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Fig. 4 | Breast Cancer Research

Fig. 4

From: The fibroblast Tiam1-osteopontin pathway modulates breast cancer invasion and metastasis

Fig. 4

OPN inhibition prevents effects of Tiam1-deficient fibroblasts on breast cancer cell invasion, migration, and cancer stem cell-like populations. a-d Inhibition with OPN silencing. a Number of projections/spheroid for SUM1315 breast cancer cells and indicated mammary fibroblasts in 3D mixed cell spheroid co-culture. At least 130 spheroids were counted for each condition; results represent duplicate experiments. b Transwell migration of SUM1315 after isolation from mixed cell spheroid co-cultures with indicated mammary fibroblasts. Cell counts were averaged across nine high-power fields for biologic triplicates. c SUM1315 breast cancer cells isolated from 3D co-cultures with indicated mammary fibroblasts were cultured at low density under ultra-low adherence conditions and tumorspheres were quantitated under light microscopy. d Populations of SUM1315 breast cancer cells were quantified by flow cytometry for expression of indicated cell surface markers using fluorophor-conjugated antibodies after isolation from 3D co-cultures with indicated fibroblasts. For fibroblasts in A-D, shC sLuc = control silencing retroviral hairpin vector and luciferase silencing lentiviral hairpin vector; shTiam shLuc = Tiam1 and luciferase silencing hairpins; shTiam shOPN = Tiam1 and OPN silencing hairpins. eh Inhibition with OPN antibody. Results of mixed cell co-cultures established as in Fig. 1a with SUM1315 breast cancer cells and either control (shC) or Tiam1-deficient (shTiam) fibroblasts, incorporating IgG or OPN antibody as indicated. e Number of projections/spheroid. At least 180 spheroids were counted for each condition; results represent duplicate experiments. f Transwell migration of SUM1315 after isolation from mixed cell spheroid co-cultures with indicated mammary fibroblasts. Cell counts were averaged across nine high-power fields. g SUM1315 breast cancer cells isolated from 3D co-cultures with indicated mammary fibroblasts were cultured at low density under ultra-low adherence conditions and tumorspheres were quantitated under light microscopy. h Populations of SUM1315 breast cancer cells were quantified by flow cytometry for expression of indicated cell surface markers using fluorophor-conjugated antibodies after isolation from 3D co-cultures with indicated fibroblasts. For b, c, d, f, g, and h results represent duplicate experiments, each with biologic triplicates

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