Cell Surface Proteomics Identifies Molecules Functionally Linked to Tumor Cell Intravasation

Abstract

In order to better understand the molecular and cellular determinants of tumor cell intravasation, our laboratory has generated a pair of congenic human HT-1080 fibrosarcoma variants (i.e. HT-hi/diss and HT-lo/diss) differing 50 -100-fold in their ability to intravasate and disseminate. To investigate the molecular differences underlying the distinct dissemination capacities of these HT-1080 variants, we performed a comparative analysis of the cell surface proteomes of HT-hi/diss and HT-lo/diss. Cell membrane proteins were enriched by biotinylation and avidin precipitation and analyzed by tandem mass spectrometry employing multidimensional protein identification technology. By this approach, 47 cell surface-associated molecules were identified as differentially expressed between the HT-1080 intravasation variants. From these candidates, four targets (i.e. TIMP-2, NCAM-1, JAM-C, and tissue factor (TF)) were selected for further biochemical validation and in vivo functional verification. Western blot analysis of the cell surface enriched fractions confirmed the proteomic array data, demonstrating that, in vitro, TIMP-2 protein was increased in the HT-lo/diss variant, whereas NCAM-1, JAM-C, and TF levels were increased in the HT-hi/diss variant. Corresponding in vivo differences in levels of TIMP-2, JAM-C, and TF were demonstrated in primary tumors grown in the chick embryo. Finally, functional inhibition of one selected protein (i.e. TF) by small interfering RNA silencing or ligation with a function-blocking antibody significantly reduced HT-hi/diss intravasation, thus clearly implicating TF in the early steps of tumor cell dissemination. Overall, our cell surface proteomic analysis provides a powerful tool for identification of specific cell membrane molecules that contribute functionally to intravasation and metastasis in vivo.One of the early and possibly rate-limiting steps in cancer progression from a localized tumor to systemic metastatic disease is intravasation (i.e. the entry of metastatic cells into the vasculature) (1-5). Molecules involved in intravasation represent attractive therapeutic targets, since preventing or inhibiting this process would confine tumor cells to their primary site and provide a more focused target for clinical intervention (6). To identify cellular attributes that functionally contribute to tumor cell intravasation and metastasis, including escape from the primary site, invasion of local stoma, and entry into the vasculature, we have employed a pair of congenic human fibrosarcoma HT-1080 cell variants, differing 50 -100-fold in their ability to intravasate and disseminate (HT-hi/diss and HT-lo/diss) while having similar capacities to form primary tumors (7). These cell variants display a distinct differential during spontaneous metastasis but behave comparably in experimental metastasis models where cells are inoculated intravenously and only the later steps of the metastatic cascade are recapitulated. Therefore, comparative analysis of the HT-hi/diss and HT-lo/diss variants can...

Tissue Factor and PAR2 Signaling in the Tumor Microenvironment

“…A role for TF in the extravasation from the TME was recently uncovered in a model of spontaneous metastasis from the chicken chorioallantoic membrane (CAM) 63. Tumor cells selected for more efficient metastasis in this assay showed increased TF expression, detected by cell surface proteomic profiling.…”

Tissue factor in cancer progression and angiogenesis

“…TF expressed by tumor cells also triggers multiple pathways that directly support tumor progression. TF promotes intravasation [4] and downstream coagulation activation orchestrates thrombin-, platelet- and fibrin-dependent cancer cell survival pathways important for metastatic tumor dissemination [5]. Host and tumor cell TF further play partially overlapping roles to enhance tumor growth and to shape the tumor microenvironment [6].…”

Two Novel Functions of Hyaluronidase-2 (Hyal2) Are Formation of the Glycocalyx and Control of CD44-ERM Interactions

“…Hyal2 also anchors tumor growth factor-␤1 to the cell surface and mediates some of its pro-apoptotic effects (15). Conversely, Hyal2 may facilitate cancer progression: Hyal2 overexpression in murine astrocytoma cells accelerates intracerebral, but not subcutaneous, tumor formation (16); the in vitro invasion capacity of several breast cancer cell lines correlates with Hyal2 expression (17,18); and a cell surface proteomics study demonstrates that Hyal2 is enriched 13-fold in a highly invasive HT-1080 fibrosarcoma cell clone relative to a congenic variant with a low potential for intravasation and metastasis (19). A better understanding of all these divergent events necessitates more information regarding the action of Hyal2 at the cellular level.…”