Abstract

The development of effective cancer vaccines remains challenged by tumor heterogeneity and insufficient T-cell activation. Whole-cell vaccines, which deliver a broad spectrum of tumor-associated antigens, represent a promising approach, especially when their immunogenicity is enhanced via strategies such as immunogenic cell death (ICD). Here, we developed a novel whole-cell vaccine, termed MDLSD, by combining mitoxantrone (MTX)-induced ICD with the TLR9 agonist SD-101. In murine Lewis lung carcinoma (LLC) models, MDLSD demonstrated superior prophylactic and therapeutic efficacy. Mechanistically, MTX-induced dying tumor cells (MDL) promoted dendritic cell (DC) phagocytosis, while the addition of SD-101 was essential for driving complete DC maturation through potent upregulation of CD80. Consequently, only the combined MDLSD vaccine robustly primed antigen-specific T cells in vivo , eliciting elevated IFN-γ secretion. Prophylactic vaccination with MDLSD conferred 100 % protection against initial tumor challenge and durable immunity upon rechallenge. In a therapeutic setting, MDLSD treatment achieved a 55 % complete regression rate, significantly suppressing established tumor growth and prolonging survival. This potent antitumor immunity was driven by MDLSD-enhanced Th1/Tc1 polarization, evidenced by increased T-bet, IFN-γ, and TNF-α, along with boosted cytolytic function via Granzyme B in both systemic and tumor-infiltrating T cells. Furthermore, mice cured by MDLSD developed long-term immunological memory, characterized by a recall response dominated by IFN-γ + CD8 + and TNF-α + T cells, enabling rejection of secondary tumor challenges. Collectively, our findings illustrate that the MDLSD vaccine synergizes the antigenic breadth of ICD with potent TLR9 stimulation to elicit robust DC activation, polyfunctional T-cell responses, and durable immunological memory, offering a compelling strategy for cancer immunotherapy.