Immunogenic potential of colorectal tumor cells after treatment with X-radiation and hyperthermia (2011)

Cancer is one of the leading causes of death worldwide, both in developed and developing countries, resulting in millions of deaths each year. Among cancer entities, a recently published global cancer statistic showed that colorectal cancer is the third and second most common cancer in men and women, respectively [1]. On the one hand, cancer therapies should stop the proliferation of tumor cells and kill them, and on the other hand stimulate a specific immune response against residual cancer cells, stem cells, and micro-metastases. Common anti-tumor therapies consist of surgery, radiotherapy with ionizing radiation (X-ray), and chemotherapy, but various immune therapies and hyperthermia are used in addition in multimodal treatment concepts today. The positive therapeutic effects of temperature-controlled hyperthermia (HT), when combined with standard tumor therapies, have been shown in multiple randomized clinical trials (summarized in [2]). Biological research has revealed that HT induces radiosensitization. This synergistic mode of action is mainly based on the complementary targets of both X-Ray and HT treatment modalities. HT increases radiation-induced DNA damages and leads to selective destruction of tumor cells in hypoxic or acidic tumor parts. While X-radiation alone mainly induces a proliferative stop of the tumor cells and therefore local tumor control, X-ray combined with HT may induce systemic immune activating effects. Cell death induction by X-ray plus HT is worth analyzing because dying and dead cells are important modulators of the immune system, contributing to anti-inflammatory or pro-inflammatory responses depending on the respective form of cell death. While apoptotic cells are usually non- or even anti-inflammatory in order to avoid chronic autoimmunity, necrotic cells are able to induce strong immune responses. In this dissertation, we analyzed the impact of single and combined treatments of colorectal tumor cells with ionizing radiation (X-ray with a single dose of 5 or 10 Gy) and moderate hyperthermia (HT - 41.5 °C for 1 h) on the amount and form of tumor cell death. Furthermore, we analyzed the release of danger signals and the in vivo immunogenicity of the treated tumor cells. We showed that the amount of potentially immunogenic cell death forms was highly increased in colorectal tumor cell lines when the cells were treated with a combination of X-ray and HT. Those combined treatments led to an increased release of the danger signals HMGB1 and HSP70. While X-ray alone led to slightly diminished phagocytosis of treated tumor cells by macrophages and by dendritic cells, treating tumor cells with X-ray plus HT significantly increased phagocytosis. In addition, pro-inflammatory cytokines were released in higher amounts when HT plus X-ray treated tumor cells were phagocytosed, in comparison to X-ray-only treated cells. Furthermore, immature dendritic cells were shown to mature and become activated when incubated with supernatants of X-ray plus HT treated cells. An in vivo clonogenic assay revealed that cells treated with the combined therapies showed significant tumor growth retardation. Finally, immunogenicity of X-ray plus HT treated tumor cells was shown to be sustained in a xenogeneic immunization assay. Taken together, X-ray applied together with HT generate a beneficial tumor cure microenvironment and might lead to the induction of tumor-specific adaptive immune responses. We therefore conclude that hyperthermia is a very potent radiosensitizer, not only in regards to enhanced reduction of tumor clonogenicity, but also in regards to enhanced induction of immunogenic tumor cell death forms and stimulation of the immune system. Combining X-radiation with hyperthermia has multiple immune activating effects, which might be responsible for the beneficial impact on local and (in special cases) systemic tumor control seen in various clinical trials. [1] A. Jemal et al., 2011 [2] P. Schildkopf et al., 2010