Human tumor vessels express tumor vascular markers (TVM), proteins that are not expressed in normal blood vessels. Antibodies targeting TVMs could act as potent therapeutics. Unfortunately, preclinical in vivo studies testing anti-human TVM therapies have been difficult to do due to a lack of in vivo models with confirmed expression of human TVMs. We therefore evaluated TVM expression in a human embryonic stem cell–derived teratoma (hESCT) tumor model previously shown to have human vessels. We now report that in the presence of tumor cells, hESCT tumor vessels express human TVMs. The addition of mouse embryonic fibroblasts and human tumor endothelial cells significantly increases the number of human tumor vessels. TVM induction is mostly tumor-type–specific with ovarian cancer cells inducing primarily ovarian TVMs, whereas breast cancer cells induce breast cancer specific TVMs. We show the use of this model to test an anti-human specific TVM immunotherapeutics; anti-human Thy1 TVM immunotherapy results in central tumor necrosis and a three-fold reduction in human tumor vascular density. Finally, we tested the ability of the hESCT model, with human tumor vascular niche, to enhance the engraftment rate of primary human ovarian cancer stem–like cells (CSC). ALDH+ CSC from patients (n = 6) engrafted in hESCT within 4 to 12 weeks whereas none engrafted in the flank. ALDH ovarian cancer cells showed no engraftment in the hESCT or flank (n = 3). Thus, this model represents a useful tool to test anti-human TVM therapy and evaluate in vivo human CSC tumor biology. Cancer Res; 73(12); 3555–65. ©2013 AACR.

The tumor vasculature expresses numerous genes not expressed in normal vasculature (1–5). This is, in part, due to the increased expression of genes associated with physiologic angiogenesis, as many tumor vascular antigens are also upregulated in angiogenic tissues (1, 6, 7). However, if the angiogenic signature is the primary difference between tumor vasculature and normal vasculature, one might anticipate a significant overlap between vascular profiles of different tumor types. Indeed this is not the case; the vascular expression profile of different tumor types appears to be distinct (3, 5, 7–10). This is consistent with murine studies suggesting physiologic and pathologic angiogenesis have distinct gene signatures (6) and indicates that the influence of the cancer cell on the tumor microenvironment may play a role in the induction of tumor specific vascular proteins.

Tumor vascular markers (TVM), antigens specifically expressed in tumor vessels and not expressed in normal vessels, represent a potentially important therapeutic target. In particular, those with extracellular exposure are ideal targets for immunotherapeutics (2, 10–12). As therapeutic targets, TVMs would be accessible to drug, and the restricted nature of TVM expression should limit therapy-associated side effects on normal tissues. Proof-of-principle studies in rodents showed the potency of tumor vascular–targeted therapy. Immunotherapeutics targeting a tumor vascular–specific splice variant of fibronectin showed profound restriction of tumor growth (13). More recently, antibodies targeting the anthrax receptor (Tem8) have been shown to specifically inhibit pathologic angiogenesis and restrict tumor growth (14, 15). Phase I clinical trials using an immunotherapeutic targeting the TVM FOLH1 suggest that antitumor vascular immunotherapeutics are safe and potentially efficacious (16).

Broader development of anti-TVM therapies has been hindered by the absence of an experimental system with confirmed human TVM expression with which to test potential therapies. Most mouse tumor models generate murine vessels and therefore cannot be used to test antibodies specific to human antigens. While models of human tumor vasculature have been proposed, these models have been difficult to reproduce, have limited long-term viability, and/or do not have confirmed expression of TVMs (17–19).

Beyond their role in providing nutrients to the tumor, tumor vascular cells are also a critical host component of the cancer stem–like cell (CSC) niche. Vascular cells receive angiogenic cues from CSCs and in turn provide CSCs with critical survival, proliferation, and differentiation signals (20). Thus, a model with robust human tumor vasculature could enhance the in vivo study of human CSCs, which have been surprisingly difficult to engraft in mice. The difficulty engrafting human CSCs in mice could be related to differences in the murine and human microenvironments, including the vasculature.

In the current study, we focused on detailed characterization of the vasculature using the previously reported human embryonic stem cell teratoma (hESCT) tumor model previously shown to have human vessels (21, 22). This model has the ease of standard xenograft models; however, tumor vessels are derived from the human ESCs and are therefore of human origin. It had not been clear whether these are “normal” human vessels or true “tumor vessels” that express TVMs. Here, we show that when injected with cancer cells, hESCTs have vessels expressing human TVMs. With the addition of mouse embryonic fibroblasts and primary tumor vascular cells, about 80% of the vessels in the tumor are human in origin and persist for up to 12 weeks. Using hESCT ovarian cancer and breast cancer models, we found that several TVMs are induced in a tumor-specific fashion. We then used this model to show the ability to test the therapeutic activity of anti-human tumor vascular–specific antibody therapeutics; an anti-THY1 immunotoxin delayed tumor growth and resulted in central tumor necrosis. Finally, we showed that this tumor model, with a human microenvironment, enhances the engraftment and growth of primary ovarian CSCs.

Cell culture

Use of hESCs was approved by the University of Michigan Embryonic Stem Cell Research Oversight Committee (Ann Arbor, MI). H9 hESC (WiCell Research Institute, Madison, WI) and H7-GFP hESC (a gift from Joseph Wu, Stanford University, Stanford, CA) were grown as previously described (23). Undifferentiated ESC colonies were initially passaged by manual dissection with final passages conducted with enzymatic digestion using TrypLE Select (Invitrogen). Human ovarian cancer cell line HEY1 and SKOV3 (American Type Culture Collection) were grown in RPMI containing 10% FBS. The breast cancer cell line MCF7 (a gift from Dr. Max Wicha, University of Michigan) was grown in Minimum Essential Media (MEM) containing 10% FBS and 0.01 mg/mL bovine insulin (Invitrogen). To create DsRED-expressing cells, both MCF7 and HEY1 cells were transduced with DsRED-expressing lentiviral construct (provided by the UMCC Vector core).

In vivo tumor models

NOD/SCID mice (Charles River) were housed and maintained in the University of Michigan Unit for Laboratory Animal Medicine. All studies were approved by the University Committee on the Use and Care of Animals. hESCTs were generated as previously described (21–23). Briefly, H9 hESCs were cultured on mouse embryonic fibroblasts (MEF), manually dispersed, and passaged. Approximately 5 × 105 undifferentiated H9 hESCs or H7-GFP ESCs were injected subcutaneously into the axilla of NOD/SCID mice (with or without MEFs) with 100 μL of PBS and 200 μL of Matrigel (BD Biosciences). Once hESCTs were palpable, tumor cells in 40 μL of PBS were injected intra-hESCT. A total of 2 × 105 tumor cells (HEY1-DsREd or MCF7 DsRED) were injected alone or with 5,000 VE-Cadherin+ primary human tumor vascular cells (isolated as previously described; ref. 7). For hESCTs injected with primary ovarian CSCs, 700 (n = 2), 5,000 (n = 3), or 10,000 (n = 3) primary ALDH+ ovarian cancer cells (from 6 different patients) or 10,000 ALDH cells from paired samples were injected (n = 3). All tumors were harvested when hESCT tumor volumes were about 2,000 mm3 (range, 4–12 weeks; median, 8 weeks). For flank xenografts, 5 × 105 cells were injected in 100 μL of PBS and 200 μL of Matrigel into the axilla of NOD/SCID mice. Tumors were imaged using biofluorescence (Xenogen IVIS 2000, Caliper Life Sciences). Murine tumors were APC/PTEN/p53-mutant mouse ovarian tumors (a gift from Dr. Kathy Cho, University of Michigan; refs. 24, 25).

Isolation of cancer stem cells from primary ovarian cancer specimens

Informed written consent was obtained from all patients before tissue procurement. All studies were conducted with the approval of the Institutional Review Board of the University of Michigan. All tumors were from patients with stage III or IV epithelial ovarian or primary peritoneal cancer. Tumors were mechanically dissected into single-cell suspensions, red cells lysed with ACK buffer, and cell pellets were collected by centrifugation. CSCs were then isolated from primary ovarian tumor single-cell suspensions using the ALDEFLUOR assay fluorescence-activated cell sorting (FACS) as previously described (26). Gating was established using propidium iodide (PI) exclusion for viability. ALDH/DEAB-treated cells were used to define negative gates. FACS was conducted using the BD FACSCanto II or FACSAria (Becton Dickinson) under low pressure in the absence of UV light.

Immunofluorescence and immunohistochemistry

About 8-μm-thick sections from fresh-frozen tumors were fixed in acetone for 10 minutes and then washed with PBS and blocked for 20 minutes. Primary antibody was incubated for 2 hours, washed with PBS, and incubated with secondary antibody for 1 hour. For immunofluorescence (IF), slides were washed with PBS and then mounted with Vectashield Mounting Medium for fluorescence with DAPI H-1200 (Vector Laboratories). Antibodies used for IF and immunohistochemistry (IHC) are listed in Supplementary Table S1. IHC staining was conducted using the Vectastain ABC Kit (Vector) per manufacturer's instructions. Select p53 IHC was conducted by the Histology/IHC Service at the University of Michigan.

RNA isolation and real-time PCR

Tumors were sectioned and regions of tumor with human vasculature were confirmed via IHC. Serial sections of were dissolved in TRIzol (Invitrogen) and RNA was extracted (PureLink RNA Mini Kit, Invitrogen) per manufacturer recommendations. RNA integrity was confirmed on the Agilent 2100 BioAnalyzer. PCR was carried out for 40 cycles with primers at 100 nmol/L concentrations (Supplementary Table S2). All transcripts were confirmed using 3% agarose gel electrophoresis.

Quantification of vessels

Vascular density quantification was conducted as previously described (27). Five sections from each of 3 tumors in each tumor group were evaluated. Total mCD31 and hCD31 stain, as defined by pixel density and hue, was assessed using Olympus Microsuite Biological Suite Software. The area of staining was then compared between mCD31 and hCD31 using the 2-sided Student t test. hCD31+ tumor microvascular density following anti-THY1-toxin therapy was similarly assessed. hCD31-Alexa 594 and GFP expression were used to assess human vessels either from tumor endothelial cell origin [Alexa-594+ (red) only, or from hESCT origin (red+GFP). Sections were photographed in toto and then quantitated using Olympus software as above.

Immunotoxin development and delivery

Anti-THY1-saporin immunotoxin was developed as previously described (27). About 2 μg of freshly conjugated anti-THY1 antibody and saporin toxin, or an equimolar concentration of strepavidin-saporin or unlabeled anti-THY1 antibody was incubated with 5 × 104 mesenchymal stem cells (MSC) in triplicate. After 3 days of treatment, viability cell was assessed using Trypan Blue. To test the efficacy of anti-TVM therapeutics in vivo, hESCT-HEY1 tumors were treated with no treatment (n = 3) or 2 μg of rat IgG-saporin (n = 3), or anti-THY1-saporin (n = 4). Immunotoxin was delivered intravenously every other day for 3 doses. Tumor growth was tracked using biofluorescent imaging with the Xenogen IVIS 200 Imaging System and LivingImage software provided by the Center of Molecular Imaging of the University of Michigan. Mice were monitored the day before and after treatment. This experiment was repeated with rat IgG-saporin controls (n = 3) and anti-Thy1-saporin (n = 3).

Vessels in hESCT-cancer model express TVMs in a cancer cell–dependent manner

We generated hESCT-ovarian cancers (HEY1) and hESCT-breast cancers (MCF7) as previously described (23) using DsRED-labeled cancer cells. IF showed clear, non-DsRED, human CD31+ vessels consistent with prior reports of human ESC–derived vessels (21, 22, 28, 29). Human vessels were predominantly found in a peritumoral location (Fig. 1A) and less frequently within the tumor islets and teratoma tissue. RT-PCR was carried out to determine whether the ovarian- or breast-specific TVMs were expressed in (i) HEY1 ovarian cancer cell culture, (ii) HEY1 ovarian tumor xenografts, (iii) in vivo hESCTs, or (iv) in vivo hESCT-HEY1 ovarian tumors. In parallel, we assessed the expression of ovarian- or breast cancer–specific TVMs were expressed in (i) MCF7 ovarian cancer cell culture, (ii) MCF7 breast cancer xenografts, (iii) in vivo hESCTs, or (iv) in vivo hESCT-MCF7 breast tumors. We evaluated the expression of TVMs that have been reported to be upregulated in numerous tumors including tumor endothelial marker-7 (TEM7), Integrin β3, and THY1, as well as for TVMs reported to be ovarian cancer–specific including EGFL6, P2Y-like receptor (GPR105), and F2RL1, or breast cancer–specific such as FAP, HOXB2, SFRP2, and SLITRTK6. Unfortunately all TVM mRNAs (and every gene we have tested to date) were expressed in both hESCTs and the ovarian cancer and breast cancer hESCT cancer model, thus real-time PCR (RT-PCR) suggested that TVMs were expressed in the hESCTs but was otherwise uninformative (Fig. 1B).

Figure 1.

Validation of human vasculature in the hESCT cancer model. A, co-IF showing the presence of hCD31+ (green) vascular structures in a peritumoral location with DsRed cancer cells. B, RT-PCR of TVMs expression in the indicated cancer cell line cultures, tumor cell line xenografts, hESCTs, and hESCT cancer models.

Figure 1.

Validation of human vasculature in the hESCT cancer model. A, co-IF showing the presence of hCD31+ (green) vascular structures in a peritumoral location with DsRed cancer cells. B, RT-PCR of TVMs expression in the indicated cancer cell line cultures, tumor cell line xenografts, hESCTs, and hESCT cancer models.

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We next conducted IHC to localize TVM expression within the cell line xenografts, hESCT, hESCT-HEY1 ovarian tumors, and hESCT-MCF7 breast cancers. Within hESCT controls, TVM protein expression could be identified in various developmental tissues, but expression was generally not found in vascular structures (Fig. 2). In contrast, the expression of ovarian TVMs could be detected within peritumoral vessels within the hESCT-HEY1 ovarian tumors (Fig. 2). Some vessels were clearly filled with red blood cells, indicating a connection with the murine vasculature and perfusion (Fig. 2 and data not shown). Serial sections stained with anti-hCD31 antibody confirmed these structures as human vessels (Supplementary Fig. S1). Identical results were obtained in an hESCT-SKOV3 ovarian cancer model (data not shown). Similarly, the breast cancer–specific TVMs FAP, SFRP2, SLITRK6, and SMPD3 were all expressed in the hESCT-MCF7 tumors (Fig. 2). No vascular expression of any of the TVMs was detected in flank tumor xenografts (Fig. 2) or in a murine ovarian tumor model (Supplementary Fig. S2), showing that IHC is not detecting murine tumor vessels.

Figure 2.

TVM expression in the vasculature is influenced by the cancer cells. IHC localization of ovarian cancer–specific TVMs, breast cancer–specific TVMs, and nontumor-specific general TVMs in the indicated tumors. While TVMs are expressed in various developmental tissues of the hESCT, vascular expression of TVMs is primarily seen only in the presence of cancer cells in a tumor-type–specific manner. n = 4 animals/group in 2 experiments. Black arrow indicates vessel containing red blood cells.

Figure 2.

TVM expression in the vasculature is influenced by the cancer cells. IHC localization of ovarian cancer–specific TVMs, breast cancer–specific TVMs, and nontumor-specific general TVMs in the indicated tumors. While TVMs are expressed in various developmental tissues of the hESCT, vascular expression of TVMs is primarily seen only in the presence of cancer cells in a tumor-type–specific manner. n = 4 animals/group in 2 experiments. Black arrow indicates vessel containing red blood cells.

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It remained unclear whether the distinctions in the tumor vascular expression profile observed for different tumors is related to different methodologies of TVM identification or a true distinction in the pattern of expression related to the tumor-specific microenvironment. We therefore also assessed the vascular expression of “breast” TVMs in the hESCT-HEY1 ovarian cancer model and the expression of “ovarian” TVMs within the hESCT-MCF7 breast cancer model. Interestingly, vascular expression of the “ovarian” TVMs F2RL1, GPR105, and EGFL6 was not detected in the hESCT-MCF7 breast cancer model (Fig. 2). Similarly the “breast” TVMs FAP and SFRP2 were not expressed in the vasculature of the hESCT-HEY1 ovarian tumors (Fig. 2). Rare vascular expression of the “breast” TVMs SLITRK6 and SMPD3 was detected in the hESCT-HEY1 ovarian tumor model (Fig. 2). These findings suggest that some TVMs are expressed in a cancer-specific manner and therefore likely induced by tumor cells, whereas others are more promiscuous and may identify angiogenic vessels or vasculogenesis.

Enhancing human vascular density in the hESCT cancer model

A primary goal of this study was to determine whether this model could be used to test anti-human TVM immunotherapeutics. However, initial studies showed that only a minority of resultant vessels (∼15%) were of human origin with the remainder being murine vessels (see below). To increase the use of the model for testing anti-vascular therapeutics, we attempted to increase the percentage of human tumor vessels in the hESCT cancer model. As fibroblasts in the ovarian tumor microenvironment can significantly promote angiogenesis (30), we co-injected hESCs and irradiated MEFs to create an hESCT in which to inject HEY1 ovarian cancer cells. Alternatively, hESCT + MEFs we co-injected with HEY1 ovarian cancer cells and 5,000 FACS-isolated VE-Cadherin+ primary ovarian tumor endothelial cells. Human CD31 IHC showed the greatest number of human vessels in tumors co-injected with MEFs and VE-Cadherin+ cells (Fig. 3A). Interestingly, while there were regions of the tumor, which had overlapping and interconnected human and murine vessels (Fig. 3B), most regions of the tumor were dominated by either human or murine vessels (data not shown). IHC analysis of these vessels confirmed the expression of TVMs (data not shown). Quantification of the vascular density of murine and human vessels using co-IF with human CD31 and murine CD31 revealed that while the hESCT-HEY1 ovarian cancer tumor model alone had about 15% human vessels, the addition of MEFs increased the percentage of human vessels to about 40% (P = 0.01; Fig. 3C). With the addition of VE-Cadherin+ tumor endothelial cells, nearly 80% of the tumor vessels were human (P < 0.0001; Fig. 3C). HEY1 cells co-injected with 5000 VE-Cadherin+ cells in Matrigel in the animals flank showed no human vessels (data not shown), showing that the profound human vascularity is unique to the hESCT model. To determine whether the increase in human vessels in the presence of VE-Cadherin+ cells was due to increased angiogenesis from the hESCT cells or proliferation of the VE-Cadherin+ cells, we repeated the above experiment using GFP-labeled H7 ESCs (Fig. 3D). Evaluation of tumor vessels showed that 60% to 80% of the human vessels were GFP and thus derived from the ovarian cancer VE-Cadherin+ cells, whereas the remaining 20% to 40% of human vessels were GFP+ and therefore derived from hESCT cells (Fig. 3E). These data show that the addition of human tumor vascular cells to this model leads to a dramatic increase in human tumor vasculature such that the majority of vessels present in the tumor are human in origin.

Figure 3.

Enhancing the number of human vessels in the hESCT cancer model. A, IHC of hCD31 in hESCT-HEY1, hESCT-HEY1-MEFs, hESCT-HEY1 + MEFs + VE-Cadherin+. B, IF showing interconnection of mouse and human vessels. C, quantification of mouse and human vessels in the hESCT cancer model alone, with MEFs, or with MEFS and VE-Cadherin+ cells. P values are indicated with error bars representing SDs. n = 4 animal/group. D, co-IF show hCD31 stain (red) in both hESCT-GFP cells (green), resulting in yellow hESC-derived vessels and non-GFP cells originating from VE-Cadherin isolated patient tumor endothelial cells (patient vessels). E, quantification of the percentage of hESCT-derived and patient tumor endothelial cell (TEC)-derived vessels.

Figure 3.

Enhancing the number of human vessels in the hESCT cancer model. A, IHC of hCD31 in hESCT-HEY1, hESCT-HEY1-MEFs, hESCT-HEY1 + MEFs + VE-Cadherin+. B, IF showing interconnection of mouse and human vessels. C, quantification of mouse and human vessels in the hESCT cancer model alone, with MEFs, or with MEFS and VE-Cadherin+ cells. P values are indicated with error bars representing SDs. n = 4 animal/group. D, co-IF show hCD31 stain (red) in both hESCT-GFP cells (green), resulting in yellow hESC-derived vessels and non-GFP cells originating from VE-Cadherin isolated patient tumor endothelial cells (patient vessels). E, quantification of the percentage of hESCT-derived and patient tumor endothelial cell (TEC)-derived vessels.

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Testing an anti-TVM therapeutic in the hESCT ovarian cancer model

To test the use of this model for screening anti-human TVM immunotherapeutics, we developed an immunotoxin targeting the human TVM THY1. This antigen was chosen due to the availability of commercial antibodies that can recognize the THY1 antigen in vivo. To create the immunotoxin, streptavidin-conjugated saporin toxin was coupled to a biotinylated anti-human THY1 antibody. The cytotoxicity of this immunotoxin was confirmed in vitro against THY1+ primary human MSCs; anti-THY1-immunotoxin resulted in statistically significant MSC death relative to antibody alone or saporin toxin alone controls (Fig. 4A). To test the efficacy of anti-TVM-immunotoxin in vivo, anti-THY1-saporin (n = 7 total in 2 experiments) immunotoxin or control rat IgG-saporin (n = 6 total in 2 experiments) was delivered intravenously to mice bearing hESCT-HEY1 DsRed tumors. Tumor growth was tracked with biofluorescent imaging. While rat IgG-saporin–treated tumors showed continued growth, THY1-saporin–treated hESCT-HEY1 ovarian tumors showed delayed growth and significant reduction in central tumor viability (the region dependent on human vessels; Fig. 4B and C). Following completion of therapy, growth resumed in peripheral tumor regions that were dependent on murine vessels continued to expand (Fig. 4B and data not shown). Control hESCT alone and HEY1-DsRED flank tumors showed no response to either therapy (data not shown).

Figure 4.

Testing anti-TVM therapeutics in the hESCT-HEY1 ovarian tumor model. A, quantification of cellular death of THY1 expressing MSC treated with anti-THY1-saporin immunotoxin and controls. B, biofluorescence of hESCT-HEY1 DsRed ovarian tumor before and after 2 treatments with anti-THY1-saporin immunotoxin arrows indicated time of treatment. C, biofluorescent images of hESCT-HEY1 DsRED tumors before and after treatment with the indicated immunotoxins. D, IHC images (1) and quantification (2) of human tumor vessels in control and anti-THY1-saporin–treated tumors. Error bars represent SDs.

Figure 4.

Testing anti-TVM therapeutics in the hESCT-HEY1 ovarian tumor model. A, quantification of cellular death of THY1 expressing MSC treated with anti-THY1-saporin immunotoxin and controls. B, biofluorescence of hESCT-HEY1 DsRed ovarian tumor before and after 2 treatments with anti-THY1-saporin immunotoxin arrows indicated time of treatment. C, biofluorescent images of hESCT-HEY1 DsRED tumors before and after treatment with the indicated immunotoxins. D, IHC images (1) and quantification (2) of human tumor vessels in control and anti-THY1-saporin–treated tumors. Error bars represent SDs.

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To further analyze the impact of anti-THY1 therapy on the human vessels, we quantified human tumor microvascular density using anti-hCD31 IHC. There was a 3-fold reduction in the number of human vessels in hESCT-HEY1 ovarian tumors treated with anti-THY1 saporin toxin compared with Rat IgG-saporin toxin–treated controls (Fig. 4D). These results confirm the use of this model for testing human TVM-specific therapeutics in vivo.

The hESCT model promotes the in vivo growth of primary human cancer stem cells

Human ovarian CSCs have been particularly challenging to grow in vivo. Engraftment rates of CSCs directly isolated from human ovarian tumors are only 20% to 40% in traditional flank tumor models and 5,000 CSCs typically require 6 to 12 months to create a tumor (26). We hypothesized that the human microenvironment of the hESCT model with a human tumor vascular niche could greatly enhance the growth of ovarian CSCs. Using ALDH as an ovarian CSC marker (26), we assessed the efficiency of primary human ovarian CSC engraftment in the hESCT tumor model. We injected FACS-isolated ALDH+ primary human ovarian CSCs (700–10,000) from 6 ovarian cancer patient samples into either hESCTs or subcutaneously. hESCTs were allowed to grow until they reached about 2,000 mm3 (4–12 weeks after tumor cell injection). Histochemical analysis of resected hESCT-ALDH+ CSCs showed regions consistent with papillary serous tumor growth (Fig. 5A and data not shown). To confirm these areas represent ovarian tumor cells, we exploited the recent finding that p53 is mutant in more than 95% of serous ovarian tumors (31). p53 IHC clearly identified human p53+ serous ovarian tumors in all hESCTs injected with ALDH+ ovarian cancer cells (Fig. 5A and C). Strong p53 stain was not identified in hESCTs alone or from hESCTs injected with ALDH+ cells from a benign fibroadenoma (data not shown). ALDH+ ovarian cancer cells injected subcutaneously in the flank showed no growth during this time period. Finally, we repeated this experiment, directly comparing the growth within hESCTs of ALDH+ and ALDH cells within from 3 patients. hESCTs injected with ALDH+ CSCs showed much more rapid growth than hESCTs injected with paired ALDH cells, indicating likely CSC engraftment in hESCTs (Fig. 5B). Once again, p53 IHC of resected hESCT-ALDH+ CSC tumors showed stain in regions consistent with papillary serous tumor growth (Fig. 5C). No p53 stain was noted in any of the hESCT-ALDH cell tumors, thus the “tumors” that grow in the ALDH hESCT represent benign teratoma growth. These data show primary ovarian CSC engraft in the human hESCT microenvironment more efficiently than in murine subcutaneous tissue.

Figure 5.

Growth of primary ovarian CSCs using the hESCT model. A, p53 IHC showing ovarian cancer cells initiated by ALDH+ CSCs injected within the hESCTs. hESCT alone and ALDH+ cells from a patient with a benign fibroadenoma showed no growth. B, hESCT ovarian tumor volumes from hESCTs injected with 10,000 ALDH+ or ALDH ovarian cancer cells from 3 patients. C, p53 IHC of hESCT injected with ALDH cancer cells and ALDH+ cancer cells showing p53+ papillary serous tumor growth from ALDH+ tumor only.

Figure 5.

Growth of primary ovarian CSCs using the hESCT model. A, p53 IHC showing ovarian cancer cells initiated by ALDH+ CSCs injected within the hESCTs. hESCT alone and ALDH+ cells from a patient with a benign fibroadenoma showed no growth. B, hESCT ovarian tumor volumes from hESCTs injected with 10,000 ALDH+ or ALDH ovarian cancer cells from 3 patients. C, p53 IHC of hESCT injected with ALDH cancer cells and ALDH+ cancer cells showing p53+ papillary serous tumor growth from ALDH+ tumor only.

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These data show that the hESCT cancer model expresses bona fide human tumor vessels. These vessels express not only the expected human vascular markers such as CD31 but also the tumor-type–specific TVMs such as EGFL6 and TEM7. A central rationale for the development of such model system is for the testing of novel vascular-targeted therapeutics. A major challenge for developing antibody-based therapies targeting tumor vessels has been the lack of an animal model with a human tumor microenvironment and human vessels; antibodies targeting human antigens cannot be tested in traditional animal tumor models unless the antibodies happen to cross-react between species. The model advanced here addresses this issue and thus allows screening of potential immunotherapeutics targeting human vascular antigens. Similarly, this model can also be used to test the in vivo binding activity of vascular-targeted peptides (23, 30, 32).

The generation of large numbers of human tumor vessels in this model required the addition of VE-Cadherin+ human tumor vascular cells. Interestingly, the addition of only 5,000 vascular cells led to nearly 80% human tumor vessels, about 70% of which were not derived from hESCs, suggesting that the human VE-Cadherin+ cells are proliferating within the hESCTs. Importantly, the human vessels in this model persisted throughout the period of tumor growth (4–12 weeks after cancer cell injection).

The need to add freshly isolated human tumor vascular cells could limit the widespread use of this model. However, a significant number of human vessels (∼40%) could still be generated in the absence of human tumor vascular cells with the addition of irradiated mouse embryonic fibroblasts. It is possible that the addition of other pro-angiogenic cells such MSCs (33) or tumor-associated myeloid cells (34) could further increase the percentage of human vessels. One limitation for therapeutic testing with this model, as with other murine tumor models of human vasculature, is that tumors are still ultimately dependent on the murine vasculature for blood flow. Therefore, even with the complete therapeutic elimination of the human tumor vessels, tumors regions supplied by the murine tumor vasculature will continue to grow.

While we used our model to confirm anti-vascular therapeutics, it can also potentially be used to test vascular imaging agents specifically targeting human vessels. This model allows testing the sensitivity of these compounds to detect tumor vasculature of “early-stage” tumors. We believe that this murine model offers a means to investigate the basic biology of human TVMs in vivo. Specifically, the mechanism of tumor-specific TVM induction could be addressed, due to the power to independently modulate the expression patterns of the hESC and the cancer cells themselves. This may be particularly relevant given that we observed differential induction of some tumor-specific TVMs by breast versus ovarian cancer cell lines.

Finally, the above data show that this model permits direct engraftment of primary human CSCs in a manner more efficient than subcutaneous injections. This expands upon and is consistent with previous reports showing improved growth of primary ovarian cell lines within hESCTs as compared with tumor flanks (21, 22). Our previous studies using flank models for the engraftment of ALDH+ ovarian CSCs showed engraftment rates of about 20% and tumor growth required 6 to 12 months (26). Using the hESCT model, we found 100% engraftment from as few as 700 ALDH+ primary human CSC within 4 to 12 weeks of tumor cell injection within the hESCTs. This model therefore represents a new tool to enhance the efficiency of the study of primary human CSCs. This model could potentially be further improved with the addition of cancer-associated MSCs (35). These findings emphasize the importance of the interplay between the tumor and the surrounding microenvironment and will allow a dissection of the signals within the tumor microenvironment that support cancer stem cell survival, proliferation, and differentiation.

We have confirmed the expression of human TVMs in a murine tumor model with robust human tumor vasculature. Importantly, many of these TVMs appear to be tumor-type–specific, indicating a tumor niche–dependent induction of these TVMs. This model is a useful tool to study therapeutics targeting human tumor vessels. In addition, this model with its unique human tumor microenvironment allowed 100% engraftment of primary human CSCs. The hESCT system will allow deeper probing of the role of the microenvironment-dependent induction of TVMs, their role in tumor biology and interactions in the tumor vascular/cancer stem cell niche.

No potential conflicts of interest were disclosed.

Conception and design: D. Burgos-Ojeda, K. McLean, S. Bai, R.J. Buckanovich

Development of methodology: D. Burgos-Ojeda, K. McLean, S. Bai, Y. Gong, I. Silva, K. Skorecki, M. Tzukerman, R.J. Buckanovich

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): D. Burgos-Ojeda, K. McLean, S. Bai, H. Pulaski, Y. Gong, I. Silva, R.J. Buckanovich

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): D. Burgos-Ojeda, R.J. Buckanovich

Writing, review, and/or revision of the manuscript: D. Burgos-Ojeda, K. McLean, S. Bai, K. Skorecki, R.J. Buckanovich

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): R.J. Buckanovich

Study supervision: R.J. Buckanovich

The authors thank the members of the UMCCC Flow Cytometry and Histology Cores for assistance in the experiments in the manuscript.

This work was initiated with support of the Damon Runyon Cancer Research Foundation and completed with the support of the NIH New Investigator Innovator Directors Award grant #00440377. Breast cancer studies were supported by the University of Michigan Cancer Center Support Grant CA046592. D. Burgos-Ojeda was supported by the NIH Cellular and Molecular Biology Training Grant T32-GM07315. K. Skorecki and M. Tzukerman receive research grant support from the Israel Science Foundation, and the Daniel Soref and Richard Satell Foundations at the American Technion Society.

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.

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