Increased hyaluronan at sites of attachment to mesentery by CD44 ...

Amiericanjoulrnal of Patbology, VoI. 148, A'O. 6,u/nle 1996 Cop yrigbtl American Societly for Investigativ'e Pathology

Short Communication Increased Hyaluronan at Sites of Attachment to Mesentery by CD44-Positive Mouse Ovarian and Breast Tumor Cells

Tet-Kin Yeo,* Janice A. Nagy,t Kiang-Teck Yeo,t Harold F. Dvorak,t and Bryan P. Toole* Fronm the Department oJA ulaton,l and Cellal/ar Biology,* TWas University School of Medicine, anld the Deparimenis oJ Pathology,t Beth Israel Hospital anld Harvard Medical School, Bostoni, AMassachblsetts

The mouse ovarian ascites tumor, MOT, and mammary ascites tumor, TA3/St, served as models to follow changes in hyaluronan levels during tumor growth, attachment, and invasion. Subsequent to introduction of tumor cells into the peritoneal cavity, hyaluronan accumulated intraperitoneally and at the initial sites of attachment of tumor cells and cell clumps to the mesenteric surface; the latter co-localized with sites offibrin deposition as reported earlier. Subsequently, high levels of hyaluronan accumulated throughout the interior of the mesentery. Because neither tumor cell line synthesized substantial amounts of hyaluronan in culture, the large accumulations observed in the mesenteries and ascites fluid of tumor-bearing animals most likely resultedfrom increased synthesis and secretion by peritoneal-lining mesothelial cells and/or fibroblasts in response to stimulation by the tumor cells or their products. TA3/St tumor cells were universally positivefor the hyaluronan receptor, CD44, whereas -90% of MOT tumor cells were CD44-negative. However, the great majority of MOT or TA3/St cells that initially attached to the mesentery were strongly CD44 positive. We propose that hyaluronan-rich matrix is involved in tumor cell attachment to the mesentery possibly via interaction with tumor cell surface CD44. (Am J Pathol 1996, 148:1733-1 740)

Cell-extracellular matrix interactions participate in several of the steps required for tumor cell invasion and metastasis.1'2 The polysaccharide hyaluronan is ubiquitously present in extracellular matrices in which cell migration and proliferation occur in vivo and promotes cell migration and proliferation in vitro.2 6 Metastatic tumors are frequently enriched in hyaluronan7-9 and, in vitro, tumor cells stimulate normal fibroblasts to synthesize and secrete hyaluronan.9 -12 Recently it has been shown that interactions of hyaluronan with its cell surface receptors RHAMM (receptor for hyaluronic-acid-mediated motility) and CD44 are crucial for tumor progression.13,14 Ovarian and breast cancer cells frequently exfoliate into body cavities where they induce accumulation of ascites fluid in which the tumor cells grow in suspension. Some of these cells then attach to and grow on mesenteric surfaces and, especially in the case of breast cancer, invade these tissues. In vitro, a significant subpopulation of human ovarian tumor cells has been shown to attach to mesothelium via interaction of hyaluronan with tumor cell surface CD44.15 In this study we use murine models of ovarian and breast cancer to investigate hyaluronan accumulation and the relationship of CD44 expression to hyaluronan distribution as a function of tumor cell growth and attachment to the peritoneal wall.

Supported by U.S. Public Health Service National Institutes of Health grants DE05838 and HD23681 and a grant from Anika Research to B. P. Toole, by National Institutes of Health grant CA58845 to H. F. Dvorak, and under terms of a contract from the National Foundation for Cancer Research. Accepted for publication February 21, 1996. Address reprint requests to Dr. Bryan Toole, Department of Anatomy and Cellular Biology, Tufts University School of Medicine, Boston, MA 02111.

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Materials and Methods Establishment of Tumors and Collection of Plasma, Ascites Fluid, and Mesentery Samples

serum and were cultured for an additional 18 to 24 hours in serum-free Dulbecco's minimal essential medium. Cell-free supernatants were then harvested for hyaluronan assay as above.

MOT16 and TA3/St17 tumor cells (1 x 106 cells per inoculum) were passaged weekly as ascites tumors, either in syngeneic male C3Heb/FeJ mice or in syngeneic female A/Jax mice, respectively, as described previously.18 In each experiment, four mice were injected intraperitoneally (i.p.) with 1 x 106 tumor cells suspended in 200 ,u of Hanks' balanced salt solution (HBSS). Control animals were injected with 200 ,lI of HBSS. As an additional control, some mice were injected with 2.0 ml of thioglycolate broth (Difco, Detroit, Ml) to induce inflammatory ascites. At various intervals after i.p. injection of tumor cells, platelet-poor plasma and ascites fluid were collected from tumor-bearing and control animals as described previously.18

Hyaluronan-Affinity Histochemistry and CD44 Immunohistochemistry

Quantitation of Hyaluronan The amounts of hyaluronan present in the plasma and ascites fluid samples from control and tumorbearing mice were measured using a hyaluronan radioimmunoassay kit according to the manufacturer's directions (Kabi Pharmacia Diagnostics, Uppsala, Sweden), employing 1251-labeled hyaluronic acid-binding region of aggrecan (HABR) as tracer. Briefly, 100 ,ul of hyaluronan standards and test samples were each mixed with 200 Al of 1251-labeled HABR (pre-diluted 1/30 with tracer diluent) and incubated for 60 minutes at 40C. Then 100 ,ul of hyaluronan-Sepharose was added to absorb unbound 1251-labeled HABR and the mixtures were incubated for 45 minutes at 40C..Washing solution (2 ml) was added and each mixture was centrifuged for 10 minutes at 1500 x g. The supernatants were discarded and the radioactivity in each 1251-labeled HABR-hyaluronan-Sepharose pellet was measured in a gamma counter. The amounts of hyaluronan present in the test samples were calculated from a curve generated from hyaluronan standards and expressed both as total amounts per mouse and as concentrations. Statistical significance was calculated with the Mann-Whitney test. For assay of tumor cell synthesis of hyaluronan, tumor cells were first isolated from the peritoneum and cultured in T175 flasks at 2.5 x 106 cells/ml in 100 ml of Dulbecco's minimal essential medium containing 5% fetal bovine serum. Four hours later, cells were washed three times in sterile HBSS to remove

The HABR of cartilage proteoglycan (aggrecan) binds strongly to hyaluronan and provides a useful probe for detecting hyaluronan in tissue sections.19'20 Paraffin sections (5 ,A.m) of mesentery were reacted with biotinylated HABR (a kind gift of Dr. Charles Underhill, Georgetown University), followed by incubation with avidin and color development with standard reagents from Vector Laboratories (Burlingame, CA). Two types of controls were employed. In the first, sections were incubated with testicular hyaluronidase (50 jig/ml, type IV-S, Sigma Chemical Co., St. Louis, MO) in 0.05 mol/L acetate buffer, pH 5.0, for 2 hours at 37°C before incubation with biotinylated HABR. Alternatively, biotinylated HABR was preincubated with 100 ,ug/ml hyaluronan (rooster comb, Sigma) for 30 minutes at room temperature before addition to the tissue sections. Immunohistochemical detection of CD44 was performed using a monoclonal antibody, KD27, against mouse CD44 (Developmental Hybridoma Bank NICHD, Bethesda, MD) followed by standard avidinbiotin and peroxidase reagents (Vector Laboratories). Immunohistochemistry was performed with and without pretreatment with testicular hyaluronidase. In all instances, staining of CD44 was slightly enhanced after hyaluronidase treatment, but no alteration in the pattern of staining was observed. For immunocytochemistry of tumor cells, the ascites was removed from tumor-bearing mice and centrifuged; the tumor cells were then washed thoroughly in HBSS and incubated on polylysine-coated slides for 30 minutes at 37°C. The slides were washed in phosphate-buffered saline (PBS), and the cells were fixed in 4% paraformaldehyde in PBS for 10 minutes at room temperature. After washing in PBS, the slides were air dried, reacted with the antiCD44 monoclonal antibody KD27, and processed as described above.

Results Increased Hyaluronan in Ascites Fluid of Tumor-Bearing Mice Very low levels of hyaluronan were measured in the small amounts of peritoneal fluid collected from nor-

Hyaluronan and Tumor Cell Attachment

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I

tion occurred on day 5 but at lower levels (-45 mg/I) than in MOT-bearing mice (data not shown). After day 5, total ascites hyaluronan accumulation and concentration fluctuated at somewhat lower but still significantly elevated levels until animal death, which occurred much earlier than with MOT cells (by 8 to 10 days) as the result of intraperitoneal hemorrhage. As a control, thioglycolate was injected i.p. to induce inflammatory ascites, which was collected at 4 days. Inflammatory ascites fluid contained low amounts of hyaluronan (-1 mg/I; data not shown). Plasma concentrations of hyaluronan (0.2 to 1.1 mg/I) in normal and tumor-bearing mice were insignificant compared with those found in the ascites fluid of tumor-bearing mice. The amounts of hyaluronan produced by either tumor cell line in vitro were very low, ie, 50 ,ug/l for MOT cells and 160 p,g/l for TA3/St cells over 18 to 24 hours of culture.

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per animal) were injected i.p. into syngeneic C3Heb/FeJ mice on day 0. A: The concentration (0) of hyaluronan was measured at various intervals after day 0 and compared with increasing tumor cell number (E). B: The total amount of accumulated hyaluronan was measured

and correlated with the volume ofascitesfluid accumulation (0) and with 125Ijlabeled fibrinogen (125I-F) accumulation (X). ID, injected dose. Tumor cell number, ascites fluid volume, andfibrinogen accumulation representpreviously reported data,18'2' here replotted to demonstrate their relationship to hyaluronan accumulation.

mal control C3Heb/FeJ mice (0.04 0.02 mg/I). Significantly increased levels of hyaluronan were found in ascites fluid as early as 2 days after i.p. injection of MOT tumor cells and persisted through day 14. A peak concentration of -200 mg/l was observed on day 9 (Figure 1). The total amount of hyaluronan present in tumor ascites also increased with time, along with increasing tumor cell number, vascular hyperpermeability to circulating macromolecules such as 1251-labeled fibrinogen, and increasing volume of ascites fluid. Total hyaluronan accumulation formed a broad peak between 9 and 14 days (Figure 1B); however, after day 9, as ascites fluid accumulation accelerated, hyaluronan concentration actually declined precipitously (Figure 1A). By day 16, both the concentration and the total amount of hyaluronan in MOT ascites fluid had fallen to near control levels and persisted at these low levels until animal death at -day 30. Similar changes in hyaluronan levels occurred during early periods after injection of TA3/St breast tumor cells into A/Jax mice, except that increases in amount were detected as early as day 1 after injection. Peak hyaluronan accumulation and concentra-

Increased Hyaluronan in the Mesentery during Ascites Tumor Growth Hyaluronan was detected by affinity histochemistry, using biotinylated HABR, at several intervals after i.p. injection of MOT and TA3/St cells into syngeneic mice. Only small amounts of hyaluronan staining were observed in the mesenteries of control mice, and these were confined almost entirely to the adventitia of relatively large blood vessels, along with faint staining of the mesenteric surface (Figure 2A). However, by 3 days after i.p. injection of MOT cells, abundant hyaluronan staining was observed on the mesenteric surface (Figure 2B). Mesenteric surface accumulation of hyaluronan increased further by 5 to 7 days at which times small numbers of individual or clumped tumor cells had attached to the mesentery; in addition, increasing hyaluronan deposits were found within the fatty mesentery (Figure 2, C and D). With time, cells in the adherent tumor clumps became embedded in a hyaluronan-rich meshwork, not only between tumor clumps and mesentery (Figure 2D) but also within tumor clumps (Figure 2E); earlier reports indicated that this matrix was also positive for fibrin.21 By 14 days, the mesentery was extensively infiltrated by an inflammatory (largely mononuclear) cell infiltrate; adipocytes had lost much of their lipid content and hyaluronan accumulated extensively within the mesentery and on the mesenteric surface (Figure 2F). By 21 days and later, mononuclear cell infiltration, loss of adipocyte lipid and intra-mesenteric hyaluronan accumulation increased further (Figure 2G); however, the mesenteric surface was

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Figure 2. Affinity bistochemical staining of hyaluronan in the mesenteries of control and ascites tumor-bearing mice, using biotinylated HABR. A to G: Meseniteriesfrom C3Heb1FeJ mice at day 0(A), 3 (B), 7(C and D), 12(E), 14 (F), anid 21 (G) after itjection ofMOTcells. H and l: Mesenteries from A/Jax mice at day 7 after injection qf TA.3/St cells. Sectionzs were counterstained with Meyers hematoxylin. Hyaluronan staining is brown, andl nuclei staini dark blue. A, B, C, F, and G show the progressive increase in hyaluironan deposition in the mesenteries wvith tiOne afterMOTcell infection. Arrows in C indicate clumps of MOT cevlls attached to mesentery at 7 days; otne of these clumps is seen at higher magnification in D, showing that most oJ the hyaluronan stainintg is betu'een the clump of tumor cells and the mesentery surface (arrows). E shous deposition of hyaluronan uwithin a MOT tumor clump at day 12. Arrows in H and indicate hyaluronan deposits between a cluimp of TA3/St cells and the mesentery surface and at the leading edge of TA3/St cell invasion, respectively; hyaluronani staininig is also associated wvith some cells within the invading tumor mass in 1, especially toward the mesenteric suirface. v, vessels uith hyaluronan-positive adventitia. Bar, 100 p.m (A to C antd F), 50 gum (D and G to 1), ancd 25 u m (E).


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now covered with a multicell-thick layer of adherent MOT tumor cells, many of which appeared to be injured or dead by morphological criteria, and surface hyaluronan was substantially reduced in amount. MOT tumor cells did not invade the mesentery to any significant extent. Similar results were found in mice injected i.p. with TA3/St tumor cells (Figure 2H). However, events proceeded more quickly and both hyaluronan accumulation and tumor cell attachment to the mesentery were evident as early as 1 day after i.p. tumor cell injection. Unlike their MOT counterparts, many TA3/St tumor cells invaded the mesentery. Hyaluronan was deposited at the interface between invading tumor cells and the surrounding host tissue, as well as between many of the cells within the tumor mass (Figure 21).

Expression of CD44 during Tumor Cell Attachment to Mesentery Only a minority (-10%) of MOT tumor cells freshly harvested from the peritoneal cavities of syngeneic mice stained for CD44; the great majority of otherwise indistinguishable tumor cells were CD44 negative (Figure 3A). Mesenteries from control animals did not show significant immunoreactivity except for nerve bundles, scattered adventitial cells around small muscular arteries and veins, and occasional lymphocytes (Figure 3C); the mesenteric surfaces were entirely negative. This staining pattern remained unchanged for the first 3 days after i.p. injection of MOT tumor cells. However, at 5 to 7 days, CD44-positive MOT cells began to attach to the mesentery (Figure 3, D and E). CD44 staining was also observed in many endothelial cells that lined amuscular mesenteric microvessels and in scattered infiltrating host lymphocytes. Subsequently, larger numbers of MOT cells attached to the mesentery, forming a nearly continuous layer of cells that coated the mesenteric surface to different depths; many of these tumor cells were CD44 negative (Figure 3F). In contrast to MOT cells, almost all TA3/St cells were CD44 positive before injection (Figure 3B). Not surprisingly, therefore, all of the TA3/St cells that attached to the mesenteric surface initially were also strongly CD44 positive (Figure 3G). However, there was considerable heterogeneity of staining for CD44 among the TA3/St cells that invaded the mesentery (Figure 3H).

Discussion When MOT ovarian or TA3/St breast tumor cells are injected into the peritoneal cavity, the tumor cells

subsequently behave in a manner that closely mimics that which occurs in human ovarian and breast cancer, respectively.18'22'23 Our data indicate that ascites growth of either ovarian (MOT) or mammary (TA3/St) tumor cell lines leads to a dramatic accumulation of hyaluronan in both ascites fluid and mesentery. Accumulation of hyaluronan followed kinetics that paralleled those of tumor cell growth and attachment to mesentery. These correlations, the remarkable co-localization of newly deposited hyaluronan with adherent tumor cells, and the preferential attachment of CD44-positive MOT tumor cells to mesentery strongly suggest that hyaluronan participates in tumor cell adhesion. What is the cellular source of the large amounts of hyaluronan that accumulated in the peritoneal fluid and mesenteries of ascites tumor-bearing mice? The tumor cells themselves are an obvious possibility, but in vitro, neither tumor cell line produced sufficient hyaluronan to account for the large amounts present in tumor ascites in vivo. Therefore, if tumor cells were directly responsible for the large amounts of hyaluronan present in tumor ascites, factors must have been present in vivo that greatly stimulated their synthetic capacity. Although this is possible, there are no published data that support such an expectation. A more likely possibility, and one for which there is published support, is that the tumor cells produced soluble effectors, eg, fibroblast growth factor, platelet-derived growth factor, or transforming growth factor-f and/or cell surface-associated factors that stimulate nonmalignant host cells to produce elevated amounts of hyaluronan.9- 12,24-26 One such study has demonstrated increased hyaluronan production in normal mesothelial cells cultured in vitro in response to factors secreted by tumor cells, in this case, mesothelioma cells.12 The simplest explanation of our data, then, is that MOT and TA3/St tumor cells stimulated hyaluronan production by peritoneal-lining mesothelial cells or mesenteric fibroblasts and that some of the newly synthesized hyaluronan was secreted into the ascites fluid and some was deposited locally at the mesenteric surface and subsequently throughout the mesentery. One way in which hyaluronan might contribute to tumor cell attachment arises from the observation that fibrin is also deposited at sites of tumor cell attachment and intercellular clumping in these and other tumor systems.21 Fibrin arises from the local clotting of plasma fibrinogen that has extravasated from peritoneal-lining blood vessels rendered hyperpermeable by a tumor-secreted cytokine, vascular permeability factor. 1821'27 Hyaluronan is reported to bind human fibrinogen and become incorporated

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Figure 3. Immunohistochemical staining for CD44 uith monoclonal antibody KD27. A and B: MOT(A) and TA,3/St tumor cells (B) derived from ascites. C: Control mesentery utith stainin1g oJ'f erve bundle (arrow) anid occasiontal lymphocytes. D and E: Mesentery 7 day.s after ip. inijection of I X Id MOT tuimor cells into C3He1FebeJ mouse. Attached tumor cells (arrows) are CD44 positive. F: Mesentery 12 days after injection of MOT cells. Note large iiii nbers o/ftumor cells attached to mesentery, the majority of uhich are CD44 tegative. G and H: Meseniteryfron A//ax mouse 7 days after ip. injectioni of 1 x 10' TA 3/St tumor cells. Note large numbers of CD44-positive tumnuor cells attached to the mesen7tery (G). In H, TA-3/St ttumor cells have invaded the mesenter. Tumor cells at the inivading edge (arrows), as aell as many wvithin the tunmor mass, are stronggly CD44 positive. The sections uwere counterstained with Meyer's bematoxylin. Positive stainitng Jbr hyalutrontan is broutn; nuclei stain dark bltue. Bar, 50 ,um (A, B, D, anid F to H), 100 ,u.n (C), anid 25 p.nm (E).

into the fibrin network.28 Although hyaluronan binds to fibrinogen from several species, it does not bind well to rat fibrinogen, and neither mouse fibrinogen nor fibrin has been tested.29 Nevertheless, it is possible that the clumps of tumor cells adherent to the mesenteric surface after i.p. tumor cell injection are

incorporated into a hyaluronan-fibrin latticework. According to this view, hyaluronan present in increased amounts in ascites fluid would concentrate in fibrin deposits because of its affinity for fibrin. This explanation is also consistent with the hyaluronan-mediated adherence of macrophages to the peritoneal


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surface in peritoneal delayed hypersensitivity reactions; in these reactions also the microvasculature is rendered hyperpermeable by vascular permeability factor with consequent extravasation of fibrinogen and clotting30 and hyaluronan accumulation.31'32 Whatever the mechanism, the elevated concentrations of mesenteric hyaluronan most likely facilitate tumor cell attachment. Past evidence has demonstrated that attachment of human ovarian tumor cell lines to mesothelium in vitro is mediated in part by interaction of tumor cell surface CD44 with mesothelium-derived hyaluronan.15 In the present study, most if not all of the tumor cells, both MOT and TA3/St, that attached to the mesentery during early stages of invasion were CD44 positive. Thus, it is likely that attachment of tumor cells to the mesentery surface was due at least in part to hyaluronan-CD44 interaction. It is also possible that interaction between hyaluronan and CD44positive tumor cells contributed to the formation of tumor cell aggregates that became attached to the mesenteric surface, as multivalent interaction of hyaluronan with CD44 on the surface of adjacent cells is known to cause aggregation of several cell types.3,2033,34 Also of interest was our observation that most of the MOT cells present in ascites were CD44 negative, suggesting that the relatively small proportion of CD44-positive cells (less than 10%) attached preferentially as compared with CD44-negative tumor cells. This may in turn explain the greater efficiency of attachment and invasion exhibited by the TA3/St cells, more than 90% of which expressed CD44 on their surface. A striking difference between the TA3/St and MOT cells is the highly invasive character of the former. As hyaluronan accumulation occurred in both cases, it is less clear whether hyaluronan facilitates invasion into the mesentery as well as initial attachment. However, our observation that a thin rim of hyaluronan was deposited at the interface between invading TA3/St cells and surrounding host tissue supports previous studies, which have convincingly demonstrated involvement of interactions between hyaluronan and CD44 or RHAMM in tumor progression in vivo and in tumor or transformed cell migration in

ctre2-5, 13, 14,35,36 In summary, our results strongly implicate interactions between hyaluronan and CD44 in the attachment of two very different ascites tumor cell systems, MOT and TA3/St, to the mesentery. Because fibrin deposits co-localize with hyaluronan at sites of tumor cell-mesentery interaction, fibrin may also play a role in tumor cell aggregation and attachment to mesentery, perhaps because of an interaction between

hyaluronan and fibrin.28 The tumor cells most likely stimulate nonmalignant mesenteric mesothelial cells and/or fibroblasts to synthesize hyaluronan, either by secreting cytokines12'26 or by direct interactions with mesenteric cells.9'10 Thus, the elevated hyaluronan concentrations that develop in mesentery may facilitate attachment of the tumor cells to the mesentery surface via interaction with CD44,15 incorporation of tumor cells into a hyaluronan- and fibrin-rich latticework that immobilizes the tumor cells at the mesentery surface,21 and further tumor progression via interactions with tumor cell surface CD445'13 or RHAMM.3536

Acknowledgments We thank Ms. Colleen Keyes, Heidi Kraus, Michelle Meyers, and Ying Zhou for excellent technical support.

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