On the other hand, the role of Lgr5+ISCs as cancer-initiating stem cells in colorectal cancer has been reported6, 7. There are Paneth cells and mesenchymal stromal cells (MSCs) in the cellular ISC niche in the small intestine. Our recent study suggested that αSMA+stromal cells in the small intestine supported ISCs-mediated epithelial differentiation.
Wnt signaling is essential for both stem cell maintenance and tissue regeneration in the adult organism. In particular, ectopic Wnt signaling in the Lgr5+ ISCs promotes the progression of intestinal tumors 6 and increases the proliferation and invasion of gastric adenocarcinoma cells 26. Importantly, Wnt2b, one of the Wnt ligands, is highly overexpressed from fibroblasts in the tumor microenvironment 23.
In particular, HFD-induced obesity is known to induce a low-grade inflammatory state 34 , 35 and increase the potential for intestinal tumorigenesis 30 , 32 . In the intestine, HFD-increased FFA promotes binding to nuclear PPAR-δ in ISCs. and ancestors.
The Rationale of my study
PPAR-δ signaling, activated by FFA binding, stimulates β-catenin target genes with β-catenin translocation into the nucleus.
Material and Methods
The wrapped tissues were then embedded in OCT compound (Sakura Finetek), frozen, sliced at 5 μm, and the tissue sections were fixed in −20°C acetone for 5 min. Paraffin or cryosections were permeabilized in PBS containing 0.5% Triton X-100 at RT for 20 min and blocked with 5% BSA in PBS at RT for 1 h. After MSCs were washed in PBS, cells were permeabilized in PBS containing 0.5% Triton X-100 at RT for 30 min.
Intestinal tissues were fixed in 4% PFA and embedded in paraffin and used for RNA in situ hybridization (RNAscope 2.5 HD Detection Kit, Advanced Cell Diagnostics) according to the manufacturer's instructions. Tissues were opened longitudinally, washed with cold PBS without magnesium and calcium chloride with vigorous shaking, and cut into approximately 1 cm pieces. Tissue pieces were incubated in PBS containing 1 mM EDTA at 4°C for 30 min and transferred to PBS containing 5 mM EDTA/gentamicin (Gibco) and incubated for 1 h at 4°C as previously described by others 37.
In the case of colon, tissue pieces were incubated in PBS containing 20 mM EDTA/gentamicin (Gibco) at 37 °C for 30 min. Dissociated crypt cells and MSCs were blocked with a solution containing 0.2% BSA in PBS and stained with a live/dead cell staining kit (Thermo Fisher) and antibodies; anti-CD45 (clone 30-F11, BD Biosciences), anti-CD31 (clone MEC13.3, BD Biosciences), anti-PDGFR-α (clone EPR22059-270, abcam), and Alexa fluor 488 conjugated anti-rabbit IgG (Thermo Fisher).
Result
A surviving HFD-fed mouse (one in ten mice) possessed more polyps in the colon than those of mice fed NCD and PD (Figure 5D). Next, we examined colonic crypt cell proliferation by Ki67 staining in PD- or HFD-fed mice. Interestingly, Ki67-positive proliferating cells were increased more in the proximal and distal colon region of HFD-fed mice than those of PD-fed mice, but no significant differences were found in the mid-colon region (Figure 7).
Importantly, Ki67-positive cells in the crypt floor, where ISCs exist, increased significantly in HFD-fed mice than PD-fed mice. Immunofluorescence imaging of Ki67 expression in the colon tissue of PD- or HFD-fed mice and quantification of Ki67+ cells per crypt in proximal, middle and distal regions of colon tissue. Bright field images and quantification of colonic organoids in the presence of WENR culture medium for 4 days.
In contrast, gene expression of DCS cell markers such as Reg4 and c-Kit was reduced in the colonic crypts of HFD-fed mice compared with that of PD-fed mice ( Figure 9A ). Among Wnt/β-catenin-related genes, Axin2, Cyclin D1, and APC were significantly increased in HFD-fed mice compared with those of PD-fed mice ( Figure 10A ). We also observed significantly higher nuclear β-catenin accumulation in the colon tissue of HFD-fed mice than that of PD-fed mice ( Figure 11 ).
Immunofluorescence imaging of nuclei (blue) and β-catenin (green) and quantification of nuclear β-catenin localization by confocal imaging in colonic crypts obtained from PD- or HFD-fed mice. Colonic organoids from mice were co-cultured with MSCs from PD- or HFD-fed mice. Pyrosequencing analysis for gut microbiota was determined from feces of rats fed PD or HFD.
To address the role of gut metabolites on the pathophysiological role of HFD, we treated the cecal contents of PD- or HFD-fed mice with colonic organoids obtained from naïve B6 mice. As a result, cecal contents from HFD-fed mice stimulated organoid growth more than that from PD-fed mice (Figure 15A). To determine which components in the cecal contents of HFD-fed mice promote organogenesis, we analyzed short-chain fatty acids (SCFA) in the cecal contents of mice fed PD or HFD.
First, we analyzed the level of free fatty acids in the cecum contents of PD- or HFD-fed mice and found that free fatty acid levels were significantly increased in HFD-fed mice compared with those in PD-fed mice (Figure 15B ). Free fatty acids in cecal HFD contents promote intestinal organogenesis. (A) Colonic organoids from naïve B6 mice were co-cultured for 4 days with caecal contents of PD- or HFD-fed mice.
Discussion
Therefore, HFD-induced dysbiosis such as decreasing A.muciniphila may be associated with obesity-related pathophysiology. Recent studies have reported that dietary signals regulate ISCs and their niche in the intestinal epithelium 29 - 31 . Of note, we identified that CD44 expression was significantly increased in the colonic crypts of HFD-fed mice while EGF signaling and Notch signaling related genes were mostly down-regulated (Figure 9).
Consistent with the increase in CD44, HFD increased gene expression related to Wnt signaling in the colonic crypts. Furthermore, CD44 has been reported to modulate epithelial–mesenchymal transitions associated with metastatic potential 53 . Taken together, increased expression of CD44 in the colonic crypts may be crucially associated with tumorigenesis in HFD 23 .
We aimed to identify the underlying cells that induce Wnt ligands in colonic crypts under HFD conditions. Of note, HFD promoted the numbers and Wnt2b secretion of PDGFR-α+MSCs in the colon (Figure 12). As a tumor microenvironment, cancer-associated fibroblasts (CAFs) have been found in various cancers and high numbers of these cells in tumors are associated with poor prognosis in patients and they provide evidence of functional significance and prognostic value 57 , 58 .
Therefore, we speculate that PDGFR-α+ MSCs in the colon overexpressing Wnt2b with HFD may be an important source of increased tumorigenesis. Among the metabolites in cecal contents, free fatty acid levels were predominantly increased by HFD (Figure 15). We found this direct effect of FFAs on ISCs, and as a result, FFAs such as PA, SA, OA and LM enhanced intestinal crypt organogenesis.
Overall, our study showed that the predominant expression of CD44 on colonic crypts was induced by HFD. In metabolomics and organoid analysis, we identified that HFD-induced FFA resulted in self-renewal of ISCs directly. Furthermore, the function of normal and malignant ISCs was further stimulated as a non-autonomous effect in the presence of PDGFR-α+MSCs.
Conclusion
Wnt ligands secreted by subepithelial mesenchymal cells are essential for the survival of intestinal stem cells and gut homeostasis. CD34+ mesenchymal cells are an important component of the intestinal stem cell niche in homeostasis and after injury. Deletion of the Wnt target and cancer stem cell marker CD44 in Apc(Min/+) mice attenuates intestinal tumorigenesis.
A dedifferentiation process driven by TGF-beta signaling enhances stem cell properties in human colorectal cancer. Evaluation of cancer stem cell markers CD133, CD44, CD24: association with AKT isoforms and radiation resistance in colon cancer cells. Fasting activates fatty acid oxidation to improve intestinal stem cell function during homeostasis and aging.
