FasR‑APC (Clone: DX2), anti‑MHC class I polypeptide‑related sequence (MIC)‑A/B‑FITC (Clone: 6D4), and anti‑human leukocyte antigen (HLA)‑ABC‑FITC (Clone: W6/32) (all sourced from Thermo Fisher Scientific, Inc.) for 30 min.

Non-adherent effector immune cells were plated at a concentration of 1 × 106 cells/well in a 12-well plate and treated with cordycepin (100 µM) or Cm-EE (25, 50, 100 µg/mL) for 24 h. Subsequently, cells were collected and stained with monoclonal fluorescent-conjugated antibodies, including anti-CD3 FITC (Clone: UCHT-1), anti-NKG2D APC (Clone: UCHT-4), anti-KIRs PerCP/Cy5.5 (Clone: LNK16), anti-Trail PE/Cy7 (Clone: RIK-2), and anti-FasL (Clone: NOK-1) (all from Thermo Fisher Scientific, Inc.) for 30 min. Cells were then fixed with 1% formaldehyde in 1x phosphate-buffered saline (PBS) containing 2% FBS. Stained cells were analyzed using a CytoFLEX S Flow Cytometer (BD Biosciences, NJ, USA). The expression of cell surface molecules and percentages of positive cells were determined using FlowJo software version 10 (BD Biosciences, CA, USA).

Cytokine production

Non-adherent immune cells were seeded at a density of 1 × 106 cells/well in a 12-well plate and then exposed to either 100 µM cordycepin or 100 µg/mL Cm-EE for 24 h. Following the incubation period, cell culture supernatants from each treatment condition were collected and analyzed using the LEGENDplex™ Human CD8/NK cell panel Cytokine Bead Array (BioLegend, San Diego, CA, USA). The assay was conducted according to the manufacturer’s instructions, allowing for the simultaneous evaluation of thirteen human cytokines and proteins, including IL-2, IL-4, IL-6, IL-10, IL-17 A, IFN-γ, TNF-α, soluble Fas (sFas), soluble FasL (sFasL), granzyme A (GrA), granzyme B (GrB), perforin, and granulysin.

Immune cell population analysis

The non-adherent cells were seeded at a density of 1 × 106 cells/well in 12-well plate and treated with either 100 µM cordycepin or 100 µg/mL Cm-EE for 24 h. Following treatment, cells were harvested and stained with monoclonal fluorescent-conjugated antibodies, including anti-CD3 FITC (Clone: UCHT-1), anti-CD4 APC (Clone: OKT-4), anti-CD8 APC (Clone: UCHT-4), anti-CD16 APC (Clone: LNK16), anti-CD19 APC (Clone: LT19) (all sourced from Immunotools, Friesoythe, Germany), and anti-CD56 PE (Clone: HCD56; BioLegend). Stained cells were subsequently fixed with 1% formaldehyde in 1x phosphate-buffered saline (PBS) containing 2% FBS. Immune cell populations were analyzed using a CytoFLEX S Flow Cytometer and FlowJo software version 10.

Killing assay by crystal violet staining

Briefly, MCF-7, MDA-MB-231, Huh-7 and SNU-449 cells were plated into a 96-well plate at a density of 3 × 104 cells/well one day prior to the experiment. The treatments were organized into four conditions as illustrated in Fig. 8 including (1) In the “Immune cells alone” condition, cancer cells (MCF-7, MDA-MB-231, Huh-7, SNU-449) were cocultured with non-adherent immune cells without cordycepin and Cm-EE treatment, maintaining an effector (E) to target (T) ratio of 20:1 for 24 h. (2) In the “Pretreated immune cell” condition, non-adherent immune cells were treated with 100 µM cordycepin or 100 µg/mL Cm-EE for 24 h and then cocultured with cancer cells at an E: T ratio of 20:1 for 24 h. (3) In the “Pretreated cancer cell” condition, cancer cells were treated with 100 µM cordycepin or 100 µg/mL Cm-EE for 24 h, followed by cocultured with non-adherent immune cells at an E:T ratio of 20:1 for 24 h. (4) In the “Combination” condition, cancer cells were cocultured with non-adherent immune cells in the presence of 100 µM cordycepin or 100 µg/mL Cm-EE at an E:T ratio of 20:1 for 24 h. Subsequently, the culture medium containing effector immune cells and dead target cells was aspirated. The remaining cells in the wells were stained with crystal violet dye at room temperature for 45 min, and the staining results were captured and recorded by a camera. The stained cells were then solubilized using 33% acetic acid and their absorbance was measured at 600 nm using a spectrophotometry. The percentage of cell viability was calculated using the formula: % Cell viability = [OD600 TEST / OD600 CONTROL] × 100.

Fig. 8
figure 8

Schematic depicting cordycepin and Cm-EE treatment conditions.

Full size image

Killing assay using mcherry-expressing target cells

The MCF-7, MDA-MB-231, Huh-7, and SNU-449 cell lines were engineered to stably express red fluorescence protein (RFP). These cells were then seeded into 8-well cell culture slides at a density of approximately 3 × 104 cells/well and allowed to incubate for 24 h. Subsequently, the culture media were aspirated, and non-adherent effector immune cells were introduced at an E:T ratio of 20:1 in the presence of either 100 µM cordycepin or 100 µg/mL Cm-EE for 24 h. The cells were visualized using fluorescent microscope, and the fluorescence intensity was quantified using the ImageJ program (National Institutes of Health, Bethesda, MD, USA). The percentage of cell viability was determined using the formula: % Cell viability = [(fluorescence intensity TEST / fluorescence intensity CONTROL) x 100].

Statistical analysis

All experimental results are expressed as the mean ± standard error of the mean (SEM). Significant difference was determined using Student’s t-test, two-way analysis of variance (ANOVA) and one-way ANOVA with Tukey’s multiple comparisons test, conducted with GraphPad Prism software version 8. Differences were considered significant at a threshold of p < 0.05.