Tumors develop a chaotic vascular network characterized by variable blood pressure and vascular permeability that inhibits effective drug delivery 
. Many areas within tumors contain irregular blood vessels that are leaky and allow influx of circulating blood components. Sporadic high cell density within the tumor prevents normal tissue drainage 
. This promotes the accumulation of cellular and blood proteins in the interstitial space, leading to high interstitial oncotic pressure, which inhibits the extravasation of systemic drugs 
. Ultimately the distribution of systemically circulating drugs in tumors can be unpredictable and irregular since it depends heavily on the passive extravasation of the drug from the vasculature into target tissues 
By transiently altering tumor blood vessel physiology during systemic anti-cancer treatment, tissue perfusion and drainage can be enhanced, thereby relieving interstitial hypertension 
. Prolonged treatment with anti-angiogenic drugs, such as Sunitinib or DC101, normalizes blood flow through the remaining stabilized vasculature. These treatments can improve tumor micro-hemodynamics and effectively lower the interstitial pressure. Consequently, the efficacy of concomitantly or subsequently administered drugs is enhanced due to improved vascular delivery 
. Similarly, treatment of hepatic tumors with interferon-β (IFN-β) induces tumor vessel maturation and tissue perfusion, which improves delivery of additional therapeutics 
. Altering oncogenic signaling in tumors can also be used to change their blood-flow dynamics 
. Specifically, inhibition of the PI3K pathway increases tumor perfusion and simultaneously enhances doxorubicin delivery 
. These findings indicate that the strategic use of adjuvants to transiently modify tumor blood flow and hemodynamics can facilitate drug delivery to cancer sites.
Normalizing blood flow promotes drug delivery by reducing the interstitial pressure that counteracts diffusion. However, normalizing agents can also reduce vascular permeability. Vascular permeability greatly influences the extravasation of drugs associated with carriers, including liposomes, micelles or other nanoparticles 
. Recent advances to manipulate vascular permeability exemplify how adjuvant therapy might facilitate the targeting of future and existing anti-cancer therapies to tumor tissues 
, . Unfortunately, the lack of accurate means to quantify vascular permeability is a significant hurdle to predicting its direct influence on drug localization and uptake in vivo
Classically, vascular permeability has been measured using the Miles Assay 
. This assay determines the leakage of a visible dye from the vasculature into the surrounding tissue spectrophotometrically, with the relative vascular permeability determined as the ratio of extravasated versus intravascular dye. This assay has several limitations, however, that preclude its use in many cases. It is limited to the analysis of a single time-point, which must be selected empirically from pilot experiments. Furthermore, due to the wide range of experimental approaches described in the literature, results are subject to a high degree of variability and their repeatability must be considered. Variability can be mitigated somewhat by using large tissue volumes. Consequently, these experiments are generally performed in rodent models with large group sizes 
, which is both expensive and time-consuming. As the Miles assay is limited to the determination of average permeability over an entire tissue, localized differences in vascular permeability, particularly within tumors, cannot be detected. A dynamic measure of vascular permeability would allow for the assessment of the impact of regional and temporal changes in vascular permeability on drug distribution within solid tumors.
Here, we present an integrated method to visualize and quantify the real-time dynamics of dextrans in a shell-less chick chorioallantoic (CAM) model. Regional and temporal differences in vessel permeability within the tumor microenvironment are captured at high resolution using an intravital imaging approach. The use of dextrans of different molecular weights allows for the concurrent evaluation of vascular permeability and vascular structural integrity. The dynamics of anti-cancer drugs as they move through the vasculature and into tumor tissues can be mimicked with dextrans 
. Dextrans of various molecular weights can mimic the diffusion of various sized macromolecules including macro-molecular drug carriers (~70 kDa) and antibodies (~150 kDa) into the tumor interstitial space. Large dextrans of ~2000 kDa, are sequestered within the lumen of the tumor vasculature 
. This work builds upon earlier observations in the shell-less chicken embryo model, which examined microvascular perselectivity during normal angiogenesis in the early stages of CAM development 
. These authors demonstrated a rapid reduction in microvascular permeability to FITC-dextrans of varying sizes (20–150 kDa) between days 4.5–5.5 of the normal 21-day gestation. They also demonstrated that dextran size correlated with permeability (dextran-20>dextran-40>dextran-70>dextran-150 kDa). Furthermore, while these authors report tumor permeability values for 70 kDa and 150 kDa dextrans, they did not examine it in the shell-less or ex ovo
chick model. The leakage of small versus large molecular weight dextrans from the vasculature in this model may provide a high-resolution measure of vascular permeability predictive of drug localization in vivo
The CAM is a thin, respiratory tissue for the developing chick embryo characterized by a dense, highly organized network of blood vessels 
. The physiological responses of the CAM are consistent with those of mammalian tissues 
and it has provided a physiologically relevant setting for angiogenesis research for more than a century 
. The commercial availability of fertilized eggs, the ease of embryo culture, and the robustness of the CAM model facilitate large, statistically powerful studies and make it suitable for high throughput approaches. The CAM is not fully immunocompetent in the early embryo 
, and it supports the growth of human and murine tumor xenografts 
. In addition, in the ex ovo
model, the CAM is directly accessible for experimental manipulation and imaging. Paired with a fluorescence microscopy platform, this model is well-suited for analyzing drug-induced changes in vascular permeability in tumor xenografts and their microenvironment.
We demonstrate, using this intravital imaging approach, that vascular permeability can be manipulated to modulate the extravasation of small molecules into the local tumor microenvironment. Treatment with vascular endothelial growth factor (VEGF) or a permeability enhancing peptide (PEP) fragment of IL-2 
either locally or systemically results in a temporary enhancement of vascular permeability that can be precisely monitored over time. We show that this transient increase in vascular permeability can be exploited to significantly enhance the accumulation of a chemotherapeutic drug within the tumor.