Thymus epithelial tumor bionic biological 3D printing organ-like model and construction method and application thereof

Abstract

The invention discloses a thymic epithelial tumor bionic biological 3D printing organ-like model as well as a construction method and application of the thymic epithelial tumor bionic biological 3D printing organ-like model. The TETs tumor extracellular matrix composition from the tumor tissue of a patient is determined through proteomics analysis and is used for guiding the preparation of the photocuring bio-ink. The finally prepared biological 3D printing organoid supports primary TETs cell proliferation, and the reduction degree of biophysical characteristics and molecular characteristics of the organoid is obviously superior to that of a traditional matrigel culture organoid. Furthermore, on the basis of the bionic platform, the fact that the rubitedin is a powerful candidate drug for treating TETs is screened out. And some potential curative effect prediction biomarkers are further identified. According to the method, the biological 3D printing type organ model of the TETs is constructed through a biological material design strategy driven by proteomics data for the first time, and a standardized platform is provided for precise drug screening and mechanism exploration of the TETs.

Description

Technical Field

[0001] This application belongs to the fields of biomedical engineering and tissue engineering, specifically relating to the construction of in vitro models of thymic epithelial tumors (TETs), and in particular a proteomics data-driven bio-3D printed organoid model for drug screening and biomarker identification. Background Technology

[0002] Thymic epithelial tumors (TETs), including thymic carcinoma (TC) and thymoma (THYM), are rare but exhibit significant biological heterogeneity, originating from thymic epithelial cells. Survival rates vary significantly among different TET subtypes. The 5-year overall survival rate for THYM is approximately 80%-90%, while survival for high-grade thymoma is significantly shorter. TC accounts for approximately 20%-30%, including squamous cell carcinoma and neuroendocrine carcinoma. Most patients require surgery and conventional platinum-based chemotherapy. However, the overall objective response rate (ORR) of chemotherapy is less than 40%, indicating limited efficacy. Currently, there are no approved targeted therapies or validated biomarkers available for precision treatment. In TET patients, the proportion of driver mutations leading to sensitivity to existing targeted therapies (such as EGFR or ALK gene mutations) is less than 5%, meaning that most patients lack modifiable molecular targets. Therefore, there is an urgent need to establish mechanism-related in vitro models to accelerate the exploration of TET treatments and the identification of biomarkers.

[0003] Organoids are three-dimensional (3D) cell assemblies that, compared to traditional two-dimensional cell culture, can more completely preserve the genetic heterogeneity, spatial tissue structure, and microenvironmental interactions of the primary tumor. However, traditional organoid systems are still limited by their dependence on animal-derived basement membrane extract, Matricene. Although Matricene is widely used in organoid research to simulate the function of the extracellular matrix (ECM), its poor adjustability, large batch-to-batch variability, and low stiffness make it difficult to accurately reflect the dense tumor microenvironment commonly found in solid malignant tumors. These limitations severely restrict the reproducibility, quantitative controllability, and precise reproduction of tissue-specific biophysical signals in organoids.

[0004] In recent years, bioprinting technology has emerged as a new generation of organoid manufacturing technology. Among the various bioprinting technologies, digital light processing (DLP)-based bioprinting has attracted much attention due to its cell compatibility and the precision of spatial and mechanical control over the generated models. By combining photocurable biomaterials with photoinitiators, DLP bioprinting technology can rapidly construct biomimetic tissue structures. GelMA materials maintain biocompatibility and integrin binding motifs while achieving stiffness adjustment through methacrylation. HAMA materials combine hydrophilicity and viscoelasticity, perfectly replicating the polysaccharide composition of the natural extracellular matrix. DLP bioprinting technology has been successfully applied to construct three-dimensional tumor organoids with adjustable stiffness and diverse microenvironments, covering multiple cancer types. However, research on organoids for thymic epithelial tumors (TETs) remains extremely limited. Currently, there are only a few reports using matrix gel culture, and there are no literature records of bioprinted organoid models. The lack of such models highlights a key need in current research: the urgent need to develop biomimetic precision engineering systems to explore the pathophysiological mechanisms, drug responses, and biomarker discovery of TETs in a reproducible manner. Summary of the Invention

[0005] The purpose of this invention is to address the shortcomings of existing technologies by providing a biomimetic 3D-printed organoid model of thymic epithelial tumors (TETs) and its construction method and application. In the construction method of this application, proteomics-guided bio-ink design is used to accurately simulate the ECM composition and mechanical stiffness of TETs in 3D-printed organoids, solving the technical problem that it is difficult to truly reflect the tumor microenvironment of TETs using matrix gel culture and other existing organoid construction technologies.

[0006] To achieve the above objectives, this application adopts the following technical solution: In a first aspect, this application provides a method for constructing a biomimetic 3D-printed organoid model of a thymic epithelial tumor, comprising the following steps: Based on the results of extracellular matrix proteome detection of thymic epithelial tumor cells, a photocurable bio-ink simulating the composition of the extracellular matrix was designed. The photocurable bio-ink was used to photocurable bio-3D print primary TETs cells, thus completing the construction of a TETs organoid model.

[0007] In some embodiments, the photocurable bio-ink comprises: 3-10 wt% GelMA, 0.1-2 wt% HAMA, and 0.01-0.5 wt% photoinitiator.

[0008] In some embodiments, the photoinitiator is selected from one or more of LAP, Irgacure 2959, and TPO. LAP is preferred due to its matching wavelength to the photopolymerization printer and its low cytotoxicity.

[0009] In some embodiments, the dissolving medium of the photocurable bio-ink is any one or more of DPBS buffer, PBS buffer, HBSS buffer, and basal culture medium DMEM, DMEM / F12, and RPMI 1640, in order to maintain an isotonic environment and ensure the activity of primary cells and the stability of the experimental system.

[0010] In some embodiments, the primary TETs cells include one or more of the following: primary tumor cells, primary immune cells, and primary cancer-associated stromal cells.

[0011] In some embodiments, the photopolymerizable bio-3D printed TETs organoid model includes: high-throughput bio-3D printing of a mixture formed by mixing the TETs primary cell suspension with photopolymerizable bio-ink, and photopolymerizing the mixture into a gel form to obtain the TETs organoid model.

[0012] In some embodiments, the method for preparing the primary TET cells includes the following steps: Step 1) After cleaning, the isolated human TETs tissue sample is cut into small pieces; Step 2) Digest the tissue obtained after step 1), collect the cell suspension and centrifuge and filter to obtain cell precipitate; Step 3) Resuspend the cells in basal culture medium and / or DPBS buffer.

[0013] In some embodiments, the bio-3D printing process further includes culturing the organoids in a complete cell culture medium under the following conditions: 37°C and 5% CO2.

[0014] Secondly, this application provides the application of TETs organoid models constructed using the construction method described in the first aspect in screening drugs and biomarkers.

[0015] Thirdly, this application provides the application of drugs or biomarkers screened from organoid models of TETs as described in the second aspect, wherein the screened drugs are used to prepare drugs for treating TETs, and the biomarkers are used to prepare products for predicting the prognosis of TET patients or predicting the therapeutic efficacy of TETs on specific drugs.

[0016] Compared with the prior art, this application has the following beneficial effects: 1) High biomimicry: This application uses proteomics to guide the design of bio-inks, and 3D-printed organoids accurately simulate the ECM composition and mechanical stiffness of TETs (tetrafluoroethylene terephthalates). Figure 1 ).

[0017] 2) High clinical relevance: Organoids preserve the tissue structure of the primary tumor ( Figure 3It also possesses high genome fidelity.

[0018] 3) Reliability of drug screening: The TETs organoid model constructed in this application can successfully identify effective candidate drugs for rubitidine ( Figure 4 This provides theoretical support for new therapies for rare tumors.

[0019] 4) Biomarker discovery: Identifying predictive biomarkers related to survival to advance precision medicine. Figure 6 ).

[0020] 5) High repeatability: DLP printing parameters are standardized, overcoming batch differences in the matrix adhesive, making it suitable for high-throughput applications. Attached Figure Description

[0021] Figure 1 (A) Schematic diagrams of TC and THYM tissues before and after decellularization; (B) Proteomic classification of ECM components in decellularized TC and THYM matrices; (C) Optical images of 3DP GelMA-HAMA constructs with different geometries; (D) Rheological curves of storage modulus (G′) and loss modulus (G″) of bio-ink; (E) Comparison of Young's modulus of native tumor tissue, 3DP constructs and matrix gel.

[0022] Figure 2 (A) Schematic diagram of the 3D printed organoid model construction process, including three steps: tumor tissue dissociation, DLP-based bioprinting, and in vitro culture; (BC) Representative bright-field micrographs of TC and THYM organoids in 3DP and matrix gel.

[0023] Figure 3 Immunofluorescence images of (AB)TC THYM organoids.

[0024] Figure 4 (A) Schematic diagram of the multi-stage drug screening workflow, including preliminary screening, secondary screening and validation experiments; (BC) Dose-response curves of IU-TAB-1 (B) and Ty-82 (C) cells after treatment with the first five effective compounds; (DE) Comparative effects of lurbinectedin and standard chemotherapy drugs in IU-TAB-1 (D) and Ty-82 (E) cells; (FG) Concentration-response curves of lurbinectedin in 3DP THYM (F) and TC (G) organoids.

[0025] Figure 5(A) Heatmap of gene expression profiles of Ty-82 cells in the control group and rubitidine-treated group; (B) Volcano plot showing the distribution of differentially expressed genes (DEGs) in the treatment group and control group; (C) Heatmap showing differential gene expression profiles of IU-TAB-1 cells in the control group and rubitidine-treated group; (D) Volcano plot showing upregulated and downregulated genes.

[0026] Figure 6 (A) Relative expression level of REPS2 in IU-TAB-1 cells (2D and 3D culture) after rubitidine treatment; (B) Relative expression level of CXCR4 in Ty-82 cells (2D and 3D culture) after rubitidine treatment; (C) Kaplan-Meier curves showing overall survival of patients with high or low REPS2 expression; (D) Kaplan-Meier overall survival analysis based on CXCR4 expression levels. Detailed Implementation

[0027] To make the present invention more apparent and understandable, preferred embodiments are described in detail below with reference to the accompanying drawings.

[0028] This application establishes, for the first time, a proteomic atlas of the extracellular matrix of TC and THYM tissues, providing valuable extracellular matrix landscape data for future TETs research and guiding material selection for in vitro model construction. Based on this extracellular matrix information, this application compares traditional matrix gel culture with a DLP printing strategy using composite bio-inks. The bio-3D printed TETs organoids exhibit superior structural integrity, growth performance, and genome fidelity, realistically reproducing the tissue structure, driver mutations, and copy number variation patterns of the primary tumor, fully demonstrating their high reproducibility and biological relevance. Building upon this biomimetic platform, high-throughput drug screening was further conducted, identifying rubitidine, a DNA topoisomerase II inhibitor already approved for small cell lung cancer treatment, as a potential therapeutic candidate for TETs. Through integrated molecular validation and TCGA survival correlation analysis, a transcriptomic comparative study of 2D and 3D models revealed efficacy predictive biomarkers related to drug response. These findings collectively construct a biomarker-guided framework for bioprinting, integrating extracellular matrix characterization, 3D model construction, high-throughput screening, and biomarker identification and validation.

[0029] The technical solution of this application will be further described below with reference to specific embodiments.

[0030] Unless otherwise specified, the test methods or experimental methods described in the following examples are conventional methods; unless otherwise specified, the reagents and materials are obtained from conventional commercial sources or prepared by conventional methods.

[0031] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0032] The experimental methods and materials involved in the following embodiments are as follows: 1. Human Samples and Ethical Approval The samples used in this application were obtained from patients with thymic epithelial-derived tumors at the Department of Thoracic Surgery, Shanghai Chest Hospital, including tumor tissue and adjacent normal tissue. All subjects signed informed consent forms, and the research protocol was approved by the hospital's ethics committee (No. KS25049).

[0033] 2. Tumor decellularization First, seven tumor samples stored at -80°C underwent three freeze-thaw cycles. Then, the tissues were repeatedly rinsed with deionized water until no visible blood residue remained. Next, the samples were incubated with sodium dodecyl sulfate (SDS, 1% w / v, Titan) for 2 hours under magnetic stirring at 4°C and 300 rpm. Afterward, the samples were thoroughly rinsed with deionized water to remove SDS. Subsequently, the tissues were treated with 1M sodium chloride (Titan) solution for 30 minutes under magnetic stirring at 4°C and 300 rpm. After treatment, the samples were rinsed again with deionized water to remove salt residue. Finally, the tissues were further stirred with deionized water for 1 hour under magnetic stirring at 4°C and 300 rpm.

[0034] 3. Proteomics analysis This application embodiment used seven samples for proteomics analysis. The proteomics analysis was performed by Shanghai Ouyi Biotechnology Co., Ltd.

[0035] 4. Tissue dissociation and single-cell separation Human thymic carcinoma tissue was stored in MACS tissue stock solution for no more than 24 hours prior to experimental processing. Fresh tissue was then washed three times with cold Hank's solution containing antibiotics. Mechanical dissociation was performed in centrifuge tubes containing a small amount of preheated enzyme solution. This enzyme solution consisted of Hank's solution with 1.5 mg / ml type IV collagenase, 1 mg / ml hyaluronidase, 0.1 mg / ml DNase I, and 1% PSA. Samples were cut into approximately 1–2 mm pieces using a sterile scalpel. 3 Small pieces of the cell suspension were collected. The digestion solution was transferred to a T-25 culture flask, and the suspension was placed in a cell culture incubator with continuous shaking for 30 minutes after digestion. The digestion process was terminated by adding twice the volume of dissociation termination buffer. The cell suspension was filtered through a sterile 100 μm cell sieve. The cell suspension was centrifuged at 400 × g for 5 minutes at 4 °C, and the supernatant was carefully aspirated.

[0036] 5. Cell lines and cell culture The Ty-82 cell line was derived from an undifferentiated thymic carcinoma sample (JCRB1330) and purchased from Zhejiang Meisen Cell Technology Co., Ltd. The IU-TAB-1 cell line was derived from an AB type thymoma sample and purchased from Applied Biomaterials (Abm, BC, Canada). Ty-82 cells were cultured in RPMI 1640 medium (Abm, BC, Canada) containing 20% ​​fetal bovine serum (FBS, Gibco) and 1% penicillin-streptomycin (Gibco). IU-TAB-1 cells were maintained in RPMI 1640 medium (Abm, BC, Canada) containing 10% fetal bovine serum (FBS, Gibco) and 1% penicillin-streptomycin (Gibco). All cell cultures were performed in a humidified incubator at 37°C with 5% CO2, and mycoplasma contamination was checked periodically.

[0037] 6. Establish a 3D TETs patient-derived tissue (Oragnoid) model After centrifuging the single-cell suspension at 400×g for 5 minutes, the cell pellet was resuspended in a small amount of high-grade DMEM / F-12 buffer. To maintain cell viability, the cell suspension was kept on ice before bioprinting. Methyl methacrylate gelatin (GelMA) and hyaluronic acid methacrylate (HAMA) used in the experiment were provided by Shanghai Yuju Technology, and the photoinitiator phenyl-2,4,6-trimethylbenzoylphosphofluoride (LAP) was purchased from EngineeringForLife. GelMA, HAMA, and LAP were dissolved in calcium- and magnesium-free DPBS buffer to prepare precursor solutions with concentrations of 10% (w / v), 10% (w / v), and 2% (w / v), respectively, and sterilized by filtration through a 0.22 μm syringe filter. When constructing the three-dimensional TET model using the DLP bioprinter from Shanghai Saibo Biotechnology Co., Ltd., the cell suspension was resuspended in the precursor solution, and the final concentrations of GelMA, HAMA, and LAP in the bio-ink were adjusted to 4%, 0.5%, and 0.1%, respectively. Gently pipetting was used to ensure uniform cell distribution and minimize bubble formation. Key printing parameters were set to control sample size and shape: model shape, layer thickness (500 μm), exposure time (25 seconds), and exposure intensity (30%). Bio-ink was added to 24-well plates, and printing was initiated. After printing, samples were gently rinsed with DPBS buffer and incubated in 1 mL of complete culture medium throughout the experiment. Subsequent incubation at 37°C and 5% CO2 was performed for further culture and analysis. The complete cell culture medium contained Advanced DMEM / F-12 and was supplemented with 50 ng ml⁻¹ EGF, 20 ng ml⁻¹ bFGF, 20 ng ml⁻¹ FGF-10, 50 ng ml⁻¹ Wnt-3A, 200 ng ml⁻¹ R-spondin-1, 100 ng ml⁻¹ Noggin, 10 ng ml⁻¹ IGF-1, 10 ng ml⁻¹ IL-2, 500 ng ml⁻¹ A83-01, 1X B-27, 10 μM Y-27632, 200 mM glutamax, 50 ng / ml Afamin, 100 U ml⁻¹ penicillin, and 100 μg ml⁻¹ streptomycin. The medium was changed three times a week.

[0038] 7. Immunofluorescence (IF) staining The 3D-printed thymic carcinoma organoid model was placed in a culture plate and fixed overnight at 4°C with 4% paraformaldehyde (PFA). After fixation, the PFA was removed, and the sample was washed three times with DPBS buffer. Subsequently, it was permeabilized by gentle shaking for 30 minutes at room temperature with DPBS solution containing 0.3% Triton X-100. Non-specific binding sites were blocked by incubation with blocking buffer for 2 hours at room temperature. Without intermediate washing, the sample was incubated overnight at 4°C with primary antibody diluted in blocking buffer. The primary antibodies used included: Pan-CK (MA513203, Yingjie Life) 1:100 dilution, CD117 (AF1356-SP, R&D Systems) 1:100 dilution, CD56 / NACM1 (ab313779, abcam) 1:100 dilution, p63 (ab124762, abcam) 1:100 dilution, TdT (TB4932S, Abmart) 1:100 dilution, and B-myb (18896-1-AP, Proteintech). Samples were washed with DPBS after primary antibody incubation. The samples were then incubated with species-specific secondary antibodies conjugated to Alexa Fluor 488, Alexa Fluor 594, and Alexa Fluor 647 (1:200, abcam) in blocking buffer at room temperature and protected from light for 4 hours. After washing with secondary antibody, the cells were incubated with DAPI staining solution (Beyotime) at room temperature in the dark for 10 minutes. For imaging, individual Organoid models were carefully separated from the cell culture plate and transferred to a confocal imaging dish. Images were acquired using an Olympus Spin SR10 super-resolution rotating disk confocal microscope.

[0039] 8. Flow cytometry detection TETs organoids were digested into single cells using 3DP digestive enzymes and TrypLE Express, and then resuspended for subsequent experiments. Cells were incubated at room temperature with FC blocking agent (130-091-935, Miltenyi Biotechnology) for 10–15 minutes to reduce non-specific staining. Fluorescently labeled antibodies against 7AAD (420404, Biolegend), CD45 (368512, Biolegend), CD326 (369816, Biolegend), CD8 (344704, Biolegend), and CD3 (317342, Biolegend) were added sequentially, followed by incubation in the dark for 30 minutes. Cells were washed twice with flow cytometry buffer to remove unbound antibodies. Data were detected using a BDLSLRFortessa instrument and analyzed using FlowJo software.

[0040] Drug screening in 9.2D and 3D models Chemical reagents were purchased from TargetMol, and the CellTiter-Glo (CTG) assay kit was purchased from Promega. In the initial drug screening stage, both two-dimensional (2D) and three-dimensional (3D) models were used to evaluate drug activity under different cellular environments. Before the experiment, cell lines were resuscitated and expanded, then seeded into 96-well plates at a density of 20,000 cells per well and cultured for 72 hours. After culture, the compounds were diluted and transferred to the assay plate, with a final test concentration of 10 μM and DMSO concentration controlled below 0.2%. A positive control (cell culture medium) was included in each well. After 48 hours of compound treatment, cell viability was measured according to the CTG instructions, using a microplate reader to measure luminescence values. Cell viability was standardized against the control group; compounds with viability below 50% were selected as candidate compounds for further validation.

[0041] Based on the preliminary drug screening results, dose-response assays were performed. The five most effective compounds with the lowest survival rates in each cell line were selected. The baseline concentration for the half-inhibitory concentration (WIC) test was set at nine times the Cmax value of each compound. Each concentration and control group was tested at least four times. Organoid models (2 μl per well) were seeded in 96-well plates at the same compound concentrations and doses as in the preliminary drug screening protocol. The same microplate reader and settings as in the preliminary drug screening experiments were used to measure the luminescence intensity after administration to ensure data consistency. Dose-response curves for each compound were plotted in GraphPad, and the WIC values ​​were calculated using nonlinear regression analysis. Based on the monotherapy results, the efficacy of the compound (Lurbinectedin) that was most sensitive to both cell lines and had the lowest WIC value was compared with clinical combination therapy regimens.

[0042] To validate the applicability of established cell line findings to more clinically relevant models, drug sensitivity testing was further extended to patient-derived primary cells. These primary cells were cultured into three-dimensional organoid models using existing methods and treated with gradient concentrations of rubitidine. After 48 hours of drug treatment, cell viability was quantitatively analyzed using the highly sensitive CellTiter-Glo 3D assay.

[0043] 10. RNA sequencing (RNAseq) TET cells were cultured in a 3D bioprinted model for 48 hours, followed by treatment with rubitidine for 24 hours, after which the cells were collected. The cells were released by digesting the hydrogel with a digestive enzyme working solution at 37°C for 10 minutes. After centrifugation at 400×g for 5 minutes, the supernatant was discarded, and the precipitate was resuspended in 1 mL of Trizol (Thermo Fisher Scientific) for RNA extraction.

[0044] After extraction, RNA integrity was assessed by 1.5% agarose gel electrophoresis or a fragment analyzer, and its concentration and purity were determined using NanoDrop. Subsequently, Oligo(dT)-labeled magnetic beads were used to enrich the mRNA.

[0045] The transcriptome library was constructed following the standardized procedure of the VAHTS Universal V8 RNA Sequencing Library Preparation Kit (Illumina): First, the target RNA was fragmented using an ion lysis reagent and bound to random primers; then, RNA-cDNA hybrids were formed through first-strand reverse transcription; the hybrid RNA was cleaved using a second-strand synthesis reagent, and the remaining RNA was used as primers for second-strand cDNA synthesis; the second-strand cDNA underwent end repair, A-tail ligation, and Y-adaptor ligation; adapter dimers were removed by magnetic bead screening, and the library template was enriched by PCR before the library was finally recovered using magnetic beads.

[0046] The library was quantified using Qubit 4.0 (Thermo Fisher Scientific, USA) and sequenced using Illumina NovaSeq (PE150).

[0047] 11. Whole exome sequencing (WES) Eight samples (including tumor tissue from TC and TYHM patients, adjacent non-tumor tissue, 3D-printed organoids, and Methylprednisolone cultured organoids) were subjected to whole exome sequencing (WES) by Shanghai Ouyi Biotechnology Co., Ltd.

[0048] 12. Real-time quantitative PCR (qRT-PCR) analysis Total RNA was extracted from TET cells using TRIzol reagent (Thermo Fisher Scientific, Massachusetts, USA). Reverse transcription synthesis was performed using the NuovaGen HiScript III first-strand cDNA synthesis kit (R312-01 / 02, China). Quantitative analysis was performed using NuovaGen ChamQ Universal SYBR qPCR premix (Q711-02 / 03, China) on a real-time quantitative PCR instrument. Gene expression levels were calculated using the 2^(-ΔΔCt) method.

[0049] 13. Statistical Analysis Statistical analysis was performed using Prism software (GraphPad). For details of the analysis methods, please refer to the figure captions for each figure.

[0050] Example 1: Characterization of decellularized TETs revealed different ECM features To achieve biomimetic reconstruction of the tumor microenvironment, this embodiment first characterized the extracellular matrix (ECM) components of TC and THYM tissues. After decellularization, the tissue structure transformed from a hard, flesh-colored mass into a soft, transparent matrix. Figure 1 A) confirms that cellular components were successfully removed while preserving the original structure of the extracellular matrix (ECM). For example... Figure 1 As shown in Figure B, similar proportions of structural proteins such as collagen, glycoproteins, and proteoglycans were detected in the ECM samples of both TC and THYM. Based on the characteristics of the glycoprotein-rich extracellular matrix (ECM), organoids based on a traditional matrix gel matrix were first prepared. Simultaneously, to preserve the properties of collagen and hyaluronic acid in the ECM, a photocurable bio-ink composed of gelatin methacrylate (GelMA) and hyaluronic acid methacrylate (HAMA) was developed for 3D bioprinting. Using this printing process, we successfully fabricated a pre-defined structure with precisely tunable stiffness. Figure 1 C). Rheological and hardness tests showed that the elastic modulus of the 3D-printed structure was basically consistent with that of natural tumor tissue, while the matrix adhesive was significantly softer. Figure 1 D, E).

[0051] Example 2: Establishment and characterization of 3D bioprinted thymic tumor organoids To reconstruct a thymic tumor model in vitro, this embodiment dissociates the patient's tumor tissue into a single-cell suspension and integrates it into GelMA-HAMA bio-ink for DLP bio-3D printing. Figure 2 A). The entire process comprises three main stages: tissue processing, bioprinting construction, and subsequent culture. Simultaneously, matrix gel-based organoids were also prepared as controls. Microscopic observation ( Figure 2 BC (Bio-Cosmetic Chemistry) studies showed that both TC and THYM-derived 3D-printed (3DP) organoids and matrix gel-embedded organoids formed dense spherical or lobed structures. The comprehensive experimental results indicate that bioprinted TC and THYM organoids not only maintain stable proliferative capacity and cell viability but also selectively enrich tumor-derived epithelial cells while preserving immune components reflecting the in vivo microenvironment. Compared to matrix gel-based culture systems, the 3D printing system demonstrates superior morphological fidelity and cell consistency, providing a reliable and reproducible experimental platform for subsequent drug screening and biomarker discovery.

[0052] Example 3: Organoid models printed using bio-3D technology reproduced the original tumor phenotype. Immunofluorescence analysis confirmed the tumor subtype characteristics of 3D-printed thymic epithelial-derived tumor organoids. Figure 3 ).

[0053] Example 4: Drug screening using bioprinted models showed that Lubbinectedin is an effective TET compound. This embodiment uses a bioprinted TET cell line model and its corresponding patient-derived organoids to screen compounds with potent antitumor activity through a two-stage high-throughput drug screening technique. Figure 4 A). The half-maximal inhibitory concentration (IC50) of the top five initially screened compounds was determined using a concentration gradient experiment. 50 The results showed that rubitidine exhibited significant dose-dependent cell viability inhibition in both IU-TAB-1 and Ty-82 cells, and its IC50 value was the lowest among all candidate drugs. 50 Lowest value ( Figure 4 BC). Compared with first-line chemotherapy drugs, both thymic carcinoma and thymoma cell lines showed significantly higher sensitivity to rubitidine than traditional drugs such as etoposide, carboplatin, and paclitaxel. Figure 4 DE). To further validate the antitumor efficacy under physiologically relevant conditions, a bioprinted organoid model derived from patient tissue was used for testing. This model can simulate the tumor-native microenvironment. Consistent with the cell line screening results, rubitidine significantly inhibited cell viability in both TC and THYM organoids. Figure 4 FG).

[0054] Example 5: Transcriptome analysis revealed the regulatory mechanism of lurbinectedin in TC and THYM cells. To elucidate the molecular mechanism of rubitidine's antitumor effect, RNA sequencing was performed on a 3D model of the TC cell line Ty-82 before and after drug treatment. Figure 5 The heatmap A shows the differences in gene expression between the control group and the rubitidine-treated group. Differential expression analysis showed that the drug upregulated 547 genes and downregulated 489 genes. Figure 5 B) indicates that rubitidin can induce extensive transcriptional reprogramming in TC cells.

[0055] Heat map ( Figure 5 C) showed a significant difference between the control group and the rubitidine-treated group, while the volcano plot ( Figure 5 D) revealed a wide range of transcriptional reprogramming phenomena, with 1,269 genes upregulated and 1,403 genes downregulated.

[0056] Example 6: Bioprinted 3D TET models can identify and verify biomarkers of the Lurbinectedin response. To identify potential biomarkers related to the efficacy of rubitidine, transcriptome analysis and experimental validation were integrated, and THYM and TC cell 3D models were used in the study.

[0057] In the THYM cell line IU-TAB-1, RNA sequencing data revealed that REPS2 was significantly downregulated before and after rubitidine treatment. Figure 6 A). Notably, REPS2 showed the most stable downregulation trend across all culture systems and was most strongly correlated with patient prognosis. Figure 6 C), indicating that it can serve as a reliable biomarker for assessing the efficacy of rubitidine and the prognosis of THYM. In the TC cell line Ty-82, the gene CXCR4 (C) was found to be significantly upregulated after rubitidine treatment. Figure 6 B), and significantly associated with overall patient survival (B). Figure 6 (D), indicating that CXCR4 can serve as a reliable biomarker for assessing the efficacy of rubitidine and the prognosis of TC.

[0058] This application establishes, for the first time, a 3D bioprinting organoid platform based on proteomics data for constructing models of thymic epithelial tumors (TETs), including thymic carcinoma (TC) and thymoma (THYM), enabling precise modeling, high-throughput drug screening, and biomarker discovery for clinically relevant TETs. By integrating extracellular matrix characterization with photocurable bio-ink design, the platform achieves, for the first time, the biophysical biomimetic reconstruction of the TET microenvironment, successfully restoring the histological structure, genomic characteristics, and mechanical stiffness of primary TET tumors. Using this platform, rubitidine was identified as a potent therapeutic candidate that significantly modulates tumor stress response and microenvironment remodeling. Further analysis revealed several biomarker genes related to efficacy, which have significant prognostic value, fully demonstrating the bridging role of 3D printed tumor models between drug efficacy assessment and translational biomarker identification.

[0059] Compared to traditional matrix gel-based organoid systems, bioprinted TET models exhibit superior reproducibility, structural controllability, and molecular fidelity. Proteomic analysis of decellularized TET tissues revealed that their extracellular matrix (ECM) is dominated by collagen and glycoproteins, providing guidance for the rational selection of printing materials and ensuring a high degree of mechanical property matching that of natural tissues. This design allows the model to retain subtype-specific genetic alterations such as CNV patterns in TC and THYM, while maintaining the intercellular signaling and matrix signaling pathways required for drug response fidelity. Mechanistically, transcriptomic data indicate that rubitidine's effects extend beyond classical DNA damage repair, remodeling transcriptional networks and combining metabolic stress inhibition with extracellular matrix regulation and neural interaction remodeling. These findings deepen our understanding of the mechanisms of action of transcriptional inhibitors in complex tumor systems and underscore the importance of modeling the tumor-matrix interface when evaluating drugs with diverse mechanisms of action.

[0060] From a translational medicine perspective, this application provides a dual breakthrough in both methodology and concept for the preclinical evaluation of rare thoracic malignancies. The 3D bioprinting system constructs a reproducible and human-like framework that effectively links molecular mechanisms with therapeutic effects. Notably, the differential responses observed between 2D and 3D culture systems highlight the necessity of employing microenvironment-aware strategies in biomarker discovery. Future research could extend this platform to multi-cell co-culture systems integrating immune or vascular components to capture dynamic intercellular changes under drug intervention. These groundbreaking advancements make 3D bioprinted TETs (tissue organoids) a powerful tool in precision oncology, enabling both predictive biomarker identification and providing rational drug development solutions for underserved tumor types.

[0061] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any form or substance. It should be noted that those skilled in the art can make several improvements and additions without departing from the present invention, and these improvements and additions should also be considered within the scope of protection of the present invention.

Claims

1. A method for constructing a biomimetic 3D-printed organoid model of a thymic epithelial tumor, characterized in that, Includes the following steps: Based on the results of extracellular matrix proteome detection of thymic epithelial tumor cells, a photocurable bio-ink simulating the composition of the extracellular matrix was designed. The photocurable bio-ink was used to photocurable bio-3D print primary TETs cells, thus completing the construction of a TETs organoid model.

2. The construction method according to claim 1, characterized in that, The composition of the photocurable bio-ink includes: 3~10wt% GelMA, 0.1~2wt% HAMA and 0.01-0.5wt% photoinitiator.

3. The construction method according to claim 2, characterized in that, The photoinitiator is selected from one or more of LAP, Irgacure2959 and TPO.

4. The construction method according to claim 2, characterized in that, The photocurable bio-ink is dissolved in DPBS buffer, PBS buffer, HBSS buffer, and any one or more of the basal culture media DMEM, DMEM / F12, and RPMI 1640 to maintain an isotonic environment and ensure the activity of primary cells and the stability of the experimental system.

5. The construction method according to claim 1, characterized in that, The primary TETs include one or more of the following: primary tumor cells, primary immune cells, and primary cancer-associated stromal cells.

6. The construction method according to claim 1, characterized in that, The photopolymerization bio-3D printing of TETs organoid models includes: high-throughput bio-3D printing of a mixture formed by mixing the TETs primary cell suspension with photopolymerization bio-ink, and photopolymerization of the mixture into a gel form to obtain the TETs organoid model.

7. The construction method according to claim 1, characterized in that, The method for preparing the primary TET cells includes the following steps: Step 1) After cleaning, the isolated human TETs tissue sample is cut into small pieces; Step 2) Digest the tissue obtained after step 1), collect the cell suspension and centrifuge and filter to obtain cell precipitate; Step 3) Resuspend the cells in basal culture medium and / or DPBS buffer.

8. The construction method according to claim 6, characterized in that, The bio-3D printing process further includes culturing the organoids in a complete cell culture medium under the following conditions: 37°C and 5% CO2.

9. The application of the TETs organoid model constructed by the construction method according to any one of claims 1 to 8 in screening drugs and biomarkers.

10. The application of drugs or biomarkers screened based on the TETs organoid model of claim 9, characterized in that, The screened drugs are used to prepare drugs for treating TETs, and the biomarkers are used to prepare products that predict the prognosis of TET patients or predict the therapeutic efficacy of TETs on specific drugs.

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