Effect of Amino-teropterin and of ThioTEPA on Growth

and Implantation of Free Tumor Cells fromAscites Tumors*

HORACEGOLDIE,MATTHEWWALKER,ALVINR. GRAHAM,ANDGEORGIAB. MITCHELL

(Laboratoryfar Experimental Oncologyand Department of Surgery, Meharry Medical College,Nathvitte, Tenn.)

Two facets of the malignant condition in themouse known as "ascites tumor" have been used

as a test object for studying the effect of chemicalor radioactive agents on tumor cells : accumulationof ascitic fluid (23, 26, 28) and growth of free tumor cells in this fluid (7, 9,10,16,18, 20). Each ofthese facets is only one of the intermediary linksin the chain of processes combined in the life history of an ascites tumor. At both ends of this chainare phenomena of implantation: the initial stagebegins with the implantation of inoculated cellsand their early descendants into visceral peritoneum and the mesentery (primary implantation)affecting capillaries and lymphatics and thus resulting in ascitic exudation and accumulation ofthe fluid (6). In the later stage, some of the freecells which have proliferated for numerous generations in the exúdate implant into serosa (secondary implantation), mostly at the sites of primary implantation. It appears, therefore, thatwhile reduction of the fluid volume and of the tumor cell concentration by treatment providesvaluable information on the effect of the agent,this information is not complete until the otherfacet of ascitic malignant condition—secondaryimplantation—is used as a test object for the sameagent. Accordingly, we have applied this principleof investigation in testing the effect of two chemical compounds, Amino-teropterin (4-aminopteroyl-triglutamic acid) and thioTEPA (triethylenethio-phosphoramide), in mice with ascites tumors.

MATERIALS AND METHODSTumor and mouse strain»,—S-37and S-180 were grown by

serial intraperitoneal transfers as ascites tumors in SwissAlbino mice (Swiss Albino Farms, Red Bank, N.J.). Lympho-

* This work was supported by a Grant-in-aid No. CH 25Bfrom the American Cancer Society, New York, N.Y., and byGrant No. C2080 from the National Cancer Institute, Bethes-da, Md.

Received for publication December 17, 1956.

sarcoma 6C3HED strain was maintained by the same methodin C3H mice (Roscoe Jackson Laboratory, Bar Harbor,Maine). Mice of 22-25 gm., mostly females, were used.

Tumor cell counts in the ascitic exúdate.—Technicsofinoculation, of exúdatewithdrawal, and of cell counts in theexúdate, as well as the method for calculation of tumor cellconcentration from total and differential counts, have beendescribed in our early publications (8-10,13). Standard patternof implantation (periuterine localization) was obtained by atechnic recently reported (14). Doses of approximately IO7or 10*tumor cells were inoculated whenever a massive growthwas planned ; they were reduced to 10' for obtaining a relativelylow level of free tumor cell concentration and implantation.The exúdatewas withdrawn for counta 3 or 4 days after inoculation and for microscopic examination 6-72 hours after treatment of 4-day-old growth.

Recording spontaneous implantation.—The majority of untreated control mice died within 10 days after inoculation withlarge numbers of cells and within 15 days after small inocula.In those surviving longer, the implants were often necrotic.For this reason, the mice which had not died within 10 dayswere sacrificed on the 10th day, and autopsies were performed.The extent of implant growth was graded as following: 0 = nomacroscopic implants; + = small nodules (not more than2 mm. in diameter); ++ = tumor (3-5 mm. in longer diameter); + + + = tumor larger than ++. The uniform pattern ofperiuterine localization and growth of implants provided a useful standard for estimate of their extent.

Checking the survival.—The death rate in untreated andtreated mice was compared by recording the number of micein each series surviving 10 days. After this period, individualvariations in survival, independent of treatment, and spontaneous necrosis of implants (as noted above) were frequentand interfered with interpretation of the results.

Induced implantation.—The abdominal wall of the mousewas divided into four quadrants and punctured inside eachquadrant with a 20-gauge needle. Twenty-four hours later theanimal was given intraperitoneal inoculations (thus, addinga fifth puncture) of tumor cells. At the autopsy of untreatedanimals large tumor nodules were found in the muscle belowthe peritoneum, at the sites of punctures (with few exceptions).Nodules 1-2 mm. in diameter were recorded as +; 3-5 mm.,as + + ; larger, as H—h+ ; absence of visible growth, as 0.

Experimental infiltration of subcutaneous tissue with freeascitic tumor cells.—Wehave reported previously (11, 19)sharply localized growth of scalp tumors from free cell inoculaand their suitability for quantitative estimate of normal tissueinvasion. In our present investigation we have used scalp tumors for testing the effect of an agent on the spread of subcutaneous malignant growth. Ascitic exúdate was inoculated

374

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GoLDiE et al.—Tumor Growth and Implantation 375

into the scalp just above the eyes and the nose. The intervalbetween the dates of inoculation and death was recorded.The extent of tumor spread was evaluated as following: nogrowth as 0; strictly localized growth on the top of the scalpreaching orbits, without involvement of the eyelids, as +;growth covering the whole area between the neck, the orbits,and the ears, and involving eyelids, as + + ! growth extensionon the ears, the neck, and the face, as + H—K

Detection of metastaiic tumor cells in organs by intraperitonealgrafts.—We have shown (12) that intraperitoneal inoculationof organ brei or blood from tumor-bearing mice (donors) resulted, in numerous instances, in peritoneal growth in newmice (recipients). The technic of organ grafting and its application for testing the effect of radioisotopes on metastatic tumorcells have been described elsewhere (21).

Treatment.—A. Agents:1 (a) ThioTEPA was dissolved in distilled water

on the day of administration or 1 day earlier; doses of 1 or2 mg/kg were injected in a volume of 0.5 or 1 cc., by variousroutes, (b) Amino-teropterin was given in doses of 0.5 mg/kg

or 1 mg/kg, intraperitoneally or subcutaneously.Schedule of treatment: Each compound was injected into

one series of mice in a single dose on the day following inoculation and into another series as daily doses for 2 or 3 successivedays after inoculation. For testing the effect on free tumorcells in ascitic fluid, either the intraperitoneal or subcutaneousroute was used. Experiments with induced implantation implied only intraperitoneal treatment; those with scalp tumors,only topical subcutaneous injections; attempts to preventmetastatic growth in the blood and the organs from theprimary intraperitoneal growth, systemic treatment by subcutaneous injections.

RESULTSDosage.—Doses of 1 mg/kg of Amino-teropterin

were not toxic for normal mice, but they killedabout 50 per cent of S-180 ascites tumor-bearinganimals. Doses of 0.5 mg/kg were well tolerated byboth groups and therefore accepted for standardtreatment. These results are within the range ofdosage used in mice by Burchenal et al. (2) and byGoldin et al. (22) in the treatment of leukemias,and by other investigators. (5).

Similarly, doses of 2 mg/kg of thioTEPA weresafe for normal animals and those with small subcutaneous tumors but toxic for about 50 per centof animals with ascites tumors. However, theytolerated repeated injections of 1 mg/kg. Thisamount is on the lower level of the dosage for miceapplied by Sugiura et al. (29) for treatment ofsubcutaneous mouse tumors (1-6 mg.) and used

also by other investigators (1, 3, 4, 27).Tolerance to both agents decreased with the in

crease in age of peritoneal growth.Inhibition of peritoneal growth and implantation.

—¿�Micewere given inoculations intraperitoneallyeither with large or with small numbers of cells.They were treated either with a single dose or withrepeated daily doses of a compound, starting 24

1 We appreciate the courtesy of Dr. James M. Ruegsegger,

Director, Medical Research, Lederle Laboratories, PearlRiver, N.Y., who supplied us with the agents.

hours after inoculation. Cell counts were performed on the 4th or the 5th day after inoculationand autopsies on the 10th day, unless the mousedied earlier. The data obtained with each agentare reported in Tables 1 and 2.

It appears from Series 1, Table 1, that the concentration of free tumor cells in the fluid and theextent of their implantation were considerably reduced by the treatment (Series 1A to ID and Fig.2) as compared with the controls (IE and Fig. 1).The effect was proportionate to the number of injections (1A and 1C as compared with IB and ID)but little influenced by increase of their dosageover a certain level (1A and 1C). The range ofvariations was wider for a single injection than forrepeated injections.

Series 2 shows that the same schedule of treatment inhibited the growth of free and implantedtumor cells from the small inocula more completely than after massive inoculation (Series 1). Inthese experiments higher doses were more efficient(2A and 2B as compared with 2C) in their effecton tumor growth level but not on the survival. Anattempt to decrease the dose (2E) further provedunsuccessful in spite of a low growth level.

Series 3 was intended to illustrate the role of injection route, either intraperitoneal (3A, 3C, and3E) or subcutaneous (3B, 3D, and 3F) in the results of treatment. In each group of two series,with the same dosage of the same agent but withdifferent routes, the amount of free cell proliferation and of implanted growth was lower in the intraperitoneal series, thus showing that topicaltreatment of intraperitoneal growth was more efficient than systemic treatment. However, the survival of the animals was extended by both routesabout equally, suggesting that the cause of deathin all series was a systemic effect of tumor growth.

Similar results were recorded in Series 4, withless tumor growth. In Series 4E and 4F, about one-third of all mice died earlier than did the controls,presumably owing to their unusual sensitivity toAmino-teropterin.

Trial of thioTEPA was carried out by similarmethods and with analogous tumor material. Theresults are arranged along similar lines in Table 2.

It should be noted that decrease in tumor cellconcentration in mice treated with either compound was associated as a rule with considerablereduction in volume of ascitic fluid. In severalmice the peritoneal cavity was completely "dried"

by treatment (Fig. 3). Thus, a low level of cellconcentration could not be interpreted in any instance as an effect of increased dilution.

As in the experiments with Amino-teropterin,the treatment with thioTEPA reduced the

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376 Cancer Research

amount of free and implanted growth even aftermassive inoculation (Series 1) and more completely after inoculation of smaller doses (Series 2) ; repeated injections of small doses (1 mg/kg) weremore efficient than a single injection of a higherdose (2 mg/kg) (Series 1 and 2); intraperitonealroute was more favorable than subcutaneous(Series 3). However, there were some significantdifferences in the results outlined in Tables 1 and2: (a) in the majority of animals treated intra-peritoneally with repeated thioTEPA injections,no macroscopic implants were found in the peritoneum; (6) larger doses of the agent reduced tumorcell concentration in the fluid to a much lower level(ID, IE, IF) than did small doses (1A, IB, 1C);however, the large dose (2 mg/kg) was not tolerated by a number of animals which died simultaneously with controls (IE and IG; 2D and 2E) oreven earlier (IF); (c) the difference in the resultsof subcutaneous and intraperitoneal treatment

with thioTEPA, as to free tumor cell concentration and extent of implantation, was considerablymore marked than in the experiment with Amino-teropterin; (d) in animals treated with doses of 1mg/kg of thioTEPA, the survival period was distinctly correlated with the amount of peritonealgrowth, thus suggesting that decrease in lethalitywas due to the inhibition of the primary tumor.

To check the consistency of these conclusions,an experiment with thioTEPA was carried outwith a different tumor strain—6C3HED. The results were quite analogous to those of the Sarcomaexperiment, and, moreover, in Series 5 with lowlevel growth, the inhibition of tumor growth andthe protection against death were nearly completein the majority of mice.

Serial microscopic examination of smears fromascitic fluid from treated and untreated mice revealed a decrease in the concentration of tumorcells as early as 6 hours after the intraperitoneal in-

TABLE 1

EFFECTOFAMINO-TEROPTERINONTHEGROWTHANDIMPLANTATIONOFFREETUMORCELLSIN THEPERITONEALCAVITYOFTHEMOUSE

Twenty mice in each group.

SERIE«ormemlAIB1CIDIE2ACB2CCD2E2FSASBsc3DSESF3G4A4B4C4D4E4F4GTUMORITU»S-180No.

IN

OCULATED

TUMORCELL«*10X10«ROUTEorADMIN,tLP.LP.LP.LP.DORE/IVJ.+(mgA«)1.01.0.5.5No.

SURVIVING

No. AFTER 10 No. TUMORCELlfcl]INJ-52121Untreated

controlsS-8710X10*LP.LP.LP.I.P.LP.1.01.0.5.5.2521212Untreated

controlsS-18010X10«LP.S.C.LP.S.C.I.P.S.C..5.55.S.5.5112238Untreated

controlsS-180S-180S-180S-180S-180S-180S-18010X10*10X10'10X10*10X10S10X10*10X10*10X10*I.P.S.C.LP.S.C.I.P.S.C..5.5.5.5.fi.5112233Untreated

controlsDAYS

t121417167172018201812202018161412818182020121116(tbousanda/cmm)3.2(0.8-5.8)7.7

(S. 3-14.6)5.8(0-16.4)6.4

(0.4-23.6)37.1(28.1-44.5)000.43(0-2.8)7.6

(O-IS.9)12.2(6.9-15.6)15.7(8.4-32.3)17.9

(4.8-46.8)29.8(16.5-39.6)8.1

(0-183)15.1(4.0-24.4)0.9

(o-2.5)10.5(4.7-17.4)63.3

(54.1-72.0)7.1

(0-19.2)10.4(2.5-18.2)1.1(0-4.1)6.8(0-25.3)1.8(o-2.4)3.2

(0-13.5)18.4(10.1-29.0)EXTENT

orPERITONEALIMPLANTATION**—313001244400004082000801140+161415114816141010129121513121801518016916Ü-t-

+1529120026108118170016571240016++0000400000000000040000004

* Cell counts on the inoculum were precise within 10 per cent.t I.P. = intraperitoneal; S.C. = subcutaneous.} Doses of Amino-teropterin were calculated from dilution of stock solution (5 mg/20 cc).§On successive days after inoculation.# Mice were sacrificed on the 11th day (unless they died earlier) to provide comparable material for autopsies. Some series in

cluded up to 40 mice, but for uniformity of presentation results on only twenty mice are given.|| Average tumor cell concentration for twenty mice in exúdate3-5 days after inoculation; range given in parentheses.** No implant in the peritoneal cavity, —¿�;nodules only, +; small tumors, H—(-;large tumors, + ++. (See "Material and

Methods.")

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GoLDiE et al.—Tumor Growth and Implantation 377

jection of 1 mg/kg of each compound. In fluidspecimens from the thioTEPA series tumor cellswere clumped and outnumbered by leukocytes,mostly polymorphonuclears which were also frequently clumped. Fluid smears after two dailythioTEPA injections showed large numbers ofclumped leukocytes and very few tumor cells. Inanalogous specimens from the Amino-teropterin-treated animals leukocytes were less numerous andthe tumor cells more frequent. Fluid smears fromuntreated controls often showed nearly a pureculture of tumor cells.

Prevention of experimented implantation of tumorcells from the ascitic exúdate into abdominal wall.—

The above reported data on inhibition of spontaneous implants in the mesentery and peritoneumof ascites tumor-bearing mice could be attributedeither to reduction in the number of free tumorcells available for implantation or to inactivationof tumor cells infiltrated into the abdominal wallor the mesentery in the process of implantation (orelse to both factors). To determine whether theseresults were due to the second alternative interpretation, we have tested each of the two compounds for their ability to prevent implantation

into the peritoneal wall at sites prepared by abdominal punctures, following the method of induced implantation which was described in"Materials and Methods." Animals prepared by

abdominal punctures before inoculations weretreated with Amino-teropterin or with thioTEPAby the methods illustrated in Tables 1 and 2. Theresults are arranged in Table 3.

In all untreated controls, with a few exceptions,large nodules caused by cell implantation from theexúdate were found at all five sites of punctures(Figs. 4 and 7). Different methods of treatmentwith each agent reduced variously both the number and the size of nodules (Figs. 5, 6, 10, 11). Almost complete inhibition of implantation was obtained by repeated injections of thioTEPA (3B,3C, 4A, 4B, 4C). Amino-teropterin decreased theamount of implant growth (size of nodules) in allseries (1A-1D, 2A-2C), but it did not influence sig

nificantly the extent of implantation (number ofnodules) in mice given inoculations of high numbers of cells (lA-lD). In the series with scantygrowth (2A-2C), the number of nodules was

slightly lower than that in controls (2D). Theseresults indicate that inhibition of growth from im-

TABLE 2

EFFECTOFTmoTEPA ONGROWTHANDIMPLANTATIONOFFREETUMORCELLSINTHEPERITONEALCAVITYOFTHEMOUSE

Twenty mice in each group.

SERIESorMICE1AIB1CIDIEIPIG2A2B2C2D2ESASB3C3DSE4A4B4C4D4EffAffBffC5DNo.INOCU-ROUTETOUORLATEO TUMOROFSTRAIN

CELLSADUIN.S-1806X1010 I.P.I.P.I.P.I.P.I.P.I.P.DOSE/DfJ.

No.(mg/kg)

INJ.2.0

12.022.031.011.021.0

3UntreatedcontrolsS-180

10s I.P.I.P.I.P.I.P.1.011.022.012.4

2UntreatedcontrolsS-180

10T I.P.S.C.I.P.S.C.1.021.021.031.0

3Untreatedcontrols6C8HED

5X10« I.P.I.P.S.C.I.P.1.011.021.021.0

3Untreatedcontrols6C3HED

10s I.P.I.P.S.C.I.P.1.011021.021.0

35E Untreated controlsNo.

808-

VIVING

AFTER10DATS12181410868202017141418141617761412100122020188No.TUMORCELLS(thousands/cmm)20.2(12.5-48.4)5.7(0-6.2)9.5(0-21.3)12.4(1.2-18.1)3.5(0-9.0)1.5(0-5.1)51.4(41.3-58.9)12.1(7.5-19.2)2.1(0-5.6)5.1(0-8.7)1.5(0-6.0)23.3(15.1-41.3)11.3(7.2-19.2)24.2(18.3-66

6)5.8(1.5-9.4)27.8(33.1-42.2)59.7(53.2-68.1)42.1(33.1-58.4)7.4(2.7-14.1)28.6(16.5-39.5)5.7(2.3-14.1)63.6(43.1-87.7)13.1(4.5-27.4)00027.8(15.5-34.5)EXTENT

OF PERITONEALIMPLANTATION-0121412101601217121801401520511ff1101020It200+18•868104683820613ff140Iff1512001008012+

+200000100000Iff07041700301400000++0000005000050000s000060000*

See footnotes to Table 1 and "Material and Methods."

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378 Cancer Research

planted cells was obtained regularly with Amino-teropterin and prevention of implantation onlyoccasionally, while both results were consistentlyproduced by thioTEPA in all experiments.

Inhibition of tissue invasion from sharply localized tumors.—Doses of IO7 S-180 cells from ascitic

fluid were inoculated into the scalp of three seriesof mice (See "Materials and Methods"). Twenty-

four and 48 hours later the inoculated area was infiltrated with doses of 0.5 mg/kg of Amino-terop-terin (Series 1) or with 1 mg/kg of thioTEPA(Series 2). The third series remained untreated(controls, Series 3). The size of growth and thesurvival of mice was recorded at various intervalsand the data obtained are reported in Table 4.

It can be noted from Table 4 that topical treatment with Amino-teropterin delayed but in no instance prevented extensive tumor growth in subcutaneous tissue of the scalp and did not increasethe survival span of mice to 30 days (groups 1Aand IB as compared with controls 1C); however,survival of a few mice was extended to 20 days. Onthe other hand, topical tissue infiltration withthioTEPA resulted eventually in complete inhibition and, in all cases, some inhibition of the spread.The survival span of 30 days was obtained in themajority of mice of series 2A and in several miceof 2B.

Inhibition of metastatic tumor cell growth fromintraperitoneatty grafted blood and organs.—Table 5

TABLE 3

EFFECTOFINTRAPERITONEALINJECTIONOFCHEMICALAGENTSONTUMORCELLIMPLANTATIONFROMPERITONEALEXÚDATEATTHESITESOFPERITONEALPUNCTURE

Twenty mice in each series, weighing 25 or 26 gm.

SERIESorMICE1AIB1CID2A2B2C2D3A8B3C3D4Õ4B4C4DTUMORSTRAINS-180S-87S-180S-37No.INOCULATEDTUMORCELLS10X10«10X10"10X10«loxio"

AGENT*DOSE(mg.)

No.INJ.

IT.I23

282

GROWTHAT THESITES OF PUNCTURNo. of takes t(average and

variation Size ofextremes) growth Ì

AT .5AT 5AT .5

Untreated controls

AT 5AT .5AT 1.0

Untreated controls

TTEPA 1.0 1TTEPA 1.0 2TTEPA 1.0 3

Untreated controlsTTEPA 1.0 2TTEPA 1.0 3TTEPA 2.0 1

Untreated controls* AT = Amino-teropterin; TTEPA = ThioTEPA.

f Peritoneum was punctured in each mouse at four sites (upper and lower abdomen on the right and the leftsides) 24 hours before inoculation and once (left middle abdomen) by the inoculation; range given in parentheses.

ÎSize of growth at the sites of inoculation: 0 = absence of growth; + = nodule 1 or 2 mm. in diameter;++ = 3-5 mm; H—h+ = larger than H—KRange given in parentheses.

4(2-5)3(2-5)3(2-5)5(4-5)2(1-3)

2(1-3)2(1-3)5(3-5)2(1-5)

1(0-3)1(0-1)5(4-5)1(0-1)

01(0-1)5(2-5)++(++-+

+ +)+(0-++)+(o-++)

+++(+H —¿�H-H+(o-++)+

(0-++)+(o-+)

+++(++-++-+(+-++)+(0-++)+(o-++)+++(++-++-+(o-+)00(0-+)++(+-+++)

SERIESor

MICE

1A

IB1C2A2BCC

TABLE 4TUMORGROWTHIN THESCALPAFTERTISSUEINFILTRATIONWITHAMINO-TEROPTERINORTHIOTEPA

Twenty mice in each series, all inoculated with Sarcoma 180.

AGENTAmino-terop

terin

TTEPA

DOSE No.(mg/kg) raj.

.5 2

.5 3Untreated controls

1.0 21.0 3

Untreated controls

NO. MICE SURVIVING*

20 days15

1214201817

30 days3

31

1680

AMOUNT OF GROWTH AFTER 20 DATS*t

100

14ITo

10

616

22

14* Number of mice in each group is indicated.f —¿�= no growth;-)- = top of the scalp rounded by growth; H—h= tumor extending from the forehead to the neck;

tumor overgrowth on the face and the back.

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GoLDiE et al.—Tumor Growth and Implantation 379

illustrates the incidence of peritoneal tumorgrowth in new mice after intraperitoneal grafts ofthe blood and the organs from treated and untreated tumor-bearing mice (controls). The number of mice in control groups A6 and T6 was twicethe number of experimental mice, in order to outline more consistently the trend in treated groups.The treatment was given subcutaneously, since itwas intended to influence tumor cells scattered inthe blood and the organs and not the primary intraperitoneal growth.

In control series A6 and T6 tumor growth wasobtained from 50-60 per cent of blood and lunggrafts and from 100 per cent of liver grafts. A

treated and untreated mice or rats. We have attempted to supplement the classic methods of biological assay on tumors by some additional methods based on the phenomena of cell growth andcell spread in a malignant condition known as"ascites tumor."

The most specific phenomenon in that conditionis the occurrence of free tumor cells rapidly proliferating for numerous generations in the asciticfluid. This free growth offers an easier approachthan subcutaneous tumors for testing the directand irreversible effect of an agent on the viabilityof tumor cells; the agent introduced in a solutionor in a homogenous suspension into the peritoneal

TABLE 5

INCIDENCEOFINTRAPERITONEALTUMORGROWTHIN MICE RECIPIENTSOFORGANGRAFTSFROMTREATEDANDUNTREATED(CONTROLS)

TUMOR-BEARINGMICE(DONORS)

SEMESOF

MU K

Al

A2ASA4A5Â6TATBTCTDTETF

TREATMENTorDONORSDoseit

(nw/kg)terop-.5.5.5No.inj.123

INCIDENCEOF TUMORGROWTHIN RECIPIENTSAFTER

GRAFTSOF!Blood Liver Lung

456terin

.5; 1.0.5

Untreated controls

ThioTEPA 1.01.01.02.0

1.0; 2.0Untreated controls

0000

10

59464

12

6351

M1010•¿�89

80

4t4ft

12

4854*

11Ten mice in each experimental series and twenty mice in each group of untreated controls.

All animals were given inoculations of 10X10* cells of S-S7 intraperitoneally and treated bysubcutaneous injections. Organ grafts into recipients were carried out on the Sth day afterinoculation of donors.

single dose of Amino-teropterin reduced the incidence of the latter to 50 per cent but did not influence results from other grafts (series Al). However, after repeated injections of Amino-teropterininto donors, their blood did in no instance producetumor growth in recipients. Positive results of liverand lung grafts were significantly more scarceafter three injections (A3 and AS) and slightly morescarce after two injections (A2 and A6).

In thioTEPA experiments the results in treatedseries varied only slightly (TA to TE) from thedata in untreated controls (TF).

DISCUSSIONSince the earliest attempts in experimental

chemotherapy of malignant tumors (25, 30), thetest methods have consisted of a comparison of thesize or weight of subcutaneous (or intramuscular)tumors or of the survival span of their hosts in

cavity filled with ascitic fluid is brought into directcontact with all floating tumor cells; their growthwithout organization in tissue, i.e., without stromaformation and blood supply, implies only one environmental factor—a body fluid. In our studieson radioisotopes introduced intraperitoneally intomice with Sarcoma 37 ascites tumor (9, 10), wehave demonstrated the direct effect of radioactiveagent on tumor cells by two methods: (a) morphological examination, revealing distortion of the cellstructure and in particular of mitoses; (6) biologicaltest, illustrating nonviability of tumor cells by thefailure of their growth after transfer into new mice.The final effect of the treatment, i.e., total disappearance of tumor cells, was interpreted by theresults of these tests.

In the above reported experiments with Amino-teropterin and thioTEPA, the tumor cells nearlydisappeared from the ascitic fluid after intraperi-

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380 Cancer Research

toneal treatment but not after subcutaneous injections. This difference suggests the topical effectof the agent on ascitic tumor cells.

The decrease in tumor cell concentration wasparalleled by the decrease in the amount of asciticfluid which could be attributed to a decrease in thenumber of implanting and exudation-eliciting tumor cells. This is in agreement with our data(Tables 1 and 2) on the reduced amount or evenabsence of implantation in treated animals.

Rous and Jones (24) have shown that areas ofperitoneal serosa damaged by kieselguhr offered afavorable ground for the implantation of intra-peritoneally inoculated tumor cells. Our methodof induced implantation is an application of thesame principle for quantitative test. In untreatedcontrols the implantation occurred, with a few exceptions, at all sites of punctures in the abdominalwall and developed into extensive tissue growth(Figs. 4 and 7). In both Amino-teropterin andthioTEPA groups, the amount of implant growthwas smaller than in controls (Figs. 5, 6, 10, and11); this may be attributed to a decrease in thenumber of free tumor cells capable of implantation. However, in thioTEPA experiments the incidence of takes in puncture areas was also considerably decreased (Series 3-5, Table 3; Fig. 7),and these results suggest an additional effect inherent to thioTEPA only, on tumor cell implantation. It may be presumed that thioTEPA affectsthe capacity of free tumor cells to implant or theirviability after infiltration into tissue. While thefirst interpretation cannot be excluded, the secondhypothesis is supported by the data of our experiments on inoculation of tumor cell suspension inascitic fluid into loose connective tissue of thescalp followed by topical treatment. The growthand spread of scalp tumors were inhibited bythioTEPA, and this topical effect was paralleledby the decrease of mortality. Since a similar parallel between the reduction of tumor growth and theincrease of survival span was found for thioTEPA-treated peritoneal growth (Table 2), it may beconcluded that protection by thioTEPA againstdeath is associated with the effect on primary tumor growth in the scalp and prevention of its implantation and spread.

Treatment with Amino-teropterin only delayedthe growth of scalp tumors without influencing themortality of their hosts. These results suggest thatamino-teropterin does not act on tumor cells infiltrated into tissue, and this is in agreement withour data on spontaneous and induced implantationin Amino-teropterin-treated mice.

Results of organ grafts from treated tumor-bearing animals indicated a moderate decrease in

the number of metastatic tumor cells transportedby blood and intercepted by organs. This effectwas obtained with both compounds; thus, it couldbe attributed to reduction of the primary asciticgrowth. However, only the blood from the Amino-teropterin-treated group was free from detectabletumor cells in almost 100 per cent of mice. Thesefindings suggested a specific effect of Amino-teropterin on tumor cells circulating in the blood.The effect is due perhaps to the longer persistenceof Amino-teropterin in the blood; it may be reminiscent of the specific effect of this compound inconditions essentially characterized by the occurrence of free tumor cells in the blood—the leuke-mias. It would not be unreasonable to test Amino-teropterin for its action on tumor cells circulating inthe blood of human patients. On the other hand,the inhibitory effect of thioTEPA on tumor cellsscattered in tissues suggests its use for preventionof the growth and spread of tumor cells spilledduring operation (19) or invading healthy tissueon tumor periphery.

SUMMARY1. Amino-teropterin in doses of 0.5 or 1 mg/kg

and thioTEPA (triethylenethiophosphoramide) indoses of 1 or 2 mg/kg, single or repeated on 2 or 3successive days, showed their cytotoxic activity byconsistently decreasing free tumor cell concentration (S-S7, S-180,6C3HED) in ascitic fluid and theamount of exudation. The intraperitoneal route oftreatment was considerably more efficient than thesubcutaneous.

2. Both the spontaneous implantation of freetumor cells from ascitic fluid (small or large tumorsin visceral peritoneum) and their induced implants (nodules at the sites of punctures in the abdominal wall) were reduced by thioTEPA morethan by Amino-teropterin. The effect was interpreted as the indirect result of a decrease in thenumber of free cells capable of implantation and,moreover, as the specific ability of thioTEPA toact on tumor cells in tissues. This hypothesis wassupported by the evidence of growth and spreadinhibition in scalp tumors treated topically withthioTEPA.

3. Organs and blood from treated and untreated ascites tumor-bearing mice were grafted intra-peritoneally into new mice for the detection ofmetastatic tumor cells. The incidence of growthfrom the grafts was consistently lower in treatedanimals, and this was attributed to the decrease ofcells in the primary growth. However, the absenceof viable tumor cells in the blood of animals treatedwith Amino-teropterin was considered as the immediate effect of this agent.

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FIGS. 1-3.—Sarcoma 37 implants in untreated (controls)and treated Swiss albino mice sacrificed 7 days after inoculation.

FIG. 1.—Untreated controls. Symmetrical implants in thearea of uterus body, both horns, and ovaries.

FIG. 2.—Four days after two daily injections of Amino-teropterin, 0.5 mg/kg. Small implants in the same area.

FIG. 3.—Fourdays after two daily injections of ThioTEPA,1 mg/kg. No implants.

FIGS. 4-6.—Implantation and growth of free tumor cellsfrom peritoneal exúdateat sites of five punctures in abdominalwall, 7 days after inoculation of 10 X 10aSarcoma 37 cells.

FIG. 4.—Untreated controls. Growth of five large nodules(+++, see legend to Table 3).

FIG. 5.—Micetreated on 2 successive days (2d and 3d afterinoculation) with 0.5 mg/kg of Amino-teropterin. One middle-

sized (++) and two small (+) nodules.FIG. 6.—Micetreated with two doses (1 mg/kg) of Thio

TEPA—no growth.FIGS.7-11.—Sizeand structure of nodules grown at sites of

punctures.FIG. 7.—Part of a large nodule, H & E, X120.FIGS. 8, 9.—Middle-sized (++) nodules, H & E, X29.FIGS. 10, 11.—Smallnodules.Small or middle-sized nodules occurred as a rule in treated

animals and showed two narrow light zones enclosing a widedark central zone of tumor tissue growth (Figs. 8-11). Theperipheral light zone = infiltration of peritoneal serosa with freecells. The distal light zone = abdominal muscle partly infiltrated with tumor cells. Large nodules consisting nearly entirely of tumor tissue, with only traces of muscle elements(Fig. 7).

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GoLDiE et al.—Tumor Growth and Implantation381

4. It was concluded that the use of variousmethods for testing the effect of a compound onvarious activities of tumor cells may help to outline its indications and the ways of application.

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1957;17:374-381. Cancer Res   Horace Goldie, Matthew Walker, Alvin R. Graham, et al.   Implantation of Free Tumor Cells from Ascites TumorsEffect of Amino-teropterin and of ThioTEPA on Growth and

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