The hybrid spheroid clonogenic assay for the intrinsic radio- and chemo-sensitivities of human tumors

0360-3016/92 $5.00 + .I0 Copyright 0 1992 Pergmm Press Ltd.

Inr. J Radiation Oncology RIO/ Phys Vol. 24, pp. 5 I I-517 Printed in the U.S.A. All rights reserved.

??Biology Original Contribution

THE HYBRID SPHEROID CLONOGENIC ASSAY FOR THE INTRINSIC RADIO- AND CHEMO-SENSITIVITIES OF HUMAN TUMORS CHRISTOPHER

S. LANGE, D. PHIL.,’ BOZIDAR DJORDJEVIC, AND WILLIAM A. BROCK, PH.D.~

PH.D.’

‘Department of Radiation Oncology, State University of New York Health Sciences Center at Brooklyn, Brooklyn, NY; and ‘Department of Experimental Radiotherapy, The University of Texas M. D. Anderson Cancer Center, Houston, TX The Hybrid Spheroid assay is based on packaging tumor cells into agglomerates of non-proliferating, but metabolically active, HeLa ceils. These agglomerates provide an in rive-like environment for entrapped test cells. Clonogenicity is determined by varying the number of test cells per hybrid spheroid so that some, but not all, spheroids give rise to macrocolonies. From the fraction of noncolony forming spheroids and the Poisson distribution, the average number of clonogens per spheroid can be calculated. The clonogenicity and radiation survival curves of cells derived from human tumors (of the maxilla, tongue, larynx, mouth floor, lung, breast, ovary, and colon) were so determined. Plating efficiency was increased in these normally poorly plating tumor cells, thus enabling survival measurements which are not practical using conventional methods. The Hybrid Spheroid assay has also been applied to determine the chemosensitivity of colon cancer cells. Hybrid spheroids, Intrinsic radiosensitivity, Clonogenic assay with high plating efficiency, Intrinsic chemosensitivity (5fluorouracil), Human tumors.

with an experimental treatment which may cure, say 60%, and one seeks to detect this 10% difference. With a prognostic assay, one can assume that all patients are not identical samples from the same population and, if accurate, the assay should predict which patients will fail the standard treatment. The 50% who will not fail get the standard treatment and are cured. The 50% who would have failed the standard treatment, are not randomized into it, but instead are given an alternative arm treatment, preferably one to which the prognostic assay suggests they would respond. Any cures in this second group (say 60% of this 50%, that is, 30%), are then added to the cures of the first group to produce a generally higher survival rate, in this case, 50 + 30 = 80%. Therefore, had we such an assay for tumor sensitivity, clinical trials could be more efficient and cure rates could be increased by optimizing the treatment for each individual patient.

INTRODUCTION

This paper presents radio- and chemo-sensitivity data obtained from the application of a new clonogenic assay system-the hybrid spheroid system (6, 7)-to cells obtained directly from a variety of human tumors. Since there has been some debate in the literature regarding the feasibility and utility of prognostic assays in general (16, 20,22), it will be useful first to list the extant assay systems and then to discuss in detail the advantages of the hybrid spheroid assay system. What use is a prognostic assay?

In a standard clinical trial, patients are randomized between two (or more) protocols, one of which is the current standard therapy, and the other(s) representing possible improved alternatives. The underlying model assumes that all patients have a response distributed about the same mean, and one seeks to determine which treatment cures a higher fraction of the population. Therefore, a standard treatment which yields 50% survival must be compared

Past and presently used prognostic assays Five assay systems have been proposed and investigated for their potential ability to predict in vivo radio- and/or

Parts of this paper were presented previously at the Conference on Radioresistance of Human Tumors: Mechanisms and Prediction, sponsored by McGill University and the Postgraduate Medical Board of the Montreal General Hospital, held in Montreal, Quebec, Canada, 13- 14 July 199 1. Reprint requests to: Prof. Christopher S. Lange, Department

of Radiation Oncology, SUNY Health Sciences Center at Brooklyn, 450 Clarkson Ave., Box 12 12, Brooklyn, NY I 1203. Parts of this investigation were supported by PHS grants 1 ROl CA 39045 and 1 PO1 CA 06294, both awarded by the National Cancer Institute, DHHS. Accepted for publication 22 April 1992. 511

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chemo-sensitivity. These are: (a) cloning in soft agar (either Salmon-Hamburger (IO) (SH) or Courtenay-Mills (5) (CM)), (b) the MTT (diMethylyThiazo1 diphenylTetrazolium) assay (4), (c) the CAM (cell adhesive matrix) assay ( 1,2,3), (d) the Fluorochomasia Cytoprinting assay (13, 14,26), and (e) the Hybrid Spheroid assay (6, 7). Of these five, only the first and last assay clonogenicity, and only the last has a high plating efficiency (PE). These two factors, clonogenicity and PE, are of crucial importance for the prediction of tumor cure. The reason for this is that organismal survival can be traced to the survival of stem cell clonogenicity ( 12), and tumor cure to tumor cell clonogenicity survival (15). Therefore, we have used the hybrid spheroid assay to measure the clonogenic survival of cells obtained from a series of human tumor specimens. Because of the many advantages of this system, let us examine it in more detail. The hybrid spheroid assay

In this assay a single-cell suspension of tumor cells is mixed in a known ratio with a suspension of clonogeniclyinactivated HeLa feeder cells. The later agglomerate to form spheroids composed of a mixture of HeLa feeder cells and tumor cells; hence the term Hybrid Spheroids (6, 7). By adjusting the ratio of tumor to feeder cells and by selection of appropriate size spheroids, one can use the fraction of plated spheroids which do not form colonies as the zero term of a Poisson distribution to determine the average number of clonogens per spheroid. The ratio of clonogenicities of treated to untreated populations of spheroids yields surviving fractions and survival curves for both ionizing radiations and drugs. METHODS

AND MATERIALS

Cell lines and maintenance HeLa S3 cells were obtained from Dr. A. Alfieri from the Memorial Sloan-Kettering Cancer Center, New York. HeLa cells were cultured in Eagle’s Minimal Essential Medium (MEM) supplemented with 10% Fetal Calf Serum (FCS) and antibiotics.* CHO EM9 and AA8 cell lines were originally selected by Dr. L. H. Thompson ( 19) and were subsequently maintained in wMEM supplemented with 10% FCS at the MD Anderson Cancer Center. Hybrid spheroids containing tumor cells were cultured in (u-MEM enriched with vitamins and growth factors and supplemented with 10% FCS and antibiotics (17). Cultures were incubated at 37°C in 5% COZ in air and subcultured by trypsinization twice weekly.

* All media, sera, and antibiotics were purchased from Gibco Laboratories, Grand Island, NY. + Both enzymes were purchased from Boehringer Mannheim, Indianapolis, IN.

Volume 24, Number 3, 1992

Preparation of single cell suspensions,from surgical specimens

tumor

Surgical specimens from patient tumors (material excess to the needs of Pathology and removed with informed consent) were mechanically and enzymatically dissociated under aseptic conditions to obtain single cell suspensions (3) essential for clonogenicity evaluations (16). Briefly, each tumor specimen, on ice, was placed in a IO cm petri dish with 10 ml of cold LU-MEMwith 10% FCS and cut with crossed scalpels. To the resulting mass of minced tissue we added 5 ml of 3% collagenase IV and 5 ml of 0.02% deoxyribonuclease, both+ in a-MEM, and the mixture was incubated for 6 hr at 37°C with constant stirring. The digested mass was then passed through a 100 pm pore stainless steel mesh; the filtrate was centrifuged and the pellet resuspended in 10 ml of Hank’s calcium- and magnesium-free balanced salts solution.* After viability was determined by trypan blue exclusion and the cell density determined in a hemocytometer, the suspension was ready for spheroid formation. Preparation of hybrid spheroids

A detailed description of the hybrid spheroid system has been presented elsewhere (6, 7). Briefly, clonogenicly inactivated HeLa “feeder” cells (incubated for 3 days with 10e5 M bromodeoxyuridine and 2 X 1O-’ M fluorodeoxyuridine and irradiated with 10 Gy of 250 kVp x-rays) were trypsinized and about 5 X lo6 were mixed with test cells (either CHO or tumor, obtained as described above) in proportions ranging from 1 test to 5 HeLa “feeder” cells, to 1 test to 50 HeLa “feeder” cells forming agglomerates containing both cell types. The structural basis of the agglomerates are the HeLa cells, which entrap the other (test) cells. The number of entrapped test cells in spheroids of selected size (loo- 150 pm in diameter) is determined by the ratio of feeder to test cells in the initial cell suspension and the duration of incubation of the mixture before selecting spheroids: for a given set of conditions, the clonogenicity is highly reproducible. The cell mixture was dispersed in 10 ml MEM and incubated in 10 cm bacteriological petri dishes for 20-45 hr at 37°C (most incubations were overnight). During incubation in the bacteriological petri dish, hybrid spheroids were formed, and subsequently harvested. This was achieved by passing the spheroid suspension through a column of nylon sieves with different pore sizes. Spheroids passing a 105 pm sieve, and arrested on an 88 pm sieve (selected for abundance and clarity of differentiation between future colony-formers and non-formers) were eluted in a desired

* Purchased from Gibco Laboratories, Grand Island, NY.

Assay of tumor radio- and chemo-sensitivities 0 C. S. LANGEet al.

volume of medium. Spheroids of this size contain cu. 100 cells each (107 -t 9) ( 1). Additional spheroid sizes can be selected on sieves with pore sizes of 125 (18 1 cells) and 149 pm (306 cells) for use with doses which leave too few survivors in smaller spheroids and/or with tumors having lower PE. The above-described method of cell suspension preparation and hybrid spheroid formation yields colonies consisting of cells with an epithelioid rather than a fibroblastic morphology, as seen from the cells which adhere to CAM plates (2, 3, 17) and from the cells which spread out from the plated hybrid spheroids. In addition, since the PE of tumor cells in hybrid spheroids plated in CAM plates was the same as that of tumor cells in hybrid spheroids plated in normal tissue culture plates (data not shown), and as fibroblasts do not grow on CAM plates (2) we can conclude that the colonies arising from hybrid spheroids do not consist of fibroblasts. If a significant number of fibroblasts had been present in the tumor digest cell suspension and were mistakenly counted as tumor cells, then the plating efficiency (PE) values presented below would have been artifactually reduced; hence these PE values are minimum estimates. Irradiation conditions Irradiations for the data presented in Table 1 and Figures 1, 3, and 4 were performed with a ‘37Cs-y irradiator at dose rates of 4.66 to 4.60 Gy per min. Irradiations for the data presented in Figures 2 and 5 were performed with a Philips model RT 250 x-ray machine, at 50 cm FSD and at a dose rate of 2.5 Gy/min (HVL 0.39 mm Cu, filter 2 mm Al, 250 kVp). Chemosensitivity-testing treatments Harvested hybrid spheroids were adjusted to a concentration of 30 per ml of MEM, and 9.5 ml of the suspension distributed in each of (usually) eight 10 cm bacteriological petri dishes (in which spheroid attachment is discouraged). One half ml of a 20x solution of 5-fluorouracilg (5-FU) was then added, and the dishes incubated for 1.5 hr at 37°C. The drug-containing medium was removed by centrifugation and the spheroids were washed in 40 ml of phosphate-buffered saline in 50 ml conical tubes. Washed spheroids were dispersed in 10 ml of nutrient medium (a-MEM in the case of tumor-containing spheroids), and 5 ml plated into duplicate T-25 culture flasks, gassed with 5% of CO2 in air, and positioned in the incubator for spheroid attachment. Determination of clonogenicity, plating eficiency, and survival in treated spheroids These parameters were obtained from the colonyforming ability of preselected well-separated spheroids

9:Obtained from Solo-Pak Laboratories, Franklin Park, IL.

513

marked on the bottom of the flask (using a dissecting microscope). Since only the marked spheroids were scored for colony formation, counting statistics were binomial. By manipulating the composition of the initial mixed cell suspension (see following section), a situation was obtained where some but not all spheroids formed colonies. (Spheroids composed only of feeder cells do not form colonies (6); colonies grown from 88-105 pm hybrid spheroids containing viable tumor cells are clearly distinguishable from spread abortive spheroids as the former contain several hundred to several thousand compactly growing cells, depending on the growth rate of the tumor). Taking the fraction of spheroids which had not formed colonies (after a 14 day incubation period) as the zero term of the Poisson distribution, one obtains the average number of colony-forming cells, or clonogenicity, per spheroid (6, 7). Plating efficiency for cells in spheroids was calculated by dividing the fraction of cells in spheroids which are clonogens by the fraction of cells which were from the tumor, in the original mixture from which the hybrid spheroids formed (7). This produces a minimum estimate of PE as discussed above and elsewhere (7). Survival was obtained from the decrease in clonogenicity relative to control. Survival curves were constructed from pooled survival data using the Systat program (23). Significance of differences in survival levels (at a particular dose) or in survival curve parameter values was determined using Student’s t-test. Surviving.fraction is independent ofsize of spheroid or number of clonogens It was previously shown (6) that although the number of clonogens per spheroid increases with size, the surviving fraction of clonogens in irradiated hybrid spheroids remains constant irrespective of spheroid size. Thus high dose and/or multiple fractions can be used on large spheroids and the data will be compatible with those from smaller spheroids. In our experience, optimal hybrid spheroid preparations were obtained when the ratio of tumor to HeLa-feeder cells in the initial cell suspension was 1:5. The overall fraction of tumors for which survival curves were obtained was 67%, with failures due to fungal infection or an insufficient number of clonogenic cells. In no case was a preparation obtained in which clonogenic cells yielded a 100% PE. RESULTS Plating ejiciency (PE) We compared the PE of human tumor cells in the CAM system to that of the same tumors in the Hybrid Spheroid assay by splitting tumor sample cell suspensions into two

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aliquots and testing each with one assay side-by-side. Table 1 shows that the PE of the Hybrid spheroid assay ranges between 2.4 and 7.0% (average ca. 5 k 2%) while that of the CAM assay is 0.2-1.8%. Thus the PE of the Hybrid spheroid assay some 2-35 times higher than that of the CAM assay and one to nearly two orders of magnitude higher than that of the CM assay. Trypsinization and replating in situ of the cells comprising hybrid spheroids (containing human tumor cells) results in a reduction of PE to ca. one third of that obtained when the spheroids are allowed to spread and form colonies (data, not shown, are from two tumors-larynx and pyriform sinus-and three spheroid sizes-97, 120, and 132 pm diameter). There is no such effect of trypsin on the PE of cells from established lines (data not shown). Therefore, at least part of the increased PE due to a sojourn in hybrid spheroids can be attributed to events occurring during the spreading out of the spheroid. Demonstration that the hybrid spheroid assay detects known d@erences in radiosensitivity in established cell lines Figure 1 shows that the radiosensitive EM-9 subline of the wild-type CHO-AA8 cell line was readily identifiable in blind assays in hybrid spheroids. Thus the difference in radiation response seen in hybrid spheroids reflects the radiosensitivity of these mutant cells seen in other assay systems ( 19). Reproducibility of survival curves for cells directly isolatedfrom human tumors Figure 2 shows that three replicate samples from the same specimen of a human colon carcinoma (specimen #I) have the same radio-sensitivity. A second specimen of colon carcinoma, from a different patient, (#2) is clearly more resistant. The difference between the two tumors is not readily discernable at 2 Gy, but at higher doses-cu. 3-4 Gy-the difference is easily seen. Comparison of carcinomas of the breast and tongue Figure 3 compares the responses of two breast carcinomas with those of two carcinomas of the tongue. Again,

Volume 24, Number 3, 1992 CHO

0.001

J

0

CELLS

2

IN

HYBRID

SPHEROIDS

4

6

DOSE

El

10

[Gy]

Fig. 1. Survival curves of wild-type CHO-AA8 and radiosensitive mutant EM-9 Chinese hamster ovary cell lines. Cells were irradiated in hybrid spheroids with 13’Csy-rays. Each point represents the mean of three experiments, each with two to three replicate flasks. The two cell lines were assayed with coded identification only (unknown to the experimenter). The linear-quadratic model parameters for the best fit to the combined data for each line are: (Y= 0.354262 (0.17307-0.53546 95% confidence limits) Gy-’ and /I = 0.0708065 (0.04055-0.11558 95% confidence limits) Gy-* for the EM-9 line, and (Y= 0.137631 (0.02874-0.24652 95% confidence limits) Gy-’ and @ = 0.038078 (0.02252-0.05364) Gy-* for the AA-8 line.

the difference between the two types of tumor is most clearly visible at higher doses (4.5 Gy). The difference between the two types of tumor is larger than the difference between tumors of the same type.

HUMAN

P F

COLON

CANCERS

0.100

& I?

COLON

CA

1 (3

REPLICATES)

0.010 0

1

3

2 DOSE

Table 1. PE (W) of tumor cells assayed by the CAM and hybrid spheroid methods

Type of tumor

Monolayer CAM assay

Hybrid spheroid assay

Larynx Tongue Tongue Floor of mouth Breast Mean +- S.D.

0.35 1.8 0.2 0.35 0.69 0.66 + 0.65

2.4 4.0 7.0 4.0 7.0 4.89 +- 2.05

Plating gain 6.9 2.2 35.0 11.4 11.7

4

5

[Gy]

Fig. 2. Survival curves of cells isolated directly from human colon carcinomas assayed in hybrid spheroids irradiated with 250 kVp x-rays. The three lower curves are replicates from the same surgical specimen (A). The upper curve is for a specimen from another patient (B). Error bars, where larger than the size of the symbol as drawn, are standard errors of the mean. For specimen A, two replicates were done on the same day and are indistinguishable from each other at all doses (0.5 > p > 0.25). The third replicate of specimen A was processed a few days earlier and was slightly more resistant at 4 Gy (0.029 vs. 0.0 13, p B 0.005). When specimen B was compared with specimen A (all replicates) it was found to be significantly more resistant at both 2 and 4 Gy (0.05 > p > 0.025, p G 0.005, respectively).

Assay of tumor radio- and chemo-sensitivities HUMAN

TUMORS

IN HYBRID

Chemosensitivity spheroids

SPHEROIDS

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0 C. S. LANGE et nl.

testing of tumor cells in hybrid

Hybrid spheroids are suitable for chemosensitivity testing, too. Figure 5 shows that cells in hybrid spheroids are very resistant to 5-fluorouracil(5-FW), as they are in vivo (2 1). This resistance is not seen in monolayer and is not

0.010 0

1

4

3

2 DOSE

5

due to differences in drug accessibility to the cells in spheroids (8, 11). Tumor cells directly isolated from human colon cancers (primary) respond to 5-FU in a similar fashion as do cell lines (passage) derived from such tumor cells. One can

6

[Gy]

easily grow passage lines for further study from the colonies which grow out from hybrid spheroids (this prevents selection by low PE).

Fig. 3. Survival curves of cells isolated directly from human carcinomas of the breast (inverted triangles) and tongue (circle and triangle) and assayed in hybrid spheroids irradiated with 13’Cs y-rays. Error bars are as in Figure 2. At 1.5 Gy the cell surviving fractions for breast and tongue tumors are not significantly different (0.25 > p > O.lO), but at 4.5 Gy they are (p cc 0.005).

Eflect of PE on stem cell number estimates

Radioresistance qf cells from tumors of d&‘erent histologies Figure 4 shows that, at higher doses (ca. 4.5 Gy), where

Due to the well known heterogeneity found within many human tumors, it is important that an assay system not select only a small atypical fraction of the tumor for its analysis. Although such selection cannot be precluded except for well established cell lines with PEs ca. 90- lOO%,

differences

are

most

pronounced,

radioresistance

de-

creases in the order: breast > ovary > lung > tongue > larynx > lip > mouth floor. Except for lung, which might be expected to be the most resistant, this is the same sequence as expected from historical responses from histological type tumors (9). The differences (at 4.5 Gy) are significant between the breast and ovary tumors (p < 0.01) between the ovary and mouth floor tumors (p < 0.01) and between the ovary and lung tumors (p < 0.005). The intermediate survival tumors (lung, tongue, larynx, lip) do not differ significantly among each other in their survival at 4.5 Gy, but their survival curves fall in the order shown for decreasing radioresistance.

DISCUSSION

the higher the PE, the lower the chance of such selection. Therefore, the optimal assay will be clonogenic (because sterilization of the tumor yields cures) and have high PE. The clonogenic soft agar assays had PEs of lop5 (SH) ( 10) and 0.1% (CM) (5). Although the CAM Assay is not clonogenic, the PE of cells plated on CAM dishes can be determined and, as shown in Table 1, is ca. 0.7%. The PE of cells from the same tumors was ca. 5% in the hybrid spheroid assay. Obviously, the agar colony assays, with their much lower PE, cannot be used to estimate “stem

HUMAN

COLON

CANCERS

FILLED OPEN HUMAN

TUMORS

IN HYBRID

SYMBOLS SYMBOLS

= =

PRIMARY PASSAGE

SPHEROIDS

1.000

$ 2 E p 0.100 5

2

500

2

5-FU

0.010 0

1

2

3 DOSE

4

5

6

[Gy]

Fig. 4. Survival curves of primary cells of human tumors irradiated with 13’Cs -r-rays in hybrid spheroids. The source of the tumors is listed from top to bottom in the figure in order of increasing radiosensitivity at 4.5 Gy. Error bars are as in Figure 2.

1000 CONCENTRATION

1500

21 00

[ug/ml]

Fig. 5. Survival curves of cells from two human colon carcinomas in hybrid spheroids exposed to the shown concentrations of 5fluorouracil for 1.5 hr before rinsing and plating for colony formation. Filled symbols represent primary cells directly isolated from the tumor; open symbols represent first passage cells derived from colonies grown from the primary cells directly isolated from the tumor; open symbols represent first passage cells derived from colonies grown from the primary tumors. The symbols for the passage lines are the same shape as those for their primary.

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cell number” if a change in assay (to Hybrid Spheroid) raises the estimate by 2-4 orders of magnitude. Estimation of the eflects of multifaction treatments It has been suggested that the surviving fraction at 2 Gy (or at whatever dose is used as the daily fraction) should be the best indicator of clinical effectiveness (local control) (9). However, the prediction made from such a measurement depends on exponentiating that surviving fraction (and its error bars) to the 30th power (number of fractions). Thus, a surviving fraction of 0.40 +- 0.04 (10% error bars) raised to the 30th power would yield an expected survival of 1.15 X 10-12, with 95% confidence limits between 2.74 X lo-” and 1.43 X 10-15. This represents a rather large uncertainty in the size of tumor sterilized. However, if one could give five 2 Gy fractions (one week) and then measure the surviving fraction (say (0.40)’ = 1.024 X 1Om2),the error bars would still be f 10% since they are determined by the number of colonies counted. This result would only need to be raised to the 6’h power, yielding the same surviving fraction as before, but with 95% confidence limits narrowed to between 3.43 X lo-l2 and 3.02 X lo-l3 (i.e., one vs. ca. five decades of uncertainty). With the sole exception of the hybrid spheroid assay, none of the below reviewed assays is suitable for this type of measurement (because of cell proliferation between doses in vitro; proliferation in vivo is thought to be significant only after 4 weeks and only for those types of tumor considered as candidates for accelerated fractionation ( 18, 24, 25)). Hybrid spheroids can be held with negligible cell proliferation during such a radiation regimen prior to plating for colony formation (8). Comparison with other assay systems 1. Cloning in soft agar. In both versions of this assay (SH and CM) the tumor is reduced to a single-cell suspension which is then mixed with a molten agar plus culture medium solution and immediately plated either directly on petri dishes (10) (SH) or on an agar underlay containing erythrocytes (5) (CM). The advantages of these methods are that they measure clonogenicity and appear to be specific for tumor cells (i.e., the colonies are reported or assumed to derive only from tumor cells). The disadvantages ( 16) are: (a) low Plating Efficiency (PE) (ca. 1O-5 (SH); lop3 (CM)), (b) therefore colony growth is not representative of tumor heterogeneity in vivo, (c) a low yield of positive assays (about 30%), (d) many colonies are cell groups, not clones, (e) the SH assay is not good for radiation survival estimation, (f) the CM assay is not suitable for all tumor types, and (g) the test is cumbersome. 2. The MTT assay. This assay is performed on established cell lines derived from human tumors, not on the primary tumors themselves (4). A cell suspension is seeded into 96-well plates, time is allowed for growth, and the cells are then fixed and stained with MTT. The Optical Density (OD) is then measured as an indicator of cell number. The advantages are: (a) the method can and has

Volume 24. Number 3, 1992

been used for mass screening, by histological type, of potential chemotherapeutic agents on established tumor cell lines; (b) the procedure is automated, and (c) reproducible. The disadvantages are: (a) the test is not clonogenic, the OD is proportional to cell number; (b) the assay is not good for primary tumor cells, only for derived lines which themselves represent severe selection (low PE) for what will grow in vitro. These disadvantages were sufficient to result in its abandonment, by NCI, as a screen for potential chemotherapeutic agents. 3. The CAM assay. In this assay the tumor is reduced to a single-cell suspension and the cells are plated on special commercially prepared (but not yet readily available) Cell Adhesive Matrix Plates (1, 2, 3). The cells are given time to grow, after which they are stained and the OD of stain is measured. The advantages of this assay are those of the MTT assay and: (a) it measures the growth ofprimary culture tumor cells; (b) the plating efficiency is relatively high (0.1 -I .O%); (c) about 65% of tumors grow, yielding a storable result; (d) the method does yield radiation survival curves; (e) CAM plates prevent normal fibroblast growth; and (f) the procedure is automatedOD reading has been computerized. The disadvantages are: the test is not clonogenic and the CAM culture containers are not readily available. 4. Thefluorochromasia assay. This assay measures the survival of cellular metabolic activity and membrane integrity ( 13, 14, 26). The tumor is reduced to a suspension of cell-clumps (not single cells) to which fluorescein monoacetate is added. Healthy cells take up the dye and remove the acetate, yielding a fluorescent fluorescein. The fluorescence is proportional to cell number. One measures the fluorescence of an untreated control sample (Fl) and compares this with that of a similar sample treated with the drug or drugs of choice (F2). The surviving fraction is the taken to be F2/Fl. One can also make repeated measurements on the same sample if sufficient time (a few days) is allowed for the fluorescein produced in the first measurement to be released from the cells. The advantages are: (a) the assay successfully measures the fraction of cells remaining metabolically intact after treatment with chemotherapeutic agents, and does this for individual patient primary tumors; therefore, (b) it is useful for the testing of chemotherapeutic drugs: (c) by repeated measurements one can obtain the fraction of viable (not clonogenic) cells as a function of time after (or during) treatment. The disadvantages are: (a) the assay is not clonogenie and, (b) it is not good for radiation-sensitivity testing (because radiation-sterilized tissue fragments remain metabolically active). Advantages of the hybrid spheroid assay system From the foregoing, it becomes clear that the hybrid spheroid assay system has the following advantages: (a) the test is clonogenic; (b) it has a high plating efficiency, I- 10% ( 1.5-2 orders of magnitude higher than that of the best of the other clonogenic assays); (c) a wide variety of

Assay of tumor radio- and chemo-sensitivities 0 C. S. LANGErf a/.

primary tumors can be assayed directly; (d) one can get both chemo- and radiation-survival curves; (e) measurements on samples from the same patient are repeatable and can be demonstrably different from those for samples from a different patient; (f) the colonies are compact and easily scored (unlike those often observed with primary cultures); (g) multifraction treatments are possible, without proliferation between doses; (h) ordinary culture containers are suitable; (i) binomial counting statistics yield low dose accuracy; (j) there is no need to know the exact numbers of tumor cells per spheroid; (k) no postirradiation trypsinization is necessary-whole spheroids are irradiated and plated: (1) radiosensitivity is related to histological type; (m) the hybrid spheroid assay mimics the in vivo

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behavior of tumor cells with regard to chemosensitivity assays (detection of resistant fractions). The only disadvantage is that the assay is not yet suitable in its present form for survival measurements of non-adhering cells. This deficiency will be remedied by automated placement of spheroids into individual wells of 96-well tissue-culture plates, where the appearance of numerous proliferating unattached cells must derive from individual spheroids. With these improvements, the hybrid spheroid assay will be ideal for in vitro comparison of the effects of new treatment modalities and the measurement of the sensitivity of individual patient tumors to each of the potential alternative radiation and chemotherapeutic treatments under clinical consideration (6, 7).

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