The first long-term culture-based model of the colon, which enables researchers to examine the effects of long-term colonic inflammation on cells, has been developed by Kiichiro Tsuchiya, at Tokyo Medical and Dental University, and co-workers across Japan1.
Patients with chronic inflammatory bowel disorders, such as Crohn’s disease and ulcerative colitis, are at increased risk of developing colitis-associated cancer. The cancer development most often happens around a decade after initial diagnosis because long-term inflammation can permanently and adversely transform cells in the body. Specifically, the continual stimulation by pro-inflammatory proteins changes the expression of genes and disrupts signalling processes, so cells that were once healthy become cancerous.
The new technique, explains Tsuchiya, is based on creating an organoid — a miniaturised, three-dimensional version of the colon that precisely mimics the organ’s anatomy. In 2016, the team created organoids from epithelial cells found in the lining of mice intestines.
“We knew from previous studies that intestinal epithelial cells (IECs) are permanently altered by chronic exposure to pro-inflammatory cytokines,” says Tsuchiya. “However, previous models simply induced colitis in mice or rats, and because a colitis flare-up only lasts a few weeks or months, it was difficult to study the long-term effects of inflammation on individual cells.”
Once the team had created the IEC organoids, they added a mixture of cytokines and bacterial components to the cultures. They then monitored cellular and genetic changes in the organoids over 60 weeks.
“Developing organoids for our model was fairly straightforward, but the real challenge was maintaining a steady inflammatory state for months,” says Tsuchiya. “If the inflammatory stimulation was too strong, the cells died. We spent considerable time adjusting the concentration of each inflammatory reagent to maintain the cultures.”
Their results showed that a key signalling pathway called NF-κB, which is involved in cell survival and cytokine production, was significantly amplified after 60 weeks of continuous inflammation. This amplification continued even after all cytokine stimulation had stopped. The upregulation of NF-κB signalling led to the over-expression of many of its target genes, including one that induces the production of reactive oxygen species, fuelling oxidative stress and perpetuating the inflammatory state.
“We have since cultured human colonic organoids derived from both normal intestinal tissues and pre-cancerous lesions from patients with ulcerative colitis,” says Tsuchiya. “If we can determine cellular damage at different stages of inflammatory bowel disorders, we may be able to predict the likelihood of an individual developing colitis-associated cancer.”
The program-affiliated researchers contributing to this research are from Tokyo Medical and Dental University.
- Hibiya, S., Tsuchiya, K., Hayashi, R., Fukushima, K., Horita, N. et al. Long-term inflammation transforms intestinal epithelial cells of colonic organoids. Journal of Crohn’s and Colitis 11, 621–630 (2017).| Article