[CANCER RESEARCH 59, 205–212, January 1, 1999] In Vivo Sensitivity of Human Melanoma to Tumor Necrosis Factor (TNF)-␣ Is Determined by Tumor Production of the Novel Cytokine Endothelial-Monocyte Activating Polypeptide II (EMAPII) Peter C. Wu, H. Richard Alexander, James Huang, Patrick Hwu, Michael Gnant, Adam C. Berger, Ewa Turner, Olga Wilson, and Steven K. Libutti1 Surgical Metabolism Section, Surgery Branch, National Cancer Institute [P. C. W., H. R. A., J. H., P. H., M. G., A. C. B., E. T., S. K. L.], and Hematology Section, Clinical Pathology, Clinical Center [O. W.], NIH, Bethesda, Maryland 20892 ABSTRACT of different tumor histologies (3). However, the results were disap- pointing because TNF resulted in significant systemic toxicity and no ␣ Tumor necrosis factor (TNF)- is a potent anticancer agent that seems significant antitumor effects at the maximally tolerated doses. The to selectively target tumor-associated vasculature resulting in hemor- clinical use of TNF was largely abandoned until Lienard et al. (4) rhagic necrosis of tumors without injury to surrounding tissues. The major limitation in the clinical use of TNF has been severe dose-limiting reported their initial results of isolated limb perfusion as a means of toxicity when administered systemically. However, when administered in delivering high concentrations to the extremity in patients with in isolated organ perfusion it results in regression of advanced bulky tumors. transit melanoma or unresectable sarcoma, while minimizing systemic A better understanding of the mechanisms of TNF-induced antitumor exposure. We and others have used isolated organ perfusion of the effects may provide valuable information into how its clinical use in limb or liver using TNF plus chemotherapeutic agents to treat unre- cancer treatment may be expanded. We describe here that the release of sectable tumors with dramatic responses in the majority of patients a novel tumor-derived cytokine endothelial-monocyte-activating polypep- (5–9). Despite these encouraging results, much remains unknown tide II (EMAPII) renders the tumor-associated vasculature sensitive to regarding the mechanism by which TNF exerts its effects on tumors. TNF. EMAPII has the unique ability to induce tissue factor production by tumor vascular endothelial cells that initiates thrombogenic cascades, A better understanding of this mechanism may improve the therapeu- which may play a role in determining tumor sensitivity to TNF. tic efficacy of TNF and help minimize systemic toxicity which limits We demonstrate here that constituitive overexpression of EMAPII in a wider application of this potent agent. TNF-resistant human melanoma line by retroviral-mediated transfer of There are several postulated theories to explain the mechanism of EMAPII cDNA renders the tumor sensitive to the effects of systemic TNF action of TNF. These include the stimulation of T cell-mediated in vivo, but not in vitro. This interaction between tumors and their responses resulting in the generation of CD8ϩ tumor-specific CTLs associated neovasculature provides an explanation for the focal effects of (10, 11), TNF-induced apoptosis (12–16), macrophage/granulocyte- TNF on tumors and possibly for the variable sensitivity of tumors to mediated injury (17), activation of cellular adhesion molecules (18), bioactive agents. and induction of fibrin deposition on endothelial surfaces and throm- bus formation (19–21). Most of the evidence, however, supports an INTRODUCTION indirect mechanism via the tumor vasculature rather than direct cyto- toxic effects of TNF. The clinical experience with TNF administered ␣2 TNF- induces procoagulant effects within tumor neovasculature, via isolated organ perfusion in patients with melanomas also supports resulting in endothelial fibrin deposition, localized thrombosis, and this hypothesis (22). Renard et al. (18) demonstrated in limb perfu- ischemic necrosis of responsive tumors. The mechanism responsible sions using TNF that coagulative and/or hemorrhagic necrosis of for this effect as well as the apparent variable in vivo sensitivity of tumors was specific for TNF because isolated limb perfusion with tumors to TNF is not well understood. O’Malley et al. (1) first melphalan alone failed to show this type of necrosis. Fig. 1 demon- demonstrated that the serum of animals treated with LPS contained an strates the complete obliteration of tumor neovasculature after a endogenous factor that could induce hemorrhagic necrosis of tumors hyperthermic isolated limb perfusion with TNF and melphalan, while in animals not exposed to LPS. Carswell et al. (2) later isolated a leaving the normal host vessels apparently unaffected. circulating protein from mice, pretreated with Bacillus Calmette- The distinction between tumor and host vasculature centers on the Guerin and challenged with LPS, that could induce significant hem- phenotypic difference of the vascular endothelium. In general, tumor orrhagic necrosis of methylcholanthrene A-induced fibrosarcomas vessel endothelium is more prone to thrombosis and capillary leakage (Meth A) that was termed “TNF.” Systemic administration of TNF to (20, 23). Cytokines present in the microenvironment of solid tumors mice bearing s.c. MethA tumors resulted in marked hemorrhagic necrosis of the tumors without observable effects on nontumor tissue. may alter the endothelium of tumor vessels influencing characteristics This observation formed the basis for several clinical trials using TNF of tumor growth and metastatic spread (24–27). Specifically, tumor- once the recombinant protein was made available. derived cytokines may play fundamental roles in determining tumor Systemic TNF was used in multiple clinical trials against a variety phenotypes relating to tumor angiogenesis, tumor progression, meta- static potential, and sensitivity to therapeutic agents. EMAPII is a cytokine that may influence interactions between Received 8/20/98; accepted 10/29/98. The costs of publication of this article were defrayed in part by the payment of page tumor cells and associated neovasculature. EMAPII was first de- charges. This article must therefore be hereby marked advertisement in accordance with scribed by Kao et al. (28, 29), using the MethA tumor as a model for 18 U.S.C. Section 1734 solely to indicate this fact. 1 To whom requests for reprints should be addressed, at Surgery Branch, National Cancer studying host-tumor responses and the effects of tumor-derived fac- Institute, NIH, Building 10, Room 2B17, 10 Center Drive MSC 1502, Bethesda, MD tors. The protein was isolated from MethA supernatant by its ability 20892-1502. Phone: (301) 496-5049; Fax: (301) 402-1788; E-mail: Steven–[email protected]. to activate endothelial cells inducing tissue factor procoagulant activ- 2 The abbreviations used are: TNF, tumor necrosis factor; LPS, lipopolysaccharide; EMAPII, endothelial-monocyte-activating polypeptide II; LTR, long terminal repeat; ity and up-regulating leukocyte adhesion molecules P-selectin and MTT, 3-(4,5-dimethyl-2-thiazoyl)-2, 5-diphenyl-2H tetrazolium bromide; TNFR1, TNF E-selectin. EMAPII is a polypeptide synthesized as a Mr 34,000 p55 receptor; MethA, methylcholanthrene A-induced fibrosarcoma; HUVEC, human umbilical vein endothelial cell; MRA, magnetic resonance angiogram; IRES, internal precursor and cleaved to produce an active Mr 22,000 mature protein. ribosomal entry site. Because EMAPII was isolated from a TNF-sensitive tumor and in- 205 Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1999 American Association for Cancer Research. EMAPII AND TNF SENSITIVITY IN HUMAN MELANOMA Fig. 1. MRA of a patient diagnosed with an unresectable Ewing’s sarcoma of the left forearm who underwent a hyperthermic isolated limb per- fusion with melphalan and TNF. By comparing baseline and follow-up MRAs, accurate assessment of vessel integrity and patency are made possible. a, a large forearm tumor diagnosed as Ewing’s sarcoma. b, the baseline preoperative MRA dem- onstrating the extensive neovasculature of the tu- mor and normal major vessels of the upper limb. c, another MRA taken 16 days after perfusion, dem- onstrating complete obliteration of the tumor vas- culature while the normal major arm vessels remain patent. duced tissue factor production by endothelial cells, it seemed to be a selection. Presence and directionality of the EMAPII insert in the pWU-EII putative TNF-sensitizing agent. Marvin et al. (30) treated mice bear- construct were confirmed by PCR amplification and restriction digest. ing TNF-resistant tumors with intratumor injection of recombinant Stable Transduction of EMAPII Into a Low-expressing Melanoma EMAPII and rendered the tumors sensitive to systemic TNF. The Line. The pWU-EII retroviral vector and pSAMEN vector control were each effect was confined to the tumor vasculature resulting in thrombohe- transfected into the amphotropic packaging cell line PA317 by calcium phos- phate precipitation (Invitrogen). The viral supernatants from the PA317 pro- morrhage and tumor regression. ducer cells were used to infect the Gibbon ape ecotropic packaging cell line The identification of tumor cytokines, such as EMAPII, that spe- PG13 with polybrene at a concentration of 8 ␮g/ml. PG13 viral supernatants cifically activate endothelial cells suggests a novel mechanism of TNF were then used to infect the human wild-type Pmel tumor line, which expresses sensitivity. If tumors express variable levels of EMAPII, overexpres- the lowest levels of EMAPII. Transduced Pmel