Application of alpha-1, 2-high mannose type CD133 as tumor stem cell marker of intrahepatic cholangiocarcinoma

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to application of alpha-1, 2-high mannose type CD133 as a tumor stem cell sorting marker of intrahepatic cholangiocarcinoma. The lectin Cyanovirus-N (CVN) used in the invention is a 11kDa protein, is derived from Nostoc ellipsoporum, and has a specific recognition function on a terminal alpha-1, 2-Man part on high mannose (Man-8 or Man-9) glycan, the carbohydrate chain structure difference between intrahepatic cholangiocarcinoma tumor stem cells and tumor non-stem cells is studied, and the specific recognition function on the terminal alpha-1, 2-Man part on the non-stem cells of the intrahepatic cholangiocarcinoma tumor stem cells and the non-stem cells of the intrahepatic cholangiocarcinoma tumor non-stem cells is realized. The invention provides a novel marker alpha-1, 2-high mannose type CD133 protein for precise targeting of tumor stem cells of intrahepatic cholangiocarcinoma, and provides application of a novel precise marker for targeting treatment of tumor stem cells by recognizing sugar chain change through lectin.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and particularly relates to the use of α-1,2-high mannose CD133 as a marker for sorting intrahepatic cholangiocarcinoma tumor stem cells, and the potential application of its upstream target MAN1C1 in the preparation of therapeutic drugs targeting intrahepatic cholangiocarcinoma tumor stem cells. Background Art

[0002] According to records, intrahepatic cholangiocarcinoma (iCCA) is a highly heterogeneous hepatobiliary malignancy with bile duct cell differentiation characteristics, accounting for 10%-20% of primary liver cancer, and its incidence is on the rise worldwide. Studies have shown that iCCA is usually asymptomatic in the early stages, and most iCCA patients are diagnosed in the late stages, with limited available treatment options, leading to poor clinical outcomes. In current clinical practice, radical resection remains the preferred treatment strategy for iCCA, but 60% of surgical patients will experience postoperative tumor recurrence and metastasis, and the overall survival rate of patients after 5 years is only 7-20%. Therefore, in order to improve the survival outcomes of iCCA patients, it is urgent to conduct in-depth research on the molecular mechanisms that regulate the occurrence and development of iCCA in all aspects to find potential targets, while exploring new targets for early screening and diagnosis of intrahepatic cholangiocarcinoma to provide new and effective treatment strategies for iCCA patients.

[0003] Research has revealed that cancer stem cells (CSCs) are a small group of cells that exist in tumors and have the characteristics of self-renewal, high tumorigenicity, multidirectional differentiation, and resistance to chemotherapy and radiotherapy. In recent years, the research results of multiple research groups published in top journals such as Nature and Cell have confirmed that cancer stem cells play an important role in tumor recurrence and metastasis, just like "seed cells". Therefore, targeted therapy and diagnosis of cancer stem cells are considered to be one of the most promising cancer treatments. It has been reported that a variety of CSCs markers have been found in human cholangiocarcinoma, including CD133, CD44, and CD90. In human cholangiocarcinoma subtypes, CSCs account for more than 30% of the tumor mass. CD133, as a marker of cancer stem cells, has 9 N-linked glycosylation sites. The currently recognized CD133 antibody used to sort cancer stem cells is also speculated to be targeted at its glycosylated epitopes. Previous studies have shown that the glycosylation of CD133 changes during the differentiation of tumor stem cells. However, due to the variable epitope masking caused by the differences in the glycosylation of CD133 and the fact that some normal stem cells also express CD133, this similarity greatly limits the application of CD133 in the targeted identification of tumor stem cells and treatment.

[0004] Studies have shown that glycosylation is the most common post-translational modification. The occurrence and development of tumors are always accompanied by abnormal protein glycosylation, which in turn regulates the malignant behavior of tumor cells. Therefore, sugar chains are expected to become clinical diagnostic markers and therapeutic targets. Many cell surface glycoproteins involved in tumor progression have N-glycosylation modifications. Lectins can specifically bind to sugar chains and can therefore be applied to the identification of tumor stem cells.

[0005] Based on the foundation and current status of the prior art, the inventors of the present application intend to provide the use of α-1,2-high mannose CD133 as a marker for sorting intrahepatic cholangiocarcinoma tumor stem cells, as well as the potential application of its upstream target MAN1C1 in the preparation of therapeutic drugs targeting intrahepatic cholangiocarcinoma tumor stem cells. Summary of the invention

[0006] The purpose of the present invention is to provide the use of α-1,2-high mannose type CD133 as a marker for sorting intrahepatic cholangiocarcinoma tumor stem cells based on the foundation and current status of the prior art, as well as the potential application of its upstream target MAN1C1 in the preparation of therapeutic drugs targeting intrahepatic cholangiocarcinoma tumor stem cells.

[0007] In the present invention, CD133 antibody and biotinylated cyanobacteria lectin Cyanovirin-N (CVN) are combined to sort out intrahepatic cholangiocarcinoma tumor stem cells, providing a more accurate marker for sorting and identifying intrahepatic cholangiocarcinoma tumor stem cells, namely, α-1,2-high mannose type CD133 protein.

[0008] The lectin Cyanovirin-N (CVN) used in the present invention is an 11kDa protein derived from the blue-green algae Nostoc ellipsosporum, which has a specific recognition function for the terminal α-1,2-Man part on the high mannose (Man-8 or Man-9) polysaccharide. It is speculated that when the cell becomes cancerous, the oligosaccharide chain of the glycoprotein on the membrane of the intrahepatic bile duct cancer stem cell may change. This abnormal change in glycosylation leads to the malignant transformation of the cell, which in turn causes the self-renewal of the cancer cell. Therefore, it is expected that the lectin can be used to identify this sugar chain change and provide a new type of precise marker for targeted tumor stem cell therapy.

[0009] Specifically, in order to achieve the above object, the present invention adopts the following technical solutions:

[0010] Primary intrahepatic cholangiocarcinoma cells were isolated from intrahepatic cholangiocarcinoma tumor tissues of two patients (Patient#1 and Patient#4) by isolating primary cells, and then CD133+ and CD133- primary intrahepatic cholangiocarcinoma cells were obtained by sorting using Miltenyi Biotec's CD133-coupled magnetic beads. CD133+ cells were further incubated in a buffer containing biotinylated CVN antibodies for 30 minutes, and cells were incubated on ice for 15 minutes with streptavidin-coupled microbeads, and CVN-positive cells were sorted using MiltenyiBiotec columns. Finally, three groups of cells were obtained, namely CD133- cells, CD133+α-1,2-Man- cells, and CD133+α-1,2-Man+ cells. Through cell sphering and limiting dilution experiments to detect stem cell characteristics, statistical analysis found that the CD133+α-1,2-Man+ cell population has stronger self-renewal ability and stemness, and has tumor stem cell characteristics: it can form cell spheres and grow in suspension in serum-free stem cell culture medium; it highly expresses stem cell markers Nanog and POU5F1; in tumor formation experiments, as few as 500 CD133+α-1,2-Man+ cells can form tumors in nude mice. 50 pairs of paired tissue sections of intrahepatic cholangiocarcinoma patients were collected, and immunohistochemistry experiments were performed with lectin CVN to observe whether there is enrichment of α-1,2-high mannose N-glycans in the tissues. The results showed that compared with adjacent tissues, the content of α-1,2-high mannose N-glycans in tumor tissues was significantly increased, which also shows that α-1,2-high mannose N-glycans can be used as a potential marker for intrahepatic cholangiocarcinoma tumor stem cells.

[0011] The present invention also provides a potential drug target, mannosidase MAN1C1, which can inhibit the stemness, spheroidization and self-renewal ability of intrahepatic cholangiocarcinoma tumor stem cells by overexpressing MAN1C1 in intrahepatic cholangiocarcinoma tumor stem cells, thereby effectively inhibiting tumor growth. When detecting the expression of CD133 protein in specimens of patients with intrahepatic cholangiocarcinoma, it was found that the molecular weight of CD133 was reduced in tumor specimens of some patients (Patient#4), indicating that glycosylation had changed. Further detection of changes in various glycosyltransferases revealed that low expression of mannosidase MAN1C1 promoted the formation of CD133α-1,2-high mannose structure, enhanced the stemness of tumor stem cells, and led to malignant progression of tumors. Therefore, MAN1C1 can also be used as an effective therapeutic target in subsequent clinical applications.

[0012] The beneficial effects of the present invention include:

[0013] 1) The present invention uses the specific sugar chain on the tumor stem cell glycoprotein CD133 as a marker to sort cells. This method is different from the previous targeted tumor stem cell marker antibody sorting method. It can achieve the sorting purpose more accurately and is innovative. The present invention clarifies the difference in sugar chain structure between tumor stem cells and tumor non-stem cells. On the basis of the original sorting using the stemness-related marker CD133, the α-1,2-high mannose N-sugar chain is used as a new marker for tumor stem cells. Intrahepatic cholangiocarcinoma tumor stem cells are sorted in combination with the lectin CVN. Cells expressing α-1,2 high mannose CD133 have tumor stem cell characteristics. Tumor stem cells in intrahepatic cholangiocarcinoma can be identified more accurately, and are not limited to stemness genes on the cell surface, providing a new research direction for the diagnosis and treatment of intrahepatic cholangiocarcinoma.

[0014] 2) The present invention is based on the fact that low expression of glycosyltransferases such as mannosidase MAN1C1 leads to the formation of CD133α-1,2 high-mannose N-glycan structures in tumor stem cells, and overexpression of MAN1C1 can significantly inhibit the proliferation and self-renewal ability of intrahepatic cholangiocarcinoma tumor stem cells. Therefore, targeting the molecular target of MAN1C1 is expected to provide new ideas for the subsequent clinical research on anticancer drugs targeting intrahepatic cholangiocarcinoma tumor stem cells, and promote the development of the field of intrahepatic cholangiocarcinoma treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Low expression of mannosidase MAN1C1 leads to CD133 mainly having α-1,2-high mannose sugar chain structure and high expression level of stemness genes, among which,

[0016] (A) Western blot and Lectinblot were used to analyze the molecular weight changes and N-glycan structure changes of CD133 in specimens from patients with intrahepatic cholangiocarcinoma; IP: first, immunoprecipitation was performed with anti-CD133 antibody, and then immunoblot IP detection was performed with anti-CD133 antibody or biotinylated lectin CVN or PHA-L. Lectin CVN recognized α-1,2-high mannose; lectin PHA-L recognized β-1,6-GlcNAc;

[0017] (B) QRT-PCR analysis of the relative expression levels of mRNA of high-mannose N-glycan-related enzyme genes (MAN1A1, MAN1A2, MAN1C1, MGAT1, MGAT2, MGAT4A, MGAT4B, and MGAT5) in specimens from patients with intrahepatic cholangiocarcinoma;

[0018] (C) QRT-PCR analysis of the mRNA levels of stemness genes POU5F1 and Nanog in 12 patients with intrahepatic cholangiocarcinoma (CD133-, 4 patient specimens that did not express CD133; CD133+α-1,2-Man+, 4 patient specimens that expressed CD133 and α-1,2-Man; CD133+α-1,2-Man-, 4 patient specimens that expressed CD133 but did not express α-1,2-Man);

[0019] (D) QRT-PCR analysis of the relative expression levels of MAN1C1 mRNA in CD133+α-1,2-Man+ and CD133+α-1,2-Man- cells sorted from patient specimens using CD133 antibody and biotinylated CVN.

[0020] (E) Western blot was used to detect the changes in MAN1C1 protein levels in CD133+α-1,2-Man+ and CD133+α-1,2-Man- cells in patient specimens. GAPDH was used as a loading control.

[0021] (F) Flow cytometry analysis of the positive expression rate of α-1,2-high mannose N-glycans on the surface of CD133+α-1,2-Man+ cells expressing FLAG or MAN1C1-FLAG. Isotype control cells were stained without fluorescent secondary antibody.

[0022] Figure 2 The CD133+α-1,2-Man+ double positive cells isolated from tumor specimens of patients with intrahepatic cholangiocarcinoma have the characteristics of tumor stem cells, among which,

[0023] (A) After isolating tumor cells from the tumor tissues of patients 1 and 4, CD133+ and CD133- cells were sorted using CD133-coupled magnetic beads. The obtained CD133+ cells were incubated with biotinylated CVN in rotation, and then incubated with streptavidin-coupled magnetic beads in rotation. The cell suspension was then added to the separation column to elute the cells, which were CD133+α-1,2-Man- cells. After removing the magnetic stand, the cells coupled to the magnetic beads were eluted as CD133+α-1,2-Man+ cells. The sorted CD133+α-1,2-Man+ cells were able to grow in suspension in the stem cell culture medium in the form of spheres. The scale bar is 100μm.

[0024] (BC) QRT-PCR analysis of the relative mRNA expression levels of stem cell markers POU5F1 and Nanog in the three groups of cells.

[0025] (D) In ​​a cell limiting dilution experiment, the three groups of cells (0-25) obtained by the above sorting were serially diluted and inoculated into a 96-well plate (20 wells for each dilution) and cultured in stem cell culture medium. The x-axis represents the number of cells inoculated per well, and the y-axis represents the sphere formation efficiency.

[0026] Figure 3 Overexpression of MAN1C1 inhibits the self-renewal of intrahepatic cholangiocarcinoma cancer stem cells,

[0027] (A) MAN1C1 was overexpressed in sorted CD133+α-1,2-Man+ cells using the lentivirus-mediated method. After a period of culture and screening, the cell sphere formation assay detected that the overexpression of MAN1C1 in CD133+α-1,2-Man+ cells expressing MAN1C1-FLAG reduced the formation of tumor stem cell spheres compared with the control group cells expressing FLAG.

[0028] (B) Statistical results of the cell sphere number in the sphere formation experiment.

[0029] (CD) Cell limiting dilution experiment, serial dilutions of MAN1C1-overexpressing or FLAG-expressing control CD133+α-1,2-Man+ cells (0-25) were seeded into 96-well plates (20 wells per dilution) and cultured in stem cell culture medium. The x-axis represents the number of seeded cells, and the y-axis represents the sphere formation efficiency.

[0030] (E) The tumor initiation ability of CD133+α-1,2-Man+ cells expressing MAN1C1-FLAG and FLAG was compared in a limiting dilution tumor formation assay in immunodeficient mice (5000, 1000, or 500 tumor cells per mouse were injected orally). Mice were sacrificed post-mortem or 180 days after cell injection and tumor formation was examined. The table shows the efficiency of cells in glioma formation in immunodeficient mice: number of mice that developed tumors / total number of mice injected with tumor cells.

[0031] Figure 4 Intrahepatic cholangiocarcinoma tumor tissue is rich in α-1,2-high mannose N-glycans, and the expression of α-1,2-high mannose N-glycans is closely related to the development of intrahepatic cholangiocarcinoma.

[0032] (A) Statistical results of immunohistochemical staining scores of α-1,2-high mannose glycan modification in cancer and adjacent tissue microarrays from 50 patients with intrahepatic cholangiocarcinoma;

[0033] (B) The immunohistochemical staining score of α-1,2-high mannose type glycan modification in patients with postoperative lymph node metastasis was higher than that in patients without lymph node metastasis;

[0034] (C) Patients with high expression of α-1,2-high mannose sugar chains had a shorter overall survival than those with low expression of α-1,2-high mannose sugar chains. DETAILED DESCRIPTION

[0035] The specific embodiments provided by the present invention are described in detail, but the implementation of the present invention is not limited thereto. The specific conditions and experimental methods not specified in this embodiment are usually implemented according to the conventional conditions described in the Molecular Cloning Experiment Guide, or with reference to the conditions provided by the reagent manufacturer.

[0036] Example 1 Immunoprecipitation and Lectin blot detection of CD133 N-glycan structural changes in intrahepatic cholangiocarcinoma patient tissues

[0037] The present invention studies the expression of CD133 N-sugar chain structure in intrahepatic cholangiocarcinoma patient tissues, selects fresh intrahepatic cholangiocarcinoma tumor specimens, lyses the patient tissues, extracts the lysate supernatant, uses CD133 antibody to immunoprecipitate CD133 protein in the tissues, and then uses Lectin blot to detect the sugar chain structure of CD133 protein, comprising the following steps:

[0038] 1. Expression, Purification and Biotin Labeling of CVN

[0039] 1) Transform BL21 competent cells with the constructed pET28a-pelB-CVN-6his expression plasmid (kana resistance);

[0040] 2) Pick a single clone into LB medium, culture it at 37°C, 220 rpm / min overnight, then add it to LB medium at a ratio of 1:100, culture it at 37°C, 220 rpm / min until OD = 0.6, add IPTG with a final concentration of 1 mM, induce it at 18°C, 180 rpm / min overnight, 12 h, CVN is expressed in the supernatant;

[0041] 3) Purification using Ni-NTA beads;

[0042] 4) After purification, 0.2 mg of CVN protein requires 0.000792 mmol of biotin (Sigma). The CVN protein is added with the biotin reagent and incubated on ice for 2 hours.

[0043] 5) Desalting column to remove excess biotin groups and byproducts (SpinOUT TM GT-600);

[0044] 6) The collected desalted protein solution is the biotinylated protein CVN-biotin from which excess biotin reagent and NHS have been removed.

[0045] 2. Lectin blot detection of the sugar chain structure of CD133

[0046] 1). Prepare glycoprotein samples;

[0047] 2). Run the sample on SDS-PAGE gel until the dye front reaches the bottom of the gel;

[0048] 3). Thoroughly wet the PVDF membrane with a small amount of methanol, prepare the membrane transfer device, and then transfer the gel;

[0049] 4. After transferring the protein from the gel to the membrane, remove the membrane and rinse with TBST buffer;

[0050] 5). Place the membrane in 15 mL of blocking solution (3% (w / v) BSA in TBST buffer) and shake slowly for 2 h;

[0051] 6). Rinse the membrane with TBST buffer, add biotinylated lectin and incubate overnight at 4 degrees;

[0052] 7). Take out the membrane and wash it in TBST buffer for 3 times, 15 min each time;

[0053] 8). After washing, add HRP-coupled streptavidin and incubate for 2 hours;

[0054] 9). Take out the membrane and wash it in TBST buffer for 3 times, 15 min each time;

[0055] 10). Add developer (mix solution A and solution B in a 1:1 ratio) to the washed membrane and then use a chemiluminescence instrument to develop and take pictures;

[0056] The results are as follows Figure 1 As shown, the molecular weight of CD133 protein decreased in patient#4 tissue, indicating that glycosylation changed, mainly α-1,2-high mannose sugar chain structure that can be specifically recognized by CVN. There were a small amount of α-1,2-high mannose sugar chains in CD133 protein in patient#1 tissue, and most of them were complex N-sugar chain structures bound by PHA-L. Further Western blot, QRT-PCR and flow cytometry detection confirmed that the formation of CD133 protein α-1,2-high mannose structure was mainly caused by the low expression of mannosidase MAN1C1, and the expression of stemness genes was higher in the tissues of patients with high expression of CD133 α-1,2-high mannose structure (***P<0.001).

[0057] Example 2: Using CD133 antibody and CVN lectin that specifically binds to α-1,2-high mannose N-sugar chains to sort intrahepatic cholangiocarcinoma cells and detecting the stemness of the sorted cells by sphering experiment, limiting dilution experiment and QRT-PCR, including the following steps:

[0058] (1) Methods for isolating and culturing primary intrahepatic cholangiocarcinoma cells

[0059] The intrahepatic cholangiocarcinoma tumor tissue specimens used in this example were obtained from patients who underwent surgery at Zhongshan Hospital Affiliated to Fudan University with approval from the Zhongshan Hospital Ethics Committee after obtaining informed consent. Within 1 hour after surgical resection, the tumor specimens were washed and enzymatically decomposed into single cells.

[0060] Cells were cultured in stem cell culture medium, DMEM / F12 medium (Gibco) containing B27 (Gibco) lacking vitamin A, 7.5% NaHCO3, 100 μg / mL penicillin and 50 μg / mL streptomycin (Gibco), 2 μg / mL heparin (Sigma), 20 ng / mL EGF (Proteintech) and 20 ng / mL b-FGF (Proteintech), under the conditions of 5% CO2-95% air, saturated humidity and 37°C.

[0061] 1. Preparation of enzymatic reagents

[0062]

[0063] 2. Place the tumor tissue in a 60 mm culture dish in 3 ml of pre-cooled HBSS balance solution;

[0064] 3. Wash twice in 3 ml pre-cooled HBSS balanced liquid to remove blood stains;

[0065] 4. Place the tumor tissue in a new 60 mm culture dish in 3 mL of pre-cooled HBSS balanced liquid and cut it into 1 mm pieces using small forceps, surgical and ophthalmic scissors. 3 Small pieces;

[0066] 5. Transfer the above tissue fragments to a 15 mL centrifuge tube and centrifuge at 2000 rpm for 10 minutes at 4 degrees;

[0067] 6. Discard the supernatant, add 5-10 mL of digestion liquid, transfer to a 60 mm dish, and digest in a 37°C incubator for 60 minutes (if it is very viscous, add DNAase I). Repeatedly pipette with a 5 mL Pasteur pipette every 10 minutes;

[0068] 7. Centrifuge at 2000 rpm for 10 minutes, add about 5 mL of pre-cooled red blood cell lysis buffer (NH4CL buffer) and lyse red blood cells on ice for 10 minutes;

[0069] 8. Centrifuge at 2000 rpm for 10 minutes at 4 degrees, remove the supernatant, add 5 mL of HBSS buffer, and centrifuge at 2000 rpm for 10 minutes;

[0070] 9. Discard the supernatant, add 1 mL of HBSS buffer, filter with a 70-mesh screen, transfer the filtrate to a 1.5 mL EP tube, centrifuge at 2000 rpm for 10 minutes at room temperature, discard the supernatant, and resuspend with stem cell culture medium for culture;

[0071] 10. Count the obtained primary cells and transfer them to a constant temperature incubator at 37°C and 5% CO2 (v / v) for culture.

[0072] (2) MACS magnetic bead sorting of CD133+α-1,2-Man+ double positive cells

[0073] 1. Cell counting.

[0074] 2. Centrifuge the cell suspension at 300 g for 10 minutes and completely remove the supernatant.

[0075] 3. Every 10 7 Resuspend the cell pellet in 60 μl of buffer.

[0076] 4. Every 10 7 Add 20 μl FcR blocking reagent per cell.

[0077] 5. Every 10 7 Add 20 μl CD133 MicroBeads per cell.

[0078] 6. Mix well and rotate slowly and continuously for 15 minutes in a MACSmix tube rotator in a 4 degree refrigerator. 7 Resuspend cells in 1-2 mL of buffer per 1 cell volume.

[0079] 7. Centrifuge at 300g for 10 min, discard the supernatant and resuspend the cells in 500μl buffer.

[0080] 8. Install the sorting column device, rinse the sorting column, and then slowly add the cell suspension. The cells that flow out of the sorting column are CD133- cells.

[0081] 9. Remove the magnetic stand and transfer the sorting column to the collection centrifuge tube. Wash the sorting column three times with 500 μl Buffer to obtain the collection fluid of CD133+ cells.

[0082] 10. Incubate CD133+ cells in a buffer containing biotinylated CVN antibody for 30 minutes.

[0083] 11. Incubate cells with streptavidin-coupled microbeads on ice for 15 minutes.

[0084] 12. Sort CVN-positive cells using Miltenyi Biotec columns. The sorting process is the same as that for CD133-positive cells.

[0085] 13. The obtained CD133-, CD133+α-1,2-Man+, and CD133+α-1,2-Man- cells were cultured in stem cell culture medium.

[0086] (3) Detection of stemness of sorted cells

[0087] 1. Spheroidization experiment: The three groups of cells were centrifuged at 1500 rpm for 5 min.

[0088] 2. Discard the old culture medium, add stem cell culture medium, and centrifuge at 1500 rpm for 5 minutes.

[0089] 3. Add stem cell culture medium to resuspend and count the cells.

[0090] 4. Add 5,000 cells to each well of a six-well plate into stem cell culture medium to culture tumor stem cells.

[0091] 5. On the seventh day, observe the formation of tumor stem cells into spheres and take photos for record.

[0092] 6. Limiting dilution experiment: Serially diluted cells were seeded into 96-well plates (20 wells for each dilution) and cultured in stem cell culture medium.

[0093] 7. Add fresh culture medium (20 μl) to each well every 3 days.

[0094] 8. After 10 days of incubation, count the wells with spheres and plot against the number of cells seeded per well to calculate the sphere formation efficiency.

[0095] The results are as follows Figure 2 The sorted CD133+α-1,2-Man+ double positive cells were able to form spheres, had higher mRNA expression levels of stem cell marker genes (***P<0.001), and each well could form neurospheres at about 10 cells, while CD133+α-1,2-Man- cells required more cells to form spheres. In summary, the sorted CD133+α-1,2-Man+ double positive cells have tumor stem cell characteristics and functions, and the α-1,2-high mannose N-sugar chain of the cell surface glycoprotein CD133 can be used as a potential tumor stem cell marker for intrahepatic cholangiocarcinoma.

[0096] After the sorted CD133+α-1,2-Man+ double positive cells overexpress MAN1C1, the cell sphering and limiting dilution experiments are used to detect the proliferation and self-renewal ability of the cells, and the animal experiment is used to detect the tumor initiation ability. The steps include:

[0097] (1) Detection of the spheroidization ability of CD133+α-1,2-Man+ double positive cells after overexpression of MAN1C1 The specific experimental steps of the spheroidization experiment and limiting dilution experiment were the same as above, and cell spheroids with a diameter greater than 100 μm were considered valid results. Figure 3 As shown in AD, after overexpression of MAN1C1, the diameter and number of cell spheres formed by CD133+α-1,2-Man+ cells were significantly smaller than those of the control group, and more cells were needed to form spheres, indicating that the expression of MAN1C1 inhibited the proliferation and self-renewal of tumor stem cells (***P<0.001).

[0098] (2) To examine the effect of overexpression of MAN1C1 on the tumor initiation ability of CD133+α-1,2-Man+ cells, a cell limiting dilution tumor formation assay was performed on immunodeficient mice, and tumor formation was detected histologically.

[0099] 1. Count the CD133+α-1,2-Man+ cells overexpressing MAN1C1 and the control cells.

[0100] 2. Resuspend 5000, 1000 or 500 cells in 200 μl PBS and inject into the liver of nude mice using a 1 mL syringe.

[0101] 3. The animals were killed after 180 days and the number of tumors was counted. Figure 3 As shown in E, the CD133+α-1,2-Man+ cells in the control group can form tumors in the mouse in situ model at a low cell number (minimum 500 cells) and have a strong tumor initiation ability, while overexpression of MAN1C1 inhibits the tumorigenic ability of intrahepatic cholangiocarcinoma tumor stem cells.

[0102] Example 4 Immunohistochemistry detected the enrichment of α-1,2-high mannose sugar chains in 50 pairs of clinical intrahepatic cholangiocarcinoma patient tissue chips and statistically analyzed the relationship between the α-1,2-high mannose sugar chain histochemical staining score and the patient's prognosis. The steps include:

[0103] (1) Immunohistochemical analysis of α-1,2-Man in intrahepatic cholangiocarcinoma tissue microarray

[0104] 1. A total of 50 pairs of intrahepatic cholangiocarcinoma specimens were included in this experiment. Tissue chip processing: dewaxing, hydrating tissue sections: xylene 3min×2 times; anhydrous ethanol 3min×2 times; 95% ethanol 3min×1 time; 70% ethanol 3min×1 time; 50% ethanol 3min×1 time; rinse the tissue specimens in tap water for a while; then put them in ddH2O and let them stand for 5min.

[0105] 2. Antigen repair: Place the high-temperature resistant container containing antigen repair solution into the autoclave, immerse the histochemical specimen, cover the autoclave tightly, raise the temperature to 123°C, cut off the power supply, maintain it at 121°C-123°C for 5 minutes, open the safety valve, take out the high-temperature resistant container, cool to room temperature, take out the slices, and wash with ddH2O for 5 minutes × 3 times.

[0106] 3. Block endogenous peroxidase: incubate in H2O2 solution diluted with ddH2O (concentration of 3%) for 10 min, wash with ddH2O for 5 min×2 times, and wash with TBST buffer (0.025% Tween-20) for 5 min×1 time.

[0107] 4. Blocking: Wipe off excess water on the histochemical specimen, gently draw a frame around the slice with a water-blocking pen, drop it in blocking solution (TBS + 0.3% Triton X-100 + 5% serum), and incubate at room temperature for 2 hours.

[0108] 5. Primary antibody: Remove the blocking solution from the specimen, add primary antibody (diluted with TBS buffer containing 5% serum), incubate at room temperature for 3 hours or at 4°C overnight, and wash with TBST buffer (0.025% Tween-20) for 5 minutes × 2 times.

[0109] 6. Secondary antibody: wipe off excess water, add secondary antibody (diluted with TBS buffer containing 5% serum), incubate at room temperature for 30 minutes, and wash with 0.025% Tween-20 TBS buffer for 5 minutes × 2 times.

[0110] 7. Color development: Add DAB solution for histochemistry onto the specimen and observe the degree of color development under a microscope. When a positive reaction occurs, rinse in tap water to block the reaction.

[0111] 8. Contrast staining: drop hematoxylin on the histochemical specimen, counterstain for 1 minute, rinse with running water, differentiate with 1% hydrochloric acid-ethanol for 10 seconds, and rinse with running water.

[0112] 9. Dehydration: 50% ethanol for 3 min, 70% ethanol for 3 min, 95% ethanol for 3 min, anhydrous ethanol for 3 min, xylene for 3 min × 2 times.

[0113] 10. Allow the tissue specimens to dry completely in a ventilated cabinet.

[0114] 11. Sealing: neutral resin sealing.

[0115] The staining results were scored and evaluated based on the staining intensity and positive rate. The scoring criteria for staining intensity were: 0 for negative, 1 for weak positive, 2 for moderate positive, and 3 for strong positive; the scoring criteria for staining positive rate were: 0% for 0, 1-11% for 1, 11-50% for 2, and more than 50% for 3. The score of staining intensity was multiplied by the score of positive rate to obtain the comprehensive score of the tissue section. The results are shown in Figure 2. Figure 4 As shown in A, according to the scoring evaluation, the immunohistochemical staining score of α-1,2-high mannose type N-glycan (CVN positive) in tumor tissue sections was significantly higher than that in adjacent cancer tissues.

[0116] (2) Prognosis of patients with intrahepatic cholangiocarcinoma and α-1,2-Man immunohistochemical staining score

[0117] The results are as follows Figure 4 As shown in BC, the postoperative lymph node metastasis and overall survival of the patients were statistically analyzed, and it was found that some patients with high expression of α-1,2-Man had lymph node metastasis and poor overall survival.

[0118] In summary, CD133+ intrahepatic cholangiocarcinoma cells enriched with α-1,2-high mannose N-glycans on the cell surface were obtained by lectin CVN sorting, and it was verified that this subpopulation of cells has tumor stem cell characteristics, which provides a theoretical basis for α-1,2-high mannose CD133 protein as a marker of intrahepatic cholangiocarcinoma tumor stem cells. Overexpression of MAN1C1 can significantly inhibit the proliferation and self-renewal ability of intrahepatic cholangiocarcinoma tumor stem cells, which is expected to provide new ideas for the subsequent clinical research on anticancer drugs targeting intrahepatic cholangiocarcinoma tumor stem cells.

Claims

1. Application of α-1,2-high mannose CD133 as a tumor stem cell marker for intrahepatic cholangiocarcinoma. In the application, the sugar chain structure of CD133 protein is detected by Lectin blot through the following steps: S1: Transform BL21 competent cells with the constructed pET28a-pelB-CVN-6his expression plasmid (kana resistance); S2: Pick a single clone into LB medium, culture it at 37°C, 220 rpm / min overnight, then add it to LB medium at a ratio of 1:100, culture it at 37°C, 220 rpm / min until OD=0.6, add IPTG with a final concentration of 1 mM, induce it at 18°C, 180 rpm / min overnight, 12 h, CVN is expressed in the supernatant; S3: Purification using Ni-NTA beads; S4: 0.2 mg of purified CVN protein requires 0.000792 mmol of biotin (sigma). CVN protein is added with biotin reagent and incubated on ice for 2 hours; S5: Desalting column to remove excess biotin groups and byproducts (SpinOUT TM GT-600); S6: The collected desalted protein solution is the biotinylated protein CVN-biotin from which excess biotin reagent and NHS have been removed.

2. The use according to claim 1, It is characterized in that The method of detecting the sugar chain structure of CD133 by Lectin blot comprises the following steps: S1: Preparation of glycoprotein samples; S2: Run on SDS-PAGE gel until the dye front reaches the bottom of the gel; S3: Wet the PVDF membrane thoroughly with a small amount of methanol, prepare the transfer device, and then transfer the gel; S4: After transferring the protein from the gel to the membrane, remove the membrane and rinse it with TBST buffer; S5: Place the membrane in 15 mL of blocking solution (3% (w / v) BSA in TBST buffer) and shake slowly for 2 h; S6: Rinse the membrane with TBST buffer, add biotinylated lectin and incubate overnight at 4°C; S7: Take out the membrane and wash it in TBST buffer for 3 times, 15 min each time; S8: After washing, add HRP-conjugated streptavidin and incubate for 2 h; S9: Take out the membrane and wash it in TBST buffer for 3 times, 15 min each time; S10: Add developer (mix solution A and solution B in a 1:1 ratio) onto the washed membrane and then develop with a chemiluminescence instrument to take pictures; S11: The α-1,2-high mannose structure of CD133 protein was clarified by further Western blot, QRT-PCR and flow cytometry.

3. The use according to claim 2, It is characterized in that Intrahepatic cholangiocarcinoma cells were sorted using CD133 antibody and CVN lectin that specifically binds to α-1,2-high mannose N-sugar chains, and the stemness of the sorted cells was detected by spheroidization assay, limiting dilution assay and QRT-PCR, including the following steps: S1: Cells were cultured in stem cell culture medium, DMEM / F12 medium (Gibco) containing B27 (Gibco) lacking vitamin A, 7.5% NaHCO3, 100 μg / mL penicillin and 50 μg / mL streptomycin (Gibco), 2 μg / mL heparin (Sigma), 20 ng / mL EGF (Proteintech) and 20 ng / mL b-FGF (Proteintech), in an atmosphere of 5% CO2-95% air, saturated humidity and 37°C; S2: Preparation of enzymatic reagents S3: Place the tumor tissue in a 60 mm culture dish in 3 ml of pre-cooled HBSS balance solution; S4: Wash twice in 3 ml pre-cooled HBSS balanced liquid to remove blood stains; S5: Place the tumor tissue in a new 60 mm culture dish in 3 mL of pre-cooled HBSS balanced liquid and cut it into 1 mm pieces using small forceps, surgical and ophthalmic scissors. 3 Small and large pieces; S6: Transfer the above tissue fragments to a 15 mL centrifuge tube and centrifuge at 2000 rpm for 10 minutes at 4 degrees; S7: Discard the supernatant, add 5-10 mL of digestion solution, transfer to a 60 mm dish, and digest for 60 minutes at 37 degrees incubator (if it is very viscous, add DNAase I) and repeatedly pipette with a 5 mL Pasteur pipette every 10 minutes; S8: Centrifuge at 2000 rpm for 10 minutes, add about 5 mL of pre-cooled red blood cell lysis buffer (NH4CL buffer) and lyse red blood cells on ice for 10 minutes; S9: Centrifuge at 2000 rpm for 10 minutes at 4 degrees, remove the supernatant, add 5 mL of HBSS buffer, and centrifuge at 2000 rpm for 10 minutes; S10: Discard the supernatant, add 1 mL of HBSS buffer, filter with a 70-mesh screen, transfer the filtrate to a 1.5 mL EP tube, centrifuge at 2000 rpm for 10 minutes at room temperature, discard the supernatant, and resuspend with stem cell culture medium for culture; S11: Count the obtained primary cells and transfer them to a constant temperature incubator at 37° C. and 5% CO 2 (v / v) for culture.

4. The use according to claim 3, It is characterized in that The obtained primary cells were sorted by MACS magnetic beads to obtain CD133+α-1,2-Man+ double positive cells, including the following steps: S1: cell counting; S2: Centrifuge the cell suspension at 300 g for 10 minutes; completely remove the supernatant; S3: Every 10 7 Resuspend the cell pellet with 60 μl buffer per cell; S4: Every 10 7 Add 20 μl FcR blocking reagent to each cell; S5: Every 10 7 Add 20 μl CD133 MicroBeads to each cell; S6: Mix well and rotate continuously at a slow speed for 15 minutes using a MACSmix tube rotator in a 4°C refrigerator; 7 Resuspend cells in 1-2 mL of buffer per cell. S7: Centrifuge at 300 g for 10 min, discard the supernatant, and resuspend the cells in 500 μl buffer; S8: Install the sorting column device, rinse the sorting column, and then slowly add the cell suspension; the cells that flow out of the sorting column are CD133- cells; S9: Remove the magnetic stand and transfer the separation column to the collection centrifuge tube, wash the separation column three times with 500 μl buffer to obtain the collection fluid of CD133+ cells; S10: CD133+ cells were incubated in a buffer containing biotinylated CVN antibody for 30 min; S11: Incubate cells with streptavidin-coupled microbeads on ice for 15 min; S12: CVN-positive cells were sorted by Miltenyi Biotec column, and the sorting process was the same as that of CD133-positive cells; S13: The obtained CD133-, CD133+α-1,2-Man+, and CD133+α-1,2-Man- cells were cultured in stem cell culture medium.

5. The use according to claim 4, It is characterized in that Detecting the stemness of the sorted cells includes the following steps: S1: sphering experiment: the three groups of cells were centrifuged at 1500 rpm for 5 min; S2: discard the old culture medium, add stem cell culture medium, and centrifuge at 1500 rpm for 5 minutes; S3: Add stem cell culture medium to resuspend and count cells; S4: Add 5000 cells to each well of a six-well plate into stem cell culture medium to culture tumor stem cells; S5: On the seventh day, observe the formation of tumor stem cells into spheres and take photos for record; S6: Limiting dilution experiment: Serially diluted cells were seeded into 96-well plates (20 wells per dilution) and cultured in stem cell culture medium; S7: fresh medium (20 μl) was added to each well every 3 days; S8: After 10 days of incubation, the wells with spheres were counted and plotted against the number of cells seeded per well to calculate the sphere formation efficiency.

6. The use according to claim 5, It is characterized in that Detection of CD133+α-1,2-Man+ double positive cells overexpressing leucosidase MAN1C1 comprises the following steps: S1: Analysis of data obtained from sphering experiments and limiting dilution experiments; S2: To examine the effect of overexpression of MAN1C1 on the tumor initiation ability of CD133+α-1,2-Man+ cells, a cell limiting dilution tumor formation assay was performed in immunodeficient mice, and tumor formation was detected histologically; S3: After counting CD133+α-1,2-Man+ cells overexpressing MAN1C1 and control cells; S4: 5000, 1000 or 500 cells were resuspended in 200 μl PBS and injected into the liver of nude mice using a 1 mL syringe; S5: The mice were killed after 180 days and the number of tumors was counted.

7. The use according to claim 6, It is characterized in that Detecting the enrichment of α-1,2-high mannose sugar chains includes the following steps: S1: Processing of the obtained tissue microarray: dewaxing and hydrating tissue sections: xylene for 3 min × 2 times; Anhydrous ethanol 3 min × 2 times; 95% ethanol 3 min × 1 time; 70% ethanol for 3 min × 1 time; 50% ethanol for 3 min × 1 time; rinse the histochemical specimen in tap water for a while; then put it in ddH2O and let it stand for 5 min; S2: Antigen repair: Place the high-temperature resistant container containing antigen repair solution into the autoclave, immerse the tissue specimen, cover the autoclave tightly, raise the temperature to 123°C, cut off the power supply, keep it at 121°C-123°C for 5 minutes, open the safety valve, take out the high-temperature resistant container, cool it to room temperature, take out the slices, and wash them with ddH2O for 5 minutes × 3 times; S3: Block endogenous peroxidase: incubate in H2O2 solution diluted with ddH2O (concentration 3%) for 10 min, wash with ddH2O for 5 min × 2 times, and wash with TBST buffer (0.025% Tween-20) for 5 min × 1 time; S4: Blocking: Wipe off excess water on the histochemical specimen, gently draw a frame around the slice with a water-blocking pen, drop it in blocking solution (TBS + 0.3% Triton X-100 + 5% serum), and incubate at room temperature for 2 hours; S5: Primary antibody: remove the blocking solution on the specimen, add primary antibody (diluted with TBS buffer containing 5% serum), incubate at room temperature for 3 hours or at 4°C overnight, and wash with TBST buffer (0.025% Tween-20) for 5 minutes × 2 times; S6: Secondary antibody: wipe off excess water, add secondary antibody (diluted with TBS buffer containing 5% serum), incubate at room temperature for 30 minutes, and wash with 0.025% Tween-20 TBS buffer for 5 minutes × 2 times; S7: Color development: Add DAB solution for histochemistry onto the histochemistry specimen and observe the color development under a microscope. When a positive reaction occurs, wash it in tap water to block the reaction. S8: Staining: drop hematoxylin on the histochemical specimen, counterstain for 1 min, rinse with running water, differentiate with 1% hydrochloric acid-ethanol for 10 s, and rinse with running water; S9: Dehydration: 50% ethanol for 3 min, 70% ethanol for 3 min, 95% ethanol for 3 min, anhydrous ethanol for 3 min, xylene for 3 min × 2 times; S10: Dry the tissue specimens in a ventilated cabinet until they are fully dried; S11: Sealing: neutral resin sealing; S12: Score and evaluate the staining results based on the staining intensity and positive rate.

8. The use according to claim 6, It is characterized in that The mannosidase MAN1C1 is used in preparing drug targets for inhibiting the stemness, spheroidization and self-renewal capabilities of intrahepatic cholangiocarcinoma tumor stem cells.

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