Preclinical evaluation of a new anthracycline derivative, SM-5887 by subrenal capsule assay

SM-5887, a new totally synthetic anthracycline derivative was evaluated the antitumor activity by subrenal capsule assay (SRCA) and the activity was compared to that of doxorubicin. The method of SRCA was the same originally developed by Bogden et al and 200 mg/kg of bredinin was administered subcutaneously 3 times every 2 days in order to induce immunosuppression of mice. Mice were given a single intravenous dose of either 30 mg/kg of SM-5887 or 15 mg/kg of doxorubicin. Fourty-four cases out of 64 were evaluable in this assay. The mean tumor growth inhibition rate (TGIR) in either SM-5887 or doxorubicin group was similar and the TGIRs of these two groups correlated well (y = 10.4 + 0.67x, p less than 0.01). Using more than 50% decrease of TGIR to define "sensitive", the chemosensitivity rates of SM-5887 and doxorubicin were 23% (10/44) and 25% (11/44) respectively. SM-5887 was effective to sarcomas and ovarian cancer especially. These results suggest that SM-5887 might have a similar antitumor activity compared to doxorubicin in clinical use.     

Study on the chemosensitivity of liver cell carcinoma by subrenal capsule assay

Chemosensitivity of liver cell carcinoma was studied by subrenal capsule assay. The method of assay was based on Bogden's one, but the antitumor activity was evaluated by tumor growth inhibition rate (TG-IR). The anticancer agent with more than 50% TG-IR was judged as positive in the chemosensitivity test. Of 3 human hepatoma cell lines transplanted in the subcutaneous space of nude mice, all of 3 were evaluable. The positive rates of ADR, MMC, CDDP, 5-FU and CPA were 66.7%, 100%, 66.7%, 100% and 0%, respectively. Of 24 patients who provided fresh tumor specimens for the assay, 12 (50%) were evaluable. The positive rates of ADR, MMC, CDDP, 5-FU and CPA were 25%, 16.7%, 16.7%, 33.3% and 8.3%, respectively. Our study suggested that 5-FU, MMC and ADR were comparatively active against the hepatoma cell, CDDP was less active than these 3 agents, CPA was inactive. These results seem to justify the use of current anticancer agents against hepatic cell carcinoma and indicate the usefulness of SRC assay for selecting chemotherapeutic agents against liver cell carcinoma.     

A 6-day subrenal capsule assay for predictive testing of primary esophageal cancers

Subrenal Capsule Assay (SRCA) as a chemosensitivity test was performed on 14 esophageal squamous cell carcinomas in order to select a more effective form of chemotherapy. Of the 14 assays, 12 were evaluable. Mice were treated with anticancer agents (e.g. Cisplatin, Bleomycin, Methotrexate, Vindesine) on days 1 and 3 after transplantation, and on day 6, the sensitivities were determined. Fresh esophageal cancers yielded an evaluable assay rate of 74%. The implant grew progressively for six days in the remaining group of control mice. Histologically, host cell infiltration at the border of the implant was observed from day 3 after transplantation, and cells had degenerated or had been partially replaced by scar tissue by day 6. The results of chemosensitivity tests differed according to the anticancer agent used or from case to case. Clinically, correspondence between the assay results and clinical results was obtained in 5 out of 7 cases. SRCA is a new promising chemosensitivity test which is clinically useful, and the present results indicated the feasibility of its use in developing an effective chemotherapy for esophageal cancer.     

Clinical trials of the subrenal capsule assay

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.     

A six-day subrenal capsule assay for predictive testing of primary human tumors

We carried out a total of 36 in vivo chemosensitivity tests in 33 cases of human malignant tumor using the subrenal capsule assay, developed by A.E. Bogden et al. Of the 36 assays, 31 were evaluable. The chemosensitivity of each tumor varied individually. UFT, 5-fluorouracil, mitomycin-C and adriamycin were administered to gastrointestinal cancer patients regularly, but our SRC-assay showed a high sensitivity rate for UFT and 5-fluorouracil but a low sensitivity rate for mitomycin-C and adriamycin. Nine patients had clinically evaluable lesions and a correlation between the assay results and clinical response existed in 6 cases. The true positive rate was 50% (3/6), the true negative rate 100% (3/3), and the overall predictive accuracy 66% (6/9). This study suggested that 6-day SRC assay is useful for selecting effective anti-tumor agents for the treatment of cancer patients.     

Fundamental evaluation of the subrenal capsule assay as an in vivo chemosensitivity test

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.     

Expression of the human transferrin receptor in subrenal capsule assay in the mouse

The expression of a cell proliferation marker, the human transferrin receptor, was studied in ten human gastrointestinal tumors prior to and after implantation under the renal capsule in the mouse (1-6 days). These data were compared to the increase in tumor size in situ, and to the infiltration of inflammatory cells. All tumors studied expressed the transferrin receptor prior to implantation. Forty of 47 implants expressed the receptor, the strongest expression occurring on day 4, accompanied by a reorganization of tumor tissue to a morphology similar to that before implantation. On days 5 and 6 the expression of the transferrin receptor declined. Implants showed maximal increase in size on days 1 and 2, decreased in size on days 3 and 4, and increased again on days 5 and 6. The increase in the size of the implants on days 5 and 6 was accompanied by considerable infiltration of inflammatory cells, and was probably mostly a result of invading host cells and inflammation. If size alone is used as a criterion for tumor proliferation in this subrenal capsule assay, day 4 seems to be the most appropriate for evaluation. This is supported by a strong expression of a proliferation marker, the human transferrin receptor, during this time.     

Experimental study of the 6-day subrenal capsule assay by cyclophosphamide pretreatment

For the purpose to suppress the host immune reaction in subrenal capsule assay (SRCA) using normal immunocompetent mice, the effects of cyclophosphamide (CPM) pre-treatment (CPM 180 mg/kg day-1 s.c.) were compared with cyclosporine A (CSA) treatment (CSA 60 mg/kg s.c. daily). CPM and CSA suppressed host reaction until day 6 and day 12, respectively. However, in the histological evaluation of day 6 tumor, there was no difference between two groups. Mouse serum levels of CPM were measured by a bioassay as indicated by the cytotoxicity against P388 cells, to evaluate its residual activity against implanted tumor. No cytotoxicity was observed in the serum of 36 hr. after CPM injection. The antitumor activities of several chemotherapeutic agents against serially transplanted human esophageal cancer xenograft (IMEs-1) in untreated control group and CPM pre-treated group of SRCA were compared with the subcutaneous transplantation assay in nude mice. In all agents, the drug activities in CPM pre-treated group were similar to that in nude mice assay system. The increase of toxicity in CPM pre-treated group was very light. The immunosuppressive activity of CPM were also tested in 14 clinical samples. CPM suppressed the host reaction satisfactorily in all samples. These results suggest the usefulness of CPM pretreatment for suppressing the host reaction in SRCA.     

Chemotherapeutic response of squamous cell carcinoma xenografts (subcutaneous and subrenal capsule assay)

Chemotherapeutic response of two squamous cell carcinoma xenograft lines (established from the primary and metastatic lesion of a tongue carcinoma) was studied using SC and SRC assays (as well as immunocompetent and -suppressed recipients in the latter assay). The two assays provided similar ranking of drugs, in the sense that in each instances two of the three (cyclophosphamide, 5-fluorouracil, vinblastine) most active agents were identical. The host response in immunocompetent recipients supports the need for histology to prove the proper quality of the implanted tumor tissue in order to be used for drug evaluation.     

Comparison between macroscopic and microscopic evaluation of tumour responsiveness using the subrenal capsule assay

Using the subrenal capsule assay in normal mice, a histologic evaluation was made of 8 human primary ovarian tumours and 3 human colon, 2 lung and 5 ovarian carcinomas growing in serial passage in nude mice. The results of the evaluation indicated that there is a tumour- and drug-dependent correlation between the macroscopically and microscopically evaluated effects, with cyclophosphamide demonstrating excellent concordance but adriamycin and cisplatin both demonstrating consistently more tumour cell killing on histologic analysis than could be appreciated macroscopically. Leukocyte infiltration and fibrosis were greatly increased by the latter 2 drugs, leading to unrepresentative macroscopic measurements. Variable amounts of host cell infiltration can also be demonstrated in the untreated control when normal mice are used. The use of nude mice decreases the discrepancy between macroscopic and microscopic evaluation.     

A study of 6-day subrenal capsule assay with cyclophosphamide pretreatment--histological evaluation of clinical samples

In order to suppress the host immune reaction in subrenal capsule assay (SRCA), effect of cyclophosphamide (CPM) was evaluated histologically in 25 fresh clinical samples. In 21 out of 25 clinical samples, the extent of lymphocyte infiltration was minimal, and in all samples the host reaction was suppressed significantly by CPM pretreatment. However, tumor cells were well preserved in only 10 out of 20 samples obtained from esophageal cancer patients. With the samples obtained from gastric and colorectal cancer patients, there were no tumor cells in the subrenal space on day 6. Persistence of tumor cells was well recognized in the samples having rich tumor and less stromal cells. The xenografts with moderately to well-preserved cancer cells showed an increase of the tumor size as expressed by delta TS. Though many xenografts showed a positive delta TS, preservation of tumor cells was poor. In each of the drug treated groups, a decrease of the tumor size and the histological effects were well correlated. It is suggested that 6-day SRCA with CPM pretreatment is useful as a chemosensitivity test, especially for the esophageal cancer whose tumor cells were well preserved.     

Evaluation of chemosensitivity in the 6-day subrenal capsule assay

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.     

Comparative study on the anticancer activities of KW2149 and mitomycin C against human tumor xenografts using subrenal capsule assay

The anticancer activity of KW2149, a new derivative of mitomycin C (MMC), was investigated against 5 human tumor xenografts derived from digestive organs using 4-day subrenal capsule assay (SRCA). Normal immunocompetent mice were used in this assay. For the comparative study, KW 2149 and MMC were administered intraperitoneally for 3 days after implantation, and the anticancer activity and the weight loss of mice were evaluated. The total doses were determined as 1/2, 1/3 and 1/4 of LD50 value of each anticancer agent. The anticancer activities of the two drugs were almost the same with no significant difference in 3 xenografts. Thus, it may be suggested the difference of the anticancer spectrum between the two drugs. The anticancer activity of KW2149 indicated higher correlation with the administered doses as compared with MMC. The toxicity of KW2149 was almost the same as MMC according to the weight loss of mice.     

Therapeutic efficacy of fosfomycin and cisplatin alone or in combination on human urogenital cancer by subrenal capsule assay

Experimental therapies of human urogenital cancer with fosfomycin and cisplatin were performed by subrenal capsule assay. The subrenal capsule assay was applied for 10 cell lines, consisted of 4 renal cell carcinomas, 2 renal pelvic carcinomas, 2 carcinomas of the urinary bladder, one prostatic carcinoma, and one testicular tumor. The efficacy of each drug was evaluated by measuring the tumor growth, and compared the mean tumor volume of treated with that of untreated groups. The growth inhibition rate in fosfomycin group was 60% and in cisplatin group was 80%, whereas that of the combination therapy of both fosfomycin and cisplatin was 50%. This indicates that the combination therapy reduced the antitumor effect. Further, the histological findings also showed a similar pattern in both kidney and tumor tissues between the treated and untreated groups.     

Rapid growth of human cancer cells in a mouse model with fibrin clot subrenal capsule assay

Rapid in vivo growth of cultured human cancer or leukemia cells was achieved by implantation into the subrenal capsule of mice. A solid structure, necessary for accurate implantation and measurement of tumor growth in this model, was provided by stepwise addition of fibrinogen and thrombin to the tumor cells, leading to rapid enzymatic formation of a solid tumor-fibrin matrix. Human leukemia and epithelial cancers increased in volume between 6- and 40-fold when measured 6-10 days after implantation into normal or immunosuppressed mice. Immunosuppression of host CD-1 mice was achieved by cyclosporine given daily after tumor implantation, cyclophosphamide given preimplantation combined with cyclosporine, or whole-body irradiation given preimplantation. Confirming the validity of tumor measurements, tumor histology in the immunosuppressed mice revealed cell proliferation, invasion, and neovascularization. Similarly, no artifactual measurement of tumor growth was observed by nonviable cancer cells, implanted after in vitro exposure to a known cytotoxic concentration of thiotepa. This model provides an economical, short-term technique for the in vivo study of human tumor growth, for the evaluation of new cancer therapies, and for in vitro - in vivo drug activity correlations in specific types of human cancer or leukemia cell lines.     

Fundamental study of subrenal capsule assay by measuring specific activity of succinate dehydrogenase]

It may not show accurate results if subrenal capsule assay (SRCA) is made only by measuring tumor size, because of infiltration of host inflammation cells resulted from host immune reaction. We developed a new method which make possible an accurate determination of chemosensitivity by measuring specific activity of succinate dehydrogenase (SD) of the tumor cells implanted in the subrenal capsular space. With reference to SDI test, the assay condition for measuring specific activity of SD was determined. A comparative study was carried out in which malignant tumors of the oral cavity serially transplanted in nude mice were tested with SRCA and subcutaneous transplantation assay in nude mice. Chemosensitivity to peplomycin (PEP), CDDP and 5-fluorouracil (5-FU) evaluated SSDI method and nude mouse assay showed a high correlation than those evaluated by TGIR method and nude mouse assay. The overall predictive accuracy compared with nude mouse assay was 72.2% by TGIR method and 88.9% by SSDI method. SSDI method seemed to be a useful method to evaluate the chemosensitivity in SRCA.     

复发耐药性非霍奇金淋巴瘤小鼠肾包膜下移植肿瘤药敏测定的临床意义

目的:探讨小鼠肾包膜下移植肿瘤药敏测定(SRCA)在复发耐药NHL治疗中的作用.方法:对25例CHOP方案化疗后出现复发耐药的NHL病人进行了SRCA药敏测定,并依据SRCA结果进行化疗.结果:NHL肿瘤细胞在小鼠肾包膜下能够快速生长,移植成功率100%;复发耐药NHL对CTX、VCR、ADM表现为耐药,而对VP_(16)、MTX、DDP敏感性较高,其中对VP_(16)的敏感性显著高于CTX和VCR(P<0.05);依据药敏结果化疗,25例病人6例CR、13例PR,总有效率76%;其中21例接受由VP_(16)、MTX、DDP和强的松组成的方案治疗,6例CR,11例PR,有效率达81%.结论:NHL适宜用SRCA法进行药敏测定;复发耐药NHL的治疗可以VP_(16)、MTX和DDP为基础;化疗前进行SRCA药敏测定,能够减少用药盲目性,提高有效率.     

Chemotherapy responsiveness of human breast tumors in the 6-day subrenal capsule assay: An update

Summary Feasibility of utilizing the 6-day subrenal capsule (SRC) assay to screen drugs against fresh surgical explants of human tumors was confirmed by testing six clinically active chemotherapeutic agents against 141 human breast cancers. Response rates of the six drugs obtained in the assay compared favorably with clinical response rates for the same drugs as reported by Carter (5). The evaluable assay rate for breast tumors was 92% as compared to 89% for gynecologic tumors. Innate drug resistance was indicated with 16 of 57 tumors (28%) which did not respond to any of the six agents tested. Differences in responsiveness of tumors to each agent in a potential three-drug combination of either CMF or CAF suggest that the effectiveness of multiagent therapy might be enhanced if the individual agents of a potential drug combination were selected on the basis of tumor sensitivity to each of the agents in a predictive assay. Although cross-resistance between L-PAM and cytoxan was demonstrated and was statistically significant, 31% of these tumors responded individually to either one or the other agent, suggesting caution in extrapolating concomitance in activity between these two alkylators.Presented in part at the 4th Annual San Antonio Breast Cancer Symposium in November 1981     

Analysis of chemosensitivity against a human breast cancer xenograft (MX-1) with the subrenal capsule assay in BDF1 mice

The antitumor activity of 27 anticancer drugs against a human breast cancer tumor (MX-1) has been studied using the subrenal capsule assay developed by A.E. Bogden et al. in 1978. Without immunosuppression with cyclophosphamide, BDF1 mice transplanted with MX-1 were treated with various drugs. The antitumor activity was evaluated by the tumor growth inhibition rate on day 6 after treatment. Among the 27 anticancer drugs tested, 10 compounds (37%) which showed more than 75% tumor growth inhibition rate were considered to be active. On the other hand, 8 compounds out of 27 drugs (30%) which showed less than 50% tumor growth inhibition rate were considered to be inactive. When the antitumor activity between the subrenal capsule assay in BDF1 mice and the subcutaneous transplantation assay in nude mice were compared, both assays were well correlated (r = 0.787, p less than 0.001). These results suggest that the antitumor activity of drugs can be evaluated faster, cheaper and easier by the subrenal capsule assay compared with the subcutaneous transplantation assay in nude mice.