CN110079501B - Mouse breast cancer circulating tumor cell line and establishing method thereof - Google Patents
Mouse breast cancer circulating tumor cell line and establishing method thereof Download PDFInfo
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Abstract
The invention provides a mouse breast cancer circulating tumor cell line 4T1-CTC, which is preserved in a China center for type culture Collection in 2019, 4 months and 10 days, wherein the preservation number is CCTCC NO: C201930. the invention also provides a method for separating and culturing the circulating tumor cells of the breast cancer of the mouse. The circulating tumor cells obtained by separation and culture have uniform quality, are identified by immunofluorescence, proliferation, invasion migration and tumorigenic capacity experiments to accord with the characteristics of the circulating tumor cells, are experimental objects for researching tumor metastasis in laboratories better, and provide good research models for clinically predicting breast cancer metastasis and detecting chemotherapeutic drug sensitivity. The method for separating and culturing the circulating tumor cells is simple, low in cost and easy to operate, and the separated cells can be passed, so that a good research tool is provided for researching the characteristics of the circulating tumor cells and the tumor metastasis mechanism in a laboratory. The invention can be widely applied to the research of tumor metastasis.
Description
Technical Field
The invention belongs to the technical field of biotechnology, and particularly relates to a mouse breast cancer circulating tumor cell line and a method for separating and culturing breast cancer circulating tumor cells.
Background
Breast cancer is the most common female malignant tumor and is the main cause of female tumor-related death, according to statistics in 2015, about 27 ten thousand new cases of breast cancer and about 7 ten thousand patients die in China, tumor metastasis is the main cause of death of tumor patients, and circulating tumor cells play a key role in the tumor blood vessel metastasis process. A large number of studies at home and abroad prove that circulating tumor cells in blood of a breast cancer patient are closely related to clinical staging, prognosis and tumor metastasis and relapse of the patient. Circulating tumor cells are listed as a biological index for breast cancer prognosis evaluation in the U.S. AJCC guideline of 2018. Therefore, the early capture of the circulating tumor cells has important guiding function for the prognosis judgment, the curative effect evaluation and the individual treatment of the patients.
The detection of the circulating tumor cells has the advantages of small wound and easy repetition, so the capture, separation and culture technology of the circulating tumor cells is a key link. So far, reports of establishment of mouse breast cancer circulating tumor cell lines are not seen.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides a mouse breast cancer circulating tumor cell line and a method for separating and culturing breast cancer circulating tumor cells.
The invention provides a mouse breast cancer circulating tumor cell line 4T1-CTC, which is preserved in a China center for type culture Collection in 2019, 4 months and 10 days, wherein the preservation number is CCTCC NO: C201930.
the invention provides a method for separating and culturing circulating tumor cells of mouse breast cancer, which comprises the following steps:
(1) Taking peripheral blood of a breast cancer in-situ metastatic tumor mouse model into a sodium citrate anticoagulant to obtain anticoagulated blood, gently transferring the anticoagulated blood into a centrifuge tube containing mouse peripheral blood lymphocyte separation liquid, centrifuging, sucking plasma and a leucocyte layer into a sterile centrifuge tube, rinsing with sterile PBS liquid, and centrifuging to obtain cell sediment;
(2) Transferring the cells obtained in step (1) into a culture flask containing 10% FBS in DMEM1640 medium, at 37 deg.C, 5% 2 Culturing in a cell culture box; and after culturing for 24-48 h, replacing the culture solution every 48h, and removing the suspended white blood cells by replacing the culture solution to obtain the cells growing adherent to the skin, namely the circulating tumor cells of the breast cancer of the mouse.
Preferably, in step (1), the method for establishing the mouse model of breast cancer metastasis in situ comprises the following steps: resuspending the breast cancer cell line 4T1 single cells in logarithmic growth phase with sterile PBS, inoculating to a mouse model, injecting to a mouse model mammary gland fat pad, and feeding for 35-40 days until the mouse model is in a lean and failure state.
Preferably, in step (1), the mouse model is an SPF-grade BALB/c mouse.
Preferably, in step (2), 1% streptomycin is further added into the DMEM1640 medium.
Preferably, in the step (2), the subculture is started when the cell density of the mouse breast cancer circulating tumor cell line to be grown adherently reaches 70-80%.
Preferably, the specific method of subculturing is as follows: discarding the culture solution, rinsing with sterile PBS, digesting with 0.25% trypsin solution at room temperature, adding DMEM1640 culture solution containing 10% FBS to blow out the cells to prepare single cell suspension when the cells become round, transferring the cell suspension to a culture flask, standing at 37 deg.C and 5% CO 2 And continuing culturing in the incubator.
Preferably, the cryopreservation method of the circulating tumor cells of the mouse breast cancer comprises the following steps: digesting the circulating tumor cells of the breast cancer of the mouse in the logarithmic growth phase into single cells by using a 0.25% trypsin solution at room temperature, centrifuging to obtain cell precipitates, slowly adding a cell cryopreservation solution, standing at 4 ℃ for 1h, standing at-20 ℃ for 2h, standing at-80 ℃ overnight, and storing in liquid nitrogen the next day.
Preferably, the cell freezing medium consists of DMEM1640 culture medium, fetal bovine serum and dimethyl sulfoxide, and the weight ratio of DMEM-1640: fetal bovine serum: volume ratio of dimethyl sulfoxide = 6.
Preferably, the method for recovering the circulating tumor cells of the mouse breast cancer comprises the following steps: thawing the frozen cells in a 37 deg.C water bath, centrifuging to obtain cell precipitate, adding 10% FBS-containing DMEM-1640 medium, and adjusting the concentration to 37 deg.C, 5% CO 2 Culturing in a cell culture box, and replacing the culture solution every 48 h.
The invention has the beneficial effects that:
the circulating tumor cells obtained by separation and culture have uniform quality, are identified by immunofluorescence, proliferation, invasion migration and tumorigenic capacity experiments to accord with the characteristics of the circulating tumor cells, are experimental objects for researching tumor metastasis in laboratories better, and provide good research models for clinically predicting breast cancer metastasis and detecting chemotherapeutic drug sensitivity. The method for separating and culturing the circulating tumor cells is simple, low in cost and easy to operate, and the separated cells can be passaged, so that a good research tool is provided for researching the characteristics of the circulating tumor cells and a tumor metastasis mechanism in a laboratory. The invention can be widely applied to the research of tumor metastasis.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention and not to limit the invention. In the drawings:
FIGS. 1 and 2 are captured mouse breast cancer circulating tumor cells 4T1-CTC.
FIGS. 3 and 4 are graphs identifying whether the obtained circulating tumor cells are of epithelial origin.
FIG. 5 is a comparison of the proliferative capacity of the obtained circulating tumor cells (4T 1-CTC) compared to their parental, in situ tumor cells.
FIG. 6 is a comparison of the clonal sphere forming ability of the obtained circulating tumor cells (4T 1-CTC) with their parental, in situ tumor cells.
FIG. 7 is a statistical analysis of the clonogenic capacity of the obtained circulating tumor cells (4T 1-CTC) with their parental cells, tumor cells in situ.
FIG. 8 is a comparison of the migratory capacity of the obtained circulating tumor cells (4T 1-CTC) with their parental and in situ tumor cells by the Transwell migration assay.
FIG. 9 is a comparison of the invasive capacity of the obtained circulating tumor cells (4T 1-CTC) with their parental and in situ tumor cells by the Transwell invasion assay.
Figures 10 and 11 are graphs comparing circulating tumor cells (4T 1-CTC) to their parental and in situ tumor cells for subcutaneous tumorigenicity in vivo subcutaneous tumorigenicity experiments.
Detailed Description
The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples are commercially available unless otherwise specified. In the quantitative tests in the following examples, three replicates were set up and the results averaged.
Example 1
(I) the related experiment reagent and experiment article related to the implementation
(1) 200mL of mouse peripheral blood lymphocyte isolate.
(2) Sodium citrate anticoagulant.
(3) DMEM1640 medium (Hyclone).
(4) FBS (fetal bovine serum) (BI), DMEM1640 medium containing 10% FBS: DMEM1640 culture was mixed in FBS at a volume ratio of 9.
(5) PBS (0.01M).
(6) 0.25% trypsin solution (Hyclone).
(7) Cell cryopreservation solution: 6mLDMEM1640 culture medium, 3mLFBS and 1mLDMSO (Sigma) are mixed evenly, stored at 4 ℃ and prepared for use.
(II) establishment of breast cancer in-situ metastatic tumor animal model
1.1 laboratory mouse and cell line preparation
(1) The amplification method of the breast cancer cell line 4T1 comprises the following steps: murine breast cancer cells 4T1 were purchased from Shanghai cell Bank of Chinese academy of sciences, cultured in DMEM1640 medium containing 10% FBS, and 1% streptomycin was added to the cells at 37 ℃ for 5% CO 2 Culturing in a cell culture box, performing passage amplification until the cell density reaches 80%, discarding the culture medium supernatant, rinsing twice with sterile PBS, adding trypsin for about 3min, resuspending with DMEM1640 medium containing 10% FBS, and performing passage amplification of cells according to 1.
(2) SPF grade BALB/C mice, 10 in one group, were purchased and housed in the SPF environment of the animal center of the university of langzhou according to SPF grade animal housing standards with free access to water.
1.2 in situ tumor inoculation of mammary gland
1.2.1 Material preparation: medical gloves, masks, autoclaved surgical instruments, surgical racks and alcohol.
1.2.2 detailed procedures
(1) Counting the cells to ensure that the number of cells is sufficient to 2X 10 cells per cell 6 And (4) diluting the amplified 4T1 cells in the logarithmic growth phase with sterile PBS according to 150 mu L of each cell, and uniformly mixing to obtain a single cell suspension.
(2) Fixing the mouse, finding a fourth pair of mammary glands, disinfecting the fourth pair of mammary glands on the right side by using an alcohol cotton ball, slightly lifting the fourth pair of mammary glands on the right side by using forceps, inserting the syringe to a mammary gland fat pad in parallel, fixing the needle head by using the forceps after inserting the needle, slowly injecting cell suspension, and slightly pressing the injection part by using the alcohol cotton ball after injection to prevent bleeding infection.
(3) After further feeding for 35 to 40 days, BALB/C mice appeared in a lean state and were ready for blood sampling.
(III) Breast cancer in situ tumor cell isolation and culture Process
Anesthetizing a tumor-bearing mouse with ether, stripping a tumor tissue of the tumor-bearing mouse with a pair of sterile surgical scissors and tweezers, placing the tumor tissue in a culture dish, cutting off surface fat and necrotic tissue with a surgical knife, rinsing with sterile PBS for 2 times, cutting the surgical knife into 3mm small blocks in a crossed manner, transferring the tissue blocks into a centrifuge tube with the surgical tweezers, adding sterile PBS, rinsing for 2 times, transferring the tissue into a sterile conical flask, adding 100mL of enzymatic hydrolysate at 37 ℃ for digestion, shaking once every 20min, immediately stopping digestion when the digestive juice is turbid, filtering with a 40 mu m cell filter screen, centrifuging at 1000rpm and 5min, rinsing a culture medium for 2 times, centrifuging to obtain cell precipitates, culturing the suspended cells with DMEM1640 basis weight containing 10 FBS, culturing the suspended cells at 37 ℃,5 ℃ CO and culturing 2 And (5) culturing in a cell culture box.
(IV) separation and culture process of circulating tumor cells
(1) Prior to blood draw, 100 μ L of sodium citrate anticoagulant was aspirated into the vial using a 1mL sterile syringe.
(2) Blood is taken from heart, and the volume ratio of blood sample to sodium citrate anticoagulant is 9.
(3) Gently transferring the anticoagulated blood into a centrifuge tube containing 3mL of mouse peripheral blood lymphocyte separation liquid, centrifuging for 800g for 20min, gently sucking blood plasma and leukocyte layers into a sterile centrifuge tube, rinsing twice with sterile PBS liquid for 250g for 5min, and obtaining cell sediment.
(4) And rinsing with sterile PBS again, and centrifuging at room temperature for 250g for 5min to obtain cell sediment.
(5) Transferring the washed cells to a flask containing 10% FBS-containing DMEM1640 medium, incubating at 37 deg.C, and 5% CO 2 When the cells are cultured in a cell culture box, 1 percent of double antibody (streptomycin) is added into a culture medium for preventing bacterial contamination.
(6) After 24 hours of culture, the culture medium was replaced with 10% FBS-containing DMEM1640 medium, then the medium was replaced every 48 hours, and from 7 to 10 days after the first replacement of the medium, leukocytes suspended in the cell culture flask were gradually eliminated by the replacement of the medium, and tumor cells were proliferated to form clones, and the first passage was performed when the clones (first generation cells) reached 70% to 80%. The adherently grown cells, namely circulating tumor cells of a breast cancer in-situ metastasis tumor model BALB/c mouse are obtained at the moment and named as a mouse breast cancer circulating tumor cell line 4T1-CTC.
The subculture method comprises the following steps: discarding the culture solution, gently rinsing twice with sterile PBS, digesting with 0.25% trypsin solution at room temperature for about 1min, adding DMEM1640 culture solution containing 10% FBS after cell rounding to blow out the cells into single cell suspension, transferring the cell suspension into a culture flask, and standing at 37 deg.C for 5% CO 2 And (4) continuously culturing in the incubator, wherein the culture solution is changed once after 24 hours of culture, and the culture solution is changed once after 48 hours.
The cultured 4T1-CTC of the mouse breast cancer circulating tumor cell line is preserved in a China typical culture preservation center in 2019, 4 months and 10 days, wherein the preservation unit is CCTCC for short, the preservation unit address is Wuhan university No. 299 in Wuhan district, wuhan city, hubei province, and the preservation number is CCTCC NO: C201930.
(7) With respect to cryopreservation and resuscitation
Freezing and storing: digesting the breast cancer circulating tumor cells in logarithmic growth phase into single cells by using 0.25% trypsin solution at room temperature, centrifuging for 5min at 1000rpm to obtain cell precipitates, dropwise and slowly adding the prepared cell cryopreservation solution, standing at 4 ℃ for 1h, standing at-20 ℃ for 2h, standing at-80 ℃ for overnight, and storing in liquid nitrogen the next day.
And (3) resuscitation: freezing the frozen powderMelting the cells in water bath at 37 deg.C, centrifuging at 1000rpm for 5min to obtain cell precipitate, adding DMEM-1640 medium containing 10% FBS, and making cell precipitate at 37 deg.C 2 Culturing in a cell culture box, and replacing the culture solution every 48 h.
The experimental results show that: the cells can be normally passaged and frozen.
(V) identification of circulating tumor cells
4T1 breast cancer cells are epithelial-derived cells, and whether circulating tumor cells express epithelial keratin or not is identified through cellular immunohistochemistry and cellular immunofluorescence.
And (3) cellular immunohistochemistry: preparing a sterile circular glass slide, putting the circular glass slide into a 24-hole plate, and collecting a mouse breast cancer circulating tumor cell line 4T1-CTC CCTCC NO: c201930 with cell density of 3X 10 3 Density inoculation to each well, the next day after the cell density reached 60%, taking out, discarding cell culture supernatant, washing with PBS 3 times, adding 1ml of precooled 4% paraformaldehyde per well for fixation for 20min, discarding, washing with PBS 3 times, 0.05% Triton 100 permeabilizing membrane 15min, discarding, washing with PBS 3 times, 3% H 2 0 2 Incubating with deionized water for 5-10min, washing with PBS for three times, 3min each time, adding dropwise normal goat serum working solution, incubating for 15min, and removing serum on the surface of the slide. The broad-spectrum keratin antibody (1. The next day, PBS rinse 3 times, each time 3min, drop biotinylated secondary antibody working solution to incubate at 37 ℃ for 15min, PBS rinse 3 times, each time 3min. Adding horseradish enzyme labeled streptavidin working solution dropwise, incubating at 37 deg.C for 15min, and rinsing with precooled PBS for 3 times, each time for 3min. And (3) dropwise adding a DAB color developing agent under a microscope, observing that cells turn yellow, stopping color development, dropwise adding tap water, fully rinsing, sleeving nuclei with hematoxylin for 10s, rinsing with tap water for three times, differentiating with a differentiation solution for 1s, rinsing with tap water for 3 times, 3min each time, returning ammonia water to blue for 2s, and rinsing with tap water for 3 times, 3min each time. The mixture is dehydrated by sequentially passing through ethanol with concentration gradient (80% once, 95% twice, 100% three times, each time for 1 min). Xylene is transparent for 3 times, each time for 3min, and the gel is sealed with neutral gum and photographed by microscope observation.
Cellular immunofluorescence: sterile circular slides were prepared and placed in a 24-well plate and the well-grown, trapped mouse mammary carcinoma was circulatedTumor cell line 4T1-CTC CCTCC NO: c201930 at 3X 10 3 Cell density, inoculation of 500 μ l per well, taking out after the next day cell density reaches 60%, discarding cell culture supernatant, washing with sterile PBS for three times, adding 1mL4% paraformaldehyde into each well, fixing for 20min, discarding, washing with PBS for 3 times, 0.05% Triton 100 permeabilizing membrane for 15min, discarding, washing with PBS for 3 times, 3% H 2 0 2 Incubating with deionized water for 5-10min, washing with PBS for three times, 3min each time, adding dropwise normal goat serum working solution, incubating for 15min, and removing serum on the surface of the slide. The broad-spectrum keratin antibody (1. The next day, 3 times for 3min in PBS, 200. Mu.l FITC-labeled fluorescent secondary antibody (1.
The identification results describe: the morphology of the captured circulating tumor cells is similar to that of the 4T1 parent and the in situ morphology, (for example, figure 1 and figure 2), and the results of the broad-spectrum keratin marking by the cellular immunohistochemistry and the cellular immunofluorescence indicate that the captured cytokeratin is positive, (for example, figure 3 and figure 4), which indicates that the circulating tumor cells belong to the epithelial source.
FIG. 1 and FIG. 2 show the captured mouse breast cancer circulating tumor cell 4T1-CTC.
FIGS. 3 and 4 are graphs identifying whether the obtained circulating tumor cells are of epithelial origin.
(VI) circulating tumor cell proliferation, clonotype formation ability, migration and invasion ability, and subcutaneous tumorigenicity study (3 replicates).
1. The proliferative capacity of circulating tumor cells was compared to their parental and in situ 4T1 cells.
The cells in the logarithmic phase of growth were digested with 0.25% trypsin solution, prepared into a single cell suspension using 1640 medium containing 10% FBS, and adjusted to a cell density of 5X 10 3 Per mL, 200 μ l per well (1000 cells per well), mouse breast cancer circulating tumor cell line 4T1-CTC CCTCC NO: c201930 and its parent and in situ 4T1 cell are respectively planted in 96-well plate, time gradient is 1/2/3/4 day, each group has 8 negative wells, 20 mul 5mg/mL MTT solution is added into each well, incubation is carried out for 4h at 37 ℃, supernatant is discarded, each well is stopped for 10min by adding 150 mul DMSO, and the microplate reader has 490nm wavelengthThe filters detect the OD values of each well, and the average value of the OD values of each well was taken as the vertical axis and time as the horizontal axis, and a growth curve was plotted, and the procedure was repeated three times.
And (4) analyzing results: the proliferation capacity of the 4T1-CTC cells is lower than that of the 4T1 parent cells and 4T1 in situ cells (as shown in figure 5).
FIG. 5 is a comparison of the proliferation potency of the obtained circulating tumor cells (4T 1-CTC) compared to their parental, in situ tumor cells.
2. Comparing the clonal sphere forming abilities of circulating tumor cells and their parents and in situ 4T1 cells.
Cloning ball formation experiment, respectively adding 4T1 parent, in situ and mouse breast cancer circulating tumor cell line 4T1-CTC CCTCC NO: c201930 inoculating 700 cells per well into six-well plate, terminating by the eighth day, rinsing twice with PBS, fixing with 4% paraformaldehyde for 10min, staining with 0.5% crystal violet for 10min, rinsing twice with PBS, and taking pictures.
And (4) analyzing results: the clonogenic capacity of the 4T1-CTC cells was lower than that of the 4T1 parental cells and 4T1 in situ cells (see FIG. 6, 7).
FIG. 6 is a comparison of the clonal sphere forming ability of the obtained circulating tumor cells (4T 1-CTC) with their parental, in situ tumor cells.
FIG. 7 is a statistical analysis of the clonal sphere forming ability of the obtained circulating tumor cells (4T 1-CTC) and their parental cells, in situ tumor cells.
The transwell migration method compares the migratory capacity of circulating tumor cells with the 4T1 parent and 4T1 in situ cells.
Transwell migration experiment: 1X 10 resuspended in serum-free Medium 5 The cells in log phase were seeded into the upper chamber of the Transwell chamber, 500. Mu.l of medium containing 10% serum was added to the lower chamber, and after 36h, the cells that did not pass out of the upper chamber of the Transwell chamber were stopped by gently rubbing them with a cotton swab, fixed in 4% paraformaldehyde for 10min, stained in 0.5% crystal violet for 15min, rinsed twice with PBS, photographed under a microscope and counted in the number of the passed-out cells.
And (4) analyzing results: the 4T1-CTC cells had lower migratory capacity than the 4T1 parental cells and the 4T1 in situ cells (see fig. 8).
FIG. 8 is a comparison of the migratory capacity of the obtained circulating tumor cells (4T 1-CTC) with their parental and in situ tumor cells by the Transwell migration assay.
The transwell invasion method compares the invasive capacity of circulating tumor cells with that of parental and in situ 4T1 cells.
Transwell invasion experiments: mu.l of matrigel (1 5 The cells in log phase were seeded into the upper chamber of the Transwell chamber, 500. Mu.l of medium containing 10% serum was added to the lower chamber, and after 48h, the cells that did not pass out of the upper chamber of the Transwell chamber were stopped by gently rubbing them with a cotton swab, fixed in 4% paraformaldehyde for 10min, stained in 0.5% crystal violet for 15min, rinsed twice with PBS, photographed under a microscope and counted in the number of the passed-out cells.
And (4) analyzing results: the 4T1-CTC cells have lower migratory capacity than the 4T1 parent cells and the 4T1 in situ cells (see FIG. 9).
FIG. 9 is a comparison of the invasive capacity of the obtained circulating tumor cells (4T 1-CTC) with their parental and in situ tumor cells by the Transwell invasion assay.
5. Comparing the subcutaneous tumorigenicity of circulating tumor cells and their parents, in situ 4T1 cells.
Circulating tumor cells and their parents and in situ 4T1 cells are 2X 10 6 One/each was inoculated to the fourth pair of fat pads of BALB/C female mice, and after tumors had formed, tumor volumes were measured every two days to plot tumor growth curves.
And (4) analyzing results: the subcutaneous tumorigenicity of 4T1-CTC cells was lower than that of 4T1 parental cells and 4T1 in situ cells (see FIGS. 10 and 11).
Figures 10 and 11 are graphs comparing circulating tumor cells (4T 1-CTC) to their parental and in situ tumor cells for subcutaneous tumorigenicity in vivo subcutaneous tumorigenicity experiments.
In order to maintain the characteristics of the circulating tumor cells, the circulating tumor cells in the above experiments were all the first three generations of cells.
The experimental results of the invention are combined, and the experimental results prove that the mouse breast cancer circulating tumor cell obtained by the first separation and culture of the invention, namely the mouse breast cancer circulating tumor cell line 4T1-CTC CCTCC NO: c201930, which is described as circulating tumor cells of a breast cancer in-situ metastasis model BALB/C mouse, the isolated culture method disclosed by the invention is simple and easy to operate, and is beneficial to providing a better experimental object for researching a related mechanism of tumor metastasis through researching the characteristics of the circulating tumor cells in a laboratory.
Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that modifications may be made to the embodiments described above, or equivalents may be substituted for elements thereof. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (1)
1. The mouse breast cancer circulating tumor cell line 4T1-CTC is preserved in the China center for type culture Collection in 2019, 4 months and 10 days, and the preservation number is CCTCC NO: C201930.
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