A simple and broad-spectrum tumor whole-cell vaccine based on proteasome inhibitors and its preparation method

Abstract

The present invention provides a simple and broad-spectrum tumor whole-cell vaccine based on a proteasome inhibitor and a preparation method thereof, wherein the vaccine comprises mouse cancer cells pretreated with bortezomib; the mouse cancer cells comprise one or more of pancreatic cancer cells Panc02, liver cancer cells Hepa1‑6, breast cancer cells 4T1, and colon cancer cells CT26. The present invention pretreats tumor cells with the proteasome inhibitor bortezomib, and prepares a simple and broad-spectrum mouse tumor whole-cell vaccine based on this, which has good biosafety and high protection efficiency against a variety of cancers.

Description

Technical Field

[0001] The present invention belongs to the technical field of pancreatic cancer prevention and treatment, and specifically relates to a simple and broad-spectrum tumor whole-cell vaccine based on a proteasome inhibitor and a preparation method thereof. Background Art

[0002] In recent years, under the guidance of systemic treatment programs, the condition of some cancer patients has been well controlled, but the tumor still cannot be cured. In the field of cancer prevention and treatment, tumor vaccines have received widespread attention as a new immune prevention and treatment strategy.

[0003] There are many types of tumor vaccines, such as whole-cell vaccines, peptide vaccines, genetically engineered vaccines, etc. However, the current research on tumor-specific antigens has not been fully clarified, which has partly led to the inability of many tumor vaccines to be widely used. Whole-cell tumor vaccines have unique advantages in this regard. This type of vaccine contains a relatively rich tumor-related antigens, which may provide a way for the widespread promotion of current tumor vaccines. However, this type of vaccine requires a certain amount of time in the early stages to explore and optimize specific methods of in vitro processing and preparation. At present, there is a lack of an efficient method on the market for rapid evaluation of the early in vitro preparation of whole-cell tumor vaccines to simplify the preparation of this type of vaccine, thereby promoting broad-spectrum applications. Summary of the invention

[0004] In order to overcome the deficiencies in the prior art, the present invention provides a simple and broad-spectrum tumor whole-cell vaccine based on proteasome inhibitors and a preparation method thereof. The vaccine of the present invention improves the immunogenicity of tumor cells by pretreatment, so that the body improves the immune presentation ability to these tumor cells, thereby promoting the immune protection effect.

[0005] The present invention provides a simple and broad-spectrum tumor whole-cell vaccine, which comprises mouse cancer cells pretreated with the proteasome inhibitor bortezomib; The mouse cancer cells include one or more of pancreatic cancer cells Panc02, liver cancer cells Hepa1-6, breast cancer cells 4T1, and colon cancer cells CT26.

[0006] Preferably, the pretreatment with the proteasome inhibitor bortezomib is that the pancreatic cancer cells Panc02 are treated with 40, 60 and 80 nM bortezomib for 18 h, or the liver cancer cells Hepa1-6 are treated with 20, 40 and 60 nM bortezomib for 18 h, or the breast cancer cells 4T1 are treated with 100, 120 and 140 nM bortezomib for 48 h, or the colon cancer cells CT26 are treated with 150, 200 and 250 nM bortezomib for 48 h.

[0007] Preferably, the pretreatment with the proteasome inhibitor bortezomib is that the pancreatic cancer cells Panc02 are treated with 60 nM bortezomib for 18 h, or the liver cancer cells Hepa1-6 are treated with 40 nM bortezomib for 18 h, or the breast cancer cells 4T1 are treated with 120 nM bortezomib for 48 h, or the colon cancer cells CT26 are treated with 250 nM bortezomib for 48 h.

[0008] The present invention provides the application of the simple and broad-spectrum tumor whole-cell vaccine in one or more aspects of preventing pancreatic cancer, liver cancer, breast cancer and colon cancer.

[0009] Beneficial effects of the present invention: The present invention pre-treats tumor cells with the proteasome inhibitor bortezomib, and prepares a simple and broad-spectrum tumor whole-cell vaccine based on this. The vaccine has good biosafety and high protection efficiency against various cancers. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 .Schematic diagram of the preparation and evaluation scheme of whole-cell tumor vaccines, where a is the in vitro preparation schematic diagram of whole-cell tumor vaccines, b is the in vitro evaluation schematic diagram of whole-cell tumor vaccines, and c is the in vivo evaluation schematic diagram of whole-cell tumor vaccines.

[0011] Figure 2 .CCK8 was used to detect the cell viability of each cell after treatment with different concentrations of bortezomib at different administration times, where a is the cell viability of Panc02 cells, b is the cell viability of Hepa1-6 cells, c is the cell viability of 4T1 cells, and d is the cell viability of CT26 cells; the results are expressed as the mean ± standard deviation of a representative experiment, *two-tailed t-test P<0.05, **P<0.01, ***P<0.001, ****P<0.0001; ns: no difference, where the upper point of the bar graph represents the data point, and the upper and lower lines are the standard deviation.

[0012] Figure 3 Trypan blue staining was used to evaluate the cell death rate of each cell after treatment with different concentrations of bortezomib at different administration times, where a is the cell death rate of Panc02 cells, b is the cell death rate of Hepa1-6 cells, c is the cell death rate of 4T1 cells, and d is the cell death rate of CT26 cells; the results are expressed as the mean ± standard deviation of a representative experiment, *two-tailed t-test P<0.05, **P<0.01, ***P<0.001, ****P<0.0001; ns: no difference, where the upper point of the bar graph represents the data point, and the upper and lower lines are standard deviations.

[0013] Figure 4. Flow cytometry analysis of the percentage of dying cells of each cell type after treatment with a specific concentration of bortezomib at a specific administration time, where a is the percentage of dying cells of Panc02 cells, b is the percentage of dying cells of Hepa1-6 cells, c is the percentage of dying cells of 4T1 cells, and d is the percentage of dying cells of CT26 cells; the results are expressed as the mean ± standard deviation of a representative experiment, *** two-tailed t-test P<0.001, ****P<0.0001, where the upper point of the bar graph represents the data point, and the upper and lower lines are standard deviations.

[0014] Figure 5 .The clone formation experiment verified the in vitro clone formation ability of each cell a after treatment with a specific concentration of bortezomib at a specific administration time, where a was the in vitro clone formation ability of Panc02 cells, b was the in vitro clone formation ability of Hepa1-6 cells, c was the in vitro clone formation ability of 4T1 cells, and d was the in vitro clone formation ability of CT26 cells; the results are expressed as the mean ± standard deviation of a representative experiment, *** two-tailed t-test P<0.001, ****P<0.0001, where the upper points of the bar graphs represent data points, and the upper and lower lines are standard deviations.

[0015] Figure 6 .Transwell assay was used to analyze the migration and invasion abilities of each cell line after treatment with specific concentrations of bortezomib at specific administration times, where a represents the migration and invasion abilities of Panc02 cells, b represents the migration and invasion abilities of Hepa1-6 cells, c represents the migration and invasion abilities of 4T1 cells, and d represents the migration and invasion abilities of CT26 cells. The results are expressed as the mean ± standard deviation of a representative experiment, ***P<0.001 by two-tailed t-test, ****P<0.0001, where the upper points on the bar graphs represent data points, and the upper and lower lines represent standard deviations.

[0016] Figure 7 .Evaluate the protective efficiency of whole-cell tumor vaccines in vitro. Vaccinated Panc02, Hepa1-6, 4T1, and CT26 cells were subcutaneously injected into different groups of C57 mice (n=7-10), and the death time of each C57 mouse was recorded, where a is the curve of the percentage of tumor-free mice vaccinated with Panc02 cells, b is the curve of the percentage of tumor-free mice vaccinated with Hepa1-6 cells, c is the curve of the percentage of tumor-free mice vaccinated with 4T1 cells, and d is the curve of the percentage of tumor-free mice vaccinated with CT26 cells; the results are expressed as the mean ± standard deviation of a representative experiment, ***P<0.001 by two-tailed t test, ****P<0.0001. DETAILED DESCRIPTION

[0017] The technical solution of the present invention is further explained below by examples, but the protection scope of the present invention is not limited in any form by the examples.

[0018] Bortezomib used in the present invention was purchased from a commercial reagent.

[0019] In the present invention, different mice were selected for in vivo evaluation experiments based on the source backgrounds of different mouse cancer cells, as follows: Pancreatic cancer cell Panc02 uses male C57 mice. Panc02 cells are a mouse pancreatic cancer cell line mainly derived from C57 mice. Specifically, they are obtained by inducing type III pancreatic ductal adenocarcinoma by implanting 3-methylcholanthrene (3-MCA)-saturated cotton thread into the pancreas.

[0020] Hepa 1-6 hepatoma cells were cultured in male C57 mice. Hepa 1-6 cells are a mouse hepatoma cell line mainly derived from C57 mice, specifically a derivative strain generated from BW7756 hepatoma cells induced in C57 mice.

[0021] The breast cancer cell 4T1 uses female BALB / c mice. 4T1 cells are a mouse breast cancer cell line mainly derived from BALB / c mice. They are triple-negative breast cancer cell lines, specifically cells with resistance to 6-thioguanine screened from spontaneous breast tumors of BALB / c mice.

[0022] Colon cancer cell CT26 was cultured in male BALB / c mice. CT26 cells are a mouse colon cancer cell line mainly derived from BALB / c mice, and are specifically obtained by inducing undifferentiated colon cancer with N-nitroso-N-methylcarbamate (NNMU).

[0023] Unless otherwise specified in the present invention, "mouse" will be used to refer to the specific strain used for the corresponding cancer cells.

[0024] Example 1 This example shows the screening criteria for bortezomib concentration and time during the preparation of whole cell tumor vaccines.

[0025] 1.CCK8 detection: Discard the DMEM complete medium in the culture flasks of mouse pancreatic cancer cells Panc02, liver cancer cells Hepa1-6, breast cancer cells 4T1, and colon cancer cells CT26, add 1 mL PBS to rinse, then add 1 mL trypsin to digest, add 1 mL DMEM complete medium after about 1 min to terminate the digestion, then transfer the cell suspension to a 15 ml centrifuge tube, centrifuge at 1000 rpm for 4 min, and discard the supernatant; add about 1 mL Resuspend the cells in DMEM complete medium, count them using a cell counting plate, and supplement the corresponding DMEM complete medium according to the obtained cell number so that Panc02 is about 40,000 cells / mL, Hepa1-6 is about 30,000 cells / mL, 4T1 is about 50,000 cells / mL, and CT26 is about 50,000 / mL. Add 100 μL of cell suspension to each well of a 96-well plate (each cell type is spread on 3 96-well plates); after the cells adhere to the wall overnight, discard the medium, wash once with PBS, and add the medium with the corresponding bortezomib concentration (0, 20, 40, 60, 80, 100 for Panc02). and 120nM, Hepa1-6 is 0, 20, 40, 60, 80 and 100nM, 4T1 is 0, 80, 100, 120, 140, 160, 180, 200 and 220nM, CT26 is 0, 50, 100, 150, 200, 250, 300, 350 and 400nM), this time is recorded as time 0, and the first detection is carried out after the corresponding time (Panc02 is 6h, Hepa1-6 is 6h, 4T1 is 18h, CT26 is 24h); the specific process is: discard the culture medium, rinse once with PBS, add 90μL complete culture medium and 10μL CCK8 reagent to each well, incubate in the incubator for 1h, read the corresponding OD value at a wavelength of 450nm on the microplate reader, and perform corresponding statistical analysis. Subsequently, two 96-well plates for each cell type were tested and analyzed according to the above process at corresponding time (18h and 24h for Panc02, 18h and 24h for Hepa1-6, 24h and 48h for 4T1, and 36h and 48h for CT26) (the control 0nM of the four cell types is the volume of DMSO required to add the maximum concentration of drugs in each group).

[0026] according to Figure 2 In this experiment, the corresponding drug treatment concentrations and times for the four cell types were initially selected, specifically Panc02 was treated for 18 h at 40, 60 and 80 nM ( Figure 2 a), Hepa1-6 was treated with 20, 40 and 60 nM for 18 h ( Figure 2 b), 4T1 was treated with 100, 120 and 140 nM for 48 h ( Figure 2 c), CT26 was treated with 150, 200 and 250 nM for 48 h ( Figure 2 d) in.

[0027] 2. Trypan blue staining: The four types of cells in good condition were digested (see above for the specific process) and then inoculated into a 6-well plate. After the cells adhered overnight, a bortezomib culture medium of corresponding concentration was added and cultured for a corresponding time (see the screening results of CCK8 detection in Example 1 for the specific concentration range and time), the supernatant was collected and digested, centrifuged and counted (see above for the specific process, except that an appropriate amount of trypan blue reagent needs to be added to the counting suspension), and statistical analysis was performed subsequently (the control treatment was the same as in Example 1).

[0028] according to Figure 3 In this experiment, based on the cell death rate of about 50%, the drug treatment concentrations of 4 types of cells were selected, which are roughly as follows: Panc02 is 60nM ( Figure 3 a), Hepa1-6 was 40 nM ( Figure 3 b), 4T1 is 120nM ( Figure 3 c), CT26 is 250nM ( Figure 3 d) in.

[0029] 3. Flow cytometry: The corresponding cells were inoculated into a 6-well plate, and after overnight attachment, a corresponding concentration of bortezomib medium was added and cultured for a corresponding time (for specific drug concentrations, see the results in Example 2, and for specific drug treatment time, see Example 1), the supernatant was collected and digested and centrifuged (for specific procedures, see above); 500 μL 1×binding buffer (5×binding buffer diluted with double distilled water) was added to each 1.5 mL EP, and the corresponding apoptosis reagent dye (5 μl Annexin V dye and 10 μl 7AAD dye) was added, resuspended by pipetting, incubated on ice in the dark for 5 min, and the expression of the corresponding Annexin V and 7AAD was detected by flow cytometry, and statistical analysis was performed (the control treatment was the same as in Example 1).

[0030] according to Figure 4 This experiment preliminarily verified that under the above-mentioned corresponding concentration and time conditions, the proportions of "live" cells (Annexin V-, 7AAD-) and "dying" cells (Annexin V+, 7AAD-) in the four types of cells were roughly equal. The values ​​in the typical representative figure are: Panc02 is 45.0% and 38.2% respectively ( Figure 4 a), Hepa 1-6 were 43.6% and 41.1% ( Figure 4 b), 4T1 is 42.1% and 44.1% respectively ( Figure 4 c), CT26 were 40.1% and 36.7% ( Figure 4d in the figure); and use this drug treatment concentration and time as the conditions for subsequent tumor whole cell vaccine preparation and evaluation.

[0031] Example 2 This example shows the in vitro evaluation results under corresponding conditions during the preparation of whole-cell tumor vaccines.

[0032] 1. Clone formation: Pre-treat the four types of cancer cells according to the treatment concentration and time obtained in Example 1, collect the supernatant, centrifuge, count and adjust to a reasonable concentration (according to the method of Example 1), and inoculate the cells into a 6-well plate (Panc02 is about 500 cells / well, Hepa1-6 is about 300 cells / well, 4T1 is about 500 cells / well, and CT26 is about 400 cells / well); observe regularly for 10-14 days, and change the medium every 2 days; terminate the culture when cell clumps appear visible to the naked eye or when the number of cells in most colonies is greater than or equal to 50 cells under a microscope; discard the supernatant, add PBS to rinse 3 times, and fix with 1 mL 4% paraformaldehyde for 20-30 minutes; discard the supernatant, add PBS to rinse 3 times, and stain with 1 mL 0.1% crystal violet for 20-30 minutes; discard the supernatant, add PBS to rinse 3 times, and take pictures after drying (the DMSO treatment of the control group is the same as in Example 1).

[0033] according to Figure 5 In this study, we found that the in vitro clone formation ability of four mouse cancer cells was significantly decreased after bortezomib pretreatment. Specifically, after bortezomib pretreatment under selected conditions, the in vitro clone number of four mouse cancer cells was significantly decreased, especially Panc02 ( Figure 5 a) and Hepa 1-6 ( Figure 5 In b), the number of clones is only in the single digit, while the corresponding control group has hundreds of clones.

[0034] 2. Migration: Four types of cancer cells were pretreated according to the treatment concentration and time obtained in Example 1, the supernatant was collected, centrifuged, counted and adjusted to a reasonable concentration (according to the method of Example 1), and the required cells were resuspended in serum-free DMEM medium (Panc02 was about 50,000 cells / well, Hepa1-6 was about 50,000 cells / well, 4T1 was about 100,000 cells / well, and CT26 was about 100,000 cells / well), 100 μL of the cell suspension was inoculated into the upper chamber of the transwell, 700 μL of DMEM medium containing 10% serum was added to the lower chamber, and the cells were cultured in an incubator; after the corresponding time (Panc02 was about 24h, Hepa1-6 was about 24h, 4T1 was about 48h, and CT26 was about 48h), the culture was terminated; the culture medium was discarded, the cells were rinsed 3 times with PBS, and the upper chamber was fixed and stained (see Example 1 for the specific process); the upper surface of the upper chamber was wiped with a moist cotton swab to remove the cells that did not penetrate the membrane, and the images were taken with a microscope (the DMSO treatment of the control group was the same as in Example 1).

[0035] according to Figure 6 In this experiment, it was found that the migration ability of four mouse cancer cells was significantly decreased after bortezomib treatment. Specifically, after bortezomib pretreatment under selected conditions, the number of four mouse cancer cells penetrating the transwell chamber membrane was significantly reduced.

[0036] 3. Invasion: The corresponding steps of Example 2 were followed, except that 10% concentration of matrix gel (Corning 356231) was added to the upper chamber in advance and placed in an incubator. After the matrix gel solidified, the excess liquid was discarded and the corresponding cell suspension was added. The subsequent operations were the same as those of the migration experiment.

[0037] according to Figure 6 In this experiment, it was found that the invasive ability of four mouse cancer cells was significantly reduced after bortezomib treatment. Specifically, after bortezomib pretreatment under selected conditions, the number of four mouse cancer cells penetrating the transwell chamber membrane containing the matrix gel film was significantly reduced.

[0038] Example 3 This example shows the results of in vivo evaluation of whole cell tumor vaccines.

[0039] 1. Safety assessment of whole-cell tumor vaccines: 1.1 Cell preparation The four types of cancer cells were appropriately expanded and cultured, and the cells were treated according to the conditions in Example 1, the supernatant was collected, centrifuged, and counted (quality control was performed using trypan blue staining, and the death rate was about 50%), and the concentration was adjusted to an appropriate level (about 80W / 50μL for Panc02, about 80W / 50μL for Hepa1-6, about 200W / 50μL for 4T1, and about 180W / 50μL for CT26). 1.2 Construction of subcutaneous tumor model: The mice were divided into two groups, namely the unvaccinated group and the vaccinated group. The hair at the tumor-bearing site under the left forelimb of the mice was shaved with a razor. The unvaccinated group was injected with PBS, and the vaccinated group was inoculated with the prepared tumor whole-cell vaccine. The specific operation was to use an insulin needle to extract an appropriate amount of cells (Panc02 is about 80W, Hepa1-6 is about 80W, 4T1 is about 200W, and CT26 is about 180W) and inject them subcutaneously (this time is marked as day 0). The mice were raised normally and the occurrence of tumors was observed regularly. If no tumor was formed in the tumor-bearing site of the mice after 7 days, it can be preliminarily considered that the prepared tumor whole-cell vaccine is safe.

[0040] In this experiment, the tumor whole-cell vaccine prepared under the conditions described in the previous experiments was safe and reliable, providing a solid preliminary foundation for further evaluation of its in vivo protective efficiency.

[0041] 2. Evaluation of the protective efficacy of whole-cell tumor vaccines On the 7th day, the hair at the tumor-bearing site in the contralateral axilla of the mice was removed, and the untreated normal tumor cells prepared in advance were subcutaneously injected (Panc02 was about 15W, Hepa1-6 was about 20W, 4T1 was about 40W, and CT26 was about 40W). The mice were fed normally, and the occurrence of tumors was observed and recorded every day. A curve of the proportion of tumor-free mice was prepared to evaluate the protective efficiency of the whole-cell tumor vaccine.

[0042] according to Figure 7 This experiment further demonstrated that the mouse tumor whole-cell vaccine prepared based on bortezomib pretreatment has significant protective efficacy in a variety of cancers, specifically Panc02 (unvaccinated group n=9, vaccinated group n=9, Figure 7 a) The protection efficiency was 66.7% (6 / 9), Hepa 1-6 (unvaccinated group n=9, vaccinated group n=9, Figure 7 b) The protection efficiency was 88.9 (8 / 9), 4T1 (unvaccinated group n=7, vaccinated group n=8, Figure 7 c) The protection efficiency was 87.5% (7 / 8), CT26 (unvaccinated group n=10, vaccinated group n=10, Figure 7 The protective efficiency in d is 80% (8 / 10). The above results indicate that we have successfully prepared a simple and broad-spectrum mouse tumor whole-cell vaccine based on bortezomib.

[0043] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. A simple and broad-spectrum tumor whole-cell vaccine, characterized in that: These included murine cancer cells pretreated with the proteasome inhibitor bortezomib; The mouse cancer cells include one or more of pancreatic cancer cells Panc02, liver cancer cells Hepa1-6, breast cancer cells 4T1, and colon cancer cells CT26.

2. The simple and broad-spectrum tumor whole-cell vaccine according to claim 1, characterized in that: The cells were pretreated with the proteasome inhibitor bortezomib, which included pancreatic cancer cells Panc02 treated with bortezomib at 40, 60 and 80 nM for 18 h, liver cancer cells Hepa1-6 treated with bortezomib at 20, 40 and 60 nM for 18 h, breast cancer cells 4T1 treated with bortezomib at 100, 120 and 140 nM for 48 h, or colon cancer cells CT26 treated with bortezomib at 150, 200 and 250 nM for 48 h.

3. The simple and broad-spectrum tumor whole-cell vaccine according to claim 1, characterized in that: The proteasome inhibitor bortezomib pretreatment is that pancreatic cancer cells Panc02 are treated with 60nM bortezomib for 18h, or liver cancer cells Hepa1-6 are treated with 40nM bortezomib for 18h, or breast cancer cells 4T1 are treated with 120nM bortezomib for 48h, or colon cancer cells CT26 are treated with 250nM bortezomib for 48h.

4. Use of the simple broad-spectrum tumor whole-cell vaccine according to claim 1 in preventing one or more of pancreatic cancer, liver cancer, breast cancer and colon cancer.

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