Prevention of Human Bladder Tumor Cell Implantation in an in Vitro Assay

THE JOURNAL OJ UROLOGY

Copyright© 1987 by The Vlilliarns -& Vlilkins Co.

OF HUIV!AN BLADDER TUMOR CELL IMPLANTATION IN VITRO ASSAY DOV

* AVIVA T. HOROWITZ, ISRAEL VLODAVSKY, AMOS SHAPIRO AND

SHOSHANA BIRAN From the Department of Urology and the Department of Radiation and Clinical Oncology, Hewbrew University Hadassah Medical School, Jerusalem, Israel

ABSTRACT

The high recurrence rate of bladder tumors can be reduced by prevention of tumor cell reimplantation on denuded urothelium following transurethral resection. This can be achieved by intravesical chemotherapy immediately after the resection of the bladder tumors. We have demonstrated, in an in-vitro system, the process of human bladder tumor cell implantation on a naturally produced extracellular matrix (ECM) which simulates the exposed bladder basement membrane and submucosa. Using this model we examined the efficacy of various cytotoxic agents in preventing tumor cell adhesion to the ECM. Human bladder tumor cell implantation was prevented following exposure of the cells to distilled water, epodyl or mitocycin C, and significantly reduced following one hour incubation in cisplatinum and doxorubicin. The maximal effect for each of these cytotoxic agents was reached within 30 to 60 minutes of treatment. Mitomycin C reached maximal effect within 10 minutes. In contrast, thiotepa did not cause a significant reduction in cell adherence to ECM as compared to untreated control cells. Bladder cancer accounts for 4.4 % of the new cases of cancer uw,"uv.,,,u each year. 1 The incidence of tumor recurrence fol-

the initial therapy for superficial bladder tumors is relatively high and ranges from 40-85%. 2 Therefore, every effort should be made to reduce the incidence of local recurrence. Since implantation of tumor cells largely contributes to the high incidence of recurrence of superficial bladder tumors, 3 it would seem that intravesical chemotherapy initiated shortly after transurethral resection might reduce tumor recurrence by destroying floating tumor cells or preventing their adhesion to the basement membrane. Various agents such as heparin or EDT A have been shown to have an inhibitory effect on cellular adhesion, but have never been used for intravesical instillation postoperatively. Hinman 4 suggested instillation of 0.02% solution of phenol for one hour at the end of the to kill floating tumor cells, but did not any clinical or experimental proof of its efficacy, and method was not .,,.,om,,-,,,., for prophylactic use. Recent studies have provided evidence for the potential usefulness of one dose instillation of various ""1,nt.-.v,,, agents Zincke and his the nt1·av,es1,cal instillation of a mg. uwJceµct, or 50 mg. doxorubicin versus water, at the time of transurethral resection. three to four months after operation 71 % of the control had recurrences. In c-rn,tr'1d 30% to ""'m"'"-,-,"'"'"' of the patients treated and doxorubicin had recurrences. Bernard et al. 6 found that instillation of 90 mg. Thiotepa into the bladder for 30 minutes immediately after TUR, reduced the incidence of recurrent bladder tumors. Eleven patients (58 %) in the group of 19 treated with thiotepa had tumor recurrence, whereas 31 patients (97%) of the 32 patients in the control group had recurrence within two to five years. Because it is unlikely that a single intravesical dose of thiotepa or doxorubicin would alter the development of new tumors, the decrease in recurrence rate Accepted for publication October 15, 1986. *Requests for reprints: Dept. of Urology, Hadassah University Hospital, P.O.B. 12000, Jerusalem 91120, Israel. Supported by PHS grant CA 30289 from the National Cancer Institute, DHHS, and by the Joint Research Fund of the Hebrew University and Hadassah.

in the treated groups was, in all likelihood, due to prevention of tumor cell implantation onto the urothelium. No side effects were caused the early instillation of the cytotoxic agents. The possibility exists that other cytotoxic agents might be more effective in preventing tumor cell implantation. In a previous study 7 we presented an in-vitro model system in which bladder tumor cells were plated on a naturally produced extracellular matrix (ECM). Since this ECM resembles the bladder basement membrane in its macromolecular composition and morphological appearance, this model has been used to investigate the process of human bladder tumor cell implantation. We have found that tumor cell attachment and flattening on an exposed basement membrane occurs within one hour and reaches a maximum value within 24 hours. The present study was undertaken to use the previously described in-vitro cell system to evaluate the efficacy of various cytotoxic agents in the inhibition of tumor cell implantation, and to determine the time and duration of their effect. MATERIALS AND METHODS

Superficial low bladder tumors from 20 ~n·,rn·nr~ were obtained during transurethral resection, without using electrical diathermy. The tissue specimens were minced with a scalpel into small pieces, suspended in culture medium and divided into samples of 15 to 30 mg. tissue/2 ml. The cell suspension contained mostly small aggregates composed of 20 to 50 and single cells. Culture medium. For the in vitro implantation assay the cells were suspended and maintained in a serum free medium composed of a 1:1 (v:v) mixture of Ham's F12 and Dulbecco's modified Eagle's medium H-21 (Grand Island Biological Co., N.Y.) and supplements, as described by Biran et al. 8 Cytotoxic drugs. Thiotepa (Lederle Lab. Division, American Cyanamid Co., N.Y.), cisplatin (Abie, Israel), mitomycin C (Kyowa Hakko Kogyo Co., Japan) and doxorubicin (Farmitalia Carlo Erba Ltd., Italy) were obtained in a powder form and reconstituted in phosphate buffered saline, pH 7.2 (PBS) to a concentration of one mg./ml. Ethoglucid (epodyl, ICI, England) was diluted with PBS to a concentration of 1 % and stored at -70C until used.

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Effect of cytotoxic agents on the rate of cell implantation (adhesion assay). Samples of tumor cells were exposed to one of the following cytotoxic agents: thiotepa (1 mg./ml.), epodyl (1 %), cisplatin (1 mg./ml.), mitomycin C (1 mg./ml.), doxorubicin (1 mg./ml.) or distilled water, or one of the control fluids: culture medium, PBS, saline and 1.1 % glycin in water. Each agent was examined in 10 different tumor samples. After one hour of incubation at 37C the cells were washed three times in PBS, suspended in culture medium and plated on ECM-coated 35 mm. culture dishes, 15 to 30 mg. of tissue per dish. The dishes were then incubated for 24 hours in a 10% CO 2 humidifed incubator at 37C. The viability of cells in each sample was determined by the trypan blue exclusion test, prior and immediately after exposure to the various agents, and at the end of 24 hours incubation in culture medium on ECM. The fraction of living cells was determined for each sample and expressed as a percentage of the total cell count. Attachment of tumor cells to the ECM and subsequent flattening were visualized by phase microscopy. To determine the extent of cell implantation, the culture dishes were rinsed three times with PBS, the unattached cells were washed away by a gentle pipetting and the firmly attached cells were then dissociated with STV (0.05% trypsin, 0.02 versene solution in PBS) and counted. Four dishes were scored for each determination, and the variations between different determinations did not exceed ±15% of the mean. The maximal effect of distilled water and the various cytotoxic agents on the implantation of cells was evaluated after various treatment periods. For this purpose, samples of tumor cells were incubated with one of these agents for five, 10, 20, 30 or 60 minutes, suspended in culture medium and plated on ECM. 24 hours later the firmly attached cells were counted as described above.

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RESULTS

Cells from each of 20 specimens of superficial, low grade human bladder tumors were tested in-vitro to evaluate different means to minimize tumor cell implantation. Cell viability was determined immediately after the one hour exposure to the various control media and cytotoxic agents, and 24 hours afterwards. The extent of tumor cell implantation was determined 24 hours after seeding on ECM. The median cell viability after tissue processing was 90%. Exposure (1 h, 37C) of the cells to various control media, PBS, culture medium, saline or 1.1 % glycin in water, had no significant effect on cell viability. 24 hours later 83% (range 80 to 86%) of the cells were still viable as determined by the trypan blue exclusion test. The viability of cells immediately after incubation with each of the cytotoxic drugs was 75 to 86%. While there was no significant cell death immediately after the one hour incubation with any of the cytotoxic drugs, a marked decrease in cell viability was noted 24 hours following the treatment with either epodyl (from 86% to 5%), cisplatin (from 80% to 29%) doxorubicin (from 85% to 35%) or mitomycin C (from 75% to 10%). In contrast, there was no significant difference between cells suspended in control media or treated with thiotepa and tested for cell viability 24 hours afterwards (83% versus 76%, respectively). 77% of the tumor cells which had been incubated in distilled water were lysed within one hour, and almost all the remaining viable cells failed to adhere to the ECM. The few cells which did adhere to the ECM within 24 hours appeared swollen and contained large vacuoles (fig. 2C). Both control and thiotepa-treated cells exhibited a similar degree of intensive cell attachment and flattening as visualized by phase microscopy (figs. 2A, 2B). In contrast, no attachment was observed with cells that were first treated with epodyl or cisplatin.

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The number of non-treated cells from each tumor sample which attached to ECM was variable from 1.2 to 18.6 X 104 cells per one mg. of tumor tissue. The quantitative effect of cytotoxic drugs and distilled water on tumor cell implantation as compared to culture medium or PBS is shown in fig. 1. The adhesion rate of non-treated cells in culture medium, from each tumor sample, was considered 100% attachment. The rate of cell adherence after incubation in PBS was similar to culture medium. Thiotepa did not significantly inhibit tumor cell implantation during the first 24 hours after treatment (80.5 ± 11.7% as compared to 88.8 ± 15.2% after incubation in PBS). Implantation was significantly reduced by distilled water (O. 72 ± 0.36% attachment), doxorubicin (5.9 ± 1.2%) and by cisplatin (1.8 ± 0.7%). Tumor cell adherence was almost completely prevented following exposure of the cells to epodyl (0.12 ± 0.1 % attachment) or mitomycin C (0.36 ± 0.2%). Mitomycin C, epodyl, cisplatin, doxorubicin and water were much more effec-

Fm. 2. 1-1m,mis;1c,,. appearance of tumor cell attachment to ECM as visualized by phase microscopy X200. A, 24 hours after incubation in culture medium. B, hours after one hour treatment with 1 mg./ml. thiotepa. C, 24 hours after one hour treatment with 1 mg./ml. mitomycin C. D, 24 hours after one hour treatment with distilied water.

tive than in the prevention of tumor ceH implantation in vitro (p While there was no Qrc,n,,n,'"' significant or •,;vater~ difference between ~,.,,,,,.w,uc, doxorubicin vvas -·,-,,w.c,~-.. ··J The effect of of the ,~,d-r.1·,rw, agents on their ECM is shown in 3. In most cases a maximal effect was reached after 30 to 60 minutes. A more effect was noted after incubation with 14% after 10 adherence was reduced to 1.8% as 43% in doxorubminutes incubation in water, 27.5% in icin and 69% in ~.,.,_..-····--'" mSCUSSWN

Previous studies have demonstrated the tic intravesical chemotherapy to reduce the recurrence rate of bladder tumors following definitive treatment with TUR. In most of these studies intravesical chemotherapy was started more than 24 hours after the TUR, and used in multiple doses. Therefore, they were not aimed specifically against tumor reimplantation. Only thiotepa and doxorubicin have been used, in a very few clinical trials, for prevention of tumor implantation by a single dose immediately after surgery. Although there may be differences between the results of an in-vitro screening test of anti cancer agents and the results of their clinical application, the assay used in our study seems to

mimic the sequence of events during transurethral resection of a bladder tumor followed a single intravesical instillation of a to to prevent implantation of this test resides of viable uvu"'""The in the similar structural and of the sub-urothelial basement membrane ECM Our indicated that attempts to prevent turnor initiated in the first hour after transurethral resection of the bladder tumors. This can be achieved rJoo,-,,,m,m the Viable HU
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which have been used for intravesical chemotherapy in many previous studies. Since prevention of tumor cell implantation should occur with the use of only a single intravesical instillation, we did not examine the dose response effect of each drug. We used each cytotoxic agent in the concentration in which it had been used in most previous clinical studies. While several clinical studies have indicated a significant reduction in the recurrence rate of superficial tumors following prophylactic multiple intravesical instillations of thiotepa, 3 in other studies the benefit of thiotepa seemed to be controversial1 2 • 13 or even absent. 14- 16 Our results show that one mg./ml. thiotepa has no significant action against tumor cell implantation on to a naturally produced basement membrane. These results support the conclusion of a recent prospective clinical trial1 7 in which newly diagnosed superficial bladder tumors were given thiotepa instillation at the time of TUR. There was no difference between the recurrence rates in these and in a control group. On the other hand, exposure of tumor cells to mitomycin C or epodyl prevented the attachment of the tumor cells and caused their death within 24 hours. Similar results were obtained using cisplatinum, whereas doxorubicin was a little less effective. Although intravesical prophylaxis with cisplatinum was found to be unsatisfactory by Blumenreich et al., 14 others found its prophylactic effect to be equal to that of thiotepa and doxorubicin. 19 In both studies multiple intravesical instillations were started 15 to 30 days after surgery, and therefore could not be expected to prevent tumor reimplantation. In conclusion, we believe that our present study indicates that immediate post-operative intravesical instillation of distilled water for more than 30 minutes may be sufficient for the prevention of tumor implantation. Since previous clinical studies did not find any ill effects following intravesical instillation of chemotherapeutic agents within the first post-operative hour, 5 • 6 one dose of a cytotoxic agent is the best alternative for the prevention of tumor implantation. Mitomycin C, one mg./ ml., was found to have the best and more rapid effect. Epodyl 1% and cisplatinum were also found to be highly effective, doxorubicin less so, and thiotepa was found to be without effect.

Incubation p~riod (min) REFERENCES

FIG. 3. Effect of various preincubation periods of human bladder tumor cells with cytotoxic agents, on attachment of tumor cells to ECM 24 hours later.

desirable for intravesical treatment in the immediate postoperative period. On the other hand, intravesical use of cytotoxic drugs has been shown to be free of any serious side effects. 10 · 11 Although the antineoplastic effect of most cytotoxic agents which have been used intravesically is related to their preferential toxicity to the nucleic acids, we have found that some agents-mitomycin C, epodyl, cisplatinum and doxorubicinhave a remarkable inhibitory effect on the adhesion of tumor cells to the ECM. The mechanism of their action is not clear. It is most probably not due to inhibition of DNA synthesis alone, but may be due to an inhibitory effect on the synthesis of the adhesive proteins (laminin, fibronectin and glucosaminoglycans). Although there was some correlation between the inhibition of cell attachment and the rate of cellular death within 24 hours, the antiadhesion effect appeared much earlier and was more complete than the cell destruction. Therefore, while the cytotoxic agent cannot ensure complete destruction of all the floating tumor cells in the bladder, it can probably prevent implantation of nearly all the tumor cells. Since anchoragedependent cells such as bladder epithelial cells are not able to function and survive in suspension, inhibition of their attachment to the basement membrane may result in the subsequent death of the floating tumor cells. We have compared the efficacy of various cytotoxic drugs

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