Tumor-educated B Lymphocytes Promote Breast Cancer Metastasis Via Pathogenic Antibodies

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 antibodiesIn 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 cancerTo 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 metastasisWe 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.