A novel model for evaluating therapies targeting human tumor vasculature and human cancer stem-like cells

Abstract

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.

Figure 1. Validation of human vasculature in the hESCT-cancer model

(A) Co-IF demonstrating 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, hESCT, and hESCT-cancer models.

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 non-tumor 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 two experiments. Black arrow indicates vessel containing red blood cells.

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 inter-connection 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 standard deviations. n=4 animal/group. (D) Co-IF demonstrating 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 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 two 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 (2) quantification of human tumor vessels in control and Anti-THY1-saporin treated tumors. Error bars represent standard deviations.

Figure 5. Growth of primary ovarian CSCs using the hESCT model

(A) p53 IHC demonstrating ovarian cancer cells initiated by ALDH⁺ CSC injected within the hESCT. hESCT alone and ALDH⁺ cells from a patient with a benign fibroadenoma demonstrated no growth. (B) hESCT-ovarian tumor volumes from hESCT injected with 10,000 ALDH⁺ or ALDH⁻ ovarian cancer cells from three patients. (C) p53 IHC of hESCT injected with ALDH⁻ cancer cells and ALDH⁺ cancer cells demonstrating p53⁺ papillary serous tumor growth from ALDH⁺ tumor only.