Tu2005 Epidermal Growth Factor Receptor Is Overexpressed in Esophageal Biopsies of Children With Eosinophilic Esophagitis: An Immunohistochemical Analysis

Abstract

Figure 1. Characterization of different MDSCs subsets in healthy controls and RAC patients. (A) Flow cytometric profile of peripheral blood (upper and mid rows) and tumor cells (lower row) from healthy control and RAC patient. X100 light microscopy image of representative cell in each subset is presented. (B) Flow cytometric analysis of different circulating MDSCs subsets in healthy controls and RAC patients. The figure represents 3 different subjects in each group; *-P<.05, *-P<.01. (C) Sonographic evaluation of locally advanced RAC before (left) and after (right) neoadjuvant therapy. T-Tumor; P-Prostate Background: Interaction between tumor and microenvironment is an important factor in tumor progression. Cancer stem cells (CSCs) in tumor, known as contributing cells to tumor recurrence and metastasis, also interact with a niche in the microenvironment which controls their maintenance and differentiation. We investigated the CSC-promoting effect of factors released from myofibroblasts in microenvironment and its molecular mechanism. Methods: To investigate the effect of factors secreted from myofibroblast on CSC expansion, we performed a flow cytometric analysis of CD133+CD44+ cells in Caco2 cells which were cultured in conditioned media (CM) of 18CO(pericryptal myofibroblast) cells. To identify contributing gene expressions, we utilized mRNA expressional microarray for detection of differential gene expressions between Caco2 cells and 18CO-cocultured Caco2 cells. The secreted factors from the 18CO cells were analyzed using cytokine antibody array, and anti- IL6/IL-8 antibody and anti-HES1 antibody were used for neutralization and Western blot analysis. Results: In mRNA expressional microarray comparing Caco2 cells and 18CO- cocultured Caco2 cells, HES1 expression, the target of Notch signaling, significantly increased in 18CO-cocultured Caco2 cells, compared to Caco-2 cells. Caco2 cells cultured in 18CO CM showed a significant increase in CD133+CD44+ cells and HES1 expression, compared to Caco2 cells cultured in the control media. Significant amounts of IL6 and IL8 were detected in 18CO CM, compared to the control media. 18CO CM-induced increase of CD133+CD44+ cells was attenuated by neutralizing antibodies to IL6 and IL8. Furthermore, neutralizing antibodies to IL6 and IL8, and STAT3 inhibitor reduced the expression of HES1 induced in Caco2 cells cultured in 18CO CM. Conclusion: The increase of CSCs by myofibroblast could be mediated by IL6/IL8-induced HES1 activation in tumor microenviron- ment. The IL6/IL8-mediated Notch/HES1 pathway in interaction between the CSCs and their microenvironment might be a target in drug development for colorectal cancer treatment. Objectives/Aims: Hydrogen sulfide (H 2S) plays a role in angiogenesis and cell proliferation— key components of tumorigenesis and metastasis. In mammals, this gasotransmitter is pro- duced mainly by cystathionine gamma-lyase (CSE) and cystathionine-β-synthase (CBS). We have shown previously that colorectal cancer (CRC) cell lines express high levels of CBS, but not CSE, and as a result, have increased levels of H 2S when compared to the normal epithelial. Thus, our objective was to identify the major source of CBS in CRC tumors and understand its pathophysiological contribution to the tumor progression. Methods: Immunostaining followed by in-situ confocal analysis was performed on CRC derived tumor tissue and adjacent normal control tissue, fixed in 1% paraformaldehyde. Non-tumorigenic colonic epithelial cell line, NCM356, and colon cancer-derived epithelial cell lines, HCT116, were also used in this study. Results: Using in-situ confocal microscopy, we first confirm our previous findings—observed on mRNA and total protein (Western blot) levels—that CBS expression is increased within CRC tumor. The increase in CBS was mostly observed within the basolateral side of epithelium and tumor stroma. Furthermore, higher CBS expression was mostly found in cells co-expressing epithelial marker, EpCAM, and stem- like cell marker, Oct-3/4, suggesting that cancer stem-like cells are mostly responsible for the increase in CBS-mediated H 2S production. We also confirmed this observation using CRC cancer cell line, HCT116, and demonstrated that CBS is mainly found within the cytosol. In contrast, normal epithelial cell, NCM356, lacked expression of CBS. Interestingly, we observed that cells expressing CBS in tumor stroma in-situ, unlike the normal control, bear an EpCAM+/α-SMA+/Oct-3/4+ phenotype. This suggests that within the CRC tumor stroma, the major cells expressing CBS are cells undergoing epithethial-mesenchymal transi- tion (EMT), preserving their cancer stem-like properties. Conclusion: Taken together, our previous and current data suggest that increase in expression of CBS within the tumor, and