Radiation-induced rescue effect on human breast carcinoma cells is regulated by macrophages

Abstract

The susceptibility of cancer cells to DNA damages is influenced by their microenvironment. For example, unirradiated neighbors of irradiated cells can produce signals that reduce DNA damages. This phenomenon, known as Radiation-Induced Rescue Effect (RIRE), has profound implications on the efficacy of radiotherapy. Using bystander cells co-cultured with mock-irradiated cells as a control, we demonstrated, for the first time, two types of RIRE. Conditioned medium from naïve by stander cells, i.e., cells not exposed to irradiated cells, could mitigate UV-induced DNA damages in human breast carcinoma MCF7 cells, as judged by phospho-H2AX and 53BP1 immunostaining. This protective effect could be further enhanced by the prior treatment of bystander cells with factors from UV-irradiated cells. We named the former effect "basal RIRE" and the latter "active RIRE" which were cell type-dependent. As bystanders, MCF7 showed a significant active RIRE, whereas THP1-derived macrophages showed a strong basal RIRE but no active RIRE. Interestingly, RIRE of macrophages could further be modulated by polarisation. The basal RIRE of macrophages was abolished by M1 polarisation, while M2 and Tumour Associated Macrophages (TAM) demonstrated pronounced basal and active RIRE. When mixtures of MCF7 cells and polarised macrophages were used as bystanders, the overall RIRE was dictated by macrophage phenotypes: RIRE was suppressed by M1 macrophages but significantly enhanced by M2 and TAM. This study shows a previously unappreciated role of the innate immune system in RIRE. Depending on polarised phenotypes, macrophages in the tumour microenvironment can interfere with the effectiveness of radiotherapy by adjusting the RIRE magnitudes.

Keywords: DNA damage; M1/M2 macrophages; Radiation-induced rescue effect (RIRE); Tumour-associated macrophages (TAMs); UV.

Fig. 1

Schematic diagram showing the procedures for the differentiation and polarisation of THP1 cells into four different macrophage subtypes, namely, M0, M1, M2, and TAM.

Fig. 2

Schematic diagram showing the experimental design of this study. Inset illustrates the nomenclature of the samples. After irradiation, the originators were co-cultured with bystanders in a medium represented by pink. Subsequently, the bystanders were transferred to a new medium, referred to as the conditioned medium, indicated by green. See text for detailed explanations.

Fig. 3

(a) Representative images from immunofluorescence staining with anti-phospho-histone H2A.X and anti-53BP1 antibodies in MCF7 cells post-UVC irradiation. CM experiments with MCF7 cells as Bystanders were carried out in the following conditions, (I) IR-CM(MCF7)-IR, (II) UIR-CM(MCF7)-IR, (III) CM(MCF7)-IR. Control experiments included (IV) IR-FM and (V) UIR-FM. Scale bar = 25 μm. (b) Graph representing relative γH2AX staining relative to that in UIR-FM cells, which is set as the baseline (dotted lines) for these conditions. (c) Graph representing relative 53BP1 staining (relative to UIR-FM) for these conditions. ∗P < 0.05; ∗∗P < 0.01 and error bars represent mean ± SD.

Fig. 4

(a) Representative images from immunofluorescence staining with anti-phospho-histone H2A.X and anti-53BP1 antibodies in MCF7 cells post-UVC irradiation. CM experiments with M0 cells as Bystanders were carried out in the following conditions, (I) IR-CM(M0)-IR, (II) UIR-CM(M0)-IR, (III) CM(M0)-IR. Control experiments included (IV) IR-FM and (V) UIR-FM. Scale bar = 25 μm. (b) Graph representing relative γH2AX staining (linear values relative to UIR-FM) for these conditions. (c) Graph representing relative 53BP1 staining (linear values relative to UIR-FM) for these conditions. ∗P < 0.05; ∗∗P < 0.01 and error bars represent mean ± SD.

Fig. 5

(a) Representative images from immunofluorescence staining with anti-phospho-histone H2A.X and anti-53BP1 antibodies in MCF7 cells post-UVC irradiation. CM experiments with M1 cells as Bystanders were carried out in the following conditions, (I) IR-CM(M1)-IR, (II) UIR-CM(M1)-IR, (III) CM(M1)-IR. Control experiments included (IV) IR-FM and (V) UIR-FM. Scale bar = 25 μm. (b) Graph representing relative γH2AX staining (linear values relative to UIR-FM) for these conditions. (c) Graph representing relative 53BP1 staining (linear values relative to UIR-FM) for these conditions. ∗P < 0.05; ∗∗P < 0.01 and error bars represent mean ± SD.

Fig. 6

(a) Representative images from immunofluorescence staining with anti-phospho-histone H2A.X and anti-53BP1 antibodies in MCF7 cells post-UVC irradiation. CM experiments with M2 cells as Bystanders were carried out in the following conditions, (I) IR-CM(M2)-IR, (II) UIR-CM(M2)-IR, (III) CM(M2)-IR. Control experiments included (IV) IR-FM and (V) UIR-FM. Scale bar = 25 μm. (b) Graph representing relative γH2AX staining (linear values relative to UIR-FM) for these conditions. (c) Graph representing relative 53BP1 staining (linear values relative to UIR-FM) for these conditions. ∗P < 0.05; ∗∗P < 0.01 and error bars represent mean ± SD.

Fig. 7

(a) Representative images from immunofluorescence staining with anti-phospho-histone H2A.X and anti-53BP1 antibodies in MCF7 cells post-UVC irradiation. CM experiments with TAMs as Bystanders were carried out in the following conditions, (I) IR-CM(TAM)-IR, (II) UIR-CM(TAM)-IR, (III) CM(TAM)-IR. Control experiments included (IV) IR-FM and (V) UIR-FM. Scale bar = 25 μm. (b) Graph representing relative γH2AX staining (linear values relative to UIR-FM) for these conditions. (c) Graph representing relative 53BP1 staining (linear values relative to UIR-FM) for these conditions. ∗P < 0.05; ∗∗P < 0.01 and error bars represent mean ± SD.

Fig. 8

The Fig. represents the data obtained from MCF7 cells treated with CM obtained from Bystanders of MCF7 and different macrophage subtypes (M0, M1, M2, and TAMs) with or without MCF7, which were previously co-cultured with Originators. (a) Representative images from immunofluorescence staining with anti-phospho-histone H2A.X and anti-53BP1 antibodies in MCF7 cells post-UVC irradiation. Scale bar = 25 μm ∗P < 0.05; ∗∗P < 0.01 and error bars represent mean ± SD. (b) The graph represents relative γH2AX staining (linear values relative to UIR-FM) for IR-CM-IR (macrophage) and IR-CM-IR (macrophage and MCF7). (c) The graph represents the average fluorescent intensities of γH2AX for the above-said conditions. (d) The graph represents relative γH2AX and 53BP1 staining (linear values relative to UIR-FM) for IR-CM-IR (MCF7).