Anti-tumor N1 phenotype neutrophil derived from bone marrow progenitor cells as well as preparation method and application thereof

Abstract

The invention discloses a preparation method of bone marrow progenitor cell-derived anti-tumor N1 phenotype neutrophil, which comprises the following steps: (1) infecting bone marrow progenitor cells by adopting ER-Hoxb8 retrovirus, and carrying out flow sorting to obtain ER-Hoxb8 progenitor cells with continuous proliferation capacity in the presence of 4-OHT culture conditions; (2) inducing and differentiating the ER-Hoxb8 progenitor cells into neutrophils by using G-CSF, and carrying out flow sorting and purification on the obtained neutrophils; and (3) stimulating the neutrophil by using IL-12 to obtain the neutrophil with the anti-tumor N1 phenotype. The invention also discloses the obtained anti-tumor N1 phenotype neutrophil and application thereof. The engineered neutrophils (IL-12-Neutrophils) prepared by the preparation method disclosed by the invention have an N1 phenotype, have an obvious killing effect on PANC02 cells, and can be used for treating pancreatic cancer.

Description

Technical Field

[0001] The present invention relates to anti-tumor N1 phenotype neutrophils derived from bone marrow progenitor cells and a preparation method and application thereof, belonging to the technical field of biomedicine. Background Art

[0002] Pancreatic ductal adenocarcinoma (PDAC) remains a highly lethal malignancy due to its aggressive nature and lack of effective treatments, with an average 5-year overall survival rate of less than 10%. Over the past decade, immunotherapy, especially immune checkpoint blockade (ICB), has been shown to enhance T cell-mediated immune responses. Currently, ICB therapy is becoming the most promising treatment option for patients with advanced skin cancer, lung cancer, and colorectal cancer. Unfortunately, recent clinical trials have reported that PDAC is resistant to almost all single-agent ICBs and double-agent ICB therapies using anti-programmed death-1 (PD-1) antibodies and anti-cytotoxic T lymphocyte-4 (CTLA-4) antibodies due to genetic heterogeneity and a highly fibrotic and immunosuppressive tumor microenvironment (TME). This highlights the urgent need to find new actionable immune targets to improve the treatment of PDAC.

[0003] The TME of PDAC is characterized by a significant infiltration of myeloid cells, which are usually lacking in cytotoxic T lymphocytes (CTLs) and have low expression of activation markers such as GZMB and IFNG. The lack of immune cells such as T cells and NK cells is an important regulator of disease progression and poor response to ICB therapy. However, the molecular mechanisms regulating PDAC TME and T cell immunity have not been fully elucidated. Various factors have been proposed to limit naturally occurring T cell immunity, such as low expression of major histocompatibility complex class I (MHC-I) on the cell surface, low mutation load, and TME-mediated inhibition of T cell priming and function. Among them, MHC-I-mediated downregulation of antigen presentation is one of the main mechanisms for tumors to evade immune surveillance, such as genetic mutations or epigenetic silencing of antigen processing pathway (APP) genes. Therefore, restoring tumor cell MHC-I expression through autophagy inhibition or using agonistic anti-CD40 antibodies may be a potential strategy to improve the responsiveness of PDAC immunotherapy.

[0004] Innate immune cells play an important role in responding to pathological conditions and maintaining immune homeostasis. Among them, neutrophils are plastic immune cells with diverse functional phenotypes that can respond to different environmental signals. Neutrophils respond to specific stimuli in the tumor microenvironment (TME) and reversibly transform into anti-tumor N1 (pro-inflammatory, classical activation) or pro-tumor N2 (anti-inflammatory, alternative activation) phenotypes, exerting anti-tumor or pro-tumor functions, respectively. As the most abundant immune cell in the circulation, neutrophils play a central role in the early immune response after tissue damage or infection. Activated neutrophils secrete various pro-inflammatory cytokines and activate T cells through antigen presentation. At the same time, neutrophils also exhibit high migration ability and the ability to penetrate tissues that are difficult for other cells to enter (such as the blood-brain barrier BBB), and can be used as effective drug carriers. However, under the influence of the tumor suppressive microenvironment and cytokines, neutrophils in the tumor microenvironment are mostly polarized into cells with N2-like phenotypes, which play a pro-tumor function by promoting angiogenesis, extracellular matrix remodeling, metastasis, and immunosuppression. Therefore, cell therapy strategies using neutrophils need to consider regulating the intrinsic properties of neutrophils to effectively trigger anti-tumor responses.

[0005] Neutrophils are more difficult to gene edit than T cells because they express abundant pattern recognition receptors and have a short half-life and cannot proliferate in vitro. Stem / progenitor cells provide an alternative cell resource for the production of neutrophil drugs. They can be stably transduced to express target genes and then differentiated into neutrophils. The most widely used production strategy currently comes from human pluripotent stem cells iPSCs. Roberts et al. first reported the generation of neutrophils with cytolytic activity from hematopoietic stem cells in 1998. Recently, a new chemically defined feeder-free cell platform was reported, allowing CD16+ neutrophils to be stably differentiated from hiPSCs using stage-specific signal regulators in vitro. Due to their unlimited self-renewal capacity and the potential to differentiate into a variety of cell types, iPSCs are currently believed to represent an unlimited cell source for cell therapy. However, different induction and differentiation strategies can greatly affect the yield and function of the final product, and iPSCs culture conditions are harsh and the induction differentiation cycle is long. Therefore, it is necessary to establish a rapid, efficient and safe protocol to achieve the clinical application of neutrophils.

[0006] The rapid response and high mobility of neutrophils to the inflammatory environment can help overcome some of the obstacles currently faced by cell therapies, and are expected to improve tumor infiltration, alleviate the immunosuppressive TME, and activate anti-tumor immune responses. However, the use of neutrophils as a potential treatment is still in the early stages of research, and clinical data are limited, making it difficult to fully evaluate its efficacy and safety. In order to further expand its application, a series of safety issues and challenges need to be addressed. For example, activated neutrophils may release a large number of proinflammatory cytokines, such as IL-1 and IL-6, which may induce serious adverse events such as cytokine release syndrome (CRS). Compared with the high proliferation capacity of T cells, neutrophils, as terminally differentiated cells, cannot be expanded in vitro or after infusion. Therefore, it is a huge challenge to obtain a large number of autologous cells for treatment, especially from patients. In addition, the life cycle of these cells is relatively short and may lack persistence. To solve this problem, a possible strategy is to use hematopoietic stem / progenitor cells (HSPCs) to generate a sufficient number of functional neutrophils and achieve long-term tumor regression through repeated infusion.

[0007] Defects and shortcomings of the existing technology:

[0008] Cell therapy drugs such as CAR-T or CAR-NK are difficult to infiltrate into solid tumors (including PDAC). Currently, tumor-associated neutrophils (TAN) in the tumor microenvironment are mainly targeted by inhibiting the chemotaxis and recruitment of neutrophils to solid tumors, and the anti-tumor function of neutrophils themselves has not yet been used for anti-tumor treatment. In addition, neutrophils are terminally differentiated cells and the first line of defense against infection in the body. The efficiency of in vivo editing or transformation is low; it is difficult to obtain neutrophil sources; at the same time, it is necessary to optimize the application of polarizing neutrophils to the anti-tumor N1 phenotype in tumor treatment. Summary of the invention

[0009] The purpose of the present invention is to obtain anti-tumor N1 phenotype neutrophils derived from bone marrow progenitor cells.

[0010] The technical solution adopted by the present invention is:

[0011] A method for preparing anti-tumor N1 phenotype neutrophils derived from bone marrow progenitor cells, the steps comprising:

[0012] (1) Infecting bone marrow progenitor cells with ER-Hoxb8 retrovirus and obtaining ER-Hoxb8 progenitor cells that can sustainably proliferate in the presence of 4-OTH by flow cytometry;

[0013] (2) ER-Hoxb8 progenitor cells were induced to differentiate into neutrophils using G-CSF, and neutrophils were obtained by flow cytometry sorting and purification;

[0014] (3) Neutrophils are stimulated with IL-12 to obtain neutrophils with anti-tumor N1 phenotype.

[0015] Preferably, step (1) specifically comprises: culturing the bone marrow progenitor cells in IMDM complete medium for 48 hours and then transfecting the ER-Hoxb8 retrovirus.

[0016] Preferably, the bone marrow progenitor cells in step (1) are mouse bone marrow progenitor cells.

[0017] Preferably, in step (1), the ER-Hoxb8 progenitor cells are cultured in Opti-MEM complete medium, and 1 μM 4-OHT is continuously added to maintain cell proliferation ability.

[0018] Preferably, in step (2), the sustainably proliferating ER-Hoxb8 progenitor cells are washed to remove 4-OHT, and then 20 ng / ml G-CSF, 10% FBS, and 1% Pen-Strep-Glut are added to the RPMI1640 medium to prepare 1640 induction medium for inducing differentiation of ER-Hoxb8 progenitor cells. The induction medium is replaced with fresh one on the third day, and mature neutrophils are obtained on the sixth day.

[0019] Preferably, in step (3), the neutrophils are stimulated with 50 ng / ml IL-12 for more than 24 hours.

[0020] The anti-tumor N1 phenotype neutrophils prepared by the above preparation method.

[0021] And the use of the above-mentioned anti-tumor N1 phenotype neutrophils in the preparation of drugs for treating cancer.

[0022] Preferably, the cancer is pancreatic cancer.

[0023] The obtained neutrophils were identified using markers of N1 and N2 phenotypes and were found to be of N1 phenotype.

[0024] After co-incubation with PANC02 cells, it was found that the expression levels of proinflammatory cytokines and reactive oxygen species (ROS) were significantly increased.

[0025] It has a significant killing effect on PANC02 cells.

[0026] The present invention realizes an anti-tumor modification method of neutrophils derived from bone marrow progenitor cells and its application; it relates to the engineering modification of the proliferation of bone marrow progenitor cells, the preparation of engineered Hoxb8 neutrophils, the polarization of anti-tumor N1 phenotype and the therapeutic application in solid tumors, which overcomes the deficiencies and defects mentioned in the background technology, uses markers of N1 and N2 phenotypes for identification, and evaluates the anti-tumor therapeutic effect based on the killing of pancreatic cancer PANC02 cells by polarized IL-12-Neutrophils and the detection of pro-inflammatory factors after co-incubation of IL-12-Neutrophils and PANC02 cells. The results show that the prepared engineered neutrophils (IL-12-Neutrophils) have an N1 phenotype and have a significant killing effect on PANC02 cells. Moreover, the engineered neutrophils (IL-12-Neutrophils) are derived from the induced differentiation of normal Hoxb8 hematopoietic stem / progenitor cells that are dependent on 4-hydroxytamoxifen for proliferation, which is safer than HL-60 and other tumor-derived neutrophils, simpler and easier to obtain than iPSCs-derived neutrophils, and more convenient and easier to transform in vitro than the autologous immune cells of clinical patients, and the quality is more uniform; the neutrophils prepared by the present invention have the advantages of sustainable proliferation, long-term storage, safety and controllability, efficient and rapid preparation, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is the map of the ER-Hoxb8 retroviral vector.

[0028] Figure 2 Flowchart for ER-Hoxb8 progenitor cell construction and neutrophil induction.

[0029] Figure 3 Lin - The results of in vitro survival time detection of progenitor / Hoxb8-progenitor cells.

[0030] Figure 4 These are photos of cell morphology staining of WT-Neutrophils / IL-12-Neutrophils cells.

[0031] Figure 5 This is the result of identifying the polarization phenotype of WT-Neutrophils / IL-12-Neutrophils cells.

[0032] Figure 6 This is the result of identification of pro-inflammatory cytokine gene expression levels in WT-Neutrophils / IL-12-Neutrophils cells.

[0033] Figure 7This is the detection result of pro-inflammatory cytokines secreted by WT-Neutrophils / IL-12-Neutrophils cells after co-incubation with PANC02 cells.

[0034] Figure 8 This is the ROS detection result after WT-Neutrophils / IL-12-Neutrophils cells were co-incubated with PANC02 cells.

[0035] Fig. 9 This is the result of the killing efficiency test of WT-Neutrophils / IL-12-Neutrophils cells on PANC02 cells.

[0036] Fig.10 These are the test results of the therapeutic effect of WT-Neutrophils / IL-12-Neutrophils cells on ectopic pancreatic cancer model mice.

[0037] Fig.11 These are the results of the detection of WT-Neutrophils / IL-12-Neutrophils cells infiltrating tumor tissue in the ectopic pancreatic cancer model. Specific implementation methods

[0039] The present invention is further described below in conjunction with embodiments, but the description of the embodiments does not impose any limitation on the protection scope of the present invention.

[0040] Unless otherwise defined, all technical terms and scientific terms used herein have the same meaning as those generally understood by those skilled in the art of the present invention, and the terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention. In addition, although demonstrations of parameters including specific values ​​may be provided herein, it should be understood that the parameters do not need to be exactly equal to the corresponding values, but may be approximated to the corresponding values ​​within an acceptable error tolerance or design constraint.

[0041] Unless otherwise specified, the materials and instruments used in the following examples can be obtained from conventional commercial channels.

[0042] Example 1

[0043] (1) Hoxb8 progenitor cell culture: 6-8 week old female C57BL / 6J mice were euthanized, bone marrow cells were extracted, and Lin-progenitor cells were obtained using a progenitor cell separation kit (Direct Lineage Cell Depletion Kit, Miltenyi Biotec). Lin-progenitor cells have no ability to continue to proliferate and will gradually die under in vitro culture conditions (whether IMDM or Opti-MEM containing 4-OHT). Lin-progenitor cells were cultured in IMDM complete medium, the formula of which included IMDM (Iscove's, Gibco), 15% FBS (Gibco), 1% pen / strep / glutamine (Gibco), 10 ng / ml murineIL-3 (Peprotech), 20 ng / ml murineIL-6 (Peprotech), and 25 ng / ml murine SCF (Peprotech). After 48 hours, the cells were infected with ER-Hoxb8 retrovirus to induce the continuous expression of the Hoxb8 gene in progenitor cells and maintain the proliferation ability of progenitor cells. Proliferative ER-Hoxb8 progenitor cells were obtained and cultured in Opti-MEM complete medium. The Opti-MEM complete medium contained Opti-MEM, 10% FBS, 1% PSG, 10 ng / ml murine SCF, 30 μM beta mercapto ethanol, and 1 μM 4-OHT. Positive cells were purified by flow cytometry. The construction process is as follows Figure 2 shown.

[0044] The sequence information of ER-Hoxb8 retrovirus is shown in the table below, and the map of retrovirus is shown in Figure 1 shown.

[0045] ER-Hoxb8 retroviral sequence information

[0046] Gene Name: ER-Hxob8 CDS Length: 1716 bp (SEQ ID NO. 1) Genetic species: Mouse Fluorescent Labeling: EGFP Tags: No protein tag Control virus marker: EGFP

[0047] (2) Proliferating ER-Hoxb8 progenitor cells and Lin cells not infected with ER-Hoxb8 retrovirus - Progenitor cells were seeded in 96-well plates and cultured in Opti-MEM containing 4-OHT. The in vitro viability of cells was detected using CCK-8 reagent (Cat#40203ES76, Yisheng). The results are shown in Figure 3 shown.

[0048] (3) The proliferative ER-Hoxb8 progenitor cells were washed to remove 4-OHT, and then induced by adding 20 ng / ml G-CSF, 10% FBS, and 1% Pen-Strep-Glut to RPMI 1640 medium. On the third day, the induction medium was replaced with fresh medium. On the sixth day, mature Hoxb8 neutrophils were obtained. The induction process was as follows: Figure 2 As shown;

[0049] (4) PANC02 cell culture: DMEM medium supplemented with 10% fetal bovine serum, 1% penicillin and streptomycin;

[0050] (5) Preparation of anti-tumor N1 phenotype IL-12-Neutrophils: Hoxb8 neutrophils were stimulated with 50 ng / ml IL-12 for 24 hours to obtain anti-tumor N1 phenotype IL-12-Neutrophils.

[0051] (6) Morphology and karyotype detection of IL-12-Neutrophils: The cells were fixed on a glass slide, stained with Wright-Giemsa stain (Nanjing Jiancheng Biotechnology Co., Ltd., D010), and imaged under a microscope. The results are as follows: Figure 4 WT-neutrophils refer to Hoxb8-neutrophils that were not stimulated with IL-12.

[0052] (7) Identification of polarized phenotype of IL-12-Neutrophils and detection of proinflammatory cytokine gene expression: N1 markers: iNOS, Ccl3, N2 markers: Arg1, Mmp9, proinflammatory cytokine genes: TNF-α, INF-γ, IL-6, IL-1β. Total RNA was extracted from IL-12-Neutrophils using TrioReagent (Invitrogen, Carlsbad, CA, USA). Specific conditions and parameters were based on the protocol in the instruction manual, and 1 μg of total RNA was reversed into cDNA using the TransScript One-Step gDNA Removal and cDNASynthesis SuperMix (Nanjing Novigene) instruction manual. Real-time PCR was performed in a 7500 detection system using SYBR-GreenI as a double-stranded DNA-specific binding dye. The mRNA of the target gene and the mRNA of the housekeeping gene β-actin were quantified in separate tubes, and the results are shown in the table below. Figure 5-6 shown.

[0053] (8) Detection of pro-inflammatory cytokines secreted after IL-12-Neutrophils were co-incubated with PANC02 cells: IL-12-Neutrophils were co-incubated with PANC02 cells for 24 hours, and the protein content of pro-inflammatory cytokines TNF-α, IL-1β, IL-6 and IFN-γ in the conditioned medium was detected by Elisa method (Thermo Fisher, according to the specific protocol in the instruction manual). The results are as follows: Figure 7 shown.

[0054] (9) ROS detection after co-incubation of IL-12-Neutrophils cells with PANC02 cells: IL-12-Neutrophils were co-incubated with PANC02 cells for 24 h. The generated ROS were detected by adding H2DCFDA probe (Biyuntian, Shanghai, China) according to the instructions. The results are shown in Figure 8 As shown. (10) Detection of the killing efficiency of IL-12-Neutrophils cells on PANC02 cells: Target cells PANC02 (stable tumor cell line produced by Luciferase lentiviral transfection) were inoculated in a black 96-well ELISA plate at a density of 5000 / 100 μL / well. Effector cells were neutrophils from different groups, and the effector cells and target cells (E:T) ratios were 5:1, 10:1, 20:1 and 50:1 respectively. After incubation for 24 hours, the ONE-Glo TM The luciferase assay system was used for detection on a Varioskan Flash luminometer (Thermo Scientific). The experimental method was based on the instructions of the kit supplier (Promega, E6110). The results are shown in Fig. 9 shown.

[0055] (11) PANC02-Luc cells were subcutaneously inoculated on the back of 6-week-old female C57B6 / J mice to construct an ectopic pancreatic cancer model at a density of 5×10 6 cells, and the inoculation volume was 100 μl. On the 10th, 17th, and 24th days of inoculation, different groups of neutrophils were injected into the tail vein for treatment. On the 35th day of inoculation, the model mice in each group were euthanized, and the tumor tissues were collected and photographed. The tumor tissues were histologically detected using hematoxylin-eosin staining. Tumor growth was monitored throughout the treatment experiment, and the tumor size was recorded and the tumor growth curve was drawn. The results are shown in Fig.10 As shown. Among them, Fig.10 A. Schematic diagram of the process of using neutrophils to treat mice with ectopic pancreatic cancer. Fig.10 B. Tumor photograph. Fig.10C. Measure the tumor volume every 7 days, record and draw a tumor growth curve. Tumor volume = 1 / 2 × tumor long diameter × tumor short diameter 2 . Fig.10 D. Representative images of hematoxylin and eosin staining of tumor tissues.

[0056] (12) Different groups of neutrophils were injected into the tail vein of mice bearing ectopic pancreatic cancer tumors. The modeling method was the same as that of the mouse model in step (11). Different groups of neutrophils were injected into the tail vein on the tenth day of inoculation. Each group of neutrophils was labeled with a DiR probe (Cat#40757ES25, Yisheng) before injection. The fluorescent signal at the tumor site was detected using the IVIS Spectrum System (Perkin-Elmer, Waltham, MA, USA) at 12h, 36h, 72h and 168h after injection. The results are shown in Fig.11 shown.

[0057] The above embodiments are only for illustrating the technical concept and features of the present invention, and their purpose is to enable people familiar with this technology to understand the contents of the present invention and implement them accordingly. They cannot be used to limit the protection scope of the present invention. Any equivalent changes or modifications made according to the spirit of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing anti-tumor N1 phenotype neutrophils derived from bone marrow progenitor cells, characterized in that The steps include: (1) Infecting bone marrow progenitor cells with ER-Hoxb8 retrovirus and obtaining ER-Hoxb8 progenitor cells that can sustainably proliferate in the presence of 4-OHT by flow cytometry; (2) ER-Hoxb8 progenitor cells were induced to differentiate into neutrophils using G-CSF, and the obtained neutrophils were flow sorted and purified; (3) Neutrophils are stimulated with IL-12 to obtain neutrophils with anti-tumor N1 phenotype.

2. The preparation method according to claim 1, characterized in that: Step (1) specifically includes: the bone marrow progenitor cells are cultured in IMDM complete medium for 48 hours and then transfected with ER-Hoxb8 retrovirus.

3. The preparation method according to claim 1, characterized in that: The bone marrow progenitor cells in step (1) are mouse bone marrow progenitor cells.

4. The preparation method according to claim 1, characterized in that: In step (1), ER-Hoxb8 progenitor cells were cultured in Opti-MEM complete medium, and 1 μM 4-OHT was continuously added to maintain cell proliferation ability.

5. The preparation method according to claim 4, characterized in that: In step (2), the sustainably proliferating ER-Hoxb8 progenitor cells are washed to remove 4-OHT, and then 20 ng / ml G-CSF, 10% FBS, and 1% Pen-Strep-Glut are added to RPMI 1640 medium as an induction medium for induction. Fresh induction medium is replaced on the third day, and mature neutrophils are obtained on the sixth day.

6. The preparation method according to claim 1, characterized in that: In step (3), neutrophils were stimulated with 50 ng / ml IL-12 for more than 24 hours.

7. Anti-tumor N1 phenotype neutrophils prepared by the preparation method according to any one of claims 1 to 6.

8. Use of the anti-tumor N1 phenotype neutrophils according to claim 7 in the preparation of drugs for treating cancer.

9. The use according to claim 8, characterized in that: The cancer is pancreatic cancer.

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