Degradation of Basement Membrane by Murine Tumor Cells 1,2 Lance A. Llotta,? Jerome Kleinerman, 4 Phillip Catanzaro, 4, 5 and Donald Rynbrandt 4, 6, 7
It has been recognized that tumor angiogenesis is a necessary prerequiste for progressive tumor growth (1). Tumor vascularization is also the first step in the hematogenous metastatic process. Tumor cells are first seen in the tumor venous effluent after tumor vascularization, and their concentration is related to the number and size of tumor vessels present (2). The mechanism by which tumor cells penetrate the vascular wall to enter the circulation may be related to intrinsic defects in the vessel wall. On the other hand, tumor cells may elaborate enzymes that damage the vascular wall. The latter hypothesis is the subject of this study. In particular we attempted to study whether tumor cells entering the circulation possessed a greater enzymatic potential for vascular invasion than the tumor cells within the tumor mass. It has been postulated that tumor cell proteases may playa role in the invasiveness of malignant tumors (36). Vascular invasion is one component of the hematogenous metastatic process, which involves both the entry of tumor cells into the circulation (7) and the initiation of metastases (8, 9). Generally, to traverse the vascular wall, tumor cells must penetrate the endothelial basement membrane. Although electron microscope studies have shown defects in basement membrane adjacent to invading tumor cells (4), a tumor enzyme that degrades a basement membrane substrate has not previously been identified. Therefore, a major objective of this study was to examine the ability of tumor cells from a metastasizing fibrosarcoma to degrade isolated pulmonary basement membrane in vitro. It has previously been shown that perfusion of the vascular bed of an implanted fibrosarcoma (T241) is a practical method for sampling the tumor cells entering the circulation (2). The T241 tumor rapidly and reproducibly produces pulmonary metastases. In this report this model system is utilized to compare the ability of tumor cells from the primary tumor and tumor cells entering the circulation to degrade isolated pulmonary basement membrane. VOL. 58, No.5, MAY 1977
Kefalides and Dend uchis (10, 11) characterized the components of epithelial and endothelial basement membrane to be a collagenous moiety combined with a matrix glycoprotein. Intact basement membrane is highly insoluble (10). Therefore, in our study breakdown of basement membrane is assayed by the quantitation of solubilized HYP and glycoprotein from a basement membrane substrate exposed to tumor cells. Additional studies utilized purified human collagen as a substrate. MATERIALS AND METHODS
Substrates.-Pulmonary basement membrane was isolated from the lungs of mice according to the acetic acid extraction method of Kefalides and Denduchis (10). The yield was approximately 0.1 % of weight of pulmonary tissue. Prolonged digestion (48 hr) of the isolated basement membrane by bacterial collagenase yielded a glycoprotein fraction and soluble HYP (10). Amino acid analysis of the pulmonary basement membrane substrate was performed after hydrolysis in 6 N HCI at 105° C for 24 hours (12). A Beckman autoanalyzer was used (Beckman Instruments, Inc., Fullerton, Calif'.). The results were compatible, with 35% of the substrate being collagen and the remainder being glycoprotein. As shown in table 1, the substrate collagen had high 3HYP/ 4HYP and HYL/HYP ratios. The exact ratios of amino acids in basement membrane collagen vary depending on the method of extraction and the tissue source (10, 11, 13 -16). The ratios of collagen containing amino acids in the substrate used here were in the range reported by other investigators for basement membrane. Thus amino acid analysis was compatible with the conclusion that a majority of the collagen in the substrate was type IV. Purified type I human collagen was isolated from the dura of a 5-year-old girl by salt extraction and supABBREVIATIONS USED: HYP = hydroxyproline; 3HYP = 3-hydroxyproline; 4HYP = 4-hydroxyproline; HYL = hydroxylysine; PRO = proline; GL Y = glysine; LYS = lysine; HBSS = Hanks' balanced salt solution; HEPES = N -2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid; MEM = minimum essential medium; ETC = effluent tumor cells; PTC = primary tumor cells. 'Received August 12, 1976; accepted November 17, 1976. Supported in part by Public Health Service grant CA16089 from the National Cancer Institute. 3Laboratory of Pathology, National Cancer Institute (NCI), National Institutes of Health, Public Health Service, U.S. Department of Health, Education, and Welfare, Bethesda, Md. 20014. 4 Department of Pathology Research, St. Luke's Hospital, Cleveland, Ohio 44104. 5 Department of Pathology, Case Western Reserve University, Cleveland, Ohio 44106. 6 Department of Biochemistry, Case Western Reserve University. 7 We greatly appreciate the technical assistance of Claudio Gattozzi. The major portions of this study were done in the laboratory of Dr. Kleinerman. Amino acid analysis was performed in the laboratory of Dr. George R. Martin, National Institute of Dental Research, National Institutes of Health, Bethesda, Md. 2
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Downloaded from http://jnci.oxfordjournals.org/ at Carleton University on May 10, 2015
ABSTRACT-Tumor cells from the murine T241 fibrosarcoma, which rapidly and reproducibly produces pulmonary metastases, were tested in vitro for their ability to degrade isolated pulmonary basement membrane. Degradation of basement membrane substrate was quantified by the culture of the substrate with tumor cells and measurement of the solubilized hydroxyproline and hexose glycoprotein at neutral pH. It was found that tumor cells collected in the tumor venous drainage were associated with a significantly greater solubilization of basement membrane than were tumor cells obtained from the primary tumor mass. Tumor cells were also assayed for their ability to solubilize type I collagen purified from human dura. Venous effluent tumor cells solubilized collagen to a significantly greater level than primary tumor cells, spleen cells, or liver cells. These findings raised the possibility that metastasizing tumor cells may be a distinct tumor subpopulation with regard to invasive potential.-J Natl Cancer Inst58: 1427-1431, 1977.
1428
LIOTTA, KLEINERMAN , CATANZARO, AND RYNBRANDT TABLE
1.-Ratios of selected amino acids in basement membrane collagen Amino acid ratios
Basement membrane source (reference) 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.
Murine pulmonary" Murine tumor (13) Canine pulmonary (10) Bowman's capsule (10) Choroid membrane (10) Descemet's membrane (10) Canine lens capsule (10) Bovine lens capsule (14) Rat glomeruli (15) Bovine brain vessels (16)
3HYP/4HYP
HYP/PRO
HYL/HYP
HYL/LYS
HYP/GLY
HYL/GLY
0.198" 0.060 Trace 0.203 Trace 0.088 0.243 Not given Not given Not given
0.364 0.690 0.450 1.68 0.714 0.860 1.500 1.560 0.840 1.09
0.401" 0.357 0.191 0.269 0.185 0.242 0.318 0.414 0.423 0.225
0.186 0.405 0.163 1.260 0.219 0.830 2.900 3.640 0.550 1.080
0.106 0.140 0.157 0.408 0.205 0.355 0.345 0.41 0.260 0.400
0.043 0.050 0.030 0.110 0.038 0.086 0.11 0.17 0.110 0.090
n. Substrate b
used in the present study. The greatest similarity is found between this preparation and that of 2, 3, 5, and 9. These high ratios are characteristic of type IV collagen.
J
NATL CANCER INST
Basement membrane breakdown.-Isolated whole basement membrane substrate was exposed to living tumor cells in vitro. Kefalides and Denduchis (10, 11) showed that basement membrane is composed of a collagen moiety within a glycoprotein matrix. The collagen moiety has a high content of HYP and HYL (10). The predominant carbohydrate in the glycoprotein matrix is hexose (10, 11). Basement membrane breakdown was evaluated by analysis of the supernatant for soluble HYP and hexose-glycoprotein. After incubation of the basement membrane substrate with living tumor cells at 37° C at pH 7.5 for 18 hours, the mixture was centrifuged at 1,400xg at 4° C for 5 minutes. The supernatant was assayed for HYP content by the method of Bergman and Loxley (20) and for protein-bound hexose by precipitation with 95% ethanol at room temperature for 30 minutes and reaction with orcinol (21). Appropriate blanks and controls consisted of: a) basement membrane substrate incubated alone in Eagle's MEM, b) tumor cells or cell fractions incubated alone in Eagle's MEM or HBSS, and c) tumor cells incubated with basement membrane at 4° C. The values expressed in "Results" reflect total activity minus the blanks or controls. The HYP and protein-bound hexose content of the blanks were always less than 10% of the values for cells incubated with substrate at 37° C. Collagenase assay.-Cell-associated type I collagenase activity was determined by a slight modification of the method of Bergman and Loxley (20) based on the determination of liberated HYP. Cells to be assayed for collagenase activity were washed three times in HBSS and adjusted for number. Collagen substrate tubes were prepared by the centrifugation of plastic tubes (Falcon Plastics, Oxnard, Calif.) containing insoluble collagen (25 mg/3 ml in Eagle's MEM; Calbiochem) at 1,000 rpm for 20 minutes at room temperature. This procedure resulted in a packed pellet of collagen at the base of the tube. The test cells were gently centrifuged onto the collagenic substrate. The tubes were tightly capped and incubated upright at 37° C for 18 hours. Appropriate trypsin (10 JLg) controls were included to ensure that the collagen was not denatured. Finally, 1 ml of the cell-free supernatant was assayed for HYP content as described above. Assays were repeated in triplicate. Experiment series A.-Tumors were perfused 15 days VOL. 58, NO.5, MAY
1977
Downloaded from http://jnci.oxfordjournals.org/ at Carleton University on May 10, 2015
plied by the laboratory of Dr. Kohn, Pathology Department, Case Western Reserve University (17). Tumor host system.-The T241 fibrosarcoma (2) was maintained by serial passage from femoral muscle of the C57BL/6] male syngeneic mouse. Tumors were initiated with 106 mechanically disaggregated (18) tumor cells from nonnecrotic tumor fragments obtained 10-15 days after passage. Tissue culture media. -The fluid to perfuse the tumor vascular bed consisted of HBSS containing 0.05 M HEPES buffer (Calbiochem, San Diego, Calif.). All in vitro procedures were carried out either with HBSS or Eagle's MEM, with calcium and magnesium buffered with HEPES as above (Gibco, Grand Island, N.Y.). ETC.-After tumor transplantation in the murine femoral muscle, cannulation and perfusion of the femoral vasculature allowed tumor cells entering the vascular space to be collected and studied. The techniques of cannulation and perfusion have been detailed previously (2, 18). The iliofemoral artery and vein were cannulated, the tumor vascular bed was perfused at physiologic pressure, and the resident blood within the tumor was discarded. The venous effluent was collected under sterile conditions. The concentration of tumor cells and leukocytes was quantified after a fixed volume of the sample was passed through a nucleopore filter (2) and the filter was stained with Giemsa. Control perfusions consisted of: 1) perfusion of normal muscle and 2) perfusion of muscle inoculated previously with glutaraldehyde-fixed tumor cells. Macrophages were removed from the limb or tumor venous effluent by their ability to adhere to plastic petri dishes (19). Macrophages were removed after incubation for 1 1/2 hours and verified as being macrophages by their characteristic microscopic appearance and their ability to phagocytize latex particles. PTC . -These cells were obtained from non necrotic fragments of perfused tumors by mechanical disaggregation as described above. Macrophages were removed by adherence to petri dishes. Spleen cells.-We collected spleen cells from tumorbearing animals by mincing the spleen, agitating the resulting suspension in HBSS, and filtering the material through gauze. We obtained liver tissue by mincing normal liver from normal animals in HBSS.
1429
BASEMENT MEMBRANE BREAKDOWN -BY METASTASIZING TUMOR CELLS TABLE
3.-Collagenase activity of tumor cells cultured with purified human collagen Cell population a
Solubilized HYP ug>: 1O-6/cell"
Venous ETC (4) PTC (4) Effluent leukocytes" (4) Spleen cells (2) Li ver cells (2)
1.39±0.40C 0.48±0.44 0.52±0.30 0.33±0.14 0.16±0.10
Numbers in parentheses indicate No. of animals studied. " Mean z sn. C Significantly different from other cell groups atP
It has been recognized that tumor angiogenesis is a necessary prerequiste for progressive tumor growth (1). Tumor vascularization is also the first step in the hematogenous metastatic process. Tumor cells are first seen in the tumor venous effluent after tumor vascularization, and their concentration is related to the number and size of tumor vessels present (2). The mechanism by which tumor cells penetrate the vascular wall to enter the circulation may be related to intrinsic defects in the vessel wall. On the other hand, tumor cells may elaborate enzymes that damage the vascular wall. The latter hypothesis is the subject of this study. In particular we attempted to study whether tumor cells entering the circulation possessed a greater enzymatic potential for vascular invasion than the tumor cells within the tumor mass. It has been postulated that tumor cell proteases may playa role in the invasiveness of malignant tumors (36). Vascular invasion is one component of the hematogenous metastatic process, which involves both the entry of tumor cells into the circulation (7) and the initiation of metastases (8, 9). Generally, to traverse the vascular wall, tumor cells must penetrate the endothelial basement membrane. Although electron microscope studies have shown defects in basement membrane adjacent to invading tumor cells (4), a tumor enzyme that degrades a basement membrane substrate has not previously been identified. Therefore, a major objective of this study was to examine the ability of tumor cells from a metastasizing fibrosarcoma to degrade isolated pulmonary basement membrane in vitro. It has previously been shown that perfusion of the vascular bed of an implanted fibrosarcoma (T241) is a practical method for sampling the tumor cells entering the circulation (2). The T241 tumor rapidly and reproducibly produces pulmonary metastases. In this report this model system is utilized to compare the ability of tumor cells from the primary tumor and tumor cells entering the circulation to degrade isolated pulmonary basement membrane. VOL. 58, No.5, MAY 1977
Kefalides and Dend uchis (10, 11) characterized the components of epithelial and endothelial basement membrane to be a collagenous moiety combined with a matrix glycoprotein. Intact basement membrane is highly insoluble (10). Therefore, in our study breakdown of basement membrane is assayed by the quantitation of solubilized HYP and glycoprotein from a basement membrane substrate exposed to tumor cells. Additional studies utilized purified human collagen as a substrate. MATERIALS AND METHODS
Substrates.-Pulmonary basement membrane was isolated from the lungs of mice according to the acetic acid extraction method of Kefalides and Denduchis (10). The yield was approximately 0.1 % of weight of pulmonary tissue. Prolonged digestion (48 hr) of the isolated basement membrane by bacterial collagenase yielded a glycoprotein fraction and soluble HYP (10). Amino acid analysis of the pulmonary basement membrane substrate was performed after hydrolysis in 6 N HCI at 105° C for 24 hours (12). A Beckman autoanalyzer was used (Beckman Instruments, Inc., Fullerton, Calif'.). The results were compatible, with 35% of the substrate being collagen and the remainder being glycoprotein. As shown in table 1, the substrate collagen had high 3HYP/ 4HYP and HYL/HYP ratios. The exact ratios of amino acids in basement membrane collagen vary depending on the method of extraction and the tissue source (10, 11, 13 -16). The ratios of collagen containing amino acids in the substrate used here were in the range reported by other investigators for basement membrane. Thus amino acid analysis was compatible with the conclusion that a majority of the collagen in the substrate was type IV. Purified type I human collagen was isolated from the dura of a 5-year-old girl by salt extraction and supABBREVIATIONS USED: HYP = hydroxyproline; 3HYP = 3-hydroxyproline; 4HYP = 4-hydroxyproline; HYL = hydroxylysine; PRO = proline; GL Y = glysine; LYS = lysine; HBSS = Hanks' balanced salt solution; HEPES = N -2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid; MEM = minimum essential medium; ETC = effluent tumor cells; PTC = primary tumor cells. 'Received August 12, 1976; accepted November 17, 1976. Supported in part by Public Health Service grant CA16089 from the National Cancer Institute. 3Laboratory of Pathology, National Cancer Institute (NCI), National Institutes of Health, Public Health Service, U.S. Department of Health, Education, and Welfare, Bethesda, Md. 20014. 4 Department of Pathology Research, St. Luke's Hospital, Cleveland, Ohio 44104. 5 Department of Pathology, Case Western Reserve University, Cleveland, Ohio 44106. 6 Department of Biochemistry, Case Western Reserve University. 7 We greatly appreciate the technical assistance of Claudio Gattozzi. The major portions of this study were done in the laboratory of Dr. Kleinerman. Amino acid analysis was performed in the laboratory of Dr. George R. Martin, National Institute of Dental Research, National Institutes of Health, Bethesda, Md. 2
1427
J NATL CANCER INST
Downloaded from http://jnci.oxfordjournals.org/ at Carleton University on May 10, 2015
ABSTRACT-Tumor cells from the murine T241 fibrosarcoma, which rapidly and reproducibly produces pulmonary metastases, were tested in vitro for their ability to degrade isolated pulmonary basement membrane. Degradation of basement membrane substrate was quantified by the culture of the substrate with tumor cells and measurement of the solubilized hydroxyproline and hexose glycoprotein at neutral pH. It was found that tumor cells collected in the tumor venous drainage were associated with a significantly greater solubilization of basement membrane than were tumor cells obtained from the primary tumor mass. Tumor cells were also assayed for their ability to solubilize type I collagen purified from human dura. Venous effluent tumor cells solubilized collagen to a significantly greater level than primary tumor cells, spleen cells, or liver cells. These findings raised the possibility that metastasizing tumor cells may be a distinct tumor subpopulation with regard to invasive potential.-J Natl Cancer Inst58: 1427-1431, 1977.
1428
LIOTTA, KLEINERMAN , CATANZARO, AND RYNBRANDT TABLE
1.-Ratios of selected amino acids in basement membrane collagen Amino acid ratios
Basement membrane source (reference) 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.
Murine pulmonary" Murine tumor (13) Canine pulmonary (10) Bowman's capsule (10) Choroid membrane (10) Descemet's membrane (10) Canine lens capsule (10) Bovine lens capsule (14) Rat glomeruli (15) Bovine brain vessels (16)
3HYP/4HYP
HYP/PRO
HYL/HYP
HYL/LYS
HYP/GLY
HYL/GLY
0.198" 0.060 Trace 0.203 Trace 0.088 0.243 Not given Not given Not given
0.364 0.690 0.450 1.68 0.714 0.860 1.500 1.560 0.840 1.09
0.401" 0.357 0.191 0.269 0.185 0.242 0.318 0.414 0.423 0.225
0.186 0.405 0.163 1.260 0.219 0.830 2.900 3.640 0.550 1.080
0.106 0.140 0.157 0.408 0.205 0.355 0.345 0.41 0.260 0.400
0.043 0.050 0.030 0.110 0.038 0.086 0.11 0.17 0.110 0.090
n. Substrate b
used in the present study. The greatest similarity is found between this preparation and that of 2, 3, 5, and 9. These high ratios are characteristic of type IV collagen.
J
NATL CANCER INST
Basement membrane breakdown.-Isolated whole basement membrane substrate was exposed to living tumor cells in vitro. Kefalides and Denduchis (10, 11) showed that basement membrane is composed of a collagen moiety within a glycoprotein matrix. The collagen moiety has a high content of HYP and HYL (10). The predominant carbohydrate in the glycoprotein matrix is hexose (10, 11). Basement membrane breakdown was evaluated by analysis of the supernatant for soluble HYP and hexose-glycoprotein. After incubation of the basement membrane substrate with living tumor cells at 37° C at pH 7.5 for 18 hours, the mixture was centrifuged at 1,400xg at 4° C for 5 minutes. The supernatant was assayed for HYP content by the method of Bergman and Loxley (20) and for protein-bound hexose by precipitation with 95% ethanol at room temperature for 30 minutes and reaction with orcinol (21). Appropriate blanks and controls consisted of: a) basement membrane substrate incubated alone in Eagle's MEM, b) tumor cells or cell fractions incubated alone in Eagle's MEM or HBSS, and c) tumor cells incubated with basement membrane at 4° C. The values expressed in "Results" reflect total activity minus the blanks or controls. The HYP and protein-bound hexose content of the blanks were always less than 10% of the values for cells incubated with substrate at 37° C. Collagenase assay.-Cell-associated type I collagenase activity was determined by a slight modification of the method of Bergman and Loxley (20) based on the determination of liberated HYP. Cells to be assayed for collagenase activity were washed three times in HBSS and adjusted for number. Collagen substrate tubes were prepared by the centrifugation of plastic tubes (Falcon Plastics, Oxnard, Calif.) containing insoluble collagen (25 mg/3 ml in Eagle's MEM; Calbiochem) at 1,000 rpm for 20 minutes at room temperature. This procedure resulted in a packed pellet of collagen at the base of the tube. The test cells were gently centrifuged onto the collagenic substrate. The tubes were tightly capped and incubated upright at 37° C for 18 hours. Appropriate trypsin (10 JLg) controls were included to ensure that the collagen was not denatured. Finally, 1 ml of the cell-free supernatant was assayed for HYP content as described above. Assays were repeated in triplicate. Experiment series A.-Tumors were perfused 15 days VOL. 58, NO.5, MAY
1977
Downloaded from http://jnci.oxfordjournals.org/ at Carleton University on May 10, 2015
plied by the laboratory of Dr. Kohn, Pathology Department, Case Western Reserve University (17). Tumor host system.-The T241 fibrosarcoma (2) was maintained by serial passage from femoral muscle of the C57BL/6] male syngeneic mouse. Tumors were initiated with 106 mechanically disaggregated (18) tumor cells from nonnecrotic tumor fragments obtained 10-15 days after passage. Tissue culture media. -The fluid to perfuse the tumor vascular bed consisted of HBSS containing 0.05 M HEPES buffer (Calbiochem, San Diego, Calif.). All in vitro procedures were carried out either with HBSS or Eagle's MEM, with calcium and magnesium buffered with HEPES as above (Gibco, Grand Island, N.Y.). ETC.-After tumor transplantation in the murine femoral muscle, cannulation and perfusion of the femoral vasculature allowed tumor cells entering the vascular space to be collected and studied. The techniques of cannulation and perfusion have been detailed previously (2, 18). The iliofemoral artery and vein were cannulated, the tumor vascular bed was perfused at physiologic pressure, and the resident blood within the tumor was discarded. The venous effluent was collected under sterile conditions. The concentration of tumor cells and leukocytes was quantified after a fixed volume of the sample was passed through a nucleopore filter (2) and the filter was stained with Giemsa. Control perfusions consisted of: 1) perfusion of normal muscle and 2) perfusion of muscle inoculated previously with glutaraldehyde-fixed tumor cells. Macrophages were removed from the limb or tumor venous effluent by their ability to adhere to plastic petri dishes (19). Macrophages were removed after incubation for 1 1/2 hours and verified as being macrophages by their characteristic microscopic appearance and their ability to phagocytize latex particles. PTC . -These cells were obtained from non necrotic fragments of perfused tumors by mechanical disaggregation as described above. Macrophages were removed by adherence to petri dishes. Spleen cells.-We collected spleen cells from tumorbearing animals by mincing the spleen, agitating the resulting suspension in HBSS, and filtering the material through gauze. We obtained liver tissue by mincing normal liver from normal animals in HBSS.
1429
BASEMENT MEMBRANE BREAKDOWN -BY METASTASIZING TUMOR CELLS TABLE
3.-Collagenase activity of tumor cells cultured with purified human collagen Cell population a
Solubilized HYP ug>: 1O-6/cell"
Venous ETC (4) PTC (4) Effluent leukocytes" (4) Spleen cells (2) Li ver cells (2)
1.39±0.40C 0.48±0.44 0.52±0.30 0.33±0.14 0.16±0.10
Numbers in parentheses indicate No. of animals studied. " Mean z sn. C Significantly different from other cell groups atP
