Cancer Letters,50 (1990) 197-201 Elsevier Scientific Publishers Ireland Ltd.
197
Stimulation of colony formation of various human cell lines by rhGM-CSF and rhIL-3 D. Nachbaur, University
Hospital
H. Denz, of Internal
H. Zwierzina, Medicine,
Department
F. Schmalzl
and
carcinoma
H. Huber
of Haematology-Oncology,
Anichstr.
35,
A-6020
Innsbruck
(Austria)
(Received 30 August 1989) (Revision received 7 December (Accepted 18 December 1989)
1989)
Summary We studied the effects human recombinant granulocyte-macrophage colony-stimulating factor and human recombinant inter/e&n-3 on the colony formation of three human solid tumor cell lines. Using a modified doublelayer agar clonogenic assay rhGM-CSF soft enhanced colony formation of all cell lines tested in a dose dependent manner (up to twofold for the breastcancer cell line BT-20, up to 163% of the control for the hypernephroma cell line C 94 and up to 147% for the non-small cell lung cancer cell line CCL 185 at a concentration of 100 ng/ml). RhlL-3 stimulated colony formation of the cell lines C 94 and BT-20, whereas on the cell line CCL 185 rhlL-3 had no effect even at the highest dose leoel tested (100 ng/ml). Combinations of growth factors showed subadditive stimulation on two cell lines tested (BT-20, C 94). These data indicate that haematopoietic growth factors exert a growth promoting activity on certain solid tumor cells in oitro at physiological concentrations. Therefore our results suggest that the application of these factors in immunoand myelosuppressed cancer patients after high dose chemotherapy should be seen in light of a possible co-stimulation of the malignant cells.
0304.3835/90/$03.50 Published and Printed
0 1990 El sevier Scientific in Ireland
Publishers
Keywords: haematopoietic growth factors; solid tumors; colony formation; clonogenic assay. Introduction Granulocyte/macrophage colony stimulating factor (GM-CSF) and interleukin 3 (IL-3) are naturally occuring glycoproteins, which are involved in the regulation of the proliferation and differentiation of haematopoietic progenitor cells [l-3]. Beside their activity on immature myeloid cells, effects of GMCSF on mature neutrophils, monocytes and eosinophils have been described [4-71. Based on these in-vitro data GM-CSF has been tested in clinical phase I and II trials with encouraging results in patients suffering from myelodysplastic syndromes, acquired immunodeficiency syndrome (AIDS) as well as in cancer patients after myelo-suppressive chemotherapy and/or bone marrow transplantation [8- 111. IL-3, which seems to act as a “multipoietin” stimulating all three haematopoietic cell lines, is now tested in clinical phase I trials. However, as recombinant human haematopoietic growth factors are applied in the therapy of Ieucopenic cancer patients and high affinity for GM-CSF have been recently described for epithelial tumors [E!], it is Ireland Ltd.
198
Haematopoietic growth factors Rh GM-CSF (E. coli), with a specific activity of 5 x lo7 CFU/mg protein and rhIL-3 (E. coli) , with a specific activity of 1 X lo7 units/mg protein, were kindly provided by Behringwerke AG (F.R.G.).
important to investigate whether there is any effect of haematopoietins on the malignant cells. Using a modified soft agar clonogenic assay, we investigated the influence of rhGMCSF and rhIL-3 on the clonal growth of cell lines derived from hypernephroma, non-small cell lung cancer (epithelial-like) and breast cancer.
Soft agar clonogenic assay A double-layer soft agar assay as described elsewhere [13] was used with slight modifications. MC Coy’s 5A medium (Gibco) was supplemented with 15% FCS, transferrin 2 pg/ml (Sigma), insulin 2 IU/ml (Hoechst), asparagine 66 pg/ml (Merck) and used as plating medium. Base layers of 0.25 ml containing 0.3% agar (Merck) and the tumor cells at concentrations ranging from 8,00040,000 cells/ml were prepared in each well of a NUNC multi-well dish. The agar was allowed to solidify at room temperature for 20 min. Liquid overlayers (0.25 ml) containing varying concentrations of growth factors were applied. Dishes were incubated at 37 OC in a humidified atmosphere of 5% CO, in air, and colonies of more than 50 ceils were counted in an Olympus inverted-phase microscope lo-14 days after plating. All experiments were performed in triplicate and repeated at least twice to confirm the reproducibility of the results.
Materials and methods Cell lines
The cell line C 94, a gift of Dr. H. Rauschmeier (Department of Urology, University Hospital Innsbruck), originates from a primary renal cell carcinoma. Cells were maintained routinely in HAMS F-10 medium (Gibco) supplemented with 10% FCS (Gibco), 125 units/ml penicillin, 125 pg/ml streptomycin sulfate and 1.15 pg/ml amphotericin B. The cell lines BT-20 (breast cancer) and CCL 185 (non-small cell lung cancer) were purchased from ATCC (Rockville, MD, U.S.A.). Cells were grown routinely as monolayers in enriched MEM medium (Seromed) supplemented with 10% FCS (Gibco), antibiotics and antimycotics as described above. For the experiments single cell suspensions of exponentially growing cells were prepared.
Table 1. Stimulation of colony formation of human solid tumor cell lines by recombinant growth factors (GM-CSF, IL-3).
Factor
Dose (ng/mI)
Colonies (% of controls + SD.) c94
rhGM-CSF
rhIL-3
rhGM-CSF + rhlL-3 *P-value < 0.05.
1 10 100 1 19 100 10
human haematopoietic
96 123 163 87 130 182
+ f ++ + +
136 +
10 11’ 10’ 13 19’ 5’ 5’
CCL 185
BT-20
93* 106 f 147 f 96 + 105 * 103 k
104k 127 + 196 k 101 + 104 & 130 f
n.d.
7 12 24’ 11 15 6
8 16’ 3’ 3 9 18’
134 + 10’
199
Statistical
eoaluation
For the evaluation of statistical significance of the differences between expected (controls) and observed surviving fractions (SF) we used the Dunnet t-test [14]. The following definitions were applied for drug interactions: Additive: SF (A + B) = SF (A) x SF (B) Subadditive: SF (A + B) < SF (A) x SF (B) and > SF (B) when SF (A) < SF (B) Synergistic: SF (A + B) > SF (A) x SF (B) Antagonistic: SF (A + B) < SF (A) x SF(B)
-0
100
I
I
I
1
IO
100
CONCENTRATION
OF GROWTH
Orb
IL-3
Orh
Ru-CSF
+ Ill IL-3
nolml
FACTORS
Results RhGM-CSF enhanced the colony formation of all three cell lines in a dose-dependent manner (up to 192% for the cell line BT-20, up to 163% of the control for the cell line C 94 and up to 147% of the control for the cell line CCL 185 at a concentration of 100 ng/ ml) (Fig. 1 A-C and Table 1). Concerning ,the stimulation of colony formation of the hypernephroma cell line C 94, rhIL-3 at a concentration of 100 ng/ml was much more active than rhGM-CSF by increasing the clonal growth of this cell line up to 182% of the control (Fig. 1A and Table 1). On the BT-20 cell line rhIL-3 (100 ng/ml) showed only a marginal but significant stimulation up to 130% of the control, whereas on the cell line CCL 185 rhIL-3 had no effect, even at the highest dose level tested (100 ng/ ml) (Fig. 1 B,C and Table 1). Combinations of rhGM-CSF and rhIL3 at a concentration of 10 ng/ml of each factor showed subadditive stimulation of the clonal growth of two cell lines tested (BT-20, C 94) (Fig. 1 A,C). Discussion Our results demonstrate that beside their activity on myeloid precursor cells and mature blood cells haematopoietic growth factors are
I
I
1 CONCENTRATION
I
I
100
10 Of GROWTH
nplml
FACTORS
C
200
150
!
0*
/
Orb GM-CSF
lrh
e,,//,/“*
100
--
IL- 3
OrbOkCSF
+ rhll-3
1
I
1
I
10
CONCENTRATION
OF GROWTH
I
100
nolml
FACTORS
Fig. 1. (A) Effects of rhGM-CSF and rhIL-3 on colony formation of the cell line C 94. (B) Effects of rhGMCSF and rhIL-3 on colony formation of the cell line CCL 185. (C) Effects of rhGM-CSF and rhIL-3 on colony formation of the cell line BT-20 *Means statistically significant (P < 0.05) when compared to the control.
200
also able to stimulate the clonal proliferation of a variety of tumor cell lines. The clinical relevance of our in-vitro data for tumor biology and propagation as a side effect of the invivo application of rhGM-CSF and rhIL-3 in leucopenic and immunocompromised cancer patients is unclear and has to be determined. Recently, other investigators could demonstrate an in-vitro growth promoting activity of GM-CSF and IL-3 on two human colon adenocarcinoma cell lines and a human small-cell carcinoma line [15,16]. GM-CSF also stimulates the proliferation of normal human bone marrow fibroblast precursors and induce human endothelial cells to migrate and proliferate [ 17,181. Solid tumors often contain more or less cells of the host’s defense system (“tumor infiltrating leukocytes”), which are confined to the tumor stroma. These cells are mainly composed of T-lymphocytes (partially activated), cells of the monocyte/macrophage system and polymorphonuclear leucocytes [ 19,201. Activated T-lymphocytes are one of the main sources of GM-CSF and IL-3 and therefore a stimulation of tumor cell growth by a paracrine mechanism might be possible. On the other hand, activation of the immune sys(neutrophil activation, enhanced tem monocyte antibody-dependent cytotoxicity, secretion of TNF-alpha and interferon) in patients with refractory and advanced cancer under in-vivo application of GM-CSF has been reported [21,22]. The experiments also suggest that these activities might be mediated by different cell surface receptors as marked differences between rhGM-CSF and rhIL-3 were seen on the stimulation of the cell lines tested. The observed subadditive stimulating activity of combinations of GM-CSF and IL-3 is propably due to a down-modulation of receptor activity, as it is described for the haematopoietic system (for a review see Ref. 15). The expression of high affinity receptors for GMCSF has been described for human small-cell carcinoma lines [12], whereas receptors for IL-3 on non-haematopoietic cells have not been documented thus far.
Our results obtained in-vitro, demonstrating a direct growth promoting activity on several tumor cell lines, and the complexity of tumor biology and host’s defense system suggest that the application of haematopoietic growth factors after immunoand myelosuppressive chemotherapy should be seen in light of a possible co-stimulation of the malignant cells. References Sieff, C.A.
(1987)
Hematopoietic
growth factors. J. Clin.
Invest., 79, 1549-1557. Clark,
S.C.
and Kamen,
R. (1987)
stimulating factors. Science, 236, Dexter,
T.M.
(1989)
The human
1229-
Haemopoietic
colony-
1237.
growth
factors.
Br.
Med. Bull., 45, 337-349. Metcalf, D. (1986) the
The molecular biology and functions of
granulocyte-macrophage
colony-stimulating
factors
and cytokines. Blood, 67, 257-262. 5
Fleischmann, J.C.
J., Golde,
(1986)
D.W.,
Weisbart,
Granulocyte-macrophage
factor enhances
phagocytosis
R.H.,
Gasson,
colony-stimulating
of bacteria by human
neu-
trophils. Blood, 68, 708-712. 6
Fabian,
I., Baldwin,
G.C.
and Golde.
synthetic granulocyte-macrophage tor 7
enhances
neutrophil
(1987)
Bio-
colony-stimulating
fac-
cytotoxicity
leukemia cells. Leukemia,
1, 613-617.
Silberstein,
W.F.,
D.S.,
J.F.,
Golde,
K.F.
and
Owen,
D.W.,
David,
towards
Gasson,
Btna, J.C., J.R.
D.W.
J.C.,
Soberman,
(1986)
human DiPersio,
R., Austen,
Enhancement
of human
eosinophil cytotoxicity and leukotrten synthesis by biosynthetic
(recombinant)
granulocyte-macrophage
stimulating factor. J. Immunol., 8
Vadhan-Raj, W.N.,
S., Keating. M., Le Ma&e,
McCredie,
Henney,
C.
recombinant
K.,
and
stimulating
factor
Trujillo,
Gutterman,
human
Groopman, D.H.
and
in patients
J.E.,
Broxmeier,
J.U.
(1987)
D.W.
R.T.,
(1987)
human granulocyte-macrophage N. Engl. J. Med., Gabrilove,
J.L.,
DeLeo, Effect
M.F.,
Brandt, S.J., Borowitz, Gilbert,
Jones,
and Oette,
granulocyte-macrophage hematopoietic
Scher,
factor
syndrome.
318,
H.,
morbidity
carcinoma
due to
of the uroth-
1414-23.
Atwater,
R.B.,
S.K.,
Shpall,
D.H.
(1988)
Km&berg,
E.J.,
J.,
Bast, R.C.,
Effect of human
colony-stiumulating
reconstitution
Sternberg,
colony-stimulating
and associated
Peters, W.P.,
M.J., C.J.
A.,
of transitional-cell
elium. N. Engl. J. Med., 11
Oette,
of recombinant
colony-stimulating
Effect of granulocyte
factor on neutropenia chemotherapy
syn-
317, 593-599. Jakubowsky,
C. et al. (1988)
of
colony-
1545-1552.
on myelopoiesis in acquired immunodeficiency 10
H.E.,
Effects
with myelodysplastic
317,
Mitsuyasu,
Golde,
A., Hittelmann,
J.M.,
granulocyte-macrophage
dromes. N. Engl. J. Med., 9
colony-
137, 3290-3295.
after high-dose
factor
on
chemother-
201
12
13
14 15
apy and autologous bone marrow transplantation. N. Engl. J. Med., 318, 869-876. Baldwin, G.C., Gasson, J.C., Kaufman, S.E., Quan, S.G., Williams, R.E., Avalos, B.R., Gazdar, A.F., Golde, D.W. and DiPersio, J.F. (1989) Nonhematopoiettc tumor cells express functional GM-CSF receptors. Blood, 73, 1033- 1037. Nachbaur, D.M., Denz, H.A., Gastl, G., Thaler, J., Lechleitner, M. and Braunsteiner H. (1989) Combination effects of human recombinant interferon (alpha-2-arg, gamma) and cytotoxic agents on colony formation of human melanoma and hypemephroma cell lines. Cancer Len., 44,49-53. Sachs, I. (1978) Angewandte Statistik, pp. 187-193. Springer-Verlag, Berlin, Heidleberg, New York. Berdel, W.E., Danhauser-Riedl. S., Steinhauser, G. and Winton, E.F. (1989) Various Human Growth Factors (Interleukin-3, GM-CSF, late clonal growth of nonhematopoietic Blood, 73, 80-83.
16
17
Hematopoietic G-CSF) stimutumor cells.
Mantovani,
18
19
20
21
Dedhar, S., Gaboury, L., Galloway, P. and Eaves, C. (1988) Human granulocytemacrophage colony-stimulating factor is a growth factor active on a variety of cell types of nonhemopoietic origin. Proc. Natl. Acad. Sci. USA, 85,9253-9257. Bussolino, F., Wang, J.W., Defilippi, P., Turrini, F., Sanavio, F., Edgell, C.J.S., Aglietta, M., Arese, P. and
22
A.
(1989)
Granulocyte-
and
granulocyte-
macrophage-colony stimulating factors induce human endothelial cells to migrate and proliferate. Nature, 337, 471-473. Kleist, S., Berling, J., Bohle, W. and Witteking, Ch. (1987) lmmunohistological analysis of lymphocyte subpopulations infiltrating breast carcinomas and benign lesions. Int. J. Cancer, 40, 18-23. Boheim, K., Denz, H., Boheim, C., Glassl, H. and Huber, H. (1987) An immunohistologic study of the distribution and status of activation of head and neck tumor infiltrating leukocytes. Arch. Otorhinolaryngol., 244, 127 -132. Kaplan, S.S., Basford, R.E., Wing, E.J. and Shadduck, R.K. (1989) The effect of recombinant human granulocyte macrophage colony-stimulating factor on neutrophil activation in patients with refractory carcinoma. Blood, 73,636-638. Wing, E.J., Magee, D.M., Whiteside, T.L., Kaplan, S.S. and Shadduck, R.K. (1989) Recombinant human granulocyte/macrophage colony-stimulating factor enhances monocyte cytotoxicity and secretion of tumor necrosis factor alpha and interferon in cancer patients. Blood, 73, 643-646. Pegoraro, L.. Avanzi, G. and Lista, P. (1988) Growth and differentiation factors matologica, 73, 525-543.
in human
hematopoiesis.
Hae-
197
Stimulation of colony formation of various human cell lines by rhGM-CSF and rhIL-3 D. Nachbaur, University
Hospital
H. Denz, of Internal
H. Zwierzina, Medicine,
Department
F. Schmalzl
and
carcinoma
H. Huber
of Haematology-Oncology,
Anichstr.
35,
A-6020
Innsbruck
(Austria)
(Received 30 August 1989) (Revision received 7 December (Accepted 18 December 1989)
1989)
Summary We studied the effects human recombinant granulocyte-macrophage colony-stimulating factor and human recombinant inter/e&n-3 on the colony formation of three human solid tumor cell lines. Using a modified doublelayer agar clonogenic assay rhGM-CSF soft enhanced colony formation of all cell lines tested in a dose dependent manner (up to twofold for the breastcancer cell line BT-20, up to 163% of the control for the hypernephroma cell line C 94 and up to 147% for the non-small cell lung cancer cell line CCL 185 at a concentration of 100 ng/ml). RhlL-3 stimulated colony formation of the cell lines C 94 and BT-20, whereas on the cell line CCL 185 rhlL-3 had no effect even at the highest dose leoel tested (100 ng/ml). Combinations of growth factors showed subadditive stimulation on two cell lines tested (BT-20, C 94). These data indicate that haematopoietic growth factors exert a growth promoting activity on certain solid tumor cells in oitro at physiological concentrations. Therefore our results suggest that the application of these factors in immunoand myelosuppressed cancer patients after high dose chemotherapy should be seen in light of a possible co-stimulation of the malignant cells.
0304.3835/90/$03.50 Published and Printed
0 1990 El sevier Scientific in Ireland
Publishers
Keywords: haematopoietic growth factors; solid tumors; colony formation; clonogenic assay. Introduction Granulocyte/macrophage colony stimulating factor (GM-CSF) and interleukin 3 (IL-3) are naturally occuring glycoproteins, which are involved in the regulation of the proliferation and differentiation of haematopoietic progenitor cells [l-3]. Beside their activity on immature myeloid cells, effects of GMCSF on mature neutrophils, monocytes and eosinophils have been described [4-71. Based on these in-vitro data GM-CSF has been tested in clinical phase I and II trials with encouraging results in patients suffering from myelodysplastic syndromes, acquired immunodeficiency syndrome (AIDS) as well as in cancer patients after myelo-suppressive chemotherapy and/or bone marrow transplantation [8- 111. IL-3, which seems to act as a “multipoietin” stimulating all three haematopoietic cell lines, is now tested in clinical phase I trials. However, as recombinant human haematopoietic growth factors are applied in the therapy of Ieucopenic cancer patients and high affinity for GM-CSF have been recently described for epithelial tumors [E!], it is Ireland Ltd.
198
Haematopoietic growth factors Rh GM-CSF (E. coli), with a specific activity of 5 x lo7 CFU/mg protein and rhIL-3 (E. coli) , with a specific activity of 1 X lo7 units/mg protein, were kindly provided by Behringwerke AG (F.R.G.).
important to investigate whether there is any effect of haematopoietins on the malignant cells. Using a modified soft agar clonogenic assay, we investigated the influence of rhGMCSF and rhIL-3 on the clonal growth of cell lines derived from hypernephroma, non-small cell lung cancer (epithelial-like) and breast cancer.
Soft agar clonogenic assay A double-layer soft agar assay as described elsewhere [13] was used with slight modifications. MC Coy’s 5A medium (Gibco) was supplemented with 15% FCS, transferrin 2 pg/ml (Sigma), insulin 2 IU/ml (Hoechst), asparagine 66 pg/ml (Merck) and used as plating medium. Base layers of 0.25 ml containing 0.3% agar (Merck) and the tumor cells at concentrations ranging from 8,00040,000 cells/ml were prepared in each well of a NUNC multi-well dish. The agar was allowed to solidify at room temperature for 20 min. Liquid overlayers (0.25 ml) containing varying concentrations of growth factors were applied. Dishes were incubated at 37 OC in a humidified atmosphere of 5% CO, in air, and colonies of more than 50 ceils were counted in an Olympus inverted-phase microscope lo-14 days after plating. All experiments were performed in triplicate and repeated at least twice to confirm the reproducibility of the results.
Materials and methods Cell lines
The cell line C 94, a gift of Dr. H. Rauschmeier (Department of Urology, University Hospital Innsbruck), originates from a primary renal cell carcinoma. Cells were maintained routinely in HAMS F-10 medium (Gibco) supplemented with 10% FCS (Gibco), 125 units/ml penicillin, 125 pg/ml streptomycin sulfate and 1.15 pg/ml amphotericin B. The cell lines BT-20 (breast cancer) and CCL 185 (non-small cell lung cancer) were purchased from ATCC (Rockville, MD, U.S.A.). Cells were grown routinely as monolayers in enriched MEM medium (Seromed) supplemented with 10% FCS (Gibco), antibiotics and antimycotics as described above. For the experiments single cell suspensions of exponentially growing cells were prepared.
Table 1. Stimulation of colony formation of human solid tumor cell lines by recombinant growth factors (GM-CSF, IL-3).
Factor
Dose (ng/mI)
Colonies (% of controls + SD.) c94
rhGM-CSF
rhIL-3
rhGM-CSF + rhlL-3 *P-value < 0.05.
1 10 100 1 19 100 10
human haematopoietic
96 123 163 87 130 182
+ f ++ + +
136 +
10 11’ 10’ 13 19’ 5’ 5’
CCL 185
BT-20
93* 106 f 147 f 96 + 105 * 103 k
104k 127 + 196 k 101 + 104 & 130 f
n.d.
7 12 24’ 11 15 6
8 16’ 3’ 3 9 18’
134 + 10’
199
Statistical
eoaluation
For the evaluation of statistical significance of the differences between expected (controls) and observed surviving fractions (SF) we used the Dunnet t-test [14]. The following definitions were applied for drug interactions: Additive: SF (A + B) = SF (A) x SF (B) Subadditive: SF (A + B) < SF (A) x SF (B) and > SF (B) when SF (A) < SF (B) Synergistic: SF (A + B) > SF (A) x SF (B) Antagonistic: SF (A + B) < SF (A) x SF(B)
-0
100
I
I
I
1
IO
100
CONCENTRATION
OF GROWTH
Orb
IL-3
Orh
Ru-CSF
+ Ill IL-3
nolml
FACTORS
Results RhGM-CSF enhanced the colony formation of all three cell lines in a dose-dependent manner (up to 192% for the cell line BT-20, up to 163% of the control for the cell line C 94 and up to 147% of the control for the cell line CCL 185 at a concentration of 100 ng/ ml) (Fig. 1 A-C and Table 1). Concerning ,the stimulation of colony formation of the hypernephroma cell line C 94, rhIL-3 at a concentration of 100 ng/ml was much more active than rhGM-CSF by increasing the clonal growth of this cell line up to 182% of the control (Fig. 1A and Table 1). On the BT-20 cell line rhIL-3 (100 ng/ml) showed only a marginal but significant stimulation up to 130% of the control, whereas on the cell line CCL 185 rhIL-3 had no effect, even at the highest dose level tested (100 ng/ ml) (Fig. 1 B,C and Table 1). Combinations of rhGM-CSF and rhIL3 at a concentration of 10 ng/ml of each factor showed subadditive stimulation of the clonal growth of two cell lines tested (BT-20, C 94) (Fig. 1 A,C). Discussion Our results demonstrate that beside their activity on myeloid precursor cells and mature blood cells haematopoietic growth factors are
I
I
1 CONCENTRATION
I
I
100
10 Of GROWTH
nplml
FACTORS
C
200
150
!
0*
/
Orb GM-CSF
lrh
e,,//,/“*
100
--
IL- 3
OrbOkCSF
+ rhll-3
1
I
1
I
10
CONCENTRATION
OF GROWTH
I
100
nolml
FACTORS
Fig. 1. (A) Effects of rhGM-CSF and rhIL-3 on colony formation of the cell line C 94. (B) Effects of rhGMCSF and rhIL-3 on colony formation of the cell line CCL 185. (C) Effects of rhGM-CSF and rhIL-3 on colony formation of the cell line BT-20 *Means statistically significant (P < 0.05) when compared to the control.
200
also able to stimulate the clonal proliferation of a variety of tumor cell lines. The clinical relevance of our in-vitro data for tumor biology and propagation as a side effect of the invivo application of rhGM-CSF and rhIL-3 in leucopenic and immunocompromised cancer patients is unclear and has to be determined. Recently, other investigators could demonstrate an in-vitro growth promoting activity of GM-CSF and IL-3 on two human colon adenocarcinoma cell lines and a human small-cell carcinoma line [15,16]. GM-CSF also stimulates the proliferation of normal human bone marrow fibroblast precursors and induce human endothelial cells to migrate and proliferate [ 17,181. Solid tumors often contain more or less cells of the host’s defense system (“tumor infiltrating leukocytes”), which are confined to the tumor stroma. These cells are mainly composed of T-lymphocytes (partially activated), cells of the monocyte/macrophage system and polymorphonuclear leucocytes [ 19,201. Activated T-lymphocytes are one of the main sources of GM-CSF and IL-3 and therefore a stimulation of tumor cell growth by a paracrine mechanism might be possible. On the other hand, activation of the immune sys(neutrophil activation, enhanced tem monocyte antibody-dependent cytotoxicity, secretion of TNF-alpha and interferon) in patients with refractory and advanced cancer under in-vivo application of GM-CSF has been reported [21,22]. The experiments also suggest that these activities might be mediated by different cell surface receptors as marked differences between rhGM-CSF and rhIL-3 were seen on the stimulation of the cell lines tested. The observed subadditive stimulating activity of combinations of GM-CSF and IL-3 is propably due to a down-modulation of receptor activity, as it is described for the haematopoietic system (for a review see Ref. 15). The expression of high affinity receptors for GMCSF has been described for human small-cell carcinoma lines [12], whereas receptors for IL-3 on non-haematopoietic cells have not been documented thus far.
Our results obtained in-vitro, demonstrating a direct growth promoting activity on several tumor cell lines, and the complexity of tumor biology and host’s defense system suggest that the application of haematopoietic growth factors after immunoand myelosuppressive chemotherapy should be seen in light of a possible co-stimulation of the malignant cells. References Sieff, C.A.
(1987)
Hematopoietic
growth factors. J. Clin.
Invest., 79, 1549-1557. Clark,
S.C.
and Kamen,
R. (1987)
stimulating factors. Science, 236, Dexter,
T.M.
(1989)
The human
1229-
Haemopoietic
colony-
1237.
growth
factors.
Br.
Med. Bull., 45, 337-349. Metcalf, D. (1986) the
The molecular biology and functions of
granulocyte-macrophage
colony-stimulating
factors
and cytokines. Blood, 67, 257-262. 5
Fleischmann, J.C.
J., Golde,
(1986)
D.W.,
Weisbart,
Granulocyte-macrophage
factor enhances
phagocytosis
R.H.,
Gasson,
colony-stimulating
of bacteria by human
neu-
trophils. Blood, 68, 708-712. 6
Fabian,
I., Baldwin,
G.C.
and Golde.
synthetic granulocyte-macrophage tor 7
enhances
neutrophil
(1987)
Bio-
colony-stimulating
fac-
cytotoxicity
leukemia cells. Leukemia,
1, 613-617.
Silberstein,
W.F.,
D.S.,
J.F.,
Golde,
K.F.
and
Owen,
D.W.,
David,
towards
Gasson,
Btna, J.C., J.R.
D.W.
J.C.,
Soberman,
(1986)
human DiPersio,
R., Austen,
Enhancement
of human
eosinophil cytotoxicity and leukotrten synthesis by biosynthetic
(recombinant)
granulocyte-macrophage
stimulating factor. J. Immunol., 8
Vadhan-Raj, W.N.,
S., Keating. M., Le Ma&e,
McCredie,
Henney,
C.
recombinant
K.,
and
stimulating
factor
Trujillo,
Gutterman,
human
Groopman, D.H.
and
in patients
J.E.,
Broxmeier,
J.U.
(1987)
D.W.
R.T.,
(1987)
human granulocyte-macrophage N. Engl. J. Med., Gabrilove,
J.L.,
DeLeo, Effect
M.F.,
Brandt, S.J., Borowitz, Gilbert,
Jones,
and Oette,
granulocyte-macrophage hematopoietic
Scher,
factor
syndrome.
318,
H.,
morbidity
carcinoma
due to
of the uroth-
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