IN VITRO CHEMOSENSITIVITY TESTING OF

RENALCELLCANCER*

Short-term Culture Technique

M. A. BAZEED, M.D. J. SCHMIDT, M.D. T. SCHARFE, M.D. G.H. JACOBI, M.D. E. BECHT, M.D. J. W. THUROFF, M.D.

From the Department of Urology, University of Mainz Medical School, Mainz, F. R. Germany

ABSTRACT-In an attempt to solve the problem of chemosensitivity testing of renal cell carcinoma in vitro, a modified short-term culture technique was developed. The kinetic study of hy- pernephroma cells and normal renal cells showed that the uptake of H%ridine and H3-thymidine is at its maximum after eighty hours. The effect of doxorubicin, c&platinum, vinblastine, and mito- mycin C in different concentrations was tested. Tumors generally showed more resistance than sensitivity. Some tumors showed sensitivity to one or more drugs, but no one drug was persistently effective in all tumors. Our short-term culture technique solved the discrepancy between cell kinetics and test duration found in the Volm test and the problem of nongrowth in the clonogenic assau.

Ideally chemotherapy of malignant tumors should be individualized, thus saving the pa- tient the needless risk of exposure to toxic but ineffective drugs. This is even more important in the case of a tumor-like renal cell cancer, which is known to be chemotherapy-resistant. A number of techniques have been established for determining tumor sensitivity to anticancer drugs:

1. The short-term in vitro test developed by Volm and Mattern in 1976r entails incubation of a tumor cell suspension with cytotoxic agents for two hours. The resulting effects on nucleic acid metabolism are measured using H3-uridine and H3-thymidine. It is an easy, rapid test.

2. The long-term (3-4 weeks) culture with measurement of cell-killing under the effect of cytotoxic drugs in monolayer or organ culture, is more suitable for body fluids containing ma- lignant cells as in malignant ascites.

3. The clonogenic assay first published by Puck and Marcus in 19553 and improved and

*Supported by a grant from Alexander van Humboldt Founda- tion, I? R. Germany.

popularized by Hamburger and Salmon in 1977,4 involves the growth of colonies from tu- mor stem cells in semisolid media after exposure of these tumor cells to cytotoxic drugs. It is also a test of long duration (3-4 weeks) in which only about 60 per cent of the tumors grow. Fleischmann, Heston, and Fair5 concluded that the clonogenic assay is not a clinically useful nor reliable means of in vitro chemosensitivity test- ing, and renal cell carcinoma in particular is unsuited to the assay in its present form.

4. Heterotransplantation models consisting of transplantation of tumor particles subcuta- neouslp*7 or under the renal capsule8 in nude mice and attempting chemotherapy in the ani- mal or in vitro on the tumor after removal is a long-term test (3-4 weeks) and requires espe- cially equipped laboratories and expert person- nel.

Material and Methods

A total of twenty-five renal cell carcinomas and their normal renal counterparts were stud- ied, using the short-term test reported by Volm

240 UROLOGY / MARCH 1988 / VOLUME XXXI, NUMBER 3

and associates in 197gg in 10 patients and our modified assay in 15 patients.

Volm assay test The kidney was transferred under sterile con-

ditions to our immunology laboratory, where a piece of non-necrotic tumor and a sample of normal kidney without its fibrous capsule were excised. Each piece was put into a sterile petri dish and cut with two scalpels into small pieces of 3 to 4 mm in size. Then a McIlwain* tissue chopper was used to cut the pieces into 1 mm each. The tissue pieces were suspended in Hank’s solution, pipetted several times into a sharp-edged glass pipette, and the resulting cell suspension was filtered through gauze of 200 pm pore size. The cells were collected in culture tubes, centrifuged for five minutes at a rate of 800 rpm, and washed with Hank’s solution. Af- ter centrifugation, Hank’s solution was de- canted and the cells resuspended in Hank’s solu- tion. The cell count was adjusted to 500,000 cells/ml, using a Neubauer counting chamber. The cells were then portioned into test tubes in aliquots of 0.9 mL. After preincubation at 37°C for fifteen minutes in a shaking water bath, the cytotoxic agents were added to the test suspension in a volume of 50 $/test tube.

Hank’s solution was added in the same vol- ume to the control tubes. Doxorubicin (Adriamycin) and endoxan were used in 5 final concentrations in the cell suspension of 100 Fgl mL, 33.3 PglmL, 10 PglmL, 3.33 pg/mL, and 1 PglmL. After incubation for two hours, the ra- dioactive nucleic acids were added to the test tubes in a volume of 50 PL (2.5 PCi). The ap- propriate radioactive nucleic acid precursor was used for each cytostatic agent,‘O H3-uridine for doxorubicin and H3-thymidine for endoxan. After one hour incubation, 100 PL were pipet- ted from each test tube onto round filter paper and dried in a stream of warm air. The nonin- corporated radioactivity was extracted by washing twice in ice-cold 5 % Trichloro-Acetic- Acid (TCA), first for fifteen minutes and then for thirty minutes. The filters were then

Short-term culture assay Carcinomatous and normal renal tissues

were cut with two scalpels into small pieces 1 mm in size. The tissue pieces were put in a sterile lOO-mL Erlenmeyer flask with a magnetic stirrer. A solution composed of equal amounts of 0.5% trypsin and 0.1% EDTA was added for ten to fifteen minutes of trypsiniza- tion with a magnetic stirrer. The supernatant was then mixed with the same volume of com- plete medium, which contains 10% FCS (fetal calf serum) to inactivate the trypsin. This was centrifuged for five minutes at 800 rpm. The supernatant was removed and the cells washed again. Thereafter, the cells were counted and the count adjusted to lo5 cells/50 PL. Flat mi- crotiter plates with 96 wells were used, filling each well with 50 PL (lo5 cells) and 50 PL ra- dioactive uridine or thymidine (2.5 &I).

Four plates were prepared for each patient, two containing cancer cells and two with nor- mal renal cells. H3-uridine was added to one normal plate and one cancer cell plate, while H3-thymidine was added to the other two plates. The cells from the wells were harvested at different intervals to establish the kinetics of normal and hypernephroma cells. The harvest- ing machine has two functions: to suck cells from each well onto a filter paper and to wash off the radioactivity not bound to the cells. The filters were allowed to dry, Each filter was put in a scintillation tube containing 5 mL scintilla- tion fluid, and radioactivity was counted. The counts were plotted against time, and a kinetic curve of H3-uridine and H3-thymidine uptake of renal cell carcinoma and normal cells was obtained.

Chemosensitivity was tested utilizing nearly the same technique as for the kinetic study, but with the addition of chemotherapeutic drugs (Table I) to test their effect. Each line (12 wells) was devoted to testing one drug. For the first three wells, only medium was added to obtain control values. The second three wells were used to test the smallest concentration (Table I),

washed for twenty minutes in a solution of equal volumes of ethanol and ether, then in ether onlv for ten minutes. and were finally air dried. S&tillation fluid (5 mL) was addid to each filter in a scintillation vial and the incor- porated radioactivity as counts/ten min was de-

TABLE I. Cytotoxic drugs used in study and concentration in cell suspensions

Drug

Ciwlatinum

Final Concentration in -Cell Suspension (rg/mL)-

0.01 0.002 0.001 termined by B-counter. Vin?blastine 40 4 0.4

Mitomycin C 0.004 0.002 0.0004

*The Mickle Laboratory Engineering Company, England. Doxorubicin 40 4 0.4

UROLOGY / MARCH 1988 / VOLUME XxX1, NUMBER 3 241

:pfll

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900-

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OJ 0.5

cpm 1200 -

1000 -

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10 20 30 LO 50 60 69 73 TIME [HOURS]

FIGURE 1. Uptake of H3-uridine and H3-thymidine by normal renal cells and renal cell carcinoma cells from patient 506.

the third three wells for the second concentra- tion, and the fourth three wells for the highest concentration. After nearly eighty hours incu- bation, the cells were harvested and activity counted as described. The radioactivity of cell- bound H3-thymidine and H3-uridine was plot- ted against the drug concentration.

Results

The short-term Volm assay chemosensitivity test was used for tumors from 10 patients. No tumor showed significant inhibition by the che- motherapeutic drugs used (doxorubicin and en- doxan), since the maximum inhibition of H3- uridine and H3-thymidine was 10 per cent.

In our short-term culture assay, kinetic stud- ies of H3-uridine and H3-thymidine uptake by material from 5 patients showed that the up- take of H3-uridine and H3-thymidine by normal renal cells and renal cell carcinoma cells gradu- ally increased over time until a maximum was reached between seventy and one hundred eighty hours. Thereafter uptake began to de-

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cline (Fig. 1). The average time until the peak was reached was around eighty hours. Our short-term tissue culture for chemosensitivity testing was used for renal cell carcinoma and normal renal cells from 10 patients. In general, the tendency was more toward resistance than sensitivity. Six tumors showed sensitivity to one or two drugs, but not in many cases was sup- pression very significant. Cisplatinum showed 42 per cent inhibition of H3-uridine uptake in one tumor. Doxorubicin showed 39 per cent/40 per cent suppression of H3-uridine uptake in two tumors. Mitomycin C was effective in 1 pa- tient. Figures 2, 3, and 4 show the results ob- tained in patients 513, 514, and 561, respec- tively, and serve as examples.

Comment

Since the results obtained with Volm’s short- term test were not acceptable, we began inves- tigating the problem further. Kinetic studies confirmed our suspicion that the incubation time of this test may be too short for a slowly growing tumor such as renal cell carcinoma. This is in agreement with others, such as Seeber and Schmidt,” who stated that the short dura- tion of the test was one of its drawbacks. Our kinetic studies not only showed that the uptake of H3-uridine and H3-thymidine is very low in the first four hours, they also provided us with information as to the ideal time of incubation: nearly eighty hours is the average time needed for H3-uridine and H3-thymidine uptake to reach a maximum. Consequently, we designed our test with an incubation time of eighty hours. The application of the modified test to 10 cases was confirmatory that this modifica- tion solved the main problem of the short-term test.

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FIGURE 2. Effect of cytotoxic drugs on uptake of HQridine by (A) normal kidney cells, and (B) renal cell carcinoma cells from patient 513.

242 UROLOGY / MARCH 1988 / VOLUME XxX1, NUMBER 3

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Our results furthermore confirm those of Lieber and Kovach12 who used the clonogenic assay for chemosensitivity testing of renal cell carcinoma in 5 patients. In those studies, che- motherapeutic drugs (at a concentration ap- proximately ten times higher than achievable in human plasma for the first hour after adminis- tration according to current clinical protocols) were incubated with tumor cells at 37°C for one hour. Even at this high drug concentration,

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FIGURE 4. Effect of cytotoxic drugs on uptake of (A) H3-uridine and (B) H3-thymidine by normal kid- ney, and (C) of H3-uridine by renal cell carcinoma cells from patient 561.

UROLOGY / MARCH1988 i VOLUMEXXXI,NUMBER3 243

the degree of inhibition of clonogenic prolifera- tion by almost every drug tested (doxorubicin 10 PglmL, mitomycin C 4.0 pg/mL, cis- platinum 45 pg/mL, and vinblastine 0.5 PgI mL) was minimal for each tumor. Some tumors were markedly sensitive to one or two drugs in vitro, but no single drug was active in more than one tumor. Our results confirm the clinical fact that renal cell carcinoma is generally a tu- mor resistant to cytotoxic agents. It also con- firms the fact that renal cell carcinomas are het- erogeneous in their response. These facts led to the conclusion that in such a situation chemo- sensitivity must be used to identify the effective cytostatic drug, to avoid exposing patients to the unnecessary toxicity of ineffective agents.

Another question is, with what concentration of a given drug must which degree of suppres- sion be obtained to consider a tumor sensitive or resistant? Volm and associates9 were able to de- termine this for human lung and ovarian car- cinoma. They studied the effect of 10 pg/mL doxorubicin on the uptake of H3-uridine by these tumors and then treated patients with doxorubicin. Of those patients whose tumors showed suppression of H3-uridine by doxorubi- tin more than 30 per cent responded favorably. In contrast, those patients whose tumors showed suppression of less than 30 per cent were resistant to the drug.

Like all other investigations, the test has its limitations such as the inability to test drugs that need activation in the liver. The relative sensitivity cannot be determined with certainty by this assay, and measurement of uridine and thymidine uptake and their respective suppres- sion cannot identify killing of resting cells.

In conclusion, we found that, comparable to the clinical situation, the renal cell carcinoma in vitro has poor chemosensitivity. The two- hour short-term test, although effective in other tumors, is not applicable to the renal cell car- cinoma because it is a slowly growing tumor. We found that short-term tissue culture is better

for this tumor because the cell population closely resembles that of the primary tumor in contrast to the situation in the clonogenic or in vivo assay. Also, in this test, resistant drugs can be individualized safely. Above all, the assay test is fast, requiring only three days, compared with the clonogenic or in vivo assays, which take four weeks.

Department of Urology University of California

500 Parnassus Avenue, U-518 San Francisco, Californ$ 94143

(DR. THUROFF)

References

1. Volm M, and Mattern J: Individuelle Chemotherapie solider Tumoren, Med Welt 27: 1171 (1976).

2. Tanneberger S, and Bacigalupo G: Einige Erfahrungen mit der individuellen zytostatischen Behandlung maligner Tbmoren nach priitherapeutischer Zytostatika-Sensibilitats-Priifung in vi- tro (Onkobiogramm), Arch Geschwulstforsch 35: 44 (1970).

3. Puck TT, and Marcus PI: A rapid method for viable cell titration and clone production with HeLa cells in tissue culture. The use of x-irradiated cells to supply conditioning factors, Proc Nat1 Acad Sci USA 41: 432 (1955).

4. Hamburger AW, and Salmon SE: Primary bioassay of hu- man tumor stem cell, Science 197: 461 (1977).

5. Fleischmann J, Heston WDW, and Fair WR: Renal cell car- cinoma and the clonogenic assay, J Urol 130: 1060 (1983).

6. Day JW, et al: In vitro chemotherapeutic testing of urologic tumours, ibid 125: 490 (1981).

7. Kurth KH, et al: Assay-Verwertbarkeit humaner Nierentu- morlinien (abstr 7), Symposium fur experimentelle Urologie, Tu bingen, April 6-8, 1984, p 83.

8. Kurth KH, Romijin JC, Dongen JW, and Schroder FH: As- say-Verwertbarkeit von Patiententumoren: Untersuchung des Verhaltens von Nierenkarzinomen im Nierenkapsel- und Thymi- din-Assay (abstr 7), ibid, p 82.

9. Volm M, Wayss K, Kaufmann M, and Mattern J: Prethera- peutic detection of tumour resistance and the results of tumour chemotherapy, Eur J Cancer 15: 983 (1979).

10. Volm M, Kaufmann M, Mattern J, and Wayss K: Miiglich- keiten und Grenzen der pratherapeutischen Sensibilitiitstestung von Tumoren gegen Zytostatika im Kurzzeittest, Schweiz Med Wochenschr 105: 74 (1975).

11. Seeber S, and Schmidt CG: Zum Problem der prathera- peutischen Sensibilit&be.stimmung von Tumoren durch Incor- porationsstudien in vitro, Klin Wochenschr 55: 1127 (1977).

12. Lieber MM, and Kovach JS: Soft agar clonogenic assay for primary human renal carcinoma: In vitro chemotherapeutic drug sensitivity testing, Invest Urol 19: 111 (1981).

244 UROLOGY / MARCH 1988 / VOLUME XxX1, NUMBER 3