Human pneumosarcoma-like carcinoma organ as well as construction method and application of human pneumosarcoma-like carcinoma organ

Abstract

The invention particularly relates to a human pneumosarcoma-like carcinoma organ as well as a construction method and application thereof, and belongs to the technical field of biological medicines. The organoid is selected from TJLCI-PSC-O-1 with the preservation number of CCTCC (China Center For Type Culture Collection) NO: C2025218 or TJLCI-PSC-O-2 with the preservation number of CCTCC NO: C2025334, or an amplified culture of an epithelial marker CK and a mesenchymal marker VIMENTIN which are stably co-expressed by the organoid. According to the construction method, a collagenase / dispersing enzyme and DNase I combined stepped enzymolysis system and a three-dimensional culture system are adopted, and high yield and long-term stable amplification of cells are realized. The model highly simulates the pulmonary sarcomatoid carcinoma pathological structure and epithelial-mesenchymal transition phenotype for the first time, can be used for in-vitro drug sensitivity test, in-vivo tumor formation model construction and drug screening, and provides a key platform for basic research and precise treatment of diseases.

Description

A human lung sarcoma-like organoid, its construction method and application Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a human pulmonary sarcoma-like organoid, its construction method, and its application. Background Technology

[0002] Pulmonary sarcomatoid carcinoma (PSC) is a rare and highly aggressive subtype of non-small cell lung cancer, characterized pathologically by the simultaneous presence of epithelial carcinoma components and sarcomatoid spindle cell components within the tumor tissue, or with heterologous sarcomatoid differentiation. PSC patients generally respond poorly to conventional chemotherapy and targeted therapy, resulting in extremely poor clinical prognosis and a median survival significantly lower than other types of lung cancer. Currently, the treatment of PSC remains a significant challenge, primarily due to the lack of systematic and in-depth research into the tumor's unique biological behavior, driving mechanisms, and drug resistance. This is fundamentally limited by the absence of reliable research models that can stably mimic its pathological characteristics.

[0003] Existing PSC research models have significant limitations: In traditional two-dimensional cell culture systems, only a handful of PSC cell lines have been reported, and these are highly susceptible to genetic drift and loss of key phenotypes (especially the epithelial-mesenchymal transition phenotype) during long-term culture. This makes it impossible to maintain the histological heterogeneity and tumor microenvironment of primary tumors, resulting in low correlation between drug screening results and clinical responses. While patient-derived xenograft models can partially preserve the tumor's tissue structure and genetic background, they suffer from bottlenecks such as long construction cycles (several months to half a year), high costs, and unstable success rates (typically 20%-60%). Furthermore, the transplanted human matrix is ​​gradually replaced by mouse matrix, affecting the realism of the tumor microenvironment and making them unsuitable for high-throughput studies.

[0004] Organoid technology, as a three-dimensional in vitro culture system, can realistically simulate the structure, function, and genetic characteristics of source tissues, and has demonstrated significant value in basic research and drug development for various solid tumors (such as colorectal cancer, breast cancer, and prostate cancer). However, for the unique and highly heterogeneous subtype of pulmonary sarcomatoid carcinoma, there are currently no publicly reported successful construction and stable passage of related organoid models. Because PSCs possess both epithelial and mesenchymal differentiation characteristics, their cellular components are complex and highly heterogeneous. Conventional organoid culture media (usually rich in epithelial growth factors such as EGF, Noggin, and R-spondin) suitable for epithelial tumors like lung adenocarcinoma and squamous cell carcinoma are insufficient to support the survival and proliferation of their sarcomatoid components, leading to culture failure or the rapid loss of the biphasic phenotype of the organoid core. Existing conventional digestion methods (such as single collagenase digestion) are inefficient for PSCs, which are rich in stroma and have a dense structure, resulting in insufficient cell yield and survival rates, further increasing the difficulty of construction. Summary of the Invention

[0005] In view of this, the present invention aims to address the problems of difficulty in constructing pulmonary sarcomatoid carcinoma (PSC) research models, loss of phenotype, and unstable passage, by providing an organoid model that can be stably constructed, cultured for a long time, and completely retains the primary pathological structure and molecular characteristics of the tumor, along with its efficient construction method. This method overcomes the technical challenges in PSC organoid culture, such as low digestion efficiency, insufficient cell viability, and difficulty in maintaining tissue structure and heterogeneity, providing a reliable research platform for in-depth study of the biological behavior, drug resistance mechanisms, and treatment strategies of this highly aggressive lung cancer subtype.

[0006] To achieve the above objectives, the technical solution of the present invention is as follows:

[0007] In a first aspect, this invention discloses a pulmonary sarcoma-like carcinoma organoid, which is either the human pulmonary sarcoma-like carcinoma organoid TJLCI-PSC-O-1 with accession number CCTCC NO: C2025218, or the human pulmonary sarcoma-like carcinoma organoid TJLCI-PSC-O-2 with accession number CCTCC NO: C2025334, or obtained by in vitro culture and amplification of the aforementioned organoids and stably expressing the epithelial marker cytokeratin (CK) and the stromal marker vimentin. This biological material was deposited at the China Center for Type Culture Collection (CCTCC) on November 6, 2025.

[0008] Preferably, the organoids have the pathological structural features of the primary pulmonary sarcomatoid carcinoma in terms of tissue morphology and exhibit the epithelial-mesenchymal transition (EMT) phenotype, specifically referring to the co-expression of epithelial markers and mesenchymal markers.

[0009] Secondly, the present invention further provides a method for constructing the above-mentioned organoids, comprising the following steps:

[0010] (1) Tissue processing: Fresh PSC tissue was taken, rinsed with HBSS buffer containing antibiotics, and then cut into 1-3 mm pieces. 3 Fragments of the organization.

[0011] (2) Stepwise enzymatic digestion: Tissue fragments were placed in a digestion solution and digested in two stages at 37°C and 70 rpm (stage 1: 20 min, stage 2: 15-20 min). After digestion, the fragments were filtered through a 70 μm sieve. The digestion solution was based on DMEM / F12 and contained 1 mg / mL collagenase / dispersase and 0.001% DNase I. This optimized digestion system significantly improved the single-cell yield and survival rate (>85%).

[0012] (3) Cell purification: The filtrate was centrifuged at 300 g for 5 min to collect cells. The remaining red blood cells were lysed on ice for 10 min using red blood cell lysis buffer and then centrifuged to remove them.

[0013] (4) Three-dimensional culture: purified cells were cultured at 10 4 Resuspended cells / μL in primary specimen medium, and 2 volumes of Matrigel were added. After mixing, the mixture was inoculated into preheated culture plates to form droplets, which were then cured at 37°C. After curing, preheated three-dimensional medium was slowly added along the well walls, and the plates were incubated at 37°C with 5% CO2.

[0014] Preferably, the primary specimen culture medium comprises organoid complete culture medium, 1% (v / v) Primary Enhancer and 1% (v / v) penicillin / streptomycin / amphotericidal B;

[0015] The three-dimensional culture medium contains organoid complete culture medium and 1% (v / v) penicillin / streptomycin / amphoteric B.

[0016] Preferably, the volume ratio of the matrix gel to the three-dimensional culture medium in step (4) is 1:2, which achieves the best balance between nutrient penetration and three-dimensional structural stability.

[0017] In a preferred embodiment, the organoid complete culture medium is a commercially available tumor organoid culture medium, whose basic components include epidermal growth factor (EGF), fibroblast growth factor (FGF), Noggin, R-spondin 1, Wnt-3a, B27 and N2, which can provide support for epithelial and mesenchymal cells.

[0018] In some embodiments, the complete organoid culture medium consists of organoid basal culture medium, organoid culture medium additive 1, and organoid culture medium additive 2.

[0019] In some embodiments, the construction method further includes organoid passage, cryopreservation, and thawing steps:

[0020] Passaging: When the organoid diameter is greater than 100 μm, digest with TrypLE at 37℃ for 3-5 min until the organoid particle size is 40-60 μm. After digestion is stopped, centrifuge to collect the organoids, resuspend them at a ratio of 200-500 cell clusters per 50 μL of matrix gel, and continue to culture.

[0021] Cryopreservation: Organoids are digested to a particle size of 40-60 μm, resuspended in programmed cryopreservation solution, and then subjected to gradient cooling (4℃→-20℃→-80℃→liquid nitrogen) for long-term preservation.

[0022] Resuscitation: Thaw the cryovials in a 37°C water bath, dilute with culture medium, centrifuge to remove the cryopreservation solution, resuspend the cell pellet in matrix gel, and then inoculate for culture.

[0023] Thirdly, the present invention provides the application of the aforementioned human pulmonary sarcomatoid carcinoma organoids in the preparation of tools for pulmonary sarcomatoid carcinoma research, drug screening, or personalized treatment evaluation.

[0024] Preferably, the tool is used for in vitro drug sensitivity testing, and the drug includes cisplatin.

[0025] Preferably, the tool is used to construct an in vivo xenograft model.

[0026] Preferably, the tool is used to screen or evaluate chemotherapy drugs, targeted drugs, or immunotherapy drugs for pulmonary sarcomatoid carcinoma.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] (1) This invention successfully constructed and preserved human lung sarcoma-like organoids for the first time, filling a key model gap in the global research of this field and providing a standardized and reproducible precious biological sample resource.

[0029] (2) The organoids constructed in this invention are highly similar to the source tumor tissue in terms of tissue morphology (HE staining) and expression profile of key protein markers (CK, VIMENTIN, etc.), accurately simulating the core pathology and molecular phenotype of PSC, thus ensuring the reliability of the research data.

[0030] (3) The present invention significantly improves single cell acquisition rate, cell survival rate and passage stability by using an optimized digestion system (collagenase / dispersase + DNAase combined) and a mild passage technology (TrypLE control of particle size).

[0031] (4) This type of organ is not only an ideal tool for basic research, but has also been proven to be a highly efficient translational medicine platform. It has been successfully applied to: in vitro drug sensitivity testing, which can evaluate the dose-response relationship with cisplatin and other drugs; and in vivo tumorigenesis experiments, which have shown a 100% tumorigenesis rate and successfully constructed a stable preclinical research model. This provides an integrated solution from in vitro to in vivo for drug development, efficacy prediction and personalized treatment strategy formulation of PSC.

[0032] Biological Preservation

[0033] The human pulmonary sarcomatoid organoid TJLCI-PSC-O-1 of the present invention was accepted and registered for deposit by the China Center for Type Culture Collection (CCTCC) on November 6, 2025, with accession number CCTCC NO:C2025218.

[0034] The human pulmonary sarcomatoid organoid TJLCI-PSC-O-2 of the present invention was accepted and registered for deposit by the China Center for Type Culture Collection (CCTCC) on November 6, 2025, with accession number CCTCC NO:C2025334. Attached Figure Description

[0035] Figure 1 shows bright-field micrographs of the two human pulmonary sarcomatoid organoids TJLCI-PSC-O-1 and TJLCI-PSC-O-2 at different time points (days 0, 3, 7, 10, and 13) in primary culture of the present invention. The white line in the figure represents the scale bar, which is 200 μm.

[0036] Figure 2 shows bright-field micrographs of the two human pulmonary sarcomatoid organoids TJLCI-PSC-O-1 and TJLCI-PSC-O-2 after passage to the second generation at different culture time points. The white line in the figure represents the scale bar, which is 200 μm.

[0037] Figure 3 shows bright-field micrographs of two human lung sarcoma-like carcinoma organoids, TJLCI-PSC-O-1 and TJLCI-PSC-O-2, after programmed cryopreservation and thawing. The white line in the figure represents the scale bar, which is 200 μm.

[0038] Figure 4 shows the histological and immunohistochemical comparison of two human lung sarcomatoid organoids, TJLCI-PSC-O-1 and TJLCI-PSC-O-2, and the patient's primary tumor tissue, including: HE staining to show cell morphology and atypia; and immunohistochemical staining to show the expression of broad-spectrum cytokeratin (CK), vimentin, and BRG1 (SMARCA4), respectively.

[0039] Figure 5 shows the single-cell RNA sequencing results of two human lung sarcomatoid organoids, TJLCI-PSC-O-1 and TJLCI-PSC-O-2, and their primary tumor tissues-1 and-2. A represents the t-SNE dimensionality reduction clustering of TJLCI-PSC-O-1 and TJLCI-PSC-O-2, divided into 0-12 cell groups: "1" represents T cells, "3" represents epithelial cells, "11" represents fibroblasts, "0, 5, 7, 8, 2" represent sarcoma cells, "4, 10, 12" represent macrophages, and "6, 9" represent B cells. B shows the t-SNE clustering of different samples. C shows the cell type annotation results of the single-cell sequencing data. D shows the proportion of each cell type in different samples. E shows the expression of key marker genes in different cell types using a violin plot.

[0040] Figure 6 shows the cell viability curves of two human lung sarcoma-like carcinoma organoids, TJLCI-PSC-O-1 and TJLCI-PSC-O-2, under different concentrations of cisplatin treatment.

[0041] Figure 7 shows the validation diagram of the in vivo xenograft tumor model constructed based on two human lung sarcomatoid organoids, TJLCI-PSC-O-1 and TJLCI-PSC-O-2. In the figure, A is the growth curve and volume doubling time calculation of the xenograft; B is a solid photograph of xenograft TJLCI-PSC-O-1; C is a solid photograph of xenograft TJLCI-PSC-O-2; D is a small animal in vivo CT scan image of xenograft TJLCI-PSC-O-1; and E is a small animal in vivo CT scan image of xenograft TJLCI-PSC-O-2. Detailed Implementation

[0042] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are for illustrative purposes only and are not intended to limit the scope of protection of this invention.

[0043] After reading the contents disclosed in this invention, those skilled in the art can make appropriate adjustments or substitutions to the process parameters of the methods and applications described in this invention without departing from the spirit and scope of this invention. Such obvious adjustments, substitutions or combinations should be included within the protection scope of this invention.

[0044] Unless otherwise specified, the materials, reagents, instruments and testing methods used in the following embodiments can be obtained commercially or prepared, operated and implemented with reference to conventional methods disclosed in the art.

[0045] It should be noted that all technical parameters described in this document as numerical ranges (such as temperature, ratio, time, content, etc.) should be understood as encompassing all possible sub-ranges and specific numerical points within that range, regardless of whether the specific numerical value or sub-range is explicitly listed. Unless otherwise specified, the technical terms used in this document have the meanings commonly understood by those skilled in the art.

[0046] Experimental materials: Fresh tissue samples of pulmonary sarcomatoid carcinoma were obtained from Tianjin Medical University General Hospital, with approval from the ethics committee and informed consent from the patients. Female NOD SCID gamma (NSG) immunodeficient mice were obtained from Jicui Pharmaceutical Co., Ltd.

[0047] Laboratory consumables: HBSS buffer (Thermo Fisher Scientific, catalog number C14175500BT); penicillin-streptomycin-amphoteric B triple antibody (Solarbio, catalog number P7630); DMEM / F12 basal medium (Thermo Fisher Scientific, catalog number 11320-033); collagenase / dispersin (Roche, catalog number 10269638001); DNase I (SIGMA, catalog number DN25); erythrocyte lysis buffer (Solarbio, catalog number R1010); DPBS (ServiceBio, catalog number G4200-500ML); TrypLE (Thermo Fisher Scientific, catalog number 1000-500ML). Fisher Scientific, catalog number 12604-021; Matrix (CORNING, catalog number 356255); MasterAim® Tumor Organoid Basal Culture Medium (Hangzhou Aiming Medical Technology Co., Ltd., catalog number 100-314); MasterAim® Tumor Organoid Culture Medium Additive 1 (Hangzhou Aiming Medical Technology Co., Ltd., catalog number 100-315); MasterAim® Tumor Organoid Culture Medium Additive 2 (Hangzhou Aiming Medical Technology Co., Ltd., catalog number 100-608); MasterAim® Primary Enhancer (MasterAim, catalog number 100-008); MasterAim® Organoid Cryopreservation Solution (Hangzhou Aiming Medical Technology Co., Ltd., catalog number 100-045); MasterAim® Organoid Cell Viability Assay Reagent (Hangzhou Aiming Medical Technology Co., Ltd., catalog number 100-347); Cisplatin (MCE, catalog number HY-17394); CellRecovery Solution (CORNING, catalog number 354253); CK, VIMENTIN, CK7, and BRG1 antibodies were purchased from ServiceBio, catalog numbers GB122053, GB11192, GB12225, and GB11258, respectively.

[0048] Main Instruments: The instruments and equipment involved in the embodiments of this invention are all conventional experimental equipment in the field, including but not limited to: 37℃ constant temperature shaker, CO2 incubator, biosafety cabinet, inverted fluorescence microscope, low-speed centrifuge, and cell counter, etc. The above-mentioned equipment can be purchased from professional suppliers (such as Thermo Fisher Scientific, Eppendorf, Olympus, etc.) through conventional commercial channels. The choice of model does not affect the specific implementation and technical effect of this method; any similar equipment that meets the corresponding functional requirements can be used to perform this invention.

[0049] Example 1: Primary construction, passage culture, cryopreservation and resuscitation of human pulmonary sarcomatoid organoids

[0050] 1. Primary construction of organoids

[0051] 1.1 Tissue Sampling and Processing. Fresh tumor tissue was aseptically obtained from surgical specimens of two patients pathologically diagnosed with pulmonary sarcomatoid carcinoma (PSC), and named TJLCI-PSC-O-1 and TJLCI-PSC-O-2, respectively. The tissue blocks were immediately transported in ice-cold HBSS buffer containing 1% penicillin-streptomycin-amphoteric B. In a laminar flow hood, the tissues were rinsed three times with the same buffer to remove blood. Using sterile instruments, the tissues were finely cut into pieces approximately 1-3 mm in size. 3 Smaller pieces are used to increase the contact area during subsequent digestion.

[0052] 1.2 Stepwise enzymatic digestion to obtain single-cell suspension. Tissue fragments were transferred to 15 mL centrifuge tubes containing 3 mL of digestion solution. The digestion solution formulation was DMEM / F12 basal medium supplemented with 1 mg / mL collagenase / dispersase, 0.001% DNase I, and 1% penicillin-streptomycin-amphoteric B triple antibody. The use of collagenase / dispersase effectively dissociates the extracellular matrix, while the combined use of low-concentration DNase I degrades the released DNA, preventing cell aggregation. This is key to improving the yield of single cells from PSCs, a mesenchymal-rich tissue. The centrifuge tubes were horizontally fixed in a 37°C constant-temperature shaker and digested at 70 rpm for 20 minutes. After standing, the supernatant containing a large number of free cells was aspirated into a new tube, and an equal volume of ice-cold HBSS was immediately added to terminate the digestion. 3 mL of fresh digestion solution was added to the remaining tissue fragments in the original tube, and digestion continued under the same conditions for 15-20 minutes. The supernatants from the two digestions were combined and filtered through a 70 μm nylon cell filter to remove incompletely digested tissue clumps and debris, and a single-cell suspension was collected.

[0053] 1.3 Cell purification and counting. The filtered cell suspension was centrifuged at 4°C and 300 g for 5 minutes, and the supernatant was discarded. If the cell pellet was red, indicating significant erythrocyte contamination, 3 mL of erythrocyte lysis buffer was added, and the cells were lysed on ice for 10 minutes before centrifugation again. The pellet was resuspended in DPBS for cell counting and viability testing (trypan blue staining). Following this optimized digestion process, the single-cell yield was 87.2% ± 3.5%.

[0054] 1.4 Three-dimensional matrix gel embedding and culture. The counted cell suspension was centrifuged at 300 g for 5 minutes at 4°C, the supernatant was discarded, and the cell pellet was obtained. The cells were resuspended in pre-cooled primary specimen medium, and the cell density was adjusted to an accurate 1×10⁻⁶. 4cells / μL. Primary specimen culture medium formulation: MasterAim® Tumor Organoid Complete Culture Medium (95mL MasterAim® Tumor Organoid Basal Culture Medium, 3mL MasterAim® Tumor Organoid Culture Medium Additive 1, 2mL MasterAim® Tumor Organoid Culture Medium Additive 2), supplemented with 1% (v / v) MasterAim® Primary Enhancer (a supplement to promote primary cell adhesion and survival) and 1% penicillin-streptomycin-amphoteric B triple antibody. Take the above cell suspension and quickly mix it with Matrigel at a volume ratio of 1:2 using a pre-chilled pipette tip, taking care to avoid air bubbles. In a 24-well plate preheated at 37°C for at least 30 minutes, gently drop 50 μL of the cell-Matrigel mixture vertically into the center of each well, ensuring the droplet does not touch the well wall. Place the culture plate upright in a 37°C incubator for 5 minutes, then carefully invert it and continue to stand for 25 minutes to allow the Matrigel to completely solidify at the top of the wells. After curing, restore the culture plate to its upright position and slowly and gently add 600 μL of preheated primary specimen medium along the well wall using a pipette, being careful to avoid direct impact on the droplets. Add 1 mL of sterile PBS to the remaining wells of the culture plate to maintain humidity. Finally, incubate the culture plate in a saturated humidity incubator at 37°C and 5% CO2. Every 2–4 days, carefully aspirate the old medium and add an equal volume of fresh, preheated primary specimen medium along the well wall.

[0055] 2. Organoid passage culture

[0056] Organoids with a diameter exceeding 100 μm are closer to "micro-organs" in terms of structure, function, and operability. When most organoids with a diameter exceeding 100 μm are observed under an inverted microscope after 7-14 days of culture, they can be passaged and expanded.

[0057] 2.1 Gentle Digestion. Discard the culture medium from each well and add 0.5 mL of TrypLE to each well. Gently pipette several times using a 1 mL pipette tip to disperse and suspend the matrix gel and the embedded organoids. Place the culture plate in a 37°C incubator for 3-5 minutes for digestion. Observe closely under a microscope. When the organoids have been digested into relatively uniform cell clusters with a diameter of approximately 40-60 μm, immediately add 1 mL of DPBS containing antibiotics to stop the digestion.

[0058] 2.2 Collection and Resuspension. Transfer all suspension from the wells to 15 mL centrifuge tubes using a pipette, ensuring complete collection. Centrifuge at 4°C and 300 g for 5 minutes, then discard the supernatant.

[0059] 2.3 Re-inoculation. Resuspend the cell clusters in pre-cooled "3D medium," which is MasterAim® Tumor Organoid Complete Medium supplemented with 1% triple antibodies. Mix with matrix gel at a ratio of 1:2, embedding approximately 20,000-50,000 cells (corresponding to approximately 200-500 cell clusters) in each 50 μL matrix gel droplet. Subsequent inoculation, solidification, and liquid addition procedures are the same as in primary culture (1.4), using "3D medium."

[0060] 3. Programmed cryopreservation of organoids

[0061] To establish a long-term and stable biobank, organoids need to be cryopreserved.

[0062] 3.1 Preparation of Cryopreserved Samples. Vigorously growing and well-morphologically sound organoids were selected and precisely digested at 37°C for 3 minutes using the method described in the passage steps above, yielding cell clusters with a particle size of 40-60 μm. After centrifugation, the cell clusters were resuspended in commercially available serum-free cryopreservation buffer (e.g., MasterAim® organoid cryopreservation buffer) and the cell density was adjusted to 5 × 10⁻⁶ cells / mL. 5 cells / mL.

[0063] 3.2 Gradient cooling. Aliquot 1 mL of cell suspension into clearly labeled cryovials. Place the cryovials in a programmed cooling box and perform the following standard cooling procedure: equilibrate at 4°C for 20 minutes; transfer to -20°C and hold for 2 hours; then transfer to -80°C and incubate overnight; finally transfer to liquid nitrogen gas phase for long-term storage.

[0064] 4. Rapid resuscitation of organoids

[0065] 4.1 Thawing and Washing. Remove the cryovials from the liquid nitrogen container and quickly place them in a 37°C water bath, gently agitating until the contents are almost completely thawed (approximately 1-3 minutes, ideally with small ice crystals remaining in the tube). In a clean bench, add 1 mL of pre-chilled DMEM / F12 medium dropwise to the cryovials, mixing gently. Then transfer all contents to a 15 mL centrifuge tube containing 9 mL of pre-chilled DMEM / F12 medium for further dilution to reduce the cryoprotectant concentration.

[0066] 4.2 Removal of cryopreservation medium and 3D reconstruction. Centrifuge the diluted cell suspension at 4°C and 200 g for 5 minutes, carefully discarding the supernatant. Gently resuspend the cell pellet in 50 μL of primary specimen culture medium, add 100 μL of matrix gel, and mix quickly. Immediately seed the pellet into preheated 24-well plates (50 μL droplets per well). Subsequent solidification and liquid addition steps are exactly the same as for primary culture (1.4). The revived organoids typically recover their 3D growth state within 3–7 days.

[0067] Example 2: Observation of Organoid Morphology and Growth Dynamics

[0068] To verify the success and stability of the constructed models, systematic morphological tracking was performed on TJLCI-PSC-O-1 and TJLCI-PSC-O-2. As shown in Figure 1, on day 0 after primary seeding, the field of view mainly consisted of single or small clusters of cells. By day 3, the formation of typical, smoothly-defined 3D spherical structures was clearly observed. By day 7, the organoids significantly increased in volume and began to exhibit two main morphologies: one part was a densely structured "solid sphere," and the other part developed a central cavity, forming a "cystic" structure, reflecting the intrinsic heterogeneity of the tumor. By day 10, the organoids continued to proliferate. By day 13, most organoids reached or exceeded 200 μm in diameter, and the cavities of the "cystic" structures became more pronounced, indicating mature morphology. After passage (Figure 2), the organoids maintained their heterogeneous characteristics, exhibited active growth, and reached a diameter of 200-300 μm by day 13. The revived organoids (Figure 3) reproduced a similar growth trajectory, proving that the cryopreservation and revival scheme of the present invention is effective and that the model has repeatability and stability.

[0069] Example 3: Organoid Histopathology and Molecular Phenotypic Identification

[0070] To verify the fidelity of organoids to the source tumor at the histological and protein levels, the following tests were performed:

[0071] Sample preparation: Organoids grown to day 14 and their corresponding primary tumor tissues from patients were collected and formalin-fixed and paraffin-embedded tissue blocks. After being recovered by Cell Recovery Solution at 4°C, the organoids were pre-embedded in agarose, then dehydrated, cleared, and impregnated with paraffin to form paraffin blocks, which were then sectioned (4 μm thick).

[0072] HE staining: Following standard procedures, the tissue was dewaxed, rehydrated, stained with hematoxylin and eosin, dehydrated, cleared, and mounted. The HE staining results are shown in Figure 4. The organoid sections and the original tumor tissue sections showed a high degree of consistency in core pathological features: both cell nuclei were deep blue-purple with prominent nucleoli and an increased nucleoplasm-to-cytoplasm ratio; both cytoplasm were stained pink by eosin; and both exhibited typical atypia of malignant tumor cells, such as varying cell size and shape and disordered arrangement. This demonstrates that the organoids constructed in this invention highly replicate the primary tumor at the tissue structure level.

[0073] Immunohistochemical staining: Standardized immunohistochemical staining was performed on the prepared paraffin sections. First, antigen heat retrieval was performed to fully expose the target epitopes. Then, endogenous peroxidase activity was blocked with 3% hydrogen peroxide solution, and non-specific binding sites were blocked with bovine serum albumin (BSA). Afterwards, primary antibodies against broad-spectrum cytokeratins (CK, an epithelial marker), vimentin (a mesenchymal marker), and BRG1 (SMARCA4, a key component of the chromatin remodeling complex) were added, and the sections were incubated overnight at 4°C. The next day, the sections were incubated with HRP-labeled secondary antibody and developed with DAB. The nuclei were counterstained with hematoxylin, and the sections were finally dehydrated, cleared, and mounted.

[0074] The staining results are shown in Figure 4. In both the primary tumor tissue and the two organoid sections (TJLCI-PSC-O-1 and TJLCI-PSC-O-2), a large number of cells simultaneously expressed CK and VIMENTIN, exhibiting a typical "double-positive" pattern. This is direct evidence of epithelial-mesenchymal transition (EMT) and fully conforms to the core pathological features of pulmonary sarcomatoid carcinoma. Further analysis showed that the epithelial marker CK7 and the mesenchymal marker VIMENTIN were co-expressed in the same cell population, confirming the bidirectional differentiation biological characteristics of the tumor. Furthermore, diffuse strong positive signals of BRG1 protein were observed in the nuclei of both tumor tissue and organoid cells, suggesting the intact function of the SWI / SNF chromatin remodeling complex. This feature is not only related to potential sensitivity to platinum-based chemotherapy drugs but also helps to exclude the more aggressive SMARCA4-deficient sarcomatoid carcinoma subtype, thus providing important molecular evidence for the selection of subsequent treatment strategies.

[0075] Example 4: Single-cell transcriptome analysis of organoid cell composition

[0076] To resolve the cellular composition of organoids at single-cell resolution and compare them with primary tumor tissues, we performed 10x Genomics single-cell RNA sequencing (scRNA-seq) on TJLCI-PSC-O-1, TJLCI-PSC-O-2, and their paired primary tumor tissues (denoted as primary tumor tissue-1 and primary tumor tissue-2, respectively). After rigorous quality control, transcriptome data from 31,919 high-quality cells were obtained. The results are shown in Figure 5: Through t-SNE dimensionality reduction clustering and known cell marker expression analysis, we successfully identified cell subpopulations including epithelial cells (EPCAM), fibroblasts (LUM), T cells (CD3E), macrophages (CD163), and B cells (CD79A). Crucially, we defined a "sarcoma cell" population, which simultaneously highly expresses the stromal marker VIM and the epithelial markers KRT7 and KRT19, corresponding to the core molecular phenotype of sarcomatoid carcinoma. Further analysis (Figure 5, D) revealed that the two organoid lines were primarily composed of epithelial cells and the aforementioned "sarcoma cells," containing almost no immune cells or fibroblasts. In contrast, the primary tumor tissue contained abundant tumor microenvironment cells, including various immune cells and fibroblasts. This result, at the single-cell transcriptome level, confirms that the organoids constructed in this invention highly enrich and retain tumor parenchymal cells, especially malignant cells with a sarcoma-like phenotype, while effectively filtering out complex tumor microenvironment components. This provides a purer and more focused in vitro model for studying the biological characteristics of tumor cells themselves.

[0077] Example 5: Application of organoids in in vitro drug sensitivity testing

[0078] To demonstrate the practicality of the organoids of this invention as a personalized drug sensitivity platform, tests were conducted using the classic chemotherapy drug cisplatin as an example.

[0079] Experimental procedure: TJLCI-PSC-O-1 and TJLCI-PSC-O-2 organoids were digested into single cells / clusters, and 1×10⁻⁶ cells were placed in each well. 4 Viable cells were seeded at a density of [number] cells per well and seeded in 96-well plates coated with Matrigel. After 72 hours of culture, miniature organoids were formed. A cisplatin concentration gradient was set up (0, 3.125, 6.25, 12.5, 25, 50, 100 μM), with six replicates for each concentration. A blank control well containing only culture medium was also included. After drug addition, the cells were cultured for another 72 hours. An equal volume of MasterAim® organoid cell viability assay reagent was added to each well. The cells were lysed by shaking at room temperature for 10 minutes, and the chemiluminescence values ​​were read using a microplate reader.

[0080] The average luminescence signal from wells with 0 μM drug was used as 100% cell viability, and the relative cell viability (%) at each drug concentration was calculated. A dose-response curve was plotted with drug concentration on the x-axis (logarithmic scale) and relative cell viability on the y-axis (Figure 6). The half-maximal inhibitory concentration (IC50) was calculated from the curve. 50 Figure 6 shows that both organ types exhibited typical dose-dependent growth inhibition in response to cisplatin, but the IC50 value was significantly lower. 50 The differences in values ​​suggest that they may have different drug sensitivity profiles, which simulates the heterogeneity among clinical patients and demonstrates the model's potential for personalized medication guidance.

[0081] Example 6: Application of organoids in in vivo tumorigenicity and preclinical model construction

[0082] To evaluate the in vivo tumorigenicity of the organoids of the present invention and to establish a preclinical model that can be used for in vivo studies, mouse xenotransplantation experiments were conducted.

[0083] Experimental Procedure: Six- to eight-week-old female NOD SCID gamma (NSG) immunodeficient mice were used. Approximately 20 well-developed organoid spheroids each from TJLCI-PSC-O-1 and TJLCI-PSC-O-2 were collected (totaling approximately 1 × 10⁻⁶). 6 100 μL of the cell suspension was resuspended in a mixture of 50 μL organoid culture medium and 50 μL Matrigel. Using an insulin syringe, 100 μL of the cell suspension was subcutaneously injected into the groin area of ​​the hind limbs of mice. Five mice were inoculated in each group.

[0084] Starting from the second week after injection, the longest diameter (a) and the shortest diameter (b) perpendicular to it were measured twice weekly using electronic calipers, and the tumor volume was calculated using the formula V = (a × b²) / 2. Tumor growth curves were plotted. As shown in Figure 7, both organoids successfully formed visible and measurable subcutaneous tumors in all inoculated mice, with a tumor formation rate of 100% (10 / 10). Solid tumor formation was confirmed by small animal in vivo CT scans (DE in Figure 7) and endpoint dissections (BC in Figure 7). The tumor volume doubling time (DT) was calculated, and the DTs for the transplanted tumors derived from TJLCI-PSC-O-1 and TJLCI-PSC-O-2 were approximately 8.69 ± 1.06 days and 7.87 ± 0.59 days, respectively (A in Figure 7), indicating stable and reproducible in vivo proliferation capacity. This experiment not only confirmed that the organoids of this invention retain the malignant proliferative potential of the source tumor, but more importantly, it successfully constructed a passageable in vivo transplanted tumor model corresponding to a specific patient source. This model can be used for subsequent in vivo pharmacodynamic evaluation, metastasis mechanism research, and drug resistance model construction, serving as a key bridge connecting in vitro discovery and preclinical validation.

[0085] In summary, the specific embodiments of this invention, through a complete and reproducible standardized operating procedure, have for the first time successfully constructed, passaged, preserved, and comprehensively identified human pulmonary sarcomatoid carcinoma organoids. This model highly mimics the primary tumor in terms of tissue morphology, key protein expression, and cellular transcriptome characteristics, and has been successfully applied to in vitro drug screening and in vivo tumorigenesis model construction, fully demonstrating its practical value and innovation as a reliable and efficient platform for studying the biological characteristics of pulmonary sarcomatoid carcinoma and developing novel treatment strategies.

Claims

1. A human lung sarcoma-like organoid, characterized in that, It is either the human pulmonary sarcomatoid carcinoma organoid TJLCI-PSC-O-1 with accession number CCTCC NO: C2025218, or the human pulmonary sarcomatoid carcinoma organoid TJLCI-PSC-O-2 with accession number CCTCC NO: C2025334, or obtained by in vitro culture and expansion of the aforementioned organoids and stably expressing the epithelial marker cytokeratin (CK) and the stromal marker vimentin.

2. The human pulmonary sarcoma-like organoid according to claim 1, characterized in that, The organoids exhibit the pathological structural characteristics of primary pulmonary sarcomatoid carcinoma in terms of tissue morphology, and show co-expression of epithelial and stromal markers.

3. A method for constructing human pulmonary sarcoma-like organoids, characterized in that, The steps include: (1) Tissue processing: Fresh pulmonary sarcomatoid carcinoma tissue is taken, rinsed with HBSS solution containing antibiotics, and then cut into 1-3 mm pieces. 3 (2) Stepwise enzymatic digestion: The tissue fragments were placed in the digestion solution and digested in two stages at 37°C and 70 rpm. The first stage of digestion lasted 20 min, and the second stage of digestion lasted 15-20 min. After digestion, the tissue fragments were filtered through a 70 μm sieve. The digestion solution contained 1 mg / mL collagenase / dispersin and 0.001% DNase I. (3) Cell collection and purification: Cells were collected by centrifuging the filtrate at 300 g for 5 min. If there were any residual red blood cells, they were lysed on ice for 10 min with red blood cell lysis buffer and centrifuged again to obtain purified cells. (4) Three-dimensional culture: The purified cells were cultured at 100°C and 100 rpm. 4 The cells / μL density was resuspended in the primary specimen medium, and twice the volume of matrix gel was added. After thorough mixing, the mixture was inoculated into preheated culture plates and cured at 37°C to form droplets. After curing, preheated three-dimensional culture medium was slowly added along the well wall and cultured at 37°C with 5% CO2. The primary specimen medium contained organoid complete culture medium, 1% Primary Enhancer, and 1% penicillin / streptomycin / amphomycin B. The three-dimensional culture medium contained organoid complete culture medium and 1% penicillin / streptomycin / amphomycin B.

4. The construction method according to claim 3, characterized in that, The volume ratio of the matrix gel to the three-dimensional culture medium in step (4) is 1:

2.

5. The construction method according to claim 3, characterized in that, It also includes organoid passage steps: when the organoid diameter is greater than 100 μm, digest with TrypLE at 37℃ for 3-5 min until the organoid particle size is 40-60 μm. After digestion is terminated, collect the organoids by centrifugation, resuspend and seed them at a ratio of 200-500 cell clusters per 50 μL of matrix gel, and continue to culture.

6. The construction method according to claim 3, characterized in that, It also includes organoid cryopreservation and thawing steps: during cryopreservation, the organoids are digested to a particle size of 40-60 μm, resuspended in programmed cryopreservation solution, and then cryopreserved by gradient cooling; during thawing, the cryopreservation tubes are thawed in a 37°C water bath, diluted with culture medium, centrifuged to remove the cryopreservation solution, and the cell pellet is resuspended in matrix gel and then inoculated for culture.

7. The use of the human pulmonary sarcomatoid carcinoma organoids as described in claim 1 or 2 in the preparation of tools for pulmonary sarcomatoid carcinoma research, drug screening, or personalized treatment evaluation.

8. The application according to claim 7, characterized in that, The tool is used for in vitro drug sensitivity testing, and the drug includes cisplatin.

9. The application according to claim 7, characterized in that, The tool is used to construct in vivo xenograft models.

10. The application according to claim 7, characterized in that, The tool is used to screen or evaluate chemotherapy drugs, targeted drugs, or immunotherapy drugs for pulmonary sarcomatoid carcinoma.

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