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. 2014 Mar;21(3):289-97.
doi: 10.1038/gt.2013.84. Epub 2014 Jan 16.

In vivo safety, biodistribution and antitumor effects of uPAR retargeted oncolytic measles virus in syngeneic cancer models

Y Jing  1 , J Zaias  2 , R Duncan  3 , S J Russell  4 , J R Merchan  1
Affiliations

In vivo safety, biodistribution and antitumor effects of uPAR retargeted oncolytic measles virus in syngeneic cancer models

Y Jing et al. Gene Ther. 2014 Mar.

Abstract

The urokinase receptor (uPAR) is a clinically relevant target for novel biological therapies. We have previously rescued oncolytic measles viruses fully retargeted against human (MV-h-uPA) or murine (MV-m-uPA) uPAR. Here, we investigated the in vivo effects of systemic administration of MV-m-uPA in immunocompetent cancer models. MV-m-uPA induced in vitro cytotoxicity and replicated in a receptor-dependent manner in murine mammary (4T1) and colon (MC-38 and CT-26) cancer cells. Intravenous administration of MV-m-uPA to 4T1 tumor-bearing mice was not associated with significant clinical or laboratory toxicity. Higher MV-N RNA copy numbers were detected in primary tumors, and viable viral particles were recovered from tumor-bearing tissues only. Non-tumor-bearing organs did not show histological signs of viral-induced toxicity. Serum anti-MV antibodies were detected at day 14 of treatment. Immunohistochemistry and immunofluorescence studies confirmed successful tumor targeting and demonstrated enhanced MV-m-uPA-induced tumor cell apoptosis in treated compared with control mice. Significant antitumor effects and prolonged survival were observed after systemic administration of MV-m-uPA in colon (CT-26) and mammary (4T1) cancer models. The above results show safety and feasibility of uPAR targeting by an oncolytic virus, and confirm significant antitumor effects in highly aggressive syngeneic immunocompetent cancer models.

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Conflict of interest statement

Conflict of interest: The authors declare no conflict of interest.

Figures

Figure 1
Figure 1. In vitro viral infection, cytotoxicity and replication by MV-m-uPA in murine cancer cells
(A) uPAR expression in mouse cancer cells MC-38, CT-26, 4T1 and B16F10 was assessed by FACS, using murine anti-uPAR monoclonal antibodies (filled histograms) or isotype controls (open histograms). (B, C) Mouse cancer cells were infected with MV-m-uPA as indicated at an MOI= 1 and photographed 48 h after infection. Representative pictures of infected cells (B: light; C: fluorescence). Scale bar = 500 μm. Arrows indicate areas of virus induced syncytia. (D) In vitro cytopathic effects of MV-m-uPA. Murine cancer cells were infected with MV-m-uPA at an MOI=1 and viability was determined at different time points (48h, 72h, and 96h) by trypan blue exclusion and presented as percentage of controls. Bars represent averages +/- SD of triplicate experiments, p < 0.001. (E) MC-38, CT-26, and 4T1 cells were infected with MV-m-uPA (MOI = 3) and titers of virus were determined at different time points by the one-step growth curve.
Figure 2
Figure 2. In vivo biodistribution after systemic administration of MV-m-uPA
The orthotopic 4T1 tumor model was established in immunocompetent female Balb/c mice. Animals were treated and tissues processed as described in the methods section (n=5 mice per time point). At days 2, 5 and 28 days post-treatment, total RNA was extracted from frozen tumors (A), organs (B-F) and urine (G) for MV-N mRNA quantification by qRT-PCR. H. Blood samples were obtained for MV-N RNA quantification at days 2, 5, 14 and 28 after treatment (n=5 per time point). Results were expressed as copies of MV-RNA/μg of total RNA in each organ/tissue, and horizontal bars represent the mean value of the replicates. (I) Determination of serum anti-MV antibody. Serum was obtained from treated mice at 7 (no antibody detected), 14 and 28 days after treatment (n=3 per time point) for antibody determination (see methods section for details).
Figure 3
Figure 3. Histologic analysis of tumors and organs of mice treated with MV-m-uPA
(A). 4T1 tumor bearing Balb/c mice (n = 5) were given 2 doses of 1.5×106 TCID50 of MV-m-uPA or PBS via tail vein. Mice were sacrificed 5 days after virus treatment and primary tumors and major organs (lung, heart, liver, spleen and brain) were removed for histological analysis (H&E). Arrows indicate the necrotic and inflammatory areas. White arrowheads (liver) represent tumor foci. Scale bar = 400 μm. (B). Effects of MV-m-uPA in the liver of tumor bearing and tumor free mice (n=5 per group). Virus treatment and tissue processing was performed as in methods section. Representative pictures of livers in the 3 groups. Left: Tumor bearing mice treated with PBS (micrometastases detected in 5/5 mice). Center: Tumor free mice treated with virus. Right: Tumor bearing mice treated with virus (micrometastases detected in 2/5 mice, picture is shown from a mouse with positive micrometastases). Note micrometastatic foci (white arrowheads) and areas of inflammation (black arrows). Scale bar = 200 μm.
Figure 4
Figure 4. Tumor Targeting, antibody production, and induction of apoptosis in vivo
(A, B) Recovery of viable viral particles from tumor, liver, lung, heart, spleen and brain from mice at day 5 (A) and Day 28 (B) after virus treatment (n=5 mice per group). Tissues were processed and assays were preformed as in methods. Viral titers are displayed as TCID50/gram of tissue . Arrow represents the assay's limit of detection-LOD (1.26×102 TCID50/gram of tissue). (C) Immunocompetent (Balb/c) mice (n = 3 per group) bearing 4T1 tumors received two intravenous injections of either PBS or MV-m-uPA (1.5×106 TCID50). Tumors were harvested 3 days later and frozen tumor sections were used for immunostaining for measles N protein. Viral protein was detected in tumors after intravenous administration of the virus. TUNEL assay of tumors from mice treated with MV-m-uPA or PBS was performed as in materials and methods (n=3 per group). Scale bar = 200 μm. (D) Quantitative analysis of TUNEL–positive nuclei in 4 microscopic fields per section per sample (displayed as percentage of positive/total nuclei; n = 3 per group). *, P < 0.01, MV-m-uPA versus ctrl.
Figure 5
Figure 5. In vivo antitumor effects and tumor targeting in mammary and colon cancer models
(A). 4T1 cells were implanted into the mammary fat pad of female Balb/c mice. When tumors reached a mean diameter of 0.4-0.5 cm, the animals (8 per group) were treated with total doses of 1×105, 1×106, 1×107, 5×107 TCID50 of MV-m-uPA, or equal volumes of PBS (control group). Total doses were split in three separate administrations, given via tail vein every other day × 3. Tumor growth was monitored as in methods. * p < 0.0001, day 17, MV-m-uPA (5×107) vs. control. Arrow indicates the time (day) of MV treatment, relative to time of tumor cell implantation. (B). Kaplan-Mier analysis of survival of tumor bearing mice treated with vehicle control or MV-m-uPA. Mice were monitored until they reached sacrifice criteria (see materials and methods). MV-m-uPA 5×107 vs. control ** p=0.0006. (C). Murine colon cancer (CT-26) cells were implanted into the right flank of female Balb/c mice. When the tumors reached a mean diameter of 0.4-0.5 cm, the animals (8 per group) were treated and followed as in the 4T1 model. *** p < 0.0001: day 21, MV-m-uPA (5×107) vs. control. Arrow indicates the time (day) of MV treatment, relative to time of tumor cell implantation. (D). Kaplan-Mier analysis of survival of tumor bearing mice treated with vehicle control or MV-m-uPA. There was a significant prolongation of survival in the MV-m-uPA treatment group compared with control. **** p=0.0297, MV-m-uPA 5×107 vs. control.
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