Periodic Reporting for period 1 - ORIBAR (Defining the origin of Barrett’s oesophagus by exploring a submucosal gland stem cell model)
Okres sprawozdawczy: 2024-01-01 do 2025-12-31
The tube connecting the throat with the stomach is called the gullet or the oesophagus. Cancer of the oesophagus does not just appear. Years before cancer appears, the mucous membrane of the oesophagus changes form. This new mucous membrane is called ‘Barrett’s oesophagus or oesophageal metaplasia’. We do not yet understand where Barrett’s oesophagus comes from. The oesophagus has glands where Barrett’s oesophagus might come from, the so called oesophageal submucosal glands.
The change in form of the mucous membrane is the origin of cancer in many organs. Metaplasia, which means “change in form”, is termed Barrett’s oesophagus when it occurs in the oesophagus. Morphologically, i.e. with regard to tissue form, Barrett’s oesophagus resembles the mucous surface of the large intestine. It develops in individuals with reflux of acid and bile from the stomach into the oesophagus. Barrett’s oesophagus is a predominantly European disease with the highest incidences in Northwestern Europe. Obesity is a risk factor for reflux of acid and bile from the stomach into the oesophagus. The increase in obesity drives the development of Barrett’s oesophagus. As Barrett’s oesophagus is the precursor lesion of oesophageal adenocarcinoma (glandular cancer of the oesophagus), the occurrence of oesophageal adenocarcinoma is expected to rise with the increased occurrence of Barrett’s oesophagus and the increased occurrence of obesity. By 2030, it is estimated that roughly 1% of European men will be diagnosed with oesophageal cancer in their lifetime. While screening might be cost effective for the highest risk patients, it remains impractical, and overall surveillance is not cost effective. This indicates an urgent need to improve our understanding of Barrett’s oesophagus to enable better prevention and treatment for Barrett’s oesophagus and oesophageal adenocarcinoma.
The problem is that the origin of Barrett’s oesophagus is unknown, which hampers its better understanding, timely detection and treatment. Several cells of origin are proposed in literature. The first is the differentiated squamous mucosal cell which would have to de- and re-differentiate into a Barrett’s cell. However, experiments have never shown the emergence of Barrett’s oesophagus via this path. The second potential cell of origin could be a cell located at the gastro-oesophageal junction. However, this theory cannot explain the occurrence of Barrett’s oesophagus after the gastroesophageal junction has been surgically removed. The last theory explains the occurrence of Barrett’s oesophagus by mucosal, tissue specific, stem cells present in adult tissues. Under certain circumstances these cells could give rise to Barrett’s oesophagus. If stem cells undergoing an intestinal differentiation program are indeed present in the adult oesophageal tissue, one wonders where these cells might reside. Literature shows patches of the squamous mucosa, within a field of Barrett’s oesophagus, were associated with oesophageal submucosal glands. These oesophageal submucosal glands produce acid-neutralizing bicarbonate, mucins, antimicrobial agents, and growth factors. Additionally, a mutation was reported in an oesophageal submucosal gland which was continuous with a field of Barrett’s oesophagus. Therefore, the research question of this project was: do oesophageal submucosal glands contain a potential cell of origin of Barrett’s oesophagus? The hypothesis was that oesophageal submucosal glands contain a tissue specific stem cell capable of generating a clone of cells with properties similar to Barrett’s oesophagus cells. Due to the absence of adequate experimental models, this hypothesis has not been tested before in the human context. This has led to the following three research objectives: Firstly, to establish and validate the first human 2D oesophageal submucosal gland cell model. Secondly, to test whether oesophageal submucosal gland cells can generate a clone of Barrett’s oesophagus like cells. Thirdly, to define the characteristics of different oesophageal submucosal gland cell clones.
The change in form of the mucous membrane is the origin of cancer in many organs. Metaplasia, which means “change in form”, is termed Barrett’s oesophagus when it occurs in the oesophagus. Morphologically, i.e. with regard to tissue form, Barrett’s oesophagus resembles the mucous surface of the large intestine. It develops in individuals with reflux of acid and bile from the stomach into the oesophagus. Barrett’s oesophagus is a predominantly European disease with the highest incidences in Northwestern Europe. Obesity is a risk factor for reflux of acid and bile from the stomach into the oesophagus. The increase in obesity drives the development of Barrett’s oesophagus. As Barrett’s oesophagus is the precursor lesion of oesophageal adenocarcinoma (glandular cancer of the oesophagus), the occurrence of oesophageal adenocarcinoma is expected to rise with the increased occurrence of Barrett’s oesophagus and the increased occurrence of obesity. By 2030, it is estimated that roughly 1% of European men will be diagnosed with oesophageal cancer in their lifetime. While screening might be cost effective for the highest risk patients, it remains impractical, and overall surveillance is not cost effective. This indicates an urgent need to improve our understanding of Barrett’s oesophagus to enable better prevention and treatment for Barrett’s oesophagus and oesophageal adenocarcinoma.
The problem is that the origin of Barrett’s oesophagus is unknown, which hampers its better understanding, timely detection and treatment. Several cells of origin are proposed in literature. The first is the differentiated squamous mucosal cell which would have to de- and re-differentiate into a Barrett’s cell. However, experiments have never shown the emergence of Barrett’s oesophagus via this path. The second potential cell of origin could be a cell located at the gastro-oesophageal junction. However, this theory cannot explain the occurrence of Barrett’s oesophagus after the gastroesophageal junction has been surgically removed. The last theory explains the occurrence of Barrett’s oesophagus by mucosal, tissue specific, stem cells present in adult tissues. Under certain circumstances these cells could give rise to Barrett’s oesophagus. If stem cells undergoing an intestinal differentiation program are indeed present in the adult oesophageal tissue, one wonders where these cells might reside. Literature shows patches of the squamous mucosa, within a field of Barrett’s oesophagus, were associated with oesophageal submucosal glands. These oesophageal submucosal glands produce acid-neutralizing bicarbonate, mucins, antimicrobial agents, and growth factors. Additionally, a mutation was reported in an oesophageal submucosal gland which was continuous with a field of Barrett’s oesophagus. Therefore, the research question of this project was: do oesophageal submucosal glands contain a potential cell of origin of Barrett’s oesophagus? The hypothesis was that oesophageal submucosal glands contain a tissue specific stem cell capable of generating a clone of cells with properties similar to Barrett’s oesophagus cells. Due to the absence of adequate experimental models, this hypothesis has not been tested before in the human context. This has led to the following three research objectives: Firstly, to establish and validate the first human 2D oesophageal submucosal gland cell model. Secondly, to test whether oesophageal submucosal gland cells can generate a clone of Barrett’s oesophagus like cells. Thirdly, to define the characteristics of different oesophageal submucosal gland cell clones.
Oesophageal submucosal glands were micro-dissected from chemo(radio)therapy treated esophagectomy specimens. Culture conditions were optimized and quantified using MTT proliferations assays with respect to EGF, fibroblast conditioned medium, and Wnt, Noggin, and R-spondin-conditioned medium concentrations. Cell proliferation capacity were assessed by the Click-iT™ EdU kit. Cells were cultured either in Matrigel to achieve 3D cultures or were cultured in 2D fashion. Organoid morphology was assessed through bright-field microscopy, H&E staining, and immunohistochemistry.
Human oesophageal submucosal glands could be identified and dissected from full-thickness oesophageal wall specimens. Oesophageal submucosal gland cell cultures show increased proliferation when exposed to higher concentrations of EGF. The addition of medium conditioned by an immortalized oesophageal fibroblast cell line also results in increased proliferation. Oesophageal submucosal gland cells can be maintained as 3D organoids and can form a 2D attaching multilayer. Oesophageal submucosal gland cells can be harvested from the glands and cultured under these conditions for several months. Oesophageal submucosal glands cells keep their proliferative capacity after cryopreservation and passage. Oesophageal submucosal gland cells continue to proliferate, after large dilution of the original cell suspension, at extremely low seeding density. 3D cultured human oesophageal submucosal glands organoids show heterogeneity in their morphology. Notably, organoids tend either to take on a solid or a hollow spheroid form. Also, these organoids express either TP63, the marker of squamous epithelium, or CK7, the marker of glandular cells, which is also known to be expressed in Barrett’s oesophagus.
Human oesophageal submucosal glands could be identified and dissected from full-thickness oesophageal wall specimens. Oesophageal submucosal gland cell cultures show increased proliferation when exposed to higher concentrations of EGF. The addition of medium conditioned by an immortalized oesophageal fibroblast cell line also results in increased proliferation. Oesophageal submucosal gland cells can be maintained as 3D organoids and can form a 2D attaching multilayer. Oesophageal submucosal gland cells can be harvested from the glands and cultured under these conditions for several months. Oesophageal submucosal glands cells keep their proliferative capacity after cryopreservation and passage. Oesophageal submucosal gland cells continue to proliferate, after large dilution of the original cell suspension, at extremely low seeding density. 3D cultured human oesophageal submucosal glands organoids show heterogeneity in their morphology. Notably, organoids tend either to take on a solid or a hollow spheroid form. Also, these organoids express either TP63, the marker of squamous epithelium, or CK7, the marker of glandular cells, which is also known to be expressed in Barrett’s oesophagus.
In this project, the first human oesophageal submucosal gland cell model was established. Factors enhancing the cell pool growth were quantified. Oesophageal submucosal gland cells were shown to maintain their proliferative capacity after cryopreservation enabling the collaboration with international research groups in the field. Oesophageal submucosal gland organoids were shown to exhibit a diversity between clones in morphology and marker expression, including expression of CK7, a glandular mucosal marker also found in Barrett’s oesophagus.