Protease-dependent versus -independent cancer cell invasion programs: three-dimensional amoeboid movement revisited - PubMed (original) (raw)

Protease-dependent versus -independent cancer cell invasion programs: three-dimensional amoeboid movement revisited

Farideh Sabeh et al. J Cell Biol. 2009.

Abstract

Tissue invasion during metastasis requires cancer cells to negotiate a stromal environment dominated by cross-linked networks of type I collagen. Although cancer cells are known to use proteinases to sever collagen networks and thus ease their passage through these barriers, migration across extracellular matrices has also been reported to occur by protease-independent mechanisms, whereby cells squeeze through collagen-lined pores by adopting an ameboid phenotype. We investigate these alternate models of motility here and demonstrate that cancer cells have an absolute requirement for the membrane-anchored metalloproteinase MT1-MMP for invasion, and that protease-independent mechanisms of cell migration are only plausible when the collagen network is devoid of the covalent cross-links that characterize normal tissues.

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3D cancer cell invasion. Multicellular spheroids of HT-1080 fibrosarcoma cells were embedded within 3D gels of native type I collagen for 3 d. Gels were fixed in 2% glutaraldehyde/1.5% paraformaldehyde in 0.1 M sodium cacodylate buffer, freeze-fractured, and processed for SEM. Images of infiltrating cells and the surrounding ECM were digitally imaged using XStream imaging software. SASHA MESHINCHI

Figure 1.

Figure 1.

Regulation of cancer cell–invasive phenotype in 3D type I collagen by MT1-MMP. (A) Di-I–labeled multicellular spheroids (150–200 µm in diameter) of HT-1080 cells or MDA-MB-231 cells were prepared in hanging droplets (Kelm et al., 2003) after electroporation with a control siRNA, MMP-1 and MMP-2 siRNAs, MT1 siRNA (50–100 nM; siRNA sequences can be found in Li et al. [2008] and Sabeh et al. [2004]), or cotransfected with MT1 siRNA and either mouse (m)MT1-MMP or a control expression vector (0.5 µg each in PCR3.1 Uni; gift of M. Seiki, University of Tokyo, Tokyo, Japan; Sabeh et al., 2004; Li et al., 2008), and embedded in 3D type I collagen gels (prepared from acid extracts of rat tail tendon as described previously [Sabeh et al., 2004] and visualized by SEM in the left-hand panel, top corner inset; bar = 5 µm) at a final concentration of 2.2 mg/ml in the absence or presence of 10 µM GM6001. Invasion was monitored by fluorescence microscopy at 0 d (top row of panels) and by confocal microscopy at 3 d after staining with Alexa-488 phalloidin and DAPI (bottom row of panels; Di-I, red; Alexa-488 phalloidin, green; DAPI, blue). For MDA-MB-231 cells (bottom row of panels), spheroid at 0 d are shown in the insets. Bar = 100 µm. (B) MT1-MMP, MMP-1, and MMP-2 siRNAs inhibited expression of the targeted MMPs in HT-1080 cells, but had no effect on the expression of nontargeted MMPs as shown by RT-PCR. (C) Invasion distance (quantified as described previously; [Hotary et al., 2000; Sabeh et al., 2004]) from the inoculation site into the surrounding matrix was monitored at 1 d and 3 d for HT-1080, MDA-MB-231, or SUM-159 cells. In the presence of a protease inhibitor cocktail directed against serine, aspartyl, cysteinyl, and matrix metallo-proteinases (Wolf et al., 2003a), HT-1080 invasion was inhibited by 99 ± 0%. Results are expressed as mean ± SEM (n = 4). (D) HT-1080 cell spheroids were embedded in type I collagen gels in the absence or presence of the indicated doses of BB-2516 for 3 d. Insets show the spheroid at 0 d after Di-I labeling and the migration of the cells after 3 d is shown in the bottom row of companion panels. Bar = 100 µm. The percentage of inhibition of invasion at 40, 100, and 500 ng/ml (1.5 µM) BB-2516 was 59 ± 4%, 86 ± 2%, and 99 ± 0%, respectively.

Figure 2.

Figure 2.

MT1-MMP–independent tumor cell invasion through telopeptide-excised or cross-link–deficient type I collagen gels. (A) Schematic of type I collagen intermolecular cross-links. In vivo, lysyl oxidase generates aldehyde moieties within the N- and C-terminal telopeptide domains of type I collagen, which are arrayed across from ϵ-amino groups (boxed region) that spontaneously condense to conform Schiff base, aldimine cross-links in vivo. N- and C-terminal telopeptides are removed during pepsin extraction (arrows). Under acidic extraction conditions, Schiff base formation is reversed to generate the starting aldehyde and amine groups. After treatment with sodium borohydride, aldehydes are converted to alcohols that no longer support cross-linked formation. (B) Di-I-labeled multicellular spheroids of HT-1080 cells, electroporated with control or MT1 siRNA, were embedded in 3D gels prepared from either pepsin-extracted bovine dermis (Vitrogen; 2.2 mg/ml) or pepsin-extracted rat tail tendon and monitored by fluorescence microscopy at 0 d (top row of panels) and after 1 d (second row of panels; Di-I, red; Alexa-488 phalloidin, green; DAPI, blue). SEM of pepsin-extracted collagen gels is shown in the inset of a 0-d spheroid electroporated with control siRNA (left-hand panel in the first row; bar = 5 µm). GM6001 (10 µM) was added to the cultures at 0 d in the right-hand column of panels. Bar, 100 µm. In the bottom row of panels, Di-I-labeled HT-1080 spheroids were embedded in borohydride-reduced, rat tail type I collagen gels (2.2 mg/ml) at 0 d (insets) and invasion monitored 1 d later after electroporation with a control siRNA, MT1-MMP siRNA or after culture in the presence of GM6001. Collagen aldehyde content before and after borohydride reduction was determined spectrophotometrically (Williams et al., 1978; Gelman et al., 1979). After the 1-d culture period, cells were stained with Alexa-488 phalloidin and DAPI as described above. Bar, 100 µm. (C) HT-1080 or MDA-MB-231 spheroids were cultured for 1 d in 3D pepsinized or reduced type I collagen and invasion distance into the surrounding matrix quantified. HT-1080 invasion in the presence of a protease inhibitor cocktail (Wolf et al., 2003a) in pepsinized or reduced collagen gels was inhibited by 0 ± 0% and 8 ± 1%, respectively. Results are expressed as mean ± SEM (n = 4). Acid-extracted, pepsin-extracted, and reduced collagen fibrils are equally trypsin resistant (not depicted).

Figure 3.

Figure 3.

MT1-MMP drives cancer cell invasion in human mammary gland explants. (A) MDA-MB-231 Di-I–labeled spheroids (comprised of cells electroporated with a control siRNA, MT1-MMP siRNA or MT1-MMP siRNA together and a mouse MT1-MMP expression vector) were injected (30-gauge needle) into human mammary gland explants (6 × 8 x 6 mm) and the position of the cancer cell aggregates recorded by fluorescence microscopy at 0 d. Bar = 20 µm. Inset shows SEM of type I collagen fibrils in mammary gland explant. Bar = 5 µm. The inoculated explants were then cultured atop the live chick CAM for 3 d. Invasive behavior of MDA-MB-231 cells was monitored by fluorescence microscopy after labeling of mammary gland tissues with Alexa-488 phalloidin and DAPI. Bar = 200 µm. Immunofluorescent staining of mammary tissue cross sections at d 3 (inserts) reveal tracts of denatured collagen detected with monoclonal antibody HU177 (Hotary et al., 2003; Sabeh et al., 2004) (green; arrows) surrounding invasive tumor cells (red), but not MT1-MMP–silenced cancer cells (bottom panels; bar = 10 µm). (B) Invasion distance for MDA-MB-231 or SUM-159 spheroids (electroporated with control siRNA, MMP-1 and MMP-2 siRNAs, MT1-MMP siRNA or MT1-MMP siRNA in combination with a mouse MT1-MMP expression vector) was quantified after a 3-d culture period in human mammary gland explants as described. Results are expressed as mean ± SEM (n = 3). (C) Invasion distance for MDA-MB-231 or SUM-159 spheroids (electroporated with control siRNA, MMP-1 and MMP-2 siRNAs, MT1-MMP siRNA or MT1-MMP siRNA in combination with a mouse MT1-MMP expression vector) was quantified after a 3-d culture period in mouse mammary gland explants as described. Results are expressed as mean ± SEM (n = 3).

Figure 4.

Figure 4.

Collagen-invasive and degradative activities of cancer cells versus leukocytes. Light micrographs of cross sections of type I collagen gels (2.2 mg/ml) traversed by HT-1080 or human PMNs prepared as described (Huber and Weiss, 1989) for 3 d or 1 d, respectively. Collagen-invasive cells are H&E stained and marked with black arrows. Double-headed arrow marks the boundaries of the underlying collagen gel. Black bar = 100 µm. Laser confocal micrographs of HT-1080 cells cultured atop 3D gels of rhodamine-labeled type I collagen for 3 d demonstrate that invasion is associated with the formation of well-demarcated tunnels (white arrows; white bar = 50 µm). In contrast, PMNs stimulated with zymosan-activated plasma (Huber and Weiss, 1989) invade rhodamine-labeled collagen gels without perturbing matrix architecture (far right-hand panel). Invasion and collagen-degradative activities of HT-1080, PMNs, T cells, and monocytes (Reddy et al., 1995) were quantified in the absence or presence of BB-2516 (1.5 µM) as described previously (bar graphs, bottom panel) (Sabeh et al., 2004). PMN invasion was also assessed in the presence of the protease inhibitor cocktail prepared as described (Wolf et al., 2003a). Results are expressed as mean ± SEM (n = 4). (B) Acid-extracted, rat tail collagen gels were prepared at pH 5.5, 6.5, 7.5, and 8.5 as described (Raub et al., 2008) and the invasive activity of HT-1080 cells into the 3D gels was quantified in the absence or presence of BB-2516 (1.5 µM) after a 3-d culture period. Collagen at pH 5.5 and 8.5 as visualized by SEM are shown in the insets (n = 3; bar = 250 nm).

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