Background: Several stromal cell subtypes including macrophages contribute to tumor progression by inducing epithelial-mesenchymal transition (EMT) at the invasive front, a mechanism also linked to metastasis. Tumor associated macrophages (TAM) reside mainly at the invasive front but they also infiltrate tumors and in this process they mainly assume a tumor promoting phenotype. In this study, we asked if TAMs also regulate EMT intratumorally. We found that TAMs through TGF-β signaling and activation of the β-catenin pathway can induce EMT in intratumoral cancer cells. Methods: We depleted macrophages in F9-teratocarcinoma bearing mice using clodronate-liposomes and analyzed the tumors for correlations between gene and protein expression of EMT-associated and macrophage markers. The functional relationship between TAMs and EMT was characterized in vitro in the murine F9 and mammary gland NMuMG cells, using a conditioned medium culture approach. The clinical relevance of our findings was evaluated on a tissue microarray cohort representing 491 patients with non-small cell lung cancer (NSCLC). Results: Gene expression analysis of F9-teratocarcinomas revealed a positive correlation between TAM-densities and mesenchymal marker expression. Moreover, immunohistochemistry showed that TAMs cluster with EMT phenotype cells in the tumors. In vitro, long term exposure of F9-and NMuMG-cells to macrophage-conditioned medium led to decreased expression of the epithelial adhesion protein E-cadherin, activation of the EMT-mediating β-catenin pathway, increased expression of mesenchymal markers and an invasive phenotype. In a candidate based screen, macrophage-derived TGF-β was identified as the main inducer of this EMT-associated phenotype. Lastly, immunohistochemical analysis of NSCLC patient samples identified a positive correlation between intratumoral macrophage densities, EMT markers, intraepithelial TGF-β levels and tumor grade. Conclusions: Data presented here identify a novel role for macrophages in EMT-promoted tumor progression. The observation that TAMs cluster with intra-epithelial fibroblastoid cells suggests that the role of macrophages in tumor-EMT extends beyond the invasive front. As macrophage infiltration and pronounced EMT tumor phenotype correlate with increased grade in NSCLC patients, we propose that TAMs also promote tumor progression by inducing EMT locally in tumors.

The L1 cell adhesion molecule (L1CAM) is potentially involved in epithelial-mesenchymal transition (EMT). EMT marker expression is of prognostic significance in non-small cell lung cancer (NSCLC). The relevance of L1CAM for NSCLC is unclear. We investigated the protein expression of L1CAM in a cohort of NSCLC patients. L1CAM protein expression was correlated with clinico-pathological parameters including survival and markers of epithelial-mesenchymal transition. L1CAM protein expression was found in 25% of squamous cell carcinomas and 24% of adenocarcinomas and correlated with blood vessel invasion and metastasis (p < 0.05). L1CAM was an independent predictor of survival in a multivariate analysis including pT, pN, and pM category, and tumor differentiation grade. L1CAM expression positively correlated with vimentin, beta-catenin, and slug, but inversely with E-cadherin (all p-values < 0.05). E-cadherin expression was higher in the tumor center than in the tumor periphery, whereas L1CAM and vimentin were expressed at the tumor-stroma interface. In L1CAM-negative A549 cells the L1CAM expression was upregulated and matrigel invasion was increased after stimulation with TGF-beta1. In L1CAM-positive SK-LU-1 and SK-LC-LL cells matrigel invasion was decreased after L1CAM siRNA knockdown. A subset of NSCLCs with vessel tropism and increased metastasis aberrantly expresses L1CAM. L1CAM is a novel prognostic marker for NSCLCs that is upregulated by EMT induction and appears to be instrumental for enhanced cell invasion.

Background: Expression of periostin is an indicator of epithelial-mesenchymal transition in cancer but a detailed analysis of periostin expression in prostate cancer has not been conducted so far. Methods: Here, we evaluated periostin expression in prostate cancer cells and peritumoural stroma immunohistochemically in two independent prostate cancer cohorts, including a training cohort (n = 93) and a test cohort (n = 325). Metastatic prostate cancers (n = 20), hormone refractory prostate cancers (n = 19) and benign prostatic tissues (n = 38) were also analyzed. Results: In total, strong epithelial periostin expression was detectable in 142 of 418 (34.0%) of prostate carcinomas and in 11 of 38 benign prostate glands (28.9%). Increased periostin expression in carcinoma cells was significantly associated with high Gleason score (p < 0.01) and advanced tumour stage (p < 0.05) in the test cohort. Whereas periostin expression was weak or absent in the stroma around normal prostate glands, strong periostin expression in tumour stroma was found in most primary and metastatic prostate cancers. High stromal periostin expression was associated with higher Gleason scores (p < 0.001). There was a relationship between stromal periostin expression and shortened PSA relapse free survival times in the training cohort (p < 0.05). Conclusions: Our data indicate that periostin up-regulation is related to increased tumour aggressiveness in prostate cancer and might be a promising target for therapeutical interventions in primary and metastatic prostate cancer.

BACKGROUND: Molecular testing of lung adenocarcinomas (ADCs) is crucial for therapy stratification of patients. Because of the often limited diagnostic material, the authors aimed to explore the suitability of cytology smears for next‐generation sequencing (NGS) and compared the results with concurrent histological specimens or cell blocks.METHODS: A total of 16 formalin‐fixed paraffin‐embedded (FFPE) ADCs with known genetic alterations were used as the first cohort for targeted DNA and RNA sequencing. In the second cohort of 8 cases, 8 cytological smears were compared with matching histological specimens or cell blocks for the study. For NGS library amplification, commercially available panels for DNA and RNA sequencing were applied. The Ion Torrent Personal Genome Machine and the Ion Reporter workflow (version 5.0) were used for sequencing.RESULTS: All DNA libraries derived from FFPE and non‐formalin‐fixed cytological smear samples produced acceptable quality metrics, thereby enabling successful targeted DNA sequencing (100% performance). Targeted RNA sequencing failed in 1 FFPE case and 1 cytology probe by not reaching enough mapped fusion reads (92% performance rate). All previously detected mutations and gene rearrangements could be confirmed (sensitivity of 100%), whereas specificity of the DNA‐based NGS assay reached 96%.CONCLUSIONS: The results of the current study demonstrated the suitability of non‐formalin cytology specimens for the simultaneous NGS testing of lung ADCs using amplicon resequencing panels. These assays allowed for the input of cytological smears equal to concurrent histology or cell blocks and proved to be accurate in the detection of therapeutically actionable somatic mutations and gene rearrangements. Cancer Cytopathol 2017;125:30‐40. © 2016 American Cancer Society.

Background: Expression of transcription-factors as Slug and Sox9 was recently described to determine mammary stem-cell state. Sox10 was previously shown to be present also in breast cancer. Protein overexpression of Slug, Sox9 and Sox10 were associated with poor overall survival and with triple-negative phenotype in breast cancer. In this study we tested the stability of Slug, Sox9 and Sox10 expression during chemotherapy and addressed their prognostic role of in neoadjuvant treated primary breast-cancer and their correlation to pathological-response and overall survival. Methods: We analyzed immunohistochemical expression of Slug, Sox9 and Sox10 in tissue microarrays of 96 breast cancers prior to and after neoadjuvant chemotherapy. Expression was evaluated in invasive tumor cells and in tumor stroma and scored as 0, 1+, 2+ 3+. Expression-profile prior to and after chemotherapy was correlated to overall survival (Kaplan Meier) and with established clinico-pathological parameter. Results: Sox9, Sox10 and Slug were expressed in 82–96% of the tumor cells prior to chemotherapy. Slug was expressed in 97% of the cases in tumor stroma before therapy. Change in expression-profile after chemotherapy occurred only in Slug expression in tumor-cells (decreased from 82 to 51%, p = 0.0001, Fisher’s exact test). The other markers showed no significant change after chemotherapy. Stromal Sox9 expression (0 to 2+) correlated to better overall survival after chemotherapy (p = 0.004) and reached almost statistical significance prior to chemotherapy (p = 0.065). There was no correlation between Sox9 and hormone-receptor expression. In multivariate-analysis, the stromal Sox9 expression after chemotherapy proved to be an independent and better prognostic marker than hormone-receptor status. Other clinico-pathological parameter (as HER2-status or pathological-stage) showed no correlation to the analyzed markers. Conclusion: Strong stromal Sox9 expression in breast cancer after chemotherapy was found to bear negative prognostic information and was associated with shortened overall survival. Slug expression was significantly changed (reduced) in samples after neoadjuvant chemotherapy.