Isolation, Karyotype, and Clonal Growth of Heterogeneous ... · Isolation, Karyotype, and Clonal...

[CANCER RESEARCH 41, 2349-1359, June 1981]0008-5472/81 /0041-OOOOS02.00

Isolation, Karyotype, and Clonal Growth of Heterogeneous Subpopulations ofHuman Malignant Gliomas1

Joan R. Shapiro, Wai-Kwan A. Yung, and William R. Shapiro2

Department of Neurology Â¡J.R. S.. W. A. Y., W. R. S.] and. George C. Cotzias Laboratory of Neuro-Oncology [J. R. S., W. A. Y., W. R. S.J. Memorial Sloan-

Kettering Cancer Center, and Department of Neurology Â¡J.R. S., W. R. S.], Cornell University Medical College, New York, New York 10021

ABSTRACT

The histolÃ³gica! pleomorphism of human malignant gliomasas well as their variable response to therapy suggests that suchtumors are not homogeneous but are composed of a heterogeneous population of cells. To search for heterogeneity inthese tumors, we developed a protocol to identify by karyotype,isolate, and clone the subpopulations of the tumor. Freshlyresected tumors were mechanically dissociated into single cellsthat were grown in suspension and short-term monolayer cul

tures. Chromosomal preparations were collected over the first6 to 72 hr postresection. The karyotypes prepared comprisedthe "reference set" of chromosomes found in each tumor. The

dissociated single cells were also dilution plated for monolayerculture. Cells that attached were marked and isolated as cloneswhose karyotypes were compared to those in the reference setin order to identify those clones that were cellular representatives of the tumor. Expiant cultures established from the sametumor were grown in monolayer culture and cloned; theirkaryotypes were likewise compared to those in the referenceset. Eight gliomas were analyzed. Each had metaphases thatranged in chromosome number from hypodiploid to hyperploid,but the frequency distribution of the subpopulations differedamong the tumors. Defining a cellular subpopulation in thetumor as one yielding at least five karyotypically identical cellsin the reference set, we found that each tumor contained fromthree to 21 subpopulations. All but one tumor produced 100 ormore clones, and in seven tumors, more than 50% of theclones could be expanded for karyotyping; 7.6 to 25% of theseclones had karyotypes identifiable in the reference set of theirtumor. There was little relationship between the morphology ofthe clones and their karyotypes or growth kinetics. Whileexpiant cultures grew well, no clone derived from such culturescould be identified in the reference set. The protocol permitsstudies of clonal lines identifiable as cellular representatives ofa patient's tumor and not variants generated in tissue culture.

These studies confirmed that human malignant gliomas arecomprised of karyotypically heterogeneous cellular subpopulations. Preliminary work suggests that their phenotypic behavior may be similarly heterogeneous.

INTRODUCTION

Increasing evidence, both experimental and in the clinic,suggests that some cancers, notably solid tumors, are not

Received September 22, 1980; accepted March 13, 1981.' Supported by Grants CA 25956, CA 09207, and CA 08748 from the National

Cancer Institute.2 To whom requests for reprints should be addressed, at 1275 York Avenue,

New York. N. Y. 10021.

homogeneous in their cell composition but are composed ofheterogeneous cell types. This heterogeneity appears to contribute to the variable response to chemotherapy of the tumor(2-4, 7, 14) as well as to variations in its cell surface markers(10, 24, 30, 31), tumor antigens (6, 19, 39), growth rates (9,10, 13), and its capacity to produce intra- and extracellular

proteins (5). Mittleman (20, 21) demonstrated karyotypicallythe presence of clones and their progressive evolution in chemically induced tumors. Fidler and Kripke (12), Nicolson ef al.(22), and Raz ef al. (31 ) reported on the metastatic potentialand ability to select for various clones of a heterogeneoustumor population.

The evidence for heterogeneity of malignant brain tumorshas so far only been suggested by their pathological appearance. Indeed, it is the pleomorphic nature of the cell types inthe tumor that gives a glioblastoma its histologically multiformappearance. Our work in human brain tumors carried in nudemice suggested different chemosensitivities for tumors derivedfrom different patients (34, 35). One possible explanation forthis differential chemosensitivity was that contained within eachpatient's tumor were some cells that grew readily in the nude

mouse and were incidentally sensitive to different drugs.We chose an in vitro approach to explore for the presence

of heterogeneous cell populations in human gliomas. Resectedhuman glioma tissue has been studied in vitro using expiantand various tissue dissociation techniques (1, 16, 17, 26, 27,29, 33, 37, 38, 40-42). A valuable feature of establishedglioma cell lines aside from their infinite life span is their abilityto retain some of the phenotypic characteristics of the parentcell. To date, approximately 70 lines have been examined andcharacterized for various phenotypic expressions (25, 28).However, there is a major drawback to the use of long-term

cell lines as they often change and lose characteristics presentin vivo or in early passage. Conclusions about heterogeneity insuch lines are therefore made with difficulty since newly generated variant cells, with a selective advantage in vitro, couldrepopulate the cell line.

We therefore examined resected human gliomas using a newprotocol that permitted the identification and immediate isolation of dividing tumor cells. Single-cell suspensions derivedfrom dissociated tumor were karyotyped and dilution plated formonolayer culturing. This technique offered advantages overother cell culture systems established for solid tumor researchin that (a) chromosomal complements of the mitotic populationof the tumor could be examined in the first 72 hr postresection;(b) the clones derived from single cells could be isolated andexpanded 7 to 28 days postplating; and (c) a proportion ofclones isolated could be defined as cellular representatives ofthe tumor cells because they carried chromosomal complements identical to mitotic cells found in the solid tumor.

JUNE 1981 2349

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J. R. Shapiro et al.

MATERIALS AND METHODS

Culture Medium

The culture medium, Waymouth MAB 87/3, was purchasedfrom Grand Island Biological Co., Grand Island, N. Y., andsupplemented with 17 to 25% PCS3 purchased from Biofluids,

New York, N. Y. The percentage of PCS was determined by acloning assay for each serum lot. Waymouth's medium plus

20% PCS is referred to as the standard medium in this report.Conditioned medium was prepared for cloning experiments byremoving medium from a confluent monolayer of the sametumor line, diluting it with an equal volume of fresh standardmedium, and filtering it through a 0.22-j^m Millex-GS filter

(Millipore Corp., Bedford, Mass.) to remove any cell debris. Noantibiotics were used.

Cell Culture Protocol (Chart 1)

Freshly resected malignant glioma tissue from previouslyuntreated patients was placed in normal 0.9% NaCI solutionand delivered to the laboratory 0.5 to 1 hr following surgery.The tumor fragments were debrided of necrotic tissue andplaced in approximately 0.5 ml of standard medium. The fragments were minced with iris scissors into pieces no larger than1 to 2 cu mm.

Expiant Culture. Ten to 15 fragments from the mincedpreparation were placed in a 25-sq cm plastic culture flask

(Corning Glass Works, Corning, N. Y.) coated previously withstandard medium. An additional 0.5 ml of standard mediumwas first added to the flask before placing it in a 5% CO2incubator at 37Â°and added once again after 3 days of incu

bation. Expiants showing no cellular outgrowth by the seventhday were removed by suction. As each flask became semicon-fluent, the monolayer was trypsinized with trypsin-EDTA (1 x)(Grand Island Biological Co.) and plated into an additional 25-

sq cm flask. All monolayer cultures were divided (1:2) as theyreached confluency. Aliquots of cell samples were placed instandard medium containing 10% glycerol and frozen in liquidnitrogen. Chromosome preparations were done on expiantlines at passage 4.

Mechanical Dissociation into Single Cells. The remaining1- to 2-cu mm fragments were separated into 4 to 5 portions.

Each portion was minced to a fine pulp, standard medium (5.0ml) was added, and the mixture was aspirated gently 4 or 5times with a 10-ml syringe containing a 20-gauge needle. The5.0-ml cell suspension was allowed to settle for 5 min in a test

tube, and the top 3 ml were removed and gently aspirated asecond time. Microscopic examination revealed small clustersand many individual cells along with filamentous and lipidfragments. The liquid volume was increased to 10 ml, and thesuspension was allowed to settle for an additional 5 min. Thetop 5.0-ml portion contained single cells and cell debris.

Reference Karyotypes. A portion of the single dissociatedcells was divided for suspension cultures and for short-term

monolayer cultures. The suspension cultures were preparedby transferring 4 ml of the single-cell suspension to each ofseveral round-bottomed tubes to which 1 ml of standard medium was added. These tubes were incubated in 5% CO2 at

CELLCULTUREPROTOCOL

ExpiantCulture

Mechanical Dissociation

SuspensionCulture

Karyotyped6-24 hours

Short-TermMonolayer Culture

Karyotyped48-72 hours

Dilution-PlatedMonolayer Culture

REFERENCE KARYOTYPE CLONES

1The abbreviation used is: PCS, fetal calf serum.

Chart 1. Cell culture protocol. The chart depicts by flow diagram the protocolused to dissociate glioma tumors into 1 to 3-cumm fragments and single cells.The fragments are established as expiant cultures while the single cells aredivided for suspension and short-term monolayer, used for chromosome preparations, and dilution plated monolayer cultures in which clones can be isolatedand expanded for further characterization.

37Â°. Chromosome preparations were made as described below, beginning at 6 hr and continuing at 2 to 4-hr intervalsthrough 24 hr. The short-term monolayer cultures were prepared by concentrating the remaining single-cell suspensionsby centrifugation (850 rpm for 5 min) to approximately 9 x 105

cells in 1.5 ml. The cell concentrate was plated into a 25-sqcm flask and incubated in 5% CO2 at 37Â°.Chromosome prep

arations were made at 48 and 72 hr after incubation. Only inTumor M S was a sample removed at 24, 48, and 72 hr.

The chromosomes were prepared as follows. Each suspension culture was incubated for 1 to 2 hr with Colcemid (0.05iig/ml) (Grand Island Biological Co.). The cells were centri-

fuged into a pellet (850 rpm/5 min) and subjected to hypotonietreatment (0.075 M KCI) for 0.5 hr. The suspension was againpelleted (850 rpm/3 min) and resuspended in Carnoy's fixative

(absolute methanokglacial acetic acid, 3:1). The fixative waschanged 3 to 6 times before the preparation was applied toslides and allowed to air dry overnight at 37Â°. Q- (8) and G-

banding (32) were performed 2 to 7 days after fixation, withthe longer interval providing the best banding resolution. Forthe chromosome preparation from the short-term monolayer

culture, Colcemid (0.05 fig/ml) was added to the flask for 0.5to 1 hr, and the cells were harvested and prepared as above.The karyotypes from the 6 to 24-hr suspension culture chromosome preparations were combined with those from theshort-term monolayer cultures prepared at 48 and 72 hr intothe "reference set" of karyotypes for that tumor.

Clones. An additional portion of the single-cell suspension

was serially diluted (1:2 to 1:64) with standard medium. Eachdilution was plated into 3 60-mm integrid Retri dishes (FalconPlastics, Oxnard, Calif.) containing a final concentration of 1.5ml medium and cells per dish. In order to identify future clonalpopulations, each Petri dish was removed individually from theCO2 incubator after 12 to 18 hr of incubation and marked forsingle-cell attachment using an O-ring marker (EBTEC Corp.,Agawam, Mass.). Each dish was then returned to the incubator

2350 CANCER RESEARCH VOL. 41



Heterogeneity of Malignant Gliomas

and left undisturbed for 4 days. Following this incubationperiod, clusters of 3 to 5 cells were seen within some of the CD-

ring marks. All cultures were then maintained by adding 0.5 mlconditioned medium on the seventh day. When a clone of 12to 30 cells was evident within an O-ring mark, the clone wasisolated with a 6-mm (inside diameter) glass cloning ring (BÃ©lico

Glass, Inc., Vineland, N. J.) using conditioned medium andtransferred to a 96-well dish (Costar, Cambridge, Mass.).Slower-growing clones remaining in the dish continued to grow

and were undisturbed by the silicone left after the removal ofthe cloning rings. Clones were collected as late as 28 to 30days in order to obtain slower-growing cell types. When the

clones became confluent in the multiwell dishes, they weretransferred to a 24-well Linbro dish (Linbro Chemical Co.,

Hamden, Conn.) (passage 1). After incubation, they were further subdivided into 2 wells of a 24-well dish (passage 2); one

well was harvested for a chromosome preparation and thesecond was expanded for further analysis. The karyotypes ofall clonal populations were Q- and/or G-banded, and these

karyotypes were compared to the reference set. Clones withchromosomal complements identical to cells comprising thereference set were considered to be cellular representatives ofthe original tumor.

RESULTS

Tumors. Ten human gliomas were mechanically dissociatedinto fragments and single-cell suspensions. While all 10 grew

as expiants, 2 failed to yield enough mitoses in the suspensionand short-term monolayer cultures to permit banding and

yielded only a few clones in the dilution cultures. These 2 wereeliminated from further analysis and the remaining 8 tumorsconstituted the experimental group.

Chromosomal Complement of Solid Tumor Cells andShort-Term Cultures. The chromosome preparations from thesuspension cultures and short-term monolayer cultures fromone Tumor RP are shown in Table 1. The number of meta-phases analyzed from each preparation is shown for eachchromosome number. The total number of metaphases analyzed by Q- or G-banding comprise the reference set of kar

yotypes.The reference sets from all 8 gliomas were compiled into the

histogram depicted in Chart 2. Between 247 and 477 metaphases were examined for each patient (Table 2). The histogram displays the frequency distribution of chromosome numbers within each tumor. In some tumors (JV, JC, and MB) themajority of metaphases were hypodiploid or near-diploid inchromosome number, while others (MA, RP, and WF) werehyperploid, and the remaining 2 (MS and AN) were mixed withabout equal frequencies of near-diploid and tetraploid cells.The histogram further demonstrates that the majority of cellswithin each tumor fell within only a few chromosome numbers,as seen by the peaks in Chart 2. Cytogenetic analysis afterbanding often demonstrated several to many different karyo-typic deviations with the same chromosome number. One suchexample from Tumor RP is chromosome number 55 for which69 cells were analyzed as depicted in Table 3. The first 3karyotypic deviations accounted for 11, 8, and 5 of the 69cells, respectively. The cell depicted in Fig. 1 is one of the first11 cells in Table 3 (karyotype 55.XXX, +1, +2, +6, +8,

+ 11p+, -13, +20, +20, +22). All of the other deviations

were represented by 3 or fewer cells.The occurrence of a variable number of cells with identical

karyotypic deviations led us to consider the relationship between the frequency of such cells and their representation inthe patient's tumor. We sought to determine if the frequency

with which a given cell occurred served to identify a subpopulation of such cells in the tumor. We also determined the lowestnumber of identical metaphases needed to prove that a cellmust have been in the tumor and could not have arisen as avariant in the short-term tissue culture. By population frequencyanalysis (see "Discussion"), we found that cells of identical

karyotype occurring 5 times or more could not have arisenrandomly but had to represent a subpopulation of the patient's

tumor. We therefore defined a subpopulation as a group of 5cells with an identical karyotypic deviation. Table 2 lists for all8 gliomas the number of metaphases analyzed, the number ofsubpopulations per tumor along with the range of cells persubpopulation, and the proportion of the metaphases represented by subpopulations. As can be seen, the number ofsubpopulations ranged from 3 to 21 /tumor, accounting for 40to 60% of all the metaphases analyzed.

Using conventional banding techniques, most of the karyotypic deviations involved monosomy, trisomy, or tetrasomy ofone or more chromosomes. Structural abberrations were identified in all tumors, but their representation was less than 5%of the total cells analyzed. However, after in vitro cultivation of3 to 4 months, numerous structural alterations began to appear.4

Establishment of Expiants and Clonal Lines. The expiantfragments containing viable cells grew within 1 to 2 days andproduced semiconfluent monolayers within a week. The cellswere passaged twice, karyotyped, and recloned. The cloningefficiency of the 8 tumors tested at passage 2 ranged from 20to- 38%.

In the dilution-plated monolayer cultures, 200 to 300 isolatedcells from each tumor were marked for single-cell attachment.

Many of these single cells grew into clonal populations (Table4). All the tumors produced at least 100 clones except forTumor JC which yielded only 85 because of fungal contamination. We attempted to expand all of the clones for karyotyp-ing. The ratio of clones successfully expanded to those isolatedis expressed as a percentage in Table 4. For 6 tumors, thisratio was greater than 50%. We then compared the karyotypesof the clones with those in the reference set and found that 7.6to 25% of the clones carried karyotypic deviations identical tothose in the reference set (Table 4). In 4 tumors, at least 20%of the clones could be positively identified as cellular representatives of the solid tumor cell population. In several tumors,more than one clonal population with the same karyotypicdeviation was isolated from separate Petri dishes (Table 1,Footnote d). Similar results were obtained with Tumors AN, JV,and MB but not JC or WF from which only a few clones wereisolated.

Lines established from expiant cultures of Tumors AN andMS were cloned. Each line retained a specific marker chromosome identified in the reference set. Ten metaphases from

' J. R. Shapiro. W-K. A. Yung, and W. R. Shapiro. Cytogenetic analysis on

twelve human malignant gliomas, manuscript in preparation.

JUNE 1981 2351




TabReference set of karyotypes (Tumor RP)

Chrom37Â° 42a 43a 44 45a 46a 47" 53 54 55 56 57 58 59 60 61

Metaphases analyzed in chromosome preparationscollected at indicated times6 to 24-hr suspension 1

48-hr monolayer 472-hr monolayer 2

Total no. of metaphases for each chromosome no. 7

No. of metaphases with identical karyotypic 6deviations0

Clone designations C-2

1 137 4

11 5

5 5

C-17 C-11

23 14 6

1 14 3

11 6

9 16

8

10

8

C-1 C-12 C-3

33729

69

1185

C-8

a Chromosome number in which one or more subpopulations of cells were found. We defined a subpopulation or clone within a tumor by finding 5 or more6 ni a break in consecutive chromosome numbers.0 Includes only those metaphases (having a specific karyotypic deviation) seen 5 or more times.0 Clones isolated from dilution-plated monolayer cultures having identical karyotypic deviations to a subpopulation in the reference set of karyotypes. In practice,' Clones isolated from separate Petri dishes having identical karyotypic deviations. In all, 13 clones were isolated that could be identified as part of this reference

46 692n 3n

Metaphose chromosome number

924n

each of the 25 clonal populations were examined. In no instance could a karyotypic deviation be found that was identicalto any in the reference sets. While clones from Tumor ANcontained no marker chromosome, 2 clones from Tumor MScarried the marker identified in the reference set of that tumor.Although the marker chromosome was present in these 2 clonalpopulations, the overall karyotypic deviation could not bematched to any karyotype in the reference set for Tumor MS.

Morphology of Clones. Fig. 2 depicts the morphology of 6clones from Tumor M A. The cell types were variable withineach tumor, ranging from highly differentiated astrocytic-likecells with small cell bodies and processes to flat squamous-like cells to fibroblast-like cells. All karyotypes prepared from

these clones were abnormal, indicating that each cell type wasneoplastic. Although Fig. 2 was compiled from Tumor MA,similar cell types could be seen in the other tumors. Theproportion of each cell type differed among the tumors; sometumors contained more astrocytic-like cells while others werecomposed of more fibroblast-like cells.

The morphology of the cells could not reliably predict chromosome number or growth characteristics although severalgeneralizations can be made (Table 5). The astrocytic-like andthe squamous-like cells tended to have near-diploid chromosome numbers while fibroblast-like cells were most often hy-perploid. Doubling times were shorter for the fibroblast-like

cells, and many of these clones retained their vigor even afterlong periods in culture (6 months). The astrocytic-like andsquamous-like cells were slower growing. After 3 to 4 months

in tissue culture, these parental cell types were generallyovergrown by newly generated variants that also grew vigorously.

Chart 2. The distribution of cells containing specific chromosome numbers in8 human gliomas (the "reference set"). Chromosome preparations were made

on cells mechanically dissociated and grown in suspension culture (karyotyped6 to 24 hr post resection) or in short-term monolayer culture (karyotyped 48 to72 hr postresection). The chromosome preparations were Q- or Q-banded forcytogenetic analysis. Two hundred fifty or more metaphases were examined foreach patient's tumor to determine the probable number of cellular subpopulations

present at the time of resection. In our system, we designated a subpopulationas a cellular representative of the resected tumor if 5 or more karyotypes withidentical deviations were found.





le 1

12111141

4011

1 2 56955C-7131

15

15C-5"C-6"1

1247

15C-3562626581196C-103

14116323

5761

1411

6eC-4eC-1382

11131117

441118

96315C-26246

metaphases having identical karyotypic deviations. There were 21 subpopulations in this tumor.

each clone also carries the tumor designation, e.g., RPC-2.set and therefore as cellular representatives of the parent tumor.

Table 2

Subpopulations in each of 8 gliomasA subpopulation was defined as 5 or more identical karyotypes. Many sub-

populations had more than 5 cells, and the range is shown. By this definition, 40to 60% of all cells could be defined as belonging to between 3 and 21 subpop-ulations/tumor.

TumorMS

ANMARPJVJCWFMBNo.

ofmeta

phasesanalyzed477

369356355397247393264No.

ofsubpopulations/tumor1618

21211512113Range

of no.of cells/sub-population5-68

5-445-215-115-395-385-48

27-66Total

cells in all subpopu-lations/total cells

analyzedNo.268/477

220/369185/356142/355187/397145/247156/393144/264%56

60524047594055

DISCUSSION

The protocol described in this report permitted the identification and isolation of tumor cells from freshly resected humangliomas. The methods allowed us to analyze the mitotic population of the tumor and to isolate clones that we could relate tothat population using techniques that minimized tissue cultureartifact. Because much of the protocol combines methods in amanner not described previously, we shall discuss briefly certain of its technical aspects and then discuss the implicationsof the results.

Protocol. The objective of the first part of the protocol wasto examine as many cells in division at the time of tumorresection as possible. The 6- to 24-hr suspension cultures

demonstrated those cells actively in mitosis but had the disadvantages that (a) the cells died after 24 hr, (b), the numbersof mitoses were still relatively small, and (c) Q- and G-bandingwas often of poor quality. The short-term monolayer culturesestablished conditions that permitted cells to remain viable andenter division. These cultures yielded many mitoses with goodto excellent banding characteristics. However, we needed todemonstrate that these dividing cells came from the samepopulation that was found in the suspension culture and werenot cells uniquely induced to divide by in vitro cultivation.Comparing the metaphases collected from the suspensioncultures with those from the short-term monolayer cultures

confirmed that both techniques yielded identical frequencydistributions of chromosome number and karyotypic deviation.In the 6- to 24-hr suspension culture, Tumor MS yielded 33%

of its metaphases with 45 or 46 chromosomes and 21% with92 chromosomes, while the 24- to 72-hr monolayer cultures

had 34% of the metaphases with 45 or 46 chromosomes and14% with 92 chromosomes. No new peaks were seen with themonolayer cultures, and the karyotypic deviations were alsothe same. That the mitotic cells in the short-term monolayercultures represented first-division cells was demonstrated withTumors MA and WF by the addition of 5-bromodeoxyuridine

(23) which produced no metaphases with differentially stainedchromatid arms for 72 hr but did so at 160 hr.5 Thus, combining

the results from the suspension cultures with those from theshort-term monolayer cultures reliably produced data on 200

to 500 metaphases and permitted us to compile the referencesets of chromosomal complements on each tumor.

To assess the statistical significance of the observed data,we utilized a simple model based on occupancy probabilities(i.e., placing balls into urns). In this case, we assumed thateach of the 23 different chromosomes could be represented 0,1, 2, 3, or 4 times in a given cell. Further, we assumed that noless than 30 and no more than 112 chromosomes (identical ordifferent) could appear in a cell. (These assumptions werebased on the chromosomal numbers in our gliomas.) Then, thenumber of possible different cell types is:

N = Â£(x,)(x2) (x23),

where the summation is taken over all XL x2, . . . x23 such that23

30 S 2 JÃ¼S112.1

Given a sample of n cells where the occurrence of each ofthe N cell types is equally likely, we were interested in computing the probability of finding at least 2 cells that match (withrespect to type and quantity of chromosomes) by chance alone.The solution to this problem is similar to that of the "birthdayproblem" (11). Since N is clearly very large and n is small

compared to N (e.g., in cell line RP, n = 355), the probabilityof finding 2 or more matching cells is approximately zero.Consequently, we concluded that the occurrence of matching

' J. R. Shapiro, W-K. A. Yung, and W. R. Shapiro, unpublished observations.

JUNE 1981 2353




TabKaryotype findings in 69 metaphases with a modal

No. of metaphaseswith identical G-

banded chromosomecomplement11

85333322222211111111111111111111155.XXX

55.XXX55.XXX55,XXX55.XXXX55.XXX55.XXX55.XXX55.

XXX55.XX55.XXXX55.XX55,

XXX55,X55.XX55.XX55.XX55.XX55.XX55.XXXX55.XXX55.

XXX55,XXX55.XXX55,

XXX55.XXX55.XXX55.XXX55,

XXX55.XXX55,

XXX55.XXXX55.XXXX55.XXXX1+

1+ 1+ 1+1+1+1+1+1-1+

1+

1+1+1-1+

1+

1+1+1_<â€¢+

11q-+

1-1+1+

1+1+

1A2+

2

+2+2+2+2+2+2+2.+2+2+2+2+2+2+23+3+3-1-3+3+3-3+3+33p-+

3 +3+3+3-3+

3 +3+3+3-33p+B4

5+

4+5+5+4+4+4+4+4+

4+5+4+4+4+4+4-4-4+4+46+

6+6

+6-(-6+6+6+6+6+6-6+6+6-6+6+

6+6 +6+6+

6 +6+6 +6+6 +6+

6+6 +6+6 + 6C7

8 910+

7 +8+8+

8+8+8+8+

8+8+

7 + 7+8+7+7+

7 +10+8+10+

7+10+7+

8+8+10-8+

8-9+

7+8+

7+8+

8 +8-9-8

-9+8

+10+1011

12+11p+

+12+

12+12+12+

12+12+11P+

-12+11

+12+

11+12+

11p ++

12+11+12+11+11p+

+12+

11p ++12+11+1212p-+11p+

+12+11ChromosX+X

+X+X+X+

X +X+x+x+x+x+

X +X+x-X+

X +X+x+x+x+x+x+x+

x+x+x+x+x+

X+X+X +X+X + X

The deviations are recorded in relation to a 46,XX karyotype.b Some abnormal chromosomes listed in this column could not be identified while others could. Isochromosome 9 [i(9q>] was found in 3 of 69 metaphases, 7

and 2 contained chromosome fragments.c DMS, double minutes; found in 5 metaphases ranging from 2 to 10/cell (Fig. 1).d The percentage of each subpopulation found for this chromosome number.

Table 4

Clones derived from single cells mechanically dissociated from human gliomas

Freshly resected human gliomas were mechanically dissociated into singlecells that were then dilution plated. The single cells were marked and when theygrew into clusters were isolated and transferred with cloning rings. The numberof clones successfully isolated is shown in Column A. These clones were thenexpanded for karyotyping, and the number successfully expanded (i.e. passagedtwice) is shown in Column B. The clones were karyotyped and compared to thereference set of karyotypes for that tumor (Chart 1). Those clones with karyotypicdeviations identical to cells in the reference set were considered to be cellularrepresentatives of the parent tumor and are shown in Column C.

Clone designationMSANMARPJVJCWFMBANo.

ofclonesiso

lated16714718113713485143174B

No.ofclonesex

pandedforkaryotype8569101971061211796B/A(%)50.846.955.870.879.114.181.855.1C

No. ofclones

identifiedas cellular representativesoftheparent

tumor11152113123923C/B(%)12.921.720.713.411.325.07.623.9

cells could not happen by random chance. In practice, wechose the even more conservative estimate of 5 identical cellson (a) the equally remote chance that a cell might have doubleditself in the short-term cultures and (b) the fact that most of the

larger peaks contained 5 or more identical cells.The objective of the second part of the protocol was to

isolate karyotypically identified tumor cells as clonal populations capable of further expansion. Successful clonal growthwas dependent on 3 factors, (a) Freshly resected glioma cells,dissociated mechanically, grew better than cells dissociated byenzymatic digestion, (b) Waymouth 87/3 medium supportedclonal growth better than media minimum essential mediumF12, Dulbecco's reinforced Eagle's medium, and Roswell Park

Memorial Institute Tissue Culture Medium 1640. And (c) clonalgrowth was dependent on the concentration of PCS used. Allserum lots were tested for the concentration that yielded thebest clonal growth. This concentration varied but was often ashigh as 20 to 25%. In addition, we found that resected humangliomas from previously untreated patients cloned only inmonolayer cultures and not in agarose, while tumors frompatients treated with radiation therapy and chemotherapy readily grew in agarose and would not grow in monolayer cultures.5

Karyology. A full analysis of the chromosomal data already





le 3chromosome number of 55 (Tumor RP)ornegroups*13-13+

13+

13+

13+13+13+

13+

13-13+

13-13+

13D14+

14+14+

14+

14+14+14+

14+14+14+14+14+14+14+14+

14+14+14+

14+

14+

1415+

15+

15+

15+15+15+

15+

15+15+15+

15+

15+

15+15+15+15+15-15+15+15+

15+

15E16

1718+

16+16+

16+16+

17+17-18+

18+

16+17+17+18+18+

17+17+18+

17+17+

16+

17+

17+

17+17+17+17-17

+18+16

+17+

17+17+18+

16+1719+

19+

19+

19+

19+19+19+

19+19+

19+19+19+19+19+

19+19+19+

19F20+

20+20+

20+

20+

20+20 +20+

20+

20+20+

20+

20+

20+20+

20+

20+20 + 20G21

22+

22+

22+

22+22+

22+

22+

22+

21+

22+

21+22+

22+22+

22+22+

21+

22+22+22-21Marker"

chromo- %d of sub-

somes DMSCpopulationDMS

1612DMS

7<4<4<4Fragment

<4<3<3DMS

<3i(9q)

<3<3DMS

<3M1M2FragmentM1i(9q)i(9q)MlMlM1

DMS

metaphases carried an 11 p+ chromosome (Fig. 1 ), 4 carried other marker chromosomes that could in part be identified, 5 carried unidentified marker chromosomes.

Table 5Morphology, modal chromosome number, and growth characteristics of 2

series of clones from Tumors RP and MA

ClonesRPC-1RPC-2RPC-3RPC-4RPC-1

0RPC-1

2MAC-2MAC-6MAC-9MAC-1

1MorphologyAstrocytic-likeSquamous-likeSquamous-likeFibroblast-likeFibroblast-likeFibroblast-likeAstrocytic-likeFibroblast-likeAstrocytic-likeSquamous-likeModal

chromosomeno.45374789834645634549Doublingtime(hr)10412014084727814460264NDaCell

pas

sage1011681383334

ND, not done; culture became contaminated after 11 days; therefore,doubling time could not be determined.

derived from 8 gliomas is still in preparation.4 While there is

obvious interest in the specificity of the chromosomal changes,we are not prepared at this stage to comment on such changes.We included a representative karyotype (Fig. 1), banding data,and frequency distributions (Chart 2) (a) to show the feasibilityof using karyotypes to identify specific tumor cell subpopula-tions in a patient's tumor, (b) to demonstrate the good quality

karyotypes that can be made from early tissue culture preparations, and (c) to describe the heterogeneity of gliomas.

Heterogeneity. The immediate karyotyping of suspensionand short-term monolayer cultures derived from solid tumors

permitted the identification of the mitotic population of tumorcells. The reference sets from the various tumors are comparedin the histogram depicted in Chart 2. Each tumor had manysubpopulations; the chromosomal complement ranged fromhypo- to near-diploid or hyperploid in chromosome numbers.

Most of the chromosomal complements examined within the 8tumors were aberrant numerically. Although structural rearrangements occurred, they did so only in isolated cells, andthe karyotypic deviation of these cells could never be matchedto any other cell in the distribution of cell types, suggestingthat they did not represent subpopulations in the tumor.4

This karyotypic heterogeneity was also evident in the cyto-genetic studies of Mark (18) who reported that stem- and/orsideline populations could be found for each of the 50 astro-

cytomas that he analyzed. However, in our study, 200 to 500metaphases were examined to determine each reference set.Since Mark's study included only Giemsa-stained karyotypes,

he could not determine the extent of heterogeneity in cells withthe same chromosome number. Using Q- and G-banding tech

niques, our analysis usually revealed 2 or more subpopulationswith the same chromosome number (Table 3), implying extensive variability in the chromosomal complements of malignantgliomas. Although there was a wide distribution of chromosomenumbers, 2 or more major chromosomal modes were alwaysevident.

The reference sets from the patients' tumors differed from

each other. Some tumors were predominantly hyperploid, oth-

JUNE 1981 2355




ers were predominantly hypodiploid, and the remaining weresome combinations of both. Using our definition of a subpop-ulation as the occurrence in the reference set of 5 or morekaryotypically identical cells, we found that among the 8 tumorsthe numbers of such subpopulations varied from 3 to 21 foreach tumor (Table 2). Many of the subpopulations in TumorsMS and AN had a marker chromosome that permitted them tobe traced to a common stem cell (15) while the other tumorslacked appropriate markers and could not be defined in thismanner. These observations indicated that human gliomas arenot only heterogeneous in their cell populations but are different from each other.

Clones. While over 100 clones were isolated from most ofthe tumors, we chose to study only those that were karyotypically identical to cellular subpopulations in the reference sets;7.6 to 25% of the clones from a tumor were identified ascellular representatives of the tumor population. It is the phe-notypic behavior of such cells that is of immediate relevance tothe patient's tumor, although the single-cell origin of the clones

and their stability in early passage makes even those clonesnot identifiably part of the patient's tumor valuable for other

work. Morphologically, the clones varied from astrocytic-like tofibroblastic-like (Fig. 2). Preliminary experiments performed on

clones isolated from the same tumor demonstrated phenotypicvariability in growth kinetics and chemotherapeutic sensitivity(36). Correlations between chemosensitivities and karyotypicdeviations have so far not been possible but represent a majorarea of interest in our laboratory.

Expiants. In contrast to the clones derived from the dilutionplating, none of the clonal populations from expiant cultures,with the exception of 2 clones that carried specific markers,could be matched to any mitotic figure of the reference set.Two clones from the MS culture contained a marker chromosome which was identified in the reference set, suggesting thatit originated in a stem cell population existing in the tumor.Nevertheless, neither clone had a karyotypic deviation identicalto any cell so identified in the reference set. The most likelyexplanation for this observation is the selection of new cellvariants generated in vitro which have growth advantagesallowing them to overgrow other cell types.

Using expiants to obtain clonal lines representative of theoriginal tumor is thus less likely to succeed than using clonallines isolated from dissociated single cells. Furthermore, thetime period necessary to establish clonal populations usingdissociated single cells is considerably shorter than the moreclassical expiant culture which requires the formation of mono-layers, and subsequent cloning, isolation, and expansion.

Conclusion

Experimentally, these in vitro techniques may be used tostudy the dynamic behavior of tumor growth with less risk ofintroducing tissue culture artifact than has been possible byany method in the past. By isolating and expanding only thosecells identified in the reference set of karyotypes as representative tumor cells, one can be assured of dealing with cellpopulations present in the patient's tumor and not with variants

arising in cell culture. If the complexity of karyotypic evolutionitself is to be studied and correlated to specific phenotypiccharacteristics of clinical significance, it must be examined inearly-passage cells rather than in established cell lines. Thesetechniques now permit the examination of single-cell clones forsurface proteins, mechanisms of drug resistance, cell-cell in

teractions, or a multitude of other characteristics associatedwith the cancer cell as it exists at the time of tumor resection.The heterogeneous nature of human gliomas as reported heremakes such studies important if we are to understand thebiology and improve the treatment of this disease.

ACKNOWLEDGMENTS

We thank Dr. Martin L. Lesser for statistical help in the population frequencyanalysis and Doris Fok and Virginia Dato for their excellent technical assistance.

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Fig. 1. A G-banded karyotype from Tumor RP. The chromosome preparation was made on a suspension culture at 24 hr following tumor cell dissociation. Thekaryotype shows a chromosomal complement of 55,XXX, monosomic for chromosome 13. and trisomie for chromosomes 1, 2, 6, 7, 8, 11, 22, and the X andtetrasomic for chromosome 20. A marker chromosome identified as being in part a number 11p+ is indicated with an arrow. Two double minutes were seen in thiskaryotype although up to 10 were seen in 4 other metaphases. The double minutes were not seen in the karyotypes prepared from the short-term or the dilution-

plated clones of this cell.Fig. 2. A composite of the various cell types isolated as clonal populations from Tumor MA. Morphologically, the cell types were variable and included astrocytic-

like. flat and squamous-like, and fibroblast-like. These cell types could be observed in all tumors, but the proportion of each differed among tumors. The majorkaryotypic deviation is listed for each clonal population. Phase contrast, x 450. In a, MAC-4 is astrocytic-like and contains a 44, Xy chromosomal complementmissing chromosomes 20 and 22; in b, MAC-8 is also astrocytic-like and has a 44.Xy chromosomal complement missing chromosomes 9 and 19; both MAC-4 andMAC-8 clones stain strongly positive for glial fibrillary acidic protein; in e, MAC-19 is flat and squamous-like and has a chromosomal complement of 42Xy withmonoxomy for chromosome 5, 11, 19, and 22; in d. MAC-16 is a very large, squamous-like cell containing a chromosomal complement of 85 XXyy missingchromosomes 9. 11. 14. 18, and two 22's (karyotypic deviation based on a 4n cell); in e, MAC-24 is fibroblast-like with 56,Xy chromosomal complement monosomic

for chromosomes 10, 13, and 21, trisomie for chromosomes 1, 2, 5, 6, 16, 17, and 19, and tetrasomic for chromosomes 7,12, and 15. It also carries a deletion in3p- arm that has been seen in several other clones; in f. MAC-32 is a fibroblast-like cell; it has a chromosomal complement of 89,XXyy, missing chromosomes 4 and

two 22 s (karyotypic deviation based on a 4n cell).

JUNE 1981 2357




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JUNE 1981 2359



1981;41:2349-2359. Cancer Res Joan R. Shapiro, Wai-Kwan A. Yung and William R. Shapiro Subpopulations of Human Malignant GliomasIsolation, Karyotype, and Clonal Growth of Heterogeneous

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