doi: 10.1161/hc0102.101437
(Circulation. 2002;105:112.)
© 2002 American Heart Association, Inc.
Basic Science Reports |
Analysis of Mural Cell Recruitment to Tumor Vessels
From the Department of Medical Biochemistry, Göteborg University (A.A., C.B.), and the Department of Orthopedics, Sahlgrenska Hospital, Göteborg University (Ö.B.), Göteborg, Sweden; the Laboratoire de Biologie Moleculaire de la Differenciation, Université Denis Diderot Paris 7, Paris, France (H.P., D.P.); and the Institute of Animal Science, The Volcani Center, Bet Dagan, Israel (M.S.).
Correspondence to Christer Betsholtz, Department of Medical Biochemistry, Göteborg University, PO Box 440, SE 405 30 Göteborg, Sweden. E-mailchrister.betsholtz{at}medkem.gu.se
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Abstract |
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Background— Tumor blood vessels are both structurally and functionally abnormal compared with normal vessels. A limited support of mural cells may contribute to these abnormalities. Here, we characterized mural cell recruitment in 2 mouse tumor models and addressed the question of why tumor vessels fail to recruit a proper coat of mural cells.
Methods and Results— We studied mural cell recruitment to the vasculature of 2 transplantable mouse tumor models, T241 fibrosarcoma and KRIB osteosarcoma. We found that both tumors formed a vessel network with heterogeneous and highly abnormal organization of mural cells. Transplantation of tumors to mice expressing lacZ in mural cells demonstrated that these cells were host-derived. Although tumor vessel endothelium expressed PDGF-B, an embryonic mitogen for mural cells, only very few PDGFRß-positive cells were found to be associated with the developing tumor vasculature, suggesting a limited pool of recruitable mural cells. We tested whether exogenous mural cells could be recruited to tumor vessels by injecting mixtures of T241 tumor cells and embryonic mesenchymal cells isolated from mice expressing lacZ in mural cells. In the tumors that arose, lacZ-positive cells were efficiently recruited to the tumor vessels.
Conclusions— T241 and KRIB tumors show a similar highly abnormal organization of vessel-associated mural cells. T241 tumor vessels seem highly capable of recruiting exogenously added mural cells. The sparse mural cell coat of tumor vessels may result from a limited pool of mural cells available for recruitment.
Key Words: angiogenesis • cells • pericytes • muscle, smooth • growth substances
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Introduction |
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It is widely acknowledged that the growth of most cancers depends on angiogenesis and that inhibition of tumor angiogenesis may provide an efficient strategy to slow down or block tumor growth (see published reviews1–3). In contrast to normal angiogenesis as it occurs during embryonic development, wound healing, and the female estrous cycle, however, angiogenesis in tumors leads to the formation of a poorly organized vasculature characterized by tortuous and leaky vessels unable to support efficient blood flow.4,5 The tumor vasculature appears to be in a continuous state of remodeling, involving simultaneous formation and regression of vessels. A possible contributing factor is excessive production of vascular endothelial growth factor (VEGF)-A by hypoxic tumor cells. Ectopic VEGF-A expression in normal tissues promotes the transient formation of abnormal vessels.6–8 Failure of tumor vessels to recruit a normal coat of mural cells (vascular smooth muscle cells and pericytes) may also contribute to the abnormal characteristics of tumor vessels.9–11 Contact between pericytes and endothelial cells has been suggested to stabilize vessels, promote endothelial survival,12 and inhibit endothelial cell proliferation.13 It is not known why tumor vessels fail to recruit a proper coverage of mural cells. During angiogenic formation of embryonic blood vessels, platelet-derived growth factor (PDGF)-B secreted from endothelial cells stimulates the spreading of PDGF receptor (PDGFR)-ß–positive pericytes to the new vascular structures.14 Mice lacking PDGF-B or PDGFRß show a severe deficit in pericytes and develop an abnormal vasculature with many features in common with tumor vessels, such as irregular vessel diameter and increased leakiness.14–16 Here, we analyzed mural cell organization and recruitment in the developing vasculature of 2 transplantable tumor models in mice using a number of markers for such cells. We also cotransplanted tumor cells and genetically tagged mouse embryo–derived mesenchymal cells to show that the tumor vasculature recruits mural cells efficiently when present. The mural cell deficiency in tumor vessels may result from an inherent inability to properly organize mural cells, but probably also from a limitation to the pool of recruitable mural cells.
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Methods |
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Tumor and MEF Cell Culture and Transplantation
T241 fibrosarcoma, KRIB osteosarcoma, and mouse embryonic fibroblast (MEF) cells (isolated as described by Levéen et al17) were propagated in DMEM with 10% FCS and standard supplements. For tumor experiments,
106 T241 cells or mixtures of T241 and MEF cells were suspended in 100 µL PBS and injected subcutaneously on the back of C57Bl6 mice. In some experiments, the MEF cells were prelabeled with the fluorescent dye PKH26 (Sigma) according to the manufacturer’s protocols. After 13 to 14 days, the tumors, 5 to 10 mm in diameter, were removed and processed for histological, immunohistochemical, and in situ RNA hybridization analysis. KRIB osteosarcoma cells were injected into the femoral bone of female nu/nu mice, at which site tumors 10 to 15 mm in diameter developed over a 6-week period.
Tissue Processing and Staining
For regular microscopy, tumors were fixed in 4% buffered paraformaldehyde, embedded in paraffin, cut at 4-µm thickness, and stained with antibodies against 
-smooth muscle actin (SMA) (clone 1A4) or desmin (clone D33; both from DakoPatts), as described.15 Endothelial cells were stained with a primary antibody against CD31 (PharMingen) and biotinylated rabbit anti-rat IgG (DakoPatts) as secondary antibody. For confocal microscopy, frozen tumors were fixed in 4% paraformaldehyde, cryoprotected in 30% sucrose, and cut into sections 50 µm thick. For double staining, sections were first incubated with antibody against CD31, followed by washing and incubation with a biotinylated rabbit anti-rat antibody. Sections were then incubated with streptavidin-Alexa568 (Molecular Probes) and FITC-conjugated SMA antibodies (clone 1A4, Sigma). For triple staining, sections were first incubated with antibody against CD31 and ß-galactosidase (Abcam), followed by a secondary goat anti–rabbit-Alexa568 antibody. After washing, sections were incubated with biotinylated rabbit anti-rat IgG and finally with streptavidin-Alexa633 (Molecular Probes) and FITC-conjugated SMA antibodies. All steps were performed in PBS containing 1% BSA and 0.5% Tween-20. Images were obtained by scanning specimens at intervals of 0.5 to 1 µmol/L with a Leica TCS-NT microscope and projecting layers to generate a composite image. In situ hybridization and ß-galactosidase staining were done as described.14,18
mRNA Isolation and Northern Blot Analysis
Total cellular RNA was isolated from confluent cell cultures with the RNeasy Mini kit (Qiagen). Total RNA (10 µg) was size-fractionated on a denaturing gel and transferred to a BrightStar-Plus membrane with a NorthernMax blotting kit (Ambion). Hybridization with 32P-labeled cDNA probes for GAPDH, PDGF-B, and PDGFRß and PDGFR and washings were performed according to the manufacturer’s instructions (Ambion) and standard protocols.
Vessel Quantification
Vessel profiles with and without associated SMA-positive (SMA+) cells were counted on cryosections 14 µm thick stained for CD31 and SMA. Five random 0.0625-mm2 fields at x400 magnification were counted in 
4 sections of each tumor specimen (T241, n=8; KRIB, n=5). Vessel density was expressed as the average number of vessels from all fields counted in each tumor. Vessel profiles associated with lacZ-positive cells in XlacZ4 (n=3) or desmin-lacZ (n=3) mice and T241:MEF mixtures (9:1, n=4; 5:5, n=5; and 1:9, n=3) were quantified on sections double-stained for CD31 and ß-galactosidase. Five random 0.0625-mm2 fields at x400 magnification were counted in 3 to 5 sections of each tumor specimen. Association with lacZ-expressing cells was calculated as the ratio between the total number of vessel profiles and the number of profiles with associated lacZ-positive cells.
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Results |
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Tumor Vessels Show Heterogeneous Mural Cell Coverage
The vasculature was studied in tumors arising from T241 fibrosarcoma cells transplanted to the subcutaneous space of the back skin of C57Bl6 mice and from human KRIB osteosarcoma cells orthotopically transplanted to the femoral bone in nude mice. Sections were stained with antibodies against the endothelial marker CD31 to visualize the tumor vasculature (Figure 1, A and B). The osteosarcoma showed a higher vessel density than the fibrosarcoma (140±28 versus 72±27 vessel profiles/mm2). Typically, the tumor vessel profiles of both tumors were highly irregular in their morphology. Antibodies against
-SMA and desmin were used to visualize mural cells associated with the tumor vessels. Both mural cell markers revealed a heterogeneous and discontinuous endothelial-mural cell association (Figure 1, C through F). In the fibrosarcoma, 56±21% of the vessel profiles were associated with SMA+ cells. In the osteosarcoma, only 5.8±2.7% of the profiles had associated SMA+ cells. Desmin staining showed a similar picture and was, like SMA, sparser in the osteosarcoma than in the fibrosarcoma. Different regions in the same tumors were compared to see whether mural cell coverage varied between regions with different vascular densities. In Figure 2, plots of vascular densities and mural cell coverage show that the mural cell density was constant over a wide range of vascular densities.
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The mural cell coverage index, as calculated above, is influenced by mural cell density, spreading, and shape and therefore provides limited information. To get a better picture of the mural cell organization in tumor vessels, we analyzed 50-µm-thick sections by confocal microscopy. Figure 3 shows images of tumor vessels with appreciable mural cell coverage (A through D) and vessels in immediately neighboring normal tissue (E, F). Each picture represents a stack of 0.5- to 1-µm confocal scans covering 20 to 40 µm of tissue thickness. The result shows that the organization of mural cells in tumor vessels and vessels of normal tissue is dramatically different. Regional clustering of SMA+ cells is interrupted by large areas of abluminal endothelial surface without SMA+ cells or with only thin associated SMA+ cytoplasmic processes (Figure 3, A through D, and data not shown). Whereas in the normal vessels, the SMA+ cells are preferentially encircling the vessels in a highly ordered fashion (Figure 1, E and F), their organization in the tumor vessels is mostly chaotic. Some mural cells of tumor vessels appear to be loosely associated with the vessels and extend away from the endothelium (Figure 3, C and D, arrows). There are also SMA+ cells without apparent vessel association (Figure 3A, arrowheads).
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Mural Cell Recruitment Occurs From the Host
The analysis above does not reveal whether the mural cells are host- or tumor-derived. This is not a trivial question, because certain tumor cells have been suggested to have the capacity of vascular mimicry, ie, to form vascular structures.19 In addition, both types of tumor cells used are of mesenchymal origin, and it cannot be ruled out that they may acquire mural cell properties within the tumors. To investigate whether host mural cells are recruited to tumor vessels, we injected T241 cells in desmin-lacZ20 and XlacZ4 mice,21 which both express lacZ in mural cells. In tumors grown in XlacZ4 mice, lacZ expression was completely restricted to the nuclei of periendothelial cells (Figure 4, B and D). The proportion of tumor vessel profiles with associated XlacZ4+ nuclei was 10.8%. This was lower than the proportion of profiles positive for SMA (56%), but the figures are nevertheless comparable, because 80% of the SMA+ profiles seen on thin sections do not contain a nucleus (data not shown). SMA is cytoplasmic, and the mural cells associated with tumor vessels display long and often branched cytoplasmic processes (Figure 3). Double staining revealed that lacZ+ cells were invariably SMA+ (data not shown). When tumors were transplanted to desmin-lacZ mice (Figure 4, A and C), lacZ+ nuclei became associated with 10.6% of the tumor vessel profiles, which is closely similar to the result for XlacZ4. Taken together, our results suggest that the vast majority of (or all) tumor vessel mural cells are host-derived in the 2 models analyzed.
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PDGF-B and PDGFRß Expression
In situ hybridization demonstrated strong expression of PDGF-B by endothelial cells of large and small blood vessels in both tumor types, but not in tumor cells in vivo (Figure 5, A and B, and data not shown) or in vitro (Figure 5F). PDGF-B expression was likewise undetectable in blood vessels of the normal tissues surrounding the tumors (not shown), which is consistent with previous notions that PDGF-B expression is restricted to growing vessels15 and to proliferating endothelial cells in vitro.22 PDGF-B–expressing endothelial cells were intermingled with nonexpressing cells in the vessel profiles (Figure 5B), indicating that neighboring endothelial cells may have different patterns of gene expression.
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At low frequency, tumor vessel profiles were found to be associated with PDGFRß-positive cells, as detected by in situ hybridization (Figure 5C). These profiles were located mainly at the periphery of the tumor and may represent newly recruited mural cells (or progenitors). The vast majority of vessel profiles, however, lacked PDGFRß-positive cells (Figure 5D). When found, PDGFRß-positive cells were tightly associated with, but distinct from, endothelial cells, as revealed by combined PDGFRß in situ hybridization and CD31 immunohistochemistry (Figure 5E). No expression of PDGFRß was detected in the KRIB tumor cells in vitro, and only a very weak signal was detected in T241 cells (Figure 5F). In contrast, MEF cultures expressed abundant PDGFRß mRNA. PDGFR was expressed in cultured T241 cells but not in KRIB or in MEF cells.
Coinjected Embryonic Cells Become Recruited as Mural Cells by Tumor Endothelium
The abundant expression of PDGF-B mRNA by the tumor endothelium suggests that the limited ability of tumor vessels to recruit a proper coating of mural cells is not caused by lack of PDGF-B. Several other factors with direct regulatory or permissive roles in mural cell recruitment may exist, however, including other growth factors, receptors, intracellular signal transduction molecules, cell adhesion molecules, and extracellular matrix molecules. In principle, any such factor may be deficient in tumor vessels as an underlying cause of the deficient mural cell recruitment. One way to analyze the inherent capacity of tumor vessels to attract mural cells is to add genetically tagged pericytes to tumors and study the efficiency with which they are recruited to tumor vessels. T241 fibrosarcoma cells were therefore mixed with different amounts of MEF cells derived from E12.5 XlacZ4 embryos, and the resulting cell mixtures were injected subcutaneously into C57Bl6 mice. Cell suspensions containing a total of 106 cells of T241:MEF cells in ratios of 9:1, 5:5, and 1:9 were injected. Tumors grew at the same rate irrespective of whether MEF cells were coadded or not, with the exception of the T241:MEF 1:9 ratio, which initially grew at a slower rate, presumably because as little as 105 tumor cells were inoculated. After 14 days, the tumors were removed, fixed, sectioned, and double-stained with X-gal and antibody against CD31 (Figure 6C) or triple fluorescence–stained for lacZ (red), CD31 (blue), and SMA (green) (Figure 6, A and B). These stainings demonstrate XlacZ4+/SMA+ cells in tight association with the tumor vessels. Vessel-associated LacZ+ cells were found in all tumors containing MEF cells, and the number of such cells increased with increasing ratio of MEF:T241 cells in the injected mixture. In tumors resulting from 5:5 mixtures, 7.4% of the vessel profiles were associated with lacZ+ cells. In the 1:9 mixture (T241:MEF), 11.3% of the vessel profiles showed associated lacZ+ cells.
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The MEF cultures are heterogeneous mixtures of embryonic mesenchymal cells, of which 5% are lacZ+ at the time of transplantation (data not shown). In the 5:5 and 1:9 mixtures of T241 and MEF, 25 000 to 50 000 XlacZ4+ cells were thus transplanted, which corresponds roughly to the numbers found in the tumors after sectioning (data not shown). To visualize the entire progeny of the transplanted MEFs, they were prelabeled with the red fluorescent dye PKH26 before transplantation. As expected, there were abundant PKH26-positive cells in the tumors, of which the majority were not vessel-associated (Figure 6D). However, 96% of the XlacZ4+ cells (307 of 320 scored cells in tumors arising from 5:5 mixtures) were found to have periendothelial locations. This may suggest that a subpopulation of already committed mural cells, preexisting in the MEF culture and transplanted together with the tumor cells, is efficiently attracted to the tumor vessel wall. Alternatively, a limited number of undifferentiated MEFs are recruited to the vessels, to subsequently turn on the expression of the XlacZ4 marker.
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Discussion |
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The formation of a mature and functional vasculature is not only a matter of migration and proliferation of endothelial cells; the subsequent investment of the endothelial tube by vascular smooth muscle cells and pericytes is clearly also essential. Knowledge about the mechanism governing this process is limited. In a number of mutant mice, lethal vascular abnormalities have been correlated with deficient recruitment of vascular mural cells. These mutants include components of the transforming growth factor-ß,23,24 angiopoietin,25–27 and PDGF-B/Rß17,28–30 signaling pathways. Our previous analysis of PDGF-B– and PDGFRß-knockout mice revealed many abnormal features of the pericyte-deficient vessels that were similar to tumor vessels, such as variable (often increased) diameter and increased permeability.15,16 The fact that tumor vessels have been reported to be deficiently invested by mural cells9–11,31 therefore attracted our attention.
The vasculature of the 2 tumor models studied here was found to be abnormal in characteristic ways. The mural cell coverage of the abluminal endothelial surface was sparse and heterogeneous and lacked proper organization. Together with other published evidence suggesting that mural cells play a role in stabilizing microvessels and promoting endothelial maturation and survival, this reinforces the notion that lack of (proper arrangement of) mural cells may contribute to the abnormal phenotype of tumor vessels, including the irregular morphology and inefficient blood flow. The apparent inability of tumor vasculature to attract a proper coat of mural cells may give rise to an immature vessel network that depends on a continuous supply of VEGF-A for survival and in which differentiation into quiescent, organized, and functional vessels does not take place.10
The strong PDGF-B mRNA expression by the tumor endothelium suggests that the relative shortage of mural cells observed in our tumor models is caused by factors other than deficient PDGF-B expression. The small number of PDGFRß-positive mural cells found in association with tumor vessels contrasts to the situation in embryos.14,15 This may suggest that a limited pool of mural cell progenitor cells is available for recruitment to tumor vessels in the adult mouse. Alternatively, competent mural progenitors are present but fail to be recruited properly because necessary factors other than PDGF-B are missing. The ability of tumor vessels to recruit exogenously added mural cells (or their progenitors) was therefore assessed in tumors derived from mixtures of T241 cells and XlacZ4+ MEF cells. Although only a small proportion of the MEF cells were XlacZ4+ in vitro and in vivo, the majority of the lacZ+ cells were specifically recruited to the tumor vessels. This demonstrates that tumor vessels are highly capable of attracting mural cells.
The issue of mural cell recruitment to new blood vessels in the adult organism has relevance beyond the question of tumor vasculature and its abnormal features. In therapeutic angiogenesis, the goal is to produce stable and functional vessels. VEGF expression in vivo produces an angiogenic response, but the vessels formed are morphologically abnormal, poorly functional, and transient.6–8 This may be connected to an insufficient recruitment of mural cells. Addition of factors with the potential of expanding the pool of mural cell progenitors or addition of ectopic mural cell progenitors in conjunction with proangiogenic therapy may therefore be worth considering.
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Acknowledgments |
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This study was supported by grants from the Swedish Cancer Society, the Association de Recherche contre le Cancer, the Inga-Britt and Arne Lundberg Foundation, and the Novo Nordisk Foundation. A. Abramsson is supported by a fellowship from the Cardiovascular Program of the Swedish Foundation for Strategic Research. We thank Dr Holger Gerhardt for helpful comments on the manuscript.
Received August 8, 2001; revision received October 16, 2001; accepted October 17, 2001.
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References |
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P. Baluk, C. G. Lee, H. Link, E. Ator, A. Haskell, J. A. Elias, and D. M. McDonald Regulated Angiogenesis and Vascular Regression in Mice Overexpressing Vascular Endothelial Growth Factor in Airways Am. J. Pathol., October 1, 2004; 165(4): 1071 - 1085. [Abstract] [Full Text] [PDF]
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T. Chan-Ling, M. P. Page, T. Gardiner, L. Baxter, E. Rosinova, and S. Hughes Desmin Ensheathment Ratio as an Indicator of Vessel Stability: Evidence in Normal Development and in Retinopathy of Prematurity Am. J. Pathol., October 1, 2004; 165(4): 1301 - 1313. [Abstract] [Full Text] [PDF]
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T. Inai, M. Mancuso, H. Hashizume, F. Baffert, A. Haskell, P. Baluk, D. D. Hu-Lowe, D. R. Shalinsky, G. Thurston, G. D. Yancopoulos, et al. Inhibition of Vascular Endothelial Growth Factor (VEGF) Signaling in Cancer Causes Loss of Endothelial Fenestrations, Regression of Tumor Vessels, and Appearance of Basement Membrane Ghosts Am. J. Pathol., July 1, 2004; 165(1): 35 - 52. [Abstract] [Full Text] [PDF]
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 O. Stoeltzing, M. F. McCarty, J. S. Wey, F. Fan, W. Liu, A. Belcheva, C. D. Bucana, G. L. Semenza, and L. M. Ellis Role of Hypoxia-Inducible Factor 1{alpha} in Gastric Cancer Cell Growth, Angiogenesis, and Vessel Maturation J Natl Cancer Inst, June 16, 2004; 96(12): 946 - 956. [Abstract] [Full Text] [PDF]
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M. Furuhashi, T. Sjoblom, A. Abramsson, J. Ellingsen, P. Micke, H. Li, E. Bergsten-Folestad, U. Eriksson, R. Heuchel, C. Betsholtz, et al. Platelet-Derived Growth Factor Production by B16 Melanoma Cells Leads to Increased Pericyte Abundance in Tumors and an Associated Increase in Tumor Growth Rate Cancer Res., April 15, 2004; 64(8): 2725 - 2733. [Abstract] [Full Text] [PDF]
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 M. Bjarnegard, M. Enge, J. Norlin, S. Gustafsdottir, S. Fredriksson, A. Abramsson, M. Takemoto, E. Gustafsson, R. Fassler, and C. Betsholtz Endothelium-specific ablation of PDGFB leads to pericyte loss and glomerular, cardiac and placental abnormalities Development, April 15, 2004; 131(8): 1847 - 1857. [Abstract] [Full Text] [PDF]
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P. Baluk, S. Morikawa, A. Haskell, M. Mancuso, and D. M. McDonald Abnormalities of Basement Membrane on Blood Vessels and Endothelial Sprouts in Tumors Am. J. Pathol., November 1, 2003; 163(5): 1801 - 1815. [Abstract] [Full Text] [PDF]
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