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

a, Heatmap representing color-coded expression levels of commonly deregulated genes in H2030-BrM cell line when cultured under radiosensitive (purple line, adherent; light green line, co-culture with inserts) compared with radioresistant (dark green line, oncospheres; blue line, cell–cell co-culture) conditions in vitro. Only genes with false discovery rate (FDR) < 0.05 and a log₂ ratio >1 were considered. b, Representative images of S100A9 protein expression levels in metastatic lesions growing in brains from mice IC injected with H2030-BrM and E0771-BrM. Three brains were analyzed in each BrM model. Scale bars, 120 µm (H2030-BrM) and 50 µm (E0771-BrM). c, Sixty-six human brain metastases from patients with lung cancer (33 cases) or breast cancer (30 cases) or other primary tumors (3 cases) were stained for S100A9 by immunohistochemistry. Representative images are shown. Scale bar, 50 µm. Quantification of different histological scoring of cancer cells is shown in pie charts. Five cases had to be excluded; 27 of 61 were scored with no staining (score 0), 9 of 61 with weak staining (score 1), 12 of 61 with moderate staining (score 2) and 13 of 61 with strong staining (score 3). d, Quantification of S100A9 expression levels in H2030-BrM after stimulation with 100 ng ml⁻¹ recombinant CXCL1 (rCXCL1) or control. Values are shown in dot plots, and dots represent independent experiments. The line in the box corresponds to the median. The boxes go from the upper to the lower quartiles, and the whiskers go from the minimum to the maximum value (n = 7, each experimental condition). P value was calculated Wilcoxon signed rank test, two sided. e, Quantification of S100A9 expression levels in H2030-BrM after stimulation with recombinant transforming growth factor α (rTGF-α) or control. Values are shown in dot plots and dots represent independent experiments. The line in the box corresponds to the median. The boxes go from the upper to the lower quartiles, and the whiskers go from the minimum to the maximum value (n = 7, each experimental condition). P value was calculated using two-tailed t-test. f, Quantification by enzyme-linked immunosorbent assay (ELISA) of human S100A9 (hS100A9) in the supernatant of H2030-BrM grown either under adherent conditions in vitro or in organotypic cultures ex vivo. Values are shown in box-and-whisker plots, where every dot represents an independent experiment and the line in the box corresponds to the median. The boxes go from the upper to the lower quartiles, and the whiskers go from the minimum to the maximum value (n = 6, H2030-BrM adherent cultures; n = 8, H2030-BrM growing in organotypic brain cultures). P value was calculated using two-tailed Mann–Whitney test. g, Quantification of in vitro viable cell fraction after irradiation at 10 Gy and 200 ng ml⁻¹ recombinant hS100A9 or control, as determined by manual cell counting of bisbenzimide-positive 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 (n = 3, each experimental condition). P value was calculated using a two-tailed t-test. h, Representative pictures of RAGE immunohistochemistry from unirradiated or irradiated established H2030-BrM metastases in vivo. Scale bar, 50 µm. i, Quantification of the percentage of NF-κB⁺ GFP⁺ H2030-BrM cells identified by the expression of an engineered mCherry NF-κB activity reporter. Brain slices with cancer cells were evaluated 72 h after treatment with radiotherapy. Values are shown in box-and-whisker plots where each dot is a brain organotypic culture (n = 11, nonirradiated; n = 7, irradiated with a single dose of 10 Gy) and the line in the box corresponds to the median. The boxes go from the upper to the lower quartiles and the whiskers go from the minimum to the maximum value. P value was calculated using a two-tailed t-test. j, Schema of working model of radioresistance in brain metastasis.