Abstract P5-11-02: Humoral immune responses direct breast cancer towards a basal-like phenotype and increases stemness and invasive potential
Authors: Mourtada Jennifer Sims , Maosa Steficah , Opdenaker Lynn , Toney Nicole 
Published: March 2022

Abstract
Introduction: A large subset of triple negative breast cancers (TNBC) are characterized by a high degree of lymphocytic infiltration. Previous studies have shown that TNBC contain higher densities of B lymphocytes than other breast cancer subtypes. B lymphocytes infiltration occurs early in breast cancer and is associated with microinvasion. Although the exact role of B cell in growth of TNBC is unclear, B cells have been shown to induce chronic inflammatory responses in other types of cancer such as melanoma through the activation of NF-KB. Chronic inflammation is known to increase stem cell features and promote epithelial mesenchymal transition (EMT) in cancer. Therefore, we sought to examine interactions between B cells and TNBC and determine the effects on stemness and phenotype of breast cancer cells. Methods: CD20 expression was evaluated in tissue microarrays of ER- DCIS by immunohistochemistry. To mimic the B cell rich tumor microenvironment, co-culture studies were performed with the TNBC cell line SUM159 and EBV-transformed B cells using FalconTM Cell Culture Insert with a 0.4 um filter. Expression of stemness genes in tumor cells after co-culture was evaluated using Human pluripotent stem cell kit array (Qiagen). Expression of EMT and stemness genes and markers were determined by qPCR and western blotting. Self-renewal was assessed using mammosphere assays of co-cultured cells compared to controls. Invasion assays were performed in SUM159 and MCF-12A cell lines after co-culture using Corning® BiocoatTM cell culture inserts. Results: High densities of B cells were found in 9 of 36 DCIS (25%) and associated with microinvasion (p=.035). A 2-fold increase in invasion was observed in co-cultured cells compared to controls. RTqPCR showed increased expression of stemness genes OCT4 and Nanog (1.3- and 2.3-fold change respectively). Additionally, increased protein expression of stemness genes (Snail, SOX17, α-Fetoprotein AFP, Otx2, Nanog, Sox2, Oct4 and pdx-1) was observed in co-cultured cells compared to the control in the commercial array analysis. Likewise, we observed a 2- fold increase in ALDH1A3 gene expression which correlated with an increase in ALDH1A3 protein expression in co-cultured cells compared to controls. Additionally, co-cultured cells exhibited a two-fold increase in mammosphere formation compared to controls indicating an increase in self-renewal. A 7-fold increase in gene expression in the mesenchymal marker vimentin and a slight increase expression of snail was observed. This change correlated with a decrease in gene and protein expression of E-cadherin indicating an increase in EMT in co-cultured cells. Conclusion: Our study confirms the finding that B cell infiltration in stage 0 breast cancer is associated to microinvasion. Further, our in vitro studies indicate that interactions between B cells and TNBC may lead to increased invasion, stemness, and EMT. Thus, early B cell infiltration may be a driver of aggressive disease. Further study is needed to examine cross-talk between B cells and TNBC which may identify new targets for therapeutic intervention. Additionally, studies should examine CD20+ B cell infiltration as a prognostic marker for ER- DCIS progression.
Citation Format: Jennifer Sims Mourtada, Steficah Maosa, Lynn Opdenaker, Nicole Toney. Humoral immune responses direct breast cancer towards a basal-like phenotype and increases stemness and invasive potential [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-11-02.