Abstract 177: Increased stiffness of the tumor microenvironment in colon cancer leads to an increase in activin and metastatic potential
Authors: Bauer Jessica , Staudacher Jonas J. , Mancinelli Georgina , Krett Nancy , Bashar Emon , Grippo Paul , Saif M Taher A. , Jung Barbara 
Published: August 2018

Colorectal cancer (CRC) is the second deadliest cancer in the US and understanding the switch to metastatic behavior and developing therapeutic strategies to target this process are clinical challenges. Stromal cells and cancer associated fibroblasts (CAF) play a critical biophysical role in cancer progression. CAFs are contractile and mechanosensitive which remodel the tumor microenvironment (TME) by cross linking, deforming and stiffening the matrix and release growth factors, which offers a unique niche for the cancer cells to become metastatic. We hypothesize that CAF-generated mechanical forces lead to metastatic spread directly or indirectly via release of growth factors including a member of the TGFb superfamily, namely activin. We will explore the mechanistic link between fibroblast force, activin release, and the induction of EMT and compare it with the activin serum levels of a CRC patient cohort. We measured activin serum levels by ELISA assay from a retrospective cohort of twenty-eight colorectal cases and twenty-eight polyp free controls. Conditioned media from co-culture of human CRC epithelial cells (FET) and stromal cells (CCD18) was examined by ELISA assay for secreted activin and was also used to culture CRC FET cells to measure induction of metastatic behavior through increase in proteins involved in epithelial-to-mesenchymal transition (EMT). CCD18 were seeded on substrates with increasing stiffness (2kPa, 10kPa, 40kPa) and treated for 72 hours with or without TGFb to generate conditioned media. FET cells were incubated with this media and subjected to trans-well migration and metabolic activity assays.Activin levels were up regulated in serum from stage IV CRC patients. High activin levels may be a predictive marker for metastasis in this cohort, with an Area Under the Curve of 0.8163. CCD18 cells secreted a 10-fold higher level of activin than FET cells alone and co-culture of CCD18 and FET cells resulted in an additional 2-fold increase in activin. Treatment of conditional media from TGFb activated stromal cells with the activin ligand trap, follistatin, leads to a decrease in FET cell migration and reversal of EMT indicating that the activin increase is functionally relevant and critical to the pro-oncogenic stromal response by TGFb. We plated CCD18 cells 2, 10 and 40 kPa polyacrylamide (PA) substrates functionalized by fibronectin with and without TGFb. We found that, with increasing gel substrate stiffness, (a) FET became more migratory, (b) CCD18 contractility increased with increasing stiffness, (c) activin in the condition media increased with increasing force. Inhibiting activin with follistatin decreased the TGFb induced activin secretion and migration in FETs. We suggest that increased TME stiffness leads to stromal cells amplification of TGFb pro-oncogenic function through thus far unrecognized induction and utilization of activin signaling.

Citation Format: Jessica Bauer, Jonas J. Staudacher, Georgina Mancinelli, Nancy Krett, Emon Bashar, Paul Grippo, M Taher A. Saif, Barbara Jung. Increased stiffness of the tumor microenvironment in colon cancer leads to an increase in activin and metastatic potential [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 177.