Radiation Oncology Znvestzgations 1:261-269 (1994)

Proliferative Assays for the Assessment of Radiosensitivity ofTumor Cell Lines Using

96-Well Microcultures Peter Cross, M.D., Elaine s. Marshall, N.z.c.s., Bruce c. Baguley, Ph.D.,

Graeme J. Finlay, Ph.D., John H.L. Matthews, M.B.Ch.B., F.R.A.c.R., and William R. Wilson, Ph.D.

Department of Clinical Oncology, Auckland Hospital (P.C., J . H.L.M.), and Cancer Research Laboratory (E.S.M., B.C.B., G.J.F.) and Department o f Pathology (W.R. W.) , University

of Auckland School o f Medicine, Auckland, New Zealund

SUMMARY We have compared the effectiveness of three short-term proliferative assays [3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MIT) reduction, methyl- ene blue staining, and [3H]-thymidine incorporation] in predicting the results of clono- genic assay of radiosensitivity in five mammalian cell lines (SCCWI, AA8, V3, FME, and MM-96). Cells were cultured in 96-well microculture plates and a lead wedge was em- ployed to provide a range of radiation doses (%o source) up to 10 Gy. Radiation dosimetry was determined by a novel modification of the Fricke/thiocyanate dosimetry technique to allow direct determination in 96-well plates. Cells were cultured following irradiation to determine clonogenic survival curves and to calculate the survival at 2 Gy (SF,), the initial slope of the fitted linear quadratic function (a), and the mean inactivation dose 0. A broad range of radiosensitivity was obtained (SF, = 0.14-0.92) with the clonogenic assay. All three proliferation assays provided measures of radiosensitivity which correlated with clonogenicity at low radiation dose. The thymidine incorporation assay, which provided excellent linear correlation (r = 0.98) with clonogenicity for SF, and IT, used semiauto- mated methods for harvesting and scintillation counting to give sample processing times comparable to the colorimetric methods. It offered the advantages of superior assay linear- ity and dynamic range, and reduced interference by non-proliferating cells at high radiation dose. Radiat Oncol Invest 1994;1:261-269. 0 1994 Wdey-Liss, Inc.

Key wora!r: thymidine incorporation, clonogenicity, cell culture, radiation

INTRODUCTION In recent years there has been considerable interest in the development of predictive assays for the radio- sensitivity of human tumors. Much of this work has concentrated on colony-forming assays, particu- larly those described by Courtenay et al. [l], and correlations have been reported between in vitro cellular radiosensitivity and clinical radiorespon- siveness of tumors [2-71. Despite its reputation as a

standard for measuring tumor cell radiosensitivity , the clonogenic assay has been limited in clinical studies by its technical demands [8]. Proliferation assays in microcultures would offer a number of advantages over clonogenic assays providing that they gave comparable results. Such assays do not require as much laboratory time to perform and endpoints are reached earlier (within 1 week as opposed to between 2 and 4 weeks for a clonogenic

Received original June 17, 1993; revised October 25, 1993; accepted December 3, 1993. Address correspondenceheprint requests to: Peter Cross, M.D., Ottawa Regional Cancer Centre, 190 Melrose, Ottawa,

0 1994 Wiley-Liss, Inc. Ontario, Canada K1Y 4K7.

262 Cross et al.: Microculture Assessment of Radiosensitivity

assay). Furthermore, fewer tumor cells are required and exhaustive tissue disaggregation, which is dif- ficult for many tumors, is not required.

The aim of the work reported here was to eval- uate the applicability to radiosensitivity testing of three assays already in use in this laboratory, namely the methylene blue (MB) staining assay [9], the 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra- zolium bromide (MlT) reduction assay [lo], and the [3H]-thymidine (TdR) incorporation assay [ 1 11. All three proliferation assays are simple to under- take and can be performed with relatively low num- bers of cells. We have determined radiation dose- response curves for five cell lines in each of these three cell proliferation assays and compared them to survival curves obtained using clonogenic assays. To maximize the range of radiosensitivity mea- sured, the radiosensitive AA8 subline V3 was used, as well as a cell line cultured in the presence of a radioprotector (cysteamine).

MATERIALS AND METHODS

Materials Materials and sources were as follows: 5-fluoro-2’- deoxyuridine (FUdR), thymidine, hydrocortisone, penicillin, DNAase type I (crude, pancreatic), cys- teamine (Sigma Chemical Co., St. Louis, MO); alpha-modified minimal essential medium, fetal bovine serum (GIBCOIBRL New Zealand Ltd., Auckland, New Zealand); 96-well plates (Flow Laboratories, Inc., McLean, VA); [3H]-TdR (DuPont/NEN Products, Boston, MA); glass fiber filter paper, filter bags, scintillation fluid (LKB Wallac Oy, Turku, Finland); trypsin (Difco Labo- ratories, Detroit, MI); crude Streptomyces griseus pronase (Calbiochem, San Diego, CA); streptomy- cin sulfate, dimethylsulfoxide (DMSO), MB, MTT (Serva Feinbiochemica, Heidelberg, Germany).

Cell Lines and Culture Conditions The murine cell line SCCVII was provided by Dr. K. Fu (University of California, San Francisco), the Chinese hamster ovary lines AA8 and V3 were provided by Drs. L. Thompson (Lawrence Liver- more National Laboratory, USA) and G. Whitmore (Ontario Cancer Institute, Canada), respectively, and the human melanoma cell lines FME and MM-96 were provided by Drs. K. Tveit (Norwe- gian Radium Hospital, Oslo, Norway) and R. Whitehead (Ludwig Institute, Melbourne, Austra- lia), respectively. Cells were harvested from expo- nential phase monolayer cultures using trypsin (0.07%). Single cell suspensions were prepared in alpha-modified minimal essential medium supple-

Fig. 1. ate ‘ T o gamma rays.

Diagram of the graded lead wedge used to attenu-

mented with fetal bovine serum (5% or 10% vol/ vol), penicillin (100 U/ml), and streptomycin (100 pg/ml) to which insulin (2 pg/ml) and hydrocorti- sone (200 ng/ml) were added for MM-96 cells.

In order to test the linearity of assays with respect to cell number, subconfluent monolayers were dissociated enzymatically using 0.07% trypsin and 50 pg/ml DNAase, centrifuged, and. resuspended in fresh medium. Cells were counted using a hemocytometer and varying numbers of cells seeded in different columns across replicate 96-well plates (150 p.Ywell). After 2 or 3 days of incubation (depending on the cell lines used) the cells were lifted from the plastic by addition of trypsin (0.07%; 100 pl/well) and resuspended in medium (100 pl/well). The pooled contents of each column of wells were counted and these direct cell counts were compared with TdR incorporation, MB staining, and MTT reduction, as described below.

Irradiation Cells in suspension culture were counted with a hemocytometer and then transferred to 96-well mi- croculture plates (0.15 ml/well) at a cell density (typically 150 cells/well) low enough to ensure that control and irradiated cells were still in exponential growth at the time of assay. The cells were allowed to adhere to the plate for 12 hr at 37°C in a humidi- fied atmosphere of 5% CO, in air. Microwell plates were irradiated at room temperature using a 6oCo teletherapy unit (Atomic Energy of Canada Lim- ited, Ottawa, Canada) with the beam attenuated by a lead wedge (Fig. 1) to provide a dose rate gradient (0.22-1.15 Gy/min) across the plate. Dose rates were assessed by thermoluminescent dosimetry us- ing lithium fluoride chips sandwiched between wax and by Fricke/thiocyanate dosimetry (see below). Control plates were not irradiated but were other- wise treated in an identical manner. One row of

Cross et al.: Microculture Assessment of Radiosensitivity 263

wells on the control plates was used as a blank for the assays. This row normally contained medium only but in some experiments was filled with heavily irradiated (20 Gy) cells. Cultures were then grown for 5 days. Plates containing FME cells were also irradiated in the presence of the radioprotector cysteamine (10 mM). After cells had adhered to the plate, half of the medium was carefully removed and replaced with cysteamine-containing medium (20 mM). The plates were irradiated immediately, after which the medium was aspirated and the cells were washed twice with phosphate buffered saline (PBS). Fresh medium was then added to each well and the plates were returned to the incubator.

FrickefT'hiocyanate Dosimetry The Fricke dosimetry method [ 121 was modified for use in 96-well plates by addition of excess thiocy- anate after irradiation to form brick red-colored [Fe(SCN),]3-" (n = 1-6) complexes which are rel- atively stable to hydrolysis under acidic conditions [13]. This shifted the absorbance maximum from 304 nm (Fe3') to 475 nm (thiocyanate complex) [ 141, thus enabling direct determination of dose rate using a microplate photometer. NaC1-modified Fricke solution (1 mM FeSO, and 1 mM NaCl in 0.8N H,SO,) was added to wells (100 pYwell) and irradiated to give doses up to 120 Gy, leaving one well empty. Unirradiated Fricke solution was then added to the empty well to act as a blank, 100 pl of an aqueous solution of NH,SCN (1 M) was added to each well, and the absorbance at 490 nm (less than at 630 nm) was determined with an MR600 Dynatech microplate photometer (Dynatech Labo- ratories, Alexandria, VA). Formation of thiocy- anate complexes in this system was approximately half-maximal at this thiocyanate concentration (0.5 M). Thermal oxidation is known to be a potential problem in KSCN/FeS04 systems [ 121, but absor- bances relative to blanks decayed only slowly under the conditions of the assay (half-life approximately 120 min at 0.5 M thiocyanate vs. 28 min at 2.5 M thiocyanate). Any potential contribution of this in- stability was further reduced by staggering the addi- tion of thiocyanate so that all wells were read at the same time after adding this reagent. A calibration curve was constructed for each experiment by irra- diating Fricke solution (100 ml) to known doses, as determined from the absorbance at 304 nm [ 121, and making a dilution series in unirradiated Fricke solution in a 96-well plate before assaying with thiocyanate as for the unknowns. The calibration curve was linear to at least 120 Gy (r 2 0.99) with a typical slope of about 0.4 absorbance units/100 Gy.

Clonogenic Assay Subconfluent monolayers were dissociated enzy- matically (0.07% trypsin in citrate saline followed by 50 pg/ml DNAase in medium), centrifuged, re- suspended in fresh medium, and irradiated in 14 ml polystyrene tubes. Clonogenicity was determined by plating between 100 and 10,OOO cells in 60 mm culture dishes containing medium (5 ml). Cultures were incubated at 37°C for 7-10 days. Medium was then decanted and the dishes were flooded with MB in 50% aqueous ethanol for 30 min. Plates were washed with water and colonies containing greater than 50 cells were counted. [3H]-TdR Incorporation Assay 5-[MethyL3H]-TdR (20 Ci/mmol, 0.04 kCi/well) together with TdR and FUdR (each at a final con- centration of 0.1 pM) in medium were added (0.02 ml/well) 6 hr before terminating cultures. Cells were released from the plastic by adding pronase to 0.5 mg/ml and sodium EDTA to 2 mM, incubating at 37°C for 1 hr, and aspirating onto glass fiber filters using a multiple automated sample harvester (Beta Harvester, LKB Wallac Oy). The filter disks were washed for 15 sec with water, dried overnight, and the amount of tritium retained was measured by liquid scintillation counting on a Betaplate Counter (LKB Wallac Oy) [ 111. M'IT Reduction Assay MTT (0.015 ml; 5 mg/ml) dissolved in PBS was added to each well 4 hr prior to terminating the culture, after which medium was aspirated and DMSO (150 11.1) and Sorenson's glycine buffer (0.1 M glycine plus 0.1 M NaCl, pH 10.5, 20 pl) were added to each well. Plates were gently agitated for several minutes to ensure solubilization of the for- mazan crystals. The absorbance of each well at 570 nm (A570) and at 630 nm (A,,,) was measured in a mult iwel l photometer and the difference (A570 - A,,,) used to determine MTT reduction [lo]. MB Assay Medium was aspirated from each well and the cells were fixed and stained with MB (5 g/1 in 50% aqueous ethanol; 100 pYwell) for 30 min at room temperature. Wells were washed in water and air- dried. Dye was then solubilized in 1% Sarkosyl in PBS (0.1 ml/well) for 1 hr and absorbances (A,,, - A,,,) were determined in a multiwell pho- tometer [9]. Data Analysis For the purpose of analysis, the endpoints of the TdR incorporation, M'IT reduction, and MB stain-

264 Cross et al.: Microculture Assessment of Radiosensitivity

1.4 7 1.2 t 0

h

* L

P)

c 3 1.0

E

- .-

0.8 - 0 x s 0.6 -

P) u)

0 a 0.4 -

0.2 -

0.0 0 2 4 6 8 1 0 1 2

Well number

Fig. 2. Radiation dose across the 96-well plate as deter- mined by modified Fricke dosimetry (0) and thennolumi- nescent dosimetry (v). A regression line is drawn through the Fricke dosimetry data.

ing assays were treated as cell survival; i.e., surviv- ing fractions at each radiation dose were defined as follows:

cpm or absorbance of Irradiated cells - B

cpm or absorbance of Surviving fraction =

control cells - B

where B was the value measured in the blank (me- dium only) wells. Survival parameters were ob- tained by fitting linear quadratic curves through all the experimental data points. In the case of the MTT and MB assays, the relationship between ab- sorbance and cell number was not strictly linear (see below), and absorbances were therefore con- verted to equivalent cell numbers before analysis. The following survival parameters were calculated: a and p, the linear (initial slope) and quadratic coefficients, respectively, of the least-squares fitted quadratic function; SF,, surviving fraction at 2 Gy; and D, the mean inactivation dose. Least-squares curve fitting and determination of Pearson product moment correlation coefficients were calculated us- ing standard statistical methods (Sigmastat; Jandel Scientific, San Rafael, CA).

RESULTS The dose gradients measured by modified Fricke dosimetry and by thermoluminescent dosimetry agreed well with one another (Fig. 2) and subse-

t 0000

- C 0 .- - ? g 1000 0 U C

0 E

D

.-

.-

.-

5 100 I v

: V

10 100 1000 10000

Number of cells seeded

10 - L I n c 0

0)

1 2 n

0) c 0 x - 5 E

0.1 U C 0 e (0

2 0.01

Fig. 3. Relationship for FME cells between cell number and TdR incorporation (0, r = 0.97), MTT reduction (A, r = 0.98). and MB staining (0, r = 0.98). Vertical lines (where larger than the symbols) represent standard error of the mean. A similar result was obtained for SCCVII cells.

quent experiments used the dose measured by mod- ified Fricke dosimetry. The linearity of the three proliferation assays with respect to cell number was investigated by comparing TdR incorporation, MTT reduction, and MB staining after seeding dif- ferent numbers of cells. All three assays provided linear regressions on double logarithmic plots and a typical result is shown in Figure 3. Whereas TdR incorporation regression was proportional to incor- porated radioactivity (slope of regression = 0.98), the relationship was non-linear for the MTT and MB assays (slopes of regression = 0.78 and 0.73, respectively). The absorbance values were there- fore corrected to cell number using these relation- ships in the analysis of the radiosensitivity data.

The effect of irradiation on cell survival was determined for all five cell lines using the clono- genic assay and the three proliferation assays. Pre- liminary experiments were carried out on each of the cell lines with a range of numbers of inoculated cells and an incubation time of 5 days. From these data a cell seeding density was chosen for each dose (column of wells) which was within the linear range. Representative dose-response curves for the four assay methods are shown for SCCVII cells in Figure 4. In the MTT and MB assays, absorbance was measured against a blank well containing me- dium only. When absorbance from SCCVII cell cultures was measured against a blank consisting of heavily irradiated (20 Gy) cells, slightly less tailing of the survival curves was found (Fig. 4). In the TdR incorporation assays, subtraction of the blank made a negligible difference to the survival curves.

Cross et

L 0

1

0.1

0.01

0 2 4 6 8 1 0 1 2 1 4

Dose (Gy)

Fig. 4. Comparison of TdR incorporation (0, O ) , MTT reduction (0, m), and MB staining ( A , A) assays as measures of the radiation response of SCCVII cells. Results for color- imetric assays are corrected for non-linearity. Open symbols represent results obtained using cell culture medium as a blank, while closed symbols represent those using heavily irradiated cells at the initial cell concentration. Results for clonogenic assays are shown for comparison (v), and the solid line represents a least-squares fitted linear quadratic curve to the clonogenicity data.

The dose-response curves for the different as- says are compared in each cell line in Figure 5 . The parameters a, p, and SF,, and were calculated from these curves for each of the proliferation as- says (Table 1) and compared with those calculated from the clonogenic survival curves (Fig. 6). Excel- lent correlations were also obtained between the clonogenic and TdR incorporation assays for a ( r = 0 . 9 0 ; P = 0 . 0 1 5 ) , SF, ( r = 0 . 9 8 ; P < O.OOl), and D (r = 0.98; P < 0.001). Good correlations were also found between the clono- genic and M'IT reduction assays for (Y (r = 0.96), SF, (r = 0.95), and D (r = 0.83). The pronounced tailing of the dose-response curve for MM-96 cells produced a non-significant correlation between clo- nogenicity and MB staining (r = 0.84; P = 0.07) and D (r = 0.28), but the correlation for a was significant (r = 0.97) (Fig. 5). Ranking of SF, val- ues obtained from the TdR incorporation assay was identical to that using the clonogenic assay (V3 <

cysteamine).

DISCUSSION Assays measuring the incorporation of radioactive precursors have been in use for many years to study

MM-96 < SCCVII < FME < AA8 < FME +

al.: Microculture Assessment of Radiosensitiviiry 265

cell survival following exposure to cytotoxic drugs [ 1 1,15-241. There are also a number of reports in which non-clonogenic assays including TdR incor- poration [20,25], MTT reduction [26,27], and fluo- rescent cell staining [28] have been compared with the results of clonogenic assays following radia- tion. The objective of the present studies was to determine if the TdR incorporation assay, adapted to 96-well microculture plate technology, could be used to predict clonogenic measurements over a wide range of radiosensitivity. A second aim was to compare the TdR incorporation assay with the MTT and MB proliferation assays, which are commonly used to measure chemosensitivity and radiosensi- tivity in vitro.

Previous investigations of radiosensitivity us- ing 96-well trays have required irradiation of cell suspensions prior to plating [26] or have required a movement of the plate under a slotted shield [28]. The use of a shielding wedge to provide attenuation across the plate makes it possible to derive a full dose-response curve conveniently from a single plate. Furthermore, the modifications to the Fricke/ thiocyanate dosimetry method described here allow the use of a microplate photometer to measure radi- ation dose rate directly in 96-well plates.

SF,, a, and B in clonogenic assays have been reported to be related to clinical radiation response [2,3,29,30]. The TdR incorporation and MTT re- duction assays predict these parameters well for each of the cell lines used (Table 1, Figs. 5 , 6). There is excellent agreement over the first two de- cades of response, which is the relevant region for clinical prediction. The MB staining and M'IT re- duction assays also provide good agreement over the initial part of the dose-response curves, and correction for deviation from proportionality in the colorimetric detection method (Fig. 3) has the ef- fect of increasing the values for a and decreasing the values for SF, and n, improving the agreement with the clonogenicity results. However, tailing of survival curves is evident at higher radiation doses, the extent of which varies according to the cell line used (Fig. 5 ) . Tailing probably results from the presence of non-proliferating cells, and it is impor- tant in these assays to choose an incubation time which minimizes the contribution of non-clono- genic cells to the end point, but which does not allow the control cells to overgrow [27]. Perhaps because it is less sensitive to interference from non- proliferating (non-S-phase) cells, the TdR incorpo- ration assay shows less tailing at high radiation dose than do the absorbance assays. While this is a dis- tinct advantage, the contribution of this problem is minimal for all three proliferation endpoints over

266 Cross et d.: Microcultare Assessment of Radiosensitivity

FME FME + Cyateamlne 1

0.1

= e Y C 0

1p

0

v

c 0.01 - 0 2 4 6 8 1 0

M 8 1

0.1

t a '

0.01 I.I.I.I.I.I 0 2 4 6 8 1(

0 2 4 6 8 1 0

v3

0 2 4 6 8 1 0

SCCVll

0 2 4 6 8 1 0

MM-96

0 2 4 6 8 1 0

Doae (Gy)

Fig. 5. Comparison of radiation survival curves for clonogenic (0) assays with corresponding data for TdR incorporation (01, MTT reduction (0). and MB staining (A). Results for colorimetric assays are corrected for linearity. The solid line represents a least-squares fitted linear quadratic curve to the clonogenicity data.

the clinically relevant low dose region of the sur- vival curve. However, the superiority of the TdR method may be greater if large numbers of non- cycling cells are present in the primary tumor cell suspensions. Further advantages of the TdR assay over the colorimetric methods include strict linear- ity with cell number and wide dynamic range (Fig. 3).

Cell lines have provided considerable informa- tion about the radiosensitivity of tumors of different histological types and have contributed toward our understanding of important aspects of radioresis- tance such as repair mechanisms, effect of hypoxia, and inherent radiosensitivity [7,3 11. However, the ultimate goal of in vitro assays is to predict accu- rately the radioresponsiveness of an individual pa- tient's tumor so that treatment can be optimized and clinical outcome improved. While we have demon- strated that the TdR incorporation, MlT reduction,

and MB staining assays give, in general, good agreement with clonogenic assays using cell lines, the techniques cannot be applied directly to primary cultures of tumor specimens. One of the major problems in examining such primary cultures is the contribution from proliferating stromal cells such as fibroblasts. Several methods have been proposed to suppress the growth of anchorage-dependent stro- ma1 cells, such as growth on agarose [ 1,11,32-341, the use of lethally irradiated contact-sensitive cell confluent monolayers [23], and adhesive substrates with selectivity for tumor cells [7,35,36]. We have used microculture plates coated with agarose in our laboratory to measure the response to cytotoxic drugs of human melanoma cells from fresh tumor biopsies [ 1 11, and we are currently developing this technique, in conjunction with TdR incorporation assays, to assay the radiosensitivity of primary cul- tures from human tumor biopsies.

Cross et al.: Mimnrlture Assessment of Radiosensitiviv 267

Table 1. Using Clonogenic Assays With Corresponding Values Obtained With Three Types of Proliferation Assay

Line Assay ci P SF* D

Comparison of Radiation Survival Parameters for the Cell Lines

-

SCCVII TdR incorporation 0.12 0.036 0.68 2.7 M'IT reduction 0.02 0.044 0.80 3.0 MB staining 0.13 0.050 0.63 2.4 Clonogenicity 0.15 0.040 0.64 2.5

M'IT reduction 1.37 0.112 0.04 0.8 MB staining 1.64 O.OO0 0.04 0.7

AA8 TdR incorporation 0.03 0.040 0.79 3.0 M'IT reduction -0.05 0.077 0.82 2.8 MB staining 0. I 1 0.041 0.68 2.6

FME TdR incorporation 0.10 0.023 0.75 3.2 M'IT reduction 0.16 0.018 0.68 3.0 MB staining 0.18 0.027 0.63 2.6 Clonogenicity 0.03 0.060 0.74 2.7

"3 TdR incorporation 0.63 0. I70 0.14 1.2

Clonogenicity 0.98 O.Oo0 0.14 1.1

Clonogenicity 0.10 0.020 0.75 3.4

FMUcysteamine TdR incorporation 0.03 0.01 I 0.90 5.4 Clonogenicity 0.02 0.014 0.92 5.3

MB staining 0.03 0.009 0.90 5.9 Clonogenicity 0.21 0.047 0.55 2.2

MM-96 TdR incorporation 0.40 -0.004 0.46 2.3 M'IT reduction 0.45 0.004 0.40 I .9

1 .o

FME+cystearnine

0.8 -

h x Z 0.6 - 0

IY D I- -

0.4 - LN u)

0.2 -

0.01'. ' ' ' ' * . 0.0 0.2 0.4 0.6 0.8 1.0 0.0 0.2 0.4 0.6 0.8 1.0 0.0 0.2 0.4 0.6 0.8 1.0

SF, (clonogenic assay)

Fig. 6. Correlations between SF, values from clonogenic assays and those from TdR incorpora- tion, MlT reduction, and MB staining assays. F M E plus cysteamine was used with the clonogenic and TdR incorporation assays only.

ACKNOWLEDGMENTS This work was supported by the Cancer Society of New Zealand, by its Auckland Division, and by the Health Research Council of New Zealand. We

thank Isla Nixon and Allan Stewart, Department of Clinical Oncology, Auckland Hospital, for their help in designing the lead wedge and for perform- ing the thermoluminescent dosimetry measurements.

268 Cross et d.: Mimculiure Assessment of Radiosensitivity

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