A vaccine targeting basic fibroblast growth factor elicits a protective immune response against murine melanoma

Tumor growth and metastasis are closely related to angiogenesis. Basic fibroblast growth factor(bFGF) is an angiogenic factor, and up-regulated expression of bFGF plays a crucial role in the development and metastasis of melanoma. Therefore, in this study, we sought to achieve antitumor activity by immunity targeting bFGF which would inhibit tumor angiogenesis and simultaneously induce bFGF specific cytotoxic T lymphocytes to kill melanoma cells. A human bFGF protein was used as exogenous antigen, coupled with a saponin-liposome adjuvant formulation to enhance CTL response. The results showed that the immunity induced strong immune response and produced prominent anti-cancer activities. CD31 immunohistochemistry and alginate-encapsulated tumor cell assay displayed that tumor angiogenesis was effectively inhibited. Further, the higher production of IFN-γ and cytotoxic T lymphocyte killing assay suggested that the anti-cancer activities may mainly depend on cellular immune response, which could cause the inhibition of tumor angiogenesis and specific killing of tumor cells by bFGF-specific cytotoxic T lymphocytes. We concluded that immunotherapy targeting bFGF may be a prominent strategy for melanoma, and that the adjuvant formulation of saponin-liposome is very desirable in enhancing cytotoxic T lymphocytes response.     

脑膜瘤组织中bFGF及FGFR-1蛋白表达的研究

目的　探讨碱性成纤维生长因子 (basicfibroblastgrowthfactor ,bFGF)及成纤维生长因子受体 1(fibroblastgrowthfactorreceptor 1,FGFR 1)在脑膜瘤中的表达及其与脑膜瘤组织病理学和复发的关系。方法　用免疫组化技术检测bFGF、FGFR 1在不同类型的脑膜瘤组织中的蛋白表达 ,用组织病理学判断脑膜瘤的良恶性。结果　脑膜瘤细胞有不同程度的bFGF及FGFR 1表达 ,其表达阳性率与肿瘤的良恶性和复发有关。结论　bFGF和FGFR具有促进脑膜瘤细胞的增殖和生长的作用。脑膜瘤表达bFGF、FGFR的阳性率可作为鉴别肿瘤良恶性的有用指标 ,并对脑膜瘤的预后和术后复发起提示作用。     

The 18 kDa isoform of basic fibroblast growth factor is sufficient to stimulate human melanoma growth and angiogenesis

Basic fibroblast growth factor is the best-characterized autocrine growth factor in melanoma development and progression. We hypothesized that basic fibroblast growth factor might induce a more aggressive phenotype dependent on the amount of protein expressed in melanoma. Two human melanoma cell lines, M14 and 1F6, known to have low endogenous basic fibroblast growth factor expression and slow growth as subcutaneous xenografts, were stably transfected with vectors encoding either the 18 kDa or all (ALL) isoform proteins of human basic fibroblast growth factor. Different clones overexpressing the 18 kDa or ALL basic fibroblast growth factor proteins were easily obtained. Increased levels of basic fibroblast growth factor were secreted in conditioned medium and stored on the extracellular membrane. Biological activity of the overexpressed basic fibroblast growth factor was confirmed in a human umbilical vein endothelial cell proliferation assay. In 1F6 cells, overexpression of either 18 kDa or ALL basic fibroblast growth factor proteins resulted in up to two-fold shorter in-vitro doubling times (P<0.05). In addition, in vivo, both 18 kDa and ALL basic fibroblast growth factor-overexpressing 1F6 subcutaneous xenografts displayed significantly higher growth rates (P<0.05). In contrast, no major differences in in-vitro and in-vivo doubling times were observed when 18 kDa or ALL isoforms of basic fibroblast growth factor were overexpressed in M14 cells. Interestingly, basic fibroblast growth factor overexpression only affected the microvasculature in 1F6 xenografts. Although blood vessels in 1F6 parent tumors were large, 1F6 tumors overexpressing basic fibroblast growth factor contained numerous small, compressed vessels. Taken together, overexpression of the 18 kDa basic fibroblast growth factor protein only can promote autocrine melanoma cell growth and paracrine-driven angiogenesis.     

Suppression of solid tumor growth by immunoneutralizing monoclonal antibody against human basic fibroblast growth factor.

Basic fibroblast growth factor (bFGF) is a potent angiogenic mitogen. To elucidate the effect of bFGF inhibitors in vivo, anti-bFGF immunoneutralizing monoclonal antibody was prepared. One monoclonal antibody against human bFGF, obtained by cell fusion and designated 3H3, completely inhibited bFGF-induced proliferation of human umbilical vein endothelial cells at a concentration of 100 ng/ml. 3H3 did not bind to acidic fibroblast growth factor or HST1 protein, indicating high specificity for bFGF. Furthermore, the immunoneutralizing activity of 3H3 was examined in vivo. K1000 cells (a BALB/c 3T3 transformant in which the leader sequence-fused bFGF gene was transfected) were transplanted s.c. into BALB/c nude mice. Growth of the tumor cells was inhibited by i.v. treatment with 3H3 at a concentration of 200 micrograms/mouse. Histological observation showed that the antitumor effect of 3H3 was due to the inhibition of bFGF-induced angiogenesis. This experiment provides direct causal evidence for the hypothesis that tumor growth is angiogenesis dependent. This finding could also have implications for the development of novel therapeutic approaches to angiogenic solid tumors.     

Inhibition of human leukemia in an animal model with human antibodies directed against vascular endothelial growth factor receptor 2. Correlation between antibody affinity and biological activity.

Vascular endothelial growth factor (VEGF) and its receptors (VEGFR) have been implicated in promoting solid tumor growth and metastasis via stimulating tumor-associated angiogenesis. We recently showed that certain 'liquid' tumors such as leukemia not only produce VEGF, but also express functional VEGFR, resulting in an autocrine loop for tumor growth and propagation. A chimeric anti-VEGFR2 (or kinase insert domain-containing receptor, KDR) antibody, IMC-1C11, was shown to be able to inhibit VEGF-induced proliferation of human leukemia cells in vitro, and to prolong survival of nonobese diabetic-severe combined immune deficient (NOD-SCID) mice inoculated with human leukemia cells. Here we produced two fully human anti-KDR antibodies (IgG1), IMC-2C6 and IMC-1121, from Fab fragments originally isolated from a large antibody phage display library. These antibodies bind specifically to KDR with high affinities: 50 and 200 pM for IMC-1121 and IMC-2C6, respectively, as compared to 270 pM for IMC-1C11. Like IMC-1C11, both human antibodies block VEGF/KDR interaction with an IC(50) of approximately 1 nM, but IMC-1121 is a more potent inhibitor to VEGF-stimulated proliferation of human endothelial cells. These anti-KDR antibodies strongly inhibited VEGF-induced migration of human leukemia cells in vitro, and when administered in vivo, significantly prolonged survival of NOD-SCID mice inoculated with human leukemia cells. It is noteworthy that the mice treated with antibody of the highest affinity, IMC-1121, survived the longest period of time, followed by mice treated with IMC-2C6 and IMC-1C11. Taken together, our data suggest that anti-KDR antibodies may have broad applications in the treatment of both solid tumors and leukemia. It further underscores the efforts to identify antibodies of high affinity for enhanced antiangiogenic and antitumor activities.     

A structure-activity analysis of antagonism of the growth factor and angiogenic activity of basic fibroblast growth factor by suramin and related polyanions.

The ability of a series of polysulphonated naphthylureas structurally related to suramin to inhibit basic fibroblast growth factor (bFGF) or serum-stimulated growth of endothelial cells [either large vessel, human umbilical vein endothelial cells (HUVEC) or microvascular, bovine adrenal capillary endothelial (BACE) cells] and angiogenesis in vivo has been examined. The polyanions encompassed two main structural variations, namely the number of aromatic amide groups intervening between two terminal naphthyl rings and/or variation in the substitution pattern of the naphthyl rings. The polyanions were either inactive (group I) or inhibited (group II) bFGF-stimulated uptake of [3H]methylthymidine by BACE cells. Group I compounds shared a common structural feature in that they were simple binaphthyl-substituted ureas. In contrast, group II compounds all had an extended multiple ring structure with at least two aromatic groups intervening between the two terminal naphthyl rings. Compounds with either two or four intervening groups were equipotent in blocking bFGF in vitro. However, compounds with two bridging aromatic groups were 5- to 10-fold less toxic than suramin in mice, suggesting a potential for an improved therapeutic ratio. The ability of the polyanions to block bFGF-driven endothelial cell proliferation in vitro correlated with antiangiogenic activity in vivo as shown by use of the rat sponge angiogenesis model. These observations could substantially widen the anti-tumour therapeutic opportunities for this class of compound.     

Expression of fibroblast growth factor receptors in human leukemia cells.

We have previously cloned from K562 leukemia cells two novel fibroblast growth factor receptors (FGFR-3 and FGFR-4; J. Partanen et al., EMBO J., 10: 1347-1354, 1991). Here we have analyzed the mRNA expression of four different FGFRs, including the two novel genes in human leukemia cell lines. We show FGFR-1, FGFR-3, and FGFR-4 mRNAs in several leukemia cell lines at levels similar to those in solid tumor cell lines. Ligand cross-linking experiments indicate that K562 cells have receptors binding acidic FGF but not basic FGF. Expression of FGFRs in leukemia cells may reflect their presence on normal hematopoietic precursor cells or induction during leukemogenesis or cell culture.     

Basic fibroblast growth factor in an animal model of spontaneous mammary tumor progression

Although basic fibroblast growth factor (FGF2) was the first pro-angiogenic molecule discovered, it has numerous activities on the growth and differentiation of non-vascular cell types. FGF2 is both stimulatory and inhibitory, depending on the cell type evaluated, the experimental design used and the context in which it is tested. Here, we investigated the effects of manipulating endogenous FGF2 on the development of mammary cancer to determine whether its endogenous contribution in vivo is pro- or anti-tumorigenic. Specifically, we examined the effects of FGF2 gene dosing in a cross between a spontaneous breast tumor model (PyVT+ mice) and FGF2-/- (FGF KO) mice. Using these mice, the onset and progression of mammary tumors was determined. As predicted, female FGF2 WT mice developed mammary tumors starting around 60 days after birth and by 80 days, 100% of FGF2 WT female mice had mammary tumors. In contrast, 80% of FGF2 KO female mice had no palpable tumors until nearly three weeks later (85 days) at times when 100% of the WT cohort was tumor positive. All FGF KO mice were tumor-bearing by 115 days. When we compared the onset of mammary tumor development and the tumor progression curves between FGF het and FGF KO mice, we observed a difference, which suggested a gene dosing effect. Analysis of the tumors demonstrated that there were significant differences in tumor size depending on FGF2 status. The delay in tumor onset supports a functional role for FGF2 in mammary tumor progression, but argues against an essential role for FGF2 in overall mammary tumor progression.     

Quantitative analysis of basic fibroblast growth factor and vascular endothelial growth factor in human colorectal cancer.

Tumour growth is angiogenesis dependent. Some authors suggest a prognostic role of microvessel count in colorectal cancer. We tested the role of basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) in the switch to the angiogenic phenotype in 35 patients with colorectal cancer at different stages of disease. We evaluated the two angiogenic factors, by enzyme-linked immunosorbent assay (ELISA), in tumour, peritumoral mucosa, pathological mesenteric and peripheral blood. We used ten endoscopic intestinal biopsies and ten peripheral blood samples from healthy subjects as control. bFGF was significantly lower in tumour tissues and in peritumoral mucosas than in healthy mucosas, whereas VEGF was up-regulated in tumours but not in peritumoral mucosa. Both angiogenic factors were greatly increased in mesenteric blood. VEGF tumour and serum levels were significantly correlated with the stage of disease. bFGF tumour and serum concentration were not correlated with the stage of disease. The high levels of bFGF in mesenteric blood suggest that this growth factor might be abnormally released from tumour tissue and peritumoral mucosa and could function as an early effector in the switch to the angiogenic phenotype. In contrast, VEGF, whose levels show a significant correlation with the stage of disease, could act in a following step, supporting tumour progression.     

Immunohistochemical expression of basic fibroblast growth factor and fibroblast growth factor receptors 1 and 2 in the pathogenesis of lung cancer

PURPOSE: To identify the patterns of protein expression of basic fibroblast growth factor (bFGF) and FGF receptors 1 and 2 in non-small cell lung carcinoma (NSCLC) and their role in the early pathogenesis of squamous cell carcinoma (SCC) of the lung. EXPERIMENTAL DESIGN: Archived tissue from NSCLC (adenocarcinoma and SCC; n = 321) and adjacent bronchial epithelial specimens (n = 426) were analyzed for the immunohistochemical expression of bFGF, FGFR1, and FGFR2, and the findings were correlated with clinicopathologic features of the patients. RESULTS: High expression of bFGF, FGFR1, and FGFR2 was shown in most NSCLC tumors. The pattern of expression for all markers varied according to tumor histologic type and cellular localization. Cytoplasmic expression scores were significantly higher in tumors than in normal epithelia. Nuclear bFGF (P = 0.03) and FGFR1 (P = 0.02) levels were significantly higher in women than in men. Although cytoplasmic FGFR1 expression was significantly higher (P = 0.002) in ever smokers than in never smokers, nuclear FGFR1 (P = 0.0001) and FGFR2 (P = 0.003) expression was significantly higher in never smokers. Different prognostic patterns for the expression of these markers were detected for both NSCLC histologic types. Dysplastic changes showed significantly higher expression of all markers compared with squamous metaplasia. CONCLUSIONS: bFGF, FGFR1, and FGFR2 are frequently overexpressed in SCC and adenocarcinoma of the lung. bFGF signaling pathway activation may be an early phenomenon in the pathogenesis of SCC and thus an attractive novel target for lung cancer chemopreventive and therapeutic strategies.     

Immunolocalization of basic fibroblast growth factor to the microvasculature of human brain tumors.

BACKGROUND. Microvascular proliferation, a prominent feature of tumors of the central nervous system, is a prime target for anti-cancer therapy. METHODS. Because basic fibroblast growth factor (bFGF) plays a key role in the regulation of angiogenesis, surgical specimens from 52 human brain tumors were examined by immunocytochemical studies with a murine monoclonal antibody to bFGF. Sections from these tumors also were incubated with Ki-67 monoclonal antibody to measure the growth fraction. RESULTS. Immunostaining for bFGF was observed in 45 of 52 (87%) neoplasms, reacting with 97% of the malignant brain tumors and 67% of benign tumors (P < 0.01). The nonreactive tumors were a medulloblastoma and 7 of 21 (33%) benign, noninvasive, slow-growing neoplasms (1 acoustic schwannoma, 3 meningiomas, 2 pituitary adenomas, and 1 cholesteatoma). The indices of proliferation (Ki-67 labeling) were lower for the 21 benign tumors (1.2 +/- 1.1%) than the 31 malignant tumors (10.3 +/- 10.5%; P < 0.001). The bFGF was immunolocalized in the tumor cell nuclei in 23 of 52 tumors (44%) and in the cytoplasm of 8 of 52 (15%) tumors. Immunostaining to bFGF was prominent in the microvascular endothelial compartment in 84% of the malignant tumors and only 52% of benign tumors (P < 0.01). Immunostaining was not present after preabsorption of the antibody with pure human recombinant bFGF. CONCLUSIONS. The presence of bFGF predominantly within the tumor microvasculature indicates a cellular depot for this potent growth factor that mediates angiogenesis and tumorigenesis. These data support a role for bFGF in the transition from the benign to the malignant phenotype.     

Vascular endothelial growth factor, interleukin 8, platelet-derived endothelial cell growth factor, and basic fibroblast growth factor promote angiogenesis and metastasis in human melanoma xenografts

Angiogenesis is a significant prognostic factor in melanoma, but the angiogenic factors controlling the neovascularization are not well defined. The purpose of this study was to investigate whether the angiogenesis and metastasis of melanoma are promoted by vascular endothelial growth factor (VEGF), interleukin 8 (IL-8), platelet-derived endothelial cell growth factor (PD-ECGF), and/or basic fibroblast growth factor (bFGF). Cells from human melanoma lines (A-07, D-12, R-18, and U-25) transplanted to BALB/c nu/nu mice were used as tumor models. Expression of angiogenic factors was studied by ELISA, Western blotting, and immunohistochemistry. Angiogenesis was assessed by using an intradermal angiogenesis assay. Lung colonization and spontaneous lung metastasis were determined after i.v. and intradermal inoculation of tumor cells, respectively. The specific roles of VEGF, IL-8, PD-ECGF, and bFGF in tumor angiogenesis, lung colonization, and spontaneous metastasis were assessed in mice treated with neutralizing antibody. The melanoma lines expressed multiple angiogenic factors, and each line showed a unique expression pattern. Multiple angiogenic factors promoted angiogenesis in the most angiogenic melanoma lines, whereas angiogenesis in the least angiogenic melanoma lines was possibly promoted solely by VEGF. Tumor growth, lung colonization, and spontaneous metastasis were controlled by the rate of angiogenesis and hence by the angiogenic factors promoting the angiogenesis. Lung colonization and spontaneous metastasis in A-07 were inhibited by treatment with neutralizing antibody against VEGF, IL-8, PD-ECGF, or bFGF. Each of these angiogenic factors may promote metastasis in melanoma, because inhibition of one of them could not be compensated for by the others. Our observations suggest that efficient antiangiogenic treatment of melanoma may require identification and blocking of common functional features of several angiogenic factors.     

Expression of basic fibroblast growth factor and its receptor in human pancreatic carcinomas.

We examined the expression of basic fibroblast growth factor (FGF) and FGF receptor by immunohistochemistry in 32 human pancreatic ductal adenocarcinomas. Mild to marked basic FGF immunoreactivity was noted in 19 (59.4%) of the 32 tumours examined, and 30 (93.3%) of the tumours exhibited a cytoplasmic staining pattern against FGF receptor. The tumours were divided into two groups according to the proportion of positively stained tumour cells: a low expression group (positive cells < 25%) and a high expression group (positive cells > or = 25%). No statistically significant difference in tumour size, differentiation, metastases or stage was found between the low and high basic FGF expression groups. However, a significant correlation was found between FGF receptor expression level and the presence of retroperitoneal invasion, lymph node metastasis, and tumour stage. In addition, low FGF receptor expression was significantly associated with a longer post-operative survival as compared with high FGF receptor expression, whereas there was no significant difference in post-operative survival between the low and high basic FGF expression groups. Increased expression of FGF receptor is correlated with the extent of malignancy and post-operative survival in human pancreatic ductal adenocarcinomas. Thus, overexpression of FGF receptor may prove to be a more useful prognostic marker than basic FGF expression level in pancreatic cancer patients.     

Influence of angiogenin on the growth of A375 human melanoma cells and the expression of basic fibroblast growth factor

Angiogenin was isolated as a tumor angiogenic factor solely on the basis of its angiogenic activity. Its expression is essential for melanoma progression and metastasis. Many studies have mainly focused on how it induces angiogenesis, which allows further melanoma growth and metastasis. Here, we investigated the effects of angiogenin on melanoma cell growth and studied its influence on the expression and function of the basic fibroblast growth factor. We transfected the angiogenin gene in the sense and antisense orientation into A375 cells, and obtained stable angiogenin under-expressing and over-expressing transfectants. We found that in the angiogenin antisense transfectants, the cell proliferation was decreased and the basic fibroblast growth factor-induced cell proliferation was inhibited, but the expression of basic fibroblast growth factor was increased. In contrast, in the angiogenin sense transfectants, the cell proliferation was increased, and the basic fibroblast growth factor-induced cell proliferation was also increased. The expression of basic fibroblast growth factor, however, was decreased. In conclusion, we demonstrated that, besides its angiogenic activity, angiogenin also directly contributes to A375 cell proliferation and is required for the basic fibroblast growth factor to induce cell proliferation. We also demonstrated that the endogenous angiogenin expression levels affect the expression of basic fibroblast growth factor in A375 cells. By targeting angiogenin, therefore, one may find a potential therapeutic approach to human malignant melanoma.     

Modification of the volumetric growth responses and steady-state hypoxic fractions of xenografted DLD-2 human colon carcinomas by administration of basic fibroblast growth factor or suramin.

We studied the growth characteristics and hypoxic fractions of DLD-2 human colon tumours xenografted into male nude mice either in the unperturbed state or after i.p. injection (q.i.d. x 7) of basic fibroblast growth factor (0.25 mg kg-1) or suramin (50 mg kg-1). Hypoxic fractions were measured by clonogenic excision assay 1 day after administration b FGF or suramin was stopped. As compared to controls, the growth of tumours in b FGF treated mice was increased by a factor of 1.5 as indicated by the relative volumes of tumours on the day of excision. Similarly, suramin decreased the growth of DLD-2 tumours by a factor of 1.6. The percentage of hypoxic cells in control neoplasms was 42.9% (95% confidence limits 34.2-52.1%). In mice that received basic fibroblast growth factor injections, hypoxic fractions decreased to 19.1% (95% confidence limits 13.5-26.9%). In contrast, in mice treated with suramin, the percentage of hypoxic cells increased to 74.0% (95% confidence limits 65.3-83.9%). These data indicate that the biology of solid tumours can be significantly modified by alteration of growth factor status.     

Tumor promoters induce basic fibroblast growth factor gene expression in human dermal fibroblasts.

Tumor-promoting phorbol esters have been shown previously to either induce or repress the expression of numerous cellular genes, and this property is likely to be important for the in vitro and in vivo biological effects of these compounds. In this report, we demonstrate that phorbol 12-myristate 13-acetate induces the accumulation of basic fibroblast growth factor mRNA and protein in human dermal fibroblasts. In contrast, acidic fibroblast growth factor expression was unaffected by this compound. The enhancement of basic fibroblast growth factor gene expression by phorbol 12-myristate 13-acetate was blocked by the isoquinolinesulfonamide derivative H7, a potent inhibitor of protein kinase C. Two additional tumor promoters that bind to and activate protein kinase C, phorbol 12,13-didecanoate and mezerein, also increased basic fibroblast growth factor mRNA levels. Basic fibroblast growth factor is a mitogen for many cell types and can stimulate angiogenesis; thus, some tumor promoter-induced cellular responses may be mediated by this polypeptide.     

Expression of vascular endothelial growth factor and basic fibroblast growth factor in small adenocarcinomas.

The expression of two angiogenetic factors, vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF), in small adenocarcinomas (相似文献   

Localization of basic fibroblast growth factor and transforming growth factor beta 1 in the human mammary gland

The presence and distribution of basic fibroblast growth factor (bFGF) and transforming growth factor beta 1 in benign and malignant human breast tissue were determined by immunohistochemistry and immunoblotting. Peroxidase staining of biopsy specimens using a polyclonal antibody to amino acids 1-24 of bFGF and a monoclonal antibody to whole recombinant bFGF showed this growth factor to be localized in the myoepithelial cells of the benign breast. Epithelial cells and stroma were negative. In hyperplasia and intraductal carcinoma in situ staining was still seen around the perimeter of enlarged ducts. In malignant biopsies, however, staining was seen only when benign elements were present or residual myoepithelial cells and basement membrane remained. Antigen absorption and immunoblotting confirmed the antibody staining to be specific for bFGF. Transforming growth factor beta 1 was shown, using the same techniques, to be located in the periductal and intraductal stroma, closely associated with epithelial or myoepithelial cells in the benign and malignant breast. The relative localization of these two growth factors in the mammary gland may be significant in the control of breast development and/or tumor formation and progression.     

Localization of acidic and basic fibroblast growth factor mRNA in human brain tumors

Acidic and basic fibroblast growth factors (aFGF and bFGF) are closely related peptide mitogens acting on both mesoderm- and neuroectoderm-derived cells, including fibroblasts, endothelial cells and glial cells. In order to identify the expression of mRNAs for these growth factors, in situ hybridization using human aFGF and bFGF RNA probes was performed in 24 human brain tumors. The mRNAs for aFGF and bFGF were expressed in the cells of various tumors (1/1 and 1/1 astrocytoma, 2/2 and 2/2 anaplastic astrocytomas, 6/6 and 6/6 glioblastomas, 4/4 and 4/4 meningiomas, 3/3 and 3/3 schwannomas, 1/2 and 1/2 pituitary adenomas, 4/4 and 4/4 metastatic carcinomas, 0/1 an 0/1 hemangioblastoma, 0/1 and 0/1 craniopharyngioma) and were also detected in endothelial cells and surrounding neuronal cells of brain tumors. These results suggest the possibilities that aFGF and bFGF contribute to the uncontrolled growth of tumor cells and the proliferation of endothelial cells in autocrine and paracrine manners, and that the expression of mRNAs for these growth factors in the surrounding neuronal cells results in enhancement of tumor growth.