Construction method, model and system of cancer metastasis organoid model

Abstract

The application discloses a kind of construction methods, model and system of cancer metastasis organoid model.The method comprises the following steps: preparing tumor cell spheroid labeled with reporter gene and target organ organoid derived from stem cells respectively;It is placed in culture solution;The preset three-dimensional acoustic field is generated using holographic acoustic tweezers, and at least one tumor cell spheroid is moved and positioned at the preset position of target organ organoid by non-contact precise control, to form initial assembly;The initial assembly is maintained in suspension culture, so that it occurs contact, fusion and invasion, to obtain cancer metastasis organoid model.The application uses acoustic force to realize non-destructive, controllable spatial assembly with micron-level precision, and the model constructed can truly reproduce the interaction of tumor cells and target organ cells in three-dimensional environment, to provide a standardized, quantifiable and scalable in-vitro experimental platform for cancer metastasis mechanism research and anti-metastatic drug screening.

Description

Technical Field

[0001] This application relates to the fields of biomedical engineering and tumor model technology, and in particular to a method, model and system for constructing a cancer metastatic organoid model. Background Technology

[0002] Cancer metastasis is a leading cause of death in cancer patients. The process involves tumor cells detaching from the primary site, invading surrounding tissues, entering the circulatory system, and colonizing distant organs. Research into the mechanisms of cancer metastasis and the development of anti-metastatic drugs urgently requires in vitro models that can realistically simulate the tumor metastasis process.

[0003] Currently, commonly used cancer research models mainly include animal models, two-dimensional cell culture models, and three-dimensional organoid models. While animal models can simulate tumor behavior at the physiological level, they suffer from problems such as significant species variability, high cost, long experimental cycles, and poor reproducibility, and are not suitable for large-scale drug screening. Two-dimensional cell culture models are easy to operate, but they cannot reproduce the three-dimensional structural features and cell-matrix interactions within the tumor microenvironment, making it difficult to simulate the migration and invasion behavior of tumor cells. Three-dimensional organoids or tumor spheroid models can better simulate the three-dimensional structure and some functions of tumor tissue, but they are usually single structures, lacking the dynamic interaction between tumor cells and target organ cells, and cannot realistically reflect the process of tumor cell metastasis from the primary tumor site to the target organ.

[0004] In recent years, some studies have attempted to combine organoids or spheres from different sources using assembly models to simulate multi-tissue interactions. However, existing assembly techniques (such as magnetic assembly and 3D bioprinting) suffer from problems such as low positioning accuracy, significant cell damage, irreversible assembly processes, and poor reproducibility, making it difficult to achieve stable and controllable reconstruction of tumor metastasis processes.

[0005] Therefore, there is an urgent need in this field for an organoid model construction method that can accurately, controllably, and reproducibly simulate the process of tumor cells metastasizing to specific target organs in vitro, in order to support research on cancer metastasis mechanisms and screening of anti-metastasis drugs. Summary of the Invention

[0006] In view of this, this application provides a method, model, and system for constructing a cancer metastatic organoid model, addressing the technical problem that existing tumor models cannot realistically and controllably simulate the process of tumor cells metastasizing to specific target organs.

[0007] To address the aforementioned technical problems, this application adopts the following technical solution: a method for constructing a cancer metastasis organoid model, comprising: S1: Prepare tumor cell spheroids labeled with reporter genes and target organoids derived from stem cells, respectively; S2: Place tumor cell spheroids and target organoids in a container containing culture medium; S3: Using holographic acoustic tweezers to generate a preset three-dimensional sound field, at least one tumor cell sphere is moved and positioned on the surface or inside a preset location of the target organoid through non-contact precise manipulation, forming an initial assembly; S4: The initial assembly is suspended and maintained in culture to allow tumor cell spheroids to come into contact with, fuse with, and / or invade the target organoids, thereby constructing a cancer metastatic organoid model.

[0008] As a further improvement of this application, the three-dimensional sound field includes one or more combinations of symmetrical vortex sound field, asymmetrical vortex sound field and double-well sound field. By switching or combining different sound fields, the movement, rotation and positioning of tumor cell spheres can be achieved.

[0009] As a further improvement to this application, step S3 specifically includes: The tumor cell spheres are moved to the vicinity of the target organoid using the first symmetric vortex acoustic field. Asymmetric vortex acoustic fields are used to propel tumor cell spheres into contact with target organoids. Dual-well acoustic fields are used to adjust the orientation of tumor cell spheres to optimize contact.

[0010] As a further improvement of this application, after positioning is completed, a second symmetrical vortex acoustic field with an aperture larger than that of the first symmetrical vortex acoustic field is used to apply a periodic or continuous maintaining force to the initial assembly.

[0011] As a further improvement to this application, the tumor cell spheroids are liver cancer cell spheroids, and the target organoids are liver and gallbladder organoids.

[0012] As a further improvement of this application, a single hepatocellular carcinoma spheroid is assembled onto a single hepatobiliary organoid to form a single-focal metastasis model; Alternatively, multiple liver cancer cell spheroids can be assembled onto a single liver and biliary organoid to form a multifocal metastasis model.

[0013] As a further improvement to this application, the hepatobiliary organoids are obtained by differentiation of human induced pluripotent stem cells; the liver cancer cell spheroids are formed by three-dimensional culture of the HuH-7 cell line.

[0014] As a further improvement to this application, tumor cell spheroids are labeled with reporter genes.

[0015] To solve the above-mentioned technical problems, another technical solution adopted in this application is to provide a cancer metastasis organoid model, which is constructed by any of the above-mentioned methods for constructing cancer metastasis organoid models.

[0016] To address the aforementioned technical problems, another technical solution adopted in this application is: providing a system for constructing the aforementioned cancer metastasis organoid model, comprising: The holographic acoustic tweezers module is used to generate and control a three-dimensional acoustic field to manipulate tumor cell spheres in a non-contact manner. The culture module is used to contain and provide tumor cell spheroids, target organoids, and culture medium, and to maintain a suitable growth environment. The imaging module is used to observe the location and assembly process of tumor cell spheroids and target organoids in real time; The control module is connected to the holographic acoustic tweezers module, the culture module, and the imaging module respectively, and is used to control the acoustic field parameters and execute the preset control program according to the imaging information.

[0017] The beneficial effects of this application are as follows: First, the method for constructing cancer metastatic organoid models utilizes acoustic forces for non-destructive manipulation, avoiding cell damage caused by traditional physical contact and maximizing cell viability and model integrity. Second, by combining multiple acoustic field modes (such as vortex fields and double-well fields), the movement, rotation, and positioning of the spheres can be flexibly and precisely controlled, achieving the controllable construction of complex three-dimensional spatial structures. Third, the constructed model simultaneously includes tumor and normal tissue, enabling dynamic simulation of tumor cell invasion, colonization, and their interaction with the microenvironment in vitro, providing a more physiologically relevant basis for metastasis mechanism research. The platform is highly versatile and scalable. Furthermore, the entire construction process is parameterized and programmed, ensuring the model's reproducibility and standardization, making it suitable for high-throughput drug screening. Finally, the scheme is highly universal and scalable; by changing cell types and culture conditions, it can be applied to various cancer types and target organ combinations, and can simulate the impact of different pathological microenvironments on metastasis. It provides a powerful and flexible tool for cancer metastasis research and anti-metastasis drug development, enabling non-contact, high-precision three-dimensional manipulation of tumor cell spheroids and spatial assembly with functionally complete target organoids to construct highly realistic cancer metastasis organoid models. Attached Figure Description

[0018] Figure 1 This is a flowchart illustrating the method for constructing a cancer metastatic organoid model according to an embodiment of the present invention; Figure 2 This is a schematic diagram illustrating the process of constructing organoids and cell spheres; Figure 3 This is a schematic diagram illustrating the directional movement of cell spheres controlled by a sound field. Figure 4 This is a schematic diagram of the RNA sequencing analysis results of the liver cancer metastasis assembly model group and the control group; Figure 5 This is a schematic diagram illustrating the construction process of single-focal and multifocal hepatocellular carcinoma metastasis models; Figure 6 This is a schematic diagram of the system for constructing organoid models of cancer metastasis according to an embodiment of the present invention. Detailed Implementation

[0019] The technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of this application, and not all of the embodiments. Based on the embodiments of this application, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of this application.

[0020] The terms "first," "second," and "third" in this application are for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Therefore, a feature defined as "first," "second," or "third" may explicitly or implicitly include at least one of that feature. In the description of this application, "multiple" means at least two, such as two, three, etc., unless otherwise explicitly specified. All directional indications (such as up, down, left, right, front, back, etc.) in the embodiments of this application are only used to explain the relative positional relationships and movements between components in a specific orientation (as shown in the figures). If the specific orientation changes, the directional indications also change accordingly. Furthermore, the terms "comprising" and "having," and any variations thereof, are intended to cover non-exclusive inclusion. For example, a process, method, system, product, or device that includes a series of steps or units is not limited to the listed steps or units, but may optionally include steps or units not listed, or may optionally include other steps or units inherent to these processes, methods, products, or devices.

[0021] In this document, the term "embodiment" means that a particular feature, structure, or characteristic described in connection with an embodiment may be included in at least one embodiment of this application. The appearance of this phrase in various places throughout the specification does not necessarily refer to the same embodiment, nor is it a separate or alternative embodiment mutually exclusive with other embodiments. It will be explicitly and implicitly understood by those skilled in the art that the embodiments described herein can be combined with other embodiments.

[0022] Figure 1 This is a flowchart illustrating the method for constructing a cancer metastatic organoid model according to an embodiment of the present invention. It should be noted that if substantially the same result is obtained, the method of the present invention is not necessarily identical. Figure 1 The illustrated process sequence is limited. For example... Figure 1 As shown, the method for constructing this cancer metastatic organoid model includes the following steps: Step S1: Prepare tumor cell spheroids labeled with reporter genes and target organoids derived from stem cells, respectively.

[0023] Specifically, this step aims to obtain two core biological components of the model: first, standardized tumor cell spheroids that can simulate the primary tumor lesion and are easy to track and observe; and second, three-dimensional organoids that can simulate the target organ microenvironment and possess corresponding tissue structure and function. In practice: tumor cell spheroids are typically selected from cell lines of specific cancer types (such as hepatocellular carcinoma HuH-7), and are made to stably express reporter genes (such as green fluorescent protein GFP) through viral transfection or selection of stable transgenic lines. Then, they are cultured in three dimensions using ultra-low adsorption plates in hanging drops or rotation to form spheroids of uniform size, dense structure, and good cell viability. Their diameter, cell viability, and fluorescence intensity must be quality controlled. Target organoids are derived from human pluripotent stem cells (such as hiPSCs). By simulating the in vivo development process, under the induction of growth factors and chemical components added at specific times, they undergo multiple stages such as directed differentiation, morphogenesis, and functional maturation (e.g., differentiating into hepatobiliary organoids with characteristics of hepatocytes and bile duct cells), ultimately obtaining micro-tissues that highly approximate real target organs in gene expression, tissue structure, and physiological function. Standardization of this step is the foundation for subsequent high-precision assembly and model reliability. It should be understood that the tumor cell spheroids described here are live cell spheroids that have not been fixed or cleared to ensure that they have the ability to migrate, invade and proliferate during subsequent assembly and culture.

[0024] It should be noted that tumor cell spheroids are labeled with reporter genes.

[0025] like Figure 2 As shown in the illustration, this embodiment uses hepatocellular carcinoma spheroids and hepatobiliary organoids (HBOs) as examples.

[0026] The specific preparation process of liver cancer spheroids: HuH-7 liver cancer cells labeled with green fluorescent protein (GFP) were cultured in three dimensions to form uniform, highly viable spheroids. Specifically, HuH-7 liver cancer cells were seeded at a density of 1500 cells per well into 96-well plates with ultra-low adsorption, and continuous culture for 4 days constructed uniformly sized, approximately 600 μm in diameter, and densely structured live cell spheroids. The regularity of the spheroid morphology could be ensured by microscopic observation, and the density and integrity of their three-dimensional structure could be verified by transparentization after model construction.

[0027] The specific preparation process for hepatobiliary organoids (HBOs) involves differentiating mature HBOs from human induced pluripotent stem cells (hiPSCs). The specific steps are as follows: 1. First, remove the culture medium and add 1 ml / well of DPSB to rinse the cell surface. Then, remove the DPSB and add 1 ml / well of ReLeSR™ and incubate at room temperature for 1 minute. Next, remove the ReLeSR™ and place the cells at 37°C for 5 minutes. Then, add 1 ml / well of Accutase and incubate at 37°C for 5 minutes to dissociate the hiPSC clonal clusters into single cells. Then, add 3 ml / well of mTeSR™ containing 10 mM and centrifuge at 250g for 5 minutes. After centrifugation, discard the supernatant and resuspend the cells in mTeSR™ containing 50 mM Y-27632, 100 ng / ml Activin A, and 100 ng / ml BMP4. Count the cells using a erythrocytometer and seed them in ultra-low adsorption 96-well plates at a ratio of 1000 cells / well. Finally, incubate the cells in an incubator for 24 hours. On the second day, change the culture medium to contain 10 mM Y-27632. mTeSR™ containing Y-27632, 100 ng / ml Activin A, and 100 ng / ml BMP4 was cultured for another three days, with the culture medium changed daily. 2. On day 5, replace the culture medium with RPMI1640 / B27 containing 30 ng / ml FGF4, 20 ng / ml BPM2 and 25% mTeSR™ and continue culturing for 4 days, changing the culture medium daily; 3. On day 9, organoids were embedded using Matrigel. The Matrigel was first thawed overnight on ice at 4°C, then used to embed the organoids (20 μl / organoid) and carefully transferred to 48-well ultra-low adsorption plates. Note that Matrigel solidifies at room temperature (22-25°C), therefore all materials in contact with Matrigel must be kept at a low temperature and the embedding process performed quickly. The culture medium was then replaced with RPMI1640 / B27 containing 20 ng / ml HGF, 20 ng / ml KGF, 1% NEAA, 1% Gluta-MAX, and 25% mTeSR™, and cultured for another 6 days, changing the culture medium daily. 4. On day 15, replace the culture medium with HCM containing 20 ng / ml Oncostatin M (OSM), 1% NEAA, and 1% Gluta-MAX and culture continuously for 10 days, changing the culture medium daily; 5. On day 25, replace the culture medium with HCM containing 20 ng / ml OSM, 0.1 μM dexamethasone (Dex), 2 μM vitamin K2 (K2), and 5 μM lithocholic acid (LCA) and culture continuously for 10 days to obtain mature HBOs. The culture medium should be changed daily.

[0028] During differentiation, cells sequentially express early embryonic markers (SOX17 and FOXA2), early liver markers (HNF4a), hepatocytes (AFP), and bile duct-specific markers (CK7), eventually forming a complete hepatobiliary organoid structure with liver and bile duct functions, including ICG uptake and release, glycogen and lipid secretion, urea secretion, and alkaline phosphatase secretion.

[0029] The above-mentioned process of constructing tumor cell spheroids and the complete differentiation of hepatobiliary organoids can be achieved through... Figure 2 The presentation clearly demonstrates the entire chain of steps from the directed differentiation of human induced pluripotent stem cells (hiPSCs) into mature hepatobiliary organoids (HBOs) and the three-dimensional culture of HuH-7 liver cancer cells into spheres, ensuring the standardization and traceability of the preparation of each core component.

[0030] Step S2: Place the tumor cell spheroids and target organoids in a container containing culture medium.

[0031] Specifically, this step is a crucial preparatory stage for model building, aiming to provide a stable, suitable, and standardized initial operating environment for subsequent non-contact precision assembly of holographic acoustic tweezers. This includes: 1. Container Selection: Specially designed glass-bottomed culture dishes are typically used. These dishes possess excellent optical transparency to meet the requirements of real-time, high-resolution imaging guidance during the assembly of the inverted fluorescence microscope. The culture dishes must be sterile and non-cytotoxic, and their surfaces are usually treated with hydrophilicity or anti-cell adhesion to minimize non-specific adhesion of spheroids and organoids in an unmanipulated state, ensuring they can freely suspend in the culture medium. This facilitates precise capture and movement of the subsequent acoustic force field.

[0032] 2. Culture Medium Preparation: The culture medium used in this step and subsequent assembly processes must meet the short-term viability maintenance needs of both tumor cell spheroids and target organoids. A culture medium compatible with both cell types and supporting their basic metabolism is typically chosen. For example, in a liver cancer to hepatobiliary organoid metastasis model, organoid maintenance medium (such as HCM basal medium) or an optimized co-culture basal medium can be used. The culture medium needs to be preheated to 37°C, the pH adjusted to the physiological range (approximately 7.2-7.4), and necessary antibiotics added to prevent microbial contamination during the process.

[0033] 3. Sample Transfer and Placement: Using sterile, wide-mouth pipette tips or fine forceps, gently remove the single or multiple tumor cell spheroids (e.g., GFP-HuH-7 spheroids) and single target organoids (e.g., HBOs) prepared in step S1 from their respective original culture modules. Transfer them together to the central area of ​​a specially prepared culture dish containing an appropriate amount (e.g., 1-2 ml) of preheated culture medium. Key Operation: During placement, it is necessary to consciously maintain an initial distance between the spheroids and organoids (usually several spheroid diameters, such as 500-1500 micrometers). This initial distance provides a clear and undisturbed operating space for subsequent acoustic manipulation and is a prerequisite for achieving a precise "capture-move-position" sequence.

[0034] 4. Environmental Control: Quickly transfer the culture dish containing the sample to the environmental control chamber of the holographic acoustic forceps manipulation system (such as the OSOAP platform). The chamber should be pre- or immediately purged with a mixed gas containing 5% CO2 and the temperature maintained at a constant 37°C to provide a stable physiological-grade growth environment for the live cells throughout the assembly operation, which may last for several hours, ensuring that cell viability is not compromised.

[0035] Step S3: Using holographic acoustic tweezers to generate a preset three-dimensional sound field, at least one tumor cell sphere is moved and positioned on the surface or inside a preset location of the target organoid through non-contact precise manipulation to form an initial assembly.

[0036] Specifically, this embodiment solves the core problems of traditional assembly technologies (such as micromanipulation needles, magnetism, and bioprinting), such as physical contact damage, low positioning accuracy, and irreversible operation. Through purely acoustic means, it achieves micron-level resolution, real-time adjustability, and highly repeatable spatial manipulation of living cell clusters in a physiological fluid environment. This makes it possible to construct a three-dimensional biological model that can accurately simulate the initial spatial relationship between tumor cells and target organs, laying an irreplaceable technical foundation for subsequent research on dynamic metastatic biological processes.

[0037] Furthermore, the three-dimensional sound field includes one or more combinations of symmetrical vortex sound fields, asymmetrical vortex sound fields, and double-well sound fields. By switching or combining different sound fields, the movement, rotation, and positioning of tumor cell spheres can be achieved.

[0038] Specifically, holographic acoustic tweezers can synthesize various three-dimensional sound fields with distinct mechanical properties in culture medium by precisely controlling the emission parameters of each element of an ultrasonic phased array. This invention mainly utilizes the following three basic sound field modes and controls their rapid switching or spatiotemporal recombination via software to achieve comprehensive control over the movement, rotation, and positioning of cell spheroids: Symmetrical vortex acoustic fields (such as 1.6λ aperture): Their sound pressure distribution exhibits an axisymmetric ring structure, forming a stable minimum sound pressure point (i.e., an acoustic trap) at the center. When a tumor cell spheroid is placed in this acoustic field, the acoustic radiation force gradient it experiences will stably trap it at the center of the acoustic trap. By dynamically adjusting the phase distribution of the array, this acoustic trap can be continuously moved in three-dimensional space, thereby carrying the trapped spheroid for non-destructive and stable long-distance transport. This is the primary means of moving spheroids from their initial position towards the vicinity of the target organ or organoid.

[0039] Asymmetric vortex sound fields (such as 1.6λ asymmetric fields): This sound field breaks symmetry, creating a distribution with strong sound pressure on one side and weak sound pressure on the other. This asymmetry generates a net acoustic radiation force pointing towards the weaker field. Utilizing this property, the sound field can apply a directional pushing force to a sphere, causing it to move precisely along a predetermined straight trajectory. It is particularly suitable for ultimately "pushing" a sphere that has approached a target to make close contact with the surface of an organoid, achieving precise positioning at the millimeter to micrometer level.

[0040] Dual-well acoustic field: This acoustic field can generate two independent acoustic traps at close range. When a sphere is captured by it, a controllable rotational motion of the sphere can be induced by adjusting the relative position or intensity of the two acoustic traps. This function is used to fine-tune the spatial orientation of the sphere before and after contact with the organoid, so that the surface most conducive to cell adhesion fully aligns with the organoid tissue, thereby optimizing the initial contact interface and creating conditions for subsequent fusion.

[0041] like Figure 3 As shown, different three-dimensional sound fields synthesized by holographic acoustic tweezers (HAT) can achieve precise manipulation of tumor cell spheres: the symmetrical vortex sound field drives the sphere to move smoothly over a long distance through the "enveloping" effect, the asymmetrical vortex sound field achieves close and precise contact between the sphere and organoid with strong field lateral thrust, and the double-well sound field optimizes the contact posture by inducing the rotation of the sphere.

[0042] In this embodiment, by combining or rapidly switching these three sound field modes in a programmed sequence, the control system can execute complex operation commands. For example, it can first use a symmetrical vortex sound field for capture and long-distance transport, then use an asymmetrical vortex sound field for precise end-effector propulsion, and finally use a dual-well sound field to adjust the contact angle as needed, thereby completing a complete high-precision assembly process of "capture-transport-contact-adjustment".

[0043] Furthermore, step S3 specifically includes: 1. Use the first symmetrical vortex acoustic field to move the tumor cell spheres to the vicinity of the target organoid.

[0044] Specifically, after identifying the target using a microscope, the system calculates and generates a first symmetrical vortex acoustic field (e.g., with an aperture of 1.6λ), whose acoustic trap center coincides with the target tumor sphere, achieving non-contact capture. Subsequently, the system dynamically adjusts the phase of this acoustic field according to a preset path, causing the acoustic trap (along with the sphere) to move smoothly, ultimately transporting the sphere to a preset "approach point" approximately 100-200 micrometers from the surface of the target organoid. This step enables the safe, long-distance transfer of the sphere.

[0045] 2. Use an asymmetric vortex acoustic field to propel tumor cell spheres into contact with target organoids.

[0046] Specifically, once the sphere reaches the "approach point," the system immediately switches the sound field mode to an asymmetric vortex sound field. This sound field is configured so that its weaker side is precisely oriented towards the target contact point of the organoid. The directional thrust generated by the sound field then acts on the sphere, precisely pushing it in a straight line towards the organoid until physical contact occurs. This step achieves precise control of the contact action.

[0047] 3. Use a dual-well acoustic field to adjust the posture of tumor cell spheres to optimize contact.

[0048] Specifically, after contact occurs, the system can briefly activate the dual-well acoustic field. By controlling the movement of the dual wells, the contacted and fixed sphere is slightly rotated, thereby adjusting the contact surface between it and the organoid. This ensures that the area on the tumor sphere with the best cell activity or the most favorable adhesion is fully adhered to the organoid tissue, laying the foundation for subsequent stable biofusion.

[0049] Furthermore, after positioning is completed, a second symmetrical vortex acoustic field with an aperture larger than that of the first symmetrical vortex acoustic field is used to apply a periodic or continuous maintaining force to the initial assembly.

[0050] Specifically, after the spheres are positioned and form an initial assembly, cell adhesion at the interface is not yet strong. To prevent disintegration of the assembly due to environmental disturbances during transfer to the incubator or in the early stages of culture, this embodiment employs acoustic methods for temporary reinforcement: 1. After the precise positioning step is completed, the system switches the sound field to a second symmetric vortex sound field with a larger aperture (e.g., 3.8λ aperture). Compared to the first symmetric vortex sound field (1.6λ) used for movement, this sound field has a wider capture range and a gentler force field gradient.

[0051] 2. The large aperture acoustic field can gently confine the entire initial assembly (including the tumor sphere and the organoids in contact with it) within its acoustic trap range.

[0052] 3. The application method adopts a periodic pattern, such as "turning on the sound field for 15 minutes, then turning off the sound field for 15 minutes," repeating this cycle several times (e.g., 2 cycles, total duration 1 hour). This intermittent application provides the necessary mechanical stability to promote initial bioadhesion while effectively avoiding the thermal effects or potential impacts on cell metabolism that may result from prolonged continuous application of the sound field. After this maintenance phase, the initial assembly has achieved a certain level of stability and can be safely transferred to an incubator for long-term maintenance culture in step S4.

[0053] It should be noted that this embodiment utilizes HAT to generate multiple sound fields, including a 1.6λ symmetric vortex sound field, a 3.8λ symmetric vortex sound field, a 16λ asymmetric vortex sound field, and a 1.6λ double-well sound field. By controlling the amplitude and phase of the sound fields, precise movement of the liver cancer spheres in vitro is achieved. In this embodiment, the main object of manipulation is the cell sphere, which is mainly formed by tightly packed cells. Its acoustic impedance properties differ from those of a single cell and are more similar to those of macroscopic tissues. To more intuitively observe and analyze the forces acting on the cell spheres in the holographic sound field, this embodiment uses COMSOL Multiphysics 5.5 software to simulate the forces acting on the cells in the sound field. Here, the cell spheres are simplified into spherical particles, and the gradient potential energy is calculated using Gor'kov, as follows: ; ; ; ; in, This refers to the volume of a spherical particle. It is the angular frequency of the emission. It is the density of the culture medium. It is the density of spherical particles. It is the speed of sound in the culture medium. It is the speed of sound in spherical particles. It is a preset coefficient related to the compression ratio. It is a preset density-related coefficient. It's sound pressure. , , These are the partial derivatives of the sound pressure in the x, y, and z directions, respectively. It is sound radiation force. It is the gradient of acoustic radiation potential energy.

[0054] Step S4: The initial assembly is suspended and maintained in culture to allow tumor cell spheroids to come into contact with, fuse with, and / or invade the target organoids, thereby constructing a cancer metastatic organoid model.

[0055] This embodiment represents the biological realization and functional maturity stage of model construction. Its purpose is to transform the "initial assembly" with a specific spatial configuration, which is formed through precise acoustic assembly in step S3, into a functional three-dimensional biological system that can dynamically simulate key metastatic behaviors such as colonization, survival, proliferation, and invasion of tumor cells in the target organ microenvironment under suitable in vitro culture conditions.

[0056] (1) Specific implementation procedures for maintenance culture: 1. Establishment of the culture environment: After the acoustic manipulation is completed, the special culture dish containing the initial assembly is smoothly transferred from the environmental control chamber of the OSOAP platform to a standard constant temperature and humidity cell culture incubator (maintained at 37°C, 5% CO2).

[0057] 2. Culture System Setup: Optimized co-culture maintenance media are used to simultaneously support the long-term survival and function of tumor cells and target organoids. For example, in a liver cancer-hepatobiliary organoid model, hepatocyte maintenance medium (HCM) supplemented with specific growth factors (such as HGF and EGF) and nutrients (such as B27) can be used. During culture, the initial assemblies are kept suspended in the culture medium to simulate the migration environment of tumor cells in body fluids or interstitial spaces during in vivo metastasis. The culture medium needs to be completely replaced every 48 to 72 hours to continuously provide nutrients, remove metabolic waste, and maintain a stable physicochemical environment.

[0058] 3. Culture cycle management: The total culture time depends on the research objective, usually ranging from 5 to 14 days. For observing early adhesion and fusion, 5-7 days may be sufficient; if studying deep invasion or the effects of long-term drug treatment, it needs to be extended to 10-14 days or longer.

[0059] (2) The dynamic evolution of the model and biological processes: During maintenance culture, the initial physical contact will trigger a series of sequential biological events, progressively mimicking the cascade of in vivo metastasis: 1. Early Contact and Stable Adhesion: In the initial culture phase, cells at the interface between the tumor spheroids and organoids are activated, establishing stable biological connections by upregulating the expression of adhesion molecules (such as integrins and cadherins) and secreting extracellular matrix components. This process transforms the initial physical contact into robust cell-cell or cell-matrix adhesion.

[0060] 2. Intermediate Fusion and Interface Remodeling: Using an inverted fluorescence microscope (utilizing a tumor cell reporter gene, such as GFP), it can be observed in real-time or periodically that tumor cells begin to actively migrate from the edge of the spheroid and infiltrate into the organoid tissue. Simultaneously, the original boundary between the two tissues gradually blurs, resulting in structural fusion. This may be accompanied by the degradation of the local extracellular matrix (through the secretion of matrix metalloproteinases, MMPs) and the remodeling of the host tissue structure.

[0061] 3. Late-stage invasion and colonization: During longer culture periods, invasive behavior deepens further. Tumor cells can invade deeper along the internal structures of organoids (such as bile duct networks, vascular structures, or hepatic plates), forming distinct invasion fronts. They may even form discrete microsatellite foci within the organoid, mimicking the formation of micrometastases. Host cells (such as hepatocytes or stromal cells in organoids) may also respond to tumor invasion, such as through stress, apoptosis, or activation.

[0062] (3) Endpoint validation and functional definition of the model: When the culture reaches the preset endpoint, multimodal analysis is required to confirm the successful construction of the model and evaluate its status. 1. Morphological and structural verification: Live-cell dynamic imaging: The entire process was recorded using time-lapse fluorescence microscopy, and the changes in the area and depth of GFP-positive tumor signal diffusion within organoids over time were quantitatively analyzed.

[0063] Model fixation and transparency processing (for endpoint structure analysis): When the culture reaches the preset endpoint, the model can be fixed and made transparent for high-resolution three-dimensional structure observation. The specific steps are as follows: 1. Preparation of washing solution: Add 100 μl of Triton X-100 and 0.2 g of BSA to 100 μl of PBS; 2. Fixation: Transfer the sample to 3 ml of 4% paraformaldehyde and incubate at 4°C for 45 minutes; 3. Washing: Transfer the sample to PBST and incubate at 4°C for 10 minutes. Then transfer the sample to the pre-prepared washing buffer (200 μl / well in a 24-well plate) and incubate at 4°C on a shaker for 15 minutes. 4. Primary antibody incubation: Without removing the washing buffer, add 200 μl of primary antibody working solution (2X concentration) to each well and incubate overnight on a shaker at 4°C; 5. Washing: The next day, add 1 ml of washing solution to each well and wash on a shaker for 2 hours. Then, remove 1 ml of washing solution, add 1 ml of washing solution, and repeat the washing process twice. 6. Secondary antibody incubation: Aspirate 1 ml of washing buffer, add 200 μl of secondary antibody working solution (2X concentration) to each well, and incubate overnight at 4°C in the dark on a shaker. 7. Washing: Repeat step 5; 8. Nucleus staining: Aspirate 1 ml of washing buffer, add 200 μl of DAPI working solution to each well, and incubate in a shaker at 4°C in the dark for 3 hours; 9. Clearing: Carefully transfer the sample to the confocal dish using a Pasteur tube. Without touching the sample, carefully remove the surrounding liquid, then add 100 μl of FC developer and let stand at room temperature for 20 minutes. 10. Imaging: Use a confocal microscope to perform three-dimensional imaging of the sample.

[0064] It should be noted that this transparency process is only used for structural characterization after the model construction is completed, and is not applicable to the live tumor cell spheroids used for assembly in step S1. The spheroids used for assembly in step S1 must remain in a live cell state.

[0065] Endpoint 3D Imaging Analysis: The model is fixed and made transparent, then subjected to high-resolution confocal microscopy or light-sheet microscopy for 3D imaging. Through immunofluorescence multiple staining (e.g., labeling tumor-specific antigens, target organ structural proteins CK19 / ALB, and nuclear DAPI), the invasion volume, invasion distance, and spatial relationship with host tissue of tumor cells are precisely quantified in three-dimensional space.

[0066] 2. Cell viability and proliferation / apoptosis analysis: Immunohistochemistry or immunofluorescence staining was performed on model sections to detect the expression and distribution of proliferation markers (such as Ki-67) and apoptosis markers (such as Cleaved Caspase-3), and to assess the viability of cells in the model and the dynamic competitive relationship between tumor and host tissue.

[0067] 3. Molecular function verification: Whole RNA sequencing (Bulk RNA-seq): As described in the above examples, total RNA was extracted from the model group (fusion assembly) and the control group (physically isolated and co-cultured) for sequencing. Differentially expressed genes were analyzed using bioinformatics, and pathway enrichment analysis (such as KEGG and GO) was performed to confirm whether cancer metastasis-related pathways (such as epithelial-mesenchymal transition, cell migration, angiogenesis, and extracellular matrix receptor interactions) were specifically activated in the model group. This is crucial evidence to confirm the functional relevance of the model at the molecular level.

[0068] Specifically, the specific analysis results of RNA sequencing are as follows: Figure 4 As shown: Figure 4 A shows the overall distribution of differentially expressed genes between the fusion assemblies (model group) and the non-fusion assemblies (control group); Figure 4 B confirmed that the expression levels of key genes for liver cancer metastasis, such as FANCD2, RAD50, and FZD2, were significantly higher in the model group than in the control group. Figure 4 C shows that transfer-related pathways such as the ATM signaling pathway and the IncRNA-regulated classical Wnt signaling pathway are specifically upregulated in the model group, directly verifying the functional reliability of the model at the molecular level.

[0069] Secretion factor analysis: The supernatant of the model culture was collected, and the changes in the secretion levels of cytokines and chemokines related to tumor metastasis and microenvironment regulation were detected by enzyme-linked immunosorbent assay or liquid microarray technology.

[0070] This embodiment employs acoustic forces for non-destructive manipulation, avoiding the damage to cells caused by traditional physical contact and maximizing cell viability and model integrity. Secondly, by combining multiple acoustic field modes (such as vortex fields and double-trap fields), the movement, rotation, and positioning of the spheres can be flexibly and precisely controlled, enabling the controllable construction of complex three-dimensional spatial structures. Thirdly, the constructed model simultaneously includes tumor and normal tissue, dynamically simulating tumor cell invasion, colonization, and their interaction with the microenvironment in vitro, providing a more physiologically relevant platform for metastasis mechanism research. Furthermore, the entire construction process is parameterized and programmed, ensuring model reproducibility and standardization, suitable for high-throughput drug screening. Finally, this approach is highly versatile and scalable; by changing cell types and culture conditions, it can be applied to various cancer types and target organ combinations, and can simulate the impact of different pathological microenvironments on metastasis, providing a powerful and flexible tool for cancer metastasis research and anti-metastasis drug development. It achieves non-contact, high-precision three-dimensional manipulation of tumor cell spheres and spatial assembly with functionally complete target organoids, thereby constructing a highly realistic cancer metastasis organoid model.

[0071] In some embodiments, the tumor cell spheroids are liver cancer cell spheroids, and the target organoids are liver and gallbladder organoids.

[0072] Specifically, this embodiment uses hepatocellular carcinoma spheroids and hepatobiliary organoids as examples. This embodiment provides core biological components: first, HuH-7 hepatocellular carcinoma spheroids labeled with green fluorescent protein (GFP), serving as optically traceable simulated primary lesions; and second, functionally mature hepatobiliary organoids (HBOs) derived from the directed differentiation of human induced pluripotent stem cells (hiPSCs), serving as physiological structures simulating the liver target organ microenvironment. In practice, the hepatocellular carcinoma spheroids are obtained by seeding GFP-HuH-7 cells into ultra-low adsorption plates for three-dimensional suspension culture, ensuring uniform size and dense structure. The hepatobiliary organoids are prepared using a multi-stage sequential differentiation protocol, sequentially inducing hiPSCs to differentiate into endoderm, hepatic progenitor cells, hepatocytes, and bile duct cells, ultimately forming miniature organs with structures combining hepatocytes (expressing ALB, possessing synthetic and metabolic functions) and bile duct cells (expressing CK19, possessing secretory functions). This standardized preparation process ensures the consistency of activity and function of the components used in subsequent assembly and the reproducibility of the model.

[0073] Furthermore, single hepatocellular carcinoma spheroids are assembled onto a single hepatobiliary organoid to form a single-focal metastasis model; or, multiple hepatocellular carcinoma spheroids are assembled onto a single hepatobiliary organoid to form a multifocal metastasis model.

[0074] Specifically, a single-focal metastasis model refers to the precise assembly of a single hepatocellular carcinoma spheroid (such as a GFP-HuH-7 spheroid) onto a specific surface or internal location of a hepatobiliary organoid (HBO). This configuration simulates the initial or typical scenario of a single primary tumor metastasizing to the liver in clinical practice. Its advantage lies in the relatively simple model structure and the ease of controlling variables, making it very suitable for studying the one-to-one basic interaction mechanisms between tumor cells and the target organ microenvironment, such as adhesion, local invasion, the initiation of epithelial-mesenchymal transition (EMT), and the early impact of the microenvironment on the formation of a single metastatic lesion. Multifocal metastasis model: refers to the precise assembly of two or more hepatocellular carcinoma spheroids sequentially or simultaneously into different pre-defined locations on the same hepatobiliary organoid. This configuration simulates the common multiple liver metastases in clinical practice. Its research value is higher and can be used to: (1) study the interaction between metastatic lesions: such as the heterogeneity of the "seed and soil" effect, and the competitive or synergistic effects between metastatic lesions. (2) simulate more complex disease processes: assess the impact of tumor burden on organ function. (3) Conduct more rigorous drug efficacy tests: Anticancer drugs or therapies need to inhibit the growth and invasion of multiple lesions at the same time, which puts forward higher requirements for evaluating the broad spectrum and efficacy of drugs, and also makes the screening results more clinically predictive.

[0075] Furthermore, the specific construction process for single-focal and multi-focal hepatocellular carcinoma metastasis models can be found in [reference needed]. Figure 5 , Figure 5 The paper clarifies the switching logic of the sound field modes: a 1.6λ symmetrical vortex sound field is used to achieve long-distance movement of cell spheroids, a 1.6λ asymmetrical vortex sound field is used to achieve precise short-distance contact, a dual-well sound field assists in spheroid rotation to optimize the fusion effect, and finally a 3.8λ symmetrical vortex sound field is used to maintain the assembly morphology through periodic action. The single-focal model needs to maintain this sound field for 1 hour, and the multi-focal model for 2 hours, clearly demonstrating the differences in the construction of different models and the standardized operating procedures. Specifically, liver cancer is used as an example for illustration: 1. Solitary hepatocellular carcinoma: First, a HuH-7 cell sphere was moved to a predetermined location near the organoid using a 1.6λ symmetric vortex acoustic field. Then, a 1.6λ asymmetric vortex acoustic field was used to move the cell sphere onto the organoid, ensuring full contact between the two. Next, a large-aperture 3.8λ symmetric vortex acoustic field was used to maintain the morphology of the organoid assembly for 1 hour (alternating between 15 minutes of acoustic field application and 15 minutes of no acoustic field application), allowing the cell sphere and organoid to fuse to a certain extent. Subsequently, it was transferred to a cell culture incubator for longer-term culture. 2. Multiple Hepatocellular Carcinomas: The construction process and the composite acoustic field used are similar to those for constructing a single hepatocellular carcinoma. Three HuH-7 cell spheres are sequentially moved onto the organoid. After all three cell spheres have moved to their preset positions, a large-aperture 3.8λ symmetric vortex acoustic field is used to maintain the morphology of the organoid assembly for 2 hours (15 minutes of acoustic field application followed by 15 minutes of no acoustic field application, alternating between the two). During the construction of the organoid assembly model, the dual-well acoustic field can control the rotation of the cell spheres to better facilitate contact and fusion with the organoid.

[0076] Furthermore, hepatobiliary organoids were obtained by differentiation of human induced pluripotent stem cells; liver cancer cell spheroids were formed by three-dimensional culture of the HuH-7 cell line.

[0077] Specifically, human induced pluripotent stem cells (hiPSCs) possess unlimited self-renewal and multi-directional differentiation potential, and are derived from the human body (from healthy donors or patients with specific diseases), thus avoiding species differences. Organoids differentiated from hiPSCs can maximally mimic human native organs in terms of genetic background, tissue structure, and physiological function, overcoming the limitations of animal-derived organoids or immortalized cell lines in terms of related gene expression and function. This implies a multi-stage, time-controlled differentiation process, gradually guiding hiPSCs through the endoderm and hepatic progenitor cell stages by precisely adding different combinations of growth factors and small molecule compounds, ultimately maturing into complex three-dimensional miniature organs containing hepatocytes and bile duct epithelial cells. This approach ensures the functional integrity and batch-to-batch consistency of the organoids and provides the possibility for constructing disease-specific models (such as using patient-derived hiPSCs).

[0078] Hepatocellular carcinoma spheroids were formed from the HuH-7 cell line through three-dimensional culture. The HuH-7 cell line is an internationally recognized and widely used human hepatocellular carcinoma (HCC) cell line, whose genetic and phenotypic characteristics have been thoroughly studied, demonstrating good stability and reproducibility. Choosing this cell line helps standardize research results and ensure comparability with previous studies. "Formed through three-dimensional culture" refers to the process using specific culture techniques (such as ultra-low adsorption plate suspension culture, microplate culture, etc.) to allow HuH-7 cells to aggregate, proliferate, and self-assemble into three-dimensional multicellular spheroids under non-adhesive conditions. This spheroid structure better mimics the microenvironment of solid tumors, such as internal hypoxia gradients, tight cell-cell junctions, and extracellular matrix deposition. Its biological behaviors (such as drug resistance and invasive tendency) are more similar to those of in vivo tumors compared to cells cultured in a two-dimensional monolayer.

[0079] The systematic differentiation and preparation of hepatobiliary organoids (HBOs) derived from hiPSCs is a precise differentiation process that lasts 30-40 days and is divided into several stages: (1) Endoderm induction: using factors such as Activin A and BMP4. (2) Liver-oriented shaping: using factors such as FGF4 and BMP2. (3) Hepatocyte expansion and bile duct morphogenesis: using factors such as HGF and KGF, and introducing Matrigel 3D culture. (4) Functional maturation: using factors such as OSM, dexamethasone, and vitamin K2. Each stage requires specific culture medium components and medium change frequency to ultimately obtain functional HBOs that are mature in transcriptomics, protein expression (such as expression of markers such as ALB, CK19, and HNF4α), and function (such as ICG uptake and urea synthesis).

[0080] The standardized preparation of HuH-7 liver cancer cell spheroids specifically includes: (1) using a HuH-7 cell line that stably expresses a reporter gene (such as GFP). (2) obtaining a single-cell suspension by trypsin digestion and accurately counting the cells. (3) seeding the cells at an optimized cell density (such as 1500-3000 cells per well) into 96-well round-bottom plates treated with ultra-low adsorption. (4) culturing the cells under standard cell culture conditions (37°C, 5% CO2) for a specific number of days (such as 4-7 days), with regular medium changes during the period. (5) finally obtaining tumor spheroids with a controllable diameter range (such as 500-800 μm), regular morphology, and dense uniformity for subsequent assembly.

[0081] Furthermore, the present invention also provides an embodiment of a cancer metastasis organoid model, which is constructed by any of the cancer metastasis organoid model construction methods described in the above embodiments.

[0082] Furthermore, such as Figure 6 As shown, the present invention also provides an embodiment of a system for constructing cancer metastatic organoid models, the system comprising a holographic acoustic forceps module 10, a culture module 11, an imaging module 12, and a control module 13.

[0083] The holographic acoustic tweezers module 10 is used to generate and control a three-dimensional acoustic field to manipulate tumor cell spheres in a non-contact manner; the culture module 11 is used to contain and provide tumor cell spheres, target organoids, and culture medium, and maintain a suitable growth environment; the imaging module 12 is used to observe the position and assembly process of tumor cell spheres and target organoids in real time; the control module 13 is communicatively connected to the holographic acoustic tweezers module 10, the culture module 11, and the imaging module 12, respectively, and is used to control the acoustic field parameters and execute a preset manipulation program based on the imaging information.

[0084] Specifically, this embodiment provides an integrated system dedicated to implementing the aforementioned construction method, namely the Organoid-Spheroid Online Assembly Platform (OSOAP). This system integrates acoustic manipulation, live cell culture, real-time imaging, and intelligent control to form an automated, closed-loop precision operation platform, which is a key equipment guarantee for realizing the high-precision and reproducible construction of cancer metastatic organoid models of this invention.

[0085] 1. Holographic acoustic tweezers module 10: Its core function is to generate and dynamically control complex three-dimensional sound fields to provide non-contact acoustic radiation force to capture and manipulate tumor cell spheres.

[0086] The core component is an ultrasonic phased array transducer. In this embodiment, a planar or curved array composed of multiple (e.g., 256) independent array elements can be used. Each array element can be independently driven by the control system.

[0087] Supporting equipment includes a multi-channel signal generator and a power amplifier. The signal generator produces an initial electrical signal with a specific frequency, phase, and amplitude according to control commands, which, after power amplification, drives the transducer array elements to emit ultrasonic waves.

[0088] Working principle: By precisely controlling the phase delay and amplitude weight of the sound waves emitted by each array element, these sound waves interfere in the culture medium, thereby synthesizing an "acoustic hologram" with a specific mechanical distribution in three-dimensional space, such as a symmetrical vortex field, an asymmetrical vortex field, or a double-well field, to achieve the control of different motion modes (movement, pushing, rotation).

[0089] 2. Training Module 11: Function: To provide a stable and reliable physiological-grade cell growth environment for the entire assembly process and for short-term maintenance.

[0090] Core Components: **Specialized Culture Module:** Typically a glass-bottomed culture dish placed above the acoustic transducer. The glass bottom facilitates high-resolution optical imaging, and its surface is often hydrophilically treated to reduce non-specific cell adhesion. **Environment Control Unit:** A sealed miniature chamber enclosing the culture module and microscope objectives. This unit integrates a temperature control system (such as Peltier temperature control) and a gas mixing and delivery system (precisely controlling CO2 concentration, such as 5%), ensuring that the temperature remains stable at 37°C and the pH value is maintained within the physiological range throughout the assembly process, which may last for several hours.

[0091] 3. Imaging module 12: Function: Provides real-time, high-resolution visual feedback, acting as the eyes to achieve precise "what you see is what you get" control.

[0092] Core component: Inverted fluorescence microscope. Equipped with: High-precision motorized stage and objectives; LED or laser fluorescence light source for stimulating reporter genes (e.g., GFP) in tumor cell spheroids; High-sensitivity scientific-grade camera (e.g., sCMOS) for acquiring bright-field and fluorescence images.

[0093] Workflow: This module continuously acquires real-time video streams of samples in the culture module. The images are transmitted to the control module for processing to identify and track the precise location, outline, and status of tumor spheres and organoids.

[0094] 4. Control Module 13: Function: As the "brain" of the system, it realizes closed-loop automated control of perception, decision-making and execution.

[0095] Hardware foundation: High-performance computer.

[0096] Core Software (Customized Control Software): Integrates multiple key algorithm modules: Image Processing and Recognition Module: Analyzes images transmitted from the imaging module in real time, automatically identifies and locates target tumor spheres (based on fluorescence) and target organoids (based on bright-field contours), and calculates their center coordinates. Sound Field Calculation and Synthesis Module: Based on the target position and preset motion trajectory (e.g., moving from point A to point B), uses angular spectrum method or iterative optimization algorithm (e.g., Gerchberg-Saxton algorithm) to quickly calculate the required phase and amplitude distribution of each element of the transducer array to generate the required manipulating sound field. Motion Control and Feedback Module: Sends the calculated sound field parameters to the signal generator to drive the holographic acoustic tweezers module. Simultaneously, it can control the precision displacement stage for auxiliary focusing or pose fine-tuning. The entire process undergoes real-time closed-loop correction based on imaging feedback to ensure manipulation accuracy. Programmed Flow Execution Module: Users can pre-edit and select assembly programs (e.g., "Construct a single-focal model" or "Construct a three-focal model") through a graphical interface. The software will automatically call the preset sound field sequence (e.g., first use 1.6λ symmetric vortex field to move, then switch to asymmetric field contact) and control its execution timing to achieve "one-click" automated assembly.

[0097] The above are merely embodiments of this application and do not limit the scope of this patent application. Any equivalent structural or procedural changes made using the content of this application's specification and drawings, or direct or indirect applications in other related technical fields, are similarly included within the scope of patent protection of this application.

Claims

1. A method for constructing an organoid model of cancer metastasis, characterized in that, include: S1: Prepare tumor cell spheroids labeled with reporter genes and target organoids derived from stem cells, respectively; S2: Place the tumor cell spheroids and the target organoids in a container containing culture medium; S3: Using holographic acoustic tweezers to generate a preset three-dimensional sound field, and through non-contact precise manipulation, move and position at least one of the tumor cell spheres to a preset position on or inside the target organoid to form an initial assembly; S4: The initial assembly is suspended and maintained in culture to allow tumor cell spheroids to come into contact with, fuse with, and / or invade the target organoid, thereby constructing a cancer metastatic organoid model.

2. The method for constructing a cancer metastatic organoid model according to claim 1, characterized in that, The three-dimensional sound field includes one or more combinations of symmetrical vortex sound field, asymmetrical vortex sound field and double-well sound field. By switching or combining different sound fields, the movement, rotation and positioning of tumor cell spheres can be achieved.

3. The method for constructing a cancer metastatic organoid model according to claim 2, characterized in that, Step S3 specifically includes: The tumor cell spheres are moved to the vicinity of the target organoid using the first symmetric vortex acoustic field. Asymmetric vortex acoustic fields are used to propel tumor cell spheres into contact with target organoids. Dual-well acoustic fields are used to adjust the orientation of tumor cell spheres to optimize contact.

4. The method for constructing a cancer metastatic organoid model according to claim 3, characterized in that, After positioning is completed, a second symmetrical vortex acoustic field with a larger aperture than the first symmetrical vortex acoustic field is used to apply a periodic or continuous maintaining force to the initial assembly.

5. The method for constructing a cancer metastatic organoid model according to claim 1, characterized in that, The tumor cell spheroids are liver cancer cell spheroids, and the target organoids are liver and gallbladder organoids.

6. The method for constructing a cancer metastatic organoid model according to claim 5, characterized in that, A single liver cancer cell spheroid is assembled onto a single liver and biliary organoid to form a single-focal metastasis model; Alternatively, multiple liver cancer cell spheroids can be assembled onto a single liver or biliary organoid to form a multifocal metastasis model.

7. The method for constructing a cancer metastatic organoid model according to claim 5, characterized in that, The hepatobiliary organoids were obtained by differentiation from human induced pluripotent stem cells; the liver cancer cell spheroids were formed by three-dimensional culture of the HuH-7 cell line.

8. The method for constructing a cancer metastatic organoid model according to claim 1, characterized in that, The tumor cell spheroids were labeled with a reporter gene.

9. A cancer metastasis organoid model, characterized in that, It is constructed by the method for constructing a cancer metastatic organoid model according to any one of claims 1 to 8.

10. A system for constructing the cancer metastasis organoid model of claim 8, characterized in that, include: The holographic acoustic tweezers module is used to generate and control a three-dimensional acoustic field to manipulate tumor cell spheres in a non-contact manner. The culture module is used to contain and provide tumor cell spheroids, target organoids, and culture medium, and to maintain a suitable growth environment. The imaging module is used to observe the location and assembly process of tumor cell spheroids and target organoids in real time; The control module is communicatively connected to the holographic acoustic tweezers module, the culture module, and the imaging module, respectively, and is used to control the acoustic field parameters and execute preset control programs based on the imaging information.

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