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Author(s): D K Jin; K Shido; H-G Kopp; I Petit; S V Shmelkov; L M Young; A T Hooper; H Amano; S T Avecilla; B Heissig; K Hattori; F Zhang; [...]

A correction to the article "Cytokine-mediated deployment of SDF-1 induces revascularization through recruitment of CXCR4+ hemangiocytes," by D. K. Jin and colleagues is presented.

Obliterative bronchiolitis (OB) is the major limitation for long-term survival of lung allograft recipients. The exact molecular and cellular mechanisms contributing to obliterative lesion formation are unknown. Pathological characteristics of OB are epithelial damage, peribronchial inflammation, and increasing obliteration of bronchioli. Vascular endothelial growth factor (VEGF) is an angiogenic growth factor that exerts proinflammatory effects by increasing endothelial permeability and inducing expression of endothelial adhesion molecules. We investigated the role of VEGF in the development of OB in rat tracheal allografts and the role of VEGF receptors (VEGFR)-1 and -2 in the development of OB in mouse tracheal allografts. In nontreated allografts, with increasing loss of epithelium and airway occlusion, VEGF messenger RNA (mRNA) and protein expression vanished in the epithelium and increased in smooth muscle cells and mononuclear inflammatory cells compared with syngeneic grafts. Intragraft VEGF overexpression by adenoviral transfer of a mouse VEGF164 gene led to a decrease in epithelial necrosis but increased luminal occlusion by >50% compared with AdLacZ-treated rat tracheal allografts. When compared with the control immunoglobulin (Ig)G group, simultaneous treatment with antibodies against VEGFR-1 and -2 significantly lowered the degree of luminal occlusion of mouse tracheal allografts.

We hypothesised that the combination of anti-angiogenic and anti-epidermal growth factor (EFG)-receptor (R) therapies would more effectively inhibit gastric cancer growth than single-agent therapy. TMK-1 gastric cancer cells were injected into the gastric wall of nude mice to generate tumours. After 4 days, mice were randomly assigned to the following groups: control, DC101 ([vascular endothelial growth factor (VEGF)-receptor (R)-2 antibody], C225 (EGF-R antibody), or a combination of DC101 and C225. The combination therapy significantly inhibited gastric tumour growth compared with the control group, whereas the decrease in tumour growth in mice treated with DC101 or C225 alone did not reach statistical significance. All mice administered DC101 demonstrated decreased tumour vascularity and increased endothelial cell apoptosis. C225 alone did not affect angiogenesis, but inhibited tumour cell proliferation. The combination therapy led to a further decrease in tumour cell proliferation. The combination of anti-VEGF-R and anti-EGF-R therapies was effective in inhibiting gastric cancer growth. These findings support the hypothesis that inhibiting multiple biological pathways that mediate tumour growth may be an effective therapeutic strategy.

Antiangiogenic agents block the effects of tumor-derived angiogenic factors (paracrine factors), such as vascular endothelial growth factor (VEGF), on endothelial cells (EC), inhibiting the growth of solid tumors. However, whether inhibition of angiogenesis also may play a role in liquid tumors is not well established. We recently have shown that certain leukemias not only produce VEGF but also selectively express functional VEGF receptors (VEGFRs), such as VEGFR-2 (Flk-1, KDR) and VEGFR1 (Flt1), resulting in the generation of an autocrine loop. Here, we examined the relative contribution of paracrine (EC-dependent) and autocrine (EC-independent) VEGF/VEGFR signaling pathways, by using a human leukemia model, where autocrine and paracrine VEGF/VEGFR loops could be selectively inhibited by neutralizing mAbs specific for murine EC (paracrine pathway) or human tumor (autocrine) VEGFRs. Blocking either the paracrine or the autocrine VEGF/VEGFR-2 pathway delayed leukemic growth and engraftment in vivo, but failed to cure inoculated mice. Long-term remission with no evidence of disease was achieved only if mice were treated with mAbs against both murine and human VEGFR-2, whereas mAbs against human or murine VEGFR-1 had no effect on mice survival. Therefore, effective antiangiogenic therapies to treat VEGF-producing, VEGFR-expressing leukemias may require blocking both paracrine and autocrine VEGF/VEGFR-2 angiogenic loops to achieve remission and long-term cure.

Hypoxia stimulates angiogenesis through the binding of hypoxia-inducible factors to the hypoxia-response element in the vascular endothelial growth factor (Vegf) promotor. Here, we report that deletion of the hypoxia-response element in the Vegf promotor reduced hypoxic Vegf expression in the spinal cord and caused adult-onset progressive motor neuron degeneration, reminiscent of amyotrophic lateral sclerosis. The neurodegeneration seemed to be due to reduced neural vascular perfusion. In addition, Vegf165 promoted survival of motor neurons during hypoxia through binding to Vegf receptor 2 and neuropilin 1. Acute ischemia is known to cause nonselective neuronal death. Our results indicate that chronic vascular insufficiency and, possibly, insufficient Vegf-dependent neuroprotection lead to the select degeneration of motor neurons.

Tyrosine kinase receptors for angiogenic factors vascular endothelial growth factor (VEGF) and angiopoietin-1 (Ang-1) are expressed not only by endothelial cells but also by subsets of hematopoietic stem cells (HSCs). To further define their role in the regulation of postnatal hematopoiesis and vasculogenesis, VEGF and Ang-1 plasma levels were elevated by injecting recombinant protein or adenoviral vectors expressing soluble VEGF(165), matrix-bound VEGF(189), or Ang-1 into mice. VEGF(165), but not VEGF(189), induced a rapid mobilization of HSCs and VEGF receptor (VEGFR)2(+) circulating endothelial precursor cells (CEPs). In contrast, Ang-1 induced delayed mobilization of CEPs and HSCs. Combined sustained elevation of Ang-1 and VEGF(165) was associated with an induction of hematopoiesis and increased marrow cellularity followed by proliferation of capillaries and expansion of sinusoidal space. Concomitant to this vascular remodeling, there was a transient depletion of hematopoietic activity in the marrow, which was compensated by an increase in mobilization and recruitment of HSCs and CEPs to the spleen resulting in splenomegaly. Neutralizing monoclonal antibody to VEGFR2 completely inhibited VEGF(165), but not Ang-1-induced mobilization and splenomegaly. These data suggest that temporal and regional activation of VEGF/VEGFR2 and Ang-1/Tie-2 signaling pathways are critical for mobilization and recruitment of HSCs and CEPs and may play a role in the physiology of postnatal angiogenesis and hematopoiesis.

Vascular endothelial growth factor (VEGF) stimulates angiogenesis by activating VEGF receptor-2 (VEGFR-2). The role of its homolog, placental growth factor (PlGF), remains unknown. Both VEGF and PlGF bind to VEGF receptor-1 (VEGFR-1), but it is unknown whether VEGFR-1, which exists as a soluble or a membrane-bound type, is an inert decoy or a signaling receptor for PlGF during angiogenesis. Here, we report that embryonic angiogenesis in mice was not affected by deficiency of PlGF (Pgf-/-). VEGF-B, another ligand of VEGFR-1, did not rescue development in Pgf-/- mice. However, loss of PlGF impaired angiogenesis, plasma extravasation and collateral growth during ischemia, inflammation, wound healing and cancer. Transplantation of wild-type bone marrow rescued the impaired angiogenesis and collateral growth in Pgf-/- mice, indicating that PlGF might have contributed to vessel growth in the adult by mobilizing bone-marrow-derived cells. The synergism between PlGF and VEGF was specific, as PlGF deficiency impaired the response to VEGF, but not to bFGF or histamine. VEGFR-1 was activated by PlGF, given that anti-VEGFR-1 antibodies and a Src-kinase inhibitor blocked the endothelial response to PlGF or VEGF/PlGF. By upregulating PlGF and the signaling subtype of VEGFR-1, endothelial cells amplify their responsiveness to VEGF during the 'angiogenic switch' in many pathological disorders.

The vascular endothelial growth factor-A (VEGF-A), also known as the vascular permeability factor (VPF), has been shown to play an important role in malignant ascites formation. The effects of VEGF-A are mediated through flt-1 and kinase insert domain-containing receptor/fetal liver kinase (KDR/Flk-1) receptors. It has been shown that KDR/Flk-1 is a predominant receptor in solid hepatocellular carcinoma (HCC) development, but the role of this receptor in hepatic ascites formation has not yet been elucidated. In this study, we examined the role of KDR/Flk-1 in the murine MH134 hepatic malignant ascites formation by means of VEGF-A- and KDR/Flk-1-specific neutralizing antibodies (VEGF-A nAb and KDR/Flk-1 nAb, respectively). The mean volume of ascites, number of tumor cells in ascites, and the peritoneal capillary permeability were significantly suppressed by VEGF-A nAb and KDR/Flk-1 nAb treatment. These inhibitory effects of KDR/Flk-1 nAb were more potent than those of VEGF-A nAb. The autophosphorylation of KDR/ Flk-1 in the peritoneal wall was almost completely abolished by KDR/ Flk-1 nAb, whereas a certain level of activation was still shown by VEGF-A nAb treatment. Another VEGF-family, VEGF-C, which also binds KDR/Flk-1, was detected in the ascites. Furthermore, in the therapeutic experiment, although both VEGF-A nAb and KDR/Flk-1 nAb prolonged the survival rate of ascites-bearing mice, the latter showed a more significant impact on the survival of animals. These results suggest that KDR/Flk-1 is a major regulator of malignant hepatic ascites formation, and that in addition to VEGF-A, VEGF-C may also be involved in the malignant ascites formation via KDR/ Flk-1 activation.

Vascular endothelial cadherin (VE-cadherin) is an endothelial cell-specific cadherin that plays an important role in the control of vascular organization. Blocking VE-cadherin antibodies strongly inhibit angiogenesis, and inactivation of VE-cadherin gene causes embryonic lethality due to a lack of correct organization and remodeling of the vasculature. Hence, inhibitors of VE-cadherin adhesive properties may constitute a tool to prevent tumor neovascularization. In this paper, we tested different monoclonal antibodies (mAbs) directed to human VE-cadherin ectodomain for their functional activity. Three mAbs (Cad 5, BV6, BV9) were able to increase paracellular permeability, inhibit VE-cadherin reorganization, and block angiogenesis in vitro. These mAbs could also induce endothelial cell apoptosis in vitro. Two additional mAbs, TEA 1.31 and Hec 1.2, had an intermediate or undetectable activity, respectively, in these assays. Epitope mapping studies show that BV6, BV9, TEA 1.31, and Hec 1.2 bound to a recombinant fragment spanning the extracellular juxtamembrane domains EC3 through EC4. In contrast, Cad 5 bound to the aminoterminal domain EC1. By peptide scanning analysis and competition experiments, we defined the sequences TIDLRY located on EC3 and KVFRVDAETGDVFAI on EC1 as the binding domain of BV6 and Cad 5, respectively. Overall, these results support the concept that VE-cadherin plays a relevant role on human endothelial cell properties. Antibodies directed to the extracellular domains EC1 but also EC3-EC4 affect VE-cadherin adhesion and clustering and alter endothelial cell permeability, apoptosis, and vascular structure formation.

Vascular endothelial growth factor (VEGF) is the predominant regulator of colon cancer angiogenesis and is associated with a poor prognosis and the development of metastases. We hypothesized that DC101, an antibody against the VEGF receptor-2 (flk-1), may be efficacious in the therapy of colon cancer peritoneal carcinomatosis in a murine model. BALB/c mice underwent intraperitoneal injection of CT-26 colon cancer cells to generate peritoneal metastases. Mice received control solvent or DC101 for up to 60 days. In parallel studies, mice were sacrificed at sequential time points to determine the effect of DC101 on tumor angiogenesis, tumor cell proliferation and apoptosis, and endothelial cell apoptosis. Mice treated with DC101 demonstrated a 30% increase in mean survival. In addition, DC101 also led to a significant decrease in tumor vascularity, growth and tumor cell proliferation. In sequential studies, anti-VEGF-R therapy led to a progressive increase in endothelial cell apoptosis followed by an increase in tumor cell apoptosis. These findings suggest that anti-flk-1 therapy may prolong survival in patients with colon cancer carcinomatosis. The temporal studies demonstrating that anti-flk-1 therapy lead to an increase in endothelial cell apoptosis that in turn lead to an increase in tumor cell apoptosis confirms the role of VEGF as an endothelial cell survival factor.

Different forms of vascular endothelial growth factor (VEGF) and their cellular receptors (VEGFR) are associated with angiogenesis, as demonstrated by the lethality of VEGF-A, VEGFR-1 or VEGFR-2 knockout mice. Here we have used an in vitro angiogenesis model, consisting of human microvascular endothelial cells (hMVEC) cultured on three-dimensional (3D) fibrin matrices to investigate the roles of VEGFR-1 and VEGFR-2 in the process of VEGF-A and VEGF-C-induced tube formation. Soluble VEGFR-1 completely inhibited the tube formation induced by the combination of VEGF-A and TNF alpha (VEGF-A/TNF alpha). This inhibition was not observed when tube formation was induced by VEGF-C/TNF alpha or bFGF/TNF alpha. Blocking monoclonal antibodies specific for VEGFR-2, but not antibodies specifically blocking VEGFR-1, were able to inhibit the VEGF-A/TNF alpha-induced as well as the VEGF-C/TNF alpha-induced tube formation in vitro. P1GF-2, which interacts only with VEGFR-1, neither induced tube formation in combination with TNF alpha, nor inhibited or stimulated by itself the VEGF-A/TNF alpha-induced tube formation in vitro. These data indicate that VEGF-A or VEGF-C activation of the VEGFR-2, and not of VEGFR-1, is involved in the formation of capillary-like tubular structures of hMVEC in 3D fibrin matrices used as a model of repair-associated or pathological angiogenesis in vitro.

Antiangiogenic therapy can enhance radiation-induced tumor growth inhibition. However, the effects of combined antiangiogenic and radiation therapy on long-term tumor control and normal tissue response have not been reported. We treated mice bearing two different human tumor xenografts with anti-vascular endothelial growth factor receptor-2 antibody (DC101) and five dose fractions of local radiation and followed them for at least 6 months. DC101 significantly decreased the dose of radiation necessary to control 50% of tumors locally. The decrease was 1.7- and 1.3-fold for the moderately radiosensitive small cell lung carcinoma 54A and the highly radioresistant glioblastoma multiforme U87, respectively. In contrast to tumors, no increase in skin radiation reaction by the antibody was detected. Surprisingly, 44% of mice bearing 54A tumor developed clear ascites after DC101 treatment at its highest dose; this was fatal to 20% of mice. This adverse effect was seen only in mice that received whole-body irradiation 1 day before tumor implantation. The encouraging results on two human tumor xenografts suggest that vascular endothelial growth factor receptor-2 blockade merits further investigation to assess its potential as an enhancer of radiation therapy in the clinic.

Vascular endothelial-cadherin (VE-cad) is an endothelial cell-specific adhesion molecule that is crucial for proper assembly of vascular tubes. Here we show that a monoclonal antibody (BV13) directed to the extracellular region of VE-cad inhibits formation of adherens junctions and capillary-like structures by endothelial cells and blocks angiogenesis in the mouse cornea and in Matrigel plugs in vivo. Systemic administration of BV13 markedly decreases the growth of s.c. Lewis lung or human A431 epidermoid tumors and strongly suppresses the growth of Lewis lung metastases. These data demonstrate that VE-cad is essential for postnatal angiogenesis and thus validate VE-cad as a novel target for antiangiogenesis agents.

Previously we reported that when cells from the human transitional cell carcinoma cell line 253J B-V growing orthotopically within the bladder of athymic nude mice were treated with the anti-epidermal growth factor receptor monoclonal antibody C225, angiogenesis was inhibited, resulting in regression of the primary tumor and inhibition of metastasis. In this study, we evaluated whether paclitaxel enhanced this therapeutic effect of C225. In vitro, the proliferation of 253J B-V cells was inhibited more by the combination of C225 and paclitaxel than with either agent alone. In vivo therapy with C225 and paclitaxel resulted in significantly greater regression of tumors compared with either agent alone. Median bladder tumor weight was 85 mg (range, 69-133 mg) compared with 168 mg (range, 72-288 mg) after C225 alone (P < 0.05), and 273 mg (range, 83-563 mg) after paclitaxel alone (P < 0.005). The incidence of spontaneous lymph node metastasis was also reduced by the combination of C225 with paclitaxel, although this result did not significantly differ from results after the use of C225 alone. Treatment with paclitaxel and C225 down-regulated the expression of basic fibroblast growth factor, vascular endothelial cell growth factor, interleukin-8, and matrix metalloproteinase type 9 and inhibited tumor-induced neovascularity compared with untreated controls (P < 0.005). Moreover, the combination of C225 and paclitaxel enhanced apoptosis in tumor and endothelial cells compared with either agent alone (P < 0.005). These studies indicate that therapy with paclitaxel increases the ability of C225 to inhibit tumorigenicity and metastasis. This effect is mediated by inhibition of angiogenesis and induction of apoptosis.

Murine vascular endothelial cadherin-2 (VE-cad-2) is a cellular adhesion molecule that is distinct from vascular endothelial cadherin 1 (VE-cad-1) in that it does not interact with catenins and does not appear to affect cell migration or growth. In this study, we have cloned a full-length cDNA of the human homolog of VE-cad-2 and used it to map the chromosomal locus of the VE-cad-2 gene. Human VE-cad-2 maps to Chromosome (Chr) 5q31. The cDNA of human VE-cad-2 is highly homologous to mouse VE-cad-2, except for a C-terminal tail. The genomic structure of VE-cad-2 is strikingly similar to that reported for a large family of neuronal protocadherin genes mapped to Chr 5q, yet the amino acid sequences between VE-cad-2 and the protocadherins are substantially divergent. The promoter of human VE-cad-2 contains two TATA boxes and transcription initiates from a single site 3' to these elements. Similar to mouse VE-cad-2, the human gene is expressed primarily in highly vascularized tissues.

Tumor oxygenation is critical for tumor survival as well as for response to therapy, e.g., radiation therapy. Hormone ablation therapy in certain hormone-dependent tumors and antiangiogenic therapy lead to vessel regression and have also shown beneficial effects when combined with radiation therapy. These findings are counterintuitive because vessel regression should reduce oxygen tension (pO2) in tumors, decreasing the effectiveness of radiotherapy. Here we report on the dynamics of pO2 and oxygen consumption in a hormone-dependent tumor following hormone ablation and during treatment with an anti-VEGFR-2 monoclonal antibody (mAb) or a combination of doxorubicin and cyclophosphamide; the latter combination is not known to cause vessel regression at doses used clinically. Androgen-dependent male mouse mammary carcinoma (Shionogi) was implanted into transparent dorsal skin-fold chambers in male severe combined immunodeficient mice. Thirteen days after the tumors were implanted, mice were treated with antiangiogenic therapy (anti-VEGFR-2 mAb, 1.4 mg/30 g body weight), hormone ablation by castration, or doxorubicin (6.5 mg/kg every 7 days) and cyclophosphamide (100 mg/kg every 7 days). A non-invasive in vivo method was used to measure pO2 profiles and to calculate oxygen consumption rates (Q(O2)) in tumors. Tumors treated with anti-VEGFR-2 mAb exhibited vessel regression and became hypoxic. Initial vessel regression was followed by a "second wave" of angiogenesis and increases in both pO2 and Q(O2). Hormone ablation led to tumor regression followed by an increase in pO2 coincident with regrowth. Chemotherapy led to tumor growth arrest characterized by constant Q(O2) and elevated pO2. The increased pO2 during anti-VEGFR-2 mAb and hormone ablation therapy may explain the observed beneficial effects of combining antiangiogenic or hormone therapies with radiation treatment. Thus, understanding the microenvironmental dynamics is critical for optimal scheduling of these treatment modalities.

Background: Recent studies have suggested that vascular endothelial growth factor (VEGF), in addition to its proangiogenic properties, also functions as a survival factor for endothelial cells. The authors hypothesized that inhibition of VEGF activity by blockade of VEGF receptor-2 (R-2) function prevents angiogenesis and decreases tumor growth in colon carcinoma liver metastases., Methods: Spleens of mice were injected with human colon carcinoma cells producing liver metastases. After 7 days of tumor growth, groups of mice received either antibody to VEGFR-2 (DC101) or phosphate-buffered saline (control). In a follow-up experiment, a similar treatment regimen was followed except that mice were sacrificed at 1-week intervals to assess the time course of endothelial cell and tumor cell apoptosis., Results: After 21 days of therapy, the authors observed a significant decrease in vessel counts in liver metastases from human colon carcinoma in nude mice after therapy with VEGFR-2 antibody. Tumor cell apoptosis was increased significantly in the tumors of mice receiving DC101. Temporal studies with immunofluorescent double staining for the microvasculature and apoptotic cells revealed an increase in endothelial cell apoptosis that preceded an increase in tumor cell apoptosis. In vitro, treatment of human umbilical vein endothelial cells with antibody to VEGFR-2 produced a > 2.5-fold increase in endothelial cell apoptosis., Conclusions: Therapy targeting the VEGFR-2 inhibited tumor growth in a murine model of colon carcinoma liver metastasis. Surprisingly, this therapy did not only inhibit angiogenesis but also led to endothelial cell death. These findings suggest that VEGF, via VEGFR-2 signaling, functions as a survival factor for tumor endothelial cells in liver metastases from colon carcinoma., (Copyright 2000 American Cancer Society.)

Emerging data suggest that VEGF receptors are expressed by endothelial cells as well as hematopoietic stem cells. Therefore, we hypothesized that functional VEGF receptors may also be expressed in malignant counterparts of hematopoietic stem cells such as leukemias. We demonstrate that certain leukemias not only produce VEGF but also express functional VEGFR-2 in vivo and in vitro, resulting in the generation of an autocrine loop that may support leukemic cell survival and proliferation. Approximately 50% of freshly isolated leukemias expressed mRNA and protein for VEGFR-2. VEGF(165) induced phosphorylation of VEGFR-2 and increased proliferation of leukemic cells, demonstrating these receptors were functional. VEGF(165) also induced the expression of MMP-9 by leukemic cells and promoted their migration through reconstituted basement membrane. The neutralizing mAb IMC-1C11, specific to human VEGFR-2, inhibited leukemic cell survival in vitro and blocked VEGF(165)-mediated proliferation of leukemic cells and VEGF-induced leukemic cell migration. Xenotransplantation of primary leukemias and leukemic cell lines into immunocompromised nonobese diabetic mice resulted in significant elevation of human, but not murine, VEGF in plasma and death of inoculated mice within 3 weeks. Injection of IMC-1C11 inhibited proliferation of xenotransplanted human leukemias and significantly increased the survival of inoculated mice. Interruption of signaling by VEGFRs, particularly VEGFR-2, may provide a novel strategy for inhibiting leukemic cell proliferation.

A new legume lectin has been identified by its ability to specifically stimulate proliferation of NIH 3T3 fibroblasts expressing the Flt3 tyrosine kinase receptor. The lectin was isolated from conditioned medium harvested from human peripheral blood mononuclear cells activated to secrete cytokines by a crude red kidney bean extract containing phytohemagglutinin (PHA). Untransfected 3T3 cells and 3T3 cells transfected with the related Fms tyrosine kinase receptor do not respond to this lectin, which we called PvFRIL (Phaseolus vulgaris Flt3 receptor-interacting lectin). When tested on cord blood mononuclear cells enriched for Flt3-expressing progenitors, purified PvFRIL fractions maintained a small population of cells that continued to express CD34 after 2 weeks in suspension cultures containing IL3. These cultures did not show the effects of IL3's strong induction of proliferation and differentiation (high cell number and exhausted medium); instead, low cell number at the end of the culture period resulted in persistence of cells in the context of cell death. These observations led to the hypothesis that PvFRIL acts in a dominant manner to preserve progenitor viability and prevent proliferation and differentiation.

Background: Pancreatic carcinoma is associated with a poor prognosis, and treatment options for patients with this disease are limited. The epidermal growth factor (EGF) receptor and its ligands are overexpressed in human pancreatic carcinoma and may contribute to the pathophysiology of these tumors., Methods: The anti-EGF receptor monoclonal antibody IMC-C225 was used to determine the effects of EGF receptor blockade on the growth of human pancreatic carcinoma BxPC-3 cells in vitro. Athymic mice bearing established (200 mm(3)) subcutaneous BxPC-3 xenografts were treated with IMC-C225 (17 or 33 mg/kg every 3 days) alone or in combination with 5-fluorouracil (17 mg/kg twice weekly)., Results: IMC-C225 inhibited exogenous ligand-stimulated tyrosine phosphorylation of the EGF receptor on BxPC-3 tumor cells. Treatment of BxPC-3 cells with IMC-C225 inhibited DNA synthesis (23.8%) and colony formation in soft agar (45.6%). IMC-C225 treatment significantly suppressed the growth of BxPC-3 tumors compared with treatment with vehicle alone (P = 0.003). Combination therapy with IMC-C225 and the chemotherapeutic agent 5-fluorouracil enhanced the antitumor effects compared with either agent alone and resulted in regression of pancreatic tumors in several animals. Histologic examination of pancreatic tumors from mice treated with IMC-C225 showed extensive tumor necrosis that coincided with a substantial decrease in tumor cell proliferation and an increase in tumor cell apoptosis., Conclusions: These data suggest that IMC-C225 affects the growth of pancreatic tumors by inhibiting EGF receptor-dependent proliferation and survival, and demonstrates the potential for therapeutic application of IMC-C225 antibody in the treatment of human pancreatic carcinoma., (Copyright 2000 American Cancer Society.)

Vascular endothelial cell growth factor (VEGF) regulates angiogenesis and metastasis of bladder cancer (transitional cell carcinoma, TCC) through binding to VEGF receptor-2 (VEGFR-2). In this study, we evaluated whether the anti-VEGFR monoclonal antibody (Mab) DC101 in combination with paclitaxel inhibited tumorigenesis, angiogenesis, and metastasis of human TCC growing within the bladder of athymic nude mice. In vivo therapy with Mab DC101 and paclitaxel induced significant regression of bladder tumors compared with either agent alone. Median bladder weights were reduced from 601 mg in untreated controls, 422 mg in mice treated with paclitaxel alone (P < 0.005), 361 mg in mice treated with DC101 alone (P < 0.005), and 113 mg in mice that received combination therapy (P < 0.0005). Only one of nine mice developed spontaneous lymph node metastasis after combined treatment, compared with seven of seven untreated controls (P < 0.0005), six of eight after DC101 (P < 0.01), and five of eight mice after paclitaxel (P < 0.05). Combined treatment with both paclitaxel and DC101 inhibited tumor-induced neovascularity compared with all other groups (P < 0.005), without altering the expression of VEGF or flk1. Mab DC101 and paclitaxel combined enhanced apoptosis in the tumor and endothelial cells compared with other treatment (P < 0.005). These studies indicate that Mab DC101, which blocks VEGFR-2 function, has significant efficacy against human TCC, especially when combined with the chemotherapeutic agent paclitaxel. The antitumor effect was mediated by inhibition of angiogenesis and induction of both tumor cell and endothelial cell apoptosis.

Vascular endothelial growth factor (VEGF) plays an important role in the production of ascitic fluid associated with malignant tumor growth. In an experimental model for malignant ascites formation, mice were inoculated intraperitoneally with syngeneic mouse sarcoma tumor cells. Ascites development was not prevented by administering tumor necrosis factor (TNF) simultaneously with the tumor cell inoculation. When the malignant ascites was first drained and renewal of ascites was monitored, however, a TNF dose-dependent inhibition of ascitic fluid accumulation was observed. Northern blot analyses indicated transient downregulation by TNF on the expression of VEGF mRNA in tumor cells. Monoclonal antibody, (mAb) DC101 generated against the mouse VEGF receptor Flk-1 prevented the recurrence of malignant ascites in mice similar to TNF inhibition. In addition, exogenous soluble human Flt-1 used as an inhibitor of endogenous VEGF binding also inhibited ascites recurrence. These data demonstrate that the observed inhibitory effect of TNF on reestablishment of malignant ascites can be achieved equally by inhibition of the interaction of VEGF with its receptor Flk-1.

Both epidermal growth factor receptor (EGF-R) signaling mechanisms and angiogenesis have been evaluated as independent targets for therapy of human pancreatic carcinoma, but a link between the two processes has been identified only recently. This study evaluated whether EGF-R blockade therapy with anti-EGF-R antibody C225 inhibits pancreatic carcinoma growth and metastasis in an orthotopic nude mouse model via tumor-mediated angiogenesis and whether gemcitabine potentiates this effect. In vitro treatment of human pancreatic carcinoma L3.6pl cells with C225 inhibited EGF-R autophosphorylation, producing a maximum of 20% cytostasis. Treatment with C225 plus gemcitabine resulted in additive cytotoxic effects that increased with increasing gemcitabine concentrations. Dose-dependent decreases in expression of the angiogenic factors vascular endothelial growth factor and interleukin 8 (but not basic fibroblast growth factor) were observed in the C225-treated cells (mRNA and protein levels). In L3.6pl tumors established in the pancreas of nude mice, systemic therapy with C225 alone and C225 in combination with gemcitabine resulted in growth inhibition, tumor regression, and abrogation of metastasis; median tumor volume was reduced from 538 to 0.3 and to 0 mm3, respectively. Gemcitabine treatment alone reduced median tumor volume from 538 to 152 mm3. Liver metastases were present in 50% of the controls, 30% of the gemcitabine-treated animals, and 20% of C225-treated animals. No macroscopically visible liver metastases were observed in the combination treatment group. As early as 11 days after C225 treatment, the median percentage of proliferating cell nuclear antigen-positive cells was substantially reduced compared with gemcitabine treatment alone (26% versus 73%, respectively) versus controls (92%), correlating with in vivo blockade of EGF-R activation. Similarly after 11 days treatment, production of vascular endothelial growth factor and interleukin 8 was significantly lower in C225 and C225 plus gemcitabine-treated tumors versus gemcitabine-treated and control tumors. Significant differences in microvessel density were observed 18 days after C225 or combination treatments (but not gemcitabine alone) in direct correlation with the difference in percentage of apoptotic endothelial cells, as visualized by double immunofluorescence microscopy. These experiments indicate that therapeutic strategies targeting EGF-R have a significant antitumor effect on human L3.6pl pancreatic carcinoma growing in nude mice which is mediated in part by inhibition of tumor-induced angiogenesis, leading to tumor cell apoptosis and regression. Furthermore, this effect is potentiated in combination with gemcitabine.

Various conventional chemotherapeutic drugs can block angiogenesis or even kill activated, dividing endothelial cells. Such effects may contribute to the antitumor efficacy of chemotherapy in vivo and may delay or prevent the acquisition of drug-resistance by cancer cells. We have implemented a treatment regimen that augments the potential antivascular effects of chemotherapy, that is devoid of obvious toxic side effects, and that obstructs the development of drug resistance by tumor cells. Xenografts of 2 independent neuroblastoma cell lines were subjected to either continuous treatment with low doses of vinblastine, a monoclonal neutralizing antibody (DC101) targeting the flk-1/KDR (type 2) receptor for VEGF, or both agents together. The rationale for this combination was that any antivascular effects of the low-dose chemotherapy would be selectively enhanced in cells of newly formed vessels when survival signals mediated by VEGF are blocked. Both DC101 and low-dose vinblastine treatment individually resulted in significant but transient xenograft regression, diminished tumor vascularity, and direct inhibition of angiogenesis. Remarkably, the combination therapy resulted in full and sustained regressions of large established tumors, without an ensuing increase in host toxicity or any signs of acquired drug resistance during the course of treatment, which lasted for >6 months. This article may have been published online in advance of the print edition. The date of publication is available from the JCI website, http://www.jci.org.