Tumor organoid model as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to a tumor organoid model and a preparation method and application thereof. In a traditional co-culture mode, the photothermal effect of the nano material is combined with the immune checkpoint inhibitor for synergistic activation. A PD-1 antibody is loaded on a gold nanocage material with a good photothermal effect, so that an activation mode of one-time delivery and double effects is realized. Experimental data show that the photo-thermal combined immune checkpoint inhibitor can increase the cell apoptosis proportion in a model, and meanwhile, the inflammatory factor level and the T cell activation proportion are uniformly and remarkably increased. It is proved by facts that the invention constructs an immune activated organ-like model.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a tumor organoid model and a preparation method and application thereof. Background Art

[0002] Tumors have always been a major threat to human life and health, and one of the puzzles that life science researchers have been trying to find solutions to for many years. At the beginning, the study of tumors was mainly focused on animal models. Medical workers had a preliminary understanding of tumors through dissection and pathological analysis of the remains and lesion tissues of patients or cancerous animals. With the advancement of biotechnology, some scientists have tried to culture tumor cells in vitro: using surface-treated cell culture bottles and suitable culture media, tumor cells obtained from the lesion site are spread on the floor of the culture bottle in a two-dimensional pattern for growth. After passage and purification, tumor cell lines that can grow indefinitely are obtained. So far, the traditional models of tumor research: cell line models and animal models have been established. In theory, cell line models can be infinitely passaged, cheap, and simple to operate, but the two-dimensional growth model is difficult to simulate the three-dimensional structure in tumor tissue, the bionic effect is poor, and the mutation of genetic material will accumulate with the increase in the number of passages. Animal models are the models with the closest bionic effect to human tumor lesion tissues, but there are also many problems such as interspecies differences, high cost, low success rate, and complex operation. There is a huge difference in the degree of bionics between the two, which leads to the data of some drugs and materials after cell line experiments being difficult to support their experimental results in animal models.

[0003] In order to fill the technological gap between the two, organoid technology came into being: this is an in vitro tumor model with good bionic effects, which provides a three-dimensional growth environment for tumor cells. At the same time, the configuration of the culture medium retains the stemness of tumor cells and can maintain the ability of tumor cells to divide and differentiate while stably propagating them. At the same time, it is relatively low in cost, simple to operate, and has a reliable modeling success rate.

[0004] However, with the development of organoid technology, scientists have put forward new requirements for organoid models. After traditional organoids are purified through passage, many components in the tumor microenvironment are gradually lost, especially the loss of the immune microenvironment, which greatly weakens the biomimetic effect of organoids.

[0005] There are several organoid culture methods in the prior art:

[0006] Microfluidic culture solution: Microfluidic technology miniaturizes bioreactors and nanoreactors to the micron or millimeter level, and integrates them on tissue chips, achieving cell culture, regulation, coercion and analysis through precise manipulation of fluids. Specific culture media for different tumor organoids are transported in pipelines, providing nutrients to transport metabolic waste while providing a personalized growth environment (such as pH value, pressure, etc.) for a variety of tumor organoids. This culture method retains the cell subpopulations in the original tumor tissue to the greatest extent, and can also inject the targeted immune cells that need to be added into the organoid culture environment through the transport pipeline to achieve co-culture.

[0007] Air-liquid interface culture scheme: Compared with microfluidics technology, the air-liquid interface culture scheme (ALI) does not require the use of special equipment, so it is easier to try to culture. This method embeds tumor organoids or tissues into matrix gel in a transwell chamber, then places the transwell chamber into the holes of the cell culture plate, and fills the holes with the corresponding culture medium of the organoids for culture. The nutrients in the culture medium and the metabolic waste produced by the organoids will pass through the bottom of the transwell chamber, and the matrix gel medium is exposed to the air to provide sufficient oxygen for the organoids. This culture method is often used in tumor tissue biopsies because it can maintain the tumor genotype and phenotype in the original tissue, and also retains stromal cells, fibroblasts, helper T cells (Th), cytotoxic T cells (CTL), B cells, NK cells and macrophages.

[0008] Organoid culture to reconstruct the tumor microenvironment: In most existing tumor organoid cultures, the original microenvironment stromal cells and immune cells will gradually be lost. At this time, if the tumor microenvironment needs to be reconstructed, exogenous lymphocytes, T cells, CAR cells, etc. need to be supplemented into the culture system. Although the bionic effect of this culture method is not as good as the original tumor microenvironment culture program in theory, it is easy to operate and is also one of the current mainstream research directions. And because the organoids have been cultured and formed, the enrichment of tumor cells is high and the cell activity is good, the success rate of this culture method is much higher than the original culture program.

[0009] In recent years, tumor immunotherapy has made breakthrough progress and has been widely used in clinical practice. PD-1 / PD-L1 is an important immune checkpoint molecule. Under normal circumstances, PD-L1 can bind to PD-1 on T cells to inhibit the activation of T cells, thereby avoiding the occurrence of autoimmune diseases. Many tumors suppress T cell responses by highly expressing PD-L1, thereby escaping immune killing. In tumor patients, T cell activation can be significantly enhanced after treatment with PD-1 / PD-L1 blocking antibodies. In theory, the action of immune checkpoint inhibitors can enhance the activity of T cells and activate the anti-tumor ability of the patient's own immune system, while most chemotherapy drugs, because they do not have a targeted effect, can damage the body's immune system and produce serious side effects. This leads to antagonism between clinical chemotherapy and immunotherapy. Due to the lack of an immune activation model, the antagonism between treatment methods is often ignored during the development of new drugs, which greatly reduces the activation of immune checkpoint inhibitors and increases the cost of treatment for patients. Summary of the invention

[0010] In order to solve the problem that the existing organoid culture schemes for reconstructing the tumor microenvironment lack an immune activation model, the antagonism between treatment methods is often ignored during new drug development, which greatly reduces the activation of immune checkpoint inhibitors and increases the treatment costs of patients, the present invention makes the following improvements.

[0011] The purpose of the first aspect of the present invention is to provide a method for constructing a tumor organoid model.

[0012] The second aspect of the present invention aims to provide a tumor organoid model.

[0013] The purpose of the third aspect of the present invention is to provide the construction method of the first aspect of the present invention and / or the application of the tumor organoid model of the second aspect of the present invention.

[0014] In order to achieve the above-mentioned purpose of the present invention, the technical solution adopted by the present invention is:

[0015] The first aspect of the present invention provides a method for constructing a tumor organoid model, comprising the following steps:

[0016] After co-culturing tumor organoids with immune cells, gold nanomaterials were added for treatment;

[0017] The gold nanomaterial is loaded with an immune activator.

[0018] Preferably, the immune activator is a PD-1 antibody.

[0019] Preferably, the tumor organoid comprises in situ tumor cells.

[0020] Preferably, the in situ tumor cells are fresh cells initially separated from the lesion site from the patient's tissue.

[0021] Preferably, the tumor comprises gastric cancer.

[0022] Preferably, the immune cells include T cells.

[0023] Preferably, the T cells are T cells from the same source as the tumor organoids.

[0024] Preferably, the culturing step comprises:

[0025] Pre-treat the plate and add immune cells after the tumor organoids have settled.

[0026] Preferably, the ratio of tumor organoids to immune cells is 1:(10-30).

[0027] Preferably, the flat plate pretreatment includes gluing the flat plate.

[0028] Preferably, the processing steps include:

[0029] Laser irradiation is performed after the addition of gold nanomaterials.

[0030] Preferably, the laser is infrared light.

[0031] Preferably, the intensity of the laser irradiation is 0.1 to 10 W / cm 2 ; The wavelength of the laser is 700~900nm.

[0032] Preferably, the ratio of the gold nanomaterial to the culture medium is 5-20 μg / mL.

[0033] In some embodiments of the present invention, the culturing process of tumor organoids according to the first aspect of the present invention comprises the following steps:

[0034] 1) The lesion sites were initially separated from the fresh patient tissues and washed by vortexing in PBS buffer containing 10% double antibody.

[0035] 2) The tumor tissue was initially mechanically broken with ophthalmic scissors, and then 1 mL of collagenase IV (1 mg / mL) was added and digested on a 37°C metal bath shaker for 3 to 8 h until the liquid became turbid.

[0036] 3) After the EP tube is left to settle naturally, the upper turbid liquid is removed and centrifuged at 500-2000 rpm for 1-5 minutes. The supernatant is discarded and DMEM complete medium containing 10% FBS is taken to terminate digestion and wash.

[0037] 4) Mix the digested tumor cells with Matrigel at 4°C, inoculate the plate, and gel at 37°C for 20-40 min. Then gently add organoid culture medium along the wall of the well plate. For a 96-well plate, add 100 μL of organoid culture medium to each well, and for a 24-well plate, add 500 μL of organoid culture medium to each well.

[0038] 5) After seeding, place the plates in a 37°C CO2 incubator and continuously observe the growth status of the organoids under a light microscope. Change the medium after 2 to 3 days to ensure the supply of nutrients.

[0039] In some embodiments of the present invention, the immune cell isolation process of the first aspect of the present invention comprises the following steps:

[0040] 1) Peripheral blood was extracted from the patient corresponding to the organoid, and then an equal volume of PBS was added to dilute the blood concentration.

[0041] 2) Add 10-20 mL of human lymphocyte separation solution to the lower end of the peripheral blood immune lymphocyte separation tube, and then gently and slowly inject the diluted blood into the upper layer of the lymphocyte separation tube. Set the slowest acceleration and deceleration gears, and centrifuge at 800-1200 rpm for 30 minutes.

[0042] 3) After centrifugation, obvious stratification can be seen in the lymphocyte separation tube. Aspirate the middle white film layer and transfer it to a new centrifuge tube. After washing with PBS, centrifuge at 800-1200 rpm to discard the supernatant and expand the culture.

[0043] 4) Count the cells obtained and calculate the number of cells to be obtained. 5 ~10 7 The cells were cultured at a density of 100 / mL and kept in a 37°C CO2 incubator. The cells were counted before each subculture and maintained at this density.

[0044] 5) On the second day of culture, CD3 / CD28 activator was added at a culture medium volume ratio of 1:50.

[0045] 6) Repeat the subculturing operation to expand the culture of PBMCs.

[0046] In some embodiments of the present invention, the preparation process of the gold nanomaterial described in the first aspect of the present invention comprises the following steps: preparation of silver cubes, preparation of gold nanocage materials, and preparation of gold nanocages loaded with PD-1 antibodies (aPD-1@Au NCs).

[0047] Preparation of Silver Cube:

[0048] 1) Dissolve 312 mg (1.4 mmol) of CF3COOAg in 5 mL of ethylene glycol. The concentration of the mother liquor is 282 mmol / L.

[0049] 2) Dissolve 300 mg PVP (Mw=55 kDa) in 15 mL ethylene glycol. The concentration of the mother solution is 20 mg / mL.

[0050] 3) Take 5 μL of 37% HCl solution and add 20 mL of ethylene glycol. The concentration of the mother solution is 2.5 mmol / L.

[0051] 4) Dissolve 8.4 mg 0.15 mmol NaHS in 5 mL ethylene glycol and dilute 10 times. The stock solution concentration is 3 mmol / L.

[0052] 5) Add 50 mL of ethylene glycol to a 250 mL round-bottom flask and heat to 150 °C in an oil bath and maintain for 25 min.

[0053] 6) Add 0.6 mL of NaHS stock solution and maintain for 2 min.

[0054] 7) Add 5 mL of HCl-ethylene glycol and 12.5 mL of PVP mother solution and maintain for 5 minutes.

[0055] 8) Add 4 mL of CF3COOAg mother solution and take samples of 10 mL after 15 min and 30 min respectively.

[0056] 9) After 45 min, place in an ice water bath to terminate the reaction.

[0057] 10) Centrifuge the mixture at 4000 rpm for 20 min with two times acetone and discard the supernatant.

[0058] 11) Wash twice with pure water ultrasonic centrifugation at 10000 rpm for 10 min.

[0059] 12) Transfer to a 100 mL glass bottle and store in pure water at 4°C. The Ag concentration is about 0.5 mg / mL.

[0060] Preparation of gold nanocage materials:

[0061] 1) Take 10mL of 0.5mg / mL Ag cube and add 150mg PVP and 90mL pure water.

[0062] 2) Maintain vigorous stirring at 90°C for 10 min.

[0063] 3) Inject 1 mmol / L HAuCl4 at 0.7 mL / min.

[0064] 4) The wavelength of the LSPR peak stops at 808 nm, and the UV / Vis spectrophotometer scans the range of 300-900 nm.

[0065] 5) Stir at 90°C for 5 min.

[0066] 6) Wash with pure water, centrifuge at 10000 rpm for 10 min, wash three times.

[0067] 7) Wash once with NH4OH and twice with anhydrous ethanol.

[0068] 8) Take a portion of Au NCs, dry and weigh them, and store the remaining materials at 4°C with pure water as solvent.

[0069] Preparation of gold nanocages loaded with PD-1 antibody (aPD-1@Au NCs):

[0070] 1) The gold nanocage material was infiltrated in 5-20 mg / mL PD-1 antibody in a super-volume incubator at 4°C for 1 day.

[0071] 2) Wash with pure water and centrifuge at 10000rpm for 10 minutes, wash three times.

[0072] In some embodiments of the present invention, the cell culture medium of the first aspect of the present invention is configured according to the following ideas:

[0073] Steam Cell T cell expansion medium (ImmunoCult TM -XF T Cell Expansion Medium) was used as the basis, and culture media with different concentration gradients of single components were prepared with reference to the components of organoid culture medium to test the sensitivity of immune cells to the components of organoid culture medium and to determine the formula of co-culture medium.

[0074] First, small molecules that may affect T cell activity were tested. One of Nic (10mM), Nac (0.5mM), A8301 (0.25μM), Y27632 (10μM), and CHIR99021 (5μM) was added to the PRMI 1640 culture medium to detect whether these small molecules that may affect T cell activity can be added to the co-culture medium.

[0075] Then, co-culture complete medium was prepared according to the concentration of Nic (10mM), Nac (0.5mM), A8301 (0.25μM), Glutamax (1mM), HEPES (10mM), primocin (1×), B27 (1×), Y27632 (10μM), LDN193189 (0.1μM), CHIR99021 (5μM), SB202190 (1.5μM), Gastrin 1 (10nM), FGF 10 (20μg / mL), human EGF (50ng / mL), PE2 (10nM), RP001 (100ng / mL), Noggin (100ng / mL), FGF7 (5ng / mL), Neuregulin 1 (25ng / mL), FBS (5%), and cultured with the obtained PBMCs for 7 days. The PBMCs culture medium was used as a control. Detect the effect of co-culture complete medium on the activity of T cells and organoids.

[0076] In some embodiments of the present invention, the culturing step of the tumor organoid model in the first aspect of the present invention comprises the following steps:

[0077] 1) Spread Matrigel glue evenly on the bottom of the board and let it self-level overnight at room temperature.

[0078] 2) Digest the organoids rapidly at 37°C for 1 min, then terminate the digestion rapidly.

[0079] 3) Resuspend the organoids after removing the matrix gel with a small amount of organoid culture medium, and drop the resuspension on the surface of the Matrigel plate, paying attention to concentrate the drops on the flatter area in the middle as much as possible.

[0080] 4) Let stand for 6 to 8 hours to allow the organoids to settle on the surface of the matrix gel.

[0081] 5) The immune cells were then resuspended in co-culture medium and counted.

[0082] 6) Add immune cell co-culture suspension at a ratio of 1:(10-30) between the number of tumor organoid cells and immune cells.

[0083] 7) Culture the cells in a 37°C CO2 incubator and observe continuously.

[0084] In some embodiments of the present invention, the processing step of the tumor organoid model in the first aspect of the present invention comprises the following steps:

[0085] 1) Adding a gold nanocage material loaded with PD-1 antibody to the co-cultured cells; the ratio of the gold nanocage material to the culture medium is 5 to 20 μg / mL.

[0086] 2) Using laser irradiation; the intensity of the laser irradiation is 0.1 to 10 W / cm 2 ; Use infrared laser to irradiate.

[0087] The second aspect of the present invention provides a tumor organoid model, which is constructed by the method described in the first aspect of the present invention.

[0088] The third aspect of the present invention provides the construction method of the first aspect of the present invention, and / or the use of the tumor organoid model of the second aspect of the present invention in anti-tumor drug screening or in the preparation of immunotherapy efficacy evaluation products.

[0089] The beneficial effects of the present invention are:

[0090] The present invention adopts the synergistic activation of the photothermal effect of nanomaterials combined with immune checkpoint inhibitors in the traditional co-culture mode. By constructing a gold nanocage material with good photothermal effect to load PD-1 antibodies, a one-time delivery and dual-effect activation method is achieved. Experimental data show that photothermal combined with immune checkpoint inhibitors can increase the proportion of cell apoptosis in the model, while the level of inflammatory factors and the proportion of T cell activation are significantly increased. Facts have proved that the present invention constructs an immune-activated organoid model. BRIEF DESCRIPTION OF THE DRAWINGS

[0091] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein:

[0092] Figure 1 It is a schematic diagram of the design principle of the present invention.

[0093] Figure 2 These are the culture results of tumor organoids in Example 2 of the present invention, including A) a picture of gastric cancer tumor tissue, where the area marked with a red circle is the tumor lesion; B) a light microscopic photo of gastric cancer tumor organoids and a local magnified image; C) light microscopic photos of gastric cancer tumor organoids before and after cryopreservation; D) light microscopic photos of continuous culture of gastric cancer tumor organoids; E) hematoxylin staining of gastric cancer tissue, HE staining of organoids, and immunohistochemistry.

[0094] Figure 3 These are the results of co-culture of tumor organoids and immune cells in Example 3 of the present invention, including A) light microscopy photos of T cells expanded in vitro; B-E) light microscopy images of T cell (B, D) and organoid (C, E) activity under different culture medium components and statistical results on the 3rd day; F) T cell and organoid activity detection under co-culture culture medium conditions; G) fluorescent imaging of the recruitment of T cells by organoids at different times in the co-culture system (red fluorescence is T cells stained with CellTrace Far Red).

[0095] Figure 4 The results of the ELISA test of the co-cultured cells and the gold nanocage material after treatment in Example 4 of the present invention are statistical data of ELISA of A) IL-6, B) TNF-α, C) IFNγ and D) granzyme B in different treatment groups. DETAILED DESCRIPTION

[0096] The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0097] The following is a description of the relevant terms:

[0098] Organoids: A three-dimensional cell culture system, which is characterized by the addition of multiple growth factors to the culture medium formula to regulate the intrinsic signal pathways of cells while providing three-dimensional mechanical support for cell growth, and can maintain the stemness of stem cells and the stability of genetic material for a long time. Therefore, it is considered to be an in vitro model with a higher degree of biomimetic than cell lines.

[0099] Growth factor: A general term for a series of substances that regulate cell growth. They can regulate cell growth and metabolism and stimulate cell activity. Some growth factors can bind to cell receptors and affect cell signaling pathways.

[0100] Matrigel: A mature animal-derived hydrogel, a decellularized mechanism extracted from mouse osteosarcoma tissue. It is liquid at 0-4°C and begins to gel above 10°C. It can be mixed with culture medium (mainly water) in any proportion. It is usually used as a "scaffold" to provide three-dimensional support for organoids during organoid culture.

[0101] Peripheral blood mononuclear cells (PBMCs): cells obtained from peripheral blood through a density system, including lymphocytes and monocytes. Mononuclear cells are different from monocytes, and the two are inclusive.

[0102] T cell culture medium: a culture medium specifically for T cells, with commercial products. It is usually based on PRMI1640 culture medium, with 10% fetal bovine serum or other components added.

[0103] Interleukin 2 (IL-2): A cytokine mainly produced by activated T cells that can promote lymphocyte growth, proliferation, and differentiation and is often added to T cell culture medium.

[0104] CD3 / CD28 activator: It is a monoclonal antibody against CD3 / CD28, which activates T cells through the αβ-T cell receptor (TCR complex) and achieves antigen-induced activation together with TCR.

[0105] Immune microenvironment: Tumor microenvironment (TME) includes a variety of immune cell types, cancer-associated fibroblasts, endothelial cells, pericytes, and various other tissue-resident cell types. Note that traditional organoid models have basically completely lost the components of the immune microenvironment after stable passage, thus affecting their immune effects.

[0106] The following is an introduction to the culture medium components:

[0107] RPMI 1640 medium: It is a medium rich in amino acids and vitamins, with low sugar content and high amino acid content. It is suitable for the culture of lymphocytes, leukemia cells and tumor cells.

[0108] L-glutamine substitute (Glutamax): can significantly reduce the accumulation of toxic ammonia in cell culture and improve cell activity.

[0109] 4-Hydroxyethylpiperazineethanesulfonic acid (HEPES): A common zwitterionic buffer that ensures sufficient bicarbonate in the culture medium. Its buffering capacity is independent of the carbon dioxide content, and it can be operated in a low carbon dioxide environment for a long time.

[0110] Primocin: Antibiotics specifically designed to protect primary cells from microbial infection, and can fight Gram-negative bacteria, Gram-positive bacteria, mycoplasma and fungi.

[0111] B27: The serum-free supplement originally used in neural cell culture, containing antioxidants, proteins, vitamins and fatty acids.

[0112] Acetylcysteine ​​(Nac) is a mucus melter that can be used to reduce mucus thickness and as a cysteine ​​precursor, can be used to prevent heme-induced iron poisoning. It prevents DNA fragmentation and activates the Ras extracellular signal-regulated kinase ERK pathway in cells.

[0113] Epidermal Growth Factor (EGF): A common epidermal growth factor that stimulates proliferation of a variety of epidermal and epithelial cells. Signals through the c-erbB receptor.

[0114] Basic fibroblast growth factor (FGF): FGF family members can significantly increase the mitotic ratio and cell survival activity, and can effectively participate in a variety of biological processes such as embryonic development, cell growth, morphogenesis, tissue repair, tumor growth and invasion. They can keep cells in an undifferentiated state in serum-free culture medium.

[0115] A8301: A potent inhibitor of TGF-β type I receptors ALK5, ALK4 and ALK7, which can inhibit the transcriptional processes induced by the three, maintain cell growth and promote cell self-renewal.

[0116] Y-27632: An ATP (direct energy source for cells) competitive ROCK-I and ROCK-II inhibitor that triggers stem cells to selectively differentiate into the mesenchymal lineage through epithelial-mesenchymal hyperregulation.

[0117] LDN193189: LDN193189 is a potent and selective inhibitor of BMP type I receptor (BMP I). LDN-193189 effectively inhibits the transcriptional activity of BMP type I receptors ALK2 and ALK3.

[0118] Laduviglusib hydrochloride (CHIR99021): an orally active GSK-3α / β inhibitor and a potent Wnt / β-catenin signaling pathway activator.

[0119] SB202190: is a selective p38 MAPK inhibitor that inhibits both p38α and p38β2 signaling pathways.

[0120] Gastrin 1 (Gastrin 1) is an endogenous peptide produced in the stomach that can act as a selective agonist of cholecystokinin 2 (CCK2) receptors and can also stimulate gastric acid secretion.

[0121] Prostaglandin E2 (PGE2, prostaglandin E2) is a hormone-like substance that is involved in a wide range of body functions, such as contraction and relaxation of smooth muscles, dilation and constriction of blood vessels, control of blood pressure and regulation of inflammation.

[0122] Neuregulin 1 (NRG 1): NRG1 fusions lead to aberrant stimulation of ErbB2 / ErbB3 signaling. Therapies targeting the ErbB receptor family have shown efficacy in preliminary studies.

[0123] R-spondin 1 (RSPO1): a member of the R-spondin family, widely used in organoid culture.

[0124] Noggin: Noggin is a secreted homodimeric glycoprotein. Secreted Noggin is close to the cell surface due to its binding to heparin-containing proteoglycans and is an antagonist of bone morphogenetic proteins (BMPs).

[0125] Wnt-3a: It is the most representative signaling protein in the Wnt family. It plays a key role in regulating multi-effect cell self-renewal, proliferation, differentiation and motility, and is a carcinogen for gastric cancer.

[0126] FBS: Fetal bovine serum, often added to cell culture media.

[0127] Immune microenvironment (TME): It is a unique component of solid tumors, which includes a variety of immune cell types, cancer-associated fibroblasts, endothelial cells, pericytes, and extracellular matrix. Current known studies have shown that these components are closely related to the organs where tumors occur, the inherent characteristics of cancer cells, tumor staging, and patient characteristics. Most researchers believe that the occurrence of tumors originates from long-term inflammation, and tumor cells coordinate a tumor-supportive environment by recruiting and reprogramming non-cancerous host cells and by remodeling the vascular system and extracellular matrix. This dynamic process depends on the heterotypic interaction between cancer cells and resident or recruited non-cancerous cells in the TME. Therefore, the formation and dynamic changes of the TME are directly related to the occurrence and development of tumors.

[0128] Immunotherapy: A treatment method that controls and eliminates tumors by restarting and maintaining the tumor-immune cycle and restoring the body's normal anti-tumor immune response. It includes monoclonal antibody immune checkpoint inhibitors, therapeutic antibodies, cancer vaccines, cell therapy, and small molecule inhibitors.

[0129] Gold nanocage: A porous gold nanomaterial made with silver cubes as sacrificial electrodes. It has excellent photothermal effects and can adsorb a variety of biological macromolecules.

[0130] Mild-temperature PTT (mPTT) is of great value for the future clinical transformation of cancer optical therapy. Compared with traditional photothermal therapy (PTT) (>50°C), mPTT has fewer side effects and better biological effects in tumor treatment, such as loosening the dense structure of tumor tissue, enhancing blood perfusion, and improving the immunosuppressive microenvironment.

[0131] Immune checkpoint inhibitors: Immune checkpoint inhibitors (CPIs) are a new class of anti-tumor agents that enhance anti-tumor effects by blocking the interaction between inhibitory receptors expressed on T cells and related ligands and regulating the body's normal immune cell activity.

[0132] Example 1 Construction of immune microenvironment organoid model

[0133] 1.1 Construction of organoids

[0134] 1.1.1 Organoid Culture

[0135] 1) The lesion site (gastric cancer tissue) was initially separated from the fresh patient tissue, and then vortexed and washed in a PBS buffer containing 10% double antibody.

[0136] 2) The tumor tissue was initially mechanically broken with ophthalmic scissors, and then 1 mL of collagenase IV (1 mg / mL) was added and digested on a 37°C metal bath shaker for 5 h until the liquid became turbid.

[0137] 3) After the EP tube was left to settle naturally, the upper turbid liquid was removed and centrifuged at 1000 rpm for 3 minutes. The supernatant was discarded and DMEM complete medium containing 10% FBS was used to terminate the digestion and wash.

[0138] 4) The digested tumor cells were mixed with Matrigel (Corning, Cat. No. 356234) at 4°C, plated, and gelled at 37°C for 30 min. Organoid culture medium was then gently added along the wall of the well plate. For a 96-well plate, 100 μL of organoid culture medium was added to each well, and for a 24-well plate, 500 μL of organoid culture medium was added to each well.

[0139] 5) After seeding, place the plates in a 37°C CO2 incubator and continuously observe the growth status of the organoids under a light microscope. Change the medium after 3 days to ensure the supply of nutrients.

[0140] 1.1.2 Passaging of organoids

[0141] 1) When the organoids grow to the 7th day, you can see under the microscope that the volume has increased significantly and a chamber structure has appeared. At this time, you can consider subculturing.

[0142] 2) Aspirate the original culture medium, add an equal volume of TrypLE, gently blow the Matrigel droplet with a pipette tip, and transfer it to a 1.5 mL EP tube.

[0143] 3) Digestion was performed on a 37°C metal bath shaker for 15 min, followed by centrifugation at 1000 rpm for 3 min, and the supernatant was discarded to terminate the digestion.

[0144] 4) Then complete the seeding and subsequent cultivation as described in 1.1.1.

[0145] 1.1.3 Cryopreservation and thawing of organoids

[0146] 1) Add 100 μL cell culture grade DMSO and 500 μL FBS to each cryovial and mix well for later use.

[0147] 2) Take the organoids on the third day after subculturing and discard the original culture medium.

[0148] 3) Add new culture medium according to the standard of 400 μL culture medium per tube, gently blow the Matrigel droplets with the tip of the pipette, and add the mixed solution to the cryopreservation tube prepared in advance.

[0149] 4) Place in a programmed cooling box and freeze in a -80°C refrigerator.

[0150] 5) Cryopreserved cells can be stored at -80°C for 1 month and in liquid nitrogen tanks for more than 1 year.

[0151] 6) Thaw the frozen organoids quickly in a 37°C water bath.

[0152] 7) Transfer to a 1.5 mL EP tube, centrifuge at 1000 rpm for 3 min, and discard the supernatant.

[0153] 8) Wash twice with DMEM complete medium containing 10% FBS.

[0154] 9) Seed the plates and change the medium as described in 1.1.1.

[0155] 1.2 Extraction of PBMCs

[0156] Extracting PBMCs from the patient's peripheral blood is the most convenient and patient-accepted way to obtain immune cells. This operation can be performed at the same time as the patient's admission examination, preoperative examination or blood drawing during surgery to avoid increasing the patient's pain.

[0157] 1) Peripheral blood was extracted from the patient corresponding to the organoid, and then an equal volume of PBS was added to dilute the blood concentration.

[0158] 2) Add 15 mL of human lymphocyte separation solution to the lower end of the peripheral blood immune lymphocyte separation tube (about 25 mL of blood diluent corresponds to 15 mL of separation solution), and then gently and slowly inject the diluted blood into the upper layer of the lymphocyte separation tube. Set the slowest acceleration and deceleration gears and centrifuge at 1000 rpm for 30 minutes.

[0159] 3) After centrifugation, obvious stratification can be seen in the lymphocyte separation tube. The middle white film layer is aspirated and transferred to a new centrifuge tube. After washing with PBS, centrifuge at 1000 rpm to discard the supernatant and expand the culture.

[0160] 4) Count the cells obtained and calculate the number of cells to be obtained. 6 The cells were cultured at a density of 100 / mL and kept in a 37°C CO2 incubator. The cells were counted before each subculture and maintained at this density.

[0161] 5) On the second day of culture, CD3 / CD28 activator (Stemcell, catalog number: 10971-2mL) was added at a culture medium volume ratio of 1:50.

[0162] 6) Repeat the subculturing operation to expand the culture of PBMCs.

[0163] 1.3 Preparation of gold nanocages

[0164] Reduction of Ag in CF3COOAg by polyols at 150°C + The silver cube was obtained and used as a sacrificial electrode to reduce the Au in HAuCl4 with the participation of the capping agent (PVP). + , that is, gold nanocage materials can be obtained.

[0165] 1.3.1 Preparation of Silver Cube

[0166] 1) Dissolve 312 mg (1.4 mmol) of CF3COOAg in 5 mL of ethylene glycol. The concentration of the mother liquor is 282 mmol / L.

[0167] 2) Dissolve 300 mg PVP (Mw = 55 kDa) in 15 mL ethylene glycol. The concentration of the mother solution is 20 mg / mL.

[0168] 3) Take 5 μL of 37% HCl solution and add 20 mL of ethylene glycol. The concentration of the mother solution is 2.5 mmol / L.

[0169] 4) Dissolve 8.4 mg 0.15 mmol NaHS in 5 mL ethylene glycol and dilute 10 times. The stock solution concentration is 3 mmol / L.

[0170] 5) Add 50 mL of ethylene glycol to a 250 mL round-bottom flask and heat to 150 °C in an oil bath and maintain for 25 min.

[0171] 6) Add 0.6 mL of NaHS stock solution and maintain for 2 min.

[0172] 7) Add 5 mL of HCl-ethylene glycol and 12.5 mL of PVP mother solution and maintain for 5 minutes.

[0173] 8) Add 4 mL of CF3COOAg mother solution and take samples of 10 mL after 15 min and 30 min respectively.

[0174] 9) After 45 min, place in an ice water bath to terminate the reaction.

[0175] 10) Centrifuge twice the volume of acetone at 4000 rpm for 20 min and discard the supernatant.

[0176] 11) Wash twice with pure water ultrasonic centrifugation at 10000 rpm for 10 min.

[0177] 12) Transfer to a 100 mL glass bottle and store in pure water at 4°C. The Ag concentration is about 0.5 mg / mL.

[0178] 1.3.2 Preparation of gold nanocage materials

[0179] 1) Take 10mL of 0.5mg / mL Ag cube and add 150mg PVP and 90mL pure water.

[0180] 2) Maintain vigorous stirring at 90°C for 10 min.

[0181] 3) Inject 1 mmol / L HAuCl4 at 0.7 mL / min.

[0182] 4) The wavelength of the LSPR peak stops at 808 nm, and the UV / Vis spectrophotometer scans the range of 300-900 nm.

[0183] 5) Stir at 90°C for 5 min.

[0184] 6) Wash with pure water, centrifuge at 10000 rpm for 10 min, wash three times.

[0185] 7) Wash once with NH4OH and twice with anhydrous ethanol.

[0186] 8) Take a portion of Au NCs, dry and weigh them, and store the remaining materials at 4°C with pure water as solvent.

[0187] 1.3.3 Preparation of PD-1 antibody-loaded gold nanocages (aPD-1@Au NCs)

[0188] 1) The gold nanocages obtained in 1.3.2 were infiltrated in 10 mg / mL PD-1 antibody (TargetMol, Catalog No.: T76703-1mg1858168-59-8) in a shaker at 4°C for 1 day.

[0189] 2) Wash with pure water and centrifuge at 10000rpm for 10 minutes, wash three times.

[0190] 1.4 Preparation of co-culture medium and cell activity detection

[0191] This step uses Steam Cell's T cell expansion medium (ImmunoCult TM -XF T Cell Expansion Medium) was used as the basis, and culture media with different concentration gradients of single components were prepared with reference to the components of organoid culture media to test the sensitivity of immune cells to the components of organoid culture media and to determine the formula of co-culture culture media.

[0192] 1) Prepare a single-component culture medium according to the concentrations of Nic (10mM), Nac (0.5mM), A8301 (0.25μM), Glutamax (1mM), HEPES (10mM), primocin (1×), B27 (1×), Y27632 (10μM), LDN193189 (0.1μM), CHIR99021 (5μM), 202 (1.5μM), Gastrin 1 (10nM), FGF 10 (20μg / mL), human EGF (50ng / mL), PE2 (10nM), RP001 (100ng / mL), Noggin (100ng / mL), FGF7 (5ng / mL), Neuregulin 1 (25ng / mL), and FBS (5%), and culture with the PBMCs obtained in 1.2 for 7 days. The PBMCs culture medium was used as a control.

[0193] 2) After 7 days, cells were lysed using Cell TitterGlo reagent and then transferred to a black-bottom 384-well plate. CellTitterGlo can generate bioluminescence based on the ATP content in cells. The number of live cells can be quantified by measuring the luminescence intensity.

[0194] 3) According to the PBMCs activity results, Nic was eliminated, and the co-culture complete medium was prepared and sterilized by filtration using a 0.2 μm filter.

[0195] 4) Use organoids on day 2 to 3 of passage and PBMCs obtained in 1.2 to co-culture with complete culture medium for 7 days, and use Cell TitterGlo (Promega, Catalog No.: G9242) to detect cell activity.

[0196] 1.5 Construction of organoid models for immune microenvironment reconstruction

[0197] The organoid model of immune microenvironment reconstruction is mainly to explore the co-culture model of tumor organoids and immune cells. This co-culture model needs to maintain the growth of tumor organoids and immune cells, while providing the necessary three-dimensional support for the organoids.

[0198] 1) Refer to the composition of organoid culture medium, prepare single-component culture medium according to the concentration of each component, and detect the activity of immune cells.

[0199] 2) After determining the composition and concentration of each component, prepare the complete co-culture medium and prove that it can maintain the activity of organoids and immune cells.

[0200] 3) Spread Matrigel glue evenly on the bottom of the board and let it self-level overnight at room temperature.

[0201] 4) As described in 1.1.2, digest the organoids rapidly at 37°C for 1 min, then terminate the digestion rapidly. This step can remove the original matrix gel covering the organoids, but can preserve the structure of the organoids as much as possible.

[0202] 5) Resuspend the organoids after removing the matrix gel with a small amount of organoid culture medium, and drop the resuspension on the surface of the Matrigel plate, paying attention to concentrate the drops on the flatter area in the middle as much as possible.

[0203] 6) Let stand for 6 to 8 hours to allow the organoids to settle on the surface of the matrix gel.

[0204] 7) The immune cells were then resuspended in co-culture medium and counted.

[0205] 8) Add immune cell co-culture suspension at a ratio of 1:10 to 100 of tumor organoid cells to immune cells.

[0206] 9) Culture the cells in a 37°C CO2 incubator and observe continuously.

[0207] 1.6 Experimental treatment of co-culture system

[0208] 1) The control group was the co-culture system described in 1.5.

[0209] 2) In the aPD-1 group, 1.5 mg / mL of PD-1 antibody was added to the co-culture model.

[0210] 3) In the Au NCs group, 12.5 μg / mL of gold nanocage material was added to the co-culture model.

[0211] 4) aPD-1@Au NCs group is a co-culture model in which the gold nanocage material loaded with PD-1 antibody described in 1.3.3 is added.

[0212] 5) Group L: 1W / cm2 was used on the basis of co-culture 2 808nm laser irradiation.

[0213] 6) The Au NCs+L group was treated similarly to the L group on the basis of Au NCs.

[0214] 7) aPD-1@Au NCs+L group is the L group operation based on aPD-1@Au NCs.

[0215] Example 2 Construction results of digestive tract tumor organoids

[0216] Figure 2 This is a physical photograph of the tumor organoid model constructed in Example 1.

[0217] Among them, gastric cancer tissue tumor organoid model ( Figure 2 Middle B) The morphology of organoids was observed continuously for 7 days during the culture process and it was found that the organoids were stable and proliferated ( Figure 2 Middle D). Observe the light microscopic images of tumor organoids before and after cryopreservation ( Figure 2 C) shows that the freezing and thawing process has no significant effect on the morphology and structure of organoids. The results of HE staining and immunohistochemistry of tumor tissue and organoid sections show that the tumor-specific antibodies in the original tumor tissue are retained during the sampling and culture of the tumor organoid model ( Figure 2 Middle E).

[0218] Example 3 Results of establishing the co-culture system

[0219] Figure 3 This is the result of co-culture of tumor organoids and T cells in Example 1.

[0220] in, Figure 3 A in the middle is a light microscopic image of peripheral blood immune cells expanded in vitro. Representative substances added to the organoid culture medium based on T cell culture medium can maintain the activity of tumor organoids ( Figure 3 By quantitatively analyzing the cell activity of T cells in this system, the nicotinamide component in the organoid culture medium was removed in the co-culture system ( Figure 3 D). T cells and organoids were co-cultured. Cell activity detection in this system proved that the co-culture system culture medium was suitable for the growth of all components of the tumor organoid model containing the microenvironment ( Figure 3 Middle F).

[0221] The tumor immune microenvironment inhibits the attack and infiltration of immune cells on tumor tissues in vivo, and the effect of immune cells in the organoid platform is also inhibited. After 7 days of continuous observation, it can be seen that tumor organoids can recruit immune cells but have no obvious killing effect on them. ( Figure 3 Middle G).

[0222] Example 4 ELISA

[0223] Figure 4 These are the changes in the expression of cellular inflammatory factors after the different treatment groups were treated in 1.6 of Example 1.

[0224] The study found that tumor cells treated with photothermal therapy can release CSPG4 protein, activate and stimulate specific CAR-T cells, leading to the proliferation and cytokine production of these cells. Generally, IL-6, TNF-α, IFNγ and granzyme B are pro-inflammatory cytokines that play a key role in inflammatory responses. According to ( Figure 4) The levels of IL-6 and TNF-α in other groups were different compared with the control group, indicating that different treatments had an effect on the expression of inflammatory factors.

[0225] The relevant tests were performed using commercial ELISA test kits.

[0226] Experimental data show that gold nanocages loaded with PD-1 antibodies can activate effector T cells and promote the release of pro-inflammatory cytokines. In tumor treatment, it can induce the transformation of the immunosuppressive microenvironment to the immune activation microenvironment, causing a strong immune response.

Claims

1. A method for constructing a tumor organoid model, comprising the following steps: After the tumor organoids are co-cultured with immune cells, gold nanomaterials are added for treatment; the gold nanomaterials are loaded with immune activators.

2. The method according to claim 1, characterized in that: The immune activator is a PD-1 antibody.

3. The method according to claim 1, characterized in that: The tumor organoids include in situ tumor cells.

4. The method according to claim 1, characterized in that: The immune cells include T cells; Preferably, the T cells are T cells from the same source as the tumor organoids.

5. The method according to claim 1, characterized in that: The culturing step comprises: Pre-treat the plate and add immune cells after the tumor organoids have settled; Preferably, the ratio of tumor organoids to immune cells is 1:(10-100).

6. The method according to claim 1, characterized in that: The steps of the process include: Laser irradiation is performed after the addition of gold nanomaterials.

7. The method according to claim 6, characterized in that: The intensity of the laser irradiation is 0.1 to 10 W / cm 2 ; The laser is infrared light.

8. The method according to claim 6, characterized in that: The ratio of the gold nanomaterial to the culture medium is 5-20 μg / mL.

9. A tumor organoid model, characterized in that: The tumor organoid model is constructed by the method described in any one of claims 1 to 8.

10. Use of the method according to any one of claims 1 to 8 and / or the tumor organoid model according to claim 9 in anti-tumor drug screening or in the preparation of immunotherapy efficacy evaluation products.

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