Pdc cells carrying a barcode dna tag library convenient for tracking, and a preparation method and application thereof

Abstract

The present application relates to a kind of PDC cell carrying bar code DNA label of convenient tracking and its preparation method and application, belong to cell construction technical field.The present application provides a kind of PDC primary cell separation culture method, and provides a kind of method for constructing recombination PDC cell containing bar code DNA label.The PDC primary cell culture system of the present application reduces the cell clumping rate, improves cell survival rate and adhesion rate, and can be continuously passaged to more than 20 generations.PDC primary cell more faithfully retains the heterogeneity of clinical tumor sample in mutation type and tumor development mechanism, etc.In the bar code DNA label introduced in PDC cell, it can be detected by the targeted sequencing and single cell RNA sequencing of bar code DNA label sequence in cell genome DNA, so as to conveniently distinguish and high-throughput track different clone groups retained by PDX PDC cell line.

Description

Technical Field

[0001] This invention relates to the field of cell construction technology, and in particular to a PDC cell carrying a library of easily traceable barcode DNA tags, its preparation method, and its application. Background Technology

[0002] Current conventional tumor research models mainly consist of various human or murine tumor cell lines, and tumor-bearing mouse models constructed by injecting these tumor cell lines subcutaneously or orally into mice. If human tumor cell lines are used, they are generally called CDX (cell line derived xenograft) models. In the last decade or so, PDX (patient-derived xenograft) mouse models, formed by directly transplanting patient-derived tumors into immunodeficient mice, have also been increasingly widely used in tumor research.

[0003] Most conventional tumor cell lines were constructed decades ago, undergoing countless in vitro passages and transfers between different laboratories. Many of their characteristics have changed significantly since their initial construction. In particular, some cell lines have been misidentified or cross-contaminated during past storage and use, casting doubt on their true identity.

[0004] PDX models, being relatively new and having undergone only a limited number of passages in vivo, better preserve the characteristics of patient tumors, especially tumor heterogeneity, i.e., different clonal populations of tumors. The evolution of these clonal populations and their differences in sensitivity to drug therapy are important factors determining tumor progression and treatment efficacy. However, PDX models (and CDX models), because they use tumor-bearing mice for experiments, have long experimental cycles, high costs, and are more difficult to implement in vitro compared to in vitro cell line culture for gene modification and gene expression intervention.

[0005] In recent years, PDX-derived cell lines (PDCs) have been reconstructed using PDXs with accurate and complete clinical information. Compared to conventional tumor cell lines, PDCs have many characteristics that are more similar to patient tumors, and their response to drug treatment and clinical efficacy are more relevant. PDC cell lines have advantages such as accurate and complete clinical information, recent construction time, and fewer passages, resulting in better clinical relevance. Furthermore, with the continuously growing number of PDX models, large-scale PDC cell banks can be constructed to increase the representativeness of tumor cases. PDCs can be easily used for gene modification, gene expression intervention, and high-throughput drug screening. PDCs can also be used in conjunction with PDX models (or by re-injecting PDCs subcutaneously into mice or constructing tumor-bearing mouse models in situ) to conduct complete in vitro-in vivo efficacy studies.

[0006] However, existing techniques for culturing primary PDC cells result in low cell viability, demanding experimental conditions, and potential contamination by other cells. Furthermore, the microenvironment for tumor cells differs between in vivo and in vitro; unsuitable culture methods can lead to the loss of some cell clones, altering cell expression products and characteristics, which is detrimental to subsequent research. The success rate of primary cell culture varies significantly across different pathological types and individual tumor cells, limiting the widespread application of primary cell culture and thus restricting tumor research.

[0007] Furthermore, PDC retains the heterogeneity and complexity of clonal populations in PDX, resulting in different pathways and mechanisms related to tumor development and drug response being mixed together, making it difficult to distinguish them in research, and also making it difficult to analyze and predict the clonal population evolution of patient tumors using PDC or PDX models. Summary of the Invention

[0008] The purpose of this 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 its preparation method and application, which can realize high-throughput real-time tracking and analysis of multiple cell clones.

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

[0010] In a first aspect, the present invention provides a method for isolating and culturing primary PDC cells, characterized by comprising 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 culture plates with poly-L-lysine and culture cells digested in step S1 in DMEM medium containing fetal bovine serum, non-essential amino acids, L-glutamine, transferrin and epidermal growth factor to obtain PDC primary cells.

[0013] This invention improves the PDC primary cell culture system, reducing cell clumping rate, increasing cell viability and adhesion rate, allowing for continuous passage to more than 20 generations, improving the success rate of primary cell culture, facilitating the acquisition of large quantities of tumor cells, and reducing clinical sample loss.

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

[0015] Furthermore, in step S1, 3-5mm 3 Clinical tumor tissue was subcutaneously injected into immunodeficient mice, and the tumor tissue mass grew to 800–1000 mm. 3 An F1 generation PDX model was obtained, and tumor tissue blocks were extracted and processed to a size of 3–5 mm. 3The tumor fragments were 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 30–60 minutes. The main disadvantages of enzymatic digestion are the cumbersome operation steps, susceptibility to contamination, and difficulty in controlling the digestion time. If the digestion time is too short, the digestion will be incomplete, which is not conducive to cell migration and adherence to the culture vessel. If the digestion time is too long, it will cause greater damage to the cells and easily disrupt the cell membrane structure.

[0017] Preferably, the digestion time is 60 minutes, which can reduce the cell clumping rate and increase the cell viability, with a cell clumping rate of <30% and a viability of >70%.

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

[0019] Further, 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-L-lysine is 400 mg / mL, which further improves the cell adhesion rate within 7 days to greater than 60%.

[0023] Further, 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 is 4 mM, the concentration of L-glutamine is 50 mg / mL, the concentration of transferrin is 50 ng / mL, and the concentration of epidermal growth factor is 50 ng / mL. This ensures continuous passage of primary PDC cells, exceeding 20 passages, 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 acid contains 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] Secondly, the present invention provides primary PDC cells obtained by the isolation and culture method described above.

[0029] Thirdly, the present invention provides a recombinant PDC cell containing a barcode DNA tag library, wherein the PDC cell is a tumor cell isolated and cultured from 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 one of the bases A (adenine), T (thymine), C (cytosine), and G (guanine). This invention, by introducing barcode DNA tags into PDC cells, can be detected both through targeted sequencing of the barcode DNA tag sequence in the cell's genomic DNA and through single-cell RNA sequencing. This facilitates the differentiation and tracking of different clonal populations derived from PDX retained during the construction of PDC cell lines.

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

[0033] S11: Construct a recombinant vector containing a barcode DNA tag;

[0034] S12: Mix the recombinant vector and lentiviral packaging plasmid described in step S11, transfect the vector cells, and then separate the lentiviral library carrying the barcode DNA tag library.

[0035] S13: Transfect PDC cells with the lentiviral library carrying the barcode DNA tag described in step S12, and adjust the MOI of viral transfection to achieve a transfection efficiency of 1% to 10%, thereby obtaining the recombinant PDC cells. This ensures that each PDC cell is transfected with at most one lentiviral particle, thus carrying only a single barcode 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 can package viruses. The barcode DNA tag can then be inserted into the PDC cell genome.

[0037] Furthermore, the lentivirus 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 recombinant vector, PSPAX2 plasmid, and PMD2.G plasmid is 4:3:2.

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

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

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

[0043] Furthermore, the ratio of QuickShuttle-293, recombinant vector, PSPAX2 plasmid, and PMD2.G plasmid is as follows: 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 carrier cell is a HEK29T cell.

[0046] Furthermore, in step S3, blast fungicide is used for drug screening after transfection.

[0047] Fifthly, the present invention provides a method for analyzing tumor cell heterogeneity and cell tracking, which involves sequencing and analyzing the barcode DNA tags, 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, barcode DNA tags and gene expression profiles of each recombinant PDC cell were obtained through single-cell sequencing. PDC cells with similar gene expression profiles were clustered to obtain cell clusters.

[0050] Furthermore, exon sequencing was used to obtain single-base mutation information of PDC cells.

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

[0052] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0053] (1) This invention provides a method for constructing recombinant PDC cells containing barcode DNA tags, which improves the PDC primary cell culture system, reduces cell clumping rate, increases cell viability and adhesion rate, can be passaged continuously for more than 20 generations, improves the success rate of primary cell culture, and increases the representativeness of the PDC cell bank to the clinical tumor sample bank.

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

[0055] (3) This invention introduces a barcoded DNA tag library into the PDC cells, which can be detected by targeted sequencing and single-cell RNA sequencing (the tag sequence is accompanied by the transcription of the marker gene) of the barcoded DNA tag sequence in the cell's genomic DNA. This facilitates the differentiation and high-throughput tracking of different clonal populations retained from the PDX construction process in the PDC cell line. Since the PDC cell line with the introduced barcoded DNA library sequence can differentiate and track different clonal populations simultaneously, it is more convenient to conduct research on gene mechanisms related to tumor progression and drug response. Attached Figure Description

[0056] Figure 1 This graph shows the changes in cell aggregation rate and cell viability under different enzyme dissociation systems. A represents cells <1 mm. 3 The agglomeration rate of tumor tissue masses under different treatment conditions; B is <1mm. 3Cell viability of tumor tissue blocks under different treatment conditions; C = 1–2 mm 3 The agglomeration rate of tumor tissue masses under different treatment conditions; D is 1-2 mm. 3 Cell viability of tumor tissue blocks under different treatment conditions.

[0057] Figure 2 The graphs show the changes in cell adhesion rate over 7 days under different culture systems. A represents the effect of different added components on cell adhesion rate when the culture plate is coated with different concentrations of poly-L-lysine using DMEM medium containing 15% FBS as the basal medium; B represents the effect of different added components on cell adhesion rate when the culture plate is coated with different concentrations of poly-L-lysine using RPMI 1640 medium containing 15% FBS as the basal medium; C represents the effect of different added components on cell adhesion rate when the culture plate is coated with different concentrations of gelatin using DMEM medium containing 15% FBS as the basal medium; and D represents the effect of different added components on cell adhesion rate when the culture plate is coated with different concentrations of gelatin using RPMI 1640 medium containing 15% FBS as the basal medium.

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

[0059] Figure 4 The backbone plasmid is pdest-ef1-egfp-BSD.

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

[0061] Figure 6 This is a flowchart illustrating the process of isolating and culturing primary PDC cells, constructing PDC cells with introduced barcode DNA tags, and performing sequencing analysis, as described in this invention.

[0062] Figure 7 Single-cell sequencing cluster analysis diagram of pancreatic cancer PDC cells with introduced barcode DNA tags.

[0063] Figure 8 Single-cell sequencing clustering analysis diagram of lung cancer PDC cells with introduced barcode 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 conventional lung cancer cell line NCI-H520.

[0066] Figure 11Whole-exome sequencing data of conventional pancreatic cancer cell line PANC-1, pancreatic cancer PDC cells with barcoded DNA tags, and clinical pancreatic cancer samples from the TCGA database were analyzed. In the figures, A represents the single nucleotide polymorphism (SNP) mutation rate; B represents the mutation type; and C is a percentage analysis graph.

[0067] Figure 12 Whole-exome sequencing data of conventional lung cancer cell line NCI-H520, lung cancer PDC cells with barcoded DNA tags, and clinical lung cancer samples from the TCGA database were analyzed. In the figures, A represents the single nucleotide mutation rate; B represents the mutation type; and C is a percentage analysis graph. Detailed Implementation

[0068] To better illustrate the purpose, technical solution, and advantages of this invention, the invention will be further described below with reference to specific embodiments. Unless otherwise specified, other materials and reagents used in the embodiments are commercially available.

[0069] Example 1: Construction of PDC primary cells

[0070] Direct primary culture of PDCs from clinical samples has several limitations. First, the quantity of clinical tumor samples is generally small, allowing only one or two primary culture cycles; if the primary culture fails, the entire tumor sample is lost. Second, the time from surgical extubation (hospital) to primary culture (animal laboratory) of clinical tumor samples is relatively long, reducing tumor cell viability and affecting the success rate of primary culture. Third, clinical tumor samples are difficult to obtain, making it challenging to optimize primary cell culture in large batches. To improve the success rate of primary cell line establishment, the primary cell line establishment system was improved:

[0071] First, cut the obtained clinical tumor sample into 3-5mm pieces. 3 The tumor masses were inoculated subcutaneously into immunodeficient mice. The tumor masses were allowed to grow to a certain size (800–1000 mm) on the mice. 3 Fresh tumor tissue blocks were taken from tumor-bearing mice to obtain F1 generation PDX, which were then cut into 3-5 mm pieces. 3 Tumor fragments were partially preserved in liquid nitrogen (labeled as F1 generation), and the remaining tissue fragments were subcutaneously inoculated into new immunodeficient mice to obtain F2 generation PDX. This process of inoculation and preservation was then 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] It will grow to 800mm 3Fresh tumor tissue blocks from F3 generation PDX (preferably early generations) were removed from tumor-bearing mice and rinsed with sterile PBS (phosphate-buffered saline) and DMEM medium (a medium containing various amino acids and glucose). The tumor tissue blocks were then cut into small pieces (1-2 mm). 3 Then, under 37°C, the cells were digested with 20 U / mL papain and 2 U / mL neutral protease for 60 min to obtain the digested cells.

[0073] Primary PDC cells were obtained by coating culture plates with 400 ng / mL poly-L-lysine and culturing digested cells in DMEM medium 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) at 37°C. The 1×NEAA contained 8.9 ng / mL L-alanine, 14.7 ng / mL L-glutamate, 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] I. Experimental Methods

[0076] 1. In Example 1, the size of the F3 generation PDX tumor tissue block is <1mm. 3 or 1-2mm 3 The digestion time was 30 min or 60 min, and the digestion reagents were: (1) 0.25% (w / v) trypsin + 0.02% (w / v) EDTA (ethylenediaminetetraacetic acid); (2) 0.25% (w / v) trypsin + 0.02% (w / v) EDTA + 20 μg / mL DNase; (3) 0.25% (w / v) trypsin + 2 mg / mL collagenase I; (4) 0.25% (w / v) trypsin + 2 mg / mL collagenase II; (5) 0.25% (w / v) trypsin + 2 mg / mL collagenase III; (6) 0.25% (w / v) trypsin + 2 mg / mL collagenase IV; (7) 0.25% (w / v) trypsin + 2 mg / mL collagenase V; (8) 20 U / mL papain + 2 U / mL neutral protease. The viability and clumping rate of digested cells were detected.

[0077] 2. In Example 1, the reagents used to coat the culture plates were polylysine of different concentrations (100 ng / mL, 200 ng / mL, 400 ng / mL and 600 ng / mL) or gelatin of different concentrations (0.2% w / v, 0.4% w / v and 0.8% w / v). The basal culture medium was DMEM medium containing 15% (v / v) FBS or RPMI 1640 medium containing 15% (v / v) FBS. The following components were 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 adhesion rate of adherent cells after 7 days of culture was measured.

[0078] 3. In Example 1, the reagents used to coat the culture plates were poly-L-lysine at different concentrations (400 ng / mL and 600 ng / mL), and the culture media were (1) DMEM medium supplemented with 1×NEAA, 4 mM L-glutamine, 50 mg / L transferrin and 15% (v / v) FBS; and (2) RPMI 1640 medium supplemented with 1×NEAA, 4 mM L-glutamine, 50 mg / L transferrin, 50 ng / mg EGF and 15% (v / v) FBS. Cell passage was then assessed.

[0079] II. Experimental Results

[0080] 1. For example Figure 1 A to Figure 1 As shown in D, 1-2mm 3 The optimal cell clumping rate and cell viability can be obtained by treating tumor tissue blocks at 37°C with a combination of 20 U / mL papain and 2 U / mL neutral protease for 60 min, i.e., cell clumping rate <30% and viability >70%.

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

[0082] 3. For example Figure 3 As shown, when culture plates were coated with 400 ng / mL poly-L-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 showed high cell adhesion and could be passaged for 20 generations with high survival rate.

[0083] Example 3: Construction of PDC cells with barcode DNA tags

[0084] 1. Constructing shuttle plasmids carrying barcode DNA tags

[0085] Random nucleotide sequences of barcode DNA tags were synthesized to obtain a barcode DNA tag library, which was then cloned into a backbone plasmid (pdest-ef1-egfp-BSD) containing barcode DNA tag insertion sites (see diagram). Figure 4 In this study, a plasmid library carrying a barcode DNA tag (pdest-ef1-egfp-BSD-barcode, see diagram) was constructed. Figure 5 In the backbone plasmid, the 5' end restriction site of the insertion site is EcoRI, and the 3' end restriction site is BamHI, located between primer-F and primer-R. Each plasmid carries a barcode DNA tag. Table 1 shows some examples of nucleotide sequences in barcode DNA tag libraries. The barcode DNA tag is a 24 bp random DNA sequence NNNNNNCTCNNNNNNGTCNNNNNN, where N is selected from any of the bases A (adenine), T (thymine), C (cytosine), and G (guanine), which can be combined to create tens of thousands of barcode DNA tags.

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

[0087]

[0088] 2. Preparation of lentiviral libraries carrying barcode DNA tags

[0089] Seed 2×10 cells in a 10cm cell culture dish 6HEK293T cells were cultured in high-glucose DMEM medium (10% FBS) at 37°C and 5% CO2. When the cell density reached 90%, two 1.5 mL centrifuge tubes were prepared, labeled A and B. 200 μL of FBS-free high-glucose DMEM medium was added to each tube. Tube A contained 20 μL of transfection reagent (QuickShuttle-293, a transfection reagent specifically for 293T cells). Tube B contained 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 mass ratio of 4:3:2) was mixed in tubes A and B to obtain a mixture. This mixture was then added to a 10cm cell culture dish inoculated with HEK293T cells for transfection. Six hours after transfection, the medium was changed to complete medium containing 10% (v / v) FBS. At 48 hours after transfection, the supernatant was collected for the first time, and complete medium containing 10% (v / v) FBS was added, followed by culturing for another 24 hours (72 hours after transfection). The supernatant was collected a second time, and complete medium containing 10% (v / v) FBS was added again, followed by culturing for another 24 hours (96 hours after transfection). The supernatant was collected a third time. The supernatants collected in the three separate collections were mixed and concentrated to obtain a lentiviral library carrying a barcode DNA tag. The lentiviral titer and homogeneity of the barcode-tagged library were measured.

[0090] 3. Constructing PDC cells with barcode DNA tags

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

[0092] Primary PDC cells from Example 1 were seeded in a 6 cm dish (1 × 10⁻⁶ cells / cm²). 5 Cells / dish) were transfected with lentiviral libraries carrying barcode DNA tags at different MOIs and transfection reagents. Fluorescent PDC cells were sorted by fluorescence flow cytometry, and the transfection efficiency of the virus was calculated to obtain a suitable MOI so that 1% to 10% of PDC cells were transfected with the virus and carried fluorescent labels.

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

[0094] Primary PDC cells from Example 1 were seeded in a 10cm culture dish (5×10⁻⁶ cells / cm²). 6PDC cells (cells / plates) were transfected with a lentiviral library carrying a barcode DNA tag and transfection reagent. Following step (1), MOI transfection was performed after adjustment and confirmation. After transfection, blastomycin (BSD) was used for drug screening to kill PDC cells not infected with the barcode DNA tag. The surviving PDC cells infected with the barcode DNA tag were collected from the culture dish. After passage, single-cell sequencing was used to detect and identify the barcode DNA tag information carried by each PDC cell. Each PDC cell carried only a unique barcode DNA tag. PDC cells successfully incorporating the barcode DNA tag were 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 introduced barcode DNA tags were first seeded and cultured. Single-cell suspensions of PDC cells in the logarithmic growth phase were prepared for single-cell sequencing, and single-cell transcriptome sequencing data were obtained using the 10×Genomics single-cell sequencing platform. The obtained single-cell transcriptome sequencing data of the PDC model were compared with the human genome database using CellRanger software to obtain the 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 fewer detected genes (<200), cells with more detected UMIs (>100,000), and cells with a high proportion of UMI counts attributed to mitochondrial genes (>20%) were removed. The filtered expression matrix was then normalized and scaled to eliminate unwanted sources of variation driven by the number of detected UMIs and mitochondrial gene expression. Harmony was used to remove batch effects, then RunTSNE was used for linear dimensionality reduction, then FindClusters was used for clustering, and finally DimPlot was used for visualization to obtain the tsne plot.

[0098] 2. Mutation type analysis using whole-exome sequencing

[0099] PDC cells with introduced barcode DNA tags were first seeded and cultured. Genomic DNA was extracted from PDC cells in the logarithmic growth phase, and a library was constructed using Agilent V6 probes. Then, whole-exome sequencing data were obtained based on Illumina sequencing.

[0100] Figure 6The invention presents a complete flowchart of the process from isolating and culturing primary PDC cells to constructing PDC cells with introduced barcode DNA tags and performing sequencing analysis.

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

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

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

[0104] Following the methods in Examples 1 and 3, lung cancer PDC cells with introduced barcode DNA tags were obtained. Single-cell sequencing and whole-genome sequencing were then performed according to the method in Example 4. Based on the single-cell sequencing data, cluster analysis identified eight different clusters. (See...) Figure 8 .

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

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

[0107] Comparative Example 2: Single-cell sequencing analysis of the 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 in Example 4. Cluster analysis based on the single-cell sequencing data revealed low heterogeneity, with only four different clusters (clonal groups) distinguishable. Figure 10 The number of clusters obtained from lung cancer PDC cells with introduced barcode DNA tags was lower than that obtained from other 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] Whole-exome sequencing was performed on 10 pancreatic cancer PDC cells obtained in Example 1 and 10 conventional pancreatic cancer cell lines PANC-1 according to the method in Example 4. The whole-exome sequencing data were then combined with the whole-exome sequencing data of clinical samples (pancreatic cancer) in the TCGA database for analysis.

[0111] Whole-exome sequencing of conventional pancreatic cancer cell lines PANC-1 and PDC cells was performed using an Illumina HiSeq 2500 sequencer, with 125 bp paired-end reads. An Agilent v4_XT_51Mb capture kit was used to achieve 100-fold average coverage. Raw reads from each cell model were aligned to the human + mouse tandem genome (GRC37 / hg19 + GRCm38 / mm10) using BWA alignment software, and reads not aligned to the human contiguous group were filtered out. 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 calling programs (muTect, strelka). SNPs were screened using quality thresholds (quality score > 20, read depth > 10), and SNPs found in germline databases (HapMap, 1000G) were discarded. To analyze mutation rates and chromosomal abnormalities among conventional pancreatic cancer cell lines PANC-1, PDC cells, and clinical samples from the TCGA database, MutSigC and GISTIC2 were used to analyze and compare the most significantly mutated genes, significantly amplified and deleted regions identified in the TCGA database. Variants were invoked via the Mutect caller. SNPs were annotated using SnpEff and the Ensembl annotation file for the human genome (GRCh37). Based on the annotation results, the average number of mutations per million bases was calculated in conventional pancreatic cancer cell lines PANC-1, pancreatic cancer PDC cells, and clinical samples from the TCGA database, representing the background mutation frequency of the samples. The proportion of each mutation type within each group was calculated to observe trends in mutation type changes among sample groups. The number of commonly detected SNP types among different sample groups was calculated, and the results were visualized using a Venn diagram. Figure 11 A to Figure 11 C). Pancreatic cancer PDC cells retain more mutation types and related tumorigenesis and development mechanisms, which is more conducive to distinguishing and studying gene mechanisms related to tumor progression and drug response.

[0112] Comparative Example 4: Whole-exome sequencing was performed on 10 cases of lung cancer PDC cells and 10 cases of conventional lung cancer cell line NCI-H520.

[0113] Whole-exome sequencing was performed on 10 lung cancer PDC cells and 10 conventional lung cancer cell lines NCI-H520 obtained from the comparative example, following the method described in Comparative Example 3. Based on the whole-exome sequencing data, and combined with whole-exome sequencing data of clinical samples (lung cancer) from the TCGA database, analysis was conducted. Using the same analytical methods as in Comparative Example 3, the frequency of single nucleotide mutations and the proportion of each mutation type in the conventional lung cancer cell line NCI-H520, lung cancer PDC cells, and the TCGA database were statistically analyzed. Finally, the intersection was taken to obtain the common mutations (…). Figure 12 A to Figure 12 C). Lung cancer PDC cells retain more mutation types and related tumorigenesis and development mechanisms, which is more conducive to distinguishing and studying gene 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 modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

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

2. The method of claim 1, wherein the recombinant PDC cells are prepared by the steps of: The method comprises the following steps: S11: constructing a recombinant vector containing a barcode DNA tag library; S12: mixing the recombinant vector of step S11 and a lentivirus packaging plasmid, transfecting a vector cell, and then isolating a lentivirus library carrying a barcode DNA tag library; S13: transfecting PDC cells with the lentivirus library carrying a barcode DNA tag of step S12, adjusting the MOI of virus transfection to make the transfection efficiency 1% to 10%, and obtaining the recombinant PDC cells.

3. A method of analyzing tumor cell heterogeneity and cell tracking, characterized by, The barcode DNA tag and the mutation and expression profile of the gene coding region of the recombinant PDC cells of claim 1 are subjected to sequencing analysis.

4. The method of claim 3, wherein, The sequencing analysis comprises single-cell sequencing cluster analysis, coding region mutation type and proportion analysis.

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