Expression of epithelial, fibroblast and endometrial markers in the primary epithelial and fibroblast cell cultures.

<p>Total RNA was subjected to quantitative real-time PCR analysis of epithelial markers (EpCAM, cytokeratin 8, E-cadherin), fibroblast markers (alpha-smooth muscle actin (αSMA), vimentin) (<b>A</b>–<b>C</b>), estrogen receptor 1 and 2 (ER1, 2), progesterone receptor (PR), progestagen-associated endometrial protein (PAEP) and matrix metalloproteinase 1 and 9 (MMP1, 9) (<b>D</b>–<b>F</b>). Data, average; error bars, S.E.M. Data shown are representative of three independent experiments.</p

Establishment of epithelial and fibroblast cells from human endometrial cancer tissues.

<p>Hematoxylin & eosin (H and E) staining of tissues and phase contrast images of established primary cells isolated from endometrial cancer: EC6 (<b>A</b>), EC14 (<b>B</b>), EC7 (<b>C</b>) and EC11 (<b>D</b>). Magnification: 100x. Subsequently these were digested with collagenase and cultured, prior to epithelial and fibroblast cell isolation using CD326 (EpCAM) and anti-fibroblast labeled magnetic beads.</p

Role of PI3K/Akt and MAPK/Erk signaling pathways in cancer-associated fibroblast-mediated endometrial cell proliferation.

<p>(<b>A</b>) Western blot analysis of phosphorylated-Akt (Ser473) and -Erk protein expression in ECC-1 cells after treated with either normal endometrial fibroblast T-HESC cells or cancer-associated fibroblast cells (EC6-Fib, EC7-Fib, EC11-Fib and EC14-Fib). Densitometry analysis compared the relative expression level of p-Akt and p-Erk to their total protein level. (<b>B</b>) Quantitative analysis of phosphorylated-Akt (Ser473 and Thr308) and phosphorylated-Erk levels in ECC-1 cells after treated with either T-HESC or CAFs, in comparison to cells treated with control (media containing 2% FBS). ECC-1 (<b>C</b>) and EC6-Ep (<b>D</b>) cells were treated with either PI3K pathway selective inhibitor (LY294002) or Erk pathway selective inhibitor (U0126) in the presence of cancer-associated fibroblasts conditioned media (1 µg/µl) for 72 hours. Data shown are cell viability after normalized with control (media containing 2% FBS). Data, average; error bars, S.E.M. *, <i>P</i><0.05; **, <i>P</i><0.0001. Data shown are representative of two independent experiments.</p

Fibroblast isolated from endometrial hyperplasia tissue did not promote endometrial cancer cell proliferation.

<p>Hematoxylin and eosin staining (<b>A</b>) and phase contrast image (<b>B</b>) of fibroblast cells isolated from human atypical hyperplasia tissue (EH-Fib); Magnification, 100x. (<b>C</b>) Flow cytometry analysis of EH-Fib stained with epithelial marker CD326-Alexa Fluor 647 and fibroblast marker CD90-PE. (<b>D</b>) Cell viability assay determining the effects of EH-Fib conditioned media on endometrial cancer cell lines (ECC-1 and HEC-1A) and primary epithelial cells (EC6-Ep and EC14-Ep) after 72 hours treatment. Data, average; error bars, S.E.M. *, <i>P</i><0.01. Data shown are representative of three independent experiments.</p

Differential effects of normal and cancer-associated endometrial fibroblast on EC cell proliferation.

<p>ECC-1 (<b>A</b>) and HEC-1A (<b>B</b>) cell lines and EC6-Ep (<b>C</b>) and EC14-Ep (<b>D</b>) primary endometrial cancer cells were tested with conditioned media prepared from cancer-associated fibroblasts (EC6-Fib, EC7-Fib, EC11-Fib and EC14-Fib) and normal endometrial fibroblast cell line (T-HESC) for 72 hours. Cell viability was examined using MTT assay and were normalized with control (media containing 2% FBS). CAFs shown are the average of all the four cancer-associated fibroblasts tested. Data, average; error bars, S.E.M. *, <i>P</i><0.005;. **, <i>P</i><0.0001. Data shown are representative of three independent experiments.</p

EpCAM and CD90 expression in established primary culture.

<p>Flow cytometry analysis of primary epithelial and fibroblast cells isolated from endometrial cancer tissues was performed after labeling cells with CD326 antibody-conjugated with AlexaFluor647 and CD90 antibody-conjugated with PE. Epithelial endometrial cell line, ECC-1 (<b>A</b>), primary epithelial cells EC6-Ep (<b>B</b>) and EC14-Ep (<b>C</b>), normal endometrial stromal cell line, T-HESC (<b>D</b>) and primary fibroblast cells, EC6-Fib (<b>E</b>), EC7-Fib (<b>F</b>), EC11-Fib (<b>G</b>) and EC14-Fib (<b>H</b>).</p

Mechanism of action of rapamycin as PI3K/mTOR pathway inhibitor.

<p>ECC-1 cell line (<b>A</b>) and EC6-Ep primary epithelial cell (<b>B</b>) were treated with increasing dose of rapamycin for 72 hours under the influence of control media (media containing 2% FBS) or 1 µg/µl EC11-Fib conditioned media. (<b>C</b>–<b>E</b>) ECC-1 cells treated with 2 µM of rapamycin with or without 1 µg/µl EC11-Fib conditioned media for 72 hours, were stained with annexin V-PE and 7-AAD before analyzed with flow cytometry. Data, average; error bars, S.E.M. *, <i>P</i><0.0001 compared to EC11-Fib treated cells. Data shown are representative of two independent experiments.</p

Collagen induces a more proliferative, migratory and chemoresistant phenotype in head and neck cancer via DDR1

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide and includes squamous cell carcinomas of the oropharynx and oral cavity. Patient prognosis has remained poor for decades and molecular targeted therapies are not in routine use. Here we showed that the overall expression of collagen subunit genes was higher in cancer-associated fibroblasts (CAFs) than normal fibroblasts. Focusing on collagen8A1 and collagen11A1, we showed that collagen is produced by both CAFs and tumour cells, indicating that HNSCCs are collagen-rich environments. We then focused on discoidin domain receptor 1 (DDR1), a collagen-activated receptor tyrosine kinase, and showed that it is over-expressed in HNSCC tissues. Further, we demonstrated that collagen promoted the proliferation and migration of HNSCC cells and attenuated the apoptotic response to cisplatin. Knockdown of DDR1 in HNSCC cells demonstrated that these tumour-promoting effects of collagen are mediated by DDR1. Our data suggest that specific inhibitors of DDR1 might provide novel therapeutic opportunities to treat HNSCC