ARTICLES
`
`feasibility of a High-Flux Anticancer Drug
`Screen Using a Diverse Panel of Cultured
`Human Tumor Cell Lines
`
`1 Anne Monks,* Dominic Scudiero, Philip Skehan, Robert Shoemaker,
`Kenneth Paull, David Vistica, Curtis Hose, John Langley, Paul Cronise,
`Anne Vaigro-Wolff, Marcia Gray-Goodrich, Hugh Campbell, Joseph
`Mayo, Michael Boyd
`
`We describe here the development and implementation of a
`pilot-scale, in vitro, anticancer drug screen utilizing a panel
`of 60 human tumor cell lines organized into sub panels repre(cid:173)
`senting leukemia, melanoma, and cancers of the lung, colon,
`kidney, ovary, and central nervous system. The ultimate
`goal of this disease-oriented screen is to facilitate the dis(cid:173)
`covery of new compounds with potential cell line-specific
`and/or subpanel-specific antitumor activity. In the current
`is
`screening protocol, each cell
`line
`inoculated onto
`microtiter plates, then preincubated for 24-28 hours. Sub(cid:173)
`sequently, test agents are added in five IO-fold dilutions and
`the culture is incubated for an additional 48 hours. For each
`test agent, a dose-response profile is generated. End-point
`determinations of the cell viability or cell growth are per(cid:173)
`formed by in situ fixation of cells, followed by staining with a
`protein-binding dye, sulforhodamine B (SRB). The SRB
`binds to the basic amino acids of cellular macromolecules;
`the solubilized stain is measured spectrophotometrically to
`determine relative cell growth or viability in treated and un(cid:173)
`treated cells. Following the pilot screening studies, a screen(cid:173)
`ing rate of 400 compounds per week has been consistently
`achieved. [J Natl Cancer Inst 83:757-766, 1991]
`
`l !
`) l )
`! The National Cancer Institute is implementing a new inves(cid:173)
`
`tigational, in vitro, disease-oriented, drug-discovery screen.
`)_ Othe, publications desc<ibe the overnll concept and rntionfile fm
`]_
`this screen (1-3) and various aspects of its technical evolution
`(4-7). The purpose of the screen is to provide for the initial
`evaluation of more than 10 000 new substances per year for
`cytotoxic and/or growth-inhibitory activity against a wide diver(cid:173)
`sity of tumor types and to allow the detection of possible tumor-
`type-specific sensitivity. The data from such an in vitro primary
`screen will allow selection of the most sensitive cell line(s) for
`
`subsequent in vivo xenograft testing of compounds of interest.
`While the concept is simple, the technical. challenges for im(cid:173)
`plementation of such a screen are greatly magnified by the re(cid:173)
`quired scope of the program.
`The goal of the program is to implement the full-scale screen
`with a capacity for testing new substances at a rate of more than
`10 000 per year against a broadly representative panel of 100 or
`more human tumor cell lines. In moving toward this goal, we in(cid:173)
`itiated the pilot-scale screen outlined here, incorporating ex(cid:173)
`perience gained from previous laboratory-scale research and
`development efforts. With the pilot screen we have explored a
`variety of conceptual issues, as well as technical and managerial
`issues critically involved in a scale-up to the full screen. This
`paper describes our experiences with the pilot screen and its
`evolution through a variety of technical stages, including com(cid:173)
`parative evaluations of two types of tetrazolium assays ( 4 ,5) and
`an assay using the protein-binding dye sulforhodamine B (SRB)
`(6), all of which are microculture assays to measure cell
`viability and cell growth.
`The results from operation of the pilot screen have been
`evaluated both continuously and retrospectively with a par(cid:173)
`ticular view toward determining the feasibility of implementa(cid:173)
`tion of the full-scale screen.
`
`Received November 19, 1990; revised March 8, 1991; accepted March 20,
`1991.
`Sponsored in part by the National Cancer Institute, National Institutes of
`Health, Department of Health and Human Services, under contract N0ICO-
`23910 with Program Resources, Inc.
`A. Monks, D. Scudiero, C. Hose, J. Langley, P. Cronise, A. Vaigro-Wolff, M.
`Gray-Goodrich, H. Campbell (Program Resources, Inc), P. Skehan, R.
`Shoemaker, K. Paull, D. Vistica, J. Mayo, M. Boyd (Developmental
`Therapeutics Program, Division of Cancer Treatment), NCI-Frederick Cancer
`Research and Development Center, Frederick, Md.
`*Correspondence to: Anne Monks, PhD, NCI-Frederick Cancer Research and
`Development Center, Bldg 432, Rm 232, Frederick, MD 21701-1013.
`
`te
`
`Vol. 83, No. 11, June 5, 1991
`
`ARTICLES 757
`
`

`

`Materials and Methods
`
`Cell Lines
`
`The human tumor cell lines currently used in the pilot screen
`have been presented previously (7); details of their origin and
`characterization are described elsewhere (Stinson SF, Alley
`MC, Kopp WJ, et al: manuscript submitted). Parallel to the im(cid:173)
`plementation and operation of the pilot screen, a major effort
`continues for the acquisition and evaluation of new cell lines for
`possible addition or substitution into the panel. Ultimately, the
`cell line panel may contain as many as twice the current number
`of lines, with each disease-related subpanel containing 10-15
`representative lines (3). However, for purposes of the pilot
`screen, we have focused on a fixed set of 60 lines (Table 1).
`Criteria for selection of a cell line for use in the interim panel
`were as follows:
`(a) adaptability to growth on a single medium (RPMI-1640
`plus 5% fetal bovine serum and 2 mM glutamine);
`(b) a negative test for mycoplasma and mouse antibody
`production;
`(c) isoenzyme and karyotype profiles verifying the human
`origin of the cells;
`( d) mass doubling time that allows for harvesting of ap(cid:173)
`proximately 3 x 107 cells twice a week; and
`(e) suitability for use with microculture assays.
`Once a line had been established as suitable, the number of
`cells was massively expanded in a minimal number of passages,
`and the cells were cryopreserved in a large repository of am(cid:173)
`pules, each containing 1 x 106 cells, to provide a consistent,
`long-term frozen stock for future use (4). Within the primary
`drug evaluation laboratory, new cell line stock samples are
`thawed as each cell line culture used for screening approaches
`its 20th passage or if there is a noticeable change in growth,
`morphology, or drug-response characteristics. Once the growth
`of the new stock is established at the second or third passage,
`the older passage line is replaced with the new stock for use in
`the screening laboratory.
`
`Cell Line Maintenance
`
`Major considerations with respect to cell line maintenance in(cid:173)
`~ude (a) the necessity of continually producing approximately 3
`x 107 cells for biweekly inoculation for the pilot screen, (b) the
`potential for cross-contamination of cell lines, and (c) the pos(cid:173)
`sibility of low-grade microbial contamination. Individual tech(cid:173)
`nicians are therefore assigned only six specific cell lines, which
`they monitor continually for growth characteristics in tissue-cul(cid:173)
`ture flasks and microculture plates, for behavior in the microcul(cid:173)
`ture assays, and for microscopic appearance. Furthermore, the
`use of a limited number of passages from a frozen-stock vial
`also helps prevent long-term cross-contamination of a cell line.
`Cells are grown and passaged in antibiotic-free growth medium
`to ensure freedom from microbial contaminants.
`Cells are maintained in multiple T150 tissue-culture flasks.
`Cells for each inoculation day are maintained separately (no
`common reagents) and passaged on separate days to prevent
`catastrophic loss of growing cell line stocks to microbial con(cid:173)
`tamination. Additional backup flasks of cells are also main-
`
`ticularly for potent cytotoxins, for compounds of limited
`solubility, or for nonroutine detailed comparisons of selected
`compounds when previous information is available. Crude ex-
`_nts, +l
`tract~ of natural pro
`u~ts afre
`t
`sted
`aLt five thdr
`eefolfd di
`ubt~o
`1
`startmg at an upper 1m1t o
`µgm
`
`1regar ess o so u 11 y,
`25
`1
`ie, particulate matter may be present. All samples are initially
`solubilized in dimethyl sulfoxide (DMSO) or water at 400 times
`the desired final maximum test concentration. Drug stocks are
`)
`not filtered or sterilized, but microbial contamination
`is
`·•
`controlled by addition of gentamicin to the drug diluent. Multi-
`ple aliquots are stored frozen at - 70°C to provide uniform 1
`j
`samples for initial tests as well as for retests, if required. Just
`prior to preparation of the drug dilutions in cell-culture medium, ·1
`these frozen concentrates are thawed at room temperature for 5
`medium containing 50 µg/mL gentamicin to twice the desired f
`minutes. The concentrates are then diluted with complete
`r
`
`final concentrations.
`
`,
`
`Drug Incubation
`
`Immediately after preparation of these intermediate dilutions,
`100-µL aliquots of each dilution are added to the appropriate
`microtiter plate wells according to the format in Fig 1, in which
`
`bis
`2H
`Pn
`ph,
`ad1
`ad1
`the
`pl::
`de1
`sin
`
`758
`
`Journal of the National Cancer Institute
`
`Vo
`
`tained. For each cell line, the seeding density per flask is deter(cid:173)
`mined for production of a healthy culture of 70%-90% confluen(cid:173)
`cy after 7 days for continued routine maintenance or after 4 or 5
`days for the microculture assay. These seeding densities are then
`utilized twice a week to maintain sufficient cells for routine cul(cid:173)
`ture and for anticancer drug screening.
`
`Preparation and Inoculation of Cells
`
`All of the adherent cell lines are detached from the culture
`flasks by addition of 2-3 mL of 0.05% trypsin-EDTA (GIBCO
`Laboratories, Grand Island, NY). Thereafter, trypsin is inac(cid:173)
`tivated by addition of 10 mL of 5% s'erum-containing RPMI-
`1640 medium. Cells are separated into single-cell suspensions
`by a gentle pipetting action, then counted using trypan-blue ex(cid:173)
`clusion on a hemacytometer or by a Coulter counter, which is
`used when viability as determined by trypan-blue exclusion is
`routinely greater than 97% and the cells maintain suspension as
`single cells. After counting, dilutions are made to give the ap(cid:173)
`propriate cell densities for inoculation onto the microtiter plates.
`Cells are inoculated in a volume of 100 µL per well at densities
`between 5000 and 40 000 cells per well (Table 1 ); the basis for
`selection of the particular inoculation densities for each cell line
`is described in the Results section. A 100-µL aliquot of com(cid:173)
`plete medium is added to cell-free wells. Cells from all cell lines
`are counted, diluted, and inoculated onto microculture plates
`within a 4-hour period on 2 days each week. The microtiter
`plates containing the cells are preincubated for approximately
`24 hours at 37°C to allow stabilization prior to addition of drug.
`
`~
`
`)·
`
`Solubilization and Dilution of Samples
`
`For initial screening of pure compounds, each agent is
`routinely tested at five 10-fold dilutions, starting from a maxi(cid:173)
`mum concentration of 10--4 M. Alternatively, a maximum of 10-3
`M may be selected if solubility permits, if the higher upper limit
`is desired, and if sufficient compound is available. Alternative
`
`,
`
`1l
`
`0e
`
`1~
`
`initial concentrations may also be utilized for retesting, par- l
`
`A
`
`B
`
`C
`
`D
`
`E
`
`F
`
`G
`
`H
`
`Fig
`test
`D2,(cid:173)
`gr0\
`well
`ITTLCI
`
`thn
`aln
`cor
`Ag,
`fill(
`inc
`phc
`the
`ass
`the
`are
`me
`
`Mi
`
`din
`De
`tai1
`µL
`wit
`pre
`grc
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`tirr
`to I
`em
`rea
`31:
`sin
`
`

`

`2
`
`3
`
`4
`
`10
`
`11
`
`12
`
`Sulforhodamine B Assay
`
`A
`
`D1 5
`
`01, 01, 01 2
`
`01, Ce
`
`Ce
`
`02, 02, 02, 02, D25
`
`Ge Ge
`
`Ge Ge
`
`Ge Ge
`
`Ge Ge
`
`Ge Ge
`
`Ge Ge
`
`C
`
`D
`
`G
`
`H
`
`\
`
`" \
`
`\J
`
`,11
`
`Ce
`
`Ce
`
`I w w
`
`\
`
`I
`
`)
`
`Drug Blank
`(No cello)
`
`Drug Blank
`(No coils)
`
`Cell Lina
`1
`
`Coll lino
`2
`
`Coll lino
`3
`
`The detailed methodology for the SRB assay is presented
`elsewhere (6). Briefly, adherent cell cultures are fixed in situ by
`adding 50 µL of cold 50% (wt/vol) trichloroacetic acid (TCA)
`(final concentration, 10% TCA) and incubating for 60 minutes
`at 4°C. The supernatant is then discarded, and the plates are
`washed five times with deionized water and dried. One hundred
`microliters of SRB solution (0.4% wt/vol in 1 % acetic acid) is
`added to each microtiter well, and the culture is incubated for 10
`minutes at room temperature. Unbound SRB is removed by
`washing five times with l % acetic acid. Then the plates are air(cid:173)
`dried. Bound stain is solubilized with Tris buffer, and the optical
`densities are read on an automated spectrophotometric plate
`reader at a single wavelength of 515 nm.
`For suspensions of cell cultures (eg, leukemias), the method is
`the same, except that at the end of the drug-incubation period,
`the settled cells are fixed to the bottom of the microtiter well by
`gently adding 50 µL of 80% cold TCA (final concentrati~n.
`16%TCA).
`
`Data Calculations
`
`Unprocessed optical density data from each microtiter plate
`are automatically
`transferred from
`the plate reader to a
`microcomputer, where the background optical density (OD)
`measurements (ie, complete medium plus stain, minus cells) are
`subtracted from the appropriate control well values and where
`the appropriate drug-blank measurements (ie, complete medium
`plus test compound dilution plus stain, minus cells) are sub(cid:173)
`tracted from the appropriate test well values. The values for
`mean ± SD of data from replicate wells are calculated. Data are
`expressed in terms of %TIC [(OD of treated cells/OD of control
`cells) x 100], as a measure of cell viability and survival in the
`presence of test materials. Calculations are also made for the
`concentration of test agents giving a T/C value of 50%, or 50%
`growth inhibition (IC50) , and a T/C value of 10%, or 90%
`growth inhibition (IC90).
`With the SRB assay, a measure is also made of the cell
`population density at time O (the time at which drugs are added)
`from two extra reference plates of inoculated cells fixed with
`TCA just prior to drug addition to the test plates. Thus, we have
`three measurements: control optical density (C), test optical den(cid:173)
`sity (T), and optical density at time zero (T0).
`Using these measurements, cellular responses can be calcu(cid:173)
`lated for growth stimulation, for no drug effect, and for growth
`inhibition. If T is greater than or equal to T0, the calculation is
`100 x [(T-T0)/(C -T0)]. If Tis less than T0, cell killing has oc(cid:173)
`curred and can be calculated from 100 x [(T-T0)/T0] (8). Thus,
`for each drug-cell line combination, a dose-response curve is
`generated and three levels of effect are calculated. Growth in(cid:173)
`hibition of 50% (Gl50) is calculated from 100 x [(T - T0)/(C -
`T0)] = 50, which is the drug concentration causing a 50% reduc(cid:173)
`tion in the net protein increase in control cells during the drug
`incubation. The drug concentration resulting in total growth in(cid:173)
`hibition (TGI) is calculated from T = T0, where the amount of
`protein at the end of drug incubation is equal to the amount at
`the beginning. The final calculation, LC50, is the concentration
`of drug causing a 50% reduction in the measured protein at the
`end of the drug incubation, compared with that at the beginning,
`
`~
`
`~ter-
`uen-
`or 5
`then
`CUI-
`
`nae-
`'MI-
`
`lture 1
`iCO l
`ions l
`: ex-
`:h is 1t
`1n is
`n as
`ap-
`Hes.
`ities
`; for
`line
`om-
`ines
`ates
`titer
`ttely
`ug.
`
`Fig L Microtiter plate format used for screening shows three cell lines and two
`test agents (DI and D2), each inoculated at five concentrations (D1,-Dl, and
`D2,-D2,): One plate holds six dose-response experiments with quadruplicate
`growth control (Ge) wells, four control background (C8 ) wells, and duplicate test
`wells, for each dose-response set. Letters A-H and Nos. 1-12 represent the
`microtiter plate map.
`
`I
`
`three cell lines are inoculated per plate. As the microtiter wells
`already contain the cells in 100 µL of medium, the final drug
`concentration tested is 50% of that in the intermediate dilutions.
`Agents are then added immediately to the cultures in the
`microtiter plates. During development of these procedures, drug
`incubation times were 1, 2, 3, 4, or 6 days at 37°C in an atmos(cid:173)
`phere of 5% CO2 and 100% relative humidity. The plates were
`then assayed for cellular growth and viability by microculture
`assay-either by one of the two types of tetrazolium assay or by
`the SRB assay. In the current screening procedure, the cultures
`are incubated with test agents for 2 days, and the end point is
`measured by the SRB assay.
`
`Microculture Tetrazolium Assay
`
`.t IS
`
`)
`axi-
`10-3 · . .
`
`The MTT assay is based on metabolic reduction of 3-(4,5-
`dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT).
`Details of this assay have been published previously ( 4), but cer(cid:173)
`tain modifications were made for use in the pilot screen. A 50-
`µL aliquot of MTT solution (1 mg/mL) in RPMI-1640 medium,
`with no serum or glutamine, is added directly to all the ap(cid:173)
`propriate microtiter plate wells containing cells, complete
`growth medium, and tes.t agents. The culture is then incubated
`for 4 hours to allow for MTT metabolism to formazan. After this
`time, the supernatant is aspirated and 150 µL of DMSO is added
`to dissolve the formazan. Plates are agitated on a plate shaker to
`ensure a homogeneous solution, and the optical densities are
`read on an automated spectrophotometric plate reader (model
`312; Biotech Research Laboratories, Inc, Rockville, Md) at a
`single wavelength of 550 nm.
`The XTT assay is based on the metabolic reduction of 2,3-
`bis[2-methoxy-4-nitro-5-sulfopheny I ]-5-[ (pheny !amino )carbonyl]-
`2H-tetrazoli um hydroxide (XTT). The detailed methodology is
`presented elsewhere (5). Briefly, 1 mg/mL XTT is mixed with
`phenazine methosulfate (7 .65 µg/mL) immediately prior to its
`addition to microtiter plates. Fifty microliters of the mixture is
`added to each well and incubated for 2-4 hours, depending on
`the metabolizing characteristics of the individual cell lines. Each
`plate is agitated for 2-3 minutes on a plate shaker; then optical
`densities are read on an spectrophotometric plate reader at a
`single wavelength of 450 nm.
`
`l
`l
`·orm l
`
`imit
`ttive
`par(cid:173)
`tited
`cted
`ex-
`ons,
`lity,
`ially
`mes
`are
`is
`I
`ulti-
`
`Just
`um,
`or 5
`,Iete
`
`,
`j
`j
`
`ired
`
`,;•
`
`ons,
`iate
`hich
`
`itute
`
`Vol. 83, No. 11, June 5, 1991
`
`ARTICLES 759
`
`

`

`indicating a net loss of cells following drug treatment. LC50 is
`calculated from 100 x [(T-T0)/f0] = - 50.
`Values are calculated for each of these parameters if the level
`of effect is reached, but if the effect is not reached or is ex(cid:173)
`ceeded, then the value is expressed as greater or less than the
`maximum or minimum drug concentration tested.
`
`Computer Support and Automation
`
`The projected goal of the National Cancer Institute (NCI) in
`vitro drug screen is to support the initial yearly evaluation of
`10 000 or more new agents against as many as 100 or more cell
`lines. This would result in the generation of more than 1 million
`dose-response curves per year. Extensive computer support has
`necessarily been developed for each step of the screening
`process, from the shipping of an agent to the drug-preparation
`laboratory to the analysis and return of screening data to a sup(cid:173)
`plier.
`Agents to be screened are received by the drug-preparation
`laboratory, and the NCI drug identification code is entered into
`the computerized data management and monitoring system.
`After solubilization of the agents, details of the solubility and
`priority for testing are entered to update the database. In addi(cid:173)
`tion, bar-coded labels containing all the appropriate information
`are generated and attached to the storage vials containing the
`concentrates, which are then placed in interim storage at -70°C.
`Screening-laboratory staff determine
`the available
`test
`capacity for the following week and enter this information into
`the mainframe database. Then, automated assignment of test
`agents to fill the scheduled requirements is performed according
`to preassigned priority status. Alternatively, assignments or
`reassignments may be performed manually.
`Immediately prior to the day of an assignment, information is
`loaded via modem to microcomputers (Compaq 386-20 running
`an SCO XENIX operating system) in the screening laboratories.
`Labels are generated and printed by computer for each
`microculture plate. Each label contains an identification number
`that is specific to a particular cell line-drug-plate format.
`After each technical task (ie, cell inoculation, addition of
`drug, or staining), the technicians confirm successful comple(cid:173)
`tion, using computer terminals available in each tissue-culture
`laboratory. This procedure allows for cross-checking of each
`stlij) of the assay to ensure that the actual and assigned proce(cid:173)
`dures are identical. Once an identity number has been entered,
`the computer system tracks it through all the technical steps so
`that when the optical densities are read spectrophotometrically,
`dose-response data can be calculated automatically and printed
`within minutes of the plate reading. At this point, there is
`provision for the manual elimination of suspicious data points
`by insertion of "reason codes" (eg, for microbial contamination,
`improper cell inoculation, or improper drug inoculation). These
`procedures are designed to prevent entry into the mainframe
`database of dose-response data known to be flawed for specific
`reasons.
`In addition to these provisions for manual deletions to ensure
`quality control, a series of automated quality control parameters
`are performed by computer. These include "flags" for (a) varia(cid:173)
`tion exceeding 20%, as determined from the coefficient of varia(cid:173)
`tion (CV) of the mean of control optical densities (n = 4); (b)
`
`variation exceeding 25%, as determined from the CV of the
`mean of duplicate dose-response optical densities;
`(c) mean
`background optical densities greater than 10% of control; (d)
`mean control optical densities exceeding 3 SD of the mean opti(cid:173)
`cal densities for the six previous experiments for that cell line;
`and (e) a dose-response curve transecting the point of 50%
`growth inhibition more than once (reversal).
`These codes constitute a "failure" of part or all of a dose(cid:173)
`response determination and eliminate those data from further
`analysis. On a daily basis, data are copied to an on-site
`microcomputer for backup and
`laboratory analysis,
`then
`downloaded nightly to a large central computer (VAX 8820) for
`future detailed analysis, comparisons, and generation of screen(cid:173)
`ing-data reports for suppliers of the samples tested.
`
`Procedure for Submitting New Cell Lines
`
`Investigators who wish to submit cell lines for consideration
`of inclusion in the panel should contact:
`
`Mr. Richard Camalier, Biologist
`BTB, DTP, DCT, NCI-FCRDC
`Building 321, Room 7
`Fort Detrick, Frederick, MD 21702
`Telephone (301) 846-5065
`FAX (301) 846-5439
`
`Results
`
`Choice of Assay for Cell Growth and Viability
`
`For the initial feasibility studies, the microculture tetrazolium
`assays (MTT and XTT) were utilized to determine cell growth
`and viability. During the evolution of the large-scale screen,
`however, certain technical limitations of these assays became
`especially apparent. As a result, we investigated alternative ap(cid:173)
`proaches, using protein and biomass stains. We found an assay
`using sulforhodamine B (SRB), a dye that binds to basic amino
`acids of cellular macromolecules, to be promising for purposes
`of this screen (6).
`The major
`technical disadvantage ' of the microculture
`tetrazolium assays (MTT and XTT) is that the "moving-target"
`nature of the optical density, which is determined by formazan
`production, is a function of time, in addition to cell number.
`With the requirements to screen at least 10 000 agents per year
`and to compare each cell line against an agent in a single test,
`1000 or more microculture plates must be evaluated for cell
`growth inhibition and viability on the same day. This presents a
`severe logistical challenge, especially at the stage of plate read(cid:173)
`ing, as there is a very narrow window of time within which the
`tetrazolium plates must be processed.
`The MTT microculture assay is a reasonably simple, sensi(cid:173)
`tive, reproducible assay that exhibits good signal-to-noise ratios
`(4). However, the MTT procedure requires aqueous aspiration
`and DMSO-solubilization steps, making the procedure more
`time-consuming and potentially error-prone. Moreover, the use
`of large quantities of DMSO is undesirable in a busy screening(cid:173)
`laboratory environment.
`The XTT microculture assay is technically very simple and
`can be applied directly to suspension or monolayer cultures.
`
`,
`
`,,
`
`I
`
`flow
`ratio:
`coup
`anotl
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`add-
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`
`Journal of the National Cancer Institute
`
`Yo1.
`
`

`

`, However, the XIT a ay yields relatively p or signal-to-noi ·e
`raiios and require addition to the as ay medium of an electron(cid:173)
`coupling agent (phenazine meth ulfate)
`representing yet
`another potential source of experimental variation. Furthermore,
`with the XIT assay there was occasional formmion of a cry tal(cid:173)
`(ine material in the culture well , which strongly interfered with
`the assay. This phenomenon was subsequently related in part to
`CO2 variations and buffering instability (9) and was one of the
`factors leading to development of a new CO2-independent cul(cid:173)
`ture medium (10) to obviate the problem.
`The SRB assay has a stable end point, since the drug-exposed
`cells are fixed with TCA at the end of the incubation period (6).
`. Once the cells are fixed and stained, there is no critical time
`restriction for subsequent determination of the SRB optical den(cid:173)
`sities, which change less than 2% over a 7-day period if
`evaporation is restricted (data not shown). Furthermore, the
`SRB assay provides excellent signal-to-noise ratios. The techni(cid:173)
`cal manipulations required for staining are greater than for the
`tetrazolium assays, requiring up to 10 washing steps and two
`drying steps. However, these steps can be automated and, under
`the current protocol, can be readily scheduled around the ac(cid:173)
`tivities of cell inoculation, addition of drug, and in situ staining
`of cells.
`Comparisons of data from several hundred agents screened in
`parallel by MTT and SRB assays indicated that, under the ex(cid:173)
`perimental conditions employed and within the limits of the data
`analyses applied, the MTT and SRB assays generally yielded
`quite similar results (] 1). Therefore, the SRB procedure was
`subsequently selected for routine use in the pilot screen, prin(cid:173)
`cipally because of the technical advantages described above.
`
`1
`
`Assay Parameters
`
`Assay parameters refer here to the particular selections of (a)
`initial cell densities for inoculation and (b) drug incubation
`times. To avoid biasing the screen excessively toward detection
`of antiproliferative effects that might otherwise obscure interest(cid:173)
`ing patterns of differential cytotoxicity, we have focused our
`developmental efforts on screening protocols incorporating rela(cid:173)
`tively high initial inoculation densities and relatively short in(cid:173)
`cubation times. Table I shows the specific cell inoculation
`densities used for each cell line; optimal initial cell densities
`were determined for each line as described in the following sec(cid:173)
`tion.
`As the cell line panel was expanded to include new lines, it
`became apparent that many cell lines were subject to severe
`nutrient depletion by the end of a 6-day incubation period. This
`was indicated by extreme acidity of the growth medium and
`peeling of the cells from the microculture plate well surface. An
`additional consideration was the effect of incubation time on
`cell doubling times. An investigation into the growth rate of
`cells inoculated into microculture plates was undertaken . Six
`cell lines with different growth rates were selected and their
`growth in the described protocol was examined. During days I
`and 2, the doubling time increased as expected, but between
`days 3 and 4, two of the six cell lines showed negative growth
`rates, indicating that the populations were dying faster than they
`Were growing (data not shown).
`
`Vol. 83,No. ll,June5, 1991
`
`-ll
`1~·
`
`I~
`
`1
`
`he
`,an
`(d)
`1ti-
`1e;
`)%
`
`,e-
`1er
`ite
`,en
`for
`:n-
`
`on
`
`Jill
`vth
`en,
`me
`lp-
`,ay
`.no
`,es
`
`1re
`et"
`'.an
`,er.
`c:ar
`:st,
`:ell
`sa
`1d-
`the
`
`1si-
`ios
`ion
`xe
`1se
`1g-
`
`111d
`·es.
`
`:ute
`
`Table l. Human tumor cell lines and inoculation densities used in the NCI
`disease-oriented in vitro drug screen
`
`Cell line
`
`Cells/well Cell line
`
`Celts/well
`
`Celt line
`
`Cells/well
`
`Lung cancer
`NCI-H23
`NCI-H226
`NCI-H322M
`NCI-H460
`NCI-H522
`A549/ATCC
`EKVX
`HOP-18
`HOP-62
`HOP-92
`LXFL 529
`DMS 114
`DMS 273
`
`Renal cancer
`UO-31
`SNl2C
`A498
`CAKI-1
`RXF393
`RXF631
`ACHN
`786-0
`TK-10
`
`Colon cancer
`20000 HT29
`20000 HCC-2998
`HCT-116
`20000
`5000
`SW-620
`15 000
`COLO205
`10000 DLD-1
`20000 HCT-15
`20000 KM12
`15000 KM20L2
`20000
`10000 Melanoma
`20000
`LOXIMVI
`5000 MALME-3M
`SK-MEL-2
`SK-MEL-5
`SK-MEL-28
`20000
`15 000 Ml9-MEL
`20000
`UACC-62
`10000 UACC-257
`20000 MI4
`10000
`15 000
`10000
`15 000
`
`5000
`10000
`5000
`IOOO0
`15 000
`5000
`10000
`15 000
`10000
`
`5000
`20000
`20000
`10000
`10000
`IOOO0
`IOOO0
`20000
`15 000
`
`CNS cancer
`SNB-19
`SNB-75
`SNB-78
`U251
`SF-268
`SF-295
`SF-539
`XF498
`
`15 000
`20000
`20000
`7500
`15 000
`10 000
`15 000
`20 000
`
`Ovarian cancer
`OVCAR-3
`10000
`OVCAR-4
`10 000
`OVCAR-5
`20 000
`OVCAR-8
`10 000
`IGR-OV-1
`10000
`SK-OV-3
`20000
`
`Leukemia
`CCRF-CEM 40 00\J
`K-562
`5000
`MOLT-4
`30 000
`HL-60
`20000
`RPMI-8226
`20000
`SR
`30000
`
`To examine the difference between the results at I, 2, 3, and 4
`days of incubation, the SRB assay was used to perform a dose(cid:173)
`response analysis on 20 standard drugs tested in six cell lines
`(A2780, HT29, HOP-62, OVCAR-5, SN12KI, and SK-MEL-5).
`A2780 and SN12KI have since been deleted from the cell line
`panel. The 20 agents were doxorubicin, amsacrine (AMSA),
`bleomycin, cisplatin, dibromodideoxymannitol (mitobronitol),
`gossypol, melphalan, mitomycin-C, retinoic acid, vinblastine,
`actinomycin (dactinomycin), carmustine (BCNU), chromo(cid:173)
`mycin, cordycepin (3'-deoxyadenosine), fluorouracil (5-FU),
`homoharringtonine, methotrexate, phyllanthoside, tamoxifen,
`and etoposide (VP-16).
`With the assays using 1-4 days of incubation, it is possible to
`determine a signal (optical density) at the time drug is added
`(T0). Using these values, calculations can be made for the con(cid:173)
`centration of drug causing 15% growth inhibition (Gl 15), 50%
`growth inhibition (Gl50), total growth inhibition (TGI), and 50%
`cell kill (LC50). Fig 2 shows representative dose-response curves
`comparing I, 2, 3, or 4 days' exposure of six cell lines to
`doxorubicin and BCNU. Table 2 shows a summary of the cel(cid:173)
`lular response parameters calculated as a function of drug in(cid:173)
`cubation time from such curves obtained for all 20 of the drugs
`used as standards. These data indicated that growth inhibition
`(Gl, 5 and Gl50) by the selected standard clinical agents could be
`reliably determined at 2-4 days of drug exposure but that this
`determination was much less reliable after only 1 day of drug
`exposure. The more demanding end points of TGI and LC50
`were less commonly achieved than the end points of Gl 15 and
`Gl50 , but the 1- and 2-day incubations discriminated these
`parameters less well than the longer incubation periods. The
`TGI parameter was achieved in five or more cell lines in the I(cid:173)
`or 2-day assay only with doxorubicin, AMSA, BCNU, gossypol,
`
`ARTICLES 761
`
`

`

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