In the tumor microenvironment, the signals from tumor-associated fibroblasts (TAF) that suppress antitumor immunity remain unclear. Here, we develop and investigate an in vitro three-dimensional (3D) scaffold model for the novel evaluation of TAF interaction with breast tumor cells and breast specific, neu antigen (p98) reactive T cells. Breast cancer cells seeded on 3D chitosan-alginate (CA) scaffolds showed productive growth and formed distinct tumor spheroids. Antigen specific p98 T cells, but not naïve T cells, bound significantly better to tumor cells on scaffolds. The p98 T cells induced potent tumor cell killing but T helper cell cytokine function was impaired in the presence of TAF co-seeding on scaffolds. We found that the immunosuppression was mediated, in part, by transforming growth factor beta (TGF-b) and interleukin-10 (IL-10). Therefore TAF appear capable of inducing potent T cell suppression. CA scaffolds can provide clinically relevant findings prior to preclinical testing of novel immunotherapies.

Effective elicitation of endogenous immunity is associated with improved prognosis for cancer patients. Clinical evidence in hematological and solid cancers show that intratumoral injection of immunostimulatory genes primes and augments endogenous T cell responses. The ability of pro-inflammatory chemokines/cytokines to facilitate migration/activation of antigen-presenting cells (APC) and lymphocytes prompted our modeling of intratumoral delivery of a chemokine/cytokine combination for breast cancer treatment. Here, we demonstrate that expression of chemokine ligand 21 (CCL21) and interferon gamma (IFNγ) in tumors improves tumor specific T cell recruitment to tumor and activation in the tumor milieu. IFNγ and CCL21 were delivered into tumor cells via plasmids, and transfected cells were seeded to form spheroids on three-dimensional (3D) chitosan-alginate (CA) scaffolds. Co-expression of CCL21 and IFNγ, as evidenced by qRT-PCR and ELISA, induced increased recruitment, binding, and infiltration of anti-neu (p98) peptide specific T cells into the breast tumors as determined by SEM and immunofluorescence assays. The co-expression promoted recruitment of only p98 T cells, but not naïve T cells, demonstrating an antigen-restricted activation. Furthermore, the co-expression impacted T helper (Th) cell immunity, promoting an increase in secretion of pro-inflammatory Th-associated cytokine, tumor necrosis factor alpha (TNFα), and cytotoxic T lymphocyte (CTL)-associated protease, Granzyme B (GzB). Therefore, 3D CA scaffolds may be a useful breast cancer tumor microenvironment model to evaluate T cell function. Further characterization of CCL21-IFNγ mediated anti-tumor immunity will potentially benefit the development of chemokine/cytokine combination platforms as anti-cancer agents.

Purpose Infusion of HER2 specific T-cells, derived from vaccine-primed patients and expanded with IL-2/IL-12, has induced tumor regression in a minority of patients with metastatic treatment-refractory HER2+ breast cancer. We questioned whether alteration of cytokine growth factors used to culture vaccine-primed T-cells could improve anti-tumor activity. Experimental Design Using the TgMMTV-neu murine mammary tumor model we cultured T-cells derived from mice immunized with a previously defined neu class II peptide, p98–114 (neu p98), and evaluated different cytokine combinations for expansion. Results Infusion of neu p98 specific T-cell lines derived from all cytokine conditions evaluated resulted in significant anti-tumor activity compared to infused naïve splenocytes (p<0.05). T-cells cultured with IL-2/IL-21 could uniquely mediate complete regression of spontaneous mammary tumors. IL-2/IL-21 cultured neu specific T-cells demonstrated a different cytokine secretion pattern as compared to other cultured T-cells; secreting high levels of TNF-alpha and IL-17 (p<0.05). Moreover, tumor infiltrating CD8+ cells were significantly increased after the infusion of IL-2/IL-21 cultured T-cells as compared to tumors treated with T-cells expanded under other cytokine conditions (p<0.001). The anti-tumor effect of the infusion of IL-2/IL-21 cultured cells was mediated by CD8 T-cells. Depletion of TNF-alpha or IL-17, but not IFN-gamma, abrogated the tumor growth inhibition induced by the IL-2/IL-21 T-cells and markedly decreased the influx of CD8 into tumors. Finally, IL-2/IL-21 cultured human antigen specific T-cells also displayed a similar polyfunctional Th1/Th17 phenotype. Conclusion Expansion of HER2 vaccine-primed T-cells with IL-2/IL-21 may have the potential to effectively mediate tumor regression when used in adoptive transfer.

ErbB-2 has been implicated as a target for cancer-initiating cells in breast and other cancers. ErbB-2-directed peptide vaccines have been shown to be effective in prevention of spontaneous tumorigenesis of breast in neu transgenic mouse model, and cellular immunity is proposed as a mechanism for the anti-tumor efficacy. However, there has been no explanation as to how immunity suppresses tumorigenesis from the early stage carcinogenesis, when ErbB-2 expression in breast is low. Here, we investigated a peptide-based vaccine, which consists of two MHC class II epitopes derived from murine ErbB-2, to prevent the occurrence of spontaneous tumors in breast and assess immune impact on breast cancer stem cells. Female MMTV-PyMT transgenic mice were immunized with either ErbB-2 peptide vaccine, or a peptide from tetanus toxoid, or PBS in immune adjuvant. ErbB-2 peptides vaccine completely suppressed spontaneous breast tumors, and the efficacy was correlated with antigen-specific T-cell and antibody responses. In addition, immune serum from the mice of ErbB-2 vaccine group had an inhibitory effect on mammosphere-forming capacity and signaling through ErbB-2 and downstream Akt pathway in ErbB-2 overexpressing mouse mammary cancer cells. We provide evidence that multi-epitope class II peptides vaccine suppresses tumorigenesis of breast potentially by inhibiting the growth of cancer stem cells. We also suggest that a strategy of inducing strong immune responses using multi-epitope ErbB-2-directed helper vaccine might be useful in preventing breast cancer recurrence.