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[CANCER RESEARCH 47, 1398-1406, March 1, 1987]
An Animal Model for Colon Cancer Metastasis: Establishment andCharacterization of Murine Cell Lines with EnhancedLiver-metastasizing Ability1
Robert S. Bresalier,2 Erkki S. Hujanen, Steven E. Raper, F. Joseph Roll, Steven H. Itzkowitz, George R. Martin, and
Young S. KimGaslroinleslinal Research [R. S. B., S. H. I., Y. S. KJ, Cell Biology [S. E. R], and Liver Center [F. J. R.] Laboratories, Veterans Administration Medical Center;Departments of Medicine ¡R.S. B., F. J. R., S. H. I., Y. S. KJ, Surgery ¡S.E. R.], and Pathology [Y. S. K.J, the University of California, San Francisco, California94121; and Laboratory of Developmental Biology and Anomalies [E. S. H., C. R. MJ, National Institute of Dental Research, NIH, Bethesda, Maryland 20892
ABSTRACT
A detailed understanding of the pathogenesis of colon cancer metastasis has been hindered by the lack of appropriate animal models whichaccurately reflect events in this complex process. An animal model forcolon cancer metastasis is described in which spontaneously metastasiz-
ing colonie tumors are formed after injection of murine colon cancer cellsinto the cecal wall of BALB/c mice. Using this model, tumor cells withdifferent liver-metastasizing potential were selected and shown to possess
several properties known to be associated with other metastatic cell lines.The ability of tumor cells to invade a reconstituted basement membraneand to secrete type IV collagenase was directly proportional to theirmetastatic ability. In addition, liver-metastasizing cells preferentially
migrated toward liver extracts in a Boyden chamber assay, as comparedto extracts of brain or lung, and adhered rapidly to highly purified hepaticsinusoidal endothelial cells versus hepatic parenchyma! cells in vitro.This model may thus be useful for studying many aspects of the pathogenesis of colon cancer metastasis.
Received 6/27/86; revised 11/11/86; accepted 12/1/86.The costs of publication of this article were defrayed in part by the payment
of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1This work was supported by USPHS Grant CA14905 from the NationalCancer Institute, by Grant PDT-293 from the American Cancer Society, and bythe Research Service of the Veterans Administration. Presented in part at theannual meeting of the American Gastroenterological Association in New YorkCity, New York, May 1985.
*To whom requests for reprints should be addressed, at G. I. Research Lab(151M2), VA Medical Center, 4150 Clement St., San Francisco, CA 94121.
even mammary carcinoma, in the case of colon cancer thesesystems may bypass many of the complex processes of themetastatic cascade.
Parental cell line 51B was established in this laboratory from trans-phiniable murine colon carcinoma 51. The original transplantabletumor, established by Dr. T. H. Corbe«(22) by repeated s.c. injectionwith 1,2 dimethylhydrazine, forms grade 11-111carcinomas when passaged in syngeneic animals. Unlike the original transplantable tumorwhich was reponed to metastasize to regional lymph nodes and lungsfrom the s.c. site in greater than 80% of animals tested, 51B has lowermetastatic ability when injected s.c. into syngeneic BALB/c mice (seebelow). Subsequent metastatic derivatives of cell line 51B were established as described below.
Preparation of Cell Lines for InjectionAll cell lines were grown and maintained in DM1M ' supplemented
with 10% fetal bovine serum, penicillin (100 units/ml), streptomycin(100 Mg/ml), and a 7% COi environment. Early passage cell lines(passagedfewerthan 15times) wereused for all studies unless otherwisenoted. Confluent cultures were harvested by brief trypsinization (0.05%trypsin-0.02% EDTA in Hanks' balanced salt solution without calciumand magnesium), washed several times with CMF, and resuspended ata final concentration of 5 x IO7cells/ml in serum-free medium. Thepresence of single cell suspensions was continued by phase contrastmicroscopy,and cell viabilitywas determined by trypan blue exclusion.
Cecal Injection
BALB/c mice (15- to 18-g females obtained from Simonsen Laboratories, Gilroy, CA) were anaesthetized with methoxyfluorane by
inhalation, and the abdomen was prepared in a sterile fashion. A smallabdominal incision was made, and the cecum was identified and isolatedbetween sterile gauze; 5x10' viable tumor cells in 0.1 ml serum-free
at this time cecal tumors of approximately 0.5 cm were present withoutperitoneal spread of tumor. Organs including cecum, liver, mesentericlymph nodes, and lungs were divided, and paired specimens wereprocessed for histológica!examination and tissue culture.
Tissue sections of liver were immersed for 2 h in a fixature consistingof 2.5% glutaraldehyde-0.8% paraformaldehyde in 0.2 M sodium bicarbonate buffer. Tissues were osmicated with 1% osmium tetroxidecontaining 1.5% potassium cyanide for 90 min, washed in 0.2 Msodiumbicarbonate buffer for 10 min, and then were dehydrated in ethanol andembedded in Epon. Sections were stained with lead citrate and uranylacetate and examined in a Philips EM 300 microscope (Philips Electronic Instruments, Inc., Mahwah, NJ).
Culture of Metastatic Foci and Establishment of Cell Lines with Enhanced Metastatic Activity
The invasive potential of tumor cells was determined with an in vitroinvasion assay (24). Briefly, cells were tested for their ability to penetratethrough a reconstituted basement membrane matrix which was composed of purified bovine tendon type I collagen [0.2 nig/matrix (25)],type IV collagen [0.2 mg/matrix (26)], and laminin [0.2 mg/matrix(27)] compressed to form a 200- to 600-//m thick mat (Helitrex Corp.,Princeton, NJ). The barrier was placed into a modified Boyden chamberdirectly onto a type IV collagen-coated Nuclepore filter (5 ¿igprotein/filter; pore size, 1.0 ¿im).
A suspension of tumor cells (7 x 10s) in DMEM containing 2%acid-treated NuSera (28) and 0.5% BSA was introduced into the uppercompartment and DMEM containing 0.5% BSA was placed into thelower compartment of the chamber. For chemoinvasion assays (29),the medium in the lower compartment also contained partially purifiedorgan-derived attractants (prepared as in the chemotaxis assay below).Similar extracts have been used in previous studies of tumor cellchemoinvasion and chemotaxis (24, 29). After 24-36 h of incubationat 37°Cin 95% air and 5% CO2, the barrier was separated from the
underlying Nuclepore filter. The cells that had penetrated the matrixand attached to the upper surface of the Nuclepore filter were detachedwith trypsin/EDTA (0.05/0.1%) solution and counted using an electronic counter. The data are expressed as the percentage of cells whichpenetrated the matrix ±SD.All assays were performed at least intriplicate.
Chemotaxis Assay
To assess the tendency of tumor cells to be directionally attracted toorgan-specific factors, they were studied using a modified Boydenchamber assay (30). Brain, liver, and lung tissues were removed from8- to 10-week-old C57/BL female mice, and partially purified extractswere prepared as previously described (29), with slight modification.
Briefly, tissues were washed three times in DMEM containing 50Mg/mlgentamicin. After homogenization at 4°Cin a buffer ( 1 ml buffer/1 g tissue) containing 0.05 M Tris-HCl, pH 7.4, 2 M guanidine HC1,0.5 MNaCl, 5 mMW-ethylmaleimide, 5 mM EDTA, 1 mM phenylmeth-ylsulfonylfluoride, and 50 ng/m\ gentamicin, homogenates were extracted by shaking with buffer for 24 h at 4°C.Extracts were then
centrifuged at 100,000 x g for 4 h and the supernatant fractions dialyzedagainst phosphate-buffered saline. After an ammonium sulfate precipitation (0-60%), supernatants (dialyzed versus DMEM) were tested fortheir chemotactic activity. The protein level in the extracts was determined by the method of Lowry et al. (31).
For the chemotaxis assay, polyvinylpyrrolidone-free Nucleporepolycarbonate filters (pore diameter, 8 ftm; Neuroprobe, Inc.) werecoated with type IV collagen (5 ¿¿g/fi'ter)and placed into a modifiedBoyden chamber to separate the upper and lower compartments. Tissueextracts (10% solutions) in DMEM containing 0.5% BSA were introduced into the lower compartment and tumor cells (5 x 10s, detached
with 0.1% EDTA) in DMEM containing 0.5% BSA were placed intothe upper compartment. After a 5-h incubation at 37°C,the filters were
removed and stained in Hemacolor solutions 1, 2, and 3 (Harelco,Gibbstown, NJ). Filters were then placed top side up on glass slidesand cells that had not migrated were removed with cotton swabs.Migration of cells was quantitated by counting eight randomly selectedhigh power (x 400) fields/filter. Results are expressed as the averagenumber of migrated cells per high power field. All assays were done intriplicate.
Type IV Collagenase Assay
The type IV collagen-degrading metalloprotease activity secreted byselected tumor cells was measured as previously described (32, 33).Briefly, nearly confluent tumor cell monolayers were washed threetimes with DMEM and subsequently incubated in serum-free medium(DMEM) at 37°Cwith 95% air/5% CO2. After a 24-h incubation,
medium was collected and cell debris was removed by centrifugation at2000 rpm for 5 min. Medium was concentrated 100-fold with ammonium sulfate precipitation (0-60%) followed by dialysis against 0.05 MTris-HCl, pH 7.6-0.2 M NaCl, at 4°C(34). Type IV collagen degradation was assayed using soluble [3H]proline-labeled type IV procollagen
as a substrate (35). The enzyme activity was expressed as the amountof type IV collagen degradation (cpm released/IO7 cells).
Labeling of Tumor Cells. Tumor cells were labeled according to themethod of Fidler and Nicolson (36). Cells were grown as describedabove in DMEM supplemented with penicillin, streptomycin, and 10%fetal calf serum. Actively growing subconfluent cultures were incubatedfor 24 h with medium containing 0.3 n<Õ[I25l]iododeoxyuridine/ml
medium (specific activity, 2000 Ci/mmol; ICN Radiochemicals, Irvine,CA) which labeled nearly all cells as determined by autoradiography.
Preparation of Target Cells. Purified hepatocytes and hepatic sinusoidal endothelial cells were prepared by a modification of that previously described (37, 38) from the livers of 200- to 250-g male Sprague-Dawley rats. Cells were purified by collagenase perfusion, Stractandensity gradient centrifugation, and centrifugal elutriation. Equal numbers of the fractionated liver cells were plated into 35-mm culture dishes(Lux Scientific, New York, NY), and grown to confluency at 37°Cin a
humidified incubator under 2% CO2 in air. The purity of endothelialcells prepared by this method has been verified by electron microscopy,specific uptake of modified lipoproteins (acetoacetylated low-densitylipoprotein), and the presence of cytochemical and morphologicalmarkers of vascular endothelia (37).
Attachment Assay. Tumor cells labeled as above were washed fivetimes with CMF, harvested by very brief trypsinization and gentlemechanical dissociation, suspended in "cold" tissue culture medium,
and the number of cells determined using an electronic cell counter(Coulter Electronics, Hialeah, FL). Cell viability was greater than 95%as tested by trypan blue dye exclusion and single-cell suspensionsdocumented by phase microscopy. Target cells were grown to confluentmonolayers in 35-mm culture dishes as described above, washed withCMF, and overlayed with single-cell suspensions of 5 x 10s labeled
tumor cells in 2 ml DMEM/dish. Duplicate dishes not containingtarget cells were used as controls ("plastic"). Dishes were incubated for10 or 30 min at 37°Cin a humidified incubator under 7% CO2 in air.
At specified time points cultures were observed by phase microscopyfor attachment of tumor to target cells, and the overlying media andunattached cells were aspirated and collected. Cultures were washed x3 with CMF to remove unbound cells, and washes were added to thepreviously aspirated media. Tumor cells remaining bound to target cellswere collected separately by trypsinization and mechanical scraping.Collected material was then subjected to gamma counting (BeckmanInstruments, San Jose, CA). The percentage of adhesion was definedas
Counts bound (cells)Counts in medium + washes + counts bound (total counts) x 100
Statistical Analysis
The data were analyzed by an independent statistician. To take intoaccount the multiple comparisons for chemoinvasion and invasionassays run for 24 h (between the three cell lines and between the twomethods), pairwise comparisons between groups were done using theTukey's Studentized range test. Because of the inequality of variance
for the two cell lines compared at 36 h, / tests for unequal variancewere done for comparison between cell lines. For chemoinvasion versusinvasion at 36 h, a t test for equal variance was done. We usedBonferroni-adjusted P values for these tests to allow for multiplecomparisons. For the chemotaxis assay a comparison of means by oneway analysis of variance was shown to be significant at the 0.0001 level.Pairwise comparisons between groups were therefore performed usingthe Tukey's Studentized range test.
RESULTS
Liver-metastasizing Potential of Tumor Cells. Murine coloncancer cell line 51B formed poorly to moderately differentiatedcarcinomas without liver-metastasizing ability when injecteds.c. into BALB/c mice (0 of 20 animals). Similarly, this cellline did not form hepatic tumors when injected into the tailveins of additional mice (0 of 10 animals). On the other hand,cells derived from line 51B when implanted into the cecal wall
Invasion. The ability of parental cell line 51B and its metastatic derivatives 51B LiM-4 and 51B LiM-5 to penetrate areconstituted basement membrane was tested in the absence(invasion) and presence (chemoinvasion: chemotaxis plus in-
kFig. 1. Experimental cecal "primary" tumor. BALB/c mice were sacrificed 4 weeks after injection of 5 x 10* tumor cells into the cecal wall. Tumor cells (71 are
seen in relation to normal mouse colonie mucosa. Hematoxylin-eosin, x 100.
vasion) of partially purified liver extract in the lower compartment of the Boyden chamber (Fig. 5). Experiments were performed for 24 h with the three cell lines and in addition for 36h with 51B and 51B LiM-4. More cells crossed the basementmembrane barrier at the longer time and the more metastaticlines were also more invasive. This was particularly evidentwhen a liver extract was added to the lower compartment. Theliver extract increased the invasion of all tumor cell lines. Cellline 51B LiM-4 was 3-fold and 51B LiM-5 was 5.6-fold moreinvasive and chemoinvasive at 24 h than cell line 51B studiedunder the same conditions (P < 0.001; see Fig. 5). At 36 h,both invasion and chemoinvasion by cell line 51B LiM-4 wereapproximately 6-fold greater than that of 51B under the sameconditions (P < 0.005 for invasion or chemoinvasion).
Chemotaxis. The possibility that these metastatic tumor cellswere preferentially attracted to a liver factor(s) was tested inthe Boyden chamber assay using filters which had been coatedwith type IV collagen and extracts from brain, lung, and liver(Fig. 6). Liver extract stimulated the movement of 51B LiM-5tumor cells 12-fold compared 0.5% bovine serum albuminalone, and 2.2- and 2.8-fold compared to extracts of lung orbrain, respectively (P < 0.001 for migration to the presence ofliver extract versus BSA, brain, or lung extract).
In order to further define the chemotactic movement of cellsfrom line 51B LiM-5 in response to liver factors, a Zigmond-Hirsch checkerboard analysis was performed (39) (Fig. 7).Different concentrations of liver extract were placed on bothsides of the Boyden chamber. Cells in the upper chambermigrated most actively and in a dose-dependent manner whenthe concentration of liver extract was greatest in the lowerchamber (Fig. 7, data below the diagonal). This extract alsostimulated the chemokinesis or random migration of 5IB LiM-
5 cells (Fig. 7, data on the diagonal), especially at high concentrations of extract. Little increase in cell migration was observedwhen a negative gradient of extract was established (Fig. 7, dataabove the diagonal).
Collagenase Type IV. The type IV collagen-degrading metal-loprotease activity secreted by parental cell line 51B and metastatic cell lines 51B LiM-4 and 51B LiM-5 was compared usingsoluble [3H]proline-labeled type IV procollagen as the substrate(Table 2). Cell line 51B LiM-4 produced 2.5-fold (P < 0.05)and 51B LiM-5 9-fold (P < 0.005) greater collagen degradingactivity than did parental cell line 51B.
Tumor Cell Adhesion. The ability of tumor cells from metastatic line 51B LiM-3 to bind selectively to purified hepatocytesand hepatic sinusoidal endothelial cells was tested in vitro(Table 3). Tumor cells were found to bind rapidly and preferentially to hepatic sinusoidal endothelial cells when comparedto hepatocytes or a plastic control. At 10 min tumor celladhesion to endothelial cells was more than 5-fold greater thanto hepatocytes (P < 0.001). Binding to hepatocytes differedlittle from that of control.
form both micro- and macrometastases in the liver. Variousobservations discussed below suggest that this may represent apartially organ-specific metastasis.
At least some metastatic cells demonstrate a common phe-notype in their interactions with basement membranes, bindingto them, secreting proteases, and invading the extracellularmatrix (33, 40-51). Some of these properties were examinedusing the colon cancer cells developed here to determinewhether for these epithelial cancer cells invasiveness and metastatic potential were correlated. Indeed, when tested against areconstituted basement membrane composed of collagen IVand laminin, their ability to penetrate it paralleled their meta-
static potential. In addition, the cells were found to producecollagen IV-degrading enzyme levels proportional to their met-astatic activity. These studies suggest that the increased meta-static activity observed in the selected cells could be related, inpart, to their ability to pass the basement membrane barrierswhich limit the spread of cells from the cecum and from thehepatic sinusoids to the liver parenchyma.
Clinical experience and experimental studies (36, 52, 53)strongly suggest that many human and animal tumors metas-tasize to preferential sites. Although this might be in part based
on their site of entry into the circulation (52), other factorsmust be involved (1, 52). Such factors may include tissue-specific chemoattractants (29,54-59) and preferential adhesionof metastatic tumor cells to target organ parenchymal cells orendothelium. In our study liver extract stimulated the movement of highly liver-metastasizing tumor cells to a significantlygreater degree than did bovine serum albumin or extracts oflung or brain (Fig. 6). This same liver extract enhanced theinvasion of tumor cells through the reconstituted basementmembrane. These results are similar to those reported by Hu-janen and Terranova (29) who demonstrated preferential migration of melanoma, sarcoma, and breast carcinoma cells toextracts from the organs for which they show metastatic predilection. The nature of such attractants remains to be determined, but these workers found the factors in brain and liver tobe of different molecular weights. According to these results,the metastasizing tumor cells can disseminate among differenttissues based on the presence of local factors stimulating theirmigration.
Preferential organ attachment of metastatic tumor cells hasbeen studied in vitro by a number of investigators (60-66).These studies have examined adhesion of tumor cells to frozentissue sections (61), organ culture slices (60), or cultured cellmonolayers (63-66). Using highly purified monolayer culturesof target cells, we have demonstrated specific in vitro adherenceof radiolabeled metastatic colon cancer cells to hepatic sinusoidal endothelial cells but not hepatocytes (Table 3). Rat targetcells were used since highly purified murine cells cannot beeasily prepared by our method in sufficient numbers from themouse to allow such studies. Our results are not surprising,since blood-borne metastatic cells must first adhere to endothelial walls and invade through basement membranes before en-
* . • *¿ - - .Fig. 4. Regional lymph node metastasis showing tumor cells (7") similar to those of the primary tu
Fig. 5. Invasion of parental and metastatic tumor cells through a reconstitutedbasement membrane matrix composed of purified bovine type I collagen (0.2 mg/matrix), type IV collagen (0.2 mg/matrix), and laminili (0.2 mg/matrix). Tumorcell suspensions (7 x 10* cells) in DMEM containing 0.5% BSA and 2% acid-treated NuSera were placed into the upper compartment of a modified Boydenchamber and allowed to penetrate the matrix onto a type IV collagen-coatedNuclepore filter (pore diameter, 1.0 ^m). Assays were done in the presence(chemoinvasion) or absence (invasion) of partially purified liver extract (10%) inthe lower compartment of the modified Boyden chamber. Data are expressed asa percentage of cells which penetrated the matrix after 24 (left) and 36 (right) h(±SD (bars)). Data were obtained for 5IB LiM-5 at 24 h only. «,P < 0.005; ««,P < 0.001 compared to parental cell line 51B studied under the same conditions:•,P< 0.05; +, P< 0.001 chemoinvasion versus invasion.
[J.1M'xÌ50-40-30-20-10-*
ñmmMM*9.5%
BSA Brun LungInnExtract
Fig. 6. Directed migration (chemotaxis) of highly liver-metastasizing tumorcell line 51B LiM-5 to target organ extracts in a modified Boyden chamber.Tumor cells 5 x 10* in DMEM containing 0.5% BSA were added to the uppercompartment and liver, lung, or brain extracts (10%) in DMEM containing 0.5%BSA were placed in the lower compartment of the chamber. After a 5-h incubationat 37"C, type IV collagen-coated Nuclepore barrier filters (pore diameter, 8 Mm)
were removed and migration to the lower surface of the filter quantitated. Thedata are average number (±SD (bars)) of migrated cells per high power field.Cells were counted in eight high power fields, and assays were performed intriplicate. «,P < 0.001 for liver versus BSA, brain, or lung extracts.
% Liver Extract Upper Compartment
ExtractLowerCompartment5
3se\\01S1008
±3
\17±237
±648
±71a±216
±2\\.21
±427
±5511±411±3X
25±3\20
±61011
±412±117±4s36
±5\
Fig. 7. Checkerboard analysis of the response of 51B LiM-5 cells to liverextract. Different concentrations of liver extract in DMEM containing 0.5% BSAwere added to the upper and lower compartments of a chemotaxis chamber.Tumor cells in DMEM containing 0.5% BSA were introduced into the uppercompartment. Studies were carried out for 4 h at 37"C. Numbers within innerboxes, number of migrated cells per high-power field (x 400). Responses of cellsto a positive gradient are shown below the diagonal, to a negative gradient, abovethe diagonal, and in the absence of a gradient, on the diagonal. Results areaverages ±SD of triplicate experiments.
countering organ parenchymal cells. Nicolson et al. (60) foundthat B16 melanoma cells often bound to exposed blood vessels,and therefore attached to endothelial rather than parenchymalcells in organ culture. Others, however, have shown that hepa-tocyte surface molecules may be involved in the adhesion ofsome types of carcinoma cells to rat hepatocyte cultures (64)and that the mechanism of adhesion of different tumor cells to
Table 2 Type IV collagen-degrading activity of tumor cell linesType IV collagen-degrading metalloprotease activity secreted by selected tumor
cell lines. Enzyme activity is expressed as amount of type IV collagen degradation(cpm released/101 cells from [3H]proline-labeled type IV procollagen). Results
represent the mean ±SD of three separate experiments each with triplicatedeterminations.
Cellline51B
51BLÃŒM-451B LiM-5Type
IV collagen-degrading activity (cpm
released/ IO7cells)191
±8.5475 ±106a
1704 ±23*
" P < 0.005 compared to 51B.
Table 3 Percentage of tumor cell adhesion to liver-derived cellsSelective binding of metastatic tumor cells from line 51B LiM-3 to purified
hepatocytes, hepatic sinusoidal endothelial cells, or plastic control in vitro wasevaluated. Cultured tumor cells were labeled for 24 h at 37°Cin mediumcontaining 0.3 >iCi/ml [I25l]iododeoxyuridine. Tumor cells (5 x 10*) were platedonto confluent monolayers of liver-derived cells or 35-mm plastic control dishesand incubated for 10 or 30 min at 37*C. Overlying medium was collected, cultures
washed x 3, and bound cells harvested and counted. The percentage of adhesionis defined as
•Mean ±SD.bp<o.ooi.c Numbers in parentheses, number of experiments.d P < 0.005 compared to either hepatocytes or plastic.
hepatic organ target cells may be different (66). We have notyet tested whether the adhesion of our colon cancer cells tohepatic sinusoidal endothelial cells is specific for this endothe-lium in particular or endothelial cells in general. Experimentsby Alby and Auerbach (65) have demonstrated that capillaryendothelial cells derived from different organs are not alike andthat differences expressed at the cell surface of these cells canbe distinguished by tumor cells.
The model which we have described should be useful forstudying many factors which might affect the invasive activityand metastasis of colon cancer cells. Similarly, the highly liver-metastasizing cells selected may be used in this model to assesstherapeutic regimens that could lessen the spread of this disease.
ACKNOWLEDGMENTS
We thank Trish Harrington and Janice Woo for preparation of themanuscript and Mary Barker for preparation of the electron micrographs.
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