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Cell Death and Survival

Induction of PSMA and Internalization of an Anti-PSMA mAb in the Vascular Compartment

Daniel P. Nguyen, Peter L. Xiong, He Liu, Samuel Pan, Wilhem Leconet, Vincent Navarro, Ming Guo, Jonathan Moy, Sae Kim, Marigdalia K. Ramirez-Fort, Jaspreet S. Batra and Neil H. Bander
DOI: 10.1158/1541-7786.MCR-16-0193 Published November 2016

Abstract

Angiogenesis is critical for tumor growth and survival and involves interactions between cancer and endothelial cells. Prostate-specific membrane antigen (PSMA/FOLH1) is expressed in the neovasculature of several types of cancer. However, the study of neovascular PSMA expression has been impeded as human umbilical vein endothelial cell (HUVEC) cultures are PSMA-negative and both tumor xenografts and patient-derived xenograft (PDX) models are not known to express PSMA in their vasculature. Therefore, PSMA expression was examined in HUVECs, in vitro and in vivo, and we tested the hypothesis that cancer cell–HUVEC crosstalk could induce the expression of PSMA in HUVECs. Interestingly, conditioned media from several cancer cell lines induced PSMA expression in HUVECs, in vitro, and these lines induced PSMA, in vivo, in a HUVEC coimplantation mouse model. Furthermore, HUVECs in which PSMA expression was induced were able to internalize J591, a mAb that recognizes an extracellular epitope of PSMA as well as nanoparticles bearing a PSMA-binding ligand/inhibitor. These findings offer new avenues to study the molecular mechanism responsible for tumor cell induction of PSMA in neovasculature as well as the biological role of PSMA in neovasculature. Finally, these data suggest that PSMA-targeted therapies could synergize with antiangiogenic and/or other antitumor agents and provide a promising model system to test therapeutic modalities that target PSMA in these settings.

Implications: Cancer cells are able to induce PSMA expression in HUVECs, in vitro and in vivo, allowing internalization of PSMA-specific mAbs and nanoparticles bearing a PSMA-binding ligand/inhibitor. Mol Cancer Res; 14(11); 1045–53. ©2016 AACR.

Introduction

Tumor angiogenesis, the formation of new vessels from resident endothelial cells, is a critical feature in the development, survival, and progression of solid malignancies (1). While hypoxia often initiates the formation of new vessels, tumor growth is impeded by insufficient oxygen and nutrients supply and reduced metabolic exchange, leading to tumor necrosis and regression (1). Consequently, the role of angiogenesis in cancer has generated intense research over the past 20 years, reflected in the approval of several drugs targeting VEGFs such as bevacizumab and tyrosine kinase inhibitors (2).

Several lines of evidence suggest that this process is dependent on dynamic interactions between cancer and endothelial cells. The balance of tumor-derived proangiogenic and antiangiogenic factors drives neovascular development (1). Similarly, endothelial cells produce molecules that are essential to tumor progression (3). The establishment of active contacts between cancer cell and endothelium is also a prerequisite for transendothelial migration, the first step of the metastatic process (4). Altogether, there is compelling evidence that tumor cells undergo dynamic interactions with endothelial cells, which will ultimately promote tumor growth and survival, and favor vascular permeability.

Prostate-specific membrane antigen (PSMA), also known as folate hydrolase 1 and glutamate carboxypeptidase II, is a 100-kDa type 2 integral membrane protein that is the most well-established, highly restricted cell surface prostate antigen known (5). PSMA functions as a cell surface receptor and efficiently internalizes bound antibody and any payload attached to that antibody (e.g., isotopes, drugs, imaging agents, etc.). Beyond its well-documented expression in 95% of prostate cancers (6–11), PSMA is found in the neovasculature of most non-prostatic malignant, solid tumors, yet not in normal endothelium (12–20). The proportion of PSMA-positive endothelial cells is variable and the intensity of PSMA expression ranges from negative to strong in any given tumor type. These features have led to good tumor localization when targeting PSMA with radioisotopes in patients with solid, vascularized tumors (21, 22). Nevertheless, the biological relevance of PSMA in tumor angiogenesis is largely unknown. This is mainly due to the lack of endothelial model systems that express PSMA in vitro or in animal tumor models thereby precluding study (23, 24).

Here, we studied the effect of cancer cell–conditioned medium on PSMA expression in human umbilical vein endothelial cells (HUVEC) to test the hypothesis that the cancer cell–HUVEC crosstalk can induce PSMA expression. We found that conditioned media from a subset of human cancer cell lines stimulate PSMA expression at the mRNA as well as at the protein level. Importantly, HUVECs in which PSMA expression was induced were able to internalize J591, a mAb that binds an extracellular epitope of PSMA (12), as well as nanoparticles bearing PSMA-binding small-molecule ligands. Our data suggest the possibility of a role for PSMA during angiogenesis. Furthermore, they support the concept of combining PSMA-targeted therapies and antiangiogenic or other anticancer agents to synergize therapeutic effects on common cancers.

Materials and Methods

Cell lines

The following human cancer cell lines were used to generate conditioned media: (i) kidney: SK-RC-13, SK-RC-9, SK-RC-39, SK-RC-42; (ii) colon: HCT-15, SW1222, LS-180, SKC-01; (iii) breast: MDA-MB-231, CAMA-1, BT-474, MCF-7; (iv) prostate: PC-3, VCaP, CWR22Rv1, LNCaP, MDA PCa 2b (these 5 cell lines were tested for Mycoplasma contamination and authenticated by DNA Diagnostics Center prior to experiments); (v) ovary: SK-OV-6; and (vi) lung: SK-LC-2. HUVECs were obtained from Lifeline Cell Technology at passage 0. HCT-15, PC-3, CWR22Rv1, LNCaP, and SKLC-2 cells were routinely maintained in RPMI1640 (Mediatech, Inc.) supplemented with 10% FBS, 1% penicillin–streptomycin, and 2 mmol/L l-glutamine (all reagents from Gemini Bio-products). MDA PCa 2b cells were grown in F12K medium (ATCC) containing 20% FBS, 1% penicillin–streptomycin, 2 mmol/L l-glutamine, 25 ng/mL cholera toxin (Sigma-Aldrich), 10 ng/mL epidermal growth factor (BD Biosciences), 5 μmol/L phosphoethanolamine (Sigma-Aldrich), 100 pg/mL hydrocortisone (Sigma-Aldrich), 45 nmol/L selenious acid (Sigma-Aldrich) and 5 μg/mL insulin (Sigma-Aldrich). VCaP cells were grown in DMEM (ATCC) supplemented with 10% FBS, 1% penicillin–streptomycin, and 2 mmol/L l-glutamine. All other cancer cell lines were routinely maintained in Eagle minimum essential medium (ATCC) supplemented with 10% FBS and 1% penicillin–streptomycin and 2 mmol/L l-glutamine. HUVECs were obtained from Lifeline Cell Technology at passage 0 and cultured in VascuLife Basal Medium supplemented with 2% FBS, 10 mmol/L l-glutamine, 5 ng/mL recombinant human (rh) basic fibroblast growth factor, 5 ng/mL rh epithelial growth factor, 5 ng/mL rh VEGF, 15 ng/mL rh insulin-like growth factor-1, 50 μg/mL ascorbic acid, 1 μg/mL hydrocortisone hemisuccinate, and 0.75 U/mL heparin sulfate (all reagents from Lifeline Cell Technology). All cell lines were fed twice a week and split with trypsinization when the cells were confluent. All cell cultures were kept at 37°C in a humidified environment of 5% CO2 atmosphere.

Production of conditioned media

The culture media were removed from T75 flasks at near confluence, and 15 mL of fresh supplemented medium of the respective cell line was added and incubated for 3 days. The media were collected, centrifuged at 1,000 rpm for 10 minutes, and the supernatants were saved and defined as conditioned media. When not used immediately for experiments, conditioned media were frozen at −20°C for future use. Conditioned media were used either undiluted or diluted 1:1 with VascuLife Basal Medium. To obtain fractionated conditioned media, the culture media were removed from T75 flasks when the cells became confluent. The flasks were washed twice with medium without supplements and 15 mL of fresh medium without supplements were added for 24 hours. The media were then collected and centrifuged at 1,000 rpm for 10 minutes. The supernatants were filtered through 100 kDa filters (Millipore), followed by 50 kDa and 10 kDa filters, and three different fractions were collected, respectively, as follows: >100 kDa, 50–100 kDa, and 10–50 kDa fractions. The filtered fractioned conditioned media were then concentrated 50-fold. The fractionated conditioned media were diluted 1:10 with VascuLife Basal Medium.

Tube formation on Matrigel

The growth factor–reduced Matrigel (Corning Life Sciences; 50 μL per well) was coated onto glass coverslips in 24-well plates, which were placed at 37°C for 30 minutes to allow gelification. After trypsinization, HUVECs at passage 1 or 2 were resuspended in undiluted or diluted conditioned medium, and plated at 50,000 cells/mL/well. The plates were incubated at 37°C for different periods of time prior to experiments. HUVECs grown in supplemented VascuLife Basal Medium without conditioned medium were used as controls for tube formation. HUVECs were imaged by Pixera studio software under Inverted Phase Contrast Microscope (Nikon Instruments).

Immunofluorescence staining

HUVECs to be stained for PSMA expression were suspended in conditioned medium and grown on glass coverslips with Matrigel as described above. At each time point, the cells were fixed with 2% paraformaldehyde for 30 minutes, washed three times with 0.1% BSA in PBS, blocked, and permeabilized with 0.075% saponin in 2.5% BSA/PBS for 30 minutes at room temperature. The primary antibody J591, a murine mAb that was produced in our laboratory and binds the extracellular epitope of PSMA with nanomolar affinity (refs. 12, 25; 20 μg/mL), was added and allowed to bind for 60 minutes at room temperature. Subsequently, the coverslips were washed three times and the fluorescein isothiocyanate (FITC)-labeled goat anti-mouse IgG secondary antibody (Jackson ImmunoResearch Laboratories, Inc.) at 1:100 dilution was added for 45 minutes. After three washes, slides were mounted with VECTASHIELD (Vector Laboratories, Inc.) and fluorescent images were acquired using a Nikon digital camera connected to a Nikon microscope and managed by a computer equipped with NIS-Elements Basic Research software (Nikon Instruments). LNCaP cells were used as positive controls for PSMA expression (12). HUVECs grown in unconditioned medium were used as negative controls. A mouse mAb to human CD-31 (BD Biosciences) was used to confirm the human endothelial nature of HUVECs.

Western blotting

Western blotting was performed to confirm the induction of PSMA expression in HUVECs. HUVECs were grown on Matrigel in 6-well plates in conditioned media at 300,000 cells/well for 96 hours. HUVECs were recovered by incubation with dispase (Corning Life Sciences) at 37°C for 10 minutes. After washing two times with supplemented culture medium, then once with PBS, lysates were obtained using 1% Triton X-100/PBS containing Halt Protease Inhibitor Cocktail (Thermo Fischer Scientific). Protein concentrations were determined using the BCA Protein Assay kit (Thermo Fischer Scientific). Equal amounts of protein (36 μg/lane) were placed in each well on a 12% SDS-PAGE gel. After electrophoresis, the separated proteins were transferred to a nitrocellulose membrane (Sigma-Aldrich). The membranes were blocked with 5% dry milk/PBST for 60 minutes. The primary antibody J591 (3 μg/mL) or 7E11 (3 μg/mL) and mouse anti-human β-actin (1:10,000, Thermo Fischer Scientific) in 5% dry milk/PBST were combined and incubated simultaneously with the membrane for 60 minutes. After washing, a horseradish peroxidase–linked secondary antibody sheep anti-mouse IgG at 1:5,000 in 5% dry milk/PBST was added for 60 minutes. After washing, the membrane was prepared for detection using ECL Western blotting detection reagents (GE Healthcare Life Sciences).

Quantitative real time-PCR

HUVECs were plated and recovered as described above. Total RNA was extracted using Pure Link RNA Mini Kit (Invitrogen, Life Technologies). The quantity and quality of total RNA was assessed by spectrophotometry at 260 and 280 nm (Beckman Coulter). cDNAs were synthesized using SuperScriptVILO cDNA synthesis kit (Invitrogen). A real-time PCR was performed to compare the mRNA levels of PSMA in HUVECs grown in diluted conditioned medium versus diluted unconditioned medium. The primers were designed and synthesized by Invitrogen. Human β-actin was used as an internal reference. Real-time PCR reactions were carried out using the Power SYBR Green PCR Master Mix (Invitrogen). Thermal cycling was performed as follows: denaturation at 95°C for 10 minutes followed by 40 cycles of annealing at 95°C for 15 seconds and elongation at 60°C for 1 minute. Primer sequences were as follows: for PSMA 5′-CAAGCAGCCACAACAAGTATGC-3′ and 5′-GAAGGGTCCACTTTGCTTTCAA-3′; for β-actin 5′-TCATGAAGTGTGACGTGGACATC-3′ and 5′-CAGGAGGAGCAATGATCTTGATCT-3′.

J591 and Cy5 nanoparticles internalization assay

Nanoparticles were prepared using block copolymers of poly(D, L-lactide) and poly(ethylene glycol; PLA-PEG) in a nanoemulsion process. A PSMA-targeting polymer of PLA-PEG conjugated to S,S-2-[3-[5-amino-1-carboxypentyl]-ureido]-pentanedioic acid was included at a ratio of 2.5 wt%. The block copolymer and targeting polymer were 16 kDa PLA and 5 kDa PEG. Cy5 conjugated to PLA was encapsulated in the nanoparticle at a load of 1%. Briefly, polymers and Cy5 were dissolved in an organic phase (a mixture of ethyl acetate and benzyl alcohol) and combined with a water phase undergoing high-energy emulsification with a microfluidizer (Microfluidics). The resulting nanoparticle suspension was purified and concentrated with tangential flow ultrafiltration/diafiltration (Millipore) and were stored as a frozen suspension in a 10% aqueous sucrose solution. Nanoparticles were approximately 100 nm in size.

For the internalization assay, HUVECs were grown on glass coverslips in diluted conditioned media for 96 hours as described above. To assess J591 and Cy5-nanoparticles (BIND Therapeutics) internalization, the culture media were removed, and J591 (20 μg/mL) or Cy5 nanoparticles bearing a PMSA-binding ligand/inhibitor (3 mg/mL) diluted in conditioned medium from the respective cell line was added for 120 minutes at 37°C to enable internalization. In parallel, the same experiment was performed on ice to demonstrate cell surface binding only. Subsequently, in both experiments, the cells were washed three times with 0.1% BSA in PBS on ice, fixed with 2% paraformaldehyde for 30 minutes at room temperature, washed three times with 0.1% BSA in PBS, and permeabilized with 0.1% BSA and 0.075% saponin in PBS for 10 minutes at room temperature. For Cy5 nanoparticles, the slides were mounted with VECTASHIELD. For J591, the FITC-labeled goat anti-mouse IgG secondary antibody (Jackson ImmunoResearch Laboratories) at 1:100 dilution was added for 45 minutes, and the cells were washed, mounted, and visualized. The mAb 7E11, which recognizes an intracellular epitope of PSMA (26), and 1% BSA in conditioned medium were used as negative controls.

In vivo experiments

Under an Institutional Animal Care and Use Committee–approved protocol that followed the NIH guide for the care and use of laboratory animals, NOD SCID mice (Charles River) aged 6–8 weeks were injected subcutaneously with 0.5–1 × 106 cancer cells mixed with 1 × 106 HUVEC suspended in 200 μL growth factor–reduced Matrigel. Cancer cells without HUVECs suspended in Matrigel served as controls. The mice were monitored twice a week and sacrificed 2 weeks or 4 weeks after injection, and the tumors were removed. Each specimen was divided into two pieces, one being snap frozen (liquid N2) in OCT compound and stored at −80°C, the other being fixed in 10% neutral-buffered formalin overnight and embedded in paraffin. The paraffin sections were deparaffinized by placing slides in Histo-Clear followed by rehydrating though graded alcohols and washing in Tris-buffered saline-Tween 20 (TBST). For PSMA staining, the deparaffinized and rehydrated sections were placed in Target Retrieval Solution pH 9.0 (Dako) and heated in a water bath (95–99°C) for 30 minutes. For CD-31 staining, the deparaffinized and rehydrated sections were placed in 0.01 mol/L citrate retrieval solution pH 6.0 and heated in a pressure cooker for 1 minute. The sections were washed in TBST. Peroxidase block was added for 5 minutes. After washing in TBST, the mAbs 3E6 (Dako) or NCL-CD31-1A10 (Leica Biosystems) for the detection of PSMA and human CD31, respectively, were added for 60 minutes at room temperature. Antibody binding was detected using peroxidase-labeled polymer conjugated to goat anti-mouse immunoglobulins and 3,3′-diaminobenzidine substrate. The sections were visualized after counterstaining with 10% hematoxylin. An isotype-matched normal mouse IgG was used as negative control.

Results

HUVEC capacity to form tubes on Matrigel

The Matrigel tube assay explores the capacity of endothelial cells to form tubular structures. Because a previous study by Liu and colleagues suggested that PSMA expression in HUVECs is induced by conditioned medium from the breast cancer cell line MDA-MB-231 (27), we studied this cell line in the first set of experiments. HUVEC grown in their standard culture medium (supplemented VascuLife Basal Medium) developed well-formed tubular networks after 6 hours that persisted up to 84 hours (data not shown). In parallel, HUVECs seeded in conditioned medium from MDA-MB-231, used either undiluted or diluted 1:1 with VascuLife Basal Medium formed capillary-like tubular structures within 18 hours, comparable quantitatively and qualitatively to that grown in VascuLife Basal Medium. However, whereas tubes that formed in undiluted conditioned medium regressed rapidly after 48 hours, those that formed in 1:1 diluted conditioned medium persisted up to 120 hours (Fig. 1A). These findings suggested the need for components from both the HUVEC culture medium and from the conditioned medium for HUVECs to maintain the tubular networks with extended lifespan in assays using conditioned medium. Thus, we used cell line conditioned medium diluted 1:1 with VascuLife Basal Medium for the remaining experiments, hereafter referred to as conditioned medium.

Figure 1.
Figure 1.

Conditioned medium from cancer cell lines induces PSMA expression in HUVEC. A, tube formation of HUVEC incubated in conditioned medium from the MDA-MB-231 cell line at the indicated time points. B, immunofluorescence staining of PSMA on HUVEC grown in MDA-MB-231–conditioned medium at the indicated time points. LNCaP served as a positive control for PSMA expression. Immunofluorescence staining with an anti-human CD-31 antibody was performed to confirm the human endothelial nature of HUVEC. C, immunofluorescence staining of PSMA on HUVECs incubated in conditioned medium from the indicated cancer cell lines for 96 hours. Conditioned media from SK-RC-13, HCT-15, and PC-3 induced the strongest PSMA expression. Scale bar, 50 μm.

MDA-MB-231-conditioned medium induces PSMA expression in HUVEC over time

PSMA expression was evaluated by immunofluorescence staining using mAb J591 (12, 25) at different time points beginning at 18 hours (Fig. 1B). HUVECs grown in its standard, unconditioned medium failed to express PSMA at any tested time point. However, when MDA-MB-231–conditioned medium was added, PSMA was weakly expressed by HUVECs at 42 hours gradually increasing over time to reach maximal intensity at 96 hours before decreasing progressively. These data showed that PSMA expression is gradually induced by conditioned medium from the breast cancer cell line MDA-MB-231.

Conditioned media from several cancer cell lines induce PSMA expression in HUVECs

In light of the results obtained with the MDA-MB-231 cell line, we wished to expand our investigation on the induction of PSMA expression in HUVECs. To this end, we used a panel of human cell lines derived from common solid cancers. Because our previous experiments with MDA-MB-231 showed maximum induction of PSMA expression at 96 hours, we chose this time point to screen for PSMA expression in HUVECs grown in conditioned media. We found that conditioned media derived from 14 of 19 cancer cell lines were able to induce PSMA expression in HUVECs to varying degrees (Fig. 1C; Table 1). Among the non-prostate cancer cell lines, conditioned media from 10 of 14 cell lines induced PSMA expression. From the five prostate cancer cell lines, conditioned medium from MDA PCa 2b was the only one that did not induce PSMA expression in HUVECs. Conditioned media from the kidney cancer cell line SK-RC-13, the colon cancer cell line HCT-15 and the prostate cancer cell line PC-3 induced the strongest PSMA expression. Conditioned media from the following cell lines induced moderate PSMA expression: SW1222, LS-180, MDA-MB-231, CAMA-1, BT-474, VCaP, and CWR22Rv1. The conditioned media from SK-RC-9, SK-RC-39, LNCaP, and SK-OV-6 induced weak expression of PSMA. Furthermore, HUVECs grown in PSMA-inducing conditioned media tended to form tubes that were qualitatively better than those grown in noninducing conditioned medium (data not shown). However, this finding does not imply that there is a causal relationship between the quality of tube formation and PSMA expression. Taken together, this experiment showed that conditioned media from several common solid cancer cell lines are able to induce PSMA expression in HUVECs.

View this table:
Table 1.

Immunofluorescence staining of PSMA on HUVECs grown in conditioned media from the indicated cancer cell lines for 96 hours

Western blot analysis to detect PSMA expression

The expression of PSMA in HUVEC after 96-hour incubation in conditioned media from inducing and noninducing cell lines was evaluated by Western blot analysis. The induction of PSMA expression was confirmed in SK-RC-13, HCT-15, and MDA-MB-231, while no PSMA expression was detected in HUVECs grown in conditioned medium from MCF-7 or unconditioned medium (Fig. 2A).

Figure 2.
Figure 2.

A, Immunoblotting of PSMA expression in HUVECs incubated in conditioned medium from the indicated cancer cell lines, using the mAbs J591 and 7E11. Equal amounts of protein were placed in each well on a 12% SDS-PAGE gel. The results are representative of four independent experiments. B, real-time PCR of PSMA expression in HUVECs incubated in conditioned medium solution from the indicated cancer cell lines. Results are shown as the fold increase in comparison with those obtained with cells cultured in unconditioned medium. Human β-actin was used as an internal reference.

Real time-PCR to study the transcriptional regulation of PSMA

Real-time PCR was carried out to measure PSMA mRNA levels in HUVECs grown in conditioned media from inducing and noninducing cell lines. The results showed that PSMA mRNA levels increased 34.8, 23.5, and 16.5-fold in HUVECs grown in conditioned media from SK-RC-13, HCT-15, and MDA-MB-231, respectively, whereas there was a 3.7-fold increase in HUVECs grown in conditioned medium from MCF-7 compared with HUVECs grown in their standard culture medium (Fig. 2B).

Internalization of mAb J591 and Cy5 nanoparticles bearing a PSMA-binding ligand/inhibitor

PSMA is constitutively internalized from the cell surface; however, in the presence of antibody the rate of PSMA internalization increases (25, 28). This is a critical feature for the development of antibody-based agents, and therefore we examined whether internalization of J591 occurred in HUVECs expressing PSMA. After 2-hour incubation with J591 or Cy5 nanoparticles bearing a PSMA-binding ligand/inhibitor at 37°C, HUVECs expressing PSMA after induction showed strong intracellular PSMA staining, whereas incubation on ice, which impedes internalization, showed staining limited to the plasma membrane (Fig. 3A). Negative controls omitting the primary antibody (J591) or using the mAb 7E11, which recognizes an intracellular epitope of PSMA (26), showed no PSMA staining (data not shown). Cy5 nanoparticles were accumulated in the cytoplasm of HUVECs with conditioned medium, but not in HUVECs without conditioned medium (Fig. 3B). Taken together, the results demonstrated that HUVECs expressing PSMA in conditioned media are able to internalize the J591 antibody and nanoparticles bearing a PSMA-binding ligand/inhibitor.

Figure 3.
Figure 3.

A, internalization assay of J591 in HUVECs incubated in conditioned medium (CM) from the cancer cell lines SK-RC-13, HCT-15, MDA-MB-231, and MCF-7 (as the negative control) for 96 hours. After a 2-hour incubation with J591 at 4°C or at 37°C, cells were permeabilized and stained with FITC-conjugated secondary antibody to detect PSMA expression. Incubation at 4°C showed PSMA expression exclusively at the cell membrane, whereas incubation at 37°C demonstrated significant internalization of J591. Scale bar, 50 μm. B, internalization assay of Cy5 nanoparticles bearing a PSMA-binding ligand/inhibitor in HUVEC incubated in conditioned medium (CM) from SK-RC-13 or their standard culture medium for 96 hours. After a 2-hour incubation with Cy5 nanoparticles at 37°C, cells were washed, fixed with 2% paraformaldehyde, and mounted with VECTASHIELD. Cy5 nanoparticles were accumulated intracellularly in HUVECs incubated in conditioned medium (arrows), but not in HUVECs incubated in their standard culture medium. Scale bar, 200 μm. C, immunostaining of HUVECs incubated in fractionated conditioned medium from the indicated cell lines. Unfractionated conditioned medium served as a positive control. The results indicate that only the 10–50 kDa fraction induced PSMA expression in HUVEC. Scale bar, 50 μm.

The PSMA-inducing activity of conditioned media is restricted to the 10–50 kDa fraction

To begin to characterize the PSMA-inducing activity in conditioned medium, we separated conditioned media from the cell lines SK-RC-13, HCT-15, and MDA-MB-231 into three fractions. Each fraction was assayed for the ability to induce PSMA expression in HUVECs. The results indicated that only the 10–50 kDa fraction possesses PSMA-inducing activity (Fig. 3C), suggesting that tumor-related factor/s implicated in PSMA expression by neovessels are found in this molecular weight range.

In vivo experiments

We next investigated whether the induction of PSMA in HUVECs by conditioned medium was reproducible in vivo. The inducing cell line SK-RC-13 and the noninducing cell line MCF-7, as a negative control, were evaluated. Suspensions of each cancer cell line alone or mixed with HUVEC were injected into the flank of NOD SCID mice aged 6–8 weeks. Microscopic evaluation at 14 days and 28 days showed well-formed vessels that stained for human CD-31, demonstrating the contribution of HUVECs to neovascularization in xenografts of SK-RC-13 with HUVECs (Fig. 4A). Staining with mAb 3E6 showed PSMA expression in the neovasculature of xenografts of SK-RC-13 with HUVECs, and the areas corresponded to those staining for human CD-31 (Fig. 4A). SK-RC-13 xenografts without HUVECs showed neither human CD-31 nor PSMA expression (Fig. 4A). This experiment demonstrated that SK-RC-13 cells are able to induce PSMA expression in HUVECs in vivo as well as in vitro. MCF-7 (PSMA noninducing) cells coimplanted with HUVECs showed the presence of CD31-positive cells within the xenografts but these cells did not form tubes or channels nor did they express PSMA (Fig. 4B).

Figure 4.
Figure 4.

A, immunohistochemical analysis of tumors from mice implanted with SK-RC-13 cells with or without HUVEC, showing expression of PSMA and CD31 in concordant tumor areas at 14 days and 28 days, while neither molecule was detected in SK-RC-13-only tumors. B, immunohistochemical analysis of tumors from mice implanted with MCF-7 cells with or without HUVECs, showing only human CD-31 expression in mice implanted with MCF-7 cells and HUVEC, but no PSMA expression. Scale bar, 50 μm.

Discussion

PSMA is a clinically validated target for imaging and therapy of prostate cancer (5). Beyond prostate cancer, multiple previous studies have documented PSMA expression in the tumor endothelium of a wide variety of solid tumors including renal cell carcinoma, breast carcinoma, colorectal, gastric adenocarcinoma, gynecologic, and head and neck cancers and glioblastoma, etc. (12–20). Importantly, adjacent normal endothelium in these cancers does not express PSMA (12, 17, 18), suggesting that tumor-related factors may induce PSMA expression by neovessels. PSMA is thus one of, if not the most, cancer-specific neovascular targets known and, therefore, a target of particular interest. Yet, the study of PSMA expression in neovasculature has been hampered as it is not expressed on HUVEC cells in vitro nor is there evidence of its expression within xenograft models where the vasculature is host/mouse-derived (23, 24). Absence of in vitro or in vivo experimental models of PSMA-expressing neovasculature has severely impeded understanding of the regulation of PSMA expression, the biological role that PSMA may play, and development of imaging and therapeutic approaches in this setting.

We expanded the findings of Liu and colleagues (27) that showed that spent medium from a single cell line, MDA-MB-231, induced PSMA expression in HUVEC. In contrast to our study, they found that PC-3 and LNCaP conditioned media did not induce good tube formation, and PSMA mRNA increased less in MDA-MB-231 (1.76-fold vs. our finding of 16.5-fold). The explanation for the differences in findings likely relate to several differences in the methodology of the Liu and colleagues study compared with ours. We collected conditioned medium after a longer incubation time and we utilized different time points for the detection of PSMA. Liu and colleagues determined PSMA expression at a single, 18-hour time point, when PSMA just started to be detectable in our time-course study. Our results confirmed PSMA induction in 14 of 19 cancer cell lines and, not only in immunofluorescence studies, but also by Western blotting, RT-PCR, and in murine models.

Interestingly, among the prostate cancer cell lines, the strongest PSMA expression was induced by the PC-3 cell line, which itself does not express PSMA (12). Conversely, strongly PSMA-expressing cell lines such as LNCaP and MDA PCa 2b (12, 29) induced PSMA in HUVECs only weakly or not at all. A recent study documented that conditioned medium from PC-3 or LNCaP-SF, an androgen-independent LNCaP subline, causes more potent expression of angiogenesis markers in HUVECs than parental LNCaP (30). The authors concluded that in the case of prostate cancer, angiogenic potential may be increased in castration-resistant disease. Our finding that PC-3, an androgen-independent cell line, induces the strongest expression of PSMA among prostate cancer lines in HUVEC is consistent with their hypothesis.

The functional role of PSMA in the neovasculature cells remains elusive. PSMA is a glutamate carboxypeptidase with both folate hydrolase and N-acetylated α-linked acidic dipeptidase (NAALADase) activity (31, 32). Peptidases are known to be important regulators of tumor angiogenesis (33). Thus, PSMA expression in endothelial cells may provide a growth advantage to various cancers through cleavage of signaling molecules involved in angiogenesis. In support of this hypothesis, Conway and colleagues previously showed that PSMA enzymatic activity is required for endothelial cell migration through extracellular matrix (34). Our finding that HUVECs, coinoculated with the PSMA noninducing cell line MCF-7, remained CD31+/PSMA− and did not form tubes or vascular channels is consistent with Conway and colleagues' findings. Furthermore, it has been hypothesized that PSMA may facilitate angiogenesis by increasing the local availability of folic acid (35), which is necessary for the adequate function of endothelial nitric oxide synthase (36).

Our results confirm the potential of PSMA as a unique and attractive therapeutic target for solid cancers. In this regard, we found that PSMA-expressing HUVEC have the capacity to internalize J591, a mAb that recognizes the extracellular domain of PSMA (12), as well as nanoparticles bearing PSMA-binding small-molecule ligands. PSMA possesses a MXXXL internalization motif which allows constitutive, rapid, and efficient endocytic internalization that increases in the presence of antibody (25, 28). This is an important feature for the development of antibody-based agents, allowing intracellular delivery of toxins, drugs, or radioisotopes (25). In contrast to other angiogenic targets such as VEGF and integrins, which are also involved in the general process of angiogenesis (37), the tumor neovascular specificity of PSMA makes it an ideal target for cytotoxic therapies. The potential of PSMA as a vascular target molecule was highlighted by phase I trials that demonstrated specific targeting of the tumor neovasculature by 111In-labeled J591 in patients with kidney, colorectal, lung, bladder, pancreas, liver, and breast cancers, and melanoma (21). PSMA is currently undergoing evaluation in the field of antibody–drug conjugates (38), PSMA DNA immunization approaches and vaccine replicon particles (39), immunotoxins (40), retargeting of immune cells (41), prodrug activation (42, 43), nanoparticles (44, 45), small interfering RNAs (42, 46), and retargeted virotherapy (47). On a broader scale, the induction of PSMA expression in endothelial cells suggests that therapeutic effects in other cancers may be enhanced by strategies that combine targeting of both PSMA and established proangiogenic factors such as VEGF.

The induction of angiogenesis is an integral part of tumor development (1). Various in vivo models demonstrated that coinjection of cancer and endothelial cells enhances tumor growth (48, 49). Altogether, our results support an in vitro and in vivo model where tumor cells in close proximity to resident endothelial cells secrete one or more as yet unknown factors, most likely in the 10–50 kDa molecular weight range, which can initiate paracrine signaling cascades resulting in PSMA expression. This model of upregulation of PSMA by conditioned media from cancer cells provides new avenues to dissect the regulatory pathways responsible for PSMA expression in HUVEC and the possible role of PSMA in tumor neoangiogenesis. Furthermore, our animal model could prove valuable to test novel therapies directed to PSMA-positive neovessels.

In conclusion, our results show that HUVEC are able to express PSMA, presumably through induction by regulatory factors secreted by tumor cells. In addition, PSMA-expressing HUVECs have the capacity to internalize anti-PSMA mAbs. Further research using our model should determine whether PSMA plays a role in the promotion of tumor angiogenesis, thus providing a permissive environment for tumor growth and progression. Our findings also strengthen the concept of PSMA as a target for therapeutic strategies in patients with solid cancers.

Disclosure of Potential Conflicts of Interest

N.H. Bander has ownership interest (including patents) and is a consultant/advisory board member for BZL Biologics, LLC. No potential conflicts of interest were disclosed by the other authors.

Authors' Contributions

Conception and design: D.P. Nguyen, H. Liu, N.H. Bander

Development of methodology: H. Liu, V. Navarro, N.H. Bander

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): D.P. Nguyen, P.L. Xiong, V. Navarro, J. Moy, J.S. Batra, N.H. Bander

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): D.P. Nguyen, W. Leconet, M. Guo, N.H. Bander

Writing, review, and/or revision of the manuscript: D.P. Nguyen, H. Liu, W. Leconet, M. Guo, M.K. Ramirez-Fort, N.H. Bander

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): D.P. Nguyen, P.L. Xiong, S. Pan, S. Kim, J.S. Batra, N.H. Bander

Study supervision: D.P. Nguyen, H. Liu, V. Navarro, J.S. Batra, N.H. Bander

Grant Support

D. P. Nguyen is a research fellow and is supported by research grants from the Nuovo-Soldati, the Arnold U. und Susanne Huggenberger-Bischoff, the Bangerter Foundations, and the Swiss Urological Association. The Cy5-nanoparticles were a gift from BIND Therapeutics.

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.

  • Received June 6, 2016.
  • Accepted June 30, 2016.

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Induction of PSMA and Internalization of an Anti-PSMA mAb in the Vascular Compartment
Daniel P. Nguyen, Peter L. Xiong, He Liu, Samuel Pan, Wilhem Leconet, Vincent Navarro, Ming Guo, Jonathan Moy, Sae Kim, Marigdalia K. Ramirez-Fort, Jaspreet S. Batra and Neil H. Bander
Mol Cancer Res November 1 2016 (14) (11) 1045-1053; DOI: 10.1158/1541-7786.MCR-16-0193
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