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. 2021 Jun 10;12:3516. doi: 10.1038/s41467-021-23583-1

Fig. 3. Spheroid co-cultures recapitulate in vivo Endo180 impact.

Fig. 3

a, b Fibroblast-tumour cell co-culture spheroid assays. a 300 D2A1 cells alone or in combination with 600 mouse CAFs, NF#1 or 3T3 fibroblasts transfected with non-targeting control (NTC) or Endo180 (E180) targeting siRNA oligonucleotides were seeded into U-bottom low adherence plates and cultured for 8 days. Spheroid growth was assessed by spheroid area and CellTiter-Glo. Box plots shows median and 25th–75th quartiles, whiskers show minimum and maximum. Dotted lines show mean spheroid size/CellTiter-Glo value of tumour cells alone (n = 6 spheroids per condition; mean values ± SEM; panel 2, two-sided Mann–Whitney U-test; other panels, two-sided t-test). b Representative images of D2A1 spheroids alone or co-cultured with siNTC or siE180-transfected CAFs (scale bar, 200 µm). c D2A1 spheroids cultured for 8 days in serum-free conditioned medium (CM) collected from siRNA-transfected CAFs supplemented with 2% FBS. Box plots show median and 25th–75th quartiles, whiskers show minimum and maximum. Dotted line shows mean spheroid size in control DMEM plus 2% FBS (n = 5 spheroids per condition; mean values ± SEM, two-sided Mann–Whitney U-test). d D2A1 colony formation assay in the presence of CM from mock, siRNA-transfected CAFs and supplemented with 2% FBS (n = 2). e CAFs transduced with shNTC or two independent shRNAs targeting Endo180 cultured with or without CM from 4T1 cells supplemented with 2.5% FBS. Cultures were stained for fibronectin. Left panel, representative images of fibronectin fibres (scale bar, 100 µm). Right panels, quantification of matrix density, normalised endpoints and normalised branch points (n = 11–16 fields of view per condition as indicated; mean values ± SEM, two-way ANOVA, Tukey post-hoc test). f 4T1 cell growth, measured by confluency, on ECM derived from CAFs or 3T3 fibroblasts transduced with shNTC or two independent shRNAs targeting Endo180 (n = 3 wells per condition; mean values ± SEM, two-way ANOVA, non-significant in all comparisons). g D2A1 colony formation assay on ECM derived from mock transfected CAFs or CAFs transfected with siNTC or two independent siRNAs targeting Endo180 (MOCK siNTC, siE180-12, n = 3; mean values ± SEM, one-way ANOVA; siE180-09, n = 2). Non-significant (ns) P values > 0.05. Source data are provided as a Source Data file.