Rebecca E. Sohn

VEGF is essential for hypoxia-inducible factor-mediated neovascularization but dispensable for endothelial sprouting.

Although our understanding of the molecular regulation of adult neovascularization has advanced tremendously, vascular-targeted therapies for tissue ischemia remain suboptimal. The master regulatory transcription factors of the hypoxia-inducible factor (HIF) family are attractive therapeutic targets because they coordinately up-regulate multiple genes controlling neovascularization. Here, we used an inducible model of epithelial HIF-1 activation, the TetON-HIF-1 mouse, to test the requirement for VEGF in HIF-1 mediated neovascularization. TetON-HIF-1, K14-Cre, and VEGFflox/flox alleles were combined to create TetON-HIF-1:VEGFΔ mice to activate HIF-1 and its target genes in adult basal keratinocytes in the absence of concomitant VEGF. HIF-1 induction failed to produce neovascularization in TetON-HIF-1:VEGFΔ mice despite robust up-regulation of multiple proangiogenic HIF targets, including PlGF, adrenomedullin, angiogenin, and PAI-1. In contrast, endothelial sprouting was preserved, enhanced, and more persistent, consistent with marked reduction in Dll4-Notch-1 signaling. Optical-resolution photoacoustic microscopy, which provides noninvasive, label-free, high resolution, and wide-field vascular imaging, revealed the absence of both capillary expansion and arteriovenous remodeling in serially imaged individual TetON-HIF-1:VEGFΔ mice. Impaired TetON-HIF-1:VEGFΔ neovascularization could be partially rescued by 12-O-tetradecanoylphorbol-13-acetate skin treatment. These data suggest that therapeutic angiogenesis for ischemic cardiovascular disease may require treatment with both HIF-1 and VEGF.

Tumor glucose metabolism imaged in vivo in small animals with whole-body photoacoustic computed tomography.

With the increasing use of small animals for human disease studies, small-animal whole-body molecular imaging plays an important role in biomedical research. Currently, none of the existing imaging modalities can provide both anatomical and glucose molecular information, leading to higher costs of building dual-modality systems. Even with image co-registration, the spatial resolution of the molecular imaging modality is not improved. Utilizing a ring-shaped confocal photoacoustic computed tomography system, we demonstrate, for the first time, that both anatomy and glucose uptake can be imaged in a single modality. Anatomy was imaged with the endogenous hemoglobin contrast, and glucose metabolism was imaged with a near-infrared dye-labeled 2-deoxyglucose.

Detection of Rapalog-Mediated Therapeutic Response in Renal Cancer Xenografts Using 64Cu-bevacizumab ImmunoPET

The importance of neovascularization for primary and metastatic tumor growth fostered numerous clinical trials of angiogenesis inhibitors either alone or in combination with conventional antineoplastic therapies. One challenge with the use of molecularly targeted agents has been the disconnection between size reduction and tumor biologic behavior, either when the drug is efficacious or when tumor resistance emerges. Here, we report the synthesis and characterization of 64Cu-NOTA-bevacizumab as a PET imaging agent for imaging intratumoral VEGF content in vivo. 64Cu-NOTA-bevacizumab avidly accumulated in 786-O renal carcinoma xenografts with lower levels in host organs. RAD001 (everolimus) markedly attenuated 64Cu-NOTA-bevacizumab accumulation within 786-O renal carcinoma xenografts. Tumor tissue and cellular molecular analysis validated PET imaging, demonstrating decreases in total and secreted VEGF content and VEGFR2 activation. Notably, 64Cu-NOTA-bevacizumab PET imaging was concordant with the growth arrest of RAD001 tumors. These data suggest that immunoPET targeting of angiogenic factors such as VEGF could be a new class of surrogate markers complementing the RECIST criteria in patients receiving molecularly targeted therapies.

The myeloid-binding peptide adenoviral vector enables multi-organ vascular endothelial gene targeting

Vascular endothelial cells (ECs) are ideal gene therapy targets as they provide widespread tissue access and are the first contact surfaces following intravenous vector administration. Human recombinant adenovirus serotype 5 (Ad5) is the most frequently used gene transfer system because of its appreciable transgene payload capacity and lack of somatic mutation risk. However, standard Ad5 vectors predominantly transduce liver but not the vasculature following intravenous administration. We recently developed an Ad5 vector with a myeloid cell-binding peptide (MBP) incorporated into the knob-deleted, T4 fibritin chimeric fiber (Ad.MBP). This vector was shown to transduce pulmonary ECs presumably via a vector handoff mechanism. Here we tested the body-wide tropism of the Ad.MBP vector, its myeloid cell necessity, and vector-EC expression dose response. Using comprehensive multi-organ co-immunofluorescence analysis, we discovered that Ad.MBP produced widespread EC transduction in the lung, heart, kidney, skeletal muscle, pancreas, small bowel, and brain. Surprisingly, Ad.MBP retained hepatocyte tropism albeit at a reduced frequency compared with the standard Ad5. While binding specifically to myeloid cells ex vivo, multi-organ Ad.MBP expression was not dependent on circulating monocytes or macrophages. Ad.MBP dose de-escalation maintained full lung-targeting capacity but drastically reduced transgene expression in other organs. Swapping the EC-specific ROBO4 for the CMV promoter/enhancer abrogated hepatocyte expression but also reduced gene expression in other organs. Collectively, our multilevel targeting strategy could enable therapeutic biological production in previously inaccessible organs that pertain to the most debilitating or lethal human diseases.

Optical-resolution photoacoustic microscopy of the metabolic rate of oxygen in a mouse renal tumor model

We propose using noninvasive longitudinal optical-resolution photoacoustic microscopy (L-ORPAM) to quantify blood flow flux, oxygen saturation (sO2), and thereby the metabolic rate of oxygen (MRO2), for a renal tumor model in the same mouse over weeks to months. Experiments showed that the sO2 difference between the artery and vein decreased greatly due to the arteriovenous shunting effect during tumor growth. Moreover, hypermetabolism was exhibited by an increase in MRO2.

Transcriptional Targeting of Primary and Metastatic Tumor Neovasculature by an Adenoviral Type 5 Roundabout4 Vector in Mice

New approaches targeting metastatic neovasculature are needed. Payload capacity, cellular transduction efficiency, and first-pass cellular uptake following systemic vector administration, motivates persistent interest in tumor vascular endothelial cell (EC) adenoviral (Ad) vector targeting. While EC transductional and transcriptional targeting has been accomplished, vector administration approaches of limited clinical utility, lack of tumor-wide EC expression quantification, and failure to address avid liver sequestration, challenged prior work. Here, we intravenously injected an Ad vector containing 3 kb of the human roundabout4 (ROBO4) enhancer/promoter transcriptionally regulating an enhanced green fluorescent protein (EGFP) reporter into immunodeficient mice bearing 786-O renal cell carcinoma subcutaneous (SC) xenografts and kidney orthotopic (KO) tumors. Initial experiments performed in human coxsackie virus and adenovirus receptor (hCAR) transgenic:Rag2 knockout mice revealed multiple ECs with high-level Ad5ROBO4-EGFP expression throughout KO and SC tumors. In contrast, Ad5CMV-EGFP was sporadically expressed in a few tumor vascular ECs and stromal cells. As the hCAR transgene also facilitated Ad5ROBO4 and control Ad5CMV vector EC expression in multiple host organs, follow-on experiments engaged warfarin-mediated liver vector detargeting in hCAR non-transgenic mice. Ad5ROBO4-mediated EC expression was undetectable in most host organs, while the frequencies of vector expressing intratumoral vessels and whole tumor EGFP protein levels remained elevated. In contrast, AdCMV vector expression was only detectable in one or two stromal cells throughout the whole tumor. The Ad5ROBO4 vector, in conjunction with liver detargeting, provides tractable genetic access for in-vivo EC genetic engineering in malignancies.

A new model of multi-visceral and bone metastatic prostate cancer with perivascular niche targeting by a novel endothelial specific adenoviral vector

While modern therapies for metastatic prostate cancer (PCa) have improved survival they are associated with an increasingly prevalent entity, aggressive variant PCa (AVPCa), lacking androgen receptor (AR) expression, enriched for cancer stem cells (CSCs), and evidencing epithelial-mesenchymal plasticity with a varying extent of neuroendocrine transdifferentiation. Parallel work revealed that endothelial cells (ECs) create a perivascular CSC niche mediated by juxtacrine and membrane tethered signaling. There is increasing interest in pharmacological metastatic niche targeting, however, targeted access has been impossible. Here, we discovered that the Gleason 7 derived, androgen receptor negative, IGR-CaP1 cell line possessed some but not all of the molecular features of AVPCa. Intracardiac injection into NOD/SCID/IL2Rg -/− (NSG) mice produced a completely penetrant bone, liver, adrenal, and brain metastatic phenotype; noninvasively and histologically detectable at 2 weeks, and necessitating sacrifice 4-5 weeks post injection. Bone metastases were osteoblastic, and osteolytic. IGR-CaP1 cells expressed the neuroendocrine marker synaptophysin, near equivalent levels of vimentin and e-cadherin, all of the EMT transcription factors, and activation of NOTCH and WNT pathways. In parallel, we created a new triple-targeted adenoviral vector containing a fiber knob RGD peptide, a hexon mutation, and an EC specific ROBO4 promoter (Ad.RGD.H5/3.ROBO4). This vector was expressed in metastatic microvessels tightly juxtaposed to IGR-CaP1 cells in bone and visceral niches. Thus, the combination of IGR-CaP1 cells and NSG mice produces a completely penetrant metastatic PCa model emulating end-stage human disease. In addition, the metastatic niche access provided by our novel Ad vector could be therapeutically leveraged for future disease control or cure.

Early-stage tumor detection using photoacoustic microscopy: a pattern recognition approach

We report photoacoustic microscopy (PAM) of arteriovenous (AV) shunts in early stage tumors in vivo, and develop a pattern recognition framework for computerized tumor detection. Here, using a high-resolution photoacoustic microscope, we implement a new blood oxygenation (sO2)-based disease marker induced by the AV shunt effect in tumor angiogenesis. We discovered a striking biological phenomenon: There can be two dramatically different sO2 values in bloodstreams flowing side-by-side in a single vessel. By tracing abnormal sO2 values in the blood vessels, we can identify a tumor region at an early stage. To further automate tumor detection based on our findings, we adopt widely used pattern recognition methods and develop an efficient computerized classification framework. The test result shows over 80% averaged detection accuracy with false positive contributing 18.52% of error test samples on a 50 PAM image dataset.

Photoacoustic microscopy of arteriovenous shunts and blood diffusion in early-stage tumors

Angiogenesis in a tumor region creates arteriovenous (AV) shunts that cause an abnormal venous blood oxygen saturation ( sO2 ) distribution. Here, we applied optical-resolution photoacoustic microscopy to study the AV shunting in vivo. First, we built a phantom to image sO2 distribution in a vessel containing converged flows from two upstream blood vessels with different sO2 values. The phantom experiment showed that the blood from the two upstream vessels maintained a clear sO2 boundary for hundreds of seconds, which is consistent with our theoretical analysis using a diffusion model. Next, we xenotransplanted O-786 tumor cells in mouse ears and observed abnormal sO2 distribution in the downstream vein from the AV shunts in vivo. Finally, we identified the tumor location by tracing the sO2 distribution. Our study suggests that abnormal sO2 distribution induced by the AV shunts in the vessel network may be used as a new functional benchmark for early tumor detection.

Abstract 5210: Creation of endothelial-targeted adenoviral vectors for genetic engineering of the metastatic tumor microenvironment

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA The endothelium is an attractive gene therapy target for metastatic cancer, providing access to systemic tumors. Previous work has focused on either viral entry/attachment (transductional targeting), or cell type specific enhancer promoter vector transgene expression regulation (transcriptional targeting). While previous studies demonstrated endothelial targeting, the tumor wide extent and transgene expression level quantification have been uncertain. Indeed, obtaining robust and stringent tumor expression has been challenging due to: insufficient transduction, focal rather than widespread intratumoral vascular expression, viral particle liver sequestration, and host preformed immunity. We developed a set of adenoviral vectors, containing 3 kb of the human ROBO4 enhancer/promoter, that are expressed at high levels throughout tumor vasculature. We now have three vectors representing four tiers of genetic modification. Transgenes encoding fluorescent proteins allowed us to quantify endothelial expression frequency by image analysis, and expression level using whole tissue Western blotting. Our first vector was Ad5.ROBO4. Compared with Ad5.CMV, Ad5.ROBO4 evidenced complete retargeting from hepatocytes to low-level expression in host organ and tumor endothelial cells. Warfarin-mediated hepatocyte detargeting produced a striking increase in tumor and bone marrow sinusoidal endothelial expression, without change in other host organs. Our second vector is Ad.RGD.H5/H3.ROBO4. Insertion of a cyclized RGD peptide in the fiber/knob HI loop engages enhanced tumor endothelial transduction via αv/β3 and αv/β5. Swapping hexon hypervariable regions responsible for Ad Type 5-Factor X binding with corresponding regions from serotype 3 enables marked diminution of hepatocyte sequestration. Use of the ROBO4 enhancer promoter produces high-level pan-intratumoral vascular expression particularly in cancers with markedly elevated VEGF production, such as orthotopic and metastatic renal cell carcinoma, orthotopic colonic liver metastases, and prostate cancer bone, brain, and liver metastases. Most striking, was enhancement of Ad.RGD.H5/H3.ROBO4 vector expression within interior tumor regions undergoing hypoxic necrosis; regions notoriously resistant to radiation or chemotherapies. Our third vector contains a polycistronic array, enabling triple transgene expression from a single vector within the tumor vasculature. Thus, we have panel of endothelial-targeted vectors with distinctive vascular tropism for use in cancers metastatic to individual host organs. These vectors enable expression of a palette of transgenes that can manipulate the tumor microenvironment to achieve metastatic growth inhibition alone, or “staggered” with chemo- or irradiation therapies. Note: This abstract was not presented at the meeting. Citation Format: Zhi Hong Lu, Sergey Kaliberov, Lyudmila Kaliberova, Rebecca E. Sohn, Yingqui Du, David T. Curiel, Jeffrey M. Arbeit. Creation of endothelial-targeted adenoviral vectors for genetic engineering of the metastatic tumor microenvironment. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5210. doi:10.1158/1538-7445.AM2015-5210