Targeting tumors with a killer-reporter adenovirus for curative fluorescence-guided surgery of soft-tissue sarcoma.

Fluorescence-guided surgery (FGS) of cancer is an area of intense interest. However, FGS of cancer has not yet been shown to be curative due to residual microscopic disease. Human fibrosarcoma HT1080 expressing red fluorescent protein (RFP) was implanted orthotopically in the quadriceps femoris muscle of nude mice. The tumor-bearing mice were injected with high and low-dose telomerase-dependent, green fluorescent protein (GFP)-containing adenovirus OBP-401, which labeled the tumor with GFP. Fluorescence-guided surgery (FGS) or bright light surgery (BLS) was then performed. OBP-401 could label soft-tissue sarcoma (STS) with GFP in situ, concordant with RFP. OBP-401-based FGS resulted in superior resection of STS in the orthotopic model of soft-tissue sarcoma, compared to BLS. High-dose administration of OBP-401 enabled FGS without residual sarcoma cells or local or metastatic recurrence, due to its dual effect of cancer-cell labeling with GFP and killing. High-dose OBP-401 based-FGS improved disease free survival (p = 0.00049) as well as preserved muscle function compared with BLS. High-dose OBP-401-based FGS could cure STS, a presently incurable disease. Since the parent virus of OBP-401, OBP-301, has been previously proven safe in a Phase I clinical trial, it is expected the OBP-401-FGS technology described in the present report should be translatable to the clinic in the near future.


IntroductIon
Fluorescence-guided surgery (FGS) of cancer is an area of broad and intense interest [1]. In the clinic, sentinel lymph nodes have been labeled by the near-infrared (NIR) fluorescing dye indocyanine [2]. However, indocyanine does not specifically label tumor cells. 5-aminolevulinic acid (5-AA), a precursor of hemoglobin has been used to label malignant glioma with significant progressionfree survival benefit, but was not curative [3]. Folate conjugated to fluorescein isothiocyanate (FITC) was used for targeting folate receptor-α (FR-α) in ovarian cancer patients whereby deposits less than 1 mm could be resected but was not shown to be curative [4]. Urano et al. used fluorescence-guided laparoscopy to visualize and remove tumors illuminated by the probe, γGlu-HMRG. www.impactjournals.com/oncotarget invasive human ovarian cancer, which in a mouse model, expresses the probe-activating GTT enzyme, but cures were not demonstrated [5].
We previously reported the effectiveness of FGS to improve outcomes in retroperitoneal-implanted nude mouse model of human fibrosarcoma, expressing green fluorescent protein (GFP). Although tumor recurrence was reduced and disease-free survival (DFS) increased, the procedure was not curative [6].
In another study from our laboratory, after FGS of a human pancreatic cancer cell line expressing green fluorescent protein (GFP) in an orthotopic nude-mouse model, the surgical resection bed was irradiated with UVC. FGS-UVC-treated mice had increased DFS and overall survival (OS) compared to FGS-only treated mice; with DFS lasting at least 150 days, indicating the animals were cured.
GFP has been used for labeling tumors in situ for FGS. Kishimoto et al. [7] selectively-labeled tumors with GFP using a telomerase-dependent adenovirus (OBP-401) that expresses the gfp gene only in cancer cells, which generally express the telomerase enzyme in contrast to normal cells. The labeled tumors could then be resected under fluorescence guidance. Tumors that recurred after FGS maintained GFP expression [8]. Because the recurrent cancer cells stably express GFP, detection of cancer recurrence and metastasis is also possible with OBP-401 GFP labeling, in contrast to fluorescent-antibody or other non-genetic labeling. Because tumors of all types express telomerase, the genetic labeling method that uses a telomerase-dependent adenovirus to deliver GFP specifically to tumors offers the potential of widespread application, since cancers of all types express telomerase.
We report here FGS in combination with OBP-401 is curative, of an STS in an orthotopic model, with preservation of walking function.

GFP-expressing adenovirus obP-401 labels and kills sarcoma cells in vitro
Time-lapse imaging demonstrated that OBP-401 labeled RFP-expressing HT1080 sarcoma cells with GFP ( Figure 1A). OBP-401 labeled HT1080 cells with GFP in a dose-dependent manner ( Figure 1B). GFP fluorescence, after OBP-401 infection of HT1080 cells, became stronger each day from day 2 to day 7 ( Figure 1A). OBP-401 also killed HT1080 cells with GFP in a dose-dependent manner ( Figure 1C). These data indicated that OBP-401 labeled sarcoma cells with GFP, and could subsequently kill them in vitro.

orthotopic sarcoma model
An orthotopic sarcoma model was developed with HT1080 RFP cells implanted in the quadriceps femoris muscles. The orthotopically-growing sarcoma cells invaded the quadriceps femoris muscles and femoral bone similar to the clinical course of STS. Tumor growth was visualized by RFP fluorescence.

bright-light surgery results in remaining sarcoma cells in the orthotopic model
We performed bright-light surgery (BLS) on the orthotopic sarcoma model (Figure 2A). Because the sarcoma invaded muscles and the femoral bone, the tumor margin was invisible under bright light. Extensive RFPexpressing sarcoma cells remained after BLS (Figure 2A and 2D).

obP-401-FGs detects and resects residual sarcoma cells in the orthotopic sts model after bls
OBP-401 was intratumorally injected 3 days before BLS in HT1080-RFP tumors growing in the quadriceps femoris muscle ( Figure 3A). After BLS, both RFP and GFP fluorescence were detected in the surgical bed ( Figure  3B). OBP-401 enabled detection of the residual cancer cells at the single cell level ( Figure 3B). After OBP-401-FGS, there were no residual cancer cells ( Figure 3B). OBP-401 delineated the precise margin between cancer and normal tissue and whether there were residual cancer cells at the single-cell level ( Figure 3C).

obP-401 based FGs resulted in recurrence-free surgery
We compared the rate of local recurrence after OBP-401-FGS with BLS alone. Fluorescence imaging showed that 8 of 10 mice that underwent BLS had RFP-expressing, small, hard nodules 3 days after surgery, indicating that these mice had local recurrences ( Figure 5A and 5B). In contrast there was no local recurrence in eight mice which received OBP-401-FGS ( Figure 5A-5C, Table 1A). These data demonstrated that tumor visualization at the singlecell level by OBP-401 enabled complete resection and prevented local recurrence.

obP-401-FGs enables minimally invasive, function-preserving surgery for sarcoma
Next, we confirmed whether high-dose OBP-401 (2×10 8 PFU) FGS enabled minimally invasive surgery of sarcoma for preservation of muscle compared with BLS or low-dose FGS ( Figure 5A). We injected high-dose OBP-401 into tumors in the orthotopic sarcoma model. High-dose injection of OBP-401 significantly reduced the size of tumors and inhibited tibial invasion compared with control tumor or low-dose injection of OBP-401 ( Figure  4A and 4B). We minimally resected tumors without residual disease under FGS, after high-dose injection of OBP-401 ( Figure 4C). Six of 8 mice lost muscle function after FGS with low-dose OBP-401. In contrast, all mice preserved muscle function after FGS with high-dose OBP-401 ( Figure 4D). These data suggested that OBP-401-based FGS enabled muscle function preservation for sarcoma.

Inhibition of lung metastasis after high-dose obP-401 and FGs
Eight of 10 mice received BLS had large lung metastases observed at necropsy ( Figure 6A-C). Three of 9 mice which received low-dose OBP-401-FGS had small metastasis in the lung ( Figure 6, Table 1B). In contrast, none of the 10 mice which received high-dose OBP-401www.impactjournals.com/oncotarget   FGS had any lung metastasis ( Figure 6, Table 1B). Highdose OBP-401-FGS significantly prolonged the overall survival rate compared with BLS alone ( Figure 6D). These data indicated that high-dose OBP-401-based FGS controlled local recurrence and distant metastasis, thereby prolonging the survival rate.
Labeling tumors with OBP-401 has none of the weaknesses of non-genetic labeling, particularly loss of label over time and limited expression of the marker used for labeling. OBP-401 may be a general method for labeling tumors that express telomerase, which are the vast majority, and could have broad application for FGS.
The results of the present study suggest that OBP-401-based FGS has clinical potential. An appropriate starting point would be sarcoma which is often superficial and amenable to i.t. injection of OBP-401 as was done in the present study, thereby, reducing potential for systemic toxicity. Administration via the i.t. route enables the potential of simple fluorescence imaging of the tumor with portable equipment [19] even in the patient to determine optimal labeling time, as well as dose, before FGS.
The present study used both low-dose (1×10 8 PFU) and high-dose (3×10 8 PFU) OBP-401. Clinical studies can start with low-dose OBP-401 and proceed to high-dose after safety is demonstrated.

GFP-expressing telomerase-specific adenovirus
In OBP-401 the promoter element of the human telomerase reverse transcriptase (hTERT) gene drives the expression of E1A and E1B genes, linked to an internal ribosome entry site, for selective replication only in cancer cells. The GFP gene is driven by the CMV promoter inserted in OBP-401 [29].

cell line and cell culture
The human sarcoma cell line HT1080 expressing RFP (HT1080-RFP) [30] was maintained and cultured in DMEM medium with 10% fetal bovine serum (FBS) and 5% penicillin/streptomycin.

In vitro or ex vivo imaging
Time-course images of OBP-401 labeled HT1080 sarcoma were acquired with an FV1000 confocal laserscanning microscope (Olympus, Tokyo, Japan) [31]. For whole-body or whole-tumor imaging, the OV100 Small Animal Imaging System (Olympus), was used [32].

Animal experiments
Athymic (nu/nu) nude mice (AntiCancer, Inc., San Diego) were kept in a barrier facility under HEPA filtration. Mice were fed with an autoclaved laboratory rodent diet (Tecklad LM-485, Western Research Products). All animal studies were conducted in accordance with the principles and procedures outlined in the National Institutes of Health Guide for the Care and Use of Laboratory Animals under Assurance Number A3873-01. orthotopic sarcoma model RFP-expressing HT1080 cells (3 × 10 6 ) suspended in Matrigel (20 uL) were inoculated into the left quadriceps femoris muscle of 5 weeks old female athymic nude mice [33]. Tumor progression was monitored by noninvasive fluorescence imaging (OV100).

OBP-401 based fluorescence-guided surgery (obP-401-FGs)
All animal procedures were performed under anesthesia using s.c. administration of a ketamine mixture [10 μl ketamine HCl, 7.6 μl xylazine, 2.4 μl acepromazine maleate, and 10 μl PBS]. Orthotopic STS tumors labeled with GFP by OBP-401 were observed with noninvasive fluorescence imaging (OV100) before FGS was performed. After surgery, the presence of cancer cells was observed with the OV100. If there were residual cancer cells, an additional resection was performed.

statistical analysis
Data are shown as means ± SD. For comparison between two groups, significant differences were determined using the Student's t-test. For comparison of more than two groups, statistical significance was determined with a one-way analysis of variance (ANOVA) followed by a Bonferroni multiple group comparison test. Pearson chi-square analysis was used to compare the rate of local recurrence between BLS and OBP-401-FGS. Statistical analysis for disease-free survival and over-all survival was performed using the Kaplan-Meier test along with log-rank test. Pearson chi-square analysis was used to evaluate the rate of local recurrence and lung metastasis between BLS and OBP-401-FGS and the extent of limb preservation between low-dose OBP-401-FGS and highdose OBP-401-FGS. P values of < 0.05 were considered significant.