Intravasation the active entry of primary tumor cells into the vasculature

Intravasation the active entry of primary tumor cells into the vasculature remains the least studied step in the metastatic cascade. carcinoma HEp3-hi/diss. Whereas naturally-acquired or experimentally-induced MMP-1 deficiency substantially suppressed HEp3-hi/diss intravasation supplementation of recombinant MMP-1 to MMP-1-silenced primary tumors restored their impaired vascular dissemination. Surprisingly abrogation of MMP-1 production and activity did not affect significantly HEp3-hi/diss migration or matrix invasion suggesting non-collagenolytic mechanisms underlying MMP-1-dependent cell intravasation. In support of such non-collagenolytic mechanisms MMP-1 silencing in HEp3-hi/diss cells modulated the microarchitecture and integrity of the angiogenic vasculature in a novel microtumor model. Concomitantly MMP-1 deficiency led to decreased levels of intratumoral vascular permeability tumor cell intravasation and metastatic dissemination. Taking advantage of PAR1 deficiency of HEp3-hi/diss cells we further demonstrate that endothelial PAR1 is a putative non-tumor-cell/non-matrix target activation of which by carcinoma-produced MMP-1 regulates endothelial permeability and transendothelial migration. The inhibitory effects of specific PAR1 antagonists in live animals have also indicated that the mechanisms of MMP-1-dependent vascular permeability in tumors involve endothelial PAR1 activation. Together our findings mechanistically underscore the contribution of a tumor MMP-1/endothelial PAR1 axis to actual intravasation events manifested by aggressive carcinoma cells. models that accurately recapitulate the entry of tumor cells into the vasculature and also allow for quantification of the intravasation events. Furthermore real-time imaging of escaping primary tumor cells and microscopic analysis of the structure and functionality of tumor-associated vasculature remain problematic for most laboratories. Because of these modeling and methodological issues no clear signature molecules which directly contribute to the intravasation event have HMN-214 been identified. However several mechanisms have been linked to the processes and events leading up to the intravasation step such as primary tumor cell escape and migration and protease-mediated tumor cell invasion. In regard to the latter proteolytic degradation of the basement membrane and stromal matrix by specific members of the matrix metalloproteinase (MMP) family of enzymes could provide functional molecular links to tumor cell escape transendothelial migration and possibly to tumor cell-mediated active entry into the vasculature. The MMPs comprise a family of zinc-dependent endopeptidases that proteolytically modify the extracellular matrix in the Rabbit Polyclonal to AF4. primary tumors and metastatic sites as well as cleave distinct molecules on the surface of tumor and stromal cells (1-3). A number of MMP genes have been linked to development and progression of squamous cell carcinomas (SCCs) which constitute 90% of head and neck cancers the fifth leading cause HMN-214 of cancer-related deaths (4). HMN-214 The MMP genes that have been linked to SCC progression include gene which was found to be third best predictor among 25 signature genes (5) suggesting a critical role of MMP-1 protein in SCC progression Furthermore while the expression of many MMPs in primary SCCs is associated with stromal or inflammatory cells rather than carcinoma cells MMP-1 protein expression has been attributed to cancer HMN-214 cells at least in oral SCCs (5). In addition MMP-1 has shown up as one of the signature genes for the metastatic phenotype for human breast cancers (6-8) and has also been validated as part of a set of 63 genes associated with the HMN-214 progression and HMN-214 metastasis of advanced cervical carcinomas (9). All these considerations clearly warrant mechanistic study of the functional contribution of tumor-produced MMP-1 to metastasis of SCCs. To functionally analyze the role of MMP-1 in overall metastatic dissemination and specifically the intravasation step of SCCs we employed the human epidermoid carcinoma cell line HEp3 which is highly metastatic in both mice and chick embryos (10 11 A distinctive feature of the chick embryo model which is based on the grafting of human tumor cells on the chorioallantoic membrane (CAM) is that it uniquely allows for quantitative monitoring of intravasation into the CAM vasculature during spontaneous metastasis. With regard of intravasation the HEp3 cells when grafted onto the CAM at early passages give rise to primary tumors and also.