Comparative radiosensitivity of clonogenic tumor cells of the transplantable Ehrlich carcinoma in the ascitic and solid forms of growth

Survival of clonogenic cells of solid Ehrlich ascites tumor (EAT) exposed to 60Co-gamma-radiation in vitro under the oxygenation conditions was investigated and the clonogenic capacity and radiosensitivity of these cells and cells of the previously studied EAT ascitic form and Lewis solid tumor comparatively studied to elucidate how the efficiency of colony formation (ECF) would affect their radiosensitivity. ECF for solid EAT cells was 2.6 +/- 0.3%, which was lower, by about an order of magnitude, than that for ascitic form of this tumor and was nearly the same as that for Lewis tumor cells. A median cell lethal dose (D0) was practically the same for all tumors under study. It is suggested that the differences in ECF do not substantially influence the radiosensitivity of clonogenic cells of the studied tumors.     

Differential expression of fibronectin in a rat histiocytoma: possible role of fibronectin in tumor cell adhesion

The specific role of fibronectin in tumor cells has been investigated using the transplantable histiocytic tumor line AK-5 [A. Khar (1986) J. Natl. Cancer Inst. 76, 871]. These cells, capable of growth as both ascites and solid tumors in rats, can be separated into four subpopulations by buoyant density centrifugation on Percoll. These subpopulations are shown to contain different amounts of fibronectin on the cell surface when studied by immunofluorescent staining followed by cytofluorometric analysis. Cells which contain high amounts of fibronectin can grow both as ascites and as solid tumors while those cells which contain low amounts of fibronectin cannot grow as solid tumors but can grow as ascitic tumors. Pretreatment of high-fibronectin-containing cells with anti-fibronectin antibody abolished their capacity to grow as solid tumors; however, the cells retained their capacity to grow as ascitic tumors. These results have been discussed with reference to the specific role of fibronectin in tumor-forming cells.     

Conformational changes in the DNA of hybridoma cells from pristane treated mice

The effects of pristane on the DNA of hybridoma cells propagated as ascitic tumors in pristane-primed BALB/c mice were determined using flow cytometric analyses. Hybridoma cells maintained in vitro or cell isolates from solid tumors which developed in unprimed mice injected with hybridoma cells exhibited similar propidium iodide (PI) staining characteristics. In contrast, PI stained cells isolated from ascites which developed in pristane-primed mice injected with the hybridoma cells displayed significant decreases in fluorescence intensity. Diphenylamine studies and analyses of pH 10 treated cells indicated that the actual DNA content of the hybridoma cells was not altered by exposure to pristane. Furthermore, the altered staining characteristics of the ascitic tumor cells were reversible in that the fluorescence intensity after serial in vitro passage of the ascites cells was similar to that of the parent cell line which had not been exposed to pristane. In addition, there was a direct correlation between the altered PI staining characteristics and the presence of cell-associated pristane as determined by gas-liquid chromatography analyses of cell extracts. Collectively these results suggest that pristane may have a direct effect on the DNA conformation of hybridoma cells which may in turn enhance their growth as ascitic tumors. The possible role of such an altered DNA conformation in hybridoma cells on the in vivo development of ascites is discussed.     

Differential remodeling of cadherins and intermediate cytoskeletal filaments influence microenvironment of solid and ascitic sarcoma

Different forms of sarcoma (solid or ascitic) often pose a critical medical situation for pediatric or adolescent group of patients. To date, predisposed genetic anomalies and related changes in protein expression are thought to be responsible for sarcoma development. However, in spite of genetic abnormality, role of tumor microenvironment is also indispensable for the evolving neoplasm. In our present study, we characterized the deferentially remodeled microenvironment in solid and ascitic tumors by sequential immunohistochemistry and flowcytometric analysis of E-cdaherin, N-cadherin, vimentin, and cytokeratin along with angiogenesis and metastasis. In addition, we considered flowcytometric apoptosis and CD133 positive cancer stem cell analysis. Comparative hemogram was also considered as a part. Our investigation revealed that both types of tumor promoted neovascularization over time with sign of local inflammation. Invasion of neighboring skeletal muscle by solid sarcoma was more frequent than its ascitic counterpart. In contrary, rapid and earlier cadherin switching (E-cadherin to N-cadherin) in ascitic sarcoma made them more aggressive than that of solid sarcoma and helped to early metastasize distant tissue like liver through the hematogenous route. Differential cadherin switching and infidelity of cytokeratin expression in Vimentin positive sarcoma also influenced the behavior of ascitic CD133+ cancer initiating cell pool with respect to CD133+ cells housed in solid sarcoma. Therefore our study concludes that differential cadherin switching program and infidelity of intermediate filaments in part, sharply discriminate the severity and metastatic potentiality of either type of sarcoma accompanying with CD133+ cellular repertoire. Besides, tumor phenotype-based dichotomous cadherin switching program could be exploited as a future drug target to manage decompensated malignant ascitic and solid sarcoma.     

Nitric oxide complexes in the interaction between primary and secondary tumor of L5178Y lymphoma.

Heme and non-heme Fe-NO complexes were observed in regard to the growth of primary and secondary solid tumors and ascites of murine L5178Y lymphoma. The complexes were detected by electron paramagnetic resonance spectroscopy at liquid nitrogen temperature. Primary solid tumors and secondary solid tumors or ascites were inoculated on the same day, or with a delay. The primary tumor inhibited growth of the secondary solid tumor only if the latter was inoculated with a delay, which did not correlate with the change of the types, nor with the increase in the level of Fe-NO complexes detected in the tissue, suggesting a "non-immunological" character of this inhibition. In some animals with solid tumors, spontaneous ascites developed. This process resulted in a marked decrease in the level of Fe-NO complexes in the solid tumor tissue. The primary solid tumor, however, did not influence the growth of secondary ascites, but intensified NO generation in the ascites of animals with partial removal of ascitic fluid. This experimental group survived 2.2 days longer than the control group without primary solid tumor. Our research revealed that the presence of Fe-NO complexes in the interaction between primary and secondary tumor strongly depends on the form of the tumor: solid or ascitic, and that murine L5178Y lymphoma may serve as a convenient model for the research on "concomitant immunity" against in vivo growing tumors. This is the first EPR study on "concomitant immunity" in regard to tumor-tumor and tumor-ascites interactions in vivo.     

Ascitic and solid Ehrlich tumor inhibition by Chenopodium ambrosioides L. treatment

The leaves of Chenopodium ambrosioides L. [Chenopodiaceae] ('mastruz') have been indicated for the treatment of several diseases, among which the cancer. There are no results focusing the effect of C. ambrosioides treatment on tumor development in vivo. The aim of this study was to investigate the effect of treatment with C. ambrosioides on Ehrlich tumor development. Swiss mice were treated by intraperitoneal route (i.p.) with hydroalcoholic extract from leaves of C. ambrosioides (5 mg/kg) or with PBS (control group) 48 h before or 48 h later the Ehrlich tumor implantation. The tumor cells were implanted on the left footpad (solid tumor) or in the peritoneal cavity (ascitic tumor). To determine the solid tumor growth, footpad was measured each 2 days until the fourteenth day, when the feet were weighed. Ascitic tumor development was evaluated after 8 days of tumor implantation by quantification of the ascitic fluid volume and tumor cell number. The i.p. administration of C. ambrosioides extract before or after the tumor implantation significantly inhibited the solid and ascitic Ehrlich tumor forms. This inhibition was observed in ascitic tumor cell number, in the ascitic volume, in the tumor-bearing foot size and foot weight when compared to control mice. The treatments also increased the survival of tumor-bearing mice. In conclusion, C. ambrosioides has a potent anti-tumoral effect which was evident with a small dose and even when the treatment was given two days after the tumor implantation. This effect is probably related with anti-oxidant properties of C. ambrosioides.     

Nitrogen metabolism in tumor bearing mice

In experiments with whole animals infested with a highly malignant strain of Ehrlich ascites tumor cells, serial concentrations of amino acids were determined for host plasma, ascitic fluid, and tumor cells, throughout tumor development. Concentration gradients of glutamine, asparagine, valine, leucine, isoleucine, phenylalanine, tyrosine, histidine, tryptophan, arginine, serine, methionine, and taurine from the host plasma toward the ascitic liquid were established; while on the other hand, concentration gradients from the ascitic liquid toward the plasma were established for glutamate, aspartate, glycine, alanine, proline, and threonine. With the exception of aspartate the concentrations of these amino acids were highest inside the cells. Arginine was the only amino acid not detected in tumor cells. In vitro incubations of tumor cells in the presence of glutamine and/or glucose, as the energy and nitrogen sources, confirmed the amino acid fluxes previously deduced from the observed relative concentrations of amino acids in plasma, ascitic liquid, and tumor cells, suggesting that glutamate, alanine, aspartate, glycine, and serine can be produced by tumors. These findings support that changes in amino acid patterns occurring in the host system are related to tumor development.     

The modelling of the growth of Ehrlich carcinoma and teratoma T-36 with ascitic fluid and tumor-cell dialysate]

The study of the effect of ascitic fluid and dialysate of Ehrlich ascites tumor cells (M.m. less than 15 kDa) on the growth of Ehrlich carcinoma and teratoma T-36 has shown that both the ascitic fluid and dialysate can protect tumor cells in vivo. The number of animals with tumors increased from 0% in control animals to 60 and 20%, respectively, in experimental ones after transplantation i.m. of 20 x 10(3) Ehrlich tumor cells into mice. Compared to control, ascitic fluid and dialysate of Ehrlich ascites tumor cells increased the rate of tumor growth to 195 and 153%, respectively. It is suggested that this test-system simulates the effect of tumor humoral factors in vivo.     

Natural killer cell cytotoxic potential of patients with ovarian carcinoma and its modulation with virus-modified tumor cell extract

Summary We have demonstrated that cancer patients with ovarian carcinoma display deficient peripheral blood NK-cell cytotoxic potential against the K-562 target cell line. Furthermore, no NK-cell activity against the same tumor was detected in ascitic fluids of these patients. The inferior peripheral blood NK-cell cytotoxicity of ovarian carcinoma patients was significantly augmented after ID inoculation with virus-modified tumor cell extract. Similarly, NK-cell activity in the ascitic fluids was dramatically increased after IP in vivo therapy with the same tumor extract preparation. Interestingly, in some of the cancer patients the augmentation of NK-cell activity in ascitic fluids after IP injection of virus-modified tumor cells extract was associated with a clinical response of the patients, as demonstrated by regression of ascitic tumors. These studies indicate, first, that virus-modified tumor extract displays immunopotentiating activity, as reflected by its marked NK cell-augmenting potential, and secondly, that regional activation of NK cells could underlie the mechanism of regression of ascitic tumors.     

Antitumor effect of natural avermectins]

The effects of the natural avermectin complex, aversectin C and individual avermectin B1 on the growth of ascitic and solid transplantable tumors in animals were studied. The results showed for the first time that both aversectin C and avermectin B1 possessed marked antitumor activity. In subtoxic doses aversectin C significantly inhibited the growth of P388 lymphoid leukemia and Ehrlich carcinoma, both ascitic and solid ones. In some administration regimens aversectin C inhibited the tumor growth by 70 to 80%. The highest effect of aversectin C was observed after its intraperitoneal administration. Avermectin B1 inhibited the growth of solid Ehrlich carcinoma and carcinoma 755.     

The bradykinin B1 receptor antagonist R-954 inhibits Ehrlich tumor growth in rodents

The present study investigated the effects of a new bradykinin B₁ receptor antagonist, R-954, on the development of Ehrlich ascitic tumor (EAT) induced by the intraperitoneal inoculation of EAT cells in mice and the formation of a solid tumor by the subcutaneous injection of the cells in rat paw. The development of the tumor was associated with an increase in mouse total cell counts in bone marrow (10.8-fold), ascitic fluid (14.6-fold), and blood (12.6-fold). R-954 (2 mg/kg, s.c.) significantly reduced the ascitic fluid volume (63.7%) and the mouse weight gain (30.5%) after 10 consecutive days of treatment. The B₁ antagonist as well as the anti-neoplasic drug vincristine also significantly inhibited the increase in total cell count in bone marrow, ascitic fluid, and blood. R-954 reduced significantly the total protein extravasation (57.3%), the production of nitric oxide (56%), PGE₂ production (82%), and TNFα release (85.7%) in mice peritoneal cavity whereas vincristine reduced the release of these inflammatory mediators by 84-94%. The increase in paw edema after intraplantar injection of EAT cells was reduced by approximately 52% by either R-954 or vincristine treatment. In conclusion, this study presents for the first time the antitumoral activity of a new bradykinin B₁ receptor antagonist on ascitic and solid tumors induced by Ehrlich cell inoculation in mice and rats.     

Comparison of cellular phenotypes in tumor cyst and ascitic fluid from ovarian serous carcinoma.

Density gradient centrifugation was applied to isolate cell subsets from tumor cyst and ascitic fluid in eight patients with ovarian serous carcinoma. A comparison of cellular composition and immunologic reactivity of cells from the cysts and from ascitic fluid in each patient was performed. Some differences in density profiles were found, but in each case the consistency of morphologic cell forms in the primary tumor and ascites was documented. Immunophenotypic analyses of isolated cellular fractions using polyclonal and monoclonal antibodies against ovarian carcinoma-associated antigens showed significant immunologic intratumoral heterogeneity. However, there was a similarity of antigen expression in cells from the primary tumors and ascitic fluids. Our study indicated that morphologic and antigenic characterization of a given tumor could be determined in a single representative sample of ascitic fluid.     

Action of valinomycin in vivo on Ehrlich ascites tumor cells. A change in the ultrastructure of the tumor cells under the influence of valinomycin]

It was found with the help of electron microscopy that valinomycin administered to mice with Ehrlich ascitic tumors in a dose inhibiting the proliferating activity induced impairements in the mitochondria. The changes were similar to those under the effect of valinomycin in vitro. An increase in the antibiotic dose resulted in more pronounced and irreversible changes not only in the mitochondria but also in other structures of the tumor cells.     

Differing microsomal monoxygenase activity in the cells of ascitic and solid forms of transplantable tumors

Aryl hydroxylase activity has been demonstrated to depend on the pattern of tumor cell structural organization. The activity of microsomal monoxygenases in the ascitic forms of sarcoma MC-11, hepatoma 22a and Ehrlich's tumor was much lower than in the corresponding solid tumors. Aryl hydroxylase was activated after the animals received 3-MC, but the magnitude of the activity induced did not correlate with the basic activity in the different tumors. In in vitro experiments, 7,8-benzoflavone inhibited the enzyme in all the tumors, whereas metyrapone did not affect BP-hydroxylation. It is assumed that all the tumors investigated contain hemoprotein that is similar to cytochrome P1-450.     

Effect of Pfaffia paniculata (Brazilian ginseng) on the Ehrlich tumor in its ascitic form

The roots of Pfaffia paniculata (Brazilian ginseng) have been indicated for the treatment of several diseases, among which the cancer. The purpose of this study was to investigate experimentally the possible antineoplastic effect of this root. Firstly, a toxicity study was performed in which the doses of 400 and 200 mg/Kg of the powdered root were administered by gavage for 10 days to BALB/cICB mice. The mice did not lose weight during the treatment. No increase in serum alanine-aminotransferase neither histopathological alteration (liver, kidney and spleen) was observed in mice treated with P. paniculata. The effect of this root on the ascitic Ehrlich tumor in BALB/cICB mice was then investigated. Male mice received, by gavage, once a day, 200 mg/Kg of the powdered root of P. paniculata or distilled water, as control, for 20 days. This protocol started 10 days before tumor inoculation with 5 x 10(6) cells i.p., and lasted until 10 days after. The ascitic tumor was evaluated by the quantification of the volume of the ascitic fluid, relative number of tumor cells and total number of tumor cells. A decrease in the total ascitic volume was observed in P. paniculata treated mice, that was followed by a numerical decrease in the total number of Ehrlich tumor cells. These results may indicate that P. paniculata anti-inflammatory effects were responsible by the decrease in the total ascitic fluid. In addition, the presence of tumor-cell inhibitory factors in P. paniculata roots is in agreement with other in vitro studies. The mechanisms of such tumor inhibition should be further investigated.     

Comparative studies of mouse tumors with respect to their capacity for growth as “ascites tumors” and their average nucleic acid content per cell

The advantages presented by the use of the comparatively homogeneous free tumor cell suspension of the Ehrlich ascites tumor for quantitative studies on the growth and chemical composition of tumor cells and for chemotherapeutic experiments have led to the present investigation of a representative array of 26 histologically and genetically different mouse tumors which were tested for their capacity of reaching a condition similar to the Ehrlich ascites tumor. This has been found to be possible in a number of cases. Dissociated solid tumor masses were intraperitoneally inoculated into the corresponding mouse strain. Tumor growth on the peritoneum, accompanied by exudate formation, resulted in 22 out of 26 cases. The resulting exudates, which, in the case of all carcinomas and most sarcomas tested, contained very few tumor cells, transmitted the tumors upon further intraperitoneal injection in 18 instances. The tumors which developed were now regularly provoking exudate formation, and on subsequent generations the exudates have always been transplanted intraperitoneally. In the case of all carcinomas and in 2 out of 3 sarcomas tested which have passed over 10 such “fluid transplant generations” (FTG), a gradual increase in the frequency of tumor cells in the ascitic fluids (examined around median survival time) was occurring after a certain     

Development of an in vitro colony formation assay for the evaluation of in vivo chemotherapy of a rat brain tumor.

An in vitro colony formation assay for the evaluation of in vivo brain tumor therapy has been developed. When plated, disaggregated cells derived from solid tumors proliferated to form relatively homogeneous colonies after a latency period of 2 to 6 days. Increasing concentrations of fetal calf serum enhanced colony-forming efficiency (CFE) with a plateau between 7 and 16%. Supplementation with either irradiated feeder cells (10(3) to 10(5) cells per dish), or medium conditioned by 1 to 3 days of in vitro incubation with the same cell line, doubled the CFE. The density of tumor cells (untreated or previously treated with chemotherapeutic agents) did not affect the CFE when a minimum of 10(4) total cells (tumor plus feeder) were plated. Therefore, in this system the optimal experimental conditions for evaluating chemotherapy and radiotherapy require incubation of disaggregated tumor cells for 12 days in medium containing 10% of fetal calf serum and enough feeder cells to provide a minimum of 10(4) cells per dish. The CFE for untreated tumors was 18 +/- 10% (+/-S.D.), demonstrating that there is significant biological variation. The assay appeared sensitive, with reproducible results, when applied to individual chemically treated tumors. An estimate of the percentage of clonogenic cells affected by in vivo chemotherapy may be obtained by comparing the CFE of cells from treated and untreated tumors. This assay can measure up to a 5 log(10) cell kill, and it should prove to be valuable in developing more effective regimens for the treatment of solid tumors in animals and man.     

Effect of lysozyme on the growth of murine lymphoma and antineoplastic activity of cyclophosphamide]

It was shown that hen egg-white lysozyme (LM) in the dose 100 mg/kg under the daily intragastral use slightly inhibited tumor grown or did not influence significantly upon it and did not change antitumor activity of cyclophosphamide. When used at mice C57Bl/6J with the transplanted ascitic or solid T-cell lymphoma EL4 (syngeneic system). On model of the same tumors in ascitic form at mice-hybrids (C57Bl/6J x DBA2)F1 (semisingeneic system) LM significantly potentiates antitumor activity of cyclophosphamide, though it had no effect on the rate of tumor growth. Potentiation of the effect of cyclophosphamide revealed itself in more slow development of ascite, increased mean life-span and the overall survival, appearance of completely cured animals. Our clinic-laboratory studies have revealed a sharp deficit of endogenic lysozyme in the blood serum of leukemic patients and extremely low lysozyme content in lavage liquid, from leukemic patients, with pneumonia. These data suggest that LM can be useful as a food additive in the complex treatment of oncological patients for enhancing antineoplastic chemotherapy efficacy.     

Changing intermediate-sized filament patterns in metastatic hepatocellular carcinoma cells of the guinea pig

Hepatocellular carcinoma cells obtained from ascitic fluid after diethylnitrosamine treatment of Sewall Wright strain-2 guinea pigs produce solid (primary) tumors, lymph-node metastases and malignant ascites when reinjected into animals of the same strain. When brought into culture the cells settle, form multilayer cultures and can be maintained in passage. In addition to epithelium-specific cytokeratin intermediate filaments (IF), these latter cells, like most cultured cells, also contain vimentin. Hepatocellular carcinoma cells in solid tumors and in metastatic tumors retain their original keratin IF and in general do not have an additional vimentin-IF system. When the tumor cells are present in ascites they develop vimentin-IF in addition to cytokeratin filaments. Vimentin is gradually lost when these cells sediment onto the peritoneal surface and proliferate continuously to form papillary projections, or when they are detected as circumscribed metastases. It seems likely, therefore, that in this system the synthesis of an additional vimentin cytoskeleton is related to reduced cell-to-cell contact and to the ability of the cells to survive individually or as cell clusters in body fluids, without being part of a cohesive tissue.