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Inhibition of Transforming Growth Factor-ß Activity Decreases Angiogenesis in a Human Prostate Cancer-reactive Stroma Xenograft Model¹

Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas 77030

	ABSTRACT

Top ABSTRACT Introduction Materials and Methods Results and Discussion REFERENCES

 
We have shown previously that reactive stroma promotes angiogenesis and growth of LNCaP human prostate tumors in the differential reactive stroma xenograft model. Regulators of reactive stroma are not known, but transforming growth factor (TGF)-ß1 is a likely candidate. Three-way differential reactive stroma tumors were generated in the presence of TGF-ß1 latency-associated peptide (LAP) or TGF-ß1 neutralizing antibody. Tumors treated with either of those TGF-ß inhibitors exhibited a reduction in blood vessels, and blood lakes were observed in some areas. The microvessel density of LAP-treated tumors was decreased 3.5-fold relative to control tumors. Moreover, the average wet-weight of LAP-treated tumors was reduced 46% compared with control tumors. The results of this study suggest that TGF-ß regulates reactive stroma and its ability to promote angiogenesis and tumor growth.

	Introduction

Top ABSTRACT Introduction Materials and Methods Results and Discussion REFERENCES

 
Several human carcinomas are associated with a stromal reaction that creates a modified tumor microenvironment (1 , 2) . In human prostate cancer, stromal cell activation to the myofibroblast phenotype, ECM³ deposition, and elevated angiogenesis have been observed adjacent to carcinoma cells and precancerous prostate intraepithelial neoplasia lesions (3 , 4) . This reactive stroma microenvironment is similar to wound repair granulation tissue and is predicted to promote cancer progression. Indeed, we have shown that co-inoculation of human prostate stromal cells with LNCaP prostate carcinoma cells in the DRS xenograft model increases the incidence of LNCaP tumor formation, accelerates LNCaP tumor growth, and promotes angiogenesis (5) . The factors that regulate reactive stroma in cancer are not known; however, TGF-ß1 is a likely candidate because it is a key mediator of the stromal response in wound repair (6) . TGF-ß is overexpressed in human cancers that exhibit reactive stroma, including breast, colon, and prostate cancers (7, 8, 9) . In prostate cancer, initiation of the reactive stroma phenotype appeared to be concurrent with elevated expression of TGF-ß1 by precancerous prostate intraepithelial neoplasia cells (3) . Moreover, TGF-ß1 induced human prostate fibroblasts to differentiate to myofibroblasts and to express ECM markers of reactive stroma in vitro (3) . In vivo studies have shown that TGF-ß1 is sufficient to induce a stromal reaction characterized by myofibroblast activation, increased collagen production, and elevated angiogenesis (10 , 11) . Accordingly, a central hypothesis is that TGF-ß expression by neoplastic cells acts to induce the stromal reaction, which results in the formation of a reactive stroma microenvironment. The reactive stroma microenvironment is predicted to promote angiogenesis and tumor growth. To address this hypothesis, we have used the DRS xenograft model to examine the role of TGF-ß during early tumor development.

	Materials and Methods

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Cell Lines.
LNCaP human prostate carcinoma cells were purchased from American Type Culture Collection (Manassas, VA). LNCaP cells were cultured in RPMI 1640 (Life Technologies, Inc., Rockville, MD) supplemented with 10% fetal bovine serum (Hyclone, Logan, UT), 100 units/ml penicillin, and 100 µg/ml streptomycin (Sigma Chemical Co., St. Louis, MO). The HTS-2T normal human prostate stromal cell line was established in our laboratory as described previously (5) . HTS-2T cells were cultured in Bfs medium: DMEM (Life Technologies, Inc.) supplemented with 5% fetal bovine serum, 5% Nu Serum (Collaborative Research, Bedford, MA), 0.5 µg/ml testosterone (Sigma), 5 µg/ml insulin, 100 units/ml penicillin, and 100 µg/ml streptomycin. HTS-2T cells are a mix of fibroblasts and myofibroblasts, which are the cell phenotypes observed in human prostate cancer-reactive stroma (3) .

Animals.
Athymic NCr-nu/nu male homozygous nude mice, 6–8 weeks of age, were purchased from Charles River Laboratories (Wilmington, MA). All experiments were conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals.

Preparation of DRS Xenografts.
Xenograft tumors were generated according to our protocol published previously (5) . Briefly, frozen aliquots of cells (16 x 10⁶ LNCaP cells and 4 x 10⁶ HTS-2T cells) were thawed in a 37°C water bath for 1–2 min and transferred to separate 15-ml conical tubes. Cells were washed with 10 ml of culture medium (RPMI 1640 + 10% fetal bovine serum) and pelleted at 950 x g for 50 s in a clinical centrifuge (IEC model CL with rotor 809, setting 5). LNCaP cells were resuspended in 10 ml of culture medium, and HTS-2T cells were resuspended in 3 ml of culture medium. Then, cells were combined in one tube and centrifuged again. The supernatant was aspirated to 300 µl, and the cells were gently resuspended in the remaining medium. TGF-ß1 LAP (#246-LAP; R&D Systems, Minneapolis, MN), TGF-ß1 neutralizing antibody (#AF-101-NA; R&D Systems), or the appropriate control (see below) was added to the 300 µl volume. The cells were incubated on ice for 3–5 min and then combined with 0.5 ml of Matrigel (Becton Dickinson, Bedford, MA). The mixture was drawn into a prechilled 1-ml syringe fitted with a 20-gauge needle. After switching to a 25-gauge needle, 100 µl of cell suspension (2 x 10⁶ LNCaP cells and 0.5 x 10⁶ HTS-2T cells) were injected s.c. in each lateral flank. In a separate set of experiments, 100 pM (final concentration) biologically active TGF-ß1 (#101-B1–001; R&D Systems) or vehicle (BSA) control was added to the cell/Matrigel mixture before injection.

The amount of TGF-ß1 LAP or TGF-ß1 antibody added was based on the ND₅₀ for each and on the amount of TGF-ß in the Matrigel. The concentration of TGF-ß in Matrigel ranges from 1.7 to 4.7 ng/ml (Becton Dickinson); all calculations were based on 4.7 ng/ml TGF-ß. Two µg of LAP were added per 500 µl of Matrigel, which is 5.3 times the ND₅₀. As a control, BSA (Sigma) was resuspended in RPMI 1640 and added at the equivalent concentration. Seven hundred fifty ng of TGF-ß1 antibody was added per 500 µl of Matrigel, which is eight times the ND₅₀. As a control, an equivalent amount of normal chicken IgY (R&D Systems) was added.

For all conditions, tumors were allowed to develop for 10 days. Tumors were surgically dissected from surrounding skin and fascia, and then tumor weight (g) was determined. Tissues were fixed in 4% paraformaldehyde overnight at 4°C, then washed three times with PBS, and processed for histology. Tissues were embedded in paraffin, and 5-µm sections were cut and mounted onto ProbeOn Plus slides (Fisher Scientific, Pittsburgh, PA).

Hemoglobin Assay.
Upon dissection, whole tumors were transferred to microcentrifuge tubes containing 50 µl of double-distilled H₂O and then homogenized with disposable pellet pestles (Fisher Scientific). Homogenates were mixed with 0.5 ml of Drabkin’s solution (Sigma) and incubated for 15 min at room temperature. Samples were centrifuged to pellet cell debris, and the supernatant was filtered through a 0.45 µm filter. The absorbance of the filtered supernatant was measured at 540 nm, with Drabkin’s solution used as a blank. The absorption, which is proportional to total hemoglobin concentration, was normalized for tumor weight.

Immunohistochemistry and in Situ Hybridization.
Immunostaining was performed with the MicroProbe Staining System (FisherBiotech, Pittsburgh, PA) according to our protocol published previously (5) . Reagents designed for use with this capillary action system were purchased from Research Genetics (Huntsville, AL). Antimouse CD31 (platelet endothelial cell adhesion molecule-1) antibody (MEC13.3, rat monoclonal) and biotin-conjugated antirat IgG secondary antibody were purchased from BD PharMingen (San Diego, CA). VEGF antibody (#sc-152, rabbit polyclonal) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA), and biotin-conjugated antirabbit secondary antibody was purchased from Sigma. Antigen retrieval was required for both antibodies; tissue sections were incubated in 0.1% trypsin (Zymed, South San Francisco, CA) for 10 min at 37°C (CD31) or subjected to high temperature treatment in 10 mM sodium citrate buffer pH 6 (VEGF). Tissues were incubated in primary antibody overnight at 4°C (CD31, 1:50; VEGF, 1:200) and then in secondary antibody (antirat, 1:100; antirabbit, 1:15) for 45 min at 37°C. In situ hybridization to identify human stromal cells was performed with the MicroProbe System and human-specific AluI/AluII probe (Research Genetics) as described previously (5) .

Microvessel Density Analysis.
Tissue sections immunostained for CD31 were scanned at x200 to identify regions with the highest vascular density. CD31-positive vessels were counted in three high power fields (x400)/section. Two independent observers performed the counts blind. For each tumor, the counts were averaged to determine its microvessel density.

Statistical Analysis.
Tumor weight and microvessel density (control versus LAP-treated) were evaluated with an unpaired t test. Analysis was performed with GraphPad Prism for Macintosh v3.0 (GraphPad Software, San Diego, CA). P < 0.05 was considered statistically significant.

	Results and Discussion

Top ABSTRACT Introduction Materials and Methods Results and Discussion REFERENCES

 
Inhibition of TGF-ß in DRS Xenograft Tumors.
Compared with traditional xenografts, the three-way DRS xenograft system (5) represents a more complete tumor model because all critical components of the tumor are supplied; carcinoma cells, reactive stromal cells, and stromal cell-derived ECM with associated growth factors and matrix remodeling enzymes. Previous studies have shown that all of these components are required to achieve 100% tumor incidence, increased tumor growth, and early angiogenesis (5) . Thus, the DRS system provides a good model to study the regulation of reactive stroma and early prostate tumorigenesis.

Under the tumor-promoting conditions of the DRS model, approaches to studying the effect of TGF-ß on prostate tumorigenesis are limited to methods that neutralize TGF-ß activity. Accordingly, we generated three-way DRS tumors in the presence of recombinant TGF-ß1 LAP or TGF-ß1 neutralizing antibody. The LAP proregion of TGF-ß noncovalently binds to mature TGF-ß1 and inhibits its biological activity. LAP has been shown to be an effective TGF-ß1 antagonist in vitro and in vivo (12) . LNCaP human prostate carcinoma cells, HTS-2T human prostate stromal cells, and Matrigel were used to create the tumors. LNCaP cells were chosen because they are insensitive to TGF-ß growth inhibition (13) , which is similar to endogenous prostate carcinoma cells that down-regulate expression of TGF-ß receptors (14) . Although LNCaP cells produce relatively low levels of TGF-ß1 (15) , the Matrigel ECM contains TGF-ß levels above physiological concentration (2.3 ng/ml).⁴ HTS-2T reactive stromal cells are a mix of fibroblasts and myofibroblasts (5) and are responsive to TGF-ß1 in vitro (data not shown). Tumors generated in the presence of TGF-ß inhibitor or the appropriate control were allowed to grow for 10 days and then harvested. The incidence of tumor formation was 100% under all conditions.

Upon dissection from the mouse host, it was apparent that the gross morphology of tumors treated with LAP and TGF-ß1 neutralizing antibody was different from control tumors. Consistent with previous observations (5) , the three-way DRS control tumors appeared well vascularized at day 10 (Fig. 1A) . In contrast, tumors generated in the presence of either TGF-ß inhibitor were typically white with few (if any) vessels visible on the tumor surface (Fig. 1A) . Moreover, tumors generated in the presence of LAP appeared smaller than three-way control tumors. In support of this observation, the mean wet-weight of LAP-treated tumors at day 10 was reduced 46% as compared with control tumors (Fig. 1B) . This decrease in tumor size was statistically significant (P = 0.014, unpaired t test). Interestingly, our previous work has shown that the presence of stromal cells in three-way DRS tumors increases tumor weight 47% at day 10, compared with two-way DRS tumors composed of LNCaP cells and Matrigel only (5) . Together, these data suggest that the ability of the stroma to promote LNCaP tumorigenesis requires TGF-ß activity.

View larger version (25K): [in this window] [in a new window]   Fig. 1. TGF-ß1 LAP inhibits growth of three-way DRS tumors. A, gross morphology of three-way DRS control tumors and LAP-treated tumors harvested at 10 days. LAP-treated tumors appeared smaller and poorly vascularized as compared with control tumors. Scale bar, 1 mm. B, mean tumor weight of three-way DRS control tumors (n = 10) and tumors treated with LAP (n = 12) at day 10. Bars, SE. *, statistically significant decrease in weight of LAP-treated tumors compared with control tumors (P = 0.014).

View larger version (137K): [in this window] [in a new window]   Fig. 2. Histological analysis of DRS xenograft tumors. A, three-way DRS control tumors at day 10 contained large clusters of LNCaP carcinoma cells with adjacent stromal cells and numerous blood vessels (arrows). B and C, LAP-treated tumors exhibited abnormal vascularization at day 10. Tumors either had no blood vessels (B) or contained a few vessels (C, arrow) and blood lakes (C, *). The histology of tumors treated with TGF-ß1 neutralizing antibody was similar (data not shown). D, two-way L/M xenografts (generated without stromal cells) were composed of smaller LNCaP clusters and were avascular at day 10. Stain, H&E. Scale Bar, 50 µm.

View larger version (117K): [in this window] [in a new window]   Fig. 3. CD31 immunostaining of vasculature in DRS xenograft tumors. A, three-way DRS control tumors contained numerous CD31-positive blood vessels at day 10. B and C, consistent with observations in H&E-stained tissues, some LAP-treated tumors contained no blood vessels (B), whereas others (C) contained a few vessels and blood lakes (*). Blood lakes were not lined by CD31-positive endothelial cells. D, two-way L/M xenografts (generated without stromal cells) also contained few (if any) blood vessels at day 10. Scale bar, 50 µm.

To further investigate the role of TGF-ß in tumor angiogenesis, tissue sections from control and LAP-treated tumors were stained for CD31 (platelet endothelial cell adhesion molecule-1), an endothelial cell marker. CD31 immunostaining confirmed that day 10 control tumors were well vascularized (Fig. 3A) . Consistent with observations in H&E-stained tissue sections, LAP-treated tumors exhibited either no blood vessels (Fig. 3B) or a reduced number of vessels with blood lakes (Fig. 3C) . As reported previously (5) , the blood lakes were not lined by CD31-positive endothelial cells (Fig. 3C , _*). Analysis of microvessel density on CD31-stained sections revealed that the average microvessel count per x400 field in control tumors was 35 ± 3. In contrast, the average microvessel count of tumors treated with LAP was 10 ± 4 (Fig. 4) . This 3.5-fold reduction in microvessel density of LAP-treated tumors was statistically significant (P < 0.001, unpaired t test). These data show that inhibition of TGF-ß activity with LAP reduced angiogenesis in three-way DRS tumors.

View larger version (12K): [in this window] [in a new window]   Fig. 4. TGF-ß1 LAP inhibits angiogenesis in three-way DRS tumors. Microvessel density analysis of three-way DRS control tumors (n = 5) and LAP-treated tumors (n = 8) at day 10 was performed. Microvessel density of control tumors was similar to that reported previously for three-way DRS tumors (5) . Bars, SE. *, statistically significant decrease in microvessel density of LAP-treated tumors compared with control tumors (P < 0.001).

It is well established that VEGF is required for angiogenesis. Thus, immunohistochemistry was used to verify that VEGF was present in both control and LAP-treated tumors. LNCaP cells expressed VEGF under all conditions, including in two-way L/M tumors (data not shown). These data suggest that in addition to VEGF, TGF-ß and stromal components are also required for angiogenesis. In the absence of either TGF-ß or stromal cells, microvessel density was reduced, and blood lakes were eventually observed. Because VEGF induces vessel permeability, it is likely that high VEGF levels under conditions of low microvessel density result in vessel leakiness and the formation of blood lakes (17) . Accordingly, blood lake formation may be one mechanism tumors use to compensate for lack of adequate vascularization in the absence of reactive stroma and normal angiogenesis.

The two different modes of inhibiting TGF-ß activity in the DRS model produced results that were consistent with each other; however, treatment with TGF-ß1 LAP appeared to be a more effective inhibitor than the TGF-ß1 neutralizing antibody in the DRS model. Because LAP naturally binds to the ECM, it may be more stable in the DRS microenvironment. LAP would be more likely to remain in the tumor as the Matrigel loses water, whereas some of the neutralizing antibody may be lost during this process. In addition, LAP is capable of inactivating all isoforms of TGF-ß (5) . In contrast, the neutralizing antibody specifically inhibits the biological activity of TGF-ß1, showing <2% cross-reactivity with TGF-ß2 and TGF-ß3.⁵ Accordingly, treatment with the neutralizing antibody may not effectively inhibit all TGF-ß activity in DRS tumors. This may explain why the weight of tumors treated with neutralizing antibody was not significantly different from controls (data not shown). Partial inhibition of TGF-ß activity could also account for the heterogeneity of vascularization observed in treated tumors. Low TGF-ß activity may result in limited angiogenesis and formation of blood lakes, whereas complete loss of TGF-ß activity would be expected to inhibit reactive stroma and angiogenesis.

In the tumor-reactive stroma microenvironment TGF-ß likely regulates stromal cell production of growth factors, ECM components, and matrix remodeling enzymes, which contribute to driving angiogenesis and tumor progression. For example, TGF-ß increased stromal cell expression of growth factors that promote endothelial cell proliferation, migration and tube formation such as FGF-2, VEGF, and Connective tissue growth factor (18 , 19) . In addition, TGF-ß regulates stromal cell expression of collagen fibers (6) . Inhibition of collagen I synthesis in the stromal compartment of xenograft tumors resulted in reduced angiogenesis and tumor volume (20) . TGF-ß action on stromal cells also enhances angiogenesis by promoting vessel stability (21) . Endothelial cells recruit adjacent stromal cells as mural cells destined to become pericytes or smooth muscle cells. TGF-ß has been shown to regulate mural cell differentiation to pericytes/smooth muscle cells, and lack of associated mural cells leads to endothelial tube instability (reviewed in Ref. 21 ). Interestingly, we have shown that stromal cells inoculated in DRS xenografts are closely associated with blood vessels in a pericyte position (5) .

Regulation of the reactive stroma microenvironment in cancer is likely to be complex. Nevertheless, data presented in this study suggest that TGF-ß is required for stromal cell promotion of tumor growth and angiogenesis. Thus, TGF-ß appears to be a regulator of reactive stroma in cancer. These results are consistent with the central hypothesis that TGF-ß and the reactive stroma microenvironment promote angiogenesis and tumor progression. In the future, it may be possible to target components of this pathway in an effort to develop novel therapies for prostate cancer.

	ACKNOWLEDGMENTS

 
We thank Liz Hopkins and Omar D. Rodriguez for histological preparation of tissues.

	FOOTNOTES

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by NIH Grants RO1-CA58093, RO1-DK45909, SPORE CA58204, and UO1-CA84296. S. J. M. is a Hudson Scholar of the Baylor College of Medicine Medical Scientist Training Program.

² To whom requests for reprints should be addressed, at Department of Molecular and Cellular Biology, Baylor College of Medicine Houston, TX 77030. Phone: (713) 798-6220; Fax: (713) 790-1275;

³ The abbreviations used are: ECM, extracellular matrix; DRS, differential reactive stroma; TGF, transforming growth factor; LAP, TGF-ß1 latency-associated peptide; ND₅₀, neutralization dose₅₀; L/M, LNCaP/Matrigel (two-way xenografts); VEGF, vascular endothelial growth factor.

⁴ Matrigel data sheet, Becton Dickinson (Bedford, MA).

⁵ TGF-ß1 LAP and TGF-ß1 antibody data sheets, R&D Systems (Minneapolis, MN).

Received 6/26/02. Accepted 9/18/02.

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