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Chemotherapy of murine bladder cancer: In vitro and in vivo
Chemotherapy of Murine Bladder Cancer:
In Vitro and In Vivo* Harvey B. Niell, M.D. Mark S. Soloway, M.D.l Robert B. Matheny Anthony Blatnik From the Departments of Medicine and Urology, University of Tennessee Center for the Health Sciences and Research Department of the Veterans Administration Hospital, Memphis, Tennessee
Animal tumor models have been the primary means for screening investigational compounds for their antineoplastic activity. Agents with acceptable toxicity, Ioundto be effective in inhibiting tumor growth in these animal models, have then been incorporated into human trials. 1 Until recently, there has been a paucity of data on the chemotherapy of human bladder cancer. In the early 1970's, the N-[4(5-nitro-2-furyl)-2-thiazolyl]formamide (FANFT)induced mouse bladder tumor (MBT) system was developed and has served as an animal model for human bladder cancer." Cis-diamminedichloroplatinum (DDP) was found to be effective in this model, and subsequent clinical trials, partially based on this work, identified DDP to be the most effective single agent in bladder cancer.v' The results from clinical trials support the continued use of this animal model to screen prospective agents for their anti-tumor efficacy. Because animal models are both expensive and time-consuming, it is appropriate to evaluate simple methods for pre-screening agents that may be effective in transitional cell carcinoma of the bladder. An in vitro culture system has been developed that will support the clonal growth of a wide variety of human tumors." This tumor colony assay (TCA) has been used for anticancer drug testing, and several laboratories have demonstrated that this technique will predict clinical response or non-response to antineoplastic agents in man."
The TCA has been shown to support the growth of human bladder cancer.P CIanogenic growth has been produced using either surgical specimens obtained at cystoscopy or from bladder washings. Due to the low growth rates of bladder cancer reported in most laboratoriesv'" and due to the small size of these lesions at cystoscopy, the Widespread use of surgical specimens for ill vitro drug screening may prove to be impractical. \Ve have recently described the growth characteristics of four MBT cell lines in a TCA and demonstrated the feasibility of utilizing them for anticancer drug testlng.!' \Ve are presently evaluating the use of these MBT cell lines in the TCA to prescreen anticancer agents that can then be tested in the murine model. If the TCA could predict antineoplastic drug response in the animal model, it would be plausible to rapidly prescreen large numbers of investigational anticancer agents using these tumor cell lines. The purpose of this study is to determine whether the TCA is predictive of chemotherapeutic drug response in the FANFT-induced MBT murine model using thirteen standard and investigational compounds. Materials and Methods
"This work was supported by USPHS Grant CA 18643 from the National Cancer Institute through the National Bladder Cancer Project and the Medical Research Services of the Veterans Administration. [Presenter,
MBT Cell Lines. Four MBT cell lines (MBT-2, MBT-8, MBT-683, and MBT-409) were used for ill vivo and in vitro drug testing. Each of these tumors originated as an invasive bladder cancer in a female C3H/He mouse after ingestion of FANFT for an average of eleven months. These tumor lines have been maintained by serial transplantation in syngeneic mice. MBT-2,
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MBT-8, and MBT-683 are transitional cell carcinomas, while MBT-409 is a squamous carcinoma.
Murine Drug Testing. The methods for murine drug testing have been reported.v'" Briefly, 1 x 104 viable MBT cells were injected into the hind limbs of C3H/He female mice. Mice were randomized into control (ten to fifteen mice) and treatment (ten to twelve mice) groups. Toxicity studies previously determined the LD 10 for each drug. Drugs were given by intraperitoneal injection on days 7,14, and 21. Table 1 lists the in vivo drug dosages. Tumors were usually palpable twelve days after tumor inoculation, and their mean diameter was calculated biweekly. The mean tumor diameter on days 25-27 was selected for comparing control and treated groups, and statistical differences were calculated by Student's t. test. The mean survival time (MST) was determined for each group, and the percentage increase life span (ILS) calculated. A drug was considered to be effective if there was a significant decrease in the mean tumor diameter (p < 0.01) and if the ILS was increased by 10%. Although a 10% increase in the ILS is a modest change in life span, previous drug testing has suggested that this criterion is liberal enough for the murine model to identify most of the active agents used in bladder cancer. TCA Culture Methods. Cells were cultured using a modification of Hamburger and Salmon." The underlayer consisted of one ml of McCoy's media in 0.5 % agar plated on 35 mm plastic petri dishes. Tumor cells were suspended in 0.3% agar with enriched CMRL 1066 medium (GIBCO, Grand Island, New York) and 15% horse serum (Flow Laboratories, Newberry Park, California). The growth characteristics of these MBT cell lines in a TCA have previously been described.'! At the end of an in vivo drug study, tumor cells were removed from the control mice for in vitro drug testing. Tumor cells were drug tested in the TCA either immediately upon removal from the mouse or after one to three passages in monolayer culture (one passage 5-7 days). The number of tumor cells plated was 30-50,000/plate when removed from shortterm culture and 500,000/plate when cultured directly from the animal. Control and drug-treated plates were cultured in triplicate. Cultures were incubated at 37° in 5% CO 2 and 100% humidified atmosphere. Cultures were examined with an Olympus CK inverted microscope at 40 x and 100 x . Final colony counts were made ten to fourteen days after plating. Cell aggregates were determined to be colonies by finding an aggregate esti-
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mated to contain thirty cells and only counting aggregates of the same size or larger. The level of colony inhibition optimal for predicting drug sensitivity has previously been determined" to represent 90 % inhibition at the highest in vitro drug level compared to control plates.
Preparation of Cell Suspensions. Tumor tissue was aseptically excised from the mouse, minced with scissors, and resuspended in CMRL 1066 medium with 15 % horse serum. Cell clumps were further separated with scalpels and passed through 21-25g needles. If clumps were still present, the cell suspension was passed through sterile gauze. Viable nucleated cells were counted in a hemocytometer, and the viability as determined by trypan blue exclusion was between 70-90 %. Tumor suspensions produced by this method were either immediately drug tested or placed in short-term monolayer culture for subsequent study. Tumor cells maintained in short-term monolayer culture were grown in 100 mm plastic petri dishes in RPMI 1640 (GIBCO, Grand Island, New York); 0.2% sodium bicarbonate; 20% newborn calf serum (GIBCO, Grand Island, New York); 0.2 g/ml Fungizone (GIBCO, Grand Island, New York); and 50 g/rnl Garamycin (Schering, Kenilworth, New Jersey). Confluent cells were subcultured at 1:10 dilutions by ten-minute incubation with 0.175% trypsin in calcium and magnesium-free phosphatebuffered saline. Tumor cells were harvested from short-term culture by incubation for ten minutes at 37° in 0.25% trypsin in Hanks' Balanced Salt Solution. The cells were centrifuged at 1100 rpm for ten minutes, the supernatant removed, and the cells resuspended in media. The viable nucleated cell counts were usually greater than 90 % . Drug Preparation. The chemotherapeutic drugs used in these experiments were Anguidine (ANG)-National Cancer Institute; Doxorubicin hydrochloride (ADR)-Adria Laboratories, Inc., Dublin, Ohio; Bleomycin sulfate (BLEO)-Bristol Laboratories, Syracuse, New York; Chlorozotocin (CHLORO)-National Cancer Institute; Cis-diamminedichloroplatinum (DDP)Bristol Laboratories, Syracuse, New York; 5Fluorouracil (5FU)-Roche Laboratories, Nutley, New Jersey; Maytansine (MAY)-National Cancer Institute; Methotrexate (MTX)-Lederle Laboratories, Wayne, New York; Mitomycin C (MITO)Bristol Laboratories, Syracuse, New York; Epipodophyllotoxin VP-16 (VP-16)-National Cancer
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TABLE
1.
Anticancer drugs and dosages used in vivo and in vitro
In Vivo Dosage (mg/Kg)
Anticancer Drug Bleomycin Doxorubicin hydrochloride Methotrexate Epipodophyllotoxin VP-16 Cis-diamrninedichloroplatinum MitomycinC 5-Fluorouracil Maytansine Chlorozotocin Anguidine Pala Vinblastine AZQ
In Vitro Dose Range for I-Hr Drug Incubation (ltg/ml)
60 5
0.001-0.90 0.01-0.38
32 50
0.04-4.00 0.79-7.90
6
0.09-0.38
3 75 0.25 10 10 300 3 8
0.01-0.11 0.15-7.28 0.001-0.01 0.07-0.69 0.001-0.05 1.2-12.1 0.002-0.02 0.001-0.1
Institute; Vinblastine (VLB)-Eli Lilly and Co., Ind ianapolis, Indiana; N-Phosphonoacetyl-LAspartate Disodium (PALA)-National Cancer Institute; and Aziridinylbenzoquinone (AZQ)National Cancer Institute. All drugs were reconstituted in the specified diluent and then diluted to the desired concentration in 0.9 % NaCI, placed in 1.5 ml volume in 6 ml plastic Falcon centrifuge tubes and stored at - 80° C. Prior to use, drugs were thawed in a 37° C waterbath. Drug testing was done at the in oitro concentrations listed in Table 1. Due to the lack of substantial pharmacokinetic drug data in mice, we used the in vitro drug dosages recommended by Alberts et al!' for testing human tumors. In general, the one hour drug exposure concentration is calculated as 10 % of the average CXT (ltg X hr/ml) for the high in vitro drug level and one log lower for the low in vitro drug level. Cells were incubated in the presence of drug for one hour, washed in CMRL x 3, resuspended in media, and plated in enriched CMRL in 0.3% agar.
TABLE.
2.
Analysis oj Data. In vitro and in vivo data were correlated in the following manner: tumor responses to drug were listed as either sensitive (S) or resistant (R). In tabulating the data, in vitro responses were listed first, i.e., in vitrolin vivo. The true positive predictive rate of the TCA was calculated and expressed as a % using the formula [S/S(S/S + SIR)] X 100. The true negative predictive rate of the TCA was calculated and expressed as a % using the formula [R/R(RIR + RIS)] X 100.
Results The activity of thirteen anticancer drugs was evaluated using four MBT cell lines. We found that when tumor cells were removed from monolayer culture for drug testing, ill vitro growth occurs in greater than 90 % of assays. Clonal growth with tumor cells removed from the animal occurred in approximately 50 % of assays attempted. Forty-nine drug studies were evaluable for comparison. The results of drug testing using the TCA were compared to the drug responses in the animal. Table 2 defines the ability of the TCA in predicting drug response in the murine system. The true positive predictive rate of the TCA was 50 %, while the true negative predictive rate was 70 % . A comparison of in vitro vs in vivo drug responses was tabulated by the individual drug being evaluated (Table 3). The TCA predicted drug response most accurately using MTX, ANG, MAY, and PALA. It should be noted, however, that these were negative correlations and could be related to an inability of either assay (in vivo andlor in vitro) to predict activity with these agents. The TCA had a low level of predictability when evaluating VP-16 and DDP. In all four cell lines, VP-16 was predicted to be active in vitro, and a significant in vivo tumor response was not seen in any of the MBT cell lines. DDP was predicted to be inactive in vitro with three out of four cell lines, but the drug significantly inhibited the growth of all of the tumor lines ill vivo. Table 4 lists the in vitro vs in vivo responses according to the MBT tumor being tested. MBT-2 was the most resistant cell line in both assays. The pre-
Predictability of TeA in murine model
TCA Sensitive TCA Resistant Level of Murine Murine Murine Murine Tumor Colony Sensitive Resistant Sensitive Resistant Inhibition (SIS) (SIR) TPPR * (RIS) (R/R) TNPRt 90%
6
6
50%
11
70%
26
*TPPR = True positive predictive rate. rT1':PR = True negative predictive rate.
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TABLE
3.
Predi ctability of TCA in murine model by individual drug TCA Sensitive
Drug
TCA Resistant
Murine
Murine
Murine
Murine
Sensitive (SIS)
Resistant (SIR)
Sensitive (RIS)
Resistant
Correlation
(RIR)
( %)
2
50 100 50
ADR
2
~ITX
BLEO VP-16 DDP CHLORO ANG
4 1
2
I
o
4
I
3 1
I 2 1
~UTO
5FU MAY PALA
1 2
3 3 1 I
1 1
3 2 2
25 75 100 75 50 100 100 66 66
4
AZQ
VLB dictability of the TCA was roughly equivalent for the other tumors. Discussion The present study demonstrates that when the TCA assay indicates a drug is active against one of these murine bladder tumors, it will correlate 50 % of the time with the in vivo response. If the TCA indicates the drug does not produce significant cytotoxicity, itwill correlate with the concurrent in vivo response 70 % of the time. The positive predictive rate of this assay in the murine mod el is similar to that previously reported in human drug testing," while the negative predictive rate is somewhat lower. To date, the majority of clinical comparisons between the TCA and drug activity in the host have been retrospective. Until prospective clinical trials have been completed, the true predictability of the TCA in human tumors will be in question. DDP and mitomycin C significantly inhibited growth of three of the four tumor lines in the animal. The activity of DDP in murine bladder cancer parallels its effectiveness in man since clinical 'st udies indicate a 20-50 % objective response rate.":" MiTABLE
MTB Cell Line MBT-2 ~1BT-8
MBT-683 MBT-409
tornycin C has established activity in superficial bladder cancer when delivered by intravesical instillation. However, I am not aware of any large trial in advanced disease. Our animal data would support consideration of such a study. The lack of dramatic efficacy for the other eleven compounds underlines the necessity for the development and screening of additional drugs in this tumor. VP-16 and ADR were predicted to be active by the TCA and were found to be inactive in vivo. The disparity between these in vitro and in vivo results is not readily apparent. VP-16 has been active in previous experiments with the MBT-2 tumor. Since both drugs are large molecules, they may not be completely absorbed from the peritoneum, and this might result in lower blood levels than if delivered by the intravenous route. Differences in drug metabolism and cellular uptake in vitro compared to in vivo and inappropriate in vivo or in vitro drug dosages may be responsible for the lack of greater concordance. DDP was very effective in the animal but ineffective in vitro. \Ve have previously shown that its activity is enhanced by increasing the time of drug ex-
4. Predictability of TCA ill murine model by MBT cell line TCA Sensitive Murine Murine Sensitive Resistant (SIS) (SIR)
o
1
2 2
2 2
2
I
TPPR' 0 50 % 50 % 66 %
TCA Resistant Murine Murine Sensitive Resistant (RIS) (R/R) 2 3 3
3
10 6 6
4
TNPHl 83 % 66 % 66 % 57 %
°TPPR = Truc positive predictive rate. ITlIiPR = True negative predictive rate.
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posure ill vitro. 16 It is unclear whether this increase in exposure time duplicates the conditions ill vivo. This data suggests, however, that increasing the duration of DDP infusion in the clinic may improve drug efficacy. The lack of efficacy of MTX ill vitro may be due to the rescue of cells by thymidine present in the culture media, thus giving a false negative result. Since only a small number of anticancer agents with activity in bladder cancer is available, it is important to continue to search for new and more effective drugs. The TCA could serve as an invaluable tool for the rapid screening of investigational compounds. Drugs found to be active in the TCA could be tested in the murine model and, if promising, incorporated into clinical trials.
References 1. Leiter, J., Abbott, B.J. and Schepartz, S.A. : Screening data from the Cancer Chemotherapy National Service Center Screening Laboratories XXVIII. Cancer Res., 25: 1626-1769, 1965. 2. Soloway, :-'I.S., deKernion, J.B., Rose, D. and Persky, L.: Effect of chemotherapeutic agents on bladder cancer: A new animal model. Surg. Forum, 13: 542-5-14, 1973. 3. Soloway, :-'I.S. and Murphy, W.:-'I.: Experimental chcrnotherapy of bladder cancer-systemic and intravesical. Scm. Oncol., 6: 166·183, 1979. 4. Yagoda, A., \\'atson, R.C ., Conzalez-Vitale, J.C., Grabstald, H. and Whitmore, WF.: Cis-dichlorodiammine platinum (II) in advanced bladder cancer. Cancer Treat. Rep., 60: 917923, 1976.
5. IIamburger, A. and Salmon, S.E .: Primary bioassay of human myeloma stem cells. J. Clin. Invest., 60: 846-854, 1877. 6. Salmon, S.E . and \'onIloH, D.O.: III dim evaluation of anticancer drugs with the human tumor stem cell assay. Scm. Oncol., s. 377·385, 19SI. 7. Buick, R.:>:., Stanisic, T.Il. , Fry, S,E ., Salmon, S.E ., Trent , J.:-'1. and Krusovich, P.: Development of an agar-methyl eellulose elonogenic assay for cells in transitional cell carcinoma of the human bladder. Cancer Hes., 39: 5050-5056, 1979. 8. Stanisic, TJI. and Buick, RII.: An in dim elonal assay for bladder cancer: Clinical correlation with the status of the urothelium in 33 patients. J. Urol., 124: 30·33, 1980. 9. \'onlloH, D.O., Casper, J., Bradley, E., Sandback, J., Jones, B. and :-'Iakuch, R. : Association between human tumor colony-forming assay results and rcsponsc of an individual patient's tumor to chemotherapy. Amer, J . Med ., 70: 10271032, 1981. 10. Nicll, Il.B. , Soloway, :-'I.S. and Nissenkom, 1.: The clonogenic growth of cells derived from bladder barbotage in pa · ticnts with transitional cell carcinoma of the bladder: A preliminary report. J . of Urol., 127: 668-670, 1082 . II. Nicll, II.B .. Mickey, D.O. , Soloway, :-'1.5. and Wood, C.A.: Growth characteristics of lI:.[4-(5-nitro-2-furyl)-2thiazolyljformamide (FAi':FT)-induccd mouse bladder tumor lines in human tumor stcm cell assay. Cancer, 49: 323· 330, 1982. 12. Soloway, :-'I.S.: Intravesical and systemic chemotherapy of murine bladder cancer. Cancer Bes., 37: 2918·2929, 1977. 13. :\iell, H.B ., Soloway. :-'I.S., \\ood , C .A., :-'lcCallum, L.\\~ and \Yebster, K.: The use of a tumor colony assay in predicting chemotherapeutic drug response in murine bladder cancer. Cancer, in press. 14. Alberts, D.S . and Chem. II·SG.: Tabular summary of pharrnacokinetic parameters relevant to ill dtro drug assay. Prog . Clin. BioI. Iles., 48: 223-245, 1980. . 15. Soloway, :-'I.S., Einstein, A., Corder, :-'I.P., Bonney, \\~, Prout, G.R., Jr. and Coombs, J.: A comparison of cisplatin and a combination of cisplatin and cyclophosphamide in advanced urothelial cancer-A National Bladder Cancer Collaborative Group A study. Cancer, in press. 16. "iell, II .B., Wood, C.A., Mickey, 0.0: and Soloway, :-'I.S.: Time and concentration dependent inhibition of the clono genic growth of lI:-[ 4.(5-nitro-2-furyl)-2-thiazolyl]forrnamide-Inducedmurinc bladder tumor cell lines by cisdiarnrnincdichloroplatinurn (II) . Cancer Res., 42: 807-811, 1982.
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Department of Urology University of Tennessee 956 Court Avenue-Room 2H23 Memphis, Tennessee 38163 (DR. SOLOWAY)
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In Vitro and In Vivo* Harvey B. Niell, M.D. Mark S. Soloway, M.D.l Robert B. Matheny Anthony Blatnik From the Departments of Medicine and Urology, University of Tennessee Center for the Health Sciences and Research Department of the Veterans Administration Hospital, Memphis, Tennessee
Animal tumor models have been the primary means for screening investigational compounds for their antineoplastic activity. Agents with acceptable toxicity, Ioundto be effective in inhibiting tumor growth in these animal models, have then been incorporated into human trials. 1 Until recently, there has been a paucity of data on the chemotherapy of human bladder cancer. In the early 1970's, the N-[4(5-nitro-2-furyl)-2-thiazolyl]formamide (FANFT)induced mouse bladder tumor (MBT) system was developed and has served as an animal model for human bladder cancer." Cis-diamminedichloroplatinum (DDP) was found to be effective in this model, and subsequent clinical trials, partially based on this work, identified DDP to be the most effective single agent in bladder cancer.v' The results from clinical trials support the continued use of this animal model to screen prospective agents for their anti-tumor efficacy. Because animal models are both expensive and time-consuming, it is appropriate to evaluate simple methods for pre-screening agents that may be effective in transitional cell carcinoma of the bladder. An in vitro culture system has been developed that will support the clonal growth of a wide variety of human tumors." This tumor colony assay (TCA) has been used for anticancer drug testing, and several laboratories have demonstrated that this technique will predict clinical response or non-response to antineoplastic agents in man."
The TCA has been shown to support the growth of human bladder cancer.P CIanogenic growth has been produced using either surgical specimens obtained at cystoscopy or from bladder washings. Due to the low growth rates of bladder cancer reported in most laboratoriesv'" and due to the small size of these lesions at cystoscopy, the Widespread use of surgical specimens for ill vitro drug screening may prove to be impractical. \Ve have recently described the growth characteristics of four MBT cell lines in a TCA and demonstrated the feasibility of utilizing them for anticancer drug testlng.!' \Ve are presently evaluating the use of these MBT cell lines in the TCA to prescreen anticancer agents that can then be tested in the murine model. If the TCA could predict antineoplastic drug response in the animal model, it would be plausible to rapidly prescreen large numbers of investigational anticancer agents using these tumor cell lines. The purpose of this study is to determine whether the TCA is predictive of chemotherapeutic drug response in the FANFT-induced MBT murine model using thirteen standard and investigational compounds. Materials and Methods
"This work was supported by USPHS Grant CA 18643 from the National Cancer Institute through the National Bladder Cancer Project and the Medical Research Services of the Veterans Administration. [Presenter,
MBT Cell Lines. Four MBT cell lines (MBT-2, MBT-8, MBT-683, and MBT-409) were used for ill vivo and in vitro drug testing. Each of these tumors originated as an invasive bladder cancer in a female C3H/He mouse after ingestion of FANFT for an average of eleven months. These tumor lines have been maintained by serial transplantation in syngeneic mice. MBT-2,
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MBT-8, and MBT-683 are transitional cell carcinomas, while MBT-409 is a squamous carcinoma.
Murine Drug Testing. The methods for murine drug testing have been reported.v'" Briefly, 1 x 104 viable MBT cells were injected into the hind limbs of C3H/He female mice. Mice were randomized into control (ten to fifteen mice) and treatment (ten to twelve mice) groups. Toxicity studies previously determined the LD 10 for each drug. Drugs were given by intraperitoneal injection on days 7,14, and 21. Table 1 lists the in vivo drug dosages. Tumors were usually palpable twelve days after tumor inoculation, and their mean diameter was calculated biweekly. The mean tumor diameter on days 25-27 was selected for comparing control and treated groups, and statistical differences were calculated by Student's t. test. The mean survival time (MST) was determined for each group, and the percentage increase life span (ILS) calculated. A drug was considered to be effective if there was a significant decrease in the mean tumor diameter (p < 0.01) and if the ILS was increased by 10%. Although a 10% increase in the ILS is a modest change in life span, previous drug testing has suggested that this criterion is liberal enough for the murine model to identify most of the active agents used in bladder cancer. TCA Culture Methods. Cells were cultured using a modification of Hamburger and Salmon." The underlayer consisted of one ml of McCoy's media in 0.5 % agar plated on 35 mm plastic petri dishes. Tumor cells were suspended in 0.3% agar with enriched CMRL 1066 medium (GIBCO, Grand Island, New York) and 15% horse serum (Flow Laboratories, Newberry Park, California). The growth characteristics of these MBT cell lines in a TCA have previously been described.'! At the end of an in vivo drug study, tumor cells were removed from the control mice for in vitro drug testing. Tumor cells were drug tested in the TCA either immediately upon removal from the mouse or after one to three passages in monolayer culture (one passage 5-7 days). The number of tumor cells plated was 30-50,000/plate when removed from shortterm culture and 500,000/plate when cultured directly from the animal. Control and drug-treated plates were cultured in triplicate. Cultures were incubated at 37° in 5% CO 2 and 100% humidified atmosphere. Cultures were examined with an Olympus CK inverted microscope at 40 x and 100 x . Final colony counts were made ten to fourteen days after plating. Cell aggregates were determined to be colonies by finding an aggregate esti-
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mated to contain thirty cells and only counting aggregates of the same size or larger. The level of colony inhibition optimal for predicting drug sensitivity has previously been determined" to represent 90 % inhibition at the highest in vitro drug level compared to control plates.
Preparation of Cell Suspensions. Tumor tissue was aseptically excised from the mouse, minced with scissors, and resuspended in CMRL 1066 medium with 15 % horse serum. Cell clumps were further separated with scalpels and passed through 21-25g needles. If clumps were still present, the cell suspension was passed through sterile gauze. Viable nucleated cells were counted in a hemocytometer, and the viability as determined by trypan blue exclusion was between 70-90 %. Tumor suspensions produced by this method were either immediately drug tested or placed in short-term monolayer culture for subsequent study. Tumor cells maintained in short-term monolayer culture were grown in 100 mm plastic petri dishes in RPMI 1640 (GIBCO, Grand Island, New York); 0.2% sodium bicarbonate; 20% newborn calf serum (GIBCO, Grand Island, New York); 0.2 g/ml Fungizone (GIBCO, Grand Island, New York); and 50 g/rnl Garamycin (Schering, Kenilworth, New Jersey). Confluent cells were subcultured at 1:10 dilutions by ten-minute incubation with 0.175% trypsin in calcium and magnesium-free phosphatebuffered saline. Tumor cells were harvested from short-term culture by incubation for ten minutes at 37° in 0.25% trypsin in Hanks' Balanced Salt Solution. The cells were centrifuged at 1100 rpm for ten minutes, the supernatant removed, and the cells resuspended in media. The viable nucleated cell counts were usually greater than 90 % . Drug Preparation. The chemotherapeutic drugs used in these experiments were Anguidine (ANG)-National Cancer Institute; Doxorubicin hydrochloride (ADR)-Adria Laboratories, Inc., Dublin, Ohio; Bleomycin sulfate (BLEO)-Bristol Laboratories, Syracuse, New York; Chlorozotocin (CHLORO)-National Cancer Institute; Cis-diamminedichloroplatinum (DDP)Bristol Laboratories, Syracuse, New York; 5Fluorouracil (5FU)-Roche Laboratories, Nutley, New Jersey; Maytansine (MAY)-National Cancer Institute; Methotrexate (MTX)-Lederle Laboratories, Wayne, New York; Mitomycin C (MITO)Bristol Laboratories, Syracuse, New York; Epipodophyllotoxin VP-16 (VP-16)-National Cancer
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TABLE
1.
Anticancer drugs and dosages used in vivo and in vitro
In Vivo Dosage (mg/Kg)
Anticancer Drug Bleomycin Doxorubicin hydrochloride Methotrexate Epipodophyllotoxin VP-16 Cis-diamrninedichloroplatinum MitomycinC 5-Fluorouracil Maytansine Chlorozotocin Anguidine Pala Vinblastine AZQ
In Vitro Dose Range for I-Hr Drug Incubation (ltg/ml)
60 5
0.001-0.90 0.01-0.38
32 50
0.04-4.00 0.79-7.90
6
0.09-0.38
3 75 0.25 10 10 300 3 8
0.01-0.11 0.15-7.28 0.001-0.01 0.07-0.69 0.001-0.05 1.2-12.1 0.002-0.02 0.001-0.1
Institute; Vinblastine (VLB)-Eli Lilly and Co., Ind ianapolis, Indiana; N-Phosphonoacetyl-LAspartate Disodium (PALA)-National Cancer Institute; and Aziridinylbenzoquinone (AZQ)National Cancer Institute. All drugs were reconstituted in the specified diluent and then diluted to the desired concentration in 0.9 % NaCI, placed in 1.5 ml volume in 6 ml plastic Falcon centrifuge tubes and stored at - 80° C. Prior to use, drugs were thawed in a 37° C waterbath. Drug testing was done at the in oitro concentrations listed in Table 1. Due to the lack of substantial pharmacokinetic drug data in mice, we used the in vitro drug dosages recommended by Alberts et al!' for testing human tumors. In general, the one hour drug exposure concentration is calculated as 10 % of the average CXT (ltg X hr/ml) for the high in vitro drug level and one log lower for the low in vitro drug level. Cells were incubated in the presence of drug for one hour, washed in CMRL x 3, resuspended in media, and plated in enriched CMRL in 0.3% agar.
TABLE.
2.
Analysis oj Data. In vitro and in vivo data were correlated in the following manner: tumor responses to drug were listed as either sensitive (S) or resistant (R). In tabulating the data, in vitro responses were listed first, i.e., in vitrolin vivo. The true positive predictive rate of the TCA was calculated and expressed as a % using the formula [S/S(S/S + SIR)] X 100. The true negative predictive rate of the TCA was calculated and expressed as a % using the formula [R/R(RIR + RIS)] X 100.
Results The activity of thirteen anticancer drugs was evaluated using four MBT cell lines. We found that when tumor cells were removed from monolayer culture for drug testing, ill vitro growth occurs in greater than 90 % of assays. Clonal growth with tumor cells removed from the animal occurred in approximately 50 % of assays attempted. Forty-nine drug studies were evaluable for comparison. The results of drug testing using the TCA were compared to the drug responses in the animal. Table 2 defines the ability of the TCA in predicting drug response in the murine system. The true positive predictive rate of the TCA was 50 %, while the true negative predictive rate was 70 % . A comparison of in vitro vs in vivo drug responses was tabulated by the individual drug being evaluated (Table 3). The TCA predicted drug response most accurately using MTX, ANG, MAY, and PALA. It should be noted, however, that these were negative correlations and could be related to an inability of either assay (in vivo andlor in vitro) to predict activity with these agents. The TCA had a low level of predictability when evaluating VP-16 and DDP. In all four cell lines, VP-16 was predicted to be active in vitro, and a significant in vivo tumor response was not seen in any of the MBT cell lines. DDP was predicted to be inactive in vitro with three out of four cell lines, but the drug significantly inhibited the growth of all of the tumor lines ill vivo. Table 4 lists the in vitro vs in vivo responses according to the MBT tumor being tested. MBT-2 was the most resistant cell line in both assays. The pre-
Predictability of TeA in murine model
TCA Sensitive TCA Resistant Level of Murine Murine Murine Murine Tumor Colony Sensitive Resistant Sensitive Resistant Inhibition (SIS) (SIR) TPPR * (RIS) (R/R) TNPRt 90%
6
6
50%
11
70%
26
*TPPR = True positive predictive rate. rT1':PR = True negative predictive rate.
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TABLE
3.
Predi ctability of TCA in murine model by individual drug TCA Sensitive
Drug
TCA Resistant
Murine
Murine
Murine
Murine
Sensitive (SIS)
Resistant (SIR)
Sensitive (RIS)
Resistant
Correlation
(RIR)
( %)
2
50 100 50
ADR
2
~ITX
BLEO VP-16 DDP CHLORO ANG
4 1
2
I
o
4
I
3 1
I 2 1
~UTO
5FU MAY PALA
1 2
3 3 1 I
1 1
3 2 2
25 75 100 75 50 100 100 66 66
4
AZQ
VLB dictability of the TCA was roughly equivalent for the other tumors. Discussion The present study demonstrates that when the TCA assay indicates a drug is active against one of these murine bladder tumors, it will correlate 50 % of the time with the in vivo response. If the TCA indicates the drug does not produce significant cytotoxicity, itwill correlate with the concurrent in vivo response 70 % of the time. The positive predictive rate of this assay in the murine mod el is similar to that previously reported in human drug testing," while the negative predictive rate is somewhat lower. To date, the majority of clinical comparisons between the TCA and drug activity in the host have been retrospective. Until prospective clinical trials have been completed, the true predictability of the TCA in human tumors will be in question. DDP and mitomycin C significantly inhibited growth of three of the four tumor lines in the animal. The activity of DDP in murine bladder cancer parallels its effectiveness in man since clinical 'st udies indicate a 20-50 % objective response rate.":" MiTABLE
MTB Cell Line MBT-2 ~1BT-8
MBT-683 MBT-409
tornycin C has established activity in superficial bladder cancer when delivered by intravesical instillation. However, I am not aware of any large trial in advanced disease. Our animal data would support consideration of such a study. The lack of dramatic efficacy for the other eleven compounds underlines the necessity for the development and screening of additional drugs in this tumor. VP-16 and ADR were predicted to be active by the TCA and were found to be inactive in vivo. The disparity between these in vitro and in vivo results is not readily apparent. VP-16 has been active in previous experiments with the MBT-2 tumor. Since both drugs are large molecules, they may not be completely absorbed from the peritoneum, and this might result in lower blood levels than if delivered by the intravenous route. Differences in drug metabolism and cellular uptake in vitro compared to in vivo and inappropriate in vivo or in vitro drug dosages may be responsible for the lack of greater concordance. DDP was very effective in the animal but ineffective in vitro. \Ve have previously shown that its activity is enhanced by increasing the time of drug ex-
4. Predictability of TCA ill murine model by MBT cell line TCA Sensitive Murine Murine Sensitive Resistant (SIS) (SIR)
o
1
2 2
2 2
2
I
TPPR' 0 50 % 50 % 66 %
TCA Resistant Murine Murine Sensitive Resistant (RIS) (R/R) 2 3 3
3
10 6 6
4
TNPHl 83 % 66 % 66 % 57 %
°TPPR = Truc positive predictive rate. ITlIiPR = True negative predictive rate.
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posure ill vitro. 16 It is unclear whether this increase in exposure time duplicates the conditions ill vivo. This data suggests, however, that increasing the duration of DDP infusion in the clinic may improve drug efficacy. The lack of efficacy of MTX ill vitro may be due to the rescue of cells by thymidine present in the culture media, thus giving a false negative result. Since only a small number of anticancer agents with activity in bladder cancer is available, it is important to continue to search for new and more effective drugs. The TCA could serve as an invaluable tool for the rapid screening of investigational compounds. Drugs found to be active in the TCA could be tested in the murine model and, if promising, incorporated into clinical trials.
References 1. Leiter, J., Abbott, B.J. and Schepartz, S.A. : Screening data from the Cancer Chemotherapy National Service Center Screening Laboratories XXVIII. Cancer Res., 25: 1626-1769, 1965. 2. Soloway, :-'I.S., deKernion, J.B., Rose, D. and Persky, L.: Effect of chemotherapeutic agents on bladder cancer: A new animal model. Surg. Forum, 13: 542-5-14, 1973. 3. Soloway, :-'I.S. and Murphy, W.:-'I.: Experimental chcrnotherapy of bladder cancer-systemic and intravesical. Scm. Oncol., 6: 166·183, 1979. 4. Yagoda, A., \\'atson, R.C ., Conzalez-Vitale, J.C., Grabstald, H. and Whitmore, WF.: Cis-dichlorodiammine platinum (II) in advanced bladder cancer. Cancer Treat. Rep., 60: 917923, 1976.
5. IIamburger, A. and Salmon, S.E .: Primary bioassay of human myeloma stem cells. J. Clin. Invest., 60: 846-854, 1877. 6. Salmon, S.E . and \'onIloH, D.O.: III dim evaluation of anticancer drugs with the human tumor stem cell assay. Scm. Oncol., s. 377·385, 19SI. 7. Buick, R.:>:., Stanisic, T.Il. , Fry, S,E ., Salmon, S.E ., Trent , J.:-'1. and Krusovich, P.: Development of an agar-methyl eellulose elonogenic assay for cells in transitional cell carcinoma of the human bladder. Cancer Hes., 39: 5050-5056, 1979. 8. Stanisic, TJI. and Buick, RII.: An in dim elonal assay for bladder cancer: Clinical correlation with the status of the urothelium in 33 patients. J. Urol., 124: 30·33, 1980. 9. \'onlloH, D.O., Casper, J., Bradley, E., Sandback, J., Jones, B. and :-'Iakuch, R. : Association between human tumor colony-forming assay results and rcsponsc of an individual patient's tumor to chemotherapy. Amer, J . Med ., 70: 10271032, 1981. 10. Nicll, Il.B. , Soloway, :-'I.S. and Nissenkom, 1.: The clonogenic growth of cells derived from bladder barbotage in pa · ticnts with transitional cell carcinoma of the bladder: A preliminary report. J . of Urol., 127: 668-670, 1082 . II. Nicll, II.B .. Mickey, D.O. , Soloway, :-'1.5. and Wood, C.A.: Growth characteristics of lI:.[4-(5-nitro-2-furyl)-2thiazolyljformamide (FAi':FT)-induccd mouse bladder tumor lines in human tumor stcm cell assay. Cancer, 49: 323· 330, 1982. 12. Soloway, :-'I.S.: Intravesical and systemic chemotherapy of murine bladder cancer. Cancer Bes., 37: 2918·2929, 1977. 13. :\iell, H.B ., Soloway. :-'I.S., \\ood , C .A., :-'lcCallum, L.\\~ and \Yebster, K.: The use of a tumor colony assay in predicting chemotherapeutic drug response in murine bladder cancer. Cancer, in press. 14. Alberts, D.S . and Chem. II·SG.: Tabular summary of pharrnacokinetic parameters relevant to ill dtro drug assay. Prog . Clin. BioI. Iles., 48: 223-245, 1980. . 15. Soloway, :-'I.S., Einstein, A., Corder, :-'I.P., Bonney, \\~, Prout, G.R., Jr. and Coombs, J.: A comparison of cisplatin and a combination of cisplatin and cyclophosphamide in advanced urothelial cancer-A National Bladder Cancer Collaborative Group A study. Cancer, in press. 16. "iell, II .B., Wood, C.A., Mickey, 0.0: and Soloway, :-'I.S.: Time and concentration dependent inhibition of the clono genic growth of lI:-[ 4.(5-nitro-2-furyl)-2-thiazolyl]forrnamide-Inducedmurinc bladder tumor cell lines by cisdiarnrnincdichloroplatinurn (II) . Cancer Res., 42: 807-811, 1982.
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Department of Urology University of Tennessee 956 Court Avenue-Room 2H23 Memphis, Tennessee 38163 (DR. SOLOWAY)
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