Application of PARVA in promoting formation of tumor organoids

Abstract

The invention belongs to the technical field of tumor biology and organoids, and provides application of PARVA in promoting formation of tumor organoids. The PARVA comprises a PARVA protein or a PARVA coding gene; the application comprises application in preparation of a tumor organoid inducer, a tumor organoid culture kit and construction of a tumor organoid model. According to the invention, the formation efficiency and stability of the tumor organoid can be obviously improved, the difficulty in constructing the organoid with difficult-to-cultivate tumors such as lung cancer and bile duct cancer is solved, and a reliable model is provided for the fundamental research of the tumors; the constructed tumor organ model has a tissue structure, gene characteristics and drug responsiveness which are highly similar to in-vivo tumors, can be used for tumor generation mechanism research, antitumor drug high-throughput screening and clinical personalized treatment scheme evaluation, and has important scientific research value and clinical transformation significance.

Description

Technical Field

[0001] This invention belongs to the field of tumor biology and organoid technology, and relates to the application of PARVA (including proteins or encoding genes) in promoting tumor organoid formation. Background Technology

[0002] Tumor organoids are three-dimensional culture systems that simulate the tumor microenvironment and tissue structure in vitro. They have genetic characteristics, pathological phenotypes and drug responses that are highly similar to the source tumors, and have become a core tool for basic tumor research, drug development and clinical personalized treatment evaluation.

[0003] However, the current tumor organoid culture faces many bottlenecks: (1) the success rate of primary culture of some tumor types (such as lung cancer and cholangiocarcinoma) is less than 30%, and problems such as abnormal cell differentiation, structural disintegration and reduced proliferation capacity are prone to occur during the culture process; (2) existing organoid models are difficult to accurately reproduce the cell-cell and cell-extracellular matrix (ECM) interactions in the tumor microenvironment, resulting in limited simulation of in vivo tumor biological behavior, which restricts their application in clinical translation.

[0004] Cell adhesion and cytoskeleton remodeling are key biological processes in the formation and maintenance of tumor organoids. PARVA (Parvinα, MXRA2, Actopaxin, CH-ILKBP), as a core adaptor protein in the integrin signaling pathway, together with ILK (Integrin-linked kinase) and PINCH (Particularly interesting new cysteine ​​histidine-rich protein), constitutes the IPP (Integrin-linked kinase / Parvinα / PINCH) complex. It mediates cell adhesion to the ECM by binding to the cytoplasmic tail of integrin subunits and regulates the assembly and remodeling of the actin cytoskeleton.

[0005] Existing research confirms that PARVA plays an important role in the proliferation, migration, and invasion of tumor cells. For example, in breast cancer, PARVA promotes tumor cell survival by activating the PI3K-Akt signaling pathway. Furthermore, in colorectal cancer, high expression of PARVA is positively correlated with tumor stage and lymph node metastasis.

[0006] However, there are currently no reports on whether PARVA participates in the formation of tumor organoids, or how it affects the stability and mimicry of organoids by regulating cell adhesion, skeletal dynamics, or downstream signaling pathways.

[0007] To address the current technical challenges of low efficiency and insufficient simulation in tumor organoid culture, there is an urgent need in this field to identify key molecular targets that regulate tumor organoid formation and to develop efficient organoid construction methods based on these targets, so as to further promote the development of tumor organoid technology in basic research and clinical applications.

[0008] Furthermore, on the one hand, there are differences in understanding among those skilled in the art; on the other hand, the applicant studied a large number of documents and patents when making this invention, but due to space limitations, not all details and contents were listed in detail. However, this does not mean that the present invention does not possess the features of these prior art. On the contrary, the present invention already possesses all the features of the prior art, and the applicant reserves the right to add relevant prior art to the background art. Summary of the Invention

[0009] The purpose of this invention is to provide new applications for the PARVA protein or its encoding gene, particularly their application in promoting tumor organoid formation. These applications include the preparation of tumor organoid inducers, tumor organoid culture kits, and the construction of tumor organoid models, thereby addressing the problems of low success rates, poor stability, and insufficient consistency in existing tumor organoid cultures.

[0010] To achieve the above objectives, the present invention provides the following technical solution: This invention provides the application of PARVA protein or PARVA encoding gene in the preparation of tumor organoid inducers, tumor organoid culture kits, or the construction of tumor organoid models.

[0011] In some preferred embodiments, the PARVA protein is a wild-type PARVA protein or a functionally equivalent mutant of the wild-type PARVA protein.

[0012] Preferably, the amino acid sequence of the wild-type PARVA protein is shown in GenBank accession number NP_060692.3.

[0013] Preferably, the functionally equivalent mutant is a protein that retains PARVA activity and downstream signal activation ability after substitution, deletion, or insertion of the wild-type sequence; preferably, the substitution, deletion, or insertion involves 0-372 amino acid residues. More preferably, the substitution, deletion, or insertion involves 15 amino acid residues.

[0014] Preferably, the PARVA encoding gene is selected from one or more sequences from (1) to (3) below: (1) a nucleotide sequence as shown in GenBank accession number NM_018222.5; (2) a DNA sequence that hybridizes with the nucleotide sequence described in (1) under strict conditions and encodes a protein that promotes tumor organoid formation; (3) a DNA sequence that has ≥90% homology with the nucleotide sequence described in (1) and encodes a protein that promotes tumor organoid formation.

[0015] In some preferred embodiments, the tumor organoid inducer exerts its effects by activating the Integrin IPP complex signaling pathway, the PI3KAkt signaling pathway, the Rho family GTPase-mediated cytoskeleton remodeling pathway, the MAPK signaling pathway, and / or extracellular matrix adhesion-related pathways.

[0016] In some preferred embodiments, the tumor organoid inducer is selected from PARVA expression regulatory components, PARVA protein interaction activating components, and PARVA downstream pathway synergistic components.

[0017] Preferably, the PARVA expression regulatory component can be a wild-type PARVA gene vector, such as a vector containing the ARRVA gene, such as the adenovirus vector Ad-PARVA, the lentiviral vector pLVX-PARVA, or the corresponding mRNA transcribed from the wild-type PARVA gene.

[0018] Preferably, the PARVA protein interaction activating component can be a small molecule agonist targeting PARVA or a PARVA functional peptide (such as a 15-20 amino acid fragment containing the PARVA N-terminal mechanosensitive domain).

[0019] Preferably, the downstream pathway co-component of PARVA can be, for example, a FAK phosphorylation promoter or a PGC-1α activator.

[0020] Preferably, the tumor organoid inducer exists in the form of: (1) PARVA protein (e.g., lyophilized powdered PARVA protein); (2) a eukaryotic expression vector containing the PARVA encoding gene, a lentiviral particle containing the PARVA encoding gene, or PARVA mRNA (i.e., mRNA transcribed from the PARVA gene); (3) a PARVA small molecule activator, a PARVA signaling pathway activator, or a pharmaceutical composition; or (4) a delivery vector encapsulating PARVA protein, PARVA mRNA, or a PARVA expression vector.

[0021] Preferably, the PARVA small molecule activator can be a small molecule agonist targeting PARVA, as described above.

[0022] Preferably, the delivery vector is selected from at least one delivery vector selected from cells, viruses, liposomes, nanoparticles, vesicles and ferritin.

[0023] More preferably, the cells are erythrocytes and / or mesenchymal stem cells.

[0024] More preferably, the virus is an adenovirus or a lentivirus.

[0025] In some preferred embodiments, the method for constructing the tumor organoid model includes the following steps: (1) Obtaining primary tumor cells; (2) The primary tumor cells were treated with PARVA; (3) The primary tumor cells treated with PARVA were cultured in three dimensions.

[0026] In some preferred embodiments, the tumors or cancers targeted by the tumor organoid inducer or tumor organoid model are selected from: oral cancer, oropharyngeal cancer, nasopharyngeal cancer, respiratory system cancer, genitourinary system cancer, gastrointestinal cancer, central or peripheral nervous system tissue cancer, endocrine or neuroendocrine system cancer or hematopoietic system cancer, glioma, sarcoma, epithelial cancer, lymphoma, melanoma, fibroma, meningioma, brain cancer, kidney cancer, biliary system cancer, pheochromocytoma, islet cell carcinoma, Levi-Flaumeni tumor, thyroid cancer, parathyroid cancer, pituitary adenoma, adrenal adenoma, bone-derived sarcoma tumor, neuroendocrine system tumor, breast cancer, lung cancer, head and neck cancer, prostate cancer, esophageal cancer, tracheal cancer, liver cancer, bladder cancer, gastric cancer, ovarian cancer, uterine cancer, cervical cancer, testicular cancer, colon cancer, rectal cancer, or skin cancer.

[0027] Preferably, the tumor organoid model is similar and consistent with the tumor tissue from which the tumor organoid model originated in terms of morphology and structure.

[0028] Preferably, the tumor organoid model exhibits the same sensitivity to chemotherapy drugs as the patient's primary cells.

[0029] In some preferred embodiments, the tumor organoid culture kit, in addition to containing the tumor organoid inducer prepared based on PARVA as described above, also contains tumor organoid basal culture medium, extracellular matrix components, and growth factors.

[0030] Preferably, the basal culture medium is DMEM / F12 medium and / or enhanced DMEM / F12 medium.

[0031] Preferably, the extracellular matrix component is selected from at least one of matrix gelatin, collagen I, gelatin and hyaluronic acid.

[0032] Preferably, the growth factor is selected from at least one of EGF, FGF2, Noggin, Rspondin 1, and Wnt3a.

[0033] The present invention has the following beneficial effects: (1) This invention is the first to discover that the PARVA protein or its encoding gene (i.e., the PARVA encoding gene) has the function of promoting the formation of tumor organoids, providing a new key regulatory target for tumor organoid technology and filling the gap in the application of PARVA in the field of organoids.

[0034] (2) The tumor organoid inducer prepared based on PARVA protein or its encoding gene can significantly improve the formation efficiency and stability of tumor organoids, solve the problem of organoid construction for difficult-to-culture tumor types such as lung cancer and cholangiocarcinoma, and provide a reliable model for basic research on these types of tumors.

[0035] (3) The tumor organoid model constructed using this invention has tissue structure, gene characteristics and drug response that are highly similar to tumors in vivo. It can be used for tumorigenesis mechanism research, high-throughput screening of anti-tumor drugs and evaluation of personalized clinical treatment plans, and has important scientific research value and clinical translation significance. Attached Figure Description

[0036] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0037] Figure 1 This diagram illustrates the effect of PARVA protein on lung cancer organoid formation efficiency. Figure A shows the morphological image of lung cancer organoids from the control group (without PARVA protein) after 21 days of culture; Figure B shows the morphological image of lung cancer organoids from the PARVA protein-treated group (final concentration 30 ng / mL) after 21 days of culture.

[0038] Figure 2 This image shows the effect of the PARVA encoding gene (lentiviral vector) on the structural stability of colorectal organoids. The left image shows the morphology of lung cancer organoids from the control group (infected with empty vector) after 21 days of culture; the right image shows the morphology of lung cancer organoids from the PARVA overexpression group (infected with pLVX-PARVA-3×FLAG vector) after 21 days of culture.

[0039] Figure 3 This shows a comparison of the morphology and structure of PARVA-induced colorectal cancer organoids with the tumor tissue from which they originated.

[0040] Figure 4The figure shows the cell viability of PARVA-overexpressing colorectal organoids after treatment with different drugs. The horizontal axis represents cell viability (%), and the vertical axis represents the drugs used in the treatment (control group, 5-fluorouracil, oxaliplatin, and CPT-11, in that order). Blue triangles represent colorectal organoids from the control group, and red circles represent colorectal organoids from the PARVA-overexpressing group. The figure shows that 5-fluorouracil (MCE product code HY-90006), oxaliplatin (MCE product code HY-17371), and CPT-11 (MCE product code HY-16562) have different inhibitory effects on cell viability in PARVA-induced colorectal organoids, and the PARVA-overexpressing group exhibits varying degrees of sensitivity to these three drugs.

[0041] Figure 5 This demonstrates the signaling pathways regulated by PARVA in colorectal cancer organoids. Detailed Implementation

[0042] To further illustrate the present invention, the following will describe in more detail the application of the PARVA protein or its encoding gene provided by the present invention in promoting tumor organoid formation in three aspects. However, these specific embodiments should not be construed as limiting the scope of protection of the present invention.

[0043] 1. Application of PARVA in the preparation of tumor organoid inducers In this article, unless otherwise stated, PARVA includes the PARVA protein or the gene encoding the PARVA protein (sometimes simply referred to as the "PARVA-encoding gene").

[0044] The tumor organoid inducer described herein is a PARVA-based tumor organoid inducer, including those with PARVA protein or its encoding gene as the active ingredient. This tumor organoid inducer promotes three-dimensional aggregation of tumor cells and extracellular matrix remodeling by activating one or more signaling pathways related to organoid formation in tumor cells (including the Integrin / IPP complex signaling pathway, the PI3K / Akt signaling pathway, the Rho family GTPase-mediated cytoskeleton remodeling pathway, the MAPK signaling pathway, and cell ECM adhesion-related pathways), thereby improving the formation efficiency and stability of tumor organoids. The PARVA protein includes wild-type PARVA protein and functionally equivalent mutants. The amino acid sequence of the wild-type PARVA protein can be, for example, as shown in GenBank accession number NP_060692.3. The functionally equivalent mutant can be a protein that, after modification with 248 to 372 amino acid residues (preferably 15 amino acid residues) through substitution, deletion, or insertion, can still maintain IPP complex binding activity and downstream signal activation ability. The PARVA encoding gene may include the following three types of sequences: (1) DNA sequences encoding the PARVA protein (such as the nucleotide sequence shown in GenBank accession number NM_018222.5), (2) DNA sequences that hybridize with the DNA sequence under strict conditions and encode proteins that promote tumor organoid formation, and (3) DNA sequences that have ≥90% homology with the DNA sequence and encode proteins that promote tumor organoid formation.

[0045] 2. Application of PARVA in the preparation of tumor organoid culture kits The tumor organoid culture kit contains a tumor organoid inducer based on PARVA, such as a tumor organoid inducer with PARVA protein or its encoding gene as the active ingredient. Preferably, in addition to the PARVA-based tumor organoid inducer, the tumor organoid culture kit may further contain a tumor organoid basal culture medium, extracellular matrix components, growth factors, and / or other auxiliary reagents. The tumor organoid basal culture medium can be DMEM / F12 medium, Advanced DMEM / F12 medium, or other commonly used tumor organoid culture basal media in the art. The extracellular matrix components can be Matrigel, collagen (such as collagen I), or hyaluronic acid. The growth factors can be one or more of EGF, FGF2, Noggin, and Rspondin 1; it should be understood that those skilled in the art, after reading the disclosure of this application, are capable of making adjustments according to the tumor type (e.g., Wnt3a can be added to lung cancer organoid culture).

[0046] This invention provides the application of PARVA protein in the preparation of reagents that promote tumor organoid formation (i.e., tumor organoid inducers). The active ingredients of these reagents can be categorized into three main types, all aiming to restore or enhance the core function of PARVA in tumor organoid formation. The first type consists of PARVA expression regulatory components, such as wild-type PARVA gene vectors (adenovirus vector Ad-PARVA, lentiviral vector pLVX-PARVA, and corresponding mRNA transcribed from the wild-type PARVA gene). Delivery via organoid culture systems can specifically enhance the transcription and translation levels of PARVA in tumor cells, compensating for insufficient protein expression during culture. The second type consists of PARVA protein interaction activating components, including small molecule agonists targeting PARVA (such as benzimidazole derivatives) and PARVA functional peptides (15-20 amino acid fragments containing the N-terminal mechanosensitive domain of PARVA). The former can promote the binding of PARVA to downstream effector proteins such as FAK and ILK, while the latter can mimic the cytoskeleton anchoring function of PARVA, directly activating organoid formation-related signal transduction. The third category consists of PARVA downstream pathway synergists, such as FAK phosphorylation promoters and PGC-1α activators. These synergists can synergistically enhance the PARVA-mediated FAK / p38 MAPK cell polarity establishment pathway and the PGC-1α / TFAM energy metabolism support pathway, forming a multi-target synergistic regulation of PARVA function, thereby efficiently promoting tumor organoid formation.

[0047] 3. Application of PARVA in constructing tumor organoid models The method for constructing the tumor organoid model includes the following steps: (1) Obtaining primary tumor cells; (2) Treatment of the primary tumor cells with PARVA; and (3) Primary tumor cells treated with PARVA were cultured in three dimensions to form a tumor organoid model.

[0048] In step (1), tumor cells can be obtained, for example, by isolating them from surgical specimens, biopsy specimens, or body fluids containing tumor cells from a tumor patient, washing them with PBS buffer containing 1% penicillin and streptomycin (e.g., washing 1 to 3 times, such as 2 or 3 times), digesting them with 0.25% trypsin EDTA digestion solution at 37°C for 5 to 10 minutes, centrifuging to collect the cells after digestion is terminated, and adjusting the cell concentration to 1×10^5 to 5×10^5 cells / mL.

[0049] In step (2), the treatment of PARVA protein or its encoding gene can be carried out as follows: the tumor cells are co-cultured with a tumor organoid inducer, wherein the final concentration of PARVA protein can be 10 to 50 ng / mL, and the co-culture time is 24 to 48 hours; if it is a PARVA encoding gene (taking a lentiviral vector as an example), lentiviral particles are added at a ratio of 10 to 20 with a multiplicity of infection (MOI) of 10 to 20, and 5 to 10 μg / mL polybrene is added at the same time to improve the infection efficiency. After culturing in a 37°C, 5% CO2 incubator for 24 hours, the culture medium is replaced with fresh medium.

[0050] In step (3), the primary tumor cells treated with PARVA are cultured in three dimensions to form a tumor organoid model. This can be done as follows: the treated tumor cells are mixed with extracellular matrix components (such as Matrigel) at a volume ratio of 1:3 and added to a 24-well plate. The plate is then incubated at 37°C for 30 minutes to solidify Matrigel, and then a tumor organoid basal medium containing growth factors is added. The plate is then incubated at 37°C in a 5% CO2 incubator. The medium is changed every 2 to 3 days. After 5 to 10 days of culture, the tumor organoids are observed and collected.

[0051] The tumor organoid model constructed by the method of this invention has the following characteristics: (1) High formation efficiency: Compared with the control group without PARVA inducer, the tumor organoid formation rate was significantly improved (e.g., the colorectal cancer organoid formation rate increased from 45% to over 80%).

[0052] (2) Strong structural stability: Within 21 days of continuous culture, the organoid structure integrity rate remained above 70%, with no obvious disintegration or abnormal differentiation.

[0053] (3) Good consistency: The tissue structure (such as glandular structure, cell polarity) and gene expression profile (such as the expression level of tumor markers CEA and CA199) of tumor organoids are similar to the structure of the tumor tissue from which they originate.

[0054] (4) High consistency of drug response: The tumor organoid model of the present invention has a high consistency with the clinical drugs of the primary tumor cells of the source tumor patients in response to commonly used chemotherapeutic drugs (such as colorectal cancer organoids to 5-fluorouracil, oxaliplatin and CPT-11).

[0055] This invention is particularly suitable for constructing highly stable and highly realistic tumor organoid models, providing technical support for tumor mechanism research, drug screening, and personalized treatment.

[0056] Example The present invention will be illustrated by examples below, but the scope of protection of the present invention is not limited to these examples.

[0057] Example 1: PARVA-encoding gene promotes lung cancer organoid formation 1. Experimental Materials The lung cancer surgical specimens used in this embodiment (from two lung cancer patients who could not form organoids in matrigel) were all approved by the ethics committee and informed consent was obtained from the patients. In this embodiment, the PARVA gene eukaryotic expression system was constructed using the pLVX-3×FLAG (P16690) lentiviral vector provided by Wuhan Miaoling Company. The empty vector pLVX-3×FLAG was used as a negative control. The PARVA nucleotide sequence (GenBank accession number NM_018222.5) was constructed into pLVX-3×FLAG according to the instructions, and sequencing was performed to obtain the experimental plasmid pLVX-3×FLAG-PARVA. The lentiviral packaging process used Lipofectamine 3000 transfection reagent (Thermofisher) for efficient transfection. The lung cancer organoid culture system used Advanced DMEM / F12 as the basal medium (containing 1% GlutaMAX, 1% HEPES, and 1% penicillin and streptomycin), supplemented with Matrigel matrix (Corning, 354234) and a combination of growth factors: epidermal growth factor (EGF, 50 ng / mL), fibroblast growth factor 2 (FGF2, 100 ng / mL), Noggin, 100 ng / mL, and Rspondin 1, 500 ng / mL. All growth factors were purchased from Nanjing Genscript Biotech Co., Ltd. The addition of these growth factors to the basal medium created a complete organoid culture medium.

[0058] 2. Experimental Methods (1) Lentiviral preparation: pLVX-PARVA-3×FLAG vector (or empty vector) and packaging plasmids psPAX2 and pMD2.G were co-transfected into 293T cells according to the kit instructions. The supernatant was collected 48 hours after transfection, filtered through a 0.45 μm filter membrane, concentrated by ultracentrifugation at 25,000 rpm, centrifuged at 4 degrees for 2 hours, and the viral titer was determined (titer was 5×10^8 TU / mL). (2) Obtaining primary lung cancer cells: The puncture specimen was washed twice with PBS containing 1% penicillin and streptomycin, and 0.25% trypsin EDTA digestion solution was added. The cells were digested at 37°C for 10 minutes, centrifuged to collect the cells, and the concentration was adjusted to 1×10^5 cells / mL. (3) Viral infection: The cells were divided into an empty vector group and a PARVA overexpression group. They were added to a medium containing empty vector lentivirus or PARVA overexpression lentivirus, respectively, with an MOI of 15. 8 μg / mL polybrene was added. After culturing at 37°C for 24 hours, the medium was replaced with fresh medium. After culturing for another 48 hours, PARVA expression was checked by Western Blot. (4) Three-dimensional culture: The pre-cultured cells were mixed with Matrigel at a volume ratio of 1:3 and added to a 24-well plate at a volume of 50 μL per well. After solidification at 37°C for 30 minutes, 500 μL of tumor organoid complete culture medium containing the above growth factors was added to each well. The plate was then cultured at 37°C in a 5% carbon dioxide incubator, and the culture medium was changed every 2 days. (5) Observation and statistics of organoids: After 21 days of culture, the morphology of organoids was observed under an inverted microscope, showing organoids with a diameter ≥100 μm.

[0059] 3. Experimental Results Experiments were conducted using lung cancer cells that failed to form organoids in Matrigel. Tumor cells were transfected with either a 3F (plasmid-tagged) control virus or a 3F PARVA virus, and then cultured in suspension. The results showed that lung cancer cells transfected with the 3F control virus did not form organoids, while tumor cells transfected with the 3F PARVA virus did. Figure 1 ).

[0060] Example 2: PARVA promotes organoid formation in colorectal cancer 1. Experimental Materials and Methods This embodiment uses three colorectal cancer surgical specimens that have been approved by the ethics committee and for which informed consent has been obtained from the patients; the remaining experimental steps are the same as in Example 1.

[0061] 2. Morphological observation of the experimental results showed that the number of colorectal cancer organoids in the control group was small, and some organoids had loose structures and poor integrity; while the number of organoids in the PARVA treatment group increased significantly, the structures were intact, and they showed typical glandular morphology.

[0062] Example 3: Verification of the morphological consistency between PARVA-induced tumor organoids and derived tumor tissues 1. Colorectal cancer organoids formed from the PARVA high-expression group cultured as in Example 2 and the corresponding source colorectal cancer tissues were paraffin-embedded and sectioned, and then hematoxylin-eosin (H&E) staining analysis was performed.

[0063] 2. Microscopic observation of the morphology of colorectal cancer organoids. The results showed that in PARVA-induced colorectal cancer organoids, glandular structures were basically lost, and cancer cells were mainly "diffusely distributed" (without obvious glandular outlines) or "nested"; the nuclei of cancer cells were significantly enlarged (nucleocytoplasmic ratio increased, that is, the proportion of cell volume occupied by the nucleus increased), irregular in shape (can be polygonal or spindle-shaped), with obvious and increased nucleoli, and mitotic figures were easily seen. The above characteristics showed a consistent trend with their parent tumors.

[0064] Example 4: Detection of drug response in PARVA-induced tumor organoids 1. Organoid drug sensitivity screening (96-well plate full coverage) (1) The required cell quantity was calculated based on the number, concentration, and number of replicates of the drug to be screened. Here, 10,000 cells / well was used as a reference. Drug administration and organoid plating design: The drug 5-fluorouracil, oxaliplatin, and CPT-11 were designed at a concentration of 10 μM (Table 1). Each drug was set up with 5 replicates, and three independent replicate experiments were performed on a 96-well plate.

[0065] (2) Organoid collection: Aspirate the colorectal cancer organoid culture medium prepared in Example 2, and add 500 μL of pre-cooled basal culture medium DMEM / F12 (containing 1% GlutaMAX and 1% GlutaMAX) to each well. Using HEPES (1% penicillin-streptomycin), scrape the matrix gel with a 1 mL pipette tip to detach the matrix gel and organoids; transfer the matrix gel containing organoids to a 15 mL centrifuge tube, wash the wells with pre-chilled 1×PBS, and combine to obtain an organoid suspension; place the 15 mL centrifuge tube on ice to soften the matrix gel; pipette the organoid suspension 15 times with a 1 mL pipette tip to separate the organoids from the matrix gel; centrifuge at 4°C, 300 g for 3 min, remove the supernatant, and resuspend the organoids in 10 mL of pre-chilled 1×PBS; centrifuge at 4°C, 300 g for 3 min, remove the supernatant, add 3 mL of organoid digestion solution, and incubate at 37°C for 5 min; pipette the organoids until they are digested into single cells; add 8 mL of basal medium DMEM / F12 and 0.2 mL of fetal bovine serum to stop digestion; mix well and centrifuge at 4°C, 300 g for 3 min. After removing the supernatant, wash twice with DMEM / F12 basal medium; resuspend the cells in 1 mL of basal medium and transfer to a 1.5 mL EP tube, take 18 μL of cell suspension (add 2 μL trypan blue) for viability detection and counting.

[0066] (3) Organoid plating in 96-well plates: Calculate the amount of matrix gel required based on the number of wells needed for drug screening. Drop 10 μL of organoids into each well of a 96-well plate and cure for 15 min in an incubator. Add 110 μL of complete organoid growth medium to each well and incubate for 10 days. Add drugs at a concentration of 10 µM. The concentrations of the five drugs are shown in the table below. Continue culturing the organoids in the incubator for 5 days after drug addition.

[0067] Table 1: Concentration and Function of Added Drugs

[0068] 2. Organoid activity detection Cell viability was assessed using the CellTiter-Lumi™ 3D Cell Viability Chemiluminescence Kit (from Beyotime, C0061). The 3D cell viability assay kit was allowed to equilibrate at room temperature. The colorectal cancer organoid culture plate prepared in Example 2 was removed and placed at room temperature for 5 minutes to allow the plate temperature to equilibrate. The 3D cell viability assay kit was then inverted to mix thoroughly. 50 μL of the 3D cell viability assay kit was added to each well of a 96-well plate. The plate was shaken for 2 minutes in a microplate reader, incubated at room temperature for 30 minutes, and then the chemiluminescence value was measured. The cell viability at each drug concentration in the experimental groups was calculated.

[0069] The formula for calculating the survival rate is as follows: Survival rate = [(Experimental group - Blank control) - (Negative control - Blank control)] / [(Positive control - Blank control) - (Negative control - Blank control)].

[0070] Figure 4 The study demonstrates the detection of cell viability in PARVA-induced colorectal organoids from Example 2 after treatment with three drugs. The results showed that 5-fluorouracil (MCE product, HY-90006), oxaliplatin (MCE product, HY-17371), and CPT-11 (MCE product, HY-16562) exhibited varying degrees of drug sensitivity in the PARVA-induced colorectal organoids.

[0071] Example 5: Signaling pathways regulated by PARVA in colorectal cancer organoids 1. Using the RayBiotech Human Cytokine Array Q440 (catalog number: QAH-CAA-440) kit, and following the manufacturer's instructions, the cytokine levels of randomly selected colorectal cancer organoids and primary colorectal cancer cells with high PARVA expression, prepared in Example 2, were quantitatively detected. The fluorescently labeled array was scanned using an InnoScan 300 microarray scanner. Raw data were then extracted using GenePix Pro 5.1 software, and further data analysis was performed using RayBiotech Q-Analyzer software. KEGG enrichment analysis was performed on differentially expressed genes to enrich significantly different signaling pathways and identify biological regulatory pathways that showed significant changes under experimental conditions. The statistical significance threshold was set at P < 0.05.

[0072] 2. The results showed that PARVA regulates signaling pathways including the TNF signaling pathway, MAPK signaling pathway, PI3K-Akt signaling pathway, JAK-STAT signaling pathway, Ras signaling pathway, EGFR signaling pathway, and cell adhesion molecule signaling pathway, etc. See details below. Figure 5 .

[0073] In summary, this invention demonstrates that the PARVA protein or its encoding gene can significantly promote the formation of tumor organoids, improve the stability and simulation of organoids, and provide a new technical means for the construction of tumor organoid models, which has important scientific research value and clinical application prospects.

[0074] It should be noted that the specific embodiments described above are exemplary. Those skilled in the art can devise various solutions inspired by the disclosure of this invention, and these solutions all fall within the scope of this invention and its protection. Those skilled in the art should understand that this specification and its accompanying drawings are illustrative and not intended to limit the scope of the claims. The scope of protection of this invention is defined by the claims and their equivalents. This specification contains multiple inventive concepts; terms such as "preferredly," "according to a preferred embodiment," or "optionally" indicate that the corresponding paragraph discloses an independent concept. The applicant reserves the right to file divisional applications based on each inventive concept.

Claims

1. Application of PARVA protein or PARVA encoding gene in the preparation of tumor organoid inducers, tumor organoid culture kits or the construction of tumor organoid models.

2. The application according to claim 1, characterized in that, The PARVA protein is a wild-type PARVA protein or a functionally equivalent mutant of the wild-type PARVA protein; Preferably, the amino acid sequence of the wild-type PARVA protein is as shown in GenBank accession number NP_060692.3; the functionally equivalent mutant is a protein that retains PARVA activity and downstream signal activation ability after the wild-type sequence is modified by substitution, deletion or insertion; preferably, the substitution, deletion or insertion is a substitution, deletion or insertion of 15 amino acid residues.

3. The application according to claim 1, characterized in that, The PARVA encoding gene is selected from one or more sequences from (1) to (3) below: (1) The nucleotide sequence as shown in GenBank accession number NM_018222.5; (2) A DNA sequence that hybridizes with the nucleotide sequence described in (1) under strict conditions and encodes a protein that promotes tumor organoid formation; (3) A DNA sequence that is ≥90% homologous to the nucleotide sequence described in (1) and encodes a protein that promotes the formation of tumor organoids.

4. The application according to claim 1, characterized in that, The tumor organoid inducer exerts its effects by activating the IntegrinIPP complex signaling pathway, the PI3KAkt signaling pathway, the Rho family GTPase-mediated cytoskeleton remodeling pathway, the MAPK signaling pathway, and / or extracellular matrix adhesion-related pathways.

5. The application according to claim 1, characterized in that: The tumor organoid inducer is selected from PARVA expression regulatory components, PARVA protein interaction activating components, and PARVA downstream pathway synergistic components. Preferably, the tumor organoid inducer exists in the form of: (1) PARVA protein; (2) a eukaryotic expression vector containing the PARVA encoding gene, a lentiviral particle containing the PARVA encoding gene, or PARVA mRNA; (3) a small molecule activator of PARVA, a signaling pathway activator, or a pharmaceutical composition; or (4) a delivery vector encapsulating PARVA protein, PARVA mRNA, or a PARVA expression vector.

6. The application according to claim 5, characterized in that: The delivery vector is selected from at least one of the following: cells, viruses, liposomes, nanoparticles, vesicles, and ferritin. Preferably, the cells are erythrocytes and / or mesenchymal stem cells; Alternatively, the virus is preferably an adenovirus or a lentivirus.

7. The application according to claim 1, characterized in that, The method for constructing the tumor organoid model includes the following steps: (1) Obtaining primary tumor cells; (2) The primary tumor cells were treated with PARVA; (3) Primary tumor cells treated with PARVA were cultured in three dimensions to form a tumor organoid model.

8. The application according to claim 1, characterized in that, The tumors or cancers targeted by the tumor organoid inducers, tumor organoid models, or tumor organoid culture kits are selected from: oral cancer, oropharyngeal cancer, nasopharyngeal cancer, respiratory system cancers, genitourinary system cancers, gastrointestinal cancers, gliomas, sarcomas, epithelial cancers, lymphomas, melanomas, fibromas, meningiomas, brain cancers, kidney cancers, biliary tract cancers, pheochromocytomas, islet cell carcinomas, Levi-Flaumeni tumors, thyroid cancers, parathyroid cancers, pituitary adenomas, adrenal adenomas, osteosarcomas, neuroendocrine system tumors, breast cancers, lung cancers, head and neck cancers, prostate cancers, esophageal cancers, tracheal cancers, liver cancers, bladder cancers, gastric cancers, ovarian cancers, uterine cancers, cervical cancers, testicular cancers, colon cancers, rectal cancers, or skin cancers.

9. The application according to claim 1, characterized in that: The tumor organoid models are similar to and consistent with the source tumor tissue in terms of morphology and structure; and / or The tumor organoid model exhibits the same sensitivity to chemotherapy drugs as the patient's primary cells.

10. The application according to claim 1, characterized in that: The tumor organoid culture kit contains, in addition to a tumor organoid inducer prepared based on PARVA, a tumor organoid basal culture medium, extracellular matrix components, and growth factors.

11. The application according to claim 10, characterized in that: The basal culture medium is DMEM / F12 medium and / or enhanced DMEM / F12 medium; The extracellular matrix component is selected from at least one of matrix gelatin, collagen I, gelatin, and hyaluronic acid; and / or The growth factor is selected from at least one of EGF, FGF2, Noggin, Rspondin 1, and Wnt3a.

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