Anti-tumor combined treatment method for improving radiotherapy curative effect by using nintedanib

Abstract

The invention provides an anti-tumor combined treatment method for improving a radiotherapy curative effect by using nintedanib, and relates to the technical field of tumor treatment. The anti-tumor combined treatment method comprises the following steps: selecting a male mouse or a male nude mouse as an experimental animal, and subcutaneously constructing a tumor-bearing mouse model on the back of the experimental animal; when the tumor volume of the tumor-bearing mice reaches 70-100mm < 3 >, randomly dividing the tumor-bearing mice into four groups, namely a control group, a nintedanib single drug group, a radiotherapy group and a combined group; measuring the subcutaneous tumor volume and weight of the tumor-bearing mouse every three days; drawing a mouse tumor growth curve, a mouse survival curve and a mouse weight change curve according to the measured data; a tumor-bearing mouse is subjected to living body imaging, tumor tissue paraffin embedding, immunohistochemical staining, TUNEL staining and Hamp; and E, carrying out section staining and blood biochemical detection, and carrying out data analysis by utilizing corresponding analysis software, so as to evaluate the anti-tumor effect and safety of the combined treatment. According to the invention, tumor growth can be effectively inhibited.

Description

Technical Field

[0001] The present invention relates to the technical field of tumor treatment, and specifically to an anti-tumor combination treatment method for improving the efficacy of radiotherapy by using nintedanib. Background Art

[0002] Radiotherapy is widely used in the treatment of more than 50% of cancer patients due to its advantages such as less trauma, fewer contraindications, and good anti-tumor effect. As an anti-fibrotic and anti-angiogenic drug, nintedanib has anti-tumor potential and theoretically can enhance the efficacy of radiotherapy.

[0003] However, there are few studies on the combination of nintedanib and radiotherapy at present, and the synergistic anti-tumor mechanism of its combination treatment strategy is still unclear. Among the existing tumor treatment methods, single treatment methods often have limitations and cannot meet the treatment needs of all patients. Therefore, it is urgent to find a more effective combination treatment plan. Summary of the Invention

[0004] (I) Technical Problem to be Solved

[0005] In view of the deficiencies of the prior art, the present invention provides an anti-tumor combination treatment method for improving the efficacy of radiotherapy by using nintedanib, and solves the problems raised in the above background art.

[0006] (II) Technical Solution

[0007] To achieve the above object, the present invention is realized through the following technical solutions: An anti-tumor combination treatment method for improving the efficacy of radiotherapy by using nintedanib, comprising the following steps:

[0008] Step 1: Construct a tumor-bearing mouse model:

[0009] Select male mice or male nude mice as experimental animals, and construct a tumor-bearing mouse model subcutaneously on the back of the experimental animals. The tumor cells used are mouse lung cancer cell line LLC or mouse colon cancer cell line MC38;

[0010] Step 2: Group treatment:

[0011] When the tumor volume of the tumor-bearing mice reaches 70 - 100 mm 3 , randomly divide the tumor-bearing mice into four groups, namely a control group, a nintedanib monotherapy group, a radiotherapy group, and a combination group, with no less than 7 mice in each group;

[0012] Step 3: Physical measurement:

[0013] Measure the subcutaneous tumor volume and body weight of the tumor-bearing mice every 3 days. Among them, the subcutaneous tumor volume is measured by vernier caliper for the longest diameter and the shortest diameter, and the tumor volume is calculated according to the formula tumor volume = (length × width2) / 2;

[0014] Step 4: Record

[0015] Draw the mouse tumor growth curve, mouse survival curve, and mouse body weight change curve based on the measured data to evaluate the anti-tumor effect;

[0016] Step Five: Processing and Evaluation:

[0017] Evaluate the anti-tumor effect and safety of the combination therapy by performing in vivo imaging on tumor-bearing mice, paraffin embedding of tumor tissues, immunohistochemical staining, TUNEL staining, H&E section staining, blood biochemical detection, and using corresponding analysis software for data analysis.

[0018] Preferably, in Step One, male C57BL / 6 mice or male BALB / c nu-nu nude mice at 6 - 8 weeks of age and weighing 20 - 22 g are selected as experimental animals.

[0019] Preferably, in Step One, the specific steps for constructing the tumor-bearing mouse model are as follows:

[0020] (1) Amplify LLC or MC38 cells in the logarithmic growth phase with good condition. Use the cell passage method to digest the tumor cells into a cell suspension, and resuspend with PBS to remove the complete medium components;

[0021] (2) Take 20 μl of the cell suspension to calculate the cell concentration. Calculate the total number of cells according to 1×10 6 tumor cells per mouse, and adjust the cell concentration to 5×10 6 / ml using PBS;

[0022] (3) If the experimental animals are C57BL / 6 mice, use a razor to shave the skin of the planned tumor implantation area on the back in advance. Nude mice do not need to be shaved. At the same time, use an ear tag pliers to number the mice;

[0023] (4) Pipette or invert and mix the tumor cell suspension evenly. Use a 1 ml insulin syringe to aspirate the cell suspension, and subcutaneously inject 200 μl of the cell suspension on the back of the shaved or unshaved mice to form a skin bleb. Slowly rotate and pull out the needle to avoid leakage, and put the mice back into the cage and record.

[0024] Preferably, in Step Two, the mice in the control group, nintedanib monotherapy group, radiotherapy group, and combination group are treated separately:

[0025] Control group: Administer intragastric gavage with the same volume of sterile ultrapure water for 5 consecutive days, then pause for 1 day, with no more than 2 cycles;

[0026] Nintedanib monotherapy group: Administer intragastric gavage with 50 mg / kg nintedanib for 5 consecutive days, then pause for 1 day, with no more than 2 cycles;

[0027] Radiotherapy group: On the day of enrollment, the subcutaneous tumor site of the mice was irradiated with 8 Gy electron beam. When irradiating, the mice were anesthetized by intraperitoneal injection of 1.5% sodium pentobarbital solution at a dose of 50 mg / kg. The control group mice were only anesthetized.

[0028] Combined group: On the day of enrollment, the subcutaneous tumor site of the mice was irradiated with 8 Gy electron beam. When irradiating, the mice were anesthetized by intraperitoneal injection of 1.5% sodium pentobarbital solution at a dose of 50 mg / kg, and were continuously given intragastric administration of 50 mg / kg nintedanib for 5 days and then paused for 1 day, with no more than 2 cycles.

[0029] Preferably, in step five, the in vivo imaging step includes: in a light-proof environment at room temperature, D-luciferin potassium salt was configured into a 15 mg / ml working solution with sterile PBS, and 1.5% sodium pentobarbital was configured into a 15 mg / ml working solution. After configuration, both were wrapped with tin foil and stored in the dark at 4°C, and were preferably prepared and used immediately; before loading the machine, first inject the substrate into the abdominal cavity of the mice, and inject anesthetic into the abdominal cavity 3 - 5 minutes later for anesthesia; after the mice were anesthetized, image acquisition was performed, and after acquisition, the analysis software SI Imaging was used to quantitatively analyze the fluorescence intensity of the tumor.

[0030] Preferably, in step five, the paraffin embedding step of the tumor tissue includes: the mice were sacrificed by the method of excessive anesthesia, and the tumor tissue to be measured of the mice was taken out with medical instruments and placed in 4% paraformaldehyde for fixation at room temperature for 24 - 48 h; the fixed tissue was successively soaked in 75%, 85%, 90%, and 95% alcohol solutions for 2 h each, then soaked in the first and second cylinders of absolute ethanol for 40 min each, and then successively soaked in the first cylinder of xylene for 10 min, the second cylinder of xylene for 8 min, the first cylinder of wax for 1 h, and the second cylinder of wax for 1 h until it became transparent; the sample was immersed in paraffin liquid with a melting point of 52 - 60°C for at least 2 h; the sample was placed in a -20°C refrigerator to solidify into a block, the wax block was trimmed, tissue sectioning and spreading were performed, the sections were placed in a baking machine to dry, and the groups were marked.

[0031] Preferably, in step five, the immunohistochemical staining steps include: dewaxing paraffin sections in xylene I, II, and III for 5 - 10 minutes each, passing through gradient ethanol for 5 minutes each, rinsing with pure water for 1 minute; placing the sections in sodium citrate antigen retrieval solution, boiling in a microwave oven at medium heat for 10 minutes, naturally cooling to room temperature, and transferring to TBS buffer for shaking and washing for 15 minutes; incubating with 3% hydrogen peroxide solution at room temperature in the dark for 20 minutes to block endogenous peroxidase, and shaking and washing with TBS for 15 minutes; adding 10% BSA and incubating at room temperature for 30 minutes; using a histochemical pen to draw a circle around the tissue, dropping the prepared primary antibody solution, placing it flat in a wet box, and incubating overnight at 4°C; after shaking and washing with TBS for 15 minutes, dropping the corresponding secondary antibody labeled with HRP and incubating at room temperature for 1 hour; after shaking and washing with TBS for 15 minutes, dropping DAB chromogenic solution, controlling the chromogenic time, with positive being brownish - yellow, and rinsing with pure water to terminate chromogenesis; counterstaining with hematoxylin for 2 minutes, rinsing with pure water, differentiating with hydrochloric acid alcohol for 3 seconds, rinsing with pure water, counter - bluing with hematoxylin blueing solution and then rinsing with pure water again; dehydrating and clearing in gradient ethanol and xylene for 2 minutes each, finally mounting with neutral balsam, examining and photographing under a microscope, and performing quantitative analysis using Image J software.

[0032] Preferably, in step five, the TUNEL staining steps include: dewaxing paraffin sections in xylene I, II, and III for 5 - 10 minutes each, passing through gradient ethanol for 5 minutes each, rinsing with pure water for 1 minute; using a histochemical pen to draw a circle around the tissue, dropping the permeabilization solution and permeabilizing at room temperature for 15 minutes; after shaking and washing with TBS for 15 minutes, dropping a mixture of 3% BSA and 20% fetal bovine serum and sealing at room temperature for 30 minutes; removing the sealing solution and dropping an appropriate amount of TUNEL reaction solution, placing it in a wet box in the dark and incubating at 37°C for 2 hours; after shaking and washing with TBS for 15 minutes, dropping DAPI to stain the cell nuclei for 5 - 10 minutes; shaking and washing with TBS for 15 minutes and mounting with an anti - fluorescence quenching mounting medium, examining and photographing under a fluorescence microscope, and performing quantitative analysis using Image J software.

[0033] Preferably, the H&E section staining steps include: dewaxing tumor paraffin sections in xylene I, II, and III for 5 - 10 minutes each, then passing through gradient ethanol for 5 minutes each, rinsing with pure water for 1 minute; placing the sections in hematoxylin and staining for 5 minutes, rinsing with pure water for 1 minute; putting the sections into 1% hydrochloric acid aqueous solution for differentiation for 1 second, rinsing with pure water for 1 minute; putting the sections into ammonia aqueous solution for counter - bluing, rinsing with pure water and soaking for 15 minutes; putting the sections into 85% and 95% ethanol for 10 minutes each; putting the sections into eosin staining solution and staining for 5 minutes; putting the sections into absolute ethanol I, II, III cylinders, n - butanol, xylene I, II cylinders for 5 minutes each until clear; mounting with neutral balsam, observing and photographing under a microscope.

[0034] Preferably, in step five, the blood biochemical detection steps include: grasping and fixing the mouse's head, trimming the whiskers on both sides of the mouse, quickly removing the right eyeball of the mouse using an ophthalmic forceps, allowing the blood droplets from the eyeball to fall into a 1.5 ml sterile EP tube pre-added with sodium heparin, collecting no less than 500 μl of blood; allowing the blood to stand at room temperature for 30 min to separate the layers, centrifuging at 5000 rpm for 15 min in a pre-cooled centrifuge at 4°C; aspirating the supernatant into a new sterile EP tube and making marks, and setting parameters according to the instructions on a Chemray 240 fully automatic biochemical analyzer and loading the sample to detect biochemical related indicators such as alanine aminotransferase, aspartate aminotransferase, blood urea nitrogen, and creatinine.

[0035] (III) Beneficial effects

[0036] The present invention provides an anti-tumor combination treatment method using nintedanib to improve the efficacy of radiotherapy, with the following beneficial effects:

[0037] 1. In experiments on various tumor-bearing mouse models (such as LLC lung cancer model, MC38 colorectal cancer model), the combination group of nintedanib combined with radiotherapy can more effectively inhibit tumor growth compared with the single drug group and the radiotherapy group. From the tumor growth curve, survival curve, and single tumor growth curve data, the combination treatment significantly reduces the tumor growth rate and prolongs the survival period of mice.

[0038] 2. The study on MC38 tumor tissues found that the combination treatment can significantly increase the number of CD3 and CD8 positive T cells in and around the tumor, enhance immune cell infiltration, and improve the body's anti-tumor immune ability. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 It is a schematic flow chart of the treatment method of the present invention;

[0040] Figure 2 It is a schematic diagram of the tumor suppression experiment of the immune-normal LLC tumor-bearing mouse model of the present invention; wherein, A. Schematic diagram of the modeling and treatment plan; B. Tumor growth curve of LLC tumor-bearing mice; C. Survival curve of LLC tumor-bearing mice; D. Single tumor growth curve corresponding to each group of LLC tumor-bearing mice;

[0041] Figure 3 It is a schematic diagram of the tumor suppression experiment of the immune-normal MC38 tumor-bearing mouse model of the present invention; wherein, A. Tumor growth curve of MC38 tumor-bearing mice; B. Survival curve of MC38 tumor-bearing mice; C. Gross image of in vivo imaging of MC38 tumor-bearing mice on the 15th day; D. Quantitative analysis of fluorescence intensity of in vivo imaging of MC38 tumor-bearing mice on the 15th day; E. Gross image of in vivo imaging of MC38 tumor-bearing mice on the 30th day; F. Single tumor growth curve corresponding to each group of MC38 tumor-bearing mice;

[0042] Figure 4 Schematic diagram of section staining and analysis of MC38 tumor tissue of the present invention; wherein, A. Representative pictures of Ki67 staining (scale bar is 50 μm) and TUNEL staining (scale bar is 20 μm) of MC38 tumor sections; B. Quantitative analysis of positive regions of Ki67 staining of MC38 tumor sections; C. Quantitative analysis of positive regions of TUNEL staining of MC38 tumor sections;

[0043] Figure 5 Schematic diagram of section and staining analysis of MC38 tumor tissue of the present invention; wherein, A. Representative staining pictures of the tumor center and tumor margin of CD3 (scale bar is 50 μm) of MC38 tumor sections; B. Representative staining pictures of the tumor center and tumor margin of CD8 (scale bar is 50 μm) of MC38 tumor sections; C. Quantitative analysis of CD3-positive T cells at the tumor margin and D. at the tumor center; E. Quantitative analysis of CD8-positive T cells at the tumor margin and F. at the tumor center;

[0044] Figure 6 Schematic diagram of antitumor experiment of LLC tumor-bearing nude mouse model of the present invention; wherein, A. Tumor growth curve of LLC tumor-bearing nude mice; B. Survival curve of LLC tumor-bearing nude mice; C. Individual tumor growth curve corresponding to each group of LLC tumor-bearing nude mice;

[0045] Figure 7 Schematic diagram of antitumor experiment of MC38 tumor-bearing nude mouse model of the present invention; wherein, A. Tumor growth curve of MC38 tumor-bearing nude mice; B. Survival curve of MC38 tumor-bearing nude mice; C. Individual tumor growth curve corresponding to each group of MC38 tumor-bearing nude mice;

[0046] Figure 8 Schematic diagram of safety evaluation of different treatment groups of the present invention; wherein, A. Body weight curve of LLC tumor-bearing C57BL / 6 mice; B. Body weight curve of MC38 tumor-bearing C57BL / 6 mice; C. Body weight curve of LLC tumor-bearing nude mice; D. Body weight curve of MC38 tumor-bearing nude mice; E. Blood biochemical tests of each group of MC38 tumor-bearing C57BL / 6 mice;

[0047] Figure 9 Schematic diagram of H&E section staining of main organs of each group of MC38 tumor-bearing C57BL / 6 mice of the present invention. Detailed implementation manners

[0048] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0049] Embodiment 1:

[0050] As Figure 1 shown, the embodiment of the present invention provides an anti-tumor combination therapy method for improving the efficacy of radiotherapy using nintedanib, including the following steps:

[0051] Step 1: Construct a tumor-bearing mouse model:

[0052] Select male C57BL / 6 mice and male nude mice (BALB / c nu-nu) at 6-8 weeks of age and weighing 20-22 g, and construct a tumor-bearing mouse model subcutaneously on the back of the experimental animals. The tumor cells used are mouse lung cancer cell line LLC or mouse colon cancer cell line MC38. Specifically, the specific steps for constructing the tumor-bearing mouse model are as follows:

[0053] (1) Amplify LLC or MC38 cells in the logarithmic growth phase and in good condition. Use the cell passage method to digest the tumor cells into a cell suspension, and resuspend them with PBS to remove the components of the complete medium;

[0054] (2) Take 20 μl of the cell suspension to calculate the cell concentration. Calculate the total number of cells according to 1×10 6 tumor cells per mouse, and use PBS to adjust the cell concentration to 5×10 6 / ml;

[0055] (3) If the experimental animal is a C57BL / 6 mouse, use a razor to shave the skin of the planned tumor implantation area on the back in advance. Nude mice do not need to be shaved. At the same time, use an ear tag pliers to number the mice;

[0056] (4) Pipette or invert and mix the tumor cell suspension evenly. Use a 1 ml insulin syringe to aspirate the cell suspension, and inject 200 μl of the cell suspension subcutaneously on the back of the shaved or unshaved mice to form a skin bump. Slowly rotate and pull out the needle to avoid leakage. Put the mice back into the cage and record;

[0057] Step 2: Group treatment:

[0058] When the tumor volume of the tumor-bearing mice reaches 70-100 mm 3At that time, the tumor-bearing mice were randomly divided into four groups, namely the control group, the nintedanib monotherapy group, the radiotherapy group, and the combination group, with no less than 7 mice in each group. The mice in the control group, the nintedanib monotherapy group, the radiotherapy group, and the combination group were treated separately:

[0059] Control group: The mice were given intragastric administration of the same volume of sterile ultrapure water for 5 consecutive days and then paused for 1 day, with no more than 2 cycles;

[0060] Nintedanib monotherapy group: The mice were given intragastric administration of 50 mg / kg nintedanib for 5 consecutive days and then paused for 1 day, with no more than 2 cycles;

[0061] Radiotherapy group: On the day of enrollment, the subcutaneous tumor site of the mice was irradiated with 8 Gy electron beam. When irradiating, the mice were anesthetized by intraperitoneal injection of 1.5% sodium pentobarbital solution at a dose of 50 mg / kg. The mice in the control group were only anesthetized;

[0062] Combination group: On the day of enrollment, the subcutaneous tumor site of the mice was irradiated with 8 Gy electron beam. When irradiating, the mice were anesthetized by intraperitoneal injection of 1.5% sodium pentobarbital solution at a dose of 50 mg / kg, and the mice were given intragastric administration of 50 mg / kg nintedanib for 5 consecutive days and then paused for 1 day, with no more than 2 cycles;

[0063] Step three: Physical examination:

[0064] The subcutaneous tumor volume and body weight of the tumor-bearing mice were measured every 3 days. Among them, the subcutaneous tumor volume was measured by the longest diameter and the shortest diameter with a vernier caliper, and the tumor volume was calculated according to the formula tumor volume = (length × width²) / 2;

[0065] Step four: Recording:

[0066] According to the measured data, the tumor growth curve, the survival curve, and the body weight change curve of the mice were drawn to evaluate the anti-tumor effect;

[0067] Step five: Treatment and evaluation:

[0068] The anti-tumor effect and safety of the combination treatment were evaluated by in vivo imaging of the tumor-bearing mice, paraffin embedding of tumor tissues, immunohistochemical staining, TUNEL staining, H&E section staining, blood biochemical detection, and data analysis using the corresponding analysis software.

[0069] Specifically, the in vivo imaging steps include: under a light-proof environment at room temperature, using sterile PBS to prepare a 15 mg / ml working solution of D-luciferin potassium salt and a 15 mg / ml working solution of 1.5% sodium pentobarbital. After preparation, wrap them with tin foil and store them temporarily at 4°C in the dark, and try to prepare and use them immediately. Before putting the mice on the machine, first inject the substrate intraperitoneally into the mice, and then inject anesthetic intraperitoneally 3 - 5 minutes later for anesthesia. After the mice are anesthetized, image acquisition is performed, and after acquisition, use the analysis software SI Imaging to quantitatively analyze the fluorescence intensity of the tumor.

[0070] Secondly, the steps for paraffin embedding of tumor tissues include: euthanize the mice by the method of excessive anesthesia, take out the tumor tissues to be measured from the mice with medical instruments and put them into 4% paraformaldehyde for fixation at room temperature for 24 - 48 h; soak the fixed tissues in 75%, 85%, 90%, and 95% alcohol solutions for 2 h each in turn, then soak them in the first and second cylinders of absolute ethanol for 40 min each, and then soak them in the first cylinder of xylene for 10 min, the second cylinder of xylene for 8 min, the first cylinder of wax for 1 h, and the second cylinder of wax for 1 h in turn until they are in a transparent state; immerse the samples in paraffin liquid with a melting point of 52 - 60°C for at least 2 h for infiltration; place the samples in a -20°C refrigerator to solidify into blocks, trim the wax blocks, perform tissue sectioning and spreading, dry the sections on a baking machine, and label the groups.

[0071] Secondly, the immunohistochemical staining steps include: dewax the paraffin sections in xylene I, II, and III for 5 - 10 min each, through gradient alcohol for 5 min each, and rinse with pure water for 1 min; place the sections in sodium citrate antigen retrieval solution, boil them in a microwave oven at medium heat for 10 min, cool them naturally to room temperature, and transfer them to TBS buffer for shaking and washing for 15 min; incubate with 3% hydrogen peroxide solution at room temperature in the dark for 20 min to block endogenous peroxidase, and wash with TBS for 15 min; add 10% BSA and incubate at room temperature for 30 min; draw a circle around the tissue with a histochemical pen, drop the prepared primary antibody solution, place it flat in a wet box, and incubate at 4°C overnight; after washing with TBS for 15 min, drop the corresponding secondary antibody labeled with HRP and incubate at room temperature for 1 h; after washing with TBS for 15 min, drop the DAB chromogenic solution, control the chromogenic time, the positive is brownish yellow, and rinse with pure water to terminate the chromogenic reaction; counterstain with hematoxylin for 2 min, rinse with pure water, differentiate with hydrochloric acid alcohol for 3 s, rinse with pure water, and blue with hematoxylin blueing solution and then rinse with pure water again; dehydrate and clarify in gradient alcohol and xylene for 2 min each, and finally seal the slices with neutral gum, examine and photograph under a microscope, and perform quantitative analysis using Image J software.

[0072] Secondly, the TUNEL staining procedure includes: dewaxing paraffin sections in xylene I, II, and III for 5 - 10 minutes each, through gradient ethanol for 5 minutes each, and rinsing with pure water for 1 minute; using a histochemical pen to draw a circle around the tissue, dropping the membrane-breaking solution and incubating at room temperature for 15 minutes to break the membrane; after washing with TBS for 15 minutes, dropping a mixture of 3% BSA and 20% fetal bovine serum and incubating at room temperature for 30 minutes for blocking; after removing the blocking solution, dropping an appropriate amount of TUNEL reaction solution, placing it in a wet box in the dark and incubating at 37°C for 2 hours; after washing with TBS for 15 minutes, dropping DAPI to stain the cell nuclei for 5 - 10 minutes; washing with TBS for 15 minutes and using an anti-fluorescence quenching mounting medium to mount the slides, examining and photographing under a fluorescence microscope, and performing quantitative analysis using Image J software;

[0073] Secondly, the H&E section staining procedure includes: dewaxing tumor paraffin sections in xylene I, II, and III for 5 - 10 minutes each, then through gradient ethanol for 5 minutes each, and rinsing with pure water for 1 minute; placing the sections in hematoxylin for staining for 5 minutes, and rinsing with pure water for 1 minute; putting the sections into 1% hydrochloric acid aqueous solution for differentiation for 1 s, and rinsing with pure water for 1 minute; putting the sections into ammonia aqueous solution for bluing, rinsing with pure water and soaking for 15 minutes; putting the sections into 85% and 95% ethanol for 10 minutes each; putting the sections into eosin staining solution for staining for 5 minutes; putting the sections into absolute ethanol I, II, III cylinders, n-butanol, and xylene I, II cylinders for 5 minutes each until transparent; mounting with neutral gum, observing and photographing under a microscope;

[0074] Finally, the blood biochemical detection procedure includes: grasping and fixing the mouse's head, cutting off the whiskers on both sides of the mouse, quickly removing the right eyeball of the mouse using ophthalmic forceps, allowing the blood drop of the eyeball to fall into a pre-heparinized 1.5 ml sterile EP tube, and collecting no less than 500 μl of blood; letting it stand at room temperature for 30 minutes to separate the blood layers, centrifuging at 5000 rpm for 15 minutes in a pre-cooled 4°C centrifuge; aspirating the supernatant into a new sterile EP tube and making marks, setting parameters according to the instructions on a Chemray 240 fully automatic biochemical analyzer and loading samples to detect biochemical related indicators such as alanine aminotransferase, aspartate aminotransferase, blood urea nitrogen, and creatinine.

[0075] It also includes data processing:

[0076] Statistical analysis was performed using GraphPad Prism 8.0 (San Diego, USA) software, and all statistical results were expressed as mean ± standard deviation (SD). Independent t-test (Student's t test), one-way ANOVA test, or two-way ANOVA test was used for difference comparison according to the situation, and the Kaplan-Meier method was used for survival analysis of experimental animals. Statistical significance was considered when P < 0.05 (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

[0077] Example 2:

[0078] To preliminarily explore the anti-tumor efficacy of nintedanib combined with radiotherapy, this experiment used the LLC (mouse lung cancer cell line) tumor cell line to establish a tumor-bearing mouse model in C57BL / 6 mice. 50 mg / kg nintedanib by gavage and 8 Gy subcutaneous tumor site irradiation were used as treatments, and grouping treatments and records were carried out according to the control group (Control), nintedanib monotherapy group (Nintedanib, NDNB), radiotherapy alone group (Radiotherapy, RT), and combination group (Nintedanib combined with Radiotherapy, NDNB+RT) ( Figure 2 A). The results showed that in the LLC tumor-bearing mouse model, both nintedanib and radiotherapy could inhibit tumor growth to a certain extent, but the efficacy of the combination group was more prominent and definite ( Figure 2 B, Figure 2 C), and the individual tumor growth curve could better reflect the anti-tumor advantage of the combination group ( Figure 2 D).

[0079] To verify the broad-spectrum anti-tumor effect of this regimen, this study further conducted the same experiment in a MC38 (mouse colorectal cancer cell line) tumor-bearing mouse model. As shown in Figure 3 A and Figure 3 B, the combined treatment strategy of nintedanib and radiotherapy also showed excellent anti-tumor effects in this model. In vivo imaging of tumor-bearing mice on the 15th day after treatment ( Figure 3 C) and fluorescence intensity quantitative analysis ( Figure 3 D) showed that the tumor volume of mice in the combination group was much smaller than that of other groups. In vivo imaging on the 30th day after treatment showed that except for the combination group, tumor-bearing mice in the other groups reached the end point ( Figure 3 E), indicating that the combined treatment strategy significantly reduced the growth rate of tumors in mice. Individual tumor growth curves ( Figure 3F) It can be seen that the tumors of two mice in the combined group achieved complete remission. Although the influence of individual differences in mice cannot be ruled out, overall, the combined treatment strategy still has obvious advantages over single treatment.

[0080] This study further performed Ki 67 immunohistochemical staining and TUNEL staining on MC38 tumor tissues to determine the proliferation and apoptosis of cells in tumor tissues. The results showed that the cell proliferation marker Ki 67 was lower in the treatment group than in the control group, and the expression of the tumor in the combined group was the lowest ( Figure 4 A and Figure 4 B). The apoptosis indicator TUNEL also showed that the apoptosis-positive area in the combined group was significantly higher than that in the other groups ( Figure 4 A and Figure 4 C). These data further confirm the good therapeutic effect of nintedanib combined with radiotherapy in solid tumor models.

[0081] As an anti-angiogenic agent, nintedanib has been reported to improve the hypoperfusion and immune cell infiltration rate of the tumor area. In this part, we performed immunohistochemical staining of CD3 and CD8 on MC38 tumor tissue to explore the effect of the strategy of nintedanib combined with radiotherapy on the infiltration of immune cells in the tumor microenvironment. The results showed that compared with the control group, the use of nintedanib alone failed to significantly increase the CD3 and CD8 positive T cells in and around the tumor. However, after the combined use of radiotherapy, the number of CD3 and CD8 positive T cells in both the tumor and the peritumor increased significantly ( Figure 5 ). This result demonstrates that nintedanib can enhance the anti-tumor effect of radiotherapy by enhancing immune cell infiltration.

[0082] Embodiment three:

[0083] In order to explore whether the effectiveness of this combination regimen depends on the immune system function of mice, this experiment continued to construct two tumor-bearing mouse models, LLC (mouse lung cancer cell line) and MC38 (mouse colorectal cancer cell line) in immune-deficient nude mice, and still set up a control group, a nintedanib monotherapy group, a simple radiotherapy group, and a combination group for grouping and recording.

[0084] like Figure 6 and Figure 7 The results showed that in both tumor-bearing nude mouse models, nintedanib and radiotherapy could inhibit tumor growth and prolong the survival of nude mice to a certain extent, and the combination group showed a more significant anti-tumor effect.

[0085] Embodiment 4:

[0086] The previous experiments have demonstrated the effectiveness of nintedanib combined with radiotherapy in anti-tumor treatment. Next, we explored the safety of this combination method. During the experiment, the general conditions of the mice, such as diet, defecation, and urination, were closely observed and no obvious abnormalities were found. By recording the body weight change curves of the mice ( Figure 8 A-D), it can be seen that there was no statistically significant difference in body weight between the treatment groups and the control group in both the tumor-bearing mouse models with intact immune function and immune function deficiency. It can be seen that this combination regimen has no obvious toxic and side effects on mice. Further, the eyeball blood of MC38 tumor-bearing C57BL / 6 mice was collected for blood biochemical index detection. Among them, there was no statistical difference in the concentrations of alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), and creatinine (CRE) in each treatment group compared with the control group ( Figure 8 E), which indicates that the treatment with nintedanib and radiotherapy has no adverse effects on the biological functions of the heart, liver, and kidneys.

[0087] To continue to verify the safety, the important organs of each group of MC38 tumor-bearing C57BL / 6 mice, including the heart, liver, spleen, kidneys, lungs, and small intestine tissues, were embedded and stained with H&E sections. Figure 9 It was shown that no obvious pathological phenomena such as inflammation and necrosis were found in these important organs. The above results all preliminarily prove the safety of the treatment strategy of nintedanib combined with radiotherapy in vivo.

[0088] Although the embodiments of the present invention have been shown and described, for those of ordinary skill in the art, it can be understood that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. An anti-tumor combination therapy method using nintedanib to improve the efficacy of radiotherapy, characterized in that: It includes the following steps: Step 1: Establish a tumor-bearing mouse model: Select male mice or male nude mice as experimental animals, and establish a tumor-bearing mouse model subcutaneously on the back of the experimental animals. The tumor cells used are mouse lung cancer cell line LLC or mouse colon cancer cell line MC38; Step 2: Group treatment: When the tumor volume of tumor-bearing mice reaches 70-100 mm 3 , the tumor-bearing mice are randomly divided into four groups: a control group, a nintedanib monotherapy group, a radiotherapy group, and a combination group, with no less than 7 mice in each group; Step 3: Physical examination: Measure the subcutaneous tumor volume and body weight of the tumor-bearing mice every 3 days. Among them, the subcutaneous tumor volume is measured by vernier caliper for the longest diameter and the shortest diameter, and the tumor volume is calculated according to the formula tumor volume = (length × width²) / 2; Step 4: Record: Draw the mouse tumor growth curve, mouse survival curve, and mouse body weight change curve based on the measured data to evaluate the anti-tumor effect; Step 5: Treatment and evaluation: Evaluate the anti-tumor effect and safety of the combination therapy by performing in vivo imaging, paraffin embedding of tumor tissues, immunohistochemical staining, TUNEL staining, H&E section staining, and blood biochemical detection on the tumor-bearing mice, and using the corresponding analysis software for data analysis.

2. The anti-tumor combination therapy method for improving the efficacy of radiotherapy by using nintedanib according to claim 1, characterized in that: In Step 1, select male C57BL / 6 mice or male BALB / c nu-nu nude mice aged 6-8 weeks and weighing 20-22 g as experimental animals.

3. An anti-tumor combination therapy method for improving the efficacy of radiotherapy using nintedanib according to claim 2, characterized in that: In Step 1, the specific steps for establishing the tumor-bearing mouse model are as follows: (1) Amplify LLC or MC38 cells in the logarithmic growth phase with good status, digest the tumor cells into cell suspension by cell passage method, and resuspend with PBS to remove the components of the complete medium; (2) Take 20 μl of cell suspension to calculate the cell concentration. Calculate the total number of cells according to 1×10 6 tumor cells per mouse, and adjust the cell concentration to 5×10 6 / ml using PBS; (3) If the experimental animal is a C57BL / 6 mouse, use a razor to shave the skin of the planned tumor implantation area on the back in advance. Nude mice do not need to be shaved. At the same time, use an ear tag pliers to number the mice; (4) Pipette or invert and mix the tumor cell suspension evenly, suck the cell suspension with a 1 ml insulin syringe, and inject 200 μl of the cell suspension subcutaneously on the back of the mice with or without skin preparation to form a skin bump. Slowly rotate and pull out the needle to avoid leakage, and put the mice back into the cage and record.

4. An anti-tumor combination therapy method for improving the efficacy of radiotherapy using nintedanib according to claim 1, characterized in that: In Step 2, treat the mice in the control group, nintedanib monotherapy group, radiotherapy group, and combination group separately: Control group: Continuously give intragastric administration of the same volume of sterile ultrapure water for 5 days and then pause for 1 day, not exceeding 2 cycles; Nintedanib monotherapy group: Continuously give intragastric administration of 50 mg / kg nintedanib for 5 days and then pause for 1 day, not exceeding 2 cycles; Radiotherapy group: On the day of enrollment, irradiate the subcutaneous tumor site of the mice with 8 Gy electron beam. When irradiating, use 1.5% pentobarbital sodium solution to anesthetize the mice by intraperitoneal injection at a dose of 50 mg / kg. The control group mice are only anesthetized; Combination group: On the day of enrollment, irradiate the subcutaneous tumor site of the mice with 8 Gy electron beam. When irradiating, use 1.5% pentobarbital sodium solution to anesthetize the mice by intraperitoneal injection at a dose of 50 mg / kg, and continuously give intragastric administration of 50 mg / kg nintedanib for 5 days and then pause for 1 day, not exceeding 2 cycles.

5. An anti-tumor combination therapy method for improving the efficacy of radiotherapy using nintedanib according to claim 1, characterized in that: In Step 5, the in vivo imaging step includes: in a light-proof environment at room temperature, preparing a 15 mg / ml working solution of D-luciferin potassium salt with sterile PBS, preparing a 15 mg / ml working solution of 1.5% sodium pentobarbital, wrapping both with tin foil after preparation and storing them temporarily in the dark at 4°C, and using them as soon as possible after preparation; before loading onto the machine, first intraperitoneally inject the substrate into the mouse, and then inject anesthetic intraperitoneally for anesthesia 3 - 5 minutes later; after the mouse is anesthetized, perform image acquisition, and after acquisition, use the analysis software SI Imaging to perform fluorescence intensity quantitative analysis on the tumor.

6. The anti-tumor combination therapy method for improving the efficacy of radiotherapy by using nintedanib according to claim 5, characterized in that: In Step 5, the paraffin embedding step of the tumor tissue includes: sacrificing the mouse by the method of excessive anesthesia, taking out the tumor tissue to be measured from the mouse with medical instruments and placing it in 4% paraformaldehyde for fixation at room temperature for 24 - 48 h; the fixed tissue is successively soaked in 75%, 85%, 90%, and 95% alcohol solutions for 2 h each, then soaked in the first and second cylinders of absolute ethanol for 40 min each, and then successively soaked in the first cylinder of xylene for 10 min, the second cylinder of xylene for 8 min, the first cylinder of wax for 1 h, and the second cylinder of wax for 1 h until it becomes transparent; immersing the sample in a paraffin liquid with a melting point of 52 - 60°C for at least 2 h; placing the sample in a -20°C refrigerator to solidify into a block, trimming the wax block, performing tissue sectioning and spreading, drying the sections on a baking machine, and labeling the groups.

7. An anti-tumor combination therapy method for improving the efficacy of radiotherapy using nintedanib according to claim 6, characterized in that: In Step 5, the immunohistochemical staining step includes: dewaxing the paraffin sections in xylene I, II, and III for 5 - 10 min each, passing through gradient alcohols for 5 min each, and rinsing with pure water for 1 min; placing the sections in a sodium citrate antigen retrieval solution, boiling in a microwave oven on medium heat for 10 min, naturally cooling to room temperature, and transferring to a TBS buffer solution for shaking and washing for 15 min; incubating with 3% hydrogen peroxide solution at room temperature in the dark for 20 min to block endogenous peroxidase, and shaking and washing with TBS for 15 min; adding 10% BSA and incubating at room temperature for 30 min; using a histochemical pen to draw a circle around the tissue, dropping the prepared primary antibody solution, placing it flat in a wet box, and incubating overnight at 4°C; after shaking and washing with TBS for 15 min, dropping the corresponding secondary antibody labeled with HRP and incubating at room temperature for 1 h; after shaking and washing with TBS for 15 min, dropping the DAB chromogenic solution, controlling the chromogenic time, with positive being brownish yellow, and rinsing with pure water to terminate chromogenesis; counterstaining with hematoxylin for 2 min, rinsing with pure water, differentiating with hydrochloric acid alcohol for 3 s, rinsing with pure water, and blueing with hematoxylin blueing solution and then rinsing with pure water again; dehydrating and making transparent in gradient alcohols and xylene for 2 min each, finally mounting with neutral balsam, examining and photographing under a microscope, and performing quantitative analysis using Image J software.

8. An anti-tumor combination therapy method for improving the efficacy of radiotherapy using nintedanib according to claim 7, characterized in that: In step five, the TUNEL staining procedure includes: dewaxing paraffin sections in xylene I, II, and III for 5 - 10 minutes each, passing through gradient ethanol for 5 minutes each, rinsing with pure water for 1 minute; using a histochemical pen to draw a circle around the tissue, adding permeabilization solution and incubating at room temperature for 15 minutes for membrane permeabilization; washing with TBS for 15 minutes, then adding a mixture of 3% BSA and 20% fetal bovine serum and incubating at room temperature for 30 minutes for blocking; removing the blocking solution, adding an appropriate amount of TUNEL reaction solution, incubating in the dark in a wet box at 37°C for 2 hours; washing with TBS for 15 minutes, then adding DAPI to stain the cell nuclei for 5 - 10 minutes; washing with TBS for 15 minutes, mounting with an anti-fluorescence quenching mounting medium, examining and photographing under a fluorescence microscope, and performing quantitative analysis using Image J software.

9. An anti-tumor combination therapy method for improving the efficacy of radiotherapy using nintedanib according to claim 8, characterized in that: In step five, the H&E section staining procedure includes: dewaxing tumor paraffin sections in xylene I, II, and III for 5 - 10 minutes each, then passing through gradient ethanol for 5 minutes each, rinsing with pure water for 1 minute; placing the sections in hematoxylin for staining for 5 minutes, rinsing with pure water for 1 minute; putting the sections into 1% hydrochloric acid aqueous solution for differentiation for 1 s, rinsing with pure water for 1 minute; putting the sections into ammonia aqueous solution for bluing, rinsing with pure water and soaking for 15 minutes; putting the sections into 85% and 95% ethanol for 10 minutes each; putting the sections into eosin staining solution for staining for 5 minutes; putting the sections into absolute ethanol I, II, III tanks, n-butanol, and xylene I, II tanks for 5 minutes each until transparent; mounting with neutral gum, observing and photographing under a microscope.

10. An anti-tumor combination therapy method for improving the efficacy of radiotherapy using nintedanib according to claim 9, characterized in that: In step five, the blood biochemical detection procedure includes: grasping and fixing the mouse's head, cutting off the whiskers on both sides of the mouse, quickly removing the right eyeball of the mouse using ophthalmic forceps, allowing the blood drop of the eyeball to fall into a pre-heparinized 1.5 ml sterile EP tube, collecting no less than 500 μl of blood; allowing the blood to stand at room temperature for 30 minutes to separate the layers, centrifuging at 5000 rpm for 15 minutes in a pre-cooled 4°C centrifuge; aspirating the supernatant into a new sterile EP tube and making a label, setting parameters according to the instructions on a Chemray 240 automatic biochemical analyzer and loading the sample for detecting biochemical related indicators such as alanine aminotransferase, aspartate aminotransferase, blood urea nitrogen, and creatinine.

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