Immunotherapy against breast cancer targeting Stat3 triggers NK/CD4+ T-cell mediated antitumoral response and inhibits tumor growth in therapeutic administration Tumor Immunology

TKACH M,; CORIA L. ; ROSEMBLIT C,; RIVAS MA,; BEGUELIN W, ; PROIETTI CJ,; DIAZ FLAQUÉ MC, ; CHARREAU EH,; ELIZALDE PV,; CASSATARO J,; SCHILLACI R

Miami

Congreso; Basic and Clinical advances; 2010

We have demonstrated that progestins induce Stat3 activation in human breast cancer cell lines and in the murine progestin-dependent C4HD cells. The evidence that disruption of Stat3 signaling in cancer cells can overcome tumor immune evasion led us to design an immunotherapy based on serial administration of irradiated breast cancer cells transfected with a dominant negative (DN) form of Stat3 (DNStat3-C4HD cells). This immunization provides protection against C4HD tumor growth in a prophylactic protocol. Therefore, our aims are: 1) disclosing the lymphocyte subsets involved in the antitumoral response and 2) evaluating whether therapeutic administration of DNStat3-C4HD cells could inhibit tumor progression. To achieve our first objective, immunized BALB/c mice were in vivo depleted of CD4+, CD8+, or NK cells with monoclonal antibodies before tumor challenge. Depletion of CD4+ T or NK cells completely abrogated resistance to tumor challenge induced by immunization with DNStat3-C4HD cells (p<0.01 and p<0.001, respectively). However, depletion of CD8+ T cells did not affect C4HD tumor rejection. CD4+ T cells from mice immunized with DNStat3-C4HD cells showed an increased IFNg production, determined by intracytoplasmic staining and flow cytometry analysis, when co-culture with wild-type C4HD cells. As we have already demonstrated that only splenocytes from mice injected with DNStat3-C4HD cells were effective in lysing C4HD in vitro we asked whether NK cells were responsible for that cytotoxic effect. NK cells isolated from spleens of mice immunized with DNStat3-C4HD cells showed an increased cytotoxicity against YAC-1 cell line and C4HD cells compared to mice immunized with empty vector-C4HD cells performing a 51Cr release assay (27.5 ± 2.5% vs 5.2 ± 1.9% and 15.7± 2.4 vs 5.3± 2.7, respectively, 5:1 effector-to-target ratio, p<0.05). We also identified the cell subsets responsible for this cytotoxic effect in vitro by depleting NK cells, CD4+ or CD8+ T cell populations from mouse splenocytes by immunomagnetic separation before performing the 51Cr release assay. Depletion of NK cells from splenocytes of mice immunized with DNStat3-C4HD cells resulted in total suppression of cytotoxic activity (p<0.05). In contrast, depletion of CD4+ or CD8+ cells from this splenocytes did not affect cytotoxicity significantly.             In order to evaluate if this vaccination is effective in the protection against growth of an established breast tumor, we designed a therapeutic protocol based on the administration of DNStat3-C4HD cells on days 5 and 19 after tumor inoculation (tumor volume=0.5mm3 at day 5). As control, we injected empty vector-transfected C4HD cells. At day 35, our results showed that mice immunized with DNStat3-C4HD cells experienced significant inhibition of tumor growth (46.6 ± 9% as compared with mice injected with empty vector-C4HD cells). The histopathological features analyzed by H&E staining of histological sections showed that tumors from animals immunized with DNStat3- C4HD cells presented a lower histological grade and a significantly lower mitotic index as compared to control tumors. Our results suggest that administration of tumor immunogens derived from DNStat3 breast cancer cells generate a significant CD4+ T cell and NK cell-dependent anti tumoral immune response and significant therapeutic effect against C4HD tumor growth.