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. 1997 Apr 7;137(1):231–245. doi: 10.1083/jcb.137.1.231

Figure 5.

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.