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Cell, Tumor, and Stem Cell Biology |
Departments of 1 Radiation Oncology and 2 Pathology, University of California, San Francisco, California; 3 Life Sciences Division, Ernest Orlando Lawrence Berkeley National Laboratory, Berkeley, California; 4 School of Natural Sciences, University of California, Merced, California; and 5 Department of Mathematics and Statistics, San Diego State University, San Diego, California
Requests for reprints: Catherine Park, University of California in San Francisco/Mt. Zion Cancer Center, 1600 Divisadero Street H1031, San Francisco, CA 94143-1708. Phone: 415-353-7186; Fax: 415-353-9883; E-mail: park{at}radonc17.ucsf.edu.
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Abstract |
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Introduction |
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Using a three-dimensional lrECM cell culture model, which emulates a more physiologically relevant microenvironment (16), we showed previously that down-modulation of ß1 integrin and growth factor signaling pathways resulted in reversion of the malignant phenotype (17), leading to growth arrest and reformation of tissue polarity (18). In addition, ß1 integrin and growth factor signaling were found to be integrated in the context of the three-dimensional lrECM but not on tissue culture plastic (18, 19).
We reasoned that a modified version of this culture model could provide an accurate surrogate for testing therapies for human breast cancer cells and tumors. We developed the modified three-dimensional lrECM assay and show that inhibition of ß1 integrin results not only in antiproliferative and proapoptotic effects in malignant cell lines in three-dimensional cultures, but that these results were recapitulated also in vivo. ß1 integrin inhibition preferentially affected malignant cells both in culture and in vivo; the nonmalignant acini and normal tissues were not affected, and remarkably, there was little or no toxicity to the animals.
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Materials and Methods |
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ß1 integrin and HER-2 inhibitory antibodies. AIIB2, a ß1 integrin function-blocking antibody (originally a gift from C. Damsky, UCSF) was isolated and prepared from a hybridoma cell line (Sierra Biosources, Milipitas, CA). AIIB2 is a rat monoclonal IgG1 that was originally isolated from a human choriocarcinoma hybridoma that specifically binds ß1 integrin extracellular domain (21–23). Experiments using F(ab')2 fragments of enzyme-digested AIIB2 indicated that the epitope-binding portion of the antibody was active and resulted in down-modulation of ß1 integrin–mediated signaling (17, 19). AIIB2 was added to culture medium on alternate days. Herceptin is a humanized monoclonal antibody against the erbB2 or HER-2 receptor (24) that was used (20 µg/mL) to treat SKBR3 cells on day 6. Control cultures for all experiments were treated with the same concentration of nonspecific IgG.
Immunofluorescence. Cells from three-dimensional cultures were fixed onto a glass slide using 4% paraformaldehyde or methanol/acetone. Nonspecific sites were blocked with 0.5% casein/PBS solution for 1 hour at room temperature. Primary ß1 integrin monoclonal rat anti-mouse antibody (PharMingen, San Diego, CA; 1:100) was diluted in blocking buffer and was applied for 1 hour at room temperature in a humidified chamber. Slides were washed in PBS containing 0.1% bovine serum albumin, before incubating in secondary antibody conjugated to FITC (Molecular Probes, Eugene, OR) for 1 hour in a dark humidified chamber at room temperature. The slides were then washed and counterstained with 4',6-diamidino-2-phenylindole before mounting with Vectashield mounting medium (Vector Laboratories, Burlingame, CA).
Confocal microscopy. Confocal images were acquired by using a Zeiss LSM 410 inverted laser scanning confocal microscope equipped with an external argon/krypton laser. Using a Zeiss Fluor x40 (1.3 numerical aperture) objective, images were captured at the colony midsection. Relative immunofluorescence intensity of images was standardized by comparing only cultures that were processed identically and stained in the same experiment.
Western immunoblot. Cells propagated in three-dimensional lrECM were first treated with ice-cold PBS/EDTA [0.01 mol/L sodium phosphate (pH 7.2) containing 138 mmol/L sodium chloride and 5 mmol/L EDTA] to isolate the cells and then lysed in radioimmunoprecipitation assay buffer as previously described (17). Equal amounts of protein were loaded onto reducing SDS gels. After transfer onto nitrocellulose membrane (Invitrogen, Carlsbad, CA), blots were blocked with 5% nonfat milk and probed. Primary antibodies used include ß1 integrin, clone 18 (1:1,000), phospho-FAK, clone 14 (1:1,000; BD Transduction Laboratories, Lexintgon, KY); phospho-ß1 integrin (1:1,000; Biosource, Camarillo, CA); ß-actin, clone AC-15 (1:5,000; Sigma, St. Louis, MO). Blots were washed, incubated with secondary antibody, and exposed to X-ray film.
Fluorescence-activated cell sorting analysis. Cells were propagated on tissue culture plastic and harvested using 0.25% trypsin. After resuspending in 1 mL DMEM/F-12 media with trypsin inhibitor, cells were spun down and washed in 1x PBS, 5% fetal bovine serum, and 0.1% sodium azide on ice. Cells were incubated with primary antibody (AIIB2, 1:10) at 4°C for 30 minutes to 1 hour, washed, and incubated with a fluorescein-conjugated IgG secondary antibody (1:100) for 30 minutes. After washing, 1 mL of 1% paraformaldehyde solution was added to the pellet and suspended immediately. Cells were analyzed using a Beckman-Coulter EPICS XL-MCL Analyzer. System II Data Acquisition and Display software, version 2.0 was used for data analysis.
Apoptosis and proliferation assays. Apoptosis was assayed in cell culture using a commercially available kit (In Situ Cell Death Detection kit, fluorescein; Roche, Nutley, NJ) designed to detect terminal deoxynucleotidyl transferase (TdT)–mediated nick end labeling (TUNEL). Cells were fixed in 4% paraformaldehyde and permeabilized in cold 0.1% Triton X-100 in 0.1% sodium citrate. After washing in PBS, cells were incubated in TUNEL reaction mixture at 37°C for 60 minutes, washed, and mounted. Proliferation was detected by indirect immunofluorescence of Ki-67 nuclear antigen. Cells were fixed in methanol/acetone and blocked using 10% goat serum, then incubated in primary rabbit antibody against Ki-67, clone MIB-1 (1:200; Novocastra Laboratories, Norwell, MA) for 1 hour and washed before FITC-conjugated anti-rabbit secondary antibody (The Jackson Laboratory, Bar Harbor, ME) was applied. Nuclei were counterstained with DAPI.
For assay of apoptosis in paraffin-embedded tissues, Apoptag In Situ Apoptosis Detection kit (Intergen, Burlington, MA) was used to detect TUNEL reaction. Paraffin-embedded xenograft tumors were sectioned at 5- to 10-µm-thick sections. Sections were deparaffinized and rehydrated using xylene and ethanol washes. Tissues were then treated with proteinase K at room temperature, washed, and quenched using 3% hydrogen peroxide. Buffer solution was applied, and sections were incubated in TdT enzyme at 37°C for 1 hour. Stop/wash buffer was used before anti-digoxigenin peroxidase conjugate was applied. Proliferation was assayed in paraffin-embedded tissues using indirect immunohistochemistry. Sections were deparaffinized as above and blocked using 10% normal horse serum, then incubated with mouse monoclonal antibody against Ki-67 (Oncogene, San Diego, CA) overnight at 4°C, and washed in PBS. They were then serially incubated with biotinylated anti-mouse antibody, and steptavidin-horseradish peroxidase and 3,3'-diaminobenzidine (DAPI) medium. After counterstaining with hematoxylin, sections were dehydrated in serial concentrated ethanol and xylene and mounted. Cells were scored by counting the total number of nuclei in five high-power microscopic fields (x40) using a x10 objective, or a minimum of 200 nuclei per tumor section.
Tumor growth and toxicity assessment in vivo. Female nu–/– mice were obtained from Charles River (Wilmington, MA) or Taconic (Germantown, NY) and housed five per cage with chow and water ad libitum in a controlled animal barrier. Animals were injected s.c. with 5 to 10 x 106 T4-2 cells or 107 MCF-7 cells into the upper back posterior to the right front limb. Estradiol pellets were inserted s.c. above the tail for animals bearing MCF-7 xenografts. AIIB2 antibody or nonspecific rat IgG was injected into the i.p. cavity biweekly beginning on day 4 or day 28 after cell implantation. Tumor dimensions (width, height, and depth) were measured biweekly. At the time of sacrifice, animals were euthanized, and tumors were harvested and either immediately frozen in ornithine carbamyl transferase or fixed in formalin. Serum was collected using cardiac puncture techniques.
Animals were monitored for evidence of toxicity by measuring weight, assessing overall activity, and necropsy. Additional toxicity studies were done using ß1 integrin inhibitory antibody, clone Ha 2/5 (PharMingen), which specifically recognizes murine ß1 integrin. Antibody was administered at doses of 1 to 20 mg/kg biweekly over 4 weeks. All experimental procedures were followed according to the UCSF, and LBNL Animal Welfare Committees approved policies and guidelines.
Statistical analysis. For each dose of AIIB2 or control IgG in culture, pairwise differences in Ki-67 or TUNEL were tested among the six cell lines using Student's t test (25). Multivariate ANOVA was used for analysis of tumor volume at each time point. For each dose of AIIB2 or control IgG in vivo, pairwise Student's t test or 
2 comparison was used to analyze differences between TUNEL and Ki-67 expression. MINITAB (Minitab, Inc., State College, PA) statistical software was used for all calculations.
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Results |
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When cultured on top of three-dimensional lrECM gels with 5% Matrigel conditioned media, nonmalignant breast cells undergo morphogenesis and, after 6 days, form acini with polarized cells oriented around a central lumen with a well organized basement membrane, recapitulating normal acinar structures found in vivo (Fig. 1A; for review, see ref. 28). In contrast, all malignant breast cell lines tested (T4-2, MDA-MB-231, SKBR3, BT474, and MCF-7) continued to proliferate and formed disorganized tumor colonies (Fig. 1A). Our previous studies have shown that ß1 integrin inhibitory monoclonal antibody, AIIB2, or its F(ab')2 fragments applied to single cells were capable of down-modulating ß1 integrin signaling pathways (17, 19). In the present studies, breast cancer cell lines were propagated in three-dimensional lrECM until colonies were formed (4 days) and were then treated with AIIB2 at doses ranging from 0.08 to 0.24 mg/mL, or with isotype-matched nonspecific rat IgG1 as control (Fig. 1B). Using confocal microscopy, we show that ß1 integrin was appropriately localized to the basolateral surfaces of the S-1 cells within the acini, as is the case in vivo. In contrast, it was diffusely distributed around the surfaces of each cell within T4-2 and MDA-MB-231 colonies in three-dimensional lrECM, and little expression was seen on three of the other cell lines (Fig. 1B).
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In contrast, the nonmalignant cell line S-1 formed acinar structures when cultured on top of three-dimensional lrECM for 6 days and, unlike colonies made of malignant cells, did not undergo increased apoptosis or cytostasis upon addition of AIIB2 regardless of the dose used (Fig. 1C and D). Similar results were obtained for S-1 cells treated at day 4 (data not shown). In addition, there was no significant change in the distribution of the size or number of total colonies (data not shown). Previous studies have shown that AIIB2 applied to single S-1 cells induce apoptosis (27); however, in the present study, we show that when S-1 cells are in the context of organized structures, they are resistant to apoptosis. This indicated that the signaling context of ß1 integrin is critical to response to AIIB2 treatment: nonmalignant mammary epithelial cells with intact cell-cell and cell-ECM interactions were resistant to the inhibitor. These results confirm and extend studies of conventional apoptotic and chemotherapeutic agents tested previously in the single-cell assay in three-dimensional lrECM (29).
Coexpression of total ß1 integrin, phosphorylated ß1 integrin, and phosphorylated 397FAK among breast cell lines cultured in three-dimensional lrECM. ß1 Integrin expression detected by immunofluorescence was characterized by basolateral localization in nonmalignant S-1 acinar structures and disorganized and aberrant expression in the malignant cell lines. To further characterize ß1 integrin expression, we analyzed cell lysates for total ß1 integrin levels using Western immunoblotting. Total ß1 integrin expression corresponded to that detected using immunofluorescence; three cell lines (S-1, T4-2, and MDA-MB-231) showed relatively higher levels of ß1 integrin compared with SKBR3, BT474, and MCF-7 (Fig. 2A). In addition, fluorescence-activated cell sorting (FACS) analysis confirmed the surface expression of ß1 integrin reflected that detected using immunofluorescence and Western blot (Fig. 2B). We concluded that ß1 integrin expression was variable, and response to ß1 integrin inhibitory antibody did not seem to correlate with total levels of ß1 integrin expression in individual cell lines.
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SKBR3 colonies respond to a combination of AIIB2 and Herceptin. The SKBR3 cell line overexpresses erbB2 (HER-2), a member of the EGF family of growth factor receptors. ß1 integrin has been shown to cooperate with other members of the EGF family, such as erbB1 (19); however, the relationship between HER-2 and ß1 integrin signaling is not well understood. We reasoned that HER-2 signaling was one factor that could contribute to the decreased cytostatic response of SKBR3 cells treated with AIIB2. Herceptin is a monoclonal antibody directed against HER-2 and has a significant role in treatment of patients with HER-2 overexpressing breast cancer (33). Therefore, we tested the effect of Herceptin and AIIB2 in combination in SKBR3 cells. Compared with colonies treated with nonspecific control IgG, SKBR3 colonies treated with AIIB2 or Herceptin alone showed a proportional decrease in Ki-67–positive cells (44.8% for AIIB2 and 39.1% for Herceptin). However, colonies that were treated with both AIIB2 and Herceptin had an augmented proportional decrease in Ki-67–positive cells (68.8%; P < 0.05, 2; Supplementary Fig. S1).
ß1 integrin inhibition preferentially affects larger tumor masses with a global redistribution in colony size and morphology. To determine the effect of treatment on the colony population as a whole, we counted the total number of cells and then scored for individual colonies by size. Using T4-2 cells as a prototype, we found that ß1 integrin inhibition resulted in a significant decrease in total cell number (Fig. 3A, mean ± SE; P < 0.05, 2). To further examine how the treatment influenced the global composition of colonies in the population, we counted the number of cells within each colony after 3 days of treatment. The mean colony size decreased from 12 to 6 cells with treatment reflected by the distribution of the size of colonies (Fig. 3B, mean ± SE; P < 0.05, t test). Similar results were seen for all other cancer cell lines (data not shown). To investigate the time course and mechanism of these changes, we counted the average number of Ki-67– and TUNEL-positive nuclei in the T4-2 cultures as a function of time after addition of AIIB2 (Fig. 3C, mean ± SE; P < 0.05, t test). The number of proliferating cells decreased dramatically even within 1 hour after addition of AIIB2, indicating an immediate growth arrest. The percentage of TUNEL-positive nuclei increased from 24 to 72 hours.
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2). After 4 weeks, animals were sacrificed, serum was analyzed for AIIB2 levels, and tumors were analyzed for histology. Compared with controls, treated animals had a dose-dependent level of AIIB2 detectable in serum samples (Fig. 4A'', mean ± SE; P < 0.05, 2). Representative micrographs of the same coregistered region of a tumor stained with H&E, Ki-67, and TUNEL are shown (Fig. 4B).
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2). In addition, tumors treated with 5 mg/kg AIIB2 had significantly higher percentage of caspase-3–positive cells (11 ± 2%) compared with controls (3.13 ± 0.7%; P < 0.05, 2). Similar results were obtained for MCF-7 xenografts treated with AIIB2 in vivo (data not shown).
AIIB2 is effective against established tumors in vivo. To further evaluate the efficacy of AIIB2 in vivo, we allowed MCF-7 cells to continue to grow for 
4 weeks and then randomized animals to receive nonspecific rat IgG1, 1 mg/kg AIIB2, or 5 mg/kg AIIB2 for four additional weeks. Compared with controls, treated animals had significantly less tumor growth (Fig. 5A). In addition, histologic analysis showed that treated tumors had significantly fewer Ki-67–positive cells compared with controls (Fig. 5B, mean ± SE; P < 0.001, 2) and significantly decreased TUNEL-positive nuclei (Fig. 5B, mean ± SE; P < 0.01, 2). Similar results were found for T4-2 xenografts treated in vivo (data not shown).
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Although AIIB2 seems to cross react with murine ß1 integrin,6 we sought to further evaluate the potential toxicity of broad ß1 integrin inhibition in vivo. Therefore, we used clone Ha2/5, a ß1 integrin function-blocking antibody that recognizes murine ß1 integrin. Adult female nu–/– mice were treated with serially increasing doses of antibody from 1 to 20 mg/kg over 4 weeks via biweekly i.p. injection. There were no differences in body weight, activity, overall appearance, or examination at necropsy among animals receiving antibody compared with controls, and no evidence of toxicity among any groups (data not shown).
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Discussion |
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The use of three-dimensional cultures provides a physiologically relevant context in which to emulate cells in vivo (35, 36) and has been used previously to investigate novel mechanisms of drug resistance in cancer cells that are demonstrable specifically only in a three-dimensional setting when the appropriate basement membrane molecules are present (37, 38). To model the differences between normal and malignant tissues, we took advantage of the ability of a nonmalignant cell line, HMT-3522-S-1, to undergo normal morphogenesis in three-dimensional lrECM, in contrast to malignant cells that continue to form disorganized invasive colonies. This allowed us to examine the effects of ß1 integrin inhibition on the morphology of cancer cell colonies as a population and to distinguish the potential effects on nonmalignant acini. We had shown previously that nonmalignant cells that were treated with ß1 integrin inhibition as single cells were susceptible to apoptosis (27, 39). However, the response of cells within acinar-like tissue structures where ß1 integrin function is relatively intact has not been investigated. We found that in response to 3 days of AIIB2 treatment, all but one malignant cell line in three-dimensional lrECM showed a dramatic loss in total number of cells, coupled with a significant increase in the percentage of apoptotic cells and a significant decrease in the percentage of proliferating cells. In contrast, S-1 cells that formed polar acinar-like structures were entirely resistant to AIIB2. These results indicate that most malignant cells that form colonies in three-dimensional lrECM rely on ß1 integrin signaling for proliferation and survival, whereas in the context of an organized structure, cells were either no longer dependent on ß1 integrin signaling for survival, or that ß1 integrin was not accessible to the antibody.
Further analysis of cell cultures during and after AIIB2 treatment revealed that the largest cancer cell colonies were being affected, resulting in a global change in the morphology and distribution of proliferating cells, reflected in a decrease in mean colony size. This pattern of multiple residual "tumor foci" was seen also in vivo (data not shown). The morphologic characteristics of these clones were distinctly different from the untreated tumors, as were features of cell-cell and cell-ECM interactions. These results have implications for clinical treatment. ß1 Integrin has been implicated in mediating resistance to cytotoxic chemotherapies (9, 10), and inhibition of different tumor types may enhance response by abrogating resistance. In addition, ionizing radiation was shown to up-regulate ß1 integrin in cancer cells (40, 41), and our preliminary studies of ß1 integrin inhibition combined with ionizing radiation are promising and may lead to novel strategies for combinatorial therapies to eradicate or further reduce tumor viability in vivo.
Several promising biological therapies aimed at signaling pathways have entered clinical trials; however, despite evidence of response to treatment, useful biomarkers have frequently been difficult to validate (1, 42, 43). For example, the current treatment of cancers with EGFR inhibition illustrates the complexity of some molecular targets and the lack of robust predictive markers that would aid in the selection of individuals for treatment (42). The mechanisms that are involved in cytostasis and apoptosis associated with ß1 integrin inhibition in malignant cells are likely to involve interactions between multiple signaling pathways. For example, our previous studies have shown that ß1 integrin signaling pathway integrates and cooperates with the EGFR signaling pathway via mitogen-activated protein kinase and phosphatidylinositol 3-kinase (18, 19). In the present study, we found that ß1 integrin expression on the six breast cell lines used was variable. We probed the cell lines for p-ß1 integrin and p-397FAK to investigate potential markers for ß1 integrin signaling activity. Interestingly, p-ß1 integrin expression was inversely correlated with total ß1 integrin, suggesting that either species is required for ß1 integrin signaling to occur. Although p-397FAK is a requisite protein for focal adhesion formation, its role in ß1 integrin signaling in the context of the cell lines we investigated remains unclear. We recognize that ß1 integrin signaling involves several steps, including activation, heterodimerization, ligand binding, and clustering (44, 45); these functional aspects of ß1 integrin signaling activity may not be reflected by the level of receptor expression and/or status of any single signaling protein alone. The major factor that distinguished the nonmalignant S-1 cells and the malignant cell lines is the organization and polarity of ß1 integrin localization, indicating that the context of signaling may be the most important feature that enhances the therapeutic window. Studies are ongoing to investigate which pathways may be the most robust predictors of response in to ß1 integrin inhibition in the clinical setting.
SKBR3 cells were less responsive to ß1 integrin inhibition compared with other cancer cell lines. This cell line is characteristically devoid of E-cadherin and overexpresses growth factor receptor HER-2 features that could contribute to uncoupling of ß1 integrin signaling and survival (46, 47). Interestingly, BT474 cells, which overexpress HER-2 and estrogen receptor (ER), remain sensitive to AIIB2. In contrast, SKBR3 cells overexpress HER-2 but are ER negative, a phenotype that has implicated growth factor signaling pathways with resistance to tamoxifen (48). Herceptin, a monoclonal antibody against HER-2, has been shown to down-modulate the HER-2 receptor, resulting in cytostasis (24). We found that the addition of Herceptin to AIIB2 in SKBR3 cells in three-dimensional lrECM resulted in a significantly decreased percentage of Ki-67–positive cells compared with cultures treated with AIIB2 alone. These data indicate that an additive cytostatic effect is achieved by using the combination of inhibitory antibodies. Further investigations of the features of SKBR3 that may confer resistance to AIIB2 are warranted and may help identify subsets of tumors that may respond to a combination of ß1 integrin inhibition and Herceptin or hormonal therapy.
We found that ß1 integrin inhibition was effective in both T4-2 and MCF-7 xenografts in nude mice in vivo, confirming our results in three-dimensional lrECM. Similar to the response observed in culture, tumor xenografts treated in vivo showed decreased proliferation and increased apoptosis compared with controls in the animals that received treatment beginning 4 days after tumor implantation. In animals where the tumors were treated after 4 weeks of implantation, there was a significant decrease in tumor size and proliferation and a decrease in apoptosis in treated animals compared with controls. The decrease in observed TUNEL-positive cells in the larger tumors could be due to the increased amounts of necrosis, an alternate mechanism of cell death, seen in larger tumors (data not shown).
Toxicity studies using AIIB2 and clone Ha2/5 revealed no discernible toxicity in animals, even with 20 mg/kg doses. These results indicate that ß1 integrin signaling confers growth and survival advantages in cancer cells in vivo that can be discriminated from normal ß1 integrin signaling by AIIB2. Other mechanisms also should be considered. For example, immune-mediated secondary effects of the antibody have been shown to play a significant role in antibody-mediated therapies (47). We have previously shown that the F(ab')2 fragments of AIIB2 are active in three-dimensional lrECM assays (17), and others have shown that AIIB2 binds to a region of ß1 integrin extracellular domain between two putative ligand binding sites that are thought to induce a conformational change (48), resulting in down-modulation of signaling. The activity or presence of Fc-directed immune response in vivo has not been isolated from the activity of the F(ab')2 region per se. These studies, in addition to humanization of the AIIB2 clone, are necessary next steps towards clinical drug development.
In summary, ß1 integrin inhibition using monoclonal antibody AIIB2 results in cytostasis and apoptosis in malignant breast cancer colonies but not normal tissue structures propagated on top of three-dimensional lrECM gels. The three-dimensional lrECM assay appropriately distinguishes the difference in response between normal structures and malignant colonies and reveals global changes in morphology associated with treatment. In addition, AIIB2 inhibits breast cancer growth in vivo by eliciting increased apoptosis and decreased proliferation, with no discernible toxicity to animals. We conclude that ß1 integrin inhibition using monoclonal antibodies is a promising approach to breast cancer treatment, and that the modified three-dimensional lrECM assay and protocol is an appropriate assay for testing differences in malignant and normal cell response to targeted therapeutic agents.
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Acknowledgments |
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
We thank Donghui Wang and Evelyn Yao for expert technical assistance.
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Footnotes |
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Received 8/29/05. Revised 11/12/05. Accepted 11/18/05.
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2/ß1 in peritoneal dissemination of a human gastric cancer xenograft model. Int J Oncol 2001;18:809–15.[Medline]1/ß1 and 6/ß1 integrin heterodimers mediate cell attachment to distinct sites on laminin. J Cell Biol 1990;110:2175–84./ß heterodimeric receptors for laminin and type IV collagen. J Cell Biol 1988;107:1241–52.This article has been cited by other articles:
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) of colonies cultured in three-dimensional lrECM. Phalloidin was used to stain actin filaments (
), and DAPI was used to stain individual nuclei (
). Bar, 13 µm for all cell lines shown. C, whereas there were no significant changes in Ki-67 or TUNEL expressing cells among nonmalignant S-1 cells after ß1 integrin inhibition, malignant cell lines, except SKBR3, had a significant decrease in Ki-67 expressing cells and a significant increase in TUNEL-positive cells after antibody treatment. Columns, mean (n = 3); bars, SE. P < 0.05, t test.
) or 5 mg/kg AIIB2 (
) were significantly smaller than control animals (
). Points, mean (n = 9); bars, SE. A', average serum concentration (µg/mL) of AIIB2 at the time of sacrifice was significantly higher in animals receiving AIIB2 compared with control vehicle. Columns, mean; bars, SD. A'', average number of animals in each treatment group that had histologic evidence of tumor at the time of sacrifice was significantly lower among treated animals compared to controls. Columns, mean for three separate experiments; bars, SE. *, P < 0.03, 
