The Cancer Cell in Vitro: A Review · Some of Alexis Car rel's earliest experiments also included attempts to culture cancer tissues and compare their behavior with that of normal

The Cancer Cell in Vitro: A Review

J.PAUL

(Biochemistry Department, Glasgow Univervity, Glasgow, Scotland)

Cancer is clearly the result of a defect of thehomeostatic mechanisms which maintain the balance among cells in an organism. Since the natureof these mechanisms is not understood it is not surprising that nothing is known of the lesion which

causes the disease. Recent research indicates that,

whereas the reaction of the host may contribute tothe establishment and growth of a cancer, there isno doubt that the primary lesion occurs in the cell.The tissue culture method has therefore been employed to study the cell lesion in isolation fromthe host.

The implications of tissue culture in cancerresearch were appreciated very early, and in 1906Beebe and Ewing (7) attempted to grow an â€œinfective canine lymphosarcomaâ€• in the blood of infected and uninfected dogs. Some of Alexis Carrel's earliest experiments also included attempts toculture cancer tissues and compare their behaviorwith that of normal tissues. For technical reasonslittle of this early work provided useful information, and perhaps the earliest significant observation was Carrel's (11) finding in 1926 that the Roussarcoma virus would survive in embryo chicktissues in vitro. In the following years each successive theory made its impact on cancer studies intissue culture. Thus, Earle (@,3,24) and his colleagues initiated investigations into the effects ofchemical carcinogens on cultured animal tissue,while Warburg's (92) hypothesis that cancer wasdue to a lesion of the respiratory enzymes in thecell was tested by Goldblatt and Cameron (34),and recently the virus theory has commandedmost interest.

The literature describing the use of tissue culture in cancer research has already been admirablyreviewed in detail up to the year 1958 (48, 49, 63,64). The object of this article is to consider thepresent situation critically and to suggest someconclusions which may be drawn. Only those re

ports which are relevant to the discussion are mentioned, and recent review papers are referred towhere possible.

Received for publication September 25, 1961.

GENERAL BEHAVIOR OF CULTUREDNORMAL AND CANCER TISSUE

Organized and unorganized growth.â€”Certaincommonplace phenomena of cultured tissues areprobably relevant to the cancer problem.

In the first place, as is well known, if fragmentsof tissue are explanted they immediately tend tobecome disorganized as a result of cell migrationwhich follows a definite pattern. Cells of the reticuloendothelial system, being most highly motile,escape first, but they rarely exhibit mitosis. Awave of migration of fibroblasts follows, and thesecells begin to multiply as soon as they leave theexplant. If epithelial cells are present they migratelater as a sheet, and mitosis may also commence inthem. Finally, many of the highly specialized cellshardly migrate at all and may be recognized withinthe thinned-out explant. When pieces of tissuehave become disorganized in this manner theemigrating cells show little tendency to react with

one another. Cells from adjacent explants willmigrate toward one another and will intermingleintimately whether they are from the same ordifferent organs.

In contrast, if tissue architecture is maintainedby careful manipulation and migration is discouraged, as is done in organ culture, then thereis no tendency to become disorganized, and

continuation of normal function can be convincingly demonstrated (26, 81, 96).

These simple observations themselves speak forsome kind of homeostatic mechanism at the tissuelevel, and may be significant, as will be discussedlater, that cancer cells, unlike normal cells, mayexhibit infiltration in organized cultures.

Primary explants.â€”It is now clear that cellsgrowing out from primary explants do not undergoany radical dedifferentiation. Cells in the migrating zone tend to preserve something of their characteristic morphology, and in some instances thisis of use in classifying and diagnosing tumors. For

instance, Murray and Stout (66) have shown thatan early diagnosis of sympathicoblastoma can bemade by tissue culture, since the neuroblasts send

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432 Cancer Research Vol. @22,May 1962

out neurites which can often be seen in @4hoursand are almost always recognizable in 48 hours(Fig. 1). The same investigators employed tissueculture to help define the cellular origin of certaintumors such as the hemangiopericytoma (glomustumor) (Fig. @),liposarcoma, and rhabdomyosarcoma.

Apart from these specific morphological features there seem to be no general properties whichdistinguish normal cells from tumor cells in primary explants, with one possible exception. Thisconcerns the phenomenon of surface inhibition(2), which consists of a momentary cessation ofsurface movement when two normal fibroblasts

CHART 1.â€”The development of aneuploidy and malignancy(tested by reinoculation) during the â€œtransformationâ€• of aculture of normal embryonic mouse skin cells. From Levan(52) after Levan and Biesele (58). (Reproduced by courtesyof Dr. Levan.)

come into contact. Abercrombie and Ambrose (1)found that it was not displayed by Sarcoma 37 orSarcoma 180 cells, either toward similar cells ortoward normal cells cultured simultaneously. Inview of the variation in behavior among similarcells, it might be premature to draw any generalconclusions from these observations, but they areof such importance that they clearly merit repetition and extension to other cell types.

It should be emphasized that the belief thatcancer tissues grow more readily in culture thando normal tissues is a fallacy. In fact, Gey (32),from his extensive experience, indicates that theopposite is often the case.

â€œTransforma/ion.â€•â€”The emergence of a@ cell

strain from a primary culture may occur withoutany change in growth pattern, but this course of

events is rather exceptional. More usually, after aninitial period of rapid growth the rate of multiplication decreases, and the cells enter a more or lessstationary state, when their survival may be indoubt. Most cultures then die out, but at sometime during this period there may be a sudden outburst of growth at several places in the culture.This appearance of a rapidly growing strain ofcells from an otherwise slowly growing culture isreferred to as â€œtransformation.â€•It is of particularinterest in a discussion of the cancer problem, sinceit is associated with changes which resemblemalignant changes.

Several mechanisms have been considered(apart from the simulation of transformation by

accidental contamination of a primary culturewith an established strain). It probably involvesthe selection of cells which are particularly suitedto the environment. These favored cells may already be present at the time of isolation, or theymay arise in some cases as a result of mutations orthe redistribution of genetic material. Again, it hasbeen suggested that the period of delay may

@â€˜ simply be necessary for the induction of some

enzymes. Whatever the cause it is the manner in2' which a cell strain usually arises, and the strain:@ which results will usually grow indefinitely in cul

ture if properly handled.Cell strains.â€”Established cell strains resemble

one another in many respects, as one might expectsince the conditions of cell culture are highlyselective. Nevertheless, three general morpho

logical types are readily distinguishedâ€”namely,fibroblastic, epithelial, and lymphocytic; and thepractised observer can often recognize further differences among individual lines. The principalpoints of resemblance are similarities in growthrates and nutritional requirements (19) , thecapacity to grow in relatively anaerobic conditionswith a high rate of aerobic glycolysis, and thetendency to develop unusual chromosomal complements.

Of these perhaps the most interesting is thetendency to develop chromosomal abnormalities.This has been studied in detail by Levan andBiesele (53) and Hsu and Klatt (40), and theyhave pointed out that the first tendency is fortetraploid cells to accumulate in the population(Chart 1). Later cells with a chromosome numberbetween diploid and tetraploid appear, andeventually cells with a typical number of chromosomes, very often in the triploid range, predominate. Ultimately, both normal and tetraploid cellsmay disappear entirely. The general result of thisprocess is that almost all cell strains have an unusual complement of chromosomes. The few excep

PassaqeNÂ°

Number of chromosomes452x



Type of

animal

inoculatedNo.cells in

ocnlatedCell

Hne@ in which tumors

were obtained with thisminimuminoculumCortisone-treated

hamsters1010210@10@KB,

HeLa, S-180 (All malignant origin).

Embryonic intestine.Normalliver.Normal

hamsters102

10@

106KB,

HeLa, J-111, Wilms6, 5-180 (All of nialignant origin).

Normal human amnion,embryonic intestine.

Normal human liver,lungs, thymus, endometrium, normal mouselymph-node, strain L929, normal monkey kidney.

PAuiâ€”Cancer Cell in Vitro 433

tions are nearly all skin fibroblasts, which tend toretain the normal diploid number for reasonswhich are not clear. Chromosomal variations almost certainly arise as a result of nondisjunctionduring mitosis, and they probably persist becausethey provide the cells with a selective advantagein the abnormal environment of a tissue culturemedium.

There have been persistent suggestions thatthese chromosomal changes may reflect the onsetof malignancy Levan and Hauschka (54) pointedout that in a series of ascites tumors the chromosome complement was almost never the normaldiploid number. This observation led them to propound the stemline theory, which proposes thatas the cells in a tumor escape from normal controlsthey develop the capacity to adapt genetically andacquire the chromosome complement most suited

to survival in a given environment. This theorywas reinforced by observations of Levan andBiesele (58) in tissue cultures. They found that theestablishment of an unusual chromosomal modewas frequently associated with the onset ofmalignancy, as tested by reinoculation (Chart 1).

Other workers have, however, failed to observe acorrelation between the onset of malignancy andchromosÃ mal variation. For instance, Hsu andKlatt (40) showed that changes in chromosomalcomplement of cultures of the Novikoff hepatomawere actually associated with loss of malignancyand transplantability. The evidence now suggeststhat chromosomal variation occurs in almost allestablished cell lines but is not necessarily associated with the onset of malignancy.

The possibility remains that specific chromosomal abnormalities may be associated with theonset of malignancy. For instance, Nowell andHungerford (68) have observed that in humanchronic myeloid leukemia blood cells characteristically carry a minute chromosome which Baikieet al. (6) consider to be a deletion of the long armof chromosome 21 or 22. Similarly, Yerganian,Leonard, and Gagnon (100) have found that theonset of malignant transformation in vitro (astested by reinoculation) in the cells of the Chinesehamster is associated with alterations in themorphology of the X1 chromosome. Clearly,chromosomal rearrangements in cell strains mayor may not result in changes in a specific chromosome which may be important in tissue homeostasis. Hence, unless each chromosome is individu

ally studied the significance of the observationsmay be missed. Incidentally, it is possible thatthese observations may provide visual evidence forthe deletion hypothesis, which will be referred tolater.

TRANSPLANTABILITY OF CELLSTRAINS OF NORMAL AND

TUMOR ORIGIN

Coriell, McAllister, and Wagner (13) reportedthat of a number of cell strains only HeLa cellsproduced invasive metastasizing tumors in cortisone-treated rats. On the other hand, Moore (59)

found no clear-cut difference between cell lines ofnormal and tumor origin in similar experiments.The discrepancy may be explained by the findingsof Foley and Handler (28, 29) (Table 1), who inoculated known numbers of cells into the cheekpouches of normal and cortisone-treated goldenhamsters. They found that in cortisone-treated

TABLE 1

TUMOR FORMATION BY CULTURED CELLLINES INOCULATED INTO THE

[email protected] CHEEK POUCH

Foley and Handler (29)

animals they could regularly produce tumors withall cell lines, including those of normal origin,when inocula larger than 10,000 cells were used.In the normal hamster tumors could regularly beproduced with all cell lines with inocula of morethan one million. However, when the inocula werereduced below these levels only cell lines arisingfrom malignant tissue would produce tumors. Forinstance, the KB, HeLa, and S-180 strains produced tumors with inocula of less than a thousandcells in cortisone-treated hamsters and withinocula as low as 10,000 cells in normal hamsters.Furthermore, when the tumors were allowed togrow for 9 days, those derived from â€œmalignantâ€•cell lines were usually healthy and uninfiltratedby host cells, whereas those originating fromâ€œnormalâ€•cell lines were localized and infiltrated



LacticacidPHformed/glucose

used6.60.06.80.027.00.067.20.27.40.827.60.57.80.758.00.85

Cai.z TYPEPER

CENT OXYGEN IN

GASPHASE0

20StrainL

HeLaHLM1.88

7.327.60 11.003.91 5.62

434 Cancer Research Vol. @22,May 1962

by inflammatory cells. These observations mdicate a fundamental quantitative difference between normal and malignant cells in their relationship with the host and are strictly in line with

observations made with transplantable tumors by

Andervont and Shimkin (5).

Southam, Moore, and Rhoads (82) undertookto inoculate human tumor cell lines into humanvolunteers, consisting of a group of healthy mdividuals on the one hand and a group of cancerpatients in the last stages of the disease on theother. The healthy volunteers displayed a much

TABLE 2

EFFECT OF PH ON THE CARB0.HYDRATE METABOLISM

OF HIM CEu@s

the cell. Similar observations were reported byother workers employing cultured normal andtumor tissues (17, 18, 43, 95). When later investi

gators re-examined this question, however, several

reports appeared claiming that many tumors

produced little or no lactate and sometimes nontumor tissues actually had higher rates of aerobicglycolysis than did tumor tissues cultivated in

vitro (30, 44, ,55, 90). These and other discrepanciesstimulated some investigators to question Warburg's theory and culminated in a public debate

by Weinhouse, Warburg, Burke, and Schade inthe columns of Science (93). A clue to the natureof the discrepancy was obtained by Paul andPearson (72), who found that the metabolism ofnewly explanted normal tissues could fluctuatewidely within the course of a few days. A similarobservation was made by M. Harris (37) and laterby others (4, 16, 31, 91). Recent studies (14, 69,71) have demonstrated that several factors, especially the leakage of intermediary metabolitesfrom cells, the pH and glucose concentration in the

TABLE 3

EFFECT OF LOWERED OXYGEN TEN.SION DURING CULTURE ON REsPIRATION SUBSEQUENTLY MEASURED INAm

(@l02 used/hr/cell X 10')

higher resistance to the development of tumors atthe site of inoculation.

These experiments indicate both that culturedcells of tumor origin are more easily transplantedto foreign hosts than cells of normal origin andthat the successful establishment of tumor cells oninoculation depends to some extent on the state

of the host.

METABOLIC BEHAVIOR OF NORMALAND MALIGNANT CELLS

The metabolic behavior of cancer cells is ofspecial interest, because rational attempts atchemotherapy may be based on discovering fundamental differences between them and their normalcounterparts. The observations which have cornmanded greatest interest in this field were madeby Warburg, Posener, and Negelein (92), who reported that cancer cells had a higher rate ofaerobic glycolysis than did normal cells (i.e., whenincubated in an atmosphere of air they produced alarger amount of lactic acid and used more glucose) . These workers also found that frequentlycancer cells had a lower rate of respiration. On thebasis of these observations, Warburg propounded

his well known theory that cancer was due to anirreversible lesion of the respiratory mechanism of

medium, and the oxygen tension and carbon dioxide tension to which cells are exposed duringcultivation can all result in radical changes in themetabolic patterns observed (Table 2). The mostinteresting observation in relation to Warburg'shypothesis was that the metabolic pattern dietated by cultivation in a given environment persisted for some hours after the stimulus was removed (Table 3). Consequently, cells grown in arather low oxygen tension show a higher rate ofglycolysis and a lower rate of respiration in the

usual experimental conditions. Similar observa

tions have been made very recently (3) by anothergroup. Since a low oxygen tension is characteristically found within tumors Paul (71) suggeststhat these observations provide an adequate explanation for the rate of aerobic glycolysis intumor cells.



PAULâ€”Cancer Cell in Vitro 435

In a few other respects significant metabolic differences have been claimed between normal andmalignant cells. In particular, Hirschberg and hiscolleagues (39) showed that azaguanine deaminase occurred in very small amounts in human

glioblastoma but was present in rather largeamounts in normal human brain. Unfortunately,when an attempt was made to exploit this observation in the treatment of patients with glioblastoma no significant chemotherapeutic effect wasobtained. Another instance of a difference of thiskind was claimed by Jacquez and his colleagues(42), who found that a transaminase was absent insome mouse tumors, although it was present incomparable normal tissues. This observation has

not been applied in chemotherapy. Jacobson (41)has demonstrated a difference between normal andleukemic blood cells in their metabolism of folicacid which may be exploitable. A somewhat different kind of differential effect has been reported byLeslie et a!. (51) in the effect of insulin on carbohydrate metabolism.

Some interest attaches to the recent demonstration (94) that certain enzymes involved in thesynthesis of DNA increase greatly in amount be

fore a resting culture begins to grow and remain athigh levels until growth ceases. The addition tothe medium of the substrate (thymidine) for someof the enzymes stimulates a further increase.These observations provide strong evidence forthe existence of feedback mechanisms of the kindsuggested by Potter and Auerbach (74) and similar to those which have been demonstrated inmicroorganisms by Yates and Pardee (99) amongothers. The importance of these mechanisms inrelation to cancer is that they may be involved inthe normal control of growth.

THE INDUCTION OF MALIGNANCYIN CULTURED CELLS

Whereas established cell strains do not necessarily give rise to tumors on reinoculation, thereare many well authenticated instances of primarily nonmalignant strains developing the capacityto initiate tumors at some time in their history.The opposite has also been observed. The bestdocumented examples are described by Gey et al.(33), Sanford et al. (77, 78), and De Bruyn (15).In Gey's laboratory the 14P cell strain was grownfrom a culture of normal rat fibroblasts and wasrepeatedly tested by re-inoculation into rats ofthe strain from which it was originally derived. Itproduced no tumors for about 2@years. One of thesublines then began to produce a rather high mcidence of tumors. After a further 10 years of cultivation, however, this capacity was lost again.

Two interesting examples have been reported bySanford et a!. from Earle's laboratory. The firstconcerns the strain L cell (which was originallyisolated from the C3H mouse and treated withmethylcholanthrene). At first this cell line produced tumors in a high percentage of C3H mice,but after some years of cultivation the incidenceof tumor production fell. Perhaps the most interesting case of all, reported from the same labora

tory, concerns a line of subcutaneous fibroblastsderived from C3H mice. The line was at one stagecloned (i.e., a single cell was isolated, and all subsequent cultures were derived from this). Twodivergent sublines eventually emerged, one ofwhich produced a very high incidence of tumors onre-inoculation into mice, whereas the other produced very few.

De Bruyn also observed a loss of malignancyduring prolonged cultivation. Her original MBstrains produced tumors in a large proportion of

inoculations, but some sublines subsequently derived from them were incapable of producing anytumors at all.

It is clear from this work that cells in continuous cultivation can spontaneously develop or lose

the capacity to produce cancers.Tumor-producing viruses.â€”Carrel (11) observed

that the Rous virus could be propagated in chickembryo tissue culture, and Halberstaedter andDoljanski (36) and Manaker and Groupe (56)made similar observations. The problem was takenfurther by Rubin and Temin (76, 88), who platedout the virus on monolayers of disaggregated chickembryo cells and were able to observe foci,recognized by the development of rapidly dividingcells of altered morphology, which tended to pileup on top of one another in distinction from thenormal embryonic cells which formed a monolayer.This kind of investigation opened the way toquantitative studies of tumor viruses.

Current interest in this field has been greatlystimulated by the observation of Stewart andEddy (85, 86) that a virus extracted from an infectious mouse tumor can produce a great varietyof tumors when re-inoculated into the animal andcan also produce necrotic lesions in cultured mousecells. The SE polyoma virus, as it is called, hasseveral interesting properties. Thus, it can infectrelated species including the rabbit, rat, andhamster as well as the mouse. Also in some cases,after the tumor has been initiated, the virus canno longer be detected. Finally, the virus itself isstrongly antigenic, and antibodies to it may develop in infected animals.

These observations suggest that the onset ofmalignancy during the prolonged cultivation of



486 Cancer R,ese.ardz Vol. 22, May 1962

cell strains may frequently be due to accidentalinfection with a virus. Perhaps this interpretationshould be accepted with caution, however, inview of the loss of malignancy which can also occur during cultivation.

There have been reports of the passage of othertumor viruses through tissue culture. In particular, Coman (12) reported passage of the Shopepapilloma virus through rabbit skin, and Lasfargue@ et al. (45) reported passage of the Bittner

virus through cultures of mouse mammary tissue.Chev2ical oarei,wgens.â€”Most chemical carcino

gens, though not all, are substances which areknown to increase the incidence of mutation&Earle and Voegtlin's (24) carefully conductedstudies on the effect of chemical carcinogens oncultured cells revealed that treatment of subcutaneous fibroblasts with 20-methyleholanthreneresulted in gross abnormalities of a kind frequentlyobserved in tumors. However, these workerssystematically studied the transplantability ofcells after different periods of treatment with 20-methyicholanthrene and found that after aninitial increase it progressively diminished untilafter about a year it was very low indeed. Duringthis time the morphological abnormalities withinthe cultures continued and even increased.

A related study was carried out by Lasnitzki(46), who observed hypertrophy and a greatly increased niitotic rate in organ cultures of mouseprostate tissue treated with 20-methyicholanthrene (Fig. 3). However, Sauerteig (80) repeatedthe experiments and attempted to transplant thealtered cultures into suitable hosts but was unable to produce tumors by this means. The malignant nature of the changes must therefore remain in some doubt, although they very closelyresembled precancerous changes seen in theanimal. Lasnitzki (47) has also shown that treatment of lung organ cultures with 3,4-benzpyreneproduces changes which might be described asprecancerous.

Metabolic stress.â€”Goldblatt and Cameron (34),in a classic experiment, attempted to test Warburg's theory of carcinogenesis by exposing cul

tures to intermittent anaerobiosis during 2@

years' continuous cultivation. Morphological ab

normalities appeared in the treated cultures, andon inoculation into animals some tumors were produced. The significance of these observations isnow less certain than it appeared to be at the time,in the light of more recent facts. Adebonojo et al.(3) were unable to demonstrate increased malignancy by treating the L cell with intermittent

anaerobiosis although the enzyme pattern of thecells changed.

Radiw@ion.â€”Treatment of cell cultures withx-rays produces multiple chromosomal abnormalities (8). Some cells fail to divide and eventually

die. The survivors show many kinds of abnormality, including giant cell formation, bizarre mitoses,

and unusual morphology. To the author's know!edge no attempt has been made to test such cultures for malignancy by animal inoculation.

INTERACTIONS BETWEEN NORMALAND CANCER CELLS

It was pointed out earlier that cells from explants of different tissues intermingle indiscrimi

nately. Leighton (47) has performed similar experiments in cellulose sponges which provide a

three-dimensional matrix and has observed thesame behavior. Similar results were obtained whenthe cells were inoculated into portions of urnbilical cord, which might be regarded as a morenormal kind of matrix (50). This invasive behaviorof normal tissues is unexplained, and it contrastswith Moscona's (60-62) observations that, whencells from embryonic tissues have been disaggregated by trypsin, they can reaggregate and segregate into organized structures. In an extension ofhis experiments Moscona (61) found that S-91mouse melanoma cells similarly tended at first tosegregate from other kinds of cells with whichthey were mixed in reaggregates. However whenthe aggregates were cultured for some days longerthe melanoma cells infiltrated normal cells in theneighborhood (Fig. 4). Moscona observed that amucinous material (which he calls ECM) was necessary for reaggregation, and that, after cultivation in a disorganized manner for some days, cells

lost the capacity to reaggregate. It is tempting tospeculate that material of this kind may be necessary for tissue organization and may be related to

the organ-specific antigens which have been foundto be absent in cancers (67).

Experiments carried out by Wolff (98) provide

further information about the interaction between normal and tumor tissues. He observedthat fragments of normal mouse embryonic tissuesinvaded chick tissues when grown in contact with

them in organ culture but did not destroy them.In contrast, when the 5-180 sarcoma was culturedin contact with organ cultures of chick tissues thesarcoma cells invaded and destroyed the fragments.

These experiments illuminate another interesting relationship between certain tumor cells andnormal cells. Santesson (79) observed that many




mouse epithelial tumors grew better in the presence of connective tissue. Schleich (82) alsoshowed that the Yoshida ascites sarcoma of the ratwould only grow satisfactorily in vitro in the pres

ence of fibroblasts. De Bruyn (15) demonstrateda similar relationship in connection with her MBstrains; and finally Wolff (98), in the experiments

referred to above, observed that although the5-180 sarcoma was very difficult to grow in cultureby itself in his conditions nevertheless it grew verywell in contact with fragments of normal organsand could be passaged in this manner throughmany subcultures.

ONCOLYTIC AGENTS

Since the rational approach to cancer therapyhas so far proved unproductive, there have beenmany purely empirical attempts to evolve therapeutic agents specific for cancer cells, and culturedcells have been used as test objects. In somestudies, such as those of Biesele (9, 10) andEichorn et al. (25), an attempt was made to screena large series of metabolic analogs for their toxicityagainst lines of cancer cells on the one hand andnormal cells on the other. On the whole, little dif

ferential toxicity was found. Eagle and Foley(20, 21) conclude that these agents exhibit a genera! cytotoxicity for all kinds of growing cells andthat their value as oncolytic agents is simply ameasure of their cytotoxicity. This conclusion isin accord with the proposition that there is no

general qualitative difference between the metabolism of cancer and normal cells.

Oncolytic viruses.â€”Certain viruses have beenfound to have some specificity for certain cancercells both in vitro and in vivo. However, it is impracticable to use these as therapeutic agents because, as Moore (57) points out, the patient develops viral antibodies more quickly than thevirus destroys the tumor.

Tuaor antibodies.â€”Attempts have been madeto produce antibodies against tumor cells, andsome limited success has been obtained. For instance, Prehn and Main (75) found that specificantibodies were produced against methyleholanthrene-induced tumors. On the other hand, theseworkers found that antibodies were not producedagainst spontaneous tumors. This is probably aspecial case, methyleholanthrene combining withcellular proteins to form new antigens. In themajority of cases where antibodies have been pro

duced against tumor cells it has been found thatthey are equally effective against some of thenormal cells of the host.

CONCLUSIONS CONCERNING THENATURE OF THE CANCER

PROCESSCancer always involves a local breakdown of the

tissue homeostatic mechanisms. Consequently,cancer cells behave rather like tissue culture cells;and similarities between them, far from implyingthat all cultured cells become malignant, serve toemphasize that cancer cells achieve almost cornplete autonomy and become, in effect, tissue cultures in i,it'o. When this proposition is accepted itbecomes clear that some of the phenomena whichoccur readily in cultured cells can probably be exeluded as being specific for the cancer process.Thus, many morphological and metabolic abnormalities and the emergence of chromosomalvariations are more likely to be results of the cancer process than etiological factors.

The fundamental lesion involved in the breakdown of the homeostatic mechanisms is not understood. It may involve the surface properties ofcells (particularly their mutual adhesiveness), theproduction of cell-specific antibodies (35), or

again some kind of feedback control of cell growthand migration operating through metabolic pathways. Probably multiple mechanisms are involved,and each of these may be controlled by one orseveral genes. The â€œdeletionâ€•theory (73) proposesthat cancer is a consequence of successive somaticmutations which result in the loss of all thesemechanisms and permit the cell to become independent of local control. It may not be necessaryto postulate successive multiple mutations. Someof the control mechanisms may be relativelycomplicated and may, for instance, consist ofseveral enzymes involved in the synthesis of aspecific substance. Each enzyme may be con

trolled by one or more genes, and consequently asingle lesion affecting any one of the genes or theprotein-synthesizing systems they control mightresult in a breakdown of the whole system and theestablishment of a cancer. A mechanism of thiskind would be in accord with current thinking inthe field of molecular biology and would not conffict with the known facts.

Much of what is known of the etiology of cancerwould fit a theory which implicates damage to thegenetic apparatus of the cell. Thus, a proportionof cancers might arise spontaneously by somaticmutation. On the other hand, specific mutagensmight not only increase the incidence of randommutations but might be expected specifically toincrease certain kinds of mutations. Finally,

viruses might operate by a process analogous totransduction. The point of convergence of all



438 Cancer Research Vol. 22, May 1962

these factors would nevertheless be expected to bethe same general homeostatic mechanism.

Without further evidence it is idle to speculatebeyond this point, except to point out the practicalimplications of these conclusions. If the mechanism of cancer is of the nature suggested above,then it seems unlikely that chemotherapy of thetype at present envisaged is likely to be effective.With a deeper understanding of the homeostaticmechanisms it is possible that a more specific formof therapy, perhaps by nucleic acids, may be possible in the future. For the time being, however,preventive measures and, when the disease isestablished, eradication by surgery or irradiationwill probably continue to be the only effectivemeans of dealing with cancer.

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FIG. 3.â€”Photomicrographs of sections of mouse prostategland maintained in organ culture. X115. Stained H. & E.a: Grown for 21 days in normal medium. b: Grown for 11 dayswith 4 @ig/ml methylcholanthrene, followed by 10 days innormal medium. (Courtesy of Dr. I. Lasnitzki.)

FIG. 4.â€”Infiltration of 7-day disaggregated chick cells by

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1962;22:431-440. Cancer Res J. Paul

in Vitro: A ReviewThe Cancer Cell

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