Alexandra Borboa

Real-time analysis of the kinetics of angiogenesis and vascular permeability in an animal model of wound healing

The use of engineered tissue for the treatment of a variety of acute to chronic wounds has become a clinical standard, and a better understanding of the cellular mechanisms of re-vascularization and barrier integrity could enhance clinical outcomes. Here, we focus on the characterization of the re-vascularization of acellular grafts such as Integra in an animal model to better understand the physiological properties of blood vessels growing in the collagen-glycosaminoglycan matrix vs. wound margins. While Integra has been extensively studied in pre-clinical models, the re-modeling mechanisms of the capillary bed under these matrices are not well understood. Therefore, our first objective was to quantify the kinetics of re-vascularization. The second objective was to assess changes in vascular permeability (VP) of the wound bed compared to normal adjacent skin. The third objective was to establish a non-invasive and quantitative assay for the measurement of VP to facilitate the rapid and reproducible characterization of vascular integrity. Using an excisional wound model in mice, we characterize the appearance, growth, and maturation of blood vessels in an Integra graft over 28 days after surgery. Initial appearance of blood vessels in the graft was observed at 7 days, with angiogenesis peaking between 7 and 14 days. The onset of VP coincided with the increase in re-vascularization of the wound bed and there was a sustained elevation of VP that declined to baseline by 28 days. We propose a non-invasive strategy to assess VP of the wound capillary bed will facilitate a better understanding of the cell and molecular basis of angiogenesis in wound healing.

Glioma-induced remodeling of the neurovascular unit

The normal BBB (blood-brain barrier) consists of a series of structures collectively known as neurovascular units, or NVU, that are composed of endothelial cells and astrocyte endfeet separated by a basal lamina at their interface. The integrity of the BBB and specifically endothelial tight junctions is maintained by interactions between these different components and the local microenvironment of the NVU. Central nervous system cancers such as gliomas disrupt the integrity of the BBB and this compromise is associated with increased tumor growth and invasion of the surrounding brain parenchyma. Because the relationship between glioma-induced BBB breakdown and glioma invasion remains poorly understood, and the host microenvironment can influence tumor cell migration, we used immunohistochemical techniques to characterize tumor associated BBB remodeling. Using an orthotopic xenograft model of glioma, we demonstrate that tumor cells induce specific changes in the composition of the basal lamina and in astrocytic components of the NVU. We suggest that these changes may be essential to understand the capacity of gliomas to regulate BBB integrity and as such, glioma invasion into brain parenchyma.

Non-invasive quantification of brain tumor-induced astrogliosis

BackgroundCNS injury including stroke, infection, and tumor growth lead to astrogliosis, a process that involves upregulation of glial fibrillary acidic protein (GFAP) in astrocytes. However, the kinetics of astrogliosis that is related to these insults (i.e. tumor) is largely unknown.ResultsUsing transgenic mice expressing firefly luciferase under the regulation of the GFAP promoter (GFAP-luc), we developed a model system to monitor astrogliosis upon tumor growth in a rapid, non-invasive manner. A biphasic induction of astrogliosis was observed in our xenograft model in which an early phase of activation of GFAP was associated with inflammatory response followed by a secondary, long-term upregulation of GFAP. These animals reveal GFAP activation with kinetics that is in parallel with tumor growth. Furthermore, a strong correlation between astrogliosis and tumor size was observed.ConclusionsOur results suggest that non-invasive, quantitative bioluminescent imaging using GFAP-luc reporter animal is a useful tool to monitor temporal-spatial kinetics of host-mediated astrogliosis that is associated with glioma and metastatic brain tumor growth.

Thrombin-processed Ecrg4 recruits myeloid cells and induces antitumorigenic inflammation

BACKGROUND Extensive infiltration of brain tumors by microglia and macrophages is a hallmark of tumor progression, and yet the overall tumor microenvironment is characterized by an immunosuppressive phenotype. Here we identify esophageal cancer-related gene 4 (Ecrg4) as a novel thrombin-processed monocyte chemoattractant that recruits myeloid cells, promotes their activation, and leads to a blockade of tumor progression. METHODS Both xenograft glioma and syngeneic glioma models were used to measure orthotopic tumor progression and overall survival. Flow cytometry and immunohistochemical analyses were performed to assess myeloid cell localization, recruitment, and activation. RESULTS Ecrg4 promotes monocyte recruitment and activation of microglia in a T-/B-cell-independent mechanism, which leads to a reduction in glioma tumor burden and increased survival. Mutational analysis reveals that the biological activity of Ecrg4 is dependent on a thrombin-processing site at the C-terminus, inducing monocyte invasion in vivo and in vitro. Furthermore, tumor-induced myeloid cell recruitment is impaired in Ecrg4 knockout mice, leading to increased tumor burden and decreased survival. CONCLUSIONS Together, these results identify Ecrg4 as a paracrine factor that activates microglia and is chemotactic for monocytes, with potential as an antitumor therapeutic.

The noninvasive, quantitative, in vivo assessment of adenoviral-mediated gene delivery in skin wound biomaterials

Because there are few reports using gene delivery in clinically-approved synthetic matrices, we examined the feasibility of using a noninvasive imaging system to study the kinetics of luciferase gene expression when delivered in an adenoviral vector. Using a mouse model of full thickness injury, we quantified the kinetics of gene expression, determined the optimal dose of particle delivery, and established the temporal importance of drug delivery in obtaining optimal gene expression. Specifically, we found that the ideal time to deliver adenovirus to a graft is during the early phase of graft wound closure (days 0-3 post-operatively) for a peak of gene expression to occur 7 days after delivery. Under these conditions, there is a saturating dose of 6 x 10(8) adenoviral particles per graft. In light of these findings, we examined whether the efficacy of delivery could be increased by modulating the composition of the grafts. When a collagen gene-activated matrix (GAM) containing basic fibroblast growth factor (FGF2) was compared to matrix alone, a significant increase in gene expression is observed when identical amounts of vector are delivered (p<0.05). Taken together, these results show how a noninvasive and quantitative assessment of gene expression can be used to optimize gene delivery and that the composition of matrices can dramatically influence gene expression in the wound bed.

Epidermal growth factor targeting of bacteriophage to the choroid plexus for gene delivery to the central nervous system via cerebrospinal fluid.

Because the choroid plexus normally controls the production and composition of cerebrospinal fluid and, as such, its many functions of the central nervous system, we investigated whether ligand-mediated targeting could deliver genes to its secretory epithelium. We show here that when bacteriophages are targeted with epidermal growth factor, they acquire the ability to enter choroid epithelial cells grown in vitro as cell cultures, ex vivo as tissue explants or in vivo by intracerebroventricular injection. The binding and internalization of these particles activate EGF receptors on targeted cells, and the dose- and time-dependent internalization of particles is inhibited by the presence of excess ligand. When the phage genome is further reengineered to contain like green fluorescent protein or firefly luciferase under control of the cytomegalovirus promoter, gene expression is detectable in the choroid plexus and ependymal epithelium by immunohistochemistry or by noninvasive imaging, respectively. Taken together, these data support the hypothesis that reengineered ligand-mediated gene delivery should be considered a viable strategy to increase the specificity of gene delivery to the central nervous system and bypass the blood-brain barrier so as to exploit the biological effectiveness of the choroid plexus as a portal of entry into the brain.

Abstract A21: Thrombin-processed Ecrg4 recruits myeloid cells and induces anti-tumorigenic inflammation

Myeloid cells regulate tumor angiogenesis and promote tumor invasion, yet few strategies exist to exploit their presence to reverse the overall immunosuppressive tumor microenvironment. Based on bioinformatic mining of sentinel genes encoding biologically active peptides, we have recently identified Ecrg4 (Esophageal Cancer-Related Gene 4), as a novel secreted pro-inflammatory mediator of macrophage activation. While higher Ecrg4 expression levels correlate with improved survival in cancers of the esophagus, prostate and breast, indicating a role as a candidate tumor suppressor gene, we have demonstrated that the biological activity of Ecrg4 in cancer functions through a paracrine mechanism, in which Ecrg4 regulates myeloid cells in brain tumors. Moreover, mutational analysis reveals that the biological activity of Ecrg4 is dependent on a thrombin-processing site at the C-terminus, inducing monocyte invasion in vivo and in vitro, demonstrating its role as a novel chemoattractant. Here we establish Ecrg4 as a thrombin-processed chemoattractant using subcutaneous tumor models. Ecrg4 gene delivery or Ecrg4 (133-148) peptide administration to tumor reduced melanoma progression through mobilization and activation of myeloid cells to tumor site. Furthermore, in Ecrg4-knockout mice, myeloid cell recruitment is impaired, leading to increased tumor burden. Together, these results identify Ecrg4 as a paracrine factor that is chemotactic for monocytes, with translational potential as an anti-tumor therapeutic. Citation Format: Jisook Lee, Alexandra Borboa, Andrew Baird, Brian Eliceiri. Thrombin-processed Ecrg4 recruits myeloid cells and induces anti-tumorigenic inflammation. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr A21. doi:10.1158/1538-7445.CHTME14-A21

Abstract A19: Ecrg4 downregulation in glioma enables transformed cells to escape immunosurveillance by tumor-associated macrophages/microglia

Tumor-associated microglia generally promote tumor invasion, adopting an immunosuppressive phenotype that is distinct from their pro-inflammatory functions. Based on bioinformatic mining of novel sentinel genes encoding biologically active peptides, we have recently identified Ecrg4, as a novel secreted pro-inflammatory mediator of macrophage activation. While downregulation of Ecrg4 expression occurs in various tumor cells, in this study, we demonstrate that the biological activity of Ecrg4 in cancer functions through a paracrine mechanism, where tumor cell-exposed Ecrg4 acts upon tumor-associated microglia. In orthotopic glioma models, tumor cells expressing Ecrg4 induce proliferation and morphological changes in tumor-associated microglia. These Ecrg4-mediated effects on microglia correlate with a reduction in tumor burden and prolonged survival. Together, these results identify Ecrg4 as a novel mediator of glioma progression functioning through a paracrine mechanism that regulates the inflammatory state of tumor-associated microglia/macrophages. Citation Format: Jisook Lee, Xitong Dang, Alexandra Borboa, Raul Coimbra, Andrew Baird, Brian Eliceiri. Ecrg4 downregulation in glioma enables transformed cells to escape immunosurveillance by tumor-associated macrophages/microglia. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr A19.

Non-invasive detection of spatio-temporal activation of SBE and NFAT5 promoters in transgenic reporter mice following stroke.

The characterization of molecular responses following cerebral ischemia‐induced changes in animal models capable of undergoing real‐time analysis is an important goal for stroke research. In this study, we use transgenic mice to examine the activation of two different promoters in a firefly luciferase reporter mouse analyzable through a non‐invasive bioluminescent imaging system. In the first model, we examine the middle cerebral artery occlusion (MCAO)‐induced activation of Smad‐binding elements (SBE), a downstream target of Smad 1/2/3 transcription factors, in which SBEs regulate the expression of the fluc reporter. We observed that MCAO induces a bilateral activation (i.e., both ipsilateral and contralateral brain hemispheres) of the SBE‐luc reporter with a peak at 24 h. In the second model, we examined MCAO‐induced activation of the osmolarity‐sensitive promoter nuclear factor of activated T‐cell 5 (NFAT5) and identified a peak reporter expression 72 h post‐MCAO in the ipsilateral but not contralateral hemisphere. In each of these models, the assessment of post‐MCAO fluc‐expression provided both a quantitative measure (i.e., radiance in photons/sec/cm2/steradian) as well as qualitative localization of the molecular response following focal ischemic injury.