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From the Department of Pathology, Northwestern University Medical School, Chicago, Illinois
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Introduction |
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In contrast, Camps and colleagues5 reported marked stimulatory effects on the growth of cancer cells in vivo by stromal cells of variable origins. These stromal cells included a rat prostatic fibroblast cell line, NbF-1; a mouse nontumorigenic fibroblast cell line, 3T3; a mouse mammary fibroblast cell line, C-1271, either irradiated or nonirradiated; a human bone fibroblast cell line, MS, derived from an osteogenic sarcoma; and rat urogenital sinus mesenchymal cells.6,7 Despite these extensive studies, it remains unclear whether or not the reported positive influence demonstrated by these prostatic and bone marrow fibroblasts apply to human prostate cancer because of the usage of cells that are clearly abnormal or of nonhuman origin.
The present investigation was performed in an attempt to clarify these seemingly contradictory results between the in vitro and in vivo studies. We tested the hypothesis that stromal cells of the prostate regulate the growth of androgen-independent prostatic carcinoma cells. We used a three-dimensional co-culture system as an in vitro model and athymic nude mice as an in vivo model. The former will be an in vitro system that would simulate best the in vivo growth system. The stromal cells derived from the normal adult prostate, bone marrow, and skin were used. These cells were nontumorigenic as they failed to form tumors in athymic nude mice. Our studies demonstrate that hepatocyte growth factor (HGF) produced by prostate stromal cells is a major growth factor that stimulates the growth of androgen-independent prostate cancer.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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All human tissues used in the present investigation were collected according to the protocol approved by the Institutional Review Board of Northwestern University. We used three human prostatic carcinoma cell lines; LNCaP is androgen-sensitive, and PC-3 and CA-7T2 are androgen-insensitive. CA-7T2 is a prostatic carcinoma cell clone established in our laboratory from a radical prostatectomy specimen. A portion of prostate tissue suspicious for carcinoma was incised, and one-half of the sliced tissue was submitted for immediate microscopic examination on cryostat sections. After establishment of the diagnosis of adenocarcinoma (Gleason score, 3 + 3), the remaining half of the tissue was used for primary culture. The tissue was cut into multiple minute cubicles, placed on a plastic surface, and grown in keratinocyte serum-free medium supplemented with 50 µg/ml bovine pituitary extract, 5 ng/ml epidermal growth factor (EGF), 100 µg/ml streptomycin, and 100 U/ml penicillin (Life Technologies, Inc., Gaithersburg, MD). As soon as outgrowths formed around the tissue fragments, infection with a retrovirus vector containing the HPV16 E6 gene (LXSN16E6; kindly provided by Dr. Denise Galloway, University of Washington, Seattle, WA) was attempted by the polybrene method. After selection of cells in medium containing Geneticin (G418; 800 µg/ml; Life Technologies., Inc.), cells were injected subcutaneously (s.c.) in athymic male nude mice. A portion of a tumor that developed after 3 months was returned to primary culture as described above. Cell clones were obtained by the limited-dilution method, and one of the clones, designated as CA-7T2, was used in the present study. CA-7T2 cells expressed neither androgen receptor nor prostate-specific antigen, were androgen-insensitive, and formed an undifferentiated carcinoma in athymic nude mice.
Prostate-derived stromal (P-ST) cells were derived from a cancer-free focus of a prostatectomy specimen removed for cancer. Bone marrow-derived stromal (BM-ST) cells were cultured from the bone marrow of a healthy male donor; heparinized bone marrow aspirates were centrifuged on a Ficoll-Hypaque (Pharmacia, Uppsala, Sweden) gradient, and the interface cells were cultured.8 Skin-derived stromal (SK-ST) cells were established from the normal abdominal skin of an adult man. All of the cells except CA-7T2 cells were maintained in RPMI 1640 (Life Technologies, Inc.) containing 10% fetal bovine serum (FBS), 100 units/ml penicillin, and 100 µg/ml streptomycin (Life Technologies, Inc.), and incubated in a humidified atmosphere of 95% air and 5% CO2 at 37°C. For maintenance in the laboratory, CA-7T2 cells were grown in keratinocyte serum-free medium supplemented with 50 µg/ml bovine pituitary extract and 5 ng/ml EGF (Life Technologies. Inc.).
In Vivo Tumorigenicity Assay
PC-3 cells (5 x 105) or CA-7T2 cells (1 x 106) were suspended in 0.1 ml of serum-free RPMI 1640 medium with or without stromal cells (5 x 105 cells for PC-3 cells and 1 x 106 cells for CA-7T2 cells) and injected at two sites s.c. in the flanks of male athymic nude mice (n = 6). In each mouse, the left flank received carcinoma and stromal cells, and the right flank received carcinoma cells alone. In the first experiment, tumors formed were measured weekly, and the assay was terminated at 4 and 6 weeks after inoculation of PC-3 and CA-7T2 cells, respectively. Tumor volumes were calculated with the following formula: volume = length x width x height x 0.5236. Representative portions of all tumors were submitted for routine microscopic examination.
In a second experiment, which was conducted by the identical protocol, CA-7T2 cells were inoculated with or without P-ST cells, and two mice each were killed 48 hours, 96 hours, 1 week, 2 weeks, and 3 weeks later. The injection sites were dissected and submitted for microscopic examination.
In a third experiment, PC-3 cells (5 x 105) were suspended in a mixture of 50 µl of matrigel (Becton Dickinson Labware, Franklin Lakes, NJ) and 50 µl of serum-free RPMI 1640 medium with or without recombinant human HGF (50 ng; R&D Systems, Minneapolis, MN) and injected s.c. in the flanks of male athymic nude mice (n = 6). Fifty µl of serum-free RPMI 1640 with or without HGF (50 ng) were inoculated to peritumor sites 2 days, 4 days, and 6 days later. Tumors formed were measured weekly, and the assay was terminated at 6 weeks after inoculation of PC-3 cells. Tumor volumes were calculated as above.
Three-Dimensional Collagen Gel Culture
Collagen gels were prepared as reported previously.9
In brief, eight volumes of rat tail type I collagen suspension (Becton
Dickinson Labware) were mixed with one volume of 10-fold concentrated
RPMI 1640 (Sigma, St. Louis, MO) and one volume of reconstruction
buffer (2.2 g of NaHCO3, 4.77 g of Hepes in
100 ml of 0.05 N NaOH). Collagen gel with or without stromal cells
(5 x 104
cells/well) was poured into a
24-well plate (0.5 ml/well; see Figure 2
). After incubation for 30
minutes at 37°C to permit complete gelation, a second collagen layer
containing carcinoma cells (5 x 104
cells/well) was placed on top of the first layer. After gelation, RPMI
1640 containing 10% FBS was added. FBS was necessary to support the
growth of both cancer cells and stromal cells in a 4-day culture. The
medium was changed every other day. After incubation for 4 days, each
gel layer was removed separately, and carcinoma cells contained in the
upper layer were recovered by treatment with 0.1% collagenase I
(Worthington Biochemical Corp., Freehold, NJ) and 0.5% trypsin-5.3
mmol/L ethylenediaminetetraacetic acid (Life Technologies, Inc.) and
counted with a hemocytometer. A preliminary study indicated that there
was no crossover of cells to the adjacent collagen layer. In some
experiments, polyclonal rabbit anti-human HGF antibody (20 µg/ml; R&D
Systems), or recombinant human HGF (0 to 100 ng/ml) was added to the
above cultures.
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Cells grown in monolayers were harvested at early confluency. RNA was prepared by lysing of cells in hypotonic buffer containing Nonidet P-40 (Sigma), followed by removal of nuclei. Cytoplasmic RNA was reverse-transcribed by Moloney murine leukemia virus reverse transcriptase (Life Technologies, Inc.) at 42°C for 60 minutes with use of random primers (5 µmol/L; Life Technologies, Inc.). Subsequently, 1 µl of the products was subjected to PCR amplification. PCR was performed as follows. The final concentration of deoxynucleotide triphosphates and primers in the reaction mixture was 200 µmol/L and 1 µmol/L, respectively. Taq DNA polymerase (Cetus Perkin-Elmer, Norwalk, CT) was added to the mixture at a final concentration of 0.05 unit/ml, and the reaction was carried out in a DNA Thermal Cycler (Cetus Perkin-Elmer). After preliminary testing to determine the optimal PCR condition for semiquantitation, we chose 5 µg of RNA and 30 cycles for PCR amplification for HGF, keratinocyte growth factor (KGF), and transforming growth factor-ß1 (TGF-ß1); 35 cycles for interleukin-6 (IL-6) and EGF; 22 cycles for vascular endothelial growth factor (VEGF); and 20 cycles of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The primers were purchased or synthesized according to previous reports: human HGF,10 KGF,11 IL-6, EGF, and TGF-ß1 (all from Clontech, Palo Alto, CA), VEGF,12 c-met/HGF receptor,13 and GAPDH.14
Collection of Conditioned Medium (CM) from PC-3 and Stromal Cells
Stromal cells were cultured to subconfluency in flasks containing
RPMI 1640 medium with 10% FBS. Cells were washed twice with Hanks’
balanced salt solution (Life Technologies, Inc.) and downshifted to
serum-free RPMI 1640 medium for starvation. After 24 hours, medium was
replaced by serum-free medium. Cells were then cultured for an
additional 48 hours. The CM was collected and clarified by
centrifugation. The protein concentration of CM was adjusted to 
5
µg/ml by addition of serum-free medium.
PC-3 cells were seeded on a 6-well plate in RPMI 1640 medium and 10% FBS. When they became subconfluent, medium was changed to serum-free medium. After 48 hours, the CM was collected and clarified by centrifugation.
Measurement of Secreted HGF
HGF protein in CM was measured by the Quantikine Human HGF Immunoassay kit (R&D Systems) according to the manufacturer’s protocol. CM was collected from collagen gel cultures and monolayer cultures.
Immunohistochemical Demonstration of c-MET and HGF Proteins in Athymic Nude Mouse Tumors
Sections were stained for the immunohistochemical expression of cytokeratin (AE1/AE3; DAKO, Carpinteria, CA), vimentin (V9; Oncogene, Cambridge, MA), HGF (AF-294-NA; R&D Systems), and c-Met (C-12; Santa Cruz Biotechnology, Santa Cruz, CA). The avidin-biotin-peroxidase complex method with a Vectastain ABC kit (Vector Laboratories, Burlingame, CA) was used.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Stromal cells of all sources showed a typical polar spindle shape and showed staining reactions which are indicative of myofibroblastic differentiation. Cells in passages 4 to 8 were used in the subsequent experiments.
We assessed the effect of the presence of stromal cells on the growth
of PC-3 and CA-7T2 cells in athymic nude mice. All sites injected with
tumor cells developed tumors. Co-inoculation of P-ST cells
significantly enhanced the growth of both types of tumor cells, the
PC-3 tumors 1.6-fold (P < 0.01) and CA-7T2
tumors 4.4-fold (P < 0.001). BM-ST cells
stimulated PC-3 growth slightly only during the first 2 weeks. SK-ST
cells showed no effect (Figure 1A)
. Both
BM-ST and SK-ST cells enhanced the growth of CA-7T2 tumors mildly but
significantly (Figure 1B)
. As compared to PC-3 cells that grow rapidly,
CA-7T2 cells were slow in growth and therefore extracellular matrix
produced by the stromal cells may have demonstrated growth stimulatory
effect better than in fast growing PC-3 cells. By microscopic
examination, the tumors were undifferentiated carcinomas with
amphophilic cytoplasm, a centrally placed round nucleus, and a
prominent nucleolus. Mitoses were frequent. A small amount of
connective tissue stroma traversed the mass. The tumors formed after
co-inoculation of carcinoma cells and stromal cells were
indistinguishable from the tumors formed by carcinoma cells alone.
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We used a three-dimensional co-culture system (Figure 2)
with the assumption that it resembled
the in vivo growth conditions. The presence of P-ST cells in
the collagen gel stimulated the growth of PC-3 and CA-7T2 cells
3.2-fold and 1.8-fold, respectively. Such an effect was not
demonstrated by either BM-ST or SK-ST cells. The lesser response by
CA-7T2 cells may be because of the use of RPMI 1640, which is not an
optimal culture medium for these cells (see Materials and Methods).
Stromal cells did not increase in number during the culture period
(data not shown).
Expression of Growth Factors by Stromal Cells
The above findings suggested that stromal cells secreted growth
factors which stimulated the growth of carcinoma cells. Expression of
several growth factors was examined by semiquantitative RT-PCR in cells
grown on a plastic surface (Table 1)
.
P-ST cells expressed HGF mRNA at a much higher level than did the
stromal cells of other types (Figure 3)
,
but VEGF expression was the least among the three cell types. There was
no significant difference in the expression of KGF, IL-6, EGF, and
TGF-ß1. Expression of HGF mRNA was not detected in any prostatic
carcinoma cells (Figure 3)
.
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90%
(Table 2)
. We tested whether this reduction was because of consumption
of HGF by PC-3 cells. Exogenous HGF (50 ng/ml) was added to collagen
culture in the presence or absence of PC-3 cells. In the culture
without cells, HGF was decreased to 22, 18, and 16 ng/ml on days 1, 2,
and 3, respectively. On the other hand, in the culture with PC-3 cells,
HGF was decreased to 17, 13, and 10 ng/ml on days 1, 2, and 3,
respectively. The concentration of HGF was 40% less in the culture
with PC-3 cells. We interpreted the data to indicate that the reduction
of HGF in the culture without cells was because of attachment to
collagen matrix and that PC-3 indeed consumed a large quantity of HGF. Expression of c-met/HGF Receptor in Prostatic Carcinoma Cells
c-met mRNA was detected in PC-3 and CA-7T2
cells, but not in LNCaP cells (Figure 4)
.
To examine the effect of c-met expression on the growth of
carcinoma cells, we co-cultured these cells with P-ST cells in collagen
gel. The growth of PC-3 and CA-7T2 cells was stimulated significantly
in the presence of P-ST cells. In contrast, the growth of LNCaP cells
was not affected by P-ST cells (data not shown).
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We next tested the effect of exogenous HGF on the growth of PC-3
cells. A positive response to HGF was observed in a dose-dependent
manner (Figure 5)
. Anti-HGF neutralizing
antibody significantly inhibited the stimulatory effect of P-ST cells
(Figure 6)
.
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In the in vivo tumorigenicity study described above and
depicted in Figure 1
, co-inoculation of P-ST cells stimulated the
growth of cancer cells. In vitro assays identified HGF
released from stromal cells as a principal growth factor supporting
tumor cell growth. However, histological examination failed to
demonstrate the presence of spindle cells of presumed prostate origin.
To elucidate the role of P-ST cells, we repeated the in vivo
study. Groups of mice were killed 48 hours, 96 hours, 1 week, 2 weeks,
and 3 weeks after inoculation of CA-7T2 cells with or without P-ST
cells, and the tissue removed from injection sites was examined
microscopically. PC-3 cells were not used in this experiment
because they expressed vimentin, which makes identification of cancer
cells difficult.
The tissue removed 48 hours after injection of CA-7T2 and P-ST cells
demonstrated a sharply demarcated mass (Figure 7,D–F)
. It showed three distinct
histological zones; the center consisted of necrotic cells (designated
zone a). It was surrounded by a broad cellular zone (designated as zone
b) consisting of spindle cells mixed with degenerated and viable tumor
cells. Zone b was surrounded by a rim (designated as zone c) consisting
of basophilic viable tumor cells. The anaplastic large cells in zones b
and c were positive for cytokeratin (Figure 7E)
and reacted positively
with the antibody that recognizes human but not mouse c-Met protein
(Figure 7M)
and were arranged in an anastomosing network. The spindle
cells in zone b were confirmed to be P-ST cells because of their
positive immunohistochemical reaction to the anti-human vimentin
antibody, which does not recognize mouse vimentin (Figure 7F)
. These
cells stained positively with the anti-HGF antibody (Figure 7N)
.
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revealed a sharply demarcated cystic mass which
consisted of a large necrotic zone a and an outer cellular zone c
made up of viable carcinoma cells. Zone b of spindle cells was
absent.
The mass removed 96 hours after co-inoculation of carcinoma and P-ST
cells again showed a three-zone architecture (Figure 7, G–I)
; zone a
consisted of necrotic cells and zone b was predominately made up of
P-ST cells with a small quantity of tumor cells arranged in a
trabecular pattern. Zone c consisted of tumor cells only.
The mass removed 96 hours after inoculation of carcinoma cells alone
was still primarily cystic (zone a) and had a narrow rim of zone c
consisting of viable tumor cells. Zone b structure was absent (data not
shown). When examined 1 week after co-inoculation of carcinoma and P-ST
cells, carcinoma cells continued to proliferate, forming a wide zone c
(Figure 7, J–L)
. Spindle cells of P-ST origin were no longer
recognizable. Spindle cells found inside and outside of zone c were
unstained by the anti-vimentin antibody, indicating that they were of
host origin (Figure 7L)
.
The mass removed 1 week after injection of carcinoma cells alone was much smaller than the corresponding mass formed after co-inoculation of cells and still exhibited a large cystic center (data not shown). By 2 weeks, the cystic center had been filled with spindle cells of host origin, and the microscopic appearance of tumors was no longer distinguishable between the two groups. P-ST cells could not be recognized (data not shown).
Effect of Exogenous HGF on in Vivo Tumor Growth
We examined the effect of exogenous HGF on PC-3 tumor
growth. Exogenous HGF was injected to peritumor sites only during the
first week in an attempt to recapitulate the effect of short-lived P-ST
cells. PC-3 tumor growth was significantly enhanced by treatment with
HGF (Figure 8)
.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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HGF is a potent mitogen that was originally isolated from serum because
of its ability to stimulate the growth of hepatocytes in
vitro.15-17
Based on in vitro and
in vivo assays, c-Met/HGF has been proposed as an
autocrine/paracrine factor for carcinomas of various organs including
the lung,18,19
breast,20-22
pancreas,23,24
stomach,25
gall
bladder,26
urinary bladder,27
and
prostate.13
In the normal prostate gland, c-Met is
expressed only in basal cells.28
In carcinomas, c-Met
protein expression as detected by immunohistochemistry is variable, but
the frequency of c-Met expression increases in cancers of advanced
stage.28
These observations suggest possible involvement
of c-Met/HGF in androgen-dependent and -independent prostate cancers
and, in particular, in advanced cases. Our in vitro data
provide strong support for this possibility. Furthermore,
co-inoculation of P-ST and carcinoma cells enhanced tumor growth
in vivo significantly. Although enhancement of carcinoma
growth by stromal cells was reported previously,5-7
these
stromal cells were not derived from normal adult prostate. When the
injection sites were examined immunohistochemically with anti-HGF and
anti-c-Met antibodies, we observed that P-ST cells were positive for
HGF and were in close contact with carcinoma cells that expressed c-Met
protein (Figure 6, M and N)
.
The following discussion is offered in an attempt to elucidate the mechanism by which P-ST cells stimulated the growth of cancer cells in vivo. Based on the observation that irradiated NbF-1 fibroblasts of rat prostate could support growth of PC-3 cells, Camps et al5 suggested that extracellular matrix elaborated by fibroblasts might be able to initiate and/or promote epithelial cell proliferation during tumor formation. This possibility exists in the present study in view of the fact that the growth of CA-7T2 cells (but not of PC-3 cells) was mildly stimulated by co-inoculation of BM-ST and SK-ST cells. It is not likely, however, that the promoting role demonstrated by P-ST cells is merely because of extracellular matrix, because BM-ST cells and SK-ST cells did not show any promoting effect on growth of PC-3 cells. If matrix production were the sole mechanism supporting cancer cell growth, all stromal cells should be equally effective. In their studies, Gleave et al6,7 did not observe any effect with two stromal cell lines; one was normal adult lung fibroblast CCD16 and another rat kidney fibroblast NRK.
In the present study, P-ST cells were short-lived, as they were detectable only during the first week, apparently because of the fact that they are nonimmortalized cells. This finding suggests that they are involved in expanding the carcinoma cell population during the early phase of growth by providing HGF.
The cancer cell lines used in the present study are different from clinical androgen-independent prostate carcinomas in that they do not express androgen receptor or secrete prostate-specific antigen.29 Immunohistochemical studies by several groups indicate that androgen receptor is expressed in almost all carcinoma samples including those from metastatic carcinomas. At the same time, however, in all tumor foci immunostaining for androgen receptor is heterogeneous and reveals cells that fail to stain. Furthermore, Gleason score, stage of tumor, or endocrine therapy did not affect the heterogeneity in the staining pattern.30-32 Thus, prostate carcinomas appear always to consist of a mixture of androgen receptor-positive and -negative cells. The latter type of cells may represent an androgen-independent group of cells as do the cancer cell lines used in the present investigation.
We (unpublished data) as well as others10,28 have shown that localized prostate cancer (but not normal prostate columnar) cells express immunohistochemically demonstrable c-Met protein in a high frequency and furthermore that immunohistochemically demonstrable HGF is also frequently expressed in localized and metastatic prostate carcinomas.33 These findings suggest possible involvement of c-Met/HGF not only in androgen-responsive (-dependent) carcinoma but androgen-independent advanced carcinoma as well. Recently, we have reported that androgen-dependent CWR22 human prostate carcinoma cells respond with significant growth to HGF exposure in vitro, and that CWR22R tumor cells derived from CWR22 tumors regrown in male host mice34 after castration produce and secrete HGF.35 Thus, it is quite possible that growth stimulation mediated by c-Met/HGF exists in androgen-dependent and -independent tumors. The fact that c-met mRNA is up-regulated in the rat prostate after castration10 lends support to c-Met/HGF as an alternative or additional mechanism supporting androgen-independent tumor growth.
In conclusion, results of the present study indicate that HGF plays a significant role in the growth of androgen-independent prostate cancer. Our proposed mechanism of HGF action in a paracrine manner may explain the regrowth of androgen-independent prostate cancer at primary site, but may be insufficient to account for the growth at metastatic sites, notably in bone marrow, where HGF release by stromal cells is minimal. In the present study, BM-ST cells did not stimulate the growth of PC-3 carcinoma cells in vitro and in vivo. HGF derived from the systemic circulation might support the growth of bone marrow metastasis. Alternatively, metastatic prostate carcinoma cells might support themselves by an autocrine mechanism with c-Met/HGF.33 Additional studies are clearly needed to prove if c-Met/HGF is indeed involved in prostate carcinoma aside from the androgen/androgen receptor system. If c-Met/HGF proved to be involved, c-Met protein can be a target in suppressing the growth of prostate cancer, for example, treatment with a four-kringle antagonist for HGF (NK4).36
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Footnotes |
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Supported by National Institutes of Health Grants CA 14649 and CA 33511, and the Joseph L. Mayberry, Sr., Research Fund.
Accepted for publication May 23, 2000.
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, P < 0.01 compared with
respective controls.
