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.

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.

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.

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.

In vivo quantification of lymph viscosity and pressure in lymphatic vessels and draining lymph nodes of arthritic joints in mice.

Rheumatoid arthritis (RA) is a chronic inflammatory joint disease with episodic flares. In TNF-Tg mice, a model of inflammatory-erosive arthritis, the popliteal lymph node (PLN) enlarges during the pre-arthritic 'expanding' phase, and then 'collapses' with adjacent knee flare associated with the loss of the intrinsic lymphatic pulse. As the mechanisms responsible are unknown, we developed in vivo methods to quantify lymph viscosity and pressure in mice with wild-type (WT), expanding and collapsed PLN. While no differences in viscosity were detected via multiphoton fluorescence recovery after photobleaching (MP-FRAP) of injected FITC-BSA, a 32.6% decrease in lymph speed was observed in vessels afferent to collapsed PLN (P < 0.05). Direct measurement of intra-lymph node pressure (LNP) demonstrated a decrease in expanding PLN versus WT pressure (3.41 ± 0.43 vs. 6.86 ± 0.56 cmH2O; P < 0.01), which dramatically increased to 9.92 ± 1.79 cmH2O in collapsed PLN. Lymphatic pumping pressure (LPP), measured indirectly by slowly releasing a pressurized cuff occluding indocyanine green (ICG), demonstrated an increase in vessels afferent to expanding PLN versus WT (18.76 ± 2.34 vs. 11.04 ± 1.47 cmH2O; P < 0.01), which dropped to 2.61 ± 0.72 cmH2O (P < 0.001) after PLN collapse. Herein, we document the first in vivo measurements of murine lymph viscosity and lymphatic pressure, and provide evidence to support the hypothesis that lymphangiogenesis and lymphatic transport are compensatory mechanisms to prevent synovitis via increased drainage of inflamed joints. Furthermore, the decrease in lymphatic flow and loss of LPP during PLN collapse are consistent with decreased drainage from the joint during arthritic flare, and validate these biomarkers of RA progression and possibly other chronic inflammatory conditions.

Fluoxetine modulates breast cancer metastasis to the brain in a murine model.

BACKGROUND: Despite advances in the treatment of primary breast tumors, the outcome of metastatic breast cancer remains dismal. Brain metastases present a particularly difficult therapeutic target due to the "sanctuary" status of the brain, with resulting inability of most chemotherapeutic agents to effectively eliminate cancer cells in the brain parenchyma. A large number of breast cancer patients receive various neuroactive drugs to combat complications of systemic anti-tumor therapies and to treat concomitant diseases. One of the most prescribed groups of neuroactive medications is anti-depressants, in particular selective serotonin reuptake inhibitors (SSRIs). Since SSRIs have profound effects on the brain, it is possible that their use in breast cancer patients could affect the development of brain metastases. This would provide important insight into the mechanisms underlying brain metastasis. Surprisingly, this possibility has been poorly explored. METHODS: We studied the effect of fluoxetine, an SSRI, on the development of brain metastatic breast cancer using MDA-MB-231BR cells in a mouse model. RESULTS: The data demonstrate that fluoxetine treatment increases the number of brain metastases, an effect accompanied by elevated permeability of the blood-brain barrier, pro-inflammatory changes in the brain, and glial activation. This suggests a possible role of brain-resident immune cells and glia in promoting increased development of brain metastases. CONCLUSION: Our results offer experimental evidence that neuroactive substances may influence the pathogenesis of brain metastatic disease. This provides a starting point for further investigations into possible mechanisms of interaction between various neuroactive drugs, tumor cells, and the brain microenvironment, which may lead to the discovery of compounds that inhibit metastasis to the brain.

Restricted diffusion of calretinin in cerebellar granule cell dendrites implies Ca²âº-dependent interactions via its EF-hand 5 domain.

Ca²âº-binding proteins (CaBPs) are important regulators of neuronal Ca²âº signalling, acting either as buffers that shape Ca²âº transients and Ca²âº diffusion and/or as Ca²âº sensors. The diffusional mobility represents a crucial functional parameter of CaBPs, describing their range-of-action and possible interactions with binding partners. Calretinin (CR) is a CaBP widely expressed in the nervous system with strong expression in cerebellar granule cells. It is involved in regulating excitability and synaptic transmission of granule cells, and its absence leads to impaired motor control. We quantified the diffusional mobility of dye-labelled CR in mouse granule cells using two-photon fluorescence recovery after photobleaching. We found that movement of macromolecules in granule cell dendrites was not well described by free Brownian diffusion and that CR diffused unexpectedly slow compared to fluorescein dextrans of comparable size. During bursts of action potentials, which were associated with dendritic Ca²âº transients, the mobility of CR was further reduced. Diffusion was significantly accelerated by a peptide embracing EF-hand 5 of CR. Our results suggest long-lasting, Ca²âº-dependent interactions of CR with large and/or immobile binding partners. These interactions render CR a poorly mobile Ca²âº buffer and point towards a Ca²âº sensor function of CR.

Stromal matrix metalloprotease-13 knockout alters Collagen I structure at the tumor-host interface and increases lung metastasis of C57BL/6 syngeneic E0771 mammary tumor cells.

BACKGROUND: Matrix metalloproteases and collagen are key participants in breast cancer, but their precise roles in cancer etiology and progression remain unclear. MMP13 helps regulate collagen structure and has been ascribed largely harmful roles in cancer, but some studies demonstrate that MMP13 may also protect against tumor pathology. Other studies indicate that collagen's organizational patterns at the breast tumor-host interface influence metastatic potential. Therefore we investigated how MMP13 modulates collagen I, a principal collagen subtype in breast tissue, and affects tumor pathology and metastasis in a mouse model of breast cancer. METHODS: Tumors were implanted into murine mammary tissues, and their growth analyzed in Wildtype and MMP13 KO mice. Following extraction, tumors were analyzed for collagen I levels and collagen I macro- and micro-structural properties at the tumor-host boundary using immunocytochemistry and two-photon and second harmonic generation microscopy. Lungs were analyzed for metastases counts, to correlate collagen I changes with a clinically significant functional parameter. Statistical analyses were performed by t-test, analysis of variance, or Wilcoxon-Mann-Whitney tests as appropriate. RESULTS: We found that genetic ablation of host stromal MMP13 led to: 1. Increased mammary tumor collagen I content, 2. Marked changes in collagen I spatial organization, and 3. Altered collagen I microstructure at the tumor-host boundary, as well as 4. Increased metastasis from the primary mammary tumor to lungs. CONCLUSIONS: These results implicate host MMP13 as a key regulator of collagen I structure and metastasis in mammary tumors, thus making it an attractive potential therapeutic target by which we might alter metastatic potential, one of the chief determinants of clinical outcome in breast cancer. In addition to identifying stromal MMP13 is an important regulator of the tumor microenvironment and metastasis, these results also suggest that stromal MMP13 may protect against breast cancer pathology under some conditions, a finding with important implications for development of chemotherapies directed against matrix metalloproteases.

Early impact of social isolation and breast tumor progression in mice.

Evidence from cancer patients and animal models of cancer indicates that exposure to psychosocial stress can promote tumor growth and metastasis, but the pathways underlying stress-induced cancer pathogenesis are not fully understood. Social isolation has been shown to promote tumor progression. We examined the impact of social isolation on breast cancer pathogenesis in adult female severe combined immunodeficiency (SCID) mice using the human breast cancer cell line, MDA-MB-231, a high ß-adrenergic receptor (AR) expressing line. When group-adapted mice were transferred into single housing (social isolation) one week prior to MB-231 tumor cell injection into a mammary fat pad (orthotopic), no alterations in tumor growth or metastasis were detected compared to group-housed mice. When social isolation was delayed until tumors were palpable, tumor growth was transiently increased in singly-housed mice. To determine if sympathetic nervous system activation was associated with increased tumor growth, spleen and tumor norepinephrine (NE) was measured after social isolation, in conjunction with tumor-promoting macrophage populations. Three days after transfer to single housing, spleen weight was transiently increased in tumor-bearing and non-tumor-bearing mice in conjunction with reduced splenic NE concentration and elevated CD11b+Gr-1+ macrophages. At day 10 after social isolation, no changes in spleen CD11b+ populations or NE were detected in singly-housed mice. In the tumors, social isolation increased CD11b+Gr-1+, CD11b+Gr-1-, and F4/80+ macrophage populations, with no change in tumor NE. The results indicate that a psychological stressor, social isolation, elicits dynamic but transient effects on macrophage populations that may facilitate tumor growth. The transiency of the changes in peripheral NE suggest that homeostatic mechanisms may mitigate the impact of social isolation over time. Studies are underway to define the neuroendocrine mechanisms underlying the tumor-promoting effects of social isolation, and to determine the contributions of increased tumor macrophages to tumor pathogenesis.

Engineering superficial zone features in tissue engineered cartilage.

A major challenge in cartilage tissue engineering is the need to recreate the native tissue's anisotropic extracellular matrix structure. This anisotropy has important mechanical and biological consequences and could be crucial for integrative repair. Here, we report that hydrodynamic conditions that mimic the motion-induced flow fields in between the articular surfaces in the synovial joint induce the formation of a distinct superficial layer in tissue engineered cartilage hydrogels, with enhanced production of cartilage matrix proteoglycan and Type II collagen. Moreover, the flow stimulation at the surface induces the production of the surface zone protein Proteoglycan 4 (aka PRG4 or lubricin). Analysis of second harmonic generation signature of collagen in this superficial layer reveals a highly aligned fibrillar matrix that resembles the alignment pattern in native tissue's surface zone, suggesting that mimicking synovial fluid flow at the cartilage surface in hydrodynamic bioreactors could be key to creating engineered cartilage with superficial zone features.

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.

Quantum dots spectrally distinguish multiple species within the tumor milieu in vivo.

A solid tumor is an organ composed of cancer and host cells embedded in an extracellular matrix and nourished by blood vessels. A prerequisite to understanding tumor pathophysiology is the ability to distinguish and monitor each component in dynamic studies. Standard fluorophores hamper simultaneous intravital imaging of these components. Here, we used multiphoton microscopy techniques and transgenic mice that expressed green fluorescent protein, and combined them with the use of quantum dot preparations. We show that these fluorescent semiconductor nanocrystals can be customized to concurrently image and differentiate tumor vessels from both the perivascular cells and the matrix. Moreover, we used them to measure the ability of particles of different sizes to access the tumor. Finally, we successfully monitored the recruitment of quantum dot-labeled bone marrow-derived precursor cells to the tumor vasculature. These examples show the versatility of quantum dots for studying tumor pathophysiology and creating avenues for treatment.

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.

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.

ß-Adrenergic receptors (ß-AR) regulate VEGF and IL-6 production by divergent pathways in high ß-AR-expressing breast cancer cell lines.

Activation of ß-adrenergic receptors (ß-AR) drives proangiogenic factor production in several types of cancers. To examine ß-AR regulation of breast cancer pathogenesis, ß-AR density, signaling capacity, and functional responses to ß-AR stimulation were studied in four human breast adenocarcinoma cell lines. ß-AR density ranged from very low in MCF7 and MB-361 to very high in MB-231 and in a brain-seeking variant of MB-231, MB-231BR. Consistent with ß-AR density, ß-AR activation elevated cAMP in MCF7 and MB-361 much less than in MB-231 and MB-231BR. Functionally, ß-AR stimulation did not markedly alter vascular endothelial growth factor (VEGF) production by MCF7 or MB-361. In the two high ß-AR-expressing cell lines MB-231 and MB-231BR, ß-AR-induced cAMP and VEGF production differed considerably, despite similar ß-AR density. The ß(2)-AR-selective agonist terbutaline and the endogenous neurotransmitter norepinephrine decreased VEGF production by MB-231, but increased VEGF production by MB-231BR. Moreover, ß(2)-AR activation increased IL-6 production by both MB-231 and MB-231BR. These functional alterations were driven by elevated cAMP, as direct activation of adenylate cyclase by forskolin elicited similar alterations in VEGF and IL-6 production. The protein kinase A antagonist KT5720 prevented ß-AR-induced alterations in MB-231 and MB-231BR VEGF production, but not IL-6 production. Conclusions ß-AR expression and signaling is heterogeneous in human breast cancer cell lines. In cells with high ß-AR density, ß-AR stimulation regulates VEGF production through the classical ß-AR-cAMP-PKA pathway, but this pathway can elicit directionally opposite outcomes. Furthermore, in the same cells, ß-AR activate a cAMP-dependent, PKA-independent pathway to increase IL-6 production. The complexity of breast cancer cell ß-AR expression and functional responses must be taken into account when considering ß-AR as a therapeutic target for breast cancer treatment.

Two-photon fluorescence correlation microscopy reveals the two-phase nature of transport in tumors.

Transport parameters determine the access of drugs to tumors. However, technical difficulties preclude the measurement of these parameters deep inside living tissues. To this end, we adapted and further optimized two-photon fluorescence correlation microscopy (TPFCM) for in vivo measurement of transport parameters in tumors. TPFCM extends the detectable range of diffusion coefficients in tumors by one order of magnitude, and reveals both a fast and a slow component of diffusion. The ratio of these two components depends on molecular size and can be altered in vivo with hyaluronidase and collagenase. These studies indicate that TPFCM is a promising tool to dissect the barriers to drug delivery in tumors.

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.