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Regular Articles |
From the Department of Anatomy and Cellular Biology,*
Tufts University School of Medicine; and the Molecular Pathology Unit
and Cancer Center,
Massachusetts General
Hospital, Boston, Massachusetts
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In a past study, we showed that hyaluronan accumulates in the ascites, and at initial sites of attachment and invasion of tumor cells at the mesothelial surface of the peritoneal wall, after introduction of murine ovarian tumor cells or mammary carcinoma cells into the peritoneal cavity of syngeneic mice.4 Several types of malignant solid tumors contain elevated levels of hyaluronan, a ubiquitous glycosaminoglycan that contributes both to the structure of extracellular matrix and to cell-matrix interactions that influence cell behavior.5,6 The enrichment of hyaluronan in tumors can result from increased production by tumor cells themselves7,8 or from interactions between tumor cells and surrounding stromal cells that induce increased production by the latter.9-11 High levels of hyaluronan correlate with tumor spread and with poor survival rates in human patients with a variety of tumor types,12-15 and experimental evidence in animal models directly implicates hyaluronan in solid tumor progression.16-20 In the present study our objective was to determine whether hyaluronan also contributes to ascites growth and tumor cell invasion of the peritoneal wall.
We have shown that stable transfection of TA3/St murine mammary carcinoma cells with cDNA encoding soluble CD44 prevents formation of metastatic nodules in the lung after introduction of the TA3/St cells into the vasculature.17 In that study, soluble CD44 presumably acted as a competitive inhibitor of crucial hyaluronan-protein interactions because transfection with mutant soluble CD44 that does not bind hyaluronan had no effect on invasion and metastasis. This supposition was confirmed by experiments showing that soluble CD44 transfection prevents hyaluronan-mediated clustering of endogenous membrane CD44 that is in turn required for binding of gelatinase B (MMP-9) to the tumor cell surface and for invasiveness.21 In the present study, we show that stable transfection of TA3/St cells with soluble CD44 not only inhibits tumor invasion but also prevents tumor cell proliferation in ascites and that this inhibition is because of a direct effect on growth characteristics of the tumor cells rather than, or in addition to, an indirect effect on other events in vivo. These changes in tumor cell growth characteristics depend on hyaluronan interactions because mutated soluble CD44 that does not bind hyaluronan does not cause these changes.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The TA3/St cell line was established from an ascites subline originally derived from a spontaneous mouse mammary adenocarcinoma.2,22 TA3/St cells were maintained by weekly passages in the peritoneal cavities of syngeneic, 4- to 6-week-old female A/Jax mice or in culture in Dulbecco’s modified Eagle’s medium (DMEM; Life Technologies, Inc., Rockville, MD) supplemented with 10% fetal bovine serum (FBS; HyClone Laboratories Inc., Logan, UT). Transfected TA3/St cells were cultured in DMEM supplemented with 10% FBS and 0.5 mg/ml geneticin (G418 sulfate, Life Technologies, Inc., Grand Island, NY).
Transfection of TA3/St Cells with Soluble CD44 Constructs
Soluble CD44 constructs were prepared and analyzed as described previously.17 For transfection, TA3/St cells were treated with either pCR3-Uni eukaryotic expression vector alone (InVitrogen Corp., San Diego, CA) or pCR3-Uni vector containing cDNAs encoding soluble CD44 isoforms, in the presence of lipofectamine. These isoforms included either variant exons v8-v10 or v6-v10, where v10 is a new insert containing a stop codon, thus leading to truncation before the transmembrane domain;17,23 v6-v10 was used with or without the R43A mutation that leads to loss of hyaluronan binding capacity.17,24 G418-resistant colonies were selected and seven clones were chosen for further study: two transfectants containing v6-v10 (v6-v10a and v6-v10b), one containing v8-v10, two containing the v6-v10 mutant (v6-v10 R43A), and two mock transfectants containing vector only. The transfectants were analyzed by reverse transcriptase-polymerase chain reaction, fluorescence-activated cell sorting, and Western blotting as described previously17 to confirm that each clone expresses the appropriate CD44 protein. All transfectants and wild-type cells produced similar amounts of surface-associated, standard, and variant CD44 isoforms. However the soluble CD44 transfectants, including the mutant soluble CD44 transfectants, also produced soluble, secreted CD44.17
Tumorigenicity Assay
TA3/St cells in log phase growth were trypsinized, washed with DMEM containing 10% FBS, and resuspended in Hanks’ balanced salt solution (HBSS; Life Technologies, Inc.) for counting. Suspensions of TA3/St cell lines were injected, using a 25-gauge needle, into the peritoneal cavities of 4- to 6-week-old female A/Jax mice (The Jackson Laboratory, Bar Harbor, ME) at 1 x 106 cells/200 µl HBSS each, and allowed to grow in vivo for a period of 7 to 19 days. For each cell line and time point, six mice were given injections. Mice were observed daily for signs of ascites tumor development and monitored twice daily after the tumor symptoms appeared: abdominal bloating, decreased movement, loss of grooming behavior, and hunched posture. If mice were not expected to survive overnight they were sacrificed before conclusion of the experimental protocol. Mice that did not exhibit the above symptoms were sacrificed according to experimental parameters. The peritoneal walls from each of the mice were removed, cut into strips (~4 mm x 8 mm), and fixed in 4% paraformaldehyde (Tousimis, Rockville, MD) in phosphate-buffered saline (PBS) for histological analysis.
Histology
Fixed strips of peritoneal wall were washed in PBS, dehydrated through 30%, 70%, 95%, 100% ethanol and xylene, and then embedded in paraffin wax (Fisher, Columbia, MD). Sections (5 µm) were cut, mounted on poly-L-lysine (Sigma)-coated slides, and stained with Mayers modified hematoxylin (Poly Scientific Research, Bay Shore, NY) or hematoxylin and eosin (Richard-Allen Medical, Richland, MI) after deparaffinization in xylene and rehydration through 100%, 95%, 70%, 35% ethanol, PBS, and water.
In Vivo Cell Proliferation Assay
Transfected TA3/St cells in log phase growth were trypsinized, washed with DMEM containing 10% FBS, and resuspended in HBSS for counting. Suspensions of the transfected TA3/St cells were seeded into the peritoneal cavities of female A/Jax mice at 2 x 106 cells/200 µl HBSS each and allowed to grow in vivo for a period of 2 to 15 days. At each of five different time points (2, 5, 7, 10, and 15 days), groups of six mice were sacrificed and cells were harvested from the peritoneal cavities with two 6-ml intraperitoneal lavages of calcium- and magnesium-free PBS (PBS-; Life Technologies, Inc.). Harvested cells were then washed three times with PBS-, using low-speed centrifugation with each wash to remove any red blood cells that were withdrawn along with tumor cells from the peritoneal cavity; the tumor cells formed a pellet while the red blood cells remained in the supernatant during these centrifugations. The washed cells were counted in a Coulter Counter (Coulter Electronics, Hialeah, FL) by diluting aliquots of cells resuspended in PBS- to concentrations between 200 to 20,000 cells/ml.
Cell-Cycle Analyses
Transfected and wild-type TA3/St cells were grown intraperitoneally for 7 days, harvested, and washed as described in the previous section. They were then resuspended in 70% EtOH and kept at -20°C until all samples had been collected for cell-cycle analysis. After removal from the freezer, the cells were washed twice with PBS-, resuspended in PBS- containing 0.1 mmol/L EDTA, pH 7.4, 50 mg/ml propidium iodide, 50 mg/ml RNase A (Boehringer Mannheim, Indianapolis, IN), and 1% Triton X-100, and incubated overnight at 4°C. Cell samples were then analyzed by fluorescence-activated cell sorting in a FACScan (Becton Dickinson, Mountain View, CA).
Transfected and wild-type cells were cultured in vitro in DMEM plus 10% FBS, then harvested during log phase of growth, and analyzed in the same way as above.
In Vitro Cell Proliferation Assay
Each cell line, in log growth phase, was trypsinized, washed with DMEM containing 10% FBS, and resuspended in the same media for culture. Cells were plated at 5 x 104 cells per well in 6-well plates (60-mm wells) and allowed to grow in 4 ml of medium at 37oC for 1 to 5 days. Every 24 hours, triplicate wells for each cell line were trypsinized, washed with DMEM, and resuspended in PBS-. The harvested cells were then counted in a Coulter Counter after dilution in PBS- to concentrations of 200 to 10,000 cells/ml.
Soft Agar Assay
Soft agar assays were performed in 60-mm dishes containing 2 ml of 1.2% agarose diluted with 2x DMEM containing 20% FBS to yield a final agarose concentration of 0.6%. Cells were harvested from monolayer culture in log growth phase by trypsinization, washing, and resuspension in DMEM containing 10% FBS for counting. The cells were then suspended in 0.33% agarose in DMEM containing 10% FBS and plated at 5000 cells/well on top of the 0.6% agarose base. After each agarose layer was allowed to solidify (10 minutes at 25oC), three additional 1-ml volumes of 0.33% agarose were layered on top of the cells. Each cell line was plated in triplicate and grown at 37oC for 28 days. Total numbers of colonies per well containing >30 cells or >200 cells per colony were counted separately using a microscope grid. The two classes of colony size were assessed by counting cells in numerous colonies under the microscope and correlating these numbers with colony size. The two classes could be distinguished readily because the great majority of colonies were found to contain between 30 and 100 cells; the large colonies (>200 cells) were very easily distinguished from the majority of colonies (30 to 100 cells) and there were virtually no colonies with <30 cells.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Stable transfectants overexpressing the naturally occurring soluble CD44 isoforms, v6-v10 and v8-v10,23 the mutant isoform, v6-v10 R43A,24 or vector alone were selected and analyzed for CD44 production and secretion as described in Materials and Methods. All cell lines produced similar amounts of membrane-bound CD44. Only the soluble CD44 transfectants, including the mutant soluble CD44 transfectants, produced secreted CD44; clones were selected that produced similar amounts of soluble CD44.17 Binding of hyaluronan to the soluble CD44 transfectants and their adhesion to a hyaluronan substratum were shown previously to be reduced compared to wild-type TA3/St cells and vector controls.17,21 Stable transfectants producing mutated soluble CD44 (v6-v10 R43A) exhibited high levels of hyaluronan binding and adhesion to hyaluronan, similar to wild-type and vector controls.17,21
Overexpression of Soluble CD44 Inhibits Growth in Ascites and Peritoneal Wall Invasion by TA3/St Mammary Carcinoma Cells
Syngeneic A/Jax mice were injected intraperitoneally, at 1 x
106
cells per animal, with wild-type TA3/St cells
or with TA3/St transfectants expressing soluble CD44 isoforms, mutant
soluble CD44 (v6-v10 R43A), or vector alone. Tumor growth and invasion
were assessed as described in Methods. Because results obtained in
pilot experiments were virtually identical for the wild-type cells,
both vector transfectants and both mutant soluble CD44 transfectants,
only one of each control transfectant was examined in detail. Three
soluble CD44 transfectant clones, two expressing v6-v10 and one
expressing v8-v10, were examined in detail. Table 1
summarizes the results of one such
experiment in which ascites accumulation, tumor growth, and tumor
invasion were compared in the above manner in groups of six animals
that were injected with either wild-type TA3/St cells, one of the
vector transfectants, one of the mutant soluble CD44 transfectants, or
the two v6-v10 soluble CD44 transfectants. Identical results to those
shown in Table 1
for the two v6-v10 transfectants were obtained for the
v8-v10 soluble CD44 transfectant in other similar experiments. The
results of these experiments are discussed below.
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. However, no tumor
cells were observed attached to the peritoneal walls of mice injected
with the v6-v10 or the v8-v10 soluble CD44 transfectants (Figure 1B)
.
Mice injected with wild-type, vector-transfected, or mutant soluble
CD44-transfected cells exhibited widespread tumorigenesis and invasion
of the mesothelium and muscle layers of the peritoneal walls by 14 to
19 days (Figure 2
; A, C, and D). In
contrast, mice injected with v6-v10 or v8-v10 soluble CD44-transfected
cells showed little or no tumor cell attachment to the mesothelium and
no invasion of the peritoneal wall (Figure 2B)
.
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. Vector-transfected and soluble CD44
R43A-transfected cells grew rapidly for the first 10 days after
injection; however, most of these animals then became terminally ill
and were sacrificed before day 15. Consequently only one to two animals
remained for analysis at the 15-day point in these cases (in all other
situations at least six animals were analyzed at each of the five time
points). For the first 5 to 7 days, the soluble CD44 transfectants grew
significantly, but at a diminished rate compared to the controls.
Subsequently, between 10 and 15 days postinjection, their numbers
became reduced to baseline (Figure 3)
. This rise and fall in number of
cells is particularly evident for the transfectant overexpressing the
v8-v10 soluble CD44 isoform, where the ascites cell number rose to
~14 x 106
at day 7 after injection but
fell back to baseline by 15 days (Figure 3)
. None of the animals
carrying the soluble CD44 transfectants accumulated ascites and most
survived indefinitely without any signs of tumor formation in the
peritoneal wall. A small number of these animals slowly developed solid
tumors outside the peritoneum near the site of tumor cell injection,
presumably arising from cells that leaked from the peritoneum during
injection or healing.
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Wild-type and transfected TA3/St cells were harvested at 7-days
postinjection from the peritoneal cavity of mice injected with 2
x 106
cells. These cells were then analyzed by
fluorescence-activated cell sorting to establish a cell-cycle profile
for each cell line in vivo. Cells transfected with soluble
CD44 isoforms exhibited G1 arrest, whereas
wild-type, vector-transfectant, and soluble CD44 R43A-transfected cells
demonstrated a cell-cycle profile typical of an asynchronously cycling
cell population (Figure 4
; Table 2
). The proportion of cells in
G0/G1 for each population
was calculated to be approximately 30 to 40% for the various control
populations compared to 75 to 85% for the soluble CD44 transfectants
(Table 2)
.
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. In a further attempt to understand the fate of the soluble CD44-transfected TA3/St cells after inoculation into the peritoneum, we allowed the transfectants to grow in vivo for 5 days, checked that they had gone into G1 arrest, as found above, then placed the cells in culture to see whether they would recover. The soluble CD44-transfected cells failed to attach and grow, whereas vector controls grew in similar fashion to that before inoculation in vivo (data not shown). Thus we conclude that, in ascites, the soluble CD44 transfectants irreversibly entered G1 arrest, subsequently died, and were cleared from the peritoneum.
Soluble CD44-Transfected TA3/St Mammary Carcinoma Cells Have Lost the Capacity for Anchorage-Independent Growth in Vitro
It is not clear from the results obtained above whether overexpression of soluble CD44 has a direct effect on tumor cell growth or whether its effect was an indirect consequence of another event in vivo. Thus we sought additional evidence to discriminate between these two possible explanations.
First, proliferation of the soluble CD44 transfectants and control
cells was compared in monolayer culture in tissue culture wells. Each
cell line grew at approximately the same rate during a 5-day period
(Figure 5)
and exhibited similar
cell-cycle profiles (Table 2)
.
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. The wild-type,
vector-transfected, and mutant soluble CD44-transfected cells formed
many times more colonies than the soluble CD44 transfectants, and the
colonies formed by the control cells were larger than those few
colonies formed by the soluble CD44 transfectants (Table 3)
.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A particularly striking finding of this study was the failure of TA3/St
transfectants overexpressing soluble CD44 to form ascites tumors. For
each of the three soluble CD44 transfectants tested, growth took place
for several days in the peritoneum subsequent to inoculation. However,
the rate of growth of the soluble CD44 transfectants was slower than
for controls and the former cells went into G1
arrest; the control cells, however, continued to increase in number to
a point that became fatal for the host animals. Growth of the soluble
CD44-transfected cells not only ceased but the numbers of cells in the
ascites decreased back to an insignificant level. Depending on the
particular transfectant, 3 to 14 million cells per mouse were lost from
the peritoneum between 5 and 15 days postinoculation, implying that the
soluble CD44 transfectants not only went into G1
arrest but also subsequently died and were cleared from the peritoneal
cavity. In a parallel study, we have compared the ability of the
soluble CD44-transfected and vector-transfected TA3/St cells studied
herein to form metastases in the lung after intravenous
injection.17
In that study, overexpression of soluble CD44
was shown to induce apoptosis subsequent to entry of the cells into
lung tissue, and consequently formation of metastatic nodules was
dramatically inhibited. In the current study it is also probable that
the soluble CD44 transfectants became apoptotic, although we were
unable to capture the cells for analysis during the window of time
between appearance of apoptotic characteristics and clearance of the
cells from the ascites. TA3/St cells transfected with mutated soluble
CD44 (R43A) behaved like vector-transfected controls (Figure 3)
,
indicating that an hyaluronan-mediated interaction is involved in these
effects of soluble CD44 on growth.
Interestingly, the cell number reached in the peritoneum for the soluble CD44 transfectants, 5 to 7 days after inoculation, was sufficient for widespread attachment to the peritoneal wall to occur in the case of the controls. However, no attachment of soluble CD44 transfectants was detected. This observation suggests that perturbed hyaluronan-CD44 interactions lead both to altered growth characteristics within the ascites and to inhibition of peritoneal wall implantation. Previous studies have also implicated interactions between tumor cell surface CD44 and mesothelial cell-derived hyaluronan in tumor cell attachment to the peritoneal wall.4,25,26
Although decreased attachment of the soluble CD44 transfectants to the peritoneal wall is consistent with past findings, the altered growth characteristics of soluble CD44 transfectants within the ascites were not predicted. Overproduction of soluble CD44 could influence any one of several events necessary for ascites tumor growth. For example, hyaluronan binds to fibrinogen;27 thus, excess soluble CD44 may disrupt formation in the ascites of a provisional matrix rich in fibrin and hyaluronan that is important for tumor progression.2,3 Hyaluronan-CD44 interactions may also be involved in angiogenesis,28,29 in which case soluble CD44 could again be potentially disruptive. Consequently we attempted to determine whether or not perturbation of endogenous tumor cell surface hyaluronan interactions by soluble CD44 gives rise to direct inhibitory effects on tumor cell growth. We have shown that the soluble CD44 transfectants, but not the mutant soluble CD44 transfectant, have lost their ability to exhibit anchorage-independent growth in soft agar, a commonly used indicator of the transformed state of cells.30,31 Thus it would seem that endogenous hyaluronan produced by the tumor cells themselves serves an important function in anchorage-independent growth. This conclusion is supported by recent experiments showing that increased expression of hyaluronan, driven by transfection with cDNA for hyaluronan synthase, leads to acquisition of the ability to grow in soft agar.19 However, it is unlikely that the effect of overexpression of soluble CD44 is because of changes in hyaluronan synthesis because none of the parent or transfected cell lines produce large amounts of hyaluronan. Rather, it is more likely that soluble CD44 disrupts the organization of endogenous pericellular hyaluronan with respect to its interactions with CD44 or other hyaluronan-binding proteins that are important for the transformed behavior of the parent and control cells, eg, CD44-mediated docking of MMP-9 (see below). Also, in vivo, both parent and soluble CD44 transfectants induce high hyaluronan levels in surrounding stromal tissue,17 indicating that this is not the underlying difference in their behavior in vivo.
Recent work from one of our laboratories21 has demonstrated binding of MMP-9 to CD44 at the surface of TA3/St murine mammary carcinoma and MC human melanoma cells. This binding of MMP-9 to CD44 is dependent on hyaluronan-induced clustering of CD44 in the plasma membrane. Overexpression of soluble CD44 disrupts clustering of endogenous membrane CD44 and thus inhibits complex formation with MMP-9. Complex formation between CD44 and MMP-9 has also been observed in other mammary carcinoma cell lines.32 Docking of MMP-9 at the surface of TA3/St cells promotes its activity, possibly via protection from tissue inhibitors of MMPs, which in turn leads to enhanced tumor invasion and angiogenesis.21,33 Cell surface-bound MMP-9 acts, at least in part, by activating latent transforming growth factor-ß1 (TGF-ß) which then stimulates new blood vessel formation in vitro and in vivo.33 In similar fashion to solid tumors, ascites tumor growth is accompanied by extensive angiogenesis within the peritoneal wall.3 Thus, it is possible that TGF-ß, activated in the above manner by MMP-9, stimulates peritoneal angiogenesis and thus ascites tumor growth. However, promotion of angiogenesis would not explain the involvement of hyaluronan in anchorage-independent growth in vitro, as discussed above. The effects of TGF-ß on growth characteristics are complex but, in many cases, loss of responsiveness to inhibitory effects of TGF-ß is associated with malignancy.34,35 Although TGF-ß is usually thought of as a tumor suppressor, it promotes late stages of carcinoma progression.36,37 TGF-ß also induces anchorage-independent growth in fibroblasts38 and in immortalized, nontumorigenic epithelial cells.39 Thus it is conceivable that TGF-ß is at least partially responsible for the effects seen herein. Alternatively MMP-9, or another metalloproteinase bound to CD44 in an analogous manner, might cause release of a factor from the tumor cell surface that stimulates transformation and/or tumor growth directly.40 Thus, hyaluronan- and CD44-dependent presentation of a metalloproteinase at the cell surface could explain the role of hyaluronan in tumor cell growth characteristics and the effects of overexpression of soluble CD44 demonstrated in the present study. Irrespective of the underlying mechanism, our findings lead to the conclusion that hyaluronan interactions at the cell surface are, at least under some circumstances, crucial to tumor cell growth characteristics in vitro and in vivo.
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Acknowledgements |
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Footnotes |
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Supported by United States Army Medical Research and Materiel Command fellowship DAMD17–96-1–6060 (to R. M. P.), by National Institutes of Health grants CA55735 and GM48614 (to I. S.), and by National Institutes of Health grant CA73839 and a grant from Mizutani Foundation for Glycoscience (to B. P. T.).
Accepted for publication March 8, 2000.
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5ß1 integrin in normal rat kidney fibroblasts. A necessary effect for induction of anchorage-independent growth. J Biol Chem 1999, 274:30139-30145This article has been cited by other articles:
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