Measuring single-cell density.

We have used a microfluidic mass sensor to measure the density of single living cells. By weighing each cell in two fluids of different densities, our technique measures the single-cell mass, volume, and density of approximately 500 cells per hour with a density precision of 0.001 g mL(-1). We observe that the intrinsic cell-to-cell variation in density is nearly 100-fold smaller than the mass or volume variation. As a result, we can measure changes in cell density indicative of cellular processes that would be otherwise undetectable by mass or volume measurements. Here, we demonstrate this with four examples: identifying Plasmodium falciparum malaria-infected erythrocytes in a culture, distinguishing transfused blood cells from a patient's own blood, identifying irreversibly sickled cells in a sickle cell patient, and identifying leukemia cells in the early stages of responding to a drug treatment. These demonstrations suggest that the ability to measure single-cell density will provide valuable insights into cell state for a wide range of biological processes.

Impairment of red blood cell deformability by tumor growth.

Red blood cell deformability is a major determinant of capillary blood flow. In mice with L1210 leukemia and with Lewis lung carcinoma, red blood cell deformability was significantly decreased during tumor growth. In mice with L1210 leukemia, deformability was significantly decreased by day 5 after transplantation and progressively decreased through day 9. Terminally, red blood cell deformability returned to normal or above normal values. In mice with Lewis lung carcinoma, significant decreases in deformability were noted 21--28 days after transplantation and persisted throughout the remainder of the tumor course. Impaired capillary blood flow, secondary to abnormal red blood cell deformability, is therefore associated with advanced cancer.

A model for prediction of chemotherapy response to 5-fluorouracil based on the differential distribution of 5-[18F]fluorouracil in sensitive versus resistant lymphocytic leukemia in mice.

The distribution of 5-[18F]fluorouracil has been compared in two variants of the same tumor in c57bL X DBA/2 F1 mice: solid L1210 lymphocytic leukemia tumor susceptible to 5-fluorouracil treatment and the same tumor, made resistant to the drug over a 34-generation span. Significant differences in 5-[18F]fluorouracil distribution were observed, most notably in the tumor:blood ratios at 12 hr postinjection. The drug-responsive tumor showed a 20:1 concentration ratio, whereas the drug-resistant tumor only had a 4:1 concentration ratio. We postulate that these differences, observed here in this animal tumor model, may be a reflection of similar ratio differences in humans. This technique may allow, by noninvasive quantification of tumor:blood ratios following administration of 5-[18F]fluorouracil to man, the differentiation of those human tumors that are likely to respond to drug therapy from those in which the response will be minimal or nil.

Blood thymidine level and iododeoxyuridine incorporation and reutilization in DNA in mice given long-acting thymidine pellets.

A long-acting thymidine pellet consisting of 190 mg of cholesterol and 60 mg of thymidine has been developed for the study of thymidine metabolism and reutilization in vivo. Implantation of such a pellet s.c. in adult mice will maintain the blood plasma concentration of thymidine at levels between 40 and 8 X 10(-6) M, which are from 36 to 7 times those of normal mice, for periods up to 48 hr. During this period, in vivo uptake and reutilization of [125I]iododeoxyuridine, a thymidine analog, into intestinal and tumor DNA were almost completely suppressed. While iododeoxyuridine reutilization is not large in normal proliferative tissue even in the absence of pellet implants, reutilization of over 30% was measured in large, rapidly growing ascites tumors. The inhibition of iododeoxyuridine incorporation by elevated thymidine blood levels is directly proportional to serum concentration. This appears to be due to a thymidine pool in rapid equilibrium with blood thymidine. This pool is at least 10 times larger than the 4-nmole pool of extracellular thymidine.

Erythrocyte entrapment of daunomycin by amphotericin B without hemolysis.

Amphotericin B is a polyene that binds to sterols and perforates cell membranes. An antileukemic drug such as daunomycin added exogenously is impermeable to the red cell membrane. However, when the cells are incubated with a low concentration of amphotericin B, daunomycin is entrapped in the red cells without hemolysis or alteration in the chemical parameter of the erythrocytes. The erythrocyte has been used as a carrier vehicle to enhance the cytotoxic activity of daunomycin against L1210 leukemic cells. In comparison to control preparations, the greatest increase in survival was obtained in vivo when the erythrocytes with entrapped daunomycin were given to C57BL X DBA/2 F1 mice bearing L1210 cells.

Effects of the combined arginase and canavanine treatment on leukemic cells in vitro and in vivo.

It was previously demonstrated in in vitro experiments that canavanine (Cav), a natural toxic arginine analogue of plant origin, is a promising candidate for augmenting the antineoplastic effects of arginine starvation. We demonstrated herein that recombinant human arginase, an arginine degrading enzyme, abrogated growth and significantly increased Cav cytotoxicity toward cultured L1210 murine leukemic cells. Cav co-treatment further reduced cells viability in a time-dependent manner and significantly promoted apoptosis induction. In the pilot study we also evaluated for the first time the potential toxicity of the combined arginine deprivation and Cav treatment in healthy mice. Administration of Cav alone or in combination with pegylated cobalt-containing human arginase (Co-hARG) did not evoke any apparent toxic effects in these animals, with no significant behavioural and survival changes after several weeks of the treatment. The therapeutic effects of the combination of Co-hARG and Cav were provisionally evaluated on the highly aggressive murine L1210 leukemia, which is semi-sensitive to arginine deprivation as a monotreatment. Combination of two drugs did not result in significant prolongation of the survival of leukemia-bearing mice. Thus, we have shown that the proposed combinational treatment is rather non-toxic for the animals. It has to be further evaluated in animal studies with alternative tumor models and/or drug doses and treatment modalities.

Pharmacokinetic studies in mice of two new thioxanthenones (183577 and 232759) that showed preferential solid tumor activity.

Two new thioxanthenones, 183577 and 232759, have rekindled interest in the development of representatives from this class of structures as useful anticancer agents. Although the mechanism of action is unknown, both compounds demonstrated a similar spectrum of solid tumor selectivity. 232759 was selected for clinical development because it showed no hepatotoxicity in preliminary studies, whereas 183577 showed hepatotoxicity but only at the maximum tolerated dose (MTD). The limiting toxicity for the clinical candidate was myelosuppression in preliminary studies. Plasma and tissue drug levels, as well as protein binding, were studied in mice using optimal administration times at the MTD for each drug (for 183577, this was a 4-h infusion at 1350 mg/m2 and for 232759, it was a 5-min injection at 240 mg/m2), as well as at one-half the MTD for the clinical candidate. The drugs were 96-100% bound by plasma proteins. The peak drug concentrations, half-life, and area under the concentration-time curve in plasma for 183577 were 3483 ng/ml, 465 min, and 2018 microgram/ml. min, respectively. The peak drug concentration, half-life, and area under the concentration-time curve in plasma for 232759 were 5257 ng/ml, 44 min, and 276 microgram/ml. min, respectively, at the MTD and 2810 ng/ml, 40 min, and 110 microgram/ml. min at one-half the MTD. In all instances of simultaneous measurements, drug concentrations were equal or higher in tissues than they were in plasma. Unlike the plasma and kidney concentrations of 183577, the liver concentrations did not show a declining trend over the 8-h observation period. Declines in plasma, liver, kidney, and tumor levels of 232759 were detected over the 8-h observation period. The sustained high 183577 concentration in liver is believed to be responsible for its prolonged half-life and hepatotoxicity. Evidence for metabolism of the parent drugs was based on the finding of additional peaks on the high-pressure liquid chromatography tracings. Future studies will focus on identification and antitumor studies of these presumed metabolites in hopes of a better understanding of the solid tumor activity profiles and toxic effects of these compounds.

Suppression of mouse erythroid colony formation by L1210 leukemia cells.

The effect of L1210 transplantable leukemic cells on in vitro formation of erythroid colonies from CD2F1 mouse bone marrow progenitor cells (CFU-E) was investigated. Clonal cell culture was carried out by a methylcellulose technique. Human urinary erythropoietin served as the stimulator. After 44 hours of incubation aggregates of eight or more erythroid cells were scored as colonies. The number of CFU-E which could be demonstrated in marrow cells from mice that had been injected intravenously 6 days before with 5 x 10(4) L1210 cells was far below that obtained from normal marrow cells. When 1.3 x 10(5) marrow cells from leukemic mice or L1210 ascites cells were cultured with an equal amount of normal cells, the number of CFU-E expressed was reduced by 51% and by 86%, respectively, relative to controls with normal cells only. Neither lethally irradiated L1210 cells (4500 rad) nor L1210 cell conditioned media suppressed erythroid colony formation. It is suggested that in L1210 leukemia erythropoiesis is decreased because of a cell-to-cell inhibitory action of the leukemia cells on CFU-E.

Hemin stimulating effect on colony formation of leukemic and bone marrow cells.

The hemin enhancing activity on colony formation of leukemic and normal bone-marrow (BM) cells is described. The colony growth of Friend erythroleukemic cells (FL) and mastocytoma cells (M) was markedly enhanced. On the other hand, myeloid leukemic cells (P) and normal bone-marrow cells (BM) were only slightly affected. Inhibition of colony formation was observed with lymphoid leukemic cells (L). For M and BM cells, the horse serum could be replaced by BSA with preservation of hemin enhancing activity.

Reversal by cytidine of cyclopentenyl cytosine-induced toxicity in mice without compromise of antitumor activity.

Among nine compounds surveyed, cytidine was found to be the most effective in reversing the antiproliferative effects of cyclopentenyl cytosine (CPEC) on human T-lymphoblasts (MOLT-4) in culture. Cytidine, at concentrations of 1-25 microM, enabled cells to maintain normal logarithmic growth when added up to 12 hr after exposure to a 200 nM concentration of the oncolytic nucleoside, CPEC. The most abundant CPEC metabolite, CPEC-5'-triphosphate, is a potent [K1 approximately 6 microM] inhibitor of CTP synthetase (EC 6.3.4.2). Accumulation of this inhibitor resulted in a depletion of CTP levels to 17% of their original cellular concentration. Exogenous cytidine reversed CPEC-induced cellular cytotoxicity by suppressing the formation of CPEC-5'-triphosphate by 70%, and by partially replenishing intracellular CTP to at least 60-70% of its original concentration. In vivo, cytidine (500 mg/kg) administered intraperitoneally 4 hr after each daily dose of CPEC (LD10-LD100) for 9 days reduced the toxicity and abolished the lethality of CPEC to non-tumored mice. Of greater practical importance is the finding that, under these experimental conditions, cytidine did not curtail the antineoplastic properties of CPEC in L1210 tumor-bearing mice. Moreover, the concentration range over which CPEC exhibited antineoplastic activity was extended with cytidine administration.

Identification of vesicle properties that enhance the antitumour activity of liposomal vincristine against murine L1210 leukemia.

The influence of vesicle lipid composition, size and drug-to-lipid ratio on the antitumour activity of liposomal vincristine was assessed in the murine L1210 ascitic leukemia model. A pH gradient-dependent entrapment procedure was used to encapsulate vincristine and allowed such vesicle properties to be independently varied. Free vincristine delivered i.v. at the maximum tolerated dose (2.0 mg/kg) resulted in a 27.8% increase in the life span (ILS) of mice inoculated i.p. with L1210 cells. Encapsulation of the drug in egg phosphatidylcholine/cholesterol vesicles did not significantly increase the antitumour efficacy of vincristine (ILS, 38.9%). In contrast, administration of vincristine entrapped in vesicles composed of distearoylphosphatidylcholine (DSPC)/cholesterol resulted in ILS values as high as 133%. This enhanced antitumour activity of the DSPC/cholesterol formulations was sensitive to the size of the liposomes; increasing the vesicle size from 100 nm to 1 micron decreased the ILS from 133.3% to 55.6% at a drug dose of 2.0 mg/kg. Decreasing the drug-to-lipid ratio from 0.1:1 to 0.05:1 (w/w) had negligible effects on the activity of liposomal vincristine; however, a further decrease in the drug-to-lipid ratio to 0.01:1 (w/w) decreased the antitumour potency at all drug doses studied. Pharmacology studies indicated that the antitumour activities of free and various liposomal forms of vincristine correlated well with the residence time of the drug in the circulation. These studies indicate that efforts to enhance the therapeutic activity of vincristine through liposome encapsulation must address not only the circulation lifetime of the vesicle systems but also the capacity of the liposomes to retain entrapped drug in vivo.

Antitumor activity of FTC-092, a masked 5-trifluoromethyl-2'-deoxyuridine derivative.

1-(3-O-Benzyl-2-deoxy-beta-D-ribofuranosyl)-5-trifluoromethyl-2,4(1H,3)- pyrimidinedione (FTC-092), a fluorinated pyrimidine derivative, appeared to be effective against various transplantable tumors in mice following oral administration, and its activity was superior to that of several other antitumor fluorinated pyrimidines. The ED50 value for FTC-092 the dose effective in achieving 50% inhibition of tumor growth against the solid form of sarcoma 180 was 13.3 mg/kg daily, whereas those for 5-trifluoromethyl-2'-deoxyuridine (CF3dUrd), the parent compound of FTC-092, for 1-(2-tetrahydrofuryl)-5-fluorouracil (Tegafur, FT), the prodrug of 5-fluorouracil (FUra), and for FUra were 64.1, 122, and 28 mg/kg daily, respectively. The therapeutic indices (LD10/ED50) of FTC-092, CF3dUrd, FT, and FUra were 4.39, 1.7, 1.35, and 1.65, respectively. FTC-092 itself is not an active agent. After it has been absorbed from the gastrointestinal tract, FTC-092 undergoes a gradual biotransformation, mainly via the action of liver microsomes, releasing CF3dUrd over a long period. The levels of CF3dUrd in the stomach and small intestine of mice after the oral administration of FTC-092 were undetectable, whereas those following the administration of CF3dUrd at the same dose were high for a period of several hours. In contrast, the CF3dUrd level generated in plasma after the administration of FTC-092 remained at a high level for a longer period than did that observed on the administration of CF3dUrd. The low levels of CF3dUrd measured in stomach and small-intestine tissues and the maintenance of CF3dUrd in blood over long periods after the administration of FTC-092 are features that favor the possible clinical application of FTC-092.

Increased protective activity against acid precipitation of poly(U) in the serum of tumor-bearing mice.

Transplantation of leukemia L1210 cells into DBA/2 mice and of Ehrlich ascites tumor cells into BALB/C mice resulted in a significant increase of protective activity against acid precipitation of poly (U) in the serum. The increase was observed as early as one day after the tumor transplantation and seems to be connected with cancer growth, since inoculation of L1210 cells into BALB/C mice did not affect the protective activity, evidently as a result of their well established inability to cause cancer in this strain. Furthermore, no increase of activity was observed when bacteria were inoculated into mice, or when the latter were partially hepatectomized. The results suggest that the protective activity against acid precipitation of poly (U) could prove to be a tumor marker for the early detection of cancer growth.

Enzyme activity in mice with transplantable leukemia.

Activity of cobalt activated acylase, gamma-glutamyltransferase, leucylaminopeptidase and alanylaminopeptidase in serum and liver of mice with transplantable leukemias (L1210, L1210/ara-C, L1210/CH3-G, AKSL-4, plasmacytoma ADJ-PC-5) were determined. Adenosinotriphosphatase, 5'-nucleotidase and alkaline phosphatase were histochemically localized in lymphatic nodes and spleen. Among the investigated enzymes the rise in serum activity of cobalt activated acylase and gamma-glutamyltransferase was demonstrated. A substantial increase of leucylaminopeptidase and alanylaminopeptidase was shown in the liver. A decrease in the histochemical reactions of all the studied enzymes was observed.

The influence of recombinant human tumor necrosis factor-alpha, alone and in combination with cyclophosphamide or methotrexate, on leukemia L1210 and normal hematopoiesis in mice.

We investigated the influence of rh-TNF administered as a single agent or in combination with CY or MTX on the survival time of mice inoculated with lymphoid leukemia L1210 and the effects of similar treatment on normal hematopoiesis in mice. The MST of rh-TNF--treated mice was longer than that of control animals. The longest survivals were observed in mice treated with 250 and 275 micrograms/kg of rh-TNF. Groups of mice receiving a combination of rh-TNF at doses of 225 or 250 micrograms/kg and MTX lived longer than animals treated with these agents separately. We observed the longest survival time of mice treated with combined administration of rh-TNF at a dose of 250 micrograms/kg and CY, but survival time was not significantly prolonged compared with mice receiving only CY. Additional studies were performed to examine the influence of rh-TNF administered as a single agent or in combination with toxic doses of CY or MTX on the number of granulocytes, lymphocytes, erythrocytes with hematocrit values and hemoglobin concentration, and platelets in peripheral blood, and the number of mononuclear cells as well as multipotential stem cells (CFU-GEMM) in bone marrow. Rh-TNF caused dose-dependent suppression of mononuclear cells and multipotential stem cells in bone marrow. The addition of MTX to rh-TNF caused no enhanced suppression of any of the above mentioned hematological parameters. In contrast, the addition of CY to rh-TNF suppressed erythrocytes and hematocrit values, as compared with rh-TNF alone.(ABSTRACT TRUNCATED AT 250 WORDS)

[Quantitative evaluation of the surface membrane receptors of leukemic cells to blood serum transcobalamin-II]. / Kolichestvennaia otsenka retseptorov poverkhnostnoi membrany leikoznykh kletok k transkobalaminu-II syvorotki krovi.

Serum protein transcobalamin II (TC-II) is responsible for transport of cobalamins into mammalian cells. A method of quantitative estimation of plasma membrane receptors of hemopoietic cells to TC-II cobalamin complex is suggested. Analysis of mouse leukemia L1210 cells includes the saturation of radiolabelled ligand-receptor complex with papain. The number of receptors and 57CoCNCbl content in one cell is determined by differentiated radioactivity count of solubilized protein complexes and of cytoplasm.

[Influence of benzyl penicillin on cytotoxicity and level of metabolic activity of cyclophosphamide in mice]. / Wplyw benzylopenicyliny na cytotoksycznosc i poziom aktywnych metabolitów cyklofosfamidu u myszy.

The influence of benzylpenicillin on cytotoxicity of cyclophosphamide (CF) against leukaemia L 1210 was studied. The level of CF metabolites in mouse serum was traced. The benzylpenicillin appeared to enhance cytotoxic activity of CF and leads to increase of active metabolites when CF applied in high doses (300 mg/kg).

[Method of flow impulse cytofluorometry in the UV spectrum for the study of tumor cells in the whole blood]. / Metod protochnoi, impul'snoi tsitofliuorometrii vi UF dlia registratsii opukholevykh kletok v tsel'noi krovi.

Under consideration is the principal opportunity of using impulse cytofluorometers working in the UV spectrum for recording tumor cells in blood. The results of model experiments evidence the advantages of this method, however at present its sensitivity is not adequate enough.

[Study of the DNA structure of peripheral blood leukocytes, proliferating lymphoid and tumor cells according to the rate of alkaline denaturation of DNA lysates]. / Izuchenie struktury DNK leikotsitov perifericheskoi krovi, proliferiruiushchikh limfoidnykh i opukholevykh kletok po skorosti shchelochnoi denaturatsii DNK lizatov.

Direct fluorometry was used to identify quantitative differences in the DNA structure of human peripheral blood leukocytes and proliferating lymphoid cells. The differences should be taken into account in the study of varied damaging actions.

[Studies on cell kinetics in leukemia using flow cytometry].

Cell kinetics were studied in human leukemia (acute leukemia, chronic myelocytic leukemia and other myelocytic malignancies) and in mouse leukemia (L 1210 cells). Flow cytometry method was employed for the analysis of DNA distribution of cells stained with propidium iodide, using a cytofluorograf (System 50, Ortho Instruments). The DNA per cell frequency distribution histograms (DNA histogram) in normal human lymphocytes from peripheral blood showed one main peak of diploid, which corresponded to the DNA from cells in G1 phase of the cell cycle, and other small portions, which corresponded to the DNA from cells in S, G2 and M compartments (S + G2 + M). The percentage of the number of cells in the S + G2 + M phase to the total cells from normal controls was 2.1%. In the case of untreated acute myelocytic leukemia (AML), the percentages of cells in the S + G2 + M phase from peripheral blood and bone marrow were 2.4% and 7.1%, respectively. In bone marrow from treated AML, the percentage of cells in the S + G2 + M phase increased to 14.8%. In the case of untreated acute lymphocytic leukemia, the percentages of the S + G2 + M phase from peripheral blood and bone marrow were 1.9% and 7.1%, respectively. After the treatment of chronic myelocytic leukemia, the percentages of the S + G2 + M phase in peripheral blood and bone marrow were 5.8% and 12.2%, respectively. The DNA histogram in untreated L 1210 cells showed two peaks. One of the peak was consisted of the DNA from cells in G1 phase and another was from cells in the S + G2 + M phase. The proportion of the S + G2 + M phase in the L 1210 cells treated with antitumor drugs significantly increased compared to the untreated cells. The findings indicate that the increase in the proportion of the S + G2 + M phase in the cells from treated human leukemia was a result of the therapeutic effects of antitumor drugs. The study of cell kinetics may provide benefits to know the effect of antitumor drugs during the treatment of human leukemia.