agar and Ovarian-Neoplasms

Drug-tolerant persister cancer cells (PCCs) play an important role in the development of multidrug resistance (MDR) to anti-cancer drugs. This is due to the strong link between PCCs formation and epithelial-mesenchymal transition (EMT), as well as the low numbers of PCCs. In addition, PCC removal by traditional cytotoxic agents is poor due to the intrinsic high MDR activity in these cells. As a novel programmed cell death pathway, ferroptosis shows high potency to eliminate cells at the EMT state via manipulating intracellular redox homeostasis. The aim of this work was to utilize triggered ferroptotic polymer micelles for PCCs removal and MDR reversal both in vitro and in vivo. The micelles were made of arachidonic acid-conjugated amphiphilic copolymer that can enable rapid cargo release upon free radical-triggering in the tumor microenvironment. A potent ferroptotic inducer, RSL3 was encapsulated in the micelles to target the glutathione peroxidase 4 (GPX4). In the model resistant human ovarian adenocarcinoma cells, the RSL3 micelles were 30-fold more toxic than activatable control micelles due to the ferroptotic machinery. The lipid peroxidation-induced intracellular glutathione level reduction also made a contribution, which enhanced the potency of RSL3 for ferroptosis induction and enabled the drug-loaded micelles all-active. As an index of PCCs population, the level of CD133

Topics: AC133 Antigen; Agar; Animals; Antineoplastic Agents; Cell Differentiation; Cell Line, Tumor; Drug Resistance, Multiple; Drug Resistance, Neoplasm; Female; Ferroptosis; Homeostasis; Humans; Lipid Peroxidation; Mice; Mice, Inbred BALB C; Mice, Nude; Micelles; Neoplasm Transplantation; Neoplastic Stem Cells; Ovarian Neoplasms; Oxidation-Reduction; Polymers; Reactive Oxygen Species

Colony formation in soft agar is the gold-standard assay for cellular transformation in vitro, but it is unsuited for high-throughput screening. Here, we describe an assay for cellular transformation that involves growth in low attachment (GILA) conditions and is strongly correlated with the soft-agar assay. Using GILA, we describe high-throughput screens for drugs and genes that selectively inhibit or increase transformation, but not proliferation. Such molecules are unlikely to be found through conventional drug screening, and they include kinase inhibitors and drugs for noncancer diseases. In addition to known oncogenes, the genetic screen identifies genes that contribute to cellular transformation. Lastly, we demonstrate the ability of Food and Drug Administration-approved noncancer drugs to selectively kill ovarian cancer cells derived from patients with chemotherapy-resistant disease, suggesting this approach may provide useful information for personalized cancer treatment.

Topics: Adenosine Triphosphate; Agar; Antineoplastic Agents; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cell Transformation, Neoplastic; Chemistry, Pharmaceutical; Drug Design; Drug Screening Assays, Antitumor; Female; Fibroblasts; Flow Cytometry; High-Throughput Screening Assays; Humans; Open Reading Frames; Ovarian Neoplasms

spindlin1, a novel human gene recently isolated by our laboratory, is highly homologous to mouse spindlin gene. In this study, we cloned cDNA full-length of this novel gene and send it to GenBank database as spindlin1 (Homo sapiens spindlin1) with Accession No. AF317228. In order to investigate the function of spindlin1, we studied further the subcellular localization of Spindlin1 protein and the effects of spindlin1 overexpression in NIH3T3 cells. The results showed that the fusion protein pEGFP-N1-spindlin1 was located in the nucleus and the C-terminal is correlated with nuclear localization of Spindlin1 protein. NIH3T3 cells which could stably express spindlin1 as a result of RT-PCR analysis compared with the control cells displayed a complete morphological change; made cell growth faster; and increased the percentage of cells in G2/M and S phase. Furthermore, overexpressed spindlin1 cells formed colonies in soft agar in vitro and formed tumors in nude mice. Our findings provide direct evidence that spindlin1 gene may contribute to tumorigenesis.

Topics: Agar; Animals; Base Sequence; Blotting, Western; Cell Cycle; Cell Cycle Proteins; Cell Division; Cell Line, Tumor; Cell Nucleus; Cell Proliferation; Cloning, Molecular; COS Cells; DNA, Complementary; Electrophoresis, Polyacrylamide Gel; Exons; Expressed Sequence Tags; Female; Flow Cytometry; G2 Phase; Gene Expression Profiling; Green Fluorescent Proteins; Humans; Introns; Mice; Mice, Nude; Microscopy, Fluorescence; Microtubule-Associated Proteins; Molecular Sequence Data; NIH 3T3 Cells; Open Reading Frames; Ovarian Neoplasms; Phosphoproteins; Plasmids; Polymerase Chain Reaction; Protein Structure, Tertiary; Reverse Transcriptase Polymerase Chain Reaction; S Phase; Transfection

Morphofunctional peculiarities of tumor cells from 15 endometrial adenocarcinomas and 2 ovarian tumors have been investigated at the ultrastructural level. These cells could develop two types of colonies in soft agar: those with histotypical differentiation (numerous microvilli, well developed tight junctions, desmosomes, secretory granules), and those without it (absence of epithelial features, ability of tumor cells to produce filamentous extracellular matrix and striated collagen fibrils which are characteristic of fibroblastic cells). The addition of progesterone and tamoxifen to cell cultures resulted in rising the level of cell differentiation in the colonies. The fact that endometrial and ovarian cancer cells can express the properties specific of connective tissue cells may suggest a multipotention of the Mullerian epithelium derivatives to shed light on the histogenesis of the mixed Mullerian tumors of uterus.

Topics: Adenocarcinoma; Agar; Cell Transformation, Neoplastic; Culture Media; Cystadenocarcinoma; Endometrial Neoplasms; Female; Granulosa Cell Tumor; Humans; Methotrexate; Microscopy, Electron; Morphogenesis; Ovarian Neoplasms; Progesterone; Tamoxifen; Tumor Cells, Cultured

Clonogenic growth (defined as the formation of greater than or equal to 5 colonies per 5 x 10(5) viable nucleated cells per plate) of ovarian cancer specimens assessed in our clonogenic assay system was significantly associated with the proportion of tumor cells in the suspensions plated (N = 87; P = 0.0006), although there was no quantitative relationship with the corresponding plating efficiencies. An inverse correlation was observed between monocytes/macrophages/mesothelial cells (M) proportion and clonogenic growth (P = 0.013). These associations were most evident when only effusions were considered. Univariate analyses identified tumor cell content, M proportion and, to a lesser degree, granulocyte content as the only factors out of 12 examined to be correlated with colony formation. Multivariate analysis using a logistic regression model identified the proportion of tumor cells as the only significant factor predicting clonogenic growth in vitro (P = 0.0006). The overall accuracy of prediction for growth or non-growth was 63.2%.

Topics: Agar; Analysis of Variance; Cell Count; Cell Division; Centrifugation; Female; Granulocytes; Humans; In Vitro Techniques; Neoplastic Stem Cells; Ovarian Neoplasms; Phagocytes; Probability; Regression Analysis; Tumor Stem Cell Assay

Concurrent Cloning Efficiencies (CE) of both ascites and solid tumor samples from 36 patients with ovarian carcinoma were studied using the soft agar assay. The CE of both were highly variable (range, 0-1.234% and 0-0.802%, respectively). There was marked intrapatient and interpatient heterogeneity in the CE. Of the 36 tested, comparative CE were evaluable in 29. CE was 0 in both solid tumor and ascites in one patient. CE was 0 in four other ascites samples from four patients. In other 24, the relative CE of solid tumor/ascites from each patient ranged from 0.066 to 435. In the 29 patients with samples of ascites and a solid tumor evaluable for concurrent CE, the median colony counts of solid tumors was more than tenfold higher than ascites. The solid tumors obtained from 31 patients had a significantly higher CE than tumor cells obtained from ascites samples from 32 patients. Solid tumors were significantly better than ascites for in vitro testing based on the data that 75% (27/36) of solid tumors and only 31% (11/36) of ascites formed greater than or equal to 30 colonies. The drug sensitivity profiles of tumor cells from a solid tumor and ascites of the same patient appear similar. Based on these observations, it may be more cost and labor effective to do soft agar in vitro chemotherapy assays using a solid tumor than ascites in ovarian carcinoma.

Topics: Agar; Ascites; Carcinoma; Colony-Forming Units Assay; Drug Screening Assays, Antitumor; Female; Humans; Ovarian Neoplasms; Tumor Stem Cell Assay

We studied the factors that control the capacity of tumor cells to grow in soft agar. And, we analyzed the effect of cell-free ascites (CFA) obtained from ovarian cancer patients in combination with various media on the cloning efficiency of H-134 and OVCAR-3nu ovarian carcinoma cell lines and the WiDr colon carcinoma cell line. Seven batches of CFA consistently enhanced the soft agar growth of tumor cells more efficiently than tested sera. The addition of charcoal-treated bovine serum albumin (BSA) lowered the amount of CFA required for optimal tumor cell growth. As little as 1.25 ng of epidermal growth factor (EGF)/ml further improved OVCAR-3nu soft agar growth in combination with all of the amounts of CFA tested. Thus, neither BSA nor EGF seems to account for the colony-stimulating effect of ascites on tumor cells. Four batches of CFA were tested for stimulating soft agar growth of normal rat kidney (NRK-49F) fibroblasts; all induced colonies of different morphologies. This effect was potentiated by EGF, which suggests the presence of several transforming growth factor-like activities in CFA. The results show that differences in cloning efficiency of tumor cells of one or two orders of magnitude can be found between standard (anchorage-dependent growth-supporting) media and media optimalized for soft agar growth, such as CFA in the presence of EGF. This paper will discuss the similarity in effects of CFA on various tumor cells and NRK cells, and possible implications of the stimulatory effects of CFA.

Topics: Agar; Animals; Ascites; Carcinoma; Cell Division; Cells, Cultured; Clone Cells; Colonic Neoplasms; Female; Humans; Neoplastic Stem Cells; Ovarian Neoplasms; Rats

To characterize in vitro activity of 2-fluoro-Ara AMP and its relation to the activities of cisplatin and doxorubicin, 28 specimens from patients wit gynecologic tumors (predominantly ovarian) were tested in a soft agar assay. Twenty-six of 28 (93%) grew when the medium was supplemented with four hormones (epidermal growth factor, hydrocortisone, estradiol-17, and insulin). Normal bone marrow cells were utilized as a biologic control to define in vitro concentrations of the three drugs. Tumors were exposed continuously to three different concentrations of each drug. 2-fluoro-Ara AMP was tested against 26 tumors, cisplatin against 24, and doxorubicin against 14. In vitro sensitivity was defined as greater than or equal to 50% colony inhibition at a drug concentration within the bone marrow inhibitory range. Seven of 26 (27%) tumor specimens were sensitive to 2-fluoro-Ara AMP. Among these, four tumors were derived from previously treated patients. However, in the 2-fluoro-Ara AMP concentration range (0.26 micrograms/ml to 0.78 micrograms/ml) tested, five of eight (62.5%) tumors from untreated patients achieved IC50 compared to only seven of 18 (39%) tumors from treated patients. Five of six (83%) specimens demonstrated cross-sensitivity between cisplatin and 2-fluoro-Ara AMP. Seventeen of 18 (94%) specimens demonstrated cross-resistance between cisplatin and 2-fluoro-Ara AMP, and 13 of 13 (100%) specimens demonstrated cross-resistance between 2-fluoro-Ara AMP and doxorubicin. A higher proportion of tumors from previously untreated patients achieved greater than or equal to 50% colony inhibition when exposed to 2-fluoro-Ara-AMP or cisplatin than did those from previously treated patients.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Agar; Arabinonucleotides; Bone Marrow Cells; Cisplatin; Clone Cells; Colony-Forming Units Assay; Culture Media; Culture Techniques; Doxorubicin; Female; Genital Neoplasms, Female; Humans; Ovarian Neoplasms; Vidarabine Phosphate

Radiation survival curves have been generated for 3 human tumor cell lines as a means of comparing and evaluating the validity of human tumor soft-agar clonogenic assays. The assays investigated were the Hamburger-Salmon, Courtenay-Mills, Courtenay-Mills plus additions, soft agar (no additions), and soft agar plus additions. The additions were formulated to supplement the media used in soft agar assays of primary ovarian and cervical carcinoma specimens. Supplementing the media with additions led to a 2- to 3-fold increase in PE of CaSki cells but had no effect on the PEs of ME180 and OWI cells. Radiation survival curves were similar in all assays for CaSki and OWI but differed for ME180 cells. For ME180 cells, the Courtenay-Mills and soft agar assays plus additions produced the most radioresistant curves (Do = 2.2 Gy); the cells were more responsive when assayed by the Hamburger-Salmon method (Do = 1.5 Gy), and the soft agar and Courtenay-Mills assays gave the most radiosensitive curves (Do = 1.2 Gy). These results demonstrate that the PE of human tumor cell lines may be increased with no effect on radiation survival; radiation survival may be altered without changes in PE and neither may be altered by applying modifications and supplements to existing clonogenic assays.

Topics: Adenocarcinoma; Agar; Carcinoma, Squamous Cell; Cell Line; Cell Survival; Colony-Forming Units Assay; Female; Humans; Neoplasms; Ovarian Neoplasms; Tumor Stem Cell Assay; Uterine Cervical Neoplasms

In the present study we describe the establishment and characteristics of a new human tumor cell line (OV-1063) positive for carcinoembryonic antigen (CEA) originating from ovarian metastatic tumor cells. Analysis of the cultured cells during their in vitro adaptation period revealed while the primary culture exhibited a low proportion of CEA-positive cells, this proportion increased with culture passages and eventually more than 90% of the cells in the established line were CEA-positive. Thus, during the period of adaptation to in vitro growth, a selection for CEA-positive cells took place but the amount of CEA secreted per each positive cell seemed to be constant. Several tumor-associated characteristics were found positive on the established OV-1063 cell line. The in vitro growing cell line exhibited an abnormal chromosome pattern with a near-trisomy karyotype for some chromosomes, colony formation in soft agar as well as positive staining with a monoclonal antibody B38.1. Culture supernatants of the OV-1063 cells contained significant amounts of CEA as well as CA-125 antigen which is an ovarian-carcinoma-associated antigen.

Topics: Agar; Antigens, Neoplasm; Antigens, Tumor-Associated, Carbohydrate; Ascitic Fluid; Carcinoembryonic Antigen; Cell Line; Culture Media; Cystadenocarcinoma; Female; Humans; Karyotyping; Keratins; Microscopy, Phase-Contrast; Middle Aged; Ovarian Neoplasms

A new human ovarian cancer cell line has been initiated which clones without agar in vitro. The cell line has been characterized by growth of a tumor resembling the primary tumor in a nude mouse, by human lactic dehydrogenase isozyme pattern, by human karyotype, and by lack of contamination by other cell lines. Initial studies have demonstrated the presence of epidermal growth factor receptors in this cell line. The utility of this line for clonogenic studies is demonstrated by dose-response studies with doxorubicin, cisplatinum, and 13-cis-retinoic acid.

Topics: Agar; Aged; Animals; Antineoplastic Agents; Carcinoma, Papillary; Cell Division; Cell Line; ErbB Receptors; Female; Humans; Isoenzymes; Karyotyping; Mice; Mice, Nude; Neoplasm Transplantation; Ovarian Neoplasms; Receptors, Cell Surface; Tumor Stem Cell Assay

Topics: Adenocarcinoma; Agar; Cells, Cultured; Colony-Forming Units Assay; Culture Media; Cystadenoma; Female; Humans; In Vitro Techniques; Ovarian Neoplasms; Tumor Stem Cell Assay

Topics: Agar; Culture Techniques; Female; Filtration; Fluorescent Antibody Technique; Histological Techniques; Humans; Ovarian Neoplasms; Polyethylene Glycols; Urogenital Neoplasms

Four families of human tumour cell lines--one of uterine, and three of ovarian origin--were established at early passage level from primary biopsy specimens of terminal patients by the propagation of anchorage-independent agar clones in liquid culture. All cell lines exhibited unique and stable characteristics and retained their ability to clone in agar. However, considerable heterogeneity was evident in clonogenic capacity, karyotype, and responsiveness to growth-promoting substances even among progeny of single agar clones isolated from the one biopsy specimen. These cell lines will be made available for further study upon request.

Topics: Agar; Aged; Biopsy; Carcinoma; Cell Line; Culture Media; Cystadenocarcinoma; Cytological Techniques; Female; Humans; Middle Aged; Neoplasms; Neoplasms, Experimental; Ovarian Neoplasms; Uterine Neoplasms

Human ovarian cancer cells from ten patients were cultured in the agar double layer assay as described by Hamburger and Salmon and in a methylcellulose monolayer system. The assays were compared under the same experimental conditions. The rate of positives (defined as greater than 30 colonies/dish) was 75% in the methylcellulose assay and 69% in the agar double layer. Plating efficiency ranged in the methylcellulose assay between 0.021% and 0.089% and in the agar double layer from 0.015% to 0.094%. Cytological and cytochemical staining of cells obtained from colonies in both test systems and of the tumour cells prior to plating revealed the same morphology. The methylcellulose monolayer system requires less additives than necessary in the agar double layer system. Furthermore, it is easier to handle with respect to the plating procedure and less time consuming. In addition, the effect of the anti-oestrogen tamoxifen on colony formation was tested. The dose response curves for colony formation with tamoxifen proved to be identical in both systems. At a concentration of 10(-6) M an inhibition of colony formation of more than 70% of controls was observed in the agar and in the methylcellulose system.

Topics: Agar; Clone Cells; Colony-Forming Units Assay; Female; Humans; Methylcellulose; Ovarian Neoplasms; Tamoxifen; Tumor Stem Cell Assay

Single time point assessment is usually employed in the Human Tumour Cloning System as the only parameter for in vitro growth. This does not seem to give a fair expression of the dynamic biological properties of tumour growth and time dependent effects, e.g. of cytotoxic drugs. We studied the time course of colony formation in temporal growth patterns (TGPs) and compared this method of growth evaluation with conventional single time point assessment in 57 samples of ovarian tumour cultures in the HTCS. A first advantage of the use of TGPs is that more cultures become evaluable, as this assessment over time can detect a rise in the number of colonies in dishes where colony-like clumps have initially been seeded. Thus only 28 of the cultures were evaluable for single time point assessment, whereas 57 were available for TGP evaluation. Growth was more often seen at TGP evaluation (14/57) than at single day assessment (8/57). Evaluation of growth over the course of time potentially allows detection of sensitivity to drugs. Furthermore TGPs reflect the dynamics of biological growth. These features cannot be studied in single time point assessment.

Topics: Agar; Cells, Cultured; Cisplatin; Colony-Forming Units Assay; Doxorubicin; Female; Humans; Neoplastic Stem Cells; Ovarian Neoplasms; Time Factors; Tumor Stem Cell Assay

One hundred and thirty three specimens from mammary and ovarian adenocarcinoma and from melanoma were cultured according to an agar/agar clonogenic assay. Melanoma and ovarian cancers exhibited a 70 per cent rate of success for culture; 50 per cent of the mammary adenocarcinomas were successfully cultured. Fifty-nine ovarian cancers were cultured in order to test the in vitro effectiveness of Cisplatinum and Adriamycin. Thirty percent of cultured tumors gave rise to relevant chemograms. The chemoresistance measured in vitro was correlated to the ineffectiveness of the patient's treatment. In contrast, we were unable to predict chemosensitivity. Taking into account the technical difficulties encountered in these assays, human tumor clonogenic assays cannot at present be proposed as a routine procedure in the prediction of the effectiveness of chemotherapeutic treatments. Nevertheless, they must be developed in order to determine the spectrum of activity of new antineoplastic agents on various human tumors.

Topics: Adenocarcinoma; Agar; Antineoplastic Agents; Breast Neoplasms; Carcinoma, Intraductal, Noninfiltrating; Cisplatin; Colony-Forming Units Assay; Cyclophosphamide; Doxorubicin; Drug Resistance; Female; Humans; Melanoma; Melphalan; Ovarian Neoplasms; Tumor Stem Cell Assay

Methods and evaluation of the human tumor stem cell assay (HTSCA) are described. Advantages and disadvantages of the test system are elaborated. The in vitro/in vivo correlation in the drug screening of human ovarian carcinomas shows that the prediction of sensitivity to a cytotoxic agent is only possible in 64%. Prediction of drug resistance, however, seems to be possible in 95%. The number of patients that profit from the HTSCA seems to be only less than 10%. Our investigations describe the influence of various hormones and antiestrogens on the colony formation of human ovarian carcinoma cells. Tamoxifen and his major metabolite 4-hydroxy-tamoxifen were the most active agents. Both compounds inhibit the colony survival (70% at pharmacological concentrations) of 60% of the screened ovarian carcinomas. A significant correlation to the quantitative level of estrogen or progesterone receptors could not be proved. Colony formation of ovarian carcinoma cells was compared in the HTSCA as described by Hamburger and Salmon and in a methylcellulose-monolayer system. Our results show that the colony formation corresponds to the results of the original HTSCA: Cloning ovarian carcinoma cells in the methylcellulose-monolayer, however, seems to be technically easier and faster.

Topics: Agar; Antineoplastic Agents; Carcinoma; Cells, Cultured; Colony-Forming Units Assay; Drug Resistance; Estradiol; Female; Hormones; Humans; Medroxyprogesterone; Medroxyprogesterone Acetate; Methylcellulose; Ovarian Neoplasms; Progesterone; Prognosis; Tamoxifen; Tumor Stem Cell Assay

Topics: Agar; Animals; Biopsy; Breast Neoplasms; Cell Survival; Colonic Neoplasms; Colony-Forming Units Assay; Culture Techniques; Female; Humans; Male; Melanoma; Melphalan; Mice; Neoplasms; Ovarian Neoplasms; Rectal Neoplasms; Tumor Stem Cell Assay

Topics: Agar; Antineoplastic Agents; Ascites; Cell Survival; Cells, Cultured; Clone Cells; Colony-Forming Units Assay; Doxorubicin; Drug Resistance; Female; Humans; Ovarian Neoplasms; Pleural Effusion; Tumor Stem Cell Assay

Human tumors were cultured by the two-layer soft-agar technique and the time course of tumor colony development was evaluated during periods of up to 6 weeks in culture. All colony counting was performed with an automated tumor colony counter (Omnicon; Bausch and Lomb, Inc, Rochester, NY, USA). This instrument provided colony counts per culture plate in six size categories from greater than 60 microns diameter colonies to greater than 149 microns diameter colonies. Six to 24 culture plates were used for each "growth curve", generally 24. Control (non-drug-treated) cultures were obtained from 117 tumors, of which 25 also provided enough cells to allow evaluation of the time course of colony development after exposure to cytostatic agents. The development of colonies in non-drug-treated plates usually demonstrated a lag phase, a logarithmic growth phase to maximum colony development and a subsequent deterioration of colonies. In spite of clumps seeded into the agar, real colony growth could be recognized by frequent colony counting of culture dishes, although the temporal patterns of growth were sometimes different if pure single-cell suspensions were compared with suspensions containing clumps from the same tumor. Drug pre-incubation caused changes in the temporal pattern of colony growth as well as in the total number of colonies. Some cultures showed drug sensitivity when evaluated at certain time points while evaluation at later time points showed only borderline drug effect or none at all. The potential utility of tumor colony growth curves in the clinical applications of tumor colony cultures is discussed.

Topics: Agar; Animals; Antineoplastic Agents; Breast Neoplasms; Carcinoma, Small Cell; Colony-Forming Units Assay; Cystadenoma; Female; Humans; Male; Ovarian Neoplasms; Prostatic Neoplasms; Rats; Skin Neoplasms; Staining and Labeling; Time Factors; Tumor Stem Cell Assay

The correlation of the colony growth of cells disaggregated from human melanoma, sarcoma, lung, and ovarian carcinomas were studied in four different semisolid tissue culture assays: (a) the soft agar assay of Pluznik and Sachs; (b) the soft agar assay of Hamburger and Salmon; (c) the soft agar-methyl cellulose assay of Buick et al.; and (d) the methyl cellulose assay of Ogawa et al. There was no colony growth of tumor cells achieved in 15 of 15 cases assayed in Ogawa's methyl cellulose assay. The plating efficiency of the above mentioned tumors was similar in the assays of Pluznik and Sachs, Hamburger and Salmon, and Buick et al. However, the tumor take rate differed among these three systems. The assay of Buick et al. appears potentially useful for analysis of the biology of human tumors.

Topics: Adenocarcinoma; Agar; Cell Division; Cells, Cultured; Cytological Techniques; Female; Humans; Lung Neoplasms; Melanoma; Methylcellulose; Ovarian Neoplasms; Sarcoma

The factors controlling the growth of human tumor cells in soft agar are poorly understood. However, it has been demonstrated that serum provides factors which promote anchorage-independent growth. We tested 58 tumor specimens, which were obtained from patients with adenocarcinoma of the lung, colon, ovary or squamous cell carcinoma, for their ability to form colonies in soft agar in serum-free or serum-supplemented media. The cells were unable to replicate, and none of the hormones or growth factors tested: insulin (I), transferrin (T), selenium (S), estradiol (E), hydrocortisone (H) or epidermal growth factor (EGF) could substitute for serum. Examination of the serum dose-response curves indicated that growth factors reduced the serum concentrations needed to support anchorage-independent growth. The addition of the supplements and the lowering of serum concentrations increased cloning efficiencies (C.E.) in 38/51 trials, when cells were able to grow initially. The addition of ITS increased C.E. in 18/21 cases, HITES in 15/17 cases and EGF in 12/18 cases as compared to controls. ITS and HITES increased the number of colonies only when serum was the limiting factor. EGF, however, increased the number of colonies even when serum was not the limiting factor. The ability of the supplements to enhance growth could not be correlated to tumor type or initial cloning efficiencies. However, in only 1/25 cases were cells that were unable to form colonies under standard conditions induced to form colonies in the presence of the growth factors. Normal and tumor-derived human fibroblasts did not form colonies in soft agar in the presence of these growth factors. The results suggest that human tumor cells may require the presence of serum-derived factors for growth in soft agar.

Topics: Adenocarcinoma; Agar; Blood; Carcinoma, Squamous Cell; Cell Division; Clone Cells; Colonic Neoplasms; Epidermal Growth Factor; Estradiol; Female; Humans; Hydrocortisone; Insulin; Lung Neoplasms; Ovarian Neoplasms; Selenium; Transferrin

In vitro growth of tumor cells may provide a way of testing the effectiveness of chemotherapeutic agents in the treatment of cancer. In 1980 and 1981, operations for gynecologic malignancy were performed on 610 Mayo Clinic patients, and malignant tissue and fluids were obtained from 204 cancers that involved the vulva, uterus, fallopian tubes, and ovaries. These yielded 76 clonogenic stem-cell preparations; and various chemotherapeutic agents were tested against these 76 tumors on soft agar. Considered in this study were the overall process of culturing the samples of tumors and, especially, the data from the preparations that showed sufficient growth of tumor cells for testing. Our guiding concerns were the usefulness of this method to gynecologists and the possible benefits to patients.

Topics: Agar; Antineoplastic Agents; Carcinoma; Clone Cells; Culture Media; Drug Resistance; Female; Genital Neoplasms, Female; Humans; In Vitro Techniques; Ovarian Neoplasms

Replacement of enriched CMRL 1066 medium by cell-free ascites from tumour patients in the human tumour clonogenic assay described by Hamburger and Salmon (1977) increased plating efficiency for ovarian cancer cells by a median of 8-fold (range 0.4-1012 fold). In 40 experiments, two cases had a lower plating efficiency when cultured in cell-free ascites, 10 grew neither in standard medium nor in cell-free ascites and in two cases, growth was only observed in cell-free ascites. With standard medium, we observed 53% growth (greater than 5 colonies/dish) and 41% evaluable for chemosensitivity testing (greater than 30 colonies/dish). With cell-free ascites as culture medium, these figures were 71% and 63%, respectively. While under standard conditions the highest plating efficiency obtained was 0.25%, in 21% of the experiments done with cell-free ascites a plating efficiency higher than 1% was reached. We conclude that cell-free ascites is able to stimulate proliferation of ovarian cancer cells in agar and that the use of it extends the applicability of the clonogenic assay.

Topics: Agar; Ascitic Fluid; Cell Count; Cell Division; Clone Cells; Culture Media; Female; Humans; Ovarian Neoplasms

114 biopsy specimens from 70 patients with ovarian carcinoma at all stages of disease were submitted for assessment of clonogenic capacity in agar. A highly significant correlation was found between agar clonogenicity and patient survival after biopsy. However, problems related to inherent tumour heterogeneity, quality of sample and tissue disaggregation indicate that this technique may have limited applicability in the routine assessment of patients. Only 41 biopsy specimens (36%) from 31 patients (44.3%) complied with the prerequisite criteria for agar clonogenic assessment, namely: (a) the confirmed presence of malignant cells in the biopsy, (b) the ability to prepare a single-cell suspension, and (c) adequate viable cell numbers for assay. Furthermore, although the dominant patterns of agar clonogenic growth could be identified and correlated with stage of disease, the heterogeneity in both initial clonogenic capacity and "self-renewal" capacity assessed by the ability of primary clones to propagate in liquid culture and reclone in agar was too inconsistent for the assay to be used as a prognostic index for the individual patient.

Topics: Agar; Biopsy; Clone Cells; Female; Humans; Ovarian Neoplasms; Prognosis

Studies were performed with a semisolid agar culture system to determine the in vitro sensitivity of human ovarian carcinoma to human leukocyte interferon (IFN-alpha) and standard chemotherapeutic agents (adriamycin, cis-platinum, hexamethylmelamine, melphalan, and velban). Growth in culture occurred in 67% of samples derived from ascitic fluid and 71% of these exhibited reduction in tumor colony number by greater than or equal to 25% in response to 300 units interferon/ml. The responsiveness of the ascitic fluid samples to interferon ran parallel to the overall responsiveness to the other agents tested. Sensitivity to interferon was not related to the histology or grade of the tumor or to the stage of the disease. As compared with cell suspensions prepared from ascitic fluid samples, solid tumor samples had markedly lower viability, 39% vs 89%, and had more tumor cells, 81% vs 28%. Also, colonies derived from solid tumor samples were less sensitive to interferon and more sensitive to cis-platinum and adriamycin than were ascitic fluid-derived colonies. Three of four ascitic fluid samples showed a reduction in tumor colony number of greater than or equal to 25%, whereas none of the solid tumor samples obtained from the same donors were affected by interferon to that degree. Retrospective analysis of drug testing (exclusive of interferon) for in vitro: in vivo correlations revealed that in 58% of 12 evaluable situations, when a single drug (or at least one of a group of drugs) gave a positive response in vitro, stabilization or regression of the tumor occurred in vivo after treatment with the drug. These studies prove the utility of the semisolid agar culture system for assessing the antiproliferative effects of interferon against ovarian carcinoma, and will be of utility in the future for assessing whether various types of interferon have the same degree, range, and mechanism of action of antitumor effect.

Topics: Agar; Antineoplastic Agents; Ascitic Fluid; Colony-Forming Units Assay; Female; Humans; Interferons; Neoplasms, Experimental; Ovarian Neoplasms

We studied the in vitro growth characteristics of 10 solid-tumor samples of patients with ovarian carcinoma using a semisolid agar culture technique. Tumor cell colonies were observed in 8 of 10 samples, but sufficient number of tumor colonies to evaluate the effects of interferon and other antitumor agents were obtained in only four samples. As compared with cell suspensions prepared from ascitic fluid samples, solid-tumor samples had markedly lower viability, 39% vs 89%, and had more tumor cells, 81% vs 28%. Also, whereas the maximum increase in tumor-colony number occurred during the first week of growth in both solid- and ascitic-fluid-derived samples grown concurrently from the same donors, increase in tumor colony number was sustained for longer periods in ascitic-fluid-derived cultures. The ascitic-fluid-derived tumor colonies were more sensitive to the antiproliferative effects of interferon than colonies derived from solid-tumors. At a concentration of 300 units/ml incorporated into the agar for the duration of the culture, three of four ascitic fluid samples showed a reduction in tumor colony number by greater than or equal to 25%, whereas none of the solid-tumor samples were affected by the interferon to that degree. In contrast, solid-tumor samples showed greater response to cis platinum and Adriamycin than did ascitic-fluid-derived cultures. Such studies and observations are critical in designing clinical trials for the use of interferon in the treatment of malignancy and the judicious selection of patients and route of administration most likely to provide optimal results, especially in view of present critical shortages in availability of interferon.

Topics: Adult; Agar; Aged; Ascites; Carcinoma; Cisplatin; Clone Cells; Culture Media; Doxorubicin; Female; Humans; Interferons; Middle Aged; Ovarian Neoplasms

To determine the sensitivity in vitro of human ovarian carcinoma cells to the antiproliferative effects of human leukocyte interferon, 18 samples of ascitic fluid from 15 affected patients were cultured in semi-solid agar by the technique of Hamburger and Salmon. Cultures were examined at weekly intervals after initiation and the number and size of each tumor colony recorded. Growth as defined by increase in total tumor colony number with time was obtained in 45% of the samples, or 53% of the patients, or 67% of the ascitic fluid samples with tumor cells demonstrated by Pap smear. No growth occurred in samples from recently treated patients or in samples devoid of tumor cells as assessed by Pap smear. Response to interferon directly incorporated into the agar culture system or preincubated with cells prior to their inclusion in culture was defined as reduction in total colony number by greater than or equal to 50% and partial response by greater than or equal to 25% reduction. The response rate for samples, The nontreated controls of which showed evidence of growth, was 71%, and that of samples that contained tumor colonies with no increase in growth during the culture period was 75%. Sensitivity to interferon was not related to the histology or grade of the tumor or to the stage of the disease. In general the responsiveness of the tumor cells to interferon ran parallel to the overall responsiveness to a variety of other chemotherapeutic agents. As this culture system has been proven by other investigators to be predictive of in vivo resistance to antitumor drugs with considerable accuracy and also to be predictive of in vivo response to a lesser degree, it will be important to determine whether similar relationships between in vitro and in vivo sensitivity obtain for interferon.

Topics: Adult; Agar; Aged; Antineoplastic Agents; Ascitic Fluid; Cell Differentiation; Cell Division; Drug Resistance; Female; Humans; In Vitro Techniques; Interferons; Methods; Middle Aged; Ovarian Neoplasms

Cytogenetic analysis of human tumors is rapidly coming of age as a useful tool in the diagnosis and prognosis of cancer. With the use of chromosome-banding analysis, an increasing display of tumors containing cytologically recognizable nonrandom chromosome change is accumulating. Additionally, with avenues of assessing drug resistance and sensitivity, current cytogenetic analyses are yielding clinically relevant information. Finally, the blending of cytogenetics with biochemistry and cell biology is yielding an impressive array of research tools for the subcellular study of cancer. DNA hybridization, gene cloning, autoradiography of various drug and hormonal chromosomal binding sites, gene mapping, and numerous other related techniques are providing fresh insights into the genetic basis of cancer. It seems clear that during the next decade substantial progress in understanding chromosome structure and function in normal cells as well as tumor cells will occur. Technical advances in the methodology for growing and harvesting tumor cells for detailed cytogenetic analysis will undoubtedly be important in this effort. Application of the described soft agar colony technique may provide a useful tool for study of the genetics of human solid tumors.

Topics: Agar; Chromosome Aberrations; Chromosome Disorders; Chromosomes, Human, 1-3; Clone Cells; Culture Media; Female; Humans; Karyotyping; Mitosis; Neoplasms; Ovarian Neoplasms; Urinary Bladder Neoplasms

Topics: Agar; Antineoplastic Agents; Ascitic Fluid; Cell Division; Cells, Cultured; Clone Cells; Doxorubicin; Female; Fluorouracil; Humans; Injections, Intraperitoneal; Ovarian Neoplasms

Topics: Adult; Agar; Antineoplastic Agents; Ascitic Fluid; Cell Survival; Clone Cells; Culture Media; Drug Evaluation, Preclinical; Exudates and Transudates; Female; Humans; Ovarian Neoplasms

Topics: Adenocarcinoma; Adenocarcinoma, Mucinous; Agar; Cell Division; Chromosome Aberrations; Cisplatin; Clone Cells; Cystadenocarcinoma; Drug Resistance; Endometriosis; Female; Humans; Methods; Neoplasms, Experimental; Ovarian Neoplasms; Phagocytes