Page 77 - ASME_NEMB_2016_Program

TRACK 6 TRACK 6 Technical Program

propose to use the ratio of hydroxylated bradykinin to bradykinin (hyp-BK/ ture induces the loss of endothelial intercellular junctions, thereby favoring
BK) in the blood as a reliable hypoxic marker for predicting the prognosis of immune cell infiltration. Immune cells have been demonstrated to employ
anticancer treatment for pancreatic cancer. adhesive membrane proteins to target inflamed vasculature, locally increase
vascular permeability, and extravasate across inflamed endothelium during
Materials and Method: We first tested the regulation of hypoxia on P4HA1 in leukocyte diapedesis. Inspired by the physiology of circulating leukocytes,
multiple pancreatic cancer cell lines by Western blot, quantitative real-time we recently developed a procedure to transfer leukocyte membranes onto
PCR, and chromatin immunoprecipitation (ChIP). An in vitro assay for eval- biocompatible Multistage Nanovectors (MSV) [2], yielding Leukolike Vectors
uating the activity of P4HA1, combined with nano-trap and MALDI-MS, was (LLV) [3]. The LLV coating contains bioactive adhesive molecules, such as
established to correlate the hyp-BK/BK ratio and hypoxia. Finally, clinical lymphocyte function-associated antigen-1 (LFA-1) and macrophage-1 antigens
serum samples from pancreatic cancer patients before any treatment were (Mac-1), which are critical in interacting with inflamed endothelium and have
evaluated for the levels of hyp-BK/BK, which was subject to correlation anal- been shown to increase blood vessel permeability by activating the ICAM-1
ysis to these patients’ prognosis after anticancer therapies. pathway. Herein, we provide a comprehensive analysis demonstrating that
the transfer of leukocyte membrane proteins onto LLV maintains their bio-
Results: Hypoxic pancreatic cancer cells showed elevated levels P4HA1, as logical functions. We demonstrated that the LLV effectively interacted with
shown by increased mRNA and protein levels of P4HA1 and HIF-1 (hypox- receptors on the endothelial surface, facilitating enhanced transport into the
ia-inducible factor-1). ChIP and immunofluorescence microscopy confirmed subendothelial space. We demonstrated that using the same mechanisms as
that P4HA1 was subject to direct regulation by HIF-1. Importantly, immuno- leukocytes, LLV preferentially accumulated at the cell border under physio-
precipitated P4HA1 from COLO-357 cells cultured under hypoxic conditions logical flow conditions in vitro, and was able to increase tumor blood vessel
produced significantly higher levels of hyp-BK/BK. Furthermore, when we permeability in vivo. We demonstrated that the purified leukocyte membrane
included a HIF-1-specific inhibitor, digoxin, under the hypoxic conditions, the coating maintained an active role in the interaction with tumor associated
ratio was significantly reduced, suggesting that hypoxia directly affected the blood vessels, leading to an increase in vascular diffusion into the suben-
readout of hyp-BK/BK. We could demonstrate that the hyp-BK/BK ratio pro- dothelial space. Furthermore, these steps are critical to further define and
vided much more consistent results compared with the use of either hyp-BK exploit the leukocyte features that are fundamental for drug delivery. Among
or BK alone, as multiple cycles of freezing/thawing and prolonged storage of these, the targeting and bioactivity towards inflamed vasculature provides
samples would not affect the readout of the hyp-BK/BK ratio. Measurement promising clinical significance, as this is a characteristic of a multitude of dis-
of hyp-BK/BK in 22 clinical pancreatic cancer samples has shown that there eases, including cancer.
was a significant correlation between high levels of hyp-BK/BK and poor
treatment response. The marker is specific to the pancreatic cancer be- [1] Maeda H, Wu J, Sawa T, Matsumura Y, Hori K. Tumor vascular permea-
cause significantly elevated level of hyp-BK/BK was only observed in clinical bility and the EPR effect in macromolecular therapeutics: a review. Journal
blood samples from the cancer patients, but not in normal or pancreatitis of controlled release : official journal of the Controlled Release Society.
samples. 2000;65:271-84.
[2] Tasciotti E, Liu X, Bhavane R, Plant K, Leonard AD, Price BK, et al. Meso-
Conclusions: Hypoxia positively regulates HIF-1 and its downstream effector porous silicon particles as a multistage delivery system for imaging and ther-
P4HA1 in pancreatic cancer cells. Consequently, the major P4HA1 catalytic apeutic applications. Nature nanotechnology. 2008;3:151-7.
product hyp-BK may be released into the blood. The ratio of hyp-BK to [3] Parodi A, Quattrocchi N, van de Ven AL, Chiappini C, Evangelopoulos M,
the unmodified BK (hyp-BK/BK) in the blood may serve a hypoxia-specific Martinez JO, et al. Synthetic nanoparticles functionalized with biomimetic
marker and could perform consistently in clinical sample measurement by leukocyte membranes possess cell-like functions. Nature nanotechnology.
MALDI-TOF MS. Results from clinical measurement support the notion that 2013;8:61-8.
the pre-therapy levels of hyp-BK/BK are significantly elevated in pancreatic
cancer patients who later showed poor response to anticancer therapies.
6-3
Leukocyte Membrane Cloaking Enables Synthetic Carriers to NANO AND MICROFLUIDICS
Target and Overcome Tumor Vasculature
Grand Ballroom 5:00pm - 8:00pm
Poster Presentation. NEMB2016-6054
Local and Sustained Delivery of Tamoxifen for the Prevention of
Michael Evangelopoulos, Roberto Palomba, Alessandro Parodi, ER+ Breast Cancer Using a Nanochannel Delivery Platform
Claudia Corbo, Mauro Ferrari, Houston Methodist Research In-
stitute, Houston, TX, United States, Francesco Salvatore, CEINGE Poster Presentation. NEMB2016-5961
Biotecnologie avanzate, Naples, Italy, Ennio Tasciotti, The Method-
ist Hospital Research Institute, Houston, TX, United States Eugenia Nicolov, Carly Filgueira, Andrea Ballerini, R. Lyle Hood,
Giacomo Bruno, Priya Jain, Alessandro Grattoni, Houston Meth-
The lack of an efficient targeted transport of chemotherapeutics remains
one of the primary reasons for poor cancer patient survival. Over the past odist Research Institute, Houston, TX, United States
decades, nanomedicine has provided several delivery platforms able to en-
hance the delivery of chemotherapeutics. Nanoparticles provide an efficient A high incidence (~75%) of primary breast cancers are estrogen receptor
means to modulate the biodistribution of injectable drugs and to favor their positive (ER+), and a large fraction of these patients can pursue chemopre-
accumulation at tumor sites. Nanoparticles accumulation mainly relies on ventive therapies. However due to adverse side effects, only 5% to 20%
exploiting the superior permeability of tumor vasculature, a phenomenon of the tens of thousands of women at high risk who could benefit from
commonly referred to as the enhanced permeability and retention (EPR) ef- chemotherapeutics enroll in preventive treatment. There is a clear need for
fect [1]. However, the EPR effect is not characteristic of all tumor vasculature, alternative preventive strategies that minimize side effects and improve en-
prompting a deeper investigation into alternative vector-associated modifi- rollment and compliance. Selective estrogen receptor modulators, such as
cations and tumor characteristics. In particular, cancer vasculature provides tamoxifen (TMX), have been shown to reduce ER+ breast cancer incidence
several opportunities to develop targeted therapies by leveraging the adhe- by up to 50% among high-risk women. Importantly, along with raloxifene, it
sive proteins over-expressed on the inflamed vasculature, such as intercel- is one of only two FDA-approved drugs for breast cancer prevention. TMX
lular adhesion molecule-1 (ICAM-1). In tumor-associated vasculature, ICAM-1 has already been in use for over 40 years and has a proven record in pre-
overexpression fosters the recruitment of immune cells to the inflamed tis- and post-menopausal women. However, the drug is marred by side effects, 77
sue in an attempt to restore homeostasis. The activation of the ICAM-1 path- the most common being symptoms of menopause. Further, women treated
way through surface interactions between leukocytes and inflamed vascula- systemically and chronically with TMX were found to have an increased inci-