标题: Transplanted Bone Marrow Cells Preferentially Home To The Vessels Of In Situ Gen [打印本页] 作者: 江边孤钓 时间: 2009-3-5 10:22 标题: Transplanted Bone Marrow Cells Preferentially Home To The Vessels Of In Situ Gen
a Departments of Hematology/Oncology and* I4 l8 H: L0 x5 c2 e
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c Neurosurgery, Freiburg University Medical Center, Freiburg, Germany;# d; V: Y) s! r( T* Z" `
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Key Words. Tumor angiogenesis ? Bone marrow transplantation l8 a5 {8 Y6 w# I) J' d
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Alexandros Spyridonidis, M.D., Freiburg University Medical Center, Hugstetterstrasse 55, 79106 Freiburg, Germany. Telephone: 49-761-2703364; Fax: 49-761-2703660; e-mail: spyridonidis@mm11.ukl.uni-freiburg.de. W1 K2 o- V8 y& R9 K
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ABSTRACT % h7 M( }1 Y+ @, v4 D5 d; A3 F) D2 f M$ C& V3 w' P5 P
The formation of new blood vessels is required for the growth and spread of tumors . Bone marrow transplantation (BMT) studies in animals challenged with s.c. implanted tumors indicate that bone marrow (BM)-derived cells migrate to the tumors and incorporate into the tumor vessels . Initial studies have suggested that these BM-derived vessel-associated cells (BM-VCs) represent BM-derived endothelial cells which participate in new vessel formation through the mechanism of vasculogenesis . More recently, a study claimed that BM-VCs are not differentiated endothelial cells but rather hematopoietic cells that home into the tumor vasculature and, at least a subset of them promotes angiogenesis . Nevertheless, these studies could demonstrate the functional role of BM cells in tumor angiogenesis. Gene manipulation or elimination of BM cells that home into the tumor vascular bed resulted not only in inhibition of tumor neoangiogenesis but also in impaired tumor growth .0 J6 z& T1 K+ _# i
7 e- r2 k% b* `- S4 J AHowever, because of the lack of suitable in vivo models, previous studies evaluating the presence and the role of BM cells recruited and incorporated into the tumor vasculature could not reliably evaluate the in vivo situation during tumorigenesis and tumor growth. Exogenous, s.c. implanted tumors differ from orthotopic, spontaneously in situ developed tumors in many ways such as morphology, tumor growth pattern, vessel morphology as well as response to angiogenic and antiangiogenic stimuli . The tissue bed in which the exogenous tumor is implanted may also differ from the stroma in which a tumor is generated in situ. Particular characteristics of these different stroma, like blood supply, morphology and functionality of the already existing vascular bed, extracellular matrix composition and cytokine milieu may affect the recruitment of BM cells and their ability to home and incorporate into functional vessels . Moreover, needle stab tissue injury, which is unavoidable during the s.c. implantation of the tumor, may affect the migration of BM cells in the tumor vasculature.% p+ t; y# ]: Y* u
7 p0 G9 R7 O; c9 h y1 tFemale transgenic mice carrying the polyoma virus middle T oncogene under the transcriptional control of the mouse mammary tumor virus promoter (MMTV-PyVT) develop palpable mammary adenocarcinomas as early as 5 weeks of age . Male transgene carriers also develop adenocarcinomas at later time points. We have taken advantage of the in situ generation of tumors in this transgenic mouse model to determine whether BM-derived cells preferentially and substantially home to and incorporate into the tumor vessels. This information is essential before one uses BM as a vehicle for transport of anti-angiogenetic signals into the tumor vascular bed. 2 c \0 V+ q& ~, F$ L2 G8 G1 h6 d, j# d) f2 r' T
MATERIALS AND METHODS 0 ^1 q9 }0 \1 z" i C * J" T) u A1 c+ b8 d1 x1 P5 uUnfractionated BM from ROSA 26 mice was used as a hematopoietic graft to rescue irradiated MMTV-PyVT transgenic mice (n = 12) or their WT congenics FVB (n = 21). At transplantation all recipients were between 3 to 4 weeks in age and had no palpable tumors. Two transgenic and four WT mice died within a median of 20 days after transplantation, probably due to infections or engraftment failure. No clinical signs of graft-versus-host-reactions to skin were observed. All evaluable MMTV-PyVT mice (n = 10) developed tumors in the mammary pad that histologically proved to be adenocarcinomas. Female MMTV-PyVT recipients (n = 6) developed palpable tumors in a median of 60 days after BMT (range 46–65), whereas male recipients (n = 6) exhibited palpable tumors in a median of 192 days after transplantation (range 159–415). Counting from the day of birth, all MMTV-PyVT BM recipients developed tumors at time points similar to their nontransplanted female and male counterparts, indicating that the irradiation and the BMT had no influence on tumor cell development and growth. MMTV-PyVT mice were sacrificed when the tumors were easily palpable. FVB controls were sacrificed either at the same time points, or were observed for periods of up to 600 days post-transplantation. Complete autopsies revealed no tumor formation in the FVB mice at any time point after BMT. All transplanted mice, including those followed up to 1 year after transplantation, revealed a stable >80% hematopoietic engraftment as determined by FACS enumeration of H-2Kb cells (ROSA 26 specific MHC class I allele) in retro-orbital blood samples or BM samples (Fig. 1). ) z$ Z6 e! U2 G2 O' R/ z; W3 q4 ]) L1 q( @
Figure 1. Hematopoietic engraftment after allogenic BMT in mice. FACS analysis of peripheral blood from a representative MMTV-PyVT mouse (FVB strain) tranplanted with unfractionated BM harvested from ROSA 26 mice (C57BL6 strain). A) The analysis gate used to exclude dead cells and debris. B) Negative control. C) Incubation of blood cells with an antibody against H-2b PE (which recognizes C57BL6 strain) and with an antibody against H-2q FITC (which recognizes FVB strain) showing hematopoietic engraftment greater than 90%.8 L) x% e$ Q/ p$ i, a9 ]
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To assess whether BM cells were recruited to the tumors and incorporated into the vessels, we performed double stains with enzymatic lacZ stain followed by immunohistochemistry with anti-mouse CD31 antibodies. The specifity and sensitivity of the lacZ/CD31 stain were tested in organ biopsies of ROSA 26 mice and FVB or MMTV-PyVT nontransplanted mice (Fig. 2). In the ROSA 26 mice all cells from aorta tissue as well as colon or liver sections stained blue. In contrast, blue stain was not seen in colon and liver biopsies from FVB or MMTV-PyVT mice. The immunohistochemical stain with anti-CD31 antibodies established the presence of vessel structures with typical morphologies in colon, liver and in higher densities in the MMTV-PyVT tumors. The intratumoral vessel density was not different in the transplanted versus the nontransplanted MMTV-PyVT mice. We used strict criteria for the identification of BM-VCs that homed and incorporated into the tumor vasculature. These included co-staining of lacZ and CD31, strong CD31 immunostaining, distinctive elongated morphology, presence of the double-stained cell in line with a structure having typical vessel morphology, and absence of tumor necrosis in the area of examination. Since tumor necrosis areas revealed a large number of lacZ hematopoietic cells, they were excluded from analysis. In contrast, lacZ cells were only rarely seen in non-necrotic areas (Fig. 2E). Donor-derived cells were evaluated for co-expression of the pan-hematopoietic marker CD45. LacZ cells found to line structures with typical vessel morphology were uniformly CD45 negative, although the CD45 antibodies strongly stained the lacZ cells within the necrotic areas. Therefore, no triple CD45/CD31/lacZ stain was performed.' _) u' ?0 p, L! `
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Figure 2. Sensitivity and specifity of the double lacZ stain/CD31 stain. A, B) Positive controls: colon (A) and liver (B) section of a ROSA 26 mouse. All cells stained blue while the endothelial cells co-stained with CD31. C, D) Negative controls: All cells of colon tissue (C) or mammary tumors (D) from MMTV-PyVT mice were stained negative for lacZ while the CD31 stain revealed the vessel structures. E) Tumors developed in MMTV-PyVT mice previously transplanted with lacZ whole BM. Tumor necrosis areas ( ) revealing a large number of lacZ hematopoietic cells were excluded from analysis. In contrast, lacZ cells were only rarely seen in non-necrotic areas (***). Original magnification, x20.$ G5 J' J$ N" E
; L: p% D! z1 e# j3 FIn order to estimate the degree of recruitment and incorporation of BM cells into the tumoral vessels, approximately 300 vessel profiles were analyzed per tumor tissue section. Approximately 1.3% of the tumor vessel walls were found to contain lacZ /CD31 BM-VCs (Table 1). In most of the cases, only one lacZ /CD31 cell was found incorporated into the vessel (Fig. 3). However, in some cases a more robust incorporation of BM cells in the tumor vessel was observed (Fig. 3C). : E+ y* N4 |& }: V* m/ W: K+ ?' a6 T5 l: l
Table 1. Incorporation of BM cells into the vasculature of tumor-bearing mice" Q4 i8 b( `4 r' E- O! r) n
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Figure 3. Incorporation of BM-derived cells into the intratumoral vessels. lacZ/CD31 double staining of tumors developed in MMTV-PyVT mice previously transplanted with lacZ BM. In most of the cases, only one lacZ /CD31 cell was found incorporated into the vessel (A, B). However, in some cases a more robust incorporation of BM cells in the tumor vessel was observed (C). Scale bar, 20 μm.% m# \; V6 o; |/ o
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We treated ROSA 26 BM donors with VEGF delivered i.p. for 5 days before we harvested the BM cells. Four female MMTV-PyVT mice were transplanted with VEGF-conditioned BM. Palpable tumors developed with a median of 74 days post-transplantation, which did not differ from the tumor development observed in the other MMTV-PyVT female mice. BM cell recruitment and incorporation into the tumor vasculature of the mice transplanted with VEGF-conditioned BM were at a degree similar to that found in the MMTV-PyVT mice receiving non-conditioned BM graft (Table 1).- [' c! S' {, i e7 s% z1 U
& b& V# p6 W( \) o; Y9 qThe incorporation of BM cells into vessels outside the tumor was tested in colon and liver sections from the 14 MMTV-PyVT mice transplanted with BM harvested from VEGF-treated and nontreated ROSA 26 donors (Table 1). No lacZ /CD31 cells were found in any of the tissue sections of any of the mice examined. In addition, mice sacrificed at later time points after transplantation (~ 300 days) did not show any incorporation of BM cells into the extratumoral vessels.: t: K( Y. f! j' p2 \& I3 K
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DISCUSSION v$ }" y, {( }& d E- C4 V 5 F. ~# J% W4 LWe thank Professor R. Mertelsmann for supporting this study, critical discussions, and contributions to the manuscript. We acknowledge the excellent work of Ingrid Huber (stains), Sabine Enger (animal work), and Li DeLima-Hahn (FACS). Professor C. Peters provided us the transgenic animal strain.5 ?" |8 y7 j8 y- m
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