Antibodies blocking adhesion and matrix binding domains of laminin-332 inhibit tumor growth and metastasis in vivo.

Laminin-332 (LN-332), which is essential for epithelial cell adhesion and migration, is up-regulated in most invasive carcinomas. Association between LN-332 and carcinoma cell integrins and stroma collagen is thought to be important for tumor growth and metastasis. Here, we show that function blocking LN-332 antibodies interfering with cellular adhesion and migration in vitro evoke apoptotic pathways. The antibodies also target epithelial tumors in vivo. Antibodies against the cell binding domain of the alpha3 chain of LN-332 inhibited tumor growth by up to 68%, and antibodies against the matrix binding domains of the beta3 and gamma2 chains significantly decreased lung metastases. The LN-332 antibodies appear to induce tumor cell anoikis and subsequent programmed cell death and reduce migration by interfering with tumor cell matrix interactions.

Selective binding of integrins from different renal cell types to the NC1 domain of alpha3 and alpha1 chains of type IV collagen.

BACKGROUND: Cells interact with type IV collagen (Col IV) via integrins through the triple-helical and NC1 domains. We examined interactions of human glomerular and proximal tubular epithelial cells with recombinant alpha1 and alpha3 NC1 chains of Col IV, to explore the ability of different cell types to interact with Col IV of different trimer composition. METHODS: Interactions of TSV-40-immortalized human glomerular epithelial cells (HGECs), HPV-16-immortalized human proximal tubular epithelial (HK-2) cells and primary human mesangial cells (MES) with recombinant alpha1 and alpha3 NC1 chains of Col IV were examined by affinity chromatography and solid-phase binding assays. The expression of integrin-regulated metalloproteinases was examined by zymography. RESULTS: HGECs bound to both alpha3 and alpha1(IV)NC1, albeit there was preferential binding to alpha3(IV)NC1, through the alpha3beta1 and alpha2beta1 integrin receptors; HK-2 cells and MES bound almost exclusively to alpha1(IV)NC1 via the alpha3beta1/alphavbeta3 and alpha1beta1/alpha2beta1 receptors, respectively. It was demonstrated that the expression of MMP-2 and MMP-9 by HGECs was down-regulated in the presence of alpha3(IV)NC1. CONCLUSIONS: The observed data indicate that the isoform NC1 chains of Col IV serve for selective, integrin-mediated cell binding which probably triggers different signaling mechanisms, resulting in the activation of specific transcription factors and the modulation of gene expression.

Laminin-511 but not -332, -111, or -411 enables mouse embryonic stem cell self-renewal in vitro.

We tested specific laminin (LN) isoforms for their ability to serve as substrata for maintaining mouse embryonic stem (ES) cells pluripotent in vitro in the absence of leukemia inhibitory factor or any other differentiation inhibitors or feeder cells. Recombinant human LN-511 alone was sufficient to enable self-renewal of mouse ES cells for up to 169 days (31 passages). Cells cultured on LN-511 maintained expression of pluripotency markers, such as Oct4, Sox2, Tert, UTF1, and Nanog, during the entire period, and cells cultured for 95 days (17 passages) were used to generate chimeric mice. LN-332 enabled ES cells proliferation but not pluripotency. In contrast, under the same conditions LN-111, Matrigel, and gelatin caused rapid differentiation, whereas LN-411 and poly-d-lysine did not support survival. ES cells formed a thin monolayer on LN-511 that differed strikingly from typical dense cluster ES cell morphology. However, expression of pluripotency markers was not affected by morphological changes. The effect was achieved at low ES cell density (<200 cell/mm(2)). The ability of LN-511 and LN-332 to support ES cell proliferation correlated with increased cell contact area with those adhesive substrata. ES cells interacted with LN-511 via beta1-integrins, mostly alpha6beta1 and alphaVbeta1. This is the first demonstration that certain extracellular matrix molecules can support ES cell self-renewal in the absence of differentiation inhibitors and at low cell density. The results suggest that recombinant laminin isoforms can provide a basis for defined surface coating systems for feeder-free maintenance of undifferentiated mammalian ES cells in vitro. Disclosure of potential conflicts of interest is found at the end of this article.

Recombinant non-collagenous domain of alpha2(IV) collagen causes involution of choroidal neovascularization by inducing apoptosis.

Vascular endothelial cells receive proangiogenic or antiangiogenic signals from components of extracellular matrix (ECM) depending upon the situation and many molecular signals can have opposite effects in different vascular beds. Tissue inhibitor of metalloproteinase 1 is antiangiogenic in several tissues, but promotes retinal neovascularization. When cleaved from native collagens, several of the non-collagenous domains (NC1) of basement membrane collagens have antiangiogenic effects in some tissues, but this is context dependent for the NC1 of the alpha 1 chain of collagen IV. It is critical to examine effects in several well-defined model systems before assuming that an ECM component is universally antiangiogenic. In this study, we examined the effects of a recombinant fragment of NC1 of the alpha 2 chain of type IV collagen (alpha2(IV)NC1) in a well-characterized model of ocular neovascularization. Intravitreous or periocular injections of alpha2(IV)NC1 caused selective apoptosis of endothelial cells participating in neovascularization resulting in suppression of neovascularization when the peptide was given prior to onset of new vessel sprouting. Importantly, when the peptide was given after neovascularization had already developed, it caused the new vessels to regress. This suggests that alpha2(IV)NC1, which has previously been shown to suppress tumor angiogenesis in xenograft models, is also a strong antiangiogenic agent in the choroid and is a therapeutic candidate for treatment of neovascular age-related macular degeneration.