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Experimental Therapeutics, Preclinical Pharmacology |
Department of Pathology and Laboratory Sciences, Medical College of Pennsylvania and Hahnemann University, Philadelphia, Pennsylvania 19102-1192 [M. E. S., M. W.]; and National Cancer Institute, NIH, Bethesda, Maryland [J. R.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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30%). ELISAs showed that the HPCA-5aHPV-18 cells normally secreted low levels of tissue inhibitor of metalloproteinase (TIMP)-2, matrix metalloproteinase (MMP)-2, and MMP-9 but relatively high levels of TIMP-1. In contrast, HPCA-10aHPV-18 cells secreted high levels of MMP-2 and MMP-9 (>40 pg/µg protein) but low levels of TIMP-1 and TIMP-2 (<5 pg/µg protein). Interleukin 10 (IL-10) (15 ng/ml) induced TIMP-1 production (>15 pg/µg protein) but reduced MMP-2 and MMP-9 secretion (<5 pg/µg protein) by the HPCA-5aHPV-18 and HPCA-10aHPV-18 cells. IL-10 (15 ng/ml) and MMP-9/MMP-2 antibodies all blocked induction of microvessel formation in the coculture experiments. In contrast, IL-10 receptor antibodies and TIMP-1 antibodies countered IL-10’s effects and promoted angiogenesis. The data demonstrated that IL-10 stimulation of TIMP-1 and inhibition of MMP-2 and MMP-9 secretion by prostate tumor cells can control induction of angiogenesis in vitro. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Although the process of angiogenesis is complex and only partially understood, most experimental efforts have indicated that cancer cells promote angiogenesis, probably via production of a variety of paracrine growth factors (i.e., basic fibroblast growth factor, insulin-like growth factor, epidermal growth factor, and platelet-derived growth factor). For example, tumor cells and endothelial cells have been shown to produce either platelet-derived growth factor A or B and to stimulate their own growth, the growth of vascular smooth muscle cells, or the growth of fibroblasts and to modulate angiogenesis and, ultimately, tumor growth (4) . Further studies delineating the mechanisms of paracrine and autocrine induction of angiogenesis are needed to understand the role of cell-cell interactions, growth factors, cytokines, extracellular matrix, and proteases in the process of tumor-induced microvessel formation.
Using cell culture methods developed by Peehl (5) , we have established primary cell lines from microdissected regions of fresh human prostate cancer tissue from three different patients bearing high Gleason score 5 glands (cell lines HPCA-10a,b,c-HPV18) and four different patients carrying low Gleason score 3 glands (cell lines HPCA-5a,b,c,d-HPV18; Ref. 6 ). In all cases, the cells were immortalized with HPV-182 retrovirus construct provided by Dr. J. S. Rhim according to methods of Rhim et al. (7) and passaged three to four times prior to use in the studies described here. Characterization of the cell lines by immunolabeling with prostate-specific antigen, keratin, and vimentin antibodies confirmed the luminal epithelial origin of the cell lines (6) .
Here, we have compared the ability of different primary tumor lines to induce microvessel formation by human bone marrow endothelial cells. The data showed that aggregates of HPCA-5aHPV-18 or HPCA-10aHPV-18 cells induced HBMCE-1 cells to form microvessels in three-dimensional collagen 1 gels. ELISAs showed that the HPCA-5aHPV-18 cells secreted relatively high levels of TIMP-1 and low levels of MMP-2 and MMP-9. Conversely, the HPCA-10aHPV-18 cells secreted very low levels of TIMP-1 and relatively high levels of MMP-2 and MMP-9. Experimental studies revealed that IL-10 induced TIMP-1 (not TIMP-2) production and inhibited MMP-2 and MMP-9 secretion by the tumor lines to block induction of microvessel formation. In addition, TIMP-1 and IL-10 receptor antibodies blocked IL-10’s inhibitory effects and enabled tumor cell-induced angiogenesis. Independent experiments showed that TIMP-1 antibodies induced angiogenesis, whereas MMP-2 and MMP-9 antibodies blocked angiogenesis in untreated cocultures. Taken together, the data demonstrated that the ability of prostate epithelial cells to induce angiogenesis correlated inversely with the amounts of TIMP-1 secreted and directly with the amounts of MMP-2 and MMP-9 secreted by the epithelial cells in the coculture experiments.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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4–8 days incubation (10)
. In addition, the HBMEC-1 cells stained positive for factor VIII, the presence of Weible-Palade bodies and was positive for low-density lipoprotein uptake (10)
. HBMEC-1 and epithelial cultures were maintained in Ham’s F-12K medium (Sigma Chemical Co., St. Louis, MO) supplemented with 10% horse serum, 50 units/ml penicillin, 50 µg/ml streptomycin, and 50 µg/ml gentamicin sulfate and kept in 5% CO2 at 37°C. The primary tumor lines were establish by microdissection of glandular structures from human prostate (courtesy of Dr. Fernando Garcia) and immortalized with HPV-18 and characterized according to previously described methods (6)
. The benign prostate epithelial cells (1519 MCLX, passage 34) were generously provided by Dr. R. Bright (NIH, National Cancer Institute, Bethesda, MD). HPV-18-immortalized basal cells (passages 6–10) from HGPIN glands were cultured according to methods of Peehl (5)
and characterized as basal cells by 34ß1 antibody staining. The tumor lines were maintained in complete minimal growth epithelial medium (Clonetics, San Diego, CA) according to Peehl (5)
.
Endothelial Cell Microvessel Formation Assay.
Type I collagen gels were prepared by simultaneously raising the pH and the ionic strength of a cold collagen 1 (100 µg/ml) solution as described previously (11)
. HBMEC cells were trypsinized and plated onto collagen 1 gel coated 24-well plate (Falcon, Piscataway, NJ) with a cell density of 1 x 104/cm2 in Ham’s F-12K medium (Clonetics) containing 2% FCS. A top layer of collagen I was added that contained tumor cell aggregates (0.5–1.0-mm-diameter) prepared according to Nicosia and colleagues (12
, 13)
. Cultures were kept in an incubator at 37°C with 5% CO2 for 1 and 2 days. Fresh medium and drug were added on day 1, or the experiment was terminated. Cultures were fixed with 3% formaldehyde in phosphate buffer (11)
.
Microvessel formation in the cultures was evaluated by measuring the microvessels formed near the surface of the tumor aggregates, as described previously by Nicosia et al. (13
, 14)
. Specifically, the number and length of the tubular structures >100 µm in each area (2.0 x 1.3 mm2 area) were measured. Each experiment was performed in triplicate wells and repeated three times to obtain a sufficient number of observations for statistical analysis.
Under these conditions, the endothelial cells normally did not form microvessels independent of the tumor aggregates after incubation periods of 1–2 days. Also, cell doubling time was 30–40 h and we estimate that, on average, there were approximately one to two cell doublings after 1 and 2 days by the different cell lines. Trypan blue exclusion assays indicated that there was <2% cell death during the 1- and 2-day incubation intervals.
ELISAs.
ELISAs (A490 nm) were performed according to methods previously described by our laboratory (15
, 16)
using polyclonal antibodies specific for MMP-2 (Mr 72,000), and MMP-9 (Mr 92,000) and monoclonal antibodies specific for TIMP-1 (Mr 28,000) and TIMP-2 (Mr 21,000; Ref. 16
). Standard curves were previously plotted for each antibody comparing the absorbance (A490 nm) for increasing amounts of purified antigen (1–120 ng; Ref. 16
). The standard curves were used to measure the amounts of each protein present in the medium of the cultures (16)
.
Specifically, each antibody was used at a dilution of 1:200 and the absorbance (A490 nm) recorded for three different dilutions (0.5, 0.75, and 1.0 µg/ml) of the conditioned medium from the cultures. Following subtraction of the absorbance levels for background levels of secondary antibody binding, the amounts of antigen present were determined using the standard curves. All values were normalized for 1.0 µg/ml total protein before values from triplicate wells and at least three experiments were averaged. All values were then expressed as ng/µg protein in the medium (16)
. Protein levels were measured by methods of Bradford (17)
. All cytokine experiments were carried out as described previously (18)
.
Materials.
All reagents, unless specified otherwise, were reagent grade and were purchased from Sigma. Tissue culture supplies (Fisher Scientific, Pittsburgh, PA). Type I collagen (Collaborative Research, Bedford, MA). IL-10 plus IL-10 and IL-10 receptor antibodies (courtesy of Dr. Narula Sawant, Schering-Plough, Kenilworth, NJ). TIMP-1, TIMP-2, MMP-2, and MMP-9 antibodies were developed in this laboratory and were characterized previously (16)
.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Fig. 1
showed that the low- and high-grade tumor lines (HPCA-5aHPV-18 and HPCA-10aHPV-18) both induced extensive microvessel formation by the HBMCE-1 cells (Fig. 1, a and b)
. Note that the microvessels largely aligned perpendicular to the surface of the tumor cell aggregates and abutted on the surface of the aggregates. It would appear that endothelial cell adhesion to the surface of the tumor cells enabled the vessel attachment to the tumor aggregate (Fig. 1
, arrow). Under the experimental conditions and incubation periods of 1–2 days, the endothelial cells independently formed a few presumptive microvessels; however, few definitive vessels were found in these cultures (Fig. 1c)
. Other studies showed that following prolonged incubation intervals of 4–6 days, the HBMCE-1 cells tended to also form microvessels independent of the tumor aggregates.
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showed that the number and length of the microvessels increased from day 1 to 2 by 30% on average in experiments with either the HPCA-5aHPV-18 or the HPCA-10aHPV-18 cultures (seeded at 1 x 105 cells/ml). The HPCA-5aHPV-18 line (i.e., derived from Gleason score 3 glands) induced fewer numbers of microvessels of a greater length compared to the HPCA-10aHPV-18 line (i.e., derived from Gleason score 5 glands) on days 1 and 2. The addition of TIMP-1 antibodies to the cocultures of HBMCE-1 cells with either HPCA-5aHPV-18 or HPCA-10aHPV-18 lines increased the average number and length of microvessels formed by 50% on day 2. In similar experiments, the addition of MMP-2 and MMP-9 antibodies significantly decreased the degree of microvessel formation to background levels by day 2, indicating TIMP-1 was inhibiting microvessel formation, whereas MMP-2 and MMP-9 were promoting angiogenesis (Table 1)
.
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demonstrated that IL-10 (15 ng/ml) added to the medium on days 0 and 1 significantly reduced the induction of microvessel formation by both the HPCA-5aHPV-18 and the HPCA-10aHPV-18 lines (to an average length of 8–9 µm x 1000/2.6 mm2) after 2 days incubation. Control experiments showed that IL-10 antibodies (1:500 dilution; data not shown) or IL-10 receptor antibodies (1:200 dilution; Table 2
), added along with IL-10, blocked IL-10’s effects, and the length of the microvessels as reduced to levels reported for the untreated cocultures in Table 1
(i.e., on days 1 and 2). Likewise, the addition of TIMP-1 antibodies (1:200 dilution) to the cultures indirectly blocked the effects of IL-10, and the extent of microvessel formation was similar to that reported for untreated cocultures exposed to TIMP-1 antibodies (see Table 1
).
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demonstrated the relative amounts of TIMP-1 (ng/µg protein) secreted by HPCA-5aHPV-18, HPCA-10aHPV-18, and HBMCE-1 cells alone and in coculture experiments in which the cells were incubated for 0, 1, and 2 days (Fig. 2
, columns a, b, and c, and e, and e) in the absence (columns a, b, and c) and presence of 15 ng/ml IL-10 (columns d, and e). The data showed that the HPCA-5aHPV-18 cells alone or in coculture with HBMCE-1 cells secreted relatively high levels of TIMP-1 after 1 and 2 days (i.e., 8–22 ng/µg protein; Fig. 2
, columns b and c). In the presence of 15 ng/ml IL-10, the levels of TIMP-1 were elevated 2–3-fold after 1 and 2 days (Fig. 2
, columns d and e). In comparison, the HPCA-10aHPV-18 cells alone or in coculture with HBMCE-1 cells secreted considerably less TIMP-1 (i.e., <4 ng/µg after 1 and 2 days; Fig. 2
, columns a and b) than the HPCA-10aHPV-18 cells. However, 15 ng/ml IL-10 stimulated a 5–6-fold increase in the amounts of TIMP-1 secreted after 1 and 2 days (i.e., >18 ng/µg; Fig. 2
, columns d and e). Note that HBMCE-1 cells alone secreted barely detectable levels of TIMP-1 after 0, 1, and 2 days (Fig. 2
, columns a–c), and IL-10 had no effect on the TIMP-1 levels secreted by these cells after 1 and 2 days (Fig. 2
, columns d and e). This is probably because HBMCE-1 cells were plated at light densities of 1 x 104 cm2 (i.e., 1000 cells/well). By comparison, the TIMP-2 levels were low or barely detectable (i.e., <2 ng/µg) in all three cell lines, and IL-10 had no effect on the levels of expression by 0, 1, and 2 days, indicating TIMP-2 might not play a modulatory role in the process of angiogenesis.
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, ELISAs were carried to measure the amounts of MMP-2 (ng/µg protein) secreted by HPCA-10aHPV-18, HPCA-5aHPV-18, and HBMCE-1 cells alone and in coculture experiments in which the cells were incubated for 0 days (Fig. 3
, column a), 1 day (columns b and d) and 2 days (columns c and e) in the absence (columns a–c) and presence of IL-10 (columns d and e). The data showed that the HPCA-10aHPV-18 cells (alone or in coculture with HBMCE-1 cells) secreted relatively high levels of MMP-2 after 1 and 2 days (i.e., 8–34 ng/µg protein; Fig. 3
, columns b and c). In the presence of 15 ng/ml IL-10, the levels of MMP-2 were reduced to 4 ng/µg by 1 and 2 days (Fig. 3
, columns d and e). In comparison, the HPCA-5aHPV-18 cells alone or in coculture with HBMCE-1 cells secreted little or no MMP-2 (i.e., <4 ng/µg after 3 and 4 days; Fig. 3
, columns a and b). Also, 15 ng/ml IL-10 produced little or no change in the amounts of MMP-2 secreted after 1 and 2 days (i.e., <4 ng/µg; Fig. 3
, columns d and e). Note that the HBMCE-1 cells alone secreted barely detectable levels of MMP-2 after 0, 1, and 2 days (Fig. 3
, columns a–c), and IL-10 had no effect on the MMP-2 levels secreted after 1 and 2 days (Fig. 3
, columns d and e).
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, ELISAs further showed the amounts of MMP-9 (ng/µg protein) secreted by HPCA-5aHPV-18, HPCA-10aHPV-18, and HBMCE-1 cells alone and in coculture experiments in which the cells were incubated for 0 days (Fig. 4
, column a), 1 day (columns b and d), and 2 days (columns c and e) in the absence (columns a–c) and presence of 15 ng/ml IL-10 (columns d and e). The data showed that the HPCA-5aHPV-18 cells alone or in coculture with HBMCE-1 cells secreted little or no MMP-9 after 0, 1, and 2 days (Fig. 4
, columns a–c). In the presence of 15 ng/ml IL-10, the levels of MMP-9 remained undetectable after 1 and 2 days (Fig. 4
, columns d and e). In comparison, the HPCA-10aHPV-18 cells alone or in coculture with HBMCE-1 cells secreted relatively high levels of MMP-9 (i.e., 4–5 ng/µg after 1 day and 9–11 ng/µg after 2 days; Fig. 4
, columns b and c). However, 15 ng/ml IL-10 reduced the levels of MMP-9 secreted to <1 ng/µg after 1 and 2 days (Fig. 4
, columns d and e). Note that the HBMCE-1 cells alone secreted barely detectable levels of MMP-9 after 0, 1, and 2 days (Fig. 4
, columns a–c), and IL-10 had no effect on the MMP-9 levels secreted after 1 and 2 days (Fig. 4
, columns d and e).
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, ELISAs further showed that following the exposure of the cells to IL-10 (15 ng/ml for 1 or 2 days) in the presence of IL-10 receptor antibodies the amounts of MMP-2 expressed increased slightly in HPCA-5aHPV-18 (Fig. 5
, column a) and HPCA-10aHPV-18 (column b) cultures after 1 or 2 days (i.e., relative to untreated cells). In these experiments, the coculture of the epithelial cells with HBMCE-1 cells had little or no effect on the levels of MMP-2 produced after 2 days (Fig. 5)
. Similar results were observed for MMP-9, but the levels of MMP-9 were significantly less than the MMP-2 levels and equivalent to that detected by 1 and 2 days in Fig. 4
.
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, ELISAs revealed that following the exposure of the cells to IL-10 (15 ng/ml for 1 or 2 days) in the presence of IL-10 receptor antibodies the amounts of TIMP-1 expressed were comparable to untreated HPCA-5aHPV-18 (Fig. 6
, column a); and HPCA-10aHPV-18 (column b) cultures after 1 and 2 days (compare Figs. 2
and 6
). Here, the coculture of the epithelial cells with HBMCE-1 cells had little or no effect on the levels of TIMP-1 produced after 2 days. Similar results were observed for TIMP-2, but the levels of TIMP-2 were low or not detectable (data not shown).
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In Fig. 7
, light microscopic pictures illustrated that extensive microvessel formation was associated with the HPCA-10aHPV-18 tumor aggregates in 2-day cocultures incubated in the presence of 15 ng/ml IL-10 plus TIMP-1 antibodies (Fig. 7a)
or IL-10 receptor antibodies (Fig. 7b)
. In contrast, the extent of microvessel formation was reduced in the presence of 10 ng/ml IL-10 (Fig. 7c)
and almost nonexistent in the presence of 15 ng/ml IL-10 (Fig. 7d)
or MMP-2 plus MMP-9 antibodies (Fig. 7e)
. Note that MMP-2 and MMP-9 antibodies independently reduced the extent of microvessel formation 75% and 25%, respectively. Finally, in cocultures of the basal cells from HGPIN and HBMCE-1 cells, little or no microvessel formation was observed either independent of the basal cell aggregates or in association with the aggregates (Fig. 7f)
.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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50%), whereas MMP-2 plus MMP-9 antibodies reduced the extent of microvessel formation to near zero. More importantly, IL-10 (15 ng/ml) significantly reduced the induction of microvessel formation by both the HPCA-5aHPV-18 and the HPCA-10aHPV-18 lines. The ELISAs clearly showed that the principle effect of IL-10 was to induce a significant increase in TIMP-1 secretion, whereas simultaneously reducing the amounts of MMP-2 and MMP-9 secretion by both the HPCA-5aHPV-18 and the HPCA-10aHPV-18 lines. The effects of IL-10 were further verified in experiments in which IL-10 induction of angiogenesis, and protein secretion was blocked by antibodies specific for IL-10, the IL-10 receptor, and TIMP-1.
One interesting aspect of the results was that the data in Figs. 3
and 4
show that the HPCA-10aHPV-18 cells produced 4–5 times more MMP-2 that the HPCA-5aHPV-18 cells (as well as substantially more MMP-9). This increase correlated with a 2-fold increase in the number of vessels in the angiogenesis assay (Table 1)
. However, the number of vessels observed in the coculture experiments did not increase 4–5-fold. This difference might reflect the inefficiency of the system, the recruitment of endothelial cells or limitations in the number of MT1-MMP receptors expressed by HBMCE cells. Because the average length of the microvessels was about the same in the presence of HPCA-5aHPV-18 and HPCA-10aHPV-18 cultures, the process of microvessel elongation may involve processes unrelated to MMP-2 levels.
Although the mechanisms by which tumor cells regulate TIMP and MMP expression are poorly understood, we suggest that, in part, regulation may depend on the differential effects of cytokines and the receptor-mediated signal pathways (18)
. In an earlier study, we examined the influence of five different cytokines (IL-2, IL-4, IL-6, IL-10, and IFN-
) on TIMP-1, TIMP-2, MMP-2, and MMP-9 expression in three different HPCA-5a,b,c-HPV18 lines and four different HPCA-10a,b,c,d-HPV18 lines (6)
. Qualitative and quantitative ELISA and Northern blot analysis revealed that IL-10 and, to a lesser extent, IL-4 and IL-6 at dosages of 15 ng/ml stimulated significant increases in the levels of TIMP-1 expression in all the cell lines by 16–36 h. In contrast, the MMP-2 levels were reduced significantly in all three lines by 24–36 h (i.e., the time frame of the experiments described here). In addition, other studies of PC-3 ML subclones showed that similar dosages of IL-10 also stimulated TIMP-1 expression (16)
. In agreement with these results, Lacraz et al. (19)
found that IL-10 and IL-4 inhibited the production of MMP-9 in human lymphocytes. IL-10 also stimulated TIMP-1 synthesis. Their data indicated that IL-10 might control MMP-9 and TIMP-1 expression at a pretranslational phase, although steady-state and half-life mRNA studies were not carried out to assess whether IL-10 affected mRNA stability. In further agreement with our results, Lacraz et al. (19)
also reported that the TIMP-2 levels were not altered by the cytokines tested, but the levels were low or undetectable on average. Interestingly, Lacraz et al. (19)
also found that IL-10 failed to influence MMP or TIMP production by human fibroblasts. Our data indicate that IL-10 also does not influence the expression of these genes in endothelial cells, suggesting cell type-specific receptor-dependent responses were required.
IL-10 is normally expressed by tumor tissue (20)
, and we have found by reverse transcription-PCR analysis of 25 different prostate cancer RNA preparations that IL-10 was normally expressed in prostate cancer cells.3
IL-10’s biological effects on tumor growth have ranged from modulating tumor growth (via indirect effects on the immune system) to inhibiting tumor angiogenesis and metastasis. Huang et al. (21)
found that human melanoma A375P cells transfected with a murine IL-10 cDNA exhibited reduced growth and metastatic abilities that correlated with a significant decrease in neovascularity of the tumors. IL-10 produced by the A375P-IL-10 cells was found to down-regulate expression of vascular endothelial growth factor, IL-1ß, tumor necrosis factor-
, IL-6, and MMP-9 in activated macrophages that normally infiltrated the tumor tissues (11)
. The authors suggested that the production of IL-10 by tumor cells might inhibit macrophage-derived angiogenic factors to block tumor growth and metastasis indirectly. Alternatively, IL-10 might sensitize tumor cells to natural killer cells that blocked metastasis as shown in a murine model of breast cancer (22)
. In similar studies, Richiter et al. (23)
reported that IL-10 blocked tumor growth, apparently by blocking angiogenesis and macrophage penetration of the tumor tissue. Kunda et al. (22)
also found with studies of IL-10-transfected murine mammary tumor cell lines that tumor growth was completely inhibited and metastasis was reduced by 90% in syngeneic BALB/ccByJ mice. Recently, we showed that IL-10 expression by IL-10-transfected human prostate PC-3 ML tumor cells (a bone- metastasizing subclone of PC-3 cells) inhibited tumor growth and metastasis to the liver, lung, peritoneum and bone marrow following orthotopic implantation in the prostate gland or i.v. injection via the tail vein of severe combined immunodeficient mice (24)
. IL-10 expression and inhibition of metastasis was also directly correlated with a significant increase in mouse survival rates (i.e., to >90%), indicating that IL-10 might be a important adjuvant therapy for treatment of primary cancer and the prevention of tumor invasion and metastasis. As the in vitro results presented here suggest, IL-10’s activity might be to stimulate TIMP-1 production, block MMP secretion, and, subsequently, interfere with tumor-induced angiogenesis. Thus, studies are currently in progress to measure TIMP and MMP levels relative to angiogenesis and tumor growth and metastasis by the HPCA-10aHPV-18 lines in severe combined immuno- deficient mice.
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FOOTNOTES |
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1 To whom requests for reprints should be addressed, at Medical College of Pennsylvania and Hahnemann University, Mail Stop 435, Department of Pathology and Laboratory Sciences, 15th and Vine Streets, Philadelphia, PA 19102-1192. 
2 The abbreviations used are: HPV, human papillomavirus; TIMP, tissue inhibitor of metalloproteinase; MMP, matrix metalloproteinase; IL, interleukin.
Received 7/23/98; revised 10/ 8/98; accepted 10/26/98.
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