Abstract 3766: Embryonal metastasis assay, an in vivo test of circulating tumorspheres to detect metastatic predisposition
Authors: Geck Peter , Kubilus James , Makarovskiy Andrew , Denes Viktoria 
Published: August 2019

Abstract
INTRODUCTION One of the unsolved problems of cancer is its progression into systemic, metastatic disease. Malignant cells are released into circulation very early (circulating tumor cells or CTCs), but distant metastases are disseminated only by cancer stem cells (CSCs) that survive circulation. We investigated a unique feature of CSCs, formation of cell clusters (spheroids or tumorspheres) in vitro. We found that it also takes place in vivo, cancer tumorspheres circulate in blood and we showed that they carry cancer stem cells and strongly correlate with metastatic disease. In this project we developed a filtration method to collect live circulating cancer stem cell in tumorspheres and searched for a method to establish a prognostic assay to predict their metastatic potential.
METHODS The conventional approaches use knockout mice to assay metastasis (NOD-SCID mice, etc.), but he high cost, the complex procedure and the lengthy assay (5 weeks) ruled them out as a routine clinical assay. Our goal was to establish an in vivo translational assay to detect metastatic predisposition early, with a chance to prevent systemic progression. We found that the rapidly progressing environment in the chicken embryo offers critical advantages to test the metastatic potential of a patient’s cancer. Easy access to chorioallantois circulation and to the embryo, the lack of immune response, the rapid turnaround time (5 days) and the technical simplicity rendered the new embryonal metastatic assay a promising and cost effective translational method. We used tumorspheres of both highly metastatic and non-metastatic cell lines. For image analysis a highly metastatic cell line (MDA-MB-436) was labeled by Green Fluorescent Protein. To model the technique of labeling real life patient spheroids (DiI membrane dye and protein tracing by CFDA-SA), we used the highly metastatic MDA-MB-231 and the non metastatic MCF7 and T47D cell lines. For quantitative analysis of embryonal metastasis we used methods applied in microchimerism detection. Embryonal DNA/RNA was extracted 5-9 days after cancer spheroid implantation and used qPCR to detect human sequences.
RESULTS In the embryonal metastatic assays, fluorescent and confocal imaging detected metastatic cells in the embryo. DiI and CFDA-SA labeling of native tumorspheres indicated a level of toxicity and needed to be optimized. Molecular detection of human microchimerism in the embryos was extremely sensitive and generated quantitative metastatic data.
CONCLUSIONS The results indicate that the embryonal metastasis assay using molecular microchimerism detection has the potential for a sensitive and quantitative method to assess the metastatic predisposition of a cancer patient. The method needs only an incubator, DNA extraction and a thermocycler, easily available in most hospitals and offers cost effective, relevant in vivo prognostic data within days.
Citation Format: Peter Geck, James Kubilus, Andrew Makarovskiy, Viktoria Denes. Embryonal metastasis assay, an in vivo test of circulating tumorspheres to detect metastatic predisposition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3766.