IN VITRO Volume 15, No. 6, 1979 All rights reserved �9

I D E N T I F I C A T I O N O F M O U S E M A M M A R Y A D I P O S E C E L L S BY M E M B R A N E A N T I G E N S

KAREN THOMPSON AND S. ABRAHAM

Bruce Lyon Memorial Research Laboratory, Children's Hospital Medical Center, 51st and Grove Streets, Oakland, California 94609

(Received September 7, 1978; accepted November 27, 1978)

SUMMARY

Antisera produced to mammary adipose cells from midpregnant BALB/c females can be used to distinguish mammary adipose cells from mammary epithelial cells and fibroblasts. The mammary adipose membrane antigen detected by indirect immunofluorescence was found in adipose cells from (a) mammary glands of virgin, midpregnant and lactating mice; (b) mammary fat pads that had been surgically cleared of glandular elements; and (c) epididymis. In all tissues, this cell-surface antigen was removed by the enzymatic action used to dissociate the cells from the tissues and was shown to be fully restored when cells were cultured for 48 hr.

Key words: cultured mammary adipose cells; adipose cell-specific antigen; immuno- fluorescence.

INTRODUCTION

We previously have developed immunofluores- cence techniques for identifying mammary epi- thelial cells in culture (1). These techniques were aimed at identifying cell-surface antigens of mouse (MMEC) and human mammary epithelial cells. Antibodies were produced to intact viable cells (1) or to the defatted milk fat globule (2), and, after appropriate absorption, these antisera could be used to distinguish mammary epithelial cells from other cell types.

The object of the present study was to define a specific antigen of the mouse mammary adipose cell (MMAC) that could be used to assess the role of the adipose cell in mammary-gland function. By following a similar protocol to that used in our previous studies (1), a heteroantiserum to mam- mary adipose cells was produced in rabbits. After absorption with mouse blood cells, mouse fibro- blasts and mouse mammary epithelial cells, this antisera (anti-MMAC) recognized mammary adi- pose cells in an indirect immunofluorescence as- say. Mammary adipose cells from the glands of virgin, midpregnant and lactating mice, and adi- pose cells from cleared mammary fat pads bound this antiserum, whereas mouse mammary epithel- ial cells and mouse fibroblasts did not.

MATERIALS AND METHODS

Animals and Tissue Sources

All mice were of the BALB/c strain. Mammary tissues were obtained from virgin (60- to 80-day- old), 14- to 16-day pregnant, and 7- to 17-day lactating mice. In addition, glands of 3-week-old female mice were surgically cleared of the glandu- lar elements by the method of DeOme et al. (3), and the cleared fat pads taken when the mice were 90 to 100 days of age. The New Zealand White adult rabbits (2 to 3 kg) used for the production of antibodies were obtained from Big Pine Rabbitry (Saratoga, Calif.). All mice were bred in our vivarium. Epididymal fat tissue, when used, was surgically removed from BALB/c males.

Cell Dissociation and Culture Techniques

MMEC and MMAC from midpregnant and lactating mice were obtained routinely by dis- sociation of mammary-gland tissue by the method previously described (1,4), which used collagen- ase, hyaluronidase and pronase. The adipose cells were separated from mammary epithelial cells, fibroblasts and other connective tissue cells by centrifugation. The floating cell layer was washed with fresh medium to remove fat and remaining

441

442 THOMPSON AND ABRAHAM

connective tissue cells. Adipose cells from cleared mammary fat pads and from the epididymis were obtained by dissociation of tissue with collagenase by the method of Rodbell (5). In some cases, adi- pose cells from mammary glands of virgin and midpregnant mice were dissociated by the Rod- bell method {5) using collagenase alone. They were separated and washed as described above.

Primary cultures of mammary epithelial cells were incubated with rotation as suspension cul- tures as previously described {1}. Primary suspen- sion cultures of adipose cells from all sources were initiated at 1 x 10 ~ cells per ml and maintained without rotation in tissue-culture tubes or Erich- meyer flasks containing 10% fetal bovine serum in Waymouth's medium (MB 752/lj plus penicil- lin and streptomycin (lk All cell cultures were incubated for 48 hr before harvest.

Preparation and Purification of Antibodies

Anti-MMAC. MMAC obtained from midpreg- nant mice and cultured in suspension for 48 hr were washed in Waymouth's medium without fetal bovine serum and then used for production of antibodies in rabbits according to a protocol similar to that previously described (1). The immunization schedule involved three weekly i.p. injections of approximately 20 x 10' cells followed by bimonthly booster injections with the same number of cells. Sermn was collected from bleed- ings 1 week after injections. Antibody titers were maintained by the booster injection and serum collected as described for up to 1 year from a sin- gle rabbit. ),-Globulin fractionation with 40% am- monium sulfate followed by dialysis against physiologically buffered saline was carried out. The ),-globulin fraction then was absorbed with mouse whole blood cells, MMEC and mouse fetal flbroblasts {MFC) (1). The completeness of absorption was tested on MMEC, MFC and other mouse tissue cells by indirect immuno- fluorescence {1). The final anti-MMAC prepara- tion subsequently was frozen in 0.2-ml aliquots and kept at -20 ~ C until used. The antibody preparation is stable for at least 2 years when stored under these conditions.

Indirect membrane immunofluorescence assays of the various unfixed cell preparations were car- ried out as previously described (1). Briefly, an aliquot containing 2 to 10 x 106 cells in Way- mouth's medium with 0.5% bovine serum al- bumin {BSA, 35% solution; Miles-Pentex Bio- chemicals} and 10 -2 sodium azide was incubated with 25 t~l anti-M_MAC or control normal rabbit

y-globulin for 20 min at 37 ~ C. After the incuba- tion period, the cells were washed twice with Waymouth's medium plus BSA and sodium azide. The second amplifying layer of fluorescent- labeled goat antiserum against rabbit ),-globulin (FIGaRyo, 20 mg protein per ml; Antibodies, Inca was diluted 1:5; 25 pl was added to the cells and incubated for 20 min at 37 ~ C. The cells were pelleted by centrifugation and washed twice as de- scribed above. Wet-mount preparations of the la- beled cells were made in the suspension medium {0.5% BSA in Waymouth's medium with 10 -2 M

sodium azide). They were examined under a Leitz Dialux microscope with fluorescent epi-illumina- tion within 18 hr after staining.

RESULTS

A summary of the results obtained with indirect immunofluorescence is given in Table 1 and Fig. 1. Heteroantisera raised in rabbits to cultured mammary adipose cells obtained from mammary glands of midpregnant BALB/c females reacted specifically with adipose cells from the former source as well as with those obtained from cleared mammary fat pads {Fig. 1A,B) and from virgin and lactating mice (Table lk Spleen cells, cul- tured mammary epithelial cells {Table 1), and fetal flbroblasts (Table 1, Fig. 1D) did not react with the absorbed anti-MMAC preparation. In addition, the epithelial cell-specific antisera pre- viously described {1) did not react in indirect immunofluorescence with mammary adipose cells (Fig. 1Ck

The free-fat droplets {Table 1} that appear as birefringent spheres in phase-contrast microscopy do not fluoresce, indicating that the anti-MMAC does not recognize free fat or substances dispersed in it. The adipose membrane antigen detected by this heteroantiserum is removed from the cells by the enzyme treatments used for isolation of these single-cell preparations of adipose cells (Table 1). We have found that it is necessary to culture the adipose cells for at least 48 hr during which time the cell-surface antigens are regenerated. Similar findings have been reported from this laboratory {1) for mammary epithelial cells. Both the colla- genase (5) and the collagenase plus hyaluronidase followed by pronase {4} methods remove the adi- pose cell-surface antigen. Mammary epithelial ceils isolated by the same enzyme methods do not react with the anti-MMAC either immediately on isolation or after 48 hr in suspension culture (Table 1). Adipose cells from another tissue source, epididymis from BALB/c males {MAC),

MAMMARY ADIPOSE MEMBRANE ANTIGEN 443

FIG. 1. Indirect fluorescence staining reactions detected with FIGaRy G as the amplifying layer and absorbed rabbit y-globulin. Top row: the fluorescence staining reaction; bottom row: the same field under phase-contrast microscopy. A, MMAC from midpregnant mice ~suspension-cultured for 48 hr~ plus rabbit anti-adipose cells and F1GaRrG. B, MMAC from cleared mammary fat pads (suspension- cultured for 48 hr) plus rabbit anti-adipose cells and F1GaRy G. C, MMAC from midpregnant mice ~suspension-cultured for 48 hr) plus rabbit anti-epithelial cells and F1GaRy G (controlL D, MFC from fetal mice icultured as monolayer for 7 days, then suspension-cultured for 48 hr) plus rabbit anti- adipose cells and F1GaRy(;. Photographs were taken with Tri-X pan black-and-white film; exposure times were 1 min for fluorescence and 0.5 see for phase contrast, x600.

react with anti-MMAC, but only weakly, indicat- ing the anti-MMAC may have selective mam- mary tissue reactivity (Table 1 ).

DISCUSSION

This study demonstrates that mammary adi- pose cells have characteristic cell antigens that can be used for identifying these cells by immuno- fluorescence with viable cell preparations. The fact that these antigens can he removed by en- zyme treatments that leave intact viable cells cap- able of regenerating these lost antigens in culture suggests that they are either on the cell surface or close to it.

Other studies (by microcytotoxicity, immuno- fluorescence and electron microscopy) of cell- surface structure have delineated many differ- entiation antigens. For example, in mouse lym-

phoid cells, thymus leukemia (TL) antigen (6) and thymus-derived antigens (Thy. 1) (7) represent differentiation antigens defining subpopulations of thymus-derived lymphocytes, whereas surface membrane immunoglobulin (SmIg) (8) distin- guishes the bone-marrow-derived lymphocytes. In many of these cases, intact cell populations were used for production of these alloantisera and heteroantisera which are able to delineate these lymphocyte differentiation antigens. In our study, an enriched population of mammary adipose cells was used for the preparation of anti-MMAC. The adipose cells obtained from mammary glands of midpregnant BALB/c females were separated from mammary epithelial cells, fibrohlasts and other connective tissue cells by centrifugation. Mammary tissue from midpregnant mice pro- vides a large amount of tissue, and subsequently good yields of both mammary adipose and epi-

444 THOMPSON AND ABRAHAM

TABLE 1

SPECIFICITY OF RABBIT ANTI-BALB/C MMAC

Cell Type Mouse Dissociation Cell Designation a Source Method b State Fluorescent Staining

% In tens i t y c MMAC Pregnant gland Collagenase,

hyaluronidase and

MMAC Pregnant gland

MMAC Lactating gland

MMAC Pregnant gland MMAC Virgin gland MMAC Cleared fat pad MAC Epididymis Free fat droplets Pregnant and/or

lactating gland

pronase 48-hr suspension culture 75-85 +4 Collagenase,

hyaluronidase and pronase freshly isolated cells 0 0

CoUagenase, hyaluronidase and pronase 48-hr suspension culture 75-85 +4

Collagenase 48-hr suspension culture 70-80 +4 Collagenase 48-hr suspension culture 65-75 +4 Collagenase 48-hr suspension culture 70-80 +4 Collagenase 48-hr suspension culture 65-70 +2 Collagenase and/or 48-hr suspension culture

hyaluronidase and or freshly isolated pronase

MMEC Pregnant gland Collagenase, hyaluronidase and pronase

MMEC Pregnant gland Collagenase MFC Fetus Trypsin

Lymphoid cells Spleen Mechanical

cells 0 0

48-hr suspension culture 0 0 48-hr suspension culture 0 0 monolayer culture, 7

days; then 48-hr suspension culture 0 0

freshly isolated cells 0 0

a MMAC = mouse mammary adipose cells; MAC ---= mouse adipose cells; MMEC = mouse mammary epithelial cells; MFC = mouse fibroblasts.

b Collagenase dissociation according to Rodbell (5); collagenase plus hyaluronidase plus pronase and trypsin (1 L e Intensity was judged on a scale of 0 to +4 (most intenseL

thelial cells, and thus was the tissue of choice for antibody production. The adipose cells float and are easily removed and washed several times in medium to remove nonadipose cells that pellet at the bottom of the tube.

Adipose cells were isolated from cleared mam- mary fat pads and tested by indirect immuno- fluorescence with anti-MMAC and F1GaRy G in order to rule out (a) the possibility that the mam- mary adipose cells from the virgin, midpregnant and/or lactating glands were adsorbing cytophilic material from epithelial cells, and (b) the possible effects of different hormonal environments in the cases of the midpregnant and lactating glands. Since no difference was found in either the fluorescent staining intensity or the percentage of adipose cells staining in virgin gland compared to midpregnant or lactating gland (Table 1), hor- monal influences do not play a significant role.

Different enzyme treatments were used for isolation of adipose cells from mammary glands of virgin mice, cleared mammary fat pads and epi-

didymis (coUagenase only) than were used for their isolation from mammary glands of midpreg- nant mice (collagenase, hyaluronidase and pro- nase). Thus adipose cells from the mammary glands of midpregnant mice also were isolated by the collagenase procedure. In all cases, freshly dissociated adipose cells were negative. After cul- ture, adipose cells did react with the anti-MMAC although, in the case of epididymal adipose cells, the reaction was weaker (Table 1).

From our study, it is evident that mammary adipose cells possess a characteristic cellular anti- gen(s) as defined by this heteroantiserum. Immunofiuorescence and/or immunoelectron microscopic methods based on the specific adi- pose membrane antigenic components delineated in this study may serve as a marker for adipose cell identification. This anti-MMAC eventually may be helpful in studies to distinguish liposar- comas from other types of soft-tissue sarcomas.

To date, mammary epithelial cells (1), mam- mary adipose cells and mammary fibroblasts (1)

MAMMARY ADIPOSE MEMBRANE ANTIGEN 445

can be distinguished from each other by immuno- fluorescence assays based on the respective cell- specific antigens. These antisera may be helpful in studies aimed at assessing the role of cell-cell interaction in mammary-gland function.

REFERENCES

1. Thompson, K., R. Ceriani, D. Wong, and S. Abra- ham. 1976. Immunologic methods for the iden- tification of cell types. I. Specific antibodies that distinguish between mammary gland epithelial cells and fibroblasts. J. Nat. Cancer Inst. 57: 167-172.

2. Ceriani, R., K. Thompson, J. A. Peterson, and S. Abraham. 1977. Surface differentiation antigens of human mammary epithelial cells carried on the human milk fat globule. Proc. Nat. Acad. Sci. 74: 582-586.

3. DeOme, K. B., L. J. Fauikin, Jr., H. A. Bern, and P. B. Blair. 1959. Development o~ mammary tu-

mors from hyperplastic alveolar nodules trans- planted into gland-free mammary fat pads of fe- male C3H mice. Cancer Res. 19: 515-520.

4. Weipjes, G. J., and F. J. Prop. 1970. Improved method for preparation of single cell suspensions from mammary glands of adult virgin mouse. Exp. Cell Res. 61: 451-454.

5. Rodbell, M. 1964. Metabolism of isolated fat cells. I. Effects of hormones on glucose metabolism and lipolysis. J. Biol. Chem. 239: 375-380.

6. Old, L. J., E. A. Boyse, and E. Stockert. 1963. Antigenic properties of experimental leukemias. J. Nat. Cancer Inst. 31: 977-986.

7. Reif, A.E., and J. M. Allen. 1964. The AKR thymus antigen and its distribution in leukemias and nervous tissue. J. Exp. Med. 120: 413-433.

8. Raff, M. C., M. Sternberg, and R.B. Taylor. 1970. Immunoglobulin determinants on the sur- face of mouse lymphoid cells. Nature 225: 553-554.

We appreciate the exceptional technical assistance of Alexander Lucas and the excellent typing of Glynis Can" and Jolyce Hardesty. This work was sup- ported by National Cancer Institute Grant Nos. CA11736 and CA18946 and Biomedical Research Support Grant No. RR05467 from the National Insti- tutes of Health, DHEW.