G. Karagkounis1,2, J. Zhao3, X. Li3, M. F. Kalady1,2 1Cleveland Clinic,Stem Cell Biology And Regenerative Medicine,Cleveland, OH, USA 2Cleveland Clinic,Colorectal Surgery,Cleveland, OH, USA 3Cleveland Clinic,Immunology,Cleveland, OH, USA
Introduction:
Our group has previously shown that interleukin-17 (IL-17) supports colorectal cancer stem cells, enhancing their viability and growth. Studying these effects in normal colonic mucosa is challenging due to the limited availability of in vitro models. Recently developed complex culture systems allowing the maintenance and expansion of multicellular structures derived from colonic mucosa, also known as colon organoids or epithelioids, have allowed new insight in colon tumorigenesis and response to inflammation. The goal of this study was to use colon organoids to investigate whether IL-17 promotes stemness in normal colonic mucosa.
Methods:
Freshly isolated mucosa without evidence of malignancy was collected from human colon according to an established IRB-approved protocol. Samples from different areas of the colon were cultured independently and expanded in media containing R-Spondin, Noggin, EGF and Wnt3a. Each cultured sample was split in two grossly equal fractions that were subsequently treated with either IL-17 (100ng/ml) or vehicle control. After 24 hours, the organoids were harvested and mRNA was isolated. Real-time quantitative PCR (RT-qPCR) was used to measure mRNA expression levels for interleukin-6 (IL-6), a known target of IL-17 used as positive control, as well as stem-cell markers ALDH1, and CD44. Paired t-test was used to compare expression levels between stimulated and control organoids.
Results:
Organoid cultures were established successfully and expanded for 10 days before experiments were performed. IL-17 stimulation resulted in significant upregulation of IL-6 expression compared to control (fold change 18.1, p=0.003). In addition, IL-17 induced CD44 (fold change 1.5, p=0.04), and ALDH1 (fold change 1.7, p=0.03).
Conclusion:
In this proof-of-concept study, inflammatory stimulation in the form of IL-17 induced stem-cell associated genes in normal colonic mucosa. Colon organoids enable in vitro studies of tumorigenesis and allow the assessment of mucosal response to various stimuli. These findings highlight the potential of this model and provide an opportunity for further studies to elucidate the role of IL-17 in the development of cancer.