Fluid Accumulation During Initial Stages of Ascites Tumor Growth*

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 Iâ€• 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 100â€”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 24â€”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 87Â°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 0.3â€”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 3000â€”5000 pulses in a thin (0.9â€”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, 8â€”10weeks of age and weighing 20â€”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 73Â°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 (Pâ€•) 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 (4Â°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.

â€˜57

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 â€œI 5- 0 x.â€”x 20s10 TumorCeIIs* (3) z 25 I'- . o-â€”-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.) â€˜5

CHART 1.â€”Effect of ascites tumor cells on the intraperi z toneal accumulation of radioactive human serum albumin or I-. mouse serum proteins after intravenous injection. Xâ€”X, z I0 Ui human serum albumin, 20 X 10 tumor cells (6); Sâ€”@, C., mouse serum proteins, 20 X 10@tumor cells (4); X-â€”X, Ui human serum albumin, NaCl (6) ; â€¢â€”@, 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.â€”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 2â€”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. There was a direct relationship the maximal 7 per cent of injected activity that between cell number and amount of ascitic fluid accumulated in 1 hour with 20 X 10@tumor cells. present at this time. Since such small volume changes cannot be de

tected with conventional technics (26), the volume activity was associated with the ascites tumor cells of fluid in the peritoneal cavity at time of sacrifice under the conditions of these experiments. Cen has been taken as 1 ml., the amount of saline in trifugation at 800 X g for 30 minutes, to produce jected at sacrifice, despite the fact that the normal â€œcell-freeâ€•ascitic fluid, removed less than 1 per peritoneal fluid (0.13 ml. [12]) and the residuum of cent of the total activity from the supernatant tumor or saline inoculation (< 0.2 ml.), as well as fluid. the above changes, added a constant small error. That the accumulation of serum proteins is a The I's' which accumulated in the peritoneal local phenomenon, involving only the peritoneal cavity was still firmly bound to its protein moiety. cavity and not the body as a whole, was indicated Precipitation of protein with 0.5 N trichloroacetic by the following experiment. Ehrlich ascites tumor acid removed over 99 per cent of the total activity cells were implanted intraperitoneally or subcu present in the ascitic fluid. Little or none of this taneously 4 days before sampling. In the sub TABLE 1 RECOVERY OF P@' iN CF1 MICE 2 HouRs AYI'ER INTRA I0.0 VENOUS INJECTION OF MOUSE JODINATED SERUM PROTEIN

5- 8.0 PER CENT INJECTED DOSE/GM > I- Pen 4.) No. toneal Gaoue MICE fluid Liver Kidney Muscle 0 6.0 U 5- Control 4 0 1.2 3.3 1.3 U â€˜Ii Tumorcells,sub- 3 0 1.4 3.4 1.4 z cutaneous* S40 Tumor cells, in- 3 5.9 1.4 3.0 1.3 5-z traperitoneal4 U 4.) * 20 X 10@ cells injected 4 days before sampling. 2.0 U a. TABLE 2

0 INTRAPERITONEAL ACCUMULATION OF P@' IN CF1 MICE 4.0 8.0 I!.0 I6.O 2 HoURS AFTER INTRAVENOUS INJECTION OF Hu HOURS AFTER LABELED PROTEIN MANIODINATED SERUMALBUMIN(RISA)

CHART 3.â€”Immediate effect of Ehrlich ascites tumor cells centexpeniNo. Per on the intraperitoneal accumulation of intravenously ad No.injectedinentimiceactivity59197.39Â±0.36482.79Â±0.118152.05Â±0.03 ministered labeled human serum albumin. Number of mice at Group each point in parentheses; cells and control fluid injected at Tumor cells (20X10@)t Ascitic plasmat arrow. S@et

S Mean Â± standard error of mean. t Injectedintraperitoneally1hourbeforesampling.

cutaneous site the tumor cells grew as a solid mass z without the formation of an ascites. The accumu lation of 1131j@ liver, kidney, and muscle of the tumor-bearing mice did not differ significantly -I 4 from that in normal mice (Table 1). It should be I,) noted that the peritoneal cavities of normal and

0 subcutaneously implanted tumor-bearing mice U I- contained no detectable activity. This differs from 0 U -) the situation previously described, in which 2 per z cent of the injected activity was normally present 5, at 2 hours. The discrepancy is due to the relative amounts of activity and to differences in the tech @ 24 0 for sample preparation in the two instances; an 0 4.0 8.0 12.0 6.0 20.0 TUMOR CELLS a infinite thickness preparation was employed in the present instance, rather than a thin-film prepara CHART 4.â€”Intraperitoneal accumulation of labeled human tion which has greater sensitivity. serum albumin as a function of Ehrlich ascites tumor inoculum size. Labeled protein injected intravenously 2 hours before Ascitic plasma alone did not lead to an appre sampling; tumor cells injected intraperitoneally 1 hour befoie ciable increase in the accumulation of labeled pro sampling. Number of mice at each point in parentheses. tein (Table 2). Although the average amount of

activity present at 2 hours was slightly higher in Fuller's earth, an inorganic irritant, also led to mice injected with reasonably cell-free ascitic fluid an increased accumulation of labeled protein than in saline-injected controls (2.79 per cent vs. (Chart 5). This increased concentration of serum 2.05 per cent), the difference was not statistically protein was, however, transient and could not be significant. As a corollary, tumor cells suspended elicited when 24â€”48hours had passed between the in either ascitic plasma or saline induced the same implantation of Fuller's earth and the injection of degree of labeled colloid accumulation. The fresh labeled protein. In contrast, the accumulation in ascitic plasma used in these experiments was ob duced by ascites cells continued throughout the tamed by centrifugation of ascites tumor fluid. It course of tumor growth. was assumed that the fluid was reasonably cell When the ascites tumor cells were lysed with free, since centrifugation was carried out at 300 X distilled water, neither the centrifuged resus pended cell fragments, the cell-free supernatant

12.0 liquid, nor a suspension containing both corn ponents was capable of inducing a significantly

5- 50.0 increased accumulation of labeled protein in the

5- â€˜3 4 8.0 8.0 a â€œI I- U 6.0 U z I-. 5- 4.0 z > 6.0 U I- 2.0 a. C.) 4

0 0 0 5.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 8.0 Ui I-. HOURS AFTER LABELED PROTEIN C.) 4.0 Ui CHART 5.â€”Effect of Fuller@s earth and homologous hepatic z cells on intraperitoneal accumulation of intravenously in jected labeled human serum albumin. Cells, Fuller's earth, and I- z saline injected intraperitoneally at arrow. Number of mice at Ui each point in parentheses. C, 2.0 Ui 0. g for 30 minutes and only the upper layers of the resultant supernatant fraction were used. No cells were found in random scanning through several 0 smears made of the fluid. Repeated intraperitoneal 0 1.0 2.0 3.0 4.0 5.0 injections (two injections/day for 7 days) of a HOURS AFTER LABELED PROTEIN lyophilized ascitic plasma concentrate, in amounts CHART 6.â€”Effect of lysed Ehrlich ascites tumor cells on equivalent to 23 ml/day of unconcentrated cell the intraperitoneal accumulation of labeled human serum free fluid, gave no gross evidence of ascites forma albumin given intravenously. Xâ€”X, 20 X 10' tumor cells tion. in saline (7); L@â€”â€”Li, 20 X 11$ tumor cells in water (4); A 10 per cent brei of mouse liver cells injected +â€”â€”+, 20 X 1O@tumorcells in water (Supernate)(7); oâ€”o, 20 X 10@tumor cells in water (Cellular debris) intraperitoneally (0.@2ml.) caused the accumula (4); L1------EJ@ â€œCell-freeâ€•ascitic fluid (3); â€¢â€”.-S, saline tion of slightly, but not significantly, more activity (7). Cells and fractions injected intraperitoneally at arrow. than ascitic plasma or saline as shown in Chart'5. Number of mice at each point in parentheses. Although the mean value of the activity accumu @ lated by the hepatic cells at hours (3.98 Â±0.56 peritoneal cavity as compared with the controls per cent) was greater than that by the â€œcell-freeâ€•(Chart 6). ascitic fluid (@2.93Â±0.68 per cent), the distribu Although a variety of ascites tumor cells have tion of the individual values about the mean was been grown intrapleurally with the formation of such that the difference was not statistically sig fluid (9) , we were unable to produce a serous fluid nificant. Since the hepatic cells approached the response to the presence of Ehrlich ascites tumor neoplastic cells in size and concentration, one cells in the pleural cavity. Instead, the cells formed might assume that the mere presence of living cells solid nodules either on the parietal or visceral in the peritoneal cavity is not the initiating cause pleura, and fluid was limited to at most one or two of the extra fluid and protein accumulation. bloody drops in the angle of the diaphragm. There

was no evidence of hydrothorax with 20 X 106 peritoneally was reflected in the lower level of cells in two separate experiments involving ten activity accumulated in the blood. animals each, although respiration was obviously An attempt was made to partially block the ef embarrassed by the bulk of the tumor. Animals ferent peritoneal lymphatics by the intraperitoneal given intraperitoneal injections of samples of the injection of a 15 per cent suspension of India ink same cells developed the usual ascitic response. (1 ml/mouse). Within the 4 hours that elapsed be This inability to evoke an accumulation of fluid in tween the administration of the India ink and the a serous-lined body cavity similar to the perito labeled protein injections, carbon was found out neum, and the demonstration of the importance of lining the lymphatic vessels or as discrete masses the liver in the development of ascites in dogs with in the angles of the peritoneum. The peritoneal partial occlusion of the vena cava (10), led us to fluid itself was clear and colorless. There was a sig investigate the role of the liver in fluid formation nificantly greater accumulation of iodine-labeled by ascites tumor cells. Mice were partially hepatectomized by a modi TABLE 3 fication of the technic devised for use in rats by EFFECT OF EHRLICH ASCITES TUMOR CELLs ON THE RE Brues et al. (2). This operation removes 70 Â±0.7 TENTION OF LABELED HUMAN SERUM ALBUMIN IN THE PERITONEAL CAVITY OF THE MOUSE

No. Per cent expeni- No. injected Group ments mice activity5 Control 4 12 8.71Â±0.78 S 8 10.63Â±0.66 > Tumor cells, 5X10't Tumor cells, 20X10't 3 8 14.04Â±2.19 U 4 S Mean Â± standard error of mean. a t Tumorcellsinjectedintraperitoneally2hoursbefore U sampling; labeled protein intraperitoneally, 3 hours before z sampling.

5- z TABLE 4 U a. EFFECT OF INDIA INK BLOCKADE ON ACCUMULATION OF INTRAVENOUSLY INJECTED HUMAN SERUM ALBU MIN IN NORMAL AND TUMOR-BEARING MICE 2.0 3.0 4.0 5.0 6.0 PER CENT INJECTED ACTIVITY5 HOURS AFTER LABELED PROTEIN sox 10' GROUP tumor celist No tumor cells (â€˜HART 7.â€”Effect of ascites tumor cells on intraperitoneal accumulation of intravenously injected human serum albumin Lymphatic blocks 8.16Â±1.73 4.59Â±0.54 24 hours after partial hepatectomy. Cells and saline injected No block 7.48Â±1.26 1.70Â±0.19 intraperitoneally at arrow. Number of mice at each point in * Mean Â± standard error of the mean. parentheses. Five animals in each group. t Injected intraperitoneally I hour before sampling and 1 per cent of the liver. The accumulation of labeled hour after the intravenous injection of labeled protein. @ 1 ml. 15 per cent India ink per mouse 6 hours before serum albumin in the peritoneal cavity of the par sampling. tially hepatectomized tumor-bearing animal (8.@2 per cent) was of the same magnitude as that in the protein after intravenous injection in mice with normal tumor-bearing mouse (7.1 per cent) at 2 lymphatics blocked by carbon particles than in hours (Chart 7), suggesting that formation of the normal nonbiockaded animals. Despite this differ ascites fluid was not primarily dependent upon ence in the control groups, the level of protein leakage from the liver. In terms of the respective accumulation in the tumor-bearing blocked and controls, however, the accumulation in the pres nonbiocked groups did not differ statistically ence of tumor cells was less in the partially hepa (Table 4). tectomized group. This is perhaps not surprising, It is of interest to note, in view of Loewenthal since the postoperative state no doubt compro and Jahn's theory of direct mechanical lymphatic mised tumor cell function. It is possible, also, that blockade by ascites tumor cells (15), that 90 per similar mechanisms are responsible for protein cent of 5 X 106 and 95 per cent of 20 X 106 in accumulation in both instances. jected tumor cells could be recovered after I hour When labeled serum albumin was injected in of incubation in the peritoneal cavity (Table 5). traperitoneally, the presence of ascites tumor cells The protein accumulated in the two instances was increased the retention of labeled protein in the 3.7 per cent and 7.0 per cent, respectively, of that cavity (Table 3). This increased retention intra injected intravenously.

DISCUSSION Basically, ascites formation, whatever the initiat The development of an ascites tumor involves ing mechanism, depends on an imbalance between proliferation of tumor cells and fluid. These experi inflow and outflow in the peritoneal cavity. ments show that the accumulation of protein in The intraperitoneal accumulation of injected the ascitic fluid at any stage of tumor growth can iodoalbumin may be analyzed by reference to a be accounted for completely by transfer of plasma schematic three-compartment model composed of protein to the peritoneal cavity from the blood, vascular bed, peritoneal cavity, and lymphatics. without having to postulate formation of ascites These compartments communicate with one an fluid protein by other mechanisms. The means by other through the intervening walls. Accumulation which this transfer from plasma may be induced in the central peritoneal compartment could occur are considered below. with an increased inflow from the vascular bed, a Ascites cell protein is formed from free amino decreased outflow into the lymphatic compart acids rather than from extracellular protein (3). ment, or a combination of both factors. An in The present experiments are in accord with this creased inflow could result from a greater vascular view, since no radioactivity is incorporated into permeability or an increased blood supply and the cells. Further, there is little proteolysis in the flow, i.e., a greater surface available. A decreased ascitic fluid in the presence of cells (3). From these outflow could result from an active retention of the findings, the assumption is made that the cells do protein by the tumor or other intraperitoneal cells not withdraw protein from the ascitic fluid in any or a direct decrease in lymph drainage. Considera substantial amount. tion of the data shows that interference with out flow is an important factor in protein accumula TABLE S tion by ascites tumor cells. The extent to which SERUMPROTEIN ACCUMULATIONANDRECOVERYOFEmt other factors, e.g., altered permeability, may con LICH ASCITES TUMOR CELLs 1 HoUR AFTER tribute to the over-all phenomenon is not so clearly INTRAPER1TONEAL INCUBATION in Vito defined. A decreased outflow is implicated for the

Serum protein accumu following reasons: No. tumor cells listed, per cent No. (10@)tumor 1. The protein concentration of ascites tumor inoculated' total activityf cells recoveredt fluid is not greater than that of normal peritoneal @@ 5x106 S .67 Â±0.44 1@â€¢ ? fluid, i.e., 2.6 gm. per cent. Increased permeability 20X 10' 7. 01Â±0. 21 might be expected to produce a peritoneal fluid a Tumor cells injected intraperitoneally I hour before sam pling and 1 hour after intravenous injection of labeled protein. with a protein concentration closer to that of t MeanÂ±standarderrorofmean. plasma, namely 4.2 gm. per cent. Four animals in each group. 2. The blocking of lymphatics with carbon in creases the intraperitoneal accumulation of in It is difficult to assess the turnover time of pro travenously injected labeled protein in normal tein in the peritoneal fluid of normal mice. In view mice, but not in tumor-bearing mice. This implies of the fact that 90 per cent or more of intraperi that both carbon particles and tumor cells accom toneally injected radioactive albumin is removed plish the same end, blockage of lymphatic drain within 3 hours, a turnover time of 4 hours might age. If the effect of the tumor cells were to increase not be amiss. In the accumulation of activity in inflow into the peritoneal space, the accumulation the peritoneal cavity from the blood, factors of of radioactive protein in the two situations should both entrance and egress are operative, and the be additive. total amount of activity passing through the cay 3. Direct measurements show that 86 per cent ity is undoubtedly greater than the 2 per cent max of radioactive protein implanted intraperitoneally imum at 2 hours. Certainly, in light of the sharp with 20 X 10@tumor cells is removed in 3 hours, as exponential decay of intravascular radioactivity, compared to 91 per cent in the controls. While the the fact that the tumor cells are given at 1 hour difference is small, it is statistically significant. after the injection of the labeled albumin would Possible causes of decreased outflow may now suggest that the accumulation in the presence of be considered in some detail. Decreased outflow by ascites tumor cells is less than a maximal value. attachment to the surface of, or incorporation into, Studies by Whipple's group show that a similar the cytoplasmic substance of the ascites tumor turnover of ascites fluid protein occurs in dogs cell is unlikely, since very little (< 1 per cent) of (17â€”19). the activity in the peritoneal cavity is associated The altered physiological processes leading to with the neoplastic cells. On the other hand, lym ascites formation have been studied in a variety of phatics may be involved in the decreased outflow. pathological conditions (1, 16, 17, 22, 23, 28). As Cunningham (6) and, later, Courtice and Sim

monds (4) have stressed in their reviews, lym strated microscopically (22). Supportive evidence phatics represent virtually the sole pathway of is adduced from the observation that urea, which absorption in the peritoneal cavity. Nearly all dissolves fibnin, reverses this process when added absorption takes place through the serosa of the with the irritant. Such a mechanism is unlikely in diaphragm (4). Although the capillary bed is ca the accumulation of peritoneal fluid which accom pable of a limited back-diffusion from the pen panies ascites tumor growth. Addition of urea to toneal cavity (7), lymphatics represent the main the tumor cell inoculum, as well as interference route by which protein leaves. with normal clotting mechanisms by previous in While there is, therefore, some reason to think jections of heparin or dicoumarol, did not alter the in terms of an effect by the tumor cells on the func pattern of protein accumulation with ascites cells tion of penitoneal lymphatics, it is not clear how (Table 6). this effect is achieved. Three mechanisms suggest TABLE 6 themselves : blockade of lymphatic drainage by the tumor cells themselves; blockade of lymphatics by EFFECT OF UREA AND ANTICOAGULANTS ON THE ACCUMULATION OF INTRAVENOUSLY INJECTED fibnin clots engendered by the presence of the tu HUMAN SERUM ALBUMIN iN NORMAL AND Tu mor cells or of their products; or interference with MOR-BEARING MICE the normal permeability or contractility of lym Per cent phatics. No. injected Early tissue infiltration is characteristic of lym mice Treatment activity 15 Tumor 7.41Â±0.35 phomas growing in the ascitic form (26), and such 5 Tumor plus urea' 7.16Â±0.38 invasion has been reported to precede fluid forma 5 Tumor plus heparint 7. 28 Â±0.S2 tion in some sarcomas adapted to ascitic growth 5 Tumor plus dicoumarol@ 7.56Â±0.48 15 Saline 2.01Â±0. 12 (31, 3@2).It is considered unlikely, however, that 5 Urea' 2.48Â±0.42 the increased intraperitoneal concentration of la 5 Heparint 2.78Â±0.61 beled colloid in the tumor-bearing mouse results 5 Dicoumarol@ 2.98Â±0.31 primarily from mechanical obstruction of the Tumor cells injected intraperitoneally I hour be fore sampling; labeled protein, 2 hours before, intra lymph vessels by tumor cells, for the following rea venously.

sons. Ninety to 95 per cent of the free ascites tu S o@3 ml. of 50 per cent urea intraperitoneally with mor cells can be recovered from the ascitic fluid tumor cells. after their presence has elicited an abnormal ac t 1,000 USP units intravenously with RISA; 500 cumulation of labeled protein in the penitoneal USP units intraperitoneally with tumor cells. @ 8 mg/kg intraperit.oneally, 2 and S days before cavity (Table 5). Klein and RÃ©vÃ©sz(12)have inoculation with the tumor. shown that infiltration of serosal surfaces could not be demonstrated microscopically, even with inocu Lymphatic permeability is easily increased (20, la as high as 40 X 106 cells, before the 5th day of 21), but clear evidence for a decrease in permeabil growth, during which period considerable fluid had ity is lacking. A spontaneous rhythmic contractil accumulated in the peritoneal cavity. Hepatic ity has been observed in the peripheral lymphatic cells, although similar in size and concentration to vessels of mice, and vasoconstrictor drugs induce the ascites tumor cells, do not evoke an extra ac lymphatic spasms in such vessels (30). The inabil cumulation of protein . Such homologous cells ity of ascitic fluid or lyophilized concentrates of would, superficially, be as capable of mechanical ascitic fluid to induce increased accumulation of blockade as their neoplastic counterparts. Previous colloids would suggest that a chemical mediator of observations of the logarithmic nature of ascites this type is not involved in the present instance. tumor cell growth and extrapolation of such curves Such a mediator may, of course, exist in minute to the original inoculum size, within limits of error, quantity or in a labile form and, hence, resist at time zero (24) also suggest that lymphatic detection by this procedure. blockade is not due to mechanical obstruction by Up to this point, we have been concerned with the tumor cells per se. the decreased outflow from the peritoneal cavity A superficially similar accumulation of protein and the processes that might play a part in effect occurs in the transient response to an intraperi ing such a decrease. A concomitant increase in col toneal injection of Fuller's earth (Chart 5). With bid entering the peritoneal compartment from the this substance, representative of a whole class of vascular bed is not excluded by these data. One of cellular irritants, the retention of protein and the more obvious ways in which such an accession other markers in the inflamed peritoneal cavity of could occur is by increasing vascular permeability. the rabbit has been ascribed to the occlusion of Mankin and Lowell (16) and Kark et at. (11) be lymphatics by fibnin clots which can be demon lieve that they have demonstrated an increase in

membrane permeability in human ascites, and label is within normal limits in tumor-bearing mice Schoenberg and his colleagues (29), as well as Ber after removal of 70 per cent of the liver. son and Yalow (1), have attempted to quantitate 8. The arguments for attributing ascitic fluid this defect in man by means of I'31-labeled human accumulation to lymphatic blockade and possible serum albumin. Although these authors recognize mechanisms for such blockade are discussed. that a decreased outflow from the peritoneal cavity could be a factor in producing these effects, it is ACKNOWLEDGMENTS dismissed as unlikely. It seems clear that, in the The author wishes to thank Dr. Harvey M. Patt for his initial fluid accumulation produced by ascites tu unfailing advice and encouragement throughout the investiga mor cells in mice, decreased outflow plays a part. tion and Mrs. Marian H. Husain for her generous assistance. 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Robert L. Straube

Cancer Res 1958;18:57-65.

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