Spasmolytic Polypeptide-expressing Metaplasia Cells Research Articles

SOX9 Governs Gastric Mucous Neck Cell Identity and Is Required for Injury-Induced Metaplasia

Acute and chronic gastric injury induces alterations in differentiation within the corpus of the stomach called pyloric metaplasia. Pyloric metaplasia is characterized by the death of parietal cells and reprogramming of mitotically quiescent zymogenic chief cells into proliferative, mucin-rich spasmolytic polypeptide-expressing metaplasia (SPEM) cells. Overall, pyloric metaplastic units show increased proliferation and specific expansion of mucous lineages, both by proliferation of normal mucous neck cells and recruitment of SPEM cells. Here, we identify Sox9 as a potential gene of interest in the regulation of mucous neck and SPEM cell identity in the stomach. We used immunostaining and electron microscopy to characterize the expression pattern of SRY-box transcription factor 9 (SOX9) during murine gastric development, homeostasis, and injury in homeostasis, after genetic deletion of Sox9 and after targeted genetic misexpression of Sox9 in the gastric epithelium and chief cells. SOX9 is expressed in all early gastric progenitors and strongly expressed in mature mucous neck cells with minor expression in the other principal gastric lineages during adult homeostasis. After injury, strong SOX9 expression was induced in the neck and base of corpus units in SPEM cells. Adult corpus units derived from Sox9-deficient gastric progenitors lacked normal mucous neck cells. Misexpression of Sox9 during postnatal development and adult homeostasis expanded mucous gene expression throughout corpus units including within the chief cell zone in the base. Sox9 deletion specifically in chief cells blunts their reprogramming into SPEM. Sox9 is a master regulator of mucous neck cell differentiation during gastric development. Sox9 also is required for chief cells to fully reprogram into SPEM after injury.

Chief cell plasticity is the origin of metaplasia following acute injury in the stomach mucosa

ObjectiveMetaplasia arises from differentiated cell types in response to injury and is considered a precursor in many cancers. Heterogeneous cell lineages are present in the reparative metaplastic mucosa with response...

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

Background & AimsMetaplasia 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.MethodsDrug- 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.ResultsOur 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.ConclusionsAQP5-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.

Proliferation and Differentiation of Gastric Mucous Neck and Chief Cells During Homeostasis and Injury-induced Metaplasia

Adult zymogen-producing (zymogenic) chief cells (ZCs) in the mammalian gastric gland base are believed to arise from descending mucous neck cells, which arise from stem cells. Gastric injury, such as from Helicobacter pylori infection in patients with chronic atrophic gastritis, can cause metaplasia, characterized by gastric cell expression of markers of wound-healing; these cells are called spasmolytic polypeptide-expressing metaplasia (SPEM) cells. We investigated differentiation and proliferation patterns of neck cells, ZCs, and SPEM cells in mice. C57BL/6 mice were given intraperitoneal injections of high-dose tamoxifen to induce SPEM or gavaged with H pylori (PMSS1) to induce chronic gastric injury. Mice were then given pulses of 5-bromo-2'-deoxyuridine (BrdU) in their drinking water, followed by chase periods without BrdU, or combined with intraperitoneal injections of 5-ethynyl-2'-deoxyuridine. We collected gastric tissues and performed immunofluorescence and immunohistochemical analyses to study gastric cell proliferation, differentiation, and turnover. After 8 weeks of continuous BrdU administration, fewer than 10% of homeostatic ZCs incorporated BrdU, whereas 88% of neck cells were labeled. In pulse-chase experiments, various chase periods decreased neck cell label but did not increase labeling of ZCs. When mice were given BrdU at the same time as tamoxifen, more than 90% of cells were labeled in all gastric lineages. After 3 months' recovery (no tamoxifen), ZCs became the predominant BrdU-labeled population, whereas other cells, including neck cells, were mostly negative. When we tracked the labeled cells in such mice over time, we observed that the proportion of BrdU-positive ZCs remained greater than 60% up to 11 months. In mice whose ZCs were the principal BrdU-positive population, acute injury by tamoxifen or chronic injury by H pylori infection resulted in SPEM cells becoming the principal BrdU-positive population. After withdrawal of tamoxifen, BrdU-positive ZCs reappeared. We studied mice in homeostasis or with tamoxifen- or H pylori-induced SPEM. Our findings indicated that mucous neck cells do not contribute substantially to generation of ZCs during homeostasis and that ZCs maintain their own census, likely through infrequent self-replication. After metaplasia-inducing injury, ZCs can become SPEM cells, and then redifferentiate into ZCs on injury resolution.

Decrease in MiR-148a Expression During Initiation of Chief Cell Transdifferentiation.

Gastric chief cells differentiate from mucous neck cells and develop their mature state at the base of oxyntic glands with expression of secretory zymogen granules. After parietal cell loss, chief cells transdifferentiate into mucous cell metaplasia, designated spasmolytic polypeptide-expressing metaplasia (SPEM), which is considered a candidate precursor of gastric cancer. We examined the range of microRNA (miRNA) expression in chief cells and identified miRNAs involved in chief cell transdifferentiation into SPEM. Among them, miR-148a was strongly and specifically expressed in chief cells and significantly decreased during the process of chief cell transdifferentiation. Interestingly, suppression of miR-148a in a conditionally immortalized chief cell line induced up-regulation of CD44 variant 9 (CD44v9), one of the transcripts expressed at an early stage of SPEM development, and DNA methyltransferase 1 (Dnmt1), an established target of miR-148a. Immunostaining analyses showed that Dnmt1 was up-regulated in SPEM cells as well as in chief cells before the emergence of SPEM in mouse models of acute oxyntic atrophy using either DMP-777 or L635. In the cascade of events that leads to transdifferentiation, miR-148a was down-regulated after acute oxyntic atrophy either in xCT knockout mice or after sulfasalazine inhibition of xCT. These findings suggest that the alteration of miR-148a expression is an early event in the process of chief cell transdifferentiation into SPEM.

Metaplastic Cells in the Stomach Arise, Independently of Stem Cells, via Dedifferentiation or Transdifferentiation of Chief Cells

Spasmolytic polypeptide-expressing metaplasia (SPEM) develops in patients with chronic atrophic gastritis due to infection with Helicobacter pylori; it might be a precursor to intestinal metaplasia and gastric adenocarcinoma. Lineage tracing experiments of the gastric corpus in mice have not established whether SPEM derives from proliferating stem cells or differentiated, post-mitotic zymogenic chief cells in the gland base. We investigated whether differentiated cells can give rise to SPEM using a nongenetic approach in mice. Mice were given intraperitoneal injections of 5-fluorouracil, which blocked gastric cell proliferation, plus tamoxifen to induce SPEM. Based on analyses of molecular and histologic markers, we found SPEM developed even in the absence of cell proliferation. SPEM therefore did not arise from stem cells. In histologic analyses of gastric resection specimens from 10 patients with adenocarcinoma, we found normal zymogenic chief cells that were transitioning into SPEM cells only in gland bases, rather than the proliferative stem cell zone. Our findings indicate that SPEM can arise by direct reprogramming of existing cells-mainly of chief cells.

Maturity and age influence chief cell ability to transdifferentiate into metaplasia.

Previous investigations have indicated that spasmolytic polypeptide-expressing metaplasia (SPEM) in the stomach arises from transdifferentiation of chief cells. Nevertheless, the intrinsic properties of chief cells that influence transdifferentiation have been largely unknown. We now report that the ability to transdifferentiate into SPEM is impaired in chief cells that lack full functional maturation, and as chief cells age, they lose their ability to transdifferentiate. Thus chief cell plasticity is dependent on both cell age and maturation.

Establishment of novel in vitro mouse chief cell and SPEM cultures identifies MAL2 as a marker of metaplasia in the stomach.

Oxyntic atrophy in the stomach leads to chief cell transdifferentiation into spasmolytic polypeptide expressing metaplasia (SPEM). Investigations of preneoplastic metaplasias in the stomach are limited by the sole reliance on in vivo mouse models, owing to the lack of in vitro models for distinct normal mucosal lineages and metaplasias. Utilizing the Immortomouse, in vitro cell models of chief cells and SPEM were developed to study the characteristics of normal chief cells and metaplasia. Chief cells and SPEM cells isolated from Immortomice were cultured and characterized at both the permissive (33°C) and the nonpermissive temperature (39°C). Clones were selected on the basis of their transcriptional expression of specific stomach lineage markers (named ImChief and ImSPEM) and protein expression and growth were analyzed. The transcriptional expression profiles of ImChief and ImSPEM cells were compared further by using gene microarrays. ImChief cells transcriptionally express most chief cell markers and contain pepsinogen C and RAB3D-immunostaining vesicles. ImSPEM cells express the SPEM markers TFF2 and HE4 and constitutively secrete HE4. Whereas ImChief cells cease proliferation at the nonpermissive temperature, ImSPEM cells continue to proliferate at 39°C. Gene expression profiling of ImChief and ImSPEM revealed myelin and lymphocyte protein 2 (MAL2) as a novel marker of SPEM lineages. Our results indicate that the expression and proliferation profiles of the novel ImChief and ImSPEM cell lines resemble in vivo chief and SPEM cell lineages. These cell culture lines provide the first in vitro systems for studying the molecular mechanisms of the metaplastic transition in the stomach.

Macrophages Promote Progression of Spasmolytic Polypeptide-Expressing Metaplasia After Acute Loss of Parietal Cells

Loss of parietal cells causes the development of spasmolytic polypeptide-expressing metaplasia (SPEM) through transdifferentiation of chief cells. In the presence of inflammation, SPEM can advance into a more proliferative metaplasia with increased expression of intestine-specific transcripts. We used L635 to induce acute SPEM with inflammation in mice and investigated the roles of inflammatory cells in the development of SPEM. To study the adaptive immune system, Rag1 knockout, interferon-γ-deficient, and wild-type (control) mice received L635 for 3 days. To study the innate immune system, macrophages were depleted by intraperitoneal injection of clodronate liposomes 2 days before and throughout L635 administration. Neutrophils were depleted by intraperitoneal injection of an antibody against Ly6G 2 days before and throughout L635 administration. Pathology and immunohistochemical analyses were used to determine depletion efficiency, metaplasia, and proliferation. To characterize SPEM in each model, gastric tissues were collected and levels of Cftr, Dmbt1, and Gpx2 mRNAs were measured. Markers of macrophage polarization were used to identify subpopulations of macrophages recruited to the gastric mucosa. Administration of L635 to Rag1 knockout, interferon-γ-deficient, and neutrophil-depleted mice led to development of proliferative SPEM and up-regulation of intestine-specific transcripts in SPEM cells, similar to controls. However, macrophage-depleted mice given L635 showed significant reductions in numbers of SPEM cells, SPEM cell proliferation, and expression of intestine-specific transcripts, compared with control mice given L635. In mice given L635, as well as patients with intestinal metaplasia, M2 macrophages were the primary inflammatory component. Results from studies of mouse models and human metaplastic tissues indicate that M2 macrophages promote the advancement of SPEM in the presence of inflammation.

Establishment and characterization of novel in vitro models of gastric chief cell and metaplastic lineages

Investigations of preneoplastic metaplasias in the stomach are limited by the lack of cell models for mature mucosal cells and metaplasias. We developed in vitro models of chief cells and spasmolytic polypeptide‐expressing metaplasia (SPEM) cells to study the transdifferentiation of chief cells into SPEM. Utilizing the Immortomouse, which is completely transgenic for tempertature‐dependent TAg, chief cells were isolated and cultured from untreated mice. SPEM cells were isolated from mice treated with L635, an acute SPEM induction model. mRNA and protein expression profiles were used to characterize the cultures. ImChief cultures express most chief cell markers with immunostaining for pepsinogen and Rab3D‐containing granules. After 2 weeks at 39°C, ImChief cells cease proliferating and resemble a more differentiated state similar to in vivo chief cells. ImSPEM cells express the SPEM markers TFF2 and HE4 and produce HE4‐containing granules. They lack expression of most other lineage specific markers. ImSPEM cells retain their proliferative ability for at least 1 month at 39°C, resembling the in vivo properties of SPEM cells. Our results indicate that the expression and proliferation profiles of the novel ImChief and ImSPEM cultures resemble in vivo chief and SPEM cell lineages. These cultures provide the first in vitro system for studying the molecular mechanisms of the metaplastic transition in the stomach.Department of Veterans Affairs Merit Review Award and NIH RO1 DK071590

Gastric Cancer: Increased Numbers of Myeloid-Derived Suppressor Cells Are Found in Gastric Cancer Patients

Gastric cancer is one of the most common neoplasms, ranking as the second leading cause of cancer-related deaths worldwide. The predominant factor associated to gastric cancer is Helicobacter pylori infection, which leads to a chronic inflammatory response and subsequent oxyntic atrophy. Parietal cell loss result in two types of metaplasia, the intestinal metaplasia (IM) characterized by the presence of cells with goblet cell morphology and spasmolytic polypeptide-expressing metaplasia (SPEM) that shows morphological characteristics of the deep antral glands and express trefoil factor 2 (spasmolytic polypeptide). Although metaplastic lesions are considered neoplastic precursors, their direct association to cancer is still in debate. Similar to other cancers, gastric cancers are marked by global gene expression alterations. MicroRNAs (miRNAs) are small noncoding RNAs involved in the post-transcriptional regulation of gene expression and an increasing number of studies has been showing their aberrant expression in cancer. In order to identify miRNAs involved in the early stages of gastric cancer we performed a miRNA profiling on laser capture micro-dissected IM and SPEM cells from patient lesions. Using a qRT-PCR approach for quantitation of 754 human miRNAs, we identified 77 miRNAs differentially expressed in the metaplastic samples (greater than two-fold) in comparison to normal chief cells. The highest number of dysregulated miRNAs was observed in IM,which showed 45 up-regulated and 25 down-regulatedmiRNAs. In SPEM, 28 miRNAs were found up-regulated (21 of them also up-regulated in IM), whereas no down-regulation was detected. Some of the up-regulated miRNAs have already been associated to cancer in general (miR-26-a, miR-191, miR-155), as well as specifically to gastric cancer (miR-18b, miR-196b and miR-106a). However, the regulation of some of the top up-regulated miRNAs revealed here including miR-802, miR-922 and miR-622 are still poorly characterized in cancer. In a comparison with a previous mRNA profiling study performed on a similar group of samples, we observed that 108 out of the 568 mRNAs found up-regulated in IM are predicted targets of the 26 microRNAs down-regulated in IM, revealing a potential mechanism for the regulation of those genes in gastric metaplasia. Particularly interesting are 4 members of the miR-30 family, with 31 predicted mRNA targets amongst those up-regulated in IM. Although miR-30 family members have been described as down-regulated in gastric cancer, no data on their targets in this neoplasia is currently available. An extended characterization of the microRNAs identified here will provide a better understanding of their function in gastric metaplasia and progression to neoplasia, as well as might reveal useful early stage biomarkers or therapeutic targets.

The Diagnostic Value of Combining DNA Promoter Hypermethylation With Autofluroscene Spectrum Analysis of Gastric Juice in Gastric Cancer

Gastric cancer is one of the most common neoplasms, ranking as the second leading cause of cancer-related deaths worldwide. The predominant factor associated to gastric cancer is Helicobacter pylori infection, which leads to a chronic inflammatory response and subsequent oxyntic atrophy. Parietal cell loss result in two types of metaplasia, the intestinal metaplasia (IM) characterized by the presence of cells with goblet cell morphology and spasmolytic polypeptide-expressing metaplasia (SPEM) that shows morphological characteristics of the deep antral glands and express trefoil factor 2 (spasmolytic polypeptide). Although metaplastic lesions are considered neoplastic precursors, their direct association to cancer is still in debate. Similar to other cancers, gastric cancers are marked by global gene expression alterations. MicroRNAs (miRNAs) are small noncoding RNAs involved in the post-transcriptional regulation of gene expression and an increasing number of studies has been showing their aberrant expression in cancer. In order to identify miRNAs involved in the early stages of gastric cancer we performed a miRNA profiling on laser capture micro-dissected IM and SPEM cells from patient lesions. Using a qRT-PCR approach for quantitation of 754 human miRNAs, we identified 77 miRNAs differentially expressed in the metaplastic samples (greater than two-fold) in comparison to normal chief cells. The highest number of dysregulated miRNAs was observed in IM,which showed 45 up-regulated and 25 down-regulatedmiRNAs. In SPEM, 28 miRNAs were found up-regulated (21 of them also up-regulated in IM), whereas no down-regulation was detected. Some of the up-regulated miRNAs have already been associated to cancer in general (miR-26-a, miR-191, miR-155), as well as specifically to gastric cancer (miR-18b, miR-196b and miR-106a). However, the regulation of some of the top up-regulated miRNAs revealed here including miR-802, miR-922 and miR-622 are still poorly characterized in cancer. In a comparison with a previous mRNA profiling study performed on a similar group of samples, we observed that 108 out of the 568 mRNAs found up-regulated in IM are predicted targets of the 26 microRNAs down-regulated in IM, revealing a potential mechanism for the regulation of those genes in gastric metaplasia. Particularly interesting are 4 members of the miR-30 family, with 31 predicted mRNA targets amongst those up-regulated in IM. Although miR-30 family members have been described as down-regulated in gastric cancer, no data on their targets in this neoplasia is currently available. An extended characterization of the microRNAs identified here will provide a better understanding of their function in gastric metaplasia and progression to neoplasia, as well as might reveal useful early stage biomarkers or therapeutic targets.

Global Profiling Study Reveals MicroRNA Dysregulation in Human Gastric Metaplastic Lineages

Gastric cancer is one of the most common neoplasms, ranking as the second leading cause of cancer-related deaths worldwide. The predominant factor associated to gastric cancer is Helicobacter pylori infection, which leads to a chronic inflammatory response and subsequent oxyntic atrophy. Parietal cell loss result in two types of metaplasia, the intestinal metaplasia (IM) characterized by the presence of cells with goblet cell morphology and spasmolytic polypeptide-expressing metaplasia (SPEM) that shows morphological characteristics of the deep antral glands and express trefoil factor 2 (spasmolytic polypeptide). Although metaplastic lesions are considered neoplastic precursors, their direct association to cancer is still in debate. Similar to other cancers, gastric cancers are marked by global gene expression alterations. MicroRNAs (miRNAs) are small noncoding RNAs involved in the post-transcriptional regulation of gene expression and an increasing number of studies has been showing their aberrant expression in cancer. In order to identify miRNAs involved in the early stages of gastric cancer we performed a miRNA profiling on laser capture micro-dissected IM and SPEM cells from patient lesions. Using a qRT-PCR approach for quantitation of 754 human miRNAs, we identified 77 miRNAs differentially expressed in the metaplastic samples (greater than two-fold) in comparison to normal chief cells. The highest number of dysregulated miRNAs was observed in IM,which showed 45 up-regulated and 25 down-regulatedmiRNAs. In SPEM, 28 miRNAs were found up-regulated (21 of them also up-regulated in IM), whereas no down-regulation was detected. Some of the up-regulated miRNAs have already been associated to cancer in general (miR-26-a, miR-191, miR-155), as well as specifically to gastric cancer (miR-18b, miR-196b and miR-106a). However, the regulation of some of the top up-regulated miRNAs revealed here including miR-802, miR-922 and miR-622 are still poorly characterized in cancer. In a comparison with a previous mRNA profiling study performed on a similar group of samples, we observed that 108 out of the 568 mRNAs found up-regulated in IM are predicted targets of the 26 microRNAs down-regulated in IM, revealing a potential mechanism for the regulation of those genes in gastric metaplasia. Particularly interesting are 4 members of the miR-30 family, with 31 predicted mRNA targets amongst those up-regulated in IM. Although miR-30 family members have been described as down-regulated in gastric cancer, no data on their targets in this neoplasia is currently available. An extended characterization of the microRNAs identified here will provide a better understanding of their function in gastric metaplasia and progression to neoplasia, as well as might reveal useful early stage biomarkers or therapeutic targets.

Abstract 914: The role of CD44 variant in the development of spasmolytic polypeptide-expressing metaplasia (SPEM) and gastric cancer

Abstract CD44 is a marker of tumor-initiating cells in gastric cancer. However, the cause or consequence of elevated CD44 in such immature tumor cells is not known. Gastric carcinogenesis involves a transition from normal mucosa to gastritis, which then leads to metaplasia and eventually adenocarcinoma. Although the emergence of metaplasia or dysplasia is a key event of inflammation-related gastric carcinogenesis, the precise mechanism underlying the transition from normal gastric epithelium to metaplasia and cancer remains unsolved. Spasmolytic polypeptide (known as trefoil factor family 2)-expressing metaplasia (SPEM) results from a metaplastic change of gastric epithelial cells in response to chronic inflammation. Here we show that the trefoil factor family 2 (TFF2)+ metaplastic glands as well as gastric tumor glands contain CD44 variant isoform-expressing (CD44v+) cells in K19-Wnt1/C2mE mouse, a genetic mouse model for gastric tumorigenesis. Furthermore, the analysis of three transgenic mouse lines K19-Wnt1, K19-C2mE and K19-Wnt1/C2mE revealed that de novo expression of CD44v in SPEM cells is triggered by prostaglandin E2-mediated signaling. Isolated CD44-expressing tumor cells in K19-Wnt1/C2mE mice by fluorescence-activated cell sorting (FACS) manifested increased expression of SPEM-related genes, suggesting that metaplastic epithelium might give rise to both of CD44v+ tumor cells and CD44v+ SPEM cells in mice. To investigate the role of CD44 in the development of SPEM and gastric tumor in mice, we generated CD44-/- K19-Wnt1/C2mE mice. We found that CD44 ablation significantly suppresses the development of SPEM and gastric tumor, suggesting that CD44v+ cells play a key role in gastric tumorigenesis. Furthermore, we found that the emergence of CD44v+ SPEM glands adjacent to cancer is tightly associated with the CD44v-expressing gastric cancer formation in human. Our present study thus suggest that SPEM cells might be susceptible for oncogenesis due to their proliferative capacity and that CD44v+ SPEM cells are cells-of-origin for CD44v+ tumor-initiating cells in human gastric adenocarcinoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 914. doi:10.1158/1538-7445.AM2011-914

Gene Expression Profiling of Metaplastic Lineages Identifies CDH17 as a Prognostic Marker in Early Stage Gastric Cancer

Intestinal metaplasia (IM) and spasmolytic polypeptide-expressing metaplasia (SPEM) are precursors to gastric carcinogenesis. We sought to identify molecular biomarkers of gastric metaplasias and gastric cancer by gene expression profiling of metaplastic lesions from patients. Complementary DNA microarray analysis was performed on IM and SPEM cells isolated from patient samples using laser capture microdissection. Up-regulated transcripts in metaplastic lesions were confirmed by immunostaining analysis in IM, SPEM, and gastric cancer tissues. Proteins that were highly expressed specifically in gastric cancer tissues were analyzed for their association with survival in a test set (n = 450) and a validation set (n = 502) of samples from gastric cancer patients. Compared with normal chief cells, 858 genes were differentially expressed in IM or SPEM samples. Immunostaining was detected for 12 proteins, including 3 new markers of IM (ACE2, LGALS4, AKR1B10) and 3 of SPEM (OLFM4, LYZ, DPCR1). Of 13 proteins expressed in IM or SPEM, 8 were expressed by 17%-50% of human gastric cancer tissues (MUC13, OLFM4, CDH17, KRT20, MUC5AC, LGALS4, AKR1B10, REG4). Expression of CDH17 or MUC13 correlated with patient survival in the test and validation sets. Multivariate analysis showed that CDH17 was an independent prognostic factor in patients with stage I or node-negative disease. We identified several novel biomarkers for IM, SPEM, and gastric cancer using gene expression profiling of human metaplastic lesions. Expression of CDH17 and MUC13 was up-regulated in gastric cancer tissues. CDH17 is a promising prognostic marker for early stage gastric cancer.

Spasmolytic polypeptide expressing metaplasia to preneoplasia in H. felis-infected mice

Background & Aims: The emergence of oxyntic atrophy and metaplastic cell lineages in response to chronic Helicobacter pylori infection predisposes to gastric neoplasia. We have described a trefoil factor family 2 (TFF2; spasmolytic polypeptide) expressing metaplasia (SPEM) associated with gastric neoplasia in both rodent and human fundus. To examine the relationship of SPEM to the neoplastic process in the H. felis-infected C57BL/6 mouse, we have now studied the association of SPEM-related transcripts with preneoplasia. Methods: SPEM-related transcripts were identified by microarray analysis of amplified cRNA from SPEM, and surface mucous cells were isolated by laser capture microdissection from the same gastric sections from male C57BL/6 mice infected with H. felis for 6 months. Expression of SPEM-related transcripts was assessed by in situ hybridization and quantitative RT-PCR, as well as immunohistochemistry for prothymosin α. Results: Eleven SPEM-related transcripts were identified as detectable only in SPEM. The expression of the SPEM-related transcripts was validated by in situ hybridization and quantitative PCR. One transcript, the noncoding RNA Xist, was only identified in SPEM cells from the infected male mice. Ten of the 11 transcripts as well as TFF2 were also expressed in regions of gastritis cystica profunda. Immunocytochemistry for one of the identified proteins, prothymosin α, demonstrated prominent nuclear staining in SPEM and gastritis cystica profunda. Conclusions: The expression of SPEM-related transcripts in regions of gastritis cystica profunda suggests that SPEM represents a precursor lineage for the development of dysplasia in this animal model of gastric carcinogenesis.

Differential protein analysis of spasomolytic polypeptide expressing metaplasia using laser capture microdissection and two-dimensional difference gel electrophoresis.

Full analysis of cellular protein constituents is a valuable tool in the evaluation of tissues. Traditional methods of evaluation, however, are time-consuming and difficult to reproduce. Two-dimensional difference gel electrophoresis (2D-DIGE), a recently developed proteomic system, affords the ability to compare and evaluate protein extracts from multiple sources. Coupled with laser capture microdissection (LCM), this technology is a powerful tool in comparing the protein profiles of separate pure cell populations. Proteins are labeled in vitro with reactive cyanine dyes that fluoresce at differential wavelengths, and after comigration on two-dimensional gels, differing protein populations become apparent. The unique aspect of this technology is the ability to identify and quantify proteins from separate preparations without issues of gel-to-gel differences. These techniques coupled with the systems for robotic acquisition of specific spots on the gel, tryptic digestion, and MALDI mass spectrometry permit identification of proteins differentially expressed in two pure cell populations. The authors used these new technologies to analyze the protein constituents of spasmolytic polypeptide expressing metaplasia (SPEM), a gastric mucosal metaplasia (fundic antralization or pseudopyloric metaplasia) that develops in the atrophic fundus mucosa of mice infected with Helicobacter felis and in humans infected with Helicobacter pylori. In addition, SPEM has been identified in the atrophic mucosa surrounding a high percentage of gastric adenocarcinomas and may represent a precursor lineage of malignancy. This technology recognized 28 differentially expressed proteins between SPEM and surface cells. Identification of novel SPEM-related proteins would allow the development of new immunohistochemical antibodies to further study this important metaplasia.