Abstract 90: Metastatic melanoma and cervical tumor cell clusters can exit blood vessels through angiopellosis augmenting tumor formation ability
Authors: Allen Tyler , Amu Emmanuel , Asad Dana , Cheng Ke 
Published: August 2018

Abstract
Introduction Accumulating data suggests metastatic primary tumor cells can travel through the circulation and form secondary tumors as multicellular clusters. However, whether these clusters must first dissociate into single cells prior to exiting of blood vessels remains largely unknown. Of late, researchers have identified a mechanism of cell extravasation (exiting blood vessel), termed angiopellosis (Allen 2016). However, whether tumor cell clusters possess the ability to exit vessles using the angiopellosis method is unstudied. We hypothesized that circulating tumor cell (CTC) clusters could exit blood vessels while maintaining a multicellular phenotype, through angiopellosis.
Methodology To test this, we used an in vivo zebrafish larvae model using the tg(fli1a:egfp) line, in which blood vessels exclusively fluoresce. First, we infused fluorescent human melanoma (A375), mouse melanoma (B16F10), and human cervical (HeLa) cells into the circulation of the larvae. Next, we used lightsheet microscopy to observe the method the tumor cells used to exit blood vessels. Additionally, mouse melanoma (B16F10) cells were intravenously infused into the circulation of C57BL/6J mice as either tumor clusters or individual cells. The mice were imaged for metastatic lung foci, to determine cluster extravasation potential.
Experimental results The intravital lightsheet microscopy allowed us to determine that tumor cells can utilize angiopellosis to exit blood vessels in the zebrafish model, both as individual CTCs and CTC clusters. Extravasating CTCs exited blood vessels through angiopellosis at 94%, with only 6% exiting through the diapedesis method (n=30). CTCs which exited as a cluster through angiopellosis were observed to form tumor masses at an 88% higher rate compared to individual tumor cells in vivo, over the course of 96 hours. In the murine model, CTCs clusters which were infused via tail vein (n=4) showed significantly higher metastatic lung foci in comparison to individual CTCs that were infused (n=3). Following isolation and analysis of metastatic CTCs, multiple genes were shown to be differentially expressed in CTC clusters in both the zebrafish and murine models.
Discussion Here, we show circulating tumor cell clusters possess the ability to exit blood vessels through the recently identified angiopellosis. We determined tumor cell clusters exhibit an augmented ability to proliferate in vivo, while individually tumor cells are more prone to dormancy. Our results challenge the belief that circulating tumor cell clusters must first disassociate to exit the circulation, and posits an alternative model. We propose tumor cells both travel and exit the circulation while maintaining a multicellular phenotype, using angiopellosis, stimulating proliferation-augmenting genetic/molecular alterations. This has been termed the Cancer Exodus Hypothesis.
Citation Format: Tyler Allen, Emmanuel Amu, Dana Asad, Ke Cheng. Metastatic melanoma and cervical tumor cell clusters can exit blood vessels through angiopellosis augmenting tumor formation ability [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 90.