comparative studies of the metastatic potential of three

Br. J. exp. Path. (1979) 60, 499

COMPARATIVE STUDIES OF THE METASTATIC POTENTIAL OFTHREE TRANSPLANTABLE RAT MAMMARY CARCINOMAS

OF SPONTANEOUS ORIGIN

N. WILLMOTT*, E. B. AUSTIN AND R. W. BALDWIN

From the Cancer Research Campaign Laboratories, University of Nottingham, University Park,Nottingham NG7 2RD

Received for publication March 9, 1979

Summary.-The metastatic potential of 3 spontaneously arising mammarycarcinomas (Sp4, Spl5 and Sp22) has been examined when transplanted in the formof a viable cell suspension into the tissue of origin. Primary tumours were excised atdifferent times after implantation and it was found that the metastatic potential of theimmunogenic tumour Sp4 was directly correlated with the size of the primary tumourwhen excised. By contrast, the incidence of metastases from the non-immunogenictumours Sp15 and Sp22 was similar irrespective of the size of the primary tumour onexcision. The pattern of metastasis also differed between the tumours, although herethere was no relation to immunogenicity. Thus, resection en bloc of large primarySp4 or Spl5 tumours plus regional lymph nodes coutd be completely curative, signi-fying initial spread of tumours via the lymphatics and only subsequently via the bloodstream. On the other hand, resection en bloc of primary Sp22 tumours plus regionallymph nodes at a similar stage of primary tumour development was never curative,signifying early spread via the blood stream.Other studies showed that the metastatic potential of mammary carcinoma Sp4

was an innate characteristic of the tumour and not related to the tissue of implanta-tion since in addition to metastasizing from the mammary pad it metastasized whenimplanted either s.c. or intradermally in a region devoid ofmammary tissue. Further-more, a rat sarcoma Mc7 showed a negligible tendency to metastasize when implantedeither in the mammary pad or in the s.c. tissue, where it had been induced withmethylcholanthrene.

THERE IS GENERAL AGREEMENT thatmany existing animal models for cancertherapy are inadequate, particularly asregards the status of the host with respectto the tumour (Hewitt, 1978; Alexander,1977). Such deficiencies include the use ofhighly immunogenic tumours such as thoseof viral origin or chemically induced, orthe administration of immunotherapeuticagents so as to alter the tumour-hostrelationship in a therapeutically unrealisticway. In a recent paper Alexander (1977)recanted a considerable amount of pre-vious work with highly immunogenic,chemically induced tumours, and made a

plea for the use of tumours that metas-tasize spontaneously from primary im-plants. Other workers have emphasizedthe desirability of using spontaneouslyarising tumours of low immunogenicity asa model of human cancer for rather longer(Hewitt, 1978).The argument about the relevance of

animal models has largely centred on theimmunogenicity of the tumour in thesyngeneic host, and it does appear, notunexpectedly, that experimental tumoursof low immunogenicity are harder to con-trol by immunotherapeutic manoeuvresthan are tumours of high immunogenicity

* Correspondence to Dr N. Willmott, Department of Clinical Oncology, 1 Horselethill Road, University ofGlasgow, Glasgow G12 9LY.

N. WILLMOTT, E. B. AUSTIN AND R. W. BALDWIN

(Pimm et al., 1978). However, as theimmunogenicity of human cancer in theautochthonous host is necessarily un-certain, the debate as to whether clinicallyrelevant animal tumours should be im-munogenic or not is perhaps unlikely to beresolved in the immediate future. Otheraspects of human cancer are less conten-tious; thus, an issue on which all are agreedis that metastases constitute both thesine qua non of malignancy and the majortherapeutic problem in cancer manage-ment, since established surgical and radio-logical treatment can in many cases dealwith localized tumours (Sugarbaker, 1952;Sugarbaker and Ketcham, 1977). There-fore, rather than immunogenicity as thecriterion for clinical relevance of animalmodels, perhaps metastatic potentialwould be preferable.

Before investigations into the therapyof tumour metastases from transplantablespontaneously arising rat mammary car-cinomas implanted into the breast pad(Greager and Baldwin, 1978), preliminarystudies were carried out with 3 such tu-mours: (a) to define at which point in theirdevelopment the primary tumours metas-tasized; (b) to record the pattern ofmetastasis; and (c) to examine whetherthe capacity to metastasize was related tothe site of tumour implantation or was aninnate property of the tumour. Apart fromtheir use as models of cancer therapy, thesestudies have some bearing on the biologyof metastases and are therefore describedhere in detail.

MATERIALS AND METHODS

Rats. Rats were from the departmentalcolony of WAB/Not inbred rats. They werenormally housed in plastic cages with sawdustbedding and fed standard diet (Oxoid) and waterad libitum. After surgery, cellulose wadding wasused as bedding, since this appeared to reducepostoperative mortality.Tumours.-Carcinomas arose without deliber-

ate induction in the mammary tissue of femaleWAB/Not inbred rats and were transplanted bytrocar graft to syngeneic recipients of the samesex as the primary host. For all 3 tumours (Sp4,Spl5 and Sp22) the LD5o was < 1000 cells inunprimed rats; however, in appropriately

immunized rats up to 2 x 104 Sp4 cells could berejected, whereas rats similarly immunized withSpl5 or Sp22 could not reproducibly reject cellinocula greater than the LD50 (Baldwin andEmbleton, 1969, 1974; Willmott et al., inpress).Sarcoma Mc7 arose after a s.c. injection of

3-methylcholanthrene in a female rat (Baldwinand Pim, 1973). It has an LD50 of 1-5 x 105 cells,but appropriately immunized rats could rejectchallenge inocula of up to 1 x 107 cells (Pimmand Baldwin, 1977).

Single-cell suspensions of all tumours wereprepared by trypsin digestion of freshly excisedtumour tissue. Only preparations of > 800oviability as assessed by trypan-blue exclusionwere used.

Surgery. The surgical procedures describedbelow were developed by Dr J. A. Greagerduring his stay in this department.

Simple excision of the primary tumour wascarried out under ether anaesthesia. In all casesthe primary tumour plus s.c. mammary, con-nective and fatty tissue were removed as far asthe axillary lymph node. After cleaning with70% alcohol the wound was closed with sutureclips.En bloc excision of primary tumour and

regional (axillary and brachial) lymph nodeswas also performed under anaesthesia. Bloodvessels associated with the axillary node wereexposed by excision of the pectoralis muscle,and haemostasis was maintained with 6/0chromic catgut. After cleaning with 70%alcohol the wound was closed with both 3/0nylon sutures and suture clips.

After surgery 2 primary tumour diameterswere measured at right angles and the meanrecorded. In addition, it was noted whether ornot the regional lymph node was palpable.Postmortem procedure. Rats the primary

tumours of which had been removed wereobserved for at least 100 days after operationand when they became sick (lethargic, losingweight, or developing respiratory distress) theywere killed and examined by necropsy. Onlygross observations were recorded such as theregional lymph-node status and presence oftumour in the thorax. Tertiary thoracic meta-stases manifested themselves in one or more ofthe following ways: tumour nodules on the lungsurface; amorphous tumour mass in the pleuraor pericardium; involvement of the para-tracheal/parathymic lymph nodes. Occasionallykidney metastases were observed.

RESULTS

Incidence of metastasis as a function oftumour size

There is debate, mostly on the philo-

500

METASTATIC POTENTIAL OF TRANSPLANTABLE RAT MAMMARY CARCINOMA 501

sophical level, as to whether human cancermetastasizes early when consisting of anidus of cells, or at some later stage (Fisheret al., 1978). The fact that cures can beobtained by removal ofthe primary tumourwould indicate to some that it remainslocalized until a certain point in itsdevelopment; however, to others this isinterpreted as meaning that the tumourhas metastasized but that control mechan-isms to eliminate metastatic foci areoperative. Whilst this dispute cannot beresolved in human cancer patients, studieson animal models selected for theirsimilarity to human cancer may be of use.Thus, Hewitt and Blake (1977) have shownthat occult regional lymph-node metastasesoccurred with a high frequency in 6tumour systems. as assayed by removal ofthe lymph nodes draining tumours andsubsequent transplantation to syngeneichosts. However, in the autochthonous hostregional lymph node metastases arose lessfreqtuently after excision of the primary.The inference here is that a primary tumournormnally sheds metastatic cells, but con-trol mechanisms of unknown characterexist.One possible control mechanism might

be the elimination of antigenic metastaticcell foci; therefore, we have examined theincidence of lethal metastases, eithersecondlary or tertiary, of mammary car-cinomas Sp4, Sp15 and Sp22 as a functionof the size of the primary tumour atexcision, to see whether the immuno-genicity of the tumour influenced the stageof primnary tumour development at whichmetastasis occurred.Rats were injected under the region of

the first or second nipples of the rightpectoral mammary pad with 1-5 x ] 04 cellsof Sp4, SpI]5 and Sp22 and observed untiltheir tumours were readv for excision. Thestages of primary tumour development atwhich excision was performed are definedas follows: Stage 1 1-1b5 cm mean dia-meter; Stage II 1 5-2K5 cm mean dia-meter; and Stage III-> 25 cm meandiameter. After excision of the tumour therats were left until the effects of secondary

TABLE I.-Incidence of Lethal MammaryCarcinoma Metastases as a Function ofPrimary Tumour Size at Excision

TumourTumouir ,

size SP4 SP15Stage 1

<S1a5 cmStage II

1-5-2-5cmStage III

> 2-5 cm

SP2-

4/16 7/7 5/7

7/1:3 6/8 6/7*

16/22 10/10 8/9* One rat -with pulmonary metastases also hadl a

recurrence at the site of primary tumouir excision.

and/or tertiary tumour spread made itnecessary to kill them.

It can be seen from Table I that therewas a marked difference in metastaticpotential of small (< 1.5 cm diameter)primary tumours. Sp4 metastasized in25% of cases (4/1]6), whereas Spl5 andSp22 metastasized in 100Oo (7/7) and 710o"(5/7) of cases respectively. Furthermore, itwas observed that larger (> 2-5 cm dia-meter) Sp4 primary tumours metastasizedwith greater frequency (73%o) than smallertumours. This behaviour is in contrastwith that of Spl5 and Sp22 tumours,which metastasized in over 70%o of casesirrespective of size. Since Sp4 is the onlyimmunogenic tumour, it is reasonable tosuppose that immunological mechanismsplay a part in controlling metastases, atleast those shed from small primarytumours.

Patterns of metastasis from mammarycarcinomas

It became evident during the course ofthe preceding study that although therewas an inverse correlation between themetastatic potential of small (< 1.5 cmdiameter) tumours and their immuno-genicity (Table I), the pattern of secondaryand tertiary spread showed no such corre-lation. For example, with mammary car-cinoma Sp4 (Table II) in almost 100l,o(17/18) of cases secondary metastasis tothe regional lymph node occurred and in66% of cases (12/18) tertiary thoracic


TABLE II.-Pattern of Mammary Car- Sp4 metastasizes initially to the regionalcinoma Metastases on Necropsy lymph node and then at some later stage

Tumour enters the blood stream, possibly, by_____________ analogy with human breast cancer spread,

Metastasis SP4 SP15 SP22 at the junction of the lymph vesselRegional efferent from the axilla and the vessels of

lymph node 17/18 9/9 4/11iMediastinum only 2/18 0/9 / the venous crculaton (nternal jugularLuing surface + subelavian vein confluence). By contrast

medliastinum 12/18 2/9 11/11 the pattern of metastatic spread of Sp22All tumours were Stage III on excision. suggests early entry into the blood streamAfter excision rats were observed until ill, tben irrespective of a malignant axillary lymph

examined by necropsy. node. To examine whether secondary

metastases to the axillary lymph nodemnetastasis occurred. Tertiary metastases were necessary for tertiary metastases towere manifest as either lung nodules, the lung, en-bloc resection of both Sp4,presumably blood-borne, or malignant Sp15 and Sp22 primary tumours plusparathymic/tracheal lymph nodes, pre- regional lymph nodes was performed at asumably via retrograde permeation from similar stage of primary tumour develop-the subelavian trunk. Features of note ment (StageIII, see TableI). The resultswere that secondary metastasis to regional in Table III show that this surgical pro-lymph nodes was not invariably associated cedure was completely curative in 6/6with tertiary metastases to the thorax cases for Sp4 mammary tumours, confirm-(Table II) (some rats came to necropsy ing that a malignant regional lymphnodeonly with enlarged axillary lymph nodes) was necessary as a focus for the furtherand that when tertiary metastases in the dissemination of this tumour, and wasform of lung nodules did appear it was curative in 4/6 cases after en-bloc resectionalways in the form of 1-10 discrete tumour of primary Sp15 tumours and regionalfoci. The metastatic patterns of Spl5 and lymph nodes. By contrast the same sur-Sp22 differed considerably; thus, although gical procedure was completely ineffectiveall 9 rats with primary Spl5 tumours with rats bearing Sp22 mammary tumours,developed regional lymph-node meta- all 5 rats eventually succumbing tostases, only 2 had developed tertiary tertiary lung metastases, demonstratingmetastases in the form ofa few lung nodules that dissemination from the primaryvisible at necropsy, despite the presence tumour via the blood stream occurredof grossly malignant regional lymph independently of the presence of a malig-nodes. The metastatic pattern of mam- nant regional lymph node.mary carcinoma Sp22 was different again: Although the location of metastaticless than 50%0 (4/11) of rats were found tohave regional lymph-node metastases atnecropsy, whereas 100% (11/11) suc- TABLES Iva Effect ofRaditcaln Sprtery oncumbed to tertiary thoracic metastases. Survival* of Rats Bearing SpontaneousHowever, these metastases were different Mlammary Tumoursfrom those observed with mammary car- Tumour SP4 SP15 SP22cinomas Sp4 and Spl5. For example, Sp4 Incidence ofand Spl5 tertiary metastases were most palpable RLNtoften seen as a few (1-10) lung nodules, at surgery 5/6 1/6 05whereas the salient feature of Sp22 tertiary long-termmetastases was their large number, the survivors

lung surface often being virtually covered after surgery 6/6 4/6 0/5with a "shower" of metastatic lesions. *Observed for at least 100 days, after excision of

primary tumour, for illness due to metastatic spread.The patterns of metastasis suggest that t RLN Regional lymph node.

a-02


lesions showed no relation to the immuno-genicity of tumours studied, the kineticsof metastatic growth as measured by thetime elapsing between surgery, and thepoint at which morbidity due to meta-stases became evident, did (Table IV).Thus, when rats bearing Sp4, Spl5 andSp22 tumours of equivalent size weretreated by simple excision of the primary,metastases from Sp4 primary tumourstook almost twice as long to producemorbidity in their hosts (69 days) thanmetastases from Sp] 5 and Sp22 primaries(35 and 38 days respectively). It shouldhowever be noted that, although ratsbecame ill from Spl5 and Sp22 metastasesat similar times after surgery, the meta-static lesions were different (see Tables IIand III). Since this makes the comparisonof growth rates of metastases somewhatcrude, where possible the growth rates ofmalignant regional lymph-node metastasesafter excision of Sp4, Spl5 and Sp22 wasmeasured and again it was found that Sp4metastases grew the most slowly (data notshown).

Effect of site of implantation on metastaticpotential of a spontaneous miammarycarcinoma and a methylcholanthrene-indaucedsarcomaDepending on the site of implantation

the growth characteristics of some experi-mental tumours can differ (Auerbach et al.,1978; Bartlett, Kreider and Purnell, 1976).While the reason for this is not known,physiological factors such as availableblood supply have been suggested. Byanalogy the presence or absence of bloodor lymph vessels in a particular tissue couldwell determine whether a transplanted

TABLE IV.-Excisioncinomas

-Survival Time of Rats afterof Primary Mammary Car-

Tumour Sp4 Sp15 Sp,),)Survival timet

(days+s.e.) 69+7 35+3 38+ 2No. rats 18 9 11

* Excised tumours were all Stage III, i.e. > 2-5 cmmean diameter.

t Time at which rat became ill due to effects ofmetastases.

tumour is disseminated or not; we havetherefore examined the metastatic poten-tial of mammary carcinoma Sp4 in its tis-sue of origin, the mammary pad, and alsoin s.c. tissue and cutaneous dermis farremoved from mammary tissue, such asthat adjacent to the lower spine. Sp4 cells(2 x 104) were injected into each site andallowed to grow until the tumours were> 2-5 cm in mean diameter, whereuponithev were excised and the rats observedfor development of metastases. It can beseen from Table V that Sp4 metastasizes toregional lymph nodes from all of thesesites and it would therefore appear that,at least for this tumour, metastatic poten-tial is an innate property and not de-pendent on the environment in which thetumour may find itself. This is substan-tiated by the fact that when a 3-methyl-cholanthrene-induced sarcoma Mc7 (I x106 cells) was injected into both themammary pad and into its site of originthe s.c. tissue excision of the tumour when> 2X5 cm in mean diameter was curative invirtually all cases.

DISCUSSIONRecently the need for more basic re-

search into the nature of the metastatic

TABLE V.-Relation of Site of -Implantation to Metastatic Potential of Tumours

Means of

Ince)M

AINammiTumour induction Site of origin pad

MIammary None AMammary tissue 4/5carcinoma Sp4

Sarcoma Mc 7 AMethylcholanthrene Subcutaneous tissue 0/5* Tumours excised when > 2-5 cm mean diameter (Stage III).

2idence of RLN metastasis afterxcision* of tumour growing in

ary Subcutaneous Cutanecustissue dermis4/6 4/5

1/6 NT


process has been emphasized (Baldwin,1978). Whilst this is undoubtedly desirablethere are unfortunately few animal tumourmodels that spontaneously metastasize,and1 even fewer that meet the rigorouscriteria set down by Hewitt for accept-ability as models of human cancer (Hewitt,1978). For example, both the Lewis lungcarcinoma and Bl6 melanoma in C57B1mice are over 20 years old and moreoverhave been transferred numerous timesfrom laboratory to laboratory. Indeed,some investigators have found the B 16melanoma unexpectedly remains localized(Paslin, 1977), which may be due to itsinitroduction into a new mouse colony.-No such artefacts can have influenced ourresults, as all the tumours arose withoutdeliberate induction in rats of the depart-ment's inbred colony within the past 12years and have been passaged in these ratssince then. Moreover, by the use of frozentissue, tumours of less than 50 in-vivopassage generations have been used inthese studies.

Since the last century observers havenoted that the metastatic process maynmanifest itself in a variety of forms. Forinistance, some human tumours metasta-size early, others late; some appear as asingle secondary tumour whilst others areseen as metastatic "showers"; and certainttimours appear to metastasize preferen-tially to particular organs (Joachim et al.,197(6). In these studies we have separatedthe concept of metastatic potential (i.e.the capacity to metastasize) from that ofmetastatic pattern (i.e. the sites at whichmetastases appear) and asked whethereither correlates with the immunogenicityof the tumours. It was found that small)rimary tumours (1 1-5 cm in mean(liameter) of the immunogenic tumourSp4 metastasized in only 25%o of cases,w7hereas tumours of equivalent size oflittle or no immunogenicity (Spl5 andSp22) metastasized in over 7000 of cases.Thus there is an inverse correlation be-tween the immunogenicity and the meta-static potential of the 3 tumours examined.This correlation mav reveal a causal rela-

tionship since there are numerous reportsthat treatment with agents known todepress immune responses has increasedthe metastatic potential of tumours. Forexample, primary implants of tumoursthat normally remain localized can be in-duced to metastasize by treatment withcortisone (Agosin et al., 1952), anti-lymphocyte serum (Fisher, Soliman andFisher, l 970a) or X-irradiation (VonEssen and Kaplan, 1952), although a moreselective depletion of immune responses bythymectomy and X-irradiation did notlead to increased metastatic potential of anon-immunogenic tumour (Jamasbi andNettesheim, 1977). Furthermore, localpostoperative irradiation of breast-cancerpatients may lead to an increased incidenceof distant metastases as first evidence ofrecurrence (Fisher et al., 1970b).Thus there appears to be support for

the view that the metastatic potential of atumour depends inter alia on the abilityof the host to respond immunologically toit, although the work of Hewitt and Blake(1977) with non-immunogenic mousetumours shows that some aspects of thedestruction of metastatic tumour cells lieoutside classical immunological mechan-isms.What then of the selectivity of meta-

static cells for various sites? It is apparentfrom Tables II and III that mammarycarcinomas Spl 5 and Sp22 exhibit verydifferent patterns of metastases, Sp 15"preferring" the lymphatic system andSp22 "preferring" the blood stream,althoug,h both tumours are essentiallynon-immunogenic. This observationprompts the question: how does the phe-nomenon of organ selection occur? Twopossible mechanisms have been suggested:Coman, Eisenberg and McCutcheon (1949)have stated that "scarcity of metastasis inan organ is explained by scarcity oftumourcells reaching that organ", and there aresome studies showing wide distribution ofmetastatic lesions after injection of tu-mour cells into the arterial circulation(Coman et al., 1949). However, more com-mon are reports of a ubiquitous distribu-

504


tion of "tagged" tumour cells, but anorgan-specific growth of metastases(Sugarbaker and Ketcham, 1977). Fur-thermore, with 3-methylcholanthrene-in-duced tumours that apparently do notmetastasize, as judged by prolonged sur-vival after excision of the primary, tumourcells can be demonstrated by bioassay inthe spleen and blood (Kim, 1966; Wexler,Sindelar and Ketcham, 1976). Assumingthen that many if not all tumours metas-tasize, why are different metastatic pat-terns observed? The difference betweenSp15 and Sp22 cannot be ascribed toimmunological intervention as both areessentially non-immunogenic. Dose-res-ponse aspects of metastasis formationmight be considered since circulatingmetastatic tumour cells are only presentin small numbers (Griffiths and Salsbury,1965) and, although this would accountfor the fact that tumours with high growththresholds (such as those induced with3-methylcholanthrene) do not commonlyexhibit palpable metastases (Table V), itdoes not account for the differences inmetastatic pattern between Sp 15 andSp22, since both have similar LD50 valuesat least when implanted in the s.c. site.

In all probability the selectivity ofmetastases for certain organs is due notonly to factors inherent in the type oftumour used ("the seed") but also influen-ces exerted by the organs of the host("the soil"). Thus it has been postulatedthat a tumour-cell-endothelial-cell bondis important in the formation of a meta-stasis (Green and Harvey, 1964). If thisis the case, comparisons of primary andmetastatic cell lines from the same tumourand metastatic cell lines from differenttumours may provide insight into the basicbiology of metastasis.

Lastly, these studies have described ina comparative fashion the metastaticbehaviour of 3 tumour systems and havelaid the basis for attempts at their therapy.For example, Spl5 and Sp22 metastasesgrow rapidly to kill the host, whereas Sp4metastases grow more slowly (Table IV);therefore it might be expected that drugs

affecting rapidly proliferating cells wouldbe more effective against Spl5 and Sp22metastases. On the other hand Sp4 isimmunogenic and an immunological ap-proach to therapy might be considered.In fact the slow growth of Sp4 metastasesmay make it a good model for experi-mental therapy studies since it is the slow-growing human tumours that are refrac-tory to current therapies (Zubrod, 1978).

The work reported here forms part of aprogramme supported by the NationalCancer Institute (Contract No. NOI-CB-64042 Tumour Immunology Program). AW'eare indebted to Mr H. B. Hewitt and DrM. V. Pimm for a critical appraisal of themanuscript.

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comparative studies of the metastatic potential of three

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