Marker for differential diagnosis of colon cancer stem cells and identification method thereof

Abstract

The invention relates to the technical field of biomedicine, in particular to a marker for identifying and diagnosing colon cancer stem cells and an identification method of the marker, the marker is CLDN2 protein, the CLDN2 protein is composed of an amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence which has at least 90% sequence identity with the SEQ ID NO: 1 and keeps the activity of the colon cancer stem cell marker, and the marker is CLDN2 protein. It is found and confirmed that CLDN2 can be used as a specific marker for differential diagnosis of colon cancer stem cells for the first time, especially can effectively distinguish colon cancer stem cells from normal intestinal stem cells, and solves the technical problem that an existing marker is difficult to distinguish tumor stem cells from normal stem cells. The invention discloses a molecular mechanism that CLDN2 promotes beta-cat nin nuclear translocation by regulating ubiquitination modification of a K63 site of LASP1 so as to regulate and control functions of the colon cancer stem cells.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to a marker for the differential diagnosis of colorectal cancer stem cells and a differential diagnosis method thereof. More specifically, the present invention relates to using Claudin 2 (CLDN2) as a marker for the differential diagnosis of colorectal cancer stem cells, and a method for the differential diagnosis, isolation, and functional analysis of colorectal cancer stem cells based on this marker. Background Art

[0002] Lung cancer is one of the malignant tumors with the highest incidence and mortality rates globally. According to the latest global cancer burden data released by the International Agency for Research on Cancer (IARC) of the World Health Organization, in 2020, the number of newly diagnosed lung cancer cases globally was approximately 2.2 million, and the number of deaths was approximately 1.8 million, accounting for 11.4% and 18.0% of all cancers, respectively. The early symptoms of lung cancer are not obvious. Approximately 70% of patients are already in the locally advanced or metastatic stage at the time of diagnosis, and the 5-year survival rate is less than 20%. For patients with early-stage lung cancer (stage I), the 5-year survival rate can reach 70%-80%. Therefore, early diagnosis and accurate typing are of great significance for improving the prognosis of lung cancer patients.

[0003] Colorectal cancer is one of the malignant tumors with relatively high incidence and mortality rates globally. Although the survival rates of colorectal cancer at all stages globally have increased through strategies such as surgical treatment and adjuvant chemotherapy and radiotherapy, nearly 50% of colorectal cancer patients will still experience tumor recurrence later. Research has shown that tumor recurrence is considered to be caused by the proliferation of resident cancer stem cells (CSCs). Cancer stem cells can regulate various intrinsic disease resistance mechanisms, including tumor metastasis, drug resistance, and the recurrence of the disease after initial treatment. Therefore, accurately identifying and targeting cancer stem cells is considered an effective strategy for preventing tumor recurrence.

[0004] In the field of cancer stem cell research, a variety of stem cell markers have been discovered. CD44 was the first surface marker molecule of solid tumor stem cells to be discovered and confirmed. It was initially discovered in breast cancer and later confirmed to be expressed in tumor stem cells of various solid tumors such as brain cancer and melanoma. However, CD44 is also highly expressed in stem cells of many normal tissues (Wang L et al., The Role of CD44 and Cancer Stem Cells, Methods Mol Biol, 2018, 1692:31-42). CD133 can be used as a marker for stem cells of various malignant tumors, but it can also be used as a marker molecule for normal hematopoietic stem cells and endothelial progenitor cells (Soleimani A et al., Emerging roles of CD133 in the treatment of gastric cancer, a novel stem cell biomarker and beyond, Life Sci, 2022, 293:120050). Lgr5 (also known as Gpr49) is an important marker for intestinal stem cells and was initially discovered as a target gene of the Wnt signal in colorectal cancer cells and small intestinal crypt cells (Fatehullah A et al., A tumour-resident Lgr5(+) stem-cell-like pool drives the establishment and progression of advanced gastric cancers, Nat Cell Biol, 2021, 23(12):1299-1313). ALDH1 is an aldehyde dehydrogenase. As a marker for CSCs, it is difficult to distinguish CSCs from normal stem cells (Tomita H et al., Aldehyde dehydrogenase 1A1 in stem cells and cancer, Oncotarget, 2016, 7(10):11018-32).

[0005] The above research shows that although there are currently many classical marker molecules that can distinguish colon cancer stem cells (CRC-CSCs) from non-CRC-CSCs colon cancer epithelial cells, they cannot effectively identify CRC-CSCs from normal tissue stem cells, resulting in poor treatment targeting. Therefore, there is an urgent need to find more specific surface marker molecules for CRC-CSCs to provide new options for targeted treatment of colon cancer.

[0006] In addition, existing studies have shown that EGFR-induced high expression of CLDN2 promotes the tumorigenicity of colon cancer cells (Fang H et al., EGFR inhibitor gefitinib regulates barrier function in human epidermal keratinocytes via the modulation of the expression of claudins, Int J Mol Med, 2019, 43(3):1522-1530), CLDN2 promotes liver metastasis of colon cancer and the survival of early cancer cells (Dhawan P et al., Claudin-2 expression increases tumorigenicity of colon cancer cells: role of epidermal growth factor receptor activation, Oncogene, 2011, 30(29):3234-47); CLDN2 promotes the growth and metastasis of colorectal cancer by inhibiting NDRG1 transcription (Wei M et al., Claudin-2 promotes colorectal cancer growth and metastasis by suppressing NDRG1 transcription, Clin Transl Med, 2021, 11(12):e667). However, most of these studies are limited to the phenotypic study of colorectal cancer, and there is no report on the regulatory mechanism of CLDN2 on CRC-CSCs.

[0007] The heterogeneity of tumor tissue cells is a very important issue, which makes the growth, spread and response to treatment of tumors very complex. Single Cell Sequencing is a "powerful tool" for the phenotypic analysis of cell subsets, which can analyze the transcriptome expression of each cell in tumor tissue, analyze the gene expression patterns of different cell subsets in tumor tissue, understand the heterogeneity inside the tumor, so as to better understand the biological characteristics of the tumor and discover potential therapeutic targets (Lee EY et al., Functional interrogation of lymphocyte subsets in alopecia areata using single-cell RNA sequencing, Proc Natl Acad Sci USA, 2023, 120(29):e2305764120).

[0008] Therefore, the present invention aims to discover and confirm a novel marker that can specifically label colorectal cancer stem cells rather than normal intestinal stem cells through single-cell sequencing and various biological techniques, and to elucidate the molecular mechanism of its regulation of the function of colorectal cancer stem cells, providing new targets and strategies for the diagnosis and treatment of colorectal cancer. Summary of the Invention

[0009] The object of the present invention is to provide a marker for the differential diagnosis of colorectal cancer stem cells and a method for its differential diagnosis. This marker can specifically distinguish colorectal cancer stem cells from non-colorectal cancer stem cells, especially can distinguish colorectal cancer stem cells from normal intestinal stem cells, providing a new target for the precise diagnosis and targeted treatment of colorectal cancer.

[0010] To achieve the above object, the present invention provides the following technical solutions:

[0011] In the first aspect of the present invention, a marker for the differential diagnosis of colorectal cancer stem cells is provided. The marker is the CLDN2 protein, which is composed of the amino acid sequence shown in SEQ ID NO:1, or is composed of an amino acid sequence having at least 90% sequence identity with SEQ ID NO:1 and maintaining the activity of the colorectal cancer stem cell marker.

[0012] Preferably, the CLDN2 protein is highly expressed specifically in colorectal cancer stem cells and is lowly expressed in normal colorectal stem cells.

[0013] In an embodiment of the present invention, the second extracellular loop region of the CLDN2 protein contains the amino acid sequence AFGSSVKATVFAVSFLYLLVVVNGVIQCTV, and the second extracellular loop region is the antigenic epitope for identifying colorectal cancer stem cells.

[0014] The present invention further provides that the CLDN2 protein promotes the nuclear translocation of β-catenin by inducing the ubiquitination modification of the K63 site of the LASP1 protein, thereby regulating the self-renewal ability of colorectal cancer stem cells.

[0015] In another embodiment of the present invention, the marker further includes Lgr5 and / or CD44 protein, and the co-expression of CLDN2 with Lgr5 and / or CD44 is used to improve the specificity of the identification of colorectal cancer stem cells.

[0016] The second aspect of the present invention provides a method for the differential diagnosis of colorectal cancer stem cells, including the following steps:

[0017] (a) Obtain a sample to be detected;

[0018] (b) Detect the expression level of the CLDN2 protein in the sample;

[0019] (c) Compare the expression level of the CLDN2 protein with a preset threshold;

[0020] (d) When the expression level of the CLDN2 protein is higher than the preset threshold, it is determined to be positive for colon cancer stem cells.

[0021] Preferably, the method further includes the step of detecting the expression levels of Lgr5 and / or CD44, and calculating the colon cancer stem cell score by the following formula:

[0022] CCRSS = 0.45 × CLDN2 expression score + 0.35 × Lgr5 expression score + 0.20 × CD44 expression score;

[0023] Wherein when the score is greater than 0.58, it is determined to be positive for colon cancer stem cells.

[0024] In a specific embodiment of the present invention, the method for detecting the expression level of the CLDN2 protein in the test sample is selected from immunohistochemistry, flow cytometry, immunofluorescence or enzyme-linked immunosorbent assay.

[0025] The present invention also provides a method for classifying colon cancer patients into a low-risk group (<0.40), a medium-risk group (0.40 - 0.75) and a high-risk group (>0.75) according to the CLDN2 expression level, for evaluating the prognosis of patients and guiding the selection of treatment regimens.

[0026] In addition, the present invention also provides a method for labeling cells with an anti-CLDN2 antibody, separating CLDN2-positive cells by magnetic bead sorting or flow cytometry sorting method, and culturing the separated CLDN2-positive cells in a serum-free medium, wherein the serum-free medium contains B27 supplement, epidermal growth factor, fibroblast growth factor and insulin, for enriching colon cancer stem cells.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] 1. The present invention first discovers and confirms that CLDN2 can be used as a specific marker for the differential diagnosis of colon cancer stem cells, especially can effectively distinguish colon cancer stem cells from normal intestinal stem cells, and solves the technical problem that it is difficult for existing markers to distinguish tumor stem cells from normal stem cells.

[0029] 2. The present invention reveals the molecular mechanism by which CLDN2 promotes the nuclear translocation of β-catenin by regulating the K63-site ubiquitination modification of LASP1, thereby regulating the function of colon cancer stem cells, providing a theoretical basis for targeting the CLDN2 signaling pathway to treat colon cancer.

[0030] 3. The present invention establishes a prognostic evaluation and stratified treatment system for colon cancer patients based on the expression level of CLDN2, providing a new strategy for the precision treatment of colon cancer.

[0031] 4. The present invention develops a method for isolating and enriching colon cancer stem cells based on CLDN2, providing technical support for the in vitro study of colon cancer stem cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 Schematic diagram of the single-cell sequencing analysis of colon cancer and adjacent tissues and the screening results of CLDN2.

[0033] A: Single-cell sequencing cell atlas of colon cancer and adjacent tissues. B: Cluster diagram of malignant cell populations. C: Bubble diagram of genes highly specifically expressed in the Lgr5+ cell population. D: Immunomagnetic bead sorting of Lgr5+ and Lgr5- cell populations in colon cancer and adjacent tissues, and qPCR detection of the expression of differential genes. E: qPCR detection of the expression of differential genes in DLD1 adherent cells, DLD1 spheroid stem cells, primary colon epithelial cell adherent cells, and spheroid stem cells (***p<0.001).

[0034] Figure 2 Schematic diagram of the detection of the expression of CLDN2 in colon cancer stem cells and adjacent stem cells.

[0035] A: qRT-PCR detection of the mRNA expression of CLDN2 in colon cancer and adjacent colon tissues (n = 90, ***p<0.001). B: Analysis of the relationship between the expression intensity of CLDN2 and the survival rate of 90 colon cancer patients. C: 10xGenomics spatial transcriptome sequencing CLDN2+ and Lgr5+ SPOT dot distribution maps. D-E: Staining of colon cancer and adjacent colon tissue sections with Lgr5 (red), CLDN2 (green), and DAPI (blue), and statistical analysis of the percentage of co-expressed cells of Lgr5 and CLDN2. F: Flow cytometry sorting of the percentage of CLDN2+ cells in Lgr5+ / - cells of colon cancer tissues and adjacent tissues. G: Flow cytometry sorting of the percentage of CLDN2+ cells in Lgr5+ / - cells of colon cancer cell lines.

[0036] Figure 3 Schematic diagram of the detection of the expression of CLDN2 in colon cancer tissues and the function in colon cancer cells.

[0037] A-B: Sorting of CLDN2+ / - cells, observing, photographing, and counting after suspension culture for 7 days (n = 3). C-D: Tumor size and weight of mice. E: Percentage of tumor-bearing mice. F: Tumor volume (***p<0.001).

[0038] Figure 4Schematic diagram showing that CLDN2 is a specific marker for CRC-CSCs superior to Lgr5.

[0039] A-B: Organoid formation and statistics of primary colon epithelial cells of Lgr5+ / - and CLDN2+ / - . C-D: Specifically knockout Lgr5 and CLDN2, induce intestinal mucosal inflammation in mice with 2.5% DSS for 7 days, sacrifice the mice on the 14th day after returning to normal diet, the ability of the latter's intestinal tissue to repair inflammatory damage is stronger than that of the former, and the length change is statistically analyzed. E: Specifically knockout Lgr5 and CLDN2, observe the repair of inflammatory damage by in vivo imaging of small animals (n = 3, ***p<0.001).

[0040] Figure 5 Schematic diagram for detecting the effect of CLDN2 on the nuclear entry of β-catenin.

[0041] A: Multicolor immunofluorescence experiment to detect the expression of β-catenin in the cytoplasm and nucleus in exogenous overexpression of CLDN2 and the control group. B: Western blot to detect the expression of related proteins in exogenous knockdown of CLDN2 and the control group. C: Immunofluorescence to detect the expression of CLDN2 and β-catenin in colon cancer tissues. D: qPCR to detect the effect of exogenous overexpression of CLDN2 on key genes downstream of β-catenin (n = 3, ***p<0.001).

[0042] Figure 6 Schematic diagram for detecting the effect of LASP1 on the nuclear entry of β-catenin.

[0043] A: Proteomic detection shows that overexpression of CLDN2 enhances the binding of LASP1 to β-catenin. B: Knockdown of LASP1 reduces the promoting effect of CLDN2 on the nuclear translocation of β-catenin. C: Overexpression of CLDN2 can promote the expression of LASP1. D: Knockdown of TRIM15 reduces the nuclear translocation of β-catenin. E: Knockdown of TRIM15 can reduce the K63-site ubiquitination of LASP1. Detailed implementation manners

[0044] The present invention will be further described in detail below in conjunction with embodiments, but the implementation manners of the present invention are not limited thereto.

[0045] In the present invention, unless otherwise specified, the technical terms or scientific terms used should have the ordinary meanings understood by those with ordinary skills in the field to which the present invention belongs.

[0046] The "about" or "approximately" used in the present invention means within a reasonable error range, and this error range refers to ±10%.

[0047] Example 1: Identification of the specific marker CLDN2 of colorectal cancer stem cells based on single-cell sequencing

[0048] To search for marker genes that are specifically highly expressed in colorectal cancer stem cells (CRC-CSCs), we performed single-cell sequencing analysis on colorectal cancer tissues and adjacent tissues. With the approval of the Ethics Committee of Affiliated Hospital of Jining Medical University and the informed consent of patients and their families, colorectal cancer tissues and adjacent tissue specimens of patients after surgery (the adjacent tissues were more than 5 cm away from the edge of the cancer tissue) were taken and sent to Shanghai OE Biotech Co., Ltd. for single-cell sequencing.

[0049] A total of 11 cell clusters were identified by single-cell sequencing, and malignant cell clusters and Lgr5+ / - CRC-CSCs-like cell clusters were identified by CNV analysis. Ten genes highly expressed in the CRC-CSCs-like cell clusters were calculated (including: cldn2, lgr5, cyp4x1, xact, casc19, lcn15, uca1, linc01186, z83843.1, ppm1h) (as shown in Figure 1 A-C). To verify the single-cell sequencing data of colorectal cancer and adjacent tissues of patients, we used Lgr5 immunomagnetic beads to sort colorectal cancer and adjacent tissues and detected the expression of differential genes. The results showed that the expression of cldn2 in Lgr5+ colorectal cancer cells was significantly higher than that in Lgr5- colorectal cancer cells and Lgr5+ / - colorectal epithelial cells (as shown in Figure 1 D).

[0050] Next, we enriched stem cell-like cells (sphere-forming cells) in vitro in serum-free medium and found that among the 10 candidate genes, only cldn2 was significantly higher expressed in CRC-CSCs-like cells than in non-CRC-CSCs-like cells (as shown in Figure 1 E), indicating that cldn2 may be specifically highly expressed in CRC-CSCs.

[0051] To further confirm the expression of CLDN2 in colorectal cancer tissues and its clinical significance, we analyzed the expression level of CLDN2 in tissues of 90 colorectal cancer patients by qRT-PCR. The results showed that compared with normal colorectal tissues, CLDN2 was significantly highly expressed in colorectal cancer tissues (P<0.001) (as shown in Figure 2 A). Kaplan-Meier survival analysis showed that high expression of CLDN2 was significantly associated with poor prognosis of colorectal cancer patients (P<0.001) (as shown in Figure 2 B).

[0052] We also used multi-color immunofluorescence to analyze the co-expression of CLDN2 and the classical stem cell marker Lgr5 in colon cancer tissues. The results showed that the cell subsets with high expression of CLDN2 were basically consistent with those with high expression of the CRC-CSCs marker gene Lgr5, but there was little expression of CLDN2 in Lgr5+ cells in normal colonic epithelial tissues (as shown in Figure 2 C-E).

[0053] Flow cytometry analysis further confirmed that in Lgr5+ colon cancer cells, the expression of CLDN2 was significantly higher than that in Lgr5- cells, while in normal colonic epithelial tissues, there was no significant difference in CLDN2 expression between Lgr5+ and Lgr5- cells (as shown in Figure 2 F-G).

[0054] In summary, in this example, through single-cell sequencing and various verification experiments, CLDN2 was identified for the first time as a specific marker of colon cancer stem cells, and its specificity was superior to the traditional Lgr5 marker, and it could effectively distinguish colon cancer stem cells from normal intestinal stem cells.

[0055] Example 2: Effects of CLDN2 on the functions of colon cancer stem cells

[0056] To clarify the regulatory effect of CLDN2 on the functions of colon cancer stem cells, we conducted a series of in vitro and in vivo functional experiments.

[0057] First, we sorted CLDN2+ and CLDN2- colon cancer cells by flow cytometry and performed in vitro suspension culture experiments. The results showed that compared with CLDN2- cells, CLDN2+ cells had stronger spheroid formation ability, and the number and volume of the formed spheroids were significantly increased (P<0.001) (as shown in Figure 3 A-B).

[0058] Next, we injected CLDN2+ and CLDN2- colon cancer cells subcutaneously into immunodeficient mice to observe their in vivo tumorigenic ability. The results showed that compared with CLDN2- cells, the tumor volume and weight formed by CLDN2+ cells were significantly increased (P<0.001), and the tumorigenesis rate was also significantly increased (as shown in Figure 3 C-F).

[0059] In addition, we also compared the differences between CLDN2 and traditional stem cell markers such as Lgr5 and CD44 in identifying CRC-CSCs. We sorted primary colonic epithelial cells with CLDN2+ / -、Lgr5+ / -、CD44+ / - respectively for organoid culture experiments. The results showed that Lgr5+ primary colonic epithelial cells could form intestinal organoids, while CLDN2+ cells could not (as shown in Figure 4as shown in A - B). More importantly, mice with intestinal tissue - specific knockout of Lgr5 were unable to repair after inflammatory injury, while specific knockout of CLDN2 had little effect on intestinal injury repair (as Figure 4 shown in C - E). These data indicate that CLDN2 is not a specific marker for normal intestinal stem cells.

[0060] To further verify the specificity of CLDN2 in differentiating colon cancer stem cells from normal intestinal stem cells, we used a dextran sulfate sodium (DSS) - induced murine colitis model. The results showed that in normal intestinal tissues, intestinal epithelial stem cells mainly expressed Lgr5 but not CLDN2; while in colon cancer tissues, stem cells highly expressed both Lgr5 and CLDN2. This finding provides strong evidence that CLDN2 can specifically label colon cancer stem cells rather than normal intestinal stem cells.

[0061] In summary, this example demonstrated through in - vitro and in - vivo functional experiments that CLDN2 can specifically label colon cancer stem cells with strong self - renewal ability and tumorigenic ability, and its specificity is superior to traditional stem cell markers, enabling effective differentiation between colon cancer stem cells and normal intestinal stem cells.

[0062] Example 3: Molecular mechanism of CLDN2 promoting the function of colon cancer stem cells

[0063] To explore the molecular mechanism of CLDN2 regulating the function of colon cancer stem cells, we detected the effect of CLDN2 on core signaling pathways. First, we detected the effect of CLDN2 on β - catenin nuclear translocation by Western blot and immunofluorescence analysis. The results showed that overexpression of CLDN2 significantly promoted the translocation of β - catenin from the cytoplasm to the nucleus (as Figure 5 shown in A), while knockdown of CLDN2 inhibited the nuclear entry of β - catenin (as Figure 5 shown in B). In colon cancer tissues, β - catenin was expressed in the nucleus in cells with high expression of CLDN2, while in cells without CLDN2 expression, β - catenin was mostly localized in the cytoplasm (as Figure 5 shown in C).

[0064] In addition, overexpression of CLDN2 significantly promoted the expression of β - catenin downstream target genes such as axin2, c - myc, cd44, etc. (as Figure 5 shown in D). These results indicate that CLDN2 may regulate the function of colon cancer stem cells by promoting β - catenin nuclear translocation to activate the Wnt / β - catenin signaling pathway.

[0065] To reveal the molecular mechanism by which CLDN2 promotes β-catenin nuclear translocation, we used proteomic techniques to detect the proteins that interact with β-catenin. The results showed that the binding ability of β-catenin to LASP1 in the CLDN2 overexpression group was more than 4-fold stronger than that in the control group (as shown in Figure 6 Figure A). Knockdown of LASP1 expression blocked the promoting effect of CLDN2 on β-catenin nuclear translocation (as shown in Figure 6 Figure B), suggesting the crucial role of LASP1 in the function of CLDN2.

[0066] We found that exogenous overexpression of CLDN2 had no obvious effect on the transcriptional expression of LASP1, but significantly affected its protein expression (as shown in Figure 6 Figure C). Further studies showed that CLDN2 might stabilize the LASP1 protein by inhibiting the ubiquitination and degradation of LASP1. Through immunoprecipitation and Western blot experiments, we found that CLDN2 could enhance the K63-site ubiquitination modification of LASP1, and this modification was beneficial to the stability and function of the LASP1 protein.

[0067] We also found that TRIM15 might be the E3 ubiquitin ligase that regulates the ubiquitination of LASP1. After knocking down TRIM15, the nuclear translocation of β-catenin decreased (as shown in Figure 6 Figure D), and at the same time, the ubiquitination level of the K63 site of LASP1 decreased (as shown in Figure 6 Figure E).

[0068] In summary, this example reveals the molecular mechanism by which CLDN2 promotes β-catenin nuclear translocation by regulating the K63-site ubiquitination modification of LASP1, thereby activating the Wnt / β-catenin signaling pathway to regulate the function of colorectal cancer stem cells. This discovery provides a new potential target for treating colorectal cancer by targeting the CLDN2-LASP1-β-catenin signaling axis.

[0069] Example 4: Application of CLDN2 in the Diagnosis and Prognosis Evaluation of Colorectal Cancer

[0070] Based on the aforementioned experimental results, we further explored the application value of CLDN2 as a biomarker in the diagnosis and prognosis evaluation of colorectal cancer.

[0071] We collected tissue samples from 392 colorectal cancer patients and 98 healthy controls, and detected the expression level of CLDN2 by immunohistochemistry. The results showed that the sensitivity of CLDN2 as a biomarker for CRC-CSCs was 90.5% (95% CI: 86.7 - 94.3%), and the specificity was 87.3% (95% CI: 83.1 - 91.5%).

[0072] When CLDN2 was detected in combination with the traditional markers Lgr5 and CD44, the sensitivity of differential diagnosis of colon cancer stem cells increased to 95.8% (95% CI: 92.9 - 98.7%), and the area under the ROC curve (AUC) reached 0.924 (95% CI: 0.886 - 0.962). According to multiple regression analysis, we established a colon cancer stem cell scoring system (CCRSS): CCRSS = 0.45 × CLDN2 expression score + 0.35 × Lgr5 expression score + 0.20 × CD44 expression score, and a score greater than 0.58 was determined to be positive for colon cancer stem cells.

[0073] Follow-up analysis showed that compared with the low-expression group of CLDN2, the overall survival in the high-expression group of CLDN2 was significantly shortened (HR = 2.47, 95% CI: 1.82 - 3.36, P < 0.001). However, the prognostic prediction effect was better based on the triple assessment of the CCRSS scoring system (HR = 3.18, 95% CI: 2.24 - 4.52, P < 0.001).

[0074] In addition, we also found that the expression level of CLDN2 was closely related to the sensitivity of colon cancer patients to chemotherapy. The chemotherapy effective rate in the high-expression group of CLDN2 was only 24.6%, while that in the low-expression group of CLDN2 reached 58.9% (P < 0.001, OR = 0.23, 95% CI: 0.15 - 0.36). This indicates that CLDN2 can be used as a biomarker for predicting the sensitivity of colon cancer to chemotherapy.

[0075] Based on the above results, we established a prognostic assessment and stratified treatment system for colon cancer patients. According to the expression level of CLDN2, patients were divided into three risk groups: low-risk group (CCRSS < 0.40), with a predicted 5-year survival rate > 80%; medium-risk group (CCRSS 0.40 - 0.75), with a predicted 5-year survival rate of 50 - 80%; high-risk group (CCRSS > 0.75), with a predicted 5-year survival rate < 50%. For patients in the high-risk group, we recommend combined targeted therapy or immunotherapy; for patients in the medium-risk group, an intensified chemotherapy regimen is recommended; and for patients in the low-risk group, a standard treatment regimen is used.

[0076] In an independent validation cohort (142 colon cancer patients), the C-index of this prognostic prediction model reached 0.81 (95% CI: 0.75 - 0.87), the slope of the calibration curve was 0.94 (the ideal value is 1.0), and the decision curve analysis showed that it was superior to the traditional TNM staging within the threshold probability range of 20 - 80%.

[0077] In summary, this example demonstrates that CLDN2 has high diagnostic accuracy and prognostic evaluation value as a colon cancer stem cell marker, providing a new strategy for the individualized and precise treatment of colon cancer.

[0078] Example 5: Method for isolating and enriching colon cancer stem cells based on CLDN2

[0079] Based on the property of CLDN2 as a specific marker for colon cancer stem cells, we developed a method for isolating and enriching colon cancer stem cells based on CLDN2.

[0080] First, we prepared single-cell suspensions from fresh colon cancer tissues or cell lines. The specific method was as follows: Under sterile conditions, the tissue was cut into small pieces of 1 - 2 mm, and added to DMEM / F12 medium containing collagenase IV (1.5 mg / ml), hyaluronidase (20 μg / ml), and DNase I (1 μg / ml), and digested in a shaker at 37 °C for 1.5 hours; then filtered through a 70 μm cell strainer, and ACK lysis buffer was used to remove red blood cells when necessary; finally, the cell viability was detected by trypan blue exclusion method, and the viable cell rate was required to be ≥80%. 3 Small pieces, added to DMEM / F12 medium containing collagenase IV (1.5 mg / ml), hyaluronidase (20 μg / ml), and DNase I (1 μg / ml), digested in a shaker at 37 °C for 1.5 hours; then filtered through a 70 μm cell strainer, and ACK lysis buffer was used to remove red blood cells when necessary; finally, the cell viability was detected by trypan blue exclusion method, and the viable cell rate was required to be ≥80%.

[0081] Next, we labeled the single-cell suspensions with CLDN2 antibody and isolated CLDN2-positive cells by magnetic bead sorting or flow cytometry sorting. For magnetic bead sorting, we incubated the single-cell suspensions with biotinylated anti-CLDN2 antibody (5 μg / ml) at 4 °C for 30 minutes, then added streptavidin magnetic beads (Miltenyi Biotec, 130 - 048 - 101) and incubated at 4 °C for 15 minutes, and finally performed magnetic sorting using an MS sorting column (Miltenyi Biotec, 130 - 042 - 201). For flow cytometry sorting, we labeled the cells with PE-labeled anti-CLDN2 antibody, set appropriate fluorescence thresholds for sorting, and required the CLDN2-positive rate to be ≥90% and the viability to be ≥90% after sorting.

[0082] The sorted CLDN2-positive cells were cultured in serum-free medium to enrich colon cancer stem cells. The composition of the serum-free medium was: DMEM / F12 (1:1) basal medium, supplemented with B27 supplement (1:50), human recombinant EGF (10 ng / ml), human recombinant FGF-basic (20 ng / ml), insulin (4 mg / ml), bovine serum albumin (0.4%), penicillin (100 U / ml), and streptomycin (100 μg / ml). The cells were seeded in an ultra-low attachment 6-well plate at a density of 5000 - 10000 cells / ml and cultured at 37 °C and 5% CO2 for 7 - 14 days to observe the spheroid formation. The spheroid formation efficiency (number of spheroids / number of seeded cells × 100%) can be used as an evaluation index for stem cell characteristics.

[0083] In addition, we have also developed a rapid detection system for CLDN2 based on a microfluidic chip. The system includes a microfluidic chip for 8-channel parallel detection. Each channel contains a microcolumn array modified with anti-CLDN2 antibody, which can efficiently capture CLDN2-positive cells. The system has the advantages of fast detection speed (<30 minutes), low sample requirement (50 μl per channel), high sensitivity (detection limit <50 CLDN2-positive cells / ml), etc., and is suitable for the rapid detection of clinical samples.

[0084] The CLDN2-positive cells isolated by the above method can be used in a variety of downstream experiments, such as in vitro functional studies, drug sensitivity tests, single-cell sequencing analysis, etc., providing an important tool for the research of colorectal cancer stem cells.

[0085] Example 6: Preparation and verification of anti-CLDN2 monoclonal antibody

[0086] To better apply CLDN2 as a biomarker for colorectal cancer stem cells, we have developed a monoclonal antibody that specifically recognizes the CLDN2 protein.

[0087] First, we selected the second extracellular loop region (151-179aa) of the CLDN2 protein as the antigenic epitope and synthesized the peptide sequence AFGSSVKATVFAVSFLYLLVVVNGVIQCTV. The peptide was prepared by Fmoc solid-phase synthesis method with a purity ≥95%, and then it was conjugated with KLH carrier protein as the immunogen and conjugated with BSA as the screening antigen.

[0088] Next, 6-8-week-old female BALB / c mice (SPF grade) were selected for immunization. The first immunization used 100 μg antigen / CFA emulsion, and then the booster immunization was performed every 14 days using 50 μg antigen / IFA emulsion. The serum antibody titer was monitored by indirect ELISA. When the titer OD value >1.0 (control group <0.2), the spleens of the mice were taken to prepare splenocytes.

[0089] Then, the splenocytes were mixed with SP2 / 0 myeloma cells in the logarithmic growth phase at a ratio of 5:1, and cell fusion was carried out using the PEG method. Hybridoma cells were screened by HAT selective medium, and monoclonal cells were obtained by limiting dilution method.

[0090] The screened hybridoma clones were identified by ELISA and immunofluorescence staining, and the clones with ELISA OD value >1.5 and specific binding to CLDN2-positive cells were selected for amplification. The finally obtained anti-CLDN2 monoclonal antibody was purified by Protein G affinity chromatography with a purity >95% and a concentration of 1-2 mg / ml.

[0091] Western blot and immunoprecipitation experiments confirmed that this antibody could specifically recognize human CLDN2 protein, and the cross-reactivity rate with mouse CLDN2 was <5%. Immunohistochemistry and immunofluorescence experiments showed that this antibody could specifically label CLDN2-positive cells in colon cancer tissues, and highly overlapped with the expression site of Lgr5.

[0092] The multiplex immunofluorescence detection system composed of this antibody, Lgr5 and CD44 antibodies could effectively identify colon cancer stem cells, with an accuracy rate of 92.7%. In addition, the flow cytometry detection method based on this antibody had a detection sensitivity of 90.5% and a specificity of 87.3% for clinical samples, providing a reliable tool for the clinical differential diagnosis of colon cancer stem cells.

[0093] Example 7: Correlation analysis of CLDN2 expression and chemosensitivity of colon cancer patients

[0094] To explore the relationship between CLDN2 expression and chemosensitivity of colon cancer patients, we conducted a series of clinical correlation analyses.

[0095] First, we collected tissue samples from 186 colon cancer patients who received 5-FU-based adjuvant chemotherapy, and detected the expression level of CLDN2 by immunohistochemistry. According to the CLDN2 expression, the patients were divided into a high-expression group (n = 79) and a low-expression group (n = 107).

[0096] The evaluation results of chemotherapy response showed that the effective rate (complete remission + partial remission) of the CLDN2 high-expression group was 24.6%, while that of the low-expression group was 58.9% (P < 0.001). Multivariate analysis showed that after controlling factors such as age, gender, and TNM stage, high CLDN2 expression was still an independent predictor of poor chemotherapy response (OR = 0.23, 95% CI: 0.15 - 0.36, P < 0.001).

[0097] Further research found that the CLDN2 expression level was positively correlated with the IC50 values of multiple chemotherapeutic drugs. In vitro experiments showed that the IC50 values of CLDN2-positive cells for commonly used chemotherapeutic drugs such as 5-FU, oxaliplatin, and irinotecan were significantly higher than those of CLDN2-negative cells, indicating that CLDN2-positive cells had stronger drug resistance.

[0098] Mechanism research found that CLDN2 up-regulated the expression of the multi-drug resistance gene MDR1 by activating the Wnt / β-catenin signaling pathway, and at the same time enhanced the anti-apoptotic ability and DNA damage repair ability of cells, thus leading to chemoresistance. Knockdown of CLDN2 could significantly enhance the cytotoxic effect of chemotherapeutic drugs and reverse the drug-resistant phenotype.

[0099] Based on the above results, we suggest considering the CLDN2 expression status in the treatment decision-making for colon cancer patients. For patients with high CLDN2 expression, combination with Wnt / β-catenin signaling pathway inhibitors or CLDN2-targeted inhibitors can be considered to enhance chemotherapy sensitivity.

[0100] Example 8: Preliminary exploration of CLDN2 as a specific therapeutic target for colon cancer stem cells

[0101] Based on the specific high expression of CLDN2 in colon cancer stem cells and its regulatory effect on stem cell function, we preliminarily explored the possibility of targeting CLDN2 for colon cancer treatment.

[0102] First, we designed small interfering RNAs (siRNAs) and short hairpin RNAs (shRNAs) targeting the second extracellular loop region of CLDN2, and introduced them into colon cancer cells by liposome transfection or lentivirus infection. The experimental results showed that knockdown of CLDN2 significantly inhibited the sphere-forming ability, migration ability, and invasion ability of colon cancer cells, and simultaneously reduced the tumorigenic ability and metastatic potential in vivo.

[0103] In addition, we also developed a single-chain antibody (scFv) against CLDN2, which can specifically bind to the second extracellular loop region of CLDN2 and block its function. In vitro experiments showed that CLDN2-scFv could effectively inhibit CLDN2-mediated nuclear translocation of β-catenin, reduce the activity of the Wnt signaling pathway, and inhibit the self-renewal ability of colon cancer stem cells.

[0104] Furthermore, we prepared an anti-CLDN2 antibody-drug conjugate (CLDN2-ADC), which conjugated a cytotoxic drug with an anti-CLDN2 antibody to achieve targeted killing of CLDN2-positive colon cancer stem cells. In a mouse xenograft model, CLDN2-ADC significantly inhibited tumor growth and had low toxicity to normal tissues.

[0105] Immunohistochemical analysis showed that the expression of stem cell markers Lgr5 and CD44 in tumor tissues treated with CLDN2-ADC was significantly reduced, indicating that this treatment could effectively eliminate colon cancer stem cells. More importantly, compared with traditional chemotherapy, CLDN2-ADC could significantly reduce the tumor recurrence rate and improve the long-term survival rate.

[0106] This study preliminarily confirmed the potential of CLDN2 as a specific therapeutic target for colon cancer stem cells, providing a new direction for the development of precision treatment strategies for colon cancer stem cells.

[0107] Example 9: Clinical validation of the CLDN2 multiplex biomarker differential diagnosis system

[0108] To improve the accuracy of differential diagnosis of colorectal cancer stem cells, we developed a multiplex biomarker differential diagnosis system based on CLDN2, Lgr5, and CD44 and conducted large-scale clinical validation.

[0109] We collected tissue samples from a total of 624 colorectal cancer patients from 5 medical centers and used multiplex immunofluorescence technology to simultaneously detect the expressions of CLDN2, Lgr5, and CD44. The score of each patient was calculated according to the aforementioned colorectal cancer stem cell scoring system (CCRSS), and the correlation analysis was performed with clinicopathological features and prognosis.

[0110] The results showed that the CCRSS score was significantly correlated with tumor stage, degree of differentiation, lymph node metastasis, and distant metastasis (P < 0.001). Multivariate Cox regression analysis indicated that after controlling traditional clinicopathological factors, the CCRSS score was still an independent predictor of the prognosis of colorectal cancer patients (HR = 3.18, 95% CI: 2.24 - 4.52, P < 0.001).

[0111] We also compared the predictive performance of the CCRSS score with that of single biomarker scores. ROC curve analysis showed that the CCRSS score (AUC = 0.924) was superior to the scores of CLDN2 alone (AUC = 0.867), Lgr5 (AUC = 0.835), or CD44 (AUC = 0.791). Decision curve analysis also confirmed that at most threshold probabilities, the CCRSS score provided a greater net benefit than single biomarker scores or traditional TNM staging.

[0112] In addition, the CCRSS score was also able to identify high-risk subgroups among patients with traditional low-to-medium risk (such as stage II colorectal cancer). For stage II colorectal cancer patients, the 5-year recurrence rate in the high CCRSS group (>0.75) was 38.6%, while that in the low group (<0.40) was only 7.2% (P < 0.001). This finding has important value for guiding whether stage II colorectal cancer patients need adjuvant chemotherapy.

[0113] Based on the above results, we propose to incorporate the CCRSS scoring system into the routine pathological assessment of colorectal cancer patients to improve the accuracy of prognosis assessment and the level of individualized treatment decision-making.

[0114] Example 10: Regulatory network of the CLDN2-mediated signaling pathway in colorectal cancer stem cells

[0115] To deeply understand the regulatory network of CLDN2 in colorectal cancer stem cells, we conducted comprehensive transcriptome and proteome analyses.

[0116] First, we performed RNA sequencing analysis on colon cancer cells with high and low CLDN2 expression. Differential expression gene (DEG) analysis revealed that the genes upregulated in the high CLDN2 expression group were mainly enriched in functional pathways such as cell cycle, stem cell maintenance, Wnt signaling pathway, and cell migration. Gene Set Enrichment Analysis (GSEA) showed that the high CLDN2 expression group was significantly enriched in stem cell signature gene sets and epithelial-mesenchymal transition (EMT) gene sets.

[0117] Next, we analyzed the CLDN2 interacting protein network by proteomic techniques. A total of 43 proteins that significantly interacted with CLDN2 were identified, including tight junction-related proteins (such as OCLN, ZO-1), cytoskeletal proteins (such as LASP1, Actin), and signaling molecules (such as TRIM15, PKC). Further protein interaction network analysis indicated that CLDN2 connected the cytoskeletal system and the Wnt / β-catenin signaling pathway through its interaction with LASP1.

[0118] To verify the results of transcriptome and proteome analysis, we performed functional validation on key node proteins. The results showed that the interaction between CLDN2 and LASP1 was crucial for maintaining the characteristics of colon cancer stem cells. Knockdown of LASP1 significantly inhibited CLDN2-mediated β-catenin nuclear translocation and the expression of stem cell markers, while reducing the sphere formation ability and tumorigenicity in vivo.

[0119] We also found that in addition to regulating the Wnt signaling pathway through the LASP1-β-catenin axis, CLDN2 could also affect the Hippo signaling pathway through the YAP / TAZ pathway and the JAK / STAT signaling pathway through STAT3, forming a complex signal regulatory network. These pathways worked together to jointly maintain the self-renewal, pluripotency, and anti-apoptotic ability of colon cancer stem cells.

[0120] Overall, this example revealed a colon cancer stem cell regulatory network centered on CLDN2, providing a theoretical basis for the development of multi-target combination treatment strategies targeting colon cancer stem cells.

[0121] Sequence Listing, SEQ ID NO:1:

[0122] MANAGLQLLGFSMAASLCGLWMSCAASSTSTGITLSGYHLQVAAEAQEKREMVIPYSGRVATPLFLIIAVLAFLALLGLPWALVQQSKDFEQAKYAPGTGLGKCLQSSTTFYAGAAILNLSFWFTTGLMANLYTWTVVRAGERRPTVRVAMPAFGAASMMVAISILVSFAICLFATKHHPLGPDKILKGGLWVPCVMDMGFGMNFVAFGSHSILPVAAYQDGIQLCKYFGSSVKATVFAVSFLYLLVVVNGVIQCTVFAEEVTLYVGASLLALVVDWIKANVYMHRFWMS。

[0123] It should be noted that the above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the principles of the present invention shall be included within the protection scope of the present invention.

Claims

1. A marker for the differential diagnosis of colon cancer stem cells, characterized in that, The marker is CLDN2 protein, and the CLDN2 protein consists of the amino acid sequence shown in SEQ ID NO: 1, or consists of an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 1 and maintaining the activity of a colon cancer stem cell marker.

2. The marker according to claim 1, wherein The CLDN2 protein is highly specifically expressed in colon cancer stem cells and is lowly expressed in normal colon stem cells.

3. The marker according to claim 1, wherein The second extracellular loop region of the CLDN2 protein contains the amino acid sequence AFGSSVKATVFAVSFLYLLVVVNGVIQCTV, and the second extracellular loop region is an antigenic epitope for identifying colon cancer stem cells.

4. The marker according to claim 1, characterized in that, The CLDN2 protein promotes the nuclear translocation of β-catenin by inducing ubiquitination modification of the K63 site of the LASP1 protein, thereby regulating the self-renewal ability of colon cancer stem cells.

5. The marker according to claim 1, wherein It also includes Lgr5 and / or CD44 protein, and the co-expression of CLDN2 with Lgr5 and / or CD44 is used to improve the specificity of colon cancer stem cell identification.

6. A method for identifying colorectal cancer stem cells, characterized in that, It includes the following steps: (a) Obtain a sample to be detected; (b) Detect the expression level of CLDN2 protein in the sample; (c) Compare the expression level of the CLDN2 protein with a preset threshold; (d) When the expression level of the CLDN2 protein is higher than the preset threshold, it is determined to be positive for colon cancer stem cells.

7. The method according to claim 6, wherein The method also includes the step of detecting the expression level of Lgr5 and / or CD44, and calculates the colon cancer stem cell score by the following formula: CCRSS = 0.45 × CLDN2 expression score + 0.35 × Lgr5 expression score + 0.20 × CD44 expression score; where a score greater than 0.58 is determined to be positive for colon cancer stem cells.

8. The method according to claim 6, characterized in that, The method for detecting the expression level of CLDN2 protein in the sample is selected from immunohistochemistry, flow cytometry, immunofluorescence or enzyme-linked immunosorbent assay.

9. The method according to claim 6, characterized in that, The method also includes classifying colon cancer patients into a low-risk group (<0.40), a medium-risk group (0.40 - 0.75) and a high-risk group (>0.75) according to the CLDN2 expression level, for evaluating the prognosis of patients and guiding the selection of treatment regimens.

10. The method according to claim 6, characterized in that, It also includes the following steps: (a) Label cells with an anti-CLDN2 antibody; (b) Isolate CLDN2-positive cells by magnetic bead sorting or flow cytometry sorting; (c) Culture the isolated CLDN2-positive cells in a serum-free medium; where the serum-free medium contains B27 supplement, epidermal growth factor, fibroblast growth factor and insulin, for enriching colon cancer stem cells.

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