Cytokine therapy can induce tumor regression in cancer patients but systemic administration of cytokines is accompanied with severe toxicity. Loco-regional delivery represents an effective and less toxic alternative to systemic injection. However; the requirement for frequent repeated injections of recombinant cytokine or the logistical difficulties associated with gene-modification have limited wide-spread use of loco-regional therapy. A simpler alternative local delivery strategy involves the use of controlled-release cytokine depot formulations. These formulations provide the advantage that physiological doses of cytokines are directly released to the tumor microenvironment in a sustained manner. Anti-tumor efficacy of IL-2; IL-12; GM-CSF or TNFalpha-encapsulated polymer microspheres has been evaluated in syngeneic murine and human tumor /SCID mouse xenograft models. A single intra-tumoral injection of these formulations; particularly that of IL-12 in combination with GM-CSF or TNFalpha; promoted the regression of established primary tumors; induced systemic anti-tumor T- and NK-cell responses and achieved complete eradication of disseminated disease. Cellular and molecular analysis of post-therapy tumor microenvironment demonstrated that treatment promoted the activation of tumor-associated T-effector/memory cells; the elimination of CD4+ CD25+ Foxp3+ T-suppressors and the de novo priming of tumor-specific CD8+ T-effector cells. Long-term monitoring of post-therapy tumors revealed that reversal of intra-tumoral immune suppression was transient and that T-suppressor cells rapidly re-infiltrated tumors. Repeated treatment resurrected anti-tumor activity; however, therapeutic efficacy declined with each treatment cycle. The observed loss of therapeutic efficacy was associated with a progressive intensification of the post-treatment T-suppressor cell rebound. In contrast; depletion of T-suppressor cells with low dose chemotherapy prior to each cycle of treatment resulted in a dramatic enhancement of long-term therapeutic efficacy leading to complete remissions. Clinical implications of these findings are discussed herein.