Separation of Cells From a Murine Fibrosarcoma on the Basis of Size. I. Relationship Between Cell Size and Age as Modified by Growth In Vivo or In Vitro 1,2,3 D. J. Grdina,

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L. J. Peters,

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S. Jones,

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and E. Chan

ABSTRACT -Cells from a murine fibrosarcoma (FSa) that had been Induced by 3-methylcholanthrene were separated and synchronized on the basis of cell size by centrifugal elutrlatlon. More than 10· tumor cells were elutrlated either Immediately following disaggregation of a solid tumor or after 48 hours of Incubation in vitro, Among the tumor cells elutriated immediately after disaggregation, the tumor suspension contained up to 32% normal diploid cells. Normal cells were not detected, however, in the suspension obtained after In vitro Incubation. Recovery of cells after elutrlatlon was 85%, and cell viability was greater than 95%. Selected fractions of cells were pooled, and average cell volumes were determined. The (modal) size of FSa cells after Incubation in vitro for 48 hours Increased from 980 to 1,620 /-13. These size differences were also reflected In the size of the cells comprising the pooled elutrlated subpopulations. Modal volumes ranged from 740 to 1,300 /-13 for FSa cells separated without prior in vitro Incubation as compared with 880-2,150 /-13 for those cells separated with prior In vitro Incubation. With the use of the method of flow mlcrofluorometry, the cell cycle parameters and the relative synchrony of each of the separated populations were determined. Cells incubated In vitro for 2 days were more efficiently synchronized than were cells separated immediately following excision of the tumor. The latter situation reflects the heterogeneity of size of cells in the same phase of the division cycle present in the tumor growing In vivo. The increased efficiency of elutriation In separating synchronized cohorts from cell populations incubated in vitro was probably due to the induction of a more homogeneous class of cells as a result of exposure to a uniform environment.-J Nail Cancer Inst 61: 209-214, 1978.

Many factors determine whether an iv disseminated tumor cell will form a metastasis. They may be related to the tumor host or intrinsic properties of the tumor cell (1). Aspects of the tumor cell that have received little study in this regard include the interrelated parameters of tumor cell size and age in the division cycle. To study properties related to the cycle of tumor cells in vivo, the ability to separate large numbers of cells derived from disaggregated solid tumors into subpopulations homogeneous with respect to their relative position in the cell cycle is advantageous. Cell size increases steadily and exponentially with age during the cell cycle (2). Partially synchronized subpopulations of cells have been successfully separated from asynchronous populations following vdocity centrifugation (3) and velocity sedimentation at unit gravity (4). Each of these methods has certain disadvantages: The first method is limited as to the resolution achieved and the number of cells that can be separated; the second method is time consuming and requires large volumes of gradient material. Recently, a method employing centrifugal elutriation has been described to separate and synchronize murine L-P59 fibroblasts grown in vitro (5). The method is rapid, and VoL. 61. NO. I. JULY 1978

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many cells can be separated without loss of viability. In this communication we describe the usefulness of this method in separating and synchronizing tumor cells from a murine FSa pursuant to a study of their in vivo lung colony-forming efficiency. Separated cell populations were characterized with respect to their sedimentation rates and average cell volumes. The method of FMF was used to determine the relative DNA content and thus the age distribution of cells in each of the separated su bpopulations.

MATERIALS AND METHODS Mice.-Female C3HflBu mice from our specific pathogen-free breeding colony were 9-10 weeks old at the initiation of each experiment. They were kept 5 per cage and were maintained on a sterile food diet and a 12-hour light-dark cycle. Tumor.- The tumor used was a 3-methylcholanthrene-induced FSa immunogenic in its syngeneic host (6). Tumors were grown from 5X105 tumor cells; each experimental mouse was given an im injection in both hind legs. All experiments were performed with the use of fifth-generation tumors having average diameters of 10-12 mm. ABBREVIATIONS lISEO: CV=coefficient(s) of variation; FCS=fetal calf serum; FMF=flow microfluorometry; FSa=fibrosarcoma(s); NDA=2· naphthol-6,8-disulfonic acid; NDC=normal diploid cells. Received October 7, 1977; accepted February 21, 1978. Supported by Public Health Service grants CA18628, CAl 7769, and CA06294 from the National Cancer Institute. , Animals used in this study were maintained in facilities approved by the American Association for Accreditation of Laboratory Animal Care and in accordance with current regulations and standards of the U.S. Department of Agriculture and U.S. Department of Health, Education, and Welfare, National Institutes of Health. • Section of Experimental Radiotherapy, The University of Texas System Cancer Center, M. D. Anderson Hospital and Tumor Institute, 6723 Bertner Ave., Houston, Tex. 77030. 5 Present address: Section of Experimental Pathology, The Univer· sity of Texas System Cancer Center. 6 This work was conducted with the technical assistance of Mr. Mike Lesem, Section of Experimental Radiotherapy, The University of Texas System Cancer Center. We thank Mr. J. Oro for performing the FMF analysis at the Physics Department, The University of Houston. We also acknowledge the use in the FMF analysis of computer subroutine STEPT written by J. P. Chandler, Department of Computing and Information Sciences, Oklahoma State University, Stillwater, Oklahoma, and distributed by the Quantum Chemistry Program Exchange. 1

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Tumor cell suspension.-In each experiment, 6 tumors were excised and single-cell suspensions were prepared by their being minced and trypsinized according to a method described in (7). Cell viability, determined by phase-contrast microscopy, was routinely greater than 95%. The yield of viable cells was about 108 /g of excised tumor tissue. The tumor suspensions were either separated immediately by centrifugal elutriation or incubated in vitro for 48 hours prior to separation. When incubated in vitro for 48 hours before separation, 1.5X107 viable cells were seeded into 32ounce glass culture bottles (10 bottles/expt) and incubated at 37° C in a water-saturated atmosphere of 5% C02 and air. The growth medium was modified by McCoy's 5-A medium supplemented with 20% FCS (8). After 24 hours, the supernatant containing floating cells was carefully discarded, and 20 ml of fresh medium was added to each culture bottle. The attached tumor cells were then incubated for an additional 24 hours prior to separation by centrifugal elutriation. Cell separation.-FSa cells were separated by centrifugal elutriation with the use of a Beckman JE-6 elutriator rotor (Beckman Instruments, Inc., Cedar Grove, N.J.) (9). The system was sterilized with 70% ethanol and maintained at 4° C (5). In the separation a modified McCoy's 5-A medium supplemented with 5% FCS was used. Also included was DNase (Sigma Chemical Co., St. Louis, Mo.) at a final concentration of 0.1 mg/ml and 5 mM NDA. NDA is nontoxic to cells and was added to minimize cell clumping (10). Between 108 and 2X10 8 cells were suspended in 20 ml of medium and were introduced into the elutriator chamber at a flow rate of 5.4 mllminute, with a rotor speed of 1,525 rpm. Throughout the separation of the cells, the rotor speed was held constant while the flow rates were varied by equal increments of 2 mllminute from 5.4 to 25.4 mllminute. Collected were II fractions as well as a final wash of the rotor (fraction 12). With the exception of the first fraction (70 ml), all fractions were of 50-ml volumes. "Sedimentation velocity," as used here, is the measured sedimentation velocity (mm/hr) divided by the centrifugal acceleration in the elutriator (in multiples of "g," the Earth's gravitational acceleration) and is equal to the sedimentation velocity at unit gravity (5). Cell counting and volume analysis.-FSa cells were counted with the use of a model ZBI Coulter counter (Coulter Electronics, Garden Grove, CaliL) fitted with a 70-1-' diameter aperture. The volume distribution of cells was determined by means of the Coulter counter and a multichannel analyzer (Channelyzer II, Coulter Electronics) and X- Y recorder. The system was calibrated with the use of latex beads (18.04 I-' in diameter) supplied by Coulter Electronics. The "average" cell volume was designated as the volume corresponding to the modal channel number of the volume distribution of each sample. Cells were also counted with a hemacytometer. Viability was determined by phase-contrast microscopy. FMF.- The DNA content of viable FSa cells was measured by FMF with a laser wavelength setting of 457 nm (11). Cells were fixed in 70% ethanol and then

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stained with 50 mg mithramycin/ml (Mithracin; Pfizer Inc., New York, N.Y.) in solution with MgCb (7.5 mM) and 12.5% aqueous ethanol. [Mithramycin is a DNAspecific fluorescent dye that preferentially forms complexes with native DNA (12).] Computer analysis of FMF-DNA histograms.- The mathematical model used to fit these FMF data is described elsewhere (Johnston DA, White RA, Barlogie B: Submitted for publication). The CV of the Gl-phase tumor cell populations in percent was calculated as 100 times the standard deviation divided by the mean channel number of the fluorescent peak. This parameter can be influenced by both the variability of the fluorescence of the stained DNA in the G1-phase cells and instrumental variations. The CV as defined above ranged from 6 to 9%. The DNA histograms were at times offset either to the right or left of the origin on the abscissa. As a result, the mean of the Grphase peak was not always positioned at twice the channel number of the mean of the G1-phase tumor peak. We estimated the percent of normal cell contamination in each of the cell suspensions by determining the area under the G1-phase normal peak and dividing it by the area under the total DNA histogram.

RESULTS Centrifugal Elutrlatlon of Tumor Cells Cell suspensions made from FSa were separated either immediately or after 48 hours of incubation in vitro. Most of the tumor cells removed and separated immediately from the tumor sedimented between fractions 3 and 8. The average sedimentation velocity of these cells was calculated to be 10.7 mm/hour / g. In contrast, tumor cells incubted in vitro sedimented faster. These cells had an average sedimentation velocity of 15.1 mm/hour/g, and most of the cells were collected in fractions 6-10. Representative sedimentation profiles of FSa cells separated after either condition are presented in text-figure 1 for comparison. The recovery of cells after centrifugal elutriation was over 85%, and the viability of these cells was 95% or greater. No difference in recovery was observed between FSa cells separated either directly from the tumor or following in vitro incubation. In all experiments fractions 1 and 12 were discarded. Fraction 1 contained subcellular debris and damaged cells. Fraction 12 had both large and small cells that were washed out of the rotor at the end of the run. Average cell volume, along with sedimentation rate, increased with increasing fraction number (see text-fig. 1), indicating that separation was being achieved by exploiting differences in cell size. The largest size variation was observed among FSa cells incubated in vitro. Modal volumes ranged from 740 1-'3 for fraction 3 cells to 2,400 1-'3 for fraction 11 cells. The modal volume of the whole population was 1,620 p.3. In contrast, FSa cells separated without prior in vitro incubation varied in volume from 600 1-'3 to 1,400 1-'3, and the modal volume of the population was 980 1-'3. VoL. 61. NO. I. JULY 1978

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FSa Cells Sedimentation Velocity (mm/hr /"g") 5

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Separation of cells from a murine fibrosarcoma on the basis of size. I. Relationship between cell size and age as modified by growth in vivo or in vitro.

Separation of Cells From a Murine Fibrosarcoma on the Basis of Size. I. Relationship Between Cell Size and Age as Modified by Growth In Vivo or In Vit...
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