胚胎干细胞在Lewis肺癌中的体内外抗肿瘤作用研究
|
摘要
肺癌是当今世界对人类健康和生命威胁最大的恶性肿瘤,尽管采取一系列综合治疗手段,肺癌患者的生存率依然没有得到明显提高,据统计,肺癌患者总的1年生存率低于30%,5年生存率低于16%。目前在临床上广泛采用的传统治疗方式如外科治疗、化疗和放疗等治疗效果不理想,即使近年来迅速发展的分子靶向治疗和个体化治疗等效果也不尽如人意,研究显示导致这种结果的主要原因是由于肺癌的远处转移和治疗后的局部复发,因此如果能发现一种既具有直接杀伤肿瘤作用,又能够提高患者机体免疫功能、消灭循环系统微转移和远处转移的治疗方法或药物,无疑是肺癌患者的福音,而这些特点恰恰是肿瘤免疫治疗的优点所在。
从免疫学角度而言,肿瘤的发生发展是肿瘤与机体免疫系统之间相互作用的结果,机体的免疫系统是体内一个功能强大而复杂的调控和执行网络,正常情况下具有明确抗原性的肿瘤细胞能被免疫识别并清除,然而肿瘤细胞通过在功能和结构方面发生一系列变化,使其能够逃避机体免疫系统的监视和杀伤作用。例如,肿瘤细胞通过改变其表面抗原的表达,使其免疫原性减弱,从而导致机体无法对其产生特异性的免疫应答；肿瘤细胞表面MHC分子缺失、表达下调或变异,导致抗原呈递过程障碍,从而不能被细胞毒性淋巴T细胞(CTL)识别并进一步影响肿瘤细胞的体内清除；多数肿瘤细胞协同刺激分子表达异常,导致细胞免疫中缺乏协同作用的第二信号传递,从而逃避机体免疫系统的攻击；肿瘤负荷引起体内的树突状细胞(DC)功能缺陷,因而抗原呈递能力变差,无法正常激活初始T细胞造成肿瘤的免疫耐受等；并且多数肿瘤细胞在体内能分泌多种免疫抑制因子,如转化生长因子β(TGF-p)、白细胞介素10(interleukin-10, IL-10)、血管内皮生长因子(vascular endothelial growth factor, VEGF)、唾液酸粘蛋白(sialomucin)和巨噬细胞迁移抑制因子(macrophage migration inhibitory factor,MIF)等,抑制体内T细胞分化和分泌功能,破坏Th1/Th2细胞因子的平衡状态,从而有利于肿瘤细胞在体内的生长和增殖。在肿瘤免疫治疗的临床实践中人们发现,正是由于肿瘤细胞表面抗原分子表达的改变、肿瘤本身对机体的免疫抑制作用以及肿瘤细胞免疫逃逸的存在使其表面标记和抗原能够逃脱机体的免疫监视,因而难以引发有效的免疫应答。
人们经多年的研究发现肿瘤细胞与胚胎干细胞在很多重要的生物学行为方面都有着非常惊人的相似性,例如两者均具有快速增殖和分化能力、侵袭能力,并且能够在一定程度上逃逸机体的免疫监视,都在细胞表面表达类似的细胞因子,细胞内或核内具有许多共同的分子信号转导通路,并且在基因和蛋白水平共有很多相似的表达产物,尤为重要的是两者共同具有区别于成熟体细胞的、类似的细胞表面标记/表面抗原。研究发现胚胎干细胞及其衍生细胞来源的抗原,能够在体内有效激活CD4+和CD8+的T淋巴细胞,并且有研究证实,胚胎干细胞表面的MHC-I类分子和其他蛋白抗原可以在体内诱发T淋巴细胞介导的、细胞毒性淋巴细胞作为效应细胞的的免疫反应。
我们认为这种机体由于接种胚胎干细胞后诱导产生的稳定而持久的免疫反应,因为表面抗原的类似,可以交叉作用于肿瘤细胞,从而使机体具备了有效的抗肿瘤免疫,这为肿瘤的免疫治疗打开了新的思路。尽管当时机制尚不明了,德国学者Schone等曾研究指出,啮齿类动物经接种早期胚胎组织可以明显抑制移植性肿瘤的生长。此后,经过一系列研究人们发现这样的现象,接种早期胚胎组织不仅可以抑制动物体内移植性肿瘤的生长,还可以预防由于物理、化学等致癌因素导致的肿瘤发生,这种抗肿瘤免疫作用对肿瘤的发生和发展均具有明显的抑制作用。而且最近的研究显示,这种胚胎组织/胚胎干细胞诱导的抗肿瘤免疫效应具有广谱的抗肿瘤作用,可能对多种肿瘤均具有显著的抑制作用；并且,这种作用被证实并不是一种机体针对外来异物发生的“异物反应”,人们推测其可能是由多种效应细胞和细胞因子参与构成的针对胚胎干细胞表面抗原的特异性免疫应答。
在另一方面,胚胎干细胞抗肿瘤作用的体外研究也取得了一系列令人惊喜的研究成果。例如,Raof和Postovit等的研究显示胚胎干细胞的生长微环境具有诱导肿瘤细胞重新编程和良性转化的作用,并且能够在一定程度上抑制体内局部肿瘤的发生和转移瘤的出现；Giuffrida等研究发现,胚胎干细胞在体外环境中能够分泌一些可溶性的抗肿瘤因子,具有抑制多种肿瘤细胞的生长的作用,并且能够阻滞肿瘤细胞的细胞周期；Lee等证实胚胎干细胞微环境能够改变肿瘤细胞的生长方式,诱导恶性肿瘤获得一定的良性表型,从而具有抗肿瘤作用；Kulesa等研究发现,胚胎神经嵴发育微环境能够使高转移黑色素瘤向正常表型转化,恶性程度降低并表现为其起源细胞的细胞学形状；最近的研究结果表明,胚胎干细胞能在体外环境中诱导分化成为具有激发机体抗肿瘤免疫作用的抗原呈递细胞-树突状细胞,或者分化成为具有直接杀伤肿瘤细胞作用的免疫效应细胞,例如自然杀伤细胞(natural killer cell,NK)、单核细胞和粒细胞等。
本课题在总结前人研究工作经验的基础上,从体外和体内两方面深入研究胚胎干细胞在Lewis肺癌中的抗肿瘤作用,并在整体、细胞和分子水平探索其抗肿瘤作用的具体作用机制：在体外研究中,通过建立胚胎干细胞-Lewis肺癌细胞体外非接触式Transwell共培养体系,更直观、更直接地观察和检测胚胎干细胞/胚胎干细胞微环境对肿瘤细胞的抑制作用,研究共培养条件下Lewis肺癌细胞的重要生物学行为改变,主要包括细胞周期、细胞凋亡和肿瘤细胞迁移、侵袭能力特别是其成瘤能力等重要生物学行为的变化；在体内研究中,通过构建Lewis肺癌小鼠动物模型并模拟肿瘤发生发展的不同阶段,分别研究胚胎干细胞在预防移植性肿瘤的发生及肿瘤进展中的治疗作用两方面的相关作用机制,并从整体水平、效应细胞、细胞因子和肿瘤组织局部病理变化等多个方面探索胚胎干细胞在体内诱导抗肿瘤免疫过程中可能的作用机制和途径,为肺癌的免疫治疗和胚胎干细胞抗肿瘤作用研究开辟新的研究领域,为肺癌的预防及治疗带来新的希望。
第一部分胚胎干细胞抗Lewis肺癌作用的体外研究
第一节胚胎干细胞对Lewis肺癌细胞体外增殖的抑制作用
[研究目的]
研究胚胎干细胞在体外细胞共培养条件下对Lewis肺癌细胞的抑制作用,探索胚胎干细胞体外抗肿瘤作用机制。
[研究方法]
通过建立C57/BL6小鼠胚胎干细胞-Lewis肺癌细胞体外Transwell非接触式共培养体系,研究共培养条件下Lewis肺癌细胞的重要生物学行为改变,主要包括细胞周期、细胞凋亡,肿瘤细胞侵袭和迁移能力以及细胞增殖水平的变化。
[结果]
Lewis肺癌细胞在与胚胎干细胞经非接触式共培养后,与对照组相比,实验组Lewis肺癌细胞体外增殖受到显著抑制,大部分Lewis肺癌细胞周期被阻滞在G2/M期,凋亡的细胞数目明显增多,并且肿瘤细胞迁移和侵袭能力明显下降,结果具有显著的统计学意义(P<0.05)。
[结论]
胚胎干细胞具有显著的体外抗肿瘤作用,包括能够抑制肿瘤细胞增殖,阻滞肿瘤细胞周期、促进肿瘤细胞凋亡,并且明显改变肿瘤细胞的侵袭和迁移能力,在一定程度上抑制其恶性表型。
第二节胚胎干细胞对Lewis肺癌细胞成瘤能力的影响
[研究目的]
研究Lewis肺癌细胞在与胚胎干细胞在体外共培养后对其成瘤能力影响,并探讨这一过程中相关的作用机制。
[研究方法]
通过建立C57/BL6、鼠Lewis肺癌动物模型,研究与胚胎干细胞共培养后Lewis肺癌细胞的体内成瘤能力的改变,主要检测实验组与对照组小鼠皮下移植性肿瘤体积和重量的变化,并通过TUNEL方法检测肿瘤组织内细胞肿瘤细胞凋亡情况。
[结果]
经与胚胎干细胞共培养后,Lewis肺癌细胞的成瘤能力显著下降,主要表现在实验组小鼠皮下肿瘤体积的增大和重量的增加均受到显著的抑制,并且实验组小鼠肿瘤组织中细胞凋亡的程度明显增加,结果具有显著的统计学意义(P<0.05)。
[结论]
胚胎干细胞在体外共培养过程中能够明显抑制Lewis肺癌细胞的成瘤能力,显著抑制其在体内实体肿瘤的生长,并诱导细胞凋亡增加,成瘤能力作为典型的恶性表型受到抑制,这种作用可能与胚胎干细胞能够在体外抑制Lewis月肺癌细胞群体中肿瘤干细胞的活性有关。
第二部分胚胎干细胞抗Lewis肺癌作用的体内研究
第一节胚胎干细胞对Lewis肺癌发生的预防作用研究
[研究目的]
研究实验小鼠经接种胚胎干细胞后,诱导体内抗肿瘤免疫反应的发生机制,及其对肿瘤发生的预防作用。
[研究方法]
将γ射线灭活的胚胎干细胞经皮下注射进入C57/BL6小鼠体内完成免疫接种,在重复接种3次后检测小鼠免疫指标水平,主要通过流式细胞术检测外周血淋巴细胞(CD3+、CD4+、CD8+T细胞和CD19+B细胞)数量及比例、ELISA检测血清细胞因子(IL-2、IL4和IFN-γ)水平,接种Lewis肺癌细胞后,监测小鼠肿瘤生长情况并绘制肿瘤生长曲线,待动物实验结束后,检测脾脏淋巴细胞增殖情况,剥离肿瘤组织、秤重并制作组织切片,IHC检测肿瘤组织内CTL淋巴细胞浸润程度,TUNEL检测肿瘤组织细胞凋亡情况。
[结果]
与对照组相比,经胚胎干细胞免疫接种的实验组小鼠外周血CD3+/CD8+T淋巴细胞绝对值和比例明显升高、血清Th1型抗肿瘤细胞因子(IL-2和IFN-y)水平显著升高,脾脏T淋巴细胞增殖显著,肿瘤发生率明显降低(P<0.05)；即使在有肿瘤生长的实验组小鼠,其实体肿瘤生长速度受到显著抑制,病理学显示其肿瘤组织CTL淋巴细胞浸润明显增加,肿瘤组织内部细胞凋亡具有显著性差异(P<0.05)。
[结论]
接种胚胎干细胞能诱导机体产生显著的抗肿瘤预防作用,小鼠体内抗肿瘤细胞免疫水平明显提高,能够有效地抑制小鼠移植性肿瘤的发生。
第二节胚胎干细胞对Lewis肺癌进展的治疗作用研究
[研究目的]
研究胚胎干细胞在小鼠肿瘤模型中诱导抗肿瘤免疫反应的发生机制,及其对肿瘤发展的治疗作用。
[研究方法]
采用C57/BL6小鼠皮下接种Lewis肺癌细胞建立肿瘤动物模型,并利用接种时间的长短和皮下瘤块的生长情况模拟不同的肿瘤发展时期,将γ射线灭活的胚胎干细胞经皮下注射进入小鼠体内(共3次间隔7天)进行免疫治疗,通过流式细胞术检测小鼠外周血淋巴细胞数量和比例变化、ELISA检测血清细胞因子水平,监测皮下肿瘤生长情况并绘制肿瘤生长曲线,待动物实验结束后剥离肿瘤组织称重,通过IHC检测脾脏淋巴细胞增殖情况和肿瘤组织淋巴细胞浸润程度,TUNEL检测肿瘤细胞凋亡情况。
[结果]
在肿瘤模型小鼠中接种胚胎干细胞能够刺激机体CD3+/CD8+T淋巴细胞增殖,血清Thl型抗肿瘤细胞因子(IL-2和IFN-γ)水平显著升高(P<0.05),荷瘤鼠的免疫抑制状态得到改善,实体肿瘤的生长得到显著抑制,病理学显示实验组小鼠脾脏T淋巴细胞增殖明显增多,肿瘤组织局部有更多CTL淋巴细胞浸润,肿瘤细胞凋亡程度具有显著性差异(P<0.05)。
[结论]
在小鼠肿瘤模型中接种胚胎干细胞能显著缓解机体的免疫抑制状态,诱导抗肿瘤免疫的产生,对实体肿瘤的生长和进展具有显著的抑制作用。
Lung cancer is the most dangerous threat to human health and life nowadays. Despite taking a comprehensive range of treatment, the survival rate of patients is still not significantly improved, the overall1-year survival rate is less than30%, while the5-year survival less than16%. The clinical treatment effects of widely used traditional therapies, such as surgical treatment, chemotherapy and radiotherapy is not ideal, even the effects the rapidly developed molecular targeted therapy and individualized treatment is not satisfied, the reason may be due to the distant metastasis of lung cancer and local recurrence after treatment. So if some treatment or drugs have not only the direct anti-tumor effect, but also improving immune function and eradicating the circulatory system micrometastasis and distant metastasis, are undoubtedly the gospel of patients with lung cancer, and these characteristics are precisely the advantages of tumor immunotherapy.
From the point of view of immunology, tumor development is the result of the interaction between the tumor and the immune system, the body's immune system is a powerful and complex regulatory body and performmance network, under normal circumstances, the tumor cells have clear antigenic markers can be immune recognition and clear away, however, the tumor cells undergo a series of changes in functional and structural aspects, to enable them to evade the immune system surveillance and killing effect. For example, by altering the expression of surface antigens, the immunogenicity of tumor cells weakened, resulting weak specific immune response produced in the body; by missing cell-surface MHC molecules, or by the downward or mutation of their expression, tumor cells could inhibit the antigen-presenting process in vivo, which can not be recognized by cytotoxic lymphoid T cells (CTL) and further affect tumor cells clearance in vivo; most tumor cells are abnormal in costimulatory molecule expression, which lead to lack of synergy in the cellular immune signaling, thereby evading the body's immune system attacks; the body's functional defects caused by tumor burden, lead to dendritic cells (DC) apoptosis, and thus the antigen-presenting ability becomes poor and unable to properly activate naive T cells which cause tumor immune tolerance in vivo; most tumor cells in the body can secrete a variety of immune suppression factors, such as transforming growth factor-β (TGF-β), interleukin-10(IL-10), vascular endothelial growth factor (VEGF), sialomucin and macrophages cell migration inhibitory factor (MIF), etc., which could greatly inhibit the differentiation and secretory function of T lymphocytes cells in the body, and destroy the balance state of Th1/Th2cytokines, thus contributing to the growth and proliferation of tumor cells in vivo.
It was found that in clinical studies of tumor immunotherapy, because of the tumor inhibition on the body immune system and the altered expression of the tumor cell surface antigen molecules, cancer cells could escape from the body immune surveillance, and it was difficult to trigger an effective immune response in vivo. While ESCs and their derivative cells could effectively activate CD4+and CD8+T lymphocytes, and there were studies confirming that the MHC-I molecules and other protein antigens on the ESCs surface could induce T lymphocytes mediated cytotoxic lymphocytes as effecter cells in immune response in vivo. More importantly, the stable and durable immune response induced by embryonic stem cells, could cross-react with cancer cells because of the similar surface antigen, so the body had achieved effective anti-tumor immunity, which could find a new way of thinking for cancer immunotherapy.
Looking back to the history of using ESCs antigen in tumor immunotherapy, we found that anti-tumor therapies involving embryonic tissues or ESCs had been carried out for many years. Although the mechanism was not clear, Schone had pointed out that animals vaccinated with embryonic tissues could significantly inhibit the growth of transplanted tumors many years ago. Since then, a series of studies had confirmed that inoculation by embryonic tissues could not only inhibit the growth of transplantable tumors of animals, but also could prevent tumor formation induced by cancer-causing factors, both tumor occurrence and development could be significantly inhibited. Researchers from Louisville University Cancer Center have found that the experimental mice inoculated with ESCs could prevent the occurrence of planting and induced tumors in2006, and this was the first time declaration of the antitumor effects of ESCs. The study from a Chinese scholar Yi Li has shown that, mice inoculated with human embryonic stem cells could also produce anti-tumor immunity against colon cancer cells, they believed that this kind of anti-tumor effect was a broad-spectrum one, and common anti-cancer vaccines could be developed on this basis. Recent researches from Kavitha Yaddanapudi confirmed that the induced anti-tumor immunity by embryonic stem cells was not foreign body reaction, which may be specific immune responses against ESCs constituted by a variety of effecter cells and cytokines.
On the other hand, there have been a lot of studies on the anti-tumor effects of embryonic stem cells in vitro, these studies also has shown us a series of satisfactory results. Raof and Postovit et al declared that the micro-environment of embryonic stem cells could induce tumor cell re-programming and positive transformation, and be able to inhibit the occurrence and metastasis of local tumor in vivo to a certain extent; Giuffrida and other researchers found that embryonic stem cells in vitro environment are capable of secreting a soluble anti-tumor factor, having the role of inhibition of growth of various tumor cells, and can block the cell cycle of the tumor cells; Lee et al confirmed that embryonic stem cell microenvironment was able to change the mode of growth of tumor cells, inducing malignancies obtained certain benign phenotype, which has anti-tumor effects; Kulesa et al found that the development of embryonic neural crest microenvironment enables highly metastatic melanoma to normal phenotypic transformation, reduce the degree of malignancy and the performance of their cells of origin cytology shape; recent research results show that embryonic stem cells can be induced to differentiate into and to stimulate the body's anti-tumor immunity antigen presenting cells-dendritic cells, or directly kill tumor cells and differentiate into immune effector cells, such as natural killer cells (Natural killer cell, NK), monocytes and granulocytes in vitro environment.
This study was built on the summary of previous researches, and took a deep look into anti-tumor effects and mechanisms of ESCs from two aspects in vitro and in vivo: observing and detecting the ESCs direct inhibitory effect on cancer cells and the interaction between ESCs and cancer cells by the establishment of ESCs-Lewis lung carcinoma cells co-culturing system in vitro; building tumor mouse model to explore the mechanism of induced anti-tumor immunity by ESCs from the aspects of effecter cells, cytokines, and local pathological changes of tumor tissue. This study will open up new research areas of immunotherapy for lung cancer and the anti-tumor effect of ESCs, and bring new hopes for the prevention and treatment of lung cancer.
CHAPTER I The anti-Lewis Lung Carcinoma effects of ESCs in vitro
SECTION I The inhibition effects of embryonic stem cells on Lewis lung carcinoma proliferation
OBJECTIVE:To study the inhibition effects of ESCs on Lewis lung carcinoma cells under co-cultured condition in vitro, and to explore the possible mechanisms of ESCs against cancer.
METHODS:Building the C57/BL6mouse ESCs-Lewis lung carcinoma cells non-contact Transwell co-culture system in vitro, studying the important changes of Lewis lung carcinoma cells in biological behavior, including cell cycle, apoptosis, and tumor cell invasion ability under co-culturing condition.
RESULTS:After the non-contact co-culture with ESCs, the cell proliferation of Lewis lung carcinoma cells was significantly inhibit, cell cycle was in stagnation of G2/M phase and the cell apoptosis increased significantly, and the cell invasion ability was significantly decreased, all results showed substantially statistical significance (P<0.05).
CONCLUSION:The embryonic stem cells have significant anti-tumor effects in vitro, including the ability to inhibit tumor cell proliferation and to promote apoptosis of tumor cells, and significantly change the invasion ability of the tumor cells.
SECTION II The anti-tumor effects of embryonic stem cells in the treatment of Lewis lung development
OBJECTIVE:To study the inhibition effects of ESCs on the tumorigenic ability of Lewis lung carcinoma cells under co-cultured condition in vitro, and to explore the possible mechanisms of this kind of effects.
METHODS:Through the establishment of C57/BL6mice with Lewis lung cancer animal model to study tumorigenicity change of Lewis lung carcinoma cells after co-cultured with embryonic stem cells in vivo. Tumor growth of mice in experimental group and the control group were detected, and the tumor cell apoptosis is detected by the TUNEL method.
RESULTS:The tumor formation ability of Lewis lung cancer cells after co-cultured with embryonic stem cells was significantly decreased, the increase of subcutaneous tumor volume and weight of the experimental group mice was subject to significant inhibition, and the apoptosis of the experimental group mice tumor tissue was significantly increased, all the results have statistical significance (P<0.05).
CONCLUSION:In vitro, the tumor formation ability of Lewis lung carcinoma cells was significantly inhibit due to co-culture with embryonic stem cells, the tumor growth was significantly inhibit in vivo entities and the cell apoptosis was increased. The tumorigenic ability was a typical ability of malignant cells; the possible mechanism may be due to the inhibition effect of embryonic stem cells on cancer stem cells.
CHAPTER II The anti-Lewis Lung Carcinoma effects of ESCs in vivo
SECTION I The vaccination effects of embryonic stem cells in Lewis lung carcinoma occurrences
OBJECTIVE:To study after embryonic stem cells inoculation, the mechanism of induced anti-tumor immune response in mice, and its role in the prevention of Lewis lung carcinoma occurrences in vivo.
METHODS:Embryonic stem cells (y-rayed) were injected subcutaneously into the C57/BL6mice as immunization (three times at7days interval), and then the number and proportion of peripheral blood lymphocytes (CD3+, CD4+, CD8+T cells and CD19+B cells) were detected by flow cytometry, serum-free cell factor (IL-2, IL4and of IFN-y) level was detected by ELISA. After Lewis lung carcinoma cells inoculation, subcutaneous tumor growth was closely monitored and the tumor growth curve was draw. When the animal experiments were finished, spleen lymphocyte proliferation was detected, the tumor tissues were isolated and weighed, tissue sections were embedded and CTL lymphocyte infiltration within the tumor tissue was detected by IHC, cancer cell apoptosis in tumor tissue was detected by TUNEL.
RESULTS:The absolute value and proportion of peripheral CD3+/CD8+T lymphocytes in experimental mice was significantly higher compared with the control group, serum level of anti-tumor cytokine (IL-2and IFN-y) was significantly elevated and splenic T lymphocytes proliferation was obviously increased, while the tumor incidence rate was significantly lower (P<0.05); tumor growth in experimental mice and the tumor growth rate was significantly inhibited, pathological tumor CTL lymphocytes infiltration within the tumor tissues were significantly increased and cell apoptosis was significantly different (P<0.05).
CONCLUSION:The vaccination by embryonic stem cells can induce significant anti-tumor effect in mice; the anti-tumor immune levels were significantly increased, which can effectively prevent the occurrence of transplanted tumors in mice.
SECTION II The anti-tumor effects of embryonic stem cells in the treatment of Lewis lung development
OBJECTIVE:To explore the possible mechanism of embryonic stem cells induced anti-tumor immune response in mouse tumor models, and its therapeutic effects on tumor development.
METHODS:The C57/BL6mice were inoculated subcutaneously with Lewis lung carcinoma cells to establish tumor animal models, and the tumor inoculation time and growth of tumors were used to simulate different tumor development period. The y-ray inactivated embryonic stem cells were injected subcutaneously into mice as immunotherapy (three times at7days interval), the changes in number and proportion of peripheral blood lymphocytes were detected by flow cytometry and mice serum cytokine levels was checked by ELISA. The subcutaneous tumor growth was closely monitored and tumor growth curve was made, the tumors were isolated and weighted and the CTL lymphocytes infiltration was checked by IHC, spleen lymphocytes proliferation was detected tumor cell apoptosis was checked by TUNEL up to the end of the animal experiments.
RESULTS:Inoculation of embryonic stem cells in tumor mice model was able to stimulate the CD3+/CD8+T lymphocyte proliferation, significantly increase the serum level of Thl-type anti-tumor cytokines (IL-2and of IFN-y)(P<0.05). The immunosuppression status in bearing mice was significantly improved and the growth of solid tumors was obviously inhibited. Pathology experiments showed that spleen T lymphocyte proliferation in experimental mice was significantly increased, more CTL lymphocytes infiltration was detected in tumor tissues, and there was a significant difference in the degree of tumor cell apoptosis (P<0.05).
CONCLUSION:Inoculation of embryonic stem cells in a tumor mice model could significantly improve the body immunosuppression status and induce anti-tumor immunity which could significantly inhibit the solid tumor growth and progress.
从免疫学角度而言,肿瘤的发生发展是肿瘤与机体免疫系统之间相互作用的结果,机体的免疫系统是体内一个功能强大而复杂的调控和执行网络,正常情况下具有明确抗原性的肿瘤细胞能被免疫识别并清除,然而肿瘤细胞通过在功能和结构方面发生一系列变化,使其能够逃避机体免疫系统的监视和杀伤作用。例如,肿瘤细胞通过改变其表面抗原的表达,使其免疫原性减弱,从而导致机体无法对其产生特异性的免疫应答；肿瘤细胞表面MHC分子缺失、表达下调或变异,导致抗原呈递过程障碍,从而不能被细胞毒性淋巴T细胞(CTL)识别并进一步影响肿瘤细胞的体内清除；多数肿瘤细胞协同刺激分子表达异常,导致细胞免疫中缺乏协同作用的第二信号传递,从而逃避机体免疫系统的攻击；肿瘤负荷引起体内的树突状细胞(DC)功能缺陷,因而抗原呈递能力变差,无法正常激活初始T细胞造成肿瘤的免疫耐受等；并且多数肿瘤细胞在体内能分泌多种免疫抑制因子,如转化生长因子β(TGF-p)、白细胞介素10(interleukin-10, IL-10)、血管内皮生长因子(vascular endothelial growth factor, VEGF)、唾液酸粘蛋白(sialomucin)和巨噬细胞迁移抑制因子(macrophage migration inhibitory factor,MIF)等,抑制体内T细胞分化和分泌功能,破坏Th1/Th2细胞因子的平衡状态,从而有利于肿瘤细胞在体内的生长和增殖。在肿瘤免疫治疗的临床实践中人们发现,正是由于肿瘤细胞表面抗原分子表达的改变、肿瘤本身对机体的免疫抑制作用以及肿瘤细胞免疫逃逸的存在使其表面标记和抗原能够逃脱机体的免疫监视,因而难以引发有效的免疫应答。
人们经多年的研究发现肿瘤细胞与胚胎干细胞在很多重要的生物学行为方面都有着非常惊人的相似性,例如两者均具有快速增殖和分化能力、侵袭能力,并且能够在一定程度上逃逸机体的免疫监视,都在细胞表面表达类似的细胞因子,细胞内或核内具有许多共同的分子信号转导通路,并且在基因和蛋白水平共有很多相似的表达产物,尤为重要的是两者共同具有区别于成熟体细胞的、类似的细胞表面标记/表面抗原。研究发现胚胎干细胞及其衍生细胞来源的抗原,能够在体内有效激活CD4+和CD8+的T淋巴细胞,并且有研究证实,胚胎干细胞表面的MHC-I类分子和其他蛋白抗原可以在体内诱发T淋巴细胞介导的、细胞毒性淋巴细胞作为效应细胞的的免疫反应。
我们认为这种机体由于接种胚胎干细胞后诱导产生的稳定而持久的免疫反应,因为表面抗原的类似,可以交叉作用于肿瘤细胞,从而使机体具备了有效的抗肿瘤免疫,这为肿瘤的免疫治疗打开了新的思路。尽管当时机制尚不明了,德国学者Schone等曾研究指出,啮齿类动物经接种早期胚胎组织可以明显抑制移植性肿瘤的生长。此后,经过一系列研究人们发现这样的现象,接种早期胚胎组织不仅可以抑制动物体内移植性肿瘤的生长,还可以预防由于物理、化学等致癌因素导致的肿瘤发生,这种抗肿瘤免疫作用对肿瘤的发生和发展均具有明显的抑制作用。而且最近的研究显示,这种胚胎组织/胚胎干细胞诱导的抗肿瘤免疫效应具有广谱的抗肿瘤作用,可能对多种肿瘤均具有显著的抑制作用；并且,这种作用被证实并不是一种机体针对外来异物发生的“异物反应”,人们推测其可能是由多种效应细胞和细胞因子参与构成的针对胚胎干细胞表面抗原的特异性免疫应答。
在另一方面,胚胎干细胞抗肿瘤作用的体外研究也取得了一系列令人惊喜的研究成果。例如,Raof和Postovit等的研究显示胚胎干细胞的生长微环境具有诱导肿瘤细胞重新编程和良性转化的作用,并且能够在一定程度上抑制体内局部肿瘤的发生和转移瘤的出现；Giuffrida等研究发现,胚胎干细胞在体外环境中能够分泌一些可溶性的抗肿瘤因子,具有抑制多种肿瘤细胞的生长的作用,并且能够阻滞肿瘤细胞的细胞周期；Lee等证实胚胎干细胞微环境能够改变肿瘤细胞的生长方式,诱导恶性肿瘤获得一定的良性表型,从而具有抗肿瘤作用；Kulesa等研究发现,胚胎神经嵴发育微环境能够使高转移黑色素瘤向正常表型转化,恶性程度降低并表现为其起源细胞的细胞学形状；最近的研究结果表明,胚胎干细胞能在体外环境中诱导分化成为具有激发机体抗肿瘤免疫作用的抗原呈递细胞-树突状细胞,或者分化成为具有直接杀伤肿瘤细胞作用的免疫效应细胞,例如自然杀伤细胞(natural killer cell,NK)、单核细胞和粒细胞等。
本课题在总结前人研究工作经验的基础上,从体外和体内两方面深入研究胚胎干细胞在Lewis肺癌中的抗肿瘤作用,并在整体、细胞和分子水平探索其抗肿瘤作用的具体作用机制：在体外研究中,通过建立胚胎干细胞-Lewis肺癌细胞体外非接触式Transwell共培养体系,更直观、更直接地观察和检测胚胎干细胞/胚胎干细胞微环境对肿瘤细胞的抑制作用,研究共培养条件下Lewis肺癌细胞的重要生物学行为改变,主要包括细胞周期、细胞凋亡和肿瘤细胞迁移、侵袭能力特别是其成瘤能力等重要生物学行为的变化；在体内研究中,通过构建Lewis肺癌小鼠动物模型并模拟肿瘤发生发展的不同阶段,分别研究胚胎干细胞在预防移植性肿瘤的发生及肿瘤进展中的治疗作用两方面的相关作用机制,并从整体水平、效应细胞、细胞因子和肿瘤组织局部病理变化等多个方面探索胚胎干细胞在体内诱导抗肿瘤免疫过程中可能的作用机制和途径,为肺癌的免疫治疗和胚胎干细胞抗肿瘤作用研究开辟新的研究领域,为肺癌的预防及治疗带来新的希望。
第一部分胚胎干细胞抗Lewis肺癌作用的体外研究
第一节胚胎干细胞对Lewis肺癌细胞体外增殖的抑制作用
[研究目的]
研究胚胎干细胞在体外细胞共培养条件下对Lewis肺癌细胞的抑制作用,探索胚胎干细胞体外抗肿瘤作用机制。
[研究方法]
通过建立C57/BL6小鼠胚胎干细胞-Lewis肺癌细胞体外Transwell非接触式共培养体系,研究共培养条件下Lewis肺癌细胞的重要生物学行为改变,主要包括细胞周期、细胞凋亡,肿瘤细胞侵袭和迁移能力以及细胞增殖水平的变化。
[结果]
Lewis肺癌细胞在与胚胎干细胞经非接触式共培养后,与对照组相比,实验组Lewis肺癌细胞体外增殖受到显著抑制,大部分Lewis肺癌细胞周期被阻滞在G2/M期,凋亡的细胞数目明显增多,并且肿瘤细胞迁移和侵袭能力明显下降,结果具有显著的统计学意义(P<0.05)。
[结论]
胚胎干细胞具有显著的体外抗肿瘤作用,包括能够抑制肿瘤细胞增殖,阻滞肿瘤细胞周期、促进肿瘤细胞凋亡,并且明显改变肿瘤细胞的侵袭和迁移能力,在一定程度上抑制其恶性表型。
第二节胚胎干细胞对Lewis肺癌细胞成瘤能力的影响
[研究目的]
研究Lewis肺癌细胞在与胚胎干细胞在体外共培养后对其成瘤能力影响,并探讨这一过程中相关的作用机制。
[研究方法]
通过建立C57/BL6、鼠Lewis肺癌动物模型,研究与胚胎干细胞共培养后Lewis肺癌细胞的体内成瘤能力的改变,主要检测实验组与对照组小鼠皮下移植性肿瘤体积和重量的变化,并通过TUNEL方法检测肿瘤组织内细胞肿瘤细胞凋亡情况。
[结果]
经与胚胎干细胞共培养后,Lewis肺癌细胞的成瘤能力显著下降,主要表现在实验组小鼠皮下肿瘤体积的增大和重量的增加均受到显著的抑制,并且实验组小鼠肿瘤组织中细胞凋亡的程度明显增加,结果具有显著的统计学意义(P<0.05)。
[结论]
胚胎干细胞在体外共培养过程中能够明显抑制Lewis肺癌细胞的成瘤能力,显著抑制其在体内实体肿瘤的生长,并诱导细胞凋亡增加,成瘤能力作为典型的恶性表型受到抑制,这种作用可能与胚胎干细胞能够在体外抑制Lewis月肺癌细胞群体中肿瘤干细胞的活性有关。
第二部分胚胎干细胞抗Lewis肺癌作用的体内研究
第一节胚胎干细胞对Lewis肺癌发生的预防作用研究
[研究目的]
研究实验小鼠经接种胚胎干细胞后,诱导体内抗肿瘤免疫反应的发生机制,及其对肿瘤发生的预防作用。
[研究方法]
将γ射线灭活的胚胎干细胞经皮下注射进入C57/BL6小鼠体内完成免疫接种,在重复接种3次后检测小鼠免疫指标水平,主要通过流式细胞术检测外周血淋巴细胞(CD3+、CD4+、CD8+T细胞和CD19+B细胞)数量及比例、ELISA检测血清细胞因子(IL-2、IL4和IFN-γ)水平,接种Lewis肺癌细胞后,监测小鼠肿瘤生长情况并绘制肿瘤生长曲线,待动物实验结束后,检测脾脏淋巴细胞增殖情况,剥离肿瘤组织、秤重并制作组织切片,IHC检测肿瘤组织内CTL淋巴细胞浸润程度,TUNEL检测肿瘤组织细胞凋亡情况。
[结果]
与对照组相比,经胚胎干细胞免疫接种的实验组小鼠外周血CD3+/CD8+T淋巴细胞绝对值和比例明显升高、血清Th1型抗肿瘤细胞因子(IL-2和IFN-y)水平显著升高,脾脏T淋巴细胞增殖显著,肿瘤发生率明显降低(P<0.05)；即使在有肿瘤生长的实验组小鼠,其实体肿瘤生长速度受到显著抑制,病理学显示其肿瘤组织CTL淋巴细胞浸润明显增加,肿瘤组织内部细胞凋亡具有显著性差异(P<0.05)。
[结论]
接种胚胎干细胞能诱导机体产生显著的抗肿瘤预防作用,小鼠体内抗肿瘤细胞免疫水平明显提高,能够有效地抑制小鼠移植性肿瘤的发生。
第二节胚胎干细胞对Lewis肺癌进展的治疗作用研究
[研究目的]
研究胚胎干细胞在小鼠肿瘤模型中诱导抗肿瘤免疫反应的发生机制,及其对肿瘤发展的治疗作用。
[研究方法]
采用C57/BL6小鼠皮下接种Lewis肺癌细胞建立肿瘤动物模型,并利用接种时间的长短和皮下瘤块的生长情况模拟不同的肿瘤发展时期,将γ射线灭活的胚胎干细胞经皮下注射进入小鼠体内(共3次间隔7天)进行免疫治疗,通过流式细胞术检测小鼠外周血淋巴细胞数量和比例变化、ELISA检测血清细胞因子水平,监测皮下肿瘤生长情况并绘制肿瘤生长曲线,待动物实验结束后剥离肿瘤组织称重,通过IHC检测脾脏淋巴细胞增殖情况和肿瘤组织淋巴细胞浸润程度,TUNEL检测肿瘤细胞凋亡情况。
[结果]
在肿瘤模型小鼠中接种胚胎干细胞能够刺激机体CD3+/CD8+T淋巴细胞增殖,血清Thl型抗肿瘤细胞因子(IL-2和IFN-γ)水平显著升高(P<0.05),荷瘤鼠的免疫抑制状态得到改善,实体肿瘤的生长得到显著抑制,病理学显示实验组小鼠脾脏T淋巴细胞增殖明显增多,肿瘤组织局部有更多CTL淋巴细胞浸润,肿瘤细胞凋亡程度具有显著性差异(P<0.05)。
[结论]
在小鼠肿瘤模型中接种胚胎干细胞能显著缓解机体的免疫抑制状态,诱导抗肿瘤免疫的产生,对实体肿瘤的生长和进展具有显著的抑制作用。
Lung cancer is the most dangerous threat to human health and life nowadays. Despite taking a comprehensive range of treatment, the survival rate of patients is still not significantly improved, the overall1-year survival rate is less than30%, while the5-year survival less than16%. The clinical treatment effects of widely used traditional therapies, such as surgical treatment, chemotherapy and radiotherapy is not ideal, even the effects the rapidly developed molecular targeted therapy and individualized treatment is not satisfied, the reason may be due to the distant metastasis of lung cancer and local recurrence after treatment. So if some treatment or drugs have not only the direct anti-tumor effect, but also improving immune function and eradicating the circulatory system micrometastasis and distant metastasis, are undoubtedly the gospel of patients with lung cancer, and these characteristics are precisely the advantages of tumor immunotherapy.
From the point of view of immunology, tumor development is the result of the interaction between the tumor and the immune system, the body's immune system is a powerful and complex regulatory body and performmance network, under normal circumstances, the tumor cells have clear antigenic markers can be immune recognition and clear away, however, the tumor cells undergo a series of changes in functional and structural aspects, to enable them to evade the immune system surveillance and killing effect. For example, by altering the expression of surface antigens, the immunogenicity of tumor cells weakened, resulting weak specific immune response produced in the body; by missing cell-surface MHC molecules, or by the downward or mutation of their expression, tumor cells could inhibit the antigen-presenting process in vivo, which can not be recognized by cytotoxic lymphoid T cells (CTL) and further affect tumor cells clearance in vivo; most tumor cells are abnormal in costimulatory molecule expression, which lead to lack of synergy in the cellular immune signaling, thereby evading the body's immune system attacks; the body's functional defects caused by tumor burden, lead to dendritic cells (DC) apoptosis, and thus the antigen-presenting ability becomes poor and unable to properly activate naive T cells which cause tumor immune tolerance in vivo; most tumor cells in the body can secrete a variety of immune suppression factors, such as transforming growth factor-β (TGF-β), interleukin-10(IL-10), vascular endothelial growth factor (VEGF), sialomucin and macrophages cell migration inhibitory factor (MIF), etc., which could greatly inhibit the differentiation and secretory function of T lymphocytes cells in the body, and destroy the balance state of Th1/Th2cytokines, thus contributing to the growth and proliferation of tumor cells in vivo.
It was found that in clinical studies of tumor immunotherapy, because of the tumor inhibition on the body immune system and the altered expression of the tumor cell surface antigen molecules, cancer cells could escape from the body immune surveillance, and it was difficult to trigger an effective immune response in vivo. While ESCs and their derivative cells could effectively activate CD4+and CD8+T lymphocytes, and there were studies confirming that the MHC-I molecules and other protein antigens on the ESCs surface could induce T lymphocytes mediated cytotoxic lymphocytes as effecter cells in immune response in vivo. More importantly, the stable and durable immune response induced by embryonic stem cells, could cross-react with cancer cells because of the similar surface antigen, so the body had achieved effective anti-tumor immunity, which could find a new way of thinking for cancer immunotherapy.
Looking back to the history of using ESCs antigen in tumor immunotherapy, we found that anti-tumor therapies involving embryonic tissues or ESCs had been carried out for many years. Although the mechanism was not clear, Schone had pointed out that animals vaccinated with embryonic tissues could significantly inhibit the growth of transplanted tumors many years ago. Since then, a series of studies had confirmed that inoculation by embryonic tissues could not only inhibit the growth of transplantable tumors of animals, but also could prevent tumor formation induced by cancer-causing factors, both tumor occurrence and development could be significantly inhibited. Researchers from Louisville University Cancer Center have found that the experimental mice inoculated with ESCs could prevent the occurrence of planting and induced tumors in2006, and this was the first time declaration of the antitumor effects of ESCs. The study from a Chinese scholar Yi Li has shown that, mice inoculated with human embryonic stem cells could also produce anti-tumor immunity against colon cancer cells, they believed that this kind of anti-tumor effect was a broad-spectrum one, and common anti-cancer vaccines could be developed on this basis. Recent researches from Kavitha Yaddanapudi confirmed that the induced anti-tumor immunity by embryonic stem cells was not foreign body reaction, which may be specific immune responses against ESCs constituted by a variety of effecter cells and cytokines.
On the other hand, there have been a lot of studies on the anti-tumor effects of embryonic stem cells in vitro, these studies also has shown us a series of satisfactory results. Raof and Postovit et al declared that the micro-environment of embryonic stem cells could induce tumor cell re-programming and positive transformation, and be able to inhibit the occurrence and metastasis of local tumor in vivo to a certain extent; Giuffrida and other researchers found that embryonic stem cells in vitro environment are capable of secreting a soluble anti-tumor factor, having the role of inhibition of growth of various tumor cells, and can block the cell cycle of the tumor cells; Lee et al confirmed that embryonic stem cell microenvironment was able to change the mode of growth of tumor cells, inducing malignancies obtained certain benign phenotype, which has anti-tumor effects; Kulesa et al found that the development of embryonic neural crest microenvironment enables highly metastatic melanoma to normal phenotypic transformation, reduce the degree of malignancy and the performance of their cells of origin cytology shape; recent research results show that embryonic stem cells can be induced to differentiate into and to stimulate the body's anti-tumor immunity antigen presenting cells-dendritic cells, or directly kill tumor cells and differentiate into immune effector cells, such as natural killer cells (Natural killer cell, NK), monocytes and granulocytes in vitro environment.
This study was built on the summary of previous researches, and took a deep look into anti-tumor effects and mechanisms of ESCs from two aspects in vitro and in vivo: observing and detecting the ESCs direct inhibitory effect on cancer cells and the interaction between ESCs and cancer cells by the establishment of ESCs-Lewis lung carcinoma cells co-culturing system in vitro; building tumor mouse model to explore the mechanism of induced anti-tumor immunity by ESCs from the aspects of effecter cells, cytokines, and local pathological changes of tumor tissue. This study will open up new research areas of immunotherapy for lung cancer and the anti-tumor effect of ESCs, and bring new hopes for the prevention and treatment of lung cancer.
CHAPTER I The anti-Lewis Lung Carcinoma effects of ESCs in vitro
SECTION I The inhibition effects of embryonic stem cells on Lewis lung carcinoma proliferation
OBJECTIVE:To study the inhibition effects of ESCs on Lewis lung carcinoma cells under co-cultured condition in vitro, and to explore the possible mechanisms of ESCs against cancer.
METHODS:Building the C57/BL6mouse ESCs-Lewis lung carcinoma cells non-contact Transwell co-culture system in vitro, studying the important changes of Lewis lung carcinoma cells in biological behavior, including cell cycle, apoptosis, and tumor cell invasion ability under co-culturing condition.
RESULTS:After the non-contact co-culture with ESCs, the cell proliferation of Lewis lung carcinoma cells was significantly inhibit, cell cycle was in stagnation of G2/M phase and the cell apoptosis increased significantly, and the cell invasion ability was significantly decreased, all results showed substantially statistical significance (P<0.05).
CONCLUSION:The embryonic stem cells have significant anti-tumor effects in vitro, including the ability to inhibit tumor cell proliferation and to promote apoptosis of tumor cells, and significantly change the invasion ability of the tumor cells.
SECTION II The anti-tumor effects of embryonic stem cells in the treatment of Lewis lung development
OBJECTIVE:To study the inhibition effects of ESCs on the tumorigenic ability of Lewis lung carcinoma cells under co-cultured condition in vitro, and to explore the possible mechanisms of this kind of effects.
METHODS:Through the establishment of C57/BL6mice with Lewis lung cancer animal model to study tumorigenicity change of Lewis lung carcinoma cells after co-cultured with embryonic stem cells in vivo. Tumor growth of mice in experimental group and the control group were detected, and the tumor cell apoptosis is detected by the TUNEL method.
RESULTS:The tumor formation ability of Lewis lung cancer cells after co-cultured with embryonic stem cells was significantly decreased, the increase of subcutaneous tumor volume and weight of the experimental group mice was subject to significant inhibition, and the apoptosis of the experimental group mice tumor tissue was significantly increased, all the results have statistical significance (P<0.05).
CONCLUSION:In vitro, the tumor formation ability of Lewis lung carcinoma cells was significantly inhibit due to co-culture with embryonic stem cells, the tumor growth was significantly inhibit in vivo entities and the cell apoptosis was increased. The tumorigenic ability was a typical ability of malignant cells; the possible mechanism may be due to the inhibition effect of embryonic stem cells on cancer stem cells.
CHAPTER II The anti-Lewis Lung Carcinoma effects of ESCs in vivo
SECTION I The vaccination effects of embryonic stem cells in Lewis lung carcinoma occurrences
OBJECTIVE:To study after embryonic stem cells inoculation, the mechanism of induced anti-tumor immune response in mice, and its role in the prevention of Lewis lung carcinoma occurrences in vivo.
METHODS:Embryonic stem cells (y-rayed) were injected subcutaneously into the C57/BL6mice as immunization (three times at7days interval), and then the number and proportion of peripheral blood lymphocytes (CD3+, CD4+, CD8+T cells and CD19+B cells) were detected by flow cytometry, serum-free cell factor (IL-2, IL4and of IFN-y) level was detected by ELISA. After Lewis lung carcinoma cells inoculation, subcutaneous tumor growth was closely monitored and the tumor growth curve was draw. When the animal experiments were finished, spleen lymphocyte proliferation was detected, the tumor tissues were isolated and weighed, tissue sections were embedded and CTL lymphocyte infiltration within the tumor tissue was detected by IHC, cancer cell apoptosis in tumor tissue was detected by TUNEL.
RESULTS:The absolute value and proportion of peripheral CD3+/CD8+T lymphocytes in experimental mice was significantly higher compared with the control group, serum level of anti-tumor cytokine (IL-2and IFN-y) was significantly elevated and splenic T lymphocytes proliferation was obviously increased, while the tumor incidence rate was significantly lower (P<0.05); tumor growth in experimental mice and the tumor growth rate was significantly inhibited, pathological tumor CTL lymphocytes infiltration within the tumor tissues were significantly increased and cell apoptosis was significantly different (P<0.05).
CONCLUSION:The vaccination by embryonic stem cells can induce significant anti-tumor effect in mice; the anti-tumor immune levels were significantly increased, which can effectively prevent the occurrence of transplanted tumors in mice.
SECTION II The anti-tumor effects of embryonic stem cells in the treatment of Lewis lung development
OBJECTIVE:To explore the possible mechanism of embryonic stem cells induced anti-tumor immune response in mouse tumor models, and its therapeutic effects on tumor development.
METHODS:The C57/BL6mice were inoculated subcutaneously with Lewis lung carcinoma cells to establish tumor animal models, and the tumor inoculation time and growth of tumors were used to simulate different tumor development period. The y-ray inactivated embryonic stem cells were injected subcutaneously into mice as immunotherapy (three times at7days interval), the changes in number and proportion of peripheral blood lymphocytes were detected by flow cytometry and mice serum cytokine levels was checked by ELISA. The subcutaneous tumor growth was closely monitored and tumor growth curve was made, the tumors were isolated and weighted and the CTL lymphocytes infiltration was checked by IHC, spleen lymphocytes proliferation was detected tumor cell apoptosis was checked by TUNEL up to the end of the animal experiments.
RESULTS:Inoculation of embryonic stem cells in tumor mice model was able to stimulate the CD3+/CD8+T lymphocyte proliferation, significantly increase the serum level of Thl-type anti-tumor cytokines (IL-2and of IFN-y)(P<0.05). The immunosuppression status in bearing mice was significantly improved and the growth of solid tumors was obviously inhibited. Pathology experiments showed that spleen T lymphocyte proliferation in experimental mice was significantly increased, more CTL lymphocytes infiltration was detected in tumor tissues, and there was a significant difference in the degree of tumor cell apoptosis (P<0.05).
CONCLUSION:Inoculation of embryonic stem cells in a tumor mice model could significantly improve the body immunosuppression status and induce anti-tumor immunity which could significantly inhibit the solid tumor growth and progress.
引文
1. Jemal A, Bray F, Center MM, Ferlay J, Ward E, Forman D:Global cancer statistics. CA Cancer J Clin 2011,61(2):69-90.
2. Available at http://www.cancer.gov/cancertopics/types/lung. NC Institute 2009.
3. Bunn PA, Jr., Thatcher N:Systemic treatment for advanced (stage Ⅲb/Ⅳ) non-small cell lung cancer:more treatment options; more things to consider. Conclusion. Oncologist 2008,13 Suppl 1:37-46.
4. Hirschowitz EA, Yannelli JR:Immunotherapy for lung cancer. Proc Am Thorac Soc 2009,6(2):224-232.
5. Vanneman M, Dranoff G:Combining immunotherapy and targeted therapies in cancer treatment. Nat Rev Cancer 2012,12(4):237-251.
6. Xiong G, Husseiny MI, Song L, Erdreich-Epstein A, Shackleford GM, Seeger RC, Jacket D, Hensel M, Metelitsa LS:Novel cancer vaccine based on genes of Salmonella pathogenicity island 2. Int J Cancer 2010,126(11):2622-2634.
7. Murray MJ, Lessey BA:Embryo implantation and tumor metastasis:common pathways of invasion and angiogenesis. Semin Reprod Endocrinol 1999, 17(3):275-290.
8. Pardal R, Clarke MF, Morrison SJ:Applying the principles of stem-cell biology to cancer. Nat Rev Cancer 2003,3(12):895-902.
9. Cabanillas R, Llorente JL:The Stem Cell Network model:clinical implications in cancer. Eur Arch Otorhinolaryngol 2009,266(2):161-170.
10. Reya T, Clevers H:Wnt signalling in stem cells and cancer. Nature 2005, 434(7035):843-850.
11. Li L, Neaves WB:Normal stem cells and cancer stem cells:the niche matters. Cancer Res 2006,66(9):4553-4557.
12. Huang EH, Heidt DG, Li CW, Simeone DM:Cancer stem cells:a new paradigm for understanding tumor progression and therapeutic resistance. Surgery 2007, 141(4):415-419.
13. Okamoto OK:Cancer stem cell genomics:the quest for early markers of malignant progression. Expert Rev Mol Diagn 2009,9(6):545-554.
14. Rubio D, Garcia-Castro J, Martin MC, de la Fuente R, Cigudosa JC, Lloyd AC, Bernad A:Spontaneous human adult stem cell transformation. Cancer Res 2005, 65(8):3035-3039.
15. Al-Hajj M, Wicha MS, Benito-Hernandez A, Morrison SJ, Clarke MF:Prospective identification of tumorigenic breast cancer cells. Proc Natl Acad Sci U S A 2003, 100(7):3983-3988.
16. Li C, Lee CJ, Simeone DM:Identification of human pancreatic cancer stem cells. Methods Mol Biol 2009,568:161-173.
17. O'Brien CA, Pollett A, Gallinger S, Dick JE:A human colon cancer cell capable of initiating tumour growth in immunodeficient mice. Nature 2007,445(7123):106-110.
18. Chu R, Clanton DJ, Snipas TS, Lee J, Mitchell E, Nguyen ML, Hare E, Peach RJ: Characterization of a subpopulation of colon cancer cells with stem cell-like properties. Int J Cancer 2009,124(6):1312-1321.
19. Murase M, Kano M, Tsukahara T, Takahashi A, Torigoe T, Kawaguchi S, Kimura S, Wada T, Uchihashi Y, Kondo T et al: Side population cells have the characteristics of cancer stem-like cells/cancer-initiating cells in bone sarcomas. Br J Cancer 2009, 101(8):1425-1432.
20. Wang S, Garcia AJ, Wu M, Lawson DA, Witte ON, Wu H:Pten deletion leads to the expansion of a prostatic stem/progenitor cell subpopulation and tumor initiation. Proc Natl Acad Sci USA 2006,103(5):1480-1485.
21. Adikrisna R, Tanaka S, Muramatsu S, Aihara A, Ban D, Ochiai T, Irie T, Kudo A, Nakamura N, Yamaoka S et al: Identification of pancreatic cancer stem cells and selective toxicity of chemotherapeutic agents. Gastroenterology 2012, 143(1):234-245 e237.
22. Zhu J, He J, Liu Y, Simeone DM, Lubman DM:Identification of glycoprotein markers for pancreatic cancer CD24+CD44+ stem-like cells using nano-LC-MS/MS and tissue microarray. JProteome Res 2012,11(4):2272-2281.
23. de Sousa EMF, Guessous I, Vermeulen L, Medema JP:[Cancer stem cells and future therapeutic implications]. Rev Med Suisse 2011,7(289):774-777.
24. Jordan CT:Targeting the most critical cells:approaching leukemia therapy as a problem in stem cell biology. Nat Clin Pract Oncol 2005,2(5):224-225.
25. Copland M, Fraser AR, Harrison SJ, Holyoake TL:Targeting the silent minority: emerging immunotherapeutic strategies for eradication of malignant stem cells in chronic myeloid leukaemia. Cancer Immunol Immunother 2005,54(4):297-306.
26. Boyd AS, Higashi Y, Wood KJ:Transplanting stem cells:potential targets for immune attack. Modulating the immune response against embryonic stem cell transplantation. Adv Drug Deliv Rev 2005,57(13):1944-1969.
27. Drukker M, Katchman H, Katz G, Even-Tov Friedman S, Shezen E, Hornstein E, Mandelboim O, Reisner Y, Benvenisty N:Human embryonic stem cells and their differentiated derivatives are less susceptible to immune rejection than adult cells. Stem Cells 2006,24(2):221-229.
28. Li Y, Zeng H, Xu RH, Liu B, Li Z:Vaccination with human pluripotent stem cells generates a broad spectrum of immunological and clinical responses against colon cancer. Stem Cells 2009,27(12):3103-3111.
29. Dong W, Du J, Shen H, Gao D, Li Z, Wang G, Mu X, Liu Q:Administration of embryonic stem cells generates effective antitumor immunity in mice with minor and heavy tumor load. Cancer Immunol Immunother 2010,59(11):1697-1705.
30. Brewer BG, Mitchell RA, Harandi A, Eaton JW:Embryonic vaccines against cancer: an early history. Exp Mol Pathol 2009,86(3):192-197.
31. LeMevel BP, Wells SA, Jr.:Foetal antigens cross-reactive with tumour-specific transplantation antigens. Nat New Biol 1973,244(136):183-184.
32. Sikora K, Stern P, Lennox E:Immunoprotection by embryonal carcinoma cells for methylcholanthrene-induced murine sarcomas. Nature 1977,269(5631):813-815.
33. Yaddanapudi K, Mitchell RA, Putty K, Willer S, Sharma RK, Yan J, Bodduluri H, Eaton JW:Vaccination with embryonic stem cells protects against lung cancer:is a broad-spectrum prophylactic vaccine against cancer possible? PLoS ONE 2012, 7(7):e42289.
34. Giuffrida D, Rogers IM, Nagy A, Calogero AE, Brown TJ, Casper RF:Human embryonic stem cells secrete soluble factors that inhibit cancer cell growth. Cell Pwlif 2009,42(6):788-798.
35. Raof NA, Raja WK, Castracane J, Xie Y:Bioengineering embryonic stem cell microenvironments for exploring inhibitory effects on metastatic breast cancer cells. Biomaterials 2011,32(17):4130-4139.
36. Postovit LM, Margaryan NV, Seftor EA, Kirschmann DA, Lipavsky A, Wheaton WW, Abbott DE, Seftor RE, Hendrix MJ:Human embryonic stem cell microenvironment suppresses the tumorigenic phenotype of aggressive cancer cells. Proc Natl Acad Sci USA 2008,105(11):4329-4334.
37. Mohib K, Wang L:Differentiation and characterization of dendritic cells from human embryonic stem cells. Curr Protoc Immunol 2012, Chapter 22:Unit 22F 11.
38. Ohlsson LB, Varas L, Kjellman C, Edvardsen K, Lindvall M:Mesenchymal progenitor cell-mediated inhibition of tumor growth in vivo and in vitro in gelatin matrix. Exp Mol Pathol 2003,75(3):248-255.
39. Woll PS, Grzywacz B, Tian X, Marcus RK, Knorr DA, Verneris MR, Kaufman DS: Human embryonic stem cells differentiate into a homogeneous population of natural killer cells with potent in vivo antitumor activity. Blood 2009, 113(24):6094-6101.
40. Yan S:New Milestone in the development of Clinical Oncology Molecular Targeted Therapy. Anti-tumor Pharmacy 2011,1(1):1-5.
41. Keller G:Embryonic stem cell differentiation:emergence of a new era in biology and medicine. Genes Dev 2005,19(10):1129-1155.
42. Postovit LM, Seftor EA, Seftor RE, Hendrix MJ:A three-dimensional model to study the epigenetic effects induced by the microenvironment of human embryonic stem cells. Stem Cells 2006,24(3):501-505.
43. Miller SJ, Lavker RM, Sun TT:Interpreting epithelial cancer biology in the context of stem cells:tumor properties and therapeutic implications. Biochim Biophys Acta 2005,1756(1):25-52.
44. Postovit LM, Seftor EA, Seftor RE, Hendrix MJ:Influence of the microenvironment on melanoma cell fate determination and phenotype. Cancer Res 2006, 66(16):7833-7836.
45. Lee LM, Seftor EA, Bonde G, Cornell RA, Hendrix MJ:The fate of human malignant melanoma cells transplanted into zebrafish embryos:assessment of migration and cell division in the absence of tumor formation. Dev Dyn 2005,233(4):1560-1570.
46. Kulesa PM, Kasemeier-Kulesa JC, Teddy JM, Margaryan NV, Seftor EA, Seftor RE, Hendrix MJ:Reprogramming metastatic melanoma cells to assume a neural crest cell-like phenotype in an embryonic microenvironment. Proc Natl Acad Sci U S A 2006,103(10):3752-3757.
47.常艳,魏伟：细胞共培养及其应用的研究进展.中国临床药理学与治疗学2009,14(7)：3.
48.罗云,孙桂波,秦蒙,姚帆,孙晓波：细胞共培养技术在医药研究中的应用.中国中药杂志2012,37(22)：5.
49. Lowe SW, Lin AW:Apoptosis in cancer. Carcinogenesis 2000,21(3):485-495.
50. Bissell MJ, Labarge MA:Context, tissue plasticity, and cancer:are tumor stem cells also regulated by the microenvironment? Cancer Cell 2005,7(1):17-23.
51. Whiteside EJ, Jackson MM, Herington AC, Edwards DR, Harvey MB:Matrix metalloproteinase-9 and tissue inhibitor of metalloproteinase-3 are key regulators of extracellular matrix degradation by mouse embryos. Biol Reprod 2001, 64(5):1331-1337.
52. Yasuda M, Tanaka Y, Tamura M, Fujii K, Sugaya M, So T, Takenoyama M, Yasumoto K.: Stimulation of betal integrin down-regulates ICAM-1 expression and ICAM-1-dependent adhesion of lung cancer cells through focal adhesion kinase. Cancer Res 2001,61(5):2022-2030.
53. Liotta LA:Cancer cell invasion and metastasis. Sci Am 1992,266(2):54-59,62-53.
54. Glennie S, Soeiro Ⅰ, Dyson PJ, Lam EW, Dazzi F:Bone marrow mesenchymal stem cells induce division arrest anergy of activated T cells. Blood 2005, 105(7):2821-2827.
55. Rasmusson Ⅰ, Ringden O, Sundberg B, Le Blanc K:Mesenchymal stem cells inhibit lymphocyte proliferation by mitogens and alloantigens by different mechanisms. Exp Cell Res 2005,305(1):33-41.
56. Sato K, Ozaki K, Oh I, Meguro A, Hatanaka K, Nagai T, Muroi K, Ozawa K:Nitric oxide plays a critical role in suppression of T-cell proliferation by mesenchymal stem cells. Blood 2007,109(1):228-234.
57. Meisel R, Zibert A, Laryea M, Gobel U, Daubener W, Dilloo D:Human bone marrow stromal cells inhibit allogeneic T-cell responses by indoleamine 2,3-dioxygenase-mediated tryptophan degradation. Blood 2004,103(12):4619-4621.
58. Lu YR, Yuan Y, Wang XJ, Wei LL, Chen YN, Cong C, Li SF, Long D, Tan WD, Mao YQ et al:The growth inhibitory effect of mesenchymal stem cells on tumor cells in vitro and in vivo. Cancer Biol Ther 2008,7(2):245-251.
1. Available at http://www.cancer.gov/cancertopics/types/lung. NC Institute 2009.
2. Swisher SG, Roth JA:Clinical update of Ad-p53 gene therapy for lung cancer. Surg Oncol Clin NAm 2002,11(3):521-535.
3. Balda FV:[Chemotherapy in areas of persistent malaria transmission and resistence of Plasmodium falciparum to chloroquine in Panama]. Bol Oficina Sanit Panam 1975,78(6):531-541.
4. Postovit LM, Seftor EA, Seftor RE, Hendrix MJ:Influence of the microenvironment on melanoma cell fate determination and phenotype. Cancer Res 2006, 66(16):7833-7836.
5. Lee LM, Seftor EA, Bonde G, Cornell RA, Hendrix MJ:The fate of human malignant melanoma cells transplanted into zebrafish embryos:assessment of migration and cell division in the absence of tumor formation. Dev Dyn 2005,233(4):1560-1570.
6. Kulesa PM, Kasemeier-Kulesa JC, Teddy JM, Margaryan NV, Seftor EA, Seftor RE, Hendrix MJ:Reprogramming metastatic melanoma cells to assume a neural crest cell-like phenotype in an embryonic microenvironment. Proc Natl Acad Sci U S A 2006,103(10):3752-3757.
7. Rosato A, Zoso A, Dalla Santa S, Milan G, Del Bianco P, De Salvo GL, Zanovello P: Predicting tumor outcome following cancer vaccination by monitoring quantitative and qualitative CD8+T cell parameters. JImmunol 2006,176(3):1999-2006.
8. Mayo JG:Biologic characterization of the subcutaneously implanted Lewis lung tumor. Cancer Chemother Rep 21972,3(1):325-330.
9. Yu F, Jiao Y, Zhu Y, Wang Y, Zhu J, Cui X, Liu Y, He Y, Park EY, Zhang H et al: MicroRNA 34c gene down-regulation via DNA methylation promotes self-renewal and epithelial-mesenchymal transition in breast tumor-initiating cells. JBiol Chem 2012,287(1):465-473.
10. Ning N, Pan Q, Zheng F, Teitz-Tennenbaum S, Egenti M, Yet J, Li M, Ginestier C, Wicha MS, Moyer JS et al: Cancer stem cell vaccination confers significant antitumor immunity. Cancer Res 2012,72(7):1853-1864.
11. Al-Hajj M, Becker MW, Wicha M, Weissman I, Clarke MF:Therapeutic implications of cancer stem cells. Curr Opin Genet Dev 2004,14(1):43-47.
12. Jordan CT:Targeting the most critical cells:approaching leukemia therapy as a problem in stem cell biology. Nat Clin Pract Oncol 2005,2(5):224-225.
13. Copland M, Fraser AR, Harrison SJ, Holyoake TL:Targeting the silent minority: emerging immunotherapeutic strategies for eradication of malignant stem cells in chronic myeloid leukaemia. Cancer Immunol Immunother 2005,54(4):297-306.
1. Triolo VA:Nineteenth Century Foundations of Cancer Research Advances in Tumor Pathology, Nomenclature, and Theories of Oncogenesis. Cancer Res 1965, 25:75-106.
2. Brewer BG, Mitchell RA, Harandi A, Eaton JW:Embryonic vaccines against cancer: an early history. Exp Mol Pathol 2009,86(3):192-197.
3. Vogelstein B, Kinzler KW:Cancer genes and the pathways they control. Nat Med 2004,10(8):789-799.
4. Petruzzelli GJ:The biology of tumor invasion, angiogenesis and lymph node metastasis. ORL JOtorhinolaryngol Relat Spec 2000,62(4):178-185.
5. Daftary GS, Taylor HS:Molecular markers of implantation:clinical implications. Curr Opin Obstet Gynecol 2001,13(3):269-274.
6. Paria BC, Song H, Dey SK:Implantation:molecular basis of embryo-uterine dialogue. Int J Dev Biol 2001,45(3):597-605.
7. Stoika RS, Panchuk RR, Stoika BR:[The similarities and the differences of embryogenesis and carcinogenesis]. Ontogenez 2004,35(2):85-90.
8. Chen H, Egan JO, Chiu JF:Regulation and activities of alpha-fetoprotein. Crit Rev Eukaryot Gene Expr 1997,7(1-2):11-41.
9. Ryu JS, Lee HJ, Cho JH, Han HS, Lee HL:The implication of elevated carcinoembryonic antigen level in pleural fluid of patients with non-malignant pleural effusion. Respirology 2003,8(4):487-491.
10. Ambrosini G, Adida C, Altieri DC:A novel anti-apoptosis gene, survivin, expressed in cancer and lymphoma. Nat Med 1997,3(8):917-921.
11. Megliorino R, Shi FD, Peng XX, Wang X, Chan EK, Tan EM, Zhang JY:Autoimmune response to anti-apoptotic protein survivin and its association with antibodies to p53 and c-myc in cancer detection. Cancer Detect Prev 2005,29(3):241-248.
12. Engelen MJ, de Bruijn HW, Hollema H, ten Hoor KA, Willemse PH, Aalders JG, van der Zee AG:Serum CA 125, carcinoembryonic antigen, and CA 19-9 as tumor markers in borderline ovarian tumors. Gynecol Oncol 2000,78(1):16-20.
13. Vukotic V, Cerovic S, Kozomara M, Lazic M:The predictive value of PSA in diagnosis of prostate cancer in non screened population. Acta Chir Iugosl 2005, 52(4):81-87.
14. Sarandakou A, Malamitsi-Puchner A, Protonotariou E, Rigopoulou U, Papagianni B, Creatsas G:Indicative markers of cell proliferation and apoptosis during the perinatal period. Am J Perinatol 2003,20(6):283-288.
15. Wilczynski JR:Cancer and pregnancy share similar mechanisms of immunological escape. Chemotherapy 2006,52(3):107-110.
16. Misirlioglu M, Page GP, Sagirkaya H, Kaya A, Parrish JJ, First NL, Memili E: Dynamics of global transcriptome in bovine matured oocytes and preimplantation embryos. Proc Natl Acad Sci USA 2006,103(50):18905-18910.
17. Morita R, Hirohashi Y, Torigoe T:Mechanism of immune escape of cancer and its regulation. Rinsho Ketsueki 2009,50(5):375-380.
18. Xie K, Abbruzzese JL:Developmental biology informs cancer:the emerging role of the hedgehog signaling pathway in upper gastrointestinal cancers. Cancer Cell 2003,4(4):245-247.
19. Wu F, Stutzman A, Mo YY:Notch signaling and its role in breast cancer. Front Biosci 2007,12:4370-4383.
20. Serr DM, Padeh B, Mashiach S, Shaki R:Chromosomal studies in tumors of embryonic origin. Obstet Gynecol 1969,33(3):324-332.
21. Hellstrom 1, Pierce GE, Hellstrom KE:Human tumor-specific antigens. Surgery 1969, 65(6):984-989.
22. Buttle GA, Eperon J, Menzies DN:Induced Tumour Resistance in Rats. Lancet 1964, 2(7349):12-14.
23. LeMevel BP, Wells SA, Jr.:Foetal antigens cross-reactive with tumour-specific transplantation antigens. Nat New Biol 1973,244(136):183-184.
24. Sikora K, Stern P, Lennox E:Immunoprotection by embryonal carcinoma cells for methylcholanthrene-induced murine sarcomas. Nature 1977,269(5631):813-815.
25. Li Y, Zeng H, Xu RH, Liu B, Li Z:Vaccination with human pluripotent stem cells generates a broad spectrum of immunological and clinical responses against colon cancer. Stem Cells 2009,27(12):3103-3111.
26. Yaddanapudi K, Mitchell RA, Putty K, Willer S, Sharma RK, Yan J, Bodduluri H, Eaton JW:Vaccination with embryonic stem cells protects against lung cancer:is a broad-spectrum prophylactic vaccine against cancer possible? PLoS ONE 2012, 7(7):e42289.
27. Rosato A, Zoso A, Dalla Santa S, Milan G, Del Bianco P, De Salvo GL, Zanovello P: Predicting tumor outcome following cancer vaccination by monitoring quantitative and qualitative CD8+ T cell parameters. J Immunol 2006,176(3):1999-2006.
28. van Herck H, Baumans V, Brandt CJ, Hesp AP, Sturkenboom JH, van Lith HA, van Tintelen G, Beynen AC:Orbital sinus blood sampling in rats as performed by different animal technicians:the influence of technique and expertise. Lab Anim 1998,32(4):377-386.
29. Mayo JG:Biologic characterization of the subcutaneously implanted Lewis lung tumor. Cancer Chemother Rep 21972,3(1):325-330.
30. Al-Hajj M, Clarke MF:Self-renewal and solid tumor stem cells. Oncogene 2004, 23(43):7274-7282.
31. Stonehill EH, Bendich A:Retrogenetic expression:the reappearance of embryonal antigens in cancer cells. Nature 1970,228(5269):370-372.
32. Baldwin RW, Glaves D, Vose BM:Embryonic antigen expression in chemically induced rat hepatomas and sarcomas. Int J Cancer 1972,10(2):233-243.
33. Baldwin RW, Glaves D, Pimm MV, Vose BM:Tumour specific and embryonic antigen expression of chemically induced rat tumours. Ann Inst Pasteur (Paris) 1972, 122(4):715-728.
34. Gold P, Freedman SO:Specific carcinoembryonic antigens of the human digestive system. J Exp Med 1965,122(3):467-481.
35. Gold P, Freedman SO:Demonstration of Tumor-Specific Antigens in Human Colonic Carcinomata by Immunological Tolerance and Absorption Techniques. J Exp Med 1965,121:439-462.
36. Horna P, Sotomayor EM:Cellular and molecular mechanisms of tumor-induced T-cell tolerance. Curr Cancer Drug Targets 2007,7(1):41-53.
37. Cohen J:Public health. HPV's peculiarities, from infection to disease. Science 2005, 308(5722):619.
38. Klavins JV, Mesa-Tejada R, Weiss M:Human carcinoma antigens cross reacting with anti-embryonic antibodies. Nat New Biol 1971,234(48):153-154.
39. Begley J, Ribas A:Targeted therapies to improve tumor immunotherapy. Clin Cancer Res 2008,14(14):4385-4391.
40. Rosenberg SA, Yang JC, Restifo NP:Cancer immunotherapy:moving beyond current vaccines. Nat Med 2004,10(9):909-915.
41. Ikeda H, Chamoto K, Tsuji T, Suzuki Y, Wakita D, Takeshima T, Nishimura T:The critical role of type-1 innate and acquired immunity in tumor immunotherapy. Cancer Sci 2004,95(9):697-703.
42. Teitz-Tennenbaum S, Wicha MS, Chang AE, Li Q:Targeting cancer stem cells via dendritic-cell vaccination. Oncoimmunology 2012, 1(8):1401-1403.
43. Galluzzi L, Senovilla L, Vacchelli E, Eggermont A, Fridman WH, Galon J, Sautes-Fridman C, Tartour E, Zitvogel L, Kroemer G:Trial watch:Dendritic cell-based interventions for cancer therapy. Oncoimmunology 2012,1(7):1111-1134.
44. Xiong G, Husseiny MI, Song L, Erdreich-Epstein A, Shackleford GM, Seeger RC, Jackel D, Hensel M, Metelitsa LS:Novel cancer vaccine based on genes of Salmonella pathogenicity island 2. Int J Cancer 2010,126(11):2622-2634.
45. Johnsen A, France J, Sy MS, Harding CV:Down-regulation of the transporter for antigen presentation, proteasome subunits, and class I major histocompatibility complex in tumor cell lines. Cancer Res 1998,58(16):3660-3667.
46. Errington F, Bateman A, Kottke T, Thompson J, Harrington K, Merrick A, Hatfield P, Selby P, Vile R, Melcher A:Allogeneic tumor cells expressing fusogenic membrane glycoproteins as a platform for clinical cancer immunotherapy. din Cancer Res 2006,12(4):1333-1341.
1. Chouaib S, El Hage F, Benlalam H, Mami-Chouaib F:[Immunotherapy of cancer: promise and reality]. Med Sci (Paris) 2006,22(8-9):755-759.
2. Wiemann B, Starnes CO:Coley's toxins, tumor necrosis factor and cancer research: a historical perspective. Pharmacol Ther 1994,64(3):529-564.
3. Nauts HC, Fowler GA, Bogatko FH:A review of the influence of bacterial infection and of bacterial products (Coley's toxins) on malignant tumors in man; a critical analysis of 30 inoperable cases treated by Coley's mixed toxins, in which diagnosis was confirmed by microscopic examination selected for special study. Acta Med Scand Suppl 1953,276:1-103.
4. Croci DO, Salatino M:Tumor immune escape mechanisms that operate during metastasis. Curr Pharm Biotechnol 2011,12(11):1923-1936.
5. Whiteside TL, Mandapathil M, Szczepanski M, Szajnik M:Mechanisms of tumor escape from the immune system:adenosine-producing Treg, exosomes and tumor-associated TLRs. Bull Cancer 2011,98(2):E25-31.
6. Xiong G, Husseiny MI, Song L, Erdreich-Epstein A, Shackleford GM, Seeger RC, Jackel D, Hensel M, Metelitsa LS:Novel cancer vaccine based on genes of Salmonella pathogenicity island 2. Int JCancer 2010,126(11):2622-2634.
7. Yang L, Pang Y, Moses HL:TGF-beta and immune cells:an important regulatory axis in the tumor microenvironment and progression. Trends Immunol 2010, 31(6):220-227.
8. Gutzmer R, Li W, Sutterwala S, Lemos MP, Elizalde JI, Urtishak SL, Behrens EM, Rivers PM, Schlienger K, Laufer TM et al: A tumor-associated glycoprotein that blocks MHC class Ⅱ-dependent antigen presentation by dendritic cells. J Immunol 2004,173(2):1023-1032.
9. Konishi J, Yamazaki K, Azuma M, Kinoshita I, Dosaka-Akita H, Nishimura M:B7-H1 expression on non-small cell lung cancer cells and its relationship with tumor-infiltrating lymphocytes and their PD-1 expression. Clin Cancer Res 2004, 10(15):5094-5100.
10. Chaux P, Favre N, Bonnotte B, Moutet M, Martin M, Martin F:Tumor-infiltrating dendritic cells are defective in their antigen-presenting function and inducible B7 expression. A role in the immune tolerance to antigenic tumors. Adv Exp Med Biol 1997,417:525-528.
11. Ikeda H, Chamoto K, Tsuji T, Suzuki Y, Wakita D, Takeshima T, Nishimura T:The critical role of type-1 innate and acquired immunity in tumor immunotherapy. Cancer Sci 2004,95(9):697-703.
12. Yoshino S, Yoshimura K, Suzuki N, Iida M, Yoshida S, Maeda Y, Maeda K, Hazama S, Oka M:[Immunoregulatory effects of PSK on the Thl/Th2 balance and regulatory T-cells in patients with colorectal cancer]. Gan To Kagaku Ryoho 2010, 37(12):2234-2236.
13. Leitch J, Fraser K, Lane C, Putzu K, Adema GJ, Zhang QJ, Jefferies WA, Bramson JL, Wan Y:CTL-dependent and -independent antitumor immunity is determined by the tumor not the vaccine. J Immunol 2004,172(9):5200-5205.
14. Qin H, Zhou C, Wang D, Ma W, Liang X, Lin C, Zhang Y, Zhang S:Specific antitumor immune response induced by a novel DNA vaccine composed of multiple CTL and T helper cell epitopes of prostate cancer associated antigens.Immunol Lett 2005,99(1):85-93.
15. Rousalova I, Krepela E:Granzyme B-induced apoptosis in cancer cells and its regulation (review). Int J Oncol 2010,37(6):1361-1378.
16. Rosenblatt J, Vasir B, Uhl L, Blotta S, Macnamara C, Somaiya P, Wu Z, Joyce R, Levine JD, Dombagoda D et al: Vaccination with dendritic cell/tumor fusion cells results in cellular and humoral antitumor immune responses in patients with multiple myeloma. Blood 2011,117(2):393-402.
17. Li Y, Liang J, Liu WB, Xu XQ, Zhao YR:[Influence of chemotherapy on Thl/Th2 cytokine switching in stomach cancer patients]. Zhonghua Zhong Liu Za Zhi 2004, 26(12):732-734.
18. Chen M, Hao X, Zhang Y, Lu B, Wu Y, Xu Y, Chen J, Yu W:[The clinical significance of detection of Th1/Th2 cell cytokines in lung cancer]. Zhongguo Fei Ai Za Zhi 2004, 7(3):214-217.
19. Yamazaki K, Yano T, Kameyama T, Suemitsu R, Yoshino I, Sugio K:Clinical significance of serum TH1/TH2 cytokines in patients with pulmonary adenocarcinoma. Surgery 2002,131(1 Suppl):S236-241.
20. Hirschowitz EA, Yannelli JR:Immunotherapy for lung cancer. Proc Am Thorac Soc 2009,6(2):224-232.
21. Whiteside TL:Immune suppression in cancer:effects on immune cells, mechanisms and future therapeutic intervention. Semin Cancer Biol 2006,16(1):3-15.
22. Sioud M:Does our current understanding of immune tolerance, autoimmunity, and immunosuppressive mechanisms facilitate the design of efficient cancer vaccines? Scand J Immunol 2009,70(6):516-525.
23. Johnsen A, France J, Sy MS, Harding CV:Down-regulation of the transporter for antigen presentation, proteasome subunits, and class I major histocompatibility complex in tumor cell lines. Cancer Res 1998,58(16):3660-3667.
24. Rosato A, Zoso A, Dalla Santa S, Milan G, Del Bianco P, De Salvo GL, Zanovello P: Predicting tumor outcome following cancer vaccination by monitoring quantitative and qualitative CD8+T cell parameters. J Immunol 2006,176(3):1999-2006.
25. van Herck H, Baumans V, Brandt CJ, Hesp AP, Sturkenboom JH, van Lith HA, van Tintelen G, Beynen AC:Orbital sinus blood sampling in rats as performed by different animal technicians:the influence of technique and expertise. Lab Anim 1998,32(4):377-386.
26. Ni C, Huang J:Dynamic regulation of cancer stem cells and clinical challenges. Clin Transl Oncol 2013,15(4):253-258.
27. Magee JA, Piskounova E, Morrison SJ:Cancer stem cells:impact, heterogeneity, and uncertainty. Cancer Cell 2012,21(3):283-296.
28. Al-Hajj M, Wicha MS, Benito-Hernandez A, Morrison SJ, Clarke MF:Prospective identification of tumorigenic breast cancer cells. Proc Natl Acad Sci U S A 2003, 100(7):3983-3988.
29. Li C, Lee CJ, Simeone DM:Identification of human pancreatic cancer stem cells. Methods Mol Biol 2009,568:161-173.
30. O'Brien CA, Pollett A, Gallinger S, Dick JE:A human colon cancer cell capable of initiating tumour growth in immunodeficient mice. Nature 2007,445(7123):106-110.
31. Chu P, Clanton DJ, Snipas TS, Lee J, Mitchell E, Nguyen ML, Hare E, Peach RJ: Characterization of a subpopulation of colon cancer cells with stem cell-like properties. Int J Cancer 2009,124(6):1312-1321.
32. Murase M, Kano M, Tsukahara T, Takahashi A, Torigoe T, Kawaguchi S, Kimura S, Wada T, Uchihashi Y, Kondo T et al: Side population cells have the characteristics of cancer stem-like cells/cancer-initiating cells in bone sarcomas. Br J Cancer 2009, 101(8):1425-1432.
33. Wang S, Garcia AJ, Wu M, Lawson DA, Witte ON, Wu H:Pten deletion leads to the expansion of a prostatic stem/progenitor cell subpopulation and tumor initiation. Proc Natl Acad Sci U S A 2006,103(5):1480-1485.
34. Chaffer CL, Brueckmann I, Scheel C, Kaestli AJ, Wiggins PA, Rodrigues LO, Brooks M, Reinhardt F, Su Y, Polyak K et al: Normal and neoplastic nonstem cells can spontaneously convert to a stem-like state. Proc Natl Acad Sci U S A 2011, 108(19):7950-7955.
35. Chang HH, Chen BY, Wu CY, Tsao ZJ, Chen YY, Chang CP, Yang CR, Lin DP: Hedgehog overexpression leads to the formation of prostate cancer stem cells with metastatic property irrespective of androgen receptor expression in the mouse model. J Biomed Sci 2011,18:6.
36. Sell S:Stem cell origin of cancer and differentiation therapy. Crit Rev Oncol Hematol 2004,51(1):1-28.
37. Sell S:Leukemia:stem cells, maturation arrest, and differentiation therapy. Stem Cell Rev 2005,1(3):197-205.
38. Sell S:Potential gene therapy strategies for cancer stem cells. Curr Gene Ther 2006, 6(5):579-591.
39. Sell S:Cancer and stem cell signaling:a guide to preventive and therapeutic strategies for cancer stem cells. Stem Cell Rev 2007,3(1):1-6.
40. A1-Hajj M, Becker MW, Wicha M, Weissman Ⅰ, Clarke MF:Therapeutic implications of cancer stem cells. Curr Opin Genet Dev 2004,14(1):43-47.
41. Jordan CT:Targeting the most critical cells:approaching leukemia therapy as a problem in stem cell biology. Nat Clin Pract Oncol 2005,2(5):224-225.
42. Copland M, Fraser AR, Harrison SJ, Holyoake TL:Targeting the silent minority: emerging immunotherapeutic strategies for eradication of malignant stem cells in chronic myeloid leukaemia. Cancer Immunol Immunother 2005,54(4):297-306.
43. Dong W, Du J, Shen H, Gao D, Li Z, Wang G, Mu X, Liu Q:Administration of embryonic stem cells generates effective antitumor immunity in mice with minor and heavy tumor load. Cancer Immunol Immunother 2010,59(11):1697-1705.
44. Drukker M, Katchman H, Katz G, Even-Tov Friedman S, Shezen E, Hornstein E, Mandelboim O, Reisner Y, Benvenisty N:Human embryonic stem cells and their differentiated derivatives are less susceptible to immune rejection than adult cells. Stem Cells 2006,24(2):221-229.
45. Drukker M, Benvenisty N:The immunogenicity of human embryonic stem-derived cells. Trends Biotechnol 2004,22(3):136-141.
46. Fairchild PJ, Cartland S, Nolan KF, Waldmann H:Embryonic stem cells and the challenge of transplantation tolerance. Trends Immunol 2004,25(9):465-470.
47. Boyd AS, Higashi Y, Wood KJ:Transplanting stem cells:potential targets for immune attack. Modulating the immune response against embryonic stem cell transplantation. Adv Drug Deliv Rev 2005,57(13):1944-1969.
48. Begley J, Ribas A:Targeted therapies to improve tumor immunotherapy. Clin Cancer Res 2008,14(14):4385-4391.
49. Rosenberg SA, Yang JC, Restifo NP:Cancer immunotherapy:moving beyond current vaccines. Nat Med 2004,10(9):909-915.
50. Shurin MR, Lu L, Kalinski P, Stewart-Akers AM, Lotze MT:Th1/Th2 balance in cancer, transplantation and pregnancy. Springer Semin Immunopathol 1999, 21(3):339-359.
51. Koestenbauer S, Zech NH, Juch H, Vanderzwalmen P, Schoonjans L, Dohr G: Embryonic stem cells:similarities and differences between human and murine embryonic stem cells. Am JReprod Immunol 2006,55(3):169-180.
52. Li Y, Zeng H, Xu RH, Liu B, Li Z:Vaccination with human pluripotent stem cells generates a broad spectrum of immunological and clinical responses against colon cancer. Stem Cells 2009,27(12):3103-3111.
2. Available at http://www.cancer.gov/cancertopics/types/lung. NC Institute 2009.
3. Bunn PA, Jr., Thatcher N:Systemic treatment for advanced (stage Ⅲb/Ⅳ) non-small cell lung cancer:more treatment options; more things to consider. Conclusion. Oncologist 2008,13 Suppl 1:37-46.
4. Hirschowitz EA, Yannelli JR:Immunotherapy for lung cancer. Proc Am Thorac Soc 2009,6(2):224-232.
5. Vanneman M, Dranoff G:Combining immunotherapy and targeted therapies in cancer treatment. Nat Rev Cancer 2012,12(4):237-251.
6. Xiong G, Husseiny MI, Song L, Erdreich-Epstein A, Shackleford GM, Seeger RC, Jacket D, Hensel M, Metelitsa LS:Novel cancer vaccine based on genes of Salmonella pathogenicity island 2. Int J Cancer 2010,126(11):2622-2634.
7. Murray MJ, Lessey BA:Embryo implantation and tumor metastasis:common pathways of invasion and angiogenesis. Semin Reprod Endocrinol 1999, 17(3):275-290.
8. Pardal R, Clarke MF, Morrison SJ:Applying the principles of stem-cell biology to cancer. Nat Rev Cancer 2003,3(12):895-902.
9. Cabanillas R, Llorente JL:The Stem Cell Network model:clinical implications in cancer. Eur Arch Otorhinolaryngol 2009,266(2):161-170.
10. Reya T, Clevers H:Wnt signalling in stem cells and cancer. Nature 2005, 434(7035):843-850.
11. Li L, Neaves WB:Normal stem cells and cancer stem cells:the niche matters. Cancer Res 2006,66(9):4553-4557.
12. Huang EH, Heidt DG, Li CW, Simeone DM:Cancer stem cells:a new paradigm for understanding tumor progression and therapeutic resistance. Surgery 2007, 141(4):415-419.
13. Okamoto OK:Cancer stem cell genomics:the quest for early markers of malignant progression. Expert Rev Mol Diagn 2009,9(6):545-554.
14. Rubio D, Garcia-Castro J, Martin MC, de la Fuente R, Cigudosa JC, Lloyd AC, Bernad A:Spontaneous human adult stem cell transformation. Cancer Res 2005, 65(8):3035-3039.
15. Al-Hajj M, Wicha MS, Benito-Hernandez A, Morrison SJ, Clarke MF:Prospective identification of tumorigenic breast cancer cells. Proc Natl Acad Sci U S A 2003, 100(7):3983-3988.
16. Li C, Lee CJ, Simeone DM:Identification of human pancreatic cancer stem cells. Methods Mol Biol 2009,568:161-173.
17. O'Brien CA, Pollett A, Gallinger S, Dick JE:A human colon cancer cell capable of initiating tumour growth in immunodeficient mice. Nature 2007,445(7123):106-110.
18. Chu R, Clanton DJ, Snipas TS, Lee J, Mitchell E, Nguyen ML, Hare E, Peach RJ: Characterization of a subpopulation of colon cancer cells with stem cell-like properties. Int J Cancer 2009,124(6):1312-1321.
19. Murase M, Kano M, Tsukahara T, Takahashi A, Torigoe T, Kawaguchi S, Kimura S, Wada T, Uchihashi Y, Kondo T et al: Side population cells have the characteristics of cancer stem-like cells/cancer-initiating cells in bone sarcomas. Br J Cancer 2009, 101(8):1425-1432.
20. Wang S, Garcia AJ, Wu M, Lawson DA, Witte ON, Wu H:Pten deletion leads to the expansion of a prostatic stem/progenitor cell subpopulation and tumor initiation. Proc Natl Acad Sci USA 2006,103(5):1480-1485.
21. Adikrisna R, Tanaka S, Muramatsu S, Aihara A, Ban D, Ochiai T, Irie T, Kudo A, Nakamura N, Yamaoka S et al: Identification of pancreatic cancer stem cells and selective toxicity of chemotherapeutic agents. Gastroenterology 2012, 143(1):234-245 e237.
22. Zhu J, He J, Liu Y, Simeone DM, Lubman DM:Identification of glycoprotein markers for pancreatic cancer CD24+CD44+ stem-like cells using nano-LC-MS/MS and tissue microarray. JProteome Res 2012,11(4):2272-2281.
23. de Sousa EMF, Guessous I, Vermeulen L, Medema JP:[Cancer stem cells and future therapeutic implications]. Rev Med Suisse 2011,7(289):774-777.
24. Jordan CT:Targeting the most critical cells:approaching leukemia therapy as a problem in stem cell biology. Nat Clin Pract Oncol 2005,2(5):224-225.
25. Copland M, Fraser AR, Harrison SJ, Holyoake TL:Targeting the silent minority: emerging immunotherapeutic strategies for eradication of malignant stem cells in chronic myeloid leukaemia. Cancer Immunol Immunother 2005,54(4):297-306.
26. Boyd AS, Higashi Y, Wood KJ:Transplanting stem cells:potential targets for immune attack. Modulating the immune response against embryonic stem cell transplantation. Adv Drug Deliv Rev 2005,57(13):1944-1969.
27. Drukker M, Katchman H, Katz G, Even-Tov Friedman S, Shezen E, Hornstein E, Mandelboim O, Reisner Y, Benvenisty N:Human embryonic stem cells and their differentiated derivatives are less susceptible to immune rejection than adult cells. Stem Cells 2006,24(2):221-229.
28. Li Y, Zeng H, Xu RH, Liu B, Li Z:Vaccination with human pluripotent stem cells generates a broad spectrum of immunological and clinical responses against colon cancer. Stem Cells 2009,27(12):3103-3111.
29. Dong W, Du J, Shen H, Gao D, Li Z, Wang G, Mu X, Liu Q:Administration of embryonic stem cells generates effective antitumor immunity in mice with minor and heavy tumor load. Cancer Immunol Immunother 2010,59(11):1697-1705.
30. Brewer BG, Mitchell RA, Harandi A, Eaton JW:Embryonic vaccines against cancer: an early history. Exp Mol Pathol 2009,86(3):192-197.
31. LeMevel BP, Wells SA, Jr.:Foetal antigens cross-reactive with tumour-specific transplantation antigens. Nat New Biol 1973,244(136):183-184.
32. Sikora K, Stern P, Lennox E:Immunoprotection by embryonal carcinoma cells for methylcholanthrene-induced murine sarcomas. Nature 1977,269(5631):813-815.
33. Yaddanapudi K, Mitchell RA, Putty K, Willer S, Sharma RK, Yan J, Bodduluri H, Eaton JW:Vaccination with embryonic stem cells protects against lung cancer:is a broad-spectrum prophylactic vaccine against cancer possible? PLoS ONE 2012, 7(7):e42289.
34. Giuffrida D, Rogers IM, Nagy A, Calogero AE, Brown TJ, Casper RF:Human embryonic stem cells secrete soluble factors that inhibit cancer cell growth. Cell Pwlif 2009,42(6):788-798.
35. Raof NA, Raja WK, Castracane J, Xie Y:Bioengineering embryonic stem cell microenvironments for exploring inhibitory effects on metastatic breast cancer cells. Biomaterials 2011,32(17):4130-4139.
36. Postovit LM, Margaryan NV, Seftor EA, Kirschmann DA, Lipavsky A, Wheaton WW, Abbott DE, Seftor RE, Hendrix MJ:Human embryonic stem cell microenvironment suppresses the tumorigenic phenotype of aggressive cancer cells. Proc Natl Acad Sci USA 2008,105(11):4329-4334.
37. Mohib K, Wang L:Differentiation and characterization of dendritic cells from human embryonic stem cells. Curr Protoc Immunol 2012, Chapter 22:Unit 22F 11.
38. Ohlsson LB, Varas L, Kjellman C, Edvardsen K, Lindvall M:Mesenchymal progenitor cell-mediated inhibition of tumor growth in vivo and in vitro in gelatin matrix. Exp Mol Pathol 2003,75(3):248-255.
39. Woll PS, Grzywacz B, Tian X, Marcus RK, Knorr DA, Verneris MR, Kaufman DS: Human embryonic stem cells differentiate into a homogeneous population of natural killer cells with potent in vivo antitumor activity. Blood 2009, 113(24):6094-6101.
40. Yan S:New Milestone in the development of Clinical Oncology Molecular Targeted Therapy. Anti-tumor Pharmacy 2011,1(1):1-5.
41. Keller G:Embryonic stem cell differentiation:emergence of a new era in biology and medicine. Genes Dev 2005,19(10):1129-1155.
42. Postovit LM, Seftor EA, Seftor RE, Hendrix MJ:A three-dimensional model to study the epigenetic effects induced by the microenvironment of human embryonic stem cells. Stem Cells 2006,24(3):501-505.
43. Miller SJ, Lavker RM, Sun TT:Interpreting epithelial cancer biology in the context of stem cells:tumor properties and therapeutic implications. Biochim Biophys Acta 2005,1756(1):25-52.
44. Postovit LM, Seftor EA, Seftor RE, Hendrix MJ:Influence of the microenvironment on melanoma cell fate determination and phenotype. Cancer Res 2006, 66(16):7833-7836.
45. Lee LM, Seftor EA, Bonde G, Cornell RA, Hendrix MJ:The fate of human malignant melanoma cells transplanted into zebrafish embryos:assessment of migration and cell division in the absence of tumor formation. Dev Dyn 2005,233(4):1560-1570.
46. Kulesa PM, Kasemeier-Kulesa JC, Teddy JM, Margaryan NV, Seftor EA, Seftor RE, Hendrix MJ:Reprogramming metastatic melanoma cells to assume a neural crest cell-like phenotype in an embryonic microenvironment. Proc Natl Acad Sci U S A 2006,103(10):3752-3757.
47.常艳,魏伟：细胞共培养及其应用的研究进展.中国临床药理学与治疗学2009,14(7)：3.
48.罗云,孙桂波,秦蒙,姚帆,孙晓波：细胞共培养技术在医药研究中的应用.中国中药杂志2012,37(22)：5.
49. Lowe SW, Lin AW:Apoptosis in cancer. Carcinogenesis 2000,21(3):485-495.
50. Bissell MJ, Labarge MA:Context, tissue plasticity, and cancer:are tumor stem cells also regulated by the microenvironment? Cancer Cell 2005,7(1):17-23.
51. Whiteside EJ, Jackson MM, Herington AC, Edwards DR, Harvey MB:Matrix metalloproteinase-9 and tissue inhibitor of metalloproteinase-3 are key regulators of extracellular matrix degradation by mouse embryos. Biol Reprod 2001, 64(5):1331-1337.
52. Yasuda M, Tanaka Y, Tamura M, Fujii K, Sugaya M, So T, Takenoyama M, Yasumoto K.: Stimulation of betal integrin down-regulates ICAM-1 expression and ICAM-1-dependent adhesion of lung cancer cells through focal adhesion kinase. Cancer Res 2001,61(5):2022-2030.
53. Liotta LA:Cancer cell invasion and metastasis. Sci Am 1992,266(2):54-59,62-53.
54. Glennie S, Soeiro Ⅰ, Dyson PJ, Lam EW, Dazzi F:Bone marrow mesenchymal stem cells induce division arrest anergy of activated T cells. Blood 2005, 105(7):2821-2827.
55. Rasmusson Ⅰ, Ringden O, Sundberg B, Le Blanc K:Mesenchymal stem cells inhibit lymphocyte proliferation by mitogens and alloantigens by different mechanisms. Exp Cell Res 2005,305(1):33-41.
56. Sato K, Ozaki K, Oh I, Meguro A, Hatanaka K, Nagai T, Muroi K, Ozawa K:Nitric oxide plays a critical role in suppression of T-cell proliferation by mesenchymal stem cells. Blood 2007,109(1):228-234.
57. Meisel R, Zibert A, Laryea M, Gobel U, Daubener W, Dilloo D:Human bone marrow stromal cells inhibit allogeneic T-cell responses by indoleamine 2,3-dioxygenase-mediated tryptophan degradation. Blood 2004,103(12):4619-4621.
58. Lu YR, Yuan Y, Wang XJ, Wei LL, Chen YN, Cong C, Li SF, Long D, Tan WD, Mao YQ et al:The growth inhibitory effect of mesenchymal stem cells on tumor cells in vitro and in vivo. Cancer Biol Ther 2008,7(2):245-251.
1. Available at http://www.cancer.gov/cancertopics/types/lung. NC Institute 2009.
2. Swisher SG, Roth JA:Clinical update of Ad-p53 gene therapy for lung cancer. Surg Oncol Clin NAm 2002,11(3):521-535.
3. Balda FV:[Chemotherapy in areas of persistent malaria transmission and resistence of Plasmodium falciparum to chloroquine in Panama]. Bol Oficina Sanit Panam 1975,78(6):531-541.
4. Postovit LM, Seftor EA, Seftor RE, Hendrix MJ:Influence of the microenvironment on melanoma cell fate determination and phenotype. Cancer Res 2006, 66(16):7833-7836.
5. Lee LM, Seftor EA, Bonde G, Cornell RA, Hendrix MJ:The fate of human malignant melanoma cells transplanted into zebrafish embryos:assessment of migration and cell division in the absence of tumor formation. Dev Dyn 2005,233(4):1560-1570.
6. Kulesa PM, Kasemeier-Kulesa JC, Teddy JM, Margaryan NV, Seftor EA, Seftor RE, Hendrix MJ:Reprogramming metastatic melanoma cells to assume a neural crest cell-like phenotype in an embryonic microenvironment. Proc Natl Acad Sci U S A 2006,103(10):3752-3757.
7. Rosato A, Zoso A, Dalla Santa S, Milan G, Del Bianco P, De Salvo GL, Zanovello P: Predicting tumor outcome following cancer vaccination by monitoring quantitative and qualitative CD8+T cell parameters. JImmunol 2006,176(3):1999-2006.
8. Mayo JG:Biologic characterization of the subcutaneously implanted Lewis lung tumor. Cancer Chemother Rep 21972,3(1):325-330.
9. Yu F, Jiao Y, Zhu Y, Wang Y, Zhu J, Cui X, Liu Y, He Y, Park EY, Zhang H et al: MicroRNA 34c gene down-regulation via DNA methylation promotes self-renewal and epithelial-mesenchymal transition in breast tumor-initiating cells. JBiol Chem 2012,287(1):465-473.
10. Ning N, Pan Q, Zheng F, Teitz-Tennenbaum S, Egenti M, Yet J, Li M, Ginestier C, Wicha MS, Moyer JS et al: Cancer stem cell vaccination confers significant antitumor immunity. Cancer Res 2012,72(7):1853-1864.
11. Al-Hajj M, Becker MW, Wicha M, Weissman I, Clarke MF:Therapeutic implications of cancer stem cells. Curr Opin Genet Dev 2004,14(1):43-47.
12. Jordan CT:Targeting the most critical cells:approaching leukemia therapy as a problem in stem cell biology. Nat Clin Pract Oncol 2005,2(5):224-225.
13. Copland M, Fraser AR, Harrison SJ, Holyoake TL:Targeting the silent minority: emerging immunotherapeutic strategies for eradication of malignant stem cells in chronic myeloid leukaemia. Cancer Immunol Immunother 2005,54(4):297-306.
1. Triolo VA:Nineteenth Century Foundations of Cancer Research Advances in Tumor Pathology, Nomenclature, and Theories of Oncogenesis. Cancer Res 1965, 25:75-106.
2. Brewer BG, Mitchell RA, Harandi A, Eaton JW:Embryonic vaccines against cancer: an early history. Exp Mol Pathol 2009,86(3):192-197.
3. Vogelstein B, Kinzler KW:Cancer genes and the pathways they control. Nat Med 2004,10(8):789-799.
4. Petruzzelli GJ:The biology of tumor invasion, angiogenesis and lymph node metastasis. ORL JOtorhinolaryngol Relat Spec 2000,62(4):178-185.
5. Daftary GS, Taylor HS:Molecular markers of implantation:clinical implications. Curr Opin Obstet Gynecol 2001,13(3):269-274.
6. Paria BC, Song H, Dey SK:Implantation:molecular basis of embryo-uterine dialogue. Int J Dev Biol 2001,45(3):597-605.
7. Stoika RS, Panchuk RR, Stoika BR:[The similarities and the differences of embryogenesis and carcinogenesis]. Ontogenez 2004,35(2):85-90.
8. Chen H, Egan JO, Chiu JF:Regulation and activities of alpha-fetoprotein. Crit Rev Eukaryot Gene Expr 1997,7(1-2):11-41.
9. Ryu JS, Lee HJ, Cho JH, Han HS, Lee HL:The implication of elevated carcinoembryonic antigen level in pleural fluid of patients with non-malignant pleural effusion. Respirology 2003,8(4):487-491.
10. Ambrosini G, Adida C, Altieri DC:A novel anti-apoptosis gene, survivin, expressed in cancer and lymphoma. Nat Med 1997,3(8):917-921.
11. Megliorino R, Shi FD, Peng XX, Wang X, Chan EK, Tan EM, Zhang JY:Autoimmune response to anti-apoptotic protein survivin and its association with antibodies to p53 and c-myc in cancer detection. Cancer Detect Prev 2005,29(3):241-248.
12. Engelen MJ, de Bruijn HW, Hollema H, ten Hoor KA, Willemse PH, Aalders JG, van der Zee AG:Serum CA 125, carcinoembryonic antigen, and CA 19-9 as tumor markers in borderline ovarian tumors. Gynecol Oncol 2000,78(1):16-20.
13. Vukotic V, Cerovic S, Kozomara M, Lazic M:The predictive value of PSA in diagnosis of prostate cancer in non screened population. Acta Chir Iugosl 2005, 52(4):81-87.
14. Sarandakou A, Malamitsi-Puchner A, Protonotariou E, Rigopoulou U, Papagianni B, Creatsas G:Indicative markers of cell proliferation and apoptosis during the perinatal period. Am J Perinatol 2003,20(6):283-288.
15. Wilczynski JR:Cancer and pregnancy share similar mechanisms of immunological escape. Chemotherapy 2006,52(3):107-110.
16. Misirlioglu M, Page GP, Sagirkaya H, Kaya A, Parrish JJ, First NL, Memili E: Dynamics of global transcriptome in bovine matured oocytes and preimplantation embryos. Proc Natl Acad Sci USA 2006,103(50):18905-18910.
17. Morita R, Hirohashi Y, Torigoe T:Mechanism of immune escape of cancer and its regulation. Rinsho Ketsueki 2009,50(5):375-380.
18. Xie K, Abbruzzese JL:Developmental biology informs cancer:the emerging role of the hedgehog signaling pathway in upper gastrointestinal cancers. Cancer Cell 2003,4(4):245-247.
19. Wu F, Stutzman A, Mo YY:Notch signaling and its role in breast cancer. Front Biosci 2007,12:4370-4383.
20. Serr DM, Padeh B, Mashiach S, Shaki R:Chromosomal studies in tumors of embryonic origin. Obstet Gynecol 1969,33(3):324-332.
21. Hellstrom 1, Pierce GE, Hellstrom KE:Human tumor-specific antigens. Surgery 1969, 65(6):984-989.
22. Buttle GA, Eperon J, Menzies DN:Induced Tumour Resistance in Rats. Lancet 1964, 2(7349):12-14.
23. LeMevel BP, Wells SA, Jr.:Foetal antigens cross-reactive with tumour-specific transplantation antigens. Nat New Biol 1973,244(136):183-184.
24. Sikora K, Stern P, Lennox E:Immunoprotection by embryonal carcinoma cells for methylcholanthrene-induced murine sarcomas. Nature 1977,269(5631):813-815.
25. Li Y, Zeng H, Xu RH, Liu B, Li Z:Vaccination with human pluripotent stem cells generates a broad spectrum of immunological and clinical responses against colon cancer. Stem Cells 2009,27(12):3103-3111.
26. Yaddanapudi K, Mitchell RA, Putty K, Willer S, Sharma RK, Yan J, Bodduluri H, Eaton JW:Vaccination with embryonic stem cells protects against lung cancer:is a broad-spectrum prophylactic vaccine against cancer possible? PLoS ONE 2012, 7(7):e42289.
27. Rosato A, Zoso A, Dalla Santa S, Milan G, Del Bianco P, De Salvo GL, Zanovello P: Predicting tumor outcome following cancer vaccination by monitoring quantitative and qualitative CD8+ T cell parameters. J Immunol 2006,176(3):1999-2006.
28. van Herck H, Baumans V, Brandt CJ, Hesp AP, Sturkenboom JH, van Lith HA, van Tintelen G, Beynen AC:Orbital sinus blood sampling in rats as performed by different animal technicians:the influence of technique and expertise. Lab Anim 1998,32(4):377-386.
29. Mayo JG:Biologic characterization of the subcutaneously implanted Lewis lung tumor. Cancer Chemother Rep 21972,3(1):325-330.
30. Al-Hajj M, Clarke MF:Self-renewal and solid tumor stem cells. Oncogene 2004, 23(43):7274-7282.
31. Stonehill EH, Bendich A:Retrogenetic expression:the reappearance of embryonal antigens in cancer cells. Nature 1970,228(5269):370-372.
32. Baldwin RW, Glaves D, Vose BM:Embryonic antigen expression in chemically induced rat hepatomas and sarcomas. Int J Cancer 1972,10(2):233-243.
33. Baldwin RW, Glaves D, Pimm MV, Vose BM:Tumour specific and embryonic antigen expression of chemically induced rat tumours. Ann Inst Pasteur (Paris) 1972, 122(4):715-728.
34. Gold P, Freedman SO:Specific carcinoembryonic antigens of the human digestive system. J Exp Med 1965,122(3):467-481.
35. Gold P, Freedman SO:Demonstration of Tumor-Specific Antigens in Human Colonic Carcinomata by Immunological Tolerance and Absorption Techniques. J Exp Med 1965,121:439-462.
36. Horna P, Sotomayor EM:Cellular and molecular mechanisms of tumor-induced T-cell tolerance. Curr Cancer Drug Targets 2007,7(1):41-53.
37. Cohen J:Public health. HPV's peculiarities, from infection to disease. Science 2005, 308(5722):619.
38. Klavins JV, Mesa-Tejada R, Weiss M:Human carcinoma antigens cross reacting with anti-embryonic antibodies. Nat New Biol 1971,234(48):153-154.
39. Begley J, Ribas A:Targeted therapies to improve tumor immunotherapy. Clin Cancer Res 2008,14(14):4385-4391.
40. Rosenberg SA, Yang JC, Restifo NP:Cancer immunotherapy:moving beyond current vaccines. Nat Med 2004,10(9):909-915.
41. Ikeda H, Chamoto K, Tsuji T, Suzuki Y, Wakita D, Takeshima T, Nishimura T:The critical role of type-1 innate and acquired immunity in tumor immunotherapy. Cancer Sci 2004,95(9):697-703.
42. Teitz-Tennenbaum S, Wicha MS, Chang AE, Li Q:Targeting cancer stem cells via dendritic-cell vaccination. Oncoimmunology 2012, 1(8):1401-1403.
43. Galluzzi L, Senovilla L, Vacchelli E, Eggermont A, Fridman WH, Galon J, Sautes-Fridman C, Tartour E, Zitvogel L, Kroemer G:Trial watch:Dendritic cell-based interventions for cancer therapy. Oncoimmunology 2012,1(7):1111-1134.
44. Xiong G, Husseiny MI, Song L, Erdreich-Epstein A, Shackleford GM, Seeger RC, Jackel D, Hensel M, Metelitsa LS:Novel cancer vaccine based on genes of Salmonella pathogenicity island 2. Int J Cancer 2010,126(11):2622-2634.
45. Johnsen A, France J, Sy MS, Harding CV:Down-regulation of the transporter for antigen presentation, proteasome subunits, and class I major histocompatibility complex in tumor cell lines. Cancer Res 1998,58(16):3660-3667.
46. Errington F, Bateman A, Kottke T, Thompson J, Harrington K, Merrick A, Hatfield P, Selby P, Vile R, Melcher A:Allogeneic tumor cells expressing fusogenic membrane glycoproteins as a platform for clinical cancer immunotherapy. din Cancer Res 2006,12(4):1333-1341.
1. Chouaib S, El Hage F, Benlalam H, Mami-Chouaib F:[Immunotherapy of cancer: promise and reality]. Med Sci (Paris) 2006,22(8-9):755-759.
2. Wiemann B, Starnes CO:Coley's toxins, tumor necrosis factor and cancer research: a historical perspective. Pharmacol Ther 1994,64(3):529-564.
3. Nauts HC, Fowler GA, Bogatko FH:A review of the influence of bacterial infection and of bacterial products (Coley's toxins) on malignant tumors in man; a critical analysis of 30 inoperable cases treated by Coley's mixed toxins, in which diagnosis was confirmed by microscopic examination selected for special study. Acta Med Scand Suppl 1953,276:1-103.
4. Croci DO, Salatino M:Tumor immune escape mechanisms that operate during metastasis. Curr Pharm Biotechnol 2011,12(11):1923-1936.
5. Whiteside TL, Mandapathil M, Szczepanski M, Szajnik M:Mechanisms of tumor escape from the immune system:adenosine-producing Treg, exosomes and tumor-associated TLRs. Bull Cancer 2011,98(2):E25-31.
6. Xiong G, Husseiny MI, Song L, Erdreich-Epstein A, Shackleford GM, Seeger RC, Jackel D, Hensel M, Metelitsa LS:Novel cancer vaccine based on genes of Salmonella pathogenicity island 2. Int JCancer 2010,126(11):2622-2634.
7. Yang L, Pang Y, Moses HL:TGF-beta and immune cells:an important regulatory axis in the tumor microenvironment and progression. Trends Immunol 2010, 31(6):220-227.
8. Gutzmer R, Li W, Sutterwala S, Lemos MP, Elizalde JI, Urtishak SL, Behrens EM, Rivers PM, Schlienger K, Laufer TM et al: A tumor-associated glycoprotein that blocks MHC class Ⅱ-dependent antigen presentation by dendritic cells. J Immunol 2004,173(2):1023-1032.
9. Konishi J, Yamazaki K, Azuma M, Kinoshita I, Dosaka-Akita H, Nishimura M:B7-H1 expression on non-small cell lung cancer cells and its relationship with tumor-infiltrating lymphocytes and their PD-1 expression. Clin Cancer Res 2004, 10(15):5094-5100.
10. Chaux P, Favre N, Bonnotte B, Moutet M, Martin M, Martin F:Tumor-infiltrating dendritic cells are defective in their antigen-presenting function and inducible B7 expression. A role in the immune tolerance to antigenic tumors. Adv Exp Med Biol 1997,417:525-528.
11. Ikeda H, Chamoto K, Tsuji T, Suzuki Y, Wakita D, Takeshima T, Nishimura T:The critical role of type-1 innate and acquired immunity in tumor immunotherapy. Cancer Sci 2004,95(9):697-703.
12. Yoshino S, Yoshimura K, Suzuki N, Iida M, Yoshida S, Maeda Y, Maeda K, Hazama S, Oka M:[Immunoregulatory effects of PSK on the Thl/Th2 balance and regulatory T-cells in patients with colorectal cancer]. Gan To Kagaku Ryoho 2010, 37(12):2234-2236.
13. Leitch J, Fraser K, Lane C, Putzu K, Adema GJ, Zhang QJ, Jefferies WA, Bramson JL, Wan Y:CTL-dependent and -independent antitumor immunity is determined by the tumor not the vaccine. J Immunol 2004,172(9):5200-5205.
14. Qin H, Zhou C, Wang D, Ma W, Liang X, Lin C, Zhang Y, Zhang S:Specific antitumor immune response induced by a novel DNA vaccine composed of multiple CTL and T helper cell epitopes of prostate cancer associated antigens.Immunol Lett 2005,99(1):85-93.
15. Rousalova I, Krepela E:Granzyme B-induced apoptosis in cancer cells and its regulation (review). Int J Oncol 2010,37(6):1361-1378.
16. Rosenblatt J, Vasir B, Uhl L, Blotta S, Macnamara C, Somaiya P, Wu Z, Joyce R, Levine JD, Dombagoda D et al: Vaccination with dendritic cell/tumor fusion cells results in cellular and humoral antitumor immune responses in patients with multiple myeloma. Blood 2011,117(2):393-402.
17. Li Y, Liang J, Liu WB, Xu XQ, Zhao YR:[Influence of chemotherapy on Thl/Th2 cytokine switching in stomach cancer patients]. Zhonghua Zhong Liu Za Zhi 2004, 26(12):732-734.
18. Chen M, Hao X, Zhang Y, Lu B, Wu Y, Xu Y, Chen J, Yu W:[The clinical significance of detection of Th1/Th2 cell cytokines in lung cancer]. Zhongguo Fei Ai Za Zhi 2004, 7(3):214-217.
19. Yamazaki K, Yano T, Kameyama T, Suemitsu R, Yoshino I, Sugio K:Clinical significance of serum TH1/TH2 cytokines in patients with pulmonary adenocarcinoma. Surgery 2002,131(1 Suppl):S236-241.
20. Hirschowitz EA, Yannelli JR:Immunotherapy for lung cancer. Proc Am Thorac Soc 2009,6(2):224-232.
21. Whiteside TL:Immune suppression in cancer:effects on immune cells, mechanisms and future therapeutic intervention. Semin Cancer Biol 2006,16(1):3-15.
22. Sioud M:Does our current understanding of immune tolerance, autoimmunity, and immunosuppressive mechanisms facilitate the design of efficient cancer vaccines? Scand J Immunol 2009,70(6):516-525.
23. Johnsen A, France J, Sy MS, Harding CV:Down-regulation of the transporter for antigen presentation, proteasome subunits, and class I major histocompatibility complex in tumor cell lines. Cancer Res 1998,58(16):3660-3667.
24. Rosato A, Zoso A, Dalla Santa S, Milan G, Del Bianco P, De Salvo GL, Zanovello P: Predicting tumor outcome following cancer vaccination by monitoring quantitative and qualitative CD8+T cell parameters. J Immunol 2006,176(3):1999-2006.
25. van Herck H, Baumans V, Brandt CJ, Hesp AP, Sturkenboom JH, van Lith HA, van Tintelen G, Beynen AC:Orbital sinus blood sampling in rats as performed by different animal technicians:the influence of technique and expertise. Lab Anim 1998,32(4):377-386.
26. Ni C, Huang J:Dynamic regulation of cancer stem cells and clinical challenges. Clin Transl Oncol 2013,15(4):253-258.
27. Magee JA, Piskounova E, Morrison SJ:Cancer stem cells:impact, heterogeneity, and uncertainty. Cancer Cell 2012,21(3):283-296.
28. Al-Hajj M, Wicha MS, Benito-Hernandez A, Morrison SJ, Clarke MF:Prospective identification of tumorigenic breast cancer cells. Proc Natl Acad Sci U S A 2003, 100(7):3983-3988.
29. Li C, Lee CJ, Simeone DM:Identification of human pancreatic cancer stem cells. Methods Mol Biol 2009,568:161-173.
30. O'Brien CA, Pollett A, Gallinger S, Dick JE:A human colon cancer cell capable of initiating tumour growth in immunodeficient mice. Nature 2007,445(7123):106-110.
31. Chu P, Clanton DJ, Snipas TS, Lee J, Mitchell E, Nguyen ML, Hare E, Peach RJ: Characterization of a subpopulation of colon cancer cells with stem cell-like properties. Int J Cancer 2009,124(6):1312-1321.
32. Murase M, Kano M, Tsukahara T, Takahashi A, Torigoe T, Kawaguchi S, Kimura S, Wada T, Uchihashi Y, Kondo T et al: Side population cells have the characteristics of cancer stem-like cells/cancer-initiating cells in bone sarcomas. Br J Cancer 2009, 101(8):1425-1432.
33. Wang S, Garcia AJ, Wu M, Lawson DA, Witte ON, Wu H:Pten deletion leads to the expansion of a prostatic stem/progenitor cell subpopulation and tumor initiation. Proc Natl Acad Sci U S A 2006,103(5):1480-1485.
34. Chaffer CL, Brueckmann I, Scheel C, Kaestli AJ, Wiggins PA, Rodrigues LO, Brooks M, Reinhardt F, Su Y, Polyak K et al: Normal and neoplastic nonstem cells can spontaneously convert to a stem-like state. Proc Natl Acad Sci U S A 2011, 108(19):7950-7955.
35. Chang HH, Chen BY, Wu CY, Tsao ZJ, Chen YY, Chang CP, Yang CR, Lin DP: Hedgehog overexpression leads to the formation of prostate cancer stem cells with metastatic property irrespective of androgen receptor expression in the mouse model. J Biomed Sci 2011,18:6.
36. Sell S:Stem cell origin of cancer and differentiation therapy. Crit Rev Oncol Hematol 2004,51(1):1-28.
37. Sell S:Leukemia:stem cells, maturation arrest, and differentiation therapy. Stem Cell Rev 2005,1(3):197-205.
38. Sell S:Potential gene therapy strategies for cancer stem cells. Curr Gene Ther 2006, 6(5):579-591.
39. Sell S:Cancer and stem cell signaling:a guide to preventive and therapeutic strategies for cancer stem cells. Stem Cell Rev 2007,3(1):1-6.
40. A1-Hajj M, Becker MW, Wicha M, Weissman Ⅰ, Clarke MF:Therapeutic implications of cancer stem cells. Curr Opin Genet Dev 2004,14(1):43-47.
41. Jordan CT:Targeting the most critical cells:approaching leukemia therapy as a problem in stem cell biology. Nat Clin Pract Oncol 2005,2(5):224-225.
42. Copland M, Fraser AR, Harrison SJ, Holyoake TL:Targeting the silent minority: emerging immunotherapeutic strategies for eradication of malignant stem cells in chronic myeloid leukaemia. Cancer Immunol Immunother 2005,54(4):297-306.
43. Dong W, Du J, Shen H, Gao D, Li Z, Wang G, Mu X, Liu Q:Administration of embryonic stem cells generates effective antitumor immunity in mice with minor and heavy tumor load. Cancer Immunol Immunother 2010,59(11):1697-1705.
44. Drukker M, Katchman H, Katz G, Even-Tov Friedman S, Shezen E, Hornstein E, Mandelboim O, Reisner Y, Benvenisty N:Human embryonic stem cells and their differentiated derivatives are less susceptible to immune rejection than adult cells. Stem Cells 2006,24(2):221-229.
45. Drukker M, Benvenisty N:The immunogenicity of human embryonic stem-derived cells. Trends Biotechnol 2004,22(3):136-141.
46. Fairchild PJ, Cartland S, Nolan KF, Waldmann H:Embryonic stem cells and the challenge of transplantation tolerance. Trends Immunol 2004,25(9):465-470.
47. Boyd AS, Higashi Y, Wood KJ:Transplanting stem cells:potential targets for immune attack. Modulating the immune response against embryonic stem cell transplantation. Adv Drug Deliv Rev 2005,57(13):1944-1969.
48. Begley J, Ribas A:Targeted therapies to improve tumor immunotherapy. Clin Cancer Res 2008,14(14):4385-4391.
49. Rosenberg SA, Yang JC, Restifo NP:Cancer immunotherapy:moving beyond current vaccines. Nat Med 2004,10(9):909-915.
50. Shurin MR, Lu L, Kalinski P, Stewart-Akers AM, Lotze MT:Th1/Th2 balance in cancer, transplantation and pregnancy. Springer Semin Immunopathol 1999, 21(3):339-359.
51. Koestenbauer S, Zech NH, Juch H, Vanderzwalmen P, Schoonjans L, Dohr G: Embryonic stem cells:similarities and differences between human and murine embryonic stem cells. Am JReprod Immunol 2006,55(3):169-180.
52. Li Y, Zeng H, Xu RH, Liu B, Li Z:Vaccination with human pluripotent stem cells generates a broad spectrum of immunological and clinical responses against colon cancer. Stem Cells 2009,27(12):3103-3111.