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Differential Growth Regulation of Human Bladder Tumor Cells (J82) in Culture by Normal Bladder Cells from Different Aged Human Donors
0022-534 7/83/1303-0593$02.00/0
Vol. 130, September Printed in U.S.A.
THE JOURNAL OF UROLOGY
Copyright© 1983 by The Williams & Wilkins Co.
Original Articles DIFFERENTIAL GROWTH REGULATION OF HUMAN BLADDER TUMOR CELLS (J82) IN CULTURE BY NORMAL BLADDER CELLS FROM DIFFERENT AGED HUMAN DONORS SUSUMU KAGAWA*
AND
DAVID KIRKt
From the Huntington Medical Research Institutes, Pasadena, California
ABSTRACT
This study used an agarose gel culture system to study growth regulatory interactions between normal and neoplastic human bladder cells_ The effects of normal human fibroblasts on tumor cell cloning efficiency in agarose was studied using 2 experimental protocols. In 1 system, the fibroblasts proliferated as anchored cells beneath the agarose, and in the other the fibroblasts were nongrowing cells embedded within the agarose as nonanchored cells. In contrast to the largely inhibitory effects of anchored fetal fibroblasts on tumor cell growth, their nonanchored counterparts were exclusively stimulatory. Similar experiments with normal fibroblasts derived from the bladders of increasingly older donors showed a progressive decline in their capacity to inhibit tumor cell growth in agarose. Further experiments modulating the growth rate of neonatal fibroblasts with fetal bovine serum have indicated that the diverse tumor responses elicited by anchored versus nonanchored cells, as well as those observed for the aged bladder fibroblasts, were a function of fibroblast growth rate. We concluded that there was an inverse regulatory relationship between the growth of normal and tumor cells; rapidly growing fibroblasts were inhibitory whereas nongrowing fibroblasts stimulated tumor cell growth in agarose culture. The stromal component of stromal-epithelial organ systems is known to have a vital role both in controlling the normal organogenesis of the vertebrate embryo 1 and in maintaining normal histological differentiation of adult tissues. 2 Recent in vitro studies have detected interesting growth interactions between normal human lung fibroblasts and an established human prostatic carcinoma cell line. 3' 4 The researchers concluded that the normal lung cells had the distinct capability to regulate the growth of the prostatic tumor cells in agar gel culture. 4 However, these studies used heterologous cell types, and the interactions were not characterized with respect to either the tissue source or donor age of the fibroblasts. We have extended this work to include a study of cellular growth interactions exclusive to normal/neoplastic human bladder cell types. We chose as the malignant cell type a wellcharacterized cell line (J82) from a human bladder transitional cell carcinoma. 5 Since growth in agarose is the best available in vitro correlate for tumorigenesis in vivo,6 we studied the effects of normal human bladder fibroblasts on the clonal growth of J82 cells in agarose. Our primary aim was to determine if the in vitro growth regulation observed for the heterotypic cell combinations4 was applicable to a homotypic normal neoplastic human bladder cell culture system. Secondly, since cancer incidence increases with old age, we investigated the role of donor age on the ability of normal bladder fibroblasts (NBF) to regulate the bladder tumor cells in culture. Accepted for publication May 3, 1983. Supported in part by Mr. Francis L. Moseley. * Current address: Department of Urology, School of Medicine, Tokushima University, Kuramoto-2, Tokushima, Japan. t Requests for reprints: Huntington Medical Research Institutes, 99 N. El Molino Avenue, Pasadena, California 91101. 593
MATERIALS AND METHODS
Human bladder tumor cells (J82) were derived initially from a transitional cell carcinoma,5 and used between passage levels 54-78. Normal human fibroblast cultures were derived from histologically normal bladder specimens obtained at surgery or autopsy. Bladder explant cultures were initiated as previously described 7 from specimens obtained from a fetus (NBF-147), a 50-year-old woman (NBF-152) and a 70-year-old man (NBF32). Fibroblast lines were used at early passage levels (1-4). The growth medium (GM) for the explant of primary tissue and the maintenance of monolayer cultures was PFMR-4 7 supplemented with 5 per cent fetal bovine serum (FBS, Irvine Scientific) and antibiotics (penicillin, 100 I.U./ml.; kanamycin, 100 µg./ml.). All cell lines were mycoplasma-free as determined by scanning electron microscopy. 8 Fibroblasts were interacted with tumor cells in agarose either as anchored or nonanchored cells as described previously. 4 Agarose was made up with PFMR-4 supplemented with 20 per cent FBS and antibiotics as before. In the anchored system, fibroblasts were seeded in GM in 60 mm. dishes and allowed to attach overnight. They were then covered with a hard agarose base (0.5 per cent, 1.5 ml.) which was in turn overlaid with a soft agarose layer (0.3 per cent, 3 ml.) containing the tumor cells (5 x 103 ). In the nonanchored system, separate layers (1.5 ml.) of tumor cells and nonanchored fibroblasts in soft agarose were physically separated by a hard agarose layer (1.5 ml.). Tumor cell growth in both the anchored and nonanchored systems was measured in terms of colony forming efficiency (CFE) which was defined as the percentage of seeded cells that formed colonies. Colonies were automatically enumerated and sized using a Hamamatsu Image Analyzer System. Only colonies greater than 90 µ in diameter were counted (average
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KAGAWA AND KIRK
diameter of the J82 cells in agarose was between .15-20 µ). All cultures were set up in triplicate and scored for CFE after 1014 days in culture. Growth rates of mass and clonal monolayer cultures were expressed as population doublings per day (PD/D). The PD/D of mass cultures was determined by taking the log2 of the net increase in cell number and dividing by the number of days in culture. For clonal cultures, the PD/D was obtained by dividing the log2 of the mean cell number per colony by the number of days in culture. Average cell generation time (g) was obtained by taking the reciprocal of the PD/D ( g
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Coverslip cultures of fibroblasts were prepared by seeding 5 X 103 cells in 1.5 ml. of GM in 24-well Costar dishes containing 13 mm. glass coverslips. Cells were allowed to attach overnight before the coverslips were used in an experiment. For radioactive incorporation measurements, cultures were pulsed for 3 hours with tritiated thymidine (1.0 µCi/ml., 74.9 Ci/mmol.) in thymidine-free PFMR-4 supplemented with 20 per cent FBS. After pulsing, coverslip cultures were rinsed in saline, fixed in absolute ethanol, rinsed in 5 per cent trichloroacetic acid at 4C (30 min. X 2), further rinsed in ethanol and air dried. Processed coverslips were counted in 10 ml. ofBeckman's Ready-Solv GP using a Beckman LS9000 liquid scintillation spectrometer.
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Fm. 1. Effect of increasing the cell inoculum of different anchored human bladder fibroblasts on the relative colony forming efficiency (CFE) of J82 cells in agarose. e, NBF-147; D, NBF-152; 0, NBF-32. (Absolute control CFE's of the 3 experiments represented in fig. 1 ranged from 0.5 to 16.3%).
RESULTS
Normal human bladder fibroblasts derived from differentaged donors were compared for their effects in modulating the clonal growth of human bladder tumor cells (J82) using an agarose culture interaction system. The fibroblasts were interacted with the tumor cells either as anchored cells (a proliferating monolayer beneath the agarose/tumor cell layer) or as nonanchored cells (nonproliferating cells embedded in a soft agarose layer separated from the agarose/tumor cell layer by a hard agarose layer). Increasing the inoculum of anchored cells caused a biphasic growth response of J82 for all 3 fibroblast lines (fig. 1), small inocula producing greater growth stimulation than higher inocula. Although all 3 fibroblast lines caused initial enhancement of J82 growth at low cell inocula, higher cell inocula showed marked differences between the different fibroblast lines. There was an inverse relationship between the donor age of the different lines and the growth response of the J82 tumor cells in agarose. The effect of a 4.5 X 104 fibroblast inoculum on J82 growth (fig. 1) showed a 50 per cent inhibition with fetal donor cells (NBF-147), no effect with 50-year-old donor cells (NBF-152) and a 165 per cent stimulation with 70year-old donor cells (NBF-32). Hence, using the anchored fibroblast system, fetal bladder cells (NBF-147) largely exhibited a strong inhibitory effect on J82 whereas cells from the oldest donor (NBF-32) resulted in a consistent growth stimulatory effect. Cells from the median aged donor (NBF-152) manifested intermediate effects, causing a moderate inhibition of J82. The effects of nonanchored bladder fibroblasts (embedded within the agarose) were dramatically different from their parallel anchored counterparts. All fibroblast lines, as nonanchored cells, were exclusively stimulatory to J82 (fig. 2). However, kinetics of the dose-dependent stimulations were different for the different lines. Maximal growth stimulation was much greater for the fetal cells (NBF-147, 6-fold) compared to the other 2 lines (NBF-152, NBF-32; both approximately 3-fold). In addition, the fibroblast inoculum necessary to effect a halfmaximal J82 growth response was greater for the fetal cells (NBF-147; 6.5 x 104 ) than for the other lines (NBF-152, NBF32; both 2.5 X 104 ). It is important to notice that the differential effects observed for the anchored fibroblasts on J82 (fig. 1) are associated with differences in the growth rates of the fibroblast lines. Growth curves of cells seeded at 105 per 60 mm. dish in 4 ml. of PFMR4 supplemented with 20 per cent FBS (nutrient regime as used in fig. 1) showed the fetal cells (NBF-147) to have a much
•
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FIG. 2. Effect of increasing the cell inoculum of different nonanchored human bladder fibroblasts on the relative colony forming efficiency (CFE) of J82 cells in agarose. e, NBF-147; D, NBF-152; 0, NBF-32. (Cultures were set up simultaneously with their anchored counterparts as described in fig. 1, and shared the same controls).
shorter cell generation time (28.9 hours) than the 50-year-old donor cells (NBF-152, 46.9 hours). To investigate further the role of fibroblast growth rate in regulating J82 growth in agarose we have used FBS as a means to modulate fibroblast growth rate. The growth of the fetal (NBF-147) and tumor cells (J82) both showed a dose-dependent serum response in monolayer culture (fig. 3). Growth rates for both lines were maximal in 20 per cent FBS although the serum requirement for half-maximal growth was lower for J82 (1 per cent, FBS) than for NBF-147 ( 4 per cent, FBS). We subsequently studied growth interactions between anchored NBF-14 7 and J82 at a range of FBS concentrations. An NBF-147 cell inoculum of 105 per dish was selected since it was known to cause approximately a 75 per cent inhibition of J82
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NORMAL-NEOPLASTIC CELL INTERACTIONS
growth in 20 per cent FBS (fig. 1). Upon reducing the serum concentration, the fibroblast effect changed from inhibitory to J82 to stimulatory in a clear biphasic fashion (fig. 4). Whereas maximum inhibition of J82 was observed with 15 per cent FBS (p < 0.001), maximum stimulation was observed with 5 per cent FBS (p < 0.05). The null-point, that is, the point where the fibroblasts had no effect on the growth of J82, was observed with 10 per cent FBS (fig. 4). An experiment was specifically designed to determine if the
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Fm. 3. Serum titrations of J82 (e) and NBF-147 cells (0) in monolayer culture. NBF-147 cells were seeded at 5 X 104 per 60 mm. dish in 4 ml. of growth medium. After 20 hours, growth medium was supplemented with different serum concentrations and cell counts on trypsinized cultures determined on day 4. (Under these seeding conditions the growth rate in 20 per cent FBS was linear for at least 7 days.) J82 were seeded at 500 cells per 60 mm. dish and set up in different serum concentrations as for NBF-147. After 7 days clones were fixed in methanol and stained with Weigert's Iron Hematoxylin.
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inhibition of J82 by NBF-147 at high serum levels could be due to the nonspecific effects of nutrient deprivation and culture toxicity. Conceptually, this was approached by monitoring the growth of "indicator" NBF cells which were cultured concomitantly in the presence of the J82/NBF co-cultures. The growth of the indicator cells was deemed to "indicate" the growth status of the nutrient medium throughout the course of the experiment. The experiment was set up as follows. Agarose cultures of J82 were set up using the anchored fibroblast protocol in 20 per cent FBS with an NBF-147 inoculum of 3 x 105 cells per dish, an inoculum which was known to cause essentially a 100 per cent inhibition of J82 (fig. 1). In addition, glass coverslip cultures ofNBF-147 (indicator cells) were added on top of the agarose containing the J82 cells, with the cell monolayer facing up. The NBF-147/J82 agarose co-cultures containing the NBF-147 coverslip cultures were refed every 2 days with 1.5 ml. of fresh liquid PFMR-4 medium supplemented with 20 per cent FBS. Cultures were periodically pulsed with tritiated thymidine and the coverslips removed and measured for tritium incorporation into NBF-147 cells. The incorporation measured in the NBF-147 coverslip cultures (indicator NBF147 cells) was thus a measure of the prevailing nutrient conditions within the anchored NBF-147/J82 agarose co-cultures. Initially (day 2), NBF-147 indicator cells in the presence of anchored NBF-147/J82 co-cultures showed a marked growth stimulation (X 2.39 control, fig. 5) which decreased with increasing time in culture. After 9 days, the indicator NBF-147 cells showed a 40 per cent inhibition whereas at this time the J82 cells showed a 95.7 per cent inhibition (data not shown). This direct demonstration of a differential inhibition of J82 cells clearly showed that adverse culture conditions account for no more than 40 per cent of the observed inhibition. The remaining 60 per cent of the inhibition represented the specific effect of NBF cells on J82 cells. In contrast, indicator NBF-147 cells alone caused a marginal stimulation of J82 clonal forming efficiency (data not shown). This increase in the growth of the J82 tumor cells was in turn
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FBS!!Jo~ Fm. 4. Effect of fetal bovine serum concentration on the interaction of NBF-147 cells on J82 cells in agarose. NBF-147 cells were seeded at 105 per 60 mm. dish and set up for the anchored fibroblast system as described in MATERIALS AND METHODS. Net fibroblast effects on J82 colony forming efficiency ( CFE) were determined by subtracting the control J82 colony forming efficiency (CcFE) from the colony forming efficiency of J82 in the NBF-147/J82 co-cultures (FcFE) for each serum level. Differences were tested for significance using a Student's t test.
DAYS Fm. 5. Relative incorporation of tritiated thymidine into indicator NBF-147 coverslip cultures. 0, in the presence ofNBF-147/J82 agarose co-cultures set up as described for the anchored fibroblast system. e, in the presence of J82 alone in agarose. Coverslips were set up in triplicate and the incorporation expressed as percentage of controls (NBF-147 coverslip cultures added to the agarose system as described but without either J82 or anchored NBF-147 cells). The standard errors ranged from 2.5 to 18% of mean values.
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KAGAWA AND KIRK
associated with a stimulation of the NBF indicator cells (fig. 5), a similar stimulation having been observed previously in another normal/tumor cell co-culture study. 3 DISCUSSION
This study showed that, using a similar agarose system, human fetal bladder fibroblasts regulated the growth of human bladder tumor cells in a similar fashion to that reported for human lung neonatal fibroblasts on human prostatic carcinoma cells. 4 In both cases nonanchored cells stimulated tumor cell growth in agarose whereas anchored cells inhibited the tumor cells. The dose-response curves for the inhibitions and stimulations also showed similar kinetics for both the bladder and the lung/prostatic cell systems. This similarity in the effects of fetal bladder fibroblasts and neonatal lung fibroblasts on regulating the growth of 2 widely different tumor cell types in culture would suggest that such regulatory properties are perhaps general features of fetal or neonatal fibroblasts. The reason for the diverse growth responses of the anchored versus the nonanchored fibroblasts is unknown. What is known, however, is that the fibroblast inhibition of the tumor cells is specific for the tumor cell and is not attributable to either medium depletion or a general toxic effect. The differential effects of the fibroblasts may be related to their different proliferative states; the nonanchored cells embedded within the agarose were nonproliferative, whereas the anchored cells proliferated under the agarose at a growth rate of 85.3 per cent of that observed for growth in an equivalent liquid medium. 4 Modulation of the growth rate of anchored neonatal bladder fibroblasts by FBS confirmed that fibroblast growth rate is an important factor in determining the nature of the tumor cell response. Rapidly-dividing fibroblasts inhibit, whereas slower or nongrowing fibroblasts stimulate. The inverse relationship observed between fibroblast and tumor cell growth rate explains the stimulatory effects of the anchored bladder fibroblast lines from the older donors (NBF-152, NBF-32) compared to the inhibitory effects of the fetal derived line (NBF-147). The differential response of the tumor cells to the older donor bladder fibroblasts is clearly correlated with a slower growth rate of these fibroblast lines in culture compared to fetal-derived cells. The biphasic responses described above suggest that the diverse tumor growth responses result from a concentration gradient of the active factor(s). High concentrations of this factor(s) inhibit tumor growth in agarose whereas lower concentrations are stimulatory. The simplest explanation is to postulate the involvement of at least 1 active fibroblast-
derived factor, the production of which is positively correlated with fibroblast growth rate. Our studies showed that fetal bladder fibroblasts possess an ability to inhibit bladder tumor cells in culture, an ability that is progressively lost when bladder fibroblasts are derived from increasingly older donors. It is at least tempting to suggest a correlation between this loss in inhibitory potential of the aged fibroblasts with the increasing. incidence of cancer associated with old age. It is interesting that the mammalian embryonic environment can have normalizing effects on certain malignant cells. Malignant embryonal carcinoma cells, when introduced into a normal blastocyst, are observed to behave as normal cells in the subsequent development of the blastocyst.9 It may be that the inhibitory potential of fetal or neonatal fibroblasts described here is one aspect of this normalizing phenomenon. Admittedly, the agarose interaction culture system used here is highly artificial and oversimplistic in terms of representing tumor biology in vivo. It does, however, permit an experimental assay to characterize further and purify the active fibroblast factor(s) which can modulate tumor growth in culture. REFERENCES
1. Grobstein, C.: Mechanisms of organogenetic tissue interactions. Nat. Cancer Inst. Monogr. 26: 279, 1967. 2. Tarin, D.: Tissue interactions and the maintenance of histological structure in adults. In: Tissue Interactions in Carcinogenesis. Edited by D. Tarin. New York: Academic Press, chapt. 3, p. 81, 1972. 3. Kirk, D. and Kaighn, M. E.: Non-reciprocal interactions in normalneoplastic human cells. A quantitative, kinetic approach to cell interactions in vitro. Cell Biol. Int. Rep., 4: 599, 1980. 4. Kirk, D., Szalay, M. and Kaighn, M. E.: Modulation of growth of a human prostatic cancer cell line (PC-3) in agar culture by normal human lung fibroblasts. Cancer Res., 41: 1100, 1981. 5. O'Toole, C., Price, Z. H., Ohnuki, Y. and Unsgaard, B.: Ultrastructure, karyology and immunology of a cell line originated from a human transitional-cell carcinoma. Brit. J. Cancer, 38: 64, 1978. 6. San, R. H. C., Laspia, M. F., Soiefer, A. I., MasLansky, C. J., Rice, J.M. and Williams, G. M.: A survey of growth in soft agar and cell surface properties as markers for transformation in adult rat liver epithelial-like cell cultures. Cancer Res., 39: 1026, 1979. 7. Lechner, J. F., Babcock, M. S., Marnell, M., Narayan, K. S. and Kaighn, M. E.: Normal human prostate epithelial cell cultures. Methods Cell Biol. 21B: 195, 1980. 8. Brown, S., Teplitz, M. and Revel, J. P.: Interaction of mycoplasmas with cell cultures, as visualized by electron microscopy. Proc. Natl. Acad. Sci. USA, 71: 464, 1974. 9. Mintz, B. and Illmensee, K.: Normal genetically mosaic mice produced from malignant teratocarcinoma cells. Proc. Natl. Acad. Sci. USA, 72: 3585, 1975.
Vol. 130, September Printed in U.S.A.
THE JOURNAL OF UROLOGY
Copyright© 1983 by The Williams & Wilkins Co.
Original Articles DIFFERENTIAL GROWTH REGULATION OF HUMAN BLADDER TUMOR CELLS (J82) IN CULTURE BY NORMAL BLADDER CELLS FROM DIFFERENT AGED HUMAN DONORS SUSUMU KAGAWA*
AND
DAVID KIRKt
From the Huntington Medical Research Institutes, Pasadena, California
ABSTRACT
This study used an agarose gel culture system to study growth regulatory interactions between normal and neoplastic human bladder cells_ The effects of normal human fibroblasts on tumor cell cloning efficiency in agarose was studied using 2 experimental protocols. In 1 system, the fibroblasts proliferated as anchored cells beneath the agarose, and in the other the fibroblasts were nongrowing cells embedded within the agarose as nonanchored cells. In contrast to the largely inhibitory effects of anchored fetal fibroblasts on tumor cell growth, their nonanchored counterparts were exclusively stimulatory. Similar experiments with normal fibroblasts derived from the bladders of increasingly older donors showed a progressive decline in their capacity to inhibit tumor cell growth in agarose. Further experiments modulating the growth rate of neonatal fibroblasts with fetal bovine serum have indicated that the diverse tumor responses elicited by anchored versus nonanchored cells, as well as those observed for the aged bladder fibroblasts, were a function of fibroblast growth rate. We concluded that there was an inverse regulatory relationship between the growth of normal and tumor cells; rapidly growing fibroblasts were inhibitory whereas nongrowing fibroblasts stimulated tumor cell growth in agarose culture. The stromal component of stromal-epithelial organ systems is known to have a vital role both in controlling the normal organogenesis of the vertebrate embryo 1 and in maintaining normal histological differentiation of adult tissues. 2 Recent in vitro studies have detected interesting growth interactions between normal human lung fibroblasts and an established human prostatic carcinoma cell line. 3' 4 The researchers concluded that the normal lung cells had the distinct capability to regulate the growth of the prostatic tumor cells in agar gel culture. 4 However, these studies used heterologous cell types, and the interactions were not characterized with respect to either the tissue source or donor age of the fibroblasts. We have extended this work to include a study of cellular growth interactions exclusive to normal/neoplastic human bladder cell types. We chose as the malignant cell type a wellcharacterized cell line (J82) from a human bladder transitional cell carcinoma. 5 Since growth in agarose is the best available in vitro correlate for tumorigenesis in vivo,6 we studied the effects of normal human bladder fibroblasts on the clonal growth of J82 cells in agarose. Our primary aim was to determine if the in vitro growth regulation observed for the heterotypic cell combinations4 was applicable to a homotypic normal neoplastic human bladder cell culture system. Secondly, since cancer incidence increases with old age, we investigated the role of donor age on the ability of normal bladder fibroblasts (NBF) to regulate the bladder tumor cells in culture. Accepted for publication May 3, 1983. Supported in part by Mr. Francis L. Moseley. * Current address: Department of Urology, School of Medicine, Tokushima University, Kuramoto-2, Tokushima, Japan. t Requests for reprints: Huntington Medical Research Institutes, 99 N. El Molino Avenue, Pasadena, California 91101. 593
MATERIALS AND METHODS
Human bladder tumor cells (J82) were derived initially from a transitional cell carcinoma,5 and used between passage levels 54-78. Normal human fibroblast cultures were derived from histologically normal bladder specimens obtained at surgery or autopsy. Bladder explant cultures were initiated as previously described 7 from specimens obtained from a fetus (NBF-147), a 50-year-old woman (NBF-152) and a 70-year-old man (NBF32). Fibroblast lines were used at early passage levels (1-4). The growth medium (GM) for the explant of primary tissue and the maintenance of monolayer cultures was PFMR-4 7 supplemented with 5 per cent fetal bovine serum (FBS, Irvine Scientific) and antibiotics (penicillin, 100 I.U./ml.; kanamycin, 100 µg./ml.). All cell lines were mycoplasma-free as determined by scanning electron microscopy. 8 Fibroblasts were interacted with tumor cells in agarose either as anchored or nonanchored cells as described previously. 4 Agarose was made up with PFMR-4 supplemented with 20 per cent FBS and antibiotics as before. In the anchored system, fibroblasts were seeded in GM in 60 mm. dishes and allowed to attach overnight. They were then covered with a hard agarose base (0.5 per cent, 1.5 ml.) which was in turn overlaid with a soft agarose layer (0.3 per cent, 3 ml.) containing the tumor cells (5 x 103 ). In the nonanchored system, separate layers (1.5 ml.) of tumor cells and nonanchored fibroblasts in soft agarose were physically separated by a hard agarose layer (1.5 ml.). Tumor cell growth in both the anchored and nonanchored systems was measured in terms of colony forming efficiency (CFE) which was defined as the percentage of seeded cells that formed colonies. Colonies were automatically enumerated and sized using a Hamamatsu Image Analyzer System. Only colonies greater than 90 µ in diameter were counted (average
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KAGAWA AND KIRK
diameter of the J82 cells in agarose was between .15-20 µ). All cultures were set up in triplicate and scored for CFE after 1014 days in culture. Growth rates of mass and clonal monolayer cultures were expressed as population doublings per day (PD/D). The PD/D of mass cultures was determined by taking the log2 of the net increase in cell number and dividing by the number of days in culture. For clonal cultures, the PD/D was obtained by dividing the log2 of the mean cell number per colony by the number of days in culture. Average cell generation time (g) was obtained by taking the reciprocal of the PD/D ( g
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Coverslip cultures of fibroblasts were prepared by seeding 5 X 103 cells in 1.5 ml. of GM in 24-well Costar dishes containing 13 mm. glass coverslips. Cells were allowed to attach overnight before the coverslips were used in an experiment. For radioactive incorporation measurements, cultures were pulsed for 3 hours with tritiated thymidine (1.0 µCi/ml., 74.9 Ci/mmol.) in thymidine-free PFMR-4 supplemented with 20 per cent FBS. After pulsing, coverslip cultures were rinsed in saline, fixed in absolute ethanol, rinsed in 5 per cent trichloroacetic acid at 4C (30 min. X 2), further rinsed in ethanol and air dried. Processed coverslips were counted in 10 ml. ofBeckman's Ready-Solv GP using a Beckman LS9000 liquid scintillation spectrometer.
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RESULTS
Normal human bladder fibroblasts derived from differentaged donors were compared for their effects in modulating the clonal growth of human bladder tumor cells (J82) using an agarose culture interaction system. The fibroblasts were interacted with the tumor cells either as anchored cells (a proliferating monolayer beneath the agarose/tumor cell layer) or as nonanchored cells (nonproliferating cells embedded in a soft agarose layer separated from the agarose/tumor cell layer by a hard agarose layer). Increasing the inoculum of anchored cells caused a biphasic growth response of J82 for all 3 fibroblast lines (fig. 1), small inocula producing greater growth stimulation than higher inocula. Although all 3 fibroblast lines caused initial enhancement of J82 growth at low cell inocula, higher cell inocula showed marked differences between the different fibroblast lines. There was an inverse relationship between the donor age of the different lines and the growth response of the J82 tumor cells in agarose. The effect of a 4.5 X 104 fibroblast inoculum on J82 growth (fig. 1) showed a 50 per cent inhibition with fetal donor cells (NBF-147), no effect with 50-year-old donor cells (NBF-152) and a 165 per cent stimulation with 70year-old donor cells (NBF-32). Hence, using the anchored fibroblast system, fetal bladder cells (NBF-147) largely exhibited a strong inhibitory effect on J82 whereas cells from the oldest donor (NBF-32) resulted in a consistent growth stimulatory effect. Cells from the median aged donor (NBF-152) manifested intermediate effects, causing a moderate inhibition of J82. The effects of nonanchored bladder fibroblasts (embedded within the agarose) were dramatically different from their parallel anchored counterparts. All fibroblast lines, as nonanchored cells, were exclusively stimulatory to J82 (fig. 2). However, kinetics of the dose-dependent stimulations were different for the different lines. Maximal growth stimulation was much greater for the fetal cells (NBF-147, 6-fold) compared to the other 2 lines (NBF-152, NBF-32; both approximately 3-fold). In addition, the fibroblast inoculum necessary to effect a halfmaximal J82 growth response was greater for the fetal cells (NBF-147; 6.5 x 104 ) than for the other lines (NBF-152, NBF32; both 2.5 X 104 ). It is important to notice that the differential effects observed for the anchored fibroblasts on J82 (fig. 1) are associated with differences in the growth rates of the fibroblast lines. Growth curves of cells seeded at 105 per 60 mm. dish in 4 ml. of PFMR4 supplemented with 20 per cent FBS (nutrient regime as used in fig. 1) showed the fetal cells (NBF-147) to have a much
•
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500
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FIG. 2. Effect of increasing the cell inoculum of different nonanchored human bladder fibroblasts on the relative colony forming efficiency (CFE) of J82 cells in agarose. e, NBF-147; D, NBF-152; 0, NBF-32. (Cultures were set up simultaneously with their anchored counterparts as described in fig. 1, and shared the same controls).
shorter cell generation time (28.9 hours) than the 50-year-old donor cells (NBF-152, 46.9 hours). To investigate further the role of fibroblast growth rate in regulating J82 growth in agarose we have used FBS as a means to modulate fibroblast growth rate. The growth of the fetal (NBF-147) and tumor cells (J82) both showed a dose-dependent serum response in monolayer culture (fig. 3). Growth rates for both lines were maximal in 20 per cent FBS although the serum requirement for half-maximal growth was lower for J82 (1 per cent, FBS) than for NBF-147 ( 4 per cent, FBS). We subsequently studied growth interactions between anchored NBF-14 7 and J82 at a range of FBS concentrations. An NBF-147 cell inoculum of 105 per dish was selected since it was known to cause approximately a 75 per cent inhibition of J82
595
NORMAL-NEOPLASTIC CELL INTERACTIONS
growth in 20 per cent FBS (fig. 1). Upon reducing the serum concentration, the fibroblast effect changed from inhibitory to J82 to stimulatory in a clear biphasic fashion (fig. 4). Whereas maximum inhibition of J82 was observed with 15 per cent FBS (p < 0.001), maximum stimulation was observed with 5 per cent FBS (p < 0.05). The null-point, that is, the point where the fibroblasts had no effect on the growth of J82, was observed with 10 per cent FBS (fig. 4). An experiment was specifically designed to determine if the
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Fm. 3. Serum titrations of J82 (e) and NBF-147 cells (0) in monolayer culture. NBF-147 cells were seeded at 5 X 104 per 60 mm. dish in 4 ml. of growth medium. After 20 hours, growth medium was supplemented with different serum concentrations and cell counts on trypsinized cultures determined on day 4. (Under these seeding conditions the growth rate in 20 per cent FBS was linear for at least 7 days.) J82 were seeded at 500 cells per 60 mm. dish and set up in different serum concentrations as for NBF-147. After 7 days clones were fixed in methanol and stained with Weigert's Iron Hematoxylin.
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inhibition of J82 by NBF-147 at high serum levels could be due to the nonspecific effects of nutrient deprivation and culture toxicity. Conceptually, this was approached by monitoring the growth of "indicator" NBF cells which were cultured concomitantly in the presence of the J82/NBF co-cultures. The growth of the indicator cells was deemed to "indicate" the growth status of the nutrient medium throughout the course of the experiment. The experiment was set up as follows. Agarose cultures of J82 were set up using the anchored fibroblast protocol in 20 per cent FBS with an NBF-147 inoculum of 3 x 105 cells per dish, an inoculum which was known to cause essentially a 100 per cent inhibition of J82 (fig. 1). In addition, glass coverslip cultures ofNBF-147 (indicator cells) were added on top of the agarose containing the J82 cells, with the cell monolayer facing up. The NBF-147/J82 agarose co-cultures containing the NBF-147 coverslip cultures were refed every 2 days with 1.5 ml. of fresh liquid PFMR-4 medium supplemented with 20 per cent FBS. Cultures were periodically pulsed with tritiated thymidine and the coverslips removed and measured for tritium incorporation into NBF-147 cells. The incorporation measured in the NBF-147 coverslip cultures (indicator NBF147 cells) was thus a measure of the prevailing nutrient conditions within the anchored NBF-147/J82 agarose co-cultures. Initially (day 2), NBF-147 indicator cells in the presence of anchored NBF-147/J82 co-cultures showed a marked growth stimulation (X 2.39 control, fig. 5) which decreased with increasing time in culture. After 9 days, the indicator NBF-147 cells showed a 40 per cent inhibition whereas at this time the J82 cells showed a 95.7 per cent inhibition (data not shown). This direct demonstration of a differential inhibition of J82 cells clearly showed that adverse culture conditions account for no more than 40 per cent of the observed inhibition. The remaining 60 per cent of the inhibition represented the specific effect of NBF cells on J82 cells. In contrast, indicator NBF-147 cells alone caused a marginal stimulation of J82 clonal forming efficiency (data not shown). This increase in the growth of the J82 tumor cells was in turn
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FBS!!Jo~ Fm. 4. Effect of fetal bovine serum concentration on the interaction of NBF-147 cells on J82 cells in agarose. NBF-147 cells were seeded at 105 per 60 mm. dish and set up for the anchored fibroblast system as described in MATERIALS AND METHODS. Net fibroblast effects on J82 colony forming efficiency ( CFE) were determined by subtracting the control J82 colony forming efficiency (CcFE) from the colony forming efficiency of J82 in the NBF-147/J82 co-cultures (FcFE) for each serum level. Differences were tested for significance using a Student's t test.
DAYS Fm. 5. Relative incorporation of tritiated thymidine into indicator NBF-147 coverslip cultures. 0, in the presence ofNBF-147/J82 agarose co-cultures set up as described for the anchored fibroblast system. e, in the presence of J82 alone in agarose. Coverslips were set up in triplicate and the incorporation expressed as percentage of controls (NBF-147 coverslip cultures added to the agarose system as described but without either J82 or anchored NBF-147 cells). The standard errors ranged from 2.5 to 18% of mean values.
596
KAGAWA AND KIRK
associated with a stimulation of the NBF indicator cells (fig. 5), a similar stimulation having been observed previously in another normal/tumor cell co-culture study. 3 DISCUSSION
This study showed that, using a similar agarose system, human fetal bladder fibroblasts regulated the growth of human bladder tumor cells in a similar fashion to that reported for human lung neonatal fibroblasts on human prostatic carcinoma cells. 4 In both cases nonanchored cells stimulated tumor cell growth in agarose whereas anchored cells inhibited the tumor cells. The dose-response curves for the inhibitions and stimulations also showed similar kinetics for both the bladder and the lung/prostatic cell systems. This similarity in the effects of fetal bladder fibroblasts and neonatal lung fibroblasts on regulating the growth of 2 widely different tumor cell types in culture would suggest that such regulatory properties are perhaps general features of fetal or neonatal fibroblasts. The reason for the diverse growth responses of the anchored versus the nonanchored fibroblasts is unknown. What is known, however, is that the fibroblast inhibition of the tumor cells is specific for the tumor cell and is not attributable to either medium depletion or a general toxic effect. The differential effects of the fibroblasts may be related to their different proliferative states; the nonanchored cells embedded within the agarose were nonproliferative, whereas the anchored cells proliferated under the agarose at a growth rate of 85.3 per cent of that observed for growth in an equivalent liquid medium. 4 Modulation of the growth rate of anchored neonatal bladder fibroblasts by FBS confirmed that fibroblast growth rate is an important factor in determining the nature of the tumor cell response. Rapidly-dividing fibroblasts inhibit, whereas slower or nongrowing fibroblasts stimulate. The inverse relationship observed between fibroblast and tumor cell growth rate explains the stimulatory effects of the anchored bladder fibroblast lines from the older donors (NBF-152, NBF-32) compared to the inhibitory effects of the fetal derived line (NBF-147). The differential response of the tumor cells to the older donor bladder fibroblasts is clearly correlated with a slower growth rate of these fibroblast lines in culture compared to fetal-derived cells. The biphasic responses described above suggest that the diverse tumor growth responses result from a concentration gradient of the active factor(s). High concentrations of this factor(s) inhibit tumor growth in agarose whereas lower concentrations are stimulatory. The simplest explanation is to postulate the involvement of at least 1 active fibroblast-
derived factor, the production of which is positively correlated with fibroblast growth rate. Our studies showed that fetal bladder fibroblasts possess an ability to inhibit bladder tumor cells in culture, an ability that is progressively lost when bladder fibroblasts are derived from increasingly older donors. It is at least tempting to suggest a correlation between this loss in inhibitory potential of the aged fibroblasts with the increasing. incidence of cancer associated with old age. It is interesting that the mammalian embryonic environment can have normalizing effects on certain malignant cells. Malignant embryonal carcinoma cells, when introduced into a normal blastocyst, are observed to behave as normal cells in the subsequent development of the blastocyst.9 It may be that the inhibitory potential of fetal or neonatal fibroblasts described here is one aspect of this normalizing phenomenon. Admittedly, the agarose interaction culture system used here is highly artificial and oversimplistic in terms of representing tumor biology in vivo. It does, however, permit an experimental assay to characterize further and purify the active fibroblast factor(s) which can modulate tumor growth in culture. REFERENCES
1. Grobstein, C.: Mechanisms of organogenetic tissue interactions. Nat. Cancer Inst. Monogr. 26: 279, 1967. 2. Tarin, D.: Tissue interactions and the maintenance of histological structure in adults. In: Tissue Interactions in Carcinogenesis. Edited by D. Tarin. New York: Academic Press, chapt. 3, p. 81, 1972. 3. Kirk, D. and Kaighn, M. E.: Non-reciprocal interactions in normalneoplastic human cells. A quantitative, kinetic approach to cell interactions in vitro. Cell Biol. Int. Rep., 4: 599, 1980. 4. Kirk, D., Szalay, M. and Kaighn, M. E.: Modulation of growth of a human prostatic cancer cell line (PC-3) in agar culture by normal human lung fibroblasts. Cancer Res., 41: 1100, 1981. 5. O'Toole, C., Price, Z. H., Ohnuki, Y. and Unsgaard, B.: Ultrastructure, karyology and immunology of a cell line originated from a human transitional-cell carcinoma. Brit. J. Cancer, 38: 64, 1978. 6. San, R. H. C., Laspia, M. F., Soiefer, A. I., MasLansky, C. J., Rice, J.M. and Williams, G. M.: A survey of growth in soft agar and cell surface properties as markers for transformation in adult rat liver epithelial-like cell cultures. Cancer Res., 39: 1026, 1979. 7. Lechner, J. F., Babcock, M. S., Marnell, M., Narayan, K. S. and Kaighn, M. E.: Normal human prostate epithelial cell cultures. Methods Cell Biol. 21B: 195, 1980. 8. Brown, S., Teplitz, M. and Revel, J. P.: Interaction of mycoplasmas with cell cultures, as visualized by electron microscopy. Proc. Natl. Acad. Sci. USA, 71: 464, 1974. 9. Mintz, B. and Illmensee, K.: Normal genetically mosaic mice produced from malignant teratocarcinoma cells. Proc. Natl. Acad. Sci. USA, 72: 3585, 1975.