Increased immunogenicity of colon cancer cells by selective depletion of cytochrome C.

We and others have previously reported in an in vivo rat colon cancer cell model that cell death precedes and is necessary for the development of a specific antitumoral immune response. To sensitize colon cancer cells to death, we depleted cytochrome c by stable transfection with an antisense construct. Cytochrome c depletion sensitizes human and rat colon cancer cells to a nonapoptotic, nonautophagic death induced by various stimuli. This increased sensitization to a necrosis-like cell death may be related to a decrease in cellular ATP levels and an increase in reactive oxygen species production caused by cytochrome c depletion. In vivo, depletion of cytochrome c decreases the tumorigenicity of colon cancer cells in syngeneic rats without influencing their growth in immune-deficient animals. Furthermore, decreased expression of cytochrome c in tumor cells facilitates in vivo "necrotic" cell death and the induction of a specific immune response. These results delineate a novel strategy to sensitize colon cancer cells to chemotherapy and to increase their immunogenicity in immuno-competent hosts.

Kinetics of tumor cell apoptosis and immune cell activation during the regression of tumors induced by lipid A in a rat model of colon cancer.

Despite the wide range of available therapies, human colon cancers remain difficult to cure. Evidence for efficient antitumoral immune responses to be raised is now widely accepted, and numerous strategies exploiting the host immune system have been developed. A treatment based on the lipid-A derivative OM-174 has been developed in our laboratory. OM-174 induces the rejection of tumors established by injection of PROb colon cancer cells in syngeneic BDIX rats. Our immunohistochemistry study demonstrated that OM-174 treatment is associated with tumor cell apoptosis. Caspase 3 activation was detected 24 h after the first OM-174 injection. Six days after the beginning of the treatment, dendritic cells were the first immune cells that invaded tumor nodules. When dendritic cells came into contact with apoptotic tumor cells, an increased expression of the costimulatory molecules B7-1 and B7-2 was detected at the surface of these cells. Five days later, macrophages were found in the tumor nodules. Lymphocytes organized into a crown surrounding the nodules that progressively regressed during the treatment. T lymphocytes were not in contact with tumor cells or apoptotic cells at any time point. The kinetics of tumor cell apoptosis induced by OM-174, as well as the appearance of innate followed by adaptative immune cells in the tumor nodules, were compatible with cell activation and the development of immune response.

Freshly isolated bone marrow cells induce death of various carcinoma cell lines.

In some carcinomas such as digestive tract carcinomas, bone marrow infiltration by tumor cells is a frequent event but usually remains a micrometastatic disease and rarely induces overt bone lesions. The mechanisms responsible for the control of these metastases in the bone marrow remain poorly known. We show that freshly isolated bone marrow cells from human, murine and rat origin rapidly kill a wide range of syngeneic or xenogeneic carcinoma cell lines in culture. Further analysis of this cytotoxic process in the rat indicated that neither resident bone marrow macrophages nor NK cells were responsible for this cytotoxic effect that was restricted to a subpopulation of bone marrow cells expressing CD90 (Thy-1), a marker of hemopoietic precursors. The tumoricidal activity of these cells did not require long-term culture nor addition of exogenous cytokines or growth factors. A subset of CD90+ cells that rapidly differentiates into CD163(ED2)-expressing macrophages was observed to be responsible for tumor cell killing. These macrophages induced a non-apoptotic death of tumor cells, a process that required both a direct interaction with the tumor cell and nitric oxide (NO) production through the activation of inducible nitric oxide-synthase (iNOS). This ability of pluripotent hemopoietic stem cells to rapidly differentiate into macrophages capable of killing invasive tumor cells may account for the limited expansion of micrometastases of some carcinomas in the bone marrow.

An atypical caspase-independent death pathway for an immunogenic cancer cell line.

REGb cell line, a highly immunogenic tumor cell variant isolated from a rat colon cancer, yields regressive tumors when injected into syngeneic hosts. We previously demonstrated that REGb tumor immunogenicity was related to the capacity of releasing dead cells in vivo. Also, in vitro, REGb cell monolayers release dead cells, especially when cultured in serum-free medium. In the current study, we show that the release of dead cells results from an atypical death process associating features of necrosis and apoptosis. In spite of features considered as hallmarks of caspase-dependent apoptosis, including chromatin fragmentation and DNA oligonucleosomal cleavage, caspases are not activated and caspase inhibitors are ineffective to prevent REGb cell death. In contrast with a number of other types of cell death, the spontaneous death of REGb cells in culture depends on de novo protein synthesis as this death is blocked by low doses of the mRNA translation inhibitor cycloheximide. This unusual mode of cell death that associates necrotic and apoptotic features could provide optimal conditions for triggering a specific immune response.