Cell Biology International Reports, Vol. 4, No. 6, June 1980 609

LOSS OF FIBRONECTIN IN HUMAN BREAST CANCER

LABAT-ROBERT J.“, BIREMBAUT P.**, ADNET J-J.** MERCANTINI F.* and ROBERT L.*

* Lab. Biochimie du Tissu Conjonctif (GR CNRS 40), Inst. Rech. Maladies Vasculaires, Fat. Med., Univ. Paris-Val de Marne, 94010 CRETEIL (France).

** Lab. Pol Bouin, Service d'Histologie et d'Anatomie Pathologique, Fat. Med., 51100 REIMS (France)

* On leave from Dept. Biochem., Medical School, Univ. of Pavia, PAVIA (Italy)

Abstract

The distribution of fibronectin was studied in normal human breast tissue and in breast tumors. In normal tissues and in benign tumors, fibronectin was present in basal laminae and on the cellu- lar surfaces of myoepithelial and secretory cells. A partial loss of (pericellular) fibronectin was seen in atypical dystrophies, and a complete disappearance of pericellular and basement membrane- bound fibronectin was observed in invasive adenocarcinoma.

Fibronectin designates a polymorphic glycoprotein or a family of glycoproteins found in biological fluids and tissues of vertebrates (Vaheri et al., 1978 ; Yamada and Olden, 1978). This glycoprotein has been identified in cultures of adherent vertebrate cells (Ruoslahti and Vaheri, 1974 ; Vaheri et al., 1976) and as a component of the extracellular matrix of cultured epithelial cells (Chen et al., 1977 ; Pearlstein et al., 1976 ; Quaroni et al., 1978). The distribution of fibronectin in normal human tissues has been recently described (Stenman and Vaheri 1978). Its role is still somewhat controversed. It seems to be involved in cell to cell adhesion as for instance the reaggregation of dissociated sponge cells (Labat-Robert et al., 1979) as well as in cell-matrix inter- action (Vaheri and Mosher 1978). The cell surface-associated fibronectin was reported to be lost in a score of malignant, trans- formed cells in culture (Vaheri et al., 1976). A decreased produc- tion of fibronectin in human astrocytomas and sarcomas (Vaherie et al., 1976 ;Mosher et al., 1977 ;Vaheri and Ruoslahti 1975) was also reported. Recently, human epithelial cell lines derived both from carcinomatous and non-malignant tissues were characterized with respect to the presence and distribution of fibronectin by immuno-

01980 Academic Press Inc. (London) Ltd.

610 Cell Biology International Reports, Vol. 4, No. 6, June 1980

Fig. l/a

Fig. l/b

Figure 1

Indirect immunofluorescent staining of human breast tiss (a) Fibrocystic disease. Fibronectin is visualised peric

around epithelial cells in acini (EC +), within peri basement membranes (BM) and in the stroma (S). (x 1 tical staining pattern is seen in normal breast tiss shown).

(b) Pericanalicular fibroadenoma. Strong pericellular st visible also on the vascular and pericanalicular bas membranes (BM) and in the stroma (S). (x 600).

,ue, :ellularly canalicular ZOO). Iden-

;ue (not

zaining, cement

Cell Biology International Reports, Vol. 4, No. 6, June 1980 611

fluorescence microscopy (Smith et al., 1979). These authors found fibronectin in extracellular matrix of cell lines derived from non- malignant tissues and primary carcinomas. In contrast, fibronectin was absent or poor in cultured cells from metastatic carcinomas (Smith et al., 1979).

No such systematic studies were undertaken to our knowledge on solid human tumors. The purpose of this work is to demonstrate the modified distribution and loss of fibronectin in malignant human breast tissues. No such changes were observed in benign tumors.

Materials and Methods

All tissue specimens were obtained as fresh surgical sam- ples. 1 normal breast (supernumerary gland), 5 fibrocystic diseases, 5 fibroadenomas (3 pericanalicular and 2 intracanalicular) and 9 carcinomas (7 invasive adenocarcinomas ; 1 infiltrating lobular cancer, 1 comedo-carcinoma) were selected (see Table I).

Anti-fibronectin serum was obtained in rabbits using a highly purified human plasma cold insoluble globulin preparation obtained by the gelatin-affinity chromatography procedure of Vuento and Vaheri (1979). The antisera were further purified by affinity chromatography on plasma proteins devoid of fibronectin linked to a CNBr-Sepharose column, followed by passage on a gelatin-Sepharose column.

Acetone-fixed (-2O"C, 20 mn) frozen sections were used for immunofluorescence staining. Fixed sections were washed twice for 10 mn in phosphate-buffered saline (PBS) and treated for 30 mn with rabbit anti-fibronectin serum diluted 1:5 to 1:50 in PBS. The PBS washes were repeated and fluorescein-isothiocyanate conjugated sheep anti-rabbit immunoglobulin (Laboratoires de 1'Institut Pasteur, Paris, France) diluted 1:5 in PBS was soaked on the sec- tions for 30 mn at room temperature. After two 10 mn washes with PBS, the preparation was mounted using a solution of Verona1 buffe- red glycerol. In control experiments, the addition of the anti- fibronectin rabbit serum was preceeded by the addition of purified human plasma fibronectin. No fluorescence was observed in these conditions. For routine histological examination, the conventional techniques were used : paraffin inclusion and hematoxylin-eosin- safran stain, orcein stain for elastin, Gomorri's reticulin stain and Masson's trichrome stain.

Results

In the normal mammary glands, fibronectin was visualized in acini and ducts within the basal lamina and on the cellular surfaces of myoepithelial and secretory cells. A much fainter fluorescent staining was observed in the stroma as thin strands around collagen bundles. Fibronectin was also visualized on the surfaces of fibroblasts and in the basement membranes of blood vessels.

In benign breast tumors such as fibroadenomas and in the fibrocystic diseases of the breast, the distribution of fibronect in

612 Cell Biology International Reports, Vol. 4, No. 6, June 1980

Fig. 2/a

Fig. 2/b

Figure 2

Indirect immunofluorescent staining of human breast tissue. (a) Invasive adenocarcinoma. Nearly complete loss of specific fibro-

nectin staining. Some weak residual staining can be seen in the stroma (S), especially around malignant cells (MC) (x 300).

(b) Zone of severe atypical hyperplasia at the periphery of an invasive adenocarcinoma. Notice the complete loss of pericellu- lar labeling of fibronectin (IA). Persistence of irregular, discontinuous staining of the basement membranes (x 600).

Cell Biology In terna tional Reports, Vol. 4, No. 6, June 1980 613

Fig. 2/c

Figure 2

(c) Comedo-carcinoma. Loss of pericellular staining of fibronectin. Persistence of irregular and partial staining of basement membranes (BM) and around some myoepithelial cells and fibro- blasts (arrow). No staining in the zone of infiltrating cancer (IC) (x 1 200).

was essentially unchanged (Fig. l/a, b). The cellular surfaces of myoepithelial and secretory cells were clearly labeled by the immunofluorescence staining. Numerous bands of fibronectin are visi- ble in the stroma, especially in fibrocystic disease.

This typical distribution of fibronectin is progressively modified in infiltrating adenocarcinomas. In the infiltrating areas of carcinoma, fibronectin was not seen, except in blood vessels and as faintly staining strands in the stroma (Fig. 2/a). Interesting changes in the distribution of fibronectin were seen in zones of severe atypical hyperplasia and in the non-infiltrating comedo- carcinoma (Fig. 2/b, c). Fibronectin was found as irregular, discon- tinuous strips within the basal lamina and on the cellular surface of some persistent myoepithelial cells but there was no pericellular labeling of severe dysplastic and malignant cells of the ducts.

The above findings clearly show the existence of an impor- tant modification of the distribution of fibronectin in solid epithelial neoplastic proliferations of the human mammary glands which were confirmed in about twenty individual cases (see Table I).

TAB

LE I

-

Dis

tribu

tion

of

fibro

nect

in

in

norm

al

hum

an b

reas

t tis

sue,

in

be

nign

an

d m

alig

nant

br

east

tu

mor

s.

Indi

rect

im

mun

oflu

ores

cent

st

aini

ng

perfo

rmed

w

ith

anti-

hum

an

plas

ma

CIG

. Th

e in

tens

ity

of

stai

- ni

ng

was

sc

ored

se

para

tely

fo

r ba

sem

ent

mem

bran

es,

cell

mem

bran

es

(per

icel

lula

r) an

d th

e fib

rous

st

rom

a.

Not

ice

the

pers

iste

nce

of

stai

ning

in

al

l th

ree

zone

s in

be

nign

br

east

di

seas

es

and

the

disa

ppea

ranc

e of

pe

ricel

lula

r an

d ba

sem

ent

mem

bran

e st

aini

ng

in

inva

sive

ad

enoc

arci

nom

as.

Site

of

di

strib

utio

n an

d in

tens

ity

of

fluor

esce

nce

Pat

ient

s

B.A

.

Lit

D:W

: D

.W.

D.W

.

E F:A

: D

.D.

P.P

. R

.D.

M.L

. D

.T.

A"*

);-

C:L

: M

.R.

P.P

. F.

V.

P.G

.

Lesi

ons

Sup

ernu

mer

ary

glan

d Fi

broc

ystic

di

seas

e Fi

broc

ystic

di

seas

e Fi

broc

ystic

di

seas

e Fi

broc

ystic

di

seas

e Fi

broc

ystic

di

seas

e Fi

broa

deno

ma

peric

anal

icul

ar

Fibr

oade

nom

a pe

rican

alic

ular

Fi

broa

deno

ma

peric

anal

icul

ar

Fibr

oade

nom

a in

traca

nalic

ular

Fi

broa

deno

ma

intra

cana

licul

ar

Inva

sive

ad

enoc

arci

nom

a In

vasi

ve

aden

ocar

cino

ma

Inva

sive

ad

enoc

arci

nom

a In

vasi

ve

aden

ocar

cino

ma

Inva

sive

ad

enoc

arci

nom

a In

vasi

ve

aden

ocar

cino

ma

Inva

sive

ad

enoc

arci

nom

a C

omed

o-ca

rcin

oma

Med

ulla

ry

carc

inom

a In

vasi

ve

lobu

lar

carc

inom

a

Bas

emen

t m

embr

anes

P

eric

eilu

lar

(epi

thel

ial

cells

)

tt tt tt tt t tt tt tt tt tt t t

Stro

ma

tt tt tt tt tt tt tt tt tt tt tt t t t t t t t tt tt t

Cell Biology International Reports, Vol. 4, No. 6, June 1980 615

Stenman and Vaheri (1978) found a weak fluorescence in basement membranes of acini and galactophoric ducts of normal breast tissue. In our experiment, the distribution of fibronectin in normal and dystrophic mammary glands and in fibroadenomas is identical to that found in normal breast tissue : the staining was observed in basal lamina and on cell surfaces.

In malignant tissues, the loss of fibronectin in infiltra- ting zones is well correlated to the absence of basal laminae as shown by Gomorri's stain for reticulin. Of particular interest is the absence of pericellular labeling in severe atypical hyperplasia and in malignant intra-ductal processes with the only persistance of irregular strips of fibronectin within the basement membranes.

Discussion

At least two distinct patterns of modified fibronectin distribution can be distinguished. The first is characterized by a loss of pericellular fibronectin but a persistance of an irregular and discontinuous labeling in the extracellular matrix as seen in the severe dysplasia, at the edge of infiltrating adenocarcinomas and in comedocarcinomas. The second more advanced ,modification seen in invasive growth is characterized by a greatly reduced or absent fibronectin staining, with only very weak stromal staining.

The disappearance of pericellular fibronectin in severe atypical hyperplasia revealed by immunofluorescence staining could be of practical interest in the early detection of malignant lesions.

In conclusion, the progressive disappearance of pericellu- lar and basement membrane localised fibronectin is well correlated to the invasive growth of breast carcinomas ; the loss of peri- cellular staining by immunofluorescence in dysplastic lesions may be a useful indication for tracing eventual malignant transforma- tions and incipiant invasion.

Acknowledgements

The skillful technical assistance of M. J.C. Derouette and of J. Arnaudies is thankfully acknowledge. Supported by C.N.R.S. (GR N" 40).

References

Chen, L.B., Maitland, N., Gallimore, P.H. and McDougall, J.K. (1977) Detection of the large external transformation sensitive protein on some epithelial cells. Exp. Cell Res., 106, 39-46.

Labat-Robert, J., Lethias, C.

Pavans de Ceccatty, M., Robs, L., Auger, C., and Garrone, R. (1979) Surface glycoproteins of

sponge cells : presence of a fibronectin-like protein on diffe- rentiated sponge cell membranes, its role in cell aggregation. In : Schauer et al. Stuttgart.

(eds) Glycoconjugates, pp. 431&432, G. Thieme,

616 Cell Biology International Reports, Vol. 4, No. 6, June 1980

Mosher, D.F., Saksela, O., Keski-Oja, L. and Vaheri, A. (1977) Distribution of a major surface-associated glycoprotein, fibro- nectin, in cultures of adherent cells. J. Supramol. Struct., 6, 551-557.

-

Pearlstein, E., Hynes, R.O., Franks, L.M. and Hemmings, V.J. (1976) Surface proteins and fibrinolytic activity of cultured mammalian cells. Cancer Res. 36, 1475-1480.

Quaroni, A., Isselbacher, K.J. and Ruoslahti, E. (1978) Fibronectin synthesis by epithelial crypt cells of rat small intestine, Proc. Nat. Acad. Sci. USA, 75, 5548-5552.

Ruoslahti, E. and Vaheri, A. (m74) Novel human serum protein from fibroblast plasma membrane, Nature 248, 789-791.

Smith, H.S., Riggs, J.L. and Mosesson,M.W. (1979) Production of fibronectin by human epithelial cells in culture. Cancer Res., 39, 4138-4144.

Steiiiiian, S. and Vaheri, A. (1978) Distribution of a major connec- tive tissue protein, fibronectin, in normal human tissues. J. Exp. Med., 147, 1054-1064.

Vaheri, A. and Ruoslahti, E. (1975) Fibroblast surface antigen pro- duced but not retained by virus-transformed human cells. J. Exp. Med., 142, 530-535.

Vaheri, A., Ruoslahti, E., Westermark, B. and Ponten, J. (1976) A common cell type specific surface antigen in cultured human glial cells and fibroblasts loss in malignant cells. J. Exp. Med. 143, 64-72.

Vaher'i, A. and Mosher, D.F. (1978) High molecular weight cell surface-associated glycoprotein (fibronectin) lost in malignant transformation. Biochem. Biophys. Acta 516, l-25.

Vuento, M. and Vaheri, A. (1979) Purificatz of fibronectin from human plasma by affinity chromatography under non-denaturing conditions. Biochem. J. 183, 331-337.

Yamada, K. and Olden, K. (lm) Fibronectin-adhesive glycoproteins of cell surface and blood. Nature, 275, 179-184.

Received: 31st March 1980 Accepted: 21st April 1980