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Colorectal cancer is a major cause of mortality worldwide. Chemotherapy and radiation remain standard treatment for locally advanced disease, with current immune-targeting therapies applying to only a small subset of patients. Expression of the immuno-oncology target indoleamine 2,3 dioxygenase 1 (IDO1) is associated with poor colorectal cancer clinical outcomes but is understudied as a potential treatment target. In this study, we examined the interaction between the IDO1 pathway and radiotherapy in colorectal cancer. We used human and mouse colorectal cancer cell lines, organoids, mouse syngeneic colorectal cancer tumor graft models, and colorectal cancer tissues from patients who received radiotherapy. IDO1 activity was blocked using the clinical IDO1 inhibitor epacadostat and by genetic disruption. We found that radiation induced IDO1 overexpression in colorectal cancer through type I and II IFN signaling. IDO1 enzymatic activity directly influenced colorectal cancer radiation sensitivity. IDO1 inhibition sensitized colorectal cancer to radiation-induced cell death, whereas the IDO1 metabolite kynurenine promoted radioprotection. IDO1 inhibition also potentiated Th1 cytokines and myeloid cell–modulating factors in the tumor microenvironment and promoted an abscopal effect on tumors outside the radiation field. Conversely, IDO1 blockade protected the normal small intestinal epithelium from radiation toxicity and accelerated recovery from radiation-induced weight loss, indicating a role in limiting side effects. These data demonstrated that IDO1 inhibition potentiates radiotherapy effectiveness in colorectal cancer. The findings also provide rationale and mechanistic insight for the study of IDO1 inhibitors as adjuvant therapy to radiation in patients with locally advanced sporadic and colitis-associated colorectal cancer.

The development and physiologic role of small intestine (SI) vasculature is poorly studied. This is partly due to a lack of targetable, organ-specific markers for in vivo studies of two critical tissue components: endothelium and stroma. This challenge is exacerbated by limitations of traditional cell culture techniques, which fail to recapitulate mechanobiologic stimuli known to affect vessel development. Here, we construct and characterize a 3D in vitro microfluidic model that supports the growth of patient-derived intestinal subepithelial myofibroblasts (ISEMFs) and endothelial cells (ECs) into perfused capillary networks. We report how ISEMF and EC-derived vasculature responds to physiologic parameters such as oxygen tension, cell density, growth factors, and pharmacotherapy with an antineoplastic agent (Erlotinib). Finally, we demonstrate effects of ISEMF and EC co-culture on patient-derived human intestinal epithelial cells (HIECs), and incorporate perfused vasculature into a gut-on-a-chip (GOC) model that includes HIECs. Overall, we demonstrate that ISEMFs possess angiogenic properties as evidenced by their ability to reliably, reproducibly, and quantifiably facilitate development of perfused vasculature in a microfluidic system. We furthermore demonstrate the feasibility of including perfused vasculature, including ISEMFs, as critical components of a novel, patient-derived, GOC system with translational relevance as a platform for precision and personalized medicine research.

Colorectal cancer is a major cause of mortality worldwide. Chemotherapy and radiation remain standard treatment for locally advanced disease, with current immune-targeting therapies applying to only a small subset of patients. Expression of the immuno-oncology target indoleamine 2,3 dioxygenase 1 (IDO1) is associated with poor colorectal cancer clinical outcomes but is understudied as a potential treatment target. In this study, we examined the interaction between the IDO1 pathway and radiotherapy in colorectal cancer. We used human and mouse colorectal cancer cell lines, organoids, mouse syngeneic colorectal cancer tumor graft models, and colorectal cancer tissues from patients who received radiotherapy. IDO1 activity was blocked using the clinical IDO1 inhibitor epacadostat and by genetic disruption. We found that radiation induced IDO1 overexpression in colorectal cancer through type I and II IFN signaling. IDO1 enzymatic activity directly influenced colorectal cancer radiation sensitivity. IDO1 inhibition sensitized colorectal cancer to radiation-induced cell death, whereas the IDO1 metabolite kynurenine promoted radioprotection. IDO1 inhibition also potentiated Th1 cytokines and myeloid cell–modulating factors in the tumor microenvironment and promoted an abscopal effect on tumors outside the radiation field. Conversely, IDO1 blockade protected the normal small intestinal epithelium from radiation toxicity and accelerated recovery from radiation-induced weight loss, indicating a role in limiting side effects. These data demonstrated that IDO1 inhibition potentiates radiotherapy effectiveness in colorectal cancer. The findings also provide rationale and mechanistic insight for the study of IDO1 inhibitors as adjuvant therapy to radiation in patients with locally advanced sporadic and colitis-associated colorectal cancer.

Background & Aims Inflammation, injury, and infection up-regulate expression of the tryptophan metabolizing enzyme indoleamine 2,3-dioxygenase 1 (IDO1) in the intestinal epithelium. We studied the effects of cell-specific IDO1 expression in the epithelium at baseline and during intestinal inflammation in mice. Methods We generated transgenic mice that overexpress fluorescence-tagged IDO1 in the intestinal epithelium under control of the villin promoter (IDO1-TG). We generated intestinal epithelial spheroids from mice with full-length Ido1 (controls), disruption of Ido1 (knockout mice), and IDO1-TG and analyzed them for stem cell and differentiation markers by real-time polymerase chain reaction, immunoblotting, and immunofluorescence. Some mice were gavaged with enteropathogenic Escherichia coli (E2348/69) to induce infectious ileitis, and ileum contents were quantified by polymerase chain reaction. Separate sets of mice were given dextran sodium sulfate or 2,4,6-trinitrobenzenesulfonic acid to induce colitis; intestinal tissues were analyzed by histology. We utilized published data sets GSE75214 and GDS2642 of RNA expression data from ilea of healthy individuals undergoing screening colonoscopies (controls) and patients with Crohn’s disease. Results Histologic analysis of small intestine tissues from IDO1-TG mice revealed increases in secretory cells. Enteroids derived from IDO1-TG intestine had increased markers of stem, goblet, Paneth, enteroendocrine, and tuft cells, compared with control enteroids, with a concomitant decrease in markers of absorptive cells. IDO1 interacted non-enzymatically with the aryl hydrocarbon receptor to inhibit activation of NOTCH1. Intestinal mucus layers from IDO1-TG mice were 2-fold thicker than mucus layers from control mice, with increased proportions of Akkermansia muciniphila and Mucispirillum schaedleri. Compared to controls, IDO1-TG mice demonstrated an 85% reduction in ileal bacteria (P = .03) when challenged with enteropathogenic E coli, and were protected from immune infiltration, crypt dropout, and ulcers following administration of dextran sodium sulfate or 2,4,6-trinitrobenzenesulfonic acid. In ilea of Crohn’s disease patients, increased expression of IDO1 correlated with increased levels of MUC2, LYZ1, and aryl hydrocarbon receptor, but reduced levels of SLC2A5. Conclusions In mice, expression of IDO1 in the intestinal epithelial promotes secretory cell differentiation and mucus production; levels of IDO1 are positively correlated with secretory cell markers in ilea of healthy individuals and Crohn’s disease patients. We propose that IDO1 contributes to intestinal homeostasis.