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[Cancer Research 63, 5707-5711, September 15, 2003]
© 2003 American Association for Cancer Research

Advances in Brief

Tumor Distribution of Bromodeoxyuridine-labeled Cells Is Strongly Dose Dependent1

Alastair H. Kyle, Lynsey A. Huxham, Jennifer H. E. Baker, Helen E. Burston and Andrew I. Minchinton2

Department of Medical Biophysics, British Columbia Cancer Research Centre, Vancouver, British Columbia, V5Z 1L3 Canada


   ABSTRACT
Top
 ABSTRACT
Introduction
 Materials and Methods
 Results
 Discussion
 REFERENCES
 
Bromodeoxyuridine (BrdUrd) is used extensively to measure the fraction of proliferating cells in tumors. Unlike endogenous markers of proliferation such as proliferating cell nuclear antigen (PCNA) and Ki-67, BrdUrd is exogenously administered and reaches the tumor via vasculature where it must then diffuse throughout the tissue to label S-phase cells. In this study, we examine the dose dependence of BrdUrd on the tumor distribution of labeled cells in histological sections. Analysis of the distribution of labeled cells in SiHa tumor xenografts showed that a dose between 400 and 1000 mg/kg was required to label cells 150 µm from blood vessels, approaching the border of necrosis. Lower doses resulted in only the cells close to blood vessels being labeled. Interestingly, cells residing furthest from blood vessels still labeled albeit at half the level of cells situated proximal to the tumor vasculature. Results were compared with the penetration of BrdUrd seen in vitro using multilayered cell culture (MCC), a three-dimensional tissue culture model of solid tumors. Using MCC, an exposure of 100 µM BrdUrd for 1 h was required for labeling of S-phase cells 150 µm into the tissue, whereas cells adjacent to the edge of the tissue could be adequately labeled with just 5 µM BrdUrd for 1 h. The area under the curve for a 100 mg/kg BrdUrd dose in mice was found to be ~30 µM·h.


   Introduction
Top
 ABSTRACT
 Introduction
Materials and Methods
 Results
 Discussion
 REFERENCES
 
BrdUrd3 is a thymidine analogue that is incorporated into DNA during the S-phase of the cell cycle. Originally investigated as a radiation sensitizer (1, 2, 3, 4) , it has found use as a marker of cell proliferation particularly in cancer (5, 6, 7, 8) where immunohistochemical detection of BrdUrd-DNA adducts (9) makes it a simpler alternative to autoradiographic techniques using tritiated thymidine. Unlike endogenous markers for cellular proliferation such as PCNA or Ki-67, BrdUrd is exogenously administered and must reach proliferating cells via the vasculature. Upon leaving the vascular compartment, it then diffuses through cellular and extracellular compartments. In order for BrdUrd to act as an effective marker of cellular proliferation, it must be able to distribute within the extravascular compartment with enough uniformity or at high enough concentrations so that proliferating cells at any distance from a blood vessel are adequately labeled.

In contrast to most normal tissues, where the microvessel density is relatively high, the extravascular compartment of solid tumors often poses a significant barrier to the penetration of molecules supplied by the blood. The increased separation of blood vessels in tumors, caused by the proliferation of cancer cells, leads to a reduction in the ability of molecules supplied from the blood to reach all cells within the tissue (10, 11, 12) . Instability of tumor blood flow may also contribute to this effect (13, 14, 15, 16) .

With these factors in mind, we chose to evaluate the penetration of BrdUrd over a range of doses into tissue using two methods. First, through direct measurement of the relation between BrdUrd-labeled cells and position relative to blood vessels as seen in cryosections from human SiHa xenograft tumors grown in mice. Then, to address the effect of decreasing proliferation with distance from vasculature on BrdUrd labeling, tissue penetration experiments were carried out using MCCs, an in vitro model of the extravascular compartment of solid tumors (17, 18, 19) , in a novel configuration where the effect of decreasing proliferation with depth into tissue can be circumvented.


   Materials and Methods
Top
 ABSTRACT
 Introduction
 Materials and Methods
Results
 Discussion
 REFERENCES
 
Monolayer Culture.
SiHa (human cervix squamous cell carcinoma) cells were purchased from American Type Culture Collection. Cells were grown in monolayers using minimum essential media (Life Technologies, Inc., Burlington, Ontario, Canada) supplemented with 10% fetal bovine serum (Life Technologies, Inc.) and passaged every 3–5 days upon reaching confluence.

Mice and Tumors.
Female nod/SCID mice were bred and maintained in our institutional animal facility in accordance with the Canadian Council on Animal Care guidelines. The experiments described in this article were approved by the Animal Care Committee of the University of British Columbia. Mice were allowed free access to standard laboratory rodent food and water. SiHa cells (50 µl of 20 x 106 cells/ml) were implanted s.c. into the sacral region of the mice. The mice were used at 12–16 weeks of age and ranged in weight from 20–26 g. The weight of the excised tumors was 70 ± 30 mg (mean ± SD).

Tumor Penetration Assay.
Tumor-bearing mice were administered 5-bromo-2-deoxyuridine, BrdUrd (Sigma Chemical, Oakville, Ontario, Canada), i.p. at 25, 50, 100, 400, 1000, and 2000 mg/kg 2 h before sacrifice. BrdUrd was administered as a 30 mg/ml solution in saline. As a marker of blood perfusion, mice were i.v. administered 75 µl of 0.6 mg/ml carbocyanine (Molecular Probes, Eugene, OR) dissolved in 75% DMSO 5 min before sacrifice. After excision, tumors were cooled to -20°C on an aluminum block, embedded in OCT medium (Tissue-TEK, Torrance, CA) and stored at -20°C until sectioning.

MCC.
The surface of the tissue culture insert membrane (CM 12 mm, pore size 0.4 µm; Millipore, Nepean, ON, Canada) was coated with 150 µl of collagen (rat tail type I; Sigma), dissolved in 0.01 M HCl and diluted 1:4 with 60% ethanol to 0.75 mg/ml, and allowed to dry overnight. Approximately 5 x 105 SiHa cells in 0.5 ml of growth media were then inoculated onto the coated surface of the membrane and incubated for 18 h to allow the cells to attach. Silicone o-rings placed around each insert were used to suspend them in a Teflon frame that had circular holes for six inserts. The frame was then completely immersed in 150 ml of stirred media. Cultures were incubated for 3 days with continual gassing (5% CO2, balance air) at 37°C.

MCC Penetration Assay.
BrdUrd was allowed to penetrate into MCCs from one side using a Plexiglas jig in which the membrane side of each tissue culture insert was closed off. This was achieved by clamping the bottom of each insert against a flat block of Plexiglas with a layer of Parafilm (American National Can, Chicago, IL) sandwiched in between to ensure a watertight seal. Slots for the insert legs were machined into the block to allow the insert membrane to press directly against it. During the penetration assay, growth medium was supplemented with excess glucose (10 g/liter D-glucose; Sigma). Once placed in the jig, MCCs were allowed to equilibrate for 45 min, and then BrdUrd was added to the growth medium at concentrations of 1, 5, 25, and 100 µM. Control MCCs were exposed to 25 µM BrdUrd from both sides under otherwise similar conditions. After an exposure of 1 h, the cultures were removed, rinsed in PBS, embedded in OCT, and stored at -20°C until sectioning.

Pharmacokinetic Assay.
BrdUrd was administered i.p. at a dose of 100 mg/kg to six SCID mice. Blood samples of ~20 µl were taken from the tail vein of the mice at 5, 10, 20, 30, 60, and 120 min after administration. For the 5–30 min time points, the mice were divided into two groups, and sampling was alternated between groups at each time point. All mice were sampled for the 60 and 120 min time points. After each time point, blood samples were weighed, diluted with 500 µl of methanol, vortexed for 20 s, and stored at 4°C. Upon completion of all time points, samples were centrifuged at 2500 x g for 10 min, and 450 µl of the supernatant were removed and evaporated to dryness using a centrifugal evaporator (1 h at 40°C; Labconco, Kansas City, MO). Samples were then reconstituted in 50 µl of 0.1 M ammonium acetate pH adjusted to 3.5.

High-Pressure Liquid Chromatography.
Chromatographic analysis was carried out with Waters equipment (Mississauga, Ontario, Canada), including a model 510 pump, model 712 WISP injector, and model 996 photodiode array detector. A Symmetry C18 column (3.9 x 150 mm) was used for sample separation. With a mobile phase consisting of a mixture of 25% acetonitrile and 0.1 M ammonium acetate (pH 3.5) (0.15:0.85) flowing at 1.0 ml/min, whereby BrdUrd eluted after 9.2 min. Samples of 30 µl were injected, and absorbance detection was carried out at 280 nm. BrdUrd stability and detection linearity in spiked blood were verified down to 1 µM.

BrdUrd Immunohistochemistry.
Tumor and MCC cryosections (10-µm thick) were air-dried for 24 h and then fixed in a 1:1 mixture of acetone-methanol for 10 min at room temperature. Slides were then washed in distilled water for 10 min and treated with 2 M HCl at room temperature for 1 h followed by neutralization for 5 min in 0.1 M sodium borate. Slides were then washed in distilled water and transferred to a PBS bath. Subsequent steps were each followed by a 5-min wash in PBS. BrdUrd incorporated into DNA was detected using a 1:200 dilution of monoclonal mouse anti-BrdUrd (clone BU33; Sigma) followed by 1:100 dilution of antimouse peroxidase conjugate antibody (Sigma) and 1:10 dilution of metal-enhanced 3,3'-diaminobenzidine substrate (Pierce, Rockford, IL). Slides were then counterstained with hematoxylin, dehydrated, and mounted using Permount (Fisher Scientific, Fair Lawn, NJ).

Image Acquisition.
The imaging system consisted of a fluorescence microscope (Zeiss III RS; Oberkochen, Baden-Württemberg, Germany), a cooled monochrome CCD videocamera (model 4922; Cohu, San Diego, CA), frame grabber (Scion, Frederick, MD), a custom built motorized x-y stage and customized NIH-Image software (public domain program developed at the United States NIH, available online4 ) running on a G3 Macintosh computer. The motorized stage allowed for tiling of adjacent microscope fields of view. Using this system, images of entire tumor sections were captured, typically 25–50 mm2 in size at a resolution of 1 pixel/µm2. Two cryosections were imaged for each MCC, making a ~2 mm2 imaged tissue area. For tumor cryosections, images of carbocyanine fluorescence within the sections were obtained before BrdUrd immunostaining using a 450–480-nm excitation filter and a 525-nm long pass emission filter. Once immunostained and mounted, the slides were then revisited and bright field images of BrdUrd-positive staining obtained.

Image Analysis: Tumors.
Using the NIH-Image software application and user supplied algorithms, images of carbocyanine fluorescence and BrdUrd/tissue staining from each tumor section were overlaid, and areas of necrosis and staining artifacts were removed. On the fluorescence image, carbocyanine-positive regions were then identified by selecting all pixels that were >20% of the maximum carbocyanine intensity seen on each image. Carbocyanine-positive regions that were <20 µm2 in size were considered artifacts and removed from the analysis. On the bright field images, BrdUrd-positive staining was identified by selecting pixels that were 2.5 SDs above tissue background levels. Measuring the distance from each point in the tissue to the nearest carbocyanine-positive pixel and noting if it were BrdUrd positive or negative, then determined the relation between proliferation and distance to the nearest blood vessel. The data were tabulated so as to determine the fraction of BrdUrd-positive pixels of the total number pixels found at each distance to a blood vessel.

Image Analysis: MCCs.
BrdUrd-positive staining was identified as above. For the MCCs that were sealed off from one side during exposure to BrdUrd, the edges of the MCCs were traced out, and then the position of each point in the tissue was measured relative to the two edges (expressed as a fraction between 0 and 1) and tabulated along with whether or not the point was BrdUrd positive. The fraction of tissue positive for BrdUrd of the total tissue found at each position within the MCC, divided into 30 steps, was then calculated. For MCCs that were exposed to BrdUrd from both sides, the fraction of BrdUrd-positive pixels of the total number of pixels found at each distance away from the nearest edge was determined.


   Results
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
Discussion
 REFERENCES
 
BrdUrd Penetration into Tumor Xenografts.
The distribution of BrdUrd around tumor microvessels was assessed via simultaneous detection of BrdUrd-DNA adducts and carbocyanine-stained blood vessels in tumor cryosections. BrdUrd was injected i.p at four doses 100, 400, 1000, and 2000 mg/kg to multiple mice, and single mice were dosed at 25 and 50 mg/kg. Images of whole tumor cryosections were captured for each marker and then overlaid. Fig. 1, A–DCitation shows sample areas from images of the 100-2000 mg/kg dosed mice. Results of analysis to determine the distribution of BrdUrd labeling relative to tumor vasculature from images of entire cryosection are shown in Fig. 1, E–HCitation . At all doses, a trend of decreasing BrdUrd staining with distance away from vasculature was seen. As dose increased, an increase was seen in the intensity of staining and the number of cells exhibiting staining. This was interpreted as being attributable to the higher plasma BrdUrd concentrations and the longer period over which labeling could be expected to occur at the higher doses. An increase was also seen in the penetration of BrdUrd away from the vasculature with increasing dose. From the distribution plots, the depth at which proliferation appeared to drop to half-maximum was determined as 60 ± 10 (n = 2), 120 ± 10 (n = 2), 135 ± 25 (n = 4), and 110 ± 20 µm (n = 4) (average ± maximum deviation) for the 100, 400, 1000, and 2000 mg/kg doses, respectively, the 400 and 1000 mg/kg groups each contained a tumor that did not follow the general trend and was excluded from the half-maximum calculations. The half-maximum values from the highest three doses were within experimental error of each other, however, peak staining levels were significantly higher for the 1000 and 2000 mg/kg doses. Single mice dosed with 25 and 50 mg/kg continued the trend toward reduced staining and penetration (data not shown). Results indicated that a dose between 400 and 1000 mg/kg was required for maximal immunohistochemical staining of S-phase cells located far from blood vessels in this tumor model.



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  		Fig. 1. SiHa tumor cryosections showing S-phase-labeled cells (grayscale) relative to vasculature (magenta) as a function of BrdUrd dose. (A) 100, (B) 400, (C) 1000, and (D) 2000 mg/kg BrdUrd. Fluorescent images of carbocyanine-labeled vasculature were digitally captured and overlaid on subsequent bright-field images of cryosections immunostained to show BrdUrd-labeled S-phase cells and counterstained with hematoxylin. Inlays show magnified regions. Scale bars: 150 mm. Line graphs show the distribution of BrdUrd labeling as a function of distance to nearest visible blood vessel at the four doses, (E) 100, (F) 400, (G) 1000, and (H) 2000 mg/kg BrdUrd. Each line represents data from an individual tumor.

 
BrdUrd Pharmacokinetics in SCID Mice.
The plasma concentration of BrdUrd in SCID mice was measured at several times after i.p administration of 100 mg/kg. BrdUrd blood concentration versus time is shown in Fig. 2Citation . The rate of decay was found to be exponential over the first 2 h with a half-life of 19 ± 1 (SE) min. The peak level was determined as 58 ± 12 (SE) µM, and the area under the curve for the first 2 h was ~30 µM·h.



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  		Fig. 2. BrdUrd plasma kinetics in SCID mice after i.p. administration of 100 mg/kg. Plasma levels follow an exponential decay over the first 2 h. The half-life was determined to be 19 ± 1 (SE) min, peak plasma levels were 58 ± 12 (SE) µM, and the area under the curve for the first 2 h was ~30 µM·h.

 
BrdUrd Penetration into MCCs.
The penetration of BrdUrd into MCCs was assessed via immunohistochemical detection of BrdUrd-DNA adducts in cultures exposed to BrdUrd at various concentrations. Exposure of each MCC to BrdUrd was made from one side to observe penetration of BrdUrd through the entire width of the culture and enable comparison of BrdUrd staining on either edge, where cells were expected to display similar rates of proliferation. Sample areas of MCCs exposed to 1, 5, 25, and 100 µM BrdUrd for 1 h are shown in Fig. 3, A–DCitation . For comparison, an additional MCC was exposed to 25 µM from both sides, Fig. 3ECitation . The images show that 100 µM BrdUrd is required to label S-phase cells on the far edge of a 150-µm thick culture. Results from analysis of the relation between BrdUrd labeling and position within the cultures, performed on images of the entire cryosections, are shown in panels Fig. 3, F–JCitation . Data shows that at 100 µM, BrdUrd-labeled cells are uniformly distributed within the culture and match the labeling seen after exposure to 25 µM BrdUrd from both sides.



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  		Fig. 3. Grayscale images of 3-day-old MCCs showing the distribution of S-phase-labeled cells as a function of BrdUrd concentration during a 1-h exposure. (A) 1, (B) 5, (C) 25, and (D) 100 µM BrdUrd from the left side of the cultures. E shows labeling after exposure to 25 µM BrdUrd from both sides of the culture. S-phase cells detected via immunohistochemical staining of BrdUrd-DNA adducts. Cryosections were counterstained with hematoxylin. Scale bars: 150 µm. Line graphs show the distribution of BrdUrd labeling for each of the exposures (F–J). Data are expressed as a function of position relative to each edge of the cultures, 0 indicating cells adjacent the left edge and 1 indicating the cells adjacent the right edge of the cultures. Each line represents data from an individual cryosection.

 
To observe the effect of exposure time on the penetration of BrdUrd at low doses, additional MCCs were exposed to BrdUrd for 24 h. In this case, the cultures were exposed to BrdUrd from both sides to avoid loss of proliferation on the far edge because of sealing of the culture over the extended period. MCCs were exposed to 1 µM BrdUrd for 2 h, 1 µM BrdUrd for 24 h, and 1 µM BrdUrd for 24 h followed by 200 µM BrdUrd for 1–2 h to detect S-phase cells not labeled by the 24-h exposure at the low concentration. During the 24-h exposure, BrdUrd concentrations were monitored by high-performance liquid chromatography and maintained at 1 ± 0.5 µM. Sample areas of the immunostained MCC cryosections are shown in Fig. 4, A–DCitation . Because of the additional day of growth, the MCCs are thicker than those from the 1-h exposures, and a loss of proliferation occurs toward their centers. Results indicate a marked increase in BrdUrd penetration after exposure to 1 µM BrdUrd for 24 h versus 2 h. However, labeling in the central region of the MCCs was seen to increase after exposure to 200 µM BrdUrd for 2 h, suggesting inadequate penetration of 1 µM BrdUrd even after 24 h. Analysis of the distribution of BrdUrd labeling as a function of distance to the nearest edge of the tissue is shown in Fig. 4ECitation . A marked increase in labeling is seen throughout the MCCs after exposure to BrdUrd for 24 h, and the data also show an increase in labeling, at a depth of 150 µm, upon exposure to 200 µM BrdUrd.



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  		Fig. 4. Grayscale images of 4-day-old MCCs showing distribution of BrdUrd-labeled S-phase cells after extended exposure from both sides under various conditions. A shows results after 1 µM BrdUrd for 2 h, B shows 1 µM BrdUrd for 24 h, C shows 1 µM BrdUrd for 24 h followed by 200 µM BrdUrd for 1 h, and D shows 1 µM BrdUrd for 24 h followed by 200 µM BrdUrd for 2 h. Cryosections were counterstained with hematoxylin. Scale bars: 150 µm. Line graphs, E, show the distribution of BrdUrd labeling from analysis of multiple MCCs; 1 µM BrdUrd for 2 h ({square}), 1 µM BrdUrd for 24 h ({circ}), and 1 µM BrdUrd for 24 h followed by 200 µM BrdUrd for either 1 h ({diamondsuit}) or 2 h ({blacktriangleup}). Data are expressed as a function of distance to nearest edge of the cultures. Error bars show SD.

 

   Discussion
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
REFERENCES
 
The tissue distribution of BrdUrd administered i.p. over a range of doses was evaluated using a human tumor xenograft grown in mice and indicated that a dose of between 400 and 1000 mg/kg was required to adequately label S-phase cells located distal to vasculature. We compared the results from tumor cryosections with data from MCC experiments using a new method that circumvented the problem of gradients in proliferation seen in vivo. MCCs exposed to increasing concentrations of BrdUrd for 1 h showed a trend in labeling distribution versus exposure that was consistent with that seen from the tumor data, although with higher overall penetration. MCC results indicated that a BrdUrd exposure of ~100 µM for 1 h was required to label S-phase cells residing 150 µm from the site of exposure. It appeared that at the highest two doses, both in the tumor and MCC studies, the maximal rate of BrdUrd incorporation into DNA was being achieved throughout the tissue. At these doses, the actual distribution of free BrdUrd was unknown and may still have exhibited a gradient. A relationship between increasing dose of BrdUrd and increasing labeling has previously been shown (20) and has generally been attributed to increased staining intensity and increased number of cells entering S-phase during the longer exposure to relevant BrdUrd levels. This study shows that there is an additional penetration component, characterized by an increase in the labeling of cells far from blood vessels with increasing BrdUrd dose.

The finding that a dose of BrdUrd between 400 and 1000 mg/kg was required to label S-phase cells furthest from tumor vasculature in cryosections was initially surprising because most flow cytometry studies are performed with doses of 100 mg/kg or lower. Flow cytometry can have a higher signal to noise ratio compared with immunohistochemical studies as is illustrated by data from the literature for SiHa xenografts showing a labeling index of ~25% for a 90 mg/kg dose of BrdUrd (21) , which is comparable with results shown here for the 1000 mg/kg dose. However, a BrdUrd-unlabeled population of cells containing S-phase levels of DNA is commonly observed in flow cytometric studies examining tumor cell proliferation (6, 7, 8) . The presence of the unlabelled cells has generally been interpreted as indicating noncycling cells with intermediate levels of DNA rather than unlabeled and proliferating cells. BrdUrd labeling of tumor tissue in vitro has been shown in some instances to increase the labeling index detected via flow cytometry (7) . Also, a study by Rodrigez using iododeoxyuridine in the HCT-116 xenograft, which exhibits similar vascular density to SiHa xenografts, found that after continuous infusion for 5 days at 100 mg/kg/day, immunohistochemical staining clearly showed incomplete tissue staining whereas flow cytometric data indicated close to a 90% labeling index (22) . It is interesting to note that human studies involving BrdUrd as a diagnostic tool typically use much lower doses of the agent, generally 100–250 mg/patient, (~1–3 mg/kg; Refs. 7 , 23 , 24 ) compared with the 400-1000 mg/kg needed in our studies in mice. It is also noteworthy that several commercially available kits for the labeling of proliferation in vivo recommend doses of <60 mg/kg i.p. Our findings here suggest that while these doses may be adequate for labeling proliferation in some normal tissues, they are inadequate for labeling of proliferating cells in tumors.

Using MCCs a 1-h exposure time to BrdUrd was chosen to match the exposure for typical bolus injection seen in mice. Results indicate that BrdUrd concentration must be kept >100 µM for 1 h to adequately label S-phase cells 150 µm from the site of exposure. The reason for poor penetration at lower concentrations is interpreted as being attributed to consumption of BrdUrd by the tissue. Extended exposure to 1 µM BrdUrd (Fig. 4)Citation improved its tissue penetration relative to the 1-h data but was still unable to produce maximal labeling in the cells furthest removed from the site of exposure. In general, results from MCC-based studies are expected to underestimate in vivo tissue penetration because of their planar geometry, which poses less of a barrier to penetration than the cylindrical geometry that often characterizes the tissue surrounding blood vessels in solid tumors. Overall, the MCC data showed that BrdUrd penetration, and not solely the reduction in the rate of proliferation away from blood vessels, was involved in determining the labeling pattern seen in the tumor xenografts.

The clinical testing of BrdUrd as a radiation sensitizer in the 1980–1990s generally used continuous infusion over several days, with doses of ~1000 mg/m2/day (equivalent to ~25 mg/kg/day). At these dose levels, BrdUrd reached steady state plasma concentration of ~1 µM (25) . In the MCC system, a 24-h exposure to 1 µM BrdUrd did not produce maximal labeling of proliferating cells distant from the site of exposure, indicating suboptimal exposure. Because the MCCs pose less of a barrier to penetration than many tumors, it is possible that an extended exposure to 1 µM BrdUrd may be insufficient to fully sensitize proliferating cells located distant from vasculature.

MCCs have in the past been used to evaluate drug penetration via measurement of flux through the cultures (12 , 26, 27, 28, 29) . The data presented here represents a new application of MCCs in the evaluation of drug penetration via immunodetection of drug-cell interactions within histological sections. Evaluating a drug’s penetration via its effect is usually confounded by intrinsic changes in cells with depth into tissue (e.g., reduced proliferation, drug sensitivity, hypoxia, necrosis; Refs. 30 , 31 ). In this study, this problem was circumvented by temporarily sealing off one side of the culture during exposure from the other side and then examining the cell layers adjacent to each surface. The benefit of this approach was to allow comparison of BrdUrd labeling in cells known to have comparable rates of proliferation. This approach could be used to evaluate the penetration of anticancer agents based on their effect on cells located at different depths in tissue using immunohistochemical assays for proliferation or cell death.


   FOOTNOTES
 
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 with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by the National Cancer Institute of Canada with funds from the Canadian Cancer Society. A. H. K. is a Michael Smith Foundation for Health Research Scholar. Back

2 To whom requests for reprints should be addressed, at British Columbia Cancer Research Centre, 601 West 10th Avenue, Vancouver, British Columbia, V5Z 1L3 Canada. E-mail: minc{at}interchange.ubc.ca Back

3 The abbreviations used are: BrdUrd, bromodeoxyuridine; MCC, multilayered cell culture. Back

4 Internet address: http://rsb.info.nih.gov/nih-image/. Back

Received 6/13/03. Revised 7/17/03. Accepted 7/21/03.


   REFERENCES
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
 REFERENCES
 

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