Tumor stroma engraftment of gene-modified mesenchymal stem cells as anti-tumor therapy against ovarian cancer

Abstract

Background aims: Many ovarian cancers originate from ovarian surface epithelium, where they develop from cysts intermixed with stroma. The stromal layer is critical to the progression and survival of the neoplasm and consequently is recruited into the tumor microenvironment.

Methods: Using both syngeneic mouse tumors (ID8-R) and human xenograft (OVCAR3, SKOV3) tumor models, we first confirmed that intraperitoneally injected circulating mesenchymal stem cells (MSCs) could target, preferentially engraft and differentiate into α-smooth muscle actin-positive myofibroblasts, suggesting their role as "reactive stroma" in ovarian carcinoma development and confirming their potential as a targeted delivery vehicle for the intratumoral production of interferon-β (IFN-β). Mice with ovarian carcinomas then received weekly intraperitoneal injections of IFN-β expressing MSCs.

Results: Intraperitoneal injections of IFN-β expressing MSCs resulted in complete eradication of tumors in 70% of treated OVCAR3 mice (P = 0.004) and an increased survival of treated SKOV3 mice compared with controls (P = 0.01). Similar tumor growth control was observed using murine IFN-β delivered by murine MSCs in ID8-R ovarian carcinoma. As a potential mechanism of tumor killing, MSCs produced IFN-β-induced caspase-dependent tumor cell apoptosis.

Conclusions: Our results demonstrate that ovarian carcinoma engrafts MSCs to participate in myofibrovascular networks and that IFN-β produced by MSCs intratumorally modulates tumor kinetics, resulting in prolonged survival.

Figure 1. IFNβ and MSC-IFNβ inhibit proliferation of ovarian carcinoma cells in vitro

(A) OVCAR3, (C) SKOV3, (E) HEY (G) ID8-R (C57BL/6J MOSEC, treated with murine IFNβ cells were cultured in the presence of increasing concentrations of IFNβ. The effect of IFNβ is expressed as proliferation relative to control cells that were not exposed to the IFNβ. Results are represented as the mean ± SEM. (B) OVCAR3, (D) SKOV3, (F) HEY and (H) ID8-R cells were co-cultured with MSC-βgal (mMSC-ffLuc for murine model) or MSC-IFNβ (mMSC-mIFNβ for murine model) in a 10:1 ratio. Cells were counted, and their relative number in co-cultures was determined by flow cytometry. Results (mean ± SEM) are expressed as the percentage of control cells (cultured alone).

Figure 2. Pharmacokinetics and mechanisms of IFNβ based killing

(A) Determination of IFNβ serum levels after an IP injection of MSC-IFNβ (black line) versus an IP injection of 40,000IU IFNβ (grey line). After IP injection of 5×10⁵ MSC-IFNβ blood was drawn at various time points and serum was assessed for IFNβ via ELISA. (B) OVCAR3 or (C) SKOV3 cells were co-cultured with MSC-IFNβ, recombinant IFNβ protein or MSC alone in the presence or absence of a pan-caspase inhibitor. Mitochondrial membrane potential (ΔΨM) and externalization of phosphatydil serine were quantified by flow cytometery.

Figure 3. Fate of engrafted MSC in the ovarian tumor microenvironment

Fate of MSC engrafted in SKOV3 ovarian tumors in vivo was determined by immunohistochemistry on tumor sections. Frozen tissue sections harvested from day 82 ovarian carcinomas that were exposed to IP-circulating MSC or negative control ( IP injected PBS) were stained with human anti-α-smooth muscle actin and anti-desmin to show human myofibroblast incorporation within the tumor sections. FAP and FSP staining within the tumor sections depicts the activated fibroblasts within the tumor microenvironment. Dark red-brown staining indicates a positive result.

Figure 4. IP administration of MSC-IFNβ significantly increases survival in mice with ovarian carcinomas

Mice with established ovarian carcinomas (n=10 for each cell line) were treated with five once weekly IP injections of 5×10⁵ MSC-IFNβ or MSC-βgal. (A) Kaplan-Meier survival curves for OVCAR3 mice. (B) Survival curves for SKOV3 mice. (C) To detect intratumoral IFNβ, OVCAR3 or SKOV3 tumor sections (10×) one and three days post IP injection of 1×10⁶ MSC-IFNβ were stained with anti-IFNβ antibody as described in the methods. Dark red-brown staining indicates production of IFNβ by MSC. Densitometric quantitation of stained tumor sections was performed to assess levels of IFNβ production and integrated density values are displayed as percentage above background in the lower left corner of images.

Figure 5. Assessment of MSC engraftment and therapy for ID8-R ovarian carcinomas in a syngenic C56BL/6J mouse model by bioluminescent imaging

BLI images of mice bearing ID8-R tumors: addition of renilla-specific substrate afford detection of ID8-R tumor, whereas addition of D-luciferin allows specific detection of firefly luciferase expressing mMSC. (A) Bioluminescence of ID8-R tumors (RENLuc) in mice that received 5 (1×/week) of 1×10⁶ mMSC-mIFNβ at day 53. (B) Bioluminescence of ID8-R tumor (RENLuc) in control mice (no treatment-tumor alone) at day 53. (C) Bioluminescence of ID8-R tumor (RENLuc) in mice that received 5 (1×/week) of 1×10⁶ mMSC-FFLuc (mMSC control) at day 53. (D) Detection of mMSC-FFLuc (Firefly-specific substrate) in mice that received 5 (1×/week) of 1×10⁶ mMSC-ffLuc, taken on day 54, one day after panel C was imaged. (E) Tumor growth over time as assessed by average photon counts/tumor within each group shows a significant decrease in tumor size by BLI of the MSC-IFNβ treated mice compared to the control. (F) Detection of selected co-localization of either mMSC or mouse embryonic fibroblasts (MEF) in vivo. C56Bl/6J mice (n=5) with 58 days established ID8-R ovarian tumors (F-1, F-3) were injected with either 5 × 10⁵ C56Bl/6J MEF(F-2) or mMSC (F-4). Mice were imaged on day 15 post-injection to detect either tumor (renilla-specific), or treatment (firefly-specific). A representative mouse from each group is shown.