A major barrier to successful antitumor vaccination is tolerance of high-avidity T cells specific to tumor antigens. In keeping with this notion, HER-2/neu (neu)-targeted vaccines, which raise strong CD8+ T cell responses to a dominant peptide (RNEU420-429) in WT FVB/N mice and protect them from a neu-expressing tumor challenge, fail to do so in MMTV-neu (neu-N) transgenic mice. However, treatment of neu-N mice with vaccine and cyclophosphamide-containing chemotherapy resulted in tumor protection in a proportion of mice. This effect was specifically abrogated by the transfer of neu-N–derived CD4+CD25+ T cells. RNEU420-429-specific CD8+ T cells were identified only in neu-N mice given vaccine and cyclophosphamide chemotherapy which rejected tumor challenge. Tetramer-binding studies demonstrated that cyclophosphamide pretreatment allowed the activation of high-avidity RNEU420-429-specific CD8+ T cells comparable to those generated from vaccinated FVB/N mice. Cyclophosphamide seemed to inhibit regulatory T (T reg) cells by selectively depleting the cycling population of CD4+CD25+ T cells in neu-N mice. These findings demonstrate that neu-N mice possess latent pools of high-avidity neu-specific CD8+ T cells that can be recruited to produce an effective antitumor response if T reg cells are blocked or removed by using approaches such as administration of cyclophosphamide before vaccination.

Purpose: Given the complex tumor microenvironment, targeting multiple cellular components may be the most effective cancer treatment strategy. Therefore, we tested whether antiangiogenic and immune-based therapy might synergize by characterizing the activity of DC101, an antiangiogenic monoclonal antibody specific for vascular endothelial growth factor receptor-2 (VEGF-R2), alone and with HER-2/neu (neu)^targeted vaccination. Experimental Design: Neu-expressing breast tumors were measured in treated nontolerant FVB mice and immune-tolerant neu transgenic (neu-N) mice. Neu-specific and tumor cellŝ pecific immune responses were assessed by intracellular cytokine staining, ELISPOT, and CTL assays.Results: DC101 decreased angiogenesis and increased tumor cell apoptosis. Although DC101 increased serum levels of the immunosuppressive cytokine VEGF, no evidence of systemic immune inhibition was detected. Moreover, DC101 did not impede the influx of tumor-infiltrating lymphocytes. In FVB mice, DC101 inhibited tumor growth in part through a T cell^dependent mechanism, resulting in both increased tumor-specific CD8 + T cells and tumor regression. Combining DC101 with neu-specific vaccination accelerated tumor regression, augmenting the lytic activity of CD8 + cytotoxicTcells. In tolerant neu-N mice, DC101only delayed tumor growth without inducing frank tumor regression or antigen-specific T-cell activation. Notably, mitigating immune tolerance by inhibiting regulatoryTcell activity with cyclophosphamide revealed DC101-mediated augmentation of antitumor responses in vaccinated neu-N mice. Conclusions: This is the first report of DC101-induced antitumor immune responses. It establishes the induction of tumor-specificT-cell responses as one consequence of VEGF-R2 targeting with DC101. These data support the development of multitargeted cancer therapy combining immune-based and antiangiogenic agents for clinical translation.

Previous work suggested that functional voltage-gated Na(+) channels (VGSCs) are expressed specifically in strongly metastatic cells of rat and human prostate cancer (PCa), thereby raising the possibility that VGSC activity could be involved in cellular behavior(s) related to the metastatic cascade. In the present study, the possible role of VGSCs in the lateral motility of rat PCa cells was investigated in vitro by testing the effect of modulators that either block or enhance VGSC activity. Two rat PCa cell lines of markedly different metastatic ability were used in a comparative approach: the strongly metastatic MAT-LyLu and the weakly metastatic AT-2 cell line, only the former being known to express functional VGSCs. Using both electrophysiological recording and a motility assay, the effects of two VGSC blockers (tetrodotoxin and phenytoin) and four potential openers (veratridine, aconitine, ATX II, and brevetoxin) were monitored on (a) Na(+) channel activity and (b) cell motility over 48 h. Tetrodotoxin (at 1 microM) and phenytoin (at 50 microM) both decreased the motility index of the MAT-LyLu cell line by 47 and 11%, respectively. Veratridine (at 20 microM) and brevetoxin (at 10 nM) had no effect on the motility of either cell line, whilst aconitine (at 100 microM) and ATX II (at 25 pM) significantly increased the motility of the MAT-LyLu cell line by 15 and 9%, respectively. Importantly, at the concentrations used, none of these drugs had effects on the proliferation or viability of either cell line. The results, taken together, would suggest strongly that functional VGSC expression enhances cellular motility of PCa cells. The relevance of these findings to the metastatic process in PCa is discussed.

The pathogenic nature of cancer is attributed, at least in part, to the ability of tumors cells to induce systemic and local mechanisms of immune tolerance. However, we previously reported that tumor-free survival in up to 100% of tolerized HER-2/neu transgenic mice can be achieved by administration of neu-specific mAb concurrently with a HER-2/neu-expressing, GM-CSF-secreting whole cell vaccine. In this report, we show that one mechanism of improved antitumor activity induced by the combination of these 2 neu-targeted interventions was enhanced Fc-mediated activation of APCs. Specifically, in vivo studies demonstrated localization of radiolabeled neu-specific mAb at the vaccine site. Subsequently, increased accumulation of neu-specific mAb at the vaccine-draining lymph node correlated with increased vaccine cell uptake by DCs in vivo. This led to enhancement of CD8 + neu-specific T cell function in terms of proliferation, cytokine production, and central memory development. Thus, the administration of a neu-specific mAb with a neu-targeted GM-CSF-secreting tumor vaccine enhanced induction of neu-specific CD8 + T cells through Fc-mediated activation of DCs. This multimodality attack on the same tumor antigen may have the potential to overcome tolerance to self antigens and weaken the immunosuppressive networks within the tumor microenvironment.

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