Abstract 928: Myofibroblasts induce poorly differentiated tumor-like spheroids in small intestinal organoids by Wnt-independent mechanism
Authors: Pastula Agnieszka , Janssen Klaus-Peter , Hauck Stefanie , Schmid Roland M. , Quante Michael 
Published: July 2016

Abstract
Recently, three dimensional (3D) organoid cultures were established from different organs; however those organoid cultures are exclusively composed of epithelial cells, thus missing the stromal niche. The later one is believed to play a crucial role during all stages of tumor development and resistance to therapy. One of the most abundant and the most important stromal cell in the tumor microenvironment is a myofibroblast. The contribution of myofibroblasts to tumor progression is well established, however little is known about their role during epithelial cell homeostasis and tumor initiation.
The niche was reconstructed by combining small intestinal (SI) crypts together with different types of mesenchymal cells in 3D culture systems. As mesenchymal cells the following cells were used: murine small intestinal fibroblasts, murine gastric carcinoma associated fibroblasts (CAFs), human fetal esophageal fibroblasts and human cardia myofibroblasts. While SI organoid culture is almost exclusively composed of budding structures, in the co-culture with intestinal myofibroblasts ∼50% of crypts were growing as non-budding spheroids. Those spheroids were positive for Ki-67, and independent of external R-Spondin, EGF and Noggin. Surprisingly, no differences between CAFs and other mesenchymal cells could be observed. Gene expression analysis revealed that myofibroblasts significantly upregulated Sox-9 (∼3 fold) and CD44 (∼4-fold) mRNA levels, and promoted proliferation rather than differentiation in the SI organoids. Clonogenicity assay showed that in the co-culture crypt cells exhibited about 3 times greater self-renewal capacity. Interestingly, by morphology and decreased number of PAS positive cells, the spheroids resembled intestinal organoids from Apc +/1638N mouse tumors. However, in the spheroids induced by co-culture the expression of Axin-2 was not increased, and Wnt inhibitor studies (IWP-2, C59) provided additional evidence that spheroid induction can be mediated by other mechanism than canonical Wnt. Indirect co-culture and conditioned media experiments revealed that spheroid formation was mediated by soluble factors. Mass spectrometric analysis of the secretome from the co-culture suggested the involvement of extracellular matrix-receptor pathway and focal adhesion pathway.
Taken together, our studies demonstrate that the microenvironment shapes intestinal crypts in a similar way as cell-autonomous genetic alterations. The data point out to the contribution of myofibroblasts to tumor initiation phenotype in the intestinal crypts by a mechanism independent of canonical Wnt. The precise signaling pathway remains to be elucidated in the future. Increased knowledge on the cellular communication between stroma and epithelium can contribute to the development of anti-cancer therapeutics that target not only cancer cells, but also the stromal niche.
Citation Format: Agnieszka Pastula, Klaus-Peter Janssen, Stefanie Hauck, Roland M. Schmid, Michael Quante. Myofibroblasts induce poorly differentiated tumor-like spheroids in small intestinal organoids by Wnt-independent mechanism. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 928.