Human tumors transplanted into nude BALB/c-nu mice have been used to test the sensitivity of the tumors to various anticancer agents. Three cancer cell lines from the stomach and one from the colon were transplanted into nude mice to establish a standard assay method for selection of effective drugs on individual tumors using the criteria of the Japanese Association of Sensitivity Determination for Carcinostatic Agents. From the LD values of anticancer drugs in nude mice, the appropriate doses of drugs were 6 mg/kg of MMC X 1(i.p.), 50 mg/kg of 5-FU q4d X 3 (i.p.), 120 mg/kg of CPA (i.p.), 30 mg/kg of ACNU (i.p.) 8 mg/kg of CDDP (i.p.) and 8 mg/kg of ADM (i.v.). At 3 weeks after initial treatment, the inhibition rate (IR) of the tumor was calculated from the formula IR = (1-T/C) X 100%, where T is the mean tumor weight of the treated group and C is the mean weight of the untreated group at that time. The tumors respond well to the anticancer agents when IR is more than 58%.

It is difficult to predict the adequate doses of effective drugs which must be administered in cancer chemotherapy. We have developed a successive colony-forming assay in order to analyze changes of drug sensitivity in human cancer cell lines, and it is used as a model of cancer chemotherapy. Presently, widely used methods include the clonog ceni assay as in vitro sensitivity test established by Salmon and Hamberger et al., for revealing effective drugs. However, formation of a second colony using initial colony-forming cells has not yet been performed. We therefore analyzed the changes occurring in drug sensitivity in 2nd and 3rd colony-forming assays. Using a human lung adenocarcinoma cell line PC-9 (established by Hayata at the Surgical Department in Tokyo Medical School), we measured the changes in sensitivity to CDDP and its derivatives. After picking up the colony-forming cells by micropipette and culturing them in culture medium, we tried to incubate them for 1 hour with the anticancer drug, and then replate them in soft agar. This technique is called "2nd colony-forming assay". We were able to carry this out three times. The results obtained showed that the anti-tumor effect of 254S was the same as that of CDDP, whereas that of CBDCA was less than either of them. Although these tendencies were noticeable even in the 2nd colony-forming assay, the sensitivities to CDDP and its derivatives were diminished in the 3rd colony-forming assay.

The chemosensitivity of human cancer lines is thought to be expressed as a result of contributions by various interacting factors. Multiple regression analyses were performed in order to clarify the weighting of factors responsible for the chemosensitivity of 15 human cancers xenografted into nude mice. Inhibition rates of 11 anticancer agents predetermined for each line of human cancer were used as the criterion variables. As the explanatory variables, 9 parameters characteristic of each cancer or cancer-bearing mouse were selected as follows; grade of differentiation, vascularity, percentage necrosis, volume doubling time, labeling index, LDH activity, tissue/serum LDH ratio, thymidine phosphorylase activity and serum CEA. By applying this analysis with stepwise deletion, the estimated multiple regression equations for drug sensitivity were clarified for each drug. Although all equations were composed of different factors and their partial repression coefficients varied from drug to drug, those among analogous drugs such as FT-207 and UFT, or MMC and M-83 had similar factors. The equations for M-83, ACNU and ADR consisted of a number of parameters with a sufficiently high coefficient of determination of over 80%. Even in cases of MXT that showed no significant factor upon simple correlation analysis, an equation with 7 factors revealed a coefficient of determination of 0.83. The estimated values of effectiveness for these drugs showed remarkable coincidence with each actual value. For some drugs, the in vivo mode of action was inferred through this analysis.

In the present paper we discussed whether a new method of chemosensitivity test could be developed using FCM (flow cytometry) and applied to malignant brain tumor cells labelled with BrdU monoclonal antibody. For this purpose, a basic study was performed with an ACNU-resistant C6 cell line and a sensitive one to see if this method was able to detect the difference of sensitivity between these cell lines. After 8 hours treatment with ACNU (concentration: 10 micrograms/ml), ACNU-sensitive cells revealed on LI (labeling index) of 46%, which was very high in comparison with controls, whereas the figure for ACNU-resistant cells was 34%, which was almost the same LI value as non-treated control cells. This means that change in the BrdU labeling index after chemotherapy in vitro can be used to determine the chemosensitivity of malignant brain tumors when FCM is employed. Furthermore, this method can detect the chemosensitivity of each clone in polyclonal human tumors which is impossible for the HCSA method, since the latter can reveal only the chemosensitivity as a whole in such tumors.

The antitumor activity of several chemotherapeutic agents against a total of 30 clinical samples obtained from 30 human tumors from the esophagus (12), stomach (10), colorectum (6) and lung (2), were tested by subrenal capsule assay (SRCA) using normal immuno-competent BDF, mice. The antitumor activity was evaluated by changes in both tumor size (delta TS method) and tumor growth inhibition rate (TGIR method). Among the 30 tumors, 15 were also tested by human tumor clonogenic assay (HTCA) and the results obtained in the two assays were compared. In the SRCA, adequate growth of the tumor in the control group for evaluation of the response of the treated group was obtained from 27 out of 30 tumors (90%). With activity criteria set at delta TS less than or equal to -1.0 dmm and TGIR greater than or equal to 50%, 41% of the drugs tested were active in delta TS while 27% were active in TGIR. When relationships between antitumor activities evaluated by delta TS and TGIR was compared, both activities were well correlated (r = -0.64). Correlations between tumor responses in the HTCA and in the SRCA were tested in 6 tumors treated with 20 drugs. The overall accuracy was 60% by the TGIR method and 70% by the delta TS method, respectively.

We carried out in vitro anticancer drug sensitivity tests a total of 24 times in 15 cases of childhood leukemias and lymphomas using the microplate culture and crystal violet staining method. We then compared them with the clinical effects. As a result, the in vitro efficacy rate at onset of cases was 40%, which was less than that for relapsed cases, which was 100%. Especially, cases of ALL at onset all failed. As for the relation between in vitro and in vivo effects, cases for which it was possible to make evaluation both in vitro and in vivo numbered 16. The results for these cases were; true positive rate, 71%; true negative rate, 50%; predictive accuracy, 69%. The true positive rate was higher than the usual rate. As for the efficiency of each anticancer drug, in vitro predictive accuracy for ADR, Ara-C and 4 HCP (CPM) was higher than for other drugs. From these results, it became clear that our method is very useful for the screening of effective anticancer drugs.

Subrenal capsule assay (SRC assay) was investigated to evaluate the usefulness as an in vivo chemosensitivity test of anticancer agents. The pathological study on the growth of the implanted tumor and host response indicated that the assay had to be done by four-day assay. The analysis of the isotope incorporation into the implanted tumor supported this results. As determination of tumor sensitivity by the microscopic measurement showed the large standard deviation, the DNA and protein content was determined for the evaluation of sensitivity by percent inhibition of the DNA/protein content (%DNA/protein). Ninety five fresh tumor specimens were examined and evaluated by relative variation of the calculated tumor weight (delta TW/TW0), while 64 specimens by %DNA/protein. The evaluability rates using delta TW/TW0 and %DNA/protein were 84.2% and 87.5%, respectively. All over predictive accuracy between the clinical responses and the results of the assay evaluated by delta TW/TW0 was 78.6%, while 81.8% was obtained by %DNA/protein. From these results, the potential utility of SRC assay examined on 4 day for determining chemosensitivity by %DNA/protein seems to be beneficial for clinical use.

We have applied the MTT dye reduction assay to the anticancer drugs sensitivity test using short-term microplate cultures. The tumor cells were cultured with the anticancer drugs for 2 and 4 days. After culture, MTT dye was placed in each microwell and culture was carried out again for 4 more hours. The formazans generated by living cells were dissolved in acidified isopropyl alcohol and the absorbances of each well were measured at a wavelength of 540 nm. When tables of cytotoxicity indices classified into anticancer drugs, concentrations and durations of culture for each type of leukemic cell were made, it became possible to compare each drug and to select the effective ones. This assay is simple, precise, rapid, has no washing steps and is convenient for handling a large volume of material. We apply this assay in clinical practice.

A soft agar colony formation assay, so called human tumor clonogenic assay (HTC assay) similar to that originally described by Salmon and colleagues, was utilized to measure the sensitivity of a total of 85 urologic malignancies including 36 urothelial cell carcinomas, 41 renal cell carcinomas, 5 testicular tumors, and 3 Wilms' tumors to anticancer drugs. In addition, the results obtained were compared with those of a novel dye exclusion method (NDE assay) described by Weisenthal and colleagues. The NDE assay was utilized to measure the sensitivity of a total of 63 urologic malignancies including 28 urothelial cell carcinomas, 25 renal cell carcinomas, 6 testicular tumors, and 4 Wilms' tumors to anticancer agents. In both assay series, the concentration of anticancer drugs tested was approximately one tenth of the maximum serum level achievable after single bolus injection. The colony forming rate inhibition of 70% or more in the HTC assay and the cell survival rate of 30% or less in the NDE assay were defined as "sensitive." Sixteen of the 36 urothelial cell carcinomas, 11 of the 41 renal cell carcinomas, and 1 of the 5 testicular tumors had both more than 30 colonies grown in control plates and enough cells in the specimens to provide at least one drug sensitivity testing. In urothelial cell carcinomas, 3 out of 13 tumors were "sensitive" to adriamycin, 3 out of 16 to cis-platinum, and 4 out of 15 to carboquone. In renal cell carcinomas, 2 out of 9 tumors were "sensitive" to adriamycin, 4 out of 11 to vinblastine, and none of 4 to Interferon alpha.(ABSTRACT TRUNCATED AT 250 WORDS)

The in vivo chemosensitivity test for bladder cancer, using the human bladder cancer xenografts (BT-8 and BT-11 strains) in nude mice (BALB/c) and the BBN-BT-1 bladder cancer strain in BALB/c hetero-mouse which was induced by peroral long-period administration of N-butyl-N-(4-hydroxybutyl) nitrosamine and transplantable into the subcutaneous of mouse, were examined especially in respect to the difference of chemosensitivity between young and old straining and the prospective propriety for clinical application. The subrenal capsule assay (SRC), was also compared with subcutaneous transplantation. Cis-diamminedichloroplatinum (II) and 5-FU were effective for all three strains and adriamycin and cyclophosphamide were effective for the BT-8 and BT-11 strains. Bleomycin, peplomycin and vinca alkaloids were more effective for the BT-11 strain than the BT-8 strain. The chemosensitivity of several anti-cancer drugs for the young BT-8 and BT-11 strains was almost equal to that of the old. A 68-year-old male with bladder cancer metastasized to lung and lymph nodes, whose primary tumor was transplanted to mice and established as the BT-11 strain in 1980, was treated with the VPM-CisCF combination chemotherapy which was evaluated as an effective therapy for this strain experimentally, and responded well to this therapy. As in this case, the results of nude mice experiments are valuable in clinical application. The chemosensitivity test in vivo for individual primary tumors should be done by SRC, and in SRC nude mice should be used instead of conventional mice until immunoreactive rejection can be prevented.

We have developed a simple in vitro chemosensitivity test for breast cancer. Tumor tissues were chopped finely with razor blade. The cell clumps were pounced into tissue culture medium, which contained a specified level of concentration of anticancer drugs, and incubated at 37 degrees C for four to eight hours. Nine to 10 kinds of drugs were tested on each specimen. After incubation, the clumps were pumped down. The cells were smeared and stained with Giemsa for microscopic examination. The individual tumors showed different sensitivities towards various drugs, and the typical morphological changes observed in their nuclei; were karyorrhexis and karyopyknosis.