Application of lipopolysaccharide in preparation of product for inducing phenotypic transformation of neutrophil or treating tumors

Abstract

The invention discloses application of lipopolysaccharide in preparation of a product for inducing phenotype transformation of neutrophil or treating tumors, the lipopolysaccharide can induce the neutrophil to improve surface anti-tumor markers, reduce surface tumor-promoting markers and promote expression of costimulatory molecules and MHC molecules, so that the neutrophil phenotype transformation can be induced, and the neutrophil phenotype transformation can be treated. Therefore, the neutrophil is promoted to be converted into the neutrophil with anti-tumor immunophenotype and the antigen presenting cell-like neutrophil, the immune system of an organism is activated, tumor cells are directly or indirectly killed, tumor growth and metastasis are inhibited, the lifetime of tumor-bearing mice is prolonged, and tumors can be thoroughly cured; in addition, the lipopolysaccharide can induce local inflammation of tumors, and then guide and drive aggregation and accumulation of neutrophils and activate the cytotoxic function of the neutrophils, so that the purpose of treating large-volume tumors is achieved.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to an application of lipopolysaccharide in preparing a product for inducing neutrophil phenotype transformation or treating tumors. Background Art

[0002] Tumors seriously threaten human life and health, and have brought huge medical burdens to modern society. Traditional treatments cannot control the recurrence and metastasis of tumors, and it is difficult to completely cure tumors; immunotherapy such as immune checkpoint inhibition is limited by the immunosuppressive tumor microenvironment, and the treatment efficiency is low. At the same time, due to the imperfect early diagnosis technology of tumors and the rapid development of tumors, most patients are in the middle and late stages when diagnosed, and there are few treatment options for large-volume tumors, high treatment difficulty, short patient survival time, and low survival rate. Therefore, effective treatment methods for large-volume tumors are urgently needed. Cell therapy is an emerging type of tumor treatment method. Among them, adoptive cell therapy extracts self-or allogeneic immune cells, edits their genes or amplifies them in vitro, obtains specific or broad-spectrum killer cells, and finally injects them into the patient's body to kill tumor cells and achieve the purpose of treating tumors. At present, adoptive cell therapy based on various immune cells (dendritic cells, T lymphocytes, natural killer cells, etc.) has shown good tumor treatment capabilities in many clinical and preclinical studies.

[0003] Neutrophils differentiate and develop in the bone marrow and then enter the blood or tissues. They are the most abundant white blood cells in the blood and play an important role in nonspecific cellular immunity. When inflammation occurs in the body, neutrophils are recruited to the inflammatory site, destroying the cell membranes of biological pathogens such as bacteria and nearby tissues, engulfing the lysed cells and then decomposing them by a large number of lysosomes in the cells. At present, neutrophils are widely used as carriers for drug delivery in the body through the "hitchhiking" pathway. The principle is to induce inflammation in the tumor site in advance using radiotherapy, photothermal therapy, etc., and then deliver therapeutic drugs to the tumor site through the inflammatory chemotaxis of neutrophils in the body. The role of neutrophils in tumors is relatively complex. TANs can be divided into tumor-promoting N2 type and anti-tumor N1 type. In the tumor microenvironment, TANs directly or indirectly act on tumor cells by releasing a variety of pro-inflammatory, immunomodulatory and angiogenic factors to promote or inhibit the occurrence, progression and metastasis of tumors, and play a very important role in tumor immune escape and immunotherapy. Anti-tumor neutrophils highly express adhesion molecules ICAM-1 (CD54) and classic activation and degranulation markers CD63 on their surfaces, promote adhesion of inflammatory sites (tumors), and play an important role in controlling tumor progression and metastasis and regulating the body's immune response. In addition to regulating the transformation of TANs to an anti-tumor phenotype to kill tumor cells, studies have found that TANs can be transformed into antigen-presenting cell-like neutrophils by enhancing the expression of surface CD80, CD86, and MHC molecules to exert antigen-presenting functions. At the same time, in order to fully exert the anti-tumor function of neutrophils, it is usually necessary to reduce or maintain a low level of tumor-promoting markers (PD-L1, CD62L, Siglec F, SIRPα, etc.). In addition, neutrophils can also kill cancer cells by releasing elastase, nitric oxide synthase, and reactive oxygen species. However, neutrophils have a short lifespan and usually die spontaneously after exerting their killing effect, which limits the number of neutrophils in the body and is insufficient to produce a continuous killing effect on tumors.

[0004] In view of this, the present invention is proposed. Summary of the invention

[0005] The first purpose of the present invention is to provide an application of lipopolysaccharide in the preparation of a product for inducing neutrophil phenotype transformation. The lipopolysaccharide in the present invention can induce neutrophils to increase surface anti-tumor markers, reduce surface pro-tumor markers, promote the expression of co-stimulatory molecules (CD80, CD86) and MHC molecules, thereby promoting the transformation of neutrophils into neutrophils with anti-tumor immune phenotypes and antigen-presenting cell-like neutrophils, activating the body's immune system, directly or indirectly killing tumor cells, inhibiting tumor growth and metastasis, prolonging the survival of tumor-bearing mice, and completely curing tumors.

[0006] The second purpose of the present invention is to provide an application of lipopolysaccharide combined with neutrophils in the preparation of a product for treating tumors. In the present invention, lipopolysaccharide can induce local inflammation of the tumor, thereby driving the aggregation and accumulation of neutrophils and activating their cytotoxic function, causing them to transform into anti-tumor phenotypes (N1 type TANs) and antigen-presenting cell-like neutrophils, thereby achieving the purpose of treating large tumors.

[0007] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are particularly adopted:

[0008] The first aspect of the present invention provides a use of lipopolysaccharide in preparing a product for inducing the transformation of neutrophils into anti-tumor immune phenotypes and / or antigen presenting cell-like neutrophils.

[0009] Preferably, the neutrophils are extracted from bone marrow or peripheral blood.

[0010] Preferably, the product comprises lipopolysaccharide and saline.

[0011] Preferably, the concentration of the lipopolysaccharide is 0.1-0.5 mg / mL.

[0012] The second aspect of the present invention provides an application of lipopolysaccharide combined with neutrophils in the preparation of a product for treating tumors.

[0013] Preferably, the neutrophils are extracted from bone marrow or peripheral blood.

[0014] Preferably, the tumor includes colorectal cancer and melanoma.

[0015] The third aspect of the present invention provides a product for treating tumors, which comprises lipopolysaccharide injection and neutrophil injection.

[0016] Preferably, the concentration of the lipopolysaccharide injection is 0.1-0.5 mg / mL; the concentration of the neutrophil injection is (1-8)×10 6 Pieces / mL.

[0017] Compared with the prior art, the beneficial effects of the present invention include at least:

[0018] The lipopolysaccharide in the present invention can induce neutrophils to increase surface anti-tumor markers, reduce surface pro-tumor markers, promote the expression of co-stimulatory molecules (CD80, CD86) and MHC molecules, thereby promoting the transformation of neutrophils into neutrophils with anti-tumor immune phenotypes and antigen-presenting cell-like neutrophils, activating the body's immune system, directly or indirectly killing tumor cells, inhibiting tumor growth and metastasis, prolonging the survival of tumor-bearing mice, and completely curing tumors.

[0019] The lipopolysaccharide in the present invention can induce local inflammation of the tumor, thereby driving the aggregation and accumulation of neutrophils and activating their cytotoxic function, so that they are transformed into anti-tumor phenotypes (N1-type TANs) and antigen-presenting cell-like neutrophils, thereby achieving the purpose of treating large-volume tumors; the lipopolysaccharide and neutrophils of the present invention have a certain healing ability on tumor-bearing mice of different tumor models, the process is simple, the therapeutic effect is good, and it has the potential to be applied to tumor patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following is a brief introduction to the drawings required for the specific embodiments or the description of the prior art. In all the drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, the elements or parts are not necessarily drawn according to the actual scale.

[0021] Figure 1 The ability of LPS and BMTNs in Example 2 of the present invention to induce neutrophils to transform into anti-tumor phenotypes and antigen-presenting cell-like cells after co-incubation with BMTNs in vitro for different time periods (1 h, 2 h and 12 h);

[0022] Figure 2 The in vivo chemotactic ability of the neutrophils isolated in Example 3 of the present invention to the tumor inflammatory environment of tumor-bearing mice induced by lipopolysaccharide;

[0023] Figure 3 The ability of inducing neutrophils to transform into antigen presenting cell-like and anti-tumor phenotypes in mice after the lipopolysaccharide combined with neutrophils is used to treat tumor-bearing mice in Example 4 of the present invention;

[0024] Figure 4 The tumor treatment effect of lipopolysaccharide combined with neutrophils in Example 5 of the present invention in large-volume colorectal cancer model mice;

[0025] Figure 5 The ability of lipopolysaccharide combined with neutrophils to prevent tumor lung metastasis in large-volume colorectal cancer model mice after treating tumor-bearing mice in Example 6 of the present invention;

[0026] Figure 6 The H&E staining results of the main organ tissue sections of mice in each group after the tumor was treated with lipopolysaccharide combined with neutrophils in Example 7 of the present invention;

[0027] Figure 7 The serum biochemical levels of mice in each group after the tumor was treated with lipopolysaccharide combined with neutrophils in Example 7 of the present invention;

[0028] Figure 8 This is the tumor treatment effect of lipopolysaccharide combined with neutrophils in Example 8 of the present invention in large-volume melanoma model mice. DETAILED DESCRIPTION

[0029] The following embodiments of the technical solution of the present invention are described in detail in conjunction with the embodiments. The following embodiments are only used to more clearly illustrate the technical solution of the present invention, and are therefore only used as examples, and cannot be used to limit the protection scope of the present invention.

[0030] It should be noted that, unless otherwise specified, the technical terms or scientific terms used in this application should have the common meanings understood by those skilled in the art to which the present invention belongs.

[0031] The embodiment of the present invention provides an application of lipopolysaccharide in preparing a product for inducing the transformation of neutrophils into anti-tumor immune phenotypes and / or antigen presenting cell-like neutrophils.

[0032] The lipopolysaccharide in the present invention can induce neutrophils to increase surface anti-tumor markers, reduce surface pro-tumor markers, promote the expression of co-stimulatory molecules (CD80, CD86) and MHC molecules, thereby promoting the transformation of neutrophils into neutrophils with anti-tumor immune phenotypes and antigen-presenting cell-like neutrophils, activating the body's immune system, directly or indirectly killing tumor cells, inhibiting tumor growth and metastasis, prolonging the survival of tumor-bearing mice, and completely curing tumors.

[0033] In one embodiment, the lipopolysaccharide is derived from Escherichia coli 055:B5.

[0034] In one embodiment, the neutrophils are extracted from bone marrow or peripheral blood.

[0035] In one embodiment, the neutrophils are prepared by the following method:

[0036] (1) Obtain bone marrow / fresh anticoagulated peripheral blood (processed within 2 hours after ex vivo);

[0037] (2) Lysis of red blood cells: For bone marrow, use red blood cell lysis buffer; for peripheral blood, use the red blood cell sedimentation solution in the kit;

[0038] (3) Obtaining separation liquid layers and sample layers with clear interfaces: for bone marrow, prepare Percoll separation liquids of different densities, prepare 100% Percoll separation liquid according to the ratio of Percoll separation liquid: 10×PBS = 9:1, and then use 1×PBS to dilute in proportion to obtain 55%, 65%, and 78% Percoll separation liquids, respectively. Slowly add the Percoll separation liquids of different densities to the centrifuge tube in order from large to small density to form a gradient density separation liquid, and finally spread the bone marrow single cell suspension after lysing red blood cells on the liquid surface to keep the interface clear; for peripheral blood, use a peripheral blood neutrophil separation kit, add 4 mL of reagent A, 2 mL of reagent C, and peripheral blood after sedimentation of red blood cells in the centrifuge tube in order to keep the interface clear;

[0039] (4) Centrifugation: room temperature, horizontal rotor 750-1000g, centrifugation for 20-30 min, speed increase 4-6, speed decrease 1. The larger the volume of cell suspension, the greater the centrifugal force required and the longer the centrifugation time.

[0040] (5) Washing: After centrifugation, two layers of milky white cells appear in the centrifuge tube. Use a syringe to carefully extract the lower neutrophil buffy coat layer into a new centrifuge tube, add 10 mL of PBS or cell washing solution, and centrifuge at 250 g for 10 min to wash the cells. Wash 2 to 3 times.

[0041] (6) Discard the supernatant, resuspend the cells, count them, and set aside.

[0042] In one embodiment, the product comprises lipopolysaccharide and saline.

[0043] In one embodiment, the concentration of the lipopolysaccharide may be any concentration between 0.1 and 0.5 mg / mL, specifically 0.1 mg / mL, 0.2 mg / mL, 0.4 mg / mL or 0.5 mg / mL.

[0044] Another embodiment of the present invention provides an application of lipopolysaccharide combined with neutrophils in the preparation of a product for treating tumors.

[0045] The lipopolysaccharide in the present invention can induce local inflammation of the tumor, thereby driving the aggregation and accumulation of neutrophils and activating their cytotoxic function, so that they are transformed into anti-tumor phenotypes (N1-type TANs) and antigen-presenting cell-like neutrophils, thereby achieving the purpose of treating large-volume tumors.

[0046] In the present invention, the method of inducing local tumor inflammation by lipopolysaccharide can be by direct intratumoral injection of lipopolysaccharide derived from Escherichia coli 055:B5, nanoparticle encapsulation delivery, bioorthogonal or other methods of delivering lipopolysaccharide to the local tumor to induce inflammation.

[0047] In one embodiment, the neutrophils are extracted from bone marrow or peripheral blood.

[0048] In one embodiment, the tumor comprises colorectal cancer and melanoma.

[0049] Yet another embodiment of the present invention provides a product for treating tumors, the product comprising lipopolysaccharide injection and neutrophil injection.

[0050] In one embodiment, the concentration of the lipopolysaccharide injection can be any concentration in the range of 0.1 to 0.5 mg / mL; the concentration of the neutrophil injection can be any concentration in the range of (1 to 8)×10 6 Any concentration of / mL.

[0051] The technical solution of the present invention is further described in detail below through specific embodiments.

[0052] Example 1

[0053] This embodiment is a method for extracting neutrophils, which includes the following steps:

[0054] (1) Obtain bone marrow / fresh anticoagulated peripheral blood (processed within 2 hours after ex vivo);

[0055] (2) Lysis of red blood cells: For bone marrow, use red blood cell lysis buffer; for peripheral blood, use the red blood cell sedimentation solution in the kit;

[0056] (3) Obtaining separation liquid layers and sample layers with clear interfaces: for bone marrow, prepare Percoll separation liquids of different densities, prepare 100% Percoll separation liquid according to the ratio of Percoll separation liquid: 10×PBS = 9:1, and then use 1×PBS to dilute in proportion to obtain 55%, 65%, and 78% Percoll separation liquids, respectively. Slowly add the Percoll separation liquids of different densities to the centrifuge tube in order from large to small density to form a gradient density separation liquid, and finally spread the bone marrow single cell suspension after lysing red blood cells on the liquid surface to keep the interface clear; for peripheral blood, use a peripheral blood neutrophil separation kit, add 4 mL of reagent A, 2 mL of reagent C, and peripheral blood after sedimentation of red blood cells in the centrifuge tube in order to keep the interface clear;

[0057] (4) Centrifugation: room temperature, swing-out rotor, 850 g, 30 min, speed up 4, speed down 1;

[0058] (5) Washing: After centrifugation, two layers of milky white cells appear in the centrifuge tube. Use a syringe to carefully extract the lower neutrophil buffy coat layer into a new centrifuge tube, add 10 mL of PBS or cell washing solution, and wash the cells by centrifugation at 250 g for 10 min. Wash three times.

[0059] (6) Discard the supernatant, resuspend the cells, count them, and dilute them to 6×10 6 / mL for future use.

[0060] Example 2

[0061] This example is a study on the ability of LPS to induce BMTNs to transform into antigen presenting cell-like and anti-tumor phenotypes in vitro:

[0062] BMTNs were extracted according to the method of Example 1 and inoculated into 24-well plates (1×10 5 cells / well), LPS (derived from Escherichia coli 055:B5, 5 μg / mL) was added and incubated for 1 h, 2 h, and 12 h. Cells were collected, and neutrophils were labeled with CD11b and Ly6G. Neutrophils (CD11b + 6G + ) The expression of CD80, CD86, MHCⅠ, MHCⅡ, CD54, CD63, CD62L, and TNF-α on the surface of the cells. The test results are as follows: Figure 1 As shown;

[0063] Depend on Figure 1 It can be seen that LPS can upregulate the proportion of neutrophil surface antigen presentation related markers (CD80, CD86, MHCⅠ, MHCⅡ) ( Figure 1 LPS upregulates the adhesion molecule CD54 on the surface of neutrophils ( Figure 1 E) and the classical activation and degranulation marker CD63 ( Figure 1 The ratio of LPS to F) effectively improves the adhesion of the tumor site, promotes neutrophil degranulation, and releases lysozyme and other substances to eliminate tumor cells. At the same time, LPS can effectively reduce the expression of the tumor-promoting marker CD62L ( Figure 1 Middle G). The proportion of anti-tumor related markers on the surface of neutrophils increased with the increase of incubation time ( Figure 1 AF), while the proportion of tumor-promoting markers decreased with the extension of incubation time ( Figure 1 In addition, LPS can effectively increase TNF-α + Neutrophil ratio ( Figure 1 Middle H), TNF-α at 12h + The decrease in the proportion of neutrophils may be due to the secretion of TNF-α outside the cells to exert its effect.

[0064] Example 3

[0065] This example is to verify the active tropism of neutrophils to the lipopolysaccharide-induced tumor inflammatory microenvironment in vivo;

[0066] A colorectal cancer mouse model was established using 6-8 week old female Balb / c mice. When the tumor volume reached 250-300 mm 3 At the same time, lipopolysaccharide derived from Escherichia coli 055:B5 was injected intratumorally at a dose of 0.5 mg / kg and a concentration of 0.2 mg / mL to induce a local inflammatory environment of the tumor; 4 hours later, the neutrophils extracted in Example 1 were labeled with DiR cell dye and injected into tumor-bearing mice through the tail vein. At the same time, a control group in which lipopolysaccharide was not used to induce inflammation was set up, and a small animal living imager was used to take pictures and observe the distribution of neutrophils in the mice at different time points (1, 8, 16, 24, 48, and 72 hours). After 72 hours of filming, the mice were euthanized, and the heart, liver, spleen, lungs, kidneys, and tumors were removed. The small animal living imager was used to take pictures and observe each in vitro organ and tumor, and the distribution of neutrophils in each major organ and tumor was analyzed;

[0067] like Figure 2 As shown, Figure 2 A in the middle is a representative photo of the in vivo imaging of small animals at different time points in the control group (Ctrl) and the inflammation-induced group (LPS). Figure 2 B is the corresponding fluorescence quantitative analysis bar graph of the tumor site (n=3). In the Ctrl group, since no inflammation was induced in the tumor site, neutrophils did not accumulate in the tumor site. However, in the LPS group, a more obvious fluorescence signal could be observed in the tumor site 16 hours after cell injection. The fluorescence intensity of the tumor site continued to increase with time and tended to stabilize after 48 hours, indicating that the inflammatory environment guidance can induce the targeted accumulation of neutrophils. Figure 2 Center C is the in vitro imaging of major organs and tumors. The image shows that in addition to accumulating in the tumor site, neutrophils in the LPS group are mainly concentrated in the liver and spleen, while the fluorescent signals of the Ctrl group are all concentrated in the liver and spleen.

[0068] Example 4

[0069] This example is to study the ability of neutrophils in the tumor site to transform into antigen presenting cell-like and anti-tumor phenotypes after treatment of tumor-bearing mice.

[0070] A colorectal cancer mouse model was established using 6-8 week old female Balb / c mice. When the tumor volume reached 250-300 mm 3The mice were randomly divided into 4 groups, including negative control group (Ctrl), group injected with neutrophils alone (bone marrow-derived BMTNs), group injected with lipopolysaccharide alone (LPS), and LPS+BMTNs group, with 3 mice in each group. In the LPS group and LPS+BMTNs group, lipopolysaccharide derived from Escherichia coli 055:B5 was first injected intratumorally at a dose of 0.5 mg / kg and a concentration of 0.2 mg / mL to induce a local inflammatory environment in the tumor; 4 hours later, the neutrophils extracted in Example 1 were intravenously injected into the recipients (BMTNs, LPS+BMTNs), 100 μL / mouse, 6×10 5 Cells / piece.

[0071] 48 hours after cell injection, mice were killed by cervical dislocation, and tumors in each group were ground and filtered through a 70 μm mesh to lyse red blood cells to obtain a single cell suspension. Corresponding antibody staining and flow cytometry were used to detect changes in neutrophil surface markers in the tumor site, as follows: Marking of tumor site leukocytes (CD45 + ) and distinguish the neutrophil population from the leukocyte population (CD11b + 6G + ), and further detected the proportions of CD80, CD86, MHCⅠ, MHCⅡ, CD54, CD63, CD62L, and TNF-α on the surface of neutrophils.

[0072] like Figure 3 As shown in Figure AG, compared with the Ctrl group and the BMTNs group, the LPS group and the LPS+BMTNs group significantly upregulated the antigen presentation-related surface markers (CD80, CD86, MHCⅠ, MHCⅡ) on the surface of neutrophils ( Figure 3 AD) expression, increase anti-tumor phenotype markers such as adhesion molecule CD54 ( Figure 3 E) and the classical activation and degranulation marker CD63 ( Figure 3 F) expression ratio, reducing the expression of tumor-promoting marker CD62L ( Figure 3 Middle G), effectively increasing the proportion of TNF-α in neutrophils ( Figure 3 The results showed that lipopolysaccharide combined with neutrophils can induce the transformation of neutrophils into antigen-presenting cell-like and anti-tumor phenotypes in vivo in the treatment of tumors.

[0073] Example 5

[0074] This example is to study the ability of lipopolysaccharide-induced local tumor inflammation to drive neutrophil aggregation to treat primary large-volume colorectal cancer:

[0075] A colorectal cancer mouse model was established using 6-8 week old female Balb / c mice. When the tumor volume reached 250-300 mm3 The mice were randomly divided into 5 groups, including negative control group (Ctrl), group injected with neutrophils alone (BMTNs from bone marrow), group injected with lipopolysaccharide alone (LPS), group injected with LPS+BMTNs, and group injected with LPS+NEs (from peripheral blood), with 6 mice in each group. In the LPS group, LPS+BMTNs group, and LPS+NEs group, lipopolysaccharide derived from Escherichia coli 055:B5 was first injected intratumorally at a dose of 0.5 mg / kg and a concentration of 0.2 mg / mL to induce a local inflammatory environment of the tumor; 4 hours later, the neutrophils extracted in Example 1 were intravenously injected into the recipients (BMTNs, LPS+BMTNs, LPS+NEs), 100 μL / mouse, 6×10 5 The day of administration was recorded as day 0, and the tumor volume and mouse weight were recorded every two days. The tumor volume was calculated as length × width 2 / 2, when the tumor volume is greater than 1500mm 3 The mice were euthanized at 4 000 rpm and the survival curves were recorded and plotted.

[0076] like Figure 4 As shown, Figure 4 Figure A is a curve showing the change of mouse tumor volume over time. The results show that the injection of neutrophils alone or lipopolysaccharide alone has no significant inhibitory effect on tumor growth, while the treatment method of injecting neutrophils after lipopolysaccharide-induced tumor inflammatory microenvironment showed a significant ability to inhibit tumor growth. Figure 4 B in the middle is the corresponding mouse weight curve, indicating that the cell therapy method has no significant effect on mouse weight. Figure 4 C is the mouse survival curve. All mice in the control group died within 2 weeks (tumor volume greater than 1500mm 3 ), the LPS+BMTNs group significantly prolonged the survival of tumor-bearing mice, and 4 out of 6 mice in the LPS+BMTNs group were cured, indicating that the new cell therapy provided by the present invention has the potential to completely cure large-volume colorectal cancer. Since peripheral blood is easier to obtain than bone marrow, and neutrophils are the cells with the highest proportion in the blood, considering the operability of practical clinical applications, neutrophils extracted from peripheral blood are used to replace neutrophils obtained from bone marrow. The experimental results show that 2 out of 6 mice in the LPS+NEs group were cured, indicating that neutrophils extracted from peripheral blood also have a good healing effect, and at the same time have more realistic clinical significance, and can replace bone marrow neutrophils in practical applications.

[0077] Example 6

[0078] This example is to study the ability of lipopolysaccharide-induced local tumor inflammation to drive neutrophil aggregation to inhibit tumor metastasis after tumor treatment:

[0079] After 60 days of cell therapy, the cured mice in Example 3 and healthy mice (Ctrl) of the same age were injected with CT26 mouse colorectal cancer cells (2×10 5 The tumor lung metastasis experiment was conducted to study the ability of the treatment method of the present invention to inhibit tumor metastasis. The mice were euthanized 20 days after the injection, and the lungs were taken out, fixed with Bouin's fixative, and photographed.

[0080] like Figure 5 As shown, there were multiple obviously larger tumor nodules in the lungs of mice in the Ctrl group, indicating lung metastasis, while the lung surfaces of the cured mice in the LPS+BMTNs group and LPS+NEs group were smooth and no metastasis occurred, indicating that the new cell therapy provided by the present invention can effectively prevent tumor metastasis.

[0081] Example 7

[0082] This example is to verify the biological safety of the lipopolysaccharide combined with neutrophils in treating tumors;

[0083] In Example 5, blood was collected from the eye sockets one week after cell therapy and centrifuged (3000 rpm, 10 min), and various biochemical indicators were tested to determine whether the liver (ALT, AST), kidney (UREA), and heart (LDH) were normal. The organs of the mice (heart, liver, spleen, lung, and kidney) were taken and the tissue sections were observed by H&E staining.

[0084] like Figure 6 As shown, no obvious abnormalities were found in the tissue staining of the main organs (heart, liver, spleen, lung, and kidney) of mice in each group. Figure 7 As shown, compared with the control group, there was no significant change in the main biochemical indices of mice in each group.

[0085] Example 8

[0086] This example is to study the ability of lipopolysaccharide-induced local tumor inflammation to drive neutrophil aggregation and phenotypic transformation to treat primary large-volume melanoma:

[0087] Melanoma mouse models were established using 6-8 week old female C57BL / 6 mice. When the tumor volume reached 200 mm 3The mice were randomly divided into 4 groups, including negative control group (Ctrl), group injected with neutrophils alone (bone marrow-derived BMTNs), group injected with lipopolysaccharide alone (LPS), and group injected with LPS+BMTNs, with 6 mice in each group. In the LPS group and LPS+BMTNs group, lipopolysaccharide derived from Escherichia coli 055:B5 was first injected intratumorally at a dose of 0.5 mg / kg and a concentration of 0.2 mg / mL to induce a local inflammatory environment in the tumor; 4 hours later, the neutrophils extracted in Example 1 were intravenously injected into the recipients (BMTNs, LPS+BMTNs), 100 μL / mouse, 6×10 5 The day of administration was recorded as day 0, and the tumor volume and mouse weight were recorded every two days. The tumor volume was calculated as length × width 2 / 2, when the tumor volume is greater than 1500mm 3 The mice were euthanized at 4 000 rpm and the survival curves were recorded and plotted.

[0088] like Figure 8 As shown, Figure 8 Figure A is a curve showing the change of mouse tumor volume over time. The results show that the injection of neutrophils alone or lipopolysaccharide alone has no significant inhibitory effect on tumor growth, while the injection of neutrophils after lipopolysaccharide-induced tumor inflammatory microenvironment showed a significant ability to inhibit tumor growth. Figure 8 B in the middle is the corresponding mouse weight curve, and there is no obvious change in the mouse weight, indicating that the present invention has high biosafety. Figure 8 C is the mouse survival curve. All mice in the control group died within 10 days (tumor volume greater than 1500mm 3 ), the LPS+BMTNs group significantly prolonged the survival of tumor-bearing mice, and 2 out of 6 mice were cured, indicating that the new cell therapy provided by the present invention also has the potential to completely cure large-volume melanoma.

[0089] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein by equivalents. These modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention, and they should all be included in the scope of the claims and specification of the present invention.

Claims

1. The use of lipopolysaccharide in the preparation of products for inducing the transformation of neutrophils into anti-tumor immune phenotypes and / or antigen-presenting cell-like neutrophils.

2. The use according to claim 1, characterized in that: The neutrophils are extracted from bone marrow or peripheral blood.

3. The use according to claim 1, characterized in that: The product includes lipopolysaccharide and normal saline.

4. The use according to claim 1, characterized in that: The concentration of the lipopolysaccharide is 0.1-0.5 mg / mL.

5. Application of lipopolysaccharide combined with neutrophils in the preparation of tumor treatment products.

6. The use according to claim 5, characterized in that: The neutrophils are extracted from bone marrow or peripheral blood.

7. The use according to claim 5, characterized in that: Such tumors include colorectal cancer and melanoma.

8. A product for treating tumors, characterized in that: The products include lipopolysaccharide injection and neutrophil injection.

9. The product for treating tumors according to claim 8, characterized in that: The concentration of the lipopolysaccharide injection is 0.1-0.5 mg / mL; the concentration of the neutrophil injection is (1-8)×10 6 Pieces / mL.

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