异体树突状细胞与骨肉瘤细胞融合瘤苗的制备及其抗骨肉瘤主动免疫效应实验的研究
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摘要
骨肉瘤(osteosarcoma)是儿童和青少年最常见的原发性恶性骨肿瘤。20世纪70年代以前,主要治疗方法是截肢和关节离断,从外科治疗到出现肺转移的平均时间为8个月,5年生存率<25%。随着外科技术和影像学的发展,新辅助化疗的进步,骨肉瘤治愈率有了明显改进,5年生存率达到80%。但目前的治疗手段仍无法解决肿瘤病人的转移、复发和药物耐药等情况,因此,有必要探索一种新的、更为有效的治疗途径。随着对肿瘤发病机制和免疫逃避机制的认识,免疫治疗尤其是主动性免疫治疗为骨肉瘤的综合治疗提供了新的手段。
制备针对肿瘤特异性抗原(tumor-specific antigens, TSA)或肿瘤相关抗原(tumor-associated antigen, TAA)的治疗性瘤苗是目前肿瘤免疫治疗中的研究热点。其抗肿瘤免疫的重要目的就是诱导针对肿瘤抗原的特异性细胞毒性T淋巴细胞(cytocoxic T lymphocyte,CTL)反应,而CTL的激活依赖于抗原提呈细胞(antigen-presenting cell,APC)提供的抗原多肽和共刺激信号。在抗原提呈细胞中,尤以树突状细胞(dendritic cell,DC)的抗原提呈功能最强,可启动并调节免疫应答,有效刺激T、B淋巴细胞活化,在抗肿瘤、抗感染、移植排斥以及自身免疫性疾病发病过程中起重要作用。
本研究以骨肉瘤全细胞提供肿瘤抗原,采用细胞电融合技术将肿瘤抗原通过DC呈递表达于T淋巴细胞表面。全细胞瘤苗的最大的优点就在于融合细胞能够呈递肿瘤细胞所有的抗原,包括已知的和未知的抗原,鉴于目前大多数人类和动物的肿瘤细胞(包括骨肉瘤细胞)抗原类型仍未得到明确的鉴定,用全肿瘤细胞作为瘤苗提供抗原的有效组分仍不失为一类简单、有效的方法。电融合技术是当前生物学界重要的开创性研究手段之一,在制备单克隆抗体、哺乳动物克隆实验研究及肿瘤的免疫治疗方面均获得了广泛的应用,和传统的化学融合方法(如聚二乙醇PEG法)相比,电融合方法是一种更为高效的手段,融合效率通常可以提高1～2个数量级,这在很大程度上弥补了DC数量较少的缺陷,该技术也使两个来源于异体的细胞相互融合成为可能,目前已被广泛应用于瘤苗制备领域。
研究工作者之所以如此积极地进行异体融合瘤苗的尝试,是因为在临床应用中自体瘤苗的成功制备通常会受到疾病本身的困绕。首先,对患者自体的肿瘤细胞进行原代培养具有某种不确定性,特别是在保守治疗和以微波、射频等热疗手段处理后的肿瘤组织,失去进一步体外培养的可能;另外,目前肿瘤的治疗方案往往是手术、化疗及放疗等相结合的综合治疗,肿瘤患者的机体经过一系列治疗副效应的累加,患者的血象及血细胞质量通常难以满足进一步的免疫治疗需要,其营养状况也基本处于过消耗状态。而异体的树突状细胞可以从储备的健康志愿者外周血单核细胞获得,又可避免使所有肿瘤患者在接受免疫治疗前都必须提供相当量的自身外周血的缺陷,避免了“雪上加霜”。
实验一UMR-106细胞系易感动物的筛选和大鼠骨肉瘤及其肺转移模型的建立
目的探讨从普通的封闭群动物中筛选出对UMR-106大鼠骨肉瘤细胞系易感的SD大鼠,并建立其近交系的方法,以及大鼠骨肉瘤及其肺转移模型的建立方法。
方法取10对4周龄封闭群饲养的SD大鼠,分别在右股外侧皮下接种1×10~7个大鼠骨肉瘤细胞UMR-106,观察成瘤情况,选取存活的成瘤的雌、雄大鼠,6周后同笼交配,约4周妊娠期后,产下幼鼠,4周后重复肿瘤细胞皮下接种过程……依照此方法重复共4次,获得第4代UMR-106细胞系易感的近交繁殖SD大鼠;取30只4周龄近交繁殖的SD大鼠随机分为3组,将UMR-106细胞分别以1×10~6、5×10~6、1×10~7个数量注射入大鼠右股外侧皮下。接种后,每周观察肿瘤生长情况,测量肿瘤直径,绘制肿瘤生长曲线,连续6周。至第6周末,取肿瘤及双侧肺,病理取材,HE染色,光镜下观察。
结果4周龄封闭群饲养的SD大鼠,以1×10~7个数量接种肿瘤细胞后,肿瘤发生率仅为30%,筛选易感SD大鼠并近交繁殖4代后,肿瘤发生率达到100%,肺转移率达70%;而1×10~6及5×10~6组肿瘤生长情况不佳,均未出现肺转移。
结论封闭群饲养的SD大鼠具有一定的遗传差异,经反复筛选并近交繁殖后,遗传性状恢复稳定,成瘤能力得以保持。大鼠骨肉瘤细胞系UMR-106具有较强的致瘤性及较高的肺转移率,应用该细胞系建立的动物模型为骨肉瘤综合治疗奠定了动物实验基础。
实验二大鼠骨髓单核细胞为来源的树突状细胞的体外定向诱导、培养及表型鉴定
目的以大鼠骨髓单核细胞(Bone Marrow Mononuclear Cell,BMMC)为来源,建立体外诱导、培养DC的方法,并检测该培养过程中,树突状细胞的表型变化情况。
方法从大鼠骨髓细胞中分离出单核细胞,经淋巴细胞分离液梯度离心、聚苯乙烯细胞培养板贴壁纯化后,加入含rGM-CSF和rIL-4的RPMI-1640培养液联合诱导DC分化,诱导d6加入nrhTNF-α促进DC成熟。对经标抗大鼠OX62单抗免疫磁珠分离纯化后的DC,分别以倒置显微镜、电子显微镜观察其形态变化,采用流式细胞仪每隔3日检测DC的表型变化情况。
结果大鼠骨髓单核细胞经诱导培养9d后,在倒置显微镜和电镜下显示出典型的树突状细胞形态和超微结构;流式细胞术分析显示在DC诱导培养过程中,细胞表面的DC功能相关抗原均呈递增性表达,第0天为OX62 1.78%,MHCⅡ13.68%,CD80 4.77%,CD86 5.77%;第3天为OX62 32.31%,MHCⅡ32.14%,CD80 23.68%,CD86 15.44%;第6天为OX62 63.13%,MHCⅡ68.36%,CD80 43.06%,CD86 54.78%;第9天为OX62 72.41%,MHCⅡ84.71%,CD80 79.06%,CD86 74.80%;而大鼠骨肉瘤相关性抗原CD44在DC始终呈阴性表达。
结论以大鼠骨髓单核细胞为来源,联合应用rGM-CSF、rIL-4及nrhTNF-α,在体外可成功诱导培养出具有典型形态、及表型特征的DC,DC的表面分子随着诱导培养时间的推进,呈递增性改变,该研究为进一步研究DC在肿瘤免疫治疗中的应用打下了基础。
实验三异体树突状细胞与骨肉瘤细胞融合瘤苗的制备及表型分析目的以Wistar大鼠树突状细胞和SD大鼠骨肉瘤细胞UMR-106为来源,应用细胞融合仪建立体外制备异体融合瘤苗方法,并检测融合细胞的表型变化情况。
方法将Wistar大鼠骨髓来源的树突状细胞和SD大鼠骨肉瘤细胞UMR-106以5:1比率混合并悬浮于含10%胎牛血清的完全培养基中,离心后,将细胞悬浮于无血清的培养基中。调整细胞浓度后,将0.5ml含5×10~6个混合细胞的悬液置于ECM 2001型细胞融合仪的特制电击槽内。融合过程施加两步骤电脉冲,第一步是施加50V交流脉冲,将细胞彼此紧密接触;既而施加250V直流脉冲,使相邻细胞的细胞膜穿孔,细胞质彼此交通,来实现细胞之间的融合。整个施加电脉冲的过程重复一遍,以确保电融合效率。静止5分钟后,将融合细胞移入完全培养基,37℃条件下过夜培养。再收集贴壁细胞,经标抗大鼠OX62单抗免疫磁珠分离纯化后,分别以倒置显微镜、电子显微镜观察其形态变化,采用流式细胞仪检测融合瘤苗的表型变化情况。
结果异体树突状细胞和骨肉瘤细胞在电脉冲作用下,实现了细胞间的融合,在扫描电镜和透射电镜下,融合细胞表现出兼具两类细胞外形特点的形态学特征和典型的多细胞核超微结构;流式细胞术分析显示,经过免疫磁珠分离纯化的融合细胞表型分子较两类成分细胞均发生了变化,其同时高表达骨肉瘤细胞表型分子CD44(50.02%)和树突状细胞表型分子OX62(83.55%),双标阳性细胞比率高达49.43%。
结论应用细胞融合仪可以将同种异体来源的树突状细胞和骨肉瘤细胞在电脉冲作用下制备融合瘤苗,融合细胞兼具有树突状细胞与骨肉瘤细胞的形态学及表型特征,异体融合瘤苗有望在针对骨肉瘤的免疫治疗中发挥作用。
实验四异体融合瘤苗体外诱导的抗骨肉瘤免疫效应实验
目的本研究旨在检验异体融合瘤苗体外诱导T淋巴细胞增殖能力,以及骨肉瘤细胞特异性的细胞毒T淋巴细胞(CTL)的杀瘤效果。
方法经标抗大鼠OX62单抗免疫磁珠分离纯化的异体融合瘤苗,接受30Gy的60Co照射后,与SD大鼠骨髓来源的T淋巴细胞作共同培养,旨在刺激T细胞的增殖,并采用MTT法测定细胞毒T淋巴细胞的杀瘤活性。将10裸鼠右后肢皮下接种UMR-106细胞1×10~6/只,3日后随机分组。治疗组以1×10~7/只皮内注射活化的T淋巴细胞,连续3次,4周后观察成瘤情况。
结果T淋巴细胞和异体融合瘤苗(DOF)经过共同培养,T细胞得以显著增殖。与生理盐水和树突状细胞对照组相比,异体融合瘤苗诱导的UMR-106特异性CTL杀瘤效果显著。裸鼠接种UMR-106细胞28天后,治疗组、对照组肿瘤体积分别为(945±125)mm3和(4867±375)mm3,与对照组相比,治疗组移植瘤的生长受到明显抑制(p<0.05)。
结论该研究为异体树突状细胞与骨肉瘤细胞融合瘤苗可以在体外有效的刺激T淋巴细胞增殖,并成功地诱导杀瘤效果显著的UMR-106特异性CTL提供了证据。从而表明异体融合瘤苗为以树突状细胞为基础的免疫治疗领域提供了新的策略,针对临床骨肉瘤患者具有广阔的应用前景。
实验五异体融合瘤苗体内诱导的抗骨肉瘤主动免疫效应实验
目的本研究旨在探讨以Wistar大鼠树突状细胞和SD大鼠骨肉瘤细胞UMR-106为来源制备的融合瘤苗诱导的特异性抗骨肉瘤免疫学效应及其应用潜能。
方法预防接种实验以10只SD大鼠作为1组,收集筛选过的异体融合细胞,经30Gy的60Co照射后作为肿瘤疫苗,洗1遍,调整细胞浓度,于第0天和第14天在实验组每只大鼠腹股沟皮内注射1×10~6个融合细胞,同时设立4个对照组,分别以生理盐水、经过照射的自身融合的肿瘤细胞、自身融合的DC以及后二者的混合物对大鼠进行皮内接种,1周之后(即第21天)采用盲法在实验组和对照组大鼠右后肢外侧皮下均注射致死量(1×10~7个)的骨肉瘤细胞,10周之后观察动物生存状况。再次以致死剂量的骨肉瘤细胞(1×10~7个)对存活下来的大鼠进行皮下注射,7周之后观察动物生存状况以检验该预防保护作用是否具有长效性。主动免疫治疗实验于第0天在每只SD大鼠右后肢外侧皮下接种1×10~7个UMR-106细胞建立大鼠的骨肉瘤动物模型,分别于第3、7、14天在大鼠腹股沟皮内注射瘤苗进行免疫治疗,绘制动物生存曲线。
结果10周之后,在预防接种了异体融合瘤苗的实验组,70%的大鼠抵抗了致死剂量骨肉瘤细胞的攻击,而得以存活。经过预防接种异体融合瘤苗而得以存活的大鼠,全部抵抗了第2次致死剂量的骨肉瘤细胞的免疫攻击,7周之后继续得以存活。异体融合瘤苗主动免疫治疗的效果根据注射融合细胞的数量的不同而出现差异,1×10~6组疗效较差,仅有10%的个体得以存活,而该剂量在预防接种实验中已经能够取得良好的疗效;2×10~6组疗效显著,60%的荷瘤大鼠瘤体萎缩、消失而得以长期存活。
结论预防接种异体融合瘤苗的实验组产生了强大的抵抗骨肉瘤细胞攻击的预防保护作用,该作用在存活下来的动物机体产生了长效的免疫记忆应答。异体融合瘤苗还可以发挥良好的主动免疫治疗作用,但是治疗剂量与预防接种实验相比需要有所提高。实验结果表明大鼠骨肉瘤细胞系UMR-106与同种异基因树突状细胞的融合瘤苗可诱发有效的特异性抗骨肉瘤免疫学效应,该方法有望在针对抗原表型尚未明确的其它肿瘤的免疫治疗中获得推广应用。
Osteosarcoma is one of the most common primary malignant bone tumors mostly in youth and adolescents. The main treatment for osteosarcoma was surgical excision including amputation and joint amputation before the1970’s. The average time is 8 months from surgical treatment to lung metastasis. Less than 25% of patients had a 5-year survival rate. With the development of surgery and screenage technology and achievement of alliance of neoadjuvant chemotherapy, radiotherapy and the immunotherapy, the osteosarcoma therapy has achieved great achievement, with 5-year disease-free survival exceeds 80%. However, the metastasis, recurrent and chemotherapy-resistant cases has not been solved. Therefore, novel strategies to prevent proliferation of malignant cells are urgently needed.
A promising approach may be the prophylactic vaccination directed against a tumor-associated antigens(TAA)or tumor-special antigens(TSA)from the whole tumor cells. The major objective of active specific immunotherapy is to generate tumor- specific cytotoxic T lymphocyte (CTL) responds, but to induced CTL responds depend on the use of professional antigen presenting cells loaded with tumor antigen. In the antigen-presenting cells, dendritic cell (DC)has been considered to be the most potent professional antigen-presenting cell (APC)that can initiates primary immune responses. Its ability to stimulate and regulate T- and B-cell responses makes DC ideally suited to serve as an adjuvant for the purpose of cancer immunotherapy. DC also can be involved in the pathogenesis of graft versus host disease or host versus graft disease after transplantation as well as immunization against viral infections and immunosuppression in autoimmune diseases.
Considering the expected emergence of antigen-loss variants and the lack of known tumor-specific antigens in most cancers, in this study, the DC-based vaccine is generated using whole osteosarcoma cell body through electrofusion process. One important advantage of immunization with the whole tumor cell is the potential to induce an immune response against all possible tumor antigens, known or unknown. Electrical cell fusion is an essential step in some of the most innovative methods in modern biology, such as the production of monoclonal antibodies, the cloning of mammals, and vaccination against cancer. Compared with the chemically induced cell fusion via polyethylene glycol (PEG), electrical cell fusion is a method with higher efficiency. More importantly, this technique makes it practical to combine two allogeneic APC or tumor cell together.
The allogeneic fusion hybrids are particularly attractive because they would enable the use of allogeneic DC or tumor cells, both providing the costimulatory fusion or MHC molecules required to induce CTL in naive T-cell precursors. Both of the options seem to project a practical advantage. For in a clinical setting, it is not easy to cultivate original tumor cell from osteosarcoma patients, who have suffered from the complications of expectant treatment, thermotherapy by microwave or radio-frequency, et al. Allogeneic tumor cells can save the necessary process of original tumor cell collection and make cell cultivation easier. While allogeneic DC can be generated conveniently from stored peripheral mononuclear cells from normal healthy volunteers from the general population, which avoids the strike to draw additional peripheral blood from cancer patients, who have been very weak.
Part I Bolting of UMR-106 Rat Osteosarcoma Cell Line Susceptible Population and Establishment of Animal Model of Rat Osteosarcoma and Its Lung Metastasis
Objective To introduce a method of bolting of UMR-106 rat osteosarcoma cell line susceptible population and breeding its inbred strain, and establish animal model of rat osteosarcoma and its lung metastasis.
Methods 10 couples 4-week-old SD rats from closed population were prepared and the UMR-106 osteosarcoma cell line was injected subcutaneously into the SD rat thigh lateral at the amount of 1×10~7. The survival couples who were susceptible to the osteosarcoma cell line were selected, and mated six weeks later. After pregnancy and delivery, the offspring emerged, who would also be challenged with UMR-106 cell line 4 weeks later……The whole process were be repeated for 4 times and as a matter of course, the the 4th generation rats from inbred strain was obtained, who were more susceptible to the osteosarcoma cell line. 30 SD rats from inbred strain of 4 weeks old were divided into 3 groups. The UMR-106 cell line was injected subcutaneously into the SD rat thigh lateral at the amount of 1×10~6、5×10~6 and 1×10~7. Neoplastic diameters were measured every week after inoculation to build the tumor growth curve. 6 weeks later, the rat’double lungs and tumors were prepared. Pathological study was made under microscope by HE stains.
Results The 4-week-old rats from closed population seemed unsusceptible to the UMR-106 cell line and only 30% of them were infected. After bolting and propagation, the 4th generation rats from inbred strain became so susceptible to the osteosarcoma cell line that in the1×10~7 group all tumors developed and 70% of them metastasized to lungs, while the tumors of 1×10~6 and 5×10~6 groups didn’t develop well,nor lung metastasis were observed.
Conclusions The SD rats from closed population showed up certain genetic diversity, while their offspring from inbred strain became identical and demonstrated no diversity in oncogenicity. The UMR-106 cell line has strong carcinogenic capability and high lung metastasis frequency. The animal models depending on it offer a method for the animal experiments of comprehensive therapy to osteosarcoma.
PartⅡInduction, Proliferation and Identification of Dendritic Cells from Rat Bone Marrow Mononuclear Cells in vitro
Objective To establish a method to induce dendritic cells from the bone marrow mononuclear cell of rats in vitro,and to identify the process of phenotypes’variation on DC.
Methods BMMCs were isolated by Ficoll-Hypaque density gradient separation and then cultured in RPMI1640-10% FCS medium in polystyrene flask under the condition of rGM-CSF and rIL-4 at 37℃and 5% CO2. On d6 of culture, immature DC were washed and suspended in medium containing nrhTNF-αto generate mature DC. On d9 after purified by monoclonal antibody OX62 and magnetic beads, DC was identified by morphological features under both inversion microscope and electron microscope. While DC’surface antigens expression was analyzed by FACS every three days.
Results After culture and induction,DC displayed typical morphology with elongated dendritic processes viewed by inversion microscope as well as electron microscope. DC expressed increasing level function associated surface antigens,including OX62 1.78%,MHCⅡ13.68%,CD80 4.77%,CD86 5.77% on d0; OX62 32.31%,MHCⅡ32.14%,CD80 23.68%,CD86 15.44% on d3; OX62 63.13%,MHCⅡ68.36%,CD80 43.06%,CD86 54.78%; and OX62 72.41%,MHCⅡ84.71%,CD80 79.06%,CD86 74.80%on d9. While the rat osteosarcoma associated surface antigen CD44 was always negatively expressed on DC.
Conclusions Mature DC with typical morphology and surface antigens expression could be generated from rat bone marrow mononuclear cells under the condition of rGM-CSF, rIL-4 and nrhTNF-α,and DC’s surface antigens expression was increased progressively with culture days added, which presents the feasibility for further clinical application of DC in the immunotherapy of cancer.
PartIII Preparation and Identification of Allogeneic DC-osteosarcoma Electrofusion Vaccine
Objective To establish a method to prepare allogeneic DC-osteosarcoma electrofusion vaccine with the Wistar rats’bone marrow derived dendritic cells and SD rats derived osteosarcoma cell line UMR-106,and to identify the phenotypes’variation on the fusion hybrids.
Methods Allogeneic DC and tumor cells were mixed at a 5:1 ratio and suspended in 0.3 M glucose solution containing 0.1mM Ca (CH3COO)2, 0.5mM Mg (CH3COO)2, and 10% bovine serum albumin. After centrifugation, the cells were resuspended in the same fusion medium without bovine serum albumin. Routinely, 0.5ml of cell suspension containing 6×10~6 cells were processed using a specially designed electroporation cuvette. For electrofusion, a pulse generator (model ECM 2001) was used. Electrofusion involves two independent but consecutive steps. The first reaction is to bring cells in close contact by dielectrophoresis, which can be accomplished by exposing cells to an alternating (ac) electric field of relatively low strength. Cell fusion can then be triggered by applying a single square wave pulse of 250V to induce reversible cell membrane breakdown in the zone of membrane contact. The entire process was repeated a second time to maximize fusion efficiency. The fusion mixture was allowed to stand for 5 min before suspending in complete medium and then incubated at 37℃overnight. The electrofusion products were purified by monoclonal antibody OX62 (a DC marker not expressed on tumor cells) sticking to the magnetic beads (Miltenyi Biotec) and then was identified by morphological features under both inversion microscope and electron microscope. The allogeneic DC- osteosarcoma products’surface antigens expression was analyzed by FACS and co-staining against both kinds of markers allows for the detection of double-positive hybrids.
Results The allogeneic DC-osteosarcoma vaccine were achieved through electrofusion pulse, and the electrofusion products displayed typical morphology of both component cells viewed by scan electron microscope as well as multi-nucleus ultrastructure viewed by transmission electron microscope. FACS analysis of a purified fusion products by monoclonal antibody OX62 and magnetic beads showed both tumor cell marker and DC marker were highly expressed, for CD44 in the case of osteosarcoma was 50.02%, OX62 in the case of DC was 83.55%, while the percentage of double-positive cell was 49.43%.
Conclusions This study demonstrated it was feasible to generate a large number of allogeneic DC-osteosarcoma hybrid cells by the electrofusion technique. The electrofusion products displayed typical morphology and phaenotype of both component cells. The allogeneic tumor vaccine affords a promising new approach for the immunotherapy of osteosarcoma.
Part IV In vitro Induction of Anti-tumor CTL Effect against Osteosarcoma Cell Line by Allogeneic DC-Osteosarcoma Fusion Vaccine
Objective This study was designed to investigate the potentiality to induce T cell proliferation and osteosarcoma-specific cytotoxic T lymphocytes of tumor vaccine produced by electrofusion between rat osteosarcoma cells and allogeneic DC.
Methods The allogeneic DC-osteosarcoma fusion cells were purified by monoclonal antibody OX62 and magnetic beads and then irradiated with 30 Gy with radioactive ray of 60Co to ensure inactivation of the tumor cells and DC. Coculture of SD bone marrow derived T lymphocytes with the tumor vaccine was to induce T cell proliferation. Then cytotoxic T lymphocytes assay was assessed according to results of MTT assay. 10 athymic mice were challenged subcutaneously with 1×10~6 UMR-106 cells and then divided into two groups randomly 3 days later. The tumor-bearing athymic mice were immunized with 1×107 activated T lymphocytes for 3 times. The condition of tumor development was observed for 4 weeks since tumor challenge.
Results After T cells were cultured with allogeneic DC-osteosarcoma fusion cells, effective activation of T cells was observed. The immunization using allogeneic DC-osteosarcoma vaccine induced UMR106-spcific CTL responses which were statistically significant (P<0.05) compared with corresponding control groups using saline or DC. 28 days later, the experiment of athymic mice showed that the induced CTL inhibited the growth of implanted tumor in athymic mice, and the volum of athymic mice’s implanted tumor was (945±125)mm3 in treatment group, and (4867±375)mm3 in control group respectively(p<0.05).
Conclusions The present study provided valid evidence of the potentiality of allogeneic DC-osteosarcoma fusion cells to induce effective T cell proliferation and osteosarcoma-specific cytotoxic T lymphocytes. The fusion cells may thus represent a promising strategy for DC-based immunotherapy of osteosarcoma and play certain role in the clinical treatment and prevention.
Part V Specific Active Antitumor Effects of Tumor Vaccine Produced by Electrofusion between Osteosarcoma Cell Line and Allogeneic Dendritic Cell in Rats in vivo.
Objective This study was designed to investigate the potency and antitumor effects of allogeneic tumor vaccine produced by electrofusion between SD rats derived osteosarcoma cell line UMR-106 and Wistar rats’bone marrow derived DC.
Methods In this study of immunization against tumor challenge, groups of 10 rats were immunized intradermally with 1×10~6 electrofused cells after irradiated with 30Gy 60Co-ray on days 0 and 14. One week after the second immunization (day 21), the rats were challenged double-blind with a lethal dose of tumor cells and were then monitored for tumor growth and survival over time. For the tumor challenge, vaccinated SD rats were prepared and the UMR-106 osteosarcoma cell line was injected subcutaneously into the SD rat thigh lateral at the amount of 1×10~7. In contrast to the results obtained with the fusion products, immunization with physiological saline, tumor cells that underwent the electrofusion process alone, DC that underwent electrofusion alone, or a mixture of these two populations was also made to induce antitumor protection. To determine whether the immunization could result in a long-term immunological memory, rats injected with UMR106-allogeneic DC fusion products that survived an UMR-106 tumor challenge were rechallenged with a second lethal dose of tumor cells 10 weeks later and then monitored for tumor growth and survival again for 7 weeks. In the active immunization therapeutic model, tumor cells were injected on day 0 and vaccination with electrofusion products was done on days 3, 7, and 14. Then the animals’survival curve was carried out.
Results In the foregoing study of immunization against tumor challenge, 70% of the rats immunized with 1×10~6 electrofused cells were typically able to reject tumor challenge and remained tumor-free. And then all of the survival rats were able to reject this secondary tumor challenge and remain to live on 7 weeks later. In the active treatment study, little or no antitumor protection was observed in the rats treated with1×10~6 electrofused cells, a dose sufficient to achieve substantial tumor protection in pretreatment studies. However, substantial antitumor protection (60% long-term survivors) was obtained with a higher dose of the vaccine (2×10~6 electrofused cells).
Conclusions Preimmunization with irradiated electrofusion products were found to provide partial to complete protection from tumor challenge in the UMR-106 tumor model. Vaccinated survivors developed long immunological memory. The therapeutic potential of this type of approach was suggested by the ability of UMR106-DC electrofusion products to induce tumor rejection in a substantial percentage of hosts bearing pre-established tumor cells. These results tended to indicate that treatment with electrofused tumor cells and allogeneic DC might be capable of inducing a potent antitumor response and could conceivably be applied to a wide range of cancer indications for which tumor-associated antigens have not been identified.
制备针对肿瘤特异性抗原(tumor-specific antigens, TSA)或肿瘤相关抗原(tumor-associated antigen, TAA)的治疗性瘤苗是目前肿瘤免疫治疗中的研究热点。其抗肿瘤免疫的重要目的就是诱导针对肿瘤抗原的特异性细胞毒性T淋巴细胞(cytocoxic T lymphocyte,CTL)反应,而CTL的激活依赖于抗原提呈细胞(antigen-presenting cell,APC)提供的抗原多肽和共刺激信号。在抗原提呈细胞中,尤以树突状细胞(dendritic cell,DC)的抗原提呈功能最强,可启动并调节免疫应答,有效刺激T、B淋巴细胞活化,在抗肿瘤、抗感染、移植排斥以及自身免疫性疾病发病过程中起重要作用。
本研究以骨肉瘤全细胞提供肿瘤抗原,采用细胞电融合技术将肿瘤抗原通过DC呈递表达于T淋巴细胞表面。全细胞瘤苗的最大的优点就在于融合细胞能够呈递肿瘤细胞所有的抗原,包括已知的和未知的抗原,鉴于目前大多数人类和动物的肿瘤细胞(包括骨肉瘤细胞)抗原类型仍未得到明确的鉴定,用全肿瘤细胞作为瘤苗提供抗原的有效组分仍不失为一类简单、有效的方法。电融合技术是当前生物学界重要的开创性研究手段之一,在制备单克隆抗体、哺乳动物克隆实验研究及肿瘤的免疫治疗方面均获得了广泛的应用,和传统的化学融合方法(如聚二乙醇PEG法)相比,电融合方法是一种更为高效的手段,融合效率通常可以提高1～2个数量级,这在很大程度上弥补了DC数量较少的缺陷,该技术也使两个来源于异体的细胞相互融合成为可能,目前已被广泛应用于瘤苗制备领域。
研究工作者之所以如此积极地进行异体融合瘤苗的尝试,是因为在临床应用中自体瘤苗的成功制备通常会受到疾病本身的困绕。首先,对患者自体的肿瘤细胞进行原代培养具有某种不确定性,特别是在保守治疗和以微波、射频等热疗手段处理后的肿瘤组织,失去进一步体外培养的可能;另外,目前肿瘤的治疗方案往往是手术、化疗及放疗等相结合的综合治疗,肿瘤患者的机体经过一系列治疗副效应的累加,患者的血象及血细胞质量通常难以满足进一步的免疫治疗需要,其营养状况也基本处于过消耗状态。而异体的树突状细胞可以从储备的健康志愿者外周血单核细胞获得,又可避免使所有肿瘤患者在接受免疫治疗前都必须提供相当量的自身外周血的缺陷,避免了“雪上加霜”。
实验一UMR-106细胞系易感动物的筛选和大鼠骨肉瘤及其肺转移模型的建立
目的探讨从普通的封闭群动物中筛选出对UMR-106大鼠骨肉瘤细胞系易感的SD大鼠,并建立其近交系的方法,以及大鼠骨肉瘤及其肺转移模型的建立方法。
方法取10对4周龄封闭群饲养的SD大鼠,分别在右股外侧皮下接种1×10~7个大鼠骨肉瘤细胞UMR-106,观察成瘤情况,选取存活的成瘤的雌、雄大鼠,6周后同笼交配,约4周妊娠期后,产下幼鼠,4周后重复肿瘤细胞皮下接种过程……依照此方法重复共4次,获得第4代UMR-106细胞系易感的近交繁殖SD大鼠;取30只4周龄近交繁殖的SD大鼠随机分为3组,将UMR-106细胞分别以1×10~6、5×10~6、1×10~7个数量注射入大鼠右股外侧皮下。接种后,每周观察肿瘤生长情况,测量肿瘤直径,绘制肿瘤生长曲线,连续6周。至第6周末,取肿瘤及双侧肺,病理取材,HE染色,光镜下观察。
结果4周龄封闭群饲养的SD大鼠,以1×10~7个数量接种肿瘤细胞后,肿瘤发生率仅为30%,筛选易感SD大鼠并近交繁殖4代后,肿瘤发生率达到100%,肺转移率达70%;而1×10~6及5×10~6组肿瘤生长情况不佳,均未出现肺转移。
结论封闭群饲养的SD大鼠具有一定的遗传差异,经反复筛选并近交繁殖后,遗传性状恢复稳定,成瘤能力得以保持。大鼠骨肉瘤细胞系UMR-106具有较强的致瘤性及较高的肺转移率,应用该细胞系建立的动物模型为骨肉瘤综合治疗奠定了动物实验基础。
实验二大鼠骨髓单核细胞为来源的树突状细胞的体外定向诱导、培养及表型鉴定
目的以大鼠骨髓单核细胞(Bone Marrow Mononuclear Cell,BMMC)为来源,建立体外诱导、培养DC的方法,并检测该培养过程中,树突状细胞的表型变化情况。
方法从大鼠骨髓细胞中分离出单核细胞,经淋巴细胞分离液梯度离心、聚苯乙烯细胞培养板贴壁纯化后,加入含rGM-CSF和rIL-4的RPMI-1640培养液联合诱导DC分化,诱导d6加入nrhTNF-α促进DC成熟。对经标抗大鼠OX62单抗免疫磁珠分离纯化后的DC,分别以倒置显微镜、电子显微镜观察其形态变化,采用流式细胞仪每隔3日检测DC的表型变化情况。
结果大鼠骨髓单核细胞经诱导培养9d后,在倒置显微镜和电镜下显示出典型的树突状细胞形态和超微结构;流式细胞术分析显示在DC诱导培养过程中,细胞表面的DC功能相关抗原均呈递增性表达,第0天为OX62 1.78%,MHCⅡ13.68%,CD80 4.77%,CD86 5.77%;第3天为OX62 32.31%,MHCⅡ32.14%,CD80 23.68%,CD86 15.44%;第6天为OX62 63.13%,MHCⅡ68.36%,CD80 43.06%,CD86 54.78%;第9天为OX62 72.41%,MHCⅡ84.71%,CD80 79.06%,CD86 74.80%;而大鼠骨肉瘤相关性抗原CD44在DC始终呈阴性表达。
结论以大鼠骨髓单核细胞为来源,联合应用rGM-CSF、rIL-4及nrhTNF-α,在体外可成功诱导培养出具有典型形态、及表型特征的DC,DC的表面分子随着诱导培养时间的推进,呈递增性改变,该研究为进一步研究DC在肿瘤免疫治疗中的应用打下了基础。
实验三异体树突状细胞与骨肉瘤细胞融合瘤苗的制备及表型分析目的以Wistar大鼠树突状细胞和SD大鼠骨肉瘤细胞UMR-106为来源,应用细胞融合仪建立体外制备异体融合瘤苗方法,并检测融合细胞的表型变化情况。
方法将Wistar大鼠骨髓来源的树突状细胞和SD大鼠骨肉瘤细胞UMR-106以5:1比率混合并悬浮于含10%胎牛血清的完全培养基中,离心后,将细胞悬浮于无血清的培养基中。调整细胞浓度后,将0.5ml含5×10~6个混合细胞的悬液置于ECM 2001型细胞融合仪的特制电击槽内。融合过程施加两步骤电脉冲,第一步是施加50V交流脉冲,将细胞彼此紧密接触;既而施加250V直流脉冲,使相邻细胞的细胞膜穿孔,细胞质彼此交通,来实现细胞之间的融合。整个施加电脉冲的过程重复一遍,以确保电融合效率。静止5分钟后,将融合细胞移入完全培养基,37℃条件下过夜培养。再收集贴壁细胞,经标抗大鼠OX62单抗免疫磁珠分离纯化后,分别以倒置显微镜、电子显微镜观察其形态变化,采用流式细胞仪检测融合瘤苗的表型变化情况。
结果异体树突状细胞和骨肉瘤细胞在电脉冲作用下,实现了细胞间的融合,在扫描电镜和透射电镜下,融合细胞表现出兼具两类细胞外形特点的形态学特征和典型的多细胞核超微结构;流式细胞术分析显示,经过免疫磁珠分离纯化的融合细胞表型分子较两类成分细胞均发生了变化,其同时高表达骨肉瘤细胞表型分子CD44(50.02%)和树突状细胞表型分子OX62(83.55%),双标阳性细胞比率高达49.43%。
结论应用细胞融合仪可以将同种异体来源的树突状细胞和骨肉瘤细胞在电脉冲作用下制备融合瘤苗,融合细胞兼具有树突状细胞与骨肉瘤细胞的形态学及表型特征,异体融合瘤苗有望在针对骨肉瘤的免疫治疗中发挥作用。
实验四异体融合瘤苗体外诱导的抗骨肉瘤免疫效应实验
目的本研究旨在检验异体融合瘤苗体外诱导T淋巴细胞增殖能力,以及骨肉瘤细胞特异性的细胞毒T淋巴细胞(CTL)的杀瘤效果。
方法经标抗大鼠OX62单抗免疫磁珠分离纯化的异体融合瘤苗,接受30Gy的60Co照射后,与SD大鼠骨髓来源的T淋巴细胞作共同培养,旨在刺激T细胞的增殖,并采用MTT法测定细胞毒T淋巴细胞的杀瘤活性。将10裸鼠右后肢皮下接种UMR-106细胞1×10~6/只,3日后随机分组。治疗组以1×10~7/只皮内注射活化的T淋巴细胞,连续3次,4周后观察成瘤情况。
结果T淋巴细胞和异体融合瘤苗(DOF)经过共同培养,T细胞得以显著增殖。与生理盐水和树突状细胞对照组相比,异体融合瘤苗诱导的UMR-106特异性CTL杀瘤效果显著。裸鼠接种UMR-106细胞28天后,治疗组、对照组肿瘤体积分别为(945±125)mm3和(4867±375)mm3,与对照组相比,治疗组移植瘤的生长受到明显抑制(p<0.05)。
结论该研究为异体树突状细胞与骨肉瘤细胞融合瘤苗可以在体外有效的刺激T淋巴细胞增殖,并成功地诱导杀瘤效果显著的UMR-106特异性CTL提供了证据。从而表明异体融合瘤苗为以树突状细胞为基础的免疫治疗领域提供了新的策略,针对临床骨肉瘤患者具有广阔的应用前景。
实验五异体融合瘤苗体内诱导的抗骨肉瘤主动免疫效应实验
目的本研究旨在探讨以Wistar大鼠树突状细胞和SD大鼠骨肉瘤细胞UMR-106为来源制备的融合瘤苗诱导的特异性抗骨肉瘤免疫学效应及其应用潜能。
方法预防接种实验以10只SD大鼠作为1组,收集筛选过的异体融合细胞,经30Gy的60Co照射后作为肿瘤疫苗,洗1遍,调整细胞浓度,于第0天和第14天在实验组每只大鼠腹股沟皮内注射1×10~6个融合细胞,同时设立4个对照组,分别以生理盐水、经过照射的自身融合的肿瘤细胞、自身融合的DC以及后二者的混合物对大鼠进行皮内接种,1周之后(即第21天)采用盲法在实验组和对照组大鼠右后肢外侧皮下均注射致死量(1×10~7个)的骨肉瘤细胞,10周之后观察动物生存状况。再次以致死剂量的骨肉瘤细胞(1×10~7个)对存活下来的大鼠进行皮下注射,7周之后观察动物生存状况以检验该预防保护作用是否具有长效性。主动免疫治疗实验于第0天在每只SD大鼠右后肢外侧皮下接种1×10~7个UMR-106细胞建立大鼠的骨肉瘤动物模型,分别于第3、7、14天在大鼠腹股沟皮内注射瘤苗进行免疫治疗,绘制动物生存曲线。
结果10周之后,在预防接种了异体融合瘤苗的实验组,70%的大鼠抵抗了致死剂量骨肉瘤细胞的攻击,而得以存活。经过预防接种异体融合瘤苗而得以存活的大鼠,全部抵抗了第2次致死剂量的骨肉瘤细胞的免疫攻击,7周之后继续得以存活。异体融合瘤苗主动免疫治疗的效果根据注射融合细胞的数量的不同而出现差异,1×10~6组疗效较差,仅有10%的个体得以存活,而该剂量在预防接种实验中已经能够取得良好的疗效;2×10~6组疗效显著,60%的荷瘤大鼠瘤体萎缩、消失而得以长期存活。
结论预防接种异体融合瘤苗的实验组产生了强大的抵抗骨肉瘤细胞攻击的预防保护作用,该作用在存活下来的动物机体产生了长效的免疫记忆应答。异体融合瘤苗还可以发挥良好的主动免疫治疗作用,但是治疗剂量与预防接种实验相比需要有所提高。实验结果表明大鼠骨肉瘤细胞系UMR-106与同种异基因树突状细胞的融合瘤苗可诱发有效的特异性抗骨肉瘤免疫学效应,该方法有望在针对抗原表型尚未明确的其它肿瘤的免疫治疗中获得推广应用。
Osteosarcoma is one of the most common primary malignant bone tumors mostly in youth and adolescents. The main treatment for osteosarcoma was surgical excision including amputation and joint amputation before the1970’s. The average time is 8 months from surgical treatment to lung metastasis. Less than 25% of patients had a 5-year survival rate. With the development of surgery and screenage technology and achievement of alliance of neoadjuvant chemotherapy, radiotherapy and the immunotherapy, the osteosarcoma therapy has achieved great achievement, with 5-year disease-free survival exceeds 80%. However, the metastasis, recurrent and chemotherapy-resistant cases has not been solved. Therefore, novel strategies to prevent proliferation of malignant cells are urgently needed.
A promising approach may be the prophylactic vaccination directed against a tumor-associated antigens(TAA)or tumor-special antigens(TSA)from the whole tumor cells. The major objective of active specific immunotherapy is to generate tumor- specific cytotoxic T lymphocyte (CTL) responds, but to induced CTL responds depend on the use of professional antigen presenting cells loaded with tumor antigen. In the antigen-presenting cells, dendritic cell (DC)has been considered to be the most potent professional antigen-presenting cell (APC)that can initiates primary immune responses. Its ability to stimulate and regulate T- and B-cell responses makes DC ideally suited to serve as an adjuvant for the purpose of cancer immunotherapy. DC also can be involved in the pathogenesis of graft versus host disease or host versus graft disease after transplantation as well as immunization against viral infections and immunosuppression in autoimmune diseases.
Considering the expected emergence of antigen-loss variants and the lack of known tumor-specific antigens in most cancers, in this study, the DC-based vaccine is generated using whole osteosarcoma cell body through electrofusion process. One important advantage of immunization with the whole tumor cell is the potential to induce an immune response against all possible tumor antigens, known or unknown. Electrical cell fusion is an essential step in some of the most innovative methods in modern biology, such as the production of monoclonal antibodies, the cloning of mammals, and vaccination against cancer. Compared with the chemically induced cell fusion via polyethylene glycol (PEG), electrical cell fusion is a method with higher efficiency. More importantly, this technique makes it practical to combine two allogeneic APC or tumor cell together.
The allogeneic fusion hybrids are particularly attractive because they would enable the use of allogeneic DC or tumor cells, both providing the costimulatory fusion or MHC molecules required to induce CTL in naive T-cell precursors. Both of the options seem to project a practical advantage. For in a clinical setting, it is not easy to cultivate original tumor cell from osteosarcoma patients, who have suffered from the complications of expectant treatment, thermotherapy by microwave or radio-frequency, et al. Allogeneic tumor cells can save the necessary process of original tumor cell collection and make cell cultivation easier. While allogeneic DC can be generated conveniently from stored peripheral mononuclear cells from normal healthy volunteers from the general population, which avoids the strike to draw additional peripheral blood from cancer patients, who have been very weak.
Part I Bolting of UMR-106 Rat Osteosarcoma Cell Line Susceptible Population and Establishment of Animal Model of Rat Osteosarcoma and Its Lung Metastasis
Objective To introduce a method of bolting of UMR-106 rat osteosarcoma cell line susceptible population and breeding its inbred strain, and establish animal model of rat osteosarcoma and its lung metastasis.
Methods 10 couples 4-week-old SD rats from closed population were prepared and the UMR-106 osteosarcoma cell line was injected subcutaneously into the SD rat thigh lateral at the amount of 1×10~7. The survival couples who were susceptible to the osteosarcoma cell line were selected, and mated six weeks later. After pregnancy and delivery, the offspring emerged, who would also be challenged with UMR-106 cell line 4 weeks later……The whole process were be repeated for 4 times and as a matter of course, the the 4th generation rats from inbred strain was obtained, who were more susceptible to the osteosarcoma cell line. 30 SD rats from inbred strain of 4 weeks old were divided into 3 groups. The UMR-106 cell line was injected subcutaneously into the SD rat thigh lateral at the amount of 1×10~6、5×10~6 and 1×10~7. Neoplastic diameters were measured every week after inoculation to build the tumor growth curve. 6 weeks later, the rat’double lungs and tumors were prepared. Pathological study was made under microscope by HE stains.
Results The 4-week-old rats from closed population seemed unsusceptible to the UMR-106 cell line and only 30% of them were infected. After bolting and propagation, the 4th generation rats from inbred strain became so susceptible to the osteosarcoma cell line that in the1×10~7 group all tumors developed and 70% of them metastasized to lungs, while the tumors of 1×10~6 and 5×10~6 groups didn’t develop well,nor lung metastasis were observed.
Conclusions The SD rats from closed population showed up certain genetic diversity, while their offspring from inbred strain became identical and demonstrated no diversity in oncogenicity. The UMR-106 cell line has strong carcinogenic capability and high lung metastasis frequency. The animal models depending on it offer a method for the animal experiments of comprehensive therapy to osteosarcoma.
PartⅡInduction, Proliferation and Identification of Dendritic Cells from Rat Bone Marrow Mononuclear Cells in vitro
Objective To establish a method to induce dendritic cells from the bone marrow mononuclear cell of rats in vitro,and to identify the process of phenotypes’variation on DC.
Methods BMMCs were isolated by Ficoll-Hypaque density gradient separation and then cultured in RPMI1640-10% FCS medium in polystyrene flask under the condition of rGM-CSF and rIL-4 at 37℃and 5% CO2. On d6 of culture, immature DC were washed and suspended in medium containing nrhTNF-αto generate mature DC. On d9 after purified by monoclonal antibody OX62 and magnetic beads, DC was identified by morphological features under both inversion microscope and electron microscope. While DC’surface antigens expression was analyzed by FACS every three days.
Results After culture and induction,DC displayed typical morphology with elongated dendritic processes viewed by inversion microscope as well as electron microscope. DC expressed increasing level function associated surface antigens,including OX62 1.78%,MHCⅡ13.68%,CD80 4.77%,CD86 5.77% on d0; OX62 32.31%,MHCⅡ32.14%,CD80 23.68%,CD86 15.44% on d3; OX62 63.13%,MHCⅡ68.36%,CD80 43.06%,CD86 54.78%; and OX62 72.41%,MHCⅡ84.71%,CD80 79.06%,CD86 74.80%on d9. While the rat osteosarcoma associated surface antigen CD44 was always negatively expressed on DC.
Conclusions Mature DC with typical morphology and surface antigens expression could be generated from rat bone marrow mononuclear cells under the condition of rGM-CSF, rIL-4 and nrhTNF-α,and DC’s surface antigens expression was increased progressively with culture days added, which presents the feasibility for further clinical application of DC in the immunotherapy of cancer.
PartIII Preparation and Identification of Allogeneic DC-osteosarcoma Electrofusion Vaccine
Objective To establish a method to prepare allogeneic DC-osteosarcoma electrofusion vaccine with the Wistar rats’bone marrow derived dendritic cells and SD rats derived osteosarcoma cell line UMR-106,and to identify the phenotypes’variation on the fusion hybrids.
Methods Allogeneic DC and tumor cells were mixed at a 5:1 ratio and suspended in 0.3 M glucose solution containing 0.1mM Ca (CH3COO)2, 0.5mM Mg (CH3COO)2, and 10% bovine serum albumin. After centrifugation, the cells were resuspended in the same fusion medium without bovine serum albumin. Routinely, 0.5ml of cell suspension containing 6×10~6 cells were processed using a specially designed electroporation cuvette. For electrofusion, a pulse generator (model ECM 2001) was used. Electrofusion involves two independent but consecutive steps. The first reaction is to bring cells in close contact by dielectrophoresis, which can be accomplished by exposing cells to an alternating (ac) electric field of relatively low strength. Cell fusion can then be triggered by applying a single square wave pulse of 250V to induce reversible cell membrane breakdown in the zone of membrane contact. The entire process was repeated a second time to maximize fusion efficiency. The fusion mixture was allowed to stand for 5 min before suspending in complete medium and then incubated at 37℃overnight. The electrofusion products were purified by monoclonal antibody OX62 (a DC marker not expressed on tumor cells) sticking to the magnetic beads (Miltenyi Biotec) and then was identified by morphological features under both inversion microscope and electron microscope. The allogeneic DC- osteosarcoma products’surface antigens expression was analyzed by FACS and co-staining against both kinds of markers allows for the detection of double-positive hybrids.
Results The allogeneic DC-osteosarcoma vaccine were achieved through electrofusion pulse, and the electrofusion products displayed typical morphology of both component cells viewed by scan electron microscope as well as multi-nucleus ultrastructure viewed by transmission electron microscope. FACS analysis of a purified fusion products by monoclonal antibody OX62 and magnetic beads showed both tumor cell marker and DC marker were highly expressed, for CD44 in the case of osteosarcoma was 50.02%, OX62 in the case of DC was 83.55%, while the percentage of double-positive cell was 49.43%.
Conclusions This study demonstrated it was feasible to generate a large number of allogeneic DC-osteosarcoma hybrid cells by the electrofusion technique. The electrofusion products displayed typical morphology and phaenotype of both component cells. The allogeneic tumor vaccine affords a promising new approach for the immunotherapy of osteosarcoma.
Part IV In vitro Induction of Anti-tumor CTL Effect against Osteosarcoma Cell Line by Allogeneic DC-Osteosarcoma Fusion Vaccine
Objective This study was designed to investigate the potentiality to induce T cell proliferation and osteosarcoma-specific cytotoxic T lymphocytes of tumor vaccine produced by electrofusion between rat osteosarcoma cells and allogeneic DC.
Methods The allogeneic DC-osteosarcoma fusion cells were purified by monoclonal antibody OX62 and magnetic beads and then irradiated with 30 Gy with radioactive ray of 60Co to ensure inactivation of the tumor cells and DC. Coculture of SD bone marrow derived T lymphocytes with the tumor vaccine was to induce T cell proliferation. Then cytotoxic T lymphocytes assay was assessed according to results of MTT assay. 10 athymic mice were challenged subcutaneously with 1×10~6 UMR-106 cells and then divided into two groups randomly 3 days later. The tumor-bearing athymic mice were immunized with 1×107 activated T lymphocytes for 3 times. The condition of tumor development was observed for 4 weeks since tumor challenge.
Results After T cells were cultured with allogeneic DC-osteosarcoma fusion cells, effective activation of T cells was observed. The immunization using allogeneic DC-osteosarcoma vaccine induced UMR106-spcific CTL responses which were statistically significant (P<0.05) compared with corresponding control groups using saline or DC. 28 days later, the experiment of athymic mice showed that the induced CTL inhibited the growth of implanted tumor in athymic mice, and the volum of athymic mice’s implanted tumor was (945±125)mm3 in treatment group, and (4867±375)mm3 in control group respectively(p<0.05).
Conclusions The present study provided valid evidence of the potentiality of allogeneic DC-osteosarcoma fusion cells to induce effective T cell proliferation and osteosarcoma-specific cytotoxic T lymphocytes. The fusion cells may thus represent a promising strategy for DC-based immunotherapy of osteosarcoma and play certain role in the clinical treatment and prevention.
Part V Specific Active Antitumor Effects of Tumor Vaccine Produced by Electrofusion between Osteosarcoma Cell Line and Allogeneic Dendritic Cell in Rats in vivo.
Objective This study was designed to investigate the potency and antitumor effects of allogeneic tumor vaccine produced by electrofusion between SD rats derived osteosarcoma cell line UMR-106 and Wistar rats’bone marrow derived DC.
Methods In this study of immunization against tumor challenge, groups of 10 rats were immunized intradermally with 1×10~6 electrofused cells after irradiated with 30Gy 60Co-ray on days 0 and 14. One week after the second immunization (day 21), the rats were challenged double-blind with a lethal dose of tumor cells and were then monitored for tumor growth and survival over time. For the tumor challenge, vaccinated SD rats were prepared and the UMR-106 osteosarcoma cell line was injected subcutaneously into the SD rat thigh lateral at the amount of 1×10~7. In contrast to the results obtained with the fusion products, immunization with physiological saline, tumor cells that underwent the electrofusion process alone, DC that underwent electrofusion alone, or a mixture of these two populations was also made to induce antitumor protection. To determine whether the immunization could result in a long-term immunological memory, rats injected with UMR106-allogeneic DC fusion products that survived an UMR-106 tumor challenge were rechallenged with a second lethal dose of tumor cells 10 weeks later and then monitored for tumor growth and survival again for 7 weeks. In the active immunization therapeutic model, tumor cells were injected on day 0 and vaccination with electrofusion products was done on days 3, 7, and 14. Then the animals’survival curve was carried out.
Results In the foregoing study of immunization against tumor challenge, 70% of the rats immunized with 1×10~6 electrofused cells were typically able to reject tumor challenge and remained tumor-free. And then all of the survival rats were able to reject this secondary tumor challenge and remain to live on 7 weeks later. In the active treatment study, little or no antitumor protection was observed in the rats treated with1×10~6 electrofused cells, a dose sufficient to achieve substantial tumor protection in pretreatment studies. However, substantial antitumor protection (60% long-term survivors) was obtained with a higher dose of the vaccine (2×10~6 electrofused cells).
Conclusions Preimmunization with irradiated electrofusion products were found to provide partial to complete protection from tumor challenge in the UMR-106 tumor model. Vaccinated survivors developed long immunological memory. The therapeutic potential of this type of approach was suggested by the ability of UMR106-DC electrofusion products to induce tumor rejection in a substantial percentage of hosts bearing pre-established tumor cells. These results tended to indicate that treatment with electrofused tumor cells and allogeneic DC might be capable of inducing a potent antitumor response and could conceivably be applied to a wide range of cancer indications for which tumor-associated antigens have not been identified.
引文
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