agar and Melanoma

We introduce a new single-head multimodal optical system that integrates optical coherence tomography (OCT), 18 MHz ultrasound (US) tomography and Raman spectroscopy (RS), allowing for fast (<2 min) and noninvasive skin cancer diagnostics and lesion depth measurement. The OCT can deliver structural and depth information of smaller skin lesions (<1 mm), while the US allows to measure the penetration depth of thicker lesions (≥4 mm), and the RS analyzes the chemical composition from a small chosen spot (≤300 μm) that can be used to distinguish between benign and malignant melanoma. The RS and OCT utilize the same scanning and optical setup, allowing for co-localized measurements. The US on the other side is integrated with an acoustical reflector, which enables B-mode measurements on the same position as OCT and RS. The US B-mode scans can be translated across the sample by laterally moving the US transducer, which is made possible by the developed adapter with a flexible membrane. We present the results on custom-made liquid and agar phantoms that show the resolution and depth capabilities of the setup, as well as preliminary ex vivo measurements on mouse models with ∼4.3 mm thick melanoma.

Topics: Agar; Animals; Biopsy; Melanoma; Mice; Spectrum Analysis, Raman; Tomography, Optical Coherence

Overcoming the notorious apoptotic resistance of melanoma cells remains a therapeutic challenge given dismal survival of patients with metastatic melanoma. However, recent clinical trials using a BRAF inhibitor revealed encouraging results for patients with advanced BRAF mutant bearing melanoma, but drug resistance accompanied by recovery of phospho-ERK (pERK) activity present challenges for this approach. While ERK1 and ERK2 are similar in amino acid composition and are frequently not distinguished in clinical reports, the possibility they regulate distinct biological functions in melanoma is largely unexplored.. Rather than indirectly inhibiting pERK by targeting upstream kinases such as BRAF or MEK, we directly (and near completely) reduced ERK1 and ERK2 using short hairpin RNAs (shRNAs) to achieve sustained inhibition of pERK1 and/or pERK2.. Using A375 melanoma cells containing activating BRAFV600E mutation, silencing ERK1 or ERK2 revealed some differences in their biological roles, but also shared roles by reduced cell proliferation, colony formation in soft agar and induced apoptosis. By contrast, chemical mediated inhibition of mutant BRAF (PLX4032) or MEK (PD0325901) triggered less killing of melanoma cells, although they did inhibit proliferation. Death of melanoma cells by silencing ERK1 and/or ERK2 was caspase dependent and accompanied by increased levels of Bak, Bad and Bim, with reduction in p-Bad and detection of activated Bax levels and loss of mitochondrial membrane permeability. Rare treatment resistant clones accompanied silencing of either ERK1 and/or ERK2. Unexpectedly, directly targeting ERK levels also led to reduction in upstream levels of BRAF, CRAF and pMEK, thereby reinforcing the importance of silencing ERK as regards killing and bypassing drug resistance.. Selectively knocking down ERK1 and/or ERK2 killed A375 melanoma cells and also increased the ability of PLX4032 to kill A375 cells. Thus, a new therapeutic window is open for future clinical trials in which agents targeting ERK1 and ERK2 should be considered in patients with melanoma.

Topics: Agar; bcl-2-Associated X Protein; Benzamides; Caspases; Cell Death; Cell Line, Tumor; Cell Proliferation; Cell Survival; Diphenylamine; Drug Resistance, Neoplasm; Drug Screening Assays, Antitumor; Gene Knockdown Techniques; Gene Silencing; Humans; Indoles; Melanoma; Membrane Potential, Mitochondrial; Mitochondria; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Neoplasm Proteins; Phosphorylation; Poly(ADP-ribose) Polymerases; Protease Inhibitors; RNA, Small Interfering; Sulfonamides; Tumor Stem Cell Assay; Vemurafenib

The objective of this study was to use the established xenograft model of human melanoma (C8161.9) to test a pharmacological approach to the effect of the metastasis suppressor KISS1. A KISS1 analog was used to inhibit the metastatic development of C8161.9 cells in nude mice. Further experiments were performed to test the validity of the C8161.9 model and test the connection between KISS1 expression and loss of metastatic potential. New clones of C8161.9 cells were obtained, with or without KISS1 expression, and were tested for metastasis formation. The absence of benefit in survival with the KISS1 analog compared with PBS prompted us to revisit the C8161.9 model. We found that the cells expressing KISS1, used in the previous study and obtained by transfection and single-cell cloning, were defective for both formation of orthotopic tumors and metastases. In mixing experiments, these cells could not suppress orthotopic tumor growth of KISS1-negative C8161.9 cells, suggesting that the suppression of metastasis by C8161.9-KISS1 cells may be intrinsic to the selected clone rather than related to KISS1 expression. Isolation of clones from parental C8161.9 cells in soft agar yielded cell populations that phenotypically and genotypically mimicked the KISS1-positive clone. In addition, new clones expressing KISS1 did not show any decrease in metastatic growth. These data demonstrate the heterogeneity of cell types in the C8161.9 cell line and the high risk of artifact linked to single-cell selection. A different xenograft model will be necessary to evaluate the use of KISS1 analogs as antimetastatic therapy.

Topics: Agar; Animals; Antineoplastic Agents; Cell Line, Tumor; Cell Survival; Culture Media, Conditioned; Enzyme-Linked Immunosorbent Assay; Humans; Kisspeptins; Melanoma; Mice; Mice, Nude; Neoplasm Metastasis; Neoplasm Transplantation; Nucleic Acid Hybridization

Utilizing gene microarray profiling of melanoma samples, we have recently identified a novel gene overexpressed in both thick primary and metastatic melanomas. This gene, progestagen-associated endometrial protein (PAEP), has never before been implicated in the oncogenic processes of melanoma, with its true function in oncogenesis and tumour progression relatively unknown. Overexpression of the PAEP gene in freshly procured thick primary and metastatic melanoma samples (58%) and daughter cell lines (77%) is confirmed by quantitative RT-PCR, immunohistochemistry, Western blotting and mass spectrometric analysis. We suggest that PAEP gene overexpression is involved with melanoma tumour progression as well as an aggressive phenotype. Transfection of melanoma cells with PAEP small interfering RNA (siRNA) reveals a significant decrease in soft agar colony formation and a marked inhibition of both cell migration and cell invasion. Furthermore, we establish stable melanoma transfectants via PAEP lentiviral small hairpin RNA (shRNA), examine their growth characteristics in a murine xenograft model and reveal that tumour growth is significantly inhibited in two separate melanoma cell lines. Our data strongly implicate the PAEP gene as a tumour growth promoter with oncogenic properties and a potential therapeutic target for patients with advanced melanoma.

Topics: Agar; Animals; Cell Line, Tumor; Cell Movement; Cell Proliferation; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Gene Silencing; Glycodelin; Glycoproteins; Humans; Lentivirus; Melanoma; Mice; Neoplasm Invasiveness; Pregnancy Proteins; Reproducibility of Results; RNA, Small Interfering; Skin Neoplasms; Transcription, Genetic; Tumor Stem Cell Assay; Xenograft Model Antitumor Assays

Melanoma accounts for only about 4% of all skin cancer cases but most of skin cancer-related deaths. Standard systemic therapies such as interferon (IFN) have not been adequately effective in the management of melanoma. Therefore, novel approaches are needed for prevention and treatment of this disease. Chemoprevention by naturally occurring agents present in food and beverages has shown benefits in certain cancers including nonmelanoma skin cancers. Here, employing 2 human melanoma cell lines (A-375 amelanotic malignant melanoma and Hs-294T metastatic melanoma) and normal human epidermal melanocytes (NHEM), we studied the antiproliferative effects of epigallocatechin-3-gallate (EGCG), the major polyphenolic antioxidant present in green tea. EGCG treatment was found to result in a dose-dependent decrease in the viability and growth of both melanoma cell lines. Interestingly, at similar EGCG concentrations, the normal melanocytes were not affected. EGCG treatment of the melanoma cell lines resulted in decreased cell proliferation (as assessed by Ki-67 and PCNA protein levels) and induction of apoptosis (as assessed cleavage of PARP, TUNEL assay and JC-1 assay). EGCG also significantly inhibited the colony formation ability of the melanoma cells studied. EGCG treatment of melanoma cells resulted in a downmodulation of anti-apoptotic protein Bcl2, upregulation of proapoptotic Bax and activation of caspases -3, -7 and -9. Furthermore, our data demonstrated that EGCG treatment resulted in a significant, dose-dependent decrease in cyclin D1 and cdk2 protein levels and induction of cyclin kinase inhibitors (ckis) p16INK4a, p21WAF1/CIP1 and p27KIP1. Our data suggest that EGCG causes significant induction of cell cycle arrest and apoptosis of melanoma cells that is mediated via modulations in the cki-cyclin-cdk network and Bcl2 family proteins. Thus, EGCG, alone or in conjunction with current therapies, could be useful for the management of melanoma.

Topics: Agar; Anticarcinogenic Agents; Apoptosis; bcl-2-Associated X Protein; Caspase 3; Caspase 7; Caspase 9; Caspases; Catechin; Cell Cycle; Cell Cycle Proteins; Cell Line, Tumor; Cell Proliferation; Cell Separation; Cell Survival; Cyclin-Dependent Kinase Inhibitor p16; Cyclin-Dependent Kinase Inhibitor p21; Dose-Response Relationship, Drug; Down-Regulation; Flow Cytometry; Humans; Immunoblotting; Immunohistochemistry; In Situ Nick-End Labeling; Ki-67 Antigen; Melanocytes; Melanoma; Membrane Potentials; Mitochondria; Poly(ADP-ribose) Polymerases; Proto-Oncogene Proteins c-bcl-2; Time Factors

Losses of heterozygosity involving chromosomes 9 and 10 are frequent events in the development and progression of cutaneous malignant melanoma. To investigate whether specifically deleted chromosomal regions encode tumor suppressor genes (TSGs), we introduced normal chromosome 10 into the tumorigenic human metastatic melanoma cell line UACC-903 by microcell fusion. In addition, two chromosome 9 derivatives that were microdeleted in the region of the p16INK4A/p15INK4B locus were transferred to determine whether an additional melanoma TSG or TSGs reside on chromosome 9p, as indicated by previous melanoma allele loss studies. In comparison to parental cells, microcell hybrids generated with chromosomes 9 (microdeleted) and 10 displayed reduced anchorage-independent growth in soft agar and markedly reduced tumorigenicity in athymic (nu/nu) mice. These data define a TSG or TSGs that function independently of p15/p16 on chromosome 9 and provide evidence for a TSG (or TSGs) on chromosome 10 that may be important in melanoma development.

Topics: Agar; Animals; Carrier Proteins; Cell Cycle Proteins; Cell Division; Chromosome Deletion; Chromosome Mapping; Chromosomes, Human, Pair 10; Chromosomes, Human, Pair 9; Cloning, Molecular; Cyclin-Dependent Kinase Inhibitor p15; Cyclin-Dependent Kinase Inhibitor p16; Female; Genes, p16; Genes, Tumor Suppressor; Humans; Melanoma; Mice; Mice, Nude; Neoplastic Stem Cells; Phenotype; Skin Neoplasms; Tumor Cells, Cultured; Tumor Suppressor Proteins

The three-dimensional growth of cultured tumor cell lines (HT29, a colon adenocarcinoma cell line; M21, a melanoma cell line; KB, a nasopharyngeal carcinoma cell line) has been investigated in an agar culture system with a fibrin matrix in vitro. The tumor cells developed to tumors 3 x 3 mm in diameter after 10 days in culture in vitro. This size was large enough to allow histologic examination. The tumor cells located in the surface area of the three-dimensional tumor seemed to grow well. However, the tumor cells in the center degenerated or did not proliferate, indicating a lack of nutrition and/or anoxia in the center. The histologic comparison between the xenografted tumors on nude mice and the three-dimensional tumors in vitro suggests that the structures of the three-dimensional tumors were comparable, especially in the surface area, to the xenografted tumors. Furthermore, the antitumor effect of mitomycin C on the three-dimensional tumors was found to be dose-dependent.

Topics: Adenocarcinoma; Agar; Animals; Cell Division; Cell Hypoxia; Colonic Neoplasms; Culture Techniques; Extracellular Matrix; Fibrin; Humans; KB Cells; Melanoma; Mice; Mice, Nude; Mitomycin; Transplantation, Heterologous; Tumor Cells, Cultured

Mycoplasmal infection of cell cultures remains a significant threat to diagnostic and research procedures. In certain defined situations, curing of mycoplasmal infected cultures is a reasonable exercise. Four methods of curing were compared: treatment with BM-cycline, 5 bromouracil, use of specific antisera and treatment of infected cells suspended in soft agar with antibiotics. Antisera treatments were of low efficiency of curing: 50%. None of nine infected cell lines treated with 5-bromouracil were consistently cured of mycoplasmas. The use of BM-cycline was effective for some, but not all lines and required long periods of treatment, 12-21 days. 35 naturally or deliberately infected cultures were treated in soft agar a total of 119 times. This procedure which consisted of suspending infected cultures in soft agar containing appropriate antibiotics resulted in successful mycoplasmal elimination 118/119 times. This soft agar technique took 1-3 days. In separate studies, it was shown that certain Mycoplasma fermentans strains were resisted to this and other curing methods. This may be due to their intracellular location. Such strains may be more amenable to antibiotics that penetrate mammalian cells. It is concluded that the soft agar technique is a rapid, efficient and reliable method to eliminate cell culture mycoplasmas.

Topics: Agar; Animals; Bromouracil; Cells, Cultured; Fibroblasts; Humans; Lymphocytes; Lymphoma; Melanoma; Methods; Mice; Monocytes; Mycoplasma; Tumor Cells, Cultured

An immunohistochemical and ultrastructural study of human melanoma colonies grown in soft agar for up to 50 days was performed. Three morphological variants of developing tumor colonies are reported: 1) large light colonies, 2) small dark colonies, and 3) smooth-edged colonies. The large light colony variant is the most frequently observed in the soft agar assay (approximately 70%), followed by the dark colony variant (approximately 27%), and the smooth-edged colony variant (approximately 3%). Major morphological characteristics are associated with each variant, as shown with light microscopy (LM) and transmission electron microscopy (TEM). Both LM and TEM analyses demonstrated that the large light colony variant was hypomelanotic and contained a microfibrillar extracellular matrix (ECM). The small dark colony variant was found to be hypermelanotic and contained a less demonstrable ECM. The smooth-edged variant has an encapsulated periphery, no demonstrable ECM, and tightly packed cells with desmosome-like junctions. In order to characterize further the ECM in the most commonly observed variant, the large light colony, specific antibodies to fibronectin (FN) and collagen types IV and V (COLs IV and V) were applied and observed with immunofluorescence microscopy and immunoperoxidase. In paraffin sections of melanoma colonies, FN was observed associated with both the cell surface and the ECM. However, no specific staining was seen for COLs IV and V. In addition, ruthenium red was used to preserve and selectively bind to glycosaminoglycans (GAGs) and proteoglycans (PGs). TEM studies reveal GAG-like granules stained with ruthenium red in the fibrillar ECM and a dotted, punctate staining of the cell surface. Understanding the biological and architectural composition of developing melanoma tumor colonies in soft agar could contribute to the development of more efficient chemotherapeutic strategies.

Topics: Agar; Cell Adhesion; Collagen; Extracellular Matrix; Fibronectins; Humans; Immunohistochemistry; Melanoma; Microscopy, Electron; Neoplasm Metastasis; Tumor Cells, Cultured

Definition of survival and measurement of colony size in soft agar assays is important in establishing in vitro radiation survival curves. Conventionally, survival is assessed according to colony-forming ability. The distinction between small colonies that are abortive and those that are viable often involves a difficult and arbitrary choice for the investigator. We have examined the effect of different minimum colony sizes (greater than or equal to 25, greater than or equal to 50, greater than or equal to 75, and greater than or equal to 100 cells) on ionizing radiation survival curves for cells from established murine (CCL 53.1) and human (M1RW5) melanoma cell lines as well as from short-term human melanoma cell strains (C8146A, C8146C, C8161, C83-2C, C82-7A1, and C8442) and patient biopsy (83-4). Single cell suspensions were plated in the upper layer of the agar bilayer and cells were irradiated by single dose X rays. Giant cells did not form in colonies containing 50 or more cells. D0 values were highest (D0 values, from 390 to 100 cGy) for cells forming smaller colonies (greater than or equal to 25 cells, greater than or equal to 4-5 doublings) and lowest (D0 values, from 190 to 50 cGy) for cells forming larger colonies (greater than or equal to 100 cells, greater than or equal to 6-7 doublings). Therefore, apparent radiosensitivity was dependent on colony size selected for analysis. Precise measurement of colony size was important in establishing radiation survival curves because errors in determining the colony size will alter apparent radiosensitivity of cells. These results should help define the biological meaning of tumor colony growth in semisolid medium, and alter the interpretation of survival curves which measure sensitivity to agents using this assay.

Topics: Agar; Animals; Cell Count; Cell Survival; Colony-Forming Units Assay; Culture Media; Humans; Melanoma; Melanoma, Experimental; Mice; Mice, Inbred DBA; Neoplastic Stem Cells; Radiation Tolerance; Radiation, Ionizing; Tumor Cells, Cultured; Tumor Stem Cell Assay

Topics: Agar; Cell Count; Cell Separation; Cells, Cultured; Humans; Melanoma; Neoplasm Metastasis; Neoplastic Cells, Circulating

Topics: Agar; Animals; Cell Survival; Colony-Forming Units Assay; Female; Hematoporphyrin Photoradiation; Hematoporphyrins; Melanoma; Mice; Mice, Inbred C57BL; Tumor Stem Cell Assay

Clonogenic assays currently define colonies as multicellular growth units above an arbitrarily designated cutoff size rather than by the biological function of different-sized growth units. To define the cutoff size between clusters and colonies in terms of the biological function of the cells within the growth units, we directly measured the self-renewal and proliferative capacity of cells from different-sized melanoma colonies. Primary colonies formed from cells of two patients were removed, pooled according to size, and replated, and the frequency and size distribution of the secondary colonies were analyzed. Cells from primary melanoma colonies that resulted from four to eight population doublings had similar extensive proliferative and self-renewal characteristics. The results demonstrated that self-renewal was not limited to cells in large colonies and suggested that the cutoff may be below 16 cells/growth unit. These data support the use of relatively small multicellular growth units to define colonies and measure highly proliferative human melanoma tumor cells. In addition, these methods may allow the determination of the cutoff size for other tumor types in terms of the biological function of cells rather than arbitrarily designating a cutoff size.

Topics: Agar; Cell Division; Cells, Cultured; Cytological Techniques; Humans; Melanoma; Neoplastic Stem Cells; Stem Cells

Three distinct dissemination-related phenotypes have been distinguished among cell subpopulations of the mouse B16 melanoma: tumorigenicity, spontaneous metastasis from subcutaneous tumors, and organ colonization following intravenous injection of cells. From a progenitor clone (G3) of tumorigenic but nonmetastatic and noncolonizing (null) cells that underwent phenotypic diversification in vitro and in vivo, 4 subclones were obtained: G3.5 (culture-generated metastatic), G3.12 (tumor-generated metastatic), G3.15 (culture-generated null), and G3.26 (tumor-generated colonizing). The growth potentials of the parent clone and derived subclones were investigated comparatively in in vivo assays (tumorigenicity, tumor growth rate, and lung colonization potential), monolayer culture assays (generation time, saturation density, clonogenicity, and rate of detachment by trypsin), and in soft agar. In overall growth potential, G3.26 greater than G3.12 greater than G3, G3.5 greater than G3.15. These results indicate that metastatic populations of the B16 melanoma are not the most rapidly and effectively growing cells obtainable from that tumor.

Topics: Agar; Animals; Cell Division; Cell Movement; Cells, Cultured; Clone Cells; Female; Lung Neoplasms; Melanoma; Mice; Mice, Inbred C57BL; Microscopy, Phase-Contrast; Models, Biological; Neoplasm Metastasis; Neoplasm Transplantation; Neoplastic Stem Cells; Stem Cells

To investigate the influence of culture conditions on the in vitro responses of tumour cells to anticancer drugs, the sensitivities observed with the soft agar methods of Hamburger & Salmon (1977) (H-S) and of Courtenay & Mills (1978) (C-M) were compared. In all cases the ID50 values were determined from dose-response curves. Six human tumour cell lines exposed to 10 different agents, and 9 patients' melanomas exposed to 5 different agents, were examined. In the studies of cell lines the H-S method gave higher sensitivity values than the C-M method in 38 out of 52 cases, whereas in 14 cases the results were the same. In the patients' tumours the H-S method gave higher sensitivity in 21 of 35 cases, equal sensitivity in 11, and lower sensitivity in 3 cases. In many instances the ID50 values obtained with the two test systems differed by factors of 10 or more, both in the case of cell lines and tumour specimens. Systematic alterations in the culture conditions indicated that the presence or absence of rat erythrocytes is the most important factor responsible for the differences observed. Also, other factors, such as supplements (in the H-S method) and the use of different serum types, appeared to influence both colony growth and chemosensitivity.

Topics: Agar; Animals; Antineoplastic Agents; Breast Neoplasms; Cell Count; Cell Line; Cell Survival; Culture Media; Dose-Response Relationship, Drug; Erythrocytes; Humans; Melanoma; Neoplasms; Oxygen; Rats; Temperature

Multicellular spheroids were grown from cells derived directly from a human melanoma xenograft propagated in athymic mice. The histological appearance of the spheroids was similar to that of the parent xenograft. The spheroids were heated in culture medium (42.5-44.5 degrees C); growth delay and single cell survival measured in soft agar were used as end points. There was a good correlation between the results obtained with these two end points, indicating that growth delay depended mainly on cell survival. Large spheroids (200 +/- 12 microns in diameter) were found to be more heat sensitive than small ones (100 +/- 5 microns in diameter), probably because the physiological conditions in large spheroids were more favorable for cell inactivation. The cells were more resistant when heated as spheroids than as single cells. This effect was not a secondary effect of differences in cell-cycle distribution. Spheroids were also found to be more heat resistant than xenografted tumors. In the tumors, heat treatment caused vascular damage which resulted in delayed cell death due to hypoxia and/or nutrient deficiency. It is concluded that spheroids seem well suited for studies of primary heat-induced cytotoxic effects. However, they appear not to mirror the complex heat response of tumors since that response also includes secondary effects, related to heat-induced reduced perfusion.

Topics: Agar; Animals; Cell Division; Cell Survival; Hot Temperature; Humans; Melanoma; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Transplantation; Transplantation, Heterologous

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

The photosensitizing/cytotoxic effects of four porphyrin compounds derived from hematoporphyrin were compared using two human tumor cell lines with a soft-agar colony-forming assay. Reproducible dose- and time-dependent increases in reproductive cell death were observed for each porphyrin tested. Two fractions derived from hematoporphyrin derivative (HPD) were found to be better photosensitizers than HPD itself, indicating the potential value of this in vitro assay for detecting the most active component from a heterogeneous mixture.

Topics: Agar; Cell Line; Cell Survival; Colony-Forming Units Assay; Hematoporphyrin Derivative; Hematoporphyrins; Humans; Light; Lung Neoplasms; Melanoma; Radiation-Sensitizing Agents

Accurate descriptions of the kinetics of cell growth in semi-solid agar clonogenic systems have been difficult because the number of cells in colonies of different sizes is largely unknown. We stained and removed tumor cell colonies from agar, directly counted their cells, and established equations to quantitate the number of cells within colonies of different sizes. We used these equations to quantitate, in terms of cell number and volume, the total amount and kinetics of clonogenic cell proliferation from biopsies of human melanoma and cell lines of several different tumor types. Daily observations of cells in agar and serial photography indicated a 0- to 4-day delay in the onset of proliferation in agar followed by rapid growth and then abrupt cessation of proliferation. We quantified the extent of proliferation of cells from melanoma biopsies of seven patients and 11 cell lines after they were allowed to proliferate in agar until they stopped. Approximately 10% of cells divided one to five times while only 0.01% divided six to nine times. The total number of cells within the colonies at the end of growth was different while the total volume of cells within the colonies per plate was similar; approximately 10(9) microns 3 cellular volume per plate represents an upper limit for proliferation within the closed, nonrefed bilayer agar system. Previous replating studies using the same biopsy cells have shown that clonogenic melanoma cells can self-renew and have more proliferative capacity than that expressed during primary colony formation. Thus, the clonogenic assay only measured initial proliferative capacities. Furthermore, variable delays in the onset of proliferation may contribute to the heterogeneity of colony size within clonogenic assays.

Topics: Agar; Cell Cycle; Cells, Cultured; Clone Cells; Culture Media; Humans; Melanoma

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; Biopsy; Breast Neoplasms; Cell Survival; Clone Cells; Culture Techniques; Female; Humans; Lung Neoplasms; Male; Melanoma; Methylcellulose; Microscopy, Electron; Neoplasms; Pleural Effusion

An investigation was made of the cell populations that occur in the sequential strata of human melanoma ( MM96 ) colonies. Colonies were either grown for the full duration of culture in a 'physiological' atmosphere of 5% O2 (unperturbed colonies), or grown in this atmosphere followed by a final incubation in a hypoxic atmosphere of less than 0.1% O2. Both autoradiographic and ultrastructural studies indicated that cell changes similar to those which occur successively in monolayer cultures experiencing nutritional deficiency, exist concurrently in the sequential strata of the larger unperturbed colonies. At the margin with the necrotic core, approximately half of the cells showed morphological changes associated with death by apoptosis. The other half were undergoing necrosis. Observations on colonies incubated for the final 24 or 48 h in a hypoxic (less than 0.1% O2) atmosphere showed that many of these cells, although otherwise well preserved, developed oedema complicated by cytoskeletal rupture and extrusion of areas of damaged cytoplasm within membrane-bound vesicles. Although sudden-onset hypoxia did not appear to precipitate cell death in small colonies previously lacking a necrotic core, large colonies suffered a marked reduction in the width of their viable rims. Cell death under this circumstances was by necrosis, the same mode of death as occurs with infarction. The study indicated that apoptosis was associated with sub-acute cell death as occurs with progressive nutrient depletion and catabolite accumulation, whereas necrosis was associated with acute cell death as seen in previously compromised cells subject in addition to sudden-onset hypoxia.

Topics: Agar; Aged; Cell Line; Cell Survival; Female; Humans; Kinetics; Melanoma; Microscopy, Electron; Necrosis; Oxygen; 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

Topics: Agar; Animals; Cell Count; Cell Division; Cell Line; Clone Cells; Cytological Techniques; Dactinomycin; Female; Humans; Melanoma; Melphalan; Mice; Stimulation, Chemical

The effect of agar sterilized by either boiling or autoclaving on human melanoma colony formation in soft agar was compared using cells from 17 biopsies of metastatic malignant melanoma. The frequency of colony formation was significantly increased for cells grown in boiled agar in 8 samples (47%), unchanged in 8 samples (47%), and decreased in only one sample (6%). There were increases in both cluster and colony formation for the melanomas which had augmented colony formation when grown in boiled agar. There was also qualitative morphological improvement, including rounder, smoother cells and less extracellular debris surrounding the colonies. These data suggest that melanoma colony formation is enhanced when cells are grown in agar which has been sterilized by boiling rather than autoclaving.

Topics: Agar; Cell Division; Clone Cells; Cytological Techniques; Humans; Melanoma; Sterilization

A previously unpublished reaction of precipitation in agarose between histone and non-histone proteins extracted in saline buffer from nuclei of human skin tumors, is reported. Two bands of precipitation similar to those in an immunodiffusion reaction between NHP and specific antisera, were observed. The reaction described is very similar to the affinodiffusion reaction of glycoproteins and lectins in agarose.

Topics: Agar; Animals; Carcinoma; Chromosomal Proteins, Non-Histone; Fibroma; Histones; Humans; Immunodiffusion; Melanoma; Rats; Rats, Inbred Strains; Skin Neoplasms

The cloning efficiencies of a murine melanoma cell line (S91 CCL 53.1) and a human melanoma cell strain (C8146c) were inhibited by dexamethasone (DEX), prostaglandin A1 (PGA1), and beta-all-trans-retinoic acid (RA) in a dose-dependent manner. Murine melanoma tumor colony-forming units (MTCFU) were inhibited more than 99% by DEX (1 X 10(-7) M) and RA (1 X 10(-7) M) with a concentration needed to produce a 50% reduction in colony formation for both hormones of 5 X 10(-9) M. Combinations of DEX and RA effected a synergistic inhibition on colony formation, which was reflected by a 11/2 log reduction in the hormone concentration needed to produce a greater than 99% inhibition of colony formation. When PGA1 was added to DEX and RA, a greater than additive reduction in colony formation was observed. Human MTCFU from cell strain C8146c were inhibited more than 85% at an RA concentration of 1 X 10(-7) M, but they were reduced only to 40% of control at a DEX concentration of 1 X 10(-6) M. DEX-RA produced an additive inhibition of colony formation. Addition of submaximal amounts of PGA1 to DEX-RA combinations or to either hormone alone resulted in synergistic reduction of human MTCFU. These results demonstrated that the proliferative potential of human and murine melanomas can be simultaneously regulated by DEX, PGA1, and RA.

Topics: Agar; Animals; Antineoplastic Combined Chemotherapy Protocols; Cell Division; Cell Line; Dexamethasone; Dose-Response Relationship, Drug; Drug Evaluation, Preclinical; Humans; Melanoma; Mice; Prostaglandins A; Tretinoin

Extracts of conditioned medium (CM) from Hs0294 human malignant melanoma cells stimulate [3H]thymidine incorporation and an increase in cell number in serum-depleted Hs0294 cells. This activity is acid and heat stable, nonproteolytic, protease sensitive, contains disulfide bonds and elutes broadly from a Bio-Gel P-30 column with an approximate molecular weight range of 6,000 to 25,000. Hs0294 CM also stimulates [3H]thymidine incorporation in nonmalignant human nevus cells and normal rat kidney fibroblast cells but not in human fibroblasts. There was only limited competition with 125I-epidermal growth factor in binding assays. Hs0294 CM extracts stimulate anchorage-independent growth in normal rat kidney fibroblast cells in soft agar but not in Hs0294 cells, nevus cells, or human fibroblasts. This second activity elutes from the Bio-Gel P-30 column in two positions with apparent molecular weights of 27,000 and 11,000.

Topics: Agar; Animals; Cell Count; Cell Division; Cell Line; Culture Media; DNA; Epidermal Growth Factor; Growth Substances; Humans; Kidney; Melanoma; Molecular Weight; Nerve Growth Factors; Nevus; Rats

In an attempt to diagnose a suspected amelanotic melanoma tumour, we examined a variety of tissue and cell samples from one patient at the ultrastructural level, which consisted of single cell suspensions of tumour cells with and without DOPA treatment, tumour cells after culture in agar with and without DOPA treatment, and single tumour cells hetero-transplanted into a nude mouse. Premalanosomes were not observed in sections of the amelanotic tumour with routine electron microscopy. Osmophilic-dense bodies, suggestive of melanosomes, were noted in the single cells in suspension treated with DOPA and cells grown in agar without DOPA treatment. Definitive premelanosomes, with an identifiable striated matrix, were only observed in cells grown into colonies in agar and treated with DOPA. Positive L-DOPA reaction products were noted in the golgi complex, endoplasmic reticulum closely related to the golgi (GERL), and in vacuoles from cells grown in agar. As controls, Cloudman S91 53.1 melanoma cells were evaluated as single cells in suspension or as colonies after culture in agar, both with and without DOPA treatment. Premelanosomes were always observed in this established melanoma cell line while DOPA-treated cells contained positive L-DOPA reaction products. The overall findings identified the tumour as amelanotic melanoma and indicated that both DOPA treatment and culture in agar were needed for the demonstration of premelanosomes.

Topics: Adult; Agar; Animals; Biopsy; Cells, Cultured; Humans; Levodopa; Male; Melanoma; Mice; Mice, Nude; Microscopy, Electron; Neoplasm Transplantation; Transplantation, Heterologous

Colony growth in soft agar of human melanoma cells from biopsy material, cell lines and xenografts was evaluated. Colony forming potential is constantly very low in all studied types of tumor tissue, however slight increase in clonogenic potential was seen during subsequent xenograft passages, but within the range of less than 1%. Results of presented studies suggest that the number of colonies does necessarily express the degree of colony forming potential and the culture preparation and conditions as well. It appears that there is no reliable correlation between the number of colonies in soft agar and the clinical course of melanoma patients under study.

Topics: Agar; Animals; Biopsy; Cells, Cultured; Humans; In Vitro Techniques; Melanoma; Mice; Skin Neoplasms; Transplantation, Heterologous; Tumor Stem Cell Assay

Topics: Agar; Alprostadil; Animals; Cell Adhesion; Cell Division; Cell Line; Cyclic AMP; Cyclic GMP; Melanocyte-Stimulating Hormones; Melanoma; Mice; Prostaglandins A; Prostaglandins E

Clonogenic assays have been widely adopted for the investigation of hematopoietic and human tumor stem cell biology. Inasmuch as specific, whole colonies need to be analyzed morphologically, we used various methods for fixing and embedding individual colonies in situ that allowed macroscopic, light microscopic (LM), immunofluorescence, and transmission electron microscopic (TEM) evaluation of the intact colony. Melanoma colonies stained with Masson's Trichrome, hematoxylin and eosin (H&E), periodic acid-Schiff, Best's carmine, Page-Green method for inclusion bodies, and Snook's reticulum revealed cellular and extracellular components by LM. Ultrastructural studies revealed specific cellular organelles and extracellular components. Immunofluorescence studies demonstrated cell-surface fibronectin, a high molecular weight, adhesive glycoprotein. Myeloma colonies contained a heterogeneous cell population and produced amyloid fibers that were observed by TEM. Fixation and embedding the colonies in agar for TEM has several advantages over centrifugation methods and other conventional techniques for collecting cells in that (a) an entire specific colony can be studied, (b) there is excellent preservation of the cell and its spatial orientation in the colony, and (c) the extracellular matrix (ECM) of the colony is preserved for immunohistochemical analysis.

Topics: Agar; Amyloid; Clone Cells; Extracellular Space; Fibronectins; Fluorescent Antibody Technique; Humans; Melanoma; Methods; Microscopy, Electron; Multiple Myeloma

An in vitro soft agar technique was used to culture human malignant melanoma cells from 61 solid tumors, 17 lymph nodes, 11 effusions, and four bone marrow specimens from 93 patients with malignant melanoma. Colonies grew in soft agar from 64 (69%) of the 93 specimens. Fifty-five percent of the specimens cultured formed greater than or equal to 30 colonies per 500,000 nucleated cells plated. Light microscopy, electron microscopy, tumor marker, and athymic nude mouse studies provided evidence the colonies were composed of malignant melanoma cells. Drug sensitivity studies utilizing the cloning technique showed similarities between in vitro results and the general clinical experience noted with the same drugs. The human tumor cloning system represents a new model for future basic biology and clinical studies of human malignant melanoma.

Topics: Agar; Animals; Antineoplastic Agents; Cell Count; Cell Survival; Clone Cells; Cytological Techniques; Drug Evaluation, Preclinical; Humans; Melanoma; Mice; Mice, Nude; Skin Neoplasms; Time Factors

Tumor cells from a human melanoma xenograft show better colony growth and plating efficiency in soft agar when incubated in a 5% O2 atmosphere that at a higher concentration of 20% O2. Exposure to low drug concentrations under a 20% (but not 5%) O2 atmosphere stimulated plating efficiency. Enhanced sensitivity (4- to 20-fold) to a variety of chemotherapeutic agents was seen in cells incubated under low but not the higher O2 concentration. These studies suggest the important role of O2 concentration in tumor colony growth, plating efficiency, and drug cytotoxicity for human solid tumors in vitro.

Topics: Agar; Antineoplastic Agents; Cell Division; Cell Survival; Clone Cells; Dose-Response Relationship, Drug; Humans; Melanoma; Neoplasms, Experimental; Oxygen

Topics: Agar; Antineoplastic Agents; Cell Division; Cell Line; Cells, Cultured; Colonic Neoplasms; DNA Replication; Drug Evaluation, Preclinical; Female; Humans; Kinetics; Melanoma; Neoplasm Metastasis; Neoplasms

Topics: Agar; Bone Marrow; Carcinoma, Squamous Cell; Clone Cells; Cytoplasm; Desmosomes; Head and Neck Neoplasms; Humans; Melanoma; Microscopy, Electron; Neuroblastoma; Skin Neoplasms

A procedure was developed to directly measure the self-renewal capacity of clonogenic cells from biopsies of metastatic human malignant melanoma. A culture of colony-forming cells was performed with bilayer agar in microtiter wells. The number of live tumor cells from biopsies of melanoma tissue was determined and was used to calculate plating efficiencies. Sequential photography showed that cells did not migrate in agar, thereby documenting that all the cells within colonies were direct descendants of clonogenic cells. A calibrated pneumatically controlled micropipet attached to a micromanipulator was used to quantitatively remove melanoma colonies without removing adjacent cells or agar. Plucked primary colonies were mechanically disaggregated into single cells; viability was greater than 95% as determined by trypan blue dye exclusion. Dose-related formation of secondary colonies was observed after replating of cells from pooled primary colonies. Cells from individual colonies were replated, and secondary colonies formed. These techniques allowed a simple and direct assessment of the self-renewal capacity of colony-forming melanoma cells.

Topics: Agar; Cell Division; Cells, Cultured; Clone Cells; Culture Media; Culture Techniques; Humans; Kinetics; Melanoma; Neoplasm Metastasis

Human melanoma xenografts in immune-deprived mice have been used to assess the value of the agar diffusion chamber for chemosensitivity testing. Tumor cells were treated with melphalan, Adriamycin, or methyl trans-1-(2-chloroethyl)-3-(4-methylcyclohexyl)-1-nitrosourea either as solid tumors growing in mice or as suspensions in agar in i.p. diffusion chambers. Survival of clonogenic human tumor cells was measured by the agar diffusion chamber assay in both cases. Cell survival curves were log-linear for treatment of tumor cells in vivo or in the chambers. For melphalan the slopes of survival curves were significantly greater for treatment in the chambers than as solid tumors in vivo, but for methyl trans-1-(2-chloroethyl(-3-(4-methylcyclohexyl)-1-nitrosourea or Adriamycin, they were indistinguishable. Experiments with [14C]melphalan showed that the levels of drug achieved were less inside the diffusion chambers than in the tumors in vivo so that the sensitivity of tumor cells to melphalan was much greater when they were treated in chambers. The differences in drug exposure and in cellular chemosensitivity between chambers and tumors suggest caution in the interpretation of drug testing using this system, but the log-linear nature of the dose-response curves is an important feature which may be useful in the eventual development of optimal chemosensitivity testing systems.

Topics: Agar; Animals; Antineoplastic Agents; Cell Survival; Diffusion; Doxorubicin; Drug Evaluation, Preclinical; Humans; Melanoma; Melphalan; Mice; Neoplasm Transplantation; Semustine; Transplantation, Heterologous

Two soft-agar methods for assaying chemosensitivity of human cancers in vitro were compared with respect to colony morphology, plating efficiency (PE) and chemosensitivity of human melanomas. In 9 xenografts and 9 patients' biopsy specimens Method A (essentially that of Courtenay & Mills, 1978) gave considerably higher PE that Method B (essentially that of Hamburger & Salmon, 1977) and, in contrast to Method B, the number of colonies was proportional to the number of cells plated. Evidence was obtained that the observed differences in PE could be attributed to the low O2 concentration and the presence of rat red blood cells in Method A. Colony morphology was similar in the 2 assays. When cells from 4 xenografted melanomas were treated in vitro with DTIC, CCNU, vinblastine and abrin, and the inhibition of colony formation was assayed concurrently in the 2 soft-agar methods, the tumour cells appeared to be more sensitive to 3 of the drugs in Method B than in A. The results demonstrate that chemosensitivity data obtained with the 2 assays cannot be directly compared.

Topics: Abrin; Agar; Animals; Cell Survival; Cells, Cultured; Dacarbazine; Drug Evaluation, Preclinical; Erythrocytes; Humans; Lomustine; Melanoma; Oxygen Consumption; Rats; Vinblastine

As part of a programme to study the predictive clinical value of a soft agar assay for measuring chemosensitivity of human melanomas in vitro, we have observed the effect of three disaggregation methods on the yield of tumor cells, plating efficiency in soft agar and chemosensitivity. The yields and plating efficiencies obtained, as well as sensitivity to DTIC, CCNU, vinblastine and abrin, were about the same whether collagenase/pronase/DNase-treatment, trypsin/EDTA-treatment or mechanical treatment was used. When melanoma xenografts of different sizes were studied, an inverse relationship between tumor size and plating efficiency was found, whereas chemosensitivity was unaffected by tumor size. The highest plating efficiencies of melanoma cells, both from patient biopsies and from xenografts, were obtained when red blood cells were present and a low oxygen concentration (5%) was used. The results demonstrate that, in the case of melanomas, the fraction of tumor cells that are clonogenic in vitro depends on the size of the tumors, and even more so on the culture conditions used. An important finding was that chemosensitivity in vitro appears to be unaffected by the disaggregation method and by tumor size.

Topics: Abrin; Agar; Cell Adhesion; Cell Count; Cells, Cultured; Erythrocytes; Humans; Lomustine; Melanoma; Oxygen; Vinblastine

Colony-forming capacity of human melanoma cells was investigated in five established cell lines and isolated cells from seven metastatic or recurrent tumors. A single-cell suspension was added to 0.3% agar medium, and colonies were observed over 7 to 21 days. With the five cell lines, colony formation occurred in all cases and was usually accelerated by 2-mercaptoethanol, but there was wide variation in cloning efficiency between the cell lines (5 to 80%), in the appearance of the colonies, and in their recloning capacity. In those cell lines with low cloning efficiency, some colonies grew attached to the culture dishes, and these had a high recloning capacity. In the melanotic cell lines, colonies originated from slightly or nonpigmented cells which became more pigmented during incubation. Stimulation of colony growth by 2-mercaptoethanol led to reduced pigmentation of colonies, whereas theophylline caused increased melanin production and decreased colony size. With freshly obtained tumor specimens, both the frequency of colonies (0.01 to 0.26%) and the growth rate were much reduced compared to the cell lines. Both could be increased by addition of 2-mercaptoethanol to the medium or by separation of the tumor cell suspension on a density gradient before culture.

Topics: Agar; Cell Line; Cells, Cultured; Clone Cells; Culture Media; Humans; Melanoma; Mercaptoethanol; Pigmentation

The effect of mechanical and enzymatic disaggregation on human malignant melanoma, soft-tissue sarcoma and lung carcinoma colony growth in soft agar was studied. The enzymatic disaggregation was advantageous in most cases of melanoma and sarcoma, giving a larger number of colonies and increasing the probability of achieving growth in soft agar. Enzymatically treated pulmonary carcinoma cell populations had lower clonogeneic potential, especially in the case of anaplastic carcinomas. Morphological studies showed that the cells growing in soft-agar colonies had the same characteristics as those of the original tumor. A linear relationship was obtained between the number of enzymatically and mechanically treated tumor cells plated and the number of colonies. Delayed plating decreased the number of colonies.

Topics: Agar; Carcinoma; Cell Aggregation; Clone Cells; Cytological Techniques; Humans; Lung Neoplasms; Melanoma; Microbial Collagenase; Sarcoma; Soft Tissue Neoplasms; Time Factors

Studies using 2 different cloning assays to grow colonies and measure cell survival after treatment of human melanoma xenografts are reported and reviewed. Clonogenic cell survival curves were constructed for 5 melanoma xenografts with a clinically relevant range of drugs, using a soft agar diffusion chamber assay. Cells in colonies were similar to human melanoma cells in morphology, histochemistry, ultrastructure and antigenicity and xenograft tumours could be grown from the colonies. The survival curves were compatible with the known clinical patterns of response of malignant melanoma. The sensitivities of the xenografts correlated with the response of the same tumour in the patient when assessable. In 2 xenograft lines, it proved possible to grow colonies in the lungs of immune deprived mice. Studies of drug sensitivity using this lung colony assay agreed closely with the soft agar assay. It is concluded that the measurement of clonogenic cell survival can be a valuable endpoint in the assessment of the response of some xenograft tumours to therapy. The agreement between the 2 assays in which colonies were grown under widely different conditions decreases the likelihood that cells cloned represent an atypical subpopulation.

Topics: Agar; Animals; Cell Survival; Clone Cells; Humans; Lung; Melanoma; Methods; Mice; Mice, Inbred CBA; Neoplasm Transplantation; Neoplasms, Experimental; Transplantation, Heterologous

Human malignant melanoma can form tumor colonies in soft agar, and at least two major morphological variants can be identified. Host cells play an important role in human melanoma colony formation, and their effects on the colony variants are selective. Neuroendocrine compounds also modulate expression of the melanoma TCFU pigmentary variants. This system appears suitable for evaluation of the response of melanoma TCFUs to chemotherapy, retinoids, heat, and radiation.

Topics: Agar; Cell Separation; Cells, Cultured; Clone Cells; Culture Media; Drug Evaluation; Growth Substances; Hormones; Humans; Lymphocytes; Melanoma; Neoplasm Metastasis; Phagocytes; Vitamin A

The resistance of a human melanoma cell line (MM96) to both ultraviolet and ionizing irradiation was compared by two different methods of cloning, on plates and in agar. A high level of resistance to both ultraviolet (D0 = 320 ergs/sq mm) and ionizing irradiation (D0 = 4300 rads) was observed when viability of cells was determined by cloning in agar. In contrast, melanoma cells were found to be as sensitive as were other cells when viability after irradiation was determined by cloning on plastic plates. The difference in sensitivity to radiation between the two methods of cloning can be explained in a model involving damage to membranes as well as to DNA. At least for ionizing radiation, this effect is not restricted to melanoma cells since a HeLa subline, HeLa-QB1, showed a similar response. In contrast, a human lymphoblastoid line (JHP) cloned in agar was sensitive under these conditions (D0 = 120 rads).

Topics: Agar; Cell Survival; Clone Cells; Gamma Rays; Melanoma; Neoplasms, Experimental; Plastics; Radiation Tolerance; Ultraviolet Rays; X-Rays

A soft agar colony assay has been developed for the B16 mouse melanoma and the Lewis lung tumour. The special features of the technique are the use of a gas phase with 5% O2 instead of air and the addition of rat red blood cells. Single cell suspensions are prepared by trypsinization from the solid tumour and the cells are plated out in 0-3% agar over a layer of 0-5% agar in 30-mm Petri dishes. After 8 to 15 days' incubation in 5% O2, colonies of more than 50 cells are produced. Plating efficiencies of between 30 and 50% are usually obtained. The addition of up to 10(4) heavily irradiated tumour cells gives some further improvement in plating efficiency for the B16 melanoma but not for the Lewis lung tumour. Applications of the technique to measure cell survival in the two tumours after treatment with cytotoxic drugs and radiation are reported. The scatter of experimental points is relatively small, and in comparative experiments good agreement has been obtained with results using in vivo assay techniques.

Topics: Agar; Animals; Cell Count; Cell Survival; Clone Cells; Cyclophosphamide; Cytological Techniques; Erythrocytes; Gamma Rays; Lung Neoplasms; Melanoma; Mice; Neoplasms, Experimental; Oxygen; Rats; Time Factors

Topics: Abdominal Neoplasms; Agar; Aged; Antigens, Neoplasm; Breast Neoplasms; Cell Division; Cells, Cultured; Clone Cells; Colonic Neoplasms; Female; Humans; Immune Sera; Immunity, Cellular; Kidney Neoplasms; Liposarcoma; Lymphocytes; Male; Melanoma; Methods; Middle Aged; Neoplasms; Prostatic Neoplasms; Rectal Neoplasms; Thyroid Neoplasms

Multi-cell spheroids were grown in soft agar. When each spheroid was cultured in a large volume of medium, frequently renewed, all spheroids eventually reached a dormant phase at a diameter of approximately 3-4 mm and a population of approximately 10(6) cells. In the dormant spheroid, newly generated cells at the periphery balanced those lost by necrosis in the center. We propose that this dormant phase is due to a gradual reduction in the ratio of surface area to volume: a size is achieved beyond which there is insufficient surface area for the spheroid to eliminate catabolites and absorb nutrients. Thus, in the face of unlimited space and of new medium, three-dimensional cell populations become self-regulating. This phenomenon contrasts with standard tissue culture in which cell populations, living on a flat plane in two dimensions, will not stop growing in the face of unlimited space and new medium because the ratio of surface area to volume remains constant. These experiments provide a mechanism for our observations in vivo: before vascularization, solid tumors live by simple diffusion as three-dimensional spheroids or ellipsoids. They become dormant at a diameter of only a few millimeters; once vascularized, they are released from this dormant phase and begin exponential growth. Thus, tumor dormancy resulting from absence of angiogenesis in vivo, may operate by the same mechanism responsible for dormancy of spheroids in vitro.

Topics: Agar; Animals; Cell Count; Cell Division; Cricetinae; Culture Techniques; Leukemia, Experimental; Lung; Melanoma; Mice; Time Factors

Topics: ABO Blood-Group System; Adolescent; Adult; Agar; Aged; Antigen-Antibody Complex; Antigen-Antibody Reactions; Antigens, Neoplasm; Cell Division; Cell Survival; Cells, Cultured; Clone Cells; Cytotoxicity Tests, Immunologic; Female; Humans; Immune Sera; Immunity, Cellular; Lymphatic Metastasis; Lymphocytes; Male; Melanoma; Middle Aged

Topics: Agar; Animals; Antineoplastic Agents; Carcinoma; Cell Line; Cells, Cultured; Disease Models, Animal; Humans; Leukemia L1210; Melanoma; Mice; Mice, Inbred Strains; Mouth Neoplasms; Neoplasm Transplantation; Neoplasms, Experimental; Oncogenic Viruses; Plant Extracts

Topics: Adolescent; Adult; Agar; Aged; Catechol Oxidase; Child; Child, Preschool; Dihydroxyphenylalanine; Electrophoresis; Female; Gels; Humans; Immune Sera; Immunodiffusion; Immunoelectrophoresis; Male; Melanoma; Middle Aged; Nevus, Pigmented; Skin; Skin Neoplasms; Tissue Extracts; Tyrosine

Topics: Agar; Animals; Catechol Oxidase; Electrophoresis; Gels; Glycoproteins; Hydroxybutyrate Dehydrogenase; Isocitrate Dehydrogenase; Isoenzymes; L-Lactate Dehydrogenase; Lipoproteins; Melanoma; Mice; Neoplasm Transplantation; Neoplasms, Experimental; Proteins