Patient Derived Colonoids as Drug Testing Platforms–Critical Importance of Oxygen Concentration

Abstract

Treatment of inflammatory bowel disease (IBD) is challenging, with a series of available drugs each helping only a fraction of patients. Patients may face time-consuming drug trials while the disease is active, thus there is an unmet need for biomarkers and assays to predict drug effect. It is well known that the intestinal epithelium is an important factor in disease pathogenesis, exhibiting physical, biochemical and immunologic driven barrier dysfunctions. One promising test system to study effects of existing or emerging IBD treatments targeting intestinal epithelial cells (IECs) is intestinal organoids (“mini-guts”). However, the fact that healthy intestinal epithelium is in a physiologically hypoxic state has largely been neglected, and studies with intestinal organoids are mainly performed at oxygen concentration of 20%. We hypothesized that lowering the incubator oxygen level from 20% to 2% would recapitulate better the in vivo physiological environment of colonic epithelial cells and enhance the translational value of intestinal organoids as a drug testing platform. In the present study we examine the effects of the key IBD cytokines and drug targets TNF/IL17 on human colonic organoids (colonoids) under atmospheric (20%) or reduced (2%) O2. We show that colonoids derived from both healthy controls and IBD-patients are viable and responsive to IBD-relevant cytokines at 2% oxygen. Because chemokine release is one of the important immunoregulatory traits of the epithelium that may be fine-tuned by IBD-drugs, we also examined chemokine expression and release at different oxygen concentrations. We show that chemokine responses to TNF/IL17 in organoids display similarities to inflamed epithelium in IBD-patients. However, inflammation-associated genes induced by TNF/IL17 were attenuated at low oxygen concentration. We detected substantial oxygen-dependent differences in gene expression in untreated as well as TNF/IL17 treated colonoids in all donors. Further, for some of the IBD-relevant cytokines differences between colonoids from healthy controls and IBD patients were more pronounced in 2% O2 than 20% O2. Our results strongly indicate that an oxygen concentration similar to the in vivo epithelial cell environment is of essence in experimental pharmacology.

Profound gene expression changes in the epithelial monolayer of active ulcerative colitis and Crohn’s disease

“…Beyond ATF3, no gene expression differences were verified on a protein level, and further verification would be required in follow-up studies of our initial observations. Indeed, we have previously published several studies where the initial hypotheses were formed on basis of the data presented in full for the first time here, thus representing a thorough validation of parts of the data [14,25,26].…”

Tofacitinib Downregulates TNF and Poly(I:C)-Dependent MHC-II Expression in the Colonic Epithelium

“…Pinch biopsies from patients were collected in 10% buffered formalin and histopathology was evaluated as described before ( 44 ). Colonoids for functional assays were established using pinch biopsies collected from uninflamed mucosa at the hepatic flexure ( 28 , 43 ). Patient characteristics are listed in Table 1 .…”

“…Colonoid cultures were established and grown in 20% O 2 and 5% CO 2 at 37°C as described in Skovdahl et al. ( 43 ), based on protocols of Mahe et al ( 40 ), and Jung et al. ( 45 ).…”

“…To extract RNA from colonoids, the RNeasy mini kit (#74106, Qiagen, Hilden, Germany) was used as per the manufacturer’s instructions. RNA sequencing (RNA-seq) of colonoids was performed as described before ( 43 ). RNA-seq libraries were generated using SENSE total RNASeq library prep kit with RiboCop rRNA depletion (Lexogen GmbH, Vienna, Austria).…”

“…The roles of antigen presentation by IECs have mostly been studied in epithelial cancer cell lines which do not carry the same cellular composition as in vivo IECs, or in animal models ( 12 , 14 , 24 , 25 , 38 ) that do not fully reflect IBD pathobiology ( 39 ). A promising model for addressing human IBD-pathobiology and IBD-drug response in intestinal epithelium is intestinal epithelial organoids that recapitulate in vivo IECs-specific functional characteristics ( 28 , 40 – 43 ). Recently, Wosen et al.…”