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Endothelial cells enhance spontaneous metastasis of human lung carcinoma cells in athymic mice
251
Cancer Letters,51 (1990) 251-257
Elsevier Scientific Publishers Ireland Ltd.
Endothelial cells enhance spontaneous lung carcinoma cells in athymic mice N.M.
Varki,
B. Viswanathan
and
metastasis
of human
T. Vu
Department of Medicine, Cancer Biology Program of the University of California, Jolla, CA 92093 (U.S.A.)
San Diego, Cancer Center,
T-012, La
(Received 22 August 1989) (Accepted 9 March 1990)
Summary
events that are necessdry for establishment of
The process of adhesion to endothelial cells is an imporfanf step in the progression to metastatic disease. The use of human neoplastic cell lines (now increasingly used in the study of the mechanisms of metastasis) in studying interactions with normal endothelial cells is therefore pertinent. In this report, the enhanced ability of a metastatic variant, MV522, of a human lung carcinoma cell line to
adhere to endothelial cell monolayers is demThe ability to spontaneously onstrated. metastasize from subcutaneous sites in athymic mice, of in vitro selected endothelial adhesiue subpopulations of MV522 is also shown. Keywords: endothelium; athymic mice.
human
neoplasm;
Introduction
The process of metastasis, whereby subpopulations of malignant cells become established in sites distant from the primary neoplasm, has been the subject of intense research [ll, 181. It has been shown that only certain specially endowed neoplastic cells from within a heterogenous primary neoplasm are able to complete the complex cascade of
metastatic foci. Neoplastic cells invade and enter the circulation and interact with a variety of host defenses and only a small fraction survive to reach distant organs. At these sites, a subpopulation of the surviving malignant cells then proceeds to attach to the endothelial cells lining the capillaries, burrow through the underlying matrix and eventually, if the host organ is permissive, establish as metastatic foci. Models for studying the interactions between malignant cells and normal endothelium have been developed D,2,710,12,13,15,16,19], and these have been used to study the mechanisms of the interactive processes. Most of these earlier studies used animal neoplasms. We recently described the establishment of a model system of a spontaneously metastasizing (from subcutaneous sites in athymic mice) human lung carcinoma cell line [17]. Since adhesion to endothelial cells is a critical step in the sequence of events that results in metastases, we wanted to determine if the parental human lung carcinoma cells, UCP3, and its derived metastatic variant, MV522, would show a differential ability to attach to confluent monolayers of endothelial cells derived from various sources. We did not examine the relationship of tumor cells with subendothelial matrices. When an enhanced ability to adhere
0 1990 Elsevier Scientific Publishers Ireland Ltd. 0304-3835/90/$03.50 Published and Printed in Ireland
to endothelial monolayers was shown by the metastatic variant MV522, we investigated the spontaneous metastatic ability of endothelium adhesive (as well as endothelium nonadhesive) subpopulations of MV522, when injected into subcutaneous sites of athymic mice. Materials and methods
Animals Healthy 3- to 4-week-old athymic female mice were obtained from commercial vendors and kept in laminar flow hoods, under pathogen-free conditions. The animals were tested routinely for infectious agents as part of the quality control. Cell lines
The parental human lung carcinoma cell line, UCP3, and the metastatic variant, MV522, were described earlier [17]. Calf pulmonary arterial endothelial ceils, CPAE, were obtained from ATCC, Rockville, MD. Adult bovine aortic endothelial cells, ABAE (originally obtained from Dr. D. Gospodarowicz, University of California, San Francisco), human carotid artery endothelial cells, HEC, and rabbit aortic endothelial cells, REC, were obtained from Dr. J. Berliner and Dr. T. Drake, University of California, Los Angeles. Mouse brain capillary endothelial cells, MBCE, were obtained from Dr. P. Cancilla, University of California, Los Angeles. The endothelial cells had all been screened and tested and proven to be of endothelial origin. The neoplastic cell lines were tested and confirmed to be human in origin and not contaminated with murine cells (161. The neoplastic cells were passaged in vitro, twice a week using RPM1 1640 medium supplemented with 10% fetal calf serum, 2 mmol of glutamine and 25 pg/ml gentamycin (Gibco, Grand Island, NY). The endothelial cells were obtained from the different investigators when confluent. CPAE was passaged once a week in RPM1 media, when needed, and was not used above passage 15.
Adhesion assays Modifications of a previously described method [ 11 were used. The neoplastic cells were seeded into 100-mm tissue culture dishes (Lux, Miles Scientific, Naperville, IL) at concentrations of lo7 cells, and were labeled with [3H]glucosamine for at least 4 days to assure maximum incorporation (10 rCi/ml, 40 Ci/ mmol (American Radiolabeled Chemicals, Inc., 11612 Bowling Green Drive, St. Louis, MO). The endothelial ceils were seeded into 24-well tissue culture dishes (Costar, 205 Broadway, Cambridge, MA) and were used after 5 days or whenever they were confluent. At the time of the assay, the labeled neoplastic cells were harvested with a 0.02% EDTA solution. Equal numbers of neoplastic cells from the parental cell line and from the metastatic variant MV522 were allowed to attach to the endothelial monolayers for varying periods of time in a humid incubator. Unattached neoplastic cells were washed off using phosphate buffered saline (PBS, pH 7.0). The labeled neoplastic cells (attached to the endothelial cell monolayers) were then lysed in 0.1 N NaOH overnight. After the addition of 0.1 N HCI which neutralized the base, scintillation cocktail was added and the radioactivity determined on the scintillation counter. The control consisted of similar numbers of neoplastic cells that were allowed to attach to plastic for the same length of time, assuming that maximum numbers of neoplastic cells would attach to plastic and that only a certain subpopulation of neoplastic cells attach to the endothelial cell would monolayers. Data presentation. The percent adhesion was calculated according to the formula: dpm sample - dpm blank dpm maximum - dpm blank
x 100
where the dpm (disintegrations per min) of neoplastic cells attached to plastic was used to obtain the maximum value and a mean of 8 samples was used for all data points. When
253
necessary, Statworks PA).
statistical analysis was done using (Cricket Software, Philadelphia,
In uitro selection for endothelial adhesive subpopulations Calf pulmonary endothelial cells (CPAE) were used for these selections. In one experiment, the endothelial cells were seeded into T25 flasks (or 100-mm tissue culture dishes) and allowed to attain confluency. Neoplastic cells of the metastatic variant MV522 were also seeded into T25 flasks (or lOO-mm tissue culture dishes) and allowed to attain confluence. The neoplastic cells were then detached from the flasks using a 0.02% EDTA solution, counted to determine cell numbers, and overlayed on the monolayers of endothelial cells, at a 1:l ratio, for a period of 6 h in the humid incubator. The non-adherent neoplastic cells were drained off and discarded. The adherent neoplastic cells, along with the CPAE to which they were adherent, were detached from plastic, using a sterile 0.02% EDTA solution. The cells were washed in PBS, resuspended and injected in 200~~1 aliquots into the subcutaneous flank area of healthy 3-4 week old female athymic mice. Other mice were injected with 10’ CPAE cells as a control. In other experiments, the non-adherent neoplastic cells were drained and collected (endothelium non-adherent cells, ENA). The monolayers were washed again with media, to ensure removal of nonadherent cells. The adherent neoplastic cells (EA) as well as the ENA cells were centrifuged to a pellet, resuspended and used for injection into athymic mice, as described earlier. In an other experiment, the non-adherent tumor cells were collected and serially selected over 3 separate monolayers of endothelial cells to obtain ‘truly non-adherent‘ tumor cell subpopulations before injection into athymic mice. After the first round of selection, the nonadherent tumor cells remained nonadherent with high viabilities. In an other experiment, endothelium adherent cells (EA) and non-adherent cells (ENA)
were selected as described above. Groups of athymic mice were then injected with either: (a) EA tumor cells attached to the underlying endothelial cells; or (b) ENA tumor cells mixed with equivalent numbers of endothelial cells as in (a). In order to obtain continuous cell lines for detailed analysis, EA and ENA ceils were selected as described above and then passaged in vitro. The derived cell lines were named EAC and ENAC (endothelium adherent cells and endothelium non-adherent cells). Metastasis survey The athymic mice which received the S.C. injections were monitored weekly for 8 weeks. The S.C. tumors that grew were measured weekly using calipers and the tumor volume was calculated according to the formula [3] : 1 x w2 Tumor volume
= 2
The average tumor volume was then calculated and the data used for in vivo growth curves. At the end of 8 weeks the mice were killed and subjected to complete autopsies. The organs were first viewed under a dissecting microscope and then fixed in 10% buffered formalin for processing towards paraffin embedding. Histologic sections were made of liver, spleen, pancreas, kidneys adrenals, brain, lymph nodes and lungs. Sections from each organ were then stained with hematoxylin/eosin and examined under the microscope. Metastatic foci were then evaluated by size and number, as seen microscopically and measured using an optical micrometer disc within the eyepiece of the Olympus BH2 microscope. Results Differential adhesion of parental UCP3 cells and metastatic variant MV522 cells to monolayers of endothelial cells Initial experiments examined the ability of
254
equal numbers of [3H]glucosamine labeled parental UCP3 cells and metastatic variant MV522 cells, (spec. act. being similar) to adhere to confluent monolayers of endothelial cells in vitro. The endothelial cells used were derived from different sources: rabbit aortic endothelium, human carotid artery endothelium, adult bovine aortic endothelium and mouse brain capillary endothelium. Thus, the initial study used endothelial cells derived mostly from large arteries. The results (Fig. 1.) indicate that MV522 shows an enhanced ability to adhere to endothelial cell monolayers derived from various sources. However, the adhesion of the metastatic variant cells to endothelial cell monolayers derived from brain capillaries, appears even more striking. On statistical analyses, the difference in adhesion seen with the MBCE cell line was significant (P < 0.05). Histologic examination of brain sections, obtained from autopsies of the athymic mice, however, have thus far failed to reveal metastatic foci. The xenogeneic origin of the endothelial cells did not appear to affect the ability to attach. Since the metastatic variant MV522 was derived from pulmonary metastases, its ability RELATIVE
ADHESION
OF NEOPLASTIC
12 I
CELLS -
10 ’b
8
?
6
0 -
4
x
2 0 REC
HEC ENDOTHELIAL
ABAE
MBCE
CELLS
Fig.1. Adhesion of equal numbers of [3H]glucosamine labeled parental UCP3 cells and metastatic variant MV522 cells (spec. act. being similar) to confluent monolayers of endothelial cells derived from various sources. REC = rabbit aortic endothelial cells; HEC = human carotid artery endothelial cells; ABAE = adult bovine aortic endothelial cells; and MBCE = mouse brain capillary endothelial cells. Y-axis bars represent standard deviation.
to adhere to endothelial cells derived from pulmonary arterial endothelium was compared with that of the parental UCP3. Lung microvessel endothelia were not used. Confluent monolayers of calf pulmonary artery endothelial cells (CPAE), were used in these assays. Although there was a lot of interassay variability, it was evident that the metastatic variant MV522 demonstrated an enhanced ability to adhere. Time course experiments showed that the enhanced ability to adhere, shown by the metastatic variant MV522 cells, was evident as early as 30 min into the assay (not shown). These experiments also showed that approximately 5 h of contact between tumor cells and confluent monolayers of endothelial cells were necessary, in order to obtain a stable subpopulation of adhesion variant tumor cells. Therefore, in the experiments that were designed to isolate an adhesion variant subpopulation of tumor cells (see below), tumor cells were allowed to attach to confluent monolayers of endothelial cells for at least six hours. The adhesion to endothelial cells appeared to be a saturable event in these in vitro assays, with the highest percentage of cells adhering when used at lo5 tumor cells/well of a 24-well plate. Many of these experiments used neoplastic cells at the plateau phase of the cell cycle. If labeled and used at mid-log phase, the metastatic variant cells did not seem to have as yet developed the ability to demonstrate the enhanced adhesion to the confluent monolayers of endothelial cells (not shown). In addition, if the endothelial cells were used for adhesion assays 3-4 weeks after becoming confluent, the results were very variable. In vitro selection for endothelial adhesive tumor subpopulation and metastatic surveys
Metastatic variant MV522 cells and confluent monolayers of calf pulmonary arterial endothelial cells, were used to select for endothelial adhesive subpopulations of tumor cells, as described in Methods. UCP3 cells were not used in these experiments. The CPAE cells proved nontumorigenic, whereas the EA and ENA cells were tumorigenic. As
255
shown in Table 1, athymic mice that received subpopuiations of endothelium adhesive MV522 cells developed significant pulmonary metastatic disease. The microscopic appearance of some of the extremely large metastases is demonstrated in Fig. 2. There were no metastases to any of the other organs. The endothelial adherent tumor cells were able to establish as large metastatic foci even though only approximately 2 x lo6 cells were used in the innoculum. Earlier studies with the MV522 used 5 x lo6 before large foci of metastases were seen. The in vitro passaged cell lines, EAC and ENAC (obtained after selection over endothelial monolayers) showed no appreciable differences in their in vitro or in vivo growth and metastatic ability. The group of animals that received ENA tumor cells mixed with endothelial cells in numbers proportional to the EA cells/endothelial cells showed no increase in incidence of pulmonary metastatic disease. Discussion
The property of adhesion to endothelial ceils and sub-endothelial matrices, shown by Table 1.
subpopulations of metastatic cells, may be important in aiding in the metastatic disease process [l]. The interaction of metastatic tumor cells with endothelial cells and subendothelial basement membranes has been described in a variety of model systems. Intense efforts have also been expended in recent years on the phenomenon of adhesion of metastatic tumor cells to artificial matrices, composed of the material that is found normally in the region of the subendothelial matrix. These studies are extremely valuable in elucidating the mechanisms whereby neoplastic cells invade. However, the fact remains that metastatic subpopulations of cells do encounter intact monolayers of endothelial cells in vivo, during the process of metastasis. The attachment of the metastatic cells to these endothelial cells is the event that must occur before the neoplastic cells can invade subendothelial matrices. In this report we show that primary and metastatic variant human carcinoma cells show a differential ability to adhere to confluent monolayers of endothelial cells. The metastatic variant cells showed an enhanced ability to adhere, and the xenogeneic origin of many of
Capacity shown by endothelial adhesive and nonadhesive spontaneously metastasize from subcutaneous sites in athymic mice.
subpopulations
(derived in vitro from MV522) to
Exp. No.
No. of cells injected
Total incidence”
No. of pulmonary foci
1 2
2 2 3 2 5 5 2 2
2/2 4/4 2/4 2/4 2/5 275 4/5 2/5
> 5oa l-> 5@ o- 12b o--2b o-9 O-9 >504 o- 12b
3 4 5
x x x x x x x x
106EA 106EA lo6 ENA lo6 EN& lo6 EACd lo6 ENACd 106EA lo6 ENA + CPAE’
“Microscopic foci greater than 200 pm in diameter, grossly visible. bMicroscopic foci less than 200 pm in diameter. Serially selected for nonadherence to endothelial cell monolayers (see Materials and methods). dCell lines developed in vitro after endothelial adherent and nonadherent subpopulations were selected in the usual manner. ‘Explained in Materials and methods.
256
Fig. 2.
Microscopic picture of grossly visible foci of metastases seen in the lungs of the host athymic mice u rhen EA (:ells were carried as subcutaneous xenotransplants. (Scale bar = 100 pm)
the endothelial cells did not seem to make a difference. The adhesion to capillary endothelium was more striking. This finding is consistent with earlier reports [15] which have observed that metastatic variant neoplastic cells show an enhanced ability to adhere to capillary endothelium as opposed to endothelium derived from large arteries. It has been shown that the adhesive interactions of malignant cells with organ-derived microvessel, but not large vessel, endothelial cells, correlated with their origin, metastatic properties, and organ preference of metastasis [2]. In this series of experiments, we did not examine this selective tumor cell adhesion mechanisms in organ-specific metastasis. We attempted instead to derive highly metastatic subpopulations of cells, using the property
shown by the metastatic variant MV522 to adhere preferentially to endothelial cell monolayers. A similar phenomenon has been reported in earlier studies whereby it was shown that hybrids formed by fusion of normal thymocytes with lymphoma clones of low tumorigenic potential demonstrated a high tumorigenic capacity [4]. Fusion of plasma membrane vesicles from high metastatic clones with low metastatic has been shown to result in high metastatic ability [14]. These results are different from those obtained by many laboratories now involved in the interesting study of tumor suppressor genes [6]. A recent report observed that tumor cells are protected from natural killer cell mediated lysis by adhesion to endothelial cells [5], whereby natural surveillance mechanisms might be circumvented.
257
Whether a similar or a different mechanism exists in this model system awaits further analysis .
8
Acknowledgements 9
We thank Lisa Bodecker for help with preparation of the manuscript. This study was supported by Research Grants USPHS lR23 CA43009 and lRO1 CA43716 from the National Cancer Institute, Bethesda, Maryland, and an Institutional grant from the American Cancer Society.
10
11
12
References 13 Alby, L. and Auerbach, R. (1984) Differential adhesion of tumor cells to capillary endothelial cells in vitro. Proc. Natl. Acad. Sci. USA, 81,5739-5743. Auerbach, R., Lu, W.C., Pardon, E. Gumkowski, F.,
14
Kaminska. G. and Kaminski, M. (1987) Specificity of adhesion between murine tumor cells and capillary endothelium: An in vitro correlate of preferential metastasis in vivo. Cancer Res., 47,1492-1496. Giavazzi, R., Campbell, D.E., Jessup, J.M., Cleary, K. and Fidler, I.J. (1986) Metastatic behavior of tumor cells isolated from primary and metastatic human colorectal carcinomas implanted into different sites in nude mice. Cancer Res., 46,1928-1933. Hays, E.F., Weinroth, S.E., Macleod, C.L. and Kitada, S. (1986) Tumorigenicity of T lymphoma/T lymphoma hybrids and T lymphoma/normal cell hybrids. Int. J. Can-
17
cer, 38,597-601. Kaminski, M. and Auerbach. R. (1988) Tumor cells are protected from NK-cell mediated lysis by adhesion to endothelial cells. Int. J. Cancer, 41,847-849.
18
Klein, G. (1987) The approaching era of the tumor suppressor genes. Sci., 238,15391544. Korach, S., Poupon, M.F., Du-Villard, J.A. and Becker, M. (1986) Differential adhesiveness of rhabdonyosarcoma-
15
16
19
derived cloned metastatic cell lines to vascular endothelial monolayers. Cancer Res., 46,3624-3629. Kramer, R.H. and Nicolson, G.L. (1979) Interactions of tumor cells with vascular endothelial cell monolayers: A model for metastatic invasion. Proc. Natl. Acad. Sci. USA, 76,5704-5708. Kramer, R.H., Vogel, K.G. and Nicolson, G.L. (1982) Solubiliiation and degradation of subendothelial matrix glycoproteins and proteoglycans by metastatic tumor cells. J. Biol. Chem., 257,2678-2686. Nicolson, G.L. (1982) Metastatic tumor cell attachment and invasion assay utilizing vascular endothelial cell monolayers. J. Histochem. Cytochem., 30,214-220. Nicolson, G.L. (1988) Cancer metastasis: Tumor cell and host organ properties important in metastasis to specific secondary sites. Biochem. Biophys. Acta, 948, 175-224. Ormerod, E.J., Everett, C.A. and Hart, I.R. (1988) Adhesion characteristics of human melanoma cell lines of varying metastaticpotential. lnt. J. Cancer, 41, 150-154. On, F.W., Adamson, LY. and Young, L. (1986) Promotion of pulmonary metastasis in mice by bleomycin-induced endothelial injury. Cancer Res., 46,891-897. Poste, G. and Nicolson, G.L. (1980) Arrest and metastasis of blood borne tumor cells are modified by fusion of plasma membrane vesicles from highly metastatic cells. Proc. Natl. Acad. Sci. USA, 77,399-403. Poste, G. and Nicolson, G.L. (1984) In vitro systems for studying the interactions of metastatic tumor cells with endothelial cells and subendothelial basement membranes. In: Biology of Endothelial Cells, pp. 438. Editor: E.A. Jaffe. Tao, T.W. and Johnson, L.K. (1982) Altered adhesiveness of tumor cell surface variants with reduced metastasizing capacity-reduced adhesiveness to vascular wall components in culture. Int. J. Cancer, 30, 763-766. Varki, N.M., Roome, L., Sparkes, R.S. and Miller, J.E. (1987) Microscopic metastasis of a human lung carcinoma cell line in athymic nude mice: Isolation of a metastatic variant. Int. J. Cancer, 40,46-52. Weiss, L. (1985) In: Principles of Metastasis. Academic Press, New York. Weiss, L., Orr, F.W. and Honn, K.Y. (1988) Interactions of cancer cells with the microvasculature during metastasis. Fed. Am. Sot. Exp. Biol. J., 2, 12-21.
Cancer Letters,51 (1990) 251-257
Elsevier Scientific Publishers Ireland Ltd.
Endothelial cells enhance spontaneous lung carcinoma cells in athymic mice N.M.
Varki,
B. Viswanathan
and
metastasis
of human
T. Vu
Department of Medicine, Cancer Biology Program of the University of California, Jolla, CA 92093 (U.S.A.)
San Diego, Cancer Center,
T-012, La
(Received 22 August 1989) (Accepted 9 March 1990)
Summary
events that are necessdry for establishment of
The process of adhesion to endothelial cells is an imporfanf step in the progression to metastatic disease. The use of human neoplastic cell lines (now increasingly used in the study of the mechanisms of metastasis) in studying interactions with normal endothelial cells is therefore pertinent. In this report, the enhanced ability of a metastatic variant, MV522, of a human lung carcinoma cell line to
adhere to endothelial cell monolayers is demThe ability to spontaneously onstrated. metastasize from subcutaneous sites in athymic mice, of in vitro selected endothelial adhesiue subpopulations of MV522 is also shown. Keywords: endothelium; athymic mice.
human
neoplasm;
Introduction
The process of metastasis, whereby subpopulations of malignant cells become established in sites distant from the primary neoplasm, has been the subject of intense research [ll, 181. It has been shown that only certain specially endowed neoplastic cells from within a heterogenous primary neoplasm are able to complete the complex cascade of
metastatic foci. Neoplastic cells invade and enter the circulation and interact with a variety of host defenses and only a small fraction survive to reach distant organs. At these sites, a subpopulation of the surviving malignant cells then proceeds to attach to the endothelial cells lining the capillaries, burrow through the underlying matrix and eventually, if the host organ is permissive, establish as metastatic foci. Models for studying the interactions between malignant cells and normal endothelium have been developed D,2,710,12,13,15,16,19], and these have been used to study the mechanisms of the interactive processes. Most of these earlier studies used animal neoplasms. We recently described the establishment of a model system of a spontaneously metastasizing (from subcutaneous sites in athymic mice) human lung carcinoma cell line [17]. Since adhesion to endothelial cells is a critical step in the sequence of events that results in metastases, we wanted to determine if the parental human lung carcinoma cells, UCP3, and its derived metastatic variant, MV522, would show a differential ability to attach to confluent monolayers of endothelial cells derived from various sources. We did not examine the relationship of tumor cells with subendothelial matrices. When an enhanced ability to adhere
0 1990 Elsevier Scientific Publishers Ireland Ltd. 0304-3835/90/$03.50 Published and Printed in Ireland
to endothelial monolayers was shown by the metastatic variant MV522, we investigated the spontaneous metastatic ability of endothelium adhesive (as well as endothelium nonadhesive) subpopulations of MV522, when injected into subcutaneous sites of athymic mice. Materials and methods
Animals Healthy 3- to 4-week-old athymic female mice were obtained from commercial vendors and kept in laminar flow hoods, under pathogen-free conditions. The animals were tested routinely for infectious agents as part of the quality control. Cell lines
The parental human lung carcinoma cell line, UCP3, and the metastatic variant, MV522, were described earlier [17]. Calf pulmonary arterial endothelial ceils, CPAE, were obtained from ATCC, Rockville, MD. Adult bovine aortic endothelial cells, ABAE (originally obtained from Dr. D. Gospodarowicz, University of California, San Francisco), human carotid artery endothelial cells, HEC, and rabbit aortic endothelial cells, REC, were obtained from Dr. J. Berliner and Dr. T. Drake, University of California, Los Angeles. Mouse brain capillary endothelial cells, MBCE, were obtained from Dr. P. Cancilla, University of California, Los Angeles. The endothelial cells had all been screened and tested and proven to be of endothelial origin. The neoplastic cell lines were tested and confirmed to be human in origin and not contaminated with murine cells (161. The neoplastic cells were passaged in vitro, twice a week using RPM1 1640 medium supplemented with 10% fetal calf serum, 2 mmol of glutamine and 25 pg/ml gentamycin (Gibco, Grand Island, NY). The endothelial cells were obtained from the different investigators when confluent. CPAE was passaged once a week in RPM1 media, when needed, and was not used above passage 15.
Adhesion assays Modifications of a previously described method [ 11 were used. The neoplastic cells were seeded into 100-mm tissue culture dishes (Lux, Miles Scientific, Naperville, IL) at concentrations of lo7 cells, and were labeled with [3H]glucosamine for at least 4 days to assure maximum incorporation (10 rCi/ml, 40 Ci/ mmol (American Radiolabeled Chemicals, Inc., 11612 Bowling Green Drive, St. Louis, MO). The endothelial ceils were seeded into 24-well tissue culture dishes (Costar, 205 Broadway, Cambridge, MA) and were used after 5 days or whenever they were confluent. At the time of the assay, the labeled neoplastic cells were harvested with a 0.02% EDTA solution. Equal numbers of neoplastic cells from the parental cell line and from the metastatic variant MV522 were allowed to attach to the endothelial monolayers for varying periods of time in a humid incubator. Unattached neoplastic cells were washed off using phosphate buffered saline (PBS, pH 7.0). The labeled neoplastic cells (attached to the endothelial cell monolayers) were then lysed in 0.1 N NaOH overnight. After the addition of 0.1 N HCI which neutralized the base, scintillation cocktail was added and the radioactivity determined on the scintillation counter. The control consisted of similar numbers of neoplastic cells that were allowed to attach to plastic for the same length of time, assuming that maximum numbers of neoplastic cells would attach to plastic and that only a certain subpopulation of neoplastic cells attach to the endothelial cell would monolayers. Data presentation. The percent adhesion was calculated according to the formula: dpm sample - dpm blank dpm maximum - dpm blank
x 100
where the dpm (disintegrations per min) of neoplastic cells attached to plastic was used to obtain the maximum value and a mean of 8 samples was used for all data points. When
253
necessary, Statworks PA).
statistical analysis was done using (Cricket Software, Philadelphia,
In uitro selection for endothelial adhesive subpopulations Calf pulmonary endothelial cells (CPAE) were used for these selections. In one experiment, the endothelial cells were seeded into T25 flasks (or 100-mm tissue culture dishes) and allowed to attain confluency. Neoplastic cells of the metastatic variant MV522 were also seeded into T25 flasks (or lOO-mm tissue culture dishes) and allowed to attain confluence. The neoplastic cells were then detached from the flasks using a 0.02% EDTA solution, counted to determine cell numbers, and overlayed on the monolayers of endothelial cells, at a 1:l ratio, for a period of 6 h in the humid incubator. The non-adherent neoplastic cells were drained off and discarded. The adherent neoplastic cells, along with the CPAE to which they were adherent, were detached from plastic, using a sterile 0.02% EDTA solution. The cells were washed in PBS, resuspended and injected in 200~~1 aliquots into the subcutaneous flank area of healthy 3-4 week old female athymic mice. Other mice were injected with 10’ CPAE cells as a control. In other experiments, the non-adherent neoplastic cells were drained and collected (endothelium non-adherent cells, ENA). The monolayers were washed again with media, to ensure removal of nonadherent cells. The adherent neoplastic cells (EA) as well as the ENA cells were centrifuged to a pellet, resuspended and used for injection into athymic mice, as described earlier. In an other experiment, the non-adherent tumor cells were collected and serially selected over 3 separate monolayers of endothelial cells to obtain ‘truly non-adherent‘ tumor cell subpopulations before injection into athymic mice. After the first round of selection, the nonadherent tumor cells remained nonadherent with high viabilities. In an other experiment, endothelium adherent cells (EA) and non-adherent cells (ENA)
were selected as described above. Groups of athymic mice were then injected with either: (a) EA tumor cells attached to the underlying endothelial cells; or (b) ENA tumor cells mixed with equivalent numbers of endothelial cells as in (a). In order to obtain continuous cell lines for detailed analysis, EA and ENA ceils were selected as described above and then passaged in vitro. The derived cell lines were named EAC and ENAC (endothelium adherent cells and endothelium non-adherent cells). Metastasis survey The athymic mice which received the S.C. injections were monitored weekly for 8 weeks. The S.C. tumors that grew were measured weekly using calipers and the tumor volume was calculated according to the formula [3] : 1 x w2 Tumor volume
= 2
The average tumor volume was then calculated and the data used for in vivo growth curves. At the end of 8 weeks the mice were killed and subjected to complete autopsies. The organs were first viewed under a dissecting microscope and then fixed in 10% buffered formalin for processing towards paraffin embedding. Histologic sections were made of liver, spleen, pancreas, kidneys adrenals, brain, lymph nodes and lungs. Sections from each organ were then stained with hematoxylin/eosin and examined under the microscope. Metastatic foci were then evaluated by size and number, as seen microscopically and measured using an optical micrometer disc within the eyepiece of the Olympus BH2 microscope. Results Differential adhesion of parental UCP3 cells and metastatic variant MV522 cells to monolayers of endothelial cells Initial experiments examined the ability of
254
equal numbers of [3H]glucosamine labeled parental UCP3 cells and metastatic variant MV522 cells, (spec. act. being similar) to adhere to confluent monolayers of endothelial cells in vitro. The endothelial cells used were derived from different sources: rabbit aortic endothelium, human carotid artery endothelium, adult bovine aortic endothelium and mouse brain capillary endothelium. Thus, the initial study used endothelial cells derived mostly from large arteries. The results (Fig. 1.) indicate that MV522 shows an enhanced ability to adhere to endothelial cell monolayers derived from various sources. However, the adhesion of the metastatic variant cells to endothelial cell monolayers derived from brain capillaries, appears even more striking. On statistical analyses, the difference in adhesion seen with the MBCE cell line was significant (P < 0.05). Histologic examination of brain sections, obtained from autopsies of the athymic mice, however, have thus far failed to reveal metastatic foci. The xenogeneic origin of the endothelial cells did not appear to affect the ability to attach. Since the metastatic variant MV522 was derived from pulmonary metastases, its ability RELATIVE
ADHESION
OF NEOPLASTIC
12 I
CELLS -
10 ’b
8
?
6
0 -
4
x
2 0 REC
HEC ENDOTHELIAL
ABAE
MBCE
CELLS
Fig.1. Adhesion of equal numbers of [3H]glucosamine labeled parental UCP3 cells and metastatic variant MV522 cells (spec. act. being similar) to confluent monolayers of endothelial cells derived from various sources. REC = rabbit aortic endothelial cells; HEC = human carotid artery endothelial cells; ABAE = adult bovine aortic endothelial cells; and MBCE = mouse brain capillary endothelial cells. Y-axis bars represent standard deviation.
to adhere to endothelial cells derived from pulmonary arterial endothelium was compared with that of the parental UCP3. Lung microvessel endothelia were not used. Confluent monolayers of calf pulmonary artery endothelial cells (CPAE), were used in these assays. Although there was a lot of interassay variability, it was evident that the metastatic variant MV522 demonstrated an enhanced ability to adhere. Time course experiments showed that the enhanced ability to adhere, shown by the metastatic variant MV522 cells, was evident as early as 30 min into the assay (not shown). These experiments also showed that approximately 5 h of contact between tumor cells and confluent monolayers of endothelial cells were necessary, in order to obtain a stable subpopulation of adhesion variant tumor cells. Therefore, in the experiments that were designed to isolate an adhesion variant subpopulation of tumor cells (see below), tumor cells were allowed to attach to confluent monolayers of endothelial cells for at least six hours. The adhesion to endothelial cells appeared to be a saturable event in these in vitro assays, with the highest percentage of cells adhering when used at lo5 tumor cells/well of a 24-well plate. Many of these experiments used neoplastic cells at the plateau phase of the cell cycle. If labeled and used at mid-log phase, the metastatic variant cells did not seem to have as yet developed the ability to demonstrate the enhanced adhesion to the confluent monolayers of endothelial cells (not shown). In addition, if the endothelial cells were used for adhesion assays 3-4 weeks after becoming confluent, the results were very variable. In vitro selection for endothelial adhesive tumor subpopulation and metastatic surveys
Metastatic variant MV522 cells and confluent monolayers of calf pulmonary arterial endothelial cells, were used to select for endothelial adhesive subpopulations of tumor cells, as described in Methods. UCP3 cells were not used in these experiments. The CPAE cells proved nontumorigenic, whereas the EA and ENA cells were tumorigenic. As
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shown in Table 1, athymic mice that received subpopuiations of endothelium adhesive MV522 cells developed significant pulmonary metastatic disease. The microscopic appearance of some of the extremely large metastases is demonstrated in Fig. 2. There were no metastases to any of the other organs. The endothelial adherent tumor cells were able to establish as large metastatic foci even though only approximately 2 x lo6 cells were used in the innoculum. Earlier studies with the MV522 used 5 x lo6 before large foci of metastases were seen. The in vitro passaged cell lines, EAC and ENAC (obtained after selection over endothelial monolayers) showed no appreciable differences in their in vitro or in vivo growth and metastatic ability. The group of animals that received ENA tumor cells mixed with endothelial cells in numbers proportional to the EA cells/endothelial cells showed no increase in incidence of pulmonary metastatic disease. Discussion
The property of adhesion to endothelial ceils and sub-endothelial matrices, shown by Table 1.
subpopulations of metastatic cells, may be important in aiding in the metastatic disease process [l]. The interaction of metastatic tumor cells with endothelial cells and subendothelial basement membranes has been described in a variety of model systems. Intense efforts have also been expended in recent years on the phenomenon of adhesion of metastatic tumor cells to artificial matrices, composed of the material that is found normally in the region of the subendothelial matrix. These studies are extremely valuable in elucidating the mechanisms whereby neoplastic cells invade. However, the fact remains that metastatic subpopulations of cells do encounter intact monolayers of endothelial cells in vivo, during the process of metastasis. The attachment of the metastatic cells to these endothelial cells is the event that must occur before the neoplastic cells can invade subendothelial matrices. In this report we show that primary and metastatic variant human carcinoma cells show a differential ability to adhere to confluent monolayers of endothelial cells. The metastatic variant cells showed an enhanced ability to adhere, and the xenogeneic origin of many of
Capacity shown by endothelial adhesive and nonadhesive spontaneously metastasize from subcutaneous sites in athymic mice.
subpopulations
(derived in vitro from MV522) to
Exp. No.
No. of cells injected
Total incidence”
No. of pulmonary foci
1 2
2 2 3 2 5 5 2 2
2/2 4/4 2/4 2/4 2/5 275 4/5 2/5
> 5oa l-> 5@ o- 12b o--2b o-9 O-9 >504 o- 12b
3 4 5
x x x x x x x x
106EA 106EA lo6 ENA lo6 EN& lo6 EACd lo6 ENACd 106EA lo6 ENA + CPAE’
“Microscopic foci greater than 200 pm in diameter, grossly visible. bMicroscopic foci less than 200 pm in diameter. Serially selected for nonadherence to endothelial cell monolayers (see Materials and methods). dCell lines developed in vitro after endothelial adherent and nonadherent subpopulations were selected in the usual manner. ‘Explained in Materials and methods.
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Fig. 2.
Microscopic picture of grossly visible foci of metastases seen in the lungs of the host athymic mice u rhen EA (:ells were carried as subcutaneous xenotransplants. (Scale bar = 100 pm)
the endothelial cells did not seem to make a difference. The adhesion to capillary endothelium was more striking. This finding is consistent with earlier reports [15] which have observed that metastatic variant neoplastic cells show an enhanced ability to adhere to capillary endothelium as opposed to endothelium derived from large arteries. It has been shown that the adhesive interactions of malignant cells with organ-derived microvessel, but not large vessel, endothelial cells, correlated with their origin, metastatic properties, and organ preference of metastasis [2]. In this series of experiments, we did not examine this selective tumor cell adhesion mechanisms in organ-specific metastasis. We attempted instead to derive highly metastatic subpopulations of cells, using the property
shown by the metastatic variant MV522 to adhere preferentially to endothelial cell monolayers. A similar phenomenon has been reported in earlier studies whereby it was shown that hybrids formed by fusion of normal thymocytes with lymphoma clones of low tumorigenic potential demonstrated a high tumorigenic capacity [4]. Fusion of plasma membrane vesicles from high metastatic clones with low metastatic has been shown to result in high metastatic ability [14]. These results are different from those obtained by many laboratories now involved in the interesting study of tumor suppressor genes [6]. A recent report observed that tumor cells are protected from natural killer cell mediated lysis by adhesion to endothelial cells [5], whereby natural surveillance mechanisms might be circumvented.
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Whether a similar or a different mechanism exists in this model system awaits further analysis .
8
Acknowledgements 9
We thank Lisa Bodecker for help with preparation of the manuscript. This study was supported by Research Grants USPHS lR23 CA43009 and lRO1 CA43716 from the National Cancer Institute, Bethesda, Maryland, and an Institutional grant from the American Cancer Society.
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