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Altered expression pattern of integrin alphavbeta3 correlates with actin cytoskeleton in primary cultures of human breast cancer.

Havaki S, Kouloukoussa M, Amawi K, Drosos Y, Arvanitis LD, Goutas N, Vlachodimitropoulos D, Vassilaros SD, Katsantoni EZ, Voloudakis-Baltatzis I, Aleporou-Marinou V, Kittas C, Marinos E - Cancer Cell Int. (2007)

Bottom Line: In the present study, short-term primary breast cancer cell cultures were developed.In the former, F-actin cytoskeleton appeared well-formed, consisting of numerous and thicker stress fibers, compared to normal epithelial cells.Furthermore, electron microscopy showed increased integrin alphavbeta3 immunogold localization in epithelial breast cancer cells over the area of stress fibers at the basal cell surface.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory of Histology and Embryology, Medical School, University of Athens, 75 Mikras Asias Str, Goudi, Greece. shavaki@med.uoa.gr

ABSTRACT

Background: Integrins are transmembrane adhesion receptors that provide the physical link between the actin cytoskeleton and the extracellular matrix. It has been well established that integrins play a major role in various cancer stages, such as tumor growth, progression, invasion and metastasis. In breast cancer, integrin alphavbeta3 has been associated with high malignant potential in cancer cells, signaling the onset of widespread metastasis. Many preclinical breast cancer studies are based on established cell lines, which may not represent the cell behavior and phenotype of the primary tumor of origin, due to undergone genotypic and phenotypic changes. In the present study, short-term primary breast cancer cell cultures were developed. Integrin alphavbeta3 localization was studied in correlation with F-actin cytoskeleton by means of immunofluorescence and immunogold ultrastructural localization. Integrin fluorescence intensities were semi-quantitatively assessed by means of computerized image analysis, while integrin and actin expression was evaluated by Western immunoblotting.

Results: In the primary breast cancer epithelial cells integrin alphavbeta3 immunofluorescence was observed in the marginal cytoplasmic area, whereas in the primary normal breast epithelial cells it was observed in the main cell body, i.e. in the ventrally located perinuclear area. In the former, F-actin cytoskeleton appeared well-formed, consisting of numerous and thicker stress fibers, compared to normal epithelial cells. Furthermore, electron microscopy showed increased integrin alphavbeta3 immunogold localization in epithelial breast cancer cells over the area of stress fibers at the basal cell surface. These findings were verified with Western immunoblotting by the higher expression of integrin beta3 subunit and actin in primary breast cancer cells, revealing their reciprocal relation, in response to the higher motility requirements, determined by the malignant potential of the breast cancer cells.

Conclusion: A model system of primary breast cancer cell cultures was developed, in an effort to maintain the closest resembling environment to the tumor of origin. Using the above system model as an experimental tool the study of breast tumor cell behavior is possible concerning the adhesion capacity and the migrating potential of these cells, as defined by the integrin alphavbeta3 distribution in correlation with F-actin cytoskeleton.

No MeSH data available.


Related in: MedlinePlus

Epifluorescent micrographs of epithelial cell of primary culture of breast cancer tissue. (Fig. 4A) Bright aggregations of integrin alphavbeta3 immunofluorescence – indicating integrin clustering – were observed along the periphery of the ventral surface of the cell (arrow), while some of them were formed at the leading edge of the advancing lamellipodium (double arrows). Less bright fine granular integrin alphavbeta3 immunofluorescence was also observed along the cell surface (arrowheads). (Fig. 4B) F-actin cytoskeleton, as visualized by rhodamine-conjucated phalloidin, appeared well developed, constituted by numerous thick stress fibers, which were arranged towards the integrin alphavbeta3 immunolocalizations, as demonstrated also in the merged images (Fig. 4C). [750×]
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Figure 4: Epifluorescent micrographs of epithelial cell of primary culture of breast cancer tissue. (Fig. 4A) Bright aggregations of integrin alphavbeta3 immunofluorescence – indicating integrin clustering – were observed along the periphery of the ventral surface of the cell (arrow), while some of them were formed at the leading edge of the advancing lamellipodium (double arrows). Less bright fine granular integrin alphavbeta3 immunofluorescence was also observed along the cell surface (arrowheads). (Fig. 4B) F-actin cytoskeleton, as visualized by rhodamine-conjucated phalloidin, appeared well developed, constituted by numerous thick stress fibers, which were arranged towards the integrin alphavbeta3 immunolocalizations, as demonstrated also in the merged images (Fig. 4C). [750×]

Mentions: The primary epithelial breast cancer cells appeared larger in size than the normal ones (Figs. 3, 4). The pattern of integrin alphavbeta3 immunofluorescence was mainly observed at the marginal region of the cells at the sites of focal contacts, contributing to the adherence of the cancer cells onto the substrate. Thus, integrin alphavbeta3 immunofluorescence revealed a pattern of bright aggregations distributed along the periphery of the ventral surface of the cells (Figs. 3A, 4A, arrows), or at the leading edge of the advancing lamellipodium (Fig. 4A, double arrows). In addition, integrin alphavbeta3 localization was also recorded as fine granular fluorescence dispersed along cell-cell contacts (Fig. 3A, arrowhead), or at the cell's periphery (Fig. 4A, arrowhead). F-actin cytoskeleton fluorescence in epithelial breast cancer cells showed numerous and thicker stress fibers than those observed in normal cells. When the cells were in contact, the stress fibers were oriented vertically to the boundary between them (Fig. 3B) or towards areas of integrin immunofluorescence (Fig. 4B). The merged images demonstrated the correlation of the distribution of the stress fibers with the integrin alphavbeta3 immunofluorescence, indicating that the orientation of stress fibers is determined by/depending on the formation of integrin clustering (Fig. 3C, 4C).


Altered expression pattern of integrin alphavbeta3 correlates with actin cytoskeleton in primary cultures of human breast cancer.

Havaki S, Kouloukoussa M, Amawi K, Drosos Y, Arvanitis LD, Goutas N, Vlachodimitropoulos D, Vassilaros SD, Katsantoni EZ, Voloudakis-Baltatzis I, Aleporou-Marinou V, Kittas C, Marinos E - Cancer Cell Int. (2007)

Epifluorescent micrographs of epithelial cell of primary culture of breast cancer tissue. (Fig. 4A) Bright aggregations of integrin alphavbeta3 immunofluorescence – indicating integrin clustering – were observed along the periphery of the ventral surface of the cell (arrow), while some of them were formed at the leading edge of the advancing lamellipodium (double arrows). Less bright fine granular integrin alphavbeta3 immunofluorescence was also observed along the cell surface (arrowheads). (Fig. 4B) F-actin cytoskeleton, as visualized by rhodamine-conjucated phalloidin, appeared well developed, constituted by numerous thick stress fibers, which were arranged towards the integrin alphavbeta3 immunolocalizations, as demonstrated also in the merged images (Fig. 4C). [750×]
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2116995&req=5

Figure 4: Epifluorescent micrographs of epithelial cell of primary culture of breast cancer tissue. (Fig. 4A) Bright aggregations of integrin alphavbeta3 immunofluorescence – indicating integrin clustering – were observed along the periphery of the ventral surface of the cell (arrow), while some of them were formed at the leading edge of the advancing lamellipodium (double arrows). Less bright fine granular integrin alphavbeta3 immunofluorescence was also observed along the cell surface (arrowheads). (Fig. 4B) F-actin cytoskeleton, as visualized by rhodamine-conjucated phalloidin, appeared well developed, constituted by numerous thick stress fibers, which were arranged towards the integrin alphavbeta3 immunolocalizations, as demonstrated also in the merged images (Fig. 4C). [750×]
Mentions: The primary epithelial breast cancer cells appeared larger in size than the normal ones (Figs. 3, 4). The pattern of integrin alphavbeta3 immunofluorescence was mainly observed at the marginal region of the cells at the sites of focal contacts, contributing to the adherence of the cancer cells onto the substrate. Thus, integrin alphavbeta3 immunofluorescence revealed a pattern of bright aggregations distributed along the periphery of the ventral surface of the cells (Figs. 3A, 4A, arrows), or at the leading edge of the advancing lamellipodium (Fig. 4A, double arrows). In addition, integrin alphavbeta3 localization was also recorded as fine granular fluorescence dispersed along cell-cell contacts (Fig. 3A, arrowhead), or at the cell's periphery (Fig. 4A, arrowhead). F-actin cytoskeleton fluorescence in epithelial breast cancer cells showed numerous and thicker stress fibers than those observed in normal cells. When the cells were in contact, the stress fibers were oriented vertically to the boundary between them (Fig. 3B) or towards areas of integrin immunofluorescence (Fig. 4B). The merged images demonstrated the correlation of the distribution of the stress fibers with the integrin alphavbeta3 immunofluorescence, indicating that the orientation of stress fibers is determined by/depending on the formation of integrin clustering (Fig. 3C, 4C).

Bottom Line: In the present study, short-term primary breast cancer cell cultures were developed.In the former, F-actin cytoskeleton appeared well-formed, consisting of numerous and thicker stress fibers, compared to normal epithelial cells.Furthermore, electron microscopy showed increased integrin alphavbeta3 immunogold localization in epithelial breast cancer cells over the area of stress fibers at the basal cell surface.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory of Histology and Embryology, Medical School, University of Athens, 75 Mikras Asias Str, Goudi, Greece. shavaki@med.uoa.gr

ABSTRACT

Background: Integrins are transmembrane adhesion receptors that provide the physical link between the actin cytoskeleton and the extracellular matrix. It has been well established that integrins play a major role in various cancer stages, such as tumor growth, progression, invasion and metastasis. In breast cancer, integrin alphavbeta3 has been associated with high malignant potential in cancer cells, signaling the onset of widespread metastasis. Many preclinical breast cancer studies are based on established cell lines, which may not represent the cell behavior and phenotype of the primary tumor of origin, due to undergone genotypic and phenotypic changes. In the present study, short-term primary breast cancer cell cultures were developed. Integrin alphavbeta3 localization was studied in correlation with F-actin cytoskeleton by means of immunofluorescence and immunogold ultrastructural localization. Integrin fluorescence intensities were semi-quantitatively assessed by means of computerized image analysis, while integrin and actin expression was evaluated by Western immunoblotting.

Results: In the primary breast cancer epithelial cells integrin alphavbeta3 immunofluorescence was observed in the marginal cytoplasmic area, whereas in the primary normal breast epithelial cells it was observed in the main cell body, i.e. in the ventrally located perinuclear area. In the former, F-actin cytoskeleton appeared well-formed, consisting of numerous and thicker stress fibers, compared to normal epithelial cells. Furthermore, electron microscopy showed increased integrin alphavbeta3 immunogold localization in epithelial breast cancer cells over the area of stress fibers at the basal cell surface. These findings were verified with Western immunoblotting by the higher expression of integrin beta3 subunit and actin in primary breast cancer cells, revealing their reciprocal relation, in response to the higher motility requirements, determined by the malignant potential of the breast cancer cells.

Conclusion: A model system of primary breast cancer cell cultures was developed, in an effort to maintain the closest resembling environment to the tumor of origin. Using the above system model as an experimental tool the study of breast tumor cell behavior is possible concerning the adhesion capacity and the migrating potential of these cells, as defined by the integrin alphavbeta3 distribution in correlation with F-actin cytoskeleton.

No MeSH data available.


Related in: MedlinePlus