Immunohistochemical Study of the Expression of Human Milk ...cancerres.aacrjournals.org/content/canres/44/5/2016.full.pdf · Formalin-fixed, paraffin-embedded blocks of hu man tissues

[CANCER RESEARCH 44, 2016-2022, May 1984]

Immunohistochemical Study of the Expression of Human Milk Fat GlobuleMembrane Glycoprotein 701

Ashraf Imam, Clive R. Taylor, and ZoltÃ¡nA. TokÃ©s

Departments of Biochemistry [A. I., 2. A. T.] and Pathology [C. R. T.], University of Southern California, School of Medicine and Comprehensive Cancer Center, LosAngeles, California 90033

ABSTRACT

Human milk fat globule membrane, which is said to derivefrom apical plasma membrane of secretory epithelial cells inbreast, was analyzed by sodium dodecyl sulfate-twordimensionalgel electrophoresis. More than 35 components were detected inthe gels. One of the major glycoproteins with an apparentmolecular weight of 70,000, human milk fat globule membraneglycoprotein, was purified to homogeneity. The pattern of distribution of this glycoprotein in tissues was studied using polyclonalrabbit antibodies to the purified component. The localization ofthe antigen was accomplished by an indirect immunoperoxidasestaining method. Normal mammary epithelial cells display thisantigen mostly on the apical plasma membrane, whereas poorlydifferentiated breast carcinoma cells retained it predominantly inthe cytoplasm. These observations suggest that the properinsertion of this glycoprotein into an apical membrane domainmay be impaired in malignant tumor cells. In addition, a smallpopulation of tumor cells in each case examined failed to expressdetectable amounts of this component, indicating the presenceof antigenic heterogeneity among the tumor cell population.

INTRODUCTION

The mechanisms leading to morphological and functional alterations in the plasma membrane of malignant cells in breasttissue are fundamental issues that remain unanswered. Constituents of this membrane domain may play an important role incell-to-cell recognition and in the maintenance of defined tissue

structures (18). Lack of progress toward the molecular characterization of these components has in part been attributable totheir unavailability in purified form. A readily available source ofthe macromolecules from plasma membranes of mammary epithelial cells is the milk fat globule (10). The cream fraction of milkconsists of fat droplets which are stabilized by an externalmembrane. This membrane layer, known as the MFGM,2 is

derived mainly from the apical domain of mammary secretoryepithelium (2,4,15,16,17,19,20). Therefore, MFGM representsa valuable source of material for purification of membrane components and for investigation of their expression as a function ofaltered tissue architecture under various pathological conditions(7, 9).

Here we report the observation of the complexity of proteincomponents found in human MFGM as determined by 2-dimen-

sional gel electrophoresis. Polyclonal rabbit antibodies have beengenerated against the purified component MFGM-gp70 and used

1Supported in part by Grant CA 24645 from the National Cancer Institute.2The abbreviations used are: MFGM, milk fat globule membranes; MFGM-gp70,

milk fat globule membrane glycoprotein with a molecular weight of 70,000; SDS,sodium dodecyl sulfate; PBS, 0.02 M sodium phosphate buffer, pH 7.2, containing0.15 M NaCI; NP40, Nonidet P-40.

Received April 19, 1982; accepted January 26, 1984.

to study the pattern of expression for this glycoprotein in normaland malignant human tissue.

MATERIALS AND METHODS

Purification of MFGM-gp70. A major glycoprotein was purified from

the human MFGM. The purification procedures have been describedrecently and consisted of an extraction of MFGM with MgCI2 followedby lectin affinity chromatography and gel filtration in the presence ofprotein-disaggregating agents (8). The purified glycoprotein yields a

single band under reducing conditions on SDSipolyacrylamide gel electrophoresis and has an estimated molecular weight of 70,000 (8). Theglycoprotein was designated MFGM-gp70.

Production and Absorption of Antisera to MFGM-gp70. New Zea

land White rabbits were immunized with the purified glycoprotein. Immunization was initiated s.c. in the nuchal area with 1.0 ml of a mixtureof MFGM-gp70 (0.2 mg/ml) in 50% (v/v) complete Freund's adjuvant.

Secondary and subsequent booster immunizations were given biweeklywith incomplete Freund's adjuvant. Fifteen days after the last booster

injection, the rabbits were bled from their ear veins.The isolation of immunoglobulins from both the specific antiserum and

preimmune rabbit serum was carried out by precipitation with 40%saturation of ammonium sulfate at 4Â°.Such fractions were redissolvedin PBS and dialyzed against several changes of the same buffer at 4Â°.

The dialyzed fraction from specific antiserum was absorbed with amembrane preparation of normal human kidney. The membrane preparation was made by the method of Colcher et al. (5). One ml of theantiserum was mixed with 2 mg of protein of the membrane preparation.Following a 2-hr incubation at 4Â°, the suspension was centrifuged at

10,000 x g for 15 min, and the absorbed antiserum was recovered. Theabsorbed immunoglobulins were subsequently used for both immuno-

precipitation and immunocytochemical localization of this glycoprotein.lodination of Human Milk Fat Globule Membrane. MgCI? extract of

MFGM (8) was iodinated by the method of Greenwood ef al. (6). Briefly,50 ng of the extract in 50 n\ of 0.05 M sodium phosphate buffer, pH 7.4,were added to 0.5 mCi (0.49 PIM) of carrier-free sodium iodide (Amer-

sham/Searie Corp., Arlington Heights, IL). The reaction was initiated byadding 10 /t\ of freshly prepared chloramine-T (4 mg/ml in PBS) and was

allowed to proceed for 30 sec at room temperature with gentle shaking.The reaction was terminated by adding 25 /Â¿Iof sodium metabisulfite (12mg/ml in PBS). The iodinated protein was separated from free iodide bypassing the reaction mixture through a Sephadex G-50 column which

was equilibrated initially with PBS containing 0.5% (w/v) bovine serumalbumin and subsequently with PBS containing 0.1% (w/v) SDS.

Immunoprecipitation of MFGM-gp70. 125l-labeledproteins from hu

man MFGM and the immunoglobulin fraction of absorbed antiserum toMFGM-gp70 were used for immunoprecipitation. The labeled protein(approximately 10 ng of protein containing 1 x 105 cpm) was mixed with

50 n\ of immunoglobulin fraction from either specifically absorbed anti-serum or preimmune rabbit serum. The mixtures were incubated at 4Â°

for 16 hr. Following the incubation, a 10-fil suspension of Sepharose 48

coupled to Protein A from Staphylococcus (Pharmacia Fine Chemicals,Piscataway, NJ) was added to each reaction mixture. The samples wereincubated for a further period of 20 min and centrifuged at 4000 x g for5 min. Following the removal of supernatant by aspiration, the pellet was

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Expression of MFGM-gp70 in Breast Tissue

washed 3 times with PBS containing 10 HIMEDTA and 0.5% (w/v) NP40.The amount of radioactivity was determined in a Packard Auto-Gamma

scintillation spectrometer.SDS-Polyacrylamide Gel Electrophoresis. The material immunopre-

cipitated with the specific antiserum was subsequently analyzed bySDS:polyacrylamide gel electrophoresis. The pellet was solubilized in0.05 M Tris-HCI buffer, pH 6.8, containing SOS (membrane protein:SDSratio, 1:2.5, w/w) and 10 mM 2-mercaptoethanol, boiled for 3 min, and

centrifuged. The supernatant was subjected to electrophoresis in 7.5%polyacrylamide slab gels in the presence of SDS by the method ofLaemmli (11 ). Constant current of 30 ma was applied to the slab gel for3 to 4 hr until the dye front approached to within 1 cm of the end. Thegel was fixed and stained with 0.25% (w/v) Coomassie blue in a solutioncontaining 50% (v/v) isopropyl alcohol and 10% (v/v) acetic acid. De-

staining was performed in a solution containing 10% (v/v) isopropylalcohol and 10% (v/v) acetic acid. Stained gels were dried on a WhatmanNo. 3MM filter paper under reduced pressure, and the radioactivecomponents were visualized by fluorography.

Two-Dimensional Gel Electrophoresis. Both unfractionated MFGMand the purified MFGM-gp70 (8) were solubilized in 0.05 M Tris-HCI

buffer, pH 6.8, containing SDS (SDS:membrane protein ratio was maintained at 2.5:1, w/w) and 10 mM 2-mercaptoethanol. Subsequently, urea

(final concentration, 8 M) and NP40 (final concentration, 2%, v/v) wereadded to each sample. The above samples were diluted in equal volumeof solution containing 2% (v/v) NP40, 8 M urea, 10 mM 2-mercaptoethanol, and 20% (v/v) glycerol. The samples thus prepared were electrofo-cused in polyacrylamide gels by the method of O'Farrell (14). The samples

were focused at 100 V for 1 hr, 200 V for 1 hr, 400 V for an additional 2hr and, finally, 800 V for 2 more hr. After electrofocusing. the gels wereeither directly placed into 10% polyacrylamide slab gel of the Laemmlisystem (11 ) and subjected to electrophoresis or were preequilibrated in0.1 M Tris-HCI buffer, pH 6.8, containing 2.5% (w/v) SDS and 10 mM 2-

mercaptoethanol for 2 to 3 hr at room temperature before subjection tothe second-dimensional electrophoresis. The gels were stained with

Coomassie blue and destained as described above.Indirect Immunoperoxidase Staining Method. Immunoperoxidase

staining of the formalin-fixed, paraffin-embedded human tissues was

performed using the immunoglobulin fraction from the absorbed antiserato MFGM-gp70. The peroxidase-antiperoxidase staining method was

used (9, 17). Tissue sections were incubated for 30 min with 100 n\ ofdiluted immunoglobulin fraction (1 to 10 ^g of protein/ml) from eitherspecifically absorbed antiserum or preimmune rabbit serum. This concentration of antibodies was chosen on the basis of minimum nonspecificbackground staining.

For each experiment, 2 different controls were performed to ensurethe specificity of the reaction. These included the use of preimmunerabbit serum and the specific antiserum absorbed with MFGM-gp70 (1

mg/ml).Tissue Specimens. Formalin-fixed, paraffin-embedded blocks of hu

man tissues from normal and malignant breast lesions (infiltrating ductalcarcinoma), colon, kidney, liver, lung, prostate glands, salivary glands,stomach, sweat glands, spleen, thyroid glands, and uterus were obtainedfrom the surgical pathology files of the University of Southern California/Los Angeles County Medical Center. Sections stained with hematoxylinand eosin were reviewed to confirm the diagnosis. Additional 5-nm

sections were cut for the immunocytochemical studies.

RESULTS

Analysis of Proteins from Human MFGM by 2-Dimensional

Gel Electrophoresis. In order to separate the membrane proteins of MFGM by 2-dimensional gel electrophoresis, it was

paramount to establish a procedure to achieve a maximumsolubilization of the proteins. This was achieved by utilizing adetergentprotein ratio of 2.5. The unfractionated membranepreparations from isolated MFGM showed more than 35 Coo

massie blue-positive components by 2-dimensional gel electro

phoresis. Fig. 1 represents an example of stained gel. Theapparent pi values of most of these components ranged from 5to 8. One of the major glycoproteins, MFGM-gp70, which yieldsa single band under reducing conditions on 1-dimensional gel

electrophoresis (8), migrated as an unresolved streak, indicatingcharge heterogeneity (Fig. 2). The apparent isoelectric pointsranged from 6.0 to 6.4.

Purification of MFGM-gp70. The yield of this purified glyco-

protein was 0.5 to 1.0 mg from 100 ml of human milk. The finalpreparation was subjected to 2-dimensional gel electrophoresis

to ascertain its purity (Fig. 2).Immunoprecipitation and Slab Gel Electrophoresis of La

beled Proteins. The 125l-labeledproteins from MgCI2 extract of

MFGM revealed 4 major bands on SDS:polyacrylamide slab gels(Fig. 3, Lane A), which corresponded to the previously identifiedcomponents designated Bands 3, 12, 16, and 19 (8). The estimated molecular weights of these glycoproteins were 155,000,70,000, 52,000, and 39,000.

A component from the 125l-labeled protein extract was specif-

â€¢ISOELECTROFOCUSING"

MW)

205

11697

1

V)

Fig. 1. Two-dimensional gel electrophoretic patterns of proteins from humanMFGM. The separation of proteins from MFGM is shown. The gel was stained withCoomassie blue. Routinely, more than 35 Coomassie blue-positive componentswere seen in gels. The position of molecular weight markers is at the extremeacidic portion of the gel.

|G" X MU )

205

11697

â€¢ISOELECTROFOCUSING

ÃŒ

;/i

Fig. 2. Two-dimensional gel electrophoretic pattern of purified MFGM-gp70.The sample was prepared and analyzed as described in the text. Although theglycoprotein yields a single band on one-dimensional gel electrophoresis, it migratedas an unresolved spot with apparent isoelectric points that ranged from 6.0 to 6.4.

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A. Imam et al.

<MFGM-gp7O> ^fr

B.B>

B

Fig. 3. SDS-polyacrylamide gel electrophoresis of immunoprecipitated MFGM-gp70 as visualized by fluorography. Lane A, '25l-labeled components of the MgCI2

extract from human MFGM; Lane B, immunoprecipitated MFGM-gp70; Lane C,evidence that none of the components are precipitated by the preimmune serum.o.e., position of the tracking dye bromophenol blue.

ically precipitated with Â¡mmunoglobulin fraction of an absorbedantiserum to MFGM-gp70. This component migrated to the same

position as did Band 12 on the gel (Fig. 3, Lane B), establishingthe specificity of the antibodies. No immunoprecipitation wasobtained between the extract and preimmune rabbit serum (Fig.3, Lane C).

Immunocytochemical Localization of MFGM-gp70 on Hu

man Tissues. The glycoprotein retains its antigenic sites duringthe buffered formalin fixation and paraffin embedding of tissues.Such a stability facilitates the study of the distribution of thisantigen by immunocytochemical method in routinely preparedhistological sections. In normal and lactating breast tissues,MFGM-gp70 was predominantly localized on the luminal mem

brane of the epithelial cells lining the ducts (Fig. 4). Epithelialcells from lactating breast tissue stained more intensely than didthose from nonlactating breast. No significant staining of cytoplasm was observed. MFGM-gp70, under these conditions, wasnot detected in myoepithelial cells, fat and stromal cells, endo-

thelial cells, erythrocytes, lymphocytes in breast tissues, and innormal skin, lung, prostate, thyroid, and spleen tissues. However, the antibodies did bind with a varying degree of intensityto the luminal membrane of columnar epithelial cells in normalcolon, epithelial cells lining the collecting tubules of kidney,salivary gland, stomach, eccrine sweat gland, and sebaceousgland. Staining of either normal or neoplastic cells was notobserved when the specific antiserum was absorbed with

MFGM-gp70 or replaced by preimmune rabbit serum. The anti-

sera were subsequently absorbed with a membrane preparationof human renal medulla. Although the absorbed antisera failedto bind to cells in normal colon, salivary gland, and stomach,abolition of staining of cells in kidney, sweat glands, and sebaceous glands was not achieved.

Infiltrating ductal carcinomas of the breast revealed variablepatterns of MFGM-gp70 distribution. Mammary carcinoma cells

that were morphologically well differentiated showed more intense staining than did poorly differentiated cells. Staining wasmostly restricted to the luminal membrane of the epithelial cellsin well-differentiated mammary carcinomas that maintained a

ductal growth pattern (Fig. 5). Their pattern of staining wassimilar to that of normal and benign breast lesions. Staining ofboth moderately and poorly differentiated tumor cells was observed in a single lesion (not illustrated). In contrast, mammaryepithelial cells from 16 patients with poorly differentiated infiltrating ductal carcinomas showed less intense staining that waspredominantly localized in the cytoplasm (Fig. 6). Representativesections of these specimens contain a population of tumor cellswhich failed to display detectable amounts of the antigen. Thenegative cells ranged from 5 to 20% in a given tissue section.The observation indicates the cellular heterogeneity which maybe already present in a primary tumor site.

Metastatic mammary carcinoma cells in all of the 15 lymphnode specimens showed clumps of malignant cells displayingMFGM-gp70 in the cytoplasm (Fig. 7). In addition, stained breast

cancer cells were demonstrated in previously confirmed casesof metastasis to lung and liver, respectively (Figs. 8 and 9). Table1 summarizes the patterns of expression in 82 breast tissuespecimens. Of these 76 cases, 8 were from normal tissues, 5were benign tissues, 48 were primary infiltrating ductal carcinomas, and 15 were tissues from metastasis to lymph nodes.

At 2.5 to 5.0 ng of antibodies/tissue section, staining wasobserved in poorly differentiated stomach, colon, and renal ad-

enocarcinomas. Further dilution of antibodies reduced the intensity of immunoperoxidase staining in all of these tissues, including the breast. However, preferential staining of breast andsebaceous gland was observed only at 0.5 to 1.0 /Â¿gof antibodies/tissue section. These observations suggest that the majorityof the epitopes recognized by the antisera are not organ specific.

DISCUSSION

A rather complex pattern of membrane proteins from MFGM

Table 1Pattern of MFGM-gp70 expression in normal and malignant breast epithelial cells

Histological grading was performed according to the criteria of Bloom andRichardson (3). Sections were scored for the intensity staining on a scale from -

to3+.

Normal breastcellsBenignbreastcellsWell-differentiated

(Grade 1)cells(infiltratingductalcarcinoma)Moderately

differentiated (Grade2)cells(infiltrating ductal carci

noma)Poorlydifferentiated cells (infiltrat

ing ductalcarcinoma)MÃ©tastasesto lymph nodeOn

the cellmembrane3+a(8/8)3+

(5/5)3+(20/20)-

(6/20)2+

(11/12)-(1/12)-

(0/16)1+

(3/15)In

the cytoplasm-

(0/8)-(0/5)1+

(14/20)2+

(10/20)-(2/12)2+

(16/16)2+

(15/15)Not

detectable-(0/8)-

(0/5)-(0/20)-

(0/20)-(0/16)-(0/15)

a -, absence of staining; 1+, weak staining; 2+, moderate staining; 3+, intense

staining.





can be recognized by 2-dimensional gel electrophoresis. At least35 Coomassie blue-positive components can be detected. Some

of the major glycoprotein components were resolved into closelyrelated isotypes. Such microheterogeneity appears to be characteristic of glycoproteins, as demonstrated earlier (1). The complexity of pattern of the membrane proteins on gel may in partbe due to genetic variation among the individual donors, sincethe pooled milk was used for the preparation of MFGM.

The purified MFGM-gp70, which yielded a single band underreducing conditions on 1-dimensional slab gel electrophoresis,

migrated as an extended streak with apparent isoelectric pointsranging from 6.0 to 6.4 on 2-dimensional gel electrophoresis. No

other component was detected in the purified preparation. Unlikeother major glycoproteins of MFGM, this component could notresolved into distinct isotypes. The specificity of the antiserumwas established by showing that only MFGM-gp70 precipitated

by reacting the antiserum with a mixture of radiolabeledMgCI2:extract of the membrane. Therefore, the antibodies represent a specific reagent to study patterns of distribution ofMFGM-gp70 in mammary epithelial cells as a function of normal

stages of differentiation and pathogenesis.Prior to absorption, the antiserum to the purified membrane

component stained epithelial cells in normal tissues that includedbreast, colon, kidney, salivary gland, sebaceous gland, andstomach. In breast, luminal membrane of epithelial cells lining theducts and lobules were positive. Under these conditions, myo-

epithelial cells, cells in connective tissue, erythrocytes, and lymphocytes were negative. Since the cells which line the collectingtubules of kidney were strongly positive, the antiserum wasabsorbed with a membrane preparation of renal medulla. Theimmunoglobulin fraction of the absorbed antiserum remainedreactive to epithelial cells in normal breast, colon, kidney, andsebaceous gland. An additional absorption of the antiserum witheither the extract or a similar extract from salivary gland did notenhance its specificity. However, a narrow range of concentrations of the antibodies (0.5 to 1.0 /Â¿g/section)showed a preferential binding to the malignant cells in breast. At this concentration of antibodies, the only 2 other organs that remained reactivewere kidney and sebaceous gland. It is possible that shedantigen(s) may be passively adsorbed on the luminal membraneof the epithelial cells, lining the tubules of kidney. An increase inthe amounts of antibodies applied eliminated the preferentialstaining of the breast cells. Therefore, it appears that a few, ifany, epitopes on MFGM-gp70 which are recognized by the

polyclonal antiserum may be specific for the cells in the breast.The observations suggest that the glycoprotein could be ashared constituent of secretory epithelial cells which may bedevelopmentally related. Verification of the possible existence ofspecific epitope for breast would require the generation of monoclonal antibodies to this glycoprotein.

One of the applications of the antibodies would be their usefor the investigation of antigenic heterogeneity in the populationsof tumor cells both at the primary and metastatic sites. Normalbreast tissue showed the presence of heterogeneous expressionof antigens in different ducts and lobules although, within anyparticular duct, the expression on adjacent cells was remarkablyuniform. The observation of more marked antigenic heterogeneity of tumor cells is not attributed to artifacts in either tissueprocessing or staining method. An apparent trend of decreasingexpression of this component was observed from well to less or

poorly differentiated tumor cells in tissue sections. The negativecells were least frequent in sections from well-differentiated

carcinomas of the infiltrating duct and represented less than 10%of the population. In poorly differentiated cases, such cells comprised 20 to 30% of the total cell population. No clearly demonstrable increase in the extent of antigenic heterogeneity wasobserved among the metastatic cells in distant organs. However,a detailed study may help to ascertain whether any selectiveadvantage is gained by those cells, which lack in the expressionof the glycoprotein, in the processes of mÃ©tastases.

A correlation was observed between the patterns of MFGM-

gp70 expression on individual cells and the degree of differentiation of mammary carcinoma cells. In both normal epithelium andwell-differentiated malignant cells, the antigen was consistently

and predominantly present on the luminal membranes of thecells. By contrast, in poorly differentiated tumor cells, it waslocalized mainly in the cytoplasm. The differences in the patternsof distribution suggest the possibility that the spread of malignantcells at the primary site and the development of mÃ©tastasesmaybe related to an altered expression of this and possibly othercomponents in the plasma membrane. Indeed, studies haveshown that alterations in the expression of cell surface glycoproteins occur in malignant breast cells (7, 9, 12, 13). It is conceivable that the inability of the structural glycoproteins to be insertedinto the plasma membrane may attribute to the morphologicaldisorientation of malignant cells in tissue. The understanding ofthe mechanism for the inability to insert this antigen in the correctdomain of plasma membrane remains a challenging question.

REFERENCES

1. Anderson, N. L., and Anderson, N. G. Microheterogeneity of serum transferin,haptoglobulin, and a2-HS glycoprotein examined by high-resolution 2-dimensional electrophoresis. Biochem. Biophys. Res. Commun., 88: 258-264.1979.

2. Bargmann, W., Reisedauer, K., and Knoop, A. Z. Ãœberdie Morphologie derMilchsekretion. II. Zugleichene Kritik am Schema der sekretions Morphologie.Zellforsch. Mikrosk. Anat., 53: 545-568, 1961.

3. Bloom, H. J. G., and Richardson, W. Histological grading and prognosis inbreast cancer. Br. J. Cancer, 11: 359-377, 1957.

4. Ceriani, R. L., Thompson, K., Peterson, J. A., and Abraham, S. Surfacedifferentiation antigens of human mammary epithelial cells carried on the humanmilk fat globule. Proc. Nati. Acad. Sci. USA, 74: 582-586, 1977.

5. Colcher, D., Horan-Hand, P., Nuti, M., and Schlom, J. A spectrum of monoclonal antibodies reactive with human mammary tumor cells. Proc. Nati. Acad.Sci. USA, 78: 3199-3203,1981.

6. Greenwood, F. C., Hunter, W. M., and Glover, J. S. The preparation of 13'l-

labeled human growth hormone of high specific radioactivity. Biochem. J., 89:114-123,1963.

7. Howard, D. R., and Batsakis, J. G. Cytostructural localization of a tumor-associated antigen. Science (Wash. DC), 270: 201-203, 1980.

8. Imam, A., Laurence, D. J. R., and Neville, A. M. Isolation and characterizationof a major glycoprotein from milk fat globule membrane of human breast milk.Biochem. J., 793: 47-54, 1981.

9. Imam, A., and TokÃ©s,Z. A. Immunoperoxidase localization of a glycoproteinon plasma membrane of secretory epithelium from human breast. J. Histo-chem. Cytochem., 29: 581-584, 1981.

10. Keenan, T. W., Moore, D. J., Olson, D. E., Yunghans, W. N., and Patton, S.Biochemical and morphological comparison of plasma membrane and milk fatglobule membrane from bovine mammary gland. J. Cell Biol., 44: 80-93,1970.

11. Laemmli, U. K. Cleavage of structural proteins during the assembly of the headof bacteriophage T4. Nature (Lond.), 227: 680-685,1970.

12. Lerner, N., Anglin, J., and Nordquist, R. Cell surface antigens from humanbreast tumor cells. J. Nati. Cancer Inst., 60: 39-44, 1978.

13. Nordquist, R., Anglin, J., and Lerner, M. Antigen shedding by human breast-cancer cells in vitro and in vivo. Br. J. Cancer, 37: 776-779, 1978.

14. O'Farrell, P. H. High-resolution 2-dimensional electrophoresis of proteins. J.

Biol. Chem., 250: 4007-4021, 1975.15. Patton, S., and Keenan, T. W. The milk fat globule membrane. Biochim.

Biophys. Acta, 475: 273-309. 1975.16. Patton, S., and Trams, E. G. The presence of plasma membrane enzymes on

the surface of bovine milk fat globule. FEBS Lett., 74: 230-232,1971.

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A. Imam et al.

17. Taylor, C. R. Immunoperoxidase techniques. Arch. Pathol. Lab. Med., 102: 1983.113-121,1978. 19. Wooding, F. B. P. The mechanism of secretion of the milk fat globule. J. Cell

18. TokÃ©s,Z. A., Gendler, S. J., Imam, A., Rullano, T., and Ross, K. Deciphering Sci., 9: 805-821,1971.cancer-associated glycoproteins which may have diagnostic relevance. In: P. 20. Wooding, F. B. P. The structure of the milk fat globule membrane. J. Ultrastruct.Moloy and G. L. Nicolson (eds.), Cellular Oncology: New Approaches in Res., 37: 388-400,1971.Biology, Diagnosis, and Treatment, pp. 28-62. New York: Praeger Press,

sapS*v "Cv'~fe'rt^K^"â€¢-.

Fig. 4. Immunohistological localization of MFGM-gp70 in normal human breast tissue. The peroxidase-antiperoxidase staining method with antiserum to MFGM-gp70is described in the text. Normal duct from lactating breast showing the presence of MFGM-gp70 on the luminal membrane. Myoepithelial cells, fat cells, and stremai cellsare unstained. Mayer's hemalum countersign; x 500.

Fig. 5. Well-differentiated carcinoma of the breast. The well-differentiated gland shows a pattern of MFGM-<jp70 expression on the luminal membrane which is similarto that seen in normal breast lesions (see Fig. 4). The poorly differentiated cells show MFGM-gp70 in their cytoplasm, as indicated by the arrows. The stroma is completelynegative. Mayer's hemalum counterstain: x 312.





Fig.6. Poorly differentiated carcinoma of the breast. Poorly differentiated infiltrating ductal carcinoma of breast shows the presence of MFGM-gp70 within thecytoplasm. The stromal components are not stained. Mayer's hemalumcounterstain; x 321.

Fig.7. Metastatic breast carcinoma cells in axillary lymph node. The clumps of malignant cells show the presence of MFGM-<jp70in their cytoplasm in a patternwhich is similar to that of poorly differentiated malignantcells (see Fig. 6). Lymphocytes were clearly negative. Mayer's hemalumcounterstain; x 400.

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A. Imam et al.

>Fig.8. Metastatic breast carcinoma cells in lung. Malignant breast cells showing the presenceof MFGM-gp70 in their cytoplasm. Note that some of the tumor cells

displayed less antigen, as indicated by arrows. Mayer's hemalumcountersign; x 312.Fig.9. Metastatic breast carcinoma cells in liver. Breast tumor cells forming clumps in the liver showing the presence of MFGM-gp70 in the cytoplasm. Mayer's

hemalumcounterstain; x 312.




1984;44:2016-2022. Cancer Res Ashraf Imam, Clive R. Taylor and Zoltán A. Tökés Fat Globule Membrane Glycoprotein 70Immunohistochemical Study of the Expression of Human Milk

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Immunohistochemical Study of the Expression of Human Milk ...cancerres.aacrjournals.org/content/canres/44/5/2016.full.pdf · Formalin-fixed, paraffin-embedded blocks of hu man tissues