Culture medium for constructing ROS1 gene rearrangement type non-small cell lung cancer organoids and application thereof

Abstract

The application discloses a culture medium for constructing a ROS1 gene rearrangement type non-small cell lung cancer organoid and application thereof, and belongs to the technical field of organoid models. The culture medium comprises a culture medium basic component, a specific factor component and an additive component. The obtained ROS1 gene rearrangement type non-small cell lung cancer organoid has the advantages of high survival rate, sufficient quantity, sustainable subculture and the like, and retains a pathological morphology consistent with an original tumor tissue. The application aims to overcome technical bottlenecks of a small amount of puncture samples and high culture difficulty, and provides a stable and reliable in-vitro research platform for individualized drug use guidance, drug resistance mechanism research and new type targeted drug screening of the ROS1 gene rearrangement type non-small cell lung cancer, so as to prolong a survival cycle of a patient and improve a life quality of the patient.

Description

Technical Field

[0001] This invention belongs to the field of organoid model technology, specifically relating to a culture medium for constructing organoids from ROS1 gene rearranged non-small cell lung cancer and its application. Background Technology

[0002] ROS1-rearranged non-small cell lung cancer (ROS1+ NSCLC) is a rare but molecularly distinctive driver gene-positive subtype of lung cancer, accounting for approximately 1%-2% of all NSCLC cases. This subtype is more common in young patients with lung adenocarcinoma who have no or a light smoking history. Its pathological features and clinical behavior are similar to those of ALK-rearranged NSCLC, but the molecular mechanisms and responses to targeted therapy differ significantly. The ROS1 gene, located at 6q22, encodes an orphan receptor tyrosine kinase belonging to the insulin receptor superfamily. Its gene rearrangement leads to the formation of fusion proteins between the N-terminal fusion chaperone (such as CD74, SLC34A2, SDC4, EZR, etc.) and the C-terminal kinase domain, resulting in ligand-independent constitutive activation, which in turn drives tumorigenesis and development. Currently, targeted therapy for ROS1+ NSCLC has made significant progress, with objective response rates of 60%-80% achieved by tyrosine kinase inhibitors such as crizotinib, entrectinib, and loratinib. However, acquired resistance is unavoidable, and the median progression-free survival is usually only 15-20 months. In particular, for patients with secondary resistance mutations (such as G2032R, L2026M, D2033N), subsequent treatment options are extremely limited. Furthermore, there is significant heterogeneity in the sensitivity and resistance mechanisms of TKIs among different fusion partner types, leading to great uncertainty and blind spots in clinical drug use.

[0003] The aforementioned challenges highlight the urgency of developing precise in vitro drug sensitivity models. Patient-derived organoids, as three-dimensional culture models that faithfully preserve the primary tumor tissue structure, cell population heterogeneity, and key signaling pathway activity, provide an ideal platform for preclinical drug sensitivity mapping analysis. Using organoids cultured from ROS1+ NSCLC samples for drug sensitivity testing not only provides clinicians with intuitive in vitro evidence before treatment, predicting patients' actual sensitivity to specific chemotherapy regimens and enabling personalized medicine, but also allows for in-depth exploration of effective therapeutic targets for different fusion partner types and drug-resistant mutation populations, which is of great significance for improving the precision treatment of ROS1+ NSCLC.

[0004] Currently, the main clinical sampling method for ROS1+ NSCLC samples is biopsy, which is based on two main reasons: First, ROS1+ NSCLC patients are often diagnosed at an intermediate or advanced stage, making it difficult to obtain surgical specimens; second, biopsy is minimally invasive, simple to perform, and can safely and effectively obtain sufficient tumor tissue from lung lesions or metastatic sites for molecular testing or culture.

[0005] However, the construction and culture of ROS1+ NSCLC organoids derived from biopsies are far more difficult than those of other common NSCLC subtypes, mainly due to two technical barriers:

[0006] The first barrier: Constitutive activation of ROS1 fusion kinase leads to imbalance in downstream signaling pathways, triggering the apoptosis program.

[0007] ROS1 fusion proteins mediate structural activation of the kinase domain through the oligomerization domain of the N-terminal chaperone, sustaining high-intensity activation of multiple downstream signaling pathways, including the MAPK / ERK, PI3K / AKT, and JAK / STAT pathways. Studies have shown that ROS1 fusion proteins activate the RAS-RAF-MEK-ERK cascade via the SHP2-GRB2-SOS1 complex, leading to a significant increase in ERK phosphorylation levels. More importantly, ROS1 signaling intensity exhibits a double-edged sword effect on cell fate: moderate ROS1 activation promotes cell proliferation, while excessive activation triggers apoptosis. Research confirms that different ROS1 fusion variants exhibit varying subcellular localization due to differences in the N-terminal chaperone, resulting in differentiated MAPK pathway activation capabilities—SDC4-ROS1 and SLC34A2-ROS1 are localized in endosomes, strongly activating the MAPK pathway; while CD74-ROS1 is localized in the endoplasmic reticulum, exhibiting relatively weaker MAPK pathway activation. This fusion partner-dependent signaling bias makes it difficult to adapt single culture conditions to patient samples with different ROS1 rearrangements.

[0008] In organoid culture environments, ROS1+ tumor cells, deprived of the in vivo microenvironment, experience further uncontrolled constitutive activation of the ROS1 fusion protein due to the loss of regulation from stromal cells, cytokines, and mechanical stress. Overactivated MEK / ERK signaling is continuously amplified through a positive feedback loop, simultaneously activating the p38MAPK stress pathway, leading to mitochondrial membrane depolarization, cytochrome c release, and a caspase cascade reaction, ultimately triggering apoptosis. This mechanism profoundly reveals the inherent vulnerability of ROS1+ tumor cells in in vitro culture: their survival is highly dependent on the precise regulation of ROS1 kinase activity; any culture conditions leading to abnormal signal intensity can disrupt the balance between proliferation and apoptosis, resulting in culture failure.

[0009] The second barrier: low cell count, lack of matrix, and mechanical stress damage in the puncture sample further exacerbate the apoptotic tendency of ROS1+ tumor cells.

[0010] Clinical diagnosis of ROS1+ NSCLC is highly dependent on molecular testing, and patients are often diagnosed at an advanced stage. Tissue samples are mainly obtained through CT-guided percutaneous lung biopsy or bronchoscopic ultrasound-guided biopsy. Compared with surgically removed specimens, biopsy samples have the following inherent limitations: (1) Extremely limited cell count: biopsy tissue usually contains only 10³-10⁻⁶ cells. 4 (1) Tumor cells, while organoid culture requires a sufficient number of starting cells to form cell clusters and establish paracrine signaling networks; (2) Complete loss of extracellular matrix: The puncture process causes tumor cells to separate from the natural matrix, lose integrin-mediated survival signals, and trigger anodic apoptosis; (3) Mechanical and metabolic stress damage: The shearing force of the puncture needle leads to cell membrane damage, reactive oxygen species (ROS) bursts and mitochondrial dysfunction, further activating the p38MAPK and JNK stress pathways; The aforementioned dual barriers create a synergistic lethal effect in organoid culture: overactivation of the ROS1 fusion protein makes tumor cells extremely sensitive to changes in the microenvironment, and the lack of matrix in the puncture sample and stress damage further amplify this sensitivity. Under conventional culture conditions, ROS1+ tumor cells undergo rapid apoptosis due to ERK hyperphosphorylation and continuous p38MAPK activation. Coupled with insufficient initial cell quantity, this results in extremely low organoid formation rates (usually below 20%) and poor passage stability. Existing lung cancer organoid culture systems (such as conventional media containing FGF7, FGF10, Noggin, and SB202190) can support the culture of some NSCLC subtypes, but they have not been optimized for the specific signaling characteristics of the ROS1+ subtype. While SB202190, as a p38 inhibitor, can alleviate stress damage, it cannot resolve the proliferation-apoptosis imbalance caused by MEK / ERK overactivation; and MEK inhibitors (such as Trametinib) completely block the MAPK pathway at tumor therapeutic doses, thus inhibiting necessary pro-survival signals. In addition, existing patents mostly focus on optimizing the composition of general lung cancer organoid culture media or improving culture methods, without distinguishing specific molecular subtypes, and without systematically designing for the heterogeneity of ROS1 rearranged fusion partners and the special characteristics of puncture samples, resulting in low success rates of ROS1+ NSCLC organoid culture, which seriously restricts the clinical translation of this technology.

[0011] Therefore, developing an organoid construction technology that is specifically designed for puncture-derived ROS1+ NSCLC samples, can precisely regulate the downstream signal intensity of ROS1, takes into account the heterogeneity of fusion partners, and is highly efficient and stable is of great practical significance for breaking through existing technical bottlenecks and establishing an in vitro model platform for ROS1+ NSCLC precision medicine.

[0012] This invention is proposed based on this urgent need. It mainly focuses on optimizing the signaling pathway (the synergistic effect of low-concentration MEK inhibitor Trametinib combined with p38 inhibitor SB202190 and ROCK inhibitor) to precisely control the culture medium formula, thereby achieving high survival rate, high yield and accurate drug sensitivity prediction of ROS1+ NSCLC organoids. Summary of the Invention

[0013] The technical problem to be solved by this invention is how to solve the problems of low survival rate and poor morphology in the culture of ROS1 gene rearranged non-small cell lung cancer organoids derived from puncture samples.

[0014] The present invention solves the above-mentioned technical problems through the following technical means: The first aspect of the present invention provides a culture medium for constructing organoids from ROS1 gene rearranged non-small cell lung cancer, comprising basic culture medium components, specific factor components, and additive components. The basic components of the culture medium include Advanced DMEM / F12, 1-5 mM GlutaMAX, 50-100 U / mL penicillin-streptomycin, and 10-25 mM HEPES; The specific factor components include 100-500 nM A 83-01, 10-50 ng / mL FGF-7, 20-100 ng / mL FGF-10, 1-10 nM Trametinib, 100-500 nmSB202190, 200-500 ng / mL R-spondin 1, and 20-100 ng / mL Noggin; The additive components include one of the following: Composition A: 5-30 µM Y-27632, 1-2 × B27 reagent, 100-200 μg / mL Primocin, 1-2 mM N-Acetylcysteine, 4-6 mM N-Cotinamide; Composition B: 1-2 × B27 reagent, 100-200 μg / mL Primocin, 1-2 mM N-Acetylcysteine, 4-6 mM N-icotinamide.

[0015] When the additive components include component A, the entire culture medium is denoted as culture medium A; When the additive components include component B, the entire culture medium is referred to as culture medium B.

[0016] illustrate: SB202190 is a p38 MAPK inhibitor that blocks the overactivation of ROS1 fusion kinase and the p38MAPK apoptosis pathway induced by puncture stress, rescues cell viability and increases organoid formation rate. Trametinib is a MEK1 / 2 inhibitor that precisely regulates the overactivated MEK / ERK pathway downstream of the ROS1 fusion protein at low concentrations, restoring the proliferation-apoptosis balance and avoiding cell death caused by complete blockade. N-acetylcysteine ​​is an antioxidant and a glutathione precursor that neutralizes the reactive oxygen species (ROS) bursts generated by the overactivation of ROS1 fusion kinase, protects mitochondrial function, and prevents oxidative stress-induced apoptosis. Combination 1: SB202190 + Trametinib (core "dual-pathway regulation" combination) This is the core innovative combination of this formulation. SB202190 blocks the p38MAPK stress-induced apoptosis pathway, while Trametinib precisely inhibits MEK / ERK overactivation. Together, they address the "double-edged sword" effect of ROS1 fusion kinase, alleviating cytotoxicity caused by overactivation while preserving essential survival-promoting signals. This allows ROS1+ tumor cells to restore proliferation-apoptosis balance in in vitro culture, increasing organoid formation rate from less than 20% to over 60%. This combination is the core innovation that distinguishes this formulation from general lung cancer organoid culture, directly targeting the molecular characteristics of the ROS1+ subtype for precise intervention, rather than simply applying a standard formulation. Combination 2: N-acetylcysteine ​​+ SB202190 (synergistic protection against oxidative stress and apoptosis) N-acetylcysteine ​​eliminates ROS bursts at the metabolic level, while SB202190 blocks stress-induced apoptosis at the signaling level. Together, they form a dual "metabolic-signaling" protective network, effectively combating mechanical damage to the punctured sample and oxidative stress caused by excessive ROS1 activation, and significantly improving the survival rate of the initiating cells.

[0017] R-spondin 1 is an LGR5 ligand that enhances stem cell properties by activating the Wnt / β-catenin signaling pathway, maintaining organoid potential (OFP) and promoting epithelial cell self-renewal and long-term expansion. Noggin is a BMP signaling antagonist that blocks BMP2 / 4 / 7-induced epithelial differentiation and apoptosis, maintains the undifferentiated state of stem cells, and synergistically enhances organoid formation efficiency with Wnt activators. A83-01 is an effective inhibitor of Smad2 phosphorylation and TGF-β-induced epithelial-mesenchymal transition. It also inhibits Smad2 phosphorylation, thereby maintaining the self-renewal and proliferation of tumor stem cells. Therefore, A83-01 plays an important role in promoting cell proliferation, preventing cell differentiation, maintaining stem cell stemness, and inhibiting apoptosis and senescence. Combination 3: R-spondin 1 + Noggin + A83-01 (the "Three Musketeers" for maintaining dryness) R-spondin 1 activates the Wnt / β-catenin pathway, Noggin inhibits BMP differentiation signaling, and A83-01 blocks TGF-β growth inhibition. The three work together to construct a "proliferation-anti-differentiation-anti-EMT" microenvironment, maintain the stem cell-like characteristics of ROS1+ tumor cells, and prevent organoid function loss due to differentiation during culture.

[0018] FGF-7 is an epithelial cell-specific mitogen that maintains basal stem cell proliferation by activating the FGFR2IIIb receptor, supporting the survival and clone formation of airway and alveolar epithelial cells. FGF-10 is a paracrine factor derived from the mesenchyme. It maintains the stemness of distal airway stem cells through FGFR1 / 2 signaling, and promotes branching morphogenesis and the complexity of organoid three-dimensional structures. Combination 4: FGF-7 + FGF-10 (synergistic support for epithelial stem cells) FGF-7 maintains basal cell proliferation through FGFR2IIIb, while FGF-10 supports distal airway stem cells through FGFR1 / 2. Both cover different epithelial subpopulations, ensuring the heterogeneity of organoid cell populations and faithfully reproducing the cellular composition of the primary tumor.

[0019] B-27 is a serum-free culture medium additive that provides lipids, antioxidants and trace element complexes to replace serum in order to maintain cell survival and reduce differentiation-inducing signals, providing basic nutritional support for organoid culture. Nicotinamide, as an NAD+ precursor, can enhance cellular energy metabolism, maintain epithelial cell proliferation and self-renewal by inhibiting sirtuins activity, and promote organoid formation efficiency. Combination 5: B-27 + Nicotinamide + N-acetylcysteine ​​(Basal metabolic support system) B-27 provides comprehensive nutrition, Nicotinamide enhances NAD+-dependent metabolic activity, and N-acetylcysteine ​​maintains redox homeostasis. These three components constitute the "metabolic base" of organoid culture, providing energy and redox support for the precise regulation of signaling pathways.

[0020] A second aspect of the present invention provides a method for preparing the above-mentioned culture medium, comprising the following steps: mixing the components evenly to obtain the culture medium.

[0021] A third aspect of the present invention proposes the application of the above-mentioned culture medium in the construction of ROS1 gene rearranged non-small cell lung cancer organoids.

[0022] Preferably, the ROS1 gene rearranged non-small cell lung cancer is derived from a biopsy sample or a non-biopsy sample.

[0023] A fourth aspect of the present invention provides a method for constructing organoids from ROS1 gene rearranged non-small cell lung cancer derived from biopsy, comprising the following steps: (1) Sample preprocessing: The ROS1 gene rearranged non-small cell lung cancer puncture samples were cleaned, then cut into small meat-like pieces, digested, filtered, centrifuged, and the sample precipitate was collected. (2) Pre-culture of cell suspension: Add the above-mentioned culture medium A to the sample precipitate for culture, and after forming a cell suspension, mix it with Matrigel, drop it into the well plate, and incubate it statically. (3) Culture in different culture media: Add the above-mentioned culture medium A to the well plate (2) for culture, and then replace it with the above-mentioned culture medium B for culture, and you will get the desired result.

[0024] Preferably, in (1), the cleaning is as follows: the sample is transferred to the sample cleaning solution and incubated at 100~300 rpm and 20~27℃ for 20~40 min, and the sample cleaning solution is replaced every 3~8 min.

[0025] Preferably, in (1), digestion is performed by transferring small pieces of minced meat into the sample digestion solution and digesting at 100-300 rpm and 35-38°C for 3-8 minutes.

[0026] Preferably, in (1), filtration is performed using a 50~80μm filter screen; centrifugation is performed at 450~550×g for 5~10min.

[0027] Preferably, in (2), the static incubation is: static incubation at 35~38℃ for 25~40min.

[0028] Preferably, in (3), culture medium A is cultured for 1 to 3 days; culture medium B is cultured for 5 to 16 days.

[0029] The fifth aspect of the present invention provides for the ROS1 gene rearranged non-small cell lung cancer organoids constructed by the above-described construction method.

[0030] The sixth aspect of this invention proposes the application of the above-mentioned ROS1 gene rearranged non-small cell lung cancer organoids in personalized medicine guidance, drug resistance mechanism research, or targeted drug screening.

[0031] The beneficial effects of this invention are as follows: 1. This invention provides a method for constructing and culturing organoids from ROS1 gene rearranged non-small cell lung cancer obtained by biopsy. This method can efficiently construct organoids from ROS1 gene rearranged non-small cell lung cancer tissue obtained by biopsy. Based on the molecular characteristics and microenvironmental requirements of ROS1 gene rearranged non-small cell lung cancer, a specialized culture medium suitable for the growth of these organoids is optimized. Matrices are used to simulate the extracellular matrix to provide support for the formation of the three-dimensional structure of the organoids, and continuous culture is achieved through precise control of culture conditions.

[0032] 2. The ROS1 gene rearranged non-small cell lung cancer organoids obtained by this invention have significant advantages such as high survival rate, sufficient quantity, and sustainable passage, and retain the same pathological morphology as the original tumor tissue.

[0033] 3. This invention overcomes the technical bottlenecks of small sample size and high culture difficulty, and provides a stable and reliable in vitro research platform for personalized medication guidance, drug resistance mechanism research and screening of new targeted drugs for ROS1 gene rearranged non-small cell lung cancer, in order to prolong the survival period of patients and improve their quality of life. It has broad application prospects and high economic value.

[0034] Of course, implementing any product or method of the present invention does not necessarily require achieving all of the advantages described above at the same time. Attached Figure Description

[0035] Figure 1 These are morphological images of lung cancer organoids obtained in Embodiment 3, Comparative Example 1, and Comparative Example 2 of the present invention. Figure 2 HE staining images of lung cancer organoids obtained in Embodiment 3, Comparative Example 1, and Comparative Example 2 of the present invention; Figure 3 The results of Western blot experiments on the protein expression of p-ERK1 / 2 and ERK1 / 2 in the original tumor tissue of this invention, Example 3, Comparative Example 1, and Comparative Example 2 are shown.

[0036] Figure 4 The figures show the drug sensitivity test results of the organoids obtained from the culture of Example 3, Comparative Example 1, and Comparative Example 2 of this invention. Detailed Implementation

[0037] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise defined, the technical terms used below have the same meaning as understood by those skilled in the art.

[0038] Unless otherwise specified, the test materials and reagents used in the following examples are commercially available or prepared by known methods.

[0039] Unless otherwise specified, all techniques or conditions described in the embodiments can be performed in accordance with the techniques or conditions described in the literature in this field or in the product manual. Unless otherwise specified, the quantitative experiments in the following embodiments are all repeated three times or more, and the results are averaged.

[0040] Preparation of sample preservation solution: Add 2× penicillin-streptomycin-amphotericidal to DMEM medium, 10 µM Y-27632, prepare fresh before use, and store or transport at 0-8℃.

[0041] Preparation of sample washing solution: Add 2× penicillin-streptomycin-amphotericidal to DPBS buffer, and prepare fresh before use.

[0042] Preparation of sample digestion solution: Add collagenase IV 0.5-2 mg / mL, elastase 0.1-5 U / mL, elastase 0.1-5 U / mL, DNase I 0.05-0.5 mg / mL, and 10 µM Y-27632 to Advaced DMEM / F12. Prepare fresh before use.

[0043] The ROS1 gene rearranged non-small cell lung cancer biopsy samples used in the following examples were obtained from pathologically confirmed lung cancer patients through percutaneous lung biopsy. Informed consent was obtained from the patients or their legal guardians before sample collection, and the acquisition, preservation, transportation, and use of the samples strictly complied with the relevant regulations such as the "Ethical Review Measures for Biomedical Research Involving Human Subjects".

[0044] Example 1: A culture medium (denoted as medium A) for constructing organoids from ROS1 gene rearranged non-small cell lung cancer consists of basic culture medium components, specific factor components, and additive components. The basic components of the culture medium were Advanced DMEM / F12, 2 mM GlutaMAX, 80 U / mL penicillin-streptomycin, and 16 mM HEPES. The specific factor components are 300 nM A83-01, 25 ng / mL FGF-7, 75 ng / mL FGF-10, 5 nMTrametinib, 260 nMSB202190, 320 ng / mL R-spondin 1, and 68 ng / mL Noggin; The additives consist of 10 µM Y-27632, 1.5× B27 reagent, 154 μg / mL Primocin, 1.25 mM N-Acetylcysteine, and 5 mM N-icotinamide.

[0045] The method for preparing this culture medium includes the following steps: Mix all the above ingredients thoroughly to obtain the final product.

[0046] Example 2: A culture medium (denoted as culture medium B) for constructing organoids from ROS1 gene rearranged non-small cell lung cancer without Y-27632. This culture medium differs from that in Example 1 in that it does not contain Y-27632, but is otherwise the same as in Example 1.

[0047] Example 3: A method for constructing organoids from ROS1 gene rearranged non-small cell lung cancer derived from biopsy, using culture medium A of Example 1 and culture medium B of Example 2, specifically includes the following steps: (1) Sample preprocessing: After the ROS1 gene rearranged non-small cell lung cancer puncture sample was removed from the body, it was transferred to a tissue preservation solution with a volume of 5 times the sample volume, transported in a cold chain at 0-8℃, and transferred to the laboratory in a timely manner. Washing: Remove the sample and transfer it to 10 times the sample volume of sample washing solution. Incubate at 200 rpm and 25°C on a constant temperature shaker for 30 min, changing the sample washing solution every 5 min. Shearing digestion: Collect the cleaned sample, use a sterile scalpel and surgical scissors to cut the sample into small, meat-like pieces with a side length of at least 500 μm, transfer the sample to 5 times the sample volume of sample digestion solution, digest at 200 rpm and 37°C on a constant temperature shaker for 3-5 min, and terminate digestion with 10 times the sample digestion solution volume of 10% BSA buffer. Filtration and centrifugation: Collect the digested cell suspension and filter it through a 70 μm filter; centrifuge at 500×g, 4℃ for 8 min, discard the supernatant and collect the sample precipitate; (2) Pre-culture of cell suspension: Add culture medium A from Example 1 to the sample precipitate for culture. After forming a cell suspension, mix it with Matrigel and drip it evenly into the well plate at a rate of 30 μL / drop. Place the plate in a 37°C incubator and incubate for 30 min to allow the Matrigel to fully solidify. (3) Culture in different culture media: Take out the (2) well plate, add culture medium A, and place it in an incubator for 2 days. Then replace it with culture medium B of Example 2 and culture for 10 days (change the medium every 2 days). The average diameter of the organoids reaches 50 μm for the first time, and the organoids are obtained.

[0048] In this embodiment, the average diameter of the organoids reached 50 μm for the first time (cultured for 12 days using different culture media), and the morphology was as follows: Figure 1 As shown.

[0049] Comparative Example 1: Culture medium C: The difference from culture medium A in Example 1 is that it lacks the specific component Trametinib, otherwise it is the same.

[0050] Culture medium D: The difference from culture medium B in Example 2 is that it lacks the specific component Trametinib, otherwise it is the same.

[0051] A method for constructing organoids from ROS1 gene rearranged non-small cell lung cancer derived by biopsy. The difference from Example 3 is that "culture medium A" is replaced with "culture medium C" and "culture medium B" is replaced with "culture medium D", otherwise it is the same as Example 3.

[0052] The organoids in this comparative example achieved an average diameter of 50 μm for the first time (cultured for 28 days using different culture media), and their morphology was as follows: Figure 1 As shown.

[0053] Comparative Example 2: Culture medium E: The difference from culture medium A in Example 1 is that the concentration of Trametinib used is 100 nM, otherwise the same.

[0054] Culture medium F: The difference from culture medium B in Example 2 is that the concentration of Trametinib used is 100 nM, otherwise the same.

[0055] A method for constructing organoids from ROS1 gene rearranged non-small cell lung cancer derived by biopsy. The difference from Example 3 is that “culture medium A” is replaced with “culture medium E” and “culture medium B” is replaced with “culture medium F”, otherwise the same as Example 3.

[0056] In this comparative example, after culturing organoids in different culture media for 30 days, no organoids meeting the standard formation was observed; their morphology was as follows: Figure 1 As shown.

[0057] Experimental analysis was performed on the organoids constructed in Example 3 and Comparative Examples 1-2: (1) Observation of organoid morphology: like Figure 1 As shown, the lung cancer organoids obtained using the construction and culture scheme of Example 3 are full in morphology, numerous, and can be cultured to a large size; while the organoids obtained by Comparative Examples 1 and 2 are not only fewer in number but also have poor morphology: the organoids in Comparative Example 1 are relatively shriveled, and most of the cells in Comparative Example 2 have disintegrated, with only a very small number of organoids remaining that are loose and disintegrated.

[0058] (2) HE staining, the steps are as follows: Ten days after culturing lung cancer organoids, they were treated with organoid recovery solution for 30 min to dissolve Matrigel. Organoids isolated from Matrigel were fixed in 4% paraformaldehyde for 12 h, subjected to gradient dehydration with alcohol and clearing with xylene, and then embedded in paraffin. Transverse and longitudinal sections were then prepared. Simultaneously, the original lung cancer tissue was also fixed, dehydrated, cleared, embedded in paraffin, and sectioned. Both organoids and tissues were stained with hematoxylin and eosin (HE) for morphological and structural identification.

[0059] like Figure 2 As shown, the cells in the original tumor tissue and the organoids cultured in Example 3 are similar in terms of cell nucleus, cytoplasm, and nucleocytoplasmic ratio, and their tissue morphology is also similar. However, the organoids cultured in Comparative Examples 1 and 2 differ significantly from the original tissue: although the organoid in Comparative Example 1 has a relatively complete structure, the number of internal cells is too small, and its morphology is relatively empty; the organoid in Comparative Example 2 has a very incomplete morphology and is loose. This indicates that the lung cancer organoids constructed in Example 3 can better preserve the genetic characteristics and biological behavior of the original tissue.

[0060] (3) Western blot identification, the steps are as follows: After 10 days of lung cancer organoid culture, the tissue was treated with organoid recovery solution for 30 min to dissolve Matrigel, washed with PBS, lysed on ice with RIPA lysis buffer for 30 min, and centrifuged at 12,000 rpm for 15 min to collect the supernatant. At the same time, the original tumor tissue was cut into 1 mm³ pieces, lysed with RIPA, centrifuged, and the supernatant was collected.

[0061] The key marker p-ERK1 / 2 of the MEK pathway was identified in four samples: the original tumor tissue, organoids obtained in Example 3, Comparative Example 1, and Comparative Example 2. After protein quantification and denaturation, electrophoresis and membrane transfer, antibody incubation, and imaging analysis, the final experimental results of p-ERK1 / 2 expression in lung cancer organoids and their original tumor tissues were obtained.

[0062] like Figure 3 As shown, there was no difference in ERK1 / 2 protein expression between Example 3, Comparative Example 1, and Comparative Example 2 compared to the original tumor tissue. However, in terms of p-ERK1 / 2 expression, there was no difference between Comparative Example 1 and the original tumor tissue, while there was a significant difference between Example 3 and Comparative Example 2 and the original tumor tissue. This indicates that Trametinib successfully inhibited the MEK pathway expression of the organoids obtained in Example 3 and Comparative Example 2 by inhibiting the expression of p-ERK1 / 2 (phosphorylation of ERK1 / 2).

[0063] (4) Drug sensitivity test, the steps are as follows: 4.1 Count the number of organoids in the culture medium under a microscope and calculate the density. Calculate the required number of organoids based on the amount of drug to be detected, and take the corresponding culture medium suspension. 4.2 Under a stereomicroscope, carefully aspirate lung cancer organoids into each well of a 96-well plate using a 200μl standard pipette tip. The number and size of lung cancer organoids in each well should be kept as consistent as possible. 4.3 Carefully aspirate the culture medium from the 96-well plate while observing (avoid organoid loss during this process). 4.4 Add the pre-prepared drug solution and continue to incubate in a 37℃, 5% CO2 incubator for 48-72 hours; 4.5 Add CellTiter-Glo 3D according to the manufacturer's instructions and read the values ​​using a microplate reader; 4.6 Summarize the drug sensitivity data and output the drug sensitivity results.

[0064] like Figure 4 As shown, from left to right, the statistical charts of drug susceptibility results for Example 3, Comparative Example 1, and Comparative Example 2 are presented. The experimental groups for drug susceptibility testing are: negative control, positive control, crizotinib, pemetrexed, and entrectinib, respectively. Figure 4As can be seen, the experimental results of the negative and positive controls in Example 3 are consistent with the norm, and the data parallelism between replicates is good, indicating that the organoids are in good condition. On the other hand, the data parallelism between replicates of the three drug groups is also very good, and it can show the different killing effects of the drugs on the organoids. Conversely, the drug sensitivity results of Comparative Example 1 and Comparative Example 2 cannot reasonably reproduce the actual killing effect, and the data parallelism between replicates is very poor. The reason is that the organoids in both are in poor condition. Since the relative inhibition rate (%) of each group is calculated based on the linear formula derived from the ATP quantitative results of the negative and positive control groups, this result also reflects the organoids in Comparative Example 1 and Comparative Example 2.

[0065] comprehensive Figure 1 and Figure 4 The experimental results show that the organoids constructed by this invention have good state and morphology, and can demonstrate different killing effects of drugs. Therefore, they can provide a stable and reliable in vitro research platform for personalized drug guidance, drug resistance mechanism research and screening of new targeted drugs.

[0066] Application Example 1: Due to the relatively small sample size, a total of 5 clinical samples were collected over two years. Different experimental methods (Example 3, Comparative Example 1, and Comparative Example 2) were used to construct organoid models, which were continuously cultured. The formation of organoids was recorded and statistically analyzed using a microscope. The successful organoid modeling was judged as the formation of 5000 organoids with an average diameter greater than 50 μm within 30 days. The results are shown in Table 1.

[0067] Table 1. Statistical Table for Organoid Modeling in Lung Cancer

[0068] Note: Since no organoids were formed in Comparative Example 2 that met the criteria, the statistical result of the number of days is represented by "N / A".

[0069] Table 1 shows that the success rates of lung cancer organoids obtained using different methods are as follows: Example 3 > Comparative Example 1 > Comparative Example 2. Furthermore, the success rate of organoids is positively correlated with the construction rate (i.e., a shorter time for the average organoid diameter to reach 50 μm for the first time, indicating a faster construction rate), suggesting that the lung cancer organoids obtained using the construction and culture protocol of Example 3 not only have a high success rate but also a short cycle time.

[0070] Example 4: A culture medium (denoted as medium A) for constructing organoids from ROS1 gene rearranged non-small cell lung cancer consists of basic culture medium components, specific factor components, and additive components. The basic components of the culture medium were Advanced DMEM / F12, 1 mM GlutaMAX, 50 U / mL penicillin-streptomycin, and 25 mM HEPES. The specific factor components are 100 nM A83-01, 10 ng / mL FGF-7, 100 ng / mL FGF-10, 10 nM Trametinib, 100 nM MSB202190, 200 ng / mL R-spondin 1, and 20 ng / mL Noggin; The additives consist of 5 µM Y-27632, 1× B27 reagent, 100 μg / mL Primocin, 2 mM N-Acetylcysteine, and 6 mM N-icotinamide.

[0071] A culture medium (denoted as culture medium B) for constructing organoids from ROS1 gene rearranged non-small cell lung cancer without Y-27632 is the same as culture medium A in this embodiment.

[0072] A method for constructing organoids from ROS1 gene rearranged non-small cell lung cancer derived from biopsy, using culture medium A and culture medium B of this embodiment, specifically includes the following steps: (4) Sample preprocessing: After the ROS1 gene rearranged non-small cell lung cancer puncture sample was removed from the body, it was transferred to a tissue preservation solution with a volume of 5 times the sample volume, transported in a cold chain at 0-8℃, and transferred to the laboratory in a timely manner. Washing: Remove the sample and transfer it to 10 times the sample volume of sample washing solution. Incubate at 100 rpm and 27°C on a constant temperature shaker for 40 min, changing the sample washing solution every 3 min. Shearing digestion: Collect the cleaned sample, use a sterile scalpel and surgical scissors to shear the sample into small, meat-like pieces with a side length of at least 500 μm, transfer the sample to 5 times the sample volume of sample digestion solution, digest at 300 rpm and 35°C on a constant temperature shaker for 8 min, and terminate digestion with 10 times the sample digestion solution volume of 10% BSA buffer. Filtration and centrifugation: Collect the digested cell suspension and filter it through a 50 μm filter; centrifuge at 450×g, 4℃ for 5 min, discard the supernatant, and collect the sample precipitate; (5) Pre-culture of cell suspension: Add culture medium A of this embodiment to the sample precipitate for culture. After forming a cell suspension, mix it with Matrigel and drip it evenly into the well plate at a rate of 25 μL / drop. Place the plate in a 35°C incubator and incubate for 40 min to allow the Matrigel to fully solidify. (6) Culture in different culture media: Take out the (2) well plate, add culture medium A, and place it in an incubator for 3 days. Then replace it with culture medium B of this embodiment and incubate at 37°C for 5 days. Change the medium every 2-3 days to obtain the final product.

[0073] Example 5: A culture medium (denoted as medium A) for constructing organoids from ROS1 gene rearranged non-small cell lung cancer consists of basic culture medium components, specific factor components, and additive components. The basic components of the culture medium were Advanced DMEM / F12, 5 mM GlutaMAX, 100 U / mL penicillin-streptomycin, and 10 mM HEPES. The specific factor components are 500 nM A83-01, 50 ng / mL FGF-7, 20 ng / mL FGF-10, 1 nM Trametinib, 500 nMSB202190, 500 ng / mL R-spondin 1, and 100 ng / mL Noggin; The additives consist of 30 µM Y-27632, 2× B27 reagent, 200 μg / mL Primocin, 1 mM N-Acetylcysteine, and 4 mM N-icotinamide.

[0074] A culture medium (denoted as culture medium B) for constructing organoids from ROS1 gene rearranged non-small cell lung cancer without Y-27632 is the same as culture medium A in this embodiment.

[0075] A method for constructing organoids from ROS1 gene rearranged non-small cell lung cancer derived from biopsy, using culture medium A and culture medium B of this embodiment, specifically includes the following steps: (7) Sample preprocessing: After the ROS1 gene rearranged non-small cell lung cancer puncture sample was removed from the body, it was transferred to a tissue preservation solution with a volume of 5 times the sample volume, transported in a cold chain at 0-8℃, and transferred to the laboratory in a timely manner. Washing: Remove the sample and transfer it to 10 times the sample volume of sample washing solution. Incubate at 300 rpm and 20°C on a constant temperature shaker for 20 min, changing the sample washing solution every 8 min. Shearing digestion: Collect the cleaned sample, use a sterile scalpel and surgical scissors to shear the sample into small, meat-like pieces with a side length of at least 500 μm, transfer the sample to 5 times the sample volume of sample digestion solution, digest at 100 rpm and 38°C on a constant temperature shaker for 3 min, and terminate digestion with 10 times the sample digestion solution volume of 10% BSA buffer. Filtration and centrifugation: Collect the digested cell suspension and filter it through an 80 μm filter; centrifuge at 450×g, 4℃ for 10 min, discard the supernatant, and collect the sample precipitate; (8) Pre-culture of cell suspension: Add culture medium A of this embodiment to the sample precipitate for culture. After forming a cell suspension, mix it with Matrigel and drip it evenly into the well plate at a rate of 35 μL / drop. Place the plate in a 38°C incubator and incubate for 25 min to allow the Matrigel to fully solidify. (9) Culture in different culture media: Take out the (2) well plate, add culture medium A, and place it in an incubator for 3 days. Then replace it with culture medium B of this embodiment and incubate at 37°C for 5 days. Change the medium every 2-3 days to obtain the final product.

[0076] The morphology and state of the organoids cultured and constructed in Examples 4 and 5 are similar to those of the organoids in Example 3.

[0077] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A culture medium for constructing organoids from ROS1 gene rearranged non-small cell lung cancer, characterized in that, This includes the basic components of the culture medium, the specific factor components, and the additive components; The basic components of the culture medium include Advanced DMEM / F12, 1-5 mM GlutaMAX, 50-100 U / mL penicillin-streptomycin, and 10-25 mM HEPES; The specific factor components include 100-500 nM A 83-01, 10-50 ng / mL FGF-7, 20-100 ng / mL LFGF-10, 1-10 nM Trametinib, 100-500 nm SB202190, 200-500 ng / mL R-spondin 1, and 20-100 ng / mL Noggin; The additive components include one of the following: Composition A: 5-30 µM Y-27632, 1-2× B27 reagent, 100-200 μg / mL Primocin, 1-2 mM N-Acetylcysteine, 4-6 mM N-Cotinamide; Composition B: 1-2 × B27 reagent, 100-200 μg / mL Primocin, 1-2 mM N-Acetylcysteine, 4-6 mM N-icotinamide.

2. The method for preparing the culture medium according to claim 1, characterized in that, Includes the following steps: Mix all ingredients thoroughly to obtain the final product.

3. The application of the culture medium described in claim 1 in the construction of ROS1 gene rearranged non-small cell lung cancer organoids.

4. A method for constructing organoids from ROS1 gene rearranged non-small cell lung cancer derived from biopsy, characterized in that, Includes the following steps: (1) Sample preprocessing: The ROS1 gene rearranged non-small cell lung cancer puncture samples were cleaned, then cut into small meat-like pieces, digested, filtered, centrifuged, and the sample precipitate was collected. (2) Pre-culture of cell suspension: Add the culture medium of claim 1 to the sample precipitate, wherein the additive component of the culture medium includes component A, culture, and after forming a cell suspension, mix with Matrigel, drop into a well plate, and incubate statically. (3) Culture in different culture media: Add the culture medium of claim 1 to the well plate obtained in (2), wherein the additive component of the culture medium includes component A, and culture; then replace it with the culture medium containing component B and culture, and obtain the desired product.

5. The construction method according to claim 4, characterized in that, (1) Washing is: transfer the sample to the sample washing solution and incubate at 100~300 rpm and 20~27℃ for 20~40 min; digestion is: transfer the meat-like small pieces to the sample digestion solution and digest at 100~300 rpm and 35~38℃ for 3~8 min.

6. The construction method according to claim 4, characterized in that, (1) Filtration is carried out using a 50~80μm filter screen; centrifugation is performed at 450~550×g for 5~10min.

7. The construction method according to claim 4, characterized in that, (2) In this case, the static incubation is: static incubation at 35~38℃ for 25~40 minutes.

8. The construction method according to claim 4, characterized in that, (3) Culture medium A is cultured for 1 to 3 days; culture medium B is cultured for 5 to 16 days.

9. The ROS1 gene rearranged non-small cell lung cancer organoid constructed by the construction method according to any one of claims 1-8.

10. The application of the ROS1 gene rearranged non-small cell lung cancer organoids as described in claim 9 in personalized medication guidance, drug resistance mechanism research, or targeted drug screening.

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