Disclosure of Invention
The technical problems to be solved are as follows:
The invention screens the animal lactobacillus which can effectively treat melanoma, can strongly activate CD4+ and CD8+ T lymphocytes in tumor microenvironment, reverse tumor immunosuppression microenvironment, and play a good anti-tumor role.
The technical scheme is as follows:
An application of lactobacillus animalis in preparing antitumor drugs is characterized in that the lactobacillus animalis is lactobacillus animalis (Lactobacillus animalis) ATCC35046.
Lactobacillus animalis strain (Lactobacillus animalis) ATCC35046 was purchased from North Nanoea-Beijing North Nanoea Biotechnology institute. After the frozen lactobacillus plantarum ATCC35046 is recovered in a solid culture medium, single colony is picked up to be activated in a liquid culture medium, then the culture is expanded, after the lactobacillus plantarum ATCC35046 grows to have the OD value of about 0.8 (2X 108 CFU/mL) in the logarithmic phase, the lactobacillus plantarum ATCC35046 is obtained through centrifugation, and after PBS is washed for 3 times, the lactobacillus plantarum is resuspended in PBS for later use.
The tumor is melanoma.
The action mechanism is that the lactobacillus plantarum ATCC35046 is administrated subcutaneously through the tissue around the tumor, and the lactobacillus plantarum ATCC35046 can strongly activate the CD4+ and the CD8+ T lymphocytes in the tumor microenvironment by promoting the maturation of dendritic cells, so that the tumor cells are effectively killed, and the effect of tumor immunotherapy is achieved.
Specifically:
In the invention, the Lactobacillus plantarum ATCC35046 and the mouse macrophage RAW 264.7 are incubated in vitro, and the RT-qPCR technology proves that the Lactobacillus plantarum ATCC35046 can obviously promote the macrophages to express cytokines TNF-alpha, IL-1 beta and IL-6 on the mRNA level. It is shown that Lactobacillus animalis stimulates macrophage activation and secretes cytokines with anti-tumor effects.
In the invention, the lactobacillus plantarum ATCC35046 is incubated with the dendritic cells derived from the mouse bone marrow in vitro, and through flow cytometry, the fact that the lactobacillus plantarum ATCC35046 can promote the expression of MHC-II and CD86 on the surfaces of the dendritic cells is verified, and the lactobacillus plantarum ATCC35046 can effectively promote the differentiation and maturation of the dendritic cells. Maturation of dendritic cells helps to further activate T lymphocytes.
The invention takes a C57BL/6 mouse as an experimental animal, and establishes a mouse melanin tumor model by subcutaneously implanting B16-F10 cells. When the average tumor volume reached about 50mm3, lactobacillus animalis ATCC35046 (4×108 CFU/mouse) was injected into the subcutaneous tissue around the tumor, while untreated PBS group and lactococcus lactis NZ9000 treated group (2×109 CFU/mouse) were set as controls, the first administration was recorded as day 0, and tumor volume changes were recorded beginning, and on day 12, experimental mice with tumor volume exceeding 1500mm3 appeared, thus ending the treatment, taking a weigh of tumor tissue, photographing, and then performing tissue section detection analysis after formaldehyde fixation. The experimental results showed that on day 12, the average tumor volume of the treated group of Lactobacillus plantarum ATCC35046 was reduced by 78% by 561mm3 compared to the untreated PBS control group, the average tumor weight of the treated group of Lactobacillus plantarum ATCC35046 was 0.66g lower than the PBS control group by 75%, and 1/3 of the mice in the treated group of Lactobacillus plantarum ATCC35046 had complete tumor regression. Meanwhile, on day 12, the average tumor weight of the Lactobacillus plantarum ATCC35046 treated group was 0.13g lower, and was 37% lower than that of the lactococcus lactis NZ9000 treated group. Further, H & E and TUNEL staining results showed that large area necrosis of mouse tumor tissue occurred and a large number of tumor cells were apoptotic after treatment with lactobacillus animalis ATCC 35046. The results show that the lactobacillus plantarum ATCC35046 can inhibit the growth of the mouse melanoma more effectively, the tumor inhibiting activity of the lactobacillus plantarum ATCC35046 is obviously superior to that of the lactobacillus plantarum, and the effective dose of the lactobacillus plantarum ATCC35046 is 1/5 of that of the lactobacillus plantarum. Of particular note, 1/3 of the mice in the treatment group with Lactobacillus plantarum ATCC35046 had their tumor completely resolved, which fully demonstrates that Lactobacillus plantarum ATCC35046 has the advantages of low dosage and good anti-tumor effect.
The invention also observes the infiltration condition of T lymphocytes in the melanin tumor tissue after the treatment of the animal lactobacillus ATCC35046 through immunofluorescence staining of the paraffin section of the tumor tissue. The results showed that the infiltration of CD4+ T and CD8+ T cells in tumor tissue was significantly increased in the Lactobacillus plantarum ATCC35046 treated group compared to the placebo group. The lactobacillus zoonotis ATCC35046 can reverse tumor immunosuppression microenvironment by promoting infiltration of T lymphocytes in tumor tissues, and enhance anti-tumor immune response, thereby effectively playing a role in inhibiting tumor growth.
The beneficial effects are that:
1. The present invention surprisingly found that Lactobacillus animalis ATCC35046 was effective in inhibiting the growth of melanoma, and that 1/3 of mice had complete tumor regression. Melanoma is a skin tumor with highest mortality rate, and the current treatment methods have limited effects. Most probiotics are used alone, and cannot achieve an effective anti-tumor effect. The invention uses the reported probiotics with a certain anti-tumor effect, namely lactococcus lactis NZ9000 as a control group, and proves that the anti-tumor effect of the animal lactobacillus ATCC35046 is better than that of the lactococcus lactis NZ9000 (with obvious difference), and the effective dose of the probiotic lactobacillus lactis NZ9000 is 1/5 of that of the lactococcus lactis NZ 9000. Of particular note, as shown in fig. 4D, 1/3 of the mice in the lactobacillus plantarum ATCC35046 treatment group had complete tumor regression, which is a significant finding in the field of probiotics for tumor treatment, demonstrating that the particular lactobacillus plantarum ATCC35046 produced unexpected technical effects.
2. The Lactobacillus plantarum ATCC35046 can be prepared into freeze-dried powder injection for subcutaneous injection around tumor or transdermal administration, and is directly applied to the surfaces of tumor and surrounding skin. The lactobacillus animalis ATCC35046 adopted by the invention is a probiotic which can be used for fermenting dairy products and has the advantages of safety and no toxicity.
3. A significant advantage of the present invention is that it can exert a highly effective tumor-inhibiting effect by Lactobacillus animalis ATCC35046 alone without the need for combination with other immunotherapies. The therapeutic effect was comparable to the effect reported in literature for the use of (Griffin ME,Espinosa J,Becker JL,Luo JD,Carroll TS,Jha JK,Fanger GR,Hang HC.Enterococcus peptidoglycan remodeling promotes checkpoint inhibitor cancer immunotherapy.Science.2021Aug 27;373(6558):1040-1046.) enterococcus in combination with anti-PD-1 antibody for treatment of the murine melanoma model (fig. 1B, 15 days after treatment), the average tumor volume of the enterococcus combination with anti-PD-1 antibody treatment group was about 150mm3, and the average tumor volume was about 73% reduced compared to the untreated control group (about 550mm3).
Terminology
TNF-alpha tumor necrosis factor-alpha
IL-1 beta interleukin-1 beta
IL-6 Interleukin-6
MHC-II major histocompatibility Complex-II
CD86 leukocyte differentiation antigen 86
H & E hematoxylin-eosin staining method
TUNEL in situ terminal transferase labelling technique
Detailed Description
The following examples will assist those skilled in the art in a more complete understanding of the invention, but are not intended to limit the invention in any way.
Lactobacillus animalis (Lactobacillus animalis) ATCC35046 and lactococcus lactis (Lactococcus lactis) NZ9000 strains were purchased from North Nanoorganism-Beijing North NanoCritical Biotechnology institute. Lipopolysaccharide (LPS) was purchased from Sigma.
EXAMPLE 1 determination of the growth curve of Lactobacillus animalis ATCC35046
The method comprises measuring growth curve of Lactobacillus plantarum ATCC35046 by turbidimetry, taking out frozen Lactobacillus plantarum ATCC35046, inoculating onto MRS solid medium by three-area line method, and standing in 30 deg.C incubator for culturing. After colonies are formed in the culture dish, single colonies are picked up into 10mL of liquid culture medium, and the culture medium is subjected to standing incubation at 30 ℃ for 14-16 hours to serve as seed culture solution. 13 triangular flasks with 20mLMRS liquid culture medium were numbered 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12h for a total of 13 time points. 200 mu L of seed culture solution is sucked by a sterile straw, added into a numbered triangular flask, kept stand at 30 ℃ for incubation, then the triangular flask is taken out according to corresponding time and stored in a 4 ℃ refrigerator, finally the OD value of the culture solution at all time points is measured at 600nm wavelength, and a growth curve is drawn.
As a result, as shown in FIG. 1, lactobacillus animalis ATCC35046 entered the logarithmic phase within 4-6 hours and gradually entered the plateau phase after 8 hours. The OD corresponding to the arrival of Lactobacillus plantarum ATCC35046 at the log phase of growth was about 0.8.
EXAMPLE 2 determination of the total number of colonies corresponding to the arrival of Lactobacillus plantarum ATCC35046 and lactococcus lactis NZ9000 at the log phase
In order to determine the total number of bacterial colonies corresponding to the logarithmic phase of the Lactobacillus plantarum ATCC35046, 200 mu L of the seed culture solution in the example 1 is absorbed into 20mL of fresh liquid culture medium, namely, 100 times of the seed culture solution is expanded for continuous culture, the seed culture solution is continuously diluted by 10 times after the seed culture solution grows to the logarithmic phase, namely, the OD value is about 0.8, 1mL of bacterial solution with three continuous dilutions of 10-2、10-4 and 10-6 is selected for carrying out plate coating operation, a solid culture dish is subjected to static incubation at 30 ℃, and after bacterial colonies grow out, a proper culture dish is selected for calculating the average bacterial colony number. The culture medium of lactococcus lactis NZ9000 (L.lactis) was M17, and the total number of colonies corresponding to the log phase of lactococcus lactis (L.lactis) was determined by the same method as that of Lactobacillus animalis ATCC35046.
As a result, when Lactobacillus plantarum ATCC35046 grew to an OD of 0.8, the total number of colonies was 2X 108 CFU/mL. The OD value of lactococcus lactis NZ9000 reaching the log phase of growth was about 0.8, and the total number of colonies corresponding thereto was 1X 109 CFU/mL.
EXAMPLE 3 Lactobacillus animalis ATCC35046 stimulates macrophage activation and secretion of associated cytokines
The method comprises collecting Lactobacillus plantarum ATCC35046 in logarithmic growth phase (2×8 CFU/mL), centrifuging to obtain thallus, and suspending in antibiotic-free DMEM cell culture medium. Macrophage RAW 246.7 was inoculated in a 6-well plate at a concentration of 1×105 cells/mL (2×105 cells/well), and then the prepared lactobacillus zoonotii ATCC35046 suspension was added to the 6-well plate (multiplicity of infection moi=25), while a negative control group (control) to which only medium was added and a positive control group to which LPS (100 ng/mL) was added were set. After incubation for 4 hours in a 37 ℃ cell incubator, the supernatant is discarded, and after washing 3 times with PBS, fresh DMEM medium containing antibiotics is replaced, after further incubation for 6 hours, cellular RNA is extracted, and the expression levels of IL-1 beta, IL-6 and TNF-alpha at mRNA levels are detected by using an RT-qRCR method.
As a result, lipopolysaccharide (LPS) is a characteristic component of the cell wall of gram-negative bacteria, and LPS and its lipid A moiety can induce activation of immune cells such as macrophages and dendritic cells by binding to Toll-like receptor 4 (TLR 4). LPS is selected as a positive control, so that on one hand, experimental conditions can be optimized, and on the other hand, the LPS can be used as a reference for evaluating the activation intensity of immune cells in an experimental test group. As shown in Table 1 and FIG. 2, the expression of the proinflammatory cytokine mRNA, such as IL-1β, TNF-. Alpha.and IL-6, was extremely significantly increased in macrophages after stimulation by Lactobacillus plantarum ATCC35046, as compared to the negative control group. Meanwhile, compared with a positive control drug (LPS), the compound has obvious rise. It was demonstrated that Lactobacillus plantarum ATCC35046 can stimulate macrophage activation and secrete cytokines with anti-tumor effects.
TABLE 1 analysis of cytokine significance differences Table (P <0.05 indicates the presence of significant differences)
| IL-1β | TNF-α | IL-6 |
| control VS L.animalis | P<0.001 | P<0.001 | ns |
| control VS LPS | P<0.5 | P<0.5 | P<0.5 |
EXAMPLE 4 Lactobacillus animalis ATCC35046 promotes maturation of dendritic cells
The method comprises collecting Lactobacillus plantarum ATCC35046 in logarithmic growth phase (2×8 CFU/mL), centrifuging to obtain thallus, and suspending in antibiotic-free RPMI-1640 cell culture solution containing 10% foetal calf serum. Mouse bone marrow-derived dendritic cells were cultured by cervical removal of 6-8 week C57BL/6 male mice, removal of bone marrow from the tibia and femur of the mice and resuspension of cells using RPMI-1640 complete medium (containing L-glutamine (2 mM), 2-mercaptoethanol (50. Mu.M), streptomycin (100. Mu.g/mL), penicillin (100U/mL), fetal bovine serum (10%) and GM-CSF (20 ng/mL)), plating the cell suspension at a concentration of 2X105 cells/mL in a 100mM bacterial culture dish (10 mL medium volume) and recording as day 0, culturing until day 3 additional 10mL fresh RPMI-1640 complete medium, half-changing the liquid until days 6 and 8, culturing until day 10, centrifuging to collect cells and counting. Then, the prepared lactobacillus animalis ATCC35046 suspension was added to the 48-well plate (multiplicity of infection moi=25) by changing to RPMI-1640 medium containing 10% fetal bovine serum and spreading it in the 48-well plate at a concentration of 1×106 cells/mL (5×105 cells/well), while setting a negative control group (control) to which only the medium was added and a positive control group to which LPS (1 μg/mL) was added. After incubation at 37 ℃ for 24 hours, the medium was removed and washed 3 times with PBS, replaced with fresh RPMI-1640 medium containing antibiotics and 10% fetal bovine serum, and after further incubation for 12 hours, the cells were collected for flow analysis. Specific method for determining MHC-II and CD86 by flow cytometry comprises adding monoclonal antibodies of APC-antiCD C, PE-anti MHC-II and FITC-anti CD86 to cell suspension, incubating at 4deg.C for 30min in the absence of light, centrifuging, washing to remove unbound free antibody, and detecting by flow cytometry after resuspension of cells with 200 μ LPBS.
As shown in Table 2 and FIG. 3, the Lactobacillus plantarum ATCC35046 can significantly promote the surface expression of MHC-II and CD86 on dendritic cells compared with the negative control group, the Lactobacillus plantarum ATCC35046 significantly promotes the surface expression of MHC-II and CD86 on dendritic cells compared with LPS, and the Lactobacillus plantarum ATCC35046 can effectively induce the maturation of dendritic cells, and the dendritic cells can further activate T lymphocytes as professional antigen presenting cells.
TABLE 2 analysis of the significant differences in MHC-II and CD86 expression on the surface of dendritic cells
| MHC-II | CD86 |
| control VS L.animalis | P<0.001 | P<0.001 |
| control VS LPS | P<0.001 | P<0.001 |
EXAMPLE 5 Lactobacillus animalis ATCC35046 was effective in inhibiting the growth of melanoma in mice
Male C57BL/6 mice, weight 22-24g, B16-F10 melanoma cells were subcutaneously implanted into the backs of the mice, after the average tumor volume was about 50mm3, the mice were randomly divided into 3 groups, namely PBS group, lactobacillus plantarum ATCC35046 (L.animalis) treatment group and lactococcus lactis NZ9000 (L.lactis) treatment group, each group of 12 mice, the logarithmic phase Lactobacillus plantarum ATCC35046 bacterial solution (2X 108 CFU/mL) and lactococcus lactis NZ9000 bacterial solution (1X 109 CFU/mL) were taken, and after the mice were resuspended in PBS, the cells were subjected to subcutaneous injection at the tumor week, the injection volume of each mouse was 100. Mu.L, the injection amount of Lactobacillus plantarum ATCC35046 was 4X 108 CFU/mouse, and the injection amount of lactococcus lactis NZ9000 was 2X 109 CFU/mouse. The first administration was recorded as day 0, and then was again administered on days 1,7 and 8, while the tumor growth volume change of the mice was monitored at regular time, and when the tumor volume of the experimental mice reached 1500mm3, the experiment was ended, and after euthanizing the treated mice, the tumors were taken out and weighed, and photographed.
As a result, most probiotics alone cannot achieve an effective antitumor effect and usually require a combination with immunotherapy. For example, literature (Griffin ME,Espinosa J,Becker JL,Luo JD,Carroll TS,Jha JK,Fanger GR,Hang HC.Enterococcus peptidoglycan remodeling promotes checkpoint inhibitor cancer immunotherapy.Science.2021Aug27;373(6558):1040-1046.) reports the effect of enterococcus in combination with anti-PD-1 antibody on treatment of melanoma in mice, the average tumor volume of the enterococcus in combination with anti-PD-1 antibody treated group after 15 treatment was about 150mm3, and the average tumor volume was reduced by about 73% compared to untreated control group (about 550mm3) (FIG. 1B). In this study, as shown in tables 3, 4 and fig. 4, there was no significant difference in the body weight of mice throughout the course of treatment, in the treated group and in the untreated group. On day 12, the average tumor volume of the treated group of Lactobacillus plantarum ATCC35046 was reduced by 78% by 561mm3 compared with the untreated PBS control group, and the average tumor weight of the treated group of Lactobacillus plantarum ATCC35046 was reduced by 0.66g compared with the PBS control group by 75%, which is equivalent to the effect reported in the above documents. Meanwhile, in the Lactobacillus plantarum ATCC35046 treated group, 1/3 of the mice had complete tumor regression (FIG. 4D). The above results fully demonstrate that Lactobacillus plantarum ATCC35046 alone can exert a highly potent tumor-inhibiting effect without the need for combination with other immunotherapies. Meanwhile, the invention takes the reported probiotics with a certain anti-tumor effect, namely lactococcus lactis NZ9000 as a control group, and the average tumor weight of the animal lactobacillus ATCC35046 treatment group is 0.13g lower than that of the lactococcus lactis NZ9000 treatment group on the 12 th day, and the average tumor weight is reduced by 37 percent. The results show that the lactobacillus plantarum ATCC35046 can inhibit the growth of the mouse melanoma more effectively, the tumor inhibiting activity of the lactobacillus plantarum ATCC35046 is obviously superior to that of the lactobacillus plantarum, and the effective dose of the lactobacillus plantarum ATCC35046 is 1/5 of that of the lactobacillus plantarum. Of particular note, 1/3 of the mice in the Lactobacillus plantarum ATCC35046 treated group had complete tumor regression. Fully shows that the Lactobacillus plantarum ATCC35046 has the advantages of low dosage and good anti-tumor effect.
Table 3 average body weight, average tumor volume, and tumor regression rate results for day 12 mice (n=12)
| Group of experiments | PBS group | Group L.lactis | Group L.animalis |
| Body weight (g) | 24 | 24 | 25 |
| Tumor weight (g) | 0.88 | 0.35 | 0.22 |
| Tumor volume (mm3) | 716 | 156 | 155 |
| Tumor regression rate | 0 | 0 | 4/12 |
Table 4 table of the significance differences between average body weight, average tumor weight and average tumor volume of day 12 mice
| Weight of body | Tumor weight | Tumor volume |
| control VS L.animalis | ns | P<0.01 | P<0.001 |
| control VS L.lactis | ns | P<0.5 | P<0.001 |
EXAMPLE 6H & E staining and TUNEL staining analysis of murine melanoma tumor tissue
Method after the tumor tissue taken out in example 3 was fixed with 10% paraformaldehyde for 24 hours, paraffin was embedded, paraffin sections were prepared, and then H & E staining and TUNEL staining were performed.
As shown in FIG. 5, the H & E results show that the Lactobacillus plantarum ATCC35046 treated group has large-area necrosis area of tumor tissues, swelling necrosis of tumor cells and fragmentation of cell nuclei, and TUNEL results show that the apoptosis degree of the tumor cells is very serious after the Lactobacillus plantarum ATCC35046 is treated. The results indicate that lactobacillus animalis ATCC35046 can promote necrosis and apoptosis of tumor cells, thereby inhibiting growth of tumors.
EXAMPLE 7 Lactobacillus animalis ATCC35046 significantly promotes infiltration of CD4+ and CD8+ T cells in melanoma tumor tissue
The method comprises dewaxing and rehydration of paraffin sections of tumor tissue in example 6, antigen retrieval with citrate buffer at ph=6, then incubation with specific anti-CD 4 and CD8 primary antibodies at 4 ℃ overnight, washing the primary antibodies, incubating with PE-labeled anti-IgG (h+l) secondary antibodies for 30min at room temperature, washing the secondary antibodies, counterstaining with DAPI, mounting, and observation with a fluorescence microscope.
As a result, as shown in FIG. 6, the infiltration amount of CD4+ and CD8+ T cells in tumor tissues was increased in the Lactobacillus plantarum ATCC35046 treated group compared with the PBS group. The lactobacillus zoon ATCC35046 is shown to activate immune response in tumor by promoting infiltration of T cells in tumor tissue, thereby playing a role of killing tumor cells and achieving the effect of inhibiting tumor growth.