The Interstitial Fluid of Solid Tumors · The interstitial fluid of a solid tumor, consisting of the liquid phase interposed between the newly formed vascular walls of the tumor and

The Interstitial Fluid of Solid Tumors

PIETRO M. GULLING,SHIRLEYH. CLARK, AND FLORA H. GRANTHAMTumor-Host Relations Section, Laboratory of Biochemistry, National Cancer Institute, Bethesda, Md.

SUMMARY

The interstitial fluid of a solid tumor, consisting of the liquid phase interposedbetween the newly formed vascular walls of the tumor and the plasma membrane of theneoplastic cells, has been sampled from a chamber enclosed into the neoplastic mass.The "tension" of the interstitial fluid of normal and neoplastic tissues was sufficient

to produce a continuous outflow from the sampling device. In some tumors, but notin all of them, the pressure needed to stop this outflow was twice as large as that required for the normal subcutaneous tissue, and it was similar to that measured in theskin after venous obstruction.

The sampling technic has been described in detail. Each sample was comparedwith the blood serum flowing into and out of the neoplastic mass, with the interstitialfluid obtained by the same technic from the normal subcutaneous tissue, with theperitoneal fluid, and with the lymph of the thoracic duct of normal and tumor-bearingrats. The fluids were first tested for sterility and then analyzed for total protein, non-protein N, urea, free amino acids, glucose, lactic acid, total sterols, lipid phosphorus,chloride, sodium, and potassium.

The main purpose was a better characterization of the milieu in which the neoplasticcells live. The results refer to small tumors (5-10 gm.) of Walker carcinoma 256,Fibrosarcoma 4956, Hepatoma 5123, Hepatoma 7974, and Novikoff hepatoma.

The interstitial fluid of the tumor was characterized by a very low content of freeglucose and high levels of lactic acid. Data are reported on the passage of glucosefrom the blood into the interstitial fluid of tumors, and they suggest that this passageoccurs by means of a transfer mechanism and not by simple diffusion. A "fermentative type" of interstitial fluid, peculiar to the tumor, was produced also in tissues with

out any sign of neoplasia being present.The concentration of proteins was about 33 per cent lower in the interstitial fluid

than in blood serum of tumors. This reduction was constant for each tumor, persistedduring growth, and disappeared only when the sampling device was surrounded bynecrotic tissue. The Â«-globulinswere reduced, but the albumin-globulin ratio of theinterstitial fluid was equal to that of blood serum.

The free amino acid level was generally higher in the tumor fluid than in serum ofblood entering or leaving the neoplastic mass, but the behavior was not uniform, andno conclusion could be drawn.

The concentration of cholesterol and lipid phosphorus in the interstitial fluid oftumors was about one-fourth of that of blood serum; however, the values in the tumorand in normal subcutaneous fluid were similar.

The gross composition of tumor fluid was maintained as constant as that of thoraciclymph, normal peritoneal fluid, or subcutaneous fluid despite the presence of necroticfoci and the known changes in metabolism of the neoplastic cell population.

This paper presents data which permit a comparison of the host. As a working hypothesis, the neoplasticbetween the interstitial/cfluid (IF) of a solid tumor and mass was considered to be a three-compartment system:(a) the blood flowing into Â¡mdout of the neoplastic mass, compartment I, the vascular bed; compartment III, the(6) the normal subcutaneoWjF, (c) the peritoneal fluid, neoplastic cells; and compartment II, the space betweenand (d) the lymph of the host. The purpose is to charac- the cellular membrane and the vascular wall (Fig. 1).terize the milieu in which a neoplastic cell population lives Compartment II consists of two major componentsâ€”theand grows and to analyze the differences and the similari- frame of collagen fibers and the "interstitial fluid," theties between this milieu and that of normal cell populations semi-liquid phase which bathes the cells.

Received for publication November8,1963. The terms "extracellular fluid" or "environmental fluid"

780

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GULLINGet al.â€”Interstitial Fluid of Solid Tumors 781

can be used as synonyms for interstitial fluid, as long as itis understood that they refer to the fluid phase of compartment II. In the literature, this has not always been thecase. Fluids found in different cavitiesâ€”e.g., the cere-brospinal fluid, the intraocular fluid, and the fluids of thejointsâ€”are generally considered as examples of "extracellular fluids." The "interstitial fluid" is almost exclu

sively known by the analysis of lymph, and, by tradition,the terms lymph or interstitial fluid are often used interchangeably. However, the lymph is only that part of theinterstitial fluid which passes into the lymphatic system,and it is very poorly understood what type of "selection,"

if any, is exerted by the walls of the lymphatic vessels.When a neoplastic mass is studied a new set of condi

tions have to be considered, since the neoplastic mass doesnot have lymphatic channels from which the "lymph ofthe tumor" can be sampled. A halo of edematous tissueis often present around neoplastic nodules grown subcu-taneously, and the "environmental fluid" of the tumor can

be obtained when pressure is exerted on this area. Whentumors grow hi the spaces of the joints or diffuse ontothe pleural surface, a certain amount of serum-like material, often hemorrhagic, can be collected in the cavities.This fluid could be considered the "extracellular fluid"

of the tumor. In some cases, cavities are found in theneoplastic masses, often as a result of necroses and sometimes as a peculiar feature of tumors, like the cystoade-nomas. Again, the fluids contained in these cavities canbe considered "extracellular." If a neoplastic mass is

sectioned, fluid will soon appear on the surface. Thishas been considered "interstitial" fluid of the tumor (36),

although a large component of it was probably derivedfrom disrupted cells.

More examples can be found in which the terms "interstitial," "extracellular," and "environmental" can be ap

plied to fluids in contact with neoplastic masses, butwhether these samples are representative of the fluid whichbathes the living neoplastic cells is a debatable question.In this paper, the term "interstitial fluid" will be used to

indicate the fluid between the cells of a tumor and collectedfrom the sampling device enclosed in the tumor mass asdescribed below. We believe that this is the fluid of thesemi-liquid phase of compartment II.

MATERIALS AND METHODS

ANIMALSANDTUMORS

Rats of both sexes, hi a weight range of 150-200 gm.,were used. The animals were fed a Purina LaboratoryChow diet1 from birth throughout the experimental period.

The following tumors were used: Walker carcinoma 256(35) and Novikoff hepatoma2 (26) carried in Sprague-Dawley rats, random-bred Ime; Hepatoma 51233 (25) andFibrosarcoma 49564 carried in Buffalo/N inbred line; and

Hepatoma 7974 carried in the ascites form in Japanese/Nline (18, 31).

1Ralston Purina Company, St. Louis, Missouri.* From 459th to 488th transplant generation.*From 35th to 48th transplant generation.4 From 90th to 106th transplant generation.

THE SAMPLINGDEVICE

The collection of IF was based on the following principle:Neoplastic cells of a tumor mass growing against a porousmembrane (0.45 n pore diameter, or less) were unable topass through the membrane, but the fluid bathing the cellscould diffuse through and be collected. (Fig. 1) A diffusionchamber was built in order to sample this fluid. A luciteorpolystyrene ring was cut 9.3 mm. inner diameter, 12.8mm. outer diameter, and 2.0 mm. thick. A hole with adiameter of 0.8 mm. was drilled through the ring, andpolyethylene tubing (PESO),5 40 cm. long, was forcedinto it. The end of the catheter facing inside the ring wasbeaded with heat and pulled tightly against the ring tocomplete the seal. Filters were cemented to both flatsides of the ring with MF cement formulation No. I.6Millipore filters, type TW (0.45 n pore diameter),6 polyporemembrane filters type AM-6 (0.45 /upore diameter),7 andS and S membrane filters (less than 0.1 p pore diameter)8were used in each experiment to test the possible influenceof the type of filter used on the results obtained (Fig. 5).

The choice of the chamber's size was determined by

the amount of fluid to be collected, the size of the host,the capacity of the tumor to grow and incorporate thechamber within its mass, and the possibility to drain thefluid. IF drained from the host only when the diameterof the diffusion chamber was 9 mm. or larger; when thediameter was smaller IF penetrated into the chamber butdid not drain.

Two mahi types of chambers were used, the "draining"and the "closed" type (Fig. 5). A continuous flow was

obtained for a period of days from the draining chambers,whereas IF was collected from the closed chamber bydirect sampling from the cavity. The closed chambercould be left in situ, and another sample of IF could becollected after a suitable lapse of tune.

In most instances well constructed chambers containedonly the fluid and in rare cases very small amounts ofjelly-like material. Defective chambers were filled withconnective tissue and neoplastic cells which penetrated,most often, through holes left by imperfect sealing of thefilter to the ring.

The absence of cells was ascertained in the followingway The fluid from the chamber was centrifuged withthe Beckman-Spinco 152 Microfuge hi polyethylene microtest tubes at about 2000 r.p.m. for 5 minutes. Then thebottom of the micro-tube was cut, the content was smearedon a slide, fixed with 90 per cent ethanol, and stained witha Giemsa solution.9 The fluid sampled never containedcells when, after drainage, the chambers appeared empty.This simplified the procedure by making a microscopicexamination of each sample unnecessary.

THE SAMPLINGPROCEDURE

When the afferent and efferent blood of the neoplasticmass was compared with the tumor IF, the draining chamber was used in conjunction with "tissue-isolated" trans-

5 Clay Adams, New York, N. Y.6Millipore Filter Co., Bedford, Mass.7 Gelman Instrument Co., Chelsea, Michigan.8 Schleicher and Schuell Co., Keene, N. H.9 National Aniline Division, Allied Chemicals, Rochester, N. Y.



782 Cancer Research Vol. 24, June 1964

plants (15). The chemical determinations were done onthree specimens obtained at sacrificeâ€”the arterial bloodfrom the aorta, the venous blood from the tumor vein, andtumor IF. The collection of all three samples requiredonly about 5 minutes, and this was considered to be "simultaneous" sampling. When the IF was drained con

tinuously several samples were collected, and the lastIF sample was formed by the fluid contained in the chamber.

When the tumor IF was compared with subcutaneousIF, blood from the aorta was sampled first, followed by theinterstitial fluid of the tumor and of the normal subcutaneous area in this order. Again the sampling was consideredsimultaneous.

Lymph was sampled by the cannulation of the thoracicduct (3). Normal and tumor-bearing animals were usedfor this purpose, and the collection lasted 24 or 48 hours.The tumors were transplanted subcutaneously and weighedbetween 10 and 20 gm. when the lymph was sampled.

The peritoneal fluid was obtained either with the sampling device used for tumor IF or directly with a pipette.In the second case the fluid was harvested at sacrifice,immediately centrifuged at about 1000 X g, and the cellswere discarded. Blood was sampled from the abdominalaorta at sacrifice. Serum was separated by centrifuga-tion at room temperature within 15 minutes from thesampling and analyzed either immediately or few dayslater (storage at -20Â° C.).

PREPARATIONOF TUMORS

Rats were used in our experiments, but the samplingtechnic can be applied to any animal of sufficient size.The animal was kept in a restraining cage10during collection of IF from the draining chamber (Fig. 6) and leftfree when bearing the closed chamber.

The sampling device was placed into the "tissue-isolated" preparation (15) when the tumor had grown to

about 3 gm. The final result was an actively growingneoplasm isolated from the surrounding tissues, connectedto the host only by one artery and one vein, and includinga sampling chamber which drained the IF (Fig. 2).

When tumor IF was compared with subcutaneous IF,a subcutaneous transplant was used (Figs. 3, 4), and twosampling chambers were placed into the same animal. Apouch of the skin was prepared in the left scapular region,about 300 mg. of tumor fragments were introduced intothe pouch along with the chamber, and the PESO tubingwas driven through a tunnel of the subcutaneous tissueto the base of the tail, fixed to the skin by a stitch, andplaced into the collecting vial. By the same procedurea second chamber was placed in the sacral region, but notumor was transplanted. The amount of tumor fragments placed around the chamber varied with the growthrate of the neoplasm. For Walker carcinoma 256 andNovikoff hepatoma, 200 mg. of material was sufficient,but for slowly growing tumors, such as Hepatoma 5123,400 mg. were necessary. The size of the inoculum wasimportant with respect to the completeness of the chamber's inclusion and the rapidity at which necrosis devel

oped. With the proper amount of inoculum all tumors10The cage was built by J. Rowland, NIH Instrument Service.

grew well around the diffusion chamber and covered itrapidly. However, when the inoculum was too smallslowly growing tumors had the tendency to form relativelylarge nodules at one end of the chamber before the rest wascompletely enclosed in the neoplastic mass. When theinoculum was too large, rapidly growing tumors formed alarge mass in a few days, and the necrotic processes aroundthe chamber rapidly became extensive.

MORPHOLOGYOF THE TISSUESAROUNDTHESAMPLINGCHAMBER

Chamber in hypodermis and peritoneal cavity.â€”Duringthe first 4-5 days after implantation an acute inflammatory process of a moderate degree was present around thechamber in the hypodermis.

After the first week, and for 60 days thereafter (thelongest observation period), the morphologic picture remained static. Only the hypodermis showed an increasein cellularity and in collagen bundles for a relatively smallarea adjacent to the chambers (Fig. 8).

To investigate the cell population surrounding the chamber, two groups of three chambers each were examined,at 3 and 30 days after implantation, respectively. Threefields of 100 cells each were counted along a strip of 0.5mm. adjacent to each chamber. At the 3d day after theimplant the cell population around the chamber consistedof fibroblasts (17 per cent), lymphocytes and large mono-nuclear cells (77 per cent), granulocytes (4 per cent),macrophages (0.6 per cent), and the mitotic figures wereabout 1 per cent. At the 30th day the fibroblasts formed85 per cent of the cell population, the lymphocytes andlarge mononuclear cells 14 per cent, the macrophages 0.5per cent, and the mitotic figures were less than 0.05 percent. Residues of hemorrhage were present at the 3dday, absent at the 30th. There was no morphologicaldifference when Millipore, Polypore, or S-S filters wereused. Despite the difficulty in recognizing all cell typesin histologie slides, the difference in cell population between the 3d and the 30th day was clear and the over-all"reaction" around the chamber limited.

When the diffusion chamber was placed into the peritoneal cavity the mesentery enveloped it, and for a longperiod of time surprisingly little "reaction" was observed.

Preparations correctly handled never showed a granuloma,and only very rarely were a few giant multinucleated cellsobserved against the filters.

Chamber in tumors.â€”When the diffusion chamber wasplaced in the hypodermis and fragments of neoplastictissue were implanted in the pouch, the tumor grew in theform of islands attached to the chamber, and these budslater aggregated into one large mass which enclosed thechamber completely (Figs. 3, 4). When the diffusionchamber was placed into the "tissue-isolated" preparation,

the surgically split tumor grew to seal the wound and toinclude the chamber completely (Fig. 2).

The neoplastic cell population of the tumor mass showedthe same characteristics in the subcutaneous and the"tissue-isolated" transplants. The cells were in contact

with the porous membrane without interposition of anyfibrous or granulomatous material (Fig. 7). In the "tissue-isolated" tumors, where some hemorrhage wa*. pro-




duced in the placement of the chamber, erythrocytescould be found during the first few days mixed with neo-plastic cells, but they soon disappeared, and the chamber'sfluid became serum-like in color. Necrosis was presentin the form of foci scattered all over the neoplastic mass.Around the chamber it started as a dissociation of thecells, followed by the appearance of a fibrillar precipitatein the interstitial spaces and by an extensive cellular damage. In both the subcutaneous and "tissue-isolated"

transplants the presence of the chamber did not appreciably increase the amount of necrosis.

The time-sequence of the onset and progression of thenecrotic process depends on the tumor type and on thekind of transplant. For Walker carcinoma 256 growingin the hypodermis, necrosis was negligible for the first 5days, appeared at the 6th or 7th day, and became extensive at the 10th day. Hepatoma 5123 enclosed thechamber in about 3 weeks and rarely presented extensivenecrosis at this time; intraparenchymal hemorrhages weremore frequent. Novikoff hepatoma behaved like Walkercarcinoma 256, and Fibrosarcoma 4956 like Hepatoma5123.

In all the tumors studied there was a definite timeinterval during which the diffusion chamber was completely enveloped by the neoplastic cells and the necroticprocess was small. This was obviously the best time tocollect the IF, because the tissue around the chamber wasa representative sample of the neoplastic cell population.

COLLECTIONANDSTERILITYOF SAMPLESDuring drainage, tumor IF was kept at 0Â°C. The

amount collected during a 24-hour period varied from 0.2to 1.0 ml. and formed one sample. The size of a samplefrom a closed 9-mm. chamber was about 100 ni., and morethan one sample could be collected. An interval of atleast 3 hours was needed to obtain ca. 20 Â¡tl.of IF.

The sterility of the sample was carefully checked, andcontaminated fluid was discarded. Normal proceduresfor the sterile treatment of the animal during surgery werefollowed. The chambers and the material were sterilizedwith ethylene oxide (27) or by dry heat (3 days at 80Â°C.).

The restraining cages were washed in strong detergentsand changed every 24 hours. Every IF sample used forchemical analysis was tested for bacterial growth. TheNIH-thioglycollate medium11 was used, and the cultureswere kept under surveillance for at least 10 days. Besidethe systematic control of IF samples with thioglycollatemedium, spot-checking was done for PPLO contamination.The PPLO enrichment broth,11 BWI medium,12 and VSmedium were used (I).13 All samples tested were negative.

One essential factor for the sterility of the sample is thesterility of the tumor. The pouch of the skin containingthe "tissue-isolated preparation" could be easily infected,

but an accurate suture and two drops of CrysticillineSquibb14 inside the pouch were generally sufficient tominimize the risk.

11Bacto-NIH Thioglycollate Broth (B 257), Difco Manual.12Baltimore Biological Laboratory, Baltimore, Maryland.15We are indebted to Dr. V. Young and Mr. C. H. Zierdt (NIH

Clinical Center, Microbiology Service) for the PPLO cultures.11Squibb and Sons, New York, N. Y.

ASSAYS

The following tests were performed : Total proteins weredetermined colorimetrically by the biuret reagent (13, 21)or by the micro-Kjeldahl method, before and after tri-chloroacetic acid precipitation. The electrophoretic analysis of IF and serum proteins was conducted with theModel RD-2 Paper Electrophoresis System, ProcedureB15(buffer solution: Veronal pH 8.6, ionic strength, 0.075;Paper, S-S 2043-A mgl strips; current, 2.5 ma/cell; duration, 18 hours; heat denaturation, 30 minutes at 120Â°C.;stain, 0.1 per cent by weight bromophenol blue in metha-nol. Scanned in Model RB Analytrol after treatmentwith NH4Ã›H). Nonprotein nitrogen was determined bythe micro-Kjeldahl method after protein precipitation withtungstic acid, following the procedure of Folin and Wu(9). Urea was determined by the diacetyl monoximemethod (10). Free amino nitrogen was assayed by theninhydrin reaction following the procedure of Yemm andCocking (44) and by the l-fluoro-2,4-dinitrobenzene reaction following the procedure of Rapp (29). Glucose wasoxidized in the presence of glucose oxidase (C. F. Boeh-ringer and Soehne of Manheim, Germany), and the hydrogen peroxide was measured by the chromogen formedin the presence of horse radish peroxidase and o-toluidine(30). Lactic acid was converted to acetaldehyde and determined colorimetrically by the reaction with p-phenyl-phenol following the Barker and Summerson procedure(2). Total sterols were extracted with ethanol-acetoncmixture (1:1), the esterified fraction was saponified withKOH, and the sterols were precipitated as digitonides andpurified. The digitonides were dissolved in acetic acidand determined with an anthrone reagent (38). Phos-pholipids were extracted in boiling acetone and alcoholmixture (1:1), digested in sulfuric acid and hydrogenperoxide, and the inorganic phosphate was determinedcolorimetrically (blue molybdenum complex) (17). Chloride was determined by the method of Schales and Schales(32), based on precipitation as mercuric chloride and backtitration of the excess of mercuric ions with "s-diphenylcarbazone." Sodium and potassium were determined byflame photometry (34).16

RESULTS

SOMEGENERALPROPERTIESOF THEINTERSTITIALFLUID

Color.â€”Subcutaneous IF had a color varying from apale to a dark yellow, and rats with dark fur had yellowerfluids. Tumor IF was pale yellow, like serum, in all neoplasms. Hemoglobin was present in the IF only immediately after a draining chamber was placed into the tumor;it disappeared in about 2 days and reappeared in allcases when the sampling chamber was enclosed in necrotictissue.

Specific gravity.â€”The density of IF was measured withthe method of gravity analysis (14). From the sameanimal aortic serum and IF, from closed and drainingchambers, were tested simultaneously and immediately

15Beckman-Spinco Division, Palo Alto, Calif.'Â«We are indebted to Mr. R. Koegel and to Mrs. Ilona Losonczy

for these determinations.




TABLE 1SPECIFICGRAVITYOF THE INTERSTITIALFLUIDMEASUREDBY THE GRAVITYMETHOD

Each value is an average on observation of four animals Â±standard error.

FLUIDInterstitial

fluidAortic serumWALEER

CARCINOMA(SDBOJT.IMPLANT)Draining

chamber1.014

Â± 0.0007

1.019 Â± 0.0005Closed

chamber*1.015

Â±0.0002

1.020 Â±0.0002SUBCUTANEOUS

TISSUE(NORMAL)Draining

chamber1.014

Â± 0.0005

1.025 Â± 0.0002Closed

chamber*1.016

Â±0.001

1.024 Â±0.0005HEPATOMA

7974 (ASCITESFORM)1.017

Â±0.01(ascites fluid) t

1.020 Â±0.005

* Drained once 3 days after implant.t Clear, serum-like fluid taken from draining chamber at 5 days after tumor transplant. The animal

was killed by the tumor in about 10 days.

after sampling. The IF fluid was always lighter than theaortic serum of the animal. No appreciable differencewas apparent between the closed and the draining chambers or between the normal subcutaneous and the tumorIF. The ascites fluid generally had a specific gravityslightly higher than IF from solid tumors (Table 1).

Draining pressure.â€”The draining pressure was measured with a mercury manometer connected to the PESOtubing of the chamber. The observation period lasted3 or 4 days and was terminated when no change in themercury level was observed for 24 hours. The pressureneeded to block the drainage of IF from the diffusionchamber placed in the normal subcutaneous tissue variedfrom 7 to 9 mm. of mercury; however, when the chamberwas in a tumor, the pressure varied between 8 and 16 mm.of Hg. There was no difference between the subcutaneous and the "tissue-isolated" transplants, but the drain

ing pressure from the neoplastic tissue showed a largervariation than that from normal animals. If these valuesreflect the "tension" of the neoplastic interstitial fluid,

some tumors have a tension higher than that of the subcutaneous tissue.

PROTEINS

The total content of proteins in tumor IF was constantlyless than that in blood serum. This was true for all fourtumors studied, including Novikoff hepatoma, whichalways yielded slightly pink IF samples. The reductionvaried from one animal to another, but in 23 cases testedfrom four different types of tumors the average reductionwas 33 per cent. In twenty out of 21 cases serum of thevenous blood leaving the tumor contained a slightly largeramount of total proteins than did that of the arterialblood; the average increase was 8 per cent (Table 2).

The variation in concentration of IF proteins duringtumor growth was studied following two procedures. Inthe first, "tissue-isolated" transplants of Walker car

cinoma received chambers at the 6th day after transplantation, and IF was collected from the 10th to the 13thday. This yielded four 24-hour samples for each animal.Over a 4-day period the total protein content remainedremarkably constant in some animals, hi others variedup to about 35 per cent. The level of total proteins wasalways below that of the aortic blood (Table 3).

In the second experiment the subcutaneous transplantsof Walker carcinoma were utilized, and two draining

TABLE 2TOTALPROTEINSOF INTERSTITIALFLUID OF TUMORSAND

BLOODSERUM(gm/100 ml)

TumorWalker

ca.256Fibrosarcoma

4956Novikoff

hepatomaHepatoma

5123AverageRat

no.12345678123456123451

23Tumor

weight(gm.)6.27.48.28.711.212.112.215.64.66.89.39.410.611.36.2

6.810.420.610.84.5

5.76.3Blood

flowinginto tumor5.24.84.63.84.13.94.34.15.94.44.96.84.84.54.6

4.14.94.46.65.54.85.24.8Blood

lowing outof tumor4.85.24.64.2â€”4.04.54.26.14.65.57.05.25.55.35.34.86.95.9

5.25.85.2Tumor

IF1.82.62.72.82.91.62.23.24.22.53.73.23.32.84.1

3.54.34.44.13.5

3.04.13.2

chambers were implanted in each animal, one with thetumor and one away from it. Arterial blood was withdrawn from the carotid artery before the insertion of thechambers and at sacrifice. The interstitial fluid collected 24 hours after transplantation of both chamberscontained amounts of total proteins equal to that of theaortic serum. The protein content was reduced in thesubsequent days to a level about 50 per cent of the aorticserum in the subcutaneous fluid, and about 40 per centin the tumor fluid. The protein content of the subcutaneous fluid remained roughly constant for the subsequent 2weeks, but the protein content of the tumor IF increased




TABLE 3PROTEINCONTENTOF TUMORIF DURINGCONTINUOUS

SAMPLING(WALKERCA. 256)(gm/100 ml)

RATNo.12345INTERSTITIAL

FLUIDDays

ofcollection13.73.83.54.13.323.73.32.13.33.232.43.12.82.63.442.83.42.43.13.8ARTERIALSERUMAt

SACRIFICE(4THDAY)4.65.14.45.45.0

TOTAL PROTEINSOF SUBCUTANEOUSAND TUMOR// DURINGTUMOR GROWTH.

5_i

I4â€¢**.(/>Â£ 3

fein

O o

o o

e ARTERIAL SERUMâ€¢SUBCUT IFo TUUOR IF

24 6 8 IO 12 K

DAYSAFTER TUMORTRANSPLANT16

CHART1.â€”Decreaseof protein content in tumor and subcutaneous interstitial fluid (IF) during the first days after transplant.Note the stabilization during tumor growth at about 40 per cent ofthe serum value and the increase in protein content of the tumorIF when extensive necroses ensues after second week of transplant.

during the 3d week when the mass was very large. Atthis stage the chamber was enclosed in a cavity full offluid and necrotic tissue as well as enmeshed in a fibrin-like material (Chart 1).

The effect of drainage on IF proteins.â€”When a closedchamber was placed hi a pouch of the subcutaneous tissue,alone or surrounded by tumor fragments, the proteinsderived from destroyed tissues penetrated hito the chamberwith the first fluid. These proteins were excluded fromany contact with the cells of the inflammatory process(28, 33) and furthermore could not be eliminated bydrainage. An experiment was devised to test whetherthe drainage of closed chambers had any effect on theprotein content of the sample. Six groups of five animalseach were used. In the first four groups, each rat received subcutaneously two closed chambers, one with andthe second without tumor; the fifth and sixth group, ascontrols, received only one closed chamber without tumor.The animals were killed at different intervals of time, andone single sample was taken from each closed chamberexcept from Group IV. Each of the five rats of thisgroup had the chamber emptied at the fifth day afterimplant and left in situ so that a second sample was taken

24 hours later. Protein content of tumor IF from closedchambers was roughly equal to that of the blood serumand independent of the length of time the chamber hadbeen implanted. However, when the chamber wasdrained once, the fluid which penetrated later had a protein content about 35 per cent lower than that of bloodserum, as previously found in continuously drainingchambers. The difference in protein content betweendraining and closed chambers is therefore limited to thefirst sample, which probably reflects the consequence ofthe destruction of proteins during the implant (Table 4).

The electrophoreticpattern of tumor IF was studied fromtumors grown "tissue-isolated" and with chambers drain

ing continuously; the closed chambers, drained once,yielded similar values (Table 5). Fibrinogen was notfound in any sample of tumor IF, and the interstitialfluid from subcutaneous or neoplastic tissue never coagulated. The relative proportions of the electrophoreticcomponents remained the same as that found in aorticserum, except for a-globulins. In all the nine animalsstudied the a-globulin fraction was smaller in tumor IFthan in aortic serum; however, in some cases this reduction was very small and the average found in all the determinations was 23 per cent. The reduction of the Â«-globulins alone does not account in every case for theconstant low level of total proteins in tumor IF comparedwith the aortic serum. The albumin:globulin ratio oftumor IF was similar to that of the afferent blood.

NONPROTEINNITROGEN

The total content of nonprotein nitrogen was studiedonly in Walker carcinoma 256. In a group of ten animalsIF, collected from draining chambers, was compared withthe afferent and efferent blood of the neoplastic mass. IFsamples were collected over a 24-hour period, and the

TABLE 4INFLUENCEOF DRAINAGEON THE PROTEINCONTENTOF

INTESTINALFLUID(gm/100 ml)

GroupDaysafterchamber

implantSubcutaneousIFTumor IFAortic serum

Closed Chambers

IIIIIIVVI571010304.6Â±0.3*4.8

Â±0.34.5Â±0.46.0

Â±0.46.0Â±0.25.

5Â±0.15.2Â±0.44.1

Â±0.2â€”5.7

Â±0.15.1Â±0.14.4Â±0.26.0Â±

0.46.2Â±0.2

Closed Chambers Drained Once

IV:Firstextrac

tionSecondex

traction566.3

Â±0.54.1

Â±0.26.5

Â±0.44.2

Â±0.2â€”6.6 Â±0.3

Total of 30 animals, six sets of five each (see text).* Â±standard error.




TABLE 5ELECTBOPHOKBTICPATTEBNOF THE INTERSTITIALFLUID OF TUMORSANDBLOODSERUM

Tumor IF was obtained from draining chambers fitted with TW millipore filters. The tumors ranged from 6 to 12 gm. in weight

TUIKUFibrosarcoma4956Novikoff

hepa-tomaHepatoma

5123Walker

carcinomaAverages

ingmperliterRATNO.123456789BLOOD

FLOWING INTOTUMORTotalÂ«

gm/148.968.147.944.943.765.543.352.449.251.5Relative

per cent of totalbromphenolblueAlbu-min45475147424244495022.7Globulins"i101046131014990116161419151516111413.2ÃŸ20172022192117212210.6â€¢Y89127111281055.0BLOOD

FLOWING OUT OFTUMORTotalÂ«

gm/154.570.452.454.847.768.757.257.853.457.4Relative

per cent of totalbrompbenolblueAlbu

min49524847475638535528.2Globulinsori674510112017ai1216131615111611012.6ÃŸ21182322191421252511.7T1071110885845.0TUMOR

IFTotalÂ«

gm/136.531.433.228.343.640.836.741.140.036.9Relative

per cent of totalbromphenolblueAlbu

min49514445364740515716.4Globulins011067891011811at811991810105117.2ÃŸ2324282726243226179.7T108121011971143.6

Albumin-globulin ratios : blood flowing into tumor = 0.79, blood flowing out of tumor = 0.96, tumor IF = 0.80.* Biuret reaction.

blood was withdrawn at the end of the collection period.In most of the animals IF and the blood flowing out of thetumor contained an amount of nonprotein nitrogen somewhat lower than that of the arterial blood. However, thereduction was neither remarkable nor constant. Duringtumor growth the nonprotein nitrogen of tumor IF compared with the subcutaneous IF showed no special pattern.

Urea, a major component of the nonprotein nitrogenfraction, was determined in two different experimentalconditions. In the first, samples of IF collected from thetumor over a 24-hour period were compared with theblood flowing into and out of "tissue-isolated" transplants.

The concentration of urea in the interstitial fluid did notsignificantly differ between: (a) a hepatoma and a carcinoma or a sarcoma, (o) a large neoplastic mass and asmall one, and (c) tumor IF and the serum of afferentand efferent blood. The content in tumor IF was oftensmaller than in blood (Table 6).

In a second experiment urea was determined in threegroups of animals bearing closed chambers during growthof Walker carcinoma 256 or Hepatoma 5123. Threesamples were collected from each animal, tumor IF, subcutaneous IF, and aortic serum, and in all three the valueswere similar, although relatively large variations werefound from one animal to another.

Free amino acid content of tumor IF was compared withthat of the afferent and efferent blood of the neoplasticmass. The IF was collected over a 24-hour period, andthe two samples of blood were taken at the end of thecollection. Tumor IF was also sampled simultaneously

TABLE 6UREAOF TUMOBIF ANDBLOODSERUM

(mg/100 ml serum)

Ratno.12345678910111213TumorWalker

carcinoma256Walkercarcinoma256Walkercarcinoma256Walkercarcinoma256Walkercarcinoma256Walkercarcinoma256Hepatoma

5123Hepatoma5123Hepatoma5123NovikoffhepatomaNovikoffhepatomaFibrosarcoma4956Fibrosarcoma

4956Tumor

weight(gm.)5.96.58.711.212.515.64.56.36.95.66.76.48.7Blood

flowinginto thetumor77306624254320163553303655Blood

flowingout ofthetumor78â€”7825â€”4420163247323556Interstitial

fluid59327019193614122031253552

with the blood, but three samples of tumor IF from threeanimals had to be pooled to have enough material forthe analysis.

Tumor IF showed in most of the cases a free aminoacid level higher than that of the blood flowing into thetumor, and the levels of the afferent and efferent bloodwere not appreciably or consistently modified (Table 7).

The mercase of free amino acids was also observed in asecond experiment in which tumor IF was compared withsubcutaneous IF and aortic serum. Arterial blood was




withdrawn at sacrifice, always in the late afternoon, fromanimals fed only overnight. In this way, the conditionsin reference to the diet were standardized. The freeamino acid level of tumor IF and subcutaneous IF werefound higher than that of the aortic serum, both in normaland tumor-bearing animals at various times after thechamber implantation (Table 8).

GLUCOSE

The content of free glucose of tumor IF was comparedwith that of the afferent and efferent blood of neoplasticmasses. The determinations were done on the threesamples taken simultaneously, and for this reason theanimal received food and water ad libitum during theexperiment. In the three types of tumor studied the

TABLE 7FREE AMINOACIDSOF THE INTERSTITIALFLUID OP

TUMORSANDBLOODSERUM(mg/100 ml serum)

TumorWalker

carcinoma256Hepatoma

5123Fibrosarcoma

4956Ratno.123451

234123Tumor

weight(gm.)5.96.510.611.212.55.5

6.08.28.46.27.58.6Blood

flowinginto thetumor5.86.46.45.76.14.3

4.85.04.14.64.14.8Blood

flowingout of the

tumor6.5â€”6.36.16.14.5

5.04.44.44.74.24.3Tumor

IF6.07.36.18.27.84.8

5.97.03.44.14.85.2

TABLE 8FREE AMINOACIDSOF THE INTERSTITIALFLUID DURING

TUMORGROWTH(mg/100 ml)

Tumor*Hepatoma

5123Fibrosarcoma

4956ControlsTumor

age(days after

transplant)15301020302102030Subcuta

neousIF7.66.86.15.55.85.36.36.55.5TumorIFt7.87.16.77.27.9â€”â€”â€”Aorticserum6.0

Â±0.6ÃŽ5.1Â±0.25.3

Â±0.25.4Â±0.44.

6Â±0.24.0Â±

0.15.1Â±0.34.8Â±0.34.2

Â±0.3

TABLE 9GLUCOSE CONTENT OP THE INTERSTITIAL FLUID OF TUMORS

ANDBLOODSERUM(mg/100 ml)

TumorWalker


5123Fibrosarcoma

4956Rat

no.12345671234123Tumorweight(gm.)8.28.79.711.212.112.215.64.95.45.95.97.79.49.5Bloodflowingintotumor188200148184126172186224218156244224190180Blood

flowingoutof

tumor126146â€”13610012290132168132118108100TumorIF0000460024000020

* Total of 45 animals, nine sets of five each.t Equal amounts of IF were pooled in each group.Ã®Â±standard error.

amount of free glucose in the interstitial fluid was equalor below the level of detection. The blood leaving thetumor constantly contained less glucose than did thearterial blood. The average reduction observed was 35per cent, with a maximum of 60 per cent. Size, histologietype, and growth rate of the neoplasia did not play anyappreciable role (Table 9).

Free glucose during tumor growth was compared in tumorIF, subcutaneous IF, and aortic serum at three intervals:(a) when the neoplastic tissue was just beginning to growhi the connective tissue around the chamber, (6) when thetumor was sufficiently large to cover the diffusion chamber completely, and, finally (c) when the neoplastic masseswere relatively large. The control groups received onlyone diffusion chamber hi the subcutaneous tissue of thesacral region, and the interstitial fluid was analyzed atintervals from 48 hours after the inclusion of the chamberup to 1 month.

When the neoplastic tissue was present around the diffusion chambers, even in very small amounts, free glucosedisappeared from the interstitial fluid. This was a constant observation when Millipore, Polypore, or S-S verydense filters were used to form the walls of the samplingchamber (Table 10). In the subcutaneous tissue free oftumor, glucose was always present during the first daysof sampling and at levels slightly lower than that of theaortic serum (10-20 per cent). In the subsequent days,the concentration of glucose in the IF of the subcutaneousor intraperitoneal area remained at the same levels whenS-S or Polypore filters were used but rapidly decreasedwhen the sampling chamber was formed by T W Milliporefilters and reached levels of a few mg. per 100 ml. bythe 10th day (Table 10). When sterilization of the chambers was done by heat instead of ethylene oxide, the resultsdid not change.

Hyperglycemia.â€”Walker carcinoma 256 was grown




subcutaneously around a closed chamber for 10 days.Then a PESO catheter was introduced into the left common carotid artery of the tumor-bearing rat and carriedthrough the skin on the surface for easy sampling. Theanimal was placed in a restraining cage and left for about1 hour to recover from the anesthesia. Then a 0.5-ml.sample of arterial blood was withdrawn, followed by asubcutaneous injection of 200 mg. of dextrose in 1.0 ml.saline. Four more of these injections were given, respectively, 30, 60, 120, and 180 minutes after the first.Aortic blood was sampled at 30, 120, 180, and 300 minutes after the first withdrawal. Interstitial fluid from thechamber enclosed in the tumor was sampled just beforethe first dextrose injection and at 60, 120, 210, and 300minutes after.

Hyperglycemia reached about 300 mg 100/ml serum in2 hours and persisted roughly at the same level for 3 morehours. Free glucose was absent in the interstitial fluidbefore dextrose was injected and started to appear whenthe glycemia was around 250 mg/100 ml serum. Despitethe leveling off of the glycemia at about 300 mg. from the2d hour onward, the concentration of free glucose in theinterstitial fluid continued to rise from 60 to 200 mg.(Chart 2).

In preliminary trials to find the right conditions for theexperiment just described, it was observed that a singleintraperitoneal injection of dextrose, which increased theglycemia up to 350 mg/100 ml serum for about 15 minutes,failed to produce the appearance of free glucose in theinterstitial fluid. Furthermore, when glucose was maintained in serum at 230 mg/100 ml, free glucose did notappear hi the tumor IF even after 4 hours of continuoushyperglycemia. It seems, therefore, that: (a) the diffusion chamber placed into the tumor contained a fluiddevoid of measurable amounts of free glucose, (6) thereappearance of free glucose in the fluid was obtainedwhen a hyperglycemia greater than 230 mg/100 ml serum

TABLE 10GLUCOSECONTENTOF THE INTERSTITIALFLUID OF

WALKERCA. 256 DURINGGROWTH(mg/100 ml)

FREE GLUCOSEOF//7 DURINGHYPERGLYCEMIA(WALKERCo.)

Tumor*Walker

carcinoma256ContrÃ´lÃ¢tControls^Days

afterchamberimplant5710251530230Tumor

weight(gm.)1.19.421.0â€”â€”â€”â€”â€”â€”SubcutaneousIF120

Â±20Â§55Â±125Â±0.3109

Â±1486Â±32Â±0.14Â±0.3101

Â±1287Â±6Tumor

Vf12

Â±13Â±0.53Â±0.6â€”â€”â€”â€”â€”â€”Aortic

serum172

Â±6172Â±6150Â±5143

Â±2148Â±2174Â±1185Â±4138

Â±3140Â±6

! '200MGGLUCOSESUBCUT. H

2 3TIME IN HOURS

CHART2.â€”Passageof free glucose into interstitial fluid of tumorduring hyperglycemia.

TABLE 11LACTICACID OF THE INTERSTITIALFLUID OF TUMORS

ANDBLOODSERUM(mg/100 ml)

TumorWalker


5123Fibrosarcoma

4956Controls*Rat

no.12345671

234123123456Tumor

weight(gm.)8.29.211.411.512.213.515.64.5

5.77.47.87.79.512.87.07.38.413.614.015.2Blood

flowinginto

tumor115421741251617112563

1307181654075681118069102109Blood

flowingout oftumor101â€”179â€”160â€”11865

1295071886876128123102120139123Tumor

IF23615024023022813320580

14011710995170120â€”â€”â€”â€”â€”â€”

Rats bearing Walker carcinoma 256 without sampling cham-

* Total of 45 animals, nine sets of five each.t TW Millipore filters.Ã®S-S filters.Â§Â±Standard error of means.

ber.

lasted more than 1 hour, and (c) the increase of free glucose in the interstitial fluid continued when the hyperglycemia was kept constant and above a certain level.

In a second experiment a sampling chamber enclosedin a 7-day-old tumor grown subcutaneously was emptiedfirst and later filled with 200 jul.of a 0.2 per cent solution




of dextrose in water. After 10, 30, and 60 minutes, 10-fd. samples from the chamber were analyzed. Freeglucose was present at 10 and 30 minutes, but absent at60 minutes in all of the three animals used in the experiment. These results showed that, when glucose wasplaced in the chamber, it diffused into the tissues. Therefore, the absence of glucose in the interstitial fluid, asshown in Tables 9 and 10, was not due to the samplingsystem but to the actual disappearance of free glucosefrom the tissue fluids surrounding the chamber.

In a third experiment, tumor IF was collected fromsubcutaneous transplants of 6-day-old Walker carcinoma,and glucose was added to this fluid to make a final concentration of 1 per cent. The mixture was incubated at37Â°C. for 2 hours, at which time no change was observed

in the glucose concentration.

LACTATE

Lactate content was determined in samples collectedsimultaneously with the afferent and efferent blood andalso in samples collected over a period of 2-4 days. Tumor IF always had a concentration of lactic acid higherthan that of the blood entering the neoplastic mass. InWalker carcinoma 256 and Fibrosarcoma 4956 the averageincrease was about 100 per cent, but in Hepatoma 5123 '

it was only about 25 per cent. Relatively large variationsfrom animal to animal were observed. However, whensamples were collected from one tumor over a period of afew days, the variations were relatively small and distributed at random when compared with the age of thetumor (Table 11). In control animals bearing Walkercarcinoma 256 without any sampling chamber, the venousblood was consistently higher than the arterial blood, asshown by Cori (6). However, this was not always thecase in tumors bearing a sampling chamber. The totalamount of lactic acid collected by the sampling deviceover a 24-hour period amounted to a few milligrams anddid not account for the amount of lactic acid which thevenous blood carries away when the arterial-venous differences are high, as indicated in Table 11.

LÃ¡clateproduction during tumor growth was comparedin the interstitial fluids coming from the subcutaneousarea and from the tumor growing subcutaneously in thesame host. At 5, 7, and 10 days after the implant theanimals were killed in groups of five and the interstitialfluid of both chambers was sampled simultaneously withthe aortic serum. Animals with only one chamber without tumor were used as controls.

The lactic acid concentration in the tumor IF was alwayshigh when the neoplastic tissue was present and did notincrease as the tumor grew. In the subcutaneous IF ofnormal or tumor-bearing rats the lactic acid concentration did not change with tune when S-S filters were usedbut did mercase when TW Millipore filters formed thewalls of the sampling chambers. This increase reachedthe level observed in Hepatoma 5123 at 10 days andremained roughly constant thereafter. Furthermore,lactic acid increased while free glucose decreased (Tables10-12), and the lactic acid content of the aortic serum oftumor-bearing animals was generally higher than that of

TABLE 12LACTICACIDOF TUMORANDSUBCUTANEOUS

INTERSTITIALFLUID(mg/100 ml)

Tumor*Walker

carcinoma256ContrÃ´lÃ¢tControlsÂ§Days


weight(gm.)1.2

Â±0.2t9.0Â±121.0Â± 3Tumor

IF120

Â±3125Â±9113Â±4Subcu

taneousIF56

Â±182Â±2103Â±359

Â±2101Â±7114Â±432

Â±236Â±3Aortic

serum51

Â±463Â±577Â±624

Â±131Â±326Â±327

Â±230Â±3

* Total of 40 animals, eight sets of five each,t Â±Standard error of means,Ã®TW Millipore filters.Â§S-S filters.

TABLE 13TOTALCHOLESTEROLCONTENTOF THE INTERSTITIAL

FLUID OF TUMORSANDBLOODSERUM(mg/100 ml)

Tumor*Walker


5123Fibrosarcoma

4956Tumor

weightrange(gm.)8.7-15.64.5-6.14.9-6.8Blood

flowing into

tumor57

Â±7t60

Â±655

Â±3Blood

flowing out of

tumor62

Â±960

Â±864

Â±7Tumor

W5

Â±125

Â±39

Â±2

* Three groups of five animals each with draining chambers,t Â±Standard error.

rats bearing the diffusion chamber without tumors (Tables11, 12).

CHOLESTEROLANDLIPID PHOSPHORUS

The total content of cholesterolof tumor IF was markedlylower than that of blood; however, there was no appreciable difference between the blood flowing into and outof the neoplastic mass (Table 13). This difference variedfrom one tumor to another, but the type or size of theneoplastic mass apparently did not play an important role.Tumor and subcutaneous IF were also compared in thesame animal. The reduction in cholesterol content waspresent in both (Table 14), and the degree of reductionwas less in the closed than in the draining chambers (Tables13, 14).

The total lipid phosphorus content of the tumor IF was,in all cases, about one-fourth or less than that of the blood,and no significant difference was detected between bloodflowing into and out of the neoplastic mass (Table 15).Histologie type and size of the tumor did not appear toplay any appreciable role in changing the lipid phosphoruscontent of tumor IF.




TABLE 14TOTAL CHOLESTEROLCONTENT OF TUMOR AND SUBCUTANEOUSINTERSTITIAL FLUID

Mg/100 ml.Closed chambers were used

Tumor*Walker

carcinomaControlsDays


weight(gm.)1.2

Â±0.3f8.6Â±1.020.4Â± 4.0Tumor

IF28.8

Â±1.735.9Â±4.522.4Â±3.9Subcutaneous

IF23.0

Â±4.335.8Â±4.028.0Â±4.931.9

Â±3.833.5Â±3.552.7Â±7.446.0Â±5.3Aortic

serum66.6

Â±4.162.8Â±0.779.6Â±12.474.7

Â±6.554.9Â±3.061.4Â±7.991.9Â± 14.3

* Total of 35 animals, seven groups of five animals each,

t Â±standard error.

The comparison between subcutaneous and tumor IFshowed equal levels of lipid phosphorus in both fluids(Table 16). The over-all concentration in these fluidswas roughly one-fourth that of serum, and only when theneoplastic masses were large, did the IF concentrationreach values one-half of that found in serum.

SODIUM,POTASSIUM,ANDCHLORIDE

The content of sodium, potassium, and chloride wasanalyzed only with the closed chambers in animals bearing Walker carcinoma 256. It has been impossible toobserve any clear difference in the distribution of theseions at different intervals of tumor growth or betweentumor and subcutaneous IF or aortic serum (Table 17).In few instances tumor IF showed an increase in Na+, butthis was neither constant nor significant. It should bepointed out that the tumors analyzed were relativelysmall, because necrosis was avoided as much as possible.

In the ascites fluid of Japanese/N rats bearing Hepatoma7974, the three ions behaved as in the interstitial fluid ofsolid Walker carcinoma 256.

LYMPHANDPERITONEALFLUID

To complete the characterization of tumor IF the lymphand peritoneal fluid of normal and tumor-bearing ratswere analyzed during the period the animals lived in theconditions required by our experimental approach.

Lymph was sampled from the thoracic duct of sixSprague-Dawley rats kept in restraining cages with foodand water ad libitum, three with Walker carcinoma 256growing subcutaneously, and three without tumor. Fromeach animal a 24-hour sample was collected in a vial keptat 0Â°C., starting the day after the cannulation. Each

sample was centrifuged at low speed to precipitate thecells and the fibrin clot. Then, from three samples ofnormal lymph, equal amounts were measured, pooled,and used for the determinations. The same procedurewas repeated for the lymph from tumor-bearing animalsand for the blood taken from the aorta of each animal atthe end of the collection period. The results of triplicatedeterminations on pooled samples were considered torepresent the behavior of the thoracic lymph in our animals during IF sampling (Table 18).

TABLE 15LIPID PHOSPHORUSOF THE INTERSTITIAL FLUID OF

TUMORS AND BLOOD SERUM(mg/100 ml)

Tumor*Walker


5123Fibrosarcoma4956Tumor

weightrange(gm.)6.0-12.34.5-6.96.4-9.2Blood

flowingintotumor4.4

Â±0.7f4.4

Â±0.26.3

Â± 0.3Blood

flowingout oftumor4.3

Â±1.04.7

Â±0.26.0

Â± 0.4Tumor

IF0.7

Â±0.11.5

Â±0.21.2

Â± 0.2

* Three groups of four animals each with draining chambers,

t Â±standard error.

TABLE 16LIPID PHOSPHORUSOF TUMOR AND SUBCUTANEOUS

INTERSTITIAL FLUID

(mg/100 ml)

Group*Walker

carcinoma256ControlDays


weight(gm.)1.54.824.1â€”â€”â€”â€”TumorIf1.91.04.6â€”â€”â€”â€”Subcutaneous

IF1.01.63.42.22.01.61.7Aorticserum5.9

Â±0.3f5.5Â±0.29.0Â±0.64.9

Â±0.35.0Â±0.34.7Â±0.34.8Â± 0.4

* Total of 35 animals, seven groups of five each. Equal

amounts of IF pooled in each set.t Â±standard error.

Compared with tumor IF, the lymph from the thoracicduct showed the same general behavior of the nitrogen-containing compounds. The free amino acid and ureafractions were higher in the lymph than in the aorticserum. As expected, glucose and lactic acid in the lymph




TABLE 17SODIUM,POTASSIUM,ANDCHLORIDECONTENT

Meq/liter serum or interstitial fluid

GROUP*Walker

ca.256ControlsDAYSAFTEÂ»

CHAMBERIMPLANT5710225SODIUMTumorIF155

Â±7f150Â±5145Â±8Subcut.

IF146

db2140Â±2142Â±2155

Â±5155Â±5Aortic

serum143

Â±2144Â±2143Â±1148

Â±7153Â±2POTASSIUMTumor

IF4.1

Â±0.24.8Â±0.14.3Â±0.2Subcut.

IF5.0

Â±0.14.1Â±0.14.6Â±0.24.1

Â±0.24.3Â±0.1Aortic

scrum4.2

Â±0.34.1Â±0.14.2Â±0.24.1

Â±0.54.3Â±0.1CHLORIDETumor

IF100

Â±0.7106Â±0.4105Â±1Subcut.

IF103

Â±0.5110Â±1.4106Â±1109

Â±1106Â±1Aortic

serum101

Â±1.4104Â±1.5102

Â±0.7110

Â±3106dh 1

* Total of 25 animals in groups of five each set.t Â±standard error.

behaved as in blood serum and opposite to the tumor IF.The lipid fractions were obviously higher in lymph thanin serum and in tumor IF. Sodium, potassium, and chloride did not show any remarkable change.

Peritoneal fluid was drained with sampling chambersinserted in the abdominal cavity of three normal Sprague-Dawley rats and six Japanese/N rats bearing Hepatoma7974. The ascites fluid was collected at the 5th day aftertransplant, directly from the chamber and simultaneouslyfrom the abdominal cavity with a pipette. These lastsamples were centrifuged at low speed to separate thecells, and the supernatant was used for the determinations.Animals with bloody ascites fluid were discarded. Thesamples were pooled in equal amounts for each group as itwas done for the lymph (Table 19).

The protein level was lower in the peritoneal fluid thanin serum, but, in contrast to tumor IF, the ascites fluidhad a free amino acid concentration lower than serum andthe normal peritoneal fluid. Glucose was down to negligible levels in the ascites fluid, but in normal peritonealfluid it was only slightly less than in the serum. Whenthe draining chamber was formed by TW Millipore filterand left in the peritoneal cavity of normal rats for morethan 10 days, glucose disappeared from the sampled fluidas it did from the subcutaneous IF. Lactic acid concentration increased as glucose decreased, as it did in thesubcutaneous fluid (values in parentheses in Table 19).When S-S filters were used the disappearance of glucoseand the increase of lactic acid did not occur. Cholesteroland lipid phosphorus were lower than in serum; sodium,potassium, and chloride ions did not show an appreciablechange.

DISCUSSION

The results described are useful if one can show thatthe sampling technic is reliable and the tumor IF can becharacterized in regard to the other body fluids.

To our knowledge only two other technics have beenused to sample the interstitial fluid. One, developed byMaurer (23), utilized capillary tubes forced gently parallelto the fibers of frog muscle. Manery (22) doubted thatthe technic could actually sample interstitial fluid, yetCreese and co-workers (7) showed that the procedureapplied to muscle could give useful information. We were

TABLE 18LYMPHFROMTHE THORACICDUCT (24-HouR SAMPLE)

COMPOUNDTotal

proteinsNonprotein-NUrea-NFree

aminoacidGlucoseLactic

acidTotallipidsCholesterolLipid

phosphorusSodiumPotassiumChlorideUNITSgm/100

mlmg/100mlmg/100mlmg/N/100mlmg/100

mlmg/100mlgm/100mlmg/100mlmg

P/100mlmeq/1meq/1meq/1RATSWITHOUT

TUMORLymph3.4168.443.06.31477.12.7911415.81316.1101Aortic

serum5.4566.615.85.814627.50.13885.41405.01.3RATS

BEARINGWALKERCARCINOMALymph2.5550.829.75.520626.73.2815019.21316.098Aortic

serum5.3156.113.35.112530.80.491134.91375.0106

Equal amounts of each sample pooled from three animals foreach determination. Aortic serum taken from the same animalsat the end of lymph collection. Cells separated by centrifugationat room temperature. (Sprague-Dawley rats.)

unable to obtain reliable results with this technic appliedto tumors.

SylvÃ©nand co-workers (4, 36) sampled fluid at theperiphery or at the center of a tumor mass by means ofglass capillaries. The "interstitial fluid" of the tumor was

considered to be the fluid phase collected in small pouchesproduced by careful compression of the tissue with bluntinstruments. The amount of cell destruction and vascular leakage produced by this procedure is probablysmall when the fluid is collected from the periphery of thetumor, but it is large when the neoplastic mass has to besplit and fluid has to be collected from a cut surface.

Vorzel] and Zajicek (39) separated by centrifugation thefluid from the solid cellular component of material extracted by needle-biopsy of human tumors. They calledthe liquid phase of their preparation, "extracellular fluid."

The extent of cellular damage and vascular leakage inherent in this procedure led us to believe that this "extra-




cellular fluid" does not represent the fluid phase of com

partment II, as defined in the introduction, and thereforethe procedure could not serve our purposes.

Our procedure may have two major disadvantages.The first one is the possibility that the sampling chamberacts as a "selecting" device. Three facts point to the

contrary : (a) when the chambers were placed directly into

TABLE 19PERITONEALFLUID (MILLIPOBECHAMBERS)

COMPOUNDTotal

proteinsNonprotein-NUrea-NFree

aminoacidGlucoseLactic

acidCholesterolLipid

phosphorusSodiumPotassiumChlorideUNITSgm/100

mlmg/100mlmg/100mlmg/100mlmg/100mlmg/100mlmg/100mlmg

P/100mlmeq/1meq/1meq/1NORMALDRAINED*3.8731.823.66.3108

(5)20(108)28.40.751555.099HEPATOMA

7974fDrainedasci

tes3.0930.320.94.2315622.31.71425.4106Centri-fugedascitest3.2427.816.64.0415822.31.01695.3106

* Equal amounts of three samples taken from three animals andpooled before the determination.

t Total of six animals. Equal amounts of samples pooled.Ascites fluid not hemorrhagic.

} Cells separated at 1000 X g (max.) at room temperature.For values in parentheses see text.

a beaker containing serum no difference was observedbetween the inside and outside samples, (o) increase ordecrease of concentration was observed equally withfilters of different make and pore diameter, and (c) whenthe ascites fluid was drained out of a chamber it had thesame composition as the fluid pipetted directly from theperitoneal cavity.

The second disadvantage is the effect of the samplingdevice on the tissues. For instance a "fermentative"

type of interstitial fluid was observed after a few days inthe subcutaneous or peritoneal samples when TW Milli-pore filters were used, but not with S-S filters. In regardto this disadvantage one can stress two facts: the absenceof a granulomatous tissue around the chamber and thepossibility to use various kinds of filters to single outartifacts.

As a side issue, attention is called to the production of a"fermentative" type of IF in non-neoplastic tissues. The

increase of fermentation was interpreted as a gradualdamage of cell respiration with production of cancer as anend-result (5, 19, 20). In our experiments a relation withthe carcinogenic properties of the filters (11, 12) is possible, but fermentation substituted for respiration (40)long before any malignancy could be expected.

On the assumption that our procedure offers a reliablesample of the fluid phase of compartment II, the lacticacid and glucose concentrations and the over-all "tension"

appear to differentiate tumor IF from the other bodyfluids (Table 20). The disappearance of glucose from theinterstitial fluid whenever neoplastic cells were present is,in a way, contrasted by the average reduction of only about35 per cent of glucose in the efferent compared with the

TABLE 20

COMPOUNDTotal

proteinsFree

aminoacidsFree

glucoseLactic

acidTotal

cholesterolLipid

phosphorusUNITSgm/100

mlmg

N/100mlmg/100

mlmg/100

mlmg/100

mlmg

P/100mlDRAINING

CHAMBERSBlood

flowinginto the

tumor4.8

Â±0.1*5.2

Â±0.2188

Â±895

Â±9(90Â±8)60

Â±45.0

Â±0.4Blood

flowingout ofthetumor5.2

Â±0.25.1

Â±0.2123

Â±6100

Â±12(122Â±4)Â§64

Â±55.0

Â±0.3Tumor

IF3.2

Â±0.15.9

Â±0.44161

Â±118

Â±21.1

Â±0.1CLOSED

CHAMBERSTumor

IF5.0

Â±0.3(4.2Â±0.2)t7.3

Â±0.26119

Â±329

Â±32.5

Â±1.0Subcutaneous

IF4.6

Â±0.3(4.1Â±0.2)f6.3

Â±0.2105

Â±12(5)Ã®56

Â±1(109)t29

Â±42.0

Â±0.8Aortic

serum5.0

Â±0.1(6.6Â±0.3)f5.3

Â±0.3154

Â±551

Â±470

Â±56.8

Â±1.0PERITONEAL

FLUIDNormal

drained3.806.3108(5)Ã®20(108

)Ã®280.8Ascites

form ofhepatoma7974Drained3.094.13156221.2Centri-

fuged3.244.04158221.0LYMPHFROM

THORACICDUCT(24-hr.

SAMPLE)RATS

BEARINGWALKER

CA.2562.555.5125271504.9

* Â±standard error.t After the chambers were drained once.ÃŽWhen the sampling chambers were left for more than 10 days, and TW millipore filters formed the walls.Â§No draining chamber present in the tumors.




afferent blood of the tumor. If the hypothesis of the threecompartments is accepted, one can suppose that compartment II is like a container from, which glucose is veryrapidly removed; hence, its level is constantly low. Asmall concentration of glucose has also been found in theascites fluid and has been attributed to the inability of thevessels of the peritoneal cavity to supply more glucose (8,19). In our system, however, compartment I has approximately 30 times more glucose than compartment II; therefore, one must assume the existence of some barrier at thelevel of the capillary wall. The data of Chart 2 supportthis hypothesis. The passage of glucose into compartment II can be interpreted as due to "overload" of the

regulatory system by hyperglycemia of a sufficient magnitude and of sufficient duration. A short circulationtime could also explain the high gradient of glucose between compartments I and II. However, this appearsunlikely in view of the low blood flow measured (16) andthe probable low capillary pressure of the tumors as indicated by the protein concentration of tumor IF. Furthermore, it does not explain the response to hyperglycemia.A rapid "transformation" of glucose in the interstitial

space could offer another explanation. We have no dataon this point, but, in view of the fact that glucose furnishes energy to the cells, the obvious transformation onecan forsee is an initial phosphorylation. This couldhardly occur outside the cell, because it is generally agreedthat phosphorylated glucose does not easily penetratethrough the cell membrane. The presence of glycolysisin the interstitial fluid in vivo seems unlikely in view ofthe fact that in vitro tumor IF failed to utilize any glucose.

The increase in lactic acid content of tumor IF was expected. If the lactate concentration of tumor IF indicatesthe glycolyzing capacity of a cell population, our data invivo confirmed the low glycolytic activity of Hepatoma5123 in vitro, reported to be due to a lack of glucokinase(47).

The high "tension" of tumor IF recalls the values

measured after venous obstruction (24) and the findingsof Young and co-workers on Brown-Pearce carcinoma(46).

The similarities between tumor IF and other body fluidsin proteins, cholesterol, and lipid phosphorus content appear relevant to the question of necrosis. One is accustomed to imagine the necrotic process as a destruction oftissues with consequent leakage of various substances intothe interstitial spaces. The data obtained, however, indicate that the milieu around the viable neoplastic cellsis rather well controlled and the necrotic tissue is somehowsegregated from the viable one. How this is accomplishedis not clear.

The uniformity of urea concentration in plasma andHepatoma 5123 interstitial fluid probably indicates thatthis tumor, morphologically well differentiated and with"minimal deviations" from the enzymatic pattern of the

liver (25, 42), is unable to form urea in vivo.Sodium retention by the tumor (37) was not found,

probably because our tumors were much smaller than 10per cent of the body weight.

The electrophoretic pattern of tumor IF, although insufficient to establish the existence of a difference in por-

tein distribution between compartments I and II, do suggest this possibility (41, 43, 45).

The gradient between blood and tumor IF for glucosecould be different from the gradient maintained by thevascular wall for total sterols and lipid phosphorus. Glucose is utilized as soon as it reaches the interstitial space,and a transfer mechanism could act simply as a rate-limiting device. Sterols and lipid phosphorus are not utilizedrapidly, and the gradient could be maintained by someselecting mechanism. The data presented here only suggest this possibility, and the "permeability" of the vascu

lar wall of the tumor vessels will be discussed in a forthcoming paper.

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16. . Studies on the Exchange of Fluids between Host andTumor. II. The Blood Flow of Hepatomas and Other Tumorsin Rats and Mice. J. Nati. Cancer Inst., 27:1465-91, 1961.

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FIG. 1.â€”Diagram representing the three-compartment hypothesis: vascular space, neoplastic cell, and the area in between.Neoplastic cells growing against a filter with average pores of0.45 Mor less did not pass through the filter but the fluid bathingthe cells could diffuse and be collected.

FIG. 2.â€”Basic preparation for the sampling of interstitial fluidof "tissue-isolated" transplants. Hepatoma 5123 connected tothe host with a peduncle bearing one artery and one vein andenclosing the diffusion chamber.

A.W. = abdominal wall of the rat.V.P. = vascular peduncle of the tumor.C = catheter coming from the sampling device enclosed into

the tumor. Note the interstitial fluid inside the catheter.FIG. 3.â€”Four-day-old subcutaneous transplant of Walker

carcinoma 256 growing around chamber. Note how the tumoradheres to the filter.

FIG. 4.â€”Cross-section of a Walker carcinoma 256 grown sub-cutaneously and enclosing the sampling device for the collectionof the interetitital fluid.



Necrotic area

Interstitia

(luidpassing

throughmembrane

7!).-)



Fio. 5.â€”Onthe left, draining chamber before being assembledand ready for implantation. In the center, closed chamber beforeand after being assembled. On the right, PE 260catheters readyfor sampling and heat-sealed after the sampling.

Flo. 6.â€”Animalsin the cages during collection. The tubingfrom the diffusion chamber enters a vial kept at 0Â°C. in a fluidrunning from a refrigerating bath (arrows).

FIG. 7.â€”Neoplastic cells against the micropore filter. N =nylon reinforcing net. F = filter. Note the contact of theneoplastic cells to the filter without interposition of a granulom-atous tissue (X 300).

FIG. 8.â€”Subcutaneous tissue of the scapular region 20 daysafter a millipore filter chamber was implanted. Arrows point tothe area where connective tissue and filter were in contact. Notethe "mild reaction". (X 50).

796



797



1964;24:780-797. Cancer Res Pietro M. Gullino, Shirley H. Clark and Flora H. Grantham The Interstitial Fluid of Solid Tumors

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The Interstitial Fluid of Solid Tumors · The interstitial fluid of a solid tumor, consisting of the liquid phase interposed between the newly formed vascular walls of the tumor and