Tumorigenicity and Metastasis of Human Breast Carcinoma ...cancerres.aacrjournals.org/content/canres/50/3/717.full.pdf · Tumorigenicity and Metastasis of Human Breast Carcinoma Cell
This document is posted to help you gain knowledge. Please leave a comment to let me know what you think about it! Share it to your friends and learn new things together.
Transcript
[CANCER RESEARCH 50, 717-721. Februar) I, 1990)
Tumorigenicity and Metastasis of Human Breast Carcinoma Cell Lines in NudeMice1
Janet E. Price,2 Aristidis Polyzos,3 Ruo Dan Zhang, and Lisa M. Daniels
Department of Cell Biology, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030
ABSTRACT
There are few reports describing experimental models of the growthand metastasis of human breast carcinomas. This article discusses thetumorigenic and metastatic properties of two estrogen receptor-negativebreast carcinomas injected into nude mice.
Our results suggest that the estrogen receptor-negative MDA-MB-435 cell line injected in the m.f.p. of nude mice could be a valuable toolfor analysis of the cellular and molecular basis of the metastasis ofadvanced breast cancer.
INTRODUCTION
Since the initial observation made by Rygaard and Povlsen(1) that a human colon adenocarcinoma grew progressively innude mice, this athymic mutant strain of mouse has beenfrequently used in human cancer research. Numerous studieshave reported that, although not all human tumors can besuccessfully xenografted, the histology and biochemical properties of the tumors that do grow in nude mice closely resemblethose of the original tumor specimens (2). A major use of nudemice is to prescreen chemotherapeutic agents that act againsthuman tumors proliferating in vivo, rather than using in vitroassays or murine tumor models (3, 4).
The nude mouse also provides a means to study the biologyof human tumor metastasis (5). Whereas some early reportsnoted that the incidence of metastasis of xenografted humantumors in the nude mouse was low (2, 6), more recent studiesreveal that the metastatic phenotype can be expressed in thismodel system. Variables affecting whether metastasis occursinclude the health and housing conditions of the mice (7, 8)and the routes of tumor cell inoculation employed (5, 9) in
Received 7/7/89; revised 10/5/89; accepted 10/24/89.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.
1Supported in part by a grant from Triton Biosciences. Inc.2To whom requests for reprints should be addressed, at The University of
Texas M. D. Anderson Cancer Center. Department of Cell Biology. 1515 Hoi-combe Blvd.. Box 173. Houston. TX 77030.
'Current address: Athens University School of Medicine, 1st Department of
Propedeutic Medicine, Laiko General Hospital. 115 27 Athens. Greece.
addition to the intrinsic properties of the tumors under investigation (10, 11). Many human tumors can proliferate wheninjected s.c. into nude mice, but metastasis from this site is rare(2, 6).
Human breast carcinomas have a low tumor take rate in nudemice (2), and there are few reports of metastasis of thesexenografted tumors. ER"-positive MCF-7 cells, growing in the
Animals. Female athymic nude mice (NCr-/iu/nu), 6 to 8 weeks old,were obtained from Simonsen Laboratories (Gilroy, CA). The animalswere housed under specific pathogen-free conditions.
Cell Lines. MDA-MB-231 and MDA-MB-435 were generous giftsfrom Dr. R. Cailleau, Department of Medicine, M. D. Anderson CancerCenter (Houston, TX). Both are ER-negative cell lines isolated fromthe pleural effusions of patients with breast carcinoma (20, 21). HT-29is a human colon carcinoma cell line (22). A375 is a human melanomacell line (23), and SN12C was established from a renal cell carcinoma(13). All the cell lines were confirmed as human by karyotypic orisoenzyme analysis.
All cell lines were examined for and found to be free of reovirus type3, pneumonia virus of mice, mouse adenovirus, murine hepatitis virus,lymphocytic choriomeningitis virus, ectromelia virus, k-virus, Theiler's
virus. Sendai virus, and láclatedehydrogenase virus (assayed by Microbiological Associates, Walkersville, MD) and Mycoplasma (tested usinga Gen-Probe Mycoplasma detection kit; Gen-Probe, Inc., San Diego,CA).
Cell Culture. The cells were maintained in monolayer culture inEagle's minimum essential medium supplemented with 5r< fetal bovine
'The abbreviations used are: ER. estrogen receptor; HBSS, Hanks' balanced
serum, sodium pyruvate, L-glutamine (2 HIM),nonessential amino acids,and 2x vitamin solution (GIBCO, Grand island, NY). The cultureswere incubated at 37°Cin a humidified atmosphere of 5% CO2 and
95% air. Tumor cells were harvested for passage or inoculation bywashing the monolayer with HBSS, followed by a brief incubation in0.25% trypsin and 0.02% EDTA. The tissue culture flask was tappedto dislodge the cells, which were then resuspended in minimum essentialmedium supplemented with 5% fetal bovine serum. Tumor cells intended for inoculation into mice were washed by centrifugation andresuspended in HBSS. Cell number and viability were determined bystaining a small volume of cell suspension with 0.2% trypan blue salinesolution and examining the cells in a hemocytometer.
Tumor Cell Inoculation. In preparation for inoculation into the m.f.p.,the mice were anesthetized with Metofane (Pitman Moore Inc., Washington NJ) and a 5-mm incision was made in the skin over the lateralthorax. The m.f.p. was exposed, anda volume of 0.1 ml of cell inoculumwas injected into the tissue through a 27-gauge needle. By exposing thefat pad, we were able to ensure that the cells were injected into thetissue and not into the s.c. space. Tumor cells were also injected s.c.(lateral flank) or i.v. (lateral tail vein) in separate experimental groups.The growth of m.f.p. and s.c. tumors was monitored by weekly examination, and growth rates were determined from caliper measurementsof two diameters. In some experiments the tumors were excised at amean diameter of 1.5 cm; in other experiments, they were excised atvariable times after cell inoculation. The mice were anesthetized, thetumors were removed, and the skin incisions were closed with woundclips. Experiments were terminated 4 weeks after tumor excision orwhen the mice were moribund. For the i.v. injection experiments, themice were killed 12 weeks after injection or when moribund.
Growth of Human Breast Carcinoma Cells on Cytodex Beads. Cyto-dex-3 beads (Pharmacia) were rehydrated, following the manufacturer'sinstructions, and sterilized. Beads (2 x 104/rnl) and cells of the twobreast carcinoma cell lines (2 x 105/rnl) were plated in 10 ml of culturemedium in 100-mm Petri dishes (i.e., not tissue culture-treated dishes;the tumor cells adhered to the beads and not to the dishes). After 3 to5 days, when the beads were covered with cells, a sample was taken andtrypsinized to estimate the numbers of cells per bead. For MDA-MB-231, this number was 75 cells/bead and for MDA-MB-435, 28 cells/bead. Beads coated with cells were washed with HBSS and injected i.v.into groups of nude mice, approximately 5 x IO3 beads in 0.2 ml of
HBSS/mouse. Eight to 10 weeks after injection, the mice were killedand the lungs were examined for tumor colonies.
Ê10,"
£0.75
1O 0.5
| 0.25C
I
iI 0.75
1 2 3 t 5 6Time (weeks)
t 0.5
•¿�0.25
2 3Tine (weeks)
s10| 0.75
S 05
1- 0.25§I 1 2 3
Time (weeks)
Fig. 1. Growth curves of four human tumor cell lines injected into two sitesin nude mice. Tumor cells (10' cells/site) were injected s.c. (•)or into the m.f.p.
(A) and tumor growth was measured for up to 10 weeks. The cell lines wereMDA-MB-435. breast carcinoma: A37S, melanoma: HT-29, colon carcinoma:and SN12C, renal cell carcinoma.
' ^ >•-t'-•-.
i'to " «' â„¢¿�•¿�"* '• - » , i ,Vjl
®£&$:
Fig. 2. MDA-MB-435 tumor in the mammary footpad of a nude mouse, x100.
RESULTS
Site of Tumor Cell Injection: s.c. versus m.f.p.. To test whetherthe site of injection influenced the growth of human breastcarcinoma cell lines in nude mice, we injected cells s.c. and intothe m.f.p. of mice and compared the tumor take and subsequentgrowth rates. For two human breast carcinoma cell lines, MDA-MB-231 and MDA-MB-435, three inoculum doses were used.At the two higher doses (IO6 and 5 x IO6 cells), tumors devel
oped in all mice at both sites of injection. However, at thelowest cell dose (10s cells), tumors grew in 2 of 5 mice injected
s.c., whereas 5 of 5 mice injected into the m.f.p. developedtumors; this result was the same for both cell lines in tworepeated experiments. Fig. 1 shows that the latent interval forappearance of MDA-MB-435 m.f.p. tumors was less than thatfor s.c. tumors, following injection of IO5 cells; the growthcurves for MDA-MB-231 tumors were similar to those ofMDA-MB-435 shown here. At the two higher cell doses, tumors
appeared in both sites after similar intervals.To determine whether the trophic effect of the m.f.p. was
specific for breast carcinoma cells, we tested the growth of threedifferent human tumor cell lines injected s.c. or into the m.f.p.For a melanoma (A375), a colon carcinoma (HT-29), and arenal cell carcinoma (SN12C), there was no difference in tumortake or growth rate following injection into the m.f.p., ascompared with growth of the same number of cells (IO5 cells)
injected s.c. (Fig. 1). A375 and SN12C cells produced tumorsin all mice given injections in either site (5 of 5), and HT-29cells produced tumors in 4 of 5 mice given injections s.c. orinto the m.f.p. The experiment was performed twice, withidentical results. Based on the results from these experimentswith MDA-MB-231 and MDA-MB-435, we adopted the m.f.p.as the site of injection in developing the model of human breastcarcinoma metastasis in nude mice.
The histology of the MDA-MB-435 cell line is that of apoorly differentiated adenocarcinoma (Figs. 2 and 3). When
Fig. 3. MDA-MB-435 s.c. tumor in the lateral flank of a nude mouse, x 100.
Table 1 Metastasis of MDA-MB-435: excision of the mammary fat pad tumorsat different times
Thirty female nude mice were given injections of 5 x 10' MDA-MB-435 cellsinto the m.f.p. At 4, 8, and 12 weeks after injection, the tumors were excisedfrom a group of 10 mice. At 16 weeks after the initial injection, all the mice werekilled and examined for metastasis.
involved included axillary, inguinal, iliac, cervical, and mediastinal.' Found in histological sections.
tumors growing in the m.f.p. and s.c. sites were compared, themost striking differences were the poorer vascularity and moreextensive necrosis in the s.c. tumors and a reduced fibrouscapsule around the m.f.p. tumors.
Metastasis from Tumors Growing in the Mammary Fat Pad.Both MDA-MB-231 and MDA-MB-435 cell lines formed progressively growing tumors following injection of cells (10* toIO6cells) into the m.f.p. Of 7 mice with MDA-MB-231 tumors
The ability of the MDA-MB-435 cells and the metastasis-derived variants to form lung colonies was assessed by injectingIO6 cells i.v. via the tail vein. None of the cell lines showed
marked ability to colonize the lungs, producing few lung colonies in only 2 or 3 of 10 mice injected in each group.
Experimental Lung Metastasis by Human Breast CarcinomaCells Injected as Single Cells or Attached to Cytodex Beads. Therationale for injecting breast carcinoma cells cultured on theCytodex beads was to establish whether the cells could proliferate in the lungs once they were arrested by physical trappingof the large beads (lOO-^m diameter). The results in Table 3show that MDA-MB-231 cells delivered to the lungs of nudemice on the beads proliferated and formed macroscopic lesionsin 7 of 7 mice injected. The estimated dose of MDA-MB-231cells injected on the beads was 4x10' cells; double this cell
MDA-MB-231 and MDA-MB-435 cells were cultured on Cytodex-3 beads.Mice were given i.v. injections of 5 x 10'beads in 0.2 ml HBSS or of monocellularsuspensions of 10' cells of the different cell lines.
CelllineMDA-MB-231.single cells (10')
MDA-MB-231 on beads (4 x 10')"MDA-231-Beadl*, single cells(10')MDA-MB-435.
single cells (10')MDA-MB-435 on beads (1.4 x 10')°MDA-435-Beadl*. single cells (10')MDA-435-PM1', single cells (10')Experimental
lungmetastasisNo.
of coloniesIncidence(range)0/10
7/7 90(5-150)0/51/10
0(0-8)5/6 30 (0-70)5/5 17(10-36)5/6 11 (0-23)
" Estimated cell dose on the Cytodex beads.* Cell lines established from the lung tumors in mice given injections of cells
growing on the beads.'Cell line established from lung colonies in mice given injections of MDA-
MB-435 cells.
had a higher lung colonization potential than the original cellline. Thus, for the MDA-MB-435 cell line, growth in the lungof nude mice appeared to select for cells with enhanced lung-colonizing properties; these cells were possibly only a smallsubset of the parent cell line, indicated by the result that 1 ofthe 10 mice given injections of single cells developed lungcolonies (Table 3).
Essentially, the experiments with the Cytodex beads showedthat the human breast carcinoma cells were capable of growthin the nude mouse lungs and that the poor lung colonizationafter i.v. injection of IO6 single cells was possibly due to few
cells arresting in the lungs, rather than an inability to grow inthis organ.
DISCUSSION
Use of athymic nude mice has allowed many and diversestudies of human tumor biology (1-3), including in relativelyrecent years the analysis of metastatic properties (6, 9-15). Inthis report, we describe a new experimental model of themetastasis of an ER-negative human breast carcinoma cell linefollowing implantation and growth in the m.f.p. of nude mice.
We thank Patherine Greenwood for preparing the manuscript.
REFERENCES
10.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
1. Rygaard. J.. and Povlsen. C. O. Heterotransplantation of a human malignanttumor to nude mice. Acta Pathol. Microbio!. Scand., 77: 758-760, 1969.
2. Giovanella. B. C.. and Fogh, J. The nude mouse in cancer research. Adv. 32.Cancer Res., 44:69-\20. 1985.
3. Ovejera. A. A., and Houchens, D. P. Human tumor xenografts in athymicnude mice as a preclinical screen for anticancer agents. Semin. Oncol., 8: 33.386-393, 1981.
4. Houchens. D. P., and Ovejera, A. A. Models for human tumor therapy in
1990;50:717-721. Cancer Res Janet E. Price, Aristidis Polyzos, Ruo Dan Zhang, et al. Lines in Nude MiceTumorigenicity and Metastasis of Human Breast Carcinoma Cell