Spasmolytic polypeptide-expressing metaplasia (SPEM) in the gastric oxyntic mucosa does not arise from Lgr5-expressing cells

Abstract

Objective: Metaplastic lineages in the oxyntic mucosa of the stomach are critical preneoplastic precursors of gastric cancer. Recent studies have demonstrated that spasmolytic polypeptide-expressing metaplasia (SPEM) in the mouse oxyntic mucosa arises from transdifferentiation of mature gastric chief cells. Other investigations of intestinal progenitor cells have shown that cells demonstrating transcriptional activity for leucine-rich repeat containing G-protein-coupled receptor 5 (Lgr5) in the intestine, colon and gastric antrum function as adult stem cells. We have now investigated whether cells demonstrating Lgr5 transcriptional activity in the oxyntic mucosa of mice might be responsible for development of metaplasia.

Design: Lgr5-EGFP-IRES-Cre(ERT2/+);Rosa26R mice were used to examine the distribution of Lgr5 transcriptionally active cells in the normal oxyntic mucosa as well as after treatment with DMP-777 or L-635 to induce acute SPEM. Lineage mapping was performed to determine if Lgr5-expressing cells gave rise to SPEM.

Results: Cells expressing transcriptional activity for Lgr5 in the oxyntic mucosa were present as scattered rare cells only along the lesser curvature of the stomach. These cells also stained for markers of chief cells (intrinsic factor and pepsinogen) but never showed any staining for proliferative markers (Ki-67). In Lgr5-EGFP-IRES-Cre(ERT2/+);Rosa26R mice induced with tamoxifen, treatment with either DMP-777 or L-635 to induce acute oxyntic atrophy caused induction of SPEM, but no lineage mapping into SPEM from Lgr5-expressing cells was observed.

Conclusion: The results indicate that, while chief cells with Lgr5 transcriptional activity are present along the lesser curvature of the gastric oxyntic mucosa, they are not responsible for production of metaplasia.

Figure 1. Cells with Lgr5 transcriptional activity in the mouse stomach

Stomachs from Lgr5-EGFP-IRES-Cre^ERT2/+ mice were immunostained for GFP expression. A. Circumferential section of the gastric oxyntic region shows GFP immunostaining cells at the bases of glands only on the lesser curvature. Bar size=700 µm. B. Higher magnification of GFP-staining cells along the lesser curvature of the oxyntic region. Note no GFP-staining cells along the greater curvature of the gastric oxyntic region. Bar size=100 µm. C. Circumferential section of the antrum stained for GFP shows stained cells at the bases of glands throughout the antrum. Bar size=100 µm. D. Higher magnification of GFP-staining cells in the antrum. Bar size=100 µm.

Figure 2. Cells with Lgr5 transcriptional activity in the oxyntic mucosa are phenotypic chief cells

Sections of stomach from Lgr5-EGFP-IRES-Cre^ERT2/+ mice were dual immunostained for either (A) GFP and pepsinogen II, (B) GFP and intrinsic factor or (C) GFP and Mist1. The immunostaining shows that the GFP-expressing cells all also labeled with pepsinogen, intrinsic factor and Mist1. Bar size=50 µm.

Figure 3. Induction of SPEM in treatment of Lgr5-EGFP-IRES-Cre^ERT2/+ mice with DMP-777 or L635

Sections of the gastric oxyntic region from untreated mice, mice treated with DMP-777 for 14 days or mice treated with L635 for 3 days were dual immunostained for GFP and TFF2. Dual labeling overlays of GFP and TFF2 staining are shown in the third column. Triple labeling overlays with GFP, TFF2 and DAPI are shown in the fourth column. The staining demonstrates that treatment with either drug led to a decrease in immunostaining for GFP. Also none of the GFP staining cells stained for TFF2 in the untreated stomach or in sections with SPEM. Bar size=50 µm.

Figure 4. GFP immunohistochemical staining in oxyntic region and antrum

GFP staining of cells expressing Lgr5 transcriptional activity in sections of oxyntic and antral mucosa was performed using peroxidase-labeled secondary antibodies and DAB chromagen. Sections of oxyntic mucosa and antrum were stained from untreated Lgr5-EGFP-IRES-Cre^ERT2/+ mice and Lgr5-EGFP-IRES-Cre^ERT2/+ mice treated with either DMP-777 for 14 days or L-635 for three days. The quantitation of GFP positive cells in sections from the oxyntic mucosa is shown at the bottom (*p<0.01 compared with untreated mice). Bar size=100 µm.

Figure 5. Lgr5-transcriptional activity does not map into SPEM

Lgr5-EGFP-IRES-Cre^ERT2/+; Rosa26R^LacZ mice were treated with 4 doses of tamoxifen and then following a 10 day hiatus mice were either maintained without further treatment, treated with 14 days of DMP-777 or treated with 3 days of L-635. At sacrifice, stomachs were fixed in glutaraldehyde and then incubated with X-gal as whole mounts. The sections demonstrate that only scattered cells were observed at the bases of oxyntic glands even after treatment and no X-gal staining was observed in SPEM. In antrum, multiple clonal X-gal-positive antral glandular lineages were visible in untreated Lgr5-EGFP-IRES-Cre^ERT2/+ mice and Lgr5-EGFP-IRES-Cre^ERT2/+ mice treated with either DMP-777 for 14 days or L-635 for three days. The quantitation of X-gal positive cells in sections from the oxyntic mucosa is shown at the bottom (*p<0.01 compared with untreated mice). Bar size=100µm

Figure 6. Bacterial β-galactosidase immunohistochemical staining in the oxyntic mucosa and antrum of Lgr5-EGFP-IRES-Cre^ERT2/+ mice

Lgr5-EGFP-IRES-Cre^ERT2/+; Rosa26R^LacZ mice were treated with 4 doses of tamoxifen and then following a 10 day hiatus mice were either maintained without further treatment, treated with 14 days of DMP-777 or treated with 3 days of L-635. The sections demonstrate that only scattered β-galactosidase (red) positive cells were observed at the bases of oxyntic glands even after treatment and no β-galactosidase staining was observed in SPEM. However, β-galactosidase (red) positive antral glandular lineages were visible in untreated Lgr5-EGFP-IRES-Cre^ERT2/+ mice and Lgr5-EGFP-IRES-Cre^ERT2/+ mice treated with either DMP-777 for 14 days or L-635 for three days. Bar size=50µm