Abstract A32: Il-6 initiates a self-sustaining signaling loop to promote nurturing tumor microenvironment
Authors: Karakasheva Tatiana A. , Soni Monica , Waldron Todd J. , Lin Eric W. , Hicks Philip D. , Klein-Szanto Andres , Wong Kwok K. , Bass Adam J. , Rustgi Anil K. 
Published: October 2016

Desmoplasia, or deposition of connective tissue proper in the stroma, is a key feature of esophageal squamous cell carcinoma (ESCC), a common malignancy worldwide. The cellular component of connective tissue proper is comprised of fibroblasts, and cancer-associated fibroblasts (CAFs) are widely recognized as a major constituent of the tumor microenvironment (TME). The purpose of this study was to investigate the cross-talk between CAFs and tumor cells in ESCC, as well as identify factors that mediate this cross-talk and evaluate these factors as therapeutic targets in ESCC and potentially other squamous cell carcinomas.

We have observed that co-culture with CAFs prompts ESCC cells to acquire a more mesenchymal phenotype and express potent mediators of cell migration and invasion, such as matricellular proteins (periostin, fibrillin, osteonectin) and growth factors (EGF, HGF, VEGF). Furthermore, co-culture of ESCC cells with CAFs promotes tumor cell migration in our 3D organotypic culture, while co-transplantation of ESCC cells with fibroblasts leads to enhanced tumor growth in vivo, compared to tumors transplanted without fibroblasts.

In order to identify potential mediators of ESCC-CAF cross-talk, we have performed a comprehensive cytokine array and found that interleukin 6 (IL-6) is significantly overexpressed in conditioned media from co-culture of esophageal CAFs and ESCC cells, compared to mono-culture. IL-6 is known to play important roles in the development of multiple types of cancer, mostly via activation of the STAT3 signaling pathway. To confirm relevance of our in vitro findings, we have performed immunohistochemical staining of tissue samples from ESCC patients and found that, compared to normal esophagus, expression of IL-6 is enhanced in ESCC in both epithelial cells and fibroblasts.

Interestingly, knockdown of IL-6 resulted in altered morphology of ESCC cells in 3D organotypic culture. This was accompanied by restored membrane localization of E-cadherin and reduced activation of STAT3 signaling. In order to investigate the importance of STAT3 and MEK/ERK signaling (the latter being another pathway activated by IL-6 in cancer) on ESCC cell biology, we have treated 3D organotypic cultures with stattic and trametinib (inhibitors of STAT3 and MEK, respectively). This treatment resulted in impaired invasion, reduced proliferation and enhanced apoptosis of ESCC cells, accompanied by decreased expression of the basal cell marker p63. Furthermore, treatment with stattic and trametinib leads to reduced secretion of IL-6 by these cultures. These effects were more pronounced upon treatment with combination of stattic and trametinib.

Since we found IL-6 to be important for ESCC progression in vitro, we have conducted a therapeutic study utilizing tocilizumab (an FDA-approved anti-human IL-6R neutralizing antibody). Subcutaneous xenograft tumors comprised of human ESCC and CAF cell lines, were treated with tocilizumab, which resulted in suppressed tumor growth, accompanied by decreased activation of STAT3 and ERK within the tumor, compared to control IgG-treated tumors. Furthermore, tocilizumab-treated animals had reduced numbers of splenic monocytic immature myeloid cells (CD11b+Ly-6C+), which is in agreement with previously published findings regarding MDSCs in murine models of ESCC.

In summary, our findings indicate that IL-6, at least in part, is responsible for migration, proliferation, survival and poor differentiation of ESCC cells, promoted by CAFs in the TME. We also report that these effects of IL-6 are mediated by STAT3 and ERK signaling. Finally, we demonstrate that inhibition of IL-6 signaling suppresses ESCC tumor growth in vivo. This is the first report of anti-tumorigenic activity of tocilizumab in ESCC, which makes it a promising candidate for ESCC therapy.

Supported by NCI P01 CA098101.

Citation Format: Tatiana A. Karakasheva, Monica Soni, Todd J. Waldron, Eric W. Lin, Philip D. Hicks, Andres Klein-Szanto, Kwok K. Wong, Adam J. Bass, Anil K. Rustgi. Il-6 initiates a self-sustaining signaling loop to promote nurturing tumor microenvironment. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr A32.