Heterogeneous breast tumoroids: An in vitro assay for investigating cellular heterogeneity and drug delivery

Breast tumors are typically heterogeneous and contain diverse subpopulations of tumor cells with differing phenotypic properties. Planar cultures of cancer cell lines are not viable models of investigation of cell-cell and cell-matrix interactions during tumor development. This article presents an in vitro coculture-based 3-dimensional heterogeneous breast tumor model that can be used in drug resistance and drug delivery investigations. Breast cancer cell lines of different phenotypes (MDAMB231, MCF7, and ZR751) were cocultured in a rotating wall vessel bioreactor to form a large number of heterogeneous tumoroids in a single cell culture experiment. Cells in the rotating vessels were labeled with Cell Tracker fluorescent probes to allow for time course fluorescence microscopy to monitor cell aggregation. Histological sections of tumoroids were stained with hematoxylin and eosin, progesterone receptor, E-cadherin (E-cad), and proliferation marker ki67. In vitro tumoroids developed in this study recapture important features of the temporal-spatial organization of solid tumors, including the presence of necrotic areas at the center and higher levels of cell division at the tumor periphery. E-cad-positive MCF7 cells form larger tumoroids than E-cad-negative MDAMB231 cells. In heterogeneous tumors, the irregular surface roughness was mainly due to the presence of MDAMB231 cells, whereas MCF7 cells formed smooth surfaces. Moreover, when heterogeneous tumoroids were placed onto collagen gels, highly invasive MDAMB231 cell-rich surface regions produced extensions into the matrix, whereas poorly invasive MCF7 cells did not. The fact that one can form a large number of 1-mm tumoroids in 1 coculture attests to the potential use of this system at high-throughput investigations of cancer drug development and drug delivery into the tumor.     

Angiogenesis and vascular remodelling in normal and cancerous tissues

Vascular development and homeostasis are underpinned by two fundamental features: the generation of new vessels to meet the metabolic demands of under-perfused regions and the elimination of vessels that do not sustain flow. In this paper we develop the first multiscale model of vascular tissue growth that combines blood flow, angiogenesis, vascular remodelling and the subcellular and tissue scale dynamics of multiple cell populations. Simulations show that vessel pruning, due to low wall shear stress, is highly sensitive to the pressure drop across a vascular network, the degree of pruning increasing as the pressure drop increases. In the model, low tissue oxygen levels alter the internal dynamics of normal cells, causing them to release vascular endothelial growth factor (VEGF), which stimulates angiogenic sprouting. Consequently, the level of blood oxygenation regulates the extent of angiogenesis, with higher oxygenation leading to fewer vessels. Simulations show that network remodelling (and de novo network formation) is best achieved via an appropriate balance between pruning and angiogenesis. An important factor is the strength of endothelial tip cell chemotaxis in response to VEGF. When a cluster of tumour cells is introduced into normal tissue, as the tumour grows hypoxic regions form, producing high levels of VEGF that stimulate angiogenesis and cause the vascular density to exceed that for normal tissue. If the original vessel network is sufficiently sparse then the tumour may remain localised near its parent vessel until new vessels bridge the gap to an adjacent vessel. This can lead to metastable periods, during which the tumour burden is approximately constant, followed by periods of rapid growth.     

Modulation of the "blood-tumor" barrier improves immunotherapy

Blood vessels inside tumors are crucial for cancer survival and progression but equally contribute to the tumor's intrinsic resistance to therapy. Abnormal blood flow in the local tumor environment acts as a physiological barrier to the delivery of chemotherapeutic agents. Furthermore, tumor vasculature can also act as a barrier for immune cell migration into the tumor parenchyma. Much has been made of anti-angiogenic therapies that specifically inhibit vessel growth. However, recent findings demonstrate that the chaotic architecture of tumor blood vessels can be reversed which in turn normalizes blood flow and physical parameters in the tumor environment. Importantly, vessel normalization also improves lymphocyte migration into tumor tissue and immune destruction. Identification of regulator of G protein signaling 5 (RGS5) as a key modulator of the vascular barrier in tumor progression and regression has brought new insights into the molecular basis of vessel normalization and opens new therapeutic opportunities.     

Role of Constitutive Behavior and Tumor-Host Mechanical Interactions in the State of Stress and Growth of Solid Tumors

Mechanical forces play a crucial role in tumor patho-physiology. Compression of cancer cells inhibits their proliferation rate, induces apoptosis and enhances their invasive and metastatic potential. Additionally, compression of intratumor blood vessels reduces the supply of oxygen, nutrients and drugs, affecting tumor progression and treatment. Despite the great importance of the mechanical microenvironment to the pathology of cancer, there are limited studies for the constitutive modeling and the mechanical properties of tumors and on how these parameters affect tumor growth. Also, the contribution of the host tissue to the growth and state of stress of the tumor remains unclear. To this end, we performed unconfined compression experiments in two tumor types and found that the experimental stress-strain response is better fitted to an exponential constitutive equation compared to the widely used neo-Hookean and Blatz-Ko models. Subsequently, we incorporated the constitutive equations along with the corresponding values of the mechanical properties - calculated by the fit - to a biomechanical model of tumor growth. Interestingly, we found that the evolution of stress and the growth rate of the tumor are independent from the selection of the constitutive equation, but depend strongly on the mechanical interactions with the surrounding host tissue. Particularly, model predictions - in agreement with experimental studies - suggest that the stiffness of solid tumors should exceed a critical value compared with that of the surrounding tissue in order to be able to displace the tissue and grow in size. With the use of the model, we estimated this critical value to be on the order of 1.5. Our results suggest that the direct effect of solid stress on tumor growth involves not only the inhibitory effect of stress on cancer cell proliferation and the induction of apoptosis, but also the resistance of the surrounding tissue to tumor expansion.     

Vascular tumor growth and treatment: Consequences of polyclonality,competition and dynamic vascular support

 A mathematical model is presented to describe the evolution of a vascular tumor in response to traditional chemotherapeutic treatment. Particular attention is paid to the effects of a dynamic vascular support system in a tumor comprised of competing cell populations that differ in proliferation rates and drug susceptibility. The model consists of a system of partial differential equations governing intratumoral drug concentration, cancer cell density, and blood vessel density. The balance between cell proliferation and death along with vessel production and destruction within the tumor generates a velocity field which drives the expansion or regression of the neoplasm. Radially symmetric solutions are obtained for the case when only one cell type is present and when the proportion of the tumor occupied by blood vessels remains constant. The stability of these solutions to asymmetric perturbations and to a small semi-drug resistant cell population is then investigated. The analysis shows that drug concentrations which are sufficient to insure eradication of a spherical tumor may be inadequate for the successful treatment of non-spherical tumors. When the drug is continuously infused, linear analysis predicts that whether or not a cure is possible is crucially dependent on the proliferation rate of the semi-resistant cells and on the competitive effect of the sensitive cells on the resistant population. When the blood vessel density is allowed to change dynamically, the model predicts a dramatic increase in the tumors growth and decrease in its response to therapy. Received: 4 August 2000 / Revised version: 13 July 2001 / Published online: 21 February 2002     

In Vivo Nano-imaging of Membrane Dynamics in Metastatic Tumor Cells Using Quantum Dots

Changes in membrane morphology and membrane protein dynamics based on its fluidity are critical for cancer metastasis. However, this subject has remained unclear, because the spatial precision of previous in vivo imaging has been limited to the micrometer level and single molecule imaging is impossible. Here, we have imaged the membrane dynamics of tumor cells in mice with a spatial precision of 7–9 nm under a confocal microscope. A metastasis-promoting factor on the cell membrane, protease-activated receptor 1 (PAR1), was labeled with quantum dots conjugated with an anti-PAR1 antibody. Movements of cancer cells and PAR1 during metastasis were clearly observed in vivo. Images used to assess PAR1 dynamics were taken of representative cells for four stages of metastasis; i.e. cancer cells far from blood vessels in tumor, near the vessel, in the bloodstream, and adherent to the inner vascular surface in the normal tissues near tumor were photographed. The diffusion constant of PAR1 in static cells far from tumor blood vessels was smaller than in moving cells near the vessels and in the bloodstream. The diffusion constant of cells adhering to the inner vascular surface in the normal tissues was also very small. Cells formed membrane protrusion during migration. The PAR1 diffusion constant on these pseudopodia was greater than in other membrane regions in the same cell. Thus, the dynamics of PAR1 movement showed that membrane fluidity increases during intravasation, reaches a peak in the vessel, decreases during extravasation, and is also higher at locally formed pseudopodia.     

Anti-tumor activities of the angiogenesis inhibitors interferon-inducible protein-10 and the calreticulin fragment vasostatin

Tumor growth depends upon an adequate supply of oxygen and nutrients achieved through angiogenesis and maintenance of an intact tumor vasculature. Therapy with individual agents that target new vessel formation or existing vessels has suppressed experimental tumor growth, but rarely resulted in the eradication of tumors. We therefore tested the combined anti-tumor activity of vasostatin and interferon-inducible protein-10 (IP-10), agents that differently target the tumor vasculature. Vasostatin, a selective and direct inhibitor of endothelial cell proliferation, significantly reduced Burkitt tumor growth and tumor vessel density. IP-10, an "angiotoxic" chemokine, caused vascular damage and focal necrosis in Burkitt tumors. When combined, vasostatin plus IP-10 reduced tumor growth more effectively than each agent alone, but complete tumor regression was not observed. Microscopically, these tumors displayed focal necrosis and reduction in vessel density. Combination therapy with the inhibitors of angiogenesis vasostatin and IP-10 is effective in reducing the rate of tumor growth but fails to induce tumor regression, suggesting that curative treatment may require supplemental drugs targeting directly the tumor cells.     

Emergent vascular network inhomogeneities and resulting blood flow patterns in a growing tumor

Tumors acquire sufficient oxygen and nutrient supply by coopting host vessels and neovasculature created via angiogenesis, thereby transforming a highly ordered network into chaotic heterogeneous tumor specific vasculature. Vessel regression inside the tumor leads to large regions of necrotic tissue interspersed with isolated surviving vessels. We extend our recently introduced model to incorporate Fahraeus-Lindqvist- and phase separation effects, refined tissue oxygen level computation and drug flow computations. We find, unexpectedly, that collapse and regression accelerates rather than diminishes the perfusion and that a tracer substance flowing through the remodeled network reaches all parts of the tumor vasculature very well. The reason for decreased drug delivery well known in tumors should therefore be different from collapse and vessel regression. Implications for drug delivery in real tumors are discussed.     

The VEGF receptor Flt-1 spatially modulates Flk-1 signaling and blood vessel branching

Blood vessel formation requires the integrated regulation of endothelial cell proliferation and branching morphogenesis, but how this coordinated regulation is achieved is not well understood. Flt-1 (vascular endothelial growth factor [VEGF] receptor 1) is a high affinity VEGF-A receptor whose loss leads to vessel overgrowth and dysmorphogenesis. We examined the ability of Flt-1 isoform transgenes to rescue the vascular development of embryonic stem cell-derived flt-1-/- mutant vessels. Endothelial proliferation was equivalently rescued by both soluble (sFlt-1) and membrane-tethered (mFlt-1) isoforms, but only sFlt-1 rescued vessel branching. Flk-1 Tyr-1173 phosphorylation was increased in flt-1-/- mutant vessels and partially rescued by the Flt-1 isoform transgenes. sFlt-1-rescued vessels exhibited more heterogeneous levels of pFlk than did mFlt-1-rescued vessels, and reporter gene expression from the flt-1 locus was also heterogeneous in developing vessels. Our data support a model whereby sFlt-1 protein is more efficient than mFlt-1 at amplifying initial expression differences, and these amplified differences set up local discontinuities in VEGF-A ligand availability that are important for proper vessel branching.     

A novel vascular-targeting peptide for gastric cancer delivers low-dose TNFα to normalize the blood vessels and improve the anti-cancer efficiency of 5-fluorouracil

Various vascular-targeted agents fused with tumor necrosis factor α (TNFα) have been shown to improve drug absorption into tumor tissues and enhance tumor vascular function. TCP-1 is a peptide selected through in vivo phage library biopanning against a mouse orthotopic colorectal cancer model and is a promising agent for drug delivery. This study further investigated the targeting ability of TCP-1 phage and peptide to blood vessels in an orthotopic gastric cancer model in mice and assessed the synergistic anti-cancer effect of 5-fluorouracil (5-FU) with subnanogram TNFα targeted delivered by TCP-1 peptide. In vivo phage targeting assay and in vivo colocalization analysis were carried out to test the targeting ability of TCP-1 phage/peptide. A targeted therapy for improvement of the therapeutic efficacy of 5-FU and vascular function was performed through administration of TCP-1/TNFα fusion protein in this model. TCP-1 phage exhibited strong homing ability to the orthotopic gastric cancer after phage injection. Immunohistochemical staining suggested that and TCP-1 phage/TCP-1 peptide could colocalize with tumor vascular endothelial cells. TCP-1/TNFα combined with 5-FU was found to synergistically inhibit tumor growth, induce apoptosis and reduce cell proliferation without evident toxicity. Simultaneously, subnanogram TCP-1/TNFα treatment normalized tumor blood vessels. Targeted delivery of low-dose TNFα by TCP-1 peptide can potentially modulate the vascular function of gastric cancer and increase the drug delivery of chemotherapeutic drugs.     

New molecular mediators in tumor angiogenesis

Angiogenesis is essential for tumor growth and progression. It has been demonstrated that tumor growth beyond a size 1 to 2 mm³ requires the induction of new vessels. Angiogenesis is regulated by several endogenous stimulators and inhibitors of endothelial cell migration, proliferation and tube formation. Under physiological conditions these mediators of endothelial cell growth are in balance and vessel growth is limited. In fact, within the angiogenic balance endothelial cell turnover is sufficient to maintain a functional vascular wall but does not allow vessel growth. Tumor growth an progression has successfully been correlated to the serum concentration of angiogenic mediators. Furthermore, the vascular density of tumor tissues could be correlated to the clinical course of the disease in several tumor entities. Within the last years several new mediators of endothelial cell growth have been isolated e.g. angiopoietin 1, angiopoietin 2, midkine, pleiotropin, leptin and maspin. In this review we discuss the mechanisms leading to tumor angiogenesis and describe some of the newer mediators of endothelial cell stimulation and inhibition.     

Vascular wall extracellular matrix proteins and vascular diseases

Extracellular matrix proteins form the basic structure of blood vessels. Along with providing basic structural support to blood vessels, matrix proteins interact with different sets of vascular cells via cell surface integrin or non-integrin receptors. Such interactions induce vascular cell de novo synthesis of new matrix proteins during blood vessel development or remodeling. Under pathological conditions, vascular matrix proteins undergo proteolytic processing, yielding bioactive fragments to influence vascular wall matrix remodeling. Vascular cells also produce alternatively spliced variants that induce vascular cell production of different matrix proteins to interrupt matrix homeostasis, leading to increased blood vessel stiffness; vascular cell migration, proliferation, or death; or vascular wall leakage and rupture. Destruction of vascular matrix proteins leads to vascular cell or blood-borne leukocyte accumulation, proliferation, and neointima formation within the vascular wall; blood vessels prone to uncontrolled enlargement during blood flow diastole; tortuous vein development; and neovascularization from existing pathological tissue microvessels. Here we summarize discoveries related to blood vessel matrix proteins within the past decade from basic and clinical studies in humans and animals — from expression to cross-linking, assembly, and degradation under physiological and vascular pathological conditions, including atherosclerosis, aortic aneurysms, varicose veins, and hypertension.     

The Effect of Sunitinib Treatment in Human Melanoma Xenografts: Associations with Angiogenic Profiles

The effect of antiangiogenic agents targeting the vascular endothelial growth factor A (VEGF-A) pathway has been reported to vary substantially in preclinical studies. The purpose of this study was to investigate the effect of sunitinib treatment on tumor vasculature and oxygenation in melanoma xenografts with different angiogenic profiles. A-07, U-25, D-12, or R-18 melanoma xenografts were grown in dorsal window chambers and given daily treatments of sunitinib (40 mg/kg) or vehicle. Morphologic parameters of tumor vascular networks were assessed from high-resolution transillumination images, and tumor blood supply times (BSTs) were assessed from first-pass imaging movies. Tumor hypoxia was assessed with immunohistochemistry by using pimonidazole as hypoxia marker, and the gene expression and the protein secretion rate of angiogenic factors were assessed by quantitative polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. The melanoma lines differed substantially in the expression of VEGF-A, VEGF-C, and platelet-derived growth factor A. Sunitinib treatment reduced vessel densities and induced hypoxia in all melanoma lines, and the magnitude of the effect was associated with the gene expression and protein secretion rate of VEGF-A. Sunitinib treatment also increased vessel segment lengths, reduced the number of small-diameter vessels, and inhibited growth-induced increases in the diameter of surviving vessels but did not change BST. In conclusion, sunitinib treatment did not improve vascular function but reduced vessel density and induced hypoxia in human melanoma xenografts. The magnitude of the treatment-induced effect was associated with the VEGF-A expression of the melanoma lines.     

Mechanisms of hydraulic conductivity in the leaf galls of Meunieriella sp. (Cecidomyiidae) in Avicennia schaueriana (Acanthaceae): does vascularization explain the preferred sites of induction?

• Intralaminar galls of Meunieriella result from ground tissue proliferation in leaves of Avicennia schaueriana, a typical halophytic mangrove. We tested if the preferred sites of gall induction were the midribs and secondary veins (SV) at the basal leaf portion, where the galls were expected to be largest; and if the vascular system in galls and adjacent regions was altered to favour water supply in galls, thus increasing their growth.
• Gall induction sites and gall sizes were quantified according to leaf portions and regions. Anatomical and histometric analyses in vascular and ground tissues of galls and adjacent regions were compared to equivalent regions of non-galled leaves.
• The galls were largest at basal sites on leaves, the midrib and SV. More galls occurred on the apical portion of the leaf, and on the leaf blade and secondary vein regions. Changes in shape and vascular system area, number and diameter of vessel elements were detected in both galls and adjacent regions. Fewer and smaller-sized vessel elements were observed in regions proximal to the galls and inside them.
• Gall size is not related with preferred induction sites, which could be explained by factors such as thermal balance. Alterations in the vascular system indicate reduced hydraulic conductivity in the xylem in the proximal region and inside galls, leading to water leakage to gall parenchyma cells. This compensatory mechanism explains the expansion and proliferation of water storage and spongy parenchyma cells in the galls, explaining the higher growth in more vascularized regions.

     

Regulation of vascular smooth muscle cells and mesenchymal stem cells by mechanical strain

Vascular smooth muscle cells (SMCs) populate in the media of the blood vessel, and play an important role in the control of vasoactivity and the remodeling of the vessel wall. Blood vessels are constantly subjected to hemodynamic stresses, and the pulsatile nature of the blood flow results in a cyclic mechanical strain in the vessel walls. Accumulating evidence in the past two decades indicates that mechanical strain regulates vascular SMC phenotype, function and matrix remodeling. Bone marrow mesenchymal stem cell (MSC) is a potential cell source for vascular regeneration therapy, and may be used to generate SMCs to construct tissue-engineered vascular grafts for blood vessel replacements. In this review, we will focus on the effects of mechanical strain on SMCs and MSCs, e.g., cell phenotype, cell morphology, cytoskeleton organization, gene expression, signal transduction and receptor activation. We will compare the responses of SMCs and MSCs to equiaxial strain, uniaxial strain and mechanical strain in three-dimensional culture. Understanding the hemodynamic regulation of SMC and MSC functions will provide a basis for the development of new vascular therapies and for the construction of tissue-engineered vascular grafts.     

Diffusion model of tumor vascularization and growth

A diffusion model of tumor growth, vascularization and necrosis is used to analyze experimental data describing the temporal changes in tumor cell and blood vessel radial distributions in a host-tissue field transplanted with a fibrosarcoma. The experimental results showed a peak density of vessels occurring at the advancing migration front of the tumor and a decline in the vessel surface area at the tumor center with time. The peak density of tumor cells shifts away from the tumor center with time. These dynamic changes can be explained by a mathematical model which views the process as one of diffusion and proliferation in time and space. Coupled diffusion equations with nonlinear source and sink terms describe the proliferation, death, and migration of tumor cells and vascular surface area. The concept of an angiogenic factor elaborated by tumor cells is incorporated.     

Angiopoietin 1 causes vessel enlargement, without angiogenic sprouting, during a critical developmental period

Early in development, endothelial cells proliferate, coalesce, and sprout to form a primitive plexus of undifferentiated microvessels. Subsequently, this plexus remodels into a hierarchical network of different-sized vessels. Although the processes of proliferation and sprouting are well studied and are dependent on the angiogenic growth factor VEGF, the factors involved in subsequent vessel remodeling are poorly understood. Here, we show that angiopoietin 1 can induce circumferential vessel enlargement, specifically on the venous side of the circulation. This action is due to the ability of angiopoietin 1 to promote endothelial cell proliferation in the absence of angiogenic sprouting; vessel growth without sprouting has not been ascribed to other vascular growth factors, nor has specificity for a particular segment of the vasculature. Moreover, angiopoietin 1 potently mediates widespread vessel enlargement only during a brief postnatal period, in particular, prior to the fourth postnatal week, corresponding to stages in which VEGF inhibition causes widespread vessel regression. These findings show that angiopoietin 1 has a potentially unique role among the vascular growth factors by acting to enlarge blood vessels without inducing sprouting, and also define a critical window of vascular plasticity in neonatal development. Finding the key molecular factors that regulate this plasticity may prove crucial to the further development of pro- and anti-angiogenic therapies.     

Vascular remodeling marks tumors that recur during chronic suppression of angiogenesis

The potential for avoiding acquired resistance to therapy has been proposed as one compelling theoretical advantage of antiangiogenic therapy based on the normal genetic status of the target vasculature. However, previous work has demonstrated that tumors may resume growth after initial inhibition if antiangiogenic blockade is continued for an extended period. The mechanisms of this recurrent growth are unclear. In these studies, we characterized molecular changes in vasculature during apparent resumption of xenograft growth after initial inhibition by vascular endothelial growth factor blockade, "metronome" topotecan chemotherapy, and combined agents in a xenograft murine model of human Wilms' tumor. Tumors that grew during antiangiogenic blockade developed as viable clusters surrounding strikingly remodeled vessels. These vessels displayed significant increases in diameter and active proliferation of vascular mural cells and expressed platelet-derived growth factor-B, a factor that functions to enhance vascular integrity via stromal cell recruitment. In addition, remodeled vessels were marked by expression of ephrinB2, required for proper assembly of stromal cells into vasculature. Thus, enhanced vascular stability appears to characterize tumor vessel response to chronic antiangiogenesis, features that potentially support increased perfusion and recurrent tumor growth.     

Blood supply of the upper craniofacial skeleton: the search for composite calvarial bone flaps

This study investigated the blood supply of the upper craniofacial skeleton by injection studies. The major supply to the calvaria is provided by the middle meningeal artery and its branches. This vessel is difficult for the plastic surgeon to exploit in composite bone-flap design. The majority of the outer surface of the craniofacial skeleton is supplied by tiny perforators from the overlying periosteum. The vascular interconnections within the periosteum are poorly developed. For this reason, the galea and the overlying vascular network (derived from the superficial temporal, occipital, supraorbital, and supratrochlear vessels) should be left broadly attached to the bone when transferring a vascularized calvarial bone flap. Dissection of the scalp away from this vascular network should be carried out just below the hair follicles. By observing these principles, vascularized calvarial bone can be transferred on the superficial temporal, deep temporal, supraorbital, supratrochlear, or occipital vessels. Details of the use of each are discussed.