The impact of B lymphocytes in the microenvironment of triple negative breast cancer
Date
2019
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
University of Delaware
Abstract
Triple negative breast cancer (TNBC) is an aggressive breast cancer subtype which accounts for approximately 10-20% of all breast cancers. Patients tend to have poor survival, high rates of early recurrence, and high rates of distant metastasis. TNBC does not express hormone receptors or human epidermal growth factor receptor 2, and therefore cannot be targeted through targeting of those receptors. Overall, there is a lack of targeted therapy for this disease. ☐ The tumor immune microenvironment may either fight cancer or promote its growth. There is a lack of consensus regarding the function of B cells in the tumor microenvironment. B cells, which are part of the humoral immune response, make up a large portion of immune cells infiltrating into the TNBC microenvironment. Preliminary analysis of gene expression in TNBC compared to normal breast tissue revealed upregulation of the humoral immune response along with activation of pro-inflammatory nuclear factor kappa B (NFκB) signaling. Culture of TNBC cells with B cells resulted in upregulation of interleukin 1 beta (IL-1β) gene expression in TNBC cells. IL-1β is both a driver and a target of NFκB signaling, and both NFκB signaling and IL-1β contribute to invasiveness and aggressiveness in cancer. Therefore, I hypothesized that B cells promote an inflammatory microenvironment that promotes aggressiveness of TNBC. I tested this hypothesis through determination of the impact of B cells on inflammatory signaling and invasiveness in TNBC, and profiling B cells and inflammatory signaling in the microenvironment of TNBC patient tissue samples. ☐ I found that B cells upregulated IL-1β-driven NFκB signaling in TNBC cell lines derived from non-cancerous breast tissue, a primary tumor, and a metastatic site. This was demonstrated through analysis of nuclear translocation of p65 by immunofluorescence, western blot analysis of p65 phosphorylation, gene expression analysis of IL-1β and interleukin 8 (IL-8), and ELISA of IL-1β protein released into culture serum. Co-culture with B cells also increased invasiveness of all TNBC cell lines which was inhibited by blocking IL-1β. B cell co-culture also resulted in increased migratory activity of TNBC cells and increased activity of matrix metalloproteinases 2 and 9. ☐ Analysis of ductal carcinoma in situ (DCIS) and invasive TNBC patient tissue sections was done to compare in vitro findings to ex vivo patient tumors. DCIS is a non-obligate precursor to invasive TNBC, defined by abnormal growth of cells confined to the breast duct. DCIS and invasive ductal carcinoma (IDC) samples were immunohistochemically stained for mature B-cell marker CD20 and plasma cell marker CD138. Samples were also quantified for expression of IL-1β and IL-8. Spearman’s correlation analysis revealed that tumor cell expression of IL-1β correlated with increased levels of infiltrating CD20+ B cells in DCIS. No correlation was found between B cells and IL-1β in invasive TNBC, although B cells did associate with higher levels of IL-8 expression in the tumor microenvironment. However, IL-1β tumor cell expression associated with worse overall survival, breast cancer specific survival and recurrence free survival in invasive TNBC, indicating the clinical importance of investigating IL-1β as a target for treatment. ☐ I also identified a population of immunoglobulin g4 (IgG4+) B cells in TNBC that associates with significantly worse overall survival, breast-cancer specific survival, and recurrence-free survival. IgG4+ cells are immunosuppressive and therefore may hinder antitumorigenic responses. Class switching may be driven by presence of interleukin 10 (IL-10). I found that IgG4+ B cells associate with expression of IL-10 by tumor cells, and culture of TNBC cells with primary B cells resulted in increased class switching to IgG4. ☐ Taken together, this data supports the hypothesis that B cells promote an inflammatory microenvironment that promotes aggressiveness of triple negative breast cancer. I also provide support for further investigation of targeting IL-1β for TNBC treatment.
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Keywords
Biological sciences, B lymphocytes, Breast cancer, Humoral immunity, Immunology, Molecular biology