`
`Br. J. Cancer (I994). 70. 228—232 © Macmillan Press Ltd.. 1994
`
`Effects of inoculation site and Matrigel on growth and metastasis of
`human breast cancer cells
`
`L. Bao, Y. Matsumura, D. Baban, Y. Sun & D. Tarin
`
`Nuflield Department of Pathology. University of Oxford, John Radclifle Hospital, Headington, Oxford, 0X3 9DL', UK.
`
`Summary The co-injection of extracellular matrix components. such as Matrigel. with human tumour cells
`into nude mice has been reported to facilitate tumour formation and growth. but it is unknown whether such
`components exert similar effects on tumour progression and metastasis. Metastatic behaviour is known to be
`enhanced when tumour cells are implanted orthotopically. and it is inferred that full and eflicient expression of
`this phenotype may involve some interactions with local connective tissue matrix. It was therefore decided to
`investigate whether manipulation of the mesenchymal environment by co-injection of extracellular matrix
`components. in the form of Matrigel. with human brcast cancer cells into orthotopic or ectopic sites could
`augment their metastatic performance, as well as their growth at the site of inoculation. Standard aliquots of
`10‘ cells of the polyclonal human breast carcinoma cell line MDA-MB—435. and of four clonal cell lines. two
`metastatic and two non-metastatic derived from it, were injected with and without Matrigel. orthotopically or
`subcutaneously into nude mice. The latent period of tumour formation at the inoculation site as well as final
`tumour size and metastatic performance at autopsy. 140 days after inoculation. were then assessed. The
`prevalence of metastasis of the parent. polyclonal, cell line and of its metastatic clones was increased if the cell
`inoculum was mixed with Matrigel. Non-metastatic clones were not induced to become metastatic by this
`trcatment, but local
`tumour growth at
`the site of inoculation was enhanwd in all experimental groups
`receiving Matrigel. Orthotopic inoculation acted synergistically with Matrigel
`to maximise both tumour
`growth and metastatic behaviour. The composition of the local extracellular matrix at the site of tumour
`growth influenwd expression of the memmtic phenotype by cells which are constitutionally capable of this
`behaviour. but did not induce it in ones which are not. Previous reports that local tumour growth is facilitated
`by enrichment of the mesenchymal matrix are confirmed. The mechanisms by which such effects are exerted
`are worthy of study. to ascertain whether they might be subject to clinical manipulation designed to retard
`tumour growth and dissemination.
`
`
`
`The discovery that mutant athymic nude mice do not reject
`heterotransplants of human tumour tissue (Rygaard & Povl-
`sen.
`1969) provided new opportunities
`for experimental
`studies on human tumours,
`including the analysis of their
`metastatic properties. However, several subsequent reports
`noted that
`the prevalence of tumour formation by xeno—
`grafted fresh primary human tumour fragments in nude mice
`is low, approximately 30% (Sharkey & Fogh, 1984), al-
`though the ‘take rate’ with passaged tumour cell lines (Fogh
`et a1., 1977) and with tissues from metastases (Sharkey &
`Fogh, 1984)
`is about double this. Also, many tumour
`implants and cell lines. including those derived from highly
`malignant human cancers, fail to form metastases in adult
`nude mice, even if they do grow at the site of implantation
`(Sharkey & Fogh, 1978; Fidler, 1986) and the animals are
`expensive, delicate and highly susceptible to infection. These
`difficulties have impeded and delayed extensive use of nude
`mouse xenografts in research on mechanisms of human
`tumour metastasis. Even so, the goal of being able to study
`this event
`in a living host has motivated investigators to
`persist in efiorts to induce human tumour cells to re-enact
`the metastatic process in emerimental animals. Variables that
`have been found to afiect whether metastasis occurs include
`the health and housing conditions of the mice (Sharkey &
`Fogh, 1978; Neulat-Duga er a1.. 1984; Fidler, 1986), the level
`of natural killer (NK)-cell activity, age of the host (Fidler.
`1986) and the route of tumour cell inoculation (Kozlowski et
`(11.. I984; Giavazzi et a1.. 1986). in addition to the intrinsic
`properties of the tumours under investigation.
`Of
`the
`several human tumour
`types now becoming
`available for the study of metastasis in the nude mouse. one
`of the most interesting for future study is the MDA-MB—435
`cell
`line isolated from a pleural efiusion in a patient with
`brcast cancer
`(Caillou e!
`(11.. 1978). Price et
`a1.
`(1990)
`reported that orthotopic implantation of cells of this line into
`the mammary fat pad (mfp) of nude mice could enhance its
`
`Correspondence: D. Tarin
`Received ll October 1993: and in revised form 14 March 1994.
`
`tumorigenicity in this host. and these tumours were found to
`be more metastatic than those formed after subcutaneous
`inoculation. These findings confirm and extend similar obser-
`vations.
`reported in recent years (Bresalier e!
`(1].. 1987:
`Morikawa et al..
`I988). with colon carcinoma cell
`lines.
`Tumours formed by these cell
`lines following intramural
`injection in the colon are more metastatic than those result-
`ing from subcutaneous inoculation. An orthotopic microen-
`vironment evidently encourages tumour cells to express the
`malignant phenotype (See also Fidler. 1990). This raises the
`question of how such an effect might be mediated and what
`it might signify.
`In vivo. carcinoma cells are surrounded by cellular connec-
`tive tissue composed of fibroblasts. endothelium and other
`cells in a dense network of extracellular matrix proteins
`which provides them all with a three-dimensional structural
`framework and influences their behaviour. The interdepen-
`dency of these elements is illustrated by some recent experi-
`mental observations. For example. Fabra er
`a1.
`(1992)
`demonstrated that highly metastatic KMIZSM colon car-
`cinoma cells co-cuitivated with fibroblasts from the colon are
`able to demonstrate an invasive phenotype and produce type
`IV collagenase, whereas the same line cultivated with skin
`fibroblasts can not. These carcinoma cells are metastatic
`from tumours formed after intramural
`inoculation in the
`colon but not after subcutaneous inoculation.
`tumour
`Recent
`investigations have also indicated that
`growth and behaviour is influean by non-cellular elements
`of the adjacent connective tissue matrix. In these studies it
`was found that a reconstituted basement membrane deriva-
`tive termed Matrigel. composed mainly of laminin. collagen
`type IV. heparan sulphate proteoglycan and entactin. greatly
`enhances the tumorigenicity of various malignant cells.
`in-
`cluding small-cell
`lung carcinomas. Blo—FIO melanoma.
`human submandibular A253 carcinoma. prostate carcinoma
`cell lines and primary colon carcinoma cells (Fridman er al..
`1990. 1991; Pretlow et a1.. 1991) Tumour cells premixed with
`Matrigel and then injected into athymic mice consistently
`produwd tumours which grew faster and became much
`larger than tumours induced by the same cells injected with-
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`out Matrigel. Fridman er al. (1992) also showed that non-
`transforrned and non-tumorigenic NIH-3T3 cells
`formed
`lowlly invasive and highly vascularised tumours when co-
`injected with Matrigel into athymic mice. The cells isolated
`from the Matrigel-induwd tumours exhibited cellular charaCo
`teristics similar
`to that observed in NIH-3T3 cells after
`malignant transformation and were eapable of forming pul-
`monary tumour colonies when injected i.v. These studies
`suggested that
`interaction of premalignant NIH-3T3 cells
`with extracellular matrix components can contribute to the
`process of tumour progression. In the current experiments we
`studied whether Matrigel would affect the growth of tumours
`formed in nude mice by human breast mreinoma cell lines
`with different metastatic potentials. We also investigated the
`efiect of this material on the incidence of metastasis from
`tumours formed by these lines after injection subcutaneously
`or into the mfp. The results showed that Matrigel could
`enhance the growth of tumours, in both sites, regardless of
`the degree of malignancy of the cell line. Matrigel could also
`increase
`the incidence of spontaneous metastasis
`from
`tumours formed by cell clones of the MDA-MB—435 line
`which have some intrinsic metastatic eapability, but did not
`induce any metastatic behaviour in clones which had no such
`inherent tendency.
`
`Maharishi-(1m
`
`Animals
`
`Athymic nude mice (MFlNu) were obtained from the brwd-
`ing facility at the John Radclifl‘e Hospital, Oxford, UK. Mice
`were injected with tumour cells when 6—8 weeks old and
`were kept in filter-top boxes in an isolated colony. Care of
`animals in this work was conducted according to United
`Kingdom Home Oflice and Oxford University regulations.
`
`Cell culture The polyclonal human breast carcinoma cell
`line MDA—MB—435 and the clonal cell lines C1, C2, C3 and
`C4 we derived from it by a limiting dilution technique were
`maintained in Dulbecco’s modified Eagle medium (DMEM)
`supplemented with
`5% newborn calf
`serum,
`sodium
`pyruvate, L-glutamine (ZmM), non-essential amino acid and
`2 x vitamin solution (Gibco). The cultures were incubated at
`37‘C in a humidified atmosphere of 5% mrbon dioxide—95%
`air. Tumour cells were harvested by washing the monolayer
`with phosphate-buffered saline (PBS)
`followed by brief
`incubation in 0.25% trypsin—0.02% EDTA at 37'C. The
`cells were then washed by centrifugation and resuspended in
`DMEM in preparation for inoculation. Clones C1 to C4
`were chosen for use in this study on the basis of earlier
`assays of their metastatic capabilities (see below) when
`injected suspended in culture medium alone.
`
`Matrigel
`
`Matrigel was extracted from fresh pieces of the mouse Eng—
`lebreth-Holm—Swarm (El-IS)
`tumour as described previ-
`ously (Kleinman er al., 1986, 1990). Briefly, 100g of the
`tumour tissue was washed in chilled 3.4M sodium chloride
`and 0.05 M Tris—HCI, pH 7.4, containing 5 mg ml‘I protease
`inhibitor, and homogenised in 150ml of 2M urea with
`0.05 M Tris—HCl, pH 7.4. The sample was left stirring over-
`night at 4°C and was then centrifuged at 10,000 g for 30 min.
`The supernatant was collected and the solid residue was
`washed once with the same volume of buffer. Then the
`supernatant and wash were combined, dialysed against
`0.15 M sodium chloride in 0.05 M Tris-HCI, pH 7.4 (TBS),
`for 6h, and subsequently against PBS and DMEM and
`finally centrifuged at 15,000 r.p.m. for 20 min to remove any
`residual
`insoluble material. The supernatant fraction was
`stored at —20°C in small aliquots until used in the
`experiments.
`
`MATRIGEL ENHANCEMENT OF METASTASIS
`
`229
`
`Tumour cell inoculation
`
`The tumour cells were harvested and resuspended in cold
`DMEM, mixed with an equal volume of cold (4'C) liquid
`Matrigel and immediately injected s.c. or in the mfp. During
`inoculation the stock cell suspension in Matrigel was kept
`chilled in an ice bucket to ensure that it did not begin to gel,
`as the extract readily does at 37'C. Mice in control groups
`were given subcutaneous and mfp injections of 10‘ tumour
`cells in 0.1 ml of DMEM with no Matrigel. For inoculation
`into the mfp the mice were anaesthetised with Metofane and
`a 5mm incision was made in the skin over the flank. The
`
`mfp was exposed and 0.1 ml of fluid containing 106 cells was
`injected into the tissue of the gland through a 27 gauge
`needle. By exposing the fat pad, we were able to ensure that
`the cells were injected into the tissue and not into the s.c.
`space. Tumour cells were injected s.c. remote from the mfp,
`in separate groups of animals.
`
`Twnorigenicity and metastasis in vivo
`
`The tumorigenicity and spontaneous metastatic eapability of
`the cells were observed following subcutaneous and mfp
`injections of l x 10‘ cells in 0.1 ml of DMEM into the lower
`right hind flank of nude mice. The animals were monitored
`every 2—3 days for over 4 months for the presence of a
`grossly visible and palpable mass at the injection site. Loeal
`primary tumour growth was evaluated by measurement of
`mean latent period and of eventual size at 140 days after
`injection. Autopsy was performed at 140 days, or sooner if
`the tumours were large or the host was ill or distreswd.
`Metastasis formation was studied by macroscopic examina-
`tion of all major organs of inoculated mice for secondary
`tumours and by histologieal examination of major organs
`and lymph nodes. The prevalence of metastasis in each batch
`of inoculated animals and the numbers of surface deposits
`seen in the lungs and other organs of each animal were
`recorded. Tissues were fixed in 10% neutral formalin and
`paraflin embedded for histologiml confirmation of macro-
`scopic observations.
`
`Remlts
`
`Eflect of Matrigel on metastasis
`
`Spontaneous metastasis from tumours formed at the site of
`inoculation by metastatic cell
`lines was reproducibly in-
`creased by Matrigel and more so by orthotopic inoculation
`(Table 1). The details are as follows:
`
`Polyclonal parent MDA-MB-435 line The prevalence of
`metastasis from tumours formed by the polyclonal MDA-
`MB—435 cell line was greater when the original inoculum was
`premixed with Matrigel before injection s.c. or in the mfp
`(Table I).
`
`lines Cl and C2
`Clonal cell lines Via the s.c. route, cell
`without Matrigel prodmd visible lung metastases in 21%
`(C1) and 37% (C2) of injected mice, respectively, and extra-
`pulmonary (hepatic) metastases in only one mouse (C2).
`When premixed with Matrigel these cell
`lines exhibited in-
`creased metastasis to the lung and to extrapulmonary sites.
`The prevalence of pulmonary metastasis in these groups of
`animals was 47% (Cl) and 56% (C2) respectively. C2 cells
`also produwd metastases in the liver, spleen and kidney (3 of
`16 mice,
`i.e. 19%), but Cl did not. Via the mfp route,
`without Matrigel, clone C l produmd lung metastases in 44%
`and C2 in 53% of mice. Only clone C2 produced a solitary
`extrapulmonary metastasis, and this was in the liver. Meta-
`static properties were incrmsed when cells were premixed
`with Matrigel and inoculated in this site. Many mice de-
`veloped easily visible lung colonies by 4 months. C1 pro-
`duwd pulmonary metastases in 60% of mice and C2 in 79%.
`C1 did not produce any extrapulmonary deposits, but with
`
`

`

`230
`
`L. BAO et al.
`
`Table 1 Effect of Matrigel on metastasis of MDA-MBA-435 cell
`
`lines
`
`Prevalence of
`pulmonary metastasir‘ (%)
`
`
`
` Cell line Injection route No Matrigel With Matrigel Number of colonies” P‘
`
`
`
`
`
`MDA-MB435 poly
`
`MDA-MB—435 C1
`
`MDA-MB-435 C2
`
`MDA-MB—43S C3
`
`MDA-MB—435 C4
`
`s.c.
`mfp
`
`s.c.
`mfp
`
`s.c.
`mfp
`
`s.c.
`mfp
`
`s.c.
`ml'p
`
`6/29 (21)
`7/17 (41)
`
`5/23 (21)
`7/16 (44)
`
`9/24 (37)
`8/15 (53)
`
`0/19
`0/15
`
`0/12
`0/13
`
`12/23 (52)
`12/18 (67)
`
`7/15 (47)
`12/20 (60)
`
`9/16 (56)
`15/19 (79)
`
`0/13
`0/14
`
`0/17
`1/17 (6)
`
`1 (0-3)
`1
`(0—5)
`
`2 (0—4)
`3 (0—10)
`
`<005
`'
`
`2 (0—5)
`2 (075)
`
`2 (0—8)
`3 (0—9)
`
`2 (0—4)
`2 (0—6)
`
`2 (0—14)
`4 (0—19)
`
`0
`0
`
`0
`0
`
`0
`0
`
`0
`0 (0—2)
`
`<005
`‘
`
`<005
`‘
`
`NS
`
`NS
`
`'Mice with metastases. Mice with tumours.
`
`t’Median and range. cSignificance tested with 2 x 2 12 test.
`
`8
`
`20
`
`O
`
`.a O
`
`E .
`
`5.
`'5
`‘5
`Ea
`‘6
`SO
`
`E3ca
`
`:o2
`
`Fig-e 1 Effect of Matrigel on the growth of tumours formed by
`polyclonal MDA-MB—435 cell
`lines. - . Without Matrigel;
`, with Matrigel.
`
`C2 metastases were seen in the liver, spleen or kidneys in
`26% of mice.
`Cell line C3 did not form any metastases via either the s.c.
`or the mfp route. Though cells premixed with Matrigel pro-
`duwd earlier and larger tumours in both s.c. and mfp sites,
`no lung or other deposits could be found in any mice. None
`was found in animals injected by either route without Mat-
`rigel. Similarly, clone C4 produced only two pulmonary de-
`posits in a single mouse. This was injected in the mfp with
`cells mixed with Matrigel. None of the remaining animals
`injected with these cells, with or without this matrix material
`in either site, had metastases in any organ.
`
`Eflect of Matrigel on the growth of twnours formed by
`polyclonal MDA-MB—435 cells
`
`l x 10‘
`tumour growth When aliquots of
`Subcutaneous
`polyclonal MDA-MB—435 cells premixed with liquid Matrigel
`or suspended in culture medium alone were injected into
`nude mice by either s.c. or mfp routes, all animals developed
`tumours. However,
`the growth of s.c.
`tumours formed by
`cells in culture medium alone, without Matrigel, was slowest.
`No visible tumours were apparent within a period of 20 days.
`The mean time required for a tumour to reach a size of 1 cm
`(latency period) was 135 days (i 10 days). The most slowly
`growing of these tumours reached a size of 10.4mm when
`the animal was killed 140 days after inoculation. Tumours
`appeared sooner and mched larger
`final dimensions in
`animals receiving cells premixed with Matrigel subcutane-
`ously. The latency period was 120 days (it 8 days).
`
`in culture medium
`Mammary tumour growth The cells
`alone,
`injected into mfp, produwd rumours with similar
`growth to s.c. tumours formed by cells mixed with Matrigel.
`The fastest growing tumours were found in the mice injected
`in the mfp with cells premixed with Matrigel (Figure 1).
`These were visible 20 days after inoculation. The latency
`period to 1 cm diameter was 80 days (i 5 days). The largest
`tumour found in this group had reached a diameter of
`29.9mm at 140 days after inoculation.
`
`Eflect of Matrigel on the growth of tumours formed by
`MDA—MB-435 cell clones with dzflerent metastatic potentials
`Four clonal cell lines derived from the parent MDA-MB—435
`line were selected to study the effect of Matrigel on the
`growth and behaviour of tumour cells with difierent meta-
`static potentials. Clones Cl and C2 were known from our
`previous assays to be metastatic via i.v. and s.c.
`routes.
`Conversely, clone C3 was completely non-metastatic by
`either i.v. or s.c. injection, and C4 produwd only two lung
`metastases in 1 out of 28 animals (3%) (Table I). The growth
`of tumours formed by cells premixed with Matrigel was
`faster than corresponding tumours formed by cells not mixed
`with Matrigel,
`in both s.c. and mfp sites (Figure 2). The
`
`8
`
`__
`SE
`°Ezo
`Ev
`35
`:0
`8810
`2%
`
`O
`
`8
`
`8
`
`O
`
`
`
`diameter(mm)
`
`
`
`
`
`Meanfinaltumour
`
`C3
`
`,
`/
`Z
`/
`%
`g
`%
`
`7/
`/
`Z
`/
`%
`g
`%
`
`»
`
`Z
`%
`g
`%
`
`8
`
`'5...
`/
`% gs
`%
`E20
`/ 3v
`Z *25
`/ to
`% cE'°
`Z 3%
`Z 5
`
`8
`
`O
`O
`3 Mean
`
`finaltumour
`
`diameter(mm) 3
`t\\\\\\\\\\\\\\\\\\\\\\\\‘
`
`Fig-e 2 Effect of Matrigel on the growth of tumours formed by
`cloned (C 1-4) MDA-MB—435 cell lines. - , Without Matrigel;
`, with Matrigel.
`
`degree of enhancement judged by final tumour size seemed to
`be the same whether the tumours were formed by metastatic
`cell clones or by non-metastatic ones (data not shown). There
`was no evidence of correlation of growth enhancement with
`metastatic capability in these four cell clones tested.
`
`

`

`D'
`
`.
`
`Previous studies by other groups have established that when
`the reconstituted basement membrane material Matrigel
`is
`co-injected s.c. with various human and murine tumour cell
`lines or with freshly dissociated primary human tumour cells
`the prevalence and the growth rates of loeal primary tumours
`are increased (Fridman et al., 1990, 1991; Pretlow et al.,
`1991). In the present study we found that Matrigel facilitated
`not only the growth but also the metastasis of tumours
`formed by the human breast earcinoma line MDA—MB-435
`in nude mice and by some of the clones derived from it.
`From this body of data it is evident that a judicious choice of
`tumour
`cell
`line,
`site of
`inoculation and facilitatory,
`mesenchymally derived,
`tissue constituents can now enable
`an investigator reliably to observe and analyse the metastatic
`spread of human tumour cells in the body of the nude
`mouse.
`
`information available to
`there is insufficient
`At present,
`define the active components in Matrigel which affect tumour
`growth and metastasis formation. Laminin, the major consti-
`tuent of Matrigel, has been shown to accelerate the attach-
`ment, activation and growth of tumour cells (Fridman et al.,
`1990, 1991), and to increase tumour metastases when injected
`intravenously with B16F10 melanoma cells (Barsky et al.,
`1984; Terranova et al., 1984). However, laminin alone does
`not promote tumour growth as effectively as Matrigel in the
`s.c. site (Fridman et al., 1990). Collagen IV, entactin and
`heparan sulphate proteoglywn are also biologieally active
`and may contribute to the growth, adhesion, spreading and
`motility of tumour cells (Aumailley & Timpl, 1986; Clement
`et al., 1989; Chakravarti er al., 1990). Further experiments
`involving sequential addition of such components to laminin
`in the medium in which the inoculated cells are suspended
`could help to analyse which of these constituents of Matrigel
`mediates its facilitatory effects on metastasis. The physical
`consistency of Matrigel
`is also more viscous than that of
`culture medium, and this may make some contribution to its
`observed effects. It is possible that this inhibits scattering of
`tumour cells after inoculation and thereby promotes relevant
`interactions between themselves and with surrounding cells
`(see below).
`Recent studies with different human and murine tumour
`cell
`lines have shown that
`the site of inoculation ean
`influence whether distant metastases are formed (Ahlering er
`
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`YAMADA. Y. (1989). Identification of a cell surface-binding pro-
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`FABRA. A.. NAKAJIMA. M.. BUCANA. CD. & FIDLER. U. (1992).
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`
`MATRIGEL ENHANCEMENT OF METASTASIS
`
`73]
`
`al., 1987; Bresalier et al., 1987; Morikawa et al., 1988; Price
`et al., 1990), although it is not clear how the loeal
`tissue
`environment exerts this effect. The present work confirms
`that the mfp is a more favourable site than the subcutis for
`the growth of mammary tumours (Miller et al., 1981; Price et
`al., 1990), and also for the expression of metastatic ability,
`there being a higher frequency of metastasis from the mfp
`tumours. Matrigel and mfp inoculation acted synergistimlly
`to facilitate all cell lines to produce larger tumours but did
`not induce the non-metastatic clones C3 and C4 to Mome
`metastatic, although the prevalence of metastasis by the
`parent
`line and by metastatic clones C1 and C2 was in-
`creased. Such findings indieate that pulmonary metastasis
`after
`inoculation, either
`s.c. or mfp, or with Matrigel,
`primarily depends on intrinsic properties of the tumour cells
`(Fidler, 1978; Tarin & Prince, 1979), but can be modulated
`by lowl microenvironmental factors.
`Recent
`results (Steeg et al., 1988; Hayle er al., 1993)
`indicate that metastatic events occur as a result of genetic
`disturbances which allow the inappropriate expression of
`genes that are silent in most cells, enabling the cells affected
`and their progeny to disseminate from the primary site. This
`new evidence suggests that metastasis may occur as a conse-
`quence either of failure of a negative regulatory event respon-
`sible for inhibiting inappropriate cell migration and distant
`colonisation, perhaps involving the 701123 gene (Steeg er al.,
`1988), or of the activation and up—regulation of a gene
`capable of dominantly conferring the phenotype (Hayle et
`al., 1993). In any event, once this balance has been disturbed,
`it appears that microenvironmental influences, such as the
`site of growth of the tumour cells or the constitution of the
`adjacent
`tissue matrix, ean accelerate tumour growth and
`dissemination. The mechanisms by which this effect is medi-
`ated deserve further investigation, to ascertain whether they
`might be susceptible to pharmacological hindrance, which
`could have the dual clinieal benefit of retarding the growth of
`secondary tumours as well as impeding further dissemina-
`tron.
`
`We wish to thank Dr J.E. Price for the gift of the MDA-MB—435 cell
`line and our colleague Mrs L. Summerville for valuable help with
`assembly of the manuscript. This work was partially supported by
`the Anthony Placito Fund for Medieal Research of Oxford Univer-
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