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Biology |
University of California San Diego, Cancer Center and Departments of Pathology [S. G., K. K., J. H., V. U., D. T.] and Surgery [R. H.], La Jolla, California 92093; AntiCancer, Inc., San Diego, California 92111 [P. J., M. Y., R. H.]
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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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From this, it follows that tumor cell dissemination by hematogenous, lymphatic, or trans-coelomic routes is necessary but not sufficient for the formation of secondary tumors and that, unless a growing tumor colony is established in a new site, the metastatic process is not fulfilled by that cell. Mechanistically, it has long been inferred from observations on humans (1 , 4) and animals (5) that the local organ microenvironment at the site where the tumor cells come to rest influences whether metastases emerge or not. The nature of these influences deserves investigation to determine whether they can be manipulated for therapeutic benefit.
In this communication, we report a study using cloned human tumor cell lines of diametrically opposite metastatic performance (6 , 7) , which we labeled with GFP2 to visually track disseminating tumor cells of differing malignant potentials and examine their fate in different host microenvironments. Additional objectives of this work were to ascertain whether such cells die within a few days, if they fail to grow in the ectopic organ sites where they implant, and to retrieve disseminated cells which are not making metastases, so as to assess their viability, proliferative potential, and tumorigenicity. The study was also conducted to ascertain whether these human breast cancer cells make bone metastases in nude mice.
One of the main aims of this work was to focus on the spontaneous, unprompted dissemination of labeled cancer cells from an undisturbed tumor and their subsequent colonization of distant organs or dormant residence there. Some previous studies (8, 9, 10) have inoculated GFP-labeled cells directly i.v. to study their short-term fate, but there has been no previous inquiry tracking the behavior and fate of cells escaping from primary tumors by themselves over prolonged periods of time. An additional novel feature in the design of the current work was to compare the behavior of GFP-labeled tumors, which are spontaneously metastatic, with that of labeled tumors formed by an isogenic sister cell line, which is virtually unable to make secondary tumors in distant organs. Detailed characteristics of this pair of cell lines have been described previously (7) . The present study yielded unexpected new information showing that the tumors which could not make detectable secondary tumors in any organ, were nevertheless shedding tumor cells into the circulation. The genetically incorporated label showed that these remained solitary and dormant in the tissues for months. In addition, it was proven that these cells could be retrieved and would resume proliferative capability in vitro as well as tumorigenicity when reinoculated into the mammary gland.
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MATERIALS AND METHODS |
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Retroviral Transduction and Selection of GFP-expressing Tumor Cell Lines.
To achieve GFP gene transduction into the M-4A4 and NM-2C5 clonal tumor cell lines, 20% confluent cells were incubated with a 1:1 precipitated mixture of retroviral supernatants of PT67 cells and RPMI 1640 (Life Technologies, Inc.) containing 7% fetal bovine serum (Life Technologies, Inc.) for 72 h. After media replenishment, M-4A4 and NM-2C5 cells were harvested by trypsin-EDTA 72 h post-transduction and subcultured at a ratio of 1:15 into selective medium that contained 200 µg/ml G418 initially. The level of G418 was subsequently increased to 800 µg/ml stepwise. Clones expressing GFP (NM-2C5-GFP and M-4A4-GFP) were selected, combined, and propagated using conventional culture methods. To evaluate the doubling time of GFP-labeled clones, cells were seeded at 1.5 x 104 in 35-mm culture dishes, harvested, and counted using a hemocytometer every 24 h.
Orthotopic Inoculation and Primary Tumor Growth.
Female athymic mice (BALB/c nu/nu; B&K Universal, Fremont, CA) aged 8–10 weeks were housed in an isolation suite for the duration of the experiments and cared for in accordance with the standards of University of California San Diego under an approved protocol of the University of California. The tumorigenicity and spontaneous metastatic capability of the cell lines were determined by orthotopic inoculation into the mfp. One million cells in 0.05 ml of a 1:1 mixture of RPMI 1640 and extracellular matrix gel (Sigma Chemical Co., St. Louis, MO) were inoculated into the surgically exposed right flank mfp of an anesthetized mouse under direct vision through a dissecting microscope. This ensured delivery of the entire inoculum into the desired location. Animals were monitored every 2 days for 
5 months for tumor growth and general health. The rate of primary tumor growth was determined by plotting the means of two orthogonal diameters of the tumors, measured at 7-day intervals. Animals were euthanized and autopsied at 3–6 months postinoculation, the autopsy being performed when a primary tumor reached a diameter of 
2 cm. For analysis of experimental metastases, BALB/c nu/nu mice were injected with 1 x 106 cells via the tail vein. Cells were harvested using EDTA and injected in a total volume of 50 µl of PBS. Mice were euthanized 6 weeks after inoculation and examined as described below.
Evaluation of Spontaneous Metastasis Distribution, Tumor Cell Dissemination, and Cell Survival.
At autopsy, the whole animal, the primary tumor, and all of the major organs were examined under blue light of wavelength 490 nm to activate GFP fluorescence using a commercial blue light fiber optic rig (Lightools Research, Encinitas, CA). Images were captured on a thermo-electrically cooled color digital camera fixed on an adjustable rig, and the collective ensemble will be referred to as the GFP imaging system. Small foci of GFP-expressing cells detected on the dorsal surface of the left upper lobe of the lung were counted. Excised tissues were stored for subsequent analysis after either fixation in 10% neutral formalin or snap freezing in liquid nitrogen. For visualization of GFP-labeled cells within fixed lung tissue, the left upper lobe of the lung was perfused with OCT compound (Sakura, Torrance, CA), and 50-µm frozen sections were viewed immersed in saline. Light and fluorescence microscopy was carried out using a Nikon E600 microscope equipped with a GFP filter set (Chroma Technology, Brattleboro, VT) and a xenon lamp power supply. Images were processed by Metamorph Version 3.0 software (Universal Imaging Corp., Downingtown, PA).
Tumor Excision and Cell Retrieval Experiments.
To evaluate whether a concomitant primary tumor inhibits the growth of metastases in this experimental system, the tumors were surgically removed at 1 cm in diameter from 10 orthotopically inoculated mice, with subsequent autopsy 3 months later. At autopsy, surface metastases and disseminated labeled cells were counted. Recovery of viable GFP-labeled cell lines from the lungs of NM-2C5 inoculated animals was accomplished by culture of tissue explants. Lung tissue was crudely homogenized by scalpel and cultured in RPMI 1640 (Life Technologies, Inc.) containing 10% fetal bovine serum (Life Technologies, Inc.) and penicillin/streptomycin. After 72 h, debris was removed, and adherent cells were subcultured in selective medium that contained 200 µg/ml G418. The concentration of G418 was subsequently increased to 800 µg/ml stepwise over 4 weeks of culture. Viable recovered cells at this time were still GFP positive.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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800 µg/ml. The selected cells maintained uniform bright GFP fluorescence in the whole population (Fig. 1)
during continued passage after G418 selection was completed, indicating that the fluorescence was sufficiently stable to permit detection after inoculation and long-term growth in vivo. The proliferation rate of the transduced cells was not altered by the transduction procedure or by GFP expression, as evidenced by the comparison of the doubling times with the nontransduced cell lines (data not shown). The distinct morphology of the NM-2C5 and M-4A4 clones was also unchanged by the transduction procedure, with NM-2C5 cells displaying a characteristic pattern of growth in tightly clumped aggregates (7)
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2 cm in size. All inoculated animals developed mammary tumors and remained active and strong until sacrifice. The growth curves of the tumors formed by the two cell lines were compared with each other and to the growth of tumors formed by the parental cell lines by weekly orthogonal measurements (Fig. 2)
. The observation described previously that the metastatic M-4A4-derived tumors grow faster than their NM-2C5 counterparts (6
, 7)
was confirmed. The growth rate of primary tumors formed by both of the GFP-transduced cell lines was broadly similar to that of unlabeled tumors (Fig. 2)
, indicating that the behavior of the lines was not dramatically changed by the transduction procedure. NM-2C5-GFP primary tumors took between 112 and 191 days (mean 155 days) to reach the experimental end point of 2-cm tumor diameter in one plane of measurement. M-4A4-GFP tumors took between 97 and 143 days (mean 113 days) to reach the same end point size (Table 1)
. In all cases, the tumor tissue was strongly fluorescent (Fig. 3)
, demonstrating stable high level GFP expression by the constituent cells throughout extended periods of growth in vivo.
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. Although previous studies (16)
have unequivocally demonstrated that internal lesions can be detected if they are large enough and close enough to the skin of the animal, internal metastases were not visible under blue light illumination in this study. On opening the body, the surface of all of the internal organs was first examined under ordinary illumination. Low magnification microscopy revealed easily detectable macroscopic lung metastases in 5 of 8 (62%) mice inoculated with M-4A4-GFP cells (Table 1)
. Subsequent examination under blue light (490 nm) showed that all metastatic deposits detected by light microscopy exhibited green fluorescence characteristic of GFP-labeled tumor cells. Metastases were not seen in any other organs in mice bearing 4A4 tumors within the time period of this experiment, although they do occur frequently in lymph nodes (7)
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Conversely, tumors derived from the clonal cell line NM-2C5 did not produce any visible metastases in the host lungs, nor in any other organ (Table 1)
. However, microscopic examination of the surfaces of the lungs and of frozen sections of these organs under blue light revealed the unequivocal presence of many disseminated tumor cells that were invisible by conventional illumination. The most striking observation was that these were clearly present in the lungs of all animals which harbored tumors formed by NM-2C5 cells in their mammary glands, as well as in those with tumors formed by M-4A4 cells. Despite the intense fluorescence of these single tumor cells, it was impossible even at the highest magnifications to establish with certainty whether they had exited from the capillaries into the lung parenchyma, because precise visualization of the capillary endothelium within the lung was not feasible. Tiny tumor cell emboli of one to four cells, which would be undetectable macroscopically or even microscopically, were easily located because of their fluorescence (Fig. 4)
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Because of the fact that breast cancer in humans is often observed to spread to the long bones and the axial skeleton, we particularly wanted to investigate whether the cell lines that we have cloned also metastasize to these organs. GFP fluorescence greatly facilitates this analysis; metastases in skeletal sites have been revealed previously in other models of metastasis (13, 14, 15) , with bone metastases being detectable through the skin of nude mice (16) . Fluorescent examination revealed no skeletal tumor deposits in mice inoculated with either metastatic M-4A4 or nonmetastatic NM-2C5 cells. In some cases, the skeleton was cleaned of all soft tissue before illumination, but still no metastases were revealed.
Effects Of Primary Tumor Excision on Disseminated NM-2C5 Tumor Cell Dormancy.
These experiments were conducted to establish whether a concomitant primary tumor in the mammary gland can suppress metastasis formation by disseminated NM-2C5 cells. The primary tumor was removed at a size of 1 cm, after an average of 16 weeks growth, and the mice were euthanized 3 months later. No metastases were found in any of the organs of nine animals from which the primary tumors were excised (Table 1)
. However, the survival and distribution of solitary brightly fluorescing cells in the surface of the lungs were confirmed, although the numbers were diminished relative to those in mice which harbored a primary tumor throughout the experiment (Fig. 4
and Table 1
), presumably because of excision of the source from which they had been seeded. Viable cells were also recoverable in culture from the lungs of these mice showing that, although unable to proliferate, NM-2C5 cells can remain viable in the lung environment for 6 months.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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These findings establish definite biological distinctions between metastasis, tumor cell dissemination, and tumor cell dormancy in this experimental system, which may apply more generally. They also indicate that the mitotic quiescence of disseminated NM-2C5 cells is induced by the surrounding host cells, although their ancestral counterparts, which they leave behind, grow progressively in the host’s mammary gland. Indeed, the very same disseminated cells, retrieved from the lungs, retain their tumorigenicity when reinoculated back into the mammary gland. The implications of this are that cells with unlimited proliferative potential, such as tumor and stem cells, are not invincible and can be returned to a state of suspended mitotic activity by the surrounding cellular microenvironment.
It is appropriate to refer here to previous investigations (8, 9, 10) , which have described the presence of viable tumor cells that did not form tumor deposits in downstream organs after i.v. inoculation of labeled cells. The retrieval of such experimentally seeded cells, after periods of 1–30 days, and their ability to grow in vitro have also been reported (10) . However, so far as we are aware, the present article is the first account of the identification and retrieval of labeled, viable, human tumor cells, which had spontaneously disseminated themselves from an undisturbed tumor and lain dormant in the host for several months. The dissemination kinetics of such cells, escaping over prolonged periods of time, are clearly different from those in a pulse bolus inoculated into a large vein, and this experimental design provides the new data, unobtainable by other methods, that tumors (e.g., NM2C5), which do not establish distant secondary deposits, can still be shedding neoplastic cells into the vascular system. The present data are also novel because they show, for the first time, that these naturally disseminated, dormant tumor cells can still proliferate indefinitely and generate tumors in appropriate circumstances. The current study, therefore, confirms and extends these (10, 11, 12, 13, 14, 15, 16) and other (17 , 18) previous reports and offers new opportunities for the investigation of factors that affect the growth of spontaneously disseminated tumor cells in distant organs. It also documents a new model for the experimental analysis of conditions causing long-term dormancy of tumor cells that have escaped from an undisturbed tumor to other tissues.
It is worth noting that many of the cells disseminating from M4A4 tumors grew into fluorescing metastases in the lungs, although adjacent sister cells from the same tumor remained solitary and quiescent. Therefore, even in a clonally derived cell population with metastatic properties, many cells do not or cannot mobilize the organ-specific growth properties needed to generate metastases. These observations closely match those on patients treated with peritoneo-venous shunts for intractable malignant ascites, because of inoperable abdominal tumors (4) . That clinico-pathological investigation showed that tumor cells grew to form metastases in some organs but not others, although billions of viable tumor cells were infused into the systemic circulation over the lifetime of the patient. Some patients did not develop any metastases at all, although viable solitary disseminated tumor cells could be seen in the tissues of downstream organs. Others developed more metastases in organs already known to harbor deposits but not in additional organs, despite prolonged survival of the host. In these patients, the malignant cells in the ascitic fluid were clearly viable and tumorigenic, as manifested by their ability to form abundant local metastases in the peritoneal lining of the abdominal cavity and in certain specific organs in the patients after vascular dissemination. The similarity of results in the present investigation to these data indicates the potential clinical value of results from this xenogeneic experimental system.
In some tumor systems, such as the Lewis lung carcinoma, investigators (19 , 20) have provided evidence that the existence of a concomitant primary tumor in the host inhibits the growth of metastases by the release of soluble factors into the serum. However, the experiments conducted in the present investigation did not show any such effect in the breast-derived tumor system currently under study when the tumors were resected. This can therefore be rejected as a potential explanation for the failure of the disseminated tumor cells to grow in ectopic sites.
The clinical implications of the collective findings of this study and opportunities presented by this new experimental approach, combining GFP labeling and clones of differing malignant phenotypes, together introduce fresh stimulus for the analysis of the molecular mechanisms involved in metastasis and for designing its clinical control.
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FOOTNOTES |
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1 To whom requests for reprints should be addressed, at University of California San Diego, Cancer Center and Department of Pathology, La Jolla, California 92093. 
2 The abbreviations used are: GFP, green fluorescent protein; mfp, mammary fat pad.
Received 12/12/02; revised 4/10/03; accepted 4/21/03.
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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