{CANCER RESEARCH 54. 4564-4568, September 1, 1994]

Advances in Brief

Vascular Permeability and Microcirculation of Gliomas and Mammary CarcinomasTransplanted in Rat and Mouse Cranial Windows1

Fan Yuan,2 Hassan A. Salehi,2 Yves Boucher, Usha S. Vasthare, Ronald F. Tuma, and Rakesh K. Jain

Department of Radiation Oncology. Massachusetts General Hospital and Har\-ard Medical School, Boston. Massachusetts 02114 ¡F. Y., H. A. S., Y. B., R. K. J.], and

Departments of Physiology and Neurosurgery, Temple University School of Medicine, Philadelphia, Pennsylvania 19140 ¡U.S. V.. R. F. T.]

Abstract

Many brain tumors are highly resistant to chemotherapy, presumablydue to the presence of a tight blood-tumor barrier. For a better under

standing of the regulation of this barrier by the brain environment, a newintravital microscopy model was established by transplanting tumor tissueinto cranial windows in both rats and mice. The model was characterizedby RBC velocities, vessel diameters, and vascular permeabilities of varioustumors: R3230AC (a rat mammary adenocarcinoma), MCalV (a mousemammary adenocarcinoma), and U87 and HGL21 (human malignantastrocytomas). Our results showed that tumor blood flow in cranialwindows was one to three orders of magnitude lower than the blood flowin pial vessels and similar to that in dorsal skin-fold chambers observed in

previous studies. The mean vessel diameter ranged from 6.8 ±1.3 urn forHGL21 to 30.4 ±8.5 urn for MCalV. At least one order of magnitudedifference in vascular permeability to albumin was observed betweentumor lines: 0.11 ±0.05x10"' cm/s for HGL21 versus 3.8 ±1.2xlO~7

cm/s for I 87. The low vascular permeability of HGL21, which was alsoconfirmed by both sodium fluorescein and Lissamine green injections,suggests that not ail tumors are leaky to tracer molecules and that theblood-tumor barrier of this tumor still possesses some characteristics ofblood-brain barrier as observed in other intracranial tumors. The model

presented here will allow us to manipulate the vascular permeability inbrain tumors and thus may provide new information on the regulation ofthe blood-tumor barrier and new strategies for improving drug delivery in

brain tumors.

Introduction

Clinical studies have shown that many brain tumors, especiallyprimary tumors, are among the most resistant to chemotherapy, presumably due to the presence of a tight BTB3 (1). One of the unique

features of the BTB in the brain is the broad range of vascularleakiness to both small and large molecules (2, 3), i.e., from impermeable to highly permeable, as compared to peripheral tumor vesselswhich universally exhibit hyperpermeability to tracer molecules (4).This indicates that specific mechanisms may be involved in theregulation of the BTB in the brain. In normal brain, moleculesreleased from astrocytes or foot processes of glial cells may contributeto the formation of tight junctions between vascular endothelial cellsin the BBB (5-8). In brain tumors, the presence of these molecules

and their functions on BTB regulation are not clear. In addition,despite its clinical importance, there is no measurement of brain tumorvascular permeability. Only blood-to-tumor transfer coefficients,which lump permeability-surface area product and local blood flow

Received 6/20/94; accepted 7/19/94.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby marked advertisement in accordance with18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by N1H Grant R35-CA-56591. Preliminary results of this work have been

presented at the 20th Annual Meeting of the Microcirculatory Society, 1994. and the 42ndAnnual Meeting of the Radiation Research Society. 1994.

2 The first two authors contributed equally to this study.3 The abbreviations used are: BTB. blood-tumor barrier; BBB. blood-brain barrier:

SCID, severe combined immunodeficient; Rho, rhodamine; BSA, bovine serum albumin;FITC, fluorescein isothiocyanate.

together, have been obtained (reviewed in Ref.3). Although the transfer coefficients are useful in calculating fluxes of drugs into braintumors, they are inadequate for understanding mechanisms of BTB,since different mechanisms are involved in the regulation of micro-

vascular permeability, surface area, and blood flow.For a better understanding of the BTB regulation in the brain, we

developed a new model for quantification of microvascular permeabilityand local blood flow in brain tumors using intravital fluorescence microscopy and cranial window preparations in rats and mice. Four tumorlines (R3230AC, MCalV, U87, and HGL21), presumably with significant differences in microvascular permeability, were selected in ourstudy. R3230AC and MCalV are mammary adenocarcinomas from ratsand mice, respectively; both LJ87 and HGL21 are derived from humanmalignant astrocytomas (a grade III). Although leptomeningeal metastasis of breast cancer has been observed in the clinic, the metastasis ofastrocytomas to the pial surface rarely occurs. Our rationale for transplanting astrocytomas in cranial windows was based on the hypothesisthat graft rather than host parenchyma, i.e., local environment rather thanorigin of vessels, determines microvascular permeability of the graft (5,8). We report here the characterization of our brain tumor model incranial windows in terms of: (a) the RBC velocity; (b) vessel diameter;and (c) microvascular permeability.

Materials and Methods

Animal and Tumor Models. One strain of rats and two strains of micewere used in our study to establish cranial windows: 4- to 6-months old femaleFisher rats (180-230 g) for R3230AC (a rat mammary adenocarcinoma fromBiomeasure, Bogden Laboratories, Hopkinton, MA); 6-months old femaleC3H mice (30-40 g) for MCalV (a mammary adenocarcinoma spontaneouslyarose in a C3H female mouse in Dr. Herman D. Suit's Laboratory at Massa

chusetts General Hospital); and 6-months old female SCID mice (20-35 g) for

MCalV, U87 (human glioblastoma and HTB 14; ATCC, Rockville, MD), andHGL21 (human glioblastoma from a patient at Massachusetts General Hospitaland kindly provided by Drs. Herman D. Suit and Feigen Huang).

The procedure of window implantation was similar to previous studies (9).During surgery, both rats and mice were anesthetized with a cocktail ofKetamine and Xylazine (10:1, w/w). For rats, 0.001 ml cocktail/g body weightwith concentration of 100 mg/ml was injected i.m. For mice, 0.01 ml cocktail/gbody weight with concentration of 10 mg/ml was injected s.c. Surgery wasdone under aseptic conditions. The head of animals was fixed by a stereotacticapparatus. The skin on top of the frontal and parietal regions of the skull wascleaned with antimicrobial betadine solution. A longitudinal incision of theskin was made between the occiput and forehead. The skin was then cut in acircular manner on top of the skull, and the periosteum underneath was scrapedoff to the temporal crests. A 6-mm circle was drawn over the frontal andparietal regions of the skull bilaterally. Using a high speed air-turbine drill

(CH4201S; Champion Dental Products, Placentia, CA) with a burr tip size of0.5 mm in diameter, a groove was made on the margin of the drawn circle. Thisgroove was made thinner by cautious and continuous drilling of the groove tillthe bone flap became loose. Cold saline was applied during the drilling processto avoid thermal injury of the cortical regions. Using a blunt microblade, thebone flap was separated from the dura mater underneath. After removal of thebone flap, the dura mater was continuously kept moist with physiological

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PERMEABILITY AND MICROCIRCULATION IN BRAIN TUMORS

saline. After removal of the bone, a nick was made close to the sagittal sinus.Iris microscissors were passed through the nick. The dura and arachnoidmembranes were cut completely from the surface of both hemispheres, avoid

ing any damage to the sagittal sinus. A piece of the tumor tissue, 1 mm indiameter, was cut and put at the center of the window. The window was thensealed with an 8-mm cover glass by adhering to the bone using a histocom-

patible cyanoacrylate glue. The surgery took 45 min in each case and wasfollowed by positioning of the animals on a heating pad (37°C) until they

recovered from the anesthesia. The experiments were conducted between 11and 18 days after tumor transplantation, depending on the vascularization andsize (>3 mm in surface diameter) of tumors.

Tracers. Rhodamine-labeled BSA (A/,, 67,000; Molecular Probes, Eugene,OR) was used as the tracer for permeability studies in all tumors. Rho-BSAwas dissolved in IX phosphate-buffered saline (Sigma Chemical Co., St.

Louis, MO) at a concentration of 8 mg/ml. The free fluorescent dye in thesolution of Rho-BSA was removed by passing the solution through a size

exclusion column (10). For blood flow measurements, two tracers were used.FITC-labeled dextran 2 M (M„2,000,000; 50 mg/ml; Sigma) was used as a

contrast agent for measurement of RBC velocity in brain tumors (11). Fluorescent latex beads (0.8%; 1.0 ¡un;Polysciences, Warrington, PA) were usedto estimate maximum blood velocity in normal pial vessels (12). Lissaminegreen B (2%; Sigma) and sodium fluorescein (1.13 mg/ml; Sigma) were usedfor BBB studies.

Experimental Procedure. The experimental protocol was similar to ourprevious studies (10, 11, 13). In brief, the animals were anesthetized using thesame procedure as described above and then put on a polycarbonate plate withthe head fixed by the plastic modeling compound. The plate was placed on thestage of an intravital fluorescence microscope (Axioplan; Zeiss, Oberkochen,Germany) equipped with the fluorescence filter set for Rho and FITC (OmegaOptical, Inc., Brattleboro, VT), an intensified CCD video camera (C2400-88;

Hamamatsu Photonics K.K., Hamamatsu, Japan), a videocassette recorder(AG-6500; Panasonic, Secaucus, NJ), and a photomultiplier (9203B; EMI,

Rockaway, NJ). Four to five animals were used for measurement of eachparameter, and none of the animals were used twice.

Two methods were used to measure the blood velocity: the four-slit method

for RBC velocity in tumors (11) and the fluorescent bead method for estimating maximum blood velocity in normal pial vessels (12). The bead method wasused because the blood velocity in pial vessels can be two orders of magnitudehigher than the maximum velocity measurable by the four-slit method. The

details of these techniques have been described in previous studies (11, 12). Inbrief, the FITC-labeled dextran 2 M was injected (0.1 ml/25 g body weight)

through the tail vein for contrast enhancement between RBCs and plasma. Fourto six locations on the tumor surface of each animal were selected, and videoimages of blood flow at each location (0.5 x 0.5 mm2) were recorded for 1 min

by the video camera and the videocassette recorder described above andanalyzed offline by the four-slit apparatus (Microflow system, model 208C,

video photometer version; IPM, San Diego, CA) equipped with a personalcomputer (IBM PS/2, 40SX; Computerland, Boston, MA). For normal pialvessels, a bolus (0.1 ml/25 g body weight) of Rho-BSA was first administered

i.v. to illuminate the vasculature. Then, multiple injections of fluorescent beads(0.01 ml/injection) were given i.V., one injection per measurement location.Four to six locations were selected per animal. The movement of beads wasmonitored by the same video system described above under an intravitalfluorescence microscope (Universal; Zeiss, Thornwood, NY) with epi-illumi-nation by a strobe lamp (MVS-2601 Strobe and MVS-100 Trigger module, 322

Hz; EG&G, Salem, MA). For each location, the video images were recordedfor 1 min and analyzed offline by playing back the video tape frame by frameusing a S-VHS videocassette recorder (SVO-9500MD; Sony Medical Systems,

Montvale, NJ). The distance a bead moved within a vessel in a giventime interval (3.1 ms) was determined by measuring the distance between twopositions of the bead in the same track via a caliper. The maximum beadvelocity was calculated by dividing the maximum distance in more than 300tracks during the 1-minute recording by 3.1 ms. The track was selected such

that at least three consecutive positions of the same bead could be observed ina single frame.

The methods for measurement of Prjr and V/S, where Pfff is the effectivemicrovascular permeability, V is the total vascular volume, and S is the totalsurface area of vessels, at the tumor surface in cranial windows have beendescribed previously (10, 13). The time constants of BSA plasma clearance used

to calculate Pclf were obtained by curve-fitting plasma concentrations of BSA

measured in rats (14) and mice (10), respectively, to an exponential function. Theresults were 6.0 X IO3 s and 9.1 X Vf s for rats and mice, respectively. In order

to confirm the existence of tight BTBs in the transplanted tumors, we also injected

(0.1 ml/25 g body weight) small tracer molecules (sodium fluorescein and Lissamine green) through tail veins of SC1D mice bearing either MCalV or HGL21tumors. One min after tracer injection, the animals were sacrificed by overdose(0.3 ml/25 g body weight) of sodium pentobarbital solution (64.8 mg/ml; AnproPharmaceutical, Arcadia, CA). The fluorescence intensity of tumor tissue aftersodium fluorescein injections was measured by the photomultiplier. In the case ofLissamine green injection, photographs of tumor tissue were taken through cranialwindows. Mann-Whitney U test was used to compare the differences in Pfff.

Results and Discussion

Tumor Transplantation in Cranial Windows. Fig. 1, A and /(.show the overview of tumors (MCalV and HGL21, respectively) transplanted in cranial windows in SCID mice. Tumor angiogenesis startedapproximately 4 days after tumor chunk transplantation. All tumor transplantations in this study were successful. The growth rate of tumors wasnot quantified, but qualitatively U87 and HGL21 transplanted in SCIDmice grew slower than R3230AC and MCalV transplanted in rats andmice, respectively. Therefore, the experiments on gliomas were performed between days 12 and 18, whereas the mammary carcinomastransplanted in both rats and mice were used for measurements betweendays 11 and 14. The maximum survival time was around 15 days foranimals with mammary carcinomas and longer than 22 days for animalswith human gliomas. At the time of experimentation, the surface diameterof tumors was between 3 and 6 mm.

Microvascular Permeability. The effective microvascular permeability to BSA was measured in five groups: R3230AC transplanted inFisher rats; MCalV transplanted in C3H and SCID mice; and U87 andHGL21 transplanted in SCID mice. All of them except for HGL21showed hyperpermeability to Rho-BSA (Table 1). The permeabilities

of tumors (MCalV, U87, and HGL21) transplanted in SCID micewere significantly different from one another (the maximumP < 0.05), and no statistical difference between MCalV tumorstransplanted in C3H and SCID mice, respectively, was observed(P = 0.22). The much lower permeability of HGL21 to BSA sug

gested the presence of a tight BTB in this tumor line. Therefore, weinjected sodium fluorescein and Lissamine green (standard dyes fortesting BBB) into animals bearing either MCalV or HGL21 andcompared the vascular leakiness between tumors and surrounding hosttissues (e.g., bone, skin, and brain). The results of Lissamine greeninjections are shown in Fig. 1, C and D. In animals bearing MCalV,both tumor and surrounding bone and skin tissues became totallygreen after 2 min of Lissamine green injection, in contrast to hostbrain tissue (Fig. 1C). However, the color of HGL21 was much closerto that of the brain (Fig. ID). There was a green stripe at the centerof the window (Fig. ID), suggesting the leakiness of vessels in duramater surrounding the sagittal sinus or the sinus itself. Similar resultswere also observed after sodium fluorescein injection (results notshown). The leakage of the Lissamine green dye in dura mater is notsurprising, since it has no BBB (15). However, the green stripe maysuggest an overestimation of the vascular permeability of HGL21 toBSA, since the leakage of BSA from dura vessels underneath thetumor could affect the measurement. For other tumor lines, thisoverestimation is insignificant (<10%).

Mechanisms of BTB regulation in the brain are still unclear (1, 2).Previous studies have suggested that in normal tissue the transportbarrier phenotype of vascular endothelial cells should be largelydetermined by the microenvironment instead of their origin (5, 7, 8).This hypothesis could be extended further to the situation of tumortissue. One of the most important features of gliomas is that the tumor

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PERMEABILITY AND MICROCIRCULATION IN BRAIN TUMORS

'

Fig. 1. Overview of cranial windows in SCID mice with tumor transplantation: MCalV (A and C) and HGL21 (B and D). The diameter of the cranial window is 6 mm. Animalswere sacrificed 1 min after injection of Lissamine green. The photos were taken before injection (A and ß)and 1 min after animals were sacrificed (C and D). Connections betweentumor and pial vessels can be observed (A and B). There is a significant leakage of Lissamine green (Afr 577) in MCalV and the surrounding bone tissue at the border of the windows,whereas the leakage of the dye in HGL21 and the host brain tissue underneath the tumors is nondetectable. There is a green stripe at the center of the window (C and D), suggestingthe leakiness of vessels in dura mater surrounding the sagittal sinus or the sinus itself.

per se may provide both brain and tumor environments (16), whereasin brain métastases,the brain environment is established, if it exists,through diffusion of factors released by host glial cells into thetumors. The brain environment may cause the formation of BBB (5,7, 8); the tumor environment may disrupt the vascular barriers throughcytokines (e.g., vascular permeability factor) released by tumor cells(17, 18). Thus, the outcome of competition between the two environments determines the vascular permeability of brain tumors (2, 19-

22). As a comparison, tumors transplanted onto dorsal skin or granulation tissue, where the brain environment is absent, exhibit highervascular permeability than host tissue (10, 13, 14, 23).

In the present study, we have demonstrated that the tumor vessels aredirectly connected to pial vessels (Fig. 1, A and B). Thus, the vessels ofHGL21 and MCalV could either be formed during angiogenesis from piamater or exist prior to tumor tissue transplantation and then anastomosewith the host pial vessels. Vessels in tumor chunks prior to transplantationoriginated from s.c. vessels, since these tumors were derived by s.c.implantation of tumor cells which were obtained from their primary massthrough single-cell suspensions and cell culture expansions. Thus, one

can assume that the origin of vasculature in both HGL21 and MCaFV isthe same. It is difficult to imagine that the vasculature of HGL21 originates from completely different host tissue compared with MCalV, sincethe procedures of tumor passage and transplantation are identical. Theidentical site of transplantations, the similar origin of vessels, and thelarge difference in tumor parenchyma and vascular permeabilities between HGL21 and MCalV suggest that the microenvironment dictatestumor vascular permeability.

The competitive environment hypothesis discussed above mightexplain our permeability data for U87, although we cannot excludeother possibilities. U87 is derived from a malignant human astrocy-

toma, similar to HGL21, but its vascular permeability to BSA waseven higher than that of the mammary carcinomas (MCalV andR3230AC). It is possible that the influence of the U87 tumor environment overwhelms the brain environment. The large differencebetween U87 and HGL21 in terms of the vascular permeability isconsistent with the previous observations that brain tumors exhibit abroad range of vascular leakiness (2, 3). Future studies are necessaryto elucidate factors responsible for the hyperpermeability of U87.

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PERMEABILITY AND MICROCIRCULATION IN BRAIN TUMORS

Table 1 Mean vessel diameter and vascular permeability to USA

Animal(AT)"Fisher

rat(5)C3H

mouse(5)SCID

mouse(4)SCID

mouse(5)SCID

mouse (5)Tumor

lineR3230ACMCalVMCalVU87HGL21Diameter(firn)20.7±4.9 (20.3)'

(15.3-28.6)''19.0

+ 4.3 (20.7)(13.5-24.1)30.4

+ 8.5 (30.1)(21.6-39.8)13.8

±2.4 (12.8)(11.6-17.8)6.8

±1.3 (6.7)(5.1-8.2)Permeability

(X 10~7cm/s)1.7

±0.6 (1.6)(0.8-2.4)2.9

±1.5 (2.8)(1.2-5.1)1.9

±0.5 (1.9)(1.3-2.5)3.8

±1.2 (4.3)(2.4-5.0)0.11+0.05(0.09)

(0.05-0.19)

" N, the number of animals.* The mean vessel diameter estimated by 4 V/S.' Mean ±SD (median).d Range.

0.8

ja*>

a8

(1.5

0.4-

0.3-

0.1-

0.0

. . •:. %*:-"•.".* •.••JL ••*...«

••f .••f . •' •}t '

g

0 10 20 30 40 50 60

Vessel Diameter (urn)

(A)

70

0.7

0.6

0.3-

0.2-

0.010 20 30 40 50 60

Vessel Diameter (urn)

(B)

70

Arterioles

Vma,, (mm/s)

r35

30

-20

15

10

Venules

*••!•~ «.*

%••

.«.I'

50 25 0 25 50

Vessel Diameter lumi

(C)

Fig. 2. A, RBC velocities in surface vessels of MCalV in C3H mice (N = 5 and n = 108). B, RBC velocities in surface vessels of U87 in SCID mice (N = 5 and n = 111). C,the maximum bead velocities (Vmax) in vessels of tumor-free pia mater of SCID mice (N = 4 and n = 31) for arterioles and 43 for venule. N is the number of animals used and nis the total number of measurements'.

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PERMEABILITY AND MICROCÕRCULATION IN BRAIN TUMORS

Blood Flow. In addition to vascular permeability, local blood flowcan also influence the delivery of blood-borne drugs. In our study, blood

flow was measured in surface vessels (7 to 63 /im in diameter) of bothtumors and tumor-free pia mater as shown in Fig. 2. In MCalV and U87,

there was no correlation between RBC velocity and vessel diameter (Fig.2, A and B). The blood velocities of R3230AC in rats and HGL21 inSCID mice were similar to that of MCalV and U87 (results not shown),and they were all comparable with the blood velocities of LS174Ttransplanted in dorsal skin-fold chambers (11). The maximum bead

velocity in normal pial vessels increased with vessel diameter, and theincrease was steeper in arterioles than in venules (Fig. 2C). There werealso a few small vessels (5 to 7 /u,m in diameter) on the normal pialsurface with low bead velocity (<1 mm/s) which were slower than thelower limit of measurable velocity by the fluorescent bead method andthus were not measured here. The maximum bead velocity is about twicethe mean blood velocity, thus the blood flow in pial vessels (Fig. 2C) wasone to three orders of magnitude faster than that in tumor vessels of thesame diameter (Fig. 2, A and B). However, in other studies (21), the bloodflow in tumors measured indirectly by the tracer uptake method is as highas 44% of that in normal cortex. This discrepancy might be explained bythe method of tumor transplantation or blood flow measurement. In ourstudy and that using dorsal skin chamber, the blood supply of tumorscomes only from the bottom of tumors, whereas the blood supply in otherstudies of brain tumors is from all directions (21), since those tumors areembedded in the brain instead of growing on the pial surface. However,we cannot explain why the tumor blood flow in the cranial window iscomparable with that in the dorsal skin-fold chamber, because the blood

flow in pial vessels is much higher than that in normal s.c. vessels (11).Vessel Diameter. A striking difference in vessel diameter was

observed among tumor lines, as shown in Table 1. In order to comparethe vessel diameter among tumor lines and to minimize the effect ofintratumor vascular heterogeneity, we measured V/S at the tumorsurface (10, 13) and estimated the mean vessel diameter of each tumoras 4 V/S (Table 1). This is equivalent to the average vessel diameterweighted by the length of vessels. Blood pools (>0.5 mm in width)were also observed in some tumors of MCalV transplanted in bothC3H and SCID mice.

In summary, we have established a new model for intravital microscopy studies of brain tumors using a cranial window preparation. Wehave shown that the vasculature in tumors transplanted into the cranialwindow can either possess or lose some of the characteristics of BBB(Table 1; Fig. 1, C and D), depending on the tumor parenchyma.Therefore, tumor vessels in this model might be used to mimic the BTBof both brain métastasesand primary brain tumors for permeabilitystudies. In addition, the current model can be used to study interstitialpressure and leukocyte-endothelial interactions in brain tumors.

Acknowledgments

We thank Drs. Herman D. Suit and Feigen Huang for providing the HGL21tumor line and Drs. Leo E. Gerweck, Feigen Huang, Paul E. G. Kristjansen,and Francisco S. Pardo for many helpful comments.

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