A method for detecting the homing efficiency of lymphocytes in tumor-draining lymph nodes

Abstract

The present invention belongs to the technical field of biological detection, and discloses a testing method for evaluating whether the homing function of lymphocytes in lung cancer draining lymph nodes is impaired by detecting whether the expression level of VEGFD in lymph nodes is abnormal. The regulatory effect of VEGFD in the immune microenvironment of TDLN is systematically revealed by comprehensively detecting the expression level of VEGFD, observing the dedifferentiation characteristics of HEV, and evaluating the homing efficiency of lymphocytes.

Description

Technical Field

[0001] The invention belongs to the field of biological detection technology, and in particular relates to a method for detecting the homing efficiency of lymphocytes in tumor-draining lymph nodes. Background Art

[0002] Lung cancer is one of the most deadly cancers worldwide, with high morbidity and mortality rates. According to data from the World Health Organization (WHO) and the International Agency for Research on Cancer (IARC), more than 2 million people are diagnosed with lung cancer each year, and most patients are already in the middle or late stages of the disease when diagnosed. Lymph node metastasis is one of the common metastasis patterns in lung cancer patients when diagnosed, and is also a key factor leading to disease progression and poor prognosis. Studies have shown that the lymph node metastasis rate of lung cancer patients is as high as 30-50%, and this proportion is significantly increased in advanced patients. Lymph node metastasis is considered to be one of the starting points and pathways for lung cancer to spread throughout the body, and the drainage lymph nodes, as the first area invaded by tumor cells, play a core role in the metastasis and progression of lung cancer.

[0003] In the lymph node metastasis of lung cancer, the draining lymph nodes are the first lymph nodes to be affected. Draining lymph nodes are not only the direct target of tumor cells, but also carry important immune surveillance functions. Under normal circumstances, they can identify and eliminate cancer cells through the homing and activation of lymphocytes. However, due to the complex regulation in the tumor microenvironment, the immune surveillance function of lymph nodes is significantly reduced. A large amount of clinical data shows that drainage lymph node dysfunction in patients with lung cancer is closely related to tumor stage, metastasis incidence and patient survival rate.

[0004] Lymphocyte homing is the process by which lymphocytes migrate from the blood to the lymph nodes to participate in the immune response. In the draining lymph nodes (TDLN), high endothelial venules (HEVs) serve as the main channel for lymphocytes to enter the lymph nodes, maintaining normal lymphocyte homing and immune function. Dedifferentiation of HEVs will lead to a decrease in the expression of important adhesion molecules, thereby hindering the migration and aggregation of lymphocytes, resulting in impaired lymphocyte homing function and the formation of an immunosuppressive environment. The mechanism of HEV dedifferentiation and weakened lymphocyte homing function provides conditions for tumor cells to evade immune surveillance and accelerates tumor metastasis and progression.

[0005] Epidemiological studies have found that lymph node metastasis in lung cancer patients is negatively correlated with their prognosis. The more lymph nodes metastasized and the wider the range, the shorter the patient's survival. Therefore, the detection and control of lymph node metastasis is one of the key steps in the diagnosis and treatment of lung cancer.

[0006] Accurately detecting lymphocyte homing in lung cancer draining lymph nodes is of great significance for clarifying the mechanism of lung cancer lymph node metastasis. Existing lymphocyte homing detection methods mainly rely on fluorescent labeling and flow cytometry. Although these methods can quantify the number of homing lymphocytes, they are limited in dynamically monitoring lymphocyte migration paths and real-time evaluation of homing function. Summary of the invention

[0007] In view of the problems existing in the prior art, the present invention provides a method for detecting lymphocyte homing. The specific technical solution is as follows:

[0008] A method for detecting the homing efficiency of lymphocytes in tumor-draining lymph nodes, characterized by detecting whether the expression level of VEGFD is abnormal, thereby evaluating whether the homing function of lymphocytes is impaired.

[0009] In some embodiments, the detecting the expression level of VEGFD is detecting the expression level of VEGFD in lymph node cells.

[0010] In some embodiments, the detection of VEGFD expression level in TDLN is caused by tumor secretion.

[0011] In some embodiments, the method for detecting the expression level of VEGFD can be quantitatively detected by ELISA, immunohistochemistry, Western blot or qPCR method.

[0012] In some embodiments, detecting increased expression of VEGFD may lead to changes in the immune microenvironment in the lymph nodes, thereby determining that the lymphocyte homing function is impaired.

[0013] In some embodiments, detecting elevated VEGFD expression in TDLN induces dedifferentiation of HEVs, and induces lumen expansion and wall thinning of HEVs.

[0014] In some embodiments, increased VEGFD expression detected in TDLN may lead to a decrease in lymphocyte homing receptors, thereby impairing lymphocyte homing function.

[0015] In combination with all the above technical solutions, the advantages and positive effects of the present invention are: the present invention provides a method for detecting the homing function of lymphocytes in lung cancer draining lymph nodes (TDLN) regulated by VEGFD,

[0016] By comprehensively detecting the expression level of VEGFD, observing the dedifferentiation characteristics of HEV, and evaluating the homing efficiency of lymphocytes, the regulatory role of VEGFD in the immune microenvironment of TDLN was systematically revealed. This method uses multiple detection methods to conduct a detailed analysis of the lymphocyte migration pathway and the immune microenvironment in TDLN, which is suitable for basic research on lung cancer, development of anti-VEGFD targeted drugs, and clinical immunotherapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] 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 will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0018] Figure 1 The experimental design flow chart of the present invention is shown;

[0019] Figure 2 is a vector map for constructing a VEGFD overexpression lentivirus in Example 1 of the present invention;

[0020] Figure 3 is the IHC localization map of VEGFD in TDLN in Example 1 of the present invention;

[0021] Figure 4 The VEGFD overexpression cell and lung cancer mouse draining lymph node model in Example 1 of the present invention is a graph of detecting VEGFD overexpression in Lewis cells, mouse peripheral blood and TDLN, respectively. ;

[0022] Figure 5 is the CFSE-positive lymphocyte flow cytometric profile constructed in Example 2 of the present invention;

[0023] Figure 6 This is a diagram showing that the tumor secretory factor VEGFD successfully induced the proliferation of the vascular system in TDLN in Example 3 of the present invention;

[0024] Figure 7 is an immunofluorescence image showing that VEGFD promotes the proliferation of HEV in Example 3 of the present invention;

[0025] Figure 8 is a histological immunofluorescence image of the morphological changes of HEV induced by VEGFD in Example 3 of the present invention;

[0026] Fig. 9 It is a flow chart of the characteristics of the immune microenvironment in TDLN regulated by VEGFD in Example 4;

[0027] Fig.10This is a tissue immunofluorescence image showing the decrease in the number of CD4+T cells mediated by HEV in TDLN of mice in the VEGFD-OE group in Application Example 4;

[0028] Fig.11 It is an immunofluorescence image of homing and transport of CFSF-positive cells in the HEV lumen in the TDLN of VEGFD-OE lung cancer constructed in Example 5;

[0029] Fig.12 This is a flow cytometric diagram of the clustering of CFSE+ cells in the lymph nodes extracted in Example 5;

[0030] Fig.13 is a flow cytometric graph showing the difference in the homing ratio of CFSE+ cells in TDLN between the Control group and the VEGFD-OE group in Application Example 5;

[0031] Fig.14 It is the immunohistochemistry diagram of CCL19 and CCL21 in TDLN in the Control group and VEGFD-OE group in Application Example 6. DETAILED DESCRIPTION

[0032] In order to make the purpose, technical scheme and advantages of the present invention more clear, the present invention is further described in detail below in conjunction with the examples. The equipment and reagents used in each embodiment and test example can be obtained from commercial sources unless otherwise specified. The specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0033] Based on the information contained in this application, it is easy for those skilled in the art to make various changes to the precise description of the present invention without departing from the spirit and scope of the appended claims. It should be understood that the scope of the present invention is not limited to the defined process, properties or components, because these embodiments and other descriptions are only for illustrating specific aspects of the present invention. In fact, various changes that the technicians in this field or related fields can obviously make to the embodiments of the present invention are all covered within the scope of the appended claims.

[0034] In order to better understand the present invention but not to limit the scope of the present invention, all the numbers used in this application to express the amount, percentage, and other numerical values ​​should be understood as modified by the word "approximately" in all cases. Therefore, unless otherwise specified, the numerical parameters listed in the specification and the appended claims are approximate values, which may be changed according to the different ideal properties attempted to be obtained. Each numerical parameter should at least be regarded as obtained based on the reported significant figures and by conventional rounding methods. In the present invention, "about" means within 10% of a given value or range, preferably within 5%.

[0035] In the following embodiments of the present invention, when the temperature is not particularly limited, it is room temperature. Room temperature refers to the natural room temperature in four seasons without additional cooling or heating treatment, and is generally controlled at 10-30°C, preferably 15-25°C.

[0036] Unless otherwise specified, the reagents and materials used in the embodiments of the present invention can be purchased from the market.

[0037] The detailed implementation process and contents are as follows:

[0038] Example 1

[0039] Construction of the draining lymph node model of VEGFD-OE lung cancer mice

[0040] In this example, Lewis stable transfected cells of VEGFD-OE were constructed. The vector information of VEGFD can be found in Figure 2 , VEGFD sequence information is shown in Table 1. The cells were transfected with plasmids, shRNA vectors or corresponding lentiviral vectors carrying antibiotic resistance such as puromycin, neomycin or geneticin (the lentiviral MOI and plasmid transfection concentration were determined in preliminary experiments). The transfection steps were basically the same as those for transient cell transfection. The lentiviral infection enhancement reagent Polybrene could be added for lentiviral infection; 48-72 hours after transfection, the percentage of fluorescent cells in the cells was observed under a microscope, and the extracted proteins were used for Western blotting (WB) to verify the down-regulation efficiency; the old culture medium was discarded, and fresh culture medium containing the corresponding antibiotics (the semi-lethal concentration of the cells was determined in preliminary experiments) was added, and the antibiotics were maintained until the percentage of fluorescent cells reached 100% and no more cells died.

[0041] Table 1 Target gene sequence information of this example

[0042]

[0043]

[0044] Use the selected VEGFD-OE cells and normal Lewis cells to form tumors, prepare tumor cells in logarithmic growth with a cell density of about 80-90%, discard the old culture medium, wash the cells 1-2 times with PBS (to remove the serum in the cells), use trypsin to digest the cells, prepare a cell suspension, centrifuge, remove the supernatant, then resuspend the cells with PBS or serum-free culture medium and count the cells, and adjust the final concentration to 1-5×10 7 cells / ml, the inoculation site was the right foot pad of the mouse, and the inoculation volume was 50ul. After inoculation, the tumor growth curve was observed and the tumor volume was monitored. When the observation endpoint (tumor volume <1000mm 3 ) to euthanize the mice. Remove the tumor and TDLN of the mice. Figure 3 As shown in Figure 2, IHC detected the localization of VEGFD in TDLN. VEGFD was widely expressed in the node, especially in the paracortical area. WB detected the expression of VEGFD in TDLN, and Elisa detected the secretion and expression of VEGFD in peripheral blood. We confirmed that the secretion of VEGFD in peripheral blood and the expression of VEGFD in TDLN of VEGFD-OE mice were significantly increased, as shown in Figure 2. Figure 4 As shown, this indicates that the VEGFD-OE lung cancer mouse draining lymph node model was successfully established.

[0045] Example 2

[0046] Identification of CFSE-labeled lymphocytes by flow cytometry

[0047] Extract the spleen of normal mice, place it in PBS, and store it at low temperature. Grind the spleen with the tail end of a 5ml syringe piston, filter it through a 70um cell strainer, centrifuge it at 1500rpm for 5min, and remove the cell supernatant. Then add 3ml of red blood cell lysis buffer to remove red blood cells, lyse them on ice for 10-15min, then add an equal amount of PBS, centrifuge it at 1500rpm for 5min, adjust the number of cells, and use a cell counting plate or automatic cell counter to calculate the cell concentration, which is usually set to 1×10 6 Prepare CFSE reagent solution by adding DMSO to CFSE powder to adjust the concentration to 5 μM and mix well.

[0048] Then prepare the CFSE working solution, the specific ratio is: 1ml PBS: 1ulCFSE. Add 1ml of the prepared CFSE working solution to the lymphocyte suspension, mix gently, incubate on ice for 10-30 minutes (avoid light), add 5ml of 1640 medium containing 10% FBS to terminate the reaction, then centrifuge at 1500rpm for 5min, add 500ul PBS to resuspend the labeled lymphocytes, mix well and add to the flow tube, place the CFSE-labeled lymphocyte suspension in the sample tube of the flow cytometer, and ensure that the cell concentration is moderate (usually 1×10 6 -5×10 6 cells / mL). Start data acquisition and record the fluorescence intensity and scattered light information of cells when they pass through the laser beam. Record 20,000 cell events. Observe the proportion of cells that are positive in the FITC channel, which are CFSE positive cells. Figure 2 As shown in the figure, we cultured the labeled fluorescent cells and then labeled them with CFSE dye, and then flow cytometry detected the efficiency of CFSE (FITC channel) labeling primary spleen cells ( Figure 5), we identified that almost all cells were labeled with CFSE fluorescent dye (99.2% and 99.6%), which showed that spleen primary cells were basically sensitive to CFSE dye and all cells were labeled, so they could be used for the next experiment.

[0049] Example 3

[0050] Tumor-secreted factor VEGFD regulates the dedifferentiation characteristics of HEV

[0051] This example aims to explore the regulatory effect of VEGFD on the dedifferentiation characteristics of HEV. The dedifferentiation of HEV is closely related to its lymphocyte homing function, and further understand the effect of VEGFD on the lymphocyte homing function in TDLN.

[0052] As in Example 1, a VEGFD-OE lung cancer mouse TDLN model was constructed. When the ethical endpoint was reached, the mice were euthanized, the mouse lymph nodes were removed, the lymph nodes were fixed with 4% paraformaldehyde, and paraffin-embedded sections were performed. The mouse lymph node sections were fixed with 4% paraformaldehyde for 10-30 minutes. The sections were washed with PBS 3 times for 5 minutes each to remove the fixative. The sections were treated with 0.1-0.5% Triton X-100 in PBS for 10-15 minutes to enhance antibody permeability. The sections were washed again with PBS 3 times for 5 minutes each. The sections were soaked in blocking solution and incubated at room temperature for 1 hour to reduce nonspecific binding. HEV was labeled with MECA-79 antibody, MECA-79 antibody and CD31 antibody were diluted at a ratio of 1:500, and the diluted primary antibody was added to the sections to cover the entire section. Incubate overnight at 4°C to ensure that the antibody is fully bound to the target protein. Wash the sections with PBS 3 times for 5-10 minutes each to remove unbound primary antibodies. Add the diluted secondary antibody to the slices, covering the entire slice. Incubate at room temperature for 1 hour. Soak the slices in PBS containing DAPI (1μg / mL) and incubate for 5-10 minutes to stain the nuclei. Wash the slices 3 times with PBS to remove excess DAPI. Wash the slices 3 times with PBS for 5 minutes each to remove unbound secondary antibody. Use an anti-fluorescence attenuation mounting solution (such as a mounting solution containing an anti-fluorescence agent), cover the slices, and gently remove bubbles. Observe the slices using a fluorescence microscope and select the appropriate excitation wavelength and filter to obtain the best fluorescence signal. Image J software was used to measure the lumen diameter and wall thickness of HEV. Figure 6 As shown, based on the model construction of Example 1, the expression of CD31 in TDLN was detected, and CD31 was significantly increased in TDLN of VEGFD-OE group mice, which indicates that VEGFD still has a significant promoting effect on the vascular system in TDLN. Figure 7As shown in Figure 2, the number of HEVs in VEGFD-OE mice was significantly increased, indicating that VEGFD promotes the proliferation of HEVs. Figure 8 As shown, through the model construction of the present invention, the lumen morphological characteristics of HEV were analyzed. In TDLN of VEGFD-OE mice, the lumen of HEV was dilated, the wall was thinned, and it showed the characteristics of dedifferentiation.

[0053] Example 4

[0054] Tumor-secreted factor VEGFD regulates the immune microenvironment of TDLN

[0055] This example aims to study the regulatory effect of tumor secretory factor VEGFD on the immune microenvironment of TDLN and explore the mechanism of VEGFD in lung cancer metastasis and immune escape. By establishing a mouse model and a series of experimental analyses, the effect of VEGFD on the distribution and function of immune cells in TDLN was evaluated.

[0056] The model was constructed as in Example 1. When the ethical endpoint was reached, the mouse was killed and the abdominal skin was carefully opened to expose the skin and abdominal wall; the longitudinal cutting range was from short beyond the urethral opening (tail side) to the mandible (brain), and four cuts were made in the direction of all four limbs; the popliteal lymph nodes and inguinal lymph nodes were removed, and the adipose tissue was completely removed. The lymph nodes were removed and placed in PBS and stored at low temperature. The lymph nodes were placed in Cell staining buffer, gently ground with the tail end of the syringe piston, the tissue was completely crushed, and filtered with a 70um filter; then centrifuged at 700g for 5 minutes, repeated, resuspended in Cell staining buffer, counted cells, and adjusted to a concentration of 1x10^6 cells per 100uL; prepared EP tubes, mixed primary antibody staining indicator antibodies (CD3, CD4, CD8, CD45, CD11c, MHCII), paid attention to the antibody dilution ratio, the total volume was 50ul, and titration was performed in advance to determine the optimal dilution ratio. Use purified αFcγRIIB / III and αFcγRIV to block Fcγ receptors (Fcγr) IIB, III and IV to avoid nonspecific binding of staining, incubate for 15 minutes, centrifuge at 700g for 5 minutes; discard the supernatant; stain each tube with 50μl primary antibody, resuspend and incubate at 4°C in the dark for 30 minutes; then add 100ul cell staining buffer to resuspend, centrifuge at 700g for 5 minutes, and discard the supernatant; prepare EP tubes, mix primary antibody staining indicator antibodies, pay attention to the antibody dilution ratio, and the total volume is 50ul; add 160ul cellstaining buffer to resuspend, centrifuge at 700g for 5 minutes, and discard the supernatant; resuspend each tube with 200ul cell staining buffer. Start data acquisition, record the fluorescence intensity and scattered light information of cells when they pass through the laser beam, and record 20,000 cell events. Fig. 9 As shown, this example confirmed that the proportions of CD4, CD8 and DC cells were decreased in the VEGFD-OE group (10.9%, 11.9% and 2.35%, respectively), indicating that increased VEGFD in TDLN will lead to changes in the immune microenvironment in the node, thereby impairing the immune response function in the node.

[0057] In this example, we analyzed the correlation between CD4+T cells and HEV, and co-labeled lymph node sections with CD4 and MECA-79, respectively, and observed the number of HEV-mediated CD4+T cells under a fluorescence microscope. Fig.10 As shown, the proportion of CD4 T cells surrounding HEV was significantly decreased in the VEGFD-OE group, indicating that HEV-mediated lymphocyte homing function was impaired.

[0058] Example 5

[0059] Tumor-secreted factor VEGFD induces a decrease in HEV-mediated lymphocyte homing function

[0060] For example, Example 1 was used to construct lung cancer drainage lymph node models for control mice and VEGFD-OE mice, and lymphocyte homing function was tested when the tumor grew to the ethical endpoint. The specific process is to extract primary cells from the spleen of normal mice and label them with CFSE. The specific steps can be seen in Example 2. The cell concentration of the CFSE-labeled primary cells was adjusted to be consistent, and the injection volume for each mouse was 150ul, ensuring that the number of cells injected for each mouse was 1*10^6. The prepared cells were injected into the control mice and VEGFD-OE group mice through the tail vein. The injection speed should not be too fast, and the injection should be kept slow. The state of the mice was observed after the injection. Within 1-2 hours of the tail vein injection, the mice were killed, and the popliteal lymph nodes were removed and fixed in PBS (cold storage) and 4% paraformaldehyde respectively. The paraformaldehyde-fixed lymph nodes were made into frozen sections and stained with the high endothelial venule marker antibody MECA-79 at the same time. The expression of CFSE+ and MECA-79 in the lymph nodes was detected under a fluorescence microscope. Fig.11 As shown, CFSE-labeled lymphocytes are transported in high endothelial venules (green) through CFSE+ cells (orange), bind to homing receptors on the surface of high endothelial venules and enter the lymph nodes. This indicates that HEV is directly related to the homing of lymphocytes, and reduced lymphocyte homing leads to a decrease in immune cells in the nodes. The extracted lymph nodes were processed as shown in Example 4 and made into single cell suspensions. Cell staining buffer was used to maintain cell activity, and the cell concentration was adjusted to 1*10^6 cells / ml. Flow cytometry was used to analyze the proportion and distribution of labeled lymphocytes in the lymph nodes. Fig.12 As shown in the figure, after circling all lymphocytes and removing cell adhesion, the proportion of homing lymphocytes (CFSE+ cell proportion) was identified and determined as the CFSE positive cell population. Fig.13 As shown, flow cytometry detected the proportion of homing cells in TDLN of mice in the Control group and VEGFD-OE group. The proportion of CFSE+ cells in the VEGFD-OE group was significantly decreased, indicating that VEGFD induces dedifferentiation of HEV, leading to a decrease in lymphocyte homing function, providing important data support for tumor immune escape and related immunotherapy strategies.

[0061] Example 6

[0062] VEGFD regulates the downregulation of homing receptor expression in TDLN

[0063] This example aims to explore the effect of tumor-secreted factor VEGFD on the expression of lymphocyte homing receptors in TDLN. By constructing a mouse model, the regulatory effect of VEGFD on the expression of lymphocyte homing receptors (such as CCL19 and CCL21) in TDLN was analyzed to reveal its mechanism in tumor immune escape.

[0064] The specific implementation steps are as follows: as in Example 1, a mouse drainage lymph node model of the Control group and the VEGFD-OE group was constructed, the mouse lymph nodes were removed and fixed in 4% paraformaldehyde and embedded in paraffin, and the paraffin-embedded tissue was cut into 4-5 μm slices using a slicer and placed on a glass slide. The slices were dewaxed in xylene, then rehydrated with gradient ethanol (100%, 95%, 80%), and finally washed with PBS. Antigen retrieval was performed in a water bath, usually using citrate buffer (pH 6.0) or Tris-EDTA buffer (pH 9.0), heated for 20-30 minutes to enhance antibody binding. The slices were treated with blocking solution and incubated at room temperature for 1 hour to reduce nonspecific binding. CCL19 and CCL21 were diluted to 1:100 and 1:200 respectively for use. The diluted primary antibody was added to the slices, covering the entire slice, and incubated overnight at 4°C. Wash the sections with PBS three times for 5-10 minutes each to remove unbound primary antibody. Add the secondary antibody to the sections, cover the entire section, and incubate at room temperature for 1 hour. Wash the sections with PBS three times for 5-10 minutes each to remove unbound secondary antibody. Use DAB colorimetric reagent and treat the sections according to the instructions to observe the color change. Usually a brown precipitate will appear after a few minutes. Gently rinse the sections with PBS or deionized water to terminate the color development reaction. Hematoxylin can be used to stain the nuclei optionally to enhance the visualization of tissue structure. Use anti-fluorescence attenuation mounting solution to mount the sections to avoid signal attenuation caused by light. Observe the sections under a microscope and take images to record the expression and distribution of CCL19 and CCL21. Fig.14 As shown, the expression of homing receptors CCL19 and CCL21 was decreased in TDLN of the VEGFD-OE group, suggesting that VEGFD caused a decrease in lymphocyte homing receptors, which in turn led to impaired lymphocyte homing function.

[0065] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for detecting the homing efficiency of lymphocytes in tumor-draining lymph nodes, characterized in that: By detecting whether the VEGFD expression level is abnormal, we can evaluate whether the lymphocyte homing function of the tumor-draining lymph nodes is impaired.

2. The method according to claim 1, characterized in that The detection of VEGFD expression level is to detect the VEGFD expression level in lymph node cells.

3. The use according to claim 1, characterized in that: The tumor is selected from at least one of breast cancer, lung cancer, colon cancer and hepatocellular carcinoma.

4. The method according to claim 1, characterized in that The tumor is selected from lung cancer.

5. The method according to claim 1, characterized in that The method for detecting the expression level of VEGFD can be quantitatively detected by ELISA, immunohistochemistry, Western blot or qPCR method.

6. The use according to claim 1, characterized in that: The VEGFD is a secretory factor secreted by the tumor to TDLN.

7. The method according to claim 1, characterized in that The increased expression of VEGFD in TDLN promoted the dedifferentiation of HEV, the expansion of its lumen and the thinning of its wall.

8. The method according to claim 1, characterized in that Detection of increased VEGFD expression can lead to changes in the immune microenvironment within the lymph nodes, thereby determining that the lymphocyte homing function is impaired.

Images