Role of extracellular matrix assembly in interstitial transport in solid tumors.

The extracellular matrix (ECM) may contribute to the drug resistance of a solid tumor by preventing the penetration of therapeutic agents. We measured differences in interstitial resistance to macromolecule (IgG) motion in four tumor types and found an unexpected correspondence between transport resistance and the mechanical stiffness. The interstitial diffusion coefficient of IgG was measured in situ by fluorescence redistribution after photobleaching. Tissue elastic modulus and hydraulic conductivity were measured by confined compression of excised tissue. In apparent contradiction to an existing paradigm, these functional properties are correlated with total tissue content of collagen, not glycosaminoglycan. An extended collagen network was observed in the more penetration-resistant tumors. Collagenase treatment of the more penetration-resistant tumors significantly increased the IgG interstitial diffusion rate. We conclude that collagen influences the tissue resistance to macromolecule transport, possibly by binding and stabilizing the glycosaminoglycan component of the ECM. These findings suggest a new method to screen tumors for potential resistance to macromolecule-based therapy. Moreover, collagen and collagen-proteoglycan bonds are identified as potential targets of treatment to improve macromolecule delivery.

Vascular permeability in a human tumor xenograft: molecular size dependence and cutoff size.

Molecular size is one of the key determinants of transvascular transport of therapeutic agents in tumors. However, there are no data in the literature on the molecular size dependence of microvascular permeability in tumors. Therefore, we measured microvascular permeability to various macromolecules in the human colon adenocarcinoma LS174T transplanted in dorsal skin chambers in severe combined immunodeficient mice. These molecules were fluorescently labeled and injected i.v. into mice. The microvascular permeability was calculated from the fluorescence intensity measured by the intravital fluorescence microscopy technique. The value of permeability varied approximately 2-fold in the range of molecular weight from 25,000 to 160,000. These data indicate that tumor vessels are less permselective than normal vessels, presumably due to large pores in the vessel wall. The transport of macromolecules appears to be limited by diffusion through these pores. The cutoff size of the pores was estimated by observations of transvascular transport of sterically stabilized liposomes of 100-600 nm in diameter. We found that tumor vessels in our model were permeable to liposomes of up to 400 nm in diameter, suggesting that the cutoff size of the pores is between 400 and 600 nm in diameter.

Microvascular permeability and interstitial penetration of sterically stabilized (stealth) liposomes in a human tumor xenograft.

Microvascular permeability and interstitial penetration of sterically stabilized liposomes in both normal s.c. tissue and human colon adenocarcinoma LS174T xenograft were quantified by using the dorsal skin-fold chamber implanted in severe combined immunodeficient mice and intravital fluorescence microscopy. Significant extravascular accumulation was the dominant feature of liposome distribution in tumors, whereas only minimal intramural accumulation in postcapillary and collecting venules was observed in normal s.c. tissue. The extravasated liposomes in tumors distributed heterogeneously and formed perivascular clusters that did not move significantly and could be observed for up to 1 week. The effective permeability of tumor vessels to liposomes (2.0 +/- 1.6 x 10(-8) cm/s; n = 23) was six times smaller than that to bovine serum albumin (1.2 +/- 0.5 x 10(-7) cm/s; n = 6). These results provide new insights into the mechanisms of transendothelial pathways of liposomes and improvements in liposome-mediated drug delivery.

Absence of functional lymphatics within a murine sarcoma: a molecular and functional evaluation.

Despite a clinically recognized association between the lymphatics and metastasis, the biology of tumor-lymphatic interaction is not clearly understood. We report here that functional lymphatic capillaries are absent from the interior of a solid tumor, despite the presence within the tumor of the lymphangiogenic molecule vascular endothelial growth factor (VEGF)-C and endothelial cells bearing its receptor, VEGF receptor 3. Functional lymphatics, enlarged and VEGF receptor 3 positive, were detected in some tumors only at the tumor periphery (within 100 microm of the interface with normal tissue). We conclude that although lymphangiogenic factors are present, formation of functional lymphatic vessels is prevented, possibly due to collapse by the solid stress exerted by growing cancer cells.

Viscous flow of cytoplasm and red cell membrane: membrane recovery and tether contraction.

Several experiments have been designed to study the flow of membrane and measure its viscosity. In all cases, the viscous dissipation in the cytoplasm (i.e., hemoglobin) is assumed to be negligible in comparison to that in the membrane although this has not been shown analytically. Therefore, we analyze here the viscous flow or "dissipation" in membrane and hemoglobin for two particular experiments. One involves the recovery within a pipet of an aspirated portion of a membrane following the release of the aspiration pressure; the other involves the contraction of a membrane cylinder (tether) following a step change in the axial force acting on the tether. For the pipet -recovery experiment, our results indicate that the viscosity of hemoglobin must be 100 times larger before its dissipation compares to that in the membrane. For the tether-contraction experiment, hemoglobin viscosity must be 1000 to 10,000 times larger before it is significant. A preliminary result from a tether-contraction experiment gives a characteristic response time (approximately 10 sec) and a viscosity (approximately 10(-3) dyn X sec/cm) consistent with the analytical result that hemoglobin dissipation is negligible.

Lateral mobility of integral proteins in red blood cell tethers.

The red blood cell membrane is a complex material that exhibits both solid- and liquidlike behavior. It is distinguished from a simple lipid bilayer capsule by its mechanical properties, particularly its shear viscoelastic behavior and by the long-range mobility of integral proteins on the membrane surface. Subject to sufficiently large extension, the membrane loses its shear rigidity and flows as a two-dimensional fluid. These experiments examine the change in integral protein mobility that accompanies the mechanical phenomenon of extensional failure and liquidlike flow. A flow channel apparatus is used to create red cell tethers, hollow cylinders of greatly deformed membrane, up to 36-microns long. The diffusion of proteins within the surface of the membrane is measured by the technique of fluorescence redistribution after photobleaching (FRAP). Integral membrane proteins are labeled directly with a fluorescein dye (DTAF). Mobility in normal membrane is measured by photobleaching half of the cell and measuring the rate of fluorescence recovery. Protein mobility in tether membrane is calculated from the fluorescence recovery rate after the entire tether has been bleached. Fluorescence recovery rates for normal membrane indicate that more than half the labeled proteins are mobile with a diffusion coefficient of approximately 4 x 10(-11) cm2/s, in agreement with results from other studies. The diffusion coefficient for proteins in tether membrane is greater than 1.5 x 10(-9) cm2/s. This dramatic increase in diffusion coefficient indicates that extensional failure involves the uncoupling of the lipid bilayer from the membrane skeleton.

Analytical solutions for shear deformation and flow of red cell membrane.

Studies of red blood cell deformation have shown that there are a number of membrane material properties that affect the deformation process. In this paper various types of deformation are modeled using geometrical and constitutive simplifications so that the effect of intrinsic elastic and viscous membrane properties and of major geometric constraints is made obvious while other factors are ignored. To this end, numerical solutions are shunned in favor of exact analytical ("closed-form") solutions to simple and basic membrane deformation problems in order to reveal functional dependence.

Transport in lymphatic capillaries. I. Macroscopic measurements using residence time distribution theory.

We present a novel integrative method for characterizing transport in the lymphatic capillaries in the tail of the anesthetized mouse, which is both sensitive and reproducible for quantifying uptake and flow. Interstitially injected, fluorescently labeled macromolecules were used to visualize and quantify these processes. Residence time distribution (RTD) theory was employed to measure net flow velocity in the lymphatic network as well as to provide a relative measure of lymphatic uptake of macromolecules from the interstitium. The effects of particle size and injection pressure were determined. The uptake rate was found to be independent of particle size in the range of a 6- to 18-nm radius; beyond this size, the interstitial matrix seemed to pose a greater barrier. A comparison of 10 vs. 40 cmH2O injection pressure showed a significant influence on the relative uptake rate but not on the net velocity within the network (3.3 +/- 0.8 vs. 3.8 +/- 1.0 micron/s). This suggested the presence of a systemic driving force for baseline lymph propulsion that is independent of the local pressure gradients driving the uptake. This model can be used to examine various aspects of transport physiology of the initial lymphatics.

Analysis of lateral diffusion from a spherical cell surface to a tubular projection.

Cell surfaces are often heterogeneous with respect to the lateral distribution and mobility of membrane components. Because lateral mobility is related to membrane structure, measurement of a particular component's local diffusion coefficient within a distinct surface region provides useful information about the formation and maintenance of that region. Many structurally interesting cell surface features can be described as narrow tubular projections from the body of the cell. In a companion paper, we consider the thin "tethers" that can be mechanically drawn from the red blood cell membrane, and we measure the transport of fluorescent integral proteins from the surface of the cell body onto the tether. In this paper we present an analysis to describe the surface diffusion of membrane particles from a spherical shell onto a thin cylindrical process. Provision is made for different rates of diffusion within the two morphologically distinct regions. The relative role of each region in controlling the diffusive flux between regions is determined primarily by a single dimensionless parameter. This parameter incorporates the ratio of the two diffusion coefficients as well as the dimensions of each region. The analysis can be applied to a fluorescence photobleaching experiment in which the extended process is bleached. If the dimensions of the spherical cell body and the cylindrical extension are known, then the diffusion coefficients of both regions can be determined from the experimental fluorescence recovery curve.

Microvascular permeability of albumin, vascular surface area, and vascular volume measured in human adenocarcinoma LS174T using dorsal chamber in SCID mice.

A novel method was developed to measure the effective permeability of microvessels in three-dimensional tumors. Two unique features characterized our approach: (i) Texas Red (with peak excitation and peak emission wavelengths of 596 and 615 nm, respectively) was used for macromolecular labeling, to minimize the absorption of fluorescence light by hemoglobin in blood. Thus the tumor tissue could be treated approximately as a uniform medium with respect to light absorption. (ii) The light absorption and scattering in tumor tissues were accounted for in relating the fluorescence intensity to the amount of Texas Red-labeled macromolecules extravasated. The vascular permeability of Texas Red-labeled bovine serum albumin in human tumor xenograft LS174T implanted in dorsal skin-fold chamber in severe combined immunodeficient mice was measured using this method. The average permeability-surface area product per unit volume (PS/V, x 10(-4) sec-1) and the average effective permeability (P, x 10(-7) cm/sec) were found to be 1.26 +/- 0.72 and 6.06 +/- 4.30, respectively; the fractional volume of tumor vessels (Vves/V, %) was found to be 9.2 +/- 2.9, and the total surface area of vessels per unit volume (S/V, cm2/cm3) was found to be 239 +/- 82. The errors in the estimation of these parameters are discussed. The method described here is general and can be adapted to study the microvascular permeability of superficial tumors in various organs in patients or animals.

Fluorescence photobleaching with spatial Fourier analysis: measurement of diffusion in light-scattering media.

A new method for the measurement of diffusion in thick samples is introduced, based upon the spatial Fourier analysis of Tsay and Jacobson (Biophys. J. 60: 360-368, 1991) for the video image analysis of fluorescence recovery after photobleaching (FRAP). In this approach, the diffusion coefficient is calculated from the decay of Fourier transform coefficients in successive fluorescence images. Previously, the application of FRAP in thick samples has been confounded by the optical effects of out-of-focus light and scattering and absorption by the sample. The theory of image formation is invoked to show that the decay rate is the same for both the observed fluorescence intensity and the true concentration distribution in the tissue. The method was tested in a series of macromolecular diffusion measurements in aqueous solution, in agarose gel, and in simulated tissue consisting of tumor cells (45% v/v) and blood cells (5% v/v) in an agarose gel. For a range of fluorescently labeled proteins (MW = 14 to 600 kD) and dextrans (MW = 4.4 to 147.8 kD), the diffusion coefficients in aqueous solution were comparable to previously published values. A comparison of the spatial Fourier analysis with a conventional direct photometric method revealed that even for the weakly scattering agarose sample, the conventional method gives a result that is inaccurate and dependent on sample thickness whereas the diffusion coefficient calculated by the spatial Fourier method agreed with published values and was independent of sample thickness. The diffusion coefficient of albumin in the simulated tissue samples, as determined by the spatial Fourier analysis, varied slightly with sample thickness. In contrast, when the same video images were analyzed by direct photometric analysis, the calculated diffusion coefficients were grossly inaccurate and highly dependent on sample thickness. No simple correction could be devised to ensure the accuracy of the direct photometric method of analysis.These in vitro experiments demonstrate the advantage of our new analysis for obtaining an accurate measure of the local diffusion coefficient in microscopic samples that are thick (thickness greater than the microscope depth of focus) and scatter light.

Comparison of IgG diffusion and extracellular matrix composition in rhabdomyosarcomas grown in mice versus in vitro as spheroids reveals the role of host stromal cells.

The tumour extracellular matrix acts as a barrier to the delivery of therapeutic agents. To test the hypothesis that extracellular matrix composition governs the penetration rate of macromolecules in tumour tissue, we measured the diffusion coefficient of nonspecific IgG in three rhabdomyosarcoma subclones growing as multicellular spheroids in vitro or as subcutaneous tumours in dorsal windows in vivo. In subcutaneous tumours, the diffusion coefficient decreased with increasing content of collagen and sulphated glycosaminoglycans. When grown as multicellular spheroids, no differences in either extracellular matrix composition or diffusion coefficient were found. Comparison of in vitro vs in vivo results suggests an over-riding role of host stromal cells in extracellular matrix production subjected to modulation by tumour cells. Penetration of therapeutic macromolecules through tumour extracellular matrix might thus be largely determined by the host organ. Hence, caution must be exercised in extrapolating drug penetrability from spheroids and multilayer cellular sandwiches consisting of only tumour cells to tumours in vivo.

Quantitative analysis of angiogenesis and growth of bone: effect of indomethacin exposure in a combined in vitro-in vivo approach.

Nonsteroidal anti-inflammatory agents have been used experimentally and clinically to suppress a variety of physiological events, including angiogenesis and formation of bone. The exact mechanisms by which indomethacin alters skeletal tissue generation are unknown, due in part to methodological limitations. By the use of an organ culture assay and an animal model using intravital microscopy in mice bearing dorsal skinfold chambers, the effect of indomethacin on growth and angiogenesis of neonatal femora was characterized over 16 days. In both assays, femora significantly elongated with time (P < 0.05). The in vitro growth rate was more rapid than in vivo and dependent on the serum concentration, culture medium and age of mice. Although enhancing the serum content promoted cellular proliferation in organ culture, it dose-dependently suppressed femoral elongation, leading at 20% fetal calf serum to growth rates identical to those observed in vivo. Indomethacin supplementation (2 and 10 mg l-1) significantly accelerated longitudinal femoral growth in organ culture (P < 0.05), whereas in vivo indomethacin (2 mg kg-1) did not modulate either angiogenesis or elongation of bone. Our in vitro data propose a central role of serum in the regulation of bone formation. Although indomethacin altered femoral growth in vitro, our findings do not suggest that indomethacin suppresses angiogenesis or growth of bone in vivo. The complexity of physiological events in vivo may be obscuring a detectable effect.

Flow velocity in the superficial lymphatic network of the mouse tail.

The present study had two goals: 1) to establish an animal model in which a large network of the initial lymphatics of the skin can be investigated in vivo and 2) to measure effective flow velocity (defined as axial component of the flow velocity) in the lymph capillary network of the skin for the first time. A fluorescence microlymphography technique was used to stain the lymph capillaries in the superficial layer of the skin of the nude mouse tail in 10 female animals (mean age 45.8 +/- 2.4 days; mean wt 21.2 +/- 0.8 g). With the use of densitometric image analysis, effective flow velocity along the tail was measured. The network consisted of a honeycomb-like layer of hexagonally shaped meshes that could be stained in all animals. Effective lymph flow velocities were in the range of 1.4-20.4 microns/s with a mean value of 7.7 +/- 5.9 microns/s; median value was 6.2 microns/s (4.5-10.5; 25 and 75% percentiles). This new animal model allows studies of a large network of lymph capillaries in the skin and should provide new insight into the physiology and pathophysiology of the initial lymphatics.

Angiogenesis and growth of isografted bone: quantitative in vivo assay in nude mice.

BACKGROUND: Understanding the regulation of vascularization and formation of bone after skeletal trauma is essential for the development of methods to promote healing. The lack of information on the biology of bone healing led us to establish an experimental model that facilitates the in vivo assessment of angiogenesis and growth of bone. EXPERIMENTAL DESIGN: Fresh, cryopreserved (frozen in the presence or absence of 10% dimethyl sulfoxide (DMSO)) or boiled neonatal femora were transplanted into dorsal skin fold chambers in adult mice of the identical strain, and angiogenesis and growth were monitored over 16 days. Computerized analysis of brightfield and epifluorescence images was employed to characterize the process of angiogenesis. Bone formation was quantified in vivo by the use of oxytetracycline. RESULTS: Reperfusion of pre-existing blood vessels of the graft was observed only in fresh transplanted femora, whereas femora of all experimental groups elicited angiogenic response from the host tissue. The rank order of the angiogenic response was: fresh > cryopreservation with DMSO > cryopreservation without DMSO > boiled. Growth of femora was completely abolished after cryopreservation or boiling. Only fresh transplanted femora increased in length (95 microns/day) and in cartilage diameter (41 microns/day). CONCLUSIONS: Our study demonstrates that (a) angiogenesis and growth of transplanted femora can be chronically assessed using in vivo microscopy; (b) the introduction of oxytetracycline for in vivo fluorescence microscopy allows the differential quantification of bone and cartilage growth; and (c) cryoprotection using DMSO enhances restoration of angiogenic potency after freezing. We consider this assay an excellent experimental model to study in vivo effects of agents or procedures that potentially modulate angiogenesis and growth of bone.

Diffusion and partitioning of proteins in charged agarose gels.

The effects of electrostatic interactions on the diffusion and equilibrium partitioning of fluorescein-labeled proteins in charged gels were examined using fluorescence recovery after photobleaching and gel chromatography, respectively. Measurements were made with BSA, ovalbumin, and lactalbumin in SP-Sepharose (6% sulfated agarose), in phosphate buffers at pH 7 and ionic strengths ranging from 0.01 to 1.0 M. Diffusivities in individual gel beads (D) and in the adjacent bulk solution (D infinity) were determined from the spatial Fourier transform of the digitized two-dimensional fluorescence recovery images. Equilibrium partition coefficients (phi) were measured by recirculating protein solutions through a gel chromatography column until equilibrium was reached, and using a mass balance. Diffusion in the gel beads was hindered noticeably, with D/D infinity = 0.4-0.5 in each case. There were no effects of ionic strength on BSA diffusivities, but with the smaller proteins (ovalbumin and lactalbumin) D infinity increased slightly and D decreased at the lowest ionic strength. In contrast to the modest changes in diffusivity, there were marked effects of ionic strength on the partition coefficients of these proteins. We conclude that for diffusion of globular proteins through gel membranes of like charge, electrostatic effects on the effective diffusivity (Deff = phi D) are likely to result primarily from variations in phi with only small contributions from the intramembrane diffusivity.

Heating or freezing bone. Effects on angiogenesis induction and growth potential in mice.

We have characterized the effect of bone graft treatment by heating or freezing (with or without dimethyl sulfoxide (DMSO)). Tissue culture and dorsal skin-fold chambers in mice were used as sites to quantify the effect on angiogenesis, growth and calcification of neonatal femora. Fresh femora increased in both length and cartilage diameter (calcification in vivo only), but cryopreservation or heating abolished the increase in femoral dimensions. In vivo, femora of all experimental groups elicited an angiogenic response from the host tissue, which was most pronounced for fresh femora, weaker for DMSO-preserved frozen bone and poor for unprotected frozen bone and boiled femora. Freezing in the presence of a cryopreservative (DMSO) was found to preserve the angiogenic potential of frozen bone, whereas unprotected heating or freezing significantly impaired angiogenesis induction and growth potential.

Hindered diffusion in agarose gels: test of effective medium model.

The diffusivities of uncharged macromolecules in gels (D) are typically lower than in free solution (D infinity), because of a combination of hydrodynamic and steric factors. To examine these factors, we measured D and D infinity for dilute solutions of several fluorescein-labeled macromolecules, using an image-based fluorescence recovery after photobleaching technique. Test macromolecules with Stokes-Einstein radii (rs) of 2.1-6.2 nm, including three globular proteins (bovine serum albumin, ovalbumin, lactalbumin) and four narrow fractions of Ficoll, were studied in agarose gels with agarose volume fractions (phi) of 0.038-0.073. The gels were characterized by measuring the hydraulic permeability of supported agarose membranes, allowing calculation of the Darcy permeability (kappa) for each gel sample. It was found that kappa, which is a measure of the intrinsic hydraulic conductance of the gel, decreased by an order of magnitude as phi was increased over the range indicated. The diffusivity ratio D/D infinity, which varied from 0.20 to 0.63, decreased with increases in rs or phi. Thus as expected, diffusional hindrances were the most severe for large macromolecules and/or relatively concentrated gels. According to a recently proposed theory for hindered diffusion through fibrous media, the diffusivity ratio is given by the product of a hydrodynamic factor (F) and a steric factor (S). The functional form is D/D infinity = F(rs/k1/2) S(f), where f = [(rs+rf)/rf]2 phi and rf is the fiber radius. Values of D/D infinity calculated from this effective medium theory, without use of adjustable parameters, were in much better agreement with the measured values than were predictions based on other approaches. The strengths and limitations of the effective medium theory for predicting diffusivities in gels are discussed.

Transport in lymphatic capillaries. II. Microscopic velocity measurement with fluorescence photobleaching.

Despite its relevance to the physiology of lymph formation and propulsion, the instantaneous flow velocity in single lymphatic capillaries has not been measured to date. The method of fluorescence recovery after photobleaching (FRAP) was adapted for this purpose and used to characterize flow in the lymphatic capillaries in tail skin of anesthetized mice during a constant-pressure intradermal injection of fluorescein isothiocyanate-dextran (mol wt 2 x 10(6). The median lymph flow velocity was 4.7 microns/s, and the velocity magnitude ranged from 0 to 29 microns/s. The direction of flow was generally proximal, but stasis and backflow toward the site of injection was also detected. Evidence for oscillatory flow was detected in some FRAP experiments, and in separate experiments a periodicity of approximately 120 min-1, directly correlated to respiration frequency, was measured by tracking the motion of fluorescent latex microspheres (1 micron diam) introduced into the lymphatic capillary network. The velocity magnitude showed a correlation with duration of infusion but not with distance from injection site. It is speculated that the temporal decay of mean velocity magnitude could be related to the relaxation of local pressure gradients as partially collapsed vessels expand during the infusion.

Direct in vivo measurement of targeted binding in a human tumor xenograft.

Binding is crucial to the function of most biologically active molecules, but difficult to quantify directly in living tissue. To this end, fluorescence recovery after photobleaching was used to detect the immobilization of fluorescently labeled ligand caused by binding to receptors in vivo. Measurements of mAb affinity to target antigen within human tumor xenografts revealed a saturable binding isotherm, from which an in vivo carcinoembryonic antigen density of 0.56 nmol/g (5.0 x 10(5)/cell) and an association constant of Ka < or = 4 x 10(7) M(-1) were estimated. The present method can be adapted for in vivo studies of cell signaling, targeted drugs, gene therapy, and other processes involving receptor-ligand binding.