Download Doxorubicin Gradients in Human Breast Cancer

Vol. 5, 1703–1707, July 1999
Clinical Cancer Research 1703
Doxorubicin Gradients in Human Breast Cancer
Jan Lankelma,1 Henk Dekker,
Rafael Fernández Luque, Sylvia Luykx,
Klaas Hoekman, Paul van der Valk,
Paul J. van Diest, and Herbert M. Pinedo
Departments of Medical Oncology [J. L., H. D., R. F. L., S. L., K. H.,
H. M. P.] and Pathology [P. v. d. V., P. J. v. D.], Free University
Hospital, 1007MB Amsterdam, the Netherlands
ABSTRACT
Ten patients with locally advanced breast cancer were
given doxorubicin i.v., and an incision biopsy was subsequently taken. Doxorubicin autofluorescence was examined
using computerized laser scanning microscopy, and microvessels were immunostained in the same sections. Overlays of both pictures revealed doxorubicin gradients in tumor islets with high concentrations in the periphery and low
concentrations in the center of the tumor islets. Gradients
were most pronounced shortly after the injection, but they
could still be detected 24 h later. No gradients were observed
in connective tissue. This study demonstrates a serious risk
of the drug not reaching all of the cancer cells in those cases
in which the cancer cells are densely packed in islets. The
efficacy of drug treatment will thus depend on the histology
of the tumor tissue.
INTRODUCTION
Epithelial cancer is currently the most common cause of
cancer-related death. Although the normal architecture of
intact epithelium is lost, in epithelial cancer, the frequent
finding of desmosomes and occasionally tight junctions (1)
indicate that the malignant epithelium may at least partially
retain its function as a barrier, e.g., against drugs. A low
density of blood capillaries may limit tumor growth (2)
because of reduced delivery of oxygen and nutrients. However, during chemotherapy, reduced delivery of anticancer
drugs may mean a growth advantage for the most remote
tumor cells. Drug transport out of the blood capillaries and
into the tissue can occur through the cells (transcellular
pathway) and through the interstitium (paracellular pathway;
Ref. 3). Doxorubicin is one of the most effective agents for
the treatment of epithelial cancer. However, the drug rarely
eliminates all cancer cells in patients with advanced disease,
Received 10/6/98; revised 3/15/99; accepted 3/19/99.
The costs of publication of this article were defrayed in part by the
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indicate this fact.
1
To whom requests for reprints should be addressed, at Department of
Medical Oncology, Room BR232, Free University Hospital, P. O. Box
7057, 1007MB Amsterdam, the Netherlands. Phone: 31-20-444-2603;
Fax: 31-20-444-3844; E-mail: [email protected].
including breast cancer. Previously, in vitro experiments
using multicell spheroids have demonstrated doxorubicin
gradients (4, 5). This study was undertaken to investigate
whether such gradients can be found in vivo as well.
At the cellular level, P-glycoprotein (6 – 8) and multidrug
resistance protein (9, 10) can mediate drug resistance by decreasing transport to the target. Drug pumping by these proteins
across the plasma membrane resulted in lower intracellular drug
concentrations (11–14). At the tissue level, drug transport barriers have been recognized for large molecules (15, 16), which,
because of their low diffusion rate, depend largely on convective
flows for their transport. Furthermore, they may also be hampered by the high hydrostatic pressure outside the microvessels,
conditions that often occur in solid tumors (15, 16). The net
transport of small molecules (such as most drugs with a Mr ,
1000) by diffusion may slow down as a result of binding to
relatively large molecules that move at a much slower speed
(17). Moreover, when the transport rate across the plasma membrane is low in combination with strong intracellular binding,
drug transport through the cells can be slowed down even more
drastically. Then, just after i.v. injection, after a fast rise in the
blood concentration of the drug, the intracellular free drug
concentration will rise slowly during influx at the front (at the
side of the blood vessel). Because drug transport through the
plasma membrane at the back of the cell (toward the neighboring cell) depends on this intracellular free drug concentration, it
will also be slow and rise slowly (the latter is due to strong
intracellular binding). In combination with a low paracellular
transport in epithelial cancer, the result may be a nonhomogenous drug distribution in tissue. It must be noted that under
these conditions, the drug molecules are not hampered by slow
convective flows in the opposite direction driven by a pressure
gradient.
In our search for gradients in vivo, we have used computerized fluorescence microscopy, exploiting the autofluorescence of doxorubicin. Earlier reports on doxorubicin tissue
distribution in paraffin sections used either direct autofluorescence measurement by conventional fluorescence microscopy (18) or indirect measurement using antibodies against
the drug (19). The drug may be partly lost during sample
preparation with both methods. Both studies reported heterogeneous drug distribution throughout the tissue. To avoid
such drug loss, we used cryosections for measuring doxorubicin fluorescence without further contact of the sections
with solvents. In mice, Hoechst 33342 dye has been reported
to show steep gradients in biopsies alongside microvessels
(20). Previously, doxorubicin gradients in tissue have been
observed in mice, using an ovarian tumor model and i.p.
injection (21). In our search for doxorubicin gradients after
i.v. injection of the drug in patients, we overlayed digital
pictures of doxorubicin fluorescence with immunohistochemistry pictures of CD31 (staining of endothelial cells). This
procedure allowed us to determine the spatial distribution of
doxorubicin in relation to microvessels.
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1704 Doxorubicin Gradients in Human Breast Cancer
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Clinical Cancer Research 1705
Patients with locally advanced breast cancer were treated
according to a moderately high-dose chemotherapy protocol in
which doxorubicin was an important ingredient (22, 23).
MATERIALS AND METHODS
Chemotherapy. Chemotherapy consisted of moderately high-dose doxorubicin (90 mg/m2 body surface) and
high-dose cyclophosphamide (1000 mg/m2) on day 1, followed by granulocyte macrophage colony-stimulating factor
(250 mg/m2) s.c. or i.v. on days 2–11. Cycles (total, six
cycles) were repeated every 3 weeks. Tumor biopsies of
approximately 0.5 3 0.5 3 0.5 cm in size were taken under
local or general anesthesia after oral consent and snap-frozen
in liquid nitrogen. Cryosections of the whole biopsies were
cut for imaging of doxorubicin fluorescence and subsequent
immunostaining.
Doxorubicin Imaging by CLSM.2 A CLSM (type TCS
4D; Leica Heidelberg, Heidelberg, Germany) was used. Fluorescence images were made using the autofluorescence of
doxorubicin. A krypton-argon laser line (488 nm) was used
for excitation of doxorubicin, and a long pass filter (550 nm)
was used for detection of the emitted light. Images at low and
high resolution were obtained throughout the biopsy. We did
not observe any change in the doxorubicin distribution during
handling of the samples, even after keeping the sample at
room temperature for 5 h. Integrated nuclear doxorubicin
fluorescence was obtained using Leica Q500MC Quantimet
software (version V01.01; Leica Cambridge Ltd., Cambridge,
United Kingdom).
Immunohistochemical Staining. The endothelial cells
of blood vessels were stained immunohistochemically, using
the same frozen sections as for doxorubicin imaging, as
follows. The cryosections were fixed in acetone on ice or in
4% paraformaldehyde followed by acetone on ice. They were
incubated for 1 h with mouse antihuman CD31 monoclonal
antibody (clone JC/70A; DAKO A/S, Glostrup, Denmark),
followed by a 30-min incubation with rabbit antimouse biotin
complex (DAKO). Peroxidase was blocked with 3% hydrogen peroxide for 30 min before incubation with ABComplex/
horseradish peroxidase (DAKO) for 30 min. A red color was
developed in 10 –30 min with the 3-amino-9-ethylcarbarole
kit (Vector Laboratories, Inc., Burlingame, CA), and nuclei
were counterstained with hematoxylin. Hematoxylin images
were obtained by combining red, green, and blue transmission images using the same optics. Images at low and high
resolution were again obtained throughout the biopsy with
2
The abbreviation used is: CLSM, confocal laser scanning microscope/
microscopy.
Fig. 2 Mean nuclear doxorubicin fluorescence (averaged pixel intensity; expressed in arbitrary units) versus distance to the border (with the
connective tissue) of the tumor islet of a representative part of the
corresponding images in Fig. 1. A corresponds to Fig. 1D; B corresponds
to Fig. 1E; C corresponds to Fig. 1H; D corresponds to Fig. 1I.
CLSM. Overlaying of pictures (doxorubicin and immunoimages) was accomplished using Photo-Paint software (version
6.00; 1995; Corel Co., Ottawa, Canada).
Materials. Doxorubicin HCl was obtained from Montedison Nederland (Rotterdam, the Netherlands). DNase I was
purchased from Boehringer Mannheim (Mannheim, Germany).
RESULTS
Doxorubicin Distribution in Breast Cancer. In the
cryosections, doxorubicin was seen as a nuclear fluorescence
clearly distinguishable from background fluorescence (Fig. 1),
which was predominantly from the cytoplasm (Fig. 1A). For
each patient (Fig. 1, B–J), both doxorubicin distribution patterns
and CD31 immunohistochemical staining of the same area of
the same section are represented. Doxorubicin gradients were
observed most clearly in the ductal invasive carcinomas showing tumor islets in the biopsies, which were taken at 2 h after
injection (Fig. 1, D–F). These doxorubicin gradients were not
detected in the connective tissue. Also, no clear gradients were
observed in patients with invasive lobular cancer with more
connective tissue and strands of tumor cells (indian files; Fig. 1,
B and C). Occasionally, connective tissue showed bands of
Fig. 1 Doxorubicin (nuclear) fluorescence in cryosections of locally advanced breast cancer biopsies at t 5 0 (A), 2 h (B–F), and 24 h (G–J) after
the first i.v. injection of doxorubicin (dose, 90 mg/m2 body surface). A glow look-up table was used, with the lightest colors indicating the highest
doxorubicin concentrations. CD31 immunohistochemical staining combined with hematoxylin counterstaining of the very same biopsy area is shown
directly to the right of the corresponding doxorubicin fluorescence image. Each doxorubicin image is from a different patient, except for A (control)
and D, which are from the same patient.
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1706 Doxorubicin Gradients in Human Breast Cancer
Fig. 3 Doxorubicin distribution (red) at 2 h after doxorubicin injection of patient K. CD31 immunostaining was visualized with a second
FITC-labeled antibody (green). Superimposition of both colors yields yellow. The highest nuclear doxorubicin concentrations were observed near
microvessels. Magnifications: 310 (A); 340 (B and C).
fluorescence (Fig. 1, D, F, and G); however, this fluorescence
could be distinguished morphologically from typical nuclear
doxorubicin fluorescence.
Gradient Steepness. In four patients, the average nuclear doxorubicin fluorescence was plotted against the distance
from the nearby rim of the islet to demonstrate the steepness of
the gradient (Fig. 2). Less steep gradients were found 24 h after
injection, as compared to gradients at 2 h after injection.
Distribution at Lower Magnification. Fig. 3 shows an
overview over a larger part of one of the sections in pseudocolors
(red, doxorubicin; green, CD31) with the highest doxorubicin
concentrations around the microvessels (103 magnification) and
gradients extending from the capillaries (403 magnification).
Previously, doxorubicin concentrations (averaged per
weight of tissue) in biopsies have been measured by highperformance liquid chromatography (24, 25). No metabolites
could be detected in breast cancer biopsies (25), indicating that
the nuclear fluorescence comes only from doxorubicin itself.
CD31 immunohistochemical staining indicated the presence of
capillary vessels in the connective tissue. To compare the data
from tumor with structurally similar normal tissue, skin biopsies
were taken from three patients after doxorubicin administration.
Doxorubicin gradients were found in all three of the biopsies
examined (data not shown).
DISCUSSION
Generally, small molecules with Mr , 1000 show a more
rapid diffusion into tissues than large molecules, such as antibodies
or viral vectors. Under conditions of extensive intracellular binding
and a low plasma membrane transport rate, penetration in the tumor
islets of such drugs, such as doxorubicin (Mr 543.54), can be
slowed down considerably. Here we show the slow penetration of
the small-molecule drug doxorubicin in patients, resulting in gradients in clinical biopsies of solid tumors. Cells in the center of the
tumor islets, which are the most remote from the microvessels, are
exposed to lower drug concentrations in the surrounding extracellular fluid compared to the cells in the periphery of the tumor islets.
In general, the three-dimensional gradient toward the center of an
islet may be steeper than the observed two-dimensional image, due
to the fact that the cross-sections of the islets generally will not run
through the center of the islets. In that case, the distance from a cell
inside such an islet to the islet boundary will be shorter than that
observed in the two-dimensional cross-sections; in reality, the
doxorubicin concentration gradient will be even steeper than that
measured. It could be argued that when the gradient reverses during
drug clearance from the blood, the drug concentration in cells at the
periphery might become lower when compared to cells at the center
of the islet. However, back supply from the inner layers will buffer
this fall in concentration. Diffusion will be slow through both the
transcellular and the paracellular routes: it will be slow through the
paracellular route because of the small volume of the intercellular
space; and it will be slow through transcellular route because of
slow membrane passage and high intracellular binding (mainly to
DNA). To understand the generation of doxorubicin gradients,
further development of transport models determining the relative
contributions of factors affecting the total transport will be necessary.
As an alternative scheme for doxorubicin administration
per bolus injection, a continuous 96-h infusion has been applied
to patients for the treatment of metastatic breast cancer and
sarcoma. This method of administration had a favorable effect
on life-threatening heart toxicity (26, 27). The comparable clinical antitumor efficacy at the relatively low plasma concentrations during infusion, when compared to the high concentrations
during the first few hours after i.v. injection, could partly be due
to a more homogenous doxorubicin tissue distribution in cancer
islets. The kinetics of drug in the blood shows that after i.v.
injection, the doxorubicin plasma concentration declined from
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Clinical Cancer Research 1707
15 to 3 mg/liter in the 6 –96-h period after injection. During the
first few hours after injection, the concentration declines much
more rapidly from a level 2 orders of magnitude higher (24).
With low effective drug diffusion into the tumor islet, this
profile leads to the development of a gradient, especially in the
first few hours, when the outer tumor cells of an islet are
exposed to relatively high concentrations. Later, e.g., at 24 h, the
tissue gradient is less steep due to diffusion and the decline of
the plasma concentration. During continuous infusion at a comparable total dose, the plasma concentration gradually rose to 15
mg/liter at 24 h after the start of the infusion (28). In that case,
the high initial concentrations after i.v. injection are missing;
therefore, the tumor islets will experience a much more steady
concentration resulting in gradients that are less steep.
Doxorubicin-based chemotherapy of breast cancer followed by surgery (22, 23) can be an effective treatment for
locally advanced disease. To avoid possible memory effects of
previous cycles, we examined biopsies after the first doxorubicin injection. This study suggests that during chemotherapy
cycles, the doxorubicin will result in a peeling off of cells from
tumor islets. Of the 10 patients shown, 3 patients (Fig. 1, B, C,
and H) had no residual tumor cells in their breast after six cycles
of chemotherapy. Two of these patients (Fig. 1, B and C)
showed no doxorubicin gradients in their breast carcinoma. This
observation underscores tissue transport of a drug as an important component of drug resistance. This study warrants additional studies on the importance of tumor islets of breast cancer
cells as a negative prognostic marker in cancer chemotherapy.
ACKNOWLEDGMENTS
We thank Aafke Honkoop for obtaining the patient samples, and
we are most indebted to the patients who were willing to make tissue
available without personal benefit. We thank Dr. Andy Ryan for grammatical correction of the manuscript.
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Doxorubicin Gradients in Human Breast Cancer
Jan Lankelma, Henk Dekker, Rafael Fernández Luque, et al.
Clin Cancer Res 1999;5:1703-1707.
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