Tumor-Stroma Crosstalk in Bone Tissue: The Osteoclastogenic Potential of a Breast Cancer Cell Line in a Co-Culture System and the Role of EGFR Inhibition

Abstract

Although bone metastases represent a major challenge in the natural history of breast cancer (BC), the complex interactions involved have hindered the development of robust in vitro models. The aim of this work is the development of a preclinical model of cancer and bone stromal cells to mimic the bone microenvironment. We studied the effects on osteoclastogenesis of BC cells and Mesenchymal stem cells (MSC) cultured alone or in combination. We also analyzed: (a) whether the blockade of the Epithelial Growth Factor Receptor (EGFR) pathway modified their influence on monocytes towards differentiation, and (b) the efficacy of bone-targeted therapy on osteoclasts. We evaluated the osteoclastogenesis modulation of human peripheral blood monocytes (PBMC) indirectly induced by the conditioned medium (CM) of the human BC cell line SCP2, cultured singly or with MSC. Osteoclastogenesis was evaluated by TRAP analysis. The effect of the EGFR blockade was assessed by treating the cells with gefitinib, and analyzed with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and Western Blot (WB). We observed that SCP2 co-cultured with MSC increased the differentiation of PBMC. This effect was underpinned upon pre-treatment of the co-culture with gefitinib. Co-culture of SCP2 with MSC increased the expression of both the bone-related marker Receptor Activator of Nuclear Factor κB (RANK) and EGFR in BC cells. These upregulations were not affected by the EGFR blockade. The effects of the CM obtained by the cells treated with gefitinib in combination with the treatment of the preosteoclasts with the bone-targeted agents and everolimus enhanced the inhibition of the osteoclastogenesis. Finally, we developed a fully human co-culture system of BC cells and bone progenitor cells. We observed that the interaction of MSC with cancer cells induced in the latter molecular changes and a higher power of inducing osteoclastogenesis. We found that blocking EGFR signaling could be an efficacious strategy for breaking the interactions between cancer and bone cells in order to inhibit bone metastasis.

The Role of the RANKL/RANK Axis in the Prevention and Treatment of Breast Cancer with Immune Checkpoint Inhibitors and Anti-RANKL

“…Breast cancer cells can also produce RANKL and normal cells in metastatic sites, such as osteoblasts in bone [ 30 , 40 , 41 , 42 , 43 ]. RANKL has also been reported to trigger the migration of human epithelial cancer and melanoma cells expressing RANK in a concentration-dependent manner, thus implying a chemotactic mechanism of action in bones [ 30 ].…”

BOne HEalth ManagEment in Patients with Early Breast Cancer: A Retrospective Italian Osteoncology Center “Real-Life” Experience (BOHEME Study)

“…Alterations in bone homeostasis induced by cancer treatments, including ET, can lead to CTIBL, resulting in a break of normal skeletal structural integrity associated with increased bone turnover and a higher risk of skeletal fractures. The process of increased bone turnover is involved in both tumor growth and clonal expansion, with cellular interactions between tumor cells and other cell types found in the bone microenvironment, similar to the process that occurs during bone metastasization [20,21,22,25]. The “seed and soil” theory of metastasis suggests that cancer cells from the primary tumor the inherent potential to seek conditions similar to their original environment in which to grow and create new bone lesions [26,27].…”

“…Studies on preclinical models have revealed that BMAs may also impact the development of bone metastases through crosstalk between tumor and host cells within the bone marrow. This mechanism seems to have a role in the survival of tumor cells within the bone marrow, suggesting that these agents also have an impact on clinical outcome [20,21,22].…”

The Role of Caspase-4 and NLRP1 in MCF7 Cell Pyroptosis Induced by hUCMSC-Secreted Factors

“…Mesenchymal stem cell- (MSC-) based approaches are being studied extensively for the development of new cancer therapeutic strategies. Human umbilical cord mesenchymal stem cells (hUCMSCs) are widely used in research focused on cancer treatment owing to their easy availability and no ethical issues [ 11 – 13 ]. We previously demonstrated that the factors secreted by hUCMSCs induced pyroptosis in the breast cancer cell line MCF7.Furthermore, RNA sequencing studies revealed a significant increase in the expression of pyroptosis-related genes CASP4 and NLRP1 in pyroptotic MCF7cells [ 14 ].…”