Methods and Uses Related to Aquaporin-5 (AQP5)

Abstract

This paper discloses a method for identifying gastric cancer stem cells or gastric cancer stem cell population.Also described herein is a method for isolating one or more gastric cancer stem cells from a cell population, comprising contacting the cell of the cell population with an agent that binds to AQP5, isolating one or more AQP5-expressing cells that are bound to the agent, and the one or more AQP5-expressing cells are gastric cancer stem cells.In addition, this paper also describes a method for removing or eliminating AQP5+ gastric cancer stem cells, as well as a method for treating gastric cancer and a method for monitoring gastric cancer tumorigenesis or gastric cancer progression.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to Singapore Provisional Application No. 10202300699X, filed March 14, 2023, and Singapore Provisional Application No. 10202302747V, filed September 27, 2023, the contents of which are incorporated herein by reference in their entirety for all purposes.

[0002] FIELD OF THE INVENTION The present invention relates generally to the field of molecular biology. In particular, the present invention relates to the use of biomarkers for the detection and diagnosis of cancer. [Background technology]

[0003] Background of the Invention Gastric cancer is one of the leading causes of cancer-related deaths both globally and in Singapore, with a 5-year survival rate of less than 30%. Gastric cancer treatment is currently limited to traditional chemotherapy, radiation therapy, and surgical resection, but these approaches remain ineffective in ameliorating cancer recurrence in many gastric cancer patients, particularly those with diffuse gastric cancer. Treatment-resistant cancer stem cell populations are thought to be important drivers of gastric cancer recurrence. However, previously proposed gastric cancer stem cell markers either lack robust functional validation directly demonstrating the stemness of the labeled cell populations or are broadly expressed across a wide range of normal and tumor tissues, making it difficult to target these cells in patients.

[0004] Implementing the concept of targeting cancer stem cells in the treatment of various human cancers in the context of gastric cancer has been hampered to date by the paucity of gastric cancer stem cell markers, most of which have not been robustly validated using near-physiological cancer models or functional assays. Furthermore, some of the markers proposed to date are widely expressed in both normal and tumor tissues, making it difficult to develop these markers into safe therapeutic targets for patients.

[0005] Therefore, there is an unmet need for methods to identify gastric cancer stem cells. Summary of the Invention

[0006] overview In one aspect, the present disclosure relates to a method for identifying a gastric cancer stem cell or a gastric cancer stem cell population, comprising: (a1) detecting expression of aquaporin 5 (AQP5) in a cell or cell population; or (b1) detecting the expression level of AQP5 in a cell or cell population and comparing the expression level with the expression level of AQP5 in a reference cell or reference cell population, wherein detection of AQP5 expression in the cell or cell population, or expression of AQP5 at an elevated level in the cell or cell population compared to the reference cell or reference cell population, identifies the cell or cell population as a gastric cancer stem cell or a gastric cancer stem cell population.

[0007] In another aspect, the present disclosure relates to a method for isolating one or more gastric cancer stem cells from a cell population, the method comprising the steps of: (i) contacting cells of the cell population with an agent that binds to AQP5; and (ii) isolating one or more AQP5-expressing cells bound to the agent, wherein the one or more AQP5-expressing cells are gastric cancer stem cells.

[0008] In yet another aspect, the present disclosure relates to a method for depleting or eliminating AQP5+ gastric cancer stem cells that have been modified to express the DTR gene or the inducible caspase 9 (iCasp9) gene, comprising contacting the cells with diphtheria toxin (DT) or an inducer of iCasp9.

[0009] In a further aspect, the present disclosure relates to a method of ablating or eliminating AQP5+ gastric cancer stem cells, comprising contacting the cells with an agent that binds to AQP5, wherein binding of the AQP5+ cells to the agent ablates or eliminates the cells.

[0010] In one aspect, the present disclosure relates to a method of treating gastric cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of one or more agents that eliminate or ablate AQP5-expressing cells.

[0011] In another aspect, the present disclosure relates to a biomarker for gastric cancer stem cells, wherein the biomarker is AQP5.

[0012] In a further aspect, the present disclosure relates to a kit for identifying, isolating, purging, or eliminating gastric cancer stem cells, comprising an agent that binds to AQP5 and instructions for use.

[0013] In yet another aspect, the present disclosure relates to a method of inhibiting gastric cancer tumorigenesis or progression, the method comprising administering to a subject an AQP5 inhibitor.

[0014] In another aspect, the present disclosure relates to a method for promoting regression of gastric cancer, the method comprising administering to a subject an AQP5 inhibitor.

[0015] In one aspect, the present disclosure relates to a method for monitoring the progression of gastric cancer in a subject, comprising: (a) measuring the expression level of AQP5 in a sample obtained from the subject after undergoing treatment for gastric cancer; and (b) measuring the expression level of AQP5 in a control sample obtained from the subject before treatment for gastric cancer, wherein an increase in the expression level of AQP5 in the sample of step a compared to the control sample indicates that gastric cancer tumorigenesis has occurred or that the gastric cancer has progressed.

[0016] In another aspect, the present disclosure relates to a method for monitoring gastric cancer tumorigenesis in a subject, comprising: (c) measuring the expression level of AQP5 in a sample obtained from the subject; and (d) measuring the expression level of AQP5 in a reference sample obtained from the subject at a time earlier than the sample of step c), wherein an increase in the expression level of AQP5 in the sample of step c compared to the reference sample of step d indicates that gastric cancer tumorigenesis has occurred. [Brief explanation of the drawings]

[0017] The present invention will be better understood by reference to the detailed description when considered in conjunction with the following non-limiting examples and the accompanying drawings.

[0018] [Figure 1]Figure 1 shows results demonstrating that Aqp5 is overexpressed in mouse and human pyloric tumors. Figure 1A shows a schematic diagram of the Aqp5-eGFPires-CreERT2; Apcfl / fl; Ptenfl / fl; KrasLSL-G12D / +; Rosa26-tdTomatoLSL conditional genetic mouse model of pyloric cancer. Figure 1B shows fluorescent images demonstrating AQP5 expression in the pyloric region across multiple stages of tumor development in the Aqp5-Cre / APK mouse model. Figure 1C shows a bar graph quantification of the change over time in the percentage of AQP5+ cells in healthy and tumor pyloric tissue. Figure 1D shows the FACS gating strategy used for the isolation of tumor epithelial (dTom+) Aqp5- and Aqp5+ cells from primary mouse pyloric tumors. Figure 1E shows a bar graph demonstrating relative Aqp5 expression in sorted mouse tumor cell populations by qPCR. Figure 1F is a heatmap showing the top 50 differentially expressed genes (DEGs) between Aqp5+ and Aqp5- samples isolated from six independent mouse pyloric tumors. Figure 1G shows a slide showing AQP5 expression in a human gastric tissue microarray of normal stomach, intestinal-type gastric tumor, diffuse-type gastric tumor, and mixed-type gastric tumor. Staining was scored on a scale ranging from no staining to strong staining, as indicated in the legend (right). The images shown are representative examples of tissue cores showing moderate staining (normal stomach) and strong staining (tumor tissue). Figure 1H shows the FACS gating strategy used to isolate tumor epithelial Aqp5- and Aqp5+ cells from primary human pyloric tumor biopsies. Figure 1I shows a bar graph showing relative Aqp5 expression in sorted human tumor cell populations by qPCR. Figure 1J is a heatmap of the top 50 DEGs between Aqp5+ and Aqp5- samples isolated from five independent human pyloric tumors. Scale bar, 200 μm. Graphs represent mean ± SD by two-tailed unpaired t-test. [Figure 2]Figure 2 shows results demonstrating that mouse and human Aqp5+ pyloric tumor cells function as cancer stem cells. Figure 2A shows images of organoids generated from single FACS-sorted mouse Aqp5+ and Aqp5- cells at passages 0, 2, and 10. Mouse Aqp5+ cell-derived organoids maintain high growth efficiency at least until P10, while the few Aqp5- cell-derived organoids that form do not survive beyond P2. Figure 2B shows bar graphs demonstrating organoid growth efficiency of single mouse Aqp5+ and Aqp5- cells at P0, along with quantification of the highest passage number reached in each culture. Figure 2C shows fluorescent images demonstrating the expression of AQP5 and lineage markers KI67, TFF2, and CHGA in mouse Aqp5+ cell-derived organoids. Figure 2D shows whole-mount and H&E images of tumors generated from transplanted mouse Aqp5+ (left) and Aqp5- (right) pyloric tumor cells. Figure 2E shows bar graph quantification of tumor volume from transplanted Aqp5+ and Aqp5- pyloric tumor cells. Figure 2F shows images of organoids generated from single FACS-sorted human Aqp5+ and Aqp5- cells at passages 0, 2, and 10. Human Aqp5+ cell-derived organoids maintain high tumor growth efficiency at least until P10, while a small number of Aqp5- cell-derived organoids fail to survive beyond P2. Figure 2G shows bar graphs quantification of organoid growth efficiency of single human Aqp5+ and Aqp5- cells at P2 and the highest passage number reached in each culture. Figure 2H shows fluorescence images showing the expression of AQP5 and lineage markers KI67, MUC5AC, and CHGA in human Aqp5+ cell-derived organoids. Scale bars are 500 μm (Figure 2A, Figure 2F) and 100 μm (Figure 2C, Figure 2H). Graphs represent mean ± SD by two-tailed unpaired t-test. [Figure 3]Figure 3 shows results demonstrating that ablation of Aqp5+ cancer stem cells blocks tumor initiation and progression. Figure 3A shows images demonstrating that diphtheria toxin (DT) treatment of Aqp5-Cre / APK / DTR mouse pyloric tumor organoids (containing the Aqp5-2A-DTR allele) at organoid development (day 0) blocks organoid growth. Four biological replicates were used. Figure 3B shows a line graph quantifying the change in organoid number over time in control and DT-treated organoid cultures after treatment on day 0. Figure 3C shows images demonstrating that diphtheria toxin (DT) treatment of Aqp5-Cre / APK / DTR mouse pyloric tumor organoids during organoid growth / maintenance (day 3) results in rapid loss of the culture. Four biological replicates were used. Figure 3D shows a line graph quantifying the change in organoid number over time in control and DT-treated organoid cultures after day 3 of treatment. Figure 3E shows images demonstrating that intraperitoneal administration of DT at an early stage of tumor development (day 3) in mice orthotopically implanted with Aqp5-Cre / APK / DTR organoids resulted in complete tumor growth abolition. (n=5 biological replicates) Figure 3F shows images demonstrating that intraperitoneal administration of DT at a later stage of tumor development (week 4) in mice orthotopically implanted with Aqp5-Cre / APK / DTR organoids promoted tumor regression. (n=5 biological replicates) G. Strategy for ablation of Aqp5+ cells in human gastric cancer organoids. The Aqp5-2A-iCaspase9 cassette was integrated into human gastric cancer organoids to drive inducible caspase 9 expression in Aqp5+ cells. Addition of the B / B homodimerizer promotes caspase-9 dimerization and activation of the apoptotic pathway, leading to cell death. Figure 3H shows images demonstrating that treatment of Aqp5-2A-iCaspase9 human gastric cancer organoids with the dimerizer (Dim) inhibits organoid growth in vitro. Figure 3I shows a bar graph quantification of organoid numbers in control cultures and cultures treated with the dimerizer. Scale bars, 500 μm (Figure 3A, Figure 3C, Figure 3G) and 1 mm (Figure 3E, Figure 3F). Graphs represent mean ± SD by two-tailed unpaired t-test. [Figure 4] Figure 4 shows results demonstrating that Aqp5 is expressed in a subset of cells within mouse pyloric tumors. Figure 4A shows representative whole-mount and H&E images of an Aqp5-Cre / APK mouse pyloric tumor. Figure 4B shows RNAscope images of Aqp5 and Lgr5 in a healthy mouse pylorus, demonstrating their colocalization within the pyloric stem cell compartment at the fundus of the gland. Figure 4C shows a schematic diagram of the Aqp5-2ACreERT2; Apcfl / fl; Ptenfl / fl; KrasLSL-G12D / +; Rosa26-tdTomatoLSL conditional genetic mouse model of pyloric tumors (left). AQP5 levels increase over time during tumor development, resembling the expression pattern in tumors derived from the Aqp5-eGFP-ires-CreERT2 model (right). Figure 4D shows a MERSCOPE-based spatial transcriptomics image of an Aqp5-Cre / APK mouse pyloric tumor section, revealing highly heterogeneous cell types assigned by Leiden clustering and highlighting Aqp5-expressing clusters. Figure 4E shows a UMAP plot of the Aqp5-Cre / APK tumor cell clusters. Figure 4F shows a heatmap of the expression levels of selected genes in each of the eight epithelial cell clusters, demonstrating the heterogeneity of the Aqp5+ population compared to other tumor cell lineages. Figure 4G shows an RNAscope image of Aqp5 and the putative gastric cancer stem cell markers Lgr5, Cxcr4, and Cd44 in an Aqp5-Cre / APK mouse pyloric tumor section. Aqp5 expression partially overlaps with known stem cell markers and also characterizes unique tumor compartments. Scale bars, 50 μm (magnified insets in b and g) and 200 μm (Figure 4A, 4C, 4D, and 4G). [Figure 5]Figure 5 shows the results of transcriptome analysis of murine Aqp5+ and Aqp5- pyloric tumor cells. Figure 5A shows a bar graph depicting selected gene ontology (GO) terms from molecular function (MF) and biological process (BP) categories that were significantly enriched in murine Aqp5+ cells compared with Aqp5- cells. Figure 5B shows the results of gene set enrichment analysis (GSEA) of the epithelial-mesenchymal transition pathway and external encapsulating structure organization pathway (left) along with accompanying heatmaps (right) depicting the relative expression of selected genes within each pathway. Figures 5C–5F show qPCR validation results (left graph) and RNA-seq expression data (right graph) of selected genes upregulated in the transcriptome of murine Aqp5+ cells associated with the tumor microenvironment (Figure 5C), metastatic dissemination (Figure 5D), gastric cancer progression (Figure 5E), and drug resistance (Figure 5F). Figure 5G shows images of RNAscope validation of selected targets Lgr5, Pthlh, Rgs5, and Hey1, confirming their upregulation in Aqp5+ tumor cell areas. Scale bar, 100 μm. Graphs represent mean ± SD by two-tailed unpaired t-test. [Figure 6]Figure 6 shows results demonstrating that Aqp5 is expressed in a subset of cells within human pyloric tumors. Figure 6A shows a UMAP plot of cell clusters generated from scRNA-seq of healthy and tumor human gastric tissues, with epithelial cell clusters labeled. Figure 6B shows a list of selected markers for assigning identity to epithelial cell clusters. Figure 6C shows a UMAP plot demonstrating Aqp5 expression in epithelial cell clusters within normal (left) and tumor (right) human gastric tissues. Figure 6D shows a violin plot of Aqp5 expression in each epithelial cluster, demonstrating elevated levels within the tumor cell population. Figures 6E-6F show a UMAP plot (Figure 6E) and a violin plot (Figure 6F) of tumor epithelial cell clusters separated by tumor subtype (intestinal, diffuse, and mixed). Figures 6G-6H show a UMAP plot (Figure 6G) and a violin plot (Figure 6H) of tumor epithelial cell clusters separated by tumor location (antrum / pylorus, corpus, and cardia). Figure 6I shows a UMAP plot of epithelial cell clusters generated from scRNA-seq of healthy and tumor human gastric tissues from the second dataset, GSE150290. Figure 6J shows the identification of selected markers to assign identity to the epithelial cell clusters in Figure 6I. Figure 6K shows a violin plot of Aqp5 expression in each epithelial cluster identified in Figure 6I, demonstrating elevated levels within the tumor population. [Figure 7]Figure 7 shows the results of transcriptome analysis of human Aqp5+ and Aqp5- pyloric tumor cells. Figure 7A shows a bar graph depicting selected gene ontology (GO) terms from molecular function (MF) and biological process (BP) categories that were significantly enriched in human Aqp5+ cells compared to Aqp5- cells. Figure 7B shows the results of gene set enrichment analysis (GSEA) of the epithelial-mesenchymal transition pathway and the external encapsulation structure organization pathway (left) along with accompanying heatmaps (right) depicting the relative expression of selected genes within each pathway. Figures 7C–7F show qPCR validation results (left graph) and RNAseq expression data (right graph) of selected genes upregulated in the transcriptome of human Aqp5+ cells related to the tumor microenvironment (Figure 7C), cancer progression (Figure 7D), drug resistance (Figure 7E), and tumor control (Figure 7F). Figure 7G shows RNAscope validation results for selected targets AQP5, CLDN2, DCHS2, HEY2, and PLA1A, confirming their upregulation in Aqp5+ tumor cell areas. Scale bar, 100 μm. Graphs represent mean ± SD by two-tailed unpaired t-test. [Figure 8]Figure 8 shows results demonstrating that mouse Aqp5+ cells function as cancer stem cells. Figure 8A shows representative brightfield and immunofluorescence images of mouse Aqp5-Cre / APK pyloric tumor organoids generated from whole glands, demonstrating organoid morphology and gastric lineage marker expression comparable to tumor organoids formed from isolated Aqp5+ tumor cells. Figure 8B shows representative whole-mount, H&E, and IHC images of the stomach of an immunodeficient NOG mouse 4 months after orthotopic implantation of primary mouse pyloric tumor cells into the gastric submucosa, with minimal tumor cell colonization. Figure 8C shows a schematic diagram of the experimental workflow for generating orthotopic tumors from the implantation of mouse pyloric tumor organoids, isolating Aqp5+ and Aqp5- cells by FACS, and reimplanting these cells into immunodeficient mice. Figure 8D shows an image depicting the histology of mouse Aqp5-Cre / APK pyloric tumor organoids assessed by H&E (left), as well as images depicting orthotopic tumors resulting from transplantation of these tumor organoids visualized by whole mount and H&E (center and right). Figure 8E shows the FACS gating strategy for isolating transplanted tumor epithelial (dTom+) Aqp5+ and Aqp5- cells. Figure 8F shows histology images of orthotopic tumors derived from transplanted tumor epithelial Aqp5+ and Aqp5- cells. RFP immunohistochemistry highlights the location of transplanted cells within the gastric mucosa. Immunofluorescence examination of Aqp5, Muc5ac, Ki67, and Chga demonstrates that tumors derived from Aqp5+ cells maintain the expression of stem cell and lineage markers. Figure 8G shows a schematic diagram of the Aqp5-2A-CreERT2; Apcfl / fl; Ptenfl / fl; Trp53R172H / +; Rosa26-tdTomatoLSL conditional genetic mouse model of pyloric tract cancer. Figure 8H shows representative whole-mount images and immunostaining images confirming the expression of p53 mutants (center) and the presence of Aqp5+ cells in a subset of Aqp5-Cre / APT tumors (right). Figure 8I shows the FACS gating strategy for isolating tumor epithelial Aqp5+ and Aqp5- cells from Aqp5-Cre / APT tumors.Figure 8J shows a bar graph demonstrating relative Aqp5 expression in sorted tumor cell populations by qPCR. Figure 8K shows images demonstrating that more organoids can be established from single Aqp5+ cells compared to Aqp5- cells. Figure 8L shows a bar graph demonstrating the organoid growth efficiency of single mouse Aqp5+ and Aqp5- cells at P0. Scale bars: 100 μm (a), 500 μm (k), 1 mm (b, d, h). Graphs represent mean ± SD by two-tailed unpaired t-test. [Figure 9] Figure 9 shows results demonstrating that human Aqp5+ cells function as cancer stem cells. Figure 9A shows representative brightfield and immunofluorescence images of human pyloric tumor organoids generated from whole glands, demonstrating organoid morphology and gastric lineage marker expression comparable to tumor organoids formed from isolated Aqp5+ tumor cells. Figure 9B shows a schematic diagram of the Aqp5-2A-CreERT2; AAVS1-CAG-tdTomatoLSL construct inserted into human gastric cancer organoids (top). Genomic PCR performed at the Aqp5-2A-CreERT2 insertion site confirms successful insertion based on the expected band size (bottom). Figure 9C shows brightfield and fluorescent images demonstrating that 16 hours of 4-OHT induction in Aqp5-2A-CreERT2;AAVS1-CAG-tdTomatoLSL-edited organoids resulted in visible tdTomato labeling of a small pool of cells by day 2, which expanded to encompass larger organoid areas by day 10. Figure 9D shows a bar graph quantification of the expansion of the area of ​​tdTomato+ labeled clones within each organoid over time. Figure 9E shows brightfield and fluorescent images demonstrating that uninduced Aqp5-2A-CreERT2;AAVS1-CAG-tdTomatoLSL organoids do not display any visible signs of tdTomato tracking at the same time point. Figure 9F shows co-immunofluorescence images confirming the overlap of tdTomato-labeled cells with lineage markers, including MUC5AC and KI67. Scale bar, 100 μm. The graph represents the mean ± SD by two-tailed unpaired t-test. [Figure 10]Figure 10 shows results demonstrating that ablation of murine Aqp5+ cells within a range of pyloric tumor models confirms their requirement for tumor initiation and progression. Figure 10A shows a schematic diagram of the Aqp5-eGFP-ires-CreERT2; Apcfl / fl; Ptenfl / fl; KrasLSL-G12D / +; Rosa26-tdTomatoLSL conditional genetic mouse model of pyloric cancer, in which the Aqp5-2A-DTR cassette was added to promote diphtheria toxin (DT)-mediated ablation of Aqp5+ cells. Figure 10B shows representative images demonstrating that Aqp5-Cre / APK and Aqp5-Cre / APK / DTR tumors are comparable in terms of histological features and Aqp5 expression. Figure 10C shows results demonstrating that intraperitoneal administration of DT to mice 10 weeks after tamoxifen-induced pyloric tumor formation reduces tumor volume by approximately 50%. Representative whole-mount images of control and DT-treated tumors are shown on the left. Figure 10D shows a timeline of tumor tissue analysis images 12 hours after DT administration and removal in Aqp5-Cre / APK / DTR mice (top). Representative immunohistochemistry images confirm the reduction of AQP5+ cells in DT-treated Aqp5-Cre / APK / DTR tumors, along with increased CAS3 and KI67 expression in the remaining tumor mass. Figure 10E shows a timeline of tumor tissue analysis 12 hours after DT administration and removal in Aqp5-Cre / APK mice lacking the Aqp5-2A-DTR allele (top). Representative immunohistochemistry images demonstrate comparable AQP5, CAS3, and KI67 levels in control and DT-treated tumors. Figure 10F shows data demonstrating that mouse pyloric tumor organoids generated from Aqp5-Cre / APK / DTR tumors recapitulate AQP5 expression in a subset of cells. Co-immunofluorescence of AQP5 and ECAD in a representative tumor organoid is shown (top). FACS analysis shows that 9-10% of cells within Aqp5-Cre / APK / DTR organoids highly express AQP5 (bottom). A mouse pyloric tumor organoid line lacking AQP5-GFP was used as a control to set the FACS gates for the profile.Figure 10G shows results demonstrating that DT treatment of Aqp5-Cre / APK / DTR organoids reduces Aqp5 levels by 80% and 95% compared to untreated control organoids during organoid outgrowth and organoid expansion, respectively. Figure 10H shows data demonstrating that treatment of Aqp5-Cre / APK organoids lacking the Aqp5-2A-DTR allele with DT on day 0 does not affect organoid growth. Figure 10I shows results demonstrating that treatment of Aqp5-Cre / APK organoids lacking the Aqp5-2A-DTR allele with DT on day 3 does not affect subsequent organoid maintenance. Figure 10J shows data demonstrating by qPCR that treatment of Aqp5-Cre / APK organoids lacking the Aqp5-2A-DTR allele with DT does not affect Aqp5 levels during both organoid outgrowth and organoid expansion. Figures 10K-10N show a timeline highlighting the administration of DT to mice after organoid transplantation for in vivo Aqp5 cell ablation and the time points at which tissues were harvested for analysis (top). Representative H&E and AQP5 immunohistochemistry images confirm that AQP5 expression was lost and / or disrupted 1 day after ablation at either 3 days (Figure 10K) or 4 weeks (Figure 10L) after transplantation of Aqp5-Cre / APK / DTR organoids. Representative H&E and AQP5 immunohistochemistry images confirm that DT administration had no effect on tumor burden or AQP5 expression 4 weeks (Figure 10M) or 8 weeks (Figure 10N) after transplantation of Aqp5-Cre / APK organoids lacking the Aqp5-2A-DTR allele. Scale bars are 500 μm (FIGS. 10F, 10H, and 10I) and 1 mm (FIGS. 10B, 10D, 10E, 10K, 10L, 10M, and 10N). Graphs represent mean ± SD by two-tailed unpaired t-test. [Figure 11]Figure 11 shows results demonstrating that ablation of human Aqp5+ cells in a human gastric cancer organoid model confirms their requirement for tumor progression. Figure 11A shows an agarose gel image. Genomic PCR performed at the Aqp5-2A-iCaspase insertion site confirms successful insertion based on the expected band sizes. Figure 11 shows immunofluorescence images of AQP5, KI67, MUC5AC, and CHGA, confirming the presence of expected lineage markers in Aqp5-2A-iCaspase organoids. Figure 11C shows results from dimerizer treatment of 0116 Aqp5-2A-iCaspase organoids, resulting in a reduction of Aqp5 levels by approximately 60% compared to untreated control organoids. Figure 11D shows results from dimerizer treatment of unedited 0116 parent organoids, demonstrating no change in organoid development or subsequent growth when followed for 10 days in culture. Figure 11E shows images demonstrating that elimination of Aqp5+ cells in an independent GC10 Aqp5-2A-iCaspase organoid line reproduces the reduced growth rate. Figure 11F shows a bar graph demonstrating that treatment of GC10 Aqp5-2A-iCaspase organoids with a dimerizer reduces Aqp5 levels by 70% compared to untreated control organoids. Figure 11G shows a bar graph demonstrating that treatment of unedited GC10 parental organoids with a dimerizer does not alter the development or subsequent growth of organoids tracked over 10 days in culture. Figure 11H shows a bar graph demonstrating that Aqp5 levels remain unchanged in control and dimerizer-treated GC10 parental organoids by qPCR. Scale bars, 100 μm (Figure 11B) and 500 μm (Figures 11D, 11E, and 11G). Graphs represent mean ± SD by two-tailed unpaired t-test. [Figure 12]Figure 12 shows images demonstrating that targeted ablation of Aqp5+ tumor cells significantly reduces organoid and tumor growth. Figure 12A shows a schematic diagram and images demonstrating the results of diphtheria toxin (DT)-mediated targeted ablation of Aqp5+ tumor cells in pyloric tumor organoids carrying the Aqp5-2A-DTR allele. In this model, tumor cells expressing Aqp5 also express DT receptors, making them sensitive to DT administration. Adding DT to ablate Aqp5+ tumor cells in organoids at the time of seeding (day 0) or mid-organoid growth (day 3) resulted in complete lethality of the tumor organoids. Figure 12B shows a schematic diagram and images demonstrating the results of DT-mediated ablation of Aqp5+ tumor cells in pyloric tumor organoids implanted into the gastric submucosa of immunodeficient NOG mice. Ablation performed at the time of tumor initiation (day 3) completely abolished tumor formation in DT-treated mice. When removal is performed at a later stage of tumor development (week 4), DT-treated tumors display a milder tumorigenic phenotype. [Figure 13] Figure 13 shows images demonstrating minimal Aqp5 expression in most major human tissues. Figure 13A shows selected tissue microarray images from the brain, heart, kidney, and liver, demonstrating absent or weak Aqp5 staining. In contrast, Aqp5 is strongly expressed in the membranes of the salivary gland and testis. Figure 13B shows images and scoring results of 47 normal human lung tissue microarray cores, with the majority of samples showing either absent Aqp5 expression or non-membrane localization of the Aqp5 protein. [Figure 14] Figure 14 shows images demonstrating that Aqp5 is upregulated in mouse and human gastric tumors. Immunohistochemistry of Aqp5 on human (top) and mouse (bottom) gastric tissue. Aqp5 expression is restricted to the fundus of the pyloric gland in both human and mouse, but is widely and highly expressed in both intestinal and diffuse-type gastric tumors. [Figure 15]Figure 15 shows an image of a previously unknown human gastric cancer organoid line. This cell line incorporates the Flip-Puro system, which allows for conditional knockout of Aqp5 upon administration of a Cre recombinase gesicle. The loss of Aqp5 in these organoids was confirmed by Western blotting. Following Aqp5 knockout in organoid cells, decreased cell viability of the organoids was observed. [Figure 16-1]Figure 16 shows data demonstrating that Aqp5 drives gastric tumorigenesis. Figure 16A shows histology images of pyloric tumors from Aqp5 wild-type (WT) and knockout (KO) mice. Representative whole-mount images (left) and H&E images (center) are shown, along with tumor volume quantification (right). Figure 16B shows significant gene ontology (GO) terms expressed by DEGs enriched in Aqp5 WT cancer stem cells (top) and Aqp5 KO cancer stem cells (bottom). Figure 16C shows data demonstrating human gastric cancer organoids (parent) and four independent Aqp5 KO clones (clones A1, A5, C2, and C5) derived from the parental line. AQP5 is expressed in the apical membrane of the parental organoids but is absent in the KO clones (top). Reduced EdU staining is detected within the Aqp5 KO organoids compared to the parental organoids (bottom). Figure 16D shows quantification of cell proliferation within parental and Aqp5 KO organoids using an in vitro MTS colorimetric assay (left) and EdU staining assay (right). Figure 16E shows representative whole-mount images (top) and H&E images (bottom) of orthotopic tumors derived from transplanted parental and Aqp5 KO human gastric cancer organoids. Figure 16F shows quantification of tumor volume (left) and invasion rate (right) of orthotopic tumors derived from transplanted parental and Aqp5 KO human gastric cancer organoids. Figure 16G shows a schematic diagram of the FLIP-Puro cassette used to generate Cre-inducible Aqp5 KO in human gastric cancer organoids. Figure 16H shows images demonstrating that conditional KO of Aqp5 by Cre recombinase treatment in human gastric cancer organoids reduces organoid growth in vitro. Figure 16I shows the quantification of relative organoid numbers 5 days after conditional Aqp5 KO. Organoids at least 100 μm in diameter were included in the analysis. Figure 16J shows a schematic diagram of significant GO terms enriched in untreated and Cre-induced Aqp5 KO organoids at various indicated time points after induction. Scale bars: 100 μm (Figure 16C, bottom), 500 μm (Figure 16C, top, Figure 16H), and 1 mm (Figure 16A, Figure 16E, bottom). Graphs represent mean ± SD by two-tailed unpaired t-test. [Figure 16-2] A continuation of Figure 16-1 is shown. [Figure 16-3] Continuation of Figure 16-2. [Figure 16-4] Continuation of Figure 16-3. [Figure 16-5] Continuation of Figure 16-4. [Figure 17-1] Figure 17 shows results demonstrating that AQP5 promotes tumor progression in multiple human gastric cancer cell lines. Figure 17A shows the results of analyzing AQP5 function in the intestinal-type AGS gastric cancer cell line. Representative immunofluorescence images and qPCR of Aqp5 levels (left) confirm the upregulation of Aqp5 in three AQP5-overexpressing (OE) clones. In vitro assays show that the OE clones exhibited higher cell proliferation and migration rates in culture (right). Figure 17B shows the results of analyzing AQP5 function in the diffuse-type SNU601 gastric cancer cell line. Representative immunofluorescence images and qPCR of Aqp5 levels (left) confirm the upregulation of Aqp5 in three AQP5-overexpressing (OE) clones. In vitro assays show that the OE clones exhibited higher cell migration rates in culture (right). Figure 17C shows results demonstrating that AGS OE cells produce orthotopic tumors in mice that exhibit larger, more aggressive tumor characteristics. Figure 17D shows analysis of AQP5 function in the diffuse KATOIII cell line, which expresses high levels of AQP5. Representative immunofluorescence images and qPCR of Aqp5 levels confirm the loss of Aqp5 in knockout (KO) cells (left). In vitro assays show that KO cells grow slower in culture than parental cells (right). Figure 17E shows that KATOIII KO cells fail to produce orthotopic tumors in mice in the majority of cases examined. Scale bars, 500 μm (Figures 17A, 17B, 17D) and 1 mm (Figures 17C, 17E). Graphs represent mean ± SD by two-tailed unpaired t-test. [Figure 17-2] A continuation of Figure 17-1 is shown. [Figure 17-3] Continuation of Figure 17-2. [Figure 18-1]Figure 18 shows data demonstrating that AQP5 knockout in a mouse model of pyloric cancer suppresses tumor growth and exhibits an altered transcriptome profile. Figure 18A shows representative immunofluorescence images of Aqp5 WT and Aqp5 KO pyloric tumors, confirming the loss of AQP5 in the KO tumors. Figure 18B shows data demonstrating that mouse pyloric tumor organoids generated from Aqp5 WT and Aqp5 KO tumors reflect the AQP5 expression pattern in the original tumors. Figure 18C shows that Aqp5 KO tumor organoids form less invasive tumors after transplantation into immunodeficient mice compared to Aqp5 WT tumor organoids. Representative whole-mount and H&E images (left) and quantification of tumor volume and invasion rate (right) are shown. Figure 18D shows the FACS gating strategy for isolating tumor epithelial GFP+ cells (stem cells) and GFP- cells (tumor remainder) from Aqp5 WT and Aqp5 KO mouse pyloric tumors. Figure 18E shows a heatmap of the top 50 differentially expressed genes (DEGs) between GFP+ and GFP- samples isolated from three independent mouse pyloric tumors. Figure 18F shows a Venn diagram of shared and unique DEGs between Aqp5 KO and Aqp5 WT tumor profiles. Figure 18G shows the results of a reactome analysis of genes enriched in Aqp5 WT tumors, highlighting potential pathways downstream of Aqp5 function. Scale bars, 500 μm (Figure 18B) and 1 mm (Figures 18A and 18C). Graphs represent mean ± SD by two-tailed unpaired t-test. [Figure 18-2] A continuation of Figure 18-1 is shown. [Figure 18-3] Continuation of Figure 18-2. [Figure 18-4] Continuation of Figure 18-3. [Figure 18-5] Continuation of Figure 18-4. [Figure 19-1]Figure 19 shows data demonstrating that AQP5 drives tumor progression in multiple human gastric cancer organoid lines. Figures 19A, 19B, and 19C show results obtained with three independent human gastric cancer organoid lines, 0045 (Figure 19A), 0235 (Figure 19B), and 0116 (Figure 19C), used to analyze AQP5 function in vitro. Representative immunofluorescence images (left) and qPCR of Aqp5 levels (center) confirm the upregulation of Aqp5 in AQP5-overexpressing (OE) organoid lines. Of note, because Aqp5 is already strongly expressed in the parental 0116 line, the upregulation of Aqp5 in 0116 OE organoids is more modest compared to the other two OE lines. As measured by MTS proliferation assay, the 0045 OE and 0235 OE organoid lines exhibit higher cell proliferation rates in vitro compared to their respective parental lines (right). Figures 19D, 19E, and 19F show the results of orthotopic transplantation of three human gastric cancer organoid lines, 0045 (Figure 19D), 0235 (Figure 19E), and 0116 (Figure 19F), used to analyze AQP5 function in vivo. Representative whole-mount and H&E images (left) demonstrate the histological characteristics of tumors formed from transplanted organoids. Quantification of tumor volume (center) and invasion rate (right) demonstrates that OE organoids can form large tumors with more invasive characteristics compared to their respective parental lines. Figure 19G shows the results of genomic PCR performed at the Aqp5-FLIP-Puro insertion site, confirming successful insertion based on the expected band size. Figure 19H shows data showing that AQP5 levels are significantly lower in Aqp5 FLIP-Puro organoids after Cre recombinase treatment. Scale bars: 500 μm (Figures 19A, 19B, 19C) and 1 mm (Figures 19D, 19E, 19F). Graphs represent mean ± SD values ​​by two-tailed unpaired t-test. [Figure 19-2] A continuation of Figure 19-1 is shown. [Figure 19-3] Continuation of Figure 19-2. [Figure 19-4] Continuation of Figure 19-3. DETAILED DESCRIPTION OF THE INVENTION

[0019] Detailed Description Aqp5 has been implicated in promoting tumor cell proliferation and migration in human gastric cancer cell lines, but its precise mechanism of action remains unclear. Gastric tumors often express high levels of Aqp5, but its role in gastric cancer has not been thoroughly investigated in near-physiological cancer organoid and mouse models. The human gastric cancer cell line AGS, derived from an intestinal tumor, exhibits modest levels of Aqp5 in vitro (Figure 17A). Three independent Aqp5-overexpressing AGS cell lines with highly elevated Aqp5 levels were generated, and these were found to exhibit significantly increased cell proliferation and migration rates in vitro, consistent with previous studies (Figure 17A). In another diffuse-type gastric cancer cell line, SNU601, increased cell migration rates were detected in three independent Aqp5-overexpressing lines in vitro (Figure 17B). Furthermore, after orthotopic transplantation into the pylorus of immunodeficient mice, Aqp5-overexpressing AGS cells formed larger tumors that actively invaded the epithelial and muscularis layers (Figure 17C). Conversely, knockout of Aqp5 in the gastric cancer cell line KATOIII, which expresses high levels of Aqp5, reduced the cell proliferation rate in vitro (Figure 17D). While parental KATOIII cells seeded large tumors in the gastric submucosa after orthotopic transplantation, Aqp5-null KATOIII cells failed to form tumors in the majority of mice analyzed (Figure 17E). This indicates that Aqp5 may contribute to key steps in tumor establishment. Therefore, the data disclosed herein demonstrate that Aqp5 functions in promoting tumorigenic properties, namely cell proliferation and migration, in gastric cancer cell lines and further extend these findings to the in vivo situation through transplantation assays.

[0020] This disclosure describes the identification of Aqp5 as a marker for gastric cancer stem cells, identified using multiple assays across physiologically relevant mouse and human gastric cancer models. It is shown herein that the Aqp5 surface marker facilitates efficient isolation of promising gastric cancer stem cells for expression profiling, identifying additional markers and potential therapeutic vulnerabilities. Elimination of Aqp5-expressing tumor cell populations, as shown herein, is an example of a therapeutic approach used to ameliorate gastric cancer progression.

[0021] Aqp5 is a functional gastric cancer stem cell marker that drives tumorigenesis Cancer stem cells constitute a self-renewing population capable of generating differentiated tumor cell lineages and driving tumor growth. While cancer stem cell markers have been proposed for gastric tumors, to date, these have remained largely limited to animal cancer models, either broadly expressed in many normal and cancer tissues or failing robust validation by functional assays demonstrating cancer stemness in near-physiological gastric cancer models in mice and humans. Importantly, the identification of such markers will facilitate cancer stem cell isolation to elucidate downstream mechanistic functions and the development of targeted therapies against this cell population in human gastric tumors, which may support longer-term disease remission. In normal tissues, Aqp5 characterizes pyloric stem cells in mice and humans, and dysregulation of key pathways altered in gastric cancer in these Aqp5+ cells is sufficient to drive pyloric tumor formation in mouse models. Herein, using multiple representative in vitro and in vivo gastric cancer models, we demonstrate that Aqp5 is a functionally validated gastric cancer stem cell marker present in both mouse and human gastric tumors. This indicates the necessity and contribution of Aqp5+ tumor cells in maintaining tumor growth. These findings will advance our understanding of cancer stem cell models and enable the development of diagnostic tools and therapeutic strategies targeting gastric cancer.

[0022] Thus, the present disclosure describes the use of Aqp5 as a marker expressed by gastric cancer stem cell pool. In one example, the markers disclosed herein are used to promote selective targeting and / or elimination of gastric cancer stem cell populations, resulting in an effect on disease progression. In a further example, Aqp5 is used as a marker for identifying gastric cancer. In another example, a biomarker for gastric cancer stem cells is disclosed, the biomarker being AQP5. In another example, the biomarker is membrane-bound AQP5.

[0023] In another embodiment, the method for identifying gastric cancer stem cell or gastric cancer is disclosed.In one example, the method disclosed herein comprises: (a1) detecting the expression of aquaporin 5 (AQP5) in cell or cell population; or (b1) detecting the expression level of AQP5 in cell or cell population, and comparing this expression level with the expression level of AQP5 in reference cell or reference cell population, and by detecting the AQP5 expression in said cell or cell population, or the AQP5 expression in said cell or cell population is increased compared with said reference cell or reference cell population, said cell or cell population is identified as gastric cancer stem cell or gastric cancer stem cell group.

[0024] In another example, the methods disclosed herein further comprise isolating the identified gastric cancer stem cells or stem cell population.

[0025] In one example, the cells or cell populations disclosed herein are in vitro, in vivo, or ex vivo cells or cell populations. In another example, the methods disclosed herein are methods performed in vitro, in vivo, or ex vivo.

[0026] In yet another example, cell or cell population disclosed herein is gastric tumor sample, biopsy material or organoid.The example of cell disclosed herein can be but is not limited to epithelial cell.In another example, cell population disclosed herein comprises epithelial cell from gastric tumor sample or biopsy material.

[0027] In one example, the method disclosed herein comprises comparing the expression level obtained with the expression level obtained from a reference cell or cell population.This reference cell or cell population can be the cell that does not express AQP5, and optionally, the cell that does not express AQP5 is non-gastric cell, non-cancerous gastric cell, or a combination thereof.In one example, the reference cell or cell population is the cell or cell population that comprises or consists of non-cancerous gastric cell.

[0028] Aqp5 is a marker of gastric cancer stem cells in both murine and human gastric tumors. Using an established protocol for isolating Aqp5+ and Aqp5- cells from gastric tumors, we demonstrated that Aqp5+ tumor cells function as stem cells capable of seeding new tumors and repopulating tumor masses when transplanted into recipient mice. Furthermore, isolated Aqp5+ tumor cells selectively form organoids in vitro, which can be maintained in long-term culture. These findings were obtained using a wide range of in-house developed murine and human gastric cancer models. These assays collectively demonstrate the cancer stemness exhibited by the Aqp5+ tumor cell population and identify Aqp5 as a validated gastric cancer stem cell marker in a near-physiological human gastric cancer model.

[0029] Aqp5 marks a subset of epithelial cells in mouse and human pyloric tumors To study the contribution of Aqp5+ tumor cells to gastric cancer, we first characterized the expression of Aqp5 in mouse pyloric tumors, including Apc, Pten, and Kras under the Aqp5-eGFP-IRES-creERT2 driver to recapitulate key co-regulated pathways commonly observed in human gastric tumors.G12D Recombination of the conditional floxed allele in these mice resulted in the formation of Aqp5-Cre / APK pyloric tumors, classified as tubular adenocarcinomas, within 2–3 months, as previously described (Figure 1A, Figure 4A).

[0030] Thus, in one example, the cells or cell populations disclosed herein are further modified to express AQP5 in combination with an inducible gene. In one example, the inducible gene is CreERT2. In another example, the inducible gene is the inducible caspase 9 (iCasp9) gene.

[0031] Before cancer induction, Aqp5 expression was restricted to the tissue-resident stem cell compartment at the fundus of the pyloric gland, overlapping with Lgr5 (Figure 1B, Figure 4C). At later stages of cancer progression, increased numbers of Aqp5+ tumor cells were detected in the transformed pyloric epithelium, including in areas above the fundus (Figure 1B, Figure 1C). Using a separate Aqp5-2A-creERT2 pyloric cancer model in which endogenous Aqp5 expression is unperturbed, we observed the spatiotemporal pattern of Aqp5 expression during tumor progression (Figure 4C). Consistent with these histological analyses, single-cell RNA sequencing (scRNAseq) of healthy pyloric and Aqp5-Cre / APK pyloric tumors demonstrated elevated Aqp5 expression in a subset of tumor epithelial cell lineages (data not shown). Expression of Aqp5 and Lgr5 was highly restricted to the same stem cell cluster in healthy pylorus, whereas within pylorus tumors, Aqp5 was enriched in a cellular compartment significantly different from Lgr5, indicating that a smaller subset coexpressed these two markers (data not shown).

[0032] To localize these Aqp5+ cell subsets within intact pyloric tumors, we also performed imaging-based spatial transcriptomics using MERSCOPE and the constructed spatial cell map of mouse Aqp5-Cre / APK tumors (Figure 4D). This approach revealed eight clusters corresponding to tumor epithelial lineages (Figure 4E), three of which showed elevated Aqp5 transcript levels and mapped to both the glandular base and tumor compartments just above the glandular base (Figure 4D, Figure 4F). The Aqp5+ tumor lineage consisted of a Wnt-active cluster overlapping with Lgr5+ cells and a highly proliferative cluster expressing Mki67 (Figure 4F), consistent with previously obtained scRNA-seq datasets. Aqp5 expression was further validated in these mouse pyloric tumors by RNAscope in comparison with other putative gastric cancer stem cell markers Lgr5, Cxcr4, and Cd44 ( Fig. 4G ), thereby demonstrating that Aqp5 labels a previously unappreciated subpopulation of pyloric tumor cells.

[0033] In one example, this method is as disclosed herein, and this method comprises further analyzing the gastric cancer stem cell, stem cell population or AQP5 expressing tumor organoid.The method of analyzing the gastric cancer stem cell or AQP5 expressing tumor organoid can include but is not limited to transcriptome analysis (for example, RNA sequencing, single-cell RNA sequencing, spatial transcriptomics, gene ontology analysis, polymerase chain reaction analysis or combination thereof), proteomics analysis (for example, single-cell proteomics) and combination thereof, etc.In one example, further analysis is spatial transcriptomics.

[0034] To gain biological insights from Aqp5+ tumor cells, we established a strategy to isolate these pyloric tumor epithelial Aqp5+ and Aqp5- cell populations within Aqp5-Cre / APK tumors by fluorescence-activated cell sorting (FACS) (Figure 1D). The Aqp5-Cre / APK model reliably tracks the entire pyloric gland within one week after induction with Rosa26-tdTomato. LSL The reporter facilitates the use of tdTomato as a marker for isolating tumor epithelial populations and eGFP as a reporter for Aqp5 expression. Sorted eGFP+ tumor epithelial cells showed an average 9.8-fold enrichment in Aqp5 levels compared to eGFP- cells by qPCR (Figure 1E). Using this sorting strategy, we performed transcriptome profiling of Aqp5+ and Aqp5- tumor epithelial cells isolated from six independent Aqp5-Cre / APK mouse pyloric tumors (Figure 1F). This revealed selective enrichment in the transcriptome of Aqp5+ tumor cells for pathways related to extracellular matrix (ECM) remodeling, epithelial-mesenchymal transition (EMT), and drug resistance (Figure 5A, Figure 5B, Table 1)—features typically associated with cancer stem cells in other systems. Many of these upregulated genes, including Pthlh, Rgs5, and Hey1, were validated by qPCR and RNAscope in additional mouse pyloric tumor samples (Figures 5C-G, Table 2), demonstrating their robust enrichment in the Aqp5+ tumor cell population.

[0035] Therefore, in one example, cell or cell population disclosed herein is modified to express AQP5 in combination with one or more detectable labels.Such detectable labels can be, but are not limited to, fluorescent labels, tags, proteins, and combinations thereof.Non-exhaustive examples of fluorescent labels are tdTomato, GFP, eGFP, RFP, YFP, and combinations thereof.In one example, fluorescent label is eGFP.

[0036] In further examples, the gastric cancer stem cells disclosed herein express one or more of gastric cancer progression markers (e.g., but not limited to, Pthlh, Hey1, Rgs5), cancer stem cell function markers (e.g., but not limited to, Hey1, Clmp, Cyb1p1), tumor microenvironment markers (e.g., Rgs5, Adamntsl3, Itgb8), and combinations thereof.

[0037] Aqp5-expressing stem cells have previously been identified and characterized in healthy human pyloric tracts. To perform a detailed evaluation of their tumor counterparts, we analyzed Aqp5 expression using publicly available human scRNA-seq datasets of healthy gastric and gastric tumor tissues. A total of six epithelial clusters were identified (Figure 6A), including lineages enriched for gastric mucosal (Muc5ac+, Muc6+), chief cell (Pgc+), and enteroendocrine (Chga+) markers, as well as intestinal (Tff3+, Fabp1+) markers (Figure 6B). Within healthy human pyloric tracts, Aqp5 is primarily expressed in the Muc6+ mucous gland fundic cell cluster, previously associated with the gastric stem cell compartment in both mice and humans (Figure 6C). This contrasts with Aqp5 expression in human gastric tumors, where it was shown to be increased across multiple epithelial cell clusters (Figure 6C, Figure 6D). Aqp5 expression was also found to exhibit a tumor subtype-specific pattern, with the highest elevated Aqp5 levels detected in intestinal-type tumors, whereas diffuse-type tumors showed no significant difference in Aqp5 levels compared with healthy tissue (Figures 6E-6H). These results were corroborated using an independent human scRNAseq dataset, showing that Aqp5 expression was also significantly higher in gastric tumor cells than in their healthy counterparts (Figure 6I). This was further substantiated by detecting Aqp5 at the protein level using IHC performed on human gastric tissue microarrays (Figure 1G), confirming previous studies. Collectively, these analyses identify an elevated Aqp5 expression signature in human gastric tumors primarily associated with intestinal-type tumors (a feature robustly captured in the Aqp5-Cre / APK mouse pyloric cancer model disclosed herein).

[0038] Therefore, in one example, the method disclosed herein comprises detecting the expression level of AQP5.This expression level can be detected as, for example, gene expression level, protein expression level, or a combination thereof.In one example, the method is as described herein, and the step of detecting the protein expression level of AQP5 is carried out.In another example, the method is as described herein, and the step of detecting the gene expression level of AQP5 is carried out.

[0039] In another example, the protein expression level of AQP5 can be detected by methods such as, but not limited to, immunohistochemistry (IHC), flow cytometry, Western blot, and combinations thereof.In one example, the protein expression level of AQP5 is detected by immunohistochemistry.In another example, the expression level of AQP5 is gene expression level.Therefore, the gene expression level of AQP5 can be obtained or quantified, for example, by carrying out polymerase chain reaction (PCR).

[0040] Because Aqp5 is a membrane-bound protein, the sorting protocol performed here was adapted to perform antibody-based FACS isolation of Aqp5+ and Aqp5- human pyloric tumor epithelial cells from freshly collected patient tumor biopsies (Figure 1H).

[0041] Therefore, the method disclosed herein can also comprise isolating one or more identified gastric cancer stem cells.The method for isolating such cells includes but is not limited to single cell sorting, fluorescence activated cell sorting (FACS), magnetic sorting or a combination thereof.In one example, cells are isolated using fluorescence activated cell sorting (FACS).

[0042] In another example, a method for isolating one or more gastric cancer stem cells from a cell population is described, comprising the steps of: (i) contacting cells of the cell population with an agent that binds to AQP5; and (ii) isolating one or more AQP5-expressing cells bound to the agent, wherein the one or more AQP5-expressing cells are gastric cancer stem cells.

[0043] Examples of such agents that bind to AQP5 can be, but are not limited to, antibodies, drugs, small molecules, and combinations thereof. In one example, the agent that binds to AQP5 is an antibody. In one example, the agent that binds to AQP5 can affect the function of AQP5 upon binding. In another example, the antibody is a detection antibody. In another example, the antibody can be conjugated to a label, such as a detectable label. Examples of such detectable labels include, but are not limited to, fluorescent labels, cleavable labels, isolation labels, purification labels, and combinations thereof. In another example, the agent is conjugated to a compound, such as a drug or small molecule.

[0044] This FACS sorting strategy was validated by qPCR, which showed an average 10.8-fold upregulation of Aqp5 levels in the sorted Aqp5+ cell population (Figure 1I). Using this sorting approach, bulk RNAseq analysis was performed on Aqp5+ and Aqp5- human pyloric tumor cells isolated from five independent tumor samples (Figure 1J). Similar to the mouse Aqp5+ profile, human Aqp5+ tumor cells exhibited a transcriptome signature enriched for cancer stem cell-associated phenotypes, including ECM organization, EMT, and chemoresistance (Figure 7A, Figure 7B, Table 3). Selected upregulated targets, including CLDN2, HEY2, and PLA1A, were further validated by qPCR and RNAscope using additional independent patient samples (Figure 7C-Figure 7G, Table 4), confirming the reliability of the RNAseq dataset. Overall, mouse and human Aqp5+ pyloric tumor cells displayed a common cancer stem cell-enriched transcriptome profile that was distinct from the Aqp5+ healthy pyloric stem cell signature, thereby highlighting its therapeutic value.

[0045] Aqp5+ tumor cells function as cancer stem cells in mouse and human pyloric tumors Markers labeling healthy cells and cancer stem cells are known to be highly overlapping in many tumor contexts. However, many gastric cancer stem cell markers, including Lgr5, Cd44, and Cxcr4, have not been robustly validated using functional assays that directly demonstrate the stemness of the tumor cell populations they mark. Given that Aqp5 characterizes the healthy mouse and human pyloric stem cell compartment and that many reported cancer stem cell-associated pathway signatures are enriched in the transcriptomes of Aqp5+ tumor cells (Figures 5 and 7), we asked whether Aqp5 might also be a specific marker for pyloric cancer stem cell populations in mouse and human gastric tumors. To functionally test this hypothesis, Aqp5+ and Aqp5- epithelial tumor cells were isolated by FACS from Aqp5-APK mouse pyloric tumors and seeded for organoid culture. This demonstrated organoid formation by Aqp5+ tumor cells, which was maintained over the long term (at least 10 passages) (Figure 2A, Figure 2B). Furthermore, organoids derived from isolated Aqp5+ tumor cells contained multiple differentiated cell lineages and resembled organoids generated from whole pyloric tumor glands (Figure 2C, Figure 8A). In contrast, sorted Aqp5- tumor cells formed significantly fewer organoids in culture, which were rapidly lost within two passages (Figure 2A, Figure 2B). This reflects a lack of self-renewal capacity. Thus, Aqp5 was shown to characterize a functional cancer stem cell population within mouse pyloric tumors capable of long-term tumor organoid growth.

[0046] In one example, the method disclosed herein comprises further culturing the gastric cancer stem cell that is isolated under the presence of the culture medium that does not contain growth factor.In other words, in one example, the further culturing of the gastric cancer stem cell that is isolated is carried out under the condition that there is no growth factor in cell culture medium.In another example, the method disclosed herein comprises isolating gastric cancer stem cell, and the gastric stem cell that is isolated forms the tumor organoid that expresses AQP5.

[0047] In one example, the gastric cancer stem cells are mammalian cells, including, but not limited to, mouse and human cells.

[0048] Another approach to assessing cancer stem cell potential is to transplant specific cell populations into mice and assess their tumorigenic potential in vivo. As is known in the art, transplanting selected cell populations from solid tumors remains challenging, and the efficiency of tumorigenesis can vary significantly depending on the tissue type and recipient. Consistent with this observation, tumor cells isolated from Aqp5-Cre / APK tumors failed to form large tumors even after 4 months of orthotopic transplantation into the pylorus of immunodeficient mice (Figure 8B). To circumvent this issue, we transplanted Aqp5-Cre / APK pylorus tumor organoids into immunodeficient mice. Next, we obtained Aqp5+ and Aqp5- cells from orthotopic tumors sorted by FACS (Figures 8C-8E), as these cells were thought to be well adapted to the local tumor environment within their host. We demonstrated that sorted Aqp5+ and Aqp5- tumor epithelial cells from Aqp5-Cre / APK orthotopic tumors seeded new tumors in mice more efficiently than cells isolated from primary tumors, thereby providing a viable system for assessing their tumorigenic potential (Figure 8F). Across five independent pairs of mice examined, Aqp5+ tumor cells consistently produced orthotopic tumors that were larger in volume and exhibited more aggressive growth and invasive properties compared with Aqp5- tumor cells derived from the same tumor (Figure 2D, Figure 2E, Figure 8F). Furthermore, tumors derived from Aqp5+ cells were shown to contain both Aqp5+ and Aqp5- cells, including expression of multiple lineage markers (Figure 8F). This indicates that these Aqp5+ cancer stem cells have the ability to reconstitute diverse lineages within their original tumors. Finally, to assess the stemness potential of Aqp5+ cells in independent tumor contexts, we used constitutively active p53 R172HAqp5+ and Aqp5- tumor cells were isolated from a mouse model of gastric cancer incorporating the mutant (Figure 8G, Figure 8H). FACS isolation of these cells was verified by qPCR, which showed a 38-fold enrichment for Aqp5 in the sorted Aqp5+ population (Figure 8I, Figure 8J). In this cancer model, sorted Aqp5+ tumor cells also formed more organoids in vitro compared to Aqp5- cells derived from the same tumor (Figure 8K, Figure 8L), demonstrating that Aqp5+ cells can function as stem cells in this setting. Taken together, organoid generation and transplantation assays established murine Aqp5+ tumor cells as a functional gastric cancer stem cell population capable of promoting tumor growth. To assess whether human Aqp5+ tumor cells also function as cancer stem cells, isolated single Aqp5+ and Aqp5- human gastric tumor cells were isolated from primary tumor samples and plated for organoid culture. We found that human Aqp5+ tumor cells also generated tumor organoids at a higher rate than corresponding Aqp5- cells isolated from the same tumor (Figure 2F, Figure 2G). Organoids derived from Aqp5+ tumor cells exhibited long-term self-renewal (at least 10 passages) and gave rise to differentiated cell lineages similar to human organoids established from whole pyloric glands (Figure 2H, Figure 9A). This suggests that the Aqp5+ cell population retained the intrinsic stemness to repopulate its cell lineage and the characteristics of its original tumor. In contrast, a small proportion of the Aqp5- cell-derived organoids that formed failed to grow beyond an average of three passages (Figure 2F, Figure 2G).

[0049] As a further readout of the stem cell potential of human Aqp5+ tumor cell populations, we established human gastric tumor organoids with CRISPR / Cas9-mediated insertion of Aqp5-2A-CreERT2;CAG-LSL-tdTomato, which allows in vitro lineage tracking of human Aqp5+ cells and their progeny within the tumor organoids (Figure 9B). Administration of 4-OHT to these tumor organoid cultures labeled a small pool of Aqp5+ cells, which expanded to encompass an average of 51% of the organoid area after 10 days (Figure 9C-E), overlapping with differentiated gastric lineage markers (Figure 9F). In contrast, uninduced organoids did not exhibit tdTomato tracking in vitro over the same time points (Figure 9E). Thus, human Aqp5+ tumor cells from both primary patient samples and human organoid models were shown to function as stem cells directly contributing to the long-term maintenance of organoid cultures and the generation of multiple differentiated cell lineages.

[0050] Ablation of Aqp5+ tumor cells attenuates tumor progression Cancer stem cells have been implicated as a major cause of tumor recurrence because they can resist standard cancer therapies, for example, by activating drug efflux transporters. Therefore, targeting cancer stem cells has been proposed as a way to eliminate tumor burden and recurrence, but this has been hindered by the lack of well-validated and clinically relevant cancer stem cell markers. Given the functional characterization of murine and human Aqp5+ cells as a cancer stem cell population in pyloric tumors (Figure 2), we tested the effect of ablation of Aqp5+ cells in murine and human gastric tumor models as an independent functional assessment of their cancer stem cell identity. We also evaluated the efficacy of targeting the Aqp5+ cancer stem cell population as a therapeutic approach for gastric cancer.

[0051] To efficiently eliminate Aqp5+ tumor cells in mouse pyloric tumors, we integrated the Aqp5-2A-DTR allele into the Aqp5-Cre / APK pyloric cancer mouse model (Figure 10A). The Aqp5-2A-DTR cassette enabled endogenous expression of Aqp5 along with the diphtheria toxin receptor (DTR) in these Aqp5-expressing cells. Aqp5-Cre / APK / DTR tumors were indistinguishable from Aqp5-Cre / APK tumors in terms of tumor morphology, tumor burden, and the proportion of tumor-resident Aqp5+ cells (Figure 10B). To selectively target Aqp5+ pyloric tumor cells, we administered diphtheria toxin (DT) intraperitoneally to Aqp5-Cre / APK / DTR mice bearing pyloric tumors. 12 hours after the final DT administration, DT-treated tumors were observed to have significantly reduced in size (Figure 10C). We confirmed the decrease in Aqp5 expression in DT-treated tumors and the concomitant appearance of caspase 3+ apoptotic cells and Ki67+ proliferative cells in the viable tumor mass (Fig. 10D).

[0052] Thus, in one example, the methods disclosed herein further comprise eliminating or ablating gastric cancer stem cells, which is achieved using, but is not limited to, a compound selected from the group consisting of diphtheria toxin, an iCasp9 inducer (e.g., an agent that dimerizes iCasp9), and an agent that selectively binds to AQP5.

[0053] Diphtheria toxin (DT) was not shown to affect pyloric tumors in Aqp5-Cre / APK mice lacking the Aqp5-2A-DTR allele (Figure 10E) or in Aqp5-Cre / APK and Aqp5-Cre / APK / DTR mice injected with PBS without DT (Figure 10D, Figure 10E). Although administration of DT to Aqp5-Cre / APK / DTR mice effectively eliminated Aqp5+ pyloric tumor cells, Aqp5+ cells in other healthy tissues were also affected, leading to high mortality in these mice and precluding analysis of later tumor time points. To circumvent this issue, we generated tumor organoids from Aqp5-Cre / APK / DTR pyloric tumors, which maintained a small (9.2%) pool of cells expressing high levels of Aqp5 in culture (Figure 10F). Addition of DT to Aqp5-Cre / APK / DTR organoid cultures during the early stages of organoid growth was shown to eliminate the majority of Aqp5+ cells (with an 80-90% decrease in Aqp5 levels after 1 day of treatment) (Figure 10G), and also completely eliminated organoid growth across four independent lineages examined (Figures 3A and 3B). The effect of DT administration was also tested in established Aqp5-Cre / APK / DTR organoid cultures, resulting in a >90% decrease in Aqp5 levels (Figure 10G) and rapid degeneration of organoids within 1 day of treatment (no viable organoids remained after 4 days) (Figures 3C and 3D). In contrast, administering the same DT dose to Aqp5-Cre / APK organoids lacking the Aqp5-2A-DTR allele had no effect on Aqp5 expression, organoid growth, or subsequent organoid maintenance (Figures S10H-S10J), highlighting the specific effect of Aqp5+ cell depletion on the observed organoid phenotype.

[0054] Thus, in one example, elimination or removal is achieved using diphtheria toxin.

[0055] In yet another example, a method for eliminating or eliminating AQP5+ gastric cancer stem cells that have been modified to express the DTR gene or the inducible caspase 9 (iCasp9) gene is described, comprising contacting the cells with an inducer of DT or iCasp9. A non-exhaustive example of such an inducer of iCasp9 is an agent that dimerizes iCasp9.

[0056] Also described herein is a method for removing or eliminating AQP5+ gastric cancer stem cells, comprising contacting the cells with an agent that binds to AQP5, wherein binding of the AQP5+ cells to the agent removes or eliminates the cells.

[0057] To confirm these findings in a more physiological system, we established a method for orthotopically transplanting Aqp5-Cre / APK / DTR tumor organoids into the pylorus of immunodeficient mice to facilitate selective elimination of Aqp5+ cells in pyloric tumors that developed from these organoids. Because the Aqp5-2A-DTR allele is not expressed in other tissues in these mice, we were able to monitor tumor progression to more advanced stages after DT administration without the complications associated with systemic elimination of Aqp5+ cells. Orthotopic transplantation of Aqp5-Cre / APK / DTR pyloric organoids resulted in the development of pyloric tumors within 4 weeks (with signs of epithelial invasion evident by week 8) (Figure 3E, F). Intraperitoneal injection of DT into mice immediately after orthotopic implantation of Aqp5-Cre / APK / DTR tumor organoids resulted in a reduction in Aqp5-expressing cells (Figure 10K) and inhibited tumor growth even 4 weeks later (Figure 3E) across all five independent experiments. DT was also administered to established tumors in mice 4 weeks after orthotopic implantation of Aqp5-Cre / APK / DTR organoids, thereby verifying its tumor elimination efficiency (Figure 10L). In contrast to untreated tumors, tumors treated with DT showed reduced tumor burden and no signs of invasion into surrounding tissue layers (Figure 3F). Furthermore, orthotopic tumors derived from Aqp5-Cre / APK tumor organoids lacking the Aqp5-2A-DTR allele were not affected by DT administration at both early and late stages of tumor growth (Figure 10M, Figure 10N). Collectively, these results demonstrate a dependency on mouse Aqp5+ tumor cells to maintain tumor establishment and progression in both organoid and mouse models of pyloric cancer.

[0058] The effect of ablation of human Aqp5+ cells was tested in a near-physiological human gastric cancer organoid system. To this end, we generated human organoids expressing Aqp5-2A iCaspase, with Aqp5+ cells co-expressing inducible caspase 9 (iCaspase9), which promotes selective induction of caspase-mediated cell death in Aqp5+ cells upon administration of a dimerizing agent (Figure 3H, Figure 3I, and Figure 11C). The resulting Aqp5-2A iCaspase organoids exhibited comparable organoid morphology, Aqp5 expression, and cell lineage marker expression compared to unedited tumor organoids (Figure 11B).

[0059] Thus, in one example, a cell or cell population disclosed herein is modified to express AQP5 in combination with the diphtheria toxin receptor (DTR) gene or the inducible caspase 9 (iCasp9) gene.

[0060] As described herein, we generated an Aqp5-2A-iCaspase9 construct for intracellular expression. This means that cells expressing Aqp5 also produce iCaspase9. Addition of an inducer, such as AP20187 (a dimerizer), causes iCaspase9 to homodimerize to produce a functional protein, resulting in apoptosis specifically in Aqp5-expressing cells, while cells that do not express Aqp5 remain unharmed.

[0061] To eliminate human Aqp5+ cells, we administered a B / B homodimerizer to Aqp5-2AiCaspase human organoid cultures. This resulted in a 60% loss of Aqp5 expression in the treated organoids (Figure 11C). These Aqp5-2AiCaspase-treated tumor organoids remained viable but also showed a reduced growth rate in culture (Figure 3H, Figure 3I). Without being bound by theory, we attribute this to undepleted Aqp5+ cells remaining in these cultures. Unedited tumor organoids were not affected by treatment with the B / B homodimerizer, and no significant differences were observed in either Aqp5 levels or organoid size (Figure 11D). Depletion experiments were performed in an independent human gastric cancer organoid line, GC10, confirming the specific effect of depletion of Aqp5+ cells on organoid growth (Figure 11E-H). Similar to their mouse counterparts, human Aqp5+ cancer stem cells in tumor organoids were also shown to be important drivers of organoid growth, thereby highlighting their validity as therapeutic targets to drive cancer regression.

[0062] Cancer stem cell models link clinical observations of chemotherapy resistance and tumor recurrence to the activity of a dedicated stem cell pool in driving continued tumor growth, which can then be harnessed for therapeutic applications. While the cancer stem cell theory has been demonstrated in many hematological, brain, and colon cancers, the existence of such a stem cell pool in gastric tumors has not been robustly demonstrated through multiple stem cell activity assays using physiologically relevant mouse and human gastric cancer models. Herein, we demonstrate that both mouse and human pyloric tumors harbor a heterogeneous Aqp5+ cancer stem cell population that, when transplanted into the mouse stomach, can initiate long-term organoid cultures and regenerate invasive pyloric tumors. Targeted ablation of Aqp5+ cells was also shown to be sufficient to block pyloric tumor development and growth, demonstrating the central role these cells play in disease progression. These findings indicate that gastric tumors may be driven by an Aqp5+ stem cell population, thereby highlighting the potential use of Aqp5+ cancer stem cells as a therapeutic target for gastric cancer.

[0063] The use of Aqp5 as a membrane-bound, FACS-selectable gastric cancer stem cell marker is also contemplated within the scope of the present disclosure. This facilitates the specific isolation of these gastric cancer stem cells directly from native tumors, thereby enabling detailed studies of cancer stem cell biology and the mechanisms driving their tumorigenic behavior. This includes identifying pathways that promote cancer stem cell function in these Aqp5-expressing cells, as well as identifying other cancer stem cell-specific genes that represent targets that can be used to eliminate or disrupt the function of Aqp5-expressing cell populations.

[0064] Therefore, in one example, the method for detecting, isolating or identifying gastric cancer stem cells disclosed herein comprises detecting, isolating or identifying membrane-bound AQP5. In other words, in another example, the AQP5 disclosed herein is membrane-bound.

[0065] Therefore, the present disclosure also contemplates a method of treating gastric cancer in a subject in need thereof. In one example, a method of treating gastric cancer in a subject identified as having gastric cancer stem cells or gastric cancer can include administering to the subject a therapy or compound consistent with standard of care for gastric cancer. Examples of standard of care include capecitabine, Cyramza (ramucirumab), docetaxel, doxorubicin hydrochloride, Enhertu (fam-trastuzumab deruxtecan-nxki), 5-FU (fluorouracil injection), fam-trastuzumab deruxtecan-nxki, fluorouracil, Herceptin (trastuzumab), Keytruda (pembrolizumab), Lonsurf (trifluridine and tipiracil hydrochloride), mitomycin, These treatments may include, but are not limited to, nivolumab, Opdivo (nivolumab), pembrolizumab, ramucirumab, Taxotere (docetaxel), trastuzumab, trifluridine and tipiracil hydrochloride, Xeloda (capecitabine), resection / surgery / surgical resection, endoscopic mucosal resection, perioperative chemotherapy with or without radiation therapy, radiation therapy, postoperative (adjuvant) chemoradiotherapy, postoperative (adjuvant) chemotherapy, and combinations thereof. As will be appreciated by those skilled in the art, treatment may vary depending on the stage or progression of the disease once identified.

[0066] In one example, the method includes administering to a subject a therapeutically effective amount of one or more agents that eliminate or remove AQP5-expressing cells. In another example, the treatment includes curing gastric cancer, reducing the growth of gastric cancer, delaying the progression of gastric cancer, improving the subject's prognosis, or a combination thereof. In another example, the use of one or more agents that eliminate or remove AQP5-expressing cells in the manufacture of a medicament for treating gastric cancer is disclosed. In another example, one or more agents that eliminate or remove AQP5-expressing cells are disclosed for use in therapy. In a further example, one or more agents that eliminate or remove AQP5-expressing cells are disclosed for use in treating gastric cancer.

[0067] In one example, the subject is a human or a mouse. In another example, the subject is a human.

[0068] Also disclosed herein are one or more agents that eliminate or eliminate AQP5-expressing cells for use in therapy. In another example, disclosed are one or more agents that eliminate or eliminate AQP5-expressing cells for use in treating gastric cancer.

[0069] The methods disclosed herein can also be used to develop approaches to recognize, target, and eliminate Aqp5-expressing gastric cancer stem cells within tumor mass to inhibit or halt cancer progression. Detection of Aqp5-expressing gastric cancer cells within tumors can also aid in cancer diagnosis and serve as an indicator of disease stage / severity.

[0070] Also contemplated herein is a kit for identifying, isolating, eliminating or removing gastric cancer stem cells.In one example, the kit described herein comprises an agent that binds to AQP5 and instructions for use.In another example, the kit further comprises an antibody, such as a detection antibody.In another example, the antibody is conjugated to a compound, such as a detectable label, a drug, or a small molecule.In another example, the antibody binds to the intracellular domain or extracellular domain of AQP5 protein.

[0071] AQP5 has been identified as a key driver of gastric cancer progression across multiple near-physiological mouse and human models of intestinal and diffuse gastric cancer. In gastric cancer patients, AQP5 is widely overexpressed across multiple subtypes / stages of gastric tumors and their associated metastases compared with healthy gastric tissue, highlighting a potential cancer-specific role for Aqp5 in driving the tumorigenic state. Indeed, knockout of Aqp5 in our mouse and human gastric cancer models significantly reduced tumor burden and, in some cases, completely eliminated cancer development. Conversely, overexpression of AQP5 accelerates the development of tumor characteristics. Targeting Aqp5 expression and / or its downstream functions represents a promising new direction for the treatment of gastric cancer.

[0072] Gastric cancer is one of the leading causes of cancer-related deaths both globally and in Singapore, with a 5-year survival rate of less than 30%. Gastric cancer treatment is currently limited to traditional methods of chemotherapy, radiation therapy, and surgical resection, but these approaches remain ineffective in ameliorating cancer recurrence in many gastric cancer patients. Therefore, there is a need to evaluate novel therapeutic modalities, such as those targeting novel functional regulators of cancer initiation, progression, and metastasis.

[0073] Aqp5 was identified herein as a key driver of gastric cancer progression across multiple near-physiological mouse and human gastric cancer model systems. In gastric cancer patients, Aqp5 is frequently overexpressed in both intestinal and diffuse-type tumors compared to healthy gastric tissue, a phenotype that is also reflected in healthy and cancerous mouse gastric tissue (Figures 17 and 18). Using a panel of human gastric cancer cell lines, we demonstrate that both intestinal and diffuse-type cancer cell lines exhibit elevated levels of cell proliferation and migration in vitro when Aqp5 is overexpressed. Furthermore, after orthotopic implantation of these human gastric cancer cell lines into the pyloric submucosa of immunodeficient mice, Aqp5-overexpressing lines seed larger tumors exhibiting more aggressive histological features, including high levels of cell proliferation and invasion into adjacent tissues (Figure 19). Furthermore, we demonstrate that these tumor phenotypes can be recapitulated across independent human gastric cancer organoid lines. These 3D organoid models, in contrast to 2D cell line monolayer cultures, grow within an extracellular matrix-like environment that preserves morphological and molecular features resembling native epithelium. Human gastric cancer organoids xenografted into the pylorus of immunodeficient mice were able to seed pyloric tumors, and Aqp5-overexpressing organoids produced larger, more aggressive tumors that frequently invaded through the pyloric epithelium and outer muscularis. Thus, this study demonstrated that Aqp5 is a widespread driver of gastric cancer tumorigenesis across multiple gastric cancer types and models.

[0074] To further evaluate the function of Aqp5 and the potential for therapeutic strategies targeting Aqp5 expression, we established an Aqp5 knockout gastric cancer model and evaluated the effect of Aqp5 deficiency on gastric tumorigenesis (Figure 20).

[0075] Human gastric cancer cell lines lacking Aqp5 grew more slowly and had significantly impaired tumor dissemination ability when orthotopically transplanted into immunodeficient mice. Similarly, human gastric cancer organoids in which Aqp5 was knocked out via CRISPR / Cas9 developed tumors in mice that were significantly smaller and unable to invade into surrounding tissue layers. These findings confirm the role of Aqp5 in driving tumorigenesis and highlight the potential of Aqp5 ablation as a way to block tumor progression and potentially promote tumor regression.

[0076] Finally, we established an Aqp5 knockout mouse gastric cancer model in which targeted recombination of cancer-inducing alleles resulted in tumor formation selectively within the pyloric region of the stomach. Aqp5 knockout mice were phenotypically normal and showed no alterations in gastric function, but when tumorigenesis was induced, these mice developed smaller pyloric tumors. Furthermore, gastric cancer stem cells isolated from Aqp5 knockout tumors exhibited reduced stemness potential and generated fewer organoids in culture compared to their Aqp5 wild-type counterparts. Overall, Aqp5 has been shown to play a specific role in the context of gastric cancer, driving tumorigenic properties that promote disease initiation and progression. Therefore, targeting Aqp5 expression and / or regulators of its expression and function represents a promising therapeutic approach for gastric cancer patients.

[0077] Thus, in one example, a method is disclosed for inhibiting gastric cancer tumorigenesis or progression, the method comprising administering an AQP5 inhibitor to a subject. In another example, a method is disclosed for promoting gastric cancer regression, the method comprising administering an AQP5 inhibitor to a subject.

[0078] In one example, an AQP5 inhibitor is a compound that inhibits or blocks the expression and / or function of AQP5. Examples of AQP5 inhibitors include, but are not limited to, siRNA, RNAi, chimeric antigen receptors (CARs), and drugs.

[0079] In one example, the AQP5 inhibitor is an inhibitory RNA (RNAi). Examples of RNAi include, but are not limited to, siRNA, shRNA, and miRNA. In another example, the AQP5 inhibitor is an siRNA. In one example, the siRNA can be, but is not limited to, a pair of sense and antisense primers. In another example, the primers are TIFF2026508610000001.tif27150 and combinations thereof.

[0080] In one example, the drug is, but is not limited to, an enzyme inhibitor, a receptor antagonist, and a channel blocker.

[0081] A functional driver of gastric cancer progression was found to accelerate the development of tumorigenic features when overexpressed. In contrast, knocking out this driver is sufficient to inhibit gastric cancer progression and, in some circumstances, block cancer development. Therefore, targeting AQP5 expression and / or its functional regulators may serve as a therapeutic approach for the treatment of gastric cancer.

[0082] As shown herein, multiple Aqp5 knockout and Aqp5 overexpressing mouse and human gastric cancer cell lines, organoids, and xenograft mouse models have been generated. These models validate the function of Aqp5 across independent systems and multiple gastric cancer subtypes, highlighting the broad relevance of our findings to the majority of gastric cancer patients. Furthermore, without being bound by theory, the Aqp5 knockout gastric cancer genetic mouse model disclosed herein enables targeted induction of pyloric tumorigenesis in mice. This provides insight into the function of Aqp5 across the entire disease time spectrum, from onset to tumor spread within the native tissue context. Thus, the technology disclosed herein robustly demonstrates the function of Aqp5 in gastric tumorigenesis across independent, near-physiological models that capture the diversity of human gastric tumor types, highlighting the utility of targeting Aqp5 and / or regulators of Aqp5 expression and function as a therapeutic approach for the treatment of gastric cancer.

[0083] In one example, a method for monitoring the progression of gastric cancer in a subject is described, comprising: (a) measuring the expression level of AQP5 in a sample obtained from the subject after treatment for gastric cancer; and (b) measuring the expression level of AQP5 in a control sample obtained from the subject before treatment for gastric cancer, wherein an increase in the expression level of AQP5 in the sample of step (a) compared to the control sample indicates that gastric cancer tumorigenesis has occurred or that the gastric cancer has progressed.

[0084] In another example, a method for monitoring gastric cancer tumorigenesis in a subject is described, comprising: (c) measuring the expression level of AQP5 in a sample obtained from the subject; and (d) measuring the expression level of AQP5 in a reference sample obtained from the subject at a time earlier than the sample of step c, wherein an increase in the expression level of AQP5 in the sample of step c compared to the reference sample of step d indicates that gastric cancer tumorigenesis has occurred.

[0085] In one example, the expression level of AQP5 is gene expression level, protein expression level, or a combination thereof.In another example, the method described herein further comprises measuring the expression level of one or more gastric cancer progression markers.In another example, in addition to other expression levels disclosed herein, the expression level of one or more gastric cancer progression markers is measured.Examples of gastric cancer progression markers can be, but are not limited to, Pthlh, Hey1, Rgs5, and combinations thereof.

[0086] In one example, if gastric cancer tumorigenesis occurs or if gastric cancer progresses, the subject is treated with an anti-gastric cancer compound or standard of care for gastric cancer.

[0087] Those skilled in the art will be able to identify which method can be used to measure the level disclosed herein.In one example, measuring the expression level of AQP5 is by immunohistochemistry, flow cytometry, Western blot or a combination thereof.In another example, measuring the expression level of AQP5 is by polymerase chain reaction.

[0088] In one example, expression level is measured in sample.The example of this sample includes but is not limited to blood, plasma, biopsy sample, tissue sample, primary cell culture sample and primary organoid line.

[0089] The method disclosed herein relates to gastric cancer. Gastric cancer, also known as stomach cancer, is cancer that occurs in the gastric mucosa. Gastric cancer can include, but is not limited to, adenocarcinoma, lymphoma, and mesenchymal tumor. In one example, gastric cancer is, but is not limited to, intestinal gastric cancer or diffuse gastric cancer.

[0090] Identifying specific genes that are expressed only in tumors and completely absent in normal tissues is challenging. Indeed, AQP5 is not only expressed in gastric tumors but also in a small proportion of healthy tissues, including lung, salivary gland, and testis. In many of these healthy tissues, including lung, AQP5 levels are minimal / low and primarily cytoplasmic. Furthermore, mice lacking AQP5 (e.g., complete knockout mice) are viable and healthy, and only minor defects in saliva secretion have been documented in these animals, suggesting that AQP5 may not play a significant role outside of cancerous conditions. Life-threatening consequences from targeted approaches to eliminate AQP5 or its regulators are not expected, and therefore, an appropriate therapeutic window can be tailored to ensure effective targeting of AQP5-expressing gastric tumor cells while minimizing damage to other AQP-expressing healthy tissues.

[0091] This technology will facilitate the development of approaches to recognize, target, and eliminate AQP5 and / or regulators of AQP5 expression and function in gastric tumors as a means of inhibiting cancer progression. Furthermore, detecting elevated AQP5 levels in gastric tumors can serve as an indicator of disease stage / severity and aid in cancer diagnosis. The series of in vitro and in vivo gastric cancer models and AQP5 knockout gastric cancer genetic mouse models generated herein can also be used in drug testing efforts to evaluate new therapies aimed at ameliorating disease progression. Finally, this technology will enable the identification of other protein partners of AQP5 and immediately downstream pathways controlled by AQP5 that drive tumor progression, potentially expanding the list of targetable components and the range of therapeutic approaches available to gastric cancer patients.

[0092] Experimental data presented in Figures 16-20 using multiple near-physiological gastric cancer models clearly demonstrate the central role AQP5 plays in promoting gastric cancer. AQP5 knockout gastric cancer cells have been shown to be less proliferative and form smaller, less aggressive tumors in vivo. Conversely, overexpression of AQP5 in gastric cancer cells increases cell proliferation and migration in vitro and promotes tumor progression in vivo. Finally, using the AQP5 knockout gastric cancer mouse model disclosed herein, we show that in the complete absence of AQP5, mice develop smaller precancerous neoplastic lesions rather than the fully developed gastric adenocarcinomas typically observed in such cancer mouse models.

[0093] We generated a previously unknown human gastric cancer organoid line incorporating the Flip-Puro system, which allows conditional knockout of AQP5 upon administration of Cre recombinase gesicles. The loss of AQP5 in these organoids was confirmed by Western blotting. As shown in Figure 21, we found reduced cell viability of organoids after AQP5 knockout.

[0094] Aqp5 drives tumor progression in mouse and human gastric cancer models We developed an Aqp5 knockout pyloric tumor mouse model (Aqp5KO-APK) by incorporating an Aqp5 null allele alongside an Aqp5-eGFP-IRES-creERT2 cassette (resulting in the inactivation of both copies of Aqp5). Before tumor induction, Aqp5 null mice were healthy and indistinguishable from their Aqp5 wild-type counterparts. After tamoxifen administration, these Aqp5KO-APK mice developed pyloric tumors in which Aqp5 expression was completely absent (Figures 16A and 18A). Importantly, these Aqp5 null tumors were significantly smaller in volume and also displayed precancerous features of intraepithelial neoplasia, whereas wild-type tumors were classified as adenocarcinomas by the same time point (Figure 16A), indicating that Aqp5 may play a role in driving tumor progression in vivo. To recapitulate these findings in vitro, we confirmed that Aqp5-null mouse antrum tumor organoids derived from Aqp5KO-APK tumors also failed to express Aqp5 (Figure 18B). Consistent with Aqp5 knockout in native tumors, Aqp5KO tumor organoids formed tumors with a less invasive phenotype after orthotopic transplantation (Figure 18C), highlighting the role of Aqp5 in driving tumor progression in this setting.

[0095] To further analyze the function of Aqp5 in the stem cell compartment of Aqp5KO-APK pyloric tumors, we isolated putative cancer stem cells (GFP+) and the remaining tumor epithelium (GFP-) from both Aqp5 knockout and Aqp5 wild-type mouse pyloric tumors for bulk RNA sequencing using a GFP marker driven by the Aqp5 promoter (Figure 18D, Figure 18E). This yielded a cancer stem cell signature that included genes common to both Aqp5 wild-type and knockout tumors (presumably genes unaffected by Aqp5 levels), as well as differentially expressed genes unique to Aqp5 wild-type and knockout tumors (Figure 18F). Pathway analysis showed that genes unique to Aqp5 wild-type cancer stem cells were enriched in ECM organization and G protein signaling pathways (Figure 16B, Figure 18G). These profiling studies identified pathways that may function downstream of Aqp5 within the cancer stem cell compartment in driving tumor progression. Next, we investigated the function of Aqp5 in human gastric cancer organoids, which are more cellularly complex and more similar to primary tumors than cancer cell lines. We identified four independent Aqp5 knockout organoid clones and validated the loss of Aqp5 expression in these lines (Figure 16C). All four Aqp5-null organoid lines reproducibly exhibited reduced proliferation rates in vitro compared with the Aqp5 wild-type parental organoid line (Figure 16D) and produced smaller, often less invasive, tumors after orthotopic transplantation into mice (Figure 16E, Figure 16F). Conversely, Aqp5 overexpression was sufficient to increase cell proliferation in two additional human gastric cancer organoid lines in vitro (Figure 19A, B), but no significant increase was observed when Aqp5 was overexpressed in organoid lines that already expressed high levels of Aqp5 (Figure 19C), suggesting that the Aqp5-induced phenotype may be dose-dependent up to a certain threshold.After orthotopic transplantation, human organoids overexpressing Aqp5 produced larger and more invasive tumors in vivo (Figures 19D, 19E), but these differences in tumors were again negligible in lines that already expressed high levels of Aqp5 (Figure 19F).

[0096] Finally, to further analyze the mechanisms driving Aqp5 function in human gastric cancer, we generated human organoids amenable to conditional knockout of Aqp5 (Figure 1G) based on the FLIP-Puro system. Administration of the Cre recombinase gesicle altered exon splicing, generating a truncated, nonfunctional Aqp5 product (Figure 1G). Cre-Gesicle-treated human organoids no longer expressed Aqp5 and exhibited slower growth in culture (Figures 1H, 16I, and 19H), consistent with the phenotype observed in constitutive Aqp5 KO human organoid lines (Figures 1C, 16D). Next, we performed bulk RNA sequencing on Aqp5 FLIP-Puro organoids at several time points immediately following Cre recombination, and performed pathway analysis on the resulting transcriptome dataset. This revealed numerous pathways activated early in organoid growth related to Wnt signaling that were abolished immediately after Aqp5 knockout (Figure 22J), highlighting a potential mechanism of action for Aqp5 function in gastric cancer.

[0097] The invention illustratively described herein may suitably be practiced in the absence of any element or elements, limitations, or limitations not specifically disclosed herein. Thus, for example, terms such as "comprising," "including," and "containing" are to be interpreted expansively and without limitation. Furthermore, the terms and expressions employed herein are used as terms of description and not of limitation, and the use of such terms and expressions is not intended to exclude any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention as claimed. Thus, while the invention has been specifically disclosed by preferred embodiments and optional features, it should be understood that modifications and variations of the invention disclosed and embodied herein may be employed by those skilled in the art, and that such modifications and variations are considered to be within the scope of the invention.

[0098] As used in this application, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a genetic marker" includes a plurality of genetic markers, including mixtures and combinations thereof.

[0099] As used herein, the term "about," in the context of concentrations of components of a formulation, typically means ±5% of the stated value, more typically ±4% of the stated value, more typically ±3% of the stated value, more typically ±2% of the stated value, even more typically ±1% of the stated value, and even more typically ±0.5% of the stated value.

[0100] Throughout this disclosure, certain embodiments may be disclosed in a range format. It should be understood that the description in range format is merely for convenience and conciseness and should not be construed as an inflexible limitation on the disclosed range. Thus, the description of a range should be considered to have specifically disclosed not only each individual numerical value within that range, but also all possible subranges. For example, the description of a range such as 1 to 6 should be considered to have specifically disclosed subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., and each individual numerical value within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0101] Certain embodiments may be described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of this disclosure. This includes generic descriptions of embodiments with a proviso or negative limitation excluding any subject matter from the genus, whether or not the excluded matter is specifically set forth herein.

[0102] The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also constitutes part of the invention. This includes a generic description of the invention with a proviso or negative limitation excluding any subject matter from that genus, whether or not the excluded matter is specifically set forth herein.

[0103] Other embodiments are within the scope of the following claims and non-limiting examples. Furthermore, where features or aspects of the invention are described in terms of a Markush group, those skilled in the art will recognize that the invention is also being described thereby in terms of individual members or subgroups of members of the Markush group. [Example]

[0104] Experimental Section Mouse studies Aqp5-eGFP-ires-creERT2, Aqp5-2A-creERT2, and Aqp5-2A-DTR mice were generated previously. LSL (Ai14)(JAX 007914), Apc fl / fl (MGI 1857966), Pten fl / fl (MGI 2182005), Kras LSL-G12D (JAX 019104), and p53 R172H Mice containing AQP5 have also been described previously. AQP5 knockout (KO) mice were obtained from Cyagen (KOCMP-11830-Aqp5-B6N-VA). For induction of Cre recombination, 8-week-old mice were intraperitoneally injected with 4 mg of tamoxifen (Merck T5648) dissolved in sunflower oil per 30 g of mouse body weight. Diphtheria toxin (DT, Sigma D0564) was administered intraperitoneally at a dose of 0.5 μg of DT dissolved in PBS per 30 g of mouse body weight.

[0105] Orthotopic transplantation of NOD.Cg-Prkdc scid Il2rg tm1Wjl This study was performed on 1 / SzJ(NSG) mice (JAX 005557). Briefly, mice were anesthetized, and the stomach was exposed under sterile surgical conditions. Injections were performed using an insulin syringe containing the transplant material (cells or organoids) inserted into the pyloric region just below the muscularis externa. After the procedure, the wound was sutured, and the mice were administered the reversal drug atipamezole along with buprenorphine for several days after surgery.

[0106] All mouse experiments were conducted in accordance with ethical and safety regulations set by the Institutional Animal Care and Use Committee (IACUC) and A*STAR. Mice were sacrificed immediately when tumors reached the maximum allowable size of 20 mm.

[0107] Human samples Patient pyloric tumor biopsies were provided by Dr. J. So of the Department of Gastroenterology at the National University Hospital of Singapore (IRB protocol 2020-038). Informed consent was obtained from all patients. Tumor samples were collected in Advanced DMEM / F-12 medium (Invitrogen 12634-028) containing 2 mM Glutamax (Invitrogen 35050-079), 10 mM HEPES (Invitrogen 15630-056), 1 mM N-acetylcysteine ​​(Sigma A9165), and 1x antibiotic-antimycotic (Gibco 15240096). Samples were vigorously washed with HBSS before proceeding to tissue dissociation. All experiments involving human material were conducted in accordance with the ethical and safety regulations set forth by A*STAR's Institutional Review Board (IRB).

[0108] Tissue dissociation for FACS Dissociation of peripyloric tumors was performed by incubating minced tissue in Advanced DMEM / F-12 medium (Invitrogen 12634-028) containing 2 mM Glutamax (Invitrogen 35050-079), 10 mM HEPES (Invitrogen 15630-056), 2 mg / ml bovine serum albumin (BSA), and 1 mg / ml type I collagenase (Life Technologies 17100017) for 45 minutes at 37°C. To dissociate gastric glands, the minced tissue was vigorously and repeatedly pipetted with cold Advanced DMEM / F-12 buffer, filtered through a 100 μm filter mesh, and then spun down at 2500 rpm for 3 minutes at 4°C. The resulting pellet was further dissociated into a single cell suspension by incubation with TrypLE (Gibco 12604) and 10 mg / ml DNase I (Sigma D4513) for 10 min at 37° C. The digestion reaction was stopped with 30 ml of cold HBSS, and the suspension was centrifuged at 2500 rpm for 3 min at 4° C.

[0109] For mouse pyloric tumors containing eGFP-tagged AQP5, cell pellets were directly resuspended in 2% fetal bovine serum (FBS) / HBSS and passed through a 40-μm filter for cell sorting on a BD Influx cell sorter (BD Biosciences).For human pyloric tumors, single-cell suspensions after TrypLE digestion were incubated with AQP5-AF647 (1:500, Abcam ab215225), CD11b-FITC (1:200, Biolegend 101205), CD31-FITC (1:200, Biolegend 303103), and CD45-FITC (1:200, Biolegend 304054) in 2% FBS / HBSS for 45 min at 4°C.

[0110] Cells were washed twice with 2% FBS / HBSS before filtering through a 40 μm filter for FACS. Cells were resuspended in DAPI for live / dead cell gating prior to FACS. Sorted cells were collected directly into RLT Plus Buffer (Qiagen) supplemented with b-mercaptoethanol for RNA isolation or into Advanced DMEM / F-12 supplemented with 0.5% Matrigel (Corning 354253) for organoid culture.

[0111] Organoid culture Pyloric tumor organoids were grown in basal medium containing Advanced DMEM / F-12 (Invitrogen 12634-028) supplemented with 2 mM Glutamax (Invitrogen 35050-079), 10 mM HEPES (Invitrogen 15630-056), 1x N2 (Invitrogen 17502-048), 1x B27 (Invitrogen 17504-044), 1 mM N-acetylcysteine ​​(Sigma A9165), and 200 μg / ml Primocin (InvivoGen Ant-pm-1). For mouse pyloric tumor organoid cultures, the organoids were supplemented with recombinant growth factors at the following concentrations at the first passage after plating: 50 ng / ml EGF (Invitrogen PMG8043), 100 ng / ml FGF10 (Peprotech 100-26), 10 nM GAST (Sigma G9145), 100 ng / ml WNT3A (Peprotech 315-20), 1 μg / ml RSPO1 (Peprotech 120-38), and 100 ng / ml NOGGIN (Peprotech 250-38). After establishment, the cultures were switched to basal medium for long-term maintenance and tumor organoid selection. Human gastric cancer organoids HCM-BROD-0045-C16, HCM-BROD-0116-C16, and HCM-BROD-0235-C16 were purchased from ATCC. Human organoid cultures were supplemented with the above recombinant growth factors along with 2 μM A-8301 (Tocris 2939) and 10 mM nicotinamide (Sigma N0636) in a basal medium consisting of 50% WRNF-conditioned medium. Single-cell cultures were also supplemented with 10 μM Y-27632 (Tocris 1254). Approximately once per week, organoids were passaged upon confluence by dissociating them using TrypLE (Gibco 12604) and maintained in growth factor-reduced Matrigel basement membrane matrix (Corning 356231).For experiments involving orthotopic transplantation of organoids into mice, intact organoids were recovered from the Matrigel using Cell Recovery Solution (Corning 354253).

[0112] To generate gene-edited human gastric cancer organoids, the following plasmids were electroporated: HR110PA-1 hAQP5-5'arm-P2A-iCas9-IRES-Venus-3'arm, pX330 hAQP5 gRNA-1, and pX330 hAQP5 gRNA-2 to generate Aqp5-2A-iCaspase organoids. HR110PA-1 hAQP5-5'arm-CreERT2-pA-3'arm, pX330 hAQP5 gRNA-1, and pX330 hAQP5 gRNA-2 were electroporated, followed by AAVS1-2A-Blasticidin-CAG-LSL-tdTomato and pX330 AAVS1 gRNA to generate Aqp5-2A-CreERT2;LSL-tdTomato organoids. The pX330 plasmid was derived from pX330-U6-Chimeric_BB-CBh-hSpCas9 (Addgene plasmid #42230, from Feng Zhang). Organoid electroporation was performed using a NEPA21 electroporator (NEPA GENE) according to the manufacturer's recommendations. Briefly, 1 day before electroporation, 5 μM CHIR99021 (Stemgent 04-0004-10) and 10 μM Y-27632 (Tocris 1254) were added to the organoids. Organoids were dissociated using TrypLE (Gibco 12604) and washed with Opti-MEM I serum-reduced medium (Life Technologies 31985-062). The cell pellet was resuspended using a BTXpress (BTX Harvard Apparatus 45-0805) and 15 μg of each plasmid DNA per 100,000 cells, then loaded into an electroporation cuvette with a 175 V poration pulse of 5 ms duration.Successful transfectants were selected with puromycin (InvivoGen Ant-pr-1), with the exception of AAVS1-2A-Blasticidin-CAG-LSL-tdTomato organoids, which were selected with blasticidin (InvivoGen Ant-bl-1). After transfection of the Aqp5-2A-iCaspase and Aqp5-2ACreERT2 constructs, organoids were further treated with Cre recombinase Gesicle (Clontech 631449) to remove the selection cassette. Briefly, organoids were dissociated with TrypLE (Gibco 12604) and washed with 10% FBS / Advanced DMEM / F12 (Invitrogen 12634-028). Cre recombinase Gesicle (Clontech 631449) and 6 μg / ml polybrene (Sigma H9268) were added, and the suspension was centrifuged at 600 g for 1 hour at 32°C. Cells were incubated at 37°C for 6 hours before being transferred to Matrigel. After Cre Gesicle treatment, cells were grown in puromycin-free organoid medium for 1 week, and RFP-negative cells were collected by cell sorting using a BD Influx cell sorter (BD Biosciences).

[0113] To induce depletion of Aqp5+ cells, organoids were treated with 500 nM B / B homodimerizer (Clontech 635059) for Aqp5-2A-iCaspase organoids and 240 ng / ml DT for Aqp5-2A-DTR organoids. To perform lineage tracing of Aqp5+ cells in Aqp5-2A-CreERT2;LSL-tdTomato organoids, 1 μM 4-OHT was administered to organoids for 16 hours and then removed. For conditional Aqp5 KO in Aqp5-FLIP-Puro organoids, organoids were dissociated with TrypLE (Gibco 12604) and washed with 10% FBS / Advanced DMEM / F12 (Invitrogen 12634-028). Cre recombinase Gesicle (Clontech 631449) and 6 μg / ml polybrene (Sigma H9268) were added, and the suspension was centrifuged at 600 g for 1 hour at 32°C. Cells were incubated at 37°C for 6 hours before being transferred to Matrigel. After Cre Gesicle treatment, cells were grown in organoid medium without puromycin.

[0114] For conditional Aqp5 KO in Aqp5-FLIP-Puro organoids, organoids were dissociated using TrypLE (Gibco 12604) and washed with 10% FBS / Advanced DMEM / F12 (Invitrogen 12634-028). Cre recombinase Gesicle (Clontech 631449) and 6 μg / ml polybrene (Sigma H9268) were added, and the suspension was centrifuged at 600 g for 1 hour at 32°C. Cells were incubated at 37°C for 6 hours before being transferred to Matrigel. After Cre Gesicle treatment, cells were grown in organoid medium without puromycin.

[0115] To measure cell proliferation in organoids, equal numbers of cells were seeded in Matrigel in 96-well plates and allowed to grow for 3 days, followed by 2-hour incubation with the CellTiter AQueous One Solution Assay (Promega G3582). Cell proliferation was measured by measuring absorbance at 490 nm using a plate reader. Proliferation was also measured using the Click-iT EdU Cell Proliferation Kit with Alexa Fluor 594 (Life Technologies C10339) according to the manufacturer's recommendations. Whole-mount organoids were imaged using an Evos M5000 (Thermofisher).

[0116] Cell line culture All tissues were processed according to standard protocols. Briefly, fresh tissues were fixed overnight in 4% paraformaldehyde at 4°C, dehydrated, and processed into paraffin blocks. 8 μm tissue sections collected on glass slides were deparaffinized and rehydrated. For hematoxylin and eosin (H&E), FFPE sections were stained with Richard Allan hematoxylin and differentially stained with acid alcohol (1% HCl in 70% alcohol), followed by counterstaining with Scott Blue and eosin. For immunohistochemistry (IHC) and immunofluorescence (IF), antigen retrieval was performed on rehydrated slides in citric acid pH 6.1 (Dako, S169984) or pH 9.0 (Dako S236784) target retrieval solution at 121°C in a pressure cooker. The primary antibodies used were rabbit anti-AQP5 (Life Technologies PA564195), mouse anti-E-cadherin (1:200, BD Biosciences 610181), rabbit anti-KI67 (1:200, Thermofisher MA5-14520), mouse anti-RFP (1:200, Abcam 129244), and rabbit anti-vimentin (1:500, Abcam ab92547). The secondary antibodies used were mouse or rabbit EnVision+ (DAKO) for IHC and anti-mouse or anti-rabbit Alexa Fluor 488, 568, or 647 (1:500, Invitrogen) for IF.

[0117] RNA isolation and qPCR Cell lysis for RNA extraction was performed using RLT Plus buffer (Qiagen) containing β-mercaptoethanol. RNA was purified from cell extracts using the RNeasy Mini or Micro kit (Qiagen) and used for cDNA synthesis with the Superscript III kit (Life Technologies) according to the manufacturer's instructions. qPCR was performed in triplicate using GoTaq qPCR Master Mix (Promega A6002). qPCR reactions were run on a QuantStudio 7 Flex Real-Time PCR System (Applied Biosystems) and analyzed using the ΔΔCt method. For qPCR validation of RNA sequencing targets, cDNA amplification was performed using an Ovation Pico WTA system (NuGen 3302-60) according to the manufacturer's instructions to generate sufficient material for validation of a large number of targets. All qPCR primer sequences used are summarized in Tables 2 and 4.

[0118] Transcriptome profiling For bulk RNA sequencing, cells were collected directly into RLT Plus buffer (Qiagen) containing b-mercaptoethanol during FACS sorting. Total RNA was extracted using the RNeasy Micro Kit (Qiagen). Before proceeding to downstream library preparation methods, RNA integrity of all samples was confirmed using an Agilent RNA 6000 Picochip (Agilent 5067-1513) and run on an Agilent 2100 Bioanalyzer. qPCR was also performed to confirm that Aqp5 levels were enriched for Aqp5+ samples compared with the corresponding sorted Aqp5- samples. Due to the low amount of material available from sorted human cells, RNA from human samples was first amplified using the SMARTer Ultra Low RNA Kit (Clontech 634936) prior to library construction and sequencing. For library construction, mRNA was enriched using oligo(dT) beads, and double-stranded cDNA libraries were generated according to the manufacturer's instructions. The libraries were sequenced using NovaSeq PE150 (Illumina), and Illumina real-time analysis software was used for base calling to obtain FASTQ files.

[0119] Histological examination and staining methods All tissues were processed according to standard protocols. Briefly, fresh tissues were fixed overnight in 4% paraformaldehyde at 4°C, dehydrated, and processed into paraffin blocks. Eight-micrometer tissue sections collected on glass slides were deparaffinized and rehydrated. For hematoxylin and eosin (H&E), formalin-fixed, paraffin-embedded (FFPE) sections were stained with Richard Allan hematoxylin and differentially stained with acid alcohol (1% HCl in 70% alcohol), followed by counterstaining with Scott Blue and eosin. For immunohistochemistry (IHC) and immunofluorescence (IF), antigen retrieval was performed on rehydrated slides in citric acid pH 6.1 (Dako, S169984) or pH 9.0 (Dako S236784) antigen retrieval solution at 121°C in a pressure cooker. The primary antibodies used were rabbit anti-AQP5 (Life Technologies PA564195), mouse anti-CHGA (1:200, Abcam 15160), mouse anti-E-cadherin (1:200, BD Biosciences 610181), rabbit anti-KI67 (1:200, Thermofisher MA5-14520), mouse anti-MUC5AC (1:200, Leica Biosystems NCL-HGM-45-M1), mouse anti-RFP (1:200, Abcam 129244), anti-TFF2, and rabbit anti-vimentin (1:500, Abcam ab92547). Secondary antibodies used were mouse or rabbit EnVision+ (DAKO) for IHC and anti-mouse or anti-rabbit Alexa Fluor 488, 568, or 647 (1:500, Invitrogen) for IF.

[0120] For in situ hybridization (ISH) experiments, tissues were collected under RNase-free conditions and fixed in 4% paraformaldehyde for 16–24 h at room temperature. RNAscope was performed using the RNAscope 2.5 High Definition Brown Assay (ACDbio 322300) and the 2.5 High Definition Duplex Reagent Assay according to the manufacturer's instructions. The following mouse probes were used: Mm-Aqp5 (ACDbio 430021), Mm-Lgr5 (ACDbio 312171), Mm-Rgs5 (ACDbio 430181), Mm-Pthlh (ACDbio 456521), Mm-Hey1 (ACDbio 319021), Mm-Cd44 (ACDbio 476201), Mm-Cxcr4 (ACDbio 425901), positive control probe Ms-PPIB (ACDbio 313911), and negative control probe DapB (ACDbio 310043). The following human probes were used: Hs-CLDN2 (ACDbio 492051), Hs-HEY2 (ACDbio 441761), Hs-PLA1A (ACDbio 536951), Hs-DCHS2 (ACDbio 1309261), and Hs-AQP5 (ACDbio 452371).

[0121] Microscopy For image acquisition, H&E and IHC slides were captured using a Nikon Ni-E microscope with a DS-Ri2 camera. IF images were acquired using a Zeiss LSM780 laser scanning confocal microscope. Bright-field and fluorescent images of organoids were captured using a Thermofisher Evos M5000 system. Large-area images acquired with the Nikon Ni-E microscope were processed with NIS-Elements AR software (Nikon), and images acquired with the Thermofisher Evos M5000 microscope were stitched together using Adobe Photoshop. All images were processed using Fiji and Adobe Photoshop.

[0122] statistical analysis All statistical analyses were performed in GraphPad Prism. Qualitative data sets were analyzed using Fisher's exact test. Quantitative data sets were first assessed for normality using the Shapiro-Wilk test. Normal distribution variables were analyzed by an unpaired two-tailed Student's t-test for two groups and ANOVA with Dunnett's multiple comparison test for more than two groups, whereas non-normal distribution variables were analyzed using an unpaired two-tailed Mann-Whitney U test for two groups and Kruskal-Wallis test with Dunn's multiple comparison test for more than two groups. Reproducibility was confirmed by at least three independent experiments.

[0123] table Table 1. Top upregulated genes in mouse Aqp5+ pyloric tumor cells compared to Aqp5- cells. TIFF2026508610000002.tif145144TIFF2026508610000003.tif212144TIFF2026508610000004.tif211144 TIFF2026508610000005.tif211144TIFF2026508610000006.tif212144TIFF2026508610000007.tif164144

[0124] Table 2. qPCR primer sequences for target validation in mice TIFF2026508610000008.tif174144

[0125] Table 3. Top upregulated genes in human Aqp5+ pyloric tumor cells compared to Aqp5- cells. TIFF2026508610000009.tif67144TIFF2026508610000010.tif218144TIFF2026508610000011.tif212144TIFF20265086100 00012.tif212144TIFF2026508610000013.tif212144TIFF2026508610000014.tif218144TIFF2026508610000015.tif61144

[0126] Table 4. qPCR primer sequences for human target validation TIFF2026508610000016.tif126145

[0127] Array Table TIFF2026508610000017.tif210146TIFF2026508610000018.tif209146TIFF2026508610000019.tif228146

Claims

1. 1. A method for identifying a gastric cancer stem cell or a gastric cancer stem cell population, comprising: a1) detecting the expression of aquaporin 5 (AQP5) in a cell or cell population; or b1) detecting the expression level of AQP5 in a cell or cell population and comparing said expression level with the expression level of AQP5 in a reference cell or reference cell population Including, Detection of AQP5 expression in the cell or cell population, or expression of AQP5 at elevated levels in the cell or cell population compared to the reference cell or reference cell population, identifies the cell or cell population as a gastric cancer stem cell or gastric cancer stem cell population. method.

2. The method of claim 1, wherein the cell or cell population is an in vitro, in vivo, or ex vivo cell or cell population.

3. The method of claim 1 or 2, wherein the cell or cell population is a gastric tumor sample, a biopsy, or an organoid.

4. 4. The method of claim 3, wherein the cells are epithelial cells or the cell population comprises epithelial cells from a stomach tumor sample or biopsy.

5. 5. The method of any one of claims 1 to 4, wherein the cell or cell population is modified to express AQP5 in combination with one or more detectable labels.

6. 6. The method of claim 5, wherein the one or more detectable labels are fluorescent labels.

7. The method of any one of claims 1 to 6, wherein the cell or cell population is further modified to express AQP5 in combination with an inducible gene.

8. The method of claim 7, wherein the inducible gene is CreERT2.

9. 9. The method of any one of claims 1 to 8, wherein the cell or cell population is modified to express AQP5 in combination with the diphtheria toxin receptor (DTR) gene or the inducible caspase 9 (iCasp9) gene.

10. The method of any one of claims 1 to 9, wherein the expression level of AQP5 is a gene expression level, a protein expression level, or a combination thereof.

11. The method of any one of claims 1 to 10, further comprising the step of isolating the identified gastric cancer stem cells.

12. The method of claim 11, wherein the isolated gastric cancer stem cells are further cultured in the presence of a culture medium that does not contain growth factors.

13. 13. The method of claim 12, wherein the isolated gastric cancer stem cells form AQP5-expressing tumor organoids.

14. The method of any one of claims 1 to 13, further comprising a step of eliminating or removing the gastric cancer stem cells.

15. 15. The method of claim 14, wherein the gastric cancer stem cells are eliminated or removed using a compound selected from the group consisting of diphtheria toxin, an inducer of iCasp9, and an agent that selectively binds to AQP5.

16. 16. The method of any one of claims 1 to 15, wherein the reference cell or reference cell population is a cell that does not express AQP5, and optionally, the cell that does not express AQP5 is a non-gastric cell, a non-cancerous gastric cell, or a combination thereof.

17. 17. The method of any one of claims 1 to 16, wherein the identification of gastric cancer stem cells results in the subject being treated with a standard of care for gastric cancer.

18. 1. A method for isolating one or more gastric cancer stem cells from a population of cells, comprising: i) contacting cells of the cell population with an agent that binds to AQP5; ii) isolating one or more AQP5-expressing cells bound to the agent, wherein the one or more AQP5-expressing cells are gastric cancer stem cells. Including, a method.

19. The method of claim 18, wherein the agent that binds to AQP5 is an antibody.

20. 20. The method of claim 19, wherein the antibody is conjugated to a label, such as a detectable label.

21. The method of any one of claims 18 to 20, wherein the one or more gastric cancer stem cells are isolated using a method selected from the group consisting of single-cell sorting, fluorescence-activated cell sorting, and magnetic sorting.

22. The method of any one of claims 18 to 21, wherein the isolated gastric cancer stem cells are further cultured in the presence of a culture medium that does not contain growth factors.

23. 23. The method of claim 22, wherein the isolated gastric cancer stem cells form AQP5-expressing tumor organoids.

24. The method of any one of claims 18 to 23, wherein the isolated one or more gastric cancer stem cells are further analyzed.

25. The method according to any one of claims 18 to 24, wherein the analysis of the isolated gastric cancer stem cells or AQP5-expressing tumor organoids comprises transcriptome analysis.

26. A method for removing or eliminating AQP5+ gastric cancer stem cells that have been modified to express the DTR gene or the inducible caspase 9 (iCasp9) gene, comprising contacting the cells with diphtheria toxin (DT) or an inducer of iCasp9.

27. A method for removing or eliminating AQP5+ gastric cancer stem cells, comprising contacting the cells with an agent that binds to AQP5, wherein binding of the AQP5+ cells to the agent removes or eliminates the cells.

28. The method of any one of claims 1 to 27, wherein the gastric cancer stem cells are mammalian cells.

29. The method of any one of claims 1 to 28, wherein AQP5 is membrane-bound.

30. A method of treating gastric cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of one or more agents that eliminate or ablate AQP5-expressing cells.

31. 31. The method of claim 30, wherein the subject in need thereof is a human or a mouse.

32. AQP5, a biomarker for gastric cancer stem cells.

33. The biomarker of claim 32, wherein the AQP5 is membrane-bound AQP5.

34. A kit for identifying, isolating, eliminating, or ablation of gastric cancer stem cells, comprising an agent that binds to AQP5 and instructions for use.

35. 35. The kit of claim 34, wherein the agent is an antibody, optionally conjugated to a compound such as a detectable label, a drug, or a small molecule.

36. The kit of claim 35, wherein the antibody binds to the intracellular or extracellular domain of the AQP5 protein.

37. A method for inhibiting gastric cancer tumorigenesis or progression, comprising administering an AQP5 inhibitor to a subject.

38. A method for promoting regression of gastric cancer, comprising administering to a subject an AQP5 inhibitor.

39. The method of any one of claims 37 to 38, wherein the AQP5 inhibitor inhibits or blocks the expression and / or function of AQP5.

40. The method of any one of claims 37 to 39, wherein the AQP5 inhibitor is selected from the group consisting of siRNA, RNAi, chimeric antigen receptor (CAR), and a drug.

41. 41. The method of claim 40, wherein the RNAi is selected from the group consisting of siRNA, shRNA, and miRNA.

42. The method of any one of claims 40 to 41, wherein the siRNA is selected from the group consisting of sense and antisense primer pairs.

43. The primer is 43. The method of claim 42, selected from the group consisting of:

44. 41. The method of claim 40, wherein the drug is selected from the group consisting of an enzyme inhibitor, a receptor antagonist, and a channel blocker.

45. 1. A method for monitoring the progression of gastric cancer in a subject, comprising: a) measuring the expression level of AQP5 in a sample obtained from the subject after undergoing treatment for gastric cancer; and b) measuring the expression level of AQP5 in a control sample obtained from the subject before treatment for gastric cancer. Including, an increase in the expression level of AQP5 in the sample of step a compared to the control sample indicates that gastric cancer tumorigenesis has occurred or that gastric cancer has progressed; method.

46. 1. A method for monitoring gastric cancer tumorigenesis in a subject, comprising: c) measuring the expression level of AQP5 in a sample obtained from the subject; and d) measuring the expression level of AQP5 in a reference sample obtained from the subject at a time earlier than the sample of step c). Including, an increase in the expression level of AQP5 in the sample of step c compared to the reference sample of step d indicates that gastric cancer tumorigenesis has occurred; method.

47. 47. The method of any one of claims 45 to 46, wherein the subject is treated with an anti-gastric cancer compound when gastric cancer tumorigenesis occurs or when gastric cancer progresses.

48. The method of any one of claims 45 to 47, wherein the expression level of AQP5 is a gene expression level, a protein expression level, or a combination thereof.

49. 49. The method of any one of claims 45 to 48, further comprising measuring the expression level of one or more gastric cancer progression markers.

50. 50. The method of claim 49, wherein the marker is selected from the group consisting of Pthlh, Hey1, Rgs5, and combinations thereof.

51. The method of any one of claims 45 to 50, wherein the step of measuring the expression level of AQP5 is by immunohistochemistry, flow cytometry, Western blot, or a combination thereof.

52. The method of any one of claims 45 to 51, wherein the step of measuring the expression level of AQP5 is by polymerase chain reaction.

53. 53. The method of any one of claims 45 to 52, wherein said sample is selected from the group consisting of blood, plasma, a biopsy sample, a tissue sample, a primary cell culture sample, and a primary organoid line.

54. The method of any one of claims 1 to 33 and 37 to 53, wherein the gastric cancer is intestinal-type gastric cancer or diffuse-type gastric cancer.

Images