CN115645524A - A composition for inhibiting the occurrence and development of breast cancer and its application - Google Patents

Abstract

The invention provides a composition for inhibiting the occurrence and development of breast cancer and its application. The composition for inhibiting the occurrence and development of breast cancer includes reagents for inhibiting the expression of C5a and PD‑1 and/or OPN, and can be used for preparing drugs for treating breast cancer. The present invention verifies that PD-L1 antibody and/or OPN antibody combined with C5a antibody can significantly inhibit the occurrence and development of breast cancer, provides a new idea for the preparation of drugs for treating breast cancer, and is beneficial to improve the quality of life of patients.

Description

A composition for inhibiting the occurrence and development of breast cancer and its application

technical field

The invention relates to the field of biomedicine, in particular to a composition for inhibiting the occurrence and development of breast cancer and its application.

Background technique

Breast cancer is a malignant tumor that occurs in the glandular epithelial tissue of the breast. In the early stage, symptoms such as breast lumps, nipple discharge, and axillary lymph node enlargement often appear. In the late stage, cancer cells may metastasize to multiple organs and cause multiple organ lesions, which directly threaten the lives of patients. They are often called "pink killers." The global incidence of breast cancer has been on the rise since the late 1970s. According to the incidence data of breast cancer released by the National Cancer Center and the Bureau of Disease Control and Prevention of the Ministry of Health in 2012, the incidence of breast cancer in the national tumor registration area ranks first among female malignant tumors.

Although some current treatments can help most cancer patients relieve their pain, breast cancer still poses a great threat to women's life safety, resulting in a decline in the quality of life. Therefore, it is more urgent to study the occurrence and development of breast cancer and develop effective therapeutic drugs.

Existing literature (“C5aR deficiency attenuates the breast cancer development via the p38/p21 axis”, Chen et al., AGING-US (AGING), Vol. 12, No. 14, 2020) discloses the involvement of the complement C5a/C5aR pathway The pathogenesis of breast cancer has been clarified, and inhibition of the C5a/C5aR pathway is expected to help the treatment of breast cancer patients, but there are no reports on more effective drug combinations based on complement C5a for the treatment of breast cancer.

Contents of the invention

In order to overcome the defects in the prior art, the present invention provides a composition for inhibiting the occurrence and development of breast cancer and its application.

To achieve the above object, the present invention adopts the following technical solutions:

The first aspect of the present invention is to provide a composition for inhibiting the occurrence and development of breast cancer, which includes an agent for inhibiting the expression of C5a and PD-1 and/or OPN.

Further, the above composition includes C5a antibody, PD-L1 antibody and/or OPN antibody.

Further, the above composition includes C5a antibody and PD-L1 antibody.

The second aspect of the present invention is to provide the application of the above composition in the preparation of medicaments for treating breast cancer.

Further, the active ingredients of the above-mentioned medicine are C5a antibody, PD-L1 antibody and/or OPN antibody.

Further, the active ingredients of the above-mentioned medicine are C5a antibody and PD-L1 antibody.

Furthermore, the above-mentioned medicine also includes a pharmaceutically acceptable carrier or excipient.

Further, the administration forms of the above-mentioned drugs are oral administration, intravenous injection or intramuscular injection.

The present invention adopts the above technical scheme, and compared with the prior art, it has the following technical effects:

The present invention verifies that PD-L1 antibody and/or OPN antibody combined with C5a antibody can significantly inhibit the occurrence and development of breast cancer, provides a new idea for the preparation of drugs for treating breast cancer, and is beneficial to improve the quality of life of patients.

Description of drawings

Figure 1 shows the results of protein chip detection in an embodiment of the present invention; wherein, Figures A and B respectively show the differences in the expression of some proteins after C5a induces breast cancer cells;

Figure 2 shows the effect of different antibodies or antibody combinations on the recruitment of MDSCs in the conditioned medium in an embodiment of the present invention; wherein, Figure A is the result of taking pictures of an inverted microscope, and Figure B shows the statistical results of the number of migrated cells;

Figure 3 shows that in an embodiment of the present invention, the addition of OPN neutralizing antibodies inhibits the function of the conditioned medium regulated by C5a to promote osteoblast differentiation; among them, Figures A-C show the blank group and breast cancer cells induced by adding C5a respectively The differentiation function of osteoblasts in the conditioned medium group and the conditioned medium of breast cancer cells induced by C5a and the OPN neutralizing antibody group;

Figure 4 shows the results of flow cytometry analysis of T lymphocyte typing in an embodiment of the present invention; wherein, Figure A is the result of the flow cytometry experiment, and Figure B is the result of counting the number of CD4 ⁺ T cells according to Figure A;

Fig. 5 shows the inhibitory effect of each antibody or its combination on bone metastasis of breast cancer cells by fluorescence imaging technology in an embodiment of the present invention;

Fig. 6 shows the results of quantitative analysis of the total amount of fluorescence intensity of mouse motion imaging in an embodiment of the present invention; **p<0.01.

Detailed ways

The invention provides a composition for inhibiting the occurrence and development of breast cancer and its application. In the following, the present invention will be described in detail and specifically through specific embodiments and accompanying drawings, so as to better understand the present invention, but the following embodiments do not limit the scope of the present invention.

The methods in the examples are conventional methods unless otherwise specified, and the reagents used are conventional commercially available reagents or reagents prepared according to conventional methods unless otherwise specified.

Example 1

In this example, the protein chip detection method was used to screen out proteins related to changes in the bone microenvironment and immune microenvironment. The specific experimental steps and results are as follows:

1. Sample preparation: Routinely culture MCF-7 cells and transfer them to a 10cm cell culture dish. When the cell density in the culture dish reaches 90%, count the cells, and take 3×10 ⁶ cells to spread to 2 cells. 10cm cell culture dish.

2. After 12 hours, when the cells grow stably, wash the cells with PBS solution for 3 times, replace with DMEM medium without phenol red and fetal bovine serum, and add human-derived medium at a concentration of 20ng/ml in the experimental group. C5a, continue to culture for 12 hours.

3. After the cell culture, collect the cell supernatant into a 15ml centrifuge tube, then use a 0.4μm filter to filter the collected conditioned medium, and store the processed samples in a -80°C refrigerator for testing.

4. The detection of protein chips is carried out by Ruiboao (Guangzhou) Biological Co., Ltd. The detection procedure is as follows: After concentrating the collected protein samples, the samples are dropped on the glass slides coated with antibodies. Usually the reaction time is After 2 hours, the biotinylated secondary antibody was added dropwise. After 2 hours of action, streptavidin (CY3) was added to continue the reaction for 1 hour. Laser scanning was used to obtain data related to the bound protein, and the corresponding software was further used The analysis of differential proteins was carried out, and the results are shown in Figure 1.

It can be seen from Figure 1 that the most significant changes compared with the control group were osteopontin (OPN) and programmed death-ligand 1 (PD-L1), and their expression differences were particularly significant.

Example 2

This example explores the influence of different reagents on the recruitment of MDSCs cells. The specific experimental steps and experimental results are as follows:

1. Take a 24-well cell culture plate, add 600 μl of complete medium to the blank control group (ctrl); add 600 μl of medium (which contains 6 μl of conditioned medium) to the wells of the experimental group, and according to different experimental groups Do not add corresponding reagents (no additional reagents were added to the C5a-CM group, OPN antibody was added to the C5a-CM-opn ab group to make the final concentration 2 μg/mL, and PD-L1 was added to the C5a-CM-PD-L1 ab group Antibody and its final concentration was 1 μg/mL, and PD-L1 antibody (final concentration was 1 μg/mL) and OPN antibody (final concentration was 2 μg/mL) were added to C5a-CM-PD-L1 ab&-opn ab group.

2. Use tweezers to pick up the migration chamber and put it into the hole, and mark the four corners of the chamber to distinguish different experimental groups.

3. After routinely digesting and centrifuging the cells planned for the migration experiment, count the cells, and according to the different cell sizes, take different numbers of cells and place them in the upper chamber of the migration chamber, add 100 μl of liquid, and spread MDSCs for 4 ×10 ⁵ cells.

4. Carry out cell migration culture according to the cell migration time determined in the pre-experiment, and MDSCs migrate for 4 hours.

5. After migration, fix the cells with 4% paraformaldehyde for 10 minutes, wash with PBS solution, and then stain with 0.1% crystal violet, usually for 3 minutes. After staining, use tweezers to clamp the small chamber, Wash in a sink filled with tap water until the washing liquid is clear.

6. After washing, use a cotton swab to wipe off the cells on the upper layer of the chamber. When wiping, care should be taken to wipe off the surrounding cells on the upper layer of the chamber as much as possible to avoid false positive cells. After drying naturally, use an inverted microscope to take pictures and count. The result is shown in Figure 2.

It can be seen from Figure 2 that the combination of OPN and PD-L1 neutralizing antibody can play a synergistic effect, further reducing the recruitment effect of conditioned medium on MDSCs.

Example 3

This example explores the effect of OPN neutralizing antibody on the function of C5a-regulated conditioned medium to promote osteoblast differentiation. The specific experimental steps and results are as follows:

1. Take the long bones of the lower limbs of 8-week-old C57bl/6 male mice, and keep the femoral head, knee joints, and ankle joints respectively, immerse the taken tissues in 75% ethanol, and then transfer them to a biological safety cabinet for cell separation.

2. Go to the biological safety cabinet, replace with 75% ethanol and soak for 5 seconds, then immerse in PBS solution, use sterilized ophthalmic scissors to cut off the tendon, and then use gauze to remove the surface muscles of the femur and tibia. The femur and tibia were respectively immersed in 75% ethanol for 5 seconds, and then immersed in PBS solution.

3. Use a 20ml syringe to absorb 10ml of complete α-MEM modified medium and equip it with a 1ml syringe needle, use ophthalmic scissors to cut off the bone tissue at both ends of the bone marrow cavity, insert the injection needle into the bone marrow cavity, and flush the bone marrow into a 15ml centrifuge tube , flush the marrow cavity of the femur and tibia one by one according to the above method.

4. Centrifuge the bone marrow mixture washed into a 15ml centrifuge tube at 800rpm for 5min, and then use 1ml complete α-MEM modified medium to resuspend the cells.

5. Filter the resuspended cells with a 100 μm sieve, add the filtrate to a 6 cm cell culture dish, then add 3 ml complete α-MEM modified medium, and continue culturing for 72 hours.

6. After culturing for 72 hours, discard the cell supernatant, digest and centrifuge the cells, and then count the cells, take 5×10 ⁴ /well cells, spread them in a 24-well cell culture plate, and continue culturing for 24 hours .

7. Prepare osteoblast differentiation medium, the reagents used include vitamin C (1000×), dexamethasone (10 ⁴ ×) and β-glycerophosphate (100×), according to different groups, in 1ml of osteoblast differentiation medium In the conditioned medium group, 10 μl of conditioned medium after C5a-induced breast cancer cells was added, and in the neutralizing antibody group, 10 μl of conditioned medium after C5a-induced breast cancer cells and OPN neutralizing antibody were added to make it final The concentration was 2 μg/mL, and after 48 hours of continuous culture, the differentiation medium was replaced again. For mineralization staining, cells need to be cultured for 14 days.

8. After the cultured and differentiated osteoblasts were fixed with 4% paraformaldehyde, the staining solution was prepared according to the ALP staining kit, and after staining at room temperature for 15 minutes, it was replaced with PBS solution. When taking pictures, it should be noted that in order to avoid the phenomenon of liquid reflection, the PBS solution in the culture plate was removed. The staining results are shown in Figure 3.

It can be seen from Figure 3 that the function of promoting osteoblast differentiation induced by the addition of C5a and collected from the conditioned culture was inhibited by the neutralizing antibody of OPN.

Example 4

This example explores the effect of PD-L1 neutralizing antibody on the conditioned medium induced by C5a on the inhibitory function of CD4 ⁺ T lymphocytes. The specific experimental steps and results are as follows:

1. Completely remove the lower limb bones of the mouse, keep the femoral head at the upper end and the ankle joint at the lower end to ensure the integrity of the bone marrow cavity.

2. After cutting off the ligament around the knee joint, separate the femur and tibia, and remove the superficial muscles, cut off the two ends of the marrow cavity of the femur and tibia respectively, and rinse the marrow cavity with PBS solution containing 1% fetal bovine serum, collect and rinse Liquid and centrifuged (800rpm, 5min).

3. After centrifugation, use PBS solution containing 1% fetal bovine serum to resuspend the cells, count the cells, and transfer to EP tubes at 10 ⁶ /100 μl for later use.

4. Isolate and culture mouse bone marrow cells, and add the conditioned medium of C5a-induced breast cancer cells. After 48 hours of action, add neutralizing antibody to PD-L1 in the neutralizing antibody group and make the final concentration 1 μg/mL. Different subsets of T lymphocytes were incubated with flow cytometry antibodies and analyzed by flow cytometry.

5. Label the EP tube according to the blank control, antibody single staining and antibody composite staining, and then add flow cytometry antibody at 0.5μl/100μl for incubation. In order to avoid operational errors caused by adding a small amount of antibody, antibody dilution can be prepared at this time. The specific method is to calculate the required amount of antibody, dilute it 10 times with PBS solution, and then add the prepared antibody diluent according to the method of 5μl/100μl, and note that after adding the antibody, it needs to be kept at 4°C and in a light-proof environment. Incubate for 30 min.

6. After the incubation, wash 3 times with PBS solution containing 1% fetal bovine serum, during which the centrifugation condition is 1000rpm, 5min.

7. After washing, use 600 μl of PBS solution containing 1% fetal bovine serum to resuspend the cells, and filter the cells with a 100 μm sieve, and perform flow cytometric detection after the treatment.

8. If multiple tests are required, 2% paraformaldehyde solution can be used to fix for 15 minutes, and then washed with PBS solution containing 1% fetal bovine serum. Cell samples can be stored for 24 hours, and cells still need to be filtered before testing.

It can be seen from Figure 4 that after adding PD-L1 neutralizing antibody, the inhibitory function of the conditioned medium of breast cancer cells on CD4 ⁺ T cells was weakened.

Example 5

This example explores the inhibitory effect of C5a antibody, PD-L1 antibody, OPN antibody and their combination on cancer cell metastasis. The specific experimental steps and results are as follows:

1. Female balb/c mice aged 6 to 8 weeks were selected as the experimental subjects. There were 28 mice in 7 groups, 4 mice in each group. Breast cancer tumor cells 4T1 expressing luciferase were selected, and the number of cells injected into the heart was 1×10 ⁵ One per mouse, resuspend the cells in 100 μl of PBS solution, filter through a 100 μm sieve and set aside for later use. Due to loss during injection, the amount of cells prepared is usually 3 times the planned number.

2. Using Avertin intraperitoneal injection, anesthetize the balb/c mice one by one, and pay attention to the degree of anesthesia, avoid too deep anesthesia, fix the mice with satisfactory anesthesia on the operating table, and use alcohol cotton balls to disinfect the injection site Finally, select the left 2/3 intercostal space for puncture. When arterial-like pulsating blood returns, inject the tumor cell suspension slowly, usually divided into 5 injections, and place the injected mice in a temperature platform at 40°C. Up to wake up.

3. Two days after the heart injection of luciferase, drug injections were performed in groups every other day: the first group was a blank group, that is, no drug injection; the second group: C5a antibody 0.1ml/piece; the third group: OPN antibody 0.1ml / rat; the fourth group: PD-L1 antibody 0.1ml/ rat; the fifth group: C5a antibody + PD-L1 antibody, each 0.1ml/ rat.

4. Two weeks after the injection of the left ventricle, the live mouse imaging test was carried out. The specific operation process was as follows: First, the luciferase substrate (Luciferin) was injected at 100 μl/mouse by intraperitoneal injection for 5 minutes. Finally, use the isoflurane gas anesthesia system to anesthetize the mouse. After the anesthesia is satisfactory, place it on the detection table for imaging observation. Due to the difference in cell transfer ability, the gradient exposure time is usually set at 1 minute, 3 minutes, and 5 minutes. .

It can be seen from Figure 5 and Figure 6 that the C5a antibody has an inhibitory effect on the bone metastasis of breast cancer cells, and the combination of C5a and PD-L1 antibody can significantly enhance the inhibitory effect; the OPN antibody alone also has a certain inhibitory effect on the metastasis of breast cancer cells .

The specific embodiments of the present invention have been described in detail above, but they are only examples, and the present invention is not limited to the specific embodiments described above. For those skilled in the art, any equivalent modifications and substitutions to the present invention are also within the scope of the present invention. Therefore, equivalent changes and modifications made without departing from the spirit and scope of the present invention shall fall within the scope of the present invention.

Claims (8)

1. A composition for inhibiting the development of breast cancer comprising an agent that inhibits C5a, and PD-1 and/or OPN expression.

2. The composition of claim 1, comprising a C5a antibody, and a PD-L1 antibody and/or an OPN antibody.

3. The composition of claim 1, comprising a C5a antibody and a PD-L1 antibody.

4. Use of a composition according to any one of claims 1 to 3 for the manufacture of a medicament for the treatment of breast cancer.

5. The use according to claim 4, wherein the effective active ingredients of the medicament are C5a antibody, and PD-L1 antibody and/or OPN antibody.

6. The use according to claim 4, wherein the effective active ingredients of the medicament are C5a antibody and PD-L1 antibody.

7. The use of claim 4, wherein the medicament further comprises a pharmaceutically acceptable carrier or excipient.

8. The use according to claim 4, wherein the administration is oral, intravenous or intramuscular.

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