Abstract B03: Blood test of metastatic predisposition in colon cancer
Authors: Denes Viktoria , Graf Laszlo , Barna Gabor , Tegze Balint , Jakso Pal , Geck Peter 
Published: March 2017

Abstract
Introduction: In the progression of colorectal cancer, metastasis is a critical step in the development of systemic disease. Early diagnosis of metastatic progression, however, is not available. We present a novel approach to detect metastatic predisposition early, from patient blood.
Background: Metastasis is disseminated by cancer stem cells. They rapidly adapt to hypoxia, they prefer hypoxic metabolism, which is also a major factor in clinical therapy-resistance and dissemination. By studying cancer stem cell differentiation we found that they readily form spheroids (small multicellular clusters or organoids) with hypoxic centers.
Hypothesis: We propose that cancer stem cells develop hypoxic spheroids to provide a protected hypoxic micro-niche for survival. Based on the fact that spheroid formation is universally found in vitro, we asked whether cancer stem cells may also form spheroids in vivo, when they enter circulation. Our hypothesis is that in vivo circulating tumorspheres carry cancer stem cells and contribute to metastatic dissemination. To investigate the possibility of circulating cancer spheroids in colorectal and other cancers, we worked out the methodology to detect and characterize them from patient blood.
Experimental Procedures: Blood samples were collected from 20 colorectal and 86 mixed cancer cases. 16 of the 20 colorectal and 62 of the 86 mixed cancers were metastatic, respectively. We also tested 44 controls. The samples were analyzed by flow cytometry using forward scatter and SYTO16 fluorescent labels. For spheroid capture we used simple nylon mesh filters (10 um and 20 um pore size). From the collected spheroids RNA was isolated and gene expression assays were performed for stem cell, hypoxia and epithelial markers. We also studied the role of inflammation and immune check point mechanisms in dissemination.
Results: We found that spheroids with epithelial lineage develop hypoxia and carry stem cells. By using Partec and Beckman-Coulter flow cytometers we detected circulating spheroids in patient blood. In the majority of colorectal cancer cases (56%) spheroids in blood indicated metastasis, while the controls were all negative. Furthermore, in half of the mixed cancer cases spheroids in blood also correlated with metastasis. All healthy samples and non-metastatic cases were negative. A subset of spheroids was positive for immune checkpoint and platelet markers suggesting immune/inflammation mechanisms in dissemination. Follow-up clinical studies will investigate the prospective significance of the results.
Conclusions: We showed that cancer stem cells form spheroid clusters in vivo and can be detected in circulation. We found significant correlation between spheroids in blood (spheremia) and metastasis in colorectal and other cancers. The most critical new finding of our studies is that systemic dissemination of cancer involves a novel spheroid mechanism. Based on this observation we developed a simple blood test that may detect metastatic predisposition early in colon cancer progression.
Citation Format: Viktoria Denes, Laszlo Graf, Gabor Barna, Balint Tegze, Pal Jakso, Peter Geck. Blood test of metastatic predisposition in colon cancer. [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer: From Initiation to Outcomes; 2016 Sep 17-20; Tampa, FL. Philadelphia (PA): AACR; Cancer Res 2017;77(3 Suppl):Abstract nr B03.