Prediction of antifolate efficacy in a rat sarcoma model.

A methylcholanthrene-induced rat sarcoma propagated both in vitro and in vivo was used to examine the usefulness of a rapid biochemical in situ assay that measures thymidylate synthase (TS) activity in whole cells to predict sensitivity and resistance to the folate antagonists methotrexate (MTX), 10-ethyl-10-deazaaminopterin (10-EDAM) and trimetrexate (TMTX). There was an excellent correlation between the effects of these drugs on inhibiting TS and cytotoxicity as measured by a clonogenic assay, when the exposure time was 4 hr and those when the rat sarcoma cell line was employed (p less than 0.001). When tumor-cell suspensions were prepared from the rat sarcoma propagated in vivo, they were less sensitive to these antifolates, but the relative effectiveness of the 3 antifolates was similar: TMTX much greater than 10-EDAM greater than MTX. As expected, continuous exposure (10 to 12 days) produced cytotoxicity at a much lower dose of these drugs. When the 3 drugs were tested for anti-tumor effectiveness in rats bearing this tumor, the tumor regression measured was best with TMTX; 10-EDAM was intermediate in effectiveness as compared with MTX. These results indicate that the in situ TS assay and clonogenic assay may be used to predict anti-tumor efficacy of antifolates in vivo in this rat sarcoma.     

A comparison of three assays used for the in vitro chemosensitivity testing of human tumours.

In this study cell lines have been used to determine the level of correlation between three assays which are in use for in vitro prediction of human tumour chemosensitivity. The methods which were compared included a clonogenic assay, a monolayer assay and a short-term biochemical assay. The results indicated that the monolayer and clonogenic assays were either directly comparable or could be made comparable by reducing the drug exposure time in the monolayer assay. The biochemical assay also gave comparable results for 3 of the 5 drugs tested. It was concluded that although the 3 assays did not produce identical dose-response curves, the assays were equally valid when used for predictive testing because selection of cut-off points which were based on retrospective correlations between in vitro sensitivity data and response data, as established by other authors, compensated for differences in sensitivity between the assays.     

Human tumor clonogenic assay in osteosarcoma and its clinical evaluation

On twenty-three specimens from patients with osteosarcoma (biopsied tissue, resected specimens from primary and metastatic lesions), the human tumor clonogenic assay (HTCA: standard assay) as well as cultivated HTCA involving short-term cultivation of a single-cell suspension, was carried out. The percentage colony-forming ability, which was very small for standard HTCA, increased significantly when cultivated HTCA was performed. With cultivated HTCA, sensitivity tests were possible in 87% of the specimens, in contrast to only 28.6% with standard HTCA. Retrospective evaluation of the results obtained by cultivated HTCA with the clinical efficacy of anticancer drugs showed a true positive rate, 66.7%, and a true negative rate, 88.9%. This analysis revealed that drugs shown to be ineffective and that tumor cells are refractory to those drugs, so that clinical use of such drugs should be avoided.     

Susceptibility of chemoresistant murine and human tumor cells to lysis by interleukin 2-activated lymphocytes

The sensitivity of three different human and murine doxorubicin (Dx)-sensitive or -resistant pairs of tumor cells to recombinant interleukin 2 (rIL2)-activated lymphocytes was studied. In two pairs of these sublines (LoVo human colon carcinoma and B16 mouse melanoma sublines), resistance to Dx was induced in vitro, while in the third pair (9229 human metastatic melanoma clones), Dx resistance was spontaneously present in clone 9229.24. Dx-resistant cells were efficiently lysed by rIL2-activated lymphocytes in a short-term 51Cr release assay; in some experiments a trend toward higher lysis of Dx-resistant cells was present. We then tested the tumor cell growth-inhibitory activity of rIL2-activated lymphocytes in the human tumor clonogenic assay after lymphocyte-tumor coculture. Complete inhibition of tumor cell growth was obtained with five of six sublines or clones (both Dx sensitive and resistant) after 3 to 6 days of coculture at effector lymphocyte/target tumor cell ratios of 5 to 50/1; a maximum 99% inhibition was observed with the melanoma clone 9229.4 even after coculture for 6 days at an effector lymphocyte/target tumor cell ratio of 50/1. By using lower effector lymphocyte/target tumor cell coculture ratios (1, 5, 25/1), it was shown that all the three Dx-resistant cell types were significantly more affected by activated lymphocytes than their Dx-sensitive counterparts. The LoVo/DX subline was also more lysed than its Dx-sensitive counterpart LoVo/H subline by an antitumor monoclonal antibody in a complement-mediated cytotoxicity assay, despite the fact that both sublines expressed a similar amount of antigen on the cell surface. These data indicate that Dx-resistant cancer cells are more susceptible to the lysis by rIL2-activated lymphocytes than their Dx-sensitive counterparts and that a complete inhibition of their clonogenic potential can be obtained in vitro.     

Drug testing in established cell lines by flow cytometric vitality measurements versus clonogenic assay

The clonogenic assay is widely considered to be the most valid test for predicting tumor cell sensitivity to cytostatic drugs. In this study it was compared with early growth curves of human leukemic cell lines (HL-60, K562, Reh) after treatment with different types of cytostatic drugs (Adriamycin,cis-diamminedichloroplatinum(II):,1-beta-D-arabinofuranosyl- cytosine, and 5-fluorouracil) for 1 and 24 h. Following drug treatment two parallel cultures were started: a soft agar culture for the clonogenic assay; and a liquid suspension culture for vital cell counting by measuring esterase activity with fluorescein diacetate at different time points. The latter was recorded using flow cytometry during the following 3 days in 12-h intervals. For each drug concentration a survival factor was calculated from the growth curve between 24 and 72 h. This survival factor takes into account both the y intercept of the extrapolated growth curve and the slope of the growth curve. The dose-response curves resulting from either the survival factors or the clonogenic assay were always nearly identical. The results demonstrate that in established cell lines flow cytometric determination of vital cell increase rates provides a convenient alternative to the clonogenic assay for drug testing.     

Comparison of in vitro drug sensitivity by inhibition of tritium release from [5-3H]-2'-deoxyuridine and a clonogenic assay

In a search for a rapid and simple method to determine drug-induced cell lethality, the inhibition of tritium release from [5-3H]-2'-deoxyuridine (ITR) was compared with a standard clonogenic assay. Seven drugs were studied. After a 4 hr incubation period, [5-3H]-2'-deoxyuridine was added to cultures of human colon carcinoma cells (HCT-8) in vitro and four dose-response curves were generated for each drug by sampling the culture medium for tritiated H2O formation 4, 24, 48 and 72 hr later. These curves were compared to those obtained by a standard clonogenic assay. The ED50 values for 5 of the 7 drugs tested, methotrexate, 5-fluorouracil, doxorubicin, vincristine and cisplatin, as measured by inhibition of HCT-8 colony growth, were within 3 times the values observed with the metabolic assay. The ITR highly overestimated the cytotoxicity of 5-fluoro-2'-deoxyuridine: the irreversible inhibition of thymidylate synthase by this nucleoside resulted in an ED50 value 100-fold lower than that observed with the clonogenic assay. Opposite results were obtained with 5-fluorouridine. These data indicate that the ITR can be used to determine drug sensitivity of these cells to a host of compounds although it cannot be used indiscriminately for every antineoplastic agent as a cytotoxicity assay.     

Predictability of in vivo chemosensitivity by in vitro MTT assay with reference to the clonogenic assay

The MTT assay reported by Mosmann is a rapid and convenient colorimetric assay for cellular growth and survival in vitro. In this paper, the MTT assay was modified as a chemosensitivity test, and its potential was investigated. Using 10 human tumor xenografts in athymic nude mice, the predictability of in vitro antitumor effects of drugs using the MTT assay was compared with that using the clonogenic assay. The MTT assay showed excellent reproducibility, and the predictable rate in this assay was 86.7%, with 100% true-positive and 77.8% true-negative rates, almost equivalent to the 90.0% predictable rate of the clonogenic assay. This method also has several advantages with respect to rapidity, quantitation, management of many samples, and cell number required for the assay. Application of this assay to chemosensitivity testing seems to be valuable and useful.     

Pharmacodynamic aspects of in vitro and in vivo chemosensitivity tests

To determine the optimal conditions for clonogenic assay, the antitumor activities of 5-fluorouracil (5-FU) in vitro and in vivo were compared from a pharmacodynamic viewpoint in a human carcinoma xenograft-nude mouse system. In the clonogenic assay, tumor cells were exposed to 5-FU continuously for 2 weeks, and the results of the assay were evaluated in terms of colony survival rates (T/C). Tumor-bearing BALB/c male nude mice were treated with 5-FU, and the results were evaluated in terms of the lowest values of relative mean tumor weights (T/C). To compute the area under the curve (AUC) after administration of 5-FU in vitro and in vivo, agar and serum levels of 5-FU were measured by bioassay. Antitumor activities in terms of T/Cs against a gastric carcinoma strain (H-111) depended highly on the AUCs in vitro and in vivo. The T/C in vitro (z) was correlated with the AUC in vitro (w), z = 58.4 x 0.99331w, and the T/C in vivo (y) was also correlated with the AUC in vivo (x), y = 52.1 x 0.88552x. On the assumption that the T/Cs of these two regression equations were equivalent (y = z), a correlation between w and x was derived. To predict the T/C at the maximum tolerated dose of 5-FU in mice, the optimal drug concentration in vitro was calculated to be 2.7 micrograms/ml, which also proved to be suitable for seven other carcinoma strains. Our experimental system was thought to be adequate for selecting the optimal drug concentration in the clonogenic assay.     

Combined recombinant human interferon alpha 2 and cytotoxic agents studied in a clonogenic assay

A human tumor clonogenic assay has been used to test the antiproliferative effect of recombinant human leukocyte interferon alpha 2 alone and in combination with each of 8 cytotoxic agents. Cell lines derived from 6 human tumors and primary tumor cells from 13 patients have been used in these clonogenic assay studies. Results show that interferon as a single agent causes insignificant reduction in tumor cell colony survival if the short-term 1-hr cell exposure method is used; only high concentrations of interferon used in continuous cell exposure in the clonogenic assay can demonstrate a reduction in colony survival to below 50% of control values. Combinations of interferon with either doxorubicin or cisplatin frequently show additive and occasionally synergistic antiproliferative effects on tumor cell colony formation. Variations in drug concentrations and sequencing of drugs have been tested, showing that optimal antiproliferative effects of combined interferon and doxorubicin are realized when maximal concentrations of interferon and prolonged cell exposure time of both interferon and doxorubicin are employed. Combinations of interferon and doxorubicin tested in the clonogenic assay demonstrate cytotoxicity superior to that of either agent tested alone.     

Improvement of human tumor clonogenic assay using cell culture in soft part sarcoma of the extremity

The human tumor clonogenic assay (HTCA = standard HTCA) gives poor results in sarcomas compared to carcinomas. In order to overcome this problem, a cultivated HTCA, using single-cell suspensions obtained from short-term culture, was developed. In forty specimens taken from 32 patients with soft part sarcomas of the extremities, both standard and cultivated HTCA were performed. Colony-forming ability was low in standard HTCA (0.0053 +/- 0.0051%) while it was high in cultivated HTCA (0.0231 +/- 0.0186%). The number of clonogenic assays suitable for sensitivity tests was low (23.7%) in standard HTCA, while the number was much higher (70.0%) in cultivated HTCA. The histological grade of malignancy (low, moderate, high) was correlated with the growth of colonies in both methods of HTCA; cultivated HTCA, versus HTCA, increased the number of colonies produced in the high and moderate groups of malignant sarcomas. The results of the sensitivity of 8 anticancer drugs on sarcomas were almost the same between the two methods. A retrospective study on the results of the cultivated HTCA and clinical chemotherapeutic effectiveness proved a significant overall correlation rate (91.7%, p less than 0.025). It was concluded that in tumors of a low colony-forming ability such as soft part sarcomas, cultivated HTCA can provide a useful chemosensitivity test.     

A murine model to evaluate the ability of in vitro clonogenic assays to predict the response to tumors in vivo

The use of the human tumor cloning assay as a predictor of clinical response of human tumors to drugs is predicated on the hypothesis that the in vivo response of a tumor to a drug can be correlated with the in vitro response of cells derived from the tumor. To test this hypothesis, we utilized a murine tumor model in which the in vivo and in vitro responses of a tumor can be accurately and reproducibly compared. Drug activity was assessed in P388 leukemia with the standard in vivo antitumor assay (i.p. tumor/i.p. drug administration) and an in vitro assay wherein the ascites tumor cells are removed from mice, treated with a drug, and directly cloned in soft agar to measure clonogenic capacity. The response of P388 cells to analogues within four separate classes of antitumor agents, anthracyclines, anthraquinones, platinum(II) coordination complexes, and phosphinogold(I) complexes was evaluated. The clonogenic assay failed to discriminate between highly active in vivo antitumor agents and analogues with only marginal in vivo efficacy (i.e., doxorubicin and daunorubicin versus rhodomycins A and B, ametantrone versus NSC 276740, cisplatin versus transplatin, [Au(dppe)2]Cl versus [Au(depe)2]PF6. Furthermore, the in vitro clonogenic assay failed to detect carboplatin which was a highly active agent in vivo. The basis for these discrepancies was explored by a more detailed comparison of doxorubicin and rhodomycin B. In vivo or in vitro drug exposure with subsequent measurement of cell kill by the in vitro clonogenic and in vivo tumorigenic assay demonstrated that the in vitro assay overestimated the cytotoxic potency of the drugs relative to the tumorigenic assay. Treatment of tumors in vivo with doxorubicin at doses below the maximally tolerated dose in mice resulted in multiple log cell kill as measured in vitro or in vivo, whereas rhodomycin B was cytotoxic only at dose levels exceeding its maximally tolerated dose. The results indicate that a subset of tumor stem cells capable of forming colonies in soft agar are significantly more sensitive to the cytotoxic effects of anthracyclines than are in vivo tumorigenic stem cells. Cytotoxic potency as measured by an in vitro soft agar clonogenic assay is not an accurate predictor of in vivo antitumor efficacy even in a model in which ascites tumor cells are directly exposed to i.p. drug. The in vitro cytotoxicity assay is useful only as a nonselective prescreen and must be used in combination with other indicators of tumor cell selectivity and dose-limiting organ toxicity.     

Accurate prediction of experimental cancer chemosensitivity using the subrenal capsule xenograft assay

The chemosensitivity of a murine lung carcinoma grown as a xenograft under the kidney capsule of a rat was compared to the chemosensitivity of this tumor grown as a spontaneous metastasis in its syngeneic host. The chemosensitivity of the tumor to intravenously or subcutaneously administered drugs determined in the short-term xenograft assay accurately predicted the chemosensitivity of this tumor when it was growing as a spontaneous micrometastasis in its syngeneic host, indicating that in both situations the pharmacological determinants of tumor response to anticancer drugs were similar. These results suggest that tumors growing as xenografts under the kidney capsule accurately reflect the responsiveness of these tumors to chemotherapy and support further investigation of the xenograft subrenal capsule assay as a model that may be useful in predicting effective chemotherapy for human tumors.     

Relationship between the in vitro colony growth of human tumors and cytologic diagnosis

The cytologic examinations and cloning efficiencies of 47 human tumor specimens (19 ovarian carcinomas, 6 sarcomas, 5 lung carcinomas, and 17 miscellaneous tumors) were compared to evaluate the predictability of clonogenecity by cytologic diagnosis. Cytologic examination preceding clonogenic assay identified the 50% to 60% of all specimens that failed to yield in vitro chemotherapy sensitivity by this assay, with a false-negative rate of 10%. The frequency of cytologically identifiable tumor cells in the plating suspension was independent of the histologic type of tumor (ovarian carcinoma or others) and the nature of the specimen (solid tumor or malignant effusion). Nearly 66% of all specimens were cytologically negative. The in vitro sensitivity can be assessed in approximately 75% of cytologically positive specimens. Cytologic evaluation preceding soft-agar clonogenic assay of human tumors can reduce cost and effort by predicting colony-producing specimens.     

The role of MAPK pathways in the action of chemotherapeutic drugs

In this study we have investigated the role of mitogen-induced and stress-activated MAP kinase pathways in the cellular response to taxol, etoposide and ceramide in three different human cancer cell lines: HeLa cervical carcinoma, MCF7 breast cancer and A431 squamous carcinoma cells. The mitogen-induced ERK MAPKs were linked to cell proliferation and survival, whereas the stress-activated MAPKs, p38 and JNK, were connected with apoptosis. Our results show that all drugs activated MAPKs, but that the extent and kinetics of activation were different. In order to assay the biological consequences of drug-induced MAPK activation we employed selective MAPK inhibitors and measured both long-term clonogenic survival as well as short-term parameters including apoptosis, mitochondrial metabolic integrity and cell cycle progression. Our results show that drug induced toxicity is not correlated with any singular parameter, but rather a combination of effects on cell cycle and apoptosis. In certain constellations the modulation of MAPK pathways could enhance or decrease drug efficacies. These effects mainly pertained to the regulation of apoptosis and clonogenic survival, but they were highly dependent on the combination of drug and cell line without any clear patterns of correlations emerging. These results suggest that the modulation of MAPK pathways to enhance the efficacy of chemotherapeutic drugs is of limited value unless it is tailored to the specific combination of drug and cancer.     

Pharmacokinetics and plasma protein binding of two platinum cytostatics CHIP and CBDCA in rats

Summary Twenty-three specimens (biopsied tissue, resected specimens, and resected pulmonary metastases were obtained during biopsy, radical surgery, or thoracotomy from patients with osteosarcoma. Using these specimens, human tumor clonogenic assay (standard HTCA) and cultivated HTCA (HTCA using single-cell suspensions obtained from short-term cultures) were carried out. The percentage of colony-forming ability, which was very small for standard HTCA, increased significantly when cultivated HTCA was performed. With cultivated HTCA, sensitivity tests were possible in 87% of the specimens in contrast to only 28.6% using standard HTCA. We retrospectively analyzed the relationship between the results of HTCA and the clinical efficacy of agents such as methotrexate, adriamycin, cis-platinum, and vincristine, which are frequently prescribed for the treatment of osteosarcoma. This analysis revealed that if drugs are show to be ineffective by this test, they can be regarded as clinically ineffective against that specific tumor target, i. e., the tumor cells are refractory to those drugs. Thus, clinical use of such drugs should be avoided.     

In vitro evaluation of anticancer drugs in relation to development of drug resistance in the human tumor clonogenic assay

Summary The efficacy of anticancer drugs against ovarian cancer, breast cancer, and colorectal cancer has been evaluated in vitro by the human tumor clonogenic assay developed by Hamburger and Salmon. The in vitro colony assay method used in this study is a minor modification of their method and was used in 83 patients with ovarian cancer, 47 patients with breast cancer, and 13 patients with colorectal cancer. The total numbers of assays performed in vitro were 258 for ovarian cancer, 87 for breast cancer, and 38 for colorectal cancer. The average chemosensitivity rates to single agents tested were 35% and 32% in the untreated patients with ovarian and breast cancer, respectivety. In contrast to this result, the chemosensitivity rate of the untreated patients with colorectal cancer was only 16%. Consisting the clinical efficacy of anticancer drugs against these tumors, these results suggest that there is a correlation between chemosensitivity in the human tumor clonogenic assay and clinical responsiveness. In this assay the chemosensitivity in specimens from ovarian cancer patients who had had prior chemotherapy was significantly lower than in those from nonpretreated patients (P0.05). This seems to indicate the development of drug resistance after treatment with anticancer drugs. These results suggest that the human tumor clonogenic assay is a useful tool for the evaluation of antitumor effects of drugs in vitro.     

Kinetics of cellular inactivation by fractionated and hyperfractionated irradiation in Lewis lung carcinoma

The kinetics of cellular inactivation by fractionated irradiation in Lewis lung carcinoma was studied in the dose range of 2.3 to 6.5 Gy per fraction. Regimens of one and two fractions per day for 10 days were compared. The number of clonogenic tumor cells was determined using an in vitro soft-agar colony assay. Under the experimental conditions used, the fraction of tumor cells inactivated per day was only dependent on the total dose per day, i.e., the cellular response was the same whether the daily dose was given in one or two fractions. Thus, the two fraction per day regimen was more effective than expected from calculations based on acute in vivo radiation dose-survival curves. This result could be explained if the clonogenic tumor cells were less hypoxic during the two fractions per day regimen than the one fraction per day schedule.     

Development of an XTT tetrazolium salt-based assay for detection of specific hyperthermia sensitizers in a high-flux screening programme

It is now possible to search for new drugs using high-throughput screening of chemical libraries accumulated over the past few years. To detect potential new hyperthermia sensitizers, we are screening for chemical inhibitors of thermotolerance. For the screening of a large chemical library, a rapid and simple assay based on the XTT-tetrazolium salt with the addition of intermediate electron acceptor, phenazine methosulphate (PMS) as a promoter, was developed. It was found that the sensitivity of the XTT/PMS assay is sufficient for assessing thermal cell killing and thermotolerance, although it was highly dependent on cell number and type. When the formazan assay system was challenged with the bioflavonoid drug quercetin (up to 25mm) and validated against the clonogenic cell survival assay, significant decreases in thermotolerant cell viability were observed, directly reflecting inhibition of thermotolerance. Although short-term assays can, in some instances, underestimate overall cell killing, the dose dependency of inhibition of thermotolerance by quercetin recorded in this study by clonogenic and XTT/PMS assays was similar. Application of the XTT/PMS assay in chemical library screening was highly effective in differentiating potential thermotolerance inhibitors from both chemicals with lack of efficacy and from toxic compounds. Taken together, these results show that the XTT/PMS assay, when carried out under careful conditions, is well suited for primary high-flux screen of many thousands of compounds, thus opening up new areas for discovery of hyperthermia sensitizers.     

乳酸脱氢酶检测化疗药物敏感性与MTT法及集落形成试验的比较

用集落形成试验、MTT吸收光度法和乳酸脱氢酶(LDH)测定3种方法检测5种常用抗肿瘤药物——阿霉素(ADM)、5—氟脲嘧啶(5—Fu)、丝裂霉素(MMC)、长春新碱(VCR)和氨甲喋呤(MTX)对人宫颈癌Hela细胞系的细胞毒作用。显示三种方法的检测结果有良好的相关性,均以ADM和MMC最有效。在这三种方法中,以集落形成试验最敏感;MTT法成功率高,实用于临床选择有效的化疗药物;而用药后LDH值的升高则是化疗有效的可靠指标。     

Development of an XTT tetrazolium salt-based assay for detection of specific hyperthermia sensitizers in a high-flux screening programme.

It is now possible to search for new drugs using high-throughput screening of chemical libraries accumulated over the past few years. To detect potential new hyperthermia sensitizers, we are screening for chemical inhibitors of thermotolerance. For the screening of a large chemical library, a rapid and simple assay based on the XTT-tetrazolium salt with the addition of intermediate electron acceptor, phenazine methosulphate (PMS) as a promoter, was developed. It was found that the sensitivity of the XTT/PMS assay is sufficient for assessing thermal cell killing and thermotolerance, although it was highly dependent on cell number and type. When the formazan assay system was challenged with the bioflavonoid drug quercetin (up to 25mm) and validated against the clonogenic cell survival assay, significant decreases in thermotolerant cell viability were observed, directly reflecting inhibition of thermotolerance. Although short-term assays can, in some instances, underestimate overall cell killing, the dose dependency of inhibition of thermotolerance by quercetin recorded in this study by clonogenic and XTT/PMS assays was similar. Application of the XTT/PMS assay in chemical library screening was highly effective in differentiating potential thermotolerance inhibitors from both chemicals with lack of efficacy and from toxic compounds. Taken together, these results show that the XTT/PMS assay, when carried out under careful conditions, is well suited for primary high-flux screen of many thousands of compounds, thus opening up new areas for discovery of hyperthermia sensitizers.