A standard reference of CDC was purchased from Sigma-Aldrich (St. Louis, MO, USA) for authentication. Recombinant cytokines were purchased from R&D Systems (Minneapolis, MN, USA). LPS, MG132, 4^′,6-diamidino-2-phenylindole (DAPI), and ADO were sourced from Sigma-Aldrich or Cayman Chemical (Ann Arbor, MI, USA). Primary antibodies against pp65 (S536) (#3033), p65 (#8242), pI$\kappa$B (S32) (#2859), Inhibitor of Nuclear Factor kappa B (I$\kappa$B) (#4814), Akt (#4691), pAkt (S473) (#4060), IKK$\beta$ (#2684), p-p38 (T180/Y182) (#4511), pERK (T202/Y204) (#4370), and actin (#4970) were obtained from Cell Signaling Technology (Danvers, MA, USA). Antibodies for F4/80 (sc-377009) and CD68 (catalogue number sc-17832, lot number II) were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

CDC was isolated from Cordyceps militaris ARA301 (BIOARA Co., Ltd., Seoul, Korea), which was cultivated on a 100% edible insect medium. Briefly, 3 kg of C. militaris ARA301 was extracted using methanol. The crude extract was subsequently partitioned with n-hexane, ethyl acetate (EtOAc), and butanol (BuOH). The resulting fractions underwent silica gel and Diaion HP-20 chromatography. CDC was further purified through additional silica chromatography and crystallization, yielding 6.418 g of final product. The chemical structure of CDC was confirmed via ¹H/¹³C nuclear magnetic resonance (NMR) and high-resolution electrospray ionization mass spectrometry (HR-ESI-MS) ([M+H]⁺ m/z 252.1092, C₁₀H₁₃N₅O₃) [24]. Purity was validated to be $>$98% purity using high-performance liquid chromatography with ultraviolet detection (HPLC-UV) on a Hypersil GOLD C18 column (Thermo Scientific, Waltham, MA, USA, 0.5 mL/min flow rate, 30 °C, detection at 254 nm).

Bone marrow (BM) cells were isolated from male C57BL/6 mice (8–10 weeks old, Orient Bio Inc., Gyeonggi-do, Korea) and plated at 2 $\times$ 10⁶ cells/mL in 12-well plates. Primary bone marrow-derived macrophages (BMDMs) were characterized by the expression of lineage-specific markers (F4/80 and CD11b) via flow cytometry. The cells were cultured with macrophage colony-stimulating factor (M-CSF, 10 ng/mL, Sigma-Aldrich) for 6 days to generate mature macrophages. On day 6, confluent cells (representing high-density, contact-inhibited states) were treated with LPS (100 ng/mL), along with CDC and/or ADO for 24 h. In parallel, cells were re-plated at low-density (2 $\times$ 10⁵ cells/mL) to stimulate a sparse environment and treated with LPS, CDC, and/or ADO for 24 h. Additionally, RAW264.7 (TIB-71) and B16F10 (CRL-6475) cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). The authenticity of these cell lines was validated by Short Tandem Repeat profiling and they were confirmed to be negative for mycoplasma contamination before use. RAW264.7 cells were plated at 3 $\times$ 10⁵ cells/mL (low-density) and 8 $\times$10⁵ cells/mL (confluent) in 12-well plates, followed by treatment with CDC or ADO for 24 h in the presence of LPS (100 ng/mL).

BM-derived M1 macrophages or RAW264.7 cells were treated with CDC or ADO for 24 h. Cells were subsequently incubated with EZ-Cytox reagent for 30 min, according to the manufacturer’s instructions (EZ-Cytox Cell Viability Assay Kit, DoGenBio, Seoul, Korea). Cell viability was quantified by measuring optical density at 450 nm using a microplate reader (NFEC-2019-10-258101) (Molecular Devices, Sunnyvale, CA, USA) at the Ewha Fluorescence Imaging Core Center. Results were expressed relative to the vehicle-treated control.

Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA), and 2 µg of RNA were subjected to reverse transcription using MMLV Reverse Transcriptase (Promega, Madison, WI, USA). qPCR was performed using Thunderbird SYBR qPCR Mix (Toyobo, Osaka, Japan) on a StepOnePlus Real-Time PCR machine (Applied Biosystems, Carlsbad, CA, USA). Relative transcript levels were calculated using the comparative Ct (2^{-Δ⁢Δ⁢Ct}) method, with $\beta$-actin as the internal control. Primer sequences used were as follows: $\beta$-actin, 5^′-agagggaaatcgtgcgtgac-3^′ and 5^′-tggatgccacaggattcc-3^′; IL-6, 5^′-gaggataccactcccaacaga-3^′ and 5^′-aagtgcatcatcgttgttcataca-3^′; IL-1$\beta$, 5^′-caaccaacaagtgatattctccat-3^′ and 5^′-gatccacactctccagctgca-3^′; TNF$\alpha$, 5^′-cagttctatggcccagaccctca-3^′ and 5^′-acaacccatcggctggcaccac-3^′; CD68, 5^′-cctatacccaattcagggtgg-3^′ and 5^′-ctgcgccatgaatgtccac-3^′; monocyte chemoattractant protein 1 (MCP1), 5^′-cttctgggcctgctgttca-3^′ and 5^′-ccagcctactcattgggatca-3^′; and CC chemokine receptor 2 (CCR2), 5^′-tggctgtgtttgcctctctaccag-3^′ and 5^′-caagtagaggcaggatcaggctc-3^′.

Cells on poly-L-lysine-coated coverslips or culture slides (Marienfeld, Lauda-Königshofen, Germany) were treated with CDC, fixed, and stained with antibodies against NF-$\kappa$B p65, followed by nuclear counterstaining with DAPI (1 µg/mL, Sigma-Aldrich). Fluorescence images were acquired using a fluorescence microscope (Axio Observer 7, Carl Zeiss, Oberkochen, Germany) (NFEC-2021-08-272462) at the Ewha Drug Development Research Core Center. For immunoblotting, cell lysates were prepared in RIPA buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, and 0.1% sodium dodesyl sulfate), resolved by Sodium Dodesyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE), and transferred to membranes. Membranes were probed with primary and secondary antibodies, detected using an ImageQuant 800 (Cytiva, Marlborough, MA, USA), and quantified with ImageJ 1.53t software (NIH, Bethesda, MD, USA).

RAW264.7 or HEK293T cells were transfected with NF-$\kappa$B-luc or pIL-6-luc reporter constructs and RSV$\beta$ for normalization. Following 24-h treatment with CDC or ADO, luciferase and $\beta$-galactosidase activities were measured using the Luciferase Assay System kit (Promega, Madison, WI, USA) and the Galacto-Light Plus™ system (Applied Biosystems, Foster City, CA, USA), respectively. Luciferase activity was expressed as fold induction compared to control after normalization to $\beta$-galactosidase activity.

For phagocytosis, cells were incubated fluorescence-conjugated zymosan bioparticles (ab234053, Abcam, Cambridge, MA, USA) at a final concentration of 5 µg/mL for 30 min at 37 °C. Phagocytic activity was analyzed via flow cytometry (FACS Calibur, BD Biosciences, San Jose, CA, USA). For the migration assay, treated M1 macrophages were placed in the upper chamber of a 24-well Transwell insert (5 µm pore size, MERCK Millipore, Billerica, MA, USA), with B16F10 melanoma cells in the lower chamber. After 5 h of incubation, cells on the upper surface of the membrane were removed, and migrated cells were stained with 0.2% crystal violet, imaged, and quantified.

B16F10 melanoma tumor cells were labeled with carboxyfluorescein succinimidyl ester (CFSE, 5 µM) for 15 min. The CFSE-labeled tumor cells were co-cultured with macrophages pre-treated with CDC or ADO for 24 h. After co-culture, cells were stained with an anti-F4/80 antibody and analyzed by flow cytometry. Phagocytic macrophages and viable tumor cells were quantified using CellQuest software (BD Biosciences, San Jose, CA, USA).

Male C57BL6/J mice (10–12 weeks old) were anesthetized with isoflurane (3%) using a small-animal inhalation anesthetic system (3–5 min) and subcutaneously injected with B16F10 melanoma cells (5 $\times$ 10⁵ cells/mouse). Mice were randomized into experimental groups. On day 10, after tumor establishment, macrophages pre-treated with CDC or vehicle were injected intratumorally (5 $\times$ 10⁵ cells/mouse in 50 µL) under isoflurane anesthesia. Tumor growth was monitored for 14 days, after which the mice were euthanized for histological analysis.

Mice were euthanized by CO₂ inhalation (at a displacement rate of 20% of the chamber volume per minute to ensure ethical and humane sacrifice), and tumors were harvested, fixed in 10% formalin, and embedded in paraffin. Tumor sections (4 µm thick) were stained for F4/80 and CD68 with fluorescent secondary antibodies and DAPI (1 µg/mL, Sigma-Aldrich) for nuclear counterstaining. Apoptotic cell death was evaluated by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining, performed with a commercial kit (R&D Systems, 4810-30-K).

Data are presented as the mean $\pm$ standard error of the mean (SEM) from at least five independent experiments. Statistical analysis was performed using GraphPad Prism version 10.0 (GraphPad Software LLC, San Diego, CA, USA). For comparisons between two groups, Student’s t-test was employed. For comparisons involving three or more groups, one-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test was used. A significance level of p 0.05 was used. For in vivo studies, investigators were blinded to the treatment groups during tumor measurement and histological analysis.

To obtain sufficient quantities of naturally occurring CDC, we utilized the high-yielding cultivar C. militaris ARA301. CDC was successfully isolated and purified from multiple fractions, yielding a total of 6.418 g from fractions XI-9 and BII (Fig. 1A). The identity and structural integrity of the purified compound were confirmed by ¹H/¹³C NMR spectra, which were consistent with reported reference data [24]. High-resolution ESI-MS analysis confirmed the molecular formula as C₁₀H₁₃N₅O₃ (Fig. 1B). HPLC-UV analysis further verified a purity greater than 98% (Fig. 1C). To validate the in vivo anti-tumor efficacy of CDC, we administered the compound intraperitoneally to a melanoma mouse model. CDC treatment resulted in a significant inhibition of tumor growth, as evidenced by reduced tumor volume and size (Fig. 1D,E). Histological analysis of tumor tissues from CDC-treated mice revealed a marked increase in apoptotic cell death (TUNEL positive) and macrophage infiltration (F4/80 positive) (Fig. 1F). Furthermore, elevated systemic levels of pro-inflammatory cytokines were detected in the spleens of these mice, indicating a robust immune response (Fig. 1G). Collectively, these findings suggest that CDC not only inhibits tumor progression but also significantly enhances macrophage activation and recruitment within the TME, contributing to its overall anti-tumor effects.

Fig. 1.

Identification and anti-tumor activity of Cordycepin (CDC) isolated from a novel Cordyceps cultivar. (A) Isolation procedure and yield of CDC from C. militaris ARA301 cultivar. (B) High-resolution electrospray ionization mass spectrometry (HR-ESI-MS) analysis for the molecular weight determination of isolated CDC. (C) High-performance liquid chromatography with ultraviolet detection (HPLC-UV) chromatogram confirming the purity and identity of the CDC fraction. (D) Inhibitory effect of CDC on in vivo B16F10 melanoma tumor growth in a syngeneic mouse model. (E) Comparison of representative tumor sizes and weights at the study endpoint. Scale bar = 1 cm. (F) Histological analysis of tumor sections via terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining (apoptosis) and F4/80 immunohistochemistry (macrophage infiltration). Scale bar = 50 µm. (G) qPCR analysis of IL-1$\beta$ and IL-6 in the spleens of tumor bearing mice. Data are presented as mean $\pm$ standard error of the mean (SEM). Statistical significance: #p 0.05; ##p 0.005; ###p 0.0005 as determined by one-way analysis of variance (ANOVA); *p 0.05 by Student’s t-test.

Given prior reports indicating that CDC can reduce cell viability [16, 17, 25], we first evaluated its cytotoxic effects on BM-derived M1 macrophages across a range of concentrations. In confluent (100% density) cultures, CDC exhibited no significant cytotoxicity at concentrations up to 20 µM, with viability decreasing only at concentrations above 50 µM (Fig. 2A). As macrophage activation and functional states are known to be modulated by physical cues and mechanical forces [26, 27, 28], we examined whether cell density affects CDC’s efficacy. Notably, while CDC remained non-cytotoxic in confluent M1 macrophages, it significantly reduced cell numbers in low-density (approx. 30%) cultures at concentrations of 10–20 µM (Fig. 2B). A consistent pattern was observed in RAW264.7 macrophages, where CDC-induced cytotoxicity was strictly confined to low-density conditions (Fig. 2C). In contrast, the structural analog ADO resulted in no significant loss of viability in either M1 or RAW264.7 macrophages, regardless of cell density (Fig. 2D,E). These findings demonstrate that CDC-induced cytotoxicity is uniquely contingent upon cell density, suggesting that mechanically-driven changes in the microenvironment—such as altered cell-to-cell contact or cytoskeletal tension—fundamentally reconfigure the macrophage response to CDC.

Fig. 2.

Context-dependent modulation of macrophage viability by CDC and adenosine (ADO). (A) Dose-dependent effects of CDC on the viability of BM-derived M1 macrophages across a range of concentrations (0.1~100 µM). (B) Modulation of M1 macrophage viability by CDC under varying cell densities (confluent vs. low-density). (C) Cell viability modulation of RAW264.7 cells treated with CDC in confluent and low-density cultures. (D) Effects of ADO on M1 macrophage viability in confluent and low-density conditions. (E) Cell viability modulation of RAW264.7 cells following ADO treatment under confluent and low-density conditions. Data are presented as mean $\pm$ SEM. Statistical significance: ##p 0.005; ###p 0.0005 as determined by one-way ANOVA.

We next measured inflammatory cytokine expression in both confluent and low-density cultures of BM-derived M1 macrophages and RAW264.7 cells. In confluent M1 macrophages, treatment with non-cytotoxic concentrations of CDC ($\le$20 µM) significantly enhanced the expression of IL-1$\beta$, IL-6, and TNF$\alpha$, whereas ADO substantially suppressed their expression under the same conditions (Fig. 3A,B). However, a starkly different pattern emerged under low-density culture conditions; CDC suppressed the expression of IL-1$\beta$, an effect associated with the concomitant density reduction in cell viability. Notably, while ADO also suppressed IL-1$\beta$ expression in low-density conditions, it did so without altering cell viability (Fig. 3C). This density-dependent shift was consistently observed in RAW264.7 cells; CDC significantly increased pro-inflammatory cytokine production in confluent cultures but suppressed it in low-density cultures where cell viability was compromised (Fig. 3D). Unlike CDC, ADO persistently reduced cytokine production regardless of cell density or its effect on cell number (Fig. 3E). Collectively, these data demonstrate that CDC’s immunomodulatory effects are fundamentally governed by the cellular microenvironment.

Fig. 3.

Context-dependent modulation of inflammatory cytokine transcripts by CDC and ADO. (A) Regulatory effects of CDC on pro-inflammatory cytokine mRNA expression in confluent M1 macrophages. (B) Inhibitory effects of ADO on pro-inflammatory cytokine expression in confluent M1 macrophages. (C) Comparative modulation of cytokine profiles by CDC or ADO in low-density M1 macrophages. (D) Density-dependent effects of CDC on pro-inflammatory cytokine expression in RAW264.7 cells (confluent vs. low-density). (E) Suppression of pro-inflammatory cytokines by ADO in RAW264.7 cells across varying culture densities. Statistical significance: #p 0.05; ##p 0.005; ###p 0.0005 as determined by one-way ANOVA.

To elucidate the molecular mechanisms by which CDC induces immunomodulatory cytokine production, we examined the activation of the NF-$\kappa$B pathway. Immunofluorescence analysis revealed that CDC dose-dependently increased the nuclear translocation of p65 in M1 macrophages (Fig. 4A). Furthermore, immunoblot analysis demonstrated that CDC enhanced the phosphorylation of p65 (pp65) and I$\kappa$B without altering the total protein levels of p65 or I$\kappa$B (Fig. 4B). Similar increases in p65 nuclear localization and phosphorylation were observed in CDC-treated RAW264.7 cells (Fig. 4C,D), whereas ADO exhibited no significant effect on p65 phosphorylation, distinguishing CDC’s unique signaling profile (Fig. 4D). Further mechanistic analysis revealed that CDC significantly increased the total Akt expression—a key upstream regulator of the NF-$\kappa$B axis—without affecting I$\kappa$B kinase (IKK) levels or the activation of p38 and ERK MAPKs (Fig. 4E). This expansion of the total Akt pool likely facilitates the subsequent phosphorylation and nuclear localization of p65. Collectively, our findings suggest that the functional activation of macrophages by CDC is primarily driven by this Akt-dependent enhancement of the NF-$\kappa$B signaling axis, providing a molecular basis for its pro-inflammatory and anti-tumor effects.

Fig. 4.

Enhancement of p65 nuclear localization and phosphorylation by CDC. (A) Representative immunofluorescence images and quantitative analysis of p65 nuclear localization in M1 macrophages following CDC treatment. Scale bar = 50 µm (top) and 5 µm (bottom). (B) Immunoblot analysis and densitometric quantification of phosphorylated p65 (pp65), total p65, pI$\kappa$B, and total Inhibitor of Nuclear Factor kappa B (I$\kappa$B). Actin was used as a loading control. (C) Immunofluorescence staining and intensity quantification of p65 in RAW264.7 cells. Scale bar = 5 µm. (D) Protein expression levels of p-p65 and total p65 in RAW264.7 cells treated with CDC or ADO, including quantification of the p-p65/p65 ratio. (E) Immunoblot analysis of upstream signaling mediators of the Nuclear Factor kappa B (NF-$\kappa$B) pathway in M1 macrophages, with corresponding densitometric quantification of total Akt. Statistical significance: #p 0.05; ##p 0.005; ###p 0.0005 as determined by one-way ANOVA.

Consistent with its effect on NF-$\kappa$B p65 phosphorylation, CDC, not ADO, significantly increased NF$\kappa$B and IL6 promoter reporter activities in RAW264.7 cells in a dose-dependent manner (Fig. 5A,B). Furthermore, the elevated NF-$\kappa$B-luc activity induced by p65 overexpression in HEK293T cells was dose-dependently enhanced by CDC, whereas ADO showed no significant effect (Fig. 5C). These contrasting pharmacological profiles prompted us to investigate whether CDC competes with ADO for A2AR-mediated signaling. We observed that ADO inhibited CDC-induced production of IL-6 and IL-1$\beta$ in a dose-dependent manner (Fig. 5D) and markedly reduced CDC-induced phosphorylation of p65 (Fig. 5E). Conversely, CDC restored inflammatory cytokine production that had been suppressed by ADO (Fig. 5F), suggesting a competitive interaction between the two nucleoside analogs at the receptor level. To definitively identify the ADO receptor subtype involved, cells were treated with selective antagonists. Notably, the A2AR-specific antagonist ZM241385 significantly attenuated CDC-induced increases in IL-6 and IL-1$\beta$. In contrast, the A2BR-selective antagonist (PSB-603) exerted no inhibitory effect under the same conditions (Fig. 5G), confirming that the immunostimulatory effects of CDC are specifically mediated through A2AR. Collectively, these findings demonstrate that CDC engages A2AR signaling in competition with ADO, thereby promoting the Akt-dependent NF-$\kappa$B axis and enhancing the production of immunomodulatory cytokines.

Fig. 5.

Activation of A2A adenosine receptor (A2AR)-mediated signaling by CDC. (A) Reporter assay measuring NF-$\kappa$B-luc activity in RAW264.7 cells following treatment with CDC or ADO. (B) pIL-6-luc reporter activity in RAW264.7 cells treated with CDC. (C) NF-$\kappa$B-luc reporter activity in p65-transfected HEK-293T cells treated with CDC or ADO (bar graph). Immunoblot analysis confirming the uniform expression of transfected p65 protein across groups. (D) qPCR analysis for IL-1$\beta$ and IL-6 in M1 macrophages treated with ADO (5~20 µM) in the presence of CDC (10 µM). (E) Immunoblot analysis and densitometric quantification of pp65 and total p65 in M1 macrophages. (F) qPCR analysis of IL-1$\beta$ and IL-6 in M1 macrophages treated with CDC (5~20 µM) in the presence of ADO (10 µM). (G) Schematic illustration of the proposed A2AR-mediated signaling, alongside qPCR analysis of IL-1$\beta$ and IL-6 following treatment with CDC and/or the A2AR-selective antagonist ZM241385 (ZM, 10 µM) or the A2BR-selective antagonist PSB-603 (PSB, 10 µM). Statistical significance: #p 0.05; ##p 0.005; ###p 0.0005 as determined by one-way ANOVA.

Building on the engagement of A2AR–Akt–NF-$\kappa$B signaling, we assessed the functional impact of CDC on macrophage phagocytic activity and migration. Phagocytosis assays using zymosan bioparticles demonstrated that CDC significantly increased phagocytic capacity of both M1 and RAW264.7 macrophages, whereas ADO showed no significant effect (Fig. 6A). This functional enhancement was markedly suppressed by the A2AR antagonist ZM241385, confirming that CDC stimulates phagocytosis specifically through A2AR pathway (Fig. 6B). In addition to phagocytosis, we evaluated macrophage migration, a critical factor for tumor infiltration. CDC significantly promoted macrophage chemotaxis toward B16F10 melanoma cells, while ADO exhibited no effect on the migratory capacity of RAW264.7 cells (Fig. 6C). Consistent with this increased motility, CDC, but not ADO, upregulated the expression of key migration- and recruitment-associated genes, including CD68, MCP1, and CCR2 (Fig. 6D). These results indicate that CDC promotes a comprehensive anti-tumor phenotype by enhancing both macrophage phagocytosis and migration. These effects, mediated by A2AR signaling, likely facilitate the recruitment of activated macrophages to the tumor site and their subsequent clearance of malignant cells, distinguishing CDC from its endogenous analog, ADO.

Fig. 6.

Enhancement of macrophage phagocytosis and migratory capacity by CDC. (A) Representative images and quantitative analysis of phagocytic activity in M1 macrophages and RAW264.7 cells following treatment with CDC (20 µM) or ADO (20 µM). (B) Phagocytosis assay and corresponding quantification of M1 macrophages in the presence or absence of the A2AR antagonist ZM241385 (ZM, 10 µM). (C) Transwell migration assay and quantification of M1 macrophages following treatment with 20 µM CDC or ADO. Scale bar = 500 µm. (D) qPCR analysis of migration-related markers (CD68, monocyte chemoattractant protein 1 (MCP-1), and CC chemokine receptor 2 (CCR2)) in M1 macrophages treated with CDC. Statistical significance: #p 0.05; ##p 0.005; ###p 0.0005 as determined by one-way ANOVA.

Having established that CDC enhances macrophage migration and phagocytosis, we evaluated its anti-tumor effects in co-culture and syngeneic mouse models. CFSE-labelled B16F10 melanoma cells were co-cultured with M1 macrophages pre-treated with either CDC or ADO. CDC-treated M1 macrophages exhibited a significantly increased phagocytic uptake of tumor cells but ADO did not (Fig. 7A). Consequently, tumor cell viability was markedly reduced in co-cultures with CDC-pre-treated M1 macrophages, and this tumoricidal effect was further enhanced when macrophages were primed with CDC. However, ADO treatment failed to produce any significant enhancement in tumor killing (Fig. 7B,C). To assess in vivo efficacy, mice bearing subcutaneous melanomas received intratumoral injections of either vehicle-treated or CDC-treated M1 macrophages. CDC-activated M1 macrophages significantly suppressed tumor growth, resulting in a substantial reduction in both tumor volume and weight compared to vehicle-treated macrophage controls (Fig. 7D,E). Histological analysis of the tumor tissues confirmed the therapeutic mechanism: tumors receiving CDC-primed macrophages showed a marked increase in apoptotic cell death (TUNEL positive) and a higher density of CD68-positive macrophage infiltration (Fig. 7F). These findings demonstrate that CDC functionally re-programs M1 macrophages into a potent tumoricidal phenotype, enhancing their ability to infiltrate the tumor mass and execute tumor cell clearance, thereby suppressing tumor progression in vivo.

Fig. 7.

Anti-tumor efficacy of CDC-activated M1 macrophages. (A) Tumor phagocytosis assay of M1 macrophages treated with 20 µM CDC or ADO co-cultured with carboxyfluorescein succinimidyl ester (CFSE)-labeled B16F10 melanoma cells. (B,C) Tumor cell survival assay in the presence of M1 macrophages. Survival of live B16F10 cells was analyzed by flow cytometry (B) and corresponding quantification (C). (D–F) Therapeutic evaluation in a syngeneic B16F10 tumor model (C57BL/6 mice) following intratumoral injection of vehicle- or CDC-pre-treated M1 macrophages. (D) Longitudinal tumor growth curves over the experimental period. (E) Comparison of terminal tumor size and mass. Each grid square is 0.5 cm. (F) Representative images of TUNEL staining (apoptosis) and CD68 staining (macrophage infiltration) in tumor sections. Sacle bar = 50 µm. Statistical significance: ##p 0.005; ###p 0.0005 as determined by one-way ANOVA.