944 PART III Pathology metastases with a replacement growth pattern expressed the hypoxia marker CA IX or had fibrin depositions at the tumor–liver interface (Table II). Probably, the well- described mechanisms of invasive tumor growth, such as fibroblast–myofibroblast transdifferentiation, TGFb path- ways, proinflammatory signaling, hyaluronic acid action, and hypoxia-responsive gene activation, are not involved. The search for gene sets that are responsible for the pheno- type of nonangiogenesis-dependent colonization of a distant site is ongoing. Selective induction of apoptosis in hepatocytes at the interface by tumor cells might be one of the mechanisms of growth of blood-vessel-coopting metastases. The other growth patterns in the liver were characterized by destruction of the architecture of the liver parenchyma and were associated with desmoplasia and new blood vessel formation. The metastases were (desmoplastic growth pattern) or were not (pushing growth pattern) sur- rounded by a fibrotic capsule. The consequences of this heterogeneity of human liver metastases are the limited value of model systems that selec- tively reproduce the well-studied angiogenesis-dependent growth of metastases and the difficulties in analyzing the results of clinical trials applying biomodulatory drugs. Imaging of the vascular flow and leakage by contrast- enhanced CT or MR might be helpful in selecting patients with angiogenic versus nonangiogenic liver metastases. The existence of different growth patterns also stresses the supe- rior value of the endothelial cell proliferation (ECP) fraction for angiogenesis quantification compared to microvessel density [1]. Liver metastases with a replacement growth pat- tern indeed have a high microvessel density as a result of cooption of the liver vasculature but have a low ECP due to lack of ongoing angiogenesis. Growth Patterns and Angiogenesis in Primary Lung Cancer Bronchiolo-alveolar lung adenocarcinomas have a typi- cal “lepidic” growth pattern: Tumor cells replace the normal pneumocytes, thereby preserving the stromal component of the alveolar wall and coopting the capillary blood vessels. The structure of the lung parenchyma remains intact, which probably explains the growth of satellite lesions in the lung due to transportation of tumor cells by airflow. This nonan- giogenic growth has been shown to be present in more com- mon types of nonsmall-cell lung carcinomas (NSCLC) and in lung metastases [3]. In 16 percent of 500 NSCLC, the tumors were growing without parenchymal destruction and without the formation of desmoplastic stroma. In this “alve- olar” growth pattern, tumor cells were filling the alveoli as solid nests. The only blood vessels present were those in the preserved alveolar septa. The coopted blood vessels did not express the integrin alpha-v-beta-3 necessary for angiogen- esis. In another study, outcome of 283 patients with opera- ble NSCLC was studied and linked to the growth pattern of the lung tumors (Sardari Nia P. et al., 2004). Whereas the majority of the patients had a tumor with associated desmo- plasia and angiogenesis in which the alveolar architecture of the lung was not preserved, 18 percent of the patients had a NSCLC with an alveolar, nonangiogenic growth at the tumor–lung interface. The alveolar growth pattern was not associated with a specific histiotype (30% adenocarcinoma, 40% squamous cell carcinoma, and 30% large cell carci- noma and other types). In univariate analysis, T-stage, N- stage, and growth pattern predicted overall and disease-free survival. Multiple logistic regression showed that TN-stage and growth pattern were independent prognostic factors. Hazard ratios for the alveolar growth pattern were 2.0 (95% confidence interval: 1.3 to 3.2) for overall survival and 2.4 (95% confidence interval: 1.5 to 3.8) for disease-free sur- vival, if compared to NSCLC with associated desmoplasia and angiogenesis. When stage I tumors were analyzed sepa- rately (174 patients), growth pattern retained its independent prognostic value. This confirms the study of Pastorino et al. [8], which described 137 pT1N0 patients. Both a nonangio- genic type of vascular pattern and epidermal growth factor receptor expression were associated with a poorer survival rate. Assessing the growth pattern in primary NSCLC is potentially important since it can predict prognosis, but probably also the response to different treatment modalities. The growth pattern is indeed an integrative parameter con- taining information of the relationship between tumor cells and stromal cells. Surgical pathologists can easily determine the growth pattern on a standard hematoxylin–eosin stained tissue section and integrate it in the pathology report. Another consequence of the growth patterns of lung carci- nomas is that the prognostic value of microvessel density in NSCLC can only be investigated within the subgroup of angiogenic tumors. Figure 2 Replacement growth pattern in a liver metastasis of a breast adenocarcinoma. The tumor cells (left) are replacing the hepatocytes in the liver plates (right), thereby coopting the sinusoidal blood vessels. There is close apposition of tumor cells and hepatocytes at the tumor–liver interface (arrows) without induction of inflammation or fibrosis. CHAPTER 139 Tumor Growth Patterns and Angiogenesis 945 Conclusion Different growth patterns of primary and metastatic tumors are a reflection of different interactions of the cancer cells with the surrounding tissue structures. The observation of nonangiogenic growth patterns in human carcinomas challenges the hypothesis that tumor growth is always dependent on angiogenesis. It would be more correct to say that neoplastic growth depends on an adequate blood supply. If this can be obtained from a vascular bed that already exists, the tumor can grow without the formation of new blood vessels. Glossary Alveolar growth pattern: Nonangiogenic growth pattern described in primary nonsmall-cell lung cancer and in pulmonary metastases; tumor cells fill the alveoli and exploit the interalveolar capillaries for their blood supply. Fibrotic focus: Focus of exaggerated reactive tumor stroma formation in the center of a carcinoma consisting of collagen, a variable number of fibroblasts, blood vessels, and inflammatory cells; practical histopatholog- ical surrogate marker of hypoxia-driven angiogenesis in breast cancer. Nonangiogenic growth: Tumor growth without induction of angio- genesis in which tumor cells obtain adequate blood supply by exploiting a preexisting vascular bed. Replacement growth pattern: Nonangiogenic growth pattern described in liver metastases; tumor cells replace the hepatocytes in the liver plates and exploit the sinusoidal blood vessels for their blood supply. References 1. Vermeulen, P. B., Gasparini, G., Fox, S. B., Colpaert, C. G., Marson, L., Gion, M., Beliën, J. A. M., de Waal, R. M. W., Van Marck, E. A., Magnani, E., Weidner, N., Harris, A. L., and Dirix, L. Y. (2002). Second international consensus on the methodology and criteria of evaluation of angiogenesis quantification in solid human tumours. Eur. J. Cancer 38, 1564–1579. Guidelines for the estimation of ongoing angiogenesis and the amount of blood vessels in a solid tumor, integrating new concepts and mechanisms of tumor vascularization. Table II Comparison of Glandular Differentiation, Fibrin Deposition, CAIX Expression, and the Macrophage Content of Breast Cancer and Colorectal Cancer Liver Metastases. Breast Colorectal Glandular differentiation: (n = 45) (n = 28) 11 (3%) 21 (75%) 28 (17%) 5 (18%) 3 36 (80%) 2 (7%) p < 0.0001 Fibrin, central: (n = 37) (n = 24) 0 16 (43%) 6 (25%) 1 10 (27%) 4 (17%) 22 (6%) 4 (17%) 39 (24%) 10 (41%) p = 0.15 Fibrin, interface: (n = 38) (n = 25) 0 30 (79%) 11 (44%) 16 (15%) 10 (40%) 21 (3%) 1 (4%) 31 (3%) 3 (12%) p = 0.037 CA IX, central: (n = 44) (n = 24) Absent 32 (73%) 1 (4%) Present 12 (27%) 23 (96%) p < 0.0001 CA IX, interface: (n = 45) (n = 24) Absent 38 (84%) 11 (46%) Present 7 (16%) 13 (54%) p = 0.002 Global CA IX score: 14.4 ± 8.6 (0) 74.5 ± 14.9 (52.5) p < 0.0001 Macrophage count: 4.57 ± 0.28 (4.25) 8.25 ± 0.60 (7.50) p < 0.0001 Differences in categorical variables are validated by two-tail Fisher’s exact testing. Continuous variables are expressed as mean ± standard error (median). Differences are validated by Wilcoxon testing. Glandular differentiation score: 1, > 75% tubule formation; 2, 10–75% tubule formation; 3, < 10% tubule forma- tion. Fibrin deposition: Detected immunohistochemically with NYB.T2G1monoclonal antibody. 0, No staining; 1, minimal staining; 2, moderate staining; 3, extensive staining. Global CA IX score: Carbonic anhydrase IX is an endogenous marker of hypoxia. Its expression is semiquantita- tively scored as the product of the percentage of immunostained cells with an immunostaining intensity score rang- ing from 0 (no staining) to 3 (strong staining). CA IX: absent, no immunostaining; present, immunostaining in any percentage of tumor cells. Macrophage count: the relative area occupied by CD68 immunostained macrophages, quantified with the Chalkley morphometric point counting method. 946 PART III Pathology 2. Hasebe, T., Tsuda, H., Hirohashi, S., Shimosato, Y., Iwai, M., Imoto, S., and Mukai, K. (1996). Fibrotic focus in invasive ductal carcinoma: An indicator of high tumor aggressiveness. Jpn. J. Cancer Res. 87, 385–394. First report presenting the fibrotic focus as a prognostic factor in invasive breast carcinoma. 3. Pezzella, F., Pastorino, U., Tagliabue, E., Andreola, S., Sozzi, G., Gasparini, G., Menard, S., Gatter, K. C., Harris, A. L., Fox, S., Buyse, M., Pilotti, S., Pierotti, M., and Rilke, F. (1997). Non-small-cell lung carcinoma tumor growth without morphological evidence of neo- angiogenesis. Am. J. Pathol. 151, 1417–1423. Investigation of the pat- tern of vascularization in a series of 500 lung carcinomas with the description of an alveolar growth pattern characterized by the lack of parenchymal destruction and absence of both tumor associated stroma and new vessels. 4. Vermeulen, P. B., Colpaert, C. G., Salgado, R., Royers, R., Hellemans, H., Van de Heuvel, E., Goovaerts, G., Dirix, L. Y., and Van Marck, E. A. (2001). Liver metastases from colorectal adenocarcinomas grow in three patterns with different angiogenesis and desmoplasia. J. Pathol. 195, 336–342. Description of different patterns of vascularization in liver metastases with identification of a growth pattern characterized by tumor cells replacing the hepatocytes in the liver plates and exploiting the preexisting sinusoidal blood vessels. 5. Colpaert, C. G., Vermeulen, P. B., Fox, S. B., Harris, A. L., Dirix, L. Y., and Van Marck, E. A. (2003). The presence of a fibrotic focus in inva- sive breast carcinoma correlates with the expression of carbonic anhy- drase IX and is a marker of hypoxia and poor prognosis. Breast Cancer Res. Treat. 81, 137–147. 6. Colpaert, C. G., Vermeulen, P. B., van Beest, P., Goovaerts, G., Weyler, J., Van Dam, P., Dirix, L. Y., and Van Marck, E. A. (2001). Intratumoral hypoxia resulting in the presence of a fibrotic focus is an independent predictor of early distant relapse in lymph node-negative breast cancer patients. Histopathology 39, 416–426. 7. Colpaert, C. G., Vermeulen, P. B., van Beest, P., Goovaerts, G., Dirix, L. Y., Harris, A. L., and Van Marck, E. A. (2003). Cutaneous breast can- cer deposits show distinct growth patterns with different degrees of angiogenesis, hypoxia and fibrin deposition. Histopathology 42, 530–540. 8. Pastorino, U., Andreola, S., Tagliabue, E., Pezzella, F., Incarbone, M., Sozzi, G., Buyse, M., Menard, S., Pierotti, M., and Rilke, F. (1997). Immunocytochemical markers in Stage I lung cancer: Relevance to prognosis. J. Clin. Oncol. 15, 2858–2865. Capsule Biography Cecile G. Colpaert is a pathologist working in a teaching hospital. Her main interest is breast cancer and the application of research findings from the field of tumor biology in diagnostic pathology practice. Peter B. Vermeulen is a diagnostic pathologist doing translational breast cancer research mainly focused on angiogenesis and tumor–stroma interactions. CHAPTER 140 Breast Cancer Resistance Protein in Microvessel Endothelium Hiran C. Cooray and Margery A. Barrand University of Cambridge, Cambridge, United Kingdom organisms, including bacteria, plants, and animals. Collec- tively, they are responsible for transporting a multitude of diverse substrates including sugars, ions, lipids and phos- pholipids, peptides, bile acids, sterols, pigments, and xeno- biotics across membranes, thus affecting the distribution of molecules at subcellular, cellular, and tissue levels. There are many different ABC transporters. The mammalian ones have now been classified into subfamilies, termed ABCA through ABCG (http://www.humanabc.org/). In this scheme, BCRP is named as ABCG2. The reader is directed to http://www.ncbi.nlm.nih.gov/books/bookres. fcgi/mono_001/mono_001.pdf and http://arjournals. annualreviews.org/doi/pdf/10.1146/annurev.biochem. 71.102301.093055 for two comprehensive reviews on ABC transporters. Structural Organization of ABC Transporters Many of the ABC transporters are constructed as a tan- dem repeat of a basic unit containing two domains: an N- terminal transmembrane domain (TD) of 6 to 11 a-helices that provide substrate specificity to the protein, and a C- terminal nucleotide-binding domain (NBD) located in the cytoplasm that binds and cleaves ATP in order to generate the energy for substrate transport. Binding and subsequent hydrolysis of ATP result in a conformational change that causes the substrate to be translocated across the membrane. This general structure is typified by the mammalian mul- tidrug transporter, P-glycoprotein, otherwise named ABCB1 (Figure 1). Some ABC transporters including the Multidrug Resistance-associated Protein, MRP1 (ABCC1), have an additional N-terminal TD. However others contain only one NBD and one TD. This “half transporter” structure is Introduction Breast Cancer Resistance Protein (BCRP) is a transporter recently identified as a member of the ATP Binding Cassette (ABC) superfamily of transmembrane proteins (discussed in the following section). It was recognized initially in several drug-resistant tumor cell lines (as discussed later) and was subsequently identified in certain tumor tissues where its presence has been putatively associated with poor clinical response to chemotherapy. However, BCRP is found on many normal, that is, nonmalignant, cells at a number of dif- ferent sites in the body (see later discussion). These include endothelial cells lining various vasculature. Clues to the possible physiological role or roles of BCRP in these loca- tions may be derived from the many studies that are now being conducted, which examine its mechanisms of action and its substrate (discussed later) and inhibitor profiles in different cell systems or that analyze its influence on the dis- tribution of drugs in whole animals. ABC Transporters Transporters belonging to the ATP-Binding Cassette (or ABC) family of proteins have the ability to transport sub- strates across cellular membranes against a concentration gradient using the energy of ATP hydrolysis. They are so called because of their distinctive ATP-binding domains, which contain highly conserved sequences, Walker A and Walker B and an additional ABC “signature” sequence. ABC proteins constitute the largest subclass of transmem- brane proteins and are expressed ubiquitously in all living Copyright © 2006, Elsevier Science (USA). 947 All rights reserved. 948 PART III Pathology exemplified by members of several ABC subfamilies includ- ing the ABCG subfamily of which BCRP is a member. In addition, members of this subfamily have their TD and NBD in reverse orientation (see Figure 1); hence, BCRP is referred to as a “reverse” half-transporter. Half transporters have to dimerize with a partner protein (either with itself to form a homodimer or with another protein to form a het- erodimer) in order to be functional. Currently, the bulk of the evidence points to BCRP being a homodimer. Interest- ingly however, BCRP shows closest homology with the white, brown, and scarlet proteins that heterodimerize (white/brown and white/scarlet) to transport pigment pre- cursors (guanine and tryptophan) in the Drosophila eye. ABC Transporters Associated with Multidrug Resistance Drug resistance can be a serious obstacle to successful anticancer treatment with tumors often failing to respond either to initial chemotherapy (intrinsic resistance) or to subsequent rounds of treatment (acquired resistance). Stud- ies conducted in the laboratory on tumor cell lines cultured in the presence of cytotoxic drugs reveal that resistance can develop, not only to the selecting drug, but to a number of structurally and functionally dissimilar drugs as well, hence providing multidrug resistance (MDR). This MDR Figure 1 Putative membrane topology of the three main multidrug transporters. BCRP consists of one transmembrane domain (TD) and one nucleotide binding domain (NBD) and is termed a half-transporter. P-glycoprotein, like many ABC proteins, is a full transporter with a TD 1 -NBD 1 - TD 2 -NBD 2 structure. Several of the MRP family members, including MRP1, have an additional N-terminal TD linked to the P-gp-like core by a linker region (L 0 ). Note that the TD and NBD in BCRP are in reverse orientation to those of the other two ABC proteins. (N and C refer to the N and C termini of the transporter, respectively.) (see color insert) CHAPTER 140 Breast Cancer Resistance Protein in Microvessel Endothelium 949 phenomenon may involve several different types of mecha- nisms, but a common cause is the presence of multidrug ABC transporters, which prevent access of the drugs to their intracellular targets sites. They accomplish this task by either effluxing the drug out of the cell via the plasma mem- brane, or by sequestering the drug within intracellular organelles such as the endoplasmic reticulum, lysosome, or peroxisome. Identification of BCRP as a Multidrug Transporter P-gp was the first ABC transporter to be associated with MDR (around the mid-1970s) with the MRPs being later discoveries (MRP1 sequenced in 1992). It was not until 1998 that BCRP was revealed as another MDR-associated ABC transporter. It was becoming apparent that there were certain tumor cell lines that showed resistance to several drugs (mainly mitoxantrone, bisantrene, topotecan, and dox- orubicin) yet did not overexpress P-glycoprotein or MRPs. Recognition of this atypical, non-P-gp, non-MRP resistance phenotype initiated a search for another multidrug trans- porter, leading to the ultimate discovery of BCRP, otherwise termed Mitoxantrone Resistance Protein (MXR) or ABC Transporter in Placenta (ABCP). The different names of this protein derive from the fact that it was characterized and cloned from three different sources in independent laborato- ries at about the same time: from a multidrug-resistant breast cancer cell line selected in doxorubicin (MCF7/ AdrVp3000, hence BCRP), from a colon cancer line selected in mitoxantrone (S1-M1-80, hence MXR), and from human placenta (hence ABCP) [1]. Though commonly called BCRP by virtue of its initial discovery in a drug-selected breast cancer cell line, it is not clear that the protein is actually often overexpressed in breast cancers in vivo. Indeed only weak BCRP expression has been found in breast tumors. There are, however, many different human tumors in which BCRP expression has been clearly demonstrated, including tumors of the kidney, ovary, stomach, colon, thyroid, brain, endometrium, and testis; squamous tumors (lung, head and neck, and esophagus); soft tissue sarcomas; pheochromocytomas; and hepatocarci- nomas. This may reflect the distribution of BCRP in normal tissues (see later discussion). Substrate and Inhibitor Profiles of BCRP Compared to Other Multidrug Transporters The ABC transporters associated with MDR vary some- what in their mechanisms of action, substrate and inhibitor profiles, and in their tissue locations. Nevertheless there is a significant overlap in these characteristics between the transporters (Figure 2). Functional studies on both BCRP overexpressing drug-selected and BCRP-transfected cell lines show that the transporter can confer resistance to anthracyclines (dox- orubicin, daunorubicin), anthracenediones (mitoxantrone), camptothecins (topotecan, irinotecan, and its active metabo- lite, SN-38), and etoposide, but not to vincristine, taxol, or colchicine, which are classical P-gp substrates. Substrates currently recognized to be transported by BCRP are shown in Table I. Figure 2 Actions of multidrug transporters BCRP, P-gp, and MRP1 in efflux of substances from cells. Drugs can enter and leave cells by passive diffusion along a concentration gradient. Multidrug transporters provide a second route for drug exit and can drive drugs out of the cell against a con- centration gradient by exploiting the energy of ATP hydrolysis. This additional efflux reduces drug concentrations inside cells to sublethal levels. P-gp and BCRP (in the form of a dimer) can efflux drugs unmodified (mainly hydrophobic, amphipathic compounds). The MRPs require the presence of reduced glutathione (GSH) to transport unmodified drugs (predominantly organic anions) but can transport drugs following their conjugation (GST, Glutathione transferase). (see color insert) 950 PART III Pathology A point mutation affecting substrate specificity has recently been reported in BCRP. Though wild-type BCRP has an arginine at the 482nd position (at the start of the third transmembrane segment), BCRP overexpressed in certain drug-selected cell lines was shown to contain either a glycine or threonine at this site. This point mutation causes a paradigm shift in the protein’s substrate specificity—wild- type BCRP is incapable of effluxing the fluorescent dye rho- damine 123 or anthracycline drugs such as doxorubicin, but the mutants can handle both these substrates. Conversely, only wild-type BCRP transports the antifolate cytotoxic methotrexate [1]. Polymorphisms of the BCRP gene have also been described in human populations. Subcellular Location of BCRP BCRP seems to be predominantly localized to the plasma membrane of both drug-selected and transfected cell lines. This sets it apart from other half-transporters that are local- ized mainly to intracellular membranes such as the mito- chondrion (ABCB7), the peroxisome (ALD subfamily), and the endoplasmic reticulum (Tap1/Tap2). Such a location for BCRP is consistent with a putative role in the efflux of sub- strates from the cell. Furthermore, it is apparent in polarized cell lines such as BCRP-transfected MDCK-II Madine- Darby canine kidney cells that the transporter becomes localized primarily to the apical aspect of the plasma mem- brane, where it mediates the translocation of substrates from basal to apical side. However, a role for BCRP in the intra- cellular trafficking of molecules cannot be ruled out as some immunocytochemical studies have reported perinuclear staining for BCRP in several topotecan- and mitoxantrone- resistant cell lines [1]. Tissue Distribution of BCRP Compared to Other Multidrug Transporters The apical siting of BCRP on polarized cells is of partic- ular relevance to the possible role or roles of BCRP in nor- mal tissues. The protein is found in many tissues, including barrier sites, as outlined in Table II. The highest BCRP expression is found in the placenta on the syncytiotrophoblast facing the maternal circulation. This suggests a role for the protein in the elimination of sub- strates from the fetus. This has been established for mouse Bcrp1; in both wild-type and P-gp knockout mice, inhibition of Bcrp1 by GF120918 (a common inhibitor of human and mouse P-gp and BCRP) resulted in at least a twofold increase in the fetal uptake of orally administered topotecan, a BCRP substrate. BCRP is also expressed at more modest levels in the colon, small intestine, liver, ovary, and breast, where it may be concerned with elimination of material from these tissues. In a recent clinical study utilizing GF120918, it was shown that the oral bioavailability of topotecan more than doubled (from 40% to 97%) when the drug was coadministered with the inhibitor, thus underlining the functional significance of BCRP expression in the intestine. Table I Substrate Profile of BCRP Compared with P-gp and MRP1. Substrate BCRP P-gp MRP1 Paclitaxel ✓ Verapamil ✓ Colchicine ✓ Vinblastine ✓✓ Etoposide (VP-16) ✓✓✓ Daunorubicin ✓✓✓ Doxorubicin ✓✓✓ Epirubicin ✓✓✓ Mitoxantrone ✓✓✓ Methotrexate ✓ a ✓✓ Prazosin ✓✓ Topotecan ✓✓ Bisantrene ✓✓ Rhodamine-123 ✓✓ Flavopiridol ✓ Lysotracker Green ✓ SN-38 ✓ E 2 -GLU ✓✓ Estrone 3-sulfate ✓✓ LTC 4 ✓ GSH ✓ E 2 -GLU, 17b-Estradiol 17-(b-D-glucuronide); GSH, reduced glu- tathione; LTC 4 , leukotriene C 4 . Rhodamine 123 and Lysotracker Green are fluorescent dyes. a Methotrexate is effluxed only by the wild-type BCRP. Table II Tissue Distribution and Putative Functions of BCRP at These Locations. Localization Putative function Placenta—syncytiotrophoblast Protection of fetus, excretion of substrates Apical membrane of epithelium Reduced uptake/excretion of of small intestine and colon substrates into maternal circulation Liver canalicular membrane Excretion of substrates by the liver into bile Apical membrane of lobules Unknown and lactiferous ducts of breast Endothelium of capillaries and Unknown veins “Side” population of Unknown hematopoietic stem cells CHAPTER 140 Breast Cancer Resistance Protein in Microvessel Endothelium 951 This distribution of BCRP shows similarities to that of the multidrug transporter P-glycoprotein, which is also expressed in various epithelia, particularly in organs associ- ated with drug absorption and disposition, such as hepato- cyte canalicular membrane and the intestinal mucosa. P-gp is thought to provide a first line of defense against the entry of many types of xenobiotics into the body. Knockout mice deficient in functional P-gp, although viable, fertile, and without obvious histological or developmental abnormali- ties, show significantly altered pharmacokinetics (and toxicity of several drugs) [2]. The third subfamily of multidrug transporters, the MRPs, are widely distributed throughout the body in tissues including the choroid plexus, oral mucosa, small intestine, testis, and respiratory tract. Because there are several MRP homologs with overlapping substrate specificities, the importance of each for the elimi- nation of particular substances is difficult to assess. Both BCRP and P-gp are to be found on the endothelium lining the blood–brain barrier (see later discussion). The presence of multidrug transporters at such barrier sites creates “pharmacological sanctuaries” within the body, per- mitting certain organs and tissues to function in relative isolation from the rest of the body. Indeed, in P-gp knockout mice, the integrity of the blood–brain barrier is shown to be significantly compromised, with much higher brain penetra- tion of P-gp substrates such as vinblastine and ivermectin being demonstrated. The relevance of BCRP at these sites is still under investigation (see later discussion). The generation of the Bcrp1 knockout mouse [3] has thrown new light on the putative physiological function of this transporter. Though these mice were anatomically nor- mal and fertile, a defect was seen in their ability to handle a metabolite of chlorophyll, pheophorbide a, resulting in severe phototoxicity in mice exposed to light. They also exhibited a previously uncharacterized form of porphyria. Thus it became known that BCRP performs an essential function at the gut epithelium in effluxing toxic products of chlorophyll metabolism. BCRP knockout mice generated independently by Zhou et al. [4] were used to demonstrate that this transporter, rather than P-gp, is responsible for the dye efflux in the cells. This allows analysis of the “side- population,” enriched in murine hematopoietic stem cells, which have high bone-marrow repopulating activity. The role BCRP plays at this location is still to be elucidated. Expression of BCRP in Endothelia of Normal Tissues and of Tumors BCRP differs from P-gp in being expressed on the endothelial lining of vascular beds in many tissues, not just at the blood-brain barrier. Interestingly, BCRP is evident in venules and capillaries (see Table II) but not in arterioles [5]. Hence the transporter is distributed in the regions of the vasculature where the bulk of the exchange of materials between blood and tissues occurs. On endothelial cells of vasculature-supplying tumors (for example, testicular germ-cell tumors, endometrial, ovarian and colon carcino- mas, and brain tumors), antibody staining for BCRP has been described as moderate to strong, stronger indeed than on the vascular endothelium in the surrounding normal regions [6]. This raises the interesting possibility that BCRP expression is perhaps upregulated in the endothelium of blood vessels during neoplastic vasculogenesis. Localization of BCRP in the Specialized Endothelium of the Blood–Brain Barrier The presence of multidrug transporters is of particular importance in vascular endothelial cells at special barrier sites such as the blood–brain and blood–testis barriers. Here the vessels possess tight junctions that place severe restric- tions on the free paracellular diffusion of many substances seen in peripheral endothelia. Recent studies have explored the localization of BCRP in human brain material using fresh-frozen samples of both normal and tumor brain (meningiomas and gliomas) [7]. Western blot results show a higher degree of expression of BCRP protein in the gliomas over the normal and menin- gioma samples. It could be seen by immunostaining that BCRP is primarily localized to blood vessels within the brain. In the case of two meningioma samples, notable heterogeneous staining for BCRP was seen in brain parenchymal cells in addition to endothelial cells. Diestra et al. [6] also reported a higher expression of BCRP in several unspecified brain tumors over normal brain parenchyma using immunohistochemical staining with a well-characterized anti-BCRP antibody. By exploiting the powerful resolving capabilities of the confocal microscope, it has been possible to gain some understanding of the subcellular distribution of BCRP within brain microvessels. Utilizing the fact that the brain endothelial glucose transporter GLUT-1 is localized on both sides of brain endothelial cells (both luminal and abluminal membranes), dual-staining with antibodies for GLUT-1 and for BCRP revealed the main sites of BCRP expression in microvessels in both normal and tumor brain sections. The distribution of BCRP staining was seen to be inner to that of GLUT-1 in all microvessels viewed, which suggests that BCRP is localized toward the luminal membrane of human brain endothelial cells in the in vivo blood–brain barrier [7]. It is probable therefore that BCRP, localized strategically at the luminal membrane of endothelial cells, has a protective function at the blood–brain barrier in limiting entry of sub- strates into the brain. P-gp also has been localized to the luminal aspect of the brain capillary endothelium. It is already well documented that in this situation it performs what has been described as a “gatekeeper” role at the blood–brain barrier, pumping out a variety of xenobiotics that would otherwise gain access to the brain via the transcellular pathway due to their lipophilicity [2]. The number of drugs known to be excluded from the brain by P-gp is large, ranging from nonsedating 952 PART III Pathology antihistamines, antiepileptics, and beta-blockers to anti-HIV reverse transcriptase inhibitors. What additional protection BCRP may bring to bear is as yet not well defined and would require the advent of knockout mice lacking several of the MDR transporters or use of combinations of specific inhibitors so that the influence of BCRP can be distin- guished from that of P-gp in vivo. The presence and importance of MRPs at the blood–brain barrier is even less clear. This is due both to the multiplicity of transporters in this family and to existing controversies in the literature. In contrast to BCRP, MRP1 is known to be functionally active in vivo at the epithelium of the choroid plexus, regulating the distribution of several xenobiotics into the CSF. But it has not been definitively localized in vivo at the blood–brain barrier. MRP1 does, however, become upregulated in cultured brain endothelial cells [8]. This has allowed its functionality to be explored in vitro in brain endothelial cells cultured from several sources including human brain. In the case of MRP2, results of recent studies using in vivo microdialysis hint at a functional role for this protein at the rat blood–brain barrier, limiting the brain uptake of the anticonvul- sant phenytoin. However, these observations need further investigation. A homolog of BCRP has been described in porcine brain endothelial cells by Eisenblatter et al. [9]. This protein, named Brain Multidrug-Resistance Protein (BMDP), shows 86 percent amino acid identity with human BCRP and is predicted to have the typical architecture of a “reverse” half- transporter. BMDP was shown, using immunohistochem- istry, to be localized to the cell membrane of cultured porcine brain endothelial cells. A blood vessel location in vivo for the message was inferred from RNA isolation experiments in which the mRNA of BMDP appeared to be concentrated in the brain microvessels, with the levels of transcript higher in isolated capillaries than in homogenized brain tissue. High levels of BMDP transcript were also detectable in cultured porcine brain endothelial cells. These appeared approximately 30 times higher than equivalent P-gp expres- sion, suggesting that at least in the porcine endothelium, BMDP plays a more prominent role than P-gp. This was corroborated via functional studies using the radiolabeled substrate 3 H-daunorubicin (a substrate common to both P-gp and BCRP) performed on cultured porcine brain endothelial cells grown as monolayers—GF120918 (which inhibits both P-gp and BCRP) abrogated almost completely the transport of daunorubicin from the basolateral to the api- cal side of the porcine brain endothelial cell monolayer. However, specific P-gp inhibitors gave only moderate inhibition. This strongly suggests that the contribution by BMDP to transport of substrates across the porcine blood–brain barrier may be greater than by P-gp. These studies are the first to report functional BCRP in cells derived from the blood–brain barrier. Many questions still remain regarding the in vivo func- tionality of BCRP. Its ubiquitous expression in the endothe- lium of veins and capillaries of every tissue so far examined suggest that it might efflux substrates that are potentially toxic to many tissues but are incapable of passing between endothelial cells. In particular, its expression at the blood–brain barrier may also be of paramount significance to limiting the brain penetration of substrates. The vast body of research available on P-gp and members of the MRP family has pointed to a number of specific–roles performed by these transporters at various sites in the body. There is still much to be learned about BCRP and the function it may perform in the microvessel endothelium. Acknowledgments The authors thank the Cancer Research Campaign for their contribu- tions to the authors’own research work and the Cambridge Commonwealth Trust for assistance toward a studentship for HCC, who also holds an award from Universities UK. References 1. Allen, J. D., and Schinkel, A. H. (2002). Multidrug resistance and phar- macological protection mediated by the Breast Cancer Resistance Pro- tein (BCRP/ABCG2). Mol. Can. Ther. 1, 427–434. A very useful and comprehensive recent review on all aspects of BCRP. 2. Schinkel, A. H. (1999). P-Glycoprotein, a gatekeeper at the blood–brain barrier. Adv. Drug Deliv. Rev. 36, 179–194. 3. Jonker, J. W., Buitelaar, M., Wagenaar, E., van der Valk, M. A., Scheffer, G. L., Scheper, R. J., Plosch, T., Kuipers, F., Oude Elferink, R. P. J., Rosing, H., Beijnen, J. H., and Schinkel, A. H. (2002). The breast cancer resistance protein protects against a major chlorophyll- derived dietary phototoxin and protoporphyria. Proc. Natl. Acad. Sci. USA 99, 15649–15654. 4. Zhou, S., Morris, J. J., Barnes, Y., Lan, L., Schuetz, J. D., and Sorrentino, B. P. (2002). BCRP1 gene expression is required for normal numbers of side-population stem cells in mice, and confers relative protection to mitoxantrone in hematopoietic cells in vivo. Proc. Natl. Acad. Sci. USA 99, 12339–12344. 5. Maliepaard, M., Scheffer, G. L., Faneyte, I. F., van Gastelen M. A., Pijnenborg, A. C. L. M., Schinkel, A. H., van de Vijver, M. J., Scheper, R. J., and Schellens, J. H. M. (2001). Subcellular localization and dis- tribution of the breast cancer resistance protein transporter in normal human tissues. Cancer Res. 61, 3458–3464. 6. Diestra, J. E., Scheffer, G. L., Catal, I., Maliepaard, M., Schellens, J. H. M., and Scheper, R. J. (2002). Frequent expression of the multidrug resistance associated protein BCRP/MXR/ABCP/ABCG2 in human tumors detected by the BXP-21 monoclonal antibody in paraffin- embedded material. J. Pathol. 198, 213–219. 7. Cooray, H. C., Blackmore, C. G., Maskell, L., and Barrand, M. A. (2002). Localization of Breast Cancer Resistance Protein in microvessel endothelium of human brain. Neuroreport 13, 2059–2063. Establishes a luminal localization for BCRP at the human blood–brain barrier. 8. Seetharaman, S., Barrand, M. A., Maskell, L., and Scheper, R. J. (1998). Multidrug resistance–related transport proteins in isolated human brain microvessels and in cells cultured from these isolates. J. Neurochem. 70, 1151–1159. 9. Eisenblatter, T., Huwel, S., and Galla, H. J. (2003). Characterisation of the brain multidrug resistance protein (BMDP/ABCG2/BCRP) expressed at the blood–brain barrier. Brain Res. 971, 221–231. The first paper to report a porcine homolog of BCRP highly expressed in cultured endothelial cells derived from the blood–brain barrier. CHAPTER 140 Breast Cancer Resistance Protein in Microvessel Endothelium 953 Further Reading Litman, T., Brangi, M., Hudson, E., Fetsch, P., Abati, A., Ross, D. D., Miyake, K., Resau, J. H., and Bates, S. E. (2000). The multidrug- resistant phenotype associated with overexpression of the new ABC half-transporter, MXR (ABCG2). J. Cell. Sci. 113, 2011–2021. Capsule Biography Hiran C. Cooray is in the final year of his doctorate studying the expres- sion and putative roles of BCRP in human brain material and in cultured endothelial cells. Dr. Margery Barrand is a Senior Lecturer in the Department of Phar- macology in the University of Cambridge. Her group has strong research interests in multidrug transporters and, in particular, transport systems at the blood–brain barrier. [...]... N., Gerber, H P., and LeCouter, J (2003) The biology of VEGF and its receptors Nat Med 9, 6 69 676 Robert R Langley received his Ph.D from the Department of Molecular and Cellular Physiology, Louisiana Health Sciences Center, Shreveport, Louisiana, in 199 9 He received postdoctoral training in the Department of Cancer Biology at the University of Texas M.D Anderson Cancer Center in Houston, Texas, from... endothelial P- and E-selectin P-selectin and von Willebrand factor are released from Weibel–Palade bodies of endothelial cells and are presented on the endothelial surface within minutes following stimulation [6] Expression of E-selectin and a second phase of P-selectin expression depend on de novo protein synthesis and occur hours after the primary stimulus The main ligand for endothelial P-selectin... (TNF, IL-1, IFN) and angiogenic growth factors (VEGF), platelet–endothelial interactions are mediated by endothelial and platelet-specific adhesion molecules Platelet interaction with the endothelial surface and subendothelial matrix occurs in a sequence of initial short-timed contact (rolling) followed by adhesion and 97 5 Copyright © 2006, Elsevier Science (USA) All rights reserved 97 6 PART III Pathology. .. from tumor cells and lead to the activation and secretion of matrix metalloproteinase -9 (MMP -9 ) by hematopoietic and stromal elements in the bone marrow MMP -9 activation, in turn, leads to liberation of soluble KIT ligand that promotes cell proliferation and also directs the transfer of these cells into the peripheral circulation Direct evidence that HSCs and EPCs contribute to tumor neovascularization... versus baseline (Kruskal–Wallis test and Wilcoxon test) Bibliography 1 Möhle, R., Green, D., Moore, M A., Nachman, R L., and Rafii, S ( 199 7) Constitutive production and thrombin-induced release of vascular endothelial growth factor by human megakaryocytes and platelets Proc Natl Acad Sci USA 94 , 663–668 2 Pinedo, H M., Verheul, H M., D’Amato, R J., and Folkman, J ( 199 8) Involvement of platelets in tumour... regression of established primary tumors and metastatic lesions McIntosh, D P., Tan, X Y., Oh, P., and Schnitzer, J E (2002) Targeting endothelium and its dynamic caveolae for tissue-specific transcytosis in vivo: A pathway to overcome cell barriers to drug and gene delivery Proc Natl Acad Sci USA 99 , 199 6–2001 Study demonstrates that the molecular composition of lung microvascular caveolae is distinct from... activation are those that encode for the polypeptide chains of platelet-derived growth factor (PDGF) To date, four PDGF polypeptide chains have been identified that combine to form five PDGF isoforms: PDGF-AA, -AB, -BB, -CC, and -DD These isoforms exert their effects by activating two protein tyrosine kinase receptors, denoted the a-receptor and b-receptor Although each of the different PDGF isoforms has been... spindle cell sarcoma All bars = 5 mm 95 7 95 8 PART III Pathology 95 9 CHAPTER 141 Tumor versus Normal Microvasculature Figure 2 Scanning electron microscopy of normal (a–d) and tumor microvascular corrosion casts (e–h) (a) The human colonic mucosal capillary plexus (c) is arranged in a honeycomb pattern and shows numerous intercapillary connections The supplying arterioles (a) and draining veins (v) take a... proteins Mutant mice with the Id1+/-Id 3-/ - phenotype possess defective angiogenic responses and, thus, are unable to support tumor growth However, when these mice are transplanted with wild-type bone marrow or HSCs, tumor growth in the subcutaneous compartment can be restored This process appears to require cooperation of both VEGFR1 and VEGFR2 in that treatment of Id1+/-Id 3-/ - mice with neutralizing antibodies... platelets has been identified to be P-selectin binding ligand1 Binding of endothelial P-selectin by glycoprotein (GP) Ib/IX/V as a second ligand depends on previous platelet activation Platelets store P-selectin along with adhesion proteins fibronectin, fibrinogen, and von Willebrand factor (vWF) in their a-granules and release these factors within minutes following stimulation P-selectin expression on activated . ✓✓ Bisantrene ✓✓ Rhodamine-123 ✓✓ Flavopiridol ✓ Lysotracker Green ✓ SN-38 ✓ E 2 -GLU ✓✓ Estrone 3-sulfate ✓✓ LTC 4 ✓ GSH ✓ E 2 -GLU, 17b-Estradiol 1 7-( b-D-glucuronide); GSH, reduced glu- tathione; LTC 4 ,. five PDGF isoforms: PDGF-AA, -AB, -BB, -CC, and -DD. These isoforms exert their effects by activating two protein tyrosine kinase receptors, denoted the a-receptor and b-receptor. Although each. squamous cell carcinoma, and 30% large cell carci- noma and other types). In univariate analysis, T-stage, N- stage, and growth pattern predicted overall and disease-free survival. Multiple logistic