Contrasting effects of VEGF gene disruption in embryonic stem cell-derived versus oncogene-induced tumors
2003; Springer Nature; Volume: 22; Issue: 16 Linguagem: Inglês
10.1093/emboj/cdg408
ISSN1460-2075
Autores Tópico(s)Cancer, Hypoxia, and Metabolism
ResumoArticle15 August 2003free access Contrasting effects of VEGF gene disruption in embryonic stem cell-derived versus oncogene-induced tumors Alicia Viloria-Petit Alicia Viloria-Petit Molecular and Cellular Biology Research, Sunnybrook and Women's College Health Sciences Centre and Department of Medical Biophysics, University of Toronto, 2075 Bayview Avenue, Toronto, Ontario, M4N 3M5 Canada Search for more papers by this author Lucile Miquerol Lucile Miquerol Samuel Lunenfeld Research Institute, Mount Sinai Hospital, University of Toronto, 600 University Avenue, Toronto, Ontario, M5G 1X5 Canada Search for more papers by this author Joanne L. Yu Joanne L. Yu Henderson Research Centre, Experimental Thrombosis Research, McMaster University, 711 Concession Street, Hamilton, Ontario, L8V 1C3 Canada Search for more papers by this author Marina Gertsenstein Marina Gertsenstein Samuel Lunenfeld Research Institute, Mount Sinai Hospital, University of Toronto, 600 University Avenue, Toronto, Ontario, M5G 1X5 Canada Search for more papers by this author Capucine Sheehan Capucine Sheehan Molecular and Cellular Biology Research, Sunnybrook and Women's College Health Sciences Centre and Department of Medical Biophysics, University of Toronto, 2075 Bayview Avenue, Toronto, Ontario, M4N 3M5 Canada Search for more papers by this author Linda May Linda May Henderson Research Centre, Experimental Thrombosis Research, McMaster University, 711 Concession Street, Hamilton, Ontario, L8V 1C3 Canada Search for more papers by this author Jack Henkin Jack Henkin Abbott Laboratories, 100 Abbott Park Road, North Chicago, IL, 60064-4000 USA Search for more papers by this author Corrinne Lobe Corrinne Lobe Molecular and Cellular Biology Research, Sunnybrook and Women's College Health Sciences Centre and Department of Medical Biophysics, University of Toronto, 2075 Bayview Avenue, Toronto, Ontario, M4N 3M5 Canada Search for more papers by this author Andras Nagy Andras Nagy Samuel Lunenfeld Research Institute, Mount Sinai Hospital, University of Toronto, 600 University Avenue, Toronto, Ontario, M5G 1X5 Canada Search for more papers by this author Robert S. Kerbel Robert S. Kerbel Molecular and Cellular Biology Research, Sunnybrook and Women's College Health Sciences Centre and Department of Medical Biophysics, University of Toronto, 2075 Bayview Avenue, Toronto, Ontario, M4N 3M5 Canada Search for more papers by this author Janusz Rak Corresponding Author Janusz Rak Henderson Research Centre, Experimental Thrombosis Research, McMaster University, 711 Concession Street, Hamilton, Ontario, L8V 1C3 Canada Search for more papers by this author Alicia Viloria-Petit Alicia Viloria-Petit Molecular and Cellular Biology Research, Sunnybrook and Women's College Health Sciences Centre and Department of Medical Biophysics, University of Toronto, 2075 Bayview Avenue, Toronto, Ontario, M4N 3M5 Canada Search for more papers by this author Lucile Miquerol Lucile Miquerol Samuel Lunenfeld Research Institute, Mount Sinai Hospital, University of Toronto, 600 University Avenue, Toronto, Ontario, M5G 1X5 Canada Search for more papers by this author Joanne L. Yu Joanne L. Yu Henderson Research Centre, Experimental Thrombosis Research, McMaster University, 711 Concession Street, Hamilton, Ontario, L8V 1C3 Canada Search for more papers by this author Marina Gertsenstein Marina Gertsenstein Samuel Lunenfeld Research Institute, Mount Sinai Hospital, University of Toronto, 600 University Avenue, Toronto, Ontario, M5G 1X5 Canada Search for more papers by this author Capucine Sheehan Capucine Sheehan Molecular and Cellular Biology Research, Sunnybrook and Women's College Health Sciences Centre and Department of Medical Biophysics, University of Toronto, 2075 Bayview Avenue, Toronto, Ontario, M4N 3M5 Canada Search for more papers by this author Linda May Linda May Henderson Research Centre, Experimental Thrombosis Research, McMaster University, 711 Concession Street, Hamilton, Ontario, L8V 1C3 Canada Search for more papers by this author Jack Henkin Jack Henkin Abbott Laboratories, 100 Abbott Park Road, North Chicago, IL, 60064-4000 USA Search for more papers by this author Corrinne Lobe Corrinne Lobe Molecular and Cellular Biology Research, Sunnybrook and Women's College Health Sciences Centre and Department of Medical Biophysics, University of Toronto, 2075 Bayview Avenue, Toronto, Ontario, M4N 3M5 Canada Search for more papers by this author Andras Nagy Andras Nagy Samuel Lunenfeld Research Institute, Mount Sinai Hospital, University of Toronto, 600 University Avenue, Toronto, Ontario, M5G 1X5 Canada Search for more papers by this author Robert S. Kerbel Robert S. Kerbel Molecular and Cellular Biology Research, Sunnybrook and Women's College Health Sciences Centre and Department of Medical Biophysics, University of Toronto, 2075 Bayview Avenue, Toronto, Ontario, M4N 3M5 Canada Search for more papers by this author Janusz Rak Corresponding Author Janusz Rak Henderson Research Centre, Experimental Thrombosis Research, McMaster University, 711 Concession Street, Hamilton, Ontario, L8V 1C3 Canada Search for more papers by this author Author Information Alicia Viloria-Petit1, Lucile Miquerol2, Joanne L. Yu3, Marina Gertsenstein2, Capucine Sheehan1, Linda May3, Jack Henkin4, Corrinne Lobe1, Andras Nagy2, Robert S. Kerbel1 and Janusz Rak 3 1Molecular and Cellular Biology Research, Sunnybrook and Women's College Health Sciences Centre and Department of Medical Biophysics, University of Toronto, 2075 Bayview Avenue, Toronto, Ontario, M4N 3M5 Canada 2Samuel Lunenfeld Research Institute, Mount Sinai Hospital, University of Toronto, 600 University Avenue, Toronto, Ontario, M5G 1X5 Canada 3Henderson Research Centre, Experimental Thrombosis Research, McMaster University, 711 Concession Street, Hamilton, Ontario, L8V 1C3 Canada 4Abbott Laboratories, 100 Abbott Park Road, North Chicago, IL, 60064-4000 USA *Corresponding author. E-mail: [email protected] The EMBO Journal (2003)22:4091-4102https://doi.org/10.1093/emboj/cdg408 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Previous gene targeting studies have implicated an indispensable role of vascular endothelial growth factor (VEGF) in tumor angiogenesis, particularly in tumors of embryonal or endocrine origin. In contrast, we report here that transformation of VEGF-deficient adult fibroblasts (MDF528) with ras or neu oncogenes gives rise to highly tumorigenic and angiogenic fibrosarcomas. These aggressive VEGF-null tumors (528ras, 528neu) originated from VEGF−/− embryonic stem cells, which themselves were tumorigenically deficient. We also report that VEGF production by tumor stroma has a modest role in oncogene-driven tumor angiogenesis. Both ras and neu oncogenes down-regulated at least two endogenous inhibitors of angiogenesis [pigment epithelium derived factor (PEDF) and thrombospondin 1 (TSP-1)]. This is functionally important as administration of an antiangiogenic TSP-1 peptide (ABT-526) markedly inhibited growth of VEGF−/− tumors, with some ingress of pericytes. These results provide the first definitive genetic demonstration of the dispensability of tumor cell-derived VEGF in certain cases of ‘adult’ tumor angiogenesis, and thus highlight the importance of considering VEGF-independent as well as VEGF-dependent pathways when attempting to block this process pharmacologically. Introduction Angiogenesis is triggered by a change in balance between different pro- and anti-angiogenic activities that regulate the behavior of capillary endothelial cells (Hanahan and Folkman, 1996). In this regard, a pre-eminent role has been established for the vascular endothelial growth factor (VEGF)-related family of angiogenic and lymphangiogenic growth factors, which currently includes VEGF-A, -B, -C, -D, -E, and placenta growth factor (PlGF). VEGF-A is believed to play an indispensable role in angiogenesis. Indeed, targeting of the VEGF-A gene in mice resulted in early embryonic lethality due to severe structural and functional abnormalities in the developing vasculature, even when only a single VEGF-A allele was inactivated (Carmeliet et al., 1996; Ferrara et al., 1996). Embryonic lethality is also induced by targeted disruption of either of the two main VEGF receptors expressed by endothelial cells, namely VEGFR-2 (Flk-1/KDR) and VEGFR-1 (Flt-1), the former regarded as the main transducer of positive pro-angiogenic signals (Carmeliet, 2000). The profound influence of the VEGF/VEGF receptor axis on vascular development and angiogenesis is likely linked to its role as a stimulator of endothelial cell survival, mitogenesis, migration, differentiation and self-assembly, as well as vascular permeability and mobilization of endothelial progenitor cells (EPCs) from the bone marrow into the peripheral circulation (Ferrara and Gerber, 2001). There are numerous reasons to suggest that VEGF also plays an important role in ‘pathological’ forms of angiogenesis, including tumor neovascularization (Ferrara and Gerber, 2001). For instance, VEGF expression is elevated in the majority of human cancers, and in many transformed cell lines in culture (Dvorak et al., 1995). Furthermore, transforming genetic lesions such as activated oncogenes (ras, neu/HER-2 and at least 20 others) (Rak and Kerbel, 2003) and inactivated tumor suppressor genes (e.g. p53, VHL, PTEN, INK4a/p16) have a direct stimulating impact on VEGF expression in cancer cells (Bouck et al., 1996). A causative role for VEGF in tumor angiogenesis has been implicated by numerous studies (Claffey et al., 1996; Ferrara and Gerber, 2001). In this regard, particularly striking are the results involving wild-type (Ferrara et al., 1996) or oncogenically transformed embryonic stem (ES) cells (Shi and Ferrara, 1999), embryonic fibroblasts (Grunstein et al., 1999) or endocrine pancreatic cells (Inoue et al., 2002), in all of which total inactivation of the VEGF gene was found to result in severe suppression of tumorigenic and angiogenic phenotypes. These studies suggest implicitly that not only is VEGF essential and indispensable for tumor growth and neovascularization, but also that tumor cells themselves constitute the major source of VEGF. There are, however, experimental findings that seem to be at variance with the notion that VEGF is a non-redundant tumor angiogenesis factor that is predominantly produced by cancer cells. For example, appreciable VEGF expression has been detected in tumor-associated fibroblasts (Hlatky et al., 1994; Fukumura et al., 1998; Kishimoto et al., 2000) and inflammatory cells (Coussens et al., 1999). This suggests that the host stromal compartment may constitute a major source of VEGF in tumors (Fukumura et al., 1998). It has also been observed that reduction of VEGF expression in cancer cells may be functionally insignificant in certain advanced experimental tumors (Yoshiji et al., 1997), and that tumor progression is associated with expression of an increasing number of different pro-angiogenic growth factors, in addition to VEGF (Relf et al., 1997). Moreover, while the anti-tumor effects of neutralizing antibodies and pharmacological antagonists of VEGF or its receptors have been promising in many cases (Kim et al., 1992; Ferrara and Gerber, 2001), in other instances the responses have been variable, transient and sometimes rather modest in magnitude, especially in advanced tumors (Garber, 2002). Indeed, a direct analysis has been lacking as to how critical the role of tumor cell-derived versus stromal VEGF is in various tumor angiogenesis settings. The purpose of our study was to address this question by investigating two fundamentally different forms of tumorigenesis (and associated angiogenesis) occurring in either the presence or absence of the functional VEGF gene and protein expression in cancer cells. Our data indicate that teratomas derived from totipotent (genetically intact) ES cells were absolutely VEGF dependent. In contrast, adult mouse dermal fibroblasts expressing mutant ras or neu oncogenes remained tumorigenic even if rendered VEGF-null. Such tumors recruited VEGF-expressing host cells and down-regulated at least two potent angiogenesis inhibitors, such as pigment epithelium derived factor (PEDF) and thrombospondin 1 (TSP-1). Thus, VEGF production by cancer cells may be non-essential in the context of oncogene-driven tumorigenesis. Results Tumorigenic properties of VEGF-deficient ES cells We decided to test the limits of VEGF involvement in tumor angiogenesis by comparing the impact of VEGF deletion on the tumor forming capacity of ES cell-derived teratomas or their related, but adult, cell descendants transformed with mutant oncogenes. First, we employed the R1 strain of ES cells (Nagy et al., 1993). Both wild-type R1 cells (wtR1) and their VEGF-deficient counterparts (clones 44.7 and 36.8) were injected subcutaneously (s.c.) into SCID mice. As expected, inoculation of wtR1 cells resulted in the rapid growth of aggressive and highly vascularized teratomas (Ferrara et al., 1996) (Figure 1). Such tumors display a complex morphology and contain a rich network of CD31-positive host blood vessels (Yu et al., 2001). In marked contrast, R1 cells in which the VEGF gene was disrupted were unable to form tumors for at least 50 days after inoculation of as many as 7 × 106 cells (Figure 1A). As growth in vitro of both types of ES cells is not influenced by their VEGF status (data not shown), we attributed these in vivo properties of teratomas to VEGF-dependent angiogenesis. Indeed, treatment of mice harboring wild-type R1 tumors with a neutralizing antibody (DC101) directed against VEGFR-2/flk-1 resulted in nearly complete inhibition of tumor growth (Figure 1B). Collectively, these observations are in keeping with the notion that the endogenous production of VEGF and its interaction with endothelial VEGFR-2 are essential events during formation and vascularization of murine R1 teratoma. Figure 1.Dependence of ES-cell-derived mouse teratomas on VEGF/VEGFR-2-driven angiogenesis. (A) Impaired tumor formation by VEGF−/− ES cells (R1, clones 36.8 and 44.7) in comparison to their wild-type (VEGF+/+) counterparts. (B) Inhibition of R1-derived teratoma growth by anti-VEGFR-2 antibody (DC101; 800 μg every 3 days), control mice received the vehicle (PBS). Download figure Download PowerPoint Generation of VEGF-deficient oncogene-transformed fibrosarcoma cell lines In contrast to the epigenetic nature of ES cell-derived teratomas, the majority of human tumors harbor various transforming genetic alterations. In order to assess the role of VEGF in the latter type, i.e oncogene-driven tumor angiogenesis, we generated a series of oncogene-transformed VEGF-proficient (VEGF+/+) or VEGF-deficient (VEGF−/−) fibrosarcoma cell lines. As summarized in Figure 2A, 4- to 5-month-old chimeric (VEGF−/−,VEGF+/+) mice were used as donors of skin explants. Primary cultures of dermal fibroblasts were subsequently established and infected with a retrovirus expressing both ras and myc oncogenes (Soloway et al., 1996). Alternatively, VEGF−/− fibroblasts were spontaneously immortalized and the resulting cell line (MDF528) was transfected with expression vectors encoding either an activated H-ras or neu oncogene (Figure 2A). All cell lines harboring mutant oncogenes (but not the MDF528 immortalized parental cells or their 528Hp hygromycin-resistant vector transfectants) exhibited the usual features of morphological trasformation (Figure 2B), formed foci in monolayer culture and grew as colonies in semi-solid media (data not shown). VEGF status in all transformed and non-transformed cell lines was verified by testing for G418 resistance (conferred by the VEGF targeting vector), Southern analysis and direct determination of VEGF mRNA and protein expression (Figure 3A–D). Figure 2.Derivation of adult oncogene transformed VEGF-deficient fibrosarcoma cell lines. (A) Derivation of VEGF−/− and VEGF+/+ ras- and neu- transformed cell lines (see text). (B) Morphology of fibrosarcoma cells harboring ras or neu oncogenes and their non-transformed parental MDF528 counterparts; immunostaining for vimentin, a fibroblast-specific cytoskeletal marker. Download figure Download PowerPoint Figure 3.Characterization of the VEGF status in fibrosarcoma cell lines. (A) Southern analysis of the VEGF gene in tumorigenic and non- tumorigenic fibroblastic cell lines: EcoRI digest (see text and Figure 2). HR, recombined/targeted VEGF gene in VEGF−/− cell lines; Wt, wild-type VEGF gene. (B) Absence of the typical 3.7–4.5 VEGF mRNA in cell lines with targeted VEGF gene. In some cases a larger transcript was detectable as described previously (Ferrara et al., 1996). (C) Absence of VEGF in conditioned medium of MDF528-derived cell lines (VEGF ELISA) treated with VEGF inducers (various FBS concentrations and/or CoCl2). 528ras1T1 and 528ras1T2 are cell lines re-established in culture from tumors that arose after s.c. injection of 528ras1 cells. (D) VEGF levels in ras/myc-transformed clones of primary dermal fibroblasts isolated from chimeric VEGF-positive/negative mice. Transformed clones with a targeted VEGF gene were negative for VEGF protein expression, whereas their G418 sensitive (non- targeted VEGF) counterparts from the same culture produced an abundance of VEGF, particularly in the presence of CoCl2. High serum levels caused a decrease in VEGF production by VEGF+/+ transformed cell lines. Download figure Download PowerPoint Tumorigenic properties of VEGF−/− cell lines expressing activated oncogenes In marked contrast to ES cell-induced teratomas, 100% tumor take occurred in the case of all oncogene-driven fibrosarcomas regardless of their VEGF status, and this was followed by aggressive growth (Figure 4A–C; Table I) of highly vascular tumors (see Figure 7A–C). However, the overall growth kinetics of VEGF−/− ras- or neu-dependent fibrosarcomas was somewhat slower than that of their VEGF+/+ 3T3ras counterparts (Figure 4A) or 528rasmV1-7 cells engineered to re-express mouse VEGF164 (Figure 4B). The relative aggressiveness in vivo of various fibrosarcomas also correlated with their apparent degree of cellular transformation. Thus, 528neu7 cells, with lower expression of activated NEU (Figures 2B and 4D), were considerably less aggressive in vivo (Figures 4C and 6C) than more transformed 528neu3 and 528neu2 cell lines. Collectively, these results suggest that in the case of oncogene-driven tumors, malignant growth capacity in vivo is not dependent on VEGF production by cancer cells (unlike in teratomas). Figure 4.In vivo growth characteristics of VEGF−/− fibrosarcoma cell lines. (A) Three independent clones of H-ras-transformed VEGF−/− cells (528ras1, -3 and -5) form aggressive tumors in SCID mice upon s.c. inoculation in a manner comparable to that of NIH 3T3-derived tumor cell line (3T3ras, VEGFwt). Differences in tumorigenic potential of various 528ras clones correlated with their degree of morphological transformation (not shown), while injected non-transformed parental MDF528 cells were non-tumorigenic (up to 7 months). (B) Overexpression of mouse VEGF164 in 528ras1 cells (two independent clones 528rasmV1 and 528rasmV7) results in a moderately increased tumorigenic potential of these cells (106 cells injected s.c) as compared to their 528ras1 parental cells and control transfectant (528rasLZ). (C) Differential tumorigenicity of MDF528-derived cell lines overexpressing neu oncogene versus control polyclonal cell line (528Hp) transfected with the hygromycin expression vector. (D) Correlation between the level/activity (phosphorylation) of the NEU oncoprotein and the tumorigenic potential of MDF528-derived VEGF−/− fibrosarcoma cell lines [compare (C) and (D)]. Blot was exposed for longer time (WB: anti-pTyr, second strip) in order to visualize phosphorylated form of NEU in both control transfectants (528HP) and tumorigenic 528neu7 cells. Download figure Download PowerPoint Figure 5.The impact of VEGFR-2 inhibition on growth of VEGF-deficient tumors. (A) Variable responsiveness to anti-VEGFR-2 antibody (DC101; 800 μg every 3–4 days) of neu-driven fibrosarcoma tumors (528neu3 tumors exhibit 50% growth inhibition upon administration of DC101, no such responses were observed in the case of 528neu2 tumors). (B) Ras-dependent tumors (528ras1) responded weakly to treatment with DC101. (C) Reduction in mean blood vessel density (MVD) in 528neu3 tumors upon DC101 treatment (CD31 immunofluorescence). Download figure Download PowerPoint Figure 6.Stromal VEGF in oncogene-driven fibrosarcomas. (A) Southern analysis of 528ras tumors and cultured cells. Both targeted (13.1 kb) and wild-type (7.6 kb) VEGF alleles were present in DNA isolated from several tumors but not that from cultured cells (compare with Figure 3). (B) Quantification of VEGF mRNA in cultured cells and tumors (mRNA ELISA). VEGF mRNA was detected only in VEGF+/+ cells (3T3ras, B104.1.1) in culture and not in MDF528-derived transformed and non-transformed VEGF−/− cell lines. In contrast, tumor masses of both VEGF−/− (528ras1, 528neu2, 528neu3) and VEGF+/+ (B104.1.1) fibrosarcomas contained measurable quantities of VEGF mRNA (see text). (C) Differential latency of VEGF−/− tumors expressing different levels of activated neu oncogene (528neu3, high neu expressors; 528neu7, low neu expressors). (D) Degree of malignant transformation (with neu) coincides with the ability of tumor cells to trigger stromal VEGF expression (highly transformed 528neu3 cells, high stromal VEGF; moderately transformed 528neu7 cells, low levels of stromal VEGF; compare with Figure 4D). Only tumors of equivalent size (700 mm3) were compared in this assay. Download figure Download PowerPoint Figure 7.Cellular composition of VEGF-deficient fibrosarcomas. Both (A) ras- and (B and C) neu-dependent tumors are highly vascularized (528ras1, 528neu3 and 528neu7, respectively) and contain dense networks of capillaries positive for endothelial markers (CD31). (C) Lower vascular density was detected in less aggressive 528neu7 tumors (CD31) than in their 528neu3 counterparts. (D) Capillaries inside tumor masses are mostly devoid of αSMA-positive (528ras1 tumor) or (E) desmin-positive (528neu3) pericytes. Such mural cells are detectable in peri-tumoral connective tissue [(D) and (E), orange arrows]. (F) Single mast cells present at the tumor periphery (CAE staining, white arrow) but not within the tumor. (G) Diffuse staining for vimentin within the 528ras1 tumor. Stroma and connective tissue stains slightly more intensively (black arrows). (H) Confocal image of YFP positive host cells associated with tumors (528ras1 cells non-fluorescent); T, tumor interior; S, skin. (I) Fibroblastoid stromal cells in tumors growing in YFP/SCID mice. (J) YFP-positive tumor blood vessel and surrounding stroma within 528ras1 tumor; Fib, fibroblast-like stromal cells; EC, endothelial cells; RBC, red blood cells. (K and L) LacZ staining of VEGF-producing stromal cells within 528ras1 tumors growing in mice harbouring a LacZ-tagged VEGF allele. Magnifications: (I), (J) and (L), 40×; (A)–(C), (E), (F) and (K), 20×; (D) and (H), 10×. Download figure Download PowerPoint Table 1. Summary of tumorigenic properties of various VEGF−/− and VEGF+/+ fibroblastic cell lines Cell linea VEGF genotype VEGF production Tumor take 3T3ras +/+ + 4/4 5D10 +/+ + 4/4 8C2V2 +/+ + 4/4 1E3 −/− − 4/4 8G5 −/− − 4/4 MDF528 −/− − 0/10 528ras1z −/− − 4/4 528ras3z −/− − 4/4 528ras5z −/− − 4/4 3T3neu (B104.1.1) +/+ + 5/5 MDF528 hygro (528Hp) −/− − 0/10 528neu2 −/− − 5/5 528neu3 −/− − 5/5 528neu7 −/− − 5/5 aSee Materials and methods and Figure 2 for details. 3T3ras are NIH 3T3 cells transfected with mutant H-ras; 5D10, 8G5 fibroblasts infected with ras/myc retrovirus; MDF528 and MDF528 hygro/528Hp, non-transformed immortalized adult dermal fibroblasts; 528ras or 528neu, MDF528 cells transfected with H-ras or neu oncogenes, respectively. We reasoned that stromal cells could be a source of VEGF in VEGF-deficient tumors, and if so, the administration of VEGFR-2 antagonists could still suppress tumor growth. Therefore, mice harboring 528neu3 tumors were treated with the DC101 antibody (against mouse VEGFR-2), and tumor growth inhibition of up to 50% (Figure 5A), as well as ∼2-fold reduction in microvascular density, were indeed observed (Figure 5C). However, responsiveness to such treatment (DC101, 800 μg twice a week) was negligible in the case of two other fibrosarcomas (i.e. 528neu2 and 528ras1; Figure 5A and B, respectively). This suggests that activation of the VEGF/VEGFR-2 pathway by tumor stroma is not a sine qua non in fibrosarcoma. Expression of VEGF by host stromal cells recruited by oncogene-driven tumors Regardless of their responsiveness to VEGFR-2 inhibition, ample stromal cell infiltration was observed in all oncogene-driven fibrosarcomas tested. For instance, even in relatively DC101-unresponsive 528ras1 tumors, Southern analysis revealed both the wild-type (host) and the targeted VEGF alleles (the former being absent in cultured cells) (Figures 3A and 6A). Moreover, Figure 6 shows that in contrast to various 528ras and 528neu VEGF-deficient cultured cell lines, the material explanted from the corresponding tumors contained measurable quantities of VEGF mRNA (Figure 6B) and protein (Figure 6D). Although VEGF was detectable in the original tumor masses, cancer cell lines re-established from such tumors (528rasT1, 528rasT2) were still devoid of any VEGF expression, even when exposed to various serum concentrations or to cobalt chloride (Figure 3C). Interesting but presently unexplained differences in the expression of stromal VEGF and microvascular vascular densities (MVD) were noted between fibrosarcomas with different levels of oncogenic transformation (e.g. 528neu7 versus 528neu 3) (Figures 4D, 6D, and 7B and C). Overall, these results can be interpreted as an (indirect) indication that, while (cultured) tumor cells of the 528 series were completely VEGF-negative, VEGF-positive stromal cells were, in fact, relatively abundant in the corresponding in vivo tumors. To gain a more direct insight into the cellular sources of the stromal-derived VEGF, we injected 528ras or 528neu cells into syngeneic or SCID mice harboring the yellow fluorescent protein (YFP) transgene. Confocal images (Figures 7H–J) of the resulting tumors revealed a rich network of YFP-positive host cells (tumor cells being non-fluorescent) detectable at the boundary between the YFP-negative tumor cell masses and the overlaying YFP-positive skin (Figure 7H). YFP-positive cells were also present within the tumor interior and decorated vascular channels and their surroundings (Figure 7I and J). By immunostaining of the corresponding tumor sections we were able to positively identify numerous endothelial cells (CD31-positive) comprising tumor capillaries (Figure 7A–C). Those were not associated with pericytes, unlike vessels present in the surrounding connective tissues (Figure 7D and E). In addition, CAE-positive single mast cells were detected in the tumor periphery (Figure 7F). Diffused specific staining for fibroblastic markers (vimentin) was detected throughout fibrosarcomas. This staining was somewhat more intense within the connective tissue surrounding and infiltrating the tumors than within the parenchyma (Figure 7G). Direct evidence for VEGF production by stromal cells infiltrating VEGF-negative tumors came from experiments with mice harbouring the LacZ-tagged VEGF allele (Miquerol et al., 1999). Because the VEGF-LacZ cassette is under control of the endogenous, unmodified VEGF promoter, individual stromal cells expressing VEGF can be detected by LacZ staining in a more reliable manner than in other VEGF reporter systems based on the exogenous VEGF promoter (Kishimoto et al., 2000). As shown in Figure 7K and L, numerous LacZ-positive fibroblast-like stromal cells were detected throughout the tumors and in para-vascular areas. This experiment demonstrates directly that at least some elements of the tumor stroma actively produce VEGF in situ, and that detection of this growth factor in whole tumor lysates was not a result of ‘contamination’ with blood cells and/or platelets. Pleiotropic effects of ras and neu oncogenes on the angiogenic phenotype Despite the presence of VEGF-positive stroma, growth of VEGF−/− fibrosarcomas was, for the most part, only partially inhibited by the DC101 antibody, even when administered at doses that obliterated growth of ES-derived teratomas (compare Figure 5A and B with Figure 1). It follows that in oncogene-driven tumors, ‘angiogenic switching’ continues to occur despite the absence of VEGF production by tumor cells and inactivation of the endothelial VEGFR-2. This implies additional/alternative (VEGF-unrelated) pro-angiogenic mechanisms. In order to assess this aspect, we performed a targeted gene expression screen involving 23 known angiogenesis-related transcripts (Figure 8; see Materials and methods). GEArray is a version of a ‘reverse northern’ analysis where specific probes are immobilized on a membrane, which is then exposed to a pool of reverse-transcribed (radiolabeled) cellular cDNA. Such comparison between parental MDF528 cells and either ras- or neu-transformed fibrosarcoma counterparts indicated the existence of several oncogene-induced changes. Thus, acidic (aFGF),
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