3D Imaging Reveals Changes in the Neurovascular Architecture of the Murine Calvarium with Aging.

Calvarial nerves, along with vasculature, influence skull formation during development and following injury, but it remains unclear how calvarial nerves are spatially distributed during postnatal growth and aging. Studying the spatial distribution of nerves in the skull remains challenging due to a lack of methods to image and quantify 3D structures in intact bone. To visualize calvarial 3D neurovascular architecture, we imaged nerves and endothelial cells with lightsheet microscopy. We employed machine-learning-based segmentation to facilitate high-resolution characterization from post-natal day 0 (P0) to Week 80 (80wk). We found that TUBB3+ nerve density decreased with aging with the frontal bone demonstrating earlier onset age-related nerve loss than the parietal bone. In addition, nerves in the periosteum and dura mater exhibited similar yet distinct temporal patterns of nerve growth and loss. While no difference was observed in TUBB3+ nerves during skeletal maturation (P0 → 12wk), we did observe an increase in the volume of unmyelinated nerves in the dura mater. Regarding calvarial vasculature, larger CD31hiEmcn- vessel density increased with aging, while CD31hiEmcnhi vessel density was reduced. For all nerve markers studied, calvarial nerves maintained a preferential spatial association with CD31hiEmcnhi vessels that decreased with aging. Additionally, we used a model of Apert syndrome that demonstrates early coronal suture fusion to explore the impact of suture-related disease on neurovascular architecture. We identified a mild dysregulation of dural nerves and minor shifts in vessel populations. Collectively, this 3D, spatiotemporal characterization of calvarial nerves throughout the lifespan and provides new insights into age-induced neurovascular architecture.

Multimodality imaging reveals angiogenic evolution in vivo during calvarial bone defect healing.

The healing of calvarial bone defects is a pressing clinical problem that involves the dynamic interplay between angiogenesis and osteogenesis within the osteogenic niche. Although structural and functional vascular remodeling (i.e., angiogenic evolution) in the osteogenic niche is a crucial modulator of oxygenation, inflammatory and bone precursor cells, most clinical and pre-clinical investigations have been limited to characterizing structural changes in the vasculature and bone. Therefore, we developed a new multimodality imaging approach that for the first time enabled the longitudinal (i.e., over four weeks) and dynamic characterization of multiple in vivo functional parameters in the remodeled vasculature and its effects on de novo osteogenesis, in a preclinical calvarial defect model. We employed multi-wavelength intrinsic optical signal (IOS) imaging to assess microvascular remodeling, intravascular oxygenation (SO2), and osteogenesis; laser speckle contrast (LSC) imaging to assess concomitant changes in blood flow and vascular maturity; and micro-computed tomography (µCT) to validate volumetric changes in calvarial bone. We found that angiogenic evolution was tightly coupled with calvarial bone regeneration and corresponded to distinct phases of bone healing, such as injury, hematoma formation, revascularization, and remodeling. The first three phases occurred during the initial two weeks of bone healing and were characterized by significant in vivo changes in vascular morphology, blood flow, oxygenation, and maturity. Overall, angiogenic evolution preceded osteogenesis, which only plateaued toward the end of bone healing (i.e., four weeks). Collectively, these data indicate the crucial role of angiogenic evolution in osteogenesis. We believe that such multimodality imaging approaches have the potential to inform the design of more efficacious tissue-engineering calvarial defect treatments.

Vascular phenotyping of the invasive front in breast cancer using a 3D angiogenesis atlas.

OBJECTIVE: Vascular remodeling at the invasive tumor front (ITF) plays a critical role in progression and metastasis of triple negative breast cancer (TNBC). Therefore, there is a crucial need to characterize the vascular phenotype (i.e. changes in the structure and function of vasculature) of the ITF and tumor core (TC) in TNBC. This requires high-resolution, 3D structural and functional microvascular data that spans the ITF and TC (i.e. ∼4-5 mm from the tumor's edge). Since such data are often challenging to obtain with most conventional imaging approaches, we employed a unique "3D whole-tumor angiogenesis atlas" derived from orthotopic xenografts to characterize the vascular phenotype of the ITF and TC in TNBC. METHODS: First, high-resolution (8 µm) computed tomography (CT) images of "whole-tumor" microvasculature were acquired from eight orthotopic TNBC xenografts, of which three tumors were excised at post-inoculation day 21 (i.e. early-stage) and five tumors were excised at post-inoculation day 35 (i.e. advanced-stage). These 3D morphological CT data were combined with soft tissue contrast from MRI as well as functional data generated in silico using image-based hemodynamic modeling to generate a multi-layered "angiogenesis atlas". Employing this atlas, blood vessels were first spatially stratified within the ITF (i.e. ≤1 mm from the tumor's edge) and TC (i.e. >1 mm from the tumor's edge) of each tumor xenograft. Then, a novel method was developed to visualize and characterize microvascular remodeling and perfusion changes in terms of distance from the tumor's edge. RESULTS: The angiogenesis atlas enabled the 3D visualization of changes in tumor vessel growth patterns, morphology and perfusion within the ITF and TC. Early and advanced stage tumors demonstrated significant differences in terms of their edge-to-center distributions for vascular surface area density, vascular length density, intervessel distance and simulated perfusion density (p â‰ª 0.01). Elevated vascular length density, vascular surface area density and perfusion density along the circumference of the ITF was suggestive of a preferential spatial pattern of angiogenic growth in this tumor cohort. Finally, we demonstrated the feasibility of differentiating the vascular phenotypes of ITF and TC in these TNBC xenografts. CONCLUSIONS: The combination of a 3D angiogenesis atlas and image-based hemodynamic modeling heralds a new approach for characterizing the role of vascular remodeling in cancer and other diseases.

Quantification of Cerebral Vascular Autoregulation Immediately Following Resuscitation from Cardiac Arrest.

Cerebral vascular autoregulation is impaired following resuscitation from cardiac arrest (CA), and its quantification may allow assessing CA-induced brain injury. However, hyperemia occurring immediately post-resuscitation limits the application of most metrics that quantify autoregulation. Therefore, to characterize autoregulation during this critical period, we developed three novel metrics based on how the cerebrovascular resistance (CVR) covaries with changes in cerebral perfusion pressure (CPP): (i) Î¸CVR, which quantifies the CVR vs CPP gradient, (ii) a CVR-based transfer function analysis, and (iii) CVRx, the correlation coefficient between CPP and CVR. We tested these metrics in a model of asphyxia induced CA and resuscitation using seven adult male Wistar rats. Mean arterial pressure (MAP) and cortical blood flow recorded for 30 min post-resuscitation via arterial cannulation and laser speckle contrast imaging, were used as surrogates of CPP and cerebral blood flow (CBF), while CVR was computed as the CPP/CBF ratio. Using our metrics, we found that the status of cerebral vascular autoregulation altered substantially during hyperemia, with changes spread throughout the 0-0.05 Hz frequency band. Our metrics push the boundary of how soon autoregulation can be assessed, and if validated against outcome markers, may help develop a reliable metric of brain injury post-resuscitation.

Vascular-centric mapping of in vivo blood oxygen saturation in preclinical models.

Assessing intravascular blood oxygen saturation (SO2) is crucial for characterizing in vivo microenvironmental changes in preclinical models of injury and disease. However, most conventional optical imaging techniques for mapping in vivo SO2 assume or compute a single value of the optical path-length in tissue. This is especially detrimental when mapping in vivo SO2 in experimental disease or wound healing models that are characterized by vascular and tissue remodeling. Therefore, to circumvent this limitation we developed an in vivo SO2 mapping technique that utilizes hemoglobin-based intrinsic optical signal (IOS) imaging combined with a vascular-centric estimation of optical path-lengths. In vivo arterial and venous SO2 distributions derived with this approach closely matched those reported in the literature, while those derived using the single path-length (i.e. conventional) approach did not. Moreover, in vivo cerebrovascular SO2 strongly correlated (R2 > 0.7) with changes in systemic SO2 measured with a pulse oximeter during hypoxia and hyperoxia paradigms. Finally, in a calvarial bone healing model, in vivo SO2 assessed over four weeks was spatiotemporally correlated with angiogenesis and osteogenesis (R2 > 0.6). During the early stages of bone healing (i.e. day 10), angiogenic vessels surrounding the calvarial defect exhibited mean SO2 that was elevated by10 % (p < 0.05) relative to that observed at a later stage (i.e., day 26), indicative of their role in osteogenesis. These correlations were not evident with the conventional SO2 mapping approach. The feasibility of our wide field-of-view in vivo SO2 mapping approach illustrates its potential for characterizing the microvascular environment in applications ranging from tissue engineering to cancer.

State-of-the-art techniques for imaging the vascular microenvironment in craniofacial bone tissue engineering applications.

Vascularization is a crucial step during musculoskeletal tissue regeneration via bioengineered constructs or grafts. Functional vasculature provides oxygen and nutrients to the graft microenvironment, facilitates wound healing, enhances graft integration with host tissue, and ensures the long-term survival of regenerating tissue. Therefore, imaging de novo vascularization (i.e., angiogenesis), changes in microvascular morphology, and the establishment and maintenance of perfusion within the graft site (i.e., vascular microenvironment or VME) can provide essential insights into engraftment, wound healing, as well as inform the design of tissue engineering (TE) constructs. In this review, we focus on state-of-the-art imaging approaches for monitoring the VME in craniofacial TE applications, as well as future advances in this field. We describe how cutting-edge in vivo and ex vivo imaging methods can yield invaluable information regarding VME parameters that can help characterize the effectiveness of different TE constructs and iteratively inform their design for enhanced craniofacial bone regeneration. Finally, we explicate how the integration of novel TE constructs, preclinical model systems, imaging techniques, and systems biology approaches could usher in an era of "image-based tissue engineering."

In vivo phenotyping of the microvasculature in necrotizing enterocolitis with multicontrast optical imaging.

OBJECTIVE: Necrotizing enterocolitis (NEC) is the most prevalent gastrointestinal emergency in premature infants and is characterized by a dysfunctional gut microcirculation. Therefore, there is a dire need for in vivo methods to characterize NEC-induced changes in the structure and function of the gut microcirculation, that is, its vascular phenotype. Since in vivo gut imaging methods are often slow and employ a single-contrast mechanism, we developed a rapid multicontrast imaging technique and a novel analyses pipeline for phenotyping the gut microcirculation. METHODS: Using an experimental NEC model, we acquired in vivo images of the gut microvasculature and blood flow over a 5000 × 7000 µm2 field of view at 5 µm resolution via the following two endogenous contrast mechanisms: intrinsic optical signals and laser speckles. Next, we transformed intestinal images into rectilinear "flat maps," and delineated 1A/V gut microvessels and their perfusion territories as "intestinal vascular units" (IVUs). Employing IVUs, we quantified and visualized NEC-induced changes to the gut vascular phenotype. RESULTS: In vivo imaging required 60-100 s per animal. Relative to the healthy gut, NEC intestines showed a significant overall decrease (i.e. 64-72%) in perfusion, accompanied by vasoconstriction (i.e. 9-12%) and a reduction in perfusion entropy (19%)within sections of the vascular bed. CONCLUSIONS: Multicontrast imaging coupled with IVU-based in vivo vascular phenotyping is a powerful new tool for elucidating NEC pathogenesis.

VascuViz: a multimodality and multiscale imaging and visualization pipeline for vascular systems biology.

Despite advances in imaging, image-based vascular systems biology has remained challenging because blood vessel data are often available only from a single modality or at a given spatial scale, and cross-modality data are difficult to integrate. Therefore, there is an exigent need for a multimodality pipeline that enables ex vivo vascular imaging with magnetic resonance imaging, computed tomography and optical microscopy of the same sample, while permitting imaging with complementary contrast mechanisms from the whole-organ to endothelial cell spatial scales. To achieve this, we developed 'VascuViz'-an easy-to-use method for simultaneous three-dimensional imaging and visualization of the vascular microenvironment using magnetic resonance imaging, computed tomography and optical microscopy in the same intact, unsectioned tissue. The VascuViz workflow permits multimodal imaging with a single labeling step using commercial reagents and is compatible with diverse tissue types and protocols. VascuViz's interdisciplinary utility in conjunction with new data visualization approaches opens up new vistas in image-based vascular systems biology.

Advances in translational imaging of the microcirculation.

The past few decades have seen an explosion in the development and use of methods for imaging the human microcirculation during health and disease. The confluence of innovative imaging technologies, affordable computing power, and economies of scale have ushered in a new era of "translational" imaging that permit us to peer into blood vessels of various organs in the human body. These imaging techniques include near-infrared spectroscopy (NIRS), positron emission tomography (PET), and magnetic resonance imaging (MRI) that are sensitive to microvascular-derived signals, as well as computed tomography (CT), optical imaging, and ultrasound (US) imaging that are capable of directly acquiring images at, or close to microvascular spatial resolution. Collectively, these imaging modalities enable us to characterize the morphological and functional changes in a tissue's microcirculation that are known to accompany the initiation and progression of numerous pathologies. Although there have been significant advances for imaging the microcirculation in preclinical models, this review focuses on developments in the assessment of the microcirculation in patients with optical imaging, NIRS, PET, US, MRI, and CT, to name a few. The goal of this review is to serve as a springboard for exploring the burgeoning role of translational imaging technologies for interrogating the structural and functional status of the microcirculation in humans, and highlight the breadth of current clinical applications. Making the human microcirculation "visible" in vivo to clinicians and researchers alike will facilitate bench-to-bedside discoveries and enhance the diagnosis and management of disease.

HemoSYS: A Toolkit for Image-based Systems Biology of Tumor Hemodynamics.

Abnormal tumor hemodynamics are a critical determinant of a tumor's microenvironment (TME), and profoundly affect drug delivery, therapeutic efficacy and the emergence of drug and radio-resistance. Since multiple hemodynamic variables can simultaneously exhibit transient and spatiotemporally heterogeneous behavior, there is an exigent need for analysis tools that employ multiple variables to characterize the anomalous hemodynamics within the TME. To address this, we developed a new toolkit called HemoSYS for quantifying the hemodynamic landscape within angiogenic microenvironments. It employs multivariable time-series data such as in vivo tumor blood flow (BF), blood volume (BV) and intravascular oxygen saturation (Hbsat) acquired concurrently using a wide-field multicontrast optical imaging system. The HemoSYS toolkit consists of propagation, clustering, coupling, perturbation and Fourier analysis modules. We demonstrate the utility of each module for characterizing the in vivo hemodynamic landscape of an orthotropic breast cancer model. With HemoSYS, we successfully described: (i) the propagation dynamics of acute hypoxia; (ii) the initiation and dissolution of distinct hemodynamic niches; (iii) tumor blood flow regulation via local vasomotion; (iv) the hemodynamic response to a systemic perturbation with carbogen gas; and (v) frequency domain analysis of hemodynamic heterogeneity in the TME. HemoSYS (freely downloadable via the internet) enables vascular phenotyping from multicontrast in vivo optical imaging data. Its modular design also enables characterization of non-tumor hemodynamics (e.g. brain), other preclinical disease models (e.g. stroke), vascular-targeted therapeutics, and hemodynamic data from other imaging modalities (e.g. MRI).

In vivo high-resolution diffusion tensor imaging of the developing neonatal rat cortex and its relationship to glial and dendritic maturation.

Diffusion tensor imaging (DTI) is increasingly utilized as a sensitive tool for studying brain maturation and injuries during the neonatal period. In this study, we acquired high resolution in vivo DTI data from neonatal rat brains from postnatal day 2 (P2) to P10 and correlated temporal changes in DTI derived markers with microstructural organization of glia, axons, and dendrites during this critical period of brain development. Group average images showed dramatic temporal changes in brain morphology, fractional anisotropy (FA) and mean diffusivity (MD). Most cortical regions showed a monotonous decline in FA and an initial increase in MD from P2 to P8 that declined slightly by P10. Qualitative histology revealed rapid maturation of the glial and dendritic networks in the developing cortex. In the cingulate and motor cortex, the decreases in FA over time significantly correlated with structural anisotropy values computed from histological sections stained with glial and dendritic markers. However, in the sensory and visual cortex, other factors probably contributed to the observed decreases in FA. We did not observe any significant correlations between FA and structural anisotropy computed from the axonal histological marker.

Tumor Ensemble-Based Modeling and Visualization of Emergent Angiogenic Heterogeneity in Breast Cancer.

There is a critical need for new tools to investigate the spatio-temporal heterogeneity and phenotypic alterations that arise in the tumor microenvironment. However, computational investigations of emergent inter- and intra-tumor angiogenic heterogeneity necessitate 3D microvascular data from 'whole-tumors' as well as "ensembles" of tumors. Until recently, technical limitations such as 3D imaging capabilities, computational power and cost precluded the incorporation of whole-tumor microvascular data in computational models. Here, we describe a novel computational approach based on multimodality, 3D whole-tumor imaging data acquired from eight orthotopic breast tumor xenografts (i.e. a tumor 'ensemble'). We assessed the heterogeneous angiogenic landscape from the microvascular to tumor ensemble scale in terms of vascular morphology, emergent hemodynamics and intravascular oxygenation. We demonstrate how the abnormal organization and hemodynamics of the tumor microvasculature give rise to unique microvascular niches within the tumor and contribute to inter- and intra-tumor heterogeneity. These tumor ensemble-based simulations together with unique data visualization approaches establish the foundation of a novel 'cancer atlas' for investigators to develop their own in silico systems biology applications. We expect this hybrid image-based modeling framework to be adaptable for the study of other tissues (e.g. brain, heart) and other vasculature-dependent diseases (e.g. stroke, myocardial infarction).

A miniature multi-contrast microscope for functional imaging in freely behaving animals.

Neurovascular coupling, cerebrovascular remodeling and hemodynamic changes are critical to brain function, and dysregulated in neuropathologies such as brain tumors. Interrogating these phenomena in freely behaving animals requires a portable microscope with multiple optical contrast mechanisms. Therefore, we developed a miniaturized microscope with: a fluorescence (FL) channel for imaging neural activity (e.g., GCaMP) or fluorescent cancer cells (e.g., 9L-GFP); an intrinsic optical signal (IOS) channel for imaging hemoglobin absorption (i.e., cerebral blood volume); and a laser speckle contrast (LSC) channel for imaging perfusion (i.e., cerebral blood flow). Following extensive validation, we demonstrate the microscope's capabilities via experiments in unanesthetized murine brains that include: (i) multi-contrast imaging of neurovascular changes following auditory stimulation; (ii) wide-area tonotopic mapping; (iii) EEG-synchronized imaging during anesthesia recovery; and (iv) microvascular connectivity mapping over the life-cycle of a brain tumor. This affordable, flexible, plug-and-play microscope heralds a new era in functional imaging of freely behaving animals.

Brain tumors disrupt the resting-state connectome.

Brain tumor patients often experience functional deficits that extend beyond the tumor site. While resting-state functional MRI (rsfMRI) has been used to map such functional connectivity changes in brain tumor patients, the interplay between abnormal tumor vasculature and the rsfMRI signal is still not well understood. Therefore, there is an exigent need for new tools to elucidate how the blood­oxygenation-level-dependent (BOLD) rsfMRI signal is modulated in brain cancer. In this initial study, we explore the utility of a preclinical model for quantifying brain tumor-induced changes on the rsfMRI signal and resting-state brain connectivity. We demonstrate that brain tumors induce brain-wide alterations of resting-state networks that extend to the contralateral hemisphere, accompanied by global attenuation of the rsfMRI signal. Preliminary histology suggests that some of these alterations in brain connectivity may be attributable to tumor-related remodeling of the neurovasculature. Moreover, this work recapitulates clinical rsfMRI findings from brain tumor patients in terms of the effects of tumor size on the neurovascular microenvironment. Collectively, these results lay the foundation of a preclinical platform for exploring the usefulness of rsfMRI as a potential new biomarker in patients with brain cancer.

Phenotyping the Microvasculature in Critical-Sized Calvarial Defects via Multimodal Optical Imaging.

Tissue-engineered scaffolds are a powerful means of healing craniofacial bone defects arising from trauma or disease. Murine models of critical-sized bone defects are especially useful in understanding the role of microenvironmental factors such as vascularization on bone regeneration. Here, we demonstrate the capability of a novel multimodality imaging platform capable of acquiring in vivo images of microvascular architecture, microvascular blood flow, and tracer/cell tracking via intrinsic optical signaling (IOS), laser speckle contrast (LSC), and fluorescence (FL) imaging, respectively, in a critical-sized calvarial defect model. Defects that were 4 mm in diameter were made in the calvarial regions of mice followed by the implantation of osteoconductive scaffolds loaded with human adipose-derived stem cells embedded in fibrin gel. Using IOS imaging, we were able to visualize microvascular angiogenesis at the graft site and extracted morphological information such as vessel radius, length, and tortuosity two weeks after scaffold implantation. FL imaging allowed us to assess functional characteristics of the angiogenic vessel bed, such as time-to-peak of a fluorescent tracer, and also allowed us to track the distribution of fluorescently tagged human umbilical vein endothelial cells. Finally, we used LSC to characterize the in vivo hemodynamic response and maturity of the remodeled microvessels in the scaffold microenvironment. In this study, we provide a methodical framework for imaging tissue-engineered scaffolds, processing the images to extract key microenvironmental parameters, and visualizing these data in a manner that enables the characterization of the vascular phenotype and its effect on bone regeneration. Such multimodality imaging platforms can inform optimization and design of tissue-engineered scaffolds and elucidate the factors that promote enhanced vascularization and bone formation.

Quantification and tracking of genetically engineered dendritic cells for studying immunotherapy.

PURPOSE: Genetically encoded reporters can assist in visualizing biological processes in live organisms and have been proposed for longitudinal and noninvasive tracking of therapeutic cells in deep tissue. Cells can be labeled in situ or ex vivo and followed in live subjects over time. Nevertheless, a major challenge for reporter systems is to identify the cell population that actually expresses an active reporter. METHODS: We have used a nucleoside analog, pyrrolo-2'-deoxycytidine, as an imaging probe for the putative reporter gene, Drosophila melanogaster 2'-deoxynucleoside kinase. Bioengineered cells were imaged in vivo in animal models of brain tumor and immunotherapy using chemical exchange saturation transfer MRI. The number of transduced cells was quantified by flow cytometry based on the optical properties of the probe. RESULTS: We performed a comparative analysis of six different cell lines and demonstrate utility in a mouse model of immunotherapy. The proposed technology can be used to quantify the number of labeled cells in a given region, and moreover is sensitive enough to detect less than 10,000 cells. CONCLUSION: This unique technology that enables efficient selection of labeled cells followed by in vivo monitoring with both optical and MRI. Magn Reson Med 79:1010-1019, 2018. © 2017 International Society for Magnetic Resonance in Medicine.

Implications of neurovascular uncoupling in functional magnetic resonance imaging (fMRI) of brain tumors.

Functional magnetic resonance imaging (fMRI) serves as a critical tool for presurgical mapping of eloquent cortex and changes in neurological function in patients diagnosed with brain tumors. However, the blood-oxygen-level-dependent (BOLD) contrast mechanism underlying fMRI assumes that neurovascular coupling remains intact during brain tumor progression, and that measured changes in cerebral blood flow (CBF) are correlated with neuronal function. Recent preclinical and clinical studies have demonstrated that even low-grade brain tumors can exhibit neurovascular uncoupling (NVU), which can confound interpretation of fMRI data. Therefore, to avoid neurosurgical complications, it is crucial to understand the biophysical basis of NVU and its impact on fMRI. Here we review the physiology of the neurovascular unit, how it is remodeled, and functionally altered by brain cancer cells. We first discuss the latest findings about the components of the neurovascular unit. Next, we synthesize results from preclinical and clinical studies to illustrate how brain tumor induced NVU affects fMRI data interpretation. We examine advances in functional imaging methods that permit the clinical evaluation of brain tumors with NVU. Finally, we discuss how the suppression of anomalous tumor blood vessel formation with antiangiogenic therapies can "normalize" the brain tumor vasculature, and potentially restore neurovascular coupling.

Breast cancer cell cyclooxygenase-2 expression alters extracellular matrix structure and function and numbers of cancer associated fibroblasts.

Cyclooxygenase-2 (COX-2) is a critically important mediator of inflammation that significantly influences tumor angiogenesis, invasion, and metastasis. We investigated the role of COX-2 expressed by triple negative breast cancer cells in altering the structure and function of the extracellular matrix (ECM). COX-2 downregulation effects on ECM structure and function were investigated using magnetic resonance imaging (MRI) and second harmonic generation (SHG) microscopy of tumors derived from triple negative MDA-MB-231 breast cancer cells, and a derived clone stably expressing a short hairpin (shRNA) molecule downregulating COX-2. MRI of albumin-GdDTPA was used to characterize macromolecular fluid transport in vivo and SHG microscopy was used to quantify collagen 1 (Col1) fiber morphology. COX-2 downregulation decreased Col1 fiber density and altered macromolecular fluid transport. Immunohistochemistry identified significantly fewer activated cancer associated fibroblasts (CAFs) in low COX-2 expressing tumors. Metastatic lung nodules established by COX-2 downregulated cells were infrequent, smaller, and contained fewer Col1 fibers.COX-2 overexpression studies were performed with tumors derived from triple negative SUM-149 breast cancer cells lentivirally transduced to overexpress COX-2. SHG microscopy identified significantly higher Col1 fiber density in COX-2 overexpressing tumors with an increase of CAFs. These data expand upon the roles of COX-2 in shaping the structure and function of the ECM in primary and metastatic tumors, and identify the potential role of COX-2 in modifying the number of CAFs in tumors that may have contributed to the altered ECM.

Structure and Function of a Prostate Cancer Dissemination-Permissive Extracellular Matrix.

Purpose: The poor prognosis of metastatic prostate cancer continues to present a major challenge in prostate cancer treatment. The tumor extracellular matrix (ECM) plays an important role in facilitating metastasis. Here, we investigated the structure and function of an ECM that facilitates prostate cancer metastasis by comparing orthotopic tumors that frequently metastasize to poorly metastatic subcutaneous tumors.Experimental Design: Both tumors were derived from a human prostate cancer PC3 cell line engineered to fluoresce under hypoxia. Second harmonic generation (SHG) microscopy was used to characterize collagen 1 (Col1) fiber patterns in the xenografts as well as in human samples. MRI was used to determine albumin-Gd-diethylenetriaminepenta-acetate (alb-GdDTPA) transport through the ECM using a saturation recovery MR method combined with fast T1 SNAPSHOT-FLASH imaging. Cancer-associated fibroblasts (CAF) were also quantified in these tumors.Results: Significant structural and functional differences were identified in the prometastatic orthotopic tumor ECM compared to the less metastatic subcutaneous tumor ECM. The significantly higher number of CAFs in orthotopic tumors may explain the higher Col1 fiber volumes in these tumors. In vivo, alb-GdDTPA pooling was significantly elevated in metastatic orthotopic tumors, consistent with the increased Col1 fibers.Conclusions: Developing noninvasive MRI indices of macromolecular transport, together with characterization of Col1 fiber patterns and CAFs can assist in stratifying prostate cancers for aggressive treatments or active surveillance. These results highlight the role of CAFs in supporting or creating aggressive cancers, and the importance of depleting CAFs to prevent metastatic dissemination in prostate cancer. Clin Cancer Res; 23(9); 2245-54. ©2016 AACR.