Observations on a Liver Mitotic Stimulant

It is well recognized that the presence of a

cancerous growth results in certain pathologicchanges in host tissues anatomically removedfrom, and not involved by, the neoplastic process.These systemic alterations in the host includesuch changes as depression of hepatic catalaseactivity (7, 9, 17), anemia (fl), thyinic involution(W), and lymphoid hyperplasia (is, @0). There

are also reports indicating the loss of ascorbic acidand cholesterol (iO, 19), histologic changes (5),and possible hypofunction associated with adrenalgland enlargement. More recently, Annau et al. (1)have reported an increased mitotic rate in thelivers of tumor-bearing rats and mice.

It has been postulated by some that these host

changes result primarily from the excessive uptakeof some nutritional element(s) by the tumor (14),and this concept appears to be substantiated inpart by a number of nutritional and metabolicstudies in tumor-bearing hosts (6, 15, 16, @3,@,4).

On the other hand, there appears to be equallyvalid evidence to support the postulate thatsystemic changes in the host result from theelaboration of some substance(s) by the tumor(@,7,8, @1).

In vim these various systemic changes are mostprobably related and to a certain extent dependentupon each other. More striking than their possiblein viva relationships, however, is their mutual dependence upon the presence of a tumor for theirinitiation.

The following study is an attempt to exploremore fully certain facets of this tumor-host relationship. Since increased mitotic activity is soclosely associated with the malignant process itself, investigation of this aspect of the problemseemed particularly interesting, and the presentexperiments were designed primarily to study thetumor properties which result in an increasedmitotic rate in the liver tissue of cancerousanimals.

MATERIALS AND METHODS

Male and female strain CSH/He and CSHf/Hemice, @,—8months old, were used as both donorand host animals. The tumor used throughout the

experiments was the transplantable mammarytumor CSHBA. Both donor and host animalswere killed by exsanguination, and all the procedures, except the 6-hour and i@-hour time interval studies, were carried out between 9:00 AM.and 11 :00 A.M. Normal mice of comparable strain,sex, and age, killed and examined in the same manncr as the treated groups, served as controls.

In all cases liver tissue for microscopic examination was taken from the left lobe of the liver,placed immediately in 10 per cent formalin fixative, imbedded in paraffin, sectioned at 5 j@,and stained lightly with hematoxylin and eosin.The microscopic procedure consisted of examining

@5different fields at a magnification of 430X. The

total number of mitoses observed in the parenchymal cells in these @5fields was considered themitotic index of the specimen.

The first experimental group consisted of anumber of mice implanted subcutaneously with0.1 ml. of mammary tumor CSHBA. Theseanimals were kified, and their livers examined aspreviously described when their tumors attainedsizes ranging from 0.3 gm. to 9.9 gm.

For other test series, tissues removed from thedonor animals were prepared and injected in thefollowing manner:

Brei.—Tumor brei was prepared by passing the tissuethrough a tissue press, sieve size, 0.6 mm. Four-tenths ml. ofthe resulting tissue brei was injected subcutaneously in theback of each recipient. For comparison, separate homogenatesof normal tissues (liver, kidney, and embryo) were also pro..pared and injected in the same manner. The recipient animalswere sacrificedat 12, 24, 48, 72, 96, and 120hours after injection for determination of the liver mitotic index.

In another test series tumor brei prepared in the same manner was mixed with various amounts of 0.85 per cent salinesolution togive concentrations ofSO, 25, 12.5, and 6.25 percent.Allthe testanimals receiving 0.4 ml. of the various brei concentrations (100 percent thru 6.25 percent) were killed at 48 hoursand examined for mitotic activity in the liver.

Ti.auefractions.—The saline supernatant fraction of tumorsweighing 2—Sgm. was prepared by homogenization for 2 minutes with an equal volume of chilled 0.85 per cent sterile salinesolution in a chilled Waring Blendor. The brei was centrifugedat 20,000 X g for 10 minutes at 5°C. A plug of sterile cottonwas placed over the surface of the brei before centrifugationto help carry down the cells and cellular debris. The resultingsupernatant was recentrifuged at the same speed for the samelength of time, and 1 ml. of the resulting supernatant was injected subcutaneously into the back of each host animal. TheReceived for publication October 10, 1955.

Present in Tumor Tissue

RICHARD A. MALMGREN

(National Cancer Inaiitute, and Department of Pathology and Microbiology, Unirerdiy of Tennessee, MernpM@t, Term.)

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MALMGREN—Liver Mitoth, Stimulant in Tumor Tissue @3s

supernatant was examined microscopically for intact cells, anda numberdaniznals receivinginjections of 1 ml. ofthe supernatent were observed for 60 days for tumor incidence.

The nueleoprotein fraction, isolated by the streptomycintechnic (1$), and the nuclear, mitochondrial, and microsomalfractions, isolated by the sucrose method (11), were also prepared from C8RBA mammary tumor tissue. Each host animalreceived 1.0 ml. of one of the tissue fractions by subcutaneousinjection (equivalent to 0.4 cc. of original tissue). All the animals treated with the various tissue fractions were killed at 48hours for determination of the liver mitotic index.

In a final study, 1.0 ml. of the saline extract previously described was injected subcutaneously into the host animals,whichwere killed for liver examination on the 1st through the4th day after injection.

RESULTS

Mitotic counts done on sections of liver tissueobtained from normal adult strain C3H/He andCSHf/He male and female mice revealed that livercell mitoses were extremely rare. In contrast,numerous mitotic figures were observed on theliver sections obtained from strain CSH/He andCSllf/He male and female mice bearing transplanted mammary carcinoma C3HBA. The correlation between tumor size and the number ofmitotic figures observed is shown in Table 1.

TABLE 1

RELATI0NSmPBETWEENTUMORS@z@AND LIVER MrroTlc INDEX

in @4hours (mean value, 1.6). A marked rise inthe liver mitotic index to a mean value of 18 occurred in 48 hours and was followed by a gradualdecline to a mean value of 5S2 from the @dthroughthe oth day after injection. The P value of thecomparison between the @4-and 48-hour mitoticindices was less than 0.01. In contrast, the subcutaneous injection of a similar amount of normaltissue brei (liver or kidney or embryo) did not produce a significant increase in the liver mitoticindex of the treated mice at 48 and 7@ hours(Table @).

Since these results indicated that the observedhost liver mitotic response was elicited only by

TABLE 2

EFFECT OF THE SUBCUTANEOUS INJECTION OF 0.4 ML.

OF TISSUE BREI ON LivER MzTosis AT

VARIOUS TIME INTERVALS

Av. mitoticHost liver mitotic index index

1,0,0,0,8,3,2,434,85,4,44,21,66,81,l,5,4,6,l,1,11,34,0,0,0,0,6,1I,I1,12,l3,172,1,O,0,1,5,4,3,15,8,183,0,2,0,0,0,8,4,2,8,9

1,1,0,0,0,0,0,0,0,0,0,1,8,0,0,0,0,0,00,1,1,1,0,0,o,l,0,0,01,2,0,8,0,0,0,4,1,0,0

Kidney:2d day 0,2,0,0,0,0,0,0,0,0,0,0,0

Embryo:2@1day 0,0,0,0,0,0,1,0,0,1,0,0,0

tumor tissue and was due primarily to the presencerather than the establishment and growth of themalignant cells, the next experimental step wasdesigned to determine whether the injection ofvarious concentrations of tumor brei would resultin a concomitant variation in the number ofmitotic figures in the host liver. Forthis purpose,0.4 ml. of a mammary tumor brei diluted withnormal saline solution to concentrations of 50, @S,1@.5, and 6.@5 per cent was injected subcutaneous

ly into mice which were killed 48 hours after injection. Examination of the liver sections revealeda positive correlation between the concentrationof tumor brei and the number of mitotic figuresobserved in the liver, ranging from a mean valueof 3.1 mitoses with a P value of less than 0.01 atthe brei concentration of 6.@5 per cent to a meanvalue of 18.0 mitoses at a brei concentration of100 per cent (Table 3).

Another group of animals was given injectionsof various fractions of tumor tissue in an attemptto determine whether the liver mitosis stimulatingfactor was associated with a particular cellularcomponent. This test group was also killed at 48

Tissueinjected

Tumor:1st day2dday3d day4th day5th day

Liver:2dday3d day5th day

1.618.0

7.95.12.9

0.310.861.0

0.14

0.14

Tumor sizeAv.mitotic(gm.)Hostliver initoticindexindex0—I0,6,3,16,18,1910.31—226,24,2,16,26,40,24,4,9,10,416.82—S21,80,21,24,18,5,41,112,44,36,20,381.23—411,4,14,12,8,0,06.24—S0,0,2,1,0,203.75—60,0,0,2,0,00.36—70,4,01.37—80,0,008—91,00.69—1000

Tumors weighing 0—1.0gm. are capable of eliciting this liver cell response. Their effectiveness,10.3 mitotic figures per specimen, was, however,considerably less than that of @.0—8.0-gm. tumors,which resulted in a mean liver mitotic index of31.@ with a P value of less than 0.01. This abilityto increase the liver mitotic index appeared todecline gradually after the tumors reached aweight of 8 gm. or more, since the mean mitoticindex for tumors weighing 5—9.9gm. was approximately 0.@6.

The next series of tests was designed primarilyto determine whether established tumor growth,or simply the presence of tumor tissue, was implicated in this host response. Following the injection of 0.4 ml. of a mammary tumor brei, examination of liver specimens from the treated recipientsobtained 1 through S days after treatment revealed a slight increase in the liver mitotic index

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Cancer Research@234

hours for liver cell mitotic counts. As shown inTable 4, the saline extract fraction, which wasmarkedly effective in stimulating host liver cellmitosis, resulted in a mean mitotic value of 1@.6,which was significantly greater than the effectiveness of any of the other fractions at a P value ofless than 0.01. By comparison, the nuclear, nucleoprotein, mitochondrial, and microsomal fractionsdid not significantly stimulate cell division in thehost liver, and the mean mitotic value of 0.3 toi.o for these fractions was approximately equal to

TABLE 3

MIT0TIc INDEX IN THE HosT LiVER 48 HouRs AFTERTHE SUBCUTANEOUS INJECTION OF

0.4 ML. OF TUMOR BRE!

value of the liver mitotic index at 48 hours. However, it would appear that the saline extract formof the stimulant resulted in a slightly greater increase in the liver mitotic rate in @4hours and amore precipitous decline in effectiveness after 48hours (Table 5) than did the injection of tumorbrei (Table @).

On microscopic examination of liver sectionsobtained from all the test animals bearing tumorsweighing 3 gm. or less in the various experimentalseries, parenchymal cells in all stages of the mitotic

cycle were observed. Cell division was confined tothe parenchymal cells, and no atypical mitoseswere seen. This mitotic activity was not confinedto any single anatomical portion of the liver lobeexamined, although there appeared to be a greaterfrequency of mitotic figures around the portalareas. In all other respects the liver appearednormal. Microscopic examination of other organsof the tumor-bearing host, including brain,adrenal, pituitary, kidney, and salivary gland,failed to reveal any mitotic activity.

The liver sections obtained from animals bearing larger tumors (5 gm. and over) rarely showedmitotic activity but were remarkable because ofthe marked cellular infiltrate observed in thesinusoids and portal areas. This infiltrate was corn

TABLE 5

LIVER MIT0TIc INDEX AT VARIOUS TIME INTERVALSAFTER THE SUBCUTANEOUS INJECTION OF 1.0 ML.

OF A SALINE EXTRACT OP TUMOR TisstE

Time after Av. mitoticinjection Host liver mitotic index index

6 hr. 0,0,0,1,1l2hr. 0,0,1,1

1 day 18,1,2,4,8,0,2,2,0,0,0,02 days 14,4,3,1,26,10,20,14,6,24,0,0,l,12,.5,

40,8,393 days 0,0,0,0,0,3,0,0,1,O,0,0,04 days 0,0,0,0,0,0,0,0

posed primarily of megakariocytes, cells of therubricytic series, and numerous lymphocytes andpolymorphonuclear leukocytes. The extrarnedullary hematopoiesis, which was uniformly presentin the livers of animals bearing tumors of 5 gm.or larger, was not observed in normal mice or inmice bearing tumors weighing 4 gm. or less.

DISCUSSION

It is well recognized that liver tissue, which hasthe capacity for regeneration, rarely shows mitoticactivity in the normal adult animal (3). As summarized by Paget, the proliferative mechanism ofliver tissue has also been stimulated in intactadult animals by the systemic administration ofvarious agents (i8). Some of the reports concern

Percentbreiinjected

6.2512.525.050.0

100.0

Fractioninjected

MitochondriaMicrosomesNucleoproteinNuclearSaline extract

Normal tissue(liver)

Untreatedcontrols

Av. mitoticindex

3.1‘5.7

10.312.418.0

Av. mitoticindex

0.60.580.731.0

12.6

0.31

Host liver mitotic index

5,3,6,4,3,4,2,2,2,03,11,3,2,8,6,6,8,9,16,12,18,9,6,18,4,10,7,1310,25,12,12,18,9,18,7,4,l034,35,4,44,21,65,31,1,5,4,6,1,1,1

TABLE 4

EFFECT OF INJECTION OF VARious FRACTIONS OF

TUMORS ON HosT LIvER MIT0TIc INDEX48 HOURSAFTERINJECTION

Host liver mitotic index0,0,0,1,2,1,0,0,0,0,4,00,0,2,0,1,0,0,1,0,0,8,04,1,1,0,1,0,3,1,0,0,O,0,0,0,0l,1,1,0,0,0,8,0,0,1,51,3,4,14,26,10,20,14,6,24,0,0,1,

12,5,40,8,390,l,1,0,0,0,0,0,0,0,1,3,0,0,0,0,0,0,0

0.40.53.012.6

0.30

0,4,0,0,2,3,0,1,0,4,0,0,O,0,0,1,0,0,0,0.470,0,0,0,1,1,0,0,o,0,o,0,0,0,0

the mean mitotic value observed in mice receivingnormal liver tissue and in untreated control mice.Microscopic examination of the saline extractrarely revealed intact tumor cells, and only @5percent (ten out of 40) of the animals treated withthis saline extract and observed for 60 days developed tumors. In contrast, 100 per cent (40 outof 40) mice treated with a O.i per cent tumor breideveloped tumors.

In the last test group, mice receiving subcutaneous injections of 1.0 ml. of a saline extract oftumor tissue were killed at daily intervals on theist through the 5th day after treatment to determine whether the host liver mitotic responsewould be influenced differently by the extract ascompared with the brei form of the mitotic stimulant. As previously observed, the injection ofequivalent amounts of saline extract and tumorbrei resulted in a maximum increase in the mean

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MALMGREN—LiVer JIiioth@ Stimulant in Tumor Tissue @235

ing mitotic activity in the regenerating liverindicate that the factors important in inciting theincreased rate of cell division are humoral (4).Therefore, the observation that the presence ofmalignant tissues anatomically remote from thehost liver also results in an increase in the hostliver mitotic index suggests the interesting possibility that certain humoral factors capable ofstimulating liver cell division are associated withneoplastic growth.

As shown by Annau et a!. (1) with a variety oftumors in both rats and mice, an increase in thehost liver mitotic index was associated with thepresence of a malignant growth. When the presentstudy, with the transplanted mammary tumorC3HBA in strain CSH/He and C311f/He mice, con

firmed Annau's report, further investigation was directed primarily toward characterizing the properties associated with malignancy which were responsible for initiating the increase in the hostliver mitotic index.

First, consideration was given to the possibilitythat a correlation between tumor size and the hostliver mitotic index might be demonstrable if thisphenomenon were initiated by the factors implicated in either of the two hypotheses previouslyadvanced to explain the systemic alterationsoccurring in cancer-bearing animals; i.e., tumoruptake of nutritional elements or tumor elaboration of some unique substance. It seemed likelythat increasing tumor size would result in bothincreasing tumor uptake and tumor output andthat either of these forms of stimuli might be refiected by an increased rate of host liver celldivision. Consequently, the positive correlationbetween host liver mitotic rate and tumor sizeobserved for tumors weighing less than 3 gui.might be interpreted as a direct quantitative relationship between the tumor stimulus and the livercell response. The absence of this positive correlation in hosts bearing tumors of 3 gm. or larger,however, suggested that this interpretation wasperhaps incomplete and that additional and complex factors were involved in the in vivo tumorhost relationship.

In animals bearing large tumors, the absenceof liver cell mitosis and the appearance of markedextramedullary hematopoietic activity followingthe fall in the mitotic rate raises the interestingquestion whether these two host liver changes arerelated.

The mitotic figures observed in the livers of allthe test animals were in all stages of the mitoticcycle, indicating that the tumor acted as a mitoticstimulant rather than as a mitotic inhibitor, notonly because mitosis in the normal adult liver is

rare, but also because the evidence for mitoticinhibition is generally arrest at a particular mitoticphase.

On the basis of the time-dose studies, whichdemonstrated that the maximum increase in thehost liver mitotic index occurred 48 hours afterthe injection of a small amount of tumor brei andwas roughly proportional to the concentration of

the brei, it seemed apparent that the factors important in the liver cell response were transferredwith the tumor tissue. It was of particular interestto note that the brei derived from the largertumors, which were no longer capable of elicitingan increase in liver cell mitosis in their own hosts,was as effective as the brei from the smaller tumorsin stimulating the recipient's liver mitotic rate

48 hours after injection, further implicating perhaps the complex in vivo mechanism associatedwith this tumor material and its role in the apparent inactivity of tumors larger than 3 gui.Since vascularization and growth of the implantwere undoubtedly at a minimum during the first

@4-48hours, the period of maximum host livermitotic response, it also seemed likely that thismitotic stimulant was contained by the tumorcells at the time of injection and did not resultfrom the proliferative process of malignant tissue.The observation that the host liver mitotic indexdeclined significantly after reaching a maximumresponse at 48 hours and did not increase again until the tumor became established in the new hostseemed to indicate further that the initial mitoticstimulus resulted from some factor present in thetumor brei at the time of injection. The failure ofinjections of equivalent amounts of normal tissuebrei (liver, kidney, embryo) to elicit an increase inthe host liver mitotic index indicated that thisstimulating factor was a unique property of themalignant tissue.

As shown by comparisons of the liver mitosisstimulating ability of various morphologic cellfractions of the tumor tissue, the active factorswere almost exclusively confined to the salineextract portion. Since the cell content of thissaline extract was minimal and far below thatdetermined to be effective in the tumor brei concentration studies, it would suggest that this livermitotic stimulant was independent of the intacttumor cell. A comparison of the time intervalnecessary to achieve the maximum liver mitoticresponse in animals treated with the cell-freed(extract) as compared with the cell-bound (brei)form of the liver mitotic stimulant also revealedthat the saline extract form resulted in a slightlymore rapid onset and decline in effectiveness, suggesting that prior release from the tumor tissue

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Cancer Research@S6

accelerated the rate of action of the liver mitoticstimulant.

Studies on the process of mitosis itself have revealed that it is an extremely complex process,and the in vivo events initiated by the stimulantderived from tumors which culminated in an increase in the host liver mitotic index can, at best,only be postulated. That these events did, however, result from a unique tumor product, andwere not necessarily associated with the presenceand growth of tumor cells, has been determinedwith one tumor in one strain of mice. Annau'sobservation that an increase in the liver mitotic

. index resulted from the presence of a variety of

tumors in two species of cancer-bearing hosts and alimited number of observations in this laboratoryindicating that liver mitosis-stimulating factorssimilar to those demonstrated for mammarycarcinoma C8HBA are associated with sarcomaHE 8971 suggest that the active factors importantin producing this increase in the liver mitotic indexof cancer-bearing hosts are common to a numberof tumors.

SUMMARY

1. An increase in the liver mitotic index and arelationship . between tumor size and the livermitotic index was observed in strains CSH/He andCSHf/He mice bearing transplanted mammarycarcinoma CSIIBA.

@.A significant increase in the host liver

mitotic index was also observed 48 hours after theinjection of 0.4 ml. of CSHBA mammary tumorbrei. The number of mitotic figures in the hostliver increased as the concentration of tumor breiinjected was increased.

3, A saline extract of C3HBA mammary tumor

brei also resulted in a significant increase in thehost liver mitotic index at 48 hours. No increasein the host liver mitotic index was observed following the injection of the nuclear, nucleoprotein,mitochondria, and microsome cell fractions obtained from CSHBA mammary tumor tissue.

4. The subcutaneous injection of 0.4 ml. normal

C3H/He and CSHf/He tissue brei (liver, kidney, embryo) into normal strain CSH/He andC3Hf/He mice did not result in an increase in thehost liver mitotic index 1 through 3 days after injection.

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1956;16:232-236. Cancer Res   Richard A. Malmgren  TissueObservations on a Liver Mitotic Stimulant Present in Tumor

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