Preparation method and application of bone metastatic tumor organoid model

Abstract

The invention discloses a preparation method and application of a bone metastatic tumor organoid model, and belongs to the technical field of biology. The preparation method comprises the following steps: selecting an epithelial tumor cell line as a seed cell; preparing a natural structure bone matrix scaffold material; the prepared bone matrix scaffold material is subjected to pore diameter measurement, a scaffold with qualified pore diameter groups is reserved, an unqualified scaffold is eliminated, and the optimized scaffold material is soaked in gel to be attached; and putting the qualified bone matrix scaffold material into a culture container, uniformly inoculating the epithelium-derived tumor cell suspension on the surface of the bone matrix scaffold material, and carrying out static culture for 2-3 weeks to generate the bone metastatic tumor organoid model. The bone metastatic tumor organoid model prepared by the invention is applied to physical and drug therapy experiments and research on molecular mechanisms of bone metastasis.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and specifically relates to a method for preparing a bone metastatic tumor organoid model and the application of the organoid model in disease research and treatment screening. Background Art

[0002] Bone metastasis is a common complication of various cancers in the late stages, leading to severe pain, pathological fractures, and decreased quality of life in patients, seriously threatening their quality of life and treatment outcomes. Currently, bone metastasis research relies primarily on animal models and two-dimensional cell culture models.

[0003] Animal models (such as mouse tail vein injection of tumor cells or intraosseous injection) can simulate the bone metastasis process at a holistic level, but significant species differences, long experimental cycles, high costs, and ethical concerns limit their application. Furthermore, animal models are difficult to implement for high-throughput drug screening, limiting the efficiency of drug development.

[0004] Two-dimensional cell culture models are valuable for studying the basic biological behavior of tumor cells. However, cell culture on a two-dimensional surface cannot simulate the three-dimensional interaction between the tumor and the bone microenvironment, ignoring key processes such as tumor cell invasion and bone matrix degradation, making it difficult to accurately reflect the actual conditions of bone metastasis.

[0005] As an emerging in vitro research technology, organoid models can better simulate the three-dimensional structure and microenvironmental characteristics of tumors, providing a more realistic tool for studying tumor development and drug screening. However, the application of existing organoid models in bone metastasis research is still in its early stages of exploration. In particular, how to generate organoid models with high stability and efficiency remains an urgent challenge. Summary of the Invention

[0006] The present invention can create an organoid model that can truly reproduce the characteristics of tumor bone metastasis in a three-dimensional in vitro environment. By combining tumor cell lines, constructing natural bone matrix porous scaffold materials and related cultures, a bone metastasis tumor organoid model with good stability and high repeatability is generated. This bone metastasis tumor organoid model can simulate the growth characteristics of tumor cells in bone tissue in a three-dimensional in vitro environment. It can not only be used for bone metastasis mechanism research, but also for sensitivity detection and screening of anti-tumor treatment, thereby providing technical support for personalized treatment.

[0007] The technical solutions of the present invention are as follows: 1. Method for Generating Bone Metastatic Tumor Organoid Models Step 1: Selection and culture of tumor cells: The primary cells selected for this invention include, but are not limited to, tumor cell lines and primary tumor cells from patients or disease model animals. Due to the high cost, limited availability, and ethical concerns associated with in vitro culturing of primary tumor cells from patients or disease model animals, we prioritize epithelial cell lines for in vitro culturing. Our experiments have shown that, based on the tiers of bone metastasis incidence, such as breast cancer, lung cancer, liver cancer, and cervical cancer, these epithelial cell lines have a higher success rate in generating bone metastatic tumor organoid models when seeded on natural bone matrix scaffolds. For example, the breast cancer MDA-MB-231 cell line, a triple-negative breast cancer (TNBC) cell line, exhibits a significant propensity for bone metastasis. Its high expression of the CXCR4 receptor allows it to migrate to the bone microenvironment through interaction with CXCL12 (secreted by bone marrow stromal cells) in the bone matrix. This signaling axis not only promotes cell homing to the bone matrix but also enhances cell survival and proliferation by activating downstream pathways such as PI3K / AKT.

[0008] The epithelial tumor cell lines selected include, but are not limited to, breast cancer MCF-7 and MDA-MB-231 cell lines, lung cancer A549 and H460 cell lines, liver cancer HepG2 and Huh7 cell lines, cervical cancer HeLa cell line, etc.

[0009] Select a tumor cell line and culture it in a conventional incubator under sterile conditions until the logarithmic growth phase, and prepare a single cell suspension for later use.

[0010] Step 2: Preparation of bone matrix scaffold material SPM (the abbreviation of the bone matrix scaffold material SPM used in the present invention comes from the abbreviation of Scaffold-based Porous Matrix): The selection of raw materials includes, but is not limited to: vertebrae, femurs and other long bones or partial flat bones of small experimental animals; the source of natural scaffold materials requires a porous structure, and cancellous bone can meet the porous requirements. Vertebrae, femurs and other long bones or partial flat bones of small experimental animals containing cancellous bone are relatively easy to obtain, so these parts are selected.

[0011] The selected bone raw materials are further processed by conventional defatting, decalcification and other technologies to obtain natural porous structure bone matrix scaffold material SPM.

[0012] Natural bone matrix scaffolds are important materials for studying bone repair and regeneration. They provide the three-dimensional structure and microenvironment necessary for cell adhesion, growth, and differentiation. RGD (Arg-Gly-Asp)-motif proteins in the bone matrix, such as osteopontin and fibronectin, bind to integrins on the surface of cancer cells, promoting cell adhesion to the scaffold surface and colony formation. This adhesion further activates intracellular signaling (such as the FAK / Src pathway), supporting cell attachment and growth.

[0013] Step 3: Evaluation and screening of the prepared SPM: Studies have shown that small pores (micropores), with a distribution range of 100-500 μm, are beneficial for cell adhesion, matrix deposition, and nutrient exchange. Pores that are too small may limit the physical migration and proliferation space of cancer cells, inhibiting their three-dimensional diffusion. Moderate pore sizes are generally the ideal range for supporting cell growth. Excessively large pore sizes may reduce cell-matrix contact signals, which is detrimental to cancer cell types that rely on adhesion. Through extensive experiments and comparisons, we found that the key factor hindering the growth success rate of bone metastatic tumor organoid models is the pore size of the natural bone matrix scaffold, which directly affects the distribution of cell adhesion on the scaffold and the culture effect. We found that when the average pore size of the bone matrix scaffold is less than 40 μm and the pore size group greater than 140 μm accounts for 70% to 80%, the organoid growth success rate is low. When 70% to 80% of the pore size group is greater than 40 μm and less than 140 μm, the organoid growth success rate is high and the quality is stable. Therefore, we screened bone matrix scaffold materials. The pore size determination of bone matrix scaffold materials includes but is not limited to: measuring the pore size of the obtained bone matrix scaffolds using a scanning electron microscope, eliminating scaffolds with pore sizes of 70% to 80% less than 40 μm, retaining scaffolds with pore sizes of 70% to 80% in the range of 40 μm to 140 μm for standby use, and retaining scaffolds with pore sizes of 70% to 80% greater than 140 μm for subsequent optimization.

[0014] Step 4: Optimize the SPM with pore size greater than 140 μm: We use pore size optimization technology to achieve a pore population distribution in the SPM pore size range of 40 μm-140 μm.

[0015] Treatment method for SPM with pore size greater than 140 μm: select chemical composition gel, adjust to a liquid with appropriate concentration, soak SPM with pore size greater than 140 μm in the liquid gel, and remove the SPM before the liquid gel solidifies by observation. After the gel on the SPM solidifies, subsequent experimental operations are carried out. When each batch of optimized scaffolds is soaked for the first time, it is necessary to sample and measure the corresponding pore size after the gel is attached. SPM that meets the pore size distribution standard of 70% to 80% of the pore size between 40 μm and 140 μm is selected and kept for use; Preferred gels include PEG hydrogels (a hydrogel composed of polyethylene glycol (PEG) and a crosslinker to form a three-dimensional network structure through chemical or physical crosslinking), hyaluronic acid gels, and sodium alginate gels. The SPMs in this experiment are gelled to maximize cell attachment. The gel fills oversized pores with liquid form. The gel rapidly solidifies at room temperature, allowing the pore size to be reduced to a range of 40 μm-140 μm, facilitating nutrient delivery and cell migration. Furthermore, the selected gels are inherently cell-compatible, retaining the RGD sequence, which promotes cell adhesion, proliferation, and differentiation. Curing can be determined by observing the gel's color, transparency, and surface condition at room temperature. Uncured gels are typically light or translucent in color, but deepen and become evenly distributed after curing. The cured surface should be smooth and flat, free of wet reflections. Oil stains, water marks, or areas of whitening indicate uneven curing. Tackiness and wetness can also be used to determine if the gel is ready. Uncured gels feel sticky and leave a residue when lightly pressed. Fully cured gels are dry and non-sticky.

[0016] Step 5: Preparation of bone metastatic tumor organoid model: According to the experimental design, the cell density in the tumor cell suspension was adjusted according to the selected scaffold size and pore size. We found that the appropriate cell amount determines the stability and time of growth of the bone metastatic tumor organoid model. In the experiment, a 5 mL test tube was used. When the inoculated cell amount was 8×10 6 -13×10 6 When the number of cells is within the range (approximately 80% to 90% of the cell mass in a 10cm culture dish), the success rate of the bone metastatic tumor organoid model is stable and the growth cycle is appropriate; when the inoculated cell amount is lower than this range, the histomorphological observation of the grown bone metastatic tumor organoids is poor, the tumor proliferation density is poor, and there is no tumor-like tissue structure showing heterogeneous growth; and when the cell amount is greater than this range, the success rate does not change much, cells are wasted, and the culture time is also invisibly extended, which is not conducive to efficient culture.

[0017] Select SPM with a pore size distribution of 40 μm-140 μm accounting for 70% to 80% of the pore group, cut it appropriately, put it into a cylindrical glass tube, and place 8×10 epithelial tumor cells in the tube. 6 -13×10 6 Cells within a certain range are evenly inoculated on the surface of the SPM, so that the cells adhere to the scaffold and are cultured in a static manner. The culture time is usually 3 weeks, and the preferred culture time is 2 weeks, and they grow into a tumor cell bone metastasis tumor organoid model.

[0018] Step 6: Identification of bone metastatic tumor organoid models: The cultured bone metastatic tumor organoid model is identified. The identification methods include but are not limited to: HE staining after tissue embedding and sectioning and immunohistochemical staining of proteins such as Ki67, PCNA, and AFP.

[0019] 2. Application of Bone Metastatic Tumor Organoid Models 1. Apply the bone metastasis tumor organoid model generated in steps 1 to 6 in "1" above to screen the efficacy evaluation of physical therapy and drug therapy for bone metastasis tumors, including physical therapy such as magnetic therapy and drug therapy such as paclitaxel and resveratrol.

[0020] 2. Apply the bone metastatic tumor organoid model generated in steps 1 to 6 above to study the molecular mechanisms of bone metastasis, including changes in protein expression during bone metastasis progression.

[0021] The tumor cell bone metastasis tumor organoid model used in the above applications 1 and 2 can also be generated by in vitro culture of primary tumor cells from patients or disease model animals according to steps 1 to 6 described in "1" above. Beneficial effects

[0022] 1. The tumor cell bone metastasis tumor organoid model generated by the present invention has a high success rate and good stability. The present invention selects epithelial-derived tumor cell lines with significant bone metastasis tendency as seed cells, and then coats the natural bone matrix scaffold material SPM with gel, which not only ensures the porosity of the scaffold material but also realizes that 70% to 80% of the pore size of the pore group can be controlled within the distribution range of 40 μm-140 μm, ensuring the adhesion of seed cells, matrix deposition and nutrient exchange, while also utilizing the cell compatibility of the gel itself to nourish cells; the present invention ensures the growth success rate of the tumor cell bone metastasis tumor organoid model by controlling key technical links such as the selection of epithelial-derived tumor cells, the preparation of natural bone matrix scaffold materials and the optimization of the pore size group distribution in the range of 40 μm-140 μm; by finding the optimal number range of inoculated tumor cells, 8×10 6 -13×10 6The growth time of the bone metastasis tumor organoid model is guaranteed to be about 2 weeks, which reduces costs and improves efficiency. By controlling the key links in the construction process, the preparation of bone metastasis tumor organoids is more programmed and controllable, achieving the goal of stable and efficient construction of bone metastasis tumor organoid models, and being able to use bone metastasis tumor organoid models to truly reproduce the characteristics of bone metastasis tumor cells.

[0023] 2. The present invention is highly reproducible, low-cost, and suitable for efficiently constructing tumor cell bone metastasis organoid models for large-scale screening and experimentation. The epithelial-derived tumor cell lines preferred in the present invention are mature commercial products. Minimal cell quantities can be routinely passaged and expanded to reach the inoculation number after culture, thereby enabling the production of a large number of bone metastasis tumor organoid models. The pure natural bone matrix scaffold material is readily available and has low production costs. The production process of the present invention facilitates the establishment of standardized preparation methods, facilitating large-scale drug screening and other experiments.

[0024] 3. Wide application: The bone metastasis tumor organoid model generated by the method of the present invention has a wide range of application potential in bone metastasis mechanism research, drug screening and personalized treatment, and improves the clinical relevance of treatment screening results. The bone metastasis tumor organoid prepared by the present invention can use immunohistochemical staining and pathological staining methods to detect the expression of molecules involved in the bone metastasis mechanism, which is helpful for the biochemical detection of upstream and downstream molecules of the signal pathway in mechanism research; the bone metastasis tumor organoid modeling time is short, and different drugs can be added for intervention, which facilitates the efficient screening of anti-bone metastasis tumor drugs; it is also applicable to personalized intervention treatments such as magnetic therapy.

[0025] 4. Strong market potential: Based on the high efficiency and low cost of culturing bone metastatic tumor organoids using epithelial tumor cell lines, we can further build patient-derived organoid models in batches, providing technical support for improving precision medicine in clinical applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Flow chart of the steps of the method for culturing a bone metastatic tumor organoid model using the tumor cell line of the present invention.

[0027] Figure 2 This is a scanning electron microscope image of the natural porous structure scaffold with pore size meeting the requirements of the present invention.

[0028] Figure 3 (a) Scanning electron microscopy image of a natural porous structure scaffold with pore size that does not meet the requirements.

[0029] Figure 3 (b) is a scanning electron micrograph of a scaffold that does not meet the requirements and is covered with gel.

[0030] Figure 3 (c) is a scanning electron micrograph of the scaffold that meets the requirements after being coated with gel.

[0031] Figure 4 This is a HE staining image of bone metastatic tumor organoids cultured from the epithelial-derived tumor cell line of the present invention.

[0032] Figure 5 This is an immunohistochemical staining identification diagram of bone metastatic tumor organoids cultured from the epithelial-derived tumor cell line of the present invention.

[0033] Figure 6 This is an immunofluorescence identification diagram of bone metastatic tumor organoids cultured from the epithelial-derived tumor cell line liver cancer Huh7 of the present invention.

[0034] Figure 7 This is a graph showing the progression of Huh7 liver cancer bone metastatic organoids at different culture times.

[0035] Figure 8 Comparison of the progression of Huh7 liver cancer bone metastatic organoids under therapeutic intervention conditions.

[0036] Figure 9 A diagram exploring the molecular mechanisms of Mcf-7 breast cancer in situ carcinoma and Mcf-7 breast cancer bone metastasis organoids.

[0037] Figure 10 The figure shows the success rate of bone metastatic organoids derived from different cell lines in the laboratory of the present invention.

[0038] Figure 11 Comparison of organoids prepared from liver cancer Huh7 on scaffolds with different pore size ranges.

[0039] Figure 12 A graph showing the changes in culture medium during organoid growth and cessation of growth.

[0040] Figure 13 Pore ​​diameters outside the range of less than 40 μm and greater than 140 μm are unsuccessful representations.

[0041] Figure 14 The inoculated cell number is less than 8×10 6 -13×10 6 Cultured tumor bone metastasis organoids.

[0042] Figure 15 Comparison of HE staining of clinical lung adenocarcinoma specimens and lung adenocarcinoma bone metastasis organoids described in this patent.

[0043] Example The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings, which are intended to explain rather than limit the present invention.

[0044] Example 1 The types of epithelial tumor cell lines selected include but are not limited to: breast cancer MCF-7, MDA-MB-231 cell lines; or lung cancer A549, H460 cell lines; or liver cancer HepG2, Huh7 cell lines; or cervical cancer HeLa cell line.

[0045] After many experiments, it has been proved that any one of the above epithelial tumor cell lines has a high success rate of bone metastasis when preparing bone metastatic tumor organoids, such as Figure 10 As shown in the table.

[0046] Take the breast cancer MCF-7 cell line as an example: MCF-7 cell lines were obtained and digested with trypsin. The cells were then placed in an incubator at 37°C and 5% CO2 and cultured under sterile conditions until the logarithmic growth phase. Single-cell suspensions were prepared and the cell density was adjusted to 1×10 6 cells / mL; Due to the slight differences in the size of the prepared scaffolds and the required pore size distribution, the amount of cells required and the length of time required to generate bone metastatic tumor organoids each time are also slightly different. Therefore, in order to improve efficiency and ensure the success rate, the number of cells in the cell suspension needs to be adjusted to an appropriate number. After a large number of experimental results, the average number of cells inoculated per scaffold was finally selected to be 8×10 6 -13×10 6 When the cell suspension is within the optimal range, the success rate is high and the time is short. In the optimal time period, 80% to 90% of the capacity of the 5 mL culture tube / glass tube can be grown for breast cancer MCF-7 metastatic bone tumor organoid cells. Therefore, the number of tumor cells in the cell suspension used each time is adjusted to 8×10 6 -13×10 6 within the range of .

[0047] Figure 14 When the inoculated cell volume is 5×10 6 - hours, less than 8×10 6 -At this time, the organoids grown from lung cancer A549, breast cancer MCF-7, cervical cancer HeLa, etc. showed poor histomorphological observation results, with poor tumor proliferation density and lack of tumor-like tissue structure showing heterogeneous growth.

[0048] When the inoculated cell volume exceeds 13 × 10 6 The morphological effect and range of the generated organoid tissues were similar to those observed at 8×10 6 -13×10 6 The results did not change much. A large number of cells wasted cells and the culture time was also extended, which did not meet the requirements of efficient culture.

[0049] Example 2 Preparation of bone matrix scaffold SPM The raw materials that can be used to prepare natural porous structural materials are selected, such as long bones or partial flat bones such as vertebrae and femurs from small experimental animals such as rabbits and pigs. The corresponding bone tissues of the experimental animals are isolated to obtain raw materials. The obtained raw materials are further processed using a 24-hour defatting and 24-hour decalcification process, and then freeze-dried for 24 to 48 hours to obtain SPM. Screening: The SPM obtained above is measured for pore size by scanning electron microscopy and other techniques. The scaffold material with 70% to 80% of the pore size distributed in the range of 40 μm to 140 μm can be directly used for inoculation, such as Figure 2 ; Figure 13 When 70% to 80% of the pore size group of the scaffold material is evenly distributed between less than 40 μm and greater than 140 μm, the amount of organoid cells generated is small, there is almost no obvious cell aggregation area, the structure is loose and irregular, and even a complete tumor tissue cannot be formed.

[0050] Optimization, eliminate the scaffold materials with pore groups with an average distribution of 70% to 80% less than 40 μm; select the scaffold materials with an average distribution of pore groups with 70% to 80% greater than 140 μm and attach hyaluronic acid gel. The gel has a natural polysaccharide structure, strong hydrophilicity, moisturizing and biodegradability, which is beneficial to pore filling and cell nutrition. The present invention uses gel liquid to fill the oversized pores, and quickly solidifies at room temperature to achieve the characteristic of adjusting the pore size. Therefore, each time it is used, it can be observed with the naked eye whether it is in a gel state at a specific room temperature; select hyaluronic acid gel and add water to a liquid with a concentration of 10% to 30%, soak the SPM in the solution, and observe it with the naked eye at room temperature. If the solution becomes a gel state and has not solidified, take it out immediately; after the gel of the scaffold material covered with gel is solidified, test it again, and leave the scaffold with a pore size distribution range of 70% to 80% in the range of 40 μm-140 μm for freezing and standby use. Figure 3 (a) is an unqualified stent with a pore size group larger than 140 μm before attachment. Figure 3 (b) is a stent being coated with gel. Figure 3 (c) The scaffold test after being coated with gel meets the required pore size group.

[0051] Example 3 Preparation of bone metastatic tumor organoids The culture container was a 5 mL cylindrical glass tube. MCF-7 tumor cell suspension was taken and centrifuged to remove 8×10 cells. 6 Cells were evenly seeded on an SPM scaffold prepared by the method of Example 2, in which 70% to 80% of the pore size distribution ranged from 40 μm to 140 μm. The scaffold was cut into a column with a diameter of 6 mm and a thickness of about 3 mm, and allowed to stand for 2 hours to promote cell attachment.

[0052] The culture container was a 5 mL cylindrical glass tube. Huh7 tumor cell suspension was taken and centrifuged to remove 13 × 10 cells. 6 Cells were evenly seeded on an SPM scaffold prepared by the method of Example 2, in which 70% to 80% of the pore size distribution ranged from 40 μm to 140 μm. The scaffold was cut into a column with a diameter of 10 mm and a thickness of about 3 mm, and allowed to stand for 2 hours to promote cell attachment.

[0053] like Figure 12 As shown, conventional static culture is used. During the experiment, the changes in cell growth and proliferation are confirmed based on the degree of color change of the culture medium. When the liquid turns light purple after 2 to 3 days, the culture medium is replaced; when the liquid turns dark purple and transparent, the cells are growing normally; the time for tumor cells to combine with the scaffold material is 7 to 21 days. Multiple experiments have shown that 14 days is usually the best culture time. At this time, the liquid is slightly darker and more transparent than the light purple at the beginning of culture, and the tumor organoid model can be collected.

[0054] Figure 7 Figure 3 shows the growth of liver cancer bone metastasis tumor organoids at different time periods. The morphological changes of the organoids after 7 days of growth are as follows: tumor cell proliferation can be clearly observed, and there is even a phenomenon of tumor cells obviously engulfing the SPM scaffold. The overall appearance is irregular cell clumps, lacking obvious boundaries and structural characteristics; the morphological changes of the organoids after 14 days of growth are as follows: tumor tissue with a stable structural morphology is gradually formed, with cells arranged tightly, showing a high degree of organization. Organoids at this stage are suitable for subsequent drug screening and functional research; the morphological changes of the organoids after 21 days of growth are as follows: a large number of cells undergo apoptosis or necrosis, which is manifested by a decrease in cell density in the central area or the appearance of vacuoles.

[0055] The experiments in Examples 1, 2, and 3 were all performed under sterile conditions to avoid contamination.

[0056] The present invention Figure 1 The process has the potential for high stability and efficient mass industrial production: Epithelial cell lines were selected: breast cancer MCF-7, MDA-MB-231 cell lines; lung cancer A549, H460 cell lines; liver cancer HepG2, Huh7 cell lines; or cervical cancer HeLa cell lines, with a cell count of 8×10 6 -13×10 6 The cells within the range are seeds; After screening and optimization, the pore size of 70% to 80% of the pores is evenly distributed in the range of 40 μm to 140 μm, and then the SPM is cut into 6-10 mm in diameter and about 3 mm thick. Use 5 mL cylindrical culture glass tubes; The vaccines are inoculated in batches at the same time and taken out at the same time within two weeks. They can be used in batches after passing the random inspection.

[0057] Example 4 Functional validation of bone metastatic tumor organoids cultured from epithelial tumor cell lines 1. Morphological Characterization Figure 4 Microscopic observation: Bone metastatic tumor organoids cultured using the methods of Example 3, such as breast cancer MCF-7 and MDA-MB-231 cell lines; lung cancer A549 and H460 cell lines; liver cancer HepG2 and Huh7 cell lines; and cervical cancer HeLa cell line, were fixed with 4% paraformaldehyde, dehydrated using conventional gradients, and embedded in paraffin blocks before HE staining to observe the three-dimensional structure of the organoids and the distribution of tumor cells on the bone matrix scaffold.

[0058] It can be observed that the histopathological characteristics of the bone metastatic tumor organoids are relatively similar to those of their corresponding tumor tissues. The blue-stained cell nuclei are distributed in large and small sizes, indicating that the tumor cells are irregular in morphology and vary in size. Sometimes, giant cells of various morphologies may appear. In a few poorly differentiated tumors, the tumor cells are smaller, rounder, and more uniform in size than normal cells.

[0059] Figure 15 This tissue comparison between a clinical lung adenocarcinoma specimen and a lung adenocarcinoma bone metastasis organoid generated using this method shows a high degree of similarity. The chromatin in the nucleus and the nucleic acids in the cytoplasm appear in blue, while components of the cytoplasm and extracellular matrix appear in red.

[0060] Figure 6 Fluorescent labeling: The liver cancer Huh7 organoids were fluorescently labeled with the cytoskeleton dye Phalloidin and the nuclear dye DAPI. The distribution and morphology of the cells were observed under high and low magnification microscopes using a laser confocal microscope. It was observed that the organoid tissue showed obvious nuclear staining and cytoskeleton staining, indicating that the distribution of tumor cells in the organoid tissue showed a staining effect consistent with the corresponding tumor tissue, which was a further identification of the distribution consistent with the HE staining results.

[0061] 2. Molecular level validation The tissue sections of the bone metastatic tumor organoids of each tumor cell line cultured using the method of Example 3 were subjected to immunohistochemical staining to detect the expression levels of tumor-related genes such as ki67, PCNA, and AFP. Figure 5 .

[0062] The higher the positive rate of tumor marker Ki67, the faster the tumor grows. Figure 5It can be observed that Ki67 is clearly brown-stained (positive expression) in all organoid tissues, and its expression varies among different tumor types. For example, the positive staining area of ​​Ki67 in cervical cancer bone metastasis organoids obtained by inoculation with HeLa is lower than that in other organoid tissues. PCNA not only reflects the proliferation ability of tumor cells, but is also related to the infiltration and metastasis of tumor cells. Figure 5 It can be observed that PCNA is positively expressed in the cervical cancer bone metastatic organoid tissue obtained by inoculation with Hela, suggesting that it has certain tumor infiltration and metastasis capabilities; AFP is the most sensitive and specific indicator for early diagnosis of primary liver cancer. Figure 5 It can be observed that AFP showed obvious positive expression in the liver cancer bone metastasis organoid tissue obtained by inoculation of Huh7, which is also a further identification of the formation of the constructed liver cancer bone metastasis organoid.

[0063] Subsequently, RNA and protein can be extracted from organoids to detect the expression levels of bone metastasis-related proteins.

[0064] 3. Biological function verification Each tumor cell line bone metastatic tumor organoid cultured as described in Example 3 can be used to assess the invasive ability of the tumor cells in the organoid using a Transwell chamber. (Strong evidence has not yet been obtained to prove its invasive ability, but it can be indirectly reflected by PCNA expression in the above-mentioned immunohistochemical staining. PCNA not only reflects the proliferation ability of tumor cells, but is also related to the infiltration and metastasis of tumor cells.) Figure 5 It can be observed that PCNA is positively expressed in the cervical cancer bone metastatic organoid tissue obtained by inoculation with Hela, suggesting that it has certain tumor infiltration and metastasis capabilities. Example 5

[0065] Application 1: Treatment Mode Screening Test In the organoid culture system, physical therapy or drug treatment is added as an intervention test in preclinical models to observe the growth status of the organoids, such as Figure 8 , which is a comparison of the growth progress of organoids after magnetic therapy intervention in clinical physical therapy. It was found that the number of tumor cell growth was greatly reduced after magnetic therapy.

[0066] Subsequently, the consistency of in vitro test results with patients' clinical treatment responses can be compared to evaluate the predictive ability of the model.

[0067] Application 2: Molecular mechanism exploration test Many studies have shown that there are certain differences in gene and protein expression between primary tumors and metastatic tumors. There are two main clinical situations for breast cancer: one is that the primary lesion is HER2 positive and the metastatic lesion is HER2 negative. This situation is very common and most of them turn negative after anti-HER2 treatment; the other is that the primary lesion is HER2 negative and the metastatic lesion is HER2 positive. This is often caused by spatiotemporal heterogeneity. Figure 9 HER2 was detected in breast cancer bone metastasis organoids cultured on MCF-7 cells, whose primary tumor model was HER2-negative (clinically, HER2 is 1+ or 0). Immunofluorescence revealed that the bone metastasis organoids were HER2-positive. This will serve as a foundation for further research into the molecular mechanisms of bone metastasis.

[0068] This specific embodiment is merely an explanation of the invention and is not a limitation of the invention. After reading this specification, those skilled in the art may make non-creative modifications to this embodiment as needed. However, as long as they are within the scope of protection of the invention, they are protected by patent law.

Claims

1. A method for preparing a bone metastatic tumor organoid model, characterized in that: The following steps are involved: S1: Select epithelial tumor cell lines or primary tumor cells from patients or disease model animals, culture them under sterile conditions until the logarithmic growth phase, and prepare a single cell suspension for later use; S2: Select bones from small experimental animals as raw materials, perform degreasing and decalcification processes, and make natural structure bone matrix scaffold materials; S3: The pore size of the bone matrix scaffold material prepared in S2 is measured, and 70% to 80% of the pores smaller than 40 μm are eliminated, those in the range of 40 μm to 140 μm are reserved, and those larger than 140 μm are retained for optimization; S4: The bone matrix scaffold material optimized in S3 was immersed in the gel, and the bone matrix scaffold material was removed before the gel solidified by observation; S5: Measure the pore size again, and keep the pores that meet the pore size group of 70% to 80% with a diameter of 40 μm to 140 μm for future use; S6: Place a bone matrix scaffold material with a pore size of 70% to 80% of 40 μm to 140 μm in the pore size group selected in any of steps S3 and S5 into a culture container, evenly inoculate the epithelial tumor cell suspension in S1 on the surface of the bone matrix scaffold material, and statically culture for 2 to 3 weeks to generate a bone metastatic tumor organoid model.

2. The method for preparing a bone metastatic tumor organoid model according to claim 1, wherein: The epithelial tumor cell lines selected in S1 include: breast cancer MCF-7 and MDA-MB-231 cell lines, lung cancer A549 and H460 cell lines, liver cancer HepG2 and Huh7 cell lines, or cervical cancer HeLa cell line.

3. The method for preparing a bone metastatic tumor organoid model according to claim 1, wherein: The skeleton of the S2 small experimental animal includes: vertebrae or femurs or part of flat bones.

4. The method for preparing a bone metastatic tumor organoid model according to claim 1, wherein: The gel in S4 includes: PEG hydrogel, hyaluronic acid gel or sodium alginate gel.

5. The method for preparing a bone metastatic tumor organoid model according to claim 1, wherein: The bone matrix scaffold material in S6 is cut into cylinders with a diameter of 6-10 mm and a thickness of 3 mm.

6. The method for preparing a bone metastatic tumor organoid model according to claim 1, wherein: The culture container in S6 is a cylindrical container with a volume of 5 mL.

7. The method for preparing a bone metastatic tumor organoid model according to claim 1, wherein: The inoculation amount of epithelial tumor cells in S6 was 8×10 6 -13×10 6 indivual.

8. A bone metastatic tumor organoid model, characterized in that: The bone metastatic tumor organoid model is prepared according to the method for preparing the bone metastatic tumor organoid model as described in any one of claims 1 to 7.

9. The use of a bone metastatic tumor organoid model according to claim 8, characterized in that: Used to screen the efficacy evaluation of physical therapy or drug therapy for bone metastasis tumors.

10. The use of a bone metastatic tumor organoid model according to claim 8, characterized in that: Used to study the molecular mechanisms of tumor bone metastasis, including changes in protein expression during bone metastasis progression.

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