Abstract 306: Role of tumor beta-1 integrin in the tumor cell extravasation cascade

It has been shown that the tumor beta-1 integrin subunit plays an important role in primary tumor growth and metastasis. However, the specific role of beta-1 in the tumor cell extravasation cascade has not yet been clearly elucidated. This is partly due to the lack of extravasation models that possess high throughput and spatio-temporal resolution of each step in the cascade, which consists of tumor-endothelium arrest, transendothelial migration, and migration into the sub-endothelial matrix. To address this question, we employ multiple in vitro microfluidic platforms to recapitulate the extravasation microenvironment, allowing us to decipher in high spatio-temporal resolution, the specific extravasation defects associated with shRNA mediated beta-1 integrin knockdown in MDA-MB-231, MA2 and SUM 159 cell lines. These models include perfusable HUVEC microvascular networks embedded in a fibrin-collagen hydrogel and an upright HUVEC monolayer on a collagen gel that allows for detailed in-plane observation of 3D extravasation events. We first show that beta-1 knockdown drastically decreases extravasation efficiency in both assays, and further validated the defect in an in vivo mouse lung metastasis model. To explain the overall decreased ability to extravasate, we employed our assays to visualize and quantify each step in the extravasation cascade. First, we show that the retention rate of beta-1 knockdowns is significantly decreased under flow in microvascular networks. This suggests that beta-1 mediates adhesion to the endothelium, which was confirmed by a reduced adhesion rate under shear flow on a planar monolayer. Next, using the upright monolayer assay, we observed via time-lapse confocal microscopy that while beta-1 knockdowns assume a rounded morphology and extends nearly no protrusions past the endothelial barrier, their ability to open the endothelium was not significantly affected. However, beta-1 knockdowns were unable to fully invade past the endothelium and remained intercalated between endothelial cells. Further analysis revealed that extravasated beta-1 knockdown cells remained closely associated with the endothelium, while control cells migrated farther out into the sub-endothelial matrix. Immunostaining of the basement membrane protein collagen IV showed that transmigrated beta-1 knockdown cells were found trapped between the endothelial and col IV layers, suggesting that the close association with the endothelium is partly due to the inability to breach the basement membrane. Taken together, our results indicate that beta-1 is required for tumor cell extravasation by mediating tumor-endothelial adhesion and invasion into the subendothelial matrix, post-endothelial breaching. Citation Format: Michelle B. Chen, John M. Lamar, Roger D. Kamm, Richard O. Hynes. Role of tumor beta-1 integrin in the tumor cell extravasation cascade. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 306. doi:10.1158/1538-7445.AM2015-306