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National Cancer Institute, Bethesda, Maryland 20892
Chen et al. (1) , in a recent issue of Cancer Research, report on liposomal gene transfer of endostatin and angiostatin to tumor-bearing animals. In this study, the authors measured serum endostatin levels (two animals per time point) 24 and 48 h after i.v. injection of liposomes complexed to an endostatin expression plasmid "to ensure that endostatin was expressed in vivo " (1) . Serum levels were 10.8 and 33 ng/ml, respectively. Serum levels of control mice were not reported. The article implies that the antitumor effect observed may be due to increased circulating levels of endostatin perhaps produced by liposomal uptake in the lung. The authors conclude that although they observed an inhibition of tumor growth in treated animals, increased tumor targeting may be necessary.
We share the authors’ enthusiasm about antiangiogenic gene therapy and are encouraged by their demonstration of a significant antitumor effect. These data indicate that liposomal delivery systems merit further investigation. However, we would caution interpretation of the data that increased serum endostatin levels were achieved, and we suggest that the antitumor effect noted in this study was possibly due to liposomal delivery of plasmid DNA to tumor vasculature rather than systemic elevation of circulating endostatin levels.
We measured serum endostatin levels in untreated 6–8-week-old female athymic nude mice (Charles River Laboratories, Wilmington, MA) using the same murine endostatin competitive immunoassay kit as Chen et al. (Ref. 1 ; Accucyte; Cytimmune Sciences, Inc., College Park, MD). Samples were measured in duplicate in 16 animals. Mean serum endostatin level ± SD was 30.9 ± 7.4 ng/ml (range, 17.3–46.4 ng/ml). These data suggest that nude mice have baseline circulating levels of endostatin similar to the peak posttreatment level reported in the above-mentioned study. A baseline level of circulating endostatin is present in humans as well. Hefler et al. (2) reported a median serum endostatin concentration of 49.8 ng/ml (range, 25.7–115.2 ng/ml) in 35 healthy women.
The most pronounced antitumor effects reported in the study by Chen et al. (1) were shortly after 50 days of tumor growth, when tumors had reached approximately 1500 mm3. This would suggest that as the tumor vasculature increased, more liposome delivery to the tumor was possible. Measurement of tumor levels of endostatin or a suitable marker gene would have been helpful.
Whereas it has been suggested that antiangiogenic gene therapy should target tumor specifically (3) , advantages to using gene therapy to elevate the circulating levels of antiangiogenic agents have also been cited (4) . Although the level of circulating endostatin required for inhibition of angiogenesis in vivo is not known, a concentration of approximately 100 ng/ml appears to be required to inhibit endothelial cell proliferation in vitro (5) . Although Chen et al. (1) cite evidence that liposomal delivery of plasmid DNA to tumor is inefficient, it nonetheless has been reported (6) .
In conclusion, the antitumor effect observed by Chen et al. (1) represents a significant advance in the field of antiangiogenic gene therapy. We believe, however, that this effect was due to increased local concentrations of endostatin and that elevated circulating levels were not achieved within the time points measured. Although we agree with the authors that increasing the tumor specificity of liposomal vectors may enhance the antitumor effect, tumor specificity remains one of the greatest challenges to all systemic therapies for cancer. We emphasize that antiangiogenic gene therapy with the goal of elevating circulating levels of endogenous antiangiogenic agents may obviate this difficult problem.
FOOTNOTES
1 To whom requests for reprints should be addressed at, Surgery Branch, National Cancer Institute, NIH, 9000 Rockville Pike, Bethesda, MD 20892. Phone: (301) 496-5049; Fax: (301) 402-1788. 
2 To whom requests for reprints should be addressed at, Department of Pathology, University of Maryland-Baltimore, 10 South Pine Street, Baltimore, MD 21201.
Received 8/11/99. Accepted 1/ 6/00.
REFERENCES
University of Maryland, Baltimore, Maryland 21201
As Drs. A. L. Feldman and S. K. Libutti suggest (1) , tumor reduction reported in our Cancer Research article (2) may be due to local secretion of endostatin from tumor vessels transfected by the liposome:plasmid complex. It was not the intent of the Cancer Research article to suggest that systemic levels of endostatin secreted from the lung vasculature or extratumoral tissues by themselves inhibited tumor growth (2) . Because other laboratories including ours have previously shown that i.v. delivery of a plasmid encoding nonsecreted p53 inhibited tumor size (3) , we were certainly aware of the potential of systemic gene therapy to produce sufficient amounts of the therapeutic protein within the tumor. In fact, these p53 experiments provided the impetus for us to investigate whether genes that encode antiangiogenic proteins inhibit cancer. However, systemic endostatin levels produced by extratumoral tissues may have some role in reducing tumor size in our study. Furthermore, we believe that the roles of intratumoral and extratumoral-secreted endostatin in reducing tumor growth cannot be clearly defined in the absence of intratumoral vascular levels of endostatin.
The assumptions reached by Drs. Feldman and Libutti about our systemic endostatin levels being insufficient to inhibit tumor growth are based solely on their obtaining a "baseline endostatin" level (mean, 30.9 ng/ml) in untreated mice higher than the level we reported in mice injected with an endostatin plasmid (PCI-Endo). As stated in our study, the primary goal in measuring endostatin levels in vivo was to demonstrate that the transfected gene product was detectable. Correlations or the lack thereof between systemic endostatin levels and tumor reduction were not made because peak endostatin levels were not determined. We did establish that endostatin levels were elevated after i.v. injection of the liposome:PCI-Endo complex. The endostatin levels were 10 and 33 ng/ml on days 1 and 2, respectively. These endostatin levels due to PCI-Endo were determined in treated mice after subtracting the baseline value of 12 ng/ml from untreated mice. This was not clearly stated in "Materials and Methods." It is important to note that other investigators have measured serum endostatin levels lower than those we reported after injecting a cationic polymer:plasmid endostatin complex into the mouse muscle (4) . They also discovered that low systemic endostatin levels secreted by the muscle were sufficient to inhibit tumor growth. Thus, the concerns raised by Drs. Feldman and Libutti regarding whether the levels of endostatin in our report were inadequate to inhibit tumor growth are not substantiated by the results of this second gene therapy study. As stated previously, however, we cannot currently delineate the contribution of intratumoral and extratumoral-produced endostatin in reducing tumor size with i.v. delivery of the liposome:plasmid complex.
The exact nature of the "baseline endostatin" measured in the serum (or urine) of untreated mice is unknown. Endostatin, a proteolytic product of collagen XVIII, may have important roles in modulating angiogenesis in normal and neoplastic states (5) . Nevertheless, it is possible that "baseline endostatin" values in untreated mice represent an array of breakdown products of collagen XVIII containing endostatin, the majority of which contain minimal to no antiangiogenic activity. Collagen XVIII and fragments of collagen XVIII containing full-length endostatin have been found not to inhibit endothelial proliferation (5) . In addition, when 11 amino acids were added to the NH2 terminus of full-length endostatin, this modified endostatin had little or no antiangiogenic activity; however, the polyclonal antibody to endostatin (Cytimmune Sciences, Inc.) recognized this modified endostatin (data not shown). Although we suspect that most fractions of "baseline endostatin" do not have antiangiogenic activity, the function of the "baseline endostatin" levels in untreated mice will remain unclear until its antiangiogenic activity is assessed with a bioassay.
We share the enthusiasm of Drs. Feldman and Libutti for systemic delivery of antiangiogenic gene therapy. Regardless of the vector delivery system or injection route, it will be a significant advance when systemic levels of antiangiogenic gene products are able to reduce a large tumor to a microscopic dormant state. Nevertheless, we do not feel that the clinical utility of i.v. delivered gene therapy with antiangiogenic proteins will be fully realized until carriers that specifically target the tumor vessels are achieved.
Received 11/30/99. Accepted 1/ 6/00.
REFERENCES
This article has been cited by other articles:
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  A. L. Feldman, H. R. Alexander, S. M. Hewitt, D. Lorang, C. E. Thiruvathukal, E. M. Turner, and S. K. Libutti Effect of Retroviral Endostatin Gene Transfer on Subcutaneous and Intraperitoneal Growth of Murine Tumors J Natl Cancer Inst, July 4, 2001; 93(13): 1014 - 1020. [Abstract] [Full Text] [PDF]
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