Fig 2: Periostin knockdown inhibited mesenchymal cell proliferation and maintained non-invasive phenotype in TNBC tumors. (A) Tumor growth curve in MCF10DCIS tumors upon dox-inducible knockdown of periostin. Arrow indicates administration period of dox. Tumor volume was measured on the indicated days. Data are presented as mean ± standard error (n = 10). *P < 0.05, Student’s t test. (B) Quantitative PCR analysis of indicated genes in tumors collected at the end of the experiment. Samples were prepared from a mixture of 10 tumors. Relative values are presented as mean ± standard error, *P < 0.02, Student’s t test. (C) Immunostaining of CD44 and E-cadherin in tumors collected at the end of the experiment. Scale bar, 100 µm. (D) Quantitation of the epithelial area ratio in tumors collected at the end of the experiment (n = 10). E-cadherin positive area was normalized against the total human nuclei positive area. Bars represent mean, and error bars represent standard deviation. *P < 0.05, ***P < 0.001, Mann-Whitney’s test. (E) Immunostaining of CD44 and Ki67 in tumors collected at the end of the experiment. Scale bar, 100 µm. (F) The ratio of CD44high/Ki67+ cancer cells against total Ki67+ cells was calculated (n = 12). Bars represent mean, and error bars represent standard deviation. ****P < 0.0001, Mann-Whitney’s test. (G) Immunostaining of E-cadherin and Ki67 in tumors collected at the end of the experiment. Scale bar, 100 µm. (H) The ratio of E-cadherin−/Ki67+ cancer cells against total Ki67+ cells was calculated (n = 12). Bars represent mean, and error bars represent standard deviation. ****P < 0.0001, Mann-Whitney’s test. (I) Immunostaining of laminin-5 and periostin in tumors collected at the end of the experiment. Scale bar, 100 µm.

Fig 3: Knockdown of periostin inhibited mesenchymal cell proliferation and invasion, but did not affect apoptosis induction immediately after chemotherapy. (A) Immunofluorescence staining of CD44 and cleaved caspase-3 in MC10DCIS tumors. Tumors were sampled 1 or 3 days after one dose of PTX treatment. Scale bar, 100 µm. (B) The number of CD44high/cleaved Caspase-3+ cancer cells per field were counted (left panel, n = 10), and the ratio of CD44high/cleaved caspase-3+ cancer cells against total cleaved caspase-3+ cells was calculated (right panel, n = 10). Bars represent mean, and error bars represent standard deviation. **P < 0.01, ***P < 0.001, ****P < 0.0001, Mann-Whitney’s test. (C) Immunofluorescence staining of CD44 and Ki67 in MC10DCIS tumors. Tumors were sampled 1 or 3 days after one dose of PTX treatment. Scale bar, 100 µm. (D) The number of CD44high/Ki67+ cancer cells per field were counted (left panel, n = 10), and the ratio of CD44high/Ki67+ cancer cells against total Ki67+ cells was calculated (right panel, n = 10). Bars represent mean, and error bars represent standard deviation. ***P < 0.001, ****P < 0.0001, Mann-Whitney’s test. (E) Immunofluorescence staining of laminin-5, periostin, E-cadherin and MT1-MMP in tumors. Tumors were sampled 3 days after one dose of PTX treatment. Scale bar, 100 µm.

Fig 4: Periostin is required for mesenchymal cell expansion and tumor invasion in residual tumors upon chemotherapy. (A) Tumor growth curve in MCF10DCIS xenografts upon PTX treatment and periostin knockdown. Vertical arrows indicate times of PTX administration, and horizontal arrow indicates administration period of doxycycline to knock down periostin. Tumor volume was measured on the indicated days and are presented as mean ± standard error (n = 10). *P < 0.05, Student’s t test. (B) Quantitative PCR analysis of Periostin gene in tumors collected at Day 30. Samples were prepared from a mixture of 10 tumors. Relative values are presented as mean ± standard error, *P < 0.02, Student’s t test. (C) Flow cytometry analysis of tumors at Day 30 using CD44 and CD24 antibodies. Samples were prepared from the mixture of 10 tumors. Ratio of CD44high/CD24low to CD24high tumor cells is presented on the plots. (D) Immunofluorescence staining of CD44 and Ki67 in tumors collected at the end of the experiment. Scale bar, 100 µm. (E) Ratio of CD44high/Ki67+ cancer cells against total Ki67+ cells was quantified at Day 30 (n = 15). Bars represent mean, and error bars represent standard deviation. ***P < 0.001, ****P < 0.0001, Mann-Whitney’s test. (F) Immunofluorescence staining of E-cadherin and Ki67 in tumors collected at the end of the experiment. Scale bar, 100 µm. (G) The ratio of E-cadherin−/Ki67+ cancer cells against total Ki67+ cells was calculated (n = 12). Bars represent mean, and error bars represent standard deviation. ****P < 0.0001, Mann-Whitney’s test. (H) Immunofluorescence staining of laminin-5 and periostin in tumors collected at the end of the experiment. Scale bar, 100 µm.

Fig 5: Dynamic regulation of mesenchymal cell expansion and tumor invasion in residual tumors upon chemotherapy. (A) Tumor growth curve following relapse from paclitaxel (PTX) treatment in MCF10DCIS xenografts. Arrows indicate times of drug administration. Tumor volume was measured on the indicated days and presented as mean ± standard error (n = 10). (B) Flow cytometry analysis of indicated tumors using CD44 and CD24 antibodies. Samples were prepared from the mixture of 10 tumors. Ratio of CD44high/CD24low to CD24high tumor cells is presented on the plots. (C) Immunofluorescence staining of E-Cadherin and human nuclei in tumor. Scale bar, 100 µm. (D) Quantitation of the epithelial area ratio in tumors (n = 15). E-Cadherin positive area was normalized against the total human nuclei positive area. Bars represent mean, and error bars represent standard deviation. *P < 0.05, ****P < 0.0001, Mann-Whitney’s test. (E) Immunofluorescence staining of CD44 and Ki67 in tumors. Scale bar, 100 µm. (F) The number of CD44high/Ki67+ cancer cells per field were counted (left panel, n = 15), and the ratio of CD44high/Ki67+ cancer cells against total Ki67+ cells was calculated (right panel, n = 15). Bars represent mean, and error bars represent standard deviation. ***P < 0.001, ****P < 0.0001, Mann-Whitney’s test. (G) Immunofluorescence staining of E-Cadherin and Ki67 in tumors. Scale bar, 100 µm. (H) The number of E-Cadherin−/Ki67+ cancer cells per field were count (left panel, n = 15), and the ratio of E-Cadherin−/Ki67+ cancer cells against total Ki67+ cells was calculated (right panel, n = 15). Bars represent mean, and error bars represent standard deviation. ***P < 0.001, ****P < 0.0001, Mann-Whitney’s test. (I) Immunofluorescence staining of laminin-5 and periostin in tumors. Scale bar, 100 µm.