Journal: Journal of Translational Medicine
Article Title: Interleukin-1β mediates a tumor-supporting environment prompted by IGF1 in triple-negative breast cancer (TNBC)
doi: 10.1186/s12967-025-06730-w
Figure Lengend Snippet: IL-1β stimulates the reprogramming of normal fibroblasts into CAFs-like cells that in turn stimulate aggressive features in TNBC cells. A FAP expression evaluated by immunofluorescence assays in WI38 cells cultured for 72 h with conditioned medium (CM) collected from MDA-MB-231 cells previously exposed for 4 h to vehicle or 50 ng/ml IGF1 alone or in combination with 5 µg/ml IL-1R1 antagonist raleukin. Nuclei were stained by DAPI. Scale bar: 100 μm. C Representative pictures of spheroids (a single spheroid/well) grown for 6 days on agar-coated plates from WI38 cells cultured for 72 h with conditioned medium (CM) collected from MDA-MB-231 cells previously exposed for 4 h to vehicle or 50 ng/ml IGF1 alone or in combination with 5 µg/ml IL-1R1 antagonist raleukin. Scale bar: 1000 μm. E FAP expression evaluated by immunofluorescence assays in WI38 cells exposed to vehicle or IL-1β (10 ng/ml) alone or in combination with 5 µg/ml IL-1R1 antagonist raleukin. Nuclei were stained by DAPI. Scale bar: 100 μm. B , F Fluorescence intensities were quantified in 20 random fields for each condition and results are expressed as fold change of relative fluorescence units (RFU) over vehicle-treated cells (set as one-fold induction). G Representative pictures of spheroids (a single spheroid/well) grown for 6 days on agar-coated plates from WI38 cells exposed for 72 h to vehicle or IL-1β (10 ng/ml) alone or in combination with 5 µg/ml IL-1R1 antagonist raleukin. Scale bar: 1000 μm. D , H Quantification of spheroid growth. Values of vehicle-treated WI38 cells were set as 100% upon which spheroid growth was determined. I Transwell migration assays in MDA-MB-231 cells co-cultured for 8 h with WI38 cells (NFs) cultured for 72 h with conditioned medium (CM) collected from MDA-MB-231 cells, which were previously exposed for 4 h to vehicle or 50 ng/ml IGF1 alone or in combination with 5 µg/ml IL-1R1 antagonist raleukin. K Representative pictures of sodium alginate beads encapsulating MDA-MB-231 cells cultured for 10 days in bioreactors in the presence of the conditioned medium (CM) of WI38 cells (NFs) treated for 72 h with the conditioned medium (CM) collected from MDA-MB-231 cells, which were previously exposed for 4 h to vehicle or 50 ng/ml IGF1, alone or in combination with 5 µg/ml IL-1R1 antagonist raleukin. M Transwell migration assay in MDA-MB-231 cells co-cultured for 8 h with WI38 cells (NFs) previously exposed for 72 h to vehicle ( −) or IL-1β (10 ng/ml) alone or in combination with 5 µg/ml IL-1R1 antagonist raleukin. J , N Cells were counted in at least 5 random fields in three independent experiments performed in triplicate. Scale bar: 100 μm. O Representative pictures of sodium alginate beads encapsulating MDA-MB-231 cells cultured for 10 days in bioreactors with the conditioned medium (CM) of WI38 cells (NFs) exposed for 72 h to vehicle ( −) or IL-1β (10 ng/ml) alone or in combination with 5 µg/ml IL-1R1 antagonist raleukin. (L, P) Cell viability of MDA-MB-231 in the sodium alginate beads. Scale bar: 1000 μm. Values of vehicle-treated MDA-MB-231 cells were set as 100% upon which cell viability was determined. Data shown are the mean ± SD of three independent experiments performed in triplicate. (*) indicates p < 0.05. Q Cartoon depicting the molecular events and the biological responses triggered by IL-1β within the TNBC microenvironment. Created with BioRender.com
Article Snippet: Next, cells were fixed in 4% paraformaldehyde in PBS, permeabilized with 0.2% Triton X-100, washed 3 times with PBS and incubated at 4 °C overnight with the primary antibody (1:250) against fibroblast-activated protein (FAP) (H-56; Santa Cruz Biotechnology, DBA, Milan, Italy).
Techniques: Expressing, Immunofluorescence, Cell Culture, Staining, Fluorescence, Migration, Transwell Migration Assay
