| ObjectiveImmunologic adjuvants play an important role in the treatment of tumors and chronic infections. CP is an anaerobic Gram-positive bacteria that has no obvious pathogenicity to humans. Recently CP is commonly used as immunologic adjuvant. CP can enhance the non-specific immunity. Much progress has been made in the treatment of tumors and chronic infection of CP. Meanwhile, severe side effects relatively constrained its clinical application. So, employing the advanced nanotechnology to dispose of CP we prepared a neotype of immunomodulator-the product from corynebacterium parvum at nano-scale (NCPP) successfully. Our former data have elucidated that NCPP have the same effects in the treatment of tumors and chronic infection as CP while reducing the side effects of CP effectively.Chemotherapy is the primary means of tumor therapy. But side effects from the common systemic chemotherapy are so severely that patients couldn't bear. In recent years, interventional chemotherapy in the treatment of solid tumors has been widely used. In the pre-study our partners established slow intra-tumor release of drugs to improved existing chemotherapy and achieved a good anti-tumor effect.To achieve a more effective treatment of cancers, we combined NCPP with slow intra-tumor release of drugs on tumors in mice.Methods1. Combined NCPP with slow intra-tumor release of drugs inhibits B16 melanoma in mice B16 melanoma cells were cultured in RPMI1640 medium supplemented with 10% fetal bovine serum. For establishment of tumor-bearing models, tumor cells were inoculated subcutaneously in the left flank of each mouse. When the maximal diameter and the minimal diameter of tumors arrived to about 5mm×5mm~2, mice were randomly divided into four groups:①the normal saline group;②the slow intra-tumor release of drugs group;③the NCPP group;④the drug combination group. Seven days later, mice were treated again and the gross tumor volume was determined. Then 14 days later, all the tumor-bearing mice were sacrificed to dislodge the tumor tissue and the spleen to make the next experiment. Measure tumor weight, stain the tumor exemplar by HE and detected the number of T lymphocyte subsets by Flow Cytometry.2. Combined NCPP with slow intra-tumor release of drugs inhibits H22 liver tumor in miceH22 liver tumor cells were cultured in abdominal cavity of BALB/c mice. For establishment of tumor-bearing models, tumor cells were inoculated subcutaneously in the left flank of each mouse. When the maximal diameter and the minimal diameter of tumors arrived to about 5mmx5mm2, mice were randomly divided into four groups:①the normal saline group;②the slow intra-tumor release of drugs group;③the NCPP group;④the drug combination group. Seven days later, mice were treated again and the gross tumor volume was determined. Then 14 days later, all the tumor-bearing mice were sacrificed to dislodge the tumor tissue and the spleen to make the next experiment. Measure tumor weight, stain the tumor exemplar by HE and detected the number of T lymphocyte subsets by Flow Cytometry.Results1. Combined NCPP with slow intra-tumor release of drugs inhibits melanoma in mice1.1 Combined therapy inhibit melanoma tumor growth effectivelyFor research the effects of combined therapy inhibit melanoma in mice, we inoculated B16 melanoma cells subcutaneously in the left flank of each mouse. When the maximal diameter and the minimal diameter of tumors arrived to about 5mm×5mm~2, mice were randomly divided into four groups:①the normal saline group;②the slow intra-tumor release of drugs group;③the NCPP group;④the drug combination group. Seven days later, mice were treated again. The gross tumor volume was determined and tumor growth curve was drawn. The result showed that: there was no significant difference between four groups in eight days after treatment. After eight days, the tumors of NS control group grow fastest, the tumors of slow intra-tumor release of drugs group and NCPP group grow slower than NS control group, the tumors of combined therapy group grow slowest. At the same time point the difference about tumor volume is statistical significance in four groups. Tumor tissues showed that the tumor volume of combined therapy group was less than the other three groups. It showed that either NCPP or slow intra-tumor release can inhibit tumor growth, but combined therapy was more effectively.1.2 Combined therapy can kill tumor cells stronglyFor analysis of the effect of treatment group, we conducted histopathological tumor detection. The results showed that: in NS control group, although there were a few necrosis domains in the center of tumor tissue, many vigorous growth tumor cells surrounding. In slow intra-tumor release of drugs group and NCPP group, moderate necrosis domains are even distributed in tumor tissues and residual tumor cells are still growing strong. In combined therapy group, a large number of tumor necrosis region in tumor tissues, the ratio of living tumor cells decreased significantly. It showed that not only tumor volume but also the number of living cells remaining is fewer in combined therapy group than in other three groups.1.3 Combined therapy can effectively increase the proportion of CD8~+ T cells in immune organsFor analysis the impact of immunity, we detected CD4~+ and CD8~+ lymphocyte subsets in spleen by Flow Cytometry. The results showed that: there is no significant change of CD4~+ T Lymphocyte Subsets. CD8~+ T Lymphocyte Subsets in NCPP group and drug combination group increased significantly than in NS control group, especially in drug combination group. It showed that combined therapy can increase the anti-tumor effect that CD8~+ T cell-mediated. 2. Combined NCPP with slow intra-tumor release of drugs inhibits H22 liver tumor in mice2.1 Combined therapy inhibit liver tumor growth effectivelyFor research the effects of combined therapy inhibit liver tumor in mice, we inoculated H22 cells subcutaneously in the left flank of each mouse. When the maximal diameter and the minimal diameter of tumors arrived to about 5mm×5mm~2, mice were randomly divided into four groups:①the normal saline group;②the slow intra-tumor release of drugs group;③the NCPP group;④the drug combination group. Seven days later, mice were treated again. The gross tumor volume was determined and tumor growth curve was drawn. The result showed that: there was no significant difference between four groups in seven days after treatment. After seven days, the tumors of NS control group grow fastest, the tumors of slow intra-tumor release of drugs group and NCPP group grow slower than NS control group, the tumors of combined therapy group grow slowest. At the same time point the difference about tumor volume is statistical significance in four groups. Tumor tissues showed that the tumor volume of combined therapy group was less than the other three groups. It showed that either NCPP or slow intra-tumor release can inhibit tumor growth, but combined therapy was more effectively.2.2 Combined therapy can kill tumor cells stronglyFor analysis of the effect of treatment group, we conducted a histopathological tumor detection. The results showed that: in NS control group, although there were a few necrosis domains in the center of tumor tissue, many vigorous growth tumor cells surrounding. In slow intra-tumor release of drugs group and NCPP group, moderate or slight necrosis domains are distributed in tumor tissues and residual tumor cells are still growing strong. In combined therapy group, a large number of tumor necrosis region in tumor tissues and very few living tumor cells remaining. It showed that not only tumor volume but also the number of living cells remaining is fewer in combined therapy group than in other three groups.2.3 Combined therapy can effectively increase the proportion of CD4~+, CD8~+ and Foxp3~+ T cells in immune organs For analysis the impact of immunity, we detected CD4~+, CD8~+ and Foxp3~+ T lymphocyte subsets in spleen by Flow Cytometry. The results showed that: CD4~+ and CD8~+ T lymphocyte Subsets in treatment groups increased significantly than in NS control group, especially in drug combination group and slow intra-tumor release of drugs group. CD4~+Foxp3~+ T lymphocyte subsets in slow intra-tumor release of drugs group slightly higher than in NS control group, while there is no significant difference between other two groups and NS control group. It showed that: the number of CD4~+ and CD8~+ T cell can be raised by slow intra-tumor release of drugs and NCPP, especially by slow intra-tumor release of drugs. The number of Treg cells can be raised by slow intra-tumor release of drugs, while it can be decreased to the level of NS control group by NCPP.2.4 Combined therapy can extended the survival rate in mice effectivelyFor observe the survival rate of mice by several treatment methods, we carried out two weeks of observation on four groups of mice. The results showed that: in NS control group mice began to die in the 7th day and fourteen days later the survival rate is 56%. In NCPP group and slow intra-tumor release of drugs group mice began to die in the 12th day and fourteen days later the survival rate is 56%. In drug combination group no mouse died in fourteen days and the survival rate is 100%. The survival rate of mice can be significantly raised in a certain period of time by combined therapy.Conclusions1. Drug combination therapy can kill tumor cells and inhibit the growth of tumor more effectively.2. Drug combination therapy can improve survival rates in mice significantly.3. Drug combination therapy can produce a higher tumor-specific immune response, which may be related to synergies by two types of treatment.Innovation and significances1. In this study we combined NCPP with slow intra-tumor release of drugs to treat a variety of tumors in mice at the first time and got a satisfying efficacy. This study provide an experimental basis of it's clinical application and provide a new idea in the treatment for solid tumors.2. In this study we confirmed the first time that combined NCPP with slow intra-tumor release of drugs can produce a higher tumor-specific immune response which may be related to synergies by two types of treatment.3. In this study a new direction was proposed in search of efficient, safe and specific treatment of tumors.Limitations1. Clinical research about NCPP has not been carried out, so this kind of drug combination therapy can not be used for patients now.2. The possibility of implantation metastasis to pinhole must be considered. |
