Mapping the Human Appendix Using Single Cell Sequencing

Study Overview
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Brief Summary
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The overall purpose of this study is to describe the cellular composition of the human appendix and its gene expression using scRNAseq and scATACseq methods. This will potentially provide is with a complete and detailed map of the appendix´ immunological properties and its role in neuro-endocrine/metabolic functions. Our results will be held up against current knowledge of the appendix and its role in the human body and thus hopefully expand our understanding of this organ and the consequences of its removal by appendectomy.

Detailed Description
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Until recently the human appendix was viewed as a rudimentary organ without any specific function in the human body. Our current knowledge of the cellular composition of the appendix is solely based on histological examinations combined with immunohistochemical stains. These examinations have shown that the structural composition of the appendix is similar to the large intestine, with mucosa, submucosa, muscularis externa and serosa. Uniquely, the appendix has almost circumferential lymphoid follicles in the submucosa and lamina propria, and thus becomes a secondary lymphoid organ. In addition to this, the appendix contains Lieberkühn's crypts, which in contrast to the large intestine contains Paneth cells and argentaffine (enterochromaffin) neuro-endocrine cells1 2 Recently, the appendix´ role in regulation of immunologic functions has been discovered. It is believed that the great bacterial diversity in the appendix stimulates the human immune system and aids in the maturation and diversification of white blood cells, especially B-cells in the lymphoid follicles in the appendix.3 The appendix contains a larger quantity of CD5+ (B1), CD19+, Immunoglobulin-secreting IgG and secretory IgA cells compared to the large intestine. In the luminal follicle-associated epithelium, a high concentration of intra-epithelial M-cells as well as human leukocyte antigen D-related (HLA-DR) T and B cells are seen.4 The human appendix has a unique cellular composition, which plays a role in bacterial homeostasis in the large intestine5 and entero-endocrine regulation6. Furthermore, removing the appendix can drastically change incidence of certain metabolic and immune-related diseases such as type II diabetes7 and ulcerative colitis8. Despite this, only very few studies have investigated the cellular composition of the human appendix, and none of these have used single-cell (sc) RNA or DNA-sequencing to aid in our understanding of this organ. By using scRNAseq and scATACseq (Single-cell Assay of Transposase Accessible Chromatin sequencing) we will be able to map open regions in the cell's DNA and RNA, thus providing us with a unique map of the cells in the appendix as well is their gene expression9-11. ScATACseq visualizes open regions in the chromatin, generating peaks which can then be used to map DNA motifs, such as transcription factor binding sites. With the emergence of scATACseq, chromatin accessibility is in combination with gene expression data an extremely useful resource to study cell type specific regulatory DNA interactions. To further study the immunological aspects of the appendix, we will extract immune cells from the Peyer´s plaques. Lastly, full blood will be extracted to better analyse metabolic risk factors in relation to the appendix´ metabolic cellular regulation.

Official Title
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Cellular Composition of the Human Appendix Using Single-cell RNA and Single-cell ATAC-sequencing.

Conditions
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Diabetes, Appendicitis, Inflammatory Bowel Diseases

Intervention / Treatment
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* Other: Biopsy


Participation Criteria
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Eligibility Criteria
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Inclusion Criteria: Patients undergoing a right or extended right-hemicolectomy for colon cancer Patients able to read and understand danish Patients able to give informed consent Patients of Scandinavian ethnicity Exclusion Criteria: Previous large bowel resections Suspicion pre or intraoperatively of disease in the appendix Tumour <10cm from the appendiceal orifice. Known inflammatory bowel disease Immuno-modulation treatment Neo-adjuvant chemotherapy. < 18 years of age

Ages Eligible for Study
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Minimum Age: 18 Years

Sexes Eligible for Study
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All

Accepts Healthy Volunteers
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No

Study Plan
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How is the study designed?
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Arms and Interventions

| Participant Group/Arm | Intervention/Treatment |
| --- | --- |
| Colo-rectal cancer patients<br>Patients receiving right colectomy or ileo-ceacal resection for colorectal cancer | Other: Biopsy<br>* Participants included in the study, will have a full thickness biopsy of the middle of the appendix taken, after the resected bowel has been removed during surgery.<br>|

What is the study measuring?
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Primary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Profiling of open chromatin regions | Mapping of cell types, including rare cell types, using profiling of open chromatin regions. (ATAC-seq) in biopsies from the appendix | 2 years | 
| Evaluation of metabolic profile | Using bioimpedance, insulin and glucose measurements and CHiP-seq we will determine patient phenotype and epigenetics to evaluate their metabolic risk-profile and correlate this to cell types in the appendix | 2 years | 

Secondary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Appendix biofilm | By sequencing bacterial DNA in our samples, we will evaluate the mucosa-associated microbiome of the appendix. This will be correlated to the two primary outcome measure | 2 years |