A novel computational predictive biological approach distinguishes Integrin ß1 as a salient biomarker for breast cancer chemoresistance.

Chemoresistance is a primary cause of breast cancer treatment failure, and protein-protein interactions significantly contribute to chemoresistance during different stages of breast cancer progression. In pursuit of novel biomarkers and relevant protein-protein interactions occurring during the emergence of breast cancer chemoresistance, we used a computational predictive biological (CPB) approach. CPB identified associations of adhesion molecules with proteins connected with different breast cancer proteins associated with chemoresistance. This approach identified an association of Integrin ß1 (ITGB1) with chemoresistance and breast cancer stem cell markers. ITGB1 activated the Focal Adhesion Kinase (FAK) pathway promoting invasion, migration, and chemoresistance in breast cancer by upregulating Erk phosphorylation. FAK also activated Wnt/Sox2 signaling, which enhanced self-renewal in breast cancer. Activation of the FAK pathway by ITGB1 represents a novel mechanism linked to breast cancer chemoresistance, which may lead to novel therapies capable of blocking breast cancer progression by intervening in ITGB1-regulated signaling pathways.

Isolation and Culture of Mouse Hepatocytes and Kupffer Cells (KCs).

Nonalcoholic steatohepatitis (NASH) is characterized by accumulation of lipids in the hepatocytes (steatosis) and chronic inflammation. Liver resident macrophages (Kupffer cells) play a pivotal role in inducing inflammation. Cross-talk between hepatocytes and Kupffer cells (KCs) regulate both steatosis and inflammation during the pathogenesis of NASH. Isolated hepatocytes and KC serve as important tools to study mechanistic events during NASH in an in vitro setting. Because mice and humans share identical genes, primary mouse hepatocytes and KC are valuable ex vivo models for NASH studies. However, isolation of mouse liver cells is challenging and requires specific technical procedure and skills. Here, we elaborate a method for effective isolation of both primary hepatocytes and KC from adult liver of the same mouse. This protocol can be used for isolation of liver cells from both wild-type (WT) and genetically-engineered mice. The principle of the method is based on a two-step collagenase perfusion technique in which the liver is washed by perfusion, liver cells are segregated by collagenase treatment, and hepatocytes and KC are then purified and cultured. We optimized this protocol in terms of reproducibility, yield of different population of liver cells, and viability.

Mouse Bone Marrow Cell Isolation and Macrophage Differentiation.

The rapid increase in the incidence of obesity contributes to a parallel increase in nonalcoholic steatohepatitis (NASH). Monocyte-derived macrophages, recruited from the bone marrow to the liver, promote NASH-related inflammation and fibrosis. In addition, adipose tissue macrophages (ATMs) release pro-inflammatory cytokines (PICs) which stimulate adipose tissue lipolysis liberating free fatty acids (FFAs) that can accumulate in the liver as triglycerides (TGs), thereby inducing steatosis. As such, bone marrow-derived macrophages (BMDMs) function as an essential tool to study the pathogenesis of NASH. BMDMs are primary bone marrow-derived cells which are differentiated into macrophages in vitro in the presence of growth factors. Macrophage colony-stimulating factor (M-CSF) is required for the proliferation and differentiation of committed myeloid progenitors into cells of the macrophage/monocyte lineage. Here, we describe a protocol for the isolation of mouse bone marrow cells and subsequent macrophage differentiation in which bone marrow cells are cultured in the presence of M-CSF, supplemented either by conditioned medium from L929 cells or in purified form. The efficiency of the differentiation is confirmed by immunofluorescent staining of macrophage surface antigen F4/80. The BMDMs serve as an excellent ex vivo model for a variety of studies, including hepatocyte-macrophage and adipocyte-macrophage cross-talk regulating NASH.