In Vivo Sensitivity of Human Melanoma to Tumor Necrosis Factor

[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 cytotoxic 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, metastatic 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

Fig. 1. MRA of a patient diagnosed with an unresectable Ewing’s sarcoma of the left forearm who underwent a hyperthermic isolated limb perfusion 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 demonstrating the extensive neovasculature of the tumor and normal major vessels of the upper limb. c, another MRA taken 16 days after perfusion, demonstrating complete obliteration of the tumor vasculature 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 tumor lines were expanded sion of EMAPII in certain tumors may predispose the tumor vascu- under neomycin selection at 800 ␮g/ml and cloned in limiting dilution. lature to the procoagulant effects of TNF and increase their sensitivity Rapid Genomic DNA Extraction and PCR Amplification. Tumor cells to this agent when administered via systemic or regional routes. To (1 ϫ 106) were placed into 200 ␮l of DNA extraction buffer containing 0.5% determine whether EMAPII production by tumors, in fact, confers Tween 20 (Bio-Rad, Hercules, CA), 100 ␮g/ml Proteinase K (Stratagene, La Jolla, TNF sensitivity, we evaluated EMAPII levels and TNF sensitivity in CA), and 1 ϫ PCR Buffer (Perkin-Elmer Corp.) and heated at 56°C for 45 min, various human melanoma lines. In addition, we transduced a TNF- followed by 95°C for 10 min. Genomic DNA samples extracted from the wild- resistant and low EMAPII-expressing human melanoma line with a type and transduced tumor clones were screened by PCR amplification with primers specific for both the EMAPII insert and downstream retroviral IRES retroviral vector encoding the EMAPII cytokine, measured EMAPII region using the following primers: IRES forward, 5Ј-AACGTTACTGGC- overexpression by immunodetection and functional assays, and char- CGAAGCC-3Ј; IRES reverse, 5Ј-AAGGAAAACCACGTCCCCGT-3Ј; EII for- acterized the in vitro and in vivo sensitivity of the tumor to TNF. ward, 5Ј-AACTGAAACAAGAGCTAATT-3Ј; EII reverse, 5Ј-CAGGCTCTC- CTGGGAAAGCA-3Ј. MATERIALS AND METHODS PCR amplification was performed using a GeneAmp thermocycler (Perkin- Elmer Corp.) for 25 cycles consisting of a 15-s 94°C denature step, a 30-s 55°C Tumor Cell Lines and Animals. Pmel, 883, Smel, and 1286 are primary annealing step, and 2-min 72°C extension step. human melanoma lines derived from patients treated at the National Cancer Preparation of Polyclonal EMAPII Antiserum. Polyclonal EMAPII rab- Institute and cultured in DMEM supplemented with 10% FCS, 2 mML- bit antiserum (kindly provided by Dr. D. Stern, Columbia University, New glutamine, and 1% penicillin/streptomycin (Biofluids, Rockville, MD) at 37°C York, NY) was purified using the ImmunoPure IgG Purification Kit (Pierce ina5%CO2 incubator. Each of the melanoma lines was passaged no more than Chemical Co., Rockford, IL) by elution from a bound Protein A column. 10 generations and cryopreserved at regular intervals. Female athymic nude IgG-purified polyclonal EMAPII antiserum was used directly for immuno- mice were obtained from the NIH small animal facility and housed maximum staining and biotinylated using an EZ-Link Sulfo-NHS-LC-Biotinylation Kit five animals/cage in a barrier care room. Human tumors were inoculated in the (Pierce Chemical Co.) for ELISA. hind limb-flank region by s.c. injection of 1 ϫ 106 tumor cells suspended in EMAPII Immunostaining. Cells were plated overnight on coverslips and 100 ␮l of sterile HBSS. All animal protocols were approved by the Animal washed three times with PBS, fixed in 10% formalin for 20 min, 1% Triton Care and Use Committee and conducted with strict compliance to guidelines X-100 for 5 min, washed again two times with PBS, blocked with 5% established by the NIH Animal Research Advisory Committee. FCS/PBS for 10 min, and incubated for 45 min at 37°C with a 1:100 dilution Detection of EMAPII mRNA by Northern Blot. Total RNA was ex- of IgG-purified polyclonal EMAPII rabbit antisera or 1 ␮g/ml rabbit IgG tracted from cultured tumor lines using Rneasy Total RNA Kits (Qiagen, (Sigma Chemical Co.) to define background staining. After another double Chatsworth, CA), isolated on 1% agarose/5% formaldehyde gels, and hybrid- wash in PBS, the cells were incubated for 45 min at 37°C with a 1:50 dilution ized using 32P-labeled cDNA probes for EMAPII and a ␤-actin loading of fluorescein-conjugated goat antirabbit IgG (Jackson ImmunoResearch Lab- standard. EMAPII transcript was quantified using a STORM phosphoimager oratories, West Grove, PA), followed by another double wash in PBS and and ImageQuaNT software analysis package (Molecular Dynamics, Sunny- mounted with DAPI/Antifade (Oncor, Gaithersburg, MD), and visualized vale, CA). under fluorescent microscopy. Tumors excised during necropsy were fixed in Generation of the Retroviral pWU-EII Vector Construct. A cDNA 10% formalin, and EMAPII immunohistochemistry was performed using the clone of human pro-EMAPII was generated using primers derived from the same antibodies by Paragon Biotech (Baltimore, MD). GenBank sequence and RNA isolated from human monocytes using reverse EMAPII ELISA. An indirect ELISA was used to detect EMAPII protein transcription-PCR amplification with Pfu polymerase (Perkin-Elmer Corp., in cell lysates prepared from the cultured tumor lines. Briefly, cell lysates Norwalk, CT). The PCR-derived fragment was cloned into PCR2.1 using the standardized to total protein as determined by BCA Protein Assay (Pierce TA Cloning Kit (Invitrogen, Carlsbad, CA) and confirmed by cycle sequencing Chemical Co.) were diluted in carbonate buffer (Pierce Chemical Co.) and using dye-labeled terminators (Perkin-Elmer Corp.). The hEMAPII cDNA was coated onto MaxiSorp immunoplates (Nunc, Inc.) overnight at 4°C. Recom- ligated into the multiple cloning site of the retroviral vector pSAMEN under binant human EMAPII (kindly provided by Dr. D. Stern) was used as a serially the expression of an LTR promoter/enhancer with neomycin-resistance gene diluted control standard. The plates, coated with control standards and lysate 206

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1999 American Association for Cancer Research. EMAPII AND TNF SENSITIVITY IN HUMAN MELANOMA samples, were blocked for1hat24°C with SuperBlock Blocking Buffer Statistical Analysis. Tumor volumes in the in vivo experiments were (Pierce Chemical Co.), incubated for1hat4°Cwith 10 ␮g/ml biotinylated compared using ANOVA between groups. Ps given were determined using the polyclonal rabbit ␣-EMAPII, followed by another 1-h incubation at 24°C with Tukey-Kramer Test for multiple comparisons. 1 ␮g/ml Neutravidin-horseradish peroxidase conjugate (Pierce Chemical Co.). The plates were then developed using Turbo TMB ELISA substrate (Pierce RESULTS Chemical Co.), stopped after 30 min by the addition of 1N H2SO4, and absorbance was measured at a wavelength of 450 nm. Each incubation was EMAPII Expression in National Cancer Institute Human Mel- preceded by a triplicate wash in 0.1% PBS, 0.04% BSA, and Tween 20. anoma Lines. Total RNA was extracted from each of the four human Preparation of Tumor-conditioned Media and Neutralizing Antibody. melanoma lines, separated by Northern blot, and hybridized with ϫ 6 2 To produce tumor-conditioned media, 5 10 tumor cells were plated/75 cm cDNA probes for EMAPII and ␤-actin as shown in Fig. 2. Quantita- tissue culture flask in 15 ml of Medium 199 (NIH Media Services) with 1% tive analysis of relative EMAPII mRNA identified the 1286 mela- penicillin/streptomycin and incubated 24 h at 37°C. The conditioned media noma line with the highest level of EMAPII expression, an average was collected and filtered across a 0.45-um low protein-binding membrane 2.2-fold higher than the lowest expressing Pmel melanoma line. Both (Corning Costar, Cambridge, MA) to remove any cellular debris. Tumor- conditioned media was prepared for immediate use in tissue factor induction lines were analyzed for EMAPII protein content by ELISA, whereas assays avoiding freeze-thaw cycles. The polyclonal ␣-EMAPII rabbit IgG was functional EMAPII activity in tumor-conditioned media was quanti- used as neutralizing antibody at a concentration of 200 ␮g/ml and incubated tated by tissue factor induction from HUVECs. The 1286 melanoma with tumor-conditioned media for 30 min at 37°C immediately before tissue had 2.5-fold higher EMAPII protein expression compared with Pmel factor induction experiments. melanoma by ELISA of cell lysates standardized to total protein (data Tissue Factor Induction Assay. HUVECs (Clonetics, San Diego, CA) not shown). Functional assay of EMAPII protein showed a 5.8-fold were passaged in complete EGM-2 media (Clonetics) for no more than four higher induction of endothelial cell tissue factor production by 1286 generations and plated at a concentration of 5 ϫ 105 cells/well in 6-well melanoma-conditioned media compared with Pmel-conditioned me- multiwell plates (Corning Costar) and incubated 48 h at 37°C in a 5% CO2 dia (Fig. 3). The reliability and specificity of the tissue factor induc- ϳ chamber reaching 80% confluency. The cells were washed twice with sterile tion assay to detect functional EMAPII protein was confirmed by PBS and treated with 1 ml of tumor-conditioned media or 150 ng/ml recom- demonstrating that neutralizing EMAPII antibody completely abro- binant IL-1␤ (R&D Systems, Minneapolis, MN) as a positive control for gated endothelial cell activation by 1286 melanoma-conditioned me- endothelial cell activation. The treated endothelial cells were cultured at 37°C for 16 h, washed twice with sterile PBS, and harvested in 300 ␮l of PBS/well dia. using mechanical cell scrapers and stored at Ϫ70°C. Each cell suspension was EMAPII Expression Is Related to in Vivo TNF Sensitivity. To quickly thawed at 37°C and centrifuged at 2000 ϫ g for 5 min, and the determine whether the level of EMAPII expression in human mela- resultant cell pellet was resuspended in 200 ␮lof50nM Tris, 100 nM NaCl, and noma lines correlated with in vivo sensitivity to systemic recombinant 0.1% BSA. The one-stage procoagulant assay was performed by adding 100 ␮l TNF, Pmel and 1286 melanoma lines were s.c. inoculated into the of Factor VIII-deficient human plasma (George-King Biomedical, Overland hind limb-flank regions of athymic nude mice and grown to uniform Park, KA) to 100 ␮l of endothelial cell suspension and incubated for 3 min at size. When the tumors reached ϳ150 mm3, animals were treated with 37°C. The coagulation reaction was catalyzed by the addition of 100 ␮lof30 either 6 ␮g of i.v. recombinant TNF or NaCl. Blinded tumor meas- nM CaCl2, and clotting time was measured using a clinical fibrometer (Baxtor, Deerfield, IL). Standard curves were generated using recombinant human tissue-factor (American Diagnostica, Greenwich, CT), and assay sensitivity was Ͻ10 pg/ml. In Vitro MTT Cytotoxicity Assay. Sensitivity of the tumor lines Pmel, 1286, Pmel-SAMEN, and Pmel-EII transduced clones to TNF was assessed in the following manner. Cells were plated in flat-bottomed 96-well plates at a concentration of 3.0 ϫ 103 cells/well in 100 ␮l of DMEM supplemented with 10% FCS and allowed to grow for 48 h before treatment. Recombinant TNF (Knoll Pharmaceuticals, Whippany, NJ) was reconstituted in media to 20 ␮g/ml, and serial dilutions were performed to desired treatment concentrations. The TNF was added to each well in 100-␮l aliquots in six replicates, plates were incubated at 37°C for 16 h, and MTT (Sigma Chemical Co.) cytotoxicity assays were performed. Briefly, 100 ␮l of 2 mg/ml MTT was added to each sample well and incubated at 37°C for 4 h. The media was then aspirated, the formazan precipitate was solubilized in 120 ␮l of DMSO (dimethylsulfoxid; Fluka Chemika), and absorbance at 570 nm was measured. Cytotoxicity, expressed as percentage control survival, was determined by dividing treatment absorbance values by the mean of control values for each experiment and expressed as percentage values. Treatment of Human Melanomas Established in Nude Mice. Athymic nude mice were implanted with human melanoma tumors as described above. Tumor volumes were determined from caliper measurements of width, length, and height based on calculated partial spherical volume (V), V ϭ ␲h(h2 ϩ 3a2)/6, where h ϭ tumor height and a ϭ (length ϩ width)/2. When tumors had reached ϳ150 mm3, typically occurring within 3–4 weeks, mice were randomized into two groups. One group received systemic recombinant TNF (Knoll Pharmaceuticals) administered 6 ␮g/mouse via lateral tail vein injection. The other group received tail vein injections of the vehicle alone Fig. 2. Northern analysis of EMAPII expression in primary human melanoma lines. (0.9% NaCl solution and 0.5% BSA). Cytokine treatment and tumor measure- The blot shown is representative of three experiments. Total RNA extracted from four 32 ments were performed in a double-blinded fashion. Each animal was calculated primary human melanoma lines were hybridized with P-cDNA probes for EMAPII and ␤-actin as a loading standard. The amounts of bound probe were quantified by phospho- as percentage baseline volume before treatment. Animals were sacrificed and imager analysis and standardized, and relative fold EMAPII expression was determined by necropsied according to NIH animal care guidelines. normalizing to the lowest expressing Pmel melanoma line. 207

Fig. 3. Tissue factor induction assay using tumor-conditioned media (c.m.) from wild-type tumor lines to activate HUVECs. Recombinant Il-1␤ (R&D Systems) is a cytokine that activates endothelial cells and acts as a positive control for tissue factor induction at a concentration of 150 ng/ml. MethA c.m. is used as an EMAPII-conditioned media positive control. The 1286 c.m. is shown both alone and treated with 200 ␮g/ml neutralizing EMAPII antibody. The means Ϯ SE are shown of three experiments.

urements were analyzed at regular intervals after treatment to calcu- producer cells were used to infect the packaging cell line PG13 late tumor volume response after exposure to a single dose of TNF. expressing the Gibbon ape viral envelope. Stable transformants were Fig. 4, a and b, demonstrates the in vivo sensitivity of the wild-type screened by successive passages under neomycin selection and were tumors Pmel and 1286 to systemic TNF. Treatment of the 1286 cloned in limiting dilution. tumor-bearing mice with systemic TNF consistently demonstrated Pmel-EMAPII-transduced clones were screened for stable retrovi- significant tumor regression compared with the steady tumor progres- ral integration by PCR amplification using a forward primer for sion observed in controls. Tumor necrosis occurred in the TNF-treated EMAPII and a reverse primer for the downstream IRES sequence. 1286 melanoma-bearing animals within 48 h of TNF administration This generated a 950-bp fragment from genomic DNA of tumor evidenced by reduction in tumor volumes and eschar formation. When clones transduced by retroviral infection (data not shown). We similar sized Pmel tumors were treated with TNF or NaCl, no differ- screened EMAPII overexpression in the transduced clones in vitro by ence was seen. The lack of significant tumor response and no observ- immunofluorescence. Two overexpressing clones, Pmel-EII.08 and able evidence of ischemic necrosis among the TNF-treated tumor- Pmel-EII.09 compared with wild-type and vector-transduced controls bearing animals demonstrates the overall resistance of Pmel are shown in Fig. 6a. Additionally, Pmel-EII tumor clones were melanoma to systemic TNF. Thus, the level of EMAPII expression in analyzed by tissue factor induction assay to confirm that the overex- these human melanoma lines was associated with in vivo TNF sensi- pression and processing of the EMAPII protein resulted in functional tivity of the cell lines. The level of EMAPII expression in three other secreted cytokine. The tumor clones Pmel-EII.08 and Pmel-EII.09 primary melanoma lines was also found to correlate with relative in showed a 7.0- and 19.9-fold increase, respectively, in tissue factor vivo sensitivity to systemic TNF (data not shown). induction compared with wild-type Pmel and vector-transduced Pmel Standard MTT cytotoxicity assays on 1286 and Pmel tumor lines (Fig. 6b), confirming constituitive overexpression of EMAPII in the showed that neither melanoma line was sensitive to TNF in vitro (Fig. retroviral-transduced tumor clones. 4c). The contrast of TNF exerting no direct cytotoxic effect on tumor After confirming functional EMAPII overexpression in the trans- cells to the observed antitumor effects when administered in vivo, duced clones Pmel-EII.08 and Pmel-EII.09, both clones were assayed strongly suggested an indirect effect of TNF, presumably on tumor for in vitro TNF cytotoxicity by MTT assay. As seen in both wild-type neovasculature. The characterization of Pmel melanoma as a rela- Pmel and 1286 melanoma, neither tumor clone was sensitive to TNF tively low EMAPII-expressing tumor line and TNF-resistant tumor in vitro (Fig. 6c). Thus, overexpression of EMAPII in wild-type Pmel identified it as a putative target for constituitive retroviral-mediated melanoma did not change the observation that TNF has no direct overexpression of EMAPII. cytotoxic effect on these tumor cells. Constituitive Overexpression of EMAPII in Pmel Melanoma In Vivo Expression of EMAPII-transduced Tumor Clones. To Using a Retroviral Vector. To test whether the lack of TNF sensi- confirm that the EMAPII cDNA is being expressed in vivo,itis tivity of the Pmel tumor might be due to the lower levels of EMAPII necessary to screen EMAPII-transduced clones for retroviral-driven expression, we hypothesized that the retroviral-mediated transduction expression by growing each tumor clone to 150 mm3 and assessing of the EMAPII cDNA under the transcriptional control of an LTR EMAPII expression. Transduced tumor clone screening in vivo not promoter/enhancer element into the Pmel tumor wild-type genome only determines that high levels of functionally active EMAPII cyto- might result in the overexpression of EMAPII, which would lead to a kine are expressed, but also establishes that EMAPII overexpression phenotypic change from a previously TNF-resistant tumor into a can be sustained through the time intervals required for tumor growth TNF-sensitive tumor. The sequence for EMAPII was cloned into the and systemic TNF treatment. From the bulk-transduced Pmel- pSAMEN retroviral vector under the transcriptional control of the EMAPII tumors, clones Pmel-EII.08 and Pmel-EII.09 demonstrated LTR promoter/enhancer element derived from the Moloney murine constituitive EMAPII overexpression in vivo after 4 weeks (Fig. 6d), leukemia virus (Fig. 5) and used to establish an amphotropic packag- providing sufficient time for tumor growth and treatment. ing cell line in PA317, as described previously (31, 32). To increase EMAPII Overexpression Results in a TNF-sensitive Phenotype. the efficiency of transducing the Pmel human melanoma line, the viral To determine whether constituitive EMAPII overexpression would supernatants harvested from the pWU-EII bulk-transfected PA317 change the TNF-resistant phenotype of wild-type Pmel melanoma, 208

cytokine EMAPII resulted in the in vivo conversion of wild-type Pmel tumor from a TNF-resistant tumor into a TNF-sensitive tumor.

DISCUSSION The mechanism of TNF-induced tumor necrosis is not well understood. Clinical observations made from therapeutic applications of TNF have consistently yielded evidence for a tumor vascular endothelial cell-mediated response. The isolation of a tumor-derived factor that specifically activates endothelial cells by up-regulation of tissue factor expression provides a possible explanation for this observation. Contrino et al. (33) reported that functional tissue factor is expressed by the vascular endothelial cells of malignant infiltrating intraductal breast cancer, but not in benign fibrocystic breast disease, suggesting an effect of tumor cells on the associated vascular endothelium. Our studies demonstrate a similar relationship in primary human melanoma lines. The elaboration of tissue factor by tumor-associated endothelium in response to mediators, such as EMAPII, generated by tumor cells may play a fundamental role in influencing the activation of tumor neovasculature. Since the initial observation that mice bearing MethA fibrosarcoma exposed to systemic TNF develop ischemic necrosis of the tumor, application to a variety of tumor types has shown that TNF sensitivity varies markedly among different tumors. Even among primary human melanoma lines, variable in vivo TNF sensitivity was observed. In- deed, there were no reliable predictors of in vivo TNF response and the mechanism of TNF sensitivity remained elusive. The identification of the tumor-derived cytokine EMAPII, with its ability to activate endothelial cells and induce coagulation, offers a plausible explanation for the variable TNF response seen in different tumors. The level of EMAPII expression in various human melanoma lines correlates with in vivo TNF sensitivity in nude mice. EMAPII is expressed ubiquitously in eukaryotic cells. Tas et al. (34) have shown by RT-PCR that mRNA for EMAPII is detected in nearly all tumor cells, established cell lines, and primary cultures. We have also detected EMAPII mRNA from total RNA extracted from organ tissues in both tumor-bearing and normal mice (data not shown), confirming the ubiquitous transcription of EMAPII in eukaryotic cells. However, immunohistochemistry of organ tissues from a tumor-bearing mouse detects EMAPII protein within tumor and not in any other tissues (data not shown), supporting previous reports that EMAPII cytokine is produced only within tumor tissue (30). Further- more, immunohistochemistry supports the variable level of EMAPII protein expression between different tumor types.

Fig. 4. In vivo and in vitro TNF sensitivity of wild-type 1286 and Pmel melanomas. a, nude mice who received s.c. inoculations of Pmel melanoma are resistant to the antitumor effects of systemic TNF (6 ␮g), compared with NaCl controls (P is not significant). b, nude mice bearing s.c. 1286 melanoma are responsive to systemic TNF (6 ␮g), demonstrating significant tumor volume reduction as compared with controls (days 2 and 4, P Ͻ0.01; days 6 and 8, P Ͻ0.001). c, TNF exhibits no direct cytotoxic effect on either 1286 or Pmel melanoma lines in vitro by MTT cytotoxicity assay. Data are shown as means Ϯ SE of three experiments. clones Pmel-EII.08 and Pmel-EII.09 were grown in athymic nude mice to approximately 150 mm3. Tumor-bearing animals were then treated with systemic TNF or NaCl solution injection. Fig. 7 demonstrates that both tumor clones Pmel-EII.08 and Pmel-EII.09 are rendered sensitive to the effects of TNF in vivo; the response curves resemble those of the TNF-sensitive 1286 melanoma. Both wild-type Pmel and Pmel transfected with the pSAMEN vector alone showed no Fig. 5. pWU-EII retroviral vector for stable transduction of EMAPII into eukaryotic response to systemic TNF. The constituitive overexpression of the cells. 209

Fig. 6. Characterization of retroviral-mediated stable transduction of EMAPII into Pmel melanoma. a, EMAPII detection by immunofluorescence of wild-type Pmel (1), pSAMEN backbone vector transfection control (Pmel-SAMEN; 2), Pmel tumor clone 8 transduced with EMAPII (Pmel-EII.08; 3), and Pmel tumor clone 9 transduced with EMAPII (Pmel-EII.09; 4). b, endothelial tissue factor induction assay of conditioned media from transduced Pmel tumor clones measuring levels of functional EMAPII protein. Data are shown as means Ϯ SE of three experiments. c, TNF lacks direct cytotoxic effects in vitro on the transduced Pmel tumor clones. d, EMAPII immunohistochemistry of 4-week s.c. in vivo tumors established in nude mice bearing wild-type 1286 melanoma (1), wild-type Pmel melanoma (2), Pmel transduced with the pSAMEN backbone vector control (3), and Pmel tumor clone 8 transduced with pWU-EII (4). 210

Our results demonstrate that EMAPII production by tumors can influence their sensitivity to systemic TNF. The mechanism by which EMAPII renders tumor vasculature sensitive to the proinflammatory effects of TNF is not clearly understood. Previous work has demonstrated that recombinant EMAPII can up-regulate endothelial cell TNFR1 expression in a dose-dependent fashion (35). Because TNFR1 expression has been associated with the induction of endothelial cell apoptosis, it follows that EMAPII produced by tumors may determine in vivo sensitivity to TNF by up-regulating TNFR1 expression on endothelial cells triggering cell death in the presence of TNF, leading to eventual ischemic necrosis of the tumor. An alternative mechanism to explain EMAPII sensitization of tumor vascular endothelium to TNF would be an additive effect on endothelial cell tissue factor expression. TNF has been found to induce tissue factor expression on endothelial surfaces (36). In fact, the use of neutralizing TNF receptor antibodies inhibits endothelial tissue factor production induced by TNF (37). There may exist a critical threshold of tissue factor expression by tumor associated vasculature that provokes TNF-induced thrombohemorrhage and EMAPII may function by promoting this process of tumor vascular activation. The essential role of EMAPII, a tumor-derived cytokine, in determining TNF sensitivity provides further insight into the interactions between tumor cells and tumor neovasculature. The procoagulant effect on the vascular endothelial cells induced by tissue factor production in response to high levels of EMAPII elaborated by tumor cells may play an important role in tumor neovascularization, primary tumor growth, metastatic potential, and sensitivity to therapeutic agents. Clinical studies are needed to determine whether EMAPII expression in tumors can predict clinical response to TNF therapy and potentially identify patients with cytokine-responsive tumors that may respond to lower TNF doses, thus, reducing dose-related toxicity. Furthermore, novel methods of delivery of bioactive agents such as EMAPII may result in promising therapeutic approaches against human malignancies.

ACKNOWLEDGMENTS

We thank Drs. David Stern (New York, NY), Michael Nishimura (Bethesda, MD), and Steven Rosenberg (Bethesda, MD) for helpful comments and sug- gestions.

REFERENCES 1. O’Malley, W. E., Achinstein, B., and Shear, M. J. Action of bacterial polysaccharide on tumors. II. Damage of sarcoma 37 by serum of mice treated with Serratia Marcescens polysaccharide, and induced tolerance. J. Natl. Cancer Inst., 29: 1169– 1175, 1962. 2. Carswell, E. A., Old, L. J., Kassel, R. L., Green, S., Fiore, N., and Williamson, B. An Fig. 7. In vivo TNF sensitivity of tumor-bearing nude mice who received inoculations endotoxin-induced serum factor that causes necrosis of tumors. Proc. Natl. Acad. Sci. of Pmel-SAMEN retroviral vector control (P is not significant; a). b, Pmel-EII.08 tumor USA, 72: 3666–3670, 1975. clone overexpressing EMAPII (day 2, P Ͻ0.05; days 4 and 7, P Ͻ0.001). c, Pmel-EII.09 3. Fraker, D. L., Alexander, H. R., and Pass, H. I. Biologic therapy with TNF: systemic tumor clone overexpressing EMAPII (days 2, 4, and 7, P Ͻ0.001). Pmel tumor clones administration and isolation-perfusion. In: V. DeVita, S. Hellman, and S. A. Rosen- overexpressing EMAPII demonstrate a phenotypic change into TNF-sensitive tumors berg (eds.), Biologic Therapy of Cancer, pp. 329–345. Philadelphia: J. B. Lippincott, from the TNF-resistant wild-type tumor. Data are shown as means Ϯ SE of three 1995. experiments. 4. Lienard, D., Ewalenko, P., Delmotti, J. J., Renard, N., and Lejeune, F. J. High-dose recombinant tumor necrosis factor ␣ in combination with interferon ␥ and melphalan in isolation perfusion of the limbs for melanoma and sarcoma. J. Clin. Oncol., 10: A direct causal effect of EMAPII in determining sensitivity to TNF 52–60, 1992. is shown by our retroviral transduction studies. The constituitive 5. Alexander, H. R., Fraker, D. L., and Bartlett, D. L. Isolated limb perfusion for malignant melanoma. Semin. Surg. Oncol., 12: 416–428, 1996. overexpression of EMAPII in a TNF-resistant melanoma line (Pmel) 6. Alexander, H. R., Jr., Bartlett, D. L., Libutti, S. K., Fraker, D. L., Moser, T., and by retroviral-mediated transfer of the EMAPII cDNA renders the Rosenberg, S. A. Isolated hepatic perfusion with tumor necrosis factor and melphalan wild-type tumor sensitive to the effects of systemic TNF. The dem- for unresectable cancers confined to the liver. J. Clin. Oncol., 16: 1479–1489, 1998. 7. Bartlett, D. L., Ma, G., Alexander, H. R., Libutti, S. K., and Fraker, D. L. Isolated onstration of this effect in more than one transduced tumor clone, limb reperfusion with tumor necrosis factor and melphalan in patients with extremity while not absolutely ruling out, does argue against the possibility of melanoma after failure of isolated limb perfusion with chemotherapeutics. Cancer (Phila.), 80: 2084–2090, 1997. selecting a TNF-sensitive wild-type clone by chance from the tumor 8. Eggermont, A. M., Schraffordt, K. H., Klausner, J. M., Lienard, D., Kroon, B. B., line. Schlag, P. M., Ben-Ari, G., and Lejeune, F. J. Isolation limb perfusion with tumor 211

necrosis factor ␣ and chemotherapy for advanced extremity soft tissue sarcomas. 24. O’Reilly, M. S., Boehm, T., Shing, Y., Fukai, N., Vasios, G., Lane, W. S., Flynn, E., Semin. Oncol., 24: 547–555, 1997. Birkhead, J. R., Olsen, B. R., and Folkman, J. Endostatin. An endogenous inhibitor of 9. Eggermont, A. M. M., Koops, H. S., Klausner, J. M., Kroon, B. B. R., Schlag, P. M., angiogenesis and tumor growth. Cell, 88: 277–285, 1997. Lie´nard, D.v. G. A. N., Hoekstra, H. J., Meller, I., Nieweg, O. E., Kettelhack, C., 25. Hanahan, D., and Folkman, J. Patterns and emerging mechanisms of the angiogenic Ben-Ari, G., Pector, J-C., and Lejeune, F. J. Isolated limb perfusion with tumor switch during tumorigenesis. Cell, 86: 353–364, 1996. necrosis factor and melphalan for limb salvage in 186 patients with locally advanced 26. Czubayko, F., Liaudet-Coopman, E. D. E., Aigner, A., Tuveson, A. T., Berchem, soft tissue extremity sarcomas. Ann. Surg., 224: 756–765, 1996. G. J., and Wellstein, A. A secreted FGF-binding protein can serve as the angiogenic ␥ 10. Barth, R. J., Mule´, J. J., Spiess, P. J., and Rosenberg, S. A. Interferon- and tumor switch in human cancer. Nature (Lond.), 3: 1137–1140, 1997. ϩ necrosis factor have a role in tumor regressions mediated by murine CD8 tumor 27. Peverali, F. A., Mandriota, S. J., Ciana, P., Marelli, R., Quax, P., Rifkin, D. B., Valle, infiltrating lymphocytes. J. Exp. Med., 173: 647–658, 1991. G. D., and Mignatti, P. Tumor cells secrete an angiogenic factor that stimulates basic 11. Asher, A. L., Mule`, J. J., and Rosenberg, S. A. Recombinant human tumor necrosis fibroblast growth factor and urokinase expression in vascular endothelial cells. J. Cell. factor mediates regression of a murine sarcoma in vivo via Lyt-2ϩ cells. Cancer Physiol., 161: 1–14, 1994. Immunol. Immunother., 28: 153–156, 1989. 28. Kao, J., Ryan, J., Brett, G., Chen, J., Shen, H., Fan, Y-G., Godman, G., Familletti, P., 12. Donato, N. J., and Perez, M. Tumor necrosis factor-induced apoptosis stimulates p53 Wang, F., Pan, Y-C., Stern, D., and Clauss, M. Endothelial monocyte-activating accumulation and p21WAF1 proteolysis in ME-180 cells. J. Biol. Chem., 273: 5067–5072, 1998. polypeptide II: a novel tumor-derived polypeptide that activates host-response mech- 13. Van Antwerp, D. J., Martin, S. J., Kafri, T., Green, D. R., and Verma, I. M. anisms. J. Biol. Chem., 267: 20239–20247, 1992. Suppression of TNF-␣-induced apoptosis by NF-␬ B. Science (Washington DC), 274: 29. Kao, J., Houck, K., Fan, Y., Hzehnel, I., Libutti, S., Kayton, M., Grikscheit, T., 787–789, 1996. Chabot, J., Nowygrod, R., Greenberg, S., Kuang, W-J., Leung, D., Hayward, J., 14. Wang, C-Y., Mayo, M. W., and Baldwin, A. S., Jr. TNF- and cancer therapy-induced Kisiel, W., Heath, M., Brett, J., and Stern, D. Characterization of a novel tumor- apoptosis: potentiation by inhibition of NF-␬ B. Science (Washington DC), 274: derived cytokine: endothelial-monocyte activating polypeptide II. J. Biol. Chem., 784–787, 1996. 269: 25106–25119, 1994. 15. Hsu, H., Shu, H. B., Pan, M. G., and Goeddel, D. V. TRADD-TRAF2 and TRADD- 30. Marvin, M., Libutti, S., Kayton, M., Kao, J., Hayward, J., Grikscheit, T., Fan, Y., FADD interactions define two distinct TNF receptor 1 signal transduction pathways. Brett, J., Weinberg, A., Nowygrod, R., LoGerfo, P., Feind, C., Hansen, K., Schwartz, Cell, 84: 299–308, 1996. M., Stern, D., and Chabot, J. A novel tumor-derived mediator that sensitizes cytokine- 16. Boldin, M. P., Goncharov, T. M., Goltsev, Y. V., and Wallach, D. Involvement of resistant tumors to tumor necrosis factor. J. Surg. Res., 63: 248–255, 1996. MACH, a novel MORTI/FADD-interacting protease, in Fas/APO-1 and TNF recep- 31. Wang, G., Chopra, R. K., Royal, R. E., Yang, J. C., Rosenberg, S. A., and Hwu, P. tor-induced cell death. Cell, 85: 803–815, 1996. A T cell-independent antitumor response in mice with bone marrow cells retrovirally 17. Van De Viel, P. A., Bloksma, N., Kuper, C. F., Hofhuis, F. M., and Willers, J. M. transduced with an antibody/Fc-␥ chain chimeric receptor gene recognizing a human Macroscopic and microscopic early effects of tumour necrosis factor on murine meth ovarian cancer antigen. Nat. Med., 4: 168–172, 1998. A sarcoma, and relation to curative activity. J. Pathol., 75: 65–73, 1989. 32. Hoatlin, M. E., Kozak, S. L., Spiro, C., and Kabat, D. Amplified and tissue-directed 18. Renard, N., Lie´nard, D., Lespagnard, L., Eggermont, A., Heimann, R., and Lejeune, expression of retroviral vectors using ping-pong techniques. J. Mol. Med., 73: F. Early endothelium activation and polymorphonuclear cell invasion precede specific 113–120, 1995. necrosis of human melanoma and sarcoma treated by intravascular high-dose tumour 33. Contrino, J., Hair, G., Kruetzer, D. L., and Rickles, F. R. In situ detection of tissue ␣ ␣ necrosis factor (rTNF ). Int. J. Cancer, 57: 656–663, 1994. factor in vascular endothelial cells: correlation with the malignant phenotype of 19. Nawroth, P. P., and Stern, D. M. Modulation of endothelial cell hemostatis properties human breast disease. Nat. Med., 2: 209–215, 1996. by tumor necrosis factor. J. Exp. Med., 163: 740–745, 1986. 34. Tas, M., Houghton, J., Jakobsen, A., Tomachova, T., Carmichael, J., and Murray, J. 20. Nawroth, P., Handley, D., Matsueda, G., de Waal, R., Gerlach, H., Blohm, D., and Cloning and expression of human endothelial-monocyte-activating polypeptide 2 Stern, D. Tumor necrosis factor/cachectin-induced intravascular fibrin formation in (EMAP-2) and identification of its putative precursor. Cytokine, 9: 535–539, 1997. meth A fibrosarcomas. J. Exp. Med., 168: 637–647, 1988. 35. Libutti, S., Vargas, H., Wu, P., Turner, E., Bartlett, D., Eliot, H., Brown, C., Wong, 21. Clauss, M., Murray, J. C., Vianna, M., de Waal, R., Thurston, G., Nawroth, P., Gerlach, H., Bach, R., Familletti, P. C., and Stern, D. A polypeptide factor produced G., Stern, D., Fraker, D., and Alexander, H. R. Endothelial monocyte activating by fibrosarcoma cells that induces endothelial tissue factor and enhances the proco- polypeptide II (EMAP II) induces expression of tumor necrosis factor (TNF) receptor agulant response to tumor necrosis factor/cachectin. J. Biol. Chem., 265: 7078–7083, P55 (R1) by endothelial cells. Proc. Soc. Surg. Oncol., Abstract 70, 1997. 1990. 36. Tijburg, P. N. M., Ryan, J., Stern, D. M., Wollitzky, B., Rimon, S., Rimon, A., 22. Olieman, A. F. T., van Ginkel, R. J., Hoekstra, H. J., Mooyaart, E. L., Molenaar, Handley, D., Nawroth, P., Sixma, J. J., and de Groot, P. G. Activation of the W. M., and Koops, H. S. Angiographic response of locally advanced soft-tissue coagulation mechanism on tumor necrosis factor-stimulated cultured endothelial cells sarcoma following hyperthermic isolated limb perfusion with tumor necrosis factor. and their extracellular matrix. J. Biol. Chem., 266: 12067–12074, 1991. Ann. Surg. Oncol., 4: 64–69, 1997. 37. Clauss, M., Grell, M., Fangman, C., Fiers, W., Scheurich, P., and Risau, W. Syner- 23. Kao, J., Fan, Y-G., Haehnel, I., Clauss, M., and Stern, D. Endothelial-monocyte gistic induction of endothelial tissue factor by tumor necrosis factor and vascular activating polypeptides (EMAPs): tumor derived mediators which activate the host endothelial growth factor: functional analysis of the tumor necrosis factor receptors inflammatory response. Behring Inst. Mitt., 92: 92–106, 1993. (Abstract). FEBS Lett., 390: 334–338, 1996.

212

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1999 American Association for Cancer Research. In Vivo Sensitivity of Human Melanoma to Tumor Necrosis Factor (TNF)- α Is Determined by Tumor Production of the Novel Cytokine Endothelial-Monocyte Activating Polypeptide II (EMAPII)

Peter C. Wu, H. Richard Alexander, James Huang, et al.

Cancer Res 1999;59:205-212.

Updated version Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/59/1/205

Cited articles This article cites 31 articles, 12 of which you can access for free at: http://cancerres.aacrjournals.org/content/59/1/205.full#ref-list-1

Citing articles This article has been cited by 7 HighWire-hosted articles. Access the articles at: http://cancerres.aacrjournals.org/content/59/1/205.full#related-urls

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://cancerres.aacrjournals.org/content/59/1/205. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.