Irma M. Grossi

αIIbβ3 Integrin expression and function in subpopulations of murine tumors

Abstract Subpopulations of B16 amelanotic melanoma (B16a) cells, isolated by centrifugal elutriation from enzymatically dispersed solid tumors, demonstrated different abilities to form lung colonies when injected intravenously. In contrast, no differences in experimental metastasis were observed among subpopulations obtained from Lewis lung (3LL) tumors. Lung colonization by B16a and 3LL subpopulations correlated positively with observed differences (B16a) or lack of differences (3LL) in tumor cell ability to induce aggregation of homologous platelets, to adhere to subendothelial matrix or fibronectin, and with the percentage of cells in the G 2 M phase of the cell cycle. Both B16a and 3LL cells express αIIbβ3 integrin receptors; however, differences in the receptor expression level were found only among B16a subpopulations. Comparison of the amount of αIIbβ3 receptor expressed on cell surface with tumor cell ability to induce platelet aggregation (TCIPA) and to adhere to fibronectin or subendothelial matrix revealed a positive correlation. Pretreatment of tumor cells with αIIbβ3 -specific antibodies inhibited tumor cell matrix adhesion, TCIPA, and lung colony formation. We propose that αIIbβ3 integrin receptor expression, tumor cell matrix adhesion, and tumor cell-induced platelet aggregation can be important parameters to indicate the metastatic potential of some tumor cells and that the αIIbβ3 is a multifunctional receptor involved in both tumor cell-matrix and tumor cell-platelet interactions. Further, the correlation among cell cycle phase, metastatic ability, and receptor expression suggests that metastatic propensity may be transiently expressed and/or increased in some tumor cell subpopulations.

Inhibition of human tumor cell induced platelet aggregation by antibodies to platelet glycoproteins Ib and IIb/IIIa

Abstract Tumor cell induced platelet aggregation was shown to be inhibited in a dose dependent manner by preincubation of human platelets with antibodies to platelet glycoprtein lb and the llb/llla complex. Combination of antibody to lb and antibody to the llb/llla complex at concentrations which produced half maximal inhibition of platelet aggregation alone caused complete inhibition of tumor cell induced platelet aggregation. Antibodies to platelet glycoproteins lb and the llb/llla complex also inhibited platelet synthesis of thromboxane A2, but not synthesis of 12-hydroxyeicosatrienoic acid. Inhibition of tumor cell induced platelet aggregation with antibodies against platelet glycoproteins suggests a role for these glycoproteins in tumor cell-platelet interactions and possibly platelet facilitated tumor cell metastasis.

Lipoxygenase products regulate IRGpIIb/IIIa receptor mediated adhesion of tumor cells to endothelial cells, subendothelial matrix and fibronectin.

Abstract Tumor cell adhesion to endothelial cells, subendothelial matrix, and fibronectin is stimulated by the lipoxygenase metabolite of arachidonic acid, 12(S)-HETE, but not by 12(R)-HETE, 5-HETE or 15-HETE. Adhesion is also stimulated by the phorbol ester TPA, an effect inhibited by lipoxygenase but not cyclooxygenase inhibitors.TPA and 12(S)-HETE mediated adhesion is due, in part, to an integrin receptor (i.e., IRGpllb/llla) related to the platelet glycoprotein llb/llla complex and is inhibited by specific monoclonal and polyclonal antibodies against platelet llb/llla. TPA and 12(S)-HETE stimulated adhesion is also inhibited by a lipoxygenase product of linoleic acid; i.e., 13-HODE. These results suggest bidirectional control of tumor cell adhesion by lipoxygenase products of arachidonic acid (increase) and linoleic acid (decrease).

Is there a role for the tumor cell integrin alpha IIb beta 3 and cytoskeleton in tumor cell-platelet interaction?:

In vitro tumor cell-platelet interaction was examined using B16 amelanotic (B16a) melanoma cells. These tumor cells express the αIIbβ3-type cytoadhesin. Aggregation studies demonstrated that tumor cell surface αIIbβ3 mediates the recognition of platelets since pretreatment of tumor cells with antibody against αIIbβ3 prevents platelet-tumor cell interaction as well as platelet activation measured by aggregometry, platelet eicosanoid metabolism and ultrastructural analysis. In B16a cells, disruption of the microfilaments and intermediate filaments inhibits mobility of αIIbβ3 on the cell surface. Microtubules do not play a role in receptor mobility, because B16a cells do not possess well-defined microtubules in interphase and colchicine does not affect receptor mobility. Disruption of microfilaments or intermediate filaments results in an inhibition of tumor cell-platelet interaction as evidenced by aggregometry studies and ultrastructural analysis. We suggest that platelet interaction with tumor cells begins with αIIbβ3-mediated receptor recognition followed by not only platelet activation but also microfilament- and vimentin intermediate filament-dependent tumor cell activation.

Separation of high and low metastatic subpopulations from solid tumors by centrifugal elutriation.

Abstract We have isolated from murine solid tumors (B16a) subpopulations of cells possessing high and low metastatic potential. Tumors were dispersed by collagenase treatment. The resulting heterogeneous population of cells (i.e., viable and non-viable tumor cells and host cells) were separated by centrifugal elutriation. Four of the fractions (100, 180, 260, 340) contained tumor cells of high viability ( > 95%) and high purity ( < 1% host cell contamination). The four fractions were characterized by flow cytometry and found to differ in distribution of cells in G-j, S and G2. The cell populations were also found to differ in metastatic potential as determined by their ability to form lung colonies following intravenous injection. The 340 fraction was approximately 5-fold more metastatic than the 100 fraction. We also observed that cells from the 100 fraction failed to induce platelet aggregation whereas cells from the 340 fraction induced significant platelet aggregation. These observations demonstrate that cells of B16a tumors are heterogeneous for phenotypic characteristics (i.e., metastatic potential; platelet aggregation, etc.) and that their ability to induce platelet aggregation is positively correlated with metastatic potential.

Effects of 12-Hydroxyeicosatetraenoic Acid on Release of Cathepsin B and Cysteine Proteinase Inhibitors from Malignant Melanoma Cells

The cysteine proteinase cathepsin B as well as the endogenous inhibitors of this enzyme have been implicated in the progression of tumors from a premalignant to a malignant state (for review see 1,2). Activity of cathepsin B has been shown to be elevated in parallel with malignancy or metastatic potential of both human and rodent tumors. These increases in cathepsin B activity correspond in part to increases in mRNA for cathepsin B and in part to reduced regulation by the endogenous low Mr cysteine proteinase inhibitors (CPIs). The inhibition constants for the interaction between stefin A purified from human tumors and cysteine proteinases are an order of magnitude greater than those for stefin A purified from human liver. Most properties of tumor cathepsin B appear to be similar to those of cathepsin B from normal tissues. However, the subcellular distribution of cathepsin B and CPIs is altered in tumors, resulting in association of cathepsin B and CPIs with plasma membrane fractions or in release of CPIs and of high Mr forms of cathepsin B (native and latent) into the extracellular mileau.

Control of Tumor Cell Induced Endothelial Cell Retraction by Lipoxygenase Metabolites, Prostacyclin and Prostacyclin Analogues

The primary element responsible for the morbidity and mortality of malignant cancers is tumor metastasis. During hematogenous metastasis the vascular endothelium forms the primary barrier between the circulatory system and extravascular tissues. Circulating tumor cells have the potential to interact with a variety of circulating host cells and the vascular wall, however only a small fraction of these tumor cells survive hemodynamic forces and host defence mechanisms to form distant metastases (1,2). Specific interactions between circulating tumor cells and a variety of host cells (i.e., platelets, leukocytes, and endothelial cells) is believed to affect tumor cell arrest and extravasation from the circulation (3,4). These types of cell-cell interactions generally occur in the microcirculation where the blood flow rate is low and the vascular surface area to blood volume ratio is high, allowing increased cell-cell contact in the microvascular space.

Lipoxygenases, Integrin Receptors, and Metastasis

Failure of tumor cells to arrest and form stable adhesions to the endothelium may be viewed as a critical barrier to the formation of a successful metastatic lesion. This suggests that agents (i.e., receptors, platelets, hemodynamic factors, eicosanoids, etc.) which regulate initial tumor cell arrest and adhesion are of significant importance in the development (or prevention) of a successful metastasis (1).

The Lipoxygenase Metabolite of Arachidonic Acid, 12-(S)-Hete, Induces Cytoskeleton Dependent Upregulation of Integrin αIIbβ3 in Melanoma Cells

Cell-extracellular matrix interactions are essential prerequisites of normal cell function mediated by specific receptors including integrins (1). This family of transmembrane proteins are characterized by α and β heterodimers linked with S-S bonds. There are a limited number of β subunits and a large number of a units which can combine to form at least 16 unique heterodimers (1).

Role of Tumour Cell Eicosanoids and Membrane Glycoproteins IRGPIb and IRGPIIb/IIIa in Metastasis

Eicosanoids are a group of oxygenated arachidonic acid metabolites which include Prostaglandins, thromboxanes, leukotrienes, lipoxins, and various hydroperoxy and hydroxy fatty acids. Eicosanoids are implicated in diverse cellular functions such as Chemotaxis, proliferation, cell-cell signaling etc. The first committed step in the biosynthesis of each of the eicosanoids, the incorporation of molecular oxygen into polyunsaturated fatty acids, is catalyzed by one of a group of enzymes calles fatty acid oxygenases. This group includes the cyclooxygenase (COX) of Prostaglandin endoperoxide synthase (PGH) and various lipoxygenases (LOX).