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. 2022;13(1):199-217.
doi: 10.1016/j.jcmgh.2021.08.017. Epub 2021 Aug 26.

Up-regulation of Aquaporin 5 Defines Spasmolytic Polypeptide-Expressing Metaplasia and Progression to Incomplete Intestinal Metaplasia

Su-Hyung Lee  1 , Bogun Jang  2 , Jimin Min  1 , Ela W Contreras-Panta  3 , Kimberly S Presentation  1 , Alberto G Delgado  4 , M Blanca Piazuelo  5 , Eunyoung Choi  6 , James R Goldenring  7
Affiliations

Up-regulation of Aquaporin 5 Defines Spasmolytic Polypeptide-Expressing Metaplasia and Progression to Incomplete Intestinal Metaplasia

Su-Hyung Lee et al. Cell Mol Gastroenterol Hepatol. 2022.

Abstract

Background & aims: Metaplasia in the stomach is highly associated with development of intestinal-type gastric cancer. Two types of metaplasias, spasmolytic polypeptide-expressing metaplasia (SPEM) and intestinal metaplasia (IM), are considered precancerous lesions. However, it remains unclear how SPEM and IM are related. Here we investigated a new lineage-specific marker for SPEM cells, aquaporin 5 (AQP5), to assist in the identification of these 2 metaplasias.

Methods: Drug- or Helicobacter felis (H felis) infection-induced mouse models were used to identify the expression pattern of AQP5 in acute or chronic SPEM. Gene-manipulated mice treated with or without drug were used to investigate how AQP5 expression is regulated in metaplastic lesions. Metaplastic samples from transgenic mice and human gastric cancer patients were evaluated for AQP5 expression. Immunostaining with lineage-specific markers was used to differentiate metaplastic gland characteristics.

Results: Our results revealed that AQP5 is a novel lineage-specific marker for SPEM cells that are localized at the base of metaplastic glands initially and expand to dominate glands after chronic H felis infection. In addition, AQP5 expression was up-regulated early in chief cell reprogramming and was promoted by interleukin 13. In humans, metaplastic corpus showed highly branched structures with AQP5-positive SPEM. Human SPEM cells strongly expressing AQP5 were present at the bases of incomplete IM glands marked by TROP2 but were absent from complete IM glands.

Conclusions: AQP5-expressing SPEM cells are present in pyloric metaplasia and TROP2-positive incomplete IM and may be an important component of metaplasia that can predict a higher risk for gastric cancer development.

Keywords: AQP5; Gastric Cancer; IM; SPEM; TROP2.

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Figures

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Graphical abstract
Figure 1
Figure 1
AQP5 is expressed in GIF-positive chief cell after L635-induced parietal cell loss and during recovery. (A) H&E and immunofluorescence staining for AQP5, chief cell marker GIF, proliferation marker Ki67, and nuclei marker DAPI in gastric corpus tissues from mice with or without L635 treatment for 1, 2 or 3 days (D1, 2, or 3). Arrows indicate Ki67- and GIF-co-positive cells. Scale bar = 100 μm or 50 μm (expanded images). (B) Immunofluorescence staining for AQP5, GIF, Ki67, and DAPI in gastric corpus tissues from mice with recovery for 2 (D1R2) or 4 (D1R4) days after L635 treatment for 1 day. Arrow indicates Ki67- and GIF-co-positive cells. Scale bar = 100 μm or 50 μm (expanded images). (C and D) Quantification of GIF-positive gland, GIF- and AQP5-co-positive gland, or GIF- and Ki67-co-positive cell per ×20 field (n = 3–4) in consecutive L635 treatments (C) or during recovery after L635 treatment for 1 day (D). ∗P < .05, ∗∗P < .01, ∗∗∗P < .001.
Figure 2
Figure 2
AQP5 is expressed in L635- and H felis infection-induced SPEM in the gastric corpus of mice. (A) Immunofluorescence staining for AQP5, mucus marker GSII-lectin, SPEM marker CD44 variant 9 (CD44v9), and epithelial membrane marker p120 in gastric corpus tissues from mice with or without L635 treatment for 1, 2 or 3 days. Arrows indicate AQP5-expressing cells. Scale bar = 100 μm or 50 μm (expanded images). (B) Quantification of AQP5 single or AQP5 and GSII-lectin co-positive gland per ×20 field (n = 6). AQP5 expression was first observed at the base of a few glands of mice treated with L635 for 1 day without GSII expression. Over time, the numbers of AQP5 single positive or AQP5 and GSII co-positive glands were significantly increased in cells at the bases of glands. ∗P < .05, ∗∗P < .01. (C) Quantification of CD44v9 single or AQP5 and CD44v9 co-positive gland per ×20 field (n = 6). CD44v9-expressing glands were prominently observed after L635 treatment for 3 days. Most of these glands co-expressed AQP5. ∗P < .05, ∗∗∗∗P < .0001.
Figure 3
Figure 3
AQP5 is expressed in DMP777-induced SPEM in gastric corpus of mice. (A) Immunofluorescence staining for AQP5, mucus marker GSII-lectin, SPEM marker CD44v9, and epithelial membrane marker p120 in gastric corpus tissues from mice without or with DMP777 treatment for 1, 3, 7 or 14 days. Scale bar = 100 μm or 50 μm (expanded images). (B) Quantification of AQP5 single or AQP5 and GSII-lectin co-positive gland per ×20 field (n = 4–6). AQP5 expression was first observed at the base of a few glands in mice treated with DMP-777 for 3 days with small amount of GSII co-expression. Over time, the numbers of AQP5 and GSII-lectin co-positive glands were significantly increased at the bases of glands. ∗P < .05, ∗∗P < .01. (C) Quantification of CD44v9 single or AQP5 and CD44v9 co-positive glands per ×20 field (n = 4–6). CD44v9 expression was observed at the base of few glands after DMP-777 treatment for 7 days, but it was markedly increased at 14 days of treatment with AQP5 co-expression. ∗∗∗P < .001.
Figure 4
Figure 4
AQP5 is up-regulated in SPEM associated with acute acetic acid ulcers and chronic H felis infection.(A) Immunofluorescence staining for AQP5, mucus marker GSII-lectin, SPEM marker CD44v9, and nuclei marker DAPI in gastric corpus tissues from acetic acid-induced injury. Arrow indicates ulcerated gastric mucosa. Note in the expanded images 2 glands showing different expression patterns for GSII-lectin. Gland nearest to the ulcerated region, indicated by asterisk, did not show GSII-lectin, but both showed AQP5 expression. Scale bar = 100 μm or 50 μm (expanded images). (B) Immunofluorescence staining for AQP5, GSII-lectin, CD44v9, and epithelial membrane marker p120 in gastric corpus tissues from H felis-infected mice. Apical AQP5 expression is seen throughout SPEM cells. Scale bar = 100 μm or 50 μm (expanded images).
Figure 5
Figure 5
AQP5 expression is up-regulated by signaling cascade of IL33 receptor, ST2, and IL13, but not affected by xCT knockout (KO). (A) Immunofluorescence staining for AQP5, mucus marker GSII-lectin, SPEM marker CD44v9, and nuclei marker DAPI in gastric corpus tissues from L635-treated ST2 KO mice for 3 days with or without recombinant IL13 injection and quantification of glands with AQP5 expression at the base (n = 4–5). ∗∗∗P < .001. Scale bar = 100 μm or 50 μm (expanded images). (B) Immunofluorescence staining for AQP5, GSII-lectin, CD44v9, and epithelial membrane marker p120 in gastric corpus tissues from xCT KO mice treated with L635 for 3 days and quantification of gland with AQP5 expression at the base (n = 4–5). Scale bar = 100 μm or 50 μm (expanded images). (C) Schematic diagram of AQP5 expression in discrete stages of transdifferentiation of chief cells into SPEM. Release of IL13 induced by parietal cell loss is required for AQP5 expression at early stages of transdifferentiation, and AQP5 expression is continuously maintained during whole process of SPEM development. Illustration was created with BioRender.com.
Figure 6
Figure 6
AQP5 is expressed in SPEM cells induced by Kras activation in Mist1-positive chief cells. H&E and immunofluorescence staining (IF) for AQP5, mucus marker GSII-lectin, and the dysplasia marker TROP2 in gastric corpus tissues from Mist1CreERT2/+;KrasLSL-G12D/+ mice. Corpus mucosa 1 month after induction by tamoxifen showed only SPEM lesions marked by AQP5 and GSII-lectin co-positivity. Three or 4 months after Kras activation in Mist1-positive cells, corpus glands were composed of AQP5- and GSII-lectin co-positive cells at the bases of glands with TROP2-expressing cells in the upper gland. Arrows indicate SPEM cells at the base of glands (H&E) and AQP5-expressing cells (IF). Insets show higher magifications of boxed areas. No expression of AQP5 in the TROP2-positive region was noted. Scale bar = 100 μm.
Figure 7
Figure 7
AQP5 is not expressed in human normal gastric corpus but is strongly expressed in normal antrum SPEM cell lineages. Histology and immunostaining were examined in sections of human (A) normal corpus, (B) normal antrum, and (C) SPEM. Sections were stained for H&E staining for identification of overall structure of normal corpus, antrum, and SPEM in the corpus, PAS staining to confirm localization of mucous accumulation in normal or SPEM lesions, and immunofluorescence staining for AQP5, mucus marker GSII-lectin, Paneth cell marker DEFA5, proliferative cell marker Ki67, foveolar cell marker Ulex europaeus agglutinin I (UEAI), goblet cell marker TFF3, SPEM marker CD44v9, dysplasia marker TROP2, and nuclei marker DAPI. Note that PAS-positive SPEM cell lineages (arrows) were marked by expression of AQP5, GSII, and CD44v9 (C, expanded images). Scale bar = 100 μm.
Figure 8
Figure 8
AQP5 is not expressed in complete type of IM. (A) H&E staining for identification of overall structure of IM in the corpus, PAS staining to confirm localization of mucous accumulation in IM lesion, HID/AB staining to determine the subtype of IM and immunofluorescence staining for AQP5, mucus marker GSII-lectin, Paneth cell marker DEFA5, proliferative cell marker Ki67, foveolar cell marker Ulex europaeus agglutinin I (UEAI), goblet cell marker TFF3, SPEM marker CD44v9, dysplasia marker TROP2, and nuclei marker DAPI. Complete IM glands were characterized by eosinophilic granules-containing Paneth cells (H&E) stained with DEFA5 and TFF3-positive sialomucin stained blue in HID/AB staining. CD44v9 was also expressed at the base of complete IM gland. Scale bar = 100 μm. (B) Expanded images from immunofluorescence staining results in (A). Complete IM gland had Paneth and goblet cells frequently showing basolateral CD44v9 expression and proliferative activity similar to an intestinal crypt. Note absence of TROP2 and AQP5 expression. Scale bar = 50 μm.
Figure 9
Figure 9
AQP5-expressing cells were observed at base of incomplete IM glands showing TROP2 positivity. (A) H&E staining for identification of overall structure of IM in the corpus, PAS staining to confirm localization of mucous accumulation in IM lesion, HID/AB staining to determine subtype of IM, and immunofluorescence staining for AQP5, mucus marker GSII-lectin, Paneth cell marker DEFA5, proliferative cell marker Ki67, foveolar cell marker Ulex europaeus agglutinin I (UEAI), goblet cell marker TFF3, SPEM marker CD44v9, dysplasia marker TROP2, and nuclei marker DAPI. Incomplete IM glands were defined on the basis of absence of DEFA5-positive Paneth cells, morphology of PAS-positive mucin, and sulfomucin stained brown in HID/AB staining. Scale bar = 100 μm. (B) Expanded images from immunofluorescence staining results in (A). Incomplete IM glands were marked by TROP2 expression. Note AQP5-expressing cells were frequently observed at base of incomplete IM glands (arrows). Scale bar = 50 μm.
Figure 10
Figure 10
Incomplete IM glands within regions mixed IM types demonstrate AQP5-expressing cells at the base. H&E staining for identification of overall structure of the IM in the corpus, PAS staining to confirm localization and morphology of mucus in IM lesion, HID/AB staining to determine subtype of IM, and immunofluorescence staining for AQP5, mucus marker GSII-lectin, Paneth cell marker DEFA5, proliferative cell marker Ki67, foveolar cell marker Ulex europaeus agglutinin I (UEAI), goblet cell marker TFF3, SPEM marker CD44v9, dysplasia marker TROP2, and nuclei marker DAPI. Differentiation of IM subtype was determined on the basis of combination of H&E, PAS, and HID/AB staining results as described in Figures 6 and 7. Scale bar = 100 μm. (B) Expanded images from PAS and HID/AB staining results in (A) with matched immunofluorescence images. Gland delineated by dotted line indicates complete IM gland, and others are incomplete IM glands. TROP2 was strongly expressed in incomplete IM gland, which showed cells at the base with CD44v9 positivity and strong AQP5 expression. Scale bar = 50 μm. (C) Quantification of individual IM glands in corpus accompanied by AQP5-positive cells at base of the gland per each core (n = 44 for complete IM and 5 for incomplete IM; top) or quantification of individual IM glands with TROP2 expression per each core (n = 44 of complete IM and 5 of incomplete IM; bottom) ∗∗∗∗P < .0001. (D) Immunofluorescence staining for AQP5, GSII-lectin, TROP2, intestinal absorptive cell marker CD10, TFF3, CD44v9, and DAPI in whole tissue sections from human metaplasia tissues. AQP5- and GSII-lectin co-positive SPEM below TROP2-expressing incomplete IM (top) and CD10-negative and TROP2-positive incomplete IM gland (bottom). Note basal branched glands populated with SPEM cells (arrows). Scale bar = 100 μm.
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