Abstract PR14: Quantitative and multiparameter analysis of tumor cell intravasation
Authors: Deryugina Elena I. , Kiosses William B. , Quigley James P. 
Published: May 2015

Abstract
Multiple steps in cancer metastasis, including tumorigenesis, tumor angiogenesis, invasion and colonization, can be successfully examined with well-established models. However, due to deficiency in experimental model systems that would accurately recapitulate tumor cell entry into the host vasculature, intravasation remains the least studied step in the metastatic cascade. Our laboratory has developed several independent avian and murine model systems to investigate the complex process of intravasation in a quantifiable and mechanistic manner.
Chick embryo models of intravasation are based on the ability of a highly vascularized tissue, the chorioallantoic membrane (CAM), to sustain primary tumor development and spontaneous metastasis. Furthermore, the CAM serves as a repository of newly intravasated cells, which are quantified by human-specific Alu-qPCR. By employing different CAM models, the early events in spontaneous metastasis that ultimately lead to intravasation can be dissected and their individual input to overall intravasation can be quantified. These early processes include cell escape from the primary tumor, establishment of intratumoral and extratumoral angiogenic networks, invasion of adjacent stroma by escaped tumor cells, and migration of escaped tumor cells along tumor-associated blood vessels. In conjunction with confirmatory models of cancer metastasis in mice, we have identified several molecular systems that functionally contribute to the intravasation process.
To investigate the specific mechanisms whereby tumor cells enter the angiogenic blood vessels, we have developed a new high-resolution confocal microscopy model based on acquisition and multiparameter analysis of primary microtumors imaged as entire units. This model was adapted to primary tumors developing within the mesoderm of the CAM as well as primary tumors developing in the dermis of mouse ear. In both avian and murine models, the volume of microtumors, the number of tumor cells per tumor, and the total volume of tumor-associated vasculature can be determined from 3D-reconstructed Z-stacks. Most importantly, the volume of intravascular tumor cells can be quantified by colocalizing tumor-vascular signals with a stringent threshold of a minimum volume of an individual tumor cell so that the cells that are localized within the vessels could be discriminated from the cells adherent to the abluminal vessel surface. In addition, high-resolution imaging of whole microtumors allows to visualize and quantify the tumor cells that appear as entering into the vasculature or in close association with the abluminal surface of the vessels. This approach allowed us for the fist time to visualize the intravasation events in numbers rendering statistical significance. By employing unique cell and protein markers in this novel system, the vascular cells interacting with the intravasating tumor cells can be identified and the candidate molecules involved in the close tumor-vascular interactions can be probed by treating the developing microtumors with specific reagents and small molecule inhibitors.
By using these novel high-resolution models for intravasation, we have documented a majority of intravasation events within the primary tumor or in the nearest proximity to the primary tumor border. These data challenge the widely accepted concept that tumor cell escape, stromal invasion, and entry of escaped tumor cells into blood vessels adjacent to the primary tumor, are prerequisites of tumor cell intravasation. In contrast, our findings indicate that the bulk of cell intravasation events can take place within the primary tumor and prior to significant stromal invasion and migration of escaped tumor cells towards and along tumor-associated blood vessels. Our data suggest that tumor cell intravasation and metastatic spread can occur much earlier than commonly acknowledged by oncologists based on histological examination of resected tumors.
This abstract is also presented as Poster A24.
Citation Format: Elena I. Deryugina, William B. Kiosses, James P. Quigley. Quantitative and multiparameter analysis of tumor cell intravasation. [abstract]. In: Proceedings of the AACR Special Conference: The Translational Impact of Model Organisms in Cancer; Nov 5-8, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(11 Suppl):Abstract nr PR14.