Kiinische

Klin. Wschr. 56, 767-777 (1978)

W°chenhrif t © Springer-Verlag 1978

Enhancement of Mechanisms Acting Against Mammary Carcinoma: Pancreatic Trypsin Inhibitor A.A. Hakim Departments of Surgery and PhysiologyDivision of Surgical Oncology, College of Medicine, University of Illinois at the Medical Center

Verstiirkung von Mechanismen gegen Mammakarzinom: Pankreatischer Trypsininhibitor Zusammenfassung. Zellen yon menschlichem Mammakarzinom, aus dem Prim/irtumor explantiert, wurden in ,,Eagle's minimum essential medium ( M E M ) " kultiviert, unter Hinzuftigung yon Insulin-PenicillinStreptomycin, fetalem Rinderserum ( 3 5 - 4 5 % ) , und/ oder Pankreastrypsininhibitor. In M E M mit InsulinPenicillin-Streptomycin-f6talem Kfilberserum (15%) wurden die Mammakarzinomzellen des prim/iren Explantats gebildet mit einem UbermaB yon Pseudopodien-fihnlichen Fasern, die sich yon den Explantaten erstreckten und Gruppen yon Zellen verbanden und umgaben. Diese Zellen iiberlebten 3 - 5 Wochen und starben dann ab. Wenn das Medium mit Pankreastrypsininhibitor (10 ~tg/ml) angereichert wurde, wurden yon den Explantaten Mammakarzinomzellen gebildet als getrennte rundliche Zellen, welche sich zu fusiformen oder dreieckigen Zellen entwickelten, ohne verbindende oder schtitzende Strukturen. Diese Zellen tiberlebten mehrere Monate bis mehr als 1 Jahr. Nach einigen ( 1 8 - 4 8 ) Wochen der Kultivierung in Anwesenheit des pankreatischen Trypsininhibitors, wurden die menschlichen Mammakarzinomzellen vermehrt empfindlich gegenfiber den zytotoxischen Wirkungen des Serums yon Patienten nach Operation eines Mammakarzinoms. Einige dieser Kulturen reagierten auf den doppelten Effekt yon hohen und niedrigen Dosen von Oestrogenen und yon Prolactin. In Gegenwart yon Insulin synthetisierten die kultivierten Zellen alpha-Laktalbumin, welches ftir Mammagewebe charakteristisch ist. Schliisselwiirter: Proteaseinhibitoren - Mammakarzinomzellen - Pankreastrypsininhibitor - Serumcytotoxizitfit. Summary. Human mammary carcinoma cells were cultivated from primary tumor explants in Eagle's

minimum essential medium (MEM) supplemented with Insulin-Penicillin-Streptomycin-Fetal Calf Serum ( 3 5 - 4 5 % ) and/or pancreatic trypsin inhibitor. In M E M supplemented with Insulin-Penicillin-Streptomycin-Fetal Calf Serum (15%) the mammary carcinoma cells were produced from the primary explants with an abundance of pseudopodia-like fibers extending from the explants connecting and surrounding groups of cells. These cells survived 3 to 5 weeks and died. If the medium was supplemented with pancreatic trypsin inhibitor (10 gg/ml) the mammary carcinoma cells were produced from the explants as separate round bodies which developed into fusiform or triangular cells, without any interlinking or protecting structures. These cells survived from several months to over a year. After several (18 to 48) weeks of cultivation in presence of pancreatic trypsin inhibitor, the human mammary carcinoma cells became more sensitive to the cytotoxic effects of serum from post surgery patients with mammary carcinoma. Some of these cultures responded to the dual effect of high and low doses of oestrogens and to the effect of prolactin. In the presence of insulin, the cultivated cells synthesized alpha lactalbumin a characteristic of mammary tissue.

Key words: Protease inhibitors-Breast cancer c e l l s Mammary carcinoma - Pancreatic trypsin inhibitor Serum cytotoxicity.

Viable and functional mammary carcinoma cell cultures could have several applications in clinical oncology as immunotherapeutic and immunodiagnostic agents. It is generally recognized that human mammary neoplasms are very difficult to grow in tissue culture. Since the original description of a breast tumour cell line [36] very few further cell lines have been estab-

768

A.A. Hakim : Enhancement of Mechanisms Acting Against M a m m a r y Carcinoma

lished from primary biopsy material. The reasons why human mammary tumours should be so much more difficult to culture than animal tumours are not clear. One possibility which must be considered is the medium and the products immediately released from tumour primary explants. Earlier studies [17, 19] led to the isolation and characterization of a plasminogen activator from cellfree media harvested from in-vitro cultured human lung adenocarcinoma and from in-vitro cultivated mammary carcinoma cells. The activator reacted as a protease with a wide spectrum of catalytic activities. Although the pathophysiology of fibrinogen in neoplastic diseases is not well understood, fibrinogen and fibrin play an important role in the proliferation and metastases of neoplastic cells [32, 40, 48]. When stained for fibrin deposits by fluorescein label technic, normal tissues, i.e. testes, kidneys, spleen, liver and smooth muscle showed no marked extravascular fibrin deposits except for some connective tissue staining and blood vessels, whereas most breast tumors showed intense staining for fibrin aggregates throughout the tumor [32]. Using tumor-bearing rats, Day et al. [11] showed that fibrinogen itself was fixed in the rat tumor, presumed to be a fibrin. The present studies attempt in-vitro cultivation of human mammary carcinoma cells under conditions which insured inactivation of the extracellular protease activities. High concentrations of fetal calf serum ( 3 5 - 4 5 % ) and/or pancreatic trypsin inhibitor were used in combination with Insulin.

Materials and Methods Materials

-OEstradiol (A 1,3.5 (10LEstratrien_3,17 fi-diol) Sigma 23 C-0350; Estriol (A1.3.s(l°~-Estratrien-3 16a, 17fitriol) Sigma 1380; and Estrone (A 1,3,s(l°l-Estratrien-3-01-17-one) Sigma 63C-1960, were obtained from Sigma Chemical Company, St. Louis, MO. 63178 U.S.A. Pancreatic trypsin inhibitor, and h u m a n alpha macroglobulin were also purchased from Sigma. Insulin was Lilly Iletin (Eli Lilly & Company, Indianapolis, Ind. U.S.A.). A total of 30 specimen from 10 patients with m a m m a r y carcinoma were obtained immediately after surgery. These were proven to be histologically either glandular m a m m a r y , medullary or infiltrating scirrhous-type carcinoma and one from pleural cavity of a patient (PMA) with metastases of breast cancer.

Methods

In brief, the technique of tissue culture was as follows: several fragments of tissue were explanted in each 6 ounce tissue culture glass bottles. Serm~a-free medium was used only to allow the explants adhere to the glass. Then the medium consisted of modified Eagle's m i n i m u m essential medium (MEM) to which was added 1 p.g/ml crystalline insulin, 50 gg/ml sodium penicillin, 50 gg/ml

streptomycin and 100 gglml mN-2-hydroxy-ethyl piperazine-N'-2ethane sulfonic acid. Insulin concentration was that used by Trowell [48] and the medium was supplemented with 35% fetal cal serum. Every 72 h the medium was replaced by fresh medium. In experiments where the hormone or steroid effects were examined, the cells were cultured for 14 days in insulin-free medium. Fourteen to thirty days after surgery, blood was drawn from patients with m a m m a r y carcinoma, allowed to clot, and the serum was separated. Blood was also drawn from patients with malignant melanoma, and colon carcinoma as well as from healthy volunteers. The sera were immediately frozen in small aliquots. The serum cytotoxic activity was assessed by the micro-cytotoxicity technique [45]. In brief, 100 to 150 m a m m a r y carcinoma cells in 0.1 ml of MEM-insulin medium were placed in wells of Falcon microplates (Falcon plastic, Oxnard, Calit: U.S.A.). The plates were incubated at 37 ° C for 24 h so that the cells adhere to the sides. At different dilutions, serum from the cancer patients or from the healthy volunteers were added to the wells and incubation continued for additional 72 h. After incubation, the supernatant fluids were gently pipetted out, the adhering cells were fixed with methanol, stained with cresol blue and counted. The results are reported in percent of killed cells. 3H-Thymidine Incorporation: Cells from confluent growth were collected and plated in quadruplicates at uniform density (10 s per 5 ml) in Eagle's M E M supplemented with 35% fetal calf serum, 2 x glutamine, penicillin and streptomycin as given above. After 24 h incubation, the medium was replaced with fresh medium containing 1 gg/ml crystalline insulin. After 48 h, 0.5 gCi 3H-thymidine (New England Nuclear, 40 Ci retool- i) was added to each bottle. One hour later the bottles were washed with phosphatebuffered saline and the cells were collected after detachment with trypsin-EDTA solution. The cell bottons were suspended in ice water and sonicated in a Brownson Sonicator for 4 s at the lowest setting. Aliquots were used for determination of protein by the method of Lowry et al. [39] and for trichloroacetic acid precipitation. Acid-insoluble counts were collected on 0.45 ~ Millipore filters and counted in a liquid scintillation counter (efficiency 35%). The results are in D P M x 10 3 per ~tg protein per hour, as the average of quadruplicate determinations + S.D. Synthesis of alpha-lactalbumin: The in-vitro cultured m a m m a r y carcinoma cells, malignant m e l a n o m a cells and normal h u m a n skin fibroblasts were cultivated in glass bottles with Eagle's essential medium supplemented with 35% fetal calf serum, insulin (1 ggl ml), penicillin and streptomycin as described above. In several experiments fl-OEstradiol, OEstriol, OEstrone, Cortisol and prolactin (Ovine) were also added at the indicated concentration to the culture medium_ Particulate-free supernatants were prepared from lyzed-harvested cells by centrifugation at 600 g to remove cellular debris, followed by ultracentrifugation at 105,000 g. These supernatants were then submitted to the radioimmunoassay for :~-lactalbumin [35].

Results

1. In- Vitro Culture of Human Cells In preliminary attempts, explants from ten separate tumors were used in the serum-free medium with insulin, conditions whereby the explants adhered to the glass within the first 48 h. Proliferation of the cells started 10 days thereafter. Two weeks in culture, the cells became granulated and 48 to 72 h later the granules were released from the cells into the medium.

A.A. Hakim: Enhancement of Mechanisms Acting Against M a m m a r y Carcinoma

Analysis of these granules revealed high level of protease-collagenase-plasminogen-activator activity (Manuscript under preparation). The cultures survived for 21 to 35 days and disintegrated. When the medium was supplemented with fetal calf serum, it was found that the higher the serum concentration the longer the cells survived. The optimum protocol consisted of alternating media supplemented with 30 up to 45% fetal calf serum with pancreatic trypsin inhibitor (at l0 ~tg/ml). The cultures MM, ES and VF survived four months, MY and JE survived six months, and PMA is in its 14th month. All these cultures attained confluent growth. Transfers were done by allowing the cells to crowl and adhere either on sterile capillary tubes or cover glass, then transferring these to new culture bottles. Trypsination or treatment with collagenase were completely avoided because they shortened the survival period. Only bicarbonate was used to buffer the medium, addition of HEPES proved to be toxic as well as it inhibited cell proliferation. The fetal calf serum could be lowered to 1 0 - 1 5 % if the medium was supplemented with 10 gg/ml of purified pancreatic trypsin inhibitor.

2. Ceil Proliferation Patterns The outgrowth from the tissue explants in Eagle's essential medium supplemented with insulin began 72 h after the explants adhered to the glass. The pattern in Figure 1 A for the tumor explant showing filamentous-like material connecting the various cells to other and to the explant. Proliferation of the cells for additional 72 h (Fig. 1 B) produced large multiform bodies, joined to each other by the filaments, which 24 to 48 h later changed into globular groups of cells (Fig. 1 C). At this stage no filaments were observed. This pattern appeared with the explants in the preliminary experiments and with each of the seven mammary carcinomas examined in detail in the present studies.

3. Effect of Pancreatic Trypsin Inhibitor on Ceil Proliferation Patterns When cultured in MEM-insulin medium containing 35% fetal calf serum and supplemented with 10 gg/ml of pancreatic trypsin inhibitor (or human alpha macroglobulin), the tumor explants proliferated without the appearance of the filamentous material (Fig. 2A), large fusiform or triangular cells developed 96 h after the explants adhered to the glass (Fig. 2B). This change in patterns was observed with all the seven cell cultures examined. Cell cultures VF, M M and

769

PMA yielded the fusiform, whereas LW, JE, ES and MY yielded the large triangular cell type.

4. Effect of Human Serum on the Cell Cultures The activity of serum from 10 patients with mammary carcinoma, 10 with malignant melanoma, 2 with colon carcinoma and 10 healthy volunteers was examined against three sets of four groups of the short term (4 months) MM, ES, and VF; two sets of four groups of the medium term (6 months) MY and JE and 10 sets ~ of four groups of the long term (over 14 months) mammary carcinoma. Two groups were cultured in absence (only 35% fetal calf serum) and the other two groups in presence of both 35% fetal calf serum and 10 lag/ml of pancreatic trypsin inhibitor. Under similar conditions, groups of cultures of normal human skin fibroblasts were also used. All ten sera from patients with mammary carcinoma were toxic to the mammary carcinoma cells. The magnitude of toxicity varied from one serum to another. Greater toxicity was observed against mammary carcinoma cells cultivated in presence of the pancreatic trypsin inhibitor. All the sera from patients with mammary carcinoma had weak to no effect on either sets of the normal skin fibroblasts. All sera from patients with either malignant melanoma or colon carcinoma had weak or no effect on either sets of the mammary carcinoma cell cultures, but, they killed 25+_1.9% and 12+_0.9% against human skin fibroblasts cultured in presence of the pancreatic trypsin inhibitor, and 14_+1.1% and 5+_0.2% against fibroblasts cultivated under the described conditions in absence of the inhibitor, respectively. Also, the data in Table 1 show that all the sera from healthy volunteers had no statistically significant effect on either groups of skin fibroblasts cultures. Sera from healthy volunteers were statistically moderately toxic (14 +_ 1.1%) against mammary carcinoma cells cultivated in presence of pancreatic trypsin inhibitor, but statistically non-toxic (2 +_0.1) against the mammary carcinoma cells cultivated under the described conditions in absence of trypsin inhibitor.

5. Effect of Serum on the Uptake of 3H-Thymidine by In- Vitro Cultivated H u m a n Cells: The effect of serum from 10 patients with mammary carcinoma, 10 with malig1 The long term, P M A cell culture were initiaIIy started from primary explants placed in ten bottles. Separately, the cells from each bottle were then split into four groups of new bottles. Therefore forty separate bottles were carried out through each subculture

770

A.A. Hakim: Enhancement of Mechanisms Acting Against M a m m a r y Carcinoma

A

B

Fig, 1.A An explant of the m a m m a r y carcinoma on the 10th day in the standard MEM-Insulin medium. The culture shows polymorphic sack-like structures filled with granule-like and attached to each others with fiber-like structures, x 60 B The polymorphic sack-like structures containing granule-like bodies interwoven with each other by fiber-like structure on the 14th day. x 60

C

C Living culture of the m a m m a r y carcinoma ceils bursting out from the sack-like structures in the standard MEM-Insulin medium. The culture was on the 25th day. The cells are arranged in glandular aggregates, x 60

A.A. Hakim: Enhancement of Mechanisms Acting Against Mammary Carcinoma

771

A

Fig. 2. A An explant of the mammary carcinoma on the 10th day in MEM-Insulin medium supplemented with 10 gg/ml of pancreatic trypsin inhibitor. 3"he culture shows few small round cells away from the explant, and large round cells in process of leaving the explant. × 60 B Living culture of the mammary carcinoma cells in MEM-Insulin medium supplemented with pancreatic trypsin inhibitor. The culture on left (MY) on the 25th day, on right (PMA) on the 30th day. The cells were separated and arranged in sheets, x 60

772

A.A. Hakim: Enhancement of Mechanisms Acting Against Mammary Carcinoma

Table 1. Cytotoxic activity of sera from patients with mammary

Table 2. Inhibition of 3H-thymidine incorporation by in-vitro cul-

carcinoma

tured cells with serum from cancer patients

The blood was drawn 12 days after surgical treatment of the cancer patients

Serum from:

Serum from patients with

Target cells + PTI a

- PTI

Mammary carcinoma Mammary carcinoma : P.V. M.W. E.D. W.L. B.J.

T.E.C. E.J.

H.L. M.M. Y.M.L.

72-+7.0 84-+8.1 57_+4.8 65-+5.9 40-+4.6 35-+2.6 58-+4.6 69-+5.6 22±1.6 47-+3.3

40 47 25 5 23 31 42 31 2 8

-+3.8 _+4.2 -+1.7 -+0.2 -+1.7 -+2.5 -+3.5 -+2.5 -+0.1 -+0.2

+ PTI

- PTI

Skin fibroblast

0.5-+0.0 0.9_+0.1 1.3+_0.1 0.9-+0.1 1.8-+0.2 3.4-+2.3 2.0-+0.1 2.0-+0.1 1.4_+0.1 1.1 -+0.l

2.5-+0.1 1.4-+0.1 1.1-+0.1 0.4-+0.4 0.8-+0.1 1.4_+0.1 0.9-+0.1 0.7+0.1 0.5-+0.0 0.7_+0.0

Malignant melanoma (5)

1-+0.1

0.7-+0.1 25 -+1.9 14 -+1.1

Colon carcinoma (2)

2+_0.1

1 -+0.2 12 -+0.9 5 _+0.2

Normal volunteers (10)

14-+1.1

2 _+0.1 0.5_+0.1 1 -+0.1

a PTI is pancreatic trypsin inhibitor 0.12 mg/ml incubation mixture. Numbers in parentheses indicate number of patients. The data are presented as percent killed (dead ceils)

Target cells Carcinoma cells

Skin fibroblasts

+ PTI

- PTI

+ PTI

- PTI

38 49 27 32 49 38 29 19 3 9 11 2 6 9

0.9_+0.0 1.2_+0.1 0.7_+0.1 1.9±0.3 3.2+1.7 2.6_+1.1 0.9_+0.1 2.6-+1.5 0.8-+0.1 2.1_+0.6 1.9_+0.6 0.7_+0.2 0.8-+0.1 2.3-+0.2

1.3_+0.1 1.5+0.1 1.0+0.1 0.8+0.1 1.8_+0.1 1.4+_0.1 1.0_+0.1 1.5-+0.3 0.7-+0.1 1.4_+0.2 1.4_+0.7 0.9-+0.1 0.9-+0.1 0.5-+0.1

Mammary cancer patients P.V. 85 _+6.5 M.W. 94 _+5.8 E.D. 69 +4.4 L.W. 78 _+5.4 B.J. 52 ±4.5 T.E.C. 47 -+3.7 E.J. 67 -+4.9 H.L. 85 -+6.7 M.M. 33 _+1.1 Y.M.L. 45 ±2.1 P.M.A. 78 -+5.3 E.S. 93 -+4.2 V.F. 59 -+2.3 M.K. 87 -+5.3 Malignant melanoma 10 patients

+_2.9 _+3.7 _+1.9 +2.1 -+1.1 +3.1 -+2.1 -+1.5 -+0.2 -+0.1 -+1.2 -+0.1 -+0.1 -+5.3

13.9-+0.4

0.8_+0.1 5.7_+0.8 1.1-+0.1

Colon carcinoma (2)

9 -+0.6

0.3-+0.1 2.9_+0.1 1.5-+0.1

Healthy volunteers (10)

19 -+0.5

5.6-+0.6 7.3-+0.7 1.0-+0.1

PTI is pancreatic trypsin inhibitor used at 0.12 mg/ml of the incubation mixture. The data represent the average of four separate assays _+S.D. and expressed as per cent inhibition of 3H-thymidine incorporation per hour

n a n t m e l a n o m a , 2 with colon c a r c i n o m a a n d 10 healthy volunteers was e x a m i n e d on 3 H - t h y m i d i n e inc o r p o r a t i o n by seven c a r c i n o m a cell cultures. Sera from patients with m a m m a r y c a r c i n o m a inhibited the 3 H - t h y m i d i n e u p t a k e by the c a r c i n o m a cell cultures (Table 2). The m a g n i t u d e of i n h i b i t i o n varied in a wide range between 2 + 0.1 to 49 + 1.1% in cells cultured in m e d i a s u p p l e m e n t e d with 35% fetal calf serum. The m a g n i t u d e of i n h i b i t i o n was increased 2 to 10 folds if the c a r c i n o m a cells were cultivated in m e d i a s u p p l e m e n t e d with b o t h 35% fetal calf serum a n d 10 gg/ml of pancreatic trypsin inhibitor. The sera from patients with m a m m a r y c a r c i n o m a had little or n o statistically significant effect o n n o r m a l skin fibroblasts or o n m a l i g n a n t m e l a n o m a cells. A l t h o u g h sera from healthy volunteers h a d no significant effect o n 3 H - t h y m i d i n e u p t a k e by the c a r c i n o m a cell cultures in presence of 35% fetal calf serum, they inhibited the precursor i n c o r p o r a t i o n by the c a r c i n o m a cells cultivated in presence of b o t h the fetal calf serum a n d the trypsin inhibitor.

6. Effect of OEstrogens on Human Carcinoma Cell Cultures a) Effect of O E s t r o g e n s o n 3 H - T h y m i d i n e U p t a k e Since some h u m a n breast cancers a p p e a r to be oestrogen d e p e n d e n t , the effects of physiologic c o n c e n t r a tions of 17-/%OEstradiol, OEstriol a n d O E s t r o n e were e x a m i n e d on the seven c a r c i n o m a cell cultures a n d on b o t h skin fibroblasts a n d m a l i g n a n t m e l a n o m a cells. The h o r m o n e s were added b o t h u n d e r serumfree c o n d i t i o n s to preclude the effects of other, undefined trophic substances a n d u n d e r the s t a n d a r d condition in presence o f 35% fetal calf serum. As indicated by the rates of 3 H - t h y m i d i n e i n c o r p o r a t i o n in T a b l e 2 , OEstradiol, OEstriol a n d O E s t r o n e at 10 8 M more t h a n d o u b l e D N A synthesis in cells LW, D F , ES, JE a n d MY. A t higher c o n c e n t r a t i o n s , the three oestrogens (5 x 10 6 M) decreased D N A as assessed by t h y m i d i n e i n c o r p o r a t i o n to less t h a n one third of the initial level in LW, JE, ES a n d MY. This differential response to oestrogen c o n c e n t r a t i o n

A.A. Hakim : Enhancement of Mechanisms Acting Against Mammary Carcinoma

773

Table 3. 3H-Thymidine incorporation by in-vitro cultured cells Condition

Control -OEstradiol (10 - s M) -OEstradiol (5 x 10- 6 M) Estriol (10 8 M) Estriol (5 x 10 6) Estrone (10 -8 M) Estrone (5 x i0-6 M) PTI (0.1 mg/ml) PTI (0.5 mg/mg) PTI (1.0 mg/ml)

Normal fibroblasts

1 +0.4 1.5_+0,6 0.7+_0.1 0.8_+0.l 0.5_+0.1 0.5_+0.1 0.4_+0.1 0.9-+0.1 1.1_+0.1 1.0_+0.1

Malignant mammary cells L.W.

V.F.

J.E.

M.M.

P.M.A.

E.S.

M.Y.

17+4.3 47_+4.3 4_+2.3 38_+3.5 39_+3.6 41+3.9 2+0.1 18+4.4 20+4.6 19_+4.5

14_+3.7 49+-4.8 48_+4.4 43-+4.1 51+_4.4 40+_4.0 35-+3.1 17+_3.9 19+_4.2 20+_2.2

i8_+4.4 40_+5.0 5+-2.6 38_+4.5 49-+4.3 53_+4.7 6+1.1 20-+4.5 17+_4.2 16+_3.1

22_+5.2 32+4.7 30+4.1 24+_2.1 34_+3.9 38+4.3 31+-3.4 24_+3.6 27_+5,8 25_+3.4

15+- 3.7 16+3.8 14+_3.5 17_+ 1.5 20-+1.6 16_+1.8 15+_1.0 19_+3.9 21_+4.1 22+1.0

16_+4.2 43_+5.0 3+1.9 40+_4.1 44_+4.0 59-+5.1 4+0.7 19-+4.4 22_+2.8 21+1.7

20_+5.0 57_+6.0 6+_2.8 59_+4.9 64+_5.1 74_+5.3 6+-0.8 22-+3.1 21+_3.0 19_+1.1

P.M,A. cells were obtained from pleural cavity of patient with metastasis of the breast carcinoma V.F. cells were obtained from patient with medullary carcinoma. L.H. cells were obtained from a patient with glandular breast carcinoma. The ceils E.S., M.Y., J.E,, and L.W. were obtained from patients with infiltrating Scirrhoustype carcinoma The results represent the average _+s.d. of quadruplicates, and are expressed as Dpm/mg protein/hour

suggests that the same population of cells which is stimulated by the oestrogens at lower concentrations of the oestrogens is inhibited at higher concentration. Thus, four out of the seven cell cultures contain specific oestrogen receptors and respond to physiologic concentrations of hormone with an increased rate of macromolecular synthesis (Table 3). b) Effect of OEstrogens on Synthesis of Alpha Lactalbumin The effect of 17-/3-OEstradiol, on synthesis of alpha lactalbumin by human cell cultures is summarized in Table 4. /%OEstradiol stimulated synthesis of lactalbumin by seven human carcinoma cell cultures, but not by either normal skin fibroblast or by malignant melanoma cell cultures. The magnitude of the stimulation was greater than that produced by prolactin but similar in magnitude to that produced by insulin.

c) Effect of Cortisol on Synthesis of Alpha Lactalbumin Similar to insulin, OEstradiol and Prolactin, cortisol did stimulate the synthesis of alpha lactalbumin by the human carcinoma cell cultures, but not by either normal skin fibroblasts or by malignant melanoma cell cultures.

7. Effect of Certain Hormones on Synthesis of Alpha Lactalbumin a) Effect of Insulin Since cells maintained in tissue culture can be examined under carefully controlled and defined condi-

Table 4. Synthesis of alpha-lactalbumin by in-vitro cultured human cells In-vitro cultured human cells Mammary cancers L.W. J.E.

E.S. M.Y. V.F. M.M. P.M.A. Skin fibroblasts Malignant melanoma (12) b

Insulin

Cortisol

/~-OEstradiol Prolactin ~

197+12 (32.5) 190_+11 (38.7) 195+13 (52.5) 176+_11 (48.3) 135_+10 (44.3) 127_+9 (30.5) 87-+7 (41.8) 0 (65.2) 0

205+18 (27.8) 198+12 (32.1) 211_+14 (39.2) 187_+10 (37.3) 124_+9 (42.7) 118_+7 (32.3) 37+_4 (44.51) 0 (78.3) 0

255+17 (23.7) 234_+15 (28.9) 246_+15 (28.5) 210_+16 (31.9) 145+10 (43.5) 87_+7 (31.9) 45_+3 (48.8) 0 (69.5) 0

165+10 (30.5) 148_+9 (27.9) 153_+8 (35.7) 132+_7 (46.3) 87+_6 (48.6) 53_+4 (34.9) 37_+3 (49.7) 0 (81.2) 0

(72.2)

(73.2)

(76.3)

(74.7)

a Cells cultured in presence of prolactin for 14 days b Number of cultures examined. The results represent the average four separate assays ±S.D. in ng/mg protein. Numbers in parenthesis represent gg of protein per assay

tions. The seven carcinoma cell cultures as well as the normal skin fibroblast and the melanoma cell cultures were examined for the synthesis of alpha lactalbumin. Table 4 summarizes the data on synthesis of the lactalbulnin. Neither skin fibroblasts nor the malignant melanoma cell cultures were capable to synthesize lactalbumin. When insulin was deleated

774

A.A. Hakim: Enhancementof MechanismsActing Against Mammary Carcinoma

from the culture medium, synthesis of alpha-lactalbumin was dramatically decreased to below than 25 ng/ mg protein, and the cells progressively became detached into the medium and died within twenty days after insulin restriction. These experiments demonstrate that the human mammary carcinoma during cultivation in vitro maintain their physiological function only if permitted to proliferate in presence of insulin. b) Effect of Prolactin Since all the seven human carcinoma cell cultures if cultured in insulin-free medium did not synthesize alpha lactalbumin, it was of interest to find out that prolactin, similar to insulin stimulated the synthesis of alpha lactalbumin (Table 4). Neither normal skin fibroblasts nor malignant melanoma cell cultures responded to prolactin. Only the carcinoma cell cultures were capable to synthesize lactalbumin in media where insulin was replaced by prolactin.

Discussion

Experiments with in-vitro assays have well established [5, 22-24, 28] that most human malignant cells bear cell-surface antigens that are either absent from, or present at very low concentrations on, the surface of normal cells of the corresponding tissue. Some of these antigens can evoke a specific cell-mediated immunity and humoral antibodies, directed against malignant cells of corresponding tissue type [25]. The sera tumor-bearing patients frequently contain blocking factors that can specifically protect tumor cells against the host immune reactivities [27]. A second factor, unblocking factor, in the sera of some individuals rendered free of malignancy by surgery, neutralizes the serum blocking factor of patients with cancer, and toxic to cells of the same histologic type [1, 2, 26, 29]. In agreement with these reports, the ability to develop an immune response to the tumor associated antigens could be assessed by the cytotoxic activity and by the number of antibody forming cells of the lymphoid tissue and of the circulating lymphocytes. In the present studies, post-surgery serum from ten patients with mammary carcinoma were found toxic against all the seven mammary carcinoma cell cultures, but had no or extremely weak effect against normal skin fibroblasts or against malignant melanoma cells cultured in-vitro. The toxicity could be attributed by the serum immunoglobulins, antibodies directed against the tumor cells. The serum from healthy volunteers or from patients with malignant melanoma had weak or no cytotoxic effect against

the mammary carcinoma cell cultures. This is in agreement with the aboved published reports. The data also suggest that the cytotoxic activity varied from one patient to another, which suggest that the cytotoxic activity is dependent on the antibody concentration in each serum. Therefore the data suggest specificity of the antibodies directed against the mammary carcinoma cell cultures, and these cultures maintained there immunological identity during invitro cultivation. Although they continued to proliferate, when preincubated in a medium containing pancreatic trypsin inhibitor, transplantable mouse spontaneous mammary adenocarcinoma cells did not produce tumor upon transfer into normal syngeneic animals [18]. Troll et al. [47] also reported that synthetic inhibitors of proteases markedly depressed mouse skin tumorigenesis induced when the skin was painted with 7,12dimethyl benz (a) anthracene (DMBA) and croton oil. Hozuma et al. [33] also reported that leupeptin, a protease inhibitor from the culture media of actinomyces strongly inhibited mouse skin tumorigenesis induced by treatment with DMBA and croton oil. The present investigation showed that when cultured in-vitro, human mammary carcinoma cells proliferated from the tumor explants in groups of cells surrounded with filamentous fibrous material. These groups of cells were attached to each other and to the explants with similar fiber-like material. Although the exact chemical nature is under extensive investigation, these filaments could act as cellular tentacles which produce cell movement out from the tumor explants, maintain contact with each other, and form a protective film or layer around the cells. At certain stage of cellular proliferation, the filamentous layer disappears, setting free individual single cells. These tantacle-like structures were not produced when the explants were permitted to proliferate in a medium containing pancreatic trypsin inhibitor. Under these conditions, the cells proliferated as single cells, and were much more sensitive to the serum toxic factors and to destruction by the immunological systems. Therefore, these experiments suggest that formation of the filament-like structures during cell proliferation is catalyzed by a protease-like enzyme(s) released by the mammary carcinoma cells into the immediate environment. This could be a mechanism by which tumor cells escape destruction by the various immunological systems and permit them to move and metastasize into various locations. The data presented support studies by others investigators which showed that an antiproteinase, aprotinin has considerable inhibitory properties against the growth and spread of tumor cells in animal systems [37a]. The reported evidence indicated that aprotinin enhance the effec-

A.A. Hakim : Enhancement of Mechanisms Acting Against Mammary Carcinoma tiveness of the immunological system in dealing with tumor cells [37b]. The immediate chemical environment plays a central role in the proliferation and differentiation of the cell. By release of enzymes or other biologically active molecules, the cell may directly influence its own fate or that of neighboring cells, and the present studies probe into the fate on in-vitro cultivated human mammary carcinoma. Since there is some evidence that mild proteolysis of the cell surface can transiently affect the surface properties of normal cells [6, 15], the increased levels of protease in transformed cells [4, 12, 16, 34, 46] assume an enhanced significance. Hayflick and Moorhead [20] first clearly enunciated the idea that human diploid fibroblasts are limited in their division potential in-vitro. After extensive further studies Hayflick and many others [21] this once controversal finding became a generally accepted one. The behavior of fibroblasts in culture relates to the present investigations in that epithelial cells, including carcinoma cells, may not necessarily exhibit the same behavior. There is now good reason to think that cells of some rapidly renewing epithelial in-vivo may have limits on doubling potential at very high levels [10, 13, 38]. These observations have not been confirmed in in-vitro studies, perhaps in part because of the difficulties in propagating epithelial cells without fibroblast overgrowth. Chang [9] has repeatedly initiated and followed the growth of human amnion. He observed, a three-to-ten fold increase in cell number within 6 - 1 0 weeks after initiation of cultures, followed by a stationary degenerative phase of 2 0 - 42 weeks. This was followed by slow recovery, often lasting several additional months, succeeded by a second growth period and eventual final degeneration in all cases. Of particular interest is a large group of experiments which were carried out in these laboratories to explain the difficulties in propagating human cells, led to the isolation from the cell-free culture medium of an enzyme with wide range of catalytic activities [19]. This enzyme, if kept in contact with the cell culture, was suspected to cause degeneration and death of the culture. After several attempts, high concentrations of fetal calf serum alone or combined with either pancreatic trypsin inhibitor or human alpha macroglobulin of human properties (unpublished data) inactivated the enzyme and fast proliferating cultures were obtained. The present studies describe the properties of seven human mammary carcinoma cell cultures, three of which proliferated for 10 weeks, two cultures for 30 weeks and one which is in active proliferation in its 14th months. A large number

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of cultures from large number of mammary carcinoma tissue from ten additional patients are being actively proliferating and are under present study. It is generally appreciated that breast cancer is very difficult to grow in tissue culture. Cell lines in particular have been difficult to establish [36, 37], although several have been reported, there have been little agreement as to their cancerous identity. While greater numbers of short-term breast cancer cells in cultures have been reported [3, 7, 14, 42] the nature of the cellular outgrowths is not known for certain [50]. In some reports the term "breast cancer cell" refer to any cell that emerges, while is some others the cell appears to be "epithelial" with or without atypism. In the present studies the human mammary carcinoma cell cultures have been identified by their response to serum obtained from post-surgery blood samples, to oestrogens and by their capacity to synthesize lactalbumin. The exact mechanisms by which oestrogens stimulate macromolecular synthesis and cell proliferation remain unknown. But it can be speculated that these cells in tissue culture will yield themselves to studies on the possible role of cycli nucleotides in mediating cellular response to hormonal stimulation. Insulin has long been considered essential for the growth and development of mammary gland in rodents [49], and may regulate proliferation of mammary tumors [30, 31]. Administration of insulin to rats with dimethylbenz (a) anthracene-induced mammary carcinoma and in-vitro administration to tumor explants have resulted in tumor growth [8], whereas insulin deprivation in alloxan diabetes caused tumor regression even in animals supplemented with oestradiol [30]. Studies on the effect of insulin in humanmammary gland explants suggest that insulin stimulate D N A synthesis and growth [8]. Experiments with the seven human cell cultures in the present investigations demonstrated that insulin stimulated the synthesis of alpha lactalbumin, and therefore the cultures maintained their mammary identity during in-vitro cultivation. Further studies on these human mammary carcinoma cells are in progress. Whether prolactin has a role in regulation of human breast cancer lacks support [44]. Most of the evidence that prolactin stimulates human cancer is drawn from studies on animal models [41] or on human carcinomas in organ culture [35]. In the present studies, the human mammary carcinoma cell cultures were examined and found to respond to prolactin. Therefore, the carcinoma cell cultures in our studies constitute the first human cell cultures responsive to prolactin. A direct evidence for the presence of prolactin receptor in these cell cultures awaits further studies.

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Received May 5, 1976 / Accepted November, 17, 1977

Anwar A. Hakim, Ph.C., Ph.D. Longwood Drive 180 Kankakee, Illinois 60901 U.S.A.