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Effects and Mechanisms of NCPP Combined with Slow Intra-tumor Release of Drugs on Tumors in Mice

Author: GuoChun
Tutor: ZhangLiNing
School: Shandong University
Course: Medical Immunology
Keywords: Combination therapy NCPP Sustained-release chemotherapy within tumor
CLC: R73-3
Type: Master's thesis
Year: 2009
Downloads: 50
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Abstract

The purpose of immune adjuvants play an important supporting role in the treatment of tumors and chronic infections. Corynebacterium parvum (corynebacterium parvum, CP) is an anaerobic gram-positive bacteria, the human body without obvious pathogenic immune adjuvant. CP has enhanced the role of non-specific immunity, made exciting progress in the treatment of cancer and resistance to chronic infection, but clinical application is limited due to its side effects. The corynebacterium nano preparation (Non-cell Corynebacterium ParvumProduct, NCPP) is an advanced nanotechnology CP is a new kind of ultra-fine grinding and classification of high-pressure processing prepared nano immunomodulatory agents. In a previous study, we confirmed The NCPP not only reserves the CP's original anti-tumor and anti-infective effects, but also significantly reduce the side effects of CP. Chemotherapy is one of the main methods of cancer treatment, but traditional systemic chemotherapy side effects, and brought great suffering to the patient. In recent years local tumor interventional chemotherapy in the treatment of solid tumors has been widely used, under the premise of ensuring the efficacy significantly reduce the side effects of chemotherapy drugs. Our collaborators in a preliminary study of the existing local interventional chemotherapy improved intratumoral sustained release therapy, and get a good anti-tumor effect. This project intends NCPP and the combination of sustained-release therapy tumor, on the one hand, the use of chemotherapy drugs locally anti-tumor effect, the other hand, the advantage of the NCPP improve immunity, to achieve the purpose of more effective treatment of tumors. Sustained-release chemotherapy C57 BL / 6 mice inhibition of melanoma B16 melanoma cells were seeded in C57 BL / 6 mice the method 1.NCPP joint intratumoral. When the tumor grew to 5mm × 5mm ~ 2, mice were randomly divided into four groups for treatment: ① normal saline (NS) control group: intratumoral and intraperitoneal injection of NS; ② sustained-release treatment group: intratumoral injection of Ara-C slow the release liquid dinitrophenyl (DNP); ③ NCPP treatment group: intraperitoneal injection of NCPP; ④ combination therapy group: intraperitoneal injection NCPP combined with intratumoral injection of Ara-C sustained release liquid two nitrobenzene (DNP). 7 days after repeated treatments. Measured daily during treatment the mice tumor size, calculate the tumor volume. After treatment for 14 days mice were sacrificed, the separation achieved tumor tissue and spleen. Weighing tumor weight evaluation the NCPP combined with intratumoral sustained release anti-tumor effect of chemotherapy. The tumor specimens HE staining tumor necrosis circumstances. Grinding spleen spleen cells suspensions flow cytometry spleen, the number of T cell subsets. Within the sustained-release chemotherapy 2.NCPP United tumor inhibition of the BALB / c mouse H22 liver tumor H22 hepatoma cells were inoculated into BALB / c mice. When the tumor grew to 5mm × 5mm ~ 2, the mice were randomly divided into four groups for treatment of the: ① saline (NS) control group: intratumoral and intraperitoneal injection of NS; ② sustained-release treatment groups: intratumoral injection of doxorubicin over the carbon dioxide bistrifluoroacetate nitrophenyl (DNP); (3) the NCPP treatment groups: intraperitoneal injection NCPP; ④ combination therapy group: intraperitoneal injection the NCPP combined with intratumoral injection of doxorubicin over the carbon dioxide bistrifluoroacetate nitrophenyl (DNP). 7 days after repeated treatments. Measuring tumor size during the next day, calculate the tumor volume, record the survival time of the mice in each group, calculate survival rates. After treatment for 14 days mice were sacrificed, the separation achieved tumor tissue and spleen. Weighing tumor weight evaluation the NCPP combined with intratumoral sustained release anti-tumor effect of chemotherapy. HE staining of tumor specimens observed necrosis. Grinding spleen spleen cells suspensions flow cytometry spleen, the number of T cell subsets. Sustained-release chemotherapy Results 1.NCPP United tumor inhibition 1.1 C57 BL / 6 mice melanoma combination therapy can effectively suppress the growth of melanoma in order to study the sustained-release chemotherapy on mouse melanoma in the the NCPP joint tumor suppression tumor effect, we cultured B16 melanoma cells were seeded in C57 BL / 6 mice skin, below, to be long to the appropriate size (volume approximately 5mm × 5mm ~ 2), divided into four groups for treatment: ① normal saline (NS) control group: intratumoral and intraperitoneal injection of NS; ② sustained-release treatment group: intratumoral injection of Ara-C release liquid two-nitrobenzene (DNP); (3) the NCPP treatment groups: intraperitoneal injection NCPP; ④ combination therapy group: intraperitoneal injection NCPP tumor injection of sustained-release chemotherapy drugs. 7 days after repeated treatments. Daily measurement of tumor size in mice during treatment, to calculate the tumor volume and tumor growth curve. The results show: 8 days after treatment among the treatment groups did not differ significantly; 8 days after the NS control group rapid tumor growth, sustained-release treatment group and NCPP group tumor growth slowed down compared with the control group, tumor growth slowest in the combined treatment group, The statistically significant differences in tumor volume at the same time point. Mice were sacrificed and dissected tumor tissue showed that the combined treatment group, tumor volume was significantly smaller than the other three groups, separate application the NCPP, or tumor within the sustained-release chemotherapy inhibited tumor growth, but their combination treatment is better. 1.2 The combination therapy can be more strongly kill tumor cells To further assess the analysis of the efficacy of each treatment regimen group, tumor tissue pathology detection. The results showed that: in the NS group, although the tumor tissue center ischemic necrosis of the area, but the surrounding tumor cells arranged in dense into a piece of tumor cells thrive; sustained-release treatment group, and NCPP treatment group tumor tissue ministries showed moderate necrosis residual tumor cells growth still strong; necrotic area within the tumor tissue of the combined treatment group, live tumor cells was significantly reduced. The combined treatment group is not only tumor volume is small, and a relatively small number of remaining living cells. The 1.3 combination therapy can effectively raised the proportion of CD8 ~ T cells, immune organ in order to further evaluate the analysis of various treatment options for immunity, we were detected by flow cytometry spleen lymphocyte CD4 ~ and CD8 ~ group change. The results found that: mouse CD4 ~ T cells no significant change in the number of four treatment groups, but significant difference between the number of CD8 ~ T cells each group. Intratumoral sustained release treatment can be mild inhibition in vivo CD8 ~ T cell proliferation, NCPPs treatment to enhance the role of the number of CD8 ~ T cells, and the combination therapy can be more significantly improved the number of CD8 ~ T cells. Description combination therapy to enhance CD8 ~ T cell-mediated anti-tumor effects. 2.NCPP United intratumoral sustained release chemotherapy combination therapy of BALB / c mouse liver tumor inhibition 2.1 can be more effective to inhibit the growth of liver tumors to chemotherapy research the NCPP joint intratumoral sustained release of the inhibitory effect of liver tumors in mice We will cultured H22 liver tumor cells were inoculated in BALB / c mice axillary subcutaneous, to be grown to the appropriate size (volume about 5mm × 5mm ~ 2), divided into four groups for treatment: ① saline (NS) control group: intratumoral and intraperitoneal injection of NS; ② sustained-release treatment group: intratumoral injection of doxorubicin over the carbon dioxide bistrifluoroacetate nitrobenzene (DNP); (3) the NCPP treatment groups: intraperitoneal injection NCPP; ④ combination therapy group: intraperitoneal injection NCPP intratumoral injection The sustained-release chemotherapy drugs. Treatment period measured every other day in the tumor size of the mice, the tumor volume was calculated and the tumor growth curve. The results show: 7 days after treatment among the treatment groups did not differ significantly; 7 days after the NS control group rapid tumor growth, sustained-release treatment group and NCPP group tumor growth slowed down compared with the control group, tumor growth slowest in the combined treatment group, The statistically significant differences in tumor volume at the same time point. Mice were sacrificed and dissected tumor tissue showed that the combined treatment group, tumor volume was significantly smaller than the other three groups, separate application the NCPP, or tumor within the sustained-release chemotherapy inhibited tumor growth, but their combination treatment is better. The 2.2 combination therapy can be more strongly kill tumor cells in order to further analysis to assess the efficacy of each treatment regimen group, we pathology detection of tumor tissue. The results showed that: in the NS group, although the tumor tissue center ischemic necrosis of the area, but the surrounding tumor cells arranged in dense into a piece of tumor cells thrive; sustained-release treatment group, and NCPP treatment group tumor tissue ministries showed moderate or mild necrosis, but still vigorous growth of residual tumor cells; within the tumor tissue of the combined treatment group, almost all necrotic tissue, very few live tumor cells. The combination therapy group not only tumor volume, and almost no live tumor cells exist. 2.3 Combination therapy can effectively raised the immune organs CD4 ~ T cells and CD8 ~~ T cell ratio In order to further evaluate the analysis of the impact of various treatment options group immunity, we to spleen lymphocytes CD4 ~~ were detected by flow cytometry, CD8 ~~ Foxp3 ~ molecular change. The results showed that: the three treatment groups, CD4 ~ T cells and CD8 ~ T cells compared with the control group have increased, sustained-release treatment group increased the most, followed by combined treatment group. CD4 ~ Foxp3 ~ T cells release treatment group is slightly higher than the NS control group, no significant difference in the other two groups with the NS control group. The results show that: the sustained-release treatment and NCPP treatment within the tumor could increase the number of tumor-bearing mouse CD4 ~ and CD8 ~ T cells, sustained-release treatment within the tumor is the most obvious; intratumoral sustained release treatment can increase the number of Treg cells, while the United NCPP treatment After enabling Treg cell numbers decreased to the level of NS control group. The 2.4 combination therapy can effectively prolong survival in order to observe the impact of several treatments on mice survival, we have four groups of mice two weeks of observation. The results showed that: the NS control group, beginning on day 7 mice died after 14 days survival rate of 56%; NCPPs the treatment group and the sustained-release treatment group starting from the first 12 days the mice died after 14 days survival rates were 78% ; combination therapy group to 14 days no mice died, a survival rate of 100%. The results show that: the combination therapy can significantly improve the survival rate over a period of time. Sustained-release chemotherapy the conclusion 1.NCPP joint intratumoral more effectively kill tumor cells and inhibit tumor growth. The sustained-release chemotherapy 2.NCPP United tumors can significantly improve the survival rate. Sustained-release chemotherapy 3.NCPP joint tumor can produce higher tumor-specific immune response, the mechanism may be related to synergies of the two treatment methods. Innovation and significance of the study for the first time the NCPP and intratumoral sustained release therapy combination of a variety of mouse tumor treatment and get satisfaction efficacy, and provide an experimental basis for clinical application. 2 This study is the first confirmed the NCPP United intratumoral sustained release therapy can produce higher tumor-specific immune response, the mechanism may be related to synergies of the two treatment methods. 3 In this study, the search for efficient, safe, specific tumor treatment pointed out a new direction, new cancer treatment research has important theoretical significance and guidance. The limitations 1.NCPP limited to laboratory, clinical studies have not been carried out, limiting the clinical application of this therapy. Intratumoral release treatment intratumoral injections may lead to tumor cell metastasis.

Effects and Mechanisms of NCPP Combined with Slow Intra-tumor Release of Drugs on Tumors in Mice

Abstract

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