2340P - T-cell immunoglobulin and mucin-domain containing molecule-3 (Tim3) in breast cancer and the impact on penetration of blood brain barrier by cancer cells

Background

Tim3 (T-cell immunoglobulin and mucin-domain containing molecule3) plays an important role in metastatic spread of cancers. The blood brain barrier (BBB) is an essential barrier that breast cancer cells have to overcome to metastasize to the CNS. This study investigated the association/functionality of Tim3 in breast cancer cells and in the invasiveness of cerebral endothelial cells by cancer cells.

Methods

Human cerebral microvessel endothelial cells (hCMEC/D3) were used to model BBB in vitro. Tim3 knockdown was by anti-Tim3 siRNA in MCF-7 and MDA-MB-231 cell lines. Electric cell impedance sensing (ECIS) was used for assessing cellular attachment and migration. Transepithelial resistance (TEER) and paracellular permeability (PCP) were used to test the barrier functions of the cells. Cellular response to docetaxel was evaluated and shown as IC50.The effects of Tim3 knockdown on penetration through the hCMEC/D3 monolayer were also assessed.

Results

Tim3 knockdown (Tim3kd) cell models were successfully established with MCF-7 and MDA-MB-231 cells. Loss of Tim3 in these cells resulted in a decrease in both cell adhesiveness and migration. The knockdown also led to a reduced transepithelial resistance as shown by TEER assay and an increase in paracellular permeability when compared with wild type (WT) cells. It was noted that loss of Tim3 rendered both MCF-7 and MDA-MB-231 cells resistant to docetaxel (IC50 for WT and Tim3kd respectively being 0.86nM vs 2.2nM for MCF7 and 1.35nM vs 11.8nM for MDA-MB-231). In the cerebral endothelial cell invasion assay, we found that Tim3 knockdown in both MCF-7 and MDA-MB-231 resulted in a significantly reduced invasion of the breast cancer cells through the endothelium (31±8 vs 24±8 for MCF7, p=0.018; 211±48 vs 142±54 for MDA-MB-231, p<0.001, WT and Tim3kd respectively).

Conclusions

Tim3 has a marked influence on the barrier functions, adhesiveness and the sensitivity to docetaxel of breast cancer cells. Loss of Tim3 in breast cancer cells reduces tumour cell's penetration of cerebral endothelium. Collectively, Tim3 plays a key role in the maintenance of barrier functions in breast cancer and their potential breach of the blood brain barrier.

Clinical trial identification

Editorial acknowledgement

Legal entity responsible for the study

The authors.

Funding

Has not received any funding.

Disclosure

All authors have declared no conflicts of interest.