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[CANCER RESEARCH 41, 3052-3057, August 1981]0008-5472/81 /0041-0000$02.00
Comparison between Agar and Methylcellulose Cultures of HumanLeukemic Cells1
Kazuo Kubota, Harvey D. Preisler,2 Kimitaka Sagawa, and Jun Minowada
Section oÃCell Biology, Department ot Medical Oncology ¡K.K., H. D. P.], and Cell Culture Laboratory [K. S., J. M.], Roswe/l Park Memorial Institute, Buffalo, NewYork 14263
ABSTRACT
A comparison was made between the agar and methylcellu-
lose culture systems with respect to their ability to support theclonal growth of leukemic cells obtained from patients withacute myeloblastic leukemia, acute lymphoblastic leukemia,and chronic myelogenous leukemia in blastic crisis. The number of clusters and/or colonies formed and the morphology ofthe cells within them varied from patient to patient. Nevertheless, no significant difference between the two culture systemswithin given leukemic specimens was found. No significantdifferences were noted among three different conditioned media used as sources of colony-stimulating activity. Most of the
cells within clusters and colonies were identified to be immaturemembers of granulocyte-macrophage series or to be indistin
guishable from the preculture leukemic blast cells by morphological and cell surface marker studies. Cells from myeloidcrisis in chronic myelogenous leukemia grew well in the cultures, but cells from lymphoid crisis did not proliferate.
INTRODUCTION
The availability of in vitro cloning techniques has led to alarge number of studies involving bone marrow and peripheralblood specimens obtained from leukemia patients (3, 12, 13,15, 21, 22, 24, 27). These studies have used either feederlayers of normal peripheral leukocytes or conditioned media assources of CSA.3 Recently, a new in vitro method has been
reported which uses PHA-LCM to stimulate the clonal prolifer
ation of leukemic cells in methylcellulose (5, 18). This methodhas been reported to be superior to the standard cloningtechniques in that it permits the growth of leukemic stem cellswhich are morphologically identical to the original leukemicblast cells and which are capable of self-renewal (4, 6).
The present study was carried out to compare the growth ofleukemic cells in methylcellulose to the growth of leukemiccells in agar. The growth of leukemic cells in both systems wasstimulated by PHA-LCM or by medium conditioned by one of2 human tissue culture cell lines. Growth under different conditions of culture was assessed by quantitation of the clustersand colonies, by morphological and cytochemical study of the
1 Supported by USPHS Grants CA 5834, CA 24162, and CA 14413 from the
National Cancer Institute and by Grant CH 168 from the American CancerSociety.
2 To whom requests for reprints should be addressed, at Department of
Medical Oncology, Roswell Park Memorial Institute, 666 Elm St., Buffalo, N. Y.14263.
3 The abbreviations used are: CSA, colony-stimulating activity; PHA-LCM,leukocyte-conditioned medium stimulated by phytohemagglutinin; ALL, acutelymphoblastic leukemia; CML, chronic myelogenous leukemia; GCT-C-CM, conditioned medium from a fibrous histiocytoma cell line; Mc-CM, conditionedmedium from a T-cell hairy cell leukemia cell line. AML, acute myeloblasticleukemia.
Received December 22. 1980; accepted April 22. 1981.
cells within the clusters and colonies, or when possible by cellsurface marker studies. These studies demonstrated that therewere no significant differences between the agar and methyl-cellulose culture systems or between the different CSA's.
MATERIALS AND METHODS
Patients. Specimens obtained from 15 patients with AML orone of its variants, one patient with ALL, and 10 patients withCML in blastic crisis were studied. The relevant clinical dataare given in Table 1.
Preparation of Cell Suspension. Cell suspensions wereprepared by the method of Minden et al. (18) with somemodifications. Heparinized peripheral blood or bone marrowcells were obtained from the patients who had given informedconsent. After centrifugation through Ficoll-Paque (specific
gravity, 1.077) (Pharmacia Fine Chemicals, Piscataway, N. J.)at 400 x g for 30 min at room temperature, interface cellswere collected, washed, and adjusted to 2 x 107/ml in Roswell
Park Memorial Institute Tissue Culture Medium 1640 (GrandIsland Biological Co. Laboratories, Grand Island, N. Y.) with5% fetal calf serum (Microbiological Associates, Walkersville,Md.). Equal volumes of the cell suspension and 5% sheep RBC(Springville Laboratories, West Seneca, N. Y.) in Roswell ParkMemorial Institute Tissue Culture Medium 1640 with 5% fetalcalf serum were mixed, centrifuged at 500 x g for 10 min at4°,and incubated for 1 hr on ice. At the end of the incubation,the cells were gently resuspended, layered over Ficoll-Paqueagain, and centrifuged at 400 x g for 30 min at 4°.After the
interface cells were collected and washed, the cells wereexposed to hypotonie Tris-NH4CI to remove the remaining RBC.
The cells were then used for culture. When a large number ofcells were available, some were stored in a-modification ofEagle's medium (Flow Laboratories, McLean, Va.) with 5%
dimethyl sulfoxide (Fisher Scientific, Fair Lawn, N. J.) and 10%fetal calf serum at -70° (1).
Culture. The single-layer agar culture was performed usinga modification (16) of the method of Robinson et al. (26). Thecell suspension was mixed in 0.3% agar (Difco Laboratories,Detroit, Mich.), a-modification of Eagle's medium, 20% fetal
calf serum, and 10% CSA. The cells were added to give a finalconcentration of 0.2, 0.5, or 1.0 x 105/ml. A 1.0-ml aliquot
was plated into a 35- x 10-mm plastic dish (Lux Scientific,
Newbury Park, Calif.). The methylcellulose culture was performed according to the method of Iscove et al. (13) with minormodifications. The cell suspension was mixed in 0.8% methyl-cellulose (Dow Chemical, Midland, Mich.), a-modification ofEagle's medium, 20% fetal calf serum, and 10% CSA. The
cells were added at a final concentration of 0.2, 0.5, or 1.0 x105/ml. A 1.0-ml aliquot was plated into a 35- x 10-mm plastic
dish using a syringe.
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Leukemic Colony Formation
Table 1
Clinical features of 16 patients with acute leukemia and i O patients with CML in blastic crisis
Case1234567891011121314151617181920212223242526Age6273215931642644706267597162564073285140684051432239SexMFMMMMFMMMMMMFFFMMFMFMFMMMDiagnosis"AMU"AMLAMMLAMLAMLAMLAPLAMMLAMLAMMLAMLAMMLAMLAMLAPLALLCML-BC(M)CML-BC(M)CML-BC(L)CML-BC(L)CML-BC(L)CML-BC(M)CML-BC(M)CML-BC(M)CML-BC(L)CML-BC(M)CML-BC(M)Stateat the
time ofstudyUntreatedRelapseRelapseUntreatedUntreatedRelapseUntreatedRelapseRelapseUntreatedUntreatedRelapseUntreatedUntreatedUntreatedUntreated%of
blasts ofbone
marrow2684589027398260NA62781344908695NANANA76NA604952782230%
of blastsWBC of periph-
(x 1CT3) eralblood22.5112.035.98.34.4131.04.410.23.3135.06.085.0215.0210.06.243.590.0114.0137.0122.036.4198.011.656.838.518.155.9788748744259454256376464608814974836547759626647754022PlateletPh'
(x 10 3)chromosome31.0130.07.057.023.012.017.019.4185.0150.097.026.057.033.065.071.015.5424.066.0476.023.0430.030.5270.060.040.0306.0<+>NA(+)NA(+>(+)(+)(+)NA(+>NA(+)
'' Diagnosis was made by morphological criteria and cell surface marker studies.6 AMML, acute myelomonocytic leukemia; APL, acute promyelocytic leukemia; NA, not available; CML-BC(M), CML
in myeloid blastic crisis; CML-BC(L), CML in lymphoid blastic crisis.
Three kinds of conditioned medium were used as sources ofCSA in this study: (a) PHA-LCM (2), generously supplied by
Dr. C. A. Izaguirre (Institute of Medical Science, University ofToronto, and the Ontario Cancer Institute, Toronto, Ontario,Canada); (b) conditioned medium from a permanent cell line(GCT-C) established from a lung metastasis of fibrous histio-
cytoma (7), which had been maintained in our laboratory; (c)Mo-CM (10), generously supplied by Dr. D. W. Golde (Univer
sity of California School of Medicine, Los Angeles, Calif.). Theeffects of these 3 different conditioned media on the growth ofnormal human bone marrow cells in agar and in methylcelluloseare given in Table 2.
After 7 days of incubation in a CO2 incubator at 37°with 5%
CO2 in air, clusters (4 to 19 cells) and colonies (20 or morecells) were scored using an inverted microscope (Nikon, Japan).
Morphology. The morphology of the cells grown in agar gelwas examined using the whole-plate technique of Kubota et al.
(16) with minor modifications. In brief, at the end of the incubation, the agar gel was carefully transferred en bloc onto aslide glass and dried with a piece of filter paper as describedpreviously. For the methylcellulose culture, slides for morphological examinations were made either by picking up individualclusters and colonies on a slide glass or by cytocentrifuge afterharvesting entire plates. The slides were stained with Wright-Giemsa, peroxidase, or naphthol AS-D chloroacetate esterase-
staining technique.Cell Surface Marker Assay. The cell surface marker studies
were done before and after the methylcellulose cultures. Fivecases (Cases 3, 5, 6, 7, and 23) were chosen because theculture plates of those cases had few single cells betweenclusters/colonies. After 8 days of incubation, the contents of
Table 2
Effect of the 3 conditioned media on the cluster and colony formation by normalhuman bone marrow cells
The 3 conditioned media used in this study were tested to determine if theyhad colony-stimulating effects on cluster and colony formation by normal humanbone marrow cells. Nonphagocytic normal bone marrow cells (1 x 10s) (16)
were plated with 10% of one of the 3 conditioned media. After 7 days ofincubation, clusters and colonies were scored.
Agar Methylcellulose
ConditionedmediumNo
stimulatorPHA-LCMGCT-C-CMMo-CMCluster2
±0a
27 ±221 ±39 ±2Colony0
78 ±765 ±5
122 ±8Cluster26
±719 ±426 ±712
±4Colony1
± 187 ± 580 ±10
131 ± 3
Mean ±S.D. of 3 experiments done in 3 replicate cultures.
entire plates were poured into a tube containing 50 ml ofDulbecco's phosphate-buffered saline and washed twice withDulbecco's phosphate-buffered saline. The cells obtained were
used for cell surface marker studies. The cell surface markerassays used were: rosette formation with sheep erythrocytes(23); rosette formation with bovine erythrocyte-IgG antibodycomplex (23) and bovine erythrocyte-IgM antibody-complement complex (23); surface membrane immunoglobulin (19);T-cell antigen (29); la-like p28, 30 antigens (19); common ALLantigen (14); and myelomonocyte antigen (28). The assaymethods were described previously elsewhere in detail (11,19). For surface membrane immunoglobulin, fluorescein iso-thiocyanate-conjugated goat anti-human immunoglobulinchain-specific reagents (anti-ic, -A, -a, -5, -y, or -^ chain antibody) were used in direct membrane immunofluorescence test(19). For the antigen detection such as T-cell antigen (29), la-like p28, 30 antigens (19), common ALL antigen (14), andmyelomonocyte antigen (28), rabbit antibody which had been
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made to be specific by extensive and careful absorption of therespective immune serum was used as a primary reagentfollowed by fluorescein isothiocyanate-conjugated goat anti-
rabbit IgG reagent as a secondary reagent in the indirectmembrane immunofluorescence test. Enumeration of fluorescent cells was carried out under a fluorescent microscope(Leitz, Germany) at X1000. The results were expressed as apercentage of fluorescent cells in at least 200 total cells ex
amined.
RESULTS
Tables 3 and 4 report the results of the agar and methylcel-
lulose cultures of leukemic cells obtained from patients withacute leukemia. Significant cluster and/or colony growth inagar was found in 4 of 17 of nonstimulated cultures, 7 of 17 ofPHA-LCM-stimulated cultures, 7 of 16 of GCT-C-CM-stimu-lated cultures, and 7 of 16 of Mo-CM-stimulated cultures. Such
growth in methylcellulose was found in 4 of 18 nonstimulatedcultures, 7 of 17 PHA-LCM-stimulated cultures, 7 of 16 GCT-C-CM-stimulated cultures, and 8 of 17 Mo-CM-stimulated cul
tures. When the 2 semisolid supporting matrices using thesame CSA are compared, specimens which grew well in agaralso grew well in methylcellulose and specimens which grewpoorly or not at all in agar did not grow well in methylcellulose.Hence, while there were large differences in growth potentialbetween specimens, the differences were manifested regardless of the semisolid supporting matrix. When the differentCSA's are considered, there were no absolute differences
among them. When cluster and/or colony growth occurred, itwas stimulated by all of the CSA's. While the PHA-LCM tended
to stimulate greater numbers of clusters and/or colonies, thedifferences between the growth produced by the differentCSA's were not statistically significant. Consideration of the
data reported in Tables 1, 3, and 4 demonstrates that there
Table 3
Results of agar cultures of 16 patients with acute leukemiaNostimulator3Case12345678910111213141516DiagnosisAM
LAMLAMMLAMLAMI-AMI.APLAMMLAMLAMMLAMLAMMLAMLAMLAPLALLSamplePB(f)"PB(f)PB(c)BM(c)PB(f)PB(f)PB(f)PB(f)PB(f)PB(f)PB(f)BM(f)PB(f)PB(f)PB(c)PB(f)PB(f)PB(f)ClusterND0032
±1400387
±39000179
±875±19001
±1000ColonyND006
±100000002
±20000000PHA-LCMCluster002120
±172C820
±8201
053 ±542733±2462600±202±301
53 ±17126±1407
±11±10ND3
± 5Colony00266
±1154±0013
±3±0029
±10±0000ND2
±9050123922GCT-C-CMClusterND01
786 ±288754±480237
±601323±52ND00117±
3260±1603
±21±10953
±820ColonyND040
±32240±90000ND003
±3001
±10000MO-CMClusterND01806
±220548± 1480370
± 1 131553 ±52ND3
±30130±
23128±
1404
±23±20847
±803
± 2ColonyND0534
±1921126±1003
±530±28ND3
±1095
±2515±805
±13±102
±01± 1
" Cultures were set up with no stimulator, PHA-LCM, GCT-C-CM, or Mo-CM.b PB, peripheral blood; f, fresh sample; ND, not determined; AMML, acute myelomonocytic leukemia; c, cryopreserved sample; BM, bone marrow, APL, acute
promyelocytic leukemia.0 Mean ±S.D. for clusters (4 to 19 cells) and colonies (20 or more cells) per 1 X 10s cells plated from 3 replicate cultures.
Table 4
Results of the methylcellulose cultures of 16 patients with acute leukemia
No stimulator PHA-LCM GCT-C-CM Mo-CM
Case Diagnosis Sample Cluster Colony Cluster Colony Cluster Colony Cluster Colony
12345678910111213141516AMLAMLAMMLAMLAMLAMLAPLAMMLAMLAMMLAMLAMMLAMLAMLAPLALLPB(f)6PB(f)PB(C)BM(c)PB(f)PB(f)PB(f)PB(f)PB(f)PB(f)PB(f)BM(f)PB(f)PB(f)PB(C)PB(f)PB(f)PB(f)000000140
±4816±30014
±658±200000000000014
±100004
±420±8000000001366
±262°734
± 1620N866
±80598±320050
±438
±202
±21±10ND2
± 200114
±1186±0N114
±54±0038
±20
±02
±1±0ND09010824216421ND0354
±330±0N420
±352±0034
±ND02
±00771
±2±34304432422132ND026
±1030±100N1
00 ±162±20012
±6ND02
±20000ND0806
±170520±900N540
±118132±240040
±1034
±6001
±10710
±922± 2ND0474
±1061174±1420N26
±242±20038
±1232±806
±20002
± 2
a Cultures were set up with no stimulator, PHA-LCM, GCT-C-CM, or Mo-CM.6 PB, peripheral blood; f, fresh sample; AMML, acute myelomonocytic leukemia; ND, not determined; c, cryopreserved sample; BM, bone marrow; APL, acute
promyelocytic leukemia; N, too numerous to score.c Mean ±S.D. of clusters (4 to 19 cells) and colonies (20 or more cells) per 1 x 105 cells plated from 3 replicate cultures.
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Leukemic Colony Formation
was no significant correlation between the percentage of blastcells in the various specimens and the number of clusters and/or colonies produced.
The morphologies of the cells within the clusters and coloniesgrown in agar and in methylcellulose which were not stimulatedor were stimulated by any of the CSA's were similar in that they
varied from specimen to specimen. In each case, the culturedcells either had the appearance of myeloblasts, promyelocytes,or macrophages or were similar in appearance to the leukemicblast cells prior to culture. The cells within the few clusters andcolonies produced by the single ALL specimen which wasstudied contained normal-appearing mature granulocytic ele
ments.Tables 5 and 6 present the results obtained when cells from
patients in the blastic phase of CML were studied. The growthcharacteristics of these cells were similar to those of the acuteleukemia specimens. Specimens from each of the patients withmyeloid blastic crisis produced cluster and/or colony formationin vitro. As with the AML specimens described above, growthin agar and in methylcellulose were similar and the effects ofeach of the CSA's were similar as well. Cluster and/or colony
growth varied from patient to patient, and the cells within theclusters and colonies had the appearance of myeloblasts,promyeloblasts, macrophages, or the leukemic blast cells.However, the cells in some clusters and colonies in Cases 21and 23 showed granulocytic maturation.
By contrast, the peripheral blood cells obtained from 3 cases
of lymphoid blastic crisis of CML failed to produce clonalgrowth either in agar or in methylcellulose regardless of thestimulator used. Bone marrow cells obtained from Patient 24(lymphoid crisis of CML) produced a small number of clusterswhich consisted of normal-appearing mature granulocytic cells.
Patient 18 is of particular interest since his initial blasticphase was myeloid; upon intensive chemotherapy, his diseasereverted to a chronic phase but lymphoid blastic crisis eventually supervened. Peripheral blood cells were cultured inmethylcellulose during both blastic phases. Cells obtained atthe time of myeloid blastic crisis produced luxuriant growth,whereas the lymphoid blastic crisis cells failed to grow in vitro.
Table 7 provides information regarding the pre- and post-
methylcellulose culture surface marker characteristics for 4acute leukemic specimens and one myeloid blastic crisis specimen. All of the cells, prior to and after culture, were essentiallynegative for rosette formation with sheep erythrocytes, rosetteformation with bovine erythrocyte-IgM antibody-complementcomplex, T-cell antigen, and surface membrane immunoglob-
ulin. Myelomonocyte antigen was used as a positive cell surface marker to distinguish mature and immature granulocytesand macrophages from other cells (28). In 5 of 5 instances, theproportion of cells which were myelomonocyte antigen positivewas greater in the postculture than in the preculture specimens.Similarly, in 3 of 4 instances, the proportion of cells with la-likep28, 30 antigens, was greater in the postculture than in thepreculture specimens. In each instance save one (Case 3),
Table 5
Results of the agar cultures of 7 patients with CML in blastic crisisNo stimulator" PHA-LCM GCT-C-CM Mo-CM
Case Diagnosis Sample Cluster Colony Cluster Colony Cluster Colony Cluster Colony
18212223242526CML-BC(L)6CML-BC(M)CML-BC(M)CML-BC(M)CML-BC(L)CML-BC(M)CML-BC(M)PB(f)PB(f>DM(t)PB(f)PB(f)BM(C)BM(f)PB(f)PB(f)013±9°13
±197±53±1001
±21273±41007
±900000350
±910173
±77307±74293±3984±201±213±4NDND0140
±49147±503±530±121±10NDND0373
±53266±5270±3619±3029
±52±2970±1100207
±5287±4101
±2000110
±2201
80 ±43120±16587±53210±624±337±735±21
700 ±940393
±9373±5010±14129±277±1024
±9913±95
Cultures were set up with no stimulator. PHA-LCM, GCT-C-CM, or Mo-CM.b CML-BC(L), CML in lymphoid blastic crisis; PB, peripheral blood; f, fresh sample; CML-BC(M), CML in myeloid blastic crisis; BM, bone marrow; c, cryopreserved
sample; ND. not determined.c Mean ±S.D. for clusters' (4 to 19 cells) and colonies (20 or more cells) per 1 x 105 cells plated from 3 replicate cultures.
Table 6
Results of the methylcellulose cultures of 10 patients with CML in blastic crisisNo stimulator" PHA-LCM GCT-C-CM Mo-CM
Case Diagnosis Sample Cluster Colony Cluster Colony Cluster Colony Cluster Colony
17IB1920212223242626CML-BC<M)0CML-BC(M)CML-BC(L)CML-BC(L)CML-BC(L)CML-BC(M)CML-BC(M)CML-BC(M)CML-BC(L)CML-BC(M)CML-BC(M)PB(f)PB(f)PB(f)PB(f)PB(f)PB(f)BM(f)PB(f)PB(f)BM(c)BM(f)PB(f)PB(f)NN00000024
±40013
±5407±116NN0000002
±2000217
±40NN000106
±47±180
±24±01
±NDND761814241N202
±42°00014
±1833±19016
±400NDNDNDN0ND026
±3854±18106±2434±16014
±710±81
37 ±33NDN0ND014
±1814±1804
±0003
±533±29NDNDNDNDNDN106
±N240
±9±25±20
±1500±5022156426NDNDNDNDNDN134
±N232
±1±023
±440±1838112204
Cultures were set up with no stimulator, PHA-LCM, GCT-C-CM, or Mo-CM." CML-BC(M), CML in myeloid blastic crisis; PB, peripheral blood; f, fresh sample; N, too numerous to score; ND, not determined; CML-BC(L), CML in lymphoid
blastic crisis; BM. bone marrow; c. cryopreserved sample.0 Mean ±S.D. for clusters (4 to 19 cells) and colonies (20 or more cells) per 1 x 105 cells plated from 3 replicate cultures.
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Table 7
Results of the cell surface marker studies
Case356723Experi-DiagnosismentAMML
12345AML
1AML
12APL
1CML-BC(M)
123SamplePB{f)PB(f)PB(f)PB(f)PB(f)PB(c)BM(f)BM(f)BM(f)BM(c)PB(f)PB(f)PB(f)PB(f)PB(c)PB(f)PB(f)PB<f)PB(f)PB(c)PB(c)Before
orafter
culture8AfterAfterBeforeAfterAfterAfterBeforeAfterAfterAfterBeforeAfterAfterBeforeAfterBeforeAfterBeforeAfterAfterAfterStimulatorNoPHA-LCMNoNoPHA-LCMPHA-LCMNoNoPHA-LCMPHA-LCMNoNoPHA-LCMNoPHA-LCMNoPHA-LCMNoMO-CMMO-CMMO-CME"<%>000.800ND2.800NDND00ND0NDNDND0ND0EA(%)79.279.844.482.182.581.243.378.666.071.722.5NDND19.7ND8.29.114.9ND4.81.8EAC(%)0010.2<1.0<1.0ND7.703.0NDNDNDNDNDNDNDNDNDNDND6.6SmlgNDNDNDNDNDNDNDNDNDNDND00NDNDNDNDNDNDND0T-Ag00ND00NDNDND3.1NDNDNDNDNDNDNDNDNDNDNDNDla-like(%)33.370.525.078.570.090.039.292.3100.090.045.0NDND84.1ND095.095.0ND86.176.9CALL0000001.005.0ND0NDND0NDNDND0NDNDNDMAi|(%)88.880.039.293.788.8100.068.6100.085.7ND61.7100.0100.017.6ND14.953.830.1ND78.653.0
a The cell surface marker studies were done before and after the methylcellulose cultures (see text). The surface marker studies before the cultures were done
on the samples prior to T-cell depletion.6 E, rosette formation with sheep erythrocytes; EA, rosette formation with bovine erythrocyte-IgG antibody complex; EAC, rosette formation with bovine
erythrocyte-IgM antibody-complement complex; Smlg, surface membrane immunoglobulin; T-Ag, T-cell antigen; la-like, la-like p28, 30 antigens; cALL, common ALLantigen; MAg, myelomonocyte antigen; AMML, acute myelomonocytic leukemia; PB, peripheral blood; f, fresh sample; ND, not determined; c, cryopreserved sample;BM, bone marrow; APL, acute promyelocytic leukemia; CML-BC(M), CML in myeloid blastic crisis.
there were essentially no cells positive for common ALL antigen, which is the lymphoid precursor cell marker. The freshbone marrow obtained from a patient with Ph1-positive AML(Case 3) was positive for common ALL antigen. This patient's
disease was quite unusual and may have represented a leukemia of mixed type.
DISCUSSION
In the studies reported here, 2 culture systems, agar andmethylcellulose, were compared to determine which providedbetter conditions for the clonal growth of human leukemic cells.The results of the agar cultures of patients with AML or one ofits variants were consistent with other reports (12, 21, 24, 27),with the number of clusters and/or colonies varying frompatient to patient. Morphological examination using the whole-
plate technique (16) demonstrated that the clusters and colonies grown in agar which were not stimulated or which werestimulated by any of the CSA's contained immature cells of the
granulocyte-macrophage series or were similar to the leukemic
blast cells before culture. None contained mature granulocytes.The results obtained from the methylcellulose cultures of
patients with AML or one of its variants were also consistentwith other reports (3, 13). The number of clusters and/orcolonies also varied from patient to patient in the same manneras that in agar. The morphology of the cells within methylcellulose was similar to that of the cells within agar. The cellsgrown in methylcellulose which were not stimulated or whichwere stimulated by any of the CSA's had the appearance of
immature members of the granulocyte-macrophage series or
were similar to the preculture leukemic blast cells. Thesemorphological data taken together with the cell surface markerstudies suggest that the vast majority of cells which proliferatedin methylcellulose were at an immature stage of the granulocyte-macrophage series. The cytogenetic studies of Moore andMetcalf (20) and Duttera ef al. (9) demonstrated that cells with
the capacity to make clusters and/or colonies under similarconditions to those used here were derived from leukemicclone(s). These prior studies as well as the failure of the cellsgrown in the present studies to differentiate normally in vitroand the preponderance of cluster growth suggest that theclonogenic cells studied here were of leukemic origin.
In recent years, it has been reported that peripheral bloodcells from patients with AML or one of its variants stimulatedby PHA-LCM are able to form colonies consisting of leukemic
blast cells in methylcellulose and that the number of coloniesproduced was proportional to the number of circulating blastcells (4, 5, 18). Our results of the methylcellulose cultures ofAML or one of its variants were consistent with the reports withregard to the morphology of the cells within clusters andcolonies, although the plating efficiency was lower than thatreported by Minden et al. (18). We also confirmed, by multiplecell surface marker studies, that the cells grown in methylcellulose stimulated by PHA-LCM belonged to the granulocyte-
macrophage series. However, unlike the previously reportedstudies, we failed to find a significant correlation between thenumber of clusters and/or colonies formed and the concentration of morphologically identified blast cells in peripheral bloodwhich was cultured. Indeed, for such a relationship to exist,the blast cells of different patients would have to have the samecloning efficiency. This is extremely unlikely since the ability ofleukemic cells to clone in vitro varies from nil to prolific. Thisshould be studied by other investigators as well since thisrelationship was one of the bases for claiming that the PHA-LCM methylcellulose culture system was superior to the agarculture system and was a "true leukemic stem cell" culture
system (4, 5, 18).Neither cluster nor colony formation was produced by pe
ripheral blood cells obtained from the 3 cases of CML inlymphoid blastic crisis. A small number of clusters with normaldifferentiation to the granulocyte level was found in the cultureof bone marrow cells from one of these patients (Case 24).
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Leukemic Colony Formation
These findings are consistent with those reported for ALL (8,17, 21 ). Both peripheral blood and bone marrow cells obtainedfrom the 7 cases of CML in myeloid blastic crisis formedclusters and/or colonies. Morphological examinations showedthat the cells grown in agar or in methylcellulose had theappearance of immature cells of the granulocyte-macrophageseries or were similar to the leukemic blast cells before culture.The surface marker studies of the cells from the methylcellulosecultures of Specimen 23 were also consistent with the conclusion that the cells were immature members of the granulocyte-
macrophage series. The observations that the leukemic cellsof Patient 18 cloned in vitro during myeloid blastic crisis butfailed to clone under identical in vitro conditions during lymph-
oid blastic crisis demonstrate that the cells of these 2 differentblastic crises differed in functional capacity as well as inmorphology and surface marker characteristics. Therefore, itmay be possible to distinguish between lymphoid and myeloidblastic crises in CML by these culture methods.
In summary, we found no differences between the ability ofagar and methylcellulose to support cluster and/or colonyformation by myeloid leukemic cells. There were also no significant differences among the 3 conditioned media used tostimulate clonal growth in vitro. Myeloid leukemic cells obtainedfrom patients with AML and myeloid blastic crisis of CMLbehaved similarly under the conditions of our studies. Thestudies reported here suggest that the leukemic cells whichclone in vitro are indistinguishable from each other regardlessof the CSA or supporting matrix. This suggestion is compatiblewith the separately reported but essentially identical tritiatedthymidine suicide index studies reported for leukemic cellscloned in agar (25) and in methylcellulose (18). However,further studies are necessary before a definitive statement canbe made as to whether or not cells cloned in agar with CSA (7,10) are identical to those cloned in methylcellulose under thestimulation of PHA-LCM. These studies should include a com
parison of drug sensitivity, size, and density of the cells whichclone in agar and in methylcellulose and an assessment of theability of the clonogenic cells to regenerate in vitro (4, 6).
ACKNOWLEDGMENTS
The authors would like to thank C. Costanze for her excellent technicalassistance and Drs. M. Bloom, T. Doeblin, and M. Stein tor providing us with theopportunity of studying their patients.
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1981;41:3052-3057. Cancer Res Kazuo Kubota, Harvey D. Preisler, Kimitaka Sagawa, et al. Human Leukemic CellsComparison between Agar and Methylcellulose Cultures of
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