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Clin Cancer Res. Author manuscript; available in PMC 2009 Jun 15.
Published in final edited form as:
PMCID: PMC2504718
NIHMSID: NIHMS59711

Tumor Vascular Maturation and Improved Drug Delivery Induced by MethylSelenocysteine Leads to Therapeutic Synergy with Anticancer Drugs

Abstract

Purpose:

Our previously reported therapeutic synergy between naturally occurring seleno-amino acid methylselenocysteine (MSC) and anticancer drugs could not be demonstrated in vitro. Studies were carried out to investigate the potential role of MSC induced tumor vascular maturation and increased drug delivery in the observed therapeutic synergy in vivo.

Experimental Design:

Mice bearing subcutaneous FaDu human head and neck squamous cell carcinoma (HNSCC) xenografts were treated with MSC (0.2 mg/day × 14 days, p.o.). Changes in microvessel density (CD31), vascular maturation (CD31/α-SMA), perfusion (Hoechst 33342/DiOC7) & permeability (dynamic-contrast enhanced magnetic resonance imaging; DCE-MRI) were determined at the end of the 14-day treatment period. Additionally, the effect of MSC on drug delivery was investigated by determining intratumoral concentration of doxorubicin using high performance liquid chromatography (HPLC) and fluorescence microscopy.

Results:

Double-immunostaining of tumor sections revealed a marked reduction (~40%) in microvessel density accompanying tumor growth inhibition following MSC treatment along with a concomitant increase in the vascular maturation index (~30% > control) indicative of increased pericyte coverage of microvessels. Hoechst/DiOC7 staining showed improved vessel functionality and DCE-MRI using the intravascular contrast agent, albumin-GdDTPA revealed a significant reduction in vascular permeability following MSC treatment. Consistent with these observations, a 4-fold increase in intratumoral doxorubicin levels was observed with MSC pretreatment compared to administration of doxorubicin alone.

Conclusion:

These results demonstrate, for the first time, the antiangiogenic effects of MSC results in tumor growth inhibition, vascular maturation in vivo and enhanced anticancer drug delivery that are associated with the observed therapeutic synergy in vivo.

Keywords: Vascular maturation, drug delivery, antiangiogenic agent, methylselenocysteine, selenium

INTRODUCTION

Selenium is an essential trace element present in grains, meat, yeast and vegetables with an average nutritional intake of 50-350μg per day (1). A strong inverse association between selenium status and site- and sex-specific cancer mortality rates for cancers of the lung, bladder, esophagus and breast has been reported (1,2). While the use of selenium as a chemopreventive agent has been a subject of research for decades, its activity as a therapeutic agent has not been extensively investigated. We have been investigating the antitumor activity of selenium alone and in combination with chemotherapy in preclinical and clinical settings (3,4). We have previously shown that selenium administered in its organic form as methylselenocysteine (MSC) significantly potentiates efficacy of the topoisomerase-1 inhibitor, Irinotecan (Camptosar®) against human tumor xenografts (3). Similar effects were seen with docetaxel, cisplatin and oxaliplatin in a variety of drug sensitive and resistant human tumor xenografts. However, the mechanism(s) that contribute to the observed therapeutic synergy are not completely clear. Recent studies in our laboratory have revealed downregulation of proangiogenic growth factors, cyclooxygenase-2, nitric oxide synthase and hypoxia-inducible factor-1 alpha expression in human head and neck squamous cell carcinoma (HNSCC; FaDu) xenografts with combination treatment (5). Studies carried out in our laboratory using FaDu tumor cells revealed only additive effects in vitro (Rustum YM, unpublished observation) further implicating tumor vasculature in the observed enhancement of antitumor activity.

Angiogenesis is an early event in tumor progression and is critical for continued growth of solid tumors beyond a few millimeters (6). It is now widely acknowledged that inhibiting the angiogenic process is an effective way of controlling tumor growth and to this end a number of antiangiogenic agents are currently undergoing preclinical and clinical evaluation (7). Selenium has also been shown to exhibit antiangiogenic properties in vitro and in vivo (8,9). Studies carried out in human umbilical vein endothelial cells (HUVECs) have shown induction of cell death through apoptosis and reduction in matrix metalloproteinase activity (8). Selenium intake, in the form of selenized garlic or methyselenocysteine has also been shown to result in reduction of vascular endothelial growth factor (VEGF) levels in mammary carcinomas (9). However, the effects of selenium on tumor vascular maturation and blood flow have not been previously investigated.

Therefore, in this study, we examined the effect of MSC on several phenotypic and functional parameters related to tumor angiogenesis in vivo. Using subcutaneous FaDu human tumor xenografts implanted in nude mice, changes in microvessel density (MVD), pericyte coverage (vascular maturation index), vascular perfusion, permeability and tumor growth were evaluated following MSC treatment (0.2 mg/day × 14 days). Additionally, the effect of MSC treatment on intratumoral drug delivery and distribution was investigated using the autofluorescent anthracycline doxorubicin. The results obtained demonstrate, for the first time, that administration of non-toxic dose of selenium, daily for fourteen days results in a marked inhibition of angiogenesis, tumor growth inhibition while concomitantly improving vascular function and therapeutic delivery and distribution of the drug into the tumor.

MATERIALS AND METHODS

Tumor model

The human HNSCC cell line, FaDu, was originally purchased from American Type Culture Collection (Manassas, VA) and xenografts were established in six-to-eight week old female athymic nude mice (Foxn1nu, Harlan Sprague Dawley, Inc. Indianapolis, IN) as described previously (3). Tumor growth following treatment was measured (N = 6 per group) using vernier calipers and tumor volumes calculated (3). All studies were performed in accordance with protocols approved by the Institute Animal Care and Use Committee at Roswell Park Cancer Institute.

Drugs

MSC (Sigma, St. Louis, MO) was dissolved in sterile saline at a concentration of 1mg/ml) and administered orally at the maximum tolerated dose of 0.2 mg/mouse/day (3) for 14 days, beginning four days after tumor implantation. For drug delivery studies, doxorubicin (Bedford Laboratories, Bedford, OH) was administered intravenously (30 mg/kg) alone or 24 hours following administration of the last MSC dose (day 14).

Immunohistochemistry

Immunohistochemical staining for endothelial cells and pericytes was performed using CD31 and alpha-smooth muscle actin (α-SMA), respectively. We have previously described procedures for CD31-immunostaining of tumor sections in detail (10). For CD31/α-SMA double staining, 5-8μm cryosections were fixed in cold acetone (−20°C) for 15 minutes followed by PBS with Tween 20 0.05% (PBS/T) rinse. Endogenous peroxidase quenching was followed by incubation with rabbit polyclonal SMA antibody (1μg/ml or 1/500) (Abcam, Cambridge, MA) and biotinylated goat anti-rabbit secondary antibody (1/250) (Vector Labs) for 30 min. This was followed by Streptavidin complex (Zymed lab Inc., San Francisco, CA) for 30 min and chromogen DAB (Dako, Carpinteria, CA) for 5 minutes. Again a blocking step with 0.03% casein was used, followed by CD31 antibody (B.D. Biosciences Pharmingen, Franklin Lakes, NJ) at 10μg/ml for 60 min. Biotinylated anti-rat secondary antibody (B.D. Biosciences Pharmingen™, Franklin Lakes, NJ) at 1:100 was used for 30 minutes followed by Alkaline Phosphatase (Dako, Carpinteria, CA) conjugated Streptavidin Reagent for 25 min. The chromogen Fast Red was then applied for 10 minutes and the slides were counterstained with Mayer's hematoxylin (Dako, Carpinteria, CA) for 45 seconds. An isotype-matched rat IgG was used as a negative control. Endothelial cells were immunostained brown and pericytes were stained pink. There were a minimum of 5 tumors per group and 3 sections from each tumor at least 10 μm apart were used for quantification purposes.

Dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI)

To determine MSC-induced changes in vascular permeability, DCE-MRI using the intravascular contrast agent, albumin-GdDTPA was performed in a 4.7T/33-cm horizontal bore MR scanner (GE Instruments, Fremont, CA) on a separate cohort of mice (N = 4 per group). Prior to imaging, animals were anesthetized using ketamine:xylazine mixture (10:1) at a dose of 1.0 ml/100g and positioned in the scanner. We have previously described the imaging protocol for calculating changes in vascular permeability (11). Briefly, the T1-relaxation rate of tissue (R1 = 1/T1) increases linearly with contrast agent concentration. We therefore acquired serial T1-weighted images before and after contrast agent administration, using a saturation-recovery, fast spin echo (FSE) sequence with an effective time of echo period (TE) = 10 ms and TR ranging from 360-6000 ms (FOV = 32 × 32 mm, slice thickness = 1.0 mm, matrix size = 128 × 96 pixels, number of excitations (NEX) = 3) to determine the change in T1-relaxation rates (ΔR1) of different tissues (tumor, muscle, kidneys). Applying a linear regression analysis to the change in ΔR1 over time, we can acquire both a slope and a y-intercept. The slope is a measure of vascular permeability and the y-intercept of the slope (at time = zero, i.e. immediately after administration of the contrast agent) is a measure of vascular volume (11).

Determination of vessel functionality

To determine the effect of MSC on vessel functionality, the double fluorescent dye technique based on the perfusion markers, Hoechst 33342 (Sigma Laboratories, St. Louis, MO) and DiOC7 (Molecular Probes, Eugene, OR) were utilized on a separate cohort of mice (N = 4). The different fluorescence excitation and emission properties of the two dyes allows for detection of temporal and spatial fluctuations in perfusion (12,13). The experimental details of the technique have been published previously (12,13). Briefly, the fluorescent dyes were administered intravenously (Hoechst – 15 mg/kg; DiOC7 - 1 mg/kg) separated by a 20-min interval and tumors excised 5 mins after the second DiOC7 injection. Cryosections (5-10μm thick) of the tumor were used for fluorescence detection of Hoechst 33342 and DiOC7 using 10X objective of Leica Confocal microscope (Leica Microsystem Heidelberg GmbH) with excitation at 488nm and emission at 500-700 nm.

Determination of doxorubicin concentration and distribution

The effect of MSC on drug delivery (N = 11 per group) and distribution (N = 105 linear paths using 24 different sections from 4 different tumors per group) was assessed using high-performance liquid chromatography (HPLC) (14) and fluorescence microscopy (15). Doxorubicin was given at a dose of 30 mg/kg to facilitate detection and quantification of autofluorescence (15). For HPLC, the separation method was carried out on a Waters Nova-Pak C18 column equipped with Bondapak C18 guard column, with mobile phase consisting of 20% acetonitrile and 80% triethylamine acetate. The detection was by fluorescence, with excitation at 370 nm and emission at 510 nm as described previously (14). In addition to the tumor, doxorubicin concentration was determined in normal tissues, liver, kidney and small intestines from animals treated with doxorubicin alone or MSC plus doxorubicin. For fluorescence microscopy, two hours post doxorubicin administration, animals were euthanized and ~5-10 μm thick frozen sections were used. An average of 4 maximum intensity projection images with a resolution of 0.23μm were acquired under the 63X objective of Leica confocal microscope similar to procedures described previously (15). All images were obtained and digitized using identical acquisition parameters.

Image analysis

Microvessel density counts were determined by counting CD31+ endothelial cell clusters in multiple high power fields (400X) covering non-necrotic areas of the whole tumor. For determination of vessel lumen area, photomicrographs of CD31-stained sections (400X) were digitized at 1350 dpi and areas manually segmented using the region-of-interest (ROI) module of the medical imaging software, Analyze® (AnalyzeDirect, OverlandPark, KS). The vascular maturation index (VMI) was derived by calculating the total number of CD31+ α-SMA+ areas and areas positive for CD31 alone in double-stained (CD31/ α-SMA) tissue sections using Analyze® (16). For drug delivery studies, the mean intensity of doxorubicin auto fluorescence at various distances away from the blood vessels was calculated using Analyze®. At least three tumors for each group were analyzed for MVD, vessel lumen, VMI and doxorubicin studies.

Statistical analysis

All results are reported as mean ± standard error of the mean. Differences between the mean of the groups were analyzed using unpaired two-tailed student t test (GraphPad Version 4.00, GraphPad Software, San Diego, CA). P values < 0.05 were considered statistically significant.

RESULTS

MSC Lowers Tumor Microvessel Density

As shown in Figure 1A, treatment with MSC (0.2 mg/mouse/day) for 14 days resulted in a ~40% reduction in MVD (13.56 ± 4.875, P = 0.02) compared to untreated control tumors (21.26 ± 1.52). This effect was tumor specific with no significant change in MVD observed in normal mouse liver tissue (Figure 1A). Quantitative measurements of vessel size in CD31-immunostained tumor sections showed a significant reduction in vessel lumen area following MSC treatment (Figure 1B, P = 0.0009).

Figure 1Figure 1Figure 1
Effect of 14 days of MSC treatment on microvessel density (MVD) and growth of HNSCC xenografts

MSC Causes a Tumor Growth Inhibition

To determine the effect of MSC treatment (0.2 mg/mouse/day) for 14 days on tumor growth, changes in tumor volume were calculated for a period of 30 days following treatment. As shown in Figure 1C, a marked reduction (P=0.002) in tumor growth was observed following MSC treatment compared to untreated controls.

MSC Induces Tumor Vascular Maturation

As shown in Figure 2A, double-stained sections of FaDu tumors obtained from mice treated with MSC showed increased α-SMA staining compared to untreated controls. The vascular maturation index (VMI), percentage of endothelial cells associated with pericytes is used as a quantitative measure of vascular maturation (16-18). Quantitative analysis of pericyte coverage showed ~30% increase in VMI in MSC-treated FaDu tumors compared to control FaDu tumors (Figure 2B). Comparative analysis of VMI in normal liver tissue did not reveal any change in pericyte coverage indicating the selectivity of MSC-induced changes in vascular maturation.

Figure 2Figure 2Figure 2
Effect of MSC treatment on vascular maturation, permeability and function in HNSCC xenografts

MSC Reduces Tumor Vascular Leakiness

The functional consequences of MSC-induced changes in MVD and VMI on vascular permeability in FaDu xenografts following MSC treatment were assessed using noninvasive DCE-MRI. As shown in Figure 2C, DCE-MRI of untreated FaDu tumors showed increase (slope, 0.0062 ± 0.001, r2 = 0.7999) in the longitudinal relaxation rate (ΔR1) as function of time following administration of the intravascular MR contrast agent, albumin-GdDTPA indicative of significant vascular leakiness. Consistent with the results of MVD and VMI studies, linear regression analysis of ΔR1 over time in MSC-treated animals revealed a marked reduction in tumor vascular permeability (slope, 0.00023 ± 0.0004, r2 = 0.097, P = 0.0147) compared to untreated controls, a sign of improved tumor vascular normalization.

MSC Improves Tumor Vascular Function

We assessed fluctuations in blood flow in untreated controls and MSC-treated FaDu tumors using the double fluorescent dye method (12,13) based on two perfusion markers, Hoechst 33342 and DiOC7. Using this technique, studies have previously shown that vessels that experience intermittent flow show uptake of only one dye (‘mismatched’ vessels) (12,13). Fluorescence microscopy revealed minimal uptake of both dyes in control FaDu tumors, with a majority of these perfused vessels observed in the tumor periphery (Figure 3A). In contrast, MSC-treated tumors showed uniform uptake of both dyes indicative of improved vessel functionality.

Figure 3Figure 3Figure 3Figure 3
Effect of MSC treatment on drug delivery

MSC Enhances Tumor Drug Delivery

HPLC analysis (Figure 3B) revealed a 4-fold increase in doxorubicin concentration in MSC-treated FaDu tumors (0.62 ± 0.16 μg, P=0.01) compared to untreated controls (0.16 ± 0.03 μg). No significant change was seen in the plasma and normal tissue (kidneys, liver, small intestine) levels of doxorubicin in both cohorts of mice. Visualization and quantitation of doxorubicin levels using fluorescence also showed increased intensity at regions close to the vessel wall and away from the blood vessel (blue arrows) highlighting the improvement in both delivery and penetration of doxorubicin following MSC treatment (Figure 3C & 3D).

DISCUSSION

We have previously shown that administration of MSC for a period of 7 days prior to irinotecan treatment significantly enhances long-term cure rates in mice bearing subcutaneous FaDu tumors (3). Similar therapeutic synergy with MSC was seen with different chemotherapeutic drugs (e.g. taxol, taxotere, doxorubicin, cisplatin, oxaliplatin) and in different human xenografts growing in nude mice (Rustum YM, unpublished data). However, in contrast to the impressive therapeutic synergy observed in vivo, only additive effects were observed in vitro. Based on these observations, we hypothesized that tumor vasculature was a potential target of action of MSC and the antiangiogenic and vessel normalization activity of MSC was responsible at least in part for the observed therapeutic synergy. To test this hypothesis, studies were performed to determine the vascular phenotypic and functional effects of MSC in vivo using autofluorescent anticancer drug doxorubicin. These studies were aimed at understanding the mechanism(s) that contribute to the observed therapeutic synergy with selenium and chemotherapy.

First, we examined the effects of MSC on microvessel density and vessel lumen area using immunohistochemistry. Treatment with MSC (0.2 mg/mouse/day) for 14 days resulted in a significant reduction in MVD (P = 0.02) compared to untreated control tumors (Figure 1A). No change was seen in normal mouse liver tissue MVD (Figure 1A) indicating thereby that the antiangiogenic effect is tumor specific and a contributing factor in significantly delaying tumor growth (Figure 1C).

Tumor vasculature has an important role in the pathophysiology of solid tumors including tumor growth, invasion, metastasis and response to therapies. The hallmark of tumor vasculature is the morphologically abnormal vascular architecture consisting of chaotic, dilated vessels showing poor overall perfusion that resists blood flow and drug delivery in tumors. These functional characteristics of tumor vasculature contributes to an elevated tumor interstitial fluid pressure (IFP) that opposes diffusion and convection – the main form of transvascular transport of therapeutic agents in tumors (19). Thus delivery of therapeutic agents both across the blood vessel wall and interstitium is compromised in solid tumors (19). In contrast to the vasculature seen in normal tissues, tumors endothelium that lack the support of pericytes, cells that serve to stabilize blood vessels and stimulate basement membrane production (20). Recent preclinical studies using antiangiogenic agents such as bevacizumab and DC101 have shown that in addition to potent effects on tumor vascular morphology (vessel size and density), these agents also cause decrease in leakiness and increased maturation of tumor vasculature through increased recruitment of pericytes to the vascular bed (17,18). Pericytes express α-SMA, immunostaining of which is widely used as a marker for pericyte coverage in tissue sections (16-18). The vascular maturation index (VMI), percentage of endothelial cells associated with pericytes is used as a quantitative measure of vascular maturation (16-18). Quantitative analysis of pericyte coverage showed ~30% increase in VMI in MSC-treated FaDu tumors compared to control FaDu tumors (Figure 2B). Comparative analysis of VMI in normal liver tissue did not reveal any change in pericyte coverage indicating the selectivity of MSC-induced changes in tumor vascular maturation. The structural aberrations associated with tumor vasculature contribute to significant functional abnormalities including enhanced vascular permeability and temporal and spatial variations in blood flow, factors that are detrimental to tumor drug delivery and distribution (6,20). Previous studies have shown that inhibition of VEGF can result in pruning of immature vessels while contributing to the evolution of a more mature vascular phenotype, typically characterized by reduced permeability and increased perfusion. This decreases tumor IFP and restores the pressure gradient across blood vessel wall as well as tumor interstitium leading to a better tumor drug delivery and penetration (18,19,21). To determine if a similar phenomenon was occurring with MSC and to assess the functional consequences of MSC-induced changes in MVD and VMI, DCE-MRI was utilized as a noninvasive tool to assess changes in vascular permeability in FaDu xenografts following MSC treatment. Consistent with the results of MVD and VMI studies, linear regression analysis (Figure 2C) of longitudinal relaxation rate (ΔR1) as function of time following administration of the intravascular MR contrast agent, albumin-GdDTPA in MSC-treated animals revealed a marked reduction in tumor vascular permeability (slope, 0.00023 ± 0.0004, r2 = 0.097, P = 0.0147) compared to untreated controls (slope, 0.0062 ± 0.001, r2 = 0.7999). Reduction in tumor vascular leakiness is a hallmark of tumor vascular normalization.

Finally, it has been shown that the ‘normalization’ process induced by antiangiogenic therapy would, at least transiently, improve functionality of blood vessels enhancing drug delivery to tumors (17,18, 21). We therefore evaluated if pretreatment with MSC resulted in a similar improvement in perfusion and drug delivery. We first assessed fluctuations in blood flow in untreated controls and MSC-treated FaDu tumors using the double fluorescent dye method (12,13) based on two perfusion markers, Hoechst 33342 and DiOC7. Using this technique, studies have previously shown that vessels that experience intermittent flow show uptake of only one dye (‘mismatched’ vessels) (12,13). In our study, tumors treated with MSC showed an improved tumor vessel functionality indicated by uptake of both dyes while in control FaDu tumors, a majority of these perfused vessels were observed only in the tumor periphery (Figure 3A). Consistent with these observation, HPLC analysis (Figure 3B) revealed a 4-fold increase in doxorubicin concentration in MSC-treated FaDu tumors (0.62 ± 0.16 μg, P=0.01) compared to untreated controls (0.16 ± 0.03 μg). No significant change was seen in the plasma and normal tissue (kidneys, liver, small intestine) levels of doxorubicin in both cohorts of mice. Visualization and quantitation of doxorubicin levels using fluorescence also showed increased intensity at regions close to the vessel wall and away from the blood vessel (blue arrows) highlighting the improvement in both intratumoral delivery and penetration of doxorubicin following MSC treatment (Figure 3C & 3D).

In conclusion, the results of our studies have shown, for the first time, potent effects of MSC on tumor angiogenesis and vascular maturation which resulted in improved vascular function and drug delivery in HNSCC xenografts. Similar results were seen with another HNSCC xenograft A253 (data not shown). It is likely that changes in the tumor microenvironment initiated by MSC-induced tumor vascular maturation play a critical role in the reported (3) potentiation of chemotherapeutic efficacy in preclinical models. Consistent with the findings of this report, ongoing studies have revealed a 34% reduction in IFP following MSC treatment in FaDu tumors (5.58 mm Hg ± 0.83, p = 0.025) compared to untreated controls (8.87 mm Hg ± 0.961) and measurements of pO2 levels showed increased oxygenation in MSC treated FaDu tumors compared to controls (2.864 ± 0.18 vs 1.66 ± 0.24, P=0.01) (Bhattacharya, Personal Communication) consistent with our recent observation of synergy between selenium and radiation therapy (22). A decrease in tumor IFP improves delivery and penetration of therapeutics by restoring the pressure gradient across blood vessel wall as well as tumor interstitium (19,21).

While the focus on selenium in the past has been mainly for its chemopreventive properties, its use as a biological agent sensitizing tumor to subsequent treatment with anticancer drugs in advanced cancers in vivo is of recent origin. The use of organoselenium compound selenomethionine as a suicide prodrug substrate for conversion to its active metabolite methylselenol through methioninase based cancer gene therapy has been reported earlier with encouraging results in preclinical animal models (23). In contrast, our study provides evidence for use of MSC as an antiangiogenic agent that has limited tumor growth inhibition but can normalize tumor vasculature and microenvironment and thus enhance the therapeutic efficacy of a wide variety of anticancer agents when used in combination therapy. Selenium, a constituent of mammalian physiology is well tolerated and results in preclinical model systems strongly support its role as a modulator of antitumor activity and toxicity of chemotherapy (3). While the selenium dose used in this study is considerably higher than the daily dose of 200μg used in chemoprevention trials, a recent Phase I study conducted at Roswell Park Cancer Institute has shown that selenium is well tolerated at relatively high doses (7200 μg) over long periods of time in humans without serious adverse effects (24,25). At this dose the achievable plasma selenium levels are equivalent to those observed in the preclinical model system. Overall, the results of this study provide useful information for future trial design and evaluation of combination strategies involving the use of high non-toxic doses of selenium in combination with chemo- and radiation- therapies.

Acknowledgements

The authors would like to thank Dr Lakshmi Pendyala for her assistance with the HPLC quantitation and Ed Hurley for his assistance with fluorescence microscopy.

Grant Support: American Institute for Cancer Research 06A072 (A. Bhattacharya) and a Comprehensive Cancer Center Support Grant CA16056 from the National Cancer Institute.

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