Abstract 1545: Metastasis blood test by in vivo tumorsphere detection
Authors: Denes Viktoria , Graf Laszlo , Barna Gabor , Tegze Balint , Jakso Pal , Pallinger Eva , Geck Peter 
Published: July 2016

Abstract
INTRODUCTION Metastasis is the turning point in the progression of most cancers. The prognosis of systemic, disseminated cancer is usually negative, but methods for early metastasis detection are not available in the clinical practice. We present a novel approach that may have the potential to detect metastatic predisposition early from patient blood.
BACKGROUND It is almost universally accepted that metastases are disseminated by cancer stem cells. It is also known that hypoxia in a cancer is a major factor in therapy-resistance and dissemination. The findings are consistent with other observations that stem cells rapidly adapt to hypoxia and prefer hypoxic metabolism. We studied cancer stem cell differentiation and found that most stem cells (cancer and normal) readily form spheroids in suspension culture in vitro. Further studies showed that these spheroids develop hypoxic centers and are positive for stem cell markers.
HYPOTHESIS These observations suggest that in vitro spheroid formation may reflect a fundamental biological feature of cancer stem cells and they may find a perfect niche in the hypoxic microenvironments in spheroids. The fact that spheroid formation is universally found in vitro raised the possibility that stem cells may also form spheroids in vivo, and by providing a “portable” hypoxic milieu, circulating tumorspheres may be critical in metastatic dissemination. We investigated, therefore, whether cancer spheroids can enter circulation and worked out the methodology to detect them in blood.
EXPERIMENTAL PROCEDURES Blood samples were collected from 106 patients, 62 metastatic and 44 controls. The samples were analyzed by flow cytometry using forward scatter and fluorescent labels. For spheroid capture we used simple nylon mesh filters (10 um and 20 um pore size). From the collected spheroids total RNA was isolated and gene expression studies were performed for stem cell, hypoxia and other markers. Both the collected spheroids and in vitro spheroid models were analyzed by immunohistochemistry.
RESULTS We found that spheroids show complex metabolic architecture with hypoxia, stem and other markers. We tested several flow cytometers and found that the wide scatter of spheroids was not optimal in some instruments, but performed well in Partec and Beckman-Coulter systems. In our pool almost half of the metastatic samples were spheroid positive, while all healthy samples were negative. Follow up clinical studies will investigate the prospective significance of the results.
CONCLUSIONS Our data demonstrate significant correlation between cancer spheroid positivity in blood and metastasis. The results suggest that circulating in vivo spheroids may be involved in the dissemination of metastatic cancer stem cells and can be used as a new prognostic/diagnostic approach.
Citation Format: Viktoria Denes, Laszlo Graf, Gabor Barna, Balint Tegze, Pal Jakso, Eva Pallinger, Peter Geck. Metastasis blood test by in vivo tumorsphere detection. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1545.