Immunomodulatory effects of carbon ion radiotherapy in patients with localized prostate cancer

Abstract

Purpose: Radiotherapy is one of the main local treatment modalities for prostate cancer, while immunosuppressive effect induced by radiotherapy is an important factor of radiation resistance and treatment failure. Carbon ion radiotherapy (CIRT) is a novel radiotherapy technique and the immunomodulatory effect of CIRT provides the possibility of overcoming radioresistance and improving efficacy. The aim of this study was to assess the immune response evoked by CIRT in localized prostate cancer patients.

Methods: Thirty-two patients were treated by CIRT combined with or without hormone therapy and peripheral blood samples were collected before and after CIRT. Investigation of peripheral immune cell frequency, proliferation, and cytokine expression was conducted by flow cytometry, real-time quantitative PCR and ELISA.

Results: There were no significant differences in the frequencies of CD3 + , CD4 + , CD8 + T cells and NK cells after CIRT. CD4/CD8 ratio increased whereas B cells decreased. All lymphocyte subsets except regulatory T cells (Tregs) displayed increased proliferation and T cells exhibited increased functionality after CIRT, characterized by modestly increased cytokine secretion of TNF. Moreover, higher frequencies of Tregs were shown. Neither monocytic myeloid-derived suppressor cells (MDSCs) nor early MDSCs changed after CIRT. TGF-β1 gene expression decreased while IL-6 showed a non-significant trend towards a decrease. Both IL-10 gene expression and plasma TGF-β1 level were unchanged.

Conclusion: CIRT demonstrates the potential to elicit immune activation in localized prostate cancer patients, based on sparing lymphocytes, increased lymphocyte proliferation, enhanced T-cell functionality, together with limited induction of immunosuppressive cells and reduced expression of immunosuppressive cytokines.

Keywords: Carbon ion radiotherapy; Immune response; Immunomodulatory effect; Peripheral immune cells; Prostate cancer.

Fig. 1

Effects of CIRT on lymphocyte subsets. a Percentage of CD3 + T cells within viable lymphocytes, b, c percentage of CD4 + and CD8 + T cells within CD3 + T cells, d CD4/CD8 ratio, and e, f percentage of CD19 + B cells and CD56 + NK cells within viable lymphocytes are shown before and after CIRT. N = 32 CIRT patients are included. Significant differences are depicted as *p < 0.05 and **p < 0.01

Fig. 2

Proliferation of lymphocyte subsets. Percentages of Ki67 + cells within (a) CD3 + T cells, (b) CD4 + T cells, (c) CD8 + T cells, (d) B cells and (e) NK cells are shown before and after CIRT. N = 32 CIRT patients are included. Significant differences are depicted as *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001

Fig. 3

Function of T cells. Intracellular cytokine (IFN-γ and TNF) production of CD4 + (a) and CD8 + (b) T cells before and after CIRT. Representative gating for IFN-γ + and TNF + cells in the subset of CD4 + and CD8 + T cells is shown. N = 21 CIRT patients are included. Significant difference is depicted as *p < 0.05, and p < 0.1 is considered as statistical trend

Fig. 4

Effects of CIRT on Tregs. a Gating strategy of Tregs is shown. b Percentage of Tregs within CD4 + T cells, and c percentage of Ki67 + cells within Tregs are shown before and after CIRT. N = 32 CIRT patients are included. Significant difference is depicted as ***p < 0.001

Fig. 5

Effects of CIRT on MDSCs. a Gating strategy of MDSCs is shown. b, c Percentages of M-MDSCs and E-MDSCs within viable PBMCs are shown before and after CIRT. N = 32 CIRT patients are included

Fig. 6

Cytokine mRNA and protein levels and correlations between mRNA and protein levels. Cytokine gene expression levels of (a) TGF-β1, (b) IL-10 and (c) IL-6 are shown before and after CIRT. N = 32 CIRT patients are included. (d) Plasma TGF-β1 concentration is shown before and after CIRT. N = 31 CIRT patients are included. (e, f) Correlations between gene and protein levels of TGF-β1 at pre-CIRT or post-CIRT timepoint. N = 31 patients are included. Significant difference is depicted as *p < 0.05

Fig. 7

Immune changes after CIRT with or without SIB. Comparison of patients treated with CIRT with or without SIB (N = 15 or 17, respectively) for (a) percentage of CD4 + T cells within CD3 + T cells, (b) CD4/CD8 ratio, (c) CD19 + B cells within viable lymphocytes, (d) Tregs within CD4 + T cells, and (e) Ki67 + cells within Tregs. Significant differences are depicted as *p < 0.05 and **p < 0.01