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human FME melanoma cells after exposure to hypoxia and acidic pH, alone or in combination. IntJ Cancer 43:350-355, 1989 (33) BROWN JM: Evidence for acutely hypoxic cells in mouse tumours, and a possible mechanism for reoxygenation. Br J Radiol 52:650-656, 1979 (34) KALLMAN RF: The phenomenon of reoxygenation and its implications for fractionated radiotherapy. Radiology 105:135-142, 1972 (35) RAMSAY J, SUIT HD, SEDLACEK R: Experimental studies on the incidence of

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facilitate the appearance of metastases. Clin Exp Metastasis 6:121-129, 1988 TANNOCK IF: The relationship between cell proliferation and the vascular system in a transplanted mouse mammary tumour. Br J Cancer 22:258-273, 1968 WATANABE N, NIITSU Y, UMENO H, ET AL: Toxic effect of tumor necrosis factor on tumor vasculature in mice. Cancer Res 48:2179-2183, 1988 DVORAK HF, GRESSER I: Microvascular injury in pathogenesis of interferoninduced necrosis of subcutaneous tumors in mice. J Natl Cancer Inst 81:497-502, 1989 Zwi LJ, BAGULEY BC, GAVIN JB, ET AL: Blood flow failure as a major determinant in the antitumor action of flavone acetic acid. J Natl Cancer Inst 81:1005-1013, 1989 GUICHARD M: Keynote address: Chemical manipulations of tissue oxygenation for therapeutic benefit. Int J Radiat Oncol Biol Phys 16:1125-1130, 1989

Richard B. Hostetter, hazel B. Augustus, Ramy Mankarious, Kefung Chi, Domonic Fan, Carol Toth, Peter Thomas, J. Milburn Jessup*

Although the serum level of carcinoembryonic antigen (CEA) is directly associated with a poor prognosis in human colorectal carcinoma (CRC), its function is obscure. As a member of the immunoglobulin supergene family, CEA may be involved with intercellular recognition and binding and facilitate attachment of CRC to sites of metastasis. In an experimental metastasis model of CRC in athymic nude mice, a systemic injection of CEA enhanced experimental liver metastasis and implantation in liver by a weakly metastatic CRC. This CRC also selectively bound to CEA that was attached to plastic. Thus, CEA may function as an attachment factor for CRC. [J Natl Cancer Inst 82:380-385, 1990]

Carcinoembryonic antigen (CEA), an oncofetal glycoprotein, was originally described in 1965 by Gold and Freeman (/) as a tumor-associated colon cancer antigen. Now, 24 years later, its function still is not known (2). The serum concentration of CEA, however, is an important prognostic indicator because a preoperative serum CEA level greater than 5 ng/mL correlates with significantly shorter disease-free survival (3,4) in Dukes' stage B-C patients. Accordingly, micrometastases are more likely to be present in patients whose serum CEA level is increased. If CEA does not participate in the biology of a malignant neoplasm, then it may be only a passive indicator of an advanced stage of disease. On the other hand, since the nucleotide sequence of the CEA gene has homojogy with members of the immunoglobulin supergene family (5), CEA may affect cellular recognition and interaction. As a potential attachment factor, it may 80

enhance the binding of tumor cells to normal cells as they pass through the microcirculation of distant organs. When CEA is cleared by the liver, itfirstbinds to Kupffer cell membranes through a specific receptor where terminal sialic acids are removed before transfer to hepatocytes before it is degraded (6-8). During this process, CEA and asialo-CEA are displayed on liver cell membranes. Lotan and Raz (9) have reported that tumor cell-surface glycoproteins may cause homotypic aggregation of cells, and Benchimol et al. (70) have demonstrated that CEA may cause homotypic aggregation of colorectal carcinoma (CRC). Thus, CRC in hepatic sinusoids may bind to liver cells that display CEA on their membranes, perhaps through homotypic aggregation. We tested this hypothesis in a nude mouse model of experimental metastasis that has been developed as a model for liver metastasis of CRC (11,12). CEA was injected in-

Received July 25, 1989; revised October 18, 1989; accepted November 17, 1989. Supported by Public Health Service grants CA-09599, CA-44586, CA-44704, and CA-42587 (National Cancer Institute), and RR-5511-23 (Division of Research Resources), National Institutes of Health, Department of Health and Human Services; and by a grant from the Joseph and Jesse Crump Cancer Center of the Kelsey-Seybold Foundation. R. B. Hostetter, L. B. Augustus, R. Mankarious, K. Chi, J. M. Jessup (Department of Surgery), D. Fan (Department of Cell Biology), The University of Texas M. D. Anderson Cancer Center, Houston, TX. C. Toth, P. Thomas, Department of Surgery, Harvard Medical School, New England Deaconess Hospital, Boston, MA. * Correspondence to: J. Milburn Jessup, M.D., Department of Surgery, New England Deaconess Hospital, 110 Francis St., Ste. 3-A, Boston, MA 02215.

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Carcinoembryonic Antigen as a Selective Enhancer of Colorectal Cancer Metastasis

travenously (IV) to saturate liver cells with CEA prior to the intrasplenic injection of CRC. Both experimental metastasis and the implantation of a weakly metastatic CRC, KM-12c, in the liver of nude mice were enhanced by CEA pretreatment. In contrast, experimental metastasis and implantation of either a nonmetastatic or a highly metastatic CRC were not enhanced by pretreatment with CEA. Only KM-12c bound to CEA that was attached to plastic, although all CRC demonstrated apparent specific binding of CEA that was free in solution. Thus, CEA may facilitate attachment of certain CRC to cells at metastatic sites.

Materials and Methods All experimental protocols were approved by the animal resources committee. Animals

Cell Lines All CRC cell lines were established at the M.D. Anderson Cancer Center as previously described (11,13). mHC 1410 was derived from a liver metastasis and is highly metastatic. Both HC 2998 and KM-12c were established from Dukes' stage B2 primary colon carcinomas; the former is nonmetastatic, and the latter is weakly metastatic. CEA production by confluent monolayers of the CRC lines was comparable (10-20 ng/mL) when the conditioned culture media were tested in commercial CEA assay systems (Roche EIA; Hoffmann-La Roche, Nutley, NJ). All lines were mycoplasma free according to Hoechst dye staining and were cultured in complete modified Eagle medium (MEM) with 10% fetal bovine serum (FBS) (GIBCO, Grand Island, NY).

Metastatic Potential Assay Experimental metastatic potential was assessed by a modification of the method of Giavazzi et al. (12). In brief, groups of five mice were initially given dorsal tail vein injections (0.2 mL) of either Hanks' balanced salt solution (HBSS), bovine serum albumin (BSA) (200 Mg/mL in HBSS), low-dose CEA (25-50 Mg/mL in HBSS), or high-dose CEA (200-300 Mg/mL in HBSS). Thirty minutes later, the mice were anesthetized and given intrasplenic injections (5 X 105 tumor cells in 0.05 mL HBSS) by laparotomy. All groups were killed when mice in any group became moribund. The presence of liver colonies was determined by macroscopic inspection. In Vivo Tumor Cell Binding Assay Mice were injected in a manner identical to that of the metastatic potential assay except that the tumor cells were radiolabeled with [l25I]iododeoxyuridine. CRC implantation into mouse liver was measured in vivo using the assay described by Fidler and Hart (15). In brief, cultures of KM-12c, HC 2998, and mHC 1410 cells in log phase were pulse labeled for 18 hours at 37 °C with 0.3 /uCi/mL of [125I]iododeoxyuridine ([ l25 I]IdUrd, specific radioactivity 200 Ci/mmol, New England Nuclear) as previously described (76). The cells were washed twice in complete medium with 1% FBS and once in HBSS to remove unbound radioactivity, harvested by a brief trypsinization (0.25% trypsin and 0.02% EDTA for 1 min at 37 °C), and then resuspended in HBSS at 1 X 107 cells/mL for intrasplenic injection. The labeling efficiency varied with cell lines and experimental conditions. In this study, the labeling efficiencies for the cell lines KM-12c, HC 2998, and mHC 1410 were 0.2 cpm/cell, 0.3 cpm/cell, and 0.4 cpm/cell, respectively. The livers, spleens, and sera (the latter obtained by cardiac puncture) were obtained from these animals at 4 hours and counted for 125I with a gamma counter (LKB Instruments, Washington, DC).

Carcinoembryonic Antigen The CEA used in these experiments was purified from a single CRC hepatic metastasis, as previously described (8). The preparation was characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), high-pressure liquid chromatography analysis, and activity in commercial CEA assay systems. It appeared as a single band of 180 to 200 kDa on Western blot, and its behavior in experimental animals is known (8). Radiolabeling of CEA CEA (100 Mg) was radiolabeled with 1 mCi Na 125I (17 Ci/mg) (New England Nuclear, Boston, MA), using the chloramine T procedure (14). Bound label was separated from unbound by chromatography on Sephadex G-25. The product was examined by SDS-PAGE (7.5% gel). A single band of radiolabeled material was seen by autoradiography as Vol. 82, No. 5, March 7, 1990

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Six- to 8-week-old BALB/c athymic nude mice were obtained from the Animal Production Area of the National Cancer Institute-Frederick Cancer Research Facility. Mice had free access to food and water and were housed five mice per cage in laminar flow cabinets under specific-pathogen-free conditions in the barrier facilities of The University of Texas M.D. Anderson Cancer Center. The mice were age and sex matched for each experiment.

corresponding to an Mr of approximately 180,000. The labeled CEA had a specific radioactivity of approximately 6 mCi/mg.

Binding of Radiolabeled CEA to Immobilized Tumor Cells Log-phase cultures of KM-12c, HC 2998, and mHC 1410 were harvested by brief trypsinization, resuspended in MEM (10% FBS), plated at 105 cells/well in 96-well plates (Costar, Cambridge, MA), and incubated at 37 °C for 24 hours to allow the tumor cells to attach firmly to the plastic. The unbound cells were aspirated, leaving wells that were 95%-100% confluent. Radiolabeled CEA was then added at 80 ng/well (10 6 cpm/well) in quadruplicate. After a 90-minute incubation at 4 °C, the wells were washed three times with HBSS. Cotton-tipped applicators were then used to remove all adherent cells by wiping the wells and were examined for 125I content by gamma counting. Competitive inhibition of binding was tested by adding a 100-fold excess of nonradioactive CEA (8 ng/weW) simultaneously with the radioactive CEA (80 ng/well). ARTICLES


Binding of Tumor Cells to Immobilized CEA

Enhancement of Liver Metastasis With Exogenous CEA Previous studies demonstrated that Kupffer cells and hepatocytes bound CEA with maximal display of membranebound CEA 30 minutes after IV injection (S). When 40 Mg of CEA was injected IV into groups of three athymic nude mice that were then killed at different times, CEA was detectable in the serum for as long as 48 hours: 3,187 ± 579 ng/mL at 30 minutes, 2,280 ± 348 ng/mL at 60 minutes, 356 ± 38 ng/mL at 4 hours, 12 ± 1.3 ng/mL at 24 hours, and 1.9 ± 0.3 ng/mL at 48 hours. To demonstrate the effects of CEA on metastatic potential, we injected CRC cells into the spleens of nude mice 30 minutes after graded doses of CEA were injected IV. KM-12c (low metastatic potential) formed liver colonies in 2% of the 45 mice injected with 5 X 105 KM-12c cells and HBSS. In contrast, 82

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Figure 1. Effect of CEA upon experimental metastatic potential of CRC. BALB/c athymic nude mice received 0.2-mL IV injections in a dorsal tail vein with HBSS, BSA (40 fig per mouse), low-dose 'CEA (5-10 ng per mouse), or high-dose CEA (40-60 ng per mouse). Thirty minutes later 5 X 105 KM-!2c, HC 2998, or mHC 1410 CRC cells were injected intrasplenically. Approximately 6 wk later mice were killed and their livers examined for colonies. Results are expressed as the percentage of the cumulative No. of mice with liver colonies in seven experiments divided by the total No. of mice injected. Both low-dose and high-dose CEA pretreatment enhanced the production of liver colonies by KM-12c but not by HC 2998 or mHC 1410. *P < .01 and **P < .001 (compared to the HBSS control using Fisher's exact test).

Journal of the National Cancer Institute

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when 5-10 ng of CEA (low-dose CEA) was injected IV, CEA was immobilized on 96-well culture plates (Costar), 33% of 15 mice formed experimental metastases. Similarly, and tumor cells were allowed to adhere in a modifica- the injection of 40-60 ng of CEA (high-dose CEA) prometastases in 48% of 25 mice that retion of the method for adhesion to fibronectin described by duced experimental 5 ceived 5 X 10 KM-12c cells intrasplenically (fig. 1). Of the McCarthy et al. (17). CEA was dried onto 96-well plates other two cell lines, HC 2998 was nonmetastatic, and CEA (Costar) at a concentration of 20 ng/well in double-distilled did not cause it to produce metastases. Similarly, CEA preH2O (ddH2O) for 12 hours at 37 °C. BSA and alpha, acid treatment did not shorten the survival of mice bearing mHC glycoprotein (AGP) were attached in a manner similar to that 1410, which was metastatic in 100% of control mice (data of controls. The free binding sites on the plastic were blocked not shown). Thus, CEA enhanced the metastatic potential of with 4% BSA for 2 hours at 37 °C. The tumor cell lines a weakly metastatic CRC in the nude mouse model but did were harvested at log phase with rubber spatulas and were not make a nonmetastatic CRC metastatic or make a highly plated in wells at concentrations of 1,000 cells/well. After a metastatic CRC more aggressive. 1-hour incubation, cells were fixed in 1% paraformaldehyde We attempted to determine whether CRC attached to and 1% sucrose. The unbound cells were removed by three CEA on liver cells or bound CEA in circulation prior to washes with HBSS, and the cells were counted in each well implanting in liver. By incubation of CRC with CEA prior in a coded manner. An enzyme-linked immunosorbent assay to intrasplenic injection, CRC would be coated with CEA demonstrated that the CEA was immobilized on the plastic. and their binding to liver cells might be facilitated. InHomotypic Aggregation Assay terestingly, the injection of KM-12c cells that had been preincubated with CEA in vitro increased the percentage Single-cell suspensions of KM-12c, HC 2998, and mHC of mice that formed liver colonies by approximately 20% 1410 were obtained from log-phase cultures by brief (table 1). CEA pretreatment in vivo, however, added to 2+ trypsinization and were resuspended in HBSS (with Ca 2+ this effect because 89% of mice that received CEA IV and Mg ). One thousand cells per well were suspended in and CEA-pretreated KM-12c cells developed liver colonies, 96-well culture plates (Costar) at 37 °C on a rotating platcompared to 56% and 70% of mice that received either form. CEA or BSA was added simultaneously to the wells CEA-preincubated cells or CEA IV, respectively (table 1). over a concentration of 5-20 /xg/well. The plates were exThis experiment is complicated by an unusually high numamined under the microscope after 90 minutes. Tumor cell ber of liver colonies in the control mice and by the complexaggregates of more than six cells were counted in a coded ity of the interpretation of the experiment. For this reason, manner. direct binding studies were begun. Statistics CEA Enhances Tumor Cell Attachment in the Liver The two-tailed Fisher's exact test was used to assess the CRC were labeled with [125I]IdUrd and injected inmetastatic potential assay. The unpaired Student's Mest was used to analyze the effects of CEA upon radiolabeled tumor trasplenically into athymic nude mice to determine whether cell implantation in nude mouse liver. The means for CRC CEA affected the attachment of circulating tumor cells to binding either to free CEA or to immobilized CEA were liver parenchyma. The mice had been treated 30 minutes earranked and analyzed by analysis of variance (ANOVA), with significance between individual group means tested with the Newman-Keuls test. The significance level was set at 5% for 100each analysis.

Table 1. Effect of CEA on experimental metastasis KM-12c treatment* In vitro


HBSS CEA 40 Mg HBSS CEA 40 fig

HBSS HBSS CEA 40 fig CEA 40 fig

No. of mice with liver colonies

3 5 7 8

Table 2. CEA enhances attachment of CRC cells to the livers of nude mice

Total No. of mice (%)

8(38) 9(56) 10(70)

9,'•> of injected cells inmouse liver* Pretreatment HBSS CEA


HC 2998

mHC 1410

27 ±4 57 ±8

5 ±2 8±4

22 ± 3 26 ± 3

9 ( 8 9 ) ? = 0.41t •Groups of three nude mice were pretreated with either HBSS or CEA (40 fig per mouse) IV followed by intrasplenic injections of radiolabeled tumor cells. Four hr later, the animals were killed, and the livers were counted in a gamma counter. Mean ± SEM of the % of CRC cells that were injected intrasplenically. The % of injected cells was calculated by dividing the cpm in liver by the No. of cpm per cell and then dividing that result by the total No. of cells injected (5 X 105 cells).

Her with either HBSS or CEA, were killed 4 hours later to examine the implantation phase of metastasis, and their spleens, livers, and sera were analyzed for 125I. All the mice had comparable amounts of radioactivity in their sera and spleens (fig. 2A and 2B). In contrast, CEA enhanced the attachment of both KM-12c and mHC 1410 to mouse liver since the

amount of 125I was significantly increased in CEA-pretreated mice (fig. 2C). CEA did not increase the number of HC 2998 cells in mouse liver. When calculated on a per-cell basis, CEA had a greater effect on the percentage of KM-12c cells retained in mouse liver than the other two cell lines (table 2). Apparent Specific Binding of CEA to Tumor Cells

CRC must bind CEA if it is to function as an attachment factor. [125I]CEA was added at 4 °C to CRC monolayers in 96-well plates. The cells were incubated at 4 °C for 90 minutes, washed, and the amount of [125I]CEA determined in a gamma counter. KM-12c, HC 2998, and mHC 1410 bound 0.3%-0.4% of CEA at 4 °C. Similar binding was also noted at 37 °C. Titration curves for cell number and amount of CEA demonstrated a linear dose-response over the range of 4.5-90 ng/well of CEA with saturation above 90 ng/well (data not shown). Competitive inhibition of binding by excess nonradioactive CEA was also demonstrated (fig. 3). Since a 100-fold excess of nonradioactive CEA causes a 46%54% reduction in the binding of [125I]CEA to CRC, CRC demonstrate apparent specific binding of CEA. Further, the binding of CEA free in solution appears to be independent


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Carcinoembryonic antigen as a selective enhancer of colorectal cancer metastasis.

Although the serum level of carcinoembryonic antigen (CEA) is directly associated with a poor prognosis in human colorectal carcinoma (CRC), its funct...
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