Introduction: Metastasis, the spread of cancer from a primary tumor to a secondary tissue site, is the leading cause of cancer-associated deaths. Breast cancer most often metastasizes to bone, brain, and lung tissues and becomes challenging to treat once it has become metastatic, and most often patients with metastases are treated with hormone therapy or chemotherapy. Hormone therapy targets specific cell receptors and chemotherapy is toxic to all cells and patients commonly develop resistance to these treatments making them ineffective in the long term. Triple negative breast cancer (TNBC) is both difficult to treat, because of its lack of receptor expression, and it aggressively metastasizes. We focus here on bone marrow because metastatic tumors disrupt critical bone functions. Within the bone marrow microenvironment, breast cancer cells could potentially become drug resistant because of limited drug diffusion, as well as survival signals from cells and the extracellular matrix1. Of particular interest to us, mesenchymal stem cells (MSCs) derived from bone marrow have been shown to facilitate both drug resistance2 and metastasis3. In this work we are interested in studying the drug response of epidermal growth factor receptor (EGFR)-overexpressing TNBC cell lines when exposed to soluble factors from the bone marrow microenvironment.
Materials and Methods: EGFR targeting drugs (Afatinib, Erlotinib, Gefitinib, & Lapatinib) and chemotherapy (Doxorubicin) at 0-50 μM were tested on EGFR-overexpressing TNBC cell lines (MDA-MB-231, BT-549, MDA-MB-468, & Hs578T) for 48 hours in 96 well plates. These tests were done with and without MSC conditioned medium. MSC conditioned medium was made by growing MSCs for 72 hours and collecting the medium at this time point. This medium contained the soluble factors released by the MSCs. The drug response was mesured by using the CellTiter-Glo ATP assay and the concentration of drug required to kill 50% of the cells (IC50) was calculated. The growth rate of the cell lines in the two media conditions without drug was measured at 1, 2, 4, and 7 days using the CellTiter 96 proliferation assay. The cell morphology at selected conditions was analyzed with manual cell image tracing and ImageJ. In separate experiments, hTERT MSCs were treated with a very low dose of drug to create drug-treated conditioned medium, and this was used to study how pretreating the MSCs with a drug affects cancer cell response to drugs. The levels of 15 soluble factors in the conditoined medium were measured with a high-throughput bead based protein quantification system (MAGPIX).
Results and Discussion: We used a simple drug screening platform that incorporates factors from the microenvironment to show that bone marrow-derived MSC soluble factors affect cancer cell drug response. The Hs578T cell line is resistant to Doxorubcin in the presence of MSC released factors and the surviving cells have a pronounced nuclear shape change. We are working to identify the specific factors that are responsible for the observed results. The MAGPIX results show that SDF-1α may be one of the soluble factors causing this effect. We have also observed that MDA-MB-231 cells die in the MSC conditioned medium after a few days without the presence of any drug. Cancer cell drug resistance to Lapatinib has been observed with Lapatinib-treated MSC conditioned medium. This suggests that treating the MSCs with drug changes the profile of the soluble factors that they release.
Conclusions: Soluble factors released by MSCs provide protection to Hs578T cells treated with Doxorubicin. Interestingly, this same conditioned medium kills the MDA-MB-231 cells even in the absence of drug. Ongoing studies include using conditioned medium from MSCs that were treated with drug to more accurately capture what happens in vivo when a patient is dosed with a drug.
References:  Meads, MB. Clinical Cancer Research. 2008. 14(9):2519-26.  Roodhart, JM. Cancer Cell. 2011. 20(3):370-83.  Karnoub, AE. Nature. 2007. 449(7162):557-63.
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