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Research in VeterinaryScience 1995, 58, 272-276

E s t a b l i s h m e n t o f a c el l l in e ( M C M - B 2 ) f r o m a b e n i g n m i x e dt u m o u r o f c a n in e m a m m a r y g la n d

B . P . P R I O S O E R Y A N T O , S . T A T E Y A M A , R . Y A M A G U C H I , K . U C H I D A , De par tm en t o f Ve ter inary Pa tho logy ,Fac ulty o f Agrieulture, M iyazak i U niversi ty, Miyazala" 889-21, Japa n

S UMMARYA cell line was established from a b enign mixed tumour of the canine mammary gland. Light microscopy of the cells cultured onplastic dishes revealed monolayer colonies. Cells that grew with in the collagen gel matrix formed larg e three-dimensionalcolonies with a branching pattern. Immunohistochemically, h ese cells reacted intensely with anti-vimentin antiserum and mildlywith anti-desmin antiserum. Ultrastruc~tral examination revealed a large nu cleus, intraeytoplasmic organelles and intermediatefilaments, wh ich varied amon g cells. The cells possessed an abnorm al chromosome num ber, an average o f 80 per cell.Histologically, the xenografted tumour o f cultured cell s was similar to anaplastic carcinoma and reacted strongly with anti-vimentin antiserum, mildly with anti-desmin antiserum, and weakly with anti-keratin antiserum. The average chromo some num-ber o f cells form the xeno grafted tumour was the sam e as that of the original cultured cells. These findings suggest that the cellline might be de rived from stem cells or atypical cells, and that it should be u seful as a mo del for the study o f cell differentiationand proliferation in canine mam mary mmours.

M A M M A R Y t u m o u r s ar e c o m m o n i n do g s, ra n k i ng s e c o n di n p r e v a l e n c e a f t e r s k i n t u m o u r s ( B r o d e y e t a l 1 9 8 3 ,M a d e w e l l a n d T h e i l e n 1 9 8 7 , M o u l t o n 1 9 9 0 ) . A b o u t 5 0 p e rc e n t o f t h e s e t u m o u r s a r e b e n i g n , a n d o f t h e s e , th e m i x e dt u m o u r i s t h e m o s t c o m m o n t y p e . T h e y g e n e r a l l y h a v e ac o m p l e x h i s t o l o g i c a l p a t t e r n a n d h a v e b e e n t h e s u b j e c t o fm a n y r e p o r t s ( F o w l e r e t a l 1 9 7 4 , B o s t o c k 1 9 7 5 , T a t e y a m aa n d C o t c h i n 1 9 7 7 , G i l b e r t s o n e t a l 1 9 8 3 , D e s t e x h e e t a l1 9 9 3 , H e l l m e n e t a l 1 9 9 3 ) . T h e c o m p l e x c e l l c o m p o n e n t so f c a n i n e m a m m a r y t i ss u e m a y b e r e s p o n s i b l e f o r t h es eh i s to log ica l fea tu res .I t h a s b e e n w e l l d o c u m e n t e d t h a t t h e h i s t o l o g i c a l o r i g i na n d b e h a v i o u r o f c a n i n e m a m m a r y g l a n d tu m o u r s a n d s u c hf e a t u r e s a s t h e i r s t e r o i d h o r m o n e r e c e p t o r s t a t u s m a y a f f e c tt h e r i sk o f m a m m a r y c a r c i n o m a i n a m a n n e r s i m i la r to t h a ti n h u m a n b r e a s t c a n c e r ( M a r t i n e t a l 1 9 8 4 , M o u l t o n 1 9 9 0 ) .a s a re s u l t d o g tu m o u r s m a y p r o v i d e s o m e a d v a n t a g e s o v e rr o d e n t t u m o u r s f o r i n c r e a s i n g t h e u n d e r s t a n d i n g o f t h ea e t io l o g y , p a t h o g e n e s is a n d t h e r a p y o f m a m m a r y ' t u m o u r s( M a d e w e l l a n d T h e i l e n 1 9 87 ). S o m e c o m p a r a t i v e s t ud i e sh a v e a l r e a d y b e e n r e p o r t e d ( S a s a d a i r a 1 9 7 6 , C h r i s p a n dS p a n g l e r 1 9 8 0 , R a y n a u d e t a l 1 9 8 1 , M a r t i n e t a l 1 9 8 4 ) .S i n ce t h e m a m m a r y g l a n d i s a c o m p l e x o r g a n c o n s is t in go f a m i x e d p o p u l a t i o n o f c e l ls , i n c lu d i n g s t e m c e l l s ( D a n i e la n d S i l b e r s t e i n 1 9 8 7 ) , i n v i t r o s t u d i e s h a v e a l s o e x p r e s s e dt h i s c o m p l e x i t y ( D u n n i n g t o n e t a l 1 9 8 3 , S c h m i d e t a l 1 9 8 3 ,T a t e y a m a et a l 1 9 9 0 ), a n d a n u m b e r o f p e r m a n e n t c e l l l i n esh a v e b e e n e s t a b l i s h e d i n v i t r o f r o m c a n i n e m a m m a r yt u m o u r s , m o s t o f w h i c h w e r e e p i t h e li a l c e ll s ( W o l f e e t a l1 9 8 6 , V a n d e r B u r g e t a l 1 9 8 9 ) . O w e n e t a l ( 1 9 7 7 ) e s t a b -l i s h e d t w o c e l l l i n e s , w h i c h w e r e i d e n t i f i e d a s f i b r o b l a s t sa n d e p i t h e l i a l c e l l s . H o w e v e r , t h e r e h a s b e e n o n l y o n er e p o r t o f t h e e s t a b li s h m e n t o f a c a n in e m a m m a r y t u m o u rc e l l l i n e w i t h a n i n t e r m e d i a t e - t y p e m o r p h o l o g y ( c e l l l i n e2 2 9 ) s u g g e s t i n g a s t e m c e l l ( H e l l m e n 1 9 9 2 ). T h e i d e n t i f i c a -t i o n a n d e s t a b l i s h m e n t o f s t e m c e l l i s o f i n t e r e st b e c a u s e o ft h e i r r e l e v a n c e to t h e b e h a v i o u r o f m a m m a r y c a n c e r a n dt h e ir p a r t ic i p a ti o n i n t h e d e v e l o p m e n t o f t h e m a m m a r yg l a n d . T h e p u r p o s e o f t h is s t u d y w a s t o e s t a b l i s h a c e l l li n e

d e r i v e d f r o m t h e s t e m c e ll s o f a c a ni n e m a m m a r y g l a n dt u m o u r f o r f u r t h e r s t u d y o f t h e d i f f e r e n t ia t i o n a n d p r o l i f e r a -t i o n o f c e ll c o m p o n e n t s i n c a n i n e m i x e d t u m o u r s .

M A T E R I A L S A N D M E T H O D SIso la t ion o f ce l l line

A t u m o u r m a s s 3 c m x 5 c m w a s o b t a i n e d s u r g i c a l l yf r o m t h e m a m m a r y g l a n d o f a 1 0 - y e a r - o l d f e m a l e p o i n te rd o g . T h e t u m o u r m a s s h a d a p p e a r e d t w o y e a r s p r e v i o u s l ya n d a n X - r a y e x a m i n a t i o n c o n f i r m e d t h a t t h e r e w a s am e t a s t a s i s i n t h e l u n g . H i s t o l o g i c a l l y , t h e t u m o u r w a s c o m -p o s e d o f a m i x e d p o p u l a t i o n o f c e l ls w i t h c h o n d r o m u c o i dt i ss u e . T h e m a j o r c e l l u la r c o m p o n e n t s w e r e e l o n g a t e d s p i n -d l e - s h a p e d a n d p o l y g o n a l c e ll s ( F i g 1 ). A d o u b l e - l a y e r e da r c h i t e c t u r e w a s c o m m o n i n t h e e p i t h e l i a l a n d m y o e p i t h e -l i al c e l l s o f t h e a c i n i. M i t o t i c f i g u r e s w e r e o c c a s i o n a l l y v i s -i b l e . F r o m t h e s e f i n d i n g s , t h e c a s e w a s d i a g n o s e d a s ab e n ig n m i x e d t u m o u r o f th e m a m m a r y g l a n d ( M o u l to n1990).T h e t i s s u e w a s m i n c e d a n d d i g e s t e d f o r e i g h t h o u r s a t3 7 C i n a h u m i d i f i e d a t m o s p h e r e o f 5 p e r c e n t c a r b o n d i o x -i d e i n a i r w i t h 4 m g m 1 - 1 c o l l a g e n a s e ( W a k o ; 2 3 2 U m g - 1)i n D u l b e c c o ' s M o d i f i e d E a g l e ' s m e d i u m ( D M E ) a n d H a m ' sN u t r i e n t M i x t u r e F - 1 2 ( S i g m a ) c o n t a i n i n g 1 0 p e r c e n t f e t a lca l f s e ru m (F CS ) , 100 iu m 1-1 pen ic i l l in and 100 g g m 1-1s t r e p t o m y c i n . T h e d i g e s t e d t i s s u e w a s f i l t e r e d t h r o u g hn y l o n m e s h c l o t h ( 8 0 g m ) , c e n t r i f u g e d a t 1 8 4 g f o r 1 0 m i n -u t e s , a n d c u l t u r e d a s d e s c r i b e d b y T a t e y a m a e t a l ( 1 9 9 0 ) .B r i e f l y , t h e i s o l a t e d c e l l s w e r e c u l t u r e d i n 9 0 m m d i a m e t e rp l a s t i c d i s h e s ( S u m i l o n , S u m i t o m o B a k e l i t e M e d i c a l ) i nDME/F -12 m edium conta in ing 10 pe r cen t F CS , 100 iu m 1-1p e n i c i ll i n , 1 0 0 g g m 1 - 1 s t r e p t o m y c i n , 1 0 g g m 1 - 1 i n s u li n ,a n d 1 ~ g m 1 - 1 h y d r o c o r t i s o n e t o g e t h e r w i t h 1 8 x 1 8 m mc o v e r s l i p s f o r f u r t h e r i m m u n o h i s t o c h e m i c a l a n d e l e c t r o nm i c r o s c o p i c a l s t u d i e s . T h e c u l t u r e d i s h e s w e r e m a i n t a i n e di n 5 p e r c e n t c a r b o n d i o x i d e i n a i r a t 3 7 C a n d o b s e r v e d

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Canine cell line rom benign mixed tumour 273

FIG 1 Histological appearan ce of the original canine tumour showing spindle cell proliferation surround ing the alve-oil (A) and cartilage formation (B). Haematoxylin and eosin x 100

daily with a phase-contrast microscope (Nikon, DIAPHOT-TMD).The cel ls were subcultured by washing them with 0.2mM ethylenediamine te tra-acet ic acid in phosphate-buffered sal ine (EDTA-PBS)and digested with 0.05 per centtrypsin in EDTA-PBS, at 37C for seven minutes. A DME/F-12 medium containing 10 per cent FCS was added to stopthe trypsinisation. Viable cells were diluted to 5.0 x 105cells m1-1, and plated on a 50 mm dish. The cells werestocked in a med ium consist ing of DME/F-12 containing 10per cent FCS and 10 per cent dimethylsulphoxide (DMSO)and stored at -80C .Agar c loning

Freshly trypsinisised single cells (1.0 x 104) were layeredin 2 ml of a 10 per cent FCS mediu m containing 0.375 percent (w/v) agar in a 50 mm dish. This layer was placed ontop of a 2 ml baselayer of the same medium containing 0-5per cent agar. The cel ls were incubated at 37C in a humid-ified atmosphere containing 5 per cent carbon dioxide inair; after 14 days colonies larger than 10 cells were scored(Van der Burg et a l 1989).Growth in collagen

Acid-soluble type I col lagen solut ion from porcine ten-don (Cellmatrix IA; 3 mg m1-1, pH 3.0) was used andrecons t i tu ted according to the manufac ture r ' s recommen-dations. For fixed gel-embedded cultures, the cel l suspen-sion was centrifuged at 184 g for five minutes, and 1.5 mlof collagen solut ion was added to the cel l precipi ta te . Thecell suspension with collagen gel was spread on to a 35mm dish, incubated for 15 minutes, and overla id with 2 mlDME/F-12 supplem ented w ith 10 per cen t FCS, andobserved dai ly by phase-contrast microscopy. For a float-ing gel-embedded culture , the cel l cul ture was prepared asfor the f ixed ge l -embedded cul ture be fore i t was o ver la idwith DME/F-12, and the gel was then released from thedish wall.

Electron microscopyThe cel ls were grown on coversl ips, and the gel withembed ded cel ls was fixed with 1.5 per cent glutaraldehydein 0.1 M cacodylate buffer, pH 7.4, for one hour and thenwith 1"5 per cent osmium tetroxide. After dehydrat ion ingraded ethanols, the Cells were embed ded in Spurr ' s resin.Ultra thin sect ions were sta ined with uranyl acetate and leadcitra te and examined with an electron microscope (HitachiH-80 0 MU).

ImmunohistochemistryThe fol lowing primary antibodies were used: mousemonoclonal ant ibodies (mAb) against human a-smoothmuscle act in, human vimentin (Dakopatts), human oestro-gen receptor (Novocastra Laboratories) and rabbit ant iseraaga ins t human kera t in and human a -desmin (DakoCorporat ion). A coversl ip with a t tached cel ls was washedin PBS, fixed in cold aceto ne (-20C) and stored in a freezerfor 30 minutes. Th e cel ls were reacted w ith each o f the pri-mary antibodies overnight a t 4C. The at tached antibodieswere visualised by an avidin-biot in-peroxidase complex(ABC) system (Vectasta in, Vect or Labo ratories).

Growth curve and chromosom e studiesThe cel ls were plated in t ripl icate a t a concentrat ion of5-0 x 102 cells m1-1 in a 35 mm dish. The cells were cou nt-ed every day for five days. Doubling t imes were deter-mined during the expo nentia l growth phase (Campling et a l

1992). For ch romo some studies, the cel ls were treated with0.1 ~tg m1 -1 demecolcine (Sigma) for three hours,t rypsinised, washed and treated with a hypotonic solut ionof 0.075 M potassium chloride and then fixed withmethanol-acet ic acid. Preparat ions of cel ls were a ir driedand sta ined with Giemsa. Chromosomes spread from 150metaphases were counted and the results averaged (Gibsonet al 1991).

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274 B. P. Priosoeryanto, S. Tateyama, R. Yamaguchi, K. Uchida

FIG 2: Cell culture a t mid-passage (45th) showing a subconfluent mono layer with long cytoplasmic bridges (A) andduct-like structural a ppe ara nce on the collagen ge l culture (B). Phase-contrast; A x 316, B x 164

Growth in athymic nude miceA suspension o f 5 x 10 7 cells was inocula ted subcuta-neou sly into six nude mice (KSN nude SLC strain, Nippo nSLC Corporation). Seven weeks after inoculation, all themice w ere killed, and the nodules that had developed at theinjection site were processed for routine histological and

immunohistochemical examinations, and the determinationof chromosome number .

R E S U L T SLight and electron microscopy

Light microscopy of the cells grown on plastic dishesrevealed that they were elongated to polygonal in shape,had large nuclei and often two or more prominent nucleoli.They forme d monolayer colonies (Fig 2a) but did not formany organoid structures. A comm on feature of these cellswas their interconnection by long cytoplasmic bridges. Thecells that grew on the gel surface showed a monolayer or

multilayer appearance. The cells within the collagen gelformed three-dimensional colonies, organoid structureswith a branc hing pattern an d duct-like structures (Fig 2b).Ultrastructural studies revealed large nuclei, numerousfree ribosomes, endoplasmic reticulum, mitochondria andintermediate filaments (Fig 3). These morphological andultrastructural characteristics of the cultured cells weremaintained during extended periods of growth in culture formore tha n 70 passages over a year.Immunohistochemistry

The cultured cells were strongly stained with anti-vimentin mAb (Fig 4) and mildly with anti-desmin anti-serum, but were negative for the antibodies against keratinand oestrogen receptor. Two types of staining patterns forvimentin-positive cells were encountered, one running par-allel to the cell margin and another forming a networkthroughout the whole Cytoplasm. The same reactivitieswere also expressed in the cells that grew on the fixed andfloating gel cultures.

FIG 3: Ultrastructure o f the cultured cells, showing discontinuou s bord erbetween cell surface and collagen gel (CG). x 6000FIG 4: Immunohistochemical examin ation of the cultured cells showing p osi-tive reaction to vimentin antibody. Avidin biotin peroxid ase complex method x27 5

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Canine cell line fro m benign mixed tumour 275

Growth in a thymic nude miceAll the mice inoculated with the cultured cells had deve-loped a tumour mass around 0.3 cm in diameter within twoweeks o f inoculation. The tumours grew rapidly to be 1 cmx 2 cm seven weeks after inoculation, when the mice w erekilled. Histologically, the mm our was composed o f oval toelongated small and large pleomorphic cells with bizarrenuclei and numerous threads of chromatin (Fig 5). Thecytopl asm was eosinophilic and mitotic figures were fore" oseven per high power field. The immunohistochemicalexamination of xenografted tissues showed that they werestrongly reactive with anti-vimentin mAb, mildly with anti-desmin antiserum, and weakly w ith anti-keratin antiserum.In vitro immunohistochemical and chromosome studies ofcultured cells obtained from the xenografted mmourrevealed the same results as in the original cultured cells.

Growth and chromosome s tudiesA growth curve was plotted for cells growing in mediasupplemented with 10 per cent Fcs at the 69th passage (Fig6). The average time for population doubling was eighthours, and a confluent monolayer was attained after 48hours. Observations of this cell line showed that fetal calf

FIG 5: Histological ap peara nce of xeno grafted tumou r showing large round tospindle-shaped cells. The large round cells were wea kly positive for keratinwhe reas the spindle-shaped cells were negative. Haematoxylin and eosin x143

10 7

10 6

lO sZ

10 4

r~ 10 3

1 0 20 1 2 3 4 5 6 7

D A Y SFIG 6: Growth cu rve of MCM-B2 cells growing in DME/F-12 supplementedwith 10 per cent FCS at the 69th passage. Each point indicates the mea n ofthree determinations

serum was required for its growth, with the best concentra-tion being 10 per cent. The cell line was able to grow andform colonies in soft agar culture with a cloning efficienc ythat ranged fro m 4.1 to 20.2 per cent.The analysis of the chromosome numbers in 150metaphase spreads of the cells from the early (6th), middle(38th) and late (67th) passages and also from the xenog raft-ed tumour, revealed a mo dal numbe r of 80, a slight increasefrom the normal num ber (2N=78).

DISCUSSIONThe cell l ine established was name d Miyazaki Universitycanine mammary gland mixed turnout, and designatedMCM-B2. The isolation and maintenance of this turnoutcell l ine was achieved by adapting dissociative techniquesto obtain significant improvements in cell cultivation. Themethods used have been extended to other cell types with

the same results (data not shown).The MCM-B2 cells showed intense immunoreactivitywith a mA b for vimentin, which is know n to be an interme-diate filament of mesenchyma l cells in vivo (Virtanen et al1981), and m ild imm unoreactivity with anti-desmin anti-serum, but they were negative for anti-keratin antiserum.The staining pattern for vimentin was in agreement withthat of an earlier report (Den[: et al 1985). The immuno-histochemical and morphological examinations confirmedthat the morphologic al characteristics of the M CM-B 2 cellswere in almost complete c onform ity with those o f fibrob-lasts. In contrast, whe n the cells were transplanted intonude mic e, they show ed a similarity to the anaplastic carci-noma described by Hampe and Misdorp (1984), and alsoexpressed keratin intermediate filament, known as a hall-mark of epithelial cells (Henderson and Webber 1981),whereas the cultured cells obtained from xenografted tissuedid not express keratin. The authors' experiments and oth-ers (Rohol et al 1991, Hellmen 1992) have shown that cellsin culture can exhibit immunohistochem ical phenom ena notobserved in the original tissue or transplant cells, both innormal and malignant cells, for example, the expression ofkeratin. The expression of different types of intermediatefilament in cell culture, might indicate phenotypic diversity(Lichtner et al 1987) and different stages of developmentfrom stem cells , as shown in the rat mammary cell l ineRA MA 37 (Dunnington et al 1983) and huma n breast carci-nom a cell line PMC 42 (White head et al 1983a).There are pluripotent stems cells located in the mam maryend bud, defined as cap cells which can differentiate intoboth mammary ductal epithelial and myoepithelial cells(Williams and Daniel 1983). There are also unidentifiedcell types in their associated tumours, consisting of cells ina state of differentiation or of different phenotypes(Hellmen 1992). Attempts to identify mam mary stem cellsin clonal culture have been reviewed by Rudland et al(1980b) and Whit ehea d et al (1983a, b). Transplantation ofthe Ram a 25 cell l ine, whose basic morpholo gy is cuboidal,into nude mice gives rise to tumours that may containregions of both spindle-shaped and epithelial cells, againsuggesting that these cells are able to differentiate (Rudlandet al 1980a). It is possible that the MCM-B2 cells differen-tiated into epitheliaMike cells wh en transplanted into nudemice, and adopted a low er degree of differentiation duringcell culture. This idea was supported by the differentexpression of intermediate filaments with the same chro-mosome number in the original and xenografled cultured

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276 B. P. Pr iosoeryan to, S. Tateyama, R. Yamagu chi , K. U chida

c e l l s . The que s t i o n whe t he r c u l t ur e d m a m m a r y c e l l sbe c o m e l e s s d i f f e r e nt i a t e d i n v i t r o ha s no t y e t be e na n s w e r e d ( H e l l m e n 1 9 9 2 ) .

A c c o r d i n g t o th e c h r o m o s o m e n u m b e r o f th e M C M - B 2c e l l , w h i c h w a s h y p e r p l o i d , v a r i a t i o n s i n t h e c h r o m o s o m en u m b e r h a v e u s u a l l y o c c u r re d i n ca n i n e m a m m a r y m m o u r s( H e l l m e n e t a l 1 9 8 8 ) a nd a l s o i n e s t a b l i s he d c e l l l i ne s( O w e n e t a l 1 9 7 7 , No r v a l e t a l 1 9 8 4 , W o l f e e t a l 1 9 8 6 ) .The s e v a r i a t i o ns m a y be a s s o c i a t e d wi t h t he i ns t a b i l i t y o fc h r o m o s o m e s t h a t o c c u r s d u r i n g m m o u r p r o g r e s s i o n( H e l l m e n 1 9 9 2 ) .The i de nt i f i c a t i o n o f s t e m c e l l s i s i m po r t a nt be c a us e , i nr a ts , the i nc i de n c e o f t um o ur s i s c o r r e l a t e d d i r e c t ly wi t h t hede ns i t y o f t he t er m i na l e nd bud s . The s us c e pt i b i l i ty o f t hem a m m a r y g l a n d t o c a r c in o g e n s i s d u e n o t o n l y t o t h e p re s -e nc e o f t e r m ina l e nd bud s a s und i f f e r e nt i a t e d s tr uc tur e s buta l s o t o t he i r b i o l o g i c a l pr o pe r t i e s s uc h a s t he i r h i g h m i t o t i ca c t i v i t y a nd h i g h DNA l a be l l i ng i nde x . The i nc i de nc e o ft u m o u r s d e c r e a s e s m a r k e d l y w h e n t h e m a m m a r y g l a n dunde r g o e s t o t a l d i f f e r e nt i a t i o n , a nd t h i s l o we r pr o l i f e r a t i v ea c t i v i t y s e e m s t o be o n e o f the pr o pe r t i e s t ha t a c c o un t f o ri t s l o we r s us c e pt i b i l i t y t o c a r c i no g e ns ( Rus s o e t a l 1 9 7 9 ) .The po t e nt i a l t o d i f f e r e nt i a t e i n v a r i o us d i r e c t i o ns de pe ndso n t he i n t r i ns i c c har a c te r is ti cs o f t he t um o ur i n c o m bi na t i o nwi t h e nv i r o nm e nt a l s t i m ul i . I t a ppe a r s t ha t M CM - B2 , whe nt r a ns p l a nt e d i n t o nude m i c e , s ho ws a n e p i t he l i a l - l i ke c e l ld i f f e r e nt i a t i o n , t ha t m a y be i n f l ue nc e d by t he s u i t a b l e i nv i v o e n v i ro n m e n t . T h e h i s t o g e n e s i s o f M C M - B 2 i s , t h er e-f o r e , no t c le a r . Th e a v a i l a b l e i n f o r m a t i o n po i n t s t o a m ul t i -po t e nt i a l c e l l t y pe , wh i c h i s no t y e t kno wn, a r i s i ng dur i ngt u m o r i g e n e s i s i n c a n i n e m a m m a r y m i x e d t u m o u r s . Ak n o w l e d g e o f t h e m o r p h o l o g i c a l a n d t u m o r i g e n i c b e h a v -i o u r o f M C M - B 2 i n n u d e m i c e s h o u l d h e l p t o c la r i fy t h isque s t i o n a nd s uc h s t ud i e s a r e no w i n pr o g r e s s .

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