Acute vascular response was observed in all three tumor types within two hours of administration of ATO IP. Non-invasive BLI and PD US each revealed vascular shutdown with considerable recovery 24 hr later. The effect was dose dependant in terms of fractional reduction in vascular perfusion and recovery.
All three imaging techniques (BLI, FLI, and US) accurately reported tumor growth by comparison with caliper measurements. The optical approaches have the advantage of revealing even sub-palpable volumes, though they do require that cells be transfected to express reporter genes. Tight correlation was observed between each of the techniques for measurements over a period of 30 days and volumes up to 200 mm3
). Measurements were highly reproducible in individual tumors as shown for repeat measurements following saline injection IP (). Correlation between FLI and BLI has been reported previously by others, but those studies used cell lines separately transfected to express luciferase of fluorescent protein, as opposed to the double transfection examine here. Notably, Choy et al.
compared MC38-GFP and MC38-luc murine colon adenocarcinoma and reported close correlation between the methods and caliper measured tumor volume. Caceres et al.
compared MCF7 cells transfected GFP or luciferase and found close correlation between BLI and FLI, but noted that BLI was superior for evaluating metastatic spread and deeper tumors. Others have reported correlation between BLI and tumor volume based on caliper measurements or MRI 
. Recently, it was reported that high resolution ultrasound provided more accurate assessment of tumor volume than calipers, noting that skin thickness can be difficult to estimate 
In common with previous reports, ATO caused dose dependant vascular shutdown 
. We found that a dose of ATO at 5 mg/kg caused reduced vascular perfusion and the effect was increased at 7, 8, or 10 mg/kg (), but the highest dose was often lethal within 24 hours. The response of PC3 and U87 tumors was quite similar, whereas MCF7 appeared more resistant with less effect at each time point (). Extensive previous literature has shown efficacy on cultured cells of each of the lines examined here. Exposure ≥2 µM ATO reportedly induced cell death by apoptosis in MCF7 cells 
. An IC50
2.5 µM was reported for PC3 cell growth and daily doses of 5 mg/kg IP significantly delayed growth of orthotopic PC3 tumors growing as xenografts in SCID mice 
. Doses of 5 mg/kg IP daily given to mice with SC U87 gliomas caused a significant tumor growth delay when combined with irradiation (5×250 cGy) within 4 hrs, but failed to reach a significant effect when given alone (p<0.07) 
Anti vascular effects of ATO were extensively reported based on uptake of intravenously injected 86
RbCl or clearance of 99m
following direct intra tumor injection 
. A dose of 8 mg/kg yielded a 50% decrease in tumor blood perfusion at 2 hrs and 6 hrs in SCK tumors in mice with substantial recovery by 24 hrs, while FSAII tumors were somewhat more sensitive with 80% decrease at 2 hrs and only 40% recovery at 24 hrs 
. A dose as low as 2 mg/kg caused 40% reduction of 86
Rb uptake in FSaII tumors at 2 hrs 
. However, ATO alone caused no tumor growth delay. Administration of ATO (8 mg/kg) 2 to 6 hrs before hyperthermia produced significantly enhanced tumor growth delay. 99m
Tc clearance could be observed by sequential radio counting of tumors over time, whereas 86
Rb uptake was based on sacrifice and analysis ex vivo
Non-invasive assessment of vascular integrity is inherently more attractive and vascular disruption has been reported for several tumor types growing in diverse locations in various rodents. Using Laser Doppler flowmetry Hines-Peralta et al
showed effects at doses as low as 1 mg/kg against VX2 tumors implanted intra renally in rabbits 
. Orthotopic mammary R3230 tumors in rats and subcutaneous RCC tumors in mice required somewhat higher doses with blood flow reduced by about 40% following 3.5 mg/kg. Some of this difference may have resulted from the site of implantation. Several reports have used a dose of 8 mg/kg 
, though others have used 10 mg/kg 
. Compared with the early studies based on uptake or clearance of radiolabels imaging requires far fewer tumor-bearing animals. More broadly Goertz et al.
demonstrated the use of high frequency PD US to evaluate VDAs 
. Power Doppler is technically more challenging than BLI, but has several potential advantages; notably, it avoids the need for luciferase expressing cells and is therefore applicable to primary human tumor xenografts, or indeed could be applied to human clinical trials. It also reveals 3D structure of vascular extent. Here, we applied PD US to the MCF7 tumors, which appear particularly well vascularized. In some tumor types vascular perfusion appeared too sluggish to effectively apply PD US even under baseline condition (e.g.
, MTLn3 growing in rat), but infusion of contrast microbubbles did reveal vasculature and impairment following VDA administration, notably CA1P 
Dynamic BLI is particularly facile to implement and allows high throughput analysis. Three to five mice may be observed simultaneously although each imaging session does require fresh administration of luciferin. Several reports indicate that IV infusion provides higher signal, though it is quite transient and requires the technical skill of IV tail vein injection on multiple, successive occasions 
. We favor subcutaneous administration in the foreback neck region, which gives highly reproducible BLI signals (), as also reported previously 
. Traditional BLI acquires images at a single time point post administration of luciferin, but acquiring the dynamic time course of signal evolution provides a more comprehensive perspective on vascular patency. We did note that the BLI signal had not reached the maximum value for the U87-luc-mCherry 7 tumor at 16 minutes (). Thus, we extended the acquisition time for MCF7-luc-mCherry, although in this case the maximum was found at about 10 minutes (, ).
Dynamic BLI was previously used to characterize vascular disruption of MDA-MB-231-luc tumors following administration of CA4P and observations were validated by reference to dynamic contrast enhanced MRI and histology 
. Here, comparative measurements were provided by PD US and confirmed using histology. In our previous work we had used a home-built BLI system 
, which allowed observation of only one mouse at a time. The commercial system makes signal acquisition and analysis much easier and allows up to five mice to be assessed simultaneously. In terms of practical application we also note that the price of luciferin has fallen considerably and luciferase expressing tumor cell lines are becoming more readily available.
Vascular shutdown was confirmed by histology based on distribution of the perfusion marker Hoechst 33342. Two hours after ATO administration (8 mg/kg) histology showed reduced perfusion (). Similarly reduced vascular perfusion was reported for TLT (transplantable mouse liver tumors) using Patent blue dye and tumor excision following 5 mg/kg ATO 
. We observed further reduced perfusion after 4 hrs, but extensive return after 24 hrs () matching the observations in vivo
(, ). Vascular impairment judged by BLI and PD US was found to be closely matched (). Fluorescent imaging showed no response up to six hours, as expected since no vascular delivery was involved and the mCherry protein is reported to have a half life of about 24 hrs 
. An alternative FLI approach is conceivable based on infusion of a vascular label; notably we have preliminary data indicating that so-called DyCE (dynamic contrast enhanced) FLI following infusion of indocyanine green (ICG) is sensitive to tumor perfusion and responsive to vascular disruption 
. Since ICG is approved for use in patients such an approach could be clinically feasible for superficial tumors and may be worth exploration.
Arsenic trioxide was used as a model agent here based on the reports that is causes acute vascular shutdown in solid tumors 
. Moreover, ATO (TRISENOX®) is FDA approved in the United States for treatment of relapsed and refractory acute promyelocytic leukemia (APL) patients 
and there are ongoing clinical trials for solid tumors including liver, brain, lung and breast cancers (ClinicalTrials.gov). Dose limiting toxicity has been widely reported to limit potential use of ATO, but new targeted formulations have been presented 
. Evaluation of the efficacy of such materials could be facilitated by non-invasive imaging procedures. There is also active development of alternative vascular disrupting agents seeking to enhance efficacy, reduce toxicity, and logically combine with additional therapeutic modalities. Indeed, dynamic BLI was recently applied to the novel tubulin-destabilizing agents BPR0L075 and KGP265 demonstrating vascular disruption in human breast cancer xenografts 
In this study the effect of ATO was demonstrated non-invasively using optical and ultrasound imaging. The strong correlation between BLI as a pre-clinical tool and PD ultrasound as a potential clinical tool suggests the potential for both assessment of pre-clinical development of VDAs and a specific biomarker to demonstrate efficacy in patients.