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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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Apoptosis induction in HMT-3522 cells by inhibitory  β1-integrin function blocking antibodies. Apoptotic labeling indices calculated for S-1 and T4-2 cultures treated with inhibitory  β1-integrin antibodies for 4 d in 3-dimensional cultures: Greater  than 70% of S-1 cells were induced to undergo apoptosis within 4 d  of incubation with β1-integrin function blocking antibody (S-1  β1) while the level of basal apoptosis was less than 5% in the isotype controls (S-1 IgG). In contrast T4-2 cells similarly treated  (T4 β1) were refractory and had an apoptosis rate comparable to  basal levels (T4-2 IgG). Results are the mean and SE of 3–6 separate experiments of duplicates.
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Figure 4: Apoptosis induction in HMT-3522 cells by inhibitory β1-integrin function blocking antibodies. Apoptotic labeling indices calculated for S-1 and T4-2 cultures treated with inhibitory β1-integrin antibodies for 4 d in 3-dimensional cultures: Greater than 70% of S-1 cells were induced to undergo apoptosis within 4 d of incubation with β1-integrin function blocking antibody (S-1 β1) while the level of basal apoptosis was less than 5% in the isotype controls (S-1 IgG). In contrast T4-2 cells similarly treated (T4 β1) were refractory and had an apoptosis rate comparable to basal levels (T4-2 IgG). Results are the mean and SE of 3–6 separate experiments of duplicates.

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)

Apoptosis induction in HMT-3522 cells by inhibitory  β1-integrin function blocking antibodies. Apoptotic labeling indices calculated for S-1 and T4-2 cultures treated with inhibitory  β1-integrin antibodies for 4 d in 3-dimensional cultures: Greater  than 70% of S-1 cells were induced to undergo apoptosis within 4 d  of incubation with β1-integrin function blocking antibody (S-1  β1) while the level of basal apoptosis was less than 5% in the isotype controls (S-1 IgG). In contrast T4-2 cells similarly treated  (T4 β1) were refractory and had an apoptosis rate comparable to  basal levels (T4-2 IgG). Results are the mean and SE of 3–6 separate experiments of duplicates.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2139858&req=5

Figure 4: Apoptosis induction in HMT-3522 cells by inhibitory β1-integrin function blocking antibodies. Apoptotic labeling indices calculated for S-1 and T4-2 cultures treated with inhibitory β1-integrin antibodies for 4 d in 3-dimensional cultures: Greater than 70% of S-1 cells were induced to undergo apoptosis within 4 d of incubation with β1-integrin function blocking antibody (S-1 β1) while the level of basal apoptosis was less than 5% in the isotype controls (S-1 IgG). In contrast T4-2 cells similarly treated (T4 β1) were refractory and had an apoptosis rate comparable to basal levels (T4-2 IgG). Results are the mean and SE of 3–6 separate experiments of duplicates.
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