Abstract 4023: Identification of novel compounds to target dormant micrometastatic cells
Authors: Hurst Robert E. , Ihnat Michael A. , Hauser Paul J. , Bailey-Downs Lora C. , Bourgignon Lilly Y. 
Published: May 2015

Abstract
In spite of decades of research and billions of dollars, cancer-specific death rates have changed little, mainly because 90% of cancer patients who die succumb to metastatic disease. Our hypothesis is that the most vulnerable target for cancer therapy is the micrometastatic cell that leaves the primary tumor early in the disease history, lies dormant or suppressed by the normal extracellular matrix (ECM) and then emerges even decades later. We here describe a novel approach to identifying compounds that target micrometastatic cells suppressed by normal ECM. We first developed an in vitro 96-well format screen based on an earlier observation in our lab that the normal ECM suppresses the malignant phenotype. Using paired wells, one of which contains a normal ECM gel (small intestine submucosa, SISgel) and the other is just bare plastic, known cancer therapeutics show lower activity (more resistance) in cells grown on SISgel than on plastic. Our endpoint was the opposite pattern, namely more resistant on plastic. We screened 3,000 compounds in an NCI diversity set and identified 3 with the requisite activity. All had low toxicity in mice (&lt;45 mg/kg). EC50 values were in the range of 3-30 µM, depending on the cell line. Screening of an additional 10K compounds identified 2 additional compounds, but they were too toxic to mice to be useful. We evaluated the activities of these compounds in vivo using a novel flank xenograft method we developed in which GFP-labeled cancer cells are co-injected with SISgel into the flank. The SISgel reprograms the cells and substantially normalizes them, even after the SISgel is resorbed such that a flat, glowing green spot marks the suppressed cells. Histopathology confirmed a substantial normalization. Cancer cells co-injected with Matrigel show rapid tumor growth. The lead compounds significantly eliminated the suppressed cells, whereas conventional chemotherapeutics were ineffective. Using a 4T1 triple negative breast cancer model modified for physiological metastatic progression; surprisingly, 2 of the 3 lead compounds given intraperitoneally or in osmotic pumps decreased primary tumor growth more effectively than did docetaxel and resulted in small decreases in small lung micrometastases. However, as predicted, the main effect was to significantly (P&lt;0.01, one-way ANOVA/Tukey post test) reduce the formation of lung macrometastases almost to zero. Given this result, we tested the activity of these compounds against ALDH+ CD44v3+ stem cells (SC) isolated from the parental line and found that one of the compounds specifically targeted the SC (EC50 = 50 µM vs 630 µM for doxorubicin). The SC also retained their stemness on SISgel. Mechanistic studies showed a weak, late apoptotic response but depending on the compound, blocking either at G1/S or G2/M in the cell cycle. In summary we describe a novel, first in class set of compounds that target micrometastatic cells and prevent their differentiation into cancer cells that form tumors.
Citation Format: Robert E. Hurst, Michael A. Ihnat, Paul J. Hauser, Lora C. Bailey-Downs, Lilly Y. Bourgignon. Identification of novel compounds to target dormant micrometastatic cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4023. doi:10.1158/1538-7445.AM2014-4023