Abstract
Introduction
The bone marrow microenvironment regulates the self-renewal and differentiation of hematopoietic stem and progenitor cells (HSPCs), through a network dependent on cell-cell interaction. This interaction is mediated by morphogens, the extracellular matrix and cell adhesion molecules expressed and secreted by various cell types in the HSPC niche. Mesenchymal stromal cells (MSCs), as the major cellular component, maintain the stemness properties of the niche. The microenvironment thus becomes conducive for HSPCs to remain quiescent, thereby enabling long term self-renewal. Therefore, the safe haven in the bone marrow microenvironment and its constituent cell types can be targeted during tumorigenesis, thus making the niche neoplastic. Dissemination of breast cancer cells into the bone marrow has been described even in the early stages of the disease. The present study focuses on the influence of breast carcinomas on the genetic and functional profile of mesenchymal and hematopoietic progenitor cells of the bone marrow niche.
Methods
In vitro coculture models of breast cancer cell lines- MDA-MB231, MCF-7 and primary MSCs derived from the bone marrow of healthy donors were used in the study. Atomic- force microscopy based single-cell force spectroscopy (AFM-SCFS) and fluorescence based assays were used for cell adhesion experiments. Hydrogel based culture systems were used for 3-dimensional cocultures of breast cancer cells and MSCs. Hypoxic and normoxic culture conditions (0.5% and 20% oxygen respectively) were used for the experiments.
Results
The breast cancer cell lines caused a significant reduction in HSPC adhesion to MSCs (88% by MDA-MB 231 cells; p<0.005 and 73% by MCF-7 cells; p<0.005). AFM-SCFS studies also indicated a higher binding force between breast cancer cells and MSCs, as compared to HSPCs (MDA-MB231 cells-0.13nN, MCF-7 cells-0.074nN and HSPCs-0.05nN). MDA-MB231and MCF-7 cells express Intercellular adhesion molecule-1(ICAM-1), which has been shown to promote breast cancer metastasis (Hanlon et al, 2002; Rosette et al, 2005; Schröder C. et al, 2011). There was a significant difference in reduction of HSPC adhesion towards MSCs by ICAM-1 knockdown (ICAM-1 KD) tumor cells as compared to MDA-MB231 cells (84.83% by MDA-MB231 cells versus 28.11% by ICAM-1KD tumor cells, p<0.001). AFM-SCFS studies also showed a reduced binding force between ICAM-1 KD tumor cells and MSCs as compared to MDA-MB231cells (MDA-MB231 cells-0.14nN versus ICAM-1-KD tumor cells-0.05nN, p value<0.001). ICAM-1 KD studies thus showed that reduction in HSPC adhesion to MSCs by breast cancer cells was mediated through ICAM-1 signaling. A cytokine array was performed to investigate if breast cancer cell lines affect the cytokine profile of MSCs. The array showed altered expression of growth factors- Basic fibroblast growth factor (bFGF) and Platelet derived growth factor–beta (PDGF-BB) (2.2 fold upregulation and 0.5 fold downregulation in breast cancer cells- MSC cocultures respectively). Based on the array, a bFGF-mediated increase in the proliferation of MSCs and breast cancer cells in coculture was observed. The bFGF upregulation also caused an increased migration of MDA-MB231 cells towards MSCs in a transwell migration assay. An upregulation in the phosphorylation status of Akt was observed in breast cancer cells – MSC cocultures, as a downstream effect of upregulated bFGF levels. The bFGF-mediated increase in the proliferation of breast cancer cells and MSCs in coculture was shown to be dependent on the activation of PI3K-Akt pathway. The bFGF- mediated increase in the migration of MDA-MB231 cells towards MSCs was also inhibited upon addition of the PI3K blocker. Interestingly, the breast cancer cells caused a reduction in osteoblastic differentiation of MSCs by downregulation of PDGF-BB. Studies with 3-dimensional cocultures of breast cancer cells and MSCs also showed a reduction in osteoblastic differentiation of MSCs. Furthermore, long-term cocultures of breast cancer cells, HSPCs and MSCs showed reduced support for primitive HSPCs in the neoplastic niche.
Conclusions
These findings indicate a perturbed HSPC niche upon tumor invasion. The possible role of altered cytokine expression, consecutive downstream signaling in niche activation and bone turnover will be further studied using in vitro and in vivo approaches to recapitulate tumor micrometastases to the HSPC niche.
Disclosures
No relevant conflicts of interest to declare.
Periostin gene expression in neu-positive breast cancer cells is regulated by a FGFR signaling cross talk with TGFβ/PI3K/AKT pathways
