[CANCER RESEARCH 46, 4109-4115, August 1986]

Growth and Metastasis of Tumor Cells Isolated from a Human Renal CellCarcinoma Implanted into Different Organs of Nude Mice1

Seiji Naito, Andrew C. von Eschenbach, Raffaella Giavazzi, and Isaiah J. Fidler2

Departments of Cell Biology fS. N., R. G., I. J. F.J and Urology [A. V. E.J, The University of Texas M. D. Anderson Hospital and Tumor Institute at Houston,Houston, Texas 77030

ABSTRACT

The purpose of this study was to determine whether the methods forisolating tumor cells from a human renal cell carcinoma (HRCC) influence the biological behavior of the cancer cells. Renal cell carcinomaobtained from a surgical specimen was dissociated by enzymatic treatmentand cells were plated into culture dishes or injected s.c. into the kidneyof BALB/c nude mice. The resultant kidney tumor produced liver metastasis and ascites. All tumors growing in nude mice (s.c., kidney, liver,ascites) were also established in culture. The human origin of all fivelines was ascertained by karyotypic and isoenzyme analyses. Cells fromall lines were injected, s.c., ¡.p.,i.V., intrasplenically, and beneath therenal capsule of nude mice. All the lines were tumorigenic after s.c. orrenal subcapsule injection, although the rate of tumor growth variedamong the five lines. The metastatic behavior of the HRCC cells wasinfluenced by both the nature of the tumor cells and the route of injectioninto nude mice. In general, cells derived from the liver metastasis produced more métastasesin nude mice than other lines. The lines established in culture from the primary HRCC and the ascites were poorlymetastatic. Even with highly metastatic cells, i.v. injection did not yieldsignificant metastasis, but the injection of cells into the renal subcapsuleresulted in extensive metastasis to the lungs and in all peritoneal organs.

These results indicate that nude mice can be used for the isolation ofpopulations of HRCC cells with different growth and metastatic potentialand that, of the organ sites tested, tin* renal subcapsule is the most

advantageous site for implantation of HRCC cells.

INTRODUCTION

The concept that neoplasms are heterogeneous and containsubpopulations of cells having different biological behaviorpatterns, including metastatic potential, has gained wide acceptance (1-6). The outcome of cancer metastasis in rodentsand in humans has been shown to depend on the interaction ofmetastatic tumor cells with host factors, including organ environment (3-7). Most of the data on metastatic heterogeneityand biological behavior of neoplasms have been derived fromstudies in rodent systems, and little data are available on the invivo metastatic process by freshly isolated human cancers. To agreat extent this has been due to the lack of suitable models forthe isolation of appropriate human cancer cells (i.e., nonmeta-static or metastatic) and the lack of a suitable laboratory modelfor the in vivo studies of human cancer metastasis. Since Ry-gaard and Povlsen (8) reported that xenogeneic human tumorscould grow in athymic T-cell-deficient nude mice, the availability of nude mice for studies of heterotransplantation of cells ortissues has proved valuable in studying many aspects of humantumor biology (8-19). Although human tumors grown in nudemice maintained their original morphological and biochemicalcharacteristics, even highly malignant neoplasms rarely produced metastasis in the nude mouse recipient (9, 11, 13, 18).

Received 11/14/85; revised 2/17/86; accepted 4/22/86.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.

1Research supported in part by a gift from the Rudman Foundation.1To whom requests for reprints should be addressed, at the Department of

Cell Biology (HMB 173), The University of Texas M. D. Anderson Hospital andTumor Institute at Houston. 6723 Bertner Avenue. Houston. TX 77030.

Because metastasis is influenced by both tumor cell propertiesand host factors ( 1, 4-6), it is not surprising that even in nudemice the outcome of metastasis depends on the nature of thetumor cells, on experimental conditions such as route of administration or organ for implantation (20-29), and on the sourceof the mice used (15, 30, 31).

The site of tumor growth can influence the production ofmetastasis (4). Recent reports from our laboratory have shownthat the i.s.3 injection of human tumor cells derived from severalwell-established cell lines (colon carcinoma, prostatic carcinoma) (19) or tumor cells freshly isolated from human colorec-tal carcinomas (32) allowed for the expression of metastaticpotential, whereas implantation of the same cells into thesubcutis did not. One notable exception was an establishedHRCC line in which growth of cells in the spleen was notassociated with production of métastasesin the liver or lung(19).

In the present study we determined whether freshly isolatedcells from a HRCC exhibited biological heterogeneity in vitroand, after implantation in athymic nude mice, in vivo. Becausethe 5 cell lines of the HRCC were derived from 5 differentisolation conditions (culture, s.c. tumor, renal tumor, livermetastasis from the renal tumor, ascites from the renal tumor),we asked whether the cell lines exhibited biological heterogeneity, including metastatic potential. Finally, we also examinedthe issue of appropriate modeling for metastasis of HRCC. Theavailability of these 5 lines allowed us to conclude that theyconsisted of different subpopulations of cells with diversegrowth and metastatic potential and also to demonstrate thatthe implantation of HRCC cells into the kidney of nude miceprovided the most advantageous method of examining theirmalignant potential.

MATERIALS AND METHODS

Animals. Male athymic BALB/c nude mice were obtained from theAnimal Production Area of the NCI-Frederick Cancer Research Facility. Mice were used when 8 weeks old and were maintained in a laminarflow cabinet under specific-pathogen-free conditions.

Tissue Culture Conditions. All tumor cell lines were maintained onplastic as monolayer cultures in Eagle's minimum essential medium

(M. A. Bioproducts, Walkersville, MD) supplemented with 10% fetalbovine serum, sodium pyruvate, nonessential amino acids, L-glutamine,and a 2-fold vitamin solution (GIBCO, Grand Island, NY). All the celllines were examined for and were found to be free of Mycoplasma,reovirus type 3, pneumonia virus of mice, mouse adenovims, murinehepatitis virus, lymphocytic choriomeningitis virus, ectromelia virus,and lactate dehydrogenase virus (Microbiological Associates, Bethesda,MD).

Establishment of Tumor Cell Lines. The origin of the 5 lines ofHRCC is schematically presented in Fig. 1. The tumor tissue wasobtained from a primary renal tumor subsequent to a radical nephrec-tomy in a 43-year-old male. The tumor was diagnosed as a renal cellcarcinoma with extensive invasion of perinephric fat (Fig. let). Tumor

3The abbreviations used are: i.v. intrasplenic; HRCC, human renal cell carcinoma: RSC, renal subcapsule; HBSS, Ca2*- and Mg2*-free Hanks' balanced salt

solution.

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GROWTH AND METASTASIS OF HRCC IN NUDE MICE

Surgical material (HRCC) Tumor growth s.c. was measured weekly, and tumor volume wascalculated as

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tissue was dissociated enzymatically with Collagenase (type 1, 200 units/ml) and DNase (270 units/ml) (Sigma Chemical Co., St. Louis, MO)as described elsewhere (32). The procedure yielded a suspension ofsingle tumor cells or very small clumps of cells (<5) with a viability>85% (trypan blue exclusion). The cell suspension was divided into 3aliquots. One aliquot was used to produce a tissue culture line designated SN12C. The second aliquot was injected s.c. in the lateral aspectof the anterior thoracic wall ("cranial s.c. site") at 2 x IO6cells/mouse.

Two months later, one s.c. tumor, 1.5 cm in diameter, was excised anddissociated enzymatically. The resultant cells were established in cultureand the line was designated as SN12S1. The third aliquot of the originalHRCC cell suspensions was injected into the kidney of nude mice (RSCinjection; see below) at 2 x IO6 tumor cells/mouse. The mice were

killed on the same day as the mice given s.c. injections. In both nudemice the kidney tumor was large (at least 2 cm in diameter). In onemouse a grossly visible tumor nodule was found in the liver. Moreover,the peritoneum was also full of ascites fluid. Tumor cells isolated fromthe kidney tumor and the liver nodule by enzymatic dissociation wereestablished as individual cell lines in culture and designated as linesSN12K1 and SN12L1, respectively. The cell line established from theascitic cells was designated as SN12A1.

The cells from these 5 lines grew on plastic as a monolayer cultureand showed quite similar morphology (Fig. 2, b-f). The human originof all S lines was ascertained by detailed karyotypic analysis andisoenzyme determinations (Authentikit; Corning Medical, Corning,NY), and no contamination with mouse cells was found.

lH Vitro Growth Rate Determinations. Cells from ninth passagegeneration were plated at a density of 1 x IO4cells/60-mm plastic Petri

dish (Falcon Plastics, Oxnard, CA). Duplicate cultures were trypsinized,and the number of cells per dish was determined every 24 h for 5successive days with a Coulter Counter (Coulter Electronics, Inc.,Hialeah, FL). A growth curve was constructed for each cell line, andthe cell doubling time was thus calculated.

Preparation of Tumor Cell Suspension for ¡nVivo Injections. Tumorcells from ninth passage generation were harvested from subconfluentcultures by overlaying monolayers with a solution of 0.25% trypsin and0.02% EDTA. After 1 min the flasks were tapped sharply to dislodgethe cells, which were then washed in medium with serum and resus-pended in HBSS for injection. Only single cell suspensions with greaterthan 90% viability were used in the in vivo experiments.

Tumor Cell Injections s.c. Mice were given injections, at the centrals.c. site, of 1 x IO6 viable tumor cells suspended in 0.2 ml of HBSS.

Tumor volume =length x (width2)

Tumor weight values were plotted against days postinjection. Tumordoubling time was thus calculated. When s.c. tumors reached 2 cm indiameter, the mice were killed. Visceral organs were removed andrinsed in water, and the lungs were inflated and fixed in Bouin's

solution. After fixation for 24 h, lungs were examined under a dissectingmicroscope, and the number of peripheral lung tumor nodules largerthan 0.1 mm in diameter were determined. The neoplastic nature ofthese lung nodules was confirmed by histological examination. All thes.c. tumors and visceral organs with suspected metastasis were fixed in10% buffered formalin solution and examined histologically.

Tumor Cell Injections i.v. To produce pulmonary tumor colonies(experimental métastases),mice were given i.v. injections of 1 x IO6

viable tumor cells in 0.2 ml HBSS through the lateral tail vein. Eightweeks later the mice were killed, the lungs fixed in Bouin's solution,

and the number of peripheral tumor nodules was determined.Tumor Cell Injections i.p. Mice were given i.p. injections of 1 X IO6

viable tumor cells in 0.2 ml HBSS. Eight weeks after the injection oftumor cells, mice were killed and autopsied, and visceral organs werecollected and examined for the presence of tumor growth by gross andhistological criteria.

Tumor Cell Injections i.s. After the mice were anesthetized withmethoxyflurane, we injected into the spleen 1 x IO6viable tumor cells

suspended in 0.05 ml of HBSS according to the methods described indetail previously (19). Eight weeks after the injection of tumor cells,the mice were killed and autopsied. All visceral organs were examinedby gross and histological procedures to verify tumor growth.

Tumor Cell Injection into the RSC. Mice were anesthetized withmethoxyflurane and placed in the left lateral decubitus position. Avertical incision was made in the right flank through the skin andperitoneum, exposing the lateral aspect of the kidney. The kidney waslifted gently and stabilized. A 27-gauge needle was inserted into therenal parenchyma from the lower pole of the kidney and advanced untilits point reached just below the renal capsule. At this time the micewere given injections of 1 x IO6viable tumor cells in 0.05 ml HBSS. A

visible bulla formation between renal parenchyma and capsule and thelack of significant bleeding or extrarenai leaking of the injected tumorcell suspension were the criteria for a successful injection. After injection, the kidney was returned to the abdominal cavity, and the woundwas closed in one layer with wound clips. When the mice becamemoribund they were autopsied. The injected kidney was removed andfixed in 10% buffered formalin solution after measuring the weight. Allvisceral organs were fixed in formalin or Bouin's solution. Determina

tions of metastasis were carried out after gross and histological examination.

Statistical Analysis. The results of all in vivo studies were analyzedby the Mann-Whitney U test (2-tailed).

RESULTS

Growth Rate in Vitro and Tumor Growth and Metastasis afters.c. Injection. The growth rates of the cells from the five linesin vitro and in the subcutis of nude mice are shown in Table 1.The tumor doubling time in vitro did not differ significantlyamong the lines, with the exception of the SN12C line initiallyadapted to culture, which exhibited the shortest doubling time.In contrast, the median tumor doubling time of each line at as.c. site of nude mice differed, ranging from 5.2 to 11.9 days.All lines were tumorigenic, but SN 12L1 cells grew most rapidly.The growth rate of SN12K1 at the s.c. site was the slowestamong the lines. A significant difference was observed in thegrowth rate of SN12L1 and SN12C (P < 0.005), SN12K1 (P< 0.005), or SN12A1 (P < 0.005). No direct relationship wasfound between the growth rates of tumor cells in vitro and at a

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GROWTH AND METASTASIS OF HRCC IN NUDE MICE

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Fig. 2. a. histology of the original human renal tumor, b-f. Phase-contrast micrographs of the tumor cells from five lines of HRCC grow ing as monolayer cultures.b, SN12C cultured line directly derived from the surgical specimen of the HRCC; e, SN12SI derived from the HRCC cells growing as a solid tumor in the subcutisof a nude mouse; d, SN12K1 derived from the HRCC cells growing in the kidney of a nude mouse; e, SNI2L1 derived from a liver metastasis of the HRCC growingin the kidney of a nude mouse;/ SNI2A1 derived from an ascitic tumor in a nude mouse with a HRCC growing in the kidney.

s.c. site. Irrespective of the tumor cell lines, a large part of thegrowing tumor in the subcutis was necrotic. The tumors contained a large quantity of necrotic fluid, and occasionally theirsurface was ulcerated. This was especially noticeable in thetumors produced by cells from the SN12S1 line.

The metastatic behavior of the five lines after s.c. injection isalso shown in Table 1. Spontaneous metastasis to lungs of micefrom s.c. tumors was found in most of the animals. The numberof lung tumor colonies, however, differed among the various

41

groups. The highest number of lung métastaseswas producedby cells of SN12S1 (skin isolated tumor) and the lowest numberof pulmonary métastaseswas produced by cells of SN12A1(ascites-isolated tumor). Extrapulmonary métastases werefound in only one mouse given an injection of SN12K1 andone mouse given an injection of SN12L1 cells, in which metastasis to the axillary lymph node was found.

Experimental Pulmonary Metastasis. The production of experimental lung métastasesby HRCC cells injected i.v. into

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GROWTH AND METASTASIS OF HRCC IN NUDE MICE

used for the transplantation of heterogeneous human tumors.In other words, the routine transplantation of human tumorssuch as colorectal carcinoma, lung carcinoma, HRCC, andothers into the s.c. space of nude mice is relatively easy, butdoes it necessarily yield cell lines that resemble the originalhuman tumor? If a human tumor is biologically heterogeneous,some of its cells may possess a growth advantage in one anatomic site or another. The growth of HRCC in the skin or thekidney of nude mice could, therefore, favor the proliferation ofdifferent cells.

The growth rate of cells from the 5 lines varied under bothin vitro and in vivo (s.c. or kidney implantation) conditions. Nodirect correlation among growth in vitro, in the s.c. site, or inthe kidney was found. The growth rate of tumor cells in vitrocan be regulated by manipulation of the environment (i.e., byserum, media, temperature) (34). Similarly, different organenvironments influence the growth of some but not all tumorcells (4, 7, 21, 22, 24, 27-29, 33). Indeed, this specific interaction of tumor cells with host environment was the basis for theoriginal "seed and soil" hypothesis by Paget (3).

The growth of the HRCC in the skin, kidney, and livermetastasis and in the ascites form in the nude mice must haveselected for cells with different metastatic properties. We basethis conclusion on the following, (a) The ranking of cell linesfor metastatic propensity was very similar regardless of the siteof implantation. Cells of the SN12L1 line were most metastaticfollowed by cells from SN12S1,SN12K1,SN12C, and SN12A1lines. SN12L1 cells exhibited invasive and metastatic propertiesregardless of organ site for their implantation (s.c., i.V., i.p., orRSC). In contrast, cells of the SN12C or SN12A1 lines werepoorly metastatic regardless of the site of implantation, (b) Themetastatic properties, in nude mice, of the cells were stabledespite 8-week assays, and expression of full metastatic pattern(pulmonary and extrapulmonary foci) required that the HRCCcells be implanted into the kidney.

Although metastasis of human neoplasms transplanted intonude mice has been considered to be rare (9, 10, 14, 18), recentstudies have indicated that the incidence of metastasis in nudemice can be increased by simple manipulation of the route oftumor cell injection (19, 23, 24). Kyriazis et al. (23) injectedcells s.c. or i.p. from human tumor lines of larynx or coloncarcinoma into nude mice and showed that only tumors growingi.p. produced metastasis to the lung. Moreover, human tumorsimplanted in the cranial s.c. site of nude mice grew faster andmetastasized more frequently than the same tumors implantedin the posterior aspect of the trunk (24). Kozlowski et al. (19)demonstrated that the i.s. injection, but not i.v. injection, allowed the most dramatic expression of metastatic capacity inseveral human tumor cell lines with the exception of a HRCCline. Furthermore, most recent studies from our laboratory (32)demonstrated that the expression of metastatic potential offreshly isolated human colorectal carcinomas could be achievedafter i.s. injection and not after i.v., s.c., or i.m. injection.Although the implantation of cells into the spleen is mostadvantageous for human colorectal carcinoma cells (32), it wasnot for the HRCC cells (Table 4); these data agree with ourearlier data using established HRCC cell lines (19).

The full expression of metastatic potential of HRCC cellswas obtained after growth in the kidney by some but not by allthe HRCC cells. These results reemphasize that both tumorcell properties and host factors determine the outcome of metastasis (5, 6). The s.c. implantation of the HRCC cells into thenude mice produced tumors with a high degree of necrosis.Interestingly, spontaneous lung métastaseswere produced by

cells of the SN12S1 line (originally isolated from a tumorgrowing s.c.) but not from other lines.

Since the conversion from solid tumor to ascitic form by i.p.passaging of cells could result in an increased metastatic capacity of the tumor cells (35-37), we expected cells of the SN12A1line to have high metastatic potential. This was not the case,but the SN12A1 line was not derived from a solid tumorartificially converted to ascites tumor but rather from an ascitestumor that spontaneously occurred in a mouse with a tumorgrowing in the kidney. This line did produce the highest incidence of ascitic tumors after i.p. implantation, suggesting onceagain that the process for its origin was selective. Nearly 30years ago, Klein (36) demonstrated that the gradual conversionof certain solid neoplasms into ascites tumors represented theselective overgrowth of a small number of cells that differedfrom the majority of the original population with regard totheir capacity to multiply under the environmental conditionsof the peritoneal fluid. Line SN12A1 could well be a selectedpopulation from the HRCC tumor having preferential growthin the peritoneal cavity. Growth in the peritoneum, however,and metastatic potential did not correlate in our studies. Themost metastatic line, SN 12L1, did not produce a high incidenceof ascites subsequent to i.p. implantation (Table 3).

In conclusion, nude mice can be used effectively to selectHRCC variant lines with different and stable metastatic properties. The expression of metastatic potential of HRCC in nudemice depends on both the intrinsic properties of the HRCCcells and the organ site of their implantation. For renal cellcarcinoma, the kidney is the natural organ of implantationwhich allows full expression of metastatic potential.

ACKNOWLEDGMENTS

We thank Dr. Corazón Bucana for help in preparing the figures,Debora Campbell for help with tumor cell injections. Dr. KennethWishnow for supplying the surgical specimen, and Dr. Sen Pathak andAngela Goodacre for chromosome analyses.

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1986;46:4109-4115. Cancer Res   Seiji Naito, Andrew C. von Eschenbach, Raffaella Giavazzi, et al.   MiceRenal Cell Carcinoma Implanted into Different Organs of Nude Growth and Metastasis of Tumor Cells Isolated from a Human

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