4'-Epi-adriamycin (EPI) is a new derivative of adriamycin (ADM). The cytotoxic effect of EPI on the T24 cell line, an established cell line from human urinary bladder carcinoma, the distribution of the drug in blood, urine and tissues of various organs and histopathological change in the bladder mucosa in dogs following intravesical instillation of the drug, were studied. The cytotoxicity of EPI on the cultured T24 cells was examined by a colony formation method. After 2 hours exposure, EPI was slightly less cytotoxic than ADM, but showed higher cytotoxicity than mitomycin C or aclacinomycin A. The drug levels in blood, urine and tissues were measured by HPLC following bladder instillation in Beagle dogs with bilateral cutaneous ureterostomy. They were elevated in proportion to the drug concentration instilled intravesically. After 50 mg of EPI dissolved in 10 ml of physiological saline was instilled intravesically, the blood levels of EPI were not elevated significantly and reached the maximum levels of only 0.222 microgram/ml. The total amount of EPI excreted into the urine during the 10 hours after instillation was 389 micrograms which was equivalent to 0.78% of instilled EPI. The tissue levels of 50 mg of EPI after 6 hours retention were 1216 +/- 1094 micrograms/g in the bladder mucosa, 259 +/- 250 micrograms/g in the bladder muscular layer, 7.65 +/- 1.19 micrograms/g in the iliac node, 22.1 +/- 4.8 micrograms/g in the cortex of kidney, 15.1 +/- 3.8 micrograms/g in the medulla of kidney, 11.3 +/- 1.0 micrograms/g in liver and 5.80 +/- 1.20 micrograms/g in the heart. To examine the effect of the drug on the bladder mucosa, 50 mg of EPI was instilled intravesically. After 6 hours retention, bladder mucosa was observed through a microscope and a scanning electron microscope. Only exfoliation in the mucosa was observed sporadically and no histological change was observed in the submucosal layer. The above results suggest that EPI is a suitable drug for intravesical chemotherapy to bladder cancers.

The in vitro cytotoxicity of etoposide against mouse P388 leukemia cells was kinetically studied and compared with those of podophyllotoxin, the parent compound, and other antitumor agents. When P388 cells were exposed to etoposide, surviving-cell fraction decreased with etoposide concentration and exposure time. Similar results were obtained with doxorubicin, peplomycin and cisplatin. The cytotoxicity of melphalan was dependent on concentration but scarcely on exposure time. From these data, n in Cn X T = K where T, C and K are exposure time, concentration required for killing 90% of P388 cells and a constant, respectively, was calculated. Etoposide gave an n value of 1.16. n values for doxorubicin, peplomycin and cisplatin, all belonging to the Shimoyama type Ib group where the cytotoxicity of the agent depends on both concentration and exposure time, were 1.29, 1.28 and 3.03, respectively. The n value for melphalan, belonging to the type Ia group where cytotoxicity depends only on concentration, was 54.0. From these results, it was concluded that etoposide is of type Ib. The cytotoxicities of podophyllotoxin, 5-fluorouracil, cytarabine and vinblastine were greatly dependent on exposure time. Podophyllotoxin may be an agent of type II whose cytotoxicity depends only on exposure time.

Soft agar cultures of human prostatic carcinoma cells obtained from prostates with needle biopsy and metastatic lymph nodes were examined. Colony formation were observed in 2 of the 4 needle biopsy specimens and in 7 of the 10 lymphatic nodes specimens. The plating efficiency was 0.04 to 0.43%. The effect of testosterone on the clonal growth of these cells in soft agar culture was studied. In half of the cases, colony formation was increased by the addition of testosterone. Whereas in the other cases, colony formation was not influenced by the testosterone. Clinically, most of the former cases responded well to the anti-androgen therapy. Neither EGF nor hydrocortisone showed any effect on the colony formation of the prostatic cancer cells in soft agar. These results suggest that primary culture of human prostatic cancer is possible in soft agar medium, and that androgen dependency of these cells can be examined in vitro.

In vitro chemosensitivity was evaluated by SDI test in various human tumors including 1 lymph node metastasis of esophageal cancer, 10 gastric cancers, 4 colo-rectal cancers, 1 hepatoma, 2 lung cancers, 2 breast cancers and 1 gallbladder cancer. Tumor fragments cut with scissors were exposed to twelve kinds of antitumor drugs at five to ten times peak plasma concentration. After 3 days at 37 degrees C, each tumor fragment suspension was washed with phosphate-buffered saline and assayed for succinate dehydrogenase (SD) activity using 3-(4,5- dimethyl-2-thiazolyl)-2, 5-diphenyl-2H tetrazolium bromide (MTT) as a hydrogen acceptor. When the SD activity of the drug-treated cells was reduced to below 50% that of control cells, the chemosensitivity to the antitumor drug was considered positive. The chemosensitivity of each tumor varied individually. Mitomycin C or 5-fluorouracil are regularly used to treat gastric cancer patients, but, some specimens of gastric cancer in this study showed a resistance to these drugs and an unexpected sensitivity to other drugs. Our results show that the SDI test is a convenient method for clinical use and gives significant information about drug sensitivity.

The effects of the anticancer drugs Nimustine (ACNU), Aclacinomycin A (ACR), Adriamycin (ADM), Bleomycin (BLM), Cisplatin (CDDP), and 5-Fluorouracil (5-FU) on the multicellular spheroid of a chemically-induced 9L rat glioma was studied. The multicellular spheroid in which cells grow in vitro as three-dimensional aggregates represents a biological model, which is intermediate between monolayer cells in vitro and solid tumors. Spheroids were initiated in bacteriological grade petri dishes seeded with 10(6) 9L rat glioma cells, cultured for four days and thereafter transferred and further developed in a spinner flask. Spheroids of 200-400 micron diameter were sorted and exposed for 24 hours to 5-FU and one hour for other drugs. After treatment both cytotoxic effect and growth delay were analyzed. Following disaggregation using collagenase, pronase and DNAase, cytotoxic effect on multicellular spheroids was measured by colony forming assay and were compared with those effects on 9L monolayer culture cells in the exponential growth. For growth delay assay, multicellular spheroids were individually transferred to 16 mm well containing 0.4 ml agarose base and 2 ml culture medium. Spheroid size was measured twice a week and growth curves were drawn. The growth delay was determined as the treated group vs. control differences in time required to a size four times that of the initial volume. For cells both in the monolayer culture and the multicellular spheroid, the dose response curve for ADM, BLM and 5-FU was "biphasic" and that for ACNU, ACR and CDDP "shoulder-threshold" type.(ABSTRACT TRUNCATED AT 250 WORDS)

We have attempted to grow several kinds of malignant tumors using human tumor stem cell assay. Formation of colonies in vitro was seen in 65 of 132 primary tumors (49%), including 25 of 41 (61%) uroepithelial cancers, 12 of 19 (63%) renal cancers, five of 12 (42%) testicular cancers, five of 21 (24%) gastrointestinal malignancies, five of 12 (42%) lung cancers, five of 11 (45%) hematopoietic malignancies and five of 16 (31%) other malignancies. Growth sufficient for in vitro chemosensitivity tests of CDDP developed from seven cases of uroepithelial cancer, three of them (43%) were sensitive to 2.5 micrograms X hour/ml of CDDP. The specimens from a metastatic testicular tumors that received several courses of PVB chemotherapy resulted in the resistance of the in vitro chemosensitivity test at a higher dosage of CDDP. Nine cases of renal cancer had sufficient growth for an in vitro chemosesitivity test of interferon. One of them was sensitive for alpha 2 type interferon. Three of seven cases were sensitive for alpha type of interferon. To predict clinical correlation, 19 patients were tested with the same drugs used in the in vitro chemosensitivity test. The predictability resulted in more than 60% of true positive, 91% of true negative and 86% of overall predictability.

Comparative studies between the in vitro human tumor clonogenic assay (HTCA) and our original in vivo method (NM-IA) in which the final evaluation was made with 3H-TdR incorporation of tumor cells transplanted into nude mice were simultaneously performed on 44 fresh human solid tumors (22 gastric cancers, 6 breast cancers, 4 gall bladder cancers, 4 liposarcomas and 8 other tumors). Mitomycin C (MMC), 5-fluorouracil (5-FU), adriamycin (ADM) and cyclophosphamide (CPM) were tested. The evaluable rate was 73% (32/44) in HTCA and 89% (38/44) in NM-IA. Although correlation between the results of HTCA and those of MN-IA was obtained for MMC and ADM, no correlation was observed for 5-FU and CPM.

Relationships between radioresistance of uterine adenocarcinoma cells and potentially lethal damage repair (PLDR) was studied using four human uterine carcinoma cell lines (HeLa S3, HEC-59, SNG-M and SKG-3a). The magnitude of PLDR was estimated by colony formation assay using cell densed and nutrition deficient cultured cells and transplanted cells in nude mice. PLDR of cultured cells and HeLa S3 cells in nude mice almost finished by 6 hours after irradiation. The magnitude of PLDR of HeLa S3 cells in vitro was almost the same 4-fold ratio as in vivo after radiation exposure at a dose of 5 Gray. This suggests that PLDR in vitro is correlated to that in vivo. Dose-surviving relationships demonstrated that radioresistance of four cultured cells was chiefly expressed as Do and Dq values in hit-theory and the magnitude of their radioresistance was as follows: HEC-59 greater than SNG-M greater than HeLa S3 greater than SKG-3a. On the other hand, the magnitude of their PLDR was as follows: HEC-59 greater than SNG-M greater than HeLa S3 greater than SKG-3a. This proved that the magnitude of radioresistance is correlated to that of PLDR and the more radioresistant adenocarcinoma cells have greater PLDR than squamous cell carcinoma cell. In conclusion, PLD repair may be an important cause of radioresistance in human uterine adenocarcinoma cells.

Over the past year, we have attempted to grow 132 different human tumor samples (78 from urological and 54 from non urological tumors) using a soft agar colony formation assay similar to that originally described by Salmon and colleagues. Formation of colonies in vitro occurred in 44 of 78 primary urological tumors (56%), including 63% (12/19) of renal cancers, 61% (25/41) of uroepithelial cancers and 42% (5/12) of testicular cancers. The effects of in vitro chemosensitivity were analysed using the inhibition of colony growth (more than 70%) at two different cut-off doses which were equilibrated with the achievable AUC doses for the higher cut-off point and to one-tenth of AUC for the lower cut-off point. Five of 13 (38%) drugs showed an effective rate between 10% and 38% for the lower cut-off point in vitro. On the other hand, eleven of 13 (85%) drugs. Showed an effective rate between 20% and 67% for the higher cut-off point in vitro. In the tested uroepithelial cancers, none of seven for the lower cut-off and three of seven for the higher cut-off were demonstrated to be sensitive cases from the dose corresponding colony inhibition curve for cisplatinum. Nine of the renal cancers were demonstrated for the dose corresponding colony inhibition curve for Interferon. To predict clinical correlation, 16 patients (20 drugs) were treated with identical drugs which were estimated from in vitro chemosensitivity testing. The predictability results were 75%-100% true positive and 85%-100% true negative, with 87% overall predictability. This assay can therefore be used to study differences of biological character including drug sensitivity.

The author reviewed his experience to date with chemosensitivity testing of 162 gastric and 144 colorectal cancers. All human tumor clonogenic assays were performed using a double-layer soft agar system with continuous exposure of cells to one concentration of standard anticancer drugs. Significant growth was defined as greater than or equal to 30 colonies/control plate. Clinical responses were determined according to standard criteria. Forty-six per cent of gastric cancer specimens and 67% of colorectal cancer specimens plated produced significant growth in vitro. When greater than or equal to 50% inhibition of colony formation was used as the criterion for differentiating sensitivity from resistance, the assay was 67% (8/12) reliable for predicting in vivo sensitivity, and 91% (10/11) reliable for in vivo resistance.

It would be helpful for successful chemotherapy in cancer patients if a drug-sensitivity test in vitro could predict the exact response of an individual patient's tumor. We have investigated a drug-sensitivity test using human tumor clonogenic assay since 1980. In this paper, results obtained in lung cancer patients are discussed. Specimens for testing were obtained from primary tumor, metastatic mass, malignant pleural and pericardial effusion, and affected bone marrow. Drugs tested in this study were adriamycin, aclarubicin , THP-adriamycin, mitoxantrone, mitomycin C, cis-platinum, 40497 S (an active compound derived from ifosfamide), and methotrexate. Out of 88 specimens tested, 41 (47%) successfully yielded more than 30 colonies per control dish, and were able to evaluate drug-sensitivity. Of those, 32 instances were valid for examination in an in vitro-in vivo association. As a result, 3 were in vitro sensitive-in vivo sensitive, 2 were in vitro sensitive-in vivo resistant, and 27 were in vitro resistant-in vivo resistant. Accordingly, the true positive rate was 60%, and the true negative rate was 100%. In summary, the human tumor clonogenic assay appeared to be an excellent method for testing drug-sensitivity for an individual patient with lung cancer.

The human tumor clonogenic assay (HTCA) developed by Hamburger and Salmon was evaluated in 135 fresh samples of breast cancer. Successful tumor colony growth (greater than or equal to 5 colonies/plate) was obtained in 100 (74%) of the 135 samples, and adequate growth for drug testing (greater than or equal to 30 colonies/plate) in 75 (56%). With regard to the success rates of growing colonies categorized by specimen source, tumor sites, histology, prior chemotherapy and estrogen receptor (ER), specimens from solid tumors and primary tumors showed higher success rates than those from pleural effusions and metastatic tumors. The effects of prior chemotherapy, histology type and ER status on the success rate of colony formation were not significant. The overall median plating efficiency was 0.012%. Higher plating efficiencies were found in pleural effusion, metastatic tumors and samples from patients with prior chemotherapy. These findings appeared to indicate that aggressiveness of disease might be related to plating efficiency. Defining a greater than or equal to 50% inhibition of colony formation (ICF) as in vitro drug sensitivity, the in vitro response rates to anticancer drugs tested were as follows: adriamycin 33%, mitomycin C 39%, 5-fluorouracil 32%, methotrexate 42%, L-PAM 31%, cisplatin 38%, vincristine 32%, vinblastine 54%. The group of patients without prior chemotherapy showed higher sensitivity in vitro compared with the group of patients who had prior chemotherapy (38% vs. 27%). Correlation between in vitro drug sensitivity (greater than or equal to 70% ICF) and clinical response in 12 patients treated with the same drugs were analyzed retrospectively. The predictive accuracy was 0% (0/1) for true positive and 91% (10/11) for resistance. Thus, overall predictive accuracy was 83%. Based on these results, HTCA appeared to be useful chemosensitivity test for evaluation of antitumor drugs for human cancers in vitro and prediction of the chemotherapeutic effect in clinical use.

For use in routine clinical studies, modifications to Salmon and Hamburger's human tumor stem cell assay were made. A multiplate with 24 wells made the handling of a large number of samples feasible. The addition of anticancer drugs to the bottom layer of agar facilitated avoidance of exposure to drugs before cell plating and evaluation of the effect of long-acting drugs such as 5-fluorouracil. Storage of test plates including anticancer drugs in a freezer produced no loss of colony-forming activity. Specimens from 32 patients with advanced malignancies of the GI tract were tested for sensitivity to anticancer drugs. Forty-seven percent formed enough colonies for the performance of drug testing. Two patients showed sensitivity to drugs from both in vitro and in vivo results; the ascites in one disappeared while the other showed more than 50 percent regression of hepatic metastatic foci after treatment with suitable drugs. Nine patients showed resistance to drugs from both in vitro and in vivo results. Eight showed resistance to all tested drugs.