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Hormone effects on human mammary cancer in organ cultures
Hormone Effects on Human Mammary Cancer in Organ Cultures K. Aspegren, MD, Lund, Sweden
Mammary cancer is regarded as a hormonally responsive tumor, but only one of three patients with widespread disease will benefit from hormonal treatment. Prediction of response by laboratory methods would be of great value. In vitro assay of estrogen receptors of mammary cancer has been correlated to the patient’s clinical course [I]. Correlation of the activity of steroid sulfhydration by cancer tissue to clinical course has been suggested [2]. Hormonal effect on dehydrogenase activity of cancer tissue has also been used for prediction [3]. These methods measure the ability of the tissue to bind or metabolize the steroid or the steroid’s effect on intermediary cell metabolism, that is, pentose shunt activity. A disadvantage of the latter method is that it is only semiquantitative. An index of cell multiplication should be a meaningful parameter in studies of drug effects on cancer growth. Therefore, the activity of DNA synthesis during S-phase, measured as incorporation of tritiated thymidine into DNA, was used in the present study. The method has the additional advantage of being technically simple. Previously we studied steroid effects on short-term incubations of cell suspensions from human cancers [4]. Hormone effects were found in mammary cancers, but not in sarcomas. This contrasts with the situation in the rat, where 7,12-DMBA-induced mammary tumors and sarcomas respond in a similar way [5,6]. Moreover, because only inhibitory effects of steroids were seen in cell suspensions of human mammary cancers, it was thought that the more “physiologic” in vitro method, previously published by us [7], could provide more pertinent data on the hormonal responsiveness of individual tumors. From the Tomblad Institute and the Department of Surgery, University of Lund, Sweden. This work was supported by the Faculty of Medicine, University of Lund and by the John and Augusta Persson Foundation, Lund, Sweden. Reprint requests should be addressed to Knut Aspegren, MD, Department of Surgery. University Hospital, S-221 35 Lund, Sweden.
V&ml.
131, May 1976
Material and Methods The technic of organ culture of human mammary cancer has been described in detail previously [ 71.In brief, twelve small explants (approximately 1.5 mm square) are placed on strips of millipore filter supported by cellulose acetate fabric in a Carrel flask. Parker 199 medium, 2 ml, with 20 per cent human inactivated serum, heparin, antibiotics, and hormone is added and the flasks are stoppered without further gassing and incubated at 37’C for 4 to 72 hours. The medium is changed every 24 hours. During the last 4 hours of incubation, the explants are transferred to centrifuge tubes with 1 ml of their own medium, and the cultures are continued in a submerged condition. Tritiated thymidine (H3-TdR) is added to a final concentration of 2 &Yml. The cultures are shaken intermittently and terminated by washing once with cold phosphate buffer. Nucleosides and nucleotides not incorporated into DNA are extracted for 30 minutes with cold (4’C) 5 per cent trichloroacetic acid (TCA), and washed once with cold TCA. A final washing is made with absolute ethanol. The tissue samples are then dissolved in 1 ml Soluene 100, and 14 ml of scintillation fluid is added. The radioactivity is registered by liquid scintillation counting for 10 minutes. The following hormones were used at the following final concentrations: testosterone, 0.5,5, and 50 bg/ml; progesterone, 0.8,8, and 80 Mg/ml;17-&estradiol, 0.02,0.2,2, and 20 wg/ml. The hormones were dissolved in ethanol to give a final ethanol concentration of 1 per cent. The present steroid concentrations were the same as used in previous investigations [d-6] to make comparisons with the latter possible. Each tumor was cultured with at least one of the hormones. In each series, three flasks were prepared as controls (with 1 per cent ethanol only) and two flasks for each concentration of hormone. To study the effect of culture time, each tumor was cultured for 4,24,48, and 72 hours. The mammary cancers (Ml to $410) were obtained from ten randomly selected patients, aged forty-five to seventy-nine years (median, 62.5 years). All tumors were histologicahy proved mammary cancers. Pieces were taken immediately after the operative specimen had been removed, transferred to the laboratory within 15 to 30 minutes, and set up in flasks within 2 to 3 hours. Transport and
575
‘O%o 5.00Effect I” loqo wm
a seventy-seven year old man. S3 was a relapse of fibrosarcoma from the left knee joint capsule in a thirty-nine year old woman. S4 was derived from the left adductor muscle group of the leg in a thirty-six year old woman. All four sarcomas were obviously from tissues that normally are not considered steroid targets. The sarcomas were studied in exactly the same way as the mammary cancers, the same hormonal solutions and sera being used throughout. Histologic Technics. Pieces for histologic sectioning were taken from each tumor when the cultures were set up. They were fixed in formalin, embedded in paraffin wax, sectioned at 5 microns, and stained with hematoxylin and eosin. One mammary cancer was studied with autoradiography after various incubation times. These explants were sectioned at 3 microns. Alternate sections were stained with hematoxylin and eosin and with Giemsa stain. The latter were submitted to autoradiography using the stripping film method, described in detail previously [7]. Statistical Methods. Hormone effects are evaluated as increases or decreases in H3-TdR incorporation in cultures with hormone added compared with similarly made cultures without hormone (controls). Incorporation data are transformed into logic, thus making calculations easier. These log10 values are normally distributed and have a lower coefficient of variation than unlogarithmed counts per minute, as shown elsewhere [S,?]. To evaluate the statistical significance of effects registered after hormone addition, the variance caused by hormone (“between control and hormone-treated groups”) was compared with the technical error variance between replicate cultures. The latter was estimated from a total of 331 double- or triple-replicate determinations. With the use of Bartlett’s test, these estimates were compared according to groupings of tumor or hormone group. Replicate cultures deviating significantly were excluded from the calculation of the general error variance; this happened in 9 of the 331 determinations. When these deviating groups were included in studies of hormone effects, correction was made according to Welch [8]. Variance analysis was used to study the effect of hormones but also of other possible sources of variation (tumor and culture time). The possible effect of these three sources of variation was studied as statistical interaction between them. Again, variances due to such interaction were evaluated against the general error variance.
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Figure 7. Results of three mammary cancers cukured with testosterone. tktper teg insert shows mean tog10 counts per minute for control flasks of each cancer for 4 to 72 hours of cufture. Qther inserts give dose-response curves for each time of cutture of individual cancers. Dotted line fi l) represents 4 hours, dashed/dotted p - - - l ) 24 hours, 72 hours of cutdashed (: - - - -) 48 hours, and solid (-) true. Signifkance limits of F-test between %ormone et%?~V’ and “general error” are given in Figures 1 ‘through 6. Crosses (x) indicate the points of measure that do not agree with the &era1 ev variance, and their significance is marked separately: = 0.05 > p > O-01; * l ‘= 0.01 > p > 0.001; ** = p < 0.001. l
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culture procedures were performed at room temperature (approximately 25’C). Four human fihrosarcomas (Sl to S4) were used as controls. Because of shortage of tumor material, three sarcomas (Sl, S2, and S4) were cultured with more than one hormone. Sl was derived from muscles of the right scapular region in a seventy-two year old woman. S2 was a relapse of a fibrosarcoma of fascia lata of the left leg in
576
Results Z’e@osterone. Three mammary cancers (Figure 1) and three sarcomas (Figure 2) were studied. The upper left insert of Figures 1 and 2 gives the mean incorporation of H3-TdR of control flasks of each individual
tumor
for each culture
time.
The other
inserts in Figures 1 and 2 show the effects, that is, differences between mean incorporation in controls and
hormone-containing
flasks.
Hormone
were seen both in the three mammary
effects
cancers and in
The American Journal
of Surgery
__ ..~ if3
Human Mammary Cancer In Vitro
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Figure 2. Resuffs of three human sarcomas cuttured wtth testosterone (see legend for Figure 1).
Figure 3. Resutts of three mamrnafy cancers cultured with progesterone (see legend for Ftgure 1).
the three sarcomas. At the highest concentration of the hormone, the effects were simiiar, although only inhibitions were seen, and they were more pronounced at 48 and 72 hours than at 4 and 24 hours. At the lowest concentration of the hormone, significant inhibition was seen at 48 hours in Ml and at 72 hours in M3. There were no stimulations from testosterone among the mammary cancers. S2 was significantly stimulated with 0.5 and 5 pg/ml at 48 hours, but not affected at other times of culture. As is evident from the control curve, the incorporation declines from 4 to 48 hours, and the effect of the hormone was to maintain the incorporation near a previous level rather than increase activity of DNA synthesis in S-phase. Progesterone. Again, three mammary cancers, M4 to M6 (Figure 3), and three sarcomas, Sl, S2, and S4
(Figure 4), were tested. All six tumors were affected by the treatment. Again, the highest concentration provided the largest effects (inhibitions), which were usually more pronounced at 48 and 72 hours than at 4 and 24 hours. S4 was markedly inhibited also with 0.8 and 8 pg/ml at 72 hours. M4 was stimulated with 0.8 pg/ml at 48 hours, but the significance of this is weak and the finding may be random. Sl was stimulated with both 0.8 and 8 pg/ml at 48 hours, the latter stimulation being highly significant (0.01 > p > 0.001). As is evident from the growth curve of the controls (Figure 4), this sarcoma increases its incorporation after 24 hours, and the recorded stimulation represents not merely maintenance of previous levels of incorporation but actual stimulation. 17-/!?-estradiol. Four mammary cancers (M7 to MlO) (Figure 5) and three sarcomas (Sl, S3, S4)
Vobma
131, May 1976
577
Aspegren
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Figure 4. Results of three sarcomas cultured with progesterone (see legend for Figure 1).
(Figure 6) were studied. All four mammary cancers responded uniformly in that at 48 hours there were significant stimulations, especially with the two lower concentrations. As is evident from the curve of control incorporation of M9, the incorporation decreases between 24 and 48 hours (logic difference = 0.52) and the effect of the hormonal addition at this concentration at 48 hours has been a maintenance at the level of the control at 24 hours rather than a true stimulation of incorporation. However, the remaining cancers that show stimulations are truly stimulated. Inhibitions are seen in two cancers (M9 and MlO) with 20 pg/ml and in M9 also at 72 hours with 2 pg/ml. With the sarcomas, inhibitions are seen at various times with 20 pg/ml. Sl is stimulated with 0.02 pg/ml at 24 and 48 hours, and there is a tendency to stimulation with 0.2 and 2 wg/ml at the same cul-
578
ture time, although significance is not reached. As the control curves reveal, the stimulations represent real increases in incorporation over previous ‘control values. To evaluate the possible influence of other sources of variation on the effects of hormones (“tumor” and “culture time”) an analysis of secondary variance between these three sources of variation was performed. Table I gives the result for the mammary cancers and for the sarcomas. Because of high technical error in one or more double determinations of an individual tumor, all tumors could not be included in the analysis at all concentrations. There is a significant secondary interaction demonstrable with 5 pg/ml and 50 pg/ml of testosterone, 8 pglml of progesterone, and 20 wg/mlof estradiol. This means that at these concentrations the effects registered due to the hormone addition vary with culture time and tumor. To check the distribution of label in the explants, a scirrhous mammary cancer was studied. Flasks were set up as controls or with hormones added, using the highest concentration of each hormone. They were cultured for 48 hours, and during the last 4 hours, either 2 &/ml of H3-thymidine or 2 PC/ml of 5-H3-uridine was added. Alternate sections from the explants were autoradiographed and similarly studied with hematoxylin and eosin sections. The general pattern of label observed previously [ 71 was found, that is, the grains were mostly distributed over epithelial cells. Labeling with uridine was more profuse than with thymidine and distributed over cytoplasm and nucleus alike. An effort was made to quantitate the labeling in controls and hormonetreated cultures, but this was not possible because of the heterogeneous structure of the tumor and the rather scarce incorporation. To summarize the results: both mammary cancers and sarcomas reacted to hormone addition. Both stimulations and inhibitions of incorporation of H3-thymidine were seen. Labeling was found mostly within epithelial cells, which even after 72 hours of culture synthesized DNA. At least at some hormone concentrations, hormone effects differed according to tumor individuality and culture time. Comments
The results of a previous study of hormonal effects on cell suspensions of mammary cancers did not correlate with the clinical course of the disease, and only inhibitory effects were registered [4]. Therefore, the present investigation was performed using the more “physiologic” method of organ cultures. On the
TheAmerican
Journal ol Surgery
Human Mammary Cancer In Vitro
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basis of previous similar experiments on rat mammary tumor and subcutaneous sarcoma [5,6], human sarcomas were included as controls. The results of earlier, methodologic studies of human mammary cancer [7] were confirmed; the cancers could be routinely cultured for 72 hours and growth was mostly confined to epithelial cells. The results of hormone addition to cultures of mammary cancers agree well with those published by other investigators [3,9]. However, the similar results obtained with sarcomas seriously question the specificity of hormonal response in vitro, as human sarcomas are not generally recognized as hormonally sensitive in vivo. Because both stimulations and in-
0.2
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Figure 5. Results of four mammary cancers cultured with 17-fl-e&ad/o/ (see legend for Figure 1).
hibitions were seen with the two tumor types, the results cannot be explained as merely toxic effects due to the high concentrations used. These are similar to those used by other workers in this field. Moreover, the present investigation demonstrates that, in culture studies of this kind, the duration of culture may affect the results obtained with hormone addition. A previous paper [6] also showed that variation caused by technical differences can influence the effects. The study shows that the results of in vitro studies of hormonal responsiveness of cancer tissue must be interpreted with great care before they can be applied clinically.
579
Aspegren
TABLE I
log,, cm
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-;
J I ./sr I
L.50 j
Hormone Concentration
I /
(pg/ml)
Variance Analysis of Interaction between Variation due to Hormonal Effects, to Differences between Mammary Cancers and Sarcomas, and to Differences between Culture Times. Secondary Interaction Variance is Given and Compared by F-Test with General Error Variance. Number of Tumors
Degree of freedom Variance
(df)
F
5
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12
1.57
5 5
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Figure 6. Results of three sarcomas cultured with 17-Bestradiol (see legend for Figure 1).
Summary
Specimens of human breast cancers and fibrosarcomas were incubated for 4 to 72 hours as organ cultures. The effect of various concentrations of testosterone, progesterone, and 17-8-estradiol on cell survival and multiplication was measured as changes in incorporation of H3-TdR into DNA. Both types of tumor reacted to the hormones; both stimulations and inhibitions were recorded. Statistical analysis of data demonstrated possible influence of factors other than hormone treatment. The specificity of hormone sensitivity assays in vitro is questioned.
References 1. Jensen EV, Block GE, Smith S, Kyser K, DeSombre ER: Estrogen receptors and hormone dependency. Estrogen Target Tissues and Neoplasia (Dao TL; ed). Chicago and London, 1972.
580
0.02
Note: General error variance NS = p > 0.05. > p > 0.01. * = 0.025 > p > 0.001. t = 0.005
= 0.0220/379
(NS) (NS) 2.31t
df.
2. Dao TL, Libby PR: Steroid sulphate formation in human breast tumors and hormone dependency. Estrogen Target Tissues and Neopiasia (Dao TL, ed). Chicago and London, 1972. 3. Salih H. Flax H, Hobbs JR: In vitro oestrogen sensitivity of breast cancer tissue as a possible screening method for hormonal treatment. Lancet 1: 1198, 1972. 4. Aspegren K, Hgkansson L: Human mammary carcinoma studied for hormone responsiveness in short term incubations. Acta Chir Stand 140: 95, 1974. 5. Aspegren K: 7,12-DMBA-induced rat mammary tumour studied for hormonal responsiveness in vitro. I. Short-term incubations of cell suspensions. Acta Pathol Microbial Scati [A] 83: 25, 1975. 6. Aspegren K: 7.1 P-DMBA-induced rat mammary tumour studied for hormonal responsiveness in vitro. II. Organ cultures. Ada Pathol Microbial Stand [A] 83: 37, 1975. 7. Aspegren K, Danielsson H: Growth quantitation of human mammary carcinoma in organ tissue cultures. Am J Swg 128: 42, 1974. 8. Welch BL: Note to Aspin AA: Tables for use in comparisons whose accuracy involves two variances, separately estimated. Wiometrika 36: 293, 1949. 9. Riley PA, Latter T, Sutton PM: Hormone assays on breast-tumour cultures. Lancer 2: 818, 1973.
The Arwrican Journal 01 Surwry
Mammary cancer is regarded as a hormonally responsive tumor, but only one of three patients with widespread disease will benefit from hormonal treatment. Prediction of response by laboratory methods would be of great value. In vitro assay of estrogen receptors of mammary cancer has been correlated to the patient’s clinical course [I]. Correlation of the activity of steroid sulfhydration by cancer tissue to clinical course has been suggested [2]. Hormonal effect on dehydrogenase activity of cancer tissue has also been used for prediction [3]. These methods measure the ability of the tissue to bind or metabolize the steroid or the steroid’s effect on intermediary cell metabolism, that is, pentose shunt activity. A disadvantage of the latter method is that it is only semiquantitative. An index of cell multiplication should be a meaningful parameter in studies of drug effects on cancer growth. Therefore, the activity of DNA synthesis during S-phase, measured as incorporation of tritiated thymidine into DNA, was used in the present study. The method has the additional advantage of being technically simple. Previously we studied steroid effects on short-term incubations of cell suspensions from human cancers [4]. Hormone effects were found in mammary cancers, but not in sarcomas. This contrasts with the situation in the rat, where 7,12-DMBA-induced mammary tumors and sarcomas respond in a similar way [5,6]. Moreover, because only inhibitory effects of steroids were seen in cell suspensions of human mammary cancers, it was thought that the more “physiologic” in vitro method, previously published by us [7], could provide more pertinent data on the hormonal responsiveness of individual tumors. From the Tomblad Institute and the Department of Surgery, University of Lund, Sweden. This work was supported by the Faculty of Medicine, University of Lund and by the John and Augusta Persson Foundation, Lund, Sweden. Reprint requests should be addressed to Knut Aspegren, MD, Department of Surgery. University Hospital, S-221 35 Lund, Sweden.
V&ml.
131, May 1976
Material and Methods The technic of organ culture of human mammary cancer has been described in detail previously [ 71.In brief, twelve small explants (approximately 1.5 mm square) are placed on strips of millipore filter supported by cellulose acetate fabric in a Carrel flask. Parker 199 medium, 2 ml, with 20 per cent human inactivated serum, heparin, antibiotics, and hormone is added and the flasks are stoppered without further gassing and incubated at 37’C for 4 to 72 hours. The medium is changed every 24 hours. During the last 4 hours of incubation, the explants are transferred to centrifuge tubes with 1 ml of their own medium, and the cultures are continued in a submerged condition. Tritiated thymidine (H3-TdR) is added to a final concentration of 2 &Yml. The cultures are shaken intermittently and terminated by washing once with cold phosphate buffer. Nucleosides and nucleotides not incorporated into DNA are extracted for 30 minutes with cold (4’C) 5 per cent trichloroacetic acid (TCA), and washed once with cold TCA. A final washing is made with absolute ethanol. The tissue samples are then dissolved in 1 ml Soluene 100, and 14 ml of scintillation fluid is added. The radioactivity is registered by liquid scintillation counting for 10 minutes. The following hormones were used at the following final concentrations: testosterone, 0.5,5, and 50 bg/ml; progesterone, 0.8,8, and 80 Mg/ml;17-&estradiol, 0.02,0.2,2, and 20 wg/ml. The hormones were dissolved in ethanol to give a final ethanol concentration of 1 per cent. The present steroid concentrations were the same as used in previous investigations [d-6] to make comparisons with the latter possible. Each tumor was cultured with at least one of the hormones. In each series, three flasks were prepared as controls (with 1 per cent ethanol only) and two flasks for each concentration of hormone. To study the effect of culture time, each tumor was cultured for 4,24,48, and 72 hours. The mammary cancers (Ml to $410) were obtained from ten randomly selected patients, aged forty-five to seventy-nine years (median, 62.5 years). All tumors were histologicahy proved mammary cancers. Pieces were taken immediately after the operative specimen had been removed, transferred to the laboratory within 15 to 30 minutes, and set up in flasks within 2 to 3 hours. Transport and
575
‘O%o 5.00Effect I” loqo wm
a seventy-seven year old man. S3 was a relapse of fibrosarcoma from the left knee joint capsule in a thirty-nine year old woman. S4 was derived from the left adductor muscle group of the leg in a thirty-six year old woman. All four sarcomas were obviously from tissues that normally are not considered steroid targets. The sarcomas were studied in exactly the same way as the mammary cancers, the same hormonal solutions and sera being used throughout. Histologic Technics. Pieces for histologic sectioning were taken from each tumor when the cultures were set up. They were fixed in formalin, embedded in paraffin wax, sectioned at 5 microns, and stained with hematoxylin and eosin. One mammary cancer was studied with autoradiography after various incubation times. These explants were sectioned at 3 microns. Alternate sections were stained with hematoxylin and eosin and with Giemsa stain. The latter were submitted to autoradiography using the stripping film method, described in detail previously [7]. Statistical Methods. Hormone effects are evaluated as increases or decreases in H3-TdR incorporation in cultures with hormone added compared with similarly made cultures without hormone (controls). Incorporation data are transformed into logic, thus making calculations easier. These log10 values are normally distributed and have a lower coefficient of variation than unlogarithmed counts per minute, as shown elsewhere [S,?]. To evaluate the statistical significance of effects registered after hormone addition, the variance caused by hormone (“between control and hormone-treated groups”) was compared with the technical error variance between replicate cultures. The latter was estimated from a total of 331 double- or triple-replicate determinations. With the use of Bartlett’s test, these estimates were compared according to groupings of tumor or hormone group. Replicate cultures deviating significantly were excluded from the calculation of the general error variance; this happened in 9 of the 331 determinations. When these deviating groups were included in studies of hormone effects, correction was made according to Welch [8]. Variance analysis was used to study the effect of hormones but also of other possible sources of variation (tumor and culture time). The possible effect of these three sources of variation was studied as statistical interaction between them. Again, variances due to such interaction were evaluated against the general error variance.
i,.-. _.--k MI
+ 0.50 _--_----___--_-*** _-__----_____-_-•* i _--------------_-•
Lt.0 -
L.00.
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. ‘1”. .., ‘. __JL___‘L__‘ __L.
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Figure 7. Results of three mammary cancers cukured with testosterone. tktper teg insert shows mean tog10 counts per minute for control flasks of each cancer for 4 to 72 hours of cufture. Qther inserts give dose-response curves for each time of cutture of individual cancers. Dotted line fi l) represents 4 hours, dashed/dotted p - - - l ) 24 hours, 72 hours of cutdashed (: - - - -) 48 hours, and solid (-) true. Signifkance limits of F-test between %ormone et%?~V’ and “general error” are given in Figures 1 ‘through 6. Crosses (x) indicate the points of measure that do not agree with the &era1 ev variance, and their significance is marked separately: = 0.05 > p > O-01; * l ‘= 0.01 > p > 0.001; ** = p < 0.001. l
l
l
l
l
l
l
culture procedures were performed at room temperature (approximately 25’C). Four human fihrosarcomas (Sl to S4) were used as controls. Because of shortage of tumor material, three sarcomas (Sl, S2, and S4) were cultured with more than one hormone. Sl was derived from muscles of the right scapular region in a seventy-two year old woman. S2 was a relapse of a fibrosarcoma of fascia lata of the left leg in
576
Results Z’e@osterone. Three mammary cancers (Figure 1) and three sarcomas (Figure 2) were studied. The upper left insert of Figures 1 and 2 gives the mean incorporation of H3-TdR of control flasks of each individual
tumor
for each culture
time.
The other
inserts in Figures 1 and 2 show the effects, that is, differences between mean incorporation in controls and
hormone-containing
flasks.
Hormone
were seen both in the three mammary
effects
cancers and in
The American Journal
of Surgery
__ ..~ if3
Human Mammary Cancer In Vitro
‘O%o
lo9,ocm
5.50
5.00 -
Effect m log,,, cPm
Effect in loqo cpm \$
I 5.00-
ML
1
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00
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Effect m loglo cPm M5
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0
00
wlml
w/ml
Figure 2. Resuffs of three human sarcomas cuttured wtth testosterone (see legend for Figure 1).
Figure 3. Resutts of three mamrnafy cancers cultured with progesterone (see legend for Ftgure 1).
the three sarcomas. At the highest concentration of the hormone, the effects were simiiar, although only inhibitions were seen, and they were more pronounced at 48 and 72 hours than at 4 and 24 hours. At the lowest concentration of the hormone, significant inhibition was seen at 48 hours in Ml and at 72 hours in M3. There were no stimulations from testosterone among the mammary cancers. S2 was significantly stimulated with 0.5 and 5 pg/ml at 48 hours, but not affected at other times of culture. As is evident from the control curve, the incorporation declines from 4 to 48 hours, and the effect of the hormone was to maintain the incorporation near a previous level rather than increase activity of DNA synthesis in S-phase. Progesterone. Again, three mammary cancers, M4 to M6 (Figure 3), and three sarcomas, Sl, S2, and S4
(Figure 4), were tested. All six tumors were affected by the treatment. Again, the highest concentration provided the largest effects (inhibitions), which were usually more pronounced at 48 and 72 hours than at 4 and 24 hours. S4 was markedly inhibited also with 0.8 and 8 pg/ml at 72 hours. M4 was stimulated with 0.8 pg/ml at 48 hours, but the significance of this is weak and the finding may be random. Sl was stimulated with both 0.8 and 8 pg/ml at 48 hours, the latter stimulation being highly significant (0.01 > p > 0.001). As is evident from the growth curve of the controls (Figure 4), this sarcoma increases its incorporation after 24 hours, and the recorded stimulation represents not merely maintenance of previous levels of incorporation but actual stimulation. 17-/!?-estradiol. Four mammary cancers (M7 to MlO) (Figure 5) and three sarcomas (Sl, S3, S4)
Vobma
131, May 1976
577
Aspegren
lo9,0cpm SSOEffect I” kqo cm Sl l
0.50 ?I-----**.
b IIII>?ffyI+IIII -T,‘,
,+..-
.<>- .
.’ .
“. .,.,, \I
3,
----------
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___________
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I_\,
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- 1.w hours
0.8
\-.l.lB .1.75 80
8
u/ml
Effect in kqo cpm
Effect in lye cm
SL
s2 l
0.50 _______________~.~ I ____----_______**
----------------I**
--------_-------•*
j__---__________. /’
_______________.
/”
a--__,
,<...-4_ ._.-..
t#mIlT ,/
,..... ...+
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<.&R----t
‘ix.
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‘\
\ ‘.. \
;
‘$::.
__________
0.50 ----------
I;
.l.S3
- 1.w
0.B
8
80
uglml
1.
.-*
\.
-‘1;_ L.--.
__ _____&_L.~_ ” 1 -.* ___ _____\,__1._\,,.. \ ‘.. \ 1 \ \ \ \
‘\, 0.8
I
8
,
1.74
. . 1.12
80
w/ml
Figure 4. Results of three sarcomas cultured with progesterone (see legend for Figure 1).
(Figure 6) were studied. All four mammary cancers responded uniformly in that at 48 hours there were significant stimulations, especially with the two lower concentrations. As is evident from the curve of control incorporation of M9, the incorporation decreases between 24 and 48 hours (logic difference = 0.52) and the effect of the hormonal addition at this concentration at 48 hours has been a maintenance at the level of the control at 24 hours rather than a true stimulation of incorporation. However, the remaining cancers that show stimulations are truly stimulated. Inhibitions are seen in two cancers (M9 and MlO) with 20 pg/ml and in M9 also at 72 hours with 2 pg/ml. With the sarcomas, inhibitions are seen at various times with 20 pg/ml. Sl is stimulated with 0.02 pg/ml at 24 and 48 hours, and there is a tendency to stimulation with 0.2 and 2 wg/ml at the same cul-
578
ture time, although significance is not reached. As the control curves reveal, the stimulations represent real increases in incorporation over previous ‘control values. To evaluate the possible influence of other sources of variation on the effects of hormones (“tumor” and “culture time”) an analysis of secondary variance between these three sources of variation was performed. Table I gives the result for the mammary cancers and for the sarcomas. Because of high technical error in one or more double determinations of an individual tumor, all tumors could not be included in the analysis at all concentrations. There is a significant secondary interaction demonstrable with 5 pg/ml and 50 pg/ml of testosterone, 8 pglml of progesterone, and 20 wg/mlof estradiol. This means that at these concentrations the effects registered due to the hormone addition vary with culture time and tumor. To check the distribution of label in the explants, a scirrhous mammary cancer was studied. Flasks were set up as controls or with hormones added, using the highest concentration of each hormone. They were cultured for 48 hours, and during the last 4 hours, either 2 &/ml of H3-thymidine or 2 PC/ml of 5-H3-uridine was added. Alternate sections from the explants were autoradiographed and similarly studied with hematoxylin and eosin sections. The general pattern of label observed previously [ 71 was found, that is, the grains were mostly distributed over epithelial cells. Labeling with uridine was more profuse than with thymidine and distributed over cytoplasm and nucleus alike. An effort was made to quantitate the labeling in controls and hormonetreated cultures, but this was not possible because of the heterogeneous structure of the tumor and the rather scarce incorporation. To summarize the results: both mammary cancers and sarcomas reacted to hormone addition. Both stimulations and inhibitions of incorporation of H3-thymidine were seen. Labeling was found mostly within epithelial cells, which even after 72 hours of culture synthesized DNA. At least at some hormone concentrations, hormone effects differed according to tumor individuality and culture time. Comments
The results of a previous study of hormonal effects on cell suspensions of mammary cancers did not correlate with the clinical course of the disease, and only inhibitory effects were registered [4]. Therefore, the present investigation was performed using the more “physiologic” method of organ cultures. On the
TheAmerican
Journal ol Surgery
Human Mammary Cancer In Vitro
loglo cpm 5.00 Effect In blo cpm
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1
TM9
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1
M7
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0.2
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M 10 1
-
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1
- 1.00
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m/ml
basis of previous similar experiments on rat mammary tumor and subcutaneous sarcoma [5,6], human sarcomas were included as controls. The results of earlier, methodologic studies of human mammary cancer [7] were confirmed; the cancers could be routinely cultured for 72 hours and growth was mostly confined to epithelial cells. The results of hormone addition to cultures of mammary cancers agree well with those published by other investigators [3,9]. However, the similar results obtained with sarcomas seriously question the specificity of hormonal response in vitro, as human sarcomas are not generally recognized as hormonally sensitive in vivo. Because both stimulations and in-
0.2
2
20 pg I ml
Figure 5. Results of four mammary cancers cultured with 17-fl-e&ad/o/ (see legend for Figure 1).
hibitions were seen with the two tumor types, the results cannot be explained as merely toxic effects due to the high concentrations used. These are similar to those used by other workers in this field. Moreover, the present investigation demonstrates that, in culture studies of this kind, the duration of culture may affect the results obtained with hormone addition. A previous paper [6] also showed that variation caused by technical differences can influence the effects. The study shows that the results of in vitro studies of hormonal responsiveness of cancer tissue must be interpreted with great care before they can be applied clinically.
579
Aspegren
TABLE I
log,, cm
5.50;
Sl Effect
I”
loglowm 51
/ ----.S3
-;
J I ./sr I
L.50 j
Hormone Concentration
I /
(pg/ml)
Variance Analysis of Interaction between Variation due to Hormonal Effects, to Differences between Mammary Cancers and Sarcomas, and to Differences between Culture Times. Secondary Interaction Variance is Given and Compared by F-Test with General Error Variance. Number of Tumors
Degree of freedom Variance
(df)
F
5
0.0347
12
1.57
5 5
0.0460 0.0617
12 12
(NS) 2.09* 2.80t
5
0.0301
12
1.37
5 3
0.0626 0.0374
12 6
(NS) 2.84t 1.70
L.W
Testosterone 0.5
-A__-._ 0.02
0.2
2
W/ml
20
Progesterone
Effect m
b10
CPm SL
5.3 l
5 50
0.8 8 80
0.50 ___________________-,*, _______________-_---..
(NS) 17.p-estradiol
-
,.A._._-_ 0.02
Q2
7
0.0312
18
1.42
0.2
7
0.0349
18
(NS) 1.58
5 6
0.0216 0.0508
12 15
2 20 2
20 w/ml
0.02
0.2
2
20 vglml
Figure 6. Results of three sarcomas cultured with 17-Bestradiol (see legend for Figure 1).
Summary
Specimens of human breast cancers and fibrosarcomas were incubated for 4 to 72 hours as organ cultures. The effect of various concentrations of testosterone, progesterone, and 17-8-estradiol on cell survival and multiplication was measured as changes in incorporation of H3-TdR into DNA. Both types of tumor reacted to the hormones; both stimulations and inhibitions were recorded. Statistical analysis of data demonstrated possible influence of factors other than hormone treatment. The specificity of hormone sensitivity assays in vitro is questioned.
References 1. Jensen EV, Block GE, Smith S, Kyser K, DeSombre ER: Estrogen receptors and hormone dependency. Estrogen Target Tissues and Neoplasia (Dao TL; ed). Chicago and London, 1972.
580
0.02
Note: General error variance NS = p > 0.05. > p > 0.01. * = 0.025 > p > 0.001. t = 0.005
= 0.0220/379
(NS) (NS) 2.31t
df.
2. Dao TL, Libby PR: Steroid sulphate formation in human breast tumors and hormone dependency. Estrogen Target Tissues and Neopiasia (Dao TL, ed). Chicago and London, 1972. 3. Salih H. Flax H, Hobbs JR: In vitro oestrogen sensitivity of breast cancer tissue as a possible screening method for hormonal treatment. Lancet 1: 1198, 1972. 4. Aspegren K, Hgkansson L: Human mammary carcinoma studied for hormone responsiveness in short term incubations. Acta Chir Stand 140: 95, 1974. 5. Aspegren K: 7,12-DMBA-induced rat mammary tumour studied for hormonal responsiveness in vitro. I. Short-term incubations of cell suspensions. Acta Pathol Microbial Scati [A] 83: 25, 1975. 6. Aspegren K: 7.1 P-DMBA-induced rat mammary tumour studied for hormonal responsiveness in vitro. II. Organ cultures. Ada Pathol Microbial Stand [A] 83: 37, 1975. 7. Aspegren K, Danielsson H: Growth quantitation of human mammary carcinoma in organ tissue cultures. Am J Swg 128: 42, 1974. 8. Welch BL: Note to Aspin AA: Tables for use in comparisons whose accuracy involves two variances, separately estimated. Wiometrika 36: 293, 1949. 9. Riley PA, Latter T, Sutton PM: Hormone assays on breast-tumour cultures. Lancer 2: 818, 1973.
The Arwrican Journal 01 Surwry