Application of GLUT5 as colorectal cancer biomarker in development of in-vitro detection kit, prognosis model and inhibitor

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to application of GLUT5 serving as a colorectal cancer biomarker in development of an in-vitro detection kit, a prognosis model and an inhibitor. The GLUT5 is highly expressed in colorectal cancer tumor-related fibroblasts (CAFs), the expression level of the GLUT5 is remarkably related to poor prognosis of a patient, and the GLUT5 can be used as a molecular basis for auxiliary diagnosis and an independent prognosis factor. In mechanism, the GLUT5 promotes tumor progression by mediating fructose metabolism reprogramming of the CAFs. A full-chain solution from early diagnosis and precise prognosis to targeted therapy is provided for colorectal cancer, and the clinical transformation value is remarkable.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to the application of the fructose transporter GLUT5 as a diagnostic marker, independent prognostic factor and therapeutic target for colorectal cancer, especially its specific expression and functional mechanism in cancer-associated fibroblasts (CAFs). Background Art

[0002] Colorectal cancer is one of the most common malignant tumors with high morbidity and mortality worldwide. Early diagnosis and accurate prognostic assessment are crucial for improving patient survival. However, current diagnostic and prognostic markers for colorectal cancer (such as CEA and KRAS) suffer from low sensitivity (approximately 60%) and insufficient specificity (susceptibility to interference by inflammation).

[0003] Tumor development is often accompanied by remodeling of the tumor microenvironment. Cancer-associated fibroblasts (CAFs), as a crucial component of the tumor stroma, are widely involved in multiple processes, including tumor proliferation, invasion, and therapeutic resistance. In recent years, tumor metabolic reprogramming has become a research hotspot. CAFs, in particular, drive tumor progression through metabolic reprogramming under conditions of nutrient shortage. However, CAF-specific metabolic targets remain unclear.

[0004] Fructose is a common dietary sugar. Unlike glucose, fructose enters cells through the fructose-specific transporter GLUT5. Under normal circumstances, GLUT5 is mainly expressed in tissues such as the small intestine and kidney, but its expression is significantly upregulated in a variety of cancers, including colorectal cancer. This adaptive change in fructose metabolism provides tumor cells with an additional source of energy while promoting their proliferation and migration capabilities. Although the expression of GLUT5 in colorectal cancer cells has been reported, the existing technology completely ignores the expression and function of GLUT5 in CAFs, and has not established a quantitative association between the expression of GLUT5 dual cell sources (tumor cells and CAFs) and prognosis. There is also a lack of therapeutic evidence targeting GLUT5 to inhibit CAFs activation. The present invention combines clinical sample analysis with in vitro and in vivo experimental studies to reveal the key role of GLUT5 in colorectal cancer and explores its potential as a diagnostic and prognostic biomarker. Summary of the Invention

[0005] In order to solve the technical problems in the background technology, the present invention proposes an application of GLUT5 as a colorectal cancer biomarker in the development of in vitro detection kits, prognosis models and inhibitors.

[0006] The first technical solution: A biomarker for the diagnosis of colorectal cancer, wherein the biomarker is the fructose transporter GLUT5 highly expressed in tumor-associated fibroblasts (CAFs) (immunohistochemistry score ≥4 points is the positive threshold).

[0007] The second technical solution: a biomarker for the prognosis evaluation of colorectal cancer, wherein the biomarker is the fructose transporter GLUT5, which is highly expressed in both tumor cells and CAFs (Cox regression HR=1.90, 95% CI 1.14-3.17), and its expression level is negatively correlated with the patient's overall survival.

[0008] The third technical solution: Use of a reagent for detecting the above-mentioned biomarkers in the preparation of colorectal cancer diagnosis and / or prognosis evaluation products, wherein the reagent comprises an anti-GLUT5 antibody (monoclonal antibody or polyclonal antibody) or a GLUT5-specific binding probe.

[0009] Furthermore, the preparation of the colorectal cancer diagnosis and / or prognosis assessment product includes: an immunohistochemistry detection kit (for detecting GLUT5 protein in CAFs in tissue samples) and a prognostic risk stratification model (based on a GLUT5 expression quantitative scoring system).

[0010] The fourth technical solution: Use of an inhibitor targeting GLUT5 in the preparation of a drug for treating colorectal cancer, wherein the inhibitor blocks tumor growth and metastasis by simultaneously inhibiting fructose metabolic reprogramming of tumor cells and CAFs.

[0011] The inhibitor simultaneously inhibits tumor cell proliferation, reduces Ki67-positive tumor cells, activates CAFs, reduces α-SMA expression, and blocks tumor-stroma metabolic coupling.

[0012] The inhibitors include the small molecule compound 2,5-AM and anti-GLUT5 neutralizing antibodies.

[0013] Beneficial effects:

[0014] Diagnostic breakthrough: For the first time, CAFs-specific GLUT5 expression was used to achieve a highly specific diagnosis (89% vs CEA 65%).

[0015] Accurate prognostic stratification: The dual-cell-derived GLUT5 co-expression model divides patients into low-risk and high-risk groups to guide individualized treatment;

[0016] Innovative therapeutic mechanism: 2,5-AM blocks tumor-stroma metabolic coupling by simultaneously inhibiting tumor cell proliferation (reduction of Ki67-positive tumor cells) and CAFs activation (reduction of α-SMA expression);

[0017] Full process coverage: From diagnostic kits (Example 1), prognosis score cards (Example 2) to targeted drugs (Example 5), a closed-loop technical solution is formed.

[0018] The core discovery of the present invention is:

[0019] Diagnostic value: In the colorectal cancer tissue cohort, GLUT5 expression in CAFs was significantly higher than that in adjacent adjacent fibroblasts (P < 0.001, receiver operating characteristic (ROC) curve area under the curve = 0.895).

[0020] Prognostic significance: Patients with high co-expression of GLUT5 in tumor cells and CAFs (defined as immunohistochemical score ≥ 4) had a significantly lower 5-year survival rate (P < 0.01);

[0021] Therapeutic targets: GLUT5 mediates fructose-dependent metabolic reprogramming of CAFs, and the inhibitor 2,5-AM can block this process and inhibit tumor metastasis. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments are briefly introduced below.

[0023] Figure 1 Expression of GLUT5 protein in the stroma of colorectal cancer tissues and their corresponding normal tissues:

[0024] A. Immunohistochemistry results showed that GLUT5 protein was expressed in the stroma of colorectal cancer tissues;

[0025] B. Immunofluorescence double staining results confirmed the expression of GLUT5 protein in CAFs of colorectal cancer tissues;

[0026] C. Immunohistochemistry results showed that the expression of GLUT5 protein in normal colorectal tissue stroma was significantly lower than that in colorectal cancer tissue;

[0027] D Based on the Human Protein Atlas database, the expression of GLUT5 in CAFs of colorectal cancer tissues was analyzed.

[0028] E. ROC curve evaluating the diagnostic efficacy of GLUT5 protein expression level in CAFs for colorectal cancer.

[0029] Figure 2 Relationship between GLUT5 protein expression level in CAFs and clinical characteristics and prognosis of colorectal cancer patients:

[0030] A All cases were divided into two groups according to the GLUT5 immunohistochemical score in CAFs: high expression and low expression;

[0031] B The expression level of GLUT5 protein in fibroblasts of colorectal cancer tissue is related to the tumor invasion depth and clinical stage.

[0032] C. Relationship between the expression level of GLUT5 protein in fibroblasts of colorectal cancer tissue and patient prognosis.

[0033] Figure 3 Relationship between the expression level of GLUT5 protein in tumor cells and the prognosis of colorectal cancer patients:

[0034] A. Immunohistochemical staining to detect the expression of GLUT5 protein in tumor cells of colorectal cancer tissues;

[0035] B. The relationship between the expression level of GLUT5 protein in tumor cells and the prognosis of colorectal cancer patients;

[0036] C. The relationship between the co-expression of GLUT5 protein in tumor cells and CAFs and the prognosis of colorectal cancer patients;

[0037] D Multivariate analysis showed that the co-expression of GLUT5 in tumor cells and CAFs can serve as an independent biomarker for the prognosis of colorectal cancer patients.

[0038] Figure 4 Fructose promotes the proliferation and migration of tumor-associated fibroblasts:

[0039] A Schematic diagram of CAF (3T3) and CAF (MEF) induction: Conditioned medium (CM) of CT26 tumor cells was collected and acted on NIH-3T3 and MEF for 7 days respectively;

[0040] B Western blot analysis of the expression levels of α-SMA and FSP-1 in MEF and NIH-3T3 cells before and after treatment with CT26-CM;

[0041] C Schematic diagram of hCAF isolation from colorectal cancer tissue;

[0042] D IF staining of CK, Vimentin, and F-actin in HT29 cells and hCAFs;

[0043] E Schematic diagram of mCAF isolation from CT26 xenografts: CT26 / mCherry cells were injected into the mouse cecum to form tumors, fibroblasts from the xenografts were isolated, and non-fluorescent cells were sorted by flow cytometry;

[0044] F Mouse tumor fragments (top) and fluorescence images of mCAFs (bottom) collected by flow cytometry. Red fluorescent cells are CT26 / mCherry, and non-fluorescent cells are mCAFs.

[0045] G IF staining of CK, Vimentin, and F-actin in CT26 cells and mCAFs;

[0046] HPI (propidium iodide) and Calcein-AM (calcein) staining were used to determine the effect of fructose on cell survival;

[0047] I CCK8 detects the effect of fructose on cell viability;

[0048] J and K Tanswell experiment detected the effect of fructose on cell migration ability.

[0049] Figure 5 GLUT5 affects fructose's regulation of tumor-associated fibroblast function:

[0050] A. Effects of GLUT5 inhibitor 2,5-AM on fibroblast viability;

[0051] B Effects of GLUT5 inhibitor 2,5-AM on fibroblast migration ability;

[0052] C. GLUT5 was overexpressed in CAFs (3T3), and the GLUT5 mRNA expression level was detected by PCR;

[0053] D. GLUT5 was overexpressed in CAF (3T3), and the expression level of GLUT5 protein was detected by western blot;

[0054] E. Effect of overexpression of GLUT5 on fibroblast viability;

[0055] F Effect of GLUT5 overexpression on the migration ability of fibroblasts.

[0056] Figure 6 Metabolism of isotope C13-labeled fructose in fibroblasts.

[0057] Figure 7 Effects of fructose on the metabolic phenotype of fibroblasts:

[0058] A cell energy metabolism analyzer was used to measure the effect of fructose on the oxygen consumption rate (OCR) of CAF (3T3);

[0059] B cell energy metabolism analyzer was used to measure the effect of fructose on the extracellular acidification rate (ECAR) of CAF (3T3);

[0060] C The ratio of OCR to ECAR of CAF (3T3) in different culture media;

[0061] D Cell Energy Metabolism Analyzer was used to detect the mitoATP production rate of CAF (3T3) in different culture media;

[0062] E cell energy metabolism analyzer was used to detect the glycoATP production rate of CAF (3T3) in different culture media;

[0063] F Energy levels of CAFs (3T3) in different culture media;

[0064] G Calculate the ratio of CAF(3T3)mitoATP and glycoATP in different culture media;

[0065] The ATP levels of CAFs (3T3) in different culture media were detected using an ATP detection kit.

[0066] Figure 8 Effects of inhibiting fructose absorption on tumor growth and metastasis in mice:

[0067] A. Effects of knocking down GLUT5 in colorectal cancer cells and CAFs on the growth of subcutaneous transplanted tumors in mice;

[0068] B and C show the effects of knocking down GLUT5 in colorectal cancer cells and CAFs, respectively, on subcutaneous transplanted tumor tissues in mice

[0069] Effects of Ki67 and α-SMA expression levels;

[0070] Effect of DF GLUT5 inhibitor 2,5-AM on tumor lung metastasis in mice. DETAILED DESCRIPTION

[0071] In order to help those skilled in the art better understand the technical solutions of the present invention, the technical solutions in the embodiments of the present invention are clearly and completely described below. The description is only a part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work should fall within the scope of protection of the present invention.

[0072] Example 1 Evaluation of the diagnostic and prognostic value of GLUT5 in colorectal cancer

[0073] (1) Collect paraffin tissue samples and corresponding clinical pathological data from 473 patients with colorectal cancer

[0074] Paraffin tissues of colorectal cancer patients who had not received any treatment before surgery, including cancerous and adjacent tissues, were collected, and the pathological characteristics such as patient gender, age, tumor size, pathological grade, TNM stage, T stage, N stage, M stage, and patient survival were recorded in detail.

[0075] (2) Preparation of tissue microarrays

[0076] Pathological diagnosis was performed on the stained tissue sections, and the desired tissue locations were circled for tissue microarray production. The tissue array blocks were sliced ​​using a fully automated tissue slicer at a blade speed of 4 μm / rev. The array sections were then baked in a 60°C oven overnight.

[0077] (3) Dyeing

[0078] Take out a tissue chip and perform HE staining to determine whether the tissue chip is qualified and whether the sampling site is accurate. Take another tissue chip and perform immunohistochemical staining of GLUT5:

[0079] Bake the tissue sections in a 70°C oven for 1-2 hours.

[0080] Dewaxing and rehydration: Tissue sections were dewaxed and rehydrated in the following order: xylene 1 for 20 minutes, xylene II for 20 minutes, anhydrous ethanol 1 for 10 minutes, anhydrous ethanol II for 10 minutes, and 95%, 85%, and 75% ethanol for 5 minutes each. Afterwards, rinse with distilled water 2-3 times.

[0081] Antigen repair (high-pressure repair): First, pour the citrate repair solution into the pressure cooker and heat it to boiling. Then place the tissue slices into the pot. When a certain pressure is reached and the air starts to spray, count for 2-3 minutes. After the time is up, turn off the power. After cooling to room temperature, take out the slices and wash them 3 times with PBS, each time for 2-3 minutes.

[0082] Blocking: 1) Soak the sections in 3% H2O2 solution for 10 minutes to block endogenous peroxidase. Wash with PBS three times for 2-3 minutes each. 2) Add serum dropwise to the tissue sections until the sections are completely covered. Incubate at room temperature for 30 minutes to block nonspecific binding.

[0083] Primary antibody incubation: After blocking, shake off the serum on the slices, then add the prepared primary antibody (Glut5 antibody diluted 1:200 with antibody diluent) until the slice tissue is completely covered. Place the slices in a humidified chamber, equilibrate at room temperature for 30 minutes, and then incubate at 4°C overnight.

[0084] Secondary antibody incubation: Remove the humidified chamber from 4°C and equilibrate at room temperature for 1 hour. Shake off the primary antibody from the sections, then wash with PBS three times, 2-3 minutes each time. After washing, shake off the excess PBS solution on the tissue sections, add the secondary antibody dropwise until the sections are completely covered, and incubate at room temperature for 1 hour. After incubation, wash with PBS three times, 2-3 minutes each time.

[0085] DAB color development: Add the prepared DAB color development solution to the tissue section until the tissue is completely covered. The color development time is determined according to the results of observation under the microscope and usually does not exceed 10 minutes. After the color development is completed, the water is added to stop the color development.

[0086] Hematoxylin staining: Place the tissue sections in hematoxylin solution for 5 minutes and rinse with running water until the color no longer fades.

[0087] Differentiation: Differentiate tissue sections in 0.7% hydrochloric acid alcohol for 2-3 seconds, and rinse with water immediately after completion.

[0088] Anti-blueing: Anti-blue the tissue sections in 1% ammonia water for 2-3 seconds, and then wash with clean water immediately after the end.

[0089] Dehydration and Clearing: Tissue sections were dehydrated and cleared in the following order: 75%, 85%, and 95% ethanol for two minutes each, anhydrous ethanol I for 10 minutes, anhydrous ethanol II for 10 minutes, and xylene I and xylene II for 20 minutes each. Mounting: After clearing, tissue sections were placed in a fume hood and, after the xylene evaporated, mounted with neutral resin glue.

[0090] (4) Immunohistochemistry scoring and statistical methods

[0091] The expression intensity was evaluated based on a comprehensive evaluation of staining area and intensity, with expression categorized into four levels: 0 = negative; 1 = weakly positive; 2 = positive; and 3 = strongly positive. The percentage of expression in tissue sections was categorized into five levels: 0 = 0%; 1 = 1-25%; 2 = 26-50%; 3 = 51-75%; and 4 = 75-100%. The product of these scores was used to determine the score, with scores ≤ 4 indicating low expression and scores > 4 indicating high expression. SPSS statistical software was used to analyze the differential expression of GLUT5 in fibroblasts from cancerous and adjacent tissues to assess the diagnostic value of GLUT5 in colorectal cancer. Patients were divided into high and low protein expression groups. The relationship between GLUT5 expression in tumor cells and fibroblasts and clinical pathological parameters, as well as overall survival, was analyzed to assess prognostic value and whether it could serve as an independent risk factor for colorectal cancer.

[0092] (5) Diagnostic value analysis

[0093] like Figure 1 As shown in middle A, spindle cells in the tumor stroma also showed GLUT5 positive expression. Further immunofluorescence double staining of GLUT5 and α-SMA ( Figure 1 It is worth noting that fibroblasts in normal colorectal tissues express low or no GLUT5. Paired t-test results showed that the expression level of GLUT5 in CAFs of colorectal cancer tissues was significantly increased compared with that in normal tissues (P<0.01, Figure 1 In addition, the staining results of colorectal cancer in the Human Protein Atlas database also clearly show that GLUT5 is expressed in CAFs ( Figure 1 The diagnostic performance of GLUT5 expression in fibroblasts was evaluated by constructing a receiver operating characteristic (ROC) curve to analyze the diagnostic efficacy of GLUT5 expression in differentiating tumors from normal tissues. Figure 1As shown in Figure E, the area under the curve (AUC) was 0.895, with a 95% confidence interval of 0.836-0.955, indicating that GLUT5 protein expression in fibroblasts is highly capable of distinguishing colorectal tissue from normal samples. These results indicate that CAFs-specific GLUT5 expression is a highly effective diagnostic marker.

[0094] (6) Prognostic value analysis

[0095] To investigate the correlation between GLUT5 protein expression levels in CAFs and the clinical characteristics and prognosis of colorectal cancer patients, patients were divided into GLUT5 high expression group and GLUT5 low expression group according to the immunohistochemical score of GLUT5 in CAFs ( Figure 2 Statistical analysis results showed that the expression level of GLUT5 was significantly positively correlated with the T stage and clinical stage of colorectal cancer ( Figure 2 B), suggesting that the protein expression level of GLUT5 in CAFs is closely related to the progression of colorectal cancer. In addition, Kaplan-Meier analysis and log-rank test results showed that high expression of GLUT5 in fibroblasts was associated with the overall survival of colorectal cancer patients, especially for patients with advanced clinical stages (stages 3 and 4) ( Figure 2 This suggests that GLUT5 in CAFs serves as a potential biomarker, and its high expression may negatively affect patients' survival outcomes and indicate a worse prognosis.

[0096] Since tumor cells also express GLUT5, similarly, patients were divided into GLUT5 high expression group and GLUT5 low expression group according to the immunohistochemical score of GLUT5 in tumor cells ( Figure 3 A), Kaplan-Meier analysis and log-rank test were used to detect survival rates. The results showed that high expression of GLUT5 in tumor cells was also significantly associated with the overall survival of patients with colorectal cancer, especially for patients with advanced clinical stages (stages 3 and 4) ( Figure 3 This suggests that GLUT5 in tumor cells can also serve as a potential biomarker.

[0097] Since GLUT5 in CAFs and tumor cells are both related to the prognosis of colorectal cancer patients, the relationship between the co-expression of GLUT5 in fibroblasts and tumor cells and the prognosis of colorectal cancer patients was analyzed. Figure 3 As shown in C, the co-expression of GLUT5 by CAFs and tumor cells has a more significant statistical effect on the prognosis of colorectal cancer patients. In addition, the results of multivariate survival analysis showed that ( Figure 3 Middle (D), co-expression of GLUT5 is an independent prognostic biomarker in patients with colorectal cancer (P = 0.013).

[0098] Example 2 GLUT5 regulates the biological functions of CAFs

[0099] (1) CAFs model construction

[0100] The present invention uses a total of 4 cell models, and the specific construction and identification methods are as follows: Figure 4 shown.

[0101] First, the conditioned medium (CM) of tumor cells CT26 was collected and used to induce fibroblasts NIH-3T3 and mouse embryonic fibroblasts (MEF) for one week ( Figure 4 Then, the expression levels of α-SMA and FSP-1 in the cells were identified by western blot. The specific steps are as follows:

[0102] The prepared 1× SDS protein lysate was added to the cells after washing with PBS, placed on ice for 20 minutes, and then the cell lysate was heated at 95°C for 15 minutes to denature the protein. Subsequently, the supernatant was transferred to a new centrifuge tube.

[0103] Protein quantification was performed using the Nanodrop method, followed by protein electrophoresis. The starting voltage was set at 80 V, and the voltage was adjusted to 120 V when the protein marker bands were clearly separated.

[0104] After protein electrophoresis, transfer can be performed. First, place the protein gel in the transfer solution for equilibrium, then add an appropriate amount of transfer solution into the container, place the sponge, filter paper, methanol-activated PVDF membrane, gel, filter paper, and sponge in order, remove the gas, and place the transfer tank in ice water. The transfer condition is 300mA constant current for 1 hour.

[0105] After the transfer was completed, the PVDF membrane was removed, placed in 5% milk, and blocked on a shaker at room temperature for 1 h.

[0106] After blocking, the membrane was washed with 1× TBST buffer, the required bands were cut, the corresponding primary antibody was added, and the membrane was placed on a shaker at 4°C overnight.

[0107] The next day, the membrane was washed three times with 1× TBST buffer for 5 min each time, then incubated with secondary antibody at room temperature for 1 h, and then washed three times with TBST buffer for 5 min each time.

[0108] After washing the membrane, ECL mixed solution was added dropwise and luminescence development was performed using a Biorad chemiluminescence instrument.

[0109] The experimental test results are as follows Figure 4As shown in B. The results showed that the expression levels of α-SMA and FSP-1 were upregulated in NIH-3T3 and MEF cells induced with CM of CT26. The successfully constructed cells were named CAF(3T3) and CAF(MEF).

[0110] hCAFs were isolated from human colorectal cancer tissues. The specific steps are as follows ( Figure 4 Middle C):

[0111] The isolated human colorectal cancer tissue was quickly placed in PBS containing 3% double-antibody (containing penicillin 100 U / ml and streptomycin 100 μg / ml), and quickly transferred to a clean bench.

[0112] Place the tissue in a culture dish and wash it several times with PBS containing double antibodies. Then, chop the tissue with scissors and place it in a 50ml centrifuge tube. Add 5-10ml of collagenase and place it in a shaker in a 37℃ incubator for digestion for 1 hour.

[0113] The digested tissue cell suspension was filtered through a filter with a pore size of 70 μm. The filtered cell solution was placed back into a new 50 ml centrifuge tube and centrifuged at 500 rpm for 5 minutes.

[0114] After discarding the supernatant, the obtained cells were evenly dispersed with culture medium containing 10% FBS, placed in a culture dish, and cultured in a CO2 incubator.

[0115] Observe the cell adhesion and growth every day. After the cells begin to adhere to the wall, gently wash the culture dish with PBS during the culture process (CAFs can stick to the bottom of the dish, while other cells will slide off under appropriate PBS washing force), replace the culture medium, and continue to culture CAFs for 1 to 2 weeks.

[0116] Part of hCAF was collected and the expression of CK, Vimentin and F-actin in the cells was identified by immunofluorescence staining ( Figure 4 Middle D).

[0117] Finally, mCAFs were isolated from mouse CT26 xenografts as follows ( Figure 4 (E and F):

[0118] Fluorescently labeled CT26-mcherry cells were inoculated into the groin of mice. When the diameter of the transplanted tumor reached approximately 1 cm, the tumor tissue was removed in a clean bench. The cell suspension of the mouse transplanted tumor was collected and cultured using the hCAF isolation method described above.

[0119] The successfully cultured cells were flow cytometry sorted to collect cells without fluorescent labels, and the expression of CK, Vimentin and F-actin in the collected cells was identified by immunofluorescence staining ( Figure 4 Middle G).

[0120] (2) Effect of fructose on CAFs function

[0121] In the previous examples, we investigated the impact of fructose transporter GLUT5 expression in CAFs on colorectal cancer patient survival in clinical samples and established a CAF model using various approaches. To further investigate the effects of fructose and its transporter GLUT5 on the biological functions of CAFs, we conducted further studies through a series of in vitro and in vivo experiments.

[0122] First, a glucose medium containing 10 mM glucose, a fructose medium containing 10 mM fructose, and a disaccharide medium containing 10 mM glucose and 10 mM fructose were prepared using a sugar-free medium.

[0123] CAFs were digested with trypsin and seeded into 12-well plates. After the cells adhered to the wall, the culture medium was changed to sugar-free, fructose, glucose, and disaccharide medium, with three replicates for each condition. After 48 hours, the cells were stained with PI (propidium iodide) and Calcein-AM (calcein), and counted under a fluorescence microscope (dead cells were PI-positive and appeared red, while live cells were Calcein-AM-positive and appeared green) to determine the survival of CAFs in different culture media. The results showed that fructose significantly alleviated the death of CAFs induced by glucose deficiency ( Figure 4 Middle H).

[0124] At the same time, the CCK8 kit was used to determine the effect of fructose on CAF cell viability. CAFs were digested with trypsin and counted, and 1000 cells were seeded into a 96-well plate per well. After the cells adhered to the wall, 200 μL of sugar-free, fructose, glucose, and disaccharide culture medium were added to each well. After 48 hours of culture, the culture medium was discarded, and 100 μL of culture medium containing 10% CCK-8 reagent was added to each well and cultured for another 2-4 hours. After the culture was completed, the absorbance at 450nm and 650nm was detected using a microplate reader. The test results showed that compared with the sugar-free culture medium, the fructose culture medium can significantly promote the survival of CAFs ( Figure 4 Middle I).

[0125] The effect of fructose on the migration ability of CAFs was then studied using the Transwell experiment. CAFs were digested with trypsin, and the cells were resuspended in serum-free culture medium, counted, and the cell density was adjusted to 1 million / mL. A Transwell culture chamber with an 8.0 μm pore size was placed in a 24-well plate, 600 μL of culture medium containing 10% fetal bovine serum was added to the lower layer of the chamber, and 100 ul of serum-free cell suspension was added to the upper chamber. After 15 hours, the Transwell chamber was removed, fixed with 4% paraformaldehyde for 15 minutes, stained with 0.1% crystal violet for 20 minutes, and the cells on the upper layer of the microporous membrane were carefully wiped off with a cotton swab. After washing twice with PBS, the cells in the lower layer were photographed and counted under a microscope ( Figure 4 The results showed that fructose could significantly promote the migration of CAFs, and the migration ability of CAFs was the strongest in the disaccharide medium ( Figure 4 Middle K).

[0126] (3) GLUT5 functional verification

[0127] The GLUT5 inhibitor 2,5-AM was used to block the uptake of fructose by CAFs. The effects of 2,5-AM on the proliferation and migration of CAFs in fructose culture medium were analyzed using CCK8 and Transwell assays. The detection methods were as described above. The results showed that 2,5-AM could effectively block the promoting effect of fructose on the biological functions of CAFs ( Figure 5 A and B).

[0128] Construction of a GLUT5 overexpression plasmid: Full-length GLUT5 was amplified by PCR using the following primers: forward primer 5′-acctccatagaagattagtagagccaccatggagcaacaggatcagagca-3′, reverse primer 5′-ttcgaattcgctagctctagatcactgttccgaagtgaggtg-3′. The amplified product was cloned into the XbaII restriction site of the pCDH-CMV-MCS-Puro lentiviral vector. The construct was further verified by restriction digestion and DNA sequencing.

[0129] Packaging of GLUT5-overexpressing virus: Lentivirus was prepared by co-transfecting HEK293T cells with two packaging plasmids (psPAX2 and pMD2.G). 48 hours after transfection, the virus-containing supernatant was collected.

[0130] Construction of CAF (3T3) cells overexpressing GLUT5: Cancer cells were infected with lentivirus for 12 h, cultured with new culture medium for 48 h, and then cultured with 2 mg / mL puromycin for 3 days.

[0131] RT-PCR and Western blot were used to analyze the expression level of GLUT5. Figure 5 C and D).

[0132] GLUT5 expression was assessed by western blot as previously described. GLUT5 mRNA expression was assessed by RT-PCR as follows: Total RNA was extracted with TRIzol and reverse-transcribed into cDNA using HiScript IIQ RT SuperMix. qPCR was performed using AceQ qPCR SYBR Green Master Mix, and gene expression was calculated using the 2-ΔΔCt method.

[0133] The effects of overexpression of GUT5 on the proliferation and migration of CAFs in fructose culture medium were analyzed using CCK8 and Transwell assays. The assays were performed as described above, and the results showed that overexpression of GUT5 could further enhance the promoting effect of fructose on the proliferation and migration of CAFs ( Figure 5 E and F).

[0134] Example 3 Fructose metabolic flux analysis

[0135] Since fructose, a common monosaccharide, can regulate the biological functions of CAFs, it is unclear whether fructose can be absorbed and metabolized by CAFs and affect their metabolic phenotype. CAF (3T3) cells and CAF (3T3) cells overexpressing GLUT5 were cultured with isotope C13-labeled fructose, and the metabolic flux was detected by LC / MS analysis to clarify the metabolic pathway of fructose in CAFs. The specific steps are as follows:

[0136] CAF (3T3) cells and GLUT5-overexpressing CAF (3T3) cells were plated into 10-cm culture dishes 24 hours in advance, and then the culture medium was replaced with 5 mM fructose C13 and 5 mM fructose C13 + 5 mM glucose.

[0137] After 8 hours of culture, the cells were washed twice with ice-cold PBS, and then 2 mL of 80% cold methanol was added. After 5 minutes, the methanol was aspirated into a 15 mL centrifuge tube. The entire process was performed on dry ice.

[0138] Centrifuge in a 15 mL centrifuge tube at 4°C, 12,000 g for 20 minutes, collect the supernatant in another centrifuge tube, and dry it into powder under a stream of nitrogen.

[0139] The samples were sent to Tsinghua University for LC / MS analysis to compare the relative abundance of metabolites among samples.

[0140] The results showed that CAF (3T3) can absorb fructose and metabolize it through glycolysis and the tricarboxylic acid cycle (Figure 6). Overexpression of GLUT5 can further increase the intake and metabolism of fructose ( Figure 6). In addition, in the presence of glucose, the uptake and metabolism of fructose is slowed down ( Figure 6 ).

[0141] Example 4 ATP generation mechanism

[0142] The rate of ATP production was determined using the XF Real-Time ATP Rate Assay Kit (A102340-100, Agilent) according to the manufacturer's instructions. Briefly, cells were seeded at a density of 3 x 104 cells / well / 200 μL in a Seahorse XF24 cell culture microplate and placed in a 37°C, 5% CO2 incubator. After 24 hours, the cells were washed and replaced with Seahorse XFDMEM Medium (103575-100, Agilent) containing 10 mM glucose (103577-100, Agilent), 1 mM pyruvate (103578-100, Agilent), and 2 mM l-glutamine (103579-100, Agilent). The cells were incubated in a 37°C, CO2-free incubator for 1 hour to allow temperature and pH to equilibrate. The Seahorse XFe24 analyzer was calibrated and analyzed using a standard XF Real-Time ATP rate template created with WAVE software (V2.6.1). Standard drug injections were performed using 1.5 μM oligomycin (port a) and 0.5 μM rotenone / Antimycin A (port b). After analysis, the results for each well were normalized using 1 μg / mL Hoechst on a Cytation5 Multifunctional Cell Imager (BioTek).

[0143] The results are as follows Figure 7 As shown, fructose supplementation increased the oxygen consumption rate (OCR) of CAFs (3T3)( Figure 7 Meanwhile, fructose slightly increased the extracellular acidification rate (ECAR) of CAFs compared with the sugar-free medium ( Figure 7 B), and reduced the OCR / ECAR ratio ( Figure 7 Regarding ATP production, glucose significantly increased the production of glycoATP in CAFs, while fructose mainly increased the production of mitoATP ( Figure 7 Overall, fructose increased ATP levels in CAFs by significantly increasing mitoATP and only slightly increasing glycoATP ( Figure 7 F, G). ATP detection kit confirmed the increase in ATP levels caused by fructose ( Figure 7 In addition, 2,5-AM inhibited the fructose-induced increase in ATP ( Figure 7 J), while overexpression of GLUT5 further increased ATP levels in CAFs ( Figure 7 Middle K).

[0144] Example 5 Verification of GLUT5 Function in Mice

[0145] To further elucidate the effect of GLUT5 on the biological behavior of colorectal cancer in vivo, we constructed GLUT5 knockdown CT26 and CAF (3T3) cell lines using shGLUT5, and then mixed and injected 5×10 5 CT26 and 10×10 5 CAF (3T3) control cells and GLUT5 knockdown cells were used to construct a subcutaneous transplant tumor model. Four groups were set up: ① control group (empty CT26 + empty CAF), ② CT26 GLUT5-KD group, ③ CAF GLUT5-KD group, and ④ double knockdown group (CT26 KD + CAF KD). After 3 weeks, the tumor tissue was peeled off, the tumor volume was measured, and the expression levels of Ki67 (proliferation marker) and α-SMA (fibroblast activation marker) in the above tumor tissues were detected by immunohistochemical staining. At the same time, to evaluate the metastasis inhibition effect, 2×10 5 CT26 / Luciferase fluorescently labeled cells and 4×10 5 A mixed suspension of CAF (3T3) cells was prepared and mice were divided into a control group (intraperitoneal injection of normal saline) and a 2,5-AM treatment group (150 mg / kg / day intraperitoneal injection, starting from inoculation and continuing until day 14). At the end of the experiment, lung tissue was collected for ex vivo fluorescence imaging and HE staining to analyze metastatic lesions.

[0146] The results are as follows Figure 8 As shown in the results, knocking down GLUT5 in either CT26 or CAF (3T3) cells can inhibit tumor growth in mice, and knocking down GLUT5 in both CT26 and CAF (3T3) cells simultaneously has the most significant effect on tumor growth inhibition ( Figure 8 A), accompanied by a significant decrease in Ki67 and α-SMA positive cells in tumor tissues ( Figure 8 In addition, in vivo imaging of mice showed that the GLUT5 inhibitor 2,5-AM could significantly inhibit the lung metastasis of colorectal cancer cells ( Figure 8 Consistently, HE staining results also showed that 2,5-AM could significantly reduce the formation of metastatic foci of colorectal cancer cells in lung tissue ( Figure 8 (F) These results indicate that inhibiting GLUT5 can significantly inhibit the growth and metastasis of colorectal cancer cells in mice.

[0147] The above detailed description of the analytical methods involved in the present invention provides a detailed introduction. It should be noted that the above description is intended solely to help those skilled in the art better understand the methods and concepts of the present invention, and is not intended to limit the relevant content. Without departing from the principles of the present invention, those skilled in the art may make appropriate adjustments or modifications to the present invention, and such adjustments and modifications shall also fall within the scope of protection of the present invention.

Claims

1. A biomarker for diagnosing colorectal cancer, characterized in that: The biomarker is the fructose transporter GLUT5 which is highly expressed in CAFs.

2. A biomarker for colorectal cancer prognosis assessment, characterized in that: The biomarker is the fructose transporter GLUT5, which is highly expressed in both tumor cells and CAFs.

3. Use of a reagent for detecting the biomarker according to claim 1 or 2 in the preparation of a product for colorectal cancer diagnosis and / or prognosis assessment, characterized in that: The reagent includes an anti-GLUT5 antibody or a GLUT5 specific binding probe; the anti-GLUT5 antibody is a monoclonal antibody or a polyclonal antibody.

4. The use according to claim 3, characterized in that The colorectal cancer diagnosis and / or prognosis assessment product comprises: an immunohistochemistry detection kit for detecting GLUT5 protein in CAFs in tissue samples; and a prognostic risk stratification model, which is based on a GLUT5 expression quantitative scoring system.

5. Use of an inhibitor targeting GLUT5 in the preparation of a drug for treating colorectal cancer, characterized in that: The inhibitor blocks tumor growth and metastasis by simultaneously inhibiting fructose metabolic reprogramming of tumor cells and CAFs.

6. The use according to claim 5, characterized in that The inhibitor simultaneously inhibits tumor cell proliferation, reduces Ki67-positive tumor cells, activates CAFs, reduces α-SMA expression, and blocks tumor-stroma metabolic coupling.

7. The use according to claim 5, characterized in that The inhibitor is a small molecule compound 2,5-AM or an anti-GLUT5 neutralizing antibody.

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