Background
The ErbB family of receptor kinases is a group of four trans-membrane proteins (ErbB1 - ErbB4) that share similarities in structure and are involved in signaling pathways that stimulate cellular proliferation [
1]. Ligand binding induces receptor homo- and hetero-dimerization, although no ligand has been identified for ErbB2. Dimerization of the receptors stimulates their intrinsic tyrosine kinase activity resulting in receptor autophosphorylation [
2]. These phosphorylated residues serve as binding sites for molecules involved in the regulation of intracellular signaling cascades. Overexpression of ErbB receptors may occur in a wide range of epithelial cancers, including those of the breast [
3], colon [
4], head and neck [
5], kidney [
6], lung [
7,
8], pancreas [
9], prostate [
10] and esophagus [
11,
12] and has been associated with an aggressive phenotype.
Molecular targeted agents that interact with receptor tyrosine kinases on tumor cells are used increasingly in clinical oncology. There are two classes of agents, monoclonal antibodies and low-molecular-weight tyrosine kinase inhibitors. Cetuximab (chimeric mouse/human) and trastuzumab (humanized) are monoclonal antibodies that target the extracellular domain of the receptors ErbB1 [
13‐
16] and ErbB2 [
15,
17] respectively. Binding of cetuximab and trastuzumab to ErbB1 and ErbB2 respectively prevents receptor phosphorylation and activation of the kinase domain, thereby inhibiting cell proliferation [
18‐
20]. Binding of trastuzumab to its receptor also reduces shedding of the extracellular domain of ErbB2 and prevents the production of an active truncated fragment [
20‐
22]. These agents have shown therapeutic activity against colorectal cancer and breast cancer respectively and are in wide clinical use [
21,
22].
Limited penetration of drugs through tumor tissue is an important and rather neglected cause of clinical resistance to chemotherapy [
23‐
25]. Drug distribution from blood vessels within tumors depends on diffusion and and/or convection, and is inhibited by consumption in proximal cells [
23,
25‐
27]; for monoclonal antibodies consumption is due to binding to the receptor target, which is dependent on antibody dose, number of antigenic targets per cell, and the affinity of the antibody for its target [
28]. Convection depends on gradients of pressure (both hydrostatic and osmotic) between the vascular space and the interstitial space, while diffusion depends on molecular size, shape and concentration gradients [
26,
27]. Because monoclonal antibodies are large molecules they might be expected to have poor distribution from tumor blood vessels [
28]. However drugs with a long half-life in the circulation may establish a more uniform distribution in tissues even if penetration of tissue is relatively slow, whereas drugs with a short half-life may have a non-uniform distribution. Here we report a study of the distribution of the monoclonal antibodies, cetuximab and trastuzumab, in tumors that express different levels of their target receptors.
Methods
Drugs and reagents
The monoclonal antibody cetuximab (IMC-C225, Erbitux) was provided by Imclone Systems, Inc. (New York, NY, USA) as a solution at a concentration of 2 mg/ml. Trastuzumab (Herceptin) was obtained from the hospital pharmacy at a concentration of 21 mg/ml. The hypoxia-selective agent EF5 and Cy5-conjugated anti-EF5 antibody [
29,
30] were kindly provided by Dr. C. Koch, Philadelphia, PA. Blood vessels in tumor sections were visualized with a rat anti-mouse CD31 (PECAM-1) monoclonal antibody that was purchased from BD Pharmingen (Mississauga, ON, Canada) and the Cy3-conjugated goat anti-rat IgG secondary antibody was purchased from Jackson Immuno Research Laboratories, Inc. (West Grove, PA). Cetuximab and trastuzumab were recognized in tissue sections with goat anti-human IgG conjugated with horseradish peroxidase (Biosource, Montreal, Canada).
Cell lines and tumor models
Experiments were performed utilizing the ErbB1-overexpressing human epidermoid carcinoma (A431) and a human breast adenocarcinoma (MDA-MB-231), using both wild-type and
ERBB2 transfected (231-H2N) cell lines. A431 and MDA-MB-231 cells were obtained from the American Type Culture Collection (Manassas, VA, USA), while MDA-MB-231 cells transfected with
ERBB2 (231-H2N) were kindly provided by Dr. J. Medin [
31] (University of Toronto, ON, Canada). All the cell lines were maintained as monolayers in Dulbecco's Modified Eagle's Medium (DMEM), supplemented with 10% fetal calf serum (FCS), at 37°C in a humidified atmosphere of 95% air plus 5% CO
2. Tests were performed routinely to ensure that cells were free of mycoplasma. Tumors were generated by injection of ~2 × 10
6 exponentially-growing cells into the right and left flanks of 6-8 week old female athymic nude mice, purchased from Harlan Sprague-Dawley Laboratory Animal Centre (Madison, WI, USA). Mice were housed five per cage, and sterile tap water and food were given ad libitum. All procedures were carried out following approval of the Institutional Animal Care Committee.
Expression of ErbB1 and ErbB2 receptors in the xenografts was confirmed by applying cetuximab or trastuzumab to sections of tumors ex vivo, followed by their recognition using anti-human IgG as described below. Endogenous expression of ErbB1 and ErbB2 were also confirmed and assessed by diagnostic antibodies from Zymed (Clone 31G7) and Neomarkers (Clone SP3) respectively.
Experimental design
Tumor-bearing mice were divided randomly into groups of 5-6, and treatment with cetuximab or trastuzumab was initiated when the diameter of tumors was approximately 7-8 mm. One group was selected randomly as the control, and the other mice received cetuximab or trastuzumab (0.01 mg to 1.0 mg) as a single intraperitoneal (i.p.) or intravenous (i.v.) injection. Control mice were given equal volumes of PBS. Animals were killed at various intervals after injection of cetuximab or trastuzumab; they received an i.p. injection of EF5 (0.2 ml of 10 mM EF5) 2 hours before they were killed in order to identify hypoxic regions of tumors [
29,
30]. Tumors were removed and embedded with Tissue-Tek OCT (Optimal Cutting Temperature, Sakura Finetek USA Inc., Torrance, CA). The tissue boxes were gently immersed in liquid nitrogen, and then stored at -70°C.
Cryosections were prepared at 10 μm thickness and triple stained to identify cetuximab or trastuzumab, CD31 and EF5. Horseradish peroxidase (HRP) conjugated to anti-human IgG was used to recognize the therapeutic monoclonal antibodies. DAB (3,3'-diaminobenzidine) is a chromogenic substrate for HRP and it deposits a brown specific stain in the presence of HRP. Blood vessels in tissue sections were recognized by the expression of CD31 on endothelial cells. Purified rat anti-mouse CD31 monoclonal antibody was applied at a concentration of 1:500 and left overnight at 4°C. Primary antibody binding was disclosed using a Cy3-conjugated goat anti-rat IgG secondary antibody. Hypoxic regions were recognized by cyanine-5-conjugated mouse anti-EF5 (1/50) antibody.
Fluorescence microscopy
Images were tiled using an Olympus BX50 upright fluorescent microscope linked to a Photometrics CoolSnap HQ2 CCD camera, a motorized X-Y stage connected to a computer preloaded with Media Cybernetics In Vivo and Image Pro-PLUS software (Media Cybernetics, Silver Spring, MD) and a stage controller board. Tumor sections were scanned and tiled under white light and two different filters: (i) images of Cy3 fluorescence of CD31 were visualized using 530 nm to 560 nm excitation and 573 nm to 647 nm emission filter sets, while (ii) images of the Cy5 fluorescence of EF5 were visualized with 630 nm to 650 nm excitation and 665 nm to 695 nm emission filter sets. Composite images of cetuximab, CD31, and EF5 or trastuzumab, CD31 and EF5 were generated using Image Pro PLUS (version 5) and subsequently pseudo-colored. To investigate the distribution of drug in relation to distance from the nearest blood vessel or hypoxic region, images displaying anti-CD31 staining or EF5 staining were converted to black and white binary images: each image was overlayed with the corresponding field of view displaying drug intensity, resulting in an 8-bit black and white image with blood vessels or hypoxic regions identified by an intensity of 255 (white) and drug intensity ranging from 0-254 (gray scale). Areas of interest were selected from each tissue section and were on average 1600 × 1600 μm (0.4 μm2/pixel). Areas of necrosis and staining artifact were excluded.
Distributions of each monoclonal antibody in relation to distance from the nearest blood vessel and the nearest region of hypoxia in the tumor section were quantified utilizing Image Pro software. A minimum signal level just below threshold was set for each tissue section; this was based on an average background reading from regions without staining. The pixel intensity and distance to the nearest vessel or region of hypoxia for all pixels within the selected region of interest above threshold were measured with a customized algorithm. The intensity of cetuximab or trastuzumab signal was represented as mean ± SEM for all pixels at a given distance to the nearest vessel or region of hypoxia and plotted as a function of that distance.
Discussion
Cetuximab and trastuzumab have shown limited efficacy in causing remission in a proportion of patients with metastatic colorectal cancer and breast cancer respectively [
21,
22], while trastuzumab has improved survival of women with ErbB2 positive breast cancer when given as adjuvant therapy after chemotherapy [
32‐
34]. Monoclonal antibodies are large molecules, which are "consumed" by binding to receptors on the cell surface, conditions that might lead to poor penetration of tissue within solid tumors [
28]. Indeed, an early study of the distribution of a radiolabeled monoclonal antibody into multicellular spheroids suggested very slow penetration of tissue, with establishment of a steep concentration gradient [
35], and more recent studies of the penetration of drugs such as doxorubicin (which binds avidly to DNA) have shown quite poor distribution [
23‐
25]. Thus limited distribution of therapeutic agents within solid tumors is a potentially important and relatively neglected cause of drug resistance, especially in the metastatic setting. Here we have used quantitative immunohistochemistry to study the distribution within human tumor xenografts of two therapeutic monoclonal antibodies in clinical use, cetuximab and trastuzumab, to determine if their efficacy might be limited by failure to reach all of the target tumor cells in an effective concentration.
The results of our study show that distribution of both of these therapeutic antibodies is time and dose-dependent. At short intervals after injection of all doses there is a concentration gradient of staining intensity of the antibodies with increasing distance from blood vessels within tumors that strongly express the target receptor. However there is a greater change in the gradient of cetuximab intensity in A431 xenografts than of trastuzumab intensity in 231-H2N xenografts. At moderate and high doses the distribution then becomes more uniform with time, while at lower doses the heterogeneous distribution is retained. Distribution of cetuximab and trastuzumab in relation to hypoxic regions provides a better understanding of the distribution of the antibodies distal to blood vessels. There remains minimal drug distribution to hypoxic tumor cells under all conditions, which is probably due both to limited availability of drug in these regions, and to decreased expression of the ErbB receptors under hypoxic conditions.
The difference in time dependence of the distributions of the monoclonal antibodies as compared to that for doxorubicin, which is relatively independent of time after injection [
24] is most likely due to the half-lives of the drugs in the circulation: doxorubicin has a short initial half-life [
36], such that most penetration from vessels takes place quickly, whereas monoclonal antibodies have a half-life of days [
37‐
39], allowing for a more constant process of tissue penetration.
The gradients of cetuximab intensity in MDA-MB-231 xenografts, which express intermediate levels of ErbB1, are less steep than in A431 xenografts, which express higher levels of ErbB1, and homogeneity of distribution of cetuximab in MDA-MB-231 xenografts was achieved more rapidly. This is probably due to the low receptor binding of cetuximab (i.e. less consumption of drug) by proximal cells in MDA-MB-231 xenografts. Trastuzumab was not identified after injection in MDA-MB-231 xenografts, which express low levels of ErbB2.
Multiple phase I and II clinical trials have established that standard weekly dosing of cetuximab or trastuzumab in humans achieves trough serum concentrations that are usually above 50 μg/ml [
37,
38,
40‐
42]. We did not measure serum concentration of cetuximab or trastuzumab in our mice. Others have reported maximum serum levels of cetuximab of ~65 μg/ml and ~400 μg/ml cetuximab after injection of doses of 0.25 mg and 1.0 mg into mice respectively [
28,
39], similar to those reported in patients. Injection of trastuzumab was reported to lead to serum levels of about 5 ng/ml at 6-24 hours after i.p injection of a single low dose of 0.3 mg/kg into mice [
43]; if pharmacokinetics were linear this would imply doses of ~15 mg/mouse to achieve levels of 10 ug/ml in serum, but it seems unlikely that pharmacokinetics of the two antibodies would differ by such a large amount.
Several other investigators have studied the distribution of various antibodies, or antibody fragments, in tumors. Their results depend on changes in blood flow [
44] the affinity of the antibodies for their targets, but in general these authors have reported problems of heterogeneity of distribution at various times after their administration [
45‐
48]. We were able to identify two other studies of the distribution of trastuzumab (but none of cetuximab) in solid tumors. Dennis et al used intravital microscopy to detect trastuzumab, conjugated to fluorescein isothiocyanate (FITC), in relation to blood vessels of MMTV/HER2 transgenic mice (expressing high levels of ErbB2) that were constrained to grow in a transparent window chamber; they reported perivascular localization of trastuzumab at 24 hours after injection of 10 mg/kg (about 0.25 mg/mouse) [
49]. Their study suggests poorer (or slower) distribution of trastuzumab than the one reported here; a possible reason is higher expression of ErbB2 in the MMTV/HER tumors as compared to the 231-H2N xenografts investigated in our study. Baker et al used similar methods to our own, and investigated time-dependent distributions of trastuzumab in xenografts (that did or did not express ErbB2) after i.p. injection doses in the range of 4-20 mg/kg (about 0.1- 0.5 mg/mouse) [
50]. They found perivascular distribution of drug at 3 h, and that tumor margins reached saturation with trastuzumab more rapidly than the (poorly-vascularized) interior. Drug distribution became more uniform at 24 h as compared to 8 h after injection of 4 mg/kg, but some heterogeneity of trastuzumab distribution was observed in the tumor under all conditions; this is consistent with our finding of poor drug uptake in hypoxic tumor regions.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
CL designed and performed all the experiments and drafted the manuscript. IT conceived of the study, obtained funding for it and participated in its design and coordination and drafted the manuscript. Both authors read and approved the final manuscript.