In vitro chemosensitivity of human bladder cancer

In Vitro Chemosensitivity of Human Bladder Cancer

LARRY M. WEISENTHAL, MD, PHD,’ A. O’TAYO LALUDE, MD,t AND J. BERNARD MILLER, MDt

The chemosensitivity of fresh specimens of human transitional cell carcinoma was tested using a novel dye exclusion assay based upon Fast Green staining, cytocentrifugation, and H & E counterstaining. Assays were successful (three or more drugs tested) in 22/24 specimens obtained by cytoscopy and 2/2 specimens obtained by open biopsy. Three of 17 specimens had more than 70% cell kill after a 1-hour exposure to thiotepa (500 ug/ml), compared with 10/18 after exposure to mitomycin C (250 ug/ml), and 19/19 after exposure to doxorubicin (100 ug/ml). There was a trend for specimens with high histologic grades or low control viabilities in short-term culture to be more sensitive to mitomycin C than specimens with low histologic grades or high control viabilities. Comparison of these data with published clinical data suggests that intrinsic chemosensitivity may be the most important determinate of clinical response to intravesical chemotherapy with thiotepa, while drug penetration to the tumor cells may primarily determine clinical response to intravesical doxorubicin. Thiotepa may be most valuable in treating large lesions which are intrinsically sensitive, while doxorubicin may be valuable in nearly all small lesions. Improved drug distribution to tumor sites could improve intravesical therapy with doxorubicin.

Cancer 51:1490-1496. 1983.

HERE IS a 50-70% recurrence rate of superficial T bladder cancer following endoscopic resection.’ T h i ~ t e p a ~ - ~ and doxorubicin4 were found to reduce recurrence rates when given prophylactically after “complete” endoscopic resection. Such prophylactic treatment reduced recurrence rates by 50% or more, but did not eliminate recurrences. With nonresected bladder tumors, intravesical chemotherapy with thi~tepa,’.~.~ mitomycin C,’-8 or doxorubicin9-lL has been found to pro- duce responses in about 65-80% of patients. Only 45- 65% of these responses are complete, however.

Drug and patient selection for intravesical chemotherapy could possibly be improved with the use of predictive in vitro chemosensitivity assays. A number of authors have proposed chemosensitivity assays for transitional cell carcinoma based on: cl~nogenicity,’~.’~ ra- dioactive precursor incorp~ration,l~-’~ and dye exclu-

From the Veterans Administration Medical Center, Long Beach.

Supported by the U. S. Veterans Administration. * Section of Hematology-Oncology, Medical Service, V.A. Medical

Center, Long Beach, California and Department of Medicine, Uni- versity of California, Irvine.

t Section of Urology, Surgical Service, V.A. Medical Center, Long Beach, California 90822 and Department of Surgery, University of California, Irvine.

Address for reprints: Larry M. Weisenthal, MD, PhD, Section of Hematology-Oncology, Medical Service. VA Medical Center, Long Beach, CA 90822.

The authors thank Joye A. Marsden, Patricia L. Dill, and Cindy K. Campbell for technical assistance.

Accepted for publication April 19, 1982.

California and the University of California. Irvine. California.

sion.I4 To date, no reports have been published to cor- relate in vitro assays with individual patient response to intravesical therapy. One of us (L.M.W.) has recently described a novel dye exclusion assay which could be used to estimate in vitro cytotoxicity in a large variety of human tumor specimens. We attempted to determine if this assay could be used to assess cytotoxicity in vitro for transitional cell carcinoma specimens obtained by endoscopic biopsy. We also attempted to determine if in vitrocytotoxicity data were compatible with the previously-described clinical response patterns.

Materials and Methods

Tumor Cell Preparation

Human transitional cell carcinomas were placed in RPMI- 1640 medium containing 15% heat-inactivated fetal calf serum immediately after surgical excision. 24 specimens were obtained by cystoscopic resection. One fifth or less of the entire surgical specimen was used for the in vitro assay to be described below. Two specimens were obtained at the time of open surgical resections. Tissue was kept at room temperature until it was convenient to process it, generally 1-2 hours or overnight for late afternoon specimens. The tissue was teased apart with 25-gauge needles affixed to tuberculin syringes, which served as convenient “needle holders.” Bladder tumors dissociated readily, and no enzyme solutions were required. Excessive trauma was avoided, as a per- fect single cell suspension was not needed for this assay.

0008-543X/83/04 I5/ 1490 $ I . I5 0 American Cancer Society

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FIG. I . Appearance of control cultures of human transitional cell carcinoma after four days in iiquid culture. Pink stained cells are presumed to be “living” and green stained cells are presumed to be “dead.” (Green cells appearing more bluish in pho- tographs because of camera filters). The large clumps were very charac- teristic of transitional cell carcinoma and it is not known if these purely represent cell aggregates or whether they may partially represent “colo- nies” from cell multiplication (X 100).

FIG. 2. Appearance of cells from the same patients depicted in Fig. 1 after exposure to doxorubicin (100 ug/rnl) for I hour, followed by culture in liquid medium for four days. Note the striking paucity of “surviv- ing” cells ( X 100).

FIG. 3. High-power magnification of the control cells depicted in Fig. I . The histologic grade of the tumor from which these cells were derived was I1 (X400).

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concerning the patients from whom the specimens were obtained. The in vitro assay data was then retrospectively correlated with the clinical and pathologic data.

Results

Success Rate

Twenty-six specimens were received (24 endoscopic biopsies and two open biopsies). The median number of viable cells obtained after tissue dissociation was 1.3 X lo6 (range, 0.3-12.0 X lo6). The median cell viability

FIG. 4. Effect of thiotepa (2.5- 500 ug/ml, hour) on the of four-day liquid cultures of transitional carcinoma. Each point or pair of points represents an assay

on cells from a different patient.

after tissue dissociation was 39% (range, 4-99%). Two specimens with extremely low initial viabilities were “enriched” for living cells by sedimentation over Ficoll- diatrizoate (Lymphocyte Separation Medium, Litton Bionetics, Kensington, MD) which resulted in an in- creased initial viability from 4 to 35% and from 9 to 67% for the two samples, respectively. The median control viability after 4 days in liquid culture was 14% (range, 0.2-49%). It was possible to assay three or more drugs successfully in 22/24 endoscopic biopsy specimens and in 2/2 open biopsy specimens, for an overall success rate of 92%. Using duplicate cultures with lo5 cells/culture, it would have been theoretically possible to assay a median of six drugs per specimen received. The actual median number of drugs which we assayed was four drugs per specimen. Thirty percent of our specimens contained more than 3 X lo6 viable cells, which would have permitted assay of upwards of 14 drugs per spec-

course, our tissue yield could have been improved.

Doxorubicin, thiotepa, mitomycin C, and cisplatin were obtained in powder form from the Division of Cancer Treatment, the National Cancer Institute, Be-

frozen at -70” at 1OX the desired final concentration until use.

Cytotoxicity Assay Drug Eflects

theda9 MD* Drug were in ’* ’ NaC1 and imen. By utilizing than 20% of each specimen, of

This will be described in greater detail elsewhere.” In summary, 1 X lo5 viable cells were exposed to drugs for one hour in the serum-supplemented medium described above, washed twice, and cultured for 4 days in the above described liquid medium in polypropylene tubes (to prevent fibroblast growth). Cells were then concen- trated to lo5 cells/0.2 ml by means of centrifugation. Following this, 0.2 ml of 2% Fast Green dye in 0.15 M NaCl was added and the suspension was vortexed. After 10 minutes, the suspension was sedimented onto mi- croscope slides using a Cytospin@ centrifuge (Shandon Southern Instruments, Sewickley, PA) ( 1200 RPM, 8 minutes), and counterstained with a modified hematox- ylin and eosin (H & E) technique. “Living” cells stained pink with H & E (Figs. 1 and 3) and “dead” cells stained green (Figs. 1-3). The ratio of “living” cells over ‘‘living’’ cells plus “dead” cells was determined for each slide and then expressed as a percentage of the control (4 day cultures without drug). All assays were performed and quantified “blindly,” without any clinical information

Thiotepa, mitomycin C, and doxorubicin were tested using a one-hour drug exposure at several concentrations, ranging up to those achievable by intravesical administration. A 30% cell survival (70% cell kill) was ar- bitrarily considered to be the cut off between “sensitivity” and “resistance.” Of five cultures, none were “sensitive” to thiotepa at 2.4 ug/ml (Fig. 4). One of two cultures was “sensitive” to 167 ug/ml. Three of 17 cultures were “sensitive” to 500 ug/ml (2.6 X M) for one hour. Four of the 14 “resistant” cultures were obtained from patients with proven clinical resistance to intravesical thiotepa (30-60 mg in 30-60 ml for one hour) for a “true-negative” rate of 100% (four of four). There were no patients assayed who were proven re- sponders to intravesical thiotepa (the remaining patients who were assayed were either not treated with chemotherapy or were inevaluable for responsiveness).

For mitomycin C, the in vitro “response rate” was 0/8 at 1.5 ug/ml, 0 of 1 at 25 ug/ml, 0/2 at 83 ug/ml, and 10/18 at 250 ug/ml (7.5 X M) (Fig. 5A). For

No. 8 CHEMOSENS~TIVITY OF BLADDER CANCER - Weisenthal et a f .

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doxorubicin, the in vitro “response rate” was 1 / I0 at 1.2 ug/ml, 1/1 at 3.3 ug/ml, 1/2 at 10 ug/ml, 2/2 at 33 ug/ ml, and 19/19 at 100 ug/ml (1.8 X M) (Fig. 5B).

Another author, using a clonogenic assay system, re- ported cisplatin to be ineffective at a one hour exposure in vitro in a rat bladder tumor system which was sensitive to cisplatin in vivo.I8 Cisplatin was effective in vitro using a continuous exposure, however. We also found limited activity with a one-hour exposure to cisplatin in our assay system, at concentrations up to 10 ug/ml (3.4 X lop5 M) (data not shown). Therefore, this drug was tested using a continuous, four-day drug exposure. The in vitro “response rates” were 0/3 at 0.33 ug/ml, 2/7 at 1 ug/ml, 2/4 at 3.3 ug/ml, and 3/5 at 10 ug/ml (Fig. 6).

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Relationship of In Vitro Cytotoxicity to Control Cell Viability and Tumor Grade

The possible relationship between control cell viability after four days in culture and in vitro chemosensitivity to mitomycin C was studied, because of the pos- sibility that the heterogeneity in chemosensitivity may have been an artifact of extracorporeal cell viability,

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FIG. 6 . Effect of cisplatin (1-10 pg/ml, continuous 44ay exposure) 5 10

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FIGS. 5A AND 5B. (A, left) Effect of mitomycin C ( 1 5 2 5 0 pg/ml, I hour) on culture of transitional cell carcinoma. (B, right) Effect of doxorubicin (1.2-100 pglml, I hour) on cultures of transitional cell carcinoma.

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rather than reflecting true differences in chemosensitivity. Figure 7 shows that there was, indeed, a trend for the less viable specimens to be more sensitive to cell kill by mitomycin C. The correlation coefficient was only 0.43, however, 7/9 specimens with a control viability of 15% or less after four days in culture were “sensitive” to mitomycin C (250 ug/ml, one hour). Three of nine specimens with a control viability of 20% or greater were “sensitive.” This difference fell short of significance by X2 analysis (P < 0.1).

The relationship between tumor grade and in vitro chemosensitivity to mitomycin C was also evaluated (Fig. 8). Three of eight grade 1-11 tumors were “sensitive”, while five of five grade 111-IV tumors were sensitive to mitomycin C (250 ug/ml, 1 hour). This difference was significant (X2 = 5.1; P < 0.025), although the meun percentage of cell survival between the two groups was not significantly different (Fig. 8; P < 0.2 for the difference between the two means).

To determine if the apparently greater chemosensitivity of the grade 111-IV tumors was a function of control cell viability, the relationship between tumor grade and viability (preculture and after four days in culture) was examined (Fig. 9). There was a nonsignificant trend

1494 CANCER April 15 1983 Vol. 51

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FIG. 7. Relationship between cell survival aRer exposure to mitomycin C (250 ug/ml, 1 hour) and viability of the control cells after four days in liquid culture. The correlation coefficient of the linear regression line constructed from this semi-log plot was 0.42.

for grade 111-IV tumors to have a lower viability (both preculture and after four days) than the grade 1-11 tumors.

Discussion

We have shown that the presently-described assay is a technically feasible approach to determining the chemosensitivity in vitro of endoscopically-obtained human transitional cell carcinoma specimens. The predictive accuracy of the assay for human bladder cancer must now be tested by means of clinical correlations, but preliminary studies with other types of tumors did suggest

that the assay had some validity for several cycle non- specific agents.”

There appeared to be a trend for specimens which were less viable after four days in control culture to be more sensitive to cytotoxicity induced by mitomycin C. This may imply that “unhealthy” cells are less capable of repairing what would only be sublethal damage for more “healthy” cells. This relationship was far from absolute, however, and fell short of statistical significance. There did appear to be a marked variation in intrinsic chemosensitivity between samples which was largely independent of control viability. It would seem advisable to examine in other types of assays whether or not the control viability in culture is a variable which should be considered in assessing the meaning of a heterogeneity in drug sensitivity among tumors of the same type. A vital dye assay similar to the one described here and el~ewhere’~ should be used, however, to determine specifically the viability of the tumor cells compared to the viability of the nontumor cells (predominately his- tiocytes) present in most human tumor specimens.

Although our results are of a limited and preliminary nature, it is of interest to draw some speculative con- clusions after comparing published clinical results with our in vitro results. Clinical response to topical chemotherapy is most likely dependent upon both drug penetration into the tumor and on the intrinsic che-

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survival after exposure to mitomycin C (250 ug/ml, 1 hour) and the histologic grade of the tumors from which the cells were obtained.

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No. 8 CHEMOSENSITIVITY OF BLADDER CANCER - Weisenthaf et af. 1495

mosensitivity of the tumor cells to the drug. Mishina et af.' found that mitomycin C was significantly more effective in low grade tumors or in small tumors than in high grade tumors or large tumors. These authors did not relate tumor grade to tumor size, but it would not be surprising if the higher grade tumors also tended to be larger. In our assay, where differences in drug penetration were not a factor, high grade tumors were equally or more sensitive to mitomycin C than were low grade tumors (Fig. 8). In contrast to the clinical findings of Mishina et af.' with mitomycin C, Koontz et aL2 found that tumor size and grade did not significantly influence tumor response to thiotepa. Although these observations obviously need to be confirmed, it is most interesting that thiotepa is absorbed while mitomycin C is said not to be absorbed.'** It is conceivable that mitomycin C is less permeable in transitional cell epithelium than is thiotepa, accounting for the above differences in response patterns based on tumor size.

We found that all of the tumor specimens were sensitive to a concentration of doxorubicin (100 ug/ml) which is easily achieved by intravesical administration.2' The 100% in vitro response rate in 19 different specimens exceeds that attained in clinical experience, where doxorubicin is a highly but not universally effective agent.'-" It might be theorized that this discrepancy is an artifact of the particular assay system used here, but dye exclusion assays have been felt to underestimate rather than overestimate cell kill compared to other types of in vitro assay^.^^.^^

An alternative explanation is that the concentrations of doxorubicin typically given (40-80 mg/20-40 ml) are really supramaximal for cell killing and that treatment failures are primarily due to inadequate tumor perfusion. The literature suggests that normal and neoplastic transitional cell epithelium may be much less permeable to doxorubicin than to thiotepa.'-' 1.19,20 Additionally, Sutherland et aLZ4 found reduced uptake of doxorubicin in the inner cells of small EMT-6 tumor spheroids, compared to the outer cells. The extent to which topical chemotherapy is affected by differences in drug permeability may limit the usefulness of in vitro chemosensitivity assays as sole predictors of tumor responsiveness, although such assays may prove useful in elim- inating from consideration drugs which lack intrinsic activity against an individual patient's tumor.

As a final point, clinical efforts to promote greater contact between drug and tumor tissue in vivo would seem worthwhile, especially for drugs with low epithelial permeability. The use of larger perfusion volumes to promote greater drug contact has been suggested,25 and

FIG. 9. Relationship between the histologic grade and the viability of the control cells ( I ) immediately after tumor dis- persal (open circles) and (2) after four days in liquid culture (solid circles).

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a small series did report a 100% complete remission rate in 1 1 patients with small, flat lesions after treatment with 80 mg of doxorubicin given in 100 ml of saline." Alternatively, surface active solvents such as dimethyl- sulfoxide26 could conceivably promote greater penetration (albeit with more systemic toxicity) of drugs with high intrinsic activity but poor penetration.

ADDENDUM

We have found that there is a steep dose-effect curve with thiotepa between 500 pglml and I500 pg/ml. This in vifro concentration range should be tested in future clinical correlation trials.

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The use of intravesical thiotepa in the management of non-invasive carcinoma of the bladder. J Urol 1981; 125:307-3 12.

3. Byar D, Blackard C. Comparisons of placebo, pyridoxine, and topical thiotepa in preventing recurrence of stage I bladder cancer. Urology 1977; 6:556-56 I. 4. Jacobi GH, Kurth KH, Klipper KF, Hohenfellner R. On the

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8. DeFuria MD, Bracken RB, Johnson DE ef al. Phase 1-11 study of mitomycin C topical therapy for low-grade, low stage transitional cell carcinomas of the bladder: An interim report. Cancer Treat Rep

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11. Niijima T. lntravesical therapy with Adriamycin and new trends. In: Diagnostics and Treatment of Superficial Urinary Bladder Tumors. Stockholm: Montedison, Lakemedel, AB 1979; 37-44.

12. Hagen K, Daly JJ, Kamali HM, Lin JC, Yu SC, Prout GR. New assays for cytotoxic agents in human bladder cancer. Surg Forum 1979; 30560-562.

13. Buick RN, Stanisic TH, Fry SE et a/. Development of an agar- methylcellulose clonogenic assay for cells in transitional cell carcinoma of the human bladder. Cancer Res 1979; 395051-5056.

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15. Shrivastav S, Paulson DF. i n vitro chemotherapy testing of transitional cell carcinoma. Invest Urol 1980; I7:395-400.

16. Day JW, Shrivastav S, Lin G, Bona RA, Paulson DF. In v i m chemotherapeutic testing of urologic tumors. J Urol I98 I ; 2 I5:490- 492.

17. Weisenthal LM, Marsden JA, Dill PL, Campbell CK. A novel dye exclusion method for testing in vitro chemosensitivity of human tumors. Cancer Res, In press.

18. Niell HB, Wood CA, Micke DD, Soloway MS. Time dependent inhibition of mouse bladder tumor colony survival by cisplatinum in

a human tumor stem cell assay. Proc Am Assoc Cuncer Res 1981; 22:2 19.

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20. Bessman JD, Johnson RK, Goldin A. Permeability of normal and cancerous rat bladder to antineoplastic agents. Urology 1975; 6: 187- 193.

21. Eksborg S, Nilsson SO, Edsmyr F. lntravesical instillation of adriamycin: A model for the standardization of chemotherapy. Eur Urol 1980; 6:2 18-220.

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24. Sutherland RM, Eddy HA, Bareham B, Reich K, Vanantwerp D. Resistance to Adriamycin in multicellar spheroids. Int J Radiur Oncol Biol Phys 1979; 5: 1225.

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26. Lerner JH, Beckloff GL. Effects of intravesical instillation of DMSO on the urinary bladder of the dog: Preliminary report. Ann NY Acad Sci 1967; 141 (1):254-260.

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In vitro chemosensitivity of human bladder cancer