Hypofractionated Irradiation Has Immune Stimulatory Potential and Induces a Timely Restricted Infiltration of Immune Cells in Colon Cancer Tumors

Abstract

In addition to locally controlling the tumor, hypofractionated radiotherapy (RT) particularly aims to activate immune cells in the RT-modified microenvironment. Therefore, we examined whether hypofractionated RT can activate dendritic cells (DCs), induce immune cell infiltration in tumors, and how the chronology of immune cell migration into tumors occurs to gain knowledge for future definition of radiation breaks and inclusion of immunotherapy. Colorectal cancer treatments offer only limited survival benefit, and immunobiological principles for additional therapies need to be explored with preclinical models. The impact of hypofractionated RT on CT26 colon cancer tumor cell death, migration of DCs toward supernatants (SN) of tumor cells, and activation of DCs by SN were analyzed. The subcutaneous tumor of a BALB/c-CT26 mouse model was locally irradiated with 2 × 5 Gy, the tumor volume was monitored, and the infiltration of immune cells in the tumor was determined by flow cytometry daily. Hypofractionated RT induced a mixture of apoptotic and necrotic CT26 cells, which is known to be in particular immunogenic. DCs that migrated toward SN of CT26 cells particularly upregulated the activation markers CD80 and CD86 when in contact with SN of irradiated tumor cells. After hypofractionated RT, the tumor outgrowth was significantly retarded and in the irradiated tumors an increased infiltration of macrophages (CD11bhigh/F4-80+) and DCs (MHC-II+), but only between day 5 and 10 after the first irradiation, takes place. While CD4+ T cells migrated into non-irradiated and irradiated tumors, CD8+ T cells were only found in tumors that had been irradiated and they were highly increased at day 8 after the first irradiation. Myeloid-derived suppressor cells and regulatory T cells show regular turnover in irradiated and non-irradiated tumors. Tumor cell-specific anti-IgM antibodies were enhanced in the serum of animals with irradiated tumors. We conclude that hypofractionated RT suffices to activate DCs and to induce infiltration of innate and adaptive immune cells into solid colorectal tumors. However, the presence of immune cells in the tumor which are beneficial for antitumor immune responses is timely restricted. These findings should be considered when innovative multimodal tumor treatment protocols of distinct RT with immune therapies are designed and clinically implemented.

Immunomodulation by ionizing radiation—impact for design of radio‐immunotherapies and for treatment of inflammatory diseases

“…The presence of the cytotoxic T cells in the tumor was, however, limited since later on the amount of these adaptive immune cells dropped again . In the CT26 colon cancer model in BAlb/c mice, we observed a peak of CD8+ T cells in the tumor 5 days after the last irradiation with 2×5 Gy at day zero and four . From the clinical point of view, a re‐irradiation on these days with higher single doses of RT should be counter‐productive since the beneficial immune cells would be hit by the irradiation.…”

“…We examined in preclinical mouse models the kinetics of immune cell infiltration into irradiated syngenic tumors and found that the infiltration is timely restricted (see also below) and that both, adaptive and innate immune cells show up in the tumor . These observations necessitate consideration (a) of the time point when the immune cell infiltration is monitored and (b) of the interplay of the different immune cells at diverse time points during RIT.…”

“…In particular, T cells are very radiosensitive when being targeted by doses of 0.5 Gy and higher (S. Falcke et al, unpublished data). Before the increased infiltration of T cells into irradiated tumors, APCs such as DCs and macrophages increase (already 1 day after the last irradiation) . But, it must not be ignored that these data refer to examination in mouse tumors and are obtained by flow‐cytometry analyses of the whole tumor‐derived cell suspension.…”

“…89 We examined in preclinical mouse models the kinetics of immune cell infiltration into irradiated syngenic tumors and found that the infiltration is timely restricted (see also below) and that both, adaptive and innate immune cells show up in the tumor. 90 One example for the latter statement is that a repetitive immunization with melanoma cells that had been treated by RT in combination with HT increased particularly the immune regulatory CD27+/CD11b− NK cell subpopulation in draining lymph nodes (LNs).…”

“…134 In the CT26 colon cancer model in BAlb/c mice, we observed a peak of CD8+ T cells in the tumor 5 days after the last irradiation with 2×5 Gy at day zero and four. 90 From the clinical point of view, a re-irradiation on these days with higher single doses of RT should be counter-productive since the beneficial immune cells would be hit by the irradiation. In particular, T cells are very radiosensitive when being targeted by doses of 0.5 Gy and higher (S. Falcke et al, unpublished data).…”