CD109 < + > exosome targeting pancreatic cancer nerve infiltration and preparation method and application of CD109 < + > exosome

Abstract

The invention belongs to the technical field of in-vitro diagnosis or biological medicine, and particularly discloses a CD109 + exosome targeting pancreatic cancer nerve infiltration and a preparation method and application thereof.The research finds that the CD109 + exosome derived from KRASG12D mutation pancreatic cancer has the effect of targeting neurons, the CD109 + exosome is prepared in vitro in an engineering mode, and the effect of targeting pancreatic cancer nerve infiltration is achieved. The KRASG12D mutant pancreatic cancer marker is proved to have high nerve targeting in in-vivo and in-vitro models, provides a natural and modifiable drug carrying tool for treatment of pancreatic cancer nerve infiltration, can be used as a target and a prognosis marker of KRASG12D mutant pancreatic cancer, and has a wide application prospect.

Description

Technical Field

[0001] The present invention relates to the field of in vitro diagnosis or biomedical technology, and in particular to a CD109 targeting neural infiltration of pancreatic cancer. + Exosomes and preparation methods and applications thereof. Background Art

[0002] Pancreatic cancer is one of the most common digestive system malignancies in the world, with an incidence rate ranking 14th among malignant tumors and a mortality rate ranking 7th among malignant tumors. In 2020, the number of new cases and deaths of pancreatic cancer worldwide was approximately 500,000 and 470,000, respectively, and its incidence rate has been increasing year by year, seriously endangering national health and economic development. There is extensive innervation around the pancreatic tissue. Once the tumor is attached to the surface of the nerve, it is very easy to metastasize along the nerve. Neural infiltration refers to the invasion of the tumor into the epineurium, endoneurium and perineurium, or the tumor surrounds the nerve circumference by more than 33%. Studies have shown that more than 80% of pancreatic cancer patients still have extensive neural infiltration in their tumor groups, which is considered to be a precursor to pancreatic cancer metastasis and is related to the tumor recurrence rate and poor prognosis after pancreatic cancer surgery. With the deepening of related research, the molecular mechanism of pancreatic cancer neural infiltration has been studied to a certain extent, but the effect on improving the prognosis of patients with pancreatic cancer neural infiltration is not obvious. The main reason is the lack of effective drug treatment targets and predictive biomarkers. Therefore, exploring effective biomarkers and new drug treatment targets for pancreatic cancer neural infiltration is a difficult problem that needs to be solved urgently in the current clinical and basic research of pancreatic cancer.

[0003] Exosomes are extracellular vesicles with a diameter of 30-150nm that are secreted by various cells and widely exist in human body fluids. They can act as carriers of bioactive molecules such as DNA, RNA, proteins and liposomes, and participate in mediating the exchange of information between cells. Due to their good stability and targeting, the role of exosomes in tumor metastasis has attracted the attention of many scholars. Studies have found that on the one hand, tumor cells can secrete high-abundance exosomes into the tumor microenvironment, inducing the formation of a microenvironment that is conducive to tumor cell metastasis; on the other hand, stromal cells in the tumor microenvironment can also deliver biological signals through exosomes to regulate the biological characteristics of tumor cells, ultimately promoting the occurrence of tumor metastasis. However, in current research, the biological role and molecular mechanism of tumor cell-derived exosomes in the neural infiltration of pancreatic cancer are still unclear.

[0004] Engineered exosomes mainly refer to cells that have been processed by bioengineering technology, so that this group of cells can produce natural exosomes carrying specific membrane surface proteins, which have the characteristics of high tissue targeting and biocompatibility. Studies in recent years have shown that small molecules or therapeutic drugs are artificially packaged into exosomes, and can be recognized by antigenic peptides on the surface of exosomes and transmitted to specific types of cells or tissues, thereby achieving the purpose of treatment. Therefore, new targets and new predictive indicators for targeted treatment of pancreatic cancer neural infiltration based on engineered exosomes have broad clinical application prospects and provide new ideas for improving patient prognosis. Summary of the invention

[0005] The present invention provides a CD109 targeting neural infiltration of pancreatic cancer + Exosomes and their preparation method and application, the present invention studies and discovers KRAS G12D Mutated pancreatic cancer-derived CD109 + Exosomes have been shown to have high neural targeting in in vitro and in vivo models, providing a natural and transformable drug delivery tool for the treatment of pancreatic cancer neural invasion and can serve as a KRAS G12D Mutation targets and prognostic markers for pancreatic cancer have broad application prospects.

[0006] The present invention solves the technical problem by adopting the following technical solutions: An exosome targeting pancreatic cancer neural infiltration, wherein the exosome is CD109 + exosomes.

[0007] As a preferred embodiment of the present invention, the CD109 + Exosomes are derived from KRAS G12D mutations in sera from pancreatic cancer patients.

[0008] As a preferred embodiment of the present invention, the CD109 + The exosome preparation method is as follows: CD109 and LAMP2B CDS regions were digested with BamHI and EcoRI and purified on gel, CD109-LAMP2B CDS region sequence was connected to eukaryotic expression vector pCDNA3.1(+)-GNSTM-HA using T4 DNA ligase. Then, the constructed plasmid was transfected into 293t cells, cell supernatant was collected 72h after transfection, and exosomes were collected by ultracentrifugation.

[0009] The present invention also provides a drug-loaded exosome targeting neural infiltration of pancreatic cancer, characterized in that the drug-loaded exosome is the exosome targeting neural infiltration of pancreatic cancer according to any one of claims 1 to 4.

[0010] As a preferred embodiment of the present invention, the method for preparing the drug-loaded exosomes is: Cy3-labeled si-circPNIT is packaged into exosomes by electroporation.

[0011] The present invention also provides an application of exosomes targeting neural infiltration of pancreatic cancer in the preparation of a drug for treating neural infiltration of pancreatic cancer.

[0012] As a preferred embodiment of the present invention, the drug further comprises a pharmaceutically acceptable carrier, excipient or diluent.

[0013] Beneficial effects of the present invention: The present invention has found that KRAS G12D Mutated pancreatic cancer-derived CD109 + Exosomes, which have the function of targeting neurons, were engineered to produce CD109 in vitro + Exosomes have been shown to have high neural targeting in in vitro and in vivo models, providing a natural and transformable drug delivery tool for the treatment of pancreatic cancer neural invasion and can serve as a KRAS G12D Targets for mutated pancreatic cancer have broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 CD109 extracted for immunoelectron microscopy + exosomes.

[0015] Figure 2 Western blot was used to identify the highly expressed exosome markers in the extracted vesicles.

[0016] Figure 3 The extracted exosome membranes for nanoflow cytometry analysis showed CD109 expression.

[0017] Figure 4 In vivo animal experiments confirmed the use of engineered CD109 + Exosomes carrying small interfering RNA can be effectively delivered to neurons around tumors and inhibit the occurrence of neural infiltration of pancreatic cancer. DETAILED DESCRIPTION

[0018] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be described clearly and completely below. Obviously, the described embodiments are 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.

[0019] In the present invention, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0020] In the present invention, when it comes to numerical ranges, unless otherwise specified, the above numerical ranges are deemed to be continuous and include the minimum and maximum values ​​of the range, as well as each value between such minimum and maximum values. Further, when a range refers to an integer, each integer between the minimum and maximum values ​​of the range is included. In addition, when multiple ranges are provided to describe features or characteristics, the ranges can be merged. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges included therein.

[0021] In the present invention, there is no particular limitation on the specific dispersion and stirring treatment methods.

[0022] The reagents or instruments used in the present invention without indicating the manufacturer are all conventional products that can be obtained through commercial purchase.

[0023] Example 1 CD109 + Exosome sequencing sources, expanded sample validation, functions, and neural targeting 1. Verification of CD109 + Exosomes in KRAS G12D High expression in serum exosomes of pancreatic cancer patients with mutations: Peripheral blood was collected from 50 patients who underwent surgery and were diagnosed with pancreatic ductal adenocarcinoma by pathological examination in Guangdong Provincial People's Hospital from 2020 to 2021, as well as peripheral blood from normal volunteers. After peripheral blood was obtained, serum was separated at 2000 rpm for 20 min. The supernatant was transferred to a new centrifuge tube by ultracentrifugation (2000 rpm X 20 min centrifugation, then 10000 rpm X 40 min centrifugation, then 120000 rpm X 70 min centrifugation, and finally the supernatant was discarded. The exosomes at the bottom of the tube were resuspended with PBS to extract and purify the exosomes in the serum, such as Figure 2As shown, the expression of exosomal marker proteins such as CD9 and TSG101 in the extracted exosomes was detected by Western blot. Specifically, the sample was added to the gel, and when the protein sample reached the separation gel, the voltage was adjusted from 65V to 150V. After separating the target protein, the gel was removed, the protein was bound to polyvinylidene fluoride (PVDF), and transferred to the membrane at a constant voltage of 25V for 15min. After blocking the PVDF membrane with 10% BSA for 1h, it was incubated with primary antibodies such as CD9 and TSG101 at 4°C overnight. The next day, after washing 3 times with TBST, it was incubated with HRP-labeled secondary antibodies at room temperature for 1h. The bands were exposed using an enhanced chemiluminescence (ECL) kit to detect the expression of exosomal marker proteins such as CD9 and TSG101 in the extracted vesicles, and the enrichment of CD109 on the surface of the exosome membrane was detected by immunoelectron microscopy and nanoflow cytometry, thereby confirming that the extracted vesicles are CD109. + Exosomes, such as Figure 1 , Figure 3 As described above, the expression of CD109 on the surface of exosomes was detected by immunoelectron microscopy and flow cytometry. It was found that compared with exosomes from patients with other KRAS subtypes, KRAS G12D The expression of CD109 on the exosome membrane of patients with mutated pancreatic cancer serum was significantly increased. The specific experimental steps are as follows: (1) Immunoelectron microscopy: 10% FBS was added to the exosomes and incubated at room temperature for 30 minutes for blocking. After washing with PBS, anti-CD109 primary antibody was added and incubated at 4°C overnight. On the next day, 12nm gold particles coated with anti-mouse IgG were added to the exosomes and incubated at room temperature for 1 hour, followed by washing with PBS. Subsequently, 1% neutral glutaraldehyde was added to the exosome mixture, incubated at room temperature for 5 minutes, and then washed with ultrapure water. The exosomes were embedded in a mixture of 4% uranyl acetate and 2% methylcellulose at a ratio of 1:9 and observed using a transmission electron microscope at a voltage of 80kV. (2) Nanoflow cytometry: The collected exosomes were incubated with anti-CD109 primary antibody at room temperature for 1 hour, then washed with PBS to obtain the exosomes, and incubated with anti-mouse fluorescent secondary antibody at room temperature for 1 hour. Subsequently, 100 nm, 200 nm, and 500 nm yellow-green mixed magnetic beads (Polysciences) were used to gate the exosomes, and unstained exosomes, identically stained exosomes, and antibodies alone were used as negative controls. Nanoflow cytometry analysis was performed using a CytoFLEXS flow cytometer, and flow cytometric data were analyzed using FlowJo v10 software.

[0024] 2. By constructing engineered CD109 + Exosomes target neurons to inhibit neural invasion of pancreatic cancer The cell supernatants of the engineered 293T cells were collected separately and transferred to new centrifuge tubes after ultracentrifugation at 2000rpm for 20min, then transferred to new centrifuge tubes after centrifugation at 10000rpm for 40min, then centrifuged at 120000rpm for 70min, and finally the supernatant was discarded. The exosomes at the bottom of the tube were resuspended with PBS to extract and purify the exosomes in the serum. The Cy3-labeled si-circPNIT was packaged into the exosomes by electroporation. An exosome-induced orthotopic xenograft tumor model was constructed. Specifically, 1 xenograft was inoculated into the pancreatic tissue of nude mice. 10 6 One week after the inoculation of 100 / PANC-1 cells, exosomes were injected into pancreatic tumors orthotopically and then injected into tumors when the tumor volume reached 2500 mm 3 Pancreatic cancer tumor tissues were collected for multicolor immunofluorescence analysis. Figure 4 As shown, multicolor immunofluorescence showed that compared with the control group, the engineered CD109 carrying Cy3- si-circPNIT + In the exosome group, exosomes were mainly enriched around the nerves in pancreatic cancer tumor tissues. The small interfering RNA si-circPNIT carried by them could effectively reduce the fluorescence intensity of circPNIT in neurons without affecting the expression of circPNIT in other parts, confirming that the engineered CD109 + Exosomes can effectively deliver si-circPNIT to neurons around tumors. In addition, we found that Cy3- si-circPNIT engineered CD109 + In pancreatic cancer tissues of the exosome group, the degree of neural infiltration and the density of invaded neurons were significantly reduced compared with the control group, and the survival time of mice was significantly prolonged, suggesting that the use of engineered CD109 + Exosome delivery of si-circPNIT can effectively block the occurrence of neural invasion of pancreatic cancer.

Claims

1. An exosome targeting neural infiltration of pancreatic cancer, characterized in that: The exosomes are CD109 + exosomes.

2. The exosomes targeting neural infiltration of pancreatic cancer according to claim 1, characterized in that CD109 + Exosomes are derived from KRAS G12D mutations in sera from pancreatic cancer patients.

3. The exosomes targeting pancreatic cancer according to claim 1, characterized in that CD109 + The exosome preparation method is as follows: CD109 and LAMP2B CDS regions were digested with BamHI and EcoRI and purified on gel, CD109-LAMP2B CDS region sequence was connected to eukaryotic expression vector pCDNA3.1(+)-GNSTM-HA using T4 DNA ligase. Then, the constructed plasmid was transfected into 293t cells, cell supernatant was collected 72h after transfection, and exosomes were collected by ultracentrifugation.

4. A drug-loaded exosome targeting neural infiltration of pancreatic cancer, characterized in that: The drug-loaded exosomes are the exosomes targeting pancreatic cancer neural infiltration as described in any one of claims 1 to 3.

5. The drug-loaded exosomes targeting neural infiltration of pancreatic cancer according to claim 4, characterized in that: The preparation method of the drug-loaded exosomes is: Cy3-labeled si-circPNIT is packaged into exosomes by electroporation.

6. Use of the exosomes targeting pancreatic cancer neural infiltration according to any one of claims 1 to 3 in the preparation of a drug for treating pancreatic cancer neural infiltration.

7. The use according to claim 4, characterized in that: The medicine also includes a pharmaceutically acceptable carrier, excipient or diluent.

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