ILAR Journal Online, Volume 40(4) 1999: Animal Models of Inflammation

Online Issues

<< All Back-issues

<< This Issue's Table of Contents

ILAR Journal V40(4) 1999
Animal Models of Inflammation

Use of Immunodeficient Mice for the Evaluation of CXC Chemokines in the Regulation of Tumor-associated Angiogenesis
Robert M. Strieter, Christina L. Addison, Jan E. Ehlert, Michael P. Keane, John A. Belperio, Marie D. Burdick, and Douglas A. Arenberg

All authors of this article are from the Department of Internal Medicine, Division of Pulmonary and Critical Medicine, The University of Michigan Medical School, Ann Arbor, Michigan. Robert M. Strieter, M.D., is a Professor; Christina L. Addison, Ph.D., is a Post-doctoral Research Fellow; Jan E. Ehlert, Ph.D., is a Post-doctoral Research Fellow; Michael P. Keane, M.B., is an Assistant Professor; John A. Belperio, M.D., is a Research Fellow; Marie D. Burdick, B.S., is Chief Technician in Dr. Strieter's Laboratory; and Douglas A. Arenberg, M.D., is an Assistant Professor.

Introduction

The use of human tumor xenografts in immunodeficient mice has provided significant insight into the biology of tumor growth and metastasis and has enabled scientists to study the complex biology of human tumor growth more effectively. Work from our laboratory and others' supports the notion that net tumor-derived angiogenesis during tumorigenesis of human tumors is determined, in part, by an imbalance in favor of the overexpression of angiogenic compared with angiostatic CXC chemokines (Figure 1). This paradigm predicts an environment that favors anglogenesis and tumorigenesis and supports the potential for spontaneous metastases. The purpose of this article is to describe the use of immunodeficient mice as an animal model system to characterize the qualitative and quantitative presence of these angiogenic and angiostatic CXC chemokines during tumorigenesis and to determine their net contribution to human tumorigenesis and metastasis in vivo. Various cancer cell lines have been used and xenografted into immunodeficient mice (such as severe combined immunodeficiency [SCID¹] or nude mice) to create a human tumor/mouse chimeras. The results of these studies have provided evidence that an imbalance in the biology of angiogenic versus angiostatic CXC chemokines supports a significant portion of human tumor-derived angiogenesis leading to augmented tumorigenesis and spontaneous metastases. Moreover, using the immuno-deficient mouse model system has made possible the identification of potential novel strategies to manipulate therapeutically the imbalance of angiogenic compared with angiostatic CXC chemokines, which may be directly translational to human disease.

Process of Angiogenesis

Angiogenesis is the growth of new blood vessels from preexisting vessels and capillaries. This biological process is critical to a variety of physiological processes like embryo-genesis and wound repair. However, it is also a process that plays a devastating role in a number of pathological processes such as tumorigenesis (Auerbach 1981; Auerbach and others 1976; Folkman 1995, 1993, 1985; Folkman and Brem 1992; Folkman and Cotran 1976; Folkman and Klagsbrun 1987; Leibovich and Weisman 1988; Polverini 1996; Polverini and others 1977). The regulation of angiogenesis depends on a dual, yet opposing, balance of local factors that promote or inhibit neovascularization. For example, the rate of normal capillary endothelial cell turnover in adults is typically measured in months or years (Engerman and others 1967; Tannock and Hayashi 1972), suggesting a balance between angiogenic and angiostatic factors under homeostatic conditions. By contrast, the development of granulation tissue of wounds shifts the balance in favor of a predominance of angiogenic factors leading to new functioning capillaries within a matter of days (Leibovich and Weisman 1988). Angiogenesis of wound granulation tissue is locally controlled and transient. These qualities support the notion that the following process occurs in wound granulation tissue: a marked reduction in the elaboration of angiogenic factors and/or a simultaneous increase in the level of factors that inhibit neovascularization (Bouck 1992). In contrast to the precise regulation in wound repair, dysregulation of angio-genesis can lead to an imbalance in the relationship of angio-genic and angiostatic factors, which favors persistent net angiogenesis that contributes to the pathogenesis of tumor growth and metastases. The complement of angiogenic and angiostatic factors may vary among different physiological and pathological settings. However, the recognition of this dual mechanism of control is critical to gain insight into this complex process and understand the regulation of angio-genesis in association with tumorigenesis.

CXC Chemokine Family of Cytokines

A variety of factors have been described that promote angio-genesis (Auerbach 1981; Folkman 1985, 1993, 1996, 1997, 1998; Folkman and Brem 1992; Folkman and Klagsbrun 1987; Gastl and others 1997; Hotfilder and others 1997; Hui and Ignoffo 1998; Kumar and Fidler 1998; Lund and others 1998; Pluda 1997; Risau 1997; Zetter 1998; Ziche and others 1996). Perhaps the best studied of the angiogenic factors are vascular endothelial growth factor (VEGF¹)and basic fibro-blast growth factor (bFGF¹) (Gastl and others 1997; Hotfilder and others 1997; Hui and Ignoffo 1998; Kumar and Fidler 1998; Lund and others 1998; Pluda 1997; Risau 1997; Zetter 1998; Ziche and others 1996). In contrast, both angiostatin and endostatin have been found to display marked inhibition of angiogenesis (O'Reilly and others 1994, 1997). Although these factors are important in the regulation of angiogenesis in both physiological and pathophysiological processes, these molecules do not fully account for all of the modulation of the neovascular response in pathological conditions, such as tumorigenesis.

CXC chemokines are characteristically heparin binding proteins. On a structural level, they have four highly conserved cysteine amino acid residues, with the first two cys-teines separated by one nonconserved amino acid residue, hence the name CXC (Adams and Lloyd 1997; Baggiolini 1998; Baggiolini and others 1994; Baggiolini and others 1997; Balkwill 1998; Luster 1998; Rollins 1997; Strieter and Kunkel 1997; Taub and Oppenheim 1994; Walz and others 1996). Although the CXC motif distinguishes this family from other chemokine families, a second structural domain within this family dictates their angiogenic potential. The NH2-terminus of the majority of the CXC chemokines containing three amino acid residues (Glu-Leu-Arg: the "ELR" motif) precedes the first cysteine amino acid residue of the primary structure of these cytokines (Adams and Lloyd 1997; Baggiolini 1998; Baggiolini and others 1994, 1997; Balkwill 1998; Luster 1998; Rollins 1997; Strieter and Kunkel 1997; Taub and Oppenheim 1994; Walz and others 1996). The family members that contain the ELR motif (ELR⁺⁾are potent promoters of angiogenesis in physiological concentrations of 1 to 10 nM (Strieter and others 1995) (Table 1). In contrast, members that lack the ELR motif (ELR-) are potent inhibitors of angiogenesis in physiological concentrations of 500 pM to 1 nM (Strieter and others 1995). This difference suggests on a structural/functional level that members of the CXC chemokine family are unique cytokines in their ability to behave in a disparate manner in the regulation of angio-genesis. The angiogenic members include intefieukin (ILl)-8, epithelial neutrophil activating protein (ENA¹)-78, growth-related oncogenes (GRO¹-a, b, and g), granulocyte chemotactic protein-2, and NH2-terminal truncated forms of platelet basic protein (pBpI), which include connective tissue activating protein-III, beta-thromboglobulin, and neutrophil activating protein-2 (Hu and others 1993; Koch and others 1992; Strieter and others 1992, 1995).

The angiostatic (ELR-) members of the CXC chemokine family include platelet factor-4 (PF4) (Thomas and others 1970), monokine induced by interferon-g(MIG¹) (Farber 1992, 1993, 1990, 1997), and interferon (IFN¹)-g-inducible protein (IP¹)-10 (Farber 1990, 1992, 1993, 1997; Luster and Ravetch 1987; Luster and others 1985) (Table 1). Although interferon-inducible T-cell alpha chemoattractant and stromal cell-derived factor (SDF¹)-1 are additional ELR- CXC chemokines, it remains unclear whether they inhibit angiogenesis. SDF-1 has been found to induce in vitro migration of human umbilical vein endothelial cells (Gupta and others 1998). In contrast, SDF-1 has also been found to attenuate the in vivo angiogenic activity of either ELR⁺CXC chemokines, bFGF, or VEGF using the rat cornea micro-pocket (CMP¹)assay of neovascularization (Arenberg and others 1997). IP-10 can be induced by all three interferons (IFN-a, b, and g) (Farher 1990, 1992, 1993, 1997; Luster 1998; Luster and Ravetch 1987; Luster and others 1985). MIG is unique in that it is only induced by IFN-g (Farber 1992, 1993, 1990, 1997; Luster 1998; Luster and Ravetch 1987; Luster and others 1985). Therefore, interferons and other cytokines that can induce the expression of interferons (IL- 12 and IL- 18) may have a profound effect on the production of IP-10 and MIG (Table 2). Although interferons induce the production of the angiostatic CXC chemokines IP-10 and MIG, they attenuate the expression of the angiogenic CXC chemokines IL-8, GRO-g, and ENA-78 (Gusella and others 1993; Schnyder-Candrian and others 1995)(Table 2). This differential regulation of angiostatic versus angio-genic CXC chemokines by interferons may, in part, account for their previously documented inhibitory effect on angio-genesis (Angiolillo and others 1996; Coughlin and others 1998; Folkman 1997; Majewski and others 1996; Sgadari and others 1996; Vizier and others 1998; Zetter 1998).

CXC Chemokines Are Potent Regulators of Angiogenesis

PF4 (ELR-) was the first CXC chemokine reported to regulate angiogenesis. PF4 was found to inhibit bFGF-induced angiogenesis and attenuate growth of melanoma and colon carcinomas in a murine model of tumorigenesis (Hansell and others 1995; Maione and others 1990, 1991; Sharpe and others 1990). In contrast, IL-8 (ELR+) was the first CXC chemokine found to induce angiogenesis. IL-8 was shown to mediate both in vitro endothelial cell chemotactic and proliferative activity as well as in vivo angiogenesis in the absence of preceding inflammation using bioassays of angiogenesis (Hu and others 1993; Koch and others 1992; Strieter and others 1992). These findings have been substantiated by other investigations, which have shown that IL-8, similar to bFGF or VEGF, induces endothelial cell tube formation in vitro and angiogenesis in vivo (Norrby 1996; Yoshida and others 1997). These experiments prove that IL-8 has a direct effect on the endothelial cell and that this angiogenic activity is distinct from its ability to induce inflammation. These findings suggest that members of the CXC chemokine family function in a disparate manner and can behave as either potent angiogenic or angiostatic factors in regulating net neovascularization.

Based on this contention, we hypothesized that the highly conserved ELR motif of several members of the CXC chemokine family is a structural/functional domain that dictates their angiogenic activity. To test this postulate, endothelial cell chemotaxis was performed in the presence or absence of varying concentrations of ELR+ or ELR- CXC chemokines to ascertain whether CXC chemokines display disparate angiogenic activity. All of the ELR⁺CXC chemokines tested demonstrated significant endothelial cell chemotactic activity over background, whereas the endothelial cell chemotactic activity of ELR- CXC chemokines was similar to background (Strieter and others 1995). Moreover, ELR-CXC chemokines inhibited endothelial cell chemotactic response to ELR⁺CXC chemokines, bFGF, or VEGF. This was further confirmed in vivo using the CMP assay of neovascularization (Strieter and others 1995) (Table 3). To establish that the ELR motif is the critical structural/functional domain that dictates angiogenic activity for members of the CXC chemokine family, site-directed mutants were constructed that contained amino acid residue substitutions for the ELR motif of wild-type IL-8 (Strieter and others 1995). In addition, a site-directed mutant of MIG was produced containing the ELR motif immediately adjacent to the first cysteine amino acid residue of the primary structure of MIG (Strieter and others 1995). Endothelial cell chemotaxis and the CMP assay were used to assess the biological activity of these mutants. Both IL-8 mutants failed to induce endothelial cell migration; however, both mutants inhibited the maximal endothelial chemotactic activity of wild-type IL-8 and bFGF (Strieter and others 1995). In addition, both IL-8 mutants inhibited the angiogenic response of either wild-type IL-8 or ENA-78, or bFGF in the CMP assay (Strieter and others 1995). In contrast, the ELR⁺mutant of wild-type MIG induced a significant angiogenic response, and wild-type MIG inhibited the angiogenic response of this mutant in the CMP assay (Strieter and others 1995). These findings suggest a structural/functional role of the ELR motif in determining the angiogenic or angiostatic potential of CXC chemokines, supporting the hypothesis that the net biological balance between angiogenic and angiostatic CXC chemokines may play an important role in regulating overall tumor-associated angiogenesis.

ELR⁺CXC Chemokines Promote Angiogenesis Associated with Tumorigenesis

The CXC chemokines are clearly important mediators of tumorigenesis related to their angiogenic properties. Although GRO-[3 has been recently reported to inhibit angiogenesis (Cao and others 1995), the concentration used in this study was 1000-fold greater (1 to 10 gM) than that found for its angiogenic activity (1 to 10 nM) (Arenberg and others 1997; Strieter and others 1995). This finding suggests that super-physiological concentrations of GRO-b can "desensitize" the angiogenic response. Moreover, studies in melanoma support that all GROs play a significant role in mediating tumorigenesis related to both their mitogenic and angiogenic activities. For example, GRO-a, b, and g are all found to be highly expressed in human melanoma specimens (Luan and others 1997). To determine the biological significance of the presence of these ELR⁺CXC chemokines in melanoma, human GRO-a, b, and g genes were transfected into immortalized murine melanocytes (Luan and others 1997; Owen and others 1997). The continuous expression of any of these GROs in the cells transformed their phenotypic behavior, allowing them to form large colonies in soft agar in vitro and to generate tumors in vivo in both nude and SCID mice (Luan and others 1997; Owen and others 1997). The tumors that formed were highly vascular and similar to the vascularity of B16 melanoma controls (Luan and others 1997; Owen and others 1997). Passive immunization of tumor-bearing animals with specific anti-GRO antibodies resulted in a marked reduction of tumor-derived angiogenesis and inhibition of tumor growth (Luan and others 1997; Owen and others 1997). These findings support the notion that the ELR⁺CXC chemokines, such as GRO-a, b, and g, have the ability to act as potent angiogenic factors to promote tumorigenesis in melanoma.

The progression and growth of ovarian carcinoma are also dependent on successful angiogenesis, and IL-8 has been determined to play a significant role in mediating human ovarian carcinoma-derived angiogenesis and tumorigenesis in nude mice (Yoneda and others 1998). Yoneda and associates (1998) examined the expression of IL-8, bFGF, and VEGF in five different human ovarian carcinoma cell lines. All cell lines expressed similar levels of bFGF in vitro; however, these cells expressed either high or low levels of IL-8 or VEGF. High-expressing IL-8 tumors were very aggressive, resulting in a high mortality rate that was tied to marked neovascularization (Yoneda and others 1998). The expression of IL-8 was directly correlated with neovascularization and inversely correlated with survival, whereas VEGF expression was correlated only with production of ascites (Yoneda and others 1998). No correlation was found for the expression of bFGF with either tumor neovascularization or survival (Yoneda and others 1998). These studies have also been substantiated in gastric carcinoma and melanoma, in which the expression of IL-8 correlates with vascularity, tumorigenicity, and metastatic properties of these tumors (Kitadai and others 1998; Singh and others 1994).

IL-8 has been found in specimens of non-small cell lung cancer (NSCLC¹)(Smith and others 1994) and has been determined to be a significant angiogenic factor contributing to overall tumor-derived angiogenic activity (Arenberg and others 1996a; Smith and others 1994). Extending these studies to an in vivo model system of human tumorigenesis (namely, human NSCLC/SCID mouse chimera) (Arenberg and others 1996a), tumor-derived IL-8 was found to be directly correlated with tumorigenesis (Arenberg and others 1996a). Tumor-beating animals treated with neutralizing antibodies to IL-8 demonstrated a >40% reduction in tumor growth, which was paralleled by a reduction in spontaneous metastases to the lung (Arenberg and others 1996a). The attenuation of tumor growth and metastases was directly correlated with reduced tumor-derived angiogenesis. These findings have been corroborated using other NSCLC cell lines. Yatsunami and associates (1997) determined that NSCLC cell lines that constitutively express IL-8 have greater tumorigenic potential in nude mice, and this property is directly correlated with their angiogenic activity.

Although IL-8 may represent an important angiogenic CXC chemokine, ENA-78 may be a more important angiogenic CXC chemokine than IL-8 in NSCLC (Arenberg and others 1998). Human surgical specimens of NSCLC tumors were found to display a direct and significant correlation of ENA-78 protein levels with tumor neovascularization. The biological relevance of this finding was confirmed in a SCID mouse model of human tumorigenesis using human NSCLC cell lines. ENA-78 expression in tumors was directly correlated with tumor growth. Moreover, when human NSCLC tumor-bearing animals were passively immunized with anti-ENA-78, both tumor growth and spontaneous metastases were markedly attenuated. The reduction in tumor growth was accompanied by a marked decrease in tumor vascularity and an increase in apoptosis of the tumor cells. The apoptosis of these cells was not due to a direct effect of depletion of ENA-78 because there was no in vitro evidence that ENA-78 had any effect on apoptosis. This belief is consistent with the fact that angiostatic therapy of tumors is associated with increased tumor cell apoptosis (O'Reilly and others 1996, 1997). Similarly, in vivo and in vitro proliferation of NSCLC cells was unaffected by the presence of anti-ENA-78 to "deplete" endogenous ENA-78. Although there was a significant correlation of ENA-78 expression with tumor vascu-larity and tumorigenesis, neutralization of ENA-78 did not completely inhibit tumor growth. This finding potentially reflects the fact that angiogenic activity induced by NSCLC tumors is probably due to many overlapping or redundant factors acting in a parallel manner. In addition to ENA-78 and IL-8, there are perhaps other angiogenic factors that have not been accounted for, including other ELR⁺CXC chemokines (GROs or GCP-2) and/or non-CXC chemokine angiogenic factors (bFGF or VEGF). Nevertheless, the findings that ELR+ CXC chemokines represent significant an-giogenic factors in human tumors suggest that strategies targeting angiogenic CXC chemokine-mediated angiogenesis may be a novel treatment approach to various cancers.

ELR^- CXC Chemokines Attenuate Angiogenesis Associated with Tumorigenesis

The antithesis of ELR⁺is the function of ELR- CXC chemokines in regulating angiogenesis. Burkitt's lymphoma cell lines form angiogenesis-dependent tumors in nude mice (Gurtsevitch and others 1988). To determine whether IP-10 or MIG regulates the angiogenic activity of Burkitt's lym-phomas, Sgadari and associates (1996) implanted several of these tumor cell lines in nude mice. The expression of IP-10 and MIG was greater in the tumors that demonstrated spontaneous regression compared with those tumors that grew significantly in nude mice. The regression of the tumors was directly related to evidence of reduced angiogenesis, increased tumor vascular damage, and tissue necrosis. To determine whether this effect was related to IP-10 or MIG, Burkitt's lymphoma cell lines grown in nude mice were subjected to intratumor inoculation with IP-10 or MIG. Both conditions resulted in marked reduction in tumor-associated angiogenesis (Sgadari and others 1997; Teruya-Feldstein and others 1997). Although both IP-10 and MIG can induce T-cell recruitment via the interaction with their putative CXC chemokine receptor (CXCR3) (Baggiolini 1998; Baggiolini and others 1997; Farber 1997; Luster 1998; Rollins 1997), the ability of both of these ELR- CXC chemokines to inhibit angiogenesis and induce lymphoma regression in nude mice supports the thinking that these chemokines mediate their effects in a T-cell-independent manner.

Our laboratory has examined the role of IP-10 in regulating angiogenesis associated with NSCLC (Arenberg and others 1996b). The levels of IP-10 from human surgical tumor specimens were significantly higher than in normal lung tissue. The increase in IP-10 from human NSCLC tissue was entirely attributable to the higher levels of IP-10 present in squamous cell carcinoma (SCCA¹)compared with adenocarcinoma. Moreover, depletion of IP-10 from SCCA surgical specimens resulted in augmented angiogenic activity (Arenberg and others 1996b). The marked difference in the levels and bioactivity of IP-10 associated with SCCA, compared with adenocarcinoma, is pathophysiologically relevant and represents a possible mechanism for the biological differences of these two cell-types of NSCLC. Compared with SCCA of the lung, patient survival is lower, metastatic potential is higher, and evidence of angiogenesis is greater for adenocarcinoma (Carney 1988; Minna 1991; Yuan and others 1995).

The studies described above were extended to a SCID mouse model system to examine the effect of IP-10 on human NSCLC cell line tumor growth in a T- and B-cell-independent manner. SCID mice were inoculated with either A549 (adenocarcinoma) or Calu-1 (SCCA) cells (Arenberg and others 1996). The production of IP-10 from A549 and Calu-1 tumors was inversely correlated with tumor growth (Arenberg and others 1996b); however, IP-10 levels were significantly higher in the Calu-1 compared with A549 tumors. The appearance of spontaneous lung metastases in SCID mice bearing A549 tumors occurred after IP-10 levels from either the primary tumor or plasma reached a nadir. In subsequent experiments, SCID mice bearing Calu-1 tumors were treated with either neutralizing anti-IP-10 to deplete endogenous IP-10, whereas, animals bearing A549 tumors were treated with intratumor IP-10 to "reconstitute" IP-10. Depletion of IP-10 in Calu-1 tumors resulted in a two-fold increase in their size at 10 wk. In contrast, reconstitution of intratumor IP-10 in A549 tumors reduced both their size and metastatic potential, which was directly attributable to a reduction in tumor-associated angiogenesis (Figure 2). These findings support the notion that tumor-derived IP-10 is an important endogenous angiostatic factor in NSCLC, and treatment strategies designed to reconstitute IP-10 and/or other angiostatic CXC chemokine may lead to marked tumor regression.

Conclusion

Angiogenesis is regulated by an opposing balance of angio-genic and angiostatic factors. The studies described above using immunodeficient mice as animal models of human tumorigenesis have demonstrated that CXC chemokines appear to be important endogenous factors that regulate angiogenesis in association with tumorigenesis in a variety of cancers. These findings support the notion that therapy directed at either inhibition of angiogenic or augmentation of angiostatic CXC chemokine biology may be a novel approach in the treatment of human tumors. Furthermore, the ability to study these molecules in immunodeficient mice has allowed us to assess the biology of human CXC chemokines in the regulation of angiogenesis in a T- and B-cell-independent manner in the context of human tumorigenesis.

¹ Abbreviations used in this article: bFGF, basic fibroblast growth factor; CMP, cornea micropocket; ELR, Glu-Leu-Arg; ENA, epithelial neutrophil activating protein; GRO, growth-related oncogenes; IFN, interferon; IL, interleukin; IP, inducible protein; MIG, monokine induced by interferon-y; NSCLC, non-small cell lung cancer; PF4, platelet factor-4; SCCA, squamous cell carcinoma; SCID, severe combined immunodeficient; SDF, stromal cell-derived factor; VEGF, vascular endothelial growth factor.

Acknowledgments

This work was supported, in part, by National Institutes of Health grants CA72543 (D.A.A.), HL03906 (M.P.K.), CA83052, P50 HL60289, and P50 HL46487 (R.M.S.) as well as by an American Lung Association grant (D.A.A.).

References

Adams DH, Lloyd AR. 1997. Chemokines: Leucocyte recruitment and activation cytokines. Lancet 349:490-495.

Angiolillo AL, Sgadari C, Tosato G. 1996. A role for the interferon-inducible protein 10 in inhibition of anglogenesis by interleukin-12. Ann N Y Acad Sci 795:158-167.

Arenberg DA, Keane MP, DiGiovine B, Kunkel SL, Morris SB, Xue YY, Burdick MD, Glass M C, Iannettoni MD, Stricter RM. 1998. Epithelial-neutrophil activating peptide (ENA-78) is an important angiogenic factor in non-small cell lung cancer. J Clin Invest 102:465-472.

Arenberg DA, Kunkel SL, Polverini PJ, Glass M, Burdick MD, Stricter RM. 1996a. Inhibition of interleukin-8 reduces tumorigenesis of human non-small cell lung cancer in SCID mice. J Clin Invest 97:2792-2802.

Arenberg DA, Kunkel SL, Polverini PJ, Morris SB, Burdick MD, Glass M, Taub DT, lannetoni MD, Whyte RI, Stricter RM. 1996b. Interferon-g-inducible protein 10 (IP-10) is an angiostatic factor that inhibits human non-small cell lung cancer (NSCLC) tumorigenesis and spontaneous metastases. J Exp Med 184:981-992.

Arenberg DA, Polverini PJ, Kunkel SL, Shanafelt A, Stricter RM. 1997. In vitro and in vivo systems to assess role of C-X-C chemokines in regulation of angiogenesis. Methods Enzymol 288:190-220.

Auerbach R. 1981. Anglogenesis-inducing Factors: A Review. Vol 69. New York: Academic Press.

Auerbach R, Kubal L, Sidky Y. 1976. Angiogenesis induction by tumors, embryonic tissue, and lymphocytes. Cancer Res. 36:3435-3440.

Baggiolini M. 1998. Chemokines and leukocyte traffic. Nature 392:565-568.

Baggiolini M, Dewald B, Moser B. 1994. lnterleukin-8 and related chemo-tactic cytokines CXC and CC Chemokines. Adv lmmunol 55:97-179.

Baggiolini M, Dewaid B, Moser B. 1997. Human chemokines: An update. Annu Rev Immunol 15:675-705.

Balkwill F. 1998. The molecular and cellular biology of the chemokines. J Viral Hepat 5:1-14.

Bouck N. 1992. Angiogenesis: A mechanism by which oncogenes and tumor suppressor genes regulate tumorigenesis. Cancer Treat Res 63:359-371.

Cao Y, Chen C, Weatherbee JA, Tsang M, Folkman J. 1995. GRO-beta, a -C-X-C- chemokine, is an angiogenesis inhibitor that suppresses the growth of Lewis lung carcinoma in mice. J Exp Med 182:2069-77.

Carney DN. 1988. Cancers of the lungs. In: Fishman AP, editor. Pulmonary Diseases and Disorders. New York: McGraw-Hill. p 1885-2068.

Coughlin CM, Salhany KE, Wysocka M, Aruga E, Kurzawa H, Chang AE, Hunter CA, Fox JC, Trinchieri G, Lee WMF. 1998. Interleukin-12 and interleukin-18 synergistically induce murine tumor regression which involves inhibition of angiogenesis. J Clin Invest 101:1441-1452.

Engerman RL, Pfaffenenbach D, Davis MD. 1967. Cell turnover of capillaries. Lab Invest 17:738-743.

Farber JM. 1990. A macrophage mRNA selectively induced by gamma-interferon encodes a member of the platelet factor 4 family of cytokines. Proc Natl Acad Sci U S A 87:5238-5242.

Farber JM. 1992. A collection of mRNA species that are inducible in the RAW 264.7 mouse macrophage cell line by gamma interferon and other agents. Mol Cell Biol 12:1535-1545.

Farher JM. 1993. HuMIG: A new member of the chemokine family of cytokines. Biochem Biophys Res Comm 192:223-230.

Farber JM. 1997. MIG and IP-10: CXC chemokines that target lympho-cytes. J Leukoc Biol 61:246-257.

Folkman J. 1985. Tumor Angiogenesis. In: Klein G, Weinhouse S, editors. Advances in Cancer Research. Orlando: Academic Press, Inc. p 175-203.

Folkman J. 1993. Tumor angiogenesis. In: Holland JF, EF Ill, RCB Jr., Kufe DW, Morton DL, Weischelbaum RR, editors. Cancer Medicine. Philadelphia PA: Lea & Febiger. p 153-170.

Folkman J. 1995. Anglogenesis in cancer, vascular, rheumatoid and other disease. Nat Med 1: 27-31.

Folkman J. 1996. Fighting cancer by attacking its blood supply. Sci Am 275:150-154.

Folkman J. 1997. Anglogenesis and angiogenesis inhibition: An overview. EXS 79:1-8.

Folkman J. 1998. Is tissue mass regulated by vascular endothelial cells? Prostate as the first evidence. Endocrinology 139:441-442.

Folkman I, Brem H. 1992. Anglogenesis and inflammation. In: Gallin JI, Goldstein IM, Snyderman R, editors. Inflammation: Basic Principles and Clinical Correlates. 2nd ed. New York: Raven Press Ltd. p 821-839.

Folkman, J, Cotran R. 1976. Relation of vascular proliferation to tumor growth, lnt Rev Exp Pathol 16:207-248.

Folkman J, Klagsbrun M. 1987. Angiogenic factors. Science 235:442-47.

Gastl G, Hermann T, Steurer M, Zmija J, Gunsilius E, Unger C, Kraft A. 1997. Angiogenesis as a target for tumor treatment. Oncology 54:177-184.

Gupta SK, Lysko PG, Pillarisetti K, Ohlstein E, Stadel JM. 1998. Chemokine receptors in hunlan endothelial cells. Functional expression of CXCR4 and its transcriptional regulation by inflammatory cytokines. J Biol Chem 273:4282-4287.

Gurtscvitch VE, O'Conor GT, Lenoir GM. 1988. Burkitt's lymphoma cell lines reveal different degrees of tumorigenicity in nude mice. lnt J Cancer 41:87-95.

Gusella GL, Musso T, Bosco MC, Espinoza-Delgado 1, Matsushima K, Varesio L. 1993. IL-2 up-regulates but IFN-g suppresses IL-8 expression in human monocytes. J Immuno1 151:2725-2732.

Hansell P, Mainne TE, Borgstrom P. 1995. Selective binding of platelet factor 4 to regions of active anglogenesis in vivo. Am J Physiol 269:H829-836.

Hotfilder M, Nowak-Gottl U, Wolff JE. 1997. Tumorangiogenesis: A network of cytokines. Klin Padiatr 209:265-270.

Hu DE, Hori Y, Fan TPD. 1993. Interleukin-8 stimulates anglogenesis in rats. Inflammation 17:135-143.

Hui YF, Ignoffo RJ. 1998. Angiogenesis inhibitors. A promising role in cancer therapy. Cancer Pratt 6:60-62.

Kitadai Y, Haruma K, Sumii K, Yamamoto S, Ue T, Yokozaki H, Yasui W, Ohmoto Y, Kajiyama G, Fidlet IJ, Tahara E. 1998. Expression of interleukin-8 correlates with vascularity in human gastric carcinomas. Am J Pathol 152:93-100.

Koch AE, Polverini PJ, Kunkel SL, Harlow LA, DiPietro LA, Elner VM, Elner SG, Strieter RM. 1992. lnterleukin-8 (IL-8) as a macrophage-derived mediator of angiogenesis. Science 258:1798-1801.

Kumar R, Fidlet IJ. 1998. Angiogenic molecules and cancer metastasis. In Vivo 12:27-34.

Leibovich S J, Weisman DM. 1988. Macrophages, wound repair and angiogenesis. Prog Clin Biol Res 266:131-145.

Luan J, Shattuck-Brandt R, Haghnegahdar H, Owen JD, Strieter R, Burdick M, Nirodi C, Beauchamp D, Johnson KN, Richmond A. 1997. Mechanism and biological significance of constitutive expression of MGSA/ GRO chemokines in malignant melanoma tumor progression. J Leukoc Biol 62:588 597.

Lund EL, Spang-Thomsen M, Skovgaard-Poulsen H, Kristjansen PE. 1998. Tumor angiogenesis A new therapeutic target in gliomas. Acta Neurol Stand 97:52-62.

Luster AD. 1998. Chemokines--Chemotactic cytokines that mediate inflammation. N Engl J Med 338:436-445.

Luster AD, Ravetch JV. 1987. Biochemical characterization of a gamma interferon-inducible cytokine (IP- 101. J Exp Med 166:1084-1097.

Luster AD, Unkeless JC, Ravetch JV. 1985. Gamma-interferon transcriptionally regulates an early-response gene containing homology to plate-let proteins. Nature 315:672-676.

Malone TE, Gray GS, Hunt A J, Sharpe RJ. 199 I. Inhibition of tumor growth in mice by an analogue of platelet factor 4 that lacks affinity for heparin and retains potent angiostatic activity. Cancer Res 51:2077-2083. Maione TE, Gray GS, Petro J, Hunt A J, Donner AL, Bauer SI, Carson HF,

Sharpe RJ. 1990. Inhibition of angiogenesis by recombinant hutnan platelet factor-4 and related peptides. Science 247:77-79.

Majewski S, Marczak M, Szmurlo A, Jablonska S, Bollag W. 1996. Interleukin-12 inhibits anglogenesis induced by human tumor cell lines in vivo. J Invest Dermatol 106:1114-1118.

Minna JD. 1991. Neoplasms if the lung. In: Isselbacher KJ, editor. Principles of Internal Medicine. New York: McGraw-Hill. p. 1102-1110.

Norrby K. 1996. Interleukin-8 and de novo mammalian angiogenesis. Cell Prolif 29:315-323.

O'Reilly MS, Boehm T, Shing Y, Fukai N, Vasios G, Lane WS, Flynn E, Birkhead JR, OIsen BR, Folkman J. 1997. Endostatin: An endogenous inhibitor of anglogenesis and tumor growth. Cell 88:277-285.

O'Reilly MS, Holmgren L, Chen C, Folkman J. 1996. Angiostatin induces and sustains dormancy of human primary tumors in mice. Nat Med 2:689-692.

O'Reilly MS, Holmgren L, Shing Y, Chen C, Rosenthai RA, Moses M, Lane WS, Cao Y, Sage EH, Folkman J. 1994. Angiostatin: A novel angiogenesis inhibitor that mediates suppression of metastases by a Lewis lung carcinoma. Cell 79:315-328.

Owen JD, Strieter R, Burdick M, Haghnegahdar H, Nanney L, Shattuck-Brandt R, Richmond A. 1997. Enhanced tumor-forming capacity for immortalized melanocytes expressing melanoma growth stimulatory activity/growth-regulated cytokine beta and gamma proteins. Int J Cancer 73:94-103.

Pluda JM. 1997. Tumor-associated anglogenesis: Mechanisms, clinical implications, and therapeutic strategies. Semin Oncol 24:203-218.

Polverini PJ. 1996. How the extracellular matrix and macrophages contribute to angiogenesis-dependent diseases. Eur J Cancer 32A:243(I-2437.

Polverini PJ, Cotran PS, Gimbrone MA, Unanue ER. 1977. Activated macrophages induce vascular proliferation. Nature 269:804-806. Risau W. 1997. Mechanisms of angiogenesis. Nature 386:671-674.

Rollins BJ. 1997. Chemokines. Blood 90:909 928.

Schnyder-Candrian S, Strieter RM, Kunkel SL, Walz A. 1995. Interferon-a and interferon-g downregulate the production of interleukin-8 and ENA-78 in human monocytes. J Leukoc Biol 57:929-935.

Sgadari C, Angiolillo AL, Chemey BW, Pike SE, Farber JM, Koniaris LG, Vanguri P, Burd PR, Sheikh N, Gupta G, Teruya-Feldstein J, Tosato G. 1996. Interferon-inducible protein-10 identified as a mediator of tumor necrosis in vivo. Proc Natl Acad Sci U S A 93:13791-13796.

Sgadari C, Farber JM, Angiolillo AL, Liao F, Teruya-Feldstein J, Burd PR, Yao L, Gupta G, Kanegane C, Tosato G. 1997. Mig, the monokine induced by interferon-gamma, promotes tumor necrosis in vivo. Blood 89:2635-2643.

Sharpe RJ, Byers HR, Scott CF, Bauer SI, Malone TE. 1990. Growth inhibition of murine melanoma and human colon carcinoma by recombinant human platelet factor 4. J Natl Cancer Inst 82:848-853.

Singh RK. Gutman M, Radinsky R, Bucana CD, Fidler IJ. 1994. Expression of interleukin 8 correlates with the metastatic potential of human melanoma cells in nude mice. Cancer Res 54: 3242-3247.

Smith DR, Polverini PJ, Kunkel SL, Orringer MB, Whyte RI, Burdick MD, Wilke CA, Strieter RM. 1994a. Inhibition of IL-8 attenuates anglogenesis in bronchogenic carcinoma. J Exp Med 179:1409-1415.

Smith DR, Polverini PJ, Kunkel SL, Orringer MB, Whyte RI, Burdick MD, Wilke CA, Strieter RM. 1994b. Inhibition of interleukin 8 attenuates anglogenesis in bronchogenic carcinoma. J Exp Med 179:1409-1415.

Strieter RM, Kunkel SL. 1997. Chemokines and the lung. In Crystal R J, West E, Weibel E, Barnes P, editors. Lung: Scientific Foundations, 2nd ed. New York: Raven Press. p 155-186.

Strieter RM, Kunkel SL, Elner VM, Martonyi CL, Koch AE, Polverini PJ, Elner SG. 1992. Interleukin-8. A corneal factor that induces neovascularization. Am J Pathol 141:1279-1284.

Strieter RM, Polverini PJ, Kunkel SL, Arenberg DA, Burdick MD, Kasper J, Dzuiba J, Van Damme J, Walz A, Marriott D, Chan S, Roczniak S, Shanafelt A. 1995. The functional role of the ELR motif in CXC chemokine-mediated angiogenesis. J Biol Chem 270:27348-27357.

Tannock IF, Hayashi HS. 1972. The proliferation of capillary and endothelial cells. Cancer Res 32:77-82.

Taub DD, Oppenheim JJ. 1994. Chemokines, inflammation and immune system. Therapeutic Immunol 1:229-246.

Teruya-Feldstein J, Jaffe ES, Burd PR, Kanegane H, Kingma DW, Wilson WH, Longo DL, Tosato G. 1997. The role of Mig, the monokine induced by interteton-gamma, and IP-10, the interferon-gamma-inducible protein-10, in tissue necrosis and vascular damage associated with Epstein-Barr virus-positive lymphoproliferative disease. Blood 90:4099-4105.

Thomas DP, Niewiarowski S, Reain VJ. 1970. Release of adenine nucle-otides and platelet factor 4 from platelets of man and four other species. J Lab Clin Med 75:607-618.

Vizier C, Rosato A, Calderazzo F, Quintieri L, Fruscella P, Wainstok de Calmanovici R, Mantovani A, Vecchi A, Zanovello P, Collavo D. 1998. Therapeutic effect of interleukin 12 on mouse haemangiosarcomas is not associated with an increased anti-tumour cytotoxic T-lymphocyte activity. Br J Cancer 77:656-662.

Walz A, Kunkel SL, Strieter RM. 1996. CXC chemokines-an overview. In: Koch AE, Strieter RM, editors. Chemokines in Disease. Austin: R.G. Landes Co. p 1-26.

Yatsunami J, Tsuruta N, Ogata K, Wakamatsu K, Takayama K, Kawasaki M, Nakanishi Y, Hara N, Hayashi S. 1997. Interleukin-8 participates in angiogenesis in non-small cell, but not small cell carcinoma of the lung. Cancer Lett 120:101-108.

Yoneda J, Kuniyasu H, Crispens MA, Price JE, Bucana CD, Fidler IJ. 1998. Expression of angiogenesis-related genes and progression of human ovarian carcinomas in nude mice. J Natl Cancer Inst 90:447-454.

Yoshida S, OnoM, Shono T, Izumi H, Ishibashi T, Suzuki H, Kuwano M.

1997. Involvement of interleukin-8, vascular endothelial growth factor, and basic fibroblast growth factor in tumor necrosis factor alpha-depen-dent angiogenesis. Mol Cell Biol 17:4015-4023.

Yuan A, Pan-Chyr Y, Chong-Jen Y, LeeY, Yu-Tuang Y, Chi-Long C, Lee L, Sow-Hsong K, Kwen-Tay L. 1995. Tumor anglogenesis correlates with histologic type and metastasis in non-small cell lung cancer. Am J Resp Crit Care Med 152:2157-2162.

Zetter BR. 1998. Angiogenesis and tumor metastasis. Annu Rev Med 49:407-424.

Ziche M, Morbidelli L, Donnini S. 1996. Anglogenesis. Exp Nephrol 4:1-14.

Table 1 ELR⁺and ELR- CXC chemokines, which are angiogenic and angiostatic factors, respectively

Angiogenic CXC chemokines containing the ELR motif (ELR⁺)

Angiostatic CXC chemokines that lack the ELR motif (ELR-)

Table 2 Stimulus specificity for the expression and production of CXC chemokines

	Stimulus
CXC chemokines	LPS^a	TNF^a	IL^a-1	IFN^a-g
IL-8	++++	++++	++++
ENA^a-78	+++	+++	+++	----
GRO^a-a	+++	+++	+++	----
IP^a-10	+	+	+	++++
MIG^a	-	-	-	++++

^a LPS, lipopolysaccharide; TNF, tumor necrosis factor; IL, interleukin; IFN, interferon; ENA, epithelial neutrophil activating protein; GRO, growth-related oncogene; IP, inducible protein, MIG, monokine induced by interferon-g_.

Table 3 The ELR* and ELR- CXC chemokines, and their responses in the corneal micropocket (CMP) model of angiogenesis when tested alone or in combination. In addition, the effect of the ELR- CXC chemokines were tested on basic fibroblast growth factor (bFGF)- and vascular endothelial growth factor (VEGF)-induced angiogenesis

Chemokine/cytokine	Angiogenic response in the CMP assay
IL^a-8	++++
ENA^a-78	++++
GRO^a-a	++++
GRO-b	++++
GRO-g	++++
GCP^a-2	++++
PF4^a	----
IP^a-10	----
MIG^a	----
IL-8+MIG	----
ENA-78+IP-10	----
ENA-78+MIG	----
bFGF+IP-10	----
bFGF+MIG	----
VEGF+IP-10	----
VEGF+MIG	----

^a lL, interleukin; ENA, epithelial neutrophil activating protein; GRO, growth-related oncogene; GCP, granulocyte chemotactic protein; PF4, platelet factor-4; IP, inducible protein; MIG, monokine induced by interferon-g.

Figure 1 Net tumor-derived angiogenesis during tumorigenesis is determined, in part, by an imbalance in favor of the overexpression of angiogenic (ELR+), compared with angiostatic (ELR-) CXC chemokines. This paradigm predicts an environment that favors angiogenesis, and tumorigenesis and supports the potential for spontaneous metastases. In contrast, this paradigm also supports the contention that inhibition of angiogenic or augmentation of angiostatic CXC chemokine biology will lead to reduced tumor growth and metastases.

Figure 2 Intratumor injection of inducible protein (IP)-10 (1 gg every other day for 8 wk), compared with equal molar concentration of human serum albumin (HSA), inhibits A549 (adenocarcinoma) tumor growth in severe combined immunodeficiency (SCID) mice. (A) Representative photograph of a SCID mouse bearing flank A549 tumors treated with IP-10. (B) Representative photograph of a SCID mouse bearing flank A549 tumors treated with HSA.