CDCP1 Identifies a Broad Spectrum of Normal and Malignant Stem/Progenitor Cell Subsets of Hematopoietic and Nonhematopoietic Origin
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《干细胞学杂志》
a University of Tübingen, Department of Internal Medicine II, Division of Hematology, Immunology, Oncology and Rheumatology, Tübingen, Germany;
b University Children’s Hospital, Department of Hematology/Oncology, Tübingen, Germany;
c Boehringer Ingelheim Austria, Department of Exploratory Research, Research and Development, Vienna, Austria;
d University of Tübingen, Department of Internal Medicine IV, Division of Diabetes Research, Tübingen, Germany
Key Words. Stem cell marker ? Phenotype ? CDCP1
Hans-J?rg Bühring, Ph.D., Medizinische Klinik II, Otfried-Müller-Str.10, 72076 Tübingen, Germany. Telephone: 49-7071-2982730; Fax: 49-7071-292730; e-mail: hans-joerg.buehring@med.uni-tuebingen.de
ABSTRACT
Stem cells are defined by their capacity to give rise to identical progeny and to differentiate into tissue-specific effector cells. Among these, hematopoietic stem cells (HSCs) are extensively characterized, and their therapeutic potential in transplantation settings has been approved for several decades. To identify and to isolate these rare cells, monoclonal antibodies against cell surface antigens have been raised that are selectively expressed on HSCs. The most prominent markers are CD34 and CD133 (AC133 antigen, prominin) . Antibodies against these markers are now routinely employed for large-scale HSC purification to eliminate undesired cells such as tumor cells or T cells responsible for graft-versus-host disease .
In the search for novel HSC markers we have recently identified CUB-domain-containing protein 1 (CDCP1) as a promising candidate molecule . This molecule was originally identified as a novel epithelial tumor antigen by combining data derived from representational difference analysis and cDNA microarrays of lung cancer cell lines versus normal lung tissues . CDCP1 mRNA was detected in colon and breast tumors and in the erythroleukemic cell line K562. The protein is a type I transmembrane protein that contains three CUB domains and several potential glycosylation sites within the extracellular domain. The cytoplasmic region contains a short hexalysine stretch and five potential tyrosine phosphorylation sites. CUB (complement protein subcomponents C1/1r, urchin embryonic growth factor, and bone morphogenetic protein 1) domains are structurally related to immunoglobulins and play important roles in cell adhesion . Many proteins with such domains are developmentally regulated and have key functions in embryonic development . These proteins include growth factors, proteases, activators of the complement system, and proteins involved in cell adhesion or interaction with extracellular matrix components .
We have recently demonstrated that transplantation of human CDCP1+ bone marrow (BM) cells into nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice gives rise to chimeric hematopoiesis . Here we analyzed the reactivity of CDCP1-specific antibodies with normal and malignant hematopoietic cell populations and their influence in early hematopoiesis. In addition, we analyzed the CDCP1 expression on cells with mesenchymal stem cell (MSCs) and neural progenitor cell (NPC) phenotypes as well as on metastatic tissues from patients with colon and lung cancer.
MATERIALS AND METHODS
Four Monoclonal Antibodies Recognize the Extracellular Domain of CDCP1
To generate monoclonal antibodies against the extracellular domain of CDCP1, a transfectant line expressing CDCP1 protein was established and used for immunization. For this purpose, a 5x myc epitope was added to the 3'-end of the complete cDNA and the cDNA cloned into the expression plasmid pRK. NIH3T3 cells were cotransfected with this construct and with pSV2neo. The tags were inserted to identify protein expression with anti-c-myc antibodies. Cell clones overexpressing CDCP1 were selected using a myc-reactive antibody and used for immunization of a BALB/c mouse. Screening of hybridoma supernatants from immune spleen cells fused with SP2/0 myeloma cells revealed that four antibodies, termed CUB1-CUB4, selectively recognized the transfected (NIH-3T3/huCDCP1) but not the parent cell line (NIH-3T3) (Fig. 1).
Figure 1. Antibodies CUB1-CUB4 recognize CDCP1. Monoclonal antibodies against CDCP1 were raised as described in Materials and Methods and screened for their reactivity with NIH-3T3/huCDCP1 transfectant cells. Antibodies CUB1-CUB4 selectively recognize the transfectant line but not parental NIH-3T3 fibroblasts.
CDCP1-Reactive Antibodies Recognize CD34+ Cell Subsets from Various Sources
To analyze CDCP1 expression on hematopoietic cells, PB, mPB, UCB, and BM cells from healthy individuals were stained with the IgG2b antibody CUB1, visualized with PE-conjugated anti-mouse IgG2b, and analyzed on a flow cytometer. All tested PB populations (B cells, T cells, monocytes, granulocytes, erythrocytes, thrombocytes) appeared to be negative for CDCP1 (not shown). In contrast, multicolor fluorescence analyses revealed that small subpopulations of mPB, UCB, and BM were positive for CDCP1 (Fig. 2A). Using antibodies against CDCP1, CD34, CD38, and CD133, we could further demonstrate that CD34+CD38– BM cells coexpressed CDCP1 and CD133 (Fig. 2B). In contrast to CD34, CDCP1 was not expressed on cells expressing higher levels of the transferrin receptor CD71 and was only rarely found on CD10+ B-cell precursor cells (Fig. 2C). Since more mature erythroid progenitors are known to reside in the fraction expressing high levels of CD71 and B-lymphoid progenitors are CD10+, CDCP1 appears to spare immature erythroid and B-lymphoid precursor cell subsets.
Figure 2. Reactivity of antibody CUB1 with CD34+ stem/progenitor cell subsets. A) Correlated expression of CDCP1 and CD34 on BM, mPB, and UCB cells. Mononuclear cells from different sources were stained with anti-CD34-FITC (IgG1) and CUB1 plus anti-IgG2b-PE conjugate and analyzed on a FACSCalibur flow cytometer. B) CDCP1 is expressed on CD34+CD38– BM cells. BM cells were stained with CD34-PerCP (IgG1), CD38-FITC (IgG1), CD133-APC (IgG1), and CUB1 plus anti-IgG2b-PE conjugate and analyzed by flow cytometry. C) CDCP1 expression is more restricted to stem cells than CD34. The correlated expression of CD34 or CDCP1 on CD71+ and CD10+ was analyzed on mononuclear BM cells. Cells were stained with either anti-CD34-PE or CUB1 plus anti-IgG2b-PE and counter-stained with either anti-CD10-FITC or anti-CD71-FITC and analyzed by flow cytometry.
Majority of CDCP1+ BM Cells Coexpress CD34 and CD133
To analyze the coexpression of the stem cell markers CD34 and CD133 on CDCP1+ populations, BM cells were stained with CUB1 and selected by immunomagnetic separation (MACS) using anti-PE microbeads. After isolation, the cells were stained with CD34-FITC and CD133-allophycocyanin. The isolated cells were more than 94% pure and could be grouped into two populations (Fig. 3). Figure 3C shows that more than 99% of the CDCP1+ cells were positive for CD34. Most of these cells also coexpressed CD133. The fact that only 81% of these cells appeared to be positive for CD133 reflects the insufficient resolution of the CD133-APC (compared with CD133-PE) conjugate rather than the real population size. In addition to the major population with moderate CDCP1 expression, a minor population was found that expressed very high levels of CDCP1 (Fig. 3D). Interestingly, this population was negative for both CD34 and CD133. More detailed analyses revealed that this population coexpressed CD45 and HLA-DR (not shown), indicating that these cells are of hematopoietic origin.
Figure 3. The majority of CDCP1+ BM cells coexpress CD34 and CD133. BM cells were labeled with CUB1 plus anti-IgG2b-PE and stained for immunomagnetic separation with anti-PE MACS beads. Cells were selected by MACS and stained with anti-CD34-FITC and anti-CD133-APC.
CDCP1 Is an Independent Marker for the Diagnosis of Leukemia
The stem cell antigens CD34 and CD133 are widely used in the diagnosis of acute leukemia. Both markers are preferentially expressed in more immature leukemic subtypes. To explore CDCP1 surface expression on malignant hematopoietic cells and to correlate it with the expression profiles of CD34 and CD133, leukemic blasts from patients with ALL (n = 20), AML (n = 11), and CML-BC (n = 10) were stained with CDCP1-reactive antibodies CUB1 and CUB2 as well as with CD34-reactive and CD133-reactive antibodies and analyzed by flow cytometry. Table 1 shows CDCP1 expression on 4 of 20 ALL, 7 of 11 AML, and 7 of 10 CML-BC samples. By contrast, CD133 was found on 7 of 20 ALL, 8 of 11 AML, and 4 of 10 CML-BC samples, and CD34 on 15 of 20 ALL, 7 of 11 AML, and 8 of 10 CML-BC samples. This suggests that CDCP1 and CD133 are expressed on leukemic blasts at similar but lower frequencies than CD34. A predominant CD34 expression was found in ALL samples, which is in line with the fact that CD34 detects a larger cell population of normal B-lymphoid precursors than CD133 or CDCP1.
Table 1. CDCP1 is expressed on subsets of leukemic blasts
Analysis of the correlated expression of CDCP1, CD133, and CD34 revealed that CDCP1 is most frequently found on CD34+CD133+ leukemic blasts (50%; Table 1). However, some samples were only double-positive for CDCP1 and CD34 or CDCP1 and CD133, and two of the studied samples exclusively expressed CDCP1. Hence, CDCP1 is a novel independent marker for the diagnosis of leukemia.
Influence of Antibodies CUB1-CUB4 on the Differentiation of CD34+ BM Cells
To study a potential regulatory effect of antibodies CUB1-CUB4 on the differentiation of CD34+ stem/progenitor cells, colony-forming assays were performed. CD34+ BM cells were grown in a commercial semisolid methylcellulose medium in the presence of defined growth factors (see Materials and Methods) and antibodies CUB1-CUB4 (60 μg/ml) or isotype-matched control antibodies. Fourteen days after culture, the colony numbers were determined.
Figure 4A shows that all CDCP1-reactive antibodies were able to moderately stimulate the growth of erythroid colonies (n = 2). Antibody CUB1 showed the most prominent effect on the growth of erythroid colonies (1.5–2-fold increase of colony-forming units erythroid ; p 0.05) and was chosen to study the dose dependency of CFU-E growth. Figure 4B demonstrates a clear correlation between antibody concentration and colony growth. Other hematopoietic colony types were not affected (not shown). This suggests that CDCP1 may be involved in early steps of erythropoiesis.
Figure 4. A) Antibodies CUB1-CUB4 influence the growth of erythroid colonies in vitro. Ten thousand CD34+ BM cells were cultured in methylcellulose-containing medium in the presence of defined growth factors and 60 μg/ml CDCP1-reactive antibodies CUB1-CUB4 or isotype-matched control antibodies. Fourteen days after culture, the resulting colonies were evaluated. The results are presented as the mean of colony numbers ± standard deviation of three independent cultures. B) Growth stimulation of erythroid colonies by CUB1 is dose dependent. Five thousand CD34+ BM cells were cultured as described above in the presence of varying CUB1 antibody concentrations (2.2–60 μg/ml). Fourteen days after culture, the resulting colonies were evaluated and the results presented as the mean ± standard deviation of the colony numbers of two dishes.
CDCP1 Is a Marker for Cells Expressing MSC and NPC Phenotypes
To test whether CDCP1 protein is also expressed on progenitor cells of nonhematopoietic origin, commercially available MSCs and NPCs were stained with CDCP1-specific antibodies CUB1 and CUB2 and analyzed by flow cytometry. The mesenchymal phenotype was confirmed by the strong reactivity of the cells with antibodies against CD13, CD90, CD105, and CD140b, and neural progenitor phenotype by the strong expression of CD56 and CD90 (not shown). As shown in Figure 5, CDCP1 was moderately expressed on cells with both MSC and NPC phenotypes. This is in contrast to the nonreactivity of CD34 with MSCs and NPCs, and the negativity of CD133 for MSCs.
Figure 5. CDCP1 is expressed on cells with MSC and NPC phenotypes. Commercially available cells with MSC and NPC designations (CellSystems) were stained either with CUB1 plus anti-IgG2b-PE or with CUB2 plus anti-IgG2a-PE. Controls were stained with isotype-matched control antibodies. Cells were analyzed by flow cytometry.
CDCP1 Protein Expression in Other Nonhematopoietic Tissues
Polyclonal antibodies against CDCP1 were used to monitor the protein expression by immunohistochemistry in biopsies derived from colon and breast cancer patients. The results show predominant expression of CDCP1 protein in colon cancer cells, while the surrounding connective tissues were not stained (Fig. 6A). Strong reactivity of CDCP1 antibodies with tumor cells was also seen in 15 of 16 breast cancer samples. Figure 6C shows a predominant staining of epithelial cells derived from a lobular mamma carcinoma. Control staining of colon (Fig. 6B) and mamma carcinoma cells (Fig. 6D) using a rabbit IgG antibody showed no reactivity. These data are in line with the observation that CDCP1 mRNA is overexpressed in colon and breast carcinoma .
Figure 6. CDCP1 is overexpressed in colorectal cancer and breast carcinoma. Frozen sections from patients with colon tumors (A) or breast carcinoma (C) were fixed in acetone and incubated with the affinity-purified rabbit antibody CDCP1-Intra-E4I. After washing, cells were stained with biotinylated anti-rabbit IgG antibody plus avidin-peroxidase (DAKO). Neighboring tissue sections were labeled with unspecific rabbit IgG or PBS (B and D) as controls.
DISCUSSION
This work was supported by a grant from the Deutsche Forschungsgemeinschaft (SFB 510, projects A1 and A6). Samples from breast and colon cancer were kindly provided by Prof. Kurt Zatloukal, Institute for Pathology, Karl-Franzens University, Graz, Austria.
FOOTNOTES
Gao Z, Fackler MJ, Leung W et al. Human CD34+ cell preparations contain over 100-fold greater NOD/SCID mouse engrafting capacity than do CD34– cell preparations. Exp Hematol 2001;29:910–921.
Civin CI, Trischmann T, Kadan NS et al. Highly purified CD34-positive cells reconstitute hematopoiesis. J Clin Oncol 1996;14:2224–2233.
Yin AH, Miraglia S, Zanjani ED et al. AC133, a novel marker for human hematopoietic stem and progenitor cells. Blood 1997;90:5002–5012.
Miraglia S, Godfrey W, Yin AH et al. A novel five-transmembrane hematopoietic stem cell antigen: isolation, characterization, and molecular cloning. Blood 1997;90:5013–5021.
Bühring HJ, Marxer A, Lammers R et al. CD133 cluster report. In: Mason D, André P, Bensussan A et al. (eds). Leucocyte Typing VII. White Cell Differentiation Antigens. Oxford: Oxford University Press, 2002:622–623.
Gordon PR, Leimig T, Babarin-Dorner A et al. Large-scale isolation of CD133+ progenitor cells from G-CSF mobilized peripheral blood stem cells. Bone Marrow Transplant 2003;31:17–22.
Conze T, Lammers R, Ku?i S et al. CDCP1 is a novel marker for hematopoietic stem cells. Ann New York Acad Sci 2003;996:222–226.
Scherl-Mostageer M, Sommergruber W, Abseher R et al. Identification of a novel gene, CDCP1, overexpressed in human colorectal cancer. Oncogene 2001;20:4402–4408.
Duke-Cohan JS, Gu J, McLaughlin DF et al. Attractin (DPPT-L), a member of the CUB family of cell adhesion and guidance proteins, is secreted by activated human T lymphocytes and modulates immune cell interactions. Proc Natl Acad Sci USA 1998;95:11336–11341.
Bork P, Beckmann G. The CUB domain. A widespread module in developmentally regulated proteins. J Mol Biol 1993;231:539–545.
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Li SW, Sieron AL, Fertala A et al. The C-proteinase that processes procollagens to fibrillar collagens is identical to the protein previously identified as bone morphogenic protein-1. Proc Natl Acad Sci USA 1996;93:5127–5130.
Stohr H, Berger C, Frohlich S et al. A novel gene encoding a putative transmembrane protein with two extracellular CUB domains and a low-density lipoprotein class A module: isolation of alternatively spliced isoforms in retina and brain. Gene 2002;286:223–231.
Chen C, Okayama H. High-efficiency transformation of mammalian cells by plasmid DNA. Mol Cell Biol 1987;7:2745–2752.
Heider KH, Hofmann M, Horst E et al. A human homologue of the rat metastasis-associated variant of CD44 is expressed in colorectal carcinomas and adenomatous polyps. J Cell Biol 1993;120:227–233.
Greaves MF, Titley I, Colman SM et al. Report on the CD34 cluster workshop. In: Schlossman SF, Boumsell L, Gilkes W et al. (eds). Leucocyte Typing V. Oxford: Oxford University Press, 1995:840–846.
Bhatia M. AC133 expression in human stem cells. Leukemia 2001;15:1685–1688.
Jiang Y, Jahagirdar BN, Reinhardt RL et al. Pluripotency of mesenchymal stem cells derived from adult marrow. Nature 2002;418:41–49.
Jiang Y, Vaessen B, Lenvik T et al. Multipotent progenitor cells can be isolated from postnatal murine bone marrow, muscle, and brain. Exp Hematol 2002;30:896–904.
Hooper JD, Zijlstra A, Aimes RT et al. Subtractive immunization using highly metastatic human tumor cells identifies SIMA135/CDCP1, a 135 kDa cell surface phosphorylated glycoprotein antigen. Oncogene 2003;22:1783–1794.
Wimberger P, Xiang W, Mayr D et al. Efficient tumor cell lysis by autologous, tumor-resident T lymphocytes in primary ovarian cancer samples by an EP-CAM-/CD3-bispecific antibody. Int J Cancer 2003;105:241–248.
Schweizer C, Strauss G, Lindner M et al. Efficient carcinoma cell killing by activated polymorphonuclear neutrophils targeted with an Ep-CAMxCD64 (HEA125x197) bispecific antibody. Cancer Immunol Immunother 2002;51:621–629.
Thor A, Viglione MJ, Ohuchi N et al. Comparison of monoclonal antibodies for the detection of occult breast carcinoma metastases in bone marrow. Breast Cancer Res Treat 1988;11:133–145.
Feinberg H, Uitdehaag JC, Davies JM et al. Crystal structure of the CUB1-EGF-CUB2 region of mannose-binding protein associated serine protease-2. EMBO J 2003;22:2348–2359.
Hartigan N, Garrigue-Antar L, Kadler KE. Bone morphogenetic protein-1 (BMP-1). Identification of the minimal domain structure procollagen C-proteinase activity. J Biol Chem 2003;278:18045–18049.
Seiffert M, Cant C, Chen Z et al. Human signal-regulatory protein is expressed on normal but not on subsets of leukemic myeloid cells and mediates cellular adhesion involving its counter-receptor CD47. Blood 1999;94:3633–3643.(Hans-J?rg Bühringa, Selim)
b University Children’s Hospital, Department of Hematology/Oncology, Tübingen, Germany;
c Boehringer Ingelheim Austria, Department of Exploratory Research, Research and Development, Vienna, Austria;
d University of Tübingen, Department of Internal Medicine IV, Division of Diabetes Research, Tübingen, Germany
Key Words. Stem cell marker ? Phenotype ? CDCP1
Hans-J?rg Bühring, Ph.D., Medizinische Klinik II, Otfried-Müller-Str.10, 72076 Tübingen, Germany. Telephone: 49-7071-2982730; Fax: 49-7071-292730; e-mail: hans-joerg.buehring@med.uni-tuebingen.de
ABSTRACT
Stem cells are defined by their capacity to give rise to identical progeny and to differentiate into tissue-specific effector cells. Among these, hematopoietic stem cells (HSCs) are extensively characterized, and their therapeutic potential in transplantation settings has been approved for several decades. To identify and to isolate these rare cells, monoclonal antibodies against cell surface antigens have been raised that are selectively expressed on HSCs. The most prominent markers are CD34 and CD133 (AC133 antigen, prominin) . Antibodies against these markers are now routinely employed for large-scale HSC purification to eliminate undesired cells such as tumor cells or T cells responsible for graft-versus-host disease .
In the search for novel HSC markers we have recently identified CUB-domain-containing protein 1 (CDCP1) as a promising candidate molecule . This molecule was originally identified as a novel epithelial tumor antigen by combining data derived from representational difference analysis and cDNA microarrays of lung cancer cell lines versus normal lung tissues . CDCP1 mRNA was detected in colon and breast tumors and in the erythroleukemic cell line K562. The protein is a type I transmembrane protein that contains three CUB domains and several potential glycosylation sites within the extracellular domain. The cytoplasmic region contains a short hexalysine stretch and five potential tyrosine phosphorylation sites. CUB (complement protein subcomponents C1/1r, urchin embryonic growth factor, and bone morphogenetic protein 1) domains are structurally related to immunoglobulins and play important roles in cell adhesion . Many proteins with such domains are developmentally regulated and have key functions in embryonic development . These proteins include growth factors, proteases, activators of the complement system, and proteins involved in cell adhesion or interaction with extracellular matrix components .
We have recently demonstrated that transplantation of human CDCP1+ bone marrow (BM) cells into nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice gives rise to chimeric hematopoiesis . Here we analyzed the reactivity of CDCP1-specific antibodies with normal and malignant hematopoietic cell populations and their influence in early hematopoiesis. In addition, we analyzed the CDCP1 expression on cells with mesenchymal stem cell (MSCs) and neural progenitor cell (NPC) phenotypes as well as on metastatic tissues from patients with colon and lung cancer.
MATERIALS AND METHODS
Four Monoclonal Antibodies Recognize the Extracellular Domain of CDCP1
To generate monoclonal antibodies against the extracellular domain of CDCP1, a transfectant line expressing CDCP1 protein was established and used for immunization. For this purpose, a 5x myc epitope was added to the 3'-end of the complete cDNA and the cDNA cloned into the expression plasmid pRK. NIH3T3 cells were cotransfected with this construct and with pSV2neo. The tags were inserted to identify protein expression with anti-c-myc antibodies. Cell clones overexpressing CDCP1 were selected using a myc-reactive antibody and used for immunization of a BALB/c mouse. Screening of hybridoma supernatants from immune spleen cells fused with SP2/0 myeloma cells revealed that four antibodies, termed CUB1-CUB4, selectively recognized the transfected (NIH-3T3/huCDCP1) but not the parent cell line (NIH-3T3) (Fig. 1).
Figure 1. Antibodies CUB1-CUB4 recognize CDCP1. Monoclonal antibodies against CDCP1 were raised as described in Materials and Methods and screened for their reactivity with NIH-3T3/huCDCP1 transfectant cells. Antibodies CUB1-CUB4 selectively recognize the transfectant line but not parental NIH-3T3 fibroblasts.
CDCP1-Reactive Antibodies Recognize CD34+ Cell Subsets from Various Sources
To analyze CDCP1 expression on hematopoietic cells, PB, mPB, UCB, and BM cells from healthy individuals were stained with the IgG2b antibody CUB1, visualized with PE-conjugated anti-mouse IgG2b, and analyzed on a flow cytometer. All tested PB populations (B cells, T cells, monocytes, granulocytes, erythrocytes, thrombocytes) appeared to be negative for CDCP1 (not shown). In contrast, multicolor fluorescence analyses revealed that small subpopulations of mPB, UCB, and BM were positive for CDCP1 (Fig. 2A). Using antibodies against CDCP1, CD34, CD38, and CD133, we could further demonstrate that CD34+CD38– BM cells coexpressed CDCP1 and CD133 (Fig. 2B). In contrast to CD34, CDCP1 was not expressed on cells expressing higher levels of the transferrin receptor CD71 and was only rarely found on CD10+ B-cell precursor cells (Fig. 2C). Since more mature erythroid progenitors are known to reside in the fraction expressing high levels of CD71 and B-lymphoid progenitors are CD10+, CDCP1 appears to spare immature erythroid and B-lymphoid precursor cell subsets.
Figure 2. Reactivity of antibody CUB1 with CD34+ stem/progenitor cell subsets. A) Correlated expression of CDCP1 and CD34 on BM, mPB, and UCB cells. Mononuclear cells from different sources were stained with anti-CD34-FITC (IgG1) and CUB1 plus anti-IgG2b-PE conjugate and analyzed on a FACSCalibur flow cytometer. B) CDCP1 is expressed on CD34+CD38– BM cells. BM cells were stained with CD34-PerCP (IgG1), CD38-FITC (IgG1), CD133-APC (IgG1), and CUB1 plus anti-IgG2b-PE conjugate and analyzed by flow cytometry. C) CDCP1 expression is more restricted to stem cells than CD34. The correlated expression of CD34 or CDCP1 on CD71+ and CD10+ was analyzed on mononuclear BM cells. Cells were stained with either anti-CD34-PE or CUB1 plus anti-IgG2b-PE and counter-stained with either anti-CD10-FITC or anti-CD71-FITC and analyzed by flow cytometry.
Majority of CDCP1+ BM Cells Coexpress CD34 and CD133
To analyze the coexpression of the stem cell markers CD34 and CD133 on CDCP1+ populations, BM cells were stained with CUB1 and selected by immunomagnetic separation (MACS) using anti-PE microbeads. After isolation, the cells were stained with CD34-FITC and CD133-allophycocyanin. The isolated cells were more than 94% pure and could be grouped into two populations (Fig. 3). Figure 3C shows that more than 99% of the CDCP1+ cells were positive for CD34. Most of these cells also coexpressed CD133. The fact that only 81% of these cells appeared to be positive for CD133 reflects the insufficient resolution of the CD133-APC (compared with CD133-PE) conjugate rather than the real population size. In addition to the major population with moderate CDCP1 expression, a minor population was found that expressed very high levels of CDCP1 (Fig. 3D). Interestingly, this population was negative for both CD34 and CD133. More detailed analyses revealed that this population coexpressed CD45 and HLA-DR (not shown), indicating that these cells are of hematopoietic origin.
Figure 3. The majority of CDCP1+ BM cells coexpress CD34 and CD133. BM cells were labeled with CUB1 plus anti-IgG2b-PE and stained for immunomagnetic separation with anti-PE MACS beads. Cells were selected by MACS and stained with anti-CD34-FITC and anti-CD133-APC.
CDCP1 Is an Independent Marker for the Diagnosis of Leukemia
The stem cell antigens CD34 and CD133 are widely used in the diagnosis of acute leukemia. Both markers are preferentially expressed in more immature leukemic subtypes. To explore CDCP1 surface expression on malignant hematopoietic cells and to correlate it with the expression profiles of CD34 and CD133, leukemic blasts from patients with ALL (n = 20), AML (n = 11), and CML-BC (n = 10) were stained with CDCP1-reactive antibodies CUB1 and CUB2 as well as with CD34-reactive and CD133-reactive antibodies and analyzed by flow cytometry. Table 1 shows CDCP1 expression on 4 of 20 ALL, 7 of 11 AML, and 7 of 10 CML-BC samples. By contrast, CD133 was found on 7 of 20 ALL, 8 of 11 AML, and 4 of 10 CML-BC samples, and CD34 on 15 of 20 ALL, 7 of 11 AML, and 8 of 10 CML-BC samples. This suggests that CDCP1 and CD133 are expressed on leukemic blasts at similar but lower frequencies than CD34. A predominant CD34 expression was found in ALL samples, which is in line with the fact that CD34 detects a larger cell population of normal B-lymphoid precursors than CD133 or CDCP1.
Table 1. CDCP1 is expressed on subsets of leukemic blasts
Analysis of the correlated expression of CDCP1, CD133, and CD34 revealed that CDCP1 is most frequently found on CD34+CD133+ leukemic blasts (50%; Table 1). However, some samples were only double-positive for CDCP1 and CD34 or CDCP1 and CD133, and two of the studied samples exclusively expressed CDCP1. Hence, CDCP1 is a novel independent marker for the diagnosis of leukemia.
Influence of Antibodies CUB1-CUB4 on the Differentiation of CD34+ BM Cells
To study a potential regulatory effect of antibodies CUB1-CUB4 on the differentiation of CD34+ stem/progenitor cells, colony-forming assays were performed. CD34+ BM cells were grown in a commercial semisolid methylcellulose medium in the presence of defined growth factors (see Materials and Methods) and antibodies CUB1-CUB4 (60 μg/ml) or isotype-matched control antibodies. Fourteen days after culture, the colony numbers were determined.
Figure 4A shows that all CDCP1-reactive antibodies were able to moderately stimulate the growth of erythroid colonies (n = 2). Antibody CUB1 showed the most prominent effect on the growth of erythroid colonies (1.5–2-fold increase of colony-forming units erythroid ; p 0.05) and was chosen to study the dose dependency of CFU-E growth. Figure 4B demonstrates a clear correlation between antibody concentration and colony growth. Other hematopoietic colony types were not affected (not shown). This suggests that CDCP1 may be involved in early steps of erythropoiesis.
Figure 4. A) Antibodies CUB1-CUB4 influence the growth of erythroid colonies in vitro. Ten thousand CD34+ BM cells were cultured in methylcellulose-containing medium in the presence of defined growth factors and 60 μg/ml CDCP1-reactive antibodies CUB1-CUB4 or isotype-matched control antibodies. Fourteen days after culture, the resulting colonies were evaluated. The results are presented as the mean of colony numbers ± standard deviation of three independent cultures. B) Growth stimulation of erythroid colonies by CUB1 is dose dependent. Five thousand CD34+ BM cells were cultured as described above in the presence of varying CUB1 antibody concentrations (2.2–60 μg/ml). Fourteen days after culture, the resulting colonies were evaluated and the results presented as the mean ± standard deviation of the colony numbers of two dishes.
CDCP1 Is a Marker for Cells Expressing MSC and NPC Phenotypes
To test whether CDCP1 protein is also expressed on progenitor cells of nonhematopoietic origin, commercially available MSCs and NPCs were stained with CDCP1-specific antibodies CUB1 and CUB2 and analyzed by flow cytometry. The mesenchymal phenotype was confirmed by the strong reactivity of the cells with antibodies against CD13, CD90, CD105, and CD140b, and neural progenitor phenotype by the strong expression of CD56 and CD90 (not shown). As shown in Figure 5, CDCP1 was moderately expressed on cells with both MSC and NPC phenotypes. This is in contrast to the nonreactivity of CD34 with MSCs and NPCs, and the negativity of CD133 for MSCs.
Figure 5. CDCP1 is expressed on cells with MSC and NPC phenotypes. Commercially available cells with MSC and NPC designations (CellSystems) were stained either with CUB1 plus anti-IgG2b-PE or with CUB2 plus anti-IgG2a-PE. Controls were stained with isotype-matched control antibodies. Cells were analyzed by flow cytometry.
CDCP1 Protein Expression in Other Nonhematopoietic Tissues
Polyclonal antibodies against CDCP1 were used to monitor the protein expression by immunohistochemistry in biopsies derived from colon and breast cancer patients. The results show predominant expression of CDCP1 protein in colon cancer cells, while the surrounding connective tissues were not stained (Fig. 6A). Strong reactivity of CDCP1 antibodies with tumor cells was also seen in 15 of 16 breast cancer samples. Figure 6C shows a predominant staining of epithelial cells derived from a lobular mamma carcinoma. Control staining of colon (Fig. 6B) and mamma carcinoma cells (Fig. 6D) using a rabbit IgG antibody showed no reactivity. These data are in line with the observation that CDCP1 mRNA is overexpressed in colon and breast carcinoma .
Figure 6. CDCP1 is overexpressed in colorectal cancer and breast carcinoma. Frozen sections from patients with colon tumors (A) or breast carcinoma (C) were fixed in acetone and incubated with the affinity-purified rabbit antibody CDCP1-Intra-E4I. After washing, cells were stained with biotinylated anti-rabbit IgG antibody plus avidin-peroxidase (DAKO). Neighboring tissue sections were labeled with unspecific rabbit IgG or PBS (B and D) as controls.
DISCUSSION
This work was supported by a grant from the Deutsche Forschungsgemeinschaft (SFB 510, projects A1 and A6). Samples from breast and colon cancer were kindly provided by Prof. Kurt Zatloukal, Institute for Pathology, Karl-Franzens University, Graz, Austria.
FOOTNOTES
Gao Z, Fackler MJ, Leung W et al. Human CD34+ cell preparations contain over 100-fold greater NOD/SCID mouse engrafting capacity than do CD34– cell preparations. Exp Hematol 2001;29:910–921.
Civin CI, Trischmann T, Kadan NS et al. Highly purified CD34-positive cells reconstitute hematopoiesis. J Clin Oncol 1996;14:2224–2233.
Yin AH, Miraglia S, Zanjani ED et al. AC133, a novel marker for human hematopoietic stem and progenitor cells. Blood 1997;90:5002–5012.
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