Cell type analysis of lymphocyte infiltration and cytokine profiles in breast cancer development and progression
Characterization of the crosstalk between tumor cells and infiltrating immune cells in breast
Age and BMI are independently associated with inflammation. In order to characterize the interactions between immune and breast cancer cells in vitro, we develop a co-culture system which allow to study crosstalk between immune and cancer cells, in which we will consider miRNAs, cytokines and chemokines as mediators of these interactions.
Tumors are composed by a heterogeneous group of cells: malignant, normal epithelial/stromal, endothelial and a wide range of immune cells. Understanding how immune cells interact with cancer cells is of increasing interest, as cancer treatments move towards combining both targeted- and immuno- therapies in new treatment regimes. Molecules such as cytokines and chemokines are secreted by cancerous cells to promote tumor growth and progression, but they also affect the type of immune cells infiltrating the tumor and/or their differentiation. In return, the infiltrating immune cells influence tumor cells homeostasis, either directly through identification and elimination or indirectly via the secretion of small molecules or vesicles. Collectively, these local changes and interactions promote the development of the tumor microenvironment. Previously, we have performed gene expression profiling in 195 breast tumors and their corresponding matched adjacent normal tissue, as well as 43 normal tissue samples from women undergone mammoplastic reduction without cancer. We identified that age and BMI were independently associated with inflammation in normal tissue but not tumors, with increased expression of macrophages and other inflammatory cells seen in patients with high BMI (>30) and older age (>50). Adjacent normal tissue from early-onset ER-negative patients had profoundly elevated luminal and basal gene expression and decreased adipose expression compared to adjacent normal tissue from all other patients. Additionally, macrophage expression was lower in ER-positive tumors compared to adjacent normal tissue in direct proportion to increased expression of ESR1 and genes with ESR1-binding activity, suggesting elevated estrogen levels may affect tumor-resident macrophages and immune cells in these patients. We calculated a novel statistic to quantify the rewiring of gene co-expression networks and demonstrated that basal gene networks are rewired in ER-positive tumors, even though their gene expression levels are not significantly different from adjacent normal tissue. This study mainly illustrated how patient age and tumor subtype interact to affect inflammation and proliferation in breast tumors and the tumor microenvironment. In the first year of the project we have performed comprehensive miRNA analysis of the same samples and at present analyse the effect of miRNAs on the observed immune profiles. To further characterize the interactions between immune and breast cancer cells in vitro, we aim at developing a co-culture system, which allow studying crosstalk without direct contact between immune and cancer cells. Two breast cancer cell lines will be used (1) the MCF7 cell line to model Estrogen Receptor (ER) positive disease and (2) MDA-MB-231 which reflects ER negative breast cancers. CD4+ T cells, CD8+ T cells, CD19+ B cells and CD14+ monocytes will be purified from human peripheral blood using magnetic beads sorting. Following co-culturing a broad range of analysis will be pursued. In total, this project will allow to characterize the changes induced by immune cells on cancer cells and vice and versa. The ultimate goal is to identify which type of exosomes or cytokines in the cell culture media induce specific molecular changes in cancer or immune cells.
Cytokine profiling of tumor interstitial fluid of the breast and its relationship with lymphocyte infiltration and clinicopathological characteristics.
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