Extended Data Fig. 2: Contact-dependent astrocyte-released cytokines induce S100A9 secretion in cancer cells triggering NF-κB activation.

a, Heatmap depicting the gene signature in Fig. 2a scored in the comparison between H2030-BrM growing in adherent conditions in vitro and in brain organotypic cultures ex vivo. Only genes with FDR < 0.05 and a log₂ ratio >1 were considered. b, Quantification of in vitro viable cell fraction after irradiation at 10 Gy and stimulation with 100 ng/ml rCXCL1 or control, as determined by manual cell counting of bisbenzimide+ nuclei. Values are percentages of viable cells respect to unirradiated controls and shown in a dot plot where each dot represents an independent experiment and the line in the box corresponds to the median and whiskers go from the minimum to the maximum value (n = 3, each experimental condition). P value was calculated using two-tailed t test. c, Representative image of the peritumoral microenvironment where CXCL1 mRNA is labelled by RNA in situ hybridization (brown) and GFAP by immunohistochemistry (purple). The dotted line surrounds the metastasis. cc: cancer cells. Scale bar: 50 µm. The experiment was independently repeated three times with similar results. d, Representative image of CXCR2 protein in H2030-BrM brain metastasis. The dotted line surrounds the metastasis. cc: cancer cells. Scale bar: 25 µm. The experiment was independently repeated three times with similar results. e, Quantification of in vitro viable cell fraction after irradiation at 10 Gy and stimulation with rTGFα or control, as determined by manual cell counting of bisbenzimide+ nuclei. Values are percentages of viable cells respect to unirradiated controls and shown in a dot plot where each dot represents an independent experiment and the line in the box corresponds to the median median and whiskers go from the minimum to the maximum value (n = 4, each experimental condition). P value was calculated using two-tailed t test. f, Representative image of the peritumoral microenvironment where TGFα co-localizes with astrocytes (GFAP + ). Scale bar: 10 µm. The experiment was independently repeated three times with similar results. g, Representative image of EGFR immunohistochemistry in experimental H2030-BrM brain metastasis. Scale bar: 50 µm. The experiment was independently repeated three times with similar results. h, Differentially expressed genes in resistant culture preparations (2 biological independent replicas used for each radioresistant preparation (oncospheres, n = 2; co-cultures (cell–cell contact), n = 2), and for radiosensitive preparations, (adherent cultures, n = 2; co-cultures (insert), n = 2)) were used to perform Gene Ontology (GO) analysis using the EnrichR software. The volcano plot represents the significance of each gene set from GO Molecular Function versus its odds ratio. P value is computed from the Fisher exact test. Larger blue points represent significant terms (P value<0.05); smaller grey points represent non-significant terms. The darker the blue color of a point, the more significant it is. i, Quantification of RAGE expression levels in H2030-BrM 24 hours after a single dose (10 Gy) of radiation. Both adherent (grey) and oncospheres (red) are plotted together. Each circle represents independent cultures (n = 3 nonirradiated adherent cells, n = 3 nonirradiated oncospheres, n = 3 nonirradiated adherent cells, n = 2 nonirradiated oncospheres). Expression values were normalized to their respective nonirradiated control for each culture condition and the line represents the mean. P value was calculated using two-tailed t test. Nonirradiated adherent cells versus irradiated adherent cells, P = 0.1521; nonirradiated oncospheres versus irradiated oncospheres, P = 0.0522. j, 66 human brain metastases from lung cancer patients (33 cases), breast cancer patients (30 cases) or patients with other primary tumors (3 cases) were stained for RAGE by immunohistochemistry. Representative images are shown. Scale bar: 50 µm. Quantification of different histological scoring of cancer cells is shown in pie charts. 6 cases had to be excluded, 15/60 were scored with no staining, 17/60 with weak staining and 28/60 with moderate staining.