Chemosensitivity testing of human gliomas using a fluorescent microcarrier technique

Journal of Neuro-Oncology 8: 103-112, 1990. © 1990 Kluwer Academic Publishers. Printed in the Netherlands.

Laboratory Investigation

Chemosensitivity testing of human gliomas using a fluorescent microcarrier technique

A.P. Bowles, Jr. ,~ Cooley G. Pantazis, 2 William Wansley 2 and Marshall B. Allen 1 i Department of Surgery, Section of Neurosurgery*, 2 Department of Pathology, Section of Anatomic Pathology, Medical College of Georgia, Augusta, GA 30912, USA

Key words: microassay, glioma, cytodex microcarriers, hydroethidine

Abstract

This report describes a new fluorescent microcarrier cytostasis assay. Human glioma cell lines and primary cultures were attached to microcarrier tissue culture beads and treated with various chemotherapeutic drugs. After treatment, the cells were labelled with two vital fluorescent dyes in order to measure cellular viability. The uptake of hydroethidine and Hoechst 33342 was evaluated alone and in combination as probes for determining metabolic activity and cellular proliferation. Hydroethidine was found to be superior when compared to trypan blue and tritiated thymidine. The use of the microcarrier technique allows for the direct cellular measurement of fluorescence without the need of extensive extraction procedures. The fluorescent assay is a sensitive, rapid and an effective way to screen for potential antiproliferative compounds.

Introduction

Malignant gliomas have a spectrum of growth rates, yet are generally refractory to surgery, radiation therapy, and chemotherapy. Response to radiation therapy is similar among patients, yet the response to chemotherapy is variable, with only 45% of patients treated with nitrosoureas respon- ding clinically [26-28]. The median time to tumor progression for patients with glioblastoma multiforme treated with combination surgery, radiation therapy, and chemotherapy is only 57-70 weeks [13] and chemotherapy alone is completely ineffective if not accompanied by surgery or radiation therapy [11]. Chemotherapy for malignant gliomas has proven to be unsuccessful and as a consequence multiple experimental treatment modalities have been attempted clinically using combination chemotherapy. With the increasing need to evaluate new treatment modalities, a quick and reliable in

vitro assay could provide objective data for drug treatment strategies.

In an attemPt to predict the clinical response of a given patient, a number of in vitro assays have been recently developed to screen for antiproliferative activity. Although many approaches are currently used, such as in vitro radiometric and visual microscopic methods [1, 10, 11] and in vivo isotopic eval- uations [17], the two most successful approaches involve the stem cell or soft agarose assay, and the monolayer cell assay. In the stem cell assay, the clonal growth of cells are determined from the number of colonies in solidified agarose. This assay provides good survival data, yet is limited by the difficulty in establishing tumor growth and low plating efficiencies [3, 12]. The monolayer technique involves growing tumor cells in tissue culture as monolayers and then determining cell viability by a number of methods, which can be employed with the use of various dyes and radioisotopes.

104

Chromium release assays can measure cytotoxicity, or cytolysis. Tritiated thymidine incorporation measures the inhibition of DNA synthesis. An in- direct method of the measurement of metabolic activity has been developed using MTT or Tetrazo- lium salts, yet this assay has proven to be less sensitive when compared to 3H-thymidine [3]. Al- though chromium release and tritiated thymidine incorporation are standard techniques for the measurement of cytotoxicity and cytostasis, the uti- lization of radioisotopes is not without limitations, particularly in terms of cumbersome techniques, safe handling, and disposal.

With the advent of flow cytometry, vital fluorescent dyes have gained popularity, and recently a number of in vitro assays have been developed to measure cellular growth. Saiki and associates have developed an effective assay for the quantitation of the antiproliferative activity of a number of anticancer agents in vitro using hydroethidine [8, 24]. This technique is limited by the attachment of cells to tissue culture plates and the extraction of the fluorochrome by detergents in order to determine the amount of dye incorporated. In order to over- come these limitations we have developed a fluorescent microcarrier technique which can be performed in non-tissue culture vessels and also allows direct fluorescent quantitation without the need for extensive extraction procedures. In the present report we describe the development of this assay using human glioma cells.

Materials and methods

Cell cultures

Five established cell lines and two primary cultures were used in this study. The established cell lines obtained from the American Type Culture Collec- tion, consisted of: Medulloblastoma (TE 671), Anaplastic Astrocytoma (U 138), and Glioblasto- ma Multiforme (U87, T 98G, U 373). Two primary cultures were obtained from biopsied fragments of tumors from patients on our neurosurgery service. These included a Primitive Neuroectodermal Tu- mor (PNET) and a Glioblastoma Multiforme

(GM). All tumor cells were maintained by contin- uous passage in Dublecco's Minimum Essential Media (DMEM), with 10% Fetal Bovine Serum (10% FBS), supplemented with essential amino acids and vitamins, pyruvate and glutamine. Cul- tures were maintained at 37°C in an incubation chamber with a humidified atmosphere containing 5% CO2 and 95% air. All cultures were free of mycoplasma and the cells were treated when con- fluence was reached.

Cytodex microcarriers

Cytodex microcarriers (Pharmacia Fine Chemi- cals, Upsala, Sweden) are transparent, biologically inert, spherically shaped beads, of a crossed linked dextran matrix, substituted with positively charged N,N-dimethyl-aminoethyl groups. The diameter of the microcarriers is approximately 180 microns. Because of the size, surface contour and charge of the beads, cells preferentially and readily bind to the microcarriers resulting in high culture yield (Fig. 1).

Fluorochromes

A stock solution of 7 mg/ml of hydroethidine (Po- lysciences, Inc., Warrington, PA) was prepared by dissolving 1.4 mg of hydroethidine in 200/xl of N,N- dimethyl-formamide. From this, 20/xl was added to 2 ml of phosphate buffered saline (PBS) and 10/xl of the working solution was added to cell suspensions to give a final concentration of 0.7 mg/ ml. Hoechst 33342 (Polysciences, Inc.) was prepared by dissolving 1 mg of solute in 1 ml of saline and from this stock, 10/~1 were added to cell suspensions to give a final concentration of 0.16 mg/ ml. All stock solutions were freshly prepared for each experiment.

Reagents

Concentrations of BCNU or 1,3-bis(2-chloroethyl)-l-nitrosourea (Bristol-Myers) were pre-

105

Fig. 1. Cytodex microcarriers. Photomicrograph of a confluent culture of TE 671 cells onto Cytodex beads. The beads are transparent and spherically shaped, easily identifying the spindled shaped medulloblastoma cells confluently attached to the Cytodex microcarriers.

pared by dissolving sterile lyophilized 1 mg aliquots in 3.0/xl of absolute ethanol and then diluted in sterile PBS to desired concentrations. Streptozocin or 2-deoxy-2-[[(methylnitrosoamino)carbonyl] amino]- (and)-D-glucopyranose (Upjohn) was reconstituted from lyophilized aliquots with sterile PBS. Daunomycin or Daunorubicin HCL (WYETH Laboratories, Inc.) and Cisplantin (Bristol-Myers) were reconstituted from lyophilized aliquots in sterile water and Methotrexate (Ly- pho Med, Inc) in sterile PBS. All drugs were used within 2 hours after reconstitution.

Chemosensitivity assay

Freshly isolated tumor tissue (PNET, GM) was dissected under a laminar flow hood into 1.0mm cubes in a 60 mm petrid dish (Falcon Plastics). The minced tissue was then enzymatically digested in a cocktail of collagenase and trypsin (25 rag/100 ml), washed in PBS, and then added to a flask containing DMEM 10% FBS as a monocellular suspen-

sion. Frozen cell lines (TE 671, U 373, U 87, U 138, T 98G) were thawed and resuspended in DMEM 10% FBS as well. Cytodex microcarriers were preswollen in PBS for 3 hours and then washed in PBS. Usually 0.3 g of beads were added to 30 ml of PBS and then autoclaved. Approximately 6000 microcarriers (0. lrnl of beads) were incubated with 5-10 x 106 cells in a total volume of 3 ml of DMEM 10% FBS. The cell and beads were gently swirled, with anchorage of cells onto the microcarriers achieved within 4 hours. 50/xl of solution, with approximately 1 - 2 x 10 4 cells attached to 100 beads, were transferred to the individual wells of the Falcon Micro Test II plates (Falcon Plastics) and then incubated with the desired concentration of drugs. Three days later, the wells were stained with hydroethidine and/or Hoechst 33342 for one hour. The cells were transferred to the Pandex 96 Filter Well plates (Baxter, Inc.) which contain 0.2 micron millipore filters, composed of cellulose acetate, allowing for easy filtering and washing of each well. After the cells were washed in PBS three times, the cells were concentrated to the center of

106

the wells by constant vacuum, and the total particle-bound fluorescence was determined by front- surface fluorimetry using the Pandex Fluorescent Concentration Analyzer (Baxter, Inc.). Hydro- ethidine and Hoechst 33342 are discriminating fluorochromes which are taken up and accumulated in only metabolically active and viable cells [8, 15, 22, 24] staining the cytoplasm blue (hydroethidine or Hoechst 33342), or the DNA red (ethidium only). Since the relative fluorescence is proportional to the number of metabolically viable cells, the percent cytostasis activity was calculated from the for- mula:

C-E % C A = ~2S x 100%.

With the fluorescence of each well expressed as RFU's or relative fluorescence units, C represents the average RFU's of the cells treated with media alone, E the average fluorescence from cells treated with reagents, and S the background fluorescence. One hundred percent cytostasis was repre- sented by treating cells 0.2 M of nitric acid, and was confirmed by the uptake of trypan blue in 100% of the cells. The background fluorescence of the wells treated with nitric acid represents the nonspecific staining of the cellulose acetate manifold. Back- ground fluorescence was found to be less than 5% of control. For all of the experiments, the drugs and dyes with the various cell lines were treated in quadruplicate and the appropriate means and standard errors were calculated.

In vitro therapeutic dose

There are several methods for determining the in vitro concentration that may provide clinically equivalent levels. We chose a method described by Dickson and Kornblith [5, 11] which takes into account that the concentration of the drug in the tumor tissue should closely approximate the concentration of the drug uniformly distributed in plasma. With a given patient dosage, the/xg/ml concentration, representing the maximum concentration of drug attained in the blood was calculated from the following:

X mg (Drug) Y/zg (Drug) 5000 ml Plasma × 10/xg/ml - ml Plasma

Therefore, Y/xg (Drug)/ml of Media represents a close estimate of the maximum concentration that would be achieved clinically.

Tritiated thymidine incorporation

Parallel experiments were conducted to compare the metabolic state of cells determined by hydroethidine to DNA synthesis determined by tritiated thymidine as described by Schultz [25]. Cells attached to the cytodex microcarriers were incubated with various concentrations of nitric acid or BCNU for 72 hours. After incubation, in one group, the cells were stained with hydroethidine as described, and in the other group, two hours before harvest, each well was pulsed with 0.5/xCi of H- thymidine (5.0 Ci/mmol, Amersham). The cultures were harvested on glass fiber filters and the radioactive incorporation was determined by using a scintillation counter.

Results

Analysis of vital fluorochromes

In the first group of experiments, the suitability of the assay was defined in the human glioma cells. Figure 2 shows that when TE 671 cells were labelled with hydroethidine and the fluorescence was measured in red or blue light (excitation 545 nm, emission 575nm; or excitation 385nm, emission 450 nm), the relative fluorescence was proportional to the number of cells. Similar results were obtained with Hoechst 33342 alone or in combination with hydroethidine. Fluorescence plateau was achieved within 20 minutes and retained for at least three hours, however the relative fluorescence ac- tually increased with Hoechst 33342 after 2 hours (Fig. 3).

In order to determine if any fluorescence oc- curred from the Cytodex microcarriers, we incubated an equivalent number of beads in a solution of medium with either hydroethidine or Hoechst

4 0 . 0 0 0 - 03 4 , . ~ 3 6 . 0 0 0 -

~ ) 3 4 . 0 0 0 -

O 32,000- C

28,000- 0

24,000-

20,000"

U- 1 6 . 0 0 0 -

. ~ 1 2 , 0 0 0 "

'~ 8,000- (D

I~" 4 ,000 -

0

• Hoechst 33342, 385/450 ~,. Hoechst 33342 T

+ Hydroethidine, 385/450 /1~ • Hydroethidine, 545/575

I ^ I . I ~ I . I . I . I l x 1 0 z 5 x 1 0 z l x 1 0 ~ 5 x 1 0 "~ 1 x 1 0 ~ 5 x 1 0 " l x 1 0 ~

Cell Number 0 5

Fig. 2. Proportional uptake of hydroethidine and Hoechst 33342. Increasing numbers of TE 671 cells were labelled with Hoechst 33342, hydroethidine or both fluorochromes. Cells labelled with hydroethidine were monitored in either blue light (excitation 385 nm, emission 450nm), or red light (excitation 545 nm, emission 575 nm). Cells labelled with Hoechst 33342 or both hydroethidine and Hoechst were monitored in only blue light. Vertical bars represent means and SEM of four replicates.

33342 for one hour. Fluorescence was moni tored in both red and blue light, with the aid of a fluorescent

microscope. Fluorescence was not observed when

the beads were stained with either hydroethidine or

Hoechst 33342.

We also compared the ability of the fluoro-

chromes to measure cytostasis by staining cells with

one dye or both dyes and measured the emit ted

fluorescence appropriately at 450nm (blue) or 575 nm (red). TE 671 cells (2 x 10 4) attached to

cytodex microcarriers were incubated with either 0.1 M or 0.2 M of nitric acid. Percent cytostasis was

calculated for each group depending on the method of staining and values were compared between

groups and with the microscopic observations de-

picting cellular dysfunction (Table 1). Treated cells

produced depressed metabolic function, with a loss

of adherence to the beads and altered cellular morphology. Only 10% of the cells were viable when t reated with 0 .1M nitric acid as shown by the uptake of t rypan blue. Cells t reated with 0.2 M nitric acid were obli terated with precipitation of cells and media. Trypan blue was taken up in 100% of the remaining cells (0% viability).

107

40,000-]" lx10 4 cells 36000- t t e 671

3210001 ~ o ~ 28,000-1 ' I x - - ±

2 4 , 0 0 0 4 / ~ ~ ~ - ~ ~ o 2°°°°11 l o 16ooo1111 ~- 12,00011 ~' • Hoechst 33342, 385/450 >o 8ooo411 .,,.Hoechst 33342

' IW + Hydroethidine, 385/450 '~ 4,000-~ • Hydl:oethidine, 545/575 ~" o ! , , , ,

0 1 2 3 4. Hours After Labelling With Dyes

Fig. 3. Duration of retention of dyes within labelled tumor cells. 1 x 10 4 TE 671 cells were labelled with Hoechst 33342 or hydroethidine or both fluorochromes. The relative fluorescence was monitored from 1-4 hours after labelling. Cells labelled with Hoechst 33342 were monitored in blue light, excitation 385 nm and emission 450 nm. Cells labelled with hydroethidine were measured in red light, excitation 545nm and emission 575 nm. Vertical bars represent the mean and SEM of four replicates.

As shown in Table 1, when the cells were stained with hydroethidine and/or Hoechst 33342 and cytostasis values were compared, staining cells with

hydroethidine alone produced superior results.

The cytostasis values with hydroethidine were dif-

ferent, depending upon whether or not the fluores-

cence was measured in blue or red light. The per-

cent cytostasis from hydroethidine was close to

90% for both 0.1 and 0.2 M of nitric acid at 545 nm/

575 nm, with a narrow standard error of the mean. On the other hand, hydroethidine at 385nm/

450 nm produced less cytostasis, with a larger stan-

dard error. When viewed with ultraviolet microscopy, we

found that the relative blue fluorescence in the

cytoplasm was unstable and reached a brief plateau

within 10 minutes and progressively decreased in

minutes thereafter. The red fluorescence in the nucleus increased, reaching a stable plateau within 20 minutes, and remained unaltered for at least 3 hours. The cytostasis values were less satisfactory when the cells were stained with Hoechst alone or stained with both Hoechst and hydroethidine. Af- ter treating the cells with 0 .2M of nitric acid, the cytostasis values were only 66% and 35% for hy-

108

droethidine and Hoechst or Hoechst alone, respectively (Table 1).

Comparison with tritiated thymidine

In another group of experiments we compared the fluorescent measurement of metabolic activity using hydroethidine with radioactive measurements of DNA synthesis using 3H-thymidine. As shown in Table 2, when the tumor cells were either stained with hydroethidine (545 nm/575 nm) or pulsed with tritiated thymidine, the percent cytostasis activity values by both methods produced comparable results.

Analysis of human cell lines and primary cultures

In the next group of experiments we determined the antiproliferative effects of the five chemotherapeutic drugs against the seven different cell lines. Because of the superior cytostasis values obtained with hydroethidine alone (545 nm/575 rim), we employed this fluorochrome to measure cytostasis activity.

BCNU produced significant cytostasis in the three glioblastoma multiforme cell lines (T 98G, U

87, U 373) and also in the medulloblastoma cell line (TE 671) (Fig. 4, right). Differential effects were measured depending on the cell line, yet for both TE 671 and T 98G the percent cytostasis activity ranged from 40% to 60% at 50/xg/ml to 100 txg/ml of BCNU. The calculated in vitro therapeutic dose for BCNU from a patient dosage of 200 mg/m 2 is 25/xg/ml to 100 ~g/ml. At similar concentrations, the activity was less for U 373 and even smaller for U 87, with only 38% + 8 and 24% + 10 cytostasis activity respectively.

Streptozotocin, Cisplantin, and Methotrexate were less effective in promoting cytostasis (Fig. 5). Within the in vitro therapeutic dose range, Cisplan- tin generated at most 30% cytostasis in the TE 671 cell line and less than 10% in the U 138 line. Metho- trexate was equally less effective in limiting cellular growth and activity, with no more than 20% cytostasis in the U 87 and TE 671 cell lines and 0% in the U 138 cell line. Streptozotocin was somewhat improved at 900/xg/ml, with 35% + 7 cytostasis in the U 373 cell line, yet only 20 to 25% in the U 138 and TE 671 cell lines.

Surprisingly, Daunomycin which is an agent not previously used for the treatment of malignant brain tumors, produced a significant disruption of metabolic activity. As shown in Fig. 6, right, the percent cytostasis was greater than 90% in the

Table 1. Comparison of the cytostasis activity of different fluorochromes. The culture of TE 671 ceils, 2 x 104 cells/well, were incubated with 0.1 M* and 0.2 M 4 o[ nitric acid, and after incubation for 72 hours the cells were labelled with hydroethidine, or Hoechst 33342, or both fluorochromes. The relative fluorescence of fluorochromes were monitored in blue light (excitation 385 nm, emission 450 nm) and red light (545/575) and the cytostasis values were compared with the microscopic changes observed and uptake of trypan blue. The quantitative cytostasis values represent the mean and SEM of four replicates and the qualitative microscopic values represent the uptake

of trypan blue.

Fluorochrome Hydroethidine Hydroethidine Hydroethidine Hoechst 33342 (545/575) (385/450) Hoechst 33342 (385/450)

(385/450)

Quantitative * 88.7 _+ 0.6 * 71.3 _ 14.2 "18.5 _+ 16.0 * 16.2 _+ 8.3 % C A = C - E **89.3_+0.8 *'61.7+_ 8.4 **66.0+ 9.4 * ' 3 5 . 1 + 2 . 0

L-,zS-

Qualitative microscopic * 90% * 90% * 90% * 90% • * 100% ** 100% ** 100% ** 100%

TE 671. 20,000 cells/well. * 0.1 Molar concentration of nitric acid. ** 0.2 Molar concentration of nitric acid.

1004

J 90

~ 80

<

4 50- 40-

~) 30- 20- 10- 0

0

• TE 671, HE, 545/575 [] T 98G, HE, 545/575 AU 373, HE, 545/575 o U 87, HE, 545/575

i I I I I I I I I I 40 80 120 160 200

BCNU pg/ml

Fig. 4. BCNU cytostasis activity. Different glioma cell lines were incubated with increasing concentrations of BCNU from 20 to 200k~g/ml. The cytostasis activity calculated from the uptake of hydroethidine (HE) varies from 20 to 60% depending on the cell line and concentration. The in vitro therapeutic concentration for BCNU is 20 to 100mg/ml, and the corre- sponding percent cytostasis activity for the majority of the cell lines ranges from 40 to 60%. Vertical bars represent means and the SEM of four replicates.

PNET and 80% in the GM primary cultures. Cytos- tasis was equally effective in the established cell line, TE 671. Calculated values for cytostasis activity correlated well with the cellular changes observed microscopically. The cells were fewer in number with altered morphology. Cells were spherically shaped without the normal spindle conformation, less refractory with increased granularity. The majority or the cells were nonadherent to the beads, attached together as nonviable clusters.

Table 2. Measurement of cytostasis acitivity using hydroethidine or tritiated thymidine. 2 x 10 4 TE 671 cells/well attached to the Cytodex microcarriers were incubated with various concentrations of BCNU and nitric acid for 72 hours. After incubation the cells were either stained with hydroethidine or pulsed with 0.5/zCi of 3H-thymidine. The uptake of hydroethidine (monitored in red light) and the incorporation of thymidine were determined generating cytostasis values for comparison. Cytos- tasis values represent the means and SEM's of four replicates.

Treatment Metabolic activity DNA synthesis hydroethidine thymidine

BCNU 50/xg/ml 42.3% +5.1 60 % _+20.2 BCNU 100txg/ml 62.4% + 7.5 67.4% -+ 20.3 Nitric acid 0.2M 89.3% _+0.8 97.5% _+ 0.4

TE 671 20,000 cells/well.

109

Discussion

Hoechst and ethidium are DNA specific dyes which fluoresce strongly when bound to DNA. Hoechst binds to the A-T pairs and ethidium to the phosphate backbone of DNA. Hoechst 33342 dif- fers from Hoechst 33258 in that it readily enters metabolically active cells without being toxic, and has been characterized as the first non-toxic vital stain for DNA [14]. Hydroethidine is produced from the reduction of ethidium and unlike ethidi- urn, readily enters living cells without being toxic. In the cytoplasm, hydroethidine fluoresces in blue light when excited by the appropriate wavelength (excitation 385 nm/emission 450 nm). Once inter- nalized, hydroethidine becomes dehydrogenated by NADP* dependent enzymes to form the ethidi- urn, which binds to DNA and fluoresces in red light. Hoechst 33342 has been used effectively in a number of assays defining cell sorting and proliferation [14, 15, 22]. Hydroethidine has been recently used in an assay to measure the antiproliferative activity of a number of compounds in both animal and human tumors [8, 24].

In the first group of experiments we examined the ability of hydroethidine and Hoechst 33342 to be taken up by tumor cells and to measure cytostasis activity. Both dyes were effectively taken up in all the tumors, yet there was a difference in the ability of the dyes to measure cytostasis. Hydroeth- idine was effective in distinguishing metabolically active tumor cells from cells treated with drugs, yet Hoechst 33342 was less effective in differentiating cells, perhaps because of the variable uptake into the brain tumor cells and the relative increase in fluorescence with time. Staining cells with both hydroethidine and Hoechst improved the measurement of percent cytostasis, yet calculated values were inferior to values with hydroethidine alone. Staining cells with hydroethidine alone produced superior results, yet there were differences in the cytostasis values depending on whether the fluorescence was measured in blue or red light. Determi- nation of cytostasis measured in red light resulted in values with greater sensitivity, with a closer ap- proximation to the visual microscopic changes observed (trypan blue), and greater reproducibility.

110

100- 90-

g 80- 70- 60"

~ 50" ~o 40"

30" 20- 10-

,101 • TE 671, HE 545/575 ~ 90 • TE 671, HE 545/575 • U 138, HE 545/575 g 80 0 U 87, HE 545/575

70 • U 138, HE 545/575 u) 60 '~ 50

40 30 20 1

0~ ~ 110 410 100 400 0 I1 2 110 210 100 2~0 1000

A Cisplantinum pg/ml a Methotrexate pg/ml

100 . 9(3--t • TE 671, HE, 545/575

i~. 80-1 ~ U 373, HE, 845/575 70-1 • U 138, HE, 545/575

6o- t ~ 501

40"t ~ o 3o-I o 20- I ,z , & " - ~ / "

1 0

0 400 800 1200 1600 2000

C Streptozoticin pg/ml

Fig. 5. Cytostasis activity of Streptozotocin, Methotrexate and Cisplantin. C. Different glioma cell lines were incubated with different concentrations of Streptozotocin from 0 to 2000/zg/ml and the cytostasis activity calculated from the uptake of hydroethidine (HE) monitored in red light (545/575). The in vitro therapeutic concentration is 300/zg/ml. Vertical bars represent the means and SEM of four replicates. B Different cell lines were incubated with increasing concentrations of Methotrexate and the percent cytostasis activity measured. The in vitro therapeutic concentration is 20/zg/ml. A. Cell lines were incubated with increasing concentrations of Cisplantin and the percent cytostasis activity calculated. The in vitro therapeutic concentration is 6 to 40/zg/ml.

The difference in the measurements reflects the greater stability of ethidium compared to hydroethidine. The blue fluorescence in the cytoplasm, represents hydroethidine which will be progressively dehydrogenated to ethidium. Ethidium binds to DNA, fluoresces in red light, and is stable within the cell, leaking from the cell only after 3 hours. To encourage the monitoring of fluorescence from ethidium, we therefore stained cells for 1 hour.

In the next group of experiments we determined the cytostasis activity of the various drugs and found that BCNU was effective in inhibiting metabolic activity, although responses were different depending upon the cell line. BCNU is a nitrosourea with both alkylating and carbomylating proper- ties and is the most common chemotherapeutic agent used for the treatment of malignant gliomas. As a single agent, it is the most effective [12, 13], yet clinical responses are variable and poor. Stud-

':;::F , . . . . + + '

8~ so / /,

~3~ 2O . . . . . . . . PNET, HE 545/575 v 1 0 ~

0 20 40 60 80 100 Daunomycin, pg/ml

A

Fig. 6. Daunomycin cytostasis activity. B. Two primary cultures (PNET, GM) and one established cell line (TE 671) were incubated with increasing concentrations of Daunomycin and the fluorescent cytostasis activity measured. The in vitro therapeutic concentration is 40/zg/ml. Vertical bars represent the means and SEM of four replicates. A. Photomicrograph of the cell culture of TE 671 cell incubated with Daunomycin. The cells depict altered morphology, with decreased adherence to the beads, clumped together as nonviable cells.

ies examining the effects of BCNU in vitro using sensitive cell lines, have demonstrated antiproliferative effects at similar drug concentrations as our report [11].

Streptozotocin, Cisplantin, and Methotrexate were all less effective in producing cytostasis, Step- tozotocin is a 1-methyl-nitrocourea combined with glucose, with altered pharmokinetics and toxicity compared to BCNU. Streptozotocin has been rare- ly employed clinically. Cisplantin, is a heavy metal with biological activity. This cis-isomer has been found to be effective in inhibiting DNA synthesis, with significant activity in testicular and ovarian cancers, yet clinical trials in malignant gliomas have not shown consistent efficacy. Methotrexate is an antimetabolite and specifically inhibits di- hydrofolate reductase, limiting DNA, RNA, and protein synthesis. Methotrexate is the treatment of choice for gestational choriocarcinoma, and has proven successful in the treatment of lymphocytic leukemia. Methotrexate has been used for the treatment of meningeal disease from medulloblastoma and carcinomatosis, yet for the treatment of primary and recurrent malignant gliomas, methotrexate has been ineffective, and many times has been accompanied by significant toxicity. The clinical results of treatment with Streptozotocin, Cis- plantin, and Methotrexate are consistent with our in vitro results.

Daunomycin, an analog of adriamycin, is an anthracycline antibiotic which is composed of a 4-ring structure linked, via a glycoside bond, to an amino sugar. It separates stacked bases which un- coils the tertiary structure of DNA, resulting in the inhibition of DNA synthesis. Daunomycin has been successfully used to treat acute leukemias, yet prior to this report, Daunomycin had not been tested in brain tumors preclinically or clinically. Surprisingly, this antibiotic achieved an impressive response, limiting the metabolic activity in practi- cally all of the cells of three clinically very aggres- sive, malignant tumors (PNET, glioblastoma multiforme, and medulloblastoma). Daunomycin, which is an unfamiliar agent within the neuro-onc- ological drug armentarium, certainly merits further clinical investigation for efficacy.

The assay described in this report uses Cytodex

111

microcarriers to facilitate cell culture growth, without the need for expensive tissue culture plastic ware. Once attached to beads, the viability of the cells can be monitored, with the aid of Particle Concentration Fluorescence technology, so that the measurement of the incorporation of the dyes can be performed without the need of expensive extraction procedures.

Many assays are available to screen for anticancer activity, yet most are tedious, cumbersome, prolonged and involve the use of radioisotopes. The fluorescent assay described is rapid, easy, and able to screen for multiple agents at several concentrations simultaneously. The assay is comparable to the technique employing tritiated thymidine and avoids the associated problems of safe handling and disposal of radioisotopes. The clinical useful- ness of the assay remains to be determined; however, the comparable results obtained with known chemotherapeutic agents warrants additional investigation in this area. Overall, the assay is an effective way to screen for potential antiproliferative agents and may provide a useful tool in the adequate planning of new treatment modalities.

Acknowledgements

Supported in part by the American Cancer Society, Grant No. IN 147.

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Address for offprints: A.P. Bowles, Jr., Department of Surgery, Section of Neurosurgery, Medical College of Georgia, Augusta, GA 30912, USA

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Chemosensitivity testing of human gliomas using a fluorescent microcarrier technique