Martin Wartenberg

Expression of E-cadherin repressors SNAIL, ZEB1 and ZEB2 by tumour and stromal cells influences tumour-budding phenotype and suggests heterogeneity of stromal cells in pancreatic cancer

Background:There is evidence that tumour–stroma interactions have a major role in the neoplastic progression of pancreatic ductal adenocarcinoma (PDAC). Tumour budding is thought to reflect the process of epithelial–mesenchymal transition (EMT); however, the relationship between tumour buds and EMT remains unclear. Here we characterize the tumour-budding- and stromal cells in PDAC at protein and mRNA levels concerning factors involved in EMT.Methods:mRNA in situ hybridisation and immunostaining for E-cadherin, β-catenin, SNAIL1, ZEB1, ZEB2, N-cadherin and TWIST1 were assessed in the main tumour, tumour buds and tumour stroma on multipunch tissue microarrays from 120 well-characterised PDACs and associated with the clinicopathological features, including peritumoural (PTB) and intratumoural (ITB) budding.Results:Tumour-budding cells showed increased levels of ZEB1 (P<0.0001) and ZEB2 (P=0.0119) and reduced E-cadherin and β-catenin (P<0.0001, each) compared with the main tumour. Loss of membranous β-catenin in the main tumour (P=0.0009) and tumour buds (P=0.0053), without nuclear translocation, as well as increased SNAIL1 in tumour and stromal cells (P=0.0002, each) correlated with high PTB. ZEB1 overexpression in the main tumour-budding and stromal cells was associated with high ITB (P=0.0084; 0.0250 and 0.0029, respectively) and high PTB (P=0.0005; 0.0392 and 0.0007, respectively). ZEB2 overexpression in stromal cells correlated with higher pT stage (P=0.03), lymphatic invasion (P=0.0172) and lymph node metastasis (P=0.0152).Conclusions:In the tumour microenvironment of phenotypically aggressive PDAC, tumour-budding cells express EMT hallmarks at protein and mRNA levels underlining their EMT-type character and are surrounded by stromal cells expressing high levels of the E-cadherin repressors ZEB1, ZEB2 and SNAIL1, this being strongly associated with the tumour-budding phenotype. Moreover, our findings suggest the existence of subtypes of stromal cells in PDAC with phenotypical and functional heterogeneity.

MicroRNA dysregulation in the tumor microenvironment influences the phenotype of pancreatic cancer

Cellular interactions in the tumor microenvironment influence neoplastic progression in pancreatic ductal adenocarcinoma. One underlying mechanism is the induction of the prognostically unfavorable epithelial–mesenchymal-transition-like tumor budding. Our aim is to explore the expression of microRNAs implicated in the regulation of tumor budding focusing on the microenvironment of the invasive front. To this end, RNA from laser-capture-microdissected material of the main tumor, tumor buds, juxta-tumoral stroma, tumor-remote stroma, and non-neoplastic pancreatic parenchyma from pancreatic cancer cases with (n=7) and without (n=6) tumor budding was analyzed by qRT-PCR for the expression of a panel of miRNAs that are known to be implicated in the regulation of epithelial–mesenchymal transition, including miR-21, miR-183, miR-200b, miR-200c, miR-203, miR-205, miR-210, and miR-217. Here we show that at the invasive front of pancreatic ductal adenocarcinoma, specific microRNAs, are differentially expressed between tumor buds and main tumor cells and between cases with and without tumor budding, indicating their involvement in the regulation of the budding phenotype. Notably, miR-200b and miR-200c were significantly downregulated in the tumor buds. Consistent with this finding, they negatively correlated with the expression of epithelial–mesenchymal-transition-associated E-cadherin repressors ZEB1 and ZEB2 in the budding cells (P<0.001). Interestingly, many microRNAs were also dysregulated in juxta-tumoral compared to tumor-remote stroma suggesting that juxta-tumoral stroma contributes to microRNA dysregulation. Notably, miR-200b and miR-200c were strongly downregulated while miR-210 and miR-21 were upregulated in the juxta-tumoral vs tumor-remote stroma in carcinomas with tumor budding. In conclusion, microRNA targeting in both tumor and stromal cells could represent a treatment option for aggressive pancreatic cancer.

Tumour budding in pancreatic cancer revisited: validation of the ITBCC scoring system

Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal malignancy with rising incidence. Biomarkers that would help the prognostic stratification of patients are needed urgently. Although tumour budding (BD) is a strong and independent prognostic factor in PDAC it is not included in histopathology reports, due partly to the lack of a standardised scoring system. The aim of the present work is to assess the reliability and reproducibility of the BD scoring system proposed recently by the International Tumour Budding Consensus Conference (ITBCC) 2016 in a well‐characterised PDAC cohort (n = 120) with complete clinicopathological and follow‐up information.

Abstract 447: Tumor microenvironment in pancreatic cancer (PDAC): interplay between tumor cells, stromal cells and immune cells

Introduction: Tumor budding is considered to be the morphologic correlate of epithelial-mesenchymal-transition (EMT) and is an independent prognostic factor in pancreatic ductal adenocarcinoma (PDAC). Here we explore the interactions between tumor budding cells, stromal cells and immune cells in the microenvironment of the tumor buds in PDAC. Methods: Multi-punch tissue-microarrays (TMAs) containing punches from the tumor center and the tumor front, including tumor buds, from a well characterized cohort of 120 PDAC patients, were stained immunohistochemically and by mRNA-in-situ-Hybridization (mRNA-ISH)for the EMT markers E-cadherin, β-catenin, Snail, ZEB1, ZEB2, N-cadherin and Twist and for the immune cell markers CD8, CD4, Foxp3, M1- and M2-macrophages. Expression in budding- and stromal cells and immune-cell counts in the microenvironment of the tumor buds were compared with findings in the main tumor. Results: Tumor buds showed loss of E-cadherin and b-catenin and overexpressed ZEB1 and ZEB2 compared with the neoplastic cells of the main tumor both at protein and mRNA level (p Conclusions: Tumor budding cells show a shift towards EMT-promoting profiles at protein and mRNA level, compared with the neoplastic cells of the main tumor. Stromal cells surrounding tumor buds express high levels of Snail, ZEB1 and ZEB2 suggesting that some stromal cells may represent complete mesenchymally transformed tumor cells or alternatively that there is a special phenotype of cancer associated fibroblasts supporting EMT-Type tumor-budding through cellular crosstalk in the tumor microenvironment of PDAC. Moreover, there is a tumor favoring immune cell composition in the immediate microenvironment of the tumor buds. Our findings suggest a close interaction of the stromal and immune response with the EMT process in PDAC. The combined assessment of host-associated factors such as stromal and immune response and tumor-associated factors such as EMT-type tumor-budding could help us to achieve superior prognostication and patient stratification than either factor alone. Citation Format: Eva Karamitopoulou, Martin Wartenberg, Jose A. Galvan, Inti Zlobec, Alessandro Lugli, Aurel Perren. Tumor microenvironment in pancreatic cancer (PDAC): interplay between tumor cells, stromal cells and immune cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 447. doi:10.1158/1538-7445.AM2015-447