A method for culturing a mucinous pancreatic cystic tumor organoid, a detection method and system

Abstract

The application belongs to the technical field of medicine, and discloses a method and system for detecting mucinous pancreatic cystic tumor, acquires a pancreatic cyst fluid sample; performs organoid culture on the pancreatic cyst fluid to obtain a mucinous pancreatic cystic tumor organoid; acquires cell morphology data of the mucinous pancreatic cystic tumor organoid; acquires H&E staining data and IHC detection data of the mucinous pancreatic cystic tumor organoid; acquires immunofluorescence detection data of the mucinous pancreatic cystic tumor organoid; performs scRNA-Seq sequencing on the mucinous pancreatic cystic tumor organoid to complete detection of the mucinous pancreatic cystic tumor; and the method can efficiently culture organoids and be used for verifying potential existence of malignant cells in function.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of medicine, and particularly relates to a mucinous pancreatic cystic tumor organoid culture method, a detection method and a system. BACKGROUND

[0002] Pancreatic cystic lesions (PCLs) refer to tumors or non-tumor (single or multiple tumor-like) cystic lesions formed by pancreatic epithelial and (or) interstitial tissue, mainly including pancreatic pseudocysts (PPs) and mucinous pancreatic cystic neoplasms (PCNs). Mucinous pancreatic cystic neoplasms account for 10-20% of pancreatic tumors and need active treatment; and certain benign cystic tumors do not need surgical intervention. Therefore, for patients with recurrent acute pancreatitis, it is very important to distinguish whether it is a pseudocyst or a pancreatic cystic tumor.

[0003] At present, the traditional diagnosis method mainly is an imaging technology (CT, MRI, ultrasound, etc.), combined with cell smears and immunohistochemistry, and has high cost and a relatively high missed diagnosis rate. SUMMARY

[0004] The application aims to provide a mucinous pancreatic cystic tumor organoid culture method, a detection method and a system.

[0005] A mucinous pancreatic cystic tumor detection method, the method comprises the following steps:

[0006] Obtaining a pancreatic cyst fluid sample;

[0007] Performing organoid culture on the pancreatic cyst fluid to obtain a mucinous pancreatic cystic tumor organoid;

[0008] Obtaining cell morphology data of the mucinous pancreatic cystic tumor organoid;

[0009] Obtaining H&E staining data and IHC detection data of the mucinous pancreatic cystic tumor organoid;

[0010] Obtaining immunofluorescence detection data of the mucinous pancreatic cystic tumor organoid;

[0011] Performing scRNA-Seq sequencing on the mucinous pancreatic cystic tumor organoid to complete detection of the mucinous pancreatic cystic tumor.

[0012] The H&E staining data and the IHC detection data are used to judge whether the cell nucleus is abnormal, and the classic tumor marker CEA and MKI67 are expressed positively in the mucinous pancreatic cystic tumor organoid.

[0013] As a preferred technical solution, the immunofluorescence detection data is used to determine whether MUC6 and MUC5A are strongly expressed in the mucinous pancreatic cystic tumor organoid.

[0014] As a preferred technical solution, the scRNA-Seq sequencing data is used to determine whether MUC6 and MUC5A are strongly expressed in the mucinous pancreatic cystic tumor organoid.

[0015] As a preferred technical solution, the cell morphology data is used to determine whether the organoid is slowly growing and thickening from a vacuole type to form a dense spherical morphology.

[0016] The present application also relates to a mucinous pancreatic cystic tumor detection system, which comprises:

[0017] A sample acquisition unit is configured to acquire a pancreatic cyst fluid sample.

[0018] A culture unit is configured to culture the pancreatic cyst fluid into an organoid to obtain a mucinous pancreatic cystic tumor organoid.

[0019] A cell morphology data acquisition unit is configured to acquire cell morphology data of the mucinous pancreatic cystic tumor organoid.

[0020] An H&E staining data and IHC detection data acquisition unit is configured to acquire H&E staining data and IHC detection data of the mucinous pancreatic cystic tumor organoid.

[0021] An immunofluorescence detection data acquisition unit is configured to acquire immunofluorescence detection data of the mucinous pancreatic cystic tumor organoid.

[0022] A cell morphology data acquisition unit is configured to perform scRNA-Seq sequencing on the mucinous pancreatic cystic tumor organoid.

[0023] As a preferred technical solution, the immunofluorescence detection data is used to determine whether MUC6 and MUC5A are strongly expressed in the mucinous pancreatic cystic tumor organoid.

[0024] As a preferred technical solution, the scRNA-Seq sequencing data is used to determine whether MUC6 and MUC5A are strongly expressed in the mucinous pancreatic cystic tumor organoid.

[0025] As a preferred technical solution, the cell morphology data is used to determine whether the organoid is slowly growing and thickening from a vacuole type to form a dense spherical morphology.

[0026] The application also relates to a mucinous pancreatic cystic neoplasm organoid culture method, which comprises the following steps:

[0027] Pancreatic cystic fluid sample collection and processing;

[0028] Pancreatic cyst fluid organoid culture;

[0029] The culture medium of the organoid culture is as follows:

[0030] The basic culture medium is Advanced DMEM / F12, and the added components and contents are as follows: Primocin 100 μg / mL, HEPES 10 mM, GlutaMAX-I 1X, A83-01 500 nM, Y-27632 10 μM, N-acetylcysteine 1.56 mM, nicotinamide 10 mM, FGF10 10 ng / mL, B27 supplement 1X, EGF 50 ng / mL, Gastrin I 0.01 μM, Wnt3A conditioned medium 50 ng / mL, R-spondin-1 conditioned medium 250 ng / mL and Noggin conditioned medium 100 ng / mL.

[0031] As a preferred technical solution, the cleaning solution used in the processing of the pancreatic cystic fluid sample is composed of a pbs solution, 0.5% FBS and 2% double antibody.

[0032] The application has the following beneficial effects:

[0033] The application is directly derived from the organoid model of human tumor tissue, can maximize the simulation of the genetic and phenotypic characteristics of in vivo groups, and accurately reproduce the biological heterogeneity.

[0034] In the prior art, single-cell sequencing requires a large number of single cells, and the method for culturing organoids of the application can meet the cell amount required for sequencing.

[0035] The application collects pancreatic cystic fluid and cultures the pancreatic cystic fluid to generate organoids, which are used for functionally verifying the potential existence of malignant cells, helping to determine whether it is a mucinous tumor or a false cyst, helping to monitor disease progression, treatment response and adaptive therapy, and improving the treatment effect of patients. As an early cancer detection method, it has far-reaching significance for early and accurate diagnosis and prognosis guidance! BRIEF DESCRIPTION OF DRAWINGS

[0036] Figure 1 The left side is the CT scan result of patient #X, and the right side is the enhanced MRI and enhanced endoscopic ultrasound examination (EUS) result;

[0037] Figure 2Ultrasound endoscopy guided fine needle aspiration of pancreatic cystic fluid for patient #X;

[0038] Figure 3 After quality control of scRNA-seq of pancreatic cystic fluid for patient #X, 10565 core cells were identified;

[0039] Figure 4 After quality control of scRNA-seq of pancreatic cystic fluid for patient #X, 11 cell types were identified;

[0040] Figure 5 Copy number variation (CNV) level analysis of epithelial cells in scRNA-seq of pancreatic cystic fluid for patient #X;

[0041] Figure 6 In the scRNA-seq results of pancreatic cystic fluid for patient #X, TBCB, TMEM147, and ARPC2 showed different expressions in the CNV_low and CNV_high groups;

[0042] Figure 7 Gene set enrichment analysis (GSEA) of the scRNA-seq results of pancreatic cystic fluid for patient #X showed that the CNV_low group was enriched in cytoplasmic translation and peptide biosynthesis processes;

[0043] Figure 8 Pancreatic cystic fluid organoid growth chart for patient #X;

[0044] Figure 9 HE results of pancreatic cystic fluid organoids for patient #X;

[0045] Figure 10 Immunohistochemical detection of classic tumor markers P53, CEA, and MKI67 expression in pancreatic cystic fluid organoids for patient #X;

[0046] Figure 11 Immunofluorescence detection of cancer cell mucins MUC5A and MUC6 expression in pancreatic cystic fluid organoids for patient #X;

[0047] Figure 12 Expression of tumor markers CEA, MKI67, MUC5A, and MUC6 in the scRNA-seq results of pancreatic cystic fluid for patient #X;

[0048] Figure 13 Pancreatic cystic fluid organoid culture for patients #Y and #Z;

[0049] Figure 14 Pancreatic cystic fluid organoid culture for patient #a;

[0050] Figure 15 The pancreatic cystic fluid scRNA-seq cell clustering analysis results of patient #a. DETAILED DESCRIPTION

[0051] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0052] A mucinous pancreatic cystic tumor detection method, the method comprising the following steps:

[0053] Obtaining a pancreatic cyst fluid sample;

[0054] Performing organoid culture on the pancreatic cyst fluid to obtain a mucinous pancreatic cystic tumor organoid;

[0055] Obtaining cell morphology data of the mucinous pancreatic cystic tumor organoid;

[0056] Obtaining H&E staining data and IHC detection data of the mucinous pancreatic cystic tumor organoid;

[0057] Obtaining immunofluorescence detection data of the mucinous pancreatic cystic tumor organoid;

[0058] Performing scRNA-Seq sequencing on the mucinous pancreatic cystic tumor organoid to complete detection of the mucinous pancreatic cystic tumor.

[0059] As a preferred embodiment, the immunofluorescence detection data is used to determine whether MUC6 and MUC5A are strongly expressed in the mucinous pancreatic cystic tumor organoid.

[0060] As a preferred embodiment, the scRNA-Seq sequencing data is used to determine whether MUC6 and MUC5A are strongly expressed in the mucinous pancreatic cystic tumor organoid.

[0061] As a preferred embodiment, the cell morphology data is used to determine whether the organoid is slowly growing and thickening from vacuole type to form a dense spherical morphology.

[0062] A mucinous pancreatic cystic tumor detection system, the system comprising:

[0063] A sample obtaining unit, the sample obtaining unit being configured to obtain a pancreatic cyst fluid sample;

[0064] A culture unit, the culture unit being configured to perform organoid culture on the pancreatic cyst fluid to obtain a mucinous pancreatic cystic tumor organoid;

[0065] a cell morphology data acquisition unit for acquiring cell morphology data of the mucinous pancreatic cystic neoplasm organoid;

[0066] an H&E staining data and IHC detection data acquisition unit for acquiring H&E staining data and IHC detection data of the mucinous pancreatic cystic neoplasm organoid;

[0067] an immunofluorescence detection data acquisition unit for acquiring immunofluorescence detection data of the mucinous pancreatic cystic neoplasm organoid;

[0068] a cell morphology data acquisition unit for performing scRNA-Seq sequencing on the mucinous pancreatic cystic neoplasm organoid.

[0069] As a preferred embodiment, the H&E staining data and IHC detection data are used to determine whether the cell nucleus is abnormal, and the classic tumor marker CEA, MKI67 is expressed positively in the mucinous pancreatic cystic neoplasm organoid.

[0070] As a preferred embodiment, the immunofluorescence detection data are used to determine whether MUC6, MUC5A is strongly expressed in the mucinous pancreatic cystic neoplasm organoid.

[0071] As a preferred embodiment, the scRNA-Seq sequencing data are used to determine whether MUC6, MUC5A is strongly expressed in the mucinous pancreatic cystic neoplasm organoid.

[0072] As a preferred embodiment, the cell morphology data are used to determine whether the organoid is gradually thickened by vacuoles to form a dense spherical morphology.

[0073] A mucinous pancreatic cystic neoplasm organoid culture method, the culture method comprising:

[0074] pancreatic cystic fluid sample collection and processing;

[0075] pancreatic cyst fluid organoid culture;

[0076] the culture medium of the organoid culture is:

[0077] Basal medium: Advanced DMEM / F12; added components and concentrations: Primocin 100 pg / mL, HEPES 10 mM, GlutaMAX-I IX, A83-01 500 nM, Y-27632 10 pM, N-acetylcysteine 1.56 mM, nicotinamide 10 mM, FGF10 10 ng / mL, B27 supplement IX, EGF 50 ng / mL, Gastrin I 0.01 pM, Wnt3A conditioned medium 50 ng / mL, R-spondin-1 conditioned medium 250 ng / mL, Noggin conditioned medium 100 ng / mL.

[0078] As a preferred embodiment, the washing solution during the processing of the pancreatic cyst fluid sample is composed of pbs solution, 0.5% FBS and 2% biotin.

[0079] Example:

[0080] First, patient #X was determined as the main research object, and the pathological condition was as follows: a 34-year-old female (patient #X) complained of upper abdominal pain for 5 years, with a history of acute pancreatitis. CT scan showed a 5 cm diameter pancreatic cystic lesion in the pancreas, suspected to be a pancreatic pseudocyst Figure 1 Left). Subsequent enhanced MRI and enhanced endoscopic ultrasound examination (EUS) confirmed that the lesion was a mucinous cystic tumor with enhanced wall nodules Figure 1 Right).

[0081] Endoscopic ultrasound-guided fine needle aspiration of about 40 mL of cystic fluid Figure 2 ). The amylase level in the cyst fluid was 165328 U / L, the CEA level was 261.98 ng / mL, and the glucose level was 0.5 mmol / L. The pathological examination of malignant cells in the cystic fluid based on cell smears was negative.

[0082] To further verify the accuracy of the diagnosis of patient #X, about 3 mL of cystic fluid was subjected to scRNA-Seq. After quality control filtering, 10565 single cells were retained for subsequent analysis Figure 3 ). A total of 11 cell types were identified Figure 4), which are fibroblast (COL1A1, COL3A1, DCN), CD4 T (CD3D, CD3E, CD4), CD8 T (CD3D, CD3E, and CD8A), B (CD79A, MS4A1), plasma (IGHA2, IGLC2), macrophage (C1QC, C1QB, C1QA), monocyte (FCN1, S100A8), DC_CD1C (CD1C, CD1E), DC_LAMP3 (LAMP3, FSCN1), proliferating (MKI67, TOP2A) cells, and importantly, epithelial (EPCAM, KRT8, and MUC5AC) group. The copy number variation (CNV) level of epithelial cells varies from each other Figure 5 ). Compared with CNV_low group, the expression of TBCB, TMEM147, and ARPC2 is lower in CNV_high group, suggesting that it may be related to malignant progression Figure 6 ). GSEA analysis shows that CNV_low group is enriched in cytoplasmic translation and peptide biosynthesis processes, suggesting that there may be malignant cells in the pancreatic cyst fluid of patient #X Figure 7 It can be seen that only possible results can be obtained by using scRNA-Seq, and there is a certain possibility of misdiagnosis, and other detection methods need to be further confirmed.

[0083] To functionally confirm that the cystic fluid contains malignant cells, the pancreatic cyst fluid (about 3 mL) of patient #X was cultured into organoids, and the culture method was as follows:

[0084] 1. Pancreatic cystic fluid sample collection

[0085] About 40 mL of cystic fluid was aspirated under endoscopic ultrasound guidance;

[0086] The cystic fluid was stored in a sterile disposable drainage bottle or drainage bag, and additional double-antibiotic sterilization could be added at a ratio of 1:100, for example, 500 ml of malignant effusion plus 5 ml of double-antibiotic sterilization solution. The cleaning solution for processing the pancreatic cyst fluid sample was pbs+0.5% FBS+2% double-antibiotic.

[0087] 2. Pancreatic cyst fluid organoid culture

[0088] 1) Transfer the pancreatic cyst fluid sample from the collection tube / draining bag to a 50 ml centrifuge tube, and add PBS at a ratio of 1:1;

[0089] 2) Centrifuge at 4°C, 300g, for 5 min, discard the supernatant, resuspend with cleaning solution, filter with a filter screen, continue centrifugation, and discard the supernatant;

[0090] 3) If there are more red blood cells in the cell precipitate, red blood cell lysis should be performed. Specific operation: add an appropriate amount of red blood cell lysis solution to the cell precipitate, mix well by blowing, ice bath for 2-3 min, add an equal volume of neutralization solution. 4℃, 300g, centrifuge for 5 min, discard the supernatant. If there are no red blood cells, proceed to the next step directly;

[0091] 4) Resuspend the cell precipitate with 1 mL of washing solution, take 10 μL of the cell suspension for cell counting before centrifugation again, 4℃, 300g, centrifuge for 5 min, discard the supernatant;

[0092] 5) According to the cell counting, determine the number of holes that can be inoculated, add an appropriate volume of Matrigel gel under ice bath conditions, generally control 1*10 5 / 10uL / hole (48-well plate);

[0093] 6) Use a 10 μL micropipette to aspirate the cell / matrix gel mixture, and add 10 μL per hole to the center of the 48-well plate, cover the hole plate cover;

[0094] 7) Place the 48-well plate in a 37℃, 5% CO2 cell incubator, stand for 10 min, and the gel liquid will solidify; add 200 μL of organoid complete culture medium to each hole, 37℃, 5% CO2, cell incubator culture; replace the culture medium every 3-4 days.

[0095] Medium composition for pancreatic cyst fluid organoids:

[0096] The basic culture medium is: Advanced DMEM / F12; add each component and content: Primocin 100 μg / mL, HEPES 10 mM, GlutaMAX-I 1X, A83-01 500 nM, Y-27632 10 μM, N-acetylcysteine 1.56 mM, nicotinamide 10 mM, FGF10 10 ng / mL, B27 supplement 1X, EGF 50 ng / mL, Gastrin I 0.01 μM, Wnt3A conditioned medium 50 ng / mL, R-spondin-1 conditioned medium 250 ng / mL, Noggin conditioned medium 100 ng / mL.

[0097] Specifically, Primocin (100 pg / mL, Invitrogen), HEPES (10 mM, Gibco), GlutaMAX-I (1X, Gibco), A83-01 (500 nM, Tocris), Y-27632 (10 mM, AbMole), N-acetylcysteine (1.56 mM, Sigma), nicotinamide (10 mM, Sigma), FGF10 (10 ng / mL, PeproTech), B27 supplement (1X, Gibco), Epidermal Growth Factor (50 ng / mL, PeproTech), Gastrin I (0.01 mM, TocrisBioscience), Wnt3A conditioned medium (50 ng / mL, R&D Systems), R-spondin-1 conditioned medium (250 ng / mL, R&D Systems), and Noggin conditioned medium (100 ng / mL, R&D Systems).

[0098] Using the aforementioned organoid culture method, the results show that the organoids grow rapidly (day 2-day 3 organoid diameter average growth of more than 25%) Figure 8 ), and the organoids gradually grow and thicken from vacuole type to form a dense spherical shape. H&E staining highlights abnormal structures in the nucleus Figure 9 ). IHC detection shows that the classic tumor markers CEA and MKI67 are positive Figure 10 ). Immunofluorescence detection results show that Figure 11 ), MUC6 is not obviously expressed on this cystic fluid organoid sample, and MUC5A is strongly expressed Figure 11 ), and scRNA-Seq results show that MUC6 is not obviously expressed on this cystic fluid organoid sample, and MUC5A is strongly expressed Figure 12 ).

[0099] To further evaluate the feasibility of organoid technology as a new, functionally advanced diagnostic method, we expanded the scope of the study and included three additional pancreatic cystic fluid patients in the validation experiment. Similarly, we cultured organoids from the pancreatic cystic fluid of patients #Y and #Z using the aforementioned culture method, and quickly observed the formation of organoids Figure 13 ). This indicates the presence of malignant cells in the cystic fluid, which is consistent with the clinical results, as both patients # eventually returned to the hospital due to the onset of pancreatic cancer. In contrast, the culture of patient #a did not produce organoidsFigure 14 ), indicating no epithelial or malignant cells, which is consistent with the scRNA-Seq data showing the dominant cell populations in the cyst fluid were CD4 T, CD8 T and macrophages ( Figure 15 ).

[0100] In summary, pancreatic cystic fluids from patients #X, #Y and #Z can all be cultured into organoids, indicating the presence of malignant cells in the cyst fluid, which is consistent with their clinical outcomes. The culture from patient #a did not generate organoids, indicating no epithelial or malignant cells, which is consistent with the scRNA-Seq data. Therefore, the potential presence of malignant cells can be verified based on the organoid technology, which can serve as a novel diagnostic method to identify mucinous pancreatic cystic neoplasms and pseudocysts, and help subsequent pathological diagnosis.

[0101] It should be noted that the relational terms herein such as first and second and the like are used solely to distinguish one entity or action from another, without necessarily requiring or implying any actual relationship or order between such entities or actions. Moreover, the terms "comprises", "comprising", or any other variations thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but can include other elements not expressly listed or inherent to such process, method, article, or apparatus.

[0102] Although embodiments of the present application have been shown and described, it is to be understood that various modifications, substitutions, alternatives, and variations can be made in the embodiments without departing from the principles and spirits of the present application, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A mucus pancreatic cystic tumor detection system characterized by, The detection system is used for distinguishing pancreatic pseudocyst and mucinous pancreatic cystic tumor, and the system comprises: a sample acquisition unit for acquiring a pancreatic cyst fluid sample; a culture unit for culturing the pancreatic cyst fluid into a mucinous pancreatic cystic tumor organoid; a cell morphology data acquisition unit for acquiring cell morphology data of the mucinous pancreatic cystic tumor organoid; an H&E staining data and IHC detection data acquisition unit for acquiring H&E staining data and IHC detection data of the mucinous pancreatic cystic tumor organoid; an immunofluorescence detection data acquisition unit for acquiring immunofluorescence detection data of the mucinous pancreatic cystic tumor organoid; a sequencing unit for performing scRNA-Seq sequencing on the mucinous pancreatic cystic tumor organoid; the immunofluorescence detection data are used for judging whether MUC6 and MUC5A are strongly expressed in the mucinous pancreatic cystic tumor organoid; the scRNA-Seq sequencing data are used for judging whether MUC6 and MUC5A are strongly expressed in the mucinous pancreatic cystic tumor organoid; the cell morphology data are used for judging whether the organoid is slowly thickened and formed into a dense spherical morphology from a vacuole type; the organoid culture method comprises: pancreatic cyst fluid sample collection and processing; the washing solution during processing of the pancreatic cyst fluid sample is composed of pbs solution, 0.5% FBS and 2% double antibody; pancreatic cyst fluid organoid culture; 1) transfer the pancreatic cyst fluid sample from the collection tube / draining bag to a 50ml centrifuge tube, and add PBS at a ratio of 1:1; 2) centrifuge at 4°C, 300g for 5 min, discard the supernatant, add the washing solution for resuspension, filter with a filter screen, continue to centrifuge, and discard the supernatant; 3) if there are many red blood cells in the cell precipitate, red blood cell lysis should be performed; specific operation: add an appropriate amount of red blood cell lysis solution to the cell precipitate, mix well by blowing, and ice bath for 2-3 min, then add an equal volume of washing solution for neutralization; centrifuge at 4°C, 300g for 5 min, discard the supernatant; if there are no red blood cells, proceed to the next step directly; 4) resuspend the cell precipitate with 1mL of washing solution, take 10µL of the cell suspension for cell counting before centrifugation again, centrifuge at 4°C, 300g for 5 min, and discard the supernatant; 5) According to the cell count, determine the number of holes that can be inoculated, add the appropriate volume of Matrigel gel under the condition of ice bath, mix thoroughly, and control 1*10 5 / 10uL / hole; 6) use a 10µL micropipette to take the cell / Matrigel gel mixture, and add it to the center of a 48-well plate at a volume of 10µL per well, and cover the well plate cover; 7) place the 48-well plate in a 37°C, 5% CO2 cell incubator, stand for 10 min, and solidify the gel liquid; add 200µL of organoid complete culture medium to each well, and culture in a 37°C, 5% CO2 cell incubator; replace the culture medium every 3-4 days; the culture medium for the organoid culture is: Basal medium: Advanced DMEM / F12; added components and concentrations: Primocin 100 pg / mL, HEPES 10 mM, GlutaMAX-I IX, A83-01 500 nM, Y-27632 10 pM, N-acetylcysteine 1.56 mM, nicotinamide 10 mM, FGF10 10 ng / mL, B27 supplement IX, EGF 50 ng / mL, GastrinI 0.01 pM, Wnt3A conditioned medium 50 ng / mL, R-spondin-1 conditioned medium 250 ng / mL, Noggin conditioned medium 100 ng / mL.

Images