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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Generation, Endothelialization, and Microsurgical Suture Anastomosis of Strong 1-mm-Diameter Collagen Tubes.

Tissue-engineered vascular grafts that are based on reconstituted extracellular matrices have been plagued by weak mechanical strength that prevents handling or anastomosis to native vessels. In this study, we devise a method for making dense, suturable collagen tubular constructs of diameter ≤1 mm for potential microsurgical applications, by dehydrating tubes of native rat tail type I collagen and crosslinking them with 20 mM genipin. Crosslinked dense collagen tubes with 1 mm inner diameter yielded ultimate tensile strength of 342 ± 15 gF and burst pressure of 1313 ± 156 mm Hg, comparable to the strength of a rat femoral artery, and supported endothelial cell adhesion and growth. End-to-end anastomosis of 0.5-mm-diameter tubes to explanted arteries displayed anastomotic strength of 82 ± 21 gF, which is sufficient for surgical applications. In vivo implantation of cell-free tubes as interpositional grafts in the rat femoral circulation yielded stable anastomosis with blood flow for 20 min. Seeded dense collagen tubes represent a promising alternative to venous graft that can potentially be used to bridge between short artery stubs in replantation surgeries.

A 3D Culture Model to Study How Fluid Pressure and Flow Affect the Behavior of Aggregates of Epithelial Cells.

Cells are surrounded by mechanical stimuli in their microenvironment. It is important to determine how cells respond to the mechanical information that surrounds them in order to understand both development and disease progression, as well as to be able to predict cell behavior in response to physical stimuli. Here we describe a protocol to determine the effects of interstitial fluid flow on the migratory behavior of an aggregate of epithelial cells in a three-dimensional (3D) culture model. This protocol includes detailed methods for the fabrication of a 3D cell culture chamber with hydrostatic pressure control, the culture of epithelial cells as an aggregate in a collagen gel, and the analysis of collective cell behavior in response to pressure-driven flow.

Physical and Chemical Signals That Promote Vascularization of Capillary-Scale Channels.

Proper vascularization remains critical to the clinical application of engineered tissues. To engineer microvessels in vitro, we and others have delivered endothelial cells through preformed channels into patterned extracellular matrix-based gels. This approach has been limited by the size of endothelial cells in suspension, and results in plugging of channels below ~30 µm in diameter. Here, we examine physical and chemical signals that can augment direct seeding, with the aim of rapidly vascularizing capillary-scale channels. By studying tapered microchannels in type I collagen gels under various conditions, we establish that stiff scaffolds, forward pressure, and elevated cyclic AMP levels promote endothelial stability and that reverse pressure promotes endothelial migration. We applied these results to uniform 20-µm-diameter channels and optimized the magnitudes of pressure, flow, and shear stress to best support endothelial migration and vascular stability. This vascularization strategy is able to form millimeter-long perfusable capillaries within three days. Our results indicate how to manipulate the physical and chemical environment to promote rapid vascularization of capillary-scale channels within type I collagen gels.

Interstitial fluid pressure regulates collective invasion in engineered human breast tumors via Snail, vimentin, and E-cadherin.

Many solid tumors exhibit elevated interstitial fluid pressure (IFP). This elevated pressure within the core of the tumor results in outward flow of interstitial fluid to the tumor periphery. We previously found that the directionality of IFP gradients modulates collective invasion from the surface of patterned three-dimensional (3D) aggregates of MDA-MB-231 human breast cancer cells. Here, we used this 3D engineered tumor model to investigate the molecular mechanisms underlying IFP-induced changes in invasive phenotype. We found that IFP alters the expression of genes associated with epithelial-mesenchymal transition (EMT). Specifically, the levels of Snail, vimentin, and E-cadherin were increased under pressure conditions that promoted collective invasion. These changes in gene expression were sufficient to direct collective invasion in response to IFP. Furthermore, we found that IFP modulates the motility and persistence of individual cells within the aggregates, which are also influenced by the expression levels of EMT markers. Together, these data provide insight into the molecular mechanisms that guide collective invasion from primary tumors in response to IFP.

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.

Muddying the waters: a new area of concern for drinking water contamination in Cameroon.

In urban Maroua, Cameroon, improved drinking water sources are available to a large majority of the population, yet this water is frequently distributed through informal distribution systems and stored in home containers (canaries), leaving it vulnerable to contamination. We assessed where contamination occurs within the distribution system, determined potential sources of environmental contamination, and investigated potential pathogens. Gastrointestinal health status (785 individuals) was collected via health surveys. Drinking water samples were collected from drinking water sources and canaries. Escherichia coli and total coliform levels were evaluated and molecular detection was performed to measure human-associated faecal marker, HF183; tetracycline-resistance gene, tetQ; Campylobacter spp.; and Staphylococcus aureus. Statistical analyses were performed to evaluate the relationship between microbial contamination and gastrointestinal illness. Canari samples had higher levels of contamination than source samples. HF183 and tetQ were detected in home and source samples. An inverse relationship was found between tetQ and E. coli. Presence of tetQ with lower E. coli levels increased the odds of reported diarrhoeal illness than E. coli levels alone. Further work is warranted to better assess the relationship between antimicrobial-resistant bacteria and other pathogens in micro-ecosystems within canaries and this relationship's impact on drinking water quality.