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. 2020 Oct 26;10(26):12127–12143. doi: 10.7150/thno.52450

Figure 3.

Figure 3

Activation of stromal cells in vitro. A) Cell activation pathway. Normal stromal cells (HELFs and HUVECs) were cultured in conditioned medium from tumor cells (HCT116-CM), and markers of cell activation were measured. + indicates >2-fold upregulation of gene expression, +++ indicates >6-fold upregulation of expression. B) Immunofluorescence images of HELFs and CAFs (green = α-SMA; blue (DAPI) = nuclei) (scale bar = 100 µm). C) Expression of genes encoding FAP, FSP1, collagen I, and TGF-β in fibroblasts activated with CM for 72 h. D) Expression of α-SMA in fibroblasts treated with CM for 0-72 h. E) Expression of the tenascin C gene in activated CAFs (but not in normal HELFs). Cells in the control group in C, D & E are HELFs. F-G) Expression of activated marker genes TEM1 (F) and TEM8 (G) in HUVECs treated with CM for 0-72 h. H) The expression of genes encoding VEGF and biglycan in HUVECs activated with CM for 72 h. Cells in the control group in F, G & H are HUVECs. I) ELISA of VEGF secreted by HUVECs before and after activation (TECs). J) Morphology of HUVECs before and after activation (TECs) (red (phalloidin) = F-actin, scale bar = 100 µm). K) Phenotype of HUVECs before and after activation (TECs) (CD144 (green), scale bar = 100 µm). L) Expression of α-SMA in HELFs cultured on collagen-coated Petri dishes (2D environment) and 3D collagen-PCL scaffolds (3D environment) 72 h after treatment with HCT116-CM. M) Expression of E-cadherin, N-cadherin, and integrin-β1 in HELFs cultured on 3D scaffolds for 48 h. N) Characterization of cell adhesion after inhibition of integrin (scale bar = 100 µm). O) Expression of α-SMA in HELFs cultured on collagen-coated Petri dishes (2D environment), 3D collagen-PCL scaffolds (3D environment), and 3D collagen-PCL scaffolds with inhibition of integrin 72 h after treatment with HCT116-CM (scale bar = 100 µm). *p < 0.05. All values represent means ± SD, n = 5.