Cancer Letters, 1(1976) 203--206 © Elsevier/North-Holland, Amsterdam -- Printed in The Netherlands
203
HUMAN BREAST CARCINOMA: H E T E R O T R A N S P L A N T A T I O N TO NEWBORN RATS*
IVY BENJAMIN and HENRY PINKERTON
Department o f Pathology, St. Louis University School o f Medicine, 1402 South Grand Boulevard, St. Louis, Missouri 63104 (U.S.A.) (Received 5 December 1975)
SUMMARY
Cultured cells from a human breast carcinoma (HBC) were injected subcutaneously in newborn cortisone-treated Sprague--Dawley rats (CNBR). Tumors of predominantly epithelial nature appeared in all within 30 days, and widespread metastases occurred often. Cells cultured from these tumors rapidly produced similar tumors in CNBR. Rat-to-rat passage b y implanting fragments of t u m o r tissue was 100% successful in CNBR. The in vivo model described is simple and inexpensive.
MATERIALS, METHODS AND RESULTS
The cell line used in the experiments reported here was derived from an invasive duct cell carcinoma excised from a 77-year-old woman. The tissue was minced to fragments of about 1 mm 3 and dispersed in 0.25% trypsin to a point where, after centrifugation, only single cells and small clusters remained in the supernate. These were explanted in 4-cm glass Petri dishes containing 3.0 b y 2.5 mm coverslips and 5 ml of 2X basal medium of Eagle, plus 10% fetal calf serum, with conventional amounts of penicillin and streptomycin. For a b o u t 3 weeks the cells were rounded and many were floating. During the 4th week, a burst of rapidly growing cells appeared, first in monolayers, b u t soon becoming multilayered, with 'criss-crossing'. In 32-ounce plastic flasks, with 20 ml of Eagle's MEM, rapid growth continued. The culture has been in vitro for 18 months. Morphologically (Fig. 1) the cells are a mixture of spindle cells, triangular cells, and rounded epithelioid cells. *Supported in part by Public Health Service Contract No. 1 CP 43359 within the VirusCancer Program of the National Cancer Institute. Address correspondence to: Ivy Benjamin, Department of Pathology, St. Louis University School of Medicine, 1402 South Grand Boulevard, St. Louis, Missouri 63104, U.S.A.
204
Fig.1. Cells cultured from HBC. (A) Low population density. (B) Higher population density from same coverslip. H. and E × 450. Chromosomal analysis of the original (human t u m o r derived) culture and the culture derived from the rat t u m o r were identical. Their number ranged from 41--69, with a mode of 62. Several h u m a n chromosomes were identifiable. Endoreduplication, fragmentation, and other indications of malignancy were noted. Presumptive evidence for their epithelial nature includes the predominance of malignant epithelial cells in the tumors they produce in rats, the failure to find collagen or reticulum fibers after extensive ultrastructural studies or in coverslips stained by the Masson and Manuel methods, and evidence (see below) that carcinoma cells may indeed appear spindle-shaped under certain environmental conditions [2,6]. Subcutaneous injections of i X 106 viable cultured cells, in passages 34--36, were given to newborn Sprague--Dawley rats, immunosuppressed by 3 subcutaneous injections per week of Cortone (Merck, Sharp and Dohme). The Cortone was diluted 1:10, each injection of 0.04 ml then containing 0.2 mg of cortisone acetate. The first injection was given 1 or 3 days after the t u m o r cells. Of the first 18 CNBR's, all developed tumors (2.5--6.5 cm in diameter) within 30 days, and 6 died from metastases. Histologically, the tumors were primarily epithelial, interspersed at times with spindle cells. They often showed considerable resemblance to the human t u m o r of origin (Fig. 2).
205
Fig.2. Surgical specimen (A) and metastatic tumor in a rat (B) H and E × 450. The Manuel stain showed black reticulum fibers in the capsule and stroma, but none in the t u m o r cell islands. Cultures from the tumors in rats grew rapidly, were like the original culture, and rapidly caused tumors in CNBR. Tumors were readily transplanted serially by implanting fragments (about 1 mm 3 ) in CNBR; one of them developed lethal widespread metastases, and no regressions occurred. The take has been 100%. DISCUSSION Cultivation in vitro. The literature is voluminous. The first outstanding success was reported by Lasfargues and Ozello [6] in 1958. Their cell line (B20) is definitely epithelial and has remained unchanged. A change in the composition of the culture medium, however, resulted in conversion of this line to fibroblast-like cells, which after several months reverted to their original epithelial morphology. Cailleau et al. [2], working with cultures obtained from pleural fluids containing metastatic HBC cells, presented ultrastructural evidence that fibroblast-like cells may be epithelial in nature. Plata et al. [9] obtained a culture from HBC and cloned 2 sublines from fibroblast-like monolayers, one of which bacame established as epithelioid, while the other remained fibroblast-like, with limited viability. Buehring and Hackett [ 1] studied 5
206
established human breast tumor lines. Ultrastructural studies suggested that at least 3 (including B20) were derived from mammary epithelium. Heterotransplantation. Greene [5] in 1952 grew a few HBC's in the anterior chamber of the eye in guinea pigs. Toolan [11] in 1953 obtained poor results in irradiated rats, but in 1955 briefly reported better results in more heavily irradiated rats [ 1 2 ] . Southam [10] in 1969 had takes in 9 of 34 rats made tolerant by intravenous injections of cell suspensions as newborns and given subcutaneous injections when 1 week old. Cobb, using thymectomized and anti-thymocyte serum-treated hamsters, found lung metastasis without local tumors in one experiment [3] and tumors reaching 9 cm 3 in 7 months in 3 of 19 animals in another [ 4 ] . All of these workers implanted surgical material directly, and we find no reports of transmissible tumors. Recently, however, Ozello et al. [8] and Nordquist et al. [7] have obtained transmissible tumors in nude athymic mice. ACKNOWLEDGMENT
We are indebted to Dr. Patricia Monteleone of the Glennon Hospital, St. Louis, for the chromosomal analysis. REFERENCES 1 Buehring, G.C. and Hackett, A.J. (1974) Human breast tumor cell lines: Identity evaluation by ultrastructure. J. Natl. Cancer Inst., 53,621--629. 2 Cailleau, R., Mackay, B., Young, R.K. and Reeves, W.J., Jr. (1974) Tissue culture studies on pleural effusions from breast carcinoma patients. Cancer Res., 34, 801--809. 3 Cobb, L.M. (1972) Metastatic spread of human tumor implanted into thymectomized anti-thymocyte serum treated hamsters. Brit. J. Cancer, 26, 183--189. 4 Cobb, L.M. (1974) The hamster as a host for the growth and study of human tumor cell populations. Cancer Res., 34,958--963. 5 Greene, H.S.N. (1952) Significance of the heterologous transplantability of human cancer. Cancer, 5, 24--44. 6 Lasfargues, E.Y. and Ozello, L. (1958) Cultivation of human breast carcinomas. J. Natl. Cancer Inst., 21, 1131--1147. 7 Nordquist, R.E., Ishmael, D.R., Lovig, C.A., Hyder, D.M. and Hoge, A.F. (1975) The tissue culture and morphology of human breast tumor line BOT-2. Cancer Res., 11, 3100--3105. 8 0 z c l l o , L., Sordat, B., Merenda, C., Carrel, S., Hurlimann, J. and Mach, J.P. (1974) Transplantation of a human mammary carcinoma cell line (BT 20) into nude mice. J. Natl. Cancer Inst., 52, 1669--1672. 9 Plata, E.J., Aoki, T., Robertson, D.D., Chu, E.W. and Gerwin, B.I. (1973) An established cultured cell line HBT-39 from human breast carcinoma. J. Natl. Cancer Inst., 50,849--862. 10 Southam, C.M., Tanzi, A.F. and Ross, S.L. (1966) Growth of primary explants of human cancer in newborn rats. Cancer, 19, 1670--1682. 11 Toolan, H.W. (1953) Growth of human tumors in cortisone treated laboratory animals. The possibility of obtaining permanently transplantable human tumors. Cancer Res., 13, 389--396. 12 Toolan, H.W. (1955) Growth oi buman mammary adenocarcinomas and melanomas in rats and hamsters. Proc. Amer. Assoc. Cancer Res., 2, 52.
203
HUMAN BREAST CARCINOMA: H E T E R O T R A N S P L A N T A T I O N TO NEWBORN RATS*
IVY BENJAMIN and HENRY PINKERTON
Department o f Pathology, St. Louis University School o f Medicine, 1402 South Grand Boulevard, St. Louis, Missouri 63104 (U.S.A.) (Received 5 December 1975)
SUMMARY
Cultured cells from a human breast carcinoma (HBC) were injected subcutaneously in newborn cortisone-treated Sprague--Dawley rats (CNBR). Tumors of predominantly epithelial nature appeared in all within 30 days, and widespread metastases occurred often. Cells cultured from these tumors rapidly produced similar tumors in CNBR. Rat-to-rat passage b y implanting fragments of t u m o r tissue was 100% successful in CNBR. The in vivo model described is simple and inexpensive.
MATERIALS, METHODS AND RESULTS
The cell line used in the experiments reported here was derived from an invasive duct cell carcinoma excised from a 77-year-old woman. The tissue was minced to fragments of about 1 mm 3 and dispersed in 0.25% trypsin to a point where, after centrifugation, only single cells and small clusters remained in the supernate. These were explanted in 4-cm glass Petri dishes containing 3.0 b y 2.5 mm coverslips and 5 ml of 2X basal medium of Eagle, plus 10% fetal calf serum, with conventional amounts of penicillin and streptomycin. For a b o u t 3 weeks the cells were rounded and many were floating. During the 4th week, a burst of rapidly growing cells appeared, first in monolayers, b u t soon becoming multilayered, with 'criss-crossing'. In 32-ounce plastic flasks, with 20 ml of Eagle's MEM, rapid growth continued. The culture has been in vitro for 18 months. Morphologically (Fig. 1) the cells are a mixture of spindle cells, triangular cells, and rounded epithelioid cells. *Supported in part by Public Health Service Contract No. 1 CP 43359 within the VirusCancer Program of the National Cancer Institute. Address correspondence to: Ivy Benjamin, Department of Pathology, St. Louis University School of Medicine, 1402 South Grand Boulevard, St. Louis, Missouri 63104, U.S.A.
204
Fig.1. Cells cultured from HBC. (A) Low population density. (B) Higher population density from same coverslip. H. and E × 450. Chromosomal analysis of the original (human t u m o r derived) culture and the culture derived from the rat t u m o r were identical. Their number ranged from 41--69, with a mode of 62. Several h u m a n chromosomes were identifiable. Endoreduplication, fragmentation, and other indications of malignancy were noted. Presumptive evidence for their epithelial nature includes the predominance of malignant epithelial cells in the tumors they produce in rats, the failure to find collagen or reticulum fibers after extensive ultrastructural studies or in coverslips stained by the Masson and Manuel methods, and evidence (see below) that carcinoma cells may indeed appear spindle-shaped under certain environmental conditions [2,6]. Subcutaneous injections of i X 106 viable cultured cells, in passages 34--36, were given to newborn Sprague--Dawley rats, immunosuppressed by 3 subcutaneous injections per week of Cortone (Merck, Sharp and Dohme). The Cortone was diluted 1:10, each injection of 0.04 ml then containing 0.2 mg of cortisone acetate. The first injection was given 1 or 3 days after the t u m o r cells. Of the first 18 CNBR's, all developed tumors (2.5--6.5 cm in diameter) within 30 days, and 6 died from metastases. Histologically, the tumors were primarily epithelial, interspersed at times with spindle cells. They often showed considerable resemblance to the human t u m o r of origin (Fig. 2).
205
Fig.2. Surgical specimen (A) and metastatic tumor in a rat (B) H and E × 450. The Manuel stain showed black reticulum fibers in the capsule and stroma, but none in the t u m o r cell islands. Cultures from the tumors in rats grew rapidly, were like the original culture, and rapidly caused tumors in CNBR. Tumors were readily transplanted serially by implanting fragments (about 1 mm 3 ) in CNBR; one of them developed lethal widespread metastases, and no regressions occurred. The take has been 100%. DISCUSSION Cultivation in vitro. The literature is voluminous. The first outstanding success was reported by Lasfargues and Ozello [6] in 1958. Their cell line (B20) is definitely epithelial and has remained unchanged. A change in the composition of the culture medium, however, resulted in conversion of this line to fibroblast-like cells, which after several months reverted to their original epithelial morphology. Cailleau et al. [2], working with cultures obtained from pleural fluids containing metastatic HBC cells, presented ultrastructural evidence that fibroblast-like cells may be epithelial in nature. Plata et al. [9] obtained a culture from HBC and cloned 2 sublines from fibroblast-like monolayers, one of which bacame established as epithelioid, while the other remained fibroblast-like, with limited viability. Buehring and Hackett [ 1] studied 5
206
established human breast tumor lines. Ultrastructural studies suggested that at least 3 (including B20) were derived from mammary epithelium. Heterotransplantation. Greene [5] in 1952 grew a few HBC's in the anterior chamber of the eye in guinea pigs. Toolan [11] in 1953 obtained poor results in irradiated rats, but in 1955 briefly reported better results in more heavily irradiated rats [ 1 2 ] . Southam [10] in 1969 had takes in 9 of 34 rats made tolerant by intravenous injections of cell suspensions as newborns and given subcutaneous injections when 1 week old. Cobb, using thymectomized and anti-thymocyte serum-treated hamsters, found lung metastasis without local tumors in one experiment [3] and tumors reaching 9 cm 3 in 7 months in 3 of 19 animals in another [ 4 ] . All of these workers implanted surgical material directly, and we find no reports of transmissible tumors. Recently, however, Ozello et al. [8] and Nordquist et al. [7] have obtained transmissible tumors in nude athymic mice. ACKNOWLEDGMENT
We are indebted to Dr. Patricia Monteleone of the Glennon Hospital, St. Louis, for the chromosomal analysis. REFERENCES 1 Buehring, G.C. and Hackett, A.J. (1974) Human breast tumor cell lines: Identity evaluation by ultrastructure. J. Natl. Cancer Inst., 53,621--629. 2 Cailleau, R., Mackay, B., Young, R.K. and Reeves, W.J., Jr. (1974) Tissue culture studies on pleural effusions from breast carcinoma patients. Cancer Res., 34, 801--809. 3 Cobb, L.M. (1972) Metastatic spread of human tumor implanted into thymectomized anti-thymocyte serum treated hamsters. Brit. J. Cancer, 26, 183--189. 4 Cobb, L.M. (1974) The hamster as a host for the growth and study of human tumor cell populations. Cancer Res., 34,958--963. 5 Greene, H.S.N. (1952) Significance of the heterologous transplantability of human cancer. Cancer, 5, 24--44. 6 Lasfargues, E.Y. and Ozello, L. (1958) Cultivation of human breast carcinomas. J. Natl. Cancer Inst., 21, 1131--1147. 7 Nordquist, R.E., Ishmael, D.R., Lovig, C.A., Hyder, D.M. and Hoge, A.F. (1975) The tissue culture and morphology of human breast tumor line BOT-2. Cancer Res., 11, 3100--3105. 8 0 z c l l o , L., Sordat, B., Merenda, C., Carrel, S., Hurlimann, J. and Mach, J.P. (1974) Transplantation of a human mammary carcinoma cell line (BT 20) into nude mice. J. Natl. Cancer Inst., 52, 1669--1672. 9 Plata, E.J., Aoki, T., Robertson, D.D., Chu, E.W. and Gerwin, B.I. (1973) An established cultured cell line HBT-39 from human breast carcinoma. J. Natl. Cancer Inst., 50,849--862. 10 Southam, C.M., Tanzi, A.F. and Ross, S.L. (1966) Growth of primary explants of human cancer in newborn rats. Cancer, 19, 1670--1682. 11 Toolan, H.W. (1953) Growth of human tumors in cortisone treated laboratory animals. The possibility of obtaining permanently transplantable human tumors. Cancer Res., 13, 389--396. 12 Toolan, H.W. (1955) Growth oi buman mammary adenocarcinomas and melanomas in rats and hamsters. Proc. Amer. Assoc. Cancer Res., 2, 52.
