纳米材料在抗肿瘤免疫治疗中的应用研究
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摘要
目的：近年来,抗肿瘤免疫疗法在肿瘤治疗中的应用日益受到医学界的重视,其中树突状细胞(dendritic cells，DC)疗法是抗肿瘤免疫治疗领域中的一个重要研究方向,已在多种肿瘤的临床治疗试验中显示出较好的应用前景。然而,DC抗肿瘤疗法的疗效尚有较大改进空间,因此仍需发展新的技术和方法以进一步增强DC所诱导的抗肿瘤免疫反应。碳纳米管(Carbon nanotubes, CNT)作为一种优良的载体可携带多种生物活性分子穿越生物膜,将DNA、RNA、多肽、蛋白、药物等带到效应器官及细胞内。在本研究中,我们评估了碳纳米管对肿瘤抗原的载体效应,构建了碳纳米管与肿瘤蛋白共价复合物(CNT-TumorP),用以刺激DC,在体外观察了碳纳米管是否会影响DC对肿瘤蛋白的吞噬,并评价了碳纳米管-肿瘤蛋白共价复合物对DC所诱导的抗肿瘤免疫反应的影响。
方法：抽取健康志愿者外周血,常规分离外周血单个核细胞(peripheral blood mononuclear cells, PBMC),贴壁去悬浮法分离单核细胞后,加入含重组人巨噬细胞-粒细胞集落刺激因子(rhGM-CSF)和重组人白介素-4(rhIL-4)的RPMI 1640+10% fBS完全培养基中培养6天诱导出DC；用细胞裂解液裂解人乳腺癌细胞MCF-7,提取细胞裂解蛋白(tumor lysate protein),标记上FITC荧光,再与经过表面处理的多壁碳纳米管在催化剂碳二亚胺(EDAC)的作用下进行共价偶联；用碳纳米管-肿瘤蛋白的复合物刺激DC后,激光共聚焦显微镜下观察DC对肿瘤蛋白的吞噬,流式细胞术检测DC表面CD40、CD83、CD86、HLA-DR分子的表达水平；在体外用可溶性肿瘤抗原或碳纳米管-肿瘤抗原复合物负载的DC刺激PBMC,以MTS法检测PBMC对MCF-7细胞的特异性杀伤作用；将MCF-7细胞置于含各种浓度碳纳米管的培养基内,检测碳纳米管对细胞存活的影响。
结果：1)人外周血单个核细胞在rhGM-CSF和rhIL-4因子的协同诱导下,可获得一定数量及功能正常的未成熟树突状细胞,20m1人外周血大约可诱导出2×106个DC；2)碳纳米管与裂解蛋白共价连接后,仍具有较好的分散性及稳定性；3)流式结果显示,肿瘤蛋白与碳纳米管结合后,DC对肿瘤蛋白的吞噬增加,平均荧光强度在肿瘤蛋白浓度为1μg/ml或0.1μg/ml时,分别提升了84%和95%(p<0.05)；4)激光共聚焦显微镜显示,碳纳米管与肿瘤蛋白的复合物可被DC吞入细胞质中；5)碳纳米管对DC的表型不产生影响,不会促进DC的成熟；6)以碳纳米管-肿瘤蛋白的复合物刺激DC,可显著增强肿瘤蛋白的免疫原性,改善DC对淋巴细胞的活化,进而提升DC所诱导的淋巴细胞对靶肿瘤细胞的杀伤效应；7)碳纳米管-肿瘤蛋白的复合物所增强的抗肿瘤免疫效应具有抗原特异性,对其它细胞的杀伤作用无显著增强；8)所用碳纳米管的安全性较好,在0.0005-0.5μg/ml的浓度范围内,对MCF-7的存活不产生影响。
结论：碳纳米管与肿瘤蛋白共价偶联后,在体外能促进DC对肿瘤蛋白的吞噬,并提升DC所诱导的淋巴细胞对肿瘤细胞的杀伤作用,因而有可能在基于DC的抗肿瘤免疫治疗中具有潜在的应用价值。
目的：抗肿瘤免疫治疗作为一种重要的肿瘤辅助治疗手段,能有效提升传统治疗方法的疗效,改善病人的预后。然而,肿瘤免疫疗法中普遍存在的一个重要问题是相关肿瘤抗原的免疫原性往往较弱,导致治疗效果有限。提高肿瘤抗原免疫原性的一种直接有效的方法是应用免疫佐剂。传统铝佐剂尽管在人类疫苗中有七十余年的应用历史,但其在增强抗肿瘤免疫方面的效果一直不佳。在本研究中我们用纳米氧化铝作为肿瘤疫苗的佐剂,在小鼠体内比较纳米铝与传统铝佐剂在抗肿瘤免疫治疗中的效果,以此来评价纳米铝作为肿瘤疫苗佐剂的潜在应用价值。
方法：用H22肝癌细胞系在BALB/c小鼠体内建立皮下肿瘤模型(DayO)；H22细胞用丝裂霉素C灭活后作为肿瘤细胞疫苗；第7天(Day7)将荷瘤小鼠随机分为四个组,分别皮下注射生理盐水、肿瘤细胞疫苗(TCV)、肿瘤细胞疫苗联合氢氧化铝佐剂(TCV+Alum)或肿瘤细胞疫苗联合纳米铝佐剂(TCV+Nano-alum)进行免疫治疗,第14天再进行相同免疫一次。在两次免疫治疗之后(Day14),每四天测量小鼠肿瘤生长大小。实验末期检测小鼠外周血单个核细胞对肿瘤细胞的杀伤率,并取肿瘤组织进行病理切片观察淋巴细胞浸润和肿瘤坏死。
结果：1)皮下肿瘤生长情况观察：TCV+Nano-alum组的平均肿瘤面积均明显小于对照组(p<0.05),除TCV+Alum组的肿瘤面积在第14天明显小于对照组外,其它治疗组与对照组相的平均肿瘤面积在所有检测点无差异；2)体外淋巴细胞毒性试验结果显示：对照组、TCV组、TCV+Alum组和TCV+Nano-alum组的肿瘤杀伤率分别是9.9±6.9%、30.1±3.09%、35.63±2.8%和67.96+9.54%,其中TCV+Nano-alum组显示了最强的肿瘤杀伤作用(p<0.05)；3)肿瘤组织病理切片观察：治疗组比对照组有更多的淋巴细胞浸润,且TCV+Nano-alum组更可见肿瘤坏死。
结论：纳米铝可提升肿瘤细胞疫苗的免疫治疗效果,因此有可能作为一种新型的佐剂在肿瘤的免疫治疗中发挥有效作用。
目的：半抗原二硝基氟苯(dinitrophenyl, DNP)修饰后的肿瘤细胞疫苗(tumor cell vaccine, TCV)可明显提升针对恶性黑色素瘤的免疫反应,并改善患者预后。然而,DNP瘤苗修饰法能否促进针对其它肿瘤的免疫反应,仍有待于探索。本研究的目的,是评价DNP修饰的瘤苗是否会影响人淋巴细胞对人乳腺癌细胞和人肺癌细胞的杀伤效应。并将DNP与另一类广泛应用于肿瘤细胞疫苗的修饰物——新城疫病毒Ulster株(New Castle Disease Virus of Ulster Strain, NDV Ulster)在增强淋巴细胞对肿瘤细胞杀伤作用方面的效果进行比较。
方法：人乳腺癌细胞系MCF-7和人肺癌细胞系H23用丝裂霉素C灭活,再经DNP/NDV修饰(或不修饰)后作为肿瘤疫苗；抽取健康志愿者外周血,用Ficoll密度梯度离心法分离单个核细胞(peripheral blood mononuclear cells, PBMC),将其用作杀伤肿瘤靶细胞的效应细胞。本试验将PBMC(其中富含的淋巴细胞发挥直接杀伤作用)分为三组：未经肿瘤疫苗刺激的淋巴细胞(T)经未修饰的肿瘤疫苗所刺激过的淋巴细胞(T+TCV),和经DNP(或NDV)修饰的肿瘤疫苗所刺激过的淋巴细胞(T+DNP-TCV或T+NDV-TCV)。淋巴细胞与各种肿瘤疫苗共同孵育三天后,用标准的MTS肿瘤抑制法检测淋巴细胞对靶细胞的杀伤。
结果：1)比之对照组(T组),不论是未修饰的肿瘤疫苗(T+TCV组)还是DNP修饰过的肿瘤疫苗(T+DNP-TCV组),均诱导淋巴细胞产生了针对MCF-7或H23细胞更强的杀伤作用；2)在MCF-7为靶细胞时,DNP修饰过的肿瘤疫苗(T+DNP-TCV组)所诱导的肿瘤杀伤,要明显高于未经修饰的肿瘤疫苗(T+TCV组)；3)DNP与NDV Ulster相比,两者增强抗肿瘤免疫反应的程度是相似的,且产生的杀伤作用不针对非肿瘤靶细胞。
结论：DNP修饰的肿瘤疫苗能显著增强人淋巴细胞对乳腺癌细胞的特异性杀伤作用。提示DNP肿瘤疫苗修饰法除了可用于治疗恶性黑色素瘤之外,也可能在针对其它人类肿瘤的免疫治疗中发挥作用。
Objective:The significance of anti-tumor immunotherapy as an adjuvant cancer treatment has been well recognised. Among the practical immunotherapy technigues, dendritic cells (DC)-based therapy is considered an important approach that has attracted mainstream attention. Some of the prior clinical studies on DC-based therapy have generated promising results in treating malignancies such as glioblastoma or malignant melanoma. Nevertheless, the outcome of DC-based anticancer immunotherapy still needs further improvement. An important issue of DC-based therapy, therefore, is to develop new technologies that can further enhance the efficacy for DC stimulation by available tumor antigens. Carbon nanotubes (CNT) have been shown to have potential applications in multiple biomedical fields, especially as transporters for delivery of various bioactive molecules such as peptides, proteins, DNAs, RNAs, or drugs. In this study, we investigated whether the conjugates of CNT and tumor protein (CNT-TumorP) would influence the endocytosis of tumor antigen by human DC in vitro. We also evaluated the effects of CNT-TumorP on the capacity of DC to induce anticancer cytotoxicity in human lymphocytes.
Methods:Human DC were cultured from fresh peripheral blood of healthy donors per standard protocol employing rhGM-CSF and rhIL-4. MCF7 human cancer cells were lysed and tumor lysate protein (TumorP) was extracted. The tumor protein was conjugated to the oxidized CNT in the presence of the coupling agent N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide (EDAC). Tumor protein (TumorP) was labeled with fluorescein-5-isothiocyanate (TumorP-FITC), and then covalently conjugated to CNT (CNT-TumorP-FITC). Two groups of DC were co-incubated with TumorP-FITC and CNT-TumorP-FITC separately. The endocytosis of tumor protein by DC was evaluated by flow cytometry and confocal microscopy. Moreover, expression of CD40, CD83, CD86 and HLA-DR on immature DC after co-incubation with CNT, TumorP or CNT-TumorP was evaluated by flow cytometry. The lymphocytes'specific cytotoxiciy to MCF-7 was then evaluated after stimulated with CNT-TumorP loaded DC using a standard MTS cytotoxicity assay. Additionally, the toxicity of CNT in a wide range of concentrations was studied using MCF-7 cell line as target.
Results:1) Approximately 2×106 immature DC were obtained by a routine protocol employing rhGM-CSF and rhIL-4, with mononuclear cells isolated from 20ml of peripheral human blood.2) The dispersibility and stability of CNT were well preserved after the conjugation.3) The flow cytometry analysis revealed that endocytosis of tumor protein by DC was significantly enhanced when TumorP was conjugated to CNT (p<0.05). The enhancements of mean fluorescence intensity were 84% and 95% for TumorP concentration of 1μg/ml and 0.1μg/ml, respectively.4) Confocal microscopy indicated that the CNT-PRO was taken into the DC.5) CNT showed no effect on the phenotype of DC and did not induce DC maturation.6) CNT-TumorP enhanced the function of DC to induce anticancer response in lymphocytes.7) The anticancer response, induced by CNT-TumorP, was specific to the tumor cells from which the TumorP was extracted, with no obvious cytotoxicity against other cell lines.8) CNT, through a range of concentration from 0.0005μg/ml to 0.5μg/ml, showed no influence on viability of MCF-7 cells.
Conclusion:The conjugates of carbron nanotubes and tumor protein could enhance the endocytosis of tumor antigen by human DC and the capability of DC to induce anticancer response in vitro. The results suggest that CNT-based nanotechnology may have a prospective role in the development of more efficacious DC-based anticancer immunotherapy.
Objective:Anti-tumor immunotherapy has the potential to significantly improve the prognosis of cancer treatment, though the efficacy of immunotherapy generally needs further improvement. One way to improve the efficacy is using immune adjuvants, but the adjuvants for anticancer immunotherapy have to be more potent than for prophylactic vaccines. The identification of novel and effective adjuvants is thus of practical importance for anticancer immunotherapy. Conventional aluminum has been used as adjuvant for more than 70 years in human vaccines; however, its efficacy for enhancing antitumor immune response is limited. Nanotechnology has the potential to generate new solutions for multiple biomedical problems. In this work, we studied whether aluminum oxide nanoparticles (nano-alum) would enhance the efficacy of an anti-tumor immunotherapy that employs tumor cell vaccine (TCV), in a murine model bearing the H22 liver cancer, to assess nano-alum's applicability as adjuvant in common anticancer immune regimens.
Methods:BALB/c mice were inoculated with 2×106 live hepatoma H22 cells subcutaneously on day 0. Tumor cell vaccine (TCV) was prepared by inactivating H22 cells with Mitomycin C. On day 7, the mice with subcutaneous tumors were randomly divided into four groups:1) the control group,2) the TCV treatment group, 3) the TCV plus conventional alum (TCV+alum) treatment group, and 4) the TCV plus nano-alum (TCV+nano-alum) treatment group. On day 7 and 14, the treatment groups received two subcutaneous doses of designed immune treatment agents for triggering anti-tumor reactions. Tumor dimensions were monitored every 4 days after the second vaccination. At the end of the study, the cytotoxicity of mouse peripheral blood lymphocytes against H22 cells was evaluated in vitro. Histological slides of the tumor tissues were prepared for evaluation tumor necrosis and lymphocyte infiltration.
Results:1) The average tumor size of the TCV+Nano-alum group was significantly smaller than that of the control group (p<0.05). Except for the TCV+Alum group which had smaller tumor size in comparison with the control group on day 14, no difference of the average tumor sizes was observed between the other groups.2) The cytotoxicity of the lymphocytes against H22 cells was 9.9±6.9% for the control group, 30.1±3.09% for the TCV group,35.63±2.8% for the TCV+alum group, and 67.96±9.54% for the TCV+nano-alum group. TCV+nano-alum group generated significantly higher anticancer cytotoxicity compared to the other treatment groups (p<0.05).3) Compared to the control group, there was more lymphocyte infiltration in the treatment groups by histological studies. Moreover, the tumor tissue of the TCV+nano-alum group developed obvious necrosis.
Conclusion:Aluminum oxide nanoparticles enhanced the anticancer immune response induced by tumor cell vaccine. The results suggest that nano-alum may potentially serve as an effective adjuvant in anticaner immunotherapies.
Objective:It has been reported that hapten dinitrophenyl (DNP) modified tumor cell vaccines (TCV) can enhance the immune response against malignant melanoma and improve the prognosis of patients. However, whether DNP-modified TCV can also induce effective immune reactions against other types of malignancy has not been well evaluated. In this study, we investigated DNP-modified TCV in the enhancement of lymphocytes'cytotoxicity against breast and lung cancer cells in vitro. Moreover, we compared the enhancement of DNP-modified TCV mediated lymphocytes' cytotoxicity with that of another widely used tumor vaccine modification agent, New Castle Disease Virus of Ulster Strain (NDV Ulster),-modified TCV.
Methods:Human breast cancer cell line MCF7, lung cancer cell line H23 cells were inactivated by mitomycin C and modified with or without DNP/NDV. As the effector cells for the killing of targeted tumor cells, peripheral blood mononuclear cells (PBMC) from healthy human donors were obtained via standard Ficoll gradient centrifugation. Then PBMC were divided into three groups:control lymphocytes (T), tumor cell vaccine-stimulated lymphocytes (T+TCV), and DNP-or NDV-modified tumor cell vaccine-stimulated lymphocytes (T+DNP-TCV or T+NDV-TCV). After 3-day incubation of the lymphocytes with different modified tumor vaccines, cell killing was evaluated using a standard MTS cytotoxicity assay.
Results:1) Compared to the control group (T), both DNP-modified TCV and unmodified TCV caused significant lymphocyte inhibition of MCF-7 cells and H23 cells.2) DNP-modified TCV (T+DNP-TCV) induced significantly higher lymphocyte inhibition of MCF-7 cells than that of unmodified TCV (T+TCV).3) Both DNP and NDV Ulster generated similar enhancement of anti-tumor cell immune reaction but not non-tumor cells.
Conclusion:DNP-modified TCV enhances the specific cytotoxicity of lymphocytes against human breast cancer cells. Our findings suggest that DNP-modification of TCV may have the potential in immunotherapy against multiple types of human cancer in addition to malignant melanoma.
方法：抽取健康志愿者外周血,常规分离外周血单个核细胞(peripheral blood mononuclear cells, PBMC),贴壁去悬浮法分离单核细胞后,加入含重组人巨噬细胞-粒细胞集落刺激因子(rhGM-CSF)和重组人白介素-4(rhIL-4)的RPMI 1640+10% fBS完全培养基中培养6天诱导出DC；用细胞裂解液裂解人乳腺癌细胞MCF-7,提取细胞裂解蛋白(tumor lysate protein),标记上FITC荧光,再与经过表面处理的多壁碳纳米管在催化剂碳二亚胺(EDAC)的作用下进行共价偶联；用碳纳米管-肿瘤蛋白的复合物刺激DC后,激光共聚焦显微镜下观察DC对肿瘤蛋白的吞噬,流式细胞术检测DC表面CD40、CD83、CD86、HLA-DR分子的表达水平；在体外用可溶性肿瘤抗原或碳纳米管-肿瘤抗原复合物负载的DC刺激PBMC,以MTS法检测PBMC对MCF-7细胞的特异性杀伤作用；将MCF-7细胞置于含各种浓度碳纳米管的培养基内,检测碳纳米管对细胞存活的影响。
结果：1)人外周血单个核细胞在rhGM-CSF和rhIL-4因子的协同诱导下,可获得一定数量及功能正常的未成熟树突状细胞,20m1人外周血大约可诱导出2×106个DC；2)碳纳米管与裂解蛋白共价连接后,仍具有较好的分散性及稳定性；3)流式结果显示,肿瘤蛋白与碳纳米管结合后,DC对肿瘤蛋白的吞噬增加,平均荧光强度在肿瘤蛋白浓度为1μg/ml或0.1μg/ml时,分别提升了84%和95%(p<0.05)；4)激光共聚焦显微镜显示,碳纳米管与肿瘤蛋白的复合物可被DC吞入细胞质中；5)碳纳米管对DC的表型不产生影响,不会促进DC的成熟；6)以碳纳米管-肿瘤蛋白的复合物刺激DC,可显著增强肿瘤蛋白的免疫原性,改善DC对淋巴细胞的活化,进而提升DC所诱导的淋巴细胞对靶肿瘤细胞的杀伤效应；7)碳纳米管-肿瘤蛋白的复合物所增强的抗肿瘤免疫效应具有抗原特异性,对其它细胞的杀伤作用无显著增强；8)所用碳纳米管的安全性较好,在0.0005-0.5μg/ml的浓度范围内,对MCF-7的存活不产生影响。
结论：碳纳米管与肿瘤蛋白共价偶联后,在体外能促进DC对肿瘤蛋白的吞噬,并提升DC所诱导的淋巴细胞对肿瘤细胞的杀伤作用,因而有可能在基于DC的抗肿瘤免疫治疗中具有潜在的应用价值。
目的：抗肿瘤免疫治疗作为一种重要的肿瘤辅助治疗手段,能有效提升传统治疗方法的疗效,改善病人的预后。然而,肿瘤免疫疗法中普遍存在的一个重要问题是相关肿瘤抗原的免疫原性往往较弱,导致治疗效果有限。提高肿瘤抗原免疫原性的一种直接有效的方法是应用免疫佐剂。传统铝佐剂尽管在人类疫苗中有七十余年的应用历史,但其在增强抗肿瘤免疫方面的效果一直不佳。在本研究中我们用纳米氧化铝作为肿瘤疫苗的佐剂,在小鼠体内比较纳米铝与传统铝佐剂在抗肿瘤免疫治疗中的效果,以此来评价纳米铝作为肿瘤疫苗佐剂的潜在应用价值。
方法：用H22肝癌细胞系在BALB/c小鼠体内建立皮下肿瘤模型(DayO)；H22细胞用丝裂霉素C灭活后作为肿瘤细胞疫苗；第7天(Day7)将荷瘤小鼠随机分为四个组,分别皮下注射生理盐水、肿瘤细胞疫苗(TCV)、肿瘤细胞疫苗联合氢氧化铝佐剂(TCV+Alum)或肿瘤细胞疫苗联合纳米铝佐剂(TCV+Nano-alum)进行免疫治疗,第14天再进行相同免疫一次。在两次免疫治疗之后(Day14),每四天测量小鼠肿瘤生长大小。实验末期检测小鼠外周血单个核细胞对肿瘤细胞的杀伤率,并取肿瘤组织进行病理切片观察淋巴细胞浸润和肿瘤坏死。
结果：1)皮下肿瘤生长情况观察：TCV+Nano-alum组的平均肿瘤面积均明显小于对照组(p<0.05),除TCV+Alum组的肿瘤面积在第14天明显小于对照组外,其它治疗组与对照组相的平均肿瘤面积在所有检测点无差异；2)体外淋巴细胞毒性试验结果显示：对照组、TCV组、TCV+Alum组和TCV+Nano-alum组的肿瘤杀伤率分别是9.9±6.9%、30.1±3.09%、35.63±2.8%和67.96+9.54%,其中TCV+Nano-alum组显示了最强的肿瘤杀伤作用(p<0.05)；3)肿瘤组织病理切片观察：治疗组比对照组有更多的淋巴细胞浸润,且TCV+Nano-alum组更可见肿瘤坏死。
结论：纳米铝可提升肿瘤细胞疫苗的免疫治疗效果,因此有可能作为一种新型的佐剂在肿瘤的免疫治疗中发挥有效作用。
目的：半抗原二硝基氟苯(dinitrophenyl, DNP)修饰后的肿瘤细胞疫苗(tumor cell vaccine, TCV)可明显提升针对恶性黑色素瘤的免疫反应,并改善患者预后。然而,DNP瘤苗修饰法能否促进针对其它肿瘤的免疫反应,仍有待于探索。本研究的目的,是评价DNP修饰的瘤苗是否会影响人淋巴细胞对人乳腺癌细胞和人肺癌细胞的杀伤效应。并将DNP与另一类广泛应用于肿瘤细胞疫苗的修饰物——新城疫病毒Ulster株(New Castle Disease Virus of Ulster Strain, NDV Ulster)在增强淋巴细胞对肿瘤细胞杀伤作用方面的效果进行比较。
方法：人乳腺癌细胞系MCF-7和人肺癌细胞系H23用丝裂霉素C灭活,再经DNP/NDV修饰(或不修饰)后作为肿瘤疫苗；抽取健康志愿者外周血,用Ficoll密度梯度离心法分离单个核细胞(peripheral blood mononuclear cells, PBMC),将其用作杀伤肿瘤靶细胞的效应细胞。本试验将PBMC(其中富含的淋巴细胞发挥直接杀伤作用)分为三组：未经肿瘤疫苗刺激的淋巴细胞(T)经未修饰的肿瘤疫苗所刺激过的淋巴细胞(T+TCV),和经DNP(或NDV)修饰的肿瘤疫苗所刺激过的淋巴细胞(T+DNP-TCV或T+NDV-TCV)。淋巴细胞与各种肿瘤疫苗共同孵育三天后,用标准的MTS肿瘤抑制法检测淋巴细胞对靶细胞的杀伤。
结果：1)比之对照组(T组),不论是未修饰的肿瘤疫苗(T+TCV组)还是DNP修饰过的肿瘤疫苗(T+DNP-TCV组),均诱导淋巴细胞产生了针对MCF-7或H23细胞更强的杀伤作用；2)在MCF-7为靶细胞时,DNP修饰过的肿瘤疫苗(T+DNP-TCV组)所诱导的肿瘤杀伤,要明显高于未经修饰的肿瘤疫苗(T+TCV组)；3)DNP与NDV Ulster相比,两者增强抗肿瘤免疫反应的程度是相似的,且产生的杀伤作用不针对非肿瘤靶细胞。
结论：DNP修饰的肿瘤疫苗能显著增强人淋巴细胞对乳腺癌细胞的特异性杀伤作用。提示DNP肿瘤疫苗修饰法除了可用于治疗恶性黑色素瘤之外,也可能在针对其它人类肿瘤的免疫治疗中发挥作用。
Objective:The significance of anti-tumor immunotherapy as an adjuvant cancer treatment has been well recognised. Among the practical immunotherapy technigues, dendritic cells (DC)-based therapy is considered an important approach that has attracted mainstream attention. Some of the prior clinical studies on DC-based therapy have generated promising results in treating malignancies such as glioblastoma or malignant melanoma. Nevertheless, the outcome of DC-based anticancer immunotherapy still needs further improvement. An important issue of DC-based therapy, therefore, is to develop new technologies that can further enhance the efficacy for DC stimulation by available tumor antigens. Carbon nanotubes (CNT) have been shown to have potential applications in multiple biomedical fields, especially as transporters for delivery of various bioactive molecules such as peptides, proteins, DNAs, RNAs, or drugs. In this study, we investigated whether the conjugates of CNT and tumor protein (CNT-TumorP) would influence the endocytosis of tumor antigen by human DC in vitro. We also evaluated the effects of CNT-TumorP on the capacity of DC to induce anticancer cytotoxicity in human lymphocytes.
Methods:Human DC were cultured from fresh peripheral blood of healthy donors per standard protocol employing rhGM-CSF and rhIL-4. MCF7 human cancer cells were lysed and tumor lysate protein (TumorP) was extracted. The tumor protein was conjugated to the oxidized CNT in the presence of the coupling agent N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide (EDAC). Tumor protein (TumorP) was labeled with fluorescein-5-isothiocyanate (TumorP-FITC), and then covalently conjugated to CNT (CNT-TumorP-FITC). Two groups of DC were co-incubated with TumorP-FITC and CNT-TumorP-FITC separately. The endocytosis of tumor protein by DC was evaluated by flow cytometry and confocal microscopy. Moreover, expression of CD40, CD83, CD86 and HLA-DR on immature DC after co-incubation with CNT, TumorP or CNT-TumorP was evaluated by flow cytometry. The lymphocytes'specific cytotoxiciy to MCF-7 was then evaluated after stimulated with CNT-TumorP loaded DC using a standard MTS cytotoxicity assay. Additionally, the toxicity of CNT in a wide range of concentrations was studied using MCF-7 cell line as target.
Results:1) Approximately 2×106 immature DC were obtained by a routine protocol employing rhGM-CSF and rhIL-4, with mononuclear cells isolated from 20ml of peripheral human blood.2) The dispersibility and stability of CNT were well preserved after the conjugation.3) The flow cytometry analysis revealed that endocytosis of tumor protein by DC was significantly enhanced when TumorP was conjugated to CNT (p<0.05). The enhancements of mean fluorescence intensity were 84% and 95% for TumorP concentration of 1μg/ml and 0.1μg/ml, respectively.4) Confocal microscopy indicated that the CNT-PRO was taken into the DC.5) CNT showed no effect on the phenotype of DC and did not induce DC maturation.6) CNT-TumorP enhanced the function of DC to induce anticancer response in lymphocytes.7) The anticancer response, induced by CNT-TumorP, was specific to the tumor cells from which the TumorP was extracted, with no obvious cytotoxicity against other cell lines.8) CNT, through a range of concentration from 0.0005μg/ml to 0.5μg/ml, showed no influence on viability of MCF-7 cells.
Conclusion:The conjugates of carbron nanotubes and tumor protein could enhance the endocytosis of tumor antigen by human DC and the capability of DC to induce anticancer response in vitro. The results suggest that CNT-based nanotechnology may have a prospective role in the development of more efficacious DC-based anticancer immunotherapy.
Objective:Anti-tumor immunotherapy has the potential to significantly improve the prognosis of cancer treatment, though the efficacy of immunotherapy generally needs further improvement. One way to improve the efficacy is using immune adjuvants, but the adjuvants for anticancer immunotherapy have to be more potent than for prophylactic vaccines. The identification of novel and effective adjuvants is thus of practical importance for anticancer immunotherapy. Conventional aluminum has been used as adjuvant for more than 70 years in human vaccines; however, its efficacy for enhancing antitumor immune response is limited. Nanotechnology has the potential to generate new solutions for multiple biomedical problems. In this work, we studied whether aluminum oxide nanoparticles (nano-alum) would enhance the efficacy of an anti-tumor immunotherapy that employs tumor cell vaccine (TCV), in a murine model bearing the H22 liver cancer, to assess nano-alum's applicability as adjuvant in common anticancer immune regimens.
Methods:BALB/c mice were inoculated with 2×106 live hepatoma H22 cells subcutaneously on day 0. Tumor cell vaccine (TCV) was prepared by inactivating H22 cells with Mitomycin C. On day 7, the mice with subcutaneous tumors were randomly divided into four groups:1) the control group,2) the TCV treatment group, 3) the TCV plus conventional alum (TCV+alum) treatment group, and 4) the TCV plus nano-alum (TCV+nano-alum) treatment group. On day 7 and 14, the treatment groups received two subcutaneous doses of designed immune treatment agents for triggering anti-tumor reactions. Tumor dimensions were monitored every 4 days after the second vaccination. At the end of the study, the cytotoxicity of mouse peripheral blood lymphocytes against H22 cells was evaluated in vitro. Histological slides of the tumor tissues were prepared for evaluation tumor necrosis and lymphocyte infiltration.
Results:1) The average tumor size of the TCV+Nano-alum group was significantly smaller than that of the control group (p<0.05). Except for the TCV+Alum group which had smaller tumor size in comparison with the control group on day 14, no difference of the average tumor sizes was observed between the other groups.2) The cytotoxicity of the lymphocytes against H22 cells was 9.9±6.9% for the control group, 30.1±3.09% for the TCV group,35.63±2.8% for the TCV+alum group, and 67.96±9.54% for the TCV+nano-alum group. TCV+nano-alum group generated significantly higher anticancer cytotoxicity compared to the other treatment groups (p<0.05).3) Compared to the control group, there was more lymphocyte infiltration in the treatment groups by histological studies. Moreover, the tumor tissue of the TCV+nano-alum group developed obvious necrosis.
Conclusion:Aluminum oxide nanoparticles enhanced the anticancer immune response induced by tumor cell vaccine. The results suggest that nano-alum may potentially serve as an effective adjuvant in anticaner immunotherapies.
Objective:It has been reported that hapten dinitrophenyl (DNP) modified tumor cell vaccines (TCV) can enhance the immune response against malignant melanoma and improve the prognosis of patients. However, whether DNP-modified TCV can also induce effective immune reactions against other types of malignancy has not been well evaluated. In this study, we investigated DNP-modified TCV in the enhancement of lymphocytes'cytotoxicity against breast and lung cancer cells in vitro. Moreover, we compared the enhancement of DNP-modified TCV mediated lymphocytes' cytotoxicity with that of another widely used tumor vaccine modification agent, New Castle Disease Virus of Ulster Strain (NDV Ulster),-modified TCV.
Methods:Human breast cancer cell line MCF7, lung cancer cell line H23 cells were inactivated by mitomycin C and modified with or without DNP/NDV. As the effector cells for the killing of targeted tumor cells, peripheral blood mononuclear cells (PBMC) from healthy human donors were obtained via standard Ficoll gradient centrifugation. Then PBMC were divided into three groups:control lymphocytes (T), tumor cell vaccine-stimulated lymphocytes (T+TCV), and DNP-or NDV-modified tumor cell vaccine-stimulated lymphocytes (T+DNP-TCV or T+NDV-TCV). After 3-day incubation of the lymphocytes with different modified tumor vaccines, cell killing was evaluated using a standard MTS cytotoxicity assay.
Results:1) Compared to the control group (T), both DNP-modified TCV and unmodified TCV caused significant lymphocyte inhibition of MCF-7 cells and H23 cells.2) DNP-modified TCV (T+DNP-TCV) induced significantly higher lymphocyte inhibition of MCF-7 cells than that of unmodified TCV (T+TCV).3) Both DNP and NDV Ulster generated similar enhancement of anti-tumor cell immune reaction but not non-tumor cells.
Conclusion:DNP-modified TCV enhances the specific cytotoxicity of lymphocytes against human breast cancer cells. Our findings suggest that DNP-modification of TCV may have the potential in immunotherapy against multiple types of human cancer in addition to malignant melanoma.
引文
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