Radiation-induced amphiregulin drives tumor metastasis
The anti-tumor effect of radiotherapy (RT) beyond the treatment field - the abscopal effect - has garnered much interest. By contrast, the potentially harmful impact of radiation in promoting metastasis is less well studied. Here, we show that RT induces the expression of epidermal growth factor receptor (EGFR) ligand amphiregulin (AREG) in tumor cells, which reprograms EGFR+ myeloid cells toward an immunosuppressive phenotype and reduces phagocytosis. This stimulates distant metastasis growth in both patients and pre-clinical murine tumor models. The inhibition of these tumor-promoting factors induced by RT may represent a novel therapeutic strategy to improve patient outcomes. Overall design: RNA-seq profliing of two separate experiments. The first experiment contains WT and WT_IR samples. WT samples contain RNA isolated from LLC tumor cells expressing RFP (LLC-RFP) cultured in vitro. WT_IR samples contain RNA isolated from LLC-RFP tumor cells cultured in vitro and treated with 4Gy irradiation. RNA was collected 12 hours after irradiation - shown in Extended Data Fig. 4j-k of the manuscript - three technical repeats for each condition. The second experiment contains files labelled WT_mono_WT_tumor, WT_mono_AREG_KO_tumor, EGFR_KO_mono_WT_tumor and EGFR_KO_mono_AREG_KO_tumor.The samples contain RNA isolated from Ly6C+ mononuclear phagocytes (MNPs) generated from bone marrow culture of EGFR+ wildtype C57BL/6 mice or conditionally Egfr-deficient mice (LysMCre Egfr-/-) - also three technical repeats of each condition. Ly6C+ MNPs were collected after 24 co-culturing bone-marrow derived cells with LLC-RFP tumor cells (LLC cells expressing RFP, labelled LLCAR+ in the manuscript) or with LLC-AR- tumor cells (LLC cells expressing RFP in which Amphiregulin has been knocked out by CRISPR gene editing). After co-culture, only Ly6C+ MNPs were sorted and used for RNA isolation. The title of each sample contains the information of the co-culture setup, for example WT_mono_WT_tumor contain RNA from Ly6C+ MNPs generated from bone marrow of EGFR+ control mice which were co-cultured with WT LLC-RFP tumor cells, whereas WT_mono_AREG_KO_tumor samples contain RNA from Ly6C+ MNPs generated from bone marrow of EGFR+ control mice which were co-cultured with LLC-AREG-KO tumor cells and the same naming system applies to the Ly6C+ MNPs from EGFR- (LysmCre Egfr-/-) mice that were co-cultured either with LLC-RFP (WT) or AREG-KO LLC cells before sorting. (These data were used to generate Extended Data Fig. 8 f-i of the manuscript).