Application of 2-DG in inhibition of lymph node metastasis of pancreatic cancer

Abstract

The invention relates to application of 2-DG in inhibition of lymph node metastasis of pancreatic cancer. The inhibition effect of 2-DG on glycolysis of cells is mainly investigated, glycolysis of lymphatic endothelial cells is inhibited by using 2-DG, and tight connection between lymphatic vessels is recovered, so that the effect of blocking lymphatic metastasis of pancreatic cancer is achieved.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and specifically relates to the application of 2-DG in inhibiting lymph node metastasis of pancreatic cancer. Background Art

[0002] Pancreatic cancer is a malignant tumor that occurs in the pancreas. Currently, there are limited treatment options, poor prognosis, and recurrence and metastasis are common. Pancreatic cancer often metastasizes to lymph nodes, which is very common in patients with recurrent and metastatic pancreatic cancer and is significantly associated with poor clinical prognosis. Blocking pancreatic cancer lymph node metastasis is of great significance for the treatment of pancreatic cancer.

[0003] Currently, there are no drugs available clinically that can block pancreatic cancer lymphatic metastasis by inhibiting endothelial cell glycolysis in lymphatic endothelial cells and restoring tight junctions between lymphatic vessels. Summary of the invention

[0004] The purpose of the present invention is to overcome the deficiencies of the prior art and provide an application of 2-DG in inhibiting pancreatic cancer lymph node metastasis.

[0005] The technical solution of the present invention is as follows:

[0006] Application of 2-DG in inhibiting lymph node metastasis of pancreatic cancer.

[0007] Furthermore, the concentration of 2-DG is 5 mM, and the treatment time is 24 hours.

[0008] Compared with the prior art, the present invention has the following beneficial effects:

[0009] 1. The present invention mainly investigates the inhibitory effect of 2-DG on cell glycolysis, and uses 2-DG to inhibit lymphatic endothelial cell glycolysis and restore the tight connection between lymphatic vessels, thereby achieving the effect of blocking pancreatic cancer lymphatic metastasis. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 This is a result diagram showing the reduction of pancreatic cancer lymph node metastasis in mice with systemic ZIP1 knockout in the present invention;

[0011] Figure 2 This is a result diagram showing the reduction of pancreatic cancer lymph node metastasis in the ZIP1 lymphatic endothelial-specific knockout mice of the present invention;

[0012] Figure 3 This is a result diagram showing that the lymphatic vessels of ZIP1 whole-body knockout mice in the present invention are more tightly connected, limiting lymphatic drainage;

[0013] Figure 4 This is a result diagram of upregulation of lymphatic endothelial tight junction proteins by knocking out ZIP1 in the present invention;

[0014] Figure 5 This is a graph showing the results of reduced lymphatic endothelial permeability after ZIP1 knockdown in the present invention;

[0015] Figure 6 This is a result diagram showing the reduction of glycolysis in lymphatic endothelial cells by knocking down ZIP1 in the present invention;

[0016] Figure 7 This is a result diagram showing that the glycolytic metabolite lactic acid promotes the increase of lymphatic vessel permeability in the present invention;

[0017] Figure 8 This is a result diagram of lactic acid promoting tumors to pass through the lymphatic endothelium in the present invention;

[0018] Fig. 9 This is a result diagram of the present invention in which 2-DG blocks endothelial cell glycolysis and restores tight junction expression;

[0019] Fig.10 This is a graph showing the results of 2-DG treatment to restrict lymphatic drainage using fluorescent microspheres FITC-Dextran;

[0020] Fig.11 This is a graph showing the results of 2-DG treatment to restrict lymphatic drainage using fluorescent microspheres FITC-Dextran;

[0021] Fig.12 This is a graph showing the results of 2-DG inhibiting tumor penetration through lymphatic endothelial cells in the present invention. DETAILED DESCRIPTION

[0022] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only 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 ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0023] like Figure 1-12 As shown, the application of 2-DG in inhibiting lymph node metastasis of pancreatic cancer;

[0024] Experimental Materials and Methods

[0025] 1. Experimental Animals

[0026] Zip1- / - mice with C57BL / 6 background were purchased from Cyagen (KOCMP-30791-Slc39a1-B6N; Suzhou, China). Zip1 mice with C57BL / 6 background were purchased from Cyagen (CKOCMP-30791-Slc39a1-B6J; Suzhou, China). flox / floxTo generate LEC-specific conditional Zip1 knockout mice (Zip1 ΔLYVE1 , Zip1 flox / - ;Prox1 Cre+ , Zip1 flox / fiox ;Prox1 Cre + ) mice;

[0027] The following primers confirmed tissue-specific genetic deletion of Zip1 at the mRNA level:

[0028] F1: 5'-TAGGGGTCACTTACATGAACACGA-3′

[0029] R1: 5'-CACATCCCATCCTCTGTGACACACATC-3';

[0030] Cyagen Biosciences provided mice expressing Cre recombinase under the control of the Lyvel promoter (Lyve1-Cre) and the Prox1 promoter (Prox1-Cre) in the C57BL / 6 background. flox / flox Mice were crossed with Lyve1-Cre or Prox1-Cre mice to obtain LEC-specific Zip1 ΔLyve1 、Zip1 flox / - ;Prox1 Cre + , Zip1 flox / flox ;Prox1 Cre+ Mice, all mice were raised at the SPF animal experimental center of Zhengzhou University at an average temperature of 21°C and 50% humidity, with a light / dark cycle of 12 h. Male mice were used as the research subjects because male animals have less phenotypic variation.

[0031] 2. Cell lines

[0032] The SVEC4-10 cell line was provided by Professor Zhu Mingzhao of the Chinese Academy of Sciences. The LEC cell line SVEC4-10 with low expression of ZIP 1 was constructed using CRISPR / Cas9 technology. kd and its control cell line SVEC4-10 ctrl .

[0033] Human lymphatic endothelial cells (hLEC) were purchased from iCell Bioscience and cultured in a primary endothelial cell culture system (Pri Med-iCell-002) consisting of ECM, 5% fetal bovine serum, and supplements containing 10 ng / ml Rh VEGF, 5 ng / ml Rh EGF, 5 ng / ml Rh-FGF, and 15 ng / ml Rh IGF-1.

[0034] The KPC-Luc cell line was derived from a spontaneous pancreatic cancer cell line, LSL-Kras. G12D / + ;LSL-Trp53 R172H / + ; Pdx-1-Cre(KPC), provided by Dr. Liu Xiaomeng from Fudan University.

[0035] Mouse pancreatic cancer cells Pan02 and KPC-Luc, and human pancreatic cancer PANC-1 were grown in DMEM with 10% FBS and 100 IU / ml penicillin-streptomycin, and the cells were incubated at 37° C. in an environment of 5% carbon dioxide and 95% air. Stable Pan02-GFP-Luc cell lines were constructed by transfecting Pan02 cells with GFP-Luc lentiviral vectors (Genechem, Shanghai, China), and S100A4-KO Pan02-GFP-Luc cells were generated using the CRISP / cas9 system.

[0036] 3. Lymph Node Metastasis Model

[0037] To study pancreatic cancer lymph node metastasis, a mouse popliteal lymph node metastasis model was established. Briefly, 1×10 6 GFP-Luc labeled Pan02 or KPC-Luc cells were inoculated into the footpads of 6-8 week old male mice (5-6 mice per group, repeated at least three times), and the tumor volume was monitored every 2 days. Tumor volume (V) is expressed in mm 3 The evaluation is done in units of: V = 0.5 × (L × W 2 ), L is the long diameter of the tumor, W is the short diameter of the tumor, and the mice were killed at the end of the experiment (around the 30th day after tumor bearing). The orthotopic tumors in the footpad were taken for immunohistochemical staining, and the popliteal lymph nodes and lungs were taken for bioluminescence quantification using the IVIS Spectrum Imaging System (PerkinElmer). The popliteal lymph nodes were fixed with 4% paraformaldehyde for paraffin sectioning procedures and hematoxylin-eosin (HE) staining.

[0038] 4. Lymphatic Drainage

[0039] Male mice were injected with 1 μL FITC-dextran (2 mg / mL, 70 kD) in the right ear and PBS in the left ear as a control. Thirty minutes later, cervical lymph nodes were collected from the sacrificed mice and then examined for fluorescence in the lymph nodes using an IVIS Spectrum Imaging System (PerkinElmer).

[0040] 5. Immunofluorescence staining

[0041] Tumor tissue frozen sections (6 μm) were prepared for immunofluorescence (IF) staining using the following antibodies: LYVE1 (1:200, MAB2125-100, R&D), PROX1 (1:200, AF2727, R&D), Claudin-5 (1:200, 34-1600, Invitrogen), VE-Cadherin (1:200, AF1002, R&D) and ZO-1 (1:200, 40-2200, Invitrogen), and paraffin-embedded sections were subjected to immunohistochemistry (IHC) staining.

[0042] 6. Trans-lymphatic endothelial migration

[0043] Gently detach SVEC4-10 and SVEC4-10 grown in the culture dish using trypsin ctrl and SVEC4-10 kd cells, and 1×10 5 cells (SVEC4-110, SVEC4-10 ctrl and SVEC4-10 kd ) were placed in the upper chamber of the Transwell chamber (NEST insert, 724301). After one day of culture, LECs formed a complete cell monolayer. For cell migration, 3×10 5 Pan02 cells stained with fluorescent dyes (CMFDA, 40721ES60, YEASEN) were added to the endothelial cell monolayer in a volume of 500 μL, and a medium containing high concentration of serum (20%) was added to the lower chamber of the Tran-well chamber. After 24 hours, the number of cells that migrated to the surface of the lower chamber was counted by taking pictures with a confocal microscope.

[0044] 7. Seahorse Cell Metabolism Assay

[0045] The Seahorse XF96 Cell Energy Metabolism Analyzer (Seahorse Bioscience, MA, USA) was used to measure the extracellular acidification rate (ECAR). SVEC4-10 (6000 cells / well) were seeded onto Seahorse XF96 cell culture plates and incubated overnight. The next day, the medium was removed and the cells were washed twice with 2 mL of XF basal medium containing 200 mM L-glutamine. The plates were then incubated in a non-CO2 incubator at 37 °C for 1 h. 10 mM glucose, 1 μM oligomycin, and 50 mM 2-deoxyglucose (2-DG) were automatically injected into each well in sequence, followed by 3 measurement cycles. ECAR was normalized based on Hoechst (Beyotime) staining. The assay was repeated 5–6 times, and each experiment was repeated three times independently. To measure mitochondrial respiration, blockers were sequentially added through the ports of the Seahorse Flux-Pak cartridge. Oxygen consumption rate was measured using the Seahorse XF96 assay.

[0046] 8. Zn 2+ Ingest

[0047] In order to detect Zn 2+ The absorption of 1×10 4 SVEC4-10 ctrl or SVEC4-10 kd Cells were seeded into individual wells of a 96-well black plate and incubated overnight. After loading with 2 μM FluoZin3 for 1 hour, 30 μM ZnCl2 was added to HBSS. 60 seconds after addition, FluoZin3 fluorescence was read every 30 seconds for the specified time to assess intracellular free Zn 2+ All values ​​minus the blank group value were used to evaluate the intracellular Zn 2+ The cell fluorescence time point at the beginning (F) was used as F0, and the change in intracellular Zn2+ was calculated as ΔF / F0, where ΔF = F-F0. The origin was forced to zero, and the measurement start time was 60 seconds.

[0048] 9. Assessment of the tightness of lymphatic endothelial cell monolayer

[0049] The intercellular compactness of LEC monolayers was assessed by measuring the electrical impedance of LEC monolayers using an xCELLigence System RTCA-MP instrument (ACEA Biosciences, Inc., San Diego, CA);

[0050] Specifically, 40,000 cells were seeded into 96-cell E-plates (ACEA Biosciences, Inc.) and monitored using an RTCA-MP instrument, with each group containing at least three replicates. Once the cell index value reached a peak, indicating that LECs had formed a monolayer, a stimulant was added to the culture. The cell index value before the addition of the stimulant was used as a reference point. Impedance was analyzed as a normalized cell index and expressed as relative cell impedance. All experiments were repeated at least three times.

[0051] 10. Lymphatic endothelial cell monolayer permeability test

[0052] 1×10 5 Cell lines (SVEC4-10 ctrl and SVEC4-10 kd ) or 2×10 5 Primary cells (mLECs) were seeded on the upper surface of the upper chamber of the transwell chamber. After one day of culture, LECs formed a complete monolayer. 100 μL DMEM containing 70kDFITC dextran (2 mg / mL, Sigma-Aldrich, Bornem, Belgium) was injected into the upper chamber of the transwell, and 500 μL DMEM was injected into the lower chamber. After 30 minutes in an incubator at 37°C, the culture medium in the lower chamber was mixed, and 100 μL of the culture medium was added to a 96-well plate (each sample was repeated 3 times), and the fluorescence (Ex: 488 nm, Em: 530 nm) was measured using SpectraMAX i3X (Molecular Devices, CA, USA).

[0053] 11. Glucose analog uptake experiment

[0054] 2-[N-(7-nitrobenzene-2-oxa-1,3-dihydroxy-4-yl)amino]-2-deoxyglucose (2-NBDG) is a glucose analog. ctrl / SVEC4-10 kd Cells were seeded in 24-well plates, and after overnight, the culture medium was discarded and the cells were washed with PBS (pH 7.4). Low-glucose medium supplemented with 2-NBDG (100 μM, N13195, Life Technologies) was then added and incubated at 37 ° C for 45 minutes. Next, the cells were washed with PBS and trypsin was added, the cells were harvested, centrifuged at 1500 rpm for 5 minutes at 4 ° C, washed twice with ice PBS, and kept on ice. A control sample lacking 2-NBDG was used to set a blank in the flow cytometer and set the 2-NBDG detection parameters.

[0055] 12. Western blotting

[0056] Cells were lysed with RIPA (Solarbio, China) lysis buffer and lysates were collected. Proteins were separated by 10% SDS-PAGE and transferred to nitrocellulose (NC) membranes. The membranes were incubated with the primary antibody and detected with HRP-conjugated secondary antibodies (ABclonal). The following primary antibodies were used: ZIP1 (1:1000, AL0-AZT-001; Alomone, Israel), claudin-5 (1:1000, 34-1600, Invitrogen, USA), VE-Cadherin (1:1000, 13368, Abcam, UK), ZO-1 314335, OriGene, USA), β-actin (1:1000, AC026, ABclonal, CN).

[0057] 13. Statistics

[0058] Statistical analysis was performed using GraphPad Prism 8.3.0. Unpaired Student's t-test (two-sided) was used to compare statistical significance between two groups. One-way ANOVA was used to compare multiple groups, and two-way ANOVA was used to compare curves. Data are presented as mean ± standard error (SEM). Sample size is indicated where appropriate. Asterisks (*) indicate statistical significance (*p < 0.05; **p < 0.01; ***p < 0.001).

[0059] 1. Results:

[0060] 1. ZIP1 systemic knockout mice show reduced lymph node metastasis of pancreatic cancer

[0061] Establishment of a mouse pancreatic cancer lymph node metastasis model ( Figure 1 AB), mouse pancreatic cancer Pan02-GFP-Luc cells were inoculated into the footpads of mice (ZIP1+ / +, ZIP1- / -). After 35 days, the ipsilateral and contralateral popliteal lymph nodes of the mice were taken to detect lymph node metastasis. The results showed that tumor metastasis could be detected in the ipsilateral popliteal lymph nodes of most control mice (ZIP1+ / +), accounting for 5 / 6, while the lymph node metastasis of knockout mice (ZIP1- / -) accounted for 3 / 6, which was significantly reduced (CE). The results show that pancreatic cancer lymph node metastasis is reduced in ZIP1 systemic knockout mice.

[0062] from Figure 1 It can be seen that systemic knockout of ZIP1 can reduce lymph node (LN) metastasis of pancreatic cancer;

[0063] in Figure 1A and B in the figure are schematic diagrams of animal experiments on popliteal lymph node metastasis in pancreatic cancer mice. Pan02-GFP-Luc cells were inoculated into the footpads of Zip1+ / + and Zip1- / - mice. After 35 days, the mice were killed and tumor metastasis was examined.

[0064] Figure 1 C in the figure indicates the detection of popliteal lymph node metastasis on the ipsilateral side of footpad tumors in Zip1+ / + and Zip1- / - mice by small animal imaging bioluminescence (n=6 in each group);

[0065] Figure 1 D in the figure represents the bioluminescence intensity for quantifying the above LN transfer;

[0066] Figure 1 E in Figure 1 represents the quantitative analysis of pancreatic cancer lymph node metastasis (LNM) in three independent animal experiments (n=18 per group).

[0067] 2. ZIP1 lymphatic endothelial-specific knockout mice reduce pancreatic cancer lymph node metastasis

[0068] Construction of mice with specific knockout of ZIP1 in lymphatic endothelium (ZIP1f / f; Lyve-Cre, Zip1 ΔLyvel Pan02-GFP-Luc cells were inoculated to detect popliteal lymph node metastasis. The results showed that compared with the control (ZIP1f / f), lymphatic endothelial-specific knockout of ZIP1 (Zip1 ΔLyvel ) Reduce tumor lymph node metastasis ( Figure 2 ).

[0069] like Figure 2 As shown, pancreatic cancer LN metastasis in mice was reduced when ZIP1 was specifically deleted in lymphatic endothelial cells;

[0070] in Figure 2 A is a control Zip1 detected by small animal imaging bioluminescence flox / flox Mouse and lymphatic endothelial cell-specific deletion of ZIP1 ΔLyve1 The mouse footpad tumor had lymph node metastasis to the popliteal fossa on the same side;

[0071] Figure 2 Part B in the figure represents the quantification of the bioluminescence intensity of lymph node metastasis (n=5 mice per group);

[0072] Figure 2 Part C in Figure 3 shows the quantification of bioluminescence intensity of lymph node metastasis (LNM) in two independent animal experiments (n=12 in each group).

[0073] 3. The lymphatic tight junctions of ZIP1 knockout mice are tighter, limiting lymphatic drainage;

[0074] Pan02-GFP-Luc tumor tissues of pancreatic cancer in mice (ZIP1+ / +, ZIP1- / -) were obtained and fluorescently stained for LYVE1 (to detect lymphatic vessels) and Claudin-5 (to detect tight junctions);

[0075] The results showed that Claudin-5 was lowly expressed on the lymphatic endothelium, but after knocking out ZIP1, the expression of Claudin-5 on lymphatic endothelial cells increased ( Figure 3 AB), indicating that ZIP1 knockout upregulated the expression of tight junctions on the lymphatic endothelium;

[0076] Using fluorescent microspheres FITC-Dextran 70kD to simulate lymphatic drainage of tumor cells to lymph nodes, Zip1 + / + Mouse, Zip1 - / - FITC-Dextran 70kD was injected into the right ear of mice to detect changes in lymphatic drainage function of lymphatic vessels;

[0077] The results showed that compared with Zip1 + / + Mouse, Zip1 - / - The mice had reduced drainage of fluorescent beads to lymph nodes, suggesting that ZIP1 knockout restricted lymphatic drainage.

[0078] from Figure 3 It can be seen that Zip1 - / - Enhanced cell-cell junctions in lymphatic vessels in mouse tumors.

[0079] in Figure 3 A representative image shows Zip1 + / + and Zip1 - / - Immunostaining of LYVE1 and claudin-5 in mouse Pan02-GFP-Luc tumor sections, scale bar 150 μm.

[0080] Figure 3 B in the figure indicates the quantitative LYVE1 + Fluorescence intensity of claudin-5 on lymphatic vessels (n=20 fields per group).

[0081] Figure 3 Representative images in C show drainage to cervical lymph nodes after FITC-dextran injection into the ears of the indicated mice;

[0082] Figure 3 D in the figure indicates the quantitative drainage of FITC-dextran fluorescence to the cervical lymph nodes (Zip1 + / + n=6; Zip1 - / - n=8).

[0083] 4. ZIP1 knockout upregulates tight junction proteins in lymphatic endothelial cells

[0084] SVEC4-10 cells were infected with CRISP / cas9 and cells with low expression of ZIP1 were screened (SVEC4-10 kd );

[0085] The results showed that compared with the control cells, Claudin-5 expression was upregulated (e.g. Figure 4 shown).

[0086] 5. ZIP1 knockdown reduces lymphatic endothelial permeability

[0087] Compared with control cells (SVEC4-10 ctrl ), ZIP1 low expression cells (SVEC4-10 kd ) has reduced permeability to fluorescent microspheres FITC-Dextran 70kD (e.g. Figure 5 shown).

[0088] 6. ZIP1 knockdown reduces glycolysis in lymphatic endothelial cells

[0089] like Figure 6 As shown, compared with control cells (SVEC4-10 ctrl ), ZIP1 low expression cells (SVEC4-10 kd ) Glycolytic function is downregulated.

[0090] in Figure 6 Lymphatic endothelial cells were plated in Seahorse energy metabolism measurement culture plates, and glucose, oligomycin and 2-DG were injected sequentially to monitor SVEC4-10 ctrl and SVEC4-10 kd The extracellular acidification rate (ECAR) of the cells (an indicator of the level of glycolysis).

[0091] 7. Lactic acid, a product of glycolysis, promotes increased lymphatic permeability

[0092] Lactate stimulation of SVEC4-10 cells increased their permeability to FITC-Dextran 70kD (eg Figure 7 As shown, lactate treatment promotes the permeability of lymphatic endothelial SVEC monolayers to 70 kD FITC-dextran).

[0093] 8. Lactic acid promotes tumor penetration through the lymphatic endothelium

[0094] Lactate stimulation of SVEC4-10 cells promoted pancreatic cancer cell Pan02-GFP-Luc to penetrate the lymphatic monolayer.

[0095] like Figure 8As shown, lactate promotes Pan02 tumor cells to cross the lymphatic endothelium. Lymphatic endothelial cells were plated on the upper layer of Transwell chamber, and SVEC monolayer was treated with different concentrations of lactate. Pan02 tumor cells migrated through the endothelial cells to the lower layer of the chamber (n=15 fields of view per group). Scale bar, 50 μm.

[0096] 9. 2-DG blocks endothelial cell glycolysis and restores tight junction expression

[0097] Stimulation of SVEC4-10 with the glycolysis inhibitor 2-deoxyglucose (2-DG) upregulated the expression of tight junction protein Claudin-5 (e.g. Fig. 9 shown).

[0098] 10. 2-DG treatment restricts lymphatic drainage with fluorescent microspheres FITC-Dextran

[0099] The glycolysis inhibitor 2-DG was injected subcutaneously into the right ear of mice. 12 hours later, the fluorescent microspheres FITC-Dextran 70kD were injected to simulate the metastasis of tumor cells to the lymph nodes. 30 minutes later, the cervical lymph nodes were taken to detect the drainage. The results showed that 2-DG treatment inhibited the drainage of fluorescent microspheres FITC-Dextran into the lymph nodes, suggesting that 2-DG can inhibit the metastasis of tumors to lymph nodes.

[0100] Fig.10 and Fig.11 FITC-Dextran fluorescent microspheres were shown to limit lymphatic drainage by 2-DG treatment.

[0101] in Fig.10 Representative images show drainage of FITC-Dextran microspheres into cervical lymph nodes. Mice were injected with 1 μL of 2-DG (0, 10, 100 mM) on the ear. Twelve hours later, 70 kD FITC-dextran was injected on the ear, and 30 minutes later, the ipsilateral cervical lymph nodes were harvested and examined for drainage of fluorescent microspheres.

[0102] Fig.11 Represents the quantitative fluorescence of FITC-dextran microspheres in cervical lymph nodes (n≥5 per group).

[0103] 11. 2-DG inhibits tumor penetration through lymphatic endothelial cells

[0104] Treatment of SVEC4-10 cells with 2-DG inhibited the permeation of pancreatic cancer cells Pan02-GFP-Luc into lymphatic monolayers.

[0105] like Fig.12As shown, 2-DG inhibits Pan02 tumor cells from passing through the lymphatic endothelium. Lymphatic endothelial cells were plated on the upper layer of the Transwell chamber. After the cells grew into a monolayer, the control group used normal culture medium, and the experimental group used culture medium containing 5mM 2-DG to treat SVEC cells for 24h. Pan02 tumor cells were added to penetrate the endothelial cells and migrate to the lower layer of the chamber (n=30 fields of view for each group). Scale bar, 50μm.

[0106] In summary, the present invention mainly investigates the inhibitory effect of 2-DG on cell glycolysis, uses 2-DG to inhibit lymphatic endothelial cell glycolysis, restores the tight connection between lymphatic vessels, and thus achieves the effect of blocking pancreatic cancer lymphatic metastasis.

[0107] Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for those skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to make equivalent substitutions for some of the technical features therein. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. 2-DG in inhibiting pancreatic cancer lymph node metastasis.

2. The use of 2-DG in inhibiting pancreatic cancer lymph node metastasis according to claim 1, characterized in that: The concentration of 2-DG was 5 mM, and the treatment time was 24 hours.

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