A method for preparing a transplanted tumor tissue and its application

Abstract

The present invention discloses a preparation method and application of transplanted tumor tissues, belonging to the field of biotechnology. Also disclosed is a reagent combination for preparing transplanted tumor tissues, including PMA and interleukins, wherein the interleukins include interleukin 2, interleukin 10 and interleukin 12. By using the reagent combination, kit and method of the present invention to prepare transplanted tumor tissues and further prepare quality control products, complex operations are not required, nor are a large number of consumables, culture equipment and culture space needed, which is very convenient for large-scale production; the transplanted tumor tissues of the present invention not only have stable sources and properties, but also the transplanted tumors have tissue-like morphology. When prepared into quality control products, they can provide more information in pathological interpretation. They can not only be used to monitor the process of immunohistochemistry experiments, but also assist in interpreting pathological results, and have very important clinical application value.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and specifically, relates to a method for preparing a transplanted tumor tissue and its application. Background Art

[0002] The tumor mutation rate caused by gene fusion mutations is low, and it is quite difficult to obtain quality control materials. Taking the anaplastic lymphoma kinase (ALK) fusion gene as an example, the mutation rate of this fusion gene mutation in non-small cell lung cancer is about 4%, and the common fusion form is ALK-EML4. Targeted drug treatment for non-small cell lung cancer with positive ALK fusion mutations can effectively resist drug resistance and prolong the survival period of patients, which is one of the most important targeted treatment indicators. However, due to the low fusion mutation rate, positive pathological samples are not easily obtained, and the characteristics between positive tissues in different batches are very unstable, making it difficult to be used as a stable quality control product for long-term use. The cultivation of cell line quality control materials requires a large amount of culture medium, serum, culture containers, and incubator space, with high costs, long acquisition cycles, and low yields. Technical personnel need to perform expansion and maintenance operations throughout the entire cell growth cycle (usually harvesting a batch every 2 - 3 weeks, with a batch yield of about 200M cells, which can form cell clusters of about 5 mm 3 ), the cell type is single, without morphological characteristics, and it has little value in pathological interpretation. Similar problems exist for quality control products for tumor pathological detection of various fusion gene mutation types. Summary of the Invention

[0003] To solve at least one of the above technical problems, the present invention induces and processes positive cell line NCI-H2228 isolated and established from non-small cell lung cancer with ALK-EML4 fusion gene mutations and negative cell line Jurkat of T lymphocyte tumor not related to the ALK gene, and then injects them into nude mice to form tumor bodies in vivo. After harvesting and identification, the tumor bodies are cut into small pieces and re-implanted into nude mice to form tumors, and through such secondary transplantation operations, transplanted tumors with faster growth rates and more stable characteristics are obtained. The expression of biomarker proteins in the cytoplasm of the tumor cells of the transplanted tumor has a high similarity with the ALK-EML4 fusion gene mutation markers to be detected in pathological tissue samples, and the expression abundance is stable, thus completing the present invention. Specifically, the technical solution of the present invention is as follows:

[0004] The first aspect of the present invention provides a reagent combination for preparing a transplanted tumor tissue, including PMA and interleukins, wherein the interleukins include interleukin 2, interleukin 10, and interleukin 12.

[0005] PMA, also known as TPA, with the full name of Phorbol-12-myristate-13-acetate (PMA), or 12-O-Tetradecanoylphorbol 13-acetate (TPA), is one of the most commonly used phorbol esters, which can be used to stimulate the proliferation of lymphocytes and the expression of cytokines in vitro. Interleukin, abbreviated as IL, refers to lymphokines that interact between white blood cells or immune cells and is a type of cytokine. In the present invention, the inventors unexpectedly found that the compounding of PMA and three interleukins can significantly promote tumorigenesis, which is unpredictable to those skilled in the art.

[0006] In some preferred embodiments of the present invention, the mixing ratio of interleukin-2, interleukin-10, and interleukin-12 is 1:1:1.

[0007] The second aspect of the present invention provides a method for preparing a transplanted tumor tissue, comprising the following steps:

[0008] S1, culturing the cells for tumorigenesis in the corresponding complete medium, passaging 2 - 3 times, controlling the cell density between 80% - 90% during each passage, controlling the passage ratio at 1:3, and performing passage or changing the medium about once every 3 days according to the cell state;

[0009] S2, adding PMA, interleukin-2, interleukin-10, and interleukin-12 to the complete medium, resuspending the cells to obtain a cell suspension, recovering the culture for 1 - 3 h, and injecting the cell suspension into the armpit of a nude mouse;

[0010] S3, normally raising the nude mouse and observing the tumorigenesis situation, cutting off the subcutaneous mass and trimming to remove the excess connective tissue, and the obtained tissue is the transplanted tumor tissue.

[0011] In the present invention, the corresponding complete medium refers to a medium that can satisfy the growth and reproduction of the corresponding cell line. Usually, a complete medium is obtained by adding substances such as serum and antibiotics to a basal medium.

[0012] In some embodiments of the present invention, the cells for tumorigenesis include target positive cells and / or target negative cells. In some specific embodiments of the present invention, for immunohistochemical detection of the ALK-EML4 fusion gene mutation, the target positive cells are the positive cell line NCI-H2228 isolated and established from non-small cell lung cancer with the ALK-EML4 fusion gene mutation; the target negative cells are the negative cell line Jurkat of T lymphocyte tumor not related to the ALK gene. The two types of cells are respectively prepared into transplanted tumor quality control products according to the above method, that is, the positive quality control product and the negative quality control product for the detection of the ALK-EML4 fusion gene mutation are obtained, which can significantly improve the accuracy and specificity of using immunohistochemistry to detect the ALK-EML4 fusion gene mutation.

[0013] In the present invention, after subculturing for 2 to 3 generations in step S1, when the cells under the microscope show a uniform polygonal shape, a smooth and plump cell membrane edge, a cytoplasm without vacuoles and no floating cell debris in the culture medium, it indicates that the cells have fully recovered and can be expanded for use.

[0014] In some embodiments of the present invention, between steps S2 and S3, it further includes a step of synchronizing the cells: transferring the cells to a basal medium without fetal bovine serum and its substitutes for starvation treatment for 16 to 24 hours, and then transferring the cells to a complete medium for culture, and collecting the cells in the mitotic phase. Performing starvation treatment can control all cells in the same cell cycle, so it is also called synchronization treatment. When transferred to a complete medium for culture again, it will stimulate the cells to grow rapidly, and they can be in the vigorous mitotic phase after 2 hours. At this time, the tumorigenicity of the cells is higher than that of ordinary cells without any treatment.

[0015] In some embodiments of the present invention, in step S2, the final concentration of PMA is 50 to 500 ng / mL; the final concentrations of interleukin-2, interleukin-10, and interleukin-12 are 50 to 100 ng / mL respectively. In some preferred embodiments of the present invention, after adding PMA, interleukin-2, interleukin-10, and interleukin-12 to the complete medium, the concentration of PMA is 100 ng / mL; the concentrations of interleukin-2, interleukin-10, and interleukin-12 are 80 ng / mL respectively.

[0016] In some embodiments of the present invention, after step S3, it further includes a step of cutting the obtained transplanted tumor tissue into pieces, washing it, and transplanting it onto another nude mouse, then raising it again and obtaining the transplanted tumor tissue, repeating at least once. Repeated inoculation can obtain a satisfactory tumorigenesis rate and tumorigenesis speed within a certain number of times. In the present invention, re-inoculation once has already had a significant effect.

[0017] The third aspect of the present invention provides the use of the reagent combination described in the first aspect of the present invention in transplanted tumor tissues.

[0018] Further, the reagent combination is used to prepare the transplanted tumor tissue based on any of the methods described in the second aspect of the present invention.

[0019] Further, a quality control product is prepared using the obtained transplanted tumor tissue, and the preparation method of the quality control product includes:

[0020] a) The obtained transplanted tumor is trimmed to remove peripheral blood vessels, fat, and connective tissue, and then cut into approximately 150 mm 3 regular blocks. After washing with running water for 5 min, the blocks are immersed in 50 mL of 10% neutral formalin buffer solution and fixed at room temperature for 48 h.

[0021] b) After 48 h, wash with running water for 5 min, dehydrate with 50% ethanol for 4 h, 75% ethanol for 4 h, 95% ethanol for 16 h, and absolute ethanol for 4 h. After dehydration is completed, immerse in 50 mL of xylene for 1 h; after 1 h, immerse the tissue blocks in a 65 °C wax bath for 16 h; after 16 h, embed the tissue blocks into wax block boxes, place them in a -20 °C cooler to cool and demold, and then store the wax blocks at 4 °C in the dark for temporary storage.

[0022] c) Take out the transplanted tumor wax blocks and place them in a -20 °C freezer for 1 h to harden. After the hardened wax blocks are properly fixed on a microtome, first set the section thickness to 5 μm to trim the wax blocks until the transplanted tumor tissue is completely exposed on the surface, and then set the section thickness to 2 μm for continuous sectioning.

[0023] Optionally, the quality control product is a slide quality control product. Further, place the sections on the water surface of a spreading machine to fully expand them, then pick them up to the center position of the glass slide, and place them on a section rack and bake in an oven at 67 °C for 2 h to ensure that the transplanted tumor tissue is firmly attached to the glass slide.

[0024] Optionally, the transplanted tumor tissue quality control product is a suspension quality control product. Further, the preparation method of the suspension includes: Collect 200 sections into 50 mL centrifuge tubes respectively, place the centrifuge tubes in a -80 °C ultra-low temperature freezer for 2 h, then quickly crush the wax slices with a glass rod, add 30 mL of xylene and shake at room temperature to dissolve the wax slices; centrifuge the solution at 3000 rpm for 7 min, discard the supernatant, add 30 mL of absolute ethanol to resuspend the cell precipitate, centrifuge at 2000 rpm for 5 min and wash once, discard the supernatant and repeat washing once with absolute ethanol, then resuspend the cells with 20 mL of 95% ethanol; pass the cell suspension through a 200-mesh sieve to remove the undissolved paraffin and larger cell clumps, and then store it at -20 °C for temporary storage.

[0025] The fourth aspect of the present invention provides a transplanted tumor tissue prepared by using the method described in the second aspect of the present invention.

[0026] The fifth aspect of the present invention provides the use of the transplanted tumor tissue described in the fourth aspect of the present invention in the preparation of a quality control product for immunohistochemical detection.

[0027] The sixth aspect of the present invention provides a quality control product prepared by using the transplanted tumor tissue described in the fourth aspect of the present invention.

[0028] The seventh aspect of the present invention provides an immunohistochemical detection kit, which is characterized by comprising the quality control product described in the sixth aspect of the present invention.

[0029] Advantages of the present invention

[0030] Compared with the prior art, the present invention has the following advantages:

[0031] The transplanted tumor quality control product prepared by using the reagent combination, kit and method of the present invention has a high similarity with the target to be detected in the sample, and the expression abundance is stable.

[0032] When preparing the transplanted tumor quality control product by using the reagent combination, kit and method of the present invention, after inoculating nude mice, except for feeding, no complex manual operations are required, and no large amount of consumables, culture equipment and culture space are needed (after inoculating a tumor body with stable traits, the growth cycle is only 1 week, and each nude mouse can produce about 1 tumor body of 400 mm 3 or so), which is very convenient for large-scale production.

[0033] The transplanted tumor quality control product of the present invention not only has a stable source and traits, but also the transplanted tumor has a tissue-like morphology, which can provide more information in pathological interpretation. It can not only be used to monitor the immunohistochemistry experiment process, but also assist in interpreting pathological results, and has very important clinical application value.

[0034] The section transplanted tumor quality control product of the present invention also contains stroma, lymphocytes and connective tissue in the section, which is similar to the morphology of actual pathological sections. As a quality control product, this section can judge whether there are mistakes in the immunohistochemical experiment process resulting in untrustworthy staining results according to the staining intensity and location. At the same time, it also has morphological characteristics, which will have a positive reference and comparison significance for the interpretation of the final results. Description of the drawings

[0035] Figure 1 Shows the growth curves of NCI-H2228 cells and Jurkat cells, ① first passage; ② second passage.

[0036] Figure 2 Shows the cell morphology of NCI-H2228 cells before synchronization and 24 h after synchronization.

[0037] Figure 3The immunohistochemical staining results of the negative cell Jurkat xenograft tumor and the Jurkat cell line are shown.

[0038] Figure 4 The immunohistochemical staining results of the positive cell NCI-H2228 xenograft tumor and the NCI-H2228 cell line are shown.

[0039] Figure 5 The immunohistochemical staining results of the positive cell NCI-H2228 xenograft tumor suspension and the negative cell Jurkat xenograft tumor suspension are shown. Detailed implementation manners

[0040] Unless otherwise specified, implied from the context or in accordance with the convention of the prior art, all parts and percentages in this application are based on weight, and the testing and characterization methods used are synchronized with the filing date of this application. Where applicable, any patents, patent applications or published content referred to in this application are incorporated herein by reference in their entirety, and their equivalent family patents are also incorporated by reference, particularly the definitions of relevant terms in the art disclosed in these documents. If the definition of a specific term disclosed in the prior art is inconsistent with any definition provided in this application, the definition of the term provided in this application shall prevail.

[0041] The numerical ranges in this application are approximate values, and thus may include values outside the ranges unless otherwise specified. The numerical ranges include all values from the lower limit value to the upper limit value increased by 1 unit, provided that there is at least a 2-unit interval between any lower value and any higher value. For ranges containing values less than 1 or containing fractions greater than 1 (such as 1.1, 1.5, etc.), 1 unit is appropriately regarded as 0.0001, 0.001, 0.01 or 0.1. For ranges containing single digits less than 10 (such as 1 to 5), 1 unit is usually regarded as 0.1. These are merely specific examples of what is intended to be expressed, and all possible combinations of the values between the lowest and highest values listed are considered to be clearly recited in this application.

[0042] The terms "comprising", "including", "having" and their derivatives do not exclude the presence of any other components, steps or processes, and are independent of whether or not these other components, steps or processes are disclosed in the present application. To remove any doubt, unless expressly stated otherwise, all compositions in the present application using the terms "comprising", "including" or "having" may contain any additional additives, excipients or compounds. In contrast, the term "consisting essentially of" excludes any other components, steps or processes from the scope of anything recited below the term, except for those necessary for operability. The term "consisting of" does not include any components, steps or processes not specifically described or listed. Unless expressly stated otherwise, the term "or" refers to the individual members listed or any combination thereof.

[0043] In order to make the technical problems, technical solutions and beneficial effects solved by the present invention more clear and understandable, the present invention will be further described in detail below in conjunction with embodiments.

[0044] Embodiment

[0045] The following examples are used herein to demonstrate the preferred embodiments of the present invention. Those skilled in the art will understand that the techniques disclosed in the following examples represent techniques that the inventors have found can be used to implement the present invention, and thus can be regarded as preferred embodiments for implementing the present invention. However, those skilled in the art should understand from this specification that many modifications can be made to the specific embodiments disclosed herein and still obtain the same or similar results without departing from the spirit or scope of the present invention.

[0046] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs, and all materials cited herein and their cited materials will be incorporated by reference.

[0047] Those skilled in the art will recognize or, through routine experimentation, will be able to appreciate many equivalent techniques to the specific embodiments of the invention described herein. These equivalents will be included in the claims.

[0048] For the molecular biology experimental methods not specifically described in the following embodiments, they are all carried out according to the specific methods listed in "Molecular Cloning: A Laboratory Manual" (Fourth Edition) (J. Sambrook, M.R. Green, 2017), or according to the kit and product instructions. For other experimental methods, unless otherwise specified, they are all conventional methods. The instrument and equipment used in the following embodiments, unless otherwise specified, are all conventional laboratory instrument and equipment; the test materials used in the following embodiments, unless otherwise specified, are all purchased from conventional biochemical reagent stores.

[0049] Example 1 Cell culture and selection of injection site

[0050] a) Preheat RPMI 1640 medium (complete medium) containing fetal bovine serum at a final concentration of 10%. Add 9 mL of the complete medium to a 15 mL centrifuge tube.

[0051] b) Prepare 2 L of warm water at 39 °C. Take out one tube of 1 mL of NCI-H2228 cells (Guangzhou Saiku Biotechnology, CC0244) and Jurkat cells (Guangzhou Saiku Biotechnology, CC1902) stored in liquid nitrogen and place them in the warm water, continuously stirring at a constant speed. Melting the cells in 1 min, and add the cell suspension to the centrifuge tube prepared in step a). Centrifuge at 900 rpm for 5 min.

[0052] c) Discard the supernatant, add 15 mL of complete medium, resuspend evenly, and then take 1×10 6 cells and inoculate them into one 75 cm 2 cell culture flask respectively and place it in the cell culture incubator to start culturing. At the same time, take another 5×10 4 cells, mix them evenly and inoculate them into 3 wells of a 24-well plate to prepare for counting and drawing the cell growth curve.

[0053] d) After 24 h, record that the number of NCI-H2228 cells in the 24-well plate is 1.2×10 5 cells, and the number of Jurkat cells is 1.8×10 5 ; after 48 h, record that the number of NCI-H2228 cells in the 24-well plate is 3×10 5 cells, and the number of Jurkat cells is 6.3×10 5 ; after 72 h, record that the number of NCI-H2228 cells in the 24-well plate is 1.4×10 6 cells, and the number of Jurkat cells is 2.8×10 6 , and draw the cell growth curve. The cell density in the cell culture flask is 80%, and subculture can be carried out.

[0054] e) For NCI-H2228, discard the original medium in the culture flask, add 5 mL of PBS buffer to wash once. Add 1 mL of EDTA-trypsin to digest the cells. After 2 min, the cells detach from the bottom of the culture flask and the digestion is completed. Then add 5 mL of complete medium to terminate the reaction, and resuspend the cells into a 15 mL centrifuge tube, centrifuge at 900 rpm for 5 min, discard the supernatant, and add 5 mL of fresh medium to resuspend the cells; for Jurkat, collect the cells into a 50 mL centrifuge tube, centrifuge at 900 rpm for 5 min, discard the supernatant, and add 5 mL of fresh medium to resuspend the cells.

[0055] f) Take 1×10 6 cells each of NCI-H2228 cells and Jurkat cells and inoculate them into one new 75 cm2 The cell culture flask containing complete medium was placed in the cell culture incubator to start culturing. At the same time, another 5×10 4 cells were mixed and then seeded into three wells of a 24-well plate respectively to prepare for the second drawing of the cell growth curve.

[0056] g) After 24 h, the number of NCI-H2228 cells in the 24-well plate was recorded as 1.3×10 5 cells, and the number of Jurkat cells was 1.8×10 5 ; after 48 h, the number of NCI-H2228 cells in the 24-well plate was recorded as 3.2×10 5 cells, and the number of Jurkat cells was 6.4×10 5 ; after 72 h, the number of NCI-H2228 cells in the 24-well plate was recorded as 1.4×10 6 cells, and the number of Jurkat cells was 2.7×10 6 , and the curves drawn according to the data of the two cell growth curves highly overlapped ( Figure 1 ). Under the microscope, NCI-H2228 cells presented uniform-sized polygons, with smooth and plump cell membrane edges, uniform cytoplasm without vacuoles, and Jurkat cells grew in clusters, with smooth and plump cell membrane edges, and the cell density was close to 85%, so expansion could be carried out.

[0057] h) For NCI-H2228, the original medium in the culture flask was discarded, and 5 mL of PBS buffer was added to wash once. 1 mL of EDTA-trypsin was added to digest the cells. After 2 min, the cells detached from the bottom of the culture flask and the digestion was completed. Then 5 mL of complete medium was added to terminate the reaction, and the cells were resuspended in a 15 mL centrifuge tube, centrifuged at 900 rpm for 5 min, the supernatant was discarded, and 5 mL of fresh medium was added to resuspend the cells; for Jurkat, the cells were collected into a 50 mL centrifuge tube, centrifuged at 900 rpm for 5 min, the supernatant was discarded, and 5 mL of fresh medium was added to resuspend the cells.

[0058] i) Two types of cells were seeded into eight 175 cm 6 culture flasks containing complete medium at 2×10 2 cells per flask respectively and placed in the cell culture incubator to start culturing.

[0059] j) After 72 h, under the microscope, NCI-H2228 cells presented uniform-sized polygons, with smooth and plump cell membrane edges, uniform cytoplasm without vacuoles, and Jurkat cells grew in clusters, with smooth and plump cell membrane edges, and the cell density was close to 90%, and preparation for collection was made.

[0060] k) For NCI-H2228 cells, discard the original culture medium in the culture flask. Add 5 mL of PBS buffer to each flask to wash the cells once. Then add 2 mL of EDTA-trypsin to digest the cells. After 2 minutes, the cells detach from the bottom of the culture flask and the digestion is complete. Add 5 mL of complete culture medium to terminate the reaction. Pipette and collect all the cells from the culture flasks into a 50-mL centrifuge tube, and centrifuge at 900 rpm for 5 minutes. For Jurkat cells, collect them into a 50-mL centrifuge tube and centrifuge at 900 rpm for 5 minutes.

[0061] l) Discard the supernatant of NCI-H2228 and Jurkat cells. Add 2 mL of PBS buffer to resuspend and count the cells. Dilute the cells to 1×10 9 / mL. Take 1 mL of the cell suspension from each and store it temporarily at 4°C for preparing to inject nude mice.

[0062] Prepare 16 six-week-old female BALB / c nude mice. Take them out one by one from the sterile package. Hold the tail of the nude mouse with the ring finger and little finger of one hand, and hold the neck of the mouse with the index finger and middle finger. Place the nude mouse abdomen-down on the grid rack, let the four limbs of the nude mouse naturally grasp the grid rack, and fully expose the back. Then use a syringe to slowly inject 200 μL of cells into the subcutaneous area under the left forelimb axilla of the nude mouse. Wait for 3 - 4 seconds and then slowly withdraw the syringe from the subcutaneous area to prevent the cells from flowing out of the injection site. After completion, put the nude mouse back into the sterile package, and inject a total of 4 mice. Change the position to the subcutaneous area of the left hindlimb groin and inject 200 μL of cells in the same way. Inject a total of 4 mice. The nude mice injected with NCI-H2228 cells are labeled as Ma1+, Ma2+, Ma3+, Ma4+ (axilla), Mg1+, Mg2+, Mg3+, Mg4+ (groin); the mice injected with Jurkat cells are labeled as Ma1-, Ma2-, Ma3-, Ma4- (axilla), Mg1-, Mg2-, Mg3-, Mg4- (groin).

[0063] m) Raise the nude mice normally and start observing the tumor formation 5 days after injection.

[0064] After 10 days, subcutaneous masses formed in Ma3+, Mg2+, Ma1-, and Ma4-. Observe and measure them every day. At 14 days, excise subcutaneous masses of about 10 mm from Ma1- and Ma4- and wash them 10 times with PBS buffer. After trimming and removing the excess connective tissue, measure the tumor volumes as Ma1-: 10 mm × 8 mm × 5 mm, Ma4-: 11 mm × 7 mm × 7 mm; at 18 days, excise subcutaneous masses of about 10 mm from Ma3+ and wash them 10 times with PBS buffer. After trimming and removing the excess connective tissue, measure the tumor volume as 9 mm × 6 mm × 5 mm; the subcutaneous mass of Mg2+ still did not reach the 10-mm standard at 24 days. After excision, it was found that the mass was an inflammatory cyst and no tumor was formed. The tumor formation results are shown in Table 1:

[0065] Table 1 Influence of Cell Seeding Location on Tumorigenesis

[0066]

[0067] It can be seen that the method of injecting cells through the armpit is significantly superior to the method of injecting through the groin in terms of tumorigenesis rate.

[0068] Example 2 Cell Synchronization Treatment

[0069] a) Refer to steps a) to h) of Example 1.

[0070] b) Two types of cells were seeded into 12 cell culture flasks with a specification of 175 cm 6 at a density of 2×10 2 cells per flask and placed in a cell culture incubator to start culturing.

[0071] c) After 56 h, under the microscope, NCI-H2228 cells presented uniform-sized polygons, with smooth and plump cell membrane edges and uniform cytoplasm without vacuoles. Jurkat cells grew in clusters, with smooth and plump cell membrane edges and a cell density close to 65%. Synchronization treatment was ready to be carried out.

[0072] d) For NCI-H2228 cells, the original culture medium in the culture flask was discarded, and 30 mL of RPMI 1640 medium (basic medium) without fetal bovine serum was added to each flask; for Jurkat cells, they were collected into a 50 mL centrifuge tube and centrifuged at 900 rpm for 5 min. The supernatant was discarded, and the cells were resuspended in 12 mL of basic medium and evenly divided into the original culture flasks, with 29 mL of basic medium added to each flask. NCI-H2228 cells and Jurkat cells were placed in the incubator for synchronization treatment for 16 h, 20 h, and 24 h (the cell states of NCI-H2228 cells before and after synchronization treatment are shown in Figure 2 ).

[0073] e) After 16 h, 20 h, and 24 h respectively, the supernatant of NCI-H2228 and Jurkat cells was discarded, and 20 mL of fresh complete medium was added to each flask and placed in the incubator for recovery culture for 2 h.

[0074] f) After recovery, the original culture medium in the culture flask of NCI-H2228 was discarded, and 5 mL of PBS buffer was added to wash once. 1 mL of EDTA-trypsin was added to digest the cells. After 2 min, the cells detached from the bottom of the culture flask and the digestion was completed. Then 5 mL of complete medium was added to terminate the reaction, and the cells were resuspended in a 15 mL centrifuge tube and centrifuged at 900 rpm for 5 min. The supernatant was discarded, and 5 mL of fresh medium was added to resuspend the cells; after recovery, Jurkat cells were collected into a 50 mL centrifuge tube and centrifuged at 900 rpm for 5 min, the supernatant was discarded, and 5 mL of fresh medium was added to resuspend the cells.

[0075] g) Discard the supernatant of NCI-H2228 and Jurkat cells, add 2 mL of PBS buffer to resuspend and count the cells, dilute the cells to 1000 M / mL, take 1 mL of cell suspension each and place it at 4 °C for temporary storage for injection into nude mice.

[0076] h) Prepare a total of 24 six-week-old female BALB / c nude mice. Take them out from the sterile package, hold the tail of the nude mouse with the ring finger and little finger of one hand, hold the neck of the mouse with the index finger and middle finger, place the nude mouse abdomen down on the grid rack, let the four limbs of the nude mouse naturally grasp the grid rack, and after the back is fully unfolded and exposed, slowly inject 200 μL of cells into the axillary subcutaneous area of the left forelimb of the nude mouse with a syringe. Wait for 3 - 4 seconds and then slowly withdraw the syringe from the subcutaneous area to prevent the cells from flowing out of the injection port. After completion, put the nude mouse back into the sterile package, and inject a total of 4 mice. The nude mice injected with NCI-H2228 cells are labeled as 16h: Ma5+, Ma6+, Ma7+, Ma8+; 20h: Ma9+, Ma10+, Ma11+, Ma12+; 24h: Ma13+, Ma14+, Ma15+, Ma16+; the nude mice injected with Jurkat cells are labeled as 16: Ma5-, Ma6-, Ma7-, Ma8-; 20h: Ma9-, Ma10-, Ma11-, Ma12-; 24h: Ma13-, Ma14-, Ma15-, Ma16-.

[0077] i) Raise the nude mice normally and start observing the tumor formation situation 5 days after injection.

[0078] After 9 days, Ma6+, Ma7+, Ma9+, Ma12+, Ma13+, Ma15+, Ma5-, Ma6-, Ma10-, Ma11-, Ma14-, and Ma15- formed subcutaneous masses, which were observed and measured daily. On day 12, the approximately 10-mm subcutaneous masses of Ma5-, Ma6-, Ma10-, Ma11-, Ma14-, and Ma15- were excised, washed 10 times with PBS buffer, and after trimming to remove excess connective tissue, the tumor volumes were measured as follows: Ma5: 11 mm × 7 mm × 7 mm, Ma6-: 10 mm × 5 mm × 6 mm, Ma10-: 9 mm × 6 mm × 7 mm, Ma11-: 10 mm × 6 mm × 7 mm, Ma14-: 11 mm × 5 mm × 7 mm, Ma15-: 8 mm × 9 mm × 7 mm. On day 14, the approximately 10-mm subcutaneous masses of Ma6+, Ma7+, Ma9+, Ma12+, Ma13+, and Ma15+ were excised, washed 10 times with PBS buffer, and after trimming to remove excess connective tissue, the tumor volumes were measured as follows: Ma6+: 12 mm × 8 mm × 7 mm, Ma7+: 10 mm × 7 mm × 7 mm, Ma9+: 9 mm × 7 mm × 7 mm, Ma12+: 11 mm × 5 mm × 7 mm, Ma13+: 10 mm × 6 mm × 8 mm, Ma15+: 12 mm × 5 mm × 5 mm. As shown in Table 2:

[0079] Table 2 Effects of cell synchronization treatment on tumorigenesis

[0080]

[0081] As can be seen from Table 2, the cells treated with synchronization were more stable and consistent in tumorigenesis time and slightly increased the tumorigenesis rate. Different synchronization times had no effect on the final number of tumors formed and the tumorigenesis time.

[0082] Example 3 Cell PMA Activation Treatment

[0083] a) Refer to steps a) to h) of Example 1.

[0084] b) Refer to steps b) to c) of Example 2.

[0085] c) For NCI-H2228 cells, discard the original culture medium in the culture flask, and add 30 mL of RPMI 1640 medium without fetal bovine serum (basic medium) to each flask of cells; for Jurkat cells, collect them into a 50-mL centrifuge tube, centrifuge at 900 rpm for 5 min, discard the supernatant, add 12 mL of basic medium to resuspend the cells, and evenly divide them into the original culture flasks, adding 29 mL of basic medium to each flask. NCI-H2228 cells and Jurkat cells were placed in an incubator for synchronization treatment for 24 h (for reasonable time arrangement).

[0086] d) After 24 h, discard the supernatant of NCI-H2228 and Jurkat cells. Add 20 mL of fresh complete medium and different concentrations of PMA (50 ng / mL, 100 ng / mL, 500 ng / mL) to each flask and place them in an incubator for 2 h of recovery culture.

[0087] e) Refer to steps f) to h) of Example 2. The nude mice injected with NCI-H2228 cells are labeled as:

[0088] 50 ng / mL PMA: Ma17+, Ma18+, Ma19+, Ma20+;

[0089] 100 ng / mL PMA: Ma21+, Ma22+, Ma23+, Ma24+;

[0090] 500 ng / mL PMA: Ma25+, Ma26+, Ma27+, Ma28+;

[0091] The nude mice injected with Jurkat cells are labeled as:

[0092] 50 ng / mL PMA: Ma17-, Ma18-, Ma19-, Ma20-;

[0093] 100 ng / mL PMA: Ma21-, Ma22-, Ma23-, Ma24-;

[0094] 500 ng / mL PMA: Ma25-, Ma26-, Ma27-, Ma28-.

[0095] f) The nude mice are normally fed, and the tumor formation is observed 5 days after injection.

[0096] Ma17+, Ma19+, Ma21+, Ma22+, Ma24+, Ma17-, Ma18-, Ma19-, Ma21-, Ma22-, Ma23-, Ma24- and Ma26- all formed subcutaneous masses after 7 days, which were observed and measured daily. On day 11, the approximately 10-mm subcutaneous masses of Ma17-, Ma18-, Ma19-, Ma21-, Ma22-, Ma23-, Ma24-, Ma26- were excised, washed 10 times with PBS buffer, and after trimming to remove excess connective tissue, the tumor volumes were measured as Ma17-: 12 mm × 8 mm × 9 mm, Ma18-: 10 mm × 8 mm × 8 mm, Ma19-: 9 mm × 8 mm × 7 mm, Ma21-: 11 mm × 6 mm × 8 mm, Ma22-: 11 mm × 8 mm × 7 mm, Ma23-: 10 mm × 9 mm × 7 mm, Ma24-: 12 mm × 8 mm × 9 mm, Ma26-: 9 mm × 8 mm × 10 mm. On day 13, the approximately 10-mm subcutaneous masses of Ma6+, Ma7+, Ma9+, Ma12+, Ma13+, Ma15+ were excised, washed 10 times with PBS buffer, and after trimming to remove excess connective tissue, the tumor volumes were measured as Ma17+: 9 mm × 8 mm × 11 mm, Ma19+: 12 mm × 9 mm × 9 mm, Ma21+: 10 mm × 8 mm × 8 mm, Ma22+: 12 mm × 9 mm × 7 mm, Ma24+: 10 mm × 9 mm × 11 mm. As shown in Table 3:

[0097] Table 3 Effects of PMA treatment on tumorigenesis

[0098]

[0099] As can be seen from Table 3, the tumorigenesis rate of the cells was significantly increased after activation with an appropriate concentration of PMA, and the tumorigenesis time could be accelerated.

[0100] Example 4 Activation treatment of cell mixture with interleukin

[0101] a) Refer to steps a) to h) of Example 1.

[0102] b) Two types of cells were inoculated into 16 cell culture flasks with a specification of 175 cm 6 at 2×10 2 cells per flask and placed in a cell culture incubator to start culturing.

[0103] c) After 56 h, observation under a microscope showed that NCI-H2228 cells presented uniform-sized polygons, with smooth and plump cell membrane edges and uniform cytoplasm without vacuoles. Jurkat cells presented clustered growth, with smooth and plump cell membrane edges and a cell density close to 65%. Synchronization treatment was ready to be carried out.

[0104] d) For NCI-H2228 cells, discard the original culture medium in the culture flask, and add 30 mL of RPMI 1640 medium (basic medium) without fetal bovine serum to each flask of cells; for Jurkat cells, collect them into a 50 mL centrifuge tube, centrifuge at 900 rpm for 5 min, discard the supernatant, add 16 mL of basic medium to resuspend the cells, evenly divide them into the original culture flasks, and add 29 mL of basic medium to each flask. Incubate NCI-H2228 cells and Jurkat cells in an incubator for synchronization treatment for 24 h.

[0105] e) After 24 h, discard the supernatant of NCI-H2228 and Jurkat cells. Divide 16 flasks of each type of cell into 4 groups, and add 20 mL of complete medium containing 80 ng / mL IL-2 (interleukin-2), 80 ng / mL IL-10 (interleukin-10), 80 ng / mL IL-12 (interleukin-12), and a mixture of 80 ng / mL IL-2, 80 ng / mL IL-10, and 80 ng / mL IL-12 to each group, then place them in an incubator for culturing for 2 h.

[0106] f) Refer to steps f) to g) of Example 2

[0107] g) A total of 32 six-week-old female BALB / c nude mice were taken out from the sterile package. Hold the tail of the nude mouse with the ring finger and little finger of one hand, hold the neck of the mouse with the index finger and middle finger, place the nude mouse abdomen down on the grid rack, let the four limbs of the nude mouse naturally grip the grid rack, and after the back is fully unfolded and exposed, slowly inject 200 μL of cells into the subcutaneous area of the left anterior limb axilla of the nude mouse with a syringe. Wait for 3 - 4 seconds and then slowly withdraw the syringe from the subcutaneous area to prevent the cells from flowing out of the injection port. After completion, place the nude mouse back into the sterile package, and a total of 4 mice were injected. The nude mice injected with NCI-H2228 cells were labeled as:

[0108] 80 ng / mL IL-2: Ma28+, Ma29+, Ma30+, Ma31+;

[0109] 80 ng / mL IL-10: Ma32+, Ma33+, Ma34+, Ma35+;

[0110] 80 ng / mL IL-12: Ma36+, Ma37+, Ma38+, Ma39+;

[0111] 80 ng / mL IL-2, IL-10, IL-12 mixture: Ma40+, Ma41+, Ma42+, Ma43+;

[0112] The nude mice injected with Jurkat cells were labeled as:

[0113] 80 ng / mL IL-2: Ma28-, Ma29-, Ma30-, Ma31-;

[0114] 80 ng / mL IL-10: Ma32-, Ma33-, Ma34-, Ma35-;

[0115] 80 ng / mL IL-12: Ma36-, Ma37-, Ma38-, Ma39-;

[0116] 80 ng / mL IL-2, IL-10, IL-12 mixture: Ma40-, Ma41-, Ma42-, Ma43-;

[0117] h) The nude mice were normally raised, and the tumor formation was observed starting 5 days after injection.

[0118] On the 9th day, subcutaneous masses formed in Ma28+, Ma31+, Ma32+, Ma33+, Ma36+, Ma39+, Ma40+, Ma41+, MMa43+, Ma28-, Ma29-, Ma32-, Ma36-, Ma39-, Ma41-, Ma42- and Ma43-. They were observed and measured daily. On the 12th day, subcutaneous masses of about 10 mm were excised, washed 10 times with PBS buffer, and after trimming to remove excess connective tissue, the tumor volumes were measured as Ma28-: 10 mm × 8 mm × 6 mm, Ma29-: 9 mm × 5 mm × 6 mm, Ma32-: 9 mm × 8 mm × 7 mm, Ma36-: 9 mm × 10 mm × 7 mm, Ma39-: 11 mm × 6 mm × 6 mm, Ma41-: 8 mm × 9 mm × 7 mm, Ma42-: 9 mm × 6 mm × 7 mm, Ma43-: 12 mm × 8 mm × 5 mm. On the 14th day, subcutaneous masses of about 10 mm were excised from Ma6+, Ma7+, Ma9+, Ma12+, Ma13+, Ma15+ and washed 10 times with PBS buffer. After trimming to remove excess connective tissue, the tumor volumes were measured as Ma28+: 10 mm × 8 mm × 7 mm, Ma31+: 11 mm × 7 mm × 7 mm, Ma32+: 9 mm × 8 mm × 8 mm, Ma33+: 10 mm × 5 mm × 6 mm, Ma36+: 11 mm × 6 mm × 6 mm, Ma36+: 10 mm × 5 mm × 5 mm, Ma39+: 10 mm × 6 mm × 6 mm, Ma40+: 11 mm × 9 mm × 5 mm, Ma41+: 8 mm × 9 mm × 8 mm, MMa43+: 8 mm × 8 mm × 7 mm. As shown in Table 4:

[0119] Table 4 Effects of mixed interleukins on tumor formation

[0120]

[0121] As can be seen from Table 4, the application of a single interleukin has no significant effect on the tumorigenesis rate and tumorigenesis time; the combined use of interleukins can increase the tumorigenesis rate.

[0122] Example 5 Cell Synchronization Treatment, Activation with PMA and Combined Interleukins

[0123] a) Refer to steps a) to h) of Example 1.

[0124] b) Two types of cells were inoculated into 8 cell culture flasks with a specification of 175 cm 6 at a density of 2×10 2 cells per flask and placed in a cell culture incubator to start culturing.

[0125] c) After 56 h, under a microscope, NCI-H2228 cells showed uniform-sized polygons, with smooth and plump cell membrane edges and uniform cytoplasm without vacuoles. Jurkat cells showed clustered growth, with smooth and plump cell membrane edges and a cell density close to 65%. Synchronization treatment was ready to be carried out.

[0126] d) For NCI-H2228 cells, the original culture medium in the culture flask was discarded, and 30 mL of RPMI 1640 medium (basic medium) without fetal bovine serum was added to each flask; for Jurkat cells, they were collected into a 50 mL centrifuge tube and centrifuged at 900 rpm for 5 min. The supernatant was discarded, and the cells were resuspended with 16 mL of basic medium and evenly divided into the original culture flasks, with 29 mL of basic medium added to each flask. NCI-H2228 cells and Jurkat cells were placed in an incubator for synchronization treatment for 24 h.

[0127] e) After 24 h, the supernatant of NCI-H2228 and Jurkat cells was discarded, and 20 mL of fresh complete medium containing 100 ng / mL PMA and 80 ng / mL of combined interleukins was added to each flask and placed in an incubator for recovery culture for 2 h.

[0128] f) Refer to steps f) to g) of Example 2.

[0129] g) A total of 8 six-week-old female BALB / c nude mice were taken out of the sterile package. Hold the tail of the nude mouse with the ring finger and little finger of one hand, hold the neck of the mouse with the index finger and middle finger, place the nude mouse abdomen-down on the grid rack, let the four limbs of the nude mouse naturally grip the grid rack, and after the back is fully unfolded and exposed, slowly inject 200 μL of cells subcutaneously into the axilla under the left forelimb of the nude mouse with a syringe. After waiting for 3 - 4 seconds, slowly withdraw the syringe from the subcutaneous tissue to prevent the cells from flowing out of the injection port. After completion, put the nude mouse back into the sterile package, and a total of 4 were injected. The nude mice injected with NCI-H2228 cells were labeled as Ma44+, Ma45+, Ma46+, Ma47+; the nude mice injected with Jurkat cells were labeled as Ma44-, Ma45-, Ma46-, Ma47-.

[0130] h) The nude mice were normally raised, and the tumor formation was observed 5 days after injection.

[0131] Seven days later, subcutaneous masses formed in Ma44+, Ma45+, Ma46+, Ma47+, Ma44-, Ma45-, Ma46-, Ma47-. Observe and measure them every day. On the 11th day, subcutaneous masses of about 10 mm were excised from Ma44-, Ma45-, Ma46-, Ma47- and washed 10 times with PBS buffer. After trimming and removing the excess connective tissue, the tumor volumes were measured as Ma44-: 9 mm × 7 mm × 9 mm, Ma45-: 10 mm × 5 mm × 5 mm, Ma46-: 9 mm × 8 mm × 8 mm, Ma47-: 8 mm × 10 mm × 7 mm. On the 13th day, subcutaneous masses of about 10 mm were excised from Ma44+, Ma45+, Ma46+, Ma47+ and washed 10 times with PBS buffer. After trimming and removing the excess connective tissue, the tumor volumes were measured as Ma44+: 9 mm × 10 mm × 6 mm, Ma45+: 10 mm × 5 mm × 5 mm, Ma46+: 10 mm × 8 mm × 5 mm, Ma47+: 8 mm × 9 mm × 9 mm. As shown in Table 5:

[0132] Table 5 Effects of cell synchronization treatment, PMA combined with mixed interleukin activation on tumor formation

[0133]

[0134] Cell synchronization treatment and activation will significantly increase the tumor formation rate and shorten the tumor formation time, as shown in Table 6.

[0135] Table 6 Effects of synchronization treatment, PMA treatment, mixed interleukin treatment, and combined use on tumor formation

[0136]

[0137] It can be seen that the combined use of multiple optimization methods significantly shortens the tumor formation time and increases the tumor formation rate.

[0138] Example 6 Tumor Tissue Re-inoculation

[0139] a) After trimming the NCI-H2228 positive tumor and Jurkat negative tumor obtained in Example 5 to remove connective tissue in an ice box, they were cut into small pieces about 1 mm 3 in size and washed 10 times with 10 mL of sterile PBS to prepare for subcutaneous inoculation of tumor pieces into nude mice.

[0140] b) Eight 6-week-old female BALB / c nude mice were taken out one by one from the sterile package, anesthetized with chloral hydrate, placed supine on the dissection stand, and their limbs, caudal vertebrae, and cervical vertebrae were fixed to fully expose the chest and abdomen. The chest and abdomen of the nude mice to be inoculated were sterilized thoroughly with 75% ethanol. An incision about 5 mm long was made under the left armpit with an ophthalmic scissors, and the skin and fascia at the incision were carefully peeled apart with forceps. Three tumor pieces were buried and the incision was sutured. The wound was disinfected again and the nude mice were put back into the sterile package. The mice were checked every 0.5 h to confirm that they had woken up and were in normal condition. Four nude mice were inoculated with positive NCI-H2228 tumor pieces and labeled as Mx1+, Mx2+, Mx3+, Mx4+; four nude mice were inoculated with negative Jurkat tumor pieces and labeled as Mx1-, Mx2-, Mx3-, Mx4-.

[0141] c) The nude mice were fed normally, and the tumor formation was observed 5 days after inoculation.

[0142] Five days later, subcutaneous masses were formed in all 8 nude mice. They were observed and measured every day. On the 9th day, subcutaneous masses about 10 mm in size were excised from Mx1-, Mx2-, Mx3-, Mx4- and washed 10 times with PBS buffer. After trimming to remove excess connective tissue, the tumor volumes were measured as Mx1-: 9 mm × 6 mm × 6 mm, Mx2-: 9 mm × 8 mm × 5 mm, Mx3-: 10 mm × 6 mm × 7 mm, Mx4-: 11 mm × 6 mm × 7 mm; on the 11th day, subcutaneous masses about 10 mm in size were excised from Mx1+, Mx2+, Mx3+, Mx4+ and washed 10 times with PBS buffer. After trimming to remove excess connective tissue, the tumor volumes were measured as Mx1-: 10 mm × 8 mm × 6 mm, Mx2-: 9 mm × 5 mm × 5 mm, Mx3-: 10 mm × 7 mm × 7 mm, Mx4-: 10 mm × 6 mm × 8 mm. As shown in Table 7:

[0143] Table 7 Tumor Formation after Re-inoculation

[0144]

[0145] As can be seen from Table 7, the tumor formation rate and tumor formation speed of repeated inoculation of tumor masses are both higher than those of cell inoculation. The tumor masses obtained by re-inoculation can also be re-inoculated multiple times, and satisfactory tumor formation rates and tumor formation speeds can be obtained within a certain number of times. In this example, significant effects have been achieved after re-inoculation once.

[0146] Example 7 Comparison between transplanted tumor sections and cell lines

[0147] a) The transplanted tumors obtained in Example 5 were trimmed to remove peripheral blood vessels, fat, and connective tissue, and then cut into regular blocks of about 150 mm 3 After rinsing with running water for 5 min, they were immersed in 50 mL of 10% neutral formalin buffer solution at room temperature for fixation for 48 h.

[0148] b) After 48 h, they were rinsed with running water for 5 min, dehydrated with 50% ethanol for 4 h, 75% ethanol for 4 h, 95% ethanol for 16 h, and absolute ethanol for 4 h. After dehydration was completed, they were immersed in 50 mL of xylene for transparency for 1 h. After 1 h, the tissue blocks were immersed in a wax bath at 65 °C for 16 h. After 16 h, the tissue blocks were embedded in a wax block box. After being placed at -20 °C for cooling and demolding, the wax blocks were stored temporarily at 4 °C in the dark.

[0149] c) After taking out the wax blocks of positive cell transplanted tumors and negative cell transplanted tumors and putting them at -20 °C for hardening for 1 h, after the hardened wax blocks were properly fixed on a microtome, first set the section thickness to 5 μm to trim the wax blocks until the transplanted tumor tissue was completely exposed on the surface, and then set the section thickness to 2 μm for continuous sectioning. After the sections were completely spread on the water surface of a spreading machine, they were fished to the center position of the glass slide.

[0150] d) The sections were placed on a section rack and baked in an oven at 67 °C for 2 h to ensure that the transplanted tumor tissue was firmly attached to the glass slide, and at the same time, the paraffin melted and dripped from the sections.

[0151] e) After 2 h, the baked sections were put into a dewaxing solution cylinder to wash away the remaining paraffin, and then gradually hydrated by immersing them in absolute ethanol, 95% ethanol, 75% ethanol, 50% ethanol, and pure water for 5 min each.

[0152] f) The hydrated sections were immersed in an alkaline repair solution with a pH of 9.0 and boiled for repair at high temperature for 30 min, and then naturally cooled to room temperature.

[0153] g) The sections were rinsed with running water for 1 min and placed in 3% hydrogen peroxide for treatment for 10 min to remove the activity of endogenous peroxidase.

[0154] h) After the completion of the enclosure, the sliced samples are rinsed with running water for 1 minute, then the water is shaken off and the samples are laid flat on the sample loading rack. 100 μL of ALK monoclonal antibody (Hangzhou Bailin Biotechnology Co., Ltd., Cat#BX50187 BP6165) is added dropwise to cover the entire range of the transplanted tumor slices, and incubated at room temperature for 30 minutes.

[0155] i) After the incubation is completed, rinse with running water for 1 minute, shake off the water, lay flat on the sample loading rack, and add 100 μL of secondary antibody (Hangzhou Bailin Biotechnology Co., Ltd., Cat#BX10001) dropwise to cover the entire range of the transplanted tumor slices, and incubate at room temperature for 30 minutes.

[0156] j) Rinse with running water for 1 minute, shake off the water, lay flat on the sample loading rack, and add 100 μL of chromogenic solution (Hangzhou Bailin Biotechnology Co., Ltd., Cat#BX10002) dropwise to cover the entire range of the transplanted tumor slices, and incubate at room temperature for 2 minutes.

[0157] k) Rinse with running water for 1 minute, and gradually dehydrate the stained slices by immersing them in 50% ethanol, 75% ethanol, 95% ethanol, and absolute ethanol for 5 minutes each.

[0158] l) Air-dry the slices, add neutral balsam dropwise, and then cover with a cover glass to observe the staining results under a microscope.

[0159] The results of the immunohistochemistry experiment showed that the transplanted tumor of negative cells Jurkat and the Jurkat cell line also showed no positive (brown) staining ( Figure 3 ); compared with the control of the transplanted tumor of positive cells NCI-H2228 and the NCI-H2228 cell line, they had the same staining intensity and cell localization, and at the same time had a compact tissue morphology that the cells did not have ( Figure 4 ). In addition to tumor cells, there were also stromal cells and various epithelial cells. The rich cell types and tissue morphology could not only be used as a quality control product in the immunohistochemistry experiment process, but also could be directly compared with pathological tissue samples as a reference for judging the diagnostic results.

[0160] Observed under a microscope, in the tumor tissue sections, the medium to strong cytoplasmic staining of the tumor tissue was consistent with the subcellular localization of AKL. At the same time, the sections also contained stroma, lymphocytes, and connective tissue, which were similar to the morphology of actual pathological sections. As a quality control product, this section could judge whether there were mistakes in the immunohistochemistry experiment process resulting in untrustworthy staining results according to the staining intensity and localization. At the same time, it also had morphological characteristics, which would have a positive reference and comparison significance for the interpretation of the final results.

[0161] Example 8 Application of the Suspension of Transplanted Tumors

[0162] a) The transplanted tumors obtained in Example 5 were trimmed to remove peripheral blood vessels, fat, and connective tissue, and then cut into regular blocks about 150 mm in size. After rinsing with running water for 5 min, they were immersed in 50 mL of 10% neutral formalin buffer solution and fixed at room temperature for 48 h. 3 After 48 h, they were rinsed with running water for 5 min, dehydrated with 50% ethanol for 4 h, 75% ethanol for 4 h, 95% ethanol for 16 h, and absolute ethanol for 4 h. After dehydration was completed, they were immersed in 50 mL of xylene for 1 h for clearing. After 1 h, the tissue blocks were immersed in a wax bath at 65 °C for 16 h. After 16 h, the tissue blocks were embedded in wax block boxes. After being placed at -20 °C for cooling and demolding, the wax blocks were stored at 4 °C in the dark for temporary storage.

[0163] b) After 48 h, they were rinsed with running water for 5 min, dehydrated with 50% ethanol for 4 h, 75% ethanol for 4 h, 95% ethanol for 16 h, and absolute ethanol for 4 h. After dehydration was completed, they were immersed in 50 mL of xylene for 1 h for clearing. After 1 h, the tissue blocks were immersed in a wax bath at 65 °C for 16 h. After 16 h, the tissue blocks were embedded in wax block boxes. After being placed at -20 °C for cooling and demolding, the wax blocks were stored at 4 °C in the dark for temporary storage.

[0164] c) After taking out the wax blocks of positive cell transplanted tumors and negative cell transplanted tumors and placing them at -20 °C for hardening for 1 h, after properly fixing the hardened wax blocks on the microtome, first set the section thickness to 5 μm to trim the wax blocks until the transplanted tumor tissue was completely exposed on the surface, and then set the section thickness to 1 μm for continuous sectioning. 200 sections were respectively collected into 50 mL centrifuge tubes.

[0165] d) The centrifuge tubes were placed at -80 °C for cryopreservation for 2 h, and then the wax slices were quickly mashed with a glass rod. 30 mL of xylene was added and shaken at room temperature to dissolve the wax slices. The solution was centrifuged at 3000 rpm for 7 min. The supernatant was discarded, and 30 mL of absolute ethanol was added to resuspend the cell pellet. It was centrifuged at 2000 rpm for 5 min for washing once. After discarding the supernatant and repeating the washing once with absolute ethanol, the cells were resuspended with 20 mL of 95% ethanol. The cell suspension was passed through a 200-mesh sieve to remove the undissolved paraffin and larger cell clumps, and then stored at -20 °C.

[0166] e) Refer to Patent CN202210394699.9 to prepare positive transplanted tumor suspension and negative transplanted tumor suspension.

[0167] f) After thoroughly mixing the suspension, 1 μL was respectively taken and dropped on the left and right sides of the test sample section, and then placed in an oven at 67 °C for baking for 1 h.

[0168] g) After 1 h, the baked sections were placed in a dewaxing solution cylinder to wash away the remaining paraffin, and then gradually hydrated by immersing in absolute ethanol, 95% ethanol, 75% ethanol, 50% ethanol, and pure water for 5 min each.

[0169] h) The hydrated sections were immersed in an alkaline repair solution with a pH of 9.0 and boiled for repair at high temperature for 30 min, and then naturally cooled to room temperature.

[0170] i) The sections were rinsed with running water for 1 min and placed in 3% hydrogen peroxide for 10 min to remove the activity of endogenous peroxidase.

[0171] j) After the completion of the enclosure, the sliced specimens are rinsed with running water for 1 min, then the water is shaken off and the specimens are laid flat on the sample loading rack. 100 μL of ALK monoclonal antibody (Cat#BX50187 BP6165, Hangzhou Bailin Biotechnology Co., Ltd.) is added dropwise so that the antibody covers the entire range of the specimens to be tested and the transplanted tumor suspension, and incubated at room temperature for 30 min.

[0172] k) After the incubation is completed, rinse with running water for 1 min, shake off the water, lay flat on the sample loading rack, and add 100 μL of secondary antibody (Cat#BX10001, Hangzhou Bailin Biotechnology Co., Ltd.) dropwise so that the secondary antibody covers the entire range of the specimens to be tested and the transplanted tumor suspension, and incubate at room temperature for 30 min.

[0173] l) Rinse with running water for 1 min, shake off the water, lay flat on the sample loading rack, and add 100 μL of chromogenic solution (Cat#BX10002, Hangzhou Bailin Biotechnology Co., Ltd.) dropwise so that the chromogenic solution covers the entire range of the specimens to be tested and the transplanted tumor suspension, and incubate at room temperature for 2 min.

[0174] m) Rinse with running water for 1 min, and gradually dehydrate the stained sliced specimens by immersing them in 50% ethanol, 75% ethanol, 95% ethanol, and absolute ethanol for 5 min each in gradient.

[0175] n) Air-dry the sliced specimens, add neutral balsam dropwise, and then cover with a cover glass to observe the staining results under a microscope.

[0176] The results of the immunohistochemistry experiment showed that the transplanted tumor suspension of negative cells Jurkat showed no positive staining; the transplanted tumor suspension of positive cells NCI-H2228 showed moderate cytoplasmic staining, and the performance of the negative suspension and the positive suspension was consistent with the expected performance ( Figure 5 ), indicating that the process of this immunohistochemistry experiment was correct and the experimental results were reliable.

[0177] The reference value of the suspension form of the transplanted tumor for the interpretation of pathological results is limited. However, since it is on the same slice as the specimens to be tested, it is more accurate and rigorous as an in-slice experimental process monitoring. At the same time, due to its liquid form, it can achieve a fast and efficient application method, giving it an irreplaceable advantage in high-throughput batch detection.

[0178] All the documents mentioned in the present invention are cited in this application for reference as if each document was individually cited for reference. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

Claims

1. A method for preparing transplanted tumor tissues, characterized in that, it comprises the following steps: S1, culturing the cells for tumorigenesis in the corresponding complete medium and subculturing 2 to 3 times; S2, adding phorbol 12-myristate 13-acetate, interleukin-2, interleukin-10 and interleukin-12 into the complete medium so that the concentration of phorbol 12-myristate 13-acetate is 100 ng / mL; the concentrations of interleukin-2, interleukin-10 and interleukin-12 are 80 ng / mL respectively, resuspending the cells to obtain a cell suspension, recovering the culture for 1 to 3 h, and injecting the cell suspension into the axilla of nude mice; S3, normally raising the nude mice and observing the tumorigenesis situation, cutting off the subcutaneous mass and trimming to remove the redundant connective tissue, and the obtained tissue is the transplanted tumor tissue.

2. The method according to claim 1, characterized in that, between step S2 and step S3, it further comprises a step of synchronizing the cells: transferring the cells to a basal medium without fetal bovine serum and its substitutes for starvation treatment for 16 to 24 h, then transferring the cells to the complete medium for culture, and collecting the cells in the mitotic phase.

3. The method according to claim 1 or 2, characterized in that, after step S3, it further comprises a step of cutting the obtained transplanted tumor tissue into pieces, washing and transplanting it onto another nude mouse, raising again and obtaining the transplanted tumor tissue, and repeating at least once.

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