CN116121194A - Drug-resistant cell line for lung cancer immunotherapy and preparation method and application thereof - Google Patents
Drug-resistant cell line for lung cancer immunotherapy and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a lung cancer immunotherapy drug-resistant cell line, a preparation method and application thereof, and belongs to the field of biological medicine. The lung cancer immunotherapy drug-resistant cell line is preserved in China center for type culture Collection, the preservation date is 2023, 1 month and 8 days, and the preservation number is CCTCCNO: C202307. Through a cell functional experiment, the in vitro proliferation and migration capability of the drug-resistant cell line is found to be unchanged; the mouse tumor-bearing experiment shows that the drug-resistant cell line has enhanced in vivo proliferation capability and obviously increased tolerance to PD-1 antibody treatment, can be used for deeply researching a molecular mechanism of lung cancer PD-1 antibody treatment resistance and developing related antitumor drugs, and provides new materials for screening molecular markers of lung cancer PD-1 antibody treatment resistance, designing and evaluating novel antitumor drugs, developing novel tumor treatment schemes and the like.
Description
Technical Field
The invention relates to the field of biological medicine, in particular to a lung cancer immunotherapy drug-resistant cell line, a preparation method and application thereof.
Background
Primary lung cancer is the most common malignant tumor in our country. Wherein the non-small cell lung cancer (non small cell lung cancer, NSCLC) accounts for about 80% -85%, and the rest is small cell lung cancer (small cell lung cancer, SCLC). The incidence and mortality of lung cancer in China are both at position 1 in malignant tumors. Because the early lung cancer has no obvious symptoms, most patients clinically have advanced symptoms when seeing a doctor, and the survival rate of the whole patients with advanced lung cancer is not high in 5 years. Statistical analysis results of 94703 NSCLC patients reported by AJCC 8 th edition tumor stage handbook show that the 5-year survival rate of the IV-stage lung cancer patients is only less than 5%, and the median survival period is only 7 months.
The treatment of advanced lung cancer is currently dominated by radiotherapy and chemotherapy. Advanced NSCLC for patients with driver-negative, the platinum-containing two-drug combination regimen is a standard first-line chemotherapy regimen. In recent years, immunotherapy typified by immune checkpoint inhibitors (such as PD-1 mab and the like) has been demonstrated to improve survival in lung cancer patients. Advanced lung cancer feasible Caririnotecan (PD-1 mab), palbociclib (PD-1 mab), tirelib (PD-1 mab), xindi (PD-1 mab) or atili (PD-L1 mab) combined pemetrexed-based platinum-containing two-drug chemotherapy are suggested in the 2022 edition of primary lung cancer diagnosis and treatment guide.
Although immunotherapy has progressed rapidly in recent years, we have to face the problem of: 1. the response rate of immunotherapy is not high. Only a few tumors have high response rates: such as hodgkin's lymphoma (87%), melanoma (40% -70%), while NSCLC response is very low (only 20%). 2. Some patients develop resistance shortly after administration, even in some patients who initially respond well, tumor recurrence is caused by acquired resistance. Overall, it has been shown that PD-1 mab has an exact therapeutic effect on advanced lung cancer, but its therapeutic effect is still limited by drug resistance issues.
The intrinsic mechanisms mainly involved in current drug resistance studies against PD-1 antibodies are mainly: MAPK pathway, PI3K signaling pathway, WNT/beta-catenin signaling pathway, IFN-gamma signaling pathway, etc. With the occurrence of immunotherapy drug resistance, it is important to research more effective drug targets. At present, no lung cancer cell strain resistant to PD-1 antibodies exists. Therefore, construction of PD-1 antibody resistant lung cancer cell lines is particularly important.
Disclosure of Invention
The invention aims to provide a lung cancer immunotherapy drug-resistant cell line, and a preparation method and application thereof, so as to solve the problems in the prior art. The drug-resistant cell line obtained by the invention has stable PD-1 antibody drug resistance, unchanged in vitro proliferation and migration capacity and higher scientific research and clinical application values.
In order to achieve the above object, the present invention provides the following solutions:
the invention provides a lung cancer immunotherapy drug-resistant cell line CMT167-R, wherein the drug-resistant cell line CMT167-R is preserved in China center for type culture Collection, the preservation date is 2023 and 1 month 8 days, and the preservation number is CCTCC NO: C202307.
Further, the construction method of the drug-resistant cell line CMT167-R comprises the following steps:
(1) Constructing a mouse subcutaneous tumor model by using a lung cancer cell line CMT167, treating by using an immune checkpoint PD-1 antibody, and screening tumors with the largest volume after treatment;
(2) Separating and screening tumor tissues with the largest volume under the sterile condition, cutting, culturing and passaging in a culture medium to obtain primary tumor cells, continuously constructing a mouse subcutaneous tumor model by using the primary tumor cells, treating by using an immune checkpoint PD-1 antibody, and screening tumors with the largest volume after treatment;
(3) Repeating step (2) until there is no significant difference in tumor volume of the immune checkpoint PD-1 antibody-treated group compared to the control group, and screening for tumors with the largest immune checkpoint PD-1 antibody-treated group volume;
(4) Separating the tumor tissue obtained in the step (3) under the aseptic condition, cutting, culturing in a culture medium and stably passaging to obtain the lung cancer immunotherapy drug-resistant cell line CMT167-R.
Further, in step (1), the lung cancer cell line is a murine lung cancer cell line.
Further, in step (2), the medium is DMEM medium containing 10% fetal bovine serum.
Further, in the step (4), the condition of the cultivation is 37℃CO 2 The volume concentration was 5%.
The invention also provides application of the lung cancer immunotherapy drug-resistant cell line CMT167-R in screening anti-tumor drug targets.
The invention also provides application of the lung cancer immunotherapy drug-resistant cell line CMT167-R in screening of antitumor drugs.
The invention also provides an application of the lung cancer immunotherapy drug-resistant cell line CMT167-R in preparing an anti-tumor drug.
The invention also provides application of the lung cancer immunotherapy drug-resistant cell line CMT167-R in screening and evaluating detection reagents of anti-tumor effects.
The invention also provides application of the lung cancer immunotherapy drug-resistant cell line CMT167-R in preparing a detection reagent for evaluating the anti-tumor effect.
The invention discloses the following technical effects:
the invention constructs a subcutaneous tumor model by using a mouse-derived lung cancer cell line CMT167 and a C57BL/6 mouse, separates primary tumor cells from PD-1 antibody-resistant mouse tumors, cultures and passages the primary tumor cells, and obtains a lung cancer PD-1 antibody-resistant cell line CMT167-R. Through a cell functional experiment, the in vitro proliferation and migration capacity of CMT167-R cells is found to be unchanged; mouse tumor-bearing experiments found that CMT167-R cells had enhanced in vivo proliferative capacity and significantly increased tolerance to PD-1 antibody treatment. The obtained lung cancer PD-1 antibody drug-resistant cell line CMT167-R provides a usable biological material for deeply researching a molecular mechanism of lung cancer PD-1 antibody therapeutic drug resistance, exploring a method for reversing immunotherapy drug resistance and developing related antitumor drugs; the application of the invention can screen the molecular markers of the lung cancer PD-1 antibody treatment drug resistance, design and evaluate novel antitumor drugs, develop novel tumor treatment schemes and the like, and has higher scientific research and clinical application values.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a diagram showing relevant experiments obtained in the construction process of a lung cancer PD-1 antibody drug-resistant cell line; wherein A is an experimental design flow chart for constructing a subcutaneous tumor model by using CMT167 cells and C57BL/6 mice to screen lung cancer PD-1 antibody drug-resistant cell lines; b is a tumor growth curve of mice after subcutaneous tumor administration, black lines are control IgG administration group mice, gray lines are PD-1 antibody administration group mice, and a number 5 mouse shown by gray dotted lines is selected for the next operation; c is the cell clone form after the separated tumor tissue is sheared and inoculated into a culture dish for culturing for 48 hours;
FIG. 2 is a graph showing experiments relating to the proliferation and migration capacity of a lung cancer PD-1 antibody-resistant cell line in vitro; wherein A is a clone formation experimental diagram of a lung cancer PD-1 antibody drug-resistant cell line CMT167-R and a control cell line CMT 167; b is a statistical analysis chart of the clone formation quantity; c is a migration capability microscopic image of a lung cancer PD-1 antibody drug-resistant cell line CMT167-R and a control cell line CMT 167; d is a statistical analysis chart of the number of tumor cells undergoing migration;
FIG. 3 is a graph showing the experimental results of the in vivo proliferation capacity of a lung cancer PD-1 antibody-resistant cell line; wherein A is subcutaneous tumor pictures of a lung cancer PD-1 antibody drug-resistant cell line CMT167-R in a C57BL/6 model, and experimental groups are a drug-resistant cell line CMT167-R tumor-bearing group and a control cell line CMT167 tumor-bearing group; b is the increase of subcutaneous tumor of lung cancer PD-1 antibody drug-resistant cell line CMT167-R tumor-bearing mice in C57BL/6 modelThe graph shows the graph, black lines are drug-resistant cell groups, gray lines are control cell groups, the abscissa is time (in days), and the ordinate is tumor volume (in mm) 3 ) The method comprises the steps of carrying out a first treatment on the surface of the C is a subcutaneous tumor weight statistical diagram of a lung cancer PD-1 antibody drug-resistant cell line CMT167-R tumor-bearing mouse in a C57BL/6 model, the abscissa is an experimental group, and the ordinate is tumor weight (unit: g);
FIG. 4 is a graph showing the experimental results of the drug resistance of the lung cancer PD-1 antibody-resistant cell line CMT 167-R; wherein A is an experimental design flow chart for verifying the drug resistance effect of a lung cancer PD-1 antibody drug-resistant cell line CMT167-R by using a C57BL/6 mouse subcutaneous tumor model; b is a volume diagram of subcutaneous tumor of a lung cancer PD-1 antibody drug-resistant cell line CMT167-R tumor-bearing mouse, and the experiment is divided into four groups, namely a drug-resistant cell group CMT167-R+IgG, a drug-resistant cell treatment group CMT167-R+anti-PD-1, a control cell group CMT167+IgG and a control cell treatment group CMT167+anti-PD-1; c is the proliferation curve graph of subcutaneous tumor of lung cancer PD-1 antibody drug-resistant cell line CMT167-R tumor-bearing mice, the abscissa is time (unit: day), and the ordinate is tumor volume (unit: mm) 3 ) The method comprises the steps of carrying out a first treatment on the surface of the D is a survival time analysis chart of a lung cancer PD-1 antibody drug-resistant cell line CMT167-R subcutaneous tumor-bearing mouse, the abscissa is time (unit: day), and the ordinate is the residual survival percentage.
Detailed Description
Various exemplary embodiments of the invention will now be described in detail, which should not be considered as limiting the invention, but rather as more detailed descriptions of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In addition, for numerical ranges in this disclosure, it is understood that each intermediate value between the upper and lower limits of the ranges is also specifically disclosed. Every smaller range between any stated value or stated range, and any other stated value or intermediate value within the stated range, is also encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the invention described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from consideration of the specification of the present invention. The specification and examples of the present invention are exemplary only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are intended to be inclusive and mean an inclusion, but not limited to.
The invention constructs a subcutaneous tumor model by using mouse-derived lung cancer cell lines CMT167 and C57BL/6 mice, separates primary tumor cells from PD-1 antibody-resistant mouse tumors, cultures and passages the primary tumor cells to obtain a PD-1 antibody-resistant lung cancer cell line CMT167-R (shown as A in figure 1), and is specifically as follows:
(1) culturing lung cancer CMT167 cells under aseptic condition, collecting tumor cells in logarithmic growth phase according to cell passage method, re-suspending cells with aseptic 1×PBS and counting, and adjusting cell concentration to 1×10 7 /ml. (2) A subcutaneous tumor model was constructed by inoculating 100. Mu.l of the cell suspension subcutaneously into the back of C57BL/6 mice using a 1ml syringe. (3) After tumor is carried for 7 days, a vernier caliper is used for measuring the long diameter a and the short diameter b of the tumor, and the volume V=1/2 ab is calculated according to the formula 2 Tumor volumes were calculated. Selecting the volume of 100+/-20 mm 3 Within the range, mice were randomly divided into 2 groups, and anti-PD-1 mab or control IgG treatment was administered by intraperitoneal injection, 1 dose per 3 days, at 200 μg/dose, 3 doses total. (4) Tumor volumes of mice were measured and calculated every 3 days, and tumor proliferation curves of mice were drawn 25 days after tumor loading. anti-I-The largest volume of mouse tumors in the PD-1 mab-treated group are considered to have PD-1 resistance potential. (5) The mouse tumor tissue was isolated in a sterile operating table and transferred to a cell culture dish, washed 3 times with sterile 1 XPBS, the waste liquid was discarded, and after adding 1ml of complete DMEM medium containing 10% fetal bovine serum, the tumor tissue was cut to a volume of about 1mm with sterile scissors 3 To 10ml of DMEM complete medium, penicillin/streptomycin was added, and the mixture was placed at 37℃and 5% CO 2 Culturing in an incubator for 48 hours, and then carrying out passage and freezing storage on the adherent cells. (6) Collecting tumor cells in logarithmic growth phase of primary cells obtained above by cell passage method, re-suspending cells with sterile 1×PBS and counting, and adjusting cell concentration to 1×10 7 /ml. 100ul of the cell suspension was inoculated subcutaneously into the back of C57BL/6 mice using a 1ml syringe, and the C57BL/6 mice subcutaneous tumor-bearing model was reconstructed. The above screening procedure was repeated 4 times. Until there was no significant difference in tumor size in the PD-1 antibody-treated group compared to the IgG control group. (7) Separating tumor tissue with maximum volume of PD-1 antibody treatment group, washing with sterile 1×PBS for 2 times, discarding the waste liquid, adding 1ml DMEM complete medium, and cutting tumor tissue into volume of about 1mm with sterile scissors 3 To 10ml of DMEM complete medium, penicillin/streptomycin was added, and the mixture was placed at 37℃and 5% CO 2 Culturing and passaging in an incubator. Thus obtaining the stable lung cancer PD-1 drug-resistant cell line CMT167-R. (8) The subcutaneous tumor model is respectively constructed by using a lung cancer cell line CMT167 and a lung cancer PD-1 antibody drug-resistant cell line CMT167-R, the PD-1 antibody treatment is carried out, the tumor volume change is detected, the tumor can still grow rapidly after the lung cancer PD-1 antibody drug-resistant cell line CMT167-R tumor-bearing mice are used, no significant difference exists between the lung cancer cell line CMT167-R and an IgG treatment group, and the lung cancer cell line of the mice has the PD-1 antibody drug resistance.
Example 1C 57BL/6 mice subcutaneous tumor model of lung cancer CMT167 cells
Culture of CMT167 cells
The CMT167 cells frozen in liquid nitrogen (purchased from ECACC, european Standard cell Collection center) were removed, rapidly placed in a 37℃constant temperature water bath to allow rapid dissolution, the cell suspension was transferred to a 15ml centrifuge tube in a sterile ultra clean bench, and 2 was addedml DMEM medium, centrifuged at 1000rpm for 5min at room temperature, the supernatant discarded, the cells resuspended in 10ml DMEM complete medium containing 10% foetal calf serum and transferred to cell culture dishes, placed at 37℃in 5% CO 2 Culturing and passaging in a cell culture box.
2. Construction of mouse subcutaneous tumor model
Taking CMT167 cells in logarithmic growth phase, digesting and centrifuging with pancreatin, discarding supernatant, resuspending tumor cells with sterile 1×PBS, counting cells, and adjusting cell concentration to 1×10 with sterile 1×PBS 7 /ml. A6-week-old C57BL/6 female mouse (available from Beijing Vitrehua laboratory animal technologies Co., ltd.) was selected, the back hair of the mouse was shaved off with a shaver, and 100. Mu.l of the cell suspension was inoculated subcutaneously into the back of the mouse with a sterile 1ml syringe, to construct a mouse lung cancer subcutaneous tumor model.
Example 2 screening of lung cancer subcutaneous tumor model PD-1 antibody treatment of drug resistant mouse tumors
On day 7 of tumor-bearing of C57BL/6 mice, the long diameter a and the short diameter b of the tumors are measured by a vernier caliper, and the volume V=1/2 ab 2 Tumor volumes were calculated. Selecting the volume of 100+/-20 mm 3 Mice within range were randomly divided into 2 groups of 10 mice each. The experimental group was given immune checkpoint PD-1 antibody (purchased from BioXcell company), and the control group was given isotype control IgG antibody (purchased from BioXcell company). The administration was by intraperitoneal injection, 1 dose every 3 days, 200. Mu.g/dose each, and 3 doses. Tumor long diameter a and short diameter b were measured every 3 days using vernier calipers and volume v=1/2 ab according to the formula 2 Tumor volume was calculated and 25 days after tumor loading, mice were plotted for tumor proliferation (as in B in fig. 1). Among them, mouse number 5 in the PD-1 antibody-treated group had the largest tumor volume and was considered to have PD-1 resistance potential. The mice were sacrificed for future use by cervical dislocation.
EXAMPLE 3 acquisition of tumor primary cells of Lung cancer PD-1 antibody-resistant mice
The sacrificed mice were soaked in 75% alcohol for 5 minutes, tumor tissues were separated with sterile scissors and forceps in a sterile super clean bench, and the whole tumor tissues peeled off were placed in 10cm cell culture dishes. Washing with sterile 1 XPBS three times, discarding the waste liquid, adding 1ml DMEM containing 10% foetal calf serum, and culturing completelyCulture medium, cutting tumor tissue to about 1mm with sterile scissors 3 Adding 8.5ml DMEM complete culture medium and 500 μl penicillin/streptomycin double antibody, mixing with a light shaking culture dish, and placing at 37deg.C and 5% CO 2 The culture was performed in an incubator for 48 hours (as shown in C in FIG. 1). The supernatant was discarded, washed three times with sterile 1×pbs, and culture was continued with addition of 10ml DMEM complete medium until passage of the cells.
EXAMPLE 4 acquisition of a Stable drug resistant cell line for Lung cancer PD-1 antibody
Taking the primary tumor cells obtained in example 3, continuing to culture and passaging, collecting the primary tumor cells in logarithmic growth phase, re-suspending the cells with sterile 1×PBS and counting, and adjusting the cell concentration to 1×10 7 /ml. 100 μl of the cell suspension was inoculated subcutaneously into the back of C57BL/6 mice using a 1ml syringe as in example 1, the C57BL/6 mice subcutaneous tumor model was reconstructed, and the screening procedures of example 2 and example 3 were repeated until there was no significant difference in tumor size between the PD-1 antibody-treated group and the IgG control group. Separating tumor tissue with maximum volume of PD-1 antibody treatment group, washing with sterile 1×PBS for 2 times, discarding the waste liquid, adding 1ml of DMEM complete medium containing 10% fetal bovine serum, cutting tumor tissue into volume of about 1mm with sterile scissors 3 To 9.5ml of DMEM complete medium, 500. Mu.l of penicillin/streptomycin was added and the mixture was placed at 37℃with 5% CO 2 Culturing and passaging in an incubator. Thus obtaining the stable PD-1 drug-resistant cell line CMT167-R.
The stable drug-resistant cell line is named as a mouse lung cancer cell CMT167-R, is preserved in China center for type culture Collection (university of Chinese, wuhan) at 2023, and has a preservation number of CCTCC NO: C202307.
example 5 in vitro proliferation and migration Capacity of lung cancer PD-1 antibody-resistant cell lines
The lung cancer cell line CMT167 in logarithmic growth phase and the lung cancer PD-1 antibody drug-resistant cell line CMT167-R obtained in example 4 were taken respectively, cells were collected by centrifugation after pancreatin digestion, cell pellet was washed 2 times with sterilized 1 XPBS, cell pellet was resuspended in 2ml DMEM medium (serum-free) after centrifugation, and cells were counted.
For clone shapeIn the experiments, the cell concentration was adjusted to 5X 10 with DMEM medium (serum-free) 2 /ml. 1ml of the cell suspension is taken and connected into a 6-hole plate, 800 mu l of DMEM culture medium and 200 mu l of fetal bovine serum are added, and the mixture is placed at 37 ℃ and 5% CO 2 Culturing in an incubator for 7 days. Cells were washed 2 times with sterile 1 XPBS, the supernatant was discarded, 1ml of 4% paraformaldehyde was added for 30 minutes, the supernatant was discarded, 1ml of 1% crystal violet was added for 30 minutes, the supernatant was discarded, and the remaining crystal violet dye was carefully washed with clear water. Photographs were taken with a microscope and subjected to clone counting and statistical analysis.
For cell migration experiments, the cell concentration was adjusted to 1X 10 with DMEM medium (serum free) 5 /ml. 200. Mu.l of the cell suspension was inoculated into the upper layer of a Transwell chamber, and 800. Mu.l of DMEM complete medium containing 10% fetal bovine serum was added to the lower layer, and the mixture was placed at 37℃with 5% CO 2 Culturing in an incubator for 48 hours. Taking out the cell, washing the cell with 1 XPBS for 2 times, discarding the supernatant, placing the cell in 4% paraformaldehyde for 30 minutes, discarding the supernatant, placing the cell in 1% crystal violet for 30 minutes, discarding the supernatant, carefully washing the cell with clear water, and carefully wiping the non-migrated tumor cells in the cell with a cotton swab. Photographs were taken with a microscope and cell counts and statistical analysis were performed.
As can be seen from FIG. 2, the in vitro proliferation capacity of the lung cancer PD-1 antibody-resistant cell line CMT167-R is not different from that of the control cell line CMT167 (FIGS. 2A and B; p > 0.05). Meanwhile, the in vitro migration capacity of the lung cancer PD-1 antibody-resistant cell line CMT167-R was not different from that of the control cell line CMT167 (C and D of FIG. 2; p > 0.05).
Example 6 verification of the in vivo proliferation Capacity of Lung cancer PD-1 antibody resistant cell lines
The lung cancer cell line CMT167 and the lung cancer PD-1 antibody drug-resistant cell line CMT167-R obtained in example 4 are used for constructing a subcutaneous tumor model of a C57BL/6 mouse, and the construction method is the same as that of example 1. The experiments were divided into two groups, a drug-resistant cell line CMT167-R tumor-bearing group and a control cell line CMT167 tumor-bearing group, each group comprising 6 mice. Subcutaneous tumor tissue was isolated and volume was measured in each group of mice 25 days after tumor bearing (a of fig. 3); from day 7, the long diameter a and the short diameter b of each group of mouse tumors were measured every 3 days using vernier calipers, and the volume v=1/2 ab was calculated according to the formula 2 Tumor volume was calculated and a mouse tumor proliferation curve was plotted (B of fig. 3); the subcutaneous tumor tissue of each group of mice was weighed and statistically analyzed (C of fig. 3).
As can be seen from fig. 3: on day 25 mice were sacrificed, the tumor volume and weight of lung cancer PD-1 antibody resistant cell line CMT167-R tumor bearing mice were significantly greater than the control cell line CMT167 tumor bearing group (B and C of fig. 3; p < 0.01).
Example 7 verification of the drug resistance effects of the lung cancer PD-1 antibody-resistant cell line CMT167-R
C57BL/6 mice subcutaneous tumor model is respectively constructed by using a lung cancer cell line CMT167 and a PD-1 antibody drug-resistant cell line CMT167-R obtained in example 4, and the construction method is the same as that of example 1; PD-1 antibody treatment is carried out, tumor volume change is detected, and the drug resistance effect of a lung cancer PD-1 antibody drug-resistant cell line CMT167-R is verified, wherein an experimental flow chart is shown in FIG. 4A. The experiments were divided into four groups, namely, drug-resistant cell group CMT167-R+IgG, drug-resistant cell treatment group CMT167-R+anti-PD-1, control cell group CMT167+IgG, control cell treatment group CMT167+anti-PD-1, 6 mice each, and the specific treatment method and tumor volume detection method were the same as in example 2. Subcutaneous tumor tissue was isolated and volume was measured in each group of mice 25 days after tumor bearing (B of fig. 4); from day 7, the long diameter a and the short diameter b of each group of mouse tumors were measured every 3 days using vernier calipers, and the volume v=1/2 ab was calculated according to the formula 2 Tumor volumes were calculated and mouse tumor proliferation curves were plotted (C of fig. 4). The survival time of each group of subcutaneous tumor-bearing mice was recorded and statistically analyzed (D of fig. 4).
From the results shown in FIG. 4, it can be seen that the tumor growth rate of the drug-resistant cell line CMT167-R tumor-bearing mice treated with the PD-1 antibody was still high, and the tumor growth rate was not significantly different from that of the isotype control IgG-treated group, indicating that the drug-resistant cell line had PD-1 antibody resistance (B and C; p >0.05 in FIG. 4), and that the survival period of the drug-resistant cell line CMT167-R tumor-bearing mice treated with the PD-1 antibody was not significantly different from that of the isotype control IgG-treated group (D; p >0.05 in FIG. 4).
In conclusion, the invention can obtain the corresponding PD-1 antibody drug-resistant cell line CMT167-R by adopting a mouse in-vivo model screening method for lung cancer cells CMT 167. Through a cell functional experiment, the in vitro proliferation and migration capacity of a lung cancer PD-1 antibody drug-resistant cell line CMT167-R is found to be unchanged; the mouse tumor-bearing experiment shows that the in vivo proliferation capacity of the lung cancer PD-1 antibody drug-resistant cell line CMT167-R is enhanced and the tolerance to PD-1 antibody treatment is obviously increased. Successfully obtain a lung cancer PD-1 antibody drug-resistant cell line CMT167-R.
According to the method, the lung cancer PD-1 antibody drug-resistant cell line can be prepared through multiple experiments, so that a person skilled in the art can repeat the technical scheme provided by the invention, the technical problem to be solved by the invention can be solved, and the effect of the technical scheme is achieved.
The above embodiments are only illustrative of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and various modifications and improvements made by those skilled in the art to the technical solutions of the present invention should fall within the protection scope defined by the claims of the present invention without departing from the design spirit of the present invention.
Claims (10)
1. The lung cancer immunotherapy drug-resistant cell line CMT167-R is characterized in that the drug-resistant cell line CMT167-R is preserved in China center for type culture Collection, the preservation date is 2023 and 1 month 8 days, and the preservation number is CCTCC NO: C202307.
2. The drug resistant cell line CMT167-R of claim 1, wherein the construction method comprises the steps of:
(1) Constructing a mouse subcutaneous tumor model by using a lung cancer cell line CMT167, treating by using an immune checkpoint PD-1 antibody, and screening tumors with the largest volume after treatment;
(2) Separating and screening tumor tissues with the largest volume under the sterile condition, cutting, culturing and passaging in a culture medium to obtain primary tumor cells, continuously constructing a mouse subcutaneous tumor model by using the primary tumor cells, treating by using an immune checkpoint PD-1 antibody, and screening tumors with the largest volume after treatment;
(3) Repeating the step (2) until the tumor volume of the immune checkpoint PD-1 antibody treatment group is not significantly different from that of the control group, and screening the tumor with the largest treatment group volume;
(4) Separating the tumor tissue obtained in the step (3) under the aseptic condition, cutting, culturing in a culture medium and stably passaging to obtain the lung cancer immunotherapy drug-resistant cell line CMT167-R.
3. The drug resistant cell line CMT167-R of claim 2, wherein in step (1) the lung cancer cell line is a murine lung cancer cell line.
4. The drug resistant cell line CMT167-R of claim 2, wherein in step (2) the medium is DMEM medium containing 10% fetal bovine serum.
5. The drug resistant cell line CMT167-R of claim 2, wherein in step (4) the conditions of the culture are 37 ℃, CO 2 The volume concentration was 5%.
6. Use of the lung cancer immunotherapy drug-resistant cell line CMT167-R according to claim 1 for screening anti-tumor drug targets.
7. Use of the lung cancer immunotherapy drug-resistant cell line CMT167-R according to claim 1 for screening anti-tumor drugs.
8. Use of the lung cancer immunotherapy drug-resistant cell line CMT167-R according to claim 1 for the preparation of an antitumor drug.
9. Use of the lung cancer immunotherapy drug-resistant cell line CMT167-R of claim 1 for screening test reagents for assessing anti-tumor effects.
10. Use of the lung cancer immunotherapy drug-resistant cell line CMT167-R of claim 1 for the preparation of a test reagent for assessing anti-tumor effects.
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