Comparison of blind deconvolution- and Patlak analysis-based methods for determining vascular permeability.

This study describes a computational algorithm to determine vascular permeability constants from time-lapse imaging data without concurrent knowledge of the arterial input function. The algorithm is based on "blind" deconvolution of imaging data, which were generated with analytical and finite-element models of bidirectional solute transport between a capillary and its surrounding tissue. Compared to the commonly used Patlak analysis, the blind algorithm is substantially more accurate in the presence of solute delay and dispersion. We also compared the performance of the blind algorithm with that of a simpler one that assumed unidirectional transport from capillary to tissue [as described in Truslow et al., Microvasc. Res. 90, 117-120 (2013)]. The algorithm based on bidirectional transport was more accurate than the one based on unidirectional transport for more permeable vessels and smaller extravascular distribution volumes, and less accurate for less permeable vessels and larger extravascular distribution volumes. Our results indicate that blind deconvolution is superior to Patlak analysis for permeability mapping under clinically relevant conditions, and can thus potentially improve the detection of tissue regions with a compromised vascular barrier.

Dynamics of Interstitial Fluid Pressure in Extracellular Matrix Hydrogels in Microfluidic Devices.

In order to understand how interstitial fluid pressure and flow affect cell behavior, many studies use microfluidic approaches to apply externally controlled pressures to the boundary of a cell-containing gel. It is generally assumed that the resulting interstitial pressure distribution quickly reaches a steady-state, but this assumption has not been rigorously tested. Here, we demonstrate experimentally and computationally that the interstitial fluid pressure within an extracellular matrix gel in a microfluidic device can, in some cases, react with a long time delay to external loading. Remarkably, the source of this delay is the slight (∼100 nm in the cases examined here) distension of the walls of the device under pressure. Finite-element models show that the dynamics of interstitial pressure can be described as an instantaneous jump, followed by axial and transverse diffusion, until the steady pressure distribution is reached. The dynamics follow scaling laws that enable estimation of a gel's poroelastic constants from time-resolved measurements of interstitial fluid pressure.

Relatively Rare Populations of Invasive Cells Drive Progression of Heterogeneous Tumors.

Introduction: Breast tumors often display an astonishing degree of spatial and temporal heterogeneity, which are associated with cancer progression, drug resistance, and relapse. Triple-negative breast cancer (TNBC) is a particularly aggressive and heterogeneous subtype for which targeted therapies are scarce. Consequently, patients with TNBC have a poorer overall prognosis compared to other breast cancer patients. Within heterogeneous tumors, individual clonal subpopulations may exhibit differences in their rates of growth and degrees of invasiveness. We hypothesized that such phenotypic heterogeneity at the single-cell level may accelerate tumor progression by enhancing the overall growth and invasion of the entire tumor. Methods: To test this hypothesis, we isolated and characterized clonal subpopulations with distinct morphologies and biomarker expression from the inherently heterogeneous 4T1 mouse mammary carcinoma cell line. We then leveraged a 3D microfluidic tumor model to reverse-engineer intratumoral heterogeneity and thus investigate how interactions between phenotypically distinct subpopulations affect tumor growth and invasion. Results: We found that the growth and invasion of multiclonal tumors were largely dictated by the presence of cells with epithelial and mesenchymal traits, respectively. The latter accelerated overall tumor invasion, even when these cells comprised less than 1% of the initial population. Consistently, tumor progression was delayed by selectively targeting the mesenchymal subpopulation. Discussion: This work reveals that highly invasive cells can dominate tumor phenotype and that specifically targeting these cells can slow the progression of heterogeneous tumors, which may help inform therapeutic approaches. Supplementary Information: The online version contains supplementary material available at 10.1007/s12195-023-00792-w.

Microfluidic models of vascular functions.

In vitro studies of vascular physiology have traditionally relied on cultures of endothelial cells, smooth muscle cells, and pericytes grown on centimeter-scale plates, filters, and flow chambers. The introduction of microfluidic tools has revolutionized the study of vascular physiology by allowing researchers to create physiologically relevant culture models, at the same time greatly reducing the consumption of expensive reagents. By taking advantage of the small dimensions and laminar flow inherent in microfluidic systems, recent studies have created in vitro models that reproduce many features of the in vivo vascular microenvironment with fine spatial and temporal resolution. In this review, we highlight the advantages of microfluidics in four areas: the investigation of hemodynamics on a capillary length scale, the modulation of fluid streams over vascular cells, angiogenesis induced by the exposure of vascular cells to well-defined gradients in growth factors or pressure, and the growth of microvascular networks in biomaterials. Such unique capabilities at the microscale are rapidly advancing the understanding of microcirculatory dynamics, shear responses, and angiogenesis in health and disease as well as the ability to create in vivo-like blood vessels in vitro.

Determination of vascular permeability coefficients under slow luminal filling.

This Communication describes a method to obtain the permeability product (permeability coefficient normalized by vascular dimensions) from time-lapse intensity data for which the introduction of labeled solute into the vasculature does not occur at a sharply defined time. This method has an error of ~10% across a wide range of filling times and noise levels, and is particularly well-suited for situations in which the permeability coefficient is greater than 10(-6)cm/s. We show that it is applicable whether the increase in vascular solute concentration is sustained or transient.

Adipose Cells Induce Escape from an Engineered Human Breast Microtumor Independently of their Obesity Status.

Introduction: Obesity is associated with increased breast cancer incidence, recurrence, and mortality. Adipocytes and adipose-derived stem cells (ASCs), two resident cell types in adipose tissue, accelerate the early stages of breast cancer progression. It remains unclear whether obesity plays a role in the subsequent escape of malignant breast cancer cells into the local circulation. Methods: We engineered models of human breast tumors with adipose stroma that exhibited different obesity-specific alterations. We used these models to assess the invasion and escape of breast cancer cells into an empty, blind-ended cavity (as a mimic of a lymphatic vessel) for up to sixteen days. Results: Lean and obese donor-derived adipose stroma hastened escape to similar extents. Moreover, a hypertrophic adipose stroma did not affect the rate of adipose-induced escape. When admixed directly into the model tumors, lean and obese donor-derived ASCs hastened escape similarly. Conclusions: This study demonstrates that the presence of adipose cells, independently of the obesity status of the adipose tissue donor, hastens the escape of human breast cancer cells in multiple models of obesity-associated breast cancer. Supplementary Information: The online version contains supplementary material available at 10.1007/s12195-022-00750-y.

Methods for Forming Human Lymphatic Microvessels In Vitro and Assessing their Drainage Function.

This chapter describes methods to engineer human lymphatic microvessels in vitro and to assess their fluid and solute drainage capacities. The lymphatics are formed within micropatterned type I collagen gels that contain a blind-ended channel for the growth of lymphatic endothelial cells. Because the vessels have one blind end and one open end each, they mimic the terminal structure of the native lymphatic microvascular tree. The solute drainage rates that are measured from the engineered lymphatics in vitro can be directly compared with published results from intact vessels in vivo. Practical considerations to increase the accuracy of the drainage assays are discussed.

Role of Lymphatic Endothelium in Vascular Escape of Engineered Human Breast Microtumors.

Introduction: Lymphatic vasculature provides a route for metastasis to secondary sites in the body. The role of the lymphatic endothelium in mediating the entry of breast cancer cells into the vasculature remains unclear. Methods: In this study, we formed aggregates of MDA-MB-231 human breast carcinoma cells next to human microvascular lymphatic endothelial cell (LEC)-lined cavities in type I collagen gels to model breast microtumors and lymphatic vessels, respectively. We tracked invasion and escape of breast microtumors into engineered lymphatics or empty cavities under matched flow rates for up to sixteen days. Results: After coming into contact with a lymphatic vessel, tumor cells escape by moving between the endothelium and the collagen wall, between endothelial cells, and/or into the endothelial lumen. Over time, tumor cells replace the LECs within the vessel wall and create regions devoid of endothelium. The presence of lymphatic endothelium slows breast tumor invasion and escape, and addition of LEC-conditioned medium to tumors is sufficient to reproduce nearly all of these inhibitory effects. Conclusions: This work sheds light on the interactions between breast cancer cells and lymphatic endothelium during vascular escape and reveals an inhibitory role for the lymphatic endothelium in breast tumor invasion and escape. Supplementary Information: The online version contains supplementary material available at 10.1007/s12195-022-00745-9.

Adipose Stroma Accelerates the Invasion and Escape of Human Breast Cancer Cells from an Engineered Microtumor.

INTRODUCTION: Approximately 20-25% of human breast tumors are found within an adipose, rather than fibrous, stroma. Adipose stroma is associated with an increased risk of lymph node metastasis, but the causal association between adipose stroma and metastatic progression in human breast cancer remains unclear. METHODS: We used micropatterned type I collagen gels to engineer ~3-mm-long microscale human breast tumors within a stroma that contains adipocytes and adipose-derived stem cells (ASCs) (collectively, "adipose cells"). Invasion and escape of human breast cancer cells into an empty 120-µm-diameter lymphatic-like cavity was used to model interstitial invasion and vascular escape in the presence of adipose cell-derived factors for up to 16 days. RESULTS: We found that adipose cells hasten invasion and escape by 1-2 days and 2-3 days, respectively. These effects were mediated by soluble factors secreted by the adipose cells, and these factors acted directly on tumor cells. Surprisingly, tumor invasion and escape were more strongly induced by ASCs than by adipocytes. CONCLUSIONS: This work reveals that both adipocytes and ASCs accelerate the interstitial invasion and escape of human breast cancer cells, and sheds light on the link between adipose stroma and lymphatic metastasis in human breast cancer. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12195-021-00697-6.

Microfluidic Biomaterials.

Since their initial description in 2005, biomaterials that are patterned to contain microfluidic networks ("microfluidic biomaterials") have emerged as promising scaffolds for a variety of tissue engineering and related applications. This class of materials is characterized by the ability to be readily perfused. Transport and exchange of solutes within microfluidic biomaterials is governed by convection within channels and diffusion between channels and the biomaterial bulk. Numerous strategies have been developed for creating microfluidic biomaterials, including micromolding, photopatterning, and 3D printing. In turn, these materials have been used in many applications that benefit from the ability to perfuse a scaffold, including the engineering of blood and lymphatic microvessels, epithelial tubes, and cell-laden tissues. This article reviews the current state of the field and suggests new areas of exploration for this unique class of materials.

Interstitial Hypertension Suppresses Escape of Human Breast Tumor Cells Via Convection of Interstitial Fluid.

INTRODUCTION: Interstitial hypertension, a rise in interstitial fluid pressure, is a common feature of many solid tumors as they progress to an invasive state. It is currently unclear whether this elevated pressure alters the probability that tumor cells eventually escape into a neighboring blood or lymphatic vessel. METHODS: In this study, we analyze the escape of MDA-MB-231 human breast tumor cells from a ~3-mm-long preformed aggregate into a 120-µm-diameter empty cavity in a micromolded type I collagen gel. The "micro-tumors" were located within ~300 µm of one or two cavities. Pressures of ~0.65 cm H2O were applied only to the tumor ("interstitial hypertension") or to its adjacent cavity. RESULTS: This work shows that interstitial hypertension suppresses escape into the adjacent cavity, but not because tumor cells respond directly to the pressure profile. Instead, hypertension alters the chemical microenvironment at the tumor margin to one that hampers escape. Administration of tumor interstitial fluid phenocopies the effects of hypertension. CONCLUSIONS: This work uncovers a link between tumor pressure, interstitial flow, and tumor cell escape in MDA-MB-231 cells, and suggests that interstitial hypertension serves to hinder further progression to metastatic escape. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s12195-020-00661-w) contains supplementary material, which is available to authorized users.

Matrix degradation and cell proliferation are coupled to promote invasion and escape from an engineered human breast microtumor.

Metastasis, the leading cause of mortality in cancer patients, depends upon the ability of cancer cells to invade into the extracellular matrix that surrounds the primary tumor and to escape into the vasculature. To investigate the features of the microenvironment that regulate invasion and escape, we generated solid microtumors of MDA-MB-231 human breast carcinoma cells within gels of type I collagen. The microtumors were formed at defined distances adjacent to an empty cavity, which served as an artificial vessel into which the constituent tumor cells could escape. To define the relative contributions of matrix degradation and cell proliferation on invasion and escape, we used pharmacological approaches to block the activity of matrix metalloproteinases (MMPs) or to arrest the cell cycle. We found that blocking MMP activity prevents both invasion and escape of the breast cancer cells. Surprisingly, blocking proliferation increases the rate of invasion but has no effect on that of escape. We found that arresting the cell cycle increases the expression of MMPs, consistent with the increased rate of invasion. To gain additional insight into the role of cell proliferation in the invasion process, we generated microtumors from cells that express the fluorescent ubiquitination-based cell cycle indicator. We found that the cells that initiate invasions are preferentially quiescent, whereas cell proliferation is associated with the extension of invasions. These data suggest that matrix degradation and cell proliferation are coupled during the invasion and escape of human breast cancer cells and highlight the critical role of matrix proteolysis in governing tumor phenotype.

Evaluation of 1-mm-diameter endothelialized dense collagen tubes in vascular microsurgery.

Although much progress has been made in engineering vascular grafts for large- and small-diameter arterial repair or bypass, the extension of these results to the microsurgical size scale has been challenging. Here, we evaluated the use of dense collagen tubes (outer diameter 1 mm, inner diameter 0.5 mm) for vascular microsurgery as interpositional grafts to the femoral artery of Lewis rats. These tubes were formed by dehydrating tubular collagen gels around a mandrel, crosslinking them with genipin, seeding with syngeneic endothelial cells, and culturing before implantation by suture anastomosis. The retention of a confluent endothelial lining inside the tubes after mock surgical handling depended strongly on the crosslinker concentration and culture time. Optimized preparation conditions enabled retention of endothelium after mock surgical handling in ~80% of tubes and maintenance of patency 7 days after implantation in ~40% of grafts. Histological analysis showed the development of granulation tissue and the presence of CD31-positive structures on the inner and outer surfaces of implants. This study provides a proof-of-principle demonstration that endothelialized dense collagen tubes can remain patent for up to 7 days after vascular microsurgery, and points to the importance of mild scaffold crosslinking for maintaining firm endothelial adhesion.

Matrix Pore Size Governs Escape of Human Breast Cancer Cells from a Microtumor to an Empty Cavity.

How the extracellular matrix (ECM) affects the progression of a localized tumor to invasion of the ECM and eventually to vascular dissemination remains unclear. Although many studies have examined the role of the ECM in early stages of tumor progression, few have considered the subsequent stages that culminate in intravasation. In the current study, we have developed a three-dimensional (3D) microfluidic culture system that captures the entire process of invasion from an engineered human micro-tumor of MDA-MB-231 breast cancer cells through a type I collagen matrix and escape into a lymphatic-like cavity. By varying the physical properties of the collagen, we have found that MDA-MB-231 tumor cells invade and escape faster in lower-density ECM. These effects are mediated by the ECM pore size, rather than by the elastic modulus or interstitial flow speed. Our results underscore the importance of ECM structure in the vascular escape of human breast cancer cells.

Tissue Engineering of the Microvasculature.

The ability to generate new microvessels in desired numbers and at desired locations has been a long-sought goal in vascular medicine, engineering, and biology. Historically, the need to revascularize ischemic tissues nonsurgically (so-called therapeutic vascularization) served as the main driving force for the development of new methods of vascular growth. More recently, vascularization of engineered tissues and the generation of vascularized microphysiological systems have provided additional targets for these methods, and have required adaptation of therapeutic vascularization to biomaterial scaffolds and to microscale devices. Three complementary strategies have been investigated to engineer microvasculature: angiogenesis (the sprouting of existing vessels), vasculogenesis (the coalescence of adult or progenitor cells into vessels), and microfluidics (the vascularization of scaffolds that possess the open geometry of microvascular networks). Over the past several decades, vascularization techniques have grown tremendously in sophistication, from the crude implantation of arteries into myocardial tunnels by Vineberg in the 1940s, to the current use of micropatterning techniques to control the exact shape and placement of vessels within a scaffold. This review provides a broad historical view of methods to engineer the microvasculature, and offers a common framework for organizing and analyzing the numerous studies in this area of tissue engineering and regenerative medicine. © 2019 American Physiological Society. Compr Physiol 9:1155-1212, 2019.

Bonding of macromolecular hydrogels using perturbants.

This work describes a method to bond patterned macromolecular gels into monolithic structures using perturbants. Bonding strengths for a variety of solutes follow a Hofmeister ordering; this result and optical measurements indicate that bonding occurs by reversible perturbation of contacting gels. The resulting microfluidic gels are mechanically robust and can serve as scaffolds for cell culture.

Effect of cyclic AMP on barrier function of human lymphatic microvascular tubes.

This work examines the effect of cyclic AMP (cAMP) on the in vitro barrier function of tubes of human dermal lymphatic microvascular endothelial cells (LECs). Under baseline conditions, the barrier function of LEC tubes was weak, with diffusional permeability coefficients to bovine serum albumin and 10 kDa dextran of 1.4(-0.6)(+0.9)x10(-6) cm/s and 1.7(-0.5)(+0.8)x10(-6) cm/s (geometric mean+/-95% CI), respectively, and 1.2+/-0.5 (mean+/-95% CI) focal leaks per mm. Exposure to low concentrations (3 microM) of a cell-permeant analog of cAMP did not alter the barrier function. Exposure to higher concentrations (80 and 400 microM) and/or the phosphodiesterase inhibitor Ro-20-1724 (20 microM) lowered permeabilities and the number of focal leaks, and increased the selectivity of the barrier. Decreased permeabilities were accompanied by an increase in continuous VE-cadherin staining at cell-cell borders. Exposure to 1 mM 2',5'-dideoxyadenosine, an inhibitor of adenylate cyclase, did not increase permeabilities. LECs expressed the lymphatic-specific master transcription factor Prox-1, regardless of whether barrier function was weak or strong. Our results indicate that the permeability of LEC tubes in vitro responds to cAMP in a manner similar to that well-described for the permeability of blood microvessels.

Engineering of microscale vascularized fat that responds to perfusion with lipoactive hormones.

Current methods to treat large soft-tissue defects mainly rely on autologous transfer of adipocutaneous flaps, a method that is often limited by donor site availability. Engineered vascularized adipose tissues can potentially be a viable and readily accessible substitute to autologous flaps. In this study, we engineered a small-scale adipose tissue with pre-patterned vasculature that enables immediate perfusion. Vessels formed after one day of perfusion and displayed barrier function after three days of perfusion. Under constant perfusion, adipose tissues remained viable and responded to lipoactive hormones insulin and epinephrine with lipid accumulation and loss, respectively. Adipocyte growth correlated inversely with distance away from the feeding vessel, as predicted by a Krogh-type model.

Design principles for lymphatic drainage of fluid and solutes from collagen scaffolds.

In vivo, tissues are drained of excess fluid and macromolecules by the lymphatic vascular system. How to engineer artificial lymphatics that can provide equivalent drainage in biomaterials remains an open question. This study elucidates design principles for engineered lymphatics, by comparing the rates of removal of fluid and solute through type I collagen gels that contain lymphatic vessels or unseeded channels, or through gels without channels. Surprisingly, no difference was found between the fluid drainage rates for gels that contained vessels or bare channels. Moreover, solute drainage rates were greater in collagen gels that contained lymphatic vessels than in those that had bare channels. The enhancement of solute drainage by lymphatic endothelium was more pronounced in longer scaffolds and with smaller solutes. Whole-scaffold imaging revealed that endothelialization aided in solute drainage by impeding solute reflux into the gel without hindering solute entry into the vessel lumen. These results were reproduced by computational models of drainage with a flow-dependent endothelial hydraulic conductivity. This study shows that endothelialization of bare channels does not impede the drainage of fluid from collagen gels and can increase the drainage of macromolecules by preventing solute transport back into the scaffold. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 106-114, 2018.

Fabrication of microfluidic hydrogels using molded gelatin as a sacrificial element.

This paper describes a general procedure for the formation of hydrogels that contain microfluidic networks. In this procedure, micromolded meshes of gelatin served as sacrificial materials. Encapsulation of gelatin meshes in a hydrogel and subsequent melting and flushing of the gelatin left behind interconnected channels in the hydrogel. The channels were as narrow as approximately 6 microm, and faithfully replicated the features in the original gelatin mesh. Fifty micrometre wide microfluidic networks in collagen and fibrin readily enabled delivery of macromolecules and particles into the channels and transport of macromolecules from channels into the bulk of the gels. Microfluidic gels were also suitable as scaffolds for cell culture, and could be seeded by human microvascular endothelial cells to form rudimentary endothelial networks for potential use in tissue engineering.