PMC

Bi-directional communication between MSCs and mammary cancer cells in stiff tumor microenvironment.

A, Growth assay of MSCs cultured with 4T1-conditioned media (CM) on floating or attached collagen gels of 1 mg/mL collagen. n = 3 experiments. B, Morphology assay of MSCs. Green: F-actin. n = 30 cells in 3 experiments. C, Western blot of αSMA, vimentin, and β-actin in MSCs. n = 3 experiments. D, Fluorescence images of nuclei, αSMA, and vimentin in MSCs. E, Phase contrast images of MSCs cultured with CM on polyacrylamide gels of 0.01, 0.16, 0.32% BIS concentration and the corresponding stiffness of the gels. F, Western blot of αSMA, vimentin, and β-actin in MSCs. n = 3 experiments. Statistical significance was determined as . Mean±S.E. are shown. Bar = 100 µm. MSCs were established from BALBc mice. F: floating collagen gels, A: attached collagen gels.

A, Growth assay of MSCs cultured with 4T1-conditioned media (CM) or control DMEM (DM) on floating collagen gels of 1, 2.5, or 4 mg/mL collagen. n = 3 (1, 4 mg/mL), 2 (2.5 mg/mL) experiments. B, Morphology assay of MSCs. Green: F-actin. Cells were quantitated for overall area, n = 30 cells in 3 (1, 4 mg/mL), 2 (2.5 mg/mL) experiments. C, Viability assay of MSCs using calcein AM. n = 3 experiments. D, Western blot of αSMA, vimentin, and β-actin in MSCs. n = 4 (1, 4 mg/mL) or n = 3 (2.5 mg/mL) experiments. Statistical significance was determined as . Mean±S.E. are shown. Bar = 100 µm. MSCs were established from BALBc mice.

A, Western blot of YAP and β-actin in MSCs cultured with 4T1-conditioned media (CM) on floating collagen gels of 1 or 4 mg/mL collagen. n = 3 experiments. B, Western blot of YAP and β-actin in MSCs cultured with CM on floating (F) or attached (A) collagen gels of 1 mg/mL collagen. n = 4 experiments. C, Western blot of YAP and β-actin in MSCs cultured with CM on polyacrylamide gels of 0.01, 0.16, 0.32% BIS concentration. n = 3 experiments. D, Fluorescence images of nuclei, YAP, and F-actin in MSCs. Relative intensity of YAP in nuclei was quantified in n = 30 cells in 3 experiments. E, Luciferase assay of a YAP reporter construct in MSCs. n = 3 experiments. F, Western blot of 2P-MLC, 1P-MLC, and β-actin in MSCs. n = 3 experiments. G, Western blot of 2P-MLC, 1P-MLC, and β-actin in MSCs cultured with CM on polyacrylamide gels of 0.01 and 0.32% BIS concentration. n = 3 experiments. Statistical significance was determined as . N.S.: no significance with 95% confidence interval. Mean±S.E. are shown. Bar = 100 µm. MSCs were established from BALBc mice.

A, Western blot of αSMA, YAP, 2P-MLC, 1P-MLC, and β-actin in non-treated (NT) or 10 µM H1152-treated MSCs cultured with 4T1-conditioned media (CM) on attached collagen gels of 1 mg/mL collagen. n = 3 experiments. B, Fluorescence images of nuclei, YAP, and F-actin in NT or 10 µM H1152-treated MSCs. Relative intensity of YAP in nuclei was quantified from n = 24 cells in 3 experiments. C, Western blot of αSMA, YAP, 2P-MLC, 1P-MLC, and β-actin in EGFP- or YAP-MSCs cultured on a collagen-coated plastic dish for 1 day. n = 3 experiments. D, Western blot of αSMA, YAP, 2P-MLC, 1P-MLC, and β-actin in empty- or shYAP-MSCs cultured with CM on attached collagen gels of 1 mg/mL collagen. n = 3 experiments. Statistical significance was determined as . N.S.: no significance with 95% confidence interval. Mean±S.E. are shown. Bar = 100 µm. MSCs were established from BALBc mice.

A, Growth assay of 4T1 cells cultured with control DMEM (NT), conditioned media of MSCs cultured with 4T1-conditioned media on floating collagen gels of 1 mg/mL collagen (1F), on floating collagen gels of 4 mg/mL collagen (4F), or on attached collagen gels of 1 mg/mL collagen (1A), n = 9 samples in 3 experiments. B, Volume of tumors 7 days after injection. WT or COL mice were injected with 4T1 cells only (4T1) or 4T1 cells with empty-MSCs (4T1-MSCs). n = 8 tumors in 4 mice (4T1), 10 in 5 mice (4T1-MSCs). C, Proteomics analysis of conditioned media from MSCs. D, Western blot of PSAP and β-actin in conditioned media (CM) or cell lysate of MSCs cultured with 4T1-conditioned media on floating (F) or attached (A) collagen gels of 1 mg/mL collagen. CM: n = 3 technical replicates. Cell lysate: n = 3 experiments. E, qPCR of PSAP, MMP13, and MMP3 in MSCs. n = 3 experiments. F, Western blot of YAP, PSAP, and β-actin in CM or cell lysate of EGFP- or YAP-MSCs cultured with 4T1-conditioned media on floating collagen gels of 1 mg/mL collagen. CM: n = 6 samples in 3 experiments. Cell lysate: n = 6 samples in 2 experiments. G, qPCR of PSAP in control EGFP or YAP-MSCs on a collagen-coated plastic dish. n = 4 experiments. Statistical significance was determined as . N.S.: no significance (P>0.05) with Wilcoxon rank sum test (B). or no significance with 95% confidence interval (D, F, and G). Mean±S.E. are shown. MSCs were established from BALBc mice.

A, Growth assay of 4T1 cells cultured with control DMEM (NT) or DMEM with exogenously added 20 ng/mL PSAP (PSAP) for 4 days. n = 9 samples in 3 experiments. B, Growth assay of 4T1 cells cultured with conditioned media from EGFP- or YAP-MSCs for 4 days. n = 12 samples in 4 experiments. C, Western blot of phosphorylated Akt at T308 (pAkt-T308) and total-Akt (Akt) in 4T1 cells cultured with DMEM (NT) or DMEM with PSAP (PSAP) for 15 min. n = 4 experiments. D, Western blot of phosphorylated Akt at S473 (pAkt-S473) and Akt in 4T1 cells cultured with DMEM (NT), DMEM with PSAP (PSAP), or RPMI with 10% FBS (FBS) for 15 min. E, Fluorescence images of nuclei and phosphorylated histone-H3 at S10 (p-histone-H3) in 4T1 cells cultured with DMEM exogenously added with DMSO (NT-DMSO), DMEM with PSAP+DMSO (PSAP−DMSO), or DMEM with PSAP+LY294002 (PSAP−LY) for 1 day. F, Relative ratio of phosphorylated histone-H3 at S10 positive (p-histone-H3+) 4T1 cells in e. n = 3 experiments. G, Survival assay of 4T1 cells. n = 9 samples in 3 experiments. H, Volume of tumors 7 days after injection. COL mice were injected with 4T1 cells with shNS-MSCs, shPSAP1-MSCs, or shPSAP2-MSCs. n = 6 tumors in 3 mice (upper panel), n = 8 tumors in 4 mice (lower panel, shNS-MSCs), or n = 4 tumors in 2 mice (lower panel, shPSAP2-MSCs). I, Number of metastatic lung lesions 21 days after injection. n = 3 mice (upper panel), n = 4 mice (lower panel, shNS-MSCs), or n = 2 mice (lower panel, shPSAP2-MSCs). J, Kaplan-Meier curves of relapse free survival in relation to the gene expression of PSAP in grade 1 breast cancer patients. Statistical significance was determined as . Mean±S.E. are shown. MSCs were established from BALBc mice. Bar = 100 µm.