Brachytherapy in a Single Dose of 10Gy as an "in situ" Vaccination

Abstract

Radiotherapy (RT) is one of the major methods of cancer treatment. RT destroys cancer cells, but also affects the tumor microenvironment (TME). The delicate balance between immunomodulation processes in TME is dependent, among other things, on a specific radiation dose. Despite many studies, the optimal dose has not been clearly determined. Here, we demonstrate that brachytherapy (contact radiotherapy) inhibits melanoma tumor growth in a dose-dependent manner. Doses of 10Gy and 15Gy cause the most effective tumor growth inhibition compared to the control group. Brachytherapy, at a single dose of ≥ 5Gy, resulted in reduced tumor blood vessel density. Only a dose of 10Gy had the greatest impact on changes in the levels of tumor-infiltrating immune cells. It most effectively reduced the accumulation of protumorogenic M2 tumor-associated macrophages and increased the infiltration of cytotoxic CD8⁺ T lymphocytes. To summarize, more knowledge about the effects of irradiation doses in anticancer therapy is needed. It may help in the optimization of RT treatment. Our results indicate that a single dose of 10Gy leads to the development of a robust immune response. It seems that it is able to convert a tumor microenvironment into an "in situ" vaccine and lead to a significant inhibition of tumor growth.

Keywords: brachytherapy; dose; tumor microenvironment; tumor vasculature; “in situ” vaccination.

Figure 1

Inhibition of B16-F10 tumor growth in response to brachytherapy. Mice with well-developed tumors ((A): 60–70 mm³; n = 5; (B) 180–200 mm³; n = 5) were treated with different doses (2–15Gy) of brachytherapy. Brachytherapy in a single dose of 2Gy slightly inhibits the growth of B16-F10 tumors compared to the control group. A single dose of 5Gy reduced tumors growth compared to the 2Gy and untreated groups of mice. The most effective tumor growth inhibition was observed in the group of mice that received a brachytherapy in a single dose of 10Gy or 15Gy. * p < 0.003 compared to compared to 2Gy and control groups, the ANOVA followed by the Tukey’s post hoc test. Photographs were taken on the 16^th (A) and 28^th (B) days of therapy. (C) Individual tumor follow-up.

Figure 1

Inhibition of B16-F10 tumor growth in response to brachytherapy. Mice with well-developed tumors ((A): 60–70 mm³; n = 5; (B) 180–200 mm³; n = 5) were treated with different doses (2–15Gy) of brachytherapy. Brachytherapy in a single dose of 2Gy slightly inhibits the growth of B16-F10 tumors compared to the control group. A single dose of 5Gy reduced tumors growth compared to the 2Gy and untreated groups of mice. The most effective tumor growth inhibition was observed in the group of mice that received a brachytherapy in a single dose of 10Gy or 15Gy. * p < 0.003 compared to compared to 2Gy and control groups, the ANOVA followed by the Tukey’s post hoc test. Photographs were taken on the 16^th (A) and 28^th (B) days of therapy. (C) Individual tumor follow-up.

Figure 2

The effect of brachytherapy on tumor blood vessels in B16-F10 tumors. On the 16^th day of brachytherapy, mice were sacrificed and tumors were excised for the immunohistochemical staining of blood vessels. α-SMA and CD31 stainings were used to identify pericyte-covered tumor vessels (α-SMA⁺CD31⁺ vessels, percentage of CD31⁺ vessels; n = 4–5; 10 visual fields per tumor section; magnification 20×). Brachytherapy affects the structure and density of tumor blood vessels in treated mice in a dose-dependent manner (percentage of CD31⁺ vessels; n = 4–5; 10 visual fields per tumor section; magnification 20×). The doses of 5Gy to 15Gy reduce the area of vessels (CD31⁺) by about 50% compared to the control group. Significant changes in the vessel structure were also observed, but only after treatment with 5Gy the vessels had a thicker layer of pericytes (αSMA⁺) adjacent to their surface. * p < 0.00001 compared to 2Gy and control groups; ** p < 0.005 compared to other groups, Kruskal Wallis test.

Figure 3

The effect of brachytherapy on the accumulation of tumor-associated macrophages (TAMs). On the 16^th day of therapy, the mice were sacrificed and tumors were excised for IHC analyzes. Tumors were stained with antibodies against CD206 and F4/80. Brachytherapy at a single dose of 10Gy has the greatest impact on the level of TAMs in B16-F10 tumors: the area of TAMs (F4/80⁺) increased more than four times, while the area of TAM M2 macrophages (F4/80⁺CD206⁺) was more than 3.5 times smaller compared to the control group. The areas of the TAM and M2 TAM macrophages were determined from three tumors per group; in each tumor 7 visual fields were analyzed (magnification 20×). * p < 0.003 compared to the control; ** p < 0.008 compared to the control, Kruskal Wallis test.

Figure 4

The effect of brachytherapy on the immune cells infiltration in the tumors. Six days after brachytherapy, the mice were sacrificed and tumor materials were collected for IHC and flow cytometry analyses. (A) Tumor cross-sections were stained with an antibody against CD8a. Brachytherapy at a single dose of 10Gy has the greatest effect on the infiltration of cytotoxic CD8⁺ lymphocytes in B16-F10 tumors. The level of tumor-infiltrating CD8⁺ T lymphocytes was increased almost sevenfold compared to the control group. The area of CD8⁺ lymphocytes was determined in four tumors per group; in each tumor, 10 visual fields were analyzed (magnification 20×). * p < 0.02 compared to 2Gy and control groups; ** p < 0.00001 compared to other groups, Kruskal Wallis test. (B) The level of T lymphocytes was determined in homogenous single-cell suspensions obtained from tumors (n = 4). The percentages of CD4⁺, CD8⁺ T lymphocytes, and NK cells were determined in the total viable CD45⁺ cells. Brachytherapy at a single dose of 10Gy increased the level of tumor-infiltrating CD8⁺ T lymphocytes (three times) compared to the control group. A higher level of NK cells was noted after a single dose of 15Gy brachytherapy. * p < 0.02 compared to the 5Gy, 15Gy, and control groups, Kruskal Wallis test; ** p < 0.007 compared to the other groups, the ANOVA followed by the Tukey’s post hoc test.