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Cancer immunotherapy often involves the use of engineered molecules to selectively bind and activate T cells located within tumour tissue. Fundamental to the success of such treatments is the presence or recruitment of T cells localised within the tumour microenvironment. Advanced organ-on-a-chip systems provide an in vitro setting in which to investigate how novel molecules influence the spatiotemporal dynamics of T cell infiltration into tissue, both in the context of anti-tumour efficacy and off-tumour toxicity. While highly promising, the complexity of these systems is such that mathematical modelling plays a crucial role in the quantitative evaluation of experimental results and maximising the mechanistic insight derived. We develop a mechanistic, mathematical model of a novel microphysiological in vitro platform that recapitulates T cell infiltration into epithelial tissue, which may be normal or transformed. The mathematical model describes the spatiotemporal dynamics of infiltrating T cells in response to chemotactic cytokine signalling. We integrate the model with dynamic imaging data to optimise key model parameters. The mathematical model demonstrates a good fit to the observed experimental data and accurately describes the distribution of infiltrating T cells. This model is designed to complement the in vitro system; with the potential to elucidate complex biological mechanisms, including the mode of action of novel therapies and the drivers of safety events, and, ultimately, improve the efficacy-safety profile of T cell-targeted cancer immunotherapies.

Background & Aims: The mucus layer in the human colon protects against commensal bacteria and pathogens, and defects in its unique bilayered structure contribute to intestinal disorders, such as ulcerative colitis. However, our understanding of colon physiology is limited by the lack of in vitro models that replicate human colonic mucus layer structure and function. Here, we investigated if combining organ-on-a-chip and organoid technologies can be leveraged to develop a human-relevant in vitro model of colon mucus physiology. Methods: A human colon-on-a-chip (Colon Chip) microfluidic device lined by primary patient-derived colonic epithelial cells was used to recapitulate mucus bilayer formation, and to visualize mucus accumulation in living cultures noninvasively. Results: The Colon Chip supports spontaneous goblet cell differentiation and accumulation of a mucus bilayer with impenetrable and penetrable layers, and a thickness similar to that observed in the human colon, while maintaining a subpopulation of proliferative epithelial cells. Live imaging of the mucus layer formation on-chip showed that stimulation of the colonic epithelium with prostaglandin E2, which is increased during inflammation, causes rapid mucus volume expansion via an Na-K-Cl cotransporter 1 ion channel–dependent increase in its hydration state, but no increase in de novo mucus secretion. Conclusions: This study shows the production of colonic mucus with a physiologically relevant bilayer structure in vitro, which can be analyzed in real time noninvasively. The Colon Chip may offer a new preclinical tool to analyze the role of mucus in human intestinal homeostasis as well as diseases, such as ulcerative colitis and cancer. Keywords: Organ Chip, Intestine, Organoid, Microfluidic, Goblet Cell, Inflammatory Bowel Disease

Abstract Background Species-specific differences in tolerance to infection are exemplified by the high susceptibility of humans to enterohemorrhagic Escherichia coli (EHEC) infection, whereas mice are relatively resistant to this pathogen. This intrinsic species-specific difference in EHEC infection limits the translation of murine research to human. Furthermore, studying the mechanisms underlying this differential susceptibility is a difficult problem due to complex in vivo interactions between the host, pathogen, and disparate commensal microbial communities. Results We utilize organ-on-a-chip (Organ Chip) microfluidic culture technology to model damage of the human colonic epithelium induced by EHEC infection, and show that epithelial injury is greater when exposed to metabolites derived from the human gut microbiome compared to mouse. Using a multi-omics approach, we discovered four human microbiome metabolites—4-methyl benzoic acid, 3,4-dimethylbenzoic acid, hexanoic acid, and heptanoic acid—that are sufficient to mediate this effect. The active human microbiome metabolites preferentially induce expression of flagellin, a bacterial protein associated with motility of EHEC and increased epithelial injury. Thus, the decreased tolerance to infection observed in humans versus other species may be due in part to the presence of compounds produced by the human intestinal microbiome that actively promote bacterial pathogenicity. Conclusion Organ-on-chip technology allowed the identification of specific human microbiome metabolites modulating EHEC pathogenesis. These identified metabolites are sufficient to increase susceptibility to EHEC in our human Colon Chip model and they contribute to species-specific tolerance. This work suggests that higher concentrations of these metabolites could be the reason for higher susceptibility to EHEC infection in certain human populations, such as children. Furthermore, this research lays the foundation for therapeutic-modulation of microbe products in order to prevent and treat human bacterial infection.

Summary: The intestinal epithelium serves as an essential barrier to the outside world and is maintained by functionally distinct populations of rapidly cycling intestinal stem cells (CBC ISCs) and slowly cycling, reserve ISCs (r-ISCs). Because disruptions in the epithelial barrier can result from pathological activation of the immune system, we sought to investigate the impact of inflammation on ISC behavior during the regenerative response. In a murine model of αCD3 antibody-induced small-intestinal inflammation, r-ISCs proved highly resistant to injury, while CBC ISCs underwent apoptosis. Moreover, r-ISCs were induced to proliferate and functionally contribute to intestinal regeneration. Further analysis revealed that the inflammatory cytokines interferon gamma and tumor necrosis factor alpha led to r-ISC activation in enteroid culture, which could be blocked by the JAK/STAT inhibitor, tofacitinib. These results highlight an important role for r-ISCs in response to acute intestinal inflammation and show that JAK/STAT-1 signaling is required for the r-ISC regenerative response.

Microfluidic organ-on-a-chip models of human intestine have been developed and used to study intestinal physiology and pathophysiology. In this article, we review this field and describe how microfluidic Intestine Chips offer new capabilities not possible with conventional culture systems or organoid cultures, including the ability to analyze contributions of individual cellular, chemical, and physical control parameters one-at-a-time; to coculture human intestinal cells with commensal microbiome for extended times; and to create human-relevant disease models. We also discuss potential future applications of human Intestine Chips, including how they might be used for drug development and personalized medicine. Keywords: Organs-on-Chips, Gut-on-a-Chip, Intestine-on-a-Chip, Microfluidic

The cellular and molecular mechanisms underlying adaptive changes to physiological stress within the intestinal epithelium remain poorly understood. Here, we show that PTEN, a negative regulator of the PI3K→AKT→mTORC1-signaling pathway, is an important regulator of dormant intestinal stem cells (d-ISCs). Acute nutrient deprivation leads to transient PTEN phosphorylation within d-ISCs and a corresponding increase in their number. This release of PTEN inhibition renders d-ISCs functionally poised to contribute to the regenerative response during re-feeding via cell-autonomous activation of the PI3K→AKT→mTORC1 pathway. Consistent with its role in mediating cell survival, PTEN is required for d-ISC maintenance at baseline, and intestines lacking PTEN have diminished regenerative capacity after irradiation. Our results highlight a PTEN-dependent mechanism for d-ISC maintenance and further demonstrate the role of d-ISCs in the intestinal response to stress.

Inflammatory bowel disease (IBD) is a chronic condition driven by loss of homeostasis between the mucosal immune system, the commensal gut microbiota, and the intestinal epithelium. Our goal is to understand how these components of the intestinal ecosystem cooperate to control homeostasis. By combining quantitative measures of epithelial hyperplasia and immune infiltration with multivariate analysis of inter- and intracellular signaling, we identified epithelial mammalian target of rapamycin (mTOR) signaling as a potential driver of inflammation in a mouse model of colitis. A kinetic analysis of mTOR inhibition revealed that the pathway regulates epithelial differentiation, which in turn controls the cytokine milieu of the colon. Consistent with our in vivo analysis, we found that cytokine expression of organoids grown ex vivo, in the absence of bacteria and immune cells, was dependent on differentiation state. Our study suggests that proper differentiation of epithelial cells is an important feature of colonic homeostasis because of its effect on the secretion of inflammatory cytokines.

Lgr5-expressing intestinal stem cells (ISCs) renew the adult gut epithelium by producing mature villus cells (VCs); the transcriptional basis for ISC functions remains unclear. RNA sequencing analysis identified transcripts modulated during differentiation of Lgr5+ ISCs into VCs, with high expression of the intestine-restricted transcription factor (TF) gene Cdx2 in both populations. Cdx2-deleted mouse ISCs showed impaired proliferation and long-term inability to produce mature lineages, revealing essential ISC functions. Chromatin immunoprecipitation sequencing analysis of CDX2 in Lgr5+ ISCs, coupled with mRNA profiling of control and Cdx2−/− ISCs, identified features of CDX2 regulation distinct from VCs. Most CDX2 binding in ISCs occurs in anticipation of future gene expression, but whereas CDX2 primarily activates VC genes, direct ISC targets are activated and repressed. Diverse CDX2 requirements in stem and differentiated cells may reflect the versatility of TFs that specify a tissue in development and control the same tissue in adults.