A method for constructing an aneuploid ovarian cancer cell model

Abstract

The application discloses a construction method of aneuploid ovarian cancer cell model. The construction method of the aneuploid ovarian cancer cell model comprises the following steps: co-culturing cytochalasin D and ovarian cancer cells in a first culture medium to obtain the aneuploid ovarian cancer cell model. The cytochalasin D is used to induce the ovarian cancer cells, which can induce the diploid and near-diploid ovarian cancer cells into aneuploid ovarian cancer cells. After the aneuploid ovarian cancer cells are inoculated into nude mice, the nude mice have tumorigenicity, still maintain a high proportion of aneuploid ovarian cancer cells, and can be used for researching the occurrence and development mechanism of ovarian cancer and tumor intervention, and can also be used for researching the curative effect of a drug for treating ovarian cancer in vitro, in vivo and in clinic, and providing new test materials for researching the occurrence, development, drug resistance, metastasis and biomarker of ovarian cancer.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a method for constructing an aneuploid ovarian cancer cell model. Background Technology

[0002] Chromosomal instability (CIN) refers to the increased frequency of chromosome addition or deletion in cells, leading to continuous changes in karyotype. It is a manifestation of genetic / genomic instability at the chromosomal level. Depending on the type of chromosomal change, it includes structural and numerical instability. Numerical instability refers to the cell's inability to correctly distribute replicated chromosomes during mitosis, resulting in the cell gaining or losing entire chromosomes. Chromosomal instability can accelerate the loss of heterozygotes of tumor suppressor genes and / or effectively amplify proto-oncogenes, thereby leading to tumorigenesis.

[0003] Aneuploidy refers to a chromosome number that is not an integer multiple of that of gametes. It is caused by chromosomal instability resulting in the gain or loss of chromosomes or chromosome arms, and is a prominent feature of malignant tumors. Approximately 90% of solid tumors and 75% of hematologic malignancies contain aneuploid cells, and 30%–60% of tumors have undergone genome-wide doubling. The occurrence of aneuploidy has a significant impact on tumorigenesis and development. Due to the vulnerability of tumor cells caused by aneuploidy in terms of energy metabolism and genomic stability, it may become a new target for tumor control and treatment.

[0004] Most ovarian cancer patients are diagnosed at an advanced stage, resulting in poor prognosis and treatment outcomes. As a copy number variation-driven tumor, more than 50% of epithelial ovarian cancers undergo genome-wide doubling events, exhibiting aneuploid karyotypes. Studies have confirmed that advanced high-grade serous ovarian cancer has a higher ploidy than early-stage patients, and the more prevalent aneuploidy events in advanced ovarian cancer suggest a poor prognosis. However, the role of aneuploid cells and genome-wide doubling events in the development and progression of ovarian cancer, and their implications for disease intervention, still require further investigation.

[0005] Given the importance of establishing aneuploid cells in ovarian cancer research, this application provides a technique for inducing and applying aneuploid cells, offering a valuable in vivo and in vitro model for further exploring the mechanisms of ovarian cancer development and tumor intervention. Summary of the Invention

[0006] In view of the shortcomings of the prior art described above, the purpose of this invention is to provide a method for constructing an aneuploid ovarian cancer cell model to solve the problems in the prior art.

[0007] To achieve the above and other related objectives, the present invention is obtained through the following technical solution.

[0008] One objective of this invention is to provide a method for constructing an aneuploid ovarian cancer cell model, comprising the following steps:

[0009] Cytochalasin D and ovarian cancer cells were co-cultured in the first culture medium to obtain the aneuploid ovarian cancer cell model.

[0010] In some embodiments, the ovarian cancer cells are selected from human ovarian cancer cells or mouse ovarian cancer cells.

[0011] In some embodiments, the ovarian cells are epithelial ovarian cells.

[0012] In some specific embodiments, the human ovarian cancer cells are selected from one or both of A2780 and OVCAR8.

[0013] In some specific embodiments, the murine ovarian cancer cells are selected from OV2944-HM1.

[0014] In some embodiments, the amount of cytochalasin D added is 1.5 to 5.0 μg / mL, based on the total volume of the first culture medium and cytochalasin D.

[0015] In some embodiments, the co-culture time is 43 to 53 hours.

[0016] In some embodiments, the co-culture temperature is 25–42°C.

[0017] In some embodiments, the first culture medium does not contain FBS.

[0018] In some embodiments, the co-culture further includes post-treatment, which includes washing and continuing culturing in a second culture medium.

[0019] In some specific implementations, the continued culture time is 19 to 29 hours.

[0020] In some specific embodiments, the temperature for continued cultivation is 25–42°C.

[0021] In some specific embodiments, the second culture medium contains FBS.

[0022] In some more specific embodiments, the concentration of FBS in the second culture medium is 5–15 wt%.

[0023] The second objective of this invention is to provide an aneuploid ovarian cancer cell model obtained by the construction method described above.

[0024] The third objective of this invention is to provide a method for constructing an aneuploid ovarian cancer animal model, comprising the following steps:

[0025] Animals were given an effective dose of the aneuploid ovarian cancer cell model described above to obtain the aneuploid ovarian cancer animal model.

[0026] In some embodiments, the effective dose is 1×10 6 ~1×10 8 One cell per animal.

[0027] In some embodiments, the animal is selected from mammals.

[0028] In some specific embodiments, the animal is a mouse.

[0029] The fourth objective of this invention is to provide the use of the aneuploid ovarian cancer cell model as described above or the aneuploid ovarian cancer animal model constructed by the construction method described above in at least one of the following:

[0030] 1) Screening for drugs to prevent and / or treat ovarian cancer;

[0031] 2) To study the selection of treatment options and / or prognostic assessment for ovarian cancer;

[0032] 3) Study the pathogenesis of ovarian cancer.

[0033] The fifth objective of this invention is to provide a method for screening candidate drugs for the prevention and / or treatment of ovarian cancer, wherein the candidate drugs are applied to an aneuploid ovarian cancer cell model as described above or an aneuploid ovarian cancer animal model constructed by the construction method described above.

[0034] In some embodiments, the method may further include screening for candidate drugs for the prevention and / or treatment of ovarian cancer based on changes in aneuploid ovarian cancer cell models or aneuploid ovarian cancer animal models in which the candidate drugs have been administered.

[0035] This invention has discovered that cytochalasin D can induce ovarian cancer cells into aneuploid ovarian cancer cells, such as tetraploid and octoploid cells, through induction. The aneuploid ovarian cancer cells formed after induction grow in a monolayer adherent state, losing contact inhibition, exhibiting a shrunken, elongated spindle-shaped morphology, and increased volume. Passaging maintains their aneuploidy. When aneuploid ovarian cancer cells are inoculated into nude mice, they exhibit tumorigenicity, and approximately 30% of the tumor tissue remains aneuploid. Therefore, the aneuploid ovarian cancer cell model and animal model of this application can be used to study the mechanisms of ovarian cancer development and tumor intervention. They can also be used to study the in vitro, in vivo, and clinical efficacy of drugs for the prevention and / or treatment of ovarian cancer (especially chromosomal instability-targeted antitumor drugs), and to provide new experimental materials for the study of ovarian cancer occurrence, development, drug resistance, metastasis, and biomarkers.

[0036] Compared with the prior art, the present invention has the following beneficial effects:

[0037] 1) Aneuploid cells obtained using the cell model constructed in this application exhibit monolayer adherent growth, loss of contact inhibition, and a shrunken, elongated spindle-shaped cell morphology with increased volume.

[0038] 2) The aneuploid cells obtained by the cell model constructed in this application grow well in vitro and can maintain their aneuploidy after passage. After being inoculated into nude mice, they exhibit tumorigenicity in nude mice, and a high proportion of aneuploid cells are still maintained in the tumor tissue of nude mice.

[0039] 3) The chromosome karyotype analysis results of aneuploid cells obtained using the cell model constructed in this application are polyploid. Attached Figure Description

[0040] Figure 1 The images shown are morphological diagrams of human ovarian cancer cells A2780 in the embodiments of the present invention and human ovarian cancer cells OVCAR8 in the embodiment 2 after treatment with cytochalasin D for 48 hours.

[0041] Figure 2 The image shown is a comparison of the cell morphology of diploid human ovarian cancer cells A2780 and aneuploid cells A2780-aneuploid under a light microscope in Example 1 of the present invention.

[0042] Figure 3 The image shown is a karyotype of diploid human ovarian cancer cells A2780 before induction and after induction with cytochalasin D in Example 1 of the present invention.

[0043] Figure 4The image shown is a karyotype of human ovarian cancer cells OVCAR8 in Example 2 of the present invention before induction and after induction with cytochalasin D.

[0044] Figure 5 The image shown is a karyotype of mouse ovarian cancer cells OV2944-HM1 before induction and after induction with cytochalasin D in Example 3 of the present invention.

[0045] Figure 6 The images shown are flow cytometry analysis diagrams of human ovarian cancer cells A2780 (Example 1), human ovarian cancer cells OVCAR8 (Example 2), and mouse ovarian cancer cells OV2944-HM (Example 3) before induction and after induction with cytochalasin D.

[0046] Figure 7 The image shown is a diagram of the morphological analysis of the cell nuclei of an ex vivo tumor tissue specimen from a nude mouse in Example 4 of this invention.

[0047] Figure 8 The image shown is a karyotype diagram of primary cultured nude mouse tumor cells in Example 4 of this invention.

[0048] Figure 9 The diagram shows the ploidy values ​​of primary cultured nude mouse tumors in Example 4 of this invention.

[0049] Figure 10 The images shown are of the naked mouse body and tumor in Embodiment 4 of this invention. Detailed Implementation

[0050] This invention prepares an aneuploid ovarian cancer cell model by using cytochalasin D to block the cytokinesis process of an ovarian cancer cell line. The model was then validated using a nude mouse subcutaneous xenograft tumor model. The aneuploid ovarian cancer cells of this invention demonstrated tumorigenicity, and digital pathological analysis of tumor tissue and karyotype analysis of primary tumor cells confirmed that this in vivo tumorigenic model remains an aneuploid model. Based on this, the invention was completed.

[0051] The first aspect of the present invention provides a method for constructing an aneuploid ovarian cancer cell model, comprising the following steps:

[0052] Cytochalasin D and ovarian cancer cells were co-cultured in the first culture medium to obtain the aneuploid ovarian cancer cell model.

[0053] In this invention, the ovarian cancer cells are selected from human ovarian cancer cells or mouse ovarian cancer cells. Preferably, the ovarian cells are epithelial ovarian cells; more preferably, the human ovarian cancer cells are selected from one or both of A2780 and OVCAR8, and the mouse ovarian cancer cells are selected from OV2944-HM1.

[0054] In this invention, based on the total volume of the first culture medium and cytochalasin D, the dosage of cytochalasin D added is 1.5–5.0 μg / mL. Preferably, the dosage of cytochalasin D added can be 1.5–2.5 μg / mL, 2.0–3.5 μg / mL, 3.0–4.5 μg / mL, or 4.0–5 μg / mL; in a preferred embodiment, it is 3.0 μg / mL.

[0055] In this invention, the first culture medium does not contain FBS. The first culture medium is RPMI 1640 cell culture medium, but other similar culture media can also be used for culturing.

[0056] In this invention, the first culture medium contains an antibiotic with a final concentration of 1%, wherein the antibiotic is selected from one or both of penicillin and streptomycin, preferably penicillin and streptomycin.

[0057] In this invention, the co-culturing time is 43–53 hours. Preferably, the co-culturing time can be 43–48 hours, 46–50 hours, or 49–53 hours; in a preferred embodiment, it is 48 hours. The co-culturing temperature is 25–42°C. Preferably, the co-culturing temperature can be 25–32°C, 30–38°C, or 35–42°C; in a preferred embodiment, it is 37°C.

[0058] In this invention, the co-culture further includes post-treatment, which includes washing and continued culturing in a second culture medium. The purpose of washing is to remove cytochalasin D; any means to achieve this purpose can be used, such as washing with PBS. The number of washes can be selected as needed, for example, 4-8 washes, 6-7 washes. The aneuploid ovarian cancer cells obtained by this invention can achieve long-term growth and stable passage in vitro. Generally, the second culture medium is RPMI 1640 cell culture medium or MEM-α cell culture medium, but other similar media can also be used for culturing. Various methods that can be expected to be used to culture this aneuploid ovarian cancer cell are included in this invention. For example, the second culture medium contains FBS; preferably, the concentration of FBS in the second culture medium is 5-15 wt%, for example, 5-10 wt%, or 5-13 wt%, or 8-15 wt%, or 10 wt%. As another example, the second culture medium contains an antibiotic at a final concentration of 1%, the antibiotic being selected from one or both of penicillin and streptomycin, preferably penicillin and streptomycin. The continued incubation period is 19–29 hours, for example, 19–25 hours, 23–28 hours, 25–29 hours, or 24 hours. Preferably, the continued incubation temperature is 25–42°C, for example, 25–32°C, or 30–38°C, or 35–42°C, or 37°C. The humidity during continued incubation is saturated humidity, and the gaseous environment during continued incubation is 5% CO2 / 95% air.

[0059] The aneuploid ovarian cancer cells of this invention can grow and stably passage in vitro for a long time. They are epithelial-like, lose contact inhibition, and have non-holistic chromosomes, such as tetraploid and octoploid. The chromosome data are concentrated at 80-92, 92-110, and 149-179. After inoculation into nude mice, the tumor formation rate is over 100%, and primary culture can still maintain about 30% of aneuploid cells.

[0060] Another aspect of the present invention provides an aneuploid ovarian cancer cell model obtained by the construction method described above.

[0061] In this invention, the aneuploid ovarian cancer cell model is used for research on the occurrence, development, or metastasis of ovarian cancer.

[0062] Another aspect of the present invention provides a method for constructing an aneuploid ovarian cancer animal model, comprising the following steps:

[0063] Animals were given an effective dose of the aneuploid ovarian cancer cell model described above to obtain the aneuploid ovarian cancer animal model.

[0064] In this invention, the effective dose is 1×10⁻⁶. 6 ~1×108 1 cell / animal introduced. Preferably, 1 × 10⁻⁶. 7 One cell per animal introduced.

[0065] In this invention, the animal is selected from mammals. Preferably, it is a mouse. More preferably, it is a Balb / c-nu female nude mouse, aged 4-6 weeks. Those skilled in the art can choose a suitable method to construct an aneuploid ovarian cancer animal model, for example, digesting aneuploid ovarian cancer cells into a single-cell suspension using trypsin, and introducing the single-cell suspension into the animal. The method of introducing the animal is a method commonly used by those skilled in the art, such as subcutaneous injection. For example, in one specific embodiment, the aneuploid ovarian cancer cells are digested with trypsin to form a cell suspension, and the cell suspension is subcutaneously injected into the nude mouse through the mid-posterior axilla.

[0066] Another aspect of the present invention provides the use of the aneuploid ovarian cancer cell model as described above or the aneuploid ovarian cancer animal model constructed by the construction method described above in at least one of the following:

[0067] 1) Screening for drugs to prevent and / or treat ovarian cancer;

[0068] 2) To study the selection of treatment options and / or prognostic assessment for ovarian cancer;

[0069] 3) Study the pathogenesis of ovarian cancer.

[0070] The pathogenesis of ovarian cancer described in this invention includes the assessment of the occurrence, development, drug resistance, and metastasis of ovarian cancer, as well as the screening of biomarkers for ovarian cancer.

[0071] Another aspect of the present invention provides a method for screening candidate drugs for the prevention and / or treatment of ovarian cancer, wherein the candidate drug is applied to an aneuploid ovarian cancer cell model as described above or an aneuploid ovarian cancer animal model constructed by the construction method described above.

[0072] This invention may further include: screening candidate drugs for the prevention and / or treatment of ovarian cancer based on changes in ovarian cancer cell models or aneuploid ovarian cancer animal models treated with the candidate drug. Generally, after administration of the candidate drug, candidate drugs for the prevention and / or treatment of ovarian cancer can be screened based on changes in aneuploid ovarian cancer cell models and / or aneuploid ovarian cancer animal models treated with the candidate drug. For example, aneuploid ovarian cancer cell models and / or aneuploid ovarian cancer animal models treated with the candidate drug can be compared with control models (e.g., aneuploid ovarian cancer cell models and / or aneuploid ovarian cancer animal models not treated with the candidate drug, and near-diploid ovarian cancer cell models and / or near-diploid ovarian cancer animal models treated with the candidate drug) to observe changes in the aneuploid ovarian cancer cell models and / or aneuploid ovarian cancer animal models treated with the candidate drug (e.g., whether the aneuploid ovarian cancer cell models and / or aneuploid ovarian cancer animal models treated with the candidate drug show significant sensitivity to ovarian cancer), thereby enabling the screening of candidate drugs.

[0073] This invention utilizes cytochalasin D to induce ovarian cancer cells, which can induce diploid, near-diploid, and human or mouse-derived ovarian cancer cells into aneuploid ovarian cancer cells, such as tetraploid and octaploid. The aneuploid ovarian cancer cells formed after induction grow in a monolayer adherent structure, lose contact inhibition, exhibit a shrunken, elongated spindle-shaped morphology, and increase in size. Passaging maintains their aneuploidy. When aneuploid ovarian cancer cells are inoculated into nude mice, they exhibit tumorigenicity, and approximately 30% of the tumor tissue remains aneuploid. Therefore, the aneuploid ovarian cancer cell model and animal model of this application can be used to study the mechanisms of ovarian cancer development and tumor intervention. They can also provide new experimental materials for studying the efficacy of in vitro, in vivo, and clinical antitumor drugs (especially chromosomal instability-targeted antitumor drugs), as well as for research on the occurrence, development, drug resistance, metastasis, and biomarkers of ovarian cancer. Furthermore, they contribute to the further development of aneuploidy in ovarian cancer research.

[0074] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification.

[0075] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention. Test methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions or as recommended by the respective manufacturers.

[0076] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, apparatus, and materials used in the embodiments, based on the knowledge of the prior art possessed by one of ordinary skill in the art and the description of this invention, any prior art methods, apparatus, and materials similar to or equivalent to those described, apparatus, and materials in the embodiments of this invention may be used to implement the present invention.

[0077] In the following examples of this application, each 100 mL of RPMI 1640 medium and MEM-α medium contains 1 mL of stock solution, which contains 10,000 units / mL of penicillin and 10,000 μg / mL of streptomycin.

[0078] In the following embodiments of this application, cytochalasin D (or Cyto D.) was purchased from Aladdin Company (Cytochalasin D, C102396), dissolved in DMSO to form a cytochalasin D solution with a concentration of 1 mg / mL, and stored at -20°C as a stock solution; the chemical formula of cytochalasin D is as follows:

[0079]

[0080] Example 1: Aneuploidy Induction of Diploid Human Ovarian Cancer Cell Line A2780

[0081] In this Example 1, the human ovarian cancer cell line A2780 was used as the target cell line. Cytochalasin D was used to induce it into aneuploid cells, including the following steps:

[0082] 1.1 Drug treatment

[0083] Once the confluence of human ovarian cancer cell line A2780 reached 60%, the medium was replaced with RPMI 1640 medium (containing 1% penicillin and streptomycin) without FBS. After treatment with cytochalasin D at a final concentration of 3.0 μg / mL for 48 hours, cell morphology was observed. Results are shown in [Figure number missing]. Figure 1 .

[0084] 1.2 Cleaning

[0085] After drug withdrawal, the cells were washed 6-7 times with PBS to remove drug residues, and then replaced with RPMI 1640 medium containing 10% FBS (1% penicillin + streptomycin). 24 hours after drug withdrawal, the cells increased in size and took on an elongated spindle shape, yielding aneuploid cells (A2780-aneuploid).

[0086] from Figure 1 It can be seen that after 48 hours of treatment with cytochalasin, diploid human ovarian cancer cells A2780 shrank and became rounded, and stopped proliferating.

[0087] The diploid human ovarian cancer cells A2780 and aneuploid cells A2780–aneuploid induced by cytochalasin D were observed under a light microscope in this embodiment. The results are shown in [Figure number missing]. Figure 2 .

[0088] from Figure 2 It can be seen that after drug withdrawal, the cells resumed mitosis, and the A2780-aneuploid cells generated after induction increased in size and were elongated spindle-shaped.

[0089] After adherence, aneuploid cells were digested with 0.25% trypsin and then subjected to ploidy identification, which included G-banding karyotype detection and flow cytometry to identify the karyotype. Results are shown in [Figure number missing]. Figure 3 and Figure 6 .

[0090] Figure 3 This is a karyotype of human ovarian cancer cells A2780 from Example 1 before induction and after induction with cytochalasin D.

[0091] from Figure 3 It can be seen that, compared with before induction, the number of chromosomes in aneuploid cells induced by cytochalasin D doubled. After counting and statistical analysis of 100 cell karyotypes, the number of chromosomes in the cells was determined to be 80-92.

[0092] from Figure 6 As can be seen from the flow cytometry results, the DNA content of aneuploid tumor cells induced by cytochalasin D doubled, the number of chromosomes in aneuploid cells doubled, and the cells were nearly tetraploid karyotypes.

[0093] The in vitro culture conditions for aneuploid ovarian cancer cells (A2780–aneuploid) were as follows: RPMI1640 cell culture medium containing 10% FBS and 1% penicillin-dextrose antibody, culture temperature of 37℃, gas environment of 5% CO2 / 95% air, and saturated humidity.

[0094] Example 2: Aneuploidy induction of near-diploid human ovarian cancer cell line OVCAR8

[0095] In this Example 2, the human ovarian cancer cell line OVCAR8 was used as the target cell line, and it was induced into aneuploid cells using cytochalasin D, including the following steps:

[0096] 2.1 Drug treatment

[0097] Once the cell confluence reached 60%, the medium was replaced with FBS-free RPMI 1640 medium (1% penicillin-dextrose antibody), and cytochalasin D was added to a final concentration of 3.0 μg / mL. The cells were then treated at 37°C for 48 hours. Cell morphology was then observed, and the results are shown below. Figure 1 .

[0098] 2.2 Cleaning

[0099] After drug withdrawal, wash the cells 6-7 times with PBS to remove drug residue, and replace with RPMI 1640 medium containing 10% FBS (1% penicillin-dextrose antibody). 24 hours after drug withdrawal, the cell volume increased.

[0100] Adherent aneuploid cells were digested with 0.25% trypsin and then subjected to ploidy identification, which included karyotype detection and flow cytometry analysis of the karyotype. Results are shown below. Figure 4 and Figure 6 .

[0101] from Figure 1 It can be seen that after 48 hours of treatment with cytochalasin, the human ovarian cancer cell line OVCAR8 shrank and became rounded, and stopped proliferating.

[0102] Figure 4 This is a karyotype of human ovarian cancer cells OVCAR8 in this embodiment before induction and after induction with cytochalasin D.

[0103] from Figure 4 It can be seen that the G-banding karyotype detection results show that, after counting and statistically analyzing 100 cell karyotypes, chromosome karyotype analysis determined that the number of chromosomes in the cells was 92-110.

[0104] from Figure 6 As can be seen from the flow cytometry results, the DNA content of aneuploid tumor cells induced by cytochalasin D doubled, the number of chromosomes in aneuploid cells doubled, and the cells were nearly tetraploid karyotypes.

[0105] The in vitro culture conditions for aneuploid ovarian cancer cells were as follows: RPMI 1640 cell culture medium containing 10% FBS and 1% penicillin-dextrose antibodies, culture temperature of 37℃, gas environment of 5% CO2 / 95% air, and humidity of saturated humidity.

[0106] Example 3: Induction of aneuploidy in mouse ovarian cancer cells OV2944-HM1

[0107] In this Example 3, murine ovarian cancer cells OV2944-HM1 were used as the target cells. Cytochalasin D was used to induce them into aneuploid cells, including the following steps:

[0108] 3.1 Drug treatment

[0109] When the confluence of mouse ovarian cancer cell line OV2944-HM1 reached 60%, the medium was replaced with RPMI 1640 medium (1% penicillin-dextrose antibody) without FBS. After treatment with cytochalasin D at a final concentration of 3.0 μg / mL for 48 h, the cells were observed to shrink and become rounded.

[0110] 3.2 Cleaning

[0111] After drug withdrawal, the cells were washed 6-7 times with PBS to remove drug residues, and then the medium was replaced with MEM-α medium containing 10% FBS (1% penicillin-dextrose antibody). 24 hours after drug withdrawal, the cells increased in size and took on an elongated spindle shape.

[0112] After adherence, aneuploid cells were digested with 0.25% trypsin and then subjected to ploidy identification, which included G-banding karyotype detection and flow cytometry to identify the karyotype. Results are shown in [Figure number missing]. Figure 5 and Figure 6 .

[0113] Figure 5 This is a karyotype of mouse ovarian cancer cells OV2944-HM1 before induction and after induction with cytochalasin D in this embodiment.

[0114] from Figure 5 The G-banding karyotype analysis showed that 100 cell karyotypes were counted and analyzed. Chromosomal karyotype analysis determined the number of chromosomes in the cells to be 149-179.

[0115] from Figure 6 As can be seen from the flow cytometry results, the DNA content of aneuploid tumor cells induced by cytochalasin D doubled, the number of chromosomes in aneuploid cells doubled, and the cells were nearly octoploid karyotypes.

[0116] Cell culture: Aneuploid ovarian cancer cells induced were cultured in vitro under the following conditions: MEM-α cell culture medium containing 10% FBS and 1% penicillin-dextrin, culture temperature of 37℃, gas environment of 5% CO2 / 95% air, and humidity of saturated humidity.

[0117] Example 4: Construction of a subcutaneous tumor model in nude mice

[0118] In this Example 4, the aneuploid ovarian cancer cells obtained in Example 1 were used to construct a nude mouse subcutaneous tumor model, including the following:

[0119] 4.1 Cell Culture

[0120] In vitro cell culture conditions: RPMI 1640 cell culture medium containing 10% FBS and 1% penicillin antibiotics, culture temperature of 37℃, gas environment of 5% CO2 / 95% air, and saturated humidity.

[0121] 4.2 Model Construction

[0122] Adherent cells were digested into a single-cell suspension using 0.25% trypsin. 4-6 week old female nude mice (Balb / c-nu) were selected, and 100-150 μL of the cell suspension (containing 1×10⁻⁶ cells) was subcutaneously injected into the mid-posterior axilla of the mice. 7 A subcutaneous tumor model was constructed in nude mice using 3 cells, and tumor growth was observed for 2 weeks. The experimental group was injected with induced aneuploid human ovarian cancer cells A2780 (labeled as hyperploid or hyperploid injected), while the control group was injected with uninduced diploid human ovarian cancer cells A2780 (labeled as diploid or diploid injected). Each group consisted of 3 nude mice.

[0123] 4.3 Primary Culture

[0124] Nude mice were sacrificed after tumor formation to obtain fresh, excised tumor specimens. These specimens were washed with PBS to remove connective and necrotic tissue. The tumor tissue was minced and digested into a single-cell suspension using type IV collagenase. After filtration, the suspension was washed with PBS and centrifuged to obtain the cell pellet. The cell suspension was seeded onto the walls of T25 culture flasks and incubated statically (culture conditions as in 4.1).

[0125] Morphological analysis and ploidy identification: Fresh tumor resection specimens were obtained from outside the body, embedded in paraffin, sectioned, and the cell nuclear morphology was analyzed by digital pathology; adherent cells from primary culture were digested and subjected to chromosome karyotype analysis and chromosome counting to obtain cell ploidy data.

[0126] Figure 7 This is a diagram showing the morphological analysis of cell nuclei in an ex vivo tumor tissue specimen from a nude mouse in this embodiment.

[0127] from Figure 7 It can be seen that the nude mouse tumor primary cells have smaller nuclei, lighter color, and lower integrated optical density (IOD) values; while the tetraploid cells have larger nuclei, darker color, and higher IOD values, indicating that the ovarian cancer cells induced in vitro still maintain their polyploidity after tumor formation in vivo.

[0128] Figure 8 This is a diagram of the karyotype of cells from primary culture of tumor tissue in nude mice in this embodiment.

[0129] from Figure 8 It can be seen that the primary nude mouse tumor cells derived from diploid cells are generally diploid, with a chromosome number of 46; while the primary nude mouse tumor cells derived from aneuploid ovarian cancer cells in Example 1 are nearly tetraploid, with a chromosome number of 80-92.

[0130] Figure 9This is a numerical diagram of cell ploidy in primary culture of nude mouse tumors in this embodiment.

[0131] Figure 10 These are photographs of the naked mouse's gross body and tumor in this embodiment.

[0132] from Figure 9 It can be seen that primary tumor cells from nude mice derived from diploid cells are almost entirely diploid, with tetraploid tumor cells accounting for less than 5%; while primary tumor cells from nude mice derived from tetraploid cells contain a certain proportion of tetraploid cells, with tetraploid tumor cells accounting for more than 30%. Figure 9 In the figure, the horizontal axis 1, 2 and 3 are the numbers of the three nude mice, and the vertical axis is the percentage of polyploid cells in the primary cultured tumor cells of the nude mice.

[0133] from Figure 10 It can be seen that tumors grew in all nude mice after inoculation with aneuploid cells, with a tumor formation rate of 100%, indicating that the aneuploid ovarian cancer cells of the present invention have a strong tumor formation ability and a tumor formation rate of 100%, making them suitable for use as clinical tumor cell models.

[0134] In summary, this invention utilizes cytochalasin D to induce ovarian cancer cells, which can induce diploid, near-diploid, and human or mouse-derived ovarian cancer cells into aneuploid ovarian cancer cells, such as tetraploid and octoploid cells. The aneuploid ovarian cancer cells formed after induction exhibit monolayer adherent growth, lose contact inhibition, and show a shrunken, elongated spindle-shaped cell morphology with increased volume. Passaging maintains their aneuploidy. When aneuploid ovarian cancer cells are inoculated into nude mice, they exhibit tumorigenicity, and approximately 30% of the tumor tissue remains aneuploid. Therefore, the aneuploid ovarian cancer cell model and animal model of this application can be used to study the mechanisms of ovarian cancer development and tumor intervention. They can also be used to prevent and / or study the efficacy of drugs for treating ovarian cancer (especially chromosomal instability-targeted anti-ovarian cancer drugs) in vitro, in vivo, and clinically. Furthermore, they provide new experimental materials for the study of ovarian cancer occurrence, development, drug resistance, metastasis, and biomarkers, and contribute to further in-depth research in the field of aneuploidy within ovarian cancer.

[0135] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by those skilled in the art without departing from the spirit and technical concept disclosed in the present invention should still be covered by the claims of the present invention.

Claims

1. A method for constructing an aneuploid ovarian cancer cell model, characterized in that, Includes the following steps: Cytochalasin D and ovarian cancer cells were co-cultured in the first culture medium to obtain the aneuploid ovarian cancer cell model described above. The ovarian cancer cells are selected from human ovarian cancer cells or mouse ovarian cancer cells. The human ovarian cancer cells are selected from one or both of A2780 and OVCAR8, and the mouse ovarian cancer cells are selected from OV2944-HM1. The first culture medium does not contain FBS. Based on the total volume of the first culture medium and cytochalasin D, the amount of cytochalasin D added is 1.5 to 5.0 μg / mL, and the co-culture time is 43 to 53 h. The co-culture also includes post-treatment, which includes washing and continued culturing in a second culture medium containing FBS; the concentration of FBS in the second culture medium is 5-15 wt%, and the chromosomes of the aneuploid ovarian cancer cell model are 80-110 or 149-179.

2. The construction method as described in claim 1, characterized in that, The co-culture temperature is 25–42°C.

3. The construction method as described in claim 2, characterized in that, The continued culture time is 19–29 hours; And / or, the temperature for continued cultivation is 25–42°C.

4. An aneuploid ovarian cancer cell model obtained by the construction method according to any one of claims 1-3.

5. A method for constructing an animal model of aneuploid ovarian cancer, characterized in that, Includes the following steps: An aneuploid ovarian cancer animal model is obtained by administering an effective dose of the aneuploid ovarian cancer cell model as described in claim 4 to animals, wherein the animals are selected from mammals.

6. The construction method as described in claim 5, characterized in that, The effective dose is 1×10 6 ~1×10 8 One cell per animal.

7. The construction method as described in claim 5, characterized in that, The mammal in question is a mouse.

8. The use of the aneuploid ovarian cancer cell model as described in any one of claims 1-3 or the aneuploid ovarian cancer animal model constructed by the construction method as described in any one of claims 5-7 in at least one of the following: 1) Screening for drugs to prevent and / or treat ovarian cancer; 2) To study the selection of treatment options and / or prognostic assessment for ovarian cancer; 3) Study the pathogenesis of ovarian cancer.

9. A method for screening candidate drugs for the prevention and / or treatment of ovarian cancer, characterized in that, The candidate drug is administered to an aneuploid ovarian cancer cell model as described in any one of claims 1-3 or to an aneuploid ovarian cancer animal model constructed using the construction method described in any one of claims 5-7.

10. The method as described in claim 9, characterized in that, Also includes: Candidate drugs for the prevention and / or treatment of ovarian cancer are screened by observing changes in aneuploid ovarian cancer cell models or aneuploid ovarian cancer animal models that have been treated with candidate drugs.

Images