Microengineering 3D Collagen Matrices with Tumor-Mimetic Gradients in Fiber Alignment.

Collagen fibers in the 3D tumor microenvironment (TME) exhibit complex alignment landscapes that are critical in directing cell migration through a process called contact guidance. Previous in vitro work studying this phenomenon has focused on quantifying cell responses in uniformly aligned environments. However, the TME also features short-range gradients in fiber alignment that result from cell-induced traction forces. Although the influence of graded biophysical taxis cues is well established, cell responses to physiological alignment gradients remain largely unexplored. In this work, fiber alignment gradients in biopsy samples are characterized and recreated using a new microfluidic biofabrication technique to achieve tunable sub-millimeter to millimeter scale gradients. This study represents the first successful engineering of continuous alignment gradients in soft, natural biomaterials. Migration experiments on graded alignment show that HUVECs exhibit increased directionality, persistence, and speed compared to uniform and unaligned fiber architectures. Similarly, patterned MDA-MB-231 aggregates exhibit biased migration toward increasing fiber alignment, suggesting a role for alignment gradients as a taxis cue. This user-friendly approach, requiring no specialized equipment, is anticipated to offer new insights into the biophysical cues that cells interpret as they traverse the extracellular matrix, with broad applicability in healthy and diseased tissue environments.

Expanding the applicability of multiphoton fluorescence recovery after photobleaching by incorporating shear stress in laminar flow.

Significance: Multi-photon fluorescence recovery after photobleaching (MPFRAP) is a nonlinear microscopy technique used to measure the diffusion coefficient of fluorescently tagged molecules in solution. Previous MPFRAP fitting models calculate the diffusion coefficient in systems with diffusion or diffusion in laminar flow. Aim: We propose an MPFRAP fitting model that accounts for shear stress in laminar flow, making it a more applicable technique for in vitro and in vivo studies involving diffusion. Approach: Fluorescence recovery curves are generated using high-throughput molecular dynamics simulations and then fit to all three models (diffusion, diffusion and flow, and diffusion and shear flow) to define the limits within which accurate diffusion coefficients are produced. Diffusion is simulated as a random walk with a variable horizontal bias to account for shear flow. Results: Contour maps of the accuracy of the fitted diffusion coefficient as a function of scaled velocity and scaled shear rate show the parameter space within which each model produces accurate diffusion coefficients; the shear-flow model covers a larger area than the previous models. Conclusion: The shear-flow model allows MPFRAP to be a viable optical tool for studying more biophysical systems than previous models.

Microengineering 3D Collagen Matrices with Tumor-Mimetic Gradients in Fiber Alignment.

In the tumor microenvironment (TME), collagen fibers facilitate tumor cell migration through the extracellular matrix. Previous studies have focused on studying the responses of cells on uniformly aligned or randomly aligned collagen fibers. However, the in vivo environment also features spatial gradients in alignment, which arise from the local reorganization of the matrix architecture due to cell-induced traction forces. Although there has been extensive research on how cells respond to graded biophysical cues, such as stiffness, porosity, and ligand density, the cellular responses to physiological fiber alignment gradients have been largely unexplored. This is due, in part, to a lack of robust experimental techniques to create controlled alignment gradients in natural materials. In this study, we image tumor biopsy samples and characterize the alignment gradients present in the TME. To replicate physiological gradients, we introduce a first-of-its-kind biofabrication technique that utilizes a microfluidic channel with constricting and expanding geometry to engineer 3D collagen hydrogels with tunable fiber alignment gradients that range from sub-millimeter to millimeter length scales. Our modular approach allows easy access to the microengineered gradient gels, and we demonstrate that HUVECs migrate in response to the fiber architecture. We provide preliminary evidence suggesting that MDA-MB-231 cell aggregates, patterned onto a specific location on the alignment gradient, exhibit preferential migration towards increasing alignment. This finding suggests that alignment gradients could serve as an additional taxis cue in the ECM. Importantly, our study represents the first successful engineering of continuous gradients of fiber alignment in soft, natural materials. We anticipate that our user-friendly platform, which needs no specialized equipment, will offer new experimental capabilities to study the impact of fiber-based contact guidance on directed cell migration.

Matching an immersion medium's refractive index to a cell's cytosol isolates organelle scattering.

Angularly-resolved light scattering has been proven to be an early detector of subtle changes in organelle size due to its sensitivity to scatterer size and refractive index contrast. However, for cells immersed in media with a refractive index close to 1.33, the cell itself acts as a larger scatterer and contributes its own angular signature. This whole-cell scattering, highly dependent on the cell's shape and size, is challenging to distinguish from the desired organelle scattering signal. This degrades the accuracy with which organelle size information can be extracted from the angular scattering. To mitigate this effect, we manipulate the refractive index of the immersion medium by mixing it with a water-soluble, biocompatible, high-refractive-index liquid. This approach physically reduces the amount of whole-cell scattering by minimizing the refractive index contrast between the cytosol and the modified medium. We demonstrate this technique on live cells adherent on a coverslip, using Fourier transform light scattering to compute the angular scattering from complex field images. We show that scattering from the cell: media refractive index contrast contributes significant scattering at angles up to twenty degrees and that refractive index-matching reduces such low-angle scatter by factors of up to 4.5. This result indicates the potential of refractive index-matching for improving the estimates of organelle size distributions in single cells.

Multiphoton Phosphorescence Quenching Microscopy Reveals Kinetics of Tumor Oxygenation during Antiangiogenesis and Angiotensin Signaling Inhibition.

PURPOSE: The abnormal function of tumor blood vessels causes tissue hypoxia, promoting disease progression and treatment resistance. Although tumor microenvironment normalization strategies can alleviate hypoxia globally, how local oxygen levels change is not known because of the inability to longitudinally assess vascular and interstitial oxygen in tumors with sufficient resolution. Understanding the spatial and temporal heterogeneity should help improve the outcome of various normalization strategies. EXPERIMENTAL DESIGN: We developed a multiphoton phosphorescence quenching microscopy system using a low-molecular-weight palladium porphyrin probe to measure perfused vessels, oxygen tension, and their spatial correlations in vivo in mouse skin, bone marrow, and four different tumor models. Further, we measured the temporal and spatial changes in oxygen and vessel perfusion in tumors in response to an anti-VEGFR2 antibody (DC101) and an angiotensin-receptor blocker (losartan). RESULTS: We found that vessel function was highly dependent on tumor type. Although some tumors had vessels with greater oxygen-carrying ability than those of normal skin, most tumors had inefficient vessels. Further, intervessel heterogeneity in tumors is associated with heterogeneous response to DC101 and losartan. Using both vascular and stromal normalizing agents, we show that spatial heterogeneity in oxygen levels persists, even with reductions in mean extravascular hypoxia. CONCLUSIONS: High-resolution spatial and temporal responses of tumor vessels to two agents known to improve vascular perfusion globally reveal spatially heterogeneous changes in vessel structure and function. These dynamic vascular changes should be considered in optimizing the dose and schedule of vascular and stromal normalizing strategies to improve the therapeutic outcome.

Second-Harmonic Generation Imaging Reveals Changes in Breast Tumor Collagen Induced by Neoadjuvant Chemotherapy.

Breast cancer is the most common invasive cancer in women, with most deaths attributed to metastases. Neoadjuvant chemotherapy (NACT) may be prescribed prior to surgical removal of the tumor for subsets of breast cancer patients but can have diverse undesired and off-target effects, including the increased appearance of the 'tumor microenvironment of metastasis', image-based multicellular signatures that are prognostic of breast tumor metastasis. To assess whether NACT can induce changes in two other image-based prognostic/predictive signatures derived from tumor collagen, we quantified second-harmonic generation (SHG) directionality and fiber alignment in formalin-fixed, paraffin-embedded sections of core needle biopsies and primary tumor excisions from 22 human epidermal growth factor receptor 2-overexpressing (HER2+) and 22 triple-negative breast cancers. In both subtypes, we found that SHG directionality (i.e., the forward-to-backward scattering ratio, or F/B) is increased by NACT in the bulk of the tumor, but not the adjacent tumor-stroma interface. Overall collagen fiber alignment is increased by NACT in triple-negative but not HER2+ breast tumors. These results suggest that NACT impacts the collagenous extracellular matrix in a complex and subtype-specific manner, with some prognostic features being unchanged while others are altered in a manner suggestive of a more metastatic phenotype.

Spatiotemporal blood vessel specification at the osteogenesis and angiogenesis interface of biomimetic nanofiber-enabled bone tissue engineering.

While extensive research has demonstrated an interdependent role of osteogenesis and angiogenesis in bone tissue engineering, little is known about how functional blood vessel networks are organized to initiate and facilitate bone tissue regeneration. Building upon the success of a biomimetic composite nanofibrous construct capable of supporting donor progenitor cell-dependent regeneration, we examined the angiogenic response and spatiotemporal blood vessel specification at the osteogenesis and angiogenesis interface of cranial bone defect repair utilizing high resolution multiphoton laser scanning microscopy (MPLSM) in conjunction with intravital imaging. We demonstrate here that the regenerative vasculature can be specified as arterial and venous capillary vessels based upon endothelial surface markers of CD31 and Endomucin (EMCN), with CD31+EMCN- vessels exhibiting higher flowrate and higher oxygen tension (pO2) than CD31+EMCN+ vessels. The donor osteoblast clusters are uniquely coupled to the sprouting CD31+EMCN+ vessels connecting to CD31+EMCN- vessels. Further analyses reveal differential vascular response and vessel type distribution in healing and non-healing defects, associated with changes of gene sets that control sprouting and morphogenesis of blood vessels. Collectively, our study highlights the key role of spatiotemporal vessel type distribution in bone tissue engineering, offering new insights for devising more effective vascularization strategies for bone tissue engineering.

Intratumoral heterogeneity of second-harmonic generation scattering from tumor collagen and its effects on metastatic risk prediction.

BACKGROUND: Metastases are the leading cause of breast cancer-related deaths. The tumor microenvironment impacts cancer progression and metastatic ability. Fibrillar collagen, a major extracellular matrix component, can be studied using the light scattering phenomenon known as second-harmonic generation (SHG). The ratio of forward- to backward-scattered SHG photons (F/B) is sensitive to collagen fiber internal structure and has been shown to be an independent prognostic indicator of metastasis-free survival time (MFS). Here we assess the effects of heterogeneity in the tumor matrix on the possible use of F/B as a prognostic tool. METHODS: SHG imaging was performed on sectioned primary tumor excisions from 95 untreated, estrogen receptor-positive, lymph node negative invasive ductal carcinoma patients. We identified two distinct regions whose collagen displayed different average F/B values, indicative of spatial heterogeneity: the cellular tumor bulk and surrounding tumor-stroma interface. To evaluate the impact of heterogeneity on F/B's prognostic ability, we performed SHG imaging in the tumor bulk and tumor-stroma interface, calculated a 21-gene recurrence score (surrogate for OncotypeDX®, or S-ODX) for each patient and evaluated their combined prognostic ability. RESULTS: We found that F/B measured in tumor-stroma interface, but not tumor bulk, is prognostic of MFS using three methods to select pixels for analysis: an intensity threshold selected by a blinded observer, a histogram-based thresholding method, and an adaptive thresholding method. Using both regression trees and Random Survival Forests for MFS outcome, we obtained data-driven prediction rules that show F/B from tumor-stroma interface, but not tumor bulk, and S-ODX both contribute to predicting MFS in this patient cohort. We also separated patients into low-intermediate (S-ODX < 26) and high risk (S-ODX ≥26) groups. In the low-intermediate risk group, comprised of patients not typically recommended for adjuvant chemotherapy, we find that F/B from the tumor-stroma interface is prognostic of MFS and can identify a patient cohort with poor outcomes. CONCLUSIONS: These data demonstrate that intratumoral heterogeneity in F/B values can play an important role in its possible use as a prognostic marker, and that F/B from tumor-stroma interface of primary tumor excisions may provide useful information to stratify patients by metastatic risk.

Chronic Stress Exposure Suppresses Mammary Tumor Growth and Reduces Circulating Exosome TGF-ß Content via ß-Adrenergic Receptor Signaling in MMTV-PyMT Mice.

Preclinical models of breast cancer have established mechanistic links between psychological stress and cancer progression. However, epidemiological evidence linking stress and cancer is equivocal. We tested the impact of stress exposure in female mice expressing the mouse mammary tumor virus polyoma middle-T antigen (MMTV-PyMT), a spontaneous model of mammary adenocarcinoma that mimics metastatic hormone receptor-positive human breast cancer development. MMTV-PyMT mice were socially isolated at 6 to 7 weeks of age during premalignant hyperplasia. To increase the potency of the stressor, singly housed mice were exposed to acute restraint stress (2 hours per day for 3 consecutive days) at 8 to 9 weeks of age during early carcinoma. Exposure to this dual stressor activated both major stress pathways, the sympathetic nervous system and hypothalamic-pituitary-adrenal axis throughout malignant transformation. Stressor exposure reduced mammary tumor burden in association with increased tumor cleaved caspase-3 expression, indicative of increased cell apoptosis. Stress exposure transiently increased tumor vascular endothelial growth factor and reduced tumor interleukin-6, but no other significant alterations in immune/inflammation-associated chemokines and cytokines or changes in myeloid cell populations were detected in tumors. No stress-induced change in second-harmonic generation-emitting collagen, indicative of a switch to a metastasis-promoting tumor extracellular matrix, was detected. Systemic indicators of slowed tumor progression included reduced myeloid-derived suppressor cell (MDSC) frequency in lung and spleen, and decreased transforming growth factor ß (TGF-ß) content in circulating exosomes, nanometer-sized particles associated with tumor progression. Chronic ß-adrenergic receptor (ß-AR) blockade with nadolol abrogated stress-induced alterations in tumor burden and cleaved caspase-3 expression, lung MDSC frequency, and exosomal TGF-ß content. Despite the evidence for reduced tumor growth, metastatic lesions in the lung were not altered by stress exposure. Unexpectedly, ß-blockade in nonstressed mice increased lung metastatic lesions and splenic MDSC frequency, suggesting that in MMTV-PyMT mice, ß-AR activation also inhibits tumor progression in the absence of stress exposure. Together, these results suggest stress exposure can act through ß-AR signaling to slow primary tumor growth in MMTV-PyMT mice.

Second-harmonic generation directionality is associated with neoadjuvant chemotherapy response in breast cancer core needle biopsies.

Neoadjuvant chemotherapy (NACT) is routinely administered to subsets of breast cancer patients, including triple negative (TN) or human epidermal growth factor receptor 2-positive (HER2+) cancers. After NACT and subsequent surgical resection, 5% to 30% of patients have no residual invasive carcinoma, termed pathological complete response. Unfortunately, many patients experience little-to-no response after NACT and unnecessarily suffer its side effects. Methods are needed to predict an individual patient's response to NACT. Core needle biopsies, taken before NACT, consist of tumor cells and the surrounding extracellular matrix. We performed second-harmonic generation (SHG) imaging of fibrillar collagen in core needle biopsy sections as a possible predictor of response to NACT. The ratio of forward-to-backward scattering (F/B) SHG was assessed in the "tumor bulk" and "tumor­host interface" in HER2+ and TN core needle biopsy sections. Patient response was classified post-treatment using the Residual Cancer Burden (RCB) score. In HER2+ biopsies, RCB class was associated with F/B derived from the tumor­stromal interface, but not tumor bulk. F/B was not associated with RCB class in TN biopsies. These findings suggest that F/B from needle biopsy sections may be a useful predictor of which patients will respond favorably to NACT, with the potential to help reduce overtreatment.

Tissue effects of intra-tissue refractive index shaping (IRIS): insights from two-photon autofluorescence and second harmonic generation microscopy.

Intra-tissue refractive index shaping (IRIS) is a novel, non-ablative form of vision correction by which femtosecond laser pulses are tightly focused into ocular tissues to induce localized refractive index (RI) change via nonlinear absorption. Here, we examined the effects of Blue-IRIS on corneal microstructure to gain insights into underlying mechanisms. Three-layer grating patterns were inscribed with IRIS ~180 µm below the epithelial surface of ex vivo rabbit globes using a 400 nm femtosecond laser. Keeping laser power constant at 82 mW in the focal volume, multiple patterns were written at different scan speeds. The largest RI change induced in this study was + 0.011 at 20 mm/s. After measuring the phase change profile of each inscribed pattern, two-photon excited autofluorescence (TPEF) and second harmonic generation (SHG) microscopy were used to quantify changes in stromal structure. While TPEF increased significantly with induced RI change, there was a noticeable suppression of SHG signal in IRIS treated regions. We posit that enhancement of TPEF was due to the formation of new fluorophores, while decreases in SHG were most likely due to degradation of collagen triple helices. All in all, the changes observed suggest that IRIS works by inducing a localized, photochemical change in collagen structure.

Embryo movements regulate tendon mechanical property development.

Tendons transmit forces from muscles to bones to enable skeletal motility. During development, tendons begin to bear load at the onset of embryo movements. Using the chick embryo model, this study showed that altered embryo movement frequency led to changes in elastic modulus of calcaneal tendon. In particular, paralysis led to decreased modulus, whereas hypermotility led to increased modulus. Paralysis also led to reductions in activity levels of lysyl oxidase (LOX), an enzyme that we previously showed is required for cross-linking-mediated elaboration of tendon mechanical properties. Additionally, inhibition of LOX activity abrogated hypermotility-induced increases in modulus. Taken together, our findings suggest embryo movements are critical for tendon mechanical property development and implicate LOX in this process. These exciting findings expand current knowledge of how functional tendons form during development and could guide future clinical approaches to treat tendon defects associated with abnormal mechanical loading in uteroThis article is part of the Theo Murphy meeting issue 'Mechanics of development'.

Chronic cigarette smoke exposure drives spiral ganglion neuron loss in mice.

Tobacco use is associated with an increased risk of hearing loss in older individuals, suggesting cigarette smoke (CS) exposure may target the peripheral auditory organs. However, the effects of CS exposure on general cochlear anatomy have not previously been explored. Here we compare control and chronic CS exposed cochleae from adult mice to assess changes in structure and cell survival. Two-photon imaging techniques, including the imaging of second harmonic generation (SHG) and two-photon excitation fluorescence (TPEF) from native molecules, were used to probe the whole cochlear organ for changes. We found evidence for fibrillar collagen accumulation in the spiral ganglion and organ of Corti, consistent with fibrosis. Quantitative TPEF indicated that basal CS-exposed spiral ganglion neurons experienced greater oxidative stress than control neurons, which was confirmed by histological staining for lipid peroxidation products. Cell counts confirmed that the CS-exposed spiral ganglion also contained fewer basal neurons. Taken together, these data support the premise that CS exposure induces oxidative stress in cochlear cells. They also indicate that two-photon techniques may screen cochlear tissues for oxidative stress.

Chemotherapeutic drug-specific alteration of microvascular blood flow in murine breast cancer as measured by diffuse correlation spectroscopy.

The non-invasive, in vivo measurement of microvascular blood flow has the potential to enhance breast cancer therapy monitoring. Here, longitudinal blood flow of 4T1 murine breast cancer (N=125) under chemotherapy was quantified with diffuse correlation spectroscopy based on layer models. Six different treatment regimens involving doxorubicin, cyclophosphamide, and paclitaxel at clinically relevant doses were investigated. Treatments with cyclophosphamide increased blood flow as early as 3 days after administration, whereas paclitaxel induced a transient blood flow decrease at 1 day after administration. Early blood flow changes correlated strongly with the treatment outcome and distinguished treated from untreated mice individually for effective treatments.

Antidepressant use and risk of central nervous system metastasis.

Selective serotonin reuptake inhibitors (SSRIs), a class of antidepressants, were found to increase central nervous system (CNS) metastasis in mice. Our study investigated in humans whether antidepressants, and specifically SSRIs, increased the relative odds of CNS metastasis. We identified 189 cases of CNS metastasis amongst breast cancer, melanoma, and non-Hodgkin lymphoma subjects who were diagnosed with CNS metastasis or infiltration between January 1, 2005 and September 30, 2013 and 756 controls (patients without CNS metastasis or infiltration). Using logistic regression, we estimated the relative odds of CNS metastasis associated with antidepressant use adjusting for relevant covariates. The prevalence of antidepressants was 28.6 % in cases and 27.5 % in controls, whereas SSRIs were used in 16.9 % of cases and 17.3 % of controls. Among all patients, antidepressants were not associated with CNS metastasis or infiltration. No consistent patterns of association were observed in the analyses of other cancer subsets or exposure measures, with the possible exception of an increased risk of CNS metastasis associated with 'any SSRI use' among breast cancer patients (OR = 1.73, 95 % CI = 0.75, 4.04). We did not observe clear patterns of association, which may be due in part to the small sample size in many of our analyses.

Second harmonic generation microscopy reveals altered collagen microstructure in usual interstitial pneumonia versus healthy lung.

BACKGROUND: It is not understood why some pulmonary fibroses such as cryptogenic organizing pneumonia (COP) respond well to treatment, while others like usual interstitial pneumonia (UIP) do not. Increased understanding of the structure and function of the matrix in this area is critical to improving our understanding of the biology of these diseases and developing novel therapies. The objectives herein are to provide new insights into the underlying collagen- and matrix-related biological mechanisms driving COP versus UIP. METHODS: Two-photon second harmonic generation (SHG) and excitation fluorescence microscopies were used to interrogate and quantify differences between intrinsic fibrillar collagen and elastin matrix signals in healthy, COP, and UIP lung. RESULTS: Collagen microstructure was different in UIP versus healthy lung, but not in COP versus healthy, as indicated by the ratio of forward-to-backward propagating SHG signal (FSHG/BSHG). This collagen microstructure as assessed by FSHG/BSHG was also different in areas with preserved alveolar architecture adjacent to UIP fibroblastic foci or honeycomb areas versus healthy lung. Fibrosis was evidenced by increased col1 and col3 content in COP and UIP versus healthy, with highest col1:col3 ratio in UIP. Evidence of elastin breakdown (i.e. reduced mature elastin fiber content), and increased collagen:mature elastin ratios, were seen in COP and UIP versus healthy. CONCLUSIONS: Fibrillar collagen's subresolution structure (i.e. "microstructure") is altered in UIP versus COP and healthy lung, which may provide novel insights into the biological reasons why unlike COP, UIP is resistant to therapies, and demonstrates the ability of SHG microscopy to potentially distinguish treatable versus intractable pulmonary fibroses.

Spatiotemporal Analyses of Osteogenesis and Angiogenesis via Intravital Imaging in Cranial Bone Defect Repair.

Osteogenesis and angiogenesis are two integrated components in bone repair and regeneration. A deeper understanding of osteogenesis and angiogenesis has been hampered by technical difficulties of analyzing bone and neovasculature simultaneously in spatiotemporal scales and in 3D formats. To overcome these barriers, a cranial defect window chamber model was established that enabled high-resolution, longitudinal, and real-time tracking of angiogenesis and bone defect healing via multiphoton laser scanning microscopy (MPLSM). By simultaneously probing new bone matrix via second harmonic generation (SHG), neovascular networks via intravenous perfusion of fluorophore, and osteoblast differentiation via 2.3-kb collagen type I promoter-driven GFP (Col2.3GFP), we examined the morphogenetic sequence of cranial bone defect healing and further established the spatiotemporal analyses of osteogenesis and angiogenesis coupling in repair and regeneration. We showed that bone defect closure was initiated in the residual bone around the edge of the defect. The expansion and migration of osteoprogenitors into the bone defect occurred during the first 3 weeks of healing, coupled with vigorous microvessel angiogenesis at the leading edge of the defect. Subsequent bone repair was marked by matrix deposition and active vascular network remodeling within new bone. Implantation of bone marrow stromal cells (BMSCs) isolated from Col2.3GFP mice further showed that donor-dependent bone formation occurred rapidly within the first 3 weeks of implantation, in concert with early angiogenesis. The subsequent bone wound closure was largely host-dependent, associated with localized modest induction of angiogenesis. The establishment of a live imaging platform via cranial window provides a unique tool to understand osteogenesis and angiogenesis in repair and regeneration, enabling further elucidation of the spatiotemporal regulatory mechanisms of osteoprogenitor cell interactions with host bone healing microenvironment.

Single- and two-photon fluorescence recovery after photobleaching.

Fluorescence recovery after photobleaching (FRAP) is a microscopy technique for measuring the kinetics of fluorescently labeled molecules and can be applied both in vitro and in vivo for two- and three-dimensional systems. This introduction discusses the three basic FRAP methods: traditional FRAP, multiphoton FRAP (MPFRAP), and FRAP with spatial Fourier analysis (SFA-FRAP). Each discussion is accompanied by a description of the mathematical analysis appropriate for situations in which the recovery kinetics is dictated by free diffusion. In some experiments, the recovery kinetics is dictated by the boundary conditions of the system, and FRAP is then used to quantify the connectivity of various compartments. Because the appropriate mathematical analysis is independent of the bleaching method, the analysis of compartmental connectivity is discussed last, in a separate section.

The role of the lymphatic system in inflammatory-erosive arthritis.

Rheumatoid arthritis (RA) is a prevalent inflammatory joint disease with enigmatic flares, which causes swelling, pain, and irreversible connective tissue damage. Recently, it has been demonstrated in murine models of RA that the popliteal lymph node (PLN) is a biomarker of arthritic flare, as it "expands" in size and contrast enhancement during a prolonged asymptomatic phase, prior to when it "collapses" with accelerated synovitis and joint erosion. This PLN collapse is associated with adjacent knee flare, decreases in PLN volume and contrast enhancement, lymphatic pulse and pumping pressure, and an increase in PLN pressure. Currently, it is known that PLN collapse is accompanied by a translocation of B cells from the follicles to the sinuses, effectively clogging the lymphatic sinuses of the PLN, and that B cell depletion therapy ameliorates arthritic flare by eliminating these B cells and restoring passive lymphatic flow from inflamed joints. Here we review the technological advances that have launched this area of research, describe future directions to help elucidate the potential mechanism of PLN collapse, and speculate on clinical translation towards new diagnostics and therapies for RA.