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PubMed1982

[Human tumor stem cell assay].

Human tumor stem cell assay is an in vitro colony-forming technique. Double soft agar layers are used for culture tumor cells and cell lethality is judged by the numbers of colony formation in this assay. Single-cell suspension made from various malignant materials in cancer patients is placed in culture after exposing to various anticancer agents for one hour and incubated for two weeks. Antitumor effects of various anticancer agents against individual patients are evaluated by % inhibition of colony formation. Of 57 tumor specimens 42 (74%) formed at least five colonies per plate (per 0.5 x 10(6) cells). The colony-forming rates of various malignancies are as follows: breast cancer 14/15 (93%), ovarian cancer 8/10 (80%), stomach cancer 5/13 (38%), sarcoma 4/5 (80%), lung cancer 1/4 (25%), colon cancer 3/3, each of pancreas cancer, leukemia and primary unknown adenocarcinoma 2/2, malignant lymphoma 1/1. The median plating efficiency (number of colonies/number of nucleated cells plated) is 0.02% (range: 0.001-0.3%). High correlation between human tumor stem cell assay results and response of an individual patient's tumor to chemotherapy is reported by Salmon and Von Hoff. Human tumor stem cell assay is useful tool for the high prediction of chemosensitivity response.

Antineoplastic Agents↗
PubMed1982

[Colony formation of solid tumors in in vitro colony assay (human tumor stem cell assay)].

Colony forming ability of solid tumor cells was studied in a tumor colony assay (human tumor stem cell assay). In 50 cases of solid tumors, cloning efficiencies of 5 X 10(5) cells plated were as follows: breast cancer 12/12 (100%), colon cancer 10/11 (91%), ovarian cancer 9/9 (100%), sarcomas 7/9 (78%), gastric cancer 3/6 (50%), endometrial cancer 2/2 and pancreatic cancer 1/1. An overall cloning efficiency was 88% (44/50) and this rate is higher than those reported in literatures. Ovarian cancer showed the highest plating efficiency of 0.07% (number of colonies/number of cells plated X 100%) in various solid tumors tested. Subsequently, plating efficiencies of colon and breast cancer were 0.03 and 0.01%, respectively. In the cases of sarcomas and gastric cancer, low plating efficiencies were seen (0.008%, 0.003%). The overall rate succeeded colony growth of solid tumors was somewhat higher in enzymatically treated tumor cells, that is, cloning efficiencies in mechanical and enzymatic methods were 85 and 90%, respectively. The enzymatic disaggregation is an advantageous method in gastric cancer and sarcomas. Various solid tumors can be formed colonies in soft agar and chemosensitivity test using in vitro colony assay is expected in solid tumors.

Breast Neoplasms↗
PubMed1984

Storage of chemotherapy drugs for use in the human tumor stem cell assay.

Tumor chemosensitivity assays require the frequent in vitro use of antineoplastic drugs. Economy and convenience are greatly enhanced if these drugs are stored in aliquots for use as needed. This study investigated the stability of eight common antineoplastic agents at -60 degrees C. Activity was measured as inhibition of colony formation in a commonly used soft agar culture system using an experimentally induced murine sarcoma. Our results indicate no loss of activity with these drugs under the specified storage conditions.

Animals↗
PubMed1984

Potentials and drawbacks of the human tumor stem cell assay.

The human tumor stem cell assay (HTSCA) provides a means of performing drug sensitivity measurements on human tumor cells in primary culture. Results from such assays offer potential for improving cancer chemotherapy by identifying drugs useful for treatment of individual patients' tumors and through application to screening new compounds for antitumor activity. While existing data supports the potential of the assay in both areas, the assay also poses significant drawbacks. Many of these drawbacks relate to technical aspects of the assay and can be eliminated or reduced by further assay development. In this paper, we describe some of the technical drawbacks in detail and some approaches which have been successful in minimizing them. Continued advances in this area should make it possible to more fully realize the potential of the human tumor stem cell assay.

Animals↗
PubMed1982

Human colorectal carcinoma: patterns of sensitivity to chemotherapeutic agents in the human tumor stem cell assay.

The human tumor stem cell assay (HTSCA) was applied to 103 primary and metastatic colorectal carcinomas. Thirty-four carcinomas could not be evaluated for colony formation (clonogenicity) because of microbial contamination. Of the remaining 69 carcinomas, 18 (26%) demonstrated clonal growth in vitro. Colony formation did not correlate with the clinicopathologic stage of the tumor, the histological grade of the tumor, the method used to disperse the solid tumors into single cells (mechanical or enzymatic), or cell viability (exclusion of trypan-blue) prior to plating. The in vitro chemotherapy sensitivity data of the 18 tumors which formed colonies indicated that most tumors were generally resistant to chemotherapeutic drugs and that a few tumors were sensitive to multiple agents. Use of the human tumor stem cell assay may facilitate an individualized approach to clinical chemotherapy of colorectal carcinoma for certain patients.

Adenocarcinoma↗
PubMed1981

Chemotherapy of ovarian cancer directed by the human tumor stem cell assay.

The human tumor stem cell assay (HTSCA) has been used to study the in vitro sensitivity rates of anticancer drugs used in the treatment of 115 patients with previously untreated and relapsing ovarian cancer. The data from these studies have identified patterns of cross resistance and residual sensitivity between these agents, and have allowed the prospective selection of single agents possessing in vitro activity for the treatment of 32 patients with relapsing disease. cis-Platinum and vinblastine were the most active agents in vitro against ovarian TCFUs from both previously untreated and relapsing patients. Prior therapy with even one drug was associated with the acquisition of resistance to several classes of compounds (e.g., melphalan resistance was almost always associated with in vitro adriamycin resistance, P less than 0.001). A clinical trial yielding similar data would have required nearly 450 evaluable ovarian cancer patients. In 11 of 32 patients in vitro testing predicted sensitivity to single agents: eight of these had partial remissions for a predictive accuracy of 73%. In 33 instances the HTSCA had 100% accuracy in predicting the lack of clinical response. Thus, the HTSCA for advanced ovarian cancer appears to have a similar predictive accuracy rate to the estrogen receptor assay for predicting the response to hormonal therapy for disseminated breast cancer.

Antineoplastic Agents↗
PubMed1992

Cloning of human tumor cell lines in porous glass capillary tubes: a further development of the human tumor stem cell assay.

The conventional human tumor stem cell assay for cloning tumor cells for drug sensitivity testing is limited by its inability to test drug combinations. In an attempt to overcome this limitation, we cloned tumor cell lines within porous glass capillary tubes. In contrast to plastic porous tubes, the porous glass membranes were transparent, and colony formation could be judged on an inverted microscope. Human as well as animal cell lines showed sufficient colony growth. Colonies formed within these porous tubes were homogeneously distributed, and their morphology was similar to those formed in the common stem cell assay. Cloning efficiency and colony size depended on the mean pore diameter of the glass membrane, with best colony growth within tubes with a pore diameter ranging from 8.5 nm to 14 nm. A linear relationship between number of cells seeded and number of grown colonies could be demonstrated for the cell lines MDA-231 and Colo 201. Colony growth achieved within porous glass capillary tubes is comparable to that achieved in Petri dishes and in nonporous tubes. We conclude that the porous capillary cloning system meets the basic suppositions for a quantitative cloning assay. Moreover, the porosity of the glass membrane offers the possibility of variable perfusion of medium and drugs. Further investigations will focus on various perfusion modalities and chemosensitivity testing.

Animals↗
PubMed1985

The human tumor stem cell assay revisited.

The human tumor stem cell assay (HTSCA) is a bilayer soft agar system for growing fresh human tumor specimens in vitro to determine drug sensitivity and improve our understanding of tumor biology. Recent clinical correlations of 60% accuracy for predicting a positive clinical response and a 90% accuracy for predicting a lack of response to therapeutic agents suggest promising clinical usefulness. However, the clinician should be aware of the assay's inherent pitfalls, such as heterogeneity of the tumor specimen, inability to obtain pure single-cell suspensions, low cloning efficiency, unusual drug dose-dependent survival curves, uncertain validity of in vitro pharmacology, non-standardized criteria for in vitro sensitivity, and the variability of in vitro results. A brief summary of the concepts, potential, and limitations of this assay are discussed.

Animals↗
PubMed1983

The role of the human tumor stem cell assay in medical oncology.

The human tumor stem cell assay is a technique that allows in vitro sensitivity testing of antineoplastic agents against cells from tumor specimens removed from patients. This assay predicts clinical response to drugs and permits individualization of chemotherapy. It is more accurate in predicting drug resistance than drug sensitivity. Although there are technical problems with the assay, it has been successfully applied to nearly every possible type of human solid tumor and can be performed using cells from malignant effusions as well. Screening of new agents for activity against a range of human tumor specimens may prove to be the most important application of the human tumor stem cell assay.

Antineoplastic Agents↗
PubMed1981

Pharmacologic studies of anticancer drugs with the human tumor stem cell assay.

To optimize the human tumor stem cell assay (HTSCA) for clinical and research purposes we have carried out in vitro pharmacology studies. Useful observations were made in four areas. (1) Drug assay design: The predictive accuracy of the HTSCA depends on the in vitro testing of drug concentrations of less than 10% of those which are pharmacologically achievable with standard in vivo drug doses. The use of unrealistically high in vitro concentrations can accurately predict clinical drug resistance, but is likely to yield high false-positive rates of clinical response prediction. (2) Drug scheduling: For certain schedule-dependent drugs, as well as those with a prolonged plasma half-life and those used according to a repeated daily schedule, prolonged in vitro exposure (rather than 1 h) may be needed to provide an adequate in vitro design. For an accurate prediction of sensitivity of tumor colony-forming units (TCFUs) to continuous drug contact in the agar, concentrations should be in the range of 1/300 that used for the standard 1-h exposure prior to plating. (3) Drug combinations: In preliminary studies of combination chemotherapy in vitro we commonly observed at least additive effects with low doses of cis-platinum plus either vinblastine or adriamycin. (4) Drug bioactivation: Rat liver microsomes or S-9 fraction were used to activate cyclophosphamide for in vitro effect, and satisfactory dose-response curves were observed for the inhibition of TCFUs. Such pharmacologic studies will be required for a wide variety of standard and new agents and will probably become a regular aspect of investigation of new anticancer drugs.

Antineoplastic Agents↗
PubMed1983

Effects of cloned human leukocyte interferons in the human tumor stem cell assay.

Clonogenic tumor cells from fresh biopsies of human cancers were cultivated in vitro and tested for sensitivity by continuous exposure to pharmacologically achievable concentrations of either of two highly purified human leukocyte interferon subtypes (IFN-alpha A and IFN-alpha D) prepared by recombinant DNA methods. The interferons were compared on a weight basis at concentrations of 0.4 and 4.0 ng/ml (equivalent to 80 and 800 units of interferon activity for IFN-alpha A and 2.0 and 20 units for IFN-alpha D). Inhibition of tumor colony-forming units (50% of control or less) was observed in 38.1% of the 273 tumors tested against IFN-alpha A, and in 16% of the 71 tumors tested against IFN-alpha D. Of the tumor types with at least ten samples tested against IFN-alpha A, the percentage of cases exhibiting inhibition was as follows: melanoma (51.7%), lung cancer (50%), myeloma (33.4%), ovarian cancer (33.9%), sarcoma (33.3%), adenocarcinoma of unknown primary (30.4%), breast cancer (28%), acute leukemia (30.8%), and renal cancer (23%). More marked inhibition (30% of control or less) was observed in 18.7% of all tumors tested against IFN-alpha A. Of 60 melanomas tested, 18 (30%) exhibited marked in vitro inhibition of growth with IFN-alpha A. Although a smaller number of tumors (71) were tested against IFN-alpha D on a weight basis, it appeared, in general, to be slightly less active than IFN-alpha A (p less than 0.01), and only 8% of tumors tested exhibited marked inhibition over the same dosage range of interferon. Comparison of the dose-response curves for the 68 tumors tested simultaneously against both interferons did not reveal marked interpatient differences in the inhibition curves, although IFN-alpha D was slightly less active overall. Tumors exhibiting at least 50% inhibition of tumor colony formation also proved to be sensitive to a significantly larger number of cytotoxic drugs (tested simultaneously) than the tumors not inhibited with interferon (p less than 0.0001 for IFN-alpha A). We conclude that the in vitro clonogenic assay may aid in targeting tumor types most likely to exhibit interferon sensitivity and assist in case selection for entry into clinical trials with cloned interferons.

Antineoplastic Agents↗
PubMed1984

Use of the human tumor stem cell assay to study chemotherapy sensitivity in cancer of the cervix.

Two questions concerning chemotherapy of cervical cancers are addressed in this study. 1) Can the human tumor stem cell assay identify active chemotherapeutic agents?; and 2) Can the human tumor stem cell assay predict which patient's tumors will demonstrate clinical responsiveness to chemotherapy? Sixty-seven of 76 cervical cancer specimens (88.2%) were grown in the human tumor stem cell assay, resulting in 159 separate drug assays. Based on the known activity of standard chemotherapeutic agents demonstrated in clinical trials, cisplatin, bleomycin, vincristine, and mitomycin C were chosen for testing. In vitro sensitivity patterns, seen at one-tenth of the peak plasma drug concentration, were similar to those seen in historic clinical trials. Retrospective correlations between in vitro drug sensitivity data and patient responses to chemotherapy are available from 13 patients, demonstrating clinically helpful predictive capabilities of the assay. Two conclusions were made: 1) The human tumor stem cell assay can determine single agent activity, which is often dose related; and 2) Useful correlations with clinical responses were observed.

Adenocarcinoma↗
PubMedDate not supplied

New drugs in ovarian cancer and malignant melanoma: in vitro phase II screening with the human tumor stem cell assay.

The successful development of a soft agar clonogenic assay for human tumor stem cells provides an in vitro technique with a high degree of accuracy for predicting in vivo clinical response to standard anticancer drugs. We used this system to conduct an "in vitro phase II trial" in human ovarian cancer and melanoma. This approach can potentially identify active phase I--II drugs suitable for treatment of given tumor types for specific patients and eliminates the need to subject patients (who would be predicted not to respond) to toxic side effects. In vitro sensitivity for new agents was operationally defined as at least a 70% reduction of tumor colony-forming units (TCFU) at concentrations which are readily achievable pharmacologically. The new agents AMSA and vindesine (as well as vinblastine) appeared to have activity in melanoma, while PALA and thymidine were inactive. Pentamethylmelamine, mitomycin C, methyl-GAG, and AMSA were relatively ineffective in ovarian cancer. Vinblastine and vindesine had definite activity. The human tumor stem cell assay may thus provide the basis for a useful alternative to the current clinical phase II testing approach for identifying antitumor activity of new agents. Validation of this concept with correlative in vitro and in vivo phase II trials of new agents in patients with tumor types predicted to be sensitive is clearly warranted.

Antineoplastic Agents↗
PubMed1983

Tumor stem cell assay for detecting metastases of human lung cancer.

We applied a tumor stem cell assay using an enriched double-layered soft agar system for the detection of metastatic sites of lung cancer. Lung cancer colonies grew from 7 of 10 effusions cytologically positive for tumor cells and 7 of 10 bone marrow aspirates cytologically and histologically positive for tumor cells. Twenty-six of 29 bone marrow aspirates cytologically and histologically negative for tumor cells showed no colony growth. However, the remaining three bone marrow aspirates, which were obtained from patients with small cell lung cancer, formed colonies in soft agar. These results indicate that the tumor stem cell assay is useful for detecting metastatic sites of lung cancer.

Bone Marrow↗
PubMed1984

Antiproliferative activity of human interferons against ovarian cancer cells grown in human tumor stem cell assay.

Fresh biopsies from 14 of 22 (64%) ovarian carcinomas cultivated in the human tumor stem cell assay (HTSCA) were sensitive (greater than 70% inhibition in cell growth) to human interferons (HuIFNs). To achieve 70% inhibition of colony growth, 500 units/ml of a naturally produced IFN-alpha or IFN-alpha A were required in 71% of the sensitive specimens. The antiproliferative potencies of five IFNs were evaluated including two native alpha IFNs, two highly purified cloned subtypes of IFN-alpha, IFN-alpha D and IFN-alpha A, and one native fibroblast-derived beta interferon (IFN-beta). The antiviral activity of the IFN-alpha as determined by a human cell target correlated with their relative antiproliferative action. IFN-alpha D had minimal inhibitory effect at the highest concentration tested, while three IFN-alpha with high antiviral activities were equivalent with respect to growth inhibition in the HTSCA. Although instability could not be eliminated as a contributing factor, IFN-beta had significantly less growth inhibitory potency for cells from ovarian cancers when compared simultaneously with native IFN-alpha in the human tumor stem cell assay (HTSCA). Assuming direct antiproliferative effects are primary, future clinical trials evaluating IFN-alpha in ovarian cancer may require high titers of IFN.

Cell Division↗
PubMed1980

Applications of the human tumor stem cell assay to new drug evaluation and screening.

The potential applications of the human tumor stem cell assay to primary drug screening and Phase I-II new drug evaluation are of very broad scope and have not been completely addressed in this exploratory analysis. As in most other areas of science, careful and stepwise testing in many laboratories will be required to confirm or refute the utility of these approaches. Of the two areas discussed, the applications to new drug screening are still most uncertain and subject to change. From my perspective, in vitro growth conditions, for clonogenic human tumor cells, are perhaps at a similar stage of development as culture techniques for bacterial cells were in the 1930s. Availability of those techniques, and a combination of good ideas, serendipidy, and hard work led to the subsequent development of penicillin and other antibiotics which now can be used to cure most forms of infectious disease. The value of a simple and reproducible assay for screening therefore cannot be overemphasized. Applications of the tumor stem cell assay to Phase I-II drug evaluation already show preliminary signs of utility. Given this background and the more limited preclinical data on other agents now under study, broadened testing of this approach to new drug screening appears warranted. If use of an in vitro approach such as this proves successful for both primary and secondary screening, it would not be surprising if significant qualitative and quantitative changes occurred in the areas of new drug development and cancer clinical trials.

Antineoplastic Agents↗
PubMed1988

Detection of small cell lung cancer bone marrow metastases by tumor stem cell assay.

Detection of small cell lung cancer (SCLC) bone marrow (BM) metastases has a prognostic implication in itself and a therapeutic interest in the setting of autologous bone marrow transplantation. In a prospective study involving 68 bone marrow samples, we compared SCLC detection results obtained using a tumor stem cell assay and those obtained using conventional morphology by light microscopy. In agar, tumoral cells were stimulated either by Salmon's conditioned medium or by epidermal growth factor (EGF). Tumoral clonogeneic cells were detected in 11 cases, although 7 of these were considered negative when investigated by light microscopy. On the contrary, metastases were detected by morphology in 8 instances where no growing colonies were revealed by tumor stem cell assay. The choice of stimulating factor did not seem critical since the number of colonies was similar in all the cases. We conclude that the tumor stem cell assay is useful in the detection of small cell lung cancer bone marrow metastases, particularly in cases where these metastases have failed to be recognized through light microscopy investigation. However, the sensitivity of this assay is low and should be complemented by other techniques such as immunodetection.

Bone Marrow Cells↗
PubMed1987

An improved human tumor stem cell assay in ovarian cancer.

To evaluate the effect of improved growth rates of ovarian cancers in the human tumor stem cell assay and its value in predicting clinical chemotherapy response, we studied 59 assays in 54 patients. A total of 81.6% of solid specimens and 85.7% of ascites specimens were successfully cultured and yielded an overall growth rate of 82.9%. Simultaneous primary and metastatic cultures were concordant for chemosensitivity in 80% (n = 16). The patients were evaluated for previous chemotherapy, residual volume of tumor, histologic type, and grade, and these were not statistically different between clinical responders and nonresponders. In vivo-in vitro correlations were made in 27 patients and yielded a predictive response of 13% and predictive resistance of 86% at 70% colony inhibition and 31% and 71% at 50% colony inhibition. Improved growth rates therefore did not result in better predictive correlations. The reported experience in ovarian cancer is summarized and the current status of the human tumor stem cell assay is reviewed.

Colony-Forming Units Assay↗