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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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β1-inhibitory antibody treatment of tumor cells leads to the formation of reverted acini. (a–a′′) Confocal fluorescence microscopy images of F actin: Both the S-1 (a) and T4-β1 reverted acini (a′′) showed basally localized nuclei (propidium iodide) and organized  filamentous F-actin (FITC), while T4-2 mock-treated colonies (T4-2 IgG) had disorganized, hatched bundles of actin and pleiomorphic  nuclei (a′). (b–b′′) Confocal immunofluorescence microscopy images of E-cadherin (FITC) and β-catenin (Texas red): In S-1 (b) and  T4-β1 reverted acini (b′′), E-cadherin and β-catenins were colocalized and superimposed at the cell–cell junctions. (c) Quantitative  analysis of tumor cell conversion efficiency by β1-integrin function blocking antibodies: Greater than 95% of S-1 and T4-β1 colonies  were scored as organized, while 97% of T4-2 IgG colonies were considered disorganized. Other reversion criteria, such as scoring for actin  and cadherin organization also yielded comparable results (not shown). (d) Cell number per colony in S-1 acini, T4-2 IgG colonies and T4β1 acini from 3–5 experiments: Both S-1 and T4-β1-revertant acini contained 6–8 cells, while T4-2 IgG tumor colonies contained 18–22 cells  when scored after 10–12 d (d). (e) Percent of thymidine-labeled cells in S-1, T4-2 IgG, and T4-β1 colonies: While a high percentage of T4-2  IgG colonies (greater than 30%) were still actively growing, the T4-2 β1 revertant acini had a greatly reduced growth rate similar to that  observed in the S-1 acini. (f) Immunoblot of cyclin D1 levels in S-1, T4-2, T4-2 IgG, and T4-β1 colonies: The level of D1 cyclin was  clearly decreased to the level of S-1 acini after β1 inhibitory antibody treatment. (g and g′) Collagen IV deposition as an indicator of  basement membrane organization: T4-β1 reverted acini (g′) deposited an organized collagen IV-containing BM at the cell-ECM junctions,  similar to that observed in S-1 acini (see Fig. 1 b). Note the contrast with T4-2 mock–treated tumor colonies (g). (Comparable results  were obtained for laminin, not shown.) All cultures were analyzed after 10–12 d inside EHS. Bars: (a–a′′and b–b′′) 16 μm; (g and g′) 25 μm.
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Figure 5: β1-inhibitory antibody treatment of tumor cells leads to the formation of reverted acini. (a–a′′) Confocal fluorescence microscopy images of F actin: Both the S-1 (a) and T4-β1 reverted acini (a′′) showed basally localized nuclei (propidium iodide) and organized filamentous F-actin (FITC), while T4-2 mock-treated colonies (T4-2 IgG) had disorganized, hatched bundles of actin and pleiomorphic nuclei (a′). (b–b′′) Confocal immunofluorescence microscopy images of E-cadherin (FITC) and β-catenin (Texas red): In S-1 (b) and T4-β1 reverted acini (b′′), E-cadherin and β-catenins were colocalized and superimposed at the cell–cell junctions. (c) Quantitative analysis of tumor cell conversion efficiency by β1-integrin function blocking antibodies: Greater than 95% of S-1 and T4-β1 colonies were scored as organized, while 97% of T4-2 IgG colonies were considered disorganized. Other reversion criteria, such as scoring for actin and cadherin organization also yielded comparable results (not shown). (d) Cell number per colony in S-1 acini, T4-2 IgG colonies and T4β1 acini from 3–5 experiments: Both S-1 and T4-β1-revertant acini contained 6–8 cells, while T4-2 IgG tumor colonies contained 18–22 cells when scored after 10–12 d (d). (e) Percent of thymidine-labeled cells in S-1, T4-2 IgG, and T4-β1 colonies: While a high percentage of T4-2 IgG colonies (greater than 30%) were still actively growing, the T4-2 β1 revertant acini had a greatly reduced growth rate similar to that observed in the S-1 acini. (f) Immunoblot of cyclin D1 levels in S-1, T4-2, T4-2 IgG, and T4-β1 colonies: The level of D1 cyclin was clearly decreased to the level of S-1 acini after β1 inhibitory antibody treatment. (g and g′) Collagen IV deposition as an indicator of basement membrane organization: T4-β1 reverted acini (g′) deposited an organized collagen IV-containing BM at the cell-ECM junctions, similar to that observed in S-1 acini (see Fig. 1 b). Note the contrast with T4-2 mock–treated tumor colonies (g). (Comparable results were obtained for laminin, not shown.) All cultures were analyzed after 10–12 d inside EHS. Bars: (a–a′′and b–b′′) 16 μm; (g and g′) 25 μm.

Mentions: Since T4-2 cells had both a higher total level and an elevated ratio of cell surface β1- to β4-integrins, we wondered whether the aberrant malignant behavior may be a reflection of the changes in these integrins. Accordingly, we examined the consequences of treatment in 3-D with varying concentrations of a previously characterized rat monoclonal β1-integrin antibody (clone AIIB2) which has been shown to inhibit ligand binding (Werb et al., 1989). This antibody caused massive apoptosis in S-1 cells, as shown previously (Howlett et al., 1995), while T4-2 cells were refractory (Fig. 4). Remarkably however, in addition to resistance to apoptosis, almost all the antibody-treated T4-2 tumor cells assumed a morphology which was indistinguishable from that observed in S-1 cultures and was discernible as early as 4 d after incubation. When examined by light microscopy after 12 d, these cultures appeared as if they had truly reverted to a “nonmalignant” phenotype. We therefore cryosectioned the colonies and examined their morphology by immunofluorescence confocal microscopy. As markers of normal acinar formation, we examined both cytoskeletal organization and superimposition and distribution of cadherins and catenins. Sections of S-1 acini revealed uniform and polarized nuclei (stained with propidium iodide; red), well-organized filamentous actin (FITC phalloidin; green) (Fig. 5 a), and uniformly superimposed E-cadherin and β-catenin at the lateral cell–cell junctions (Fig. 5 b). In contrast, untreated or IgG-treated tumor cells had polymorphic nuclei and a grossly disorganized actin cytoskeleton, visualized as random, hatched bundles (Fig. 5 a′). Additionally, E-cadherin and β-catenin were no longer colocalized (Fig. 5 b′). In contrast, β1-treated T4-2 cells (referred to as T4-β1) revealed striking rearrangements of cytoarchitecture as demonstrated by their well-organized actin (Fig. 5 a′′), and cytokeratin 18 intermediate filament (not shown) networks. Furthermore, organized adherens junctions became evident in T4-β1 acini (Fig. 5 b′′) and were accompanied by the re-establishment of E-cadherin–catenin complexes (not shown). These changes were shown to occur in greater than 95% of the tumor colonies treated with blocking antibody, as quantified by analyzing the numbers of disorganized vs organized spheroids in relation to the S-1 and the mock-treated T4-2 cells (Fig. 5 c). Viability and growth assays conducted on cells grown as monolayers ruled out toxicity (not shown). Interestingly, while treatment with the inhibitory β1-integrin antibody reduced cell adhesion and retarded the rate of cell proliferation when T4-2 cells were grown in two-dimension, culture in a three-dimensional reconstituted basement membrane was required for complete expression of the reverted phenotype (not shown).


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)

β1-inhibitory antibody treatment of tumor cells leads to the formation of reverted acini. (a–a′′) Confocal fluorescence microscopy images of F actin: Both the S-1 (a) and T4-β1 reverted acini (a′′) showed basally localized nuclei (propidium iodide) and organized  filamentous F-actin (FITC), while T4-2 mock-treated colonies (T4-2 IgG) had disorganized, hatched bundles of actin and pleiomorphic  nuclei (a′). (b–b′′) Confocal immunofluorescence microscopy images of E-cadherin (FITC) and β-catenin (Texas red): In S-1 (b) and  T4-β1 reverted acini (b′′), E-cadherin and β-catenins were colocalized and superimposed at the cell–cell junctions. (c) Quantitative  analysis of tumor cell conversion efficiency by β1-integrin function blocking antibodies: Greater than 95% of S-1 and T4-β1 colonies  were scored as organized, while 97% of T4-2 IgG colonies were considered disorganized. Other reversion criteria, such as scoring for actin  and cadherin organization also yielded comparable results (not shown). (d) Cell number per colony in S-1 acini, T4-2 IgG colonies and T4β1 acini from 3–5 experiments: Both S-1 and T4-β1-revertant acini contained 6–8 cells, while T4-2 IgG tumor colonies contained 18–22 cells  when scored after 10–12 d (d). (e) Percent of thymidine-labeled cells in S-1, T4-2 IgG, and T4-β1 colonies: While a high percentage of T4-2  IgG colonies (greater than 30%) were still actively growing, the T4-2 β1 revertant acini had a greatly reduced growth rate similar to that  observed in the S-1 acini. (f) Immunoblot of cyclin D1 levels in S-1, T4-2, T4-2 IgG, and T4-β1 colonies: The level of D1 cyclin was  clearly decreased to the level of S-1 acini after β1 inhibitory antibody treatment. (g and g′) Collagen IV deposition as an indicator of  basement membrane organization: T4-β1 reverted acini (g′) deposited an organized collagen IV-containing BM at the cell-ECM junctions,  similar to that observed in S-1 acini (see Fig. 1 b). Note the contrast with T4-2 mock–treated tumor colonies (g). (Comparable results  were obtained for laminin, not shown.) All cultures were analyzed after 10–12 d inside EHS. Bars: (a–a′′and b–b′′) 16 μm; (g and g′) 25 μm.
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Related In: Results  -  Collection

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Figure 5: β1-inhibitory antibody treatment of tumor cells leads to the formation of reverted acini. (a–a′′) Confocal fluorescence microscopy images of F actin: Both the S-1 (a) and T4-β1 reverted acini (a′′) showed basally localized nuclei (propidium iodide) and organized filamentous F-actin (FITC), while T4-2 mock-treated colonies (T4-2 IgG) had disorganized, hatched bundles of actin and pleiomorphic nuclei (a′). (b–b′′) Confocal immunofluorescence microscopy images of E-cadherin (FITC) and β-catenin (Texas red): In S-1 (b) and T4-β1 reverted acini (b′′), E-cadherin and β-catenins were colocalized and superimposed at the cell–cell junctions. (c) Quantitative analysis of tumor cell conversion efficiency by β1-integrin function blocking antibodies: Greater than 95% of S-1 and T4-β1 colonies were scored as organized, while 97% of T4-2 IgG colonies were considered disorganized. Other reversion criteria, such as scoring for actin and cadherin organization also yielded comparable results (not shown). (d) Cell number per colony in S-1 acini, T4-2 IgG colonies and T4β1 acini from 3–5 experiments: Both S-1 and T4-β1-revertant acini contained 6–8 cells, while T4-2 IgG tumor colonies contained 18–22 cells when scored after 10–12 d (d). (e) Percent of thymidine-labeled cells in S-1, T4-2 IgG, and T4-β1 colonies: While a high percentage of T4-2 IgG colonies (greater than 30%) were still actively growing, the T4-2 β1 revertant acini had a greatly reduced growth rate similar to that observed in the S-1 acini. (f) Immunoblot of cyclin D1 levels in S-1, T4-2, T4-2 IgG, and T4-β1 colonies: The level of D1 cyclin was clearly decreased to the level of S-1 acini after β1 inhibitory antibody treatment. (g and g′) Collagen IV deposition as an indicator of basement membrane organization: T4-β1 reverted acini (g′) deposited an organized collagen IV-containing BM at the cell-ECM junctions, similar to that observed in S-1 acini (see Fig. 1 b). Note the contrast with T4-2 mock–treated tumor colonies (g). (Comparable results were obtained for laminin, not shown.) All cultures were analyzed after 10–12 d inside EHS. Bars: (a–a′′and b–b′′) 16 μm; (g and g′) 25 μm.
Mentions: Since T4-2 cells had both a higher total level and an elevated ratio of cell surface β1- to β4-integrins, we wondered whether the aberrant malignant behavior may be a reflection of the changes in these integrins. Accordingly, we examined the consequences of treatment in 3-D with varying concentrations of a previously characterized rat monoclonal β1-integrin antibody (clone AIIB2) which has been shown to inhibit ligand binding (Werb et al., 1989). This antibody caused massive apoptosis in S-1 cells, as shown previously (Howlett et al., 1995), while T4-2 cells were refractory (Fig. 4). Remarkably however, in addition to resistance to apoptosis, almost all the antibody-treated T4-2 tumor cells assumed a morphology which was indistinguishable from that observed in S-1 cultures and was discernible as early as 4 d after incubation. When examined by light microscopy after 12 d, these cultures appeared as if they had truly reverted to a “nonmalignant” phenotype. We therefore cryosectioned the colonies and examined their morphology by immunofluorescence confocal microscopy. As markers of normal acinar formation, we examined both cytoskeletal organization and superimposition and distribution of cadherins and catenins. Sections of S-1 acini revealed uniform and polarized nuclei (stained with propidium iodide; red), well-organized filamentous actin (FITC phalloidin; green) (Fig. 5 a), and uniformly superimposed E-cadherin and β-catenin at the lateral cell–cell junctions (Fig. 5 b). In contrast, untreated or IgG-treated tumor cells had polymorphic nuclei and a grossly disorganized actin cytoskeleton, visualized as random, hatched bundles (Fig. 5 a′). Additionally, E-cadherin and β-catenin were no longer colocalized (Fig. 5 b′). In contrast, β1-treated T4-2 cells (referred to as T4-β1) revealed striking rearrangements of cytoarchitecture as demonstrated by their well-organized actin (Fig. 5 a′′), and cytokeratin 18 intermediate filament (not shown) networks. Furthermore, organized adherens junctions became evident in T4-β1 acini (Fig. 5 b′′) and were accompanied by the re-establishment of E-cadherin–catenin complexes (not shown). These changes were shown to occur in greater than 95% of the tumor colonies treated with blocking antibody, as quantified by analyzing the numbers of disorganized vs organized spheroids in relation to the S-1 and the mock-treated T4-2 cells (Fig. 5 c). Viability and growth assays conducted on cells grown as monolayers ruled out toxicity (not shown). Interestingly, while treatment with the inhibitory β1-integrin antibody reduced cell adhesion and retarded the rate of cell proliferation when T4-2 cells were grown in two-dimension, culture in a three-dimensional reconstituted basement membrane was required for complete expression of the reverted phenotype (not shown).

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