Title Page
Abstract
Contents
Chapter 1. Introduction 6
1.1. Study Background 6
1.2. Purpose of Research 8
Chapter 2. Body 9
2.1. Materials and Methods 9
2.1.1. Collection of fecal samples 9
2.1.2. 16S bacterial rRNA analysis 9
2.1.3. Sample preparations of CRC and colon epithelium for organoid culture 10
2.1.4. Organoid culture medium 10
2.1.5. Organoid culture 11
2.1.6. STR profiling 11
2.1.7. Treatment of cell-free microbial supernatant with organoids 12
2.1.8. RLU measurement for Cell viability assay 12
2.1.9. High-throughput Screening 13
2.1.10. RNA sequencing 13
2.1.11. Gas chromatography 14
2.2. Results 15
2.2.1. Schematic Diagram 15
2.2.2. Demographics 15
2.2.3. Metagenomic analysis 15
2.2.4. Establishment of colon normal and tumor organoid lines 16
2.2.5. Addition of bacterial supernatants to organoid culture media 16
2.2.6. Transcriptomic impact of the bacterial metabolome 17
2.2.7. Component analysis of bacterial culture supernatants 18
2.3. Discussion 19
Chapter 3. Conclusions 21
Bibliography 22
Abstract in Korean 63
Table 1. Species that are more abundant in healthy controls vs. CRC patients 34
Figure 1. Schematic diagram of the study. The overall flow chart was miniaturized. 27
Figure 2A. TNM classification and the AJCC stage of CRC (n=40) 28
Figure 2B. Alpha (intra-sample) diversity from healthy controls and CRC patients was analyzed by Chao1, Shannon, and Simpson methods 29
Figure 2C. Microbial taxonomic distribution data from healthy controls and CRC patients were patterned by Bray-Curtis, weighted... 30
Figure 2D. The microbial distribution of individual samples at the phylum level 31
Figure 2E. The linear discriminant analysis (LDA) effect size (LEfSe) analysis. The LDA score was calculated to present the portions of... 32
Figure 2F. Cladogram 33
Figure 3A. Bright field image of established CRC organoid lines 35
Figure 3B. Bright field image of established normal colon epithelium organoid lines 36
Figure 4A. SNU-7237-TO was treated with each microbial supernatant, organoid plain medium, and bacteria plain medium. The... 37
Figure 4B. SNU-7390S3-TO was treated with each microbial supernatant, organoid plain medium, and bacteria plain medium. The... 38
Figure 4C. Viable tumor organoid cells after treatment with culture metabolites visualized as a bar plot based on the relative light unit... 39
Figure 4D. Viable normal colon epithelium organoid cells after treatment with culture metabolites visualized as a bar plot based on... 40
Figure 4E. Perimeters in the captured images of SNU-7237-TO which is treated by each bacterial supernatant, bacterial culture... 41
Figure 5A. RNA sequencing analysis. The Bifidobacterium group separated from the CRC-abundant group (Parabacteroides... 43
Figure 5B. RNA sequencing analysis 44
Figure 6A. Component analysis. B. adolescentis and B. Longum were considered as healthy controls while data of P. distasonis and... 45
Figure 6B. The concentration of six short-chain fatty acids in each subjected sample 46
Figure 6C. Metabolomic distances between normal and cancer patients were performed for PCA (left) and OPLS-DA (right)... 47
Supplementary Figure 1A. Relative abundance of Blautia obeum between fecal samples from healthy controls and CRC patients 48
Supplementary Figure 1B. Relative abundance of Blautia producta between fecal samples from healthy controls and CRC patients 49
Supplementary Figure 1C. Relative abundance of Dorea longicatena between fecal samples from healthy controls and CRC patients 50
Supplementary Figure 1D. Relative abundance of Faecalibacterium prausnitzii between fecal samples from healthy controls and CRC patients 51
Supplementary Figure 1E. Relative abundance of Clostridium celatum between fecal samples from healthy controls and CRC patients 52
Supplementary Figure 1F. Relative abundance of Bifidobacterium longum between fecal samples from healthy controls and CRC patients 53
Supplementary Figure 1G. Relative abundance of Bifidobacterium adolescentis between fecal samples from healthy controls and CRC patients 54
Supplementary Figure 1I. Relative abundance of Lactobacillus ruminis between fecal samples from healthy controls and CRC patients 55
Supplementary Figure 1J. Relative abundance of Ruminococcus callidus between fecal samples from healthy controls and CRC patients 56
Supplementary Figure 2A. Relative abundance of Prevotella stercorea between fecal samples from healthy controls and CRC patients 57
Supplementary Figure 2B. Relative abundance of Parabacteroides distasonis between fecal samples from healthy controls and CRC patients 58
Supplementary Figure 2C. Relative abundance of Bacteroides uniformis between fecal samples from healthy controls and CRC patients 59
Supplementary Figure 2D. Relative abundance of Bacteroides ovatus between fecal samples from healthy controls and CRC patients 60
Supplementary Figure 2E. Relative abundance of Bacteroides fragilis between fecal samples from healthy controls and CRC patients 61
Supplementary Figure 2F. Relative abundance of Bacteroides caccae between fecal samples from healthy controls and CRC patients 62