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Tumor Biology |
Edwin L. Steele Laboratory [W. L. M., D. F., T. G., F. Y., R. K. J.], Department of Radiation Oncology [M. A.], Center for Imaging and Pharmaceutical Research [V. P. T.], Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114; Department of Radiobiological Sciences, Beth Israel-Deaconess Medical Center, Boston, Massachusetts 02114 [W. L. M.]; and Department of Gene Regulation and Differentiation, National Research Center for Biotechnology, Branschweig, Germany [H. A. W.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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7 nm in diameter) and extravasation of polyethylene glycol-stabilized long-circulating liposomes (100–400 nm) and latex microspheres (800 nm) were determined by intravital microscopy. Vascular permeability was found to be temporally heterogeneous. VEGF superfusion (100 ng/ml) significantly increased vascular permeability to albumin in normal s.c. vessels, whereas a 30-fold higher dose of VEGF (3000 ng/ml) was required to increase permeability in pial vessels, suggesting that different tissues exhibit different dose thresholds for VEGF activity. Furthermore, VEGF superfusion (1000 ng/ml) increased vascular permeability to albumin in a hypopermeable human glioma xenograft in cranial window, whereas VEGF superfusion (10–1000 ng/ml) failed to increase permeability in a variety of hyperpermeable tumors grown in dorsal skin chamber. Interestingly, low-dose VEGF treatment (10 ng/ml) doubled the maximum pore size (from 400 to 800 nm) and significantly increased the frequency of large (400 nm) pores in human colon carcinoma xenografts. PlGF-1, PlGF-2, or bFGF did not show any significant effect on permeability or pore size in tumors. These findings suggest that exogenous VEGF may be useful for augmenting the transvascular delivery of larger antineoplastic agents such as gene targeting vectors and encapsulated drug carriers (typical range, 100–300 nm) into tumors. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We chose VEGF, also referred to as vascular permeability factor (9, 10, 11) , because: (a) it is 50,000 times more potent than histamine in increasing permeability (9 , 10) ; (b) it has a short biological half-life (11) ; (c) its receptors are predominantly expressed on neovasculature such as that found in the tumor (11 , 12) ; and, (d) morphometric studies have shown that chronic VEGF exposure increases the number of transvascular pathways such as VVOs, open junctions, and fenestrae in both tumor and normal vessels (1 , 13, 14, 15) . Although VEGF and its receptors are overexpressed in the majority of tumors investigated (11) , their distribution is heterogeneous throughout a given tumor (12 , 16) . Tumor regions with low levels of VEGF and/or receptor expression may exhibit decreased microvascular permeability, pore size, and pore numbers. Thus, the exogenous addition of VEGF to hypopermeable vessels could be used to increase transvascular transport in these regions. PlGF-1 and PlGF-2, members of the VEGF family (17 , 18) , and bFGF have been shown to potentiate some of the activity of VEGF (19 , 20) . We evaluated the change in effective permeability, pore cutoff size, and number of transvascular pathways of normal and tumor vessels over time as well as following superfusion with either VEGF, bFGF, PlGF-1, or PlGF-2 alone or PlGF-1 or PlGF-2 in combination with VEGF. These approaches should help enhance the transvascular transport of large macromolecules or nanoparticles such as viral and nonviral vectors for gene therapy as well as encapsulated drug carriers.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Intravital Microscopy
Microcirculatory parameters of normal s.c. and pial tissue as well as of tumors grown in dorsal chambers and cranial windows were studied with an intravital microscope (Axioplan, Zeiss, Oberkochen, Germany) equipped with filter sets for rhodamine (excitation peak at 541 nm, emission peak at 572 nm), cyanine-5 (excitation peak at 649 nm, emission peak at 670 nm), and FITC (excitation, 450–490 nm; emission peak 515–545 nm); an intensified CCD video camera (C2400-88; Hamamatsu Photonics K.K., Hamamatsu, Japan); photomultiplier tube (9203B; EMI, Rockaway, NJ); super VHS video recorder (SVO-9500 MD; Sony, Tokyo, Japan), and thermal video printer (P67U, Mitsubishi, Somerset, NJ). In all experiments, transillumination or epi-illumination (100 W mercury lamp, model 770; Opti-Quip, Inc., Highland Mills, NY) was used to locate and stereotactically record the vessel region of interest using video photo documentation to enable relocalization after a given time interval or superfusion protocol. To minimize the variation in microvascular parameters due to spatial heterogeneity, the same vessels were observed for each experiment before and after the particular treatment (as described below). For each experiment, the relative change from the pretreatment baseline is reported. To facilitate relocalization of vessels after a given time period or treatment (as described below), images of the tumor vessels to be studied were recorded at x2.5, x5, x10, and x20 objectives using a thermal video printer (P67U; Mitsubishi, Somerset, NJ), and marker coordinates were recorded at each magnification.
Permeability.
Albumin permeability (P) was determined as described previously (24)
. Animals were anesthetized with a s.c. injection of 90 mg of Ketamine (Parke-Davis, Morris Plains, NJ) and 9 mg of Xylazine (Fermenta, Kansas City, MO) per kg of body weight. Background intensity was measured under epi-fluorescence illumination and bolus injection of fluorescently labeled BSA (7 nm in diameter) was administered via the tail vein (0.15 ml over 20 s). Fluorescence intensity was measured at 2-min intervals for 21 min at 10 s/observation. The value of P was calculated as P = (1- HT) x V/S {1/(I0 - Ib) x dI/dt + 1/K}, where I is the average fluorescence intensity of the whole image, I0 is the value of I immediately after all vessels are filled by fluorescent BSA, Ib is the background intensity, HT is the average hematocrit in tumor vessels, V and S are the total volume and surface area of vessels within the tissue volume captured by the microscope image, and K is the time constant of plasma clearance of BSA. If the linear curve-fitting of the fluorescence intensity over time had a correlation coefficient of 
0.7, the slope was assumed to be zero. In addition, we used the average time constant of plasma clearance of BSA (K = 9.1 x 103 sec) in calculating P (3)
. Thus, the variation in K among animals may result in a negative value of P when -1/(I0-Ib) x dI/dt is close to 1/K. In this case, we assumed P = 0 (this applied to only a few measurements in normal pial vessels)
Pore Cutoff Size.
As described previously (1)
, incrementally larger rhodamine-labeled, PEG-stabilized, long-circulating liposomes (100–400 nm) and latex microspheres (800 nm) were injected i.v., and the cutoff size for nanoparticle extravasation was determined. To determine the frequency of occurrence of a transvascular pathway of a particular size or larger, the rhodamine-labeled, PEG-stabilized liposome of the size of interest was injected iv, and areas of extravasation were quantified.
Tracers.
Dextran (Mr 2,000,000) labeled with FITC (Sigma, St. Louis, MO) was used for vessel localization and measurements of vascular surface area. Albumin labeled with either cyanine-5 (Amersham, Arlington Heights, IL) or tetramethylrhodamine (Molecular Probes, Eugene, OR) was used when measuring permeability. PEG-stabilized long-circulating latex microspheres (800 nm), and 100 and 400 nm PEG-stabilized, long-circulating liposomes labeled with tetramethylrhodamine were prepared as described previously (25)
for studies of pore cutoff size and transvascular pathway frequency. Particle size was determined using a Coulter N4 MD Particle Size Analyzer (Coulter, Inc., Hialeah, FL).
Experimental Procedure
Temporal Heterogeneity of Tumor Microvascular Permeability.
LS174T human colon adenocarcinoma tumor xenograft was grown in the SCID mouse dorsal skinfold chamber. To determine temporal variation, vascular permeability of each location was measured twice. Permeability to albumin labeled with either cyanine-5 or rhodamine was determined at an initial time point and again at the same, stereotactically located vessels of the tumor with the alternate color-labeled albumin at 1, 3, 5, 12, 19, or 24 h after the initial measurement (n = 4 tumors, for each time interval). All vessels analyzed were well perfused throughout the measurements.
Effect of Topical Superfusion on Normal s.c. and Cranial Microvascular Permeability and Pore Size.
Topical superfusion of VEGF was performed as in our previous studies (26)
, instead of local or systemic injection, to ensure uniform concentration of VEGF around hypo- and hyperpermeable vessels. This uniformity was necessary to test our hypothesis. Experiments were conducted 7–10 days after implantation of dorsal skinfold chambers or cranial windows. The removal of the coverslip may affect permeability. To account for this effect, we performed control measurements after removal of the coverslip and compared the same vessels after the desired treatment in all studies. The coverslip was carefully removed, avoiding hemorrhage and inflammation, to expose the microvasculature and then replaced loosely. The 0.2-mm2 tissue region of interest was located microscopically. Permeability to albumin labeled with either cyanine-5 or rhodamine was determined initially. For superfusion, the coverslip was again removed, and 100 µl of VEGF165 (R & D Systems, Minneapolis, MN; 10, 100, 1000, or 3000 ng/ml), bFGF (R & D Systems; 100 µl at 100 ng/ml), PlGF-1, and PlGF-2 (5 µg; Ref. 18
), or PBS (Sigma) for control was topically applied to these exposed vessels (n = 5 for each superfusion). After 20–40 min, the coverslip was replaced, and the permeability to albumin labeled with the alternate tracer was measured at the exact same vessel region (26)
. To study larger size pores, the extravasation of rhodamine-labeled (100 nm), long-circulating liposomes was observed 24 h after i.v. infusion.
Effect of Topical Superfusion on Tumor Microvascular Permeability.
Tumors were grown as described above. The experiments were then conducted as described for normal microvasculature. One hundred µl of VEGF (10, 100, or 1000 ng/ml), bFGF (1 µg), PlGF-1 (5 µg), PlGF-1 (5 µg) with VEGF (100 µl at 10 ng/ml), or PBS (control) was topically superfused (n = 5 for each superfusion).
Effect of Topical Superfusion on Vascular Pore Cutoff Size and Frequency of Transvascular Pathways.
LS174T tumor xenografts grown in the dorsal chamber were shown to have a pore cutoff size of 400 nm (25)
. These tumors were superfused with 100 µl of VEGF (10 ng/ml), bFGF (1 µg), PlGF-1 (5 µg), PlGF-1 (5 µg) with VEGF (1 ng/ml), or PBS (n = 5 for each superfusion) as described above. Immediately afterward, rhodamine-labeled, 800-nm, long-circulating microspheres were infused i.v. The extravasated microspheres were observed adjacent to tumor microvessels after 24 h, allowing clearance from the bloodstream.
The frequency of tumor transvascular pathways was observed in LS174T tumors grown in dorsal skinfold chambers. Rhodamine-labeled, 400-nm, long-circulating lipososomes were infused i.v. Areas with low levels of extravasation were stereotactically localized and recorded 24 h later, allowing for clearance from the bloodstream. VEGF (100 µl, 10 ng/ml) or PBS was topically superfused, and rhodamine-labeled, 400-nm, long-circulating liposomes were again infused i.v. After 24 h, the same vessels with previously low extravasation were again observed for extravasation of the long-circulating liposomes (n = 5 for each superfusion).
Statistics
Data presented are expressed as mean ± SE. We used paired t tests to compare albumin permeability, pore cutoff size, and frequency of transvascular transport pathways of a particular size before and after the superfusion. In measurements of temporal changes in tumor permeability, we determined 95% confidence intervals by computing the standard errors of the slope of linear regression fitted to the appropriate part of the extravasation curve. Vessel areas did not change significantly over time throughout the studies, as tested with a series of separate measurements using the t test. t tests were also used to compare the effect of the dye and dye type and sequence used for permeability measurements.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Within the same region of the tumor, change in permeability to albumin ranges from 9.18 x 10-7 cm/s to -5.33 x 10-7 cm/s over 24 h. Permeability estimates and confidence intervals for particular animals and time points demonstrate that in 32% of the animals, the changes from baseline were statistically significant at the Bonferroni-corrected level of P = 0.0026 (data not shown). However, there is no temporal trend in these permeability changes, as evidenced by the line of weighted least squares (both coefficients are not significantly different from zero; Fig. 1
).
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VEGF Superfusion Differentially Affects Normal Cranial versus s.c. Microvascular Permeability and Pore Size.
Normal s.c. and pial vessels exhibit low permeability to albumin as compared with tumor vessels. To quantify the permeabilizing effect of VEGF on the normal vessels, we topically superfused s.c. vessels of the dorsal chamber and pial vessels of the cranial window. Topical superfusion is ideal for rapid and homogeneous distribution of a mediator of interest (26)
. However, measurement artifacts may result from coverslip removal. Therefore, we used the data after coverslip removal and PBS superfusion as controls. Control superfusion with PBS did not significantly increase microvascular permeability of the normal s.c. and pial vessels (data not shown). VEGF superfused at 100 ng/ml significantly increased the effective permeability to albumin in s.c. vessels (Fig. 2A)
. On the other hand, 3000 ng/ml VEGF superfusion were required to significantly increase effective permeability of the pial vessels (Fig. 2B)
. The threshold level of VEGF required to increase vascular permeability varied depending on the tissue. The high threshold in pial vessel may be due to the presence of the blood brain barrier in these vessels. Microvascular permeability to albumin in normal s.c. vessels [5.4 ± 1.7 x 10-8 cm/s (n = 5)] did not significantly increase after bFGF superfusion (100 ng/ml; 7.4 ± 6.1 x 10-8 cm/s).
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7 nm in intact s.c. vessels and lower in pial vessels. After coverslip removal and PBS superfusion, 11 ± 4.6% of s.c. vessels were permeable to 100-nm, long-circulating liposomes. Neither VEGF (100 ng/ml and 10 ng/ml) nor bFGF (1 µg), alone or in combination with VEGF (10 ng/ml), significantly increased the percentage of s.c. vessels permeable to the 100-nm liposomes (13 ± 4.2%, 21 ± 9.5%, 16 ± 2.3%, and 18 ± 3.8%, respectively). Similarly, neither PlGF-1 nor PlGF-2 (5 µg) alone resulted in a significant increase in the extravasation of 100-nm, long-circulating liposomes from these vessels (22 ± 1.3% and 13 ± 8.5%, respectively). However, 78 ± 2.6% of the vessels became permeable to 100-nm, long-circulating liposomes (P < 0.005) when PlGF-1 (5 µg) was superfused along with low level of VEGF (10 ng/ml).
VEGF Superfusion Increases Tumor Microvascular Permeability in Hypopermeable Tumors.
To determine whether VEGF could increase the effective permeability of tumor microvasculature, we topically superfused various tumors with VEGF (10, 100, and 1000 ng/ml). Superfusion with VEGF (1000 ng/ml) significantly increased microvascular permeability to albumin from 0.96 ± 0.55 to 4.3 ± 1.5 x 10-7 cm/s (P < 0.0285, paired t test) in the relatively hypopermeable HGL-21 human glioma grown in the cranial window (Fig. 3B)
. However, permeability was not significantly increased with VEGF superfusion in the LS174T human colon adenocarcinoma, HCa-1 murine hepatoma, or ST-8 murine fibrosarcoma tumors grown in the dorsal chamber (Fig. 3A)
. Microvascular permeability was similar before and after removal of the coverslip in the LS174T, HCa-1, and ST-8 tumors (1)
. Superfusion with 100 ng/ml VEGF alone or in combination with 5 µg of PlGF-1 did not significantly increase permeability in the HGL-21 tumor (Fig. 3B)
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. Superfusion of lower levels of VEGF (1 ng/ml) with PlGF-1 (5 µg) and bFGF (1 µg) did not significantly increase the number of vessels permeable to the 800-nm microspheres, 11 ± 5.1 and 15 ± 4.2%, respectively. Extravasation of 800-nm microspheres from PBS superfused vessels was probably due to slight mechanical disruption of the vessel during coverslip removal because no 800-nm microspheres were seen to extravasate from vessels in nonsuperfused tumors, without coverslip removal, in parallel control experiments (data not shown). Although most of the tumor vessels allowed the extravasation of 400-nm microspheres in LS174T tumors (25)
, hypopermeable regions were found in which only 18 ± 3.8% (0–35%) of vessels were permeable to these microspheres. We identified tumor vessels with low levels of 400-nm particle extravasation and superfused them with VEGF (10 ng/ml). A significantly increased fraction of these vessels (56 ± 3.5%) was permeable compared with the fraction of permeable vessels obtained after superfusion with PBS (23 ± 3.8%; Figs. 5
and 6
).
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In Vivo VEGF Function Is Modulated by the Microenvironment.
Before determining how exogenous VEGF modulates transvascular transport in tumors, we examined its effect on normal s.c. and pial vessels. Interestingly, VEGF did not induce similar responses in pial and s.c. vessels at the same concentrations. Higher concentrations of VEGF were required to increase pial vessel permeability, suggesting that the threshold level for the permeabilizing activity of VEGF may be modulated by the local microenvironment. These results are consistent with our previous findings that vessels of tumors grown in cranial windows are less permeable to low molecular weight tracers (3
, 7)
and/or exhibit smaller pore cutoff size (1)
compared with vessels in s.c. space. Conversely, the vessels in LS174T are permeable to BSA in both dorsal skin chamber and cranial window settings (5)
. In addition, neutralization of VEGF with an anti-VEGF antibody reduces the microvascular permeability in LS174T by a similar proportion in both locations (5)
. These data suggest that both the host organ site and the tumor type are important determinants of VEGF function in vivo.
The threshold levels of VEGF may differ for the various activities of VEGF. Chronic administration of low dose VEGF (10 ng) induced open interendothelial junctions and fenestrae but did not induce angiogenesis, which required 50–200 ng of VEGF (14)
. Four to 10-fold increase in chronic VEGF expression by transfected tumors were associated with hemorrhage, whereas mice injected intracerebrally with either 500 or 1000 ng of VEGF165 showed no vascular changes or evidence of angiogenesis in 7 days (39)
. Thus, relatively high levels of VEGF supplied chronically appear to be necessary for angiogenesis in the cranial microenvironment. Embryonic lethality in heterozygous VEGF gene-deleted animals suggests a threshold level of VEGF is needed for embryonic angiogenesis (40
, 41)
. Furthermore, we have found recently that the tumors derived from HIF-1
gene knock-out embryonal stem cells exhibit reduced angiogenesis but similar vascular permeability compared with wild-type embryonal stem cell-derived tumors (42)
. Our results support the emerging concept that different threshold levels of VEGF are required for its different physiological functions and that the threshold levels may be dependent on microenvironmental factors such as the expression of VEGF receptors, cofactors, inhibitors, and other growth factors.
These observations, taken together with the short half-life of VEGF, led us to conjecture that acute application of VEGF at concentrations below the threshold level required for angiogenesis should merely permeabilize hypopermeable vessels and not augment angiogenesis or tumorigenesis locally or at distant sites. We did not notice enhanced angiogenesis or tumorigenicity after any superfusion. In further support of this notion, Dvorak and colleagues (10 , 43 , 44) demonstrated that VEGF-induced vascular permeability lasted <30 min. We did not see a decrease in permeability within 27 min; however, 1 h after superfusion, permeability to albumin had returned to levels expected for nontreated vessels (n = 4; data not shown).
Although we observed a significant increase in BSA (7 nm) permeability in s.c. vessels at 100 ng/ml VEGF, we did not see an increase in pore cutoff size; there was no extravasation of 100- and 200-nm long-circulating liposomes. On the other hand, despite a significant increase in pore cutoff size in LS174T tumor vessels by VEGF (10 ng/ml) superfusion, there was no significant change in vascular permeability to BSA. Thus, BSA permeability does not necessarily correlate with pore cutoff size. Collectively, these data suggest two distinct transport pathways that may be differentially regulated by VEGF: large open gaps for nano-particle transport (1)
and VVOs for macromolecular transport. Furthermore, permeability regulation is different in normal and tumor vessels. In support of this finding, Yuan et al. (5)
noted previously that VEGF neutralization reduced tumor microvascular permeability and suggested that the reduction of VEGF levels might result in the closure of fenestrae without affecting normal junctions between endothelial cells. Careful ultrastructual studies, in conjunction with intravital microscopy studies, are now needed to confirm these hypotheses.
VEGF Can Enhance Permeability in Hypopermeable Tumors.
Previously, we showed that normal vessels adjacent to tumors maintained a pore cutoff size identical to that of normal tissue (1)
. Here, we have shown that lower concentrations of VEGF affect tumor and normal vessels differently. Specifically, VEGF increased pore cutoff size and the number of large pores only in tumor vessels. This result suggests the potential for selective intervention and manipulation of the tumor vasculature.
A threshold level of VEGF required to increase/maintain vessel permeability may exist. Therefore, in hyperpermeable tumors such as LS174T and/or in regions of tumors that express VEGF, exogenous VEGF is not expected to increase albumin microvascular permeability (1) . On the other hand, VEGF would increase albumin permeability in relatively hypopermeable tumors, such as HGL-21 glioblastoma, and/or in hypopermeable regions of a relatively permeable tumor. In support of these results, we have observed decreases in the permeability of hyperpermeable tumor vessels with anti-VEGF neutralizing antibodies (5) , and these changes were reversible with exogenous addition of VEGF (data not shown).
The acute effect of VEGF superfusion on larger transvascular pathways, allowing the extravasation of nanoparticles, was dramatic. Yuan et al. (45)
noted that maximal liposome-extravasation occurs in about 1 day in untreated tumors. Because PEG coating allows liposomes to circulate for a relatively long time, they require 24 h to be cleared from the circulation. In this study, it appears that the maximal extravasation occurs within the first few hours after VEGF superfusion. This, taken together with the short half-life of VEGF in vivo, suggests that changes in transvascular pore size and frequency probably occur immediately after VEGF superfusion and that these changes are transient.
Finally, we investigated the effect of bFGF, another potent angiogenesis stimulator, and PlGF-1 and PlGF-2, members of the VEGF family, on vascular permeability. Although chronic exposure to bFGF (0.5–2 µg) over 10 to 20 days has been shown to induce open junctions (14) , acute bFGF exposure increased neither the pore cutoff size in normal or LS174T microvasculature nor endothelial permeability in vitro (46) or in vivo (47) . PlGF-1 and PlGF-2 have been shown previously not to affect vascular permeability on their own (48 , 49) but to potentiate the effect of low dose VEGF in normal skin vessels (48) . The synergism between PlGF (Flt-1 ligand) and VEGF (Flt-1 and Flk-1 ligand) may be due to competition between PlGF and VEGF for Flt-1 receptors, increasing the amount of VEGF available to the Flk-1 receptor, which is believed to mediate VEGF functions (12 , 48) . In agreement with previous findings, we found that PlGF-1 acted synergistically with low levels of VEGF to increase the size of the transvascular pathways in normal s.c. vessels. However, superfusion with PlGF-1 with lower concentrations of VEGF did not increase the macromolecular permeability in the HGL-21 tumor vessels or the pore cutoff size in the LS174T tumor. Thus, tumor and normal vessels seem to respond differently to PlGF-1 in combination with VEGF.
Several novel antineoplastic agents, such as monoclonal antibodies (5–9.5 nm), gene therapy vectors (100–1000 nm), and encapsulated carriers (100–400 nm) have fallen short of expectations, partly due to transport barriers intrinsic to the tumor microvascular physiology (7 , 22 , 50) . For an antineoplastic agent to be effective, it must reach all regions of a solid tumor in therapeutically effective concentrations. The regions of a tumor that receive suboptimal amounts of a therapeutic agent are likely to cause tumor regrowth and relapse. The amount of an agent delivered to the tumor cells from the vasculature is partly dependent on the permeability of the vessel wall, which in turn is partly determined by the number, size, and distribution of transvascular pathways. A greater understanding of the spatial and temporal heterogeneity in transvascular transport, such as presented here, should aid in optimizing the delivery of these larger antineoplastic agents.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by Outstanding Investigator Grant R35-CA56591 from the National Cancer Institute (to R. K. J.). W. L. M. was supported by a National Cancer Institute Training Grant to the Department of Radiology at Beth Israel Deaconess Medical Center. 
2 Present address: Department of Biomedical Engineering, Duke University, Durham, NC 27708.
3 To whom requests for reprints should be addressed, at Department of Radiation Oncology, Massachusetts General Hospital, 100 Blossom Street, Cox-7, Boston, MA 02114. Phone: (617) 726-4083; Fax: (617) 726-4172; E-mail: jain{at}steele.mgh.harvard.edu
4 The abbreviations used are: VEGF, vascular endothelial growth factor; bFGF, basic fibroblast growth factor; BSA, bovine serum albumin; PEG, polyethylene glycol; PlGF, placenta growth factor; SCID, severe combined immune-deficient; VVO, vesicular vacuolar organelle.
Received 12/ 7/98. Accepted 6/15/99.
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