Chieko Koike

Functional role of sialyl Lewis X and fibronectin-derived RGDS peptide analogue on tumor-cell arrest in lungs followed by extravasation.

Our study demonstrates that synthetic sialyl Lewis X (SLex) as a ligand for selectins and fibronectin‐derived RGDS peptide analogue[Ar(DRGDS)3] inhibits lung metastases produced by i.v. co‐injection of B16‐BL6 melanoma cells. To investigate the inhibitory mechanisms in a living animal, we performed positron‐emission tomography (PET) analysis after i.v. injection of [2‐18F]2‐fluoro‐2‐deoxy‐D‐glucose‐labeled tumor cells with or without liposomal SLex or Ar(DRGDS)3. The real‐time PET measurement for the first 120 min, started immediately after injection, showed that tumor‐cell arrest, i.e., accumulation in the target organ (lung) was remarkably inhibited by liposomal SLex, but not inhibited by Ar(DRGDS)3 or liposomal Me‐SLex, which is not recognized by selectins. In contrast, AR(DRGDS)3 inhibited the invasion of B16‐BL6 cells into reconstituted basement membrane (Matrigel) following tumor arrest, whereas SLex‐ or Me‐SLex‐entrapped liposomes did not affect tumor invasion. In the metastatic processes containing tumor‐cell lodgement and arrest in the target organ followed by extravasation (invasion), SLex resulted in the inhibition of initial arrest of tumor cells, presumably tumor‐endothelium interaction, while Ar(DRGDS)3 achieved inhibition of tumor invasion into basement membrane at later steps of the cascade, consequently leading to inhibition of metastasis. Thus, tumor‐cell arrest in lungs in the metastatic processes must be precisely and properly controlled by different adhesion molecules at different stages, which are similar to those observed in leukocyte‐endothelium interaction.

Role of integrin αvβ3 in the early phase of liver metastasis: PET and IVM analyses

To clarify the function of integrin αvβ3 in the early stage of liver metastasis, we investigated the interactions of metastatic cells with their target organ under the actual blood flow by using positron emission tomography (PET). The cells used were CHO-K1 cells and their transfectants bearing human integrin αvβ3 cDNA (αvβ3-CHO-K1 cells). The liver accumulation of αvβ3-CHO-K1 cells was significantly higher than that of CHO-K1 cells after injection via the portal vein, whereas no significant difference was observed in the lung accumulation after tail vein injection, suggesting a specific interaction of αvβ3-CHO-K1 cells with the hepatic sinusoids. Furthermore, to clarify the precise location of each cell in the liver, i.e., to determine whether individual cells were intravascularly localized or had extravasated, we performed intravital fluorescence microscopy (IVM) on the liver by using stable transfectants bearing the green fluorescent protein (GFP) gene, namely, GFP-CHO-K1 and GFP-αvβ3-CHO-K1 cells. Both types of cells remained in the hepatic blood vessels 1 h after injection via the portal vein. On the other hand, expression of integrin αvβ3 promoted the cells to reach the extravascular region after 24 h. These results suggest the possibility that the specific accumulation of αvβ3-CHO-K1 cells in the liver is followed by migration of the cells into the extravascular region. Interestingly, the adhesion of the two types of cells to hepatic sinusoidal endothelial cells in vitro did not correspond to in vivo accumulation of these cells. Therefore, integrin αvβ3 may function to promote extravasation of integrin αvβ3-expressing tumor cells in liver through a process possibly mediated by vitronectin produced by this organ.

Liposomal Arg-Gly-Asp analogs effectively inhibit metastatic B16 melanoma colonization in murine lungs

Analogs of a synthetic peptide having the L-arginine-L-glycine-L-aspartic acid (RGD) sequence have been found to decrease metastatic colonization. To enhance the metastasis-suppressing efficacy of these analogs, we sought to stabilize these analogs and to prolong their circulation time by incorporating them into a liposomal formulation. Various structures of RGD analogs grafted to hydrophobic groups were synthesized and then incorporated into liposomes. Liposomes composed of distearoylphosphatidylcholine, cholesterol, dipalmitoylphosphatidylglycerol and appropriate RGD analogs were injected intravenously along with B16BL6 murine melanoma cells into mice. Liposomal RGD (0.6 mumol of the analog equivalent to ca. 200 micrograms RGD peptides) inhibited lung colonization up to 76%. This dose is an order of magnitude lower than that for comparable inhibition reported for free RGD. Multi-dose administration of liposomal RGD (0.15 mumol of the analog) also inhibited the spontaneous lung metastasis of cells from a primary tumor site of B16BL6 cells subcutaneously implanted into the footpad of mice. Taken together, our data indicate that liposomal RGD may serve as a useful anti-metastatic agent.

REAL-TIME PET ANALYSIS OF METASTATIC TUMOR CELL TRAFFICKING IN VIVO AND ITS RELATION TO ADHESION PROPERTIES

Although a number of studies have indicated that highly metastatic cells tend to adhere more to target endothelium in vitro than low or non-metastatic cells, direct evidence about the correlation between cellular adhesiveness and organ disposition of the cells has not been obtained. Using positron emission tomography (PET), we have developed a novel technique that enables the non-invasive detection of the real-time tumor cell trafficking. The present study shows the correlation between trafficking of murine large cell lymphoma RAW117 and the adhesion properties of the cells in vitro. Cells accumulated in the liver time-dependently, and accumulation of RAW117-H10, liver metastatic subline cells, was more intense than that of RAW117-P, the parental cells, indicating that the metastatic potential is correlated with the in vivo accumulation of the cells in the target tissue. To examine whether the adhesion properties of the cell membrane determine the cell trafficking, we performed PET analysis after altering the adhesion properties on the cell membrane by means of cellular protein kinase C modulation, since the modulation of this enzyme is known to alter the surface adhesion molecules, i.e., those of the integrin superfamily. The treatment of RAW117-P with 12-O-tetradecanoylphorbol 13-acetate, which caused augmentation of adhesion to hepatic sinusoidal microvessel endothelial cells (HSE) in vitro, enhanced the hepatic accumulation of the cells in vivo. On the contrary, treatment of RAW117-H10 with the protein kinase C inhibitor H-7, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride, which reduced the adhesion activity of the cells to HSE, suppressed their accumulation in the liver, although the suppression was observed only during the first 30 min after administration of the cells. These data suggest that the adhesion properties of metastatic lymphoma cells are critical for the accumulation of these cells in the target tissue.

Role of sialylglycoconjugate(s) in the initial phase of metastasis of liver-metastatic RAW117 lymphoma cells.

To elucidate the early events of blood‐borne metastasis under actual blood flow, real‐time trafficking of RAW117 large cell lymphoma cells, namely parental RAW117‐P and liver‐metastatic RAW117‐H10 cells, was investigated using positron emission tomography (PET). Both types of cells accumulated in the liver immediately after injection via the portal vein, and were eliminated from the liver time‐dependently. The elimination rate of RAW117‐H10 cells, however, was slower than that of RAW117‐P cells, suggesting that RAW117‐H10 cells interact more strongly with hepatic sinusoidal endothelium than the parental cells. This result correlated with the metastatic potential of these cells: RAW117‐H10 cells metastasized in the liver to a greater extent than RAW117‐P cells after injection via this route. To investigate the role of sialylglycoconjugates in the interaction of RAW117‐H10 cells with the hepatic endothelium after injection via the portal vein, the trafficking of RAW117‐H10 cells was examined after the cells had been treated with sialidase. The elimination rate of RAW117‐H10 cells from liver was observed to be greatly accelerated by sialidase treatment. To elucidate what kind of sialylglycoconjugates is related to this phenomenon, we analyzed the distribution of sialyl Lewis A and sialyl Lewis X antigens of both sublines of RAW117 by using flow cytometry. RAW117‐H10 cells were found to express a much higher level of sialyl Lewis A than RAW117‐P cells, whereas the amount of sialyl Lewis X did not differ significantly. These findings suggest that some sialylglycoconjugates, perhaps sialyl Lewis A in particular, play an important role in the initial interaction of RAW117‐H10 cells with the hepatic endothelium, leading to metastasis.

Application of liposomes for cancer metastasis

Abstract Metastasis is established by a complex cascade of activities, and adhesion of tumor cells to endothelia or to extracellular matrix is one of the critical steps in the metastatic cascade. Therefore, agents that suppress such interaction may serve as anti-metastatic drugs. We previously established a non-invasive method to determine metastatic tumor cell trafficking by use of positron emission tomography (PET). In this method, positron-labeled metastatic cells are injected into bloodstream to determine tumor cell biodistribution in real-time from immediately after injection in a living animal. Here, to elucidate the involvement of cellular surface adhesion molecules in metastatic process, we investigated the effect of liposomalized sialyl Lewis X (sLe X ) as well as l -arginine- l -glycine- l -aspartic acid (RGD)-related peptide on the trafficking of B16BL6 melanoma cells and on metastatic potential. The trafficking of B16BL6 cells after injection into the tail vein was highly affected by liposomal sLe X , but only little by RGD-related peptide, suggesting that the adhesion of metastatic cells to the target is initially mediated via selectin, and integrin-mediated adhesion may occur the later stages. Furthermore, liposomal sLe X suppressed experimental metastasis suggesting that adhesion via selectin is an important step for metastasis. Next, to enhance the metastasis-suppressing efficacy, liposomalizaton of RGD was attempted, since RGD-related peptides have been found to suppress metastasis. Various structures of RGD analogs grafted to hydrophobic groups were synthesized and then incorporated into liposomes. Some liposomalized RGD markedly inhibited lung colonization at the concentration of an order of magnitude lower than that for comparable inhibition reported for free RGD. The present study indicates that liposomal application is useful for both clarifying the mechanism of metastasis and the development of anti-metastatic pharmaceutics.

How platelet aggregation affects B16BL6 melanoma cell trafficking

In blood‐borne metastasis, intravasated metastatic tumor cells are thought to localize at the target site via a series of processes involving platelet aggregation, adhesion to endothelium, and invasion through the basal membrane. In the present study, we examined how platelet aggregation contributes to the trafficking of metastatic tumor cells in vivo by use of an inhibitor of platelet aggregation. Highly invasive B16BL6 melanoma cells were labeled with [2‐18F]2‐fluoro‐2‐deoxy‐d‐glucose and injected into mice to determine cell trafficking non‐invasively by positron emission tomography. Both platelet aggregation inhibitor cyclo(RSarDPhg), which could not inhibit metastasis, and metastatic inhibitor cyclo(GRGDSPA) suppressed the accumulation of B16BL6 cells in the lung by about 12%, suggesting that platelet aggregation partly affects cell trafficking but not to a great extent, and that platelet aggregation is not the essential step for B16BL6 cell arrest in targets.