Induction of anti-tumor immunity in mice using a syngeneic endothelial cell vaccine.

Tumor endothelium could represent a novel target for active and passive immunotherapies of cancer. Here, we show that endothelial cells can be used as a vaccine in mice. In this study, three endothelial cell vaccine preparations from syngeneic (SVR), allogeneic (ISOS-1) and xenogeneic (ISO-HAS) sources were used to vaccinate mice. All mice developed humoral immune responses to endothelial cells and showed lower basal serum VEGF levels (37-45% lower) compared with unvaccinated control mice. Mice receiving the syngeneic SVR vaccine showed substantial inhibition of tumor growth after B16F10 melanoma challenge (50% of the mice in this group were tumor-free). The tumors that developed in the few mice in the syngeneic group had lower microvessel density counts (4-5 fold) compared with the other groups. The data suggests an in vivo antiangiogenic effect as the potential mechanism for the anti-cancer effect. In summary, further studies using other tumor models to demonstrate broad protection of this novel type of antiangiogenic vaccine are warranted.

Polyclonal antibodies to xenogeneic endothelial cells induce apoptosis and block support of tumor growth in mice.

In this study, we demonstrate that vaccination of rabbits with murine endothelial cells yields polyclonal immunoglobulin (IgG) with potent antiangiogenic activity. The mechanism of this response appears to be through apoptosis of endothelial cells in vitro. Induction of polyclonal IgG in a xenogeneic host may be useful in passive immunotherapy of a variety of cancers. In fact, the antibody showed antitumor activity in three mouse tumor models (murine B16F10 melanoma, murine SVR angiosarcoma, and human DLD-1 colorectal adenocarcinoma). The polyclonal antibody generated here demonstrated utility in radioimaging of tumors in vivo, using positron emission tomography (PET) imaging, and suggested an antitumor effect in vivo. The results suggest that the antitumor effect in vivo may be related to antiangiogenic effects. Furthermore, anti-endothelial cell antibodies such as these could be useful reagents in isolating specific targets that comprise and induce the antiangiogenic effect.

Combination angiostatin and endostatin gene transfer induces synergistic antiangiogenic activity in vitro and antitumor efficacy in leukemia and solid tumors in mice.

Angiostatin and endostatin are potent endothelial cell growth inhibitors that have been shown to inhibit angiogenesis in vivo and tumor growth in mice. However, tumor shrinkage requires chronic delivery of large doses of these proteins. Here we report synergistic antitumor activity and survival of animals when these factors are delivered in combination to tumors by retroviral gene transfer. We have demonstrated this efficacy in both murine leukemia and melanoma models. Complete loss of tumorigenicity was seen in 40% of the animals receiving tumors transduced by the combination of angiostatin and endostatin in the leukemia model. The synergy was also demonstrated in vitro on human umbilical vein endothelial cell differentiation and this antiangiogenic activity may suggest a mechanism for the antitumor activity in vivo. These findings imply separate pathways by which angiostatin and endostatin mediate their antiangiogenic effects. Together, these data suggest that a combination of antiangiogenic factors delivered by retroviral gene transfer may produce synergistic antitumor effects in both leukemia and solid tumors, thus avoiding long-term administration of recombinant proteins. The data also suggest that novel combinations of antiangiogenic factors delivered into tumors require further investigation as therapeutic modalities.

New molecular targets and biological therapies in sarcomas.

The treatment of patients with soft tissue and bone sarcomas has dramatically improved over the last decade. This improvement has been brought about through advances in diagnosis, surgical techniques, conformal radiotherapy, and combination chemotherapy. Further advances in the management of the diverse spectrum of sarcoma patients will reflect tailoring of therapy based on molecular abnormalities. The role of cytogenetics and molecular analysis of fusion or mutated genes in diagnosis, prognosis, and design of biological treatments is discussed. An example of this approach has been the recent success in treatment of patients with gastrointestinal stromal tumours expressing mutant c-kit with a specific tyrosine kinase inhibitor, STI571. Molecular rearrangements may also serve as targets for designing specific immunotherapies with the fusion gene product. The use of biological therapies with signal transduction inhibitors, angiogenesis inhibitors, matrix metalloproteinase inhibitors, immunotherapy, differentiation inducers, and gene therapy could complement existing treatments for long-term control of disease. As these newer biological agents take form, clinical trial design will undergo change to reflect the chronic nature of these therapies.

Statin-AE: a novel angiostatin-endostatin fusion protein with enhanced antiangiogenic and antitumor activity.

The combination of angiostatin and endostatin has been shown to have synergistic antiangiogenic and antitumor effects when the genes for these proteins are delivered to tumor cells by retroviral gene transfer. Here we report the construction of a murine angiostatin-endostatin fusion gene (Statin-AE) which shows enhanced antiangiogenic activity on human umbilical vein endothelial cell (HUVEC) tube formation in vitro compared with angiostatin or endostatin alone. Similarly, the fusion gene demonstrates antiangiogenic effects in vivo and antitumor activity in a B16F10 melanoma model when co-delivered by retroviral packaging cell inoculation in mice. The fusion gene demonstrates significantly greater inhibition of tumor growth compared with angiostatin, endostatin or the combination of genes.

Altered expression of cytochrome P450 mRNA during chemical-induced hepatocarcinogenesis and following partial hepatectomy.

Levels of various cytochrome P450 proteins have been reported to be decreased to varying degrees in chemically induced hepatocyte nodules and following partial hepatectomy (PH). By screening a rat liver lambda ZAP cDNA expression library with antibodies raised against a partially purified preparation of cytochrome P450 isolated from untreated male Fischer 344 rats, we have isolated a 1.1-kb cDNA. This cDNA was sequenced for 139 bases from the 5' end of the sense strand and comparison of the resulting sequence with the sequences in Gene Man DNA data bank revealed 95% homology of the sequenced portion with male-specific rat cytochrome P450 (M-1, CYP IIC11). The 32P-labeled cDNA was used as a hybridization probe on RNA blots (Northern blots) prepared with total RNA from rat livers obtained post PH, from aflatoxin B1(AFB1)-induced rat liver tumors and from rat liver nodules induced with a combination of diethylnitrosamine/acetylaminofluorene/PH (DEN/AFF/PH). At 36 and 72 hr post PH, the mRNA level was decreased by > 93%. Relative to the corresponding control livers, the mRNA level was also decreased by 97% in the liver nodules and by 57% in AFB1-induced liver tumors. The RNA blots derived from the liver nodules and AFB1-induced liver tumors were also probed with a cDNA probe (R17) that recognizes other cytochromes P450 (CYP IIB1/CYP IIB2). The mRNA corresponding to CYP IIB1/CYP IIB2 was also depressed 92% in the nodules and 65% in the tumors. These results clearly indicate that the depression of both CYP IIC11 and IIB1/IIB2 in the hepatic nodules and the tumors is related to the inhibition of transcription and/or enhanced degradation of the mRNA.

Interaction of the cyclophosphamide metabolite, acrolein, with lactate dehydrogenase-m homolog.

The cyclophosphamide metabolite, acrolein, binds to both nucleic acids and proteins. In this report, acrolein is shown to interact preferentially with a rat liver 35KD DNA-binding protein. The 35KD protein was purified to homogeneity from a hepatic microsomal fraction using (NH4)2SO4 precipitation, DEAE-Sepharose chromatography, cation exchange HPLC, and DNA-Sepharose affinity chromatography. The protein was identified as a homologue of lactate dehydrogenase (LDH) M subunit on the basis of partial amino acid sequencing, amino acid composition and immunological methods. The interaction of this protein with acrolein may have implications for tissue-specific toxicities and/or oncoselectivity of cyclophosphamide.

32P-postlabeling analysis of binding of the cyclophosphamide metabolite, acrolein, to DNA.

The cyclophosphamide metabolite, acrolein, was reacted with 2'-deoxyguanosine-3'-monophosphate, and two adducts were detected by high performance liquid chromatography and 32P-postlabeling assay. These adducts were resistant to dephosphorylation by nuclease P1 and could be isolated and detected from calf thymus DNA that had been reacted in vitro with acrolein. A combination of HPLC purification and enzymatic digestion of normal nucleotides by nuclease P1 allowed for the detection of these adducts in hepatic DNA from mice treated with cyclophosphamide. The level of the two adducts in the hepatic DNA, as determined by 32P-postlabeling, was one adduct per 2.7-4.1 x 10(7) normal nucleotides.