Method for co-culturing double tumor species and human embryonic stem cells to enhance curative effect of supernatant and expand tumor spectrum

Abstract

The invention provides a method for enhancing the curative effect of supernatant and expanding the tumor spectrum through co-culture of double tumor species and human embryonic stem cells. A traditional co-culture supernatant for treating tumors by human embryonic stem cells is still limited to a single tumor mode at present, and the prepared supernatant is insufficient in cancer suppression efficiency and treatment range. A large number of studies show that ovarian cancer and breast cancer have significant common points in multiple aspects, a direct contact co-culture system of double tumor species (ovarian cancer and breast cancer) and human embryonic stem cells is constructed, and the human embryonic stem cells are stimulated to generate rich and efficient cancer suppression factors by improving the diversity of tumor antigens. Experimental results show that co-culture of the double tumor species enhances the anti-tumor curative effect of the supernate and expands the tumor treatment spectrum of the supernate, a new technical path is provided for development of broad-spectrum anti-tumor biological treatment, an important foundation is laid for multi-cancer-species collaborative intervention and efficient biological therapy, and the method has wide application prospects.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to a method for enhancing the efficacy of the supernatant and expanding the treatment tumor spectrum by co-culturing two tumor types with human embryonic stem cells. Background Art

[0002] Ovarian cancer (OC) is a malignant tumor that commonly occurs in middle-aged and elderly women, and its incidence increases with age. Ovarian cancer is a malignant tumor of the reproductive system that seriously affects the health of women in China. There are usually no obvious symptoms in the early stage of ovarian cancer, and the earliest reason for seeking medical treatment is often the appearance of ascites or distant metastasis symptoms. Due to the long latency period and hidden early symptoms of ovarian cancer, it is difficult to detect it early, and most cases are already in the advanced stage when diagnosed.

[0003] In recent years, the incidence of breast cancer has been showing a continuous upward trend globally and has become one of the most common malignant tumors in women.

[0004] Both of these cancers are closely related to women's health, and their incidence rates are continuously rising globally, becoming important diseases threatening women's health. A large number of studies have shown that ovarian cancer and breast cancer have significant commonalities in many aspects. There are significant commonalities in the pathogenesis of ovarian cancer and breast cancer: both are hormone-dependent, share DNA repair defects caused by BRCA1 / 2 gene mutations, and abnormal activation of key signaling pathways such as PI3K / AKT / mTOR. These commonalities make the two types of tumors show cross characteristics in the metastasis pattern and treatment resistance mechanism. Therefore, co-culturing human embryonic stem cells with two types of cancer cells can dynamically simulate the stromal-epithelial interaction in the tumor microenvironment, capture the common targets of the two types of cancer cells in stemness maintenance and metabolic reprogramming through a direct co-culture model, and then screen broad-spectrum treatment strategies that can simultaneously block the progression of ovarian cancer and breast cancer, providing an innovative research platform for the development of precision therapies based on the common mechanisms of tumors.

[0005] Ovarian cancer and breast cancer show significant convergence in the metastasis mechanism. This study first established a system using direct contact co-culture, providing a scientific basis for the development of cross-cancer combined treatment strategies. By further studying the application potential of these common mechanisms in the treatment of cancer with co-culture supernatant, improving the anti-cancer efficiency, and expanding the scope of applicable tumor types, it provides an innovative method for the development of pan-cancer biotherapy strategies, builds a technical platform for the research and development of multi-tumor collaborative treatment strategies and highly efficient biotherapy means, and shows broad clinical application potential. Through the co-culture mode of two cancer types, the superimposed effect of tumor suppression efficacy and the diversified extension of treatment targets are realized, laying a technical foundation for constructing a biological intervention plan covering multiple malignant tumors, and having a profound development prospect in the field of oncology. Summary of the Invention

[0006] To solve the problems in the prior art, the present invention innovatively proposes a method for co-culturing two tumor types (breast cancer, ovarian cancer) with human embryonic stem cells to enhance the efficacy of the supernatant and expand the treatment tumor spectrum.

[0007] Due to the lack of simulation of tumor microenvironment interaction in the traditional co-culture system, the anti-cancer efficiency is limited, and the co-culture supernatant has specificity, with a limited treatment spectrum, and it cannot solve the treatment problems of breast cancer-ovarian cancer co-occurrence in clinical practice. The inventor found that when human embryonic stem cells are directly co-cultured with ovarian cancer and breast cancer cells in a three-cell contact manner, the two cancer cells can stimulate human embryonic stem cells and trigger anti-cancer biological effects. When the supernatant of this co-culture system is used for tumor treatment research, it is found that compared with the traditional single-cancer co-culture system (that is, the culture system containing only ovarian cancer or only breast cancer), the co-culture supernatant obtained by the method of the present invention shows significantly enhanced inhibitory efficiency in inhibiting key malignant phenotypes such as tumor migration, invasion, and proliferation. This achievement has opened up a new strategic path for the combined treatment of double tumors, showing important clinical transformation value in the field of breast cancer-ovarian cancer combined treatment, and laying a scientific foundation for the development of biotherapy with broad-spectrum anti-tumor effects. In the co-culture process of the present invention, a special embryonic stem cell culture medium is used to maintain the totipotency of human embryonic stem cells. Human embryonic stem cells are co-cultured with ovarian cancer and breast cancer at an appropriate cell ratio. It is found that during the co-culture process, if the ratio of the three types of cells is inappropriate, the addition time points of the two types of tumor cells are inaccurate, or the collection time and concentration are not suitable, the co-culture supernatant of human embryonic stem cells cannot produce a strong biological effect of inhibiting tumor growth.

[0008] Unless otherwise specified, the parts mentioned in the present invention are all parts by weight, and the percentages are all mass percentages.

[0009] To achieve the above object, the technical solution of the present invention is as follows:

[0010] A method for co-culturing two tumor types with human embryonic stem cells to enhance the efficacy of the supernatant and expand the treatment tumor spectrum, characterized by including the following steps:

[0011] Pre-coat Matrigel matrix glue to prepare a special culture plate for human embryonic stem cells;

[0012] Use the special culture plate for human embryonic stem cells prepared by pre-coating Matrigel matrix glue in step (1) to inoculate human embryonic stem cells;

[0013] Digest and passage human embryonic stem cells;

[0014] Take ovarian cancer and breast cancer cells in the logarithmic growth phase. After aspirating the culture medium, add DPBS buffer to wash the cells, digest them with EDTA at 32 - 42 °C for 1 minute and 30 seconds - 2 minutes, aspirate the supernatant, and resuspend the ovarian cancer and breast cancer cells into a single cell suspension with human embryonic stem cell complete medium. Add the two types of cells to the human embryonic stem cell culture system according to the ratio of 1:1:1 - 1:1:5 of human embryonic stem cells in the culture system, and co-culture the two types of cells in a CO2 cell incubator at 37 °C;

[0015] Put the co-culture system of the two types of cells into a CO2 cell incubator and collect the co-culture supernatant after 48 - 96 hours.

[0016] The human embryonic stem cells and pancreatic cancer cells described in the present invention are both established cell lines.

[0017] The special medium for human embryonic stem cells is PGM1 human pluripotent stem cell medium (Beijing Saibei Bio CA1007500), Y - 27632 (MCE USA HY10071).

[0018] In the process of stimulating embryonic stem cells, the present invention will use the specific PGM1 human pluripotent stem cell medium to keep embryonic stem cells in an undifferentiated state as much as possible.

[0019] The Matrigel matrix gel is produced by Corning (354277). The preparation method of the special culture plate for human embryonic stem cells is as follows: After thawing the Matrigel matrix gel on ice, dilute it with DPBS buffer according to the ratio (Matrigel:DPBS / PBS buffer = 1:80 - 100) and spread it into the culture plate, and place it in a 37 °C, 5% CO2 incubator for 4 - 5 hours until the matrix gel solidifies and forms, then it can be used.

[0020] The method for digesting and passaging human embryonic stem cells is as follows: Select human embryonic stem cells in the logarithmic growth phase. After aspirating the culture medium, add DPBS buffer to wash the cells for 20 - 30 seconds. Add 700 - 1000 μL of cell dissociation reagent, stem cell digestive solution, to each well, and place it in the incubator for 5 - 7 minutes. Observe under the microscope that there are obvious gaps between most cells inside the clone, and observe with the naked eye that the cell colony becomes opaque and white, indicating that the cell digestion time is ideal. Aspirate the stem cell digestive solution, add stem cell medium, gently blow down the cell mass with a pipette, slightly disperse it, and add it to the special culture plate for human embryonic stem cells that has been pre - paved with 1 mL of human embryonic stem cell complete medium.

[0021] When selecting human embryonic stem cells for sub - culture, select culture wells with a coverage rate of 50% - 60%, smooth clone edges and no differentiated cells, and the passage number is 2 - 10 generations.

[0022] The method for inoculating human embryonic stem cells is as follows: take the well plate in step (1), aspirate and discard the basic working solution for preparing Matrigel matrix gel, add DPBS buffer to wash the prepared matrix gel, add stem cell medium, and then evenly inoculate the cell mass suspension in step (2) into the prepared Matrigel matrix gel well plate, and change the medium after 24 h.

[0023] Ovarian cancer and breast cancer cells are used to stimulate embryonic stem cells, which is characterized in that: take ovarian cancer and breast cancer cells in the logarithmic growth phase, digest them with 0.25% Trypsin-EDTA (37 °C, 1 min 30 s - 2 min), centrifuge (5 min, 1000 rpm / min), resuspend them with complete medium for human pluripotent stem cells into single cells, and add them to human embryonic stem cells with 60% confluence at a ratio of ovarian cancer cells: breast cancer cells: human embryonic stem cells of 1:1:1 - 1:1:5, and place them in an incubator at 37 °C with 5% CO2 for culture.

[0024] Collect the co-culture medium: after co-culturing for 48 h - 96 h, collect the supernatant with a centrifuge tube.

[0025] Centrifuge at low temperature at 3000 rpm for 20 min, then filter with a 0.22 μm microporous filter membrane, aliquot and store at 80 °C.

[0026] Beneficial effects

[0027] Human embryonic stem cells are stem cells that can be cultured in vitro and have developmental totipotency, with the abilities of highly self-renewing, unlimited proliferation, and multi-directional differentiation. The inventor unexpectedly found that when human embryonic stem cells are directly co-cultured with ovarian cancer and breast cancer cells, the microenvironment of human embryonic stem cells is stimulated by various tumor cells, and a stronger anti-cancer biological effect will be produced, and it has characteristics for both types of tumors. Therefore, the supernatant can be extracted and applied to the treatment of ovarian cancer and breast cancer.

[0028] The co-culture supernatant of the present invention not only significantly improves the anti-cancer efficiency of the traditional co-culture supernatant, but also shows significant therapeutic effects in the proliferation, migration, and invasion of ovarian cancer and breast cancer cells, indicating that it has great potential in innovative biological therapeutic drugs for ovarian cancer and breast cancer. Brief description of the drawings

[0029] Figure 1 It is a graph showing the in vitro proliferation inhibition results of the co-culture supernatant of two cancer types on ovarian cancer cells;

[0030] Figure 2 It is a graph showing the in vitro proliferation inhibition results of the co-culture supernatant of two cancer types on breast cancer cells;

[0031] Figure 3It is the inhibitory effect of the co-culture supernatant of two cancer types on the cloning ability of ovarian cancer cells, specifically the cell plate cloning experiment diagram and the experimental statistical result diagram;

[0032] Figure 4 It is the inhibitory effect of the co-culture supernatant of two cancer types on the cloning ability of breast cancer, specifically the cell plate cloning experiment diagram and the experimental statistical result diagram;

[0033] Figure 5 It is the inhibitory effect of the co-culture supernatant of two cancer types on the migration ability of ovarian cancer cells, specifically the microscopic diagram of the transwell experiment result of ovarian cancer cells and the experimental statistical result diagram;

[0034] Figure 6 It is the inhibitory effect of the co-culture supernatant of two cancer types on the invasion ability of ovarian cancer cells, specifically the microscopic diagram of the transwell experiment result of ovarian cancer cells and the experimental statistical result diagram;

[0035] Figure 7 、 8 It is the inhibitory effect of the co-culture supernatant of two cancer types on the migration and invasion abilities of breast cancer cells, specifically the microscopic diagram of the transwell experiment result of breast cancer cells and the experimental statistical result diagram;

[0036] Figure 9 It is that the supernatant of human embryonic stem cells after being stimulated by two cancer types promotes the apoptosis of ovarian cancer cells, specifically the experimental diagram of detecting the expression of apoptosis index Bax / Bcl-2 in ovarian cancer cells by qRT-PCR. Specific implementation manners

[0037] The present invention will be specifically described below through specific examples. It should be noted here that the following examples are only used to further illustrate the present invention and cannot be construed as limiting the protection scope of the present invention. Those skilled in the art can make some non-essential improvements and adjustments to the present invention according to the above-mentioned invention content. The raw materials and reagents used in the present invention are all commercially available products. Among them, in the examples, ovarian cancer is cell lines (SKOV3, A2780), and breast cancer is cell lines (BT549, MDA-MB-231), all of which are established cell lines and are preserved in the Laboratory of Biochemistry and Molecular Pharmacology, Chongqing Medical University; in the examples, human embryonic stem cells are established stem cell lines, specifically, the human embryonic stem cell H9 cell line is purchased from the Chinese Academy of Sciences Stem Cell Bank.

[0038] Example 1

[0039] Preparation of co-culture supernatant of human embryonic stem cells with ovarian cancer and breast cancer cells

[0040] Preparation of a special culture plate for human embryonic stem cells by pre - coating with Matrigel matrix gel: Pre - coat the diluted Matrigel (DPBS:Matrigel = 50:1) in six - well plates at a volume of 1 mL / well, and place the plates back into a 37°C constant - temperature CO₂ cell culture incubator for 4 - 5 hours;

[0041] Digestion and passage: Take out the complete medium for human pluripotent stem cells and equilibrate it to room temperature. Take out the culture dish coated with Matrigel matrix gel, aspirate the coating solution and add an appropriate amount of complete medium for human pluripotent stem cells, and place it in a 37°C constant - temperature CO₂ cell culture incubator. Select human embryonic stem cells in good condition (i.e., hESCs with a cell coverage rate of about 80%, large stem cell colonies, regular clone edges and no differentiated cells). After aspirating the medium, add 2 mL of DPBS to wash the cells twice. After aspiration, add 1 mL of digestion solution for human pluripotent stem cells, and then place the cells back into a 37°C constant - temperature CO₂ cell culture incubator for incubation. Under the microscope, when most of the clone edges begin to detach from the bottom of the dish and there are obvious gaps between most of the cells inside the clone, and the cell colonies become opaque and white to the naked eye, it indicates that the cell digestion time is ideal. After aspirating the digestion solution, add 1 mL of complete medium for human pluripotent stem cells to each well, and gently scrape the cells with a cell scraper to make the cells into small cell clumps. Inoculation: Take out the well - plates pre - coated with Matrigel, and then evenly inoculate the cell clump suspension into the well - plates with Matrigel matrix gel prepared.

[0042] Co - culture: Take ovarian cancer and breast cancer cells in the logarithmic growth phase. After aspirating the medium, add DPBS buffer to wash the cells twice, digest with EDTA at 37°C for 1 min 30 s - 2 min, aspirate the supernatant, and resuspend the cancer cells into a single - cell suspension with

[0043] complete medium for human pluripotent stem cells. According to the proportion of 1 / 3 of the number of ovarian cancer and breast cancer cells to the number of human embryonic stem cells in the culture system, add them into the human embryonic stem cell culture system, and co - culture the two types of cells in a 37°C constant - temperature CO₂ cell culture incubator. Harvest of co - culture supernatant: Preparation of co - culture supernatant: Collect the co - culture supernatant at 48 - 96 h with a 15 - mL centrifuge tube, centrifuge, and then filter with a 0.22 - µm microporous filter membrane. The filtered supernatant is aliquoted and stored in 5 - mL sterile EPs and stored at 80°C.

[0044] Example 2

[0045] The co - culture supernatant of human embryonic stem cells with ovarian cancer and breast cancer cells can inhibit the growth of ovarian cancer cells

[0046] The co - culture supernatant of human embryonic stem cells with ovarian cancer and breast cancer cells can inhibit the growth of ovarian cancer cells

[0047] 1. Cell proliferation assay

[0048] Ovarian cancer and breast cancer cells in the logarithmic growth phase were seeded into 96-well plates at a density of 5×10 3 cells per well. 100 μL of co-culture supernatants with volume fractions of 0%, 20%, 40%, 60%, and 80% were added respectively. The 96-well plates were placed in an incubator at 37°C. After 24 h, 10 μL of CCK8 working solution was added to each well and incubated for about 2 h. The absorbance value of each well was measured at 450 nm using a microplate reader. Cell survival rate = (OD of experimental group - OD of blank group) / (OD of control group - OD of blank group) × 100%. The results showed ( Figure 1-2 ), compared with the blank control group, hESC supernatant, and traditional co-culture method (CO CM), the co-culture supernatant of double cancer types (dCO CM) could significantly inhibit the proliferation of ovarian cancer and breast cancer cells, and the inhibitory effect was concentration-dependent.

[0055] 2. Cell colony formation assay

[0056] Ovarian cancer and breast cancer cells in the logarithmic growth phase were digested, resuspended, and the cell concentration was adjusted to 500 - 1000 cells / mL. They were added to 6-well plates according to the groups, 3 mL per well. After the cells adhered for 24 h, the culture medium was replaced with the corresponding co-culture supernatant and hESC supernatant, and cultured for 15 d. Cell colonies were visible to the naked eye. The culture medium was aspirated, washed twice with PBS, fixed with 4% paraformaldehyde for 20 min, stained with 0.1% crystal violet for 10 min, and photographed. The results showed ( Figure 3-4 ), compared with the blank control group, hESC supernatant, and traditional co-culture method (CO CM), the co-culture supernatant of human embryonic stem cells and double cancer type cells (dCO CM) had stronger ability to inhibit the migration of ovarian cancer and breast cancer cells in vitro.

[0057] 3. Migration assay

[0058] Ovarian cancer cells in the logarithmic phase were digested with trypsin, centrifuged and collected. Ovarian cancer cells / breast cancer cells were resuspended as single cells with 5A basal medium / DMEM basal medium and seeded into the upper chamber of Transwell at a density of 1×10 5 cells per chamber (blank control group, hESC supernatant, CO CM, and dCO CM). Then, DMEM medium containing 20% fetal bovine serum was added to the lower chamber. After incubation for 16 h, the adherent cells were stained with a dye solution containing 0.05% crystal violet. Under an optical microscope, five random areas (200×) of stained cells were selected for counting and photographing. The results showed ( Figure 5-6), compared with the blank control group, hESC supernatant, and traditional co-culture method (CO CM), the co-culture supernatant of human embryonic stem cells and dual-cancer cells (dCO CM) has a stronger ability to inhibit the migration of ovarian cancer and breast cancer cells in vitro.

[0059] 4. Invasion experiment

[0060] Add 100 μl of Matrigel bottom working solution (Matrigel: DPBS = 1:50) to the upper chamber of Transwell, and incubate it in a 37 °C cell culture incubator for 4 - 5 h until it forms. Log-phase ovarian cancer cells are digested with trypsin, centrifuged and collected. The ovarian cancer cells / breast cancer cells are resuspended in 5A basal medium / DMEM basal medium as single cells, and inoculated into the upper chamber of Transwell at 1×10 5 cells / well (blank control group, hESC CM, CO CM, and dCO CM). Then, add 5A / DMEM medium containing 20% fetal bovine serum to the lower chamber. After incubating for 24 hours, stain the adherent cells with a dye solution containing 0.05% crystal violet. Under an optical microscope, randomly select five areas (200×) of stained cells for counting and photography. The results show that ( Figure 7-8 ) compared with the control group, the co-culture supernatant of human embryonic stem cells and dual-cancer cells (dCO CM) has a stronger ability to inhibit the invasion of ovarian cancer and breast cancer cells in vitro.

[0061] 5. qRT-PCR experiment

[0062] Use 6 cm 2 culture dishes to culture ovarian cancer cells (SKOV3). When the cell density reaches 60% - 70%, change the medium to the conditioned medium of the blank control group, hESC CM, CO CM, and dCO CM, and continue to culture for 72 h. Use an RNA extraction kit (ES Science RNA-Quick Purification Kit) to extract the total RNA of the cells. After measuring the concentration and purity, reverse transcribe 1 μg of RNA into cDNA according to the reverse transcription instructions. qRT-PCR uses the SYBR Green method. Each 10 μL reaction system contains 5 μL SYBR Green Mix, 1 μL of upstream and downstream primers, 2 μL of cDNA template, and 2 μL of DEPC water. Amplification program: pre-denaturation at 95 °C for 30 s, 40 cycles (95 °C for 5 s, 60 °C for 30 s), and melting curve analysis. Using GAPDH as an internal reference, analyze the data using the 2 -ΔΔCt method. The results show that ( Figure 9 ) the expression of Bax mRNA in the experimental group is significantly up-regulated, and the expression of Bcl-2 mRNA is down-regulated (*p < 0.05; **p < 0.01).

Claims

1. A method for enhancing the efficacy of the supernatant and expanding the therapeutic tumor spectrum by co-culturing two tumor species with human embryonic stem cells, characterized in that It includes the following steps: (1) Pre-coat with human pluripotent stem cell basement matrix gel to prepare a special culture plate for human embryonic stem cells; (2) Use the human pluripotent stem cell basement matrix gel pre-coated in step (1) to prepare a special culture plate for human embryonic stem cells for inoculating human embryonic stem cells; (3) Passage human embryonic stem cells; (4) Stimulate embryonic stem cells with two tumor cell lines: Take ovarian cancer cells and breast cancer cells in the logarithmic growth phase, wash the cells 2 times with PBS, then digest them with 0.25% Trypsin-EDTA (37 °C, 1 min 30 s - 2 min), and resuspend the cells with an equal volume of 5A complete medium and DMEM complete medium. After centrifugation (5 mins, 1000 rpm / min), discard the supernatant and resuspend with 5A basal medium and DMEM basal medium into single cells, and add them to human embryonic stem cells with 60% confluence at a ratio of ovarian tumor cells: breast cancer cells: human embryonic stem cells of 1:1:1 - 1:1:1.

5. (5) Place the co-culture system in an incubator at 37 °C with 5% CO2 for culturing; replace the fresh PGM1 medium every day, and collect the co-culture supernatant after 48 - 96 h.

2. The method according to claim 1, wherein: The special medium for human embryonic stem cells is PGM1 human pluripotent stem cell medium (Beijing Saibei Biology CA1007500), Y-27632 (MCE, USA HY10071).

3. The method according to claim 1, wherein: The Matrigel matrix gel is produced by Corning (354277). The preparation method of the special culture plate for human embryonic stem cells is: thaw the matrigel matrix gel on ice, dilute it with DPBS buffer at a ratio (matrigel: DPBS / PBS buffer = 1:80 - 100), then spread it into the culture plate, and place it in an incubator at 37 °C with 5% CO2 for 4 - 5 h until the matrix gel solidifies and forms a shape, then it can be used.

4. The method according to claim 2, wherein: The method for digesting and passaging human embryonic stem cells is to select human embryonic stem cells in the logarithmic growth phase, aspirate the old medium, add stem cell digestive solution, place it in the incubator for about 5 - 7 min, observe the white cell clumps with the naked eye, aspirate the stem cell digestive solution, add an appropriate amount of stem cell medium, and blow and beat the cell colonies with a pipette fanwise to make them into several cell clumps.

5. The method according to claim 4, characterized in that: The method for inoculating human embryonic stem cells is to take the well plate in step (1), aspirate the basic working solution, add PGM1 medium, and then evenly inoculate the cell clump suspension in step (2) into the pre-prepared special culture plate for stem cells. Replace the fresh PGM1 medium every day and collect the co-culture supernatant.

6. The method according to claim 5, characterized in that: The state of human embryonic stem cells is embryonic stem cells in the logarithmic growth phase. When the cell confluence reaches 50 - 60%, the clone edges are regular, there are no differentiated cells, and the cell colonies are not completely fused, showing a relatively dispersed state.

7. The method according to claim 1, wherein: Collect the co-culture supernatant of the two cancer cell lines stimulating embryonic stem cells. The co-culture time is 48 h - 96 h. Collect the supernatant with a centrifuge tube, centrifuge at low temperature at 3000 rpm * 20 min, then filter it with a 0.22 μm microporous filter membrane, and store it at 80 °C after aliquoting.

8. Use of the supernatant of co-culture of human embryonic stem cells prepared by the method according to any one of claims 1-7 with double cancer types (ovarian cancer and breast cancer) in the preparation of drugs against ovarian cancer and breast cancer.

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