In vivo anti-metastatic effects of uPAR retargeted measles virus in syngeneic and xenograft models of mammary cancer

Abstract

Purpose The urokinase receptor (uPAR) plays a critical role in breast cancer (BC) progression and metastases, and is a validated target for novel therapies. The current study investigates the effects of MV-uPA, an oncolytic measles virus fully retargeted against uPAR in syngeneic and xenograft BC metastases models. Methods In vitro replication and cytotoxicity of MVs retargeted against human (MV-h-uPA) or mouse (MV-m-uPA) uPAR were assessed in human and murine cancer and non-cancer mammary epithelial cells. The in vivo effects of species-specific uPAR retargeted MVs were assessed in syngeneic and xenograft models of experimental metastases, established by intravenous administration of luciferase expressing 4T1 or MDA-MD-231 cells. Metastases progression was assessed by in vivo bioluminescence imaging. Tumor targeting was evaluated by qRT-PCR of MV-N, rescue of viable viral particles and immunostaining of MV particles in lungs from tumor bearing mice. Results In vitro, MV-h-uPA and MV-m-uPA selectively infected, replicated and induced cytotoxicity in cancer compared to non-cancer cells in a species-specific manner. In vivo, MV-m-uPA delayed 4T1 lung metastases progression and prolonged survival. These effects were associated with identification of viable viral particles, viral RNA and detection of MV-N by immunostaining from lung tissues in treated mice. In the human MDA-MB-231 metastases model, intravenous administration of MV-h-uPA markedly inhibited metastases progression and significantly improved survival, compared to controls. No significant treatment related toxicity was observed in treated mice. Conclusions The above preclinical findings strongly suggest that uPAR retargeted measles virotherapy is a novel and feasible systemic therapy strategy against metastatic breast cancer.

Molecular Effects of Stromal-Selective Targeting by uPAR-Retargeted Oncolytic Virus in Breast Cancer

“…As expected, from prior reports by us [23, 24], MV-h-uPA and MV-m-uPA infected human MDA-MB 231 and murine 4T1 tumor cells in a species specific manner, while non-cancer epithelial cells (HMEC, NMuMG) were not susceptible to viral infection (Fig. 1).…”

“…Similar species specific effects were observed in human HT-29 and T47D, as well as murine CT-26 cancer cells. As previously reported [24], MV-h-uPA was more selective to human cancer than non-cancer epithelial cells (HMEC) than MV-GFP.…”

“…To detect and visualize apoptosis, slides were washed twice with PBS (after fixation), permeabilized with 0.2% TritonX-100 for 20 minutes at room temperature and after 2 additional washes with PBS, sections were probed with label solution (for negative controls) or TUNEL reaction mix, following manufacturer’s instructions ( In Situ Cell Death Detection Kit-TMR red; Roche Applied Science, Indianapolis, IN) [24, 26]. …”

Potential and clinical translation of oncolytic measles viruses

“…Retargeted viruses, via single-chain antibodies, have been developed, thus far, against tumor cell specific targets including: CD38, epidermal growth factor (EGFR), EGFR mutant vIII, alpha folate receptor [91–93], CD133 [94], insulin-like growth factor receptor 1 [93], CEA [95], CD20 [80], prostate-specific antigen [85], and the urokinase receptor [96,97]. MV strains displaying cytokines, such as IL-13 and thus targeting the glioma specific interleukin-13 receptor alpha2, were also successfully created [98].…”

In vivo antitumor activity by dual stromal and tumor-targeted oncolytic measles viruses

“…We have previously developed and characterized novel, species specific oncolytic MVs fully retargeted against the human or murine urokinase receptor (uPAR) [17,18], a GPI-anchored cell surface receptor, which is overexpressed in tumor and particularly in stromal cells, and whose role in tumor-stromal interactions and cancer progression are well established [19][20][21][22][23][24][25]. We demonstrated that systemic 10 3 10 4 10 5 10 6 10 3 10 4 10 5 10 6 10 3 10 4 10 5 10 6 10 3 10 4 10 5 10 6 10 3 10 4 10 5 10 6 10 3 10 4 10 5 10 6 10 2 10 3 10 4 10 5 10 2 10 3 10 4 10 5 10 2 10 3 10 4 10 5 10 2 10 3 10 4 10 5 10 2 10 3 10 4 10 5 10 2 10 3 10 4 10 5 10 2 10 3 10 4 10 5 administration of species specific, human (MV-huPA) or murine (MV-muPA) uPAR retargeted MVs is safe, successfully targets tumor tissues over non-cancer tissues, and is associated with significant tumor delaying effects in primary or metastatic, xenograft, and syngeneic cancer models, respectively [18,26]. Taking advantage of the species specificity of MV-muPA, we recently reported that (murine) stromal-selective MV-muPA was associated with direct stromal targeting in a human breast cancer xenograft (where human cancer cells are not permissive to the murine targeted virus), leading to modulation of murine stromal gene expression, indirect effects on tumor cell gene expression, and measurable in vivo tumor growth delay by MV-muPA [27].…”