Development of cellular resistance to multiple types of anticancer agents has been recognized as one of the major obstacles for the effective cancer chemotherapy. Increased expression of mdr 1 mRNA seems to be a common mechanism for multidrug resistance (MDR) in human malignant cells. The product of the mdr 1 gene is P-glycoprotein. The predicted membrane orientation of the protein and homology with bacterial active transport proteins, and capability of the protein to bind hydrophobic anticancer agents are consistent with the function of P-glycoprotein as an energy-dependent efflux pump responsible for MDR phenotype. Most of the hydrophobic agents which overcome MDR are cationic and amphipathic. These agents interact with certain polar lipids and inhibit also the binding of hydrophobic anticancer agents with P-glycoprotein. They might directly bind to the binding site of anticancer agents on P-glycoprotein and competitively inhibit the binding of anticancer agents. Alternatively, they might bind to polar lipids of membrane vesicles and indirectly inhibit the binding ability of the protein to anticancer agents by perturbing the membrane function.

Subrenal capsule assay was performed on 35 fresh cancer tissue samples (13 stomach, 7 colon, 8 breast and 7 others) against MMC, 5-FU, ADM, CPA, MTX and cDDP employing normal immunocompetent ddY mice following Bogden's original method. Evaluability was 86% (30/35). Sensitivity was between 32 and 46% for each of the drugs except cDDP. Some organ-specific variations were noted between gastro-colic cancers and breast cancers with regard to chemosensitivity spectrum and also individual cases of the same histological type did not show a uniform one. Gastric cancer had the highest activity. The assay/clinical correlations were evaluable in 10 cases where 6 cases on active agents achieved two PR, two NC and PD, while all 4 cases on non-active agents showed PD. Histological analysis of implants, however, disclosed the following inherent problems: First, there was heterogeneity in tissue fragments due to the variable amounts of stroma; Second, apparent host reactions were seen in the untreated groups on the 6th day, accompanying marked damage to cancer cells. In contrast, in drug-treated groups, host reactions in most of them were suppressed and cancer cells were seen frequently in considerable amounts. This fact implies the mechanism that SRCA is based on, in spite of the xenograft immune; Third, there was some discordance between the macroscopic and microscopic findings. SRCA is assumed to be appliable to individual clinical cases up to a certain level which should be determined by histological analysis as well as by accumulation of clinically correlated cases.

Clinical trials of 4-day subrenal capsule assay (SRC assay) were carried out. One hundred and forty-one cases were investigated in order to evaluate the clinical utility of the assay. A total evaluability rate of 81.0% and a response rate of 36.5% were obtained in the SRC assay. The overall predictive accuracy between the tumor sensitivity of the assay and the clinical response was 82.1%. The percentage inhibition of %DNA/protein content of the implanted tumor, as a new predictor of the tumor growth inhibition, also indicated a good prediction rate for the assay. Correlation between the sensitivity test and the end results after chemotherapy in cases of inoperable gastric cancer classified as stage IV was investigated, retrospectively. Comparison of the survival curves between the patients treated with sensitive agents and those with insensitive agents exhibited a significant advantage for the former (p less than 0.01). These results suggest the utility of the SRC assay for clinical use, but histological studies exhibited certain limitations of this assay due to the existence of early host rejection of the implanted tumor. The utility of the SRC assay should be finally evaluated using more histological assessments and clinical trials.

Subrenal Capsule Assay (SRCA) developed by Bogden et al. is a new in vivo sensitivity test for anticancer agents. However, the presence of host reaction on about day 6 is largest problem for this method, and now many researchers are scrutinizing or introducing several modifications for this assay chiefly by immunosuppressing procedures. As one of the means to measure the actual change of the tumor volume and minimize the misreading of host reactions, we introduced three dimensional measurements of the grafts (volume method) instead of original mean diameter measurement. Using this method, prospective quantitative study was made. The results of 16 assays with 15 cases of miscellaneous malignancies showed 94% evaluability and in 22 treatments predictive accuracy was 77%. Assay/clinical correlation coefficient was 0.38 and coincidence of ranking order of the effectiveness was 6/6, with good contrast to those of 78%, 56%, 0.00 and 1/3, by original method respectively. Clinical responses more than PR were obtained in 9 of 15 cases. Two cases are still in CR for two years by assay directed single agent treatments. Many other minor modifications were also adopted and evaluated by quantitative observation of assay/clinical correlations. SRCA with our modifications seemed to have utilities than original.

The antitumor activity of chemotherapeutic agents can be evaluable faster by subrenal capsule assay (SRCA), and the activity ratings between SRCA and subcutaneous transplantation assay in nude mice were correlated well. Thus, SRCA can be utilized as a disease-oriented screening in vivo instead of a human tumor-nude mice system in the future. The original SRCA method developed by A.E. Bogden is a chemosensitivity test which is carried out in easy procedure. But because of infiltration of host cells derived by a host immune reaction, the chemosensitivity results in SRCA may be modified and so further work must be attempted to get exact results of chemosensitivity and good clinical correlation. Although, SRCA has a high possibility of application on an individual basis for prediction of chemosensitivity and it was discussed in this paper.

Histological analyses of the subrenal capsule assay (SRCA) carried out with modification of Bogden's original method revealed that the major portion of cancer tissue implanted were almost replaced by the host immune cells and reactive tissue by day 6. For the purpose to find a more improved condition, the following experiments were performed using human cancer lines xenografted in nude mice. No prominent differences in the host reaction to xenograft were found between 3 kinds of immunocompetent mice. Subrenal space of the athymic nude mice allowed cancer tissue to grow well until 15 days after inoculation, and then chemosensitivity test to judge the growth inhibition rates. The similar condition as in nude mice was achieved in CDF1 mice by daily injection of 60 mg/kg of cyclosporin A.

We attempted to examine the practicality and the usefulness of 6-day subrenal capsule assay (SRCA) as a predictive chemosensitivity test. The success rate of this assay was 81% in 150 cases of various cancers. The drug sensitivity was evaluated by the inhibition rate of tumor growth (TGIR) on day 6 after drug treatment without immunosuppression of mice. We analyzed the clinical response in comparison with the results of SRCA in 38 cases in which transplanted tumor showed over 1.0 tumor growth rate (TGR). The average TGIR in these cases attained CR, PR, MR, NC and PD clinically were 60, 60, 49, 53 and 40%, respectively. The TGIR of CR and PR cases were significantly greater than that of PD cases (p less than 0.05). These results in SRCA correspond to clinically observed responses, using a greater than or equal to 45% inhibition in TGIR to define 'sensitivity'. Of 26 patients sensitive in SRCA, 18 patients (69%) showed true positive and 75% of patients (12 cases) resistant in the assay also failed to respond clinically (true negative). In the cases with less than 1.0 in TGR, a correlation between clinical responses and TGIR was not found. Based on these results, 6-day SRCA without immunosuppresive procedures can be utilized as a predictive chemosensitivity test for cancers which can grow over 1.0 in TGR in this assay system.

A study on the effect of anti-tumor agents combined with caffeine on sarcoma cells was carried out by clonogenic assay. The materials used were an established line of human osteosarcoma cells (OST strain) and twelve surgically resected or biopsied specimens. Caffeine showed a marked synergistic effect on sarcoma cells with the DNA-damaging agents, ADR, CDDP, CPM and MMC in terms of colony inhibition. In particular, 0.2 micrograms/ml CDDP with 2 mM caffeine showed a considerable synergistic effect on human sarcoma cells. Among the 12 cases, more than 50% colony inhibition was observed in 7 cases which were treated with this combination of CDDP with caffeine. Furthermore, a combination of 0.02 micrograms/ml CDDP (1/100 of peak plasma concentration) with 2 mM caffeine also showed more than 50% colony inhibition. Therefore, we assumed that caffeine was able to reduce the necessary dose of anti-tumor agent in some way. We stress that caffeine seems to be a very useful synergistic drug for causing lethality in sarcoma cells in combination with various DNA-damaging agents which are not effective on sarcoma cells.

We examined the value of the CSF-HU preparation (colony-stimulating factor derived from human urine: P-100) to prevent or treat granulocytopenia induced by anticancer chemotherapy. Subjects were urogenital cancer patients who underwent two courses of the same chemotherapy regimen. Among these patients, we selected the subjects whose leucocyte counts were decreased to less than 2000/mm3 after the first course of anticancer chemotherapy. P-100 was administered from the following day of the end of the second course of chemotherapy at a dose of 8,000,000 units/day by intravenous drip infusion for 7 successive days. According to the global evaluation by consideration of changes in leucocyte and granulocyte counts, the utility rate by physicians was 52.4% (22/42), and that by a committee was 50.0% (18/36). No difference was seen in utility rate and efficacy rate according to P.S., cancer types and antineoplastic drugs used. Side effects were noted only in 2.3% (1/44) which was mild and transient fever. These findings suggest that P-100 is a very useful drug for prevention and/or treatment of granulocytopenia following cancer chemotherapy.

For the purpose to study reasonable treatment for recurrent gliomas, in vitro immunochemosensitivity tests were performed by using human malignant glioma cell line (ONS-12) and its ACNU-resistant cell line (ONS-12/ACNU), which were established in our laboratory. ONS-12/ACNU cells showed a cross-resistance to Ara-C, but not for cisplatin and methotrexate. The lymphokine-activated killer (LAK) cells induced in vitro from the peripheral blood lymphocytes (PBL) of healthy subjects, showed stronger cytotoxicity to ONS-12/ACNU than ONS-12 cells. From these data, selection of appropriate anti-tumor agents on the in vitro sensitivity tests was a most useful method for the treatment of recurrent gliomas, and the adoptive immunotherapy with LAK cells may be useful for ACNU-resistant gliomas.

Xenografts of human tumors in nude mice have more advantages to evaluate drug response than conventional experimental models. However, this method has difficulties in establishing tumor growth and in evaluating drug response in short period. In order to evaluate drug response in short time and to apply it on clinical study, we examined a newly devised method of sensitivity test in which tumor tissues were inoculated subrenal capsules of nude mice. The drug doses were decided from LD50 values of nude mice. Drug effects were estimated ten to twelve days after tumor implantation. Subrenal capsule implants grew early without latent period in nude mice, but in BDF1 normal mice those were diminished and disappeared within four to seven days after inoculation. The results of drug response in this method almost agreed with the method of subcutaneous inoculation in which drug effects were estimated three weeks after tumor inoculation.