Development of a novel air-liquid interface airway tissue equivalent model for in vitro respiratory modeling studies.

The human airways are complex structures with important interactions between cells, extracellular matrix (ECM) proteins and the biomechanical microenvironment. A robust, well-differentiated in vitro culture system that accurately models these interactions would provide a useful tool for studying normal and pathological airway biology. Here, we report the development and characterization of a physiologically relevant air-liquid interface (ALI) 3D airway 'organ tissue equivalent' (OTE) model with three novel features: native pulmonary fibroblasts, solubilized lung ECM, and hydrogel substrate with tunable stiffness and porosity. We demonstrate the versatility of the OTE model by evaluating the impact of these features on human bronchial epithelial (HBE) cell phenotype. Variations of this model were analyzed during 28 days of ALI culture by evaluating epithelial confluence, trans-epithelial electrical resistance, and epithelial phenotype via multispectral immuno-histochemistry and next-generation sequencing. Cultures that included both solubilized lung ECM and native pulmonary fibroblasts within the hydrogel substrate formed well-differentiated ALI cultures that maintained a barrier function and expressed mature epithelial markers relating to goblet, club, and ciliated cells. Modulation of hydrogel stiffness did not negatively impact HBE differentiation and could be a valuable variable to alter epithelial phenotype. This study highlights the feasibility and versatility of a 3D airway OTE model to model the multiple components of the human airway 3D microenvironment.

Imaging of Fibroblast Activation Protein Alpha Expression in a Preclinical Mouse Model of Glioma Using Positron Emission Tomography.

Glioblastoma multiforme (GBM) is the most aggressive glioma of the primary central nervous system. Due to the lack of effective treatment options, the prognosis for patients remains bleak. Fibroblast activation protein alpha (FAP), a 170 kDa type II transmembrane serine protease was observed to be expressed on glioma cells and within the glioma tumor microenvironment. To understand the utility of targeting FAP in this tumor type, the immuno-PET radiopharmaceutical [89Zr]Zr-Df-Bz-F19 mAb was prepared and Lindmo analysis was used for its in vitro evaluation using the U87MG cell line, which expresses FAP endogenously. Lindmo analysis revealed an association constant (Ka) of 10-8 M-1 and an immunoreactivity of 52%. Biodistribution studies in U87MG tumor-bearing mice revealed increasing radiotracer retention in tumors over time, leading to average tumor-to-muscle ratios of 3.1, 7.3, 7.2, and 8.3 at 2, 24, 48 and 72 h, respectively. Small animal PET corroborated the biodistribution studies; tumor-to-muscle ratios at 2, 24, 48, and 72 h were 2.0, 5.0, 6.1 and 7.8, respectively. Autoradiography demonstrated accumulated activity throughout the interior of FAP+ tumors, while sequential tumor sections stained positively for FAP expression. Conversely, FAP- tissues retained minimal radioactivity and were negative for FAP expression by immunohistochemistry. These results demonstrate FAP as a promising biomarker that may be exploited to diagnose and potentially treat GBM and other neuroepithelial cancers.

Near Infrared Fluorescent Nanoparticles Derived from Hyaluronic Acid Improve Tumor Contrast for Image-Guided Surgery.

Tumor tissue that remains undetected at the primary surgical site can cause tumor recurrence, repeat surgery, and treatment strategy alterations that impose a significant patient and healthcare burden. Intraoperative near infrared fluorescence (NIRF) imaging is one potential method to identify remaining tumor by visualization of NIR fluorophores that are preferentially localized to the tumor. This requires development of fluorophores that consistently identify tumor tissue in different patients and tumor types. In this study we examined a panel of NIRF contrast agents consisting of polymeric nanoparticle (NP) formulations derived from hyaluronic acid (HA), with either physically entrapped indocyanine green (ICG) or covalently conjugated Cy7.5. Using orthotopic human breast cancer MDA-MB-231 xenografts in nude mice we identified two lead formulations. One, NanoICGPBA, with physicochemically entrapped ICG, showed 2.3-fold greater tumor contrast than ICG alone at 24 h (p < 0.01), and another, NanoCy7.5100-H, with covalently conjugated Cy7.5, showed 74-fold greater tumor contrast than Cy7.5 alone at 24 h (p < 0.0001). These two lead formulations were then tested in immune competent BALB/c mice bearing orthotopic 4T1 breast cancer tumors. NanoICGPBA showed 2.2-fold greater contrast than ICG alone (p < 0.0001), and NanoCy7.5100-H showed 14.8-fold greater contrast than Cy7.5 alone (p < 0.0001). Furthermore, both NanoICGPBA and NanoCy7.5100-H provided strong tumor enhancement using image-guided surgery in mice bearing 4T1 tumors. These studies demonstrate the efficacy of a panel of HA-derived NPs in delineating tumors in vivo, and identifies promising formulations that can be used for future in vivo tumor removal efficacy studies.

Tumor-associated stromal cells as key contributors to the tumor microenvironment.

The tumor microenvironment is a heterogeneous population of cells consisting of the tumor bulk plus supporting cells. It is becoming increasingly evident that these supporting cells are recruited by cancer cells from nearby endogenous host stroma and promote events such as tumor angiogenesis, proliferation, invasion, and metastasis, as well as mediate mechanisms of therapeutic resistance. In addition, recruited stromal cells range in type and include vascular endothelial cells, pericytes, adipocytes, fibroblasts, and bone-marrow mesenchymal stromal cells. During normal wound healing and inflammatory processes, local stromal cells change their phenotype to become that of reactive stroma. Under certain conditions, however, tumor cells can co-opt these reactive stromal cells and further transition them into tumor-associated stromal cells (TASCs). These TASCs express higher levels of proteins, including alpha-smooth muscle actin, fibroblast activating protein, and matrix metalloproteinases, compared with their normal, non-reactive counterparts. TASCs are also known to secrete many pro-tumorigenic factors, including IL-6, IL-8, stromal-derived factor-1 alpha, vascular endothelial growth factor, tenascin-C, and matrix metalloproteinases, among others, which recruit additional tumor and pro-tumorigenic cells to the developing microenvironment. Here, we review the current literature pertaining to the origins of recruited host stroma, contributions toward tumor progression, tumor-associated stromal cells, and mechanisms of crosstalk between endogenous host stroma and tumor cells.