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Reversion of the malignant phenotype of human breast cells in three-dimensional culture and in vivo by integrin blocking antibodies.

Weaver VM, Petersen OW, Wang F, Larabell CA, Briand P, Damsky C, Bissell MJ - J. Cell Biol. (1997)

Bottom Line: A stimulatory beta1-integrin antibody proved to be ineffective.The observed phenotypes were reversible when the cells were disassociated and the antibodies removed.Our results illustrate that the extracellular matrix and its receptors dictate the phenotype of mammary epithelial cells, and thus in this model system the tissue phenotype is dominant over the cellular genotype.

View Article: PubMed Central - PubMed

Affiliation: Ernest Orlando Lawrence Berkeley National Laboratory, California 94720, USA.

ABSTRACT
In a recently developed human breast cancer model, treatment of tumor cells in a 3-dimensional culture with inhibitory beta1-integrin antibody or its Fab fragments led to a striking morphological and functional reversion to a normal phenotype. A stimulatory beta1-integrin antibody proved to be ineffective. The newly formed reverted acini re-assembled a basement membrane and re-established E-cadherin-catenin complexes, and re-organized their cytoskeletons. At the same time they downregulated cyclin D1, upregulated p21(cip,wat-1), and stopped growing. Tumor cells treated with the same antibody and injected into nude mice had significantly reduced number and size of tumors in nude mice. The tissue distribution of other integrins was also normalized, suggesting the existence of intimate interactions between the different integrin pathways as well as adherens junctions. On the other hand, nonmalignant cells when treated with either alpha6 or beta4 function altering antibodies continued to grow, and had disorganized colony morphologies resembling the untreated tumor colonies. This shows a significant role of the alpha6/beta4 heterodimer in directing polarity and tissue structure. The observed phenotypes were reversible when the cells were disassociated and the antibodies removed. Our results illustrate that the extracellular matrix and its receptors dictate the phenotype of mammary epithelial cells, and thus in this model system the tissue phenotype is dominant over the cellular genotype.

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Characterization of the HMT-3522 human breast cancer model. (a and a′) Phase contrast micrographs of nonmalignant S-1  cell colonies (a) and tumorigenic T4-2 cell colonies (a′) viewed directly inside EHS for morphology: S-1 cells formed spherical structures  reminiscent of true acini (a), whereas T4-2 cells formed large irregular colonies (a′). (b and b′) Immunostaining for basement membrane  components: S-1 acini (b) stained for basement membrane proteins at the cell-ECM junctions as expected, while basement membrane  deposition in tumor colonies (b′) was clearly disorganized and no longer polarized. Comparable results were obtained for laminin (not  shown). (c and c′) Confocal fluorescence microscopy of cryosectioned colonies immunostained for E-cadherin: S-1 acini (c) stained for  E-cadherin primarily at the cell–cell junctions as is typically observed in normal breast sections. The T4-2 colonies (c′) showed punctate,  dispersed membrane and intracellular staining. (d) The β-catenin–E-cadherin interaction ratio as a measure of adherens junction assembly: There was a 50% decrease in β-catenin protein coprecipitating with E-cadherin in T4-2 cells as compared to S-1 acini. Data are expressed as a ratio of β-catenin to E-cadherin densitometric measurements from duplicates of two separate experiments. Similar results  were observed for α-catenin (not shown). (e) Immunoblots of total levels of E-cadherin, α-catenin, and β-catenin: There were comparable levels of all three adherens proteins in S-1 acini and T4-2 colonies. (f and g) Percent of thymidine and Ki-67 labeling in S-1 acini and  T4-2 colonies, expressed as thymidine and Ki-67 labeling indices from 3–4 separate experiments: Greater than 90% of the S-1 acini were  growth arrested (f), and had exited the cell cycle (g) whereas T4-2 colonies were still actively growing (f) and continued to cycle (g). All  cultures were analyzed after 10–12 d inside EHS. Bars: (a and a′) 16 μm; (b and b′) 25 μm; (c and c′) 8 μm.
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Figure 1: Characterization of the HMT-3522 human breast cancer model. (a and a′) Phase contrast micrographs of nonmalignant S-1 cell colonies (a) and tumorigenic T4-2 cell colonies (a′) viewed directly inside EHS for morphology: S-1 cells formed spherical structures reminiscent of true acini (a), whereas T4-2 cells formed large irregular colonies (a′). (b and b′) Immunostaining for basement membrane components: S-1 acini (b) stained for basement membrane proteins at the cell-ECM junctions as expected, while basement membrane deposition in tumor colonies (b′) was clearly disorganized and no longer polarized. Comparable results were obtained for laminin (not shown). (c and c′) Confocal fluorescence microscopy of cryosectioned colonies immunostained for E-cadherin: S-1 acini (c) stained for E-cadherin primarily at the cell–cell junctions as is typically observed in normal breast sections. The T4-2 colonies (c′) showed punctate, dispersed membrane and intracellular staining. (d) The β-catenin–E-cadherin interaction ratio as a measure of adherens junction assembly: There was a 50% decrease in β-catenin protein coprecipitating with E-cadherin in T4-2 cells as compared to S-1 acini. Data are expressed as a ratio of β-catenin to E-cadherin densitometric measurements from duplicates of two separate experiments. Similar results were observed for α-catenin (not shown). (e) Immunoblots of total levels of E-cadherin, α-catenin, and β-catenin: There were comparable levels of all three adherens proteins in S-1 acini and T4-2 colonies. (f and g) Percent of thymidine and Ki-67 labeling in S-1 acini and T4-2 colonies, expressed as thymidine and Ki-67 labeling indices from 3–4 separate experiments: Greater than 90% of the S-1 acini were growth arrested (f), and had exited the cell cycle (g) whereas T4-2 colonies were still actively growing (f) and continued to cycle (g). All cultures were analyzed after 10–12 d inside EHS. Bars: (a and a′) 16 μm; (b and b′) 25 μm; (c and c′) 8 μm.

Mentions: Only small differences in morphology and growth rates could be observed between S-1 and T4-2 cells on tissue culture plastic (not shown), but profound differences became evident after only 4 d following their culture within a 3-D reconstituted basement membrane. Within 10 d, S-1 cells underwent morphogenesis and formed organized acini reminiscent of those formed by cells from reduction mammoplasty, while T4-2 cells formed large, loosely disorganized invasive colonies of cells similar to primary tumor cells tested in this assay previously (Fig. 1, a and a′; Petersen et al., 1992). In addition, S-1 cells were able to basally deposit and organize a basement membrane, as shown by immunostaining of type IV collagen (Fig. 1 b, and laminin, not shown), thereby demonstrating that the cells were able to form polarized structures. The irregular T4-2 colonies, while staining for basement membrane components had no discernible organized basement membrane (Fig. 1, b and b′). The failure of T4-2 cells to undergo morphogenesis was also indicated by their compromised cell–cell adhesion. This was shown by the absence of lateral E-cadherin immunostaining (Fig. 1, c and c′), an increase in cytoplasmic localization of E-cadherin (cell fractionation and extraction studies, not shown) and reduced interaction of α- and β-catenins with E-cadherin (co-immunoprecipitation, Fig. 1 d). Nevertheless, the two cell types expressed essentially the same levels of the three cell adhesion proteins (Fig. 1 e) indicating that the malignant conversion was associated with compromised assembly of adherens junctions rather than with down-regulation of these adhesion proteins. Coincident with the formation of acini, S-1 cells became growth arrested and exited the cell cycle, as demonstrated by negligible thymidine incorporation and low immunostaining for Ki-67. Consistent with their loss of structural organization, the tumorigenic T4-2 cells failed to growth arrest by these criteria (Fig. 1, f and g).


Reversion of the malignant phenotype of human breast cells in three-dimensional culture and in vivo by integrin blocking antibodies.

Weaver VM, Petersen OW, Wang F, Larabell CA, Briand P, Damsky C, Bissell MJ - J. Cell Biol. (1997)

Characterization of the HMT-3522 human breast cancer model. (a and a′) Phase contrast micrographs of nonmalignant S-1  cell colonies (a) and tumorigenic T4-2 cell colonies (a′) viewed directly inside EHS for morphology: S-1 cells formed spherical structures  reminiscent of true acini (a), whereas T4-2 cells formed large irregular colonies (a′). (b and b′) Immunostaining for basement membrane  components: S-1 acini (b) stained for basement membrane proteins at the cell-ECM junctions as expected, while basement membrane  deposition in tumor colonies (b′) was clearly disorganized and no longer polarized. Comparable results were obtained for laminin (not  shown). (c and c′) Confocal fluorescence microscopy of cryosectioned colonies immunostained for E-cadherin: S-1 acini (c) stained for  E-cadherin primarily at the cell–cell junctions as is typically observed in normal breast sections. The T4-2 colonies (c′) showed punctate,  dispersed membrane and intracellular staining. (d) The β-catenin–E-cadherin interaction ratio as a measure of adherens junction assembly: There was a 50% decrease in β-catenin protein coprecipitating with E-cadherin in T4-2 cells as compared to S-1 acini. Data are expressed as a ratio of β-catenin to E-cadherin densitometric measurements from duplicates of two separate experiments. Similar results  were observed for α-catenin (not shown). (e) Immunoblots of total levels of E-cadherin, α-catenin, and β-catenin: There were comparable levels of all three adherens proteins in S-1 acini and T4-2 colonies. (f and g) Percent of thymidine and Ki-67 labeling in S-1 acini and  T4-2 colonies, expressed as thymidine and Ki-67 labeling indices from 3–4 separate experiments: Greater than 90% of the S-1 acini were  growth arrested (f), and had exited the cell cycle (g) whereas T4-2 colonies were still actively growing (f) and continued to cycle (g). All  cultures were analyzed after 10–12 d inside EHS. Bars: (a and a′) 16 μm; (b and b′) 25 μm; (c and c′) 8 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2139858&req=5

Figure 1: Characterization of the HMT-3522 human breast cancer model. (a and a′) Phase contrast micrographs of nonmalignant S-1 cell colonies (a) and tumorigenic T4-2 cell colonies (a′) viewed directly inside EHS for morphology: S-1 cells formed spherical structures reminiscent of true acini (a), whereas T4-2 cells formed large irregular colonies (a′). (b and b′) Immunostaining for basement membrane components: S-1 acini (b) stained for basement membrane proteins at the cell-ECM junctions as expected, while basement membrane deposition in tumor colonies (b′) was clearly disorganized and no longer polarized. Comparable results were obtained for laminin (not shown). (c and c′) Confocal fluorescence microscopy of cryosectioned colonies immunostained for E-cadherin: S-1 acini (c) stained for E-cadherin primarily at the cell–cell junctions as is typically observed in normal breast sections. The T4-2 colonies (c′) showed punctate, dispersed membrane and intracellular staining. (d) The β-catenin–E-cadherin interaction ratio as a measure of adherens junction assembly: There was a 50% decrease in β-catenin protein coprecipitating with E-cadherin in T4-2 cells as compared to S-1 acini. Data are expressed as a ratio of β-catenin to E-cadherin densitometric measurements from duplicates of two separate experiments. Similar results were observed for α-catenin (not shown). (e) Immunoblots of total levels of E-cadherin, α-catenin, and β-catenin: There were comparable levels of all three adherens proteins in S-1 acini and T4-2 colonies. (f and g) Percent of thymidine and Ki-67 labeling in S-1 acini and T4-2 colonies, expressed as thymidine and Ki-67 labeling indices from 3–4 separate experiments: Greater than 90% of the S-1 acini were growth arrested (f), and had exited the cell cycle (g) whereas T4-2 colonies were still actively growing (f) and continued to cycle (g). All cultures were analyzed after 10–12 d inside EHS. Bars: (a and a′) 16 μm; (b and b′) 25 μm; (c and c′) 8 μm.
Mentions: Only small differences in morphology and growth rates could be observed between S-1 and T4-2 cells on tissue culture plastic (not shown), but profound differences became evident after only 4 d following their culture within a 3-D reconstituted basement membrane. Within 10 d, S-1 cells underwent morphogenesis and formed organized acini reminiscent of those formed by cells from reduction mammoplasty, while T4-2 cells formed large, loosely disorganized invasive colonies of cells similar to primary tumor cells tested in this assay previously (Fig. 1, a and a′; Petersen et al., 1992). In addition, S-1 cells were able to basally deposit and organize a basement membrane, as shown by immunostaining of type IV collagen (Fig. 1 b, and laminin, not shown), thereby demonstrating that the cells were able to form polarized structures. The irregular T4-2 colonies, while staining for basement membrane components had no discernible organized basement membrane (Fig. 1, b and b′). The failure of T4-2 cells to undergo morphogenesis was also indicated by their compromised cell–cell adhesion. This was shown by the absence of lateral E-cadherin immunostaining (Fig. 1, c and c′), an increase in cytoplasmic localization of E-cadherin (cell fractionation and extraction studies, not shown) and reduced interaction of α- and β-catenins with E-cadherin (co-immunoprecipitation, Fig. 1 d). Nevertheless, the two cell types expressed essentially the same levels of the three cell adhesion proteins (Fig. 1 e) indicating that the malignant conversion was associated with compromised assembly of adherens junctions rather than with down-regulation of these adhesion proteins. Coincident with the formation of acini, S-1 cells became growth arrested and exited the cell cycle, as demonstrated by negligible thymidine incorporation and low immunostaining for Ki-67. Consistent with their loss of structural organization, the tumorigenic T4-2 cells failed to growth arrest by these criteria (Fig. 1, f and g).

Bottom Line: A stimulatory beta1-integrin antibody proved to be ineffective.The observed phenotypes were reversible when the cells were disassociated and the antibodies removed.Our results illustrate that the extracellular matrix and its receptors dictate the phenotype of mammary epithelial cells, and thus in this model system the tissue phenotype is dominant over the cellular genotype.

View Article: PubMed Central - PubMed

Affiliation: Ernest Orlando Lawrence Berkeley National Laboratory, California 94720, USA.

ABSTRACT
In a recently developed human breast cancer model, treatment of tumor cells in a 3-dimensional culture with inhibitory beta1-integrin antibody or its Fab fragments led to a striking morphological and functional reversion to a normal phenotype. A stimulatory beta1-integrin antibody proved to be ineffective. The newly formed reverted acini re-assembled a basement membrane and re-established E-cadherin-catenin complexes, and re-organized their cytoskeletons. At the same time they downregulated cyclin D1, upregulated p21(cip,wat-1), and stopped growing. Tumor cells treated with the same antibody and injected into nude mice had significantly reduced number and size of tumors in nude mice. The tissue distribution of other integrins was also normalized, suggesting the existence of intimate interactions between the different integrin pathways as well as adherens junctions. On the other hand, nonmalignant cells when treated with either alpha6 or beta4 function altering antibodies continued to grow, and had disorganized colony morphologies resembling the untreated tumor colonies. This shows a significant role of the alpha6/beta4 heterodimer in directing polarity and tissue structure. The observed phenotypes were reversible when the cells were disassociated and the antibodies removed. Our results illustrate that the extracellular matrix and its receptors dictate the phenotype of mammary epithelial cells, and thus in this model system the tissue phenotype is dominant over the cellular genotype.

Show MeSH
Related in: MedlinePlus