Replication of patient- and sex-specific hallmarks of IBD in a human organ chip reveals stromal fibroblasts as drivers of inflammation, fibrosis, and enhanced cancer risk
By Benjamin Boettner
(BOSTON) — Inflammatory bowel disease (IBD), which comprises the inflammatory conditions Crohn’s disease and ulcerative colitis, affects about 1.6 million Americans, many of whom cannot be effectively treated. This is mostly due to a lack of understanding of what exactly causes the increased inflammation, fibrosis, and compromised intestinal barrier that underlie this disease and its manifold symptoms, ranging from severe abdominal pain, to diarrhea, weight loss, rectal bleeding, and anemia, to anxiety and depression. Many of these symptoms also tend to be stronger in women than in men and they increase their risk of preterm birth during pregnancy, making IBD a particular concern in Women’s Health. In addition, IBD patients have a significantly increased incidence of cancers in their intestinal tract.
The dearth of effective treatment options for IBD patients is mirrored by the utter lack of human in vitro models that can mimic the disease’s complex pathological features, help untangle causes and effects, and advance new therapies. Commonly used mouse models fall short of recapitulating human IBD because of differences in physiology, immunology, and gender-specific responses.
“IBD is a chronic inflammatory disease that is driven by uncontrolled inflammation, causing intestinal tissue to lose its integrity and build up pathological features. But which processes in the intestinal system trigger these responses in the first place and, over time, exacerbate the breakdown of the intestinal barrier and provoke fibrosis, the inflammation-induced thickening and scarring of intestinal tissue that occurs in IBD, largely remains a black box,” explains Alican Özkan, Ph.D., the first author of a new study that gives new impetus to IBD research.
In the study, a multi-disciplinary research team led by Wyss Institute at Harvard University Founding Director Donald Ingber, M.D., Ph.D., worked with clinicians at McGill University in Canada and Massachusetts General Hospital who provided IBD and healthy tissue biopsies from the colon region of the same patients to create donor-specific microfluidic organ-on-a-chip (Organ Chip) models of colon that replicate major hallmarks of IBD in vitro in an unprecedented way. Their approach pinpointed stromal fibroblasts, a type of cell in the connective tissue that underlies the intestinal epithelial lining, and mechanical forces associated with peristalsis motions as new drivers of IBD progression. For the first time, they also were able to demonstrate a direct impact of pregnancy hormones on IBD severity in female IBD patient chips and recapitulate the enhanced initiation of cancer formation in IBD tissues compared to healthy tissues in vitro. The findings are published in Nature Biomedical Engineering.
“Our human Colon Chips lined by living epithelial and stromal cells isolated from the same patients present an important breakthrough in IBD research because they enable us to control many different potentially contributing factors, including various cell types, hormonal exposures, and peristalsis motions, individually and in combination, which allowed us to gain new insight into key drivers of IBD development and progression,” said Ingber. “To my knowledge, this is the first model that has recapitulated in vitro the disease exacerbations that pregnant women with IBD often can experience. Perhaps even more importantly, we showed that our system enables studying the earliest stages of cancer formation within human tissues growing in an organ-relevant context in vitro. This allowed us to replicate the increased propensity of intestinal cells to undergo cancer formation that is observed IBD patients and discover that IBD-associated stromal fibroblasts help to drive this process through aberrant stromal-epithelial interactions.” Ingber is also the Judah Folkman Professor of Vascular Biology at Harvard Medical School and Boston Children’s Hospital, and the Hansjörg Wyss Professor of Biologically Inspired Engineering at Harvard John A. Paulson School of Engineering and Applied Sciences.
IBD is a chronic inflammatory disease that is driven by uncontrolled inflammation causing intestinal tissue to lose its integrity and build up pathological features. But which processes in the intestinal system trigger these responses in the first place and, over time, exacerbate the break-down […] largely remains a black box.
From taking fresh inventory of IBD…
To build donor-specific IBD and healthy control chips that recreate the tissue-tissue interface seen in the actual intestinal wall, the team grew and expanded epithelial cells derived from inflamed and unaffected regions from patients’ colons in organoid cultures. They then dissociated the organoids into their component cells and cultured them in one of two parallel channels of a microfluidic organ chip the size of a memory stick. In the other channel, which is separated from the epithelial channel by a porous membrane, they created a stroma-like tissue using fibroblasts derived from respective inflamed or unaffected colon regions from the same patient tissue specimens. Both tissue compartments were independently perfused with media, simulating the flow of intestinal fluids and blood, respectively; and they could communicate with each other by sending and receiving molecules that passed through the pores of the separating membrane. Importantly, these human Colon Chips could also be subjected to peristalsis-like mechanical motions by cyclically stretching and contracting the membrane and adherent tissues in this flexible device, and human immune cells could be perfused or circulated through the device.
Colon Chips constructed with IBD-affected epithelial and fibroblast cells (IBD Chips), as opposed to Colon Chips constructed with healthy cells from the same patients (Healthy Chips), showed typical hallmarks of IBD, including a decreased height of the epithelial wall and leakage of unwanted molecules through the normally tight intestinal barrier, thinning of the secreted mucus layer that protects the intestinal wall from various insults, increased production of inflammatory molecules, and enhanced fibrosis (accumulation of fibrillar collagens by IBD stromal fibroblasts). The gene expression patterns of cells in IBD Chips also resembled those known for Crohn’s disease and ulcerative colitis. Moreover, when the researchers perfused immune cells through the stromal channel, they migrated through stromal tissue, passed through the porous membrane, and entered into the epithelial tissue in IBD Chips mimicking inflammation seen in vivo, whereas few immune cells did this in Healthy Chips. Interestingly, many of these IBD features were also exacerbated when the researchers applied peristalsis-like motions.
… to new IBD drivers
Importantly, their chip design enabled the team to address a long-standing question: which intestinal cell type acts as the principal driver of IBD. Colon Chips were created containing “tissue recombinants” by lining them with IBD epithelial cells derived from IBD-affected tissues and healthy fibroblasts from a healthy region of the same patient’s colon, and vice versa. When they did this, they found that IBD-affected stromal fibroblasts were the major driver of many of the classic features of IBD, including barrier disruption and enhanced inflammation. “Our study for the first time demonstrates that diseased fibroblasts are sufficient to cause healthy epithelial cells to take on many features of IBD. Previously, researchers have used inflammatory molecules to induce these alterations without knowing precisely which cells actually produced them,” said Özkan, who now is a Senior Scientist at Abbvie Immunology Discovery.
Modeling women-specific IBD
The symptoms of IBD are often exacerbated in women, particularly during pregnancy, and physicians often warn women with IBD who seek to become pregnant to first ensure their disease is under control. If doctors could effectively prevent this exacerbation, risks of miscarriage, preterm birth and newborns’ low birthweights could be avoided. To address whether ovarian hormones directly exacerbate IBD in pregnant women by acting on the colon, rather than indirectly through other changes they cause in the body, the team flowed a cocktail of pregnancy-associated hormones at concentrations through the chips that corresponded to those in the first trimester of pregnancy. Exposure to pregnancy hormones enhanced inflammation and dramatically increased the level of fibrosis in the IBD Chips, but not in Healthy Chips. Thus, these findings offer a potential new avenue for finding therapeutic interventions in women with IBD who seek to become pregnant.
Window to study human cancer initiation in vitro and its enhancement in IBD
IBD patients have a higher risk of developing cancer. But how exactly this dismal shift occurs is still enigmatic. An understanding of the changes in epithelial cells that drive the earliest stages of this transition when they are present in living tissues within the intestines of IBD patients would open new opportunities for researchers to develop cancer-preventing interventions. Unfortunately, it has not been possible to study how human cancers initiate within a relevant tissue and organ context in vitro, until now.
To my knowledge, this is the first model that has recapitulated in vitro the disease exacerbations that pregnant women with IBD often can experience. Perhaps even more importantly, we showed that our system enables studying the earliest stages of cancer formation within human tissues growing in an organ-relevant context in vitro.
To ask whether their IBD Chips could be used to investigate the transition from IBD to cancer, the Wyss team exposed IBD and Healthy Chips to a carcinogen, known as ENU, working in collaboration with researchers at Queen Mary University of London and Cancer Research UK Cambridge Institute in Great Britain. The IBD Chips exhibited a significantly higher sensitivity to ENU than Healthy Chips. The levels of inflammatory molecules they produced were elevated and signaling pathways known to be associated with cancer initiation became activated. Even more impressively, the colon cells in the IBD chips accumulated diverse types of gene mutations and chromosomal duplications, whereas this was not seen in Healthy Chips. “By treating IBD Chips lined with tissue recombinants with ENU, we again found a dominant function for IBD fibroblasts, which potently induced the expression of the early-stage colorectal cancer marker (CEACAM5) in healthy epithelial cells exposed to this carcinogen,” said Özkan. “This highlights the relevance of fibroblasts as important drivers of IBD-associated cancers as well as many other features of this disease.”
“These findings are very exciting as, to our knowledge, they are the first to demonstrate early cancer progression in a preclinical human Organ Chip model, and they open the door to a much more detailed investigation of this transition as well as the development of targeted interventions, particularly in IBD where patients with the disease have an increased propensity for cancer formation,” said Ingber. “This personalized patient-to-chip approach that serves as both a mechanistic tool and a testbed for new therapeutics in a personalized way will hopefully lead to new and more effective approaches to mitigate painful features of these diseases, as well as to prevent cancer formation, in both men and women with IBD in the future.”
Other authors on the study are Gwenn Merry, David Chou, Ryan Posey, Anna Stejskalova, Karina Calderon, Megan Sperry, Joshua Piatok, Viktor Horvath, Lorenzo Ferri, Emanuela Carlotti, Stuart McDonald, Douglas Winton, Rocco Riccardi, Liliana Bordeianou, Sean Hall, and Girija Goyal. The study was supported by the Wyss Institute for Biologically Inspired Engineering at Harvard University, Cancer Research UK (award #C19767/A27145), as well as by the NIH training grants #5T32DK007199-44 and 5T32EB016652-10 to Özkan).