PDC (Polycrystalline Diamond Compact) cell carrying bar code DNA (Deoxyribonucleic Acid) tag library convenient to track as well as preparation method and application thereof

Abstract

The invention relates to a PDC (Polycrystalline Diamond Compact) cell carrying a bar code DNA (Deoxyribose Nucleic Acid) tag convenient to track as well as a preparation method and application thereof, and belongs to the technical field of The invention provides an isolated culture method of PDC primary cells, and provides a method for constructing recombinant PDC cells containing bar code DNA tags. According to the PDC primary cell culture system, the cell caking rate is reduced, the cell viability and the adherence rate are improved, and continuous passage to 20 generations or above can be achieved. The PDC primary cells faithfully retain the heterogeneity of clinical tumor samples in the aspects of mutation types, tumor generation and development mechanisms and the like. The bar code DNA tag introduced into the PDC cell can be detected by carrying out targeted sequencing and single cell RNA sequencing on a bar code DNA tag sequence in cell genome DNA, so that different clone groups reserved from PDX of the PDC cell line are conveniently distinguished and tracked in a high-throughput manner.

Description

Technical Field

[0001] The present invention relates to the technical field of cell construction, and in particular to a PDC cell carrying a barcode DNA label library for easy tracking, and a preparation method and application thereof. Background Art

[0002] Conventional tumor research models currently available primarily consist of various human or mouse tumor cell lines, as well as tumor-bearing mouse models constructed by injecting these cell lines subcutaneously or orthotopically into mice. Human tumor cell lines are typically referred to as CDX (cell line-derived xenograft) models. In the past decade or so, PDX (patient-derived xenograft) models, created by directly transplanting patient-derived tumors into immunodeficient mice, have also become increasingly widely used in tumor research.

[0003] Most of the current conventional tumor cell lines were established decades ago and have undergone countless in vitro passages and transfers between laboratories. Many of their characteristics have diverged significantly from when they were first established. In particular, some cell lines have been subject to misidentification or cross-contamination during past storage and use, raising doubts about their true identity.

[0004] Because PDX models are relatively recent and have only undergone a limited number of passages in vivo, they better retain the characteristics of the patient's tumor. This is especially true of tumor heterogeneity, which refers to the different clonal populations of the tumor. The evolution of these clonal populations and their differences in sensitivity to drug treatment are important factors in determining tumor progression and treatment efficacy. However, because PDX models (and CDX models) are used in tumor-bearing mice, their experimental cycles are long and costly, and operations such as genetic modification and gene expression intervention are more difficult to perform than in vitro cultured cell lines.

[0005] In recent years, tumor cell lines (PDX-derived cellline, PDC for short) have been reconstructed using PDX with accurate and complete clinical information. Compared with current conventional tumor cell lines, many of its characteristics are closer to patient tumors, and its response to drug treatment is more relevant to clinical efficacy. PDC cell lines have the advantages of accurate and complete clinical information, new construction time, and few passages, and their clinical relevance is better. At the same time, with the help of the continuously growing number of PDX models, large-scale PDC cell banks can be constructed to increase the representativeness of tumor cases. PDC can be easily used for genetic modification, gene expression intervention, high-throughput drug screening and other operations. PDC can also be used in conjunction with PDX models (or PDC re-injected into the mouse subcutaneously or in situ to construct a tumor-bearing mouse model) to carry out complete in vitro and in vivo efficacy studies.

[0006] However, existing techniques for culturing primary PDCs suffer from low cell survival rates, demanding experimental conditions, and a certain degree of contamination from other cells. Tumor cells exist in different microenvironments both in vivo and in vitro. Inappropriate culture methods can easily lead to the loss of some cell clones, altering cell expression products and properties, hindering subsequent research. The success rates of primary cell culture methods vary significantly across pathological types and individual tumors, limiting their widespread application and hindering ongoing cancer research.

[0007] Moreover, PDC retains the heterogeneity and complexity of the clonal groups in PDX, which leads to the mixing of different pathways and mechanisms related to tumor development and drug response, making it difficult to conduct differentiated studies and to analyze and predict the clonal evolution of patient tumors through PDC or PDX models. Summary of the Invention

[0008] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a PDC cell carrying a barcode DNA tag library that is easy to track, as well as a preparation method and application thereof, which can realize high-throughput real-time tracking analysis of multiple cell clones.

[0009] To achieve the above object, the technical solution adopted by the present invention is:

[0010] In a first aspect, the present invention provides a method for isolating and culturing primary PDC cells, characterized in that it comprises the following steps:

[0011] S1: Tumor tissue was isolated from the PDX model and digested with papain and neutral protease to obtain digested cells;

[0012] S2: Coat a culture plate with poly-lysine and culture the cells digested in step S1 with DMEM medium containing fetal bovine serum, non-essential amino acids, L-glutamine, transferrin and epidermal growth factor to obtain PDC primary cells.

[0013] The present invention improves the PDC primary cell line culture system, reduces the cell clumping rate, improves the cell viability and adhesion rate, and can sustainably passage to more than 20 generations, thereby increasing the success rate of primary cell culture, facilitating the acquisition of large numbers of tumor cells, and reducing clinical sample loss.

[0014] Furthermore, in step S1, the PDX model is of F2-F3 generation.

[0015] Furthermore, in step S1, 3 to 5 mm 3 Clinical tumor tissue was inoculated subcutaneously into immunodeficient mice, and the tumor tissue mass grew to 800-1000 mm 3 , the F1 generation PDX model was obtained, and the tumor tissue was removed and processed into 3-5 mm 3The tumor mass was inoculated subcutaneously into new immunodeficient mice to obtain the F2 generation PDX model. The above inoculation method was repeated to obtain the F3 generation PDX model.

[0016] Furthermore, in step S1, the digestion time is 30 to 60 minutes. The main disadvantages of the enzymatic digestion method are that the operation steps are cumbersome, it is easy to be contaminated, and the digestion time is difficult to control. If the digestion time is too short, the digestion is incomplete, which is not conducive to the cells crawling out of the adherent culture. If the digestion time is too long, it will cause greater damage to the cells and easily destroy the cell membrane structure.

[0017] Preferably, the digestion time is 60 min, which can reduce the cell clumping rate and improve the cell viability. The cell clumping rate is less than 30% and the cell viability is greater than 70%.

[0018] Furthermore, in step S1, the size of the tumor tissue is 1 to 2 mm 3 .

[0019] Furthermore, in step S1, the concentration of papain is 10-20 U / mL, and the concentration of neutral protease is 1-4 U / mL. Preferably, the concentration of papain is 20 U / mL, and the concentration of neutral protease is 2 U / mL.

[0020] Furthermore, in step S2, the concentration of polylysine is 100-600 mg / mL.

[0021] Preferably, the concentration of polylysine is 200-600 mg / mL.

[0022] Most preferably, the concentration of poly-lysine is 400 mg / mL, which further increases the cell attachment rate to greater than 60% within 7 days.

[0023] Furthermore, in step S2, the concentration of fetal bovine serum is 5-15%, the concentration of non-essential amino acids is 1×-5×, the concentration of L-glutamine is 2-4 mM, the concentration of transferrin is 2-10 mg / mL, and the concentration of epidermal growth factor is 10-50 ng / mL.

[0024] Preferably, the concentration of fetal bovine serum is 15%, the concentration of 1× non-essential amino acids, the concentration of L-glutamine is 4 mM, the concentration of transferrin is 50 mg / mL, and the concentration of epidermal growth factor is 50 ng / mL. This can ensure that primary PDC cells can be continuously passaged for more than 20 generations with high cell viability.

[0025] Furthermore, the non-essential amino acids include L-alanine, L-glutamic acid, L-asparagine, L-aspartic acid, L-proline, L-serine and glycine.

[0026] Furthermore, the non-essential amino acids contain 8.9-44.5 ng / mL L-alanine, 14.7-73.5 ng / mL L-glutamic acid, 13.2-66 ng / mL L-asparagine, 13.3-66.5 ng / mL L-aspartic acid, 11.5-57.5 ng / mL L-proline, 10.5-52.5 ng / mL L-serine and 7.5-37.5 ng / mL glycine.

[0027] Preferably, the 1× non-essential amino acids contain 8.9 ng / mL L-alanine, 14.7 ng / mL L-glutamic acid, 13.2 ng / mL L-asparagine, 13.3 ng / mL L-aspartic acid, 11.5 ng / mL L-proline, 10.5 ng / mL L-serine and 7.5 ng / mL glycine.

[0028] In a second aspect, the present invention provides primary PDC cells obtained by the isolation and culture method.

[0029] In a third aspect, the present invention provides a recombinant PDC cell, wherein the recombinant PDC cell contains a barcode DNA tag library, and the PDC cell is a tumor cell isolated and cultured from a tumor tissue of a PDX model.

[0030] Furthermore, the PDC cells are primary PDC cells obtained by the isolation and culture method.

[0031] Furthermore, the recognition sequence portion of the barcode DNA tag library is a library composed of a 24bp random DNA sequence NNNNNNCTCNNNNNNGTCNNNNNN, where N is selected from any base of A (adenine), T (thymine), C (cytosine), and G (guanine). The present invention introduces barcode DNA tags into PDC cells, which can be detected by targeted sequencing of the barcode DNA tag sequence in the cell genomic DNA or by single-cell RNA sequencing, making it easy to distinguish and track different clonal groups derived from PDX retained during the construction of the PDC cell line.

[0032] In a fourth aspect, the present invention provides a method for preparing the recombinant PDC cells, comprising the following steps:

[0033] S11: Construction of a recombinant vector containing a barcoded DNA tag;

[0034] S12: mixing the recombinant vector and the lentiviral packaging plasmid described in step S11, transfecting the carrier cells, and isolating the lentiviral library carrying the barcoded DNA tag library;

[0035] S13: Transfecting PDC cells with the lentiviral library carrying the barcoded DNA tag described in step S12, adjusting the MOI of viral transfection to achieve a transfection efficiency of 1% to 10%, to obtain the recombinant PDC cells. This ensures that each PDC cell is transfected with at most one lentiviral particle, thereby carrying only a single barcoded DNA tag.

[0036] Furthermore, in step S11, the backbone vector used to construct the recombinant vector containing the barcode DNA tag can be a conventional commercially available vector that can normally transcribe and translate genes and package viruses, and the barcode DNA tag can be inserted into the PDC cell genome.

[0037] Furthermore, the lentiviral packaging plasmids are PSPAX2 plasmid and PMD2.G plasmid.

[0038] Furthermore, the mass ratio of the recombinant vector, PSPAX2 plasmid and PMD2.G plasmid is: recombinant vector, PSPAX2 plasmid and PMD2.G plasmid = 3-5:2-3:1.5-2.

[0039] Furthermore, the ratio of the recombinant vector, PSPAX2 plasmid and PMD2.G plasmid is 4:3:2.

[0040] Furthermore, in step S12, the transfection time is 48 to 96 hours.

[0041] Furthermore, in step S12, the culture supernatants are collected at 48h, 72h, and 96h after transfection and mixed to obtain a lentiviral library carrying a barcoded DNA tag library.

[0042] Furthermore, in step S2, the transfection reagent used is QuickShuttle-293.

[0043] Furthermore, the ratio of the QuickShuttle-293, recombinant vector, PSPAX2 plasmid and PMD2.G plasmid is: QuickShuttle-293: recombinant vector: PSPAX2 plasmid: PMD2.G plasmid = 10-20 μL: 3-5 μg: 2-3 μg: 1.5-2 μg.

[0044] Furthermore, QuickShuttle-293: recombinant vector: PSPAX2 plasmid: PMD2.G plasmid = 10 μL: 4 μg: 3 μg: 2 μg.

[0045] Furthermore, in step S2, the vector cell is a HEK29T cell.

[0046] Furthermore, in step S3, drug screening is performed using blasticidin after transfection.

[0047] In a fifth aspect, the present invention provides a method for analyzing tumor cell heterogeneity and cell tracking, and sequencing and analyzing the barcode DNA tags and gene coding region mutations and expression profiles of the recombinant PDC cells.

[0048] Furthermore, the sequencing analysis includes single-cell sequencing cluster analysis, coding region mutation type and proportion analysis.

[0049] Furthermore, the barcoded DNA tags and gene expression profiles of each recombinant PDC cell were obtained through single-cell sequencing, and PDC cells with similar gene expression profiles were clustered to obtain cell clusters.

[0050] Furthermore, the single-base mutation information of the PDC cells' genes was obtained by exon sequencing.

[0051] Furthermore, the tumor includes pancreatic cancer or lung cancer.

[0052] Compared with the prior art, the present invention has the following beneficial effects:

[0053] (1) The present invention provides a method for constructing recombinant PDC cells containing barcoded DNA tags, which improves the PDC primary cell line culture system, reduces the cell clumping rate, improves the cell viability and adhesion rate, and can be sustainably passaged for more than 20 generations, thereby improving the success rate of primary cell culture and increasing the representativeness of the PDC cell bank to the clinical tumor sample library.

[0054] (2) The PDC primary cells of the present invention have strong heterogeneity. Compared with conventional tumor cell lines, they more faithfully retain the heterogeneity of clinical tumor samples in terms of mutation types and tumor occurrence and development mechanisms, which is more conducive to the study of differentiated gene mechanisms related to tumor progression and drug response.

[0055] (3) The present invention introduces a barcode DNA tag library into the PDC cells, which can be detected by targeted sequencing and single-cell RNA sequencing of the barcode DNA tag sequence in the cell genomic DNA (the tag sequence will be accompanied by marker gene transcription), thereby facilitating the differentiation and high-throughput tracking of different clonal groups retained from the PDC cell line during the PDX construction process. Since the PDC cell line introduced with the barcode DNA library sequence can distinguish and simultaneously track different clonal groups, it is more convenient to conduct research on the gene mechanism related to tumor progression and drug response. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1 The graph below shows the changes in cell aggregation rate and cell viability under different enzyme dissociation systems. 3 Agglomeration rate of tumor tissue blocks under different treatment conditions; B is <1 mm 3Cell viability of tumor tissue under different treatment conditions; C is 1-2 mm 3 Agglomeration rate of tumor tissue blocks under different treatment conditions; D is 1-2 mm 3 Cell viability of tumor tissue explants under different treatment conditions.

[0057] Figure 2 Figure 2 shows the changes in cell adhesion efficiency over 7 days under different culture systems. Figure A shows the effects of different additives on cell adhesion efficiency when the basal medium is DMEM with 15% FBS and the culture plates are coated with different concentrations of poly-lysine. Figure B shows the effects of different additives on cell adhesion efficiency when the basal medium is RPMI 1640 with 15% FBS and the culture plates are coated with different concentrations of poly-lysine. Figure C shows the effects of different additives on cell adhesion efficiency when the basal medium is DMEM with 15% FBS and the culture plates are coated with different concentrations of gelatin. Figure D shows the effects of different additives on cell adhesion efficiency when the basal medium is RPMI 1640 with 15% FBS and the culture plates are coated with different concentrations of gelatin.

[0058] Figure 3 The graph shows the changes in cell passage number under the culture system where the cell attachment rate exceeds 45% after 7 days.

[0059] Figure 4 This is the pDEST-EF1-EGFP-BSD backbone plasmid.

[0060] Figure 5 The pdest is a plasmid carrying a barcoded DNA tag (pdest-ef1-egfp-BSD-barcode).

[0061] Figure 6 This is a flow chart of the present invention for isolating and culturing primary PDC cells, constructing PDC cells with introduced barcode DNA tags, and performing sequencing analysis.

[0062] Figure 7 This is a cluster analysis diagram of single-cell sequencing of pancreatic cancer PDC cells with introduced barcoded DNA tags.

[0063] Figure 8 This is a cluster analysis diagram of single-cell sequencing of lung cancer PDC cells with introduced barcoded DNA tags.

[0064] Figure 9 This is a single-cell sequencing cluster analysis diagram of the conventional pancreatic cancer cell line PANC-1.

[0065] Figure 10 This is a single-cell sequencing cluster analysis diagram of the common lung cancer cell line NCI-H520.

[0066] Figure 11This figure shows whole-exome sequencing data analysis for the conventional pancreatic cancer cell line PANC-1, pancreatic cancer PDC cells with barcoded DNA tags, and pancreatic cancer clinical samples from the TCGA database. A represents the single-base mutation rate; B represents the mutation type; and C represents the percentage analysis.

[0067] Figure 12 This figure shows whole-exome sequencing data analysis for the conventional lung cancer cell line NCI-H520, lung cancer PDC cells with barcoded DNA tags, and lung cancer clinical samples from the TCGA database. A represents the single-base mutation rate; B represents the mutation type; and C represents the percentage analysis chart. DETAILED DESCRIPTION

[0068] To better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below with reference to specific examples. Other materials, reagents, etc. used in the examples, unless otherwise specified, can be obtained from commercial sources.

[0069] Example 1 Construction of PDC primary cells

[0070] There are many limitations to directly culturing PDCs from clinical samples. First, the amount of clinical tumor samples is generally very small, and only one or two primary cultures can be performed. If the primary culture fails, the tumor sample will be lost. Second, it takes a long time for clinical tumor samples to be removed from the body (in the hospital) and processed for primary culture (in the animal laboratory), and the activity of tumor cells will decrease, affecting the success rate of primary culture. Third, clinical tumor samples are not easy to obtain, and it is difficult to optimize batch primary cell culture. In order to improve the success rate of primary cell culture establishment, the primary cell culture system has been improved:

[0071] First, the clinical tumor samples were cut into 3-5 mm 3 The tumor mass was inoculated into the subcutaneous tissue of immunodeficient mice. 3 ), fresh tumor tissue blocks were removed from tumor-bearing mice to obtain F1 generation PDX, which were cut into 3-5 mm 3 The tumor mass is partially preserved in liquid nitrogen (labeled as F1 generation), and the remaining tissue mass is inoculated subcutaneously into new immunodeficient mice to obtain F2 generation PDX. This inoculation and preservation operation is repeated to obtain F3 generation PDX, and so on to obtain higher generation PDX. In this way, a large number of PDX samples derived from clinical tumor samples can be obtained.

[0072] Will grow to 800mm 3Fresh tumor tissue blocks of F3 generation PDX (early generation should be used as much as possible) were removed from tumor-bearing mice and washed with sterile PBS (phosphate buffered saline) solution and DMEM medium (a medium containing various amino acids and glucose). The tumor tissue blocks were first cut into small pieces (1-2 mm 3 ), and then digested with 20 U / mL papain and 2 U / mL neutral protease at 37°C for 60 min to obtain digested cells.

[0073] Culture plates were coated with 400 ng / mL poly-lysine and the digested cells were cultured at 37°C in DMEM supplemented with 15% (v / v) FBS (fetal bovine serum), 1× NEAA (non-essential amino acids), 4 mM L-glutamine, 50 mg / L transferrin, and 50 ng / mL EGF (epidermal growth factor) to obtain primary PDC cells. The 1× non-essential amino acids contained 8.9 ng / mL L-alanine, 14.7 ng / mL L-glutamic acid, 13.2 ng / mL L-asparagine, 13.3 ng / mL L-aspartic acid, 11.5 ng / mL L-proline, 10.5 ng / mL L-serine, and 7.5 ng / mL glycine.

[0074] Example 2 Effects of different culture methods on primary PDC cells

[0075] 1. Experimental Methods

[0076] 1. In Example 1, the size of the F3 generation PDX tumor tissue block is <1 mm 3 or 1-2 mm 3 The digestion time was 30 min or 60 min, respectively. The digestion reagents were: (1) 0.25% (w / v) pancreatic enzyme + 0.02% (w / v) EDTA (ethylenediaminetetraacetic acid); (2) 0.25% (w / v) pancreatic enzyme + 0.02% (w / v) EDTA + 20 μg / mL DNA enzyme; (3) 0.25% (w / v) pancreatic enzyme + 2 mg / mL collagenase I; (4) 0.25% (w / v) pancreatic enzyme + 2 mg / mL collagenase II; (3) 0.25% (w / v) pancreatic enzyme + 2 mg / mL collagenase III; (4) 0.25% (w / v) pancreatic enzyme + 2 mg / mL collagenase IV; (5) 0.25% (w / v) pancreatic enzyme + 2 mg / mL collagenase V; (6) 20 U / mL papain + 2 U / mL neutral protease. The viability and clumping rate of the digested cells were detected.

[0077] 2. In Example 1, the reagents used to coat the culture plate are polylysine at different concentrations (100 ng / mL, 200 ng / mL, 400 ng / mL, and 600 ng / mL) or gelatin at different concentrations (0.2% w / v, 0.4% w / v, and 0.8% w / v), the basal culture medium is DMEM medium containing 15% (v / v) FBS or RPMI 1640 medium containing 15% (v / v) FBS, and the following components are added to the culture medium: (1) 1×NEAA+2 mM L-glutamine; (2) 1×NEAA+3 mM L-glutamine; (3) 1×NEAA+4 mM L-glutamine; (4) 1×NEAA+4 mM L-glutamine+50 mg / L transferrin; (5) 1×NEAA+4 mM L-glutamine+50 mg / L transferrin+50 ng / mL EGF. The attachment rate of cells cultured for 7 days was detected.

[0078] 3. In Example 1, the culture plate was coated with different concentrations of poly-lysine (400 ng / mL and 600 ng / mL), and the culture medium was (1) DMEM supplemented with 1×NEAA, 4 mM L-glutamine, 50 mg / L transferrin, and 15% (v / v) FBS; and (2) RPMI 1640 supplemented with 1×NEAA, 4 mM L-glutamine, 50 mg / L transferrin, 50 ng / mg EGF, and 15% (v / v) FBS. Cell passage was tested.

[0079] 2. Experimental Results

[0080] 1. If Figure 1 A to Figure 1 As shown in D, 1 to 2 mm 3 The best cell clumping rate and cell viability were obtained after the tumor tissue blocks were treated with 20 U / mL papain and 2 U / mL neutral protease at 37°C for 60 minutes, that is, the cell clumping rate was less than 30% and the cell viability was greater than 70%.

[0081] 2. If Figure 2 A to Figure 2 As shown in Figure D, under the same culture conditions, the cell attachment rate is higher when the culture plate is coated with poly-lysine than when it is coated with gelatin. The cell attachment rate is even higher after the addition of EGF.

[0082] 3. If Figure 3 As shown, culture plates were coated with 400 ng / mL poly-lysine and cells were cultured in DMEM medium supplemented with 1×NEAA, 4 mM L-glutamine, 50 mg / L transferrin, 50 ng / mL EGF and 15% (v / v) FBS. The cells had a high attachment rate and could be continuously passaged for 20 generations with a high survival rate.

[0083] Example 3 Construction of PDC cells with introduced barcoded DNA tags

[0084] 1. Construction of shuttle plasmid carrying barcoded DNA tags

[0085] The nucleotide sequence of the random barcode DNA tag was synthesized to obtain the barcode DNA tag library, and the library was cloned into the backbone plasmid containing the barcode DNA tag insertion site (pdest-ef1-egfp-BSD, see the map). Figure 4 ) was used to construct a plasmid library carrying a barcoded DNA tag (pdest-ef1-egfp-BSD-barcode, see the map for details). Figure 5 In the backbone plasmid, the 5' restriction site of the insertion site is EcoRI, and the 3' restriction site is BamHI, located between primer-F and primer-R. Each plasmid carries a barcoded DNA tag. Table 1 shows some examples of nucleotide sequences for barcoded DNA tag libraries. The barcoded DNA tag is a 24-bp random DNA sequence: NNNNNNCTCNNNNNNGTCNNNNNN, where N is selected from any base of A (adenine), T (thymine), C (cytosine), and G (guanine). Tens of thousands of barcoded DNA tags can be combined.

[0086] Table 1 Examples of barcoded DNA tag library sequences (partial display)

[0087]

[0088] 2. Preparation of a Lentivirus Library Carrying Barcoded DNA Tags

[0089] 2×10 cells were seeded in a 10 cm cell culture dish. 6HEK293T cells were cultured in a high-glucose DMEM medium with 10% FBS at 37°C under 5% CO2. When the cell density reached 90%, two 1.5 mL centrifuge tubes were prepared and labeled as tube A and tube B. 200 μL of high-glucose DMEM medium without FBS was added to each tube. 20 μL of transfection reagent (QuickShuttle-293, a special transfection reagent for 293T cells) was added to tube A. 8 μg of a plasmid library carrying a barcoded DNA tag (pdest-ef1-egfp-BSD-barcode, 6 μg of PSPAX2 plasmid, and 4 μg of PMD2.G plasmid (pdest-ef1-egfp-BSD-barcode, PSPAX2 plasmid, and PMD2.G in a 4:3:2 mass ratio) was mixed from tubes A and B to obtain a mixture, which was then dripped onto a 10 cm dish of HEK293T cells for transfection. Six hours after transfection, the culture medium was changed to complete medium containing 10% (v / v) FBS. 48 hours after transfection, the supernatant was collected for the first time and supplemented with complete medium containing 10% (v / v) FBS for an additional 24 hours (72 hours after transfection). The supernatant was collected for the second time and supplemented with complete medium containing 10% (v / v) FBS for an additional 24 hours (96 hours after transfection). The supernatants from these three collections were combined and concentrated to generate a lentiviral library carrying barcoded DNA tags. The lentiviral titer and homogeneity of the barcoded tagged library were determined.

[0090] 3. Construction of PDC cells with introduced barcoded DNA tags

[0091] (1) MOI (multiplicity of infection) setting and viral library transfection

[0092] The PDC primary cells of Example 1 were plated in a 6 cm dish (1×10 5 cells / dish), added lentiviral libraries carrying barcoded DNA tags and transfection reagents at different MOIs for transfection, and fluorescent PDC cells were sorted by fluorescence flow cytometry. The transfection efficiency of the virus was calculated to obtain the appropriate MOI so that 1% to 10% of the PDC cells were transfected with the virus and carried the fluorescent label.

[0093] (2) Screening of PDC cells with barcoded DNA tags

[0094] The PDC primary cells of Example 1 were plated in a 10 cm culture dish (5×10 6cells / dish), adding a lentiviral library carrying a barcoded DNA tag and a transfection reagent for transfection, adjusting and confirming the MOI of transfection according to step (1), and using blasticidin (BSD) for drug screening after transfection to kill PDC cells that are not infected by the lentivirus carrying the barcoded DNA tag, collecting the surviving PDC cells infected with the lentivirus carrying the barcoded DNA tag in the culture dish, and after subculturing, using single-cell sequencing to detect and identify the barcoded DNA tag information carried by them. The PDC cells only carry a unique barcoded DNA tag. The PDC cells that have successfully introduced the barcoded DNA tag are used for subsequent experiments and cryopreservation.

[0095] Example 4 Sequencing analysis of PDC cells with introduced barcode DNA tags

[0096] 1. Cluster analysis using single-cell sequencing

[0097] PDC cells with barcoded DNA tags were first inoculated and cultured. PDC cells in the logarithmic growth phase were then prepared into a single cell suspension for single-cell sequencing. Single-cell transcriptome sequencing data were obtained using the 10× Genomics single-cell sequencing platform. The single-cell transcriptome sequencing data of the PDC model were aligned with the human genome library using cellranger software to obtain a human cell-gene expression profile matrix. The R package Seurat (version 5) was used for data processing and visualization. For the single-cell transcriptome sequencing data of the PDC model, cells with a low number of detected genes (<200), cells with a high number of detected UMIs (>100,000), and cells with a high proportion of UMI counts attributed to mitochondrial genes (>20%) were deleted. The filtered expression matrix was then normalized and scaled to eliminate unnecessary sources of variation driven by the number of detected UMIs and mitochondrial gene expression. Harmony was used to remove batch effects, and then RunTSNE was used for linear dimensionality reduction. Then, FindClusters was used for clustering, and finally DimPlot was used to visualize the tsne graph.

[0098] 2. Whole exome sequencing for mutation type analysis

[0099] The PDC cells with introduced barcoded DNA tags were first inoculated and cultured. The PDC cells with introduced barcoded DNA tags in the logarithmic growth phase were taken, and genomic DNA was extracted. A library was constructed using Agilent V6 probes, and then whole-exome sequencing data was obtained based on Illumina sequencing.

[0100] Figure 6The complete flow chart of the present invention is shown, from isolating and culturing primary PDC cells, to constructing PDC cells with introduced barcode DNA tags, and performing sequencing analysis.

[0101] Example 5 Analysis of the heterogeneity and barcode tracking characteristics of pancreatic cancer PDC cells introduced with barcode DNA tags

[0102] According to the methods of Examples 1 and 3, pancreatic cancer PDC cells with introduced barcode DNA tags were obtained, and single-cell sequencing was performed on them according to the method of Example 4. Based on the single-cell sequencing data, 12 different clusters (clonal groups) were distinguished by cluster analysis, see Figure 7 .

[0103] Example 6 Analysis of the heterogeneity and barcode tracking characteristics of lung cancer PDC cells introduced with barcode DNA tags

[0104] According to the methods of Examples 1 and 3, lung cancer PDC cells with barcoded DNA tags were obtained, and single-cell sequencing and whole-genome sequencing were performed on them according to the method of Example 4. Based on the single-cell sequencing data, eight different clusters were distinguished by cluster analysis, as shown in FIG. Figure 8 .

[0105] Comparative Example 1 Single-cell sequencing analysis of conventional human pancreatic cancer cell line PANC-1

[0106] Single-cell sequencing was performed on the conventional human pancreatic cancer cell line PANC-1 according to the method of Example 4. Cluster analysis based on the single-cell sequencing data revealed low heterogeneity, with only three different clusters (clonal groups) being identified ( Figure 9 ), which is lower than the number of clusters obtained by pancreatic cancer PDC cells introduced with barcoded DNA tags.

[0107] Comparative Example 2 Single-cell sequencing analysis of conventional human lung cancer cell line NCI-H520

[0108] Single-cell sequencing was performed on the conventional human lung cancer cell line NCI-H520 according to the method of Example 4. Cluster analysis based on the single-cell sequencing data found that the heterogeneity was low and only 4 different clusters (clonal groups) could be distinguished ( Figure 10 ), which is lower than the number of clusters obtained by introducing barcoded DNA tags into lung cancer PDC cells.

[0109] Comparative Example 3: Whole exome sequencing was performed on 10 pancreatic cancer PDC cells and 10 conventional pancreatic cancer cell lines PANC-1.

[0110] According to the method of Example 4, whole exome sequencing was performed on 10 pancreatic cancer PDC cells obtained in Example 1 and 10 conventional pancreatic cancer cell lines PANC-1. Based on their whole exome sequencing data, analysis was performed in combination with the whole exome sequencing data of clinical samples (pancreatic cancer) in the TCGA database.

[0111] Whole-exome sequencing of conventional pancreatic cancer cell lines PANC-1 and PDC cells was performed on an Illumina HiSeq 2500 sequencer using 125-bp paired-end reads and an Agilent v4_XT_51Mb capture kit to achieve 100x average coverage. Raw reads from each cell model were aligned to the human and mouse concatenated genomes (GRC37 / hg19+GRCm38 / mm10) using BWA alignment software, and reads that did not align to human contigs were filtered. Local realignment around indels and recalibration of base quality scores were performed using the GATK pipeline. SNP analysis of cell line samples was performed using multiple callers (muTect, strelka). SNPs were filtered using quality thresholds (quality score >20, read depth >10), and SNPs found in germline databases (HapMap, 1000G) were discarded. In order to analyze the mutation rate and chromosomal abnormalities between conventional pancreatic cancer cell lines PANC-1, PDC cells and clinical samples on the TCGA database, MutSigC and GISTIC2 were used to analyze the most significantly mutated genes, significantly amplified and deleted regions identified in the TCGA database, and a comparative analysis was performed. Variants were called by Mutect caller. SNPs were annotated using SnpEff and the Ensembl annotation file for the human genome (GRCh37). Based on the annotation result file, the average number of mutations per million bases in conventional pancreatic cancer cell lines PANC-1, pancreatic cancer PDC cells, and clinical samples on the TCGA database were counted to represent the background mutation frequency of the samples. The proportion of each mutation type within the group was counted to observe the trend of changes in mutation types between sample groups. The number of SNP types detected in common between different sample groups was calculated, and the results were visualized using a Venn diagram ( Figure 11 A to Figure 11 C) Pancreatic cancer PDCs retain more mutation types and related tumorigenesis and development mechanisms, which is more conducive to the study of the genetic mechanisms related to tumor progression and drug response.

[0112] Comparative Example 4: Whole exome sequencing of 10 lung cancer PDC cells and 10 conventional lung cancer cell lines NCI-H520

[0113] According to the method of Comparative Example 3, 10 lung cancer PDC cells obtained in the embodiment and 10 conventional lung cancer cell lines NCI-H520 were subjected to whole exome sequencing. Based on the whole exome sequencing data, combined with the whole exome sequencing data of clinical samples (lung cancer) in the TCGA database, analysis was performed. The same analysis method as in Comparative Example 3 was used to count the frequencies of single nucleotide mutations in the conventional lung cancer cell line NCI-H520, lung cancer PDC cells and the TCGA database, as well as the proportion of each mutation type. Finally, the intersection was taken to obtain the common mutation ( Figure 12 A to Figure 12 C) Lung cancer PDCs retain more mutation types and related tumorigenesis and development mechanisms, making them more conducive to distinguishing between the genetic mechanisms related to tumor progression and drug response.

[0114] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A method for isolating and culturing primary PDC cells, characterized in that: The following steps are involved: S1: Tumor tissue was isolated from the PDX model and digested with papain and neutral protease to obtain digested cells; S2: Coat a culture plate with poly-lysine and culture the cells digested in step S1 with DMEM medium containing fetal bovine serum, non-essential amino acids, L-glutamine, transferrin and epidermal growth factor to obtain PDC primary cells.

2. The separation and culture method according to claim 1, wherein In step S1, the digestion time is 30 to 60 minutes.

3. The separation and culture method according to claim 1, wherein In step S1, the size of the tumor tissue is 1 to 2 mm 3 .

4. The separation and culture method according to claim 1, wherein In step S2, the concentration of polylysine is 100-600 mg / mL.

5. Primary PDC cells obtained by the isolation and culture method according to any one of claims 1 to 4.

6. A recombinant PDC cell, characterized in that: The recombinant PDC cells contain a barcode DNA tag library, and the PDC cells are tumor cells isolated and cultured from tumor tissues of a PDX model.

7. The recombinant PDC cell according to claim 6, characterized in that The PDC cells are the primary PDC cells according to claim 5.

8. The method for preparing the recombinant PDC cells according to claim 6 or 7, characterized in that: The following steps are involved: S11: Construction of a recombinant vector containing a barcoded DNA tag library; S12: mixing the recombinant vector and the lentiviral packaging plasmid described in step S11, transfecting the carrier cells, and isolating the lentiviral library carrying the barcoded DNA tag library; S13: Transfecting PDC cells with the lentiviral library carrying the barcode DNA tag described in step S12, adjusting the MOI of viral transfection so that the transfection efficiency is 1% to 10%, and obtaining the recombinant PDC cells.

9. A method for analyzing tumor cell heterogeneity and cell tracking, characterized in that: The barcode DNA tags, gene coding region mutations and expression profiles of the recombinant PDC cells according to claim 6 or 7 are sequenced and analyzed.

10. The method according to claim 9, wherein The sequencing analysis includes single-cell sequencing cluster analysis, coding region mutation type and proportion analysis.

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