ARTICLE |
Correspondence to: Eberhard Spiess, Biomedizinische Strukturforschung 0195, Deutsches Krebsforschungszentrum, PO Box 101949, D69009 Heidelberg, Germany.
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Summary |
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We used the nondestructive procedures of confocal laser scanning microscopy in combination with computer-assisted methods to visualize tumor cells in the process of penetrating collagen gels. Three independent sets of images were collected. The image information of all data sets was combined into one image, giving a three-dimensional (3D) impression at high light microscopic resolution and sensitivity. We collected information about the extracellular matrix using the reflection mode, the cell surface/morphology by staining with the fluorescent dye DiOC6(3), and the distribution of cathepsin B by Cy-3- labeled immunolocalization. The specific aim of our study was visualization of the spatial relationship of cell organelles as far as they contain the enzyme cathepsin B to cell morphology and motility in a 3D model of extracellular matrix. The majority of the enzyme was localized pericellularly, with no visible relationship to the direction of movement. However, substantial amounts also appeared in intramatrix pseudopodia and associated with the extracellular face of the plasma membane, which may be indicative either of secretion and/or epicellular activity. Our approach has general applicability to study of the spatial relationships of cell compartments and their possible reorganization over time. This could open new horizons in understanding cell structure and function. (J Histochem Cytochem 45:975-983, 1997)
Key Words: confocal microscopy, 3D image reconstruction, tumor cell, cathepsin B, localization
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Introduction |
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Biological structures have a dynamic three-dimensional (3D) organization. However, our understanding of structures and functions and their interrelations in cells is almost exclusively based on two-dimensional images and is therefore incomplete. In medical sciences, 3D imagery of whole organs or large anatomic regions of the body is possible by a number of noninvasive methods. Moreover, at the molecular level, spatial reconstitution of molecules or molecular assemblies is a common, although complicated procedure. At the cellular level, knowledge of spatial extension or interaction of structures at the light and electron microscopic level was obtained by destructive sectioning methods. Confocal laser scanning microscopy is now a well-established method to obtain optical sections of spatially extended biological objects (
Such a methodology is an ideal tool to study cell locomotion (movement). In situ, this is a 3D process. It involves complex rearrangements of the intracellular compartments, intercellular interactions, and interactions of cells with the extracellular matrix. Movement of cells through natural barriers (invasion) is a basic phenomenon in living organisms. In higher organisms, such invasive processes occur during all stages of development and maintenance of life. Of particular interest is the movement of tumor cells that leads eventually to metastasis. It is postulated that penetration of cells into the extracellular matrix involves a proteolytic cascade that includes the plasminogen activator system and collagenases (
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Materials and Methods |
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Cells and Cell Culture
Two human lung tumor cell lines were used in our investigations: SB-3, which is derived from a metastasis of an adenocarcinoma (
Experimental Setup
A chamber (12-mm inner diameter) with a glass bottom fitting to the microscope stage was used for the study of cells (
The model for the interstitial matrix we used is collagen G (Biochrom). It is extracted from calfskin collagen and consists of 90% collagen Type I and 10% collagen Type III. We mixed 8 parts of collagen G with 1 part 7.5% NaCl and one part 0.2 N NaOH in 200 mM HEPES, pH 12.0, under sterile conditions. The solution (40 µl) was pipetted onto the bottom of the chambers and allowed to polymerize at 37C for 1 hr. The polymerized gels had a thickness of 100-300 µm.
Suspended in 200 µl of full medium, 104 cells were seeded into a chamber, which was transferred to a plastic petri dish and incubated for 24 hr under standard culture conditions.
Immunofluorescence
The cells were fixed in situ by methanol (-20C) for 5 min, washed two times with PBS for 5 min, and then for 5 min with PBS containing 0.1% BSA (Sigma Chemical; Deisenhofen, Germany). For detection of cathepsin B, the cells were incubated with the monoclonal antibody Ab-1 IM27 (Oncogene Science; Uniondale, NY, obtained from Dianova, Hamburg, Germany) diluted 1:50 in PBS for 45 min at room temperature (RT) in a humid atmosphere. This antibody was raised by the manufacturer to a peptide sequence of the mature part of the protein and should therefore detect propeptide and mature peptide. This specificity has been proved by others (
Confocal Laser Scanning Microscopy
Confocal laser scanning microscopy (CLSM) was performed with an inverse LSM 410 UV (Carl Zeiss; Jena, Germany). To minimize lens aberrations, which can interfere considerably with acurate imaging, we used a water immersion objective (C-Apochromat 40x/1,2 W Korr; 220 µm working distance, Carl Zeiss) and corrected for individual coverslip thickness. The refractive index of the matrix surrounding the cells was taken into account for calculation of the real physical dimensions in the 3D reconstructions. Series of 60 or 79 optical sections were produced (0.5 µm section distance; scanning speed 2 sec per image; pinhole setting 12). The gel was visualized via reflection mode with the 488-nm exitation wavelength of an argon ion laser. The same wavelength in combination with a 510-525 bandpass filter for detection of cell surfaces stained by DiOC6(3), and the 543 nm excitation of a helium-neon laser together with a 590 longpass filter, were used to record cathepsin B stained by Cy-3-labeled antibodies. Color-coded depth representation of image series is a feature of the CSLM 410 software for rapid demonstration of 3D extension of objects.
Three-dimensional Reconstruction
A program written in the computer language "C" was developed to allow automatic image processing using the native file format of the CLSM as well as compressed files. The command line driven program allows linear and nonlinear filtering as well as application of several edge detection algorithms (
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Results |
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The ability of cells to invade the extracellular matrix depends on their ability to interact with the matrix. In vivo, this eventually leads to directed movement of cells to particular sites. We studied penetration into reconstituted collagen by human lung tumor cells SB-3 and LCLC103H in 3D in vitro model systems. The cells were seeded on collagen G, which is a model substrate for interstitial space extracelluar matrix, and incubated for 24 hr before they were fixed in situ and stained for observation. In this experimental setup there is, other than gravity, no gradient along which the cells can move, and the depth of matrix by far exceeds the cell size. Experiments performed with beads resembling cells in size and mass showed that the surface strength of the gels is sufficient to support their mass. Therefore, we can assume that penetration into the gels depends on the active movement of the cells. An overview of SB-3 cells seeded on a gel is given in Figure 1A as a depth-coded reconstruction of a series of 32 consecutive images. It is clear that the activities of the individual cells were quite different. Some remained inactive on the surface of the gel. Active cells developed pseudopodia and moved into the gel. A 3D reconstruction of an SB-3 cell and the collagen matrix is shown in Figure 1B-F. A series of 60 images was taken in the three recording modes and the images were combined by superposition. The physical dimensions of the recorded cuboid are 74.3 x 74.3 x 29.7 µm. The gel is given in white (reflection mode recording), the cell surface in blue [DiOC6(3) fluorescence staining], and cathepsin B in red (Cy-3 fluorescence indirect immunolocalization). The 3D shape of the cell is emphasized by contour lines (red) that were calculated for a fraction of the optical sections. Two different perspectives were selected to show the entire extension of the scanned cuboid (Figure 1B, view from the top; Figure 1C, view from the side and rotated clockwise for 90° with respect to Figure 1B). The collagen matrix is a 3D, randomly distributed fiber network. In the direct environment of the cell, an organization of the fibers with respect to cellular morphology can be found (Figure 1B). Fibers in contact with the prominent pseudopodium are oriented parallel to its extension. A radial orientation of fibers to the cell body is seen as well. This was a general observation. The fibers appear to be of different thicknesses. Reconstructions performed with higher resolution indicate that thick fibers are composed of several thin fibers that are closely apposed. The surface of the matrix is inclined with respect to the optical axis of the system (Figure 1C). This inclination is due to the parameters of the chamber and the surface tension of the collagen solution.
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The surface of the cell appears to be very smooth. In contrast to other microscopic procedures, we can now image the complete surface of a cell. In the penetrating cells we found differences between the apical and basal surfaces. The apical surface had a predominantly smooth topography but the basal surface was strewn with small pocks. This polarity indicates a substrate-oriented cell activity. To exclude errors based on the optical system and image reconstruction procedure, we compared this result with scanning electron micrographs that provide a higher resolution. We found the same appearance of the apical cell surface in these images, but scanning electron microscopy does not allow visualization of the basal surface. The thin filopodia, present on the surface of cells when visualized by scanning electron microscopy, were absent in the reconstructions of light optical scans. These structures must be below the resolution limit of the reconstruction. Penetration of the cell into the collagen gel becomes visible in Figure 1D, in which we omitted the collagen matrix, thus opening the view on parts of the cell that were previously hidden. The cell generated a prominent pseudopodium stretched horizontally over the surface of the matrix and also extending into it. Isolated cell structures embedded in the matrix (Figure 1B) can now be identified either as parts of pseudopodia or as isolated vesicles. These vesicles provide evidence of the cell's exploratory activities by stationary motility or locomotion in the gel.
A comparison of the apparent cell morphology in Figure 1B-D reveals that most of the volume of the bell-shaped cell is embedded in the matrix. Therefore, without any gradient other than gravity, there was a net movement of the cell in the vertical direction.
The intracellular localization of cathepsin B for this cell is shown in Figure 1E and Figure 1F in top and side views, respectively. In Figure 1E the same point of view is shown as in Figure 1B, but the cubus is slighty tilted around the x-axis. For these images the gel was removed and the color of the cell surface was attenuated. The enzyme appears in sheet-like and vesicular structures highly concentrated in the central part of the cell. The spatial arrangement of this central assembly reveals a polarity. One side is smooth and concave (Figure 1F). This curvature suggests a close apposition of this face to the nucleus, which is thus negatively imaged. The opposite face appears to be dissolved into vesicular structures (Figure 1E). The entire staining pattern we observed should represent such parts as the endoplasmic reticulum, the Golgi complex, the trans-Golgi network, and lysosomes that contain cathepsin B, pro- and/or mature forms, respectively. Further cathepsin B-containing structures appear dispersed all over the cell. Figure 1E shows that cathepsin B could also be found in pseudopodia.
Cells of the line LCLC103H are shown in Figure 2. Figure 2A shows cells on a gel in a color-coded depth reconstruction from a series of 32 sections. These cells behaved similarly to the SB-3 populations shown in Figure 1A. In Figure 2B-D, two cells on such a gel are presented in a reconstruction from a series of 79 sections. The attenuation of the gel in Figure 2C reveals the movement of the cells into the gel. Figure 2D-F show particularly the distribution of cathepsin B in views from the top (Figure 2D), and the side (Figure 2E and Figure 2F). The pseudopodium marked by an arrow in Figure 2D is seen at a higher magnification in Figure 2E and Figure 2F. The cells appear very flat, and the translucent surface opens the view on the cathepsin B-containing organelles that also appear in the pseu-dopodium. Reconstruction of the plasma membrane reveals external cell surface-associated enzyme (Figure 2F). In these cells, the cathepsin B distribution is very similar to that found in SB-3 cells. However, the plasma membrane-associated cathepsin B was observed much more frequently. In addition, the calculation of isosurfaces allowed precise quantitative determination of volumes. The total volume of the SB-3 cell shown in Figure 1B was determined to be about 15 µm3 and the volume of the cathepsin B-containing structures to be about 1.183 µm3. This is about 8% of total cell volume.
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Discussion |
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Confocal laser scanning microscopy is now a well- established method for obtaining optical sections of cells (
In the present work we took an in vitro approach to study human lung cancer cells SB-3 and LCLC103H penetrating collagen in a 3D model. Such experimental methods are applied to study the invasive behavior of tumor cells (
For the 3D reconstitution work, we used the software package AVS (
The reconstructions render a complete image of the in vitro situation at a defined moment in time. Interaction of the cell with the gel matrix becomes obvious by the rearrangement of gel fibers from an originally random pattern to a cell-oriented pattern in the close vicinity of the cell, but the general organization of the gel was not changed by these activities. The cells developed pseudopodia stretching in vertical and horizontal directions into the gels and on their surfaces. Vesicles enclosed in the gel but several micrometers away from the cell body also indicate cellular activity. We assume that the gel collapsed after the retraction of pseudopodia. Loss or shedding of cellular material is a well-known phenomenon (
The main goal of our investigations concerned the localization of cathepsin B. The antibody we used for identification binds to the proform and the mature form of the enzyme. Therefore, we imaged all organelles, specifically those parts of them that are involved in synthesis, processing, trafficking, and functions of cathepsin B. Endoplasmic reticulum and the Golgi complex showed a perinuclear localization with limited extension. They were so closely apposed to the nucleus that the shape of the nucleus itself was perceptible as a negative image. The 3D reconstruction delineates the morphologies of these organelles in a resolution beyond that of classical light microscopy. They appear densely packed as layers of lacunae that dissolve at the distal part of the complex in vesicular structures. The localization of cathepsin B-containing structures, presumably vesicles, in the cytoplasm and associated with the plasma membrane confirms earlier findings concerning such localization in these and other cells (
The final conclusion of our work addresses its future prospects. The extremely sensitive instrumentation, the construction of a broad range of physically distinctive dyes, and the development of specific antibodies to cell constituents will now easily extend our comprehension of the spatial relationships of cell structure and function.
An animated video sequence of these results is available on request.
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Acknowledgments |
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Supported by grant Ca58 of the German-Israeli cooperation DKFZ-NCRD and the Klinisch-Biomedizinischer Forschungsverbund of the Bundesministerium für Bildung, Wissenschaft, Forschung und Technologie, Bonn, Germany (ES, ARS). Financial support by the Deutsche Forschungsgemeinschaft, Bonn, under grant Ac 37/ 9-1 is gratefully acknowledged (TP, HA).
We thank Dennis Strand (Deutsches Krebsforschungszentrum, Heidelberg) for critically revising the manuscript. A-RS and ES gratefully acknowledge the introduction to and continuous support in confocal microscopy by Herbert Spring (same institution).
Received for publication July 17, 1996; accepted February 6, 1997.
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Literature Cited |
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![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Albini A, Iwamoto Y, Kleinman HK, Martin GR, Aaronson SA, Kozlowski JM, McEwan RN (1987) A rapid in vitro assay for quantitating the invasive potential of tumor cells. Cancer Res 47:3239-3245[Abstract]
Albrecht-Buehler G (1977) Phagokinetic tracks of 3T3 cells: parallel between the orientation of track segments and of cellular structures which contain actin or tubulin. Cell 12:333-339[Medline]
Ashkar GP, Modesting JW (1978) The contour extraction problem with biomedical application. Comput Graphics Image Process 7:331-335
Bepler G, Koehler A, Kiefer P, Havemann K, Beisenherz K, Jaques G, Haeder M (1988) Characterization of the state of differentiation of six newly established human non-small lung cancer cell lines. Differentiation 37:158-171[Medline]
Berquin IM, Sloane BF (1994) Cysteine proteases and tumor progression. Perspec Drug Discov Design 2:371-388
Brakenhoff GJ, Van Spronsen EA, Van der Voort HTM, Nanninga N (1989) Three dimensional confocal fluorescence microscopy. In Taylor DL, Wang Y, eds. Methods in Cell Biology. San Diego, Academic Press, 379-398
Brix K, Lemansky P, Herzog V (1996) Evidence for extracellularly acting cathepsins mediating thyroid hormone liberation in thyroid epithelial cells. Endocrinology 137:1963-1974[Abstract]
Buck MR, Karustis DG, Day NA, Honn KV, Sloane BF (1992) Degradation of extracellular-matrix proteins by human cathepsin B from normal and tumour tissues. Biochem J 282:273-278[Medline]
Carlsson K. (1991) The influence of specimen refraction index, detector signal integration and non uniform scan speed on the imaging properties in confocal microscopy. J Microsc 163:167-178
DiMilla PA, Stone JA, Quinn JA, Albelda SM, Lauffenburger DA (1993) Maximal migration of human smooth muscle cells on fibronectin and type IV collagen occurs at an indermediate attachment strength. J Cell Biol 122:729-737[Abstract]
Dolo A, Ginestra G, Ghersi H, Nagase H, Vitorelli ML (1994) Human breast carcinoma cells cultured in the prescence of serum shed membrane vesicles rich in gelatinolytic activities. J Submicrosc Cytol Pathol 26:173-180[Medline]
Elliot E, Sloane BF (1996) The cysteine protease cathepsin B in cancer. Perspect Drug Discov Design 6:12-32
Erdel M, Spiess E, Trefz G, Boxberger H-J, Ebert W (1992) Cell interactions and motility in human lung tumor cell lines HS-24 and SB-3 under the influence of extracellular matrix components and proteinase inhibitors. Anticancer Res 12:349-360[Medline]
Erdel M, Trefz G, Spiess E, Habermaas S, Spring H, Lah T, Ebert W (1990) Localization of cathepsin B in two human lung cancer cell lines. J Histochem Cytochem 38:1313-1321[Abstract]
Foley JD, van Dam A, Feiner SK, Hughes JF, Phillips RL (1994) Introduction to Computer Graphics. New York, Addison-Wesley
Honn KV, Timar J, Rozhin J, Bazaz R, Sameni M, Ziegler G, Sloane BF (1994) A lipoxygenase metabolite, 12-(S)-HETE, stimulates protein kinase C-mediated release of cathepsin B from malignant cells. Exp Cell Res 214:120-130[Medline]
Huang TS, Yang GJ, Tang GY (1979) A fast two-dimensional median filtering algorithm. Acoust Speech Sign Process 27:13-18
Kirschke H, Barrett AJ, Rawlings ND (1995) Proteinases1: lysosomal cysteine proteinases. Protein Profile 2:1587-1643
Kobayashi H, Schmitt M, Goretzki L, Chucholowski N, Calvete J, Kramer M, Günzler WA, Jänicke F, Graeff H (1991) Cathepsin B efficiently activates the soluble and the tumor cell receptor-bound form of the proenzyme urokinase-type plasminogen activator (pro-uPA). J Biol Chem 266:5147-5152
Lah TT, Buck MR, Honn KV, Crissman JD, Rao NC, Liotta LA, Sloane BF (1989) Degradation of laminin by human tumor cathepsin B. Clin Exp Metastasis 7:461-468[Medline]
Lorensen WE, Cline HE (1987) Marching cubes: a high resolution 3D surface construction algorithm. Comput Graphics 21:163-169
Lucas L, Gilbert N, Ploton D, Bonnet N (1996) Visualization of volume data in confocal microscopy: comparison and improvements of volume rendering methods. J Microsc 181:238-252[Medline]
Masters BR, Farmer MA (1993) Three-dimensional confocal microscopy and visualization of in situ cornea. Comput Med Imaging Graphics 17:211-219[Medline]
McCarthy JB, Furcht LT (1984) Laminin and fibronectin promote the haptotactic migration of B16 mouse melanoma cells in vitro. J Cell Biol 98:1474-1480[Abstract]
Messerli JM, Van der Voort HTM, Rungger-Brändle E, Pierrard JC (1993) 3-dimensional visualization of multi-channel volume data: the amSFP algorithm. Cytometry 14:725-735[Medline]
Mignatti P, Rifkin DB (1993) Biology and biochemistry of proteinases in tumor invasion. Physiol Rev 73:161-195
Porwol T, Merten E, Opitz N, Acker H (1996) 3D imaging of rhodamine 123 fluorescence distribution in human melanoma cells by means of confocal laser scanning microsocopy. Acta Anat, 157:116-125[Medline]
Rozhin J, Sameni M, Ziegler G, Sloane BF (1994) Pericellular pH affects distribution and secretion of cathepsin B in malignant cells. Cancer Res 54:6517-6525[Abstract]
Schoenberger OL, Beikirch S, Trefz G, Drings P, Ebert W (1987) Proteolytic activity of human tumor cells deriving from bronchial squamous cell carcinoma, pulmonary metastasis of rhabdomyosarcoma and pleural metastasis of mesothelioma. Eur J Respir Dis 71:434-439[Medline]
Sheehan B, Fuller SD, Pique ME, Yeager M (1996) AVS software for visualization in molecular microscopy. J Struct Biol 116:99-106[Medline]
Sloane BF, Moin K, Sameni M, Tait LR, Rozhin J, Ziegler G (1994) Membrane association of cathepsin B can be induced by transfection of human breast epithelial cells with c-Ha-ras oncogene. J Cell Sci 107:373-384
Sloane BF, Rozhin J, Hatfield JS, Crissman JD, Honn KV (1987) Plasma-membrane associated cysteine proteinases in human and animal tumors. Exp Cell Biol 55:209-224[Medline]
Spiess E, Brüning A, Gack S, Ulbricht B, Spring H, Trefz G, Ebert W (1994) Cathepsin B activity in human lung tumor cell lines: ultrastructural localization, pH sensitivity, and inhibitor status at the cellular level. J Histochem Cytochem 42:917-929
Strohmaier A-R, Spring H, Spiess E (1996) Three-dimensional analysis of the substrate-dependent invasive behavior of a human lung tumor cell line with a confocal laser scanning microscope. Histochem Cell Biol 105:179-185[Medline]
Sylven B, Snellman O, Sträuli P (1974) Immunofluorescent studies on the occurence of cathepsin B1 at tumor cell surfaces. Virchows Arch Cell Pathol 17:97-112
Taylor DD, Black PH (1986) Shedding of plasma membrane fragments. Neoplastic and developmental importance. In Steinberg M, ed. Developmental Biology. New York, Plenum Press, 33-57
Thalmann N, Thalmann D (1991) New Trends in Animation and Visualization. Chichester, John Wiley & Sons
Thompson EW, Lippman ME, Dickson RB (1991) Regulation of basement membrane invasiveness in human breast cancer model systems. Mol Cell Endocrinol 82:C203-C208[Medline]
Trefz G, Erdel M, Spiess E, Ebert W (1990) Detection of cathepsin B, plasminogen activators and plasminigen activator inhibitor in human non-small lung cancer cell lines. Biol Chem Hoppe Seyler 371:617-624[Medline]
Ulbricht B, Hagmann W, Ebert W, Spiess E (1996) Differential secretion of cathepsins B and L from normal and tumor human lung cells stimulated by 12(S)-hydroxy-eicosatetraenoic acid (12(S)-HETE). Exp Cell Res 226:255-263[Medline]
Van der Voort HTM, Brakenhoff GJ (1990) 3D image formation in high aperture fluorescence confocal microscopy: a numerical analysis. J Microsc 158:43-54
Van der Voort HTM, Messerli JM, Noordmans HJ, Smeulders AWM (1993) Volume visualization for interactive microscopic image analysis. Bioimaging 1:20-29
Vassalli J-D, Pepper MS (1994) Membrane proteases in focus. Nature 370:14-15[Medline]
Visser TD, Brakenhoff GJ (1992) Refractive index and distance measurements in 3-D microscopy. Optik 90:17-19
Wilhelms J, Van Gelder A (1990) Topological considerations in isosurface generation. Comput Graphics 24:79-86
Wright SJ, Centonze VE, Sricker SA, DeVries PJ, Paddock SW, Schatten G (1993) Introduction to confocal microscopy and three-dimensional reconstruction. Methods Cell Biol 38:1-45[Medline]