[CANCER RESEARCH 43, 5451-5455, November 1983]

New Method to Quantitate Clonogenic Tumor Cells in the BloodCirculation of Mice1

Norio Suzuki

Section of Radiobiology, The Johns Hopkins Oncology Center, Baltimore, Maryland 21205

ABSTRACT

A bioassay method to quantitate "clonogenic" tumor cells

released into the blood circulation from murine primary tumorsis described. The method uses preirradiation of the thorax of thetumor-bearing mice, followed 22 hr later by preparation and

culture of a lung cell suspension which contains filtered tumorcells from the blood. The malignant cells form colonies. Ourresults indicate that the number of "clonogenic" tumor cells in

the blood circulation can be quantitated efficiently using only afew mice. This is a major advantage, since previous studies werelimited by small amounts of available blood and somewhat uncertain techniques for identification of blood-borne tumor cells.The present method allows us to evaluate "clonogenic" tumor

cells and tumorigenicity, which microscopical identification oftumor cells after filtration of a small amount of blood does notpermit.

INTRODUCTION

Prevention or control of metastasis could be achieved byblocking the metastatic processes either at tumor cell releasefrom the primary sites or transportation or trapping and growthat the secondary sites. The tumor cell release has been of generalconcern (3,4, 7,11-13,16,17, 24-27) in studies on metastasis,

but the studies have been limited by lack of adequate methods.Numerous investigations of over 20 years on patients' blood

failed to establish a positive correlation between the presence oftumor cells in the blood and prognosis (17). However, the significance of the results from these clinical studies is fairly limitedbecause most studies (17) used very small amounts of bloodrelative to the total human blood volume, relied on cytologicalidentification of tumor cells trapped on membranes, and did notinclude evaluation of clonogenicity and tumorigenicity of the cells.We need an efficient and reliable method to assay released"clonogenic" tumor cells, with which we can (a) quantify theCTCR2 processes, (b) determine the response of the CTCR

processes to treatment (e.g., local irradiation of primary tumors),and (c) evaluate the significance of the CTCR processes inspontaneous metastasis.

We have initiated studies to establish better methods to evaluate "clonogenic" tumor cells in the blood using completely

different approaches from the microscopic identification of membrane-trapped tumor cells commonly used in the past. In this

1This investigation was supported by Grant CA06973 awarded by the National

Cancer Institute, Department of Health and Human Services.2The abbreviations used are: CTCR or CTCR processes, clonogenic-tumor-cell-

release, the present method concerns "clonogenic" tumor cells released and not

all the cells released, which include dead or dying cells. Processes of CTCR mayinvolve "clonogenic" tumor cells ready to be released and/or related structures

such as blood vessels; SLME, spontaneous lung metastasis efficiency from tumorsinoculated i.m. in the leg; FMC, flow cytometry.

Received March 14,1983; accepted August 4, 1983.

report, we describe a practical and reliable method to quantitate"clonogenic" tumor cells in the blood circulation.

MATERIALS AND METHODS

Tumors and Mice. FSA1231 and FSA1233 were isolated by soft agarcloning from a methylcholanthrene-induced fibrosarcoma and have beencharacterized in the past several years (18-23). The cells are stored in

a liquid nitrogen freezer. Every month, old cultures were replaced withnew cells from the frozen stock. The cells were grown in McCoy's

Medium 5A supplemented with 15% fetal bovine serum in 32-oz pre

scription bottles. The NFSA2ALM1 was recently established in ourlaboratory from a spontaneous fibrosarcoma NFSA (1). These cells werecultured in Fischer's medium supplemented with 10% horse serum. Male

10-week-old C3H/H3J mice, syngeneic hosts to these tumors, were

purchased from The Jackson Laboratory, Bar Harbor, Maine.Lung-mediated Assay of Blood-borne Clonogenic Tumor Cells.

The mice were inoculated i.m. in a hind leg with 5 x 105 of either

FSA1231 or FSA1233 cells suspended in 0.1 ml medium. The cellsuspensions were prepared from late log phase in vitro cultures. Themice were irradiated with 150 grays locally at the thorax, 40 days afterthe inoculation, using a 137Cs i-ray irradiator at 11.5 Gy/min under

anesthesia with pentobarbital sodium (40 mg/kg). Anesthetized mice(maximum, 7 mice at a time; a dose-flattening filter was installed) were

taped on a Lucite plate to locate their thorax along a slit of a collimator.Radiation dose (midline dose) and dose distribution (adjustment of collimator and mouse position) were controlled by film and thermoluminesc-

ence dosimetry. In case of NFSA2ALM1, mice were inoculated with 2 x104 cells, and thorax irradiation was given 31 days later. The thorax

irradiation was intended to eradicate tumor cells already metastasizedto the lung and also to enhance lung trapping and retention of tumorcells (2, 8, 10, 14, 28). The mice (3 mice/group) were killed immediately(0 hr-control) or 22 hr after irradiation. The lungs were removed and

rinsed with cold 0.9% NaCI solution, minced with scissors, incubated for1 hr at 37°with protease (types IX, 2 mg/ml) and DNase I (1 mg/ml)(both from Sigma Chemical Co., St. Louis, Mo.) in Puck's Saline G and

then stirred for 30 min at room temperature. The whole preparation waswashed 3 times by centrifugation. The cell suspensions were placed in150-sq cm flasks (Corning Glass Works, Corning, N. Y.) containing 20ml McCoy's Medium 5A supplemented with 15% fetal bovine serum

(Grand Island Biological Co., Grand Island, N. Y.). Heavily irradiated (120Gy) FSA1231, FSA1233, or NFSA2ALM1 cells from culture were alsoincluded as feeder cells (106/flask). An additional 10 ml of medium was

added 2 days later, and thereafter medium was changed every 3 to 4days when medium became acidic, with careful handling to avoid disrupting colonies. Colonies were stained 2 weeks later with 0.5% crystalviolet solution in 95% ethanol.

SLME Assay. The tumor cell suspensions were prepared from latelog phase in vitro cultures and inoculated into a leg i.m. at 5 x 105 forFSA1231 and FSA1233 cells and 2x10" for NFSA2ALM1 cells, and the

mice were killed 42 and 33 days later, respectively. In these conditions,all animals develop primary tumors. Lung nodules were scored macro-scopically after overnight fixation in Bouin's fluid (18, 21 -23).

Cell Counting and Volume Analysis. As a routine procedure, thecells from culture were always monitored for cell number and modal peakposition of cell volume distribution. This and FCM analysis of the cell

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N. Suzuki

suspensions assure reproducibility of the experiments. Cell counts andvolume distribution analysis were carried out with a Model ZBI CoulterCounter and a Channelyzer II Multichannel analyzer and plotter (CoulterElectronics, Hialeah, Fla.). The system was calibrated with latex beads.The average cell volume for cells in a given sample was calculated fromthe modal channel number of the volume distribution (18, 23).

FCM. Cells were first fixed with 70% ethanol and stained with mith-

ramycin (Mithracin; Charles Pfizer and Co., Inc., New York, N. Y.) forDNA content analysis according to the method described by Crissmanand Tobey (5), as used earlier (18-20). The staining solution contained

mithramycin (50 ^g/ml) and 7.5 HIM MgCI2 in 12.5% aqueous ethanol.FCM analysis was performed using FACS II (Becton Dickinson, Sunnyvale, Calif.) with laser wavelength setting of 457.9 nm.

RESULTS

Lung-mediated Assay of Blood-borne Tumor Cells. Fig. 1shows tumor cell colonies in 150-sq cm flasks. AlthoughFSA1231 and FSA1233 can grow in soft agar-containing me

dium, which decreases contaminating normal cell growth (20),regular surface culture was also satisfactory and had the addedadvantage of easier colony counting. As shown in Fig. 1, normalcells in the background were very limited (thorax was preirra-

diated with 150 Gy) and did not disturb quantitation of tumor cellcolonies, which were larger and more dense (Flasks A, B, andC). Microscopically, also there was an obvious difference between the tumor cell colonies and colony-like (this does not seem

to be reproductive growth) normal cell growth; tumor cells wererandomly overlapping each other while normal cells were in onelayer of diffuse growth (Fig. 2).

Identification of the Colonies. These colonies were identifiedas tumor cells by DNA content distribution by FCM, sinceFSA1231 and FSA1233 have G, DNA content of about 1.6 and3.1 relative to Gìnormal cells (19) (Chart 1), and by tumorigenicityin the syngeneic host mice. Cells in flasks from control and 22-

hr groups were once trypsinized and propagated for severaladditional days prior to injection. The trypsinized cell suspensionfrom each flask was centrifuged and resuspended in 0.4 ml ofmedium and then injected into the leg of 3 mice (10-week-old

male C3H/HeJ, unirradiated mice; 0.1 ml/mouse). The remainingcells were used for DNA content determination by FCM (Chart1). The cells from control flasks showed little growth in thesecondary cultures while the cell cultures from 22-hr flasks werevery vigorous. None of the mice given injections of control cellsdeveloped tumors, but all the mice with cells from 22-hr flasksdeveloped tumors.

Cell Recovery and Retention. To estimate recovery of blood-borne tumor cells with the current method, 4 FSA1231 tumor-bearing mice were given i.v. injections of FSA1231 cultured cells,5 x 10"/mouse, right after 150-Gy thorax irradiation, which was

then followed by immediate killing and preparation of lung cellsuspensions with the standard method. Small portions of the cellsuspension were plated in 75-sq cm flasks (4 flasks/group) with

heavily irradiated FSA1231 feeder cells. The colony numberswere 152 ±20 (S.D.) from a portion of the lung cell suspensioncalculated to contain 1.8 x 104 injected cells, i.e., 0.8% recovery.

If that figure is corrected for clonogenicity of the injected cellsuspension (15% plating efficiency for FSA1231), the correctedrecovery rate through lung trapping, mincing, digesting, andculture is 5.3%. Similar studies but using no tumor bearing mice(therefore, no additional tumor cell supply to the initially injectedamount) to test retention were performed by making lung cellsuspensions at 0 or 22 hr after i.v. injection. Recovery percentage (mean of 6 to 10 10-cm Retri dishes) at 0 and 22 hr were,

respectively: 0.46 ±0.07, 0.53 ±0.06 (NFSA2ALM1); 1.87 ±0.51, 1.22 ± 0.23 (FSA1231); 1.72 ± 0.76, 2.30 ± 1.29(FSA1233). These results indicate that the number of clonogenictumor cells (i.v. injected and once trapped at the lung) remainsat the same level after 22 hr, although some increase by celldivision and decrease by cell loss may be involved.

Application to Different Tumor Systems. As shown in Table1, the present method was proven effective in 3 different tumorsystems. The SLMEs were determined at 42 days (FSA1231and FSA1233) and 33 days (NFSA2ALM1) after i.m. inoculationof the tumor cells (host mice start to die around these times).The lung-mediated assay of blood-borne tumor cells was per

formed 2 days prior to the killing times for SLME assay. The

Chart 1. DNA content profile of the cellsfrom the colonies. Cellular DNA content profileswere determined by FCM of the trypsinized cellsuspensions of the primary cultures (A and B)and the secondary cultures (C and D ) of thecolonies derived from the blood-borne tumor

cells; FSA1231 (A and C) and FSA1233 (B and0) Normal cell peaks at around 20 channelsare decreased during cultures indicating poorgrowth of normal cells derived from once heavilyirradiated lung tissues.

500

400

300

CO200_l

UJ 100

3o

UJ00

200

100 -

\J,.

100 200 0 100

CHANNEL NUMBER (DNA CONTENT)

200

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Lung-mediated Assay of Clonogenic Blood-borne Tumor Cells

Table 1

Tumor cell colonies, SLMEs and tumor volumes

TumorsFSA1231

FSA1233NFSA2ALM1Colonies"35.3

± 8.7e

15.7± 5.787.0 ±13.0Lung

metastasis positive/

totalmice25/70(36)"

10/98 (10)15/15(100)SLMEsLung

metastasisnodules/mouse0.89

±0.22 (0-9)"

0.21 ±0.09 (0-8)28.4 ±7.2 (1-90)Tumor/volumes6

(cumm)5,800±200 (22)'

9,500 ±400 (26)3,400 ±100(15)

* Colonies, Clonogenic tumor cells released into the blood and trapped in the lung during 22 hr. Colony

numbers per 3 mice. The experiments were performed parallel to SLME experiments, i.e., 150 Gy of thoraxirradiation was given 2 days prior to the killing dates for SLME.

" Tumor volumes were measured 1 day prior to SLME assay. Volumes were calculated by *-/6 x a x öx

c (3 diameters measured by a caliper).c Mean ±S.E. of 3 experiments." Numbers in parentheses, percentage.e Numbers in parentheses, range.' Numbers in parentheses, number of mice measured.

respective colony number and SLME for FSA1231, FSA1233,and NFSA2ALM1 indicate a positive correlation between thenumber of Clonogenic tumor cells in the blood and SLME. Theprimary tumor size did not correlate among different tumorsystems with the number of Clonogenic blood-borne tumor cells

or with the SLME.Radiation Response of CTCR Processes. Local irradiation

of primary tumors (NFSA2ALM1 at 33 days postinoculation) with10 Gy was immediately followed by a 150-Gy thorax irradiationand the routine lung-mediated CTCR assay for 22 hr. In 2

separate experiments, the mice (3 mice/group) irradiated locallywith 10 Gy had only 13,13 Clonogenic blood-borne tumor cells

compared with 99, 75 for the control mice with no local irradiation. This indicates that the local irradiation of the primary tumorswith 10 Gy drastically reduced CTCR in this system. Thus, thepresent method could be usable to measure radiation responseof CTCR processes. The CTCR processes affected by irradiationmay be Clonogenic tumor cells ready to be released and/orrelated structures such as blood vessels.

DISCUSSION

The small size of a mouse limits the amount of blood whichcan be drawn for analysis. Therefore, detection or direct measurement of tumor cells in the blood of tumor-bearing mice is

very difficult. Some of the studies reported in the past includeinjection of blood into secondary hosts, i.e., s.c. injection or lungcolony formation by i.v. injection (27), filtration of tumor cellsfrom blood on membranes with cytological identification (3, 11-

13,16,17).In the present method, during the 22-hr incubation time after

thorax irradiation, about 1300 ml of blood would be filtered bythe lungs if a 1-ml/min blood flow is assumed (11). Therefore,

the method is applicable to a system with less frequent tumorcells in the blood. Similarly, the blood is filtered over a period oftime, instead of instantaneous sampling; therefore, we may beable to determine the release rate of Clonogenic tumor cells fromthe primary tumors and compare release rates from primarytumors of various types and under different conditions. Thismethod does not require complex operations such as insertionof cannulae into the tumor blood vessels and perfusion of tumorsand similarly avoids potential perturbations of the animal andtumor by such techniques. Additionally, we are now able toquantitate Clonogenic tumor cells in the blood by counting colo

nies grown in culture and to isolate viable tumor cells from thecolonies for further analysis.

Earlier reports indicate that tumor cells injected i.v. as a bolusinto healthy or tumor-bearing mice (2, 6-10, 14, 15, 28) are

trapped indiscriminately at the lung, with the majority of the cellsdisappearing in a few days leaving only a minor portion of thecells retained alive; this process could be selective, and the initialindiscriminate trapping may not have significance for eventualdevelopment of lung nodules (6, 7). Cell cycle stage, cell size,and cell clumps may affect the trapping and retention at the lung(10, 12, 18). However, preirradiation of the thorax may producemore efficient retention of all tumor cells at the lung by reducingsubsequent clearance (2, 8, 10, 14, 28). Methods of isolatingtumor cells from lung tissues into culture have been widely usedin various studies (7, 21), and the FSA tumor cells are known toovergrow the contaminating normal cells from the lung (21).Further, in the present method, the thorax is heavily irradiatedwith 150 Gy. Therefore, it seems unlikely that normal cells fromwithin the irradiated volume could survive; the majority of normalcells are killed.

Presently, we know neither the exact trapping-retention rate

of spontaneously released tumor cells at the lung after 150 Gynor the recovery rate of the spontaneously released tumor cellsafter mincing, digesting, and culturing. However, the presentmethod is readily usable to quantify relative differences of CTCRefficiency (e.g., to determine the effect of irradiation of primarytumors on its CTCR processes). In order to quantify the absolutenumber of released "Clonogenic" tumor cells or release efficiency,

correction factors for lung trapping efficiency, retention or lossrate in the lung, and recovery rate through mincing, digestingwith enzymes, and colony formation in vitro have to be determined for each tumor system. The corrected recovery rate usingcultured FSA1231 cells was 5.3%, which, we think, can beimproved further. Regarding retention data determined by directmeasurement of the Clonogenic tumor cells trapped in the lungafter i.v. injection, the difference between the present method(the number of Clonogenic tumor cells once trapped at the lungremained at the same level after 22 hr) and radioisotope-labeled

tumor cell studies (most of the radioactivity, 90 to 99%, disappears in 1 day) cannot be explained by cell division of trappedtumor cells (at most, a 2- to 4-fold increase is reasonablyexpected). "Clonogenicity" means colony-forming ability either invitro or in vivo, while radioisotope labeling (e.g., [125l]iododeox-

yuridine) simply indicates that the cells incorporated iododeox-

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N. Suzuki

yuridine or synthesized DNA at that time and these cells may bedying in a few cell divisions thereafter. This raises a fundamentalquestion concerning the radioisotope-labeled tumor cell method.

Is the major decline (90 to 99%) of radioactivity at the initial 1day really caused by disappearance of the "clonogenic" tumor

cells trapped at the lung? These problems are under investigation.

While a conventional conclusion elucidated from the numerousclinical studies has been that the mere existence of tumor cellsin the blood circulation is not critical for prognosis or metastasisdevelopment (17), the present method may serve to reevaluatethis by determining whether the number of "clonogenic" tumor

cells released from the primary into the blood is important forspontaneous metastasis.

Thus, we think that the current method could be usable tostudy the effects of various treatments on CTCR processes(tumor cells ready to be released and/or related structures suchas blood vessels) or released tumor cells. With additional studies,the present method may be useful to further clarify the role ofCTCR processes in metastasis and the significance of tumorcells in the blood circulation.

ACKNOWLEDGMENTS

I would like to thank Scott Kuperman for excellent technicalassistancewith theexperiments, Michael O'Neill and Dr. W-C. Lam for radiation dosimetry, and Dr.

Ralph E. Durandfor critical readingof the manuscript and for the use of a computerprogram for chart drawing.

Animals used in this study were maintainedin facilitiesapproved by the AmericanAssociation for Accreditation of Laboratory Animal Care and in accordance withcurrent United States Department of Agriculture and Department of Health andHuman Services, NIH, regulations and standards.

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Fig. 1. Colonies developed from lung-trapped blood-borne tumor cells. A, FSA1231, 22 hr; S, FSA1233, 22 hr; C, NFSA2ALM1. 22 hr; corresponding 0-hr controls atbottom. Feeder cells alone (not shown) did not develop any growth.

Fig. 2. Microscopic pictures of the colonies. A part of a FSA1233 derived colony (left) and a colony-like growth of normal cells (right) with their higher magnificationsat the bottom. Crystal violet, (op, x 10; bottom, x 40.

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1983;43:5451-5455. Cancer Res Norio Suzuki Circulation of MiceNew Method to Quantitate Clonogenic Tumor Cells in the Blood

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