Hickie, R. A.

Meyskens, F. L.

Sipes, N. J.

Thomson, S. P.

English

PubMed

Meyskens, F L, Jr

Thomson, S P

Hickie, R A

Sipes, N J

eScholarship - University of California

UC IrvineUC Irvine Previously Published WorksTitlePotential biological explanation of stimulation of colony growth in semi-solid agar by cytotoxic agents.Permalinkhttps://escholarship.org/uc/item/2bk7k5tzJournalBritish journal of cancer, 48(6)ISSN0007-0920AuthorsMeyskens, F L, JrThomson, S PHickie, R Aet al.Publication Date1983-12-01 Peer reviewedeScholarship.org Powered by the California Digital LibraryUniversity of CaliforniaBr. J. Cancer (1983), 48, 863-868Short CommunicationPotential biological explanation of stimulation of colonygrowth in semi-solid agar by cytotoxic agentsF.L. Meyskens, Jr., S.P. Thomson, R.A. Hickiel & N.J. SipesCancer Center Division and Department of Internal Medicine, University of Arizona, Tucson, Arizona 85724,USA and 'Department of Pharmacology, University of Saskatchewan, Saskatoon. Canada S7N OWO.Growth of single cells into colonies in semisolidmedium has been widely used both as a measure ofthe clonogenic potential of normal and transformedcells (Park et al., 1971; Thomson & Rauth, 1974;Courtenay, 1976; Metcalf, 1977; Buick et al., 1977;Hamburger et al., 1977; Salmon, 1980) and as an invitro marker for cellular transformation(Macpherson & Montagnier, 1964; MacAllister &Reed, 1968). Growth of cells in semisolid mediumalso has been used to measure the effect of variouscytotoxic and non-cytotoxic agents on clonogenictumour cells (Salmon et al., 1978; Tveit et al., 1980,1982; Von Hoff et al., 1981; Meyskens et al., 1981).The relation between the number of cells platedand the formation of clusters and colonies, definedhere as growth units, should be clearly defined toassure valid interpretation of perturbations ofclonogenic cells. The frequency and extent ofproliferation to form growth units has beenregarded as a function of the clonogenic capacity ofthe tumour sample together with the conditions ofculture. However, many of the effects of theconditions of culture remain undefined. Therefore,because clonogenic assays are generally closed nonre-fed systems, we have examined the relationshipbetween the number of cells plated, the number ofgrowth units formed, the relative frequency ofgrowth units containing different numbers of cells,and the total number of cells formed within thegrowth units. We found that the cloning efficiencyand proliferative characteristics of clonogenic cellsin agar is significantly determined by the number ofcells plated.Murine melanoma cells (CCL 53.1) were grownin medium (FIO plus 10% horse and 2% heat-inactivated foetal calf-serum) as monolayers inplastic Falcon flasks. Treatment with Tryodessolution removed the cells and producedsuspensions of single cells. Different numbers ofcells were plated in 1.0ml of medium containing0.3% agar (Bacto) over 1.0ml of 0.5% agar inmedium in 35mm diameter Petri dishes. The plateswere incubated in a humidified, 5% Co2, airatmosphere at 37°C for 18 days. Every group ofcells (containing greater than one cell) was countedin randomly selected 1, 2.5, or 6.25mm2 areas. Themean +s.e. number of growth units per area wasmultiplied by 908 mm2 per 35mm culture dish toobtain the total number of growth units per culturedish. The number of growth units of differentdiameters was calculated using the relativefrequency of growth units by size, obtained bydirect measurement of 200 growth units per platewith a micrometer, and multiplying by the totalnumber of growth units per plate.A nomogram was constructed to determine thenumber of cells per growth unit. This was done bydirect visual observation in growth units containing<8 cells. For larger growth units stacking andcrowding of cells prevented accurate counting so5-10 growth units at each 10pm interval diameterwere plucked with a micromanipulator and the cellscounted in stained preparations. The total numbersof cells within the growth units were calculated bymultiplying the frequency of growth units of eachsize by the number of cells per growth unit andsumming. The estimate of the number of populationdoublings is a minimum number as it was assumedthat all progeny cells were capable of doubling andno cell loss occurred.We examined both the size and frequency of thegrowth units in relation to the number of murinemelanoma cells plated. The relationship between thenumber of cells plated and the distribution ofgrowth units by size is shown in Figure 1A,B. Thesize of the growth units decreased as the number ofcells plated increased, which was true even at lownumbers of cells plated per dish (Figure LA). Forexample, when 150,000 cells were plated, 40% of theclonogenic cells produced groups of cells < 30gm indiameter (4 cells). Only 2.0% of the clonogenic cellsgave rise to colonies >80pm in diameter. As thenumber of cells plated was decreased, the number oflarger growth units increased. For example, when500 cells were plated, only 10% of colonies wereThe Macmillan Press Ltd., 1983Correspondence: F.L. Meyskens.Received 10 May 1983; accepted 6 September 1983.864 F.L. MEYSKENS et al.Growth unitsaDiarrof cl)0 20 4.0neter (jim) No. cells No. populationolony (>) (>) doublings (>)30 4 240 8 350 16 470 32 590 64 6110 128 7140 256 8190 515 9No. of murine melanoma cells (ccl 53.1 x 103/plate)5.'4.0Co320x200)06z100 2.5 5.0 10.0 15.0No. of murine melanoma cells(ccl 53.1 x 104/plate)Figure 1 Relationship between the number of murine melanoma cells (CCL 53.1) plated and the number anddiameter of growth units. a: Illustrates 0 to 104 cells plated. b: Extension of range to 15 x 104 cells plated.Relative s.e. averaged 6.75% of the means and were omitted for clarity. Symbols for b the same as for a.86.4Co0xU,0)06zl1COLONY SIZE IN AGAR 865< 90 pm in diameter (64 cells) and > 77% ofcolonies were > 140pm in diameter (256 cells). Foreach number of cells plated, we also calculated thetotal number of cells in growth units. Whether50,000 or 100 cells were plated the total number ofcells in the growth units was nearly the same (8-10 x 105) after 18 days of culture. At the highestnumber of cells plated (150,000), the number of cellsin the growth units was actually decreased (to4 x 105), probably due to exhaustion of nutrientsby the large number of single cells plated initiallywhich consumed nutrients before even small growthunits were formed. These data are consistent withthe study of KHT tumour cells plated in agar byThomson & Rauth (1974). They found that colonyformation and size decreased when large numbersof cells were plated.The observations detailed above are not peculiarto cells from this murine melanoma line. We haveperformed similar experiments with cells fromseveral cell lines of human origin (ovarian, HEY;multiple myeloma, RPMI 8226; adenocarcinoma ofcolon, WIDR; small cell carcinoma of lung,NC1417; melanomas, 81-46A, 82-7A) and with cellsfrom biopsies of melanoma tissues from 5 patientsand obtained the same general results. For example,the cloning efficiency of 60 pm diameter growthunits of the murine melanoma line was essentiallyconstant until >25,000 cells were plated (Figure.IB, 2A) then it decreased rapidly. The other celltypes examined also maintained relatively constantcloning efficiencies for 60pm diameter growth unitsat lower number of cells plated (Figure 2A,B) andhad marked decreases in cloning efficiencies athigher numbers of cells plated. Note that the rangeof constant cloning efficiencies varied among thecell types. Furthermore, cloning efficienciesdecreased at both high and low numbers of cellsplated for 4 to 5 melanoma biopsies (Figure 2B).This general decrease in cloning efficiency seen withlarge numbers of cells plated is consistent with thedata of Tveit et al. (1982). They showed that plating2x l04 tumour cells ml-' instead of 2x I05 to5x 10 gave relatively higher cloning efficiencies;27% of melanoma tumours had cloning efficiencies>1%.A significant effect of these observations is thatlinearity and non-linearity in the formation ofgrowth units depends on the size of the growth unitand the number of cells plated. For example, for25,000 cells plated formation of growth units<70pm in diameter (32 cells, minimum of 5population doublings) was in the linear range, butfor larger growth units the frequency of formationwas in the non-linear range. Thus the larger thegrowth unit, the shorter the range of linearity forgrowth unit formation.This limited range of linearity has a substantialeffect for the interpretation of clonogenic assays. Asexpected from the above observations, we foundsignificant effects of using different growth unit sizecriteria on dose-response curves. We studied theeffect of melphalan on growth unit formation using5,000 murine cell line cells per plate. There was adose-dependent inhibition of 60pm diameter growthunit formation (Figure 3A). However, using largerdiameter growth units, there was no inhibition and"stimulation" with the lower doses of melphalan.The 0.5pgmlP' dose greatly inhibited formation of60pm diameter growth units to 18% survival whileit "stimulated" the 149pm diameter growth units to264% survival. Higher doses inhibited all growthunit formation.These results corroborate our description of therelation between number of cells plated andformation of growth units (Figure 1A,B).Specifically, at 5,000 murine melanoma cells perplate the formation of 60pm diameter growth unitswas in the linear range. Thus agents that inhibit aportion of the cells from proliferating, such as lowdoses of melphalan, should decrease the number of60pm diameter growth units, just as we observed.However, at 5,000 cells per plate the formation of149 pm diameter growth units was not in the linearrange (Figure IA). Thus, partial decreases in theeffective cell number should increase 149pmdiameter growth unit formation, presumably byallowing the remaining cells to consume thenutrients which would have been used by theinhibited cells and their progeny. Thus this wouldonly be an apparent stimulation which is actuallydue to agent induced partial inhibition. This"stimulation" of large growth units was observed atlower doses of melphalan (Figure 3A). Furthermore,if the agent inhibits enough to lower the effectivecell concentration into the linear range for the largecolonies, then inhibition should occur and weobserved it for 1.0 and 10.0 pg ml-1 melphalandoses. Similar results were found for cells from amelanoma biopsy tested with actinomycin D(Figure 3B), with the low dose causing a"stimulation" that increased with the size of growthunits used to define the survival curve. The limitedrange of linearity may also affect the analysis ofagents that stimulate proliferation of clonogeniccells. If the number of cells plated is near the pointwhere cloning efficiency decreases then a stimulatingagent may produce an apparent "inhibition" ofgrowth unit formation.Our data suggest that the apparent cloningefficiency and proliferative characteristics ofclonogenic cells in agar are significantly determinedby the number of cells plated. Thus theinterpretation of parameters of clonogenicity suchas cloning efficiency, extent of proliferation, andsurvival curves of cells which grow in semisolidmedium should be interpreted with considerablecaution.B.J.C.-F866 F.L. MEYSKENS et al.C).20)CD0u2103 5 x 103 104 5 x 104 105 5 X 105No. of cells plated0-1a)4-0-._0).CCb40-20-103 5 x 104 105 5 x 105No. of cells platedFigure 2 Relationship between the number of cells plated and cloning efficiency of >60Qm diameter growthunits. a: Cells from 7 cell lines, 0 myeloma, 8226; 0 ovarian, HEY (R. Buick); * colon, WIDR; EC lung,NCI 417 (D. Carney); melanoma, A C8146A, A C827A and e murine CCL 53.1 respectively. b: Cells frombiopsies of human malignant melanoma, 0 patient 826, 0 patient 8157, * patient 8056, C1 patient 827A,and A patient 8054. Mean + s.e. are shown; no error bar means s.e. was smaller than symbol.COLONY SIZE IN AGAR 867a250 200- b200-150150-o 0~~~~~~~~~~~~~~~~o10050-500 0.01 0.1 0.5 1.0 10.0 00005 0.10Melphalan (pg mlF1) Actinomycin D (pg mlI1)Figure 3 The effect of growth unit size on survival curves. Growth unit size and frequency assay by FAS IIautomated image analysis system, growth unit diameter . 60 um, 0; > 104pm, 0; _ 124pm, *; . 149 pm, O;mean ± s.e. shown. a: Effect of melphalan on growth unit formation from a murine melanoma cell line, CCL. b:Effects of Actinomycin D on growth unit formation from biopsy cells of human melanoma, patient 8140.Mean + s.e. are shown, no error bar means s.e. was smaller than symbol.We thank J. Trent and B. Durie for helpful comments,Desmond Carney for cell line NCI-417 and Ronald Buickfor HEY cell line, and R. Markmann, J. Buckmeier and L.Kimball for secretarial assistance and graphic preparation.This work was supported by grants from AmericanCancer Society (PDT * 184) and the National CancerInstitute (CA17094, CA27502).ReferencesBUICK, R.N., TILL, J.E. & McCULLOCH, E.A. (1977).Colony assays for proliferative blast cells circulating inmyeloblastic leukemia. Lancet, i, 862.COURTENAY, V.D. (1976). A soft agar colony assay forLewis lung tumor and B16 melanoma taken directlyfrom the mouse. Br. J. Cancer, 34, 39.MAcALLISTER, R.M. & REED, G. (1968). Colony growth inagar of cells derived from neoplastic and non-neoplastic tissues of children. Pediat. Res., 2, 356.MACPHERSON, I. & MONTAGNIER, L. (1964). Agarsuspension culture for the selective assay of cellstransformed by polyoma virus. Virology, 23, 291.METCALF, D. (1977). Hematopoeitic colonies: In vitrocloning of normal and leukemic cells. Berlin: Springer-Verlag.MEYSKENS, F.L. Jr., MOON, T.E., DANA, B. & 6 others(1981). Quantitation of drug sensitivity by humanmetastatic melanoma colony forming units. Br. J.Cancer, 44, 787.PARK, C.H., BERGSAGEL, D. & McCULLOCH, E.A. (1971).Mouse myeloma tumor stem cells; a primary cellculture assay. J. Natl. Can. Inst., 46, 411.SALMON, S.E. (1980). Cloning of Human Tumor StemCells. New York: Alan R. Liss.868 F.L. MEYSKENS et al.SALMON, S.E., HAMBURGER, A.W., SOEHNLEN, B.J.,DURIE, B.G.M., ALBERTS, D.S. & MOON, T.E. (1978).Quantitation of differential sensitivity of human tumorstem cells to anti-cancer drugs. N. Engl. J. Med., 298,1321.THOMSON, J.E. & RAUTH, A.M. (1974). An in vitro assayto measure the viability of KHT tumor cells notpreviously exposed to culture conditions. Radiat. Res.,58, 262.TVEIT, K.M. FODSTAD, O., LOTSBERG, J., VAAGE, S. &PIHL, A. (1982). Colony growth and chemosensitivityin vitro of human melanoma biopsies. Relationship toclinical parameters. Int. J. Cancer, 29, 533.TVEIT, K.M., FODSTAD, O., OLSUES, S. & PIHL, A. (1980).In vitro sensitivity of human melanoma xenografts tocytotoxic drugs. Correlations with in vitrochemosensitivity. Int. J. Cancer, 26, 717.VON HOFF, D.D., CASPER, J., BRADLEY, E., JONES, D. &MAKUCH, R. (1981). Association between humantumor colony forming assay results and response of anindividual patient's tumor to chemotherapy. Am. J.Med., 70, 1021.

Potential biological explanation of stimulation of colony growth in semi-solid agar by cytotoxic agents.

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Potential biological explanation of stimulation of colony growth in semi-solid agar by cytotoxic agents.

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