Methods and uses involving aquapoprotein-5 (AQP5)

Abstract

Disclosed herein are methods of identifying gastric cancer stem cells or populations of gastric cancer stem cells. Also described herein are methods of isolating one or more gastric cancer stem cells from a population of cells, comprising contacting the cells of the population of cells with an AQP5-binding agent, isolating one or more AQP5-expressing cells bound to the agent, where the one or more AQP5-expressing cells are gastric cancer stem cells. Further described herein are methods of removing or eliminating AQP5 + gastric cancer stem cells as well as methods of treating gastric cancer and methods of monitoring gastric cancer neogenesis or gastric cancer progression.

Description

[0001] Cross-reference with related applications

[0002] This application claims priority to Singapore Provisional Application No. 10202300699X, filed on March 14, 2023, and Singapore Provisional Application No. 10202302747V, filed on September 27, 2023, the entire contents of which are incorporated herein by reference for all purposes. Technical Field

[0003] This invention generally relates to the field of molecular biology. In particular, this invention relates to the use of biomarkers for the detection and diagnosis of cancer. Background of the Invention

[0005] Gastric cancer is one of the leading causes of cancer-related deaths globally and in Singapore, with a five-year survival rate of less than 30%. Currently, treatment for gastric cancer is limited to traditional methods such as chemotherapy, radiotherapy, and surgical resection, but these methods are ineffective in improving cancer recurrence in many gastric cancer patients, especially those with diffuse gastric cancer. Therapy-resistant cancer stem cell populations are considered a key driver of gastric cancer recurrence. However, the gastric cancer stem cell markers proposed to date either lack robust functional validation directly demonstrating the stem potential of the labeled cell populations, or are expressed in large quantities in both normal and tumor tissues, making targeting these cells in patients challenging.

[0006] The implementation of the concept of targeting cancer stem cells for the treatment of various human cancers in the context of gastric cancer has previously been hampered by the lack of gastric cancer stem cell biomarkers, most of which have not been reliably validated using near-physiological cancer models and functional assays. Furthermore, some previously proposed biomarkers are widely expressed in both normal and tumor tissues, making it challenging to develop these biomarkers into safe therapeutic targets for patients.

[0007] Therefore, the need for methods to identify gastric cancer stem cells has not yet been met.

[0008] Overview

[0009] In one aspect, this disclosure relates to a method for identifying gastric cancer stem cells or gastric cancer stem cell populations, comprising a1) detecting the expression of aquaporin 5 (AQP5) in cells or cell populations; or b1) detecting the expression level of AQP5 in cells or cell populations and comparing the expression level with the expression level of AQP5 in reference cells or reference cell populations, wherein the cells or cell populations are identified as gastric cancer stem cells or gastric cancer stem cell populations when AQP5 expression is detected in cells or cell populations, or when the expression level of AQP5 in cells or cell populations is increased compared to that in reference cells or reference cell populations.

[0010] In another aspect, this disclosure relates to a method for isolating one or more gastric cancer stem cells from a cell population, comprising i) contacting cells in the cell population with an agent that binds to AQP5; and ii) isolating one or more AQP5-expressing cells that bind to the agent, wherein one or more AQP5-expressing cells are gastric cancer stem cells.

[0011] In another aspect, this disclosure relates to a method for removing or eliminating AQP5+ gastric cancer stem cells modified to express the DTR gene or the inducible Caspase9 (iCasp9) gene, comprising contacting the cells with a diphtheria toxin (DT) or an inducer of iCasp9.

[0012] On the other hand, this disclosure relates to a method for removing or eliminating AQP5+ gastric cancer stem cells, comprising contacting the cells with an agent that binds to AQP5, wherein the binding of the AQP5+ cells to the agent causes the cells to be removed or eliminated.

[0013] In one aspect, this disclosure relates to a method of treating gastric cancer in a subject in need of it, comprising administering to the subject a therapeutically effective amount of one or more agents that eliminate or remove cells expressing AQP5.

[0014] On the other hand, this disclosure relates to biomarkers for gastric cancer stem cells, wherein the biomarker is AQP5.

[0015] On the other hand, this disclosure relates to a kit for identifying, isolating, eliminating or removing gastric cancer stem cells, comprising an agent that binds to AQP5 and instructions for use.

[0016] In another aspect, this disclosure relates to a method for inhibiting the occurrence or progression of gastric cancer tumors, the method comprising administering an AQP5 inhibitor to a subject.

[0017] On the other hand, this disclosure relates to a method for promoting the regression of gastric cancer, the method comprising administering an AQP5 inhibitor to a subject.

[0018] In one aspect, this disclosure relates to a method for monitoring the progression of gastric cancer in a subject, the method comprising: a) measuring the expression level of AQP5 in a sample obtained from a subject after receiving gastric cancer treatment; and b) measuring the expression level of AQP5 in a control sample obtained from the subject before gastric cancer treatment; wherein, compared with the control sample, an elevated expression level of AQP5 in the sample of step a indicates that gastric cancer tumorigenesis has occurred or that gastric cancer has progressed.

[0019] In another aspect, this disclosure relates to a method for monitoring the occurrence of gastric cancer tumors in a subject, the method 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 point earlier than that of the sample obtained in step c; wherein the expression level of AQP5 in the sample obtained in step c is elevated compared to that of the reference sample obtained in step d, indicating that the occurrence of gastric cancer tumors has occurred.

[0020] Brief description of the attached figures

[0021] The invention will be better understood when considered in conjunction with the detailed description and non-limiting examples and accompanying drawings, in which:

[0022] Figure 1 The results showed that Aqp5 was overexpressed in pyloric tumors in mice and humans. Figure 1 A shows Aqp5-eGFPires-CreERT2; Apc fl / fl Pten fl / fl Kras LSL-G12D / + Rosa26-tdTomato LSL A schematic diagram of a mouse model of conditional inheritance of pyloric cancer. Figure 1 B shows a fluorescence image indicating that AQP5 is expressed in the pylorus of the Aqp5-Cre / APK mouse model at multiple stages of tumor development. Figure 1 C shows a bar graph quantifying the change in the proportion of AQP5 cells over time in healthy tissue and tumor pyloric tissue. Figure 1 D shows the FACS gating strategy for isolating tumor epithelial (dTom+) Aqp5- cells and Aqp5+ cells from primary mouse pyloric tumors. Figure 1 E shows a bar graph depicting the relative expression of Aqp5 in a sorted mouse tumor cell population by qPCR. Figure 1 F is a heatmap showing the top 50 differentially expressed genes (DEGs) between Aqp5+ and Aqp5- samples isolated from 6 independent mouse pyloric tumors. Figure 1 G shows a slide representing AQP5 expression in a microarray of human gastric tissue, indicating normal stomach, intestinal-type gastric tumors, diffuse gastric tumors, and mixed gastric tumors. Staining was graded from unstained to strongly stained, as shown in the legend (right). The image shown represents the tissue core exhibiting moderate staining (in normal stomach) and strong staining (in tumor tissue). Figure 1 H shows the FACS gating strategy for isolating tumor epithelial Aqp5- and Aqp5+ cells from primary human pyloric tumor biopsy tissue. Figure 1 I shows a bar graph depicting the relative expression of Aqp5 in a sorted human tumor cell population using qPCR. Figure 1J is a heatmap of the top 50 DEGs from Aqp5+ and Aqp5- samples isolated from 5 independent human pyloric tumors. Scale bar, 200 μm. The figures represent mean ± standard deviation, analyzed using a two-tailed unpaired t-test.

[0023] Figure 2 The results show that mouse and human Aqp5+ pyloric tumor cells function as cancer stem cells. Figure 2 Image A shows images of organoids generated by mouse Aqp5+ and Aqp5- cells sorted by single FACS at passages 0, 2, and 10. Mouse Aqp5+ cell-derived organoids maintained high growth efficiency at least up to P10, while a small number of Aqp5- cell-derived organoids were formed, which failed to survive after P2. Figure 2 B shows a bar graph, which displays data indicating the outgrowth efficiency of individual mouse Aqp5+ and Aqp5- cells at P0, as well as the quantification of the maximum number of passages reached for each culture. Figure 2 C shows a fluorescence image indicating the expression of AQP5 and the lineage markers KI67, TFF2, and CHGA in mouse AQP5+ cell-derived organoids. Figure 2 D shows the full specimens and H&E images of tumors derived from transplanted mouse Aqp5+ (left) and Aqp5- (right) pyloric tumor cells. Figure 2 E shows a bar graph quantification of tumor volume from transplanted Aqp5+ pyloric tumor cells and Aqp5- pyloric tumor cells. Figure 2 F shows images of organoids derived from generation 0, 2, and 10 human Aqp5+ and Aqp5- cells sorted from single FACS. Human Aqp5+ cell-derived organoids maintained high growth efficiency at least up to P10, while a small number of Aqp5- cell-derived organoids failed to survive after P2. Figure 2 G is a bar graph showing data on organoid growth efficiency of individual human Aqp5+ and Aqp5- cells at P2, as well as a quantification of the highest number of passages achieved by each culture. Figure 2 H is a fluorescence image indicating the expression of AQP5 and the lineage markers KI67, MUC5AC, and CHGA in human AQP5+ cell-derived organoids. Scale bar, 500 μm. Figure 2 A, Figure 2 F) and 100μm ( Figure 2 C Figure 2 H). The figure represents the mean ± standard deviation, and a two-tailed unpaired t-test was used.

[0024] Figure 3 The results showed that removing Aqp5+ cancer stem cells blocked tumor development and progression. Figure 3 A shows multiple images illustrating the results of treating Aqp5-Cre / APK / DTR mouse pyloric tumor organoids (integrating the Aqp5-2A-DTR allele) with diphtheria toxin (DT) during organoid initiation (day 0) to block organoid growth. n = 4 biological replicates. Figure 3 B is a line graph that shows the quantitative change in the number of organoids over time in the control and DT-treated organoid cultures after treatment on day 0. Figure 3 Image C shows the results of treatment of Aqp5-Cre / APK / DTR mouse pyloric tumor organoids with diphtheria toxin (DT) during organoid growth / maintenance (day 3), resulting in rapid reduction of cultures. n = 4 biological replicates. Figure 3 D shows a line graph that quantifies the change in organoid number over time in control and DT-treated organoid cultures after day 3 of treatment. Figure 3 E shows multiple images demonstrating that intraperitoneal administration of DT to mice with orthotopically transplanted Aqp5-Cre / APK / DTR organoids resulted in complete absence of tumor growth during the early stages of tumorigenesis (day 3). n = 5 biological replicates. Figure 3 F shows multiple images illustrating tumor regression driven by intraperitoneal administration of DT to mice orthotopically transplanted with Aqp5-Cre / APK / DTR organoids at late tumor growth stage (week 4). n = 5 biological replicates. g, Strategy for removing Aqp5 cells from human gastric cancer organoids. The Aqp5-2A-iCaspase9 cassette was integrated into human gastric cancer organoids to drive the expression of Aqp5+ intracellular inducible Caspase9. The addition of B / B homodimers promoted Caspase9 dimerization and activation of apoptosis pathways leading to cell death. Figure 3 H shows multiple images depicting the in vitro inhibition of organoid growth in human gastric cancer organoids treated with Dimer (Dim). Figure 3 Figure 1 shows a bar graph quantifying the number of organoids in cultures of control and dimer-treated cultures. Scale bar, 500 μm. Figure 3 A, Figure 3 C Figure 3 G) and 1mm ( Figure 3 E, Figure 3 F). The figure represents the mean ± standard deviation, and a two-tailed unpaired t-test was used.

[0025] Figure 4 The results showed that Aqp5 was expressed in a cell subset within mouse pyloric tumors. Figure 4 A shows a representative full specimen and H&E images of a pyloric tumor in Aqp5-Cre / APK mice. Figure 4Image B shows RNAscope results of Aqp5 and Lgr5 in the pylorus of healthy mice, which demonstrates their co-localization in the pyloric stem cell compartment at the glandular base. Figure 4 C displays Aqp5-2ACreERT2; Apc fl / fl Pten fl / fl Kras LSL-G12D / + Rosa26-tdTomato LSL A schematic diagram of a conditionally inherited mouse model of pyloric cancer (left). During tumor development, AQP5 levels increase over time and are similar to the expression patterns of tumors derived from the Aqp5-eGFP-ires-CreERT2 model (right). Figure 4 D shows a MERSCOPE-based spatial transcriptomics image of pyloric tumor slices from Aqp5-Cre / APK mice, which reveals highly heterogeneous cell types assigned by Leiden clusters, with Aqp5 expression clusters highlighted. Figure 4 E shows a UMAP map of the Aqp5-Cre / APK tumor cell cluster. Figure 4 F shows a heatmap of the expression levels of selected genes in each of the eight epithelial cell clusters, which demonstrates the heterogeneity of the Aqp5+ population relative to other tumor cell lineages. Figure 4 G shows RNAscope images of Aqp5 and putative gastric cancer stem cell markers Lgr5, Cxcr4, and Cd44 within pyloric tumor sections from Aqp5-Cre / APK mice. Aqp5 expression partially overlaps with known stem cell markers and also marks unique tumor compartments. Scale bar, 50 μm (b, g – magnified inset) and 200 μm ( Figure 4 A, Figure 4 C Figure 4 D、 Figure 4 G).

[0026] Figure 5 The transcriptome analysis results of mouse Aqp5+ pyloric tumor cells and Aqp5- pyloric tumor cells are shown. Figure 5 A shows a bar graph depicting gene ontology (GO) terms selected from the categories of molecular function (MF) and biological process (BP), which are significantly enriched in mouse Aqp5+ cells compared to Aqp5- cells. Figure 5 B shows the results of gene set enrichment analysis (GSEA) of the epithelial-mesenchymal transition and external encapsulation pathways (left), with an accompanying heatmap showing the relative expression of selected genes within each pathway (right). Figure 5 C to Figure 5 F shows the upregulated tumor microenvironment in the transcriptome of mouse Aqp5+ cells. Figure 5 C) Transfer and dissemination ( Figure 5 D) Gastric cancer progression ( Figure 5 E) and drug resistance ( Figure 5 F) qPCR validation results (left) and RNAseq expression data (right) of the selected genes. Figure 5 G shows RNAscope validation images of selected targets Lgr5, Pthlh, Rgs5, and Hey1, confirming their upregulation in the Aqp5+ tumor cell region. Scale bar, 100 μm. Figures represent mean ± standard deviation, analyzed using a two-tailed unpaired t-test.

[0027] Figure 6 The results show that Aqp5 is expressed in a cell subset within human pyloric tumors. Figure 6 A shows a UMAP map of cell clusters generated by scRNAseq from healthy human gastric tissue and human tumor-bearing gastric tissue, in which epithelial cell clusters are labeled. Figure 6 B shows a list of selected markers used to assign identity to epithelial cell clusters. Figure 6 C shows a UMAP plot illustrating Aqp5 expression in epithelial cell clusters within normal human gastric cells (left) and human tumor gastric cells (right). Figure 6 D shows a violin plot of Aqp5 expression in each epithelial cluster, which shows the elevated levels in the tumor cell population. Figure 6 E to Figure 6 F shows a UMAP map of tumor epithelial cell clusters separated according to tumor subtype (intestinal, diffuse, and mixed). Figure 6 E) and violin diagram ( Figure 6 F). Figure 6 G to Figure 6 H shows a UMAP map of tumor epithelial cell clusters separated according to tumor location (antrum / pylorus, body, and cardia). Figure 6 G) and violin diagram ( Figure 6 H). Figure 6 I shows a UMAP map of epithelial cell clusters generated by scRNAseq from healthy human gastric tissue and human tumor gastric tissue from the second dataset GSE150290. Figure 6 J shows the method used to give Figure 6 Identification of selected markers for the assignment of epithelial cell clusters in I. Figure 6 K showed Figure 6 Violin plots of Aqp5 expression in each epithelial cluster identified in I, showing elevated levels within the tumor population.

[0028] Figure 7 The transcriptome analysis results of human Aqp5+ pyloric tumor cells and Aqp5- pyloric tumor cells are shown. Figure 7A shows a bar chart depicting gene ontology (GO) terms selected from the categories of molecular function (MF) and biological process (BP), which are significantly enriched in human Aqp5+ cells compared to Aqp5- cells. Figure 7 B shows the results of gene set enrichment analysis (GSEA) of the epithelial-mesenchymal transition and external encapsulation pathways (left), with an accompanying heatmap showing the relative expression of selected genes within each pathway (right). Figure 7 C to Figure 7 F shows the upregulated activity in the human Aqp5+ cell transcriptome related to the tumor microenvironment (F). Figure 7 C) Cancer progression ( Figure 7 D) Drug resistance ( Figure 7 E) and tumor regulation ( Figure 7 F) qPCR validation results (left) and RNAseq expression data (right) of the selected genes. Figure 7 G shows the RNAscope validation results for the selected targets AQP5, CLDN2, DCHS2, HEY2, and PLA1A, confirming their upregulation in the Aqp5 tumor cell region. Scale bar, 100 μm. Figures represent mean ± standard deviation, analyzed using a two-tailed unpaired t-test.

[0029] Figure 8 The results show that mouse Aqp5+ cells function as cancer stem cells. Figure 8 Image A shows representative bright-field and immunofluorescence images of mouse Aqp5-Cre / APK pyloric tumor organoids generated from the entire gland, which demonstrate organoid morphology and expression of gastric lineage markers comparable to tumor organoids formed from isolated Aqp5+ tumor cells. Figure 8 B shows a representative full specimen, H&E, and IHC images of the stomach of immunodeficient NOG mice 4 months after orthotopic transplantation of primary mouse pyloric tumor cells into the gastric submucosa, with very few tumor cells established. Figure 8 C shows a schematic diagram of the experimental workflow for generating orthotopic tumors from mouse pyloric tumor organoids, isolating Aqp5+ and Aqp5- cells via FACS, and then transplanting these cells into immunodeficient mice. Figure 8 Image D shows the histology (left) of mouse Aqp5-Cre / APK pyloric tumor organoids as assessed by H&E, and the in situ tumors (middle and right) resulting from transplantation of these tumor organoids as observed by whole specimens and H&E. Figure 8 E shows the FACS gating strategy for isolating transplanted tumor epithelial (dTom+) Aqp5+ cells and Aqp5- cells. Figure 8F shows histological images of orthotopic tumors derived from transplanted tumor epithelial Aqp5+ and Aqp5- cells. Immunohistochemistry of RFP highlights the location of transplanted cells within the gastric mucosa. Immunofluorescence of Aqp5, Muc5ac, Ki67, and Chga indicates that the Aqp5+ cell-derived tumors maintain the expression of stemness and differentiation lineage markers. Figure 8 G displays Aqp5-2A-CreERT2; Apc fl / fl Pten fl / fl Trp53 R172H / + Rosa26-tdTomato LSL A schematic diagram of a conditionally inherited mouse model of pyloric cancer. Figure 8 H shows representative whole specimen and immunostaining images that confirm p53 mutant expression (middle) and the presence of Aqp5+ cells in the Aqp5-Cre / APT tumor subgroup (right). Figure 8 I shows a FACS gating strategy for isolating tumor epithelial Aqp5+ cells and Aqp5- cells from Aqp5-Cre / APT tumors. Figure 8 J shows a bar chart depicting the relative Aqp5 expression of sorted tumor cell populations by qPCR. Figure 8 The images shown by K indicate that a greater number of organoids can be generated from a single Aqp5+ cell compared to Aqp5- cells. Figure 8 L shows bar graphs illustrating the organoid growth efficiency of individual mouse Aqp5+ and Aqp5- cells at P0. Scale bars: 100 μm (a), 500 μm (k), and 1 mm (b, d, h). Figures represent mean ± standard deviation, analyzed using a two-tailed unpaired t-test.

[0030] Figure 9 The results show that human Aqp5+ cells function as cancer stem cells. Figure 9 Image A shows representative bright-field and immunofluorescence images of human pyloric tumor organoids generated from the entire gland, demonstrating organoid morphology and expression of gastric lineage markers comparable to tumor organoids formed from isolated Aqp5+ tumor cells. Figure 9 B shows Aqp5-2A-CreERT2 and AAVS1-CAG-tdTomato inserted into human gastric cancer organoids. LSL A schematic diagram of the construct (top image). Genomic PCR was performed at the insertion site of Aqp5-2A-CreERT2, and the insertion was confirmed to be successful based on the expected band size (bottom image). Figure 9 C shows bright-field and fluorescence images, which demonstrate the activity of Aqp5-2A-CreERT2, AAVS1-CAG-tdTomato LSLThe 16-hour 4-OHT induction in the edited organoids resulted in a small cluster of cells with tdTomato markers visible on day 2, which expanded to cover a larger organoid region on day 10. Figure 9 D shows a bar graph quantifying the expansion of tdTomato+ labeled clonal regions within each organoid over time. Figure 9 E shows bright-field and fluorescence images, which reveal uninduced Aqp5-2A-CreERT2, AAVS1-CAG-tdTomato LSL The organoids never showed any visible traces of tdTomato at the same time point. Figure 9 F shows an image of co-immunofluorescence, which confirms the overlap of tdTomato-labeled cells and differentiation lineage markers (including MUC5AC and KI67). Scale bar, 100 μm. Figures represent mean ± standard deviation, analyzed using a two-tailed unpaired t-test.

[0031] Figure 10 The results showed that the removal of mouse Aqp5+ cells in a series of pyloric tumor models confirmed that they are required for tumor development and progression. Figure 10 A shows Aqp5-eGFP-ires-CreERT2, Apc fl / fl Pten fl / fl Kras LSL-G12D / + Rosa26-tdTomato LSL A schematic diagram of a conditionally inherited mouse model of pyloric cancer, in which an Aqp5-2A-DTR box is added to promote the removal of diphtheria toxin (DT)-mediated Aqp5+ cells. Figure 10 Image B shows representative images demonstrating that Aqp5-Cre / APK and Aqp5-Cri / APK / DTR tumors are comparable in histological features and Aqp5 expression. Figure 10 The results shown in C indicate that intraperitoneal administration of DT to mice 10 weeks after tamoxifen-induced pyloric tumor formation resulted in a reduction of tumor volume by approximately 50%. Representative full-specimen images of the control and DT-treated tumors are shown on the left. Figure 10 D shows the timeline of DT administration and analytical images of tumor tissue 12 hours after tumor removal in Aqp5-Cre / APK / DTR mice (top). Representative immunohistochemical images confirm the loss of AQP5+ cells in DT-treated Aqp5-Cre / APK / DTR tumors, accompanied by increased expression of CAS3 and KI67 in the remaining tumor burden. Figure 10E shows the timeline of DT administration and the analysis of tumor tissue 12 hours after removal of tumor tissue from Aqp5-Cre / APK mice lacking the Aqp5-2A-DTR allele (top). Representative immunohistochemical images show that control and DT-treated tumors are comparable in terms of AQP5, CAS3, and KI67 levels. Figure 10 Data shown in F indicate that mouse pyloric tumor organoids derived from Aqp5-Cre / APK / DTR tumors encapsulate AQP5 expression across cell subsets. Co-immunofluorescence of AQP5 and ECAD is shown in representative tumor organoids (top panel). FACS analysis revealed that 9%–10% of cells in Aqp5-Cre / APK / DTR organoids highly expressed AQP5 (bottom panel). A mouse pyloric tumor organoid line lacking AQP5-GFP was used as a control for setting FACS gating in the atlas. Figure 10 G presented the results, which showed that DT treatment of Aqp5-Cre / APK / DTR organoids resulted in a reduction of Aqp5 levels by 80% and 95% respectively compared to untreated control organoids during the organoid growth and organoid expansion phases. Figure 10 Data from H showed that DT treatment of Aqp5-Cre / APK organoids lacking the Aqp5-2A-DTR allele on day 0 did not affect organoid growth. Figure 10 The results shown in Figure 1 indicate that DT treatment of Aqp5-Cre / APK organoids lacking the Aqp5-2A-DTR allele on day 3 does not affect subsequent organoid maintenance. Figure 10 The data presented by J, obtained via qPCR, showed that DT treatment of Aqp5-Cre / APK organoids lacking the Aqp5-2A-DTR allele did not affect Aqp5 levels during organoid growth and expansion phases. Figure 10 K to Figure 10 N shows the timeline of DT administration to mice after prominent organoid transplantation to remove Aqp5 cells in vivo and the points at which harvested tissue was analyzed (top panel). Representative H&E and AQP5 immunohistochemical images confirm that, 3 days after graft removal ( Figure 10 K) or 4 weeks ( Figure 10 One day after transplantation of Aqp5-Cre / APK / DTR organoids lacking the Aqp5-2A-DTR allele, AQP5 expression was lost and / or impaired. Representative H&E and AQP5 immunohistochemical images confirmed that AQP5 expression was lost and / or impaired 4 weeks after transplantation of Aqp5-Cre / APK organoids lacking the Aqp5-2A-DTR allele. Figure 10 M) or 8 weeks ( Figure 10 N) DT administration does not affect tumor burden or AQP5 expression. Scale bar, 500 μm ( Figure 10 F, Figure 10 H, Figure 10I) and 1mm Figure 10 B. Figure 10 D、 Figure 10 E, Figure 10 K, Figure 10 L, Figure 10 M, Figure 10 N). The mean ± standard deviation of the graph was analyzed using a two-tailed unpaired t-test.

[0032] Figure 11 The results showed that the removal of human Aqp5+ cells in a human gastric cancer organoid model confirmed that they are required for tumor progression. Figure 11 Image A shows the agarose gel image. Genomic PCR was performed at the insertion site of Aqp5-2A-iCaspase, and successful insertion was confirmed based on the expected band size. Figure 11 Immunofluorescence images of AQP5, KI67, MUC5AC, and CHGA are shown, confirming the presence of the expected lineage markers in Aqp5-2A-iCaspase organoids. Figure 11 C shows that dimer treatment of 0116Aqp5-2A-iCaspase organoids resulted in a reduction of Aqp5 levels by approximately 60% compared to untreated control organoids. Figure 11 D shows the results of dimer treatment on unedited 0116 parental organoids, which was shown not to alter organoid development in cultures or subsequent growth over 10 days. Figure 11 E shows an image in a standalone GC10Aqp5-2A-iCaspase organoid cell line, where the removal of Aqp5+ cells again indicates a reduced growth rate. Figure 11 F shows a bar graph indicating that dimer treatment of GC10 Aqp5-2A-iCaspase organoids resulted in a 70% reduction in Aqp5 levels compared to untreated control organoids. Figure 11 G is a bar graph that shows that dimer treatment of unedited GC10 parental organoids does not alter organoid development in cultures and or subsequently, growth over 10 days. Figure 11 H shows a bar graph, which, as demonstrated by qPCR, shows that Aqp5 levels remained unchanged in both control and dimer-treated GC10 parental organoids. Scale bar, 100 μm ( Figure 11 B) and 500μm ( Figure 11 D、 Figure 11 E, Figure 11 G). The figure shows the mean ± SD, using a two-tailed unpaired t-test.

[0033] Figure 12 The images shown indicate that targeted removal of Aqp5+ tumor cells severely hinders organoid and tumor growth. Figure 12Figure A shows a schematic diagram and images illustrating the targeted removal of Aqp5-carrying pyloric tumor cells from organoids mediated by diphtheria toxin (DT). In this model, Aqp5-expressing tumor cells also express the DT receptor, making them sensitive to DT administration. Adding DT at inoculation (day 0) or mid-organoid growth (day 3) to remove Aqp5-carrying tumor cells from the organoids resulted in complete lethality of the tumor organoids. Figure 12 B shows a schematic diagram and images of the DT removal results of Aqp5+ tumor cells from pyloric tumor organoids transplanted into the gastric submucosa of immunodeficient NOG mice. Removal performed at tumor initiation (day 3) completely eliminated tumor formation in DT-treated mice. When removal was performed later in tumor growth (week 4), DT-treated tumors showed a milder tumorigenesis phenotype.

[0034] Figure 13 The images shown indicate that Aqp5 expression is low in most major human tissues. Figure 13 Image A shows microarray images of selected tissues from the brain, heart, kidneys, and liver, revealing little or no Aqp5 staining. In contrast, Aqp5 is strongly expressed on the membranes of the salivary glands and testes. Figure 13 B shows images and scoring results of the microarray cores of 47 normal human lung tissues, the vast majority of which showed the absence of Aqp5 expression or non-membrane localization of Aqp5 protein.

[0035] Figure 14 The images shown indicate that Aqp5 is upregulated in gastric tumors in mice and humans. Immunohistochemistry of Aqp5 in human (top) and mouse (bottom) gastric tissues. Aqp5 expression is confined to the glandular basal layer in the pyloric glands of humans and mice, but is widely and highly expressed in intestinal and diffuse gastric tumors.

[0036] Figure 15 Images of a previously unknown human gastric cancer organoid cell line are presented. This cell line integrates the Flip-Puro system and conditionally knocks out Aqp5 upon administration of Cre recombinase cellular nanovesicles (gesicles). The loss of Aqp5 in these organoids was confirmed by Western blotting. Knockout of Aqp5 in the organoid cells showed reduced cell viability.

[0037] Figure 16 The data shown indicates that Aqp5 drives the development of gastric tumors. Figure 16 Image A shows histological images of pyloric tumors in Aqp5 wild-type (WT) and knockout (KO) mice. Images of representative whole specimens (left) and H&E (middle) are shown, along with quantification of tumor volume (right). Figure 16B shows the important gene ontology (GO) items presented in the DEG enriched in Aqp5 WT cancer stem cells (top) and Aqp5 KO cancer stem cells (bottom). Figure 16 C shows data for human gastric cancer organoids (parental) 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 absent in the KO clones (top panel). Reduced EdU staining was detected in the Aqp5 KO organoids compared to the parental line (bottom panel). Figure 16 D shows the quantification of cell proliferation in parental and Aqp5 KO organoids using in vitro MTS colorimetric assay (left) and EdU staining assay (right). Figure 16 E shows representative full specimens (top) and H&E (bottom) images of orthotopic tumors derived from transplanted parental and Aqp5 KO human gastric cancer organoids. Figure 16 F shows the quantitative results of tumor volume (left) and invasion rate (right) of orthotopic tumors derived from transplanted parental and Aqp5 KO human gastric cancer organoids. Figure 16 G shows a schematic diagram of the FLIP-Puro box used to generate Cre-inducible Aqp5 KO in human gastric cancer organoids. Figure 16 The image shown in H indicates that conditional KO of Aqp5 treated with Cre recombinase in human gastric cancer organoids reduced in vitro organoid growth. Figure 16 I shows the quantitative results of the relative number of organoids at a time point of 5 days after conditional Aqp5 KO. Organoids with a diameter of at least 100 μm were included in the analysis. Figure 16 J shows a schematic diagram of key GO terms enriched in untreated and Cre-induced Aqp5 KO organoids at different indicative time points after induction. Scale bar, 100 μm ( Figure 16 C– (below), 500μm ( Figure 16 C – Up, Figure 16 H) and 1mm ( Figure 16 A, Figure 16 E–below). The figure represents the mean ± standard deviation, using a two-tailed unpaired t-test.

[0038] Figure 17 The results show that AQP5 drives tumor progression in various human gastric cancer cell lines. Figure 17 A shows the analysis results of AQP5 function in the intestinal AGS gastric cancer cell line. Representative immunofluorescence images and qPCR of Aqp5 levels (left) confirmed the upregulation of Aqp5 in three Aqp5 overexpression (OE) clones. In vitro assays showed that the OE clones had higher cell proliferation and cell migration rates in culture (right). Figure 17B shows the analysis of AQP5 function in the diffuse SNU601 gastric cancer cell line. Representative immunofluorescence images and qPCR of Aqp5 levels (left) confirmed the upregulation of Aqp5 in three Aqp5 overexpression (OE) clones. In vitro assays showed that the OE clones had higher cell migration rates at culture time (right). Figure 17 The results shown by C indicate that AGS OE cells established larger in situ tumors in mice that exhibited more aggressive tumor characteristics. Figure 17 D shows the analysis of AQP5 function in the diffuse KATOⅢ cell line expressing 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 proliferate more slowly in culture compared to parental cells (right). Figure 17 E showed that KATOⅢKO cells failed to establish in situ tumors in mice in most of the examined cases. Scale bar, 500 μm ( Figure 17 A, Figure 17 B. Figure 17 D) and 1mm ( Figure 17 C Figure 17 E). The figure represents the mean ± standard deviation, and a two-tailed unpaired t-test was used.

[0039] Figure 18 The data shown indicate that knocking out AQP5 in a mouse model of pyloric cancer inhibits tumor growth and reveals altered transcriptomic profiles. Figure 18 Image A shows representative immunofluorescence images of Aqp5 WT and Aqp5 KO pyloric tumors, confirming the loss of Aqp5 in KO tumors. Figure 18 Data shown in B indicate that mouse pyloric tumor organoids derived from Aqp5 WT and Aqp5 KO tumors reflect the expression pattern of AQP5 in the original tumor. Figure 18 C shows that, compared with Aqp5 WT tumor organoids, Aqp5 KO tumor organoids formed less invasive tumors after transplantation into immunodeficient mice. Representative whole specimens and H&E images (left) are shown, along with quantification of tumor volume and invasiveness (right). Figure 18 D shows the FACS gating strategy used to isolate tumor epithelial GFP+ (stem) cells and GFP- (the remainder of the tumor) cells from pyloric tumors in Aqp5 WT and Aqp5 KO mice. Figure 18 E shows a heatmap of the top 50 differentially expressed genes (DEGs) between GFP+ and GFP- samples isolated from three independent mouse pyloric tumors. Figure 18 F shows a Venn diagram of common and unique DEGs between the Aqp5 KO and Aqp5 WT tumor spectrum. Figure 18G shows the results of reactome analysis of genes enriched in Aqp5 WT tumors, highlighting potential downstream pathways of Aqp5 function. Scale bar, 500 μm ( Figure 18 B) and 1mm Figure 18 A, Figure 18 C). The figure represents the mean ± standard deviation, and a two-tailed unpaired t-test was used.

[0040] Figure 19 The data shown indicate that AQP5 drives tumor progression in various human gastric cancer organoid cell lines. Figure 19 A, Figure 19 B and Figure 19 C shows the results of three independent human gastric cancer organoid cell lines 0045 used for in vitro analysis of AQP5 function. Figure 19 A), 0235 Figure 19 B) and 0116 Figure 19 Results obtained in (C). Representative immunofluorescence images (left) and qPCR of Aqp5 levels (middle) confirmed the upregulation of Aqp5 in the Aqp5-overexpressing (OE) organoid cell lines. Notably, the upregulation of Aqp5 in the 0116OE organoid was smaller compared to the other two OE cell lines, as Aqp5 is already strongly expressed in the parental 0116 cell line. As measured by MTS proliferation assays, the 0045OE and 0235OE organoid cell lines showed higher cell proliferation rates in vitro than their respective parental cell lines (right). Figure 19 D、 Figure 19 E and Figure 19 F shows three human gastric cancer organoid cell lines 0045 used for in vivo AQP5 functional analysis. Figure 19 D), 0235 Figure 19 E) and 0116 ( Figure 19 F) Orthotopic transplantation results. Representative whole specimens and H&E images (left) depict the histological features of tumors formed from transplanted organoids. Quantification of tumor volume (middle) and invasiveness (right) indicates that OE organoids are capable of forming large tumors with more aggressive characteristics compared to their respective parental cell lines. Figure 19 G shows the results of a genomic PCR performed at the Aqp5-FLIP-Puro insertion site, confirming successful insertion based on the expected band size. Figure 19 Data shown in H indicate that AQP5 levels were significantly reduced in Aqp5 FLIP-Puro organoids after Cre recombinase treatment. Scale bar, 500 μm ( Figure 19 A, Figure 19 B. Figure 19 C) and 1mm ( Figure 19 D、 Figure 19 E, Figure 19F). The figure represents the mean ± standard deviation, and a two-tailed unpaired t-test was used.

[0041] Detailed description

[0042] Although Aqp5 is involved in driving tumor cell proliferation and migration in human gastric cancer cell lines, its exact mechanism of action remains unclear. Aqp5 is frequently expressed at high levels in gastric tumors, but its role in gastric cancer has not been fully explored in near-physiological cancer organoids and mouse models. The human gastric cancer cell line AGS, derived from intestinal tumors, exhibits moderate levels of Aqp5 in vitro. Figure 17 A). Three independent Aqp5-overexpressing AGS cell lines were generated, with highly elevated Aqp5 levels, and they were found to exhibit significantly increased cell proliferation and migration rates in vitro. Figure 17 A), which is consistent with previous studies. Increased cell migration was detected in vitro in three independent Aqp5-overexpressing cell lines, specifically in the isolated diffuse gastric cancer cell line SNU601. Figure 17 B). Furthermore, Aqp5-overexpressing AGS cells formed larger tumors after being implanted into the pylorus of immunodeficient mice via the original translocation, aggressively invading the epithelial and muscle layers (B). Figure 17 C). Conversely, knockout of Aqp5 in the KATOⅢ gastric cancer cell line, which expresses high levels of Aqp5, resulted in decreased cell proliferation rate in vitro. Figure 17 D). Although parental KATOⅢ cells seeded large tumors in the gastric submucosa after orthotopic transplantation, Aqp5-knockout KATOⅢ cells failed to establish tumors in most of the analyzed mice. Figure 17 E) indicates that Aqp5 may play a role in a key step in tumor establishment. Therefore, the data presented in this paper demonstrate that Aqp5 plays a role in tumorigenic traits that promote cell proliferation and migration in gastric cancer cell lines, and these findings are further extended to the in vivo environment through transplantation assays.

[0043] This disclosure describes the identification of Aqp5 as a biomarker for gastric cancer stem cells, which has been identified using various assays in physiologically relevant mouse and human gastric cancer models. This paper demonstrates that the Aqp5 surface biomarker facilitates the efficient isolation of promising gastric cancer stem cells for expression profiling to identify other biomarkers and potential therapeutic attackability. The elimination of Aqp5-expressing tumor cell populations, as illustrated herein, is an example of a therapeutic approach for improving gastric cancer progression.

[0044] Aqp5 is a functional biomarker for gastric cancer stem cells that drives tumorigenesis.

[0045] Cancer stem cells constitute a self-renewing population capable of generating differentiated tumor cell lineages and promoting tumor growth. In gastric tumors, biomarkers for cancer stem cells have been proposed, but to date, these biomarkers have largely remained confined to animal cancer models, widely expressed in many normal and cancerous tissues, or have failed to be robustly validated by functional assays demonstrating the potential of cancer stem cells in near-physiological mouse and human gastric cancer models. Crucially, the identification of such biomarkers could facilitate the isolation of cancer stem cells to elucidate their downstream functional mechanisms and contribute to the development of targeted therapies against this cell population in human gastric cancer, potentially supporting long-term disease remission. In normal tissues, Aqp5 marks pyloric stem cells in mice and humans, but in these Aqp5+ cells, dysregulation of a major pathway altered in gastric cancer is sufficient to drive pyloric tumor formation in mouse models. Here, using several representative in vitro and in vivo gastric cancer models, we demonstrate that Aqp5 is a functionally validated biomarker for gastric cancer stem cells present in mouse and human gastric tumors. This demonstrates the necessity and contribution of Aqp5+ tumor cells in maintaining tumor growth. These findings have improved our understanding of cancer stem cell models, enabling the development of diagnostic tools and treatment strategies targeting gastric cancer.

[0046] Therefore, this disclosure describes the use of Aqp5 as a biomarker for expression in gastric cancer stem cell repositories. In one instance, the biomarker disclosed herein is used to facilitate selective targeting and / or elimination of gastric cancer stem cell populations, thereby influencing disease progression. In another instance, Aqp5 is used as a biomarker for identifying gastric cancer. In yet another instance, a biomarker for gastric cancer stem cells is disclosed, wherein the biomarker is AQP5. In yet another instance, the biomarker is membrane-bound AQP5.

[0047] In another example, a method for identifying gastric cancer stem cells or gastric cancer is disclosed. In one example, the method disclosed herein includes a1) detecting the expression of aquaporin 5 (AQP5) in cells or cell populations; or b1) detecting the expression level of AQP5 in cells or cell populations and comparing the expression level with the expression level of AQP5 in reference cells or reference cell populations, wherein the detection of AQP5 expression in cells or cell populations, or an increase in the expression level of AQP5 in cells or cell populations compared to reference cells or reference cell populations, identifies said cells or cell populations as gastric cancer stem cells or gastric cancer stem cell populations.

[0048] In another instance, the method disclosed herein also includes isolating the identified gastric cancer stem cells or stem cell populations.

[0049] In one instance, the cells or cell populations disclosed herein are in vitro, in vivo, or isolated cells or cell populations. In another instance, the methods disclosed herein are methods performed in vitro, in vivo, or in vitro.

[0050] In yet another instance, the cells or cell populations disclosed herein are gastric tumor samples, biopsy tissues, or organoids. Examples of cells disclosed herein may include, but are not limited to, epithelial cells. In yet another instance, the cell populations disclosed herein include epithelial cells derived from gastric tumor samples or biopsy tissues.

[0051] In one instance, the method disclosed herein includes comparing the obtained expression level with an expression level obtained from a reference cell or cell population. Such a reference cell or cell population may be cells that do not express AQP5, optionally wherein the cells that do not express AQP5 are non-gastric cells, non-cancerous gastric cells, or a combination thereof. In one instance, the reference cell or cell population is a cell group comprising or composed of non-cancerous gastric cells.

[0052] Aqp5 is a biomarker for gastric cancer stem cells in mouse and human gastric tumors. Using a pre-defined protocol, Aqp5+ and Aqp5- cells were isolated from gastric tumors, demonstrating that Aqp5+ tumor cells function as stem cells, capable of seeding new tumors and reconstructing tumor masses upon transplantation into mouse recipients. Furthermore, the isolated Aqp5+ tumor cells selectively formed organoids in vitro, which could be maintained in long-term culture. These findings were obtained using a series of internally developed mouse and human gastric cancer models. These assays collectively demonstrate the cancer stem cell potential presented by the Aqp5+ tumor cell population and identify Aqp5 as a biomarker for gastric cancer stem cells, which has been validated in near-physiological human gastric cancer models.

[0053] Aqp5-labeled epithelial cell subsets in mouse and human pyloric tumors

[0054] To investigate the role of Aqp5+ tumor cells in gastric cancer, Aqp5 expression was first characterized in mouse pyloric tumors. As previously described, conditionally recombinant Apc, Pten, and Kras were fluxed between LoxP sites under the Aqp5-eGFP-IRES-creERT2 driver. G12D Alleles were used to outline a major co-dysregulated pathway prevalent in human gastric tumors, resulting in the formation of Aqp5-Cre / APK pyloric tumors, classified as tubular adenocarcinoma, in these mice within 2 to 3 months. Figure 1 A, Figure 4 A).

[0055] Therefore, in one instance, the cells or cell populations disclosed herein were further modified to express AQP5 that binds to an inducible gene. In one instance, the inducible gene is CreERT2. In another instance, the inducible gene is the inducible Caspase9 (iCasp9) gene.

[0056] Aqp5 expression is confined to the tissue-resident stem cell compartment at the pyloric gland base before cancer induction, and overlaps with Lgr5. Figure 1 B. Figure 4 C). In later stages of cancer progression, increasing numbers of Aqp5+ tumor cells were detected in the transformed pyloric epithelium, including in the region above the glandular base (C). Figure 1 B. Figure 1 C). Because the Aqp5-eGFP-IRES-creERT2 box disrupts endogenous Aqp5 expression, the spatiotemporal pattern of Aqp5 expression during tumor progression was observed using an alternative Aqp5-2A-creERT2 pyloric cancer model, in which endogenous Aqp5 expression was undisturbed. Figure 4 C). Consistent with these histological analyses, single-cell RNA sequencing (scRNAseq) of healthy pylorus and Aqp5-Cre / APK pyloric tumors showed elevated Aqp5 levels in a subset of tumor epithelial cell lineages (data not shown). While the expression of Aqp5 and Lgr5 was highly confined to the same stem cell cluster in healthy pylorus, Aqp5 was shown to be enriched in pyloric tumors in cell compartments largely different from those of Lgr5, with smaller subsets co-expressing both markers (data not shown).

[0057] To localize these Aqp5+ cell subsets within intact pyloric tumors, imaging-based spatial transcriptomics was performed using MERSCOPE, and a spatial cellular map of mouse Aqp5-Cre / APK tumors was constructed. Figure 4 D). This method revealed eight (8) clusters corresponding to tumor epithelial lineages ( Figure 4 E), of which three (3) clusters showed elevated Aqp5 transcription levels, which were mapped to the glandular base and tumor compartments immediately above the glandular base (E). Figure 4 D、 Figure 4 F). The Aqp5+ tumor lineage consists of Wnt-active clusters overlapping with Lgr5+ cells and highly proliferating clusters expressing Mki67. Figure 4 F), which is consistent with previously obtained scRNAseq data. RNAscope further validated Aqp5 expression in these mouse pyloric tumors relative to other putative gastric cancer stem cell markers Lgr5, Cxcr4, and Cd44. Figure 4 G), thus indicating that Aqp5 marks a previously unassessed subset of pyloric tumor cells.

[0058] In one instance, the method, as disclosed herein, includes further analysis of isolated gastric cancer stem cells, stem cell populations, or tumor organoids expressing AQP5. Methods for analyzing isolated gastric cancer stem cells or tumor organoids expressing AQP5 may include, but are not limited to, omics analyses such as transcriptomics analyses (e.g., RNA sequencing, single-cell RNA sequencing, spatial transcriptomics, gene ontology analysis, polymerase chain reaction analysis, or combinations thereof), proteomics analyses (e.g., single-cell proteomics), and combinations thereof. In one instance, the further analysis is spatial transcriptomics.

[0059] A strategy was established to separate Aqp5+ and Aqp5- cell populations of pyloric tumor epithelium within Aqp5-Cre / APK tumors using fluorescence-activated cell sorting (FACS) to extract biological insights from Aqp5+ tumor cells. Figure 1 D). The Aqp5-Cre / APK model contains Rosa26-tdTomato LSL A reporter gene, which robustly traces the entire pyloric gland within one week post-induction, was used to facilitate the isolation of tumor epithelial populations using tdTomato as a marker, and eGFP was used as a reporter gene for Aqp5 expression. qPCR showed that sorted eGFP+ tumor epithelial cells exhibited an average enrichment of 9.8-fold in Aqp5 levels compared to eGFP- cells. Figure 1 E). Using this sorting strategy, transcriptomic analysis was performed on Aqp5+ and Aqp5- tumor epithelial cells isolated from six independent Aqp5-Cre / APK mouse pyloric tumors. Figure 1 F). This reveals the selective enrichment of extracellular matrix (ECM) remodeling, epithelial-mesenchymal transition (EMT), and drug resistance-related pathways in the transcriptome of Aqp5+ tumor cells. Figure 5 A, Figure 5 B, Table 1), these characteristics are often associated with cancer stem cells in other systems. Many of these upregulated genes, including Pthlh, Rgs5, and Hey1, were validated by qPCR and RNAscope using additional mouse pyloric tumor samples. Figure 5 C to Figure 5 G (Table 2) shows their significant enrichment in Aqp5+ tumor cell populations.

[0060] Therefore, in one instance, the cells or cell populations disclosed herein are modified to express AQP5 coupled with one or more detectable markers. Such detectable markers can be, but are not limited to, fluorescent markers, tags, proteins, and combinations thereof. Incomplete examples of fluorescent markers include tdTomato, GFP, eGFP, RFP, YFP, and combinations thereof. In one instance, the fluorescent marker is eGFP.

[0061] In yet another instance, the gastric cancer stem cells disclosed herein express one or more markers of gastric cancer progression (e.g., but not limited to Pthlh, Hey1, Rgs5), cancer stem cell function (e.g., but not limited to Hey1, Clmp, Cyb1p1), tumor microenvironment (e.g., Rgs5, Adamntsl3, Itgb8), and combinations thereof.

[0062] Aqp5-expressing stem cells have previously been identified and characterized in the pylorus of healthy individuals. To provide a detailed evaluation of their tumor equivalents, Aqp5 expression was analyzed using publicly available human scRNAseq datasets from healthy stomach and gastric tumor tissues. A total of six (6) epithelial clusters were identified. Figure 6 A) includes lineages rich in gastric mucosal (Muc5ac+, Muc6+), chief cell (Pgc+), and enteroendocrine (Chga+) markers, as well as intestinal (Tff3+, Fabb1+) markers. Figure 6 B). In the pylorus of healthy individuals, Aqp5 is primarily expressed in the basal cell clusters of Muc6+ mucinous glands, and has previously been associated with gastric stem cell compartments in mice and humans. Figure 6 C). This contrasts with Aqp5 expression in human gastric tumors, which is shown to be increased in multiple epithelial cell clusters (C). Figure 6 C Figure 6 D). Aqp5 expression was also observed to show a tumor subtype-specific pattern, with the greatest increase in Aqp5 levels detected in intestinal tumors, while diffuse tumors showed no significant difference in Aqp5 levels compared to healthy tissues. Figure 6 E to Figure 6 H). These results were confirmed using an independent human scRNA-seq dataset, in which Aqp5 was also significantly higher in gastric tumor cells than in its healthy equivalent (H). Figure 6 I). This was further confirmed by detecting Aqp5 at the protein level using IHC performed on a human gastric tissue microarray. Figure 1 G), confirming previous work. In summary, these analyses identified that elevated Aqp5 expression signatures in human gastric tumors are primarily associated with intestinal-type tumors, and these signatures were well captured in the Aqp5-Cre / APK mouse pyloric cancer model presented in this paper.

[0063] Therefore, in one instance, the method disclosed herein includes detecting the expression level of AQP5. This expression level can be detected, for example, as a gene expression level, a protein expression level, or a combination thereof. In one instance, the method, as described herein, includes a step of detecting the protein expression level of AQP5. In another instance, the method, as described herein, includes a step of detecting the gene expression level of AQP5.

[0064] In another instance, the protein expression level of AQP5 can be detected using methods such as, but not limited to, immunohistochemistry (IHC), flow cytometry, Western blotting, and combinations thereof. In one instance, immunohistochemistry is used to detect the protein expression level of AQP5. In another instance, the expression level of AQP5 is the gene expression level. Therefore, the gene expression level of AQP5 can be obtained or quantified, for example, by performing polymerase chain reaction (PCR).

[0065] Since Aqp5 is a membrane-bound protein, the sorting protocol described in this paper is suitable for antibody-based FACS separation of Aqp5+ human pyloric tumor cells and Aqp5- human pyloric tumor epithelial cells from freshly collected patient tumor biopsy tissues. Figure 1 H).

[0066] Therefore, the methods disclosed herein may also include the step of isolating one or more identified gastric cancer stem cells. Methods for isolating these cells include, but are not limited to, single-cell sorting, fluorescence-activated cell sorting (FACS), magnetic sorting, or combinations thereof. In one example, fluorescence-activated cell sorting (FACS) is used to isolate cells.

[0067] In another example, a method for isolating one or more types of gastric cancer stem cells from a cell population is described, comprising i) contacting cells of the cell population with an agent that binds to AQP5; and ii) isolating one or more AQP5-expressing cells that bind to the agent, wherein one or more AQP5-expressing cells are gastric cancer stem cells.

[0068] 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 binding to AQP5 is an antibody. In one example, the agent binding to AQP5 may affect the function of AQP5 once bound. In another example, the antibody is a detection antibody. In yet another example, the antibody may be conjugated to a label, such as a detectable label. Examples of such detectable labels are, but are not limited to, fluorescent labels, cleavable labels, separation labels, purification labels, and combinations thereof. In yet another example, the agent is conjugated to a compound (such as a drug or small molecule).

[0069] This FACS sorting strategy was validated by qPCR, which showed that Aqp5 levels were upregulated by an average of 10.8-fold in the sorted Aqp5+ cell population. Figure 1 I). Using this sorting method, batch RNAseq analysis was performed on isolated Aqp5+ and Aqp5- human pyloric tumor cells from five (5) independent tumor samples. Figure 1J). Similar to the mouse Aqp5+ profile, human Aqp5+ tumor cells exhibit transcriptomic features enriched with cancer stem cell-related phenotypes, including ECM tissue, EMT, and chemotherapy resistance. Figure 7 A, Figure 7 B, Table 3). Using additional independent patient samples, the selected upregulated targets, including CLDN2, HEY2, and PLA1A, were further validated by qPCR and RNAscope. Figure 7 C to Figure 7 Table 4 (G) confirms the reliability of the RNAseq dataset. In summary, mouse and human Aqp5+ pyloric tumor cells exhibit a common cancer stem cell-enriched transcriptome profile, which differs from the characteristics of healthy Aqp5+ pyloric stem cells, thus highlighting their therapeutic value.

[0070] Aqp5+ tumor cells function as cancer stem cells in pyloric tumors in mice and humans.

[0071] Biomarkers for healthy and cancer stem cells are known to highly overlap across many tumor settings. However, many gastric cancer stem cell biomarkers, including Lgr5, Cd44, and Cxcr4, have not been effectively validated by functional assays that directly demonstrate the stem potential of the tumor cell populations they label. Given that Aqp5 marks healthy mouse and human pyloric stem cell compartments, and that many reported cancer stem cell-related pathway signatures are enriched in the transcriptome of Aqp5-derived tumor cells (…),… Figure 5 and Figure 7 The study inquired whether Aqp5 could also serve as a specific biomarker for pyloric cancer stem cell populations within gastric tumors in mice and humans. To functionally test this hypothesis, Aqp5+ and Aqp5- epithelial tumor cells were isolated from pyloric tumors in Aqp5-APK mice using FACS and seeded for organoid culture. This revealed that long-term (at least 10 passages) maintenance of Aqp5+ tumor cells resulted in the formation of organoids. Figure 2 A, Figure 2 B). Furthermore, organoids derived from isolated Aqp5+ tumor cells comprise multiple differentiated cell lineages that resemble organoids derived from intact pyloric tumor glands. Figure 2 C Figure 8 A). In contrast, sorted Aqp5-tumor cells formed significantly fewer organoids in cultures, and these organoids were rapidly lost within two generations. Figure 2 A, Figure 2 B) reflects a lack of self-renewal capacity. Therefore, Aqp5 has been shown to mark a functional cancer stem cell population capable of long-term growth of tumor organoids within mouse pyloric tumors.

[0072] In one instance, the method disclosed herein includes a step of further culturing isolated gastric cancer stem cells in the presence of a culture medium free of growth factors. In other words, in one instance, the step of further culturing isolated gastric cancer stem cells is performed in the absence of growth factors in the cell culture medium. In another instance, the method disclosed herein includes a step of isolating gastric cancer stem cells, wherein the isolated gastric stem cells form tumor organoids expressing AQP5.

[0073] In one instance, gastric cancer stem cells are mammalian cells. Examples of such mammalian cells are, but are not limited to, mouse (mouse) cells and human cells.

[0074] Another approach to assessing the potential of cancer stem cells is to transplant specific cell populations into mice to evaluate their tumorigenetic capacity in an in vivo context. As is known in the art, transplanting sorted cell populations from solid tumors remains challenging, and the efficiency of tumorigenesis varies significantly depending on tissue type and recipient. Consistent with this observation, tumor cells isolated from Aqp5-Cre / APK tumors failed to establish significant tumor formation even after 4 months of in vivo transplantation into the pylorus of immunodeficient mice. Figure 8 B). To address this issue, Aqp5-Cre / APK pyloric tumor organoids were transplanted into immunodeficient mice. Aqp5+ and Aqp5- cells sorted from the orthotopic tumor via FACS were derived from these mice. Figure 8 C to Figure 8 E), because these cells are thought to be better adapted to the local tumor environment within their host. Results showed that Aqp5+ and Aqp5- tumor epithelial cells sorted from Aqp5-Cre / APK orthotopic tumors seeded new tumors in mice more effectively than cells isolated from the primary tumor, thus providing a feasible system for assessing their tumorigenetic capacity. Figure 8 F). In the five (5) groups of independent mice examined, Aqp5+ tumor cells consistently established larger in situ tumors and exhibited more aggressive proliferative and invasive characteristics compared to Aqp5- tumor cells from the same tumor. Figure 2 D、 Figure 2 E, Figure 8 F). Tumors derived from Aqp5+ cells also showed the presence of both Aqp5+ and Aqp5- cells, including the expression of markers of multidifferentiated cell lineages (F). Figure 8 F) indicates that these Aqp5+ cancer stem cells have the ability to reconstruct different lineages within their primary tumors. Finally, Aqp5+ tumor cells and Aqp5- tumor cells were isolated from a pyloric cancer mouse model integrating the constitutively active p53R172H mutant. Figure 8 G, Figure 8H) was used to assess the stem potential of Aqp5+ cells in an independent tumor setting. FACS isolation of these cells was validated by qPCR, reflecting a 38-fold enrichment of Aqp5 in the sorted Aqp5+ population. Figure 8 I, Figure 8 J). In this cancer model, sorted Aqp5+ tumor cells were also observed to form a higher number of organoids in vitro compared to Aqp5- cells derived from the same tumor. Figure 8 K, Figure 8 This indicates that Aqp5+ cells can also function as stem cells in this context. In summary, organoid development and transplantation assays identified mouse Aqp5+ tumor cells as a functional gastric cancer stem cell population capable of driving tumor growth. To assess whether human Aqp5+ tumor cells also function as cancer stem cells, single Aqp5+ and Aqp5- human pyloric tumor cells were isolated from primary tumor samples and plated for organoid culture. It was found that human Aqp5+ tumor cells also generated tumor organoids at a higher rate than corresponding Aqp5- cells isolated from the same tumor. Figure 2 F, Figure 2 G). Aqp5+ tumor cell-derived organoids exhibit long-term self-renewal capacity (at least 10 generations) and produce differentiated cell lineages similar to human organoids derived from intact pyloric glands. Figure 2 H, Figure 9 A), indicating that the Aqp5+ cell population retained its intrinsic stem cell capacity for reconstructing cell lineages and its original tumor characteristics. In contrast, the small fraction of Aqp5- cell-derived organoids formed failed to proliferate for more than 3 generations on average. Figure 2 F, Figure 2 G).

[0075] As an additional readout of the stem cell potential of human Aqp5+ tumor cell population, human gastric tumor organoids with CRISPR / Cas9-mediated Aqp5-2A-CreERT2 and CAG-LSL-tdTomato inserts were established, enabling in vitro lineage tracing of human Aqp5+ cells and their progeny within these tumor organoids. Figure 9 B). Administration of 4-OHT to these tumor organoid cultures resulted in the labeling of a small population of Aqp5+ cells, which expanded to contain an average of 51% organoid region after 10 days. Figure 9 C to Figure 9 E), overlapping with differentiated gastric lineage markers ( Figure 9 F). In contrast, at the same time point, uninduced organoids did not show tdTomato tracerization in vitro (F). Figure 9E). Therefore, human Aqp5+ tumor cells from primary patient samples and human organoid models have demonstrated stem cell function, directly contributing to the long-term maintenance of organoid cultures and the generation of multi-differentiated cell lineages.

[0076] The removal of Aqp5+ tumor cells hindered tumor development.

[0077] Cancer stem cells are considered a major source of tumor recurrence because they are resistant to standard cancer treatments, such as those involving the activation of drug efflux transporters. Therefore, targeting cancer stem cells has been proposed as a method to eliminate tumor burden and recurrence, but this approach has been hampered by the lack of well-validated and clinically relevant cancer stem cell biomarkers. Given the functional characteristics of mouse and human Aqp5+ cells as a cancer stem cell population in pyloric tumors (…), Figure 2 The effects of Aqp5+ cell depletion in mouse and human gastric tumor models were tested as an independent functional assessment of its cancer stem cell characteristics. The efficacy of targeting Aqp5+ cancer stem cell populations as a therapeutic approach for gastric cancer was also evaluated.

[0078] To effectively remove Aqp5+ tumor cells from pyloric tumors in mice, the Aqp5-2A-DTR allele was integrated into the Aqp5-Cre / APK pyloric cancer mouse model. Figure 10 A). The Aqp5-2A-DTR box allows Aqp5 to be endogenously expressed along with the diphtheria toxin receptor (DTR) in these Aqp5-expressing cells. Aqp5-Cre / APK / DTR tumors and Aqp5-Cre / APK tumors are indistinguishable in terms of tumor morphology, tumor burden, and the proportion of Aqp5+ resident cells. Figure 10 B). To selectively target Aqp5+ pyloric tumor cells, diphtheria toxin (DT) was administered intraperitoneally to Aqp5-Cre / APK / DTR mice carrying pyloric tumors. A significant reduction in tumor size was observed twelve (12) hours after the last DT administration. Figure 10 C). This confirmed the loss of Aqp5 expression in tumors treated with DT and the simultaneous presence of Caspase3+ apoptotic cells and Ki67+ proliferating cells in the surviving tumor burden. Figure 10 D).

[0079] Therefore, in one instance, the method disclosed herein also includes the elimination or removal of gastric cancer stem cells. In one instance, elimination or removal is carried out using compounds selected from, but not limited to, the group consisting of diphtheria toxin, iCasp9 inducers (e.g., agents that dimerize iCasp9), and agents that selectively bind AQP5.

[0080] In Aqp5-Cre / APK mice lacking the Aqp5-2A-DTR allele ( Figure 10 E), or in Aqp5-Cre / APK and Aqp5-Cre / APK / DTR mice that received PBS injections without DT ( Figure 10 D、 Figure 10 E) Diphtheria toxin (DT) did not show any effect on pyloric tumors. 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 making it impossible to analyze late-stage tumor time points. To address this issue, tumor organoids from Aqp5-Cre / APK / DTR pyloric tumors were generated, which maintained a small number (9.2%) of cells with high Aqp5 expression in culture. Figure 10 F). Adding DT to Aqp5-Cre / APK / DTR organoid cultures during the initial stages of organoid growth was shown to remove most Aqp5+ cells, with Aqp5 levels decreasing by 80%-90% one day after treatment. Figure 10 G), and was also shown to completely eliminate organoid growth in four (4) independent cell lines tested. Figure 3 A, Figure 3 B). The effects of DT administration were also tested on established Aqp5-Cre / APK / DTR organoid cultures, which resulted in a >90% reduction in Aqp5 levels. Figure 10 G) and the organoids rapidly degenerated within one day of treatment, with no surviving organoids remaining after four days. Figure 3 C Figure 3 D). Conversely, administration of the same DT dose to Aqp5-Cre / APK organoids lacking the Aqp5-2A-DTR allele had no effect on Aqp5 expression or organoid growth and subsequent organoid maintenance. Figure 10 H to Figure 10 This highlights the specific effects of Aqp5+ cell removal on the observed organoid phenotype.

[0081] Therefore, in one instance, diphtheria toxin was used for elimination or removal.

[0082] In yet another example, a method for removing or eliminating AQP5+ gastric cancer stem cells 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 iCasp9 inducer is an agent that dimers iCasp9.

[0083] This article also describes a method for removing or eliminating AQP5+ gastric cancer stem cells, which involves contacting the cells with an agent that binds to AQP5, wherein the binding of AQP5+ cells to the agent causes the cells to be removed or eliminated.

[0084] To confirm these findings in a more physiological system, a method was established to facilitate the selective removal of Aqp5+ cells from pyloric tumors derived from these organoids by orthotopic transplantation of Aqp5-Cre / APK / DTR tumor organoids into the pylorus of immunodeficient mice. Because the Aqp5-2A-DTR allele is not expressed in other tissues of these mice, it was possible to monitor tumor progression to later stages after DT administration without the complications associated with systemic removal of Aqp5+ cells. Orthotopic transplantation of Aqp5-Cre / APK / DTR pyloric organoids was shown to induce pyloric tumor initiation within 4 weeks and signs of epithelial invasion at 8 weeks. Figure 3 E, Figure 3 F). In all five (5) independent experiments conducted, intraperitoneal injection of DT immediately after orthotopic transplantation of Aqp5-Cre / APK / DTR tumor organoids in mice resulted in the loss of Aqp5-expressing cells (F). Figure 10 K) and even after 4 weeks, it still suppressed tumor growth ( Figure 3 E). Four weeks after orthotopic transplantation of Aqp5-Cre / APK / DTR organoids, DT was also administered to established tumors in mice to verify the removal efficiency in these tumors. Figure 10 L). Compared to untreated tumors, tumors treated with DT showed reduced tumor burden and no signs of invasion into surrounding tissue layers. Figure 3 F). Furthermore, it was confirmed that in both early and late stages of tumor growth, orthotopic tumors derived from Aqp5-Cre / APK tumor organoids but lacking the Aqp5-2A-DTR allele were unaffected by DT administration. Figure 10 M, Figure 10 In summary, these results indicate that in organoid and mouse models of pyloric carcinoma, maintaining tumor establishment and progression depends on mouse Aqp5+ tumor cells.

[0085] The effects of removing human Aqp5+ cells were tested in a near-physiological human gastric cancer organoid system. For this purpose, human organoids expressing Aqp5-2 AiCaspase were generated, containing Aqp5+ cells co-expressing inducible caspase9 (iCaspase9), which, upon administration of a dimerizing agent, selectively induced cysteine ​​protease-mediated cell death in Aqp5+ cells. Figure 3 H, Figure 3 I and Figure 11C). Compared with unedited tumor organoids, the resulting Aqp5-2A-iCaspase organoids exhibited comparable organoid morphology, Aqp5 expression, and cell lineage marker expression (C). Figure 11 B).

[0086] Therefore, in one instance, the cells or cell populations disclosed herein were modified to express AQP5 coupled to the diphtheria toxin receptor (DTR) gene or the inducible Caspase9 (iCasp9) gene.

[0087] As described in this article, the Aqp5-2A-iCaspase9 construct was generated for expression in cells. This means that cells expressing Aqp5 will simultaneously produce iCaspase9. Upon the addition of an inducer such as AP20187 (a dimerizing agent), iCaspase9 undergoes homodimerization to produce a functional protein, leading to apoptosis-specific apoptosis in cells expressing Aqp5, while cells not expressing Aqp5 remain intact.

[0088] To remove human Aqp5+ cells, a B / B homodimer was applied to Aqp5-2AiCaspase human organoid cultures. This resulted in a 60% loss of Aqp5 expression in the treated organoids. Figure 11 C). These treated Aqp5-2A-iCaspase tumor organoids also showed reduced growth rates in cultures. Figure 3 H, Figure 3 I), but they still survived. Unbound by theory, this is thought to be due to residual, unremoved Aqp5+ cells in these cultures. Unedited tumor organoids were unaffected by B / B homodimer treatment, and no significant differences in Aqp5 levels and organoid size were observed. Figure 11 D). Removal experiments were performed in the independent human gastric cancer organoid cell line GC10, thus confirming the specific effect of Aqp5+ cell removal on organoid growth. Figure 11 E to Figure 11 H). Like their mouse counterparts, human Aqp5+ cancer stem cells in tumor organoids have also been shown to be key drivers of organoid growth, thus highlighting their effectiveness as therapeutic targets to drive cancer regression.

[0089] Cancer stem cell models link clinical observations of chemotherapy resistance and tumor recurrence with the activity of specialized stem cell banks in promoting sustained tumor growth, which can then be used for therapeutic purposes. While the cancer stem cell theory has been validated in many hematologic, brain, and colon cancers, the existence of such stem cell banks in gastric tumors requires further validation using multiple assays of stem cell activity in physiologically relevant mouse and human gastric cancer models. Here, we show that both mouse and human pyloric tumors possess unique populations of Aqp5+ cancer stem cells that, when transplanted into the mouse stomach, were able to initiate long-term organoid cultures and reconstruct invasive pyloric tumors. Targeted removal of Aqp5+ cells was also shown to be sufficient to block the initiation and growth of pyloric tumors, establishing the central role of these cells in disease progression. These findings suggest that gastric tumors can be driven by Aqp5+ stem cell populations, highlighting the potential for using Aqp5+ cancer stem cells as a target in gastric cancer therapy.

[0090] Within the scope of this disclosure, the use of Aqp5 as a membrane-bound cancer stem cell marker suitable for FACS sorting is also considered. This application facilitates the specific isolation of these gastric cancer stem cells directly from natural tumors, enabling in-depth research into cancer stem cell biology and the mechanisms driving their tumorigenetic behavior. This includes identifying pathways that promote cancer stem cell function in these Aqp5-expressing cells, as well as other cancer stem cell-specific genes that represent targets that can be used to eliminate or interfere with the function of Aqp5-expressing cell populations.

[0091] Therefore, in one instance, the method for detecting, isolating, or identifying gastric cancer stem cells, as disclosed herein, includes detecting, isolating, or identifying membrane-bound AQP5. In other words, in another instance, the AQP5 disclosed herein is membrane-bound.

[0092] Therefore, this disclosure also contemplates methods for treating gastric cancer in subjects who require it. In one instance, a method for treating gastric cancer in a subject identified as having gastric cancer stem cells or gastric cancer may include the step of administering a therapy or compound to the subject that conforms to standard treatment for gastric cancer. Examples of standard treatments may include, but are not limited to, capecitabine, cyramza (ramucirumab), docetaxel, doxorubicin hydrochloride, enhertu (Fam-trastuzumab deruxtecan-nxki), 5-FU (fluorouracil injection), ramucirumab, fluorouracil, Herceptin (trastuzumab), Keytruda (pembrolizumab), lansifol (trifluorouridine and tipyracillin hydrochloride), mitomycin, nivolumab, Opdivo (nivolumab), pembrolizumab, ramucirumab, Taxotere (docetaxel), trastuzumab, fluorouridine and tipyracillin hydrochloride, capecitabine, resection / surgery / surgical resection, endoscopic mucosal resection, perioperative chemotherapy with or without radiotherapy, radiotherapy, postoperative (adjuvant) chemoradiotherapy, postoperative (adjuvant) chemotherapy, and their combinations. As will be understood by those skilled in the art, once identified, treatment can vary depending on the stage or progression of the disease.

[0093] In one example, the method includes administering to a subject a therapeutically effective amount of one or more agents that eliminate or remove cells expressing AQP5. In another example, the treatment includes curing gastric cancer, reducing gastric cancer growth, slowing gastric cancer progression, improving the prognosis of a subject, or a combination thereof. In yet another example, the use of one or more agents that eliminate or remove cells expressing AQP5 is disclosed in the manufacture of a medicament for treating gastric cancer. In another example, one or more agents for treating gastric cancer that eliminate or remove cells expressing AQP5 are disclosed. In yet another example, one or more agents for treating gastric cancer that eliminate or remove cells expressing AQP5 are disclosed.

[0094] In one instance, the subject was either a human or a mouse. In another instance, the subject was a human.

[0095] This article also discloses one or more agents for treating AQP5-expressing cells. In another example, one or more agents for treating gastric cancer that eliminate or remove AQP5-expressing cells are disclosed.

[0096] The methods disclosed in this paper can also be used to develop methods for identifying, targeting, and eliminating gastric cancer stem cells expressing Aqp5 in tumor burden to inhibit or halt cancer progression. Detection of gastric cancer cells expressing Aqp5 within tumors can also aid in cancer diagnosis and serve as an indicator of disease stage / severity.

[0097] This article also considers kits for identifying, isolating, eliminating, or removing gastric cancer stem cells. In one example, the kit described herein contains an agent that binds to AQP5 and instructions for use. In another example, the kit also contains an antibody, such as a detection antibody. In yet another example, the antibody is conjugated to a compound such as a detectable marker, drug, or small molecule. In yet another example, the antibody binds to the intracellular or extracellular domains of the AQP5 protein.

[0098] In various near-physiological mouse and human intestinal and diffuse gastric cancer models, AQP5 has been identified as a major driver of gastric cancer progression. In gastric cancer patients, AQP5 is widely overexpressed across multiple subtypes / stages of gastric tumors and their associated metastases relative to healthy gastric tissue, highlighting the potential cancer-specific role of Aqp5 in driving tumorigenesis. Indeed, knockout of Aqp5 in our mouse and human gastric cancer models resulted in a significant reduction in tumor burden and, in some cases, complete elimination of cancer development. Conversely, AQP5 overexpression accelerated the development of tumor characteristics. Targeting Aqp5 expression and / or its downstream functions represents a promising new direction for gastric cancer therapy.

[0099] Stomach cancer is one of the leading causes of cancer-related deaths globally and in Singapore, with a five-year survival rate of less than 30%. Currently, treatment for stomach cancer is limited to traditional methods such as chemotherapy, radiotherapy, and surgical resection, but these methods remain ineffective in improving cancer recurrence in many patients. Therefore, there is a need to evaluate new treatment modalities, such as novel functional modulators that target cancer development, progression, and metastasis.

[0100] In this study, Aqp5 was identified as a major driver of gastric cancer progression in various near-physiological mouse and human gastric cancer model systems. In patients with gastric cancer, Aqp5 was frequently overexpressed in both intestinal and diffuse tumors relative to healthy gastric tissue; this phenotype was also reflected in both healthy and cancerous gastric tissues in mice. Figure 17 and Figure 18 Using a group of human gastric cancer cell lines, it was shown that both intestinal and diffuse cancer cell lines exhibited increased levels of cell proliferation and migration in vitro when Aqp5 was overexpressed. Furthermore, after orthotopic transplantation of these human gastric cancer cell lines into the pyloric submucosa of immunodeficient mice, the Aqp5-overexpressing cell lines produced larger tumors that displayed more aggressive histological features, including high levels of cell proliferation and invasion of adjacent tissues. Figure 19Furthermore, these tumor phenotypes can be replicated in independent human gastric cancer organoid cell lines. Compared to two-dimensional cell line monolayer cultures, these three-dimensional organoid models grow in an extracellular matrix-like environment that preserves morphological and molecular characteristics similar to native epithelium. Xenografting of human gastric cancer organoids into the pylorus of immunodeficient mice seeded pyloric tumors, and Aqp5-overexpressing organoids established larger, more aggressive tumors that frequently invaded through the pyloric epithelium and outer muscular layer. Therefore, this work establishes Aqp5 as a pervasive driver of gastric cancer tumorigenesis across multiple gastric cancer types and models.

[0101] To further evaluate the function of Aqp5 and the potential of therapeutic strategies targeting Aqp5 expression, an Aqp5 knockout gastric cancer model was established, and the impact of Aqp5 loss on gastric tumorigenesis was assessed (Figure 20).

[0102] In human gastric cancer cell lines lacking Aqp5, cell proliferation was slower and tumor implantation was severely impaired when orally transplanted into immunodeficient mice. Similarly, human gastric cancer organoids with CRISPR / Cas9-mediated Aqp5 knockout induced significantly smaller tumors in mice that failed to invade surrounding tissue layers. These findings confirm the role of Aqp5 in driving tumorigenesis and highlight the potential of Aqp5 elimination as a method to block tumor progression and potentially promote its regression.

[0103] Finally, an Aqp5 knockout mouse model of gastric cancer was established, in which tumors could be selectively generated within the pylorus of the stomach by inducing targeted recombination of cancer-related alleles. Aqp5 knockout mice were phenotypically normal and did not exhibit alterations in gastric function, but developed smaller pyloric tumors upon induction of tumor formation. Furthermore, gastric cancer stem cells isolated from Aqp5 knockout tumors showed reduced stem cell potential, producing fewer organoids during culture compared to Aqp5 wild-type counterparts. In conclusion, this study demonstrates that Aqp5 plays a specific role in the context of gastric cancer, driving tumorigenesis and promoting disease development and progression. Therefore, targeting Aqp5 expression and / or its regulatory factors is a feasible therapeutic approach for gastric cancer patients.

[0104] Therefore, in one instance, a method for inhibiting the occurrence or progression of gastric cancer is disclosed, which includes administering an AQP5 inhibitor to a subject. In another instance, a method for promoting gastric cancer regression is disclosed, which includes administering an AQP5 inhibitor to a subject.

[0105] In one instance, 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.

[0106] In one instance, the AQP5 inhibitor is a repressive RNA (RNAi). Examples of RNAi include, but are not limited to, siRNA, shRNA, and miRNA. In another instance, the AQP5 inhibitor is siRNA. In one instance, the siRNA can be, but is not limited to, sense and antisense primer pairs. In another instance, the primers are, but are not limited to, 5′-AAAACTCTGCGAACACGGCCCCTGTCTC-3′ (SEQ ID NO:49) and 5′-AAGGCCGTGTTCGCAGAGTTCCTGTCTC-3′ (SEQ ID NO:50); 5′-CGGUGGUCAUGAAUCGGUUTT-3′ (SEQ ID NO:51) and 5′-AACCGAUUCAUGACCACCGCA-3′ (SEQ ID NO:52); and 5′-GCGUGUGGCCAUCAUCAAATT-3′ (SEQ ID NO:53) and 5′-UUUGAUGAUGGCCACACGCTT-3′ (SEQ ID NO:54) and combinations thereof.

[0107] In one instance, the drug is, but is not limited to, an enzyme inhibitor, a receptor antagonist, and a channel blocker.

[0108] The study found that functional drivers of gastric cancer progression, when overexpressed, accelerate the development of tumorigenetic features. In contrast, knocking out these drivers attenuated gastric cancer progression and, in some cases, was sufficient to block cancer development. Therefore, this suggests that targeting the expression of AQP5 and / or its functional regulators could be a therapeutic approach for gastric cancer.

[0109] As presented in this paper, multiple mouse and human gastric cancer cell lines, organoids, and xenograft mouse models with Aqp5 knockout and Aqp5 overexpression have been generated. These models have validated the function of Aqp5 in independent systems and various gastric cancer subtypes, highlighting the broad relevance of our findings to most gastric cancer patients. Furthermore, without being bound by theory, the Aqp5 knockout gastric cancer genetic mouse model disclosed in this paper allows for targeted induction of pyloric tumor formation in mice. This provides insights into the role of Aqp5 throughout the entire disease timeline, from onset to tumor spread in the native tissue environment. Therefore, the techniques disclosed in this paper powerfully demonstrate the role of Aqp5 in gastric tumorigenesis in independent, near-physiological models that capture the diversity of human gastric tumor types, and underscore the utility of targeting Aqp5 and / or regulators of Aqp5 expression and function as therapeutic approaches for gastric cancer.

[0110] 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 gastric cancer treatment; and b) measuring the expression level of AQP5 in a control sample obtained from the subject before gastric cancer treatment; wherein an elevated AQP5 expression level in the sample of step a, compared to the control sample, indicates that gastric cancer tumorigenesis has occurred or that gastric cancer has progressed.

[0111] 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 point earlier than that of the sample obtained in step c; wherein the elevated AQP5 expression level in the sample obtained in step c compared to the reference sample obtained in step d indicates that gastric cancer tumorigenesis has occurred.

[0112] In one instance, the expression level of AQP5 is a gene expression level, a protein expression level, or a combination thereof. In another instance, the method described herein also includes measuring the expression levels of one or more biomarkers of gastric cancer progression. In yet another instance, the expression levels of one or more biomarkers of gastric cancer progression are measured in addition to the other expression levels disclosed herein. Examples of biomarkers of gastric cancer progression may be, but are not limited to, Pthlh, Hey1, Rgs5, and combinations thereof.

[0113] In one instance, if gastric cancer tumors have already developed or gastric cancer has progressed, the subject is treated with an anti-gastric cancer compound or standard gastric cancer treatment.

[0114] Those skilled in the art will be able to identify which methods can be used to measure the levels disclosed herein. In one example, the steps for measuring AQP5 expression levels are immunohistochemistry, flow cytometry, Western blotting, or a combination thereof. In another example, the steps for measuring AQP5 expression levels are polymerase chain reaction.

[0115] In one instance, the expression level in a sample is measured. Examples of such samples include, but are not limited to, blood, plasma, biopsy samples, tissue samples, primary cell culture samples, and primary organoid lines.

[0116] The methods disclosed in this article relate to gastric cancer. Gastric cancer, also known as stomach cancer, is a type of cancer that occurs in the gastric mucosa. Gastric cancer can include, but is not limited to, adenocarcinoma, lymphoma, and mesenchymal tumors. In one instance, the gastric cancer is, but is not limited to, intestinal-type gastric cancer or diffuse gastric cancer.

[0117] Identifying specific genes expressed only in tumors but completely absent in normal tissues is challenging. In fact, AQP5 is not only expressed in gastric cancer but also present in a small subset of healthy tissues, including the lungs, salivary glands, and testes. AQP5 levels are low and it is primarily found in the cytoplasm of many healthy tissues, including the lungs. Furthermore, mice lacking AQP5 (e.g., complete knockout mice) are vigorous and healthy, with only mild salivation defects recorded in these animals, suggesting that AQP5 may not play a crucial role outside of cancerous states. Targeting approaches to eliminate AQP5 or its modulators do not have life-threatening consequences; therefore, an appropriate therapeutic window can be calibrated to ensure effective targeting of gastric tumor cells expressing AQP5 while causing minimal damage to other healthy tissues expressing AQP5.

[0118] This technology facilitates the development of methods to identify, target, and eliminate AQP5 and / or regulators of AQP5 expression and function within gastric tumors as a means of inhibiting cancer progression. Furthermore, the detection of high AQP5 levels in gastric tumors can serve as an indicator of disease stage / severity and aid in cancer diagnosis. This suite of in vitro and in vivo gastric cancer models, as well as the AQP5 knockout gastric cancer genetic mouse model generated in this paper, can also be used for drug testing to evaluate novel therapies aimed at improving disease progression. Finally, this technology enables the identification of other AQP5 protein chaperones and direct downstream pathways regulated by AQP5 that drive tumor progression, expanding the list of targetable components and the range of therapeutic approaches available to gastric cancer patients.

[0119] Using various near-physiological gastric cancer models, such as Figures 16 to 2 The experimental data shown in Figure 0 highlight the central role of AQP5 in driving gastric cancer. AQP5 knockout gastric cancer cells exhibited lower proliferation and formed smaller, less aggressive tumors in vivo. Conversely, overexpression of AQP5 in gastric cancer cells increased cell proliferation and migration in vitro and accelerated tumor progression in vivo. Finally, using the AQP5 knockout gastric cancer mouse model disclosed in this paper, it was shown that in the complete absence of AQP5, mice developed smaller precancerous lesions, rather than the fully grown gastric adenocarcinoma typically observed in such cancer mouse models.

[0120] Furthermore, previously unknown human gastric cancer organoid lines integrating the Flip-Puro system have been generated, which conditionally knocks out AQP5 after administration of Cre recombinase cellular nanovesicles. The loss of AQP5 in these organoids was confirmed by Western blotting. Figure 2 As shown in Figure 1, a decrease in cell viability was found in organoids after AQP5 knockout.

[0121] Aqp5 drives tumor progression in mouse and human gastric cancer models.

[0122] A mouse model of Aqp5 knockout pyloric tumor (Aqp5KO-APK) was developed by incorporating the Aqp5 knockout allele alongside the Aqp5-eGFP-IRES-creERT2 cassette (resulting in the inactivation of two copies of Aqp5). Prior to cancer induction, the Aqp5-free mice were healthy and indistinguishable from their Aqp5 wild-type counterparts. Following administration of tamoxifen, pyloric tumors formed in these Aqp5KO-APK mice, which exhibited complete loss of Aqp5 expression. Figure 16 A, Figure 18 A). Crucially, these Aqp5 knockout tumors were also significantly smaller and exhibited precancerous features of intraepithelial neoplasia, while wild-type tumors were classified as adenocarcinoma at the same time point. Figure 16 A) indicates that Aqp5 may play a role in driving tumor progression in the in vivo environment. To reproduce these findings in vitro, Aqp5 knockout mouse pyloric tumor organoids were extracted from Aqp5KO APK tumors and confirmed that they also did not express Aqp5. Figure 18 B). Consistent with Aqp5 knockout in natural tumors, Aqp5KO tumor organoids induced tumors with a lower invasive phenotype after orthotopic transplantation. Figure 18 C) emphasizes the role of Aqp5 in driving tumor progression in this context.

[0123] To further elucidate the function of Aqp5 in the stem cell compartment of Aqp5KO-APK pyloric tumors, putative cancer stem cells (GFP+) and residual tumor epithelial blocks (GFP-) were isolated from pyloric tumors of Aqp5 knockout and Aqp5 wild-type mice using a GFP marker expressing Aqp5 promoter-driven RNA for large-scale RNA sequencing. Figure 18 D、 Figure 18 E). This yielded cancer stem cell markers, including genes common to both Aqp5 wild-type and knockout tumors (presumably genes unaffected by Aqp5 levels) and differentially expressed genes specific to both Aqp5 wild-type and knockout tumors. Figure 18 F). Pathway analysis showed that genes specific to Aqp5 wild-type cancer stem cells were enriched in ECM tissues and G protein signaling pathways. Figure 16 B. Figure 18 G). These analyses identified pathways in cancer stem cell compartments that may play a role in driving tumor progression downstream of Aqp5. Aqp5 function was then explored in human gastric cancer organoids, which provided greater cellular complexity and similarity to the primary tumor compared to cancer cell lines. Four independent Aqp5 knockout organoid clones were identified, and the loss of Aqp5 expression in these cell lines was confirmed. Figure 16 C). Compared to the wild-type parental organoid line of Aqp5 ( Figure 16D), all four Aqp5 knockout organoid cell lines reproducibly showed lower proliferation rates in vitro and established smaller tumors after original displacement implantation into mice, which were generally less invasive. Figure 16 E, Figure 16 F). Conversely, overexpression of Aqp5 was sufficient to increase in vitro cell proliferation in two other gastric cancer organoid cell lines (F). Figure 19 A, Figure 19 B), but there was no significant increase when Aqp5 was overexpressed in organoid cell lines that already expressed high levels of Aqp5. Figure 19 C), suggesting that the Aqp5-induced phenotype may be dose-dependent, up to a fixed threshold. Following orthotopic transplantation, human organoids overexpressing Aqp5 established larger tumors, which were also more aggressive in vivo. Figure 19 D、 Figure 19 E), but in cell lines that already express high levels of Aqp5, these tumor differences are again negligible. Figure 19 F).

[0124] Finally, to elucidate the mechanism driving Aqp5's role in human gastric cancer, human organoids based on the FLIP-Puro system suitable for conditional Aqp5 knockout were developed. Figure 19 G). Administration of Cre recombinase to cellular nanovesicles resulted in exon splicing alterations, producing truncated, nonfunctional Aqp5 products (G). Figure 1 G). It was confirmed that human organoids treated with Cre cell nanovesicles no longer expressed Aqp5 and grew more slowly in culture. Figure 16 H, Figure 16 I, Figure 19 H), consistent with the phenotype observed in constitutive Aqp5 KO human organoid cell lines ( Figure 16 C Figure 16 D). Then, batch RNAseq was performed on Aqp5-FLIP-Puro organoids at several time points after Cre recombination, and pathway analysis was performed on the resulting transcriptome datasets. This revealed multiple pathways activated in the early stages of organoid growth associated with Wnt signaling, which were immediately eliminated after Aqp5 knockout (Fig. 22J), highlighting the potential mechanism of action of Aqp5 in gastric cancer.

[0125] The invention described herein can be suitably practiced without any one or more elements, or one or more limitations, not specifically disclosed herein. Therefore, terms such as “comprising,” “including,” and “containing” should be interpreted expansively and without limitation. Furthermore, the terms and expressions used herein are descriptive rather than restrictive, and their use is not intended to exclude any equivalents or portions thereof of the shown and described features; however, it should be recognized that various modifications are possible within the claimed scope of the invention. Therefore, it should be understood that although the invention has been specifically disclosed by way of preferred embodiments and optional features, modifications and variations can be made to the invention disclosed herein by those skilled in the art, and such modifications and variations are considered to be within the scope of the invention.

[0126] As used herein, the singular forms “a” and “the” include plural references unless the context clearly specifies otherwise. For example, the term “genetic marker” includes multiple genetic markers, including mixtures and combinations thereof.

[0127] As used herein, the term “about” in the context of formulation ingredient concentration typically refers to ±5% of a specified value, more typically ±4% of a specified value, more typically ±3% of a specified value, more typically ±2% of a specified value, even more typically ±1% of a specified value, and even more typically ±0.5% of a specified value.

[0128] Throughout this disclosure, certain embodiments may be disclosed in a range format. It should be understood that the range format is merely for convenience and brevity and should not be interpreted as a rigid limitation on the disclosed range. Therefore, the description of the range should be considered to have specifically disclosed all possible subranges and the individual values ​​within that range. For example, a 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 individual numbers within that range, such as 1, 2, 3, 4, 5, and 6. This applies regardless of how broad the range may be.

[0129] This document may also describe certain embodiments broadly and generally. Each narrower group of species and subgenera belonging to the general disclosure also forms part of this disclosure. This includes a general description of the embodiments, accompanied by conditions or negative limitations for removing any subject from that class, regardless of whether the removed material is specifically described herein.

[0130] The invention has been described extensively and generally herein. Each narrower group of species and subgenus belonging to the generally disclosed scope also constitutes part of the invention. This includes the general description of the invention, accompanied by conditions or negative limitations that would remove any subject matter from that class, regardless of whether the removed material is specifically described herein.

[0131] Other embodiments are defined in the following claims and non-limiting embodiments. Furthermore, in the context of the description of features or aspects of the invention according to the Markush group, those skilled in the art will recognize that the invention is therefore also described in accordance with any single member or subgroup of the Markush group.

[0132] Experimental Section

[0133] Mouse work

[0134] Aqp5-eGFP-ires-creERT2, Aqp5-2A-creERT2, and Aqp5-2A-DTR mice have been previously developed. Rosa26-tdTomato mice have also been previously described. LSL (Ai14)(JAX 007914), Apc fl / fl (MGI 1857966), Pten fl / fl (MGI 2182005), Kras LSL-G12D (JAX 019104) and p53 R172H AQP5 knockout (KO) mice (KOCMP-11830-Aqp5-B6N-VA) were obtained from Cyagen. To induce Cre recombination, 8-week-old mice were intraperitoneally injected with 4 mg tamoxifen (Merck T5648) dissolved in sunflower oil at a dose of 30 g mouse body weight. Diphtheria toxin (DT; SigmaD0564) was administered intraperitoneally at a dose of 0.5 μg DT dissolved in PBS at a dose of 30 g mouse body weight.

[0135] In NOD.Cg-Prkdc scid Il2rg tm1Wjl Orthotopic transplantation was performed on / SzJ(NSG) mice (JAX 005557). In short, the mice were anesthetized, and their stomachs were exposed under aseptic surgical conditions. An insulin syringe containing the transplant material (cells or organoids) was inserted into the pyloric region directly beneath the outer muscle layer. Postoperatively, the wound was sutured, and the mice were administered the reversal drugs atemetimazole and buprenorphine within a few days postoperatively.

[0136] All mouse experiments were conducted in accordance with the ethical and safety regulations of the Institutional Committee on Laboratory Animal Management and Use (IACUC) and A*STAR. Mice were euthanized immediately when the tumor reached the maximum permissible size of 20 mm.

[0137] Human samples

[0138] Patient pyloric tumor biopsy tissue was provided by J. So of the Department of Gastroenterology, National University Hospital, 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-antifungal agent (Gibco 15240096). Samples were vigorously washed in HBSS before proceeding with tissue isolation steps. All experiments involving human materials were performed in accordance with ethical and safety regulations covered by the A*STAR Institutional Review Board (IRB).

[0139] Organism dissociation for FACS

[0140] Pyloric tumors were dissociated by incubating tissue erosions 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) at 37°C for 45 min. To release the gastric glands, the tissue erosions were repeatedly and forcefully aspirated using cold advanced DMEM / F-12 buffer, filtered through a 100 μm filter, and then centrifuged at 2500 rpm for 3 min at 4°C. The resulting precipitate was further dissociated into a single-cell suspension by incubation at 37°C for 10 min with TrypLE (Gibco 12604) and 10 mg / ml DNase I (Sigma D4513). The digestion reaction was terminated with 30 ml of cold HBSS, and the suspension was centrifuged at 2500 rpm for 3 minutes at 4°C.

[0141] For mouse pyloric tumors with AQP5 labeled with eGFP, the cell pellet was directly resuspended in 2% fetal bovine serum (FBS) in HBSS and passed through a 40 μm filter for cell sorting on a BD inflow cell sorter (BD Biosciences). For human pyloric tumors, the TrypLE-digested single-cell suspension was 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 in HBSS at 4°C for 45 min.

[0142] Cells were washed twice with 2% FBS in HBSS before FACS filtration through a 40 μm filter. Cells were resuspended with DAPI for live / dead cell gating prior to FACS. Sorted cells were collected directly in RLT Plus buffer (Qiagen) containing β-mercaptoethanol for RNA isolation, or in advanced DMEM / F-12 containing 0.5% Matrigel (Corning 354253) for organoid culture.

[0143] organoid culture

[0144] Pyloric tumor organoids were grown in a basal medium containing 2 mM Glutamax (Invitrogen 35050-079), 10 mM HEPES (Invitrogen 15630-056), 1 x N2 (Invitrogen 17502-048), 1 x B27 (Invitrogen 17504-044), 1 mM N-acetylcysteine ​​(Sigma A9165), and 200 μg / ml Primocin (InvivoGen Ant-pm-1) in advanced DMEM / F-12 (Invitrogen 12634-028). For mouse pyloric tumor organoid cultures, during the first passage after plating, the organoids were supplemented with the following recombinant growth factors at specified concentrations: 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 transferred to basal medium for long-term maintenance to select tumor organoids. 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 administered the aforementioned recombinant growth factors, along with 2 μm A-8301 (Tocris 2939) and 10 mM nicotinamide (SigmaN0636), in a basal medium consisting of 50% WRNF conditioned medium. Single-cell cultures were also given 10 μm Y-27632 (Tocris1254). Upon confluence, organoids were dissociated using TrypLE (Gibco12604), passaged approximately weekly, and maintained in a growth factor-depleted MMR basement membrane matrix (Corning 356231). For experiments involving the original displacement of organoids into mice, intact organoids were recovered from the MMR using cell recovery medium (Corning354253).

[0145] To generate gene-edited human gastric cancer organoids, the following plasmids were electroporated: HR110PA-1hAQP5-5′arm-P2A-iCas9-IRES-Venus-3′arm, pX330 hAQP5 gRNA-1, and pX330 hAQP5 gRNA-2 to generate Aqp5-2A-iCaspase organoids; HR110PA-1hAQP5-5′arm-CreERT2-pA-3′arm, pX330 hAQP5 gRNA-1, and pX330 hAQP5 gRNA-2; followed by AAVS1-2A-Blasticidin-CAG-LSL-tdTomato and pX330AAVS1 gRNA to generate Aqp5-2A-CreERT2 and LSL-tdTomato organoids. The pX330 plasmid was derived from pX330-U6-Chimeric_BB-CBh-hSpCas9 (from Feng Zhang's Addgene plasmid #42230). Electroporation of organoids was performed using a NEPA21 electroporator (NEPA GENE) as recommended by the manufacturer. Briefly, organoids were supplemented one day prior to electroporation with 5 μM CHIR99021 (Stemgent 04-0004-10) and 10 μM Y-27632 (Tocris 1254). Organoids were dissociated using TrypLE (Gibco 12604) and washed with Opti-MEM I serum-depleted medium (Life Technologies 31985-062). Before loading into electroporation reaction cups, the cell pellet was resuspended in BTXpress (BTX Harvard Instruments 45-0805) with the corresponding plasmid at a ratio of 100,000 cells to 15 μg DNA. Electroporation was performed using a 175V piercing pulse and a 5ms pulse length. Successful transfectants were selected by puromycin (InvivoGen Ant-pr-1), with the exception of AAV S1-2A-blasticin-CAG-LSL-tdTomato organoids selected by blastcin (InvivoGen Ant-bl-1). After transfection with Aqp5-2A-iCaspase and Aqp5-2ACreERT2 constructs, organoids were further treated with Cre recombinase cell nanovesicles (Clontech 631449) to remove the selection cassette. Briefly, organoids were dissociated with TrypLE (Gibco 12604) and washed with high-grade DMEM / F12 (Invitrogen 12634-028) containing 10% FBS.Cre recombinase cell nanovesicles (Clontech 631449) and 6 μg / ml polybrene (Sigma H9268) were added, and the suspension was centrifuged at 600g for 1 hour at 32°C. Cells were incubated at 37°C for 6 hours and then transferred to Matrigel. After Cre cell nanovesicle treatment, cells were grown in puromycin-free organoid medium for 1 week and sorted using a BD inflow cell sorter (BD Biosciences) to collect RFP-negative cells.

[0146] To induce Aqp5+ cell removal, Aqp5-2A-iCaspase organoids were treated with 500 nM B / B homodimer (Clontech 635059), and Aqp5-2A-DTR organoids were treated with 240 ng / ml DT. For lineage tracing of Aqp5+ cells in Aqp5-2A-CreERT2 and LSL-tdTomato organoids, organoids were treated with 1 μm 4-OHT for 16 h and then removed. For conditional Aqp5 KO in Aqp5-FLIP-Puro organoids, organoids were dissociated with TrypLE (Gibco 12604) and washed with high-grade DMEM / F12 (Invitrogen 12634-028) containing 10% FBS. Cre recombinase cell nanovesicles (Clontech 631449) and 6 μg / ml polybrene (Sigma H9268) were added, and the suspension was centrifuged at 600g for 1 hour at 32°C. Cells were incubated at 37°C for 6 hours and then transferred to a matrix gel. After Cre cell nanovesicle treatment, cells were grown in puromycin-free organoid culture medium.

[0147] For conditional Aqp5 KO in Aqp5-FLIP-Puro organoids, organoids were dissociated using TrypLE (Gibco 12604) and washed with high-quality DMEM / F12 (Invitrogen 12634-028) containing 10% FBS. Cre recombinase cell nanovesicles (Clontech 631449) and 6 μg / ml polybrene (Sigma H9268) were added, and the suspension was centrifuged at 600g for 1 hour at 32°C. Cells were incubated at 37°C for 6 hours and then transferred to a matrix gel. After Cre cell nanovesicle treatment, cells were grown in puromycin-free organoid medium.

[0148] To measure cell proliferation in organoids, the same number of cells were seeded in matrix gel in 96-well plates, allowed to grow for 3 days, and incubated for 2 hours using CellTiter AQueous Single Solution Assay (Promega G3582). Absorbance at 490 nm was measured using a microplate reader as the reading for cell proliferation. Proliferation was also measured using the Click-iT EdU Cell Proliferation Kit (Life Technologies C10339) with Alexa Fluor594, as recommended by the manufacturer. Whole-sample organoids were imaged on an Evos M5000 (Thermofisher).

[0149] Cell line culture

[0150] All tissues were processed according to standard protocols. In short, fresh tissues were fixed overnight at 4°C with 4% paraformaldehyde, dehydrated, and processed into paraffin blocks. 8μm tissue sections were collected on slides, dewaxed, and rehydrated. For hematoxylin and eosin (H&E), FFPE sections were stained with Richard Allan hematoxylin, differentiated with acidic ethanol (70% ethanol containing 1% HCl), and counterstained with Scott blue and eosin. For immunohistochemistry (IHC) and immunofluorescence (IF), antigen retrieval was performed on rehydrated slides in a 121°C autoclave in target retrieval solutions at pH 6.1 (Dako, S169984) or pH 9.0 (Dako S236784). 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.

[0151] RNA isolation and qPCR

[0152] Cells were lysed using RLT Plus buffer containing β-mercaptoethanol (Qiagen) to extract RNA. RNA was purified from cell extracts using the RNeasy mini or micro kit (Qiagen), and cDNA synthesis was performed using the Superscript III kit (Life Technologies) according to the manufacturer's instructions. qPCR was performed in triplicate using a GoTaq qPCR MasterMix (Promega A6002). qPCR reactions were performed on a QuantStudio 7Flex real-time PCR system (Applied Biosystems) and analyzed using the dual δCt 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 produce sufficient material for validation of a large number of targets. Tables 2 and 4 summarize all qPCR primer sequences used.

[0153] Transcriptome analysis

[0154] For batch RNA sequencing, cells were collected directly in RLTPlus buffer (Qiagen) containing β-mercaptoethanol during FACS sorting. Total RNA was extracted using the RNeasy micro kit (Qiagen). RNA integrity of all samples was validated using an Agilent RNA 6000 Pico chip (Agilent 5067-1513) and run on an Agilent 2100 bioanalyzer before downstream library preparation. For Aqp5+ samples, qPCR was also performed to confirm enrichment of Aqp5 levels in 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 Ultralow RNA Kit (Clontech 634936) before 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. Library sequencing was performed on a NovaSeq PE150 (Illumina) and Illumina real-time analysis software for base retrieval to obtain FASTQ files.

[0155] Histological and staining methods

[0156] All tissues were processed according to standard protocols. In short, fresh tissues were fixed overnight at 4°C with 4% paraformaldehyde, dehydrated, and processed into paraffin blocks. 8μm tissue sections were collected on slides, dewaxed, and rehydrated. For hematoxylin and eosin (H&E), formalin-fixed paraffin-embedded (FFPE) sections were stained with Richard Allan hematoxylin, differentiated with acidic ethanol (70% ethanol containing 1% HCl), and counterstained with Scott blue and eosin. For immunohistochemistry (IHC) and immunofluorescence (IF), antigen retrieval was performed on rehydrated slides in a 121°C autoclave in target retrieval solutions at pH 6.1 (Dako, S169984) or pH 9.0 (Dako S236784). The primary antibodies used were rabbit anti-AQP5 (LifeTechnologies PA564195), mouse anti-CHGA (1:200, Abcam 15160), mouse anti-E-cadherin (1:200, BDBiosciences 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). 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.

[0157] For in situ hybridization (ISH) experiments, tissues were collected under RNase-free conditions and fixed in 4% paraformaldehyde at room temperature for 16 to 24 hours. RNAscope was performed using RNAscope 2.5 High Definition Brown Assay (ACDbio 322300) and 2.5 High Definition Duplex Reagent Assay, following 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 (ACDbio441761), Hs-PLA1A (ACDbio 536951), Hs-DCHS2 (ACDbio 1309261), and Hs-AQP5 (ACDbio452371).

[0158] Microscopy

[0159] 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 fluorescence images of organoids were captured using a Thermofisher Evos M5000 system. Large-area images acquired on the Nikon Ni-E microscope were processed using NIS Elements AR software (Nikon), and images acquired on the Thermofisher Evos M5000 microscope were stitched together using Adobe Photoshop. All acquired images were processed using Fiji and Adobe Photoshop.

[0160] Statistical analysis

[0161] All statistical analyses were performed in GraphPad Prism. The Fisher exact test was used to analyze the qualitative dataset. The normality of the quantitative dataset was first assessed using the Shapiro-Wilk test. Variables following a normal distribution were analyzed using unpaired two-tailed Student's t-tests (for two groups) or ANOVA with Dunnett's multiple comparison test (for more than two groups), while variables not following a normal distribution were analyzed using unpaired two-tailed Mann-Whitney U tests (for two groups) or Kruskal-Wallis tests with Dunn's multiple comparison test (for more than two groups). Reproducibility was confirmed by at least three independent experiments.

[0162] sheet

[0163] Table 1: The most upregulated genes in mouse Aqp5+ pyloric tumor cells compared to Aqp5- cells.

[0164]

[0165]

[0166]

[0167]

[0168]

[0169]

[0170] Table 2: Primer sequences for mouse target validation qPCR.

[0171]

[0172]

[0173] Table 3: The most upregulated genes in human Aqp5+ pyloric tumor cells compared to Aqp5- cells.

[0174]

[0175]

[0176]

[0177]

[0178]

[0179]

[0180]

[0181]

[0182] Table 4: Primer sequences for human target validation qPCR.

[0183]

[0184] sequence list

[0185]

[0186]

Claims

1. A method for identifying gastric cancer stem cells or gastric cancer stem cell populations, comprising: a1) Detect the expression of aquaporin 5 (AQP5) in cells or cell populations; or b1) Detect the expression level of AQP5 in cells or cell populations and compare the expression level with the expression level of AQP5 in reference cells or reference cell populations, wherein the detection of AQP5 expression in the cells or cell populations or an increase in the expression level of AQP5 in the cells or cell populations compared with the reference cells or reference cell populations identifies the cells or cell populations as gastric cancer stem cells or gastric cancer stem cell populations.

2. The method according to claim 1, wherein the cell or cell group is an in vitro, in vivo, or ex vivo cell or cell group.

3. The method according to claim 1 or 2, wherein the cell or cell group is a gastric tumor sample, biopsy tissue, or organoid.

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

5. The method according to any one of claims 1 to 4, wherein the cells or cell populations are modified to express AQP5 coupled with one or more detectable markers.

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

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

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

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

10. The method according to 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 according to any one of claims 1 to 10, further comprising 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. The method of claim 12, wherein the isolated gastric cancer stem cells form tumor organoids expressing AQP5.

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

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

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

17. The method according to any one of claims 1 to 16, wherein the identification of gastric cancer stem cells leads to the subject receiving standard gastric cancer care treatment.

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

19. The method of claim 18, wherein the agent binding AQP5 is an antibody.

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

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

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

24. The method according to any one of claims 18 to 23, wherein one or more gastric cancer stem cells isolated 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 tumor organoids expressing AQP5 includes transcriptome analysis.

26. A method for removing or eliminating AQP5+ gastric cancer stem cells modified to express the DTR gene or the inducible Caspase9 (iCasp9) gene, comprising contacting the cells with a 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 the binding of the AQP5+ cells to the agent removes or eliminates the cells.

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

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

30. A method of treating gastric cancer in a subject who requires it, comprising administering to the subject a therapeutically effective amount of one or more agents that eliminate or remove cells expressing AQP5.

31. The method of claim 30, wherein the subject requiring it is a human or a mouse.

32. A biomarker for gastric cancer stem cells, wherein the biomarker is AQP5.

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

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

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

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

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

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

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

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

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

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

43. The method of claim 42, wherein the primers are selected from 5′-AAAACTCTGCGAACACGGCCCCTGTCTC-3′ (SEQ ID NO: 49) and 5′-AAGGCCGTGTTCGCAGAGTTCCTGTCTC-3′ (SEQ ID NO: 50); 5′-CGGUGGUCAUGAAUCGGUUTT-3′ (SEQ ID NO: 51) and 5′-AACCGAUUCAUGACCACCGCA-3′ (SEQ ID NO: 52); and 5′-GCGUGUGGCCAUCAUCAAATT-3′ (SEQ ID NO: 53) and 5′-UUUGAUGAUGGCCACACGCTT-3′ (SEQ ID NO: 54).

44. The method of claim 40, wherein the drug is selected from enzyme inhibitors, receptor antagonists, and channel blockers.

45. A method for monitoring the progression of gastric cancer in a subject, the method comprising: a) Measure the AQP5 expression level in samples obtained from subjects who received gastric cancer treatment; and b) Measure the AQP5 expression level in control samples obtained from the subjects before gastric cancer treatment; An increase in AQP5 expression level in the sample from step a compared to the control sample indicates that gastric cancer has occurred or has progressed.

46. ​​A method for monitoring the occurrence of gastric cancer tumors in a subject, the method comprising: c) Measure the expression level of AQP5 in samples obtained from the subjects; and d) Measure the expression level of AQP5 in reference samples obtained from the subject at a time point earlier than that of the sample obtained in step c; The increased expression level of AQP5 in the sample from step c compared to the reference sample from step d indicates that gastric cancer tumorigenesis has occurred.

47. The method according to any one of claims 45 to 46, wherein, If gastric cancer has already occurred or has progressed, the subject is treated with an anti-gastric cancer compound.

48. The method according to 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. The method according to any one of claims 45 to 48, further comprising measuring the expression levels of one or more biomarkers of gastric cancer progression.

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

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

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

53. The method according to any one of claims 45 to 52, wherein the sample is selected from blood, plasma, biopsy samples, tissue samples, primary cell culture samples, and primary organoid lines.

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

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