Abstract 2714: The metastatic potential of circulating cancer spheroids: Diagnostic assay development using embryonic circulation models
Authors: Geck Peter , Kubilus James , Makarovskiy Andrew , Marinelli Kathleen , Kyritsis Christine , Denes Viktoria 
Published: September 2020

Abstract
Introduction: Progression into metastatic disease is a critical step in most malignancies. Cancer cells may enter circulation very early (circulating tumor cells or CTCs), but distant metastases are disseminated only by cancer stem cells (CSCs) that survive circulation. CSCs are known to form cell clusters in vitro (spheroids/tumorspheres). We reported that tumorspheres also circulate in blood in vivo. We found that they carry cancer stem cells and strongly correlate with metastatic disease. We collected tumorspheres from blood by filtration. This project aims to test the metastatic potential of tumorspheres by developing an assay using in vivo circulation models. The goal is to establish a novel prognostic test for metastatic predisposition in cancer patients.
Methods: To develop an in vivo assay for metastasis we use the chorioallantoic circulation in the chicken embryo, with several advantages: easy access to circulation, lack of immune response, rapid turnaround time (5 to 10 days) and inexpensive and simple technique. For clinical applications we introduced three technical innovations: (i) to make fertilized chicken embryos easily available we worked out a method to store viable embryos beyond 30 days; (ii) we developed a new coverglass chamber technique for micromanipulations; and (iii) for microscopic analysis new over-drained chorioallantois preparations can accommodate high resolution objectives. To test tumorspheres in embryonic circulation we used both highly metastatic (e.g. MDA-MB-436) and non-metastatic (e.g. MCF7) cell lines with GFP label, DiI membrane dye and CFDA-SA dye. For molecular studies DNA was extracted 5 days after cancer spheroid implantation and used qPCR to detect human sequences.
Results: In traditional storage few embryos survive beyond 6 days. As fertilized eggs carry pluripotent stem cells, we optimized temperature, gas and humidity combinations to match known stem cell optimums. By suppressing metabolism, blocking embryo adhesion and controlling gas and humidity exchange we extended viable embryo storage beyond 30 days. We modified the traditional window method by forming visually clear microscopic coverglass chambers, and the new over-draining approach lowered the chorioallantois membranes and allowed high resolution imaging. The results showed invasive and hematogenic dissemination of the cancer spheroids. Molecular studies of human markers detected metastatic progression and generated quantitative data.
Conclusions: We optimized the in vivo embryonic metastasis assay. We show that it has the potential for a sensitive and quantitative method to diagnose metastatic predisposition in cancer patients. The molecular approach (DNA extraction, PCR markers) is easily up-scalable, the instrumentation is available in most hospitals and offers cost effective and critical early diagnosis of cancer progression.
Citation Format: Peter Geck, James Kubilus, Andrew Makarovskiy, Kathleen Marinelli, Christine Kyritsis, Viktoria Denes. The metastatic potential of circulating cancer spheroids: Diagnostic assay development using embryonic circulation models [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2714.