Application of infliximab in auxiliary repair of pancreatic cancer tumor vascular barrier function

Abstract

The invention belongs to the technical field of medicines, and particularly relates to application of infliximab in auxiliary repair of a pancreatic cancer tumor vascular barrier function. Research finds that infliximab can significantly repair vascular endothelial barrier injury in a pancreatic cancer environment, increase the pericyte expression quantity, reduce the expression of adhesion protein ICAM1 and vascular permeability protein Src and p-Src, and can be combined with gemcitabine to promote the chemotherapy effect of pancreatic cancer. The application shows that the infliximab and the gemcitabine have a synergistic effect in the aspect of treating the pancreatic cancer, the chemotherapy effect of the gemcitabine on the pancreatic cancer can be effectively promoted, a new way is provided for treating the pancreatic cancer, the understanding of tumor blood vessel targeted drugs and a combined treatment mechanism is deepened, a more effective anti-tumor method is explored, and the application prospect is wide. And therefore, a greater contribution is made to human health and welfare.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and specifically relates to the application of infliximab in assisting the repair of pancreatic cancer tumor vascular barrier function. Background Art

[0002] Pancreatic cancer is one of the most deadly types of cancer. It is highly malignant, has a poor prognosis, and is insidious. 80% of patients are already in the advanced stage when diagnosed. Among them, pancreatic ductal adenocarcinoma (PDAC) is the most common type of pancreatic cancer. At present, chemotherapy and surgery are still the main treatment options for PDAC. In the commonly used combination therapy regimens based on chemotherapy (gemcitabine combined with albumin paclitaxel, FOLFIRINOX), the response rate of chemotherapy in patients with advanced pancreatic cancer is low, and the single-drug response rate is less than 10%. Therefore, more effective therapeutic targets and more effective combination therapy regimens are urgently needed. At the same time, for surgical treatment, only 15%-20% of patients are eligible for surgery at diagnosis. And about 70% of early pancreatic cancer and locally advanced pancreatic cancer progress to metastatic pancreatic cancer within one year after surgical treatment / or other treatments. This is mainly because the pancreas is close to important vascular systems. Even for patients without clinical evidence of extrapancreatic spread, severe involvement or occlusion of these blood vessels by the primary tumor may make it impossible to undergo surgery. Studies have shown that high compression and poor perfusion caused by tumor vascular damage are the key to initiating tumor cell metastasis. However, there is still a lack of effective vascular targeted drugs and combined treatment options for pancreatic cancer. Therefore, exploring targets for vascular normalization and repair in the pancreatic cancer tumor microenvironment has important scientific research significance for both targeted combined treatment of pancreatic cancer and surgical resection.

[0003] However, the anti-angiogenic treatment regimen currently used may make pancreatic cancer more invasive and more prone to distant metastasis. Pancreatic cancer tissue hypoxia is more severe after anti-angiogenic treatment, and with the inhibition of angiogenesis, tissue hypoxia is further aggravated, resulting in further optimization of cells with anti-hypoxia ability in tumor cells. At the same time, the process of anti-angiogenic treatment will also cause vascular endothelial cell dysplasia and increase vascular permeability, making tumor cells more susceptible to erosion and distant metastasis. In addition, anti-angiogenic treatment will also lead to a decrease in microvascular density (MVD) in the pancreatic cancer microenvironment, which will reduce the concentration of chemotherapeutic drugs in tumor tissues and affect the treatment effect. Since the vascular expansion caused by destromalization and anti-angiogenic treatment ultimately failed to bring benefits, a treatment strategy based on the health of pancreatic cancer microvessels has emerged. At present, clinical studies have confirmed that targeted inhibition of STAT3 combined with gemcitabine can make pancreatic cancer tumor blood vessels healthy, and can increase the local concentration of chemotherapeutic drugs without interfering with pancreatic cancer stroma remodeling, and healthy blood vessels can also reduce tumor cell erosion and distant metastasis.

[0004] In summary, it is expected that the program of targeting tumor vascular normalization can overcome the current dilemma of combined anti-vascular treatment of solid tumors, correct the immunosuppressive tumor microenvironment by promoting the normalization of tumor vascular structure and function, restore drug penetration in the core area of ​​the tumor, and thus fully sensitize the effects of combined treatments such as chemotherapy, tyrosine kinase inhibitors (TKIs) and immune checkpoint inhibitors (ICIs). Therefore, targeted tumor vascular normalization therapy may become a new strategy for reshaping the tumor microenvironment and clinical anti-tumor treatment. Summary of the invention

[0005] In order to overcome the above-mentioned deficiencies of the prior art, the present invention proposes that infliximab exerts its anti-pancreatic cancer effect by assisting in repairing the pancreatic cancer tumor vascular barrier, providing a new option for the treatment of pancreatic cancer and having important practical application value in the fields of medicine and clinical anti-tumor.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is:

[0007] The first aspect of the present invention provides the use of infliximab in the preparation of anti-pancreatic cancer drugs.

[0008] Preferably, the anti-pancreatic cancer drug is a drug for repairing the pancreatic cancer tumor vascular barrier.

[0009] More preferably, the repairing of pancreatic cancer tumor vascular barrier includes repairing vascular endothelial barrier damage in the pancreatic cancer environment, increasing pericyte expression, and reducing the expression of adhesion protein ICAM1 and vascular permeability proteins Src and p-Src.

[0010] Infliximab (also known as infliximab) is a human-mouse chimeric monoclonal antibody against tumor necrosis factor (TNFα). As a TNFα antagonist, it can bind to the soluble form and transmembrane form of the key inflammatory factor TNFα in the human body with high affinity, inhibiting the binding of TNFα to the receptor, thereby making TNFα lose its biological activity to avoid its attack on human tissues. The present invention has found through research that infliximab can promote the chemotherapeutic effect of gemcitabine on pancreatic cancer by assisting in repairing the vascular barrier of pancreatic cancer tumors (including repairing the damage of the vascular endothelial barrier in the pancreatic cancer environment, increasing the expression of pericytes, and reducing the expression of adhesion protein ICAM1 and vascular permeability protein Src, p-Src), providing a new combined treatment plan for the treatment of pancreatic cancer.

[0011] Preferably, the pancreatic cancer is pancreatic ductal adenocarcinoma.

[0012] A second aspect of the present invention provides an anti-pancreatic cancer drug, which includes infliximab.

[0013] The third aspect of the present invention also provides an anti-pancreatic cancer drug, which includes infliximab and gemcitabine (Gem).

[0014] In the early stage of this invention, multiple immunofluorescence detection was performed using clinical pancreatic cancer tissues and paired adjacent tumor tissues. Statistical analysis showed that the expression of co-localized cells of vascular and pericyte markers in tumor tissues was significantly lower than that in adjacent tumor tissues, and patients with low integrity of vascular endothelial cells had a poor prognosis. In vitro angiogenesis and endothelial barrier experiments showed that the barrier of primary umbilical vein endothelial cells treated with pancreatic cancer cell conditioned medium was significantly damaged. Further studies using single-cell dynamic transcription sequencing technology showed that vascular endothelial cells mainly produced inflammatory cell subpopulations when treated with tumor cell conditioned medium. Further database sample verification combined with in vitro cell experiments and angiogenesis factor chip detection of clinical samples determined that the TNF-a pathway was the main pathway for regulatory changes in pancreatic cancer tumor endothelial cells. Therefore, specific TNF-a inhibitors were screened through in vivo and in vitro experiments, and it was finally determined that infliximab can significantly repair vascular endothelial barrier damage in the pancreatic cancer environment, increase pericyte expression, reduce the expression of adhesion protein ICAM1 and vascular permeability proteins Src and p-Src, and can combine with gemcitabine to promote the chemotherapy effect of pancreatic cancer.

[0015] Preferably, in order to enrich the application forms of the drug and make it suitable for different ranges, the drug also includes a pharmaceutically acceptable carrier.

[0016] Furthermore, the carrier is an acceptable functional pharmaceutical excipient in the pharmaceutical field, including surfactants, suspending agents, emulsifiers and some new pharmaceutical polymer materials, such as cyclodextrin, chitosan, polylactic acid (PLA), polyglycolic acid polylactic acid copolymer (PLGA), hyaluronic acid, etc. It may also include excipients such as diluents, adhesives, lubricants, disintegrants, cosolvents, stabilizers, etc.

[0017] Preferably, in order to improve the use form of the drug, the dosage form of the drug includes tablets, capsules, powders, granules and liquids. The drug preparation can be administered orally or parenterally (e.g., intravenously, subcutaneously, intraperitoneally or topically). If some drugs are unstable under gastric conditions, they can be prepared into enteric-coated tablets.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] Pancreatic cancer is highly malignant, with poor prognosis and insidious onset. 80% of patients are in the advanced stage at the time of diagnosis. Currently, surgery is still the only main way to eradicate the disease, but about 75% of patients still metastasize after surgery. At present, pancreatic cancer still lacks effective vascular targeted drugs and combined treatment plans. To this end, the present invention has found through research that infliximab can significantly repair vascular endothelial barrier damage in the pancreatic cancer environment, increase the expression of pericytes, reduce the expression of adhesion protein ICAM1 and vascular permeability protein Src, p-Src, and can promote the chemotherapy effect of pancreatic cancer in combination with gemcitabine. It shows that infliximab and gemcitabine have a synergistic effect in the treatment of pancreatic cancer, can effectively promote the chemotherapy effect of gemcitabine on pancreatic cancer, provide a new way for the treatment of pancreatic cancer, and help to deepen the understanding of tumor vascular targeted drugs and combined treatment mechanisms, explore more effective anti-tumor methods, and thus make greater contributions to human health and well-being. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Representative results of multiple immunofluorescence detection of CD31, CD34, CK19, and GSPG4 expressions in pancreatic cancer tissues and adjacent tissues (A, B).

[0021] Figure 2 Vascular endothelial cell damage in the pancreatic cancer environment is associated with poor prognosis: the expression of CD34+CD31 (A) and CD31+CD34+CSPG4+ (B) in paracancerous tissues were significantly higher than those in paracancerous tissues (p<0.05); patients with low expression of CD31+CD34+ and CD31+CD34+CSPG4+ had a poor overall survival (C, D).

[0022] Figure 3 Single-cell dynamic RNA sequencing revealed the transcriptional profile of HUVEC: A. UMAP clustering of EC, i.e. cluster C1-C8 EC populations; B. C1 cluster accounted for the highest proportion (27.49%), and C8 cluster accounted for the lowest proportion (1.37%); CD. Through cell differentiation analysis, cells in C4 had the highest cell stemness, which may be the starting point of differentiation; E. Through functional analysis, 8 EC subclusters were analyzed, C4 / C8 clustered in the inflammatory subcluster, C3 clustered in the oxidative subcluster related to oxidation / reactive oxygen function, C5 / C6 / C7 clustered in the subcluster related to cell cycle, and C1 / C2 clustered in the subcluster related to normal growth and development; F. Endothelial inflammatory cell subcluster metabolic activity increased; GI. Inflammatory cell subcluster upregulated in apoptosis, EndMT, senescence and aging functions.

[0023] Figure 4Inflammatory cells are the main cell subsets generated by endothelial cells under the influence of pancreatic cancer cells: A. The upregulated genes in clusters 1, 4 and 8 were enriched in the TNF-a pathway; B. The NF-kB and TNF-a pathways were verified by GSEs data analysis; D. The expression of angiogenic factors in pancreatic cancer tissues (n=4) and paracancerous tissues (n=4) was detected using human angiogenic factor protein array, and PCA analysis showed that the samples had typical pancreatic cancer tissues and paracancerous tissues; E. Proteins that increased by 1 times (>1.5 times) or decreased by 1 times (<0.5 times, P<0.05) were defined as differentially expressed between pancreatic cancer tissues and paracancerous tissues, and significantly upregulated angiogenic proteins were enriched in TNF-a.

[0024] Figure 5 The TNF-a inhibitor infliximab can alleviate the barrier damage of endothelial cells in the pancreatic cancer microenvironment: A, B. Representative images of in vitro angiogenesis experiments with different drug treatments. The vascular experiment showed that the tube branches and tube points of endothelial cells in the T group were reduced, while infliximab helped repair the damage and maintain blood vessels; C. The effect of infliximab on the barrier damage of pancreatic cancer endothelial cells; D. Matrigel plug detection of the angiogenic effects of TNF-a inhibitor R7050, infliximab and erlotinib in vivo; E. CD31 immunohistochemistry detection of vascular morphology.

[0025] Figure 6 The TNF-a inhibitor infliximab promotes the chemotherapeutic effect of gemcitabine in pancreatic cancer by repairing the endothelial cell barrier in the pancreatic cancer tumor microenvironment: AB. After collecting mouse tumor cells, the tumor tissue was analyzed for tumor volume. The combination of Gem and infliximab had the best effect in inhibiting tumor growth; CE. Immunoblotting showed that infliximab reduced the levels of proteins related to inflammation, adhesion and vascular permeability, especially il-6 and p-Src; F. Immunohistochemical detection of CD31 and CD34 staining showed that infliximab can preserve vascular structure better than bevacizumab, which can enhance its efficacy with gemcitabine; G. Compared with the monotherapy group, the ratio of extracellular markers in the combination treatment group was significantly increased. DETAILED DESCRIPTION

[0026] The specific embodiments of the present invention are further described below. It should be noted that the description of these embodiments is used to help understand the present invention, but does not constitute a limitation of the present invention. In addition, the technical features involved in each embodiment of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0027] The experimental methods in the following examples are conventional methods unless otherwise specified, and the experimental materials used in the following examples are commercially available unless otherwise specified.

[0028] Example 1: Low levels of vascular integrity in the pancreatic cancer microenvironment are associated with poor patient prognosis

[0029] 1.1 Experimental methods:

[0030] Pancreatic cancer clinical tissue chips purchased from Shanghai Xinchao Company (HPanA170Su04) were used as test samples. Multiple immunofluorescence detection of vascular markers CD31, CD34, CSPG4 (NG2+), tumor marker CK and fibroblast marker a-SMA in pancreatic cancer clinical samples (n=90) and paired adjacent cancer tissues (n=90) was performed. The detection steps are as follows:

[0031] ① Drying: Place the tissue chip in an oven, adjust the temperature to 63 degrees, and dry it for one hour;

[0032] ② Dewaxing: Completed in a fully automatic dyeing machine, the dewaxing time is as follows:

[0033] 2 cylinders of xylene, 15 minutes per cylinder;

[0034] 2 tanks of 100% alcohol, 7 minutes per tank;

[0035] 90% alcohol 1 tank, 5 minutes;

[0036] 80% alcohol 1 tank, 5 minutes;

[0037] 70% alcohol (1 cylinder), 5 minutes.

[0038] ③ Antigen repair: dilute 10× repair solution (servicebio, G1203-250ML) to 1× working solution, adjust the microwave oven to high heat for 3 minutes to boil, then put the slide in, adjust the microwave oven power to low heat and continue repair for 15-20 minutes (make sure the tissue is immersed in the liquid during the whole process), and cool naturally at room temperature;

[0039] ④ Blocking: Take out the slides, wash with TBST and place in a wet box, add blocking buffer, and incubate with primary antibodies [a-SMA (dilution 1:2000), CSPG4 (dilution 1:500), CK (dilution 1:10), CD34 (dilution 1:500), CD31 (dilution 1:500)] for 10 min;

[0040] ⑤ Secondary antibody incubation: Take out the slide, place it in a wet box, add secondary antibody (servicebio, G1214-100ML and G1213-100ML), incubate at room temperature for 10 minutes, and wash the slide with TBST;

[0041] ⑥ Opal dye color development: Take out the slide, place it in a wet box, add Opal dye diluent (VECTORLABORATORIESDAB SK-4100) (1:100 dilution), incubate at room temperature for 10 min, wash the slide with TBST, take out the slide, and add fluorescent antifade mounting medium (Thermo Fisher Scientific) to mount the slide;

[0042] ⑦Use TissueFAXS system (TissueGnostics, TissueFAXS Spectra S) to perform panoramic multispectral scanning of slides;

[0043] ⑧Data were imported into StrataQuest analysis software (TissueGnostics StrataQuest7.0.1.165);

[0044] ⑨Use the spectral library built for monochrome samples (Shanghai Outdo Biotech, spectraldb.db) to perform spectral decomposition on the multi-spectral scanned samples to obtain clean single-channel fluorescence signals. Analyze the expression of markers according to the intensity of fluorescence signals, and statistically analyze the proportion of intact blood vessels in pancreatic cancer and adjacent tissues. Further statistical analysis was performed in combination with clinical pathological information (pancreatic cancer samples of HPanA170Su04 chip are equipped with clinical information) to explore the correlation between the expression of intact blood vessels and TMN stage, tumor markers CA199, CA125, and patient prognosis.

[0045] 1.2 Experimental results:

[0046] Tumor endothelial cells, as an important component of the tumor microenvironment, affect the therapeutic effect of pancreatic cancer patients. In order to study the exact status of endothelial cells in pancreatic cancer tissue, the expression of tumor vascular endothelial cell markers CD31 and CD34, perivascular cell marker CSPG4 (NG2+), tumor epithelial cell marker CK+ and fibroblast marker a-SMA were detected and analyzed by multicolor immunofluorescence. The expression level of each marker was determined by the spectral characteristics of each fluorophore, and cells co-expressing CD31, CD34 and CSPG4 (NG2+) were used to represent intact blood vessels, and vascular endothelial cells lacking CSPG4 (NG2+) represented incomplete cell levels. The results showed that the density of CD31+CD34+ co-localized cells and CD31+CD34+CSPG4+ co-localized cells in pancreatic cancer tissue was significantly lower than that in adjacent tissues ( Figure 1 A, B; Figure 2A, B). Combined with clinical data (clinical information comes from the chip attached data), pancreatic cancer patients with high expression of incomplete blood vessels CD31+CD34+CSPG4- in the tumor microenvironment had a poor TNM stage (Table 1). Further cox analysis showed that CD31+CD34+CSPG4- cells were significantly positively correlated with TNM, CA125, and CA199 (Table 1). In addition, patients with low expression of CD31+CD34+ and CD31+CD34+CSPG4+ had a worse overall survival rate than patients with high expression of vascular integrity (P<0.05) ( Figure 2 C, D). Therefore, in pancreatic cancer, reduced levels of intact blood vessels are associated with poor patient prognosis.

[0047] Table 1 Correlation between the expression of CD31+CD34+CSPG4- in incomplete blood vessels and pathological information

[0048]

[0049]

[0050] Example 2: Single-cell dynamic transcriptome sequencing reveals changes in vascular endothelial cells in the microenvironment of pancreatic cancer tumor cells

[0051] 1.1 Experimental methods:

[0052] (1) Cell culture and conditioned medium preparation

[0053] Primary human umbilical vein endothelial cells (HUVEC) were purchased from CELL RESEARCH (PRI-H-00023) and cultured in 5% CO2 at 37°C in culture medium (CELL RESEARCH, ZQ-1304). All tests were performed using HUVEC cells after 2-5 passages. The pancreatic cancer cell line MIA PaCa-2 was cultured in RPMI 1640 medium containing 10% fetal bovine serum, 2mM glutamine, 100U / mL penicillin and 100μg / mL streptomycin. The control cell line hTERT-HPNE was cultured in hTERT-HPNE-specific culture medium (iCell, Cat: h102-001b).

[0054] Preparation of conditioned medium (CM): The designated cells [MIA PaCa-2 (CELL RESEARCH) and HPNE (CELL RESEARCH)] were cultured to 30%-40%. Then the growth medium was replaced with the corresponding serum-free medium and cultured for 24 hours. The supernatant was harvested when the cells reached 60%-80%. Then, according to the experimental needs, the collected supernatant was added (100uL for 96-well plates, 1.5mL for 6-well plates, and 7mL for 10cm culture dishes) to the HUVEC culture dishes that had been removed from the original culture medium, and placed in a CO2 incubator for 4 hours. All conditioned medium experiments were repeated three times.

[0055] (2) Effects of pancreatic cancer cell culture medium environment on angiogenesis of primary umbilical vein endothelial cells

[0056] In vitro angiogenesis experiments were performed using primary umbilical vein endothelial cells purchased from CELL RESEARCH (PRI-H-00023): Matrigel (Corning, 365142) was coated on a 96-well plate, and the volume of Matrigel and culture medium was 1:1 to coat the 96-well plate (Corning). The HUVECs were resuspended in the culture supernatant of pancreatic cancer MiaPaCa cells and pancreatic normal control HPNE cells, and then 8×10 4 The cells were seeded in 96-well plates covered with Matrigel at a concentration of 100 μg / ml. After incubation at 37°C and 5% CO2 for 6 h, the cells were stained with calcein AM (Dojindo Molecular Technologies, Tokyo, Japan) and observed under a fluorescence microscope. The capillary network was imaged and the number of tubes and branch points were calculated using Image J. The duct density was calculated by averaging the number of tubes in 5 random fields at 10x magnification per well.

[0057] (3) Dynamic single-cell transcriptome analysis to detect changes in the transcriptome of primary umbilical vein endothelial cells in the microenvironment of pancreatic cancer cell culture medium

[0058] Single-cell dynamic transcriptome sequencing technology can complete RNA metabolic labeling efficiently and without bias. While obtaining sample transcriptome information, it can also understand the transcriptional activity information of various types of cells at the single-cell level. The detection results are based on rich and multidimensional single-cell transcriptome information. The dimension of time is added, thereby realizing high-throughput RNA transcription dynamic monitoring, and more accurately detecting the dynamic transcription changes that occur in vascular endothelial cells during tumor cell conditioned medium treatment.

[0059] Primary umbilical vein endothelial cells were cultured in a 96-well plate with matrix gel and treated with pancreatic cancer tumor cell culture medium for 6 hours. The samples were then sent for dynamic single-cell detection (Nanjing, Syngegen Single-Cell Sequencing Company) and bioinformatics analysis.

[0060] (4) Vascular regulatory factor chip detection of pancreatic cancer clinical samples verified that the angiogenesis-related proteins in pancreatic cancer tissues were mainly enriched in the TNF-a signaling pathway

[0061] A vascular regulatory factor chip (RayBiotech, Inc. cat. no. QAH-ANG-1000) was used to perform chip detection using pancreatic cancer clinical tumor samples (n=4) and paired adjacent tumor samples (n=4) (samples purchased from Shanghai Xinchao Company, sample numbers: D20A0280, D20A0293, D20A0337, D20A0770), and then the detection data were subjected to bioinformatics analysis. The clinical sample information is shown in Table 2.

[0062] Table 2 Pathological information of pancreatic cancer clinical tumor samples

[0063]

[0064] 1.2 Experimental results:

[0065] Further, single-cell dynamic transcriptome sequencing was used to detect the regulatory changes of the transcriptome of primary umbilical cord venous blood endothelial cells treated with pancreatic cancer tumor cell culture medium within 6 hours. According to literature reports and our previous experiments (DeCicco-Skinner KL, Henry GH, Cataisson C, et al. Endothelial cell tubeformation assay for the in vitro study of angiogenesis. J Vis Exp. 2014; Wüstehube J, Bartol A, Liebler SS, et al. Cerebral cavernous malformation protein CCM1 inhibits sprouting angiogenesis by activating DELTA-NOTCH signaling. Proc Natl Acad Sci US A. 2010; 107(28): 12640-5), vascular endothelial cells are in the best state of forming small tubes in vitro for 5-6 hours. During this period, single-cell dynamic transcriptome sequencing was used to mark newly synthesized mRNA to detect the dynamic changes of transcripts in vascular endothelial cells. According to the expression of significantly differentially expressed genes, the detected endothelial cells were divided into 8 subgroups: C1-C8 ( Figure 3 A), among which C1 subgroup accounted for the highest proportion (27.49%), and C8 accounted for the lowest proportion (1.37%) ( Figure 3 B). Compared with the control group H (vascular endothelial cells treated with HPNE medium of normal pancreatic cells), the proportion of cells in the C4, C6, C7 and C8 subpopulations in the T group (vascular endothelial cells treated with pancreatic tumor cells) increased. The results of SLICE and cytotrace analysis showed that the C4 subpopulation had the highest stemness and might be the starting point of differentiation ( Figure 3 C, D). Therefore, the vascular endothelial cell subpopulations in the pancreatic cancer tumor microenvironment have changed. Next, we will focus on the C4 and C8 subpopulations. According to the functional analysis, the subpopulations were merged according to the results of the functional clustering. From the UMAP diagram, the different functional clusters were clearly distinguished. These 8 cell subpopulations can be divided into 4 functional groups, namely, inflammatory cell subpopulation C4 / C8: inflammation-related subpopulations, oxidative subpopulation C3: may be related to oxidation / reactive oxygen function, cell cycle-related C5 / C6 / C7: cell cycle-related cell subpopulations and subpopulation EC C1 / C2: normal growth and development-related functions ( Figure 3 E). Metabolic analysis showed that the pentose phosphate pathway of inflammatory cells was upregulated in the T group ( Figure 3 F), functional analysis found that the expression of apoptosis, EndMT, senescence, and senescence in the inflammatory cell subgroups was significantly upregulated ( Figure 3 GI).

[0066] Example 3: Pancreatic cancer cells affect vascular endothelial cells and upregulate inflammatory pathways represented by TNF-a

[0067] 1.1. Experimental method: The method in this part is the same as that in Example 2.

[0068] 1.2 Experimental results:

[0069] Through differential gene function enrichment analysis, it was found that the up-regulated genes of the three subgroups C1, C4, and C8 were significantly enriched in the TNF-a pathway and NF-kB pathway. Figure 4 The representative cluster4 enrichment analysis results are shown in ( Figure 4 A). GSEs data were used to perform a significant analysis of NF-kB and TNF-a in pancreatic cancer endothelial cells. The results showed that compared with the H group, the expression of TNF-a in the vascular endothelial cells of the T group was significantly different, while NF-kB was not significant ( Figure 4 B, C). Therefore, the subsequent analysis focused on the TNF-a signaling pathway. The expression of angiogenic factors in pancreatic cancer tissues (n=4) and adjacent tissues (n=4) was further detected using a human angiogenic factor protein array. Figure 4D), the results showed that the human angiogenesis antibody array can simultaneously detect the expression of 60 angiogenesis-related proteins. The angiogenesis proteins that were significantly upregulated in pancreatic cancer tissues were enriched in TNF-a, which was consistent with the results of single-cell transcriptional analysis ( Figure 4 E). This result also verifies that TNF-a is a valuable and noteworthy pathway in pancreatic cancer. Therefore, changes in inflammatory pathways represented by TNF-a mainly occur in vascular endothelial cells in the microenvironment of pancreatic cancer tumor cells, and related adhesion proteins and cytokines are significantly upregulated, indicating that vascular barrier function damage in the tumor microenvironment may be related to inflammation.

[0070] Example 4: TNF-a inhibitor infliximab can partially alleviate the barrier damage of endothelial cells in pancreatic cancer microenvironment

[0071] 1.1 Experimental methods:

[0072] (1) Angiogenesis experiment: The method is the same as that in Example 2.

[0073] (2) Western blotting experiment:

[0074] The total protein of HUVEC cells after treatment was collected, and after quantitative determination of protein content, 30 μg of protein was subjected to SDS-PAGE, then transferred to a PVDF membrane, blocked with 5% skim milk in TBST (20 mM Tris-HCl, 500 mM NaCl and 0.1% Tween-20) at room temperature for 1 h, and incubated with specific primary and secondary antibodies (the antibodies used are shown in Table 3). Finally, the protein-antibody complex was detected using the ECL advanced western blot detection kit, and the band intensity was quantitatively detected using Quantity One software (Bio-Rad).

[0075] (3) Vascular endothelial barrier detection experiment:

[0076] Primary umbilical vein endothelial cells were treated with conditioned medium from pancreatic cancer cells MIA and pancreatic control cells HPNE, and a dynamic cell monitoring system (ECIS 3247, Applied Biophysics, Troy, NY, USA) was used to dynamically monitor the significant (p<0.05) barrier damage of vascular endothelial cells after treatment with tumor cell culture medium.

[0077] (4) Using in vivo matrigel experiments in mice to verify the effects of TNF-a specific inhibitors on angiogenesis:

[0078] In vivo matrigel experiments were performed on male C57BL / 6 mice (4 weeks old). FGF reagent (FGF is a fibroblast factor, used as a positive group drug for matrigel angiogenesis in mice to promote angiogenesis) at a concentration of 50 ng / mL (MCE, HY-P73052) and F12 culture medium (Gibco, 12634010) were mixed to form a 900uL mixture and injected subcutaneously into mice as a positive control for the matrigel experiment. Matrigel mixed with pancreatic cancer tumor cell MIA culture medium was injected subcutaneously into mice. The mixed volume ratio of culture medium to matrigel was 2:7, and the injection volume was 900uL. After the matrigel mixture was implanted subcutaneously, the following 4 groups were treated: one group was a negative control group NT (Non treatment) without any drug treatment, and the other three groups were drug treatment (specific inhibitor) groups: Erlotinib 20mg / kg, R7050 6mg / kg, Infliximab 10 mg / kg, these three specific inhibitors were administered by intraperitoneal injection for one week, and then the matrigel samples were collected for HE and vascular marker staining CD31, CD34, and the vascular parameters were statistically analyzed to verify the inhibitory effect of infliximab on pancreatic cancer vascular cells.

[0079] (5) Immunohistochemistry experiment:

[0080] 1.1 Experimental methods:

[0081] ① Slice: Slice thickness is 4 microns, spread at 42℃ water temperature, and bake in 60℃ oven for 30 minutes. Note: For tissues such as bones or skin that are easy to peel off, the baking time can be appropriately extended.

[0082] ② Dewaxing to water: xylene I for 5 minutes, xylene II for 5 minutes, xylene III for 5 minutes, anhydrous ethanol for 1 minute, 95% ethanol for 1 minute, 75% ethanol for 1 minute, and distilled water for 5 minutes. Note: The specific time needs to be flexibly adjusted according to the room temperature and the freshness of the xylene. The higher the room temperature and the fresher the xylene, the shorter the corresponding time, which is based on the absence of paraffin residue on the tissue.

[0083] ③Antigen repair: EDTA microwave heat repair for 5-8 minutes, cool to room temperature. Note: For most proteins, EDTA repair solution can generally obtain satisfactory results. Trypsin repair solution or traditional citrate repair solution can also be used. If the sample is fixed for too long or the positive expression is not strong, the number and intensity of repair can be appropriately increased.

[0084] ④ Inactivation: Draw a circle with an immunohistochemistry pen to prevent the reagent from flowing out, add endogenous peroxidase blocking solution, incubate at room temperature for 10 minutes, and wash 3 times with PBS buffer, each time for 5 minutes. Note: For tissues with high endogenous peroxidase content, the incubation time can be appropriately extended or the hydrogen peroxide concentration can be increased.

[0085] ⑤ Blocking: Add blocking serum and incubate at 37℃ for 30 minutes, shake off excess serum and do not wash.

[0086] ⑥ Primary antibody incubation: add primary antibody (the antibodies used are shown in Table 3), incubate in a 37℃ wet box for 2 hours, and wash with PBS buffer 3 times, 5 minutes each time. Note: For the first experiment, it is recommended to make a concentration gradient of the primary antibody to determine the optimal dilution ratio of the primary antibody.

[0087] ⑦ Secondary antibody incubation: add HRP-labeled goat anti-rabbit, incubate at 37℃ for 30 minutes, wash with PBS buffer 3 times, 5 minutes each time. Note: The type of secondary antibody is determined according to the species of the primary antibody. For example, if the primary antibody is rabbit antibody, then the secondary antibody should be HRP-labeled goat anti-rabbit IgG.

[0088] ⑧ Color development: Use DAB color developing solution, first prepare DAB, mix 1mL B solution + 1 drop of A solution, then add DAB color developing solution, observe under the microscope until the positive result is obviously enhanced and the background is clear, then stop the color development; if the color is light, repeat the previous step to enhance the color.

[0089] ⑨Restaining: Add Mayer'S hematoxylin for 30 seconds, wash with distilled water, soak in bluing solution for 1 minute, and wash with water.

[0090] ⑩ Dehydration and transparent sealing: 75%-95%-100% alcohol gradient dehydration, 1 minute per cylinder, three cylinders of xylene transparent, 2 minutes per cylinder, and neutral gum sealing.

[0091] The expression of related indicators was based on the immunoreactivity score (IRS): the degree of staining was scored according to the staining characteristics of the target cells: no staining 0 points; light yellow 1 point; brown 2 points; dark brown 3 points. The positive rate was scored as follows: 0-5%, 0 points; 6-25%, 1 point; 26-50%, 2 points; 51-75%, 3 points; >75%, 4 points. The staining degree (0-3 points) and the positive rate (0-4 points) were multiplied to obtain the IRS.

[0092] 1.2 Experimental results:

[0093] In view of the up-regulated expression of TNF-a in vascular endothelial cells in the microenvironment of pancreatic cancer tumor cells, anti-inflammatory drugs are used to restore the barrier function of endothelial cells. Candidate TNF-a inhibitory drugs include infliximad, C87, R7050, Ben, and UCB. The most effective drugs are tested by western blot, angiogenesis assay, and cell barrier assay. Angiogenesis assay showed that the treatment of endothelial cells with pancreatic cancer tumor culture medium destroyed the barrier of endothelial cells, and infliximab significantly alleviated the damage of tumor cells to vascular endothelial angiogenesis and partially maintained the vascular morphology ( Figure 5A, B). Infliximab can also significantly restore the barrier damage of vascular endothelial cells in the microenvironment of pancreatic cancer tumor cells ( Figure 5 C). In vivo, Matrigel plug experiments were performed to further investigate the effects of TNF-a inhibitors R7050, infliximab, and erlotinib on angiogenesis. Analysis of vascular morphology and vascular density showed that the treatment group from pancreatic cell culture medium significantly increased vascular density but the vascular morphology was damaged and incomplete, while the R7050, infliximab, and erlotinib groups significantly inhibited vascular density ( Figure 5 D). The vascular morphology was detected by IHC of CD31, and the results showed that the infliximab group had a more complete microvascular morphology ( Figure 5 E). Therefore, the TNF-a inhibitor infliximab can alleviate the barrier damage of pancreatic cancer, maintain vascular morphology, inhibit the expression of adhesion proteins, and reduce excessive blood vessel growth.

[0094] Table 3 Antibodies and reagents used for western blotting and immunohistochemistry

[0095]

[0096] Example 5: TNF-a inhibitor infliximab repairs the endothelial cell barrier in the pancreatic cancer tumor microenvironment and promotes the chemotherapeutic effect of gemcitabine in pancreatic cancer

[0097] 1.1. The nude mouse subcutaneous tumor model was used to verify the inhibitory effect of the combination of infliximab inhibitor and gemcitabine on mouse tumor growth. The specific experimental method is as follows:

[0098] (1) Subcutaneous tumor formation in mice: 30 female immunodeficient mice (Nanjing Model Animal Company, Balb / c nude mice) were selected, aged 4 weeks and weighing 14-16 g. Human pancreatic cancer MIA PaCa-2 cells in the logarithmic growth phase were transferred into 75 mL culture flasks for expansion culture. After the cells were fully grown, the supernatant was discarded, washed with PBS, digested with trypsin, neutralized with complete medium, centrifuged at 800 rpm for 5 min, the supernatant was discarded, and the cells were resuspended in a mixture of PBS and Marigel at a volume ratio of 3:1. The cell density was adjusted to 1×10 by cell counting. 6 Then, 100uL of cell suspension was drawn with an insulin needle and injected subcutaneously on the right back of each nude mouse. Each nude mouse was marked and the nude mice were kept under normal conditions for 1 week.

[0099] (2) Animal grouping and dosing regimen: After one week of normal feeding, 30 Balb / c nude mice were randomly divided into control group, A group, A+B group, A+C group, B+C group, and A+B+C group according to tumor size, a total of 6 groups, of which ABC were Gemcitabine (Gem), inflixmab (bevacizumab), and Bevacizumab (infliximab) (Gemcitabine, purchased from MCE, catalog number: HY-17026; Infliximab, purchased from MCE, catalog number: HY-P9970D; Bevacizumab, purchased from MCE, catalog number: HY-P9906), 5 mice in each group. The control group was given 0.1 mL of normal saline intraperitoneally once a day for three consecutive weeks. Group A, Group A+B, Group A+C, Group B+C, and Group A+B+C were given intraperitoneal injections according to the drug concentrations and dosing frequencies of drug A 50 mg / kg Gemcitabine 0.1 mL once a week, drug B 10 mg / kg Infliximab 0.1 mL once a day, and drug C 5 mg / kg Bevacizumab 0.1 mL once a week for three consecutive weeks.

[0100] (3) Tumor measurement and recording: The general condition of mice in each group was observed for 3 weeks after administration, and the volume of tumors and the weight of mice were measured and recorded every two days from day 0 to day 20. After collecting tumor cells from mice, the tumor tissues were stained with HE and vascular endothelial markers.

[0101] (4) Western blotting experiment (specifically the same as the experimental method in Example 4) to detect the effect of TNF-a inhibitors on angiogenesis and the expression of vascular adhesion / permeability related proteins.

[0102] (5) Immunohistochemical detection: (Specifically the same as the experimental method in Example 4).

[0103] (6) Immunofluorescence detection: (Specifically the same as the experimental method in Example 1).

[0104] 1.2 Experimental results:

[0105] The combination therapy that significantly inhibited tumors was tested in a mouse subcutaneous human pancreatic cancer tumor model. Statistical analysis of mouse tumor volume revealed that infliximab could promote the chemotherapeutic effect of gemcitabine ( Figure 6 A, B). The expression of inflammation-related proteins, adhesion protein markers and vascular permeability markers was detected by immunoblotting, and it was found that infliximab could inhibit the expression of inflammatory factor IL10, adhesion protein ICAM1, FAK, p-FAK and vascular permeability markers Src, p-Src ( Figure 6 C, D, E). Immunohistochemical detection of vascular markers CD31 and CD34 revealed that infliximab was superior to Bevacizumab in maintaining vascular morphology ( Figure 6 F). Further immunofluorescence detection of pericyte expression was used to evaluate vascular integrity, and it was found that the pericyte expression in the Infliximab combination group was significantly higher than that in the single-drug group ( Figure 6 G). The above results indicate that infliximab has a repairing effect on the tumor vascular barrier function of pancreatic cancer and can increase the chemotherapeutic effect of gemcitabine on pancreatic cancer.

[0106] In summary, infliximab can promote the chemotherapeutic effect of gemcitabine on pancreatic cancer by repairing the vascular barrier of pancreatic cancer tumors (including repairing vascular endothelial barrier damage in the pancreatic cancer environment, increasing pericyte expression, and reducing the expression of adhesion protein ICAM1 and vascular permeability proteins Src and p-Src), providing a new approach for the treatment of pancreatic cancer and has broad application prospects.

[0107] The embodiments of the present invention are described in detail above, but the present invention is not limited to the described embodiments. For those skilled in the art, various changes, modifications, substitutions and variations of these embodiments are made without departing from the principles and spirit of the present invention, and still fall within the protection scope of the present invention.

Claims

1. Application of infliximab in the preparation of anti-pancreatic cancer drugs.

2. The use according to claim 1, characterized in that: The pancreatic cancer is pancreatic ductal adenocarcinoma.

3. The use according to claim 1, characterized in that: The anti-pancreatic cancer drug is a drug for repairing the vascular barrier of pancreatic cancer tumors.

4. The use according to claim 3, characterized in that: The repair of pancreatic cancer tumor vascular barrier includes repairing vascular endothelial barrier damage in the pancreatic cancer environment, increasing the expression of pericytes, and reducing the expression of adhesion protein ICAM1 and vascular permeability proteins Src and p-Src.

5. An anti-pancreatic cancer drug, characterized in that: The drugs include infliximab.

6. An anti-pancreatic cancer drug, characterized in that: The drugs include infliximab and gemcitabine.

7. An anti-pancreatic cancer drug according to claim 5 or 6, characterized in that: The medicament further includes a pharmaceutically acceptable carrier.

8. An anti-pancreatic cancer drug according to claim 5 or 6, characterized in that: The dosage forms of the drug include tablets, capsules, powders, granules and liquids.

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