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. 2009 Feb 26;113(9):1992-2002.
doi: 10.1182/blood-2008-02-133751. Epub 2008 Dec 5.

Pleiotrophin Produced by Multiple Myeloma Induces Transdifferentiation of Monocytes Into Vascular Endothelial Cells: A Novel Mechanism of Tumor-Induced Vasculogenesis

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Pleiotrophin Produced by Multiple Myeloma Induces Transdifferentiation of Monocytes Into Vascular Endothelial Cells: A Novel Mechanism of Tumor-Induced Vasculogenesis

Haiming Chen et al. Blood. .
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Abstract

Enhanced angiogenesis is a hallmark of cancer. Pleiotrophin (PTN) is an angiogenic factor that is produced by many different human cancers and stimulates tumor blood vessel formation when it is expressed in malignant cancer cells. Recent studies show that monocytes may give rise to vascular endothelium. In these studies, we show that PTN combined with macrophage colony-stimulating factor (M-CSF) induces expression of vascular endothelial cell (VEC) genes and proteins in human monocyte cell lines and monocytes from human peripheral blood (PB). Monocytes induce VEC gene expression and develop tube-like structures when they are exposed to serum or cultured with bone marrow (BM) from patients with multiple myeloma (MM) that express PTN, effects specifically blocked with antiPTN antibodies. When coinjected with human MM cells into severe combined immunodeficient (SCID) mice, green fluorescent protein (GFP)-marked human monocytes were found incorporated into tumor blood vessels and expressed human VEC protein markers and genes that were blocked by anti-PTN antibody. Our results suggest that vasculogenesis in human MM may develop from tumoral production of PTN, which orchestrates the transdifferentiation of monocytes into VECs.

Figures

Figure 1
Figure 1
PTN and M-CSF together induce VEC gene expression in fresh monocytes in vitro. Human CD14+ monocytes were purified from PB by density gradient centrifugation and anti-CD14 antibody treatment with magnetic bead selection. After 1 hour of culture, M-CSF (10 ng/mL) was added, and/or PTN (50 nM) and/or VEGF (20 ng/mL) were added twice to the cultures (after 24 hours and 5 days of culture). Cells were cultured for a total of 7 days. Total RNA was isolated from 106 cells containing monocytes treated with various cytokine combinations that were serially diluted with T cells. RT-PCR was performed with primers capable of detecting VEC gene expression (Tie-2, Flk-1, VWF, and VE-Cad [cadherin]) with a sensitivity of 1 human coronary arterial endothelial cell (HCAEC) in 106 T cells that lacked VEC gene expression to show that there was no VEC contamination within the purified fresh monocyte population. GAPDH was used as a control. Using primers for the monocyte-specific gene c-fms, monocytes were detected with a sensitivity of 1 cell diluted in 106 T cells.
Figure 2
Figure 2
MM tumor cells stimulate tube-like structure formation and Flk-1 staining in freshly obtained CD14+ cells that are blocked by anti-PTN antibodies; PTN and M-CSF induce tube-like structure formation and Flk-1 staining in fresh human monocytes in vitro that is blocked by anti-PTN antibodies. (A) BMMCs from a MM patient containing more than 90% plasma cells and a healthy control subject and U937 cells were stained with either anti-PTN or isotype-matched control antibodies (40×/objective lens, Olympus BX51; Olympus, San Diego, CA). (B top row) CD14+ cells were cocultured with BMMCs from a healthy donor or MM patient containing more than 90% tumor cells with and without anti-PTN antibody using Transwell culture plates for 14 days. Light microscopy of CD14+ cells stained with an anti–Flk-1 antibody is shown. (Middle row) CD14+ cells were exposed to M-CSF alone, PTN and M-CSF together, or the combination of PTN, M-CSF, and VEGF. The cells were stained with anti–Flk-1 antibody using immunohistochemical (IHC). (Bottom row) In a separate experiment, CD14+ cells were exposed to M-CSF alone or the combination of M-CSF and PTN with or without anti-PTN antibody.
Figure 3
Figure 3
MM cells as well as PCL serum induce VEC gene and protein expression in monocytes that is blocked by anti-PTN antibodies. (A) Freshly obtained CD14+ cells cocultured with MM BM tumor cells with and without anti-PTN antibody were analyzed for Flk-1, Tie-2, and VWF protein expression using Western blot analysis. Haceks served as a positive control. (B) U937, U266, and RPMI8226 cells were analyzed for PTN and GAPDH gene expression with RT-PCR. (C) LAGλ-1 and U266 cells were stained with either anti-PTN or isotype-matched control antibodies. (D) THP-1, U937, RPMI8226, and U266 cells were cultured for 48 hours and the culture supernatant was analyzed for PTN protein concentration by ELISA. Data for PTN graphed are the average of experiments performed in triplicate and show means plus or minus SEM. (E) Endothelial cells (HCAECs) or CD14+ cells alone or exposed to M-CSF, the PTN-producing MM cell lines U266 and RPMI8226, or serum from a healthy control subject or a patient with PCL containing high levels of PTN (3.4 ng/mL) were analyzed for gene expression using primers specific for VWF, Tie-2, Flk-1, and GAPDH with RT-PCR. (F) Endothelial cells (HCAECs) or CD14+ cells alone or exposed to M-CSF, the PTN-producing MM cell line U266, or BM from a healthy control subject or serum from the patient with PCL were analyzed for expression of the VWF, Tie-2, and Flk-1 genes in the presence of anti-PTN or isotype-matched control antibodies.
Figure 4
Figure 4
CD133 and CD45 expression in CD14+ cells alone or treated with PTN, M-CSF, or the combination of both cytokines for 7 days. (A) RT-PCR analysis of CD133 and CD45 gene expression in CD14+ cells alone or treated with PTN, M-CSF, or the combination of these cytokines. (B) Flow cytometric analysis of CD45 expression on CD14+ cells following treatment with no treatment, PTN, M-CSF, or the combination of both cytokines. The proportion of cells expressing CD45 is shown. Each experiment was performed in triplicate and error bars represent multiple assays.
Figure 5
Figure 5
To determine whether PTN induced the monocyte cell line THP-1 to express VEC genes, total RNA was isolated and separated by 1% agarose gel electrophoresis after THP-1 monocytes were cultured with PMA, PCL serum, or normal human serum or cocultured with U266 and RPMI8226. In addition, THP-1 cells exposed to U266 or PCL serum were also treated with either anti-PTN or isotype-matched control antibodies during tissue culture. RT-PCR was performed on RNA from THP-1 cells with primers specific for the Tie-2, Flk-1, VWF, and GAPDH genes. HCAECs were used as a positive control.
Figure 6
Figure 6
GFP-marked THP-1 cells (THP-1/GFP) coinjected with human LAGλ-1 MM tumor cells into C.B-17 SCID/SCID mice become incorporated into tumor blood vessels and express VEC markers. Human LAGλ-1 MM tumor cells were injected subcutaneously alone or in combination with THP-1/GFP monocytes into mice. Six weeks later, tumors were excised and immediately fixed with formalin. Tumor sections were prepared using standard histologic protocols. (A) Cells expressing GFP were determined using fluorescence and immunofluorescence was determined in sections stained with anti–human Tie-2 and anti-Dapi antibodies. (B) Another tumor blood vessel from a SCID mouse containing LAGλ-1 and THP-1/GFP cells was stained with anti–human CD144 (VE-cadherin) antibodies. Similarly, cells expressing GFP were determined using fluorescence and immunofluorescence was determined in sections stained with anti–human CD144 and anti-Dapi antibodies (100×/oil immersion, Olympus BX51; Olympus). (C) Human LAGλ-1 MM cells coinjected with THP-1 monocytes express VEC genes that are markedly reduced in the presence of anti-PTN antibodies. (i) Human LAGλ-1 MM cells were injected subcutaneously alone or in combination with THP-1 monocytes into mice. Six weeks later, tumors were excised and RNA was extracted. Expression of Tie-2, Flk-1, VWF, CD144, and GAPDH was determined using RT-PCR. (ii) Similarly, human LAGλ-1 MM or THP-1 cells were each injected subcutaneously alone or together into mice also treated twice weekly with intraperitoneal injections of either polyclonal goat anti–human PTN antibodies or control preimmune goat IgG. Six weeks later, tumors were excised and RNA was extracted. Tie-2 and GAPDH expression was determined using RT-PCR.
Figure 7
Figure 7
Microdissection of single cells lining blood vessels from LAGλ-1 tumors containing THP-1 cells shows cells with the presence of 3 different types of DNA: only human, both human and mouse, and only murine sequences. (A) Single cells were microdissected from the lining of tumor blood vessels from mice injected with LAGλ-1 and THP-1 cells (100×/objective lens, Olympus BX51; Olympus). (B) To test the sensitivity to detect human DNA with the human-specific alu primers with PCR, we serially diluted human monocytes from 104 to less than a single cell with mouse liver cells. DNA was isolated with salmon sperm DNA protection and separated on 1% agarose gel following 40 cycles of PCR with human alu–specific or mouse pf1–specific primers. The results show that human-specific alu DNA can be detected to a sensitivity of 0.01 human cell equivalents. (C) Similarly, we determined the sensitivity to detect mouse-specific pf1 DNA by serially diluting mouse liver cells in human cells (U266 MM cell line). The results show that mouse-specific pf1 DNA can be detected to a sensitivity of 0.01 mouse cell equivalents. (D) Single cells were microdissected from tumor blood vessels derived from mice coinjected with LAGλ-1 and THP-1 cells, and DNA was isolated with salmon sperm DNA protection and separated on 1% agarose gel following 40 cycles of PCR with human alu–specific or mouse pf1–specific primers. Three different patterns of PCR-amplified products were identified: human DNA alone, mouse DNA alone, and both human and mouse DNA. (E) Single cells were microdissected from the lining of tumor blood vessels from mice injected subcutaneously with LAGλ-1 + THP-1 cells. DNA was isolated with salmon sperm DNA protection and separated on 1% agarose gel following 40 cycles of PCR with human alu–specific or mouse pf1–specific primers. The lanes containing 20 and 30 cells were derived from microdissected single cells that were combined together for PCR analysis. Single cells lining the tumor blood vessels derived from mice injected with human LAGλ-1 cells alone showed the presence of only mouse DNA in tumor blood vessels, whereas mice injected with LAGλ-1 and THP-1 cells together showed the presence of both human and mouse DNA.

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