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 (CD11b^high/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.

Keywords: CD8+ T cell; antigen-presenting cells; colorectal cancer; hypofractionated radiotherapy; immunogenic radiotherapy; macrophages; tumor cell-specific IgM; tumor-infiltrating immune cells.

Figure 1

Hypofractionated irradiation reduces the colony formation and induces apoptosis and necrosis of CT26 cells. The colony formation was determined by standard colony formation assay (A). After incubation for approximately 2 weeks, the cells were fixed and colonies with >50 cells were scored. The cell death analyses were performed 24 h after single or double irradiation of CT26 colorectal tumor cells with 5 Gy. Cell death was determined by flow cytometry; apoptotic cells (gray) are defined as AxV⁺/PI⁻ cells and necrotic (black) as AxV⁺/PI⁺ cells (B). Joint data of three independent experiments, each performed in duplicates, are presented as mean ± SEM and analyzed by Student’s t-test; **p < 0.01.

Figure 2

Dendritic cells (DCs) migrate toward supernatants (SN) of CT26 cells and are particularly activated by SN of irradiated CT26 cells. Bone marrow-derived DCs from BALB/c mice (mDCs) were harvested and seeded to the upper chamber of a transmigration system (3.0 μm pore size). The lower chamber was filled with cell culture SN obtained from CT26 tumor cells 24 h after irradiation with 2 × 5 Gy on consecutive days or with SN of mock-treated cells. After 14 h of incubation at 37°C, the transmigration index (MI), reflecting the migration of mDCs toward SN of the tumor cells versus the medium only control, was determined (A) and the expression of CD80 (B) and CD86 (C) on the MHCII⁺ transmigrated cells was determined by flow cytometry. Joint data of three independent experiments are presented as mean ± SEM and analyzed by Student’s t-test; **p < 0.01.

Figure 3

The activation of dendritic cells (DCs) by supernatants (SN) of irradiated CT26 cells is independent of the migration. Bone marrow-derived DCs from BALB/c mice (mDCs) were incubated at 37°C in SN obtained from CT26 tumor cells 24 h after irradiation with 2 × 5 Gy on consecutive days, in SN of non-irradiated mock treated CT26 cells, or in medium containing lipopolysaccharide (LPS). The expression of CD80 (A) and CD86 (B) on mDCs was analyzed after 24 and 48 h via flow cytometry. Representative data of one out of three independent experiments each performed in triplicates are presented as mean ± SEM and analyzed by Student’s t-test; **p < 0.01.

Figure 4

Hypofractionated radiotherapy (RT) results in local control of CT26 colon cancer tumors in BALB/c mice. The planning of the irradiation was conducted using a computer tomography image of the irradiation box and tumor-bearing mice with Philips pinnacle software to obtain an optimal target volume. Afterward, the dosimetry of the irradiation was performed manually with a calibrated ionization chamber. To further protect the normal tissue, the gantry of the 6-MV linear accelerator was rotated to 340°. Tumors of three anesthetized mice can be irradiated locally at once and the dose distribution (colored areas) shows that only the tumor and not the rest of the mouse is exposed to radiation (A). The tumor volumes were determined daily. Up to day 4 after the first irradiation with 5 Gy, the infiltration of immune cells in the tumors was monitored in tumors of three mice from each group (B). Hypofractionated irradiation with 2 × 5 Gy resulted in good tumor control (C); *p < 0.05, **p < 0.01; n: variable: at the starting point n = 40, with three mice less each following day per treatment group.

Figure 5

The infiltration of immune cells in irradiated tumors is timely restricted. At each day of the examination period, three tumors of each group were separately enzymatically dissociated and consecutively analyzed for immune cell infiltration by flow cytometry. The amount of the indicated immune cells out of all analyzed viable cells is displayed (A–E). Data of three independent tumors are presented as mean ± SEM and analyzed by Student’s t-test; *p < 0.05, **p < 0.01.

Figure 6

Hypofractionated irradiation of colorectal tumors induces tumor-cell-specific IgM antibodies. The sera of the mice whose tumor had been irradiated with 2 × 5 Gy were analyzed for tumor cell-specific IgM antibodies. For this, serum was collected from blood samples taken at the last day of the observation period (see Figure 3). These sera were then co-incubated with viable CT26 cells. IgM antibodies bound to the tumor cells were stained with FITC-conjugated anti-mouse IgM F(ab’)₂ fragments and analyzed via flow cytometry (A). Data of three independent tumor-bearing mice are presented as mean ± SEM (B) and analyzed by Student’s t-test; **p < 0.01.