Abstract: The present invention provides compositions and methods for generating and cryopreserving dendritic cells with superior functionality in producing stronger signals to T cells, resulting in a more potent DC-based anti-tumor vaccine. The present invention includes mature, antigen loaded DCs activated by Toll-like receptor agonists that induce clinically effective immune responses, preferably when used earlier in the disease process. The DCs of the present invention produce desirable levels of cytokines and chemokines, and further have the capacity to induce apoptosis of tumor cells. The cells can be cryopreserved and thawed for later use, thereby reducing the need for repeated pheresis and elutriation processes during vaccine production. These methods can also be utilized to directly target molecules involved in carcinogenetic signaling pathways and cancer stem cells.