Context-Dependent Modulation of Macrophage Plasticity by Cordycepin Drives Tumor Regression in Melanoma

Abstract

Background and Aim: Cordycepin (CDC), an adenosine (ADO) analog from Cordyceps mushrooms, exhibits potent anti-tumor and immunomodulatory activities. However, the precise mechanisms governing its effects on macrophage plasticity remain poorly understood. This study aimed to isolate CDC from a high-yielding Cordyceps cultivar, validate its systemic anti-tumor efficacy, and elucidate the mechanobiological cues regulating CDC-driven macrophage functions under varying cell-density states.Experimental Procedure: CDC (>98% purity) was isolated and structurally characterized via nuclear magnetic resonance (NMR) and high-resolution electrospray ionization mass spectrometry (HR-ESI-MS). Primary bone marrow-derived macrophages and RAW264.7 cells, cultured under sparse (similar to 30%) or confluent (100%) conditions, were treated with CDC or ADO. We evaluated cell viability, pro-inflammatory cytokine expression, and Nuclear Factor kappa B (NF-kappa B) p65 signaling, phagocytosis, and migration. The therapeutic potential of CDC-primed macrophages was assessed via in vitro melanoma co-culture and in vivo intratumoral adoptive transfer in B16F10 tumor-bearing mice.Key Results: Systemic administration of CDC significantly inhibited melanoma growth in vivo, promoting apoptosis, enhancing macrophage infiltration. We discovered that CDC regulates macrophage functions via a density-dependent "switch": while CDC induced cytotoxicity in sparse cultures, it significantly augmented M1-like cytokine production in confluent states without compromising viability. This density-dependent activation was mediated by the A2A adenosine receptor (A2AR), triggering the Akt-NF-kappa B p65 signaling axis. Furthermore, CDC upregulated migration- and phagocytosis-associated genes, enhancing tumor cell clearance. Notably, intratumoral injection of CDC-primed macrophages markedly reduced tumor volume and size in vivo.Conclusions and Implications: CDC modulates macrophage activation through a unique mechanobiological switch. Under high-density conditions-mimicking the dense tumor microenvironment-CDC enhances A2AR-mediated NF-kappa B activation, boosting macrophage activation, recruitment, and phagocytosis to facilitate tumor regression. These findings establish CDC as a context-dependent immunomodulator capable of reprogramming macrophages toward a tumoricidal phenotype.