Fluid Accumulation during Initial Stages of Ascites Tumor Growth* ROBERT L. STRAUBE (Divis-ion of Biological and Medical Research, Argonne National Laboratiry, Lenwn!, 11/.) The accumulation of intraperitoneal fluid is a no activity remained in the supernatant fraction following characteristic feature of ascites tumor develop precipitation with trichioroacetic acid. The final solutions con tamed from 2 to 5 pc/ml and about 20 mg protein/ml. lodi ment. An increase in free tumor cell number is ac nated human serum albumin was used in some experiments. companied by a concomitant increase in peritoneal The iodinated human serum albumin (RISA)' contained from fluid volume. For some tumors, e.g., the Krebs-92 0.3 to 0.9 mc. of J131and approximately 10 mg of human serum and Ehrlich ascites carcinomas, a linear relation albumin/ml. The unbound I― activity was less than 1 per cent. ship exists between these parameters with an opti In experiments where solid tissues were collected in addition to plasma and body fluids, samples were prepared by wet diges mal concentration of approximately 100—130X tion with 1.5 N sodium hydroxide. Weighed wet samples (less 106 cells/mi. The early accumulation of fluid, i.e., than 1 gm. total weight) were minced and digested for 24—48 within a few hours after tumor inoculation, may hours in fiat-bottomed, porcelain Coors crucibles, 4.5 cm. in result from initiating mechanisms that could be diameter. This procedure dissolves the tissue completely. Dry ing was accomplished in an oven maintained at 87°C. No evi quite different from, and obscured by, secondary dence of volatilization of iodine was observed during the prepa events such as hypoproteinemia and tumor infil ration and counting of the samples. Due to the deliquescence of tration, which may help to perpetuate fluid ac the dry alkali, samples were kept in a desiccator until counted. cumulation. In experiments in which plasma and ascitic fluid alone were In an attempt to characterize the early, tumor taken, 0.1-0.2 ml. of plasma and 0.3—0.5ml. of aseitic fluid were diluted to 7.5 ml. in Coors crucibles with distilled water, to induced accumulation of intraperitoneal fluid, the insure an even film, and similarly dried. All standards and movement of blood proteins labeled with iodine samples for a single experiment were prepared at the same time 131 was followed. A quantitative relationship be and measured in the same manner. Samples were counted for tween number of cells inoculated and the maximal 3000—5000 pulses in a thin (0.9—1.2mg/sq cm) end-window Geiger-Muller tube. The usual precautions as to geometry, accumulation of radioactivity was derived. By coincidence, etc. (14), were followed. this technic, some aspects of the physiological Mice were given injections of 0.2 ml. of radioactive serum processes leading to the accumulation of fluid by protein either intravascularly, via a tail vein, or intraperito the tumor cell were studied subsequently. neally. One hour later, a known number of ascites tumor cells or other experimental or control material was injected intra MATERIALS AND METHODS peritoneally in a volume of 0.2 ml. At various intervals there CF1 female mice, 8—10weeks of age and weighing 20—28 after, animals were anesthetized with ether, and blood samples gm., were used. In any one experiment the weight range did not were obtained by severing the brachial plexus. The mice were exceed S gm. The animals were maintained on standard Rock then sacrificed by cervical fracture, 1 ml. of 0.9 per cent saline land mouse diet and water, ad libitum, in aft-conditioned quar was injected intraperitoneally, and the abdomen was massaged tars at 73°F. The Ehrlich ascites carcinoma was originally ob gently for 2 minutes to insure mixing of saline and peritoneal tamed from Dr. T. S. Hauschka in 1952. Sterile technic was fluid. The peritoneal fluid was aspirated as completely as pea used in collecting and inoculating the cells, and the tumor cell sible into heparinized tubes with a capillary pipette. Blood number was determined by the method of Patt et al. (25). The samples were centrifuged in a clinical centrifuge at 200 X g for radioactive iodine (P―) was obtained as the inorganic sodium 10 minutes, and the plasma was removed. Ascitic fluids were salt.' Reconstituted lyophilizedmouseserum was used as the iiot centrifuged unless grossly hemorrhagic. tracer vehicle. Serum proteins were iodinated by a modification In those animals in which ascites tumor growth was newly of the method used by Fine and Seligman (8) as described by established, the volume of the peritoneal fluid was assumed to Crispell, Niest, and Porter (5). After dialysis of the iodinated he 1 ml., the amount of saline injected at sacrifice. The con tribution of tumor inoculum and of the small volume of ascites protein against distilled water in the cold (4°C.) for 48 hours, formed during this interval to the total volume could not be S This work was performed under the auspices of the U.S. accurately characterized by conventionaldye dilution technics Atomic Energy Commission. The paper contains material from owing to the error of measurement. The 0.13 ml. of normal a thesis submitted in partial fulfillmentof the requirementsfor peritoneal fluid (12) also added a small constant error. the degreeof Doctor of Philosophy, University of Chicago. Total protein concentrations of plasma and peritoneal fluids i Isotope Division of the Oak Ridge National Laboratory. were computed from determinations of the total nitrogen by the standard micro-Kjeldahlmethod (27).Nonprotein nitrogen 2 Carworth Farms Laboratories. (NPN) correction was taken as 0.3 mg/mI. Received for publication July 19, 1957. a Abbott Laboratories. ‘57 Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1958 American Association for Cancer Research. 58 Cancer Research VOL. 18, JANUARY 1958 RESULTS In the recently inoculated mouse, a similar pat Peritoneal fluid was collected, undiluted, from tern of increased intraperitoneal colloid accumula twenty normal CF1 female mice by means of a tion obtained. Mice were given intravenous injec capillary pipette. The pooled sample had a protein tions of the serum protein, and 1 hour later the concentration of 2.61 gm. per cent. Ascitic plasma normal base-line concentration of intraperitoneal (pooled, ten mice), undiluted, after removal of the accumulation was measured. At this point, appro ascitic cells, had a protein content of 2.73 gui. per priate groups of animals were given injections in cent. The corresponding plasma value in these traperitoneally of varying numbers of tumor cells tumor-bearing animals (ten mice) was 4.16 gm. per in a constant volume (0.2 ml.) of fluid. Within 30 cent. minutes after tumor injection (1.5 hours after la The general pattern of accumulation of labeled beled protein), there was an alteration in the level protein in the peritoneal cavity was similar of protein accumulation in the tumor-bearing groups (Chart 3). Maximum intraperitoneal ac cumulation of the I'3'-labeled protein occurred 2 hours after the injection of the labeled protein and > 1 hour after injection of the tumor cells. There was I. 0 0 IIIII “I 5- 0 x.—x 20s10 TumorCeIIs* (3) z 25 I'- . o-—-o 4 * IO@TumorCeIIs@(3) 0 I. @ I- I' 2 E10 TumorCeIIs*(3) z > w 0 I- .x \ @___, SOIiRS* (3) C.) 20 U a. 4 0 \ *Inoculoted3days 0 4.0 8.0 2.0 6.0 20.0 24.0 w I- \ beforesampling HOURS AFTER LABELEO PROTEIN INJECTION C.) ‘5 CHART 1.—Effect of ascites tumor cells on the intraperi z toneal accumulation of radioactive human serum albumin or I-. mouse serum proteins after intravenous injection. X—X, z I0 Ui human serum albumin, 20 X 10 tumor cells (6); S—@, C., mouse serum proteins, 20 X 10@tumor cells (4); X-—X, Ui human serum albumin, NaCl (6) ; •—@, mouse serum 0. proteins, NaCl(4). Number of mice at each point in paren 5 theses; cells and control fluid injected at arrow. whether mouse serum protein or human serum 0 albumin (RISA) was used as the tracer vehicle 0 4 8 12 $6 20 24 (Chart 1). This is in agreement with the results ob HOURS AFTER LABELED PROTEIN tained by Krieger et al. (13) in comparing excretion patterns of iodinated canine and human serum CHART 2.—Effect of well established Ehrlich ascites tumors on the intraperitoneal accumulation of intravenously injected proteins in dogs. The 2-hour maxima of 7 per cent labeled human serum albumin. Number of mice at each point and 2 per cent of total injected activity in tumor in parentheses. bearing and control groups, respectively, were re producible with either heterologous or homologous a simple linear relationship between these maxi protein. mum values and tumor inoculum size (Chart 4). When 2, 4, or 20 X 106 Ehrlich ascites tumor The slope of the line indicates that, over the range cells were injected intraperitoneally into female of 2—20X 10' cells, labeled protein accumulation mice and allowed to multiply for 3 days, the in the peritoneal cavity increased by 0.25 per cent amount of labeled protein accumulation in 2 hours of the total injected activity for every 10@tumor in these well established tumors was greater than cells inoculated. that observed in normal mice. The total amount of The levels of activity observed could be ac radioactive colloid present in the abdominal cavity counted for by the accumulation of relatively small was a function of the tumor inoculum size as volumes of fluid. Only 0.07 ml. of plasma furnished shown in Chart 2.