肿瘤驯化的B淋巴细胞通过分泌病理性抗体促进乳腺癌转移及其相关机制研究
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摘要
肿瘤是严重危害人类健康的重大疾病，而肿瘤转移则是肿瘤患者死亡的主要原因。转移是恶性肿瘤的重要特征之一，是指肿瘤细胞从原发部位扩散到身体的其他部位，并再次增殖成为肿瘤病灶，其过程包括原位肿瘤的侵袭、肿瘤细胞在循环中的存活静息及在远隔转移部位的定居与克隆形成。在肿瘤转移的机制研究方面，“种子”与“土壤”假说得到了肿瘤免疫学领域研究人员的普遍认可。肿瘤微环境作为肿瘤发生的“土壤”是近年来该领域研究前沿热点之一。肿瘤细胞通过产生大量膜型和分泌型生物活性物质（Tumor-derived factors, TDFs）诱导基质和相关免疫细胞功能转化，形成稳定的肿瘤免疫微环境，保护肿瘤组织逃脱机体的免疫监视并促进肿瘤的转移。随着肿瘤的进程，肿瘤组织及远隔器官中的免疫细胞能通过与肿瘤细胞相互接触或分泌可溶性的细胞因子而改变肿瘤的侵袭及转移的能力。2005年Kaplan教授提出的肿瘤转移前微环境假说，该假说认为大量肿瘤细胞驯化的细胞以及肿瘤来源的分子先于肿瘤细胞进入待转移器官为肿瘤转移提供土壤并指示方向趋化肿瘤种子进入该器官。越来越多的实验证据支持该假说，多种免疫细胞亚群及分子被证实参与肿瘤转移前微环境的形成，包括免疫抑制性细胞如调节性T细胞（Regulatory T cells, Tregs）、肿瘤相关巨噬细胞（Tumor-associated macrophages, TAMs）、髓系来源的抑制性细胞（Myeloid-derived suppressor cells, MDSCs）等，以及一氧化氮（NO）、精氨酸酶（Arginase，ARG）等免疫抑制分子。因此，研究肿瘤免疫微环境的产生机制、如何打破抑制性肿瘤免疫微环境以及如何诱导肿瘤相关免疫细胞的功能逆转成为肿瘤免疫治疗学界关注的重点。
B淋巴细胞是在机体体液免疫中发挥关键作用的免疫细胞，广泛分布于血液、脾脏、淋巴结及其他粘膜组织中。B细胞在抗原刺激下可分化为浆细胞，合成和分泌免疫球蛋白（Immunoglobulin，Ig），主要执行机体的体液免疫功能。B细胞的主要功能是分泌抗体，肿瘤细胞因表达肿瘤抗原而能激活B细胞，进一步分化发育为浆细胞，浆细胞可分泌具有抗肿瘤作用的抗体。这些抗体可通过激活补体系统溶解肿瘤细胞、介导巨噬细胞、NK细胞发挥ADCC效应以及封闭肿瘤细胞上的某些受体（如转铁蛋白受体）以抑制肿瘤细胞生长与转移等。抗体在肿瘤中的研究大部分是寻找针对肿瘤相关抗原的特异性抗体以治疗肿瘤，抗体疗法也是目前靶向治疗肿瘤的主要方法。病理性抗体（Pathogenic antibody）概念的提出改变了人们对抗体的认识。研究证实其在抑制损伤修复、促进自身免疫病发病等方面都存在重要作用。也有研究表明B细胞所产生的抗体通过与免疫细胞Fc受体的结合对肿瘤发生起到促进作用，这也是炎症导致肿瘤的机制之一。但病理性抗体影响肿瘤免疫的研究尚处起步阶段，机理研究尚不透彻，而且对于肿瘤转移的作用也鲜有研究。
相对于T细胞而言，B细胞在肿瘤免疫中的研究还很少。1998年德国ThomasBlankenstein教授等与本实验室合作研究发现B细胞对于细胞毒性T细胞（CytotoxicT lymphocytes, CTL）抗肿瘤免疫起到抑制作用，由此，B细胞对肿瘤的负向免疫调节功能逐渐为研究者所重视，目前，多种具有免疫抑制功能的B细胞亚群陆续被发现，尤其是一类分泌IL-10的调节性B细胞（Regulatory B cells）。大量研究及临床数据显示，这些具有负向免疫调控功能的B细胞在肿瘤的发生、发展及转移中发挥了重要作用。因此，肿瘤驯化的B细胞及其所分泌的病理性抗体在肿瘤转移前微环境形成和肿瘤转移中的作用已成为肿瘤免疫学研究的热点之一。
基于以上研究进展和现状分析，我们深入研究了肿瘤微环境对于B细胞功能的驯化以及其分泌抗体的影响，并且发现了荷瘤小鼠体内存在大量肿瘤特异性抗体，这些病理性抗体能够促进肿瘤的淋巴结转移，进一步机制研究证实趋化因子及其受体SDF1α/CXCR4在其中发挥了重要的功能。通过上述研究，我们发现了B细胞及抗体参与肿瘤免疫调控的多样化功能。
我们的研究分为以下三个部分：
1、肿瘤驯化的B细胞及其分泌的病理性抗体的发现
为研究肿瘤驯化下的淋巴结微环境对于肿瘤转移的影响，我们制备了小鼠乳腺癌4T1原位种植模型，分析肿瘤引流淋巴结（Draining lymph node， DLN）中各淋巴细
胞群体尤其是B细胞数量及比例的变化。通过流式分析，我们发现伴随着肿瘤的进展DLN中B细胞比例及数量明显增加。动态监测显示，在荷瘤早期（1周内）B细胞比例开始升高，2-3周显著上升，后达到平台期，直至淋巴结转移。而淋巴结中T细胞、DC及巨噬细胞的比例则随着荷瘤时间增长比例呈下降趋势。上述实验结果在另一小鼠乳腺癌模型EMT6中进一步得到验证。以上实验表明，B细胞在肿瘤的驯化下于淋巴结中大量聚集，尤其是在DLN中，而DLN往往是乳腺癌转移的首站，提示肿瘤驯化B细胞有可能参与到乳腺癌淋巴结转移的免疫调控中。
我们进一步探讨了B细胞在淋巴结中聚集的影响因素。首先，我们利用Brdu体内增殖实验检测荷瘤后B细胞在LN中的增殖情况，研究发现，DLN中B细胞增殖的比例较正常对照LN有所增高。其次，我们对DLN中的基质细胞趋化B细胞的能力进行检测，结果显示，来源于DLN的基质细胞较之对照LN对B细胞的趋化能力明显增强。我们的研究表明，小鼠乳腺癌能够通过影响淋巴结基质微环境增强对B细胞的趋化，并促使B细胞发生增殖，使得B细胞在DLN中的数量不断增加。
为进一步明确肿瘤驯化下的B细胞性质及功能，我们检测了不同来源B细胞的表面标记、细胞因子及免疫球蛋白谱（Immunoglobulin, Ig）。流式分析发现DLN中B细胞表达的与其分型相关的标记（CD1d、CD5、IgM、IgD、CD21、CD23）与对照组相比无明显差异，而活化型标记（CD25、CD69）则在DLN B细胞当中表达增高。ELISA检测B细胞分泌的细胞因子显示各组B细胞分泌IL10、IL6、TNFα、PGE2、TGFβ、INFγ、IL12p70及IL2水平无统计学差异。体外利用LPS刺激后，对其免疫球蛋白谱检测发现，DLN B细胞分泌IgG水平较其他组明显增高，这与表达谱芯片的检测结果一致。同时，我们检测了小鼠体内Ig的表达情况，与体外结果相符，荷瘤小鼠体内IgG1、IgG2a及IgG2b水平要高于正常对照小鼠。
为了研究荷瘤小鼠体内这些病理性抗体的特征，我们用纯化后的IgG孵育肿瘤细胞，标记荧光二抗，利用流式细胞仪和激光共聚焦显微镜进行检测。流式结果显示，与正常IgG相比，肿瘤来源的IgG能够特异性结合乳腺癌细胞。同样，激光共聚焦显微镜结果也证实，肿瘤来源IgG能够与肿瘤细胞表面分子特异性结合。
以上研究表明，伴随着肿瘤进程，乳腺癌小鼠DLN中B细胞的比例及数量逐渐增多。进一步表型及细胞因子表达谱结果显示，肿瘤驯化B细胞为一群活化的B细胞，其分泌抗体的能力增强，同时荷瘤小鼠体内抗体水平也增高，提纯体内IgG研究发现，该抗体在体外能够结合肿瘤细胞，具有肿瘤特异性。以上研究为肿瘤微环境影响B细胞提供了新的认识，同时也为进一步研究肿瘤驯化B细胞及其所产生的抗体在肿瘤免疫逃逸中的作用提供了依据。
2、肿瘤驯化的B细胞与病理性抗体在乳腺癌淋巴结转移中的作用研究
为研究肿瘤驯化B细胞及其分泌的抗体对于肿瘤转移的影响，我们应用B细胞缺陷μMT-/-小鼠进行原位乳腺癌种植，并对荷瘤小鼠给予不同来源IgG回输。活体荧光成像结果显示μMT-/-小鼠荷瘤后淋巴结转移的个数较正常小鼠明显减少，但回输了病理性IgG的小鼠其肿瘤转移的能力又显著增强，而回输正常IgG则没有该效应。进一步生存实验表明，回输病理性IgG后μMT-/-小鼠的生存时间明显缩短，与正常小鼠一致。我们也检测了各组小鼠的肿瘤大小，发现回输IgG对肿瘤大小无明显影响。
我们又利用体外实验研究病理性IgG对肿瘤转移中涉及的增殖、凋亡、趋化及侵袭因素的影响。MTT法增殖实验显示，病理性IgG对于肿瘤细胞的增殖无明显影响。在化疗药物诱导的肿瘤细胞凋亡体系中，我们发现病理性IgG能够明显抑制阿霉素所诱导的肿瘤细胞凋亡，而正常对照IgG无该效应。Tranwell趋化实验显示，与空白对照及正常IgG比较，病理性IgG能够明显增强肿瘤细胞向下层淋巴结基质的趋化能力。侵袭实验也有相似结果。
以上实验表明，肿瘤驯化的B细胞不仅能够通过分泌病理性IgG显著促进肿瘤淋巴结转移，而且影响了肿瘤细胞转移的多个环节，包括抵抗凋亡、侵袭及向淋巴结趋化能力，尤其是肿瘤细胞的趋化，使其到达转移部位定居，形成转移灶。既往研究证明多种趋化因子及受体参与肿瘤转移过程，如SDF1a/CXCR4、CCL21/CCR7等，因而，我们在接下来研究肿瘤驯化的B细胞诱导肿瘤转移的具体分子机制时也重点关注了肿瘤驯化的B细胞与影响肿瘤趋化的趋化因子及受体之间的相关性。
3、肿瘤驯化的B细胞与病理性抗体促进乳腺癌淋巴结转移的机制研究
前面的结果提示，病理性IgG能够促进乳腺癌淋巴结转移，我们推测趋化因子及其受体可能参与到该过程。有研究报道，肿瘤细胞高表达趋化因子受体主要是CXCR4和CCR7，其中，SDF1α/CXCR4则是肿瘤向淋巴结转移最为重要的趋化因素。因此，我们检测了IgG刺激后肿瘤细胞这两种趋化因子受体的表达情况。定量PCR结果显示，病理性IgG能够明显刺激肿瘤细胞CXCR4mRNA水平的表达，而正常IgG无该效应，蛋白水平检测得到了同样的结果。病理性IgG是如何促进CXCR4的表达？我们在体外检测了IgG刺激后肿瘤细胞转移相关基因的表达，结果显示，Hif1α及Cox2的表达明显增高。Hif1α是肿瘤适应低氧环境所产生的主要调控因子之一，其表达的高低与肿瘤的转移及预后密切相关。有报道称Hif1α能够通过诱导CXCR4表达从而促进肿瘤细胞的迁移，而SDF1α/CXCR4则是肿瘤向淋巴结转移最为重要的趋化因素。为了验证是否病理性IgG诱导肿瘤细胞Hif1α从而促进CXCR4表达上调，我们检测了不同IgG剂量以及不同时间刺激下肿瘤细胞在mRNA及蛋白水平表达Hif1α的情况。结果提示，与CXCR4表达变化一致，肿瘤细胞在病理性IgG刺激24小时后，其表达的Hif1α的水平明显增高，且随着刺激浓度及时间的增加而不断增高，相反如果用RNA干扰阻断Hif1α的表达，CXCR4的上调幅度受到明显的抑制。
我们又对基质细胞分泌CXCR4配体SDF-1α的调控机制进行了研究，利用IgG刺激后肿瘤条件培养基培养基质细胞，检测基质分泌SDF-1α的水平。结果发现，病理性IgG刺激后的肿瘤上清能够促进SDF-1α的分泌。既往研究表明细胞因子IL-1β、TGF-β、TNF-α以及PGE2等能够调控SDF-1α的分泌。我们用ELISA检测病理性IgG刺激肿瘤细胞上清中细胞因子的种类，发现病理性IgG能够刺激肿瘤细胞分泌大量的PGE2；同时，我们检测了肿瘤细胞在病理性IgG刺激后相关基因的表达水平，发现Cox2在刺激后明显增高，病理性IgG刺激24h后随着刺激时间的增长，Cox2mRNA水平的表达明显增加。所以我们推测肿瘤细胞能够通过Cox2介导的PGE2对淋巴结基质分泌SDF-1α起到调控作用。为了进一步验证PGE2对于SDF-1α的分泌是否有调控作用，我们利用上述基质细胞的培养体系中，当干扰了肿瘤细胞Cox2的表达后SDF-1α的升高效应受到明显抑制，如果在培养体系中直接加入PGE2，又能刺激SDF-1α的分泌。因此，该结果表明Cox2介导的PGE2增高是病理性IgG刺激后的肿瘤上清促进基质细胞SDF1α分泌的影响因素。
上述研究提示病理性IgG能够刺激肿瘤细胞Cox2及Hif1α的表达，从而促进SDF1α与CXCR4这对趋化因子轴功能的发挥，病理性IgG与肿瘤细胞相互作用的具体靶点及传导途径尚不清楚；接下来，我们进一步深入研究病理性IgG介导Cox2及Hif1α表达增高具体分子机制。已有文献报道，肿瘤细胞Hif1α及Cox2的表达受到多个信号通路的调控，如NF-kB、Ras-MAPK等。为检测病理性IgG刺激肿瘤细胞后下游信号通路的活化情况，我们在该刺激体系中加入不同信号通路的阻断剂。结果显示，NF-kB通路阻断剂PDTC能够明显抑制IgG刺激的肿瘤细胞Hif1α及Cox2的上调。
接下来，我们又对病理性IgG如何刺激肿瘤细胞NF-κB通路的活化进行了研究。首先，我们排除了肿瘤细胞表面Fc受体参与该活化过程，并证实IgG与肿瘤细胞表面靶抗原的结合是引起胞内NF-κB通路活化的原因。为寻找靶抗原，我们分离了肿瘤细胞膜蛋白，通过co-IP后进行蛋白质谱检测，发现了15个候选蛋白（Adam10、Nucleolin、Itgb3、Mucin1、Hspa4、Moesin、CD151、Enolase1、Itgb5、TfR1、Erzin、Radixin、Erbb2），siRNA干扰后NF-kB活化程度下降的有Itgb3、Nucleolin、TfR1、Enolase1、Hspa4、Erbb2。为做进一步筛选，我们在293T细胞中过表达基因Itgb3、Nucleolin、TfR1、Enolase1、Hspa4、Erbb2，病理性IgG刺激后检测NF-kB报告基因表达程度，结果显示，293T细胞本身不能被IgG所刺激，在过表达Nucleolin、Hspa4、Itgb3后NF-kB表达活性明显增强。为确定靶抗原，我们通过免疫共沉淀（IP）利用Flag抗体将293T细胞中目的蛋白Nucleolin、Hspa4及Itgb3纯化，免疫印迹（IB）检测发现病理性IgG能够结合Hspa4，而不能结合Nucleolin及Itgb3。进一步共聚焦显微镜检测显示，干扰了4T1细胞Hspa4后，病理性IgG在胞膜表面的结合明显减少。以上结果提示针对靶抗原Hspa4的特异性抗体通过抗原与抗体特异性结合介导NF-kB传导通路的活化从而诱导下游的Hif1α及Cox2表达上调，进而促进SDF1α与CXCR4这对趋化因子轴发挥效应介导了转移前微环境的形成以及肿瘤细胞的定向转移，具体分子机制尚需要进一步验证与深入研究。
综上所述，我们研究发现肿瘤影响下的DLN中B细胞比例及数量显著增加。特征分析显示，这群肿瘤驯化的B细胞分泌抗体的能力明显增强，尤其是IgG1、IgG2a及IgG2b，但是该B细胞大部分表面分子及细胞因子表达谱与正常B细胞没有明显差异。对荷瘤小鼠体内病理性IgG研究显示其具有肿瘤抗原特异性，能够结合在肿瘤细胞表面。体内功能试验证实，B细胞缺失以后，荷瘤小鼠淋巴结转移的个数明显减少，而回输了病理性IgG的小鼠其肿瘤转移的能力显著增强。进一步分子机制探讨表明，病理性IgG能够通过结合特异性肿瘤抗原Hspa4，刺激下游NF-κB通路的活化，从而上调Hif1α及Cox2的表达。Hif1α的表达促进肿瘤细胞表达趋化因子受体CXCR4，而Cox2介导的PGE2分泌则诱导淋巴结基质细胞分泌趋化因子SDF1α，从而促进肿瘤转移前微环境形成并诱导肿瘤细胞向引流淋巴结的迁移。
本课题研究发现在乳腺癌发展过程中，肿瘤驯化下的B细胞及其分泌病理性抗体能够促进肿瘤淋巴结转移，并对其具体分子调控机制进行深入研究，证实在肿瘤免疫调控过程中，B细胞不仅能够通过分泌IL-10和TGFβ发挥负向免疫调控作用而且能够通过产生病理性抗体参与肿瘤转移前微环境的形成诱导肿瘤转移。该研究结果不仅拓宽了人们对于B细胞及抗体性质与功能的认识，也为肿瘤免疫逃逸和肿瘤转移提出新的作用机制，为全面认识复杂而精细的肿瘤免疫调控网络提供了新的视角，也有望为肿瘤的临床治疗及转移防治方法的寻找提供新的启示。
Tumor metastasis is the leading cause for the death of cancer patients. Metastasis is oneof the most important characteristics of malignancy, which is referred to tumor cellspreading from the primary site to other parts of the body where proliferating at the newniches. The metastasis process includes tumor invasion in situ, survival and resting oftumor cells in circulation, settlement in distant sites and colony formation. A variety offactors affect tumor metastasis, and among them, the immune microenvironment hasattracted much attention recently. Through secreting tumor derived factors (TDFs), tumorcells educate the matrix and immune cells to form a stable immunosuppressivemicroenvironment which enables tumor cells to escape from immune surveillance andpromote tumor metastasis. On the other side, immune cells in tumor tissue and distantorgans could change the invasion and metastasis of tumor through direct contacting withtumor cells or indirect secretion of cytokines/growth factors. A variety of immune cellsubsets and molecules were confirmed to participate in the crosstalk with tumor cells,including immunosuppressive cells such as regulatory T cells (Tregs), tumor-associatedmacrophages (TAMs), myeloid-derived suppressor cells (MDSC), and immunosuppressivemolecules such as IL-10, nitric oxide (NO) and arginase (ARG). However, the mechanismsfor how tumor microenvironment can educate immune cells to be immunosuppressive arenow not fully elucidated and need further investigation, so as to break immunosuppressivetumor microenvironment and reverse the function of tumor-educated immune cells for theimmunotherapy of cancer.
B cells are antibody-producing cells and widely distribute in the blood, spleen, lymph nodes and other mucosal tissues. After antigen stimulation, B cells differentiate into plasmacells, which could synthesis and secret immunoglobulin (Ig), and establish humoralimmunity. However, the role of B cells in tumor immunity remain unclear. Dr. ThomasBlankenstein’s group, in cooperation with our laboratory in1998, found that B cells couldinhibit anti-tumor cytotoxic T cells (CTL) responses. Thus, the negativeimmune-regulating function of B cells in tumor immunity gradually emerges, and a B cellsubset with immunosuppressive function has been reported, which is characterized assecretion of IL-10. These IL-10-producing B cells with negative immune regulatoryfunction play an important role in tumor development and metastasis. Therefore, B cellsand their subsets involved in tumor immunity have attracted more and more attention.
In cancer patients, B cells could be activated by tumor antigens, and furtherdifferentiated into plasma cells, which secreted antibodies with anti-tumor effect. Theseantibodies could dissolve tumor cells by activation of the complement system, or by ADCCeffect mediated by macrophages and NK cells. They can also inhibit the growth andmetastasis of tumor cells by blockade of certain receptors on tumor cells (e.g., transferrinreceptor). Antibody therapy is currently the main method of targeted therapy of cancer, andlots of scientists are looking for effective antibody against tumor-associated antigen for thetreatment of cancer. However, the proposal of pathogenic antibody has changed our viewof antibody before. The reports have confirmed that pathogenic antibodies play animportant role in the inhibition of damage-repair, promoting autoimmune diseases and soon. In term of tumor immunity, there are also studies that pathogenic antibody couldpromote tumorigenesis by binding to Fc receptors on immune cells, which could be one ofthe mechanisms of inflammation-caused tumorigenesis. However, the research onpathogenic antibodies in tumor immunity and metastasis is just at the beginning, and themechanisms underlying the tumor-promoting effect of pathogenic antibodies are not fullyilluminated.
Based on the above analysis, we studied the role of B cells and their secreted antibodiesin tumor immunity. We identified a large number of tumor-specific antibodies in the tumor-bearing mice, which promoted tumor metastasis to lymph nodes. Further studyconfirmed that chemokines and their receptors such as SDF1α/CXCR4, up-regulated bythe pathogenic antibodies played important role in the promotion of tumor metastasis bypathogenic antibodies. Our results show the new functions of B cells and their derivedpathogenic antibodies in the negative regulation of tumor immunity and promotion oftumor metastasis.
Our project is divided into the following three parts:
Part I Identification of tumor-educated B cells and pathogenic antibodies
In order to identify the role of lymph node (LN) microenvironment in the tumormetastasis, we analyzed the lymphocyte population in LN in4T1orthotopic implant tumormodel, especially the total number and proportion of B cells. We found that the proportionof B cells in DLN started to increase in the first week post tumor inoculation, andincreased dramatically in the second to third weeks, while the proportion of otherlymphocytes such as T cells, DC and macrophages showed trend to be decreased. Theresults were further confirmed in EMT6breast cancer model.
To further analyze the mechanisms of B cells accumulating in DLN, we studied theproliferation of B cells in vivo and investigated the chemotactic ability of B cells to the LNstromal cells. The proliferation of B cells increased in DLN as detected by Brduproliferation assay. Chemotactic assay indicated that B cells were easier to chemotact tothe DLN stromal cells as compared with that from normal LN. The data showed that tumorenhanced the chemotaxis of B cells to LN through the influence on lymph nodemicroenvironment, and then induced the proliferation of accumulated B cells in situ.
In order to better understand the function of tumor-educated B cells, we detected theirphenotype, cytokines and immunoglobulin profiles. Flow cytometry analysis indicated thatB cells in DLN showed no difference in the expression of traditional B cell markers (CD1d,CD5, IgM, IgD, CD21, CD23) as compared with the control group, while the activatingmarkers (CD25, CD69) were highly expressed in DLN B cells. Then the cytokine profiles of B cells, detected by ELISA, showed that B cells expressed equivalent level of IL6,TNF-α, IL-10, PGE2, TGF-β, IFN-γ, IL-12p70and IL-2as compared to that in controlgroup. After stimulated with LPS, IgG secreted by B cells derived from DLN increasedobviously as compared with other groups. We also detected the serum level of Ig intumor-bearing mice, and the data indicated the level of IgG1, IgG2a and IgG2b was higherthan the control. Further analyses of these IgG revealed that they were tumor-specific andcould bind to the membrane of tumor cells.
These data indicated that the proportion and number of B cells in LN increased withthe tumor process. Further analyses of phenotype, cytokines and Ig profiles showed that asa group of activated B cells, they had enhanced ability of secreting IgG, and the IgG levelsin the serum of tumor-bearing mice also increased. Flow cytometry and confocal studiesindicated that these IgG was tumor-specific and could bind the membrane of tumor cellsbut not to the control cells.
Part II Role of tumor-educated B cells and pathogenic antibodies in the lymph nodemetastasis of breast cancer
To study the function of B cells and antibodies in tumor metastasis, we inoculated breastcancer cells in μMT-/-mice with B cell deficiency,, and different sourced IgG wasadoptively injected afterwards. In vivo fluorescence imaging results showed that,compared with normal mice, the number of metastastic lymph nodes in μMT-/-tumor-bearing mice significantly reduced, but with pathogenic IgG injection, the numberof metastastic lymph nodes significantly increased in μMT-/-tumor-bearing mice, whileadministration of normal IgG have no effect. Further survival experiments showed that theμMT-/-mice survived significantly shorter after administration of pathogenic IgG. Inaddition, pathogenic IgG has no effect on the tumor size, indicating pathogenic IgG doesnot affect the tumor growth.
We took advantage of the in vitro experiment to study the effect of pathogenic IgG in the metastasis, proliferation, apoptosis, chemotaxis and invasion of tumor. The MTT assayshowed that pathogenic IgG did not have significant effect on tumor cell proliferation.Pathogenic IgG could inhibit tumor cell apoptosis induced by chemotherapeutic agentsdoxirubicin, while control IgG could not. The chemoattractant transwell experimentalshowed that, compared with the blank and normal IgG, pathogenic IgG significantlyenhanced tumor cells chemotaxis to the lower matrix. The invasion assay showed similarresults.
Thus, the above experiments indicated that B cells could significantly promote lymphnode metastasis by the secretion of pathogenic IgG, which promoted many profits ofmetastasis, including resistance to apoptosis, invasion and chemotaxis.Part III Mechanism of tumor-educated B cells and pathogenic antibodies to promotebreast cancer metastasis
We found that pathogenic IgG could promote the lymph node metastasis of breast cancer.In order to further explore the molecular mechanism, we examined the expression of genesrelated to metastasis after IgG stimulation in vitro, and the expression of Hif1α and Cox2was observed to be increased significantly. Hif1α, as one of the main factors of tumoradapting to hypoxia, was highly related to metastasis and prognosis of malignancy andcould induce CXCR4expression so as to promote the migration of tumor cells. SDF1α/CXCR4was the most important chemotactic factors related to the lymph node metastasis.In our study, the expression of CXCR4was significantly increased post IgG stimulation,which could be reversed by inhibiting Hif1α expression.
We have also carried out studies on the CXCR4ligand SDF1α using different tumorconditioned medium stimulated by IgG to co-culture with stromal cells. The resultsshowed that, tumor supernatant stimulated by pathogenic IgG could promote SDF1αsecretion of stromal cells. When we interfered Cox2expression of tumor cells, the increaseof SDF1α production is suppressed, which could be rescued by adding PGE2in the culture system. So we found that pathologic IgG could stimulate the tumor cells to produce PGE2and then PGE2promoted the secretion of SDF1α from stromal cells.
We further investigated the mechanisms of elevated Hif1α and Cox2with pathogenicIgG stimulation. Several signaling pathways were assessed and the results showed that theNF-kB pathway inhibitor PDTC could significantly inhibit the up-regulation of Hif1α andCox2. NF-kB reporter gene assay also confirmed pathogenic IgG could enhance thetranscriptional activity of NF-kB in tumor cells. The mechanisms leading to NF-kBactivation were further analyzed. First, we ruled out the Fc receptors on tumor cell surfaceare involved in the activation process, and confirmed that the combination of IgG andtarget antigen on the surface of tumor cells was the cause of NF-kB pathway activation.Looking for the potential tumor antigens/membrane molecules, we isolated tumor cellmembrane proteins and referred to MASS analysis after Co-IP with pathogenic antibodies.Fifteen candidate proteins were identified, including Adam10, Nucleolin, Itgb3, Mucin1,Hspa4, Moesin, CD151, Enolase1, Itgb5, TfR1, Erzin, Radixin, Erbb2. We found thatinterfering with the nucleolin, Hspa4, Itgb3decreased binding of pathogenic antibodieswith tumor cell membrane, and also attenutiated pathogenic IgG-stimulated NF-kBactivation. We also overexpressed nucleolin, Hspa4, Itgb3in293T cells (itself can not bestimulated with pathogenic IgG), and found that pathogenic IgG-stimulated NF-kBactivation was significantly enhanced. To identify the target antigen, we used anti-flagantibody to purify protein Nucleolin, Hspa4and Itgb3in293T cells byimmunoprecipitation (IP). Then immunoblotting (IB) assay found that pathogenic IgGcould bind with Hspa4but not Nucleolin and Itgb3. Further confocal microscope analysisshowed that, the binding of pathologic IgG on the tumor cell membrane decreasedsignificantly after Hspa4silence. Therefore, these results suggested that Hspa4mightmediate activation of NF-kB pathway in tumor cells by pathogenic IgG..
In summary, we found that, under tumor condition, the proportion and total number of Bcells significantly increased in the DLN. Characteristic analysis showed that most of theB-cell phenotype and cytokine secretion did not differ significantly between tumor-educated B cells and normal B cells. However, tumor-educated B cells exerted anactivated phenotype with elevated expression of CD25and CD69. These activated B cellssecreted antibodies more markedly, which was in accordance with the in vivo elevatedIgG1and IgG2b antibody levels in the tumor-beraring mice. Furthermore, these pathogenicIgG from tumor-bearing mice was tumor-specific, and capable of binding to the surface oftumor cells. In vivo function analysis confirmed that, without B cells, the number of lymphnode metastasis was significantly reduced, but administration of pathogenic IgGsignificantly promoted tumor metastasis. Mechanistic study revealed that pathogenic IgGbound to the tumor surface protein Hspa4and stimulated the downstream activation ofNF-kB B pathway, which sequentially up-regulated the Hif1α and Cox2expression. Hif1αpromoted chemokine receptor CXCR4expression on tumor cells and Cox2mediatedPGE2secretion, which then induced lymph node matrix cells to secrete chemokine SDF1α.The corporation of SDF1a and CXCR4promoted tumor cell migration to the draininglymph nodes.
In conclusion, we discovered the new role of tumor-educated B lymphocytes andpathogenic antibodies in promoting lymph node metastasis. Our study will helpunderstanding the new function of B cells and antibodies, and providing a new perspectivefor comprehensive understanding of complex and sophisticated regulating network oftumor immunity.
B淋巴细胞是在机体体液免疫中发挥关键作用的免疫细胞，广泛分布于血液、脾脏、淋巴结及其他粘膜组织中。B细胞在抗原刺激下可分化为浆细胞，合成和分泌免疫球蛋白（Immunoglobulin，Ig），主要执行机体的体液免疫功能。B细胞的主要功能是分泌抗体，肿瘤细胞因表达肿瘤抗原而能激活B细胞，进一步分化发育为浆细胞，浆细胞可分泌具有抗肿瘤作用的抗体。这些抗体可通过激活补体系统溶解肿瘤细胞、介导巨噬细胞、NK细胞发挥ADCC效应以及封闭肿瘤细胞上的某些受体（如转铁蛋白受体）以抑制肿瘤细胞生长与转移等。抗体在肿瘤中的研究大部分是寻找针对肿瘤相关抗原的特异性抗体以治疗肿瘤，抗体疗法也是目前靶向治疗肿瘤的主要方法。病理性抗体（Pathogenic antibody）概念的提出改变了人们对抗体的认识。研究证实其在抑制损伤修复、促进自身免疫病发病等方面都存在重要作用。也有研究表明B细胞所产生的抗体通过与免疫细胞Fc受体的结合对肿瘤发生起到促进作用，这也是炎症导致肿瘤的机制之一。但病理性抗体影响肿瘤免疫的研究尚处起步阶段，机理研究尚不透彻，而且对于肿瘤转移的作用也鲜有研究。
相对于T细胞而言，B细胞在肿瘤免疫中的研究还很少。1998年德国ThomasBlankenstein教授等与本实验室合作研究发现B细胞对于细胞毒性T细胞（CytotoxicT lymphocytes, CTL）抗肿瘤免疫起到抑制作用，由此，B细胞对肿瘤的负向免疫调节功能逐渐为研究者所重视，目前，多种具有免疫抑制功能的B细胞亚群陆续被发现，尤其是一类分泌IL-10的调节性B细胞（Regulatory B cells）。大量研究及临床数据显示，这些具有负向免疫调控功能的B细胞在肿瘤的发生、发展及转移中发挥了重要作用。因此，肿瘤驯化的B细胞及其所分泌的病理性抗体在肿瘤转移前微环境形成和肿瘤转移中的作用已成为肿瘤免疫学研究的热点之一。
基于以上研究进展和现状分析，我们深入研究了肿瘤微环境对于B细胞功能的驯化以及其分泌抗体的影响，并且发现了荷瘤小鼠体内存在大量肿瘤特异性抗体，这些病理性抗体能够促进肿瘤的淋巴结转移，进一步机制研究证实趋化因子及其受体SDF1α/CXCR4在其中发挥了重要的功能。通过上述研究，我们发现了B细胞及抗体参与肿瘤免疫调控的多样化功能。
我们的研究分为以下三个部分：
1、肿瘤驯化的B细胞及其分泌的病理性抗体的发现
为研究肿瘤驯化下的淋巴结微环境对于肿瘤转移的影响，我们制备了小鼠乳腺癌4T1原位种植模型，分析肿瘤引流淋巴结（Draining lymph node， DLN）中各淋巴细
胞群体尤其是B细胞数量及比例的变化。通过流式分析，我们发现伴随着肿瘤的进展DLN中B细胞比例及数量明显增加。动态监测显示，在荷瘤早期（1周内）B细胞比例开始升高，2-3周显著上升，后达到平台期，直至淋巴结转移。而淋巴结中T细胞、DC及巨噬细胞的比例则随着荷瘤时间增长比例呈下降趋势。上述实验结果在另一小鼠乳腺癌模型EMT6中进一步得到验证。以上实验表明，B细胞在肿瘤的驯化下于淋巴结中大量聚集，尤其是在DLN中，而DLN往往是乳腺癌转移的首站，提示肿瘤驯化B细胞有可能参与到乳腺癌淋巴结转移的免疫调控中。
我们进一步探讨了B细胞在淋巴结中聚集的影响因素。首先，我们利用Brdu体内增殖实验检测荷瘤后B细胞在LN中的增殖情况，研究发现，DLN中B细胞增殖的比例较正常对照LN有所增高。其次，我们对DLN中的基质细胞趋化B细胞的能力进行检测，结果显示，来源于DLN的基质细胞较之对照LN对B细胞的趋化能力明显增强。我们的研究表明，小鼠乳腺癌能够通过影响淋巴结基质微环境增强对B细胞的趋化，并促使B细胞发生增殖，使得B细胞在DLN中的数量不断增加。
为进一步明确肿瘤驯化下的B细胞性质及功能，我们检测了不同来源B细胞的表面标记、细胞因子及免疫球蛋白谱（Immunoglobulin, Ig）。流式分析发现DLN中B细胞表达的与其分型相关的标记（CD1d、CD5、IgM、IgD、CD21、CD23）与对照组相比无明显差异，而活化型标记（CD25、CD69）则在DLN B细胞当中表达增高。ELISA检测B细胞分泌的细胞因子显示各组B细胞分泌IL10、IL6、TNFα、PGE2、TGFβ、INFγ、IL12p70及IL2水平无统计学差异。体外利用LPS刺激后，对其免疫球蛋白谱检测发现，DLN B细胞分泌IgG水平较其他组明显增高，这与表达谱芯片的检测结果一致。同时，我们检测了小鼠体内Ig的表达情况，与体外结果相符，荷瘤小鼠体内IgG1、IgG2a及IgG2b水平要高于正常对照小鼠。
为了研究荷瘤小鼠体内这些病理性抗体的特征，我们用纯化后的IgG孵育肿瘤细胞，标记荧光二抗，利用流式细胞仪和激光共聚焦显微镜进行检测。流式结果显示，与正常IgG相比，肿瘤来源的IgG能够特异性结合乳腺癌细胞。同样，激光共聚焦显微镜结果也证实，肿瘤来源IgG能够与肿瘤细胞表面分子特异性结合。
以上研究表明，伴随着肿瘤进程，乳腺癌小鼠DLN中B细胞的比例及数量逐渐增多。进一步表型及细胞因子表达谱结果显示，肿瘤驯化B细胞为一群活化的B细胞，其分泌抗体的能力增强，同时荷瘤小鼠体内抗体水平也增高，提纯体内IgG研究发现，该抗体在体外能够结合肿瘤细胞，具有肿瘤特异性。以上研究为肿瘤微环境影响B细胞提供了新的认识，同时也为进一步研究肿瘤驯化B细胞及其所产生的抗体在肿瘤免疫逃逸中的作用提供了依据。
2、肿瘤驯化的B细胞与病理性抗体在乳腺癌淋巴结转移中的作用研究
为研究肿瘤驯化B细胞及其分泌的抗体对于肿瘤转移的影响，我们应用B细胞缺陷μMT-/-小鼠进行原位乳腺癌种植，并对荷瘤小鼠给予不同来源IgG回输。活体荧光成像结果显示μMT-/-小鼠荷瘤后淋巴结转移的个数较正常小鼠明显减少，但回输了病理性IgG的小鼠其肿瘤转移的能力又显著增强，而回输正常IgG则没有该效应。进一步生存实验表明，回输病理性IgG后μMT-/-小鼠的生存时间明显缩短，与正常小鼠一致。我们也检测了各组小鼠的肿瘤大小，发现回输IgG对肿瘤大小无明显影响。
我们又利用体外实验研究病理性IgG对肿瘤转移中涉及的增殖、凋亡、趋化及侵袭因素的影响。MTT法增殖实验显示，病理性IgG对于肿瘤细胞的增殖无明显影响。在化疗药物诱导的肿瘤细胞凋亡体系中，我们发现病理性IgG能够明显抑制阿霉素所诱导的肿瘤细胞凋亡，而正常对照IgG无该效应。Tranwell趋化实验显示，与空白对照及正常IgG比较，病理性IgG能够明显增强肿瘤细胞向下层淋巴结基质的趋化能力。侵袭实验也有相似结果。
以上实验表明，肿瘤驯化的B细胞不仅能够通过分泌病理性IgG显著促进肿瘤淋巴结转移，而且影响了肿瘤细胞转移的多个环节，包括抵抗凋亡、侵袭及向淋巴结趋化能力，尤其是肿瘤细胞的趋化，使其到达转移部位定居，形成转移灶。既往研究证明多种趋化因子及受体参与肿瘤转移过程，如SDF1a/CXCR4、CCL21/CCR7等，因而，我们在接下来研究肿瘤驯化的B细胞诱导肿瘤转移的具体分子机制时也重点关注了肿瘤驯化的B细胞与影响肿瘤趋化的趋化因子及受体之间的相关性。
3、肿瘤驯化的B细胞与病理性抗体促进乳腺癌淋巴结转移的机制研究
前面的结果提示，病理性IgG能够促进乳腺癌淋巴结转移，我们推测趋化因子及其受体可能参与到该过程。有研究报道，肿瘤细胞高表达趋化因子受体主要是CXCR4和CCR7，其中，SDF1α/CXCR4则是肿瘤向淋巴结转移最为重要的趋化因素。因此，我们检测了IgG刺激后肿瘤细胞这两种趋化因子受体的表达情况。定量PCR结果显示，病理性IgG能够明显刺激肿瘤细胞CXCR4mRNA水平的表达，而正常IgG无该效应，蛋白水平检测得到了同样的结果。病理性IgG是如何促进CXCR4的表达？我们在体外检测了IgG刺激后肿瘤细胞转移相关基因的表达，结果显示，Hif1α及Cox2的表达明显增高。Hif1α是肿瘤适应低氧环境所产生的主要调控因子之一，其表达的高低与肿瘤的转移及预后密切相关。有报道称Hif1α能够通过诱导CXCR4表达从而促进肿瘤细胞的迁移，而SDF1α/CXCR4则是肿瘤向淋巴结转移最为重要的趋化因素。为了验证是否病理性IgG诱导肿瘤细胞Hif1α从而促进CXCR4表达上调，我们检测了不同IgG剂量以及不同时间刺激下肿瘤细胞在mRNA及蛋白水平表达Hif1α的情况。结果提示，与CXCR4表达变化一致，肿瘤细胞在病理性IgG刺激24小时后，其表达的Hif1α的水平明显增高，且随着刺激浓度及时间的增加而不断增高，相反如果用RNA干扰阻断Hif1α的表达，CXCR4的上调幅度受到明显的抑制。
我们又对基质细胞分泌CXCR4配体SDF-1α的调控机制进行了研究，利用IgG刺激后肿瘤条件培养基培养基质细胞，检测基质分泌SDF-1α的水平。结果发现，病理性IgG刺激后的肿瘤上清能够促进SDF-1α的分泌。既往研究表明细胞因子IL-1β、TGF-β、TNF-α以及PGE2等能够调控SDF-1α的分泌。我们用ELISA检测病理性IgG刺激肿瘤细胞上清中细胞因子的种类，发现病理性IgG能够刺激肿瘤细胞分泌大量的PGE2；同时，我们检测了肿瘤细胞在病理性IgG刺激后相关基因的表达水平，发现Cox2在刺激后明显增高，病理性IgG刺激24h后随着刺激时间的增长，Cox2mRNA水平的表达明显增加。所以我们推测肿瘤细胞能够通过Cox2介导的PGE2对淋巴结基质分泌SDF-1α起到调控作用。为了进一步验证PGE2对于SDF-1α的分泌是否有调控作用，我们利用上述基质细胞的培养体系中，当干扰了肿瘤细胞Cox2的表达后SDF-1α的升高效应受到明显抑制，如果在培养体系中直接加入PGE2，又能刺激SDF-1α的分泌。因此，该结果表明Cox2介导的PGE2增高是病理性IgG刺激后的肿瘤上清促进基质细胞SDF1α分泌的影响因素。
上述研究提示病理性IgG能够刺激肿瘤细胞Cox2及Hif1α的表达，从而促进SDF1α与CXCR4这对趋化因子轴功能的发挥，病理性IgG与肿瘤细胞相互作用的具体靶点及传导途径尚不清楚；接下来，我们进一步深入研究病理性IgG介导Cox2及Hif1α表达增高具体分子机制。已有文献报道，肿瘤细胞Hif1α及Cox2的表达受到多个信号通路的调控，如NF-kB、Ras-MAPK等。为检测病理性IgG刺激肿瘤细胞后下游信号通路的活化情况，我们在该刺激体系中加入不同信号通路的阻断剂。结果显示，NF-kB通路阻断剂PDTC能够明显抑制IgG刺激的肿瘤细胞Hif1α及Cox2的上调。
接下来，我们又对病理性IgG如何刺激肿瘤细胞NF-κB通路的活化进行了研究。首先，我们排除了肿瘤细胞表面Fc受体参与该活化过程，并证实IgG与肿瘤细胞表面靶抗原的结合是引起胞内NF-κB通路活化的原因。为寻找靶抗原，我们分离了肿瘤细胞膜蛋白，通过co-IP后进行蛋白质谱检测，发现了15个候选蛋白（Adam10、Nucleolin、Itgb3、Mucin1、Hspa4、Moesin、CD151、Enolase1、Itgb5、TfR1、Erzin、Radixin、Erbb2），siRNA干扰后NF-kB活化程度下降的有Itgb3、Nucleolin、TfR1、Enolase1、Hspa4、Erbb2。为做进一步筛选，我们在293T细胞中过表达基因Itgb3、Nucleolin、TfR1、Enolase1、Hspa4、Erbb2，病理性IgG刺激后检测NF-kB报告基因表达程度，结果显示，293T细胞本身不能被IgG所刺激，在过表达Nucleolin、Hspa4、Itgb3后NF-kB表达活性明显增强。为确定靶抗原，我们通过免疫共沉淀（IP）利用Flag抗体将293T细胞中目的蛋白Nucleolin、Hspa4及Itgb3纯化，免疫印迹（IB）检测发现病理性IgG能够结合Hspa4，而不能结合Nucleolin及Itgb3。进一步共聚焦显微镜检测显示，干扰了4T1细胞Hspa4后，病理性IgG在胞膜表面的结合明显减少。以上结果提示针对靶抗原Hspa4的特异性抗体通过抗原与抗体特异性结合介导NF-kB传导通路的活化从而诱导下游的Hif1α及Cox2表达上调，进而促进SDF1α与CXCR4这对趋化因子轴发挥效应介导了转移前微环境的形成以及肿瘤细胞的定向转移，具体分子机制尚需要进一步验证与深入研究。
综上所述，我们研究发现肿瘤影响下的DLN中B细胞比例及数量显著增加。特征分析显示，这群肿瘤驯化的B细胞分泌抗体的能力明显增强，尤其是IgG1、IgG2a及IgG2b，但是该B细胞大部分表面分子及细胞因子表达谱与正常B细胞没有明显差异。对荷瘤小鼠体内病理性IgG研究显示其具有肿瘤抗原特异性，能够结合在肿瘤细胞表面。体内功能试验证实，B细胞缺失以后，荷瘤小鼠淋巴结转移的个数明显减少，而回输了病理性IgG的小鼠其肿瘤转移的能力显著增强。进一步分子机制探讨表明，病理性IgG能够通过结合特异性肿瘤抗原Hspa4，刺激下游NF-κB通路的活化，从而上调Hif1α及Cox2的表达。Hif1α的表达促进肿瘤细胞表达趋化因子受体CXCR4，而Cox2介导的PGE2分泌则诱导淋巴结基质细胞分泌趋化因子SDF1α，从而促进肿瘤转移前微环境形成并诱导肿瘤细胞向引流淋巴结的迁移。
本课题研究发现在乳腺癌发展过程中，肿瘤驯化下的B细胞及其分泌病理性抗体能够促进肿瘤淋巴结转移，并对其具体分子调控机制进行深入研究，证实在肿瘤免疫调控过程中，B细胞不仅能够通过分泌IL-10和TGFβ发挥负向免疫调控作用而且能够通过产生病理性抗体参与肿瘤转移前微环境的形成诱导肿瘤转移。该研究结果不仅拓宽了人们对于B细胞及抗体性质与功能的认识，也为肿瘤免疫逃逸和肿瘤转移提出新的作用机制，为全面认识复杂而精细的肿瘤免疫调控网络提供了新的视角，也有望为肿瘤的临床治疗及转移防治方法的寻找提供新的启示。
Tumor metastasis is the leading cause for the death of cancer patients. Metastasis is oneof the most important characteristics of malignancy, which is referred to tumor cellspreading from the primary site to other parts of the body where proliferating at the newniches. The metastasis process includes tumor invasion in situ, survival and resting oftumor cells in circulation, settlement in distant sites and colony formation. A variety offactors affect tumor metastasis, and among them, the immune microenvironment hasattracted much attention recently. Through secreting tumor derived factors (TDFs), tumorcells educate the matrix and immune cells to form a stable immunosuppressivemicroenvironment which enables tumor cells to escape from immune surveillance andpromote tumor metastasis. On the other side, immune cells in tumor tissue and distantorgans could change the invasion and metastasis of tumor through direct contacting withtumor cells or indirect secretion of cytokines/growth factors. A variety of immune cellsubsets and molecules were confirmed to participate in the crosstalk with tumor cells,including immunosuppressive cells such as regulatory T cells (Tregs), tumor-associatedmacrophages (TAMs), myeloid-derived suppressor cells (MDSC), and immunosuppressivemolecules such as IL-10, nitric oxide (NO) and arginase (ARG). However, the mechanismsfor how tumor microenvironment can educate immune cells to be immunosuppressive arenow not fully elucidated and need further investigation, so as to break immunosuppressivetumor microenvironment and reverse the function of tumor-educated immune cells for theimmunotherapy of cancer.
B cells are antibody-producing cells and widely distribute in the blood, spleen, lymph nodes and other mucosal tissues. After antigen stimulation, B cells differentiate into plasmacells, which could synthesis and secret immunoglobulin (Ig), and establish humoralimmunity. However, the role of B cells in tumor immunity remain unclear. Dr. ThomasBlankenstein’s group, in cooperation with our laboratory in1998, found that B cells couldinhibit anti-tumor cytotoxic T cells (CTL) responses. Thus, the negativeimmune-regulating function of B cells in tumor immunity gradually emerges, and a B cellsubset with immunosuppressive function has been reported, which is characterized assecretion of IL-10. These IL-10-producing B cells with negative immune regulatoryfunction play an important role in tumor development and metastasis. Therefore, B cellsand their subsets involved in tumor immunity have attracted more and more attention.
In cancer patients, B cells could be activated by tumor antigens, and furtherdifferentiated into plasma cells, which secreted antibodies with anti-tumor effect. Theseantibodies could dissolve tumor cells by activation of the complement system, or by ADCCeffect mediated by macrophages and NK cells. They can also inhibit the growth andmetastasis of tumor cells by blockade of certain receptors on tumor cells (e.g., transferrinreceptor). Antibody therapy is currently the main method of targeted therapy of cancer, andlots of scientists are looking for effective antibody against tumor-associated antigen for thetreatment of cancer. However, the proposal of pathogenic antibody has changed our viewof antibody before. The reports have confirmed that pathogenic antibodies play animportant role in the inhibition of damage-repair, promoting autoimmune diseases and soon. In term of tumor immunity, there are also studies that pathogenic antibody couldpromote tumorigenesis by binding to Fc receptors on immune cells, which could be one ofthe mechanisms of inflammation-caused tumorigenesis. However, the research onpathogenic antibodies in tumor immunity and metastasis is just at the beginning, and themechanisms underlying the tumor-promoting effect of pathogenic antibodies are not fullyilluminated.
Based on the above analysis, we studied the role of B cells and their secreted antibodiesin tumor immunity. We identified a large number of tumor-specific antibodies in the tumor-bearing mice, which promoted tumor metastasis to lymph nodes. Further studyconfirmed that chemokines and their receptors such as SDF1α/CXCR4, up-regulated bythe pathogenic antibodies played important role in the promotion of tumor metastasis bypathogenic antibodies. Our results show the new functions of B cells and their derivedpathogenic antibodies in the negative regulation of tumor immunity and promotion oftumor metastasis.
Our project is divided into the following three parts:
Part I Identification of tumor-educated B cells and pathogenic antibodies
In order to identify the role of lymph node (LN) microenvironment in the tumormetastasis, we analyzed the lymphocyte population in LN in4T1orthotopic implant tumormodel, especially the total number and proportion of B cells. We found that the proportionof B cells in DLN started to increase in the first week post tumor inoculation, andincreased dramatically in the second to third weeks, while the proportion of otherlymphocytes such as T cells, DC and macrophages showed trend to be decreased. Theresults were further confirmed in EMT6breast cancer model.
To further analyze the mechanisms of B cells accumulating in DLN, we studied theproliferation of B cells in vivo and investigated the chemotactic ability of B cells to the LNstromal cells. The proliferation of B cells increased in DLN as detected by Brduproliferation assay. Chemotactic assay indicated that B cells were easier to chemotact tothe DLN stromal cells as compared with that from normal LN. The data showed that tumorenhanced the chemotaxis of B cells to LN through the influence on lymph nodemicroenvironment, and then induced the proliferation of accumulated B cells in situ.
In order to better understand the function of tumor-educated B cells, we detected theirphenotype, cytokines and immunoglobulin profiles. Flow cytometry analysis indicated thatB cells in DLN showed no difference in the expression of traditional B cell markers (CD1d,CD5, IgM, IgD, CD21, CD23) as compared with the control group, while the activatingmarkers (CD25, CD69) were highly expressed in DLN B cells. Then the cytokine profiles of B cells, detected by ELISA, showed that B cells expressed equivalent level of IL6,TNF-α, IL-10, PGE2, TGF-β, IFN-γ, IL-12p70and IL-2as compared to that in controlgroup. After stimulated with LPS, IgG secreted by B cells derived from DLN increasedobviously as compared with other groups. We also detected the serum level of Ig intumor-bearing mice, and the data indicated the level of IgG1, IgG2a and IgG2b was higherthan the control. Further analyses of these IgG revealed that they were tumor-specific andcould bind to the membrane of tumor cells.
These data indicated that the proportion and number of B cells in LN increased withthe tumor process. Further analyses of phenotype, cytokines and Ig profiles showed that asa group of activated B cells, they had enhanced ability of secreting IgG, and the IgG levelsin the serum of tumor-bearing mice also increased. Flow cytometry and confocal studiesindicated that these IgG was tumor-specific and could bind the membrane of tumor cellsbut not to the control cells.
Part II Role of tumor-educated B cells and pathogenic antibodies in the lymph nodemetastasis of breast cancer
To study the function of B cells and antibodies in tumor metastasis, we inoculated breastcancer cells in μMT-/-mice with B cell deficiency,, and different sourced IgG wasadoptively injected afterwards. In vivo fluorescence imaging results showed that,compared with normal mice, the number of metastastic lymph nodes in μMT-/-tumor-bearing mice significantly reduced, but with pathogenic IgG injection, the numberof metastastic lymph nodes significantly increased in μMT-/-tumor-bearing mice, whileadministration of normal IgG have no effect. Further survival experiments showed that theμMT-/-mice survived significantly shorter after administration of pathogenic IgG. Inaddition, pathogenic IgG has no effect on the tumor size, indicating pathogenic IgG doesnot affect the tumor growth.
We took advantage of the in vitro experiment to study the effect of pathogenic IgG in the metastasis, proliferation, apoptosis, chemotaxis and invasion of tumor. The MTT assayshowed that pathogenic IgG did not have significant effect on tumor cell proliferation.Pathogenic IgG could inhibit tumor cell apoptosis induced by chemotherapeutic agentsdoxirubicin, while control IgG could not. The chemoattractant transwell experimentalshowed that, compared with the blank and normal IgG, pathogenic IgG significantlyenhanced tumor cells chemotaxis to the lower matrix. The invasion assay showed similarresults.
Thus, the above experiments indicated that B cells could significantly promote lymphnode metastasis by the secretion of pathogenic IgG, which promoted many profits ofmetastasis, including resistance to apoptosis, invasion and chemotaxis.Part III Mechanism of tumor-educated B cells and pathogenic antibodies to promotebreast cancer metastasis
We found that pathogenic IgG could promote the lymph node metastasis of breast cancer.In order to further explore the molecular mechanism, we examined the expression of genesrelated to metastasis after IgG stimulation in vitro, and the expression of Hif1α and Cox2was observed to be increased significantly. Hif1α, as one of the main factors of tumoradapting to hypoxia, was highly related to metastasis and prognosis of malignancy andcould induce CXCR4expression so as to promote the migration of tumor cells. SDF1α/CXCR4was the most important chemotactic factors related to the lymph node metastasis.In our study, the expression of CXCR4was significantly increased post IgG stimulation,which could be reversed by inhibiting Hif1α expression.
We have also carried out studies on the CXCR4ligand SDF1α using different tumorconditioned medium stimulated by IgG to co-culture with stromal cells. The resultsshowed that, tumor supernatant stimulated by pathogenic IgG could promote SDF1αsecretion of stromal cells. When we interfered Cox2expression of tumor cells, the increaseof SDF1α production is suppressed, which could be rescued by adding PGE2in the culture system. So we found that pathologic IgG could stimulate the tumor cells to produce PGE2and then PGE2promoted the secretion of SDF1α from stromal cells.
We further investigated the mechanisms of elevated Hif1α and Cox2with pathogenicIgG stimulation. Several signaling pathways were assessed and the results showed that theNF-kB pathway inhibitor PDTC could significantly inhibit the up-regulation of Hif1α andCox2. NF-kB reporter gene assay also confirmed pathogenic IgG could enhance thetranscriptional activity of NF-kB in tumor cells. The mechanisms leading to NF-kBactivation were further analyzed. First, we ruled out the Fc receptors on tumor cell surfaceare involved in the activation process, and confirmed that the combination of IgG andtarget antigen on the surface of tumor cells was the cause of NF-kB pathway activation.Looking for the potential tumor antigens/membrane molecules, we isolated tumor cellmembrane proteins and referred to MASS analysis after Co-IP with pathogenic antibodies.Fifteen candidate proteins were identified, including Adam10, Nucleolin, Itgb3, Mucin1,Hspa4, Moesin, CD151, Enolase1, Itgb5, TfR1, Erzin, Radixin, Erbb2. We found thatinterfering with the nucleolin, Hspa4, Itgb3decreased binding of pathogenic antibodieswith tumor cell membrane, and also attenutiated pathogenic IgG-stimulated NF-kBactivation. We also overexpressed nucleolin, Hspa4, Itgb3in293T cells (itself can not bestimulated with pathogenic IgG), and found that pathogenic IgG-stimulated NF-kBactivation was significantly enhanced. To identify the target antigen, we used anti-flagantibody to purify protein Nucleolin, Hspa4and Itgb3in293T cells byimmunoprecipitation (IP). Then immunoblotting (IB) assay found that pathogenic IgGcould bind with Hspa4but not Nucleolin and Itgb3. Further confocal microscope analysisshowed that, the binding of pathologic IgG on the tumor cell membrane decreasedsignificantly after Hspa4silence. Therefore, these results suggested that Hspa4mightmediate activation of NF-kB pathway in tumor cells by pathogenic IgG..
In summary, we found that, under tumor condition, the proportion and total number of Bcells significantly increased in the DLN. Characteristic analysis showed that most of theB-cell phenotype and cytokine secretion did not differ significantly between tumor-educated B cells and normal B cells. However, tumor-educated B cells exerted anactivated phenotype with elevated expression of CD25and CD69. These activated B cellssecreted antibodies more markedly, which was in accordance with the in vivo elevatedIgG1and IgG2b antibody levels in the tumor-beraring mice. Furthermore, these pathogenicIgG from tumor-bearing mice was tumor-specific, and capable of binding to the surface oftumor cells. In vivo function analysis confirmed that, without B cells, the number of lymphnode metastasis was significantly reduced, but administration of pathogenic IgGsignificantly promoted tumor metastasis. Mechanistic study revealed that pathogenic IgGbound to the tumor surface protein Hspa4and stimulated the downstream activation ofNF-kB B pathway, which sequentially up-regulated the Hif1α and Cox2expression. Hif1αpromoted chemokine receptor CXCR4expression on tumor cells and Cox2mediatedPGE2secretion, which then induced lymph node matrix cells to secrete chemokine SDF1α.The corporation of SDF1a and CXCR4promoted tumor cell migration to the draininglymph nodes.
In conclusion, we discovered the new role of tumor-educated B lymphocytes andpathogenic antibodies in promoting lymph node metastasis. Our study will helpunderstanding the new function of B cells and antibodies, and providing a new perspectivefor comprehensive understanding of complex and sophisticated regulating network oftumor immunity.
引文
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