Previous findings indicated that the activated leukocyte cell adhesion molecule (ALCAM) is expressed by tumors and plays a role in tumor biology. In this study, we show that ALCAM is shed from epithelial ovarian cancer (EOC) cells in vitro, leading to the generation of a soluble ALCAM (sALCAM), consisting of most of the extracellular domain. A similar sALCAM molecule was also found in the ascitic fluids and sera from EOC patients, suggesting that this process also occurs in vivo. sALCAM is constitutively produced by EOC cells, and this process can be enhanced by cell treatment with pervanadate, phorbol 12-myristate 13-acetate (PMA), or epidermal growth factor (EGF), a known growth factor for EOC. Pharmacologic inhibitors of matrix metalloproteinases (MMP) and of a disintegrin and metalloproteases (ADAM), and the tissue inhibitor of metalloproteinase-3, significantly inhibited sALCAM release by EOC cells. The ADAM17/TACE molecule was expressed in EOC cell lines and ADAM17/TACE silencing by specific small interfering RNA-reduced ALCAM shedding. In addition, inhibitors of ADAM function blocked EOC cell motility in a wound-healing assay. Conversely, a recombinant antibody blocking ALCAM adhesive functions and inducing ALCAM internalization enhanced EOC cell motility. Altogether, our data suggest that the disruption of ALCAM-mediated adhesion is a relevant step in EOC motility, and ADAM17/TACE takes part in this process, which may be relevant to EOC invasive potential.

IL-21 is an immune-stimulatory four α helix cytokine produced by activated T cells. To study the in vivo antitumor activities of IL-21, TS/A murine mammary adenocarcinoma cells were genetically modified to secrete IL-21 (TS/A-IL-21). These cells developed small tumors that were subsequently rejected by 90% of s.c. injected syngeneic mice. Five days after injection, TS/A-IL-21 tumors showed numerous infiltrating granulocytes, NK cells, and to a lesser extent CD8+ T cells, along with the expression of TNF-α, IFN-γ, and endothelial adhesion molecules ICAM-1 and VCAM-1. At day 7, CD8+ and CD4+ T cells increased together with IFN-γ, and the CXC chemokines IFN-γ-inducible protein 10, monokine induced by IFN-γ, and IFN-inducible T cell α-chemoattractant. The TS/A-IL-21 tumor displayed a disrupted vascular network with abortive sprouting and signs of endothelial cell damage. In vivo depletion experiments by specific Abs showed that rejection of TS/A-IL-21 cells required CD8+ T lymphocytes and granulocytes. When injected in IFN-γ-deficient mice, TS/A-IL-21 cells formed tumors that regressed in only 29% of animals, indicating a role for IFN-γ in IL-21-mediated antitumor response, but also the existence of IFN-γ-independent effects. Most immunocompetent mice rejecting TS/A-IL-21 cells developed protective immunity against TS/A-pc (75%) and against the antigenically related C26 colon carcinoma cells (61%), as indicated by rechallenge experiments. A specific CTL response against the gp70-env protein of an endogenous murine retrovirus coexpressed by TS/A and C26 cells was detected in mice rejecting TS/A-IL-21 cells. These data suggest that IL-21 represents a suitable adjuvant in inducing specific CTL responses.

IL-21 is an IL-2-like cytokine, signaling through a specific IL-21R and the IL-2R γ-chain. Because the TS/A mammary adenocarcinoma cells genetically modified to secrete IL-21 (TS/A-IL-21) are strongly immunogenic in syngeneic mice, we analyzed their application as vaccine. In mice bearing TS/A-parental cell (pc) micrometastases, vaccination with irradiated TS/A-IL-21 cells significantly increased the animal life span, but cured only 17% of mice. Spleen cells from cured mice developed CTL activity and produced IFN-γ in response to stimulation by the AH1 epitope of the gp70env Ag of TS/A-pc. We tested whether the low therapeutic outcome might be due to CD4+CD25+ regulatory T cells (Treg) present in TS/A-pc tumors and draining lymph nodes and whether IL-21 had any effect on these cells. Indeed, CD4+CD25+ cells suppressed IFN-γ production by splenocytes from immune mice in response to stimulation by the AH1 peptide. Low concentrations of IL-21 (10 ng/ml) failed to reverse the inhibitory activity of CD4+CD25+ cells in an allogeneic MLR, whereas 60 ng/ml rIL-21 partially restored responder T cell proliferation. IL-21R expression on CD25− lymphocytes suggested that IL-21 could be more effective in mice depleted of CD25+ cells. Depletion of Treg cells by a single dose of anti-CD25 mAb combined with TS/A-IL-21 cell vaccine cured >70% of mice bearing micrometastases, whereas anti-CD25 mAb treatment alone had no effect. Successful combined immunotherapy required NK cells, CD8+ T cells, and IFN-γ. In conclusion, immunotherapy of micrometastases by an IL-21-based cellular vaccine is strongly potentiated by CD25+ cell depletion.

Interleukin-18 (IL-18

IL‐15 and IL‐12 display anti‐tumor activity in different models and IFN‐γ has been reported as a secondary mediator of both IL‐12 and IL‐15 effects. TS/A murine adenocarcinoma cells were engineered to secrete IL‐12, IL‐15 or both cytokines. TS/A cells secreting IL‐15 (TS/A‐IL‐15) displayed a reduced tumorigenicity (50%) when implanted subcutaneously in syngeneic mice, while both TS/A‐IL‐12 and TS/A‐IL‐12/IL‐15 were rejected by 100% of animals. In contrast, TS/A‐IL‐15 and TS/A‐IL‐12 were tumorigenic in syngeneic IFN‐γ knockout mice (100% and >90% of take rate, respectively), but TS/A‐IL‐12/IL‐15 were completely rejected by 90% of these mice. All IFN‐γ‐deficient mice rejecting TS/A‐IL‐12/IL‐15 developed protective immunity against wild‐type TS/A, as indicated by re‐challenge experiments. Immunohistochemical analysis of the TS/A‐IL‐12/IL‐15 tumor rejection area in IFN‐γ‐deficient mice showed a marked reactive cell infiltration constituted of CD8+ cells, granulocytes, NK cells, macrophages and dendritic cells associated with the expression of IL‐1β, TNF‐α, GM‐CSF, MCP‐1 and MIP‐2. In vivo depletion experiments showed that rejection of TS/A‐IL‐12/IL‐15 cells required CD8+ T lymphocytes and also involved granulocytes, while CD4+ and NK cells played a minor role. These data show IFN‐γ‐independent synergistic anti‐tumor effects of IL‐12 and IL‐15, involving CD8+ cells and secondary chemokines and cytokines, such as TNF‐α.