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Published as doi: 10.1096/fj.07-9150com.
 (The FASEB Journal. 2008;22:276-284.)
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(The FASEB Journal. 2008;22:276-284.)
© 2008 FASEB

Enhanced macromolecule diffusion deep in tumors after enzymatic digestion of extracellular matrix collagen and its associated proteoglycan decorin

Mazin Magzoub, Songwan Jin and A. S. Verkman1

Departments of Medicine and Physiology, University of California, San Francisco, California, USA

1Correspondence: 1246 Health Sciences East Tower, Cardiovascular Research Institute, University of California, San Francisco, CA 94143-0521, USA. E-mail: alan.verkman{at}ucsf.edu; http://www.ucsf.edu/verklab


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Drug access to tumors is limited by diffusion through the tumor interstitium. We used a microfiberoptic epifluorescence photobleaching method to determine the role of extracellular matrix (ECM) components in macromolecule diffusion deep in tumor tissue. In subcutaneous B16 tumors in living mice, translational diffusion of 10 kDa FITC-dextran was slowed 2- to 3-fold (compared with its diffusion in water) within a depth of 0.2 mm from the tumor surface, but >10-fold beyond a depth of 1 mm. Diffusion of larger macromolecules, FITC-albumin and 500 kDa FITC-dextran, was slowed by up to 40-fold at 0.5 mm and 300-fold at 2 mm. Intratumoral collagenase (to digest collagen) or cathespin C (to digest decorin) each increased diffusion of 10 kDa FITC-dextran by ~2-fold. However, these treatments dramatically increased diffusion (>10-fold) of larger macromolecules, such as 500 kDa dextran, in deep tumor (2 mm depth). Intratumoral hyaluronidase, in contrast, slowed diffusion throughout the tumor. In vitro measurements in defined gel-like mixtures of collagen, hyaluronan, and decorin closely recapitulated results in tumors in vivo. Mathematical modeling quantified the roles of extracellular space volume fraction and dimensions, and indicated a substantial effect of cell density on diffusion in deep tumor. Our data define the determinants of diffusion in deep tumor and suggest collagen and decorin digestion to greatly facilitate macromolecule delivery.—Magzoub, M., Jin, S., Verkman, A. S. Enhanced macromolecule diffusion deep in tumors after enzymatic digestion of extracellular matrix collagen and its associated proteoglycan decorin.


Key Words: FRAP • ECS • hyluronan


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
DELIVERY OF INTRAVENOUS OR DIRECTLY injected antitumor drugs and genes to tumor cells requires their transport through the tumor extracellular space (ECS), also called the tumor interstitium. Macromolecule diffusion through solid tumors occurs largely by passive diffusion through the tumor ECS (1 , 2) . Little convective delivery occurs in solid tumors because of elevated interstitial fluid pressure as a consequence of vessel leakiness and the absence of functional lymphatics (3 , 4) . The principal independent determinants of diffusion in the ECS include extracellular matrix (ECM) composition and ECS geometry (5 6 7) . The ECM is composed mainly of type I collagen, glycosaminoglycans such as hyluronan (HA), proteoglycans such as decorin, and glycoproteins (8 , 9) .

Measurements using single and multiphoton optical methods have described the effects of matrix composition and macromolecule characteristics on diffusion at the light-accessible surface of solid tumor tissue (7 , 10 11 12 13) . In superficial tumor tissue, the diffusion of dextrans and proteins such as albumin is slowed by 2- to 10-fold compared with their diffusion in saline, depending on tumor type and macromolecule size and charge (7 , 10 , 13) . However, because of the shallow penetration of light using conventional optical methods, little information has been available about diffusion beyond ~ 0.2 mm, where the vast majority of the tumor resides. Also, prior work has largely ignored the role of proteoglycans such as decorin as a determinant of diffusion in the ECM. It has been proposed that the leucine-rich decorin modulates the alignment and spacing of collagen fibrils by binding noncovalently via its protein core to the fibrils and spanning the interfibrillar space with its glycosaminoglycan (dermatan/chrondroitin sulfate) side chains (14 , 15) .

We recently developed and validated a microfiberoptic epifluorescence photobleaching (MFEP) method that overcomes the limited light penetration in solid tissues and allows measurement of diffusion deep in tissue (16) . Here we applied MFEP to measure the diffusion of macromolecules of different sizes deep in tumor tissue in vivo and used enzymes to digest individual ECM components. In vivo experimental data were compared with in vitro diffusion measurements in artificial ECM-like gels to define the viscous determinants of ECS diffusion, and diffusion in tumor ECS was modeled mathematically to define the geometric determinants of diffusion.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Microfiberoptic epifluorescence photobleaching (MFEP)
Photobleaching measurements were done using an epifluorescence apparatus described previously (17) , with modifications. The first-order beam of an argon ion laser (488 nm, Innova 70–4, Coherent Inc., Santa Clara, CA, USA) was diffracted by an acousto-optic modulator (Brimrose Inc., Baltimore, MD, USA) and focused onto the back of a 62.5 µm core diameter multimode fiberoptic (ThorLabs, Newton, NJ, USA) with ferule connector (FC) using a x20 objective lens (numerical aperture, 0.25; Zeiss air). Bleaching was accomplished by increasing laser illumination intensity ~4000-fold, with the bleach duration set empirically (in the range 0.2–10 ms) to reduce fluorescence by 30–40%. The distal end of the fiber was stripped and chemically etched as described (16) to create a micron-sized tip. The shaft near the fiber tip was coated with aluminum (Evaporated Coatings, Inc., Willow Grove, PA, USA) to minimize light loss and prevent detection of fluorescence along the tapered fiber shaft. Sample fluorescence collected by the fiberoptic and objective lens was filtered (490 nm dichroic mirror, 510 nm long-pass filter), detected by a photomultiplier, amplified, and digitized. Fluorescence was sampled at rates of up to 1 MHz for 0.05–5 s after bleaching or at 1 Hz (shutter opened for 20 ms per acquisition) for longer periods (5–40 s).

Introduction of fluorescent dyes (FITC-dextran or FITC-BSA; 40 mg/ml) and photobleaching measurements were done using a double lumen microinjection microfiberoptic device (DMMD), which consists of two pulled glass micropipettes (each down to 4 µm diameter) held together with parallel orientation to allow insertion into solid tissue (Fig. 1 a). One lumen is used to deliver small volumes of fluorescent dye using a syringe pump (0.5–1 µl/min for 20–40 min), and the second lumen is a glass sheath through which the microfiberoptic is inserted. The distance between the microfiberoptic tip and microinjection needle tip was 1–1.5 mm. The DMMD and optical fiber were secured separately using micromanipulators, allowing independent specification of insertion depth.


Figure 1
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  		Figure 1. Microfiberoptic photobleaching method and solution measurements. a) Schematic of MFEP (microfiberoptic epifluorescence photobleaching) apparatus (left) with photographs of a double lumen device (right, top) and its insertion into tumor (right, bottom). Ar laser, argon ion laser; AOM, acoutso-optic modulator; PMT, photomultiplier. b) Left: fluorescence recovery curves of aqueous solutions containing the fluorophores used in this study: 10 kDa and 500 kDa FITC-dextran, and FITC-albumin (top). Data shown in black with fitted curves from Eq. 1 in red. 100% recovery is defined as prebleach intensity. Right: recovery half-times (t1/2) for indicated macromolecules in saline (SE, n=6). c) Recovery curves (left) and t1/2 summary (right) for diffusion of 10 kDa FITC-dextran in solutions made viscous with glycerol (SE, n=6).

Diffusion measurements in solutions and gels
Fluorescein isothiocyanate (FITC) -conjugated dextrans (10 and 500 kDa) and FITC-albumin (all from Sigma, St. Louis, MO, USA) were used for solution and in vivo measurements. Solution measurements were done by introducing the microfiberoptic tip 0.5–1.0 mm into a ~200 µl volume of the test solution. In some experiments solution viscosity was increased by addition of glycerol (0–60% w/v).

Collagen gels were prepared by first titrating a freshly prepared solution of type I collagen from rat tail (4.3 mg/ml; BD Biosciences, Bedford, MA, USA) to pH 7.4 using NaOH. Following centrifugation (15000 g, 30 min), the pellet was dissolved in an acidic solution (pH 2) and diluted in alkaline PBS/NaOH to a final concentration of 4.5% (45 mg/ml), pH 7.4. The collagen was then allowed to gel for 48 h at 37°C. Hyaluronan (HA) gels were prepared by dissolving hyaluronic acid sodium salt (from rooster comb, Sigma) by slow addition of PBS (pH 7.4) to give a final concentration of 0.05% (0.5 mg/ml). After stirring at 4°C for 12 h, the solution was allowed to gel overnight at 4°C. Decorin solutions were produced by dissolving decorin from bovine articular cartilage (Sigma) at a concentration of 0.05% (0.5 mg/ml) in PBS (pH 7.4). For gels containing mixtures of collagen and HA, collagen gels were first produced as described above, then HA was added and allowed to gel. For collagen/decorin gels, decorin was added to the collagen gels. For gels containing all three components, HA followed by decorin was added to preformed collagen gels. Photobleaching measurements in gels were done at 37°C.

Diffusion measurements in tumors in vivo
Experiments were performed on weight- and sex-matched mice that were maintained in air-filtered cages and fed normal mouse chow at the University of California, San Francisco Animal Care facility. For tumor implantation, B16F10 melanoma cells (ATCC No. CRL-6475) were cultured in modified Eagle’s medium (MEM) supplemented with 2 mM L-glutamine, sodium pyruvate (0.11 mg/ml), nonessential amino acids, penicillin (100 U/ml), streptomycin (0.1 mg/ml), and 10% fetal bovine serum (FBS). Following culture, 106 cells in 200 µl PBS were injected subcutaneously between the shoulder blades. Experiments were done 10–12 days postimplantation, at which time tumor volume was generally 1–2 cm3. For MFEP measurements, mice were anesthetized with 2,2,2-tribromoethanol (avertin, Sigma) (i.p. 125 mg/kg) and immobilized in a stereotactic frame. Additional avertin was given as needed. Temperature was monitored using a rectal probe and maintained at 37°C.

Enzymatic digestion of ECM components
Partial digestion of collagen or HA was achieved by direct intratumoral injection with high-purity bacterial collagenase (20 µl, 10%; Sigma) and bacterial hyaluronidase (20 µl; 10%; Sigma), respectively. Measurements were made ~ 3 h after enzyme injection. For digestion of decorin, tumors were injected with cathepsin C from bovine spleen (20 µl, 10%; Sigma) in 0.1 M sodium citrate buffer, pH 5.0, ~3 h prior to measurements. Cathepsin C cleaves the glycosaminoglycan (dermatan sulfate) side chains of proteoglycans (18) . The injection site was 2–3 mm away from the fluorophore injection and the fiberoptic introduction sites. For digestion of collagen, HA, or decorin in artificial gels, the enzymes (at the same concentrations used for in vivo experiments) were incubated with preformed gels at 37°C for 3 h prior to measurements. Control studies (in vivo and in vitro) were done by injection of 20 µl of PBS without enzymes.

Immunostaining of tumor sections
Tumors were removed and fixed with 4% paraformaldehyde in PBS. Tissues were infiltrated with sucrose, embedded in OCT (optimal cutting temperature compound) and frozen, and 6 µm cryostat sections were cut. Sections were blocked with 1% BSA/0.25% Triton X-100 in PBS and incubated with rabbit antiserum against type I collagen (Chemicon, Temecula, CA, USA) or goat antiserum against mouse decorin (R&D Systems, Minneapolis, MN, USA) overnight at 4°C. Secondary antibodies conjugated to Texas Red for collagen or Alexa Fluor 633 or Alexa Fluor 488 for decorin (all from Invitrogen, Eugene, OR, USA) were added at dilutions of 1:50 for collagen and 1:100 for decorin. Cell nuclei were stained with 4'-6-diamidino-2-phenylindole (DAPI). Sections were photographed with a Leica DM4000 B fluorescence microscope equipped with cooled CCD (charge-coupled device) camera (Spot, Diagnostic Instruments, Sterling Heights, MI, USA).

Immunoblot analysis
Tumors tissue was homogenized in 10 mM Tris buffer containing 250 mM sucrose and PMSF (pH 8.0). Samples (6 µg/lane) were electrophoresed on a 10% SDS-polyacrylamide gel, transferred to a nitrocellulose membrane (Amersham Biosciences, Arlington Heights, IL, USA), and incubated with rabbit anticollagen antibody (1:1000; Chemicon International) or anti-β-actin antibody (1:2000; GE Healthcare, Piscataway, NJ, USA), followed by anti-rabbit IgG horseradish peroxidase-linked antibody (1:10,000; GE Healthcare), and visualized using enhanced chemiluminescence (Amersham Biosciences). Protein band densitometry was performed (Scion Image for Macintosh; Scion, Frederick, MD, USA), normalizing to β-actin immunoreactivity.

Photobleaching data analysis
Fluorescence recovery curves, F(t), were analyzed by nonlinear least-squares regression using the semi-empirical equation (19):

Formula 1(1)
where F is the prebleach fluorescence, F0 is the fluorescence immediately after bleaching, R is the mobile fraction, {alpha} is the time exponent ({alpha}=1 for Brownian diffusion and<1 for anomalous diffusion), and t1/2 is the half-time for recovery.

Mathematical modeling of diffusion in tumor extracellular space
Diffusion in tumor ECS was modeled using a 2-dimensional assembly of Voronoi cells in which an ECS for diffusion was created by shrinkage of cellular elements (see Supplemental Material). Voronoi cells were generated from seed points determined by Gaussian-distributed random displacements from an initial evenly spaced distribution. The density of seed points was varied from 11 x 104 mm–2 to 100 x 104 mm–2 to examine the effect of cell density. Diffusion in the ECS was simulated as a random walk process. Point particles were placed initially at random positions in the ECS. At each time step, x and y displacements were sampled from normal distributions with zero mean and SD (2Do{Delta}t)1/2, where {Delta}t is the time interval between successive frames. Diffusion coefficient was 100 µm2/s, time step was 0.001–0.002 s, and total simulation time was >20 s. It was confirmed that the parameters are sufficient for diffusing molecules to travel between many cells. For each simulation condition, >350 trajectories were generated. The simulation domain size was varied from 100 x 100 µm2 to 300 x 300 µm2 for different cell densities and ECS area fractions ({alpha} varied from 0.05 to 0.5). In control simulations, 2- to 3-fold smaller step sizes (0.2 µm) did not alter the results (data not shown), confirming the appropriateness of step length. Simulations were written in Matlab 7.2 (Mathworks) and run on a PC computer. Solute diffusion coefficients were deduced from trajectories by mean-squared displacement analysis. Nonzero molecular size of the diffusing species was modeled by virtual expansion of cells to restrict the accessible x, y coordinates of diffusing molecules.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Microfiberoptic epifluorescence photobleaching (MFEP)
As diagrammed in Fig. 1a , photobleaching was done deep in normally light-inaccessible tumor tissue by insertion of a microfiberoptic with a micron-sized tip whose proximal end is optically coupled to an epi-illumination microscope using an objective lens (Fig. 1a ). The fiberoptic tip illuminated a small, approximately cone-shaped volume of tumor tissue. Photobleaching was carried out by briefly increasing illumination intensity several thousand-fold using an acousto-optic modulator, as diagrammed. We previously reported in vitro experimental analysis and mathematical modeling to validate MFEP for measurement of diffusion deep in solid tissue (16) .

Figure 1b shows representative original MFEP recovery curves obtained by immersing the fiberoptic tip in solutions containing the fluorescent dyes used in this study. For these measurements, the fiberoptic tip diameter was ~2 µm and the tapered tip shaft was coated with a very thin layer of aluminum to allow illumination and fluorescence detection only at the exposed tip. As expected, fluorescence recovery was complete, indicating fully mobile dye, and was slowed with increased molecular size. The curves were fitted well using a semi-empirical photobleaching equation (Eq. 1 ) introduced originally for spot photobleaching of a 2-dimensional array of fluorophores illuminated by a Gaussian beam profile (19) . Figure 1c shows slowed diffusion of 10 kDa FITC-dextran in solutions made viscous with glycerol. Again, fluorescence recovery was complete and the curve fits were very close. As expected, diffusion was inversely related to solution viscosity (not shown).

Slowed macromolecule diffusion deep in tumors
Figure 2 a (left) shows fluorescence recovery curves for diffusion of 10 kDa FITC-dextran with the microfiberoptic tip positioned at different depths in tumor tissue. As found for fluorescence recovery in solutions, the recovery curves in tumors were monophasic and fitted well to Eq. 1 with {alpha} = 1, indicating random (Brownian) diffusion. However, unlike the solution data, recovery curves were incomplete at greater depths in tumor tissue, suggesting dye compartmentalization in diffusion-inaccessible pockets. As summarized in Fig. 2a (right), FITC-dextran diffusion in superficial tumor (≤0.2 mm) was slowed mildly compared with that in saline (Do/D ~2- to 3-fold), but greatly slowed by >10-fold in deeper tumor (≥1 mm). Fluorescence recovery was ~100% complete for superficial tumor, but incomplete in deeper tumor (68±4% at 2 mm). Because some anticancer strategies utilize higher molecular weight agents, we investigated the dependence of diffusion on macromolecule size. As shown in Fig. 2b , the diffusion of FITC-albumin (~66 kDa) and 500 kDa FITC-dextran was slowed to a greater extent than 10 kDa FITC-dextran in tumor vs. saline even in superficial tumor. Incomplete fluorescence recovery was also found for both macromolecules (63±5% for 500 kDa FITC-dextran). Immunostaining of tumor sections showed a greater content of type I collagen and decorin in deeper regions of the tumor compared with the periphery, with substantial staining observed in most sections at depths of >0.5 mm (Fig. 2c ).


Figure 2
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  		Figure 2. Diffusion deep in tumor in vivo. a) Left: fluorescence recovery curves of 10 kDa FITC-dextran at indicated depths from the tumor surface. Right: summary of percent recovery (top) and relative diffusion in saline vs. tumor (Do/D, bottom) (SE, n=4). b) Recovery curves for FITC-albumin and 500 kDa FITC-dextran at 0.5 mm depth from the tumor surface (left), with data summary (right) (SE, n=3). c) Immunostaining of tumor sections for collagen type I (top; red) and decorin (bottom; green). Nuclei stained blue with DAPI. Scale bar, 100 µm.

Altered diffusion in tumor tissue after digestion of ECM components
Each major component of tumor ECM (type I collagen, HA, and decorin) was degraded individually by direct intratumoral enzyme injection. Collagenase treatment efficiently digests collagen in tumor ECM (12) and was found to increase macromolecule diffusion at the tumor surface (10 , 20 , 21) . As shown in Fig. 3 a, collagenase treatment for 3 h increased the diffusion of 10 kDa FITC-dextran by ~2-fold at all depths in tumor tissue. Fluorescence recovery after collagenase treatment became near complete to a depth of 2 mm. Digestion of deocrin by cathepsin C, which successively removes N-terminal dipeptides such that the glycosaminoglycan side chain remain linked only with Ala-Ser (18) , increased FITC-dextran diffusion throughout the tumor to an extent similar to that found for collagenase treatment (Fig. 3a ). In contrast, hyaluronan digestion by hyaluronidase slowed FITC-dextran remarkably throughout the tumor and significantly reduced fluorescence recovery (60±4% at 1.5 mm depth).


Figure 3
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  		Figure 3. Altered diffusion in tumors after enzymatic digestion of ECM components. a) Left three panels: fluorescence recovery curves of 10 kDa FITC-dextran at increasing depth from the tumor surface 3 h after intratumoral injections of collagenase, hyaluronidase, or cathepsin C. Right: data summary (SE, n=4 mice, mean of three measurements per depth, two sets of measurements per tumor). b) Left two panels: recovery curves for 10 kDa FITC-dextran as a function of time after intratumoral injection of collagenase or cathepsin. Right: data summary (SE, n=4). c) Collagen immunoblot. Lane 1: control tumor tissue; lane 2: 3 h after cathepsin C injection; lane 3: 3 h after hyaluronidase injection; lanes 4 –6: 1, 2, or 3 h after collagenase injection. β-actin used as a loading control.

Figure 3b shows the time course of increasing FITC-dextran diffusion at a depth of 1 mm after intratumor injection of collagenase and cathepsin C. Collagenase produced 50% of its effect by ~20 min. The cathepsin C effect was slower, which may be related in part to its greater molecular size (210 kDa) compared with collagenase (107 kDa), and consequently slowed diffusion through the ECM. Control measurements with enzyme-free PBS injected into the tumors indicated that the fluid injection itself does not affect diffusion (Fig. 3b ). Western blot analysis of tumor sections confirmed effective digestion of ECM collagen at 3 h in tumor tissue near the site of collagnease injection (Fig. 3c ). For the larger macromolecules, collagen digestion increased by ~2-fold the diffusion of albumin and 500 kDa dextran at a depth of 0.5 mm (Fig. 4 a) and significantly increased the percentage of fluorescence recovery (albumin, 94±3%; 500 kDa dextran, 84±5%). Decorin digestion also increased percentage fluorescence recovery (albumin, 91±4%; 500 kDa dextran, 76±5%). More pronounced effects of ECM digestions were found deeper in tumor tissue. Collagenase and cathepsin C treatments produced a >10-fold enhancement in the diffusion of 500 kDa dextran at a depth of 2 mm and a dramatic increase in percentage recovery (Fig. 4b ).


Figure 4
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  		Figure 4. Greatly enhanced diffusion of large macromolecules deep in tumors after digestion of collagen and decorin. a) Fluorescence recovery curves (left) and data summary (right) (SE, n=4) for FITC-albumin and 500 kDa FITC-dextran at 0.5 mm depth at 3 h after intratumoral injection of collagenase or cathepsin C. b) Fluorescence recovery curves and data summary (SE, n=3) for 500 kDa FITC-dextran at 2.0 mm depth at 3 h after intratumoral injection of collagenase or cathepsin C.

Diffusion measurements in artificial ECM-like gels
Diffusion in tumor ECS is slowed by viscous and geometric factors. We investigated viscous factors by in vitro measurements in artificial ECM-like gels and geometric factors by mathematical modeling (see below). Diffusion measurements were done in vitro in gels composed of mixtures of type I collagen, HA, and decorin at physiologically relevant concentrations (22 23 24) . In pure collagen gels (at 4.5% w/v), diffusion of 10 kDa FITC-dextran was slowed by 2- to 3-fold (Fig. 5 a), whereas diffusion was slowed little in HA gels (0.05% w/v) or decorin solutions (0.05% w/v). Much greater slowing of diffusion was seen in gels containing more than one component (at these same concentrations). Diffusion in gels composed of collagen and HA or collagen and decorin was slowed by >4-fold (Fig. 5b ). Diffusion was slowed by ~8-fold in gels containing all three components. Treatment of the three-component gels with collagenase or cathepsin C increased diffusion by >2-fold, whereas hyaluronidase greatly slowed diffusion (Fig. 5c ). These results are in semiquantitative agreement with findings in tumors in vivo.


Figure 5
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  		Figure 5. Diffusion in artificial gels containing ECM components. a) Left: fluorescence recovery of 10 kDa FITC-dextran in gels containing collagen (4.5% w/v) or HA (0.05% w/v) in PBS or a solution of decorin (0.05% w/v) in PBS. Right: data summary of relative diffusion in gels vs. saline (SE, n=4). b) Left: recovery curves for 10 kDa FITC-dextran in collagen gels containing HA, decorin, or both at the same concentration as in panel a. Right: data summary (SE, n=4). c) Recovery curves and data summary (SE, n=3) for 10 kDa FITC-dextran in collagen gels containing HA and decorin (at the concentrations used above) 3 h after incubations with collagenase, hyaluronidase, or cathepsin C.

Mathematical model of macromolecule diffusion in tumor extracellular space
The above data define the "viscous" determinants of macromolecule diffusion in the ECM by key ECM components. Mathematical modeling was done to investigate geometric restrictions to diffusion in the ECS. As described in Materials and Methods, we modeled the ECS surrounding tumor cells using a 2-dimensional assembly of Voronoi cells and random walk diffusion in which cell density, ECS fraction, ECS shape, and size of the diffusing particle could be specified. Figure 6 a shows an assembly of Voronoi cells with an ECS in which particles could diffuse. In the initial simulation, cell size was taken to be fairly homogeneous, the walls of the ECS were parallel, and point-like particles diffused freely throughout the ECS (Fig. 6a , middle panel). As shown in the left and right panels in Fig. 6a , the ECS area (volume) fraction ({alpha}) (at constant cell density) and the cell density (at constant {alpha}) were varied. Figure 6b (left) summarizes computed Do/D as a function of {alpha} for different cell densities. The simulations predicted the expected reduction in Do/D with increasing {alpha} and decreasing cell density. Of relevance to diffusion in deep tumor, at constant {alpha} there was a relatively large effect of cell density. With regard to the experimental data in Fig. 2 , these computations indicated that the hindered diffusion in deep tumor tissue can be produced by either reduced volume fraction or increased cell density. From DAPI staining of nuclei, there was on average an ~2.5-fold increased cell density in deep vs. superficial tumor tissue in the tumor model used here.


Figure 6
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  		Figure 6. Mathematical model of diffusion in tumor extracellular space. a) Assemblies of Voronoi cells with a parallel ECS shown for different ECS area fraction, {alpha} (left), and cell density (right). Inset at the right shows representative diffusive trajectory of a point particle. Parameters: cell density = 11 x 104/mm2, {alpha} = 0.1 (left); cell density = 11 x 104/mm2, {alpha} = 0.3 (middle); cell density = 25 x 104/mm2, {alpha} = 0.3 (right); box size = 100 x 100 µm2. b) Do/D from simulations as a function of {alpha} (left) and diameter of diffusing particle (dp, right) for different cell densities. c) Left three panels: schematic of ECS geometries for nonparallel ECS with different {sigma}ECS. Right: Do/D from simulations as a function of particle diameter for different {sigma}ECS.

We also modeled effects of the size of the diffusing particle, keeping {alpha} and cell density constant. These computations were done with a parallel wall ECS, as in Fig. 6a, as well as a nonparallel wall, or "heterometric" ECS (Fig. 6c , left 3 panels). Heterometricity was quantified by the SD, {sigma}ECS, of the Gaussian distribution (see Supplemental Material), with {sigma}ECS = 0 corresponding to a parallel wall ECS. Figure 6c (right) shows that, for higher {sigma}ECS, there was a greater increase in Do/D with increasing diameter of the diffusing particle, dp. This particle size dependence to Do/D results from hindrance to diffusion of larger particles in passing through the more narrow spaces of a heterometric ECS. With regard to the experimental data in Fig. 4 showing great hindrance of 500 kDa dextran deep in tumor, this computation indicates that the hindrance can arise from increased cell density and/or heterometricity.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We measured by microfiberoptic epifluorescence photobleaching the diffusion of macromolecules deep in tumors in vivo at depths much greater than those accessible with conventional surface illumination optical methods. We found mild slowing of diffusion of 10 kDa FITC-dextran in superficial tumor (≤0.2 mm), but >10-fold slowing in deeper regions (≥1 mm) (Fig. 2a ). Diffusion of larger macromolecules (albumin and 500 kDa dextran) was slowed to a somewhat greater extent in superficial tumor (Fig. 2b ), but became greatly slowed and restricted in deep tumor (Fig. 3d ). Since these larger macromolecules are comparable in size to some chemotherapeutics and viral/liposomal delivery systems, our results implicate diffusion in tumor ECS as a significant barrier to macromolecule delivery to tumor cells in deep tumor tissue, where the majority of tumor cells reside.

Digestion of type I collagen by collagenase produced an ~2-fold increase in the diffusion of 10 kDa dextran at all depths in tumor (Fig. 3a ). Collagenase treatment also increased the diffusion of 500 kDa FITC-dextran and albumin by ~2-fold, but only in superficial tumor (Fig. 3c ). This enhancement of diffusion is similar to the 2-fold increase in IgG diffusion reported in superficial tumor after collagenase treatment (20) . Collagenase treatment was also found recently to enhance the diffusion of an oncolytic herpes simplex virus in superficial tumor, increasing the area of viral distribution by ~3-fold (21) . In contrast to results in superficial tumor, we found a much greater effect of collagenase on the diffusion of large macromolecules in deep tumor. Diffusion of 500 kDa dextran was increased by >10-fold and the extent of fluorescence recovery was increased by ~4-fold, with the majority of the originally immobile macromolecules becoming mobile (Fig. 3d ). This may be due to the greater density of fibrillar collagen in the ECM deep in tumor tissue, resulting in restricted diffusion of larger molecules more than smaller molecules. Staining of human adenocarcinomas showed an ~4-fold increase in collagen content from superficial to deeper (2 mm) tumor tissue (25) , as found for the mouse tumor model used here (Fig. 2c ). These results support the potential use of collagen digestion as a strategy to enhance drug delivery deep in tumors.

We report here the first evidence for decorin as an important determinant of macromolecule diffusion in tumor ECM. ECM decorin is postulated to suppress tumor growth and spread (26 , 27) . Conversely, collagen-associated decorin in pancreatic tumors was found to attenuate the cytostatic actions of chemotherapeutic drugs (28) . Decorin colocalizes with collagen in the ECM (Fig. 2a ), and its digestion by cathepsin C increased diffusion throughout tumor tissue to an extent similar to that found for collagen digestion (Figs. 3a and 4a ). Decorin digestion produced a more substantial increase in the diffusion of 500 kDa dextran deeper in tumor tissue, similar to the finding for collagen digestion (Fig. 4b ). Decorin cleavage may produce widening of collagen interfibrillar spaces, as it has been implicated in collagen fibril-fibril interactions (29) . Interfibrillar spaces are widened in skin in decorin knockout mice (30) , and inhibition of decorin synthesis by β-D-xyloside produced large separations in fibrillar collagen in corneal stroma (31) . Our data thus support a previously unrecognized role for decorin as a determinant of diffusion in tumor ECM and suggest decorin as a target to enhance macromolecule diffusion in tumors. It remains to be determined whether other ECM collagen binding proteoglycans, such as lumican (32) , might play a similar role.

Digestion of ECM hyaluronan slowed macromolecule diffusion (Fig. 3a ), which was surprising because hyaluronidase administration was found to enhance the therapeutic efficacy of antitumor agents (33 , 34) . Hyaluronan polymerizes in cage-like structures of ~15 nm diameter that contain water-filled spaces, allowing the passage of tracers (35) . Hyaluronidase treatment has been proposed to result in collapse of the water-swelled cage structures, increasing viscous hindrance (10) . Thus, the enhanced chemotherapeutic efficacy observed with hyaluronidase treatment is likely due to mechanisms other than enhanced drug penetration in the ECM, such as inhibition of intercellular adhesion, which overrides cell contact-dependent growth, recruits cells into the cycling pool, and renders tumor cells more sensitive to cytotoxic agents (36) .

The in vitro studies here showed that collagen gels produced significant slowing of diffusion, whereas little or no slowing was found in HA gels or decorin solutions (Fig. 5a ). Addition of HA to the collagen gels produced greater slowing of diffusion than would be expected from the additive effects of the individual components (Fig. 5b ), likely because of the formation of an HA mesh bridging the collagen interfibrillar regions and reducing spaces through which macromolecules can diffuse (37) . This finding agrees with the observation that HA-rich tumors are not particularly resistant to fluid and macromolecule penetration unless the HA is stabilized by a collagen matrix (20) . Similar synergy in slowing was seen for gels consisting of mixtures of collagen and decorin (Fig. 5b ). Decorin binds strongly to type I collagen in vitro, with the phosphate-dependent interaction involving binding of collagen to the decorin protein core rather than its glycosaminoglycan side chains (14 , 38 , 39) . In the experiments here, decorin was added to preformed collagen gels because decorin has been shown to inhibit collagen gel formation (38 , 40 , 41) . Addition of HA to collagen gels followed by decorin resulted in an ~8-fold slowed diffusion (Fig. 5b ), comparable to that seen in relatively deep (~1 mm) tumor. Enzymatic digestion of the collagen or decorin in the in vitro gels resulted in an ~2-fold increase in diffusion, whereas digestion of HA substantially reduced diffusion (Fig. 5c ). These results support the conclusions from the in vivo tumor studies on roles of the individual ECM components in slowing macromolecule diffusion.

The in vivo and in vitro effects of enzymatic treatment provided information about the ECM determinants of diffusion in the ECS. Mathematical modeling was used to analyze the other major determinant of diffusion in the ECS: ECS geometry. There have been few prior attempts to model diffusion in tumor ECS. Tumor geometry has been modeled as an ordered periodic array of cubic cells (42) , as a cylindrical cord of cells surrounding a blood vessel (43) , and as an organized network of interconnected pores (44) . Representing a more comprehensive model, the computations in Fig. 6 provide quantitative data predicting effects on diffusion of ECS volume fraction, cell density, diffusing particle size, and ECS wall geometry. The computations demonstrate the role of cell density as an important, independent determinant of diffusion, which likely accounts in part for the slowed macromolecular diffusion deep in tumor tissue. The computations also defined the factors that may contribute, along with altered ECM composition, to the greatly hindered diffusion of large macromolecules in deep tumor tissue. The parameters used for modeling are in the range of those suggested by the relatively little experimental data on the subject. In fibrosarcomas, the ECS volume fraction was estimated to vary between 0.33 and 0.6, whereas in gliomas it varies between 0.2 and 0.4, and in carcinomas between 0.36 and 0.5 (22) . Within the same fibrosacromas, the ECS volume fraction was reported to vary greatly from 0.4 to 0.8 in different tumor subregions (45) ; in melanomas an intratumoral variation of 0.05–0.7 was reported (46) . Until additional experimental data become available on tumor ECS geometric parameters, the utility of the modeling done here is limited to a descriptive analysis of how various characteristics of the tumor ECS could affect diffusion.

Our data provide a rational basis for consideration of enzymatic modification of tumor matrix as a strategy to enhance drug delivery to tumor cells. However, there are some caveats and considerations for selection of enzymes and administration procedures. Binding of decorin to collagen has been shown to reduce susceptibility of collagen fibrils to degradation by collagenases, possibly due to the steric hindrance of the proteoglycan to access of collagenases (47) , which could account for the observation that collagenase treatment did not completely reverse the slowing of diffusion in the gels (Fig. 5c ). Thus, coadministration of enzymes that target both collagen and decorin might yield a greater enhancement of macromolecule diffusion in tumors in vivo than by the individual enzymes. Another important consideration is the tumor microenvironment. Due to abnormal microcirculation resulting in lactic acid accumulation and an acidic pH as low 5.8 (48) , activities of some enzymes may be inhibited. This low extracellular pH is favorable to cathepsin proteases (49) , but not ideal for collagenases, which generally have an optimal pH of 6.3 to 8.8 (50) . Finally, a potential concern of enzyme digestion is intratumoral hemorrhage, with possible increased risk of metastasis and blood loss. However, these problems were not seen in earlier studies where tumors were injected with various proteases including collagenase, dispase, and trypsin (21 , 51) .

In summary, our studies define the roles of individual ECM components as determinants of macromolecule diffusion in the ECS in deep tumor. Our data quantify the effects of collagen and HA digestion in deep tumor, and demonstrate a previously unrecognized role of decorin. The dramatic effects of enzyme treatment in enhancing large macromolecule diffusion in deep tumor support the possibility of enzymatic targeting of ECM collagen and decorin as a strategy to enhance delivery of chemotherapeutic agents to tumor cells.


   ACKNOWLEDGMENTS
 
We thank Liman Qian for mouse breeding and genotype analysis, Drs. Hara-Chikuma and Hye-Rahn Bae for help with tumor cell preparation, Dr. Javier Ruiz-Ederra for help with immunostaining, and Dr. Aaron Mills for help with tumor photography. This work was supported by grants EB00415, DK35124, EY13574, HL59198, DK72517, and HL73856 from the National Institutes of Health and by Research Development Program and Drug Discovery grants from the Cystic Fibrosis Foundation.

Received for publication June 4, 2007. Accepted for publication July 26, 2007.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Netti, P. A., Hamberg, L. M., Babich, J. W., Kierstad, D., Graham, W., Hunter, G. H., Wolf, G. L., Fischman, A., Boucher, Y., Jain, R. K. (1999) Enhancement of fluid filtration across tumor vessels: implication for delivery of macromolecules. Proc. Natl. Acad. Sci. U. S. A. 96,3137-3142[Abstract/Free Full Text]
  2. Banerjee, R. K., Sung, C., Bungay, P. M., Dedrick, R. L., van Osdol, W. W. (2002) Antibody penetration into a spherical prevascular tumor nodule embedded in normal tissue. Ann. Biomed. Eng. 30,828-839[CrossRef][Medline]
  3. Ribatti, D., Nico, B., Crivellato, E., Vacca, A. (2007) The structure of the vascular network of tumors. Cancer Lett. 248,18-23[CrossRef][Medline]
  4. DiResta, G. R., Lee, J., Healey, J. H., Levchenko, A., Larson, S. M., Arbit, E. (2000) Artificial lymphatic system: a new approach to reduce interstitial hypertension and increase blood flow, pH and pO(2) in solid tumors. Ann. Biomed. Eng. 28,543-555[CrossRef][Medline]
  5. Levick, J. R. (1987) Flow through interstitium and other fibrous matrices. Q. J. Exp. Physiol. 72,409-437[Abstract/Free Full Text]
  6. Graff, B. A., Bjørnæs, I., Rofstad, E. K. (2000) Macromolecule uptake in human melanoma xenografts: relationships to blood supply, vascular density, microvessel permeability and extracellular volume fraction. Eur. J. Cancer 36,1433-1440[CrossRef][Medline]
  7. Pluen, A., Boucher, Y., Ramanujan, S., McKee, T. D., Gohongi, T., di Tomaso, E., Brown, E. B., Izumi, Y., Campbell, R. B., Berk, D. A., Jain, R. K. (2001) Role of tumor-host interactions in interstitial diffusion of macromolecules: cranial vs. subcutaneous tumors. Proc. Natl. Acad. Sci. U. S. A. 98,4628-4633[Abstract/Free Full Text]
  8. Alberts, B., Bray, D., Lewis, J., Raff, M., Roberts, K., Watson, J. (1994) Extracellular matrix of animals. Molecular Biology of the Cell 3rd Ed. ,971-995 Garland Publishing New York.
  9. Gribbon, P M., Maroudas, A., Parker, K H., Winlove, C P. (1998) Water and solute transport in the extracellular matrix: physical principles and macromolecular determinants. Reed, R. K. Rubin, K. eds. Connective Tissue Biology: Integration and Reductionism ,95-124 Portland Press London.
  10. Alexandrakis, G., Brown, E. B., Tong, R. T., McKee, T. D., Campbell, R. B., Boucher, Y., Jain, R. K. (2004) Two-photon fluorescence correlation microscopy reveals the two-phase nature of transport in tumors. Nat. Med. 10,203-207[CrossRef][Medline]
  11. Brown, E. B., Campbell, R. B., Tsuzuki, Y., Xu, L., Carmeliet, P., Fukumura, D., Jain, R. K. (2001) In vivo measurement of gene expression, angiogenesis and physiological function in tumors using multiphoton laser scanning microscopy. Nat. Med. 7,864-868[CrossRef][Medline]
  12. Brown, E., McKee, T., diTomaso, E., Pluen, A., Seed, B., Boucher, Y., Jain, R. K. (2003) Dynamic imaging of collagen and its modulation in tumors in vivo using second-harmonic generation. Nat. Med. 9,796-800[CrossRef][Medline]
  13. Stroh, M., Zimmer, J. P., Duda, D. G., Levchenko, T. S., Cohen, K. S., Brown, E. B., Scadden, D. T., Torchilin, V. P., Bawendi, M. G., Fukumura, D., Jain, R. K. (2005) Quantum dots spectrally distinguish multiple species within the tumor milieu in vivo. Nat. Med. 11,678-682[CrossRef][Medline]
  14. Scott, J. E. (1988) Proteoglycan-fibrillar collagen interactions. Biochem. J. 252,313-323[Medline]
  15. Iozzo, R. V. (1998) Matrix proteoglycans: from molecular design to cellular function. Annu. Rev. Biochem. 67,609-652[CrossRef][Medline]
  16. Thiagarajah, J. R., Kim, J. K., Magzoub, M., Verkman, A. S. (2006) Slowed diffusion in tumors revealed by microfiberoptic epifluorescence photobleaching. Nat. Methods 3,275-228[CrossRef][Medline]
  17. Seksek, O., Biwersi, J., Verkman, A. S. (1997) Translational diffusion of macromolecule-sized solutes in cytoplasm and nucleus. J. Cell Biol. 138,131-142[Abstract/Free Full Text]
  18. Bittner, K., Liszio, C., Blumberg, P., Schönherr, E., Kresse, H. (1996) Modulation of collagen gel contraction by decorin. Biochem. J. 314,159-166[Medline]
  19. Feder, T. J., Brust-Mascher, I., Slattery, J. P., Baird, B., Webb, W. W. (1996) Constrained diffusion or immobile fraction on cell surfaces: a new interpretation. Biophys. J. 70,2767-2773[Medline]
  20. Netti, P. A., Berk, D. A., Swartz, M. A., Grodzinsky, A. J., Jain, R. K. (2000) Role of extracellular matrix assembly in interstitial transport in solid tumors. Cancer Res. 60,2497-2503[Abstract/Free Full Text]
  21. McKee, T. D., Grandi, P., Mok, W., Alexandrakis, G., Insin, N., Zimmer, J.P., Bawendi, M. G., Boucher, Y., Breakefield, X. O., Jain, R. K. (2006) Degradation of fibrillar collagen in a human melanoma xenograft improves the efficacy of an oncolytic herpes simplex virus vector. Cancer Res. 66,2509-2513[Abstract/Free Full Text]
  22. Jain, R. K. (1987) Transport of molecules in the tumor interstitium: a review. Cancer Res. 47,3039-3051[Abstract/Free Full Text]
  23. Ramanujan, S., Pluen, A., McKee, T. D., Brown, E. B., Boucher, Y., Jain, R. K. (2002) Diffusion and convection in collagen gels: implications for transport in the tumor interstitium. Biophys. J. 83,1650-1660[Medline]
  24. Theocharis, A. D., Vynios, D. H., Papageorgakopoulou, N., Skandalis, S. S., Theocharis, D. A. (2003) Altered content composition and structure of glycosaminoglycans and proteoglycans in gastric carcinoma. Int. J. Biochem. Cell Biol. 35,376-390[CrossRef][Medline]
  25. Brown, E. B., Boucher, Y., Nasser, S., Jain, R. K. (2004) Measurement of macromolecular diffusion coefficients in human tumors. Microvasc. Res. 67,231-236[CrossRef][Medline]
  26. Csordas, G., Santra, M., Reed, C. C., Eichstetter, I., McQuillan, D. J., Gross, D., Nugent, M. A., Hajnóczky, G., Iozzo, R. V. (2000) Sustained down-regulation of the epidermal growth factor receptor by decorin. A mechanism for controlling tumor growth in vivo. J. Biol. Chem. 275,32879-32887[Abstract/Free Full Text]
  27. Nash, M. A., Deavers, M. T., Freedman, R. S. (2002) The expression of decorin in human ovarian tumors. Clin. Cancer Res. 8,1754-1760[Abstract/Free Full Text]
  28. Köninger, J., Giese, N. A., di Mola, F. F., Berberat, P., Giese, T., Esposito, I., Bachem, M. G., Büchler, M. W., Friess, H. (2004) Overexpressed decorin in pancreatic cancer: potential tumor growth inhibition and attenuation of chemotherapeutic action. Clin. Cancer Res. 10,4776-4783[Abstract/Free Full Text]
  29. Scott, J. E., Dyne, K. M., Thomlinson, A. M., Ritchie, M., Bateman, J., Cetta, G., Valli, M. (1998) Human cells unable to express decorin produced disorganised extracellular matrix lacking ‘shape modules’ (interfibrillar proteoglycan bridges). Exp. Cell Res. 243,59-66[CrossRef][Medline]
  30. Danielson, K. G., Baribault, H., Holmes, D. F., Graham, H., Kadler, K. E., Iozzo, R. V. (1997) Targeted disruption of decorin leads to abnormal collagen fibril morphology and skin fragility. J. Cell Biol. 136,729-743[Abstract/Free Full Text]
  31. Hahn, R. A., Birk, D. E. (1992) Beta-D xyloside alters dermatan sulfate proteoglycan synthesis and the organization of the developing avian corneal stroma. Development 115,383-393[Abstract]
  32. Chakravarti, S., Magnuson, T., Lass, J. H., Jepsen, K. J., LaMantia, C., Carroll, H. (1998) Lumican regulates collagen fibril assembly: skin fragility and corneal opacity in the absence of lumican. J. Cell Biol. 141,1277-1286[Abstract/Free Full Text]
  33. Beckenlehner, K., Bannke, S., Spruss, T., Bernhardt, G., Schonenberg, H., Scheiss, W. (1992) Hyaluronidase enhances the activity of adriamycin in breast cancer models in vitro and in vivo. J. Cancer Res. Clin. Oncol. 118,591-596[CrossRef][Medline]
  34. Smith, K. J., Skelton, H. G., Turiansky, G., Wagner, K. F. (1997) Hyaluronidase enhances the therapeutic effect of vinblastine in intralesional treatment of Kaposi’s sarcoma. Military Medical Consortium for the Advancement of Retroviral Research (MMCARR). J. Am. Acad. Dermatol. 36,239-242[CrossRef][Medline]
  35. Masuda, A., Ushida, K., Koshino, H., Yamashita, K., Kluge, T. (2001) Novel distance dependence of diffusion constants in hyaluronan aqueous solution resulting from its characteristic nano-microstructure. J. Am. Chem. Soc. 123,11468-11471[CrossRef][Medline]
  36. Croix, B. S., Rak, J. W., Kapitain, S., Sheehan, C., Graham, C. H., Kerbel, R. S. (1996) Reversal by hyaluronidase of adhesion-dependent multicellular drug resistance in mammary carcinoma cells. J. Natl. Cancer Inst. 88,1285-1296[Abstract/Free Full Text]
  37. Shenoy, V., Rosenblatt, J. (1995) Diffusion of macromolecules in collagen and hyaluronic acid, rigid-rod–flexible polymer, composite matrices. Macromolecules 28,8751-8758[CrossRef]
  38. Pogány, G., Hernandez, D. J., Vogel, K.G. (1994) The in vitro interaction of proteoglycans with type I collagen is modulated by phosphate. Arch. Biochem. Biophys. 313,102-111[CrossRef][Medline]
  39. Svensson, L., Heinegård, D., Oldberg, A. (1995) Decorin-binding sites for collagen type I are mainly located in leucine-rich repeats 4–5. J. Biol. Chem. 270,20712-20716[Abstract/Free Full Text]
  40. Kuc, I. M., Scott, P. G. (1997) Increased diameters of collagen fibrils precipitated in vitro in the presence of decorin from various connective tissues. Connect. Tissue Res. 36,287-296[Medline]
  41. Douglas, T., Heinemann, S., Bierbaum, S., Scharnweber, D., Worch, H. (2006) Fibrillogenesis of collagen types I, II, and III with small leucine-rich proteoglycans decorin and biglycan. Biomacromolecules 7,2388-2393[CrossRef][Medline]
  42. El Kareh, A. W., Braunstein, S. L., Secomb, T. W. (1993) Effect of cell arrangement and interstitial volume fraction on the diffusivity of monoclonal antibodies in tissue. Biophys. J. 64,1638-1646[Medline]
  43. Kyle, A. H., Minchinton, A. I. (1999) Measurement of delivery and metabolism of tirapazamine to tumour tissue using the multilayered cell culture model. Cancer Chemother. Pharmacol. 43,213-220[CrossRef][Medline]
  44. Yuan, F., Krol, A., Tong, S. (2001) Available space and extracellular transport of macromolecules: effects of pore size and connectedness. Ann. Biomed. Eng. 29,1150-1158[CrossRef][Medline]
  45. Krol, A., Maresca, J., Dewhirst, M. W., Yuan, F. (1999) Available volume fraction of macromolecules in the extravascular space of a fibrosarcoma: implications for drug delivery. Cancer Res. 59,4136-4141[Abstract/Free Full Text]
  46. Jakobsen, I., Lyng, H., Kaalhus, O., Rofstad, E. K. (1995) MRI of human tumor xenografts in vivo: proton relaxation times and extracellular tumor volume. Magn. Reson. Imaging 2,97-106
  47. Geng, Y., McQuillan, D., Roughley, P. J. (2006) SLRP interaction can protect collagen fibrils from cleavage by collagenases. Matrix Biol. 25,484-491[CrossRef][Medline]
  48. Tannock, I. F., Rotin, D. (1989) Acid pH in tumors and its potential for therapeutic exploitation. Cancer Res. 49,4373-8434[Abstract/Free Full Text]
  49. Bühling, F., Waldburg, N., Reisenauer, A., Heimburg, A., Golpon, H., Welte, T. (2004) Lysosomal cysteine proteases in the lung: role in protein processing and immunoregulation. Eur. Respir. J. 23,620-628[Abstract/Free Full Text]
  50. Schomburg, D., Salzmann, M. (1991) Enzyme Handbook Springer-Verlang Berlin.
  51. Kuriyama, N., Kuriyama, H., Julin, C. M., Lamborn, K. R., Israel, M. A. (2001) Protease pretreatment increases the efficacy of adenovirus-mediated gene therapy for the treatment of an experimental glioblastoma model. Cancer Res. 61,1805-1809[Abstract/Free Full Text]



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